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Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway.

Katanov C, Lerrer S, Liubomirski Y, Leider-Trejo L, Meshel T, Bar J, Feniger-Barish R, Kamer I, Soria-Artzi G, Kahani H, Banerjee D, Ben-Baruch A - Stem Cell Res Ther (2015)

Bottom Line: CCL2 release by TNF-α-stimulated MSCs was mediated by TNF-RI and TNF-RII, through the NF-κB but not via the AP-1 pathway.Exposure of MSCs to TNF-α led to potent CCL2-induced migration of monocytic cells, a process that may yield pro-cancerous myeloid infiltrates in breast tumors.Our novel results emphasize the important roles of inflammation-stroma interactions in breast cancer, and suggest that NF-κB may be a potential target for inhibition in tumor-adjacent stromal cells, enabling improved tumor control in inflammation-driven malignancies.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, P.O. Box 39040, Tel Aviv, 6997801, Israel. christina.katanov@weizmann.ac.il.

ABSTRACT

Introduction: Breast cancer progression is promoted by stromal cells that populate the tumors, including cancer-associated fibroblasts (CAFs) and mesenchymal stem/stromal cells (MSCs). The activities of CAFs and MSCs in breast cancer are integrated within an intimate inflammatory tumor microenvironment (TME) that includes high levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β). Here, we identified the impact of TNF-α and IL-1β on the inflammatory phenotype of CAFs and MSCs by determining the expression of inflammatory chemokines that are well-characterized as pro-tumorigenic in breast cancer: CCL2 (MCP-1), CXCL8 (IL-8) and CCL5 (RANTES).

Methods: Chemokine expression was determined in breast cancer patient-derived CAFs by ELISA and in patient biopsies by immunohistochemistry. Chemokine levels were determined by ELISA in (1) human bone marrow-derived MSCs stimulated by tumor conditioned media (Tumor CM) of breast tumor cells (MDA-MB-231 and MCF-7) at the end of MSC-to-CAF-conversion process; (2) Tumor CM-derived CAFs, patient CAFs and MSCs stimulated by TNF-α (and IL-1β). The roles of AP-1 and NF-κB in chemokine secretion were analyzed by Western blotting and by siRNAs to c-Jun and p65, respectively. Migration of monocytic cells was determined in modified Boyden chambers.

Results: TNF-α (and IL-1β) induced the release of CCL2, CXCL8 and CCL5 by MSCs and CAFs generated by prolonged stimulation of MSCs with Tumor CM of MDA-MB-231 and MCF-7 cells. Patient-derived CAFs expressed CCL2 and CXCL8, and secreted CCL5 following TNF-α (and IL-1β) stimulation. CCL2 was expressed in CAFs residing in proximity to breast tumor cells in biopsies of patients diagnosed with invasive ductal carcinoma. CCL2 release by TNF-α-stimulated MSCs was mediated by TNF-RI and TNF-RII, through the NF-κB but not via the AP-1 pathway. Exposure of MSCs to TNF-α led to potent CCL2-induced migration of monocytic cells, a process that may yield pro-cancerous myeloid infiltrates in breast tumors.

Conclusions: Our novel results emphasize the important roles of inflammation-stroma interactions in breast cancer, and suggest that NF-κB may be a potential target for inhibition in tumor-adjacent stromal cells, enabling improved tumor control in inflammation-driven malignancies.

No MeSH data available.


Related in: MedlinePlus

Impact of prolonged stimulation by Tumor CM on the release of inflammatory chemokines by the resulting CAFs. Human BM-derived MSCs were cultured with Tumor CM from MDA-MB-231 cells (MDA) (A) or MCF-7 cells (B) over a prolonged period of time (~30 days; MSCs + MDA CM or MSCs + MCF-7 CM, respectively). Twenty-four hours after medium exchange to fresh Tumor CM, cell supernatants were collected and the expression of CCL2 (A1, B1), CXCL8 (A2, B2) and CCL5 (A3, B3) was determined in comparison with supernatants of MSCs that were not supplemented with CM (MSCs) and with the original Tumor CM of MDA-MB-231 or MCF-7 cells alone (MDA CM or MCF-7 CM, respectively). Chemokine expression was determined by ELISA, in the linear range of absorbance. (A1), (A2), (B1) Representatives of n = 3 independent experiments that have shown similar results. (A3), (B2), (B3) Ratios between MSCs and MSCs + Tumor CM were not consistent in different experimental repeats. Therefore, in these panels, the findings are presented as mean ± standard deviation of normalized values (MSCs were given the value of 1) obtained in relevant experimental repeats (at least n = 3).
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Fig1: Impact of prolonged stimulation by Tumor CM on the release of inflammatory chemokines by the resulting CAFs. Human BM-derived MSCs were cultured with Tumor CM from MDA-MB-231 cells (MDA) (A) or MCF-7 cells (B) over a prolonged period of time (~30 days; MSCs + MDA CM or MSCs + MCF-7 CM, respectively). Twenty-four hours after medium exchange to fresh Tumor CM, cell supernatants were collected and the expression of CCL2 (A1, B1), CXCL8 (A2, B2) and CCL5 (A3, B3) was determined in comparison with supernatants of MSCs that were not supplemented with CM (MSCs) and with the original Tumor CM of MDA-MB-231 or MCF-7 cells alone (MDA CM or MCF-7 CM, respectively). Chemokine expression was determined by ELISA, in the linear range of absorbance. (A1), (A2), (B1) Representatives of n = 3 independent experiments that have shown similar results. (A3), (B2), (B3) Ratios between MSCs and MSCs + Tumor CM were not consistent in different experimental repeats. Therefore, in these panels, the findings are presented as mean ± standard deviation of normalized values (MSCs were given the value of 1) obtained in relevant experimental repeats (at least n = 3).

Mentions: Student’s t test was used to calculate P values for ELISAs and chemotaxis assays. P <0.05 was considered significant. Adjustment for multiplicity of comparisons was done using the Benjamini–Hochberg procedure. Using this procedure, all of the significant results that were presented in the manuscript remained statistically significant after correcting for their multiplicity. The ELISA results presented in the paper show a representative experiment out of n ≥ 3 independent experiments that have shown similar trends. An exception is shown in Figure 1A3,B2,B3, where the effect of Tumor CM on chemokine release was not consistent; therefore, in these cases, the results are presented as mean ± standard deviation of the different experiments (MSCs were given the value of 1). The confocal pictures are representatives of many pictures that were taken in at least three independent experiments.Figure 1


Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway.

Katanov C, Lerrer S, Liubomirski Y, Leider-Trejo L, Meshel T, Bar J, Feniger-Barish R, Kamer I, Soria-Artzi G, Kahani H, Banerjee D, Ben-Baruch A - Stem Cell Res Ther (2015)

Impact of prolonged stimulation by Tumor CM on the release of inflammatory chemokines by the resulting CAFs. Human BM-derived MSCs were cultured with Tumor CM from MDA-MB-231 cells (MDA) (A) or MCF-7 cells (B) over a prolonged period of time (~30 days; MSCs + MDA CM or MSCs + MCF-7 CM, respectively). Twenty-four hours after medium exchange to fresh Tumor CM, cell supernatants were collected and the expression of CCL2 (A1, B1), CXCL8 (A2, B2) and CCL5 (A3, B3) was determined in comparison with supernatants of MSCs that were not supplemented with CM (MSCs) and with the original Tumor CM of MDA-MB-231 or MCF-7 cells alone (MDA CM or MCF-7 CM, respectively). Chemokine expression was determined by ELISA, in the linear range of absorbance. (A1), (A2), (B1) Representatives of n = 3 independent experiments that have shown similar results. (A3), (B2), (B3) Ratios between MSCs and MSCs + Tumor CM were not consistent in different experimental repeats. Therefore, in these panels, the findings are presented as mean ± standard deviation of normalized values (MSCs were given the value of 1) obtained in relevant experimental repeats (at least n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4469428&req=5

Fig1: Impact of prolonged stimulation by Tumor CM on the release of inflammatory chemokines by the resulting CAFs. Human BM-derived MSCs were cultured with Tumor CM from MDA-MB-231 cells (MDA) (A) or MCF-7 cells (B) over a prolonged period of time (~30 days; MSCs + MDA CM or MSCs + MCF-7 CM, respectively). Twenty-four hours after medium exchange to fresh Tumor CM, cell supernatants were collected and the expression of CCL2 (A1, B1), CXCL8 (A2, B2) and CCL5 (A3, B3) was determined in comparison with supernatants of MSCs that were not supplemented with CM (MSCs) and with the original Tumor CM of MDA-MB-231 or MCF-7 cells alone (MDA CM or MCF-7 CM, respectively). Chemokine expression was determined by ELISA, in the linear range of absorbance. (A1), (A2), (B1) Representatives of n = 3 independent experiments that have shown similar results. (A3), (B2), (B3) Ratios between MSCs and MSCs + Tumor CM were not consistent in different experimental repeats. Therefore, in these panels, the findings are presented as mean ± standard deviation of normalized values (MSCs were given the value of 1) obtained in relevant experimental repeats (at least n = 3).
Mentions: Student’s t test was used to calculate P values for ELISAs and chemotaxis assays. P <0.05 was considered significant. Adjustment for multiplicity of comparisons was done using the Benjamini–Hochberg procedure. Using this procedure, all of the significant results that were presented in the manuscript remained statistically significant after correcting for their multiplicity. The ELISA results presented in the paper show a representative experiment out of n ≥ 3 independent experiments that have shown similar trends. An exception is shown in Figure 1A3,B2,B3, where the effect of Tumor CM on chemokine release was not consistent; therefore, in these cases, the results are presented as mean ± standard deviation of the different experiments (MSCs were given the value of 1). The confocal pictures are representatives of many pictures that were taken in at least three independent experiments.Figure 1

Bottom Line: CCL2 release by TNF-α-stimulated MSCs was mediated by TNF-RI and TNF-RII, through the NF-κB but not via the AP-1 pathway.Exposure of MSCs to TNF-α led to potent CCL2-induced migration of monocytic cells, a process that may yield pro-cancerous myeloid infiltrates in breast tumors.Our novel results emphasize the important roles of inflammation-stroma interactions in breast cancer, and suggest that NF-κB may be a potential target for inhibition in tumor-adjacent stromal cells, enabling improved tumor control in inflammation-driven malignancies.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, P.O. Box 39040, Tel Aviv, 6997801, Israel. christina.katanov@weizmann.ac.il.

ABSTRACT

Introduction: Breast cancer progression is promoted by stromal cells that populate the tumors, including cancer-associated fibroblasts (CAFs) and mesenchymal stem/stromal cells (MSCs). The activities of CAFs and MSCs in breast cancer are integrated within an intimate inflammatory tumor microenvironment (TME) that includes high levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β). Here, we identified the impact of TNF-α and IL-1β on the inflammatory phenotype of CAFs and MSCs by determining the expression of inflammatory chemokines that are well-characterized as pro-tumorigenic in breast cancer: CCL2 (MCP-1), CXCL8 (IL-8) and CCL5 (RANTES).

Methods: Chemokine expression was determined in breast cancer patient-derived CAFs by ELISA and in patient biopsies by immunohistochemistry. Chemokine levels were determined by ELISA in (1) human bone marrow-derived MSCs stimulated by tumor conditioned media (Tumor CM) of breast tumor cells (MDA-MB-231 and MCF-7) at the end of MSC-to-CAF-conversion process; (2) Tumor CM-derived CAFs, patient CAFs and MSCs stimulated by TNF-α (and IL-1β). The roles of AP-1 and NF-κB in chemokine secretion were analyzed by Western blotting and by siRNAs to c-Jun and p65, respectively. Migration of monocytic cells was determined in modified Boyden chambers.

Results: TNF-α (and IL-1β) induced the release of CCL2, CXCL8 and CCL5 by MSCs and CAFs generated by prolonged stimulation of MSCs with Tumor CM of MDA-MB-231 and MCF-7 cells. Patient-derived CAFs expressed CCL2 and CXCL8, and secreted CCL5 following TNF-α (and IL-1β) stimulation. CCL2 was expressed in CAFs residing in proximity to breast tumor cells in biopsies of patients diagnosed with invasive ductal carcinoma. CCL2 release by TNF-α-stimulated MSCs was mediated by TNF-RI and TNF-RII, through the NF-κB but not via the AP-1 pathway. Exposure of MSCs to TNF-α led to potent CCL2-induced migration of monocytic cells, a process that may yield pro-cancerous myeloid infiltrates in breast tumors.

Conclusions: Our novel results emphasize the important roles of inflammation-stroma interactions in breast cancer, and suggest that NF-κB may be a potential target for inhibition in tumor-adjacent stromal cells, enabling improved tumor control in inflammation-driven malignancies.

No MeSH data available.


Related in: MedlinePlus