1 hAT-MSCs migrated to TME and evolved into TA-MSCs
Before analysing the effects of HCC-CM on hAT-MSC phenotype and glucose metabolism, we first needed to characterize the obtained cells. Flow cytometry indicated that the obtained cells were positive for the MSC surface markers CD90, CD105 and CD146 and negative for the haematopoietic marker CD45 (Figure 1a). Experiments of differentiation in vitro indicated that the obtained cells could differentiate to adipocytes, osteocytes and chondrocytes (Figure 1b). Thus, we determined that the obtained cells were indeed hAT-MSCs.
In the next experiment, the precondition was based entirely on the tumour-homing properties of MSCs. To verify the chemotaxis capacity of naïve hAT-MSCs towards the liver cancer environment, we designed in vitro chemotaxis assays. Naïve hAT-MSCs were allowed to migrate towards HCC-CM (Hep3B, Huh7 and HCCLM3) or DMEM as a control. Significant increases in the migratory capacity of hAT-MSCs towards all HCC-CM were found when compared to DMEM (Figure 1c, d). The results suggested that hAT-MSCs were actively recruited to HCCs.
Once MSCs migrated to TME, tumour cells can ‘educate’ MSCs to evolve into TA-MSCs via paracrine interaction. As a consequence, MSCs gain expression of α-SMA and Vimentin and become stellate in shape. Therefore, we decided to study the changes in hAT-MSC properties after HCC-CM stimulation (educated hAT-MSCs). After 4-8 weeks of exposure to HCC-CM, the results of Western blot showed that the expression of α-SMA and Vimentin in hAT-MSCs increased significantly (Figure 1e). To further investigate the effects of HCC-CM on morphological and cytoskeletal changes in hAT-MSCs, immunofluorescence assays were performed. The results showed that hAT-MSCs exhibited strong α-SMA expression following treatment with HCC-CM, in keeping with the morphology as cruciform or stellate shape (Figure 1f). All of the above results suggested that hAT-MSCs can be truly converted into TA-MSCs after being induced by HCC-CM.
Figure 1 Identification results of hAT-MSCs and their characteristics. (a) Surface markers of hAT-MSCs. (b) Differentiation in vitro of hAT-MSCs. (c & d) Chemotaxis assays (left) and quantitative results (right) of hAT-MSCs towards HCC-CM and normal medium; (e) α-SMA and Vimentin expression levels were evaluated by Western blot of hAT-MSCs after exposure to HCC-CM or normal medium; (f) α-SMA expression levels and distribution were evaluated by immunofluorescence of hAT-MSCs after exposure to HCC-CM or normal medium. **p<0.01, vs DMEM, n=2. Abbreviations: 3B-CM: Hep3B-conditioned medium, Huh7-CM: Huh7-conditioned medium, LM3-CM: HCCLM3-conditioned medium.
2 Phenotypic changes of hAT-MSCs after exposure to HCC-CM
The remodelling and reorganization of the cytoskeleton can impact cellular motility. Therefore, we decided to study the migration and invasion capacities of hAT-MSCs after exposure to HCC-CM. Significant increases in the migratory and invasion capacities of educated hAT-MSCs were found (Figure 2a, b).
Next, to evaluate the effect of HCC-CM on the proliferation of hAT-MSCs, CCK-8 assays and EdU staining were used. EdU assay results showed a significant reduction in EdU-positive cells after exposure to HCC-CM (Figure 2c, d). In accordance with the results of EdU assays, CCK-8 assays revealed significant suppression of cell viability after HCC-CM stimuli, except for the 24-h point (Figure 2e). In brief, proliferation of hAT-MSCs was significantly inhibited by HCC-CM.
Considering that distribution of the cell cycle and level of cell apoptosis can influence cell proliferation and viability, flow cytometry was applied to analyse hAT-MSC apoptosis and cell cycle. The results of cell apoptosis showed a markedly higher cell apoptosis rate (Annexin V +) in hAT-MSCs after exposure to HCC-CM (Figure 2f). Regarding the cell cycle, we observed that after exposure to HCC-CM, the ratio of cells in the G0/G1 phase was significantly increased while in the G2/M phase it’s significantly reduced, which suggested that HCC-CM induced G2/M phase cell cycle arrest (Figure 2g).
Finally, we determined the expression levels of apoptosis- and cell cycle-related proteins by Western blot. The results shown that the mitochondrial apoptosis pathway was activated in hAT-MSCs after exposure to HCC-CM. The results revealed an increase in the expression levels of apoptotic proteins (Bax and cleaved caspase-3) and a decrease in anti-apoptotic protein (Bcl-2) (Figure 2h). Regarding cycle-related regulatory proteins, cyclins and cyclin-dependent kinases were generally downregulated, especially the expression levels of regulatory proteins related to the G2/M phase (Cyclin A2, Cyclin B1, and CDK1) (Figure 2i).
These data demonstrate that after exposure to HCC-CM, the proliferation of hAT-MSCs was inhibited by regulating the cell cycle and activating the mitochondrial apoptosis pathway, while the migratory and invasion capacities were significantly enhanced.
Figure 2 Effects of HCC-CM on cell phenotype of hAT-MSCs. (a) Cell migration assays (left) and quantitative results (right) of hAT-MSCs after exposure to HCC-CM or normal medium; (b) Cell invasion assays (left) and quantitative results (right) of hAT-MSCs after exposure to HCC-CM or normal medium; (c & d) EdU assays (c) and quantitative results (d) of hAT-MSCs after exposure to HCC-CM or normal medium; (e) Quantitative results of OD value at 570 nm in hAT-MSCs after exposure to HCC-CM or normal medium; (f) Representative images (left) and quantification results (right) of cell apoptosis in hAT-MSCs after exposure to HCC-CM or normal medium; (g) Representative images (left) and quantification results (right) of cell cycle distribution in hAT-MSCs after exposure to HCC-CM or normal medium; (h & i) The expression levels of apoptosis-related (h) and cell cycle-related (i) proteins were evaluated by Western blot of hAT-MSCs after exposure to HCC-CM or normal medium. *p<0.05, **p<0.01, vs NC, n=2, except for CCK-8 assays (n=8).
3 Glucose metabolic changes of hAT-MSCs after exposure to HCC-CM
As we described above, the mitochondrial apoptosis pathway of hAT-MSCs was activated after exposure to HCC-CM; thus, we wondered whether the function of mitochondria could be influenced by HCC-CM. Maintenance of mitochondrial transmembrane potential is essential for normal mitochondrial function. Therefore, we used JC-1 and Mito-Tracker to stain mitochondria. The accumulation of JC-1 within the mitochondria is dependent on the electrochemical gradient, so it can be used to evaluate the MMP. However, Mito-Tracker is independent of membrane potential; thus, mitochondrial mass can be monitored with it. The staining results showed that the mitochondrial mass of hAT-MSCs was essentially unaffected after exposure to HCC-CM, while there was a marked decline in MMP (Figure 3a). These results suggest that HCC-CM induced mitochondrial dysfunction.
Mitochondria are the cellular bioenergetic centres, and mitochondrial dysfunction will lead to a metabolic switch from oxidative phosphorylation to glycolysis. Therefore, we tested whether HCC-CM modulated the glycolytic phenotype in hAT-MSCs. The results showed significant increases in glucose uptake, pyruvate level, lactate production and ATP level of hAT-MSCs after exposure to HCC-CM (Figure 3b-e). Moreover, after exposure to HCC-CM, hAT-MSCs showed markedly enhanced GLUT1, GPI, GAPDH, PGK1, LDHA and LDHB, but not HK2 and PKM2, at the mRNA and protein levels (Figure 3f, g).
These data indicate glycolysis is enhanced in hAT-MSCs after exposure to HCC-CM due to mitochondrial dysfunction.
Figure 3 Glycolysis and mitochondrial function of hAT-MSCs after exposure to HCC-CM or normal medium. (a) Representative images of mitochondrial mass (Mito-Tracker) and MMP (JC-1) in hAT-MSCs after exposure to HCC-CM or normal medium; (b-e) Quantification of relative glucose uptake (b), lactate production (c), pyruvate level (d) and ATP level (e) in hAT-MSCs after exposure to HCC-CM or normal medium; (f & g) Analysis of glycolytic gene expression in hAT-MSCs after exposure to HCC-CM or normal medium. qRT-PCR (f) and immunoblot (g). A schematic diagram of the glycolysis pathway is shown on the left (f). *p<0.05, **p<0.01, vs NC, n=3.
4 Reversal of phenotypic changes in hAT-MSCs after withdrawal of HCC-CM treatment
Our previous study showed that the fate of hAT-MSCs had been seriously threatened after exposure to HCC-CM, while cells gained the capacities of migration and invasion. Thus, we wondered whether educated hAT-MSCs would escape from TME and reverse their fate. To verify the conjecture, we decided to culture educated hAT-MSCs in normal medium for another 2-4 weeks and to evaluate the reversibility in phenotype and glucose metabolism.
Before verification, we needed to verify if the educated hAT-MSCs still retained the same characteristic of TA-MSCs after withdrawal of HCC-CM treatment. Western blot results indicated that hAT-MSCs maintained the high-level expression of α-SMA even after withdrawal of HCC-CM treatment (Figure 4a). In addition, the results of immunofluorescence suggested that hAT-MSCs still maintained their cruciform or stellate shape after withdrawal of HCC-CM treatment (Figure 4b). These findings indicate that HCC-CM can induce permanent alterations during the conversion of hAT-MSCs into TA-MSCs.
Once again, we evaluated proliferation of educated hAT-MSCs after the detachment from HCC-CM by CCK-8 assays and EdU staining. The results showed no significant difference in proliferation between the reversed hAT-MSCs and naïve hAT-MSCs (Figure 4c-e). These findings suggest the reversal of HCC-CM-induced inhibition of proliferation in hAT-MSCs after withdrawal of HCC-CM treatment.
We then used flow cytometry to analyse reversed hAT-MSC apoptosis and cell cycle. The results showed that the apoptosis rate (Annexin V +) and cell cycle distribution were not significantly different between the groups (Figure 4f, g). These data demonstrate that both activated apoptosis and cell cycle arrest returned to control levels after withdrawal of HCC-CM treatment.
Finally, we determined the expression levels of cell cycle- and apoptosis-related proteins by Western blot. The results revealed a slight increase in the expression of anti-apoptotic protein (Bcl-2) and no difference in apoptotic proteins (Bax and cleaved caspase-3) (Figure 4h). Regarding cell cycle-related regulatory proteins, all cyclins and cyclin-dependent kinases were restored, consistent with the naïve hAT-MSCs (Figure 4i).
These data indicate that HCC-CM-induced inhibition of proliferation, activation of the mitochondrial apoptosis pathway and cell cycle arrest in hAT-MSCs can be reversed and restored to normal by withdrawal of HCC-CM treatment.
Figure 4 Proliferation, apoptosis and cell cycle of hAT-MSCs after withdrawal of HCC-CM treatment. (a) α-SMA expression was evaluated by Western blot of hAT-MSCs after exposure to HCC-CM and withdrawal of HCC-CM treatment; (b) α-SMA expression and distribution were evaluated by immunofluorescence assays of hAT-MSCs after withdrawal of HCC-CM treatment; (c, d) EdU assays (c) and quantitative results (d) of hAT-MSCs after withdrawal of HCC-CM treatment; (e) CCK-8 assays and quantitative results of hAT-MSCs after withdrawal of HCC-CM treatment; (f) Representative images (left) and quantification results (right) of cell apoptosis in hAT-MSCs after withdrawal of HCC-CM treatment; (g) Representative images (left) and quantification results (right) of cell cycle distribution in hAT-MSCs after withdrawal of HCC-CM treatment; (h & i) The expression of apoptosis-related (h) and cell cycle-related (i) proteins were evaluated by Western blot of hAT-MSCs after withdrawal of HCC-CM treatment. vs NC, n=2, except for CCK-8 assays (n=8). Abbreviations: R-3B-CM: reversed-Hep3B-conditioned medium, R-Huh7-CM: reversed-Huh7-conditioned medium, R-LM3-CM: reversed-HCCLM3-conditioned medium.
5 Reversal of metabolic changes in hAT-MSCs after withdrawal of HCC-CM treatment
As we found above, the mitochondrial apoptosis pathway of hAT-MSCs was reversed after withdrawal of HCC-CM treatment; thus, we wondered whether the function of mitochondria could also be reversed. Therefore, we used JC-1 and Mito-Tracker to stain mitochondria again. The staining results showed that the mitochondrial mass of hAT-MSCs was essentially unaffected after withdrawal of HCC-CM treatment, while there was a marked enhancement in MMP (Figure 5a). These results suggest that HCC-CM-induced mitochondrial dysfunction was reversed.
Next, we tested the glycolytic phenotype in hAT-MSCs after withdrawal of HCC-CM treatment. The results showed a significant reduction in lactate production, but there were no significant differences in glucose uptake, pyruvate level and ATP level of hAT-MSCs after withdrawal of HCC-CM treatment (Figure 5b-e). Moreover, after withdrawal of HCC-CM treatment, hAT-MSCs showed markedly reduced GLUT1, GPI and PKM2, but not HK2, GAPDH, PGK1 and LDHA, at the mRNA levels (Figure 5f). At the protein levels, hAT-MSCs showed reduced levels of HK2, PGK1, PKM2 and LDHB (Figure 5g).
These data indicate that the enhanced glycolysis in hAT-MSCs was reversed after withdrawal of HCC-CM treatment.
Figure 5 Glycolysis and mitochondrial function of hAT-MSCs after withdrawal of HCC-CM treatment. (5) Representative images of mitochondrial mass (Mito-Tracker) and MMP (JC-1) in hAT-MSCs after withdrawal of HCC-CM treatment; (b-e) Quantification of relative glucose uptake (b), lactate production (c), pyruvate level (d) and ATP level (e) in hAT-MSCs after withdrawal of HCC-CM treatment; (f & g) Analysis of glycolytic gene expression in hAT-MSCs after withdrawal of HCC-CM treatment. qRT-PCR (f) and immunoblot (g). *p<0.05, **p<0.01, vs NC, n=3.
6 HCC-CM regulated the activation of the ROS/MAPK/HIF-1α signalling pathway
Given the central roles that mitochondria played in phenotypic and metabolic alterations of hAT-MSCs, as mitochondria are the major source of ROS and since ROS can determine cell fate by regulating multiple signalling pathways, we used DCFH-DA probes to measure intracellular ROS. Our results showed that ROS levels significantly increased in hAT-MSCs after exposure to HCC-CM (Figure 6a). However, after withdrawal of HCC-CM treatment, levels of ROS were restored to normal control levels (Figure 6b).
To further investigate the possible roles of ROS-associated signalling pathways, we assessed the levels of HIF-1α, MAPK (ERK1/2, JNK and p38) and AKT. We found that exposure of HCC-CM resulted in activation of the HIF-1α and MAPK pathways, but not the AKT pathway. The phosphorylation levels of JNK and p38 were increased, while that of ERK1/2 was decreased (Figure 6c). However, after withdrawal of HCC-CM treatment, the levels of HIF-1α and phosphorylated ERK, p38, and JNK were restored to normal control levels (Figure 6d).
Next, to determine whether ROS are the upstream signal molecules of MAPK and HIF-1α pathways, we used the ROS scavenger NAC to block ROS generation. When cells were pre-treated with 5 mM NAC for 24h, ROS production was severely suppressed (Figure 6e). Meanwhile, the levels of HIF-1α and phosphorylated JNK and p38 were clearly reduced, and the phosphorylation level of ERK1/2 was restored (Figure 6f).
These data suggest that the ROS/MAPK/HIF-1α signalling pathway plays an important role in HCC-CM-induced phenotypic and metabolic alterations of hAT-MSCs.
Figure 6 Effect of HCC-CM on the ROS/MAPK/HIF-1α signalling pathway. (a) Representative histogram (left) and quantification data (right) of ROS levels in hAT-MSCs after exposure to HCC-CM or normal medium; (b) Representative histogram (left) and quantification data (right) of ROS levels in hAT-MSCs after withdrawal of HCC-CM treatment; (c) HIF-1α, phosphorylated AKT, total AKT, phosphorylated JNK, total JNK, phosphorylated p38, total p38, phosphorylated ERK and total ERK protein expression of hAT-MSCs after exposure to HCC-CM or normal medium; (d) HIF-1α, phosphorylated AKT, total AKT, phosphorylated JNK, total JNK, phosphorylated p38, total p38, phosphorylated ERK and total ERK protein expression of hAT-MSCs after withdrawal of HCC-CM treatment; (e) ROS levels in hAT-MSCs after exposure to HCC-CM, normal medium or pre-treated with NAC; (f) HIF-1α, phosphorylated AKT, total AKT, phosphorylated JNK, total JNK, phosphorylated p38, total p38, phosphorylated ERK and total ERK protein expression of hAT-MSCs after exposure to HCC-CM, normal medium or pre-treated with NAC. *p<0.05, **p<0.01, vs NC, n=2.
7 HCC-CM endow hAT-MSCs with permanent secretory phenotype and tumour-promoting properties.
The fate of hAT-MSCs had been seriously threatened after exposure to HCC-CM, however it can be reversed by withdrawal of HCC-CM treatment. We wondered whether these observations have functional relevance, then we investigated the effect of HCC-CM on secretory phenotype in hAT-MSCs, the mRNA expression of a variety of cytokines, growth factors and chemokines were analysed. The expression levels of many factors, especially IL-1β, TGF-β and CCL-7, were much higher after exposure to HCC-CM than in naïve hAT-MSCs (Figure 7a). and the characteristics of factors secretion by TA‑MSCs appears to be permanent, since these cells keep producing these factors even after withdrawal of HCC-CM treatment (Figure 7b).
We next determined whether the altered mRNA expression in TA‑MSCs has functional relevance, CCK-8 and Transwell assays were used to investigate the effect of MSC-CM on proliferation and migration in HCC cells. No significant differences in proliferation and migration were observed when HCC cells were incubated with conditioned medium from naïve hAT-MSCs (N-MSC-CM), compared to the normal medium (DMEM) (Figure 7c-h). In contrast, conditioned medium form educated hAT-MSCs (E-MSC-CM) and reversed hAT-MSCs (R-MSC-CM) significantly enhanced proliferation compared to DMEM and N-MSC-CM at 96-h point (Figure 7c-e). Transwell migration assays demonstrated that E-MSC-CM and R-MSC-CM significantly increased the migration potency of HCC cells (Figure 7f-h).
Taken together, these results revealed that HCC-CM endow hAT-MSCs with permanent tumour-promoting properties.
Figure 7 Secretory phenotype and tumour-promoting properties of hAT-MSCs after exposure to and withdrawal form HCC-CM. (a & b) mRNA expression of various factors in hAT-MSCs after exposure to (a) and withdrawal form HCC-CM (b) was determined by qRT-PCR; (c-e) Quantitative results of OD value at 570 nm in Hep3B (c), Huh7 (d) and HCCLM3 (e) after exposure to N-MSC-CM, E-MSC-CM, R-MSC-CM or normal medium; (f-h) Cell migration assays (left) and quantitative results (right) of Hep3B (f), Huh7 (g) and HCCLM3 (h) after exposure to N-MSC-CM, E-MSC-CM, R-MSC-CM or normal medium. *p<0.05, **p<0.01, vs NC or DMEM, #p<0.05, ##p<0.01, vs N-MSC-CM, n=2, except for CCK-8 assays (n=8).