09/06/2023

4T1 cells

4T1 is a transplantable murine breast cancer cell line. It is widely used as a genetically identical in vitro tumour model to investigate human breast cancer. 4T1 cells are highly invasive and tumorigenic; they tend to metastasize from the primary tumour site in the mammary gland to other sites, including the liver, lymph nodes, lungs, bones, and brain, thus are ideal for studying breast cancer metastasis [1].

This article will discuss all the helpful information about 4T1 tumour cells. Notably, it will include:

General characteristics and origin of 4T1 cells
Culturing information of 4T1 cell line
4T1 cell line: Advantages & disadvantages
Research applications of 4T1 cells
4T1 cells: Research publications
Resources for 4T1 cell line: Protocols, Videos, and More

1. General characteristics and origin of 4T1 cells

Knowledge about a cell line's origin and general characteristics is essential before working with it. Here, in this section, we have mentioned the basics of 4T1 breast cancer cells. For instance, what are 4T1 cells? What are 4t1 cell line characteristics? What is the origin of the 4T1 cell line? What is the morphology of the 4T1 cell line?

4T1 is a breast tumour cell line originating from a mammary tumour that spontaneously arose in mouse BALB/c strain. These highly invasive and tumorigenic cells closely mimic human breast cancer's behaviour in terms of growth and metastatic spread. Specifically, the 4T1 tumour model investigates triple-negative breast cancer (TNBC).
4T1 cells are adherent and have an epithelial cell-like morphology.

4T1 Vs EMT-6 cell line

4T1 and EMT-6 are non-immunogenic, murine cell models for triple-negative breast cancer studies. Here, 4T1 are more aggressive and invasive tumour cells than EMT-6 with less invasive properties [2].

4T1 Vs 4T07 cells

4T07 is also a mouse cancer cell line. The major difference between 4T1 and 4T07 cells is; 4T1 cells can leave the primary tumor site and spread to form visible secondary metastases while 4T07 cells can not form visible metastases despite leaving the primary site [3].

SEM imaging of breast cancer cells.

2. Culturing information of 4T1 cell line

4T1 breast cancer cell line offers extensive research applications in the biomedical field. For culturing these cells, you should go through the following key points describing; what is 4T1 doubling time? How 4T1 cells are cultured? What is the cell seeding density for 4T1?

Key Points for Culturing 4T1 Cells

Doubling Time:	The average population doubling time reported for 4T1 breast cancer cells is 14 hours.
Adherent or in Suspension:	4T1 is an adherent cell line.
Sub-cultivation ratio:	A split ratio of 1:6 and 1:8 is recommended for the 4T1 triple-negative breast cancer cell line. For splitting, cells are rinsed with PBS and incubated with Accutase enzyme for 8 to 10 minutes. Dissociated cells are collected through centrifugation and resuspended in a fresh medium. Resuspended cells are dispensed into the new flask for growth.
Growth Medium:	RPMI 1640 medium is used for culturing 4T1 cells. 10% fetal bovine serum (FBS), 2.0 g/L NaHCO3, and 2.1 mM stable Glutamine is added into the culture medium for ideal cell growth.
Growth Conditions:	4T1 tumor cells are kept in a 37°C humidified incubator with a 5% CO₂ supply.
Storage:	4T1 cells should be stored at below -150 °C temperature, i.e., electric freezer or vapour phase of liquid nitrogen, to maintain cell viability.
Freezing Process and Medium:	CM-1 or CM-ACF are recommended for the 4T1 cell line. A slow freezing method is preferred as it protects cell viability by allowing a gradual 1°C drop in temperature.
Thawing Process:	Frozen cells vial is kept in a 37°C water bath for a few seconds until cells thaw and only a small ice clump is left. These cells are resuspended in the fresh medium and poured into the flask for culturing. After 24 hours of incubation, the media is replaced to remove freezing media components.
Biosafety Level:	Biosafety level 1 laboratory is recommended for 4T1 breast cancer cell culturing.

Murine 4T1 breast cancer cells, 20- and 10-fold magnified.

3. 4T1 cell line: Advantages & disadvantages

This section of the article will cover the advantages and disadvantages associated with the 4T1 metastatic model.

Advantages

The main advantages of the 4T1 cell line are:

Tumorigenicity	4T1 is a highly tumorigenic cell line. These cells have the ability to form a 4T1 syngeneic mouse model for breast cancer study when injected into a mouse. Therefore, the 4T1 model is ideally used to study tumour development, growth, and metastasis. In addition, it is useful for screening and evaluating therapeutic drugs.
In vitro metastatic model	4T1 model has a natural metastasis tendency that allows researchers to effectively investigate underlying mechanisms and pathways involved in the metastasis process. Moreover, 4T1 cells are 6-thioguanine resistant. This helps efficient detection of micro-metastatic cells with improved accuracy compared to other models. As it dissipates the need to weigh target organs and count nodules.

Disadvantages

The disadvantage associated with the 4T1 cell line is:

Aggressiveness/rapid growth rate

4T1 cell line is a highly aggressive triple-negative breast cancer cell line. Due to the rapid growth rate, using this cell line in long-term experiments and control experimental variables becomes challenging.

4. Research applications of 4T1 cells

The 4T1 mouse model cell line is a valuable research tool for studying cancer biology and evaluating new treatments. The major applications of 4T1 cells are listed here:

Cancer biology: 4T1 cell line is widely used to study cancer development and growth. It is an ideal in vitro model to investigate and explore different cellular and molecular mechanisms involved in tumour progression and metastasis. In addition, it may unveil the role of cell signalling pathways, microenvironment, and gene expression in these processes. Research conducted in 2019 used 4T1 cells to discover the driving mechanisms behind cancer cell invasion and migration. The results revealed that STAT3, MMP2, and MMP9 genes are involved, and inhibition of these genes can markedly restrict migration and invasion in cancer cells [4]. Similar studies have also indicated several other signalling pathways involved in tumour development and metastasis.
Cancer therapy research: The 4T1 cell line is extensively applied in cancer therapy research for screening and evaluating new treatments. A study explored the cytotoxic potential of green synthesized iron nanoparticles from rosemary extract (Rosemary-FeNPs) and pure rosemary extract on the 4T1 cell line. The research findings proposed Rosemary-FeNPs are more promising than the pure extract [5]. Similarly to this, A study conducted in 2021 examined the anti-tumour potential of ginger extract in 4T1 breast cancer cells and a 4T1 mouse model [6].

5. 4T1 cells: Research publications

This article section will cover a few interesting and most cited publications on 4T1 cells.

Potential anti-proliferative activity of AgNPs synthesized using M. longifolia in 4T1 cell line through ROS generation and cell membrane damage

This article was published in 2018 in the Journal of Photochemistry and Photobiology. The research proposed that silver nanoparticles synthesized from Madhuca longifolia exert an anti-proliferative effect in 4T1 tumour cells via cell wall degradation and ROS generation.

The cytotoxicity and apoptotic effects of verbascoside on breast cancer 4T1 cell line

This study in the BMC Pharmacology and Toxicology journal (2021) proposed that verbascoside, a natural compound, induced apoptotic effects in 4T1 breast cancer cells via TLR4 signalling.

Eupatorin suppressed tumor progression and enhanced immunity in a 4T1 murine breast cancer model

This research was published in Integrative Cancer Therapies in 2020. This study used 4T1 cells to develop a 4T1 syngeneic mouse model and explored the anticancer potential of Eupatorin using it. Eupatorin delayed tumour development in the 4T1 mouse model.

Inhibitory effect of Viola odorata extract on tumor growth and metastasis in 4T1 breast cancer model

This research published in the Iranian Journal of Pharmaceutical Research (2018) proposed that a medical herb, Viola odorata, has potential cytotoxic effects on 4T1 cells.

Docetaxel-loaded solid lipid nanoparticles prevent tumor growth and lung metastasis of 4T1 murine mammary carcinoma cells

This study proposed that docetaxel-loaded solid lipid nanoparticles (DTX-loaded SLNs) can be a promising agent to treat breast cancer and prevent metastasis as they restrict cancer growth and lung metastasis of 4T1 cells.

6. Resources for 4T1 cell line: Protocols, Videos, and More

The following are a few resources on 4T1 cells:

4T1 transfection tutorial: This video is a step-by-step guide to performing the 4T1 cell line transfection.
Subculturing a cell line: This video explains the subculturing protocol for adherent cell lines.

The following link contains the cell culture protocol for 4T1 cells:

4T1 cell line: This website link contains all the useful information regarding handling 4T1 cultures. It has culture media information, handling protocols for cryopreserved cultures and proliferating cultures.

References

Pulaski, B.A. and S. Ostrand-Rosenberg, Mouse 4T1 breast tumor model. Curr Protoc Immunol, 2001. Chapter 20: p. Unit 20.2.
Maxwell, K.G., Immunomodulation of Breast Cancer Cells for Whole Tumor Vaccination. 2016.
Walker, I., et al., Mammary tumors induce central pro-inflammatory cytokine expression, but not behavioral deficits in Balb/C mice. Scientific Reports, 2017. 7(1): p. 1-13.
Li, Y., et al., Inhibition of Stat3 signaling pathway by natural product pectolinarigenin attenuates breast cancer metastasis. Frontiers in Pharmacology, 2019. 10: p. 1195.
Farshchi, H.K., et al., Green synthesis of iron nanoparticles by Rosemary extract and cytotoxicity effect evaluation on cancer cell lines. Biocatalysis and agricultural biotechnology, 2018. 16: p. 54-62.
Gholizadeh, A.P., et al., Effect of Ginger Extract on 4T1 Breast Cancer Cell Line in Balb/c Mouse. Clinical Cancer Drugs, 2021. 8(1): p. 43-49.