T cell induced expression of Coronin-1A facilitates blood-brain barrier transmigration of breast cancer cells

Abstract

In previous work we discovered that T lymphocytes play a prominent role in the rise of brain metastases of ER-negative breast cancers. In the present study we explored how T lymphocytes promote breast cancer cell penetration through the blood brain barrier (BBB). An in vitro BBB model was employed to study the effects of T lymphocytes on BBB trespassing capacity of three different breast carcinoma cell lines. Differential protein expression was explored by comparing the proteomes of the breast cancer cells before and after co-culture with activated T lymphocytes using liquid chromatography-mass spectrometry (LC-MS). siRNA was used to silence protein expression in the breast cancer cells to study contribution to in vitro BBB passage. Furthermore, protein expression in primary breast cancer tissues was explored and related to brain-metastatic potential. Co-culturing with activated T lymphocytes or their conditioned medium (CM) resulted in increased passage through the in vitro BBB. The effects were less for cell line MDA-MB-231-B2M2 (brain affinity) as compared to MDA-MB-231 and SK-BR-7. Mass spectrometry-based proteomics revealed significant alterations in the expression of 35 proteins by the breast cancer cell lines upon T cell contact. Among the proteins is coronin-1 A, a protein related to cell motility. Knockdown of CORO1A in the breast cancer cells reduced their ability to cross the artificial BBB to 60%. The effects were significantly less for the cell line derived from breast cancer with affinity for brain. The expression of coronin-1A was confirmed by immunohistochemistry and RT-PCR of 52 breast cancer samples of patients with metastasized breast cancers, with and without brain locations. Lastly, CORO1A upregulation was validated in a publicly available mRNA expression database from 204 primary breast cancers with known metastatic sites. We conclude that T lymphocytes trigger cancer cells to express proteins including coronin-1A that enable the cancer cells to cross an in vitro BBB. In addition, a prominent role of coronin-1A in the formation of cerebral metastases in breast cancer patients is strongly suggestive by its upregulation in tissue samples of breast cancer patients with brain metastases.

Keywords: Blood–brain barrier; Brain metastasis; Breast cancer; Coronin-1A; Liquid chromatography-mass spectrometry; Proteomics.

Fig. 1

BBB passage following co-culture with T cells or T cell medium. Co-culture of breast cancer cell lines with activated T lymphocytes stimulated passage of the cancer cells through the artificial BBB. The passage of MDA-MB-231-B2M2 cells was less than for MDA-MB-231 and SK-BR-7. The stimulating effect of T lymphocyte conditioned medium (CM) was grossly similar for all three cell lines and less than the effect of direct T cell contact. MDA-MB-231: ER-/PR-/Her2-/E-cadherin-/TP53^mut breast cancer cell line derived from invasive ductal carcinoma; MDA-MB-231-B2M2: derived from MDA-MB-231 with metastatic preference for brain; SK-BR-3: Her2 + breast cancer cell line from metastasizing tumor without specific organ preference. Control = matched cancer cells without pre-treatment; +T lymphocytes = cancer cells following five days of co-culture with activated T lymphocytes; + CM = cancer cells following five days incubation with cell-free media conditioned by activated T lymphocytes. Data representative of three independent experiments. For abbreviated cell lines see M&M.

Fig. 2

Venn diagram of proteomics measurements. Total numbers of proteins measured in the cancer cell lines prior to, and incubation with activated T lymphocytes. 307 proteins were identified only in cancer cell lines after incubation with T lymphocytes.

Fig. 3

Coronin-1 A is upregulated in breast cancer cells after contact with T lymphocytes. (a) Unique peptide counts for Coronin-1 A; (b) Elevated CORO1A mRNA expression levels in MDA-MB-231 cells following direct contact with activated T lymphocytes, or incubation with T cells separated from the tumor cells with a 0.4 micron filter, or with CM of activated T lymphocytes. Control = matched cancer cells without pre-treatment; + T = cancer cells after 5 days of co-culture with activated T lymphocytes, + CM = cancer cells after 5 days incubation with cell-free media conditioned by activated T lymphocytes.

Fig. 4

CORO1A siRNA impairs the BBB passage in all three breast cancer cell lines. (a) CORO1A mRNA levels in all three breast cancer cell lines before and after CORO1A siRNA for 72 h. mRNA expression is reduced to almost undetectable in all three cell lines. Data representative of two independent experiments. (b) Fraction of breast cancer cells passing the BBB before and following siRNA CORO1A and subsequent co-culture with activated T lymphocytes. The fraction of cancer cells not silenced for CORO1A (siSham) are taken as fraction = 1. The fractions of crossing cells are significantly reduced for MDA-MB-231 and MDA-MB-231-B2M2. The fraction of SK-BR-7 cells decreased, though not significantly. Data representative of two independent experiments. (c) Coronin-1A immunofluorescence (IF) of MDA-MB-231 without (left) and after (right) co-culture with activated T lymphocytes. The cancer cells express coronin-1A upon T cell contact. (d) Coronin 1A-IF FOC intensity-levels in the breast cancer cell lines before and after 72 h of co-culture with activated T lymphocytes. For cell line MDA-MB-231 and SK-BR-7 upregulation is robust, but remains marginal in MDA-MB-231-B2M2 (data representative of three independent experiments). (e) Example of coronin-1A-IF of MDA-MB-231 siSham cells (left) and of MDA-MB-231 siCORO1A following co-culture with activated T lymphocytes (right). (f) Coronin 1A protein fraction levels before and after 72 h incubation with non-targeting siSham or siRNA CORO1A were equally reduced in all three cell lines. Data representative of three independent experiments.

Fig. 5

Coronin 1A is highly expressed in primary breast cancers from patients who developed brain metastases. (a) Coronin 1A immunohistochemistry mean-intensity levels are significantly higher in the group of breast cancers from patients who developed brain metastasis (n = 10, Brain Mets) when compared with breast cancers from patients with metastases to organs other than brain (n = 22, Other Mets), p = 0.0035. The lowest and highest boundaries of the box represent the 25 and 75 percentiles, respectively. The solid line across the box indicates the median value. Error bars indicate the 5–95 percentile. (b) CORO1A mRNA relative (REL) and fold of change (FOC) expression levels in the two groups described in a), p > 0.05. (c) Example of coronin-1A IHC of primary breast cancers from patients who developed brain metastasis (left) or metastasis to other organs than brain (right). (d) CORO1A mRNA expression levels of 204 primary breast cancers associated with different sites of metastasis: brain (n = 8); brain and other organs (n = 8); other metastatic organs (n = 169) and metastasis to the contralateral breast (n = 19). Dataset information is publicly available under GEO accession number GSE12276, EXP00013 [37].