scholarly journals Mechanophenotyping of 3D Multicellular Clusters using Displacement Arrays of Rendered Tractions

2019 ◽  
Author(s):  
Susan E. Leggett ◽  
Mohak Patel ◽  
Thomas M. Valentin ◽  
Lena Gamboa ◽  
Amanda S. Khoo ◽  
...  
Keyword(s):  
Tumor Invasion ◽  
Wound Repair ◽  
Epithelial Mesenchymal Transition ◽  
Ex Vivo ◽  
Three Dimensional ◽  
Human Patient ◽  
Mesenchymal Transition ◽  
Successive Loss ◽  
Surrounding Extracellular Matrix

ABSTRACTEpithelial tissues mechanically deform the surrounding extracellular matrix during embryonic development, wound repair, and tumor invasion. Ex vivo measurements of such multicellular tractions within three-dimensional (3D) biomaterials could elucidate collective dissemination during disease progression, and enable preclinical testing of targeted anti-migration therapies. However, past 3D traction measurements have been low throughput due to the challenges of imaging and analyzing information-rich 3D material deformations. Here, we demonstrate a method to profile multicellular clusters in a 96-well plate format based on spatially heterogeneous contractile, protrusive, and circumferential tractions. As a case study, we profile multicellular clusters across varying states of the epithelial-mesenchymal transition, revealing a successive loss of protrusive and circumferential tractions, as well as the formation of localized contractile tractions with elongated cluster morphologies. These cluster phenotypes were biochemically perturbed using drugs, biasing towards traction signatures of different epithelial or mesenchymal states. This higher-throughput analysis is promising to systematically interrogate and perturb aberrant mechanobiology, which could be utilized with human patient samples to guide personalized therapies.

scholarly journals Mechanophenotyping of 3D multicellular clusters using displacement arrays of rendered tractions

2020 ◽  
Vol 117 (11) ◽  
pp. 5655-5663 ◽  
Author(s):  
Susan E. Leggett ◽  
Mohak Patel ◽  
Thomas M. Valentin ◽  
Lena Gamboa ◽  
Amanda S. Khoo ◽  
...  
Keyword(s):  
Tumor Invasion ◽  
Wound Repair ◽  
Epithelial Mesenchymal Transition ◽  
Ex Vivo ◽  
Three Dimensional ◽  
Human Patient ◽  
Mesenchymal Transition ◽  
Successive Loss ◽  
Surrounding Extracellular Matrix

Epithelial tissues mechanically deform the surrounding extracellular matrix during embryonic development, wound repair, and tumor invasion. Ex vivo measurements of such multicellular tractions within three-dimensional (3D) biomaterials could elucidate collective dissemination during disease progression and enable preclinical testing of targeted antimigration therapies. However, past 3D traction measurements have been low throughput due to the challenges of imaging and analyzing information-rich 3D material deformations. Here, we demonstrate a method to profile multicellular clusters in a 96-well-plate format based on spatially heterogeneous contractile, protrusive, and circumferential tractions. As a case study, we profile multicellular clusters across varying states of the epithelial–mesenchymal transition, revealing a successive loss of protrusive and circumferential tractions, as well as the formation of localized contractile tractions with elongated cluster morphologies. These cluster phenotypes were biochemically perturbed by using drugs, biasing toward traction signatures of different epithelial or mesenchymal states. This higher-throughput analysis is promising to systematically interrogate and perturb aberrant mechanobiology, which could be utilized with human-patient samples to guide personalized therapies.

scholarly journals Loss of Nitric Oxide Induces Fibrogenic Response in Organotypic 3D Co-Culture of Mammary Epithelia and Fibroblasts—An Indicator for Breast Carcinogenesis

Cancers ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2815
Author(s):  
Gang Ren ◽  
Xunzhen Zheng ◽  
Vandana Sharma ◽  
Joshua Letson ◽  
Andrea L. Nestor-Kalinoski ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Culture System ◽  
Epithelial Mesenchymal Transition ◽  
Precancerous Lesions ◽  
Three Dimensional ◽  
Epidemiological Studies ◽  
Mesenchymal Transition ◽  
In Vitro Response ◽  
Mammary Epithelia

Excessive myofibroblast activation, which leads to dysregulated collagen deposition and the stiffening of the extracellular matrix (ECM), plays pivotal roles in cancer initiation and progression. Cumulative evidence attests to the cancer-causing effects of a number of fibrogenic factors found in the environment, diseases and drugs. While identifying such factors largely depends on epidemiological studies, it would be of great importance to develop a robust in vitro method to demonstrate the causal relationship between fibrosis and cancer. Here, we tested whether our recently developed organotypic three-dimensional (3D) co-culture would be suitable for that purpose. This co-culture system utilizes the discontinuous ECM to separately culture mammary epithelia and fibroblasts in the discrete matrices to model the complexity of the mammary gland. We observed that pharmaceutical deprivation of nitric oxide (NO) in 3D co-cultures induced myofibroblast differentiation of the stroma as well as the occurrence of epithelial–mesenchymal transition (EMT) of the parenchyma. Such in vitro response to NO deprivation was unique to co-cultures and closely mimicked the phenotype of NO-depleted mammary glands exhibiting stromal desmoplasia and precancerous lesions undergoing EMT. These results suggest that this novel 3D co-culture system could be utilized in the deep mechanistic studies of the linkage between fibrosis and cancer.

scholarly journals Integrin β4 as a Potential Diagnostic and Therapeutic Tumor Marker

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1197
Author(s):  
Haoyu Yang ◽  
Zixuan Xu ◽  
Yuqian Peng ◽  
Jiali Wang ◽  
Yang Xiang
Keyword(s):  
Signal Transduction ◽  
Tumor Marker ◽  
Tumor Invasion ◽  
Epithelial Mesenchymal Transition ◽  
Vital Role ◽  
Structure And Function ◽  
Mesenchymal Transition ◽  
Tumor Tissues ◽  
Integrin Β4 ◽  
And Function

Integrin β4 (ITGβ4) is a class of transmembrane adhesion molecules composed of hemidesmosomes (HDs). Its unique long intracellular domain provides intricate signal transduction functions. These signal transduction effects are especially prominent in tumors. Many recent studies have shown that integrin β4 is differentially expressed in various tumors, and it plays a vital role in tumor invasion, proliferation, epithelial–mesenchymal transition, and angiogenesis. Therefore, we categorize the research related to integrin β4, starting from its structure and function in tumor tissues, and provide a basic description. Based on its structure and function, we believe that integrin β4 can be used as a tumor marker. In clinical practice, it is described as a diagnostic marker for the targeted treatment of cancer and will be helpful in the clinical diagnosis and treatment of tumors.

scholarly journals Airway Epithelial Repair by a Prebiotic Mannan Derived from Saccharomyces cerevisiae

2017 ◽  
Vol 2017 ◽  
pp. 1-7 ◽  
Author(s):  
Christie F. Michael ◽  
Christopher M. Waters ◽  
Kim S. LeMessurier ◽  
Amali E. Samarasinghe ◽  
Chi Y. Song ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Wound Repair ◽  
Epithelial Mesenchymal Transition ◽  
Bronchial Epithelial Cells ◽  
Current Therapy ◽  
Airway Epithelial ◽  
Coated Pits ◽  
Bronchial Epithelial ◽  
Mesenchymal Transition ◽  
Mannose Receptors

In asthmatic airways, repeated epithelial damage and repair occur. No current therapy directly targets this process. We aimed to determine the effects of mannan derived from S. cerevisiae (SC-MN) on airway epithelial wound repair, in vitro. The presence of functional mannose receptors in bronchial epithelial cells was shown by endocytosis of colloidal gold-Man BSA via clathrin-coated pits in 16HBE cells. In primary normal human bronchial epithelial cells (NHBEC), SC-MN significantly facilitated wound closure. Treatment with SC-MN stimulated cell spreading as indicated by a significant increase in the average lamellipodial width of wound edge 16HBE cells. In addition, NHBEC treated with SC-MN showed increased expression and activation of Krüppel-like factors (KLFs) 4 and 5, transcription factors important in epithelial cell survival and regulation of epithelial-mesenchymal transition. We conclude that SC-MN facilitates wound repair in human bronchial epithelium, involving mannose receptors.

scholarly journals Explanting Is anEx VivoModel of Renal Epithelial-Mesenchymal Transition

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Catherine E. Winbanks ◽  
Ian A. Darby ◽  
Kristen J. Kelynack ◽  
Dodie Pouniotis ◽  
Gavin J. Becker ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
De Novo ◽  
Epithelial Mesenchymal Transition ◽  
Ex Vivo ◽  
Rat Kidney ◽  
Smooth Muscle Actin ◽  
Muscle Actin ◽  
Alpha Smooth Muscle Actin ◽  
Mesenchymal Transition ◽  
Renal Tubulointerstitial Fibrosis

Recognised by theirde novoexpression of alpha-smooth muscle actin (SMA), recruitment of myofibroblasts is key to the pathogenesis of fibrosis in chronic kidney disease. Increasingly, we realise that epithelial-mesenchymal transition (EMT) may be an important source of these cells. In this study we describe a novel model of renal EMT. Rat kidney explants were finely diced on gelatin-coated Petri dishes and cultured in serum-supplemented media. Morphology and immunocytochemistry were used to identify mesenchymal (vimentin+, α-smooth muscle actin (SMA)+, desmin+), epithelial (cytokeratin+), and endothelial (RECA+) cells at various time points. Cell outgrowths were all epithelial in origin (cytokeratin+) at day 3. By day 10, 50 ± 12% (mean ± SE) of cytokeratin+ cells double-labelled for SMA, indicating EMT. Lectin staining established a proximal tubule origin. By day 17, cultures consisted only of myofibroblasts (SMA+/cytokeratin−). Explanting is a reproducibleex vivomodel of EMT. The ability to modify this change in phenotype provides a useful tool to study the regulation and mechanisms of renal tubulointerstitial fibrosis.

scholarly journals Pancreatic Cancer Cells Respond to Type I Collagen by Inducing Snail Expression to Promote Membrane Type 1 Matrix Metalloproteinase-dependent Collagen Invasion

2011 ◽  
Vol 286 (12) ◽  
pp. 10495-10504 ◽  
Author(s):  
Mario A. Shields ◽  
Surabhi Dangi-Garimella ◽  
Seth B. Krantz ◽  
David J. Bentrem ◽  
Hidayatullah G. Munshi
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Type I Collagen ◽  
Epithelial Mesenchymal Transition ◽  
Three Dimensional ◽  
Type I ◽  
Collagen Gels ◽  
Mesenchymal Transition ◽  
Pancreatic Cancer Cells ◽  
Membrane Type

Pancreatic ductal adenocarcinoma (PDAC) is characterized by pronounced fibrotic reaction composed primarily of type I collagen. Although type I collagen functions as a barrier to invasion, pancreatic cancer cells have been shown to respond to type I collagen by becoming more motile and invasive. Because epithelial-mesenchymal transition is also associated with cancer invasion, we examined the extent to which collagen modulated the expression of Snail, a well known regulator of epithelial-mesenchymal transition. Relative to cells grown on tissue culture plastic, PDAC cells grown in three-dimensional collagen gels induced Snail. Inhibiting the activity or expression of the TGF-β type I receptor abrogated collagen-induced Snail. Downstream of the receptor, we showed that Smad3 and Smad4 were critical for the induction of Snail by collagen. In contrast, Smad2 or ERK1/2 was not involved in collagen-mediated Snail expression. Overexpression of Snail in PDAC cells resulted in a robust membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14)-dependent invasion through collagen-coated transwell chambers. Snail-expressing PDAC cells also demonstrated MT1-MMP-dependent scattering in three-dimensional collagen gels. Mechanistically, Snail increased the expression of MT1-MMP through activation of ERK-MAPK signaling, and inhibiting ERK signaling in Snail-expressing cells blocked two-dimensional collagen invasion and attenuated scattering in three-dimensional collagen. To provide in vivo support for our findings that Snail can regulate MT1-MMP, we examined the expression of Snail and MT1-MMP in human PDAC tumors and found a statistically significant positive correlation between MT1-MMP and Snail in these tumors. Overall, our data demonstrate that pancreatic cancer cells increase Snail on encountering collagen-rich milieu and suggest that the desmoplastic reaction actively contributes to PDAC progression.

scholarly journals Down-regulation of epithelial cadherin is required to initiate metastatic outgrowth of breast cancer

2011 ◽  
Vol 22 (14) ◽  
pp. 2423-2435 ◽  
Author(s):  
Michael K. Wendt ◽  
Molly A. Taylor ◽  
Barbara J. Schiemann ◽  
William P. Schiemann
Keyword(s):  
Breast Cancer ◽  
Transforming Growth Factor ◽  
Epithelial Mesenchymal Transition ◽  
Three Dimensional ◽  
Transforming Growth Factor Β ◽  
Β1 Integrin ◽  
Integrin Expression ◽  
Mesenchymal Transition ◽  
Epithelial Cadherin ◽  
Cancer Dormancy

Reduced epithelial cadherin (E-cad) is a hallmark of invasive carcinomas that have acquired epithelial-mesenchymal transition (EMT) phenotypes. Here we show that down-regulated E-cad expression induced by transforming growth factor-β (TGF-β) and EMT preceded breast cancer outgrowth in three-dimensional (3D) organotypic assays and in the lungs of mice. Pharmacological inhibitors against focal adhesion kinase prevented metastatic outgrowth of newly seeded organoids, but not that of their fully established counterparts. Interrogating the D2-HAN (hyperplastic alveolar nodule) model of breast cancer dormancy and metastasis showed that dormant D2.OR cells produced branched organoid morphologies in 3D-cultures, and expressed robust quantities of E-cad that was uncoupled from regulation by TGF-β. In contrast, metastatic D2.A1 organoids were spherical and wholly lacked E-cad expression. Interestingly, D2.A1 cells engineered to re-express E-cad formed branched organoids, down-regulated β1 integrin expression, and failed to undergo metastatic outgrowth. The tumor-suppressing function of E-cad was inactivated by increased microenvironmental rigidity, and was not recapitulated by expression of an E-cad mutant lacking its extracellular domain. Twist expression, but not that of Snail, reinitiated metastatic outgrowth in dormant D2.OR cells. Our findings show that EMT and its down-regulated expression of E-cad circumvent breast cancer dormancy in part by facilitating β1 integrin expression necessary for metastatic outgrowth.

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