Lung cancer is the leading cause of cancer-related death, primarily due to distant metastatic disease. Metastatic lung cancer cells can undergo an epithelial-to-mesenchymal transition (EMT) regulated by many transcription factors, including double-negative feedback loop between the microRNA-200 (miR-200) family and ZEB1, but the precise mechanisms by which ZEB1-dependent EMT promotes malignancy remain largely undefined. While the cell-intrinsic effects of EMT are important for tumor progression, the reciprocal dynamic crosstalk between mesenchymal cancer cells and the extracellular matrix (ECM) is equally critical in regulating invasion and metastasis. Investigating the collaborative effect of EMT and ECM in the metastatic process reveals increased collagen deposition in metastatic tumor tissues as a direct consequence of amplified collagen gene expression in ZEB1-activated mesenchymal lung cancer cells. Additionally, collagen fibers in metastatic lung tumors exhibit greater linearity and organization as a result of collagen crosslinking by the lysyl oxidase (LOX) family of enzymes. Expression of the LOX and LOXL2 isoforms is directly regulated by miR-200 and ZEB1, respectively, and their upregulation in metastatic tumors and mesenchymal cell lines is coordinated to that of collagen. Functionally, LOXL2, as opposed to LOX, is the principle isoform that crosslinks and stabilizes insoluble collagen deposition in tumor tissues. In turn, focal adhesion formation and FAK/SRC signaling is activated in mesenchymal tumor cells by crosslinked collagen in the ECM. Our study is the first to validate direct regulation of LOX and LOXL2 by the miR-200/ZEB1 axis, defines a novel mechanism driving tumor metastasis, delineates collagen as a prognostic marker, and identifies LOXL2 as a potential therapeutic target against tumor progression.

Transmission electron microscopy (TEM) of hygroscopic, permeable, and electron-absorbing biological cells has been an important challenge due to the volumetric shrinkage, electrostatic charging, and structural degradation of cells under high vacuum and fixed electron beam.(1-3) Here we show that bacterial cells can be encased within a graphenic chamber to preserve their dimensional and topological characteristics under high vacuum (10(-5) Torr) and beam current (150 A/cm(2)). The strongly repelling π clouds in the interstitial sites of graphene's lattice(4) reduces the graphene-encased-cell's permeability(5) from 7.6-20 nm/s to 0 nm/s. The C-C bond flexibility(5,6) enables conformal encasement of cells. Additionally, graphene's high Young's modulus(6,7) retains cell's structural integrity under TEM conditions, while its high electrical(8) and thermal conductivity(9) significantly abates electrostatic charging. We envision that the graphenic encasement approach will facilitate real-time TEM imaging of fluidic samples and potentially biochemical activity.

Intratumoral collagen cross-links heighten stromal stiffness and stimulate tumor cell invasion, but it is unclear how collagen cross-linking is regulated in epithelial tumors. To address this question, we used KrasLA1 mice, which develop lung adenocarcinomas from somatic activation of a KrasG12D allele. The lung tumors in KrasLA1 mice were highly fibrotic and contained cancer-associated fibroblasts (CAFs) that produced collagen and generated stiffness in collagen gels. In xenograft tumors generated by injection of wild-type mice with lung adenocarcinoma cells alone or in combination with CAFs, the total concentration of collagen cross-links was the same in tumors generated with or without CAFs, but co-injected tumors had higher hydroxylysine aldehyde-derived collagen cross-links (HLCCs) and lower lysine-aldehyde-derived collagen cross-links (LCCs). Therefore, we postulated that an LCC-to-HLCC switch induced by CAFs promotes the migratory and invasive properties of lung adenocarcinoma cells. To test this hypothesis, we created co-culture models in which CAFs are positioned interstitially or peripherally in tumor cell aggregates, mimicking distinct spatial orientations of CAFs in human lung cancer. In both contexts, CAFs enhanced the invasive properties of tumor cells in 3-dimensional (3D) collagen gels. Tumor cell aggregates that attached to CAF networks on a Matrigel surface dissociated and migrated on the networks. Lysyl hydroxylase 2 (PLOD2/LH2), which drives HLCC formation, was expressed in CAFs, and LH2 depletion abrogated the ability of CAFs to promote tumor cell invasion and migration.

The use of biodegradable beta-tricalcium phosphate (β-TCP) scaffolds holds great promise for bone tissue engineering. However, the effects of β-TCP on bone and endothelial cells are not fully understood. This study was to investigate cell proliferation and differentiation of mono- or co-cultured human bone marrow-derived mesenchymal stem cells (hBMSCs) and human umbilical vein endothelial cells (HUVECs) on a 3D porous, biodegradable β-TCP scaffold. In co-culture studies, the ratio of hBMSCs:HUVECs were 5:1, 1:1 and 1:5. Cellular morphologies of HUVECs, hBMSCs, and co-cultured HUVECs/hBMSCs on the β-TCP scaffolds were monitored using confocal and scanning electron microscopy. Cell proliferation was monitored by measuring the amount of dsDNA whereas hBMSCs and HUVECs differentiation were assessed using the osteogenic and angiogenic markers, alkaline phosphatase (ALP) and PECAM-1 (CD31), respectively. Results show that HUVECs, hBMSCs, and hBMSCs/HUVECs adhered and proliferated well on the β-TCP scaffolds. In monoculture, hBMSCs grew faster than HUVECs on the β-TCP scaffolds after 7 days, but HUVECs reached similar levels of proliferation after 14 days. In monoculture, β-TCP scaffolds promoted ALP activities of both hBMSCs and HUVECs when compared to those grown on tissue culture well plates. ALP activity of cells in co-culture was higher than that of hBMSCs in monoculture. Real-time PCR results indicate that runx2 and alp gene expression in monocultured hBMSCs remained unchanged at day 7 and 14, but alp gene expression was significantly increased in hBMSCs co-cultures in absence of distinguishing the contribution of individual cell type.

Bioprosthetic heart valve implants are beset by calcification and failure due to the interactions between the body and the transplant. Hydrogels can be used as biological blank slates that may help to shield implants from these interactions; however, traditional light-based hydrogel polymerization is impeded by tissue opacity and topography. Therefore, new methods must be created to bind hydrogel to implant tissues. To address these complications, a two-step surface-coating method for bioprosthetic valves was developed. A previously developed bioprosthetic valve model (VM) was used to investigate and optimize the coating method. Generally, this coating is achieved by first reacting surface amine groups with an NHS-PEG-acrylate while also allowing glucose to absorb into the bulk. Then, glucose oxidase, poly(ethylene glycol) diacrylate (PEGDA), and iron ions are added to the system to initiate free-radical polymerization that bonds the PEGDA hydrogel to the acrylates sites on the surface. Results showed a thin (∼8 μm), continuous coating on VM samples that is capable of repelling protein adhesion (2% surface fouling versus 20% on uncoated samples) and does not significantly affect the surface mechanical properties. Based on this success, the coating method was translated to glutaraldehyde-fixed valve tissue samples. Results showed noncontinuous but evident coating on the surface, which was further improved by adjusting the coating solution. These results demonstrate the feasibility of the proposed two-step surface coating method for modifying the surface of bioprosthetic valve replacements.