Judith Runnels

Here, using two-photon phosphorescence lifetime microscopy, the local oxygen tension in the bone marrow of live mice is found to be quite low, with spatiotemporal variations depending on the blood vessel type, distance to the endosteum, and changes in cellularity after stress. Low oxygen tension (hypoxia) is commonly thought to be a shared niche characteristic in maintaining quiescence in many stem cell types. However, local oxygen concentration, for example in the bone marrow, has never been measured directly. Charles Lin and colleagues have now developed a method based on two-photon microscopy to measure the absolute local oxygen tension (pO2) in the marrow of live animals. Using this method, they found that while vascular density is high throughout the bone marrow, overall oxygenation is quite low and there is heterogeneity in local pO2 with respect to vessel type and location. For example, surprisingly, the endosteal region is not the region of the lowest pO2. After radiation or chemotherapy, bone marrow pO2 becomes elevated and transplanted haematopoietic stem/progenitor cells do not seek out regions with the lowest pO2 for homing. Characterization of how the microenvironment, or niche, regulates stem cell activity is central to understanding stem cell biology and to developing strategies for the therapeutic manipulation of stem cells1. Low oxygen tension (hypoxia) is commonly thought to be a shared niche characteristic in maintaining quiescence in multiple stem cell types2,3,4. However, support for the existence of a hypoxic niche has largely come from indirect evidence such as proteomic analysis5, expression of hypoxia inducible factor-1α (Hif-1α) and related genes6, and staining with surrogate hypoxic markers (for example, pimonidazole)6,7,8. Here we perform direct in vivo measurements of local oxygen tension ( ) in the bone marrow of live mice. Using two-photon phosphorescence lifetime microscopy, we determined the absolute of the bone marrow to be quite low (<32 mm Hg) despite very high vascular density. We further uncovered heterogeneities in local , with the lowest (∼9.9 mm Hg, or 1.3%) found in deeper peri-sinusoidal regions. The endosteal region, by contrast, is less hypoxic as it is perfused with small arteries that are often positive for the marker nestin. These values change markedly after radiation and chemotherapy, pointing to the role of stress in altering the stem cell metabolic microenvironment.

The organization of cellular niches is known to have a key role in regulating normal stem cell differentiation and regeneration, but relatively little is known about the architecture of microenvironments that support malignant metastasis. Using dynamic in vivo confocal imaging, here we show that murine bone marrow contains unique anatomic regions defined by specialized endothelium. This vasculature expresses the adhesion molecule E-selectin and the chemoattractant stromal-cell-derived factor 1 (SDF-1) in discrete, discontinuous areas that influence the homing of a variety of tumour cell lines. Disruption of the interactions between SDF-1 and its receptor CXCR4 inhibits the homing of Nalm-6 cells (an acute lymphoblastic leukaemia cell line) to these vessels. Further studies revealed that circulating leukaemic cells can engraft around these vessels, suggesting that this molecularly distinct vasculature demarcates a microenvironment for early metastatic tumour spread in bone marrow. Finally, purified haematopoietic stem/progenitor cells and lymphocytes also localize to the same microdomains, indicating that this vasculature might also function in benign states to demarcate specific portals for the entry of cells into the marrow space. Specialized vascular structures therefore appear to delineate a microenvironment with unique physiology that can be exploited by circulating malignant cells.

Abstract The mechanisms by which multiple myeloma (MM) cells migrate and home to the bone marrow are not well understood. In this study, we sought to determine the effect of the chemokine SDF-1 (CXCL12) and its receptor CXCR4 on the migration and homing of MM cells. We demonstrated that CXCR4 is differentially expressed at high levels in the peripheral blood and is down-regulated in the bone marrow in response to high levels of SDF-1. SDF-1 induced motility, internalization, and cytoskeletal rearrangement in MM cells evidenced by confocal microscopy. The specific CXCR4 inhibitor AMD3100 and the anti-CXCR4 antibody MAB171 inhibited the migration of MM cells in vitro. CXCR4 knockdown experiments demonstrated that SDF-1–dependent migration was regulated by the PI3K and ERK/MAPK pathways but not by p38 MAPK. In addition, we demonstrated that AMD3100 inhibited the homing of MM cells to the bone marrow niches using in vivo flow cytometry, in vivo confocal microscopy, and whole body bioluminescence imaging. This study, therefore, demonstrates that SDF-1/CXCR4 is a critical regulator of MM homing and that it provides the framework for inhibitors of this pathway to be used in future clinical trials to abrogate MM trafficking.

The interaction of multiple myeloma (MM) cells with their microenvironment in the bone marrow (BM) provides a protective environment and resistance to therapeutic agents. We hypothesized that disruption of the interaction of MM cells with their BM milieu would lead to their sensitization to therapeutic agents such as bortezomib, melphalan, doxorubicin, and dexamethasone. We report that the CXCR4 inhibitor AMD3100 induces disruption of the interaction of MM cells with the BM reflected by mobilization of MM cells into the circulation in vivo, with kinetics that differed from that of hematopoietic stem cells. AMD3100 enhanced sensitivity of MM cell to multiple therapeutic agents in vitro by disrupting adhesion of MM cells to bone marrow stromal cells (BMSCs). Moreover, AMD3100 increased mobilization of MM cells to the circulation in vivo, increased the ratio of apoptotic circulating MM cells, and enhanced the tumor reduction induced by bortezomib. Mechanistically, AMD3100 significantly inhibited Akt phosphorylation and enhanced poly(ADP-ribose) polymerase (PARP) cleavage as a result of bortezomib, in the presence of BMSCs in coculture. These experiments provide a proof of concept for the use of agents that disrupt interaction with the microenvironment for enhancement of efficacy of cytotoxic agents in cancer therapy.

Detailed genomic studies have shown that cytogenetic abnormalities contribute to multiple myeloma (MM) pathogenesis and disease progression. Nevertheless, little is known about the characteristics of MM at the epigenetic level and specifically how microRNAs regulate MM progression in the context of the bone marrow milieu. Therefore, we performed microRNA expression profiling of bone marrow derived CD138(+) MM cells versus their normal cellular counterparts and validated data by qRT-PCR. We identified a MM-specific microRNA signature characterized by down-expression of microRNA-15a/-16 and overexpression of microRNA-222/-221/-382/-181a/-181b (P < .01). We investigated the functional role of microRNA-15a and -16 and showed that they regulate proliferation and growth of MM cells in vitro and in vivo by inhibiting AKT serine/threonine-protein-kinase (AKT3), ribosomal-protein-S6, MAP-kinases, and NF-kappaB-activator MAP3KIP3. Moreover, miRNA-15a and -16 exerted their anti-MM activity even in the context of the bone marrow milieu in vitro and in vivo. These data indicate that microRNAs play a pivotal role in the biology of MM and represent important targets for novel therapies in MM.

A single hematopoietic stem cell (HSC) is capable of reconstituting hematopoiesis and maintaining homeostasis by balancing self-renewal and cell differentiation. The mechanisms of HSC division balance, however, are not yet defined. Here we demonstrate, by characterizing at the single-cell level a purified and minimally heterogeneous murine Tie2+ HSC population, that these top hierarchical HSCs preferentially undergo symmetric divisions. The induction of mitophagy, a quality control process in mitochondria, plays an essential role in self-renewing expansion of Tie2+ HSCs. Activation of the PPAR (peroxisome proliferator-activated receptor)-fatty acid oxidation pathway promotes expansion of Tie2+ HSCs through enhanced Parkin recruitment in mitochondria. These metabolic pathways are conserved in human TIE2+ HSCs. Our data thus identify mitophagy as a key mechanism of HSC expansion and suggest potential methods of cell-fate manipulation through metabolic pathways.

Waldenstrom macroglobulinemia (WM) is an incurable low-grade lymphoplasmacytic lymphoma. We demonstrate up-regulated Akt activity in WM, and that Akt down-regulation by Akt knockdown and the inhibitor perifosine leads to significant inhibition of proliferation and induction of apoptosis in WM cells in vitro, but not in normal donor peripheral blood and hematopoietic progenitors. Importantly, down-regulation of Akt induced cytotoxicity of WM cells in the bone marrow microenvironment (BMM) context. Perifosine induced significant reduction in WM tumor growth in vivo in a subcutaneous xenograft model through inhibition of Akt phosphorylation and downstream targets. We also demonstrated that Akt pathway down-regulation inhibited migration and adhesion in vitro and homing of WM tumor cells to the BMM in vivo. Proteomic analysis identified other signaling pathways modulated by perifosine, such as activation of ERK MAPK pathway, which induces survival of tumor cells. Interestingly, MEK inhibitor significantly enhanced perifosine-induced cytotoxicity in WM cells. Using Akt knockdown experiments and specific Akt and PI3K inhibitors, we demonstrated that ERK activation is through a direct effect, rather than feedback activation, of perifosine upstream ERK pathway. These results provide understanding of biological effects of Akt pathway in WM and provide the framework for clinical evaluation of perifosine in WM patients.

Waldenström macroglobulinemia (WM) cells present with increased expression of microRNA-206 (miRNA-206) and reduced expression of miRNA-9*. Predicted miRNA-206- and -9*-targeted genes include histone deacetylases (HDACs) and histone acetyl transferases (HATs), indicating that these miRNAs may play a role in regulating histone acetylation. We were able to demonstrate that primary WM cells are characterized by unbalanced expression of HDACs and HATs, responsible for decreased acetylated histone-H3 and -H4, and increased HDAC activity. We next examined whether miRNA-206 and -9* modulate the aberrant expression of HDAC and HATs in WM cells leading to increased transcriptional activity. We found that restoring miRNA-9* levels induced toxicity in WM cells, supported by down-modulation of HDAC4 and HDAC5 and up-regulation of acetyl-histone-H3 and -H4. These, together with inhibited HDAC activity, led to induction of apoptosis and autophagy in WM cells. To further confirm that miRNA-9*-dependent modulation of histone acetylation is responsible for induction of WM cytotoxicity, a novel class of HDAC inhibitor (LBH589) was used; we confirmed that inhibition of HDAC activity leads to toxicity in this disease. These findings confirm that histone-modifying genes and HDAC activity are deregulated in WM cells, partially driven by the aberrant expression of miRNA-206 and -9* in the tumor clone.

Waldenstrom macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow at the time of diagnosis, implying continuous homing of WM cells into the marrow. The mechanisms by which trafficking of the malignant cells into the bone marrow has not been previously elucidated. In this study, we show that WM cells express high levels of chemokine and adhesion receptors, including CXCR4 and VLA-4. We showed that CXCR4 was essential for the migration and trans-endothelial migration of WM cells under static and dynamic shear flow conditions, with significant inhibition of migration using CXCR4 knockdown or the CXCR4 inhibitor AMD3100. Similarly, CXCR4 or VLA-4 inhibition led to significant inhibition of adhesion to fibronectin, stromal cells, and endothelial cells. Decreased adhesion of WM cells to stromal cells by AMD3100 led to increased sensitivity of these cells to cytotoxicity by bortezomib. To further investigate the mechanisms of CXCR4-dependent adhesion, we showed that CXCR4 and VLA-4 directly interact in response to SDF-1, we further investigated downstream signaling pathways regulating migration and adhesion in WM. Together, these studies demonstrate that the CXCR4/SDF-1 axis interacts with VLA-4 in regulating migration and adhesion of WM cells in the bone marrow microenvironment.

Abstract Multilevel genetic characterization of Waldenström macroglobulinemia (WM) is required to improve our understanding of the underlying molecular changes that lead to the initiation and progression of this disease. We performed microRNA-expression profiling of bone marrow–derived CD19+ WM cells, compared with their normal cellular counterparts and validated data by quantitative reverse-transcription–polymerase chain reaction (qRT-PCR). We identified a WM-specific microRNA signature characterized by increased expression of microRNA-363*/-206/-494/-155/-184/-542-3p, and decreased expression of microRNA-9* (ANOVA; P &lt; .01). We found that microRNA-155 regulates proliferation and growth of WM cells in vitro and in vivo, by inhibiting MAPK/ERK, PI3/AKT, and NF-κB pathways. Potential microRNA-155 target genes were identified using gene-expression profiling and included genes involved in cell-cycle progression, adhesion, and migration. Importantly, increased expression of the 6 miRNAs significantly correlated with a poorer outcome predicted by the International Prognostic Staging System for WM. We further demonstrated that therapeutic agents commonly used in WM alter the levels of the major miRNAs identified, by inducing downmodulation of 5 increased miRNAs and up-modulation of patient-downexpressed miRNA-9*. These data indicate that microRNAs play a pivotal role in the biology of WM; represent important prognostic marker; and provide the basis for the development of new microRNA-based targeted therapies in WM.

Abstract The interaction of multiple myeloma (MM) cells with the bone marrow (BM) milieu plays a crucial role in MM pathogenesis. Stromal cell–derived factor-1 (SDF1) regulates homing of MM cells to the BM. In this study, we examined the role of RhoA and Rac1 GTPases in SDF1-induced adhesion and chemotaxis of MM. We found that both RhoA and Rac1 play key roles in SDF1-induced adhesion of MM cells to BM stromal cells, whereas RhoA was involved in chemotaxis and motility. Furthermore, both ROCK and Rac1 inhibitors reduced SDF1-induced polymerization of actin and activation of LIMK, SRC, FAK, and cofilin. Moreover, RhoA and Rac1 reduced homing of MM cells to BM niches. In conclusion, we characterized the role of RhoA and Rac1 GTPases in SDF1-induced adhesion, chemotaxis, and homing of MM cells to the BM, providing the framework for targeting RhoA and Rac1 GTPases as novel MM therapy.

The nuclear factor-kappaB (NF-kappaB) path-way has been implicated in tumor B-cell survival, growth, and resistance to therapy. Because tumor cells overcome single-agent antitumor activity, we hypothesized that combination of agents that target differentially NF-kappaB pathway will induce significant cytotoxicity. Therapeutic agents that target proteasome and Akt pathways should induce significant activity in B-cell malignancies as both pathways impact NF-kappaB activity. We demonstrated that perifosine and bortezomib both targeted NF-kappaB through its recruitment to the promoter of its target gene IkappaB using chromatin immunoprecipitation assay. This combination led to synergistic cytotoxicity in Waldenstrom macroglobulinemia (WM) cells that was mediated through a combined reduction of the PI3K/Akt and ERK signaling pathways, found to be critical for survival of WM cells. Moreover, a combination of these drugs with the CD20 monoclonal antibody rituximab further increased their cytotoxic activity. Thus, effective WM therapy may require combination regimens targeting the NF-kappaB pathway.

Abstract We have previously shown clinical activity of a mammalian target of rapamycin (mTOR) complex 1 inhibitor in Waldenstrom macroglobulinemia (WM). However, 50% of patients did not respond to therapy. We therefore examined mechanisms of activation of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR in WM, and mechanisms of overcoming resistance to therapy. We first demonstrated that primary WM cells show constitutive activation of the PI3K/Akt pathway, supported by decreased expression of phosphate and tensin homolog tumor suppressor gene (PTEN) at the gene and protein levels, together with constitutive activation of Akt and mTOR. We illustrated that dual targeting of the PI3K/mTOR pathway by the novel inhibitor NVP-BEZ235 showed higher cytotoxicity on WM cells compared with inhibition of the PI3K or mTOR pathways alone. In addition, NVP-BEZ235 inhibited both rictor and raptor, thus abrogating the rictor-induced Akt phosphorylation. NVP-BEZ235 also induced significant cytotoxicity in WM cells in a caspase-dependent and -independent manner, through targeting the Forkhead box transcription factors. In addition, NVP-BEZ235 targeted WM cells in the context of bone marrow microenvironment, leading to significant inhibition of migration, adhesion in vitro, and homing in vivo. These studies therefore show that dual targeting of the PI3K/mTOR pathway is a better modality of targeted therapy for tumors that harbor activation of the PI3K/mTOR signaling cascade, such as WM.

Circulating tumor cells (CTCs) are of great interest in cancer research, but methods for their enumeration remain far from optimal. We developed a new small animal research tool called "Diffuse in vivo Flow Cytometry" (DiFC) for detecting extremely rare fluorescently-labeled circulating cells directly in the bloodstream. The technique exploits near-infrared diffuse photons to detect and count cells flowing in large superficial arteries and veins without drawing blood samples. DiFC uses custom-designed, dual fiber optic probes that are placed in contact with the skin surface approximately above a major vascular bundle. In combination with a novel signal processing algorithm, DiFC allows counting of individual cells moving in arterial or venous directions, as well as measurement of their speed and depth. We show that DiFC allows sampling of the entire circulating blood volume of a mouse in under 10 minutes, while maintaining a false alarm rate of 0.014 per minute. In practice, this means that DiFC allows reliable detection of circulating cells below 1 cell per mL. Hence, the unique capabilities of DiFC are highly suited to biological applications involving very rare cell types such as the study of hematogenous cancer metastasis.

Waldenström macroglobulinemia (WM) is an incurable low-grade B-cell lymphoma characterized by high protein turnover. We dissected the biologic role of the proteasome in WM using 2 proteasome inhibitors, NPI-0052 and bortezomib. We found that NPI-0052 inhibited proliferation and induced apoptosis in WM cells, and that the combination of NPI-0052 and bortezomib induced synergistic cytotoxicity in WM cells, leading to inhibition of nuclear translocation of p65NF-kappaB and synergistic induction of caspases-3, -8, and -9 and PARP cleavage. These 2 agents inhibited the canonical and noncanonical NF-kappaB pathways and acted synergistically through their differential effect on Akt activity and on chymotrypsin-like, caspaselike, and trypsinlike activities of the proteasome. We demonstrated that NPI-0052-induced cytotoxicity was completely abrogated in an Akt knockdown cell line, indicating that its major activity is mediated through the Akt pathway. Moreover, we demonstrated that NPI-0052 and bortezomib inhibited migration and adhesion in vitro and homing of WM cells in vivo, and overcame resistance induced by mesenchymal cells or by the addition of interleukin-6 in a coculture in vitro system. Theses studies enhance our understanding of the biologic role of the proteasome pathway in WM, and provide the preclinical basis for clinical trials of combinations of proteasome inhibitors in WM.

Proteasome inhibition represents a valid antitumor approach and its use has been validated in Waldenström macroglobulinemia (WM), where bortezomib has been successfully tested in clinical trials. Nevertheless, a significant fraction of patients relapses, and many present toxicity due to its off-target effects. Selective inhibition of the chymotrypsin-like (CT-L) activity of constitutive proteasome 20S (c20S) and immunoproteasome 20S (i20S) represents a sufficient and successful strategy to induce antineoplastic effect in hematologic tumors. We therefore studied ONX0912, a novel selective, irreversible inhibitor of the CT-L activity of i20S and c20S. Primary WM cells express higher level of i20S compared with c20S, and that ONX0912 inhibited the CT-L activity of both i20S and c20S, leading to induction of toxicity in primary WM cells, as well as of apoptosis through c-Jun N-terminal kinase activation, nuclear factor κB (NF-κB) inhibition, caspase cleavage, and initiation of the unfolded protein response. Importantly, ONX0912 exerted toxicity in WM cells, by reducing bone marrow (BM)–derived interleukin-6 (IL-6) and insulin-like growth factor 1 (IGF-1) secretion, thus inhibiting BM-induced p-Akt and phosphorylated extracellular signal-related kinase (p-ERK) activation in WM cells. These findings suggest that targeting i20S and c20S CT-L activity by ONX0912 represents a valid antitumor therapy in WM.

Two bacteriophage T4-induced, nucleic acid-modifying activities, 5′ polynucleotide kinase and 3′ phosphatase, are both coded by the pseT gene. Therefore, the product of this gene is an enzyme which can remove phosphates from 3′ termini and add them to 5′-hydroxyl termini and thus could be said to "shuttle" phosphates on polynucleotides. This enzyme is sometimes required for T4 true-late gene expression, probably by helping establish the required intracellular DNA structure. Our data suggest that a host gene product normally can substitute for the product of the pseT gene, making it non-essential for phage multiplication on most laboratory strains of Escherichia coli.

We describe a novel photoconversion technique to track individual cells in vivo using a commercial lipophilic membrane dye, DiR. We show that DiR exhibits a permanent fluorescence emission shift (photoconversion) after light exposure and does not reacquire the original color over time. Ratiometric imaging can be used to distinguish photoconverted from non-converted cells with high sensitivity. Combining the use of this photoconvertible dye with intravital microscopy, we tracked the division of individual hematopoietic stem/progenitor cells within the calvarium bone marrow of live mice. We also studied the peripheral differentiation of individual T cells by tracking the gain or loss of FoxP3-GFP expression, a marker of the immune suppressive function of CD4+ T cells. With the near-infrared photoconvertible membrane dye, the entire visible spectral range is available for simultaneous use with other fluorescent proteins to monitor gene expression or to trace cell lineage commitment in vivo with high spatial and temporal resolution.

Benzoporphyrin derivative monoacid (BPD-MA) photosensitization was examined for its effects on cellular adhesion of a human ovarian cancer cell line, OVCAR 3, to extracellular matrix (ECM) components. Mild BPD-MA photosensitization (approximately 85% cell survival) of OVCAR 3 transiently decreased adhesion to collagen IV, fibronectin, laminin and vitronectin to a greater extent than could be attributed to cell death. The loss in adhesiveness was accompanied by a loss of beta1 integrin-containing focal adhesion plaques (FAPs), although beta1 subunits were still recognized by monoclonal antibody directed against human beta1 subunits. In vivo BPD-MA photosensitization decreased OVCAR 3 adhesiveness as well. Photosensitized adhesion was reduced in the presence of sodium azide and enhanced in deuterium oxide, suggesting mediation by singlet oxygen. Co-localization studies of BPD-MA and Rhodamine 123 showed that the photosensitizer was largely mitochondrial, but also exhibited extramitochondrial, intracellullar, diffuse cytosolic fluorescence. Taken together, these data show that intracellular damage mediated by BPD-PDT remote from the FAP site can affect cellular-ECM interactions and result in loss of FAP formation. This may have an impact on long-term effects of photodynamic therapy. The topic merits further investigation.

We describe a new method for imaging leukocytes in vivo by exciting the endogenous protein fluorescence in the ultraviolet (UV) spectral region where tryptophan is the major fluorophore. Two-photon excitation near 590 nm allows noninvasive optical sectioning through the epidermal cell layers into the dermis of mouse skin, where leukocytes can be observed by video-rate microscopy to interact dynamically with the dermal vascular endothelium. Inflammation significantly enhances leukocyte rolling, adhesion, and tissue infiltration. After exiting the vasculature, leukocytes continue to move actively in tissue as observed by time-lapse microscopy, and are distinguishable from resident autofluorescent cells that are not motile. Because the new method alleviates the need to introduce exogenous labels, it is potentially applicable for tracking leukocytes and monitoring inflammatory cellular reactions in humans.

Abstract Purpose: We hypothesized that targeting both Akt and heat shock protein (HSP) 90 would induce cytotoxic activity against multiple myeloma (MM) cells and target the bone marrow (BM) microenvironment to inhibit angiogenesis, osteoclast formation, as well as migration and adhesion of MM cells. Experimental Design: MM cell lines were incubated with perifosine (5 and 10 μmol/L) and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG; 50 and 100 nmol/L) alone and in combination. Results: The combination of Akt inhibitor perifosine and HSP90 inhibitor 17-DMAG was synergistic in inducing MM cell cytotoxicity, evidenced by inhibition of DNA synthesis and induction of apoptosis. In addition, perifosine and 17-DMAG almost completely inhibited osteoclast formation: perifosine interfered with both early and late stages of osteoclast progenitor development, whereas 17-DMAG targeted only early stages. We next showed that combined therapy overcomes tumor growth and resistance induced by BM stromal cells and endothelial cells as well as the proliferative effect of exogenous interleukin-6, insulin-like growth factor-I, and vascular endothelial growth factor. Moreover, the combination also induced apoptosis and growth inhibition in endothelial cells and inhibited angiogenesis. Finally, we showed that the two agents prevented migration of MM cells toward stromal-derived factor-1 and vascular endothelial growth factor, which are present in the BM milieu, and also prevented adhesion of MM cells to fibronectin. Conclusions: This study provides the preclinical framework for treatment protocols targeting both the Akt and HSP pathways in MM.

Abstract We provide an overview of the methods used to label circulating cells for fluorescence detection by in vivo flow cytometry. These methods are useful for cell tracking in small animals without the need to draw blood samples and are particularly useful for the detection of circulating cancer cells and quantification of circulating immune cells. © 2011 International Society for Advancement of Cytometry.

There has been significant recent interest in the development of technologies for enumeration of rare circulating cells directly in the bloodstream in many areas of research, for example, in small animal models of circulating tumor cell dissemination during cancer metastasis. We describe a fiber-based optical probe that allows fluorescence detection of labeled circulating cells in vivo in a diffuse reflectance configuration. We validated this probe in a tissue-mimicking flow phantom model in vitro and in nude mice injected with fluorescently labeled multiple myeloma cells in vivo. Compared to our previous work, this design yields an improvement in detection signal-to-noise ratio of 10 dB, virtually eliminates problematic motion artifacts due to mouse breathing, and potentially allows operation in larger animals and limbs.

Multiple myeloma (MM), the second most common hematological malignancy, initiates from a single site and spreads via circulation to multiple sites in the bone marrow (BM). Methods to track MM cells both in the BM and circulation would be useful for developing new therapeutic strategies to target MM cell spread. We describe the use of complementary optical techniques to track human MM cells expressing both bioluminescent and fluorescent reporters in a mouse xenograft model. Long-term tumor growth and response to therapy are monitored using bioluminescence imaging (BLI), while numbers of circulating tumor cells are detected by in-vivo flow cytometry. Intravital microscopy is used to detect early seeding of MM cells to the BM, as well as residual cancer cells that remain in the BM after the bulk of the tumor is eradicated following drug treatment. Thus, intravital microscopy provides a powerful, albeit invasive, means to study cellular processes in vivo at the very early stage of the disease process and at the very late stage of therapeutic intervention when the tumor burden is too small to be detected by other imaging methods.

Circulating tumor cells (CTCs) are of great interest in cancer research because of their crucial role in hematogenous metastasis. We recently developed "diffuse <italic>in vivo</italic> flow cytometry" (DiFC), a preclinical research tool for enumerating extremely rare fluorescently labeled CTCs directly <italic>in vivo</italic>. In this work, we developed a green fluorescent protein (GFP)-compatible version of DiFC and used it to noninvasively monitor tumor cell numbers in circulation in a multiple myeloma (MM) disseminated xenograft mouse model. We show that DiFC allowed enumeration of CTCs in individual mice overtime during MM growth, with sensitivity below 1 CTC mL^{− 1} of peripheral blood. DiFC also revealed the presence of CTC clusters (CTCCs) in circulation to our knowledge for the first time in this model and allowed us to calculate CTCC size, frequency, and kinetics of shedding. We anticipate that the unique capabilities of DiFC will have many uses in preclinical study of metastasis, in particular, with a large number of GFP-expressing xenograft and transgenic mouse models.

Accurate quantification of circulating cell populations in mice is important in many areas of preclinical biomedical research. Normally, this is done either by extraction and analysis of small blood samples or, more recently, by using microscopy-based in vivo fluorescence flow cytometry. We describe a new technological approach to this problem using detection of diffuse fluorescent light from relatively large blood vessels in vivo. The diffuse fluorescence flow cytometer (DFFC) uses a laser to illuminate a mouse limb and an array of optical fibers coupled to a high-sensitivity photomultiplier tube array operating in photon counting mode to detect weak fluorescence signals from cells. We first demonstrate that the DFFC instrument is capable of detecting fluorescent microspheres and Vybrant-DiD-labeled cells in a custom-made optical flow phantom with similar size, optical properties, linear flow rates, and autofluorescence as a mouse limb. We also present preliminary data demonstrating that the DFFC is capable of detecting circulating cells in nude mice in vivo. In principle, this device would allow interrogation of the whole blood volume of a mouse in minutes, with sensitivity improvement by several orders of magnitude compared to current approaches.

Gamma irradiation and bone marrow transplantation (BMT) are established clinical procedures for the treatment of hematologic malignancies. The radiation targets cells in the bone marrow, but injury to other tissues, including the central nervous system (CNS), have been reported. Here, we examine if anti-inflammatory treatment can mitigate the radiation-induced turnover of retinal microglia and the replacement by bone marrow-derived cells (BMDCs).Two-color chimeric mice were generated by lethal irradiation of heterozygous CX3CR1-GFP mice that express GFP in microglial cells and bone marrow transplantation from universal DsRed donor mice. Mice were treated with the corticosteroid dexamethasone; a control group received no dexamethasone treatment. The populations of resident microglia (GFP+) and BMDCs (DsRed+) were quantified by serial in vivo imaging for 10 weeks after irradiation with a confocal scanning laser ophthalmoscope that we custom-built specifically for multicolor imaging of the murine retina.Ionizing radiation resulted in loss of 75% of the resident retinal microglia population after 70 days. Recruitment of BMDCs was delayed with respect to the microglia loss, resulting in a transient depletion of the total immune cell number in the retina. With dexamethasone treatment, both the loss of the resident microglia and the infiltration of BMDCs were suppressed by at least 50%.Anti-inflammatory treatment with the corticosteroidal agent dexamethasone preserves resident microglia and minimizes recruitment of BMDCs after ionizing radiation exposure and BMT.

The product of bacteriophage T4 gene 63 has two activities, one which catalyzes the attachment of tail fibers to base plates during morphogenesis (TFA) and one which catalyzes the joining of single-stranded polynucleotides (RNA ligase). The only phenotype attributed to mutations in gene 63 is a defect in attachment of tail fibers leading to fiberless T4 particles. However, it is suspected that TFA and RNA ligase are unrelated activities of the same protein since they have very different requirements in vitro. We have isolated new mutants which have lost the RNA ligase but have retained the TFA activity of the product of gene 63. These mutants exhibit defects in T4 DNA replication and late gene expression in some strains of Escherichia coli. This work allows us to draw three conclusions: (1) the TFA and RNA ligase activities are unrelated functions of the gene 63 product making this the prototype for a protein which has more than one unrelated function; (2) the RNA ligase is probably involved in DNA metabolism rather than RNA processing as has been proposed: (3) the RNA ligase and polynucleotide 5′ kinase 3′ phosphatase of T4 perform intimately related functions.

Molecular expression on the vascular endothelium is critical in regulating the interaction of circulating cells with the blood vessel wall. Leukocytes as well as circulating cancer cells gain entry into tissue by interacting with adhesion molecules on the endothelial cells (EC). Molecular targets on the EC are increasingly being explored for tissue-specific delivery of therapeutic and imaging agents. Here we use in vivo immunofluorescence microscopy to visualize the endothelial molecular expression in the vasculature of live animals. High-resolution images are obtained by optical sectioning through the intact skin using in vivo confocal and multiphoton microscopy after in situ labeling of EC surface markers with fluorescent antibodies. Other vascular beds such as the bone marrow and ocular blood vessels can be imaged with little or no tissue manipulation. Live imaging is particularly useful for following the dynamic expression of inducible molecules such as E-selectin during an inflammatory response.

Current research on multiphoton autofluorescence microscopy is primarily focused on imaging the signal from reduced nicotinamide adenine dinucleatide (NADH) in tissue. NADH levels in cells are useful reporters of metabolic information, as well as early indicators in precancer and cancer diagnosis. While NADH is typically imaged in the 400-500 nm spectral window, the amino acid tryptophan is the major source of tissue fluorescence in the Ultraviolet range. Here, we briefly review current progress in multiphoton autofluorescence imaging of live tissues and cells, and report our recent findings of <i xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">in vivo</i> mouse skin imaging based on multiphoton excited tryptophan autofluorescence. This new method enables noninvasive imaging of skin tissue at video-rate and allows for the visualization and identification of cellular components in the epidermis, dermis, and muscle layers. It is also possible to image through small blood vessels in the mouse skin and observe circulating leukocytes <i xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">in situ</i> .

Abstract Background Retinal degeneration is a disease affecting the eye, which is an immune-privileged site because of its anatomical and physiological properties. Alterations in retinal homeostasis—because of injury, disease, or aging—initiate inflammatory cascades, where peripheral leukocytes (PL) infiltrate the parenchyma, leading to retinal degeneration. So far, research on PL's role in retinal degeneration was limited to observing a few cell types at specific times or sectioning the tissue. This restricted our understanding of immune cell interactions and response duration. Methods In vivo microscopy in preclinical mouse models can overcome these limitations enabling the spatio-temporal characterization of PL dynamics. Through in vivo imaging, we assessed structural and fluorescence changes in response to a focal injury at a defined location over time. We also utilized minimally invasive techniques, pharmacological interventions, and knockout (KO) mice to determine the role of PL in local inflammation. Furthermore, we investigated PL abundance and localization during retinal degeneration in human eyes by histological analysis to assess to which extent our preclinical study translates to human retinal degeneration. Results We demonstrate that PL, especially T cells, play a detrimental role during retinal injury response. In mice, we observed the recruitment of helper and cytotoxic T cells in the parenchyma post-injury, and T cells also resided in the macula and peripheral retina in pathological conditions in humans. Additionally, we found that the pharmacological PL reduction and genetic depletion of T-cells reduced injured areas in murine retinas and rescued the blood–retina barrier (BRB) integrity. Both conditions promoted morphological changes of Cx3cr1 + cells, including microglial cells, toward an amoeboid phenotype during injury response. Interestingly, selective depletion of CD8 + T cells accelerated recovery of the BRB compared to broader depletions. After anti-CD8 treatment, the retinal function improved, concomitant to a beneficial immune response. Conclusions Our data provide novel insights into the adaptive immune response to retinal injury in mice and human retinal degeneration. Such information is fundamental to understanding retinal disorders and developing therapeutics to modulate immune responses to retinal degeneration safely.

purpose. Hyperfluorescent cells labeled with indocyanine green (ICG) have been observed in retinal and choroidal circulation using scanning laser ophthalmoscopy. It has been suggested that ICG labels leukocytes and that ICG can be used to track leukocyte movement in vivo. The purpose of this study is to identify the cell population that takes up ICG and to study their trafficking pattern in vivo by confocal fluorescence microscopy. methods. ICG was injected into the mouse tail vein, and images were taken by in vivo confocal microscopy. The trafficking pattern of ICG-labeled cells was compared with that of rhodamine 6G-labeled leukocytes. In vitro labeling of human blood cells with antibodies against cell lineage markers and with DNA stains was further used to identify the ICG-labeled cells. Antibodies against the following cell surface markers were used: CD45 (leukocytes), CD3 (T lymphocytes), CD19 (B lymphocytes), CD16 (Fc receptor), glycophorin A (erythroid lineage cells), and CD71 (transferrin receptor). results. The ICG-labeled cells were made up of two blood cell populations with distinct levels of ICG uptake. The strongly ICG-labeled cells did not roll on dermal vascular endothelium in vivo, in contrast to rhodamine 6G–labeled leukocytes. They were identified as reticulocytes because antibody staining showed that they were CD 45−, glycophorin A+ and CD 71+. The weakly ICG-labeled cells were identified as neutrophils because they were CD45+, CD16+, CD3−, and CD19−. conclusions. ICG strongly labels reticulocytes and weakly labels neutrophils. To the authors’ knowledge, this is the first report of selective staining of reticulocytes by ICG.

Transplantation of a single hematopoietic stem cell is an important method for its functional characterization, but the standard transplantation protocol relies on cell homing to the bone marrow after intravenous injection. Here, we present a method to transplant single cells directly into the bone marrow of live mice. We developed an optical platform that integrates a multiphoton microscope with a laser ablation unit for microsurgery and an optical tweezer for cell micromanipulation. These tools allow image-guided single cell transplantation with high spatial control. The platform was used to deliver single hematopoietic stem cells. The engraftment of transplants was tracked over time, illustrating that the technique can be useful for studying both normal and malignant stem cells in vivo.

An Escherichia coli B strain, B834 galU56, has been isolated which supports growth of bacteriophage T4 with cytosine in its DNA while restricting growth of T4 with hydroxymethylcytosine. This host is partially deficient in uridine diphosphoglucose as determined by the ability of DNA isolated from T4 grown on it to accept glucose in an in vitro assay. In this mutant an intact rgl restriction system recognizes unglucosylated hydroxymethylcytosine residues in phage DNA, while the absence of a functional rB restriction function prevents degradation of unmodified DNA containing cytosine.

We examine the effect of bone marrow transplantation (BMT) on retinal cell turnover by performing simultaneous cell tracking of native microglia and engrafting donor bone marrow-derived cell (BMDC) populations in the retinae of live mice using a custom-built multi-color confocal scanning laser ophthalmoscope (SLO) specifically developed for murine retinal imaging. CX3CR1GFP/+ mice whose retinal microglia express the green fluorescent protein (GFP) were exposed to a lethal dose of gamma radiation and subsequently rescued with bone marrow cells from universal DsRed donor mice. Over a time course of four months after the irradiation and BMT, progressive loss of GFP+ microglia was accompanied by delayed engraftment of DsRed+ BMDC. Morphologic examination revealed that the remaining GFP+ microglia were ramified, while engrafting DsRed+ cells exhibited both ramification and dendriform shape. Leukocyte endothelial interaction, normally absent in healthy retinal vasculature, was observed even after three months, indicating sustained inflammation long after the radiation exposure. Fluorescein angiography demonstrated that the blood-retina barrier is compromised early after irradiation. In vivo imaging provides a powerful means to study dynamic cellular processes over a broad range of timescales from seconds to months that have previously not been accessible by ex vivo analysis.

Abstract Purpose: Waldenstrom macroglobulinemia is a lymphoplasmacytic lymphoma characterized by widespread involvement of the bone marrow. Despite different options of therapy, Waldenstrom macroglobulinemia is still incurable. Src tyrosine kinase has been shown to play a central role in the regulation of a variety of biological processes, such as cell proliferation, migration, adhesion, and survival in solid tumors. We sought to determine whether the protein tyrosine kinase Src regulates adhesion, migration, and survival in Waldenstrom macroglobulinemia. Experimental Design: We tested the expression of Src tyrosine kinase in Waldenstrom macroglobulinemia and normal cells, and the effect of the specific Src inhibitor AZD0530 on the adhesion, migration, cell cycle, and survival of a Waldenstrom macroglobulinemia cell line and patient samples. Moreover, we tested the effect of AZD0530 on cytoskeletal and cell cycle signaling in Waldenstrom macroglobulinemia. Results: We show that Src is overexpressed in Waldenstrom macroglobulinemia cells compared with control B cells, and that the use of the Src inhibitor AZD0530 led to significant inhibition of adhesion, migration, and cytoskeletal signaling induced by SDF1. Moreover, inhibition of Src activity induced G1 cell cycle arrest; however, it had minimal effect on survival of Waldenstrom macroglobulinemia cells, and no significant effect on survival of normal cells. Conclusions: Taken together, these results delineate the role of Src kinase activity in Waldenstrom macroglobulinemia and provide the framework for future clinical trials using Src inhibitors in combination with other drugs to improve the outcome of patients with Waldenstrom macroglobulinemia. (Clin Cancer Res 2009;15(19):6035–41)

Sensing and enumeration of specific types of circulating cells in small animals is an important problem in many areas of biomedical research. Microscopy-based fluorescence in vivo flow cytometry methods have been developed previously, but these are typically limited to sampling of very small blood volumes, so that very rare circulating cells may escape detection. Recently, we described the development of a 'diffuse fluorescence flow cytometer' (DFFC) that allows sampling of much larger blood vessels and therefore circulating blood volumes in the hindlimb, forelimb or tail of a mouse. In this work, we extend this concept by developing and validating a method to tomographically localize circulating fluorescently labeled cells in the cross section of a tissue simulating optical flow phantom and mouse limb. This was achieved using two modulated light sources and an array of six fiber-coupled detectors that allowed rapid, high-sensitivity acquisition of full tomographic data sets at 10 Hz. These were reconstructed into two-dimensional cross-sectional images using Monte Carlo models of light propagation and the randomized algebraic reconstruction technique. We were able to obtain continuous images of moving cells in the sample cross section with 0.5 mm accuracy or better. We first demonstrated this concept in limb-mimicking optical flow photons with up to four flow channels, and then in the tails of mice with fluorescently labeled multiple myeloma cells. This approach increases the overall diagnostic utility of our DFFC instrument.

Over the past 50 years, much insight has been gained into the biology of hematopoietic stem cells (HSCs). Much of this information has been gained though isolation of specific bone marrow populations, and transplantation into irradiated recipients followed by characterization of chimeras months later. These studies have yielded insights into the function of HSCs, but have shed little light on the interactions of individual stem cells with their environment. Characterization of the behavior of single HSCs awaited the use of relatively noninvasive intravital microscopy, which allows one to identify rare cells in real time and follow them in multiple imaging sessions. Here we describe techniques used to image transplanted HSCs in the mouse calvarium using hybrid confocal/multi-photon microscopy and second harmonic imaging. For detection, fluorescently tagged HSCs are transplanted into a recipient mouse. The architecture of the bone marrow can be delineated using a combination of fluorescent probes and vascular dyes, second harmonic generation to detect the collagen signal from bone, and transgenic recipient mice containing specific fluorescent support cell populations.

Clonal heterogeneity and selection underpin many biological processes including development and tumor progression. Combinatorial fluorescent protein expression in germline cells has proven its utility for tracking the formation and regeneration of different organ systems. Such cell populations encoded by combinatorial fluorescent proteins are also attractive tools for understanding clonal expansion and clonal competition in cancer. However, the assignment of clonal identity requires an analytical framework in which clonal markings can be parameterized and validated. Here we present a systematic and quantitative method for RGB analysis of fluorescent melanoma cancer clones. We then demonstrate refined clonal trackability of melanoma cells using this scheme.

Detection and enumeration of rare circulating cells in mice are important problems in many areas of preclinical biomedical research. Recently, we developed a new method termed “diffuse fluorescence flow cytometry” (DFFC) that uses diffuse photons to increase the blood sampling volume and sensitivity versus existing in vivo flow cytometry methods. In this work, we describe a new DFFC prototype with approximately an order-of-magnitude improvement in sensitivity compared to our previous work. This sensitivity improvement is enabled by a number of technical innovations, which include a method for the removal of motion artifacts (allowing interrogation of mouse hindlegs that was less optically attenuating versus the tail) and improved collection optics and signal preamplification. We validated our system first in limb mimicking optical flow phantoms with fluorescent microspheres and then in nude mice with fluorescently labeled mesenchymal stem cells at injected concentrations of 5×10 3 cells/mL . In combination, these improvements resulted in an overall cell counting sensitivity of about 1 cell/mL or better in vivo.

Abstract 1. The microsomal mixed-function oxidase (MFO) of the pigeon liver seems to be most active around pH 6·8 (0·1 M phosphate buffer) and 45°C to epoxidize aldrin and epoxidize and hydroxylate chlordene. 2. This level of activity is higher than that reported for other avian species but lower than that for mammalian MFO. 3. The MFO system shows lower levels of cytochrome b 5 and cytochrome P-450 as reported for rats and mice; but much higher levels of NADPH-cytochrome c -reductase activity than that reported for mammals. 4. Cytochrome P-450 binds aliphatic polychlorinated cyclodienes as type I and the aromatic pyridine as type II substrates.

We describe a novel photoconversion technique to track individual cells in vivo using a commercial lipophilic membrane dye, DiR.We show that DiR exhibits a permanent fluorescence emission shift (photoconversion) after light exposure and does not reacquire the original color over time.Ratiometric imaging can be used to distinguish photoconverted from nonconverted cells with high sensitivity.Combining the use of this photoconvertible dye with intravital microscopy, we tracked the division of individual hematopoietic stem/progenitor cells within the calvarium bone marrow of live mice.We also studied the peripheral differentiation of individual T cells by tracking the gain or loss of FoxP3-GFP expression, a marker of the immune suppressive function of CD4 + T cells.With the near-infrared photoconvertible membrane dye, the entire visible spectral range is available for simultaneous use with other fluorescent proteins to monitor gene expression or to trace cell lineage commitment in vivo with high spatial and temporal resolution.

Advances in intravital microscopy (IVM) have enabled the studies of cellular organization and dynamics in the native microenvironment of intact organisms with minimal perturbation. The abilities to track specific cell populations and monitor their interactions have opened up new horizons for visualizing cell biology in vivo , yet the success of standard fluorescence cell labeling approaches for IVM comes with a “dark side” in that unlabeled cells are invisible, leaving labeled cells or structures to appear isolated in space, devoid of their surroundings and lacking proper biological context. Here we describe a novel method for “filling in the void” by harnessing the ubiquity of extracellular (interstitial) fluid and its ease of fluorescence labelling by commonly used vascular and lymphatic tracers. We show that during routine labeling of the vasculature and lymphatics for IVM, commonly used fluorescent tracers readily perfuse the interstitial spaces of the bone marrow (BM) and the lymph node (LN), outlining the unlabeled cells and forming negative contrast images that complement standard (positive) cell labeling approaches. The method is simple yet powerful, offering a comprehensive view of the cellular landscape such as cell density and spatial distribution, as well as dynamic processes such as cell motility and transmigration across the vascular endothelium. The extracellular localization of the dye and the interstitial flow provide favorable conditions for prolonged Intravital time lapse imaging with minimal toxicity and photobleaching.

The paradigm for the treatment of monoclonal gammophaties has dramatically changed: based on the understanding of the complex interaction between tumor cells and bone marrow microenvironment and the signaling pathways that are deregulated in this process, a number of novel therapeutic agents are now available. For example, 3 novel agents with a targeted anti-multiple myeloma activity, have been FDA approved for the treatment of this disease, namely bortezomib, thalidomide, and lenalidomide. The success of targeted therapy in myeloma has led to the development and investigation of more than 30 new compounds in this disease and in other plasma cell dyscrasias such as Waldenström's macroglobulinemia (WM), both in the preclinical settings and as part of clinical trials. Among them the role of proteasome inhibitors has been widely dissected providing the preclinical basis for clinical trials of combinations of proteasome inhibitors in WM.

Published in 2001, the Common European Framework of Reference for Languages (CEFR), a reference framework which informs teaching, learning and assessment in language education, appears to be increasingly recognized, referenced and utilized in language education contexts worldwide. To date however, the extent, provenance and adoption of the collected body of knowledge concerning the CEFR has yet to be systematically analysed, rendering it difficult for any conclusions to be made about its impact. A bibliometric analysis was therefore conducted to explore the CEFR from the document’s more formal origins in 1990 to the end of 2017 for the bibliometric indicators of number of publications per year, geographical location of research, highly cited works and journals with the highest number of relevant publications. The findings show that research on the CEFR has increased significantly over the examined time. The majority of publications with a focus on the CEFR are European, but numbers are increasing in geographical areas outside of Europe, and particularly in Asia. The framework is discussed in numerous types of publications covering a range of topics in language education. These findings suggest that the CEFR has been used in contexts beyond its origins and has influenced many aspects of language education around the globe. Diffusion of innovations theory suggests that the CEFR’s impact and influence is likely to increase over the next ten years in and outside of Europe and especially in Asia.

Within the past few years, major advances in the preclinical and clinical testing of novel therapeutic agents have occurred in Waldenström's macroglobulinemia (WM). These include agents that target the PI3K/Akt/mTOR pathway, PKC pathways, NF-kB signaling pathway, as well as tyrosine kinases and histone deacetylase inhibitors. In this review, we summarize the current understanding of the clinical development of these agents in WM.

Supplementary Data from Src Tyrosine Kinase Regulates Adhesion and Chemotaxis in Waldenstrom Macroglobulinemia

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; Waldenstrom macroglobulinemia is a lymphoplasmacytic lymphoma characterized by widespread involvement of the bone marrow. Despite different options of therapy, Waldenstrom macroglobulinemia is still incurable. Src tyrosine kinase has been shown to play a central role in the regulation of a variety of biological processes, such as cell proliferation, migration, adhesion, and survival in solid tumors. We sought to determine whether the protein tyrosine kinase Src regulates adhesion, migration, and survival in Waldenstrom macroglobulinemia.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; We tested the expression of Src tyrosine kinase in Waldenstrom macroglobulinemia and normal cells, and the effect of the specific Src inhibitor AZD0530 on the adhesion, migration, cell cycle, and survival of a Waldenstrom macroglobulinemia cell line and patient samples. Moreover, we tested the effect of AZD0530 on cytoskeletal and cell cycle signaling in Waldenstrom macroglobulinemia.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; We show that Src is overexpressed in Waldenstrom macroglobulinemia cells compared with control B cells, and that the use of the Src inhibitor AZD0530 led to significant inhibition of adhesion, migration, and cytoskeletal signaling induced by SDF1. Moreover, inhibition of Src activity induced G&lt;sub&gt;1&lt;/sub&gt; cell cycle arrest; however, it had minimal effect on survival of Waldenstrom macroglobulinemia cells, and no significant effect on survival of normal cells.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; Taken together, these results delineate the role of Src kinase activity in Waldenstrom macroglobulinemia and provide the framework for future clinical trials using Src inhibitors in combination with other drugs to improve the outcome of patients with Waldenstrom macroglobulinemia. (Clin Cancer Res 2009;15(19):6035–41)&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; Waldenstrom macroglobulinemia is a lymphoplasmacytic lymphoma characterized by widespread involvement of the bone marrow. Despite different options of therapy, Waldenstrom macroglobulinemia is still incurable. Src tyrosine kinase has been shown to play a central role in the regulation of a variety of biological processes, such as cell proliferation, migration, adhesion, and survival in solid tumors. We sought to determine whether the protein tyrosine kinase Src regulates adhesion, migration, and survival in Waldenstrom macroglobulinemia.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; We tested the expression of Src tyrosine kinase in Waldenstrom macroglobulinemia and normal cells, and the effect of the specific Src inhibitor AZD0530 on the adhesion, migration, cell cycle, and survival of a Waldenstrom macroglobulinemia cell line and patient samples. Moreover, we tested the effect of AZD0530 on cytoskeletal and cell cycle signaling in Waldenstrom macroglobulinemia.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; We show that Src is overexpressed in Waldenstrom macroglobulinemia cells compared with control B cells, and that the use of the Src inhibitor AZD0530 led to significant inhibition of adhesion, migration, and cytoskeletal signaling induced by SDF1. Moreover, inhibition of Src activity induced G&lt;sub&gt;1&lt;/sub&gt; cell cycle arrest; however, it had minimal effect on survival of Waldenstrom macroglobulinemia cells, and no significant effect on survival of normal cells.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; Taken together, these results delineate the role of Src kinase activity in Waldenstrom macroglobulinemia and provide the framework for future clinical trials using Src inhibitors in combination with other drugs to improve the outcome of patients with Waldenstrom macroglobulinemia. (Clin Cancer Res 2009;15(19):6035–41)&lt;/p&gt;&lt;/div&gt;

Supplementary Data from Src Tyrosine Kinase Regulates Adhesion and Chemotaxis in Waldenstrom Macroglobulinemia

Clonal Hematopoiesis of indeterminate potential (CHIP) is a condition where blood cells are produced from a few clones of hematopoietic stem cells (HSCs) carrying leukemia-associated driver mutations 1. Although selective pressures from low-grade inflammation of the bone marrow microenvironment have been shown to promote mutant cell expansion 2-7, we focus on the disease-initiating niche, which is defined as a subset of marrow cells surrounding rare HSCs. Indeed, as we have recently described, clonal expansion of acute myeloid leukemia and activated HSCs 8,9 was not uniform, and only occurred in a subset of marrow spaces harboring bone remodeling activities. To identify the disease-initiating niche for therapeutic targeting, we developed an intravital imaging protocol to visualize the rare pre-malignant clones in the minimally perturbed microenvironment and phenotype their niche. The standard practice to generate CHIP models relies on transplanting syngeneic or allogeneic cells into a sub-lethally irradiated recipient. However, concerns arise as irradiation compromises the microenvironment 10. Successful engraftment in non-conditioned mice can be achieved by injecting a high number of cells (1.5 x 10 7 cells) 11,12, but modeling early clonal expansion events from single cells remain challenging, as a crowd of cells is often observed in proximity. Here, we established a low dose (0.5-1.5 Gy), whole body or local irradiation regimen that preserves the integrity of the bone marrow microenvironment. We performed high-resolution in-vivo imaging to capture dynamics of single cells and cells undergoing early expansion. Specifically, as little as 0.5Gy irradiation enabled survival of the transplanted cells (2x10 6 GFP + healthy whole bone marrow) in non-irradiated side, and allowed direct visualization of early clonal expansion in vivo through 16 weeks . Engraftment was negligible in non-irradiated mice.Through in-vivo, video-rate tracking of Rhodamine dextran dye (70kDa) leakage from vessels into the extravascular space showed no increase in permeability 13 or signs of vessel dilation, suggesting minimal inflammation after the radiation insult (N= 3 mice, data points are individual vessel segments. Mann-Whitney U test). Preliminary findings with in-vitro cultures of mesenchymal stromal cells (MSCs) have also suggested that the MSCs retain their ability for tri-lineage differentiation after the low-dose irradiation. With this imaging protocol, we showed that hot spots of cell expansion exist in the Tet2 +/- murine model. To capture the highly localized niche factors responsible for these hot spots, we implemented image-guided live-cell labeling adapted from the previously published Image-seq protocols 9. As illustrated in Figure 1, a small channel is etched in the bone with plasma-mediated laser ablation. The micropipette was then inserted to the region of interest for delivery of fluorescent antibodies under image guidance. By administering FITC-conjugated anti-CD45 antibodies into the marrow cavities, we can label and recover ~10,000 live cells with 98% viability from two regions of interest, suitable for next generation single cell sequencing. The technique can be expanded to employ antibody cocktails to visualize and enrich particular cell lineages of interest during cell isolation, while preserving spatial information. Its compatibility with standard cell isolation protocols for FACS may help collect cellular targets that are geometrically inaccessible for cell aspiration. In conclusion, we established a working model to visualize expansion of healthy and Tet2 +/- hematopoietic cells in vivo. This can be followed by image-assisted live-cell tagging to isolate local microenvironment cells and study cell-niche coordination at high spatial precision. The imaging protocol may also be broadly applied to study microenvironment regulations in non-malignant clonal disorders.

Abstract Background: Waldenstrom’s Macroglobulinemia (WM) is an incurable low-grade lymphoplasmacytic lymphoma with a median overall survival of 5 to 6 years, and most symptomatic patients succumb to disease progression. Current therapies used in upfront or relapsed settings include the monoclonal antibody rituximab, alkylator agents (e.g. chlorambucil), and nucleoside analogues (cladribine or fludarabine). With those agents, the overall response rates (ORR) ranges from 30 to 70%. The proteasome inhibitor bortezomib has recently demonstrated about 50% ORR in patients with relapsed WM. Therefore, new therapies are needed in WM to enhance the activity of current therapeutic regimens. Perifosine (NSC 639966; Keryx Biopharmaceuticals, NY) is a novel Akt inhibitor that demonstrated significant activity in vitro and in vivo in WM. Here, we examined the effect of combinations of perifosine with other conventional therapies for WM. Methods: chlorambucil (Sigma Aldricht), dexamethasone (Sigma Aldricht), doxorubicin (Sigma Aldricht), fludarabine (Berlex), rituximab (Genentech), bortezomib (Millenium), and melphalan (Sigma Aldricht) were tested alone or in combination with perifosine in WM cell lines (BCWM.1 and WM-WSU). Cytotoxicity was measured using the MTT growth inhibition assay. ADCC was measured by calcein-AM release on a Wallack Spectrafluor (485 nm excitation/535 nm emission) in the presence or absence of effector mononuclear cells for 4hrs (pretreated 24hrs with IL-2 100UI/mL, ratio effector:target (E:T) cells 40:1). Determination of the additive or synergistic effect of the combination was calculated using the CalcuSyn software (Biosoft, MO) based on the Chou-Talalay method, with a combination index (CI)&lt;1.0 indicating synergism. Results: Perifosine induced significant cytotoxicity, with an IC50 of 5–20uM. The following doses of single agent bortezomib and cytotoxic agents induced 50% growth inhibition (IC50): bortezomib 15–20nM, fludarabine 5ug/mL, doxorubicin 0.5–1nM, and melphalan 6–10uM. Dexamethasone 100nM and chlorambucil 100uM inhibited cell growth by 20%. Rituximab (10ug/mL, 1 hr) induced 121% specific lysis by ADCC in presence of effector cells. We then studied low doses of perifosine (5uM) in combination with these drugs. Perifosine 5uM induced 20% cytotoxicity at 48hrs, that was increased to 72% with fludarabine 5ug/mL (CI=0.521), to 60% with bortezomib 10nM (CI=0.49), to 67% with doxorubicin 0.1nM (CI=0.765), to 41% with dexamethasone 100nM (CI=0.335), to 50% with melphalan 5uM (CI=0.359), and to 42% with chlorambucil 50uM (CI=0.477). Finally, perifosine 10uM in combination with rituximab (10ug/mL, 1 hour) induces 193% specific lysis by ADCC mechanism in the presence of effector cells (p&lt;0.001). Conclusion: The combination of perifosine with bortezomib, rituximab and other conventional agents used in the therapy of WM has synergistic activity. Those results provide the framework for clinical evaluation of combination of perifosine with other therapies in WM. XL and GO are co-first authors. Supported in part by an ASH Scholar Award.

Abstract Background: Waldenstrom Macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow (BM), and lymphadenopathy in 20% of the patients, implying continuous trafficking of WM cells into and out of the BM and lymph nodes. The normal process of B-cell homing is regulated by cytokines, chemokines, and adhesion molecules. One of the most extensively studied chemokines in migration is stromal derived factor SDF-1 and its receptor CXCR4. Here we study the role of chemokine receptors, and the SDF-1/CXCR4 axis on migration and adhesion in WM. Methods: Flow cytometry for CXC and CC chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CCR2, CCR4, CCR5, CCR6 and CCR7), and adhesion molecules (VLA-4 and LFA-1) on WM cell lines (BCWM.1 and WM-WSU) and patient samples was performed. Migration was determined using the transwell migration assay (Costar, NY). Cells were placed in the upper chambers of the migration assay with 1% FCS medium in the presence of serial concentrations of SDF-1 in the lower chambers. After 4 hours of incubation, cells that migrated to the lower chambers were counted. Similarly, adhesion was determined using an adhesion assay (EMD Biosciences, San Diego, CA) with 96-well plated coated with fibronectin. Immunoblotting for proteins downstream of CXCR4 was performed. The CXCR4 inhibitor AMD3100 (10–100uM, Sigma, MO) and Gi protein inhibitor pertussis toxin PTX (10–200ng/ml, Sigma, MO) were used to inhibit CXCR4 signaling. Results: The following chemokine receptors were expressed on patient CD19+WM cells with over 30% expression: CXCR1 (mean 60%), CXCR2 (mean 47%), CXCR4 (mean 47%), CXCR5 (mean 69%), CCR4 (mean 54%) and CCR6 (mean 61%). Similar expression was observed on WM cell lines. We next determined the effect of SDF-1 on migration and signaling pathways in WM. SDF-1 (10–100nM) induced migration in a bell-shaped curve with 30nM inducing maximum migration (110% compared to control). SDF-1 30nM induced a rapid activation of signaling pathways downstream of CXCR4 including pERK1/2, pAKT, and pPKC at 1 min, with maximum activation at 5min. The CXCR4 inhibitor AMD3100 inhibited migration of BCWM.1 in the presence of 30nM SDF-1, with AMD3100 10uM inhibiting migration at 59% of control, and 20 to 50uM leading to a plateau in inhibition of migration at 54% of control. AMD3100 inhibited pERK and pPKC activation, downstream of CXCR4 in a dose-dependent fashion. Similar results were observed using PTX, with inhibition of migration of WM cells at 50% compared to control. To determine the role of SDF-1 on adhesion, we first demonstrated that WM cells from patients and cell lines expressed high levels of surface VLA-4 expression (mean 95% surface expression). WM cells had an increase in adhesion to fibronectin (VLA-4 ligand) compared to BSA control. AMD3100 10uM inhibited adhesion to fibronectin (63 % of control), indicating that the SDF-1/CXCR4 axis regulates adhesion. Conclusion: CXCR4 is highly expressed on WM cells and regulates migration and adhesion, indicating a potential role in regulating WM trafficking into the BM and lymph nodes. These studies provide the preclinical framework to study CXCR4 inhibitors in the regulation of homing and adhesion in WM.

206/-494/-155/-184/-542-3p, and decreased expression of microRNA-9* (ANOVA; P < .01). We found that microRNA-155 regulates proliferation and growth of WM cells in vitro and in vivo, by inhibiting MAPK/ERK, PI3/AKT, and NF-B pathways. Potential microRNA-155 target genes were identified using geneexpression profiling and included genes involved in cell-cycle progression, adhesion, and migration. Importantly, increased expression of the 6 miRNAs significantly correlated with a poorer outcome predicted by the International Prognostic Staging System for WM. We further demonstrated that therapeutic agents commonly used in WM alter the levels of the major miRNAs identified, by inducing downmodulation of 5 increased miRNAs and up-modulation of patient-downexpressed miRNA-9*. These data indicate that microRNAs play a pivotal role in the biology of WM; represent important prognostic marker; and provide the basis for the development of new microRNAbased targeted therapies in WM. (Blood. 2009;113:4391-4402)

Abstract Abstract 3732 Poster Board III-668 Background Waldenstrom Macroglobulinema (WM) is an incurable B-cell disorder characterized by the presence of IgM monoclonal gammopathy and bone marrow infiltration of lymphoplasmacytic cells. Apart from promising advances in therapeutic strategy, response rates and treatment-free survival remain inconsistent across prescribed regimens. As we have previously shown, the PI3K/Akt pathway actively mediates tumor growth, survival, migration, and cell cycle within primary WM cells. An active target of this pathway includes the downstream mammalian target of rapamycin (mTOR). By inhibiting the mTOR pathway with RAD001 (Afinitor™, Novartis Pharmaceuticals), we hoped to constructively regulate aforementioned cellular function, and also understand from a preclinical perspective its specificity of action in our microenvironmental models, both alone and the presence of monoclonal antibody against CD20 and proteasome inhibitor. Methods WM cell lines (BCWM1) and IgM secreting cell lines MEC1 and RL were used. Bone marrow stromal cells (BMSC) were obtained from patients with WM. Peripheral blood mononuclear cells were harvested from healthy donors. Agents tested included RAD001, Rituximab and Bortezomib. Cytotoxicity, DNA synthesis, and cell cycle were measured using MTT assay, [3H]-thymidine uptake, and flow cytometry/PI staining, respectively. Antibody-dependent cellular cytoxicity (ADCC), transwell, and Matrigel assays were preformed to measure cell lysis, SDF-1 chemotaxis-induced migration, and angiogenesis, respectively. Adhesion to fibronectin has been evaluated with WM cells and BMSC in the presence of RAD001, with and without Bortezomib. Results RAD001 induced cytotoxicity and inhibition of DNA synthesis with an IC50 of 1-10 nM in BCWM1 cells at 48 hours. Similar effects were seen in IgM cell lines with an effective dose of 0.1-1 nM. In contrast, at IC50 treatment level donor cells displayed no significant cytotoxicity (&gt;85% survival). Cell cycle analysis showed corresponding G1 arrest, and angiogenesis was also inhibited. RAD001, Bortezomib, and Rituxan combinations showed synergistic cytotoxicity, which was attenuated when WM cells were co-cultured with BMSC. Migration of BCWM1 and adhesion of BCWM1 to BMSC was reduced under presence of RAD001. Conclusions These functional assays therefore show that RAD001 has significant and synergistic antitumor activity in WM which will inform the design of future clinical trials. Disclosures: Ghobrial: Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Abstract Abstract 951 INTRODUCTION: Multiple Myeloma (MM) is characterized by widespread disease at diagnosis with the presence of multiple lytic lesions and disseminated involvement of the bone marrow (BM), implying that the progression of MM involves a continuous re-circulation of the MM cells in the peripheral blood and re-entrance into the BM. Selectins are adhesion molecules expressed by activated endothelium of venules and leukocytes, and are involved in the primary interaction of lymphocytes with the endothelium of blood vessels. The binding of selectins serves as a biologic brake, making leukocyte quickly decelerate by rolling on endothelial cells, as the first step of extravasation. In this study, we have investigated the role of selectins and their ligands in the regulation of homing of MM Cells to the BM and the therapeutic implications of this role. METHODS AND RESULTS: We have used flow cytometry to characterize the expression of E, L and P-selectins and their ligands on MM cell lines, patient samples and on plasma cells from normal subjects. We found that all MM cell lines and patient samples showed high expression of L and P, but little of no E-selectin. While normal plasma cells showed low expression of all selectins and ligands.(give numbers) A pan-selectin inhibitor GMI-1070 (GlycoMimetics Inc., Gaithersburg, MD) inhibited the interaction of recombinant selectins with the selectin-ligands on the MM cells in a dose response manner. We have tested the role of the selectins and their ligands on the adhesion of MM cells to endothelial cells and found that MM cells adhered preferentially to endothelial cells expressing P-selectin compared to control endothelial cells and endothelial cells expressing E-selectin (p&lt;0.05). Moreover, we found that blockade of P-selectin on endothelial cells reduced their interaction with MM cells (p&lt;0.01), while blockade of E and L-selectin did not show any effect. Treating endothelial cells with GMI-1070 mimicked the effect of blocking P-selectin. Moreover, we found that treating endothelial cells with the chemokine stroma cell-derived factor-1-alpha (SDF1) increased their expression of P but not E or L-selectin detected by flow cytometry. Neither the blockade of each of the selectins and their ligands nor the GMI-1070 inhibited the trans-well chemotaxis of MM cells towards SDF1-alpha. However, blockade of P-selectin (p&lt;0.001) on endothelial cells by GMI-1070 inhibited the trans-endothelial chemotaxis of MM cells towards SDF1-alpha. Both adhesion to endothelial cells and activation with recombinant P-selectin induced phosphorylation of cell adhesion related molecules including FAK, SRC, Cadherins, Cofilin, AKT and GSK3. GMI-1070 decreased the activation of cell adhesion molecules induced by both recombinant P-selectin and endothelial cells. Using in vivo flow cytometry we found that both anti P-selectin antibody and GMI-1070 prevented the extravasation of MM cells out of blood vessels into the bone marrow in mice. Moreover, we found that, in a co-culture system, endothelial cells protected MM cells from bortezomib induced apoptosis, an effect which was reversed by using GMI-1070, showing synergistic effect with bortezomib. CONCLUSION: In summary, we showed that P-selectin ligand is highly expressed in MM cells compared to normal plasma cells, and that it plays a major role in homing of MM cells to the BM, an effect which was inhibited by the pan-selectin inhibitor GMI-1070. This provides a basis for testing the effect of selectin inhibition on tumor initiation and tumor response to therapeutic agents such as bortezomib. Moreover, it provides a basis for future clinical trials for prevention of MM metastasis and increasing efficacy of existing therapies by using selectin inhibitors for the treatment of myeloma. Disclosures: Patton: GlycoMimetics, Inc: Employment. Smith:GlycoMimetics, Inc: Employment. Sarkar:GlycoMimetics, Inc: Employment. Anderson:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Magnani:GlycoMimetics, Inc.: Employment. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Abstract Background. MicroRNAs (miRNAs) constitute a class of small, non-coding, 18–24 nucleotide RNAs, that act as negative regulators of gene expression. By repressing several target mRNAs, mature miRNAs play a pivotal role in regulating development, cell differentiation, apoptosis, and cell proliferation. Moreover, miRNAs have been described to play roles in both solid tumors and hematologic malignancies, however the role of miRNAs in WM has not been yet elucidated. Methods. We performed miRNA expression profiling of bone marrow-derived CD19+ WM cells, compared to their normal cellular counterparts and validated data by qRT-PCR. In vitro and in vivo functional studies were performed on miRNA-155 knockdown WM cells. Effect of miRNA-155 on signaling cascades have been evaluated by western blot and immunofluorescence. NF-kB activity has been investigated using a DNA-binding enzyme-linked immunosorbent assay-based assay. Gene expression profile analysis has been performed on both miRNA-155 knockdown- and control probe-transfected WM cells in order to identify potential miRNA-155 targeted genes. Finally correlation between miRNA signature and prognosis has been evaluated. Results. We identified a WM specific miRNA signature characterized by increased expression of miRNA-363*/-206/-494/-155/-184/-542-3p; and decreased expression of miRNA-9* (ANOVA;P&amp;lt; 0.01). Our data showed that miRNA-155 regulates proliferation and growth of WM cells in vitro and in vivo, by inhibiting MAPK/ERK, PI3/AKT, and NF-kB signaling pathways. Potential miRNA-155 target genes were identified using gene expression profiling and included genes involved in cell cycle progression, adhesion, and migration. Importantly, increased expression of the 6 miRNAs significantly correlated with a poorer outcome predicted by the International Prognostic Staging System for WM (P&amp;lt;0.01). We further demonstrated that therapeutic agents commonly used in WM (rituximab, perifosine, bortezomib) alter the levels of the major miRNAs identified, by inducing down-modulation of five increased miRNAs (all but miR-206) and upmodulation of patient-downexpressed miR-9* microRNA. Conclusion. These data indicate that miRNAs play a pivotal role in the biology of WM; represent important prognostic markers; and provide the basis for the development of new miRNA-based targeted therapies in this disease.

Abstract BACKGROUND: Multiple myeloma (MM) is a plasma cell malignancy that depends on interactions with the bone marrow (BM) microenvironment for growth and survival. In turn, adhesion of MM cells to the BM stroma provides a mechanism of resistance from standard chemotherapeutic agents. Recently, our lab has shown that by disrupting this adhesion using a selective CXCR4 inhibitor named AMD3100, MM cells are more sensitive to the proteasome inhibitor Bortezomib (Ghobrial lab, unpublished data). CXCR4 has been a particularly attractive target because its ligand SDF-1 is known to induce p42/44 MAPK, AKT, and the down-stream anti-apoptotic protein bad in MM cells, leading to increased MM growth and survival. Until recently, CXCR4 was thought to be a canonical receptor for the SDF-1 ligand. However, a second chemokine receptor for SDF-1 was subsequently discovered and named CXCR7. CXCR7 is a novel chemokine receptor that is important in cell adhesion, growth and survival in several tumor types. However, the role of CXCR7 in multiple myeloma (MM) has yet to be explored. Furthermore, the ability of SDF-1 ligand to regulate MM function via CXCR7 has not been studied. METHODS: The MM cell lines (U266, MM1.S, RPMI, OPM2, OPM1) were used. After informed consent was obtained, primary bone marrow samples from MM patients were collected. CD138 positive mononuclear cells were isolated by microbead selection. The expression of CXCR7 on MM cell lines and patient samples was confirmed using flow cytometry and RT-PCR analysis. For functional in vitro and ex-vivo assays, the CXCR7 selective antagonist 733 was used (ChemoCentryx Inc., Mountain View, CA). RESULTS: Here we show that CXCR7 was expressed on all tested MM cell lines and primary patient samples as demonstrated by flow cytometry and RT-PCR. Furthermore, CXCR7 was found to regulate SDF-1 induced MM cell adhesion, as demonstrated by in vitro assays using a small molecule compound specific for CXCR7 (733). The CXCR7 antagonist showed significant inhibition of adhesion of MM cell lines and patient samples to fibronectin, endothelial cells and stromal cells, with 50% reduction of adhesion at 5nM of the CXCR7 inhibitor, and with similar activity compared to 20uM of AMD3100 (CXCR4 inhibitor). However, unlike CXCR4, CXCR7 did not effect trans-well migration to SDF-1 chemokine. Interestingly, both receptors were found to be important for trans-endothelial migration of MM cells. Moreover, pre-treatment with 733 reduced homing of MM cells to the BM niche in vivo. Previous studies have failed to show signaling in response to CXCR7 in many tumor types. Here, we demonstrate that treatment with 733 inhibited SDF-1 induced pERK and pAKT, ribosomal pS6Kinase, pGSK3, pSTAT3, pFAK and pPAK signaling pathways, confirming a role for CXCR7 in facilitating SDF-1 signaling. This effect was further confirmed using immunofluorescence. To investigate whether CXCR7 and CXCR4 interact directly, we examined the effect of 733 and AMD3100 on CXCR4 expression and found that AMD3100 significantly inhibited CXCR4 expression, while 733 had no effect on CXCR4 expression, even in the presence of SDF-1. The CXCR7 inhibitor had no effect on the survival of MM cells using MTT and flow cytometry analysis, while high doses of 733 (1uM) had modest inhibition of proliferation. Interestingly, 733 prevented the growth advantage induced by 30nM SDF-1 at 24 hrs. CONCLUSION: Together, these results demonstrate the importance of CXCR7 in regulating MM adhesion and homing, and highlight the differential effects of CXCR4 and CXCR7 in regulating SDF-1 signaling in MM, thus providing a rationale for targeting the SDF-1/CXCR7 axis in MM.

Differential interference contrast (DIC) microscopy is a powerful technique for imaging phase objects in transparent samples but does not work with scattering samples. This Letter, to the best of our knowledge, describes a new technique for obtaining DIC-like phase-gradient images in scattering media based on differential detection of forward-scattered light, using detectors arranged in a ring configuration around the microscope objective pupil or its conjugate pupil plane. This method, called pupil plane differential detection (P2D2) microscopy, does not need polarization optics or a confocal pinhole, yet produces images that are free of speckles and interference noises. We compared the P2D2 imaging technique with reflectance confocal microscopy and demonstrated P2D2 as a simple add-on to conventional laser scanning microscopes.

Circulating tumor cells (CTCs) are of great interest in cancer research, but methods for their enumeration remain far from optimal. We developed a new small animal research tool called Diffuse in vivo Flow Cytometry (DiFC) for detecting extremely rare fluorescently- labeled circulating cells directly in the bloodstream. The technique exploits near-infrared diffuse photons to detect and count cells flowing in large superficial arteries and veins without drawing blood samples. DiFC uses custom-designed, dual fiber optic probes that are placed in contact with the skin surface approximately above a major vascular bundle. In combination with a novel signal processing, algorithm DiFC allows counting of individual cells moving in arterial or venous directions, as well as measurement of their speed and depth. We show that DiFC allows sampling of the entire circulating blood volume of a mouse in under 10 minutes, while maintaining a false alarm rate of 0.014 per minute. Hence, the unique capabilities of DiFC are highly suited to biological applications involving very rare cell types such as the study of hematogenic cancer metastasis.

Abstract We recently developed ‘Diffuse in vivo Flow Cytometry’ (DiFC), a new pre-clinical research tool for enumerating extremely rare fluorescently-labeled circulating cells directly in vivo . In this paper, we developed a green fluorescent protein (GFP) compatible version of DiFC, and used it to non-invasively monitor the circulating tumor cell (CTC) burden over time in a multiple myeloma disseminated xenograft model. We show that DiFC allowed counting of CTCs at estimated concentrations below 1 cell per mL in peripheral blood with a negligible false alarm rate. DiFC also revealed the presence of CTC clusters in circulation to our knowledge for the first time in this model, and allowed us to calculate their size, kinetics, and frequency of shedding. We anticipate that the unique capabilities of DiFC will have many applications in the study of hematogenous metastasis, and as a powerful complementary methodology to liquid biopsy assays.

Abstract Background: MM is characterized by widespread involvement of the bone marrow (BM) as the result of successful homing, engraftment and growth of myeloma cells. The BM provides protection and resistance of MM to therapeutic agents. Therefore, disruption of the interaction of MM cells with their microenvironment should lead to enhanced sensitivity to therapeutic agents. We hypothesized that disrupting CXCR4/SDF-1 axis will induce mobilization of MM cells from the BM into the circulation. Methods: MM.1S cells were co-cultured with bone marrow stromal cells (BMSCs) in the presence of AMD3100 (50uM, Sigma), bortezomib (0–2.5nM, Millennium) or combination of both; and cell proliferation was measured using [3H]-thymidine uptake. We then tested the in vivo AMD3100-induced mobilization of MM cells after they homed to the BM. MM1.S cells that had been fluorescently labeled with DiD (Invitrogen) were injected into mice through their tail veins. Beginning 24 hours later and for three subsequent days, the mice were treated with 5mg/kg AMD3100 sq daily, injected with fluorescently labeled c-kit antibody for HSC detection and immediately monitored for presence of circulating MM1-S cells or HSCs, using in vivo flow cytometry. To test whether AMD3100 induces mobilization of MM cells in established tumors, a GFP+ and luciferase+ osteotropic MM.1S cell line (Luc+GFP+MM.1S) was developed. This mouse model was used for continuous, real-time quantitation of MM cells mobilization. Bioluminescence imaging was used to determine tumor growth in vivo. Mice were treated with AMD3100 (5mg/kg, daily), bortezomib (1mg/kg biweekly) or the combination. Results: There was a significant increase in proliferation of MM cells in co-culture with BMSCs compared to MM cells alone. Moreover, AMD3100 alone did not inhibit proliferation; however, it significantly enhanced the cytotoxic effect of bortezomib in the presence of stromal cells. In vivo studies revealed that, unlike HSC, no appreciable mobilization of MM1S cells occurred after the first AMD3100 injection; however, the second AMD3100 treatment induced a fourfold increase in circulating MM cell numbers above background, while no further remarkable increase in circulating HSC was observed. The effect of AMD on an established tumor model showed that by the end of the first week of AMD3100 or bortezomib treatment, a 2-fold increase of circulating MM cells was observed compared to control mice, a trend which continued for three weeks. Moreover, compared to AMD3100 or bortezomib treatment alone, a further increase of circulating MM cells was observed in the peripheral blood of mice treated with combination of AMD3100 and bortezomib following the second bortezomib treatment. In the AMD3100 only treated mice, the counts of circulating MM cell continued to increase and tumors continued to progress, while in the AMD3100-bortezomib-treated mice, the circulating MM cell count decreased and the tumors regressed. Conclusion: These data support the hypothesis that disruption of the CXCR4/SDF-1 axis mobilizes MM cells, and that AMD3100 can be used to enhance the effects of therapeutic agents such as bortezomib.

Detection and quantification of rare circulating cells in biological tissues is an important problem and has many applications in biomedical research. Current methods normally involve extraction of blood samples and counting of cells ex vivo, or the use of microscopy-based fluorescence in vivo flow cytometry. The goal of this work is to develop an instrument for non-invasively enumerating very rare circulating cells in small animals with diffuse light with several orders of magnitude sensitivity improvement versus current approaches. In this work, we describe the design of our system and show that single, fluorescent microspheres can be detected in limb-mimicking optical flow phantoms with varying optical properties chosen to simulate in vivo conditions. Further, we demonstrate single cell counting capabilities using fluorescently (Vybrant-DiD) labeled Jurkat and Multiple Myeloma cells. Ongoing work includes in vivo testing and characterization of our system in mice.

Abstract Background: Waldenstrom Macroglobulinemia (WM) is a low-grade lymphoma characterized by widespread involvement of the bone marrow (BM) and involvement of lymph nodes and hepatosplenomegaly (HSM) in about 20% of the patients. We have recently demonstrated that the presence of HSM is one of the most important adverse prognostic factors in WM. The mechanisms of trafficking of WM cells to and from the BM and lymphoid organs is not well defined. The PI3k/Akt pathway is constitutively activated in WM, and regulates migration/homing in cancer cells and B-cells. We hypothesized that the Akt inhibitor perifosine (NSC 639966; Keryx Biopharmaceuticals, NY) modulates homing of WM cells to the BM. Methods: WM cell line (BCWM.1) was treated with perifosine 2 to 5uM for 2 hours. Surface adhesion receptors were studied using flow cytometry. The adhesion assay coated with fibronectin, a ligand of VLA-4 (EMD Biosciences, CA) was used to test in vitro adhesion. Migration was determined using the transwell migration assay (Costar, NY). We then studied homing of WM into the BM niches using in vivo flow cytometry and confocal microscopy in Balb/c mice. In brief, BCWM.1 were incubated with 5uM perifosine for 2 hrs (or control PBS). The cells were fluorescently labeled by incubation with the dialkylcarbocyanine membrane dye, “DiD” (Molecular Probes), 0.5uM dye for 30 minutes. Cells were then injected in the tail vein of the Balb/c mice, and in vivo confocal flow cytometry was performed on an artery from the ear lobe of the mice. Cell counts were obtained every 5 min. from the time of injection. In vivo confocal microscopy and two-photon microscopy was performed to study cells homing to BM vasculature of the skull (BM niches). Results: WM cells expressed very high levels of VLA-4, with a median 95% expression. BCWM.1 demonstrated increased adhesion to fibronectin-coated wells as compared to BSA-coated wells. Perifosine inhibited adhesion in a dose dependent manner with 50% decrease in adhesion at 2uM. Perifosine 10uM did not change the level of surface expression of VLA-4 after 6 and 24 hrs, indicating an effect on intracellular signaling but not on surface adhesion molecules. Perifosine 5uM significantly inhibited migration of BCWM.1 in response to SDF-1, a ligand that induces migration of WM cells. Perifosine also demonstrated significant inhibition of in vivo homing of WM cells to the BM niches. The number of cells in the peripheral circulation decreased dramatically (75% decrease) after 1 hr in the control, indicating homing, whereas there was a 40% reduction in the cells at 1hr after perifosine treatment (p=0.001). We then looked at the images obtained from the BM niches. The number of cells that homed and adhered to these areas was lower in the perifosine-treated mouse compared to the control mouse, indicating that fewer cells homed to the BM of the treated mouse. Conclusion: These results confirm that the PI3K/Akt pathway is important for migration, adhesion and homing of WM in vitro and in vivo.

An in vitro culture system in which Moloney murine leukemia virus induces immortalization of mature B lymphocytes has been developed. The cell lines derived in this way are nontumorigenic, and virus production is not required to sustain them. This system provides a new in vitro model with which to study the stepwise process of transformation by retroviruses lacking oncogenes.

Then focused on the RTK’s which were shown to be upregulated in WM and tested the effect of TKI-258 effect on their activity. Moreover, we tested the effect of TKI-258 survival, proliferation, and apoptosis of WM cells, and its effect of different signaling pathways in WM. Results: We found that epidermal growth factor receptor (EGF-R), ephrin receptor (Eph-R) and receptor tyrosine kinase–like orphan receptor 1 (ROR-1) were highly activated in WM patients compared to normal subjects (5, 10, and 14-fold increase, respectively), and were found to be upregulated also in BCWM1. TKI-258 reduced the activity of EGF-R and Eph-R, but not ROR1. Moreover, TKI-258 induced cytotoxicity, prevented proliferation and induced apoptosis in WM cells in a dose dependent manner with IC50 of 500-800 nM. Moreover, it was shown to induce cleavage of PARP, caspase-3 and caspase-9, and also reduced phosphorylations of ERK1/2. Conclusion: We identified two novel therapeutic targets for WM; EGF-R and Eph-R, and identified Eph-R as a novel target of TKI-258. Moreover, TKI-258 reduced WM progression in vitro, and we are now testing the effect of TKI-258 on WM progression in vitro and alone and in combination with other drugs.

11103 Background: Multiple myeloma (MM) is characterized by the disseminated involvement of the bone-marrow (BM), and its progression involves a continuous circulation of the MM cells (MMCs) in the peripheral blood and homing back to the BM. Selectins are adhesion molecules involved in the primary interaction of lymphocytes with the endothelial cells (ECs) of blood vessels. In this study we studied the role selectins in the pathogenesis of MM. Methods: We have characterized the expression of E, L and P-selectins and their ligands on MM cell lines, patient sample and plasma cells from normal subjects (NPCs). We have tested the effect of blockade of each of the selectins and selectin-ligands on the interaction of MMCs with ECs. Moreover, we tested the effect of a pan selectin inhibitor on MMCs adhesion to ECs, and trans-well (through filter) and trans-endothelial SDF1-induced migration in vitro, and characterized its effect on cytoskeletal signaling induced by the interaction of MMCs and ECs. Moreover, we have tested the effect of the inhibitor on homing of MMCs to the BM in mice using in vivo flow cytometry to detect the number of circulating cells, and in vivo confocal microscopy to directly visualize the homing. Results: All MM cell lines and patient samples had low expression of all selectins and high expression of L and P, but not E, selectin ligands. While NPCs showed low expression of all selectins and ligands. Blockade of L and P-selectin ligands reduced the interaction of MMCs with ECs in vitro, while blockade of E-selectin ligand or any of selectins did not show any effect. The pan-selectin inhibitor reduced the interaction of MMCs with ECs in vitro, did not alter their SDF1-induced migration through filter, but reduced significantly the migration through ECs. The inhibitor inhibited the activation of FAK and ERK induced by interaction of MMCs and ECs. Moreover, the selectin inhibitor extending the circulation time of MM cells in mice, and reduced the homing of MMCs. Conclusions: We found that L and P selectin ligands are highly expressed in MMCs compared to NPCs, and that those play a major role in homing of MMCs to the BM. Moreover, the pan-selectin inhibitor prevented the homing of MMCs to the BM. This provides a basis for testing the effect of the inhibitor on MM tumor progression and initiation. No significant financial relationships to disclose.

Abstract Abstract 1785 Poster Board I-811 Background We and Others have previously demonstrated that primary multiple myeloma (MM) cells are characterized by a specific microRNA (miRNA) signature compared to the related normal plasmacell counterpart; and that miRNAs play a crucial role in regulating MM pathogenesis. Nevertheless, miRNA changes that occur in MM cells in the context of the bone marrow microenvironment have not been previously examined. Therefore, characterization of miRNA profiling of MM cells in conjunction with bone marrow stromal cells (BMSCs) is important to better understand the underlying molecular changes that lead to initiation and progression of this disease. Methods We performed miRNA-expression-profiling of MM cell lines (MM.1S; RPMI8226) that were co-cultured with primary BMSCs obtained from 5 MM patients, using liquid phase Luminex microbead miRNA profiling (Luminex, Austin, TX). The expression patterns of unfiltered data were performed using unsupervised hierarchical clustering of samples, based on centroid linkage and 1-correlation distance metric, using dChip (www.dchip.org). To further define those miRNAs differentially expressed between groups (patients vs normal), the data were filtered on significance of differences using ANOVA test, (P &lt; 0.05). Microbead-miRNA profiling data were validated data by stem-loop qRT-PCR. To identify specific predicted miRNA-targeted mRNAs, TargetScan, PicTar, and miRanda algorithms were used. Results miRNA profiling of MM cells cultured with primary BMSCs (MM+BMSC system) differs from MM cells which were not grown in contact with primary BMSCs (MM cells alone). Specifically, we observed increased expression of miRNA-450, -432*, -299-5p, -409-3p, -29b, -542-5p, -184, -517*, -218, 128b, -142-5p and -211 (P&lt;0.05) in MM cells obtained from the MM+BMSC system, compared to MM cells alone. Stem-loop qRT-PCR was performed on matched samples and showed expression patterns similar to those observed in miRNA analysis. Using algorithms commonly used to predict human miRNA gene targets (miRanda; TargetScan; PicTar), predicted targets of the increased miRNAs included negative regulators of NFkB, PI3K/Akt/mTOR, and MAPK/ERK signaling pathways, such as PTEN, KSR2, TWEAK, and DUSP; as well as tumor suppressors (MCC, TSSC1, TUSC1, FBW7, RHOBTB), pro-apoptotic factors and cyclin-dependent kinases inhibitors. These data demonstrate that bone marrow stromal cells exert a modulatory effect on miRNA profiling in MM cells, which results in promoting MM cell growth and reducing MM cell survival. Disclosures Ghobrial: Millennium : Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Abstract Abstract 4916 Introduction Proteasome inhibition represents a valid therapeutical approach in several tumors and its use has been validated in Waldenstrom's macroglobulinemia (WM), where single-agent Bortezomib has been successfully tested in phase 2 clinical trials. Nevertheless, a significant fraction of patients relapse, or develop significant toxicity due to high toxicity in non-transformed cells. Therefore preclinical evaluation of new proteasome inhibitor with a more selective inhibition of neoplastic cells is needed in order to increase efficacy and improve patient outcome. We tested Carfilzomib, a tetrapeptide epoxyketone selective inhibitor of the chymotrypsin-like activity of the immunoproteasome and constitutive proteasome in WM. Methods WM and IgM secreting low-grade lymphoma cell lines (BCWM.1, MEC1, RL) were used. Expression of imunoproteasome and constitutive proteasome subunits (beta1, beta2, beta5; LMP2, MECL1, LMP7) were detected primary WM cells and cell lines by an ELISA-based assay. Cytotoxicity and DNA synthesis were measured by thymidine uptake and MTT, respectively. Cell signaling and apoptotic pathways were determined by Western Blot. Determination of the additive or synergistic effect of drugs combination was calculated using the CalcuSyn software based on the Chou-Talalay method. Results Primary CD19 bone-marrow derived WM cells express higher level of the immunopreoteasome as compared to the constitutive proteasome. Carfilzomib inhibited the chymotrypsin-like activity of both the immunoproteasome (LMP7) and the constitutive proteasome (beta5) and in WM cells, in a dose-dependent manner; leading to inhibition of proliferation (IC50: 5nM; 48h) and induction of cytotoxicity (IC50: 7.5nM; 48h) in WM cells. Carfilzomib mediated apoptosis in WM by increasing PARP-, caspase-9- and -3-cleavage; as well as by inducing activation of c-jun-N-terminal kinase and ER-stress in a dose-dependent manner. Moreover, combination of Carfilzomib and bortezomib induced synergistic cytotoxicity in WM cells, as shown by enhanced PARP-, caspase-9- and -3-cleavage; and synergy in inhibiting the chymotrypsin-like activity of the immunoproteasome and constitutive proteasome. Conclusion Taken together, these findings provide the pre-clinical rational for testing Carfilzomib in Waldenstrom Macroglobulinemia. Disclosures Aujay: Proteolix: Employment, Equity Ownership. Demo:Proteolix: Employment, Equity Ownership. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

The present standard of blood cell analysis is an invasive procedure requiring the extraction of patient’s blood, followed by ex-vivo analysis using a flow cytometer or a hemocytometer. We are developing a noninvasive optical technique that alleviates the need for blood extraction. For in-vivo blood analysis we need a high speed, high resolution and high contrast label-free imaging technique. In this proceeding report, we reported a label-free method based on differential epi-detection of forward scattered light, a method inspired by Jerome Mertz's oblique back-illumination microscopy (OBM) (Ford et al, <i>Nat. Meth.</i> 9(12) 2012). The differential epi-detection of forward light gives phase contrast image at diffraction-limited resolution. Unlike reflection confocal microscopy (RCM), which detects only sharp refractive index variation and suffers from speckle noise, this technique is suitable for detection of subtle variation of refractive index in biological tissue and it provides the shape and the size of cells. A custom built high speed electronic detection circuit board produces a real-time differential signal which yields image contrast based on phase gradient in the sample. We recorded blood flow in-vivo at 17.2k lines per second in line scan mode, or 30 frames per second (full frame), or 120 frame per second (quarter frame) in frame scan mode. The image contrast and speed of line scan data recording show the potential of the system for noninvasive blood cell analysis.

doi:10.1182/blood-2007-12-129395Prepublished online April 30, 2008;2008 112: 150-158€€€€Farag, Robert Sackstein and Irene M. GhobrialMoreau, Nicholas Burwick, Abdel Kareem Azab, Aldo Roccaro, Feda Azab, Antonio Sacco, Mena Hai T. Ngo, Xavier Leleu, Jack Lee, Xiaoying Jia, Molly Melhem, Judith Runnels, Anne-Sophie€

We have developed a new approach for fluorescence sensing, enumeration and tomographic localization of rare circulating cells with diffuse light. We validate our instrument using multiple-myeloma cells in optical phantoms and in mice in vivo.

B570 Centrosomal Clustering: A Novel Therapeutic Target for Multiple Myeloma MS Raab, I Breitkreutz, B Rebacz, PJ Hayden, TO Larsen, MH Clausen, JH Fruehauf, H Goldschmidt, KC Anderson, A Kraemer Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA; CCU Molecular Hematology/ Oncology, German Cancer Research Center, Heidelberg, Germany; BioCentrum, Technical University of Denmark, Kopenhagen, Denmark; Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA; National Center of Tumor Diseases, Dept. of Medicine, University of Heidelberg, Germany

e14640 Background: MicroRNAs (miRNAs) are non-coding RNAs that act as negative regulators of gene expression. They have been described to play roles in solid tumors and hematologic malignancies. However the role of miRNAs in multiple myeloma (MM) has not been yet fully described. Methods: We performed miRNA-profiling of primary bone marrow-derived CD138+ MM cells, compared to their normal cellular counterparts and validated data by qRT-PCR. In vitro and in vivo functional studies were performed on miRNA-15a- and -16–1-precursors-transfected MM cells. Effect of miRNA-15a and -16–1 on signaling cascades have been evaluated by western blot and immunofluorescence. NF-kB activity has been studied using the Active Motif TransAM kit. In vivo MM cell growth has been evaluated by either using an in vivo imaging model or bioluminescence. Angiogenesis has been studied both in vitro and in vivo using the chorioallantoic membrane model. Results: We identified a MM-specific miRNA signature characterized by down-expression of miRNA-15a, -16–1 and over-expression of miRNA-222/-221/-382/-181a/-181b (P&lt;0.01). Data were confirmed by qRT-PCR on matched samples. Predicted targets for the decreased miRNAs in MM patients included pro-angiogenic cytokines, oncogenes, cell cycle regulators, NFkB activators. Conversely, predicted targets for the increased miRNAs included cell cycle inhibitors, suppressors of cytokine signaling, and pro-apoptotic factors. We demonstrated that miRNA-15a and - 16–1 regulate proliferation and growth of MM cells. Indeed, transfected cells showed decreased DNA synthesis; decreased cyclinD1/cyclinD3/CDC25a/pRb protein expression; phase G1 cell cycle arrest. Moreover, transfected cells showed inhibition of NFkB pathway as shown by reduced p65-/p50-/p52-NFkB activities; downregulation of p-p65/p50/p52 nuclear protein level; upregulation of cttosolic phospho-IkB; and inhibited translocation of p-p65 from the cytolplasm to the nucleus. Similarly, inhibition of MM cell growth was confirmed in vivo; and anti-angiogenic properties of miRNA-15a and -16–1 were demonstrated both in vitro and in vivo. Conclusions: These data indicate that miRNAs play a pivotal role in the biology of MM; providing the basis for the development of new miRNA-based targeted therapies in this disease. No significant financial relationships to disclose.

Abstract BACKGROUND: Imaging animal models that offer serial measurement of systemic tumor progression, such as the GFP+ or bioluminescence MM model, have been limited to low resolution, gross measurements of tumor progression that are insufficient to detect individual cells, and their interaction with their microenvironment. Therefore, the need exists for development of sensitive, high resolution three-dimensional imaging methods that identify the dynamic changes that occur during tumor initiation and progression. We here show the use of in vivo fluorescence confocal microscopy to follow MM tumor initiation and progression at the cellular level using stably GFP-transfected MM1S cells in a xenograft model of MM. METHODS: 5 × 10 6 MM1S-GFP-Luc cells were injected into the tail veins of non-irradiated SCID/Beige male mice. MM cell growth in the marrow of the calvarial bone was analyzed using in vivo flow cytometry and fluorescence confocal microscopy, as previously described (Sipkins et al 2005). High-resolution images with unprecedented cellular detail were obtained through the intact mouse skull at depths of up to 250μm. To visualize the bone marrow vasculature the mice were injected with a blood pool marker (Angiosense 680 or 750) immediately before imaging, and to delineate the surface of calvarial bone, a fluorescent hydroxyapatite tag (Osteosense) was used. The validity of the imaging data was established by sacrificing select mice, and analyzing the previously imaged tissues by standard histologic and immunohistologic techniques. After MM tumors became established in the fourth week following injection, 1 mg/kg Bortezomib was administered twice weekly to a subset of the mice, these were imaged following treatment along with controls that were not treated. For all mice imaged, the number and areas of the skull where GFP+ MM cells were found were recorded. Confirmation of homing and tumor progression was also performed using CD138+ selected primary tumor cells. RESULTS: Using this model, we were able to detect and monitor individual GFP+MM cells within the bone marrow microenvironment. We demonstrate that MM.1 S cells and primary CD138+ cells exit the systemic circulation within one hour of injection, followed by specific rolling and adhesion to the vasculature of the bone marrow microenvironment. Within 4 days post after injection, the MM cells were fully engrafted along the bone marrow sinusoids, which were surrounded by bisphosphonate-rich bone structures including ostoeoblasts. Within the second week, loose clusters of a few cells began to form around the blood vessels. Growth and expansion appeared to be closely associated with the vasculature. Tumor growth dramatically increased in the third week following cell injection when areas of the parasagittal regions became completely involved with MM cells. In contrast, standard bioluminescence imaging performed concurrently detected tumor initiation only at 4 weeks post-injection, indicating that confocal microscopy is a much more sensitive technique in detecting early tumor proliferation. Imaging of bortezomib-treated mice demonstrated that tumor size and density was reduced in the skull, but even more dramatically the number of sites containing GFP+MM was greatly reduced. CONCLUSIONS: Our imaging model differs from other models due to its unprecedented resolution. Therefore it is particularly useful for following small numbers of tumor cells either early in disease progression or after therapeutic treatment. This model offers a more sensitive spatial and temporal live imaging of MM cells in the BM microenvironment and can be used to explore the dynamic interaction of MM with different structures and environments of the BM. We anticipate that this model will allow for a better understanding of the biologic effects of therapeutic agents on the growth of MM cells within the bone marrow niches.

Abstract Background: Waldenstrom’s Macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy. Insulin-like growth factor 1 (IGF1) is a polypeptide hormone that has been shown to have a proactive role in many cancer cell types, including multiple myeloma and solid tumor cells. We evaluated the role of IGF1 in WM. Methods: WM cell lines (BCWM1 and WSU-WM) and IgM secreting low-grade lymphoma cell lines (MEC1, RL) were used. Bone marrow-derived primary CD19+ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. The small molecule IGF-1R inhibitor II (Calbiochem) and the inhibitory antibody αIR3 (Calbiochem) were used. Cytotoxicity and DNA synthesis were measured by MTT assay and thymidine uptake assay, respectively. Cell signaling and apoptotic pathways were determined by Western Blot. Cell cycle and receptor analysis was obtained through flow cytometry. IGF1 levels were measured by ELISA. Receptor tyrosine kinase activity was evaluated in WM cell lines using the Luminex-microbeads-based assay. Results: We demonstrated that the IGF1 receptor (IGF1R) was expressed on WM primary patients and normal CD19+ control B cells, while IGF1 levels of serum and bone marrow samples in both patient and healthy donor samples were similar, suggesting a possible constitutive activation of the pathway downstream of IGF1R in WM and independent of receptor activity or cytokine levels. Surface IGF1R was expressed on BCWM1 cells, in serum-starved conditions, while, it was internalized with the addition of FBS that includes IGF-1. Finally, we also showed that IGF1 induces the activation of IGF1R as demonstrated by the induction of related tyrosine kinase activity. To demonstrate the protective effect of IGF1 on WM cells, IGF1 was added to serum starved BCWM1 cells, and we found it rescued nearly 100% of the cells from apoptosis in concentrations as low as 25ng/ml. The effect of IGF1R inhibitor on WM cells has been investigated and found that it blocked migration, induced cytotoxicity and decreased cell proliferation; without any effect on healthy donor peripheral blood mononuclear cells. Furthermore, the inhibitor decreased the tyrosine kinase activity of IGF1R, overcoming its initial activation in the presence of IGF1. Conclusion: IGF1 plays a complex role in WM cells with no evidence of constitutive activation of the receptor itself or higher levels of cytokine in WM marrow samples, indicating that activation of this pathway is downstream of IGF1R. Inhibition of IGF1R by a specific small molecule or antibody inhibitor led to a significant decrease in proliferation, survival, and migration in WM cells, but not in control mononuclear cells. These studies provide the framework to investigate the role of IGF1R inhibitors in clinical trials in WM.

Abstract Abstract 1825 Poster Board I-851 Purpose Recent advances in understanding of the molecular alterations that occur at the genetic and epigenetic levels in Multiple Myeloma (MM) have led to major leaps in identifying molecular pathways that regulate progression and resistance to therapeutic agents. However, despite great scientific advances at the genomic level, studies to identify signaling pathways deregulated at the functional proteomic level in MM are limited. We have previously demonstrated that Citron Rho Interacting Kinase (CRIK) is overexpressed in primary multiple myeloma (MM) cells, as compared to the normal plasma cell counterpart, using an antibody-based protein microarray technique. We therefore sought to investigate the functional role of CRIK in MM cells. Methods We determined the protein expression level of 512 polypeptides in 12 samples of newly diagnosed patients with MM using high-throughput proteomic analysis with antibody-based protein microarray. Primary CD138+ sorted MM cells were obtained from the bone marrow of patients after informed consent. MM.1S, RPMI8226, and INA6 MM cell lines were used in this study. Protein expression has been studied by immunoblotting. Gene expression analysis has been assessed using the Affymetrix U133A platform. Lentivirus was used to knockdown CRIK in MM cell lines (MM.1S, RPMI8226, INA6). DNA synthesis, cell survival, cell cycle profiling and apoptosis were assessed by thymidine uptake, MTT, PI and Annexin/PI staining and flow cytometric analysis, respectively. Results Overexpression of CRIK has been confirmed in primary CD138+ tumor cells isolated from bone marrow of 12 patients with MM, as compared to normal plasma cells obtained from healthy donors. We found that CRIK-knockdown exerted an anti-proliferative and pro-apoptotic effect only in IL-6-dependent MM cell line INA6; in contrast, no effect on proliferation and survival was observed in MM1.S and RPMI8226. Indeed, INA6 CRIK-knockdown cells were characterized by a reduction in the proliferation rate, associated with decreased S-phase and G2/M phase cell cycle arrest. Moreover, induction of cytotoxicity was also demonstrated in CRIK knockdown cells compared to scramble probe transfected or non-transfected cells. We also showed that CRIK knockdown led to cytokinesis in INA6, indicating a possible mechanism for inhibition of proliferation of these cells. We next correlated CRIK gene expression level (CIT) with prognosis using previously published gene expression datasets and found that CRIK correlated with poor prognosis. Conclusion In this study, we show that MM cells express a high level of CRIK, and that inhibition of this protein leads to significant inhibition of proliferation and survival of IL-6 dependent MM cells. Moreover, CRIK protein expression correlated with poor survival in patients with MM. Disclosures Anderson: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Abstract BACKGROUND: Tyrosine phosphorylation is a central event in the regulation of a variety of biological processes such as cell proliferation, migration, adhesion, and survival. Waldenström Macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy and characterized by widespread involvement of the bone marrow. We sought to determine whether the protein tyrosine kinase Src regulates adhesion and survival in WM. METHODS: The WM cell lines BCWM1, WM-WSU, and IgM secreting lymphoma cells lines MEC-1 and RL were used in these studies. Primary CD19+ WM cells were obtained from the bone marrow of patients after informed consent. AstraZeneca Biopharmaceuticals (London, England) provided the Src inhibitor AZD0503 (AZD). Cytotoxicity and DNA synthesis were measured using MTT assay and [3H]-thymidine uptake, respectively. Apoptosis was measured using flow cytometry with Apo 2.7 staining. Western blotting was performed to determine downstream signaling pathways. Migration was performed using the transwell migration assay. RESULTS: We demonstrated that pSrc is overexpressed in WM cells compared to control B cells. Similarly, phospho-Src protein expression was upregulated in WM cell lines, specifically BCWM.1 but not in WM-WSU. We then showed that pSrc regulates migration and adhesion in response to the chemokine SDF-1, as well as in vivo homing using in vivo flow cytometry. The use of the specific Src inhibitor AZD0530 led to significant inhibition of adhesion and migration in cell lines with pSrc activation, but not in those deficient of Src activation. Similarly, inhibition of Src activity led to significant inhibition of proliferation and survival through inhibition of STAT3, Akt, and ERK/MAPK pathways. The monoclonal antibody rituximab signals through Src kinase, and the combination of AZD0530 and rituximab was synergistic in vitro. CONCLUSION: Taken together, these studies delineate the role of Src kinase activity in WM and provide the framework for future clinical trials using Src inhibitors in combination with rituximab to improve the outcome of patients with WM.

Abstract Background. We have previously showed that PI3K/Akt is constitutively active in Waldenström Macroglobulinemia (WM) malignancies, mediating growth, survival, cell cycle regulation, and migration in primary tumor cells. Once activated, Akt phosphorylates downstream targets, including mammalian target of rapamycin (mTOR). Both PI3K/Akt and mTOR represent valid targets for antitumor therapeutic strategies. We therefore evaluated the antitumor activity of NVP-BEZ235 (Novartis, MA) in WM. Methods: WM cell lines (BCWM.1) and IgM secreting cell lines (MEK1, Namalwa) were used. Bone marrow primary CD19+ malignant cells and bone marrow stromal cells (BMSC) were obtained from WM patients. Cytotoxicity, DNA synthesis, and cell cycle were measured using MTT assay, [3H]-thymidine uptake, PI staining/flow cytometry, respectively. Effects of NVP-BEZ235 on cell signaling cascades were determined using immunoblotting and immunofluorescence. Adhesion on fibronectin has been evaluated in WM cells in the presence of NVP-BEZ235. Results: NVP-BEZ235 induced cytotoxicity and inhibited DNA synthesis with an IC50 of 20–25nM in BCWM.1 at 48 hours. Similar effects were demonstrated in all IgM secreting cell lines and in primary CD19+ WM cells, with an IC50 between 20nM and 50nM. No cytotoxicity was observed on peripheral blood mononuclear cells, indicating selective toxicity of the compound on the malignant lymphoplasmacytic clone. We observed that NVP-BEZ235 inhibited Akt (but not ERK phosphorylation) in a dose-dependent manner in BCWM.1 cells at 6 hours. Phosphorylation of GSK3α/β and ribosomal protein-S6, downstream target proteins of Akt, were also markedly inhibited. NVP-BEZ235-inhibited Akt phosphorylation was further confirmed by immunofluorescence. NVP-BEZ235 induced caspase-9, PARP cleavage and increased the release of Smac/DIABLO from the mitochondria to the cytosol, suggesting an induction of apoptosis in a caspase-dependent and –independent manner. We showed that NVP-BEZ235 inhibited adhesion of BCWM.1 cells to fibronectin in a dose-dependent fashion. Lastly, adherence to BMSCs did not confer protection to WM cells against NVP-BEZ235- induced cytotoxicity. Conclusions. These data indicate that NVP-BEZ235 has significant antitumor activity in WM, thus providing the framework for clinical trials in this disease.

Abstract PURPOSE: Recent advances in understanding of the molecular alterations that occur at the genetic and epigenetic levels in Multiple Myeloma (MM) have led to major leaps in identifying molecular pathways that regulate progression and resistance to therapeutic agents. However, despite great scientific advances at the genomic level, studies to identify signaling pathways deregulated at the functional proteomic level in MM are limited. In this study, we used a rapid and reproducible antibody-based protein microarray technique to screen the functional differences between malignant plasma cells in samples obtained from patients with MM compared to normal plasma cells (NPC) from the bone marrow of healthy volunteers. METHODS: We determined the protein expression level of 512 polypeptides in 12 samples of newly diagnosed patients with MM using high-throughput proteomic analysis with antibody-based protein microarray. Primary CD138+ sorted MM cells were obtained from the bone marrow of patients after informed consent. MM1.S was used in this study. Using immunohistochemistry and immunoblotting were confirmed. Lentivirus was used to knockdown CRIK. Gene expression datasets from the Mayo Clinic (accession number GSE 6477) and the UAMS (accession number GSE 2658) were obtained from the Gene Expression Omnibus for analysis. The Mayo dataset was generated using Affymetrix U133A platform whereas the UAMS dataset was generated using Affymetrix U133plus 2.0 platform. RESULTS: We identified four subgroups of MM using unsupervised clustering analysis. We confirmed overexpression of some of these proteins including CRIK and CDK4 using immunohistochemistry and immunoblotting. Many of these proteins are known to be deregulated in MM, indicating that this technique can accurately identify proteins that are over or under-expressed in MM in a high-throughput fashion. In addition, we identified novel proteins that are not previously known to be differentially expressed in MM, including the small GTPase member of the Rho family, CRIK protein. We then showed using knockdown of CRIK that this novel protein specifically regulates migration and adhesion in MM. There was no effect on survival of MM cells using the CRIK knockdown. Analyzing the GEP data of the 15 NPC, 46 monoclonal gammopathy of undetermined significance (MGUS) or smoldering myeloma (SMM) and 101 MM samples from the Mayo Clinic, there was a significant increase in expression of CIT from NPC to MM. Among the 351 patients entered into TTII trials from UAMS, CIT expression was similar across the different TC class. Using a cut-off normalized expression level of 1.25 (a level above expression in NPC), MM with a high CIT expression (n=81) had a significantly shorter survival than the other patients. CONCLUSION: In this study, we show that MM cells express a high level of CRIK, and that inhibition of this protein leads to significant inhibition of adhesion and migration of MM cells. In addition, CRIK protein expression correlated with CIT gene expression, with high expression in MM samples compared to NPC. In addition, high CIT expression correlated with poor survival in patients with MM.

Abstract Abstract 3843 Poster Board III-779 Background The PI3K/Akt and mTOR pathways play a pivotal role in the initiation and progression of malignancies, enhancing cell survival by stimulating cell proliferation and inhibiting apoptosis. Therefore, it is critical to examine therapeutic agents that explicitly target this pathway, specifically in tumors that harbor activation of the PI3K/Akt pathway, such as Waldenstrom macroglobulinemia (WM). Methods Primary-CD19+ bone marrow-derived WM cells, -bone marrow stromal cells, WM and IgM secreting low-grade lymphoma cell lines (BCWM.1, MEC1, RL), and primary normal CD19+ peripheral blood-derived (CD19+ PB)cells were used. Gene-expression and microRNA profiling have been performed on primary WM cells, as compared to CD19+ PB cells. Cytotoxicity, DNA synthesis, cell cycle and apoptosis were measured by thymidine uptake, MTT, PI staining, and Apo2.7/flow cytometry analysis, respectively. Cell signaling and apoptotic pathways were delineated by Western Blot and immunofluorescence analysis. In vivo homing has been assessed by in vivo flow cytometry. Results Primary bone-marrow derived WM cells are characterized by lower expression of PTEN gene and protein; higher expression of pospho(p)-Akt, p-mTOR, rictor and raptor, as compared to their normal cellular counterpart (CD19+ PB cells). We also observed that microRNA-542-3p and -494 are more highly expressed in primary WM cells as compared to normal CD19+ PB cells (P&lt;.01); and they both target PTEN, as predicted using TargetScan, PicTar, and miRanda algorithms, suggesting their role in inhibiting PTEN expression. We next assessed the effect of the dual PI3K/Akt and mTOR inhibitor NVP-BEZ235 (Novartis, Basel, Switzerland). This agent induced cytotoxicity and inhibited DNA synthesis (IC50 20-25nM) in BCWM.1 at 48 hours. Similar effects were demonstrated in all IgM secreting cell lines and in primary CD19+ WM cells (IC50 20-50nM). No cytotoxicity was observed on CD19+ PB cells, indicating selective toxicity of the compound on the malignant lymphoplasmacytic clone. NVP-BEZ235 inhibited p-Akt and p-mTOR, as well as the downstream Akt- targeted proteins GSK3a/b, p-S6R and p-p70S6, in a dose-dependent manner. Akt and mTOR in vitro kinase activity was also inhibited by NVP-BEZ235 treatment. In addition, NVP-BEZ235 inhibited both rictor and raptor, thus abrogating the rictor-induced Akt phosphorylation in WM cells. NVP-BEZ235 also induced significant cytotoxicity in WM cells in a caspase-dependent and -independent manner, through targeting the forkhead box transcription factors. Finally, NVP-BEZ235 targeted WM cells in the context of bone marrow microenvironment evidenced by significant inhibition of migration, adhesion in vitro and homing in vivo. Conclusion These studies therefore show that dual targeting of the PI3K/mTOR pathway represents a promising therapy for tumors that harbor activation of the PI3K/mTOR signaling cascade such as WM. Disclosures: Ghobrial: Millennium : Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Abstract Background: Waldenström’s Macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy. Protein kinase C (PKC) is a multigene family that that plays an important role in the regulation of cell growth and tumor progression. PKC412, a derivate of staurosporine, is a competitive inhibitor of ATP binding to PKC subtypes. This PKC inhibitor was used to examine its role in WM. Methods: WM cell lines (BCWM1 and WSU-WM) and IgM secreting low-grade lymphoma cell lines (MEC1, RL) were used. Bone marrow primary CD19+ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. Cytotoxicity and DNA synthesis were measured by MTT assay and thymidine uptake assay. Cell signaling of HSP response, VEGF signaling and AKT pathways were determined by Western Blot analysis. Cell cycle and apoptosis analysis were obtained through flow cytometry. Results: We showed that PKC412 induced cytotoxicity in WM cell lines tested (IC50 of 1uM) with no cytotoxic effect on healthy donor peripheral blood mononuclear cells at concentrations as high as 5uM. Cell proliferation and apoptosis analysis showed similar results, in a time independent manner. A G2/M arrest was observed upon cell cycle analysis, suggesting a possible mechanism for apoptosis. Similarly, the Akt pathway was investigated and was found to show a decrease in its activity in a dose dependent manner. Finally, PKC412 regulated VEGF levels in WM samples indicating an anti-angiogenic activity. Conclusion: PKC412 has a significant effect on the survival and proliferation of WM cells, including regulation of angiogenesis. These studies provide the framework for clinical evaluation of this novel PKC inhibitor in Waldenström Macroglobulinemia.

Abstract Abstract 2935 Poster Board II-911 INTRODUCTION: Waldenstrom's macroglobulinemia (WM) is a low-grade lymphoma characterized by the presence of lymphoplasmacytic lymphoma cells in the bone marrow (BM). The BM microenvironment was shown to promote growth and proliferation of WM cells. The Eph receptor family are receptor tyrosine kinases RTKs activated by ephrin which a cell-surface protein, and the interaction between the receptor on one cell and the ligand on other cells promotes the activity of the receptor. Eph receptors are known to control processes such as cell growth, proliferation, migration, and invasion, and their expression level was shown to be elevated in a wide range of solid tumors. However, their role in WM was never explored. METHODS AND RESULTS: Using phosphor-RTK array kit, we have screened the activity of 42 RTKs in CD19+ cells from 4 WM patients, WM cell line BCWM1, IgM secreting cell lines MEC1 and RL, and CD19+ cells from the BM of 4 healthy donors. We found that one of the most significant RTKs which showed high activation in all WM cells from patient sample and cells, compared to all normal cells was Eph-B2 receptor. We have used ephrin-B2, the ligand of the receptor Eph-B2, to test the signaling pathways involved in the activity of this receptor. We found, by immunoblotting, that ephrin-B2 induce phosphorylation of the receptor in a bell-curve manner, with the activity peaking in 100 ng/ml, while the expression of the total form of the receptor was unchanged over arrange of 0-1000 ng/ml of ephrin. A comparable activation was found for several cell-adhesion related proteins including FAK, SRC, paxillin, P130 and cofillin. These finding indicated a major role of the Eph-B2 receptor cell adhesion of WM cell. We have coated plates with increasing concentration of ephrin-B2 and tested the adhesion of BCWM1, MEC1 and RL to ephrin. Again, we found the adhesion peaked at the concentration of 100nM of ephrin. In contrast, ephrin-B2 had no chemotactic effect on WM cells. BM microenvironment components including BM stromal cells (BMSCs) and endothelial cells were shown to enhance the proliferation of WM cells detected by thymidine uptake assay. Ephrin-B2 was shown to be expressed on both endothelial cells and BMSCs. The inhibition of either the ephrin-B2 on endothelial cells and BMSCs, or inhibition of the Eph-B2 receptor on WM cell reduced the adhesion of WM cell to both endothelial cells and BMSCs and decreased the increase of WM cell proliferation induced by endothelial cells and BMSCs. The combination of both inhibition ephrin-B2 and Eph-B2 did not have an additive effect compared to each of them alone. Moreover, the inhibition of either ephrin-B2 or Eph-B2 reduced the activation of cell adhesion-related proteins. CONCLUSION: In conclusion, we showed that the Eph-B2/ephrin-B2 axis was activated in WM cells and that it is important for the adhesion and proliferation of WM cells induced by the BM microenvironment. These findings provide a novel therapeutic target for WM. Disclosures: Ghobrial: Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.