Abstract 1083: Cell membrane-anchored and tumor-targeted IL12 T-cell therapy destroys cancer-associated fibroblasts and disrupts extracellular matrix in heterogenous osteosarcoma xenograft models
Authors: Hu Jiemiao , Lazar Alexander , Wang Wei-Lien , Zhang Wendong , Jia Zhiliang , Ragoonanan Dristhi , Wang Jian , Xia Xueqing , Mahadeo Kris , Gorlick Richard , Li Shulin 
Published: March 2024

Abstract
Background: The extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs) play major roles in tumor progression, metastasis, and the poor response of many solid tumors to immunotherapy. CAF-targeted CAR-T cell therapy cannot infiltrate ECM-rich tumors such as osteosarcoma.
Method: In this study, we used RNA sequencing to assess whether the recently invented membrane-anchored and tumor-targeted IL12-armed (attIL12) T cells, which bind cell-surface vimentin (CSV) on tumor cells, could destroy CAFs to disrupt the ECM. We established an in vitro model of the interaction between osteosarcoma CAFs and attIL12-T cells to uncover the underlying mechanism by which attIL12-T cells penetrate stroma-enriched osteosarcoma tumors.
Results: RNA sequencing demonstrated that attIL12-T cell treatment altered ECM-related gene expression. Immunohistochemistry staining revealed disruption or elimination of high-density CAFs and ECM in osteosarcoma xenograft tumors following attIL12-T cell treatment, and CAF/ECM density was inversely correlated with T-cell infiltration. Other IL12-armed T cells, such as wild-type IL12- or tumor-targeted IL12-T cells, did not disrupt the ECM because this effect depended on the engagement between CSV on the tumor cell and its ligand on the attIL12-T cells. Mechanistic studies found that attIL12-T cell treatment elevated IFNγ production upon interacting with CSV+ tumor cells, suppressing TGFβ secretion and in turn upregulating FAS-mediated CAF apoptosis. CAF destruction reshaped the tumor stroma to favor T-cell infiltration and tumor inhibition.
Conclusions: This study unveiled a novel therapy—attIL12-T cells—for targeting CAFs/ECM. These findings are highly relevant to humans because CAFs are abundant in human osteosarcoma. What is already known on this topic: As the major source of ECM factors in tumors, the FAP+αSMA+ subtype of CAFs within tumors induce production of ECM factors, preventing antitumor T cells from accessing the core regions of tumors. What this study adds: Our group demonstrated that attIL12-T cell transfer (US Patent: 11421010) induced T-cell infiltration into and eliminated large osteosarcoma PDX tumors. Comparison of gene expression in sensitive or resistant PDX models to attIL12-T cell therapy elucidated the ECM-targeting mechanism of attIL12-T cells. Our in vitro system further determined that the CSV-attIL12 interaction mediated IFNγ upregulation, which triggered FAS induction in CAFs to induce CAF apoptosis. In sum, attIL12-T cell treatment shifts tumor stroma from a TGFβ-dependent CAF-promoting phenotype to an IFNγ-induced immune-inflamed phenotype. How this study might affect research, practice or policy: attIL12-T cell transfer is a novel anti-ECM strategy that renders ECM-rich tumors more susceptible to T cell-mediated tumor killing.
Citation Format: Jiemiao Hu, Alexander Lazar, Wei-Lien Wang, Wendong Zhang, Zhiliang Jia, Dristhi Ragoonanan, Jian Wang, Xueqing Xia, Kris Mahadeo, Richard Gorlick, Shulin Li. Cell membrane-anchored and tumor-targeted IL12 T-cell therapy destroys cancer-associated fibroblasts and disrupts extracellular matrix in heterogenous osteosarcoma xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1083.