Labban, N. | Search bioRxiv

bioRxiv2025

Fast, flexible, learning-free organoid quantification and tracking with OrganoSeg2

Organoids are routinely imaged by brightfield microscopy at low magnification, but these images are challenging to analyze quantitatively at scale. Given differences in organoid-culture format and image acquisition among research groups, there is a general need for versatile segmentation algorithms that refine for specific applications. Here, we introduce OrganoSeg2, an overhauled software that substantively advances the multi-window adaptive thresholding of its predecessor. OrganoSeg2 gives users access to additional segmentation parameters that were latent in OrganoSeg, and common operations are accelerated [~]10-fold. Using data from six organoid types, we find that the generalized segmentation accuracy of OrganoSeg2 surpasses multiple alternatives, including segmenters based on deep learning. OrganoSeg2 adds longitudinal single-organoid tracking and multicolor fluorescence quantification, which we use to examine growth trajectories and radiotherapy responses in luminal breast cancer organoids. OrganoSeg2 is shared freely as installation packages for current users and source code for future developers (https://github.com/JanesLab/OrganoSeg2). MOTIVATIONOrganoids are routinely documented with low-magnification brightfield and fluorescence images that are challenging to quantify accurately in large numbers. OrganoSeg2 is a streamlined, highly customizable segmenter that surpasses its prior version and AI-themed competitors in various organoid contexts. New longitudinal tracking and fluorescence capabilities of OrganoSeg2 are demonstrated with experiments investigating the cell-death responses of luminal breast cancer organoids to radiotherapy.

bioengineering↗

bioRxiv2024

Patient-derived response estimates from zero-passage organoids of luminal breast cancer

BackgroundPrimary luminal breast cancer cells lose their identity rapidly in standard tissue culture, which is problematic for testing hormone interventions and molecular pathways specific to the luminal subtype. Breast cancer organoids are thought to retain tumor characteristics better, but long-term viability of luminal-subtype cases is a persistent challenge. Our goal was to adapt short-term organoids of luminal breast cancer for parallel testing of genetic and pharmacologic perturbations. MethodsWe freshly isolated patient-derived cells from luminal tumor scrapes, miniaturized the organoid format into 5 {micro}l replicates for increased throughput, and set an endpoint of 14 days to minimize drift. Therapeutic hormone targeting was mimicked in these "zero-passage" organoids by withdrawing {beta}-estradiol and adding 4-hydroxytamoxifen. We also examined sulforaphane as an electrophilic stress and commercial neutraceutical with reported anti-cancer properties. Downstream mechanisms were tested genetically by lentiviral transduction of two complementary sgRNAs and Cas9 stabilization for the first week of organoid culture. Transcriptional changes were measured by RT-qPCR or RNA sequencing, and organoid phenotypes were quantified by serial brightfield imaging, digital image segmentation, and regression modeling of cellular doubling times. ResultsWe achieved >50% success in initiating luminal breast cancer organoids from tumor scrapes and maintaining them to the 14-day zero-passage endpoint. Success was mostly independent of clinical parameters, supporting general applicability of the approach. Abundance of ESR1 and PGR in zero-passage organoids consistently remained within the range of patient variability at the endpoint. However, responsiveness to hormone withdrawal and blockade was highly variable among luminal breast cancer cases tested. Combining sulforaphane with knockout of NQO1 (a phase II antioxidant response gene and downstream effector of sulforaphane) also yielded a breadth of organoid growth phenotypes, including growth inhibition with sulforaphane, growth promotion with NQO1 knockout, and growth antagonism when combined. ConclusionsZero-passage organoids are a rapid and scalable way to interrogate properties of luminal breast cancer cells from patient-derived material. This includes testing drug mechanisms of action in different clinical cohorts. A future goal is to relate inter-patient variability of zero-passage organoids to long-term outcomes.

cancer biology↗