Enrique Podaza, Jared Capuano, Hui–Hsuan Kuo, Majd Al Assaad, Geoffrey Markowitz, M. Victoria Revuelta, John Nguyen, Adriana Irizarry, Hiranmayi Ravichandran, Sarah Ackermann, Troy Kane, Jyothi Manohar, Alyssa Duren-Lubanski, Michael Sigouros, Jenna Moyer, Bhavneet Bhinder, Pooja Chandra, Murtaza Malbari, Karsten Boehnke, Juan Miguel Mosquera, V Mittal, Andrea Sboner, Hamza Gokozan, Nasser Altorki, Olivier Elemento, M. Laura Martin
Non-small cell lung cancer (NSCLC) remains a leading cause of cancer-related mortality. Despite various therapeutic options, treatment resistance is common, underscoring the need for effective combination therapies and reliable pre-clinical models for patient-specific evaluation. Here, we describe strategies for reconstituting tumor immune microenvironment (TIME) components within patient-derived tumor organoid (PDTO) cultures. We established a tumor processing pipeline that enables concurrent expansion of tumor-infiltrating lymphocytes (TILs) and PDTO generation from the same resection. We optimized scalable assays to assess IFN-γ secretion and T cell cytotoxicity with immune checkpoint inhibitors (alone or in combination) and targeted inhibitors, capturing inter-patient heterogeneity and intra-patient variations between TILs and peripheral blood mononuclear cells (PBMCs). Additionally, we developed methods for differentiating PDTO-specific tumor-associated macrophages (TAMs) and established PDTO-TAM co-culture systems to evaluate TAM effects on PDTO growth and chemotherapy sensitivity. All approaches are scalable to high-throughput levels, highlighting the value of TIME-PDTO co-cultures for therapeutic modeling and precision medicine.