Generation and characterization of 3D organoid models as tools to identify the biological underpinnings of therapy resistance and relapse in pediatric sarcomas – CHILD-SARC

Organoid can be defined as an in vitro 3D cellular cluster derived from stem/progenitor cells, in which cells spontaneously self-organize into progenitors and properly differentiated functional cell types, which recapitulate at least some functions of the organ. The establishment of cancer-derived organoids (also called tumoroids) has recently begun to emerge as a prominent and promising tool to enhance our understanding of human cancers. Indeed, human cancer-derived organoids have been observed to preserve the (i) histological architecture, (ii) genes expression profile, (iii) genomic and epigenomic landscape, (v) genetic intratumoral heterogeneity (subclonal tumoral populations), (v) cellular intratumoral heterogeneity (from CSCs to more differentiated cells) and (iv) metastatic potential of the original tumor even after long-term expansion in culture. Moreover, tumoroids can greatly expand tumoral cells from very limited amounts of starting material such as biopsies, which is a key issue considering scarcity of material in pediatric oncology field. Finally, these tumoroids are amenable for biomarkers identification, drug-screening testing and detection of gene-drug associations, but also recapitulate patient responses in clinical trials. Thus, cancer-derived organoids present wide-ranging utilities in furthering the understanding of cancer biology, resistance and relapse, and in developing personalized-medicine approaches for the disease. However, such models have not been developed for pediatric cancers yet. Thus, I aim to take advantage of my skills and knowledge, I will establish, characterize and use pediatric sarcoma-derived organoid models to decipher the biology of these tumors, and in particular their heterogeneity and plasticity in order to better understand their mechanisms of resistance and relapse. Beyond the scope of this proposal, this outbreaking pediatric sarcomas biology understanding will help to develop innovative therapeutic strategies adapted to young patients.

In the last 18 months, we developed culture conditions that allows generation and growing, in 3D culture, of RMS tumor organoids (RMS-TO) from biopsies and/or patient-derived xenografts.

Our preliminary results indicate that these RMS-TO recapitulate histological features, cellular diversity and gene expression of their corresponding parental tumours even after long-term expansion in culture. Our RMSO models provide, thus, the first RMS close-to-native system (patent pending).

patent pending, EP21305446.3

Rhabdomyosarcoma (RMS) and osteosarcoma (OS) are the most frequent soft-tissue and bone sarcoma in children and adolescents/young adults (AYA), respectively. Pediatric oncologists are responsible for providing children with the most adequate treatment and best quality of life. Yet, despite the use of multimodal treatments and the implementation of several clinical trials, survival rate for these two pathologies has not significantly evolved over the last decades. Moreover, intensive therapies are not devoid of long-term side effects including secondary malignancies. It is therefore crucial to develop more effective and less toxic treatments adapted to young patients. A critical step in achieving this, is to unravel the molecular and cellular underpinnings of pediatric sarcomas to determine their specificities. However, RMS and OS display distinct clinical behaviors and experimental models are currently lacking, thus hampering investigations into their biological and clinical features. The recent advent of stem cell-derived organoid systems provides a compelling new class of biological models to study tissue development, stem cell (SC) behavior and disease ex vivo. Organoids can be defined as in vitro 3D cellular clusters derived from stem/progenitor cells, in which cells spontaneously self-organize into progenitors and differentiated functional cell types, which recapitulate, at least partially, functions of the organ. Recently established cancer-derived organoids (also called tumoroids) are increasingly recognized as prominent and promising tools to enhance our understanding of human cancers. Indeed, human cancer-derived organoids have been observed to preserve the (i) histological architecture, (ii) gene expression profile, (iii) genomic and epigenomic landscape, (v) genetic intratumoral heterogeneity (subclonal tumoral populations), (v) cellular intratumoral heterogeneity (from cancer stem cells (CSCs) to more differentiated cells) and (iv) metastatic potential of the original tumor even after long-term expansion in culture. Moreover, tumoroids arise from the substantial proliferation of tumoral cells from very limited amounts of starting material such as biopsies, which is a key issue considering scarcity of material in the field of pediatric oncology. Thus, cancer-derived organoids present wide-ranging advantages in furthering our understanding of cancer biology, resistance and relapse, and in developing personalized-medicine approaches for the disease. However, such models have as yet not been developed for pediatric sarcomas. Increasing evidence suggests that OS and RMS are hierarchically organized tumors and possess CSCs. Thus, I strongly believe 3D-organoid models may provide unprecedented reliable RMS and OS models to improve our comprehension of these malignancies, to identify new oncogenic processes and setup new therapeutic paths. Based on my expertise and knowledge, I propose to develop and fully characterize 3D RMS and OS organoid models. These cultures will be derived directly from patient tissues and co-cultured with microenvironmental cells to closely mimic in vivo tumors. Besides filling the current void in relevant pediatric oncology models, I will use these tools to decipher the ability of pediatric sarcomas to resist treatment and to relapse, with a specific focus on their heterogeneity and plasticity, as the first steps towards the identification of new actionable targets. Beyond the scope of this proposal, our findings and models may be used to develop innovative therapeutic strategies adapted to young patients.