Tension directs cancer cell migration over fiber alignment through energy minimization.

Cell migration during many fundamental biological processes including metastasis requires cells to traverse tissue with heterogeneous mechanical cues that direct migration as well as determine force and energy requirements for motility. However, the influence of discrete structural and mechanical cues on migration remains challenging to determine as they are often coupled. Here, we decouple the pro-invasive cues of collagen fiber alignment and tension to study their individual impact on migration. When presented with both cues, cells preferentially travel in the axis of tension against fiber alignment. Computational and experimental data show applying tension perpendicular to alignment increases potential energy stored within collagen fibers, lowering requirements for cell-induced matrix deformation and energy usage during migration compared to motility in the direction of fiber alignment. Energy minimization directs migration trajectory, and tension can facilitate migration against fiber alignment. These findings provide a conceptual understanding of bioenergetics during migration through a fibrous matrix.

Obesogenic High-Fat Diet and MYC Cooperate to Promote Lactate Accumulation and Tumor Microenvironment Remodeling in Prostate Cancer.

Cancer cells exhibit metabolic plasticity to meet oncogene-driven dependencies while coping with nutrient availability. A better understanding of how systemic metabolism impacts the accumulation of metabolites that reprogram the tumor microenvironment (TME) and drive cancer could facilitate development of precision nutrition approaches. Using the Hi-MYC prostate cancer mouse model, we demonstrated that an obesogenic high-fat diet (HFD) rich in saturated fats accelerates the development of c-MYC-driven invasive prostate cancer through metabolic rewiring. Although c-MYC modulated key metabolic pathways, interaction with an obesogenic HFD was necessary to induce glycolysis and lactate accumulation in tumors. These metabolic changes were associated with augmented infiltration of CD206+ and PD-L1+ tumor-associated macrophages (TAM) and FOXP3+ regulatory T cells, as well as with the activation of transcriptional programs linked to disease progression and therapy resistance. Lactate itself also stimulated neoangiogenesis and prostate cancer cell migration, which were significantly reduced following treatment with the lactate dehydrogenase inhibitor FX11. In patients with prostate cancer, high saturated fat intake and increased body mass index were associated with tumor glycolytic features that promote the infiltration of M2-like TAMs. Finally, upregulation of lactate dehydrogenase, indicative of a lactagenic phenotype, was associated with a shorter time to biochemical recurrence in independent clinical cohorts. This work identifies cooperation between genetic drivers and systemic metabolism to hijack the TME and promote prostate cancer progression through oncometabolite accumulation. This sets the stage for the assessment of lactate as a prognostic biomarker and supports strategies of dietary intervention and direct lactagenesis blockade in treating advanced prostate cancer. SIGNIFICANCE: Lactate accumulation driven by high-fat diet and MYC reprograms the tumor microenvironment and promotes prostate cancer progression, supporting the potential of lactate as a biomarker and therapeutic target in prostate cancer. See related commentary by Frigo, p. 1742.

Using Machine Learning to Predict TP53 Mutation Status and Aggressiveness of Prostate Cancer from Routine Histology Images.

Despite years of progress, we still lack reliable tools to predict the aggressiveness of tumors, including in the case of prostate cancer. Biomarkers have been developed, but they often suffer from poor accuracy if used alone due to tumor heterogeneity. Nevertheless, some mutations, notably TP53 mutations, are highly correlated with progression. In their work in this issue of Cancer Research, Pizurica and colleagues implemented a machine learning-based model applied to routine histology and trained with prior information on TP53 mutation status. Their model output provides a quantitative prediction of TP53 mutation status while having a strong correlation with aggressiveness, showing promise as a prognostic in silico biomarker. See related article by Pizurica et al., p. 2970.

Matrix stiffness regulates tumor cell intravasation through expression and ESRP1-mediated alternative splicing of MENA.

During intravasation, cancer cells cross the endothelial barrier and enter the circulation. Extracellular matrix stiffening has been correlated with tumor metastatic potential; however, little is known about the effects of matrix stiffness on intravasation. Here, we utilize in vitro systems, a mouse model, specimens from patients with breast cancer, and RNA expression profiles from The Cancer Genome Atlas Program (TCGA) to investigate the molecular mechanism by which matrix stiffening promotes tumor cell intravasation. Our data show that heightened matrix stiffness increases MENA expression, which promotes contractility and intravasation through focal adhesion kinase activity. Further, matrix stiffening decreases epithelial splicing regulatory protein 1 (ESRP1) expression, which triggers alternative splicing of MENA, decreases the expression of MENA11a, and enhances contractility and intravasation. Altogether, our data indicate that matrix stiffness regulates tumor cell intravasation through enhanced expression and ESRP1-mediated alternative splicing of MENA, providing a mechanism by which matrix stiffness regulates tumor cell intravasation.

Coordination of non-professional efferocytosis and actomyosin contractility during epithelial tissue morphogenesis.

In developing embryos, specific cell populations are often removed to remodel tissue architecture for organogenesis. During urinary tract development, an epithelial duct called the common nephric duct (CND) gets shortened and eventually eliminated to remodel the entry point of the ureter into the bladder. Here we show that non-professional efferocytosis (the process in which epithelial cells engulf apoptotic bodies) is the main mechanism that contributes to CND shortening. Combining biological metrics and computational modeling, we show that efferocytosis with actomyosin contractility are essential factors that drive the CND shortening without compromising the ureter-bladder structural connection. The disruption of either apoptosis, non-professional efferocytosis, or actomyosin results in contractile tension reduction and deficient CND shortening. Actomyosin activity helps to maintain tissue architecture while non-professional efferocytosis removes cellular volume. Together our results demonstrate that non-professional efferocytosis with actomyosin contractility are important morphogenetic factors controlling CND morphogenesis.

Matrix stiffness enhances cancer-macrophage interactions and M2-like macrophage accumulation in the breast tumor microenvironment.

The role of intratumor heterogeneity is becoming increasingly apparent in part due to expansion in single cell technologies. Clinically, tumor heterogeneity poses several obstacles to effective cancer therapy dealing with biomarker variability and treatment responses. Matrix stiffening is known to occur during tumor progression and contribute to pathogenesis in several cancer hallmarks, including tumor angiogenesis and metastasis. However, the effects of matrix stiffening on intratumor heterogeneity have not been thoroughly studied. In this study, we applied single-cell RNA sequencing to investigate the differences in the transcriptional landscapes between stiff and compliant MMTV-PyMT mouse mammary tumors. We found similar compositions of cancer and stromal subpopulations in compliant and stiff tumors but differential intercellular communication and a significantly higher concentration of tumor-promoting, M2-like macrophages in the stiffer tumor microenvironments. Interestingly, we found that cancer cells seeded on stiffer substrates recruited more macrophages. Furthermore, elevated matrix stiffness increased Colony Stimulating Factor 1 (CSF-1) expression in breast cancer cells and reduction of CSF-1 expression on stiffer substrates reduced macrophage recruitment. Thus, our results demonstrate that tissue phenotypes were conserved between stiff and compliant tumors but matrix stiffening altered cell-cell interactions which may be responsible for shifting the phenotypic balance of macrophages residing in the tumor microenvironment towards a pro-tumor progression M2 phenotype. STATEMENT OF SIGNIFICANCE: Cells within tumors are highly heterogeneous, posing challenges with treatment and recurrence. While increased tissue stiffness can promote several hallmarks of cancer, its effects on tumor heterogeneity are unclear. We used single-cell RNA sequencing to investigate the differences in the transcriptional landscapes between stiff and compliant MMTV-PyMT mouse mammary tumors. We found similar compositions of cancer and stromal subpopulations in compliant and stiff tumors but differential intercellular communication and a significantly higher concentration of tumor-promoting, M2-like macrophages in the stiffer tumor microenvironments. Using a biomaterial-based platform, we found that cancer cells seeded on stiffer substrates recruited more macrophages, supporting our in vivo findings. Together, our results demonstrate a key role of matrix stiffness in affecting cell-cell communication and macrophage recruitment.

Weakly migratory metastatic breast cancer cells activate fibroblasts via microvesicle-Tg2 to facilitate dissemination and metastasis.

Cancer cell migration is highly heterogeneous, and the migratory capability of cancer cells is thought to be an indicator of metastatic potential. It is becoming clear that a cancer cell does not have to be inherently migratory to metastasize, with weakly migratory cancer cells often found to be highly metastatic. However, the mechanism through which weakly migratory cells escape from the primary tumor remains unclear. Here, utilizing phenotypically sorted highly and weakly migratory human breast cancer cells, we demonstrate that weakly migratory metastatic cells disseminate from the primary tumor via communication with stromal cells. While highly migratory cells are capable of single cell migration, weakly migratory cells rely on cell-cell signaling with fibroblasts to escape the primary tumor. Weakly migratory cells release microvesicles rich in tissue transglutaminase 2 (Tg2) which activate murine fibroblasts and lead weakly migratory cancer cell migration in vitro. These microvesicles also induce tumor stiffening and fibroblast activation in vivo and enhance the metastasis of weakly migratory cells. Our results identify microvesicles and Tg2 as potential therapeutic targets for metastasis and reveal a novel aspect of the metastatic cascade in which weakly migratory cells release microvesicles which activate fibroblasts to enhance cancer cell dissemination.

Extracellular Vesicles from Ocular Melanoma Have Pro-Fibrotic and Pro-Angiogenic Properties on the Tumor Microenvironment.

Uveal melanoma (UM) is the most common primary intraocular tumor and often spreads to the liver. Intercellular communication though extracellular vesicles (EVs) plays an important role in several oncogenic processes, including metastasis, therapeutic resistance, and immune escape. This study examines how EVs released by UM cells modify stellate and endothelial cells in the tumor microenvironment. The surface markers, and the concentration and size of EVs derived from UM cells or choroidal melanocytes were characterized by high-resolution flow cytometry, electron microscopy, and Western blotting. The selective biodistribution of EVs was studied in mice by fluorescence imaging. The activation/contractility of stellate cells and the tubular organization of endothelial cells after exposure to melanomic EVs were determined by traction force microscopy, collagen gel contraction, or endothelial tube formation assays. We showed that large EVs from UM cells and healthy melanocytes are heterogenous in size, as well as their expression of phosphatidylserine, tetraspanins, and Tsg101. Melanomic EVs mainly accumulated in the liver and lungs of mice. Hepatic stellate cells with internalized melanomic EVs had increased contractility, whereas EV-treated endothelial cells developed more capillary-like networks. Our study demonstrates that the transfer of EVs from UM cells leads to a pro-fibrotic and pro-angiogenic phenotype in hepatic stellate and endothelial cells.

Cancer-Associated Fibroblasts in a 3D Engineered Tissue Model Induce Tumor-like Matrix Stiffening and EMT Transition.

A tumor microenvironment is characterized by its altered mechanical properties. However, most models remain unable to faithfully recreate the mechanical properties of a tumor. Engineered models based on the self-assembly method have the potential to better recapitulate the stroma architecture and composition. Here, we used the self-assembly method based on a bladder tissue model to engineer a tumor-like environment. The tissue-engineered tumor models were reconstituted from stroma-derived healthy primary fibroblasts (HFs) induced into cancer-associated fibroblast cells (iCAFs) along with an urothelium overlay. The iCAFs-derived extracellular matrix (ECM) composition was found to be stiffer, with increased ECM deposition and remodeling. The urothelial cells overlaid on the iCAFs-derived ECM were more contractile, as measured by quantitative polarization microscopy, and displayed increased YAP nuclear translocation. We further showed that the proliferation and expression of epithelial-to-mesenchymal transition (EMT) marker in the urothelial cells correlate with the increased stiffness of the iCAFs-derived ECM. Our data showed an increased expression of EMT markers within the urothelium on the iCAFs-derived ECM. Together, our results demonstrate that our tissue-engineered tumor model can achieve stiffness levels comparable to that of a bladder tumor, while triggering a tumor-like response from the urothelium.

The Extracellular Matrix Stiffening: A Trigger of Prostate Cancer Progression and Castration Resistance?

Despite advancements made in diagnosis and treatment, prostate cancer remains the second most diagnosed cancer among men worldwide in 2020, and the first in North America and Europe. Patients with localized disease usually respond well to first-line treatments, however, up to 30% develop castration-resistant prostate cancer (CRPC), which is often metastatic, making this stage of the disease incurable and ultimately fatal. Over the last years, interest has grown into the extracellular matrix (ECM) stiffening as an important mediator of diseases, including cancers. While this process is increasingly well-characterized in breast cancer, a similar in-depth look at ECM stiffening remains lacking for prostate cancer. In this review, we scrutinize the current state of literature regarding ECM stiffening in prostate cancer and its potential association with disease progression and castration resistance.

Phenotypic Heterogeneity and Metastasis of Breast Cancer Cells.

Although intratumoral genomic heterogeneity can impede cancer research and treatment, less is known about the effects of phenotypic heterogeneities. To investigate the role of cell migration heterogeneities in metastasis, we phenotypically sorted metastatic breast cancer cells into two subpopulations based on migration ability. Although migration is typically considered to be associated with metastasis, when injected orthotopically in vivo, the weakly migratory subpopulation metastasized significantly more than the highly migratory subpopulation. To investigate the mechanism behind this observation, both subpopulations were assessed at each stage of the metastatic cascade, including dissemination from the primary tumor, survival in the circulation, extravasation, and colonization. Although both subpopulations performed each step successfully, weakly migratory cells presented as circulating tumor cell (CTC) clusters in the circulation, suggesting clustering as one potential mechanism behind the increased metastasis of weakly migratory cells. RNA sequencing revealed weakly migratory subpopulations to be more epithelial and highly migratory subpopulations to be more mesenchymal. Depletion of E-cadherin expression from weakly migratory cells abrogated metastasis. Conversely, induction of E-cadherin expression in highly migratory cells increased metastasis. Clinical patient data and blood samples showed that CTC clustering and E-cadherin expression are both associated with worsened patient outcome. This study demonstrates that deconvolving phenotypic heterogeneities can reveal fundamental insights into metastatic progression. More specifically, these results indicate that migratory ability does not necessarily correlate with metastatic potential and that E-cadherin promotes metastasis in phenotypically sorted breast cancer cell subpopulations by enabling CTC clustering. SIGNIFICANCE: This study employs phenotypic cell sorting for migration to reveal a weakly migratory, highly metastatic breast cancer cell subpopulation regulated by E-cadherin, highlighting the dichotomy between cancer cell migration and metastasis.

High type I collagen density fails to increase breast cancer stem cell phenotype.

Breast cancer is a highly frequent and lethal malignancy which metastasis and relapse frequently associates with the existence of breast cancer stem cells (CSCs). CSCs are undifferentiated, aggressive and highly resistant to therapy, with traits modulated by microenvironmental cells and the extracellular matrix (ECM), a biologically complex and dynamic structure composed mainly by type I collagen (Col-I). Col-I enrichment in the tumor-associated ECM leads to microenvironment stiffness and higher tumor aggressiveness and metastatic potential. While Col-I is also known to induce tumor stemness, it is unknown if such effect is dependent of Col-I density. To answer this question, we evaluated the stemness phenotype of MDA-MB-231 and MCF-7 human breast cancer cells cultured within gels of varying Col-I densities. High Col-I density increased CD44+CD24- breast cancer stem cell (BCSC) immunophenotype but failed to potentiate Col-I fiber alignment, cell self-renewal and clonogenicity in MDA-MB-231 cells. In MCF-7 cells, high Col-I density decreased total levels of variant CD44 (CD44v). Common to both cell types, high Col-I density induced neither markers related to CSC nor those related with mechanically-induced cell response. We conclude that high Col-I density per se is not sufficient to fully develop the BCSC phenotype.

Adenoviral protein E4orf4 interacts with the polarity protein Par3 to induce nuclear rupture and tumor cell death.

The tumor cell-selective killing activity of the adenovirus type 2 early region 4 ORF4 (E4orf4) protein is poorly defined at the molecular level. Here, we show that the tumoricidal effect of E4orf4 is typified by changes in nuclear dynamics that depend on its interaction with the polarity protein Par3 and actomyosin contractility. Mechanistically, E4orf4 induced a high incidence of nuclear bleb formation and repetitive nuclear ruptures, which promoted nuclear efflux of E4orf4 and loss of nuclear integrity. This process was regulated by nucleocytoskeletal connections, Par3 clustering proximal to nuclear lamina folds, and retrograde movement of actin bundles that correlated with nuclear ruptures. Significantly, Par3 also regulated the incidence of spontaneous nuclear ruptures facilitated by the downmodulation of lamins. This work uncovered a novel role for Par3 in controlling the actin-dependent forces acting on the nuclear envelope to remodel nuclear shape, which might be a defining feature of tumor cells that is harnessed by E4orf4.

Tissue transglutaminase 2 regulates tumor cell tensional homeostasis by increasing contractility.

Abnormal tensional cellular homeostasis is now considered a hallmark of cancer. Despite this, the origin of this abnormality remains unclear. In this work, we investigated the role of tissue transglutaminase 2 (TG2, also known as TGM2), a protein associated with poor prognosis and increased metastatic potential, and its relationship to the EGF receptor in the regulation of the mechanical state of tumor cells. Remarkably, we observed a TG2-mediated modulation of focal adhesion composition as well as stiffness-induced FAK activation, which was linked with a distinctive increase in cell contractility, in experiments using both pharmacological and shRNA-based approaches. Additionally, the increased contractility could be reproduced in non-malignant cells upon TG2 expression. Moreover, the increased cell contractility mediated by TG2 was largely due to the loss of EGFR-mediated inhibition of cell contractility. These findings establish intracellular TG2 as a regulator of cellular tensional homeostasis and suggest the existence of signaling switches that control the contribution of growth factor receptors in determining the mechanical state of a cell.

Extent of Cell Confinement in Microtracks Affects Speed and Results in Differential Matrix Strains.

During metastasis, cancer cells navigate through a spatially heterogeneous extracellular matrix (ECM). Physical properties of ECM, including the degree of confinement, influence cell migration behavior. Here, utilizing in vitro three-dimensional collagen microtracks, we demonstrate that cell-ECM interactions, specifically the degree of spatial confinement, regulate migratory behavior. We found that cells migrate faster when they are fully confined, contacting all four walls (top, bottom, and two sides) of a collagen microtrack, compared with cells that are partially confined, contacting less than four walls. When fully confined, cells exhibit fewer but larger vinculin-containing adhesions and create greater strains in the surrounding matrix directed toward the cell body. In contrast, partially confined cells develop a more elongated morphology with smaller but significantly more vinculin-containing adhesions and displace the surrounding matrix less than fully confined cells. The resulting effect of increasing cell contractility via Rho activation is dependent on the number of walls with which the cell is in contact. Although matrix strains increase in both fully and partially confined cells, cells that are partially confined increase speed, whereas those in full confinement decrease speed. Together, these results suggest that the degree of cell-ECM contact during confined migration is a key determinant of speed, morphology, and cell-generated substrate strains during motility, and these factors may work in tandem to facilitate metastatic cell migration.

Energetic costs regulated by cell mechanics and confinement are predictive of migration path during decision-making.

Cell migration during the invasion-metastasis cascade requires cancer cells to navigate a spatially complex microenvironment that presents directional choices to migrating cells. Here, we investigate cellular energetics during migration decision-making in confined spaces. Theoretical and experimental data show that energetic costs for migration through confined spaces are mediated by a balance between cell and matrix compliance as well as the degree of spatial confinement to direct decision-making. Energetic costs, driven by the cellular work needed to generate force for matrix displacement, increase with increasing cell stiffness, matrix stiffness, and degree of spatial confinement, limiting migration. By assessing energetic costs between possible migration paths, we can predict the probability of migration choice. Our findings indicate that motility in confined spaces imposes high energetic demands on migrating cells, and cells migrate in the direction of least confinement to minimize energetic costs. Therefore, therapeutically targeting metabolism may limit cancer cell migration and metastasis.

Synergic Interactions Between Hepatic Stellate Cells and Uveal Melanoma in Metastatic Growth.

Uveal melanoma (UM) is a malignant intraocular tumor that spreads to the liver in half of the cases. Since hepatic cells could play a role in the therapeutic resistance of metastatic UM, the purpose of our study was to investigate the pro-invasive role of hepatic stellate cells (HSteCs) in metastatic UM at the micro- and macro-metastatic stages. We first performed an immunostaining with the alpha-smooth muscle actin (αSMA) to localize activated HSteCs in UM liver macro-metastases from four patients. Their accumulation of collagen was assessed with Masson's Trichrome stain. Next, we inoculated metastatic UM cells alone or with human HSteCs in triple-immunodeficient mice, in order to determine if HSteCs are recruited as early as the micro-metastatic stage. The growth of metastatic foci was imaged in the liver by ex vivo fluorescence imaging. Histological analyses were performed with Masson's Trichrome and Picrosirius Red stains, and antibodies against Melan-A and αSMA. The collagen content was measured in xenografts by quantitative polarization microscopy. In patient hepatectomy samples, activated HSteCs and their pathological matrix were localized surrounding the malignant lesions. In the mouse xenograft model, the number of hepatic metastases was increased when human HSteCs were co-inoculated. Histological analyses revealed a significant recruitment of HSteCs near the micro/macrolesions, and an increase in fibrillar collagen production. Our results show that HSteCs can provide a permissive microenvironment and might increase the therapeutic resistance of metastatic UM.

Coupling Microfluidic Platforms, Microfabrication, and Tissue Engineered Scaffolds to Investigate Tumor Cells Mechanobiology.

The tumor microenvironment (TME) is composed of dynamic and complex networks composed of matrix substrates, extracellular matrix (ECM), non-malignant cells, and tumor cells. The TME is in constant evolution during the disease progression, most notably through gradual stiffening of the stroma. Within the tumor, increased ECM stiffness drives tumor growth and metastatic events. However, classic in vitro strategies to study the TME in cancer lack the complexity to fully replicate the TME. The quest to understand how the mechanical, geometrical, and biochemical environment of cells impacts their behavior and fate has been a major force driving the recent development of new technologies in cell biology research. Despite rapid advances in this field, many challenges remain in order to bridge the gap between the classical culture dish and the biological reality of actual tissue. Microfabrication coupled with microfluidic approaches aim to engineer the actual complexity of the TME. Moreover, TME bioengineering allows artificial modulations with single or multiple cues to study different phenomena occurring in vivo. Some innovative cutting-edge tools and new microfluidic approaches could have an important impact on the fields of biology and medicine by bringing deeper understanding of the TME, cell behavior, and drug effects.

Matrix stiffness regulates microvesicle-induced fibroblast activation.

Extracellular vesicles released by cancer cells have recently been implicated in the differentiation of stromal cells to their activated, cancer-supporting states. Microvesicles, a subset of extracellular vesicles released from the plasma membrane of cancer cells, contain biologically active cargo, including DNA, mRNA, and miRNA, which are transferred to recipient cells and induce a phenotypic change in behavior. While it is known that microvesicles can alter recipient cell phenotype, little is known about how the physical properties of the tumor microenvironment affect fibroblast response to microvesicles. Here, we utilized cancer cell-derived microvesicles and synthetic substrates designed to mimic the stiffness of the tumor and tumor stroma to investigate the effects of microvesicles on fibroblast phenotype as a function of the mechanical properties of the microenvironment. We show that microvesicles released by highly malignant breast cancer cells cause an increase in fibroblast spreading, α-smooth muscle actin expression, proliferation, cell-generated traction force, and collagen gel compaction. Notably, our data indicate that these phenotypic changes occur only on stiff matrices mimicking the stiffness of the tumor periphery and are dependent on the cell type from which the microvesicles are shed. Overall, these results show that the effects of cancer cell-derived microvesicles on fibroblast activation are regulated by the physical properties of the microenvironment, and these data suggest that microvesicles may have a more robust effect on fibroblasts located at the tumor periphery to influence cancer progression.