Ionizing radiation sensitizes tumors to PD-L1 immune checkpoint blockade in orthotopic murine head and neck squamous cell carcinoma

Abstract

Immunotherapy clinical trials targeting the programmed-death ligand axis (PD-1/PD-L1) show that most head and neck squamous cell carcinoma (HNSCC) patients are resistant to PD-1/PD-L1 inhibition. We investigated whether local radiation to the tumor can transform the immune landscape and render poorly immunogenic HNSCC tumors sensitive to PD-L1 inhibition. We used the first novel orthotopic model of HNSCC with genetically distinct murine cell lines. Tumors were resistant to PD-L1 checkpoint blockade, harbored minimal PD-L1 expression and tumor infiltrating lymphocytes at baseline, and were resistant to radiotherapy. The combination of radiation and PD-L1 inhibition significantly enhanced tumor control and improved survival. This was mediated in part through upregulation of PD-L1 on tumor cells and increased T-cell infiltration after RT, resulting in a highly inflamed tumor. Depletion of both CD4 and CD8 T-cells completely abrogated the effect of anti PD-L1 with radiation on tumor growth. Our findings provide evidence that radiation to the tumor can induce sensitivity to PD-L1 checkpoint blockade in orthotopic models of HNSCC. These findings have direct relevance to high risk HNSCC patients with poorly immunogenic tumors and who may benefit from combined radiation and checkpoint blockade.

Inhibition of EphB4–Ephrin-B2 Signaling Reprograms the Tumor Immune Microenvironment in Head and Neck Cancers

“…For immunocompetent mouse model studies, 5-to 6-weekold female BALB/c mice (n ¼ 7-8; Charles River Laboratories) or C57BL/6 mice (n ¼ 7-8; Jackson Laboratories) were used. Tumor cell inoculation was performed as described earlier (28). Mice were randomized at day 4 to 5 posttumor inoculation (tumor volume $ 50 mm 3 ) to receive either pcDNA3 control plasmid or TNYL-RAW-Fc plasmid (20 mg in $2 mL of PBS) via hydrodynamic injection (24) and tumor size was measured biweekly as described earlier (28).…”

“…Tumor cell inoculation was performed as described earlier (28). Mice were randomized at day 4 to 5 posttumor inoculation (tumor volume $ 50 mm 3 ) to receive either pcDNA3 control plasmid or TNYL-RAW-Fc plasmid (20 mg in $2 mL of PBS) via hydrodynamic injection (24) and tumor size was measured biweekly as described earlier (28). For combination therapy studies, mice were randomized into IgGþpcDNA3 control, IgGþTNYL-RAW-Fc, anti-PDL1þTNYL-RAW-Fc, RTþIgGþpcDNA3, RTþIgGþTNYL-RAW-Fc, RTþanti-PDL1þpcDNA3, and RTþanti-PDL1þTNYL-RAW-Fc.…”

“…IgG2b control (referred as IgG; BioXcell) and anti-PDL1 (BioXcell) were administered on day 7 to 9 after tumor inoculation by intraperitoneal method at a dose of 10 mg/kg twice a week throughout the course of experiment. RT was administered at a single dose of 10 Gy as described earlier (28). Plasmid DNA treatment was initiated on day 5 after tumor inoculation and administered as a single dose.…”

“…Tumors and spleens were processed into single-cell suspensions for flow cytometric analysis as described earlier (28) and 1 to 2 Â 10 6 live cells were plated in a 96-well plate followed by blocking with anti-CD16/32 antibody. For analysis of immune cells, cytokines, and phospho-STAT3 marker, the following conjugated antibodies were used: Alexa Fluor 700-CD45 (1:50, Clone 30-F11; catalog no.…”

“…Total RNA was collected from Tregs and macrophages using RNeasy mini prep kits (Qiagen). cDNA was prepared as described earlier (28). Aliquots (2 mL) of a 1:2 dilution of the reverse transcription reactions were subjected to quantitative realtime PCR with the following primers using a iQ real time PCR detection system (BioRad).…”