Fig. 1: TMEM doorway tumor cells show increased CSF-1 levels at active TMEM doorways.

A Cartoon denoting the identification of parameters used for analysis of CSF-1 levels in TMEM doorway tumor cells (TTCs) and TMEM doorway function. The three cells composing the TMEM doorway are indicated by the yellow triangle connecting the TMEM doorway macrophage (TM), the TMEM doorway endothelial cell (TEC) and the TTC. Intravascular dextran (green) is in the lumen of blood vessel, extravascular dextran is outside the lumen of blood vessel, and CSF-1 is directly measured in the TTC. Figure created with BioRender.com. B Panel shows inactive and active TMEM doorways, visualized by immunohistochemistry (IHC) staining for Mena, Iba-1, and endomucin. The three cells of the TMEM doorway (contained in black circle, with the three cells forming the TMEM doorway indicated with the yellow triangle) are the TMEM doorway endothelial cell (TEC, endomucin stained in blue, circled in white), TMEM doorway macrophage (TM, Iba1 stained in brown, circled in teal), and TMEM doorway tumor cell (TTC, Mena stained in pink, and circled in pink) and black arrows indicate where the cells are localized within the TMEM doorway. TMEM doorways were identified using automated analysis by VisioPharm identifying three adjacent immuno-histochemical stains. C The sequential tissue sections after the IHC in (B) were stained using immunofluorescence (IF) with antibodies against endomucin (white), dextran (green), CSF-1 (red), and nuclear stain DAPI (blue). The two sequential sections from B,C were aligned and the same TMEM doorways were matched between the two sections, as indicated by the black circle in the IHC panel (B) and white circle in the IF panels (C). The left panels demonstrate the association of CSF-1 level in the TMEM doorway tumor cell (TTC) with TMEM doorway activity. The middle panel shows the extravascular signal for dextran as a green mask and the endomucin stain as a white mask, where thresholded, positive signal for these stains was converted into a binary mask. Active versus inactive TMEM doorways were distinguished by the presence of extravascular dextran staining (non-overlapping with the endomucin stain), which indicates that the vessel had a TMEM doorway-associated vascular opening (TAVO). Scale bars = 20 μm. D Immunofluorescence measurement of CSF-1 levels in TMEM doorway tumor cells in active and inactive TMEM doorways. Active and inactive TMEM doorways were identified in the IF-stained sections as described above (see C). Next, tumor cells were identified using the Mena-positive cells at TMEM doorway (B, IHC stain). The level of CSF-1 in the TMEM doorway tumor cells was measured in both active and inactive TMEM doorways using Visiopharm. Between 82 and 134 TMEM doorways were analyzed per mouse, in 12 mice. Each point represents one TMEM doorway, bars show the mean intensity (AU) and error bars represent standard deviation. ****P < 0.0001 analyzed by Student’s t-test. E Average immunofluorescence intensity plot of CSF-1 staining within TMEM doorways compared to the average intensity of extravascular dextran within TMEM doorways in tumor tissues stained and aligned as in Fig. 1B, C. n = 12 mice, each point represents the average value per mouse. Pearson correlation coefficient r = 0.9733, p < 0.0001.