Application of Monolayer Cell Cultures for Investigating Basic Mechanisms of Photodynamic Therapy.

Conventional monolayer cell cultures continue to be an inexpensive and highly accessible model of human disease that can be easily harnessed to study the molecular and cellular mechanisms of photodynamic therapy (PDT). In this communication, a collection of informative assays for conventional cell cultures are provided to determine (1) the photosensitizer uptake kinetics and localization, (2) the efficacy of PDT using metabolism- or protein-based quantification methods, (3) the effects of PDT and combination treatments on the cell cycle, (4) the cell death pathways induced by PDT, and (5) the extent of mitochondrial membrane permeabilization of PDT and photochemotherapy combinations. For each type of assay, examples from the recent literature are provided in which novel photosensitizers, their nanocarriers, and various PDT-based combination therapies are investigated. Together, these assays are examples of approaches by which monolayer cell cultures can be used as a simple yet robust and versatile model to investigate PDT.

Microtumor Models as a Preclinical Investigational Platform for Photodynamic Therapy.

Classic preclinical investigations on the mechanisms and effects of photodynamic therapy (PDT) are typically performed in two-dimensional cell cultures that have some, albeit limited, relevance to cancer biology. Bioengineered three-dimensional (3D) culture models of cancer are gaining traction in translational oncology as microtumors recapitulate the tumor architectures and cellular heterogeneity more faithfully than conventional 2D cultures. These 3D models bridge a gap between highly relevant but low-throughput in vivo animal models and high-throughput two-dimensional cultures with low clinical relevance, and thus hold promise as preclinical testing platforms in PDT research. Here, we discuss the potential applications of organotypic cancer models for PDT research and provide two well-established methodologies for generating 3D cultures of cancer: a liquid-suspended spheroid model and an adherent microtumor culture model grown on extracellular matrix scaffolds. Particular emphasis is given to harvesting the cultures for the purpose of immunoblotting and flow cytometry.

Vitamin D Receptor Activation and Photodynamic Priming Enables Durable Low-dose Chemotherapy.

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.

Inhibition of hypoxia inducible factor 1 and topoisomerase with acriflavine sensitizes perihilar cholangiocarcinomas to photodynamic therapy.

BACKGROUND: Photodynamic therapy (PDT) induces tumor cell death by oxidative stress and hypoxia but also survival signaling through activation of hypoxia-inducible factor 1 (HIF-1). Since perihilar cholangiocarcinomas are relatively recalcitrant to PDT, the aims were to (1) determine the expression levels of HIF-1-associated proteins in human perihilar cholangiocarcinomas, (2) investigate the role of HIF-1 in PDT-treated human perihilar cholangiocarcinoma cells, and (3) determine whether HIF-1 inhibition reduces survival signaling and enhances PDT efficacy. RESULTS: Increased expression of VEGF, CD105, CD31/Ki-67, and GLUT-1 was confirmed in human perihilar cholangiocarcinomas. PDT with liposome-delivered zinc phthalocyanine caused HIF-1α stabilization in SK-ChA-1 cells and increased transcription of HIF-1α downstream genes. Acriflavine was taken up by SK-ChA-1 cells and translocated to the nucleus under hypoxic conditions. Importantly, pretreatment of SK-ChA-1 cells with acriflavine enhanced PDT efficacy via inhibition of HIF-1 and topoisomerases I and II. METHODS: The expression of VEGF, CD105, CD31/Ki-67, and GLUT-1 was determined by immunohistochemistry in human perihilar cholangiocarcinomas. In addition, the response of human perihilar cholangiocarcinoma (SK-ChA-1) cells to PDT with liposome-delivered zinc phthalocyanine was investigated under both normoxic and hypoxic conditions. Acriflavine, a HIF-1α/HIF-1ß dimerization inhibitor and a potential dual topoisomerase I/II inhibitor, was evaluated for its adjuvant effect on PDT efficacy. CONCLUSIONS: HIF-1, which is activated in human hilar cholangiocarcinomas, contributes to tumor cell survival following PDT in vitro. Combining PDT with acriflavine pretreatment improves PDT efficacy in cultured cells and therefore warrants further preclinical validation for therapy-recalcitrant perihilar cholangiocarcinomas.

Inhibition of NF-κB in Tumor Cells Exacerbates Immune Cell Activation Following Photodynamic Therapy.

Although photodynamic therapy (PDT) yields very good outcomes in numerous types of superficial solid cancers, some tumors respond suboptimally to PDT. Novel treatment strategies are therefore needed to enhance the efficacy in these therapy-resistant tumors. One of these strategies is to combine PDT with inhibitors of PDT-induced survival pathways. In this respect, the transcription factor nuclear factor κB (NF-κB) has been identified as a potential pharmacological target, albeit inhibition of NF-κB may concurrently dampen the subsequent anti-tumor immune response required for complete tumor eradication and abscopal effects. In contrast to these postulations, this study demonstrated that siRNA knockdown of NF-κB in murine breast carcinoma (EMT-6) cells increased survival signaling in these cells and exacerbated the inflammatory response in murine RAW 264.7 macrophages. These results suggest a pro-death and immunosuppressive role of NF-κB in PDT-treated cells that concurs with a hyperstimulated immune response in innate immune cells.

An overview of three promising mechanical, optical, and biochemical engineering approaches to improve selective photothermolysis of refractory port wine stains.

During the last three decades, several laser systems, ancillary technologies, and treatment modalities have been developed for the treatment of port wine stains (PWSs). However, approximately half of the PWS patient population responds suboptimally to laser treatment. Consequently, novel treatment modalities and therapeutic techniques/strategies are required to improve PWS treatment efficacy. This overview therefore focuses on three distinct experimental approaches for the optimization of PWS laser treatment. The approaches are addressed from the perspective of mechanical engineering (the use of local hypobaric pressure to induce vasodilation in the laser-irradiated dermal microcirculation), optical engineering (laser-speckle imaging of post-treatment flow in laser-treated PWS skin), and biochemical engineering (light- and heat-activatable liposomal drug delivery systems to enhance the extent of post-irradiation vascular occlusion).