Louis-Bastien Weiswald

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.

Characterization of a Large Panel of Patient-Derived Tumor Xenografts Representing the Clinical Heterogeneity of Human Colorectal Cancer

Purpose: Patient-derived xenograft models are considered to represent the heterogeneity of human cancers and advanced preclinical models. Our consortium joins efforts to extensively develop and characterize a new collection of patient-derived colorectal cancer (CRC) models. Experimental Design: From the 85 unsupervised surgical colorectal samples collection, 54 tumors were successfully xenografted in immunodeficient mice and rats, representing 35 primary tumors, 5 peritoneal carcinoses and 14 metastases. Histologic and molecular characterization of patient tumors, first and late passages on mice includes the sequence of key genes involved in CRC (i.e., APC, KRAS, TP53), aCGH, and transcriptomic analysis. Results: This comprehensive characterization shows that our collection recapitulates the clinical situation about the histopathology and molecular diversity of CRC. Moreover, patient tumors and corresponding models are clustering together allowing comparison studies between clinical and preclinical data. Hence, we conducted pharmacologic monotherapy studies with standard of care for CRC (5-fluorouracil, oxaliplatin, irinotecan, and cetuximab). Through this extensive in vivo analysis, we have shown the loss of human stroma cells after engraftment, observed a metastatic phenotype in some models, and finally compared the molecular profile with the drug sensitivity of each tumor model. Through an experimental cetuximab phase II trial, we confirmed the key role of KRAS mutation in cetuximab resistance. Conclusions: This new collection could bring benefit to evaluate novel targeted therapeutic strategies and to better understand the basis for sensitivity or resistance of tumors from individual patients. Clin Cancer Res; 18(19); 5314–28. ©2012 AACR.

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.

The Wnt Antagonist Dickkopf-1 Increases Endothelial Progenitor Cell Angiogenic Potential

Objective—To determine the role of Wnt antagonist Dickkopf (DKK) 1 in human endothelial colony-forming cells (ECFCs) in view of the emerging importance of Wnt pathways in vascular biology. Methods and Results—Endothelial progenitor cells have been proposed to be crucial in tumor neovascularization. Recombinant DKK1 has been tested in ECFC angiogenic properties in vitro. DKK1 enhanced ECFC proliferation and the capacity of ECFCs to form pseudotubes in Matrigel. These effects have been attributed to enhancement of vascular endothelial growth factor receptor 2, SDF-1, and CXCR4. DKK1 gene silencing has been realized on ECFCs and mesenchymal stem cells, and we found that DKK1 silencing in the 2 cell types decreased their angiogenic potential. We then examined the possible role of DKK1 in tumor neovasculogenesis and found that blood vessels of breast cancer tissues expressed DKK1 far more strongly in human breast tumors than in normal breast tissues. By studying 62 human breast tumors, we found a significant positive correlation between DKK1 expression and von Willebrand factor. In vivo, DKK1 strongly enhanced the vascularization of Matrigel plugs and increased tumor size in a xenograft model of human breast carcinoma in nude mice. Conclusion—DKK1 enhances angiogenic properties of ECFCs in vitro and is required for ECFC and mesenchymal stem cell angiogenic phenotypes in vivo. DKK1 also increases tumoral angiogenesis. Thus, we demonstrated a major role of DKK1 in angiogenic processes.

Exosomes confer pro-survival signals to alter the phenotype of prostate cells in their surrounding environment

Prostate cancer (PCa) is the most frequently diagnosed cancer in men. Current research on tumour-related extracellular vesicles (EVs) suggests that exosomes play a significant role in paracrine signaling pathways, thus potentially influencing cancer progression via multiple mechanisms. In fact, during the last decade numerous studies have revealed the role of EVs in the progression of various pathological conditions including cancer. Moreover, differences in the proteomic, lipidomic, and cholesterol content of exosomes derived from PCa cell lines versus benign prostate cell lines confirm that exosomes could be excellent biomarker candidates. As such, as part of an extensive proteomic analysis using LCMS we previously described a potential role of exosomes as biomarkers for PCa. Current evidence suggests that uptake of EVs into the local tumour microenvironment encouraging us to further examine the role of these vesicles in distinct mechanisms involved in the progression of PCa and castration resistant PCa. For the purpose of this study, we hypothesized that exosomes play a pivotal role in cell-cell communication in the local tumour microenvironment, conferring activation of numerous survival mechanisms during PCa progression and development of therapeutic resistance. Our in vitro results demonstrate that PCa derived exosomes significantly reduce apoptosis, increase cancer cell proliferation and induce cell migration in LNCaP and RWPE-1 cells. In conjunction with our in vitro findings, we have also demonstrated that exosomes increased tumor volume and serum PSA levels in vivo when xenograft bearing mice were administered DU145 cell derived exosomes intravenously. This research suggests that, regardless of androgen receptor phenotype, exosomes derived from PCa cells significantly enhance multiple mechanisms that contribute to PCa progression.

Structure-guided design of pyridoclax derivatives based on Noxa / Mcl-1 interaction mode

Protein-protein interactions are attractive targets because they control numerous cellular processes. In oncology, apoptosis regulating Bcl-2 family proteins are of particular interest. Apoptotic cell death is controlled via PPIs between the anti-apoptotic proteins hydrophobic groove and the pro-apoptotic proteins BH3 domain. In ovarian carcinoma, it has been previously demonstrated that Bcl-xL and Mcl-1 cooperate to protect tumor cells against apoptosis. Moreover, Mcl-1 is a key regulator of cancer cell survival and is a known resistance factor to Bcl-2/Bcl-xL pharmacological inhibitors making it an attractive therapeutic target. Here, using a structure-guided design from the oligopyridine lead Pyridoclax based on Noxa/Mcl-1 interaction we identified a new derivative, active at lower concentration as compared to Pyridoclax. This new derivative selectively binds to the Mcl-1 hydrophobic groove and releases Bak and Bim from Mcl-1 to induce cell death and sensitize cancer cells to Bcl-2/Bcl-xL targeting strategies.

Inactivation of the Kinase Domain of CDK10 Prevents Tumor Growth in a Preclinical Model of Colorectal Cancer, and Is Accompanied by Downregulation of Bcl-2

Cyclin-dependent kinase 10 (CDK10), a CDC2-related kinase, is highly expressed in colorectal cancer. Its role in the pathogenesis of colorectal cancer is unknown. This study examines the function of CDK10 in colorectal cancer, and demonstrates its role in suppressing apoptosis and in promoting tumor growth in vitro and in vivo. Modulation of CDK10 expression in colorectal cancer cell lines demonstrates that CDK10 promotes cell growth, reduces chemosensitivity and inhibits apoptosis by upregulating the expression of Bcl-2. This effect appears to depend on its kinase activity, as kinase-defective mutant colorectal cancer cell lines have an exaggerated apoptotic response and reduced proliferative capacity. In vivo, inhibiting CDK10 in colorectal cancer following intratumoral injections of lentivirus-mediated CDK10 siRNA in a patient-derived xenograft mouse model demonstrated its efficacy in suppressing tumor growth. Furthermore, using a tissue microarray of human colorectal cancer tissues, the potential for CDK10 to be a prognostic biomarker in colorectal cancer was explored. In tumors of individuals with colorectal cancer, high expression of CDK10 correlates with earlier relapse and shorter overall survival. The findings of this study indicate that CDK10 plays a role in the pathogenesis in colorectal cancer and may be a potential therapeutic target for treatment. Mol Cancer Ther; 16(10); 2292–303. ©2017 AACR.

Abstract 4169: CReMEC initiative: Characterization of patient-derived colorectal tumor models and correlation with patient profile

Well characterized models representing the heterogeneity of human colorectal cancers (CRC) are needed to develop effective therapeutic agents. Establishment of such tools will allow a better prediction of the clinical outcome, taking into account the diversity of each patient tumor phenotype and genotype. For this purpose and with the financial support of the French Ministry of Industry, we have associated efforts from hospitals, academic groups, biotech and pharmaceutical companies. From May 2007 to January 2009, 86 surgical specimens [59 primary (P) tumors, 19 metastasis (M), and 8 peritoneal carcinomatosis (C)] were collected from CRC patients (with informed consents and negative HBV, HCV, and HIVs serologies). Tumor samples were subcutaneously xenografted in nude mice. The take rate was 57% (P), 55% (C), and 82% (M) with a difference in the take rate between tumor stages (p= 0.0184). We further engrafted in nude mice, SCID mice and nude rats. No significant difference in the take rate and growth was observed between the 2 mouse strains. Most of the mouse-growing tumors (95%) were successfully engrafted in nude rats, however their growth was significantly slower showing a more pronounced stromal component when compared with mice. Characteristics of our models are in accordance with the CRC clinical heterogeneity. Sequence analysis of 54 models has been performed and mutations were observed for KRAS (46%), APC (44%), TP53 (59%), BRAF (11%), PI3KCA (11%), FBXW7 (6%), CTNNB1 (2%), EGFR (2%), and 5/21 present a MSI status. Besides molecular markers, we compared the gene copy number using CGH technology before and after engraftment. The results exhibit high similarity between early passages of xenografts and the original clinical tumor samples. The histological structure and molecular profile were preserved, indicating the relevance of these models. Nevertheless the quality control is required to follow potential molecular deviation after multiple in vivo passages. The established models are being evaluated for ex vivo and in vivo sensitivities to colon anticancer drugs (5-FU, oxaliplatin, irinotecan and cetuximab). In vivo studies performed in our panel show 14/19 models sensitive to 5-FU, 1/19 to oxaliplatin, 7/8 to irinotecan, and 6/19 to cetuximab. We will present the correlation between pharmacological studies, molecular profile and patient clinical history. Preclinical studies on patient-derived tumor models will bring benefits to evaluate novel targeted therapeutic strategies and potentially help the stratification strategy for cancer patients. 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 4169.

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.