Adipose‑derived mesenchymal stem cells exhibit tumor tropism and promote tumorsphere formation of breast cancer cells

Abstract

Mesenchymal stem cells reportedly have a marked effect on tumor growth or suppression. However, it remains uncertain whether adipose‑derived mesenchymal stem cells (ADSCs) from grafted fat can contribute to breast cancer growth and recurrence. In the present study, interactions between ADSCs and MCF‑7 breast cancer cells were evaluated in a Matrigel co‑culture system and in an in vivo nude mouse model. Results suggested that MCF‑7 cells exerted tumor tropism effects on ADSCs and this may be regulated by chemokines, such as the macrophage inflammatory protein (MIP)‑1δ and MIP‑3α. Additionally, ADSCs significantly induced tumorsphere formation in vitro and promoted tumorigenicity in vivo. RT‑qPCR analysis indicated that tumorsphere formation by MCF‑7 cells was associated with the induction of stem‑like properties, which was mediated by epithelial‑-mesenchymal transition. Together, the present findings indicated that ADSCs exhibit tropism and induce tumorsphere formation of MCF‑7 cells.

Figure 1.

Multi-lineage differentiation potential and senescence evaluation of ADSCs. (A) Oil Red O; (B) Alizarin red and (C) Alcian blue staining. (D and E) Representative microscopic fields of acid β-galactosidase (blue) staining in cells and (F) quantification of β-galactosidase-positive cells. Black arrows indicate senescent cells. Experiments were repeated 3 times. ***P<0.001. Scale bar, 100 µm. ADSCs, adipose-derived mesenchymal stem cells.

Figure 2.

Interaction between ADSCs and MCF-7 cells in vitro. (A) ADSCs and MCF-7 cells display a uniform distribution at 12 h after cell implantation. (B) At 24 h, ADSCs were touching and surrounding MCF-7 cells. (B) At 96 h, ADSCs were observed inside MCF-7 tumorspheres. Experiments were repeated 4 times. Scale bar, 100 µm. ADSCs, adipose-derived mesenchymal stem cells.

Figure 3.

MCF-7 cells induce ASC migration and chemokines expression in vitro. (A) ADSCs in upper chamber were co-cultured MCF-7 cells or AM-CM in lower chamber, while DMEM in lower chamber severed as a control and (B) quantification of the ADSCs migrated at the bottom of the insert membrane; n=5 visual fields for each condition. (C and D) Expression levels of chemokines (C) MIP-1δ and (D) MIP-3α in MCF-7 + ADSCs group and the control MCF-7/DMEM group, as measured by reverse transcription-quantitative polymerase chain reaction. Experiments were repeated 3 times. The results are presented as mean ± standard deviation; **P<0.01, ***P<0.001. Scale bar, 100 µm. ADSCs, adipose-derived mesenchymal stem cells; AM-CM, ADSCs and MCF-7 co-culture conditioned media; ASC, adipose stromal cells; MIP, macrophage inflammatory protein.

Figure 4.

Tumor tropism of ADSCs in vivo. (A and B) Tracing of ADSCs after tail veins injection using a bioluminescence imaging system. (C) Hematoxylin and eosin staining and (D) the corresponding fluorescence image of tumor sample. Experiments were repeated 3 times. Scale bar, 200 µm. ADSCs, adipose-derived mesenchymal stem cells.

Figure 5.

Scanning electron microscopy analysis of ADSCs and MCF-7 interaction. (A) Tumorspheres formation of spherical MCF-7 cells (red arrows) in Matrigel. (B) Tumorspheres were surrounded by flatly spread cells with typical ADSCs morphology (yellow arrows). (C) Intercellular connections (white arrows) between ADSCs and MCF-7 cells. Scale bar, 10 µm. ADSCs, adipose-derived mesenchymal stem cells.

Figure 6.

ADSCs enhance tumorspheres formation of MCF-7 cells. (A) Analysis of ADSC-induced tumorsphere formation effects on MCF-7 cells. (B) Quantification of tumor diameter in different groups. (C and D) Expression levels of CSC markers in AM-CM-treated MCF-7 cells and the control MCF-7 cells, as measured by reverse transcription-quantitative polymerase chain reaction. The results are presented as mean ± standard deviation; *P<0.05, **P<0.01 and ***P<0.001. Scale bar, 100 µm. ADSCs, adipose-derived mesenchymal stem cells; AM-CM, ADSCs and MCF-7 co-culture conditioned media; ASC, adipose stromal cells; CSC, cancer stem cell; OCT, Octamer-binding protein; SOX, Sex determining region Y-box.

Figure 7.

Effects of ADSCs on tumorigenesis of MCF-7 cells in vivo. (A) Nude mice were injected with ADSCs alone or mixed with MCF-7. The tumors were indicated by black arrows. (B) Quantification of tumor volume. (C) Hematoxylin and eosin staining of tumor tissue and (D) quantification of the necrotic area in tumor tissue. The results are presented as mean ± standard deviation; *P<0.05, ***P<0.001. Scale bar, 100 µm. ADSCs, adipose-derived mesenchymal stem cells.

Figure 8.

ADSCs induce EMT in MCF-7 cells. (A and B) E-Cadherin and Vimentin (A) mRNA and (B) protein expression levels were measured by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively, in MCF-7 cells alone or AM-CM treated MCF-7 cells at different time points. Scale bar, 100 µm; **P<0.01. ADSCs, adipose-derived mesenchymal stem cells; AM-CM, ADSCs and MCF-7 co-culture conditioned media; EMT, epithelial mesenchymal transition.

Figure 9.

Schematic diagram presenting the potential mechanisms for the interaction between ADSCs and MCF-7 cells. ADSCs, adipose-derived mesenchymal stem cells; AM-CM, ADSCs and MCF-7 co-culture conditioned media; CSC, cancer stem cells; EMT, epithelial mesenchymal transition; MIP, macrophage inflammatory protein; OCT, Octamer-binding protein; SOX, Sex determining region Y-box.