Radiotherapy remodels the tumor microenvironment for enhancing immunotherapeutic sensitivity

Abstract

Cancer immunotherapy has transformed traditional treatments, with immune checkpoint blockade being particularly prominent. However, immunotherapy has minimal benefit for patients in most types of cancer and is largely ineffective in some cancers (such as pancreatic cancer and glioma). A synergistic anti-tumor response may be produced through the combined application with traditional tumor treatment methods. Radiotherapy (RT) not only kills tumor cells but also triggers the pro-inflammatory molecules' release and immune cell infiltration, which remodel the tumor microenvironment (TME). Therefore, the combination of RT and immunotherapy is expected to achieve improved efficacy. In this review, we summarize the effects of RT on cellular components of the TME, including T cell receptor repertoires, different T cell subsets, metabolism, tumor-associated macrophages and other myeloid cells (dendritic cells, myeloid-derived suppressor cells, neutrophils and eosinophils). Meanwhile, non-cellular components such as lactate and extracellular vesicles are also elaborated. In addition, we discuss the impact of different RT modalities on tumor immunity and issues related to the clinical practice of combination therapy.

Fig. 1. Irradiation-mediated alterations of T cells in the TME.

A RT increases the expression of tumor-associated antigen NY-ESO-1, which binds CRT and thus activates CD8⁺ T cells to secrete more IFN-γ. Irradiation induces cytoplasmic DNA, which is sensed by cGAS, generating the second messenger cGAMP, thereby activating the STING protein. STING has been shown to induce IFN-β production by recruiting TBK1 to activate IRF3. IFN-β acts in an autocrine manner on the IFNAR of T cells, inhibiting AKT activity and promoting TCF-1 expression, which maintains the T cell stem cell-like state. However, RT activates IRF1 to promote Serpinb9 gene expression, thereby blocking CD8⁺ T cell attack. On the other hand, irradiation induces tumor cells to secrete IFNs, working on the IFNAR to activate Serpinb9 gene expression through the JAK/STAT pathway. In addition, irradiation reduces MYC expression levels and downregulates GLUT1, HK2 and LDHA genes involved in glucose uptake and glycolysis of T cells through mTORC regulation. Treg cell transcription factor Foxp3 reprograms cellular metabolism by repressing MYC. RT elicits an increase in ROS, activates NFAT and subsequently promotes IL-2 production, thereby activating effector T cells. B RT increases CD62^-CD44⁺ effector memory T cells and CD62⁺CD44⁺ central memory T cells in the spleen. Irradiation augments activated CD25⁺CD8⁺ memory T cells, CD25⁺CD4⁺ memory T cells and ICOS⁺CD4⁺ effector memory T cells in peripheral blood. C RT combined with immunotherapy (αPD-1 and αCTLA-4) facilitates the differentiation of pre-exhausted Th1-like cells into intratumoral CD4⁺ Tex cells, during which exhaustion-related and cytotoxic genes are upregulated. D RT enhances the secretion of activin A from tumor cells. Activin A binds to the corresponding receptor ActRI/ActRII, activates the receptor kinase activity and phosphorylates the intracellular mediator SMAD2/3. SMAD2/3 translocates to the nucleus and binds to the CNS1 together with the NFAT. This promotes the transcription of Foxp3. Irradiation induces ROS production, which is reported to stabilize and accumulate SENP3, thereby mediating deSUMOylation of the transcription factor BACH2 and maintaining the immunosuppressive effects of Tregs. Solid lines represent anti-tumor effects and dashed lines represent pro-tumor effects.

Fig. 2. Effects of radiotherapy on myeloid cells in the TME.

Irradiation induces an increase in HMGB1 release from tumor cells, activating TLR4 on TAMs. Activated TLR4 initiates NF-κB/AP-1 transcription factors via the MyD88/JNK signaling pathway, and triggers a pro-inflammatory-associated translational program that promotes M1-type polarization and stimulates CD8⁺ T cells. In addition, DAMPs activate NLRP3 inflammasome and launch pro-inflammatory genes, mediating M1 macrophage polarization. RT-induced DAMPs stimulate the expression of downstream type I IFNs through the cGAS/STING signaling pathway, thereby promoting the maturation of DCs (increased expression of CD80 and CD86). DCs present MHC to activate the TCR on CD8⁺ T cells. DCs also overexpress RAE1 after irradiation, which binds to NKG2D on CD8⁺ T cells. Moreover, RT-mediated elevation of DAMPs such as HMGB1, HSP70 and S100A8/9 results in increased expression of E-selectin, ICAM-1 and VCAM-1 on endothelial cells. Endothelial cells release chemokines such as IL-6, CXCL1, CXCL2 and CCL7 to recruit TANs. Irradiation increases the release of γH2AX, followed by elevated levels of chemokines CXCL1, CXCL2 and CCL5, which recruit TANs. RT induces high ROS production from TANs and inhibits PI3K/Akt phosphorylation, thereby reducing Snail expression to reverse the EMT. In addition, RT combined with αPD-L1 suppresses the TNF pathway, which has an anti-apoptotic function, thereby reducing MDSCs. RT combined with IL-12 treatment causes MDSCs to express higher MHC-II and CD86. MDSCs produce large amounts of ROS by the NOX2, which inhibits the formation of TCR and MHC antigen complexes in T cells. Irradiation inhibits the production of ROS in MDSCs to suppress this process. Solid lines represent anti-tumor effects and dashed lines represent pro-tumor effects.

Fig. 3. The value of lactate in the radiotherapy-mediated TME.

RT upregulates the expression of PKM2 and LDHA in tumor cells, which catalyze glycolysis to produce lactate. Irradiation causes a significant increase in HIF-1α activity and regulates LDHA activity. PKM2 has been reported to stabilize HIF-1α. Lactate is transported into TME via MCT. Lactate accumulation inhibits NFAT in T and NK cells, thereby reducing IFN-γ synthesis. Lactate binds to GPR81 on MDSCs to activate Akt. Subsequently, functional genes S100A8/9, Arg1 and MMPs are upregulated through the mTOR/HIF-1α/STAT3 pathway, suggesting activation of MDSCs. Tregs take up lactate in TME via MCT1, which promotes NFAT1 entry into the nucleus and induces PD-1 expression. In addition, RT causes a decrease in LDHA expression and inhibits the conversion of pyruvate to lactate in microglia. Solid lines represent anti-tumor effects and dashed lines represent pro-tumor effects.

Fig. 4. The value of EVs in the radiotherapy-mediated TME.

The IFN-I pathway in tumor cells is activated by irradiation, and tumor cells release EVs carrying dsDNA. These EVs stimulate the expression of co-stimulatory molecules CD80 and CD86 and activate the cGAS/STING pathway in DCs to secrete IFN-β. RT promotes the release of exosomes from tumor cells to activate DCs, which contain HMGB1, CRT and HSP70. Similarly, irradiated tumors upregulate HSP70, HSP90 and a potential TAA, CDCP1 protein, in EVs. DCs have an enhanced ability to phagocytose EVs and perform antigen presentation, thereby activating CD8⁺ T cells via the PI3K/Akt signaling pathway. In addition, irradiated tumor cell-derived EVs activate Aim2 inflammasome in macrophages and induce IL-1β production to stimulate DCs. Contact of EVs carrying HMGB1 with TAMs results in increased expression of activation markers CD86, CD64, CCR7 and pro-inflammatory factors TNF-α and IL-12 p70, suggesting a conversion of M2 to M1 macrophages. Solid lines represent anti-tumor effects and dashed lines represent pro-tumor effects.

Fig. 5. Radiotherapy remodels the TME in different cancer species.

RT alters the TME in a variety of cancers, including tumor suppression and tumor promotion. RT induces differentiation and maturation of DCs, increases CD8⁺ T cells and effector molecules secretion, stimulates macrophage polarization and attenuates suppressive myeloid cells. However, RT facilitates tumor progression by acidifying the TME.