2 research outputs found
Rapid Organoid Reconstitution by Chemical Micromolding
Purified populations
of cells can be reconstituted into organoids
that recapitulate aspects of their in vivo structure and function.
These organoids are useful as models of healthy and diseased tissue
in the basic sciences, in vitro screens, and regenerative medicine.
Existing strategies to reconstitute organoids from purified cells
face obstacles with respect to cell-viability, multicellular connectivity,
scalability, and compatibility with subsequent experimental or analytical
techniques. To address these challenges, we developed a strategy for
rapidly casting populations of cells into microtissues of prescribed
size and shape. This approach begins by chemically remodeling the
adhesive properties of living cells with membrane-anchored ssDNA with
modest annealing kinetics. Populations of complementary labeled cells
are then combined into microwells that rapidly mold the DNA-adhesive
cell populations into 3D aggregates of uniform size and shape. Once
formed, aggregates are removed from the molds in the presence of “capping”
oligonucleotides that block hybridization of residual surface DNA
between aggregates in suspension. Finally, transfer of aggregates
to biomimetic gels for 3D culture completes the process of reconstitution.
This strategy of chemical micromolding allows for control over aggregate
internal topology and does not perturb the natural process of self-organization
in primary human mammary epithelial cells
DataSheet1_Microenvironment-Induced Non-sporadic Expression of the AXL and cKIT Receptors Are Related to Epithelial Plasticity and Drug Resistance.zip
<p>The existence of rare cancer cells that sporadically acquire drug-tolerance through epigenetic mechanisms is proposed as one mechanism that drives cancer therapy failure. Here we provide evidence that specific microenvironments impose non-sporadic expression of proteins related to epithelial plasticity and drug resistance. Microarrays of robotically printed combinatorial microenvironments of known composition were used to make cell-based functional associations between microenvironments, which were design-inspired by normal and tumor-burdened breast tissues, and cell phenotypes. We hypothesized that specific combinations of microenvironment constituents non-sporadically impose the induction of the AXL and cKIT receptor tyrosine kinase proteins, which are known to be involved in epithelial plasticity and drug-tolerance, in an isogenic human mammary epithelial cell (HMEC) malignant progression series. Dimension reduction analysis reveals type I collagen as a dominant feature, inducing expression of both markers in pre-stasis finite lifespan HMECs, and transformed non-malignant and malignant immortal cell lines. Basement membrane-associated matrix proteins, laminin-111 and type IV collagen, suppress AXL and cKIT expression in pre-stasis and non-malignant cells. However, AXL and cKIT are not suppressed by laminin-111 in malignant cells. General linear models identified key factors, osteopontin, IL-8, and type VIα3 collagen, which significantly upregulated AXL and cKIT, as well as a plasticity-related gene expression program that is often observed in stem cells and in epithelial-to-mesenchymal-transition. These factors are co-located with AXL-expressing cells in situ in normal and breast cancer tissues, and associated with resistance to paclitaxel. A greater diversity of microenvironments induced AXL and cKIT expression consistent with plasticity and drug-tolerant phenotypes in tumorigenic cells compared to normal or immortal cells, suggesting a reduced perception of microenvironment specificity in malignant cells. Microenvironment-imposed reprogramming could explain why resistant cells are seemingly persistent and rapidly adaptable to multiple classes of drugs. These results support the notion that specific microenvironments drive drug-tolerant cellular phenotypes and suggest a novel interventional avenue for preventing acquired therapy resistance.</p