Multiscale Evaluation of Tumor Hypoxia In Orthotopic and Heterotopic Head and Neck Cancer PDX Models
Abstract
Purpose
Heterotopic flank xenografts are widely used for preclinical head and neck cancer (HNC), despite hypothesized microenvironmental differences relative to orthotopic tumors. Tumor hypoxia is a major determinant of radiation response and arises from complex interactions among vascular structure, perfusion, and cellular heterogeneity. This study investigates whether histologic metrics reflect functional tumor perfusion and oxygenation, and whether these differ between orthotopic and heterotopic HNC models, using a multiscale imaging and molecular approach.
Methods
HNC patient-derived xenografts (PDXs) were established in orthotopic (buccal) and heterotopic (flank) sites. Microvessel density (MVD) and expression of pro‑angiogenic markers (IL‑8, HGFα, VEGF‑A) were quantified via immunohistochemistry (IHC) in a preliminary cohort using whole‑slide imaging with automated scoring. A pilot study of contrast‑enhanced ultrasound (CEUS) demonstrated limited sensitivity and high variability for detecting site‑dependent perfusion differences. To overcome these limitations, multispectral photoacoustic imaging (PAI) with contrast agent administration is being employed to noninvasively quantify tumor perfusion and hemoglobin oxygen saturation (sO₂). In parallel, single‑cell RNA sequencing (scRNA‑seq) is being performed to characterize cellular heterogeneity and hypoxia‑associated transcriptional states within the tumor microenvironment.
Results
Preliminary analysis of the IHC cohort showed no apparent differences in MVD or pro-angiogenic marker expression between orthotopic and heterotopic tumors. Ongoing PAI and scRNA-seq analyses will determine whether functional hypoxia and underlying cellular states differ between implantation sites despite similar preliminary histologic profiles.
Conclusion
Although preliminary histology suggests comparable vascular profiles between orthotopic and heterotopic HNC PDXs, ongoing functional photoacoustic imaging and single‑cell transcriptomics will determine whether site‑dependent differences in perfusion, oxygenation, and hypoxia‑associated cellular states exist and whether histologic metrics capture them. By quantitatively assessing the fidelity of histologic surrogates against functional and molecular readouts, this multiscale approach advances a radiotherapy‑relevant strategy for characterizing tumor hypoxia and directly informs the design and interpretation of preclinical radiotherapy studies across xenograft implantation sites.