Cellules endothéliales circulantes et progéniteurs endothéliaux circulants (biomarqueurs de l'angiogénèse tumorale et des traitements anti-angiogéniques et anti-vasculaires.)

Malgré l efficacité thérapeutique avérée des agents anti-angiogéniques et des agents anti-vasculaires (VDA), le mécanisme d action précis des stratégies ciblant les vaisseaux sanguins tumoraux, les raisons de leur efficacité ainsi que les mécanismes de résistance à ces drogues sont encore mal compris. Il est rapidement apparu essentiel d identifier des biomarqueurs capables de refléter l angiogénèse tumorale ou les effets sur la vascularisation tumorale de ces traitements. Compte tenu de leur importance dans des pathologies vasculaires, les cellules endothéliales matures circulantes (CEC) et les progéniteurs endothéliaux circulants (CEP) ont d emblée été pressenties comme des candidats intéressants pour être des biomarqueurs de réponse aux stratégies ciblant la vascularisation tumorale. Nous avons exploré l intérêt de ces cellules en tant que biomarqueurs de l angiogénèse dans des tumeurs pédiatriques, et leur rôle en tant que biomarqueurs de traitement par des agents anti-angiogéniques chez des sujets adultes atteints de cancer. Ces travaux ont mis en lumière l intérêt des CEP et ont été à la source d un travail plus mécanistique où nous avons étudié dans différents modèles murins le rôle des CEC et CEP dans le mécanisme d action des agents anti-vasculaires et plus particulièrement le rôle fonctionnel des CEP dans la résistance à ces molécules. Par des stratégies d association d agents anti-angiogéniques aux VDA destinées à inhiber les CEP, nous montrons l augmentation de l activité anti-tumorale des VDA et offrons un rationnel mécanistique pour optimiser les schémas thérapeutiques actuels des traitements anti-vasculaires. Nos données apportent des arguments en faveur du rôle potentiel de ces cellules en tant que biomarqueurs de l angiogénèse, des traitements anti-angiogéniques et de la résistance aux traitements anti-vasculaires.Despite their therapeutic impact and clinical benefit, the mecanisms of action of anti-angiogenic agents and vascular disrupting agents (VDA), the reasons for their efficacy as well as the mechanisms underlying resistance to these drugs are not fully understood. Thus, identifying surrogate biomarkers of tumor angiogenesis and of the effects of these new therapeutic agents targeting tumor blood vessels has become a crucial objective. Because of their importance in vascular diseases, mature circulating endothelial cells (CEC) and circulating endothelial progenitor cells (CEP) were suggested to be potential candidate biomarkers of disease response and relapse to vascular targeting strategies. We investigated the role of these cells as biomarkers of tumor angiogenesis in pediatric solid tumors, as well as biomarkers of response to anti-angiogenic therapies in adult cancer patients. By revealing the particularly important role of CEP, these initial studies led to a more mechanistic study in which the cellular and molecular effects of a VDA were evaluated with regard to CEC and CEP in different mouse models; in particular, the catalytic role of CEP was explored as a mechanism of resistance to VDA. By combining anti-angiogenic agents aimed to inhibit CEP mobilized by the VDA, we demonstrate an increase in the anti-tumor activity of the VDA and offer a mechanistic rational to optimize VDA-based therapeutic strategies. Our data support the role of CEC and CEP as biomarkers of angiogenesis, of anti-angiogenic strategies and of resistance to vascular-disrupting therapies.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Tumor Evolution and Therapeutic Choice Seen through a Prism of Circulating Tumor Cell Genomic Instability

Circulating tumor cells (CTCs) provide an accessible tool for investigating tumor heterogeneity and cell populations with metastatic potential. Although an in-depth molecular investigation is limited by the extremely low CTC count in circulation, significant progress has been made recently in single-cell analytical processes. Indeed, CTC monitoring through molecular and functional characterization may provide an understanding of genomic instability (GI) molecular mechanisms, which contribute to tumor evolution and emergence of resistant clones. In this review, we discuss the sources and consequences of GI seen through single-cell analysis of CTCs in different types of tumors. We present a detailed overview of chromosomal instability (CIN) in CTCs assessed by fluorescence in situ hybridization (FISH), and we reveal utility of CTC single-cell sequencing in identifying copy number alterations (CNA) oncogenic drivers. We highlight the role of CIN in CTC-driven metastatic progression and acquired resistance, and we comment on the technical obstacles and challenges encountered during single CTC analysis. We focus on the DNA damage response and depict DNA-repair-related dynamic biomarkers reported to date in CTCs and their role in predicting response to genotoxic treatment. In summary, the suggested relationship between genomic aberrations in CTCs and prognosis strongly supports the potential utility of GI monitoring in CTCs in clinical risk assessment and therapeutic choice

Microfluidic magnetic cell sorting system for cancer diagnosis

This project is aimed at developing a new integrated microfluidic system for high throughput cell sorting and analysis. This micro-device will be able to sort rare population of cells, like circulating tumor cells (CTC) in whole blood, and analyze them by bright field and fluorescence microscopy at high magnification. Evaluating the concentration of CTC in blood will have a strong diagnosis and prognosis value, for evaluating risks of metastases and tumor cells dissemination. Cell sample flows through an array of magnetic columns that are coated with antibodies. These antibodies are specific of membrane protein that are expressed by the target cells and allow their capture in chip. Once they are trapped on columns, their shape and the expression of other proteins are observed by microscopy. As a proof of concept, Raji cells (B lymphocytes, CD19+) mixed with Jurkat cells (T lymphocytes, CD19-) with an initial concentration of 1/1000 are sorted in an array of magnetic beads functionalized with anti-CD19 antibodies. 500μL of sample are analyzed within 1 hour with a global capture yield of 55 +/- 10 %. The captured cells are then stained with immunofluorescent probes which provide membrane (anti- CD19-AlexaFluor 488) and nucleus (Hoechst) imaging. Imagining can be performed at high resolution microscopy, including 100X oil immersion objectives

Circulating Tumor Cells with Aberrant ALK-Copy Number Predicts Progression-Free Survival to Crizotinib in ALK-Rearranged Non-Small-Cell Lung Cancer Patients

Abstract The duration and magnitude of clinical response are unpredictable in ALK-rearranged non–small cell lung cancer (NSCLC) patients treated with crizotinib, although all patients invariably develop resistance. Here, we evaluated whether circulating tumor cells (CTC) with aberrant ALK-FISH patterns [ALK-rearrangement, ALK-copy number gain (ALK-CNG)] monitored on crizotinib could predict progression-free survival (PFS) in a cohort of ALK-rearranged patients. Thirty-nine ALK-rearranged NSCLC patients treated with crizotinib as first ALK inhibitor were recruited prospectively. Blood samples were collected at baseline and at an early time-point (2 months) on crizotinib. Aberrant ALK-FISH patterns were examined in CTCs using immunofluorescence staining combined with filter-adapted FISH after filtration enrichment. CTCs were classified into distinct subsets according to the presence of ALK-rearrangement and/or ALK-CNG signals. No significant association between baseline numbers of ALK-rearranged or ALK-CNG CTCs and PFS was observed. However, we observed a significant association between the decrease in CTC number with ALK-CNG on crizotinib and a longer PFS (likelihood ratio test, P = 0.025). In multivariate analysis, the dynamic change of CTC with ALK-CNG was the strongest factor associated with PFS (HR, 4.485; 95% confidence interval, 1.543–13.030, P = 0.006). Although not dominant, ALK-CNG has been reported to be one of the mechanisms of acquired resistance to crizotinib in tumor biopsies. Our results suggest that the dynamic change in the numbers of CTCs with ALK-CNG may be a predictive biomarker for crizotinib efficacy in ALK-rearranged NSCLC patients. Serial molecular analysis of CTC shows promise for real-time patient monitoring and clinical outcome prediction in this population. Cancer Res; 77(9); 2222–30. ©2017 AACR.</jats:p