Daniel Öhlund

Pancreatic stellate cells (PSCs) differentiate into cancer-associated fibroblasts (CAFs) that produce desmoplastic stroma, thereby modulating disease progression and therapeutic response in pancreatic ductal adenocarcinoma (PDA). However, it is unknown whether CAFs uniformly carry out these tasks or if subtypes of CAFs with distinct phenotypes in PDA exist. We identified a CAF subpopulation with elevated expression of α-smooth muscle actin (αSMA) located immediately adjacent to neoplastic cells in mouse and human PDA tissue. We recapitulated this finding in co-cultures of murine PSCs and PDA organoids, and demonstrated that organoid-activated CAFs produced desmoplastic stroma. The co-cultures showed cooperative interactions and revealed another distinct subpopulation of CAFs, located more distantly from neoplastic cells, which lacked elevated αSMA expression and instead secreted IL6 and additional inflammatory mediators. These findings were corroborated in mouse and human PDA tissue, providing direct evidence for CAF heterogeneity in PDA tumor biology with implications for disease etiology and therapeutic development.

Pancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy.

Fibroblasts regulate the structure and function of healthy tissues, participate transiently in tissue repair after acute inflammation, and assume an aberrant stimulatory role during chronic inflammatory states including cancer. Such cancer-associated fibroblasts (CAFs) modulate the tumor microenvironment and influence the behavior of neoplastic cells in either a tumor-promoting or tumor-inhibiting manner. These pleiotropic functions highlight the inherent plasticity of fibroblasts and may provide new avenues to understand and therapeutically intervene in malignancies. We discuss the emerging themes of CAF biology in the context of tumorigenesis and therapy.

Pancreatic cancer is a deadly malignancy that lacks effective therapeutics. We previously reported that oncogenic Kras induced the redox master regulator Nfe2l2/Nrf2 to stimulate pancreatic and lung cancer initiation. Here, we show that NRF2 is necessary to maintain pancreatic cancer proliferation by regulating mRNA translation. Specifically, loss of NRF2 led to defects in autocrine epidermal growth factor receptor (EGFR) signaling and oxidation of specific translational regulatory proteins, resulting in impaired cap-dependent and cap-independent mRNA translation in pancreatic cancer cells. Combined targeting of the EGFR effector AKT and the glutathione antioxidant pathway mimicked Nrf2 ablation to potently inhibit pancreatic cancer ex vivo and in vivo, representing a promising synthetic lethal strategy for treating the disease.

Pancreatic ductal adenocarcinoma (PDAC) has prominent extracellular matrix (ECM) that compromises treatments yet cannot be nonselectively disrupted without adverse consequences. ECM of PDAC, despite the recognition of its importance, has not been comprehensively studied in patients. In this study, we used quantitative mass spectrometry (MS)-based proteomics to characterize ECM proteins in normal pancreas and pancreatic intraepithelial neoplasia (PanIN)- and PDAC-bearing pancreas from both human patients and mouse genetic models, as well as chronic pancreatitis patient samples. We describe detailed changes in both abundance and complexity of matrisome proteins in the course of PDAC progression. We reveal an early up-regulated group of matrisome proteins in PanIN, which are further up-regulated in PDAC, and we uncover notable similarities in matrix changes between pancreatitis and PDAC. We further assigned cellular origins to matrisome proteins by performing MS on multiple lines of human-to-mouse xenograft tumors. We found that, although stromal cells produce over 90% of the ECM mass, elevated levels of ECM proteins derived from the tumor cells, but not those produced exclusively by stromal cells, tend to correlate with poor patient survival. Furthermore, distinct pathways were implicated in regulating expression of matrisome proteins in cancer cells and stromal cells. We suggest that, rather than global suppression of ECM production, more precise ECM manipulations, such as targeting tumor-promoting ECM proteins and their regulators in cancer cells, could be more effective therapeutically.

Pancreatic ductal adenocarcinoma (PDA) is notoriously aggressive and hard to treat. The tumour microenvironment (TME) in PDA is highly dynamic and has been found to promote tumour progression, metastasis niche formation and therapeutic resistance. Intensive research of recent years has revealed an incredible heterogeneity and complexity of the different components of the TME, including cancer-associated fibroblasts, immune cells, extracellular matrix components, tumour vessels and nerves. It has been hypothesised that paracrine interactions between neoplastic epithelial cells and TME compartments may result in either tumour-promoting or tumour-restraining consequences. A better preclinical understanding of such complex and dynamic network systems is required to develop more powerful treatment strategies for patients. Scientific activity and the number of compelling findings has virtually exploded during recent years. Here, we provide an update of the most recent findings in this area and discuss their translational and clinical implications for basic scientists and clinicians alike.

Abstract The prognosis for pancreatic ductal adenocarcinoma (PDAC) remains poor despite decades of effort. The abundant extracellular matrix (ECM) in PDAC comprises a major fraction of the tumor mass and plays various roles in promoting resistance to therapies. However, nonselective depletion of ECM has led to poor patient outcomes. Consistent with that observation, we previously showed that individual matrisome proteins derived from stromal cells correlate with either long or short patient survival. In marked contrast, those derived from cancer cells correlate strongly with poor survival. Here, we studied three cancer cell–derived matrisome proteins that are significantly overrepresented during PDAC progression, AGRN (agrin), SERPINB5 (serine protease inhibitor B5), and CSTB (cystatin B). Using both overexpression and knockdown experiments, we demonstrate that all three are promoters of PDAC metastasis. Furthermore, these proteins operate at different metastatic steps. AGRN promoted epithelial-to-mesenchymal transition in primary tumors, whereas SERPINB5 and CSTB enhanced late steps in the metastatic cascade by elevating invadopodia formation and in vivo extravasation. All three genes were associated with a poor prognosis in human patients and high levels of SERPINB5, secreted by cancer cells and deposited in the ECM, correlated with poor patient prognosis. This study provides strong evidence that cancer cell–derived matrisome proteins can be causal in promoting tumorigenesis and metastasis and lead to poor patient survival. Therefore, compared with the bulk matrix, mostly made by stromal cells, precise interventions targeting cancer cell–derived matrisome proteins, such as AGRN, SERPINB5, and CSTB, may represent preferred potential therapeutic targets. Significance: This study provides insights into the biological roles of cancer cell–derived matrisome proteins in PDAC and supports the notion that these proteins are protumorigenic and better therapeutic targets.

<h2>Summary</h2><h3>Background</h3> In patients undergoing resection for pancreatic cancer, adjuvant modified fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX) improves overall survival compared with alternative chemotherapy regimens. We aimed to compare the efficacy and safety of neoadjuvant FOLFIRINOX with the standard strategy of upfront surgery in patients with resectable pancreatic ductal adenocarcinoma. <h3>Methods</h3> NORPACT-1 was a multicentre, randomised, phase 2 trial done in 12 hospitals in Denmark, Finland, Norway, and Sweden. Eligible patients were aged 18 years or older, with a WHO performance status of 0 or 1, and had a resectable tumour of the pancreatic head radiologically strongly suspected to be pancreatic adenocarcinoma. Participants were randomly assigned (3:2 before October, 2018, and 1:1 after) to the neoadjuvant FOLFIRINOX group or upfront surgery group. Patients in the neoadjuvant FOLFIRINOX group received four neoadjuvant cycles of FOLFIRINOX (oxaliplatin 85 mg/m², irinotecan 180 mg/m², leucovorin 400 mg/m², and fluorouracil 400 mg/m² bolus then 2400 mg/m² over 46 h on day 1 of each 14-day cycle), followed by surgery and adjuvant chemotherapy. Patients in the upfront surgery group underwent surgery and then received adjuvant chemotherapy. Initially, adjuvant chemotherapy was gemcitabine plus capecitabine (gemcitabine 1000 mg/m² over 30 min on days 1, 8, and 15 of each 28-day cycle and capecitabine 830 mg/m² twice daily for 3 weeks with 1 week of rest in each 28-day cycle; four cycles in the neoadjuvant FOLFIRINOX group, six cycles in the upfront surgery group). A protocol amendment was subsequently made to permit use of adjuvant modified FOLFIRINOX (oxaliplatin 85 mg/m², irinotecan 150 mg/m², leucovorin 400 mg/m², and fluorouracil 2400 mg/m² over 46 h on day 1 of each 14-day cycle; eight cycles in the neoadjuvant FOLFIRINOX group, 12 cycles in the upfront surgery group). Randomisation was performed with a computerised algorithm that stratified for each participating centre and used a concealed block size of two to six. Patients, investigators, and study team members were not masked to treatment allocation. The primary endpoint was overall survival at 18 months. Analyses were done in the intention-to-treat (ITT) and per-protocol populations. Safety was assessed in all patients who were randomly assigned and received at least one cycle of neoadjuvant or adjuvant therapy. This trial is registered with ClinicalTrials.gov, NCT02919787, and EudraCT, 2015-001635-21, and is ongoing. <h3>Findings</h3> Between Feb 8, 2017, and April 21, 2021, 77 patients were randomly assigned to receive neoadjuvant FOLFIRINOX and 63 to undergo upfront surgery. All patients were included in the ITT analysis. For the per-protocol analysis, 17 (22%) patients were excluded from the neoadjuvant FOLFIRINOX group (ten did not receive neoadjuvant therapy, four did not have pancreatic ductal adenocarcinoma, and three received another neoadjuvant regimen), and eight (13%) were excluded from the upfront surgery group (seven did not have pancreatic ductal adenocarcinoma and one did not undergo surgical exploration). 61 (79%) of 77 patients in the neoadjuvant FOLFIRINOX group received neoadjuvant therapy. The proportion of patients alive at 18 months by ITT was 60% (95% CI 49–71) in the neoadjuvant FOLFIRINOX group versus 73% (62–84) in the upfront surgery group (p=0·032), and median overall survival by ITT was 25·1 months (95% CI 17·2–34·9) versus 38·5 months (27·6–not reached; hazard ratio [HR] 1·52 [95% CI 1·00–2·33], log-rank p=0·050). The proportion of patients alive at 18 months in per-protocol analysis was 57% (95% CI 46–67) in the neoadjuvant FOLFIRINOX group versus 70% (55–83) in the upfront surgery group (p=0·14), and median overall survival in per-protocol population was 23·0 months (95% CI 16·2–34·9) versus 34·4 months (19·4–not reached; HR 1·46 [95% CI 0·99–2·17], log-rank p=0·058). In the safety population, 42 (58%) of 73 patients in the neoadjuvant FOLFIRINOX group and 19 (40%) of 47 patients in the upfront surgery group had at least one grade 3 or worse adverse event. 63 (82%) of 77 patients in the neoadjuvant group and 56 (89%) of 63 patients in the upfront surgery group had resection (p=0·24). One sudden death of unknown cause and one COVID-19-related death occurred after the first cycle of neoadjuvant FOLFIRINOX. Adjuvant chemotherapy was initiated in 51 (86%) of 59 patients with resected pancreatic ductal adenocarcinoma in the neoadjuvant FOLFIRINOX group and 44 (90%) of 49 patients with resected pancreatic ductal adenocarcinoma in the upfront surgery group (p=0·56). Adjuvant modified FOLFIRINOX was given to 13 (25%) patients in the neoadjuvant FOLFIRINOX group and 19 (43%) patients in the upfront surgery group. During adjuvant chemotherapy, neutropenia (11 [22%] patients in the neoadjuvant FOLFIRINOX group and five [11%] in the upfront surgery group) was the most common grade 3 or worse adverse event. <h3>Interpretation</h3> This phase 2 trial did not show a survival benefit from neoadjuvant FOLFIRINOX in resectable pancreatic ductal adenocarcinoma compared with upfront surgery. Implementation of neoadjuvant FOLFIRINOX was challenging. Future trials on treatment sequencing in resectable pancreatic ductal adenocarcinoma should be biomarker driven. <h3>Funding</h3> Norwegian Cancer Society, South Eastern Norwegian Health Authority, The Sjöberg Foundation, and Helsinki University Hospital Research Grants.

Pancreatic cancer shows a highly aggressive and infiltrative growth pattern and is characterized by an abundant tumor stroma known to interact with the cancer cells, and to influence tumor growth and drug resistance. Cancer cells actively take part in the production of extracellular matrix proteins, which then become deposited into the tumor stroma. Type IV collagen, an important component of the basement membrane, is highly expressed by pancreatic cancer cells both in vivo and in vitro. In this study, the cellular effects of type IV collagen produced by the cancer cells were characterized.The expression of type IV collagen and its integrin receptors were examined in vivo in human pancreatic cancer tissue. The cellular effects of type IV collagen were studied in pancreatic cancer cell lines by reducing type IV collagen expression through RNA interference and by functional receptor blocking of integrins and their binding-sites on the type IV collagen molecule.We show that type IV collagen is expressed close to the cancer cells in vivo, forming basement membrane like structures on the cancer cell surface that colocalize with the integrin receptors. Furthermore, the interaction between type IV collagen produced by the cancer cell, and integrins on the surface of the cancer cells, are important for continuous cancer cell growth, maintenance of a migratory phenotype, and for avoiding apoptosis.We show that type IV collagen provides essential cell survival signals to the pancreatic cancer cells through an autocrine loop.

Pancreas cancer is a dreaded disease with high mortality, despite progress in surgical and oncological treatments in recent years. The field is hampered by a lack of good prognostic and predictive tumour biomarkers to be used during follow-up of patients.The circulating level of type IV collagen was measured by ELISA in pancreas cancer patients and controls. The expression pattern of type IV collagen in normal pancreas, pancreas cancer tissue and in pancreas cancer cell lines was studied by immunofluorescence and Western blot techniques.Patients with pancreas cancer have significantly increased circulating levels of type IV collagen. In pancreas cancer tissue high levels of type IV collagen expression was found in close proximity to cancer cells in the tumour stroma. Furthermore, pancreas cancer cells were found to produce and secrete type IV collagen in vitro, which in part can explain the high type IV collagen expression observed in pancreas cancer tissue, and the increased circulating levels in pancreas cancer patients. Of clinical importance, our results show that the circulating level of type IV collagen after surgery is strongly related to prognosis in patients treated for pancreas cancer by pancreatico-duodenectomy with curative intent. Persisting high levels of circulating type IV collagen after surgery indicates a quick relapse in disease and poor survival.Our results most importantly show that stroma related substances can be evaluated as potential cancer biomarkers, and thereby underline the importance of the tumour microenvironment also in this context.

The signal transducer and activator of transcription 3 (STAT3) protein is a master regulator of most key hallmarks and enablers of cancer, including cell proliferation and the response to DNA damage. G-Quadruplex (G4) structures are four-stranded noncanonical DNA structures enriched at telomeres and oncogenes’ promoters. In cancer cells, stabilization of G4 DNAs leads to replication stress and DNA damage accumulation and is therefore considered a promising target for oncotherapy. Here, we designed and synthesized novel quinazoline-based compounds that simultaneously and selectively affect these two well-recognized cancer targets, G4 DNA structures and the STAT3 protein. Using a combination of in vitro assays, NMR, and molecular dynamics simulations, we show that these small, uncharged compounds not only bind to the STAT3 protein but also stabilize G4 structures. In human cultured cells, the compounds inhibit phosphorylation-dependent activation of STAT3 without affecting the antiapoptotic factor STAT1 and cause increased formation of G4 structures, as revealed by the use of a G4 DNA-specific antibody. As a result, treated cells show slower DNA replication, DNA damage checkpoint activation, and an increased apoptotic rate. Importantly, cancer cells are more sensitive to these molecules compared to noncancerous cell lines. This is the first report of a promising class of compounds that not only targets the DNA damage cancer response machinery but also simultaneously inhibits the STAT3-induced cancer cell proliferation, demonstrating a novel approach in cancer therapy.

Abstract Purpose: KRAS is mutated in the majority of pancreatic ductal adenocarcinoma. MAPK and PI3K-AKT are primary KRAS effector pathways, but combined MAPK and PI3K inhibition has not been demonstrated to be clinically effective to date. We explore the resistance mechanisms uniquely employed by malignant cells. Experimental Design: We evaluated the expression and activation of receptor tyrosine kinases in response to combined MEK and AKT inhibition in KPC mice and pancreatic ductal organoids. In addition, we sought to determine the therapeutic efficacy of targeting resistance pathways induced by MEK and AKT inhibition in order to identify malignant-specific vulnerabilities. Results: Combined MEK and AKT inhibition modestly extended the survival of KPC mice and increased Egfr and ErbB2 phosphorylation levels. Tumor organoids, but not their normal counterparts, exhibited elevated phosphorylation of ERBB2 and ERBB3 after MEK and AKT blockade. A pan-ERBB inhibitor synergized with MEK and AKT blockade in human PDA organoids, whereas this was not observed for the EGFR inhibitor erlotinib. Combined MEK and ERBB inhibitor treatment of human organoid orthotopic xenografts was sufficient to cause tumor regression in short-term intervention studies. Conclusions: Analyses of normal and tumor pancreatic organoids revealed the importance of ERBB activation during MEK and AKT blockade primarily in the malignant cultures. The lack of ERBB hyperactivation in normal organoids suggests a larger therapeutic index. In our models, pan-ERBB inhibition was synergistic with dual inhibition of MEK and AKT, and the combination of a pan-ERBB inhibitor with MEK antagonists showed the highest activity both in vitro and in vivo.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a dismal prognosis that is frequently diagnosed at an advanced stage. Although less common than other malignant diseases, it currently ranks as the fourth most common cause of cancer-related death in the European Union with a five-year survival rate of below 9%. Surgical resection, followed by adjuvant chemotherapy, remains the only potentially curative treatment but only a minority of patients is diagnosed with locally resectable, non-metastatic disease. Patients with advanced disease are treated with chemotherapy but high rates of treatment resistance and unfavorable side-effect profiles of some of the used regimens remain major challenges. Biomarkers reflect pathophysiological or physiological processes linked to a disease and can be used as diagnostic, prognostic and predictive tools. Thus, accurate biomarkers can allow for better patient stratification and guide therapy choices. Currently, the only broadly used biomarker for PDAC, CA 19-9, has multiple limitations and the need for novel biomarkers is urgent. In this review, we highlight the current situation, recent discoveries and developments in the field of biomarkers of PDAC and their potential clinical applications.

Colorectal cancer (CRC) is a heterogeneous disease, with varying clinical presentations and patient prognosis. Different molecular subgroups of CRC should be treated differently and therefore, must be better characterized. Organoid culture has recently been suggested as a good model to reflect the heterogeneous nature of CRC. However, organoid cultures cannot be established from all CRC tumors. The study examines which CRC tumors are more likely to generate organoids and thus benefit from ex vivo organoid drug testing. Long-term organoid cultures from 22 out of 40 CRC tumor specimens were established. It was found that organoid cultures were more difficult to establish from tumors characterized as microsatellite instable (MSI), BRAF-mutated, poorly differentiated and/or of a mucinous type. This suggests that patients with such tumors are less likely to benefit from ex vivo organoid drug testing, but it may also suggest biological difference in tumor growth. RNA sequencing analysis of tumor sections revealed that the in vivo maintenance of these non-organoid-forming tumors depends on factors related to inflammation and pathogen exposure. Furthermore, using TCGA data we could show a trend towards a worse prognosis for patients with organoid-forming tumors, suggesting also clinical differences. Results suggest that organoids are more difficult to establish from tumors characterized as MSI, BRAF-mutated, poorly differentiated and/or of a mucinous type. We further suggest that the maintenance of cell growth of these tumors in vivo may be promoted by immune-related factors and other stromal components within the tumor microenvironment.

Recently, we identified unique processing patterns of apolipoprotein A2 (ApoA2) in patients with pancreatic cancer. Our study provides a first prospective evaluation of an ApoA2 isoform ("ApoA2-ATQ/AT"), alone and in combination with carbohydrate antigen 19-9 (CA19-9), as an early detection biomarker for pancreatic cancer. We performed ELISA measurements of CA19-9 and ApoA2-ATQ/AT in 156 patients with pancreatic cancer and 217 matched controls within the European EPIC cohort, using plasma samples collected up to 60 months prior to diagnosis. The detection discrimination statistics were calculated for risk scores by strata of lag-time. For CA19-9, in univariate marker analyses, C-statistics to distinguish future pancreatic cancer patients from cancer-free individuals were 0.80 for plasma taken ≤6 months before diagnosis, and 0.71 for >6-18 months; for ApoA2-ATQ/AT, C-statistics were 0.62, and 0.65, respectively. Joint models based on ApoA2-ATQ/AT plus CA19-9 significantly improved discrimination within >6-18 months (C = 0.74 vs. 0.71 for CA19-9 alone, p = 0.022) and ≤ 18 months (C = 0.75 vs. 0.74, p = 0.022). At 98% specificity, and for lag times of ≤6, >6-18 or ≤ 18 months, sensitivities were 57%, 36% and 43% for CA19-9 combined with ApoA2-ATQ/AT, respectively, vs. 50%, 29% and 36% for CA19-9 alone. Compared to CA19-9 alone, the combination of CA19-9 and ApoA2-ATQ/AT may improve detection of pancreatic cancer up to 18 months prior to diagnosis under usual care, and may provide a useful first measure for pancreatic cancer detection prior to imaging.

Pancreatic ductal adenocarcinoma (PDAC) has one of the worst outcomes among cancers with a 5-years survival rate of below 10%. This is a result of late diagnosis and the lack of effective treatments. The tumor is characterized by a highly fibrotic stroma containing distinct cellular components, embedded within an extracellular matrix (ECM). This ECM-abundant tumor microenvironment (TME) in PDAC plays a pivotal role in tumor progression and resistance to treatment. Cancer-associated fibroblasts (CAFs), being a dominant cell type of the stroma, are in fact functionally heterogeneous populations of cells within the TME. Certain subtypes of CAFs are the main producer of the ECM components of the stroma, with the most abundant one being the collagen family of proteins. Collagens are large macromolecules that upon deposition into the ECM form supramolecular fibrillar structures which provide a mechanical framework to the TME. They not only bring structure to the tissue by being the main structural proteins but also contain binding domains that interact with surface receptors on the cancer cells. These interactions can induce various responses in the cancer cells and activate signaling pathways leading to epithelial-to-mesenchymal transition (EMT) and ultimately metastasis. In addition, collagens are one of the main contributors to building up mechanical forces in the tumor. These forces influence the signaling pathways that are involved in cell motility and tumor progression and affect tumor microstructure and tissue stiffness by exerting solid stress and interstitial fluid pressure on the cells. Taken together, the TME is subjected to various types of mechanical forces and interactions that affect tumor progression, metastasis, and drug response. In this review article, we aim to summarize and contextualize the recent knowledge of components of the PDAC stroma, especially the role of different collagens and mechanical traits on tumor progression. We furthermore discuss different experimental models available for studying tumor-stromal interactions and finally discuss potential therapeutic targets within the stroma.

The identification of pancreatic ductal adenocarcinoma (PDAC) dysregulated genes may unveil novel molecular targets entering inhibitory strategies. Laminins are emerging as potential targets in PDAC given their role as diagnostic and prognostic markers. Here, we investigated the cellular, functional, and clinical relevance of LAMC2 and its regulated network, with the ultimate goal of identifying potential therapies.LAMC2 expression was analyzed in PDAC tissues, a panel of human and mouse cell lines, and a genetically engineered mouse model. Genetic perturbation in 2D, 3D, and in vivo allograft and xenograft models was done. Expression profiling of a LAMC2 network was performed by RNA-sequencing, and publicly available gene expression datasets from experimental and clinical studies examined to query its human relevance. Dual inhibition of pharmacologically targetable LAMC2-regulated effectors was investigated.LAMC2 was consistently upregulated in human and mouse experimental models as well as in human PDAC specimens, and associated with tumor grade and survival. LAMC2 inhibition impaired cell cycle, induced apoptosis, and sensitized PDAC to MEK1/2 inhibitors (MEK1/2i). A LAMC2-regulated network was featured in PDAC, including both classical and quasi-mesenchymal subtypes, and contained downstream effectors transcriptionally shared by the KRAS signaling pathway. LAMC2 regulated a functional FOSL1-AXL axis via AKT phosphorylation. Furthermore, genetic LAMC2 or pharmacological AXL inhibition elicited a synergistic antiproliferative effect in combination with MEK1/2is that was consistent across 2D and 3D human and mouse PDAC models, including primary patient-derived organoids.LAMC2 is a molecular target in PDAC that regulates a transcriptional network that unveils a dual drug combination for cancer treatment.

Abstract Photodynamic therapy (PDT) ideally relies on the administration, selective accumulation and photoactivation of a photosensitizer (PS) into diseased tissues. In this context, we report a new heavy-atom-free fluorescent G-quadruplex (G4) DNA-binding PS, named DBI. We reveal by fluorescence microscopy that DBI preferentially localizes in intraluminal vesicles (ILVs), precursors of exosomes, which are key components of cancer cell proliferation. Moreover, purified exosomal DNA was recognized by a G4-specific antibody, thus highlighting the presence of such G4-forming sequences in the vesicles. Despite the absence of fluorescence signal from DBI in nuclei, light-irradiated DBI-treated cells generated reactive oxygen species (ROS), triggering a 3-fold increase of nuclear G4 foci, slowing fork progression and elevated levels of both DNA base damage, 8-oxoguanine, and double-stranded DNA breaks. Consequently, DBI was found to exert significant phototoxic effects (at nanomolar scale) toward cancer cell lines and tumor organoids. Furthermore, in vivo testing reveals that photoactivation of DBI induces not only G4 formation and DNA damage but also apoptosis in zebrafish, specifically in the area where DBI had accumulated. Collectively, this approach shows significant promise for image-guided PDT.

G-quadruplex (G4) DNA structures are involved in central biological processes such as DNA replication and transcription. These DNA structures are enriched in promotor regions of oncogenes and are thus promising as novel gene silencing therapeutic targets that can be used to regulate expression of oncoproteins and in particular those that has proven hard to drug with conventional strategies. G4 DNA structures in general have a well-defined and hydrophobic binding area that also is very flat and featureless and there are ample examples of G4 ligands but their further progression towards drug development is limited. In this study, we use synthetic organic chemistry to equip a drug-like and low molecular weight central fragment with different side chains and evaluate how this affect the compound's selectivity and ability to bind and stabilize G4 DNA. Furthermore, we study the binding interactions of the compounds and connect the experimental observations with the compound's structural conformations and electrostatic potentials to understand the basis for the observed improvements. Finally, we evaluate the top candidates' ability to selectively reduce cancer cell growth in a 3D co-culture model of pancreatic cancer which show that this is a powerful approach to generate highly active and selective low molecular weight G4 ligands with a promising therapeutic window.

Abstract Endostatin is a potent inhibitor of angiogenesis that is cleaved from the basement membrane protein type XVIII collagen. Expression of endostatin has recently been shown by Western blot analysis of tissue lysates in normal pancreas and pancreas cancer tissue. We show here that the expression pattern of type XVIII collagen/endostatin is shifted from a general basement membrane staining and is mainly located in the vasculature during tumor progression. This shift in type XVIII collagen/endostatin expression pattern coincides with an up‐regulation of MMPs involved in endostatin processing in the tumor microenvironment, such as MMP‐3, MMP‐9 and MMP‐13. The circulating levels of endostatin was analyzed in patients with pancreas cancer and compared to that of healthy controls, as well as after surgical treatment or in a group of nonoperable patients after intraperitoneal fluorouracil (5‐FU) chemotherapy. The results show that patients with pancreas cancer have increased circulating levels of endostatin and that these levels are normalized after surgery or intraperitoneal chemotherapy. These findings indicate that endostatin could be used as a biomarker for pancreas cancer progression. © 2008 Wiley‐Liss, Inc.

Abstract Unstable and dysfunctional tumor vasculature promotes cancer progression and spread. Signal transduction by the pro‐angiogenic vascular endothelial growth factor (VEGF) receptor‐2 (VEGFR2) is modulated by VEGFA‐dependent complex formation with neuropilin 1 (NRP1). NRP1 expressed on tumor cells can form VEGFR2/NRP1 trans ‐complexes between tumor cells and endothelial cells which arrests VEGFR2 on the endothelial surface, thus interfering with productive VEGFR2 signaling. In mouse fibrosarcoma, VEGFR2/NRP1 trans ‐complexes correlated with reduced tumor vessel branching and reduced tumor cell proliferation. Pancreatic ductal adenocarcinoma (PDAC) strongly expressed NRP1 on both tumor cells and endothelial cells, in contrast to other common cancer forms. Using proximity ligation assay, VEGFR2/NRP1 trans ‐complexes were identified in human PDAC tumor tissue, and its presence was associated with reduced tumor vessel branching, reduced tumor cell proliferation, and improved patient survival after adjusting for other known survival predictors. We conclude that VEGFR2/NRP1 trans ‐complex formation is an independent predictor of PDAC patient survival. © 2018 The Authors. The Journal of Pathology published by John Wiley &amp; Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Objectives: The aim of this research was to study whether plasma microRNAs (miRNA) can be used for early detection of pancreatic cancer (PC) by analyzing prediagnostic plasma samples collected before a PC diagnosis. Background: PC has a poor prognosis due to late presenting symptoms and early metastasis. Circulating miRNAs are altered in PC at diagnosis but have not been evaluated in a prediagnostic setting. Methods: We first performed an initial screen using a panel of 372 miRNAs in a retrospective case-control cohort that included early-stage PC patients and healthy controls. Significantly altered miRNAs at diagnosis were then measured in an early detection case-control cohort wherein plasma samples in the cases are collected before a PC diagnosis. Carbohydrate antigen 19–9 (Ca 19–9) levels were measured in all samples for comparison. Results: Our initial screen, including 23 stage I-II PC cases and 22 controls, revealed 15 candidate miRNAs that were differentially expressed in plasma samples at PC diagnosis. We combined all 15 miRNAs into a multivariate statistical model, which outperformed Ca 19–9 in receiver-operating characteristics analysis. However, none of the candidate miRNAs, individually or in combination, were significantly altered in prediagnostic plasma samples from 67 future PC patients compared with 132 matched controls. In comparison, Ca 19–9 levels were significantly higher in the cases at <5 years before diagnosis. Conclusion: Plasma miRNAs are altered in PC patients at diagnosis, but the candidate miRNAs found in this study appear late in the course of the disease and cannot be used for early detection of the disease.

The tumor microenvironment is crucial in the initiation and progression of cancers. The interplay between cancer cells and the surrounding stroma shapes the tumor biology and dictates the response to cancer therapies. Consequently, a better understanding of the interactions between cancer cells and different components of the tumor microenvironment will drive progress in developing novel, effective, treatment strategies. Co-cultures can be used to study various aspects of these interactions in detail. This includes studies of paracrine relationships between cancer cells and stromal cells such as fibroblasts, endothelial cells, and immune cells, as well as the influence of physical and mechanical interactions with the extracellular matrix of the tumor microenvironment. The development of novel co-culture models to study the tumor microenvironment has progressed rapidly over recent years. Many of these models have already been shown to be powerful tools for further understanding of the pathophysiological role of the stroma and provide mechanistic insights into tumor-stromal interactions. Here we give a structured overview of different co-culture models that have been established to study tumor-stromal interactions and what we have learnt from these models. We also introduce a set of guidelines for generating and reporting co-culture experiments to facilitate experimental robustness and reproducibility.

BACKGROUND: A lack of disease-specific symptoms and good tumour markers makes early detection and diagnosis of pancreatic ductal adenocarcinoma (PDAC) challenging.OBJECTIVE: To analyse the tissue expression and circulating levels of four stroma-derived substances (type IV collagen, endostatin/type XVIII collagen, osteopontin and tenascin C) and four conventional tumour markers (CA 19-9, TPS, CEA and Ca 125) in a PDAC cohort.METHODS: Tissue expression of markers in normal pancreas and PDAC tissue was analysed with immunofluorescence.Plasma concentrations of markers were measured before and after surgery.Patients with non-malignant disorders served as controls.RESULTS: The conventional and stromal substances were expressed in the cancer cell compartment and the stroma, respectively.Although most patients had increased levels of many markers before surgery, 2/12 (17%) of patients had normal levels of Ca 19-9 at this stage.High preoperative endostatin/type XVIII collagen, and postoperative type IV collagen was associated with short survival.Neither the pre-nor postoperative levels of TPS, Ca 125 or CA 19-9 were associated to survival.CONCLUSIONS: PDAC is characterized by an abundant stroma.These initial observations indicate that the stroma can be a source of PDAC tumour markers that are found in different compartments of the cancer, thus reflecting different aspects of tumour biology.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by rich deposits of extracellular matrix (ECM), affecting the pathophysiology of the disease. Here, we identified galectin 4 (gal 4) as a cancer cell–produced protein that was deposited into the ECM of PDAC tumors and detected high-circulating levels of gal 4 in patients with PDAC. In orthotopic transplantation experiments, we observed increased infiltration of T cells and prolonged survival in immunocompetent mice transplanted with cancer cells with reduced expression of gal 4. Increased survival was not observed in immunodeficient RAG1−/− mice, demonstrating that the effect was mediated by the adaptive immune system. By performing single-cell RNA-sequencing, we found that the myeloid compartment and cancer-associated fibroblast (CAF) subtypes were altered in the transplanted tumors. Reduced gal 4 expression associated with a higher proportion of myofibroblastic CAFs and reduced numbers of inflammatory CAFs. We also found higher proportions of M1 macrophages, T cells, and antigen-presenting dendritic cells in tumors with reduced gal 4 expression. Using a coculture system, we observed that extracellular gal 4 induced apoptosis in T cells by binding N-glycosylation residues on CD3ε/δ. Hence, we show that gal 4 is involved in immune evasion and identify gal 4 as a promising drug target for overcoming immunosuppression in PDAC.

Screening of the general population for cancer is a matter of primary prevention reducing the burden of disease. Whilst this is successful for several cancers including breast, colon and prostate, the situation to screen and hence prevent pancreatic cancer is different. The organ is not as accessible to simple physical exam or biological samples (fecal or blood test). Neither exists a blood test such as PSA that is cost-effective. Reviewing the evidence from screening risk groups for pancreatic cancer, one must conclude that there is no rational at present to screen the general population, for a lack of appropriate tests.

Abstract The dense stroma in pancreatic cancer tumours is rich in secreted extracellular matrix proteins and proteoglycans. Secreted hyaluronan, osteopontin and type IV collagen sustain oncogenic signalling by interactions with CD44s and its variant isoform CD44v6 on cancer cell membranes. Although well established in animal and in vitro models, this oncogenic CD44‐stromal ligand network is less explored in human cancer. Here, we use a pancreatic cancer tissue microarray from 69 primary tumours and 37 metastatic lymph nodes and demonstrate that high tumour cell expression of CD44s and, surprisingly, low stromal deposition of osteopontin correlate with poor survival independent of established prognostic factors for pancreatic cancer. High stromal expression of hyaluronan was a universal trait of both primary tumours and metastatic lymph nodes. However, hyaluronan species of different molecular mass are known to function differently in pancreatic cancer biology and immunohistochemistry cannot distinguish between them. Using gas‐phase electrophoretic molecular mobility analysis, we uncover a shift towards high molecular mass hyaluronan in pancreatic cancer tissue compared to normal pancreas and at a transcriptional level, we find that hyaluronan synthesising HAS2 correlates positively with CD44. The resulting prediction that high molecular mass hyaluronan would then correlate with poor survival in pancreatic cancer was confirmed in serum samples, where we demonstrate that hyaluronan &gt;27 kDa measured before surgery is an independent predictor of postoperative survival. Our findings confirm the prognostic value of CD44 tissue expression and highlight osteopontin tissue expression and serum high molecular mass hyaluronan as novel prognostic markers in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer death that typically presents at an advanced stage. No reliable markers for early detection presently exist. The prominent tumor stroma represents a source of circulating biomarkers for use together with cancer cell-derived biomarkers for earlier PDAC diagnosis. CA19-9 and CEA (cancer cell-derived biomarkers), together with endostatin and collagen IV (stroma-derived) were examined alone, or together, by multivariable modelling, using pre-diagnostic plasma samples (n = 259 samples) from the Northern Sweden Health and Disease Study biobank. Serial samples were available for a subgroup of future patients. Marker efficacy for future PDAC case prediction (n = 154 future cases) was examined by both cross-sectional (ROC analysis) and longitudinal analyses. CA19-9 performed well at, and within, six months to diagnosis and multivariable modelling was not superior to CA19-9 alone in cross-sectional analysis. Within six months to diagnosis, CA19-9 (AUC = 0.92) outperformed the multivariable model (AUC = 0.81) at a cross-sectional level. At diagnosis, CA19-9 (AUC = 0.995) and the model (AUC = 0.977) performed similarly. Longitudinal analysis revealed increases in CA19-9 up to two years to diagnosis which indicates a window of opportunity for early detection of PDAC.

Early detection of pancreatic ductal adenocarcinoma (PDAC) is challenging, and late diagnosis partly explains the low 5-year survival. Novel and sensitive biomarkers are needed to enable early PDAC detection and improve patient outcomes. Tissue polypeptide specific antigen (TPS) has been studied as a biomarker in PDAC diagnostics, and it has previously been shown to reflect clinical status better than the 'golden standard' biomarker carbohydrate antigen 19-9 (CA 19-9) that is most widely used in the clinical setting. In this cross-sectional case-control study using pre-diagnostic plasma samples, we aim to evaluate the potential of TPS as a biomarker for early PDAC detection. Furthermore, in a subset of individuals with multiple samples available at different time points before diagnosis, a longitudinal analysis was used. We assessed plasma TPS levels using enzyme-linked immunosorbent assay (ELISA) in 267 pre-diagnostic PDAC plasma samples taken up to 18.8 years before clinical PDAC diagnosis and in 320 matched healthy controls. TPS levels were also assessed in 25 samples at PDAC diagnosis. Circulating TPS levels were low both in pre-diagnostic samples of future PDAC patients and in healthy controls, whereas TPS levels at PDAC diagnosis were significantly increased (odds ratio 1.03; 95% confidence interval: 1.01-1.05) in a logistic regression model adjusted for age. In conclusion, TPS levels increase late in PDAC progression and hold no potential as a biomarker for early detection.

In recent years, pancreatic cancer-associated stroma has been the topic of a number of studies that have proposed stromal components as promising, but still controversial, targets for novel therapeutic strategies. A new study describes how epithelial TGF-β signalling deficiency and consequential STAT3 activation act as key players in modulating the desmoplastic reaction, with implications for both clinical outcome and future drug development

&lt;div&gt;Abstract&lt;p&gt;The prognosis for pancreatic ductal adenocarcinoma (PDAC) remains poor despite decades of effort. The abundant extracellular matrix (ECM) in PDAC comprises a major fraction of the tumor mass and plays various roles in promoting resistance to therapies. However, nonselective depletion of ECM has led to poor patient outcomes. Consistent with that observation, we previously showed that individual matrisome proteins derived from stromal cells correlate with either long or short patient survival. In marked contrast, those derived from cancer cells correlate strongly with poor survival. Here, we studied three cancer cell–derived matrisome proteins that are significantly overrepresented during PDAC progression, AGRN (agrin), SERPINB5 (serine protease inhibitor B5), and CSTB (cystatin B). Using both overexpression and knockdown experiments, we demonstrate that all three are promoters of PDAC metastasis. Furthermore, these proteins operate at different metastatic steps. AGRN promoted epithelial-to-mesenchymal transition in primary tumors, whereas SERPINB5 and CSTB enhanced late steps in the metastatic cascade by elevating invadopodia formation and &lt;i&gt;in vivo&lt;/i&gt; extravasation. All three genes were associated with a poor prognosis in human patients and high levels of SERPINB5, secreted by cancer cells and deposited in the ECM, correlated with poor patient prognosis. This study provides strong evidence that cancer cell–derived matrisome proteins can be causal in promoting tumorigenesis and metastasis and lead to poor patient survival. Therefore, compared with the bulk matrix, mostly made by stromal cells, precise interventions targeting cancer cell–derived matrisome proteins, such as AGRN, SERPINB5, and CSTB, may represent preferred potential therapeutic targets.&lt;/p&gt;Significance:&lt;p&gt;This study provides insights into the biological roles of cancer cell–derived matrisome proteins in PDAC and supports the notion that these proteins are protumorigenic and better therapeutic targets.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;Suppl. Figure 4. A. Correlation plot of mRNA expression levels of LAMC2 and AXL in the PDAC human data set from ICGC. B. Correlation plot of mRNA expression levels of LAMC2 and AXL in the PDAC human data set from TCGA.&lt;/p&gt;

&lt;p&gt;List of published results of the identified proteins.&lt;/p&gt;

&lt;p&gt;List of qPCR primers.&lt;/p&gt;

&lt;p&gt;This is supplemental figure legends.&lt;/p&gt;

&lt;p&gt;This is supplemental figure legends.&lt;/p&gt;

&lt;p&gt;List of published results of the identified proteins.&lt;/p&gt;

&lt;p&gt;List of qPCR primers.&lt;/p&gt;

&lt;p&gt;Supplementary Figures 1-3&lt;/p&gt;

&lt;p&gt;Epithelial SERPINB5 staining is not prognostic.&lt;/p&gt;

&lt;p&gt;Example images for lungs after tail-vein injection and cells with TKS5 immunofluorescence staining.&lt;/p&gt;

&lt;p&gt;Supplementary Figures 1-3&lt;/p&gt;

&lt;div&gt;AbstractPurpose:&lt;p&gt;&lt;i&gt;KRAS&lt;/i&gt; is mutated in the majority of pancreatic ductal adenocarcinoma. MAPK and PI3K-AKT are primary KRAS effector pathways, but combined MAPK and PI3K inhibition has not been demonstrated to be clinically effective to date. We explore the resistance mechanisms uniquely employed by malignant cells.&lt;/p&gt;Experimental Design:&lt;p&gt;We evaluated the expression and activation of receptor tyrosine kinases in response to combined MEK and AKT inhibition in KPC mice and pancreatic ductal organoids. In addition, we sought to determine the therapeutic efficacy of targeting resistance pathways induced by MEK and AKT inhibition in order to identify malignant-specific vulnerabilities.&lt;/p&gt;Results:&lt;p&gt;Combined MEK and AKT inhibition modestly extended the survival of KPC mice and increased Egfr and ErbB2 phosphorylation levels. Tumor organoids, but not their normal counterparts, exhibited elevated phosphorylation of ERBB2 and ERBB3 after MEK and AKT blockade. A pan-ERBB inhibitor synergized with MEK and AKT blockade in human PDA organoids, whereas this was not observed for the EGFR inhibitor erlotinib. Combined MEK and ERBB inhibitor treatment of human organoid orthotopic xenografts was sufficient to cause tumor regression in short-term intervention studies.&lt;/p&gt;Conclusions:&lt;p&gt;Analyses of normal and tumor pancreatic organoids revealed the importance of ERBB activation during MEK and AKT blockade primarily in the malignant cultures. The lack of ERBB hyperactivation in normal organoids suggests a larger therapeutic index. In our models, pan-ERBB inhibition was synergistic with dual inhibition of MEK and AKT, and the combination of a pan-ERBB inhibitor with MEK antagonists showed the highest activity both &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;AbstractPurpose:&lt;p&gt;The identification of pancreatic ductal adenocarcinoma (PDAC) dysregulated genes may unveil novel molecular targets entering inhibitory strategies. Laminins are emerging as potential targets in PDAC given their role as diagnostic and prognostic markers. Here, we investigated the cellular, functional, and clinical relevance of LAMC2 and its regulated network, with the ultimate goal of identifying potential therapies.&lt;/p&gt;Experimental Design:&lt;p&gt;LAMC2 expression was analyzed in PDAC tissues, a panel of human and mouse cell lines, and a genetically engineered mouse model. Genetic perturbation in 2D, 3D, and &lt;i&gt;in vivo&lt;/i&gt; allograft and xenograft models was done. Expression profiling of a LAMC2 network was performed by RNA-sequencing, and publicly available gene expression datasets from experimental and clinical studies examined to query its human relevance. Dual inhibition of pharmacologically targetable LAMC2-regulated effectors was investigated.&lt;/p&gt;Results:&lt;p&gt;LAMC2 was consistently upregulated in human and mouse experimental models as well as in human PDAC specimens, and associated with tumor grade and survival. LAMC2 inhibition impaired cell cycle, induced apoptosis, and sensitized PDAC to MEK1/2 inhibitors (MEK1/2i). A LAMC2-regulated network was featured in PDAC, including both classical and quasi-mesenchymal subtypes, and contained downstream effectors transcriptionally shared by the KRAS signaling pathway. LAMC2 regulated a functional FOSL1–AXL axis via AKT phosphorylation. Furthermore, genetic LAMC2 or pharmacological AXL inhibition elicited a synergistic antiproliferative effect in combination with MEK1/2is that was consistent across 2D and 3D human and mouse PDAC models, including primary patient-derived organoids.&lt;/p&gt;Conclusions:&lt;p&gt;LAMC2 is a molecular target in PDAC that regulates a transcriptional network that unveils a dual drug combination for cancer treatment.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;AbstractPurpose:&lt;p&gt;The identification of pancreatic ductal adenocarcinoma (PDAC) dysregulated genes may unveil novel molecular targets entering inhibitory strategies. Laminins are emerging as potential targets in PDAC given their role as diagnostic and prognostic markers. Here, we investigated the cellular, functional, and clinical relevance of LAMC2 and its regulated network, with the ultimate goal of identifying potential therapies.&lt;/p&gt;Experimental Design:&lt;p&gt;LAMC2 expression was analyzed in PDAC tissues, a panel of human and mouse cell lines, and a genetically engineered mouse model. Genetic perturbation in 2D, 3D, and &lt;i&gt;in vivo&lt;/i&gt; allograft and xenograft models was done. Expression profiling of a LAMC2 network was performed by RNA-sequencing, and publicly available gene expression datasets from experimental and clinical studies examined to query its human relevance. Dual inhibition of pharmacologically targetable LAMC2-regulated effectors was investigated.&lt;/p&gt;Results:&lt;p&gt;LAMC2 was consistently upregulated in human and mouse experimental models as well as in human PDAC specimens, and associated with tumor grade and survival. LAMC2 inhibition impaired cell cycle, induced apoptosis, and sensitized PDAC to MEK1/2 inhibitors (MEK1/2i). A LAMC2-regulated network was featured in PDAC, including both classical and quasi-mesenchymal subtypes, and contained downstream effectors transcriptionally shared by the KRAS signaling pathway. LAMC2 regulated a functional FOSL1–AXL axis via AKT phosphorylation. Furthermore, genetic LAMC2 or pharmacological AXL inhibition elicited a synergistic antiproliferative effect in combination with MEK1/2is that was consistent across 2D and 3D human and mouse PDAC models, including primary patient-derived organoids.&lt;/p&gt;Conclusions:&lt;p&gt;LAMC2 is a molecular target in PDAC that regulates a transcriptional network that unveils a dual drug combination for cancer treatment.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;Suppl. Figure 3. A. Immunoblot of LAMC2 upon Lamc2 knockdown by a specific shRNA in 511950 and PM12167 PDAC mouse cell lines. B. Quantification of organoid number upon LAMC2 knockdown by a specific shRNA in 511950 and PM12167 PDAC mouse cell lines.C. Quantification of organoid area (AU) in same cell lines as in B. Error bars correspond to s.d. D. Immunoblot of LAMC2 upon LAMC2 knockdown in HPAFII cell line. E. Quantification of organoid number and organoid area (AU) in HPAFII cell line expressing a TET-inducible shRNA against LAMC2 (bottom) or a control shRNA (top) cultured in 3D. F. Representative images of the subcutaneous tumors from PATU8902 cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice. G. Representative images of the subcutaneous tumors from HPAFII cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice. H and I. Representative IHC of LAMC2 expression in subcutaneous tumors from PATU8902 (H) and HPAFII (I) cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice J. Representative images of the tumors derived from intrapancreatic injection of PATU8902 cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice. K. Representative images of the subcutaneous tumors from PM12167 mouse cells expressing a shRNA against Lamc2 or a control shRNA at sacrifice. L and M. Representative images of the subcutaneous (sc) tumors from PATU8902 and HPAFII expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice. ShRNA expression induced after tumor establishment at Day 8. N and O. Representative IHC of LAMC2 expression in subcutaneous tumors (Tx) from PATU8902 (H) and HPAFII (I) cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA, shRNA expression was induced at day 8 of tumor establishment. P. Quantification of Ki67 IHC staining in PATU8902 and HFAFII xenograft models: SC and Tx. Q. Quantification of cleaved caspase3 IHC staining in PATU8902 and HFAFII xenograft models: SC and Tx.&lt;/p&gt;

&lt;p&gt;Suppl. Figure 1. A. Association analysis of LAMC2 expression with clinical variables using quartile 1 of H score (23.2) as cut-off. B. Correlation of LAMC2 expression and tumor volume in a tissue array of 119 PDAC samples. C and D. Survival analysis stratified by LAMC2 expression from ICGC and TCGA datasets.&lt;/p&gt;

&lt;p&gt;Suppl. Figure 2. A. LAMC2 immunoblot in HPAFII after LAMC2 knockdown using 2 different sgRNAs. B. Cell proliferation assessed by MTS after LAMC2 knockdown using 2 sgRNAs. Error bars correspond to s.d. C. LAMC2 immunoblot in PM12167 after shRNA mediated Lamc2 knockdown. D. Cell proliferation assessed by MTS after shRNA mediated Lamc2 knockdown. Error bars correspond to s.d. E. Colony forming ability after shRNA mediated Lamc2 knockdown. Error bars correspond to s.d. F. Cell proliferation assessed by MTS, after shRNA-mediated LAMC2 knockdown in PATU8902 and HPAFII PDAC cell lines using a tet-inducible system. Error bars correspond to s.d.&lt;/p&gt;

&lt;p&gt;Suppl. Figure 1. A. Association analysis of LAMC2 expression with clinical variables using quartile 1 of H score (23.2) as cut-off. B. Correlation of LAMC2 expression and tumor volume in a tissue array of 119 PDAC samples. C and D. Survival analysis stratified by LAMC2 expression from ICGC and TCGA datasets.&lt;/p&gt;

&lt;div&gt;AbstractPurpose:&lt;p&gt;&lt;i&gt;KRAS&lt;/i&gt; is mutated in the majority of pancreatic ductal adenocarcinoma. MAPK and PI3K-AKT are primary KRAS effector pathways, but combined MAPK and PI3K inhibition has not been demonstrated to be clinically effective to date. We explore the resistance mechanisms uniquely employed by malignant cells.&lt;/p&gt;Experimental Design:&lt;p&gt;We evaluated the expression and activation of receptor tyrosine kinases in response to combined MEK and AKT inhibition in KPC mice and pancreatic ductal organoids. In addition, we sought to determine the therapeutic efficacy of targeting resistance pathways induced by MEK and AKT inhibition in order to identify malignant-specific vulnerabilities.&lt;/p&gt;Results:&lt;p&gt;Combined MEK and AKT inhibition modestly extended the survival of KPC mice and increased Egfr and ErbB2 phosphorylation levels. Tumor organoids, but not their normal counterparts, exhibited elevated phosphorylation of ERBB2 and ERBB3 after MEK and AKT blockade. A pan-ERBB inhibitor synergized with MEK and AKT blockade in human PDA organoids, whereas this was not observed for the EGFR inhibitor erlotinib. Combined MEK and ERBB inhibitor treatment of human organoid orthotopic xenografts was sufficient to cause tumor regression in short-term intervention studies.&lt;/p&gt;Conclusions:&lt;p&gt;Analyses of normal and tumor pancreatic organoids revealed the importance of ERBB activation during MEK and AKT blockade primarily in the malignant cultures. The lack of ERBB hyperactivation in normal organoids suggests a larger therapeutic index. In our models, pan-ERBB inhibition was synergistic with dual inhibition of MEK and AKT, and the combination of a pan-ERBB inhibitor with MEK antagonists showed the highest activity both &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;QPCR with human-specific primers shows that AGRN promotes EMT in primary tumors.&lt;/p&gt;

&lt;p&gt;Suppl. Figure 3. A. Immunoblot of LAMC2 upon Lamc2 knockdown by a specific shRNA in 511950 and PM12167 PDAC mouse cell lines. B. Quantification of organoid number upon LAMC2 knockdown by a specific shRNA in 511950 and PM12167 PDAC mouse cell lines.C. Quantification of organoid area (AU) in same cell lines as in B. Error bars correspond to s.d. D. Immunoblot of LAMC2 upon LAMC2 knockdown in HPAFII cell line. E. Quantification of organoid number and organoid area (AU) in HPAFII cell line expressing a TET-inducible shRNA against LAMC2 (bottom) or a control shRNA (top) cultured in 3D. F. Representative images of the subcutaneous tumors from PATU8902 cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice. G. Representative images of the subcutaneous tumors from HPAFII cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice. H and I. Representative IHC of LAMC2 expression in subcutaneous tumors from PATU8902 (H) and HPAFII (I) cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice J. Representative images of the tumors derived from intrapancreatic injection of PATU8902 cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice. K. Representative images of the subcutaneous tumors from PM12167 mouse cells expressing a shRNA against Lamc2 or a control shRNA at sacrifice. L and M. Representative images of the subcutaneous (sc) tumors from PATU8902 and HPAFII expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice. ShRNA expression induced after tumor establishment at Day 8. N and O. Representative IHC of LAMC2 expression in subcutaneous tumors (Tx) from PATU8902 (H) and HPAFII (I) cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA, shRNA expression was induced at day 8 of tumor establishment. P. Quantification of Ki67 IHC staining in PATU8902 and HFAFII xenograft models: SC and Tx. Q. Quantification of cleaved caspase3 IHC staining in PATU8902 and HFAFII xenograft models: SC and Tx.&lt;/p&gt;

&lt;p&gt;Suppl. Figure 5. A. RNA expression of KRAS, AREG, DUSP6 and FOSL1 in PATU8902 and HPAFII cells expressing a TET-inducible KRAS shRNA or a control shRNA. B. Box and whiskers plot showing Mean Fluorescence Intensity (MFI) of Areg, Dusp6, Fosl1 upon KRas inactivation in iKRAS GEM. Error bars correspond to s.d. C. Heat map of downregulated genes in HPAFII cells expressing a tet-inducible LAMC2 shRNA compared to control group (shRNA GFP). D. LAMC2 and FOSL1 mRNA expression in PATU8902 and HPAFII cells expressing a tet-inducible LAMC2 shRNA or a control shRNA. Error bars correspond to s.d. E. mRNA expression of overlapping LAMC2 and FOSL1 downregulated genes (LAMC2 -ctrl-, FGFBP1, HMGA1, AURKA, HMMR, PLK1, SNAI2 and PITPNC1) in PATU8902 and HPAFII cells expressing a TET-inducible LAMC2 shRNA or a control shRNA. F. Protein expression of phospho-ERK, ERK. Phospho-EGFR, EGFR in PATU8902 and HPAFII cells expressing a TETinducible LAMC2 shRNAs or a control shRNA. Quantification: pEGFR levels normalized to total EGFR levels and housekeeping protein levels. G. Fosl1 and AXL protein expression in PATU8902 and HPAFII cells treated with AKTi (10μM) and vehicle control. Quantification: AXL and FOSL1 levels normalized to housekeeping protein levels.&lt;/p&gt;

&lt;p&gt;Suppl. Figure 6. A. Left. Protein expression levels of phospho-ERK and ERK in HPAFII and PATU8902 cells treated with MEKi (50 nM) for 24 h. Right. Protein expression levels of phospho-AXL and AXL in HPAFII and PATU8902 cells treated with AXLi (0.2 μM) for 24 h. Quantification: pAXL levels normalized to total AXL levels and housekeeping protein levels. B. Heatmap of the Fa fraction of CAPAN2 PDAC human cell line treated with single MEKi, AXLi, or both at indicated concentrations for 72 h. C. 2D surface plot of Bliss synergy scores from B. Red regions indicate synergistic interactions.&lt;/p&gt;

&lt;p&gt;Suppl. Figure 7. A. KRAS, TP53, SMAD4 genetic alteration in the 2 PDO characterized by DNA sequencing.&lt;/p&gt;

&lt;p&gt;This file includes the Suppl. Methods, Table (1) and References&lt;/p&gt;

&lt;p&gt;This file includes the Suppl. Methods, Table (1) and References&lt;/p&gt;

&lt;p&gt;Suppl. Figure 5. A. RNA expression of KRAS, AREG, DUSP6 and FOSL1 in PATU8902 and HPAFII cells expressing a TET-inducible KRAS shRNA or a control shRNA. B. Box and whiskers plot showing Mean Fluorescence Intensity (MFI) of Areg, Dusp6, Fosl1 upon KRas inactivation in iKRAS GEM. Error bars correspond to s.d. C. Heat map of downregulated genes in HPAFII cells expressing a tet-inducible LAMC2 shRNA compared to control group (shRNA GFP). D. LAMC2 and FOSL1 mRNA expression in PATU8902 and HPAFII cells expressing a tet-inducible LAMC2 shRNA or a control shRNA. Error bars correspond to s.d. E. mRNA expression of overlapping LAMC2 and FOSL1 downregulated genes (LAMC2 -ctrl-, FGFBP1, HMGA1, AURKA, HMMR, PLK1, SNAI2 and PITPNC1) in PATU8902 and HPAFII cells expressing a TET-inducible LAMC2 shRNA or a control shRNA. F. Protein expression of phospho-ERK, ERK. Phospho-EGFR, EGFR in PATU8902 and HPAFII cells expressing a TETinducible LAMC2 shRNAs or a control shRNA. Quantification: pEGFR levels normalized to total EGFR levels and housekeeping protein levels. G. Fosl1 and AXL protein expression in PATU8902 and HPAFII cells treated with AKTi (10μM) and vehicle control. Quantification: AXL and FOSL1 levels normalized to housekeeping protein levels.&lt;/p&gt;

&lt;p&gt;Suppl. Figure 7. A. KRAS, TP53, SMAD4 genetic alteration in the 2 PDO characterized by DNA sequencing.&lt;/p&gt;

&lt;p&gt;Suppl. Figure 2. A. LAMC2 immunoblot in HPAFII after LAMC2 knockdown using 2 different sgRNAs. B. Cell proliferation assessed by MTS after LAMC2 knockdown using 2 sgRNAs. Error bars correspond to s.d. C. LAMC2 immunoblot in PM12167 after shRNA mediated Lamc2 knockdown. D. Cell proliferation assessed by MTS after shRNA mediated Lamc2 knockdown. Error bars correspond to s.d. E. Colony forming ability after shRNA mediated Lamc2 knockdown. Error bars correspond to s.d. F. Cell proliferation assessed by MTS, after shRNA-mediated LAMC2 knockdown in PATU8902 and HPAFII PDAC cell lines using a tet-inducible system. Error bars correspond to s.d.&lt;/p&gt;

&lt;p&gt;Suppl. Figure 6. A. Left. Protein expression levels of phospho-ERK and ERK in HPAFII and PATU8902 cells treated with MEKi (50 nM) for 24 h. Right. Protein expression levels of phospho-AXL and AXL in HPAFII and PATU8902 cells treated with AXLi (0.2 μM) for 24 h. Quantification: pAXL levels normalized to total AXL levels and housekeeping protein levels. B. Heatmap of the Fa fraction of CAPAN2 PDAC human cell line treated with single MEKi, AXLi, or both at indicated concentrations for 72 h. C. 2D surface plot of Bliss synergy scores from B. Red regions indicate synergistic interactions.&lt;/p&gt;

&lt;p&gt;Suppl. Figure 4. A. Correlation plot of mRNA expression levels of LAMC2 and AXL in the PDAC human data set from ICGC. B. Correlation plot of mRNA expression levels of LAMC2 and AXL in the PDAC human data set from TCGA.&lt;/p&gt;

&lt;p&gt;QPCR with human-specific primers shows that AGRN promotes EMT in primary tumors.&lt;/p&gt;

&lt;p&gt;In vitro proliferation assay for the overexpression and knockdown sets of cells.&lt;/p&gt;

&lt;p&gt;Example images for lungs after tail-vein injection and cells with TKS5 immunofluorescence staining.&lt;/p&gt;

&lt;p&gt;Xenograft tumors knocked down and overexpressing AGRN, SERPINB5, and CSTB show expected protein expression changes.&lt;/p&gt;

&lt;p&gt;Epithelial SERPINB5 staining is not prognostic.&lt;/p&gt;

&lt;p&gt;AGRN, SERPINB5, and CSTB gene expression in a panel of PDAC cell lines.&lt;/p&gt;

&lt;p&gt;In vitro proliferation assay for the overexpression and knockdown sets of cells.&lt;/p&gt;

&lt;p&gt;Xenograft tumors knocked down and overexpressing AGRN, SERPINB5, and CSTB show expected protein expression changes.&lt;/p&gt;

&lt;p&gt;AGRN, SERPINB5, and CSTB gene expression in a panel of PDAC cell lines.&lt;/p&gt;

&lt;div&gt;Abstract&lt;p&gt;Pancreatic ductal adenocarcinoma (PDAC) is characterized by rich deposits of extracellular matrix (ECM), affecting the pathophysiology of the disease. Here, we identified galectin 4 (gal 4) as a cancer cell–produced protein that was deposited into the ECM of PDAC tumors and detected high-circulating levels of gal 4 in patients with PDAC. In orthotopic transplantation experiments, we observed increased infiltration of T cells and prolonged survival in immunocompetent mice transplanted with cancer cells with reduced expression of gal 4. Increased survival was not observed in immunodeficient RAG1&lt;sup&gt;−/−&lt;/sup&gt; mice, demonstrating that the effect was mediated by the adaptive immune system. By performing single-cell RNA-sequencing, we found that the myeloid compartment and cancer-associated fibroblast (CAF) subtypes were altered in the transplanted tumors. Reduced gal 4 expression associated with a higher proportion of myofibroblastic CAFs and reduced numbers of inflammatory CAFs. We also found higher proportions of M1 macrophages, T cells, and antigen-presenting dendritic cells in tumors with reduced gal 4 expression. Using a coculture system, we observed that extracellular gal 4 induced apoptosis in T cells by binding N-glycosylation residues on CD3ε/δ. Hence, we show that gal 4 is involved in immune evasion and identify gal 4 as a promising drug target for overcoming immunosuppression in PDAC.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;Lists of genes used to calculate signature scores for cytolytic activity, T cell activation and M1 vs M2 macrophage polarization in single cell data analysis&lt;/p&gt;

&lt;p&gt;Top 30 markers of clusters in CAF subset of single cell data&lt;/p&gt;

&lt;p&gt;Supplementary Figures 1-5&lt;/p&gt;

&lt;p&gt;Supplementary Tables&lt;/p&gt;

&lt;p&gt;Metadata and single cell annotation for all 7677 cells in single cell dataset&lt;/p&gt;

&lt;p&gt;Top 30 markers of clusters in Immune subset of single cell data&lt;/p&gt;

&lt;p&gt;Raw read count, normalized expression values (TPM) and sample metadata for bulk RNA sequencing data&lt;/p&gt;

&lt;p&gt;Legends for sup figures 1-5&lt;/p&gt;

&lt;div&gt;Abstract&lt;p&gt;Pancreatic ductal adenocarcinoma (PDAC) is characterized by rich deposits of extracellular matrix (ECM), affecting the pathophysiology of the disease. Here, we identified galectin 4 (gal 4) as a cancer cell–produced protein that was deposited into the ECM of PDAC tumors and detected high-circulating levels of gal 4 in patients with PDAC. In orthotopic transplantation experiments, we observed increased infiltration of T cells and prolonged survival in immunocompetent mice transplanted with cancer cells with reduced expression of gal 4. Increased survival was not observed in immunodeficient RAG1&lt;sup&gt;−/−&lt;/sup&gt; mice, demonstrating that the effect was mediated by the adaptive immune system. By performing single-cell RNA-sequencing, we found that the myeloid compartment and cancer-associated fibroblast (CAF) subtypes were altered in the transplanted tumors. Reduced gal 4 expression associated with a higher proportion of myofibroblastic CAFs and reduced numbers of inflammatory CAFs. We also found higher proportions of M1 macrophages, T cells, and antigen-presenting dendritic cells in tumors with reduced gal 4 expression. Using a coculture system, we observed that extracellular gal 4 induced apoptosis in T cells by binding N-glycosylation residues on CD3ε/δ. Hence, we show that gal 4 is involved in immune evasion and identify gal 4 as a promising drug target for overcoming immunosuppression in PDAC.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;Supplementary Tables&lt;/p&gt;

&lt;p&gt;Top 30 markers of clusters in CAF subset of single cell data&lt;/p&gt;

&lt;p&gt;Supplementary Figures 1-5&lt;/p&gt;

&lt;p&gt;Metadata and single cell annotation for all 7677 cells in single cell dataset&lt;/p&gt;

&lt;p&gt;Legends for sup figures 1-5&lt;/p&gt;

&lt;p&gt;Top 30 markers of clusters in Immune subset of single cell data&lt;/p&gt;

&lt;p&gt;Lists of genes used to calculate signature scores for cytolytic activity, T cell activation and M1 vs M2 macrophage polarization in single cell data analysis&lt;/p&gt;

&lt;p&gt;Raw read count, normalized expression values (TPM) and sample metadata for bulk RNA sequencing data&lt;/p&gt;

Abstract Human norovirus (HuNoV) is a leading cause of acute viral gastroenteritis, but despite high impact on public health and health care, the mechanisms of viral attachment to and entry into target cells are not yet fully understood. It is well known that body fluids such as blood can transmit unrelated viruses, but recent reports also indicate that saliva and bile contribute to transmission of HuNoV. For example, human bile acids increase cell surface ceramide levels in human enteroids, which improves norovirus entry into cells resulting in enhanced replication. Bile acids can also interact directly with the norovirus capsid, but it is not known whether bile or other gastrointestinal body fluids directly affect HuNoV attachment to host cells. In this study, we investigated the effects of patient-derived gastric juice, pancreatic juice, and bile on HuNoV GII.4 virus-like particle (VLP) attachment to and entry into a human duodenal cell line, HuTu-80. We show that while gastric juice and pancreatic juice do not affect viral attachment or entry, bile – in particular hydrophobic bile acids – significantly enhance cellular attachment and subsequent entry of GII.4 VLPs into cells. In addition, we show that hydrophobic bile acids induce accumulation of viral particles in the vicinity of cells. Our results suggest the presence of a new en masse infection mechanism, where bile acids aggregate virions, and allow direct and more efficient attachment to and entry into target cells. Importance Viruses transmitted by the fecal-oral route encounter secreted host factors in gastrointestinal fluids. Some host factors can be exploited by the virus to facilitate infection. Human bile acids indirectly promote norovirus entry into and infection of human enteroids, but the direct effect of bile acids on attachment and uptake, along with the impact of other gastrointestinal fluids, remain unknown. Here, we investigated the direct effects of human body fluids on cellular attachment of norovirus VLPs. We show that human bile and hydrophobic bile acids induce an accumulation of norovirus VLPs, which is associated with significantly enhanced attachment and entry into human duodenal cell lines. These results highlight the differential effects of gastrointestinal body fluids on viral attachment and entry, while providing useful information into the complex HuNoV-host interactions that facilitate infection.

Background: High nerve density in tumors and metastasis via nerves (perineural invasion—PNI) have been reported extensively in solid tumors throughout the body including pancreatic, head and neck, gastric, prostate, breast, and colorectal cancers. Ablation of tumor nerves results in improved disease outcomes, suggesting that blocking nerve–tumor communication could be a novel treatment strategy. However, the molecular mechanisms underlying this remain poorly understood. Thus, the aim here was to identify molecular pathways underlying nerve–tumor crosstalk and to determine common molecular features between PNI-associated cancers. Results: Analysis of head and neck (HNSCC), pancreatic, and gastric (STAD) cancer Gene Expression Omnibus datasets was used to identify differentially expressed genes (DEGs). This revealed extracellular matrix components as highly dysregulated. To enrich for pathways associated with PNI, genes previously correlated with PNI in STAD and in 2 HNSCC studies where tumor samples were segregated by PNI status were analyzed. Neurodevelopmental genes were found to be enriched with PNI. In datasets where tumor samples were not segregated by PNI, neurodevelopmental pathways accounted for 12%–16% of the DEGs. Further dysregulation of axon guidance genes was common to all cancers analyzed. By examining paralog genes, a clear pattern emerged where at least one family member from several axon guidance pathways was affected in all cancers examined. Overall 17 different axon guidance gene families were disrupted, including the ephrin–Eph, semaphorin–neuropilin/plexin, and slit–robo pathways. These findings were validated using The Cancer Genome Atlas and cross-referenced to other cancers with a high incidence of PNI including colon, cholangiocarcinoma, prostate, and breast cancers. Survival analysis revealed that the expression levels of neurodevelopmental gene families impacted disease survival. Conclusion: These data highlight the importance of the tumor as a source of signals for neural tropism and neural plasticity as a common feature of cancer. The analysis supports the hypothesis that dysregulation of neurodevelopmental programs is a common feature associated with PNI. Furthermore, the data suggested that different cancers may have evolved to employ alternative genetic strategies to disrupt the same pathways . Overall, these findings provide potential druggable targets for novel therapies of cancer management and provide multi-cancer molecular biomarkers.

&lt;div&gt;Abstract&lt;p&gt;The prognosis for pancreatic ductal adenocarcinoma (PDAC) remains poor despite decades of effort. The abundant extracellular matrix (ECM) in PDAC comprises a major fraction of the tumor mass and plays various roles in promoting resistance to therapies. However, nonselective depletion of ECM has led to poor patient outcomes. Consistent with that observation, we previously showed that individual matrisome proteins derived from stromal cells correlate with either long or short patient survival. In marked contrast, those derived from cancer cells correlate strongly with poor survival. Here, we studied three cancer cell–derived matrisome proteins that are significantly overrepresented during PDAC progression, AGRN (agrin), SERPINB5 (serine protease inhibitor B5), and CSTB (cystatin B). Using both overexpression and knockdown experiments, we demonstrate that all three are promoters of PDAC metastasis. Furthermore, these proteins operate at different metastatic steps. AGRN promoted epithelial-to-mesenchymal transition in primary tumors, whereas SERPINB5 and CSTB enhanced late steps in the metastatic cascade by elevating invadopodia formation and &lt;i&gt;in vivo&lt;/i&gt; extravasation. All three genes were associated with a poor prognosis in human patients and high levels of SERPINB5, secreted by cancer cells and deposited in the ECM, correlated with poor patient prognosis. This study provides strong evidence that cancer cell–derived matrisome proteins can be causal in promoting tumorigenesis and metastasis and lead to poor patient survival. Therefore, compared with the bulk matrix, mostly made by stromal cells, precise interventions targeting cancer cell–derived matrisome proteins, such as AGRN, SERPINB5, and CSTB, may represent preferred potential therapeutic targets.&lt;/p&gt;Significance:&lt;p&gt;This study provides insights into the biological roles of cancer cell–derived matrisome proteins in PDAC and supports the notion that these proteins are protumorigenic and better therapeutic targets.&lt;/p&gt;&lt;/div&gt;

Over the years there have been a number of instances describing a minimum in the flotation recovery of hematite in the alkaline pH region when oleate is used as the collector. These cases usually coincide with the use of a commercial oleate sample containing other fatty acids and/or salts as the collector. It is the purpose of this paper to provide experimental data involving the effects of pH on the flotation of a natural hematite sample using two collectors containing oleate and compare these data to changes in surface tension and bubble characteristics of aqueous oleate systems also as a function of pH. The main aim of this paper is to highlight and address this difference in hematite flotation behaviour with oleate solution characteristics and develop a better understanding of these interactions. It is proposed that this flotation minimum is associated with a reduction in both contact angle and bubble-particle flotation rate constant under these conditions, even though the surfactant solution chemistries would suggest that flotation should still be strong in this region.

Abstract Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable model systems to interrogate pathways involved in pancreatic tumorigenesis and to probe individual responses to novel therapies are urgently needed. To that end, we established methods to culture normal and neoplastic pancreatic duct cells as three-dimensional organoid cultures. Pancreatic organoids can be rapidly generated from resected tumors or fine needle biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Following orthotopic transplant, neoplastic organoids recapitulated the full spectrum of tumor development by forming early-grade neoplasms that progressed to locally invasive and metastatic carcinomas, demonstrating the utility of organoids to model the stages of PDA tumorigenesis. Monolayer cell lines were generated from organoid cultures with high efficiency, creating a diverse collection of new PDA cell lines. To better understand pathways involved in PDA progression, we performed transcriptomic and proteomic analyses of murine organoids derived from normal pancreatic ducts, pancreatic intraepithelial neoplasias (PanINs), and PDAs. These datasets revealed expression changes associated with early and late pancreatic tumorigenesis. To identify genes dysregulated during pancreatic tumorigenesis whose depletion impaired human PDA cells, a CRISPR-Cas competition assay was employed. Taken together, pancreatic organoids offer a novel model system for studying pancreatic cancer biology and can be used to screen for genetic dependencies in PDA. Citation Format: Lindsey A. Baker, Hervé Tiriac, Vincenzo Corbo, Sylvia F. Boj, Chang-il Hwang, Iok In Christine Chio, Danielle D. Engle, Myrthe Jager, Mariano Ponz-Sarvise, Mona S. Spector, Ana Gracanin, Tobiloba Oni, Kenneth H. Yu, Ruben van Boxtel, Meritxell Huch, Keith D. Rivera, John P. Wilson, Michael E. Feigin, Daniel Öhlund, Abram Handly-Santana, Christine M. Ardito-Abraham, Michael Ludwig, Ela Elyada, Brinda Alagesan, Giulia Biffi, Georgi N. Yordanov, Bethany Delcuze, Brianna Creighton, Kevin Wright, Youngkyu Park, Folkert H.M. Morsink, I. Quintus Molenaar, Inne H. Borel Rinkes, Edwin Cuppen, Yuan Hao, Ying Jin, Isaac J. Nijman, Christine Iacobuzio-Donahue, Steven D. Leach, Darryl J. Pappin, Molly Hammell, David S. Klimstra, Olca Basturk, Ralph H. Hruban, George Johan Offerhaus, Robert G.J. Vries, Hans Clevers, David A. Tuveson. Using human patient-derived organoids to identify genetic dependencies in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B16.

Background: Breast cancer is the most common cancer and cause of cancer death among women. Localised breast cancer can be cured by surgery but the mortality remains high as some tumours metastasize early. Perlecan is a basal membrane protein, which is involved in promoting angiogenesis and invasiveness of cancers. Here, the expression pattern of Perlecan during tumour progression and related to the molecular subtypes of breast cancer (Luminal A, Luminal B, Luminal H, ErbB2 and Basal cell-like cancer) was studied. In addition, the pre- and postoperative circulating levels of Perlecan in plasma was measured in patients with breast cancer, and compared with controls.

Examensarbetet har utforts for att besvara ett antal fragestallningar fran Kraftringen angaende produktionsoptimeringen for deras kraftvarmeverk, Ortoftaverket. Tva av fragorna som kraftringen ville fa svar pa var (1) vilken admissionsdata (tryck och temperatur pa angan ut fran pannan) ar optimal for att fa ut maximal eleffekt for ett givet angflode? (2) Vilken eleffektokning fas vid stangning av avtappningarna till hogtrycksforvarmarna? For att kunna besvara dessa fragor byggdes en termodynamisk berakningsmodell i Microsoft Excel. Modellen bygger pa energibalanser for varmevaxlare, angturbin och verkningsgradsberakningar for angturbin. Resultatet av testerna med modellen visar pa att andrade admissionsdata ger en hogre effekt for vissa angfloden. For angflodet 30 kg/s fas en eleffektokning pa 120 kW om admissionsdatan andras fran 540 °C och 110 bar till 540 °C och 90 bar. Vid stangning av avtappningarna till hogtrycksforvarmarna okade eleffekten for samtliga angfloden. Okningen blev 3,5 - 4 procent. Varfor eleffekten okar nar admissionstrycket sanks beror pa minskade strypforluster i reglerventilerna vid turbininloppet. Anledningen till att eleffekten okar nar avtappningarna till hogtrycksforvarmarna stangs ar att mer massflode strommar genom turbinen.

Introduction: Pancreatic cancer is a disease with poor survival due to late presenting symptoms, aggressive growth behaviour and poor response to chemotherapy. Hyaluronan is expressed in the voluminous pancreatic cancer stroma, and has been proposed to promote malignant properties. In this study stromal hyaluronan in pancreatic cancer is studied in vivo and pre- and postoperative serum levels of hyaluronan is measured.