an-orthotopic-sciatic-nerve-xenograft-for-neurofibromatosis-type-1

An Orthotopic Sciatic Nerve Xenograft for Neurofibromatosis Type 1 Neurofibromas

Neurofibromatosis type 1 (NF1) is a genetic disorder caused by pathogenic variants in the NF1 gene, leading to the loss of neurofibromin function and the ...

PSL Research University

Paris Cité University

Tumor endothelial transdifferentiation and VEGFR1/2 expression by cancer cells have been reported in glioblastoma but remain poorly documented for many other cancer types.

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

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.

Spherical cancer models in tumor biology.

The sequence of the beta-subunit of human chorionic gonadotropin (hCGβ) varies depending on whether hCGβ is encoded by type I or type II genes. Type II genes are upregulated in trophoblast and cancer but hCGβ can be detected in the serum of nonpregnant women and healthy individuals. We aimed to determine whether monoclonal antibody (mAb) FBT11-II specifically detects hCGβ encoded by type II genes (type II hCGβ).

Specific detection of type II human chorionic gonadotropin beta subunit produced by trophoblastic and neoplastic cells.

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.

Evaluating patient-derived colorectal cancer xenografts as preclinical models by comparison with patient clinical data.

Patient-derived xenografts (PDXs) have emerged as an important platform to elucidate new treatments and biomarkers in oncology. PDX models are used to address clinically relevant questions, including the contribution of tumour heterogeneity to therapeutic responsiveness, the patterns of cancer evolutionary dynamics during tumour progression and under drug pressure, and the mechanisms of resistance to treatment. The ability of PDX models to predict clinical outcomes is being improved through mouse humanization strategies and the implementation of co-clinical trials, within which patients and PDXs reciprocally inform therapeutic decisions. This Opinion article discusses aspects of PDX modelling that are relevant to these questions and highlights the merits of shared PDX resources to advance cancer medicine from the perspective of EurOPDX, an international initiative devoted to PDX-based research.

Interrogating open issues in cancer precision medicine with patient-derived xenografts.

The placenta relies on phenotypes that are characteristic of cancer to successfully implant the embryo in the uterus during early pregnancy. Notably, it has to invade its host tissues, promote angiogenesis-while surviving hypoxia-, and escape the immune system. Similarities in DNA methylation patterns between the placenta and cancers suggest that common epigenetic mechanisms may be involved in regulating these behaviors. We show here that megabase-scale patterns of hypomethylation distinguish first from third trimester chorionic villi in the placenta, and that these patterns mirror those that distinguish many tumors from corresponding normal tissues. We confirmed these findings in villous cytotrophoblasts isolated from the placenta and identified a time window at the end of the first trimester, when these cells come into contact with maternal blood, as the likely time period for the methylome alterations. Furthermore, the large genomic regions affected by these patterns of hypomethylation encompass genes involved in pathways related to epithelial-mesenchymal transition, immune response, and inflammation. Analyses of expression profiles corresponding to genes in these hypomethylated regions in colon adenocarcinoma tumors point to networks of differentially expressed genes previously implicated in carcinogenesis and placentogenesis, where nuclear factor kappa B is a key hub. Taken together, our results suggest the existence of epigenetic switches involving large-scale changes of methylation in the placenta during pregnancy and in tumors during neoplastic transformation. The characterization of such epigenetic switches might lead to the identification of biomarkers and drug targets in oncology as well as in obstetrics and gynecology.

The early pregnancy placenta foreshadows DNA methylation alterations of solid tumors.

Anal squamous cell carcinoma (ASCC) is a rare tumour, but its incidence is increasing. Standard chemoradiotherapy fails to cure 20% of patients and no targeted therapy is currently approved for recurrent ASCC. The PI3K/Akt/mTOR pathway is frequently altered in this poorly characterised carcinoma IGF2 was identified here as a key factor in ASCC oncogenesis, as IGF2 was shown to play a crucial role in the PI3K pathway with frequent (~60%) and mutually exclusive genomic alterations in IGF2, IGF1R, PTEN and PIK3CA genes. We also demonstrated that IGF2 expression is mainly due to cancer-associated fibroblasts and that IGF2 secreted by cancer-associated fibroblasts from ASCC samples promotes proliferation of a human ASCC cell line via IGF2 paracrine signalling. Altogether, these results highlight the key role of the IGF2/PI3K axis, and the major role of cancer-associated fibroblasts in tumour growth via IGF2 secretion, suggesting a major role of IGF2/IGF1R inhibitors in ASCC therapies.

Interaction between IGF2-PI3K axis and cancer-associated-fibroblasts promotes anal squamous carcinogenesis.

Patient-derived tumor xenograft (PDX) is now largely recognized as a key preclinical model for cancer research, mimicking patient tumor phenotype and genotype. Immunodeficient mice, well-known to develop spontaneous lymphoma, are required for PDX growth. As for all animal models used for further clinical translation, a robust experimental design is strongly required to lead to conclusive results. Here we briefly report unintentional co-engraftment of mouse lymphoma during expansion of well-established PDXs to illustrate the importance of systematic check of the PDX identity to avoid misinterpretation. Besides, this quality control based on complementary approaches deserves a more detailed description in materials and methods section to ensure experimental validity and reproducibility.

Spontaneous mouse lymphoma in patient-derived tumor xenografts: The importance of systematic analysis of xenografted human tumor tissues in preclinical efficacy trials.

Intravital microscopy (IVM) is a powerful technique that enables imaging of internal tissues at (sub)cellular resolutions in living animals. Here, we present a silicone-based imaging window consisting of a fully flexible, sutureless design that is ideally suited for long-term, longitudinal IVM of growing tissues and tumors. Crucially, we show that this window, without any customization, is suitable for numerous anatomical locations in mice using a rapid and standardized implantation procedure. This low-cost device represents a substantial technological and performance advance that facilitates intravital imaging in diverse contexts in higher organisms, opening previously unattainable avenues for in vivo imaging of soft and fragile tissues.

Longitudinal high-resolution imaging through a flexible intravital imaging window.

Spheroids are three-dimensional cellular models with widespread basic and translational application across academia and industry. However, methodological transparency and guidelines for spheroid research have not yet been established. The MISpheroID Consortium developed a crowdsourcing knowledgebase that assembles the experimental parameters of 3,058 published spheroid-related experiments. Interrogation of this knowledgebase identified heterogeneity in the methodological setup of spheroids. Empirical evaluation and interlaboratory validation of selected variations in spheroid methodology revealed diverse impacts on spheroid metrics. To facilitate interpretation, stimulate transparency and increase awareness, the Consortium defines the MISpheroID string, a minimum set of experimental parameters required to report spheroid research. Thus, MISpheroID combines a valuable resource and a tool for three-dimensional cellular models to mine experimental parameters and to improve reproducibility.

MISpheroID: a knowledgebase and transparency tool for minimum information in spheroid identity.

Main prognostic factors of anal squamous cell carcinoma (ASCC) are tumor size, differentiation, lymph node involvement, and male gender. However, they are insufficient to predict relapses after exclusive radiotherapy (RT) or chemoradiotherapy (CRT).  has been associated with poor prognosis in several digestive cancers. In this study, we assessed the association between intratumoral  load and clinico-pathological features, relapse, and survival in patients with ASCC who underwent abdominoperineal resection (APR) after RT/CRT. We retrospectively analyzed surgical samples from a cohort of 166 patients with ASCC who underwent APR.  16S rRNA gene sequences were quantified using real-time quantitative PCR. We associated  load with classical clinicopathological features, overall survival (OS), disease-free survival (DFS), and metastasis-free survival (MFS) using Cox regression univariate and multivariate analyses. Tumors harboring high loads of  (highest tercile) showed longer OS and DFS (median: not reached vs. 50.1 months,  = 0.01, and median: not reached vs. 18.3 months,  = 0.007, respectively). High  load was a predictor of longer OS (HR = 0.55,  = 0.04) and DFS (HR = 0.50,  = 0.02) in multivariate analysis. High  load is an independent favorable prognostic factor in patients with ASCC who underwent APR.

Prognostic Value of  after Abdominoperineal Resection for Anal Squamous Cell Carcinoma.

The management of anal squamous cell carcinoma (ASCC) has yet to experience the transformative impact of precision medicine. Conducting genomic analyses may uncover novel prognostic biomarkers and offer potential directions for the development of targeted therapies. To that end, we assessed the prognostic and theragnostic implications of pathogenic variants identified in 571 cancer-related genes from surgical samples collected from a homogeneous, multicentric French cohort of 158 ASCC patients who underwent abdominoperineal resection treatment. Alterations in PI3K/AKT/mTOR, chromatin remodeling, and Notch pathways were frequent in HPV-positive tumors, while HPV-negative tumors often harbored variants in cell cycle regulation and genome integrity maintenance genes (e.g., frequent TP53 and TERT promoter mutations). In patients with HPV-positive tumors, KMT2C and PIK3CA exon 9/20 pathogenic variants were associated with worse overall survival in multivariate analysis (Hazard ratio (HR)  = 2.54, 95%CI = [1.25,5.17], P value = .010; HR  = 2.43, 95%CI = [1.3,4.56], P value = .006). Alterations with theragnostic value in another cancer type was detected in 43% of patients. These results suggest that PIK3CA and KMT2C pathogenic variants are independent prognostic factors in patients with ASCC with HPV-positive tumors treated by abdominoperineal resection. And, importantly, the high prevalence of alterations bearing potential theragnostic value strongly supports the use of genomic profiling to allow patient enrollment in precision medicine clinical trials.

Pathogenic alterations in PIK3CA and KMT2C are frequent and independent prognostic factors in anal squamous cell carcinoma treated with salvage abdominoperineal resection.

Existing guidelines on the preparation (Planning Research and Experimental Procedures on Animals: Recommendations for Excellence (PREPARE)) and reporting (Animal Research: Reporting of In Vivo Experiments (ARRIVE)) of animal experiments do not provide a clear and standardized approach for refinement during in vivo cancer studies, resulting in the publication of generic methodological sections that poorly reflect the attempts made at accurately monitoring different pathologies. Compliance with the 3Rs guidelines has mainly focused on reduction and replacement; however, refinement has been harder to implement. The Oncology Best-practices: Signs, Endpoints and Refinements for in Vivo Experiments (OBSERVE) guidelines are the result of a European initiative supported by EurOPDX and INFRAFRONTIER, and aim to facilitate the refinement of studies using in vivo cancer models by offering robust and practical recommendations on approaches to research scientists and animal care staff. We listed cancer-specific clinical signs as a reference point and from there developed sets of guidelines for a wide variety of rodent models, including genetically engineered models and patient derived xenografts. In this Consensus Statement, we systematically and comprehensively address refinement and monitoring approaches during the design and execution of murine cancer studies. We elaborate on the appropriate preparation of tumor-initiating biologicals and the refinement of tumor-implantation methods. We describe the clinical signs to monitor associated with tumor growth, the appropriate follow-up of animals tailored to varying clinical signs and humane endpoints, and an overview of severity assessment in relation to clinical signs, implantation method and tumor characteristics. The guidelines provide oncology researchers clear and robust guidance for the refinement of in vivo cancer models.

Article	Year
Vasculature analysis of patient derived tumor xenografts using species-specific PCR assays: evidence of tumor endothelial cells and atypical VEGFA-VEGFR1/2 signalings. BMC cancer\| PubMed ID: 24625025	2014
Spherical cancer models in tumor biology. Neoplasia (New York, N.Y.)\| PubMed ID: 25622895	2015
Specific detection of type II human chorionic gonadotropin beta subunit produced by trophoblastic and neoplastic cells. Clinica chimica acta; international journal of clinical chemistry\| PubMed ID: 25681647	2015
Evaluating patient-derived colorectal cancer xenografts as preclinical models by comparison with patient clinical data. Cancer research\| PubMed ID: 25712343	2015
Interrogating open issues in cancer precision medicine with patient-derived xenografts. Nature reviews. Cancer\| PubMed ID: 28104906	2017
The early pregnancy placenta foreshadows DNA methylation alterations of solid tumors. Epigenetics\| PubMed ID: 28678605	2017
Interaction between IGF2-PI3K axis and cancer-associated-fibroblasts promotes anal squamous carcinogenesis. International journal of cancer\| PubMed ID: 30714617	2019
Spontaneous mouse lymphoma in patient-derived tumor xenografts: The importance of systematic analysis of xenografted human tumor tissues in preclinical efficacy trials. Translational oncology\| PubMed ID: 34051622	2021
Longitudinal high-resolution imaging through a flexible intravital imaging window. Science advances\| PubMed ID: 34134982	2021
MISpheroID: a knowledgebase and transparency tool for minimum information in spheroid identity. Nature methods\| PubMed ID: 34725485	2021
Prognostic Value of after Abdominoperineal Resection for Anal Squamous Cell Carcinoma. Cancers\| PubMed ID: 35406380	2022
Pathogenic alterations in PIK3CA and KMT2C are frequent and independent prognostic factors in anal squamous cell carcinoma treated with salvage abdominoperineal resection. International journal of cancer\| PubMed ID: 37908048	2024
OBSERVE: guidelines for the refinement of rodent cancer models. Nature protocols\| PubMed ID: 38992214	2024

Virginie Dangles-Marie

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