Role of GLUT-1 in the Upregulation of PD-L1 Expression After Radiotherapy and Association of PD-L1 with Favourable Overall Survival in Hypopharyngeal Cancer

Figures & data

Figure 1 PD‑L1 and Glut-1 expression, and CD4+, CD8+ T cells infiltration (the black arrows point) in HNSCC at baseline (pre-RT) and after RT (magnification ×400). Positive expression of PD-L1 pre-RT (A) and after RT (B); Positive expression of Glut-1 pre-RT (C) and after RT (D); CD4+ T cells infiltration in tumor tissues pre-RT (E) and after RT (F); CD8+ T cells infiltration in tumor tissues pre-RT (G) and after RT (H).

Abbreviations: GLUT-1, glucose transporter 1; HNSCC, head and neck squamous cell carcinoma; PD‑L1, programmed cell death ligand 1; RT, radiotherapy.

Table 1 Patient Characteristics

Patient Characteristics	Number
Age
>60 years	31
<60 years	16
Tumor location
Pyriform sinus carcinoma	38
Posterior pharyngeal wall carcinoma	6
Postcricoid area carcinoma	3
Histologic grade
Well	12
Moderate	26
poor	9
Clinical stage
III	23
IV	24
Treatment method
Surgery+ (chemo) radiotherapy	17
Biopsy+Adjuvant Radiotherapy+Surgery	25
Biopsy+Radiotherapy	5

Figure 2 Survival analysis of patients with HNSCC. (A) Survival analysis of patients with high and low levels of PD-L1 expression. (B) Survival analysis of patients with high and low CD4+ T cell infiltration. (C) Survival analysis of patients with high and low CD8+ T cell infiltration.

Abbreviations: HNSCC, head and neck squamous cell carcinoma; PD‑L1, programmed cell death ligand 1; RT, radiotherapy.

Table 2 Multivariate Analysis of Overall Survival in Patients with Hypopharyngeal Cancer

Variables	Overall Survival
	Hazard Ratio (95% CI) P-value
PD-L1 expression (ref. low)	3.483 (1.080–11.235) 0.037
CD4+ T cell infiltration (ref. low)	4.299 (1.313–14.072) 0.016
CD8+ T cell infiltration (ref. low)	2.483 (0.721–8.550) 0.149
Glut-1 expression (ref. low)	0.955 (0.354–2.574) 0.927
Age(ref. <60 years)	1.772 (0.625–5.022) 0.282
Clinical stage(ref. Stage IV)	3.32 (1.22–9.09) 0.019
Histologic grade(ref.moderate)	0.848 (0.186–3.873) 0.832
Histologic grade(ref.high)	0.883 (0.220–3.541) 0.860
Location (ref. Posterior pharyngeal wall carcinoma)	4.586 (0.369–57.002) 0.236
Location (ref. Postcricoid area carcinoma)	1.739 (0.105–28.866) 0.700

Figure 3 Alteration of PD-L1, and CD4+, CD8+ T cells after RT compared with pre-RT. (A) PD-L1 expression in tumor cells was significantly increased after RT compared with pre-RT. (B) CD4+ T cell infiltration was markedly increased after RT. (C) CD8+ T cell infiltration was markedly increased after RT. (D) Expression of PD-L1 was correlated with the expression of GLUT1 in pre-RT specimens. (E) PD-L1 expression tended to correlate with the expression of GLUT1 after RT (**p<0.01).

Abbreviations: GLUT1, glucose transporter 1; PD‑L1, programmed cell death ligand 1; RT, radiotherapy.

Figure 4 PD-L1 and GLUT1 expression before and after radiotherapy. (A) PD-L1 mRNA expression in FaDu cells was significantly upregulated at 96 h after RT compared with the control group (p<0.001). (B) PD-L1 protein expression was also significantly upregulated at 96 h after RT (p<0.001). (C) GLUT1 mRNA expression was decreased at 48 h after RT compared with the control group (p<0.01), and it was significantly increased at 96 h after RT compared with 48 h after RT (p<0.001). (D) GLUT1 protein expression was decreased at 48 h after RT compared with the control group and the 24-h after RT group (p<0.01 and p<0.05, respectively). GLUT1 protein expression was significantly increased at 96 h after RT compared with 48 h after RT (p<0.001). (*p<0.05, **p<0.01, ***p<0.001).

Abbreviations: GLUT1, glucose transporter 1; PD‑L1, programmed cell death ligand 1; RT, radiotherapy.

Figure 5 PD-L1 expression before and after RT in siRNA-GLUT1 FaDu cells. (A) Transfection efficiency of GLUT1 siRNA by fluorescence analysis. (B) GLUT1 protein expression in siRNA-GLUT1 FaDu cells before and after RT. (C, D) PD-L1 mRNA and protein expression in siRNA-GLUT1 FaDu cells was significantly decreased at 96 h after RT compared with that recorded in FaDu cells, as presented by the two-way analysis of variance (p<0.001 and p<0.001, respectively). (***p<0.001).

Abbreviations: GLUT1, glucose transporter 1; PD‑L1, programmed cell death ligand 1; RT, radiotherapy.