Hello Olena Odnokoz

-Site of injection (Orthotopic mammary fat pad vs subcutaneous).

Compared to the subcutaneous space, the orthotopic location is able to provide the appropriate microenvironment to realistically model tumor invasiveness and one can study the metastatic cascade.

Orthotopic injection is likely to increase tumor volume. Also, vascularization of orthotopic tumors is significantly enhanced as compared to subcutaneous tumors. The involvement with local connective tissue matrix enhances metastatic behavior when tumor cells are implanted orthotopically.

While orthotopically implanted tumors can be challenging to follow, they are advantageous in that they more accurately mimic the natural environment of tumor development and provides an effective approach to investigate tumor pathophysiology and develop therapeutic strategies.

While the subcutaneous tumor models can be used as a quick screen to grossly evaluate drug efficiency studies, it is not a platform that can assess the agent in a clinically relevant site. It has limitations in reliably predicting in-vivo efficacy due to the differences in tumor microvasculature and angiogenesis between the subcutaneous pocket and the orthotopic location. But this model still remains popular in use due to its ease of use.

-Do I need Matrigel? Based on my search I don't need to mix cells with Matrigel for orthotopic mammary gland injection. For subcutaneous injection, MDA-MB-231 seems to grow very slowly and it is recommended to mix it with Matrigel 1:1.

For orthotopic mammary gland injection, you don't need to mix cells with Matrigel (unless it is a requirement in certain cases) as it most accurately recapitulates tumor stroma interactions in the primary tumor microenvironment. However, for subcutaneous injection, the co-injection of extracellular matrix components, like Matrigel, with human tumor cells into mice is recommended as it will enhance local tumor formation and growth at the site of inoculation.

-What cell number to inject? The number varies in the literature from 0.8x106 to 1X107 cells per mouse.

You may use 0.5 x 10^6 to 1 x 10^6 cells. Please note that the amount of cells depends on the cell type used. Use of 0.8 x 10^6 cells in 100ul PBS should not be a problem.

Good luck!

Hello Malcolm,

Thank you so much for your help and detailed information! I really appreciate it.

Best regards,

Olena

It sounds like you have already done a good job of researching the literature on MDA-MB-231 xenograft experiments. Here are some suggestions and feedback:

• Site of injection: The site of injection can affect the growth of tumors and their response to treatment. Orthotopic mammary fat pad injection is considered to be more clinically relevant as it mimics the natural environment of the tumor, but it can be technically more challenging. Subcutaneous injection is easier to perform, but it may not accurately represent the tumor microenvironment. Ultimately, the choice of injection site should depend on the research question and the specific goals of the experiment.
• Matrigel: The use of Matrigel can provide a better growth environment for cells, but it may also influence the response of the cells to treatment. The decision to use Matrigel should depend on the specific research question and the goals of the experiment.
• Cell number: The optimal number of cells to inject can depend on the specific experimental design and the research question. As you mentioned, the literature suggests a range of cell numbers to inject, and the choice should depend on the specific goals of the experiment.

In addition to the factors you have mentioned, other considerations could include the age and sex of the mice, the use of anesthesia during injection, and the choice of endpoint criteria. It is always a good idea to consult with experienced colleagues or a veterinarian to ensure that the experiment is well-designed and meets ethical and regulatory requirements. Good luck with your experiment!

Hi Olena,

Regarding whether you should use Matrigel or not, there are some factors to consider. Matrigel will definitely help to provide a more localized tumor generation compared to injection of PBS in both orthotopic mammary fat pad and subcutaneous injection sites. The tumour origins of Matrigel use is always a confounding factor, however results are generally accepted.

Matrigel can be a little tricky to order right now as supply is quite limited, so I would suggest checking out OkaSciences. They make Matrigel equivalents and ship next day! https://okasciences.com/shop/okamatrix/