The Effects And Mechanisms Of Tumor-associated Macrophages On Prognosis Of Patients With Lung Adenocarcinoma

Lung cancer is the leading cause of cancer mortality for human beings.Pathologically,most of the lung cancers are non-small cell lung cancer（NSCLC）and nearly half of the histological classifications are lung adenocarcinoma（LAC）.Although ther e are many advances in chemotherapy,radiation,and molecular targeted therapy,the 5-year survival rate for lung cancer patients remains quite low,one of the main reasons is the tolerance,escape or abnormality of tumor immunity.Tumor microenvironment is supposed to be vital in promoting tumor progression,recurrence and metastasis.Therefore,a deeper understanding of the status of the tumor microenvironment and the molecular mechanism of microenvironment on LAC patients’prognosis are extremely important for immunotherapy in advanced patients.Tumor-associated macrophages（TAMs）,as a heterogeneous population of immune cells,form a pivotal component of the infiltrating immune cells in cancer.TAMs have been reported to exert dual influence on tumor progression,depending on the tumor microenvironment.The relationship between the distribution of TAMs and the prognosis of LAC patients and the mechanism of its influence on the prognosis deserve further study.Recently,increasing evidence has been emerging to show that tumor cells express programmed cell death-ligand 1（PD-L1）to avoid the cytotoxic effect of activated T lymphocytes expressing programmed cell death protein 1（PD-1）,thereby promoting tumor progression.Immunotherapies targeting immune checkpoints PD-1/PD-L1 axis have been regarded as a promising therapeutic strategy for various cancer,including lung cancer,and won the Nobel prize in physiology or medicine in 2018.But why the treatment effect of some patients who received PD-1/PD-L1 antibody treatment is not ideal,the mechanisms and countermeasures are still unclear.The relationship between the distribution of TAMs and the expression of PD-1 and whether TAMs influence the prognosis of LAC patients through the expression of PD-1 are uncertain.Furthermore,the influence of TAMs combined with PD-L1 expression in tumor cells on the LAC patients’prognosis remains to be clarified.Given the importance of localization of TAMs in tumor progression and the great clinical significance of anti-PD-1/PD-L1 immunotherapy,detailed investigations of the effect of TAMs on the prognosis of LAC patients and the possible mechanisms are urgent.In this study,immunohistochemistry（IHC）was applied to evaluate the distribution of CD68⁺TAMs and CD163⁺TAMs on the specimens from 213 cases of surgically-resected LAC.It was found that the localization of TAMs in LAC was closely related to the prognosis of patients.The mechanism of TAMs affecting prognosis of LAC patients was further studied.It was confirmed that whether TAMs in the tumor stroma or islets expressed PD-1 was the key factor of their different influence on the prognosis of LAC patients.Although the expression of PD-L1 in LAC cells is highly heterogeneous,the effect of stromal PD-1⁺CD163⁺TAMs on poor prognosis of LAC patients is not dependent on the expression of PD-L1 in tumor cells.The main results and conclusions of this study are summarised in the following:1.The distribution and clinical pathological significance of TAMs in LAC（1）The distribution of TAMs in the tumor stroma and tumor islets of LACImmunofluorescence（IF）results showed that most CD68⁺TAMs co-expressed CD163,a maker used to identify tumor-promoting TAMs.The distribution of CD68⁺TAMs and CD163⁺TAMs in 213 LAC specimens was analyzed by IHC staining.The results showed that TAMs were found more frequently distributed in the tumor stroma than those in the tumor islets（p<0.0001）.（2）TAMs distribution in relation to survival of LAC patientsKaplan-Meier survival analysis showed that the number of stromal CD68⁺TAMs was associated with shorter survival of patients（p=0.0231）.A striking positive association between survival and the density of CD68⁺TAMs in the tumor islets was found（p=0.0001）.Furthermore,significant inverse association between survival and stromal CD163⁺TAM density was observed（p=0.0040）.The prognostic significance of macrophage infiltration was assessed according to the proportion and distribution of different immune phenotypes.The results showed that patients in the group with low density of stromal CD68⁺TAMs but high density of CD163⁺TAMs had the shorter survival as compared with those in the group with low density of both stromal CD68⁺TAMs and CD163⁺TAMs（p<0.0001）.Patients with the high density of total CD68⁺TAMs in the tumor islet had a better outcome,no mater the density of CD163⁺TAMs was high or low.（3）The relationship between TAMs distribution and clinicopathological characteristicsChi-square test showed that stromal CD68⁺TAMs were associated with differentiation grade（p=0.023）and histology subtype（p=0.047）.The high density of stromal CD163⁺TAMs was correlated with lymph node（LN）metastasis（p=0.007）,differentiation grade（p=0.008）and TNM stage（p=0.005）.The density of CD163⁺TAMs in tumor islets of male was higher than those of females（p=0.0001）.The univariate and multivariate Cox regression analysis demonstrated that tumor islet CD68⁺TAM density was an independent favorable predictor for survival（p=0.001 and p<0.001）of LAC patients,and high number of stromal CD163⁺TAMs is considered as an independent prognostic factor of reduced survival of LAC patients（p=0.005 and p=0.027）.2.Mechanisms of TAMs on prognosis of LAC patients（1）IL-4 induced macrophages express high level of PD-1The TAMs induction model in vitro was established by inducing mononuclear cell lines THP1 and U937 to polarize into macrophages with IL-4.The high expression of PD-1and CD163 in IL-4-induced macrophages was further confirmed by q RT-PCR,Western blot and IF staining.It is suggested that the high expression of PD-1 in TAMs can be mediated by IL-4.（2）High expression of PD-1 in stromal TAMs affects prognosis of LAC patients（1）Firstly,a subcutaneous tumor model was established by subcutaneous injection of lung cancer cell line Lewis into C57BL/6 mice.Three weeks later,the transplanted tumor was isolated and analyzed by FCM.The results showed that PD-1 was expressed by some CD11b⁺F4/80⁺macrophages in tumor tissues,while macrophages in spleen tissues expressed low level of PD-1.The dominant phenotype of mouse PD-1⁺TAMs is tumor-promoting CD206⁺MHC II^low.（2）IHC staining was used to detect the expression and distribution of PD-1 in 213cases of LAC.The results showed that PD-1⁺cells could be found in both tumor stroma and tumor islets of LAC specimens.The density of PD-1⁺cells infiltrated in tumor stroma was significantly higher than that in tumor islets（p<0.0001）.IHC and IF double-staining confirmed that CD68⁺TAMs expressed PD-1 and some CD163⁺TAMs also expressed PD-1in the tumor stroma,suggesting that stromal PD-1⁺CD163⁺TAMs is a special subset of TAMs in LAC.The Cancer Genome Atlas（TCGA）database analysis showed that the expression of PD-1 was positively correlated with the expression of CD68（p<0.0001）and CD163（p<0.0001）at m RNA level.IHC results showed that the number of PD-1⁺cells in the stroma was significantly correlated with the density of CD68⁺TAMs（p<0.0001）and CD163⁺TAMs（p<0.0001）,but the number of PD-1⁺cells in the tumor islets was not correlated with the density of TAMs.These findings indicate that the number of stromal PD-1⁺cells is closely related to the density of stromal TAMs in LAC.Kaplan-Meier survival analysis showed that the higher density of stromal PD-1⁺cells was associated with the worse prognosis（p=0.0038）.The relationship between the density of PD-1⁺TAMs and the prognosis of lung adenocarcinoma patients was further analyzed according to the number of PD-1⁺cells and the density of CD163⁺TAMs in the tumor stroma.The results showed that the survival time of LAC patients with high number of PD-1⁺cells and high density of CD163⁺TAMs was significantly shorter than those with low density of CD163⁺TAMs and PD-1⁺cells in the tumor stroma（p=0.0001）.Chi-square test showed that the density of stromal PD-1⁺cells was closely related to the degree of differentiation（p=0.0002）and histological subtype classification（p=0.0001）.The density of stromal PD-1⁺CD163⁺TAMs was associated with TNM stage（P=0.041）.Univariate and multivariate Cox regression analysis showed that PD-1⁺cell density in the tumor islets（p<0.001 and p=0.001）and stromal PD-1⁺CD163⁺TAMs（p=0.002 and p=0.035）were independent prognostic factors in patients with LAC.（3）The mechanism of TAMs expressing PD-1.RNA sequence analysis of PD-1⁺TAMs and PD-1^-TAMs from mouse transplanted tumor was performed to investigate the possible molecular mechanism of up-regulation of PD-1 expression in TAMs.The data analysis preliminarily showed that IL-4-related JAK-STAT signaling pathway was enriched in PD-1⁺TAMs,suggesting that the JAK-STAT signaling pathway may regulate the up-regulation of PD-1 expression in TAMs.3.Heterogeneous expression of PD-L1 in LAC cells（1）Heterogeneity of PD-L1 expression in LACThe expression levels of PD-L1 in different cell lines of LAC were detected by q RT-PCR,Western blot,IF staining and FCM.It was found that the expression of PD-L1 in different LAC cell lines were diverse.IHC staining was used to detect the expression of PD-L1 in 213 cases of LAC and 149 cases of SCC specimens.The expression of PD-L1was assessed by three methods:conventional positive staining percentage（1%,5%,10%or50%）,H-score with internal reference to PD-L1 expression on alveolar macrophages（AMs）,and IOD value of comprehensive staining intensity and percentage.The results showed that no matter which method was used,the expression of PD-L1 in LAC was significantly lower than that in SCC.These results indicate that the expression of PD-L1 in LAC is low and heterogeneous.Furthermore,IHC staining was used to detect the expression of PD-L1 in 70cases of paired primary and LN metastatic LAC.The results showed that when the cut-off value was 1%and 5%,the expression of PD-L1 in primary and metastatic LAC tumors,whether N1 or N2,showed higher consistency.The results reveal that there is heterogeneity in the expression of PD-L1 in different tumor tissues of the same patient.（2）The relationship between PD-L1 expression and prognosis of NSCLC patientsSurvival analysis showed that the expression of PD-L1 was not correlated with the survival of LAC patients,indicating that the prognostic significance of PD-L1 expression in LAC is not significant.4.The clinical prognostic significance of combined analysis of PD-1⁺TAMs and tumor cell PD-L1 expression in LAC（1）Microenvironment subtypes of LAC based on PD-1/PD-L1When classified according to the density of infiltrated PD-1⁺cells in the tumor islets or stroma,the major subtypes of LAC microenvironment were type II immune ignorance（low PD-L1 and low PD-1⁺cells）,accounting for 45%（96/213）and 27%（57/213）,respectively.It is suggested that immune failure may occur in nearly one third of LAC patients during the diagnosis process,which is difficult to obtain the effect of anti-PD-1/PD-L1 immunotherapy in clinical treatment.（2）The relationship between microenvironment subtypes and prognosis of LAC patientsSurvival analysis showed that type Ⅲ LAC patients with high expression of PD-L1 but low density of PD-1⁺cells had a longer survival（p=0.0001 and P=0.0021,respectively）,regardless of the density of PD-1⁺cells in the tumor islets or stroma.It is suggested that comprehensive analysis of PD-1⁺cell density and PD-L1 expression has a significant impact on the survival and prognosis of LAC patients.（3）LAC microenvironment subtypes based on PD-1⁺CD163⁺TAM/PD-L1 and their relationship with patients’prognosisCompared with type Ⅲ patients with high expression of PD-L1 and low density of stromal PD-1⁺CD163⁺TAMs,the prognosis of type IV patients with low expression of PD-L1 but high density of stromal PD-1⁺CD163⁺TAMs was poor in LAC（p<0.0001）.When PD-L1 was low expressed,the survival time of type IV patients with high density of stromal PD-1⁺CD163⁺TAMs was significantly shorter than that of type II patients with low density of stromal PD-1⁺CD163⁺TAMs（p<0.0001）,suggesting that the combined detection of PD-1 and CD163 is more effective in distinguishing two subgroups of LAC with low expression of PD-L1.These results demonstrate that the effect of stromal PD-1⁺CD163⁺TAMs on the poor prognosis of LAC patients is independent of PD-L1expression in tumor cells.In summary,our results confirm that the difference in prognostic predictions of TAMs distributed in different areas of LAC originates from the discrepancy in the expression of PD-1 on TAMs infiltrated in the tumor stroma and islets.Stromal CD163⁺TAMs express high levels of PD-1,and the density of stromal PD-1⁺CD163⁺TAMs is an independent prognostic factor for poor prognosis of LAC patients.The up-regulated expression of PD-1in TAMs may be related to activation of JAK-STAT signaling pathway.PD-L1 is heterogeneously expressed in LAC,but the effect of stromal PD-1⁺CD163⁺TAMs on the poor prognosis of LAC patients is not dependent on the expression of PD-L1 in tumor cells.