Distinctive Expression of Metastamirs in Breast Cancer Mesenchymal Stem Cells Isolated from Solid Tumor.

BACKGROUND: MSCs are a part of the tumor microenvironment, which secrete cytokines and chemokines. They can affect metastasis and the growth of tumors. metastamiRs are newly recognized regulatory elements of the metastasis pathway which are involved in epithelial-to-mesenchymal transition (EMT). OBJECTIVE: In the present study, we aimed to assess the expression profile of metastamiRs in the context of MSCs in correlation with their invasion and migration power. METHODS: tumor-isolated BC-MSCs and normal human mammary epithelial cells (HMECs) along with MCF-7, MDA-MB231, and MCF-10A cells were prepared and confirmed for their identity. The cells were assessed for CD44+CD24¯ percentage, Oct-4, and Survivin expression. GEO, KEGG, and TCGA databases were investigated to detect differential miR-expressions. Real-time PCR for 13 miRs was performed using LNA primers. Ultimately, Transwell-Matrigel assays as used to assess the level of migration and invasion. RESULTS: Our results indicated that some oncomiRs like miR-10b were upregulated in BC-MSCs, while the levels of miR-373 and miR-520c were similar to the MCF-10A. Generally, miR-200 family members were on lower levels compared to the other miR-suppressor (miR-146a, 146b, and 335). miR-31 and 193b were up-regulated in MCF-10A. The most invasiveness was observed in the MDA-MB231 cell line. CONCLUSION: We have demonstrated that the miR-expression levels of BC-MSCs are somewhat in between MCF-7 and MDA-MB231 miR-expression levels. This could be the logic behind the moderate level of invasion in BC-MSCs. Therefore, miR-therapy approaches such as miR-mimic or antagomiRs could be used for BC-MSCs in clinical cancer therapy.

Engineered extracellular vesicles expressing ICAM-1: A promising targeted delivery system for T cell modifications.

BACKGROUND: Extracellular vesicles (EVs) are natural nano-carriers that possess the required crucial features of an ideal biomolecular delivery system. However, using unmodified EVs may have some limitations such as low accumulation in target sites. Studies have established that engineering EVs against different cell surface markers can overcome most of these hurdles. METHODS: In this study, engineered EVs expressing ICAM-1/LAMP2b fusion protein on their surfaces were produced and isolated. The uptake of isolated targeted and non-targeted EVs was evaluated by imaging and flow cytometry. To assess the ability of targeted EVs to be applied as a safe carrier, pAAVS1-Puro-GFP plasmids were encapsulated into EVs by electroporation. RESULTS: The HEKT 293 cell line was successfully modified permanently by a lentiviral vector to express ICAM-1 on the surface of the derived EVs. The ELISA and western blot tests established the binding affinity of targeted EVs for recombinant LFA-1 with a remarkable difference from non-targeted EVs. Furthermore, flow cytometry results revealed noteworthy differences in the binding of LFA-1-positive, non-targeted EVs, and targeted EVs to LFA-1-negative cells. Finally, imaging and flow cytometry indicated that newly produced EVs could efficiently interact with T cells and functionally deliver loaded plasmids to them. CONCLUSION: These LFA-1-targeted EVs were able to interact with T cells as their recipient cells. They can be utilized as an ideal delivery system to transfer various biomolecules to T cells, facilitating immunotherapies or other cell-based treatments.

Isolation and characterization of a novel GRP78-specific single-chain variable fragment (scFv) using ribosome display method.

Glucose-regulated protein 78 (GRP78) is a well-characterized endoplasmic reticulum (ER) chaperon frequently overexpressed at the surface of tumor cells and associated with tumor survival, metastasis, and chemoresistance. Hence, potential GRP78 binders emerge as promising candidates for cancer therapy and diagnosis. We applied ribosome display to isolate a single-chain variable domain (scFv) specific for the C-terminal domain of a recombinant human GRP78 (CGRP). Six female BALB/c mice were immunized and then splenocyte mRNA was extracted. An scFv-ribosome display library was established by joining the amplified VH/Vκ fragments through a 72-bp linker using overlap extension PCR. Then, selection was performed by applying two rounds of eukaryotic ribosome display panning with stepwise decreased amount of CGRP. Ultimately, the selected scFv was characterized using the indirect-ELISA assay, competitive-ELISA assay, Western blotting, Surface Plasmon Resonance (SPR), and in-silico analyses. The constructed library had a length of ~ 1100 bp and the high-affinity scFvs were isolated using the outputs of the final panning round. Among 60 positive clones, GSF3 was selected and its expression, purification, and binding capacity was confirmed by SDS-PAGE and Western blotting. GSF3 exhibited an affinity of 13 × 107 M-1 to CGRP as assessed by SPR. Moreover, the in-silico analyses indicated that GSF3 binds the C-terminal domain of GRP78 through key residues engaged in antibody-antigen interactions. We found that ribosome display is a swift and reliable technique for specific and high-affinity scFv isolation. Moreover, our results suggest that GSF3 might be applied as a potential cancer immunotherapeutic and diagnostic tool if this approach is carefully followed by successful preclinical and clinical evaluations to validate the findings for further confirmation.

Radiolabeled HER2-directed exosomes exhibit improved cell targeting and specificity.

Aim: Here, we established a reliable strategy for generation and characterization of targeted radiolabeled exosomes for the detection of HER2-positive cells quantitatively. Materials & methods: Targeted exosomes (T-exos) were radiolabeled by two different radiotracers, [99mTc]Tc-HMPAO or [111In]In-oxine. The labeling efficiency and stability were assessed using exosome exclusive spin columns. HER2-positive and -negative cells were treated with [111In]In-oxine-exosomes after 3 and 24 h. Results: [111In]In-oxine labeling did not change the binding ability and general features of the exosomes. With [111In]In-oxine, 70% labeling efficiency and 78% radiochemical stability over 24 h were achieved. [111In]In-oxine-T-exos showed greater uptake by HER2-positive cells compared with untargeted exosomes. Conclusion: [111In]In-oxine-T-exos could potentially be used as an effective imaging tool for HER2 expression.

99mTc-radiolabeled HER2 targeted exosome for tumor imaging.

Exosomes represent unique features including nontoxicity, non-immunogenicity, biodegradability, and targeting ability that make them suitable candidates for clinical applications. Therefore, in this study, 99mTc-radiolabel HER2 targeted exosomes (99mTc-exosomes) were provided for tumor imaging. These exomes are obtained from genetically engineered cells and possessed DARPin G3 as a ligand for HER2 receptors. These exosomes were radiolabeled using fac-[99mTc(CO)3(H2O)3]+ synthon. The quality control showed high radiochemical purity (RCP) for 99mTc-exosomes (>96%). 99mTc-exosomes displayed a higher affinity toward SKOV-3 cells (higher HER2 expression) in comparison with MCF-7, HT29, U87-MG, A549 cell lines at different levels of HER2 expression. Trastuzumab (an antibody with a high affinity toward HER2) inhibited the binding of 99mTc-exosomes to SKOV-3 cells up to 40%. Biodistribution study in SKOV-3 tumor bearing nude mice confirmed the ability of 99mTc-exosomes for accumulation in the tumor. 99mTc-exosomes can visualize tumor in SKOV-3 tumor-bearing nude mouse. The blockage of HER2 receptors using trastuzumab (excessive amount) suggests the 99mTc-exosomes binding to the receptors and reduced the accumulation of 99mTc-exosomes in the tumor site. This suggest that 99mTc-exosomes interact with HER2 receptors and act through specific targeting.

Engineered Exosomes for Targeted Transfer of siRNA to HER2 Positive Breast Cancer Cells.

Exosomes are the best options for gene targeting, because of their natural, nontoxic, non-immunogenic, biodegradable, and targetable properties. By engineering exosome-producing cells, ligands can be expressed fusing with exosomal surface proteins for targeting cancer cell receptors. In the present study, HER2-positive breast cancer cells were targeted with a modified exosome producing engineered HEK293T cell. For this purpose, the HEK293T cells were transduced by a lentiviral vector bearing-LAMP2b-DARPin G3 chimeric gene. Stable cells expressing the fusion protein were selected, and the exosomes produced by these cells were isolated from the culture medium, characterized, and then loaded with siRNA for subsequent delivery to the SKBR3 cells. Our results showed that stable HEK293T cells produced DARPin G3 on the surface of exosomes. These exosomes can bind specifically to HER2/Neu and are capable of delivering siRNA molecules against TPD52 gene into SKBR3 cell line down-regulating the gene expression up to 70%. Present approach is envisaged to facilitate genes and drugs transfer to HER2 cancer cells providing additional option for gene therapy and drug delivery.

Inhibition of breast cancer metastasis by co-transfection of miR-31/193b-mimics.

OBJECTIVES: Various studies have been conducted to reduce the metastatic behavior of cancerous cells. In this regard, ectopic expression of anti-metastatic microRNAs by miR-mimic and miR-restoration-based therapies could bring new insights to the field. In the present study, the consequences of co-transfecting breast cancer cell lines with miR-193b and miR-31 were investigated via invasion and migration assays. MATERIALS AND METHODS: Double stranded oligonucleotide of mature miR-193b-3p and miR-31-5p were cloned into pcDNA 6.2gw/EmGFP plasmid. The resulting plasmids were used for transfection. Real time-PCR was performed to assess the expression of miR-193b and miR-31 as well as Ras homolog gene family member A (RhoA) and urokinase-type plasminogen activator (uPA) as miR targets. Scratch, Transwell migration and Matrigel invasion assays were carried out to assess the extent of migration and invasion of cell lines. RESULTS: The most significant increase in expression of miRs belonged to the single transfection of mimic-miRs in MDA-MB231. Although the co-transfection was not as successful as single transfection in miR expression, it was significantly more effective in inhibition of the cells invasive potential. CONCLUSION: Although the miR-restoration therapy based on co-transfection of two miRs could be less effective in expression of each miRNA, the resulting decrease in metastatic behavior of the cells is more significant due to collective effect of co-transfection to decrease target gene expression. Our results revealed that employing this sort of combinatorial strategies could lead to more efficient reduction in metastatic behavior. It seems that using this strategy would bring about more successful therapeutic outcomes.

Gentamicin induces efaA expression and biofilm formation in Enterococcus faecalis.

Enterococci have been ranked among the leading causes of nosocomial bacteremia and urinary tract infection. This study aimed to investigate the effect of ampicillin, vancomycin, gentamicin and ceftizoxime on biofilm formation and gene expression of colonization factors on Enterococcus faecalis. Twelve clinical isolates of E. faecalis were used to investigate the effect of antibiotics on biofilm formation and gene expression of efaA, asa1, ebpA, esp and ace. Flow system assay and Microtiter plates were used for biofilm assay. Two hundred clinical isolates were used for confirming the effect of antibiotics on biofilm formation. Ampicillin, vancomycin and ceftizoxime did not have any significant effect on biofilm formation, but gentamicin induced biofilm formation in 89% of isolates. In twelve selected isolate gentamicin increased expression of esp (+50.9%) and efaA (+33.9%) genes and reduced or maintained expression of others (asa1:-47.4%, ebpA: 0, ace:-19.2%). Vancomycin increased expression of esp (+89.1%) but reduced the others (asa1: -34.9%, ebpA:-11%, ace:-30%, efaA:-60%). Ceftizoxime increased slightly ebpA (+19.7%) and reduced others (asa1:-66.2%, esp:-35%, ace:-28.1%, efaA:-38.4%). and ampicillin strongly increased expression of ace (+231%), esp (+131%) and ebpA (+83%) but reduced others (asa1:-85.5%, efaA:-47.4%). The findings of the present study showed that antibiotics may have a role in biofilm formation and sustainability of enterococci, especially in case of gentamicin. efaA gene may have an important role, especially in antibiotic induced biofilm formation by gentamicin. Experiments with efaA mutants are needed to investigate the exact effect of efaA on biofilm formation with antibiotic induced cells.

Comparison of TGFbR2 down-regulation in expanded HSCs on MBA/DBM scaffolds coated by UCB stromal cells.

Bone marrow transplants (BMTs) are mainly limited by a low number of CD34(+) cells. The transforming growth factor-beta (TGF-ß) pathway downregulation is a key factor that increases cell self-renewal. In nature, hematopoietic stem cells (HSCs) are in a microenvironment, surrounded by cells in a three-dimensional (3D) configuration. The aim of this study is to investigate the association between a 3D culture and the delivery ratio of downregulation. Demineralized bone matrix (DBM) and mineralized bone allograft (MBA) scaffolds were coated using unrestricted somatic stem cells (USSCs) as the feeder layer. Umbilical cord blood (UCB)-CD34(+) cells were then ex vivo expanded in them and transfected by small interfering RNA (siRNA) against TGFbR2, a type 2 receptor in the TGF-ß pathway. Finally, quantitative real-time PCR, flow cytometry, and clonogenic assay were performed. In a global comparison, we observed that the highest expansion ratio, lowest expression level, and the highest CD34 marker belonged to the simple 2D culture transfected group. This suggests that TGFbR2 downregulation in a 2D culture can be done more effectively. The siRNA delivery system and the transfection ratio in an ex vivo environment, which mimicks in vivo conditions, have low efficiency. Genetic modification of the cells needs free 3D spaces to enable better transfection.

The effect of bone morphogenetic protein 4 on the differentiation of mouse embryonic stem cell to erythroid lineage in serum free and serum supplemented media.

This study was done to compare the effects of bone morphogenetic protein-4 (BMP-4) on mouse embryonic stem cells (ESC) differentiation to erythroid lineage in serum free and serum supplemented media. The embryoid bodies (EBs) cells were seeded in semisolid serum free and serum supplemented media in the presence of different concentrations of BMP-4. The erythroid colonies were assessed morphologically, ultrastructurally and by benzidine staining. The expression of the epsilon (ε), ßH1 and ßmajor globins, Runx1 and ß2m genes was evaluated by Real time PCR. The colony size and the percent of benzidine-positive colonies increased in both BMP-4 supplementd groups but the number of colonies were lower in these groups than control. Erythropoiesis related genes were expressed in both serum free and serum supplemented groups. There were not significant differences between the ratios of genes expression to ß2m in these groups except the ratio of Runx1 was significantly higher in serum free group (P<0.05). The ratio of ε and ßH1 to ß2m in EBs was higher than both BMP-4 containing groups (P<0.05) and ßmajor was not expressed in EB cells. These findings showed in serum free condition the effects of BMP-4 on the erythroid differentiation was prominent than serum supplemented group.