Abstract: Macrophages, vasculature, and fibroblasts form oncofetal niches that determine immune cell behavior and responses to immunotherapy in the tumor microenvironment (TME). How they spatially contribute to TME is still poorly understood, but the lack of immune and stromal cells in cancer organoids hamper this line of research. Therefore, we endow immune stromal compartment in cancer organoids. We established scaffold-free assembloids that fused hPSC-derived vascular immune organoids (VIOs) with either patient-derived cancer organoids (PDOs) or cancer spheroids. scRNA-seq showed a heterogenous population of macrophages and fibroblasts in the VIOs. We next characterized spatial TME features in assembloids by whole-mount immunofluorescent staining. We observed macrophage shifting from tissue-resident into cancer-infiltrating behaviour in colorectal assembloids. We observed angiogenic switch of vasculature growing towards cancer core in liver assembloids. We also observed perivascular niches and pairs of intravasating macrophage and cancer for metastasis. We further characterized extracellular matrix by Masson’s trichrome staining. We observed collagen density was elevated by agonistic CD40 myeloid immunotherapy while provoking macrophage phagocytosis and infiltrations. To resolve the oncofetal niches, we are performing Visium-HD spatial transcriptomic on lung PDO assembloids. We will investigate how do the oncofetal interactions determines anti-HER2 CAR-T efficiency.
