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Brás MM, Cruz TB, Maia AF, Oliveira MJ, Sousa SR, Granja PL, Radmacher M. Mechanical Properties of Colorectal Cancer Cells Determined by Dynamic Atomic Force Microscopy: A Novel Biomarker. Cancers (Basel) 2022; 14:cancers14205053. [PMID: 36291838 PMCID: PMC9600571 DOI: 10.3390/cancers14205053] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2022] [Revised: 10/11/2022] [Accepted: 10/12/2022] [Indexed: 11/17/2022] Open
Abstract
Simple Summary Colorectal cancer (CRC) is presently the third-most abundant and the second-most lethal cancer worldwide. Thus, there is a real and urgent need to investigate the processes behind the appearance, development, and proliferation of CRC cells. Several biochemical pathways have been investigated to understand their role in oncogene activation and tumor-suppressor gene inhibition. Despite the research increase in biochemistry, there is still a need to better understand the biophysical cues that drive the activation of signaling pathways relevant to mechanotransduction and cell transformation. The elucidation of these biological processes may help to hinder oncogenic mechanisms and to find biomarkers that could be used to design more personalized therapeutic strategies. Abstract Colorectal cancer (CRC) has been addressed in the framework of molecular, cellular biology, and biochemical traits. A new approach to studying CRC is focused on the relationship between biochemical pathways and biophysical cues, which may contribute to disease understanding and therapy development. Herein, we investigated the mechanical properties of CRC cells, namely, HCT116, HCT15, and SW620, using static and dynamic methodologies by atomic force microscopy (AFM). The static method quantifies Young’s modulus; the dynamic method allows the determination of elasticity, viscosity, and fluidity. AFM results were correlated with confocal laser scanning microscopy and cell migration assay data. The SW620 metastatic cells presented the highest Young’s and storage moduli, with a defined cortical actin ring with distributed F-actin filaments, scarce vinculin expression, abundant total focal adhesions (FAK), and no filopodia formation, which could explain the lessened migratory behavior. In contrast, HCT15 cells presented lower Young’s and storage moduli, high cortical tubulin, less cortical F-actin and less FAK, and more filopodia formation, probably explaining the higher migratory behavior. HCT116 cells presented Young’s and storage moduli values in between the other cell lines, high cortical F-actin expression, intermediate levels of total FAK, and abundant filopodia formation, possibly explaining the highest migratory behavior.
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Affiliation(s)
- M. Manuela Brás
- Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, 4200-135 Porto, Portugal
- Instituto de Engenharia Biomédica (INEB), Universidade do Porto, 4200-135 Porto, Portugal
- Faculdade de Engenharia da Universidade do Porto, 4200-465 Porto, Portugal
| | - Tânia B. Cruz
- Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, 4200-135 Porto, Portugal
- Instituto de Engenharia Biomédica (INEB), Universidade do Porto, 4200-135 Porto, Portugal
| | - André F. Maia
- Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, 4200-135 Porto, Portugal
- Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, 4200-135 Porto, Portugal
| | - Maria José Oliveira
- Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, 4200-135 Porto, Portugal
- Instituto de Engenharia Biomédica (INEB), Universidade do Porto, 4200-135 Porto, Portugal
- Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, 4050-313 Porto, Portugal
| | - Susana R. Sousa
- Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, 4200-135 Porto, Portugal
- Instituto de Engenharia Biomédica (INEB), Universidade do Porto, 4200-135 Porto, Portugal
- Instituto Superior de Engenharia do Porto (ISEP), Instituto Politécnico do Porto, 4200-072 Porto, Portugal
| | - Pedro L. Granja
- Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, 4200-135 Porto, Portugal
- Instituto de Engenharia Biomédica (INEB), Universidade do Porto, 4200-135 Porto, Portugal
| | - Manfred Radmacher
- Institute of Biophysics, University of Bremen, 28334 Bremen, Germany
- Correspondence:
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Brás MM, Sousa SR, Carneiro F, Radmacher M, Granja PL. Mechanobiology of Colorectal Cancer. Cancers (Basel) 2022; 14:1945. [PMID: 35454852 PMCID: PMC9028036 DOI: 10.3390/cancers14081945] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2022] [Revised: 04/06/2022] [Accepted: 04/07/2022] [Indexed: 11/16/2022] Open
Abstract
In this review, the mechanobiology of colorectal cancer (CRC) are discussed. Mechanotransduction of CRC is addressed considering the relationship of several biophysical cues and biochemical pathways. Mechanobiology is focused on considering how it may influence epithelial cells in terms of motility, morphometric changes, intravasation, circulation, extravasation, and metastization in CRC development. The roles of the tumor microenvironment, ECM, and stroma are also discussed, taking into account the influence of alterations and surface modifications on mechanical properties and their impact on epithelial cells and CRC progression. The role of cancer-associated fibroblasts and the impact of flow shear stress is addressed in terms of how it affects CRC metastization. Finally, some insights concerning how the knowledge of biophysical mechanisms may contribute to the development of new therapeutic strategies and targeting molecules and how mechanical changes of the microenvironment play a role in CRC disease are presented.
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Affiliation(s)
- Maria Manuela Brás
- Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, 4200-135 Porto, Portugal; (M.M.B.); (S.R.S.); (F.C.); (P.L.G.)
- Instituto de Engenharia Biomédica (INEB), Universidade do Porto, 4200-135 Porto, Portugal
- Faculdade de Engenharia da Universidade do Porto (FEUP), 4200-465 Porto, Portugal
| | - Susana R. Sousa
- Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, 4200-135 Porto, Portugal; (M.M.B.); (S.R.S.); (F.C.); (P.L.G.)
- Instituto de Engenharia Biomédica (INEB), Universidade do Porto, 4200-135 Porto, Portugal
- Instituto Superior de Engenharia do Porto (ISEP), Instituto Politécnico do Porto (IPP), 4200-072 Porto, Portugal
| | - Fátima Carneiro
- Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, 4200-135 Porto, Portugal; (M.M.B.); (S.R.S.); (F.C.); (P.L.G.)
- Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), 4200-465 Porto, Portugal
- Serviço de Patologia, Centro Hospitalar Universitário de São João (CHUSJ), 4200-319 Porto, Portugal
- Faculdade de Medicina da Universidade do Porto (FMUP), 4200-319 Porto, Portugal
| | - Manfred Radmacher
- Institute for Biophysics, University of Bremen, 28334 Bremen, Germany
| | - Pedro L. Granja
- Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, 4200-135 Porto, Portugal; (M.M.B.); (S.R.S.); (F.C.); (P.L.G.)
- Instituto de Engenharia Biomédica (INEB), Universidade do Porto, 4200-135 Porto, Portugal
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Daulagala AC, Yost J, Yeganegi A, Richardson WJ, Yost MJ, Kourtidis A. A Simple Method to Test Mechanical Strain on Epithelial Cell Monolayers Using a 3D-Printed Stretcher. Methods Mol Biol 2020; 2367:235-247. [PMID: 32789778 DOI: 10.1007/7651_2020_314] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
With the realization that mechanical forces mediate many biological processes and contribute to disease progression, researchers are focusing on developing new methods to understand the role of mechanotransduction in biological systems. Despite recent advances in stretching devices that analyze the effects of mechanical strain in vitro, there are still possibilities to develop new equipment. For example, many of these devices tend be expensive, whereas few have been designed to assess the effects of mechanical strain driven by the extracellular matrix (ECM) to epithelial cell monolayers and to cell-cell adhesion. In this chapter, we introduce a cost-efficient, user-friendly, 3D-printed stretching device that can be used to test the effects of mechanical strain on cultured epithelial cells. Evaluation of the device using speckle-tracking shows homogeneous strain distribution along the horizontal plane of membranes at 2.5% and 5% strains, supporting the reliability of the device. Since cell-cell junctions are mechanosensitive protein complexes, we hereby used this device to examine effects on cell-cell adhesion. For this, we used colon epithelial Caco2 cell monolayers that well-differentiate in culture and form mature adherens junctions. Subjecting Caco2 cells to 2.5% and 5% strain using our device resulted in significant reduction in the localization of the core adherens junction component E-cadherin at areas of cell-cell contact and its increased translocation to the cytoplasm, which in agreement with other methodologies showing that increased ECM-driven strain negatively affects cell-cell adhesion. In summary, we here present a new, cost-effective, homemade device that can be reliably used to examine effects of mechanical strain on epithelial cell monolayers and cell-cell adhesion, in vitro.
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Affiliation(s)
- Amanda C Daulagala
- Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC, USA
| | - John Yost
- Department of Surgery, Medical University of South Carolina, Charleston, SC, USA
| | | | | | - Michael J Yost
- Department of Surgery, Medical University of South Carolina, Charleston, SC, USA
| | - Antonis Kourtidis
- Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC, USA.
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Daulagala AC, Bridges MC, Kourtidis A. E-cadherin Beyond Structure: A Signaling Hub in Colon Homeostasis and Disease. Int J Mol Sci 2019; 20:E2756. [PMID: 31195621 PMCID: PMC6600153 DOI: 10.3390/ijms20112756] [Citation(s) in RCA: 72] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2019] [Revised: 05/27/2019] [Accepted: 06/01/2019] [Indexed: 12/17/2022] Open
Abstract
E-cadherin is the core component of epithelial adherens junctions, essential for tissue development, differentiation, and maintenance. It is also fundamental for tissue barrier formation, a critical function of epithelial tissues. The colon or large intestine is lined by an epithelial monolayer that encompasses an E-cadherin-dependent barrier, critical for the homeostasis of the organ. Compromised barriers of the colonic epithelium lead to inflammation, fibrosis, and are commonly observed in colorectal cancer. In addition to its architectural role, E-cadherin is also considered a tumor suppressor in the colon, primarily a result of its opposing function to Wnt signaling, the predominant driver of colon tumorigenesis. Beyond these well-established traditional roles, several studies have portrayed an evolving role of E-cadherin as a signaling epicenter that regulates cell behavior in response to intra- and extra-cellular cues. Intriguingly, these recent findings also reveal tumor-promoting functions of E-cadherin in colon tumorigenesis and new interacting partners, opening future avenues of investigation. In this Review, we focus on these emerging aspects of E-cadherin signaling, and we discuss their implications in colon biology and disease.
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Affiliation(s)
- Amanda C Daulagala
- Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425, USA.
| | - Mary Catherine Bridges
- Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425, USA.
| | - Antonis Kourtidis
- Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425, USA.
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Stewart DC, Berrie D, Li J, Liu X, Rickerson C, Mkoji D, Iqbal A, Tan S, Doty AL, Glover SC, Simmons CS. Quantitative assessment of intestinal stiffness and associations with fibrosis in human inflammatory bowel disease. PLoS One 2018; 13:e0200377. [PMID: 29995938 PMCID: PMC6040714 DOI: 10.1371/journal.pone.0200377] [Citation(s) in RCA: 51] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2018] [Accepted: 06/25/2018] [Indexed: 01/05/2023] Open
Abstract
Inflammatory bowel disease (IBD) continues to increase in prevalence in industrialized countries. Major complications of IBD include formation of fibrotic strictures, fistulas, reduced absorptive function, cancer risk, and the need for surgery. In other chronic gastrointestinal disease models, stiffness has been shown to precede fibrosis; therefore, stiffness may be a reasonable indicator of progression toward stricture formation in IBD patients. Herein, we seek to quantify tissue stiffness and characterize fibrosis in patients with IBD and to compare mechanical properties of unaffected human tissue to common animal species used for IBD studies. Inflamed and unaffected tissue from IBD patients and unaffected tissue from mice, pigs, and cows were indented using a custom device to determine the effective stiffness. Histology was performed on matched tissues, and total RNA was isolated from IBD tissue samples and used for gene expression analysis of pro-fibrotic genes. We observed an increase in the effective stiffness (steady-state modulus, SSM) (p < 0.0001) and increased expression of the collagen type I gene (COL1A1, p = 0.01) in inflamed tissue compared to unaffected areas in our IBD patient cohort. We also found that increased staining of collagen fibers in submucosa positively correlated with SSM (p = 0.093). We determined that unaffected animal bowel stiffness is significantly greater than similar human tissues, suggesting additional limitations on animal models for translational investigations regarding stiffness-related hypotheses. Taken together, our data support development of tools for evaluation of bowel stiffness in IBD patients for prognostic applications that may enable more accurate prediction of those who will develop fibrosis and more precise prescription of aggressive therapies.
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Affiliation(s)
- Daniel C. Stewart
- J. Crayton Pruitt Family Department of Biomedical Engineering, Herbert Wertheim College of Engineering, University of Florida, Gainesville, FL, United States of America
| | - Dalton Berrie
- J. Crayton Pruitt Family Department of Biomedical Engineering, Herbert Wertheim College of Engineering, University of Florida, Gainesville, FL, United States of America
- Division of Gastroenterology, Hepatology and Nutrition, College of Medicine, University of Florida, Gainesville, FL, United States of America
| | - Jian Li
- Division of Gastroenterology, Hepatology and Nutrition, College of Medicine, University of Florida, Gainesville, FL, United States of America
| | - Xinyue Liu
- Department of Pharmaceutical Outcomes and Policy, College of Pharmacy, University of Florida, Gainesville, FL, United States of America
| | - Cooper Rickerson
- J. Crayton Pruitt Family Department of Biomedical Engineering, Herbert Wertheim College of Engineering, University of Florida, Gainesville, FL, United States of America
| | - David Mkoji
- Department of Mechanical and Aerospace Engineering, Herbert Wertheim College of Engineering, University of Florida, Gainesville, FL, United States of America
| | - Atif Iqbal
- Department of Surgery, College of Medicine, University of Florida, Gainesville, FL, United States of America
| | - Sanda Tan
- Department of Surgery, College of Medicine, University of Florida, Gainesville, FL, United States of America
| | - Andria L. Doty
- Division of Gastroenterology, Hepatology and Nutrition, College of Medicine, University of Florida, Gainesville, FL, United States of America
| | - Sarah C. Glover
- Division of Gastroenterology, Hepatology and Nutrition, College of Medicine, University of Florida, Gainesville, FL, United States of America
| | - Chelsey S. Simmons
- J. Crayton Pruitt Family Department of Biomedical Engineering, Herbert Wertheim College of Engineering, University of Florida, Gainesville, FL, United States of America
- Department of Mechanical and Aerospace Engineering, Herbert Wertheim College of Engineering, University of Florida, Gainesville, FL, United States of America
- Division of Cardiovascular Medicine, College of Medicine, University of Florida, Gainesville, FL, United States of America
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Ciasca G, Papi M, Minelli E, Palmieri V, De Spirito M. Changes in cellular mechanical properties during onset or progression of colorectal cancer. World J Gastroenterol 2016; 22:7203-7214. [PMID: 27621568 PMCID: PMC4997642 DOI: 10.3748/wjg.v22.i32.7203] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/13/2016] [Revised: 07/11/2016] [Accepted: 08/01/2016] [Indexed: 02/06/2023] Open
Abstract
Colorectal cancer (CRC) development represents a multistep process starting with specific mutations that affect proto-oncogenes and tumour suppressor genes. These mutations confer a selective growth advantage to colonic epithelial cells that form first dysplastic crypts, and then malignant tumours and metastases. All these steps are accompanied by deep mechanical changes at the cellular and the tissue level. A growing consensus is emerging that such modifications are not merely a by-product of the malignant progression, but they could play a relevant role in the cancer onset and accelerate its progression. In this review, we focus on recent studies investigating the role of the biomechanical signals in the initiation and the development of CRC. We show that mechanical cues might contribute to early phases of the tumour initiation by controlling the Wnt pathway, one of most important regulators of cell proliferation in various systems. We highlight how physical stimuli may be involved in the differentiation of non-invasive cells into metastatic variants and how metastatic cells modify their mechanical properties, both stiffness and adhesion, to survive the mechanical stress associated with intravasation, circulation and extravasation. A deep comprehension of these mechanical modifications may help scientist to define novel molecular targets for the cure of CRC.
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Abstract
A variety of cell types exhibit phenotype changes in response to the mechanical stiffness of the substrate. Many cells excluding neurons display an increase in the spread area, actin stress fiber formation and larger focal adhesion complexes as substrate stiffness increases in a sparsely populated culture. Cell proliferation is also known to directly correlate with these phenotype changes/changes in substrate stiffness. Augmented spreading and proliferation on stiffer substrates require nuclear transcriptional regulator YAP (Yes associated protein) localization in the cell nucleus and is tightly coupled to larger traction force generation. In this study, we show that different types of fibroblasts can exhibit spread morphology, well defined actin stress fibers, and larger focal adhesions even on very soft collagen gels (modulus in hundreds of Pascals) as if they are on hard glass substrates (modulus in GPa, several orders of magnitude higher). Strikingly, we show, for the first time, that augmented spreading and other hard substrate cytoskeleton architectures on soft collagen gels are not correlated with the cell proliferation pattern and do not require YAP localization in the cell nucleus. Finally, we examine the response of human colon carcinoma (HCT-8) cells on soft collagen gels. Recent studies show that human colon carcinoma (HCT-8) cells form multicellular clusters by 2-3 days when cultured on soft polyacrylamide (PA) gels with a wide range of stiffness (0.5-50 kPa) and coated with an extracellular matrix, ECM (collagen monomer/fibronectin). These clusters show limited spreading/wetting on PA gels, form 3D structures at the edges, and eventually display a remarkable, dissociative metastasis like phenotype (MLP), i.e., epithelial to rounded morphological transition after a week of culture on PA gels only, but not on collagen monomer coated stiff polystyrene/glass where they exhibit enhanced wetting and form confluent monolayers. Here, we show that HCT-8 cell clusters also show augmented spreading/wetting on soft collagen gels and eventually form confluent monolayers as on rigid glass substrates and MLP is completely inhibited on soft collagen gels. Overall, these results suggest that cell-material interactions (soft collagen gels in this case) can induce cellular phenotype and cytoskeleton organization in a remarkably distinct manner compared to a classical synthetic polyacrylamide (PA) hydrogel cell culture model and may contribute in designing new functional biomaterials.
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Affiliation(s)
- M Yakut Ali
- Department of Mechanical Science and Engineering, College of Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA61801.
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