Nahum Puebla-Osorio ^1* Natalie Fowlkes ¹ Hampartsoum B. Barsoumian ¹ Gitika Srivastava ² Claudia Kettlun-Leyton ³ Sara Nizzero ² Tiffany Voss ¹ Thomas S. Riad ¹ Christina Wong ⁴ Ailing Huang ¹ Yun Hu ¹ Joylise Mitchell ⁵ Mingee Kim ⁶ Zahid Rafiq ¹ Kewen He ¹ Duygu Sezen ¹ Ethan Hsu ⁷ Fatemeh Masrorpour ¹ Aurian Maleki ⁸ Carola Leuschner ¹ Maria A. Cortez ⁹ Philipp Oertle ² Marija Plodinec ² Marko Loparic ² Janet L. Markman ⁴ James Welsh ^1*

• ¹ University of Texas MD Anderson Cancer Center, Houston, United States
• ² Artidis AG, Basel, Switzerland
• ³ MD Anderson Cancer Center Madrid, Madrid, Catalonia, Spain
• ⁴ Takeda Development Centers Americas, Lexington, Kentucky, United States
• ⁵ Texas Heart Institute, Houston, Texas, United States
• ⁶ Medical College of Wisconsin, Milwaukee, Wisconsin, United States
• ⁷ University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States
• ⁸ Rice University, Houston, Texas, United States
• ⁹ University of Texas Southwestern Medical Center, Dallas, Texas, United States

Robust infiltration of chimeric antigen receptor T (CAR-T) cellschimeric antigen receptor T cells into solid tumors is essential for an effective antitumor response and therapeutic success.In this study, we investigated the synergistic impact of low-dose radiotherapy (LDRT) administered before T-cell therapy on tumor growth and survival across two NSG mouse models harboring gastric carcinoma (GSU) or pancreatic adenocarcinoma (CAPAN-2) cells. Specifically, we employed engineered CAR-T cells targeting guanyl cyclase-C (GCC) or mesothelin.Additionally, we explored this a similar therapeutic strategy in C57BL/6 mice with MC38-gp100+ cell implants, followed by LDRT preceding the adoptive transfer of pmel+ T cells before and after LDRT. We achieved enhanced tumor control and prolonged survival in murine models by first administering LDRT followed by T-cell therapy, surpassing the outcomes of either monotherapy. This combined approach holds promise for improving responses in patients with solid tumors, leveraging radiotherapy to augment the intratumoral infiltration of effector T cells.Additionally, our study utilized atomic force microscopy (AFM) in a small cohort to assess the impact of radiotherapy preceding CAR-T cell therapy on tumor stiffness and plasticity. Our findings propose the nanomechanical signature as a potential biomarker for predicting outcomes in this sequential therapy. Our research findings underscore the transformative potential of incorporating LDRT as a precursor to cell therapies in treating solid tumors. We emphasize the capacity to augment the intratumoral infiltration of adoptively transferred T cells, thereby offering a promising avenue for reshaping, and refining current therapeutic approaches for solid tumors.