A multi-scale imaging pipeline identifies regions of NMII activation in colorectal cancer which promote tumor survival

The aim of this study is to define the spatial activation of non-muscle myosin II (NMII) in colorectal cancer (CRC) and determine how it promotes tumor survival. Our lab has identified curvature-sensing, pro-survival signaling hubs that form at the base of ROCK1/NMII-driven blebs in low-adhesion environments. Adoption of this weakly adherent, highly contractile amoeboid phenotype is associated with metabolic shifts and immunosuppression that worsen prognosis. However, these observations come largely from mouse models and have yet to be validated in human disease. We hypothesize that, in human CRC, elevated actomyosin contractility sustains tumor survival in poorly adherent regions by activating ROCK1/NMII signaling and reducing tumor-immune cell engagement. To test this hypothesis, we developed a cyclic multiplexed immunofluorescence panel (CODEX) and applied it to a CRC tissue microarray and whole-slide CRC specimens. The panel included markers of cell identity, metabolism, proliferation, and immune activation. Spatial analysis was performed using SPACEMAP, a consensus machine-learning platform that integrates multiple phenotyping algorithms to improve cell-type classification accuracy. To characterize nanoscale architecture, a 30-µm section adjacent to each CODEX-imaged slide was embedded in a hydrogel, expanded, and imaged using Axially Swept Light-Sheet Microscopy (ASLM). Expanded volumes were registered to the multiplexed immunofluorescence dataset, enabling subcellular analysis with ~80-nm isotropic resolution. 3D segmentation and morphological quantification were performed using u-Segment3D. Active NMII (pMLC2) signal in the tumor epithelium was observed in 25% of CRC cases, localizing to discrete regions with reduced cell-cell adhesion, including necrotic zones, mucin pools, luminal surfaces, and isolated tumor cells. pMLC2-high tumor cells exhibited decreased proliferation and increased mitochondrial abundance. Strikingly, both granulocytes and cytotoxic T cells also demonstrated elevated pMLC2 expression, and these pMLC2-high immune cells showed increased markers of immunosuppression. Registration of CODEX and ExASLM datasets enabled high-resolution assessment of membrane architecture, revealing distinct alterations in tumor cell morphology and cell-cell interfaces within pMLC2-high niches. Together, these findings demonstrate that CRC tumors upregulate pMLC2 within low-adhesion microenvironments, supporting tumor survival through coordinated changes in proliferation, metabolism, membrane architecture, and immune engagement. This work identifies spatially regulated actomyosin contractility as a critical survival program in CRC and highlights it as a potential therapeutic vulnerability.