Cells categorized as having the same identity can perform different activitiesSingle-cell expression data can be used to infer the activities cells take part in Activity profiles provide a basis to measure phenotypic cell-cell divergence Cell activity can quantify intra-tumor heterogeneity more fully than identity

Background: Follicular lymphoma (FL) is an incurable B-cell malignancy that constitutes a quarter of all lymphomas. Although RCHOP immuno-chemotherapy induces high rates of complete remission, almost all FL patients experience multiple relapses post-treatment. The limited understanding of treatment response heterogeneity is due to the absence of in vitro or in vivo experimental models, primarily because tumor cells heavily rely on their microenvironment to survive. In this study, we present an innovative xenograft model of primary FL cells in avian embryos, circumventing these limitations. Methods: We developed the FL-AVI-PDX model by transplanting 20 biopsy FL samples, including good (n=11) and poor clinical responders (POD24, n=9), into chicken embryos. Each set of embryos was treated with RCHOP or vehicle intravenously. We evaluated the effect of immuno-chemotherapy on tumor volume by light sheet microscopy and on tumor biology by transcriptomic analysis at the single-cell level. Results: We successfully engrafted all samples in avian embryos. We found that RCHOP treatment in ovo led to tumor volume reduction, which predicted progression-free survival in multivariate analysis, demonstrating the model's capacity to capture clinical heterogeneity at the patient level. The FL-AVI-PDX model also provided a unique opportunity to analyze the transcriptomic impact of RCHOP on FL cells using single-cell RNA sequencing. We identified a signature of 21 genes upregulated after RCHOP exposure, displaying significant intra-tumoral heterogeneity. As a proof of concept, we validated the functional involvement of BAX, a gene from the RCHOP-induced signature, as a critical effector of immuno-chemotherapy in vitro and in avian embryos. Conclusions: The FL-AVI-PDX model is a platform for functional precision oncology in primary FL cells that captures both inter- and intra-patient heterogeneity of clinical response to a complex therapeutic regimen. It offers a unique opportunity to better understand FL biology, opening perspectives for the development of new drugs.

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