Three-dimensional co-culture microfluidic model and its application for research on cancer stem-like cells inducing migration of endothelial cells

Objectives

To build a three-dimensional co-culture model in a microfluidic device for cancer research and evaluate its feasibility by investigating cancer stem-like cells (SCs) induced migration of human umbilical vein endothelial cells (ECs).

Results

The microfluidic device provided two-dimensional and three-dimensional (2D/3D) culture and co-culture environments without affecting cell viability. The device also provided an effective concentration for the chemiotaxis of cells, and to support real-time monitoring of cell behavior. In this model, SCs significantly increased the migration area of ECs with a hepatocarcinoma cell line (MHCC97H; MCs). The presence of ECs also induced both MCs and SCs invasion into Matrigel. The migration area of MCs and SCs significantly increased when co-cultured with ECs.

Conclusions

This 3D co-culture microfluidic model is a suitable model in cancer research. Compared with MCs, SCs had greater potential in inducing EC migration and interacting with ECs.

     

MicroRNA-190b confers radio-sensitivity through negative regulation of Bcl-2 in gastric cancer cells

Objectives

To determine the role of miR-190b in radio-sensitivity of gastric cancer (GC).

Results

In radio-resistant GC cells, down-regulation of miR-190b and up-regulation of Bcl-2 were observed. The protein expression of Bcl-2 was negatively regulated by miR-190b. Overexpression of miR-190b significantly decreased cell viability and enhanced radio-sensitivity of GC cells. Of note, these effects of miR-190b on GC cells radio-sensitivity were abolished by Bcl-2.

Conclusion

miR-190b confers radio-sensitivity of GC cells, possibly via negative regulation of Bcl-2.

     

SOX2 as a novel contributor of oxidative metabolism in melanoma cells

Background

Deregulated metabolism is a hallmark of cancer and recent evidence underlines that targeting tumor energetics may improve therapy response and patient outcome. Despite the general attitude of cancer cells to exploit the glycolytic pathway even in the presence of oxygen (aerobic glycolysis or “Warburg effect”), tumor metabolism is extremely plastic, and such ability to switch from glycolysis to oxidative phosphorylation (OxPhos) allows cancer cells to survive under hostile microenvironments. Recently, OxPhos has been related with malignant progression, chemo-resistance and metastasis. OxPhos is induced under extracellular acidosis, a well-known characteristic of most solid tumors, included melanoma.

Methods

To evaluate whether SOX2 modulation is correlated with metabolic changes under standard or acidic conditions, SOX2 was silenced and overexpressed in several melanoma cell lines. To demonstrate that SOX2 directly represses HIF1A expression we used chromatin immunoprecipitation (ChIP) and luciferase assay.

Results

In A375-M6 melanoma cells, extracellular acidosis increases SOX2 expression, that sustains the oxidative cancer metabolism exploited under acidic conditions. By studying non-acidic SSM2c and 501-Mel melanoma cells (high- and very low-SOX2 expressing cells, respectively), we confirmed the metabolic role of SOX2, attributing SOX2-driven OxPhos reprogramming to HIF1α pathway disruption.

Conclusions

SOX2 contributes to the acquisition of an aggressive oxidative tumor phenotype, endowed with enhanced drug resistance and metastatic ability.

     

Aligned electrospun fibers for neural patterning

Objectives

To test a 3D approach for neural network formation, alignment, and patterning that is reproducible and sufficiently stable to allow for easy manipulation.

Results

A novel cell culture system was designed by engineering a method for the directional growth of neurons. This uses NG108-15 neuroblastoma x glioma hybrid cells cultured on suspended and aligned electrospun fibers. These fiber networks improved cellular directionality, with alignment angle standard deviations significantly lower on fibers than on regular culture surfaces. Morphological studies found nuclear aspect ratios and cell projection lengths to be unchanged, indicating that cells maintained neural morphology while growing on fibers and forming a 3D network. Furthermore, fibronectin-coated fibers enhanced neurite extensions for all investigated time points. Differentiated neurons exhibited significant increases in average neurite lengths 96 h post plating, and formed neurite extensions parallel to suspended fibers, as visualized through scanning electron microscopy.

Conclusions

The developed model has the potential to serve as the basis for advanced 3D studies, providing an original approach to neural network patterning and setting the groundwork for further investigations into functionality.

     

Monitoring cancer prognosis,diagnosis and treatment efficacy using metabolomics and lipidomics

Introduction

Cellular metabolism is altered during cancer initiation and progression, which allows cancer cells to increase anabolic synthesis, avoid apoptosis and adapt to low nutrient and oxygen availability. The metabolic nature of cancer enables patient cancer status to be monitored by metabolomics and lipidomics. Additionally, monitoring metabolic status of patients or biological models can be used to greater understand the action of anticancer therapeutics.

Objectives

Discuss how metabolomics and lipidomics can be used to (i) identify metabolic biomarkers of cancer and (ii) understand the mechanism-of-action of anticancer therapies. Discuss considerations that can maximize the clinical value of metabolic cancer biomarkers including case–control, prognostic and longitudinal study designs.

Methods

A literature search of the current relevant primary research was performed.

Results

Metabolomics and lipidomics can identify metabolic signatures that associate with cancer diagnosis, prognosis and disease progression. Discriminatory metabolites were most commonly linked to lipid or energy metabolism. Case–control studies outnumbered prognostic and longitudinal approaches. Prognostic studies were able to correlate metabolic features with future cancer risk, whereas longitudinal studies were most effective for studying cancer progression. Metabolomics and lipidomics can help to understand the mechanism-of-action of anticancer therapeutics and mechanisms of drug resistance.

Conclusion

Metabolomics and lipidomics can be used to identify biomarkers associated with cancer and to better understand anticancer therapies.

     

Stimulation of mouse vibrissal follicle growth by recombinant human fibroblast growth factor 20

Objectives

To explore potential effects of recombinant human fibroblast growth factor 20 (rhFGF20) in the growth of cultured mouse vibrissal follicles.

Results

The growth of cultured mouse vibrissal follicles was significantly induced by rhFGF20 in a dose dependent pattern in the in vitro vibrissal follicle organ culture model. However, too high concentration of rhFGF20 could inhibit the growth of vibrissal follicles. We further demonstrated that rhFGF20 stimulated the proliferation of hair matrix cells and activated Wnt/β-catenin signaling pathway.

Conclusions

The rhFGF20 might be a potential therapeutic agent to treat hair loss disorders.

     

NMRProcFlow: a graphical and interactive tool dedicated to 1D spectra processing for NMR-based metabolomics

Introduction

Concerning NMR-based metabolomics, 1D spectra processing often requires an expert eye for disentangling the intertwined peaks.

Objectives

The objective of NMRProcFlow is to assist the expert in this task in the best way without requirement of programming skills.

Methods

NMRProcFlow was developed to be a graphical and interactive 1D NMR (¹H & ¹³C) spectra processing tool.

Results

NMRProcFlow (http://nmrprocflow.org), dedicated to metabolic fingerprinting and targeted metabolomics, covers all spectra processing steps including baseline correction, chemical shift calibration and alignment.

Conclusion

Biologists and NMR spectroscopists can easily interact and develop synergies by visualizing the NMR spectra along with their corresponding experimental-factor levels, thus setting a bridge between experimental design and subsequent statistical analyses.

     

Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

     

A novel trichosanthin fusion protein with increased cytotoxicity to tumor cells

Objective

To evaluate the anti-tumor effects of trichosanthin after fusion with a cell penetrating peptide, heparin-binding peptide (HBP), derived from human heparin-binding EGF-like growth factor (HB-EGF).

Results

The fusion protein of trichosanthin-HBP was expressed in Escherichia coli BL21 and purified by Ni–NTA affinity chromatography. The HBP domain had no influence on the topological inactivation activity and N-glycosidase activity of trichosanthin. Trichosanthin-HBP significantly inhibited the growth of tested cancer cells which are impervious to trichosanthin. Tumor cell apoptosis and both the mitochondrial- and death receptor-mediated apoptotic signaling pathways induced by trichosanthin-HBP were more significant than those induced by trichosanthin in HeLa cells.

Conclusion

HBP is an efficient intracellular delivery vehicle for trichosanthin and makes trichosanthin-HBP become a promising agent for cancer therapy.

     

Inhibition of integrin β3, a binding partner of kallistatin,leads to reduced viability,invasion and proliferation in NCI-H446 cells

Background

Kallistatin is a serine proteinase inhibitor and heparin-binding protein. It is considered an endogenous angiogenic inhibitor. In addition, multiple studies demonstrated that kallistatin directly inhibits cancer cell growth. However, the molecular mechanisms underlying these effects remain unclear.

Methods

Pull-down, immunoprecipitation, and immunoblotting were used for binding experiments. To elucidate the mechanisms, integrin β3 knockdown (siRNA) or blockage (antibody treatment) on the cell surface of small the cell lung cancer NCI-H446 cell line was used.

Results

Interestingly, kallistatin was capable of binding integrin β3 on the cell surface of NCI-H446 cells. Meanwhile, integrin β3 knockdown or blockage resulted in loss of antitumor activities induced by kallistatin. Furthermore, kallistatin suppressed tyrosine phosphorylation of integrin β3 and its downstream signaling pathways, including FAK/-Src, AKT and Erk/MAPK. Viability, proliferation and migration of NCI-H446 cells were inhibited by kallistatin, with Bcl-2 and Grb2 downregulation, and Bax, cleaved caspase-9 and caspase 3 upregulation.

Conclusions

These findings reveal a novel role for kallistatin in preventing small cell lung cancer growth and mobility, by direct interaction with integrin β3, leading to blockade of the related signaling pathway.

     

High-throughput and direct measurement of androgen levels using turbulent flow chromatography liquid chromatography-triple quadrupole mass spectrometry (TFC-LC-TQMS) to discover chemicals that modulate dihydrotestosterone production in human prostate cancer cells

Objectives

To develop a high-throughput screening system to measure the conversion of testosterone to dihydrotestosterone (DHT) in cultured human prostate cancer cells using turbulent flow chromatography liquid chromatography-triple quadrupole mass spectrometry (TFC-LC-TQMS).

Results

After optimizing the cell reaction system, this method demonstrated a screening capability of 103 samples, including 78 single compounds and 25 extracts, in less than 12 h without manual sample preparation. Consequently, fucoxanthin, phenethyl caffeate, and Curcuma longa L. extract were validated as bioactive chemicals that inhibited DHT production in cultured DU145 cells. In addition, naringenin boosted DHT production in DU145 cells.

Conclusion

The method can facilitate the discovery of bioactive chemicals that modulate the DHT production, and four phytochemicals are potential candidates of nutraceuticals to adjust DHT levels in male hormonal dysfunction.

     

The Characteristics Of Human Bone-Derived Cells (HBDCS) during osteogenesis in vitro

Background

The primary human bone-derived cell culture technique is used as a model to study human osteogenesis. Compared to cell line cultures, primary osteoprogenitor and osteoblast cultures provide more complex information about osteogenesis, bone remodeling and regeneration than cell line cultures.

Methods

In this study, we isolated human bone-derived cells (HBDCs) and promoted their differentiation into osteoblasts. The following parameters were evaluated: cell number and viability, total protein expression, alkaline phosphatase activity, collagenous matrix production and osteogenic genes expression, i.e., gene coding for type I collagen and alkaline phosphatase.

Results

It was proved the results show that HBDCs intensively proliferate during the first 7 days of culture followed by differentiation accompanied by an increase in alkaline phosphatase activity. Moreover, it was observed that during the differentiation of HBDCs, the expression of integrin β1 increased.

Conclusions

The process was also accompanied by changes in cell shape and rearrangement of the actin cytoskeleton and focal contacts containing FAK and the integrin β1 subunit. We suggest that the β1 integrin subunit may be a suitable new target in studies of the differentiation of primary human osteoblasts in culture.

     

Metastatic Hurthle Cell Carcinoma of the thyroid presenting as a Breast Lump: A Case Report

Background

Hurthle cell carcinoma of the thyroid is a rare form of thyroid cancer. It may present as a low grade tumour or can present as a more aggressive metastatic carcinoma. Hurthle cell carcinoma has the highest incidence of metastasis among all differentiated thyroid cancers. Most commonly haematogenous spread to lungs, bones and brain, however spread to regional lymph nodes is not uncommon. The breast is a rare site for metastasis from extramammary sources. We present the first case of breast metastasis from Hurthle cell carcinoma of the thyroid.

Case presentation

We report a 77 year old lady who had total thyroidectomy and bilateral neck dissection followed by radiotherapy for a high grade metastatic Hurthle cell carcinoma of the thyroid. Ten months later she presented to the breast clinic with left breast lump and a lump at the left axilla. Fine needle aspiration cytology of the lumps and histology after wide local excision of the breast lump confirmed metastatic Hurthle cell carcinoma.

Conclusion

The presence of breast lumps in patients with history of extramammary cancer should raise the possibility of metastasis.

     

Modeling the behavior of human induced pluripotent stem cells seeded on melt electrospun scaffolds

Background

Human induced pluripotent stem cells (hiPSCs) can form any tissue found in the body, making them attractive for regenerative medicine applications. Seeding hiPSC aggregates into biomaterial scaffolds can control their differentiation into specific tissue types. Here we develop and analyze a mathematical model of hiPSC aggregate behavior when seeded on melt electrospun scaffolds with defined topography.

Results

We used ordinary differential equations to model the different cellular populations (stem, progenitor, differentiated) present in our scaffolds based on experimental results and published literature. Our model successfully captures qualitative features of the cellular dynamics observed experimentally. We determined the optimal parameter sets to maximize specific cellular populations experimentally, showing that a physiologic oxygen level (～?5%) increases the number of neural progenitors and differentiated neurons compared to atmospheric oxygen levels (～?21%) and a scaffold porosity of ～?63% maximizes aggregate size.

Conclusions

Our mathematical model determined the key factors controlling hiPSC behavior on melt electrospun scaffolds, enabling optimization of experimental parameters.

     

Targeted metabolomic profiling of low and high grade serous epithelial ovarian cancer tissues: a pilot study

Introduction

Epithelial ovarian cancer (EOC) remains the leading cause of death from gynecologic malignancies and has an alarming global fatality rate. Besides the differences in underlying pathogenesis, distinguishing between high grade (HG) and low grade (LG) EOC is imperative for the prediction of disease progression and responsiveness to chemotherapy.

Objectives

The aim of this study was to investigate, the tissue metabolome associated with HG and LG serous epithelial ovarian cancer.

Methods

A combination of one dimensional proton nuclear magnetic resonance (1D H NMR) spectroscopy and targeted mass spectrometry (MS) was employed to profile the tissue metabolome of HG, LG serous EOCs, and controls.

Results

Using partial least squares-discriminant analysis, we observed significant separation between all groups (p?<?0.05) following cross validation. We identified which metabolites were significantly perturbed in each EOC grade as compared with controls and report the biochemical pathways which were perturbed due to the disease. Among these metabolic pathways, ascorbate and aldarate metabolism was identified, for the first time, as being significantly altered in both LG and HG serous cancers. Further, we have identified potential biomarkers of EOC and generated predictive algorithms with AUC (CI)?=?0.940 and 0.929 for HG and LG, respectively.

Conclusion

These previously unreported biochemical changes provide a framework for future metabolomic studies for the development of EOC biomarkers. Finally, pharmacologic targeting of the key metabolic pathways identified herein could lead to novel and effective treatments of EOC.

     

Contribution of cytoskeletal elements to the axonal mechanical properties

Background

Microtubules, microfilaments, and neurofilaments are cytoskeletal elements that affect cell morphology, cellular processes, and mechanical structures in neural cells. The objective of the current study was to investigate the contribution of each type of cytoskeletal element to the mechanical properties of axons of dorsal root and sympathetic ganglia cells in chick embryos.

Results

Microtubules, microfilaments, and neurofilaments in axons were disrupted by nocodazole, cytochalasin D, and acrylamide, respectively, or a combination of the three. An atomic force microscope (AFM) was then used to compress the treated axons, and the resulting corresponding force-deformation information was analyzed to estimate the mechanical properties of axons that were partially or fully disrupted.

Conclusion

We have found that the mechanical stiffness was most reduced in microtubules-disrupted-axons, followed by neurofilaments-disrupted- and microfilaments-disrupted-axons. This suggests that microtubules contribute the most of the mechanical stiffness to axons.

     

Identification of trunk mutations in gastric carcinoma: a case study

Background

Intratumor heterogeneity (ITH) poses an urgent challenge for cancer precision medicine because it can cause drug resistance against cancer target therapy and immunotherapy. The search for trunk mutations that are present in all cancer cells is therefore critical for each patient.

Case presentation

In this study, we aimed to evaluate the efficiency of multiregional sequencing for the identification of trunk mutations present in all regions of a tumor as a case study. We applied multiregional whole-exome sequencing (WES) to investigate the genetic heterogeneity and homogeneity of a case of gastric carcinoma. Approximately 83% of common missense mutations present in two samples and approximately 89% of common missense mutations present in three samples were trunk mutations. Notably, trunk mutations appeared to have higher variant allele frequencies (VAFs) than non-trunk mutations.

Conclusions

Our results indicate that small-scale multiregional sampling and subsequent screening of low VAF somatic mutations might be a cost-effective strategy for identifying the majority of trunk mutations in gastric carcinoma.