Topical calcitriol prior to photodynamic therapy enhances treatment efficacy in non-melanoma skin cancer mouse models

Abstract

Non-melanoma skin cancers (NMSCs) such as basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are the most common form of human cancer worldwide, and their incidence is increasing. Photodynamic therapy (PDT), mediated by topically applied aminolevulinic acid (ALA) and subsequent exposure to light (either a laser or a noncoherent source), is being increasingly used for the treatment of dermatological disorders, including BCC and SCC. However, therapeutic responses of NMSCs to ALA-PDT are currently not superior to standard therapies, although the latter have undesirable side effects including scarring. In this study, we report that preconditioning of skin tumors with calcitriol (active form of Vitamin D; Vit D) prior to ALA-PDT, significantly improves the treatment outcome. In BCC and UVB-induced SCC mouse models, we identified an increase in tumor-specific accumulation of ALA induced photosensitizer (protoporphyrin IX, PpIX) due to Vit D preconditioning, of up to 6-fold in vivo. In addition, increased expression of differentiation (145 fold, p < 0.02) and proliferation (42 fold, p < 0.005) markers were identified in BCC tumors, all leading to increased tumor destruction (18.3 fold, p < 0.03) with the combination approach, as compared to ALA-PDT alone. Histomorphological changes identified using hematoxylin and eosin staining, and results of TUNEL staining, together documented a beneficial effect of Vit D pretreatment upon tumor cell death. We conclude that this new combination approach with Vit D and ALA-PDT has great potential to achieve complete remission of NMSC tumors, with excellent cosmetic results and an overall beneficial impact upon patient care.

Keywords: Aminolevulinic acid; Basal Cell Carcinoma; Calcitriol; Non-invasive imaging; Non-melanoma Skin Cancer; Photodynamic Therapy; Protoporphyrin IX; Squamous Cell Carcinoma; Vitamin D.

Fig. 1

BCC and SCC mouse models, and the histomorphology of the tumors. (A), Left image shows a nodular BCC tumor developing on a PTCH1+/− K14-Cre-ER p53 flox/flox mouse at 5–6 months of age following Tamoxifen and IR treatment. Right image shows features of a typical BCC characterized by proliferating tumor nests and cleft-like retractions of the tumor stroma. (B), Left image, white arrows point to papillomatous-like SCC tumors developing on an SKH-1 hairless mouse after exposure to UVB for 20 weeks. Right image shows the histology of two SCC lesions.

Fig. 2

Ex vivo analysis of Vit D effect on PpIX synthesis, using Confocal Microscopy. (A) Left half of figure shows BCC tumors, (B) Right side of figure shows SCC tumors. For each half, the phase contrast (upper panels), fluorescence images (middle panels), and fluorescence quantification (lower panels) illustrates PpIX levels in tumor sections after 3 days of Aquaphor or Vit D treatment, followed by 4 h application of ALA. Number of tumors per treatment group is shown in parenthesis. Mean ± SEM of three representative images from each tumor; P-value from unpaired two-sided t-test.

Fig. 3

In vivo imaging of PpIX in BCC tumors using Maestro EX IVIS^®. (A), White light images, and (B), fluorescence images showing BCC tumors and their corresponding ALA induced PpIX levels after three days of pretreatment with Aquaphor and Vit D. (C), Quantification of PpIX specific fluorescence (total signal/area) calculated using Maestro EX 3.0 Image Processing Software. Mean ± SEM of three representative images from each of three tumors per treatment group is shown as fold change. Number of tumors per treatment group is shown in parenthesis. P-value is from unpaired two-sided t-test.

Fig. 4

Effect of Vit D on differentiation, proliferation, and cell death due to ALA-PDT in BCC tumors. Fluorescence images and quantification of signal in tumor sections immunostained with secondary antibodies to (A), differentiation marker, E-cadherin, (B), proliferation marker, Ki-67, and (C), apoptotic marker, TUNEL 24 h post PDT. Mean ± SEM of three representative images from each of three tumors per treatment group is shown as fold change. Number of tumors per treatment group is shown in parenthesis. P-value, from unpaired two-sided t-test.

Fig. 5

Histomorphological analysis of BCC tumors at 24 h post PDT, elucidating the tumor cell death-promoting effects of Vit D. Panels show H&E stained images of paraffin embedded tumor sections obtained from absolute controls (left panels), 24 h post-PDT with Aquaphor pretreatment (middle panels), and 24 h post PDT with Vit D treated tumors (right panels). Top and bottom panels show typical microscopic fields at low and high magnification, respectively.