Phenotyping tumor-immune microenvironment in vivo in skin cancer patients

Phenotyping of tumors into hot, altered, or cold based on assessment of only T-cell infiltration in static tumor biopsies provides suboptimal prediction of immunotherapy response. In vivo dynamic mechanisms within the tumor microenvironment such as tumor angiogenesis and leukocyte trafficking also play a central role in modulating anti-tumor immunity and therefore immunotherapy response. Here, we report novel tumor immune microenvironment (TiME) phenotyping in vivo in patients with non-invasive spatially-resolved cellular-level imaging based on endogenous contrast. Investigating skin cancers as a model, with reflectance confocal microscopy (RCM) imaging, we determined four major phenotypes with variable prevalence of vasculature (Vasc) and inflammation (Inf) features: VaschiInfhi, VaschiInflo, VascloInfhi and Vascmed/hiInflo. The VaschiInfhi phenotype correlates with high immune activation, exhaustion, and vascular signatures while VaschiInflo with endothelial anergy and immune exclusion. Automated quantification of TiME features demonstrates moderate-high accuracy and correlation with corresponding gene expression. Prospectively analyzed response to topical immunotherapy show highest response in VascloInfhi, and reveals the added value of vascular features in predicting treatment response. 14 skin cancer (basal call carcinoma, BCC) tissues from human patients