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