TY - JOUR
AU - Hutmacher, Dietmar Werner
AB - Abstract Despite positive testing in animal studies, more than 80% of novel drug candidates fail to proof their efficacy when tested in humans. This is primarily due to the use of preclinical models that are not able to recapitulate the physiological or pathological processes in humans. Hence, one of the key challenges in the field of translational medicine is to “make the model organism mouse more human.” To get answers to questions that would be prognostic of outcomes in human medicine, the mouse's genome can be altered in order to create a more permissive host that allows the engraftment of human cell systems. It has been shown in the past that these strategies can improve our understanding of tumor immunology. However, the translational benefits of these platforms have still to be proven. In the 21st century, several research groups and consortia around the world take up the challenge to improve our understanding of how to humanize the animal's genetic code, its cells and, based on tissue engineering principles, its extracellular microenvironment, its tissues, or entire organs with the ultimate goal to foster the translation of new therapeutic strategies from bench to bedside. This article provides an overview of the state of the art of humanized models of tumor immunology and highlights future developments in the field such as the application of tissue engineering and regenerative medicine strategies to further enhance humanized murine model systems. Stem Cells 2015;33:1696–1704 Adult stem cells, Adult hematopoietic stem cells, Bone marrow, Bone, CD34+, Stem cell‐microenvironment interactions, Stem cell transplantation, Tissue regeneration, Humanized mice models Introduction For many decades, the laboratory mouse has been the key surrogate model system to provide insights into the development and treatment of human diseases such as cancer. However, in the light of recent clinical trial failures of novel drug candidates, the value of mouse models to translate research findings from bench to bedside has been increasingly questioned [1]. While significant insights into the molecular and cellular basis of human cancer biology are derived from small animal models, it is well known that considerable differences exist in terms of organ physiology, cellular dynamics, and regulatory proteins, when mice are compared to humans [2, 3]. Relying solely on syngeneic murine models poses the risk of translating therapeutic strategies into the clinic that lack proven mechanisms. Even though animal studies might suggest novel drug candidates to be efficient, more than 80% of these drugs fail when tested in humans [4]. To get answers that are more predictive of results in human medicine, major basic and translational research efforts are directed toward the humanization of the model organism mouse [5]. This can be achieved by incorporation of human transgenes into the host animal or transplantation of functional human cells, tissues, or entire organs [6]. However, humanizing a mammalian species is not a clear‐cut assignment due to the multifaceted interaction of biological components and spatial scales. The term “humanized mouse” first became popular after the publication of the paper “Humanized mice: are we there yet?” from Macchiarini et al. in 2005 [7], which summarizes the application spectrum of these model organisms in biomedical research. Since then, humanized mice have been increasingly used for the study of human biological processes, malignancies, or disorders [6, 8, 9]. Although these model systems are of high value for the research community, they also have their inherent limitations and the expectations that humanized mice are the “Rosetta Stone” of translational research have not been fulfilled yet [10]. Scientists are still working on the improvement of our understanding of how to humanize the animal's genetic code, its cells and extracellular microenvironment, its tissues, or entire organs to foster the translation of new therapeutic strategies from bench to bedside. A humanized mouse is an immuno‐compromised hybrid model organism, a chimera, which is murine enough to provide a vital host environment for xenotransplanted cells but at the same time is not too much human so that a graft versus host disease (GvHD) is triggered. Without any doubt, this approach has the potential to answer specific questions in human medicine and particularly in cancer research; however at the moment, the humanization of the mouse and the development of a translatable model system is a process rather than a finalized product, since the success of this model system is growing in parallel with technological advancements in genetics, proteomics, stem cell research, tissue engineering & regenerative medicine (TE&RM), inter alia (Fig. 1). Despite their promises and potential for translational medicine, the generation of humanized mice has raised ethical issues and moral confusion within the public and the research community. Therefore, scientists have to continuously question their own experimental approaches to ensure that their respect for both human and animal dignity is maintained [11]. Ethical guidelines have to be continuously refined in parallel to the technical advances available. Open in new tabDownload slide One of the major challenges within the biomedical research community in the 21st century will be to solve problems of highly complex and integrated biological systems. Validated humanized mouse models hold great promise for improving preclinical drug testing and unravelling biological mechanisms of the human disease. However, the true potential of this platform can only be uncovered by an integrative effort of different research disciplines. Open in new tabDownload slide One of the major challenges within the biomedical research community in the 21st century will be to solve problems of highly complex and integrated biological systems. Validated humanized mouse models hold great promise for improving preclinical drug testing and unravelling biological mechanisms of the human disease. However, the true potential of this platform can only be uncovered by an integrative effort of different research disciplines. Creating “Niches” for the Engraftment of Human Cells One of the most widely used methods in cancer research is the transplantation of human xenografts into murine hosts to investigate human tumor growth, invasion, progression, and metastasis. Cotransplantation with human immune system elements allows the analysis of species‐specific interactions between human tumor cells and a humanized immune system. This requires the use of immuno‐compromised hosts that readily integrate xenografts and do not reject human cancer cells. The ability to successfully transplant human cells that can be reproducibly maintained and amplified within the murine system was first made possible with the discovery of athymic nude mice in 1966 [12]. These mice are homozygous for a mutation in the forkhead box N1 (Foxn1) gene resulting in T‐cell deficiency. Although they were shown to support human cancer cell engraftment and tumor growth, they are not suitable to host functional human cells or tissues as they exhibit an intact B‐cell activity and functioning innate immunity [13]. In 1983, Bosma et al. reported a spontaneous autosomal recessive mutation in the Prkdcscid gene (protein kinase, DNA activated catalytic polypeptide), which severely impairs lymphopoiesis in affected C.B‐17 mice [14]. The severe combined immunodeficiency syndrome occurring in the C.B‐17‐Scid mouse is clinically very similar to the disease found in humans and analogous in the mode of inheritance with impaired rearrangement processes at the JH region. The lack of both T and B cells associated with this mutation allowed for the successful transplantation of human derived hematopoietic stem cells (HSCs) [15], peripheral blood mononuclear cells (PBMCs) [16], and fetal tissues [17]. However, engraftment rates were still low due to the remaining high level of innate immunity and due to a process known as “leakiness,” which is the production of functional T and B cells during ageing [8]. In 1980, Makino et al. described nonobese diabetic mice (NOD) that are characterized by an impaired innate immunity [18]. Crossing NOD and Scid strains resulted in a mouse with defects in both the innate and adaptive immunity. The diabetic phenotype was not relevant any more as the onset of diabetes in NOD mice is predominantly T‐cell dependent [19]. This so‐called NOD/Scid mouse developed by Shultz et al. in 1995 has served as the gold standard for the engraftment of functional human cells for the last 2 decades. However, some limitations still remained even with this strain, namely the limited longevity due to the development of thymic lymphomas and the reduced but yet prevailing leakiness [9]. While the above‐mentioned mouse strains are mainly the result of sophisticated breeding strategies, state‐of‐the‐art platforms use genetic engineering to decrease immunity and increase the level of humanization within these strains. In the 1990s, RAG‐1 and RAG‐2 mutant mice were genetically engineered that lack the ability to perform V(D)J rearrangement resulting in a Scid phenotype without leakiness [20, 21]. Other targeted mutations were performed at the β2 microglobulin and perforin genes to prevent the development of natural killer (NK) cells [7, 22, 23]. Nevertheless, problems such as residual innate or adaptive immunity or the development of lymphomas in mice with Scid background still prevented the sufficient engraftment of functional human cells. These problems were not overcome until the development of immuno‐deficient mice homozygous for a targeted mutation in the interleukin‐2 receptor γ chain gene (IL‐2rg) which encodes a transmembrane receptor subunit for signal transduction of IL‐2, IL‐4, IL‐7, IL‐15, and IL‐21. These cytokines are essential for the function of leukocytes [24]. Mice carrying this mutation are characterized by severe impairment of T‐ and B‐cell development, complete lack of NK cells, and poor lymph node development [25]. Consequently, a series of highly immuno‐deficient mice have been developed in the 2000s by combining the IL‐2rγnull gene with mice carrying the Rag1null, Rag2null, or Scid mutation resulting in even more profound immunological defects (reviewed in detail by Shultz et al. [8] and Ito et al. [26]). Mice containing the IL‐2rγnull mutation allow multilineage hematopoietic cell engraftment after transplantation of human HSCs [8, 27, 28]. They have proven their efficacy in analyzing human immunological processes or key steps involved in human tumor growth or metastasis and have therefore become the new standard for humanized mouse models [8]. Modeling Human Cellular and Humoral Antitumor Immune Response Studies have demonstrated that a higher number of human cancer cells exhibit tumorigenic potential when transplanted into 2rγnull mice than into NOD/Scid mice [29]. This is mainly due to the more biologically permissive conditions found in 2rγnull mice. To recapitulate the clinical conditions more closely and to model the human antitumor immune response, immuno‐compromised mice can be engrafted with both human cancer cells and functional human immune cells [30-33]. In 2010, there were still only a limited number of papers published in which effects of human immune systems on human tumors were investigated within a murine host. However, within the last years and in parallel with the development of more permissive hosts, more and more groups have adopted these innovative concepts for their specific research questions. One method to generate humanized mice is to transplant human PBMCs. This does not require highly sophisticated cell isolation techniques and leads to a stable engraftment of activated T‐cell populations. Engrafted IL‐2rγnull mice usually develop a robust xenogeneic GvHD after a few weeks only which is even accelerated when the mice are irradiated prior to cell transplantation [34]. Despite the transplantation of human PBMCs allows only a limited investigative time window, the model has already yielded significant insights into human tumor immunology [31, 35-37]. In 2009, Ito et al. [37] transplanted human lymphoma cells subcutaneously into 6–8 weeks old NOD/Shi‐Prkdcscid Il2rctm1Sug/Jic mice. After 28 days, they treated those mice with either 1 × 107 adult PBMCs alone or in combination with KM2760, a chimeric anti‐CCR4 monoclonal antibody, for 2 weeks. KM2760 was shown to mediate robust antibody‐dependent cellular cytotoxicity and resulted in a significant increase of human tumor‐infiltrating NK cells in mice bearing lymphomas [37]. Wulf‐Goldenberg et al. [31] injected newborn NOD/C.B‐17‐Scid mice with 5 × 106 cord‐blood mononuclear cells intrahepatically after γ‐irradiation with 1 Gy. After 5 weeks, T‐cell engraftment was observed and mice were subcutaneously transplanted with human SW480 colon carcinoma cells. While the engrafted human mononuclear cells alone had only a slight negative effect on tumor growth when compared with nonengrafted mice, treatment with a bispecific EpCAM/CD3 antibody was able to enhance the antitumoral effect of human immune cells [31]. In a similar study performed by McCormack et al. [38], the efficacy of bifunctional ImmTAC‐NYE, a T‐cell receptor specific for NY‐ESO‐1 fused to an anti‐CD3 scFv, was tested in a xenograft model in which 20 × 106 CD45+CD3+ PBMCs were intravenously injected before or after subcutaneous engraftment of NY‐ESO‐1‐positive tumors. In both, the preventive and treatment group, there was a significant effect of the reagent when compared with PBS injected mice [38]. Guichelaar et al. [36] investigated the antitumor response of human PBMCs with or without human Tregs in a humanized mouse model. They injected Luciferase‐labeled human myeloma cells into the circulatory system of adult Stock (H2d)‐Rag2tm1FwaIl2rgtm1Krf mice. After 2–5 weeks of tumor cell injection, 1 × 107 PBMCs were injected intravenously to induce both a graft versus tumor reaction and GvHD. In another experimental group, the authors cotransplanted 1 × 107 Tregs. As expected, a high engraftment rate of CD4+ and CD8+ T cells was found after 2 weeks of PBMC transplantation and 50% of the mice died after 4 weeks due to GvHD. Cotransplantation of Tregs, however, resulted in a significantly longer survival rate by inhibiting GvHD while not suppressing the graft versus tumor reaction [36]. Human PBMC xenograft tumor models can also be used for the preclinical testing of human T‐cell therapies. Peripheral human T cells can be isolated and specifically targeted against an array of human tumor antigens by genetic transfer of antigen‐specific receptors such as chimeric antigen receptors [35, 39-42] or the heterodimeric T‐cell antigen receptor [43]. This platform makes it possible to test the efficacy and safety profile of targeted T‐cell therapies in vivo. However, it has to be emphasized that the translational value of these studies is limited as true antitumoral T‐cell responses can hardly be differentiated from allogeneic graft versus tumor activity due to HLA‐mismatch between human T cells and cancer cells. Furthermore, the xenogeneic GvHD occurring in these models can also influence the outcomes of these studies. A more complex method is the transplantation of CD34+ human hematopoietic stem and progenitor cells into the murine host [44]. Here, the technological approaches are quite diverse and only a few well‐controlled studies comparing the efficacy of different isolation methods, culture conditions, or engraftment routes can be found in the literature [45-47]. Human CD34+ cells can be isolated from cord blood, bone marrow, peripheral blood, and fetal liver. Isolation is usually performed by Ficoll separation and subsequent incubation with human CD34 magnetic selection beads. Currently, human cord blood cells are seen as the gold standard since they are readily available and result in higher engraftment rates as compared to cells isolated from adult bone marrow [45]. Werner‐Klein et al. recently found that although more CD34+ cells per number of mononuclear cells can be isolated from bone marrow than cord blood, approximately 2 × 105 CD34+ bone marrow cells are needed to achieve engraftment levels equivalent to 4.8 × 104 cord blood cells [45]. The group also demonstrated that in adult mice there is no statistical qualitative or quantitative difference in reconstitution capacity between cord blood cells transplanted via the intrafemoral, intrahepatic, or intravenous route [45]. When neonatal mice are used, the intrahepatic route is recommended [47], as the liver is the main hematopoietic organ at birth [48]. For bone marrow cells, no significant differences were found when transplanted by intrafemoral injection into adults or intrahepatic injection into newborn mice [45]. In 1998, Tagawa et al. [49] reconstituted 6–8‐week‐old C.B‐17‐Scid mice intraperitoneally with 5 × 106 human CD34+ cells. Although the engraftment rate was low, the authors demonstrated that IL‐2 producing gastric carcinoma cell growth was effectively inhibited by the engrafted human cell system [49]. Aspord et al. [50] injected 3–6 × 106 CD34+ cells obtained from peripheral blood of healthy donors intravenously into sublethally irradiated NOD/C.B‐17‐Scid/β2m−/− mice. Four weeks later, the mice were subcutaneously engrafted with Hs578T breast cancer cells. Repeated injections of T cells autologous to the previously injected CD34+ cells resulted in accelerated tumor growth. The authors demonstrated that this effect was mediated by CD4+ T cells [50]. In more permissive strains such as 2rγnull mice, transplantation of HSCs allows higher engraftment rates [47] and stable multilineage de novo reconstitution after only a few weeks [51] including CD4+ and CD8+ T cells [52, 53]. This model was used by Wege et al. who cotransplanted 3 × 105 human CD34+ cord blood cells together with human breast cancer cells into the liver of IL‐2rγnull mice [30, 33] to demonstrate that tumor growth was accompanied by tumor cell‐specific T‐cell activation. However, as stated above, the observed activation of human T cells might have also been induced by a MHC mismatch between the transplanted human hematopoietic and malignant cell populations [30]. There are still fundamental limitations that reduce the applicability of this platform. Despite the numbers of human T cells and myeloid cells that can be found in the bone marrow of 2rγnull mice are higher than in any other mouse strain used before, their numbers are still very low in the blood [54]. Furthermore, the percentage distribution of B and T lymphocytes and myeloids is still very different to the one found in humans [54]. The reconstituted human immune cell system found in the blood consists mainly of B cells with only a very little amount of myeloids, whereas in humans the immune cell system in the blood consists in large parts of myeloid cells [54]. The established adaptive immune system is naive [8] and an appropriate antigen‐specific T‐cell response is not possible as human T cells cannot be educated through HLA restriction within the mouse thymus [54]. Furthermore, human B cells that develop within the murine host are immature without appropriate antibody switch from IgM to IgG after immunization or infection [55]. Even after rigorous depletion of T cells, there is a risk of GvHD caused by de novo generated T cells, although the onset of the wasting disease can be expected at a later time point than after transplantation of PBMCs. The lifespan of the animals reconstituted with CD34+ cells is dependent on their strain and age, the number and reconstitution capacity of the transplanted cells, the route of transplantation, and adjuvant interventions such as conditional irradiation [56]. In currently used highly permissive 2rγnull mice, first clinical signs for GvHD can be expected when bleeding of the mice reveals anemia and engraftment levels of more than 60% of human CD45+ cells [56]. Most recently, it has been demonstrated that human‐specific cytokines are necessary for the generation of functional human immune cells and their proper hierarchical organization as some human cytokines are absent in the mouse system or characterized by low homology with murine cytokines [54]. These issues are currently being addressed by knockin of human orthologous genes [57-59] or insertion of nonorthologous genes into the IL‐2rγnull mouse genome [60-62] encoding for human‐specific cytokines or HLA antigens. Another commonly used method is the knockout of murine genes encoding for MHC class I or II molecules [34, 61]. These technologies are designed to enhance human innate and adaptive immunity within the host by expression of human‐specific factors such as M‐CDF, GM‐CSF, IL‐3, SIRPα, and SCF, to decrease the risk of GvHD and support the development of human HLA restricted T cells [63]. Recently, Rongvaux et al. [56] used Rag2−/−IL2rg−/− mice to knock in genes encoding for human IL‐3, M‐CSF, GM‐CSF, thrombopoietin and to insert SIRPα into the murine genome. Intrahepatic transplantation of CD34+ fetal liver cells after sublethal irradiation resulted in a previously undocumented high engraftment rate for CD33+ myeloid cells that were able to infiltrate human melanoma tumor xenografts in a manner resembling the human disease [56]. The third method used to engraft mice with a human immune system is the bone‐liver‐thymus (BLT) model. In this platform, developed independently by two different groups in 2006 [64, 65], fetal liver and thymus tissue are implanted under the kidney capsule and CD34+ cells, autologous to these tissues, are cotransplanted into the murine host. The fetal tissues usually engraft well within the liver and develop a functional humanized organoid. In general, the same limitations apply for this platform as for the sole transplantation of CD34+ cells described above. However, the MHC molecules expressed by the implanted human tissues make a selection process of human lymphocytes possible. This results in the development of functional human T cells capable of an antigen‐specific immune response and a higher engraftment rate of human cells in the peripheral blood than in any other model [54, 64, 65]. Vatakis et al. used this model to create a platform in which the antitumoral activity of genetically modified human HLA‐restricted CD8+ T cells can be tested [66]. Fetal liver and thymus tissue were implanted under the kidney capsule of 6–8 weeks old IL‐2rγnull mice together with 6 × 106 autologous CD34− cells and 106 autologous CD34+ cells transduced to express HLA‐A*0201‐restricted melanoma‐specific T‐cell receptor (HLA‐A*0201+MART+) [66, 67]. After 4 weeks, mice were sublethally irradiated and transplanted with 1–5 × 105 transduced CD34+ cells obtained from the same fetus to support full reconstitution. The mice were then subcutaneously implanted with HLA‐A*0201+MART+ and HLA‐A*0201−MART+ melanoma cells, respectively. Growth inhibition and tumor necrosis was only observed in HLA‐A*0201+MART+ tumors. This observation led the authors conclude that the antitumoral activity was most likely caused by a specific response generated by the genetically modified human HLA‐restricted CD8+ T cells and was not the result of a nonspecific alloreactivity [66, 67]. The success of this model demonstrates that the generation of humanized organoids can open up new vistas for the translation of research findings in preclinical animal models of tumor immunology. Convergence of Tissue Engineering and Cancer Research Current literature has presented increasing evidence that processes such as tumor initiation, progression, and metastasis are not only the result of genetic or epigenetic alterations but are indeed initiated and maintained by factors provided by the microenvironment [68-71]. A paradigm shift has taken place where tumors are not anymore seen as a mere conglomerate of homogenous cancer cells but rather as organs with a composition at least as intricate as nondiseased organ systems [72]. As it is important to have a profound knowledge about how distinct cellular and acellular components affect disease development when treating organ or tissue failure, it is likewise essential to understand the pathophysiology of the tumor with its different specialized cell types and extracellular components. To ultimately leave the view that tumors consist of cancer cells only [72], it is important to dissect the roles of the tumor microenvironment for tumor initiation, progression, and metastasis. The extracellular matrix (ECM) has long been identified as a three‐dimensional (3D) structure that influences cellular function and maintains tissue architecture. It is not only an inert “ground substance” in which cells and important biological factors such as hormones, nutrients, and growth factors are embedded but rather a responsive structure that determines mechanical tissue properties and guides cellular functions and biochemical processes. Fortunately, researchers have become aware that the ECM does not only regulate physiological processes within healthy tissues or organs but also significantly influences pathological processes such as tissue regeneration after trauma and tumorigenesis. This makes the limitations of conventional two‐dimensional cell culture experiments easy to understand. Within this context, the tissue engineering tool box makes it possible to probe the importance not only of microenvironmental cellular cues but also of 3D structural variables for cancer development under reproducible conditions [73]. Our group has previously demonstrated that tumor cells cultured in 3D matrices exhibit different morphological features and biological functions compared to cells grown in monolayer cultures [70, 74-78]. While there are increasing numbers of 3D models investigating tumor growth, there are only very few analyzing processes involved in tumor immunology (reviewed in detail in [79]). Interestingly, these few studies have demonstrated that 3D culture of cancer cells results in downregulation of tumor‐associated antigens and MHC class I molecules when compared with conventional monolayer cultures [80-83]. This in turn leads to a reduced capacity of cytotoxic T lymphocytes to recognize malignant cell clones. The observed impaired immune response in 3D cultures is also mediated by the increased production of lactic acid in multilayered architecture suppressing the proliferation of cytotoxic T cells and differentiation of monocytes toward dendritic cells [81, 84]. Taken together, 3D culture models have demonstrated that tumor cell escape mechanisms are mediated by hierarchical structures provided by the microenvironment. There is no doubt that these tissue engineered in vitro models can provide significant insights into the interdependence of tumor immunological processes and the surrounding milieu, thereby affecting response to therapy. However, these platforms certainly have limitations and many important questions remain unanswered even with 3D in vitro models. Although they may be suitable to answer specific biological questions under reproducible conditions, they are so far only able to analyze single steps of the complex disease cascade depending on the cellular and architectural repertoire available. Moreover, most 3D culture platforms are naturally derived which makes it hard to define their composition in a way to reliably exclude batch‐to‐batch variation [85]. Validated animal models need to be developed side by side with in vitro model platforms providing the opportunity to investigate the multicellular interactions and dynamic multistep processes involved in cancerogenesis. We have previously shown that the technological toolbox developed for TE&RM applications can be used to humanize xenograft models of tumor entities such as prostate [86, 87], breast [3, 88], and ovarian [75] cancer. TE&RM strategies can be also used to increase the value of existing models of tumor immunology by incorporation of human cell and extracellular matrix systems to engineer a functionally defined humanized stroma before transplantation of human hematopoietic cells or cancer cells [70]. This allows not only to test the efficacy of certain drugs against human cancer cells but also to determine their safety profile and effects on healthy human cells or tissues. As shown in the BLT model, humanized organoids can be engineered and provide a humanized stroma which makes it possible to supply human transplanted cells with factors that are essential for their engraftment and proliferation. This allows investigating the behavior of human cells within their specific niches [87]. The latter is of utmost importance as recent literature suggests that some elements of the mutual interaction between human cells and their microenvironment are species‐specific [89]. As shown by Rongvaux et al., the level of conservation between humans and mice is highly variable for different chemokines and adhesion molecules [54]. While the amino acid sequence of some human proteins is highly similar to the ones found in mice, some others exhibit only low or even no homology [54]. However, even if the protein of interest shares the same amino acid sequence between humans and the model organism, there is the possibility that there is no functional cross‐reactivity of proteins of both species. This can only be guaranteed if both the protein expressing cell and the target cell are of human origin. Future Directions As outlined above, even in the BLT model there are several limitations that prevent high throughput testing of novel drug candidates or targeted cell therapies. Advancements are under way and include the transgenic expression of HLA antigens to support the development of HLA‐restricted T cells and the knockout of murine MHC I and II molecules to decrease the risk for GvHD [8]. Although, the introduction of human transgenes encoding for human cytokines has been demonstrated to be successful in increasing engraftment rates, this has resulted in supraphysiological concentrations of these cytokines leading to artificial effects such as rapid exhaustion of HSCs [90]. A new way to solve these problems and develop a better functioning humanized immune system within mice is the generation of peripheral humanized lymphoid organs that can provide species‐specific factors necessary for the maintenance of human HSCs without the need for genetic manipulations. Recent innovations in TE&RM made it indeed possible to engineer such ectopic lymphoid constructs in mice. Lee et al. [91] implanted polyacrylamide hydrogels loaded with human BMSCs subcutaneously into IL‐2rγnull mice. The implants induced vascularized hematopoietic tissue formation and were able to attract transplanted human hematopoietic progenitors and leukemic cells; however, the engineered constructs did not recapitulate the physiological niche morphology since the transplantation of human BMSCs did not result in any bone formation. Moreover, the authors failed to prove whether the human cells were able to survive and secrete extracellular matrix proteins after transplantation into the host [91]. Groen et al. [92] demonstrated that subcutaneous implantation of calcium phosphate particles seeded with human MSCs resulted in ectopic bone formation and development of a HSC niche in RAG−/−γc−/− mice. The authors showed that transplanted human CD34+ and myeloma cells were able to home to these osseous constructs but failed to analyze whether the transplanted cells can differentiate into different hematopoietic lineages and develop a functional bone marrow compartment [92]. Although these studies intended to investigate species‐specific microenvironmental cues between the human niche and human hematopoietic cells, they provide no evidence whether the transplanted human mesenchymal cells survive and contribute to the niche formation [91, 92]. Recently, we were able to demonstrate that a humanized organ bone can be engineered that recapitulates both the function and morphology of the HSC niches [87, 88] and is supportive of the maintenance of transplanted human hematopoietic stem and progenitor cells (Fig. 2). With this scaffold‐based tissue engineering approach we can transplant a large variety of vital human cells of different origin tissue and differentiation stages after undergoing an in‐process control to guarantee sufficient quality and quantity of not only the cells seeded onto the scaffolds before implantation but also the ECM secreted. More importantly, after in vivo bone formation it is possible to precisely quantify the level of chimerism and determine to what degree the transplanted human mesenchymal cells contribute to the niche formation [87, 88]. Open in new tabDownload slide Tissue engineering of a humanized hematopoietic organ to recapitulate tumor‐immunological processes involved in bone metastasis. (A): Human mesenchymal progenitor cells are seeded on tubular bioresorbable composite scaffolds. After in vitro culture and production of human extracellular matrix components, the constructs are filled with fibrin glue and recombinant human bone morphogenetic protein (rhBMP‐7) and subcutaneously implanted into the flanks of 2rγnull mice. (B): Micro‐CT analyses show that the tissue engineered constructs recapitulate the morphological features of a physiological organ bone with a cortex like outer structure and an inner trabecular network. Histology (H&E) demonstrates that intertrabecular spaces are filled with hematopoietic cell systems indicating that the organ is functional. (C): Immunostaining for human‐specific collagen I (hsCol‐1) demonstrates that the transplanted human mesenchymal cells are capable of producing human extracellular matrix components (human matrix brown, murine pale blue). Transplanted human mesenchymal cells survive and are incorporated into the newly formed organ bone as indicated by human‐specific staining for nuclear mitotic apparatus protein‐1 (hsNuMa) (human cells brown, murine cells blue). Transplanted human CD34+ cells can engraft the animal after myeloablative irradiation and develop a humanized bone marrow compartment within the chimeric ossicles as shown by staining for human‐specific CD45 (hsCD45) (human cells brown, murine cells blue). (D): Human cancer cells can be injected directly into the humanized engineered organs or inoculated into the vascular system of the host to recapitulate the bone metastatic cascade. Histological analysis (H&E) and human‐specific immunostaining for NuMa demonstrate the replacement of the physiological bone marrow compartment by the human cancer cells (human cells brown, murine cells blue). Multinucleated cells positive for TRAP (red) represent osteoclasts indicating osteolysis (scale bars = 50 µm). Abbreviations: ECM, extracellular matrix; TRAP, tartrate resistant acid phosphatase. Open in new tabDownload slide Tissue engineering of a humanized hematopoietic organ to recapitulate tumor‐immunological processes involved in bone metastasis. (A): Human mesenchymal progenitor cells are seeded on tubular bioresorbable composite scaffolds. After in vitro culture and production of human extracellular matrix components, the constructs are filled with fibrin glue and recombinant human bone morphogenetic protein (rhBMP‐7) and subcutaneously implanted into the flanks of 2rγnull mice. (B): Micro‐CT analyses show that the tissue engineered constructs recapitulate the morphological features of a physiological organ bone with a cortex like outer structure and an inner trabecular network. Histology (H&E) demonstrates that intertrabecular spaces are filled with hematopoietic cell systems indicating that the organ is functional. (C): Immunostaining for human‐specific collagen I (hsCol‐1) demonstrates that the transplanted human mesenchymal cells are capable of producing human extracellular matrix components (human matrix brown, murine pale blue). Transplanted human mesenchymal cells survive and are incorporated into the newly formed organ bone as indicated by human‐specific staining for nuclear mitotic apparatus protein‐1 (hsNuMa) (human cells brown, murine cells blue). Transplanted human CD34+ cells can engraft the animal after myeloablative irradiation and develop a humanized bone marrow compartment within the chimeric ossicles as shown by staining for human‐specific CD45 (hsCD45) (human cells brown, murine cells blue). (D): Human cancer cells can be injected directly into the humanized engineered organs or inoculated into the vascular system of the host to recapitulate the bone metastatic cascade. Histological analysis (H&E) and human‐specific immunostaining for NuMa demonstrate the replacement of the physiological bone marrow compartment by the human cancer cells (human cells brown, murine cells blue). Multinucleated cells positive for TRAP (red) represent osteoclasts indicating osteolysis (scale bars = 50 µm). Abbreviations: ECM, extracellular matrix; TRAP, tartrate resistant acid phosphatase. The convergence of this validated tissue engineering concept with other established platforms such as the BLT model could be particularly useful to study the behavior of human primary or secondary bone tumors or hematologic malignancies within their native microenvironment under influence of human immune cells. Expanding on these promising results that are motivating a paradigm shift in the field of cancer research, we are currently working on the establishment of a humanized orthotopic cancer microenvironment to recapitulate all hallmarks of human cancer from the primary tumor to the overt bone metastasis. Such fully humanized orthotopic xenograft models of cancer will significantly improve our understanding of the relevant biological processes and will provide a preclinical platform to investigate potential therapeutics at multiple intervention points of the metastatic cascade from the primary tumor to the overt metastasis. Author Contributions B.M.H.: conception and design, data collection and assembly, literature review, and manuscript writing; F.W. and L.T.: data collection and assembly, figure preparation, and manuscript editing; J.‐P.L.: manuscript review and final approval of manuscript; D.W.H.: conception and design, manuscript writing, and final approval of manuscript. Disclosure of Potential Conflicts of Interest The authors indicate no potential conflicts of interest. References 1 Couzin‐Frankel J . The littlest patient . Science 2014 ; 346 : 24 – 27 . Google Scholar Crossref Search ADS PubMed WorldCat 2 Holzapfel BM , Thibaudeau L, Hesami P et al. Humanised xenograft models of bone metastasis revisited: Novel insights into species‐specific mechanisms of cancer cell osteotropism . Cancer Metastasis Rev 2013 ; 32 : 129 – 145 . Google Scholar Crossref Search ADS PubMed WorldCat 3 Thibaudeau L , Quent VM, Holzapfel BM et al. Mimicking breast cancer‐induced bone metastasis in vivo: Current transplantation models and advanced humanized strategies . Cancer Metastasis Rev 2014 ; 33 : 721 – 735 . Google Scholar Crossref Search ADS PubMed WorldCat 4 Perrin S . Preclinical research: Make mouse studies work . Nature 2014 ; 507 : 423 – 425 . Google Scholar Crossref Search ADS PubMed WorldCat 5 Burkhardt AM , Zlotnik A. Translating translational research: Mouse models of human disease . Cell Mol Immunol 2013 ; 10 : 373 – 374 . Google Scholar Crossref Search ADS PubMed WorldCat 6 Brehm MA , Shultz LD, Greiner DL. Humanized mouse models to study human diseases . Curr Opin Endocrinol Diabetes Obes 2010 ; 17 : 120 – 125 . Google Scholar Crossref Search ADS PubMed WorldCat 7 Macchiarini F , Manz MG, Palucka AK et al. Humanized mice: Are we there yet? J Exp Med 2005 ; 202 : 1307 – 1311 . Google Scholar Crossref Search ADS PubMed WorldCat 8 Shultz LD , Brehm MA, Garcia‐Martinez JV et al. Humanized mice for immune system investigation: Progress, promise and challenges . Nat Rev Immunol 2012 ; 12 : 786 – 798 . Google Scholar Crossref Search ADS PubMed WorldCat 9 Shultz LD , Ishikawa F, Greiner DL. Humanized mice in translational biomedical research . Nat Rev Immunol 2007 ; 7 : 118 – 130 . Google Scholar Crossref Search ADS PubMed WorldCat 10 Davies G . What is a humanized mouse? Remaking the species and spaces of translational medicine . Body Soc 2012 ; 18 : 126 – 155 . Google Scholar Crossref Search ADS WorldCat 11 Karpowicz P , Cohen CB, van der Kooy D. It is ethical to transplant human stem cells into nonhuman embryos . Nat Med 2004 ; 10 : 331 – 335 . Google Scholar Crossref Search ADS PubMed WorldCat 12 Flanagan SP . ‘Nude', a new hairless gene with pleiotropic effects in the mouse . Genet Res 1966 ; 8 : 295 – 309 . Google Scholar Crossref Search ADS PubMed WorldCat 13 Ganick DJ , Sarnwick RD, Shahidi NT et al. Inability of intravenously injected monocellular suspensions of human bone marrow to establish in the nude mouse . Int Arch Allergy Appl Immunol 1980 ; 62 : 330 – 333 . Google Scholar Crossref Search ADS PubMed WorldCat 14 Bosma GC , Custer RP, Bosma MJ. A severe combined immunodeficiency mutation in the mouse . Nature 1983 ; 301 : 527 – 530 . Google Scholar Crossref Search ADS PubMed WorldCat 15 Lapidot T , Pflumio F, Doedens M et al. Cytokine stimulation of multilineage hematopoiesis from immature human cells engrafted in SCID mice . Science 1992 ; 255 : 1137 – 1141 . Google Scholar Crossref Search ADS PubMed WorldCat 16 Mosier DE , Gulizia RJ, Baird SM et al. Transfer of a functional human immune system to mice with severe combined immunodeficiency . Nature 1988 ; 335 : 256 – 259 . Google Scholar Crossref Search ADS PubMed WorldCat 17 McCune JM , Namikawa R, Kaneshima H et al. The SCID‐hu mouse: Murine model for the analysis of human hematolymphoid differentiation and function . Science 1988 ; 241 : 1632 – 1639 . Google Scholar Crossref Search ADS PubMed WorldCat 18 Makino S , Kunimoto K, Muraoka Y et al. Breeding of a non‐obese, diabetic strain of mice . Jikken Dobutsu 1980 ; 29 : 1 – 13 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 19 Shultz LD , Schweitzer PA, Christianson SW et al. Multiple defects in innate and adaptive immunologic function in NOD/LtSz‐scid mice . J Immunol 1995 ; 154 : 180 – 191 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 20 Mombaerts P , Iacomini J, Johnson RS et al. RAG‐1‐deficient mice have no mature B and T lymphocytes . Cell 1992 ; 68 : 869 – 877 . Google Scholar Crossref Search ADS PubMed WorldCat 21 Shinkai Y , Rathbun G, Lam KP et al. RAG‐2‐deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement . Cell 1992 ; 68 : 855 – 867 . Google Scholar Crossref Search ADS PubMed WorldCat 22 Banuelos SJ , Shultz LD, Greiner DL et al. Rejection of human islets and human HLA‐A2.1 transgenic mouse islets by alloreactive human lymphocytes in immunodeficient NOD‐scid and NOD‐Rag1(null)Prf1(null) mice . Clin Immunol 2004 ; 112 : 273 – 283 . Google Scholar Crossref Search ADS PubMed WorldCat 23 Palucka AK , Gatlin J, Blanck JP et al. Human dendritic cell subsets in NOD/SCID mice engrafted with CD34+ hematopoietic progenitors . Blood 2003 ; 102 : 3302 – 3310 . Google Scholar Crossref Search ADS PubMed WorldCat 24 Leonard WJ . Cytokines and immunodeficiency diseases . Nat Rev Immunol 2001 ; 1 : 200 – 208 . Google Scholar Crossref Search ADS PubMed WorldCat 25 Goldman JP , Blundell MP, Lopes L et al. Enhanced human cell engraftment in mice deficient in RAG2 and the common cytokine receptor gamma chain . Br J Haematol 1998 ; 103 : 335 – 342 . Google Scholar Crossref Search ADS PubMed WorldCat 26 Ito R , Takahashi T, Katano I et al. Current advances in humanized mouse models . Cell Mol Immunol 2012 ; 9 : 208 – 214 . Google Scholar Crossref Search ADS PubMed WorldCat 27 Hiramatsu H , Nishikomori R, Heike T et al. Complete reconstitution of human lymphocytes from cord blood CD34+ cells using the NOD/SCID/gammacnull mice model . Blood 2003 ; 102 : 873 – 880 . Google Scholar Crossref Search ADS PubMed WorldCat 28 Traggiai E , Chicha L, Mazzucchelli L et al. Development of a human adaptive immune system in cord blood cell‐transplanted mice . Science 2004 ; 304 : 104 – 107 . Google Scholar Crossref Search ADS PubMed WorldCat 29 Quintana E , Shackleton M, Sabel MS et al. Efficient tumour formation by single human melanoma cells . Nature 2008 ; 456 : 593 – 598 . Google Scholar Crossref Search ADS PubMed WorldCat 30 Wege AK , Ernst W, Eckl J et al. Humanized tumor mice—A new model to study and manipulate the immune response in advanced cancer therapy . Int J Cancer 2011 ; 129 : 2194 – 2206 . Google Scholar Crossref Search ADS PubMed WorldCat 31 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 . Cytotherapy 2011 ; 13 : 108 – 113 . Google Scholar Crossref Search ADS PubMed WorldCat 32 Seitz G , Pfeiffer M, Fuchs J et al. Establishment of a rhabdomyosarcoma xenograft model in human‐adapted mice . Oncol Rep 2010 ; 24 : 1067 – 1072 . Google Scholar Crossref Search ADS PubMed WorldCat 33 Wege AK , Schmidt M, Ueberham E et al. Co‐transplantation of human hematopoietic stem cells and human breast cancer cells in NSG mice: A novel approach to generate tumor cell specific human antibodies . MAbs 2014 ; 6 : 968 – 977 . Google Scholar Crossref Search ADS PubMed WorldCat 34 King MA , Covassin L, Brehm MA et al. Human peripheral blood leucocyte non‐obese diabetic‐severe combined immunodeficiency interleukin‐2 receptor gamma chain gene mouse model of xenogeneic graft‐versus‐host‐like disease and the role of host major histocompatibility complex . Clin Exp Immunol 2009 ; 157 : 104 – 118 . Google Scholar Crossref Search ADS PubMed WorldCat 35 Abate‐Daga D , Lagisetty KH, Tran E et al. A novel chimeric antigen receptor against prostate stem cell antigen mediates tumor destruction in a humanized mouse model of pancreatic cancer . Hum Gene Ther 2014 ; 25 : 1003 – 1012 . Google Scholar Crossref Search ADS PubMed WorldCat 36 Guichelaar T , Emmelot ME, Rozemuller H et al. Human regulatory T cells do not suppress the antitumor immunity in the bone marrow: A role for bone marrow stromal cells in neutralizing regulatory T cells . Clin Cancer Res 2013 ; 19 : 1467 – 1475 . Google Scholar Crossref Search ADS PubMed WorldCat 37 Ito A , Ishida T, Yano H et al. Defucosylated anti‐CCR4 monoclonal antibody exercises potent ADCC‐mediated antitumor effect in the novel tumor‐bearing humanized NOD/Shi‐scid, IL‐2Rgamma(null) mouse model . Cancer Immunol Immunother 2009 ; 58 : 1195 – 1206 . Google Scholar Crossref Search ADS PubMed WorldCat 38 McCormack E , Adams KJ, Hassan NJ et al. Bi‐specific TCR‐anti CD3 redirected T‐cell targeting of NY‐ESO‐1‐ and LAGE‐1‐positive tumors . Cancer Immunol Immunother 2013 ; 62 : 773 – 785 . Google Scholar Crossref Search ADS PubMed WorldCat 39 Hudecek M , Lupo‐Stanghellini MT, Kosasih PL et al. Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1‐specific chimeric antigen receptor T cells . Clin Cancer Res 2013 ; 19 : 3153 – 3164 . Google Scholar Crossref Search ADS PubMed WorldCat 40 Song DG , Ye Q, Carpenito C et al. In vivo persistence, tumor localization, and antitumor activity of CAR‐engineered T cells is enhanced by costimulatory signaling through CD137 (4‐1BB) . Cancer Res 2011 ; 71 : 4617 – 4627 . Google Scholar Crossref Search ADS PubMed WorldCat 41 Kloss CC , Condomines M, Cartellieri M et al. Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells . Nat Biotechnol 2013 ; 31 : 71 – 75 . Google Scholar Crossref Search ADS PubMed WorldCat 42 Markley JC , Sadelain M. IL‐7 and IL‐21 are superior to IL‐2 and IL‐15 in promoting human T cell‐mediated rejection of systemic lymphoma in immunodeficient mice . Blood 2010 ; 115 : 3508 – 3519 . Google Scholar Crossref Search ADS PubMed WorldCat 43 Abate‐Daga D , Speiser DE, Chinnasamy N et al. Development of a T cell receptor targeting an HLA‐A*0201 restricted epitope from the cancer‐testis antigen SSX2 for adoptive immunotherapy of cancer . PLoS One 2014 ; 9 : e93321 . Google Scholar Crossref Search ADS PubMed WorldCat 44 Tanner A , Taylor SE, Decottignies W et al. Humanized mice as a model to study human hematopoietic stem cell transplantation . Stem Cells Dev 2014 ; 23 : 76 – 82 . Google Scholar Crossref Search ADS PubMed WorldCat 45 Werner‐Klein M , Proske J, Werno C et al. Immune humanization of immunodeficient mice using diagnostic bone marrow aspirates from carcinoma patients . PLoS One 2014 ; 9 : e97860 . Google Scholar Crossref Search ADS PubMed WorldCat 46 Lang J , Weiss N, Freed BM et al. Generation of hematopoietic humanized mice in the newborn BALB/c‐Rag2null Il2rgammanull mouse model: A multivariable optimization approach . Clin Immunol 2011 ; 140 : 102 – 116 . Google Scholar Crossref Search ADS PubMed WorldCat 47 Legrand N , Weijer K, Spits H. Experimental models to study development and function of the human immune system in vivo . J Immunol 2006 ; 176 : 2053 – 2058 . Google Scholar Crossref Search ADS PubMed WorldCat 48 Kikuchi K , Kondo M. Developmental switch of mouse hematopoietic stem cells from fetal to adult type occurs in bone marrow after birth . Proc Natl Acad Sci USA 2006 ; 103 : 17852 – 17857 . Google Scholar Crossref Search ADS PubMed WorldCat 49 Tagawa M , Goto S, Takenaga K et al. Reduced tumorigenicity of human gastric carcinoma cells engineered to produce IL‐2 in SCID mice reconstituted with peripheral blood cells from cancer patients . Cancer Lett 1998 ; 123 : 87 – 93 . Google Scholar Crossref Search ADS PubMed WorldCat 50 Aspord C , Pedroza‐Gonzalez A, Gallegos M et al. Breast cancer instructs dendritic cells to prime interleukin 13‐secreting CD4+ T cells that facilitate tumor development . J Exp Med 2007 ; 204 : 1037 – 1047 . Google Scholar Crossref Search ADS PubMed WorldCat 51 Ishikawa F , Yasukawa M, Lyons B et al. Development of functional human blood and immune systems in NOD/SCID/IL2 receptor {gamma} chain(null) mice . Blood 2005 ; 106 : 1565 – 1573 . Google Scholar Crossref Search ADS PubMed WorldCat 52 Brehm MA , Cuthbert A, Yang C et al. Parameters for establishing humanized mouse models to study human immunity: Analysis of human hematopoietic stem cell engraftment in three immunodeficient strains of mice bearing the IL2rgamma(null) mutation . Clin Immunol 2010 ; 135 : 84 – 98 . Google Scholar Crossref Search ADS PubMed WorldCat 53 Onoe T , Kalscheuer H, Danzl N et al. Human natural regulatory T cell development, suppressive function, and postthymic maturation in a humanized mouse model . J Immunol 2011 ; 187 : 3895 – 3903 . Google Scholar Crossref Search ADS PubMed WorldCat 54 Rongvaux A , Takizawa H, Strowig T et al. Human hemato‐lymphoid system mice: Current use and future potential for medicine . Annu Rev Immunol 2013 ; 31 : 635 – 674 . Google Scholar Crossref Search ADS PubMed WorldCat 55 Biswas S , Chang H, Sarkis PT et al. Humoral immune responses in humanized BLT mice immunized with West Nile virus and HIV‐1 envelope proteins are largely mediated via human CD5+ B cells . Immunology 2011 ; 134 : 419 – 433 . Google Scholar Crossref Search ADS PubMed WorldCat 56 Rongvaux A , Willinger T, Martinek J et al. Development and function of human innate immune cells in a humanized mouse model . Nat Biotechnol 2014 ; 32 : 364 – 372 . Google Scholar Crossref Search ADS PubMed WorldCat 57 Rathinam C , Poueymirou WT, Rojas J et al. Efficient differentiation and function of human macrophages in humanized CSF‐1 mice . Blood 2011 ; 118 : 3119 – 3128 . Google Scholar Crossref Search ADS PubMed WorldCat 58 Rongvaux A , Willinger T, Takizawa H et al. Human thrombopoietin knockin mice efficiently support human hematopoiesis in vivo . Proc Natl Acad Sci USA 2011 ; 108 : 2378 – 2383 . Google Scholar Crossref Search ADS PubMed WorldCat 59 Willinger T , Rongvaux A, Takizawa H et al. Human IL‐3/GM‐CSF knock‐in mice support human alveolar macrophage development and human immune responses in the lung . Proc Natl Acad Sci USA 2011 ; 108 : 2390 – 2395 . Google Scholar Crossref Search ADS PubMed WorldCat 60 Billerbeck E , Barry WT, Mu K et al. Development of human CD4+FoxP3+ regulatory T cells in human stem cell factor‐, granulocyte‐macrophage colony‐stimulating factor‐, and interleukin‐3‐expressing NOD‐SCID IL2Rgamma(null) humanized mice . Blood 2011 ; 117 : 3076 – 3086 . Google Scholar Crossref Search ADS PubMed WorldCat 61 Covassin L , Laning J, Abdi R et al. Human peripheral blood CD4 T cell‐engrafted non‐obese diabetic‐scid IL2rgamma(null) H2‐Ab1 (tm1Gru) Tg (human leucocyte antigen D‐related 4) mice: A mouse model of human allogeneic graft‐versus‐host disease . Clin Exp Immunol 2011 ; 166 : 269 – 280 . Google Scholar Crossref Search ADS PubMed WorldCat 62 Takagi S , Saito Y, Hijikata A et al. Membrane‐bound human SCF/KL promotes in vivo human hematopoietic engraftment and myeloid differentiation . Blood 2012 ; 119 : 2768 – 2777 . Google Scholar Crossref Search ADS PubMed WorldCat 63 Brehm MA , Shultz LD, Luban J et al. Overcoming current limitations in humanized mouse research . J Infect Dis 2013 ; 208 ( suppl 2 ): S125 – 130 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 64 Lan P , Tonomura N, Shimizu A et al. Reconstitution of a functional human immune system in immunodeficient mice through combined human fetal thymus/liver and CD34+ cell transplantation . Blood 2006 ; 108 : 487 – 492 . Google Scholar Crossref Search ADS PubMed WorldCat 65 Melkus MW , Estes JD, Padgett‐Thomas A et al. Humanized mice mount specific adaptive and innate immune responses to EBV and TSST‐1 . Nat Med 2006 ; 12 : 1316 – 1322 . Google Scholar Crossref Search ADS PubMed WorldCat 66 Vatakis DN , Koya RC, Nixon CC et al. Antitumor activity from antigen‐specific CD8 T cells generated in vivo from genetically engineered human hematopoietic stem cells . Proc Natl Acad Sci USA 2011 ; 108 : E1408 – 1416 . Google Scholar Crossref Search ADS PubMed WorldCat 67 Vatakis DN , Bristol GC, Kim SG et al. Using the BLT humanized mouse as a stem cell based gene therapy tumor model . J Vis Exp 2012 : e4181 . Google Scholar OpenURL Placeholder Text WorldCat 68 Ren G , Zhao X, Wang Y et al. CCR2‐dependent recruitment of macrophages by tumor‐educated mesenchymal stromal cells promotes tumor development and is mimicked by TNFalpha . Cell Stem Cell 2012 ; 11 : 812 – 824 . Google Scholar Crossref Search ADS PubMed WorldCat 69 Addadi Y , Moskovits N, Granot D et al. p53 status in stromal fibroblasts modulates tumor growth in an SDF1‐dependent manner . Cancer Res 2010 ; 70 : 9650 – 9658 . Google Scholar Crossref Search ADS PubMed WorldCat 70 Loessner D , Holzapfel BM, Clements JA. Engineered microenvironments provide new insights into ovarian and prostate cancer progression and drug responses . Adv Drug Deliv Rev 2014 ; 79-80 : 193 – 213 . Google Scholar Crossref Search ADS PubMed WorldCat 71 Shiozawa Y , Pedersen EA, Havens AM et al. Human prostate cancer metastases target the hematopoietic stem cell niche to establish footholds in mouse bone marrow . J Clin Invest 2011 ; 121 : 1298 – 1312 . Google Scholar Crossref Search ADS PubMed WorldCat 72 Hanahan D , Weinberg RA. Hallmarks of cancer: The next generation . Cell 2011 ; 144 : 646 – 674 . Google Scholar Crossref Search ADS PubMed WorldCat 73 Hutmacher DW . Biomaterials offer cancer research the third dimension . Nat Mater 2010 ; 9 : 90 – 93 . Google Scholar Crossref Search ADS PubMed WorldCat 74 Clark AK , Taubenberger AV, Taylor RA et al. A bioengineered microenvironment to quantitatively measure the tumorigenic properties of cancer‐associated fibroblasts in human prostate cancer . Biomaterials 2013 ; 34 : 4777 – 4785 . Google Scholar Crossref Search ADS PubMed WorldCat 75 Kaemmerer E , Melchels FP, Holzapfel BM et al. Gelatine methacrylamide‐based hydrogels: An alternative three‐dimensional cancer cell culture system . Acta Biomater 2014 ; 10 : 2551 – 2562 . Google Scholar Crossref Search ADS PubMed WorldCat 76 Sieh S , Taubenberger AV, Rizzi SC et al. Phenotypic characterization of prostate cancer LNCaP cells cultured within a bioengineered microenvironment . PLoS One 2012 ; 7 : e40217 . Google Scholar Crossref Search ADS PubMed WorldCat 77 Sieh S , Taubenberger AV, Lehman ML et al. Paracrine interactions between LNCaP prostate cancer cells and bioengineered bone in 3D in vitro culture reflect molecular changes during bone metastasis . Bone 2014 ; 63 : 121 – 131 . Google Scholar Crossref Search ADS PubMed WorldCat 78 Taubenberger AV . In vitro microenvironments to study breast cancer bone colonisation . Adv Drug Deliv Rev 2014 ; 79-80C : 135 – 144 . Google Scholar OpenURL Placeholder Text WorldCat 79 Hirt C , Papadimitropoulos A, Mele V et al. “In vitro” 3D models of tumor‐immune system interaction . Adv Drug Deliv Rev 2014 ; 79-80 : 145 – 154 . Google Scholar Crossref Search ADS PubMed WorldCat 80 Feder‐Mengus C , Ghosh S, Weber WP et al. Multiple mechanisms underlie defective recognition of melanoma cells cultured in three‐dimensional architectures by antigen‐specific cytotoxic T lymphocytes . Br J Cancer 2007 ; 96 : 1072 – 1082 . Google Scholar Crossref Search ADS PubMed WorldCat 81 Fischer K , Hoffmann P, Voelkl S et al. Inhibitory effect of tumor cell‐derived lactic acid on human T cells . Blood 2007 ; 109 : 3812 – 3819 . Google Scholar Crossref Search ADS PubMed WorldCat 82 Ghosh S , Rosenthal R, Zajac P et al. Culture of melanoma cells in 3‐dimensional architectures results in impaired immunorecognition by cytotoxic T lymphocytes specific for Melan‐A/MART‐1 tumor‐associated antigen . Ann Surg 2005 ; 242 : 851 – 857, discussion 858 . Google Scholar Crossref Search ADS PubMed WorldCat 83 Dangles‐Marie V , Richon S, El‐Behi M et al. A three‐dimensional tumor cell defect in activating autologous CTLs is associated with inefficient antigen presentation correlated with heat shock protein‐70 down‐regulation . Cancer Res 2003 ; 63 : 3682 – 3687 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 84 Gottfried E , Kunz‐Schughart LA, Ebner S et al. Tumor‐derived lactic acid modulates dendritic cell activation and antigen expression . Blood 2006 ; 107 : 2013 – 2021 . Google Scholar Crossref Search ADS PubMed WorldCat 85 Ranga A , Gobaa S, Okawa Y et al. 3D niche microarrays for systems‐level analyses of cell fate . Nat Commun 2014 ; 5 : 4324 . Google Scholar Crossref Search ADS PubMed WorldCat 86 Hesami P , Holzapfel B, Taubenberger A et al. A humanized tissue‐engineered in vivo model to dissect interactions between human prostate cancer cells and human bone . Clin Exp Metastasis 2014 : 1 – 12 . Google Scholar OpenURL Placeholder Text WorldCat 87 Holzapfel BM , Wagner F, Loessner D et al. Species‐specific homing mechanisms of human prostate cancer metastasis in tissue engineered bone . Biomaterials 2014 ; 35 : 4108 – 4115 . Google Scholar Crossref Search ADS PubMed WorldCat 88 Thibaudeau L , Taubenberger AV, Holzapfel BM et al. A tissue‐engineered humanized xenograft model of human breast cancer metastasis to bone . Dis Model Mech 2014 ; 7 : 299 – 309 . Google Scholar Crossref Search ADS PubMed WorldCat 89 Parmar H , Cunha GR. Epithelial‐stromal interactions in the mouse and human mammary gland in vivo . Endocr Relat Cancer 2004 ; 11 : 437 – 458 . Google Scholar Crossref Search ADS PubMed WorldCat 90 Nicolini FE , Cashman JD, Hogge DE et al. NOD/SCID mice engineered to express human IL‐3, GM‐CSF and Steel factor constitutively mobilize engrafted human progenitors and compromise human stem cell regeneration . Leukemia 2004 ; 18 : 341 – 347 . Google Scholar Crossref Search ADS PubMed WorldCat 91 Lee J , Li M, Milwid J et al. Implantable microenvironments to attract hematopoietic stem/cancer cells . Proc Natl Acad Sci USA 2012 ; 109 : 19638 – 19643 . Google Scholar Crossref Search ADS PubMed WorldCat 92 Groen RW , Noort WA, Raymakers RA et al. Reconstructing the human hematopoietic niche in immunodeficient mice: Opportunities for studying primary multiple myeloma . Blood 2012 ; 120 : e9 – e16 . Google Scholar Crossref Search ADS PubMed WorldCat Author notes The copyright line for this article was changed on September 21 after original online publication © 2015 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Concise Review: Humanized Models of Tumor Immunology in the 21st Century: Convergence of Cancer Research and Tissue Engineering
JF - Stem Cells
DO - 10.1002/stem.1978
DA - 2015-06-01
UR - https://www.deepdyve.com/lp/oxford-university-press/concise-review-humanized-models-of-tumor-immunology-in-the-21st-ANb8FIOFtP
SP - 1696
EP - 1704
VL - 33
IS - 6
DP - DeepDyve
ER -