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Recent advances in the development of transplanted colorectal cancer mouse models

  • Author Footnotes
    # Co-first authors.
    Yu-Shen Yang
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    # Co-first authors.
    Affiliations
    Department of Anaesthesiology, the Second Affiliated Hospital of Fujian Medical University, Quanzhou, Fujian Province, China
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  • Author Footnotes
    # Co-first authors.
    Chu-Yun Liu
    Footnotes
    # Co-first authors.
    Affiliations
    Department of Anaesthesiology, the Second Affiliated Hospital of Fujian Medical University, Quanzhou, Fujian Province, China
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  • Dan Wen
    Affiliations
    Department of General Surgery, the Affiliated Xinhua Hospital of Dalian University, Dalian, 116021, China
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  • Da-Zhi Gao
    Affiliations
    Department of General Surgery, the Affiliated Xinhua Hospital of Dalian University, Dalian, 116021, China
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  • Shu Lin
    Correspondence
    Reprint requests: Shu Lin, He-fan He, No. 34 North Zhongshan Road, Quanzhou, Fujian Province, 362000, China
    Affiliations
    Department of Anaesthesiology, the Second Affiliated Hospital of Fujian Medical University, Quanzhou, Fujian Province, China

    Centre of Neurological and Metabolic Research, the Second Affiliated Hospital of Fujian Medical University, Quanzhou, Fujian Province, China

    Group of Neuroendocrinology, Garvan Institute of Medical Research, Darlinghurst, Sydney, NSW 2010, Australia
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  • He-fan He
    Correspondence
    Reprint requests: Shu Lin, He-fan He, No. 34 North Zhongshan Road, Quanzhou, Fujian Province, 362000, China
    Affiliations
    Department of Anaesthesiology, the Second Affiliated Hospital of Fujian Medical University, Quanzhou, Fujian Province, China
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  • Xue-Feng Zhao
    Correspondence
    Reprint requests: Prof. Xue-Feng Zhao, No.156 Wansui Street, Shahekou District, Dalian, Liaoning 116021, China
    Affiliations
    Department of General Surgery, the Affiliated Xinhua Hospital of Dalian University, Dalian, 116021, China
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    # Co-first authors.
Open AccessPublished:July 15, 2022DOI:https://doi.org/10.1016/j.trsl.2022.07.003
      Despite progress in prevention and treatment, colorectal cancer (CRC) remains the third most common malignancy worldwide and the second most common cause of cancer death in 2020. To evaluate various characteristics of human CRC, a variety of mouse models have been established. Transplant mouse models have distinct advantages in studying the clinical behavior and therapeutic progress of CRC. Host, xenograft, and transplantation routes are the basis of transplant mouse models. As the effects of the tumor microenvironment and the systemic environment on cancer cells are gradually revealed, 3 key elements of transplanted CRC mouse models have been revolutionized. This has led to the development of humanized mice, patient-derived xenografts, and orthotopic transplants that reflect the human systemic environment, patient's tumor of origin, and tumor growth microenvironments in immunodeficient mice, respectively. These milestone events have allowed for great progress in tumor biology and the treatment of CRC. This article reviews the evolution of these events and points out their strengths and weaknesses as innovative and useful preclinical tools to study CRC progression and metastasis and to exploit novel treatment schedules by establishing a testing platform. This review article depicts the optimal transplanted CRC mouse models and emphasizes the significance of surgical models in the study of CRC behavior and treatment response.

      Abbreviations:

      CDX (cell line‑derived xenograft), CRC (colorectal cancer), EGFR (epidermal growth factor receptor), FGFR1 (fibroblast Growth Factor Receptor 1), HER2 (human epidermal growth factor receptor 2), IL2Rγ (interleukin 2-receptor common gamma chain gene), Jak3 (Janus kinase 3), NK cells (natural killer cells), NOD mice (nonobese diabetic mice), NOG mice (NOD/SCID/IL2Rγtm1Sug mice with complete loss of NK cells), NOJ mice (Jak3-deficient NOD/SCID/Jak3null mice), Nude R/J mice (BALB/c Nude Rag-2/Jak3 double-deficient mice), NSG mice (NOD/SCID/IL2Rγtm1Wjl mice with complete loss of NK cells), PDX (patient-derived xenograft), PDOs (patient-derived organoids), PI3K (phosphatidylinositol 3-kinase), RNF2 (ring finger protein 2), SCID mice (severe combined immunodeficiency), SRE (Sanguisorbae Radix extract)

      INTRODUCTION

      Despite significant efforts to prevent and treat colorectal cancer (CRC), a total of 935,173 deaths worldwide from CRC were reported in 2020.
      • Lu B
      • Li N
      • Luo CY
      • et al.
      Colorectal cancer incidence and mortality: the current status, temporal trends and their attributable risk factors in 60 countries in 2000-2019.
      The primary problem of CRC treatment is not the eradication of the primary tumor itself but rather the formation of incurable metastases. The reported 5-year incidence of stage III metastatic CRC is 28%–73%, while that of stage IV is 5%.
      • Recio-Boiles A
      • Cagir B.
      Colon Cancer. StatPearls. Treasure Island FL: © 2022.
      Therefore, the early diagnosis, detection, and treatment of CRC patients are very important. Although CRC research has made great progress over the years, there are still many critical problems to be solved, such as early detection of micro-metastases and overcoming chemotherapy resistance. To address these issues, preclinical animal models are an indispensable tool.
      In recent years, animal models of human cancer have served as important research models in gaining an in-depth understanding of the biological mechanisms of the pathogenesis and development of malignant tumors. More than any other animal model system; the mouse models have led to revolutionizing our capability to understand the molecular mechanisms of CRC initiation, progression, and treatment. Compared with other mammalian models, the mouse models have several important advantages including (1) small size, (2) inexpensive maintenance, (3) short reproductive cycle, and (4) genetic controllability.
      • Li E
      • Lin L
      • Chen CW
      • et al.
      Mouse models for immunotherapy in hepatocellular carcinoma.
      Currently, there are many preclinical mouse models, including carcinogen-induced models, genetically engineered mouse models, and transplanted CRC mouse models. However, the defects of the first two models, which include low invasiveness, time inefficiency, and uncertain formation sites, greatly confined their application in the aspect of CRC metastasis and treatment.
      • Bürtin F
      • Mullins CS
      • Linnebacher M.
      Mouse models of colorectal cancer: past, present and future perspectives.
      In contrast, transplanted CRC mouse models can overcome these limitations. Therefore, these mouse models have a vital role in the research of human CRC.
      Host, xenograft, and routes of transplantation are the pillars of transplant mice models. During many years of continuous development and growth, the host has undergone several changes from first generation to fourth generation (namely from remaining complete immune system of mice to rebuilding human functional immune system in mice). The first-, second-, third- and fourth-generation hosts respectively include immunocompetent mice, genetically immunodeficient mice, novel combined immunodeficient mice, and humanized mice. CRC samples derived from patients are used for the generation of tumor cell lines, patient-derived xenograft (PDX) and/or patient-derived organoid (PDO). For the establishment of the transplant mice model, patient-derived tumor materials are implanted into host mainly via subcutaneous, intrasplenic, or orthotopic routes. Transplant mice model has commonly been exploited to find novel biomarkers, create a drug testing platform, and develop surgical models (Fig 1).
      Fig 1
      Fig 1Schematic representation of transplant mice models and their applications. To establish the transplant mice model, patient-derived tumor materials are implanted into hosts via subcutaneous, intrasplenic, or orthotopic routes. The first-, second-, third- and fourth-generation hosts include immunocompetent, genetically immunodeficient, novel combined immunodeficient, and humanized mice, respectively. The transplant mice models have commonly been exploited to find novel biomarkers, create a drug testing platform, and develop surgical models. (Color version of figure is available online.)
      Thus, this review summarizes the recent progress of transplanted CRC mouse models and points out their advantages and disadvantages. These pieces of information can provide suitable and useful preclinical tools for studying the invasion and metastasis of CRC and developing new drugs by establishing drug test platforms. In addition, this review describes the optimal transplanted CRC mouse models and emphasizes the significance of the surgical model in the study of CRC behavior and treatment response.

      HOSTS FOR HUMAN CRC XENOGRAFT

      Immune rejection is the primary problem that needs to be addressed during xenograft. Rejection reaction is mainly mediated by immune cells, especially natural killer (NK) cells and T cells. Studies have demonstrated that T cell-mediated cellular immune responses can destroy malignant cells and mediate host-versus-graft responses when exotic cells are transplanted into the mice. In addition, recent evidence has shown that NK cells also play an important role in xenograft rejection by recognizing xenogeneic cells.
      • Candinas D
      • Belliveau S
      • Koyamada N
      • et al.
      T cell independence of macrophage and natural killer cell infiltration, cytokine production, and endothelial activation during delayed xenograft rejection.
      ,
      • Chen D
      • Weber M
      • Lechler R
      • et al.
      NK-cell-dependent acute xenograft rejection in the mouse heart-to-rat model.
      Therefore, avoiding or eliminating these cells is necessary to overcome the immune rejection of xenografts.

      Immunodeficient mice: the first attack of the mouse immune system

      Early attempts in achieving xenograft included: (1) finding sites of blunted or delayed immune responses (eg, anterior chamber of the eye,
      • Greene HS.
      Heterologous transplantation of human and other mammalian tumors.
      brain,
      • Greene HS.
      A conception of tumor autonomy based on transplantation studies: a review.
      and cheek pouch in the hamster
      • Lemon HM
      • Lutz BR
      • Pope R
      • et al.
      Survival and growth of human tissues transplanted to hamster cheek pouch.
      ); (2) using immunosuppressive agents and immune-incompetent animals (new-born mice and fetuses which are naturally immune-incompetent)
      • Gallagher FW
      • Korson R.
      Growth of human cancer (H Ep 3) in normal rats.
      • Kutner LJ
      • Southam CM.
      Growth of human cancer cells (HEp 2) in newborn rats.
      • Levin AG
      • Friberg Jr., S
      • Klein E
      Xenotransplantation of a Burkitt lymphoma culture line with surface immunoglobulin specificity.
      ; and (3) using antilymphocyte
      • Phillips B
      • Gazet JC.
      Growth of 2 human tumour cell lines in mice treated with antilymphocyte serum.
      or antithymocyte serum,
      • Cobb LM.
      Metastatic spread of human tumour implanted into thymectomized, antithymocyte serum treated hamsters.
      or thymectomy
      • Osoba D
      • Auersperg N.
      Growth of a human carcinoma of the cervix in mice thymectomized at birth.
      in different combinations. However, these strategies are limited by their innate drawbacks (Table 1). The emergence of genetically immunosuppressed mice is undoubtedly a milestone in the development of immunosuppressive measures for xenografts.
      Table 1The characteristics of animal transplant model for studies of colorectal cancer
      AnimalMeasures to overcome immune rejectionAdvantagesDisadvantagesReference
      HamsterIntraorbital engraftment1. Immunological unresponsiveness1. Difficult operation
      • Greene HS.
      Heterologous transplantation of human and other mammalian tumors.
      Intracranial engraftment1. Immunological unresponsiveness1. Inaccessible inspection
      • Greene HS.
      A conception of tumor autonomy based on transplantation studies: a review.
      Engraftment into the cheek pouch1. Easy to inspect and follow

      2. Immunologically delayed responsiveness
      1. Immune response eventually develops; 2. Very difficult to evaluate many therapeutic results obtained; 3. Low take rate; 4. Difficult serial passages
      • Lemon HM
      • Lutz BR
      • Pope R
      • et al.
      Survival and growth of human tissues transplanted to hamster cheek pouch.
      Newborn mice and fetusesUndeveloped immune system1. Immunosuppression1. Eventually acquirement of immunocompetence; 2. Higher rate of abortion; 3. A higher rate of destruction of the newborns through maternal cannibalism
      • Gallagher FW
      • Korson R.
      Growth of human cancer (H Ep 3) in normal rats.
      • Kutner LJ
      • Southam CM.
      Growth of human cancer cells (HEp 2) in newborn rats.
      • Levin AG
      • Friberg Jr., S
      • Klein E
      Xenotransplantation of a Burkitt lymphoma culture line with surface immunoglobulin specificity.
      Immunocompetent animalsWhole-body irradiation with or without corticosteroids1. Immunosuppression1. Very toxic to the animals; 2. Severe damage to the guts and bone marrow; 3. High incidence of infections; 4. A shortened life span
      • Toolan HW.
      Neoplastic properties of animal cell lines–general comments.
      Immunocompetent animalsThe use of thymectomy, ALS, or ATS in various combinations1. Immunosuppression1. Difficulty for complete removal of all the thymic tissue; 2. Maternal cannibalism; 3. Irreconcilable titration standard; 4. Toxic and allergic serum reaction; 5. Difficult reconstitution with syngeneic bone marrow
      • Phillips B
      • Gazet JC.
      Growth of 2 human tumour cell lines in mice treated with antilymphocyte serum.
      • Cobb LM.
      Metastatic spread of human tumour implanted into thymectomized, antithymocyte serum treated hamsters.
      • Osoba D
      • Auersperg N.
      Growth of a human carcinoma of the cervix in mice thymectomized at birth.
      ALS, antilymphocyte serum; ATS, antithymocyte serum.
      First proposed by Flanagan in 1966, nude mice are the oldest immunodeficient mice, which are characterized by significant hair loss due to a novel recessive gene.
      • Flanagan SP.
      Nude', a new hairless gene with pleiotropic effects in the mouse.
      The most important feature of nude mice used for cancer research is the lack of thymus and T lymphocytes, which ensures the survival and biological behavior of tumor cells after implantation.
      • Pantelouris EM.
      Athymic development in the mouse.
      ,
      • Sun L
      • Li H
      • Luo H
      • et al.
      Thymic epithelial cell development and its dysfunction in human diseases.
      Furthermore, the natural lack of hair in nude mice made subcutaneously transplanted tumors easy to observe. Therefore, following the first successful transplantation of patient-derived CRC tissue into nude mice,
      • Rygaard J
      • Povlsen CO.
      Heterotransplantation of a human malignant tumour to "Nude" mice.
      engraftment of human colorectal tumors into these rodents has been reported by many investigators.
      • Ren F
      • Li B
      • Wang C
      • et al.
      Iodine-125 seed represses the growth and facilitates the apoptosis of colorectal cancer cells by suppressing the methylation of miR-615 promoter.
      However, although only a small number of T lymphocytes are present in the peripheral blood of nude mice, they still carry intact innate immune system components, especially NK cells and B cells, thus limiting the take rate and subsequent biological process of engrafted tumors.
      • Shultz LD
      • Goodwin N
      • Ishikawa F
      • et al.
      Human cancer growth and therapy in immunodeficient mouse models.
      In 1983, another genetic immunodeficiency mouse appeared. Due to the lack of DNA-dependent protein kinase, it was called severe combined immunodeficiency (SCID) mouse, which largely prevents the development of T and B lymphocytes.
      • Bosma GC
      • Custer RP
      • Bosma MJ.
      A severe combined immunodeficiency mutation in the mouse.
      Because of the lack of functional T and B cells, SCID mice were first used to transplant human hematopoietic stem cells and peripheral blood mononuclear cells.
      • McCune JM
      • Namikawa R
      • Kaneshima H
      • et al.
      The SCID-hu mouse: murine model for the analysis of human hematolymphoid differentiation and function.
      ,
      • Mosier DE
      • Gulizia RJ
      • Baird SM
      • et al.
      Transfer of a functional human immune system to mice with severe combined immunodeficiency.
      Subsequent studies further showed that the rate of human tumor engraftment into SCID mice was higher than that in nude mice.
      • Taghian A
      • Budach W
      • Zietman A
      • et al.
      Quantitative comparison between the transplantability of human and murine tumors into the subcutaneous tissue of NCr/Sed-nu/nu nude and severe combined immunodeficient mice.
      Therefore, SCID mice are mainly used as recipients of human tumors.
      • Paine-Murrieta GD
      • Taylor CW
      • Curtis RA
      • et al.
      Human tumor models in the severe combined immune deficient (SCID) mouse.
      However, recent studies have come to the opposite conclusion that the engraftment efficiency of human gastrointestinal tumors in SCID mice is lower than that in nude mice.
      • Okada S
      • Vaeteewoottacharn K
      • Kariya R.
      Application of highly immunocompromised mice for the establishment of patient-derived xenograft (PDX) models.
      The exact mechanism behind this finding is unclear. Although the SCID mouse model holds great potential, it also has certain limitations. For example, its DNA repair mechanisms are defective and vulnerable to radiation.
      • Fulop GM
      • Phillips RA.
      The SCID mutation in mice causes a general defect in DNA repair.
      Furthermore, like nude mice,
      • Shultz LD
      • Goodwin N
      • Ishikawa F
      • et al.
      Human cancer growth and therapy in immunodeficient mouse models.
      SCID mice exhibit leakage of functional T and B cells later in life,
      • Bosma GC
      • Fried M
      • Custer RP
      • et al.
      Evidence of functional lymphocytes in some (leaky) SCID mice.
      as well as functional macrophages and high levels of NK cell activity. These shortcomings limit the efficiency of engrafting human tumors in this model.

      Novel combined immunocompromised mice: the second attack to the mouse immune system

      As mentioned earlier, immunodeficient mice provide an excellent platform for the study of human tumors, but remnant NK cells prevent homing and maintenance of the tumor cells. Thus, the engraftment efficiencies of human tumor cells in these mice are not as high as expected. To abolish NK cell activity, the novel combined immunocompromised mice were created by crossing SCID mice or nude mice with other breeds.
      NOD/SCID mice were generated by crossing nonobesity diabetes (NOD) mice with SCID mice.
      • Greiner DL
      • Shultz LD
      • Yates J
      • et al.
      Improved engraftment of human spleen cells in NOD/LtSz-scid/scid mice as compared with C.B-17-scid/scid mice.
      Due to multiple deficiencies in both innate and adaptive immunity, NOD/SCID mice proved to be well-suited for transplantation of human tumors including CRC.
      • He Y
      • Liu S
      • Newburg DS.
      Musarin, a novel protein with tyrosine kinase inhibitory activity from Trametes versicolor, inhibits colorectal cancer stem cell growth.
      However, NOD/SCID mice have some lethal defects, such as short lifespan, immune leakiness of T and B cells, and residual NK activities.
      • Santagostino SF
      • Arbona RJR
      • Nashat MA
      • et al.
      Pathology of aging in NOD SCID gamma female mice.
      The development of immunodeficient mice with targeted mutations in the interleukin 2 receptor common gamma chain gene (IL2Rγ) represents a dramatic advancement in the elimination of immune function in mice. As a common receptor complex for 6 different IL receptors, IL2Rγ plays a key role in the development of NK cells and lymphocytes by interacting with the Janus kinase 3 (Jak3) nonreceptor tyrosine kinase.
      • Notarangelo LD
      • Giliani S
      • Mazza C
      • et al.
      Of genes and phenotypes: the immunological and molecular spectrum of combined immune deficiency. Defects of the gamma(c)-JAK3 signaling pathway as a model.
      Hence, IL2Rγ- and Jak3-deficient mice exhibit common phenotypes including NK deficiency and reduced T and B cells.
      • Suzuki K
      • Nakajima H
      • Saito Y
      • et al.
      Janus kinase 3 (Jak3) is essential for common cytokine receptor gamma chain (gamma(c))-dependent signaling: comparative analysis of gamma(c), Jak3, and gamma(c) and Jak3 double-deficient mice.
      To completely overcome NK cell function, IL2Rγ/Jak3-deficient mice were used to establish the NOG/NSG/NOJ mice (NOG, NOD/SCID/IL2Rγtm1Sug; NSG, NOD/SCID/IL2Rγtm1Wjl; NOJ, NOD/SCID/Jak3null) by crossbreeding with NOD/SCID. The NOG and NSG mice show a similar genetic background with partial or complete deletion of IL2Rγ, which makes them better recipients for human CRC.
      • Samdal H
      • Olsen LC
      • Grøn KS
      • et al.
      Establishment of a patient-derived xenograft model of colorectal cancer in CIEA NOG mice and exploring smartfish liquid diet as a source of omega-3 fatty acids.
      ,
      • Zhang Y
      • Lee SH
      • Wang C
      • et al.
      Establishing metastatic patient-derived xenograft model for colorectal cancer.
      In addition to SCID, some scholars have also used nude mice to generate new types of combined immunocompromised mice. For example, Rag-2null and Jak3null mice with a BALB/c background were crossed with nude mice to establish BALB/c Nude Rag-2/Jak3 double-deficient (Nude R/J) mice.
      • Kariya R
      • Matsuda K
      • Gotoh K
      • et al.
      Establishment of nude mice with complete loss of lymphocytes and NK cells and application for in vivo bio-imaging.
      ,
      • Tanaka A
      • Takeda S
      • Kariya R
      • et al.
      A novel therapeutic molecule against HTLV-1 infection targeting provirus.
      Nude R/J mice preserved the characteristic of no fur and had a higher immune-deficient level (no NK, T and B cells) than nude mice.
      Taken together, the novel combined immunocompromised mice provide a better model for overcoming immune rejection by selectively and completely eliminating immune cells in the mice (Table 2).
      Table 2The characteristics of transplant mouse models for studies of colorectal cancer (CRC)
      AnimalGenetic alterationsAdvantagesDisadvantages
      Athymic nude mice
      • Flanagan SP.
      Nude', a new hairless gene with pleiotropic effects in the mouse.
      Spontaneous mutation of Foxn1 causing a lack of thymic tissue1. No T cells; 2. High take rate of human tumors; 3. No coat of hair; 4. easy to detect subcutaneous tumor1. No deficiency in B cells or myeloid cells; 2. T-cell functionality increases with age; 3. Not suitable for primary cell transplantation
      SCID mice
      • Bosma GC
      • Custer RP
      • Bosma MJ.
      A severe combined immunodeficiency mutation in the mouse.
      Defect in DNA protein kinase, no functional rearrangement of antigen-specific receptors1. No mature T and B cells

      2. Better engraftment compared with nude mice
      1. Short lifespan (<12 months); 2. Functional NK cell; 3. Leakage of T cells; 4. Thymic lymphoma development; 5. Radiosensitive
      NOD-SCID mice
      • Shultz LD
      • Schweitzer PA
      • Christianson SW
      • et al.
      Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice.
      Expression of the SCID mutation on the NOD strain background1. No mature T and B cells; 2. Impaired NK function; 3. Impaired macrophage and dendritic cell; 4. Very low leakiness with age; 5. Better engraftment1. High incidence of lymphomas; 2. Radiosensitive; 3. Short life span (av. 36 wks)
      NOG/NSG/NOJ mice
      • Ito M
      • Hiramatsu H
      • Kobayashi K
      • et al.
      NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse model for engraftment of human cells.
      • Shultz LD
      • Lyons BL
      • Burzenski LM
      • et al.
      Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells.
      • Okada S
      • Harada H
      • Ito T
      • et al.
      Early development of human hematopoietic and acquired immune systems in new born NOD/Scid/Jak3null mice intrahepatic engrafted with cord blood-derived CD34 + cells.
      1. NOG: NOD.Cg-PrkdcscidIl2rg tm1 Sug/Jic SCID, IL-2γ Partial deficiency

      2. NSG: NOD.Cg-PrkdcscidIl2rg tm1 Wjl/SzJ SCID, IL-2Rγ Complete deficiency

      3. NOJ: NOD.Cg-Prkdcscid Jak3tm1card SCID, Jak3 deficiency
      1.No mature T and B cells; 2. No NK cells; 3. impaired macrophage and dendritic cell; 4. High engraftment1. Need strict SPF conditions; 2. Difficult and expensive breeding
      Nude R/J mice
      • Kariya R
      • Matsuda K
      • Gotoh K
      • et al.
      Establishment of nude mice with complete loss of lymphocytes and NK cells and application for in vivo bio-imaging.
      • Tanaka A
      • Takeda S
      • Kariya R
      • et al.
      A novel therapeutic molecule against HTLV-1 infection targeting provirus.
      Rag-2/Jak3 double-deficiency1.No mature T and B cells; 2. No NK cells; 3. High engraftment; 4. Resistant to stress; 5. Easy breeding; 6. radio resistant-
      Humanized mice
      • Olson B
      • Li Y
      • Lin Y
      • et al.
      Mouse models for cancer immunotherapy research.
      • Brown ZJ
      • Heinrich B
      • Greten TF.
      Mouse models of hepatocellular carcinoma: an overview and highlights for immunotherapy research.
      • Pearson T
      • Greiner DL
      • Shultz LD.
      Creation of "humanized" mice to study human immunity.
      • Walsh NC
      • Kenney LL
      • Jangalwe S
      • et al.
      Humanized mouse models of clinical disease.
      Immunodeficient mouse engrafted with human immune systems1. Immediately available for experiment; 2. The complex human immune system and human CRC can be replicated1. Time-consuming; 2. Difficult to set up; 3. High in cost
      IL2Rγ, IL-2 receptor γ-deficient; NK cell, natural killer cell; NOD, nonobese diabetic; NOG, NOD/SCID/IL2Rγnul mice with complete loss of NK cells; NOJ mice, Jak3-deficient NOD/SCID/Jak3null mice; Nude R/J mice, BALB/c Nude Rag-2/Jak3 double-deficient mice; NSG mice, NOD/SCID/IL2Rγnul mice with complete loss of NK cells; Prkdc, protein kinase, DNA activated, catalytic polypeptide; SCID, severe combined immunodeficiency mice; SPF, specified pathogen free.

      Humanized mice: from destroying the mouse immune system to rebuilding the human immune system in mice

      Immune cells that mediate tumor-associated inflammation and immune destruction have been demonstrated to play a role in the formation and progression of human tumors. In 2022, Douglas Hanahan published a sensational review that incorporated tumor-promoting inflammation and avoidance of immune destruction into the hallmarks of cancer, which were segregated as “core hallmarks”.
      • Hanahan D.
      Hallmarks of Cancer: New Dimensions.
      Consequently, interest in tumor-immune system interactions and immunotherapy has grown over the past few years. However, there are significant differences in the immune system between humans and mice,
      • Zschaler J
      • Schlorke D
      • Arnhold J.
      Differences in innate immune response between man and mouse.
      which lead to the inability of mice to mimic the immune properties of actual cancer patients. The development of humanized mice addressed this major limitation.
      Humanized mice are referred to as immunodeficient mice engrafted with a functioning human immune system.
      • Yong KSM
      • Her Z
      • Chen Q.
      Humanized mice as unique tools for human-specific studies.
      The immunodeficient mice can be considered humanized mice after engrafting human immune systems into them. Currently, there are 3 methods used to establish humanized mice for tumor growth and tumor immunology studies according to different engraftment combinations: (1) combined engraftment of immune cells or tissues with tumor cells; (2) single patient-derived xenograft (PDX) engraftment; and (3) combined engraftment of immune cells or tissues with PDX.
      • Yong KSM
      • Her Z
      • Chen Q.
      Humanized mice as unique tools for human-specific studies.
      An ideal humanized mice model not only replicates the complex human immune system and tumor but also can be immediately available for an experiment (Table 2). Thus, much literature reported the application of humanized mice in the research of human CRC immunotherapy (relevant research has been summarized and presented in Table 3). The humanization model also has some limitations, such as high cost, low success rates, and long experiment period, which impede its spread and development. There is little doubt, however, that the discovery of "humanized" mice provides an optimal human CRC xenograft host for uncovering tumor-immune interactions, monitoring immunotherapy, and evaluating efficacy.
      Table 3Humanized mice and CRC immunotherapy
      Author (year)Mouse strainHumanized methodsXenograftRoutes of tumor transplantationMain results
      Wulf-Goldenberg et al. (2011)
      • Wulf-Goldenberg A
      • Eckert K
      • Fichtner I.
      Intrahepatically transplanted human cord blood cells reduce SW480 tumor growth in the presence of bispecific EpCAM/CD3 antibody.
      NOD/SCID miceHuman cord blood cells engraftmentSW480 cell lineSubcutaneousThe intrahepatic transplantation of cord blood stem cells into newborn mice represents a valuable model for establishing functionally active human T cells with antitumor activity.
      Sanmamed et al. (2015)
      • Sanmamed MF
      • Rodriguez I
      • Schalper KA
      • et al.
      Nivolumab and urelumab enhance antitumor activity of human T lymphocytes engrafted in Rag2-/-IL2Rγnull immunodeficient mice.
      Rag2−/−IL2Rgnull miceHuman lymphocytes engraftmentHT-29 cell lineSubcutaneousCoadministration of urelumab and nivolumab was sufficient to slow tumor growth significantly.
      Margolin et al. (2015)
      • Margolin DA
      • Myers T
      • Zhang X
      • et al.
      The critical roles of tumor-initiating cells and the lymph node stromal microenvironment in human colorectal cancer extranodal metastasis using a unique humanized orthotopic mouse model.
      NOD/SCID miceMesenteric LN stromal cells, stromal cell line HK, or CXCL12 knockdown HK cells engraftmentHT-29 cell lineOrthotopicBoth Co-TICs and LN stromal factors play crucial roles in CRC metastasis through the CXCL12/CXCR4 axis.
      Wu et al. (2019)
      • Wu Z
      • Guo HF
      • Xu H
      • et al.
      Development of a tetravalent anti-GPA33/anti-CD3 bispecific antibody for colorectal cancers.
      DKO miceUsing CDR grafting, mouse A33 was humanized as IgG1LS174T, Colo205 cell linesSubcutaneous and intraperitonealIn vivo, huA33-BsAb inhibited the colon and gastric cancer xenografts in both subcutaneous and intraperitoneal tumor models. More importantly, both microsatellite instable and stable CRC were effectively eliminated by huA33-BsAb.
      Hollandsworth et al. (2020)
      • Hollandsworth HM
      • Amirfakhri S
      • Filemoni F
      • et al.
      Near-infrared photoimmunotherapy is effective treatment for colorectal cancer in orthotopic nude-mouse models.
      Athymic nude miceLS174T tumor fragments engraftmentLS174T cellsOrthotopicPhotoimmunotherapy arrests tumor growth in colon cancer orthotopic nude-mouse models. Repeated photoimmunotherapy arrests colon cancer growth for a longer period.
      Suto et al. (2021)
      • Suto H
      • Funakoshi Y
      • Nagatani Y
      • et al.
      Microsatellite instability-high colorectal cancer patient-derived xenograft models for cancer immunity research.
      NSG micePeripheral blood mononuclear cell engraftmentPDXsSubcutaneousThe MSI-H tumor with an MMR mutation is suitable for MSI-H PDX model generation. The peripheral blood mononuclear cells humanized MSI-H PDX has the potential to be used as an efficient model for cancer immunotherapy research.
      Lee et al. (2021)
      • Lee EJ
      • Kim JH
      • Kim TI
      • et al.
      Sanguisorbae radix suppresses colorectal tumor growth through PD-1/PD-L1 blockade and synergistic effect with pembrolizumab in a humanized PD-L1-expressing colorectal cancer mouse model.
      C57BL/6J humanized PD-1 knock-in miceGenetic modificationHuman PD-L1 MC38 cellsSubcutaneousSanguisorbae Radix extract alone has anticancer effects via PD-1/PD-L1 blockade, and the combination therapy of Sanguisorbae Radix extract and pembrolizumab has enhanced immuno-oncologic effects.
      Co-TICs, colorectal cancer tumor-initiating cells; CRC, colorectal cancer; LN, lymph node; MSI-H, microsatellite instability-high; MMR, mismatch repair; PD, programmed death-1; PDL-1, programmed death-ligand 1; PDX, patients-derived xenograft.

      TUMOR MATERIALS FOR HUMAN CRC XENOGRAFT

      Distant metastasis is the main cause of high mortality in CRC patients.
      • Liu W
      • Wang HW
      • Wang K
      • et al.
      The primary tumor location impacts survival outcome of colorectal liver metastases after hepatic resection: a systematic review and meta-analysis.
      An applicable preclinical model to mimic this behavior plays a crucial role in oncology research. Existing models cannot replicate human CRC as an entity, but available transplant mouse models approximate many features of colonic invasion and metastasis. The metastatic capacity of transplanted tumors in immunodeficient mice depends on the experimental technique, the routes of transplantation, the origin of tumor materials, and the source and conditions of the mice used. Among these, the origin of tumor materials is one of the most critical factors affecting the biological phenotype of xenografts in immunodeficient mice. There are 3 sources of xenografts for colorectal tumors, including cell line-derived xenografts (CDX), PDX, and PDO. Fig 2 summarizes the characteristics of these xenografts.
      Fig 2
      Fig 2Schematic diagram for the summary of the characteristics of implantation routes and xenografts. (Color version of figure is available online.)

      CDX

      The culture of colorectal tumors provides useful experimental material for various studies. Human CRC cells propagated in immunodeficient mice have been shown to resemble tumor origins. Other important features, including morphological characteristics, the extent of tumor necrosis, isoenzyme mobility patterns, and the capacity to produce cellular products such as mucin, were also well-preserved in xenografts. The high degree of identity between CDXs and human tumors makes it a useful tool in studying human CRC.
      • Fidler IJ.
      Orthotopic implantation of human colon carcinomas into nude mice provides a valuable model for the biology and therapy of metastasis.
      Aside from the transplantation route, the invasiveness and metastasis of CDXs in immunodeficient mice are mainly related to their primary tumors. Giavazzi et al.
      • Giavazzi R
      • Campbell DE
      • Jessup JM
      • et al.
      Metastatic behavior of tumor cells isolated from primary and metastatic human colorectal carcinomas implanted into different sites in nude mice.
      investigated the growth characteristics and metastatic behavior of several tumor lines derived from primary CRC and hepatic metastases. They found that models infused with metastatic cells grew and metastasized faster than primary tumor cells. Subsequent studies have yielded similar results.
      • Flatmark K
      • Maelandsmo GM
      • Martinsen M
      • et al.
      Twelve colorectal cancer cell lines exhibit highly variable growth and metastatic capacities in an orthotopic model in nude mice.
      ,
      • Céspedes MV
      • Espina C
      • García-Cabezas MA
      • et al.
      Orthotopic microinjection of human colon cancer cells in nude mice induces tumor foci in all clinically relevant metastatic sites.
      These findings confirm that: (1) tumor lines of different origins differ in biological phenotypes after transplantation into nude mice and (2) the metastatic potential of human malignant tumors in nude mice increases with the degree of malignancy of the original tumors. Another interesting finding is that it is indeed possible to select highly metastatic cells from low metastatic CRC by in vivo passages.
      • Morikawa K
      • Walker SM
      • Jessup JM
      • et al.
      In vivo selection of highly metastatic cells from surgical specimens of different primary human colon carcinomas implanted into nude mice.
      Additional advantages of the CDX model are easy operation, high take rate, and short tumor-forming time (2–8 weeks).
      • Lee SH
      • Hong JH
      • Park HK
      • et al.
      Colorectal cancer-derived tumor spheroids retain the characteristics of original tumors.
      However, some reports have found that orthotopic injection of cell suspensions may not express the full metastatic potential and therapeutic response of the primary tumor. This is due to the deterioration of 3-dimensional tissue architecture and the loss of tumor heterogeneity.
      • Vogelstein B
      • Papadopoulos N
      • Velculescu VE
      • et al.
      Cancer genome landscapes.
      Therefore, CDXs have limited predictive value for human CRC research.

      PDX

      Nowadays, there is growing interest in PDXs as a more advanced preclinical cancer model. PDXs are usually implanted subcutaneously or orthotopically into immunodeficient mice and more accurately reflect human CRC than CDXs. Indeed, PDXs not only preserve the 3-dimensional tissue structures and cell-to-cell interactions within tumor tissues, but even in chromosomally unstable early-stage human CRC tumors, they also retain chromosomal instability, intratumor heterogeneity, and histology of the parent tumor for over 14 passages.
      • Guenot D
      • Guérin E
      • Aguillon-Romain S
      • et al.
      Primary tumour genetic alterations and intra-tumoral heterogeneity are maintained in xenografts of human colon cancers showing chromosome instability.
      Although human microvascular and stromal compounds are eventually replaced by their murine counterparts, tumor tissue remains intact in most cases.
      • Hylander BL
      • Punt N
      • Tang H
      • et al.
      Origin of the vasculature supporting growth of primary patient tumor xenografts.
      Furthermore, these mouse mesenchymal cells have also been shown to have a metabolic phenotype similar to that of humans.
      • Blomme A
      • Van Simaeys G
      • Doumont G
      • et al.
      Murine stroma adopts a human-like metabolic phenotype in the PDX model of colorectal cancer and liver metastases.
      These characteristics suggest that the PDX model is a good and valuable platform that can be used to study human tumors.
      CRC tumors have a higher tumor collection rate (> 60%) in the PDX model compared with other solid tumors, including breast cancer (10%–37%) and prostate cancer (<5%).
      • Julien S
      • Merino-Trigo A
      • Lacroix L
      • et al.
      Characterization of a large panel of patient-derived tumor xenografts representing the clinical heterogeneity of human colorectal cancer.
      However, the success rate of PDX varies in different pieces of literature, and is influenced by several factors including (1) characteristics of the tumor, such as sample type, tumor subtype, and tumor stage;
      • Katsiampoura A
      • Raghav K
      • Jiang ZQ
      • et al.
      Modeling of patient-derived xenografts in colorectal cancer.
      ,
      • Prasetyanti PR
      • van Hooff SR
      • van Herwaarden T
      • et al.
      Capturing colorectal cancer inter-tumor heterogeneity in patient-derived xenograft (PDX) models.
      ; (2) PDX preservation
      • Linnebacher M
      • Maletzki C
      • Ostwald C
      • et al.
      Cryopreservation of human colorectal carcinomas prior to xenografting.
      and incubation mode;
      • Gock M
      • Kühn F
      • Mullins CS
      • et al.
      Tumor take rate optimization for colorectal carcinoma patient-derived xenograft models.
      (3) number and size of the implanted tumor tissue; (4) implant location; and (5) recipient strain.
      • Day CP
      • Merlino G
      • Van Dyke T.
      Preclinical mouse cancer models: a maze of opportunities and challenges.
      In fact, the PDX model of human CRC was originally developed to mimic various clinical behaviors in human subjects, including local invasion and distant metastasis.
      • Fu XY
      • Besterman JM
      • Monosov A
      • et al.
      Models of human metastatic colon cancer in nude mice orthotopically constructed by using histologically intact patient specimens.
      To improve the drawbacks of the traditional PDX model, Sun et al. established a human CRC nude mouse hypermetastatic model, namely AC3488UM, and the transplanted animals had 100% spontaneous liver metastasis 10 days after transplantation.
      • Sun FX
      • Sasson AR
      • Jiang P
      • et al.
      An ultra-metastatic model of human colon cancer in nude mice.
      Nevertheless, studies in recent years have focused more on anticancer drug response, biomarker development, and improved engraftment rates in CRC PDX models.
      • Day CP
      • Merlino G
      • Van Dyke T.
      Preclinical mouse cancer models: a maze of opportunities and challenges.
      ,
      • Jung J
      • Seol HS
      • Chang S.
      The generation and application of patient-derived xenograft model for cancer research.
      Only a few pieces of literatures report in detail the clinical behavioral outcomes of PDX in mice.
      • Cho YB
      • Hong HK
      • Choi YL
      • et al.
      Colorectal cancer patient-derived xenografted tumors maintain characteristic features of the original tumors.
      PDX also has some drawbacks, including long establishment time (around 2–4 months), high cost, and the possibility that a low percentage of mutated variants occur through the passages. Nonetheless, PDXs appear to be more suitable for studying cancer biology and therapeutic development than CDXs due to the former's high fidelity to the original tumor at the genomic and transcriptomic levels.

      PDOs

      Although the development of the PDX model is insightful, it is a relatively expensive and time-consuming task. The emergence of PDOs provides a possible solution to these problems. PDOs are clusters of 3-dimensional cultured multicellular aggregates grown from patient stem cells or isolated organ progenitor cells. It has been demonstrated that PDOs not only retain the characteristics of the parent matrix but also preserve their tissue functions.
      • van de Wetering M
      • Francies HE
      • Francis JM
      • et al.
      Prospective derivation of a living organoid biobank of colorectal cancer patients.
      Moreover, PDOs can be easily maintained and genetically manipulated in vitro and dutifully exhibit features of in vivo tissues during homeostasis and disease such as CRC.
      • Sato T
      • Stange DE
      • Ferrante M
      • et al.
      Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.
      Another major advantage of PDOs is the high success rate of generation from primary CRC tissue (∼ 90% for primary cancer).
      • van de Wetering M
      • Francies HE
      • Francis JM
      • et al.
      Prospective derivation of a living organoid biobank of colorectal cancer patients.
      The high tumor formation rates upon the transplantation of PODs into the cecal (100%) or colon wall (60%) make it a good platform for establishing more efficient CRC mice models.
      • Fumagalli A
      • Suijkerbuijk SJE
      • Begthel H
      • et al.
      A surgical orthotopic organoid transplantation approach in mice to visualize and study colorectal cancer progression.
      It has been reported that PDOs with multiple cancer-related mutations were transplanted into mouse renal capsule and spleen to establish CRC and liver metastasis models.
      • Matano M
      • Date S
      • Shimokawa M
      • et al.
      Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids.
      In addition, transplantation of PDOs into mouse flanks, spleen, and kidney sub-capsules has also been reported to establish a liver seeding model.
      • Fujii M
      • Shimokawa M
      • Date S
      • et al.
      A colorectal tumor organoid library demonstrates progressive loss of niche factor requirements during tumorigenesis.
      However, these ectopic transplantation models do not allow the study of tumor invasion into the muscularis propria or extravasation into the circulation through the colon serosa. Therefore, some research groups orthotopically transplanted PDOs into colonic or rectal mucosa to study primary cancers and liver metastases.
      • Fumagalli A
      • Drost J
      • Suijkerbuijk SJ
      • et al.
      Genetic dissection of colorectal cancer progression by orthotopic transplantation of engineered cancer organoids.
      PDOs can also be engineered for desired mutations using gene-editing techniques,
      • Matano M
      • Date S
      • Shimokawa M
      • et al.
      Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids.
      which is much faster than generating germline genetically engineered mouse models. Fluorescent labels and other desired features can also be easily inserted into PDOs.
      There are some inadequacies in PDOs, such as long culture cycle, single source of cell, and unstable culture conditions. Yet, as the latest source of xenografts with the terrific fidelity and maneuverability in the CRC model, we still consider PDOs as another breakthrough in cancer research after the PDX model, holding great promise in the study of the biological behaviors and drug response of CRC.

      ROUTES OF HUMAN CRC XENOGRAFT

      In addition to the origin of tumor materials, another crucial factor determining the malignant behavior of transplanted tumor is the routes of transplantation. Human CRC transplant mouse models can be divided into 2 types: heterotopic transplantation and orthotopic transplantation. The former mainly consists of subcutaneous transplantation and intrasplenic transplantation. On the other hand, the latter was involved in intramural transplantation of the mouse colon or rectum. The characteristics of different routes are presented in Fig 2.

      Subcutaneous transplantation

      In 1969, Rygaard et al.
      • Rygaard J
      • Povlsen CO.
      Heterotransplantation of a human malignant tumour to "Nude" mice.
      reported the first case of subcutaneous transplantation of human CRC tissue, indicating that nude mice were more tumorigenic than immune. Subsequently, Kyriazis et al,
      • Kyriazis AP
      • DiPersio L
      • Michael GJ
      • et al.
      Growth patterns and metastatic behavior of human tumors growing in athymic mice.
      presented data involving the growth pattern and metastatic capacity of human colon cancer cells grown subcutaneously in nude mice. While tumor growth was seen in all subcutaneous models, they showed that none developed metastases. This is mainly due to poor angiogenesis in subcutaneously transplanted tumors caused by the fibrous capsule. Local vascular supply is a key factor affecting the biological behavior of xenografts. However, the fibrous capsule can interfere with the vascularization of the graft, resulting in tumor cell necrosis, which in turn, affects tumor growth and prevents distant metastasis.
      The reason why subcutaneous transplantation is widely used can be attributed to its unique advantages, namely, nominal surgical skills, short time, and easy observation. Due to these characteristics, subcutaneous tumor models are usually used to evaluate therapeutic effects in human cancers. Some of these models have been reported to correctly predict clinical responses in specific cancers.
      • Houghton PJ
      • Morton CL
      • Tucker C
      • et al.
      The pediatric preclinical testing program: description of models and early testing results.
      Nevertheless, this model suffers from a major disadvantage: the subcutaneous microenvironment is greatly different from that of the colon. Interactions between the tumor microenvironment and tumor grafts have been shown to determine the molecular properties and biological behavior of tumors. Subcutaneous tumor models cannot replicate the characteristics of primary CRC due to differences in the microenvironment. Extensive drug screening has shown that subcutaneous transplant models have rather limited value in predicting clinical response in humans.
      • Talmadge JE
      • Singh RK
      • Fidler IJ
      • et al.
      Murine models to evaluate novel and conventional therapeutic strategies for cancer.

      Intrasplenic transplantation

      The most common site of CRC metastasis is the liver. Hence, there is an urgent need to find a suitable CRC liver metastasis model for research. The intrasplenic transplantation mouse model is an ideal platform for studying hepatic metastasis of human tumors. The injection of human tumor cell lines into the spleen of nude mice allows for the most dramatic overall expression of metastatic potential in these cell lines compared to other transplantation routes.
      • Xu Y
      • Zhang L
      • Wang Q
      • et al.
      Comparison of different colorectal cancer with liver metastases models using six colorectal cancer cell lines.
      After intrasplenic injection, tumor cells directly enter the bloodstream and then reach the liver, where they proliferate towards secondary lesions, which prevents them from undergoing the initial stage of metastasis. These characteristics make the intrasplenic transplantation model irreplaceable in the study of human tumor liver metastasis.
      • Yang YS
      • Wen D
      • Zhao XF.
      Sophocarpine can enhance the inhibiting effect of oxaliplatin on colon cancer liver metastasis-in vitro and in vivo.
      However, the traditional intrasplenic transplantation model, also known as the whole spleen model, still has some disadvantages, such as tumor growth in the spleen and the inability to repeatedly administer drugs. To overcome these drawbacks, researchers established a mouse subcutaneous hemi-spleen depot model to treat liver malignancies through repeated portal vein injections.
      • Kasuya H
      • Kuruppu DK
      • Donahue JM
      • et al.
      Mouse models of subcutaneous spleen reservoir for multiple portal venous injections to treat liver malignancies.
      Unlike humans, mice have 2 splenic pedicles, which are located at opposite ends of the spleen. Therefore, this model divides the spleen into 2 hemi-spleens with vascular pedicles: one inoculated with tumor cells and removed after 10 minutes, and the other one transposed subcutaneously for multiple intrasplenic administrations. The hemi-spleen model solves the aforementioned problems well and is widely used in the study of the treatment of liver tumors.
      • Yang YS
      • Wen D
      • Zhao XF.
      Preventive and therapeutic effect of intraportal oridonin on BALb/c nude mice hemispleen model of colon cancer liver metastasis.
      Circumventing the initial steps of metastasis helps to improve the efficiency of liver metastasis but cannot truly reflect the metastatic process from the primary tumor to the metastatic tumor. This limits the clinical value of the intrasplenic transplantation model.

      Orthotopic transplantation

      Tumor dissemination is considered to begin when the tumor graft invades the surrounding host tissues. This initial step is very important in the metastatic cascade, as tumor cells need to undergo a series of functional and morphological changes from a nonmetastatic to a metastatic state. However, ectopic models usually bypass this initial process and lack an appropriate tumor microenvironment. Therefore, orthotopic CRC models were established to overcome the drawbacks of heterotopic models.
      The establishment of an orthotopic colon cancer model in mice after intramural cell injection was first described by Tan et al. in 1977. In their report, Tan et al. not only demonstrated the feasibility of intramural cell injection but also showed that the take rate (90%) of the cecum was higher than that of other intestinal areas.
      • Tan MH
      • Holyoke ED
      • Goldrosen MH.
      Murine colon adenocarcinoma: syngeneic orthotopic transplantation and subsequent hepatic metastases.
      Subsequently, intercecal injection of human CRC cells in immunodeficient mice was also successful and resulted in liver metastases.
      • Morikawa K
      • Walker SM
      • Jessup JM
      • et al.
      In vivo selection of highly metastatic cells from surgical specimens of different primary human colon carcinomas implanted into nude mice.
      Morikata et al. even proposed that human colon cancer cell lines must be directly injected into the cecum wall of nude mice for sustained metastasis.
      • Morikawa K
      • Walker SM
      • Jessup JM
      • et al.
      In vivo selection of highly metastatic cells from surgical specimens of different primary human colon carcinomas implanted into nude mice.
      Since then, to better reflect the original nature of human cancer and avoid spilling over of cells, many improvement strategies have been adopted. These strategies include the addition of Matrigel to the cell suspension to avoid cell extravasation
      • Klose J
      • Eissele J
      • Volz C
      • et al.
      Salinomycin inhibits metastatic colorectal cancer growth and interferes with Wnt/β-catenin signaling in CD133(+) human colorectal cancer cells.
      ; suturing of histologically intact tumor tissue to the cecum wall
      • Chunhua L
      • Donglan L
      • Xiuqiong F
      • et al.
      Apigenin up-regulates transgelin and inhibits invasion and migration of colorectal cancer through decreased phosphorylation of AKT.
      ; formation and suturing of subserosa "pockets"
      • Lin W
      • Zhuang Q
      • Zheng L
      • et al.
      Pien Tze Huang inhibits liver metastasis by targeting TGF-β signaling in an orthotopic model of colorectal cancer.
      between the mucosa and the extracecal muscularis for tumor seeding, or in situ cell microinjection.
      • Céspedes MV
      • Espina C
      • García-Cabezas MA
      • et al.
      Orthotopic microinjection of human colon cancer cells in nude mice induces tumor foci in all clinically relevant metastatic sites.
      Despite the clear advantages of intercecal transplantation, there remain some aspects that require careful consideration, including the requirement for laparotomy and limited relevance with rectal cancer. Hence, a novel animal model of rectal cancer was developed.
      • Donigan M
      • Norcross LS
      • Aversa J
      • et al.
      Novel murine model for colon cancer: non-operative trans-anal rectal injection.
      Rectal anatomy and lymph node drainage patterns in mice are similar to those in humans.
      • Cook MJ.
      The Anatomy of the Laboratory Mouse.
      The growth pattern of tumor cells in the rectal wall of mice is similar to that of clinical CRC patients,
      • Donigan M
      • Norcross LS
      • Aversa J
      • et al.
      Novel murine model for colon cancer: non-operative trans-anal rectal injection.
      suggesting the clinical relevance of this model. Furthermore, Hite et al.
      • Hite N
      • Klinger A
      • Hellmers L
      • et al.
      An optimal orthotopic mouse model for human colorectal cancer primary tumor growth and spontaneous metastasis.
      compared 3 orthotopic models, namely, intercecal injection, transanal submucosal injection, and acid enema. They found that intrarectal injection was the safest, most reproducible, and most successful orthotopic model for primary tumor growth and spontaneous metastasis in human CRC. Therefore, mouse models of rectal cancer have received increasing attention and interest. In practical applications, some modifications are made to meet the needs of specific research. Enquist et al.
      • Enquist IB
      • Good Z
      • Jubb AM
      • et al.
      Lymph node-independent liver metastasis in a model of metastatic colorectal cancer.
      used the prolapse technique to suture human CRC cell lines to the rectal mucosa to reveal the origin of liver metastases. In a recent mouse model of rectal cancer, clinical-grade titanium fiducial clips were placed on opposite sides of a rectal tumor to enable targeted delivery of short-term radiation therapy.
      • Uccello TP
      • Kintzel SA
      • Mills BN
      • et al.
      Development of an orthotopic murine model of rectal cancer in conjunction with targeted short-course radiation therapy.
      Interestingly, to avoid laparotomy and reduce the damage caused by surgery, some nonsurgical orthotopic CRC mouse models have also been established using mouse colonoscopy or other instruments.
      • Bettenworth D
      • Mücke MM
      • Schwegmann K
      • et al.
      Endoscopy-guided orthotopic implantation of colorectal cancer cells results in metastatic colorectal cancer in mice.
      For example, through a p200 pipette enema or colonoscopy, O'Rourke et al.
      • O'Rourke KP
      • Loizou E
      • Livshits G
      • et al.
      Transplantation of engineered organoids enables rapid generation of metastatic mouse models of colorectal cancer.
      and Roper et al.
      • Roper J
      • Tammela T
      • Cetinbas NM
      • et al.
      In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis.
      established distinct experimental systems for the orthotopic transplantation of CRC organoids into mice to model the adenoma-carcinoma-metastasis sequence. These approaches make it possible to rapidly characterize the cancer-associated genes and reproduce the entire spectrum of tumor progression and metastasis.
      Although the optimal site (cecum or rectum) for orthotopic transplantation remains controversial, orthotopic transplantation model mimics the environment in which human tumors grow in mice. Thus, we regard it as another milestone in cancer research with great potential in the development of precision medicine in CRC.

      APPLICATION OF TRANSPLANT MOUSE MODEL IN CRC

      As a bridge to clinical application, transplant mouse models have been widely used in various aspects of CRC therapy. Different transplant mouse models have their unique characteristics, and likewise, each model has its drawbacks. For example, CDX models do not adequately reflect patient drug responses, resulting in very low rates of clinical approval of cancer drugs (approximately <15%).
      • DiMasi JA
      • Reichert JM
      • Feldman L
      • et al.
      Clinical approval success rates for investigational cancer drugs.
      Subcutaneous transplantation models have limited predictive value for human clinical response through large drug screening.
      • Talmadge JE
      • Singh RK
      • Fidler IJ
      • et al.
      Murine models to evaluate novel and conventional therapeutic strategies for cancer.
      In contrast, the orthotopic PDX models maintain the highest concordance of drug responses between patients and mouse models, supporting their use as an optimal screening platform for anticancer drug evaluation.
      • Bertotti A
      • Migliardi G
      • Galimi F
      • et al.
      A molecularly annotated platform of patient-derived xenografts ("xenopatients") identifies HER2 as an effective therapeutic target in cetuximab-resistant colorectal cancer.
      Currently, the applications of transplant mouse models mainly involve the development of biomarkers, drug testing, and surgical modeling.

      Development of biomarkers

      Transplant mouse models can reveal novel biomarkers, thus providing reliable evidence for individualized treatment of CRC patients. Wang et al. showed that tumor-transplanted mice developed severe colonic tissue damage after upregulation of ring finger protein 2 (RNF2), suggesting that RNF2 may be a potential therapeutic target for CRC.
      • Wang J
      • Ouyang X
      • Zhou Z
      • et al.
      RNF2 promotes the progression of colon cancer by regulating ubiquitination and degradation of IRF4.
      Furthermore, the correlation between drug efficacy and molecular properties can be easily studied using transplant mouse models. In a recent study, amplification of the human epidermal growth factor receptor 2 (HER2) gene was shown to promote cetuximab resistance in a KRAS/NRAS/BRAF/PI3KCA wild-type PDX model with metastatic CRC and was found to predict responses to antiepidermal growth factor receptor (EGFR) and anti-HER2 antibodies. Subsequently, these findings were further translated into successful clinical studies.
      • Bertotti A
      • Migliardi G
      • Galimi F
      • et al.
      A molecularly annotated platform of patient-derived xenografts ("xenopatients") identifies HER2 as an effective therapeutic target in cetuximab-resistant colorectal cancer.
      Other molecular targets have also been shown to be biomarkers of cetuximab resistance, such as the MET proto-oncogene and fibroblast growth factor receptor 1 amplification, through candidate gene or comprehensive genomic analysis.
      • Bertotti A
      • Papp E
      • Jones S
      • et al.
      The genomic landscape of response to EGFR blockade in colorectal cancer.
      The application of transplant mouse models has advanced the understanding of adaptive escape mechanisms that maintain residual lesions during maximal drug response. Lupo et al. recently found that in the PDX model, EGFR-inhibited surviving metastatic CRC cells exhibited reduced EGFR ligand expression, enhanced HER2/HER3 signaling pathway activity, and sustained activation of the phosphatidylinositol 3-kinase (PI3K) pathway. They further demonstrated that Pan-HER antibodies minimized residual disease, blunted PI3K signaling, and induced long-term tumor control after treatment discontinuation in a preclinical trial.
      • Lupo B
      • Sassi F
      • Pinnelli M
      • et al.
      Colorectal cancer residual disease at maximal response to EGFR blockade displays a druggable Paneth cell-like phenotype.
      Another interesting finding was that chemotherapy preferentially eradicated actively proliferating cells and promoted the dominance of previously minor or dormant lineages in the CRC PDX model.
      • Kreso A
      • O'Brien CA
      • van Galen P
      • et al.
      Variable clonal repopulation dynamics influence chemotherapy response in colorectal cancer.
      Undoubtedly, these findings provide opportunities to pre-emptively target residual disease.
      In summary, the discovery of novel tumor biomarkers with the help of transplant mouse models is essential in promoting translational research in clinical and basic sciences. Moreover, it can facilitate individualized treatment based on tumor molecular classification.

      Drug testing

      Transplant mouse models can be used in investigating the efficacy of controversial or novel antitumor agents against CRC. Using the CDX model, Liu et al. demonstrated that a novel andrographolide derivative AGS-30 could induce apoptosis in CRC cells by activating the ros-dependent JNK signaling pathway.
      • Liu Z
      • Wu X
      • Dai K
      • et al.
      The new andrographolide derivative AGS-30 induces apoptosis in human colon cancer cells by activating a ROS-dependent JNK signalling pathway.
      Through the CDX model, our group found that the traditional Chinese medicine sophocarpine can enhance the inhibitory effect of oxaliplatin on metastatic CRC.
      • Yang YS
      • Wen D
      • Zhao XF.
      Sophocarpine can enhance the inhibiting effect of oxaliplatin on colon cancer liver metastasis-in vitro and in vivo.
      Additionally, in the KRAS-mutated metastatic CRC xenografted model, metformin was reported to inhibit tumor growth and cell viability.
      • Xie J
      • Xia L
      • Xiang W
      • et al.
      Metformin selectively inhibits metastatic colorectal cancer with the KRAS mutation by intracellular accumulation through silencing MATE1.
      Another application of transplant mice models is the coclinical trial, which is defined as a clinical trial conducted in parallel with a preclinical trial. Currently, co-clinical trials in CRC have already generated many hopeful outcomes. For example, in BRAF-mutated CRCs, PDX models were identified to faithfully replicate clinical results and permit further study of acquired resistance mechanisms.
      • Corcoran RB
      • Atreya CE
      • Falchook GS
      • et al.
      Combined BRAF and MEK inhibition with dabrafenib and trametinib in BRAF V600-mutant colorectal cancer.
      Julien and colleagues reported that by using the PDX models, they were able to reproduce the results observed in clinical trials of cetuximab in KRAS.
      • Julien S
      • Merino-Trigo A
      • Lacroix L
      • et al.
      Characterization of a large panel of patient-derived tumor xenografts representing the clinical heterogeneity of human colorectal cancer.
      Therefore, by establishing PDX models of patients enrolled in clinical trials and treating them with new drugs, prognostic biomarkers can be screened, and potential drug response mechanisms can be studied. Although not widely attempted so far, the co-clinical trial may be further utilized in the future as it can shorten the time to drug development and demonstrate individualized medicines in a preclinical setting.
      Beyond coclinical trials, transplant mouse models are also considered useful tools for studying immunotherapy. Hollandsworth et al.
      • Hollandsworth HM
      • Amirfakhri S
      • Filemoni F
      • et al.
      Near-infrared photoimmunotherapy is effective treatment for colorectal cancer in orthotopic nude-mouse models.
      verified that near-infrared photoimmunotherapy is an effective method for the treatment of CRC using humanized mice. To overcome the limitations of antibody therapy for immune checkpoint blockade, Lee et al.
      • Lee EJ
      • Kim JH
      • Kim TI
      • et al.
      Sanguisorbae radix suppresses colorectal tumor growth through PD-1/PD-L1 blockade and synergistic effect with pembrolizumab in a humanized PD-L1-expressing colorectal cancer mouse model.
      explored a novel blockade in traditional oriental medicine, called Sanguisorbae Radix extract (SRE). They demonstrated that SRE alone has anticancer effects via immune checkpoint blockade and that the combination therapy of SRE and pembrolizumab has enhanced immuno-oncologic effects. Using transplant mouse models, many new immunotherapeutic strategies are being developed, some of which have received regulatory approval or are being studied in clinical trials.

      Surgical model

      The standard treatment modality for CRC is a surgery-based holistic therapy. Although transplant mice models are a valuable tool for biomarker development and drug screening, their predictive power is limited by several factors. Among them, surgery is extremely important but often overlooked. Preclinical studies have demonstrated that resection of the primary tumor activates proliferative and metastatic pathways that accelerate the growth of microscopic or macroscopic residual tumors.
      • Ceelen WP
      • Morris S
      • Paraskeva P
      • et al.
      Surgical trauma, minimal residual disease and locoregional cancer recurrence.
      Wang et al. further demonstrated that colorectal surgery enhances tumor cell adhesion and invasion through endotoxin/LPS-related and/or β1 integrin-dependent mechanisms.
      • Wang JH
      • Manning BJ
      • Wu QD
      • et al.
      Endotoxin/lipopolysaccharide activates NF-kappa B and enhances tumor cell adhesion and invasion through a beta 1 integrin-dependent mechanism.
      A similar phenomenon was observed in mice.
      • Schackert HK
      • Fidler IJ.
      Development of an animal model to study the biology of recurrent colorectal cancer originating from mesenteric lymph system metastases.
      Therefore, surgery has an important impact on the biological behavior of postoperative residual tumors and treatment response. Evaluating this impact requires establishing a surgical model that reflects more realistic clinical practice through a combination of drug trials and surgery.
      Previously, some simple surgical models have been established for certain scientific purposes. In 1986, Giavazzi et al.
      • Giavazzi R
      • Campbell DE
      • Jessup JM
      • et al.
      Metastatic behavior of tumor cells isolated from primary and metastatic human colorectal carcinomas implanted into different sites in nude mice.
      found that CRC growth after thigh resection did not increase the incidence of metastases. In 1989, Schackert et al.
      • Schackert HK
      • Fidler IJ.
      Development of an animal model to study the biology of recurrent colorectal cancer originating from mesenteric lymph system metastases.
      found that after resection of the tumor in the mouse cecum, most models had tumor recurrence at the resection site and mesenteric regional lymph nodes. In 1995, Allendorf et al.
      • Allendorf JD
      • Bessler M
      • Kayton ML
      • et al.
      Increased tumor establishment and growth after laparotomy vs laparoscopy in a murine model.
      reported that compared with laparotomy, tumors were less likely to form after laparoscopic surgery, and the tumor was less aggressive. Although the above surgical models facilitated the in-depth understanding of tumor growth and recurrence, none of them can reflect real-life clinical situations. Interestingly, Pang et al.
      • Pang TCY
      • Xu Z
      • Mekapogu AR
      • et al.
      An orthotopic resectional mouse model of pancreatic cancer.
      recently reported a robust and reproducible technique for a mouse pancreatic cancer surgical model and showed that this model could be useful in testing both preoperative and postoperative adjuvant treatments. Thus, establishing the surgical mouse model of CRC perhaps contributes to the development of future surgery-based combination therapy in the treatment of CRC.

      CONCLUSION AND FUTURE DIRECTIONS

      Due to the high failure rate of new therapeutic strategies in clinical studies, establishing good preclinical models is crucial in translational cancer research. However, differences between species lead to biologically inadequate preclinical models that do not accurately reflect the considerable genetic and phenotypic heterogeneity of tumors. Cancer has been shown to be a systemic disease rather than an isolated disease, and it is thus influenced by both systemic (such as hormone,
      • Haynes T
      • Oprea-Ilies G
      • Manne U
      • et al.
      The interplay of pineal hormones and socioeconomic status leading to colorectal cancer disparity.
      immunity and metabolism
      • Yang X
      • Peng H
      • Luo Z
      • et al.
      The dietary carcinogen PhIP activates p53-dependent DNA damage response in the colon of CYP1A-humanized mice.
      ) and local factors (such as tumor growth environment and intra-tumoral microenvironment). Therefore, the factors with the ability to influence tumor progression, metastasis, and treatment response can be roughly divided into 3 points: systemic factors, local growth conditions of tumor cells, and intra-tumoral microenvironment. An ideal model is expected to fully replicate the impact of the above factors on tumor cells.
      The development of humanized mice, PDXs/PODs, and orthotopic transplantation provides a promising solution to the above issues. Since orthotopic transplantation approximates the environment for tumor growth and PDXs/PODs maintain the intact tumor tissue structure and intra-tumoral microenvironment as much as possible, humanized mice make it possible to re-establish the human system environment in mice. Thus, the orthotopic transplantation of PDXs/PODs into humanized mice is potentially an ideal CRC transplant model to anatomize the multifactorial etiology and progression of the tumor (Fig 3). However, there is less literature providing clear evidence demonstrating the superiority of this model to date.
      Fig 3
      Fig 3The ideal transplant mice model of colorectal cancer. The development of PDXs/PDOs, orthotopic transplantation, and humanized mice can accurately reflect the intact intratumoral microenvironment, actual context of tumor growth, and human systemic environment in immunodeficiency mice. Thus, the orthotopic transplantation of PDXs/PDOs into humanized mice is potentially the ideal CRC transplant model to anatomize the multifactorial etiology and progression of the tumor. CRC, colorectal cancer; PDXs, patients-derived xenografts; PDOs, patients-derived organoids. (Color version of figure is available online.)
      On the other hand, humanized mice were generally used to reconstitute the human immune system in mice and then study the impacts of immunity on disease. As previously mentioned, other systemic factors, such as metabolism and endocrine, also exert a regulatory effect on the tumor. However, establishing a rodent animal model that can reflect these factors is still in groping. In addition, our team have preliminarily established an early-stage CRC resection model (Supplementary Fig 1) and demonstrated the technical viability of this model. However, much work is still needed to establish the surgical model of stage II-IV CRC to evaluate the efficacy of surgery-based combination therapy. Further studies are required to address these gaps.
      In conclusion, this is the first review of CRC transplant models to provide a comprehensive assessment of their characteristics in the research of human CRC. We believe the CRC transplant model is an essential preclinical tool to broaden the personalized medicine strategy in the future. More studies for improving the graft success rate and generating humanized mice with a similar systemic environment as patients will accelerate the usage of this preclinical model.

      FUNDING

      This work was supported by Liaoning Provincial Natural Science Foundations of China (LJKZ1192), Natural Science Foundation of Fujian Province (2020J01227), Medical Innovation Science and Technology Project of Fujian Province (2020CXA047), and Science and Technology Bureau of Quanzhou (2020CT003).

      AUTHOR CONTRIBUTIONS

      Yu-Shen Yang, Xue-Feng Zhao, He-fan He, and Shu Lin: conceptualization, writing-original draft preparation, writing-editing and reviewing. Yu-Shen Yang and Chu-Yun Liu: writing-original draft preparation, writing-editing and reviewing. Dan Wen: writing-editing and review. Da-Gao Zhi: writing-editing and review. All authors contributed to the article and approved the submitted version.

      ACKNOWLEDGMENTS

      All authors have read the journal's authorship agreement. We are thankful to Dalian University Affiliated Xinhua Hospital and The Second Affiliated Hospital of Fujian Medical University for providing infrastructure facilities. We would like to thank Editage (www.editage.cn) for English language editing.
      Conflict of Interest: All authors have read the journal's policy on conflicts of interest and declare that they have no competing interests.

      Appendix. Supplementary materials

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