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.