Altering adsorbed proteins or cellular gene expression in bone-metastatic cancer cells affects PTHrP and Gli2 without altering cell growth

AbstractThe contents of this data in brief are related to the article titled “Matrix Rigidity Regulates the Transition of Tumor Cells to a Bone-Destructive Phenotype through Integrin β3 and TGF-β Receptor Type II”. In this DIB we will present our supplemental data investigating Integrin expression, attachment of cells to various adhesion molecules, and changes in gene expression in multiple cancer cell lines. Since the interactions of Integrins with adsorbed matrix proteins are thought to affect the ability of cancer cells to interact with their underlying substrates, we examined the expression of Integrin β1, β3, and β5 in response to matrix rigidity. We found that only Iβ3 increased with increasing substrate modulus. While it was shown that fibronectin greatly affects the expression of tumor-produced factors associated with bone destruction (parathyroid hormone-related protein, PTHrP, and Gli2), poly-l-lysine, vitronectin and type I collagen were also analyzed as potential matrix proteins. Each of the proteins was independently adsorbed on both rigid and compliant polyurethane films which were subsequently used to culture cancer cells. Poly-l-lysine, vitronectin and type I collagen all had negligible effects on PTHrP or Gli2 expression, but fibronectin was shown to have a dose dependent effect. Finally, altering the expression of Iβ3 demonstrated that it is required for tumor cells to respond to the rigidity of the matrix, but does not affect other cell growth or viability. Together these data support the data presented in our manuscript to show that the rigidity of bone drives Integrinβ3/TGF-β crosstalk, leading to increased expression of Gli2 and PTHrP

Matrix Rigidity Induces Osteolytic Gene Expression of Metastatic Breast Cancer Cells

Nearly 70% of breast cancer patients with advanced disease will develop bone metastases. Once established in bone, tumor cells produce factors that cause changes in normal bone remodeling, such as parathyroid hormone-related protein (PTHrP). While enhanced expression of PTHrP is known to stimulate osteoclasts to resorb bone, the environmental factors driving tumor cells to express PTHrP in the early stages of development of metastatic bone disease are unknown. In this study, we have shown that tumor cells known to metastasize to bone respond to 2D substrates with rigidities comparable to that of the bone microenvironment by increasing expression and production of PTHrP. The cellular response is regulated by Rho-dependent actomyosin contractility mediated by TGF-ß signaling. Inhibition of Rho-associated kinase (ROCK) using both pharmacological and genetic approaches decreased PTHrP expression. Furthermore, cells expressing a dominant negative form of the TGF-ß receptor did not respond to substrate rigidity, and inhibition of ROCK decreased PTHrP expression induced by exogenous TGF-ß. These observations suggest a role for the differential rigidity of the mineralized bone microenvironment in early stages of tumor-induced osteolysis, which is especially important in metastatic cancer since many cancers (such as those of the breast and lung) preferentially metastasize to bone

3D Bone Morphology Alters Gene Expression, Motility, and Drug Responses in Bone Metastatic Tumor Cells

Patients with advanced skeletal metastases arising from primary cancers including breast, lung, and prostate suffer from extreme pain, bone loss, and frequent fractures. While the importance of interactions between bone and tumors is well-established, our understanding of complex cell–cell and cell–microenvironment interactions remains limited in part due to a lack of appropriate 3D bone models. To improve our understanding of the influence of bone morphometric properties on the regulation of tumor-induced bone disease (TIBD), we utilized bone-like 3D scaffolds in vitro and in vivo. Scaffolds were seeded with tumor cells, and changes in cell motility, proliferation, and gene expression were measured. Genes associated with TIBD significantly increased with increasing scaffold rigidity. Drug response differed when tumors were cultured in 3D compared to 2D. Inhibitors for Integrin β3 and TGF-β Receptor II significantly reduced bone-metastatic gene expression in 2D but not 3D, while treatment with the Gli antagonist GANT58 significantly reduced gene expression in both 2D and 3D. When tumor-seeded 3D scaffolds were implanted into mice, infiltration of myeloid progenitors changed in response to pore size and rigidity. This study demonstrates a versatile 3D model of bone used to study the influence of mechanical and morphometric properties of bone on TIBD

Relationships between Poly(ethylene glycol) Modifications on RNA–Spherical Nucleic Acid Conjugates and Cellular Uptake and Circulation Time

Two synthetic approaches that allow one to control PEG content within spherical nucleic acids (SNAs) have been developed. One approach begins with RNA-modified gold nanoparticles followed by a backfill of PEG 2K alkanethiols, and the other involves co-adsorption of the two entities on a gold nanoparticle template. These two methods have been used to explore the role of PEG density on the chemical and biological properties of RNA–SNAs. Such studies show that while increasing the extent of PEGylation within RNA–SNAs extends their blood circulation half-life in mice, it also results in decreased cellular uptake. Modified ELISA assays show that constructs, depending upon RNA and PEG content, have markedly different affinities for class A scavenger receptors, the entities responsible, in part, for cellular internalization of SNAs. In designing SNAs for therapeutic purposes, these competing factors must be considered and appropriately adjusted depending upon the desired use

TGF-ß mediates the response of MDA-MB-231 cells to substrate rigidity.

<p>(A) PTHrP gene expression increases significantly for MDA-MB-231 cells transfected with the plasmid control (MDA-pc), while MDA-MB-231 cells transfected to express a dominant negative form of the TGF-ß Type II receptor (MDA-TβRIIcyt ) and MCF-7 cells that do not express the TGF-ß Type II receptor show no increase in expression with rigidity. (B) TGF-ß signaling was measured in MDA-MB-231 cells transfected with the 3TP-Lux TGF-ß reporter construct. This showed an increase in TGF-ß signaling when cells are grown on rigid substrates. (C) TGF-ß1 mRNA expression by MDA-MB-231 cells increased on more rigid substrates. *,# = p<0.05; **,## = p<0.01; ***,### = p<0.005 compared to 0.00045MPa value.</p

Expression and secretion of PTHrP by osteolytic, metastatic tumor cells increases with increasing substrate modulus.

<p>PTHrP mRNA normalized by 18S as measured by qPCR and secreted PTHrP as measured by IRMA for (A) MDA-MB-231 , (B) RWGT2, and (C) MCF-7 cells on substrates of increasing elastic modulus. *,# = p<0.05; **,## = p<0.01; ***,### = p<0.005 compared to 0.00045MPa value. Changes in MCF-7 cells were not significant. (D)–(F) Morphology of MDA-MB-231 cells cultured on substrates of varying elastic modulus 4 hours post-attachment. Nuclei were stained using DAPI (blue) and cells were visualized with both visible light and GFP (green). Similar effects of the modulus on cell morphology were observed for RWGT2 cells, while no effects were observed for MCF-7 cells.</p

Expression of Gli2 and by MDA-MB-231 and RWGT2 cells increases with increasing substrate modulus.

<p>Expression of Gli2 mRNA normalized by 18S as measured by qPCR for (A) MDA-MB-231 and (B) RWGT2 cells on substrates of varying elastic modulus. Gli2 signaling increased 25-fold as the modulus increased from 3.3 to 1700 MPa (data not shown). *,# = p<0.05; **,## = p<0.01; ***,### = p<0.005 compared to 0.00045MPa value.</p

Schematic of materials synthesis and characterization.

<p>(A) Synthesis of PUR networks from an LDI quasi-prepolymer (red) and a poly(ε-caprolactone-co-glycolide) triol with molecular weight M_n = 3<i>w</i>, where <i>w</i> is the equivalent weight (g eq⁻¹). (B) Experimental values of the elastic modulus <i>E</i> of PUR networks as a function of equivalent weight <i>w</i>. (C) Physical characteristics of polymer networks as calculated from rubber elasticity theory. E_m and G_m represents measured elastic and shear moduli, is the fraction of polymer in the swollen mass, M_c is the calculated molecular weight between cross-links and E_c and G_c are the calculated elastic and shear moduli. (D) Schemes for providing a uniform surface concentration of fibronectin (Fn) for polyurethane networks and polyacrylamide gels.</p