Q22 - Nuclear morphological irregularities are conserved across stromal and immune cell types from peritumor microenvironments

Introduction: : Solid tumors such as mammary and brain exert compressive forces on the surrounding host tissue over the course of tumor expansion. However, how these forces impact neighboring peritumoral cells remains poorly understood. In this study, we used an in vitro compression system to investigate the effect of tumor-mimicking compression on cells found in the breast and brain peritumor, including fibroblasts, astrocytes, and macrophages. Using image analysis tools, we demonstrate that compression induces changes in nuclear morphology that is conserved in all three cell types despite varying lineages. Future work will focus on understanding the mechanisms driving these changes and correlating them with cell phenotype and function.

Materials and

Methods: : We utilized a previously established in vitro transwell compression assay to evaluate the effects of 24 hours of tumor-mimicking compression on nuclear morphology in NIH/3T3 murine fibroblasts, immortalized human astrocytes (IHAs), and RAW264.7 murine macrophages. To quantify differences in nuclear morphological characteristics as well as perform nuclear morphometric analysis (NMA), we used the image analysis software ImageJ, with the NMA being accomplished via the NII plugin. We also utilized an unsupervised machine-learning clustering algorithm known as VAMPIRE (Visually Aided Morpho-Phenotyping Image Recognition) to determine the relative abundance of various nuclear geometries present in both groups.

Results, Conclusions, and Discussions:: Following 24 hours of compression, we observed dramatic differences between the nuclei of compressed NIH/3T3 fibroblasts, IHAs, and RAW264.7 macrophages compared to their uncompressed counterparts (Fig. 1a). We validated these observations using ImageJ, showing that compressed nuclei possessed significantly lower circularity, roundness, and solidity, while displaying significantly higher eccentricity in all three cell types (Fig. 1b). Therefore, compression seemingly promotes similar nuclear morphological changes irrespective of cell lineage, suggesting the induction of a physical mechanism that is conserved across all three cell types. In support of these findings, we demonstrated that compressed nuclei predominantly exhibit diverse morphologies, including circular, elongated, and “kidney bean”-like shapes (Fig. 2). In contrast, uncompressed nuclei were morphologically homogeneous, with most maintaining a canonical circular or elliptical shape (Fig. 2).

Furthermore, we performed NMA to numerically characterize the relative irregularity of compressed nuclei in IHAs based on the calculation of a nuclear irregularity index (NII). Our analysis revealed that the NII of compressed nuclei is significantly higher than their uncompressed counterparts while the nuclear area in both groups was similar. Among 7 morphological categories, uncompressed nuclei were classified as “normal” while compressed nuclei were classified as “irregular.” These findings suggest that compressed nuclei may either be undergoing mitotic catastrophe or another nuclear damaging event rather than acquire aberrant morphologies due to mechanically induced senescence or apoptosis. In fact, we were able to demonstrate in preliminary live-dead cell imaging experiments that these morphologies are captured in compressed live cells.

Ongoing work is aimed at identifying the mechanism(s) by which compression promotes changes in nuclear morphology (e.g., chromatin condensation and remodeling, nucleoskeletal remodeling, cytoskeletal remodeling) as well as correlating nuclear aberrancies with cell phenotype and function. Indeed, understanding the mechanisms driving compression-induced nuclear morphological changes may lead to the design of novel, tissue-agnostic anti-cancer treatment strategies to improve patient outcomes.

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