Mesenchymal-endothelial nexus in breast cancer spheroids induces vasculogenesis and local invasion in a CAM model.

Interplay between non-cancerous cells (immune, fibroblasts, mesenchymal stromal cells (MSC), and endothelial cells (EC)) has been identified as vital in driving tumor progression. As studying such interactions in vivo is challenging, ex vivo systems that can recapitulate in vivo scenarios can aid in unraveling the factors impacting tumorigenesis and metastasis. Using the synthetic tumor microenvironment mimics (STEMs)-a spheroid system composed of breast cancer cells (BCC) with defined human MSC and EC fractions, here we show that EC organization into vascular structures is BC phenotype dependent, and independent of ERα expression in epithelial cancer cells, and involves MSC-mediated Notch1 signaling. In a 3D-bioprinted model system to mimic local invasion, MDA STEMs collectively respond to serum gradient and form invading cell clusters. STEMs grown on chick chorioallantoic membrane undergo local invasion to form CAM tumors that can anastomose with host vasculature and bear the typical hallmarks of human BC and this process requires both EC and MSC. This study provides a framework for developing well-defined in vitro systems, including patient-derived xenografts that recapitulate in vivo events, to investigate heterotypic cell interactions in tumors, to identify factors promoting tumor metastasis-related events, and possibly drug screening in the context of personalized medicine.

Unraveling the role of ß1 integrin isoforms in cRGD-mediated uptake of nanoparticles bearing hydrophilized alkyne moieties in epithelial and endothelial cells.

The uptake and trafficking of NPs is impacted by several attributes such as size, shape, surface charge and importantly by surface ligands that can interact with the cell plasma membrane. We envision that NPs which can be readily modified in aqueous environments will be key to engineering patient-specific nanotherapeutics. Towards such systems that can be functionalized "on demand" in aqueous environments, an α-ω epoxy ester monomer that bears an alkyne group at the end of an oligoethylene glycol moiety was designed and synthesized. Copolymerization of this monomer with ε-caprolactone yielded polymers that present hydrophilized alkyne groups along the backbone. This enabled the direct modification of the surface of NPs, as suspensions in aqueous phase, with cell interaction peptides such cyclic-arginine-glycine-aspartic acid (cRGD) using the "click reaction". Uptake of cRGD modified NPs (cRGD-NPs) in human endothelial and tumor epithelial cells revealed that cRGD surprisingly diminished uptake in both tumor epithelial and microvascular endothelial cells by 40-50 percent in comparison to unmodified particles. Probing the mechanism of uptake revealed that the expression pattern of two isoforms of ß1 integrin impacted the uptake of cRGD-NPs differently. While the expression of high molecular weight 140 kDa form of the ß1 integrin enhanced NP uptake, the expression of low molecular 120 kDa form had an inhibitory effect. Furthermore, although, the expression of ß3 integrin was enhanced in endothelial cells and breast cancer epithelial cells, no correlation between ß3 integrin and NP uptake was observed. Additionally, in presence of clathrin and caveolae pathway inhibitors the uptake of cRGD-NPS was in general diminished with a 25-75% decrease in presence of Filipin, a caveolae inhibitor; suggesting a role for lipid rafts in the ß1 integrin-mediated uptake of cRGD-NP NPs. In sum, the polymer system described can be readily adapted to engineer other targeting peptide-based nanotherapeutics, especially for the delivery across difficult penetrate biological barriers such as the blood brain barrier. The main findings of this study have significant implication for the development of integrin targeted nanotherapeutics for anti-tumor therapy.

Renal clearance of polymeric nanoparticles by mimicry of glycan surface of viruses.

It has been shown that viral particles such as herpes simplex virus-1 and cytomegalovirus show renal clearance despite their large size (155-240 nm). Interestingly, one of the common characteristics of these viruses is their glycoprotein rich viral envelope. Since, glycosaminoglycans (GAGs) share similarities with oligosaccharide chains in the glycoproteins, we hypothesize that modification of nanoparticles (NPs) surface with naturally found GAGs could alter NP clearance characteristics by mimicking physicochemical aspects of viral glycoprotein envelope. We demonstrate that polymeric NP bearing surfaces enriched with dermatan sulfate, chondroitin sulfate, heparin sulfate, and hyaluronic acid undergo rapid renal clearance (74% of injected dose as early as 2 h) while showing reduced liver accumulation. Ultra-structural analyses suggest that the excretion of intact NPs occurs via proximal tubule secretion, but not via glomerular filtration. Finally, we demonstrate that our bioinspired NPs are able to accumulate within the epithelial tumor microenvironment despite their efficient renal clearance. Our system provides a framework to address renal toxicity associated with repeated dosing of NP and a platform to elaborate on plausible mechanism of renal clearance of virus particle.

Autophagy inhibition enhances Matrine derivative MASM induced apoptosis in cancer cells via a mechanism involving reactive oxygen species-mediated PI3K/Akt/mTOR and Erk/p38 signaling.

BACKGROUND: In the quest for new anti-cancer drugs, the drug discovery process has shifted to screening of active ingredients in traditional eastern medicine. Matrine is an active alkaloid isolated from plants of the Sophora genus used in traditional Chinese herbal medicine that exhibits a wide spectrum of biological properties and has a potential as an anti-proliferative agent. In this study, we investigated the anticancer property of MASM, ([(6aS, 10S, 11aR, 11bR, 11cS)210-Methylamino-dodecahydro-3a, 7a-diaza-benzo (de)anthracene-8-thione]), a potent derivative of matrine. METHODS: Four epithelial cancer cell lines representing the dominant cancers, namely: A549 (non-small-cell lung cancer cell line), MCF-7 and MDA-MB-231 (breast cancer cell lines), and Hela (cervical cancer cell line) were employed, and the mechanistic underpinning of MASM-induced apoptosis was investigated using flow cytometry, western blot and immunofluorescence. RESULTS: MASM, induced apoptosis via caspase 3 dependent and independent pathways, and autophagy in all the four cancer cell lines, but post-EMT (epithelial mesenchymal transition) cells showed greater sensitivity to MASM. Scavenging reactive oxygen species using N-acetylcysteine rescued all cancer cell lines from apoptosis and autophagy. Mechanistic analysis revealed that MASM induced autophagy involves inhibition of Akt signaling and the activation of Erk and p38 signaling, and inhibition of autophagy further enhanced the apoptosis induced by MASM. CONCLUSIONS: These results indicate that MASM possesses potency against cancer cells and modulating autophagy during MASM administration could be used to further enhance its therapeutic effects.

Mechanically Defined Microenvironment Promotes Stabilization of Microvasculature, Which Correlates with the Enrichment of a Novel Piezo-1+ Population of Circulating CD11b+ /CD115+ Monocytes.

Vascularization is a critical step in the restoration of cellular homeostasis. Several strategies including localized growth factor delivery, endothelial progenitor cells, genetically engineered cells, gene therapy, and prevascularized implants have been explored to promote revascularization. But, long-term stabilization of newly induced vessels remains a challenge. It has been shown that fibroblasts and mesenchymal stem cells can stabilize newly induced vessels. However, whether an injected biomaterial alone can serve as an instructive environment for angiogenesis remains to be elucidated. It is reported here that appropriate vascular branching, and long-term stabilization can be promoted simply by implanting a hydrogel with stiffness matching that of fibrin clot. A unique subpopulation of circulating CD11b+ myeloid and CD11b+ /CD115+ monocytes that express the stretch activated cation channel Piezo-1, which is enriched prominently in the clot-like hydrogel, is identified. These findings offer evidence for a mechanobiology paradigm in angiogenesis involving an interplay between mechanosensitive circulating cells and mechanics of tissue microenvironment.

Cell number in mesenchymal stem cell aggregates dictates cell stiffness and chondrogenesis.

BACKGROUND: Although mesenchymal stem/stromal cell (MSC) chondrogenic differentiation has been thoroughly investigated, the rudiments for enhancing chondrogenesis have remained largely dependent on external cues. Focus to date has been on extrinsic variables such as soluble signals, culture conditions (bioreactors), and mechanical stimulation. However, the role of intrinsic mechanisms of MSC programming-based mechanobiology remains to be explored. Since aggregation of MSCs, a prerequisite for chondrogenesis, generates tension within the cell agglomerate, we inquired if the initial number of cells forming the aggregate (aggregate cell number (ACN)) can impact chondrogenesis. METHODS: Aggregates of varying ACN were formed using well-established centrifugation approach. Progression of chondrogenic differentiation in the aggregates was assessed over 3 weeks in presence and absence of transforming growth factor-beta 1 (TGF-ß1). Mechanical properties of the cells were characterized using high-throughput real-time deformability cytometry (RT-DC), and gene expression was analyzed using Affymetrix gene array. Expression of molecular markers linked to chondrogenesis was assessed using western blot and immunofluorescence. RESULTS: Reducing ACN from 500 k to 70 k lead to activation and acceleration of the chondrogenic differentiation, independent of soluble chondro-inductive factors, which involves changes to ß-catenin-dependent TCF/LEF transcriptional activity and expression of anti-apoptotic protein survivin. RT-DC analysis revealed that stiffness and size of cells within aggregates are modulated by ACN. A direct correlation between progression of chondrogenesis and emergence of stiffer cell phenotype was found. Affymetrix gene array analysis revealed a downregulation of genes associated with lipid synthesis and regulation, which could account for observed changes in cell stiffness. Immunofluorescence and western blot analysis revealed that increasing ACN upregulates the expression of lipid raft protein caveolin-1, a ß-catenin binding partner, and downregulates the expression of N-cadherin. As a demonstration of the relevance of these findings in MSC-based strategies for skeletal repair, it is shown that implanting aggregates within collagenous matrix not only decreases the necessity for high cell numbers but also leads to marked improvement in the quality of the deposited tissue. CONCLUSIONS: This study presents a simple and donor-independent strategy to enhance the efficiency of MSC chondrogenic differentiation and identifies changes in cell mechanics coincident with MSC chondrogenesis with potential translational applications.

Hyperstimulation of CaSR in human MSCs by biomimetic apatite inhibits endochondral ossification via temporal down-regulation of PTH1R.

In adult bone injuries, periosteum-derived mesenchymal stem/stromal cells (MSCs) form bone via endochondral ossification (EO), whereas those from bone marrow (BM)/endosteum form bone primarily through intramembranous ossification (IMO). We hypothesized that this phenomenon is influenced by the proximity of MSCs residing in the BM to the trabecular bone microenvironment. Herein, we investigated the impact of the bone mineral phase on human BM-derived MSCs' choice of ossification pathway, using a biomimetic bone-like hydroxyapatite (BBHAp) interface. BBHAp induced hyperstimulation of extracellular calcium-sensing receptor (CaSR) and temporal down-regulation of parathyroid hormone 1 receptor (PTH1R), leading to inhibition of chondrogenic differentiation of MSCs even in the presence of chondroinductive factors, such as transforming growth factor-ß1 (TGF-ß1). Interestingly rescuing PTH1R expression using human PTH fragment (1-34) partially restored chondrogenesis in the BBHAp environment. In vivo studies in an ectopic site revealed that the BBHAp interface inhibits EO and strictly promotes IMO. Furthermore, CaSR knockdown (CaSR KD) disrupted the bone-forming potential of MSCs irrespective of the absence or presence of the BBHAp interface. Our findings confirm the expression of CaSR in human BM-derived MSCs and unravel a prominent role for the interplay between CaSR and PTH1R in regulating MSC fate and the choice of pathway for bone formation.

Liposomal Treatment of Cancer Cells Modulates Uptake Pathway of Polymeric Nanoparticles by Altering Membrane Stiffness.

Nanomedicines can be taken up by cells via nonspecific and dynamin-dependent (energy-dependent) clathrin and caveolae-mediated endocytosis. While significant effort has focused on targeting pathway-specific transporters, the role of nanobiophysics in the cell lipid bilayer nanoparticle uptake pathway remains largely unexplored. In this study, it is demonstrated that stiffness of lipid bilayer is a key determinant of uptake of liposomes by mammalian cells. Dynamin-mediated endocytosis (DME) of liposomes is found to correlate with its phase behavior, with transition toward solid phase promoting DME, and transition toward fluidic phase resulting in dynamin-independent endocytosis. Since liposomes can transfer lipids to cell membrane, it is sought to engineer the biophysical properties of the membrane of breast epithelial tumor cells (MD-MBA-231) by treatment with phosphatidylcholine liposomes, and elucidate its effect on the uptake of polymeric nanoparticles. Analysis of the giant plasma membrane vesicles derived from treated cells using flicker spectroscopy reveals that liposome treatment alters membrane stiffness and DME of nanoparticles. Since liposomes have a history of use in drug delivery, localized priming of tumors with liposomes may present a hitherto unexploited means of targeting tumors based on biophysical interactions.