Mesenchymal stem cells inhibit multiple myeloma cells via the Fas/Fas ligand pathway

Abstract

Introduction: Cell-based therapy represents a new frontier in the treatment of a wide variety of human diseases traditionally associated with morbidity outcomes, including those involving inflammation, autoimmunity, tissue damage, and cancer. However, the use of mesenchymal stem cells (MSCs) to treat multiple myeloma (MM) bone disease has raised concerns. Specifically, evidence has shown that infused MSCs might support tumor growth and metastasis.

Methods: In this study, we used a standard disseminated MM model in mice to identify the in vivo effects of intravenous MSC infusion. In addition, a series of in vitro co-culture assays were preformed to explore whether Fas/Fas ligand (Fas-L) is involved in the inhibitory effects of MSCs on MM cells.

Results: In the MM mouse model, treatment of MSCs with highly expressed Fas ligand (Fas-L high MSCs) showed remarkable inhibitory effects on MM indenization in terms of extending the mouse survival rate and inhibiting tumor growth, bone resorption in the lumbus and collum femoris, and MM cell metastasis in the lungs and kidneys. In addition, reduced proliferation and increased apoptosis of MM cells was observed when co-cultured with Fas-L high MSCs in vitro. Furthermore, mechanistically, the binding between Fas and Fas-L significantly induced apoptosis in MM cells, as evidenced through an increase in the expression of apoptosis marker and Fas in MM cells. In contrast, Fas-L null MSCs promote MM growth.

Conclusions: These data suggest that Fas/Fas-L-induced MM apoptosis plays a crucial role in the MSC-based inhibition of MM growth. Although whether MSCs inhibit or promote cancer growth remains controversial, the levels of Fas-L expression in MSCs determine, at least partially, the effects of MSCs on MM cell growth.

Figure 1

Effect of mesenchymal stem cell infusion in multiple myeloma model mice. (A) Experimental protocol of multiple myeloma (MM) cell and mesenchymal stem cell (MSC) injection and the design of lymphocyte Peyer’s patch adhesion molecule (LPAM) administration. (B) Six-week survival rates of animals in different groups (for each group, n = 14). The survival rate in the nontreated MM model group was dramatically decreased after 3 weeks. The MSC and L-PAM groups had almost the same survival rate, but were higher than the MM group (P <0.05). Data shown as the mean ± standard deviation (SD) of three parallel experiments. *P <0.05 versus MM group. (C) Comparison of clinical findings in each group. The number (left) and size (right) of cancroid pearls in the neck, root of tail and abdominal cavity are shown as column graphs (top panel). Data shown as mean ± SD for three parallel experiments. *P <0.05 versus MSC group; #P <0.05 versus L-PAM group. Lower panel: representative photograph of typical cancroid pearls in the base of the tail (bar = 5 mm). (D) Comparison of bone resorption in lumbus and collum femoris in each group. Radiographs of lumbus and collum femoris at 3 weeks after MM cell injection, and the bone density in each group was analyzed. Data shown as mean ± SD for four parallel experiments. *P <0.05 versus MM group; #P <0.05 versus L-PAM group. (E) Myeloma cell metastasis in the lungs and kidneys in each group. Data shown as mean ± SD for three parallel experiments. *P <0.05 versus MSC group. Left panels: light micrographs of the lungs and kidneys (hematoxylin and eosin staining, bar = 100 μm). Con, control.

Figure 2

Relationship between multiple myeloma cells and mesenchymal stem cells under co-culture conditions. (A) Multiplication of multiple myeloma (MM) cells under co-culture with mesenchymal stem cells (MSCs). (B) Western blot analyses of apoptosis markers. Expression levels of cleaved caspase-3 and caspase-8 in MM cells with or without co-culture with MSCs were analyzed through western blotting at 0, 6, 12, and 24 hours (lower table arranged to numerical value from upper data). (C) Apoptotic analysis through fluorescence-activated cell sorting (FACS) (x axis, Annexin V; y axis, 7AAD-positive cells). Apoptosis of MM cells with or without co-culture with MSCs was detected and quantified through FACS at 0, 6, 12, and 24 hours (lower table arranged from upper data). Data presented as mean ± standard deviation (SD) for two parallel experiments. #P <0.05 versus the control group (Cont.). (D) Rate of apoptotic MM cells under co-culture with MSCs. The microscope pictures represent typical reactions of MM cells in fluorescence staining, where MM cells were prestained for carboxyfluorescein diacetate, succinimidyl ester (CFSE; green) and apoptosis markers using Annexin V and 7AAD (red). Lower table arranged to numerical value from upper data. Data presented as mean ± SD for two parallel experiments. #P <0.05 versus the control group (Cont.). DAPI, 4′,6-diamidino-2-phenylindole.

Figure 3

Fas and Fas ligand analysis in multiple myeloma cells and mesenchymal stem cells under co-culture. (A) Multiplication of multiple myeloma (MM) cells under different co-culture conditions with mesenchymal stem cells (MSCs) (Cont., MM single culture; C-medium, MM culture with conditioned medium from MSC culture; Trans-well, indirect co-culture between MM and MSC; Co-cul, direct culture between MM and MSC). Each data point represents the mean ± standard deviation (SD) of two parallel experiments. #P <0.05 versus the control group (Cont.). (B) Western blotting analyses of the expression levels of Fas and Fas ligand (Fas-L) in MM cells and MSCs under co-culture condition for 0, 6, 12, and 24 hours. (C), (D) The rates of Fas-positive or Fas-L-positive cells were determined through immunofluorescence staining. (C) MM cells were prestained for carboxyfluorescein diacetate, succinimidyl ester (CFSE; green) and then post-stained for Fas (red). (D) MSCs were stained for Scar-1 (green) and subsequently stained for Fas-L (red). Bar = 300 μm. Numbers of Fas-positive MM cells and Fas-L-positive MSCs are shown. Data presented as the mean ± SD for two parallel experiments. #P <0.05 versus the control group (Cont.). DAPI, 4′,6-diamidino-2-phenylindole.

Figure 4

Effects of Fas ligand levels in mesenchymal stem cells on multiple myeloma cells under co-culture. (A) Comparison of Fas ligand (Fas-L) expression in mesenchymal stem cells (MSCs) after aspirin treatment. (B) Multiplication of multiple myeloma (MM) cells under co-culture condition with MSCs that express different levels of Fas-L. For the graph, the number of MM co-cultured without MSCs (Cont.), with normal MSCs (Co-cul), with MSCs from generalized lymphoproliferative disease mice (Co-cul(gld)) or with MSCs treated with any aspirin (Co-cul(Asp)) were counted after 0, 6, 12 or 24 hours. (C) Apoptotic analysis of MM cells under co-culture condition with MSCs expressing different levels of Fas-L (detected and quantified through fluorescence-activated cell sorting (FACS): x axis, Annexin V; y axis, 7AAD-positive cells). Data presented as mean ± standard deviation (SD) for two parallel experiments. #P <0.05 versus Con. (D) Expression levels of cleaved caspase-3 in MM cells with or without co-cultured MSCs (expressing different levels of Fas-L) at 0, 6, 12, and 24 hours. (E) Rates of apoptotic MM under co-culture with MSCs (expressing different levels of Fas-L). Data presented as mean ± SD for two parallel experiments. #P <0.05 versus the control group (Con.). CFSE, carboxyfluorescein diacetate, succinimidyl ester; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 5

Effect of mesenchymal stem cells with highly activated Fas ligand on multiple myeloma model mice. (A) Five-week survival rates of multiple myeloma (MM) model mice. (B) Comparison of the number (left) and size (right) of cancroid pearls in MM model mice after treatment with mesenchymal stem cells (MSCs) expressing different levels of Fas ligand (Fas-L). Data presented as mean ± standard deviation (SD) for three parallel experiments. *P <0.05 versus MSC group; #P <0.05 versus the control group. Right panel: representative photograph of typical cancroid pearls at the base of the tail (bar = 5 mm). (C) Distribution of cancroid pearl size in the four test groups (n = 14). (D) Apoptosis of MM cells in MM model mice. The pearls in each group were triple-stained for 4′,6-diamidino-2-phenylindole ( blue), Scar-1 (green) and Annexin V/7AAD (red). All groups treated with MSCs had Scar-1-positive cells in the pearls. However, the positive reactions of apoptosis marker in the MSC(gld) group were much lower than those observed in other groups. The MSC(Asp) group had the highest number of reactions among all groups (bar = 5μm). Lower panels stained through the terminal deoxynucleotidyl transferase-mediated UTP nick-end labeling (TUNEL) assay. Positive reactions were observed in both MSC and MSC(Asp) groups. ASP, aspirin; Con, control; gld, generalized lymphoproliferative disease.