Abstract A17: Development of a 3D tunable platform to measure the impact of patient-derived organoids on macrophage polarization

There is an increasing need to develop physiologically relevant models that recapitulate the complexity of the human tumor microenvironment (TME) to better understand and treat cancer. Macrophages are a major component of the TME, with high macrophage infiltration linked to poor prognosis in several cancer types. Tumor-associated macrophages have been reported to promote tumor progression, contribute to immune suppression, and limit the efficacy of therapy. Therefore, our goal is to develop a 3D tunable cell culture model that enables the study of macrophage and cancer cell interactions in an environment that more closely mimics the tumor milieu and to serve as a platform for screening therapies that target or reprogram macrophages. Simplistically, macrophages can be polarized into pro-inflammatory (M1) or immune-suppressive (M2) states in response to stimuli from the TME. To examine the crosstalk between cancer cells and macrophages, we first evaluated the signaling pathways and functional metabolic markers of a murine macrophage cell line J774A.1 and a human monocytic cell line THP-1. Treatment of both cell lines with M1 (LPS + IFN-g) and M2 (IL-4) inducers yielded STAT1 and STAT6 activation, respectively. Arginine metabolism by nitric oxide synthase (iNOS) in M1 macrophages and arginase (Arg-1) in M2 macrophages produces nitric oxide and polyamine metabolites. Treatment with M1 and M2 inducers yielded respective iNOS and Arg-1 expressions in J774A.1 cells, but not in THP-1 cells. However, other metabolic markers such as IDO and TGM2 were upregulated by M1 and M2 inducers in THP-1 cells, suggesting that arginine may be metabolized differently in human and mouse macrophages. Furthermore, tumor-conditioned media from murine triple-negative breast cancer 4T1 cells polarized J774A.1 cells toward M2-like macrophages, independent of STAT6 signaling. We extended these studies to co-cultures of macrophages and patient-derived tumor organoids. We established a diverse biobank of patient-derived organoids from breast and colorectal cancer tissues. Using a high-content imaging-based workflow, we examined the inter-patient effect of organoids on the polarization of THP-1 cells by staining and quantifying the expression of M1 and M2 markers. In summary, the methodologies we are employing to interrogate the involvement of macrophages in cancer could provide new insights into critical TME features that contribute to macrophage polarization and offer a venue for evaluat