Photodynamic therapy (PDT), using 5-aminolevulinic acid (ALA) to drive synthesis of protoporphryin IX (PpIX) is a promising, scar-free alternative to surgery for skin cancers, including squamous cell carcinoma (SCC) and SCC precursors called actinic keratoses (AK). In the United States, PDT is only FDA approved for treatment of AK; this narrow range of indications could be broadened if PDT efficacy were improved. Toward that goal, we developed a mechanism-based combination approach using 5-fluorouracil (5-FU) as a neoadjuvant for ALA-based PDT. In mouse models of SCC (orthotopic UV-induced lesions, and subcutaneous A431 and 4T1 tumors), pretreatment with 5-FU for 3 days followed by ALA for 4 hours led to large, tumor-selective increases in PpIX levels, and enhanced cell death upon illumination. Several mechanisms were identified that might explain the relatively improved therapeutic response. Firstly, the expression of key enzymes in the heme synthesis pathway was altered, including upregulated coproporphyrinogen oxidase and downregulated ferrochelatase. Secondly, a 3- to 6-fold induction of p53 in 5-FU pretreated tumors was noted. The fact that A431 contains a mutant form p53 did not prevent the development of a neoadjuvantal 5-FU effect. Furthermore, 5-FU pretreatment of 4T1 tumors (cells that completely lack p53), still led to significant beneficial inductions, i.e., 2.5-fold for both PpIX and PDT-induced cell death. Thus, neoadjuvantal 5-FU combined with PDT represents a new therapeutic approach that appears useful even for p53-mutant and p53-null tumors.
Photodynamic therapy (PDT) is a treatment modality that uses a specific photosensitizing agent, molecular oxygen, and light of a particular wavelength to kill cells targeted by the therapy. Topically administered aminolevulinic acid (ALA) is widely used to effectively treat cancerous and precancerous skin lesions, resulting in targeted tissue damage and little to no scarring. The targeting aspect of the treatment arises from the fact that ALA is preferentially converted into protoporphyrin IX (PpIX) in neoplastic cells. To monitor the amount of PpIX in tissues, techniques have been developed to measure PpIX-specific fluorescence, which provides information useful for monitoring the abundance and location of the photosensitizer before and during the illumination phase of PDT. This review summarizes the current state of these fluorescence detection techniques. Non-invasive devices are available for point measurements, or for wide-field optical imaging, to enable monitoring of PpIX in superficial tissues. To gain access to information at greater tissue depths, multi-modal techniques are being developed which combine fluorescent measurements with ultrasound or optical coherence tomography, or with microscopic techniques such as confocal or multiphoton approaches. The tools available at present, and newer devices under development, offer the promise of better enabling clinicians to inform and guide PDT treatment planning, thereby optimizing therapeutic outcomes for patients.
Photodynamic therapy (PDT), in which 5-ALA (a precursor for protoporphyrin IX, PpIX) is administered prior to exposure to light, is a nonscarring treatment for skin cancers. However, for deep tumors, ALA-PDT is not always effective due to inadequate production of PpIX. We previously developed and reported a combination approach in which the active form of vitamin D3 (calcitriol) is given systemically prior to PDT to improve PpIX accumulation and to enhance PDT-induced tumor cell death; calcitriol, however, poses a risk of hypercalcemia. Here, we tested a possible strategy to circumvent the problem of hypercalcemia by substituting natural dietary vitamin D3 (cholecalciferol; D3) for calcitriol. Oral D3 supplementation (10 days of a 10-fold elevated D3 diet) enhanced PpIX levels 3- to 4-fold, and PDT-mediated cell death 20-fold, in subcutaneous A431 tumors. PpIX levels and cell viability in normal tissues were not affected. Hydroxylated metabolic forms of D3 were only modestly elevated in serum, indicating minimal hypercalcemic risk. These results show that brief oral administration of cholecalciferol can serve as a safe neoadjuvant to ALA-PDT. We suggest a clinical study, using oral vitamin D3 prior to PDT, should be considered to evaluate this promising new approach to treating human skin cancer.
Cutaneous metastasis occurs more frequently in breast cancer than in any other malignancy in women, causing significant morbidity. Photodynamic therapy (PDT), which combines a porphyrin-based photosensitizer and activation by light, can be employed for breast cancer (especially cutaneous metastases) but tumor control after PDT has not surpassed traditional treatments methods such as surgery, radiation, and chemotherapy up to now. Here, we report that breast cancer nodules in mice can be effectively treated by preconditioning the tumors with 1α, 25-dihydroxyvitamin D3 (calcitriol; Vit D) prior to administering 5-aminolevulinate (ALA)-based PDT. Breast carcinoma tumors (MDA-MB-231 cells implanted subcutaneously in nude mice) received systemic Vit D (1 μg/kg) for 3 days prior to receiving ALA. The addition of Vit D increased intratumoral accumulation of protoporphyrin IX (PpIX) by 3.3 ± 0.5-fold, relative to mice receiving ALA alone. Bioluminescence imaging in vivo and immunohistochemical staining confirmed that tumor-specific cell death after ALA-PDT was markedly enhanced (36.8 ± 7.4-fold increase in TUNEL-positive nuclei; radiance decreased to 14% of control) in Vit D pretreated tumors as compared to vehicle-pretreated tumors. Vit D stimulated proliferation (10.7 ± 2.8-fold) and differentiation (9.62 ± 1.7-fold) in tumor cells, underlying an augmented cellular sensitivity to ALA-PDT. The observed enhancement of tumor responses to ALA-PDT after low, nontoxic doses of Vit D supports a new combination approach that deserves consideration in the clinical setting, and offers potential for improved remission of cutaneous breast cancer metastases.
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.
Better noninvasive techniques are needed to monitor protoporphyrin IX (PpIX) levels before and during photodynamic therapy (PDT) of squamous cell carcinoma (SCC) of the skin. Our aim was to evaluate: (1) multispectral fluorescent imaging of ultraviolet light (UV)-induced cancer and precancer in a mouse model of SCC; (2) multispectral imaging and probe-based fluorescence detection as a tool to study Vitamin D (VD) effects on aminolevulinic acid (ALA)-induced PpIX synthesis. Dorsal skin of hairless mice was imaged weekly during a 24-week UV carcinogenesis protocol. Hot spots of PpIX fluorescence were detectable by multispectral imaging beginning at 14 weeks of UV exposure. Many hot spots disappeared after cessation of UV at week 20, but others persisted or became visible after week 20, and corresponded to tumors that eventually became visible by eye. In SCC-bearing mice pretreated with topical VD before ALA application, our optical techniques confirmed that VD preconditioning induces a tumor-selective increase in PpIX levels. Fluorescence-based optical imaging of PpIX is a promising tool for detecting early SCC lesions of the skin. Pretreatment with VD can increase the ability to detect early tumors, providing a potential new way to improve efficacy of ALA-PDT.
Photodynamic therapy with aminolevulinic acid can be modified by pretreatment regimens with drugs such as 5-Fluorouracil (5-FU) or Vitamin D (calcitriol) that enhance accumulation of protoporphyrin IX (PpIX) within tumor tissue which presumably will enhance the therapeutic response to light. However, histological approaches for monitoring therapeutic responses are poorly suited for studying long term survival because large numbers of mice need to be sacrificed. To address this limitation, a non-invasive model to monitor tumor regression and regrowth has been established. Breast cancer cells, stably transfected with firefly luciferase (MDA-Luc cell line), are implanted orthotopically in nude mice (0.25 -1 x 10 6 cells/site), and monitored 0-60 min after s.c. injection of luciferin, with Xenogen in-vivo imaging system. Luminescence is detectable at day 1 post-implantation. Tumors are suitable for experimentation on day 6, when daily injections of pretreatment agents (5-FU, 300 mg/kg; calcitriol, 1 µg/kg) begin. On day 9, ALA (75 mg/kg i.p.) is given for 4 hr, followed by illumination (633 nm, 100 J/cm 2 ). Tumor luminescence post-PDT is monitored daily and compared with caliper measurements. Pretreatments (5-FU, calcitriol) by themselves do not inhibit luciferase expression, and all tumors grow at a similar rate during the pretreatment period. Results from in vivo survival experiments can be correlated to survival responses of MDA-Luc cells grown in monolayer cultures ± PDT and ± pretreatments, and additional mechanistic information (e.g. Ki67 and E-cadherin expression) obtained. In summary, this noninvasive model will permit testing of the therapeutic survival advantages of various pretreatments during cPDT.
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