M. C. Kuo1, A. N. Kothari1, Z. Mi1 1Loyola University Chicago Stritch School Of Medicine,Surgery,Maywood, IL, USA
Introduction: Cancer cells metastasize to the bone marrow (BM) to create the pre-metastatic niche. Cancer stemness (or expression of stem cell characteristics) is regulated by the tumor microenvironment (TME) and associated with self renewal, clonal maintenance, and poor clinical outcomes. Osteopontin (OPN) induces mesenchymal stem cells (MSC) in the TME adopt a cancer-associated fibroblast (CAF) phenotype to potentiate cancer growth and metastasis. The mechanisms by which cancer cells and TME regulate stemness in the BM pre-metastatic niche are unknown.
Methods: Human breast cancer cell lines, MB231-Luc (OPN+) and MCF7-Luc (OPN-), were used in an orthotopic murine xenograft model. 6-week-old female NOD scid mice were implanted with 2×106 tumor cells in the presence and absence of human MSC-GFP cells in the R4 position of the mammary fat pad. In selected instances, MCF7-Luc transfected to express OPN (MCF7[lvOPN]) were co-implanted instead of MCF7-Luc. OPN aptamer (APT), which blocks and inactivates extracellular OPN, and/or inactive mutant APT (muAPT) were utilized. After 8 weeks, animals were sacrificed and femoral BM isolated. Stem cell markers, Sox2, Oct4 and Nanog, and CAF markers, SMA and vimentin, were measured by RT-PCR using human specific primers and normalized to Luc and GFP mRNA, respectively. Relative cell number was determined by titrating cell number to Ct value of GFP or Luc in vitro. Each treatment had 3-5 mice. Statistical analysis was performed using Student’s t-test; p-values < 0.05 were considered significant.
Results:
In BM from MB231+MSC, expression of Sox2, Oct4 and Nanog was 4x greater than MB231 alone.(p<0.05) Administration of APT to block OPN decreased Sox2, Oct4 and Nanog to levels equivalent to MB231 alone and MB231+MuAPT. Total MB231 cell numbers were not different. In contrast, although CAF markers were not different among all treatments, CAF numbers in BM were 15-fold greater in MB231+MSC vs MB231 alone and MB231+MSC+APT.(p<0.05) In parallel gain-of-function studies, MCF7-Luc (which do not express OPN) co-implanted with MSC did not express increased stem cell markers in BM when compared to MCF-Luc alone. In contrast, using MCF7 expressing OPN, MCF7[lvOPN]+MSC had 5x greater Sox2, Oct4 and Nanog expression.(p<0.05) APT with MCF7[vOPN]+MSC ablated the increase in stem cell markers. Again, CAF markers were unchanged among treatments, but CAF numbers were 70x higher in MCF7[lvOPN] + MSC vs MCF7+MSC and MCF7[lvOPN]+MSC+APT.(p<0.05)
Conclusion:
In this xenograft model of human breast cancer, our results indicate: 1) both breast cancer cells and accompanying CAF from the primary site metastasize to the BM to form the pre-metastatic niche, 2) tumor-derived OPN mediates CAF migration to the BM, and 3) cancer cells exhibit significantly increased stemness in the presence of CAF in the BM. We conclude that OPN-dependent migration of CAF is required for increased cancer cell stemness in the BM pre-metastatic niche.