An investigation into the cellular uptake of indocyanine green in colorectal cancer

Intraoperative imaging uses indocyanine green (ICG) in characterising colorectal lesions. This thesis will address the hypothesis that active cellular processes are required for intracellular ICG uptake at clinically relevant concentrations in 2D and 3D culture environments. The primary objective was to discern the cellular uptake mechanism of ICG in 2D models of colorectal cancer. Observations revealed heightened ICG absorption in colon cancer cell lines compared to normal colon cells. Furthermore, it was noted that caveolin-mediated endocytosis regulated ICG uptake at both clinically relevant and higher concentrations. Subsequently, the study progressed to establishing and characterising 3D spheroids using colorectal cancer cells. To ascertain the validity of the cellular uptake mechanisms identified in 2D within a 3D context, developing a 3D spheroid model of colorectal cancer, specifically using HT-29 cells, was essential. Caveolin-mediated endocytosis was observed to support ICG uptake in 3D spheroids, aligning with findings from 2D cell models. The impact of matrix stiffness on ICG uptake in cancer cells was tested using 2D polyacrylamide gels and 3D peptide hydrogels, revealing no significant effect in the utilised models. However, it should be noted that potential effects under different stiffness conditions are possible and should be investigated in the future. The research further assessed colorectal cancer spheroids as a model for examining fluorescent probe uptake dynamics, specifically observing the gradient difference between the core and periphery as spheroids mature. Although the 3D spheroids served well in analysing ICG and novel NIR-AZA probe uptake, the homogeneity of ICG intensity throughout the spheroid was noted. The absence of a necrotic core in the spheroids may explain the lack of gradient differences in ICG intensity. Future investigations may benefit from prolonged growth periods for the spheroids to induce a necrotic core and potentially observe gradients. Overall, this study emphasises the critical role of caveolin-mediated endocytosis in facilitating ICG uptake across different cellular environments. These insights into ICG dynamics contribute to our fundamental understanding and hold promise for advancing applications in clinical research, particularly in fluorescence-guided surgery and diagnostic imaging strategies.