Graduate Research Assistant University of Akron, Ohio, United States
Introduction:: Metastasis accounts for over 90% of mortality of cancer patients. Estrogen receptor-positive breast cancers (ER+ BC) comprise about 75% of all breast cancers and respond well to targeted endocrine therapies[1]. Nevertheless, recurrence of ER+ BC in metastatic sites poses an ongoing threat to patients as risks of recurrence and death steadily increase post-diagnosis. Even after 5 years of adjuvant endocrine therapy, up to 52% of patients remain at risk of distant recurrence and death 20 years after diagnosis[2]. ER+ BC cells survival in bone marrow to form metastases accounts for about 70% of advanced disease in ER+ BC[3]. Although mesenchymal stromal cells (MSCs) in bone marrow are suggested to play a key role in this process, mechanisms of survival and resistance of ER+ BC cells to endocrine therapies are unknown. The goal of this study was to develop a 3D tissue engineered model to investigate effects of interactions of ER+ BC cells and MSCs on drug responses of cancer cells. We found that MSCs promoted resistance of ER+ BC cells to hormonal therapy by altering estrogen receptor expression on cancer cells and inducing stemness in cancer cells.
Materials and Methods:: MCF7, T47D, and HCC1428 (click beetle green luciferase) [4] were used as ER+ BC cells. Patient-derived bone marrow stromal cells (pMSCs) (ATCC) and HS5 [4] were used as MSCs. The cells were dispersed in 25 µl of human type 1 collagen either as monoculture (cancer cells alone) or as co-culture (cancer-stromal cells in a 1:9 cell density ratio). The cell-collagen suspension was incubated for one hour at 37°C for gelation, and then 25 µl of low-glucose, 1% FBS culture medium was added. Fulvestrant and Tamoxifen, two standard anti-estrogenic drugs, were used to treat the cultures at different concentrations. On day 4, measurements of bioluminescence were done from eight replicates of each condition using a plate reader, and cell viability was calculated as the bioluminescence intensity. Cancer cells were separated from stromal cells using human EpCAM microbeads (Miltenyi Biotech) and used for gene expression of different markers using qPCR. The fold change in mRNA expression was determined as 2-ΔΔCt and normalized to β-actin and GAPDH. All results were statistically analyzed to determine effects of bone marrow MSCs on ER+ BC cells.
Results, Conclusions, and Discussions:: We treated the cultures with Tamoxifen and Fulvestrant dose-dependently and determined the cellular viability. Figure 1 illustrates the viability of HCC1428 cancer cells under co-culture with HS5 or pMSC and in monoculture. Results indicate that HCC1428 cells develop resistance to the drugs when co-cultured with MSCs, suggesting that MSCs are a key regulator of anti-cancer drug resistance in ER+ BC cells. We conducted qPCR experiments and found a significant decrease in estrogen receptor gene (ESR) expression in HCC1428 cells in co-culture with MSCs (Figure 2). We also found a significant increase in CD44 and ALDH gene expression, but a small increase in CD24 gene expression, in cancer cells co-cultured with HS5 MSCs (Figure 2). Because ER+ BC cells typically proliferate through signaling of estrogen-ESR, a decrease in ESR expression implies that metastasized cancer cells might use an alternative proliferation pathway independent of estrogen. This phenomenon may explain why anti-estrogenic drug treatments become less effective against ER+ BCs. Moreover, high CD44 and ALDH and low CD24 expression has been associated with a hybrid cancer stem cell (CSC) phenotype, which exhibits properties such as high plasticity, drug resistance, and invasiveness [5]. Our results suggest that the presence of MSCs shifts ER+ BC cells towards a hybrid CSC phenotype. We engineered a tumor model to study survival and drug resistance of ER+ BC cells in a bone marrow environment. Interactions between MSCs and BC cells caused the cancer to resist anti-estrogenic drug treatments. This resistance may be caused by the activation of hybrid cancer stem cell (CSC) genes.
Acknowledgements (Optional): : NIH/ NCI (grant CA225549) and DoD (grant W81XWH-22-1-0119)
References (Optional): : [1] Spring LM, et al. JAMA Oncology 2016;2:1477–86. [2] Pan H, et al. New England Journal of Medicine 2017;377:1836–46. [3] Savci-Heijink CD, et al. Breast Cancer Research and Treatment2015;150:54757. [4] Cavnar, SP, et al., Neoplasia 2015; 17:625–633. [5] Pastushenko I, et al. Nature 2018;7702: 463-468