Virginie Dangles-Marie

Spherical Cancer Models in Tumor Biology

Three-dimensional (3D) in vitro models have been used in cancer research as an intermediate model between in vitro cancer cell line cultures and in vivo tumor. Spherical cancer models represent major 3D in vitro models that have been described over the past 4 decades. These models have gained popularity in cancer stem cell research using tumorospheres. Thus, it is crucial to define and clarify the different spherical cancer models thus far described. Here, we focus on in vitro multicellular spheres used in cancer research. All these spherelike structures are characterized by their well-rounded shape, the presence of cancer cells, and their capacity to be maintained as free-floating cultures. We propose a rational classification of the four most commonly used spherical cancer models in cancer research based on culture methods for obtaining them and on subsequent differences in sphere biology: the multicellular tumor spheroid model, first described in the early 70s and obtained by culture of cancer cell lines under nonadherent conditions; tumorospheres, a model of cancer stem cell expansion established in a serum-free medium supplemented with growth factors; tissue-derived tumor spheres and organotypic multicellular spheroids, obtained by tumor tissue mechanical dissociation and cutting. In addition, we describe their applications to and interest in cancer research; in particular, we describe their contribution to chemoresistance, radioresistance, tumorigenicity, and invasion and migration studies. Although these models share a common 3D conformation, each displays its own intrinsic properties. Therefore, the most relevant spherical cancer model must be carefully selected, as a function of the study aim and cancer type.

Evaluating Patient-Derived Colorectal Cancer Xenografts as Preclinical Models by Comparison with Patient Clinical Data

Development of targeted therapeutics required translationally relevant preclinical models with well-characterized cancer genome alterations. Here, by studying 52 colorectal patient-derived tumor xenografts (PDX), we examined key molecular alterations of the IGF2-PI3K and ERBB-RAS pathways and response to cetuximab. PDX molecular data were compared with that published for patient colorectal tumors in The Cancer Genome Atlas. We demonstrated a significant pattern of mutual exclusivity of genomic abnormalities in the IGF2-PI3K and ERBB-RAS pathways. The genomic anomaly frequencies observed in microsatellite stable PDX reproduce those detected in nonhypermutated patient tumors. We found frequent IGF2 upregulation (16%), which was mutually exclusive with IRS2, PIK3CA, PTEN, and INPP4B alterations, supporting IGF2 as a potential drug target. In addition to maintaining the genomic and histologic diversity, correct preclinical models need to reproduce drug response observed in patients. Responses of PDXs to cetuximab recapitulate also clinical data in patients, with partial or complete response in 15% (8 of 52) of PDXs and response strictly restricted to KRAS wild-type models. The response rate reaches 53% (8 of 15) when KRAS, BRAF, and NRAS mutations are concomitantly excluded, proving a functional cross-validation of predictive biomarkers obtained retrospectively in patients. Collectively, these results show that, because of their clinical relevance, colorectal PDXs are appropriate tools to identify both new targets, like IGF2, and predictive biomarkers of response/resistance to targeted therapies.

Vasculature analysis of patient derived tumor xenografts using species-specific PCR assays: evidence of tumor endothelial cells and atypical VEGFA-VEGFR1/2 signalings.

BackgroundTumor endothelial transdifferentiation and VEGFR1/2 expression by cancer cells have been reported in glioblastoma but remain poorly documented for many other cancer types.MethodsTo characterize vasculature of patient-derived tumor xenografts (PDXs), largely used in preclinical anti-angiogenic assays, we designed here species-specific real-time quantitative RT-PCR assays. Human and mouse PECAM1/CD31, ENG/CD105, FLT1/VEGFR1, KDR/VEGFR2 and VEGFA transcripts were analyzed in a large series of 150 PDXs established from 8 different tumor types (53 colorectal, 14 ovarian, 39 breast and 15 renal cell cancers, 6 small cell and 5 non small cell lung carcinomas, 13 cutaneous melanomas and 5 glioblastomas) and in two bevacizumab-treated non small cell lung carcinomas xenografts.ResultsAs expected, mouse cell proportion in PDXs -evaluated by quantifying expression of the housekeeping gene TBP- correlated with all mouse endothelial markers and human VEGFA RNA levels. More interestingly, we observed human PECAM1/CD31 and ENG/CD105 expression in all tumor types, with higher rate in glioblastoma and renal cancer xenografts. Human VEGFR expression profile varied widely depending on tumor types with particularly high levels of human FLT1/VEGFR1 transcripts in colon cancers and non small cell lung carcinomas, and upper levels of human KDR/VEGFR2 transcripts in non small cell lung carcinomas. Bevacizumab treatment induced significant low expression of mouse Pecam1/Cd31, Eng/Cd105, Flt1/Vegfr1 and Kdr/Vefr2 while the human PECAM1/CD31 and VEGFA were upregulated.ConclusionsTaken together, our results strongly suggest existence of human tumor endothelial cells in all tumor types tested and of both stromal and tumoral autocrine VEGFA-VEGFR1/2 signalings. These findings should be considered when evaluating molecular mechanisms of preclinical response and resistance to tumor anti-angiogenic strategies.

Array comparative genomic hybridization identifies high level of PI3K/Akt/mTOR pathway alterations in anal cancer recurrences

Genomic alterations of anal squamous cell carcinoma (ASCC) remain poorly understood due to the rarity of this tumor. Array comparative genomic hybridization and targeted gene sequencing were performed in 49 cases of ASCC. The most frequently altered regions (with a frequency greater than 25%) were 10 deleted regions (2q35, 2q36.3, 3p21.2, 4p16.3, 4p31.21, 7q36.1, 8p23.3, 10q23.2, 11q22.3, and 13q14.11) and 8 gained regions (1p36.33, 1q21.1, 3q26.32, 5p15.33, 8q24.3, 9q34.3, 16p13.3, and 19p13.3). The most frequent minimal regions of deletion (55%) encompassed the 11q22.3 region containing ATM, while the most frequent minimal regions of gain (57%) encompassed the 3q26.32 region containing PIK3CA. Recurrent homozygous deletions were observed for 5 loci (ie, TGFR2 in 4 cases), and recurrent focal amplifications were observed for 8 loci (ie, DDR2 and CCND1 in 3 cases, respectively). Several of the focal amplified genes are targets for specific therapies. Integrated analysis showed that the PI3K/Akt/mTOR signaling pathway was the pathway most extensively affected, particularly in recurrences compared to treatment‐naive tumors (64% vs 30%; P = .017). In patients with ASCC recurrences, poor overall survival (OS) was significantly correlated with a large number of altered regions (P = .024). These findings provide insight into the somatic genomic alterations in ASCC and highlight the key role of the druggable PI3K/Akt/mTOR signaling pathway.

Abstract IA05: PDX in colorectal cancer and carcinoma of the anal canal

Through 2 examples of digestive tumors, (colorectal cancer, “CRC”, and anal squamous cell carcinoma, “ASCC”), we will present the development and use of patient-derived tumor xenografts. Colorectal cancer remains a major cause of mortality worldwide and CRC patient death is generally attributable to metastasis development. By contrast, ASCC is a rare tumor ( We will illustrate the capacity of these PDXs to recapitulate the intratumoral clonal heterogeneity, gene expression profile, key molecular alterations, histology and drug response of the patient tumors through passages in mice. In addition to these now well-known features depicted in a large number of tumor types, we will discuss about practical situations (orthotopic engraftment, impact of mouse host, criteria for measuring drug efficacy). Finally, we will report alternative methods as ex vivo tumor spheres we have developed. Citation Format: Virginie Dangles-Marie. PDX in colorectal cancer and carcinoma of the anal canal. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr IA05.

Abstract LB-34: Similar PI3K and RTK-Ras status in patient derived colorectal cancer-xenografts and patients.

There is a recent increase in the use of patient-derived tumor xenografts (PDX) engrafted into immunodeficient mice as improved preclinical models of patient tumors. An important component in the validation of disease-specific PDXs for clinical relevance is comparing the genomic alterations and the response to standard agents. The CReMEC collection of 54 colorectal PDX has been recently reported to mimic the clinical situation for histopathological and molecular diversity of colorectal cancer. We further analyze here this colorectal PDX collection in regard to robust human patient molecular features: 1) Alterations in both PI3K and RTK-Ras pathways; 2) Role of oncogenic activation of EGFR-Ras downstream signaling on response to cetuximab. The Cancer Genome Atlas Network (TCGA) newly reported a large genome-scale analysis of 276 colorectal cancer tissue samples, showing common genetic alterations in the PI3K and RTK-RAS pathways, with mutual exclusions in the PI3K pathway. The analysis of the PDX panel by CGH array, RNA expression (microarray, real-time qRT-PCR) and exome sequencing confirmed activation with mutual exclusion in PI3K pathway (IGF2 focal amplification/overexpression; IRS2 overexpression, mutation of PIK3CA, PIK3R1 and PTEN homozygous deletion). In RTK-Ras pathway, frequencies of genomic abnormalities (ERBB2 mutation/amplification; mutation of ERBB3, NRAS, KRAS and BRAF) in PDXs are fully in line with the TCGA patient collection. The response to cetuximab of 52 subcutaneous engrafted PDX (including 24 KRAS mutated PDX) has been analyzed according to translated clinical criteria: xenograft regression as been defined as a partial response (decrease of at least 70% of the tumor volume measured at the beginning of the treatment) or as a complete response. In this unselected population, tumor regression occurred in 8 out of 52 cases (15%); all were KRAS wild type tumors. The percentage of responders was enriched up to 30% (7/23) when PTEN deletion and mutations of KRAS, BRAF, and PIK3CA are concomitantly excluded. These results completely fit with recent publication of data in patients treated with anti-EGFR antibodies. Taken together, these results demonstrate that colorectal PDXs are representative clinical colorectal tumor models. They also underline their interest as appropriate tools to identify and test new targeted therapeutics. Citation Format: Manoel Nunes, Louis-Bastien Weiswald, Patricia Vrignaud, Sophie Vacher, Edouard Turlotte, Sophie Richon, Dominique Bellet, Sergio Roman-Roman, Ivan Bieche, Virginie Dangles-Marie. Similar PI3K and RTK-Ras status in patient derived colorectal cancer-xenografts and patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-34. doi:10.1158/1538-7445.AM2013-LB-34

Abstract LB-111: Mastospheres as a new 3D ex vivo breast cancer microtumors for preclinical drug testing

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL There is increasing evidence that three-dimensional (3D) tissue culture technologies have many uses within the preclinical assays in cancer. The 3D tumor model accurately reproduces the in vivo tumor phenotype and represents an additional tool for studying tumor biology and allowing better preclinical evaluation of anticancer drugs. One of the used models involves small tumor aggregates, termed spheres, that are obtained from mechanical tumor dissociation and that have shown the superiority of 3D culture over standard two-dimensional cell culture for mimicking the tumor biology and drug response observed in vivo. The aim of this study was to generate new ex vivo 3D models from breast (BC) cancer xenografts established from patients’ tumor fragments. These ex vivo mastospheres are easily obtained from mechanically dissociated fresh human BC tissue xenografted in Nude mice, in a similar way as colospheres from human colon cancer (Weiswald et al, Br J Cancer 2009, 101:473). In contrast to mammospheres described in the literature, 3D mastospheres are obtained from tumor fragments, without enzymatic tumor dissociation, without matrix substratum and in SVF supplemented medium. From a large panel of patient-derived BC xenografts already well characterized (Marangoni et al, Clin Cancer Res 2007, 13:3989; Cottu et al, Breast Cancer Res Treat 2011; Reyal et al, Breast Cancer Res 2012, 14:R11), we get mastospheres from 26 out of 36 (72%) xenografts. Mastosphere formation is scored on day 1 after culture according to the number of spheres/mg of dissociated xenograft tissue. Within mastospheres, we clearly distinguish 3 distinct morphologies: round, grape-like and aggregates. Histological analyses show also that mastospheres were formed only with proliferating cancer cells. In a reproducible way, dissociation of a given xenograft leads to a similar score and to a similar morphology. All BC subtypes (luminal, triple negative and HER2+ tumors) give rise to mastospheres. We noted that the 5 out of 5 xenografts (100%) able to metastasise in Nude mice (lung metastasis) form mastospheres, suggesting that the capacity to give mastospheres could be related to tumour aggressiveness, as already reported with colospheres and colon cancer. These different features prompted us to test the potential of these mastospheres in chemosensitivity assays. We first demonstrated that mastospheres can be kept viable a couple of days, consistent with ex vivo assay aim. For this purpose, we tested the sensitivity to cisplatin in two xenograft models with different in vivo response. We found that the paired mastospheres tested in viability assays mimicked these different response profiles (mean of IC50: 1.2 µM versus 15.5 µM). In conclusion, according to these preliminary data, mastospheres deserve additional investigation because of their interest as new ex vivo microtumour model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-111. doi:1538-7445.AM2012-LB-111

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.