Complement-mediated Stem Cell Recruitment to Breast Cancer

Institution: Torrey Pines Institute for Molecular Studies
Investigator(s): Ingrid Schraufstatter, M.D. -
Award Cycle: 2010 (Cycle 16) Grant #: 16IB-0086 Award: $136,500
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2010)

Breast cancers invoke a similar response of the body as wounds that fail to heal. In this scenario cells in the tumor environment such as mesenchymal stem cells (MSC) exploit the normal wound healing process to benefit the tumor. It was recently shown that MSC are recruited to developing breast cancers, where they contribute to tumor growth and metastasis. The factors involved in the process of MSC recruitment and in the interaction of these cells with breast cancer cells are largely unknown. It is hypothesized here that complement activation, which is part of the body’s defense against infection, contributes to this process: it has been shown that there is persistent complement activation in breast cancers, which leads to the production of two small proteins called C3a and C5a. We recently described that MSC express receptors for C3a and C5a called C3aR and C5aR, and that MSC migrate towards C3a and C5a. It is therefore hypothesized that C3a and/or C5a produced within breast tumors recruit MSC to the tumor. Once MSC enter the tumor, C3a and C5a could play a crucial role in increasing tumor growth and metastasis, since MSC stimulated with C3a or C5a produce increased amounts of factors that support the growth of tumor cells and blood vessels and that interfere with the immune response mounted by white blood cells.

It is anticipated that C3aR-deficient mice receiving a mix of breast cancer cells with MSC from C3aR-deficient mice or C5aR-deficient mice receiving a mix of breast cancer cells with MSC from C5aR-deficient mice in the mammary fat pad will show diminished tumor growth, blood vessel growth and metastasis compared to ‘normal’ mice receiving a breast cancer cell/normal MSC mixture. For these studies, MSCs will be isolated from mouse bone marrow of normal mice or from mice that are deficient in the C3aR or the C5aR and fluorescently labeled. C3aR-deficient mice will be injected into the mammary fat pad with a breast cancer cell/C3aR-deficient MSC mixture, C5aR-deficient mice will receive a breast cancer cell/C5aR-deficient MSC mixture, and normal mice a breast cancer cell/normal MSC mixture. Additional groups of mice will receive only breast cancer cells. Tumor growth will be measured over time. After 30 days the excised tumors will be analyzed for the presence of fluorescent MSC, various white blood cell populations, and an indicator that shows that complement had been activated. Blood vessel growth will be assessed, growth factor production will be measured and tumor metastasis to the lung will be evaluated. If C3aR-deficient or C5aR-deficient mice show decreased tumor growth and metastasis, inhibitors of the C3aR of the C5aR would be a novel therapeutic target in metastatic breast cancer.

The role of MSC in tumor growth and metastasis has only recently been recognized and MSC are a novel target in breast cancer research that may lead to innovative therapeutic approaches. More specifically, we recently recognized that MSC are recruited by C3a and C5a, and that C3a and C5a induce the production of growth factors in MSC, which suggests a potential for enhanced tumor growth and metastasis. However, none of this has been shown in the context of breast cancer, or any cancer. Therefore this project is based on an entirely novel concept.

Final Report (2012)

It was the goal of this proposal to determine whether complement activation, which occurs in all breast cancers and leads to the formation of C3a and C5a, contributes to the recruitment of mesenchymal stem cells (MSC) to breast carcinomas. Since it is important to perform these experiments in animals with an intact immune system, mouse 4T1 breast cancer cells were used in BALB/c mice. MSC were isolated from wild type mice or from mice in whom the C3a receptor (C3aR) or the C5a receptor (C5aR) genes had been “knocked out “ (C3aR ko or C5aR ko) so that these cells cannot respond any longer to C3a or C5a. Following fluorescent tagging of these cells it was planned to inject them intravenously (i.v.) into mice bearing 4T1 tumors and to detect the respective recruitment of these cells to the tumors. Bone marrow MSC from wild type mice could be isolated relatively easily. However, there was some initial contamination with specific white blood cells called macrophages, but these cells die out over the following weeks and the MSC can be expanded. However unexpectedly, in the C5aR ko and to a lesser degree in the C3aR ko MSC cultures, macrophages persisted long-term - meaning for several months. Therefore, it was necessary to first deplete macrophages prior to establishing MSC cultures from these mice and to develop specific culture conditions.

Eventually it was possible to establish and characterize MSC from all 3 types of mice. The initial idea was to express green fluorescent protein (GFP) in these cells for in vivo tracking, but unfortunately GFP expression caused unwanted changes in these cells (larger cell size and slower proliferation). Therefore a different approach, labeling with a lipophilic fluorescent marker, DiR was developed, which did not have these negative effects. A preliminary experiment in which mice bearing 4T1 tumors were injected with wt MSC labeled with DiR indicated that this approach is feasible: MSC distribution can be followed over time by in vivo imaging and reproducible recruitment to the tumors can be detected. However, because of the unexpected difficulty encountered with the isolation of pure MSC from C3aR ko and C5aR ko mice, which took much time and effort to overcome, the comparative studies between the 3 groups of mice has not been performed, but cells are now available for all groups. In the future it is planned to compare wt, C3aR ko and C5aR ko mice and injecting MSC either i.v. or intraperitoneally (i.p.). Although i.p. injection was not initially planned, recent findings in this area of research indicate that MSC are most likely being recruited in this fashion. It is furthermore planned to determine the effect of co-injecting 4T1 breast cancer cells and the respective MSC (wt, C3aR ko, C5aR ko) and to determine the effect on breast cancer growth and metastasis. The hypothesis is that MSC will show a increased tumor growth and metastasis, which is expected to be diminished, when MSC from C3aR or C5aR ko mice are used, indicating that complement activation plays a detrimental role in breast cancer.