Photodynamic priming with triple-receptor targeted nanoconjugates that trigger T cell-mediated immune responses in a 3D in vitro heterocellular model of pancreatic cancer

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\u27s 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 in human PDAC cells (MIA PaCa-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

C/EBP Transcription Factors in Human Squamous Cell Carcinoma: Selective Changes in Expression of Isoforms Correlate with the Neoplastic State

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

Dual-Channel Red/Blue Fluorescence Dosimetry with Broadband Reflectance Spectroscopic Correction Measures Protoporphyrin IX Production during Photodynamic Therapy of Actinic Keratosis

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 \u3c0.9 . 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

Assessing Daylight & Low-Dose Rate Photodynamic Therapy Efficacy, Using Biomarkers of Photophysical, Biochemical and Biological Damage Metrics in Situ.

Background Sunlight can activate photodynamic therapy (PDT), and this is a proven strategy to reduce pain caused by conventional 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/cm2) 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. Graphical abstract Low-fluence daylight photodynamic therapy (PDT) has been shown to reduce pain with similar efficacy of conventional treatments. Three objective assays were performed to assess efficacy of different light treatment strategies: PpIX photobleaching, STAT3 crosslinking, and keratinocyte damage. Of these metrics, STAT3 crosslinking and keratinocyte damage showed a strong correlation to the PpIX-weighted light dose

Roles of Proteoglycans and Glycosaminoglycans in Wound Healing and Fibrosis

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

Ultraviolet B-Induced Apoptosis of Keratinocytes in Murine Skin Is Reduced by Mild Local Hyperthermia

Two components of sunlight, ultraviolet (UV) B (290-320 nm) and infrared (greater than 700 nm), each cause damage to the skin. However, we recently identified a protective response in which heat reduces UVB-induced killing of cultured keratinocytes. Here, this investigation is extended to the living epidermis. The effects of hyperthermic preconditioning upon UVB-induced apoptosis were studied morphologically with hematoxylin and eosin staining, and biochemically with TUNEL (terminal deoxynucleotide transferase nick-end labeling) to measure endonu-cleolytic cleavage of DNA in situ. Anesthetized SKH-1 hairless mice were exposed to UVB light (0 to 120 mJ/cm2), after which their skin was biopsied at 24 h and paraffin sections were stained with hematoxylin and eosin or with TUNEL. Apoptotic keratinocytes were found to increase after UVB in a dose-related manner. In contrast, if one flank of the mouse was pretreated at 40°C for 1 h and both flanks subsequently were UVB-irradiated at 6 h, the resulting formation of apoptotic cells was reduced twofold or more in the heated flank. Protection appeared by 3 h, reached a maximum at 6 h, and disappeared by 12 h. In summary, heat induces a transient protective effect that reduces UVB-mediated death of keratinocytes in skin at physiologically attainable doses of heat and UVB

Transcription Factors C/EBPα, C/EBPβ, and CHOP (Gadd153) Expressed During the Differentiation Program of Keratinocytes In Vitro and In Vivo

CCAAT-enhancer binding proteins (C/EBP) are basic region/leucine zipper (bZIP) transcription factors selectively expressed during the differentiation of liver, adipose tissue, blood cells, and the endocrine pancreas. Here we show that C/EBP isoforms are differentially expressed in the skin. BALB/MK keratinocytes incubated in 0.12 mM calcium medium undergo a differentiation program featuring growth-arrest at 24–48 h, keratin K10 gene expression beginning at 24 h, and apoptosis commencing at 48 h. Within this framework, western immunoblot analysis and immunohistochemistry reveal that C/EBPα increases 5-fold at 1–2 d and remains elevated, C/EBPβ rises 2-fold at 2–4 d and gradually falls, and CHOP rises 9-fold in the first 24 h then returns rapidly to baseline. Several products of alternative translation are observed in BALB/MK cells, i.e., 42 kDa and 30 kDa forms of C/EBPα, and 32 kDa and 20 kDa forms of C/EBPβ. By immunohistologic examination of human, rat, and mouse skin, all three transcription factors are highly expressed within epithelial compartments in a spatially restricted distribution. C/EBPα is concentrated in the upper epidermis in a predominantly cytoplasmic location within cells, whereas the highest levels of C/EBPβ and CHOP are seen in the mid-epidermis, mainly within nuclei. High levels of C/EBPβ and CHOP (but not C/EBPα) are also observed in hair follicles and sebaceous glands. The identity of these factors in the epidermis is confirmed by western immunoblot analyses. In summary, C/EBP are expressed in a differentiation-associated manner in the skin, and may play an important role in regulating one or more aspects of the epidermal differentiation program

Hyperglycemia-Induced Changes in Hyaluronan Contribute to Impaired Skin Wound Healing in Diabetes: Review and Perspective

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

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