Fig 2: Preclinical prevention trials of calcitriol against OSCC. Two studies were conducted to examine the effects of calcitriol on inhibition of disease initiation (A) and progression (B) in the 4NQO model. In the first study (A), C57BL/6 mice were administered PBS (n = 4; 4NQO) or calcitriol (n = 6; 0.1 µg i.p, Monday, Wednesday, Friday; 4NQO + calcitriol; shaded in yellow) for a total of 16 weeks concurrently with 4NQO exposure in drinking water. Non-invasive magnetic resonance imaging (MRI) was performed at week 0 prior to start of 4NQO exposure and at the end of week 16. Following completion of imaging, animals were euthanized for histologic evaluation of the incidence of premalignant and malignant lesions. (B) In the second study, C57BL/6 mice were randomized into a PBS control arm (n = 4; 4NQO) or one of three calcitriol (0.1 µg i.p, Monday, Wednesday, Friday) arms. Arm 1 (n = 5): Calcitriol was administered concurrently with 4NQO exposure in drinking water for a total of 16 weeks (4NQO + calcitriol; shaded in yellow). Arm 2 (n = 5): Calcitriol was administered 10 weeks post completion of 16 weeks of 4NQO exposure (4NQO ? calcitriol; shaded in blue). Arm 3 (n = 5): Calcitriol was administered for a period of 26 weeks concurrent with and following 4NQO exposure (4NQO + calcitriol ? calcitriol; shaded in red). Non-invasive magnetic resonance imaging (MRI) was performed at week 0 prior to start of 4NQO exposure with subsequent examinations performed at weeks 16, 20, 24 and 26 of study. At week 26, histopathologic assessment of tongue sections was performed to examine the effects of the three calcitriol regimens on the incidence of oral premalignant lesions and invasive cancer. Tongue sections were also immunostained for VDR, CYP24A1 and Ki67. Serum levels of 25 (OH)D3, 1,25 (OH)2D3 and calcium were determined using liquid chromatography-mass spectrometry (LC–MS/MS) and colorimetric assay, respectively.

Fig 3: Impact of calcitriol intervention on disease phenotype and vitamin D signaling. The panel of images represent photomicrographs of hematoxylin and eosin stained tongue sections (H&E) along with matched fields of VDR and CYP24A1 (10X magnification). Corresponding Ki67 immunostained field (40X) magnification is also shown on the right. The images for the control (4NQO alone) and the three calcitriol regimens have been shown for normal tongue epithelium (A), low-grade dysplasia (B), high-grade dysplasia (C), and invasive cancer (D).