Tesi sul tema "Syngeneic model"

Considerable data demonstrate a high prevalence of depression symptoms in patients with cancer, with some studies showing the prevalence for major depressive disorder (MDD) to be as high as 50%. Because depression researchers have found that a significant relationship exists between depression symptoms and indices of systemic inflammation and because several cancer types exploit the mechanisms of the body's inflammatory response to aid in their own progression, it was hypothesized that tumor in the body could be a cause of depression symptoms in cancer patients. Examination of this question was conducted using an immunocompetent mouse model of ovarian cancer and several measures of depressive-like and sickness behavior. Initial investigation of the model (Chapter 2) involved a series of pilot experiments that addressed methodology and demonstrated that ID8 murine ovarian carcinoma was capable of inducing elevated levels of systemic IL-6 and depressive-like behavior, specifically anhedonia as measured by a decrease in sucrose solution. In Chapter 3, a larger experiment (Experiment 1) was conducted that examined the effect of ovarian tumor on sucrose intake, food intake, body weight, locomotion, and rotarod performance. Results in the study indicated that sucrose-measured anhedonia in the model was not confounded by anorexia because tumor-bearing mice and control mice exhibited no significant difference in appetite. In Chapter 4, a second experimental factor, social housing, was added alongside tumor condition, and a second measure of depressive-like behavior, tail suspension test (TST) immobility, was added to measures from the previous experiment. The results of this second large experiment (Experiment 2) demonstrated that ovarian tumor had no significant effect on TST immobility, even though it did cause mice to exhibit less motor activity in the home cage. Housing condition did affect TST immobility. Mice that were individually-housed exhibited significantly more TST immobility than group-housed mice. Also, individually-housed mice exhibited less sucrose intake than group-housed mice. This gave rise to a significant interaction in sucrose preference among the four experimental groups where individually-housed tumor-bearing mice showed less sucrose preference than the other groups. In Chapter 5, systemic proinflammatory and antiinflammatory cytokines from both Experiment 1 and Experiment 2 were examined. Results indicated that both proinflammatory and antiinflammatory cytokines were significantly higher in tumor-bearing mice than in control mice, and these effects were largest for interleukin-6 (IL-6) and IL-10. Among tumor-bearing mice, significant correlations between IL-1β, IL-6, tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF- β) and locomotion were noted, but there was no significant correlation between cytokines and anhedonia. No significant effect of housing condition on cytokines was found. In Chapter 6, principal findings of the project are summarized and discussed with a focus on anhedonia and psychomotor slowing in MDD. Current evidence suggests that dopaminergic and glutamatergic systems in the brain may underlie anhedonic and psychomotor features in inflammation-induced depression. Thus, future investigation of the mediators between ovarian tumor and these depressive-like behaviors in the model may benefit from targeting these specific neural mechanisms.

Tumour immunity is a balance between immune mediators that promote tumor progression versus mediators that promote tumor rejection. Infiltrating lymphocytes in human colorectal cancer tissues are independent prognostic factors for a better survival and a high number of cytotoxic CD8+ T-cells have been associated with a better prognosis in terms of a longer and disease free survival for the patient. In our syngeneic rat model we induce colon carcinoma subperitoneally by injecting a colon cancer cell line BN7005, a cell line expressing the epitope (Lewis Y) for the BR96 antibody. Tumours are dissected out and treated with different fixatives and then either frozen, snap-frozen or embedded in paraffin followed by sectioning. Immunohistochemistry using monoclonal antibodies against the tumour infiltrating leukocytes was performed on the tissue. The results were seen as an infiltration of different leukocytes in the tumours.

Le cancer colorectal (CCR) est le troisième cancer le plus fréquent et le deuxième cancer le plus mortel dans le monde. En conséquence, de nouveaux biomarqueurs diagnostiques ainsi qu’une amélioration des traitements actuels sont nécessaires. Les tumeurs se développent dans des microenvironnements complexes où les cellules cancéreuses interagissent et modulent la réponse immunitaire locale pour persister et se multiplier. Les lectines de type C, exprimées notamment par les cellules de l’immunité, régulent la réponse anti-cancéreuse, et donc le développement tumoral. Parmi elles, l'immunorécepteur des cellules dendritiques (DCIR), a été montré comme jouant un rôle immunitaire majeur au cours des maladies infectieuses et auto-immunes. À l’inverse, son rôle dans l'immunité tumorale reste méconnu. L'analyse des données transcriptomiques de deux cohortes de patients atteints de CCR a révélé un lien entre une expression élevée de DCIR et une meilleure survie des patients. Dans ce contexte, l'objectif principal de ma thèse était de déterminer l'impact de DCIR sur le développement du CCR et la réponse immunitaire associée. Dans ce but, j’ai établi un modèle murin préclinique, orthotopique et syngénique du CCR consistant en l'injection intracaecale de cellules tumorales MC38 exprimant la luciférase (MC38-fLuc+) dans des souris C57BL/6. Le suivi de la croissance tumorale par bioluminescence a montré que, malgré l’acquisition initiale de tumeurs solides par toutes les souris, seulement 30% des souris ont développé un CCR progressif et mortel, tandis que les autres animaux ont spontanément rejeté leurs tumeurs. Aucun rejet des tumeurs CCR MC38 n'a été observé en l'absence d'immunité adaptative, ni lors de l'injection de cellules MC38 dans d'autres sites anatomiques. L'immunophénotypage par transcriptomique et cytométrie de flux a révélé que les souris développant des tumeurs progressives présentaient une réponse immunitaire pro-tumorale, définie par une signature caractéristique des lymphocytes T régulateurs, détectable peu après l'implantation tumorale, et par une infiltration de cellules myéloïdes suppressives connues pour favoriser la croissance tumorale. En revanche, les souris rejetant les tumeurs présentaient une signature pro-inflammatoire précoce et un microenvironnement anti-tumoral enrichi en lymphocytes T CD8+. Ainsi, nos résultats démontrent un rôle du microenvironnement spécifique du côlon dans la régulation de l'équilibre entre les réponses immunitaires anti- ou pro-tumorales et souligne l'importance d'utiliser des modèles murins orthotopiques pour les études in vivo. Dans la seconde partie de ma thèse, j’ai utilisé ce modèle murin de CCR pour comparer le développement tumoral dans des souris C57BL/6 de type sauvage ou des souris déficientes pour l’expression de mDcir1 (mDcir1-KO), un homologue murin du DCIR humain. Bien que l'absence de mDCIR1 n'ait aucune incidence sur le pourcentage de souris développant ou rejetant les tumeurs du CCR, nous avons observé que les animaux mDcir1-KO développaient des tumeurs plus importantes que les sauvages En accord avec ce résultat, nous avons constaté une infiltration plus faible de lymphocytes CD8+ cytotoxiques et une activation moindre des lymphocytes T CD4+ et CD8+ (c'est-à-dire T-BET+, CD44haut, CTLA-4+) dans les tumeurs des souris mDcir1-KO par rapport aux souris sauvages. Ainsi, nos données indiquent un rôle protecteur et anti-tumoral de DCIR pendant le développement du CCR, probablement dû à une dérégulation de l'équilibre existant entre la tumeur et la réponse immunitaire. Dans l'ensemble, cette étude ouvre la voie à la mise au point éventuelle de biomolécules pharmacologiques ciblant DCIR pour déclencher une réponse immunitaire anti-tumorale efficace dans le contexte du CCR et au-delà
Colorectal cancer (CRC) is the third most common and second deadliest cancer worldwide accounting for 900.000 deaths in 2018. Consequently, there is a strong need for new biomarkers as well as an improvement of the current treatments. Tumors develop in complex microenvironments where cancer cells constantly crosstalk with, and modulate, the local immune response to persist and replicate. C-type lectins receptors, expressed in particular by immune cells, actively regulate the immune response to cancer cells and, therefore, tumor development. Dendritic cell immunoreceptor (DCIR), a C-type lectin expressed by myeloid cells, has been shown to play a major role in immunity to infectious and autoimmune diseases. Yet, the role played by DCIR in tumor immunity remains unknown. Analysis of publicly available transcriptomic data from two cohorts of CRC patients revealed an association between high DCIR gene expression and improved survival of patients. In this context, the principal objective of my PhD thesis was to determine the role played by DCIR in the immune response during CRC development. First, I developed an orthotopic syngeneic pre-clinical CRC mouse model consisting in the intra-caecal injection of engineered MC38 tumor cells expressing firefly luciferase (MC38-fLuc+) in C57BL/6 mice. Monitoring of the tumor growth by bioluminescence revealed that, despite an initial growth of solid tumors in all the mice, only 30% of mice developed a progressive lethal CRC, while the remaining animals spontaneously rejected their solid tumor and survived more than 100 days. No rejection of tumors was observed in the absence of adaptive immunity, nor when MC38-fLuc+ cells were injected in other anatomical locations (i.e., liver and skin). Immunophenotyping by transcriptomic and flow cytometry showed that mice with progressive CRC tumors exhibited a pro-tumor immune response, characterized by a regulatory T cell pattern, discernible shortly post-tumor implantation, as well as myeloid suppressor cells that are well-known to favor tumor growth. By contrast, tumor-rejecting mice presented an early pro-inflammatory response and an anti-tumor microenvironment enriched with CD8+ T cells. Taken together, our results demonstrate a preponderant role of the colon-specific microenvironment in regulating the balance between anti- or pro-tumor immune responses and underline the importance of using orthotopic mouse models for in vivo studies. In a second part of my thesis, we used this CRC mouse model to compare the tumor development in wild-type (WT) C57BL/6 mice or mice deficient for mDcir1 (mDcir1-KO), a murine homologue of human DCIR. While the lack of mDCIR1 has no impact on the percentage of mice developing or rejecting CRC tumors, we observed that mDcir1-KO animals developed bigger tumors than their WT counterparts. In line with this result, we found a lower infiltration of cytotoxic CD8+ and decreased activation of both CD4+ and CD8+ T cells (i.e., T-BET+, CD44high, CTLA-4+) in CRC tumors from mDcir1-KO mice compared to WT mice. Altogether, our data point to a protective and anti-tumor role of DCIR during CRC development, probably due to a dysregulation of the balance existing between the tumor and the immune response. Overall, this study paves the way for the potential future development of pharmacological biomolecules targeting DCIR to trigger an efficient anti-tumor immune response in the context of CRC and beyond

Renal cell carcinoma (RCC) is the third most common genito-urinary cancer. Beyond surgery, few other treatment options for RCC exist and about one-third of the patients who have had nephrectomy develop metastasis subsequently. The treatment of the metastatic disease remains a clinical challenge. Hence, novel therapeutic options are necessary for the patients with metastatic RCC. Immunotherapy is the most common mode of treatment in RCC presently; however, it contributes to a large number of toxic side effects to the patients. The immunotherapeutic regimens currently used to treat metastatic renal cancer are recombinant human interleukin -2 (IL-2) and recombinant human interferon alpha alone or in combination. However, the uses of these high dose cytokines are limited by their toxicity and poor patient response rates. Preclinical studies in animal tumor models of RCC are required to address the newer and effective therapeutic approaches for late stage metastatic RCC. A suitable animal model for studying RCC is lacking. Hence, development of a novel animal model would largely contribute in testing newer therapeutics and combating the metastatic disease. Cyclooxygenases are group of enzymes that catalyze the conversion of arachidonic acid to prostaglandins (PGE2). It comprises of two isoforms Cox-1 and Cox-2. Previous studies have implicated the potential role of Cox-2 in carcinogenesis and the expression of Cox-2 have been reported in colorectal, lung, breast, gastric and esophageal carcinomas. Cox-2 is also highly expressed in RCC and a potential biomarker in RCC. Based on emerging clinical evidences on the role of Cox-2 in several malignancies, we hypothesize that overexpression of Cox-2 in RCC promotes tumor growth and metastasis. Selective Cox-2 inhibitors act by inhibiting PGE2 synthesis and have been shown to retard tumor formation, metastasis, and angiogenesis. They induce apoptosis and inhibit the PGE2 induced immunosupression. Thus, a specific Cox-2 inhibitor like indomethacin or NS398 would be able to inhibit the tumorigenic properties of Cox-2, thereby attenuating tumor growth and dissemination to other organs. In this context, we injected the Cox-2 engineered Renca cell lines (COX2 -ve and COX2 +ve) in the subcapsular space of kidney of a Balbc/Cr mice. This resulted in the tumor growth, which was monitored by bioluminescence imaging (BLI) for a period of three weeks post-inoculation. Metastases were evident in distant organs such as lung, liver, spleen and lymph nodes. This was expressed as luciferase activity per milligram of protein of the particular tissue normalized with the background luciferase activity per milligram of the tissue from control (non-injected mice). Thus, the animal model was established and validated in our preliminary studies. To address the potential role of PGE2 in the tumor microenvironment, tumors were harvested and then processed for assessment by histology and immunohistochemistry. Initial characterization studies included immunohistochemical assessment of tumor vasculature as elucidated by staining with specific markers for lymphatic vessels (LYVE-1), blood endothelium (CD31) and tumor infiltrating macrophages (Cd11b). Macrophage recruitment close to the LYVE-1+ structures were also determined by the double positive events obtained by staining with both CD11b and LYVE-1. A large number of peripheral (18%±2.79), intratumoral (12%±3) and marginal (7%±2.01) LYVE-1+ve structures were found in PGE2 producing tumor than in the control tumor. Although, the frequency of blood vessels in both the tumor types were unaltered, however, an increased vascular area was obtained in the COX2 +ve tumor than in COX2 -ve tumor. There was a significant increase in the frequency of infiltrating macrophages in the peripheral (25%± 3.86), intratumoral (10%± 3.93) and tumor-kidney margin of COX-2 positive tumors (10%± 2.34) than in the COX-2 negative tumors (12% ± 2.36, 0% and 0%) respectively. Frequency of the blood vessels in both the tumors were unaltered, however, a significant increase in the mean fluorescent intensity (MFI) in the peripheral region (4.3±0.1) of COX2 +ve tumor was observed when compared to COX2 -ve tumors (2.3±0.05). These preliminary studies indicate the potential role of PGE2 in promoting tumor vasculature, increased macrophage recruitment within the tumors and tumor-kidney margins. In our initial studies, a significant enrichment of LYVE-1+ve macrophages were observed in the kidney-tumor sections of the COX2 +ve mice, which might indicate that, PGE2 may promote differentiation of the macrophages into a lymphatic phenotype. Thus, this animal model would further help in thorough characterization of other immune infiltrating cells like CD8+ T cells and NK cells and thereby lead us to identify the cause of immune dysregulation in renal cell carcinoma due to RCC derived soluble factors like PGE2 and TGF-beta. Furthermore, treatment with Cox-2 inhibitors like NS-398 should retard tumor growth, metastases, immune cell dysregulation, and tumor vasculature.

In order to study the expression of MMP2, MMP 3 and MMP9 in breast cancer brain and lung metastasis, we used a syngeneic rat model of distant metastasis of ENU1564, a carcinogen-induced mammary adenocarcinoma cell line. At six weeks post inoculation we observed development of micro-metastasis in the brain and lung. Immunohistochemistry and Western blotting analyses showed that MMP 2, -3 and -9 protein expression is consistently significantly higher in neoplastic brain tissue compared to normal brain tissue. Lung metastases express abundant MMP2, -3 and -9 in neoplastic cell cytoplasm. In situ zymography revealed gelatinase activity within the brain metastasis. Gel zymography showed an increase in MMP2 and MMP3 activity in brain metastasis. Furthermore, we were able to significantly decrease the development of breast cancer brain and lung metastasis in animals by treatment with PD 166793, a selective synthetic MMP inhibitor. In addition, PD 166793 decreased the in vitro invasive cell behavior of ENU1546. TIMP2 overexpression also decreased the development of breast cancer lung metastasis in our model. Our results suggest that MMP2, -3 and -9 may be involved in the process of metastasis of breast cancer to the brain and lung. Because astrocytes have been associated with breast cancer brain metastasis we evaluated the role of astrocytes and ERK2 pathway in MMP2 up-regulation in BC brain metastasis. A significant decrease in brain metastases development, and orthotopic tumor size and weight were observed in animals inoculated with ENU1564-TIMP2 cells. These were associated with decreased MMP2 activity, as demonstrated by gel zymography. Rat astrocyte-conditioned media increased expression of MMP2 in ENU15645 cells and increased in vitro cell invasion of ENU1564 and ENU1564-TIMP2 cells. Blockage of ERK1/2 phosphorylation by treatment with PD98059 decreased the expression of MMP2 in cancer cells grown in rat astrocyte-conditioned media. We determine that MMP2 plays a role in in vivo development of breast cancer brain metastases. Additionally, we conclude that astrocytes are associated with expression of MMP2 in cancer cells via ERK1/2 signaling pathway.

Monitoring of cell transplantation in vivo is crucial to understanding how the progeny of stem cells contribute to or ameliorate disease. Although animal models enable researchers to understand the behaviour of specific cell types in great detail, they typically require invasive procedures or large numbers of animals in order to track sdisease progression or treatment efficacy over time. Positron emission tomography (PET) has emerged as a promising non-invasive method of tracking transplanted cells in preclinical research, as it enables researchers to track cells longitudinally over the lifespan of the host animal. Although existing reporter gene PET methodologies are under development, they are subject to a number of restrictions which limit their usefulness in preclinical research. These limitations include the need to transduce cells with reporter genes, which is inefficient and may lead to unwanted phenotypic changes in the cells of interest. Another major limitation for many applications is the inability of many reporter ligands to cross an intact blood-brain barrier. Described in this thesis is a novel methodology utilising the transplantation of wild-type bone marrow cells into mice lacking the Translocator Protein (18kDa; TSPO). The TSPO knockout mouse serves as a null-background model for transplantation of unmodified bone marrow cells which express endogenous TSPO. These TSPO-expressing cells are then targeted for PET imaging by a TSPO-specific radio-ligand, 18F-PBR111. PBR111 binds selectively to TSPO and can penetrate the intact blood-brain barrier, making it a promising approach for overcoming some of the limitations of reporter gene imaging. In brief, TSPO-knockout mice (and wild-type controls) were conditioned with a sub-lethal dose of gamma radiation and transplanted with nucleated bone marrow cells from C57BL/6 mice engineered to express green fluorescent protein (GFP). At various stages of engraftment ranging from 1 to 12 months post-transplant, mice were anaesthetised with 1-4% isoflurane, cannulated via the tail vein, and injected with 0.2nM of 18F-PBR111. Data was collected with an Inveon pre-clinical PET/CT camera for 50 minutes post-injection. Images were re-constructed into 20 time frames and were again re-constructed using a 3-dimensional ordered subset expectation maximization algorithm (3D-OSEM). From this, time activity curves (TACs) were extracted from regions of interest drawn on co-registered PET/CT images, corresponding to whole organs. TACs were then used to calculate standardised uptake values for each region of interest. Sections taken from collected tissues were later incubated with 125I-CLINDE in order to visualise receptor density in tissues independently of pharmacokinetic parameters associated with IV tracer injection. Sections were also stained with a monoclonal TSPO antibody in order to identify the TSPO-expressing cells at high spatial resolution. Engraftment rates in blood were assessed at 1, 3 and 6 months post transplantation using quantitative real-time PCR in order to determine whether haematopoietic reconstitution in TSPO knockout mice resembles its wild-type counterparts, which is important for establishing whether the results observed in vivo are physiologically ‘normal’ and therefore applicable to models of disease. Although engraftment rates were equivalent in wild-type and TSPO-/- background recipient mice 1 and 3 months post-transplantation, at 6 months engraftment rates in the TSPO-/- mice were significantly higher. Nonetheless, both genotypes displayed persistent engraftment of donor cells at a minimum of 6 months post-transplant, and observed up to 12 months post-transplant in the TSPO-/- recipients. This engraftment was detectable using both in vitro and in vivo methods (PET) in spleen tissue throughout the duration of the study, and in some tissues susceptible to inflammation such as lung and salivary gland at 6 months post-transplant. A pilot of longitudinal imaging also suggested this persistent migration of donor-derived cells could be detected up to at least half of the animal’s lifespan. Although some major successes in longitudinal monitoring of TSPO-expressing donor-derived cells was observed in this study, there were some notable exceptions, such as the bone marrow itself, susceptible to major spillover associated with free fluorine accumulating within bone, and regions marked by low level engraftment of cells, including brain, and lung parenchyma. For each tissue of interest, an in-depth discussion of the implications of the findings are presented, followed by an outline of the methodological challenges associated with whole-body imaging of rodents in this context. Overall, the model presented in this study has the potential for adaptation to a variety of models incorporating cell-based diseases or treatments, with the ability to track the accumulation of inflammatory cells in a variety of organs.

BACKGROUND:Tumor cell lines that can be tracked in vivo during tumorigenesis and metastasis provide vital tools for studying the specific cellular mechanisms that mediate these processes as well as investigating therapeutic targets to inhibit them. The goal of this study was to engineer imageable mouse mammary tumor cell lines with discrete propensities to metastasize to bone in vivo. Two novel luciferase expressing cell lines were developed and characterized for use in the study of breast cancer metastasis to bone in a syngeneic mouse model.RESULTS:The 4 T1.2 luc3 and 66c14 luc2 cell lines were shown to have high levels of bioluminescence intensity in vitro and in vivo after orthotopic injection into mouse mammary fat pads. The 4 T1.2 luc3 cell line was found to closely model the sites of metastases seen in human patients including lung, liver, and bone. Specifically, 4 T1.2 luc3 cells demonstrated a high incidence of metastasis to spine, with an ex-vivo BLI intensity three orders of magnitude above the commercially available 4 T1 luc2 cells. 66c14 luc2 cells also demonstrated metastasis to spine, which was lower than that of 4 T1.2 luc3 cells but higher than 4 T1 luc2 cells, in addition to previously unreported metastases in the liver. High osteolytic activity of the 4 T1.2 luc3 cells in vivo in the bone microenvironment was also detected.CONCLUSIONS:The engineered 4 T1.2 luc3 and 66c14 luc2 cell lines described in this study are valuable tools for studying the cellular events moderating the metastasis of breast tumor cells to bone.

État de l’art : La dormance tumorale est une stratégie de résistance utilisée par les cellules cancéreuses. Elle constitue un obstacle majeur dans la thérapie du cancer, puisqu’elle mène à la maladie résiduelle minimale (MRD) et augmente le risque de rechute. Bien que cliniquement significatifs, les mécanismes derrière la dormance tumorale et la MRD ne sont pas bien compris. Nous avons utilisé deux modèles murins syngéniques de leucémie myéloïde et de mélanome élaborés par le laboratoire pour explorer les profils génétiques, épigénétiques, transcriptomiques et protéomiques liés à la dormance tumorale. Par cette approche multi-omique, nous avons cherché à découvrir les processus moléculaires conduisant à la MRD et à identifier des cibles thérapeutiques potentielles.Résultats :Nous avons réalisé une analyse multi-omique complexe incluant le séquençage de l'exome entier (WES), l'analyse des variations du nombre de copies (CNV), l'immunoprécipitation de la chromatine suivie du séquençage (ChIP-seq) et des investigations du transcriptome et du protéome. L'analyse WES a identifié un chevauchement subtil de mutations génétiques entre les modèles de dormance de mélanome et de leucémie, avec de nombreuses mutations trouvées exclusivement dans les cellules de dormance. Ces signatures génétiques spécifiques suggèrent que les pressions sélectives durant la MRD peuvent conférer une résistance au microenvironnement ou aux traitements. En combinant les données CNV, les marques d'histones et les signatures d'expression génique transcriptomique avec l'analyse d'enrichissement de Gene Ontology (GO), nous avons identifié des rôles fonctionnels potentiels de ces gènes mutés et obtenu des informations sur les voies impliquées dans la MRD. De plus, en comparant les "gènes MRD murins" avec les données correspondantes aux maladies humaines provenant de bases de données publiques,nos travaux soulignent des caractéristiques communes liées à la progression de la maladie. L'analyse protéomique, combinée aux investigations génétiques multiomiques,a révélé une signature protéique distincte dans les cellules de dormance avec une implication minimale des mécanismes génétiques. L'analyse 'enrichissement des voies a mis en évidence les processus métaboliques, de différenciation et de remodelage du cytosquelette impliqués dans la MRD. Enfin, nous avons identifié 11 protéines exprimées différemment dans les cellules de dormance de ces deux pathologies. Conclusions : Notre recherche met en évidence la nature complexe de la dormance tumorale,impliquant à la fois des éléments génétiques et non génétiques. En comparant les données génomiques, transcriptomiques, protéomiques et épigénomiques, nous fournissons un aperçu approfondi du paysage moléculaire associé à la MRD. Ces résultats posent une base solide pour de futures études et suggèrent des directions prometteuses pour le développement de thérapies ciblées pour la MRD chez les patients atteints de leucémie et de mélanome. Cela souligne la nécessité de prendre en compte à la fois des facteurs génétiques et non génétiques dans les stratégies de traitement
Background : Tumor dormancy, a resistance strategy used by cancer cells, is a major impediment in cancer therapy, leading to minimal residual disease (MRD) and increasing the risk of relapse. Although clinically significant, the mechanisms behind tumor dormancy and MRD are not well understood. In this research, we employed two syngeneic murine models of myeloid leukemia and melanoma to explore the genetic,epigenetic, transcriptomic, and proteomic profiles linked to tumor dormancy. By applying a multiomics approach, we aimed to uncover the molecular processes driving MRD and identify possible therapeutic targets. Results : We performed a comprehensive omics analysis that included whole-exome sequencing (WES), copy number variation (CNV) analysis, chromatin immunoprecipitation followed by sequencing (ChIP-seq), and investigations of the transcriptome and proteome. The WES analysis identified a limited overlap of gene mutations between the melanoma and leukemia dormancy models, with many mutations found exclusively in dormant cells. These unique genetic signatures suggest that selective pressures during MRD may provide resistance to the surrounding microenvironment or treatments. Combining CNV data, histone marks, and transcriptomic gene expression signatures with Gene Ontology enrichment analysis,we identified the potential functional roles of these mutated genes and gained insights into the pathways involved in MRD. Furthermore, by comparing "murine MRD genes"with corresponding human disease data from public databases, we identified common features related to disease progression. Proteomic analysis, integrated with multi-omics genetic investigations, revealed a distinct protein signature in dormant cells with minimal involvement of genetic mechanisms. Pathway enrichment analysis pointed to the metabolic, differentiation, and cytoskeletal remodeling processes involved in MRD. Ultimately, we identified 11 proteins that were differentially expressed in dormant cells across both types of pathology. Conclusions : Our research highlights the intricate nature of tumor dormancy, involving both genetic and non-genetic elements. Through the comparison of genomic,transcriptomic, proteomic, and epigenomic data, we deliver an extensive overview of the molecular landscape associated with minimal residual disease. These findings laya solid groundwork for future studies and suggest promising directions for developing targeted therapies for MRD in leukemia and melanoma patients. This underscores the necessity of incorporating both genetic and non-genetic factors into treatment strategies

博士
國立成功大學
臨床醫學研究所
95
The promoter of human telomearse reverse transcriptase (hTERT) is inactive in normal cells, but active in the majority of cancers. The hTERT promoter can selectively drive transgene expression in telomerase-positive cancers. Here we evaluated the combination therapy of adenoviral vector Ad-hTERT-CD encoding E. Coli cytosine deaminase (CD) drived by the hTERT promoter and low-dose etoposide (0.1 μg/mL) for treating bladder cancer. The results showed that Ad-hTERT-CD conferred sensitivity to 5-fluorocytosine (5-FC) in bladder cancer cells, which could be enhanced by etoposide treatment, but not in normal cells. The up-regulated hTERT promoter was correlated with the expression of hypoxia-inducible factor (HIF)-1α. By contrast, etoposide activated p53 and down-regulated hTERT promoter activity in normal cells. Etoposide also increased adenoviral infection via enhancement of coxsackie-adenovirus receptor (CAR) expression on bladder cancer and normal cells. The combined therapy of Ad-hTERT-CD (109 plaque-forming units)/5-FC (200 mg/kg) with etoposide (2 mg/kg) synergistically suppressed tumor growth and prolonged survival in mice bearing syngeneic MBT-2 bladder tumors. This combination therapy induced complete tumor regression in 75 % of tumor-bearing mice. Furthermore, increased infiltrating CD4+ and CD8+ T cells and necrosis within the tumors were found in mice receiving combination therapy of Ad-hTERT-CD and etoposide compared with those treated with either treatment alone. Thus, the combination therapy may be an appealing therapeutic intervention for bladder cancers. In adenoviral vector system, conditionally replicating adenoviruses (CRAds), which can induce cytolysis of infected tumor cells and spread to surrounding cells, can provide better antitumor efficacy than non-replicating groups. Iressa is a selective tyrosine kinase inhibitor of EGFR, which is overexpressed in majority of human bladder cancers, to block tumor cell proliferation, survival and progression. We hypothesized that the combined therapy of Iressa and an E1B-55kD-deleted CRAd, designed Ad5GS1 which expressed intact E1A driven by hTERT promoter, exerted tumor-specific oncolysis. Ad5GS1 caused severe cytolysis in telomerase-positive human and murine bladder cancer cells, which could be enhanced by Iressa, whereas it was severely attenuated in telomerase-positive normal cells, including murine nontransformed, immortalized fibroblasts and embryonic stem cells. Most notably, after Ad5GS1 infection hTERT promoter was continuously up-regulated in murine bladder cancer cells with mutant p53. The up-regulation of hTERT promoter activity was correlated with the dosage of E1A, which was the adenoviral early gene driven by hTERT promoter. Iressa also up-regulated hTERT promoter activity in Iressa-resistant bladder cancer cells. Ad5GS1 exhibited higher oncolytic activities than an E1B-55 kD-deleted adenovirus driven by the E1A promoter in both in vitro and in vivo studies. Intratumoral administration of Ad5GS1 significantly retarded tumor growth and prolonged the survival time in immunocompetent mice bearing murine bladder tumors. The replication of Ad5GS1 was detectable in tumors for at least 15 days after treatment. Our results suggest that oncolytic effects of Ad5GS1 in telomerase-positive bladder cancer cells provides safety features for tumor-selective killing and yields a therapeutic benefit in the syngeneic murine tumor model, which is relevant to clinical settings. Furthermore, Iressa up-regulated the hTERT promoter activity to enhance the cytolytic efficacy of Ad5GS1 system. Because a majority of human tumors exhibit high telomerase activity, the combination therapy of chemotherapeutic agent and hTERT promoter-driven gene therapy may be applicable to a broad spectrum of cancers.