Oral Cancer Associated Vascular and Neural Innervation Patterns

Objectives: Oral cancer patients suffer pain at the cancer site. Pain is attributed to sensitization of primary afferent neurons by mediators released from the cancer and microenvironment. Cancers induce sprouting of nerves into the microenvironment. Metastatic oral cancer (N+) patients experience greater pain than non-metastatic cancer (N0) patients. Genes overexpressed in N+ patients experiencing the highest pain levels encode secreted and membrane proteins present in exosomes and enriched for functions in extracellular matrix organization and angiogenesis. The proteins are reported to act as oncogenes and have neuronal functions. We are investigating the vascular (microvessel density, MVD, neoangiogenesis) and neural innervation patterns in oral pre-cancers and cancers in humans and oral cancer mouse models. Vascular patterns associate with N+ oral cancer. Knowledge is lacking regarding patterns of innervation and association with tumor vasculature.
Methods: Mice (C57Bl/6) were offered 4-nitroquinoline 1-oxide (4NQO) in drinking water for 16 weeks and sacrificed at 33 weeks. For complete histological analysis, 100 serial sections were obtained from bisected and formalin fixed tongues. Sections on three adjacent slides were immunostained with antibodies to CD31 (blood vessels), GAP-43 (nerves) or double stained for CD31 and GAP-43. Immunoreactivity is being quantified using Visiopharm and ImageJ software.
Results: Oral lesions (hyperplasia, dysplasia, papillomas, and cancer) developed on the tongue. Positive CD31 immunostaining (CD31-ir) was observed in the core of papillomas, around tumor islands and penetrating within the tumor mass of the most invasive lesion. The percentage CD31-ir area at the center of papillomas (mean ± SEM = 3.794 ± 0.3962, n=3) was greater than cancers (mean ± SEM = 1.065 ± 0.1567, n=6). The difference was significant (p=0.0001, unpaired two-tailed t-test).
Conclusions: Initial studies demonstrate quantitative differences in vasculature of papillomas and cancers in the 4NQO model. Work is ongoing to evaluate patterns of vasculature and neuronal innervation in murine and human oral lesions.