Human bone marrow-derived MSCs can home to orthotopic breast cancer tumors and promote bone metastasis

Abstract

American women have a nearly 25% lifetime risk of developing breast cancer, with 20% to 40% of these patients developing life-threatening metastases. More than 70% of patients presenting with metastases have skeletal involvement, which signals progression to an incurable stage. Tumor-stroma cell interactions are only superficially understood, specifically regarding the ability of stromal cells to affect metastasis. In vivo models show that exogenously supplied human bone marrow-derived stem cells (hBMSC) migrate to breast cancer tumors, but no reports have shown endogenous hBMSC migration from the bone to primary tumors. Here, we present a model of in vivo hBMSC migration from a physiologic human bone environment to human breast tumors. Furthermore, hBMSCs alter tumor growth and bone metastasis frequency. These may home to certain breast tumors based on tumor-derived TGF-β1. Moreover, at the primary tumor level, interleukin 17B (IL-17B)/IL-17BR signaling may mediate interactions between hBMSCs and breast cancer cells.

Figure 1

hBMSCs from the human bone environment migrate to orthotopic BCC tumors in vivo. (A) A schematic of the in vivo model of hBMSC migration from the bone environment to primary BCC tumors. (B) FACS analysis of tumors explanted and digested from mice. The ratio of counts within both gates (hBMSC and BCC gates) for tumors from mice implanted with (“w/TEB”) and without TEB (“ no TEB”) were compared for each tumor type No TEB mice were implanted with unseeded scaffolds. Mice were analyzed with primary tumors of SUM1315 (i), SUM1313-BP2 (ii), MDA-MB-231 (iii) and MCF7 (iv) BCCs. Gating of fluorescence from hBMSCs and BCCs was based on labelled (positive) and unlabelled (negative) single cell controls. The y-axis (hBMSC gate counts: BCC gate counts) represents the ratio of counts within the fluorescent gates for each tumor type. Statistics utilized a one-sided Student’s t-test.

Figure 2

hBMSCs affect BCC growth in vivo. 500,000 SUM1315 (A), SUM1315-BP2 (B), MDA-MB-231 (C) and MCF7 (D) BCCs were injected alone or with 1.5×10⁶ hBMSCs into the MFP. Tumor volume was measured with bioluminescent imaging weekly. Data are normalized to bioluminescence from the first week of measurement and represented as mean + S.E.M. N=5 for all groups. Statistics performed using 2-way ANOVA or two-sided Student’s T-test on endpoint: SUM1315, *, p=0.0122; SUM1315-BP2, ***, p=0.0001; MDA-MB-231, ns, p=0.9282; MCF7, ns, p=0.0875.

Figure 3

hBMSCs affect migration and metastasis of BCCs. (A) SUM1315, SUM1315-BP2, and MDA-MB-231 BCCs were grown in hBMSC-CM for 24-hours. BCCs migrated for 6-hours on transwell migration assays. Values represent average number of cells, normalized to cancer cell samples without hBMSCs for each BCC line. (B) 500,000 BCCs were injected alone or with 1.5×10⁶ hBMSCs into the MFP of 10-week-old NOD/Scid female mice harboring implanted human bone cores. After 10 weeks mice were sacrificed and human bone cores, lungs and livers were analyzed for metastases using bioluminescence. Metastases were counted and frequency was calculated by dividing number of metastatic samples by total number of samples. N=6; SUM1315 and SUM1315-BP2 with hBMSC. N=5; SUM1315 and SUM1315-BP2 alone. N=7; MDA-MB-231.

Figure 4

IL-17B stimulation of IL-17BR can drive migration and metastasis. (A) Expression of il-17br was analyzed using qRT-PCR. Metastatic cells were compared with matched primary tumors. N=3 except for the SUM1315-BP2 mouse bone data point where too few metastases inhibited significance. (B) Serum-starved SUM1315, SUM1315-BP2 and MDA-MB-231 BCCs showed a dose-dependent reaction to IL-17B stimulation after 6-hours. No increase in migration was observed for MCF7 BCCs. (C) Serum-starved SUM1315 and MDA-MB-231 BCCs (mock infected or infected with lentivirus to overexpress il-17br cDNA) migrated for 6-hours on transwell migration assays. (D) Bioluminescent imaging of human bone fragments, lungs, and livers was used to assess metastasis frequency.