Oct freezing media

Explanted tumors were embedded in OCT freezing media (Bio‐optica). Sections (5 μm) were cut on a Cryostat, adhered to Superfrost Plus slide (Thermo Scientific), fixed with acetone, and blocked with PBS containing 0.3% Triton X‐100 (Sigma‐Aldrich) and 10% FBS. Sections were then stained with purified anti‐mouse CD31 antibody (1:200) in blocking buffer, 1 h at room temperature. Sections were washed with TBS buffer and incubated with Rat‐on‐Mouse HRP‐Polymer kit (Biocare Medical). Dako EnVision System‐HRP was used as chromogen and counterstain with Mayer's Hematoxylin (Bio‐optica). After dehydration, stained slides were mounted with Eukitt and images acquired with a Vs120 dotSlide (Olympus). Image analysis was performed using FIJI‐ImageJ (Schindelin et al, 2012, 2015). ROI color deconvolution was used to separate the DAB signal, and particle analysis was used to automatically recognize, count, and measure all the CD31‐positive spots within the total tumor area.

For the CT26 and 4T1 tumor analysis, BALB/c mice were inoculated in the deep derma in the left flank with the minimal tumorigenic dose of CT26 or 4T1 tumor cells (5 × 10⁴) at Day 0. Explanted tumors at Day 5 were embedded in OCT freezing media (Biooptica). Sections (5 μm) were cut on a Cryostat, adhered to Superfrost Plus slide (Thermo Scientific), and then imaged under the two-photon excitation microscope.
Mice subjected to tumor injection were monitored on a daily basis for signs of discomfort, including hunched posture, ruffled fur and lack of movement within the cage. The body condition score index (a qualitative assessment of an animal overall appearance based on its weight, muscle mass, and bone prominence) was used to evaluate the welfare of the mice. Mice did not present signs of distress given the short period of time between tumor injection and killing.
Three entire tumor sections from three different animals (from 100 to 300 fields of view for each section) have been analyzed for both tumor models. The method has been also applied to five different tumor sections that were acquired as a set of 6 fields of view per sample, encompassing the skin-tumor boundaries.