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.