| Objectives:With long-lasting response,immunotherapy improved survival of advanced cancer patients significantly.However,low response rates and treatment resistance in the overall population remain intractable clinical problems.Growing evidence shows organ-specific immunotherapy response are one of the reasons for the poor outcome of immunotherapy.Bone and bone marrow is the most common distant metastatic organ among malignancies,inducing skeletal related events that affect the quality of life and impair the efficacy of immunotherapy,seriously threatening the survival of patients.Therefore,it is of great clinical importance to investigate whether there is immunotherapy resistance in patients with bone metastasis,and how bone metastasis mediated immunotherapy response,so that we can provide a clinically available solution to reverse the negative affection of bone metastasis on the immune system and improve the immunotherapy efficacy in patients with bone metastasis.Methods:1.Impact of bone metastasis on immunotherapy.(1)Retrospectively recruited 415 patients with advanced cancer treated with immunotherapy(pan-cancer immunotherapy cohort)and collected information on patients’general condition,baseline information,organ metastasis site,treatment status,treatment evaluation within 4-8 weeks after the first immunotherapy,and progression-free survival(PFS).Survival curves were analyzed by Kaplan-Meier,differences by Log-rank test,risk of disease progression by Cox regression,and changes in extraosseous lesions after immunotherapy by chi-squared test.(2)Information on 197 patients with advanced non-small-cell lung cancer(NSCLC)(NSCLC immunotherapy cohort)was collected retrospectively.Data were collected,organized,and analyzed in the same manner as the pan-cancer immunotherapy cohort.(3)Using the public database data(download from TIDE),we analyzed the differences in overall survival(OS)of patients with different organ metastasis in melanoma.(4)The correlation between the incidence of bone metastasis and the objective response rate(ORR)was analyzed using the data published in literature.2.Recurrence of clinical phenomena in tumor-bearing mice to verify the effect of bone metastasis on immunotherapy.(1)5×10~5 B16-F10 cells were injected by puncture through the tibial plateau,then the tumor was harvested,HE staining was performed to verify the tumor growth inside the bone.(2)Transfected B16-F10 cell lines with lentivirus which overexpressed luciferase,stable expressing luciferase cells(B16-F10-Luc)were selected by purinomycin,implanted B16-F10-Luc intasubcutaneously(i.s)(5×10~5)and intraosseously(i.o)(2.5×10~5),the tumor growth was monitored by Small-Animal Imager.(3)Implanted B16-F10 cell lines i.s(5×10~5,SC group)and both i.s(5×10~5)and i.o(2.5×10~5)(SC+IO group),anti-αPD-L1 antibody or isotype antibody were administered on day 10(200μg in 200μL,i.p,every 3 days,3 times in total),then tumor growth was observed and recorded.(4)Exchange tumor model with MC38 cell lines i.s(5×10~5,SC group)and both i.s(5×10~5)and i.o(2.5×10~5)(SC+IO group),to repeat the same experiment schedule as B16-F10,and the tumor growth was observed and recorded.(5)Establishment of B16-F10 lung metastasis model(lung metastasis vs lung metastasis+bone metastasis):anti-αPD-L1 or isotype antibody was administered on day 10(200μg in 200μL,i.p,every 3 days,3 times in total),and lung tissues were harvested on day17,and the number of nodules in the lungs were counted.(6)Test for the influence of tumor burden on immunotherapy:unilateral side(SC group)or bilateral sides(SCSC group)were implanted with 5×10~5 MC38 cell lines subcutaneously,anti-αPD-L1 or isotype antibody was beginning at day 10,the growth of the subcutaneous tumors were observed and recorded.(7)Test the influence of tumor-specific immunity on bone metastasis associated immunotherapy resistance:implanted MC38 cell lines(5×10~5)i.s and B16-F10 cell lines(2.5×10~5)i.o,in day 10 administered anti-αPD-L1 antibody or isotype antibody as mentioned above,the subcutaneous tumors were observed and recorded.(8)The proportion and number of immune cell subgroups infiltrating the subcutaneous tumor microenvironment in SC and SC+IO group with B16-F10 tumor model were analyzed by flow cytometry.3.Effect of radiotherapy in bone lesions on immunotherapy efficacy of bone metastasis.(1)The effect of radiotherapy in reversing immunotherapy resistance in bone metastasis model.Anti-αPD-L1 antibody alone(immunotherapy group)or combined with radiotherapy(20Gy to bone tumor)(immunotherapy+RT group)on day 10 in MC38subcutaneous and intraosseous tumor-bearing model,the subcutaneous tumors were observed and recorded.(2)Survival analysis in bone metastasis subgroup of pan-cancer immunotherapy cohort,comparing PFS difference between patients with or without radiotherapy.Results:1.systematic immunotherapy resistance in patients with bone metastas is.(1)Results of the pan-cancer immunotherapy cohort analysis:415 advanced cancer patients who accepted immunotherapy were recruited retrospectively.In patients with advanced cancer,PFS of metastasis in organs were different compared to the overall population.Immunotherapy with bone metastasis shows a tendency of shorten PFS(HR=1.292[0.9332 to 1.789],p=0.0945).When grouped according to the presence or absence of bone metastasis,the PFS of patients with bone metastasis was significantly shorter than that of patients without bone metastasis(bone metastasis vs non-bone metastasis=294 days vs 390 days),bone metastasis increased the risk of disease progression by 53.6%(HR=1.536[0.99-1.98],p=0.014.The ORR for immunotherapy was significantly lower in patients with bone metastasis compared to patients without bone metastasis(15.24%vs 29.68%,p=0.0084).The survival prognosis of patients with bone metastasis was generally worse than that of patients without bone metastasis when stratified by baseline demographics(sex/age),stage of treatment(first-line or second-line),treatment regimen(monotherapy or combination therapy),or other sites of metastasis.In patients with bone metastasis,the degree of tumor regression of extra-osseous lesions was reduced after immunotherapy compared to patients without bone metastasis.(2)Analysis of NSCLC immunotherapy cohort:In advanced NSCLC,the PFS of immunotherapy also differs among organ metastasis,compared to the overall population;patients with bone metastasis showed a tendency of shorter PFS after immunotherapy(bone metastasis vs overall population=229 days vs 272days,p=0.060).When grouped by with or without bone metastasis:patients with bone metastasis showed significant shorter PFS(bone metastasis vs non-bone metastasis=229 days vs 314days,p=0.0033).And ORR in patients with bone metastasis was lower than that in patients without bone metastasis significantly(12.31%vs 32.57%,p=0.0425).Further stratification of the two groups according to baseline demographics,treatment stage,treatment regimen or presence of other comorbid metastatic sites also showed an overall increased risk of disease progression in patients with bone metastasis.In NSCLC who received immunotherapy,except in the adrenal gland,bone metastasis retarded metastasis lesions regression in brain,intrapulmonary,liver,lymph node,and pleural nodal than those in patients without bone metastasis.(3)Survival analysis in patients with advanced melanoma accepted immunotherapy from public databases showed a shorten tendency in OS in patients with bone metastasis(HR=1.556[0.9115 to 2.657],p=0.0614).When divided patients according to whether with bone metastasis or not,bone metastasis significantly shortened the OS(bone metastasis vs non-bone metastasis=426 days vs 968 days),and the risk of death increased by 1.2 times(HR=2.272[1.363 to 3.789],p=0.0436).(4)Based on the date published in literature,Pearson correlation analysis showed an overall negative tendency between bone metastasis rate and PD-L1 ORR among 18 cancer types(R=-0.4637,p=0.0526).2.Bone metastasis impairs immunotherapy efficacy in tumor-bearing mice models.(1)B16-F10 cell lines were injected through the tibial plateau,bone metastasis tumor grows faster than simultaneously implanted subcutaneous tumor.HE staining showed tumor growth within intraosseous and destroy bone significantly.(2)In vivo fluorescence imaging system was successfully constructed to monitor tumor growth i.o(2.5×10~5)and i.s(5×10~5)continuously.The detection rate of bone metastasis tumor on day 10 after tumor implantation was 100%,and no significant difference in the mean fluorescence intensity between subcutaneous tumor and bone metastasis was found.Thus,in the next experiments,SC+IO model construct followed i.o(2.5×10~5)and i.s(5×10~5)in B16-F10 and MC38 cell lines,and the treatment/observation starting time point was set on day 10 after tumor inoculation.(3)Immunotherapy efficacy in B16-F10 bone metastasis tumor-bearing model:The anti-tumor efficacy ofαPD-L1 was not significant in either SC or SC+IO group.While in SC+IO-PD-L1 group tumor curve was much closer to that in SC+IO-isotype group,and the difference between SC-PD-L1 and SC+IO-PD-L1 group reached statistical significance,suggesting that the degree of immunotherapy resistance in SC+IO group was aggravated.(4)Immunotherapy efficacy in MC38 bone metastasis tumor-bearing model:in SC group,αPD-L1 subcutaneous tumor growth retardation than isotype significantly,while tumor growth regression degree in SC+IO-PD-L1 was not as obvious that in SC-PD-L1,and the difference between SC-PD-L1 and SC+IO-PD-L1 group was statistically significant.(5)Establishment of B16-F10 lung metastasis model:lung metastasis(IV group),lung metastasis+bone metastasis(IV+IO group):AfterαPD-L1 or isotype antibody administration,lung tissues were harvested on day 17,and the number of nodules in the lungs surface were counted.Statistical data showed the number of lung metastatic tumor nodules in the IV+IO group treated with isotype antibody,showed an increasing tendency than that in the IV group(p=0.135);andαPD-L1 significantly reduced nodules in the lungs of IV group mice,the difference in the IV+IO group was not obvious,suggesting the presence of bone metastasis reduced the sensitivity of extraosseous lesions to immunotherapy.(6)Establishment of unilateral or bilateral subcutaneous MC38 tumor-bearing mouse model:Both subcutaneous tumors of unilateral and bilateral tumor-bearing models showed similar immunotherapy sensitivity toαPD-L1 antibody(p=0.0057,p=0.0009 respectively).(7)Establishment of tumor-bearing model of B16-F10 i.o+MC38 i.s:The subcutaneous MC38 tumor did not slow down tumor growth significantly afterαPD-L1antibody treatment,showing a tumor growth curve close to that of the isotype group(p=0.6604).(8)Establishment of B16-F10 i.s(SC)and i.s+i.o(SC+IO)tumor-bearing models:Subcutaneous tumors were harvested on day 10 and detected by flow cytometry.Proportion and number difference of immune subgroup cells in the SC+IO group in tumor-associated macrophages(TAM)(CD11b~+F4/80~+),myeloid-derived suppressor cells(MDSCs)(CD11b~+Gr-1~+)cells,NK cells(CD3~-NK1.1~+)and CD4~+T cells were close,and the proportion and number of CD8~+T cells were significantly reduced;the proportion of functional molecules IFN-γand TNF-αin CD8~+T cells were significantly reduced and IFN-γMFI was significantly reduced,compared with that in the SC group.3.Radiotherapy reverses immunotherapy resistance of bone metastasis.(1)In MC38 i.s+i.o tumor-bearing model,compared to immunotherapy alone,subcutaneous tumor growth was significantly retarded when immunotherapy combined with i.o tumor radiotherapy.(2)Subgroup survival analysis of the pan-cancer immunotherapy cohort:The median PFS of patients in the immunotherapy with radiotherapy group(19 cases)was significantly longer than those in the immunotherapy alone group(52 cases)(535 days vs 302 days),and the risk of disease progression was reduced by nearly 50%(HR=0.4912[0.2487 to 0.9699],p=0.0458).Conclusions:1.Patients with bone metastasis show systemic immunotherapy resistance.2.Tumor burden does not influence the efficacy of immunotherapy.3.Bone metastasis-associated immunotherapy resistance is not antigen-specific immunity depended.4.Immunotherapy resistance in bone metastasis is associated with impairment of CD8~+T cell dependent anti-tumor efficacy.5.Local radiotherapy improves the efficacy of immunotherapy in bone metastasis. |
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