Background:
Specialized metabolism of tumor cells results in the creation of an acidic, hypoxic, nutrient-depleted microenvironment coupled with a metabolic milieu that is hostile to the anti-tumor immune response. The metabolic milieu of human papillomavirus (HPV)-associated versus carcinogen-driven head and neck squamous cell carcinomas (HNSCs) is unknown. Here we present a novel analysis using mass spectrometry imaging to interrogate the metabolic landscape of head and neck cancer with the long-term goal of identifying targetable metabolic pathways to enhance anti-tumor immune responses and overcome hurdles of resistance to immunotherapy.

Methods: HNSC specimens were slow frozen over liquid nitrogen after tumor extirpation, and tumor sections were cut onto glass slides. Slides were coated in alpha-cyano-4-hydroxy-cinnamic acid (CHCA) matrix and subjected to mass spectrometry imaging using matrix-assisted laser desorption ionization (MALDI) on a Bruker SolariX XR 12T Hybrid QqFT-ICR mass spectrometer.

Results: A total of seven HPV-associated (three metastatic lymph nodes and four primary tumors) and six carcinogen-driven (primary tumors) HNSC specimens were analyzed. Metabolites significantly enriched in HPV-associated HNSC relative to carcinogen-driven HNSC were glutamine, inosine monophosphate, spermidine, spermine, xanthine, and indole-3-carboxyaldehyde. Metabolites significantly enriched in carcinogen-driven HNSC relative to HPV-associated HNSC were s-adenosylmethionine, hypoxanthine, and phosphorylcholine. Metabolites enriched among HPV-associated primary tumors relative to all other samples include O-propanoyl-D-carnitine, kynurenic acid, and valerylcarnitine. Adenosine monophosphate was enriched in HPV-associated metastatic lymph nodes compared to all other samples.

Conclusion: This is the first study to perform high resolution imaging of the metabolic milieu of head and neck cancer. Several candidate metabolites were differentially enriched in HPV-associated primary tumors or lymph nodes compared to carcinogen-driven primary HNSCs. In ongoing analyses, we are staining these MALDI slides for CD8+ T cell and macrophage markers to correlate metabolite enrichment with immune cell infiltration to evaluate for evidence of immune cell exclusion or enrichment in a metabolite-dependent context. These data aim to overcome the hurdle of resistance to immunotherapy and anti-tumor immune response.