Binner, M. | bioRxiv Science

bioRxiv2025

Colorectal cancer heterogeneity co-evolves with tumor architecture to determine disease outcome

Intratumoral heterogeneity, originating from genetic, epigenetic, and phenotypic cellular diversity, is pervasive in cancer. As these heterogeneous states employ diverse mechanisms to promote tumor progression, metastasis, and therapy resistance, emergence of cancer heterogeneity is one of the most significant barriers to curative treatment. Here we leverage deep learning approaches to develop a high-throughput image-analysis paradigm with subcellular resolution that quantifies and predicts colorectal cancer (CRC) patient outcome based on tissue architecture and nuclear morphology. We further combine this approach with spatial transcriptomics, multiplex immunohistochemistry, and patient-derived organoids to uncover a dynamic, co-evolutionary relationship between tumor architecture and cell states. We identify clinically relevant architectural interfaces in CRC tissue that diversify cellular identities by favoring distinct cancer stem cell states and thus promote evolution of tumor heterogeneity. Specifically, tissue fragmentation and associated compressive forces promote loss of classic stem cell signature and acquisition of fetal/regenerative stem cell states, which initially emerges as a hybrid state with features of epithelial-to-mesenchyme (EMT) transition. Additional tumor stroma communication then diversifies these states into distinct stem cell and EMT states at the invasive margins of tumors. Reciprocally, Wnt signaling state of tumor cells tunes their responsiveness to tissue architecture. Collectively, this work uncovers a feedback loop between cell states and tissue architecture that drives cancer heterogeneity and cell state diversification. Machine learning-based analyses harness this co-dependency, independently of mutational status or cancer stage, to predict patient outcome.

cancer biology↗

bioRxiv2019

3D microenvironment stiffness regulates tumor spheroid growth and mechanics via p21 and ROCK

Mechanical properties of cancer cells and their microenvironment contribute to breast cancer progression. While mechanosensing has been extensively studied using two-dimensional (2D) substrates, much less is known about it in a physiologically more relevant 3D context. Here we demonstrate that breast cancer tumor spheroids, growing in 3D polyethylene glycol-heparin hydrogels, are sensitive to their environment stiffness. During tumor spheroid growth, compressive stresses of up to 2 kPa built up, as quantitated using elastic polymer beads as stress sensors. Atomic force microscopy (AFM) revealed that tumor spheroid stiffness increased with hydrogel stiffness. Also, constituent cell stiffness increased in a ROCK- and F-actin-dependent manner. Increased hydrogel stiffness correlated with attenuated tumor spheroid growth, a higher proportion of cells in G0/G1 phase and elevated levels of the cyclin-dependent kinase inhibitor p21. Drug-mediated ROCK inhibition reversed not only cell stiffening upon culture in stiff hydrogels but also increased tumor spheroid growth. Taken together, we reveal here a mechanism by which the growth of a tumor spheroid can be regulated via cytoskeleton rearrangements in response to its mechanoenvironment. Thus, our findings contribute to a better understanding of how cancer cells react to compressive stress when growing under confinement in stiff environments and provide the basis for a more in-depth exploration of the underlying mechanosensory response.

bioengineering↗