2.5. Activation of HSCs by arginase in mice
To further investigate the mechanism by which arginase promoted immune evasion of HCC cells, we imaged the changes of CAFs in mice with different levels of arginase expression. An orthotopic mouse model of HCC was constructed. The expression of arginase in mice was stimulated using LPS and inhibited by BEC. Subsequently, the mice were incubated with TPEARG and Cy-FAP to imaging the changes of arginase and CAFs. The results showed that the fluorescence intensity of arginase in tumor tissues of mice stimulated with LPS was significantly higher compared to the normal or HCC group. Nevertheless, the fluorescence intensity in mice decreased after inhibition of arginase with BEC (Figure 5A). TPEARG was adequate for monitoring changes of arginase in living mice. In addition, when the content of arginase in mouse tumors was increased, the CAFs exhibited subsequently enhanced activation levels. When arginase was inhibited by the inhibitor, the amount of CAFs then decreased.
To further investigate the immune evasion caused by arginase change, we examined immune evasion related markers in each experimental group. The results exhibited that when the arginase increased, the number of CAFs also increased, which in turn led to the increase of immune evasion-promoting marker CTLA-4 (Figure 5B, Figure S7). Up-regulation of PD-L1, a widely recognized immunosuppressive factor, was also appeared in the HCC mice that arginase was high expressed (Figure 5C). Cytokines IL-10 that reported to contributed to the immunosuppressive effect exhibited up-regulation in the HCC mice that arginase was high expressed (Figure 5D). However, when the arginase was inhibited by the administration of BEC, these immunosuppressive factors were down-regulated, suggesting weaken immune evasion in HCC mice. Furthermore, pro-inflammatory effect like TNF-α and IFN-γ revealed down-regulation in the HCC mice that arginase was high expressed (Figure 5E and F). The above experimental data prove that TAMs in vivo stimulate the activation of CAFs via arginase-induced proline production, and then aggravate the immune evasion of HCC.
2.6. Signaling pathway underlying the arginase-mediated immune evasion
Ultimately, we determined genes and pathways associated with arginase-induced immune evasion. Mouse models of orthotopic HCC were constructed and treated with LPS as an arginase agonist, then the mRNA in mouse tumor tissues were tested and analyzed. As shown in Figure 6A, the expression of Oat, Scd2, Spp1, and Prelid2 genes related to arginine metabolism, lipid metabolism, and ECM production were elevated in the mice with high arginase expression, indicating elevated activation of HSCs in the tumor tissues of mice with high arginase expression. Further, genes with enhanced immune evasion functions, such as Map2k6 and Adipor2, were highly expressed when the arginase was highly expressed. Our previous work found JAK2-STAT3 signaling pathway through overproduced ROS. Here, we further analyzed the expression of signaling pathways associated with immune evasion in HCC mice. The IL-2-JAK2-STAT signaling pathway associated with oxidative stress was up-regulated when arginase was highly expressed (Figure 6B and C). From the above experimental results, we conclude a new mechanism of arginase promoting immune evasion from HCC: TAMs stimulate the activation of HSCs in the liver to disintegrate into CAFs through high expression of arginase, which in turn promote immune evasion if HCC by interfering with the JAK-STAT signaling pathway through the secretion of excessive ROS.
3. Discussion
Previous studies have shown that the interactions of CAFs and TAMs deepened their heterogeneity and promote the development of immune evasion towards a more complex scenario of TME[7b, 15]. Investigations have indicated that TAMs can induce the activation of CAFs via cytokine secretion[7a]. However, the underlying mechanisms are unclear. Given that the interaction is based on the interaction between active molecules among the CAFs and TAMs, arginase, an immunoregulatory enzyme of TAMs, is involved in the ornithine cycle that plays a crucial role in the production of proline[16]. Meantime, proline serves as a major substrate for ECM synthesis by CAFs derived from HSCs[17]. Thus, in this study, we set sights on exploring the unique roles of arginase during immune evasion. Tracing the change of arginase during the immune evasion process in the physiological environment would be beneficial to discovery the underlying mechanisms, but still challenging. Thus, we planned to develop an applicative detection strategy for real-time and in-situ tracing of arginase in living cells and in vivo.
Currently, fluorescence imaging technique is a robust approach for the detection of biologically active molecules[12, 18]. In this study, we designed and synthesized the first arginase-specific small molecule fluorescent probe, TPEARG, based on AIE principle. The microenvironmental changes after the enzyme identifies the probe molecule could be utilized by AIE principle. The advantage of this strategy lies in the ability to modulate the probe fluorescence independent of the enzyme-specific cut-off reaction. Consequently, we opted to employ the AIE principle in order to design arginase probes that rely on alterations in the microenvironment subsequent to probe identification of the enzyme’s active site. TPEARG can provide powerful assistance in the real-time and in-situ measurement of arginase change and distribution in cells and in vivo.
In this work, presented findings suggest that TAMs generate large amounts of proline by up-regulating the expression of arginase. We suspect this, in turn, may triggers the transformation of HSCs into CAFs within the HCC TME, thereby exacerbating the tumor cells’ evasion of immune surveillance. Subsequently, experimental results demonstrated that proline could enhance the fluorescence intensity of Cy-FAP in CAFs, meaning an increased activation of CAFs. By combining the two imaging probes, the interaction between arginase expression in TAMs and the activation process of CAFs was observed for the first time. Proline secreted by TAMs could cause an increased activation of CAFs, while promoting CAFs to secrete more bioactive molecules with immunosuppressive functions. To this point, we find that TAMs produce large amounts of proline through high expression of arginase, which in turn stimulates CAFs to deepen the degree of immune evasion.
Furthermore, the detailed molecular mechanism of the process was explored. The JAK-STAT signaling pathway was found to be an important mechanism deeply involved in immune evasion from cancer cells in previous work[19]. Encouragingly, the results revealed that arginase medicated the down-regulation of JAK-STAT signaling pathway in CAFs via secretion of proline by TAMs. Then, increased secretion of PD-L1 and enhanced immune evasion of HCC were showed, which validated our previous conclusion.
In summary, by utilizing a new arginase-specific probe TPEARG and our established CAFs-specific probe Cy-FAP, we presented a dual imaging strategy for simultaneous observation of TAMs and CAFs. We applied this method in cells and in vivo to measure the changes of arginase and CAFs activation. Subsequently, using our established CAFs-specific imaging platform, we further explored the detailed molecular mechanism of arginase promoting immune evasion in HCC. Results showed that TAMs promoted the activation of CAFs by elevating proline levels through high expression of arginase, and CAFs deepened TAMs-induced immune evasion of HCC by secreting immunosuppressive active molecules. This approach we developed will provide a powerful tool for the study of arginase. Results has revealed a novel role played by arginase during immune evasion, which provided a new therapeutic target for HCC.
4. Experimental Section
4.1. Materials and Reagents
All chemical reagents were purchased from Shanghai xianding biotechnology co., LTD. Analytical grade solvents were used without further purification. Mouse PD-L1 and TNF-α ELISA kits were purchased from Absin Bioscience Inc. Raw264.7, LX-2 cells and all supplies for cell culture were purchased from Boster Biological Technology co. ltd.