An orthotopic xenograft is a specialized research model created by transplanting human cells or tissue into the anatomically corresponding organ of an animal. The term itself provides clues to its meaning; “xeno” originates from the Greek word for foreign, indicating the use of material from a different species, while “orthotopic” means “correct place.” These models almost always involve an immunocompromised animal, typically a mouse, to prevent its immune system from rejecting the foreign human tissue. By placing the human cells in their native organ environment, researchers can generate a model that more closely mirrors how a disease, particularly cancer, behaves in humans.
The Orthotopic Xenograft Procedure
The creation of an orthotopic xenograft model is a meticulous process that begins with the selection of an appropriate host animal. To prevent tissue rejection, researchers use immunocompromised mice. Common strains include athymic nude mice, which lack a thymus gland and cannot produce T-cells, and Severe Combined Immunodeficiency (SCID) mice, which lack both T-cells and B-cells. For studies requiring an even more compromised immune system, researchers may use NOD-scid Gamma (NSG) mice, which also lack functional natural killer cells.
Following host selection, the graft material is prepared. This material can be derived from established human cancer cell lines or from patient-derived xenografts (PDX). PDX models involve implanting fresh tumor tissue, obtained directly from a patient’s surgery or biopsy, into the mouse. This method is thought to better preserve the original tumor’s genetic and structural characteristics. The tissue is prepared as a single-cell suspension or as very small tissue fragments.
The final step is the surgical implantation of the graft into the target organ. For example, to model pancreatic cancer, a small incision is made in the mouse’s abdomen, the pancreas is exposed, and the prepared tumor cells or tissue are carefully implanted onto it. Similarly, creating a brain tumor model may involve using stereotactic equipment to inject cancer cells into a precise location within the mouse brain. After the procedure, the animal is closely monitored for recovery and for signs of tumor growth.
Distinguishing Orthotopic from Other Xenograft Models
Orthotopic xenografts are defined by their specific implantation site, which sets them apart from other common xenograft models, most notably the subcutaneous model. In a subcutaneous xenograft, cancer cells are injected into the space just beneath the animal’s skin, usually on the flank. This method is technically simpler, faster, and allows for easy monitoring of tumor growth, as the resulting mass can be measured directly with calipers.
The fundamental difference lies in the tumor microenvironment—the complex ecosystem of blood vessels, stromal cells, and signaling molecules that surrounds a tumor. When a tumor is grown orthotopically, it develops within the architecture of its organ of origin. This placement allows it to interact with the correct tissue-specific cell types and establish a more natural blood supply, which can influence its growth and response to therapy. These interactions are largely absent in the subcutaneous space. While subcutaneous models are valuable for initial screenings, the orthotopic approach provides a more faithful representation of human disease.
Key Applications in Preclinical Research
The characteristics of orthotopic xenografts make them useful for specific areas of preclinical research. A primary application is in the study of cancer metastasis. Because the primary tumor is growing in its natural organ, it can invade surrounding tissues and spread to distant sites through the bloodstream or lymphatic system in patterns that often resemble those seen in human patients. For instance, an orthotopic pancreatic cancer model in a mouse may metastasize to the liver and peritoneum, which are common sites of spread for this disease in humans. This allows researchers to investigate the biological mechanisms of metastasis and test therapies aimed at preventing or treating it.
Another application is the evaluation of therapeutic efficacy. Testing a potential drug on a tumor that is interacting with its correct microenvironment can provide a more accurate prediction of how that drug might perform in a clinical setting. The organ-specific environment influences factors like drug delivery to the tumor through the local blood supply. For example, when testing a new drug for glioblastoma, an aggressive brain cancer, an orthotopic model is necessary to see if the drug can cross the blood-brain barrier.
These models also allow for the assessment of novel treatment modalities, such as immunotherapies. The data from orthotopic models can be more predictive of clinical outcomes, helping guide the development of new treatments and justifying the model’s complexity and cost.
Technical Considerations and Limitations
Despite their advantages, orthotopic xenograft models present several technical and biological challenges. The procedure requires microsurgical skills and is more invasive, time-consuming, and expensive than creating subcutaneous models. The success rate of tumor engraftment can also be more variable depending on the organ and the specific technique used, potentially requiring a larger number of animals for a study.
Monitoring the growth of internal tumors is a practical issue. Unlike subcutaneous tumors that are easily visible and measurable, orthotopic tumors require sophisticated imaging technologies for tracking. Researchers rely on non-invasive systems like bioluminescence imaging (BLI) or high-resolution imaging such as micro-MRI and micro-ultrasound. This equipment adds complexity and cost.
A biological limitation is that the tumor’s supporting stroma is from the host mouse, not human. This mouse-derived stroma can interact with the human cancer cells differently than a human stroma would. This can influence tumor behavior and response to certain therapies.