LDAcoop: Integrating non-linear population dynamics into the analysis of clonogenic growth in vitro.

The limiting dilution assay (LDA) is a key method to quantify clonogenic cells with self-renewing capacity in vitro, crucial for preclinical cancer research and therapy response assessment. It estimates the frequency of individual clonogenic, stem-like cells within a population based on their ability to form colonies with ≥50 cells at limiting cell numbers. Standard LDA analysis relies on linear, single-hit Poisson models, yet clonogenic growth under single-cell conditions often involves cooperative or competitive dynamics, violating this linearity assumption. Here, we present a modeling framework incorporating non-linear population dynamics into LDA analysis and introduce LDAcoop, an R-based tool for universal quantification of clonogenic cells in LDA formats. Across multiple cancer cell types, we benchmarked LDA against the colony formation assay (CFA) and show that LDA outperforms CFA, especially for patient-derived organoids, suspension cultures, and higher throughput applications. This renders the LDA format particularly suitable for larger-scale pharmacogenomic screening and drug sensitivity testing in complex models. Our results establish LDA and LDAcoop as versatile, scalable tools for robust quantification of clonogenic growth, supporting preclinical drug development and molecular precision oncology research.