Tumor microenvironment regulation - enhanced radio - immunotherapy

Highlights

• SM NPs regulated tumor microenvironment through oxygenation, ROS generation, GSH depletion, and immune activation.

• SM NPs enhanced the efficacy of radio-immunotherapy through inhibiting both primary tumors and distant untreated tumors.

• The combination of SM NPs and the anti-CTLA-4 antibody enhanced the efficacy of RT at a low dose of radiation.

Abstract

Radiotherapy (RT) is frequently utilized for cancer treatment in clinical practice and has been proved to have immune stimulation potency in recent years. However, its inhibitory effect on tumor growth, especially on tumor metastasis, is still limited by many factors, including the complex tumor microenvironment (TME). Therefore, the TME - regulating SiO₂@MnO₂ nanoparticles (SM NPs) were prepared and applied to the combination of RT and immunotherapy. In a bilateral animal model, SM NPs not only enhanced the inhibitory effect of RT on primary tumor growth, but also strengthened the abscopal effect to inhibit the growth of distant untreated tumors. As for the distant untreated tumor, 40% of mice showed complete inhibition of tumor growth and 40% showed a suppressed tumor growth. Moreover, SM NPs showed modulation functions for TME through inducing the increase in intracellular levels of oxygen and reactive oxygen species after their reaction with hydrogen peroxide and the main antioxidative agent glutathione in TME. Lastly, SM NPs also effectively induced the increase in the amounts of cytokines secreted by macrophage - like cells, indicating modulation functions for immune responses. This work highlighted a potential strategy of simultaneously inhibiting tumor growth and metastasis through the regulation of TME and immune responses by SM NPs - enhanced radio - immunotherapy.

Introduction

RT is one of the vital cancer treatment strategies in clinical practice, and one of the major radio-toxic effects of RT is DNA damage [1], [2]. In recent years, the abscopal effect of RT has been proved to stimulate immune responses, but it is seen in only 10% of cases in clinical practice [3]. Therefore, the inhibitory effect of RT on tumor metastasis is still far from satisfactory [4], [5]. Immunotherapy has emerged in recent years with promising clinical applications owing to its modulation functions for immune responses [6]. Interestingly, compared with RT alone, the abscopal effect of RT can become more common when combined with immunotherapy both in preclinical and clinical models of programmed cell death 1 (PD - 1) and cytotoxic T lymphocyte antigen 4 (CTLA - 4) inhibitors [7], [8]. However, owing to the host mechanism to remain immunologically silent after cell death caused by DNA damage, the DNA damage generated will be inactivated through many mechanisms, including cancer cell autophagy [9], apoptotic caspase activation during apoptosis [10], and digestion by host deoxyribonuclease (DNase) [10], [11]. Moreover, the efficacy of RT and immunotherapy is limited by the complex TME, which is usually characterized by hypoxia, acidic pH value [12], elevated hydrogen peroxide (H₂O₂) levels [13], and powerful antioxidative systems [14]. Hypoxia of TME contributes to tumor recurrence and poor prognosis in RT [15] and limits the efficacy of RT through the support of the development of cancer stem cells and inhibition of the radical generation [16], [17]. Specifically, hypoxic cells require a threefold higher dose of radiation than normoxic cells [18]. Moreover, a hypoxic TME can suppress anticancer immune responses through the promotion of the infiltration and accumulation of suppressor T - cells and the inhibition of the adaptive immune system, resulting in tumor angiogenesis and cancer metastasis [19], [20]. Moreover, glutathione (GSH), one of the main reducing agents of antioxidative systems in cancer cells, can scavenge reactive oxygen species (ROS), which decreases the efficacy of ROS - based therapies and promotes the expression of glutathione peroxidase 4 (GPX - 4), which further enhances the antioxidation barrier in TME, thus strongly suppressing the efficacy of RT [14]. These findings emphasize the fact that regulating TME can be a promising strategy to enhance the synergistic efficacy of radio-immunotherapy.

In recent years, TME - responsive anticancer therapies have been widely studied, which modulate TME through the unique physical and chemical properties of nanomaterials [21], [22]. They have been proved effective in improving treatment outcomes of various anticancer therapies [23], [24]. For example, H₂O₂, an ideal prodrug for Fenton reactions or Fenton-like reactions in chemodynamic therapy (CDT), can react with various nanomaterials to produce highly toxic hydroxyl radicals (·OH), including manganese (Mn) [25], [26], [27]. Recently, Mn-based nanomaterials have attracted tremendous interest in the field of anticancer therapies. Mn plays an important role in many in vivo processes, such as neuronal function, immune regulation, antioxidant defenses, and the biosynthesis of some metalloenzymes [28], [29]. Studies have shown that Mn²⁺ can be excreted rapidly by the kidneys leading to fewer side effects on the body [30]. Moreover, its unique properties in regulating TME make Mn - based nanomaterials potentially applicable to anticancer therapies. Typically, Mn - based nanomaterials can induce increase in oxygen (O₂) and ROS levels through reactions with H₂O₂ and GSH in TME, thus enhancing the efficacy of many anticancer therapies [31], [32], [33]. Notably, Mn²⁺ has been proved by recent studies to have immune stimulation potential [34], [35], [36], [37], [38]. It has been proved that Mn²⁺ is critical in the innate immune sensing of tumors and enhancing adaptive immune responses, which can promote the sensitivity of the DNA sensor of the cyclic GMP - AMP synthase (cGAS) and the stimulator of interferon genes (STING) [34], [35]. Interestingly, RT - induced DNA damage has also been proved to activate the cGAS / STING pathway and enhance immune responses [39], [40]. Therefore, Mn - based nanomaterials may be promising in enhancing the immune responses and the abscopal effect of RT. Except for Mn-based nanomaterials, various inorganic nanomaterials have been proved to stimulate immune responses [41], especially silica. In the previous studies, silica-based nanomaterials have shown significant immunogenicity, which can promote antigen presentation, cytokine secretion, CD4⁺ and CD8⁺ T cell proliferation, and effector memory T cell population [42], [43], [44], [45], [46], [47], [48], [49]. Therefore, it is considered that combining different nanomaterials with immune stimulation capacity may maximize the immune responses in immunotherapy. However, the applications of Si - Mn - based NPs to radiotherapy to synergistically inhibit both tumor growth and tumor recurrence and metastasis through the regulation of TME and immune responses are still rare. Whereas, tumor recurrence and metastasis are still challenging for tumor treatment by RT, indicating that simply aiming at enhancing primary tumors treatment is far from satisfaction, and the problems of tumor recurrence and metastasis are urgent to be solved. The Si-Mn-based NPs have been synthesized to regulate the TME to a beneficial status before exposure to radiation in radio-immunotherapy from different aspects: oxygenation, GSH depletion, ROS generation, and immune activation. Notably, due to the ROS-involved redox metabolism equilibrium, only generating ROS may trigger more GSH production and result in more ROS consumption by GSH. Here, Mn-based nanomaterials not only induce ROS generation, but also deplete GSH, which breaks the ROS-involved redox metabolism equilibrium to regulate TME. In conclusion, the Si-Mn-based NPs have not only acted as the radiosensitizer to enhance the treatment of primary tumors by RT at a low dose of radiation, but also played a role in activating immune response to inhibit the tumor recurrence and metastasis.

Herein, we prepared Si - Mn - based NPs by coating MnO₂ onto SiO₂ NPs (SM NPs) by a hydrothermal method to solve the limitation of TME - induced resistance and inhibit tumor growth and tumor metastasis in radio-immunotherapy (Scheme 1). The obtained SM NPs induced the generation of O₂, which could relieve hypoxia to reduce radiation tolerance and immune suppression, as well as depleted GSH and induced OH generation to increase the levels of ROS and lipid peroxidation (LPO) and enhance DNA damage in Lewis lung carcinoma (LLC) cells. Thus, the cancer cell killing effect of RT was improved at a low dose, at which dose radiation alone could'nt kill cancer cells. Furthermore, SM NPs stimulated antitumor immune responses through improving the secretion of interleukin 1β (IL - 1β) and tumor necrosis factor - α (TNF - α) by macrophage - like cells in vitro. Interestingly, combining low - dose radiation with SM NPs and the anti - CTLA - 4 antibody induced a significant inhibitory effect on the growth of primary tumors and distant untreated tumors. Therefore, it was demonstrated that SM NPs may provide a new strategy for radio - immunotherapy to inhibit primary tumor growth at a low dose of radiation and suppress metastatic tumor growth even at distant sites not exposed to radiation.