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Collaborators in The Netherlands have created a 3D well system integrated into biomaterials that can be used either in vitro or in vivo for molecule screening in this HOT Technical Innovation.

The research was led by Gustavo Higuera and Jeanine Hendriks at Screvo Ltd and CellCoTec, with a team at The University of Twente. Avoiding animal experimentation by using 3D systems that mimic the in vivo environment is desirable yet challenging. Optimising animal experiments to be as efficient and effective as possible is a common goal of much research.

This team envisioned the integration of such in vitro 3D cell culture mimicking methods with biocompatible materials into animal models. To do this, they create a 9-well system that can be implanted into four sites under the skin of one mouse for in vivo screening. This enables testing of up to thirty six different conditions at once.

The innovative platform was produced from a wide range of biomaterials chosen for biocompatibility instead of conventional materials like PDMS. The cell culture compatibility of wells made with copolymer PEOT-PBT was tested using human mesenchymal stem cells. They test the systemic effect of the device on surrounding tissue of twenty mice.

This innovative bridge between in vivo and in vitro experimentation has great potential to both increase throughput and minimise animal experimentation by reducing the number of animals needed. Read the article in full now as it’s free to access for the next 4 weeks*:

In vivo screening of extracellular matrix components produced under multiple experimental conditions implanted in one animal
Gustavo A. Higuera, Jeanine A. A. Hendriks, Joost van Dalum, Ling Wu, Roka Schotel, Liliana Moreira-Teixeira, Mirella van den Doel, Jeroen C. H. Leijten, Jens Riesle, Marcel Karperien, Clemens A. van Blitterswijk and Lorenzo Moroni 
DOI: 10.1039/C3IB40023A

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Today’s HOT article as recommended by the referees is from Rafael Davalos and colleagues at Virginia Tech, USA. Using contactless dielectrophoresis, the changes in the electrical properties and surface roughness of cancer cells treated with non-toxic doses of spingolipid  metabolites.

One metabolite, S1P, promotes tumour formation and the second, So, has anti-tumour properties. The electrical properties of cancer cells change as they transform from benign to malignant. Contactless electrophoresis means that there is no contact between the cells and the electrodes, ruling out challenges of traditional techniques.

Using this technique, the researchers show that treatment with So partially transforms late stage malignant mouse ovarian surface epithelial cancer cells back to a benign state. S1P as expected does not reverse the transformation.

The exploration of potential non-toxic treatments as an alternative to highly toxic chemotherapy and of drugs whose action is not affected by the presence of variable biomarkers is important. This study shows that So has the potential for such therapy and it also demonstrates contactless dielectrophoresis as a useful tool in drug efficacy studies.

As a HOT article, we’ve made it free to access for the next 4 weeks!

Sphingolipid metabolites modulate dielectric characteristics of cells in a mouse ovarian cancer progression model
Alireza Salmanzadeh, Elizabeth S. Elvington, Paul C. Roberts, Eva M. Schmelz and Rafael V. Davalos
DOI: 10.1039/C3IB00008G

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A cross-departmental team at the University of Wisconsin-Madison led by Elaine Alarid and Integrative Biology Associate Editor David Beebe use a tumour biomarker to study the surrounding microenvironment by applying microfluidics and immunofluorescence techniques in this iBiology article. Using this innovative method, they can predict how the microenvironment will influence the growth of the tumour.

The biomarker used is Estrogen Receptor-α (ERα), which is a therapeutic target in the most common form of breast cancer. There are a number of different factors involved in the ERα signalling pathway; in this article these processes are defined as the microenvironment activity.

The researchers use an array of different microenvironments in a high-throughput microfluidic co-culture model to test the different variables. Quantitative immunofluorescence imaging is used to monitor ERα levels.  Other measures used are gene expression and phosphorylation status.

They discover a previously unrecognised linear correlation between growth of the tumour and decreases in ERα protein levels, indicating activation, under most of the various signalling input conditions. This work enables ERα regulation to be a dynamic biosensor of microenvironment activity for predicting tumour growth. Two of the conditions analysed showed a different correlation with tumour growth, with the growth appearing to be independent of ERα. The group will be looking into this further as it may have implications for the effectiveness of therapy.

They envision co-culturing samples of patient tumour microenvironment with MCF-7 cells acting as a biomarker for ERα activation. Read on for more detail into the assay and the results:

Hormonally responsive breast cancer cells in a microfluidic co-culture model as a sensor of microenvironmental activity
Jessica D. Lang, Scott M. Berry, Ginny L. Powers, David J. Beebe and Elaine T. Alarid
DOI: 10.1039/C3IB20265H

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A team at Stanford University, USA, test how changing the stiffness of the body changes sensitivity of touch of the model worm C. elegans using a microelectomechanical clamp.

Touch is the least understood of the five human senses. It is obvious that force applied to the skin is an important stimulator of neurons and mechanoreceptors below the surface. The mechanics of this process are not understood. In this HOT article, the researchers test the hypothesis that body stiffness influences touch sensitivity.

The microelectromechaical (MEMS) force clamp applies a force to a freely crawling worm and the team then measured the response stimulated by the level of force. The clamp is integrated with a moving stage. The worms are sensitive to under 1 μN of force and to indentations under 1 μm deep.

The team led by Miriam Goodman and Beth Pruitt then genetically alter the wild type worms’ skin proteins, softening the skin. They also modify their ability to control body wall muscles to show the effects of changing body mechanics.  Even small changes resulted in dramatic changes in force sensitivity, but had little effect on indentation depth sensitivity. They looked into whether the worms were directly responding to the force or whether they were reacting to the production of indentations. The conclusion is that the indentation of the skin drives touch sensitivity.

In this HOT article, the researchers also use a theoretical model to understand different kinds of deformation and force transfer involved. The article also discusses how this research relates to other animals and humans.

We’ve made this HOT article free to access for the next 4 weeks*:

MEMS-based force-clamp analysis of the role of body stiffness in C. elegans touch sensation
Bryan C. Petzold, Sung-Jin Park, Eileen A. Mazzochette, Miriam B. Goodman and Beth L. Pruitt
DOI: 10.1039/C3IB20293C

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A team of researchers at the University of South Carolina are investigating how cancer cells respond to the conditions of 3D cell culture and how we can use their behaviour to study anti-cancer drugs and cancer stem cells. In their work they combine biology with mathematics and biomaterials chemistry.

In this just published Integrative Biology paper, the team led by Hexin Chen and Qian Wang, culture MCF-7 cells on 3D polycaprolactone (PCL) fibrous scaffolds. The fibers are randomly orientated as in a cancerous environment collagen fibres surround breast cancer cells randomly. The fibrous scaffolds are made by electrospinning.

They discover that the epithelial-to-mesenchymal transition of the cells, required for cancer cell migration and malignancy, is enhanced when human epithelial breast cancer cells are cultured on this 3D fibrous substrate. For 2D cultures, the population of cancer stem cells remains constant.

Elucidating the exact mechanism of how will be part of the group’s further research. The researchers will also be expanding their previous mathematical models of cancer stem cell growth in tumours to include transformation of non-stem cells to stem cells. This would give an insight into whether the increase in cancer stem cells is down to non-stem cells converting or increasing division of those cancer stem cells already present.

Expansion of breast cancer stem cells with fibrous scaffolds
Sheng Feng, Xinrui Duan, Pang-Kuo Lo, Shou Liu, Xinfeng Liu, Hexin Chen and Qian Wang
DOI: 10.1039/C3IB20255K

Cornell University researchers use a 3D collagen matrix to mimic the migration of metastatic cancer cells, shedding light on the ability of secondary cancer cells to follow tracks made by primary invasive cells.

Cancer cells use matrix metalloproteinases (MMPs) to breakdown the extracellular matrix (ECM) barring their path of migration. Tracks are created through the ECM and it is thought that whilst some cancer cells need to produce MMPs in this way, the cells that follow on can do so along these tracks without the cost of producing MMPs themselves.

The 3D collagen environment in this research mimics natural peritumoral ECM and provides physiologically relevant results. The microtracks in this study were produced precisely using micromolding, mimicking the structure of natural tracks produced by MMPs. The cells studied are metastatic MDAMB231 breast cancer cells.

Time-lapse phase-contrast microscopy is used to show that such tracks enable non-invasive cancer cells that would not otherwise be able to migrate to do so. This work also demonstrates that seeding these microtracks with cells increases the cell migration speed, which MMP inhibitors failed to reduce. The migration of even the invasive cancer cells is made easier using the microtracks. This explains why clinical treatments based on the inhibition of MMPs fail to prevent further metastasis as once the tracks are formed cells continue to migrate regardless of MMP action.

This fascinating work changes what we know about cancer metastasis and it’s is now free to access for the next 4 weeks*:

Microfabricated collagen tracks facilitate single cell metastatic invasion in 3D
Casey M. Kraning-Rush, Shawn P. Carey, Marsha C. Lampi and Cynthia A. Reinhart-King
DOI: 10.1039/C3IB20196A

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Yana Wang and Darrell Irvine at MIT, USA, gain further insight into the mechanisms of chemotaxis by analysing how chemoattractant production by tissues shapes cell migration patterns. They do this using a combination of computational modelling and experiments on chemokine gradient generation by chemokine-releasing microspheres to mimic chemokine production. This HOT article has been made free to access for the next 4 weeks*!

The researchers look at human T-cells and dendritic cells as the migration of leukocytes in response to chemokine production is not well understood. As cells approach a chemokine source, the concentration increases and the concentration gradient also increases rapidly. This causes two competing effects making the migration pattern less predictable for steep gradients. Receptor saturation and desensitization may occur if the concentration becomes too high. However, increasing gradient steepness should mean that the cell is directed more accurately towards the source.

The experimental system uses single-cell-sized hydrogel microspheres previously designed by the group that mimic secretion of cytokines by cells over several hours. These were embedded in 3D extracellular matrix gels with T-cells and dendritic cells. The two chemokines used are CCL19 and CCL21, normally involved in leukocyte trafficking in the body.

The directionality of the T-cells is independent of where they begin in the chemokine gradient and their paths were strikingly linear. The T-cells’ starting position did influence the fraction of cells that responded, with more cells at a given position responding as distance of the population from the source decreased. When few receptors were desensitized (in low concentrations), stable gradients produced swarms of T-cells around individual microspheres. Where concentrations were high around several microspheres, the populations around each microsphere were less stable and T-cells hopped between microspheres.

This article highlights the widely differing T-cell behaviour that can be produced by one chemokine when concentrations of the chemokine vary within a tissue. Differences were also found between the two chemokines, previously thought to induce similar behaviour. Read the in-depth study here:

Convolution of chemoattractant secretion rate, source density, and receptor desensitization direct diverse migration patterns in leukocytes
Yana Wang and Darrell J. Irvine
DOI: 10.1039/C3IB20249F

Diabetic retinopathy is characterised by the local expansion of capillaries in the eye (microaneurysms). This results in a change in the flow and hemodynamic forces, meaning leakage, dysfunction, fluid accumulation (edema) and eventually blindness.

In this HOT article, a team led by Yaakov Nahmias at The Hebrew University of Jerusalem, Israel in combination with researchers at Stanford University and University of Florida, USA define a critical ratio of microaneurysm to vessel diameter for identification of high risk microaneurysms in diabetic retinopathy patients. This ratio determines the changes in flow speed, shear force and pressure.

The researchers used an analytical model to characterise this complex state. They calculated the critical ratio based on a shear threshold value for endothelial cell dysfunction. Above this critical ratio, leakage of the vessel is highly likely. This means that high risk vessels could be targeted for laser-ablation treatment, limiting the amount of unnecessary damage and making treatment more effective.

The validation was carried out using confocal images of diabetic retinas. This comparison also revealed that those microaneurysms over this critical threshold also display an increase in Von Villebrand Factor (vWF) expression. This is a biomarker for endothelial dysfunction and could help to explain the non-linear relationship between microaneurysm size and leakage.

This HOT article contains work relevant to the millions of people in developed countries diagnosed with diabetes and provides further understanding of the mechanisms of retinopathy. It’s free to access for the next 4 weeks*, just click on the link below:

Non-dimensional analysis of retinal microaneurysms: critical threshold for treatment
Elishai Ezra, Eliezer Keinan, Yossi Mandel, Michael E. Boulton and Yaakov Nahmias
DOI: 10.1039/C3IB20259C

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Chemoresistance is a known problem for the effectiveness of chemotherapy. The mechanisms that reduce drug potency are not fully understood and it can be very difficult to predict when chemoresistance may occur. Recently, researchers have been looking to the expression of the genome as a predictor of the response of a patient to a certain drug. Pharmacogenomics could lead to an individually designed chemotherapy regime for each patient.

Paola Lecca at The Microsoft Research–University of Trento Centre for Computational and Systems Biology integrates in three ways in this method for inferring potential chemoresistance from genome expression. The work aims to address the correlations found between genes and enzymes that determine resistance and sensitivity to a drug. This method:

-          Integrates known network information into the model;

-          Integrates simulation of both the gene and metabolic networks into the inference network and;

-          Integrates all of the inferred networks into a large network to show the correlations between genes and metabolism.

The method used here is based on Bayesian inference, which is most helpful with a large data set and enables the inclusion of known information to give models to explain the data. The method works by assigning probabilities to the hypotheses.  This is alternated with simulation of inferred networks. This is then inputted into a correlation-based inference proceeding to integrate the networks.

Lecca concentrates on gemcitabine, a pancreatic cancer drug, to demonstrate and validate this new method. It is used to infer correlations between four genes responsible for resistance and the enzymes metabolising gemcitabine in pancreatic cells.

Read this fascinating and complex work in detail now, as this article is free to access for the next 4 weeks*:

An integrative network inference approach to predict mechanisms of cancer chemoresistance
Paola Lecca
DOI: 10.1039/C2IB20205K

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