Heterogeneity of chemokine cell-surface receptor expression in triple-negative breast cancer

Abstract

Introduction: Tumor heterogeneity is a well-established concept in cancer research. In this paper, we examine an additional type of tumor cell heterogeneity - tumor cell-surface receptor heterogeneity.

Methods: We use flow cytometry to measure the frequency and numbers of cell-surface receptors on triple negative breast cancer cell lines.

Results: We find two distinct populations of human triple-negative breast cancer cells MDA-MB-231 when they are grown in culture, one with low surface levels of various chemokine receptors and a second with much higher levels. The population with high surface levels of these receptors is increased in the more metastatic MDA-MB-231-luc-d3h2ln cell line.

Conclusion: We hypothesize that this high cell-surface receptor population is involved in metastasis. We find that the receptor high populations can be modulated by tumor conditioned media and IL6 treatment indicating that the tumor microenvironment is important for the maintenance and sizes of these populations.

Keywords: CCR5; CXCR3; CXCR4; IL6; MDA-MB-231; stem cells.

Figure 1

MB231 and cell MB231-luc ACEA motility assay. A. Plot of cell index over time for 24 hours. B. The average motility index for MB231 and MB231-luc cells at 24 h.

Figure 2

The receptor high populations in MB231 and MB231-luc cells measured by flow cytometry. A. The CXCR3 receptor high population in MB231 cells. B. The CXCR1 receptor high population in MB231 cells. C. The CXCR3 receptor high population in MB231-luc cells. D. The CXCR1 receptor high population for MB231-luc cells.

Figure 3

Percentages and quantification of receptor high populations in MB231 and MB231-luc cells. A. The percentage of cells in the CCR5 and CXCR3 receptor high populations. B. The percentage of cells in the CXCR1 and CXCR4 receptor high populations. C. Quantification of the overlap between CXCR3 and CCR5 surface receptors. The level of CXCR3 in the CCR5 low populations, the level of CXCR3 in the CCR5 high populations, and the levels of CCR5 in the CXCR3 high populations are quantified.

Figure 4

The percentages of receptor high populations of MB231 and MB231-luc cells treated with control and tumor-conditioned media (TCM). A. The CXCR3 receptor high population in MB231 treated with regular media. B. The CXCR3 receptor high population in MB231 treated with tumor-conditioned media. C. The percentages of CCR5 and CXCR3 receptor high populations in MB231 cells treated with control media or TCM. D. The percentages of CCR5 and CXCR3 receptor high populations in MB231-luc cells treated with control media or TCM.

Figure 5

Quantification of surface receptor low and high populations of cells treated with control media and tumor-conditioned media. A. Quantification of MB231 cell surface receptors. B. Quantification of MB231-luc cell surface receptors.

Figure 6

The effects of IL6 treatment on CCR5 and CXCR3 cell surface receptors on MB231 cells. A. The percentages of receptor high populations on MB231 cells treated with control or IL6. B. Quantification of the number of surface receptors in the high and low populations of MB231 cells treated with control or IL6.

Figure 7

In vivo measurements of receptor high populations in MB231-luc tumor xenografts. A. The percentage of receptor high populations in three different tumors. B. Quantification of surface receptor numbers in the low receptor population. C. Quantification of surface receptor numbers in the high receptor population.

Figure 8

Percentages of the stem cell populations in MB231 and MB231-luc cell lines. A. Gating of the CD24-low and CD44-high receptor populations. B. The percentages of stem cells in MB231 and MB231-luc cell lines treated with control media or TCM.