Does anyone had tumor inoculation issues with a high GFP expressing cell line such as RM1.PGLS?

If you have not observed palpable tumors even after increasing the cell numbers and performing subcutaneous tumor inoculation, there could be several reasons for this. Here are some possibilities to consider:

Immune response: The immune system of the host mice may be effectively clearing or suppressing the growth of the injected cells. This can happen if the cells you are injecting are recognized as foreign or if the immune response is particularly strong in the mouse strain you are using (in this case, C57BL/6 WT). To address this, you could try using immunodeficient mouse strains or consider immune modulation strategies to dampen the immune response.

Cell viability and health: Ensure that the cells you are injecting are viable and healthy. Check for proper cell culture conditions, including the use of appropriate media, supplements, and cell handling techniques. Confirm cell viability by performing a trypan blue exclusion assay or similar methods.

Injection site and technique: Verify that the injection site and technique are appropriate for subcutaneous tumor formation. The injection site should be consistent and easily accessible. Ensure that you are injecting the cells at an appropriate depth and avoiding intradermal or intramuscular injection. Double-check the needle gauge, length, and angle of injection.

Microenvironment and angiogenesis: The local microenvironment and angiogenesis play crucial roles in tumor formation and growth. It is possible that the inoculated cells are not able to establish a suitable microenvironment for tumor growth. Consider optimizing the injection site or co-injecting with other factors such as extracellular matrix components or angiogenic factors to enhance tumor formation.

Cell line characteristics: Confirm that the cell line you are using is tumorigenic in mice. It is possible that the cell line you are working with may have lost its tumorigenic potential or is not suitable for subcutaneous tumor formation. Perform a literature search or consult with experts to ensure the tumorigenicity of the specific cell line you are using.

Timing: Tumor growth may require more time to become palpable. Although one week is a reasonable timeframe to expect palpable tumors, it is possible that the tumors are still in the early stages of growth. Consider extending the observation period to allow for further tumor development.

Genetic factors: It's worth noting that individual genetic variability between animals can influence tumor formation. Even in a highly GFP positive cell line, some animals may exhibit different tumor growth kinetics or resistance to tumor formation. Consider using a larger cohort of mice to account for individual variation.

To increase the chances of tumor formation, you can explore the following strategies:

Increase the number of injected cells further to a higher concentration.
Try different injection sites, such as the flank or mammary fat pad, to evaluate their suitability for tumor formation.
Pre-condition the mice with immunosuppressive treatments or immune-modulating agents prior to cell inoculation to minimize immune rejection.
Co-inject the cells with extracellular matrix components or other factors known to enhance tumor formation and growth.
Consult with experts in the field who have experience with the specific cell line and mouse strain to gather further insights and guidance.

It's important to thoroughly assess and troubleshoot each of these factors to determine the cause of unsuccessful tumor formation and identify the best strategies to increase your chances of successful tumor inoculation.

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