Poster #020 , Sanford Burnham Prebys Medical Discovery Institute
Deciphering Immune Cell Crosstalk in the Tumor Microenvironment
Mentors: Robert Wiesheu, PhD and Kelly Kersten, PhD
Immunotherapy in melanoma patients has been successful for some patients; however, many develop resistance. To mimic melanoma patients, we used two preclinical mouse melanoma cell lines, YUMM1.7 and YUMMER1.7. YUMM1.7 tumors are immunotherapy resistant and have higher amounts of macrophages, which are known to inhibit the anti-tumor function of CD8 T-cells. In contrast, YUMMER1.7 tumors respond to immunotherapy and hold more monocytes, the precursors to macrophages. Utilizing in vitro coculture assays, we aimed to investigate how YUMM1.7 or YUMMER1.7 cancer cells contribute to the differentiation of monocytes to macrophages.
A six-well coculture assay of mouse bone marrow and tumor cells was set up over three days, four wells containing YUMM1.7 or YUMMER1.7 tumor cells or conditioned media, respectively. The last two wells contained DMEM only, one with the growth factor M-CSF as a positive control, while the other lacked M-CSF as a negative control. Analysis of monocyte and macrophage differentiation by multi-parameter flow cytometry revealed that, besides the M-CSF control, YUMM1.7 coculture as well as conditioned media alone accelerated macrophage differentiation as compared to YUMMER1.7, in line with our in vivo findings.
Furthermore, to unveil potential T-cell and monocyte/macrophage interactions in the tumor microenvironment of melanoma, we performed immunofluorescent staining on cryosections of tumors that were subcutaneously injected into mice. Current efforts are underway to refine monocyte/macrophage staining to provide insight into the colocalization with T-cells to elucidate their crosstalk during immunotherapy response. Understanding this crosstalk is key in identifying novel therapeutic targets to overcome immunotherapy resistance in patients.