Abstract P4-04-09: Mesenchymal stem cells and macrophage interactions promote inflammatory breast cancer cell invasion and self renewal
Authors: Wolfe Adam R , Atkinson Rachel , Debeb Bisrat G , Zhang Yan , Ruffel Brian , Reuben James M , Ueno Naoto T , Woodward Wendy A 
Published: May 2015

Abstract
Background: Inflammatory breast cancer (IBC) is responsible for 10% of breast cancer deaths. The hallmarks of IBC are skin involvement and a high propensity to metastasize. Our lab has shown previously, "normal" breast tissue from women with an IBC diagnosis had significantly greater macrophage infiltration and increased cells with stem cell markers compared to non IBC "normal" breast tissue. These changes were present prior to diagnosis in two patients where pre-IBC biopsies were available. Therefore, we hypothesized changes in the normal breast microenvironment prior to tumor formation contributes to the IBC phenotype.
Methods: To study our hypothesis we used a co-culture system to measure the interactions between normal macrophages (Raw 264.7 cell line), bone-marrow derived mesenchymal stem cells (MSCs), and IBC cells (SUM 149, IBC3, and KPL4). Conditioned media (CM) from MSC culture was added to macrophages overnight. The macrophages were subsequently analyzed for their surface markers and cytokine production. Reciprocally, MSCs were "educated" by macrophages by adding CM from polarized M2 macrophages to the MSC culture media at a 1:1 ratio. MSCs and cancer cells were co-cultured in trans-wells (Boyden chambers) for 24 hours. Migration and invasion in vitro was determined by adding MSCs or IBC cells to the insert of the trans-well and cultured in combination with either polarized macrophages or macrophage educated MSCs for 24 hours. After co-culture, IBC cells were analyzed for their ability to form mammospheres.
Results: Mouse macrophages polarized (using IL-4) into a M2 phenotype (Tumor associated macrophage) secreted 3 fold more IL-6 compared to unstimulated or M1 polarized macrophages. The addition of MSC CM to the macrophage culture for 24 hours polarized the macrophages into a similar M2 phenotype described above with 4-fold increase in IL-6 secretion compared to unstimulated macrophages (P&lt;0.01). When M2 macrophages at the bottom of the co-culture system were co-cultured with MSC, the number of MSCs migrating increased 2 fold with the addition of M2 macrophages compared to media alone, or unstimulated macrophages (P&lt;0.05). IBC cells showed a 2 fold enhancement of invasion towards M2 educated MSCs compared to either uneducated MSCs, or media alone (P&lt;0.05). IBC cells co cultured with M2 educated MSCs grew 3 fold more mammospheres compared to IBC cells grown with uneducated MSCs (P&lt;0.01). IBC cells added to the bottom of the co-culture system also enhanced migration of MSCs and did so to a greater extent than non-IBC cells (P&lt;0.01). Lastly, the addition of IL-6 neutralizing antibody inhibited IBC induced MSC migration.
Conclusions: Herein we demonstrate reciprocal tumor interactions between normal cells in the IBC microenvironment. MSC and macrophages can influence each other to increase the tumor promoting influence of each on IBC cells. Our results suggest IL-6 a mediator of these tumor promoting influences and is important for the IBC induced migration of MSCs. Currently we are investigating the in vivo interactions between macrophages and MSCs in an orthotopic IBC mouse model.
Citation Format: Adam R Wolfe, Rachel Atkinson, Bisrat G Debeb, Yan Zhang, Brian Ruffel, James M Reuben, Naoto T Ueno, Wendy A Woodward. Mesenchymal stem cells and macrophage interactions promote inflammatory breast cancer cell invasion and self renewal [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-04-09.