Fig. 1. TiME phenotypes were derived from RCM imaging and correlated with underlying biology and treatment response to topical toll-like receptor agonist (TLRA) imiquimod.

In vivo RCM imaging was performed on patients with clinically suspected skin cancers or rashes visiting the Dermatology Service at MSKCC. Imaging on the lesion was performed to span large field-of-view (FOV) for exhaustive sampling in tumor, peritumoral and adjacent normal areas. Tumor, inflammation, vasculature and trafficking were imaged within each lesion. Data was used for machine learning and automated quantification of inflammation density, vessel diameter and density, and frequency of leukocyte trafficking. RCM-TiME phenotypes were investigated using unsupervised analysis for basal cell carcinoma (BCC), and melanoma cohorts. The RCM phenotypes were correlated with pathology and dual immunohistochemistry (IHC) for CD3⁺ (T-cell) and CD20⁺ (B-cell) labeling of tertiary lymphoid structures. BCC phenotyping was additionally validated using multiplexed immunofluorescence (CD8⁺, FOXP3, CD68⁺, PD-1⁺, PD-L1⁺) and bulk RNA sequencing. A subset of patients with confirmed BCC diagnoses on RCM undergoing treatment with a TLRA agonist were enrolled. The patients were imaged 6 months after end of treatment to confirm tumor clearance and were classified as responders (complete tumor regression) or non-responders (incomplete or no tumor regression). Treatment response was correlated with TiME features and phenotypes. Created with www.Biorender.com RCM: reflectance confocal microscopy; BCC: basal cell carcinoma; IHC: immunohistochemistry; TLRA: toll-like receptor agonist.