Cancer Associated Fibroblasts (CAFS) Traction Dynamics and Remodeling in 3D Matrices

 Neuromics' Colorectal CAFS in Action

In a first of its kind study, the mechanical properties of our CAFS were determined. This is important because, understanding how these cell behave in vivo will help improve the efficacy of new therapies: Bashar Emon, Zhengwei Li, Md Saddam Hossain Joy, Umnia Doha, Farhad Kosari, and M Taher A Saif. (2020). A Novel Method for Sensor-Based Quantification of Single/Multi-Cellular Traction Dynamics and Remodeling in 3D Matrices. bioRxiv. doi: 10.1101/2020.09.24.311647

"Cells in vivo generate mechanical forces (traction) on surrounding 3D extra cellular matrix (ECM) and cells. Such traction and biochemical cues may remodel the matrix, e.g. increase stiffness, which in turn influences cell functions and forces. This dynamic reciprocity mediates development and tumorigenesis. Currently, there is no method available to directly quantify single cell traction and matrix remodeling in 3D. Here, we introduce a method to fulfil this long-standing need. We developed a high-resolution microfabricated sensor which hosts a 3D cell-ECM tissue formed by self-assembly. It measures cell forces and tissue-stiffness and can apply mechanical stimulation to the tissue. We measured single and multicellular force dynamics of fibroblasts (3T3), human colon (FET) and lung (A549) cancer cells and cancer associated fibroblasts (CAF05) with 1 nN resolution. Single cells show significant force fluctuations in 3D. FET/CAF co-culture system, mimicking cancer tumor microenvironment, increased tissue stiffness by 3 times within 24 hours."

Here's one of 10 videos illustrating these properties.

 CAFS are widely used and frequently published. Check them out today!

Sorting Cancer-Associated Fibroblasts (CAFs)

Featuring Our Colorectal CAFS
CAFs play a central role in the Tumor Microenvironment (TME). The TME has been identified as one of the driving factors of tumor progression and invasion. Inside this microenvironment, CAFs, a type of perpetually activated fibroblasts, have been implicated to have a strong tumor modulating effect and play a key role in areas such as drug resistance.

This makes CAFs a target for cancer therapies. The challenge is the TME is heterogeneous making it a challenge to derive homogeneously relevant populations for basic research and drug discovery. This new study uses our Colorectal CAFs to identify markers for isolating these populations. Martin Nurmik, Pit Ullmann, Fabien Rodriguez, Serge Haan, Elisabeth Letellier. In search of definitions: Cancer-associated fibroblasts and their markers. doi: 10.1002/ijc.32193.

Images: Expression of common markers in patient-derived fibroblasts. Immunofluorescent staining of primary colon cancer fibroblasts (Neuromics, #CAF05), reveals a heterogeneous expression pattern for both αSMA/ACTA2 (abcam #ab7817, 1/200) and FAP (Santa-Cruz Biotechnology #sc-65398, 1/200), while PDGFRα (abcam #ab61219, 1/200) expression in tested cells remains relatively homogenous. Nuclei of fibroblasts were stained using DAPI (DAPI Fluoromount-G® Mounting Medium). Image is representative of three independent biological experiments.Cells were imaged using a Zeiss LSM 510 Meta laser scanning confocal microscope (Carl Zeiss, Jena, Germany) with a Plan-Apochromat 63x/1.40.

The authors conclude when selecting for CAF populations using antibody-based methods such as FACS, it is essential that multiple surface markers are used in order to avoid any chance of introducing unintentional subtype bias. Other available surface markers such as PDGFRα/β work well here, as do more general fibroblast surface markers like Thy-1 cell surface antigen (CD90), provided that the cell population is also subjected to selection with negative markers,

Fatty Acids and IBD and Colitis

The Dangers of a High Fatty Acid Diets
Our westernized high-fat diet (HFD) is associated with the development of inflammatory bowel disease (IBD), Colitis and other intestinal related diseases and cancers. Here one the root causes is identified to elevated production of pro-inflammatory cytokine-IL-1β by macrophages. It is suspected that HFD sets off an autoimmune like cascade.

This cascade is triggered by NLRP3 inflammasomes. Our Apoptosis Kit-Magic Red-Real Time Cathepsin B is used to demonstrate these inflammasomes are activated via Cathepsin B Release: Shengnan Zhao, Zizhen Gong1, Jiefei Zhou1, Chunyan Tian, Yanhong Gao, Congfeng Xu, Yingwei Chen, Wei Cai and Jin Wu1Deoxycholic Acid Triggers NLRP3 Inflammasome Activation and Aggravates DSS-Induced Colitis in Mice. Front. Immunol., 28 November 2016 | http://dx.doi.org/10.3389/fimmu.2016.00536 ...Lipopolysaccharide-primed J774A.1 cells were incubated with or without DCA (100 μM, 24 h); then, the cells were stained with Lyso Tracker Green DND-26 (Invitrogen) or magic red cathepsin B fluorogenic substrate z-Arg-Arg cresyl violet (Neuromics) for 1 h, followed by Hoechst staining for half an hour. Fluorogenic signals were captured by inverted fluorescence microscope (Leica)...

Image: Lipopolysaccharide-primed J774A.1 cells were incubated with or without DCA (100 μM, 24 h); then, the cells were stained with Lyso Tracker Green DND-26 (Invitrogen) or Magic RedTM Cathepsin B fluorogenic substrate z-Arg-Arg cresyl violet (Neuromics) for 1 h, followed by Hoechst staining for half an hour. Fluorogenic signals were captured by inverted fluorescence microscope (Leica). Front. Immunol., 28 November 2016 http://dx.doi.org/10.3389/fimmu.2016.00536

We will continue to post how our customers are using our Apoptosis Kits for their discoveries here.

Cancer Associated Fibroblasts (CAFs)-Flow Cytometry and Immunostaining Results

If they Walk and Talk Like CAFs, They Are!

We are experiencing growing demand for are CAFs. They are closely associated with primary tumor cells and participate in the neoplastic process. There is reciprocal communication between CAFs and tumor cells through paracrine effects of secreted growth factors, cytokines & chemokines from both fibroblasts, tumor cells and other tumor-associated cells.

Potential users are asking how well the cells are characterized. The purpose of this posting is to share key gene expression data:

CELL LINES	CD105	CD90	CD44	CD326	CD133	FAP
SC00A1 (hMSCs)	+	+	+	-	-	-
SC00A5 (pancreatic fibroblasts)	+	+	+	-	-	-
CAF05 (colorectal CAFs)	+	+	+	-	-	+
CAF07-AD (adenocarcinoma lung CAFs)	+	+	+	-	-	+
CAF08 (Pancreatic Stellate CAFs)	+	+	+	-	-	+

Table: expression of key proteins

Images: Human Lung Adenocarcinoma CAFs stained with Vimentin, aSMA, b-Catenin and DAPI (blue),

Cancer Associated Fibroblasts Gene Expression Data(pdf - 784Kb)-Flow Cytometry and Immunostaining Results.

Questions? Please contact me directly-Pete Shuster- CEO & Owner-pshuster@neuromics.com or 612-801-1007.

The Growing Value of Cancer Associated Fibroblasts or CAFS

Balancing Supply and Demand

There is reciprocal communication between Cancer Associated Fibroblasts (CAFs) and tumor cells. They are now recognized as playing a key role in promoting the malignant process and blocking immune surveillance  They are becoming increasing important in Cancer Drug Discovery Programs.

Their growing value is making it harder for us to source resected tumors. We have been receiving advanced orders from clients who need to insure they have a stock. We have ordered tumors for the below CAFs and will be shipping to those who have place advance orders by the end of November.

Name	Catalog #	Type	Species	Applications	Size	Price
Human Colorectal Tumor CAFs Kit	CAF05	Human Primary CAFs	H	Cell Assays	1,000,000 cells	$799
Human Endometrial Adenocarcinoma CAFs	CAF01	Human Primary CAFs	H	Cell Assays	1,000,000 cells	$799
Human Ovarian Serous Tumor CAFs	CAF02	Human Primary CAFs	H	Cell Assays	1,000,000 cells	$799
Human Lung Adenocarcinoma CAFs	CAF07A	Human Primary CAFs	H	Cell Assays	1,000,000 cells	$799
Human Lung Squamous Cell Tumor CAFs	CAF07SC	Human Primary CAFs	H	Cell Assays	1,000,000 cells	$799
Human Immortalized Pancreatic CAF-Stellate Cells	CAF08	Human Primary CAFs	H	Cell Assays	500,000 Immortized Cells	$4,335
Human Pancreatic Tumor Cells Kit	PXPC3A1	Cells+Media Kit	H	Cell Assays	1,000,000 cells	$499
Conditioned Media for Ovarian Serous Tumor CAFS	CAF04	Cell Growth Media	H	Cell Assays	5 ml	$250
Conditioned Media from Endometrial CAFS	CAF03	Cell Growth Media	H	Cell Assays	5 ml	$250
VitroPlus III, low serum, complete	PC00B1-100	Cell Growth Media	H	Cell Assays	100 ml 500 ml	$65 $165

Image: Endometrial CAFs courtesy of Tiana Tonrey, Vitro Biopharma

Should have questions about availability and ship dates for any of these please call (612-801-1007) or e-mail: pshuster@neuromics.com. Thank you.
Pete Shuster, CEO, Neuromics