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Circulating tumor cells are of utmost importance among various biomarkers in liquid biopsies as a prognosis indicator of metastasis as well as in chemotherapeutic monitoring. This study introduces an efficient tool composed of soft nano/hybrid immune microgels for magnetic isolation of targeted tumor cells. The development process involves the in situ synthesis of magnetic nanoparticles within the three-dimensional matrix of thermoresponsive microgels. Surface modification and anti-EpCAM conjugation are adjusted by changing the temperature, and a conjugation efficiency of around 70% is achieved by using a protein G linker. Anti-EpCAM-conjugated nano/hybrid magnetic microgels are used to isolate EpCAM-expressing breast adenocarcinoma MCF-7 cells from culture media and whole blood with an efficiency of 75 and 70%, respectively. Furthermore, we demonstrate the ability of the hybrid microgels to isolate cancer cells with a purity of 65% and culture the cells post-isolation for further drug studies. The multifunctional hybrid microcarriers reported in this work can be potentially used for continuous monitoring of cancers and in personalized medicine.

Rhabdomyosarcoma (RMS) is the most common pediatric soft-tissue malignant tumor. Treatment of RMS usually includes primary tumor resection along with systemic chemotherapy. Two-dimensional (2D) cell culture systems and animal models have been extensively used for investigating the potential efficacy of new RMS treatments. However, RMS cells behave differently in 2D culture than in vivo, which has recently inspired the adoption of three-dimensional (3D) culture environments. In the current paper, we will describe the detailed methodology we have developed for fabricating a 3D engineered model to study alveolar RMS (ARMS) in vitro. This model consists of a thermally cross-linked collagen disk laden with RMS cells that mimics the structural and bio-chemical aspects of the tumor extracellular matrix (ECM). This process is highly reproducible and produces a 3D engineered model that can be used to analyze the cytotoxicity and autophagy induction of drugs on ARMS cells. The most improtant bullet points are as following:•We fabricated 3D model of ARMS.•The current ARMS 3D model can be used for screening of chemotherapy drugs.•We developed methods to detect apoptosis and autophagy in ARMS 3D model to detect the mechansims of chemotherapy agents., Graphical abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft-tissue malignant tumor. Despite numerous clinical trials that have investigated the efficacy of multidrug chemotherapy regimens, outcomes for high-risk RMS patients are still poor which has inspired research into new targeted therapies that molecularly target malignant RMS cells. In order to evaluate such therapies in vitro in a realistic tumor microenvironment, we have bioengineered a cell-laden 3D tissue-engineered model with uniform geometry and an uncomplicated and reproducible biofabrication process. The transparent collagen-I hydrogel promotes cell attachment and growth and allows for analysis of the cellular response to drugs through brightfield microscopy, immunocytochemistry, and live/dead viability assays.Image, graphical abstract

Glioblastoma multiforme (GBM) is a rapidly progressive and deadly form of brain tumor with a median survival rate of ~15 months. GBMs are hard to treat and significantly affect the patient's physical and cognitive abilities and quality of life. Temozolomide (TMZ)&mdash, an alkylating agent that causes DNA damage&mdash, is the only chemotherapy choice for the treatment of GBM. However, TMZ also induces autophagy and causes tumor cell resistance and thus fails to improve the survival rate among patients. Here, we studied the drug-induced programmed cell death and invasion inhibition capacity of TMZ and a mevalonate cascade inhibitor, simvastatin (Simva), in a three-dimensional (3D) microfluidic model of GBM. We elucidate the role of autophagy in apoptotic cell death by comparing apoptosis in autophagy knockdown cells (Atg7 KD) against their scrambled counterparts. Our results show that the cells were significantly less sensitive to drugs in the 3D model as compared to monolayer culture systems. An immunofluorescence analysis confirmed that apoptosis is the mechanism of cell death in TMZ- and Simva-treated glioma cells. However, the induction of apoptosis in the 3D model is significantly lower than in monolayer cultures. We have also shown that autophagy inhibition (Atg7 KD) did not change TMZ and Simva-induced apoptosis in the 3D microfluidic model. Overall, for the first time in this study we have established the simultaneous detection of drug induced apoptosis and autophagy in a 3D microfluidic model of GBM. Our study presents a potential ex vivo platform for developing novel therapeutic strategies tailored toward disrupting key molecular pathways involved in programmed cell death and tumor invasion in glioblastoma.

Statins are some of the most widely used drugs worldwide, but one of their major side effects is myotoxicity. Using mouse myoblast (C2C12) and human alveolar rhabdomyosarcoma cell lines (RH30) in both 2-dimensional (2D) and 3-dimensional (3D) cell culture, we investigated the mechanisms of simvastatin's myotoxicity. We found that simvastatin significantly reduced cell viability in C2C12 cells compared to RH30 cells. However, simvastatin induced greater apoptosis in RH30 compared to C2C12 cells. Simvastatin-induced cell death is dependent on geranylgeranyl pyrophosphate (GGPP) in C2C12 cells, while in RH30 cells it is dependent on both farnesyl pyrophosphate (FPP) and GGPP. Simvastatin inhibited autophagy flux in both C2C12 and RH30 cells and inhibited lysosomal acidification in C2C12 cells, while autophagy inhibition with Bafilomycin-A1 increased simvastatin myotoxicity in both cell lines. Simvastatin induced greater cell death in RH30 cells compared to C2C12 in a 3D culture model with similar effects on autophagy flux as in 2D culture. Overall, our results suggest that simvastatin-induced myotoxicity involves both apoptosis and autophagy, where autophagy serves a pro-survival role in both cell lines. The sensitivity to simvastatin-induced myotoxicity differs between 2D and 3D culture, demonstrating that the cellular microenvironment is a critical factor in regulating simvastatin-induced cell death in myoblasts.

Temozolomide (TMZ) is a chemotherapy agent used to treat Grade IV astrocytoma, also known as glioblastoma (GBM). TMZ treatment causes DNA damage that results in tumor cell apoptosis and increases the survival rate of GBM patients. However, chemoresistance as a result of TMZ-induced autophagy significantly reduces this anticancer effects over time. Statins are competitive inhibitors of HMG-CoA reductase, the rate-limiting enzyme of the mevalonate (MEV) cascade. Statins are best known for their cholesterol (CH)-lowering effect. Long-term consumption of statins, prior to and in parallel with other cancer therapeutic approaches, has been reported to increase the survival rate of patients with various forms of cancers. In this study, we investigated the potentiation of TMZ-induced apoptosis by simvastatin (Simva) in human GBM cell lines and patient GBM cells, using cell monolayers and three-dimensional cell culture systems. The incubation of cells with a combination of Simva and TMZ resulted in a significant increase in apoptotic cells compared to cells treated with TMZ alone. Incubation of cells with CH or MEV cascade intermediates failed to compensate the decrease in cell viability induced by the combined Simva and TMZ treatment. Simva treatment inhibited the autophagy flux induced by TMZ by blocking autophago-lysosome formation. Our results suggest that Simva sensitizes GBM cells to TMZ-induced cell death in a MEV cascade-independent manner and identifies the inhibition of autophagosome-lysosome fusion as a promising therapeutic strategy in the treatment of GBM.

Rhabdomyosarcoma (RMS) is a muscle-derived tumor. In both pre-clinical and clinical studies Temozolomide (TMZ) has been recently tested against RMS; however, the precise mechanism of action of TMZ in RMS remains unclear. Here we demonstrate that TMZ decreases the cell viability of the RH30 RMS and C2C12 cell line, where cells display evidence of mitochondrial outer membrane permeability. Interestingly, the C2C12 mouse myoblast line was relatively more resistant to TMZ-induced apoptosis. Moreover, we observed that TMZ activated biochemical and morphological markers of autophagy in both cell lines. Autophagy inhibition in both RH30 and C2C12 cells significantly increased TMZ-induced cell death. In RH30 cells, TMZ increased Mcl-1 and Bax protein expression compared to corresponding time match controls while in C2C12 Mcl-1, Bcl-2, Bcl-XL, and Bax protein expression were not changed. Baf-A1 co-treatment with TMZ significantly decrease Mcl-1 expression compared to TMZ while increase Bax expression in C2C12 cells (Bcl2 and Bcl-XL do not significantly change in Baf-A1/TMZ co-treatment). Using a three-dimensional (3D) C2C12 and RH30 culture model we demonstrated that TMZ is significantly more toxic in RH30 cells (live/dead assay). Additionally, we have observed in our 3D culture model that TMZ induced both apoptosis (cleavage of PARP) and autophagy (LC3-puncta and localization of LC3/p62). Therefore, our data demonstrate that TMZ induces simultaneous autophagy and apoptosis in both RH30 and C2C12 cells in 2D and 3D culture model, where RH30 cells are more sensitive to TMZ-induced death. Furthermore, autophagy serves to protect RH30 cells from TMZ-induced death.

The present study is intended to investigate and compare the hemodynamics in two different sizes of hemodialysis arteriovenous grafts for upper arm hemodialysis vascular access: 8-mm tapered to 6-mm at the arterial side and straight 6 mm. A computational simulation approach is presented for this study, which is validated against the available experimental and numerical pressure measurements in the literature. The imposed boundary conditions at the arterial inlet and venous outlet boundaries of the models are physiological velocity and pressure waveforms, respectively. Blood flow fields and distribution patterns of the hemodynamic indices including wall shear stress (WSS) as one of the major hemodynamic parameters of the cardiovascular system and spatial wall shear stress gradient (SWSSG) as an indicator of disturbed flow patterns and hence susceptible sites of lesion developments are analyzed and compared between the two grafts. The tapered 6- to 8-mm graft seemingly is associated with less disturbed flow patterns within the venous anastomosis (VA) and the vein downstream while benefiting from higher blood flow rates within. Also, it shows a definitive advantage in terms of WSS and SWSSG distribution patterns around the VA and throughout the vein downstream with significantly lower values, which reduce the risk of thrombosis formation and stenotic lesion developments. The only disadvantage encountered in using 6- to 8-mm tapered graft is higher values of hemodynamic parameters at the arterial junction attributable to its significantly higher mean blood flow rate within. The results clearly indicate that the tapered 6- to 8-mm graft entirely outperforms straight 6-mm graft hemodynamically as an upper arm hemodialysis vascular access graft and confirms clinical data in the literature, which suggests advantageous use of tapered 6- to 8-mm grafts in the creation of upper arm brachioaxillary hemodialysis vascular access grafts in selected groups of patients with expectably higher patency rates and lower complications.

Hemodialysis vascular access dysfunction is still considered as a major cause of morbidity and mortality in hemodialysis patients. The most common cause of this vascular access dysfunction is venous stenosis secondary to the progression of venous intimal hyperplasia at the graft-vein junction or in the draining vein. The geometry of the vessels and the local hemodynamics are believed to be contributory factors in the development of intimal hyperplasia. In the present study, the hemodynamic parameters in two types of vascular access grafts of different diameters: 6 mm and 8 mm tapered to 6 mm at the arterial site were compared. A three-dimensional CFD model was developed to simulate blood flow in the two graft types. The boundary conditions applied were a pulsatile velocity waveform at the arterial inlet and a pulsatile pressure waveform at the venous outlet. Computational fluid dynamics software was used to solve the time dependent Navier-Stokes equations under physiological conditions in both graft types. Since the cross section area and the diameter of the venous side of the tapered graft are larger than those of the straight one, the flow aligns well with the vein downstream of the graft and hence the smaller vortices are generated. Comparing the parameters at the two venous anastomosis regions also indicates that in spite of the higher flow rate in 6–8 mm tapered graft, the velocity and the wall shear stress values are lower than the values of the 6 mm straight graft.Copyright © 2010 by ASME

Hemodialysis vascular access failure considered as a primary cause of morbidity and hospital admissions for chronic hemodialysis patients. Clearly the hemodynamics within a vascular access graft and also the anastomotic geometry induced disturbed flow patterns have a substantial influence on the initiation and development of “intimal hyperplasia” the most common cause of vascular access failures. The present study is intended to investigate and compare the hemodynamics in two types of hemodialysis vascular access grafts of different diameters: 6 mm straight versus 8 mm tapered to 6 mm at the arterial site in an upper arm brachio-axillary prosthetic graft in straight configuration. Using a three-dimensional CFD model of an upper arm straight vascular access graft the two graft types have been compared in terms of overall flow pattern, wall shear stress distribution and other hemodynamic parameters. The boundary conditions applied are a physiological pulsatile velocity waveform at the arterial inlet and a pulsatile pressure waveform at the venous outlet. Results show higher values of wall shear stresses at the arterial amastomosis region of the 6-8 mm tapered graft over its 6 mm straight graft counterpart. Comparing the parameters at the two venous anastomosis regions indicates that in spite of the higher flow rate in 6-8 mm tapered graft, the velocity and the wall shear stress values are lower than the values of the 6 mm straight graft. The larger diameter and hence the larger venous anastomotic length of the tapered 6-8 mm graft let the flow align well with the vein downstream of the graft with less perturbation and thereby smaller the vortices generated and lower the wall shear stress values at the vein floor opposite to the graft outflow in comparison with the straight 6 mm graft.