An integrated microfluidic cell array for apoptosis and proliferation analysis induction of breast cancer cells

In vitro sensitivity testing of tumor cells could rationalize and improve the choice of chemotherapy and hormone therapy. In this report, a microfluidic device made from poly(dimethylsiloxane) and glass was developed for an assay of drug induced cytotoxicity. We evaluated the apoptotic and proliferation-inhibitory effects of anticancer drugs mitomycin C (MMC) and tamoxifen (TAM) using MCF-7 breast cancer cells. MMC and TAM both induced apoptosis and inhibited proliferation of MCF-7 cells in a concentration-dependent manner. MMC caused the expression of antiapoptotic protein Bcl-2 a dose-dependent reduction in MCF-7 cells. The expression of Bcl-2 did not change significantly in MCF-7 cells treated by TAM. The results in the microfluidic device were correlated well with the data obtained from the parallel experiments carried out in the conventional culture plates. The developed microfluidic device could be a potential useful tool for high content screening and high throughput screening research.     

Status of epidermal growth factor receptors family in hormone-dependent carcinomas of the breast and prostate with reference to serum lipids and lipoproteins

There are numerous growing evidences of resemblance between carcinomas of the breast and prostate. A total of 45 cases of these two hormone-dependent cancers along with appropriate controls were subjected for status of epidermal growth factor receptors as well as serum lipid profile. Paraffin embedded tissue sections from aforesald tumours were analysed by immunohistochemical staining for epidermal growth factor receptor (EGF-R), c-erbB-2 oncoprotein, estrogen receptor (ER) and progesterone receptor (PgR). Sera from same individuals were studied for serum lipid profile analysis. The study revealed that immunoexpression of all receptor proteins (EGF-R). c-erbB-2 was significantly higher in breast carcinoma. In addition, mean levels of triglycerides, total cholesterol, LDL-cholesterol were found to be significantly elevated while the level of HDL-cholesterol was observed to be lower among patients with breast cancer as compared to matched controls. Further, ER-positive breast cancer cases have significantly higher mean level of HDL-cholesterol when compared with ER-negative breast cancer patients. Contrary to this, no alteration in different serum lipid fractions was noticed among the patients with prostate cancer. However, a positive relationship was noticed between immunoexpressions of EGF-R and c-erbB-2 in prostate cancer.     

Quick chip assay using locked nucleic acid modified epithelial cell adhesion molecule and nucleolin aptamers for the capture of circulating tumor cells

The role of circulating tumor cells (CTCs) in disease diagnosis, prognosis, monitoring of the therapeutic efficacy, and clinical decision making is immense and has attracted tremendous focus in the last decade. We designed and fabricated simple, flat channel microfluidic devices polydimethylsiloxane (PDMS based) functionalized with locked nucleic acid (LNA) modified aptamers (targeting epithelial cell adhesion molecule (EpCAM) and nucleolin expression) for quick and efficient capture of CTCs and cancer cells. With optimized flow rates (10 μl/min), it was revealed that the aptamer modified devices offered reusability for up to six times while retaining optimal capture efficiency (>90%) and specificity. High capture sensitivity (92%) and specificity (100%) was observed in whole blood samples spiked with Caco-2 cells (10–100 cells/ml). Analysis of blood samples obtained from 25 head and neck cancer patients on the EpCAM LNA aptamer functionalized chip revealed that an average count of 5 ± 3 CTCs/ml of blood were captured from 22/25 samples (88%). EpCAM intracellular domain (EpICD) immunohistochemistry on 9 oral squamous cell carcinomas showed the EpICD positivity in the tumor cells, confirming the EpCAM expression in CTCs from head and neck cancers. These microfluidic devices also maintained viability for in vitro culture and characterization. Use of LNA modified aptamers provided added benefits in terms of cost effectiveness due to increased reusability and sustainability of the devices. Our results present a robust, quick, and efficient CTC capture platform with the use of simple PDMS based devices that are easy to fabricate at low cost and have an immense potential in cancer diagnosis, prognosis, and therapeutic planning.     

Expression,purification and biological effect of a novel single chain Fv antibody and protamine fusion protein for the targeted delivery of siRNAs to FGFR3 positive cancer cells

BackgroundGain-of-function of fibroblast growth factor receptor 3 (FGFR3) is involved in the pathogenesis of many tumors. More and more studies have focused on the potential usage of therapeutic single-chain Fv (ScFv) antibodies against FGFR3. RNA interference (RNAi) has been considered as a promising therapeutic method against cancer. A tool which can deliver small interference RNAs (siRNAs) into FGFR3 positive cancer cells is very promising for anti-tumor therapy.ResultsIn this study, a novel fusion protein R3P, which consists of FGFR3-ScFv and protamine, was generated in Escherichia coli by inclusion body expression strategy and Ni-NTA chromatography. Its yield reached 10 mg per liter of bacterial culture and its purity was shown to be higher than 95%. 1 μg of R3P could efficiently bind to about 2.5 pmol siRNAs and deliver siRNAs into FGFR3 positive RT112 and K562 cells. Annexin V staining results showed that R3P can deliver the amplified breast cancer 1 (AIB1) siRNAs to induce RT112 cell apoptosis.ConclusionThese results indicated that R3P was a promising carrier tool to deliver siRNAs into FGFR3 positive cancer cells and to exert anti-tumor effect.