3-D Screens Predict Breast Cancer Metastasis

Cell-ECM Interactions

3-D culturing solutions and methods are increasingly being used to created more in vivo like assays.

Here researchers use our Human Collagen IV extracellular matrix protein (ECM) to develop a simple biomaterial platform with systematic control over the ECM protein density and composition to determine if integrin binding governs how metastatic cells differentiate between secondary tissue sites: L.E. Barney, E.C. Dandley, L.E. Jansen, N.G. Reich, A.M. Mercurio, and S.R. Peyton. A cell–ECM screening method to predict breast cancer metastasis. Integr Biol (Camb). 2015 Feb 10; 7(2): 198–212. doi: 10.1039/c4ib00218k.

This publication details the creation of an in vitro fingerprint that is predictive of in vivo metastasis.

Figure: Biomaterial platform for integrin-mediated phenotyping. (a) Breast cancer cell lines with their known in vivo metastatic tropisms.  (b) Three distinct ECM microenvironments regulate integrin binding. (c) Adhesion and motility phenotypes of the MDA-MB-231 cell line. Black: ECM 1; blue: ECM 2; green: ECM 3.

Figure: Correlations between adhesion and migration responses identify potent integrin antibodies in vitro. Pairwise comparisons between adhesion and migration measurements in the (a, b) MDA-MB-231 and (c, d) SkBr3 cell lines across normal, EGF-stimulated, and integrin antibody conditions. Arrows highlight conditions where integrin antibodies increased migration metrics. Spearman correlations are indicated on each plot with two-tailed p-values. Circle: ECM 1; square: ECM 2; triangle: ECM 3; black: normal; green: EGF; blue: anti-β1 integrin; red: anti-α2 integrin; orange: anti-α6 integrin. (e, f) SkBr3 migration mechanisms are displayed via 10 random cell paths under (e) normal and (f) anti-β1 conditions. Red paths identify cells detaching and adhering elsewhere on the surface. Inset: representative images of cell morphology. Scale bar is 25 μm. (g) Individual cells that invaded into an overlaid 3D collagen gel from the ECM 1 surface after 48 hours. Bar indicates mean distance invaded of all invading cells. Inset: schematic of cells invading upward from the ECM surface into an overlaid gel.

A richer understanding of cancer metastasis is important for the development of new, more potent chemotherapies. I am hopeful 3-D/ECM assays become more embraced as a way to more accuratly determine in vivo cell behaviors.

Extracellular Matrix Environment and Chemotherapeutics

Substrate Matters!

Neuromics' has been promoting a variety of 3-D Cell Based Assay Solutions for the past several years. These include: Nanofibers, Hydrogels and Extracellular Matrix (ECM) Proteins. We have found that the adoption rate for these as standard tools for drug discovery is slower that we anticipated.

We believe substrate matters so I am pleased to share a recent publication that references use of our Collagen IV and other ECM proteins. This confirms the importance of using a more in vivo like environment in testing chemotherapeutics: Thuy V. Nguyen,Marianne Sleiman,Timothy Moriarty,William G. Herrick,Shelly R. Peyton. Sorafenib resistance and JNK signaling in carcinoma during extracellular matrix stiffening. Publication: Biomaterials. Elsevier. 13 April 2014. http://dx.doi.org/10.1016/j.biomaterials.2014.03.058.

Abstract: Tumor progression is coincident with mechanochemical changes in the extracellular matrix (ECM). We hypothesized that tumor stroma stiffening, alongside a shift in the ECM composition from a basement membrane-like microenvironment toward a dense network of collagen-rich fibers during tumorigenesis, confers resistance to otherwise powerful chemotherapeutics. To test this hypothesis, we created a high-throughput drug screening platform based on our poly(ethylene glycol)-phosphorylcholine (PEG-PC) hydrogel system, and customized it to capture the stiffness and integrin-binding profile of in vivo tumors. We report that the efficacy of a Raf kinase inhibitor, sorafenib, is reduced on stiff, collagen-rich microenvironments, independent of ROCK activity. Instead, sustained activation of JNK mediated this resistance, and combining a JNK inhibitor with sorafenib eliminated stiffness-mediated resistance in triple negative breast cancer cells. Surprisingly, neither ERK nor p38 appears to mediate sorafenib resistance, and instead, either ERK or p38 inhibition rescued sorafenib resistance during JNK inhibition, suggesting negative crosstalk between these signaling pathways on stiff, collagen-rich environments. Overall, we discovered that β1 integrin and its downstream effector JNK mediate sorafenib resistance during tumor stiffening. These results also highlight the need for more advanced cell culture platforms, such as our high-throughput PEG-PC system, with which to screen chemotherapeutics.

Figure: High-throughput biomaterial platform for drug screening. (A) The high-throughput platform consists of a black-walled, glass bottom plate, with PEG-PC gels cast in each of the inner 6x10 wells. (B) Gels can be functionalized with any protein or peptide of interest, and they support the adhesion and growth of carcinoma cells. We used this platform to test carcinoma cell response to a kinase inhibitor (sorafenib) as a function of underlying gel stiffness and ECM adhesive protein cocktail. (C) A representative graph of SkBr3 proliferation (y-axis) in response to sorafenib (x-axis) across a range of gel stiffness (colors) demonstrates the IC-50 calculation.

This confirms the importance of considering your substrate environment when developing your in vitro assays for High Content and High Throughput Drug Discovery. We will continue to provide updates.

From Zero to 3-D Cell Based Assays in 15 Minutes

Collagel Hydrogels are Designed to Match Your Cell Types.

We are pleased to add Collagel Hydrogels to our 3-D Cell Based Assay Solutions.

The are 3 different gel types that mimic the different in vivo extracellular environment that your cells experience. You will be able to get really nice adipogenesis with MSCs using our CollaGel Hydrogel Standard. CollaGel Hydrogel Soft is an ideal matrix for growing fibroblast; primary hepatocyte cultures and great for growing smooth muscle cells. CollaGel Hydrogel Soft+ is an ideal matrix for growing nervous cells, veins cells and Hydroxyapatite crystals.

Image: Primary hepatocyte culture in 3D model using our CollaGel Hydrogel. 

Using these gels, you can now form small tube-like structures in combination with Human Umbilical Vein Endothelial Cells (HUVEC) in a 3D model. Our CollaGel Hydrogel can also be used for 3D printing without worrying about the needles in your machine been broken due to the fast gelling process. Collagel Hyrogels can be used for: Stem Cell Behavior Studies Fibrosis Studies,  Cosmetic Toxicology, Hepatocyte Assays, Neuronal Branching, Wound Healing Assays, Cell Invasion Assays, Migration Assays, Cancer Cell Phenotyping and  Bioprinting.

I will be posting Collagel Hydrogel related data and images provided by our customers.