128 Merging Mass Cytometry and Flow-Assisted Cell Sorting to Simultaneously Characterize a Tumor and Its Microenvironment

Previously, we developed a mouse model which allows for inducible carcinogenesis coupled to a fluorescent reporter. This system produced tumors in immunocompromised mice, but not in immunocompetent mice, indicating that the immune system prevents tumor initiation. While no tumors formed, the transformed cells could still be detected in grafts for up to 300 days after induction and host infiltrates were detected surrounding the lesions. To identify these infiltrates and characterize the nature of this immune-mediated tumor suppression, we developed a protocol to interrogate both tumor and microenvironment simultaneously by combining mass cytometry with flow-assisted cell sorting (FACS). Mass cytometry is a method of single-cell analysis using heavy metal-tagged antibodies which are read using mass spectrometry. Current mass cytometers can measure up to 50 different mass channels simultaneously. This is advantageous over traditional flow cytometry due to minimal spectral overlap between channels, allowing for larger dimensional analysis. After harvesting and dissociating grafts, the host infiltrates are first separated from the endogenously EGFP-labeled transformed cells using FACS. Transformed cells are subjected to RNA-seq to characterize changes in gene expression, antigenicity, and immune-evasive traits relative to non-transformed cells. The unlabeled fraction of each sample is barcoded and labeled with a panel of 18 mass-tagged antibodies, allowing for an unbiased characterization of immune activity. We are applying this analysis to lesions throughout their development to measure the co-evolution of transformed cells and the immune mechanisms that control them during cancer initiation. We will also use this method to test whether emerging immunotherapies may have an effect on premalignant lesions, indicating a possible cancer prevention strategy in humans.