Differences in the tumor microenvironment are key factors affecting the effectiveness of radiotherapy and the prognosis of the disease after radiotherapy. Studies have demonstrated that tumor infiltrating T cells (TILs) increased after neoadjuvant radiotherapy and chemotherapy in cervical cancer, esophageal cancer and rectal cancer [15]. However, another study in patients with cervical cancer reported a decrease in TILs after radiotherapy [16]. Regarding these seemingly contradictory results, they may be explained by changes in the microenvironment of tumor cells at different stages of radiotherapy. In vitro research, we found that the expression of Siha cell membrane protein PD-L1 in the medium containing lactic acid was higher than that in the ordinary medium, and cells in the lactic acid-containing medium had a higher survival rate after the same dose of radiation treatment.This result has also been verified in human cervical cancer tissues. Patients with high lactate concentration in cervical cancer tissue specimens are less sensitive to radiotherapy. In addition, as the radiotherapy dose increases, patients with low pre-radiotherapy PD-L1 expression are significantly more likely to have PD-L1 increases than those with high PD-L1 expression. Therefore, we believe that radiation induces changes in the tumor microenvironment, and higher lactic acid concentration in tumor microenvironment not only affects the radiotherapy sensitivity of tumor cells, but also leads to immune evasion of tumor cells by affecting the expression of PD-L1 on the tumor cell surface. Therefore, concurrent treatment of immune checkpoint inhibitors with radiotherapy may be the optimal timing to activate anti-tumor immunity.
Research has shown that immunotherapy combined with local radiotherapy can improve the anti-tumor immune response. Moreover, compared with chemotherapy, radiotherapy has obvious advantages in tumor targeting and in reducing adverse reactions of the immune system [17]. Radiotherapy induced tumor cell death is immunogenic and can induce anti-tumor immunity, which is called immunogenic cell death (ICD) by Zitvogel and Kroemer et al. [18].The antitumor immune response is similar to the "inflammatory response". Firstly, more circulating infiltrating T cells can be recruited to gather around the tumor in a state of local vasodilation and congestion in the early stages of radiation, as it usually induces an inflammatory response around the tumor tissue. However, within the course of radiotherapy, the local inflammatory response of the tumor will gradually fade away, and it will become difficult for the peripheral lymphocytes to gather around the tumor. Therefore, TILs may decrease after radiotherapy. Secondly, radiotherapy increases inflammation in tumors by activating the NF-κB and the Type I interferon response pathways to induce expression of pro-inflammatory cytokines. This inflammation coupled with antigen released from irradiated cells facilitates dendritic cell maturation and cross-presentation of tumor antigens to prime tumor-specific T cell responses [19].However, the immune responses observed in patients treated with radiotherapy alone are generally dismal and unable to produce long-lasting protective immunity, because immune response can be suppressed by immunosuppressive cells and molecules, including regulatory T cells, M2 macrophages, immune checkpoint molecules (PD-L1, CTLA-4), etc.Therefore, we speculate that during radiotherapy there may be a window of immune activation, which may be the best therapeutic opportunity for combined immunomodulatory agents. PACIFIC study revealed that immune checkpoint inhibitors administered within 14 days after completion of radiotherapy had better progression-free survival than those who administered 14 days later [20]. In another retrospective study of 758 patients diagnosed with cancer who received radiotherapy and immunotherapy (anti-CTLA-4 inhibitors or anti-PD-1/ PD-L1 inhibitors), overall survival was higher in patients who received concurrent therapy. All these studies show that we can modify the timing of the combination of radiotherapy and immunotherapy to achieve the best combined treatment effect.
Radiotherapy can increase the infiltration of T cells in the tumor.The Warburg effect refers to the synthesis of ATP by glycolysis and oxidative phosphorylation in normal cells, while cancer cells only rely on glycolysis to generate ATP even under aerobic conditions, leading to lactic acid accumulation in the tumor microenvironment. High lactate concentration in the tumor environment blocks lactate output from T cells and interferes with T cell metabolism. Experiments have confirmed that lactate inhibits the proliferation and cytokine production of human cytotoxic T lymphocytes (CTL) by up to 95% and leads to a 50% reduction in cytotoxic activity. After irradiation, lactate levels continue to decrease and return to baseline levels in several hours. The lactate level in the surrounding environment of the tumor decreases temporarily after radiation [21], and this change can reactive the infiltrated CD8 + T cells around the tumor, while the tumor cells will upregulate PD-L1 expression correspondingly to avoid the attack of CD8 + T cells. If PD-1/ PD-L1 inhibitors are added at this time, blocking the signaling pathway can increase the killing ability of CD8 + T cells to tumor, and play an optimal synergistic effect with radiotherapy. Our research group found that the higher the concentration of lactic acid in the medium, the more tolerant tumor cells were to radiotherapy.Similarly, we also found that there was a correlation between lactic acid concentration in tissues and PD-L1 expression and lymph node metastasis in cervical cancer tissue samples, which conducted in 50 locally advanced cervical cancer patients. The results showed that the expression level of PD-L1 in tumor tissues with high lactate concentration increased significantly, and lymph node metastasis was more likely in these patients. Studies on breast cancer, head and neck cancer, non-small cell lung cancer and prostate cancer have shown that vessel density is associated with the incidence of metastasis [22].It is generally believed that the acidic overlaps with hypoxic regions. However, studies have shown that H + accumulation also occurs in the hyperproliferative zone at the tumor-mesenchymal junction, and tumor cells still undergo glycolysis under aerobic conditions, so we cannot conclude that the causal relationship between hypoxia and high lactate concentration. Therefore, the high regional lactate concentration in the tumor reflects the imbalance between output and clearance of lactate.On one hand, lactic acid in the tumor microenvironment directly activates its receptor GPR81 and initiates the PKA-TAZ signaling cascade to upregulate PD-L1[23]. On the other hand, the function of cytotoxic T cells can be inhibited by directly upregulating the expression of PD-L1. T cells exposed to lactate also showed a high activation threshold, required complete activation of the costimulatory signal of CD28, and showed negative regulatory signals by upregulation of IFN-γ-R2 and CTLA-4. Previous studies have shown that lactate can reduce the expression of cell adhesion molecules and enhance cell motility on the one hand.On the other hand, the expression of PD-L1 in tumor cells increases in the lactic acid environment, which increases the possibility of immune evasion. Therefore, tumor cells in the environment of lactic acid with enhanced motility and high incidence of immune evasion are more likely to have distant metastasis, which is consistent with our findings. Both the concentration of lactic acid and the expression level of PD-L1 in tissue samples with lymph node metastasis are higher than those without lymph node metastasis. Wolfgang F found that the lactate level in tumor tissue samples could be as high as 40umol/g.To prevent intracellular lactic acid accumulation, tumor cells transport lactic acid out of cells through monocarboxylate transporter 4. However, the distribution of this lactic acid transporter in tumor tissues is not uniform, which may lead to the presence of different acidity and alkalinity regions in tumor stroma. G protein-coupled receptor GPR81 is a lactate receptor expressed on cancer cells, which also can promote the expression of PD-L1 in lung cancer [23].
Radiotherapy can promote the transformation of "cold" tumors into "hot" tumors, leading to the increase of PD-L1 expression on the surface of cervical cancer cells. In the present study, it was found that the expression of PD-L1 increased on the 2nd and 4th days of radiotherapy, and decreased on the 10th day after completion of radiotherapy. In cervical cancer tissues, we also found that the expression of PD-L1 in tumor cells showed an upward trend at the beginning of radiotherapy, but did not increase continuously with the increase of radiation dose. In an experimental study in mice, J. Dovedi et al. [24] found that fractionated radiotherapy adaptively upregulated PD-L1 expression in tumor cells and reached a peak on the third day after the end of radiotherapy, followed by a decrease, which was consistent with our findings.These experimental data suggested that the increase in PD-L1 induced by radiotherapy is not persistent. Previous results have shown that PD-L1 binds to GSK-3β phosphorylation and subsequently loses stability through proteasomal degradation of PD-L1. A study in lung cancer showed that the inactivation of gsk-3β occurred at 96h after irradiation [25]. Combined with the changes in PD-L1 expression results identified in cervical cancer tissues treated with radiotherapy, we hypothesized that a similar response system may exist in cervical cancer cells, where PD-L1 expression enters a relatively slow growth cycle, that is, a steady state, due to the inactivation of GSK-3β. At this time, tumor cells died with the accumulation of radiation dose, so PD-L1 decreased 7 days after irradiation.
In addition, studies have shown that the level of PD-L1 expression is associated with the body’s autoimmune status, regional microenvironment, and tumor differentiation degree. Our study showed that after the same dose of radiation, the expression of PD-L1 in patients with low expression (CPS ≤ 20) would further increase, while it did not obviously change or possibly decreased in patients with high expression (CPS ≤ 20).This may be because patients with originally high PD-L1 expression may be insensitive to radiotherapy due to the high lactate concentration around them, so it is difficult for radiation rays to increase PD-L1 expression by affecting the tumor microenvironment. In clinical practice, for these patients, we can only use PD-L1 inhibitors to achieve good therapeutic effects. On the contrary, for patients with low expression of PD-L1, radiation may cause a downregulation of lactate concentration in the tumor microenvironment, thereby activating CD8 + T cells and regulating PD-L1 expression. Therefore, the initiation of PD-L1 inhibitors at the beginning of radiotherapy may be crucial to generate an effective and durable anti-tumor immune response.In melanoma, PD-L1 expression coexisting with CD8 + T-cell infiltration in tumor is hypothesized to be the mechanism of immune evasion [26]. We suggest that PD-L1 expression in tumor cells may be an adaptive response to CD8 + T cells in the local microenvironment of tumor cells. When CD8 + T cells are abundant in the tumor microenvironment, tumor cells adaptively up-regulate the expression of PD-L1 in order to escape immune attack. Radiotherapy can not only change the expression of PD-L1, but also increase the expression of CD8 + T cells in the interstitial cells, and the increase of CD8 + T cells is significantly associated with the improvement of OS and PFS after chemoradiotherapy [27].Inhibiting T cell receptor (TCR) signaling by binding to PD-1 on the surface of lymphocytes leads to apoptosis of antigen-specific T cells and maintenance of peripheral immune tolerance. Therefore, the CD8 + T cell immune response induced by radiotherapy can be attenuated by the PD-1 / PD-L1 signaling pathway, and inhibit this pathway can produce a long-lasting, broad immune response. However, since this immune change caused by radiotherapy is affected by dose distribution and time, the timing of combination therapy may directly affect the efficacy of immunotherapy.
In conclusion, this research demonstrated that radiotherapy can change the expression of PD-L1 on the surface of tumor cells, which is influenced by the concentration of lactic acid in the microenvironment of tumor cells, the course of radiotherapy, and the pre-treatment expression state of PD-L1. In clinical practice, radiotherapy can be used to change the tumor microenvironment by stimulating tumor cells to induce upregulation of PD-L1 expression on the cell surface. The effect of adding PD-1/ PD-L1 inhibitors at the beginning of radiotherapy may be better than that after radiotherapy, and it can effectively prevent immune evasion of tumor cells during treatment. This study provides a robust theoretical basis for the simultaneous use of immunotherapy and radiotherapy. Our treatment center is conducting clinical studies on the combination of immunotherapy and radiotherapy in metastatic or recurrent cervical cancer,which is expected to provide more clinical data for this regimen.