Authors: Michelle Lee, Florian Muller, Elisa Aquilanti, Baoli Hu & Ronald DePinho
Xenografting of human cancer cell lines injected subcutanously in nude mice has become the standard testing platform for dissecting mechanistic aspects of tumorigenesis and for pre-clinical drug development. However, subcutanous xenografts do not model tumors in their tissue of origin and thus may have some clinically relevant limitations. Xenografted human tumor cells directly injected into their relevant organ may present a more accurate model of human tumors. Here, we describe a rapid protocol for the injection of tumor cell lines into the mouse brain for the establishment of orthotopic tumors.
Subcutaneuous growth of human tumor cells in immunodeficient mice is a mainstay of cancer research and anti-cancer drug development. However, it is known that the microenvironment influences tumor growth as well drug response.
To better model human tumors in immunocompromised mice, cancer cells can be injected into the organ of origin (orthotopically), allowing organotypical interactions between cancer cells and the surrounding microenvironment. Orthotopic tumors may more properly recapitulate human tumor biology, as the interactions of tumor cells with normal tissue cells as well as stromal, vascular, and immunologic cells could have effects on cell survival and migration, and even developmental potential. Similarly, orthotopic tumors may recreate some physiologic barriers and could reveal important issues in drug penetration and metabolism.
Located within the microenvironment of the highly specialized and protected central nervous system, orthotopic brain tumor models may have higher clinical relevance than conventional subcutaneous xenografts for understanding tumor behavior and for predicting drug efficacy.
The procedure described below details how to perform intracranial tumor cell injection in mice. This is a major surgery and all procedures must be approved by IACUC or other animal use committee.
Prepare cells for injection:
Load Hamilton syringe with cells:
After proper setup of an efficient system, 3-4 mice injected per hour can be passed through the procedure, which includes, anesthesia, injection, and recovery.
Some typical problems include injection of cells in the sub-cutaneous areas of the skull or into the membranes (dura, pia) covering the brain, rather than the brain itself. The ideal location is in the frontal cortex, with tumors cells located within the brain parenchyma near the lateral ventricles.
As with all mouse surgical procedures requiring deep anesthesia, death from hypothermia can be an issue, which must be minimized by appropriate supportive care including monitoring and the use of a heating pad.
It is of course advisable to include a positive control when first performing this procedure. Many established glioma and non-glioma cell lines rapidly and consistently form intracranial tumors when injected in this manner, including U87 cells.
A negative control would consist of normal, immortalized but non-transformed human astrocytes.
The tumor latency is dependent on both the cell line and the number of cells injected. The typical time to onset of neurological symptoms is about 3 months but can be as long as 6 months, or even longer.
Volumetric measurement of intracranial tumors by MRI is possible but not practical, and is cost-prohibitive when using a large number of animals. Therefore, neurological symptoms, specifically abnormalities of gross motor function, are used as an endpoint for tumor latency. Neurological symptoms are indicative of a tumor that is sufficiently large to have displaced or infiltrated the mouse brain or to have caused obstruction of cerebrospinal fluid flow with resultant increased intracranial pressure.
Dormant, non-tumor forming cells can remain alive in the cerebrospinal fluid for a prolonged time after injection: using anti-human NUMA (Epitomics 3402-1) that specifically detects human but not mouse cells, we have observed residual, non-tumor forming cells as long as 6 months after orthotopic injections.
Zheng H, Ying H, Yan H, Kimmelman AC, Hiller DJ, Chen AJ, Perry SR, Tonon G, Chu GC, Ding Z, Stommel JM, Dunn KL, Wiedemeyer R, You MJ, Brennan C, Wang YA, Ligon KL, Wong WH, Chin L, DePinho RA, 'p53 and Pten control neural and glioma stem/progenitor cell renewal and differentiation.'
Bachoo RM, Maher EA, Ligon KL, Sharpless NE, Chan SS, You MJ, Tang Y, DeFrances J, Stover E, Weissleder R, Rowitch DH, Louis DN, DePinho RA. 'Epidermal growth factor receptor and Ink4a/Arf: convergent mechanisms governing terminal differentiation and transformation along the neural stem cell to astrocyte axis.' Cancer Cell. 2002 Apr;1(3):269-77.
Loi M, Di Paolo D, Becherini P, Zorzoli A, Perri P, Carosio R, Cilli M, Ribatti D, Brignole C, Pagnan G, Ponzoni M, Pastorino F., 'The use of the orthotopic model to validate antivascular therapies for cancer.' Int J Dev Biol. 2011;55(4-5):547-55.
Michelle Lee, Florian Muller, Elisa Aquilanti, Baoli Hu & Ronald DePinho, Ronald DePinho
Correspondence to: Florian Muller ([email protected])
Source: Protocol Exchange (2012) doi:10.1038/protex.2012.041. Originally published online 22 August 2012.