The Role And Clinical Significance Of Tumor Microenvironment In Laryngeal Squamous Cell Carcinoma

The tumor microenvironment plays an important role in tumor cell proliferation,tumor progression,invasion and metastasis.The tumor microenvironment is a complex microenvironment.Head and neck squamous cell carcinoma escapes the host immune system through the influence of microenvironment on the tumors,which promotes tumor metastasis,angiogenesis and growth.The present study was performed mainly to observe the infiltration characteristics of tumor infiltrating T lymphocytes(TILs)and tumor associated macrophages(TAMs)and the expression of related immune checkpoint molecules in the tumor microenvironment of laryngeal squamous cell carcinoma(LSCC).The relationship between tumor associated immune cells with the progression and prognosis of LSCC was also analysized.Objective: The characteristics of CD3+,CD4+ and CD8+ T lymphocytes infiltration in peritumoral and intratumoral regions of LSCC were observed.The relationship between TILs infiltration and clinicopathological features and prognosis of LSCC was analyzed.Next,the characteristics of CD68+ and CD163+ TAMs infiltration in peritumoral and intratumoral regions of LSCC were observed and it’s relationship with clinicopathological features and prognosis of LSCC was analyzed.Thirdly,Expression and clinical significance of TIM-3 protein and m RNA were evaluated in LSCC.Then,the co-expression of PD-1 and TIM-3 on TILs and PD-L1 on TAMs in LSCC was assessed and its’ clinical significance was evaluated.Methods: Immunohistochemistry was used to detect the infiltration of CD3+,CD4+,CD8+ TILs and CD68+ and CD163+ TAMs.Immunohistochemistry was also used to detect the expression of TIM-3 protein.RNAscope were used to detect the expression of m RNA.Multi-labeled immunofluorescence staining multispectral imaging technique was used to detect the co-expression of PD-1 and TIM-3 on CD4+ and CD8+ TIL,as well as the expression of PD-L1 on CD68+ and CD163+ TAMs in different regions of LSCC.Results: There is a large amount of infiltration of TILs and TAMs in LSCC,and the level of infiltration in peritumoeral region was obviously higher than in intratumoral region.CD4+ TIL infiltration was significantly stronger than CD8+ TIL,and CD68+ TAMs were significantly stronger than CD163+ cells in peritumoral region.There was no difference in infiltration between CD4+ and CD8+ T cells and between CD68+ and CD163+ macrophages TAMs within the tumor in intratumoral region.In the early stage of T stage of LSCC,patients with no lymph node metastasis and no recurrence had higher levels of infiltration than patients with advanced stage,lymph node metastasis and recurrence.And high levels of infiltrating CD3+ and CD8+ TILs have longer overall survival duration.The infiltration level of CD68+ and CD163+ TAMs in patients with advanced T stage and recurrence was significantly higher than that in patients with early T stage and no recurrence,and the overall survival rate of patients with high level TAMs infiltrating was lower.Multivariate analysis showed that CD8+ TILs and CD163+ TAMs in peritumoral regions were independent prognostic factors for LSCC.TIM-3 is expressed on immune cells and tumor cells in LSCC.High expression of TIM-3 protein and m RNA was associated with poor differentiation,recurrence and lymphatic metastasis of LSCC,and patients with high TIM-3 protein expression have worse outcome.Co-expression of PD-1 and TIM-3 on CD4+ and CD8+ TILs was presented in LSCC,and patients with advanced T stage,relapse and poor differentiation had higher co-expression rate.Moreover,patients with higher co-expression rates of PD-1 and TIM-3 on CD4+ TILs had worse prognosis.PD-L1 was expressed on CD68+/CD163+ TAMs in both peritumoral and intrtumoral regions of LSCC.Similarly,patients with advanced T stage,relapse and poor differentiation had higher expression rate.Moreover,patients with higher PD-L1 expression rates on CD163+ TAMs had poor outcome.Conclusion: 1.There were a large amount of infiltration of TILs and TAMs in LSCC,and the pattern of infiltration was diverse in different tumor sites,different cell types and different T stages.2.High level of TILs infiltration was associated with early T stage of LSCC and favorable prognosis.In contrast,the high infiltration of TAMs is associated with progression,metastasis and poor prognosis of LSCC.3.The high level of TIM-3 expression in LSCC was associated with progression,metastasis and poor prognosis of LSCC.4.Co-expressed PD-1 and TIM-3 on TILs,and PD-L1 expression on TAMs helped to predict poor outcome in LSCC.TILs and TAM may play a vital role in immune evasion and promoting tumor growth infiltration through co-expressing different immune checkpoint receptors.5.Combining different location,tumor infiltration immune cell types and tumor progression stages could help to better predict the prognosis of LSCC and the response to immunotherapy in LSCC...