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Transcriptomic profiling of breast cancer cells on hydrogels with different stiffness [sequencing]

ABSTRACT: Remodeling and increased stiffness of the extracellular matrix is a hallmark of solid cancers and contributes to tumor progression through increased proliferation, survival and migration. Mechanotransduction, the process in which cells sense and transduce mechanical signals, can result in transcriptional changes that in turn alter cellular behavior. In vitro culturing of MCF10ACA1a (CA1a) breast cancer cells on polyacrylamide-based hydrogels of variable stiffness revealed drastic changes in morphology, indicating a stiffness dependent induction of a malignant phenotype. We used two different techniques, DNA microarray and RNA sequencing, to characterize the transcriptome of CA1a cells on low (500 Pa) and high (8000 Pa) stiffness, corresponding to the stiffness of normal breast and breast cancer, respectively.

ORGANISM(S): Homo sapiens

PROVIDER: GSE205816 | GEO | 2023/03/13

REPOSITORIES: GEO

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Project description:Purpose: When we infect MDCK epithelial cells we low dosage of Listeria monocytogenes, we observe that at late times (24 h) post-infection the infected cells pertaining in infection foci get collectively extruded out of the epithelial monolayer forming a 3D mound. The formation of the infection mound results from the competition between two populations: the uninfected cells ("surrounders") that eventually squeeze and extrude the infected cells ("mounders") in the infected wells. The extracellular matrix where the host cells adhere appears to impact the formation of infection mounds with more mounding on glassr as opposed to soft 3 kPa substrata, as evidenced by confocal 3D microscopy. Here we built poalyacrylamide hydrogels of varing stiffness (3 kPa soft or 70 kPa stiff) and coated them with collagen I rat tail. We also placed host cells on glass collagen I-coated surfaces. We then infected for 24 h (or not) the host cells with Listeria monocytogenes to infer how matrix stiffness-dependent gene expression of MDCK cells in monolayers changes in uninfected versus infected wells, through RNA-Sequencing. Our goal was to understand how matrix stiffness impacts transcriptional regulation of host cells and whether that could modulate the collective extrusion of infected epithelial cells triggered by surrounding uninfected cells. Methods and Results: By analyzing the differentially expressed genes between all conditions we find a significant number of up- or down-regulated genes between all groups, other than between cells residing on soft versus stiff polyacrylamide hydrogels that behave similarly in terms of gene expression. Through pathway enrichement analysis, we find that cells residing on polyacrylamide hydrogels as opposed to cells residing on glass show higher upregulation of a number of innate immune signaling related pathways (e.g. MAPK, TNF) even when infection is not involved. When comparing infected versus not wells, the relative differences in gene expression for cells residing on glass are higher than for cells residing on hydrogels. However certain specific innate immune signaling pathways that regulate mound formation (https://www.sciencedirect.com/science/article/abs/pii/S1534580721000708), show higher upregulation in infection for cells on hydrogels as opposed to glass (e.g. IL-17, TNF, NF-κΒ; evidenced by pathway enrichement analysis). Finally, through pathway enrichement analysis we find that the relative change based on enrichement score when comparing uninfected versus infected settings for cells residing on glass versus on polyacrylamide hydrogels is lower for the former (i.e. for cells residing on glass) for NF-κB, TNF and IL17. However, with regards to the MAPK pathway the enrichment score of cells from infected versus uninfected wells, is much higher for cells residing on glass as opposed to polyacrylamide hydrogles. Conclusions: Infection of MDCK epithelial cells with Listeria monocytogenes significantly alters the transcriptome of the infected host cells when those are examined 24 h post infection and compared to uninfected cells on all different substrata. Compared to cells residing on glass substrates cells residing on polyacrylamide hydrogels (irrespective of whether the stiffness is low i.e. 3 kPa or high i.e. 70 kPa) show significant differences both when uninfected settings and when infected ones are compared.

Project description:In vitro cultures of primary cardiac fibroblasts (CFs), the major extracellular matrix (ECM)-producing cells of the heart, are used to determine molecular mechanisms of cardiac fibrosis. However, the supraphysiologic stiffness of tissue culture polystyrene (TCPS) automatically triggers the conversion of CFs into an activated myofibroblast-like state, and serial passage of the cells results in the induction of replicative senescence. These dramatic phenotypic switches confound interpretation of experimental data obtained with cultured CFs. In an attempt to circumvent TCPS-induced activation and senescence of CFs, we utilized poly (ethylene glycol) (PEG) hydrogels as cell culture platforms with low and high stiffness formulations to mimic healthy and fibrotic cardiac ECM, respectively. As hypothesized, low hydrogel stiffness converted activated CFs into a quiescent state with reduced abundance of a-smooth muscle actin (a-SMA)-containing stress fibers. Unexpectedly, lower substrate stiffness concomitantly augmented CF senescence, marked by elevated senescence-associated b-galactosidase (SA-b-Gal) activity and increased expression of p16 and p21, which are cyclin-dependent kinase (CDK) inhibitors and markers of senescence. Using dynamically stiffening hydrogels with phototunable crosslinking capabilities, we demonstrate that substrate-induced CF senescence is partially reversible. RNA-sequencing analysis revealed widespread transcriptional reprogramming of CFs cultured on low stiffness hydrogels, with a dramatic reduction in the expression of profibrotic genes encoding ECM proteins, and an attendant increase in expression of NF-kB-responsive inflammatory genes that typify the senescence-associated secretory phenotype (SASP). Our findings further demonstrate that alterations in matrix stiffness profoundly impact CF cell state transitions, and suggest mechanisms by which CFs change phenotype in vivo depending on the stiffness of the myocardial microenvironment to which they are exposed.

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Transcriptomic profiling of breast cancer cells on hydrogels with different stiffness [sequencing]

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