Additional file 1: Figure S1. of Multispectral imaging of formalin-fixed tissue predicts ability to generate tumor-infiltrating lymphocytes from melanoma

Schematic representation of process for TIL initiation, expansion and testing. Figure S2. Lymphocytic immune infiltrate is insufficient to predict TIL culture success. A) Representative H&E stain from 4 patients, two are classified as 3+ (Top) and two are classified as 1+ (Bottom), TIL culture status is indicated above images. B) Compares average pathology scores of lymphocytic infiltrate between tumors that grew TILs and those that did not. Figure S3. Comparison of two methods to evaluate immune infiltrate. A) Comparison between % CD8 T cell infiltrate and # of CD8 positive cells/mm^2. B) Comparison between # of CD8 positive T cells/mm^2 determined using PerkinElmer and Definiens. The comparisons were done using the identical image. Statistics were determined using Prism. Figure S4. PD-L1 Localization. A) On tumor cells B) on CD8+ T cells and C) on CD163+ macrophages. Figure S5. Time of growth is predictive of tumor reactivity. Statistics were done using unpaired nonparametric T test. Significance was established at P < 0.05. Figure S6. Comparison between FFPE (Top) and frozen (Bottom) sections. A,F) CD8; B,G) CD3; C,H) CD163; D,I) FoxP3; E,J) PD-L1. Figure S7. Heatmap representation of parameters generated from multispectral imaging with dark red indicating maximal expression and dark blue indicating minimum expression. *indicates patients from whom we failed to culture TIL. **indicates tumor sample that grew TIL that were not reactive to autologous tumor. Figure S8. Multispectral image showing individual channels: Original is the raw image with all channels taken using the Vectra imaging software. The spectrum for each fluorophore is measured with control slides and subtracted from the original image to establish individual channels and the composite. Figure S9. Representative image from individual multispectral samples. TIL status is indicated with “+” or “-“. Enumerated immune infiltrates are indicated for each sample in the lower right corner. (PPTX 38563 kb