Inducing tumors in Balb/c mice using human cancer cell lines like H1299 (a non-small cell lung carcinoma line) can be particularly challenging, especially since Balb/c mice are immunocompetent and typically reject human xenografts without severe immunosuppression.

Possible Reasons Why the Tumor Didn't Form:

1. Insufficient Immunosuppression

• While your regimen (CsA + Ketoconazole + Cyclophosphamide) does have immunosuppressive potential, Balb/c mice have a strong immune response, and human xenograft rejection is common unless mice are fully immunodeficient (e.g., nude, SCID, or NSG mice).
• CsA and Ketoconazole may not be potent enough to allow consistent engraftment of human cells.
• Cyclophosphamide helps deplete lymphocytes but may not provide durable suppression if given only once.

2. Cell Line Compatibility

• H1299 is a human NSCLC cell line, and Balb/c is a mouse strain with a fully functional immune system, so even transient immunosuppression may not be enough for engraftment.
• Successful xenograft models usually use immunodeficient mice like NOD/SCID, NSG, or athymic nude mice for human tumor cells.

3. Cell Viability or Handling

• Was the cell viability >90% before injection?
• Did you inject cells in cold PBS/Matrigel? Matrigel helps mimic ECM and aids engraftment.
• Were cells injected subcutaneously at the correct depth and site?

4. Injection Site and Dose

• 5×10⁶ cells is a reasonable dose for subcutaneous injection.
• However, if the cells are not injected properly (e.g., leakage, shallow injection, trauma), they may die off quickly.

5. Immune Rejection Over Time

• Even if early engraftment began, a rebound immune response may have eliminated the tumor cells if immunosuppression waned.

Troubleshooting Suggestions:

1. Switch to Immunodeficient Mice

• Consider switching to nude, NOD/SCID, or NSG mice for reliable human tumor xenografts.

2. Optimize Cell Preparation

• Ensure cells are freshly harvested, >90% viable, and suspended in cold PBS + Matrigel.
• Inject 100–200 µL subcutaneously with proper technique.

3. Enhance Immunosuppression (if staying with Balb/c)

• If you must use Balb/c mice, consider more aggressive or sustained immunosuppressive regimens, but this is still less reliable than using immunodeficient models.
• Alternatively, use murine-derived tumor cell lines syngeneic to Balb/c mice (e.g., CT26 for colorectal cancer, 4T1 for breast cancer).

4. Monitor Engraftment

• Use in vivo imaging (if using luciferase-labeled cells) or palpation every few days post-injection.
• Early biopsy or histology may help determine if cells persisted at the site.

Summary:

Your tumor model likely failed due to insufficient immunosuppression in a fully immunocompetent host using human cells. For reproducible results, use immunodeficient mice or switch to murine tumor cell lines syngeneic to the mouse strain. Optimizing injection conditions and cell viability is also key.