Tumor hypoxia is associated with resistance to PD-1 blockade in squamous cell carcinoma of the head and neck

Abstract

The majority of patients with recurrent/metastatic squamous cell carcinoma of the head and neck (HNSCC) (R/M) do not benefit from anti-PD-1 therapy. Hypoxia induced immunosuppression may be a barrier to immunotherapy. Therefore, we examined the metabolic effect of anti-PD-1 therapy in a murine MEER HNSCC model as well as intratumoral hypoxia in R/M patients. In order to characterize the tumor microenvironment in PD-1 resistance, a MEER cell line was created from the parental line that are completely resistant to anti-PD-1. These cell lines were then metabolically profiled using seahorse technology and injected into C57/BL6 mice. After tumor growth, mice were pulsed with pimonidazole and immunofluorescent imaging was performed to analyze hypoxia and T cell infiltration. To validate the preclinical results, we analyzed tissues from R/M patients (n=36) treated with anti-PD-1 mAb, via immunofluorescent imaging for number of CD8+ T cells (CD8), Tregs and the percent area (CAIX) and mean intensity (I) of carbonic anhydrase IX in tumor. We analyzed disease control rate (DCR), progression free survival (PFS), and overall survival (OS) using proportional odds and proportional hazards (Cox) regression. We found that anti-PD-1 resistant MEER has significantly higher oxidative metabolism, while there was no difference in glycolytic metabolism. Intratumoral hypoxia was significantly increased and CD8+ T cells decreased in anti-PD-1 resistant tumors compared with parental tumors in the same mouse. In R/M patients, lower tumor hypoxia by CAIX/I was significantly associated with DCR (p=0.007), PFS, and OS, and independently associated with response (p=0.028) and PFS (p=0.04) in a multivariate model including other significant immune factors. During PD-1 resistance, tumor cells developed increased oxidative metabolism leading to increased intratumoral hypoxia and a decrease in CD8+ T cells. Lower tumor hypoxia was independently associated with increased efficacy of anti-PD-1 therapy in patients with R/M HNSCC. To our knowledge this is the first analysis of the effect of hypoxia in this patient population and highlights its importance not only as a predictive biomarker but also as a potential target for therapeutic intervention.

Trial registration: ClinicalTrials.gov NCT04114136.

Keywords: head and neck neoplasms; immunotherapy; lymphocytes; metabolic networks and pathways; tumor microenvironment; tumor-infiltrating.

Conflict of interest statement

Competing interests: DPZ has research support (institutional) for his role a PI for studies with Merck, BMS, GSK, AstraZeneca, Aduro, Lilly, Astellas, Macrogenics, Varastem, advisory board for Blueprint Medicines. GMD declares competing financial interests and has submitted patents covering the use of PGC1α in cell therapies that are licensed or pending and is entitled to a share in net income generated from licensing of these patent rights for commercial development. GMD consults for and/or is on the scientific advisory board of BlueSphere Bio, Century Therapeutics, Novasenta, Pieris Pharmaceuticals, and Western Oncolytics/Kalivir; has grants from bluebird bio, Novasenta, Pfizer, Pieris Pharmaceuticals, TCR2, and Western Oncolytics/Kalivir; GMD owns stock in Novasenta. RLF serves on advisory boards for BMS, EMD Serono, Macrogenics, Merck, Numab Therapeutics, Pfizer. RLF receives research funding from Astra-Zeneca/MedImmune, BMS, Novasenta, Tesaro. RLF consults for Aduro and Novasenta, RLF owns stock in Novasenta.

© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1

Programmed cell death protein 1 (PD-1) blockade resistance leads to increased tumor cell oxidative metabolism and intratumoral hypoxia. (A) Schematic of PD-1 resistant murine head and neck cancer cell model, MEER cell line generation. (B) Growth curve and survival of C57/BL6 mice inoculated with parental or PD-1 resistant MEER cells intradermally then treated with 200 µg anti-PD-1 or isotype controls three times per week when tumors reached 1–3 mm. Tumor-free indicates a complete regression. Partial response (PR) indicates mice that showed tumor regression for at least two measurements. Each line represents one animal. (C) Oxygen consumption rate (OCR) trace (left) and tabulated basal OCR (right) of parental and PD-1 resistant MEER cells. (D) Extracellular acidification rate (ECAR) trace (left) and tabulated basal ECAR (right) as in (C). (E) Schematic of imaging preformed on parental and PD-1 resistant MEER cells. (F) Pimonidazole, CD8, and DAPI staining of full tumor sections from mice bearing parental and PD-1 resistant MEER tumors. Scale bar, 500 µm. (G) Tabulated results of the internal hypoxyprobe area and intensity from mice as in (F). (H) CD8+ T cell counts normalized to tumor area from mice as in (F). (I) Ratio of CD8+ T cells and Foxp3+ T cells from mice as in (F). (J) Foxp3+ T cell counts normalized to tumor area from mice as in (F). Data represent two to three independent experiments. *p

Figure 2

Hypoxia is associated with poor clinical efficacy with Programmed cell death protein 1 (PD-1) blockade therapy in patients. (A) Representative immunofluorescence of pan cytokeratin (PanCK), carbonic anhydrase IX (CAIX), and CD8 at 20× magnification of formalin-fixed paraffin-embedded (FFPE) sections from squamous cell carcinoma of the head and neck (HNSCC) patients treated with anti-PD-1 monoclonal antibodies (mAb). Scale bar, 200 µm comparing a patient with disease control (stable disease) to a patient with progression. (B) Tabulated results of percent area and mean intensity of CAIX (CAIX/I) within PanCK+ regions of FFPE sections from patients with HNSCC that progressed on (P) or had disease control (DC; defined as stable disease, partial or complete response) anti-PD-1 mAb. (C) Tabulated results of CD8+ T cell counts normalized to the PanCk+ area from patients as in (B). (D) Overall survival of patients treated with anti-PD-1 mAb based on CAIX/I. (E) Progression free survival of patients as in (C). (F) Overall survival of patients treated with anti-PD-1 mAb based on CD8+ T cell counts normalized to the PanCk area. (G) Progression free survival of patients as in (F). (H) Overall survival of patients treated with anti-PD-1 mAb monotherapy based on combination of CAIX/I and CD8+ T cell counts. (I) Progression free survival of patients as in (H). *p