5.2.4 The NOD/LtSz-scid with
IL2γcnull (NSG) Mouse
NSG mouse strain is an immunodeficient mouse that knocks out the
interleukin 2 receptor gamma chain (IL2Rγ) gene on the basis of
NOD/SCID(Ito et al., 2002). This mice strain lacks mature T and B
lymphocytes and completely loses NK cell activity(Koller & Smithies,
1989). Compared with NOD/SCID mice, the transplantation survival rate of
human cell and tissue in NSG mice is significantly improved, and at the
same time, a higher proportion of normal or cancerous human cells and
tissues can be implanted.
The NSG mouse xenograft model is considered the gold standard for
evaluating human hematologic malignancy grafts(Sontakke et al., 2016)
and is widely used to assess disease pathogenesis and explore potential
therapeutic strategies. Researchers have used the NSG mouse
xenotransplantation method to elucidate the leukemia-initiating cell
(LIC) biology of pre-B ALL in order to optimize the disease model.
Different in vivo growth requirements were determined between
adult and child ALL subtypes, and the importance of age/leukemia subtype
specific research was discovered. This animal model improves our
understanding of the biology of ALL disease(Patel et al., 2014). In
addition, the NSG model is also used to evaluate the effect of cytokines
on leukemia. Studies have shown that IL-27 can promote the proliferation
and differentiation of CD34+ cells in non-leukemia
patients(Seita et al., 2008), suggests that the cytokine may have an
effect on normal hematopoietic stem cells. Based on this, researchers
injected pediatric leukemia cell samples intravenously into NSG mice forin vivo studies to evaluate the anti-tumor activity of human
IL-27. IL-27 reduces the angiogenesis potential of tumor cells,
regulates tumor spread-related genes, and exerts an anti-tumor
effect(Zorzoli et al., 2012). In recent years, cellular immunotherapy
has proven to be an effective treatment for B-cell malignant hematologic
diseases. T cells modified with CD19-specific chimeric antigen receptors
(CARs) have significant clinical advantages for the treatment of
leukemia. NSG mice model is also an excellent tool for evaluating the
superiority of CAR-T therapy, and it has been demonstrated that a
lentiviral vector targeting CD8 (CD8-LV) could generate CD19-CAR T cells
in NSG mice(Pfeiffer et al., 2018). Recently, researchers have made
further progress by using CD4-targeted lentiviral vector (CD4-LV) to
selectively produce CD19-CAR T cells in CD4+ cells
successfully. Transplanted Nalm-6 cells which encoded by
CD19+ luciferase into NSG mice and injected activated
PBMCs cells. According to the luciferase intensity measured by thein vivo imaging system (IVIS) to evaluate the growth, spread and
regression of leukemia in the body, it was found that the mice given
CD4-LV showed faster and better tumor cell killing ability, indicating
that CD4+CAR-T cells have a stronger ability to
eliminate tumor cells(Agarwal, Hanauer, Frank, Riechert, Thalheimer &
Buchholz, 2020).
Xenograft models provide powerful tools for studying normal and
malignant human hematopoietic systems. However, current models cannot
simulate the human bone marrow (BM) microenvironment, and many human
leukemia cells cannot be xenotransplanted. In order to overcome these
limitations, scientists have used new advances in the field of
bioengineering and synthetic material development to manufacture
biological inserts or scaffolds that support the growth and
differentiation of implanted cells effectively(Abarrategi, Mian,
Passaro, Rouault-Pierre, Grey & Bonnet, 2018). Implanted ceramic
scaffolds containing mesenchymal stromal cells (MSCs) into NSG mice to
generate a human bone marrow (huBM-sc)-like niche and observed that AML
MLL-AF9+ and ALL BCR-ABL+ patient
cells were efficiently implanted, maintaining an immature primitive
cell-like phenotype in the huBM-sc ecotype compared to the murine bone
marrow (mBM) niche(Sontakke et al., 2016). Later, based on the discovery
that human bone marrow mesenchymal stromal cells (BM-MSC) transplanted
into heterotopic NSG mice can rebuild the functional hematopoietic
microenvironment(Reinisch et al., 2015), the researchers mixed human
bone marrow BM-MSC with extracellular matrix and transplanted
subcutaneously to form a humanized bone marrow microenvironment in the
ossicle after 8-10 weeks. The implantation level of normal human HSCs
and leukemia cells containing subcutaneous humanized ossicles model is
much higher than that of untreated mice, and direct intraosseous
transplantation accelerates cell implantation and leads to an increase
in the frequency of leukemia initiating cells(Reinisch et al., 2016).
The scaffold implantation approach can evaluate the multicellular
interactions between human MSCs and HSCs, and generate patient-specific
human microenvironment in mice, which can be used to unravel the human
tumor microenvironment, disease pathology and the role of drug
physiological response(Abarrategi, Mian, Passaro, Rouault-Pierre, Grey
& Bonnet, 2018).
6. Transgenic ALL
Animal Models
Traditional transgenic(Tg) animal model is to inject the DNA fragments
into the pronucleus of the fertilized egg directly, and then into the
pseudopregnant female mice(Würtele, Little & Chartrand, 2003),or
injected DNA into embryonic stem (ES) cells through
electroporation(Thomas & Capecchi, 1987). In order to more accurately
simulate the occurrence of leukemia, homologous recombination is used to
generate vectors targeting specific sites in the genome. These vectors
conditionally express genes in response to doxycycline (Tet ON/OFF
system) or Cre recombinase (using Lox/Cre system) to produce
tissue-specific expression of transgenes, and chromosomal translocations
can be produced in mice through Cre-loxP-mediated recombination(Drynan
et al., 2005; Forster et al., 2003), thereby mimicking the chromosomal
translocations that often lead to the formation of tumor-specific fusion
oncogenes in human malignancies(Corral et al., 1996). In addition to the
expression of oncogenes, oncogenic
alleles can also be ”knocked in” to the corresponding normal loci, or
”knocked out” of tumor suppressor genes to establish transgenic animal
models. In recent years, transgenic animal models have played a huge
role in the general study of ALL subtypes, including B-ALL, T-ALL, and
Burkitt’s leukemia/lymphoblastic lymphoma, facilitating the discovery of
drug targets and improving the treatment of leukemia, which will be
described in detail in the following.
6.1 BCR-ABL