Pivotal Roles Of Tumor-Draining Lymph Nodes In The Effect For Combined Immunotherapy And Radiotherapy

IntroductionRadiotherapy overcomes the challenges posed by immunotherapy resistance through providing enhanced tumor immunogenicity and modulation of the tumor microenvironment(TME).Immune checkpoint inhibitors enhance the abscopal effect of radiotherapy by improving the function of T lymphocytes.An increasing number of clinical trials and preclinical experiments have shown that immunotherapy combined with radiotherapy(iRT)is better than radiotherapy or immunotherapy alone and with a combined effect of 1+1>2.Lymph nodes are important secondary lymphoid organs that regulate the interaction and transport of many types of immune cells,such as T lymphocytes,B lymphocytes,and antigenpresenting cells.Tumor draining lymph nodes(TDLNs)are pivotal for radiotherapy-mediated immunostimulatory and abscopal effects.Although PD-1/PD-L1 blocking can activate T lymphocytes in the TME,PD-L1 antibodies can play a role in TDLNs.After radiotherapy,initial T lymphocytes are activated by mature dendritic cells(DC)in TDLNs,and the function of tumor-specific cytotoxic T lymphocytes induced by radiotherapy is affected during the loss of TDLN function.Currently,some studies have shown that TDLN dysfunction caused by surgery,radiotherapy,drug inhibitors,or gene ablation significantly weakens the anti-tumor effect of radiotherapy or therapy.Selective TDLNs radiotherapy can regulate chemokine signal transduction to reduce intratumoral immune infiltration and result in a decrease in the CD8+T/Treg ratio in the TME.However,the role and mechanism of TDLNs in the iRT-induced abscopal effect are not clear.TDLNs are the main site of tumor antigen presentation and the source of metastasis in most cases.Therefore,the type and function of immune cells in TDLNs play an important role in the tumor immune microenvironment(TIME)and may affect the clinical outcome of tumors.Different lymphocytes enter the TIME to regulate anti-tumor immune responses.Specific subsets of immune cells have been identified as key drivers of anti-tumor activity,such as CD8+T lymphocytes,tumor-associated macrophages(TAMs),and myeloid-derived suppressor cells(MDSCs).A key factor determining the success of anti-tumor immunity and immunotherapy is the efficient recruitment of immune effector molecules in the TIME,especially the activation of tumor-specific CD8+T lymphocytes.To explore the role and mechanism of TDLNs in the treatment of iRT,we put forward the following scientific hypothesis:TDLNs promote the iRT-induced anti-tumor effect by regulating the TIME.To verify this hypothesis,we established mouse primary and abscopal tumor models and TDLN resection models,evaluated changes in the abundance of immune cells in the TIME of primary and abscopal tumors after iRT treatment in different TDLN states,and further verified the important role of TDLNs in the prognosis of patients.Objectives1.To investigate the effect of TDLNs on primary and abscopal tumors during radiotherapy,immunotherapy,and iRT.2.To explore the mechanism by which TDLNs mediate the anti-tumor and abscopal effects of iRT.3.To verify the important role of TDLNs in the prognosis of patients through patient data analysis.MethodsPart I:The roles of TDLNs in radiotherapy combined with immunotherapy in mice A mouse model of the unilateral subcutaneous tumor was established using mouse MC38 colon cancer cells and B16F10 melanoma cells.The mice were randomly divided into nine groups.Radiotherapy,anti-PD-1 immunotherapy,and iRT therapy were administered in the presence or resection of TDLNs to determine the effect of iRT on primary and abscopal tumors.Then,the mouse model treated with FTY720 was established to block T-cell egress from lymphoid tissues and inhibit the function of lymph nodes in immunity.The role of TDLNs in the iRT-induced anti-tumor response was further verified.The bilateral subcutaneous tumor models of the above two cell lines were randomly divided into 20 groups to verify the effects of radiotherapy,anti-PD-1,and iRT on primary and abscopal tumors after resection of primary,abscopal,and bilateral TDLNs,respectively.The tumor models were used to determine the role of different parts of TDLNs in the anti-tumor effect of primary tumor and the abscopal effect of tumors in mice.Part II:The mechanism of TDLNs in regulating tumor immune microenvironment contributing to anti-tumor effects of iRTTo determine the role of TDLNs in iRT treatment,TDLNs from primary and abscopal sides of mice at 1,3,5,and 7 days after iRT treatment were used for RNA sequencing(RNA-Seq)analysis of differentially expressed genes and signaling pathways.To identify the immune cell subsets that play a role in TDLNs after iRT treatment and study the possible mechanism underlying the involvement of TDLNs,flow cytometry was performed to analyze the distribution of immune cells in TDLNs at different time points after iRT treatment.To clarify the changes in the TIME of primary and abscopal tumors induced by TDLN resection,tumor-infiltrating lymphocytes(TILs)were extracted from tumors and analyzed by RNA-Seq and NanoString analysis.Results showed that the differences in the enrichment of differentially expressed genes(DEGs)and signaling pathways were mainly concentrated in CD8+T cells and TAMs after TDLN removal and iRT.Flow cytometry and immunofluorescence staining were performed to determine the distribution of CD8+T cells in the TIME,the changes in the level of immune effector factors,and the role of TAM polarization in iRT therapy.Furthermore,the CD8+T-cell depletion test verified the key role of CD8+T cells in iRT treatment.Part III:The relationship between the number of resected lymph nodes and the prognosis of patients with breast cancerData on a total of 42,557 patients who were diagnosed with breast cancer and underwent surgery from January 2011 to December 2011 were collected from The Surveillance,Epidemiology,and End Results(SEER)database.The patients with an unknown number of resected axillary lymph nodes and axillary lymph node metastases were excluded,and there were 24,464 patients in the group.Follow-up was conducted until 2018.The end events were OS and breast cancer-specific survival(BCSS).The X-Tile software was used to analyze the appropriate critical point for grouping the number of negative lymph nodes,and SPSS 19.0 was used to analyze the data.The survival curve was drawn by Kaplan-Meier survival analysis,and the differences between groups were tested by the log-rank test.Values with p ≤ 0.05 were considered statistically significant.A total of 220 patients with breast cancer who underwent primary breast-conserving surgery and no axillary lymph node metastasis in Shandong Cancer Hospital from April 2013 to March 2020 were analyzed retrospectively.The changes in the indices of routine blood parameters before and after surgery were analyzed.The patients were divided into two groups according to the number of axillary lymph nodes resected.The differences in the count of white blood cells,lymphocytes,neutrophils,monocytes,and platelets before and after surgery were analyzed.SPSS 19.0 was used to analyze the data,and the nonparametric Mann-Whitney ranksum test was performed.Values with p≤0.05 were considered statistically significant.ResultsPart I:The roles of TDLNs in radiotherapy combined with immunotherapy in mice1.Determining the location of TDLNs and the mode of radiotherapy in miceTo determine the location of TDLNs,methylene blue staining was performed by subcutaneous injection into the tumor site in mice.Transparent lymph nodes were identified in the same location in the mice without methylene blue staining,and the complete lymph node structure was verified by the pathologist,indicating that our TDLN resection model was constructed successfully.On day 2 after TDLN resection or pseudoresection in mice,the primary tumor(left side)of mice was irradiated vertically for 12 Gy after anesthesia.All body parts of mice except the tumor were shielded by a 4 mm thick customized lead block.This method was used in the radiotherapy of tumors in mice.2.Intact TDLNs are required for iRT-induced anti-tumor effectsIn the mice model of unilateral subcutaneous tumor formation in MC38 and B16F10 cell tumor-bearing mice,monotherapy(radiotherapy or anti-PD-1)or iRT was performed with or without TDLN removal.The results showed that iRT enhanced the anti-tumor effect compared with radiotherapy or anti-PD-1.The anti-tumor effect of the iRT group was significantly better than that of the iRT+Resection group.Our study shows that TDLNs are implicated in the antitumor effect induced by iRT.Based on the above experimental results,TDLNs are an important hub of anti-tumor response promoted by iRT.3.Role of FTY720 drug-mediated weakening of the anti-tumor effect of iRTFTY720 was administered to both the control and iRT groups.After 48 h of radiotherapy,the level of CD3+T cells in peripheral blood was analyzed by flow cytometry.The use of FTY720 significantly reduced the infiltration of peripheral T lymphocytes.The tumor growth curve showed that FTY720 treatment significantly weakened the anti-tumor effect induced by iRT.Therefore,TDLNs are an important center of anti-tumor response induced by iRT.4.Intact TDLNs are required for iRT-induced abscopal effectsThe role of TDLNs in the abscopal effect induced by iRT was verified in the established bilateral tumor-bearing mouse model.Surgical resection of unilateral or bilateral TDLNs was performed to verify the contribution of the different parts of TDLNs to the anti-tumor and abscopal effects induced by different treatments.We found that bilateral TDLN resection significantly weakened the anti-tumor and abscopal effects of iRT on primary and abscopal tumors.Part II:Elucidation of the mechanism by which TDLNs regulate the TIME,contributing to the anti-tumor effects of iRT1.Transcriptomes of TDLNs demonstrate different repertoires after iRTTo determine the mechanism by which TDLNs exert an anti-tumor response after iRT treatment,TDLNs from primary and abscopal sides of mice at 1,3,5,and 7 days after iRT treatment were used for RNA-Seq.We found significant differences in DEGs and enriched signaling pathways between primary and abscopal tumors at different time points after iRT treatment.TDLNs of primary and abscopal tumors may promote the anti-tumor immune response in a time-dependent manner through different mechanisms.The mechanism of the specific role of TDLNs in iRT treatment needs to be verified by further experiments.2.Increase in the abundance of stem cell-like CD8+T cells in TDLNs induced by iRTTo identify the immune cell subsets in which TDLNs play a role in iRT treatment,TDLNs at different times after iRT treatment were analyzed by flow cytometry.We found that CD8+T cells did not change significantly with the increase of time after iRT treatment in both primary and abscopal TDLNs,but stem cell-like TCF1+Tim3-PD-1+CD8+CD45+T cell subsets significantly increased,then decreased and gradually returned to the normal level,indicating that stem cell subsets in TDLNs may play an important role in iRT therapy,and the specific mechanism needs further analysis of immune cells in tumor tissue.3.Transcriptomes of TILs from iRT-treated tumors demonstrate different repertoires after removal of bilateral TDLNsTo evaluate the effect of iRT on TILs,TILs were isolated from primary and abscopal tumors 7 days after iRT treatment,and RNA-Seq was performed.We found a significant statistical difference in the expression of TILs in CD8+ T cells and macrophages between primary and abscopal tumors during the presence and resection of TDLNs.Bilateral TDLN excision may affect iRT-mediated T-cell response and TAM polarization;however,the specific mechanism needs to be further verified.4.TDLN resection leads to decreased enrichment of TILs for immune-related pathways after iRT treatment of tumorsTo further clarify the mechanism of bilateral TDLN resection on iRT-mediated T-cell response and TAM polarization,we performed a Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis on the RNA-Seq data.We further compared the pathways enriched in the iRT group vs.control in primary tumors with those enriched in the iRT group vs.iRT treatment combined with TDLN resection in the primary tumors.We found two overlapping pathways,one relating to the cytokine-cytokine receptor interaction and the other to phagosomes.Using similar comparison methods to examine TILs of the abscopal tumors,we found 9 overlapping pathways,which also included those related to cytokine-cytokine receptors pathway and phagosomes in addition to the chemokine signaling pathway.Notably,metabolic and oxidative phosphorylation pathways were significantly upregulated in the TILs of abscopal tumors after iRT.The abundance of TILs was downregulated after TDLN resection,thus illustrating a potential difference between the primary and abscopal TME.5.TDLN removal impairs iRT-induced anti-tumor immune responses mediated by CD8+T cellsFirst,a unilateral MC38 cell tumor model was established and treated with radiotherapy and or anti-PD-1 with or without TDLNs.Flow cytometry showed that iRT induced CD8+Tcell infiltration in a manner dependent on the presence of intact TDLNs.Furthermore,the bilateral MC38 cell tumor model was established,and iRT treatment was performed under the condition of surgical resection or pseudo-surgical resection of bilateral TDLNs.Flow cytometry analysis and immunofluorescence staining showed that removal of TDLNs alone did not affect the infiltration of CD45+T and CD8+T cells in primary or abscopal tumor TME without iRT treatment.Moreover,iRT treatment significantly increased the infiltration of CD8+T cells and CD45+T cells,while TDLN resection decreased the infiltration of CD8+T cells and CD45+T cells.Furthermore,iRT resulted in the significant enrichment of IFNγ+and Granzyme B+T cells in primary and abscopal tumors,while this enrichment decreased significantly after TDLN resection.The role of CD8+T cells was further verified by an Anti-CD8 antibody depletion test.6.TDLNs maintain TAM polarization in primary and abscopal TME after iRT treatmentWe found that the expression of Ml and M2 TAMs in the iRT group was significantly different from that in the iRT+Res group by NanoString analysis.The bilateral MC38 cell tumor model was used to further verify the effect of different states of TDLNs on myeloid cell infiltration in the TME during iRT.Compared with the differences in the abundance of PMN-MDSCs,M-MDSCs,and DCs in primary and abscopal tumors,the infiltration of TAMs in primary and abscopal tumors decreased due to iRT,and this decrease was reversed by TDLN resection.In primary tumors,compared with iRT alone,iRT+Res led to decreased enrichment of M1 TAMs.Although no significant change in the enrichment of M2 TAMs was observed,the ratio of CD86+M1/CD206+M2 and MHCⅡ+M1/CD206+M2 was significantly increased.In abscopal tumors,iRT treatment did not change the enrichment of M1 and M2 TAMs,but the ratio of CD86+M1/CD206+M2 was significantly upregulated.Immunofluorescence staining was used to analyze the expression of TAMs in primary and abscopal tumors.Consistent with the results of flow cytometry,decreased infiltration of M1 TAMs and increased infiltration of M2 TAMs were detected in primary and abscopal tumors in the bilateral TDLN resection group of iRT.Part Ⅲ:The relationship between the number of resected lymph nodes and the prognosis of patients with breast cancer1.Poor prognosis of patients with excessive lymphadenectomyThe clinical data of patients with breast cancer without axillary lymph node metastasis in the SEER database were analyzed.Using X-Tile software,the critical value of axillary lymph node resection related to patient survival was found to be 6.The patients were divided into two groups according to the critical value:one with<6 resected lymph nodes and another with>6 resected lymph nodes.The patients in both groups were mainly in stages Ⅰ and Ⅱ,accounting for 72.61%and 24.66%of all patients,respectively.In terms of the molecular subtype,luminal A accounted for 71.1%of all patients.Similar differences in the molecular subtype were found between the two groups with a different number of negative lymph nodes.We found that patients with a higher number of resected lymph nodes had a poorer prognosis.2.Excessive lymphadenectomy leads to a decrease in lymphocyte count after surgeryThe data of 220 patients with breast cancer in Shandong Cancer Hospital were analyzed.With the increase in the number of resected lymph nodes,the difference between postoperative lymphocyte count subtraction preoperative lymphocyte count became lesser,without any significant difference in monocyte count when the number of lymph nodes was 1-11.With an increase in the number of lymph nodes,the postoperative lymphocyte count decreased;however,no significant difference in the count of white blood cells,neutrophils,monocytes,and platelets was observed before and after surgery.Conclusion1.The anti-tumor effect of iRT treatment is better than that of radiotherapy or anti-PD-1 alone.Compared with radiotherapy or anti-PD-1 alone,iRT treatment induces a more obvious abscopal effect.Intact TDLNs are necessary for the anti-tumor effect of primary and abscopal tumors induced by iRT.2.In the presence of TDLNs,iRT led to significant infiltration of functional CD8+T cells and M1 TAMs in both primary and abscopal tumors,contributing to efficient anti-tumor effects.However,the absence of bilateral TDLNs impaired the enrichment and cytotoxic capacity of CD8+T cells coupled with M2 polarization of TAMs in primary and abscopal TMEs,resulting in compromised iRT-induced tumor regression.3.Excessive lymphadenectomy is associated with a lower absolute lymphocyte count,which may have a poorer prognosis.Because cancer patients are frequently immunocompromised as a result of the disease or treatment,it may be clinically advantageous to limit the radiation dose(or degree of resection)to uninvolved contralateral lymph nodes to avoid hampering the possibility of abscopal responses.Innovation1.The mice model of TDLN deletion was successfully established by surgical resection of TDLNs,which confirmed the important role of TDLNs in radiotherapy,anti-PD-1,and iRT induced abscopal effects in primary and abscopal tumors,respectively.2.Although iRT has been widely recognized as a combination therapy for cancer,the role and mechanism of TDLNs have not been confirmed.By establishing a mice abscopal tumor model,our experiment demonstrated that TDLNs play a role in iRT mainly by regulating the activation of CD8+T cells and polarization of TAMs in TIME.3.Because patients with cancer are often immunocompromised from cancer itself or from therapy,preservation of lymphocytes(and lymph nodes)may be a highly consequential clinical goal,especially for patients who may subsequently undergo radiotherapy or immunotherapy.Using the clinical data of the patients,combined with SEER database analysis,we confirmed the important role of lymph nodes in the prognosis of patients,thereby providing important insights into the therapeutic strategies of preserving uninvolved TDLNs for low-risk patients.Limitations1.Although mice have a genetic background that is comparable to that of humans,their immune systems are not.The primary and abscopal tumor models are located on the left and right sides of the back of mice,respectively.We performed left,right,and bilateral TDLNsresection experiments to confirm the integrity of TDLNs in primary and abscopal tumors,especially the primary tumor lymph nodes,for iRT treatment.To further understand the role of TDLNs in patients with primary and metastatic cancers,more clinical data is needed.2.In this study,we investigate the effect of iRT therapy on the distribution of immune cells in primary and abscopal TDLNs at different time points.However,the mechanism by which TDLNs affect the activation of CD8+T cells and TAM polarization in the TME remains unclear.3.The condition in patients with different lymph node resection states mentioned in our experiments with iRT treatment was not present.Therefore,we only included patients with breast cancer who had different axillary lymphadenectomy statuses in the clinical data analysis and did not administer iRT therapy.