Tumor progress intercept by intervening in Caveolin-1 related intercellular communication via ROS-sensitive c-Myc targeting therapy

Abstract

Tumor-associated macrophages (TAMs) in the tumor microenvironment (TME) play an important role in the development of tumors by secreting a variety of cytokines or directly communicating with tumor cells, making TAMs-targeted therapeutic strategies very attractive. It has been reported that oncogene c-Myc is related to every aspect of the oncogenic process of tumor cells and the alternative activation of macrophages. Hence, we constructed a glycolipid nanocarrier containing ROS-responsive peroxalate linkages (CSOPOSA) for ROS-triggered release of drugs and further modified it with Ex 26 (Ex 26-CSOPOSA), a selective sphingosine 1-phosphate receptor 1 (S1PR1) antagonist, to achieve the dual-targeted delivery of the c-Myc inhibitor JQ1 via S1PR1, which is overexpressed on both tumor cells and TAMs, thereby inducing apoptosis of tumor cells, and blocking M2 polarization of macrophages. More strikingly, our studies found that JQ1 could effectively inhibit the migration of tumor cells induced by M2 macrophages-derived exosomes via blocking Caveolin-1 related intercellular exosome exchange through lncRNA H19 and miR-107. The in vivo results revealed that this dual-targeted delivery strategy effectively inhibited tumor growth and metastasis with less systemic toxicity, providing a potential method for effective tumor treatment.

Introduction

In order to improve the efficacy of tumor therapy, the trend of targeted therapy has shifted from primarily attacking the tumor cells to targeting specific areas at the tumor site [1]. Unlike tumor cells, stromal cells in the tumor microenvironment (TME) are genetically stable and thus represent an attractive therapeutic target with reduced risk of drug resistance and tumor recurrence. In the context of tumors, macrophages termed as tumor-associated macrophages (TAMs), an essential innate immune population, promote important steps in tumor progression including tumor angiogenesis, tumor cell proliferation, metastasis, the suppression of adaptive anti-tumor immunity and the regulation or limitation of the efficacy of various forms of anti-cancer therapies [2]. TAMs derive from peripheral blood monocytes recruited into TME and differentiate into classically activated M1 or alternatively activated M2 subtypes responding to signals in the microenvironment [3]. Among them, M2 macrophages, marked by CD206, typically express arginase 1 (ARG1) and high levels of cytokines, growth factors and proteases that support their pro-tumor functions [[2], [3], [4]]. Despite the phenotypic plasticity of TAMs, ultimately, the polarization of TAMs toward an immunosuppressive phenotype seems to be a common feature of most cancers [2]. TAMs in progressing tumors typically express characteristic surface molecules, such as the scavenger receptor CD163 and macrophage mannose receptor 1 (also known as CD206) [5], and exhibit properties related to stimulation of angiogenesis, suppression of adaptive immunity, and promotion of tumor growth and metastasis [[6], [7], [8]]. Therefore, TAMs-targeted therapeutic strategies by depleting them to block their pro-tumor functions, or functionally reprogramming them to restore their anti-tumor properties would be attractive strategies for treating malignant tumors.

Oncogene c-Myc is reported to be involved broadly in many cancers including triple-negative breast cancer. It is estimated that its expression is elevated in up to 70% of human cancers, and such deregulation is often associated with poor prognosis and adverse patient survival [[9], [10], [11], [12]]. Importantly, c-Myc mediates the tumorigenic mechanisms of both tumor cells and TAMs. c-Myc is overexpressed in TAMs and is associated with the alternative activation of macrophages since c-Myc controls the induction of approximately 45% of genes associated with alternative activation [13,14]. The absence of c-Myc in mice impairs the maturation of TAMs and the expression of pro-tumor cytokines, thus inhibiting oncogenesis [15]. In tumor cells, c-Myc plays a central role in almost every aspect of the oncogenic process, such as orchestrating proliferation, apoptosis, differentiation and metabolism [9]. What's more, it has been shown that c-Myc induces expression of the innate immune regulator CD47, and the immune-checkpoint protein PD-L1 to suppress the activation of innate immunity and adaptive immunity [16]. Therefore, targeting c-Myc in tumor cells and TAMs may be an effective approach to treat tumors. The BET bromodomain binds to the c-Myc promoter region and plays a key role in the expression of c-Myc. As a BET bromodomain inhibitor, JQ1 is effective in inhibiting c-Myc expression in different tumor types [17]. Since JQ1 has poor water solubility and a short half-life, the dosage of JQ1 is large in order to maintain its high concentration in cells, leading to more drug toxicity during the treatment [18]. Therefore, it is necessary to achieve a high concentration of drug molecules at the tumor site by means of active targeted drug delivery technology.

Studies have shown that sphingosine 1-phosphate (S1P) secreted by apoptotic tumor cells can induce alternative polarization of macrophages, thus further exerting its pro-tumor effect [[19], [20], [21]]. Therefore, tumor therapy should not only kill malignant tumor cells, but also interfere the effect of apoptotic tumor cells on macrophages through S1P, so as to more effectively limit the pro-tumor effect of apoptotic cells and improve the anti-tumor ability of macrophages. Sphingosine 1-phosphate receptor 1 (S1PR1), which is the binding site of S1P, belongs to G protein coupled receptor (GPCR) superfamily and overexpresses both on tumor cells and TAMs [[22], [23], [24]]. Therefore, Ex 26, the selective S1PR1 antagonist, was used to design an active targeted delivery system, which was expected to achieve dual-targeting of tumor cells and TAMs and block the effect of apoptotic tumor cells on macrophages through S1P.

Due to metabolic and signaling aberrations, tumor cells and TAMs exhibit elevated reactive oxygen species (ROS) levels [[25], [26], [27], [28]]. Therefore, it is possible to achieve efficient triggered-release of drugs by designing nanocarriers containing ROS-sensitive bonds, thus ensuring a large accumulation of drugs in tumor cells and TAMs, and furthermore improving the efficacy and reducing the toxic and side effects of drugs. It has been reported that peroxalate can easily react with oxidant H₂O₂ to form 1, 2-dioxetanedione, which rapidly decomposes into carbon dioxide [29,30]. Thus, strategically introducing peroxalate bonds within the nanoparticles can induce their degradation and release the cargos upon exposed to ROS. In this study, chitosan, a marine biological material with low toxicity and biodegradability, was used as the basic skeleton, and ROS-responsive peroxalate linkages were used as the chain bridges. The hydrophilic low-molecular weight chitosan was cross-linked with hydrophobic stearyl alcohol to construct ROS-sensitive glycolipid grafts (CSOPOSA). Using N, N′-disuccinimidyl carbonate (DSC) as a bridging agent, Ex 26-modified PEGylated ROS-sensitive glycolipid nanocarriers (Ex 26-CSOPOSA) were synthesized. The physicochemical properties and stability of the synthesized grafts were characterized. With JQ1 as the model drug, drug-loaded nanoparticles were prepared, and the drug release kinetics of drug-loaded nanoparticles under different oxidation conditions were investigated.

Taking 4T1 cells and RAW264.7 cells as model cells, the M2 macrophages model was constructed via IL-4 to investigate the uptake of micelles and drug delivery behavior of drug-loaded nanoparticles against tumor cells and M2 macrophages. The efficacy of drug-loaded nanoparticles in promoting tumor cell apoptosis and inhibiting tumor cell migration was evaluated. In addition, the effect of micelles on the alternative polarization process of macrophages was investigated and quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the expression levels of related genes in tumor cells and M2 macrophages. What's more, the co-culture system of tumor cells and M2 macrophages was constructed to evaluate the effect of drug-loaded nanoparticles on the cargo exchange between tumor cells and M2 macrophages, and the mechanism was investigated. M2 macrophages-derived exosomes (M2-exosomes) were extracted to investigate their effect on the migration ability of tumor cells and the intervention effect of drug-loaded nanoparticles on this process was evaluated. In vivo, 4T1 tumor-bearing mice were used as model animals to investigate the distribution and drug release of nanoparticles. The anti-tumor efficacy and safety on model animals with drug-loaded nanoparticles was evaluated, and the molecular mechanism was further explored, providing a new method for the treatment of tumors.

It is well known that intercellular communication between tumor cells and TAMs is associated with tumor progression and metastasis [31,32]. Exosomes are important mediators in this interaction, which transmit information from one cell to another and reprogram recipient cells [33,34]. However, existing research has focused on the characterization of exosomes in different tumor types [35]. It is urgent to further study on how to intervene in exosome-based intercellular communication, as our understanding of the cellular and molecular mechanisms that govern the biogenesis, release, uptake, and function of exosomes remains limited [36]. In this study, it was found that JQ1 could effectively inhibit the cargo exchange between tumor cells and M2 macrophages, which was partly related to the inhibition of the Caveolin-1 levels by JQ1 and JQ1-loaded nanoparticles, thus reducing the exosome uptake between tumor cells and M2 macrophages (Scheme 1). M2-exosomes were extracted and further studies showed that M2-exosomes could effectively promote the migration of tumor cells, which could be suppressed by JQ1 and JQ1-loaded nanoparticles through reducing the uptake of M2-exosomes by tumor cells. In order to clarify the molecular mechanism of c-Myc regulating the expression of Caveolin-1, cells were transfected with siRNA or miRNA mimics to knockdown or make the overexpression of specific genes, and qRT-PCR was used to measure the expression of related genes. It was found that c-Myc regulated the expression of Caveolin-1 by interfering with the expression of lncRNA H19 and miR-107.