Colorectal cancer (CRC) has a poor prognosis and urgently needs better therapeutic approaches. 5-Aminolevulinic acid (ALA) induced protoprophyrin IX (PpIX) based photodynamic therapy (PDT) is already approved in the clinic...

Cellular uptake and localisation of TPPS2a To investigate the uptake of TPPS2a within the SKOV3 and HEY cells in non-spheroid constructs (75,000 cells/construct), the constructs were incubated with TPPS2a (1μg/mL), for 20 hours. Afterwards, the constructs were washed with PBS and incubated with fresh cell culture medium without photosensitiser for a further 4 hours before imaging. The constructs were imaged with a fluorescence microscope with quasi-confocal structured illumination capability (20x objective, Apotome.2, Carl Zeiss). The optical sectioning capability of this microscope enabled imaging within the 3D gels with retention of imaging resolution. Fluorescence from the photosensitiser was recorded within the range of 600-700 nm using Alexa Fluor 647 channel (Figure S2).

In this study we explored the efficacy of combining low dose photodynamic therapy using a porphyrin photosensitiser and dactinomycin, a commonly used chemotherapeutic agent. The studies were carried out on compressed collagen 3D constructs of two human ovarian cancer cell lines (SKOV3 and HEY) versus their monolayer counterparts. An amphiphilc photosensitiser was employed, disulfonated tetraphenylporphine, which is not a substrate for ABC efflux transporters that can mediate drug resistance. The combination treatment was shown to be effective in both monolayer and 3D constructs of both cell lines, causing a significant and synergistic reduction in cell viability. Compared to dactinomycin alone or PDT alone, higher cell kill was found using 2D monolayer culture vs. 3D culture for the same doses. In 3D culture, the combination therapy resulted in 10 and 22 times higher cell kill in SKOV3 and HEY cells at the highest light dose compared to dactinomycin monotherapy, and 2.2 and 5.5 times higher cell kill than PDT alone. The combination of low dose PDT and dactinomycin appears to be a promising way to repurpose dactinomycin and widen its therapeutic applications.

Photochemical internalisation (PCI) is a means of achieving spatio-temporal control of cytosolic drug delivery using sub-lethal photodynamic therapy (PDT), with a photosensitiser that can be activated by non-ionising visible light. Various 3D models including those developed at our laboratory, where spheroids are grown in a compressed collagen matrix, have been used for studying anti-cancer drug effects. However, the use of a more biomimetic tumouroid model which consists of a relatively hypoxic central cancer mass surrounded by its microenvironment (stroma) has not yet been explored in either toxicity or phototoxicity studies involving PCI. Here, we examined the efficacy of PCI using a porphyrin photosensitiser and a cytotoxin (Saporin) on ovarian cancer tumouroids, with HEY ovarian cancer cells in the central cancer compartment, and HDF fibroblast cells and HUVEC endothelial cells in the surrounding stromal compartment. The efficacy was compared to tumouroids treated with either Saporin or PDT alone, or no treatment. PCI treatment was shown to be effective in the tumouroids (determined through viability assays and imaging) and caused a considerable decrease in the viability of cancer cells both within the central cancer mass and those which had migrated into the stroma, as well as a reduction in the cell density of surrounding HUVEC and HDFs. Post-treatment, the mean distance of stromal invasion by cancer cells from the original cancer mass following treatment with Saporin alone was 730 μm vs. 125 μm for PCI. PDT was also effective at reducing viability in the central cancer mass and stroma but required a higher photosensitiser dose and light dose than PCI. Tumouroids, as tissue mimics, are suitable models for interrogating multicellular events following pharmacological assault.