Treatment of nonmelanoma skin cancer with pro-differentiation agents and photodynamic therapy: Preclinical and clinical studies (Review).

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.

Combination of vitamin D and photodynamic therapy enhances immune responses in murine models of squamous cell skin cancer.

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.

High Sensitivity Singlet Oxygen Luminescence Sensor Using Computational Spectroscopy and Solid-State Detector.

This paper presents a technique for high sensitivity measurement of singlet oxygen luminescence generated during photodynamic therapy (PDT) and ultraviolet (UV) irradiation on skin. The high measurement sensitivity is achieved by using a computational spectroscopy (CS) approach that provides improved photon detection efficiency compared to spectral filtering methodology. A solid-state InGaAs photodiode is used as the CS detector, which significantly reduces system cost and improves robustness compared to photomultiplier tubes. The spectral resolution enables high-accuracy determination and subtraction of photosensitizer fluorescence baseline without the need for time-gating. This allows for high sensitivity detection of singlet oxygen luminescence emission generated by continuous wave light sources, such as solar simulator sources and those commonly used in PDT clinics. The value of the technology is demonstrated during in vivo and ex vivo experiments that show the correlation of measured singlet oxygen with PDT treatment efficacy and the illumination intensity on the skin. These results demonstrate the potential use of the technology as a dosimeter to guide PDT treatment and as an analytical tool supporting the development of improved sunscreen products for skin cancer prevention.

The recombinant Link module of human TSG-6 suppresses cartilage damage in models of osteoarthritis: A potential disease-modifying OA drug.

OBJECTIVE: To investigate the role of endogenous TSG-6 in human osteoarthritis (OA) and assess the disease-modifying potential of a TSG-6-based biological treatment in cell, explant and animal models of OA. DESIGN: Knee articular cartilages from OA patients were analyzed for TSG-6 protein and mRNA expression using immunohistochemistry and RNAscope, respectively. The inhibitory activities of TSG-6 and its isolated Link module (Link_TSG6) on cytokine-induced degradation of OA cartilage explants were compared. Human mesenchymal stem/stromal cell-derived chondrocyte pellet cultures were used to determine the effects of Link_TSG6 and full-length TSG-6 on IL-1α-, IL-1ß-, or TNF-stimulated ADAMTS4, ADAMTS5, and MMP13 mRNA expression. Link_TSG6 was administered i.a. to the rat ACLTpMMx model; cartilage damage and tactile allodynia were assessed. RESULTS: TSG-6 is predominantly associated with chondrocytes in regions of cartilage damage where high TSG-6 expression aligns with low MMP13, the major collagenase implicated in OA progression. Link_TSG6 is more potent than full-length TSG-6 at inhibiting cytokine-mediated matrix breakdown in human OA cartilage explants;>50% of donor cartilages, from 59 tested, were responsive to Link_TSG6 treatment. Link_TSG6 also displayed more potent effects in 3D pellet cultures, suppressing ADAMTS4, ADAMTS5, and MMP13 gene expression, which was consistent with reduced aggrecanase and collagenase activities in explant cultures. Link_TSG6 treatment reduced touch-evoked pain behavior and dose-dependently inhibited cartilage damage in a rodent model of surgically-induced OA. CONCLUSIONS: Link_TSG6 has enhanced chondroprotective activity compared to the full-length TSG-6 protein and shows potential as a disease modifying OA drug via its inhibition of aggrecanase and collagenase activity.

Significant Association of Poly-A and Fok1 Polymorphic Alleles of the Vitamin D Receptor with Vitamin D Serum Levels and Incidence of Squamous Cutaneous Neoplasia.

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.

Combination of 5-Fluorouracil with Photodynamic Therapy: Enhancement of Innate and Adaptive Immune Responses in a Murine Model of Actinic Keratosis†.

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.

Alternative Bedding Materials for Poultry: Availability, Efficacy, and Major Constraints.

The increasing demand of wood shavings (WS) and sawdust (SD) by other industries and growing concerns of potential chemical contaminants from wood products have amplified research interest in alternative bedding materials for commercial poultry. Several alternative materials-corn cob (CC), straws (ST) and hays (HA), sand (SA), shredded papers (SP), rice hulls (RH), peanut hulls (PH), and gypsum (CaSO4.2H2O2)-can replace conventional ones in poultry houses, depending on availability, cost, and ability to absorb and adsorb moisture and provide the birds enough room to exhibit their natural behaviors. Alternative materials hold a brighter future as bedding materials, but more studies about their physicochemical properties and litter management practices for optimum poultry welfare are recommended.

Current Prospects for Treatment of Solid Tumors via Photodynamic, Photothermal, or Ionizing Radiation Therapies Combined with Immune Checkpoint Inhibition (A Review).

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 Triggers Innate and Adaptive Immune Responses in a Murine Model of UV-induced Squamous Skin Pre-cancer.

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.

A non-toxic approach for treatment of breast cancer and its metastases: capecitabine enhanced photodynamic therapy in a murine breast tumor model.

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.

Painless versus conventional photodynamic therapy for treatment of actinic keratosis: Comparison of cell death and immune response in a murine model.

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.

5-Fluorouracil Enhances Protoporphyrin IX Accumulation and Lesion Clearance during Photodynamic Therapy of Actinic Keratoses: A Mechanism-Based Clinical Trial.

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.

Stratification of knee osteoarthritis: two major patient subgroups identified by genome-wide expression analysis of articular cartilage.

INTRODUCTION: Osteoarthritis (OA) is a heterogeneous and complex disease. We have used a network biology approach based on genome-wide analysis of gene expression in OA knee cartilage to seek evidence for pathogenic mechanisms that may distinguish different patient subgroups. METHODS: Results from RNA-Sequencing (RNA-Seq) were collected from intact knee cartilage at total knee replacement from 44 patients with OA, from 16 additional patients with OA and 10 control patients with non-OA. Results were analysed to identify patient subsets and compare major active pathways. RESULTS: The RNA-Seq results showed 2692 differentially expressed genes between OA and non-OA. Analysis by unsupervised clustering identified two distinct OA groups: Group A with 24 patients (55%) and Group B with 18 patients (41%). A 10 gene subgroup classifier was validated by RT-qPCR in 16 further patients with OA. Pathway analysis showed increased protein expression in both groups. PhenomeExpress analysis revealed group differences in complement activation, innate immune responses and altered Wnt and TGFß signalling, but no activation of inflammatory cytokine expression. Both groups showed suppressed circadian regulators and whereas matrix changes in Group A were chondrogenic, in Group B they were non-chondrogenic with changes in mechanoreceptors, calcium signalling, ion channels and in cytoskeletal organisers. The gene expression changes predicted 478 potential biomarkers for detection in synovial fluid to distinguish patients from the two groups. CONCLUSIONS: Two subgroups of knee OA were identified by network analysis of RNA-Seq data with evidence for the presence of two major pathogenic pathways. This has potential importance as a new basis for the stratification of patients with OA for drug trials and for the development of new targeted treatments.

Asymmetric Sensory-Motor Regeneration of Transected Peripheral Nerves Using Molecular Guidance Cues.

Neural interfaces are designed to decode motor intent and evoke sensory precepts in amputees. In peripheral nerves, recording movement intent is challenging because motor axons are only a small fraction compared to sensory fibers and are heterogeneously mixed particularly at proximal levels. We previously reported that pain and myelinated axons regenerating through a Y-shaped nerve guide with sealed ends, can be modulated by luminar release of nerve growth factor (NGF) and neurotrophin-3 (NT-3), respectively. Here, we evaluate the differential potency of NGF, glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), pleiotrophin (PTN), and NT-3 in asymmetrically guiding the regeneration of sensory and motor neurons. We report that, in the absence of distal target organs, molecular guidance cues can mediate the growth of electrically conductive fascicles with normal microanatomy. Compared to Y-tube compartments with bovine serum albumin (BSA), GDNF and NGF increased the motor and sensory axon content, respectively. In addition, the sensory to motor ratio was significantly increased by PTN (12.7:1) when compared to a BDNF + GDNF choice. The differential content of motor and sensory axons modulated by selective guidance cues may provide a strategy to better define axon types in peripheral nerve interfaces.

Fluorouracil Enhances Photodynamic Therapy of Squamous Cell Carcinoma via a p53-Independent Mechanism that Increases Protoporphyrin IX levels and Tumor Cell Death.

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.

Biomaterials and Fabrication to Optimise Scaffold Properties for Musculoskeletal Tissue Engineering.

Tissue engineering has emerged as a promising scientific field potentially yielding in vitro developed tissue to replace degenerative or injured tissues in vivo, thus avoiding the donor site morbidity associated with reconstructive surgery. Integral to the process is the role of scaffolds and the biomaterials used to form them. This review explores the concept of scaffold based tissue engineering and design considerations. The scaffold needs to have certain mechanical and architectural properties, it needs to be biocompatible and biodegradable, and allow combination with bioactive molecules. We also discuss scaffolding techniques, different biomaterial options and fabrication technologies, and future areas of development.

Infrapatellar fat pad syndrome: a review of anatomy, function, treatment and dynamics.

The infrapatellar (Hoffa's) fatpad is an important structure within the knee, whose function and role are both poorly understood. This review explores the anatomy, neural innervation, vascularity, role in biomechanics, pathology, imaging (stressing the importance of dynamic ultrasound assessment) and treatment of disorders presenting within this structure.

Noninvasive Optical Imaging of UV-Induced Squamous Cell Carcinoma in Murine Skin: Studies of Early Tumor Development and Vitamin D Enhancement of Protoporphyrin IX Production.

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.

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

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.

Pain inhibition by optogenetic activation of specific anterior cingulate cortical neurons.

Cumulative evidence from both humans and animals suggests that the anterior cingulate cortex (ACC) is important for pain-related perception, and thus a likely target for pain relief therapy. However, use of existing electrode based ACC stimulation has not significantly reduced pain, at least in part due to the lack of specificity and likely co-activation of both excitatory and inhibitory neurons. Herein, we report a dramatic reduction of pain behavior in transgenic mice by optogenetic stimulation of the inhibitory neural circuitry of the ACC expressing channelrhodopsin-2. Electrophysiological measurements confirmed that stimulation of ACC inhibitory neurons is associated with decreased neural activity in the ACC. Further, a distinct optogenetic stimulation intensity and frequency-dependent inhibition of spiking activity in the ACC was observed. Moreover, we confirmed specific electrophysiological responses from different neuronal units in the thalamus, in response to particular types of painful stimuli (i,e., formalin injection, pinch), which we found to be modulated by optogenetic control of the ACC inhibitory neurons. These results underscore the inhibition of the ACC as a clinical alternative in inhibiting chronic pain, and leads to a better understanding of the pain processing circuitry of the cingulate cortex.