Evaluation of new oral cancer model using novel CT-based soft tissue imaging

Oral carcinogenesis has frequently been modeled in mice using the tobacco carcinogen 4NQO (4-nitroquinoline-1-oxide) administered in drinking water for 8-16 weeks for a 4-6 month full study duration. While 4NQO carcinogenesis follows a sequence of hyperplasia-> dysplasia -> carcinoma in situ -> invasive cancer analogous to that of humans, lesion histology typically coexists with papillary growth and is not ulcerated. Neither 4NQO or MNU (N-methylnitrosourea) single agent models show regional lymph node metastases, a common feature of human disease. To address these shortcomings, we have been exploring a model where MNU, a more aggressive, albeit less stable carcinogen, is combined with mechanical scratching in larynx (Caicedo-Granados, Head Neck, 2013), to simulate a more faithful etiology with a shorter exposure period. Chronic irritation and/or trauma have been recognized as important factors in oral carcinogenesis particularly in studies of non-smoking patients. In our pilot study using A/J mice (n=28), scratching and MNU were applied to either lateral tongue or cheekpouch in two scratchings, combined with 6 weekly topical carcinogen applications, with a study duration of 20 weeks. An additional challenge with long carcinogenesis models is there are no obvious signs to assess early tumor formation, complicating ethical animal management and initiating interventional studies. As a result, group sizes are large (n =16-24). Therefore, we investigated, for the first time, contrast-enhanced CT imaging to follow live animals. The CT protocol was a preprogrammed 1 minute scan with a benchtop CT (Sophie G8 PET/CT, pixel = 200um) using Volume Graphics (UMN Dental School) for image reconstruction. The contrast agent was a Dy Dota chelate formulated into an ultrasmall (~ 11 nm) protein-coated nanocapsule, previously shown to target tumor, and chronically-inflamed tissues through tenascin-C targeting (Unger, Mol Can Ther, 2014). Tenascin-C is an embryonic protein that only exists in the adult in sites of chronic inflammation and lymph nodes. As our interest is in oral cavity and airway, we evaluated several routes of administration including inhalation, oral, subcutaneous, singly and in combination. We had previously tested a novel, low air-speed nebulizer as being efficacious in delivery of suspension and nanocapsule agents to rodents (Yi, Int J Pharm, 2012). We found the MNU + scratching method in A/J oral cavity shows calculable cervical lymph node metastasis and ulceration, using beta-catenin as a marker. Beta-catenin activation was observed in tongue keratinocytes only in mice receiving tongue scratching + MNU but not cheek scratching + MNU. Further, the one tongue tumor induced by week 20 was visible by contrasted CT. Given tongue background was minimal in no contrast, naïve mice, contrast-guided imaging, over 12-20 weeks, was able to follow disordering of tongue vasculature consistent with lesion formation.