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. 2016 Dec 13;7(50):82482-82492.
doi: 10.18632/oncotarget.12694.

Mesenchymal stem cells and macrophages interact through IL-6 to promote inflammatory breast cancer in pre-clinical models

Adam R Wolfe  1   2 , Nicholaus J Trenton  3 , Bisrat G Debeb  1   2 , Richard Larson  1   2 , Brian Ruffell  4 , Khoi Chu  5 , Walter Hittelman  5 , Michael Diehl  3 , Jim M Reuben  1 , Naoto T Ueno  1   6 , Wendy A Woodward  1   2
Affiliations

Mesenchymal stem cells and macrophages interact through IL-6 to promote inflammatory breast cancer in pre-clinical models

Adam R Wolfe et al. Oncotarget. .

Abstract

Inflammatory breast cancer (IBC) is a unique and deadly disease with unknown drivers. We hypothesized the inflammatory environment contributes to the IBC phenotype. We used an in vitro co-culture system to investigate interactions between normal and polarized macrophages (RAW 264.7 cell line), bone-marrow derived mesenchymal stem cells (MSCs), and IBC cells (SUM 149 and MDA-IBC3). We used an in vivo model that reproduces the IBC phenotype by co-injecting IBC cells with MSCs into the mammary fat pad. Mice were then treated with a macrophage recruitment inhibitor, anti-CSF1. MSC and macrophages grown in co-culture produced higher levels of pro-tumor properties such as enhanced migration and elevated IL-6 secretion. IBC cells co-cultured with educated MSCs also displayed enhanced invasion and mammosphere formation and blocked by anti-IL-6 and statin treatment. The treatment of mice co-injected with IBC cells and MSCs with anti-CSF1 inhibited tumor associated macrophages and inhibited pSTAT3 expression in tumor cells. Anti-CSF1 treated mice also exhibited reduced tumor growth, skin invasion, and local recurrence. Herein we demonstrate reciprocal tumor interactions through IL-6 with cells found in the IBC microenvironment. Our results suggest IL-6 is a mediator of these tumor promoting influences and is important for the IBC induced migration of MSCs.

Keywords: IL-6; inflammatory breast cancer; macrophages; mesenchymal stem cells; statins.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1. Model of Co-Culture System
Macrophages were polarized with either LPS or IL-4 to become M1 or M2 macrophages respectively. Using a Boyden chamber MSCs were seeded with M1 or M2 macrophages for 24 hours to become either MSC1s or MSC2s. MSC1s or MSC2s were then co-cultured in Boyden chambers with IBC cells for 24 hours. Following co-culture MSCs and IBC cells were analyzed for migration, invasion, and mammosphere formation.
Figure 2
Figure 2. MSCs and macrophages cross-talk through IL-6
Following co-culture of with either un-polarized (M0), LPS induced (M1), or IL-4 induced (M2), and MSCs for 24 hours, (A) the number of MSCs that passed through the transwell membrane after 24 hours was counted. Significant differences are shown as follows: *P < 0.05 for unpaired 2-tailed Student's t-test (n = 3). (B) Macrophages were grown in the presence of MSC conditioned media (CM) for 24 hours and then stained with either anti-CD206 or anti-arginase-1 antibodies and submitted for flow cytometry analysis. (C) Following 24 hours of co-culture with macrophages and MSCs, supernatant was collected for IL-6 ELISA. (D) MSC or MSC2s were co-cultured with SUM149 and IBC-3 cells for 24 hours with or without anti-IL6 antibodies. The number of MSCs that passed through the transwell membrane after 24 hours was counted. Significant differences are shown as follows: *P < 0.05 for unpaired 2-tailed Student's t-test (n = 3).
Figure 3
Figure 3. Crosstalk between educated MSCs and IBC cells increases IBC invasion and self-renewal through IL-6
Following 24 hours of co-culture with M2 macrophages, the “educated” MSC2s or parental MSCs were co-cultured with (A) SUM149 or (B) MDA-IBC3 for 24 hours with or without anti-IL6 antibodies. The number of IBC cells that invaded through the basement membrane transwell after 24 hours was counted. Significant differences are shown as follows: *P < 0.05 for unpaired 2-tailed Student's t-test (n = 3). (C) SUM149 or (D) MDA-IBC3 cells were co-cultured with MSC or MSC2s for 24 hours with or without anti-IL6 antibodies. IBC cells were then seeded in self-renewal mammosphre media promoting suspension culture conditions. Significant differences are shown as follows: *P < 0.05 for unpaired 2-tailed Student's t-test (n = 3).
Figure 4
Figure 4. Simvastatin blocks IL-6 secretion and inhibits the effects of M2 educated MSCs on IBC invasion and self-renewal
(A) MSC or MSC2s were treated with DMSO (control) or simvastatin for 4 hours and the supernatant was submitted for IL-6 ELISA. MSCs and MSC2s were either pre-treated with simvastatin or simvastatin plus IL-6 for 4 hours then co-cultured with SUM149 cells for 24 hours. (B) Following co-culture the number of SUM149 cells that invaded through the basement membrane transwell after 24 hours was counted. (C) SUM149 cells were seeded in self-renewal promoting suspension culture conditions. Significant differences are shown as follows: *P < 0.05 for unpaired 2-tailed Student's t-test (n = 3).
Figure 5
Figure 5. Inhibiting M2 macrophage recruitment in the in vivo IBC model inhibites IBC growth and skin invasion
(A) Cell suspensions of SUM149 cells and human bone marrow derived MSCs were prepared from monolayer cultures. In both groups, SUM149 and MSCs (9:1 ratio) were mixed and then co-injected into the mammary cleared fat pad as previously described. 24 hours following co-injection of tumor cells and MSCs, we started treatment with either anti-CSF1 antibody or IgG intraperitoneally. We continued with injections of these antibodies weekly up until the week before resection of the tumor. (B) Weekly tumor measurements were made. (C) The presence of skin invasion and following tumor resection the presence of local recurrence was recorded. Tumors from both groups were resected and collected for digestion. Tumor samples were either stained with (D) anti-F4/80 antibodies or (E) both anti-F4/80 and anti-CD206 antibodies. The results of 3 replicates are shown by flow cytometry. Significant differences are shown as follows: *P < 0.05 for unpaired 2-tailed Student's t-test (n = 3).
Figure 6
Figure 6. Inhibiting tumor associated macrophages reduces expression of pSTAT3 in tumor samples (A) Stitched fluorescent image (20X) of IgG tissue staining for DAPI (blue) and pSTAT3 (red)
(B) Hematoxylin and eosin stain of IgG tumor tissue with arrow showing tumor invasion into the muscular skin layer. (C) Multiplex image (20×) of anti-CSF-1 tumor tissue staining for DAPI (blue) and pSTAT3 (red). (D) Hematoxylin and eosin stain of anti-CSF1 tumor tissue showing high degree of necrosis and undifferentiation. (E) Representative fluorescent image (20×) of IgG and anti-CSF1 mouse showing DAPI, pSTAT3, and merged images. (F) Left: representative histogram of mean nuclear pSTAT3 intensity (background-subtracted) between IgG and anti-CSF-1 tumor nuclei distributions (nnuclei = 3000 in a single tissue section analyzed for each sample). Right: mean nuclear pSTAT3 intensity (background-subtracted) across multiple mice (nIgG = 2 mice, nanti-CSF1 = 3 mice; 3 sections per mouse analyzed).
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