Histological analysis

H&E histological sections were reviewed by a board-certified pathologist (D.W.) to evaluate tumor microvascular density (MVD). Blood vessels lined by endothelial cells were counted on cross-sections at 400x magnification. Twenty high power fields were counted for each sample and the mean number per high power field was calculated. The necrotic area was estimated on each section and averaged on three sections per sample.

Fluorescent histological sections were digitized using a Zeiss Axio Scan.Z1 with a CY3 channel exposure of 400 ms, and a DAPI channel exposure of 3 ms. CZI files were exported as tiff images and analyzed in ImageJ. Manual segmentations of the tumor cross-sections were used to quantify the mean fluorescent intensity (MFI) of the rhodamine lectin stain as a metric of intratumoral vasculature. The images were then binarized using Otsu’s method threshold and a measure of the number of ‘vessel’ pixels in the images were calculated as a percentage of the total number of pixels in the imaging cross-section. This metric was termed the fluorescent area percentage, which served as a surrogate measure for microvascular density.