Fusion-negative rhabdomyosarcoma 3D organoids to predict effective drug combinations: A proof-of-concept on cell death inducers.

Rhabdomyosarcoma (RMS) is the main form of pediatric soft-tissue sarcoma. Its cure rate has not notably improved in the last 20 years following relapse, and the lack of reliable preclinical models has hampered the design of new therapies. This is particularly true for highly heterogeneous fusion-negative RMS (FNRMS). Although methods have been proposed to establish FNRMS organoids, their efficiency remains limited to date, both in terms of derivation rate and ability to accurately mimic the original tumor. Here, we present the development of a next-generation 3D organoid model derived from relapsed adult and pediatric FNRMS. This model preserves the molecular features of the patients' tumors and is expandable for several months in 3D, reinforcing its interest to drug combination screening with longitudinal efficacy monitoring. As a proof-of-concept, we demonstrate its preclinical relevance by reevaluating the therapeutic opportunities of targeting apoptosis in FNRMS from a streamlined approach based on transcriptomic data exploitation.

The TLR3 L412F polymorphism prevents TLR3-mediated tumor cell death induction in pediatric sarcomas.

Toll-like receptor 3 (TLR3) is a pattern recognition receptor mainly known for its role in innate immune response to infection. Indeed, binding of double-stranded RNA (dsRNA) to TLR3 triggers a pro-inflammatory cascade leading to cytokine release and immune cell activation. Its anti-tumoral potential has emerged progressively, associated with a direct impact on tumor cell death induction and with an indirect action on immune system reactivation. Accordingly, TLR3 agonists are currently being tested in clinical trials for several adult cancers. Meanwhile, TLR3 variants have been linked to auto-immune disorders, and as risk factors of viral infection and cancers. However, aside from neuroblastoma, TLR3 role in childhood cancers has not been evaluated. Here, by integrating public transcriptomic data of pediatric tumors, we unveil that high TLR3 expression is largely associated with a better prognosis in childhood sarcomas. Using osteosarcomas and rhabdomyosarcomas as models, we show that TLR3 efficiently drives tumor cell death in vitro and induces tumor regression in vivo. Interestingly, this anti-tumoral effect was lost in cells expressing the homozygous TLR3 L412F polymorphism, which is enriched in a rhabdomyosarcomas cohort. Thus, our results demonstrate the therapeutic potential associated with the targeting of TLR3 in pediatric sarcomas, but also the need to stratify patients eligible for this clinical approach with respect to the TLR3 variants expressed.

Immune contexture of paediatric cancers.

BACKGROUND: The clinical development of immune checkpoint-targeted immunotherapies has been disappointing so far in paediatric solid tumours. However, as opposed to adults, very little is known about the immune contexture of paediatric malignancies. METHODS: We investigated by gene expression and immunohistochemistry (IHC) the immune microenvironment of five major paediatric cancers: Ewing sarcoma (ES), osteosarcoma (OS), rhabdomyosarcoma (RMS), medulloblastoma (MB) and neuroblastoma (NB; 20 cases each; n = 100 samples total), and correlated them with overall survival. RESULTS: NB and RMS tumours had high immune cell gene expression values and high T-cell counts but were low for antigen processing cell (APC) genes. OS and ES tumours showed low levels of T-cells but the highest levels of APC genes. OS had the highest levels of macrophages (CSF1R, CD163 and CD68), whereas ES had the lowest. MB appeared as immune deserts. Tregs (FOXP3 staining) were higher in both RMS and OS. Most tumours scored negative for PD-L1 in tumour and immune cells, with only 11 of 100 samples positive for PD-L1 staining. PD-L1 and OX40 levels were generally low across all five indications. Interestingly, NB had comparable levels of CD8 by IHC and by gene expression to adult tumours. However, by gene expression, these tumours were low for T-cell cytotoxic molecules GZMB, GZMA and PRF1. Surprisingly, the lower the level of tumour infiltrative CD8 T-cells, the better the prognosis was in NB, RMS and ES. Gene expression analyses showed that MYCN-amplified NB have higher amounts of immune suppressive cells such as macrophages, myeloid-derived suppressor cells and Tregs, whereas the non-MYCN-amplified tumours were more infiltrated and had higher expression levels of Teff. CONCLUSIONS: Our results describe the quality and quantity of immune cells across five major paediatric cancers and provide some key features differentiating these tumours from adult tumour types. These findings explain why anti-PD(L)1 might not have had single agent success in paediatric cancers. These results provides the rationale for the development of biologically stratified and personalised immunotherapy strategies in children with relapsing/refractory cancers.