Valérie Pierrefite‐Carle

Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies.

The mucosal immune system of mammals consists of an integrated network of lymphoid cells which work in concert with innate host factors to promote host defense. Major mucosal effector immune mechanisms include secretory antibodies, largely of immunoglobulin A (IgA) isotype, cytotoxic T cells, as well as cytokines, chemokines and their receptors. Immunologic unresponsiveness (tolerance) is a key feature of the mucosal immune system, and deliberate vaccination or natural immunization by a mucosal route can effectively induce immune suppression. The diverse compartments located in the aerodigestive and genitourinary tracts and exocrine glands communicate via preferential homing of lymphocytes and antigen-presenting cells. Mucosal administration of antigens may result in the concomitant expression of secretory immunoglobulin A (S-IgA) antibody responses in various mucosal tissues and secretions, and under certain conditions, in the suppression of immune responses. Thus, developing formulations based on efficient delivery of selected antigens/tolerogens, cytokines and adjuvants may impact on the design of future vaccines and of specific immunotherapeutic approaches against diseases associated with untoward immune responses, such as autoimmune disorders, allergic reactions, and tissue-damaging inflammatory reactions triggered by persistent microorganisms.

Suicide gene therapy is based on the introduction into tumor cells of a viral or a bacterial gene, which allows the conversion of a non-toxic compound into a lethal drug. Although suicide gene therapy has been successfully used in a large number of in vitro and in vivo studies, its application to cancer patients has not reached the desirable clinical significance. However, recent reports on pre-clinical cancer models demonstrate the huge potential of this strategy when used in combination with new therapeutic approaches. In this review, we summarize the different suicide gene systems and gene delivery vectors addressed to cancer, with particular emphasis on recently developed systems and associated bystander effects. In addition, we review the different strategies that have been used in combination with suicide gene therapy and provide some insights into the future directions of this approach, particularly towards cancer stem cell eradication.

Autophagy, a major catabolic pathway responsible of the elimination of damaged proteins and organelles, is now recognized as an anti-aging process. In addition to its basal role in cell homeostasis, autophagy is also a stress-responsive mechanism for survival purposes. Here, we review recent literature to highlight the autophagy role in the different bone cell types, i.e., osteoblasts, osteoclasts and osteocytes. We also discuss the effects of autophagy modulators in bone physiology and of bone anabolic compounds in autophagy. Finally, we analyzed studies regarding bone cell autophagy-deficient mouse models to obtain a more general view on how autophagy modulates bone physiology and pathophysiology, particularly during aging.

Osteosarcoma (OS) is the most common primary bone tumour in children and adolescents. It is a highly aggressive tumor with a tendency to spread to the lungs, which are the most common site of metastasis. Advanced osteosarcoma patients with metastasis share a poor prognosis. Despite the use of chemotherapy to treat OS, the 5-year overall survival rate for patients has remained unchanged at 65-70% for the past 20 years. In addition, the 5-year survival of patients with a metastatic disease is around 20%, highlighting the need for novel therapeutic targets. Autophagy is an intracellular degradation process which eliminates and recycles damaged proteins and organelles to improve cell lifespan. In the context of cancer, numerous studies have demonstrated that autophagy is used by tumor cells to repress initial steps of carcinogenesis and/or support the survival and growth of established tumors. In osteosarcoma, autophagy appears to be deregulated and could also act both as a pro or anti-tumoral process. In this manuscript, we aim to review these major findings regarding the role of autophagy in osteosarcoma.

Autophagy is the major catabolic process in eukaryotic cells for the degradation and recycling of damaged macromolecules and organelles. It plays a crucial role in cell quality control and nutrient supply under stress conditions. Although autophagy is classically described as a degradative mechanism, it can also be involved in some secretion pathways, leading to the extracellular release of proteins, aggregates, or organelles. The role of autophagy in cancer is complex and depends on tumor development stage. While autophagy limits cancer development in the early stages of tumorigenesis, it can also have a protumoral role in more advanced cancers, promoting primary tumor growth and metastatic spread. In addition to its pro-survival role in established tumors, autophagy recently emerged as an active player in the crosstalk between tumor and stromal cells. The aim of this review is to analyze the impact of tumoral autophagy on the microenvironment and conversely the effect of stromal cell autophagy on tumor cells.

Autophagy is a ubiquitous cellular process, allowing the removal and recycling of damaged proteins and organelles. At the basal level, this process plays a role in quality control, thus participating in cellular homeostasis. Autophagy can also be induced by various stresses, such as nutrient deprivation or hypoxia, to allow the cell to survive until conditions improve. In recent years, the role of this process has been widely studied in many pathologies such as neurodegenerative diseases or cancers. In bone tissue, various studies have shown that autophagy is involved in the survival, differentiation and activity of osteoblasts, osteocytes and osteoclasts. The evolution of this knowledge has led to the identification of new molecular pathophysiological mechanisms in bone pathologies. This review reports the current state of knowledge on the role of autophagy in 4 bone diseases: osteoporosis, which seems to be associated with a decrease in autophagy, osteopetrosis and Paget's disease where the course of the autophagic process is disturbed, and finally osteosarcoma where autophagy seems to play a protumoral role. A better understanding of the involvement of autophagy in these pathologies should eventually lead to the identification of new potential therapeutic targets.

Age-related bone loss is associated with an increased oxidative stress which is worsened by estrogen fall during menauposis. This observation has drawn attention to autophagy, a major cellular catabolic process, able to alleviate oxidative stress in osteoblasts (OB) and osteocytes (OST), two key bone cell types. Moreover, an autophagy decline can be associated with aging, suggesting that an age-related autophagy deficiency in OB and/or OST could contribute to skeletal aging and osteoporosis onset.In the present work, autophagy activity was analyzed in OST and OB in male and female mice according to their age and hormonal status. In OST, autophagy decreases with aging in both sexes. In OB, although a 95% decrease in autophagy is observed in OB derived from old females, this activity remains unchanged in males. In addition, while ovariectomy has no effect on OB autophagy levels, orchidectomy appears to stimulate this process. An inverse correlation between autophagy and the oxidative stress level was observed in OB derived from males or females. Finally, using OB-specific autophagy-deficient mice, we showed that autophagy deficiency aggravates the bone loss associated with aging and estrogen deprivation.Taken together, our data indicate that autophagic modulation in bone cells differs according to sex and cell type. The lowering of autophagy in female OB, which is associated with an increased oxidative stress, could play a role in osteoporosis pathophysiology and suggests that autophagy could be a new therapeutic target for osteoporosis in women.

Abstract Cancer stem cells (CSCs) represent a minor population of self-renewing cancer cells that fuel tumor growth. As CSCs are generally spared by conventional treatments, this population is likely to be responsible for relapses that are observed in most cancers. In this work, we analyzed the preventive efficiency of a CSC-based vaccine on the development of liver metastasis from colon cancer in a syngeneic rat model. We isolated a CSC-enriched population from the rat PROb colon carcinoma cell line on the basis of the expression of the aldehyde dehydrogenase-1 (ALDH1) marker. Comparative analysis of vaccines containing lysates of PROb or ALDHhigh cells by mass spectrometry identifies four proteins specifically expressed in the CSC subpopulation. The expression of two of them (heat shock protein 27-kDa and aldose reductase) is already known to be associated with treatment resistance and poor prognosis in colon cancer. Preventive intraperitoneal administration of vaccines was then performed before the intrahepatic injection of PROb cancer cells. While no significant difference in tumor occurrence was observed between control and PROb-vaccinated groups, 50% of the CSC-based vaccinated animals became resistant to tumor development. In addition, CSC-based vaccination induced a 99.5% reduction in tumor volume compared to the control group. To our knowledge, this study constitutes the first work analyzing the potential of a CSC-based vaccination to prevent liver metastasis development. Our data demonstrate that a CSC-based vaccine reduces efficiently both tumor volume and occurrence in a rat colon carcinoma syngeneic model.

Suicide gene therapy has been used for the treatment of a variety of cancers. We reported previously the in vitro efficacy of the Herpes Simplex Virus Thymidine kinase (HSV-tk)/ganciclovir (GCV) system to mediate cytotoxicity in oral squamous cancer cells, using transferrin (Tf)-lipoplexes, prepared from cationic liposomes composed of 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and cholesterol. In the present study, we evaluated the antitumoral efficacy mediated by this lipoplex formulation in two suicide gene therapy strategies, HSV-tk/GCV and cytosine deaminase (CD)/5-fluorocytosine (5-FC), using a syngeneic, orthotopic murine model for head and neck squamous cell carcinoma. The cellular and molecular events associated with the antitumoral response elicited by both the therapeutic approaches were investigated by analyzing tumor cell death, tumor-infiltrating immune cells and tumor cytokine microenvironment. Significant tumor reduction was achieved upon intratumoral delivery of HSV-tk or CD genes mediated by Tf-lipoplexes, followed by intraperitoneal injection of GCV or 5-FC, respectively. Enhanced apoptosis, the recruitment of NK cells, CD4 and CD8 T-lymphocytes and an increase in the levels of several cytokines/chemokines were observed within the tumors. These observations suggest that suicide gene therapy with lipoplexes modifies the tumor microenvironment, and leads to the recruitment of immune effector cells that can act as adjuvants in reducing the tumor size.

Cancer stem cells (CSCs) represent a minor population of cancer cells with stem cell-like properties which are able to fuel tumor growth and resist conventional treatments. Autophagy has been described to be upregulated in some CSCs and to play a crucial role by maintaining stem features and promoting resistance to both hostile microenvironments and treatments. Osteosarcoma (OS) is an aggressive bone cancer which mainly affects children and adolescents and autophagy in OS CSCs has been poorly studied. However, this is a very interesting case because autophagy is often deregulated in this cancer. In the present work, we used two OS cell lines showing different autophagy capacities to isolate CSC-enriched populations and to analyze the autophagy in basal and nutrient-deprived conditions. Our results indicate that autophagy is more efficient in CSCs populations compared to the parental cell lines, suggesting that autophagy is a critical process in OS CSCs. We also showed that the antipsychotic drug thioridazine is able to stimulate, and then impair autophagy in both CSC-enriched populations, leading to autosis, a cell death mediated by the Na+/K+ ATPase pump and triggered by dysregulated accumulation of autophagosomes. Taken together, our results indicate that autophagy is very active in OS CSCs and that targeting this pathway to switch their fate from survival to death could provide a novel strategy to eradicate these cells in osteosarcoma.

The cytosine deaminase gene of Escherichia coli converts the nontoxic compound 5-fluorocytosine into 5-fluorouracil (5-FU), thereby acting as a suicide gene when introduced into cancer cells, killing the cells when they are exposed to 5-fluorocytosine. We analyzed the efficacy of using cytosine deaminase-bearing cancer cells as an autologous tumor vaccine in a rat model that mimics liver metastasis from colon carcinoma.We introduced a plasmid vector containing the E. coli cytosine deaminase gene into a BDIX rat colon carcinoma cell line. Intrahepatic injection of the modified cells in syngeneic animals generates a single experimental liver "suicide tumor." We then analyzed the effect of 5-fluorocytosine treatment in terms of regression of cytosine deaminase-expressing cells in vivo as well as protection against wild-type cancer cells.Treatment with 5-fluorocytosine induced regression of cytosine deaminase-expressing (CD+) tumors, with seven of 11 treated animals being tumor free at the end of 30 days and a statistically significant difference in tumor volumes between treated and control animals (two-sided P<.0001). Intrahepatic injection of CD+ cells followed by 5-fluorocytosine treatment rendered the treated animals resistant to challenge with wild-type tumor cells, with no (zero of seven) treated animals developing wild-type tumors in contrast to all (four of four) control animals. Moreover, in animals with established wild-type liver tumors, injection of CD+ tumor cells followed by 5-fluorocytosine treatment produced a statistically significant increase in survival time (two-sided P<.0001). In vivo immunodepletion and immunohistologic analysis of experimental tumors indicate that natural killer cells are the major immune component involved in this antitumor effect.Taken together, these results suggest the potential use of suicide gene-modified tumor cells as therapeutic vaccines against liver metastasis from colon carcinoma.

Unmethylated CpG dinucleotides, present in bacterial DNA, are recognized in vertebrates via the Toll-like receptor 9 (TLR9) and are known to act as an anticancer agent by stimulating immune cells to induce a proinflammatory response. Although the effects of CpG-oligodeoxynucleotides (CpG-ODNs) in immune cells have been widely studied, little is known regarding their molecular effects in TLR9-positive tumor cells. To better understand the role of these bacterial motifs in cancer cells, we analyzed proteome modifications induced in TLR9-positive tumor cells in vitro and in vivo after CpG-ODN treatment in a rat colon carcinoma model. Proteomics analysis of tumor cells by two-dimensional gel electrophoresis followed by mass spectrometry identified several proteins modulated by bacterial CpG motifs. Among them, several are related to autophagy including potential autophagic substrates. In addition, we observed an increased glyceraldehyde-3-phosphate dehydrogenase expression, which has been shown to be sufficient to trigger an autophagic process. Autophagy is a self-digestion pathway whereby cytoplasmic material is sequestered by a structure termed the autophagosome for subsequent degradation and recycling. As bacteria are known to trigger autophagy, we assessed whether bacterial CpG motifs might induce autophagy in TLR9-positive tumor cells. We showed that CpG-ODN can induce autophagy in rodent and human tumor cell lines and was TLR9-dependent. In addition, an increase in the number of autophagosomes can also be observed in vivo after CpG motif intratumoral injection. Our findings bring new insights on the effect of bacterial CpG motifs in tumor cells and may be relevant for cancer treatment and more generally for gene therapy approaches in TLR9-positive tissues.

The bacterial cytosine deaminase (CD) gene, associated with the 5-fluorocytosine (5FC) prodrug, is one of the most widely used suicide systems in gene therapy. Introduction of the CD gene within a tumor induces, after 5FC treatment of the animal, a local production of 5-fluorouracil resulting in intratumor chemotherapy. Destruction of the gene-modified tumor is then followed by the triggering of an antitumor immune reaction resulting in the regression of distant wild-type metastasis. The global effects of 5FC on colorectal adenocarcinoma cells expressing the CD gene were analyzed using the proteomic method. Application of 5FC induced apoptosis and 19 proteins showed a significant change in 5FC-treated cells compared with control cells. The up-regulated and down-regulated proteins include cytoskeletal proteins, chaperones, and proteins involved in protein synthesis, the antioxidative network, and detoxification. Most of these proteins are involved in resistance to anticancer drugs and resistance to apoptosis. In addition, we show that the heat shock protein Hsp90beta is phosphorylated on serine 254 upon 5FC treatment. Our results suggest that activation of Hsp90beta by phosphorylation might contribute to tumor regression and tumor immunogenicity. Our findings bring new insights into the mechanism of the anticancer effects induced by CD/5FC treatment.

➤ Aseptic loosening, the most common cause of arthroplasty component failure, is due to implant wear and subsequent release of biomaterial wear particles to the bone microenvironment, leading to a chronic inflammatory response. ➤ Autophagy, a cell-cleaning process allowing the degradation of damaged material, can be upregulated in response to various stresses in which it acts primarily as a survival mechanism. In addition to the classic role of autophagy in the degradation pathway, autophagy can be involved in some secretion processes. ➤ Autophagy seems to be triggered by the presence of wear debris in the 3 main cell types involved in aseptic loosening, i.e., osteocytes, osteoblasts, and macrophages. ➤ Autophagy can mediate the secretion of proinflammatory cytokines such as interleukin (IL)-6 and IL-8 or the danger signal protein HMGB1 (high mobility group box 1). All of these proteins have been implicated in the pathogenesis of aseptic loosening. ➤ Recent studies using animal models have demonstrated that autophagy inhibition can decrease the severity of osteolysis, suggesting that transient and local autophagy modulation could be a potential therapeutic option to prevent wear debris-induced osteolysis.

In addition to its clean-up function, autophagy is considered as an innate immunity mechanism due to its role in the removal of intracellular pathogens. Toll-like receptors (TLRs) are crucial components of innate immunity involved in the recognition of a diverse array of microbial products. Recent works demonstrated that different pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) and single-strand RNA are able to induce autophagy via different TLRs in immune cells. In a recent report, we showed that bacterial CpG motifs, another PAMP, can induce autophagy in rodent and human tumor cell lines and that this process is TLR9-dependent. In addition, an increase in the number of autophagosomes can also be observed in vivo after the intratumoral injection of CpG motifs. These results extend the link between TLRs and autophagy to non-immune tumor cells and may be relevant for cancer treatment and more generally for gene therapy approaches in TLR9-positive tissues. In this addendum, we discuss the potential mechanisms and the consequences of the CpG-induced autophagy in tumor cells.

The incidence of oral tumors is increasing around the world and despite recent advances in early detection and diagnosis, current treatments are still unsatisfactory. Recent data suggest that tumor persistence and recurrence could be due to the presence of a rare cell population called cancer stem cells (CSCs), which are generally spared by traditional treatments. Therefore, identification and characterization of CSCs are extremely important to develop novel and effective treatment strategies for cancer. The aim of this study was to identify and isolate CSCs in an established murine head and neck squamous cell carcinoma (HNSCC) cell line and to investigate the influence of hypoxic conditions on the isolated cell popul­ation. Using the expression of the aldehyde dehydrogenase 1 (ALDH1) enzymatic activity, which is now recognized as a CSC marker in various tumors, we isolated a cell population expressing high levels of ALDH1 (ALDH1high) representing 1±0.6% in the murine SCC-VII cell line. These cells were injected subcutaneously in syngeneic animals to evaluate their tumorigenic properties. For the lowest injected cell dose (250 injected cells), tumor occurrence and median tumor size were higher in ALDH1high injected mice than in ALDH1low injected mice. Following an in vivo passage and culture in serum-free medium, the percentage of ALDH1high cells increased by 3‑fold in SCC-VII CSCs (oral spheres) compared to the SCC-VII cell line. This percentage was further increased when oral spheres were cultured under hypoxic conditions. In conclusion, this study reports for the first time the isolation of HNSCC CSCs in a syngeneic mouse model and the use of hypoxia as a method to further enrich the ALDH1high cell population.

Osteoporosis (OP) is a pathology characterized by bone fragility affecting 30% of postmenopausal women, mainly due to estrogen deprivation and increased oxidative stress. An autophagy involvement is suspected in OP pathogenesis but a definitive proof in humans remains to be obtained.Postmenopausal women hospitalized for femoral neck fracture (OP group) or total hip replacement (Control group) were enrolled using very strict exclusion criteria. Western blot was used to analyze autophagy level.The protein expression level of the autophagosome marker LC3-II was significantly decreased in bone of OP patients relative to the control group. In addition, the protein expression of the hormonally upregulated neu-associated kinase (HUNK), which is upregulated by female hormones and promotes autophagy, was also significantly reduced in bone of the OP group.These results demonstrate for the first time that postmenopausal OP patients have a deficit in bone autophagy level and suggest that HUNK could be the factor linking estrogen loss and autophagy decline.ClinicalTrials.gov Identifier: NCT03175874, 2/6/2017.

Uranium is a naturally occurring radionuclide ubiquitously present in the environment. The skeleton is the main site of uranium long-term accumulation. While it has been shown that natural uranium is able to perturb bone metabolism through its chemical toxicity, its impact on bone resorption by osteoclasts has been poorly explored. Here, we examined for the first time in vitro effects of natural uranium on osteoclasts. The effects of uranium on the RAW 264.7 monocyte/macrophage mouse cell line and primary murine osteoclastic cells were characterized by biochemical, molecular and functional analyses. We observed a cytotoxicity effect of uranium on osteoclast precursors. Uranium concentrations in the μM range are able to inhibit osteoclast formation, mature osteoclast survival and mineral resorption but don't affect the expression of the osteoclast gene markers Nfatc1, Dc-stamp, Ctsk, Acp5, Atp6v0a3 or Atp6v0d2 in RAW 274.7 cells. Instead, we observed that uranium induces a dose-dependent accumulation of SQSTM1/p62 during osteoclastogenesis. We show here that uranium impairs osteoclast formation and function in vitro. The decrease in available precursor cells, as well as the reduced viability of mature osteoclasts appears to account for these effects of uranium. The SQSTM1/p62 level increase observed in response to uranium exposure is of particular interest since this protein is a known regulator of osteoclast formation. A tempting hypothesis discussed herein is that SQSTM1/p62 dysregulation contributes to uranium effects on osteoclastogenesis. We describe cellular and molecular effects of uranium that potentially affect bone homeostasis.

The cytosine deaminase (CD) gene converts the nontoxic prodrug, 5-fluorocytosine (5-FC), into 5-fluorouracil (5-FU). We previously showed that injection of CD-bearing cancer cells followed by 5-FC treatment can act as an autologous tumor vaccine in a syngenic liver metastasis model in rats. In the present work, we analyzed the antitumor efficiency of a direct intratumoral injection of a CD-expressing plasmid. In rats bearing microscopic or macroscopic metastases in right and left liver lobes, an injection of a CD-expressing plasmid was performed in the left lobe tumor, followed by 5-FC treatment of the animals. A significant regression of the DNA-injected tumor was observed in 5-FC–treated rats, both in microscopic (P = .007) or advanced (P < .0001) tumor models. Moreover, this treatment also induced a potent distant bystander effect on untreated controlateral liver tumors and extrahepatic metastases, resulting in an increased survival compared with control animals in both tumor models (P < .05). In conclusion, these data suggest that direct intratumoral injection of a CD-expressing plasmid, associated to 5-FC administration, can constitute a powerful and innocuous alternative treatment for unresectable liver metastases from colon carcinoma.

Vascular endothelial growth factor is a potent pro-angiogenic growth factor which is also known to alter tumor microenvironment by inhibiting dendritic cell differentiation and promoting accumulation of myeloid-derived suppressor cells. In the present study, we analyzed the modifications induced by intratumoral expression of sFLT-1, a soluble VEGF receptor, in a rat metastatic colon carcinoma model. We generated colon cancer cell lines stably expressing sFLT-1 or a mock construct. Human umbilical vein endothelial cells cultured with conditioned medium from sFLT-1-expressing tumor cells exhibit a significantly decreased survival, demonstrating the functionality of the secreted sFLT-1. Invivo, sFLT-1 expression induced a 30% decrease in microvessel density in 15-day old experimental liver metastasis from colon carcinoma. Tumor growth was inhibited by 63% and 52% in left and right liver lobes respectively within 25 days. In these tumors, sFLT-1 expression was associated with a decreased myeloid cell infiltration and a modification in the expression of several cytokines/chemokines. Altogether, these results suggest that VEGF trapping by sFLT-1 intratumoral expression results in reduced vascularization, tumor growth inhibition and modification of immune tumor microenvironment.

Once absorbed in the body, natural uranium [U(VI)], a radionucleotide naturally present in the environment, is targeted to the skeleton which is the long-term storage organ. We and others have reported the U(VI) negative effects on osteoblasts (OB) and osteoclasts (OC), the main two cell types involved in bone remodeling. In the present work, we addressed the U(VI) effect on osteocytes (OST), the longest living bone cell type and the more numerous (> 90%). These cells, which are embedded in bone matrix and thus are the more prone to U(VI) long-term exposure, are now considered as the chief orchestrators of the bone remodeling process. Our results show that the cytotoxicity index of OST is close to 730 µM, which is about twice the one reported for OB and OC. However, despite this resistance potential, we observed that chronic U(VI) exposure as low as 5 µM led to a drastic decrease of the OST mineralization function. Gene expression analysis showed that this impairment could potentially be linked to an altered differentiation process of these cells. We also observed that U(VI) was able to trigger autophagy, a highly conserved survival mechanism. Extended X-ray absorption fine structure analysis at the U LIII edge of OST cells exposed to U(VI) unambiguously shows the formation of an uranyl phosphate phase in which the uranyl local structure is similar to the one present in Autunite. Thus, our results demonstrate for the first time that OST mineralization function can be affected by U(VI) exposure as low as 5 µM, suggesting that prolonged exposure could alter the central role of these cells in the bone environment.

Suicide gene therapy consists of the transfer into tumour cells of a "suicide" gene that can convert a non-toxic compound into a lethal drug. Expression of the cytosine deaminase gene leads to the conversion of the non-toxic compound 5-fluorocytosine to 5-fluorouracil. We have recently shown that "suicide cell based vaccination" consisting of intrahepatic injection of cytosine deaminase expressing colon cancer cells followed by 5-fluorocytosine treatment induces regression of a distant wild-type liver tumour in rats.This study was conducted to test if (i) a distant bystander effect on a liver tumour can be induced after subcutaneous suicide cell based vaccination and (ii) suicide cell based vaccination is efficient in limiting tumour dissemination to extrahepatic compartments.An aggressive variant of rat colon carcinoma cells was selected after successive passages in vitro. Rats carrying an experimental liver "metastasis" generated by injection of these cells were vaccinated by subcapsular or subcutaneous injection of cytosine deaminase expressing cells followed by 5-fluorocytosine treatment.Subcutaneous and subcapsular vaccination induced 70% regression in the median volume of the pre-established liver tumour (p=0.001) and abolished tumour dissemination compared with control animals.This study has compared for the first time the efficiency of subcutaneous and intrahepatic suicide cell based vaccination in a metastatic colorectal carcinoma model in rats. The results indicate that both modes of vaccination are equally efficient in inducing a systemic antitumour response, suggesting that this strategy is a powerful approach against the development and dissemination of metastatic colon carcinoma.

In case of an incident in the nuclear industry or an act of war or terrorism, the dissemination of plutonium could contaminate the environment and, hence, humans. Human contamination mainly occurs via inhalation and/or wounding (and, less likely, ingestion). In such cases, plutonium, if soluble, reaches circulation, whereas the poorly soluble fraction (such as small colloids) is trapped in alveolar macrophages or remains at the site of wounding. Once in the blood, the plutonium is delivered to the liver and/or to the bone, particularly into its mineral part, mostly composed of hydroxyapatite. Countermeasures against plutonium exist and consist of intravenous injections or inhalation of diethylenetetraminepentaacetate salts. Their effectiveness is, however, mainly confined to the circulating soluble forms of plutonium. Furthermore, the short bioavailability of diethylenetetraminepentaacetate results in its rapid elimination. To overcome these limitations and to provide a complementary approach to this common therapy, we developed polymeric analogs to indirectly target the problematic retention sites. We present herein a first study regarding the decontamination abilities of polyethyleneimine methylcarboxylate (structural diethylenetetraminepentaacetate polymer analog) and polyethyleneimine methylphosphonate (phosphonate polymeric analog) directed against Th(IV), used here as a Pu(IV) surrogate, which was incorporated into hydroxyapatite used as a bone model. Our results suggest that polyethylenimine methylphosphonate could be a good candidate for powerful bone decontamination action.

Uranium has been shown to interfere with bone physiology and it is well established that this metal accumulates in bone. However, little is known about the effect of natural uranium on the behavior of bone cells. In particular, the impact of uranium on osteoclasts, the cells responsible for the resorption of the bone matrix, is not documented. To investigate this issue, we have established a new protocol using uranyl acetate as a source of natural uranium and the murine RAW 264.7 cell line as a model of osteoclast precursors. Herein, we detailed all the assays required to test uranium cytotoxicity on osteoclast precursors and to evaluate its impact on the osteoclastogenesis and on the resorbing function of mature osteoclasts. The conditions we have developed, in particular for the preparation of uranyl-containing culture media and for the seeding of RAW 264.7 cells allow to obtain reliable and highly reproductive results. Moreover, we have optimized the use of software tools to facilitate the analysis of various parameters such as the size of osteoclasts or the percentage of resorbed matrix.

<h3>Background</h3> Osteoporosis (OP) is a diffuse skeletal pathology characterized by major bone fragility that affects one out of 3 women after menopause. In addition to estrogen loss, an increase in oxidative stress is now recognized as a central mechanism in this pathology. Autophagy is an intracellular process allowing the removal and recycling of damaged proteins and organelles. Thanks to the elimination of damaged mitochondria, which are the main sources of reactive oxygen species, autophagy plays a major role in reducing oxidative stress. An age-related autophagy decline, associated with various experimental arguments in animal models, suggests autophagy implication in OP. However, a definitive proof in humans remains to be obtained. <h3>Objectives</h3> Our objective was to show for the first time that there is a defect in autophagy in the bone of osteoporotic patients. <h3>Methods</h3> We have analyzed autophagy by Western Blot in bone samples from 14 postmenopausal women with OP fracture compared to 30 controls undergoing total hip replacement for osteoarthritis. <h3>Results</h3> Our results indicate that the expression level of the autophagosome marker LC3-II is significantly decreased in OP patients compared to the age-matched controls. We also observed a tendency for the accumulation of the SQSTM1/p62 protein, which is an autophagy substrate, thus supporting the previous results. Finally, we also demonstrate a significant decrease in the hormonally up-regulated neu-associated kinase (HUNK) in bone samples from OP patients compared to the control group. This kinase, whose expression is upregulated by female hormones, promotes autophagy by inhibiting the autophagy inhibitor Rubicon through phosphorylation. <h3>Conclusion</h3> Taken together, these data demonstrate for the first time that OP patients have a deficit in osteocytes autophagy and suggest the involvement of the HUNK kinase in OP pathogenesis. This study should contribute to better understand the role of autophagy in OP pathophysiology and could lead to the design of new therapeutics targeting autophagy for the management of pathological aging. <h3>Acknowledgements</h3> We thank the French Society of Rheumatology for their funding. <h3>Disclosure of Interests</h3> None Declared.

Cytosine deaminase (CD) gene of E. coli converts the non-toxic compound 5-fluorocytosine (5-FC) into 5-fluorouracil. We have introduced a vector expressing the CD gene in a rat colon carcinoma cell line. Expression of the CD gene confers 5-FC sensitivity to these cells in vitro and in vivo. In a bifocal model consisting in a simultaneous engrafment of a CD+ tumor on one lobe of the liver and a wild-type parental tumor on the opposite lobe, treatment with 5-FC results in regression of both type of tumors, indicating the existence of a distant bystander effect.

Bacterial DNA contains unmethylated cytosine-phosphate-guanine (CpG) motifs which are recognized by mammalian immune cells as a danger signal indicating an infection. These immunostimulatory properties led to the use of oligodeoxynucleotides bearing CpG motifs (CpG-ODN) for cancer treatment in preclinical and clinical studies. Although naked DNA administration presently represents 18% of the gene therapy clinical trials worldwide, most of the work regarding the effects of unmethylated CpG sequences was performed using CpG-ODN. In the present study, we analyzed early induced tumor microenvironment modifications in a rat liver metastasis model after intratumoral injection of a plasmid used in suicide gene therapy. We first showed that plasmidic CpG motifs were active, i.e. able to induce IFN-gamma secretion by rat splenocytes. Then, we compared tumor-infiltrating immune cells 24 h after injection of native or SssI-treated plasmid, in which immunostimulatory CpG motifs have been inactivated by methylation. The presence of active plasmidic CpG sequences within the tumor was associated with a decrease in the number of tumor-infiltrating conventional dendritic cells and an upregulation of the CCR7 chemokine receptor responsible for lymph node homing. We also observed an increase in plasmacytoid dendritic cells and natural killer cell infiltration within the tumors as well as an increased mRNA expression of three cytokines/chemokines (IL-1beta, IL-10 and IL-18). These data suggest that, although suicide plasmid injection without prodrug treatment is not sufficient to observe a therapeutic effect, the presence of plasmidic CpG motifs within the tumor induces the recruitment and activation of the immune cells involved in antitumor response. These early cellular and molecular events should facilitate the induction of the immune response against tumor antigens released after in situ drug production.

Despite the great promise of gene therapy, its application in humans has been hampered by the inability to efficiently and safely deliver the gene of interest to the target tissue. The severe toxic side effects observed in some clinical trials using viral vectors have regained interest in non-viral methods. These non-viral approaches are associated with low toxicity and immunogenicity as well as an easy and inexpensive manufacturing. The two major problems encountered with these vectors are the low gene transfer efficiency and the transient transgene expression. In this regard, cancer appears as a disease of choice for the use of non-viral vectors as (i) long term transgene expression is not always required, (ii) the low gene transfer efficiency can be relayed by immune system activation and/or local bystander effects. This review focuses on non-viral gene therapy applications for treatment of cancer, and particularly on naked DNA or DNA in association with liposomes (lipoplexes).

Uranium is widely spread in the environment due to its natural and anthropogenic occurrences, hence the importance of understanding its impact on human health. The skeleton is the main site of long-term accumulation of this actinide. However, interactions of this metal with biological processes involving the mineralized extracellular matrix and bone cells are still poorly understood. To get a better insight into these interactions, we developed new biomimetic bone matrices containing low doses of natural uranium (up to 0.85 µg of uranium per cm2). These models were characterized by spectroscopic and microscopic approaches before being used as a support for the culture and differentiation of pre-osteoclastic cells. In doing so, we demonstrate that uranium can exert opposite effects on osteoclast resorption depending on its concentration in the bone microenvironment. Our results also provide evidence for the first time that resorption contributes to the remobilization of bone matrix-bound uranium. In agreement with this, we identified, by HRTEM, uranium phosphate internalized in vesicles of resorbing osteoclasts. Thanks to the biomimetic matrices we developed, this study highlights the complex mutual effects between osteoclasts and uranium. This demonstrates the relevance of these 3D models to further study the cellular mechanisms at play in response to uranium storage in bone tissue, and thus better understand the impact of environmental exposure to uranium on human bone health.

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies.

Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies.

Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies.

Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies.

Suicide gene therapy consists of transferring into tumor cells a viral or bacterial gene encoding for an enzyme which converts a non-toxic product into a lethal drug.To analyze the therapeutic potential of vaccination with tumor cells expressing the bacterial cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) treatment in a rat liver metastasis model.We used a rat colon carcinoma cell line which, after subcapsular or intraportal injection in syngenic animals, generates single or multiple experimental liver metastases, respectively. We have shown that introduction of a vector expressing the CD gene in this colon carcinoma cell line results in 5-FC sensitivity (PRObCD).Intrahepatic subcapsular injection of PRObCD tumor cells, followed by 5-FC treatment, induces total regression of a wild-type tumor pre-established in the contralateral liver lobe in 45% of animals with a 96% decrease in mean volume (p < 0.0001), demonstrating the existence of a distant bystander effect. This vaccination significantly increased the survival of rats with single (log-rank p < 0.0001) or multiple (log-rank p = 0.01) liver metastasisThese results suggest that suicide gene-modified tumor cells can act as potent therapeutic vaccines against liver metastasis from colon carcinoma.

Arthroplasty surgery has become one of the most efficient procedures to restore joint function. However, implant failure due to bone loss is a major complication of joint replacement, with severe consequences both for the individual patient and for health-care systems. Peri-prosthetic loosening without concurrent infection or trauma is called aseptic loosening. It is estimated that up to 20% of all prosthetic implants will induce aseptic osteolysis, generally leading to implant loosening and surgical revision (1).

Bone tissue is constantly renewed by the concerted action of three main cell types: osteoclasts, the bone-resorbing cells; osteoblasts, the cells in charge of bone matrix production and mineralization; and osteocytes, the orchestrators of this remodeling process. Our knowledge of the role of autophagy in bone tissue has increased considerably, and this process is now recognized as being involved in bone cell differentiation and functions such as mineralization and resorption. This chapter summarizes the main results obtained in this field and highlights the crucial role of autophagy in this complex tissue.

L’autophagie est un processus cellulaire ubiquitaire, permettant l’élimination et le recyclage des protéines et organelles endommagées. Au niveau basal, ce processus joue un rôle dans le contrôle qualité, participant ainsi à l’homéostasie cellulaire. L’autophagie peut également être induite par différents stress, tels que la privation de nutriments ou l’hypoxie, afin de permettre à la cellule de survivre le temps que les conditions s’améliorent. Ces dernières années, le rôle de ce processus a été largement étudié dans de nombreuses pathologies telles que les maladies neurodégénératives ou les cancers. Dans le tissu osseux, différents travaux ont montré que l’autophagie est impliquée dans la survie, la différenciation et l’activité des ostéoblastes, des ostéocytes et des ostéoclastes. L’évolution de ces connaissances a permis de mettre en évidence de nouveaux mécanismes moléculaires physiopathologiques dans les pathologies osseuses. Cette revue rapporte l’état actuel des connaissances sur le rôle de l’autophagie dans 4 pathologies osseuses : l’ostéoporose, qui semble être associée à une diminution de l’autophagie, l’ostéopétrose et la maladie de Paget où le déroulement du processus autophagique est perturbé, et enfin l’ostéosarcome où l’autophagie semble jouer un rôle protumoral. Une meilleure compréhension de l’implication de l’autophagie dans ces pathologies devrait à terme permettre d’identifier de nouvelles cibles thérapeutiques potentielles.

Uranium has been shown to interfere with bone physiology and it is well established that this metal accumulates in bone. However, little is known about the effect of natural uranium on the behavior of bone cells. In particular, the impact of uranium on osteoclasts, the cells responsible for the resorption of the bone matrix, is not documented. To investigate this issue, we have established a new protocol using uranyl acetate as a source of natural uranium and the murine RAW 264.7 cell line as a model of osteoclast precursors. Herein, we detailed all the assays required to test uranium cytotoxicity on osteoclast precursors and to evaluate its impact on the osteoclastogenesis and on the resorbing function of mature osteoclasts. The conditions we have developed, in particular for the preparation of uranyl-containing culture media and for the seeding of RAW 264.7 cells allow to obtain reliable and highly reproductive results. Moreover, we have optimized the use of software tools to facilitate the analysis of various parameters such as the size of osteoclasts or the percentage of resorbed matrix.

Osteosarcoma (OS) is a bone cancer exhibiting a 20% survival rate for metastatic patients, which motivates the development of new therapeutic options. Among the various new treatment approaches, modulation of autophagy is the subject of rising interest. In addition to its pro-survival role in established tumors, autophagy recently emerged as an active player in the crosstalk between tumor and stromal cells. In OS, although the knockdown of key autophagy genes in human cell lines demonstrates a protumoral role of autophagy, the analysis of patient tumors indicates that lack of LC3-positive punctae at resection following neoadjuvant chemotherapy is a poor prognostic marker, suggesting that loss of autophagy is not detrimental for the tumor. In the present work, we analyzed the consequences of autophagy inactivation in OS cells both on tumor development and on bone microenvironment in an orthotopic syngeneic model. We found that inactivation of the autophagy-essential gene Atg5 in OS cells decreases their tumorigenic properties in vitro. However, these effects were no longer observed in vivo, likely due to microenvironment modifications such as overexpression of the major OS-promoting factor TGF-β or increased infiltration of Foxp3-positive and CD31-positive cells in Atg5 KO tumors. In addition, autophagy-deficient tumor cells stimulate the in vitro formation of osteoclast, the cells in charge of bone resorption which can release bone matrix-embedded growth factors thereby stimulating tumor growth. Taken together, these results suggest that Atg5 inactivation in OS cells is associated with microenvironment modifications known as poor prognosis-associated factors in OS, and could thus balance the negative cell-autonomous effects of autophagy suppression.Abbreviations: ACTB -β-actin; Atg -autophagy-related; Baf-A1 - Bafilomycin-A1; CSC -cancer stem cells; Col1A -type 1a collagen; d -day; HBSS -Hank’s balanced salt solution; LC3 -microtubule-associated protein 1 light chain 3 protein; SQSTM1/p62 -sequestosome; OB -osteoblast; OC -osteoclast; OS -osteosarcoma; TEM -transmission electron microscopy; TGF-β -transforming growth factor β; TRAP -acid phosphatase 5, tartrate-resistant.

L’ostéoporose (OP) touche une femme sur 3 après la ménopause. Outre la carence oestrogénique, l’augmentation du stress oxydatif est maintenant reconnu comme un mécanisme physiopathologique central de l’OP. L’autophagie est un processus intracellulaire permettant l’élimination et le recyclage des protéines et organelles endommagées. Le processus est initié dans le cytoplasme par la formation d’une vésicule à double membrane appelée autophagosome. Ces vésicules autophagiques sont caractérisées par la présence de la protéine de l’autophagie LC3-II insérée dans la double membrane, ainsi que SQSTM1/p62, un récepteur considéré comme un substrat de l’autophagie. Grâce à l’élimination des mitochondries endommagées, principales sources de radicaux libres oxygénés, l’autophagie joue un rôle majeur dans la réduction du stress oxydatif. Un déclin de l’autophagie lié à l’âge, associé à de nombreux arguments expérimentaux dans des modèles animaux, suggère une implication de l’autophagie dans l’OP. Cependant, une preuve définitive reste à établir chez l’Homme. L’objectif de ce travail est de déterminer pour la première fois si il existe un défaut d’autophagie dans l’OP humaine afin d’ouvrir la voie à de nouvelles approches thérapeutiques. Étude prospective monocentrique incluant des patientes ménopausées de plus de 65 ans, opérées pour une coxarthrose (groupe contrôle) ou pour une fracture du col du fémur (groupe OP) entre 2018 et 2021. Étaient exclus dans les 2 groupes les patientes présentant des traitements ou pathologies connues pour interférer avec l’autophagie, et dans le groupe OP les OP secondaires. L’os cortical du col du fémur prélevé était nettoyé, réduit en poudre, décalcifié et une extraction protéique était réalisée. Le niveau d’expression de LC3-II et SQSTM1/p62 était analysé par Western Blot. Au total, 43 patientes ont été inclues dans l’analyse : 29 témoins et 14 OP. La vitamine D est comparable dans les 2 groupes. Le niveau d’expression de la protéine LC3-II est significativement diminué chez les patientes OP par rapport aux contrôles (p = 0,02), indiquant une diminution du nombre d’autophagosomes chez les OP. Le niveau de LC3-II n’est pas corrélé à l’âge. Il existe une tendance à l’accumulation de la protéine SQSTM1/p62, qui est un substrat de l’autophagie, confirmant ainsi le résultat précédent. Ces données sont concordantes avec les données expérimentales obtenues chez les rongeurs. Ces données démontrent pour la première fois que les patientes OP présentent un déficit d’autophagie dans les ostéocytes, contribuant ainsi à mieux comprendre le rôle de l’autophagie dans la physiopathologie de l’OP. Ceci ouvre de nouvelles perspectives thérapeutiques dans l’OP via la stimulation de l’autophagie osseuse.

The bacterial cytosine deaminase (CD) gene, associated to the 5-fluorocytosine (5-FC) prodrug, is one of the more widely used suicide systems in gene therapy. Introduction of the CD gene within a tumor induces, after 5-FC treatment of the animal, a local production of 5-fluorouracil (5-FU) resulting in intratumor chemotherapy. Destruction of the gene-modified tumor is then followed by the triggering of an anti-tumor immune reaction resulting in the regression of distant wild-type metastasis. In pre-clinical studies, 5-FC is generally administered by daily intraperitoneal injections. However, when used as an anti-fungal in humans, either IV or oral administration is used. In this study, we compared oral and intraperitoneal 5-FC administration in rats bearing a wild-type and a cytosine deaminase-expressing liver tumors. The results indicate that per os 5-FC administration is as efficient as intraperitoneal for the induction of CD-expressing tumor regression and the triggering of a distant bystander effect, acting on wild-type liver tumor and extra-hepatic metastasis.

Hintergrund: Kolorektale Lebermetastasen sind ein zahlenmäßig bedeutendes und bislang therapeutisch nicht befriedigend gelöstes Problem. Die Gentherapie könnte hier einen neuen Ansatz darstellen. Methodik: Zellen einer Kolonkarzinomlinie der BD IX Ratte wurden mit dem bakteriellen Gytosin Desaminase-Gen (CD-Gen) transfiziert. Das Genprodukt ermöglicht die Umwandlung des nicht-toxischen 5-Fluorcytosin (5-FC) in 5-FU. Bei syngenetischen Ratten wurden dann im einen Leberlappen Metastasen der genveränderten Tumorzellen und im anderen Lappen Metastasen der wild-Typ-Zellen erzeugt. Ergebnisse: Es konnte gezeigt werden, daß eine anschließende 5-FC-Behandlung nicht nur zu einer Regression der CD-positiven sondern über einen Distanz-Bystander-Effekt auch der CD-negativen Tumoren führt. Dieser Effekt war auch zu beobachten, wenn wild-Typ-Metastasen präexistent waren und erst zeitlich versetzt CD-positive Tumoren generiert und mit 5-FC therapiert wurden. Schlußfolgerungen: In dem verwendeten Modell ließ sich ein therapeutischer Distanz-Bystander-Effekt in der Leber nachweisen. Somit erscheint auf diesem Wegauch eine Gentherapie bereits etablierter kolorektaler Metastasen beim Menschen prinzipiell möglich.