A proteomic approach to immune-mediated epithelial-mesenchymal transition

Abstract Background Human corneal stromal keratocytes propagated in culture media supplemented with human amnion extract (AME) can correct early corneal haze in an animal model. Clinical application of cultivated keratocytes is limited by infectious disease screening before amnion products can be used in humans. It remains unclear if AME from cryopreserved versus fresh human amnion can support human keratocyte propagation, and which components of the extract promote keratocyte growth. Methods Three placentas were collected for the preparation of fresh and cryopreserved amnion tissues followed by homogenization and protein extraction. AME protein profiles were studied using isobaric tagging for relative and absolute quantitation (iTRAQ) proteomics. Enriched gene ontology (GO) terms and functional classes were identified. Primary human keratocytes from 4 donor corneas were cultured in media supplemented with fresh AME (F-AME) or cryopreserved AME (C-AME). Cell viability, proliferation and keratocyte marker expression were examined by confocal immunofluorescence and flow cytometry. Results AME proteomics revealed 1385 proteins with similar expression levels (between 0.5- and 2-fold) between F- and C-AME, while 286 proteins were reduced (less than 0.5-fold) in C-AME. Enriched GO term and biological pathway analysis showed that those proteins with comparable expression between F-AME and C-AME were involved in cell metabolism, epithelial-mesenchymal transition, focal adhesion, cell-extracellular matrix interaction, cell stress regulation and complement cascades. Human corneal stromal keratocytes cultured with F-AME or C-AME showed similar morphology and viability, while cell proliferation was mildly suppressed with C-AME (P > 0.05). Expression of aldehyde dehydrogenase 3A1 (ALDH3A1) and CD34 was similar in both cultures. Conclusion AME from cryopreserved amnion had limited influence on keratocyte culture. It is feasible to use protein extract from cryopreserved amnion to propagate human keratocytes for potential translational applications.

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Microenvironmental Hypoxia Induces Dynamic Changes in Lung Cancer Synthesis and Secretion of Extracellular Vesicles

Cancers ◽

10.3390/cancers12102917 ◽

2020 ◽

Vol 12 (10) ◽

pp. 2917

Author(s):

Shun Wilford Tse ◽

Chee Fan Tan ◽

Jung Eun Park ◽

JebaMercy Gnanasekaran ◽

Nikhil Gupta ◽

...

Keyword(s):

Lung Cancer ◽

Extracellular Vesicles ◽

Cancer Progression ◽

Membrane Trafficking ◽

Epithelial Mesenchymal Transition ◽

Migration And Invasion ◽

Mesenchymal Transition ◽

Functional Clustering ◽

Signalling Molecules ◽

Proteomic Approach

Extracellular vesicles (EVs) mediate critical intercellular communication within healthy tissues, but are also exploited by tumour cells to promote angiogenesis, metastasis, and host immunosuppression under hypoxic stress. We hypothesize that hypoxic tumours synthesize hypoxia-sensitive proteins for packing into EVs to modulate their microenvironment for cancer progression. In the current report, we employed a heavy isotope pulse/trace quantitative proteomic approach to study hypoxia sensitive proteins in tumour-derived EVs protein. The results revealed that hypoxia stimulated cells to synthesize EVs proteins involved in enhancing tumour cell proliferation (NRSN2, WISP2, SPRX1, LCK), metastasis (GOLM1, STC1, MGAT5B), stemness (STC1, TMEM59), angiogenesis (ANGPTL4), and suppressing host immunity (CD70). In addition, functional clustering analyses revealed that tumour hypoxia was strongly associated with rapid synthesis and EV loading of lysosome-related hydrolases and membrane-trafficking proteins to enhance EVs secretion. Moreover, lung cancer-derived EVs were also enriched in signalling molecules capable of inducing epithelial-mesenchymal transition in recipient cancer cells to promote their migration and invasion. Together, these data indicate that lung-cancer-derived EVs can act as paracrine/autocrine mediators of tumorigenesis and metastasis in hypoxic microenvironments. Tumour EVs may, therefore, offer novel opportunities for useful biomarkers discovery and therapeutic targeting of different cancer types and at different stages according to microenvironmental conditions.

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Development of Cancer Vaccines Targeting Brachyury, a Transcription Factor Associated with Tumor Epithelial-Mesenchymal Transition

Cells Tissues Organs ◽

10.1159/000446495 ◽

2017 ◽

Vol 203 (2) ◽

pp. 128-138 ◽

Cited By ~ 6

Author(s):

Duane H. Hamilton ◽

Justin M. David ◽

Charli Dominguez ◽

Claudia Palena

Keyword(s):

Transcription Factor ◽

Cancer Vaccines ◽

Epithelial Mesenchymal Transition ◽

Human Tumor ◽

Tumor Resistance ◽

Epithelial Cancer ◽

Immune Effector ◽

Mesenchymal Transition ◽

Immune Mediated ◽

Tumor Dissemination

Epithelial-mesenchymal transition (EMT) is recognized as a relevant process during the progression of carcinomas towards metastatic disease. Epithelial cancer cells undergoing an EMT program may acquire mesenchymal features, motility, invasiveness, and resistance to a variety of anticancer therapeutics. Preventing or reverting the EMT process in carcinomas has the potential to minimize tumor dissemination and the emergence of therapeutic resistance. One of the strategies currently under investigation to target tumor cells undergoing EMT is the generation of a sustained immune response directed against an essential molecular driver of the process. This review focuses on the current development of immune-mediated anticancer interventions aimed at targeting a transcription factor, brachyury, associated with human tumor EMT. Also presented here is a summary of recent studies demonstrating a role for EMT in tumor resistance to immune effector cytotoxicity, and the study of novel strategies aimed at reverting the EMT to be used in combination with immune-mediated anticancer interventions.

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