CD68+ Macrophage Infiltration Associates With Poor Outcome of HPV Negative Oral Squamous Carcinoma Patients Receiving Radiation: Poly(I:C) Enhances Radiosensitivity of CAL-27 Cells but Promotes Macrophage Recruitment Through HMGB1

Abstract

Patients with human papillomavirus (HPV) negative oral squamous cell carcinoma (OSCC) generally have poor clinical outcomes and worse responses to radiotherapy. It is urgent to explore the underlining mechanisms of the distinct prognoses between HPV negative and HPV positive OSCC and to develop effective therapy strategy to increase the survival rate of HPV negative OSCC patients. We conducted a retrospective cohort of 99 resected OSCC patients to evaluate the prognosis of HPV negative and HPV positive OSCC patients receiving radiation or not. We further addressed the association of CD68⁺ macrophage infiltration with HPV status and the effects on survival of OSCC patients. We also used the TCGA-OSCC cohort for further verification. Based on the cohort study, we applied a synthetic dsRNA polymer, polyriboinosinic-polyribocytidylic acid (poly(I:C)), on CAL-27 (HPV negative OSCC cells). We co-cultured its condition medium with THP-1 derived macrophage and examined the cytokines and macrophage migration. We found that high CD68⁺ macrophage infiltration associated with poor overall survival in HPV negative OSCC patients receiving radiation. In vitro, poly(I:C) could induce apoptosis and enhance the radiosensitivity, but increase macrophage recruitment. Targeting HMGB1 could inhibit IL-6 induction and macrophage recruitment. Our findings indicated that CD68⁺ macrophage might play an important role in the outcomes of HPV negative OSCC patients receiving radiation. Our findings also suggested that radiation combined poly(I:C) might be a potential therapy strategy to increase the radiation response and prognosis of HPV negative OSCC. Notably, HMGB1 should be targeted to inhibit macrophage recruitment and enhance overall therapy effects.

Keywords: HMGB1; human papillomavirus; oral squamous cell carcinoma; poly(I:C); radiosensitivity.

Figure 1

The landscape of immune infiltration of 278 OSCC patients in TCGA cohort. (A) Heatmap of 22 types of tumor-infiltrating immune cells deconvolved using CIBERSORT in OSCC. (B) Heatmap of 7 types of tumor-infiltrating cells deconvolved using EPIC in OSCC.

Figure 2

Association of HPV status and radiation with survival of OSCC patients in our cohort. (A) Representative immunohistochemical images of p16 positive and negative OSCC (left: 100×, right: 400×). (B, C) Kaplan-Meier curves show overall survival (B) and disease-free survival (C) of HPV negative and HPV positive OSCC patients. (D, E) Kaplan-Meier curves show overall survival (D) and disease-free survival (E) of HPV negative and HPV positive OSCC patients receiving radiation or no radiation. Log-rank test and/or pair wised comparison was used for significance. RT, radiation.

Figure 3

Association of CD68⁺ macrophage infiltration and HPV status with survival of OSCC patients in our cohort. (A) Representative immunohistochemical images of high or low level of CD68⁺ macrophages in OSCC (left: 100×, right: 400×). (B) Proportion of high or low level of CD68⁺ macrophages in patients with HPV negative and HPV positive OSCC. (C, D) Kaplan-Meier curves exhibit overall survival (C) and disease-free survival (D) in high or low level of CD68⁺ macrophage infiltrated OSCC patients. (E, F) Kaplan-Meier curves exhibit overall survival (E) and disease-free survival (F) in high or low level of CD68⁺ macrophage infiltrated OSCC patients with HPV negative and HPV positive status. Log-rank test and/or pair wised comparison was used for significance. CD68-Hi, CD68-High; CD68-Lo, CD68-Low; RT, radiation.

Figure 4

Association of CD68⁺ macrophage infilatration and radiation with survival of all OSCC patients in our cohort. (A, B) Kaplan-Meier curves show overall survival (A) and disease-free survival (B) in high or low level of CD68⁺ macrophage infiltrated OSCC patients receiving radiation or no radiation. Log-rank test and/or pair wised comparison was used for significance. (C, D) Forest plots illustrate hazard ratios of subgroup univariate cox regression of overall survival (C) and disease-free survival (D). CD68-Hi, CD68-High; CD68-Lo, CD68-Low; RT, radiation.

Figure 5

Association of CD68⁺ macrophage infilatration and radiation with survival of HPV negative OSCC patients in our cohort. (A, B) Kaplan-Meier curves exhibit overall survival (A) and disease-free survival (B) in high or low level of CD68⁺ macrophage infiltrated HPV negative OSCC patients receiving radiation or no radiation. Log-rank test and/or pair wised comparison was used for significance. (C, D) Forest plots illustrate hazard ratios of subgroup univariate cox regression of overall survival (C) and disease-free survival (D). CD68-Hi, CD68-High; CD68-Lo, CD68-Low; RT, radiation.

Figure 6

Poly(I:C) enhances radiation-induced apoptosis and inhibits proliferation of CAL-27. CAL-27 cells were treated with poly(I:C) or PBS for 24 h followed by radiation. (A) Representative plots illustrate apoptosis of CAL-27 according to annexin V and/or propidium iodide (PI) staining. (B) Bar plots indicate the quantifications of early apoptotic cells ((annexin V+/PI-) of the bottom right quadrant. (C) Bar plots indicate the quantifications of late apoptotic cells (annexin V+/PI+) of the top right quadrant. (D) Bar plots indicate the quantifications of total apoptotic cells (annexin V+) of the top right and bottom right quadrants. (E) The proliferation curve shows the proliferation of CAL-27 detected by CCK-8 assay. Student’s t-test was used for significance determination of flowcytometry (mean ± SD, n = 3). ANOVA was used for significance determination of CCK-8 assay (mean ± SD, n = 5). * indicates P < 0.05, *** indicates P < 0.001 and **** indicates P < 0.0001. PIC, poly (I:C).

Figure 7

Poly(I:C) and radiation-stimulated CAL-27 alters cytokine secretion of macrophages and promotes macrophage recruitment through IL-6 and HMGB1. CAL-27 cells were treated with poly(I:C) or PBS for 24 h followed by 8 Gy or 0 Gy radiation. THP-1-derived macrophages were treated with above CAL-27 CM (or no CM control) and continued to culture for 42 h before incubation for 24 h with fresh medium. (A) The concentrations of IL-1β, IL-6, IL-17, TNF-α, IL-7, and IL-12p70 in the supernatants were detected by cytokine assay. ANOVA was used (mean ± SD, n = 5). (B) Bar plot shows HMGB1 concentrations in CAL-27 CM detected by ELISA. Mann-Whitney U-test was used (mean ± SD, n = 3). (C) Bar plot shows the concentration of IL-6 in the supernatants of THP-1-derived macrophages treated with CAL-27 CM (poly (I:C) 24 h combined 8 Gy radiation) or pretreated with HMGB1 neutralizing antibody or isotype antibody. (D, E) Representative images (×200) and quantifications of the migrated THP-1-derived macrophages treated with CAL-27 CM and IL-6 neutrolizing antibody (or isotype). (F, G) Representative images (×200) and quantifications of the migrated THP-1-derived macrophages treated with CAL-27 CM and HMGB1 neutrolizing antibody (or isotype). ANOVA was used (mean ± SD, n = 5). * indicates P < 0.05 and ** indicates P < 0.01. PIC, poly (I:C); CM, condition medium; RT, radiation. NAb, neutralizing antibody; IsoAb, isotype antibody.

Figure 8

The schematic diagram depicting the strategy of targeting HMGB1 may enhance radiosensitivity of CAL-27 cells by inhibiting macrophage recruitment induced by poly(I:C).