Quantitative Imaging of Epithelial Cell Scattering Identifies Specific Inhibitors of Cell Motility and Cell-Cell Dissociation

Loerke, Dinah; Duc, Quint le; Blonk, Iris; Kerstens, Andre; Spanjaard, Emma; Macháček, Matthias; Danuser, Gaudenz; Rooij, Johan de

doi:10.1126/scisignal.2002677

Cited by 32 publications

(31 citation statements)

References 25 publications

(31 reference statements)

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“…All cell lines were grown at 37°C in a humidified atmosphere of 5% CO 2 in air. Migration and adhesion assays of MK Itga3 fl/fl and MK Itga3 −/− (6) on a laminin-332 rich matrix deposited by Rac11-P cells were performed as previously described (20,(34)(35)(36). Murine skin cancer cell lines P1, B9, and A5 were cultured as described (23,24) (SI Materials and Methods).…”

Section: Methodsmentioning

confidence: 99%

Loss of integrin α3 prevents skin tumor formation by promoting epidermal turnover and depletion of slow-cycling cells

Sachs¹,

Secades²,

Hulst³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Progression through the various stages of skin tumorigenesis is correlated with an altered expression of the integrin α3β1, suggesting that it plays an important role in the tumorigenic process. Using epidermis-specific Itga3 KO mice subjected to the 7,12-dimethylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate two-stage skin carcinogenesis protocol, we demonstrate that efficient tumor development is critically dependent on the presence of α3β1. In the absence of α3β1, tumor initiation is dramatically decreased because of increased epidermal turnover, leading to a loss of DMBA-initiated label-retaining keratinocytes. Lineage tracing revealed emigration of α3-deficient keratinocytes residing in the bulge of the hair follicle toward the interfollicular epidermis. Furthermore, tumor growth and cell proliferation were strongly reduced in mice with an epidermis-specific deletion of Itga3. However, the rate of progression of α3β1-null squamous cell carcinomas to undifferentiated, invasive carcinomas was increased. Therefore, α3β1 critically affects skin carcinogenesis with opposing effects early and late in tumorigenesis.skin cancer | cell adhesion | cell migration | laminin receptor | hair cycling S kin cancer is the most common form of cancer among white populations, with basal cell carcinomas and squamous cell carcinomas (SSCs) being the most common subtypes. Although early detection and surgical resection can prevent most complications associated with this disease, SCCs frequently metastasize and then cannot be effectively treated. Understanding the molecular basis of skin tumorigenesis is a prerequisite for future prevention and therapy. The well-characterized 7,12-dimethylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) protocol models the multistep nature of human skin carcinogenesis in mice. Oncogenic mutations (e.g., Hras), induced by a single treatment with the carcinogen DMBA confer growth advantage to the initiated cells, which form benign papillomas under repetitive tumorpromoting treatments with the phorbol ester TPA. Subsequent progression to SCCs involves mutation of Trp53 and trisomization of chromosomes 6 and 7 (1-5).Integrins are αβ heterodimeric adhesion receptors that play an important role in maintaining epithelial integrity. In the skin, the major integrins α2β1, α3β1, and α6β4 connect the cytoskeleton of basal keratinocytes to the underlying basement membrane (6). Besides their key function in skin physiology, these integrins also have been implicated in the development and progression of SCCs (7). Mouse models in which different integrins are either overexpressed in the suprabasal epidermis or mutated in the whole animal showed altered susceptibilities to chemically induced skin tumorigenesis (8-10). Increased expression of α2β1, α3β1, and α6β4 has been observed in hyperproliferating human cancers of the head and neck (11). Integrins thus seem to play a role in initiation and promotion of tumors. Surprisingly, the role of α3β1 in basal keratinocytes in skin tumorigene...

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Section: Methodsmentioning

confidence: 99%

Loss of integrin α3 prevents skin tumor formation by promoting epidermal turnover and depletion of slow-cycling cells

Sachs¹,

Secades²,

Hulst³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…1A and provided additional information at the single-cell level. Quantifying the ratio of E-cadherin staining at individual cell-cell junctions relative to junction-proximal regions, as in previous studies related to cell adhesion (Loerke et al, 2012;Č áslavský et al, 2013), demonstrated that the overall decrease in E-cadherin expression observed by western blot was accompanied by a delocalization of E-cadherin from cell-cell junctions in response to TGFβ with or without EGF. Analysis of vimentin staining revealed that the higher total levels of vimentin expression measured by western blot reflected an increased percentage of vimentinpositive cells.…”

Section: Egf Augments Tgfβ-induced Emtmentioning

confidence: 99%

“…Weakening cell-cell junctions within preformed epithelial cell clusters can lead to disrupted cluster circularity and scatter of cells away from these clusters (Loerke et al, 2012). These changes can be monitored by measuring a cluster circularity index, calculated as 4π(area/perimeter 2 ), which has a value of 1 for a circle or 0 for a line (Č áslavský et al, 2013).…”

Section: Egf Augments Tgfβ-induced Emtmentioning

confidence: 99%

“…E-cadherin junctional localization was quantified using ImageJ and a method similar to that described in Loerke et al (2012). Each cell-cell junction was traced with a 15-pixel (1.1 μm)-wide freehand line (corresponding to the average thickness of E-cadherin stained junctions in the untreated condition), for which the area (A j ), integrated density (ID j ), and area-averaged mean intensity (M j ) were measured.…”

Section: Immunofluorescence Staining and Image Analysismentioning

confidence: 99%

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EGF augments TGFβ-induced epithelial–mesenchymal transition by promoting SHP2 binding to GAB1

Buonato

Lan

Lazzara

2015

Journal of Cell Science

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In many epithelial cells, epidermal growth factor (EGF) augments the epithelial-mesenchymal transition (EMT) that occurs when cells are treated with transforming growth factor β (TGFβ). We demonstrate that this augmentation requires activation of SH2 domain-containing phosphatase-2 (SHP2; also known as PTPN11), a proto-oncogene. In lung and pancreatic cancer cell lines, reductions in E-cadherin expression, increases in vimentin expression and increases in cell scatter rates were larger when cells were treated with TGFβ and EGF versus TGFβ or EGF alone. SHP2 knockdown promoted epithelial characteristics basally and antagonized EMT in response to TGFβ alone or in combination with EGF. Whereas EGF promoted SHP2 binding to tyrosine phosphorylated GAB1, which promotes SHP2 activity, TGFβ did not induce SHP2 association with phosphotyrosine-containing proteins. Knockdown of endogenous SHP2 and reconstitution with an SHP2 mutant with impaired phosphotyrosine binding ability eliminated the EGF-mediated EMT augmentation that was otherwise restored with wild-type SHP2 reconstitution. These results demonstrate roles for basal and ligandinduced SHP2 activity in EMT and further motivate efforts to identify specific ways to inhibit SHP2, given the role of EMT in tumor dissemination and chemoresistance.

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“…There have been several techniques developed, including segmentation of invading cells and calculation of scattered tumor area [16], calculation of nearest-neighbor distance measures between cells [10], and cellular motility [7]. As in our current work, we will develop several features that will measure different aspects of cell spread, and use these features to identify which feature is most altered in +HGF spheroids vs. controls.…”

Section: Discussionmentioning

confidence: 99%

Feature Identification for Colon Tumor Classification

2013

SIURO

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Abstract. Hepatocyte Growth Factor (HGF) has been shown to be increased in the tumor microenvironment due to increased secretion by cancer-associated stromal cells. Qualitatively, high extracellular HGF has been correlated with increased growth and dispersiveness of a tumor. In this study, we develop quantitative methods to measure HGF-induced tumor growth and dispersion. Using image processing and machine learning techniques, we effectively classify images of colon cancer tumor spheroids cultured in +/-HGF conditions. Our goals are to define features that are effective for classification and to further help biologists quantify the effect of HGF on tumor spheroids.

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Quantitative Imaging of Epithelial Cell Scattering Identifies Specific Inhibitors of Cell Motility and Cell-Cell Dissociation

Cited by 32 publications

References 25 publications

Loss of integrin α3 prevents skin tumor formation by promoting epidermal turnover and depletion of slow-cycling cells

Loss of integrin α3 prevents skin tumor formation by promoting epidermal turnover and depletion of slow-cycling cells

EGF augments TGFβ-induced epithelial–mesenchymal transition by promoting SHP2 binding to GAB1

Feature Identification for Colon Tumor Classification

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