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The architecture of co-culture spheroids regulates tumor invasion within a 3D extracellular matrix

ABSTRACT: Tumor invasion, the process by which tumor cells break away from their primary tumor and gain access to vascular systems, is an important step in cancer metastasis. Most current 3D tumor invasion assays consisted of single tumor cells embedded within an extracellular matrix (ECM). These assays taught us much of what we know today on how key biophysical (e.g. ECM stiffness) and biochemical (e.g. cytokine gradients) parameters within the tumor microenvironment guided and regulated tumor invasion. One limitation of the single tumor cell invasion assay was that it did not account for cell-cell adhesion within the tumor. In this article, we developed a micrometer scale 3D co-culture spheroid invasion assay that was compatible with microscopic imaging. Micrometer scale co-culture spheroids (1:1 ratio of metastatic breast cancer MDA-MB-231 and non-tumorigenic epithelial MCF-10A cells) were made using an array of microwells, and then were embedded within a collagen matrix in a microfluidic platform. Real time imaging of tumor spheroid invasion revealed that the spatial distribution of the two cell types within the tumor spheroid critically regulated tumor invasion. This work linked tumor architecture with tumor invasion and highlighted the importance of the biophysical cues within the bulk of the tumor in tumor invasion.

SUBMITTER: Huang Y

PROVIDER: S-EPMC7540657 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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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:Purpose: Both cardiomyocytes and cardiac fibroblasts (CF) play essential roles in cardiac development, function, and remodeling. Properties of 3D co-cultures are incompletely understood. Hence, 3D co-culture of cardiomyocytes and CF was characterized, and selected features compared with single-type and 2D culture conditions. Methods: Human cardiomyocytes derived from induced-pluripotent stem cells (hiPSC-CMs) were obtained from Cellular Dynamics or Ncardia, and primary human cardiac fibroblasts from ScienCell. Cardiac spheroids were investigated using cryosections and whole-mount confocal microscopy, video motion analysis, scanning-, and transmission-electron microscopy (SEM, TEM), action potential recording, and quantitative PCR (qPCR). Results: Spheroids formed in hanging drops or in non-adhesive wells showed spontaneous contractions for at least 1 month with frequent media changes. SEM of mechanically opened spheroids revealed a dense inner structure and no signs of blebbing. TEM of co-culture spheroids at 1 month showed myofibrils, intercalated disc-like structures and mitochondria. Ultrastructural features were comparable to fetal human myocardium. We then assessed immunostained 2D cultures, cryosections of spheroids, and whole-mount preparations by confocal microscopy. CF in co-culture spheroids assumed a small size and shape similar to the situation in ventricular tissue. Spheroids made only of CF and cultured for 3 weeks showed no stress fibers and strongly reduced amounts of alpha smooth muscle actin compared to early spheroids and 2D cultures as shown by confocal microscopy, western blotting, and qPCR. The addition of CF to cardiac spheroids did not lead to arrhythmogenic effects as measured by sharp-electrode electrophysiology. Video motion analysis showed a faster spontaneous contraction rate in co-culture spheroids compared to pure hiPSC-CMs, but similar contraction amplitudes and kinetics. Spontaneous contraction rates were not dependent on spheroid size. Applying increasing pacing frequencies resulted in decreasing contraction amplitudes without positive staircase effect. Gene expression analysis of selected cytoskeleton and myofibrillar proteins showed more tissue-like expression patterns in co-culture spheroids than with cardiomyocytes alone or in 2D culture. Conclusion: We demonstrate that the use of 3D co-culture of hiPSC-CMs and CF is superior over 2D culture conditions for co-culture models and more closely mimicking the native state of the myocardium with relevance to drug development as well as for personalized medicine.

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The architecture of co-culture spheroids regulates tumor invasion within a 3D extracellular matrix

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