Spatiotemporal Tracking of Different Cell Populations in Cancer Organoid Models for Investigations on Photodynamic Therapy

Three-dimensional (3D) in vitro models of tumors are gaining interest as versatile platforms for treatment screening. In this context, heterocellular cultures in which various cell types are co-cultured are being explored to investigate whether partner cells can influence the treatment efficacies. However, when the cells are co-cultured, it is challenging to distinguish them and it becomes impossible to identify whether the treatment affects each cell line in a similar way or if there is a certain selectivity. Here, we propose a protocol in which different cell types are pre-labeled with fluorescent reporters prior to 3D culture initiation. Subsequently, the internal architecture of the 3D cancer models can be longitudinally monitored for model characterization, and to potentially detect architectural and treatment selectivity in response to therapy. This protocol employs quantum dots as non-photobleaching dyes and two-photon excited microscopy as a widely accessible imaging modality. In combination with an appropriate image analysis workflow, this protocol will help to investigate the architectural development of heterotypic microtumor/spheroid/organoid models and possibly identify treatment efficacies on individual cell populations represented within the models.