[I have been busy with new books recently and have my job, so the revision will be slower. You can wait until the book is finished before reading]

Radiotherapy (RT) is an indispensable treatment method for lung cancer. For patients with iiia and iiib who are not suitable for surgery, radical chemoradiotherapy is the standard of treatment, which has been widely accepted. Unfortunately, tumor regrowth after radiotherapy is the main obstacle to successful disease control.

Preclinical studies have shown that radiotherapy has a dual effect on the tumor immune microenvironment at the primary and metastatic sites. On the one hand, local irradiation may activate the systemic immune response against tumor cells far away from the irradiated area, i.e., the distant segregation effect, first reported by mole       1953     .rt promotes the release of tumor antigens from dying tumor cells, upregulates mhc                                                                                                                                                                                            �

For example, rt upregulates the expression of a variety of immunosuppressive cytokines, such as tumor necrosis factor-α(tnf-α), il-6, il-10     and transforming growth factor-β(tgf-β). rt     Promote the accumulation of immunosuppressive cells, such as tumor-associated macrophages (tam), regulatory T                                                                                                                                                                                                      

In order to overcome the immunosuppressive effect of radiotherapy, various combinations of immunotherapy and radiotherapy have been proposed and tested. In the pacific study, the addition of durvalumab after standard chemoradiation and chemotherapy can prolong the progression-free survival and overall survival of patients. The huge success of the study not only changed the clinical guidelines for stage iii    Nonsclc                                                                                                                                                                                                            

The accumulation and activation of mdscs play a key role in the establishment of immunosuppression. The exact effect on mdscs is complex. Large segmentation irradiation (20    gy) inhibits the accumulation and infiltration of mdscs, while lower doses of irradiation often promote the recruitment of mdscs into tumors. In addition, preclinical and clinical studies have shown that gastrointestinal tumors, including colorectal and liver cancer, are relatively compatible after radiotherapy

The levels of mdscs are prone to decrease, while in other tumor models and clinical settings, such as glioma, lung cancer, breast cancer, head and neck squamous cell carcinoma, prostate cancer and cervical cancer, lead to a continuous increase in the number of mdscs. Cai et al. reported for the first time that mdscs’ expression was upregulated by irradiation. However, little is known about the exact role of mdscs, especially its underlying mechanism, in shaping TME after irradiation.

mdscs are a group of heteromyeloid cells with strong immunosuppressive abilities. According to phenotype and morphology, mdscs can be divided into polymorphic mdscs (pmn-mdscs, also known as granulocyte-type mdscs) and monokaryotic mdscs (m-mdscs).

Immunosuppressive activity is a key marker of mdscs. The inhibitory mechanisms of mdscs include the expression of inducible nitric oxide synthase (inos), arginase 1 (arg1) and indoleamine 2,3-dioxygenase (ido), as well as the production of nitric oxide (no) and reactive oxygen species (ros). These different mechanisms do not work at the same time. It is generally believed that pmn-mdscs and m-mdscs regulate TME through different mechanisms. In addition, the upregulation of pd-l1 is also considered to be one of the immunosuppressive mechanisms for radiation recruitment of mdscs. At present, there is no consensus on the effect of irradiation-induced mdscs on TME and the therapeutic effect of radiotherapy.

Phosphodiesterase-5 (PDE5) inhibitors, such as sildenafil and tadalafil, can block the hydrolysis of cyclic guanosine phosphate (CGMP). Latest data show that PDE5    inhibitors can promote antitumor immunity by inhibiting the activity and expression of inos and arg1 in tumor-bearing (TB) mice and patients. However, how PDE5    inhibitors affect the subpopulation of mdsc     and whether combinations of RT                                                                                                                                                                                 �

Our recent research shows that elimination of recruited mdsc     delays regeneration of Lewis lung cancer (LLC) after radiotherapy. Here, we report that local irradiation weakens anti-tumor immunity by promoting the proliferation of PMN-Mdsc    and its subsequent recruitment to TMME   . Upregulation and activation of arg1 are the main mechanisms of PMN-Mdscs-mediated T-cell inhibition after irradiation. The combination of sildenafil and radiotherapy eliminates radiation-derived immunosuppression by inhibiting                                                                                                                                                                             

In tb                                                                                                                                                                                                                                                             �

As shown in Figure 1c, the percentage of total mdscs in tumor tissues steadily increases with tumor development, rising from less than 10% (5.84±1.22%) of tumor-infiltrating lymphocytes in the first week after inoculation to more than 20% (21.71±3.22%) in the fourth week (p<0.01). In our llc                                                                                                                                                                                                           �

In order to better understand the systemic distribution of mdscs, we also studied the proportion of mdscs in peripheral blood, spleen and bone marrow. Only one week after inoculation of 11C cells, the percentage of mdscs in peripheral blood of TB mice was twice that of tumor-free mice (27.33±4.62% vs.    12.48±0.93%, p<0.05), and the percentage of mdscs after 3 weeks was 5 times that of tumor-free mice (27.33±4.62% vs.     12.48±0.93%, p<0.05). The proportion of pmn-mdscs in peripheral blood of TB mice also increased, while the proportion of m-mdscs was not related to tumor size.

pd-l1    is one of the most important checkpoint molecules for expression of tumor cells, mdscs, macrophages and dendritic cells. To determine whether the expression of mdscs in tb                                                                                                                                                                                                                                 �

The mean fluorescence intensity (mfi) of pd-l1 gradually increased from 386.8±100.9 in the first week after cell inoculation to 1,068.0±121.8 in the fourth week, a.u(      p   <0.01), which shows that pd-l1 has a potential role in mdsc     in our model.

In the spleen and bone marrow, the proportion of mdscs increased significantly as the tumor progresses, with the same pattern as mdscs in tumor tissue and peripheral blood. In addition, we observed significant splenomegaly in TB mice, which is consistent with the clustering of mdscs in the spleen that we observed.

To determine the effect of local irradiation on tumor growth and mdscs, when the tumor diameter reaches 7.5    mm, we used large segmentation RT (20    gy/f) to treat subcutaneous llc tumors. Radiotherapy resulted in a delay in tumor progression for up to one week, with a minimum volume of about 500mm on days 7 to 10, but the tumor began to regenerate. According to the tumor growth curve before and after radiotherapy, we chose the 3rd day after radiotherapy as the growth before and after radiotherapy as the growth of the day after radiotherapy, and the minimum tumor volume was the beginning of regeneration, and the 2 and 3 weeks after radiotherapy as the regeneration stage. Hematoxylin-eosin staining in the tumor tissue showed that compared with untreated tumors, there were more infiltrating inflammatory cells in the tumor after radiotherapy. Subsequent CD11b specific immunohistochemical staining showed that most of the inflammatory cells were CD11b myeloid cells, which indicated that local irradiation was

Ejaculation may lead to the accumulation of mdscs. To confirm our hypothesis, we performed flow cytometry analysis of total mdscs and these two subpopulations at different time points after local irradiation. After local irradiation, the proportion of tumor infiltrating mdscs after radiotherapy was 2 times higher than that of untreated tumors (ctrlvs.                                                                                                                                                                                                              �

To determine whether the gradual accumulation of PMN-Mdsc in irradiated tumors contributes to the regrowth of llc, or whether this accumulation is only the result of tumor growth, the use of anti-ly-6g monoclonal antibodies to consume mdsc. The application of anti-ly-6g antibodies significantly reduced the frequency of PMN-Mdsc in tumor sites and peripheral blood (p<0.05). In addition, treatment with anti-ly-6g antibodies greatly delayed regeneration after irradiation, which indicated that recruitment of PMN-Mdsc is crucial for tumor regeneration.

Although PMN-Mdscs utilize a series of mechanisms to inhibit anti-tumor immune responses, which involve many immune cells and cytokines, inhibition of cd8t cells is undoubtedly the most important. To determine whether pmn-Mdscs-induced immunosuppression depends on cd8 cells after rt                                                                                                                                                                                                                        �

As shown in Figure 3d, the percentage of cd8                                                                                                                                                                                                                                                        �

The expression of ifn-γ, cd28 and pd-1 on the internal and external surfaces. Local rt                                                                                                                                                                                                                                                 

No significant changes were observed in cd28 expression. When the anti-ly-6g antibody was given to irradiated mice, ifn-γ secretion reached the same level as that of the untreated llc mice (29.74±3.55%), while the expression of pd-1 after treatment with irradiated mice was not changed. Therefore, in this section we recommend that pmn-mdscs promote tumor regeneration after radiotherapy by inhibiting cd8                                                                                                                                                                                                     �

To determine the inhibitory mechanism of irradiation-induced mdscs, we performed immunohistochemical staining and inos and arg1 activity detection. Immunohistochemical staining of tumor sections showed that local RT enhanced the expression of arg1 but did not enhance the expression of inos. The arg1 activity assay showed that local irradiation significantly increased the activity of arg1, from 0.400.15    u/l to 3.78    0.39     u/l      ((p<0.01). In contrast, no fluorescently labeled inos activity detection showed that the fluorescence intensity of irradiated and untreated tumor tissue samples was similar.

The expression of pd-l1 is a novel immunosuppressive mechanism of mdscs     . We then asked whether pd-l1 upregulation is one of the mechanisms of immunosuppression mediated by rt                                                                                                                                                                                                                                5.3   au    vs.      1328.0      ±324.3   au,p<0.05). However, the expression of pd-l1 continued to decline after that, and the local irradiated group was significantly lower than that of the untreated group at the 3rd week after irradiation (ctrl    vs.                                                                                                                                                                                            

The above data show that the upregulation of arg1 expression is a reasonable mechanism for the inhibitory function of PMN-MDSSCs after irradiation. However, the regulation of pd-l1 and inos is not involved. To further confirm this hypothesis, the inhibitor Nor-noha was administered by gavage after radiation.10    mg/kg/d    nor-noha    effectively reduces the activity of arg1                                                                                                                                                                                               

The administration of nor-noha after RT significantly increased the proportion of cd8t cells (rt    vs.      rtnor-noha=2.420.62%    vs.   6.14   0.64,     p<0.01) and was accompanied by delayed tumor regeneration. Inos inhibitor 1400w     had no effect on tumor regeneration.

Our results show that rt promotes tumor immune evasion by upregulating the percentage of PMN-Mdsc     and arg1 activity. It is reasonable to speculate that inhibition of PMN-Mdscs     and its arg1 activity may be a new anti-tumor strategy. There is growing evidence that pde5 inhibitors can inhibit the activity and expression of inos and arg1 in tb    Mdsc    Mdsc     Mdsc      Mdsc         Mdsc                                                                                                                                                               � Sildenafil was administered 20    mg/kg/d to study whether it could promote the antitumor effect of rt     and elucidate the underlying mechanism. As we expected, sildenafil delayed tumor regeneration after irradiation, which is comparable to the positive control nor-noha. The immune characteristics of tme    show that when sildenafil was given, the proportion of pmn-mdsc    in tumors decreased from 38.66±4.24% to 23.57±2.38%.

In addition, sildenafil also significantly reduced the expression of arg1. To further examine whether sildenafil’s inhibition of mdsc                                                                                                                                                                                                                                            

In addition, when sildenafil was given, ifn-γ secreted by cd8                                                                                                                                                                                                                                                   �

Work reveals a new mechanism by which the Arg1 pathway mediates tumor regeneration after RT in PMN-MDSSCs, as shown in Figure 6. We believe that PMN-MDSSCs is the main subtype of irradiation recruitment, not m-MDSSCs. In a broad immunosuppressive mechanism, upregulation and activation of arg1 are the main mechanisms for PMN-MDSSCs to inhibit CD8T cells after radiotherapy. To overcome the immunosuppression caused by PMN-MDSSCs, we propose and demonstrate that the combined use of SLD and RT reduces PMN-MDSSCs

Recruitment and immunosuppression of S in TME activates the CD8 T cell response, resulting in delayed tumor growth. In summary, our findings provide a new solution to alleviate immunosuppression of TME to improve the therapeutic effect of RT. Although all these results were performed in LLC mouse models, it is not clear whether the same mechanism applies to other tumor models. In addition, how MDSSCs and their subtypes affect TME under different radiation regimens is still undetermined. Therefore, these issues need further research to resolve.
Chapter completed!