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Photodynamic therapy (PDT) is a nonscarring cancer treatment in which a pro-drug (5-aminolevulinic acid, ALA) is applied, converted into a photosensitizer (protoporphyrin IX, PpIX) which is then activated by visible light. ALA-PDT is now popular for treating nonmelanoma skin cancer (NMSC), but can be ineffective for larger skin tumors, mainly due to inadequate production of PpIX. Work over the past two decades has shown that differentiation-promoting agents, including methotrexate (MTX), 5-fluorouracil (5FU) and vitamin D (Vit D) can be combined with ALA-PDT as neoadjuvants to promote tumor-specific accumulation of PpIX, enhance tumor-selective cell death, and improve therapeutic outcome. In this review, we provide a historical perspective of how the combinations of differentiation-promoting agents with PDT (cPDT) evolved, including Initial discoveries, biochemical and molecular mechanisms, and clinical translation for the treatment of NMSCs. For added context, we also compare the differentiation-promoting neoadjuvants with some other clinical PDT combinations such as surgery, laser ablation, iron-chelating agents (CP94), and immunomodulators that do not induce differentiation. Although this review focuses mainly on the application of cPDT for NMSCs, the concepts and findings described here may be more broadly applicable towards improving the therapeutic outcomes of PDT treatment for other types of cancers., (© 2024 The Authors. Photochemistry and Photobiology published by Wiley Periodicals LLC on behalf of American Society for Photobiology.)

Introduction: Photodynamic therapy (PDT) is a safe, non-mutagenic, and non-scarring treatment for actinic keratoses (AK)., Background: 'Painless' photodynamic therapy (p-PDT) is a regimen for AK that employs simultaneous aminolevulinate incubation and blue light illumination. The efficacy of p-PDT resembles that of traditional PDT, but detailed mechanisms of action for p-PDT are not well understood., Methods: To characterize the inflammatory effects of the p-PDT procedure 48 h following treatment and determine the association of inflammation with precancer burden, we performed a retrospective cohort study of 104 patients with AK of face or scalp treated with p-PDT between 2017 and 2019. Patients self-reported their side effects 48 h following p-PDT and took photographs of their face and scalp. Photographs were edited to define seven anatomic regions, and erythema was scored by four investigators., Results: Ninety-eight patients provided photographs suitable for erythema evaluation. Most patients experienced 2 or more side effects and some pain 48 h post-procedure. Females experienced more pain (p = 0.01) and side effects (p = 0.002) compared to males. AK burden was positively associated with post p-PDT erythema response (p < 0.0001) at all sites, but particularly in the temples (p = 0.002) and supralabial area (p = 0.009)., Discussion: This study confirms a strong clinical inflammatory response after p-PDT. Severity of inflammation is positively associated with AK tumor burden, suggesting that post-treatment inflammation may be a pre-requisite for p-PDT efficacy. Interestingly, the results also identify certain gender-related differences in the severity of side effects experienced by patients post-PDT., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2023. Published by Elsevier B.V.)

Improved treatment outcomes for non-melanoma skin cancers can be achieved if Vitamin D (Vit D) is used as a neoadjuvant prior to photodynamic therapy (PDT). However, the mechanisms for this effect are unclear. Vit D elevates protoporphyrin (PpIX) levels within tumor cells, but also exerts immune-modulatory effects. Here, two murine models, UVB-induced actinic keratoses (AK) and human squamous cell carcinoma (A431) xenografts, were used to analyze the time course of local and systemic immune responses after PDT ± Vit D. Fluorescence immunohistochemistry of tissues and flow analysis (FACS) of blood were employed. In tissue, damage-associated molecular patterns (DAMPs) were increased, and infiltration of neutrophils (Ly6G+), macrophages (F4/80+), and dendritic cells (CD11c+) were observed. In most cases, Vit D alone or PDT alone increased cell recruitment, but Vit D + PDT showed even greater recruitment effects. Similarly for T cells, increased infiltration of total (CD3+), cytotoxic (CD8+) and regulatory (FoxP3+) T-cells was observed after Vit D or PDT, but the increase was even greater with the combination. FACS analysis revealed a variety of interesting changes in circulating immune cell levels. In particular, neutrophils decreased in the blood after Vit D, consistent with migration of neutrophils into AK lesions. Levels of cells expressing the PD-1+ checkpoint receptor were reduced in AKs following Vit D, potentially counteracting PD-1+ elevations seen after PDT alone. In summary, Vit D and ALA-PDT, two treatments with individual immunogenic effects, may be advantageous in combination to improve treatment efficacy and management of AK in the dermatology clinic., Competing Interests: Declarations of competing interest None., (Copyright © 2024. Published by Elsevier B.V.)

Each year, 3.3 million Americans are diagnosed with non-melanoma skin cancers (NMSC) and an additional 40 million individuals undergo treatment of precancerous actinic keratosis lesions. The most effective treatments of NMSC (surgical excision and Mohs surgery) are invasive, expensive and require specialised training. More readily accessible topical therapies currently are 5-fluorouracil (a chemotherapeutic agent) and imiquimod (an immune modulator), but these can have significant side effects which limit their efficacy. Therefore, more effective and accessible treatments are needed for non-melanoma cancers and precancers. Our previous work demonstrated that the small molecule N-phosphonacetyl-L-aspartate (PALA) both inhibits pyrimidine nucleotide synthesis and activates pattern recognition receptor nucleotide-binding oligomerization domain 2. We propose that topical application of PALA would be an effective NMSC therapy, by combining the chemotherapeutic and immune modulatory features of 5-fluorouracil and imiquimod. Daily topical application of PALA to mouse skin was well tolerated and resulted in less irritation, fewer histopathological changes, and less inflammation than caused by either 5-fluorouracil or imiquimod. In an ultraviolet light-induced NMSC mouse model, topical PALA treatment substantially reduced the numbers, areas and grades of tumours, compared to vehicle controls. This anti-neoplastic activity was associated with increased expression of the antimicrobial peptide cathelicidin and increased recruitment of CD8 + T cells and F4/80 + macrophages to the tumours, demonstrating both immunomodulatory and anti-proliferative effects. These findings indicate that topical PALA is an excellent candidate as an effective alternative to current standard-of-care NMSC therapies., (© 2023 The Authors. Experimental Dermatology published by John Wiley & Sons Ltd.)

Vitamin D3, a prohormone, is converted to circulating calcidiol and then to calcitriol, the hormone that binds to the vitamin D receptor (VDR) (a nuclear transcription factor). Polymorphic genetic sequence variants of the VDR are associated with an increased risk of breast cancer and melanoma. However, the relationship between VDR allelic variants and the risk of squamous cell carcinoma and actinic keratosis remains unclear. We examined the associations between two VDR polymorphic sites, Fok1 and Poly-A, and serum calcidiol levels, actinic keratosis lesion incidence, and the history of cutaneous squamous cell carcinoma in 137 serially enrolled patients. By evaluating the Fok1 (F) and (f) alleles and the Poly-A long (L) and short (S) alleles together, a strong association between genotypes FFSS or FfSS and high calcidiol serum levels (50.0 ng/ml) was found; conversely, ffLL patients showed very low calcidiol levels (29.1 ng/ml). Interestingly, the FFSS and FfSS genotypes were also associated with reduced actinic keratosis incidence. For Poly-A, additive modeling showed that Poly-A (L) is a risk allele for squamous cell carcinoma, with an OR of 1.55 per copy of the L allele. We conclude that actinic keratosis and squamous cell carcinoma should be added to the list of squamous neoplasias that are differentially regulated by the VDR Poly-A allele., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Pyoderma gangrenosum (PG) is a debilitating skin condition often accompanied by inflammatory bowel disease (IBD). Strikingly, ~40% of patients that present with PG have underlying IBD, suggesting shared but unknown mechanisms of pathogenesis. Impeding the development of effective treatments for PG is the absence of an animal model that exhibits features of both skin and gut manifestations. This study describes the development of the first experimental drug-induced mouse model of PG with concomitant intestinal inflammation. Topical application of pyrimidine synthesis inhibitors on wounded mouse skin generates skin ulcers enriched in neutrophil extracellular traps (NETs) as well as pro-inflammatory cellular and soluble mediators mimicking human PG. The mice also develop spontaneous intestinal inflammation demonstrated by histologic damage. Further investigations revealed increased circulating low density IL-1β primed neutrophils that undergo enhanced NETosis at inflamed tissue sites supported by an increase in circulatory citrullinated histone 3, a marker of aberrant NET formation. Granulocyte depletion dampens the intestinal inflammation in this model, further supporting the notion that granulocytes contribute to the skin-gut crosstalk in PG mice. We anticipate that this novel murine PG model will enable researchers to probe common disease mechanisms and identify more effective targets for treatment for PG patients with IBD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Jatana, Ponti, Johnson, Rebert, Smith, Fulmer, Maytin, Achkar, Fernandez and McDonald.)

Translation of experimental techniques from one scientific discipline to another is often difficult but rewarding. Knowledge gained from the new area can lead to long lasting and fruitful collaborations with concomitant development of new ideas and studies. In this Review Article, we describe how early work on the chemically pumped atomic iodine laser (COIL) led to the development of a key diagnostic for a promising cancer treatment known as photodynamic therapy (PDT). The highly metastable excited state of molecular oxygen, a 1 Δ g , also known as singlet oxygen, is the link between these disparate fields. It powers the COIL laser and is the active species that kills cancer cells during PDT. We describe the fundamentals of both COIL and PDT and trace the development path of an ultrasensitive dosimeter for singlet oxygen. The path from COIL lasers to cancer research was relatively long and required medical and engineering expertise from numerous collaborations. As we show below, the knowledge gained in the COIL research, combined with these extensive collaborations, has resulted in our being able to show a strong correlation between cancer cell death and the singlet oxygen measured during PDT treatments of mice. This progress is a key step in the eventual development of a singlet oxygen dosimeter that could be used to guide PDT treatments and improve outcomes.

We previously showed that a combination of differentiation-inducing agents (5-fluorouracil [5FU], vitamin D3 or methotrexate) and aminolevulinate-based photodynamic therapy (PDT) improves clinical responses by enhancing protoporphyrin IX (PpIX) photosensitizer levels and cell death. Here, we show that in addition to its previously known effects, 5FU enhances PDT-induced tumor-regressing immunity. Murine actinic keratoses were treated with topical 5FU or vehicle for 3 days prior to aminolevulinic acid application, followed by blue light illumination (~417 nm). Lesions were harvested for time-course analyses of innate immune cell recruitment into lesions, i.e. neutrophils (Ly6G+) and macrophages (F4/80+), which peaked at 72 h and 1 week post-PDT, respectively, and were greater in 5FU-treated lesions. Enhanced infiltration of activated T cells (CD3+) throughout the time course, and of cytotoxic T cells (CD8+) at 1-2 weeks post-PDT, also occurred in 5FU-treated lesions. 5FU pretreatment reduced the presence of cells expressing the immune checkpoint marker PD-1 at ~72 h post-PDT, favoring cytotoxic T cell activity. A combination of 5FU and PDT, each individually known to induce long-term tumor-targeting immune responses in addition to their more immediate effects on cancer cells, may synergize to provide better management of squamous precancers., (© 2022 The Authors. Photochemistry and Photobiology published by Wiley Periodicals LLC on behalf of American Society for Photobiology.)

Normal myofibroblast differentiation is critical for proper skin wound healing. Neoexpression of α-smooth muscle actin (α-SMA), a marker for myofibroblast differentiation, is driven by transforming growth factor (TGF)-β receptor-mediated signaling. Hyaluronan and its three synthesizing enzymes, hyaluronan synthases (Has 1, 2, and 3), also participate in this process. Closure of skin wounds is significantly accelerated in Has1/3 double-knockout (Has1/3-null) mice. Herein, TGF-β activity and dermal collagen maturation were increased in Has1/3-null healing skin. Cultures of primary skin fibroblasts isolated from Has1/3-null mice had higher levels of TGF-β activity, α-SMA expression, and phosphorylation of p38 mitogen-activated protein kinase at Thr180/Tyr182, compared with wild-type fibroblasts. p38α mitogen-activated protein kinase was a necessary element in a noncanonical TGF-β receptor signaling pathway driving α-SMA expression in Has1/3-null fibroblasts. Myocardin-related transcription factor (MRTF), a cofactor that binds to the transcription factor serum response factor (SRF), was also critical. Nuclear localization of MRTF was increased, and MRTF binding to SRF was enhanced in Has1/3-null fibroblasts. Inhibition of MRTF or SRF expression by RNA interference suppresses α-SMA expression at baseline and diminished its overexpression in Has1/3-null fibroblasts. Interestingly, total matrix metalloproteinase activity was increased in healing skin and fibroblasts from Has1/3-null mice, possibly explaining the increased TGF-β activation., (Copyright © 2022 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Introduction: Photodynamic therapy (PDT) is a non-scarring, repeatable, and safe treatment for actinic keratosis (AK), but improvements in efficacy are still needed., Background: Devices such as steel blades, needle rollers, and lasers are currently used to remove hypertrophic stratum corneum on AKs to improve PDT outcomes. However, curettage with fine sandpaper could be a gentler, effective alternative., Methods: A retrospective study was designed to compare PDT with or without sandpaper curettage. Patients were selected from a database registry of patients with face and scalp AKs (ClinicalTrials.gov NCT03319251). Patients in Group 1 underwent PDT alone (20% ALA, 15 min; blue light 417 nm, 30 min). Patients in Group 2 were pretreated with gentle sandpaper curettage prior to ALA and illumination. The two groups were compared using multivariate matching, normalizing for age, sex, initial AK counts, and time to follow-up., Results: Sixty-six patients were selected for matching analysis (n=38, PDT only; n=28, PDT+curettage). Demographics between the groups were similar (mean ± SD), including age (71.0 ± 8.3 vs. 71.0 ± 8.0 years), baseline AK count (53 ± 39 vs. 44± 32), and time to post-PDT follow-up (111 ± 28 vs. 113 ± 32 days). At follow-up, patients who received curettage showed an overall 55% improvement in scalp AK clearance compared to patients who did not receive curettage, adjusting for sex, age, time to follow-up, and baseline AK count (p = 0.0322, multivariable linear regression)., Discussion: Sandpaper curettage before PDT treatment is an easy and inexpensive method to significantly improve AK clearance rates., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Background: In mouse models of skin cancer, high-dose oral vitamin D3 (VD3; cholecalciferol) combined with photodynamic therapy (PDT) can improve the clearance of squamous precancers (actinic keratoses [AKs])., Objective: To determine whether oral VD3 can improve the clinical efficacy of a painless PDT regimen in humans with AK., Methods: The baseline lesion counts and serum 25-hydroxyvitamin D 3 levels were determined. In group 1, 29 patients underwent gentle debridement and 15-minute aminolevulinic acid preincubation with blue light (30 minutes; 20 J/cm 2 ). In group 2, 29 patients took oral VD3 (10,000 IU daily for 5 or 14 days) prior to debridement and PDT. Lesion clearance was assessed at 3 to 6 months., Results: In group 1, the mean clearance rates of facial AK were lower in patients with VD3 deficiency (25-hydroxyvitamin D 3 level < 31 ng/dL; clearance rate, 40.9% ± 42%) than in patients with normal 25-hydroxyvitamin D 3 levels (62.6% ± 14.2%). High-dose VD3 supplementation (group 2) significantly improved the overall AK lesion response (72.5% ± 13.6%) compared with that in group 1 (54.4% ± 22.8%). No differences in side effects were noted., Limitations: Nonrandomized trial design (interventional cohort matched to registry-based controls)., Conclusions: Oral VD3 pretreatment significantly improves AK clinical responses to PDT. The regimen appears promising and well tolerated., Competing Interests: Conflicts of interest None disclosed., (Copyright © 2022 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.)

Photodynamic priming ( PDP ), a collateral effect of photodynamic therapy, can transiently alter the tumor microenvironment (TME) beyond the cytotoxic zone. Studies have demonstrated that PDP increases tumor permeability and modulates immune-stimulatory effects by inducing immunogenic cell death, via the release of damage-associated molecular patterns and tumor-associated antigens. Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest of cancers with a stubborn immunosuppressive TME and a dense stroma, representing a challenge for current molecular targeted therapies often involving macromolecules. We, therefore, tested the hypothesis that PDP's TME modulation will enable targeted therapy and result in immune stimulation. Using triple-receptor-targeted photoimmuno-nanoconjugate (TR-PINs)-mediated PDP , targeting epidermal growth factor receptor, transferrin receptor, and human epidermal growth factor receptor 2 we show light dose-dependent TR-PINs mediated cytotoxicity inhuman PDA Ccells (MIAPaCa-2),co-cultured with human pancreatic cancer-associated fibroblasts (PCAFs) in spheroids. Furthermore, TR-PINs induced the expression of heat shock proteins (Hsp60, Hsp70), Calreticulin, and high mobility group box 1 in a light dose and time-dependent manner.TR-PINs-mediated T cell activation was observed in co-cultures of immune cells with the MIA PaCa-2-PCAF spheroids. Both CD4 + T and CD8 + T cells showed light dose and time-dependant antitumor reactivity by upregulating degranulation marker CD107a and interferon-gamma post-PDP. Substantial tumor cell death in immune cell-spheroid co-cultures by day 3 shows the augmentation by antitumor T cell activation and their ability to recognize tumors for a light dose-dependent kill. These data confirm enhanced destruction of heterogeneous pancreatic spheroids mediated by PDP -induced phototoxicity, TME modulation and increased immunogenicity with targeted nanoconstructs., Competing Interests: Conflict of interest statement: The authors declare no conflicts of interest regarding this article.

Background and Aims: Locally advanced pancreatic cancer (LAPC) often causes obstruction. Verteporfin photodynamic therapy (PDT) can feasibly "debulk" the tumor more safely than noncurative surgery and has multiple advantages over older PDT agents. We aimed to assess the feasibility of EUS-guided verteporfin PDT in ablating nonresectable LAPC., Methods: Adults with LAPC with adequate biliary drainage were prospectively enrolled. Exclusion criteria were significant metastatic disease burden, disease involving >50% duodenal or major artery circumference, and recent treatment with curative intent. CT was obtained between days -28 to 0. On day 0, verteporfin .4 mg/kg was infused 60 to 90 minutes before EUS, during which a diffuser was positioned in the tumor and delivered light at 50 J/cm for 333 seconds. CT was obtained on day 2, with adverse event monitoring occurring on days 1, 2, and 14. The primary outcome was presence of necrosis., Results: Of 8 patients (62.5% men, mean age 65 ± 7.9 years) included in the study, 5 were staged at T3, 2 at T2, and 1 at T1. Most (n = 4) had primary lesions in the pancreatic head. Mean pretrial tumor diameter was 33.3 ± 13.4 mm. On day 2 CT, 5 lesions demonstrated a zone of necrosis measuring a mean diameter of 15.7 ± 5.5 mm; 3 cases did not develop necrosis. No adverse events were noted during the procedure or postprocedure observation period (days 1-3), and no changes in patient-reported outcomes were noted., Conclusions: In this pilot study, EUS-guided verteporfin PDT is feasible and shows promise as a minimally invasive ablative therapy for LAPC in select patients. Tumor necrosis is visible within 48 hours after treatment. Patient enrollment and data collection are ongoing. (Clinical trial registration number: NCT03033225.)., (Copyright © 2021 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.)

Photodynamic therapy (PDT) causes selective damage to tumor cells and vasculature and also triggers an anti-tumor immune response. The latter fact has prompted the exploration of PDT as an immune-stimulatory adjuvant. PDT is not the only cancer treatment that relies on electromagnetic energy to destroy cancer tissue. Ionizing radiation therapy (RT) and photothermal therapy (PTT) are two other treatment modalities that employ photons (with wavelengths either shorter or longer than PDT, respectively) and also cause tissue damage and immunomodulation. Research on the three modalities has occurred in different "silos", with minimal interaction between the three topics. This is happening at a time when immune checkpoint inhibition (ICI), another focus of intense research and clinical development, has opened exciting possibilities for combining PDT, PTT, or RT with ICI to achieve improved therapeutic benefits. In this review, we surveyed the literature for studies that describe changes in anti-tumor immunity following the administration of PDT, PTT, and RT, including efforts to combine each modality with ICI. This information, collected all in one place, may make it easier to recognize similarities and differences and help to identify new mechanistic hypotheses toward the goal of achieving optimized combinations and tumor cures.

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., (© 2020 American Society for Photobiology.)

We investigated how loss of TSG-6 affects wound closure and skin inflammation. TSG-6 has several known biological functions, including the enzymatic transfer of heavy-chain proteins from inter-α-trypsin inhibitor to hyaluronan to form heavy-chain protein-hyaluronan complexes. TSG-6 and heavy-chain protein-hyaluronan are constitutively expressed in normal skin and increase post-wounding but are completely absent in TSG-6-null mice. Wound closure rates are significantly delayed in TSG-6-null mice relative to wildtype mice. Neutrophil recruitment is delayed in early wounds (12 hours and day 1), whereas late wounds (day 7) show elevated neutrophil accumulation. In addition, granulation phase resolution is delayed, with persistent blood vessels and reduced dermal collagen at 10 days. The proinflammatory cytokine TNFα is elevated >3-fold in unwounded TSG-6-null skin and increases further after wounding (from 12 hours to 7 days) before returning to baseline by day 10. Other cytokines examined, such as IL-6, IL-10, and monocyte chemotactic protein-1, showed no consistent differences. Reintroduction of TSG-6 into TSG-6-null wounds rescues both the delay in wound closure and the aberrant neutrophil phenotype. In summary, our study indicates that TSG-6 plays an important role in regulating wound closure and inflammation during cutaneous wound repair., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The incidence of chronic, non-healing skin wounds is accelerating, largely due to the epidemic of obesity-related Type 2 diabetes. Abnormal inflammation in wounds contributes to delayed healing. During wound repair, blood monocytes are recruited into the wound bed where they differentiate into macrophages that secrete cytokines and regulate subsequent repair events. Because the study of wound macrophages via immunohistochemistry is often unsatisfactory due to nonspecific antibody staining, the ability to isolate and analyze single cells is important for determining the phenotypes of the wound macrophages. In this article, we have expanded upon a protocol originally described by Wilson et al, 2002 [1], and optimized it for isolation of large numbers of viable macrophages from murine skin wounds that are suitable for flow cytometric cell sorting or analysis. Several parameters were found to be critical for improved macrophage yields, including: (1) The proper amount of starting material (skin tissue); (2) The optimal time for addition of Brefeldin A during enzymatic digestion; (3) Revamped guidelines for centrifugation to maximize cell pellet recovery. This optimized protocol could be further modified to perform cell sorting and flow-based immunophenotyping of any cell type involved in wound healing and inflammation., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors. Published by Elsevier B.V.)

Patients with cancer often confront the decision of whether to continue high-dose chemotherapy at the expense of cumulative toxicities. Reducing the dose of chemotherapy regimens while preserving efficacy is sorely needed to preserve the performance status of these vulnerable patients, yet has not been prioritized. Here, we introduce a dual pronged approach to modulate the microenvironment of desmoplastic pancreatic tumors and enable significant dose deescalation of the FDA-approved chemotherapeutic nanoliposomal irinotecan (nal-IRI) without compromising tumor control. We demonstrate that light-based photodynamic priming (PDP) coupled with vitamin D3 receptor (VDR) activation within fibroblasts increases intratumoral nal-IRI accumulation and suppresses protumorigenic CXCL12/CXCR7 crosstalk. Combined photodynamic and biochemical modulation of the tumor microenvironment enables a 75% dose reduction of nal-IRI while maintaining treatment efficacy, resulting in improved tolerability. Modifying the disease landscape to increase the susceptibility of cancer, via preferentially modulating fibroblasts, represents a promising and relatively underexplored strategy to enable dose deescalation. The approach presented here, using a combination of three clinically available therapies with nonoverlapping toxicities, can be rapidly translated with minimal modification to treatment workflow, and challenges the notion that significant improvements in chemotherapy efficacy can only be achieved at the expense of increased toxicity., (©2020 American Association for Cancer Research.)

The efficacy of photodynamic therapy (PDT) using aminolevulinic acid (ALA), which is preferentially taken up by cancerous cells and converted to protoporphyrin IX (PpIX), can be substantially improved by pretreating the tumor cells with vitamin D (Vit D). Vit D is one of several "differentiation-promoting agents" that can promote the preferential accumulation of PpIX within the mitochondria of neoplastic cells, making them better targets for PDT. This article provides a historical overview of how the concept of using combination agents ("neoadjuvants") for PDT evolved, from initial discoveries about neoadjuvant effects of methotrexate and fluorouracil to later studies to determine how vitamin D and other agents actually work to augment PDT efficacy. While this review focuses mainly on skin cancer, it includes a discussion about how these concepts may be applied more broadly toward improving PDT outcomes in other types of cancer., (© 2020 American Society for Photobiology.)

Background: Blue light photodynamic therapy (PDT) is effective for actinic keratosis, but many patients experience stinging pain during illumination., Objective: To compare a conventional regimen (1 hour of 5-aminolevulinic acid [ALA] preincubation, followed by blue light) versus a new modified regimen in which blue light is started immediately after ALA application., Methods: A clinical trial with a bilaterally controlled, intrapatient study design was conducted with 23 patients. Topical 20% ALA was applied to the entire face and/or scalp. On 1 side of the body, blue light was started immediately and continued for either 30, 45, or 60 minutes (simultaneous PDT). On the contralateral side, the blue light began 1 hour after ALA application and lasted 1000 seconds (conventional PDT). Pain was evaluated on a scale from 0 to 10. Actinic keratosis lesion counts were determined by clinical examination and photography., Results: All patients experienced significantly less pain during simultaneous illumination than during the conventional regimen. At 3 months after treatment, lesion clearance was nearly identical on the 2 sides, as determined by statistical testing of noninferiority ± 15% margin., Limitations: Although bilaterally controlled, the study was relatively small. Additional studies are recommended., Conclusion: The modified PDT regimen is essentially painless, yet it provides treatment efficacy similar to a conventional regimen., (Copyright © 2019 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.)

Daylight activation for photodynamic therapy (PDT) of skin lesions is now widely adopted in many countries as a less painful and equally effective treatment mechanism, as compared to red or blue light activation. However, seasonal daylight availability and transient weather conditions complicate light dose estimations. A method is presented for dose planning without placing a large burden on clinical staff, by limiting spectral measurements to a one-time site assessment, and then using automatically acquired weather reports to track transient conditions. The site assessment tools are used to identify appropriate treatment locations for the annual and daily variations in sunlight exposure for clinical center planning. The spectral information collected from the site assessment can then be integrated with real-time daily electronic weather data. It was shown that a directly measured light exposure has strong correlation (R 2 : 0.87) with both satellite cloud coverage data and UV index, suggesting that the automated weather indexes can be surrogates for daylight PDT optical dose. These updated inputs can be used in a dose-planning treatment model to estimate photodynamic dose at depth in tissue. A simple standardized method for estimating light dose during daylight-PDT could help improve intersite reproducibility while minimizing treatment times., (© 2019 American Society for Photobiology.)

Well-regulated differentiation of fibroblasts into myofibroblasts (MF) is critical for skin wound healing. Neoexpression of α-smooth muscle actin (α-SMA), an established marker for MF differentiation, is driven by TGFβ receptor (TGFβR)-mediated signaling. Hyaluronan (HA) and its receptor CD44 may also participate in this process. To further understand this process, primary mouse skin fibroblasts were isolated and treated in vitro with recombinant TGF-β1 (rTGF-β1) to induce α-SMA expression. CD44 expression was also increased. Paradoxically, CD44 knockdown by RNA interference (RNAi) led to increased α-SMA expression and α-SMA-containing stress fibers. Removal of extracellular HA or inhibition of HA synthesis had no effect on α-SMA levels, suggesting a dispensable role for HA. Exploration of mechanisms linking CD44 knockdown to α-SMA induction, using RNAi and chemical inhibitors, revealed a requirement for noncanonical TGFβR signaling through p38MAPK. Decreased monomeric G-actin but increased filamentous F-actin following CD44 RNAi suggested a possible role for myocardin-related transcription factor (MRTF), a known regulator of α-SMA transcription and itself regulated by G-actin binding. CD44 RNAi promoted nuclear accumulation of MRTF and the binding to its transcriptional cofactor SRF. MRTF knockdown abrogated the increased α-SMA expression caused by CD44 RNAi, suggesting that MRTF is required for CD44-mediated regulation of α-SMA. Finally, chemical inhibition of p38MAPK reversed nuclear MRTF accumulation after rTGF-β1 addition or CD44 RNAi, revealing a central involvement of p38MAPK in both cases. We concluded that CD44 regulates α-SMA gene expression through cooperation between two intersecting signaling pathways, one mediated by G-actin/MRTF and the other via TGFβR/p38MAPK., (© 2019 Wang et al.)

Competing Interests: Disclosures.: Dr. Maytin serves on the scientific advisory board of Sun Pharmaceutical Industries Ltd. Dr. Segal and Ms. Houlihan are employees of Sun Pharmaceutical Industries Ltd. with medial affairs responsibilities related to Levulan®-BluU photodynamic therapy.

Background: Daylight-activated PDT has seen increased support in recent years as a treatment method for actinic keratosis and other non-melanoma skin cancers. The inherent variability observed in broad-spectrum light used in this methodology makes it difficult to plan and monitor light dose, or compare to lamp light doses., Methods: The present study expands on the commonly used PpIX-weighted effective surface irradiance metric by introducing a Monte Carlo method for estimating effective fluence rates into depths of the skin. The fluence rates are compared between multiple broadband and narrowband sources that have been reported in previous studies, and an effective total fluence for various treatment times is reported. A dynamic estimate of PpIX concentration produced during pro-drug incubation and treatment is used with the fluence estimates to calculate a photodynamic dose., Results: Even when there is up to a 5x reduction between the effective surface irradiance of the broadband light sources, the effective fluence below 250 μm depth is predicted to be relatively equivalent. An effective threshold fluence value (0. 70J eff /cm 2 ) is introduced based on a meta-analysis of previously published ALA-PpIX induced cell death. This was combined with a threshold PpIX concentration (50 nM) to define a threshold photodynamic dose of 0.035 u M J eff /cm 2 ., Conclusions: The threshold was used to generate lookup tables to prescribe minimal treatment times to achieve depth-dependent cytotoxic effect based on incubation times and irradiance values for each light source., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Importance: Risk factors for the development of skin cancer after solid-organ transplant can inform clinical care, but data on these risk factors are limited., Objective: To study the association between HLA antigen mismatch and skin cancer incidence after solid-organ transplant., Design, Setting, and Participants: This retrospective cohort study is a secondary analysis of the multicenter Transplant Skin Cancer Network study of 10 649 adults who underwent a primary solid-organ transplant between January 1, 2003, and December 31, 2003, or between January 1, 2008, and December 31, 2008. These participants were identified through the Scientific Registry of Transplant Recipients standard analysis files, which contain data collected mostly by the Organ Procurement and Transplantation Network. Participants were matched to skin cancer outcomes by medical record review. This study was conducted from August 1, 2016, to July 31, 2017., Main Outcomes and Measures: The primary outcome was time to diagnosis of posttransplant skin cancer, including squamous cell carcinoma, melanoma, and Merkel cell carcinoma. The HLA antigen mismatch was calculated based on the 2016 Organ Procurement and Transplantation Network guidelines. Risk of skin cancer was analyzed using a multivariate Cox proportional hazards regression model., Results: In total, 10 649 organ transplant recipients (6776 men [63.6%], with a mean [SD] age of 51 [12] years) contributed 59 923 years of follow-up. For each additional mismatched allele, a 7% to 8% reduction in skin cancer risk was found (adjusted hazard ratio [HR], 0.93; 95% CI, 0.87-0.99; P = .01). Subgroup analysis found the protective effect of HLA antigen mismatch to be statistically significant in lung (adjusted HR, 0.70; 95% CI, 0.56-0.87; P = .001) and heart (adjusted HR, 0.75; 95% CI, 0.60-0.93; P = .008) transplant recipients but not for recipients of liver, kidney, or pancreas. The degree of HLA-DR mismatch, but not HLA-A or HLA-B mismatch, was the most statistically significant for skin cancer risk (adjusted HR, 0.85; 95% CI, 0.74-0.97; P = .01)., Conclusions and Relevance: The HLA antigen mismatch appears to be associated with reductions in the risk of skin cancer after solid-organ transplant among heart and lung transplant recipients; this finding suggests that HLA antigen mismatch activates the tumor surveillance mechanisms that protect against skin cancer in transplant recipients and that skin cancer risk may be higher in patients who received a well-matched organ.

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.

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., Competing Interests: Conflicts of interest All authors declared that there are no conflicts of interest.

Daylight-mediated photodynamic therapy (d-PDT) as a treatment for actinic keratosis (AK) is an increasingly common technique due to a significant reduction in pain, leading to better patient tolerability. While past studies have looked at different light sources and delivery methods, this study strives to provide equivalent PpIX-weighted light doses with the hypothesis that artificial light sources could be equally as effective as natural sunlight if their PpIX-weighted fluences were equalized. Normal mouse skin was used as the model to compare blue LED light, metal halide white light and natural sunlight, with minimal incubation time between topical ALA application and the onset of light delivery. A total PpIX-weighted fluence of 20 J eff cm -2 was delivered over 2 h, and the efficacy of response was quantified using three acute bioassays for PDT damage: PpIX photobleaching, Stat3 crosslinking and quantitative histopathology. These bioassays indicated blue light was slightly inferior to both sunlight and white light, but that the latter two were not significantly different. The results suggest that metal halide white light could be a reasonable alternative to daylight PDT, which should allow a more controlled treatment that is independent of weather and yet should have similar response rates with limited pain during treatment., (© 2018 The American Society of Photobiology.)

Purpose: Actinic keratoses (AK) are precancerous lesions that can progress to squamous cell carcinoma. Photodynamic therapy (PDT) and topical 5-fluorouracil (5FU) are commonly used agents for AK. Empirical reports suggest that combining them can improve the therapeutic response. However, the optimal combined regimen was not clear in terms of proper sequence, timing, and mechanism. This clinical study explored mechanisms of action for neoadjuvantal 5FU and PDT for treatment of AK. Patients and Methods: A bilaterally controlled trial (17 patients) was performed. One side of the body (face, scalp, forearms) received 5FU pretreatment for 6 days, whereas the other side served as no-pretreatment control. Methylaminolevulinate cream was applied to both sides for 3 hours, and protoporphyrin IX (PpIX) levels were measured by noninvasive fluorimetry and skin biopsy. After red light illumination, lesion clearance was assessed at 3, 6, 9, and 12 months after PDT. Results: PpIX levels were increased 2- to 3-fold in 5FU-pretreated lesions versus controls. Altered expression of heme-synthetic enzymes (coproporphyrinogen oxidase and ferrochelatase) and induction of p53 were observed, probably accounting for increased PpIX and subsequent cancer cell death. Relative clearance rates after PDT with or without 5FU pretreatment were 75% versus 45% at 3 months, and 67% versus 39% at 6 months, respectively; these differences were statistically significant. Conclusions: Serial 5FU and PDT improve AK clearance by at least two mechanisms, enhanced photosensitizer accumulation and p53 induction. Because 5FU and PDT are FDA-approved modalities, the combined regimen can be readily employed in clinical practice to reduce AK burden and reduce SCC risk. Clin Cancer Res; 24(13); 3026-35. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Background: Photodynamic therapy (PDT) is a non-scarring alternative for treating basal cell carcinoma (BCC) in patients with Basal Cell Nevus Syndrome (BCNS), also known as Gorlin syndrome. In Europe, red light (635 nm) is the predominant source for PDT, whereas in the United States blue light (400 nm) is more widely available. The objective of this study was to conduct a head-to-head comparison of blue light and red light PDT in the same BCNS patients., Methods: In a pilot study of three patients with 141 BCC lesions, 5-aminolevulinate (20% solution) was applied to all tumors. After 4 h, half of the tumors were illuminated with blue light and the remainder with red light. To ensure safety while treating this many tumors simultaneously, light doses were escalated gradually. Six treatments were administered in three biweekly sessions over 4 months, with a final evaluation at 6 months. Tumor status was documented with high-resolution photographs. Persistent lesions were biopsied at 6 months., Results: Clearance rates after blue light (98%) were slightly better than after red light (93%), with blue light shown to be statistically non-inferior to red light. Eight suspicious lesions were biopsied, 5 after red light (5/5 were BCC) and 3 after blue light (1 was BCC). Blue light PDT was reportedly less painful., Conclusion: Blue light and red light PDT appear to be equally safe and perhaps equally effective for treating BCC tumors in BCNS patients. Further studies to evaluate long-term clearance after blue light PDT are needed., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Sunlight can activate photodynamic therapy (PDT), and this is a proven strategy to reduce pain caused byconventional PDT treatment, but assessment of this and other alternative low dose rate light sources, and their efficacy, has not been studied in an objective, controlled pre-clinical setting. This study used three objective assays to assess the efficacy of different PDT treatment regimens, using PpIX fluorescence as a photophysical measure, STAT3 cross-linking as a photochemical measure, and keratinocyte damage as a photobiological measure., Methods: Nude mouse skin was used along with in vivo measures of photosensitizer fluorescence, keratinocyte nucleus damage from pathology, and STAT3 cross-linking from Western blot analysis. Light sources compared included a low fluence rate red LED panel, compact fluorescent bulbs, halogen bulbs and direct sunlight, as compared to traditional PDT delivery with conventional and fractionated high fluence rate red LED light delivery., Results: Of the three biomarkers, two had strong correlation to the PpIX-weighted light dose, which is calculated as the product of the treatment light dose (J/cm 2 ) and the normalized PpIX absorption spectra. Comparison of STAT3 cross-linking to PpIX-weighted light dose had an R=0.74, and comparison of keratinocyte nuclear damage R=0.70. There was little correlation to PpIX fluorescence. These assays indicate most of the low fluence rate treatment modalities were as effective as conventional PDT, while fractionated PDT showed the most damage., Conclusions: Daylight or artificial light PDT provides an alternative schedule for delivery of drug-light treatment, and this pre-clinical assay demonstrated that in vivo assays of damage could be used to objectively predict a clinical outcome in this altered delivery process., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

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. In the United States, PDT is only FDA approved for treatment of actinic keratoses; 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. First, the expression of key enzymes in the heme synthesis pathway was altered, including upregulated coproporphyrinogen oxidase and downregulated ferrochelatase. Second, 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, that is, 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. Mol Cancer Ther; 16(6); 1092-101. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Background: Aminolevulinic acid (ALA)-based photodynamic therapy (PDT) provides selective uptake and conversion of ALA into protoporphyrin IX (PpIX) in actinic keratosis and squamous cell carcinoma, yet large response variations in effect are common between individuals. The aim of this study was to compare pre-treatment strategies that increase the therapeutic effect, including fractionated light delivery during PDT (fPDT) and use of iron chelator desferrioxamine (DFO), separately and combined., Methods: Optical measurements of fluorescence were used to quantify PpIX produced, and the total amount of PpIX photobleached as an implicit measure of the photodynamic dose. In addition, measurements of white light reflectance were used to quantify changes in vascular physiology throughout the PDT treatment., Results: fPDT produced both a replenishment of PpIX and vascular re-oxygenation during a 2 h dark interval between the first and second PDT light fractions. The absolute photodynamic dose was increased 57% by fPDT, DFO and their combination, as compared with PDT group (from 0.7 to 1.1). Despite that light fractionation increased oedema and scab formation during the week after treatment, no significant difference in long-term survival has been observed between treatment groups. However, outcomes stratified on the basis of measured photodynamic dose showed a significant difference in long-term survival., Conclusions: The assessment of implicit photodynamic dose was a more significant predictor of efficacy for ALA-PDT skin cancer treatments than prescription of an enhanced treatment strategy, likely because of high individual variation in response between subjects.

Dermatology is a field that strives not only to alleviate skin disease (therapeutics) but also to improve the perception of wellness (cosmetics). Thus, in this special issue of Glycobiology, it seems appropriate to discuss the biology of a glycosaminoglycan, called hyaluronic acid (hyaluronan, or HA), that has become the most popular agent today for intradermal injections to improve wrinkles and other cosmetic defects. HA is a simple linear polymer in which a simple disaccharide is repeated thousands of time, thereby creating a huge hydrophilic molecule that confers a large volume of hydration and contributes to the turgor and flexibility of healthy skin. Beyond cosmetic considerations, however, HA also has important biological and physiological functions that were largely under-appreciated until recently. New research has confirmed that HA is dynamically produced by most skin cells, not only fibroblasts (the cells that make most of the skin's extracellular matrix) but also by keratinocytes in the outer protective layer (epidermis). For both fibroblasts and keratinocytes, HA plays a regulatory role in controlling cell physiology through interaction of extracellular HA with a major cell-surface receptor, CD44. This interaction mediates intracellular signaling both directly and indirectly, through CD44 interactions with the cytoskeleton and with EGF and TGFβ receptors. Furthermore, degradation of HA by specific hyaluronidase enzymes produces HA fragments that can help to regulate inflammatory processes. In this review, current knowledge about the role of HA in skin inflammation and wound healing are reviewed and possible future applications of such knowledge discussed., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Photodynamic therapy (PDT) can be a highly complex treatment, with many parameters influencing treatment efficacy. The extent to which dosimetry is used to monitor and standardize treatment delivery varies widely, ranging from measurement of a single surrogate marker to comprehensive approaches that aim to measure or estimate as many relevant parameters as possible. Today, most clinical PDT treatments are still administered with little more than application of a prescribed drug dose and timed light delivery, and thus the role of patient-specific dosimetry has not reached widespread clinical adoption. This disconnect is at least partly due to the inherent conflict between the need to measure and understand multiple parameters in vivo in order to optimize treatment, and the need for expedience in the clinic and in the regulatory and commercialization process. Thus, a methodical approach to selecting primary dosimetry metrics is required at each stage of translation of a treatment procedure, moving from complex measurements to understand PDT mechanisms in pre-clinical and early phase I trials, towards the identification and application of essential dose-limiting and/or surrogate measurements in phase II/III trials. If successful, identifying the essential and/or reliable surrogate dosimetry measurements should help facilitate increased adoption of clinical PDT. In this paper, examples of essential dosimetry points and surrogate dosimetry tools that may be implemented in phase II/III trials are discussed. For example, the treatment efficacy as limited by light penetration in interstitial PDT may be predicted by the amount of contrast uptake in CT, and so this could be utilized as a surrogate dosimetry measurement to prescribe light doses based upon pre-treatment contrast. Success of clinical ALA-based skin lesion treatment is predicted almost uniquely by the explicit or implicit measurements of photosensitizer and photobleaching, yet the individualization of treatment based upon each patients measured bleaching needs to be attempted. In the case of ALA, lack of PpIX is more likely an indicator that alternative PpIX production methods must be implemented. Parsimonious dosimetry, using surrogate measurements that are clinically acceptable, might strategically help to advance PDT in a medical world that is increasingly cost and time sensitive. Careful attention to methodologies that can identify and advance the most critical dosimetric measurements, either direct or surrogate, are needed to ensure successful incorporation of PDT into niche clinical procedures.

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 and (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., (© 2015 The American Society of Photobiology.)

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., (© 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

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.

A wound is a type of injury that damages living tissues. In this review, we will be referring mainly to healing responses in the organs including skin and the lungs. Fibrosis is a process of dysregulated extracellular matrix (ECM) production that leads to a dense and functionally abnormal connective tissue compartment (dermis). In tissues such as the skin, the repair of the dermis after wounding requires not only the fibroblasts that produce the ECM molecules, but also the overlying epithelial layer (keratinocytes), the endothelial cells, and smooth muscle cells of the blood vessel and white blood cells such as neutrophils and macrophages, which together orchestrate the cytokine-mediated signaling and paracrine interactions that are required to regulate the proper extent and timing of the repair process. This review will focus on the importance of extracellular molecules in the microenvironment, primarily the proteoglycans and glycosaminoglycan hyaluronan, and their roles in wound healing. First, we will briefly summarize the physiological, cellular, and biochemical elements of wound healing, including the importance of cytokine cross-talk between cell types. Second, we will discuss the role of proteoglycans and hyaluronan in regulating these processes. Finally, approaches that utilize these concepts as potential therapies for fibrosis are discussed.

Ulcers and chronic wounds are a particularly common problem in diabetics and are associated with hyperglycemia. In this targeted review, we summarize evidence suggesting that defective wound healing in diabetics is causally linked, at least in part, to hyperglycemia-induced changes in the status of hyaluronan (HA) that resides in the pericellular coat (glycocalyx) of endothelial cells of small cutaneous blood vessels. Potential mechanisms through which exposure to high glucose levels causes a loss of the glycocalyx on the endothelium and accelerates the recruitment of leukocytes, creating a proinflammatory environment, are discussed in detail. Hyperglycemia also affects other cells in the immediate perivascular area, including pericytes and smooth muscle cells, through exposure to increased cytokine levels and through glucose elevations in the interstitial fluid. Possible roles of newly recognized, cross-linked forms of HA, and interactions of a major HA receptor (CD44) with cytokine/growth factor receptors during hyperglycemia, are also discussed.

The CCAAT/Enhancer Binding Proteins (C/EBPs) are a family of leucine-zipper transcription factors that regulate physiological processes such as energy metabolism, inflammation, cell cycle, and the development and differentiation of several tissues including skin. Recently, a role for C/EBPs in tumor cell proliferation and differentiation has been proposed, but the incomplete characterization in the literature of multiple translational isoforms of these proteins has made interpretation of these roles difficult. Therefore, we have carefully reexamined C/EBP isoform expression in human non-melanoma skin cancers. C/EBPα, C/EBPβ, and C/EBPδ were analyzed histologically in squamous cell carcinomas (SCC). The individual isoforms of C/EBPα and C/EBPβ were examined by immunofluorescent digital imaging, western blotting and DNA binding activity (electrophoretic mobility shift analysis). Expression of all C/EBP family proteins was decreased in SCC tumors. Suppression was greatest for C/EBPα, less for C/EBPβ, and least for C/EBPδ. Western analyses confirmed that C/EBPα p42 and p30 isoforms were decreased. For C/EBPβ, only the abundant full-length isoform (C/EBPβ-1, LAP*, 55 kD) was reduced, whereas the smaller isoforms, C/EBPβ-2 (LAP, 48 kD) and C/EBPβ-3 (LIP, 20 kD), which are predominantly nuclear, were significantly increased in well- and moderately-differentiated SCC (up to 14-fold for C/EBPβ-3). These elevations correlated with increases in PCNA, a marker of proliferation. Although C/EBPβ displayed increased post-translational modifications in SCC, phosphorylation of C/EBPβ-1 (Thr 235) was not altered. C/EBP-specific DNA binding activity in nuclear and whole-cell extracts of cultured cells and tumors was predominantly attributable to C/EBPβ. In summary, two short C/EBPβ isoforms, C/EBPβ-2 and C/EBPβ-3, represent strong candidate markers for epithelial skin malignancy, due to their preferential expression in carcinoma versus normal skin, and their strong correlation with tumor proliferation.

A balanced turnover of dermal fibroblasts is crucial for structural integrity and normal function of the skin. During recovery from environmental injury (such as UV exposure and physical wounding), apoptosis is an important mechanism regulating fibroblast turnover. We are interested in the role that hyaluronan (HA), an extracellular matrix molecule synthesized by HA synthase enzymes (Has), plays in regulating apoptosis in fibroblasts. We previously reported that Has1 and Has3 double knock-out (Has1/3 null) mice show accelerated wound closure and increased numbers of fibroblasts in the dermis. In the present study, we report that HA levels and Has2 mRNA expression are higher in cultured Has1/3 null primary skin fibroblasts than in wild type (WT) cells. Apoptosis induced by two different environmental stressors, UV exposure and serum starvation (SS), was reduced in the Has1/3 null cells. Hyaluronidase, added to cultures to remove extracellular HA, surprisingly had no effect upon apoptotic susceptibility to UVB or SS. However, cells treated with 4-methylumbelliferone to inhibit HA synthesis were sensitized to apoptosis induced by SS or UVB. When fibroblasts were transfected with Has2-specific siRNA that lowered Has2 mRNA and HA levels by 90%, both Has1/3 null and WT cells became significantly more sensitive to apoptosis. The exogenous addition of high molecular weight HA failed to reverse this effect. We conclude that Has1/3 null skin fibroblasts (which have higher levels of Has2 gene expression) are resistant to stress-induced apoptosis., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

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., (© 2014 The American Society of Photobiology.)

Dosimetry for aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) photodynamic therapy of actinic keratosis was examined with an optimized fluorescence dosimeter to measure PpIX during treatment. While insufficient PpIX generation may be an indicator of incomplete response, there exists no standardized method to quantitate PpIX production at depths in the skin during clinical treatments. In this study, a spectrometer-based point probe dosimeter system was used to sample PpIX fluorescence from superficial (blue wavelength excitation) and deeper (red wavelength excitation) tissue layers. Broadband white light spectroscopy (WLS) was used to monitor aspects of vascular physiology and inform a correction of fluorescence for the background optical properties. Measurements in tissue phantoms showed accurate recovery of blood volume fraction and reduced scattering coefficient from WLS, and a linear response of PpIX fluorescence versus concentration down to 1.95 and 250 nM for blue and red excitations, respectively. A pilot clinical study of 19 patients receiving 1-h ALA incubation before treatment showed high intrinsic variance in PpIX fluorescence with a standard deviation/mean ratio of > 0.9. PpIX fluorescence was significantly higher in patients reporting higher pain levels on a visual analog scale. These pilot data suggest that patient-specific PpIX quantitation may predict outcome response.

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.

The efficacy of photodynamic therapy (PDT) for epithelial cancers is increased when PDT is combined with calcitriol (Vit D), a form of differentiation therapy (DT). Here, we describe an underlying mechanism for this effect. Differentiation-promoting agents are known to upregulate CCAAT/enhancer-binding proteins (C/EBP), powerful regulators of cellular differentiation. In subcutaneous A431 tumors in mice, pretreatment with Vit D induced the expression of C/EBPβ isoforms, and of coproporphyrinogen oxidase (CPO), a heme pathway enzyme responsible for the conversion of 5-aminolevulinic acid (ALA) into protoporphyrin IX (PpIX), the principal light-absorbing molecule during PDT. To further investigate this apparent link between C/EBPs and CPO, two cell lines (MEL and LNCaP) were exposed to differentiating agents, and levels of PpIX, C/EBPs, and CPO were measured. Differentiating agents, or transfection of C/EBP expression vectors, increased C/EBP and CPO levels in parallel. Focusing on approximately 1,300 bp of upstream CPO gene promoter, we tested the ability of recombinant C/EBPα, C/EBPβ, C/EBPδ, and C/EBPζ to bind to CPO gene sequences [electrophoretic mobility shift assay (EMSA) assays] and to affect transcriptional activity (luciferase assays). Multiple C/EBP consensus binding sites were identified (15 for mouse, 18 for human). Individual probes representing each site bound to C/EBPs with characteristic affinities (strong, moderate, or weak), but when sites were inactivated in the context of the native promoter, transcriptional activity was reduced nearly equally for strong or weak sites. Cooperative interactions between regularly spaced C/EBP sites seem critical for CPO transcriptional regulation by differentiation therapy. These results provide a mechanistic rationale for DT/PDT combination therapy for cancer.