Tracking interactions between TAMs and CAFs mediated by arginase-induced proline production during immune evasion of HCC
Chuanchen Wu, Yuantao Mao, Xinru Qi, Xin Wang, Ping Li, *Wen Zhang and Bo Tang*
C. C. Wu, Y. T. Mao, X. R. Qi, X. Wang, Prof. P. Li, W. Zhang and Prof. B. Tang
College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Institutes of Biomedical Sciences
Shandong Normal University
Jinan 250014, People’s Republic of China
mail: lip@sdnu.edu.cn, tangb@sdnu.edu.cn
Prof. B. Tang
Laoshan Laboratory
168Wenhai Middle Rd, Aoshanwei Jimo
Qingdao 266237, People’s Republic of China
E-mail: tangb@sdnu.edu.cn
Keywords: Fluorescent probe, Arginase, Tumor-associated macrophages, Immune evasion, Tumor-associated fibroblasts
Abstract: Synergistic changes between tumor-associated macrophages (TAMs) and tumor-associated fibroblasts (CAFs) aggravated immune evasion of hepatocellular carcinoma (HCC), however, the underlying molecular mechanisms remain elusive. Their continuous and dynamic interactions are subject to bioactive molecule changes. A real-time and in-situ monitoring method suitable for in vivo research of these processes would be indispensable but is scarce. In this study, a dual imaging strategy that tracing the TAMs and CAFs simultaneously was developed using a new arginase-specific probe and established CAFs-specific probe. The emerging roles of arginase in mediating CAFs activation in mice were explored. Results showed arginase up-regulation in TAMs, followed by proline increase. Subsequently, proline produced by TAMs initiated the activation of CAFs. Through the JAK-STAT signaling, CAFs up-regulated the PD-L1 and CTLA-4, ultimately promoting immune evasion of HCC. This study revealed a new mechanism by which TAMs and CAFs collaborate in immune evasion, providing new targets for HCC immunotherapy.
1. Introduction
Immune evasion of hepatocellular carcinoma (HCC) adopts multiple mechanisms to prevent antigen presentation, evade immune surveillance and clearance, promoting the continuous proliferation and further metastasis of HCC[1]. This is one of the essential characteristics of HCC progression. Importantly, immune evasion is a crucial reason for patients’ resistance to immunotherapy drugs for HCC[2]. Immune evasion poses enormous challenges for patients’ treatment and leads to a high mortality rate of HCC. Exploring the process of HCC immune evasion and revealing its related molecular mechanisms is pivotal to the development of more effective HCC treatment methods[3].
The tumor microenvironment (TME) is the pathological environment in which immune evasion of HCC progression occurs[4]. The intercellular communication in TME is vital pathway for immune evasion[5]. To reveal the detailed molecular mechanisms of HCC immune evasion, it is prerequisite to explore the interplay of cells inside TME. Tumor-associated macrophages (TAMs) and tumor-associated fibroblasts (CAFs) are essential members of TME and exhibit various immunosuppressive functions[6]. Importantly, their mutual interactions in TME can induce recruitment and activation of each other, further enhancing immune evasion[7]. Although studies have reported the importance of CAFs and TAMs in tumor progression, the intercellular interaction mechanisms between them have not been fully revealed. Especially during the process of HCC immune evasion, the synergistic effect of CAFs and TAMs yet to be explored, and the detailed molecular mechanism remains elusive.
Arginase is a highly active enzyme that mostly expressed in TAMs[8]. Arginase consumes arginine in the TME, leading to T cell exhaustion, thus plays crucial roles in TAMs-mediated immune evasion[9]. In addition, arginase participates in regulating downstream pathways for the extracellular matrix (ECM) generation in the TME[10]. CAFs are responsible for ECM generation in the TME[11]. These findings suggest a hypothesis that TAMs might regulate the activity of CAFs through arginase, thereby affecting immune evasion of HCC. However, the underlying molecular mechanisms are mysterious. Cellular interplay between CAFs and TAMs is a continuous and dynamic process that is regulated by multiple active molecules. However, commonly used in vitro characterization methods such as western blot and polymerase chain reaction (PCR) are insufficient to explore the changes of active molecules in the physiological environment. The continuous and dynamic changes of arginase during the interaction between TAMs and CAFs are unrevealed. Fluorescence imaging technique would be a powerful tool for revealing the real-time and in-situ changes of arginase in the interaction between TAMs and CAFs in vivo, but applicative imaging strategy is lacking[12].
Therefore, in this study, we have developed a dual imaging strategy that tracing the dynamic changes of TAMs and CAFs in cells and HCC mice. To fulfil this goal, a new fluorescent probe TPEARG which emits bright fluorescence upon targeting arginase was constructed. This method enables in-situ dynamic imaging of arginase changes in TAMs. By combining this imaging strategy with our established CAFs activation-specific imaging method, we have investigated the dynamic role of TAMs-derived arginase in promoting CAFs activation and facilitating immune evasion of HCC. The experimental results demonstrate that as the level of arginase in TAMs rises, the content of proline significantly increases. We have observed that the increased content of proline can significantly enhance the activation level of CAFs, which in turn affects the JAK-STAT pathway and enhances immune evasion of HCC.
2. Result
2.1. Design strategy of TPEARG and optical properties of TPEARG
Rational selection of identifying group, regulation mechanism of fluorescence and appropriate fluorophore were made to fabric the fluorescent probe that fulfill our demand. Firstly, knowing that the active site domain of arginase was specific to arginine, thus we chose arginine as the identifying group[8a]. Then, according to the steric hindrance change after the probe bound to the active site domain of the arginase, aggregation-induced emission (AIE) principle was selected as the fluorescence regulation mechanism after recognition[13]. Tetraphenylethene (TPE) is an excellent AIE fluorophore with outstanding fluorescence imaging performance. TPE structure is compact with low steric hindrance, which is conducive to the probe entering the ectodomain of enzyme, so as to achieve high specificity and high selectivity imaging detection of enzymes[14]. In light of these considerations, we incorporated an arginine moiety to a TPE fluorophore via an amide linkage to construct the fluorescent probe TPEARG. The uncombined TPEARG emit weak fluorescence due to the bond rotation. Upon interaction with the arginase, the arginine could enter in the ectodomain of enzyme, resulting in the rotation restriction of TPE, which leading to about 6-fold fluorescence increase at 450 nm (Figure 1A ).
The detailed synthesis route and methods of TPEARG were described in the supporting information. The structure of TPEARG was characterized with1H NMR, 13C NMR, and HRMS in the supporting information. The optical characterizations of TPEARG were examined in detail. Firstly, the AIE performances of TPEARG in various MeOH-H2O mixture were tested. As shown in Figure 1B, TPEARG was weakly fluorescent in MeOH because of the single molecular motion process of TPE. However, its fluorescence intensity enhanced greatly when the water fractions increased due to the formation of depositing aggregates. Moreover, the influence of viscosity on the AIE process was also measured. With the increasing of viscosity, fluorescence emission of TPEARG enhancement was observed resulted from the rotation restriction of TPE (Figure 1C). These data prove that we have successfully synthesized the TPEARG with outstanding AIE performances.
Nest, the recognition performances of TPEARG toward arginase were examined. As shown in the Figure 1D and E, upon interaction with sequential dosing of arginase, the fluorescence emission of TPEARG enhanced swiftly and gradually, which showed a linear manner (R2=0.99). The limit of detection (LOD) was calculated as 0.04 U/mL. The selectivity of TPEARG to arginase and other proteases was further verified. As revealed in Figure 1F, TPEARG emitted low fluorescence when incubated with trypsase and other proteases than that with arginase. Moreover, widespread bioactive molecules like reactive oxygen species (ROS), metal ions and amino acids exhibit minimal interferences to the fluorescence of TPEARG (Figure 1G). The above experimental results show that TPEARG perform excellent sensitivity and selectivity toward arginase.
Then, the fluorescence responses of TPEARG to arginase in the PBS from pH 6.0 to pH 8.5 were further tested. Results indicated TPEARG’s great photostability (Figure S1). The cytotoxicity and in vivo toxicity of TPEARG were measured. As shown in the Figure S2, TPEARG exhibited weak toxicity on cell proliferation and growth and organ development in mice. Hemolysis test in Figure S3 showed that TPEARG possessed minimal hemolytic activity (<3%) at tested concentrations (1 μM ~ 1 mM). These above experimental results demonstrate that TPEARG has great potential for biological applications.