2.4. Observing the activation of CAFs induced by the arginase
Given the overproduction of arginase could increase the metabolism of proline thus induced the ECM deposition in the tissue[8b, 10]. We supposed that the overproduction of arginase-induced proline in the TAMs would promote the activation of hepatic stellate cells (HSCs) to differentiate into CAFs, which further aggravated the immune evasion of HCC (Figure 4A). To verify our hypothesis, the proline concentrations in cell culture media with different levels of arginase were examined. As indicated in the Figure 4B, M2 macrophages secreted more proline than M1 macrophages, and the contents of proline decreased when the arginase was inhibited. Further, the proline concentrations depended on the expression of arginase (Figure 4C). These results suggest that the arginase profoundly impacted the produce of proline. Inspired by the above results, we further investigated whether the overproduced proline of TAMs could promote the activation of HSCs. LX-2 cells, a HSCs cell line, were incubated with DMEM (Control), DMEM from M2 macrophage, 10 μg/mL proline, 10 μg/mL aspartate, and 10 μg/mL cysteine, respectively. And the activation maker FAP was imaged using our established probe Cy-FAP. Interestingly, compared to the cells treated with aspartate or cysteine, LX-2 cells incubated with proline exhibited nearly 9-fold enhancement of fluorescence intensity (Figure 4D and E). Similarly, LX-2 cells co-cultured with medium extracted from M2 macrophages showed over 6-fold intensity increase compared with the control, aspartate or cysteine group. When the arginase increases, the activation degree of CAFs was deeper as the fluorescence of Cy-FAP got brighter. These results indicate that elevated proline produced by the M2 macrophage is responsible for the activation of CAFs.
To further prove the contribution of arginase in inducing immune evasion which was mediated by CAFs activation, we measured the PD-L1, IFN-γ and IL-10 in each cell group. Experimental data revealed that the expression of PD-L1, IFN-γ and IL-10 in LX-2 cells that co-cultured with M2 macrophages raised when compared with the LX-2 cells or M2 macrophages (Figure S6). The above experimental results reveal that M2 macrophages can stimulate the differentiation of HSCs into CAFs by arginase-induced proline secretion, thereby promoting the immune evasion of HCC.