Abstract 4712: Spatial biology of tumor-stroma assembloids reveals that nothing is lost, nothing is created, everything is reorganized

Abstract A close association between active fibroblastic foci (desmoplasia) and distant metastasis in the lung adenocarcinoma (LUAD) has been established decades ago and yet, the pro- and anti-tumor properties of cancer-associated fibroblasts (CAFs) are still being resolved in the hope of preventing LUAD progression. To better understand the role of stromal cells in progression, there is a growing interest in 3D patient-derived models for their translational and throughput advantages. In parallel, the emergence of multiplexed spatial biology platforms is advancing translational oncology forward by providing a more global understanding of the tumor microenvironment (TME). Despite the enthusiasm for these new technologies, there are no study showing the applicability and significance of using multiplexed spatial biology to characterized 3D patient-derived models. Here, we modeled spatial heterogeneity by assembling EGFR mutated patient-derived organoids (PDOs) with fibroblasts harvested from the tumor edge (tumor-adjacent fibroblasts; TAFs) or tumor core (tumor core fibroblasts; TCFs) referred here as tumor-stroma assembloids. Using these assembloids, we generated an extensive dataset with CODEX imaging technology and used a quantitative approach to dissect tumor-stroma crosstalk. Our results show that TAFs expressing the myofibroblast marker aSMA were strictly found at the periphery of the hydrogel dome containing the assembloid, while aSMA- fibroblasts were found strictly in the center. In contrast, we did not observe a similar spatial organization in TCF assembloids. Next, we used CELESTA, an unsupervised machine learning algorithm that uses prior knowledge of the cell expression and spatial information to dissect cell heterogeneity, then performed co-localization analyses. We also leveraged the EGFR mutation status of the PDOs and observed the protective effect of the fibroblasts from erlotinib. Both assembloid conditions showed drastically different tumor-stroma spatial changes including gains and losses of colocalizations even though erlotinib did not significantly change the subpopulation fractions, cell heterogeneity, nor cell density. The top tumor-stroma colocalization pairs that persisted after erlotinib involved either myofibroblasts and PanCK-Vim+MUC1+ cancer cells or were found in the center of the assembloids, shielded by a stromal barrier of myofibroblasts. Taken together, our results show that the assembloids undergo drastic spatial reorganization following erlotinib treatment even though subpopulation fractions and cell heterogeneity remain unchanged. This study provided new insights on tumor-stroma crosstalk in the LUAD and motivates the use 3D patient-derived models coupled with multiplex imaging to answer complex spatial biology questions. Citation Format: Gina Bouchard, Irene Li, Weiruo Zhang, Sylvia Plevritis. Spatial biology of tumor-stroma assembloids reveals that nothing is lost, nothing is created, everything is reorganized. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4712.