Identification and characterization of circulating tumors cells in colorectal cancer

La présence de métastases est un facteur de mauvais pronostic dans les cancers solides et une meilleure compréhension de la dissémination tumorale est nécessaire afin d'améliorer la prise en charge de ces formes avancées. Les cellules tumorales circulantes (CTC) représentent un intérêt majeur dans la pathologie tumorale, d'une part sur le plan clinique en tant que marqueur prédictif et pronostique et d'autre part sur le plan de la compréhension des mécanismes impliqués dans la formation des métastases. Les CTC sont rares et hétérogènes et restent mal caractérisées, et ce, particulièrement dans le cancer colorectal. Une partie de ces cellules aurait un phénotype de cellules initiatrices de tumeur (CIT) leur permettant de former des métastases, de résister aux traitements et par conséquent d'être responsables des rechutes. Une meilleure connaissance des CTC possédant un phénotype de CIT représente donc un enjeu majeur. L'objectif de ce travail a été d'identifier et de caractériser les CTC avec un potentiel de cellules initiatrices de tumeur dans le cancer colorectal en se basant sur les propriétés fonctionnelles des CIT. Nous avons ainsi, pour la première fois, pu établir deux modèles permettant de répondre à cet objectif. D'une part des lignées de CTC avec un fort potentiel de CIT obtenues à partir d'échantillons sanguins de patients atteints de cancer colorectal, et d'autre part, nous avons mis en place un modèle murin de dissémination tumorale par xénogreffe orthotopique permettant d'isoler les CTC.Liver or lung metastases represent a poor prognosis in colorectal cancer patients and better understanding tumor spreading became essential to improve patient care. Circulating tumor cells (CTC) is considered as a promising tool, both as prognostic marker and as tool to study mechanisms involved in metastasis development. CTCs are rare and heterogeneous and remain poorly characterized especially in colorectal cancer. It is accepted that at least some of the CTC have a tumor initiating cell (TIC) phenotype that could be responsible for metastasis, chemoresistance and consequently lead to relapse. A deep characterization of CTC became thus an urgent unmet need. The aim of this work was to identify and characterize CTC with TIC properties in colorectal cancer, on the basis of their functional properties. To reach this aim, we established for the first time and characterized CTC lines from blood sample of colorectal cancer patient, and we also developed an orthotopic xenograft mouse model in which tumoral cells are circulating in the blood

Identification and characterization of circulating tumors cells in colorectal cancer

La présence de métastases est un facteur de mauvais pronostic dans les cancers solides et une meilleure compréhension de la dissémination tumorale est nécessaire afin d'améliorer la prise en charge de ces formes avancées. Les cellules tumorales circulantes (CTC) représentent un intérêt majeur dans la pathologie tumorale, d'une part sur le plan clinique en tant que marqueur prédictif et pronostique et d'autre part sur le plan de la compréhension des mécanismes impliqués dans la formation des métastases. Les CTC sont rares et hétérogènes et restent mal caractérisées, et ce, particulièrement dans le cancer colorectal. Une partie de ces cellules aurait un phénotype de cellules initiatrices de tumeur (CIT) leur permettant de former des métastases, de résister aux traitements et par conséquent d'être responsables des rechutes. Une meilleure connaissance des CTC possédant un phénotype de CIT représente donc un enjeu majeur. L'objectif de ce travail a été d'identifier et de caractériser les CTC avec un potentiel de cellules initiatrices de tumeur dans le cancer colorectal en se basant sur les propriétés fonctionnelles des CIT. Nous avons ainsi, pour la première fois, pu établir deux modèles permettant de répondre à cet objectif. D'une part des lignées de CTC avec un fort potentiel de CIT obtenues à partir d'échantillons sanguins de patients atteints de cancer colorectal, et d'autre part, nous avons mis en place un modèle murin de dissémination tumorale par xénogreffe orthotopique permettant d'isoler les CTC.Liver or lung metastases represent a poor prognosis in colorectal cancer patients and better understanding tumor spreading became essential to improve patient care. Circulating tumor cells (CTC) is considered as a promising tool, both as prognostic marker and as tool to study mechanisms involved in metastasis development. CTCs are rare and heterogeneous and remain poorly characterized especially in colorectal cancer. It is accepted that at least some of the CTC have a tumor initiating cell (TIC) phenotype that could be responsible for metastasis, chemoresistance and consequently lead to relapse. A deep characterization of CTC became thus an urgent unmet need. The aim of this work was to identify and characterize CTC with TIC properties in colorectal cancer, on the basis of their functional properties. To reach this aim, we established for the first time and characterized CTC lines from blood sample of colorectal cancer patient, and we also developed an orthotopic xenograft mouse model in which tumoral cells are circulating in the blood

Bases fondamentales du processus métastatique

International audienceFUNDAMENTAL BASIS OF METASTATIC PROCESS: Metastatic process is described as a "dissemination of neoplastic cells in a distant secondary site, in which cells proliferate to develop a mass of cells partially differentiated". The vast majority of death in solid cancers is the consequence of metastasis development which lead to vital organ dysfunction. In the present review, either recent discoveries or controversial subjects associated with metastasis process will be discussed. Indeed epithelia-mesenchymal transition (EMT), circulating tumor cells, tumor dormancy, colonization in distant organ and cancer stem cells are tackled

Stratification of sigmoid volvulus early recurrence risk using a combination of CT features

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Essential requirement for -arrestin2 in mouse intestinal tumors with elevated Wnt signaling

International audienceβ-Arrestins (Arrb) participate in the regulation of multiple signaling pathways, including Wnt/β-catenin, the major actor in human colorectal cancer initiation. To better understand the roles of Arrb in intestinal tumorigenesis, a reverse genetic approach (Arrb(-/-)) and in vivo siRNA treatment were used in Apc(Δ14/+) mice. Mice with Arrb2 depletion (knockout and siRNA) developed only 33% of the tumors detected in their Arrb2-WT littermates, whereas Arrb1 depletion remained without significant effect. These remaining tumors grow normally and are essentially Arrb2-independent. Unsupervised hierarchical clustering analysis showed that they clustered with 25% of Apc(Δ14/+);Arrb2(+/+) tumors. Genes overexpressed in this subset reflect a high interaction with the immune system, whereas those overexpressed in Arrb2-dependent tumors are predominantly involved in Wnt signaling, cell adhesion, migration, and extracellular matrix remodeling. The involvement of Arrb2 in intestinal tumor development via the regulation of the Wnt pathway is supported by ex vivo and in vitro experiments using either tumors from Apc(Δ14/+) mice or murine Apc(Min/+) cells. Indeed, Arrb2 siRNAs decreased the expression of Wnt target genes in cells isolated from 12 of 18 tumors from Apc(Δ14/+) mice. In Apc(Min/+) cells, Arrb2 siRNAs completely reversed the increased Wnt activity and colony formation in soft agar induced by Apc siRNA treatment, whereas they did not affect these parameters in basal conditions or in cells expressing constitutively active β-catenin. We demonstrate that Arrb2 is essential for the initiation and growth of intestinal tumors displaying elevated Wnt pathway activity and identify a previously unsuspected molecular heterogeneity among tumors induced by truncating Apc mutations

Progastrin production transitions from Bmi1+/Prox1+ to Lgr5high cells during early intestinal tumorigenesis

International audienceProgastrin is an unprocessed soluble peptide precursor with a well-described tumor-promoting role in colorectal cancer. It is expressed at small levels in the healthy intestinal mucosa, and its expression is enhanced at early stages of intestinal tumor development, with high levels of this peptide in hyperplastic intestinal polyps being associated with poor neoplasm-free survival in patients. Yet, the precise type of progastrin-producing cells in the healthy intestinal mucosa and in early adenomas remains unclear. Here, we used a combination of immunostaining, RNAscope labelling and retrospective analysis of single cell RNAseq results to demonstrate that progastrin is produced within intestinal crypts by a subset of Bmi1+/Prox1+/LGR5low endocrine cells, previously shown to act as replacement stem cells in case of mucosal injury. In contrast, our findings indicate that intestinal stem cells, specified by expression of the Wnt signaling target LGR5, become the main source of progastrin production in early mouse and human intestinal adenomas. Collectively our results suggest that the previously identified feed-forward mechanisms between progastrin and Wnt signaling is a hallmark of early neoplastic transformation in mouse and human colonic adenomas

D-myo-inositol 1-phosphate as a surrogate of D-myo-inositol 1,4,5-tris phosphate to monitor G protein-coupled receptor activation.

International audiencePhospholipase C beta (PLC-beta)-coupled G protein-coupled receptor (GPCR) activities traditionally are assessed by measuring Ca2+ triggered by D-myo-inositol 1,4,5-trisphosphate (IP3), a PLC-beta hydrolysis product, or by measuring the production of inositol phosphate using cumbersome radioactive assays. A specific detection of IP3 production was also established using IP3 binding proteins. The short lifetime of IP3 makes this detection very challenging in measuring GPCR responses. Indeed, this IP3 rapidly enters the metabolic inositol phosphate cascade. It has been known for decades that lithium chloride (LiCl) leads to D-myo-inositol 1-phosphate accumulation on GPCR activation by inhibiting inositol monophosphatase, the final enzyme of the IP3 metabolic cascade. We show here that IP1 can be used as a surrogate of IP3 to monitor GPCR activation. We developed a novel homogeneous time-resolved fluorescence (HTRF) assay that correlates perfectly with existing methods and is easily amenable to high-throughput screening. The IP-One assay was validated on various GPCR models. It has the advantage over the traditional Ca2+ assay of allowing the measurement of inverse agonist activity as well as the analysis of PLC-beta activity in any nontransfected primary cultures. Finally, the high assay specificity for D-myo-inositol 1 monophosphate (IP1(1)) opens new possibilities in developing selective assays to study the functional roles of the various isoforms of inositol phosphates

Inhibition of DDR1‐BCR signalling by nilotinib as a new therapeutic strategy for metastatic colorectal cancer

Abstract The clinical management of metastatic colorectal cancer (mCRC) faces major challenges. Here, we show that nilotinib, a clinically approved drug for chronic myeloid leukaemia, strongly inhibits human CRC cell invasion in vitro and reduces their metastatic potential in intrasplenic tumour mouse models. Nilotinib acts by inhibiting the kinase activity of DDR1, a receptor tyrosine kinase for collagens, which we identified as a RAS‐independent inducer of CRC metastasis. Using quantitative phosphoproteomics, we identified BCR as a new DDR1 substrate and demonstrated that nilotinib prevents DDR1‐mediated BCR phosphorylation on Tyr177, which is important for maintaining β‐catenin transcriptional activity necessary for tumour cell invasion. DDR1 kinase inhibition also reduced the invasion of patient‐derived metastatic and circulating CRC cell lines. Collectively, our results indicate that the targeting DDR1 kinase activity with nilotinib may be beneficial for patients with mCRC

Circulating tumour cells from patients with colorectal cancer have cancer stem cell hallmarks in ex vivo culture

International audienceOBJECTIVE:Although counting of circulating tumour cells (CTC) has attracted a broad interest as potential markers of tumour progression and treatment response, the lack of functional characterisation of these cells had become a bottleneck in taking these observations to the clinic. Our objective was to culture these cells in order to understand them and exploit their therapeutic potential to the full.DESIGN:Here, hypothesising that some CTC potentially have cancer stem cell (CSC) phenotype, we generated several CTC lines from the blood of patients with advanced metastatic colorectal cancer (CRC) based on their self-renewal abilities. Multiple standard tests were then employed to characterise these cells.RESULTS:Our CTC lines self-renew, express CSC markers and have multilineage differentiation ability, both in vitro and in vivo. Patient-derived CTC lines are tumorigenic in subcutaneous xenografts and are also able to colonise the liver after intrasplenic injection. RNA sequencing analyses strikingly demonstrate that drug metabolising pathways represent the most upregulated feature among CTC lines in comparison with primary CRC cells grown under similar conditions. This result is corroborated by the high resistance of the CTC lines to conventional cytotoxic compounds.CONCLUSIONS:Taken together, our results directly demonstrate the existence of patient-derived colorectal CTCs that bear all the functional attributes of CSCs. The CTC culture model described here is simple and takes <1 month from blood collection to drug testing, therefore, routine clinical application could facilitate access to personalised medicine.CLINICAL TRIAL REGISTRATION:ClinicalTrial.gov NCT01577511

Pregnane X-receptor promotes stem cell-mediated colon cancer relapse

International audienceColorectal cancer lethality usually results from post-treatment relapse in the majority of stage II-IV patients, due to the enhanced resistance of Cancer Stem Cells (CSCs). Here, we show that the nuclear receptor Pregnane X Receptor (PXR, NR1I2), behaves as a key driver of CSC-mediated tumor recurrence. First, PXR is specifically expressed in CSCs, where it drives the expression of genes involved in self-renewal and chemoresistance. Clinically, high levels of PXR correlate with poor recurrence-free survival in a cohort of >200 stage II/III colorectal cancer patients treated with chemotherapy, for whom finding biomarkers of treatment outcome is an urgent clinical need. shRNA silencing of PXR increased the chemo-sensitivity of human colon CSCs, reduced their self-renewal and tumor-initiating potential, and drastically delayed tumor recurrence in mice following chemotherapy. This study uncovers PXR as a key factor for CSC self-renewal and chemoresistance and targeting PXR thus represents a promising strategy to minimize colorectal cancer relapse by selectively sensitizing CSCs to chemotherapy