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Aminolevulinic acid based photodynamic therapy (ALA-PDT) is a popular and efficacious treatment for actinic keratosis (AK). However, standard PDT can elicit stinging pain during illumination, and hence is not always favored by patients. In a new regimen called metronomic PDT (mPDT), similar to daylight PDT but using blue light, the illumination is delivered concurrently with ALA application rather than after a 1-hour pre-incubation (conventional regimen, cPDT). In the clinic, mPDT is not only painless but also nearly as effective as cPDT for AK lesion clearance. In this investigation, a murine AK model (generated by repeated UVB exposure) was treated with either mPDT or cPDT. Lesion clearance was followed by area measurement, and samples were harvested for mechanistic analyses. Compared to pretreatment (100%), the average lesion area was reduced to 47% and 32% in cPDT, and to 57% and 40% in mPDT at 1- and 2-weeks post PDT, respectively. Relative to untreated controls, enhanced cell death (histomorphology by H&E staining and apoptosis by TUNEL assay), and generation of Reactive Oxygen Species (ROS; CM-H2DCFDA staining) were observed in both cPDT and mPDT samples. Activation of cleaved Caspase-3 was specifically observed only in cPDT samples. Immunomodulation by inflammatory cells was observed by enhanced infiltration/retention of neutrophils and macrophages in metronomic PDT samples. Our results suggest that metronomic PDT can be just as effective as conventional PDT for treatment of AK, but the mechanisms may be quite different.

Painless photodynamic therapy (p-PDT), which involves application of photosensitizer and immediate exposure to light to treat actinic keratosis (AK) in patients, causes negligible pain on the day of treatment but leads to delayed inflammation and effective lesion clearance (Kaw et al., J Am Acad Dermatol 2020). To better understand how p-PDT works, hairless mice with UV-induced AK were treated with p-PDT and monitored for 2 weeks. Lesion clearance after p-PDT was similar to clearance after conventional PDT (c-PDT). However, lesion biopsies showed minimal cell death and less production of reactive oxygen species (ROS) in p-PDT treated than in c-PDT-treated lesions. Interestingly, p-PDT triggered vigorous recruitment of immune cells associated with innate immunity. Neutrophils (Ly6G+) and macrophages (F4/80+) appeared at 4 h and peaked at 24 h after p-PDT. Damage-associated molecular patterns (DAMPs), including calreticulin, HMGB1, and HSP70, were expressed at maximum levels around 24 h post-p-PDT. Total T cells (CD3+) were increased at 24 h, whereas large increases in cytotoxic (CD8+) and regulatory (Foxp3+) T cells were observed at 1 and 2 weeks post-p-PDT. In summary, the ability of p-PDT to eliminate AK lesions, despite very little overt cellular damage, appears to involve stimulation of a local immune response.

Aminolevulinate-based photodynamic therapy (ALA-PDT) is an effective treatment for cutaneous pre-cancers (Actinic Keratoses; AK) and Basal Cell Carcinoma (BCC), the most common skin malignancies. When administered in a conventional regimen with 1-4 h of ALA preincubation prior to light exposure, ALA-PDT elicits stinging pain during illumination that patients find objectionable. To avoid this pain, we have described a new regimen called metronomic PDT (mPDT) which is similar to daylight PDT but uses blue light (Kaw et al, J Am Acad Dermatol 2019). Metronomic PDT is not only painless but also nearly as effective as conventional PDT for AK lesion clearance. In this investigation, murine models of AK induced by repeated UVB exposure were treated with mPDT, followed by time-course analyses of immune responses in the lesions harvested. Our preliminary data showed that relative to conventional PDT, cell death (apoptosis) and generation of Reactive Oxygen Species (ROS) were compromised in mPDT samples. However, relative to untreated controls, enhanced recruitment/infiltration of immune cells that mediate innate immunity [neutrophils (Ly6G+) and macrophages (F4/80+)] was observed at early times after mPDT. Just as importantly, enhanced presence of cells regulating adaptive immune responses [T cells (CD3+, CD8+ and Foxp3+)] was observed at later times post mPDT. Activation of calreticulin and HMGB1 (markers of Damage Associated Molecular Patterns, DAMPs) were also observed in mPDT treated lesions. Our results suggest that mPDT can be just as effective as conventional PDT for treatment of skin cancer and pre-cancer, and that the therapeutic mechanisms may involve immune cell responses triggered by metronomic PDT.

Aim Breast cancer (BCA) in women is a leading cause of mortality and morbidity; distant metastases occur in ~40% of cases. Here, as an alternative to ionizing radiation therapy and chemotherapy and their associated side effects, we explored a new combination approach using capecitabine (CPBN) and aminolevulinate-based photodynamic therapy (PDT). We had previously developed a combination PDT approach in which 5-fluorouracil (5FU), a differentiation-promoting agent, increases the levels of protoporphyrin IX (PpIX) in cancer cells when given as a neoadjuvant prior to aminolevulinic acid (ALA). However, 5FU can be toxic when administered systemically at high levels. We reasoned that CPBN, a known chemotherapeutic for BCA and less toxic than 5FU (because CPBN is metabolized to 5FU specifically within tumor tissues), might work equally well as a PDT neoadjuvant. Methods Murine 4T1 BCA cells harboring a luciferase transgene were injected into breast fat pads of female nude mice. CPBN (600 mg/kg/day) was administered by oral gavage for 3 days followed by intraperitoneal ALA administration and PDT with red light (633 nm) on day 4. Tumor growth and regression were monitored in vivo using bioluminescence imaging. Histological changes in primary tumors and metastases were assessed by immunohistochemistry after necropsy. Results CPBN pretreatment of 4T1 tumors increased cellular differentiation, reduced proliferation, raised PpIX levels, enhanced tumor cell death, and reduced metastatic spread of 4T1 cells post-PDT, relative to vehicle-only controls. Conclusion The use of CPBN as a non-toxic PDT neoadjuvant for treatment of BCA represents a novel approach with significant potential for translation into the clinic.