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EMT status of circulating breast cancer cells and impact of fluidic shear stress – By Jin et al., 20


Background

Tumour metastasis is a major cause of cancer death, but scientists still don’t fully understand its mechanisms. Finding proper treatment and prevention strategies for cancer metastasis is challenging without this knowledge. Circulating tumour cells (CTCs) are cancer cells that shed from the primary tumour into the bloodstream and can form metastasis in distant body parts.

The Epithelial-to-Mesenchymal Transition (EMT) is a process in which epithelial cells lose their properties and become mesenchymal cells, facilitating metastasis. CTCs can be at the epithelial (E-CTC), intermediate epithelial/mesenchymal (E/M-CTC), or mesenchymal (M-CTC) state of the EMT. M-CTCs are often associated with poor outcomes for breast and colorectal cancer patients.

In this study, Jin and colleagues tested the hypothesis that:

  • CTCs are heterogeneous in their EMT state, which differs from primary tumour cells.

  • Hydrodynamic forces of the blood circulation regulate EMT states of CTCs.

  • EMT states define the growth and metastasis characteristics of CTCs.

Methods

To test their hypothesis, the researchers conducted experiments using an animal model of breast cancer. After 30 days of injecting cancer cells into the mammary fat pad to initiate tumour formation, the researchers euthanised the animals and collected blood samples. Next, they created a culture that eliminated the leukocytes, allowing CTCs to grow. They classified these CTC into two groups according to their characteristics: 4T1CTC-1 (predominantly epithelial) and 4T1CTC-2 (mesenchymal) cells.

Cell assay under flow conditions

The researchers used a microfluidic circulatory system to study how the hydrodynamic forces of circulating blood affect the development of EMT. The system consisted of Vena8 Fluoro+ biochip microfluidic chambers with 8 channels and a total volume of 0.8 μL. First, they washed the device with 2% BSA in PBS to prevent cells from attaching to the connection tubes and wall of the reservoir. They infused the cells at 2 × 10^6/mL into the system and allowed them to circulate for 2 h at 37˚C in a humidified 5% CO2 incubator at a flow rate of 1.908 mL/min, which generated a wall shear stress of 15 dyne/cm2 to mimic the shear stress of arterial circulation. Then, they collected the cells and analysed them for EMT markers.


Results

The scientists investigated how fluid shear stress affects the EMT state of CTCs. Here are the key discoveries from their experiments:

  • There was no difference in the expression of the epithelial marker E-cadherin and the mesenchymal marker Vimentin between cells in adherent and suspension cultures (24 h) for the 4T1 primary tumor cells, 4T1 CTC-1 cells and 4T1 CTC-2 cells. Thus, the extracellular matrix did not affect the EMT state of these cells (Fig. 4 A – B).

  • Exposure to 15 dyne/cm2 of arterial shear stress for 2 h at 37˚C reduced the expression of E-cadherin and increased the expression of Vimentin in the 4T1 primary tumour cells, 4T1 CTC-1 cells and 4T1 CTC-2 cells (Fig. 4 C-E).

  • The expression of the epithelial marker EpCAM was reduced in the 4T1 primary tumor cells and 4T1 CTC-1 cells when exposed to arterial shear stress (Fig. 4 F).

Fig. 4. Effect of shear stress on the EMT state of CTCs. (A&B) The expression of the EMT markers E-cadherin and Vimentin in 4T1primary cells,4T1CTC-1, and 4T1CTC-2 cells after 24 h in suspension culture. Left panel: representative immunoblots, and right panel: the densitometry measurements of immunoblots (n = 3, Student’s t-test, ns: no statistical difference vs. no suspension). (C, D&E) The expression of the EMT markers E-cadherin and Vimentin on 4T1primary cells, 4T1CTC-1, and 4T1CTC-2 cells after exposure to 15 dyn/cm2 of shear stress; representative immunoblots and quantitative densitometry (n = 3, Student’s t-test, *p < 0.05, **p <0.01, ***p < 0.01 vs. No shear stress). (F) The expression of EpCAM of 4T1primary cells and 4T1CTC-1 cells exposed to fluid shear stress of 15 dyn/cm2 was examined by Flow cytometry.

Other experiments in this study

To explore how CTCs differed from each other and from their parent cells, the researchers conducted a series of experiments. This included cell proliferation, migration, invasion assays, and proteomics analysis.

The main findings of these experiments were:

  • 4T1CTC-1 and 4T1CTC-2 cells had different EMT states distinct from the primary tumor cells.

  • 4T1CTC-1 cells were at an intermediate E/M state, whereas 4T1CTC-2 cells were at a mesenchymal state.

  • 4T1CTC-1 and 4T1CTC-2 cells proliferated at a lower rate than the 4T1primary tumor cells. 4T1CTC-2 cells also proliferated slower than 4T1CTC-1 cells.

  • When injected into the mammary fat pad of female mice, the tumor volume and weight of 4T1CTC-1 and 4T1CTC-2 cells were significantly smaller than those of 4T1primary tumor cells at 16 days post implants.

  • When measured in vitro, 4T1CTC-1 cells migrated slower than 4T1CTC-2 and 4T1primary tumor cells. However, 4T1CTC-1 and 4T1CTC-2 cells invaded the extracellular matrix at lower rates than the 4T1 primary tumor cells.

  • The proteomics analysis found that 4T1CTC-1 cells expressed high levels of the epithelial markers EpCAM, Cadherin-1, CK19, CK18, and CK8, whereas 4T1CTC-2 cells expressed a high level of Vimentin.

Conclusion

According to the authors, these results could lead to new discoveries regarding the EMT state of CTCs and the contribution of fluidic shear stress.

You can see more details of the experiments here.


How to get started?

Thinking about trying out similar experiments in your lab? Here's what you'll need as a minimum setup:

  • VenaFlux Pro platform – a semi-automated microfluidic platform designed for conducting studies on cell adhesion, binding, rolling, and migration under shear flow conditions that replicate in vivo flow rates.

  • Vena8 Fluoro+ Biochips – to mimic the shear stress of arterial circulation.

  • Mirus Evo Pump – a microfluidic system equipped with an 8-channel syringe pump for analysing cells under shear flow, simulating the natural flow conditions within blood vessels.

  • Microenvironmental chamber – a temperature-controlled frame that keeps the biochip at 370˚ C.

  • Inverted microscope – we supply the Zeiss AxioVert A1 with the VenaFlux Pro option or the Zeiss AxioObserver7 with the VenaFlux Elite option.

  • Digital camera – to capture images and video recordings. We supply the Prime BSI Express with the VenaFlux Pro and Elite options

  • Image Pro Cell Analysis software – for image and video analysis.

If you already have some of these items (such as the inverted microscope, camera, or cell analysis software), we recommend the VenaFlux Starter kit. We have options that suit all budgets. You can check them out on our eShop


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