Advancing Organoid-Interacting Lymphocytes Towards In Vivo Validation for Solid Tumor Treatment

Abstract

Adoptive cell therapy (ACT) is a promising cancer therapeutic strategy, however efficacy in solid tumors remains limited by the immunosuppressive tumor microenvironment, and the challenges of both expanding high-quality, tumor-reactive lymphocytes and diverse neoantigens in heterogenous tumors. Traditional methods for expanding tumor-infiltrating lymphocytes are hindered by low yield and T cell exhaustion. To address these limitations, this thesis explores a personalized platform to train circulating lymphocytes against tumor-specific antigens, creating a tumor-primed therapeutic product termed organoid-interacting lymphocytes (OILs). We developed a microfluidic device to facilitate the co-culture of murine splenocytes with CT26 colon carcinoma. Fabrication methods were iteratively refined, transitioning from optimizing an acrylic based assembly to a standardized soft lithography process to ensure structural reproducibility. Immune cell culture was optimized for conditions in the chip moving from human peripheral blood mononuclear cells (PBMC) to murine splenocyte expansion. Following ex vivo priming, the therapeutic efficacy of OILs was evaluated in a syngeneic BALB/c murine model. Tumors were established using both intraperitoneal and subcutaneous flank injection of tumor cells and monitored longitudinally using bioluminescence imaging and multispectral optoacoustic tomography. This work establishes a comprehensive pipeline that bridges microfluidic manufacturing with systemic validation. Preliminary results showed changes in OILs-treated mice compared to controls, providing initial proof-of-concept for in vivo anti-tumor activity. By utilizing patient-specific biology within a standardized platform, this work provides a blueprint for point-of-care cell therapy production.