Multimodal mechano-microscopy reveals mechanical phenotypes of breast cancer spheroids in three dimensions.

Cancer cell invasion relies on an equilibrium between cell deformability and the biophysical constraints imposed by the extracellular matrix (ECM). However, there is little consensus on the nature of the local biomechanical alterations in cancer cell dissemination in the context of three-dimensional (3D) tumor microenvironments (TMEs). While the shortcomings of two-dimensional (2D) models in replicating in situ cell behavior are well known, 3D TME models remain underutilized because contemporary mechanical quantification tools are limited to surface measurements. Here, we overcome this major challenge by quantifying local mechanics of cancer cell spheroids in 3D TMEs. We achieve this using multimodal mechano-microscopy, integrating optical coherence microscopy-based elasticity imaging with confocal fluorescence microscopy. We observe that non-metastatic cancer spheroids show no invasion while showing increased peripheral cell elasticity in both stiff and soft environments. Metastatic cancer spheroids, however, show ECM-mediated softening in a stiff microenvironment and, in a soft environment, initiate cell invasion with peripheral softening associated with early metastatic dissemination. This exemplar of live-cell 3D mechanotyping supports that invasion increases cell deformability in a 3D context, illustrating the power of multimodal mechano-microscopy for quantitative mechanobiology in situ.

Tumor spheroid elasticity estimation using mechano-microscopy combined with a conditional generative adversarial network.

BACKGROUND AND OBJECTIVES: Techniques for imaging the mechanical properties of cells are needed to study how cell mechanics influence cell function and disease progression. Mechano-microscopy (a high-resolution variant of compression optical coherence elastography) generates elasticity images of a sample undergoing compression from the phase difference between optical coherence microscopy (OCM) B-scans. However, the existing mechano-microscopy signal processing chain (referred to as the algebraic method) assumes the sample stress is uniaxial and axially uniform, such that violation of these assumptions reduces the accuracy and precision of elasticity images. Furthermore, it does not account for prior information regarding the sample geometry or mechanical property distribution. In this study, we investigate the feasibility of training a conditional generative adversarial network (cGAN) to generate elasticity images from phase difference images of samples containing a cell spheroid embedded in a hydrogel. METHODS: To construct the cGAN training and simulated test sets, we generated 30,000 artificial elasticity images using a parametric model and computed the corresponding phase difference images using finite element analysis to simulate compression applied to the artificial samples. We also imaged real MCF7 breast tumor spheroids embedded in hydrogel using mechano-microscopy to construct the experimental test set and evaluated the cGAN using the algebraic elasticity images and co-registered OCM and confocal fluorescence microscopy (CFM) images. RESULTS: Comparison with the simulated test set ground truth elasticity images shows the cGAN produces a lower root mean square error (median: 3.47 kPa, 95 % confidence interval (CI) [3.41, 3.52]) than the algebraic method (median: 4.91 kPa, 95 % CI [4.85, 4.97]). For the experimental test set, the cGAN elasticity images contain features resembling stiff nuclei at locations corresponding to nuclei seen in the algebraic elasticity, OCM, and CFM images. Furthermore, the cGAN elasticity images are higher resolution and more robust to noise than the algebraic elasticity images. CONCLUSIONS: The cGAN elasticity images exhibit better accuracy, spatial resolution, sensitivity, and robustness to noise than the algebraic elasticity images for both simulated and real experimental data.

3D Volumetric Mechanosensation of MCF7 Breast Cancer Spheroids in a Linear Stiffness Gradient GelAGE.

The tumor microenvironment presents spatiotemporal shifts in biomechanical properties with cancer progression. Hydrogel biomaterials like GelAGE offer the stiffness tuneability to recapitulate dynamic changes in tumor tissues by altering photo-energy exposures. Here, a tuneable hydrogel with spatiotemporal control of stiffness and mesh-network is developed. The volume of MCF7 spheroids encapsulated in a linear stiffness gradient demonstrates an inverse relationship with stiffness (p < 0.0001). As spheroids are exposed to increased crosslinking (stiffer) and greater mechanical confinement, spheroid stiffness increases. Protein expression (TRPV4, ß1 integrin, E-cadherin, and F-actin) decreases with increasing stiffness while showing strong correlations to spheroid volume (r2  > 0.9). To further investigate the role of volume, MCF7 spheroids are grown in a soft matrix for 5 days prior to a second polymerisation which presents a stiffness gradient to equally expanded spheroids. Despite being exposed to variable stiffness, these spheroids show even protein expression, confirming volume as a key regulator. Overall, this work showcases the versatility of GelAGE and demonstrates volume expansion as a key regulator of 3D mechanosensation in MCF7 breast cancer spheroids. This platform has the potential to further investigation into the role of stiffness and dimensionality in 3D spheroid culture for other types of cancers and diseases.

Reprogramming anchorage dependency by adherent-to-suspension transition promotes metastatic dissemination.

BACKGROUND: Although metastasis is the foremost cause of cancer-related death, a specialized mechanism that reprograms anchorage dependency of solid tumor cells into circulating tumor cells (CTCs) during metastatic dissemination remains a critical area of challenge. METHODS: We analyzed blood cell-specific transcripts and selected key Adherent-to-Suspension Transition (AST) factors that are competent to reprogram anchorage dependency of adherent cells into suspension cells in an inducible and reversible manner. The mechanisms of AST were evaluated by a series of in vitro and in vivo assays. Paired samples of primary tumors, CTCs, and metastatic tumors were collected from breast cancer and melanoma mouse xenograft models and patients with de novo metastasis. Analyses of single-cell RNA sequencing (scRNA-seq) and tissue staining were performed to validate the role of AST factors in CTCs. Loss-of-function experiments were performed by shRNA knockdown, gene editing, and pharmacological inhibition to block metastasis and prolong survival. RESULTS: We discovered a biological phenomenon referred to as AST that reprograms adherent cells into suspension cells via defined hematopoietic transcriptional regulators, which are hijacked by solid tumor cells to disseminate into CTCs. Induction of AST in adherent cells 1) suppress global integrin/ECM gene expression via Hippo-YAP/TEAD inhibition to evoke spontaneous cell-matrix dissociation and 2) upregulate globin genes that prevent oxidative stress to acquire anoikis resistance, in the absence of lineage differentiation. During dissemination, we uncover the critical roles of AST factors in CTCs derived from patients with de novo metastasis and mouse models. Pharmacological blockade of AST factors via thalidomide derivatives in breast cancer and melanoma cells abrogated CTC formation and suppressed lung metastases without affecting the primary tumor growth. CONCLUSION: We demonstrate that suspension cells can directly arise from adherent cells by the addition of defined hematopoietic factors that confer metastatic traits. Furthermore, our findings expand the prevailing cancer treatment paradigm toward direct intervention within the metastatic spread of cancer.

NONO enhances mRNA processing of super-enhancer-associated GATA2 and HAND2 genes in neuroblastoma.

High-risk neuroblastoma patients have poor survival rates and require better therapeutic options. High expression of a multifunctional DNA and RNA-binding protein, NONO, in neuroblastoma is associated with poor patient outcome; however, there is little understanding of the mechanism of NONO-dependent oncogenic gene regulatory activity in neuroblastoma. Here, we used cell imaging, biochemical and genome-wide molecular analysis to reveal complex NONO-dependent regulation of gene expression. NONO forms RNA- and DNA-tethered condensates throughout the nucleus and undergoes phase separation in vitro, modulated by nucleic acid binding. CLIP analyses show that NONO mainly binds to the 5' end of pre-mRNAs and modulates pre-mRNA processing, dependent on its RNA-binding activity. NONO regulates super-enhancer-associated genes, including HAND2 and GATA2. Abrogating NONO RNA binding, or phase separation activity, results in decreased expression of HAND2 and GATA2. Thus, future development of agents that target RNA-binding activity of NONO may have therapeutic potential in this cancer context.

The Cancer Microenvironment: Mechanical Challenges of the Metastatic Cascade.

The metastatic cascade presents a significant challenge to patient survival in the fight against cancer. As metastatic cells disseminate and colonize a secondary site, stepwise exposure to microenvironment-specific mechanical stimuli influences and protects successful metastasis. Following cancerous transformation and associated cell recruitment, the tumor microenvironment (TME) becomes a mechanically complex niche, owing to changes in extracellular matrix (ECM) stiffness and architecture. The ECM mechanically reprograms the cancer cell phenotype, priming cells for invasion. 2D and 3D hydrogel-based culture platforms approximate these environmental variables and permit investigations into tumor-dependent shifts in malignancy. Following TME modification, malignant cells must invade the local ECM, driven toward blood, and lymph vessels by sensing biochemical and biophysical gradients. Microfluidic chips recreate cancer-modified ECM tracks, empowering studies into modes of confined motility. Intravasation and extravasation consist of complex cancer-endothelial interactions that modify an otherwise submicron-scale migration. Perfused microfluidic platforms facilitate the physiological culture of endothelial cells and thus enhance the translatability of basic research into metastatic transendothelial migration. These platforms also shed light on the poorly understood circulating tumor cell, which defies adherent cell norms by surviving the shear stress of blood flow and avoiding anoikis. Metastatic cancers possess the plasticity to adapt to new mechanical conditions, permitting their invasiveness, and ensuring their survival against anomalous stimuli. Here, we review the cellular mechanics of metastasis in the context of current in vitro approaches. Advances that further expose the mechanisms underpinning the phenotypic fluidity of metastatic cancers remain central to the development of novel interventions targeting cancer.