Signaling events at TMEM doorways provide potential targets for inhibiting breast cancer dissemination

Abstract

Tumor cell intravasation is essential for metastatic dissemination, but its exact mechanism is incompletely understood. We have previously shown that in breast cancer, the direct and stable association of a tumor cell expressing Mena, a Tie2^hi/VEGF^hi macrophage, and a vascular endothelial cell, creates an intravasation portal, called a "tumor microenvironment of metastasis" (TMEM) doorway, for tumor cell intravasation, leading to dissemination to distant sites. The density of TMEM doorways, also called TMEM doorway score, is a clinically validated prognostic marker of distant metastasis in breast cancer patients. Although we know that tumor cells utilize TMEM doorway-associated transient vascular openings to intravasate, the precise signaling mechanisms involved in TMEM doorway function are only partially understood. Using two mouse models of breast cancer and an in vitro assay of intravasation, we report that CSF-1 secreted by the TMEM doorway tumor cell stimulates local secretion of VEGF-A from the Tie2^hi TMEM doorway macrophage, leading to the dissociation of endothelial junctions between TMEM doorway associated endothelial cells, supporting tumor cell intravasation. Acute blockade of CSF-1R signaling decreases macrophage VEGF-A secretion as well as TMEM doorway-associated vascular opening, tumor cell trans-endothelial migration, and dissemination. These new insights into signaling events regulating TMEM doorway function should be explored further as treatment strategies for metastatic disease.

Figure 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 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 with immunofluorescence (IF) with antibodies against endomucin (white), dextran (green), CSF-1 (red), and nuclear stain DAPI (blue). The two sequential sections from 1B and 1C were aligned and the same TMEM doorways were matched between the two sections, as indicated by the black circle in the IHC panel (1B) and white circle in the IF panels (1C) showing the same TMEM doorway after alignment. 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=20μm. D) Immunofluorescence measurement of CSF-1 levels in TMEM doorway tumor cells in active and inactive TMEM doorways. TMEM doorways were identified in the IF-stained sections as described above. Active TMEM doorways were identified by the presence of extravascular dextran which is not present in inactive TMEM doorways (see panel C). Next, tumor cells were identified using the Mena positive cells at TMEM doorway (panel 1B, IHC stain). The level of CSF-1 in the TMEM doorway tumor cells was measured in both active and inactive TMEM doorways using Visiopharm. n= 12 mice. ****P < 0.0001 analyzed by Student’s t-test.

Figure 2.. TMEM doorway macrophages secrete VEGF-A in response to CSF-1 and tumor cells secrete CSF-1.

A) VEGF-A ELISA of conditioned media obtained from BAC1.2F5 macrophages (Mϕ) treated with or without CSF-1 (3000 units/mL) for the times indicated. VEGF-A is indicated as concentration (pg/ml) of secreted protein. n=3 individual experiments done in duplicate, **p<0.01, by two-way ANOVA. B) CSF-1 ELISA of conditioned media obtained from MDA-MB 231 tumor cells (TCs). TCs were cultured for 24 hours in serum-free media and the concentration of CSF-1 (pg/mL) secreted by the TCs measured in the tumor cell conditioned media by ELISA. Control is media not exposed to tumor cells but treated in the same way as the cells. n=3 individual experiments done in duplicate. ****p<0.0001 by Student’s t-test. C) Immunofluorescence staining of serial PyMT tumor sections stained for CSF-1R (green), vasculature (CD31, magenta), macrophages (top: CD68, white; bottom: CD206, red) and DAPI (blue). The TMEM doorway is indicated with the yellow arrow, with each point of the triangle identifying each cell in the doorway, as described in Figure 1 (10). TMEM doorway cells are indicated with white arrows: endothelial cell (TEC, white circle); TMEM doorway macrophage (TM, blue circle); and TMEM doorway tumor cell (TTC, pink circle). Scale bar 30 μm. D) Quantification of the percent of CD68⁺ TMEM doorway macrophages and CD68⁺/CD206⁺ macrophages which stain positively for CSF-1R (n = 6).

Figure 3.. Tumor cell secreted CSF-1 increases CSF-1R dependent macrophage VEGF-A secretion.

A) VEGF-A ELISA of conditioned media obtained from BAC1.2F5 macrophages co-cultured with MDA-MB-231 tumor cells treated with control antibody (ctrl Ab), CSF-1R blocking Ab or CSF-1R inhibitor (GW2580), denoted as concentration (pg/mL) of secreted protein. n=3 individual experiments performed in duplicate, *p<0.05, ***p<0.001, ****p<0.0001 analyzed by two-way ANOVA. B) ELISA determination of VEGF-A (pg/mL) in medium obtained from BAC1.2F5 macrophages cultured in medium conditioned by MDA-MB-231 tumor cells (Tumor Cell CM) and treated with control Ab, CSF-1R blocking Ab (CSF-1R Ab) or CSF-1R inhibitor (GW2580). n=3 individual experiments performed in duplicate, ***p<0.001, ****p<0.0001 analyzed by two-way ANOVA. C) ELISA determination of VEGF-A (pg/mL) in medium conditioned y bone marrow macrophage (BMMs) either expressing (Csf1r^+/+) or lacking (Csf1r^−/−) CSF-1R co-cultured with MDA-MB-231 tumor cells for the times indicated. n=3 individual experiments done in duplicate, ****p<0.0001 analyzed by two-way ANOVA. D) ELISA determination of VEGF-A (pg/mL) in medium from Csf1r^+/+ or Csf1r^−/− BMMs cultured in MDA-MB-231 tumor cell conditioned medium (Tumor Cell CM) for the times indicated. n=3 individual experiments done in duplicate, ****p<0.0001 analyzed by two-way ANOVA. E) BAC1.2F5 macrophages, labelled with CellTracker^™ Green, were co-cultured with or without MDA-MB-231 tumor cells (TC), labelled with CellTracker^™ Red, and treated with either control or CSF-1R blocking antibodies for 48 hours. Cells were fixed, permeabilized and stained for VEGF-A. Macrophages were identified by the CellTracker Green label and only images of macrophages are shown. Heat map scale to right shows intensity of VEGF-A staining. Scale bar 5 μm. F) Quantitation of the VEGF-A fluorescence intensity in macrophages in (E). The amount of VEGF-A in the macrophage was quantified using ImageJ, as described in the methods section. n=3. *p<0.05, **p<0.01, analyzed by one-way ANOVA. G, H) Immuno-staining and quantification of VEGF-A intensity around TMEM doorways, obtained from PyMT mice treated with control antibody (ctrl Ab) or CSF-1R blocking Ab. Sequential tumor sections derived from PyMT tumors were stained by immunofluorescence (VEGF-A, Iba-1) and IHC (TMEM doorways-Mena, Iba-1, endomucin). TMEM doorways were identified as described in figure 1A. The circle in the IHC (black) and IF (white) panels show the same TMEM doorways obtained from the alignment of serial sections, and the three cells making up the TMEM doorway are pointed out with the yellow triangle in each panel. TMEM doorways are outlined in the circle with the endothelial cell (TE, blue stain, white circle), macrophage (TM, brown stain, teal circle) and TMEM doorway tumor cell (TTC, pink stain, pink circle) indicated with arrows. Next, the two sequential sections were aligned and TMEM doorway was identified in the IF-stained section. The immunofluorescence intensity of VEGF-A expression (green stain) in the identified TMEM doorway ROIs was measured and plotted here. N=analysis of images obtained from tissue sections from individual 11 mice, Scale bar 20 μm, not significant, analyzed by Student’s t-test.

Figure 4.. Inhibition of CSF-1R signaling reduces TAVO and trans-endothelial migration.

A) IF staining of tumor tissue from mice treated with control Ab or CSF-1R Ab were stained for CD31 (green), TMR-dextran (red), and ZO-1 (magenta). Right panel shows increased magnification of yellow box from middle panel to demonstrate overlap between CD31 and ZO-1 stains (merge/overlap indicated with white signal, and pointed out with white arrows). B) Quantification of extravascular 155 kDa dextran-TMR. *p<0.05. C) Vascular ZO-1 *p<0.05, and D) circulating tumor cells for mice treated with control Ab or CSF-1R blocking Ab in A). *p<0.05, analyzed by Student’s t-test. (B, C, D, n = 8) E) Quantitation of subluminal to luminal trans-endothelial migration (iTEM activity, which is a measure of number of intravasating cells in the intravasation direction) of MDA-MB-231 cells plated on the confluent and sealed endothelium either alone or with BAC1.2F5 macrophages. Tumor cells were treated with control antibody (ctrl Ab & DMSO), CSF-1R blocking Ab (CSF-1R Ab) or CSF-1R inhibitor (GW2580). Tumor cells were labelled with CellTracker^™ green which allowed us to identify and quantify the cells crossing. The number of MDA-MB-231 cells which crossed the endothelium were imaged using a confocal microscope and quantified using ImageJ software. n=3, *p<0.05 analyzed by one-way ANOVA. F) Quantitation of iTEM activity of MDA-MB-231 cells plated on the endothelium alone or with BMMs expressing or lacking CSF-1R expression. Tumor cells were labelled with CellTracker^™ green which allowed us to identify and quantify the cells crossing. The number of MDA-MB-231 cells which crossed the endothelium were imaged using a confocal microscope and quantified using ImageJ software. N=3, ***p< 0.001 analyzed by one-way ANOVA. G) Quantitation of iTEM activity of MDA-MB-231 cells transfected with either control siRNA or two different siRNAs targeting CSF-1 (#1, #2). Transfected cells were plated on the endothelium either alone or with BAC1.2F5 macrophages. Tumor cells were labelled with CellTracker^™ green which allowed us to identify and quantify the cells crossing. The number of MDA-MB-231 cells which crossed the endothelium were imaged using a confocal microscope and quantified using ImageJ software. n=3, **p<0.01, ***p< 0.001 analyzed by one-way ANOVA.

Figure 5.. Model of CSF-1-induced TMEM doorway opening and release of CTCs into the blood stream.

A) In the primary tumor, macrophages and tumor cells exhibit a CSF-1/EGF paracrine interaction between tumor cells and macrophages which facilitates tumor cell migration toward blood vessels in breast cancer. Tumor cells secrete CSF-1 which attracts CSF-1R expressing macrophages, which in turn secrete EGF, increasing the migration of EGFR-expressing tumor cells. The tumor cells within the loop encounter an HGF signal found in a gradient in the tumor microenvironment with the highest concentration near blood vessels. The tumor cells migrate toward the blood vessels along the HGF gradient, adding directionality to the migration of the tumor cell-macrophage paracrine loop engaged partners. B) In an inactive TMEM doorway (box), with the absence of any CSF-1 secreted by the TMEM doorway tumor cell (TTC), the VEGF-A remains within the TMEM doorway macrophage (TM). The blood vessels, including the TMEM doorway endothelial cell (TEC) within the tumor remain sealed and do not allow tumor cells to intravasate through TMEM doorways. C) In contrast, in an active TMEM doorway, CSF-1 is secreted by the TMEM doorway tumor cell and binds to macrophage CSF-1R, stimulating the TMEM doorway macrophage to secrete its VEGF-A. VEGF-A causes vascular opening (TAVO event) and allows tumor cells to intravasate through the TMEM doorway, creating CTCs, and dextran or blood to leak out of the vessel. D) These circulating tumor cells (CTCs) travel through the vasculature to secondary sites where TMEM doorways are also found in metastatic foci in lymph nodes and lungs. Thin membranous connections between macrophages and tumor cells stretch from the macrophage on extravascular side of the blood vessel through the endothelial junctions and interact with CTCs which could facilitate CTC extravasation at secondary sites. TMEM doorways in metastases could also be the sites were tumor cells re-intravasate and then seed tertiary metastases. Figure created with BioRender.com.