A deep conical agarose microwell array for adhesion independent three-dimensional cell culture and dynamic volume measurement

Thomsen, Andreas; Aldrian, Christine; Bronsert, Peter; Thomann, Yi; Nanko, N; Melin, Nicolas; Rücker, Gerta; Follo, Marie; Grosu, Anca L.; Niedermann, Gabriele; Layer, Paul G.; Heselich, Anja; Lund, Per

doi:10.1039/c7lc00832e

Cited by 68 publications

(80 citation statements)

References 52 publications

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“…This method allows for monitoring of dose-dependent effects of combinatorial treatments using cell aggregate volumes as the read out [37]. The presence of A37 alone at higher concentrations (20 μM and 40 μM) lowers cell aggregate volume.…”

Section: Resultsmentioning

confidence: 99%

“…For 3D experiments, MiaPaCa-2 cells were seeded in conical agarose microwell array (CAMA) to form non-spheroidal cell aggregates as previously described [37]. Briefly, 1000 cells were seeded into each microwell of these CAMA plates, cultured for 48 hours and treated with increasing concentrations (10 μM, 20 μM, and 40 μM) of ALDH1A1 inhibitor A37.…”

Section: Methodsmentioning

confidence: 99%

“…The cell aggregates were left to grow for 14 days. The volume changes of the cell aggregates were quantified by scanning using a high-resolution flatbed scanner (CanoScan 9000F Mark II, Canon Inc.) as previously described [37].…”

Section: Methodsmentioning

confidence: 99%

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Proteome Profiling of Primary Pancreatic Ductal Adenocarcinomas Undergoing Additive Chemoradiation Link ALDH1A1 to Early Local Recurrence and Chemoradiation Resistance

Oria

Bronsert

Thomsen

et al. 2018

Translational Oncology

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Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis with frequent post-surgical local recurrence. The combination of adjuvant chemotherapy with radiotherapy is under consideration to achieve a prolonged progression-free survival (PFS). To date, few studies have determined the proteome profiles associated with response to adjuvant chemoradiation. We herein analyzed the proteomes of primary PDAC tumors subjected to additive chemoradiation after surgical resection and achieving short PFS (median 6 months) versus prolonged PFS (median 28 months). Proteomic analysis revealed the overexpression of Aldehyde Dehydrogenase 1 Family Member A1 (ALDH1A1) and Monoamine Oxidase A (MAOA) in the short PFS cohort, which were corroborated by immunohistochemistry. In vitro, specific inhibition of ALDH1A1 by A37 in combination with gemcitabine, radiation, and chemoradiation lowered cell viability and augmented cell death in MiaPaCa-2 and Panc 05.04 cells. ALDH1A1 silencing in both cell lines dampened cell proliferation, cell metabolism, and colony formation. In MiaPaCa-2 cells, ALDH1A1 silencing sensitized cells towards treatment with gemcitabine, radiation or chemoradiation. In Panc 05.04, increased cell death was observed upon gemcitabine treatment only. These findings are in line with previous studies that have suggested a role of ALDH1A1 chemoradiation resistance, e.g., in esophageal cancer. In summary, we present one of the first proteome studies to investigate the responsiveness of PDAC to chemoradiation and provide further evidence for a role of ALDH1A1 in therapy resistance.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Proteome Profiling of Primary Pancreatic Ductal Adenocarcinomas Undergoing Additive Chemoradiation Link ALDH1A1 to Early Local Recurrence and Chemoradiation Resistance

Oria

Bronsert

Thomsen

et al. 2018

Translational Oncology

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“…For this purpose, we used a customized 3D in vitro model [53] to isolate EVs released by cell aggregates from two gastric cancer (GC) cell lines. For this purpose, we used a customized 3D in vitro model [53] to isolate EVs released by cell aggregates from two gastric cancer (GC) cell lines.…”

Section: Introductionmentioning

confidence: 99%

3D Cellular Architecture Affects MicroRNA and Protein Cargo of Extracellular Vesicles

et al. 2018

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The success of malignant tumors is conditioned by the intercellular communication between tumor cells and their microenvironment, with extracellular vesicles (EVs) acting as main mediators. While the value of 3D conditions to study tumor cells is well established, the impact of cellular architecture on EV content and function is not investigated yet. Here, a recently developed 3D cell culture microwell array is adapted for EV production and a comprehensive comparative analysis of biochemical features, RNA and proteomic profiles of EVs secreted by 2D vs 3D cultures of gastric cancer cells, is performed. 3D cultures are significantly more efficient in producing EVs than 2D cultures. Global upregulation of microRNAs and downregulation of proteins in 3D are observed, indicating their dynamic coregulation in response to cellular architecture, with the ADP‐ribosylation factor 6 signaling pathway significantly downregulated in 3D EVs. The data strengthen the biological relevance of cellular architecture for production and cargo of EVs.

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“…Similar morphology of the colonies was observed in a conventional soft agar assay (Figure B, upper panel); quantitative analysis revealed that Tspan8 overexpression causes a strong reduction of the colony number (Figure B, bottom panel), indicating diminished resistance to anoikis by the MTPa‐Tspan8 cells. Using recently characterised agarose microwell arrays , we tested cell proliferation and observed that, in contrast to 2D, the Tspan8 proliferation‐supporting effect was attenuated in a 3D environment (Figure C).…”

Section: Resultsmentioning

confidence: 99%

Tspan8 is expressed in breast cancer and regulates E‐cadherin/catenin signalling and metastasis accompanied by increased circulating extracellular vesicles

Voglstaetter

Thomsen

Nouvel

et al. 2019

The Journal of Pathology

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Tspan8 exhibits a functional role in many cancer types including pancreatic, colorectal, oesophagus carcinoma, and melanoma. We present a first study on the expression and function of Tspan8 in breast cancer. Tspan8 protein was present in the majority of human primary breast cancer lesions and metastases in the brain, bone, lung, and liver. In a syngeneic rat breast cancer model, Tspan8+ tumours formed multiple liver and spleen metastases, while Tspan8− tumours exhibited a significantly diminished ability to metastasise, indicating a role of Tspan8 in metastases. Addressing the underlying molecular mechanisms, we discovered that Tspan8 can mediate up‐regulation of E‐cadherin and down‐regulation of Twist, p120‐catenin, and β‐catenin target genes accompanied by the change of cell phenotype, resembling the mesenchymal–epithelial transition. Furthermore, Tspan8+ cells exhibited enhanced cell–cell adhesion, diminished motility, and decreased sensitivity to irradiation. As a regulator of the content and function of extracellular vesicles (EVs), Tspan8 mediated a several‐fold increase in EV number in cell culture and the circulation of tumour‐bearing animals. We observed increased protein levels of E‐cadherin and p120‐catenin in these EVs; furthermore, Tspan8 and p120‐catenin were co‐immunoprecipitated, indicating that they may interact with each other. Altogether, our findings show the presence of Tspan8 in breast cancer primary lesion and metastases and indicate its role as a regulator of cell behaviour and EV release in breast cancer. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

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A deep conical agarose microwell array for adhesion independent three-dimensional cell culture and dynamic volume measurement

Abstract: Miniaturised conical measures for cell aggregates.

Cited by 68 publications

References 52 publications

Proteome Profiling of Primary Pancreatic Ductal Adenocarcinomas Undergoing Additive Chemoradiation Link ALDH1A1 to Early Local Recurrence and Chemoradiation Resistance

Proteome Profiling of Primary Pancreatic Ductal Adenocarcinomas Undergoing Additive Chemoradiation Link ALDH1A1 to Early Local Recurrence and Chemoradiation Resistance

3D Cellular Architecture Affects MicroRNA and Protein Cargo of Extracellular Vesicles

Tspan8 is expressed in breast cancer and regulates E‐cadherin/catenin signalling and metastasis accompanied by increased circulating extracellular vesicles

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