Sophie Richon

Establishment of Human Colon Cancer Cell Lines from Fresh Tumors versus Xenografts: Comparison of Success Rate and Cell Line Features

Obtaining representative human colon cancer cell lines from fresh tumors is technically difficult. Using 32 tumor fragments from patients with colon cancer, the present study shows that prior xenograft leads to more efficient cell line establishment compared with direct establishment from fresh tumors (P < 0.05). From 26 tumor specimens, we successfully established 20 tumor xenografts in nude mice (77%); among 19 of these xenografts, 9 (47%) led to cell lines, including four from liver metastases. Only 3 of 31 tumor specimens (9.7%) grew immediately in vitro, and all were derived from primary tumors. To compare major phenotypic and genotypic characteristics of human colon cancer cell lines derived from the same tumor fragment using two protocols, the two pairs of cell lines obtained from 2 of 32 tumor fragments were extensively studied. They displayed similar morphology and were able to form compact spheroids. Chemosensitivity to 5-fluorouracil, CPT11, and L-OHP differed between cell lines obtained from patient tumors and those derived from xenografts. Matched cell lines shared a common core of karyotype alterations and distinctive additional chromosomal aberrations. Expression levels of genes selected for their role in oncogenesis evaluated by real-time quantitative PCR were found to be statistically correlated whatever the in vitro culture model used. In conclusion, xenotransplantation in mice of tumor fragments before establishment of cell lines enables generation of more novel human cancer cell lines for investigation of colon cancer cell biology, opening up the opportunity of reproducing the diversity of this disease.

Newly characterised ex vivo colospheres as a three-dimensional colon cancer cell model of tumour aggressiveness

Background:New models continue to be required to improve our understanding of colorectal cancer progression. To this aim, we characterised in this study a three-dimensional multicellular tumour model that we named colospheres, directly obtained from mechanically dissociated colonic primary tumours and correlated with metastatic potential.Methods:Colorectal primary tumours (n=203) and 120 paired non-tumoral colon mucosa were mechanically disaggregated into small fragments for short-term cultures. Features of tumours producing colospheres were analysed. Further characterisation was performed using colospheres, generated from a human colon cancer xenograft, and spheroids, formed on agarose by the paired cancer cell lines.Results:Colospheres, exclusively formed by viable cancer cells, were obtained in only 1 day from 98 tumours (47%). Inversely, non-tumoral colonic mucosa never generated colospheres. Colosphere-forming capacity was statistically significantly associated with tumour aggressiveness, according to AJCC stage analysis. Despite a close morphology, colospheres displayed higher invasivity than did spheroids. Spheroids and colospheres migrated into Matrigel but matrix metalloproteinase (MMP)-2 and MMP-9 activity was detected only in colospheres. Mouse subrenal capsule assay revealed the unique tumorigenic and metastatic phenotype of colospheres. Moreover, colospheres and parental xenograft reproduced similar CD44 and CD133 expressions in which CD44+ cells represented a minority subset of the CD133+ population.Conclusion:The present colospheres provide an ex vivo three-dimensional model, potentially useful for studying metastatic process.

Gene expression profiles of bladder cancers: evidence for a striking effect of in vitro cell models on gene patterns

In order to assess the effect of in vitro models on the expression of key genes known to be implicated in the development or progression of cancer, we quantified by real-time quantitative PCR the expression of 28 key genes in three bladder cancer tissue specimens and in their derived cell lines, studied either as one-dimensional single cell suspensions, two-dimensional monolayers or three-dimensional spheroids. Global analysis of gene expression profiles showed that in vitro models had a dramatic impact upon gene expression. Remarkably, quantitative differences in gene expression of 2–63-fold were observed in 24 out of 28 genes among the cell models. In addition, we observed that the in vitro model which most closely mimicked in vivo mRNA phenotype varied with both the gene and the patient. These results provide evidence that mRNA expression databases based on cancer cell lines, which are studied to provide a rationale for selection of therapy on the basis of molecular characteristics of a patients tumour, must be carefully interpreted.

Impact of human bladder cancer cell architecture on autologous T-lymphocyte activation

To investigate the influence of tumor cell architecture on T‐cell activation, we used an autologous human model based on 2 bladder tumor cell lines as targets for cytotoxic tumor‐infiltrating lymphocytes (TILs). These tumor cell lines were grown in vitro as either standard 2‐dimensional (2D) monolayers or 3‐dimensional (3D) spheroids. T‐cell activation was determined by measuring the production of three major cytokines (tumor necrosis factor, granulocyte/macrophage colony‐stimulating factor and interferon‐γ), known to be secreted by most activated TILs. Changes in the architecture of target cells from 2D to 3D induced a dramatic decrease in their capacity for stimulating TILs. Interestingly, neither TIL infiltration nor MHC class I, B7.1 costimulatory or lymphocyte function‐associated factor‐3 adhesion molecule downregulation played a major role in this decrease. These findings demonstrate that tumor architecture has a major impact on T‐cell activation and might be implicated in the escape of tumor cells from the immune system.

Mutational analysis of anal cancers demonstrates frequent PIK3CA mutations associated with poor outcome after salvage abdominoperineal resection

Background:A better understanding of the molecular profile of anal squamous cell carcinomas (ASCCs) is necessary to consider new therapeutic approaches, and the identification of prognostic and predictive factors for response to treatment.Methods:We retrospectively analysed tumours from ASCC patients for mutational analysis of KRAS, NRAS, HRAS, BRAF, PIK3CA, MET, TP53 and FBXW7 genes by HRM and Sanger sequencing analysis.Results:Specimens from 148 patients were analysed: 96 treatment-naive tumours and 52 recurrences after initial radiotherapy (RT) or chemoradiotherapy (CRT). Mutations of KRAS, PIK3CA, FBXW7 and TP53 genes were present in 3 (2.0%), 30 (20.3%), 9 (6.1%) and 7 tumours (4.7%), respectively. The distribution of the mutations was similar between treatment-naive tumours and recurrences, except for TP53 mutations being more frequent in recurrences (P=0.0005). In patients treated with abdominoperineal resection (APR) after relapse (n=38, median follow-up of 18.2 years), overall survival (OS) was significantly correlated with HPV16 status (P=0.048), gender (P=0.045) and PIK3CA mutation (P=0.037). The PIK3CA status retained its prognostic significance in Cox multivariate regression analysis (P=0.025).Conclusions:Our study identified PIK3CA mutation as an independent prognostic factor in patients who underwent APR for ASCC recurrence, suggesting a potential benefit from adjuvant treatment and the evaluation of targeted therapies with PI3K/Akt/mTor inhibitors in PIK3CA-mutated patients.

Clinical value of R-spondins in triple-negative and metaplastic breast cancers

Background:RSPO ligands, activators of the Wnt/β-catenin pathway, are overexpressed in different cancers. The objective of this study was to investigate the role of RSPOs in breast cancer (BC).Methods:Expression of RSPO and markers of various cancer pathways were measured in breast tumours and cell lines by qRT–PCR. The effect of RSPO on the Wnt/β-catenin pathway activity was determined by luciferase assay, western blotting, and qRT–PCR. The effect of RSPO2 inhibition on proliferation was determined by using RSPO2 siRNAs. The effect of IWR-1, an inhibitor of the Wnt/β-catenin pathway, was examined on the growth of an RSPO2-positive patient-derived xenograft (PDX) model of metaplastic triple-negative BC.Results:We detected RSPO2 and RSPO4 overexpression levels in BC, particularly in triple-negative BC (TNBC), metaplastic BC, and triple-negative cell lines. Various mechanisms could account for this overexpression: presence of fusion transcripts involving RSPO, and amplification or hypomethylation of RSPO genes. Patients with RSPO2-overexpressing tumours have a poorer metastasis-free survival (P=3.6 × 10−4). RSPO2 and RSPO4 stimulate Wnt/β-catenin pathway activity. Inhibition of RSPO expression in a TN cell line inhibits cell growth, and IWR-1 significantly inhibits the growth of an RSPO2-overexpressing PDX.Conclusions:RSPO overexpression could therefore be a new prognostic biomarker and therapeutic target for TNBC.

Abstract A9: Establishment and characterization of residual breast cancer patient-derived xenografts resistant to neo-adjuvant therapy.

Background: In HER2 positive and triple-negative breast cancer subgroups, residual disease after neoadjuvant therapy is associated with higher risk of metastatic recurrence compared to patients achieving a pathological complete response. Residual tumor analysis after neoadjuvant treatment is a major and under-explored field to identify resistance mechanisms. To develop patient-derived xenografts (PDX) of residual breast cancer we started a program of residual tumor engraftment in nude mice, following the same procedures previously published for PDX of human breast cancer (Marangoni et al, 2007 and Reyal et al, 2012). Methods: 26 residual breast tumors and 2 residual metastatic axillary lymph nodes were engrafted in swiss nude mice immediately after surgery. Expression of Ki67, HER2, PTEN, P-AKT, P-S6, MET, RET and KIT were analyzed in xenografts by immunohistochemistry, western blot and RT-PCR analyses. Brain, lungs, liver and bones of xenografts were systematically formalin-fixed to search for human metastasis. The in vivo drug response of established xenografts was determined for the following treatments: adryamicin+cyclophosphamide (AC), docetaxel, capecitabine, cisplatin, irinotecan, everolimus, trastuzumab and lapatinib (for the HER2+ PDX). PDX tumors were additionally mechanically dissociated to establish cell lines. Results: Seven PDX were established (tumor take of 25%), 5 triple-negative and 2 HER2+. Six out of seven PDX were metastatic in the lungs. Two xenografts were established from lymph node metastasis. The in vivo drug responses were concordant with the response to neo-adjuvant treatments in patients. Histological analyses showed that xenografts’ tumors recapitulated the patients’ tumor morphology. Residual tumor xenografts expressed high level of Ki67 protein and tumor latency during the first tumor passages was found to be shorter when compared to tumor latency of non pre-treated breast cancers. In 5/5 triple-negative breast cancer PDX the PTEN protein was lost and the PI3 kinase pathway activated. The mTOR inhibitor Everolimus was tested in 2 triple-negative PDX: one was resistant and one was responding, with a tumor growth inhibition of 80%. Triple-negative PDX show expression of “druggable” tyrosin kinase receptors (MET, RET, KIT) providing relevant models to test new target therapies in these models. One cell line was established from a highly metastatic triple-negative breast cancer xenograft. When re-injected into mice, the cell line was tumorigenic, however the tumor architecture was changed and the xenograft was not metastatic. Conclusions: we have established a panel of metastatic PDX models of breast cancer resistant to neo-adjuvant therapies. These models provide a valuable preclinical tool to investigate mechanisms of resistance to neo-adjuvant treatments and for the preclinical testing of new targeted agents. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A9. Citation Format: Elisabetta Marangoni, Dalila Labiod, Franck Assayag, Rania El Botty, Rana Hatem, Sophie Richon, Sophie Chateau-Joubert, Marine Carlus, Helene Bonsang-Kitzis, Alice Pinheiro, Cecile Laurent, Ivan Bieche, Fabien Reyal. Establishment and characterization of residual breast cancer patient-derived xenografts resistant to neo-adjuvant therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A9.

Abstract LB-161: Colospheres: 3D ex vivo microtumors with more aggressive phenotype than original human colon cancer tissues

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The ex vivo colospheres are a newly characterised three-dimensional colon cancer multicellular model, derived from mechanically dissociated human colon cancer tissue (Br J Cancer 2009, 101:473-82). We have previously demonstrated that this short term culture model is exclusively formed by cancer cells and associated with tumor aggressiveness. To further investigate the potential interest of colospheres as micrometastasis or cancer stem cell model, we used 3 human colon cancer xenografts directly established from 3 patient colon adenocarcinoma in Nude mice (Cancer Res 2007, 67:398-407). These xenograft tissues are able to give rise to numerous colospheres in only 3 days after mechanical dissociation. This approach allows working with reproducible biological tumor material, in which all human part is known to be cancer cells. Xenograft tissues and derived colospheres were collected for gene expression study. Expression of 78 genes, involved in stemness, proliferation, epithelial-to-mesenchymal transition and metastasis, has been studied using real-time RT-PCR. All the probes used are human specific and do not cross with mouse genome. Consequently, the comparison of gene expression profiles corresponds to the comparison of gene expression in colosphere-forming cells versus that in the cancer cell counterpart from xenograft tissue. Gene expression clustering analysis clearly showed that for the 3 colon adenocarcinoma, colospheres matched with their parent xenografts. This first point is important because it demonstrated 1) the lack of ex vivo culture artefact (which would have led to classification into 2 groups: all xenografts opposed to all colospheres); 2) the relevance of colospheres for mimicking in vivo tumor cells. Nevertheless, in all 3 pairs xenograft/colospheres, 3 genes were found overexpressed: ALDH1, KLF4 and PLAUR. This overexpression has to be put in line with high tumorigenicity of these colospheres when injected into mice (Br J Cancer 2009, 101:473-82). Gene expression increase will be confirmed at protein level. To gain also information about location of the cells expressing ALDH1, KLF4 and PLAUR, we developed an immunostaining protocol for confocal microscopy and in situ protein detection (BMC Cancer, in revision). In conclusion, these preliminary data underline the interest of colospheres as ex vivo microtumor model with cancer initiating-cell functions. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-161.