The effect of tumor derived HMBG1 on intra-tumoral B cells in esophageal squamous cell carcinoma

Abstract

Background: Tumor microenvironment (TME) is often hypoxic and characterized by diverse cell populations (tumor cells and lymphocytes). An intranuclear architectural protein termed high mobility group box chromosomal protein 1 (HMGB-1) is enriched in hypoxic environment, where it acts as a chemokine to promote recruitment of inflammatory cells. We studied the effect of tumor derived HMBG1 on B cell migration and phenotype differentiation in esophageal squamous cell carcinoma (ESCC). Methods: Immunohistochemistry (IHC) staining for HMGB-1 was performed on tissue microarray (TMA) of ESCC and correlated with survival outcome. An immunostaining scoring system corresponding to total staining intensity as follows; (strong staining score =3; moderate staining score=2; weak staining scores= 1; no staining scores= 0). Co-staining with CD20+B was also performed to study B cells distribution in the tumor. ESCC cell lines were transfected with HMGB-1 and transwell migration of B cells were studied. Results: TMA containing 78 paired primary tumors and normal tissue from ESCC patients was analyzed by IHC using an anti-HMGB1 antibody. We found that HMGB1 was expressed at a higher level as compared to paired normal tissue. Survival curves were estimated by the Kaplan-Meier method and compared with log-rank test to evaluate the relationship between biomarker HMGB1 and survival outcomes. Patients were divided into two groups based on the optimal cutoffs of low (score 0-1), high (score 2-3) staining of HMGB1, we found out that HMGB1 staining were not correlated to patient survivals (p = 0.363). We then performed double-staining and demonstrated that B cells were located in the stroma along HMGB1-stained tumor. Stable HMGB1 ESCC cell lines were established where boyden chamber and wound-healing assays demonstrated that B cells migrated and proliferated at higher rates towards HMGB1-overexpressing cell lines. Conclusions: Our findings indicate that HMGB1 was over-expressed in tumor compared with normal tissue. HMGB1 expression per se was not correlated to survival in our log-rank analysis. HMGB1 expression has probable role in B cells recruitment, migration/proliferation.