Exploiting Tertiary Lymphoid Structures in Rhabdomyosarcoma for promoting immunotherapy response

Rhabdomyosarcoma (RMS), the most common STS in pediatric, adolescent and young adult patients. RMS is a devastating malignancy; outcomes for patients with high-risk disease are dismal and unlike many pediatric cancers, have not improved over the last 3 decades. Immunotherapy holds great potential for improving outcomes in sarcoma patients. Immunotherapy has key advantages, namely specificity, memory, adaptability and low toxicity. Disappointingly, three large pediatric oncology trials utilizing immune checkpoint inhibition (ICI) for solid tumors, including RMS, demonstrated futility of single or dual ICI therapy with the exception of Hodgkin lymphoma. Importantly, deep immune profiling of specimens from patients on those trials was not performed so it is hard to point out the exact mechanisms of resistance to immunotherapy in those cases. It is clear now that current immune-oncology approaches are ineffective in RMS, but the reasons why are extraordinarily poorly understood. We have discovered that within the tumor beds of RMS, T and B cells interact to form highly organized structures similar to lymph nodes, termed tertiary lymphoid structures. We hypothesize that the generation of tertiary lymphoid structures (TLS) in RMS is essential for the generation of intratumoral immune responses, which could be exploited for the development of novel therapeutic approaches aiming to increase mature TLS formation, B cell differentiation and anti-tumor antibody production within tumors. We propose to uncover the unique signals that lead to the genesis of TLS in RMS, define their specific composition and function, and then manipulate these inducible lymphoid organs in an in vitro system with the goal of enhancing the elimination of RMS cancer cells. Our proposed aims are: Aim #1: Define the cellular and molecular mechanisms that regulate the generation of TLS in human RMS. Aim #2: Identify the phenotype, functional state and clonality of tumor infiltrating B & T cells present in TLS of human RMS. Aim #3: Engineer a 3D model of TLS formation implementing an organoid-like culture with the goal of determining whether in vitro modulation of defined stimuli under controlled conditions can regulate their development and function. Our proposed studies will exert a profound effect in the field by elucidating the mechanisms leading to the spontaneous formation of TLS in RMS. This mechanistic understanding will lead to optimized interventions aimed to promote their assembly as a novel immunotherapy, thus providing a significant advance towards the goal of personalized medicine and the cure of this devastating disease.