Immunodeficient Mice for Cancer Studies: Which host strain should I use?

It’s a straightforward question with a not-so-straightforward answer!

Four classes of immunodeficient mice

JAX distributes a variety of immunodeficient strains that can be divided into four main categories:

• “Nude” mice
• “Scid” mice
• “Rag-deficient” mice
• “Higher-order, multigenic” immunodeficient mice

A JAX Notes article thoroughly describes these offerings. Briefly, “nude” mice are homozygous for the Foxn1^nu, or “nude,” mutation. Foxn1 encodes a transcription factor required for both hair follicle and thymic development. In its absence, mice are both hairless and athymic. Because the thymus fails to form, there is no place for CD4⁺ and CD8⁺ T cells to differentiate and mature, making nude homozygotes T cell-deficient.

“Scid” mice are homozygous for the Prkdc^scid mutation. The gene Prkdc encodes the catalytic subunit of DNA-dependent protein kinase that is required for DNA repair and for sealing the double-stranded DNA breaks that occur during somatic recombination of T cell receptor (TCR) and immunoglobulin (Ig) genes. In the absence of Prkdc protein, TCR and Ig genes cannot rearrange, resulting in mice that are both T and B cell deficient.

“Rag-deficient” mice are mice that fail to express functional Rag1 or Rag2 proteins. Like the Prkdc gene, both Rag1 and Rag2 are required for somatic recombination of TCR and Ig genes, and the absence of either gene results in T and B cell deficiency. Mice that carry either the Rag1^tm1Mom or Rag2^tm1.1Cgn mutations have very similar, if not identical, phenotypes.

Finally, “higher-order, multigenic” immunodeficient mice are constructed from either Prkdc^scid or Rag-deficient mice, and carry additional immunodeficiency-enhancing mutations. Among these mice are our NSG and NRG mice, which carry a specific mutation in the interleukin 2 receptor gamma subunit gene (Il2rg^tm1Wjl) in combination with the Prkdc^scid and Rag1^tm1Mom, respectively. These mice are B, T and NK cell deficient. Additionally, because they both have NOD/ShiLtJ genetic backgrounds, they are hemolytic complement-deficient and carry alleles that adversely affect macrophage and dendritic cell functions.

Choosing a tumor cell host

Choosing the best host for a tumor study from among these strains depends on several factors, including:

• What type of tumor do you want to engraft— e.g., an established cell line, a blood-borne tumor, or a slow-growing primary tumor?
• Where will you engraft the tumor— e.g., subcutaneously or orthotopically— and how will you evaluate its growth?
• How long do you need the mice to remain engrafted?

Matching your answers to these questions to the characteristics of the mice described in the Comprehensive Immunodeficient Suite should considerably narrow your host choices.

Nude mice

In general, nude mice make ideal hosts for established, rapidly growing tumor cell lines. Because they are hairless, they don’t have to be shaved or depilated to evaluate the growth of a subcutaneous tumor. The hairless phenotype also makes following fluorescently labeled cells by whole-body imaging very easy. However, because nude mice still have B cell and robust NK cell responses, they are not suitable hosts for blood-borne cancers, such as leukemias or lymphomas. They are also poor hosts for slow-growing human or mouse primary tumor cells or heterogeneous tumor fragments.

JAX maintains live colonies of three different nude strains: NU/J (002019) inbred mice, B6-nude (000819) congenic mice, and J:NU (007850) outbred nudes. Being inbred and congenic, respectively, NU/J and B6-nudes are genetically homogeneous. Therefore, they are likely to support more consistent tumor growth. In contrast, outbred J:NU nudes may exhibit more variable tumor growth because they are genetically heterogeneous. Their outbred nature, however, makes them more vigorous than either NU/J or B6-nudes, so they may be able to withstand more invasive or severe experimental manipulation. Because both NU/J and J:NU mice are albino, they may be preferred for monitoring tumor growth by whole-body imaging.

Scid and Rag-deficient mice

If you’re going to engraft slow-growing, established, primary cell lines, or blood-borne cancers, scid and Rag-deficient mice are better choices than nude mice. Both are T and B cell-deficient and therefore more immunocompromised than nudes. Tumors that grow only stubbornly in nude mice likely will grow more robustly in either of these strains.

Of the two models, scids are probably more widely used as tumor cell hosts, possibly because they’ve been around for a long time – since the 1990s – and are widely available from most mouse vendors. However, they are prone to “leakiness,” which means they may develop low levels of serum immunoglobulins (Igs). Such leakiness is more common in B6- (001913) and BALB/c scid (001803) mice, less common in C3H scid (001131) mice, and least common in NOD- scid (001303) mice. It is not clear what effect, if any, this leakiness has on the suitability of these mice as tumor cell hosts. Studies indicate that the repertoire of antigens that the “leaky” Igs recognize is small, and data linking leakiness to tissue rejection or poor tumor growth is weak.

Of the four scid strains mentioned above, NOD scid mice typically support higher levels of engraftment. However, NOD scid mice can develop thymic lymphomas, which limit their average life span to approximately 30 weeks. Therefore, NOD scid mice may not be suitable hosts for long-term engraftment studies.

Because Prkdc protein is involved in DNA repair, scid mutants are radiation-sensitive and therefore may not be suitable hosts if they need to be irradiated. On the other hand, because the Rag proteins are not involved in DNA repair, Rag-deficient mice are radiation-resistant. Moreover, they are not “leaky.” Indeed, Rag-deficient mice have occasionally been referred to as “non-leaky scids.”

Because neither scid nor Rag-deficient mice are hairless, they need to be shaved or depilated to monitor the growth of a subcutaneous tumor.

Higher-order immunodeficient mice: Preferred hosts for human primary tumors

If you’re going to engraft human and mouse primary tumors, you’ll probably need a greater degree of immunodeficiency than that provided by nude, scid, or Rag-deficient mice. Over the years, several strains have been produced that combine additional mutations with either scid or Rag deficiency to further suppress a mouse’s immune responses. These include scid beige (Lyst^bg), NOD scid/(b2-microglobulin (B2m)-deficient (002570), and NOD Rag1/perforin 1 (Prf1)-null (004848) mice. More recently, NSG and NRG mice, which combine the scid and Rag1 mutations respectively with a deficiency in the interleukin 2 receptor gamma chain (Il2rg), have been developed that support more robust post-engraftment tumor growth that the earlier, double-mutant mice. Moreover, human primary tumors engrafted into NSG mice retain more of their native cytostructural and stromal characteristics than when they are engrafted into BALB/c scid mice. The unparalleled immune deficiency of NSG mice has made them a preferred host for establishing and passaging the human primary tumors in our Patient-Derived Xenograft (PDX) Resource, which now contains more than 200 established, low-passage models.

The only major disadvantage when using NSG or NRG as tumor hosts is that the mice do have hair and must be shaved or depilated to monitor the growth of subcutaneous tumors. Further, in at least one study, NSG mice were too permissive: melanoma cells that normally are not tumorigenic in a human patient formed tumors in this mouse. In that study, NOD scid hosts were found to more faithfully recapitulate the malignancies normally observed in human cancer patients.

In general, the more severely immunocompromised a mouse host, the more likely it is that tumor cell line or primary tumor will grow. In many cases, however, a less immunocompromised host will support adequate tumor growth. The choice of an immunodeficient host, then, depends greatly on the origin of the tumorigenic cells and their typical grow rate when transplanted into a host. For additional guidance in choosing an immunodeficient host for any sort of tumor engraftment study, please contact our Technical Information Services group.