Hypericin-mediated photodynamic therapy in two- and three-dimensional models of colorectal cancer: optimisation using a novel microfluidic device

Introduction:
Surgery, chemotherapy and radiation therapy are the main treatment options in colorectal cancer (CRC), which is a major cause of global mortality. Photodynamic therapy (PDT) is an emerging field in oncological research, involving light mediated activation of a photosensitiser and subsequent cytotoxicity. Hypericin is a promising photosensitiser, however its potential in clinical oncology is under review. Three-dimensional (3D) multicellular spheroids are recognised as better representations of cancer as compared to two-dimensional (2D) cell cultures. Conventional 3D cell culturing methods are complex procedures. Integrating 3D cell culturing into microfluidics, can scale down traditional protocols and allow precise high throughput experiments to be conducted on a microfluidic platform.

Methods:
HT29, HCT116, Caco2 colorectal cancer and HFFF2 human foetal foreskin fibroblast cell lines were used in this project. Non-cell adherent coating, ultra- low attachment plates, hanging drop and spinner flask techniques were used to generate 3D spheroids. For PDT, cell cultures were treated with Hypericin, then subjected to 1J/cm2 of light. Western blot and immunofluorescence was conducted to detect ABCG2 protein. Designs of microfluidic devices were drawn in SolidWorks and produced using 3D printing and laser cutting. Fluid flow was driven using a syringe pump. HT29 spheroids were cultured and treated with 5- Fluorouracil (5-FU) in the microfluidic device. Trypan blue, fluorescent dyes and the Lactate Dehydrogenase assay were used to analyse cytotoxicity.

Results:
The agarose-coated plate and spinner flasks methods were found to be successful methods for culturing 3D spheroids. Light and Hypericin-dose dependant reduction in cell viability was observed in all cell lines (p < 0.0001). Spheroids were more resistant than 2D cultures to Hypericin-PDT (HT29: p = 0.003, HCT116: p = 0.006) and also had a greater expression of ABCG2 protein. Inhibition of ABCG2 protein in spheroids with Ko143 resulted in an enhanced Hypericin-PDT effect compared to Hypericin-PDT alone (HT29: p = 0.04, HCT116: p = 0.01). HT29 spheroids were successfully cultured in the ‘version 3’ microfluidic device. 5-FU treatment and cytotoxic analysis was achievable ‘on-chip’ through flow.

Conclusions:
Hypericin-PDT is an effective method for treating CRC, with potential clinical application. 3D spheroids are better representations of in vivo cancers and could improve pre-clinical to clinical translation of Hypericin-PDT in CRC. Microfluidic platforms provide a convenient route for streamlining and downsizing traditional laboratory-based experiments and techniques. Combining complex 3D cell culturing methods and microfluidics can enhance the routine incorporation of spheroids in pre-clinical investigations.