Next generation seuquencing of 3D breast cancer cell culture in synthetic hydrogels presenting either a collagen mimic or laminin mimic
Ontology highlight
ABSTRACT: MDA-MB-231 (ER-) and T47D (ER+) breast cancer cells were encapsulated in synthetic hydrogels, formed with a multi-arm bioinert polymer poly(ethylene glycol), cell-degradable peptide crosslinks, and receptor-binding pendant peptides, and cultured for 3 days to assess initial cell responses to different matrix compositions. The synthetic matrices had a Young's modulus (E) ~ 0.6 kPa, in the range of bone marrow and healthy mammary tissue, and biochemcial cues to mimic binding sites found in collagen (GFOGER peptide) or laminin (IKVAV peptide). After 3 days in culture, mRNA was collected and RNA-seq performed to compare cell responses between the collagen and laminin mimetic cultures.
Project description:MDA-MB-231 (ER-) and T47D (ER+) breast cancer cells were encapsualted in synthetic hydrogels, formed with a multi-arm bioinert polymer poly(ethylene glycol), cell-degradable peptide crosslinks, and receptor-binding pendant peptides, and cultured for 3 days and up to 40 days to assess initial cell responses to different matrix compositions. The synthetic matrices had a Young's modulus (E) ~ 0.6 kPa (low wt%) and # ~ 5 kPa (high wt%), in the range of bone marrow or healthy mammary tissue and lung tissue, respectively, and Biochemcial cues to mimic binding sites found in collagen (GFOGER peptide) or laminin (IKVAV peptide). After 3 days and 40 days in culture, mRNA was collected and RNA-seq performed to compare cell responses between the collagen and laminin mimetic cultures overtime in both low wt% and high wt% matrices.
Project description:Next generation seuquencing of 3D breast cancer cell culture in synthetic hydrogels presenting either a collagen mimic or laminin mimic
Project description:Natural materials can reversibly self-assemble into hierarchical networks with diverse structural and functional properties. Recreating these dynamic architectures using peptide-based synthetic materials has been an elusive goal, hindered by challenges in relating sequence to structure to function. Here we report on the de novo discovery of short peptides based on the “tryptophan zipper” (Trpzip) motif that self-assemble into hierarchically structured hydrogels. Trpzip hydrogels have a tunable modulus and show self-healing, stress relaxation, antimicrobial properties, and biocompatibility. The low yield point allows syringe extrusion with cytoprotection, and cell harvest with a flick of the wrist. Integrating a pendant cell adhesion motif promotes human intestinal organoid growth and differentiation, with polarity control and improved shape fidelity. Considering these unique characteristics, we anticipate Trpzip hydrogels will prove a versatile reagent for biotechnology and medicine.
Project description:Next generation seuquencing of 3D breast cancer cell culture in synthetic hydrogels presenting either a collagen mimic or laminin mimic with low and high mechanical properties
Project description:Cells interact with their mechanical environment and respond in consequence. Mechanical cues can have a wide range of influences on cell behaviour, ranging from guidance of differentiation and cell fate to immune activation. The impact of substrate stiffness on primary macrophages - a key player in innate immunity and inflammation - had not been previously studied. We prepared bone marrow-derived macrophage cultures from adult rat hematopoietic stem cells exposed to M-CSF, and cultured these on polyacrylamide substrates of controlled stiffness (ranging from 50 to 0.1 kPa shear modulus, covering the range found in physiological tissues) for 3 days. The RNA from these cells was then extracted and sequenced.
Project description:Almost all solid tumors become stiff with progression of cancer. Cancer Associated Fibroblasts (CAFs), most abundant stromal cells in the tumor microenvironment (TME), are known to mediate the stiffening. While the biochemical crosstalk between CAFs and cancer cells have been widely investigated, it is yet not clear whether CAFs in stiffer TME promote metastatic progression, and if so, how. To address this question, here we explore the role of mechanical stiffness and cell traction force in regulating human colorectal CAF (CAF05 cell line) gene expressions that are relevant to metastatic progression. We cultured CAFs on 2D polyacrylamide hydrogels with increasing elastic modulus (E) of 1, 10 and 40 kPa, and quantified corresponding cell spreading area and cytoskeletal force. We performed genome-wide transcriptome analyses in these cells to identify differentially expressed genes on 40 KPa compared to those on 1 kPa substrate. The latter represents normal tissue stiffness of colon.
Project description:Almost all solid tumors become stiff with progression of cancer. Cancer Associated Fibroblasts (CAFs), most abundant stromal cells in the tumor microenvironment (TME), are known to mediate the stiffening. While the biochemical crosstalk between CAFs and cancer cells have been widely investigated, it is yet not clear whether CAFs in stiffer TME promote metastatic progression, and if so, how. To address this question, here we explore the role of mechanical stiffness and cell traction force in regulating human colorectal CAF (CAF05 cell line) gene expressions that are relevant to metastatic progression. We cultured CAFs on 2D polyacrylamide hydrogels with increasing elastic modulus (E) of 1, 10 and 40 kPa, and quantified corresponding cell spreading area and cytoskeletal force. We performed genome-wide transcriptome analyses in these cells to identify differentially expressed genes on 40 KPa compared to those on 1 kPa substrate. The latter represents normal tissue stiffness of colon.
Project description:Biologically ligands (e.g., RGDS from fibronectin: Fn-RGD) play a critical role in the development of chemically defined biomaterials. However, there has been limited progress in recent decades towards discovering novel extracellular-matrix-protein-derived ligands for translational applications. Here, by combining motif analysis of evolutionarily conserved RGD-containing regions in laminin (Ln) with functional microarray screening, we identified a Ln-derived angiogenic peptide (LDAP) that showed enhanced proangiogenic activities over commonly used Fn-RGD. Mechanistic studies using RNA-sequencing of LDAP functionalized hydrogels showed an improved angiogenic transcriptome over Fn-RGD functionalized hydrogels and high similarity to Matrigel, attributed to the ability of LDAP to engage both Ln- and Fn-binding integrins. Injectable hydrogels functionalized with LDAP, along with MMP-QK (a VEGF-mimetic peptide), exhibited increased functional recovery over decellularized extracellular matrix (dECM) and alginates functionalized with Fn-RGD and MMP-QK in a mouse ischemic hindlimb model, illustrating the power of the strategy to rapidly develop potent chemically-defined biomaterials for therapeutic applications.
Project description:Substrate elasticity may direct cell-fate decisions of stem cells. However, it is largely unclear how matrix stiffness impacts on differentiation of induced pluripotent stem cells (iPSCs) and if this is also reflected by epigenetic modifications. We have therefore cultured iPSCs on tissue culture plastic (TCP) and polydimethylsiloxane (PDMS) with different Young´s modulus (0.2 kPa, 16 kPa, or 64 kPa) to investigate the sequel on growth and differentiation towards endoderm, mesoderm, and ectoderm. Immunofluorescence and gene expression of canonical differentiation markers was hardly affected by the substrates. Notably, when we analyzed DNA methylation profiles of iPSCs or after three-lineage differentiation, we did not see any significant differences on the three different PDMS elasticities. Only when we compared DNA methylation profiles on PDMS-substrates versus TCP, we observed clear epigenetic differences, particularly upon mesodermal differentiation. Taken together, stiffness of PDMS-substrates did not impact on directed differentiation of iPSCs, whereas the moderate epigenetic differences on TCP might also be attributed to other chemical parameters.
Project description:Given the clinical need for osteoregenerative materials incorporating controlled biomimetic and biophysical cues, a novel norbornene-modified gelatin was developed with a degree of substitution of 169% compared to the number of amines present in pristine gelatin type B. It is, to the best of our knowledge, the highest substitution degree reported to date for norbornene-functionalised gelatin. Thiol-ene crosslinking exploiting thiolated gelatin as cell-interactive crosslinker resulted in networks with high (>95%) norbornene conversion. Comparing the number of physical crosslinks present, the degree of hydrolytic degradation upon modification, the network density as well as the chemical crosslinking type, the novel thiol-ene network was benchmarked against conventional gelatin-based systems in terms of the effect of biophysical cues, more specifically visco-elastic and topographical properties, affecting osteogenesis both in 2D and in 3D via a biofabrication strategy. The novel thiol-ene network outperformed conventional gelatin-based networks in terms of osteogenesis, as evidenced in 2D dental pulp stem cell seeding assays, resulting from the presentation of both a local (substrate elasticity, 25-40 kPa) and a bulk (compressive modulus, 25-45 kPa) osteogenic substrate modulus in combination with adequate fibrillar cell adhesion spacing to optimally transfer traction forces from the fibrillar ECM (as evidenced by mesh size determination with the rubber-elasticity theory) and resulting in a 1.5- and 1.7-fold increase in alkaline phosphatase activity and calcium production respectively as osteogenic markers (compared to the gold standard gelatin methacryloyl (GelMA)). Dental pulp stem cell encapsulation in extrusion-based biofabricated 3D constructs also showed a favorable response towards the novel thiol-ene network thanks to the combination of its RGD mobility (as observed from proteomic analysis), stress relaxation and substrate rigidity (bulk compressive modulus of 11-30 kPa) to enable a 1.5- and 7-fold increase in alkaline phosphatase activity and calcium production respectively as osteogenic markers (compared to the gold standard GelMA).