Invasive breast carcinoma cells from patients exhibit MenaINV- and macrophage-dependent transendothelial migration

Abstract

Metastasis is a complex, multistep process of cancer progression that has few treatment options. A critical event is the invasion of cancer cells into blood vessels (intravasation), through which cancer cells disseminate to distant organs. Breast cancer cells with increased abundance of Mena [an epidermal growth factor (EGF)-responsive cell migration protein] are present with macrophages at sites of intravasation, called TMEM sites (for tumor microenvironment of metastasis), in patient tumor samples. Furthermore, the density of these intravasation sites correlates with metastatic risk in patients. We found that intravasation of breast cancer cells may be prevented by blocking the signaling between cancer cells and macrophages. We obtained invasive breast ductal carcinoma cells of various subtypes by fine-needle aspiration (FNA) biopsies from patients and found that, in an in vitro transendothelial migration assay, cells that migrated through a layer of human endothelial cells were enriched for the transcript encoding Mena(INV), an invasive isoform of Mena. This enhanced transendothelial migration required macrophages and occurred with all of the breast cancer subtypes. Using mouse macrophages and the human cancer cells from the FNAs, we identified paracrine and autocrine activation of colony-stimulating factor-1 receptor (CSF-1R). The paracrine or autocrine nature of the signal depended on the breast cancer cell subtype. Knocking down Mena(INV) or adding an antibody that blocks CSF-1R function prevented transendothelial migration. Our findings indicate that Mena(INV) and TMEM frequency are correlated prognostic markers and CSF-1 and Mena(INV) may be therapeutic targets to prevent metastasis of multiple breast cancer subtypes.

Fig. 1. Correlation of Mena isoform expression to TMEM score

(A) TMEM microanatomic cancer cell TEM sites visualized by IHC. TC, Mena-expressing tumor cells; EC, CD31-expressing vascular endothelial cells; M, CD68-expressing macrophages. Scale bars, 300 μm (left) and 50 μm (right). (B and C) Scatter plots of relative Mena^INV transcript expression against TMEM score in the entire cohort of 100 IDCs (B) or by clinical subtype (C). Data were analyzed by rank-order correlation. The differences in slopes between subtypes were not statistically significant as shown by the regression model fit to the rank-transformed data. n, number of tumor cases.

Fig. 2. Correlation of Mena^INV-TMEM scores in different grades of human IDCs

(A) Scatter plots of relative Mena^INV transcript expression against TMEM score in IDCs of different grades. Data were analyzed with Spearman’s rank-order correlation. The differences in slopes between grades were not statistically significant as shown by the regression model fit to the rank-transformed data. (B) Representative microscopy of poorly differentiated (upper), moderately differentiated (lower), and well-differentiated (bottom) IDCs triple-stained for TMEM. TMEM sites are indicated by circles. Scale bar, 100 μm. (C) TMEM score (left panel) and relative Mena^INV transcript abundance (right panel) according to tumor grade. Median, 5th and 95th percentile for Mena^INV fold change, and TMEM score are presented for well-differentiated (n = 9), moderately differentiated (n = 40), and poorly differentiated (n = 51) tumors. Analyses were done using the Mann-Whitney U test at a statistical significance threshold of 0.008.

Fig. 3. Correlation of E-cadherin and TMEM and Mena^INV scores

(A and B) E-cadherin abundance in tumor tissues classified as either (A) low TMEM and low Mena^INV (scores <10 and <1, respectively) or high TMEM and high Mena^INV (scores >50 and >5, respectively). (C) Representative images showing staining scores of 0 (none) to 3 (strong). Data are means ± SEM from ten 40× fields scored from 10 cases in each group. P < 0.001, number of cells in the low TMEM-Mena^INV group that had moderate to strong E-cadherin staining (2 and 3) compared with that in the high TMEM-Mena^INV group. P < 0.0001, number of cells in the high TMEM-Mena^INV group that had none to weak E-cadherin staining (0 and 1) compared with that in the low TMEM-Mena^INV group. Data were analyzed with unpaired, two-tailed Student’s t tests assuming equal variances and an α level of 0.05.

Fig. 4. iTEM assays using human patient IDC cells obtained from FNA biopsies

(A) Fold increase in the number of cells from ERPR⁺ or TN cases that crossed a HUVEC monolayer in the presence of macrophages (Φ) relative to the number that crossed in the absence of macrophages. Data are means ± SEM from three experiments. **P < 0.005, ***P < 0.0005, by two-tailed Student’s t tests assuming equal variances. (B) Mena^INV or Mena11a transcript expression in cells that crossed the HUVEC monolayer relative to that in the loaded cell population. Data are from 16 patient cases. (C) Average Mena^INV and Mena11a isoform expression in iTEM-competent cells from (B) grouped by clinical subtype. Data were not significantly different by a Student’s t test. (D) Apical z-sections from the iTEM assay. Tumor cells, green; macrophages, blue; HUVEC junctions (ZO-1), red. Squares indicate dissociating HUVEC junctions, magnified below. Right: Representative z-stacks by clinical subtype. Data are representative of each from the 16 cases in (B). (E) Mena^INV and Mena11a transcript expression in iTEM-competent cells from TN human tissue transplants HT17 and HT39. Data are means ± SEM from three mice each. (F) Apical z-sections of the iTEM assay with HT17 tumor cells as in (D). Images are representative of three experiments.

Fig. 5. Mena^INV is required for macrophage-induced iTEM in MDA-MB-231 TN breast cancer cells

(A) Transcript abundance for total Mena, Mena11a, and Mena^INV in parental MDA-MB-231 cells transfected with one of three Mena^INV-targeted siRNAs. (B) iTEM of MDA-MB-231 cells after Mena^INV depletion in the presence or absence of macrophages (Φ). (C) iTEM of MDA-MB-231 cells expressing GFP-tagged Mena^INV, alone or cotransfected with Mena^INV-targeted siRNA (1). iTEM was performed in the presence of macrophages. Data in (A) to (C) are means ± SEM from three experiments. *P < 0.05, **P < 0.005, ***P < 0.0005, by two-tailed Student’s t tests assuming equal variances.

Fig. 6. CSF1R expression in iTEM-competent cells differs among breast cancer subtypes

(A) The number of macrophages associating with tumor cells during iTEM, presented as a ratio by clinical subtype. Data are means ± SEM from twelve 63× microscopic fields in two to three cases each. *P < 0.05 compared with that in ERPR⁺/HER2⁻ cases. (B and C) CSF1R transcript abundance in iTEM-competent cells from each of 15 cases analyzed across clinical subtypes (B). Data in (C) are means ± SEM by clinical subtype shown in (B). (D) Effect of mouse-specific CSF-1R antibodies or human-specific CSF-1R antibodies on iTEM of ERPR⁺/HER2⁻, ERPR⁻/HER2⁺ (HER2⁺), and TN cancers. Data are means ± SEM from three cases per subtype relative to the background (tumor cells cross without the presence of macrophages). Control, tumor cells cross in the presence of macrophages. (E and F) CSF1R transcript abundance in iTEM-competent cells from patient tumor transplants HT17 and HT39 (E), and the effect of mouse- or human-specific CSF-1R functionally blocking antibodies on iTEM capacity. Data are means ± SEM from five mice per tumor sample. **P < 0.005, ***P < 0.0005. Statistical analyses in (A), (C), (D), and (F) used two-tailed Student’s t tests assuming equal variances.

Fig. 7. Primary human macrophages increase iTEM of primary human breast cancer cells

(A) Number of iTEM-competent cells from TN human tissue transplants HT17 and HT39 in the presence or absence of primary human macrophages (Φ). (B) Representative apical z-sections of the iTEM assay demonstrating HT39 cells (green) and crossing the endothelial monolayer (red) in close proximity with macrophages (blue). (C and D) Relative Mena^INV and Mena11a (C) or CSF-1R (D) transcript abundance in iTEM-competent HT17 and HT39 cells in the presence of primary human macrophages. Data are means ± SEM from three mice for each tumor. ***P < 0.0005, two-tailed Student’s t tests assuming equal variances.

Fig. 8. Model of Mena^INV- and macrophage-mediated tumor cell invasion and TEM

ITEM-competent human breast cancer cells from all clinically relevant subtypes display Mena^INV high/Mena11a low isoform expression and perivascular macrophages promote endothelial transmigration at TMEM sites (outlined). iTEM in ERPR⁺/HER2⁻ cells is promoted by paracrine CSF-1–CSF-1R signaling, whereas iTEM in HER2⁺ and TN cells is promoted by both paracrine and autocrine CSF-1–CSF-1R signaling. R, receptor.