Chemokine and chemokine receptor expression in the tumor microenvironment of HNSCC: association with clinical staging

Increased leukocyte infiltration in the tumor microenvironment (TME) is significantly associated with improved prognosis and response to treatment in Head and Neck Squamous Cell Carcinoma (HNSCC). There is evidence that the association with prognosis depends not only on the number of infiltrating cells, but also on the cell type and phenotype. Infiltration of leukocytes in the TME is largely dependent on the chemokines and chemokine receptors expressed by neoplastic/stromal cells and leukocytes, respectively. Increased CCR4/CCL17 interactions are associated with greater infiltration of pro-tumoral M2-like macrophages, Tregs and Th2 cells and worse prognosis and poor response to treatment. The CCR5/CCL5 interaction is involved in the anti-tumoral response, as CCL5 can be secreted by M1-like (anti-tumoral) macrophages and attract Th1 and NK cells. In this study we assess the expression of selected chemokines and chemokine receptors in the TME of HNSCC and verify its association with tumor aggressiveness according to clinical TNM staging. Expression of CCL2, CCR2, CCL4, CCR4, CCL5, CCR5, CCL17, and CCL20 was determined by RT-qPCR in 47 cases of HNSCC treated at the Santa Casa of Araraquara Hospital (CONEP CAAE 53343421.0.0000.5416): 42 men, 7 women, 82,97% smokers, T1/T2=26, T3/T4=21, Nx/N0=18 and N1/N2/N3=29. Total RNA was extracted from surgical specimens and used for cDNA synthesis. The cDNA was used in qPCR reactions using the SYBR Green system and specific primers, with HRPT1 as the normalization gene. Expression of CCL2 (p = 0.02) and CCR2 (p = 0.03) was increased in tumors without nodal invasion, possibly indicating reduced aggressiveness. CCR4 expression was greater (p = 0.0232) in smaller tumors, but there was difference regarding the presence of nodal invasion (p = 0.2069). Expression of CCL20, CCL4, CCL5 and CCR5 were not significantly different when tumors were grouped by size or by presence of nodal invasion. There was a tendency for lower CCL17 expression, which may attract Treg cells, in more aggressive tumors with nodal invasion (p = 0.0914). This data will be subsequently explored with the prevalence of infiltrating M1, M2 macrophages, as well as CD8+, CD4+ T and FOXP3+ Treg cells, but it supports the relevance of chemokines and chemokine receptors in shaping leukocyte infiltration in HNSCC tumor microenvironment. The data indicates that increased expression of CCL2 and CCR2 are associated with less aggressive HNSCC in this cohort of HNSCC patients.

Corresponding author: Thaina da Silva Ferreira (e-mail: thaina.s.ferreira@unesp.br).