Dataset Information

A novel pancreatic tumour and stellate cell 3D co-culture spheroid model.

ABSTRACT:

Background

Pancreatic ductal adenocarcinoma is a devastating disease with poor outcome, generally characterized by an excessive stroma component. The purpose of this study was to develop a simple and reproducible in vitro 3D-assay employing the main constituents of pancreatic ductal adenocarcinoma, namely pancreatic stellate and cancer cells.

Method

A spheroid assay, directly co-culturing human pancreatic stellate cells with human pancreatic tumour cells in 3D was established and characterized by electron microscopy, immunohistochemistry and real-time RT-PCR. In order to facilitate the cell type-specific crosstalk analysis by real-time RT-PCR, we developed a novel in vitro 3D co-culture model, where the participating cell types were from different species, human and mouse, respectively. Using species-specific PCR primers, we were able to investigate the crosstalk between stromal and cancer cells without previous cell separation and sorting.

Results

We found clear evidence for mutual influence, such as increased proliferation and a shift towards a more mesenchymal phenotype in cancer cells and an activation of pancreatic stellate cells towards the myofibroblast phenotype. Using a heterospecies approach, which we coined virtual sorting, confirmed the findings we made initially in the human-human spheroids.

Conclusions

We developed and characterized different easy to set up 3D models to investigate the crosstalk between cancer and stroma cells for pancreatic cancer.

SUBMITTER: Norberg KJ

PROVIDER: S-EPMC7251727 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Publications

A novel pancreatic tumour and stellate cell 3D co-culture spheroid model.

Norberg K J KJ Liu X X Fernández Moro C C Strell C C Nania S S Blümel M M Balboni A A Bozóky B B Heuchel R L RL Löhr J M JM

BMC cancer 20200527 1

<h4>Background</h4>Pancreatic ductal adenocarcinoma is a devastating disease with poor outcome, generally characterized by an excessive stroma component. The purpose of this study was to develop a simple and reproducible in vitro 3D-assay employing the main constituents of pancreatic ductal adenocarcinoma, namely pancreatic stellate and cancer cells.<h4>Method</h4>A spheroid assay, directly co-culturing human pancreatic stellate cells with human pancreatic tumour cells in 3D was established and ...[more]

PMID: 32460715

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Project description:BACKGROUND:Pancreatic stellate cells (PSCs), a major component of the tumor microenvironment in pancreatic cancer, play roles in cancer progression as well as drug resistance. Culturing various cells in microfluidic (microchannel) devices has proven to be a useful in studying cellular interactions and drug sensitivity. Here we present a microchannel plate-based co-culture model that integrates tumor spheroids with PSCs in a three-dimensional (3D) collagen matrix to mimic the tumor microenvironment in vivo by recapitulating epithelial-mesenchymal transition and chemoresistance. METHODS:A 7-channel microchannel plate was prepared using poly-dimethylsiloxane (PDMS) via soft lithography. PANC-1, a human pancreatic cancer cell line, and PSCs, each within a designated channel of the microchannel plate, were cultured embedded in type I collagen. Expression of EMT-related markers and factors was analyzed using immunofluorescent staining or Proteome analysis. Changes in viability following exposure to gemcitabine and paclitaxel were measured using Live/Dead assay. RESULTS:PANC-1 cells formed 3D tumor spheroids within 5 days and the number of spheroids increased when co-cultured with PSCs. Culture conditions were optimized for PANC-1 cells and PSCs, and their appropriate interaction was confirmed by reciprocal activation shown as increased cell motility. PSCs under co-culture showed an increased expression of ?-SMA. Expression of EMT-related markers, such as vimentin and TGF-?, was higher in co-cultured PANC-1 spheroids compared to that in mono-cultured spheroids; as was the expression of many other EMT-related factors including TIMP1 and IL-8. Following gemcitabine exposure, no significant changes in survival were observed. When paclitaxel was combined with gemcitabine, a growth inhibitory advantage was prominent in tumor spheroids, which was accompanied by significant cytotoxicity in PSCs. CONCLUSIONS:We demonstrated that cancer cells grown as tumor spheroids in a 3D collagen matrix and PSCs co-cultured in sub-millimeter proximity participate in mutual interactions that induce EMT and drug resistance in a microchannel plate. Microfluidic co-culture of pancreatic tumor spheroids with PSCs may serve as a useful model for studying EMT and drug resistance in a clinically relevant manner.

Project description:BackgroundPancreatic ductal adenocarcinoma (PDAC) is a stroma-rich carcinoma, and pancreatic stellate cells (PSCs) are a major component of this dense stroma. PSCs play significant roles in metastatic progression and chemoresistance through cross-talk with cancer cells. Preclinical in vitro tumor model of invasive phenotype should incorporate three-dimensional (3D) culture of cancer cells and PSCs in extracellular matrix (ECM) for clinical relevance and predictability.MethodsPANC-1 cells were cultured as tumor spheroids (TSs) using our previously developed minipillar chips, and co-cultured with PSCs, both embedded in collagen gels. Effects of PSC co-culture on ECM fiber network, invasive migration of cancer cells, and expression of epithelial-mesenchymal transition (EMT)-related proteins were examined. Conditioned media was also analyzed for secreted factors involved in cancer cell-PSC interactions. Inhibitory effect on cancer cell invasion was compared between gemcitabine and paclitaxel at an equitoxic concentration in PANC-1 TSs co-cultured with PSCs.ResultsCo-culture condition was optimized for the growth of TSs, activation of PSCs, and their interaction. Increase in cancer cell invasion via ECM remodeling, invadopodia formation and EMT, as well as drug resistance was recapitulated in the TS-PSC co-culture, and appeared to be mediated by cancer cell-PSC interaction via multiple secreted factors, including IL-6, IL-8, IGF-1, EGF, TIMP-1, uPA, PAI-1, and TSP-1. Compared to gemcitabine, paclitaxel showed a greater anti-invasive activity, which was attributed to suppresion of invadopodia formation in cancer cells as well as to PSC-specific cytotoxicity abrogating its paracrine signaling.ConclusionsHere, we established 3D co-culture of TSs of PANC-1 cells and PSCs using minipillar histochips as a novel tumoroid model of PDAC. Our results indicate usefulness of the present co-culture model and multiplex quantitative analysis method not only in studying the role of PSCs and their interactions with tumor cell towards metastatic progression, but also in the drug evaluation of stroma-targeting drugs.

Project description:BackgroundDifferent 3D-cell culture approaches with varying degrees of complexity have been developed to serve as melanoma models for drug testing or mechanistic studies. While these 3D-culture initiatives are already often superior to classical 2D approaches, they are either composed of only melanoma cells or they are so complex that the behavior of individual cell types is hard to understand, and often they are difficult to establish and expensive.MethodsThis study used low-attachment based generation of spheroids composed of up to three cell types. Characterization of cells and spheroids involved cryosectioning, immunofluorescence, FACS, and quantitative analyses. Statistical evaluation used one-way ANOVA with post-hoc Tukey test or Student's t-test.ResultsThe tri-culture model allowed to track cellular behavior in a cell-type specific manner and recapitulated different characteristics of early melanoma stages. Cells arranged into a collagen-IV rich fibroblast core, a ring of keratinocytes, and groups of highly proliferating melanoma cells on the outside. Regularly, some melanoma cells were also found to invade the fibroblast core. In the absence of melanoma cells, the keratinocyte ring stratified into central basal-like and peripheral, more differentiated cells. Conversely, keratinocyte differentiation was clearly reduced upon addition of melanoma cells. Treatment with the cytostatic drug, docetaxel, restored keratinocyte differentiation and induced apoptosis of external melanoma cells. Remaining intact external melanoma cells showed a significantly increased amount of ABCB5-immunoreactivity.ConclusionsIn the present work, a novel, simple spheroid-based melanoma tri-culture model composed of fibroblasts, keratinocytes, and melanoma cells was described. This model mimicked features observed in early melanoma stages, including loss of keratinocyte differentiation, melanoma cell invasion, and drug-induced increase of ABCB5 expression in external melanoma cells.

Project description:Background:Three dimensional (3D) cell cultures have been an area of increasing interest and relevance across several research fields including drug discovery, developmental biology and stem cell-based therapies. However, handling 3D structures can be difficult. In particular, the replacement of liquid media and reagents in which liquid is removed using pipettes is difficult to perform as the 3D spheroids can be easily aspirated into the pipette tip. Results:We have developed the 3D-tip, a novel tool that facilitates media change and washing procedures of 3D-spheroid cultures. The 3D-tip contains a mesh with 40-?m pores allowing the aspiration of liquids including media, drugs, buffers and reagents, with the mesh acting as a barrier preventing the spheroids being aspirated into the pipette tip. After aspiration of liquids, the spheroids are gently deposited back into the culture vessel. Our results demonstrate that the 3D-tips offer superior handling of 3D-spheroid cultures in comparison to commonly used methods. We showed that the 3D-tips can easily be used on both fixed and unfixed spheroids and on cancer cell, stem cell and glial cell spheroids.In contrast with the 50/50 media exchange method, the 3D-tips allow a complete media change with minimal loss of spheroids and without damaging their morphology. Our results showed that 86.0% of spheroids remained in the chamber after changing the media using the 3D-tips. In contrast, only 45.0% of spheroids remained using the 50/50 media exchange strategy.In comparison with the centrifugation technique, the 3D-tips preserved spheroids whereas centrifugation led to the loss of spheroids and/or the alteration of the size and shape of the 3D cellular structures. We observed that 87.6 and 84.6% of the fixed and unfixed spheroids remained using the 3D-tip, respectively. In contrast, only 66.3% of the fixed spheroids and 36.4% of the unfixed spheroids were left using the centrifugation method. From a time perspective, the 3D-tips dramatically reduce the time taken for replacing media. Conclusions:This novel pipette tip is suitable for high throughput screening and automation and will revolutionise the techniques used for the production and analysis of 3D spheroids.

Dataset Information

A novel pancreatic tumour and stellate cell 3D co-culture spheroid model.

Background

Method

Results

Conclusions

Publications

A novel pancreatic tumour and stellate cell 3D co-culture spheroid model.

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