Patient-derived xenografts, the cancer stem cell paradigm, and cancer pathobiology in the 21st century.

Cancer is a heterogeneous disease manifest in many forms. Tumor histopathology can differ significantly among patients and cellular heterogeneity within tumors is common. A primary goal of cancer biologists is to better understand tumorigenesis and cancer progression; however, the complex nature of tumors has posed a substantial challenge to unlocking cancer's secrets. The cancer stem cell (CSC) paradigm for the pathobiology of solid tumors appropriately acknowledges phenotypic and functional tumor cell heterogeneity observed in solid tumors and accounts for the disconnect between drug approval based on response and the general inability of approved therapies to meaningfully impact survival due to their failure to eradicate these most important of cellular targets. First proposed to exist decades ago, CSC have only recently begun to be precisely identified due to technical advancements that facilitate identification, isolation, and interrogation of distinct tumor cell subpopulations with differing ability to form and perpetuate tumors. Precise identification of CSC populations and the complete hierarchy of cells within solid tumors will facilitate more accurate characterization of patient subtypes and ultimately contribute to more personalized and effective therapies. Rapid advancement in the understanding of tumor biology as it exists in patients requires cooperation among institutions, surgeons, pathologists, cancer biologists and patients alike, primarily because this translational research is best done with patient-derived tissue grown in the xenograft setting as patient-derived xenografts. This review calls for a broader change in the approaches taken to study cancer pathobiology, highlights what implications the CSC paradigm has for pathologists and cancer biologists alike, and calls for greater collaboration between institutions, physicians and scientists in order to more rapidly advance our collective understanding of cancer.

In vivo expansion of co-transplanted T cells impacts on tumor re-initiating activity of human acute myeloid leukemia in NSG mice.

Human cells from acute myeloid leukemia (AML) patients are frequently transplanted into immune-compromised mouse strains to provide an in vivo environment for studies on the biology of the disease. Since frequencies of leukemia re-initiating cells are low and a unique cell surface phenotype that includes all tumor re-initiating activity remains unknown, the underlying mechanisms leading to limitations in the xenotransplantation assay need to be understood and overcome to obtain robust engraftment of AML-containing samples. We report here that in the NSG xenotransplantation assay, the large majority of mononucleated cells from patients with AML fail to establish a reproducible myeloid engraftment despite high donor chimerism. Instead, donor-derived cells mainly consist of polyclonal disease-unrelated expanded co-transplanted human T lymphocytes that induce xenogeneic graft versus host disease and mask the engraftment of human AML in mice. Engraftment of mainly myeloid cell types can be enforced by the prevention of T cell expansion through the depletion of lymphocytes from the graft prior transplantation.

Knockdown of HMGA1 inhibits human breast cancer cell growth and metastasis in immunodeficient mice.

The high mobility group A1 gene (HMGA1) has been previously implicated in breast carcinogenesis, and is considered an attractive target for therapeutic intervention because its expression is virtually absent in normal adult tissue. Other studies have shown that knockdown of HMGA1 reduces the tumorigenic potential of breast cancer cells in vitro. Therefore, we sought to determine if silencing HMGA1 can affect breast cancer development and metastatic progression in vivo. We silenced HMGA1 expression in the human breast cancer cell line MDA-MB-231 using an RNA interference vector, and observed a significant reduction in anchorage-independent growth and tumorsphere formation, which respectively indicate loss of tumorigenesis and self-renewal ability. Moreover, silencing HMGA1 significantly impaired xenograft growth in immunodeficient mice, and while control cells metastasized extensively to the lungs and lymph nodes, HMGA1-silenced cells generated only a few small metastases. Thus, our results show that interfering with HMGA1 expression reduces the tumorigenic and metastatic potential of breast cancer cells in vivo, and lend further support to investigations into targeting HMGA1 as a potential treatment for breast cancer.

Chronic stress promoted the growth of ovarian carcinoma via increasing serum levels of norepinephrine and interleukin-10 and altering nm23 and NDRG1 expression in tumor tissues in nude mice.

The current study aimed to examine the effects and underlying mechanisms of chronic psychological stress on the growth of ovarian carcinoma. Human ovarian carcinoma cells SKOV-3 were subcutaneously inoculated into nude mice to establish an ectopic mouse model. The animals were experimentally stressed 6 h daily for a total of 42 days with a physical restraint system. We examined the effects of stress on the growth of tumor cells that were inoculated 7 days after the initiation of stress. The growth of SKOV-3 xenografts in the stress group showed a more rapid trend than that in the control. The mean weight of tumors that were removed at the end of the experiment increased by 71.7% in the stress group as compared to the control. In order to explore the underlying mechanisms, we first determined the serum levels of norepinephrine (NE) and interleukin 10 (IL-10) in the mice using an enzyme-linked immunoabsorbent assay (ELISA) and then analyzed protein expression profiles of SKOV-3 xenografts using a proteomics-based approach combining two-dimensional electrophoresis with ultra performance liquid chromatography-electrospray tandem mass spectrometry (nanoUPLC-ESI-MS/MS). Results demonstrated that serum levels of NE and IL-10 were obviously increased in the mice receiving 6 h of immobilization daily for 42 days. In xenografts exposed to stress, a tumor promoting protein nm23 was significantly upregulated while a tumor suppressing protein NDRG1 was obviously downregulated, which were confirmed by subsequent Western blot analysis. Results obtained in the current study suggested that chronic stress promoted the growth of ovarian carcinoma in nude mice through increasing serum levels of NE and IL-10 and altering nm23 and NDRG1 expression in tumor tissues.

Pharmacogenomic considerations of xenograft mouse models of acute leukemia.

The use of combination chemotherapy to cure acute lymphoblastic leukemia in children and acute myeloid leukemia in adults emerged for acute myeloid leukemia in the 1960s and for acute lymphoblastic leukemia in the 1980s as a paradigm for curing any disseminated cancer. This article summarizes recent developments and considerations in the use of acute leukemia xenografts established in immunodeficient mice to elucidate the genetic and genomic basis of acute leukemia pathogenesis and treatment response.

Synthetic lethality of PARP and NAMPT inhibition in triple-negative breast cancer cells.

PARP inhibitors have been proposed as a potential targeted therapy for patients with triple-negative (ER-, PR-, HER2-negative) breast cancers. However, it is as yet unclear as to whether single agent or combination therapy using PARP inhibitors would be most beneficial. To better understand the mechanisms that determine the response to PARP inhibitors, we investigated whether enzymes involved in metabolism of the PARP substrate, ß-NAD(+) , might alter the response to a clinical PARP inhibitor. Using an olaparib sensitization screen in a triple-negative (TN) breast cancer model, we identified nicotinamide phosphoribosyltransferase (NAMPT) as a non-redundant modifier of olaparib response. NAMPT is a rate-limiting enzyme involved in the generation of the PARP substrate ß-NAD(+) and the suppression of ß-NAD(+) levels by NAMPT inhibition most likely explains these observations. Importantly, the combination of a NAMPT small molecule inhibitor, FK866, with olaparib inhibited TN breast tumour growth in vivo to a greater extent than either single agent alone suggesting that assessing NAMPT/PARP inhibitor combinations for the treatment of TN breast cancer may be warranted.

Costimulation blockade in pig artery patch xenotransplantation - a simple model to monitor the adaptive immune response in nonhuman primates.

BACKGROUND: CD154 blockade-based immunosuppression successfully prevents both humoral and cellular adaptive immune responses in baboons receiving α1,3-galactosyltransferase gene-knockout (GTKO) pig organs. Using a GTKO pig artery transplantation model in baboons, we evaluated the efficacy of CD28/B7 costimulatory pathway blockade in comparison with CD154 blockade. METHODS: Baboons received artery patch grafts from GTKO pigs, with no (Group1), anti-CD154mAb-based (Group2), or CTLA4-Ig-based (Group3) immunosuppressive therapy. Anti-pig IgM and IgG antibody and cellular responses were monitored. Xenografts were immunohistologically evaluated for antibody and complement deposition, and cellular infiltration. RESULTS: Group1 baboons developed increased IgM and IgG antibody and cellular responses against GTKO antigens. In Group2, anti-CD154mAb alone prevented the development of both IgM and IgG antibody and cellular responses,but not cellular infiltration of the graft. In the single baboon that received anti-thymocyte globulin (ATG) + mycophenolate mofetil (MMF) + anti-CD154mAb, cellular infiltration of the graft was not seen. In Group3, CTLA4-Ig with ATG + MMF inhibited the cellular proliferative response to pig antigens but did not prevent the IgG response or cellular infiltration. CONCLUSIONS: (i) Artery patch transplantation is a simple model to monitor the adaptive immune response to xenografts; (ii) anti-CD154mAb prevents sensitization but not cellular infiltration (but, without anticoagulation, may result in early thrombosis of a pig xenograft); (iii) although in only one baboon, the addition of ATG and MMF prevents cellular infiltration and (iv) replacement of anti-CD154mAb by CTLA4-Ig (at the doses used), even in combination with ATG and MMF, prevents the cellular proliferative response to GTKO pig antigens but is insufficient to prevent the development of anti-pig antibodies.

Synergistic suppression of pre-perfusion of donor livers with recipient serum and cobra venom factor treatment on hyperacute rejection following liver xenotransplantation.

PURPOSE: To investigate synergistic suppression of donor liver pre-perfusion with recipient serum (RS) and cobra venom factor (CVF) treatment on hyperacute rejection (HAR) following liver xenotransplantation. METHODS: Guinea-pigs (GP, n=24) and Sprague-Dawley rats (SD, n=24) were recruited. Before transplantation, serum was collected from SD rats and used for preparation of inactivated complements. GP and SD rats were randomly assigned into four groups (n=6), respectively: RS group, CVF group, RS+CVF group and control group. Orthotopic liver xenotransplantation was performed with modified two-cuff technique. The survival time and liver function of recipients, morphological and pathological changes in rat livers were investigated. RESULTS: There was no piebald like change in the recipient livers in all experiment groups. The survival time of recipients in all experiment groups was longer than that in control group (p<0.05). Moreover, the survival time in the RS+CVF group was markedly longer than that in the RS group (p<0.01) and CVF group (p<0.05). The serum ALT level in all experiment groups were lower than that in the control group (p<0.05). Furthermore, the ALT level in the RS+CVF group was significantly lower than that in the CVF group (p<0.05) and RS group (p<0.01). The histological damages were significantly improved when compared with the control group, and the histological damages in the RS+CVF group were milder than those in the remaining groups (p<0.05) CONCLUSION: Pre-perfusion of donor liver with recipient serum and cobra venom factor treatment can exert synergistic suppressive effects on the hyperacute rejection following liver xenotransplantation.

Synergistic suppression of pre-perfusion of donor livers with recipient serum and cobra venom factor treatment on hyperacute rejection following liver xenotransplantation / Supressão sinérgica da rejeição hiperaguda no xenotransplante hepático com uso da pre-perfusão dos fígados doadores tratados com soro do receptor e com fator veneno de cobra

PURPOSE: To investigate synergistic suppression of donor liver pre-perfusion with recipient serum (RS) and cobra venom factor (CVF) treatment on hyperacute rejection (HAR) following liver xenotransplantation. METHODS: Guinea-pigs (GP, n=24) and Sprague-Dawley rats (SD, n=24) were recruited. Before transplantation, serum was collected from SD rats and used for preparation of inactivated complements. GP and SD rats were randomly assigned into four groups (n=6), respectively: RS group, CVF group, RS+CVF group and control group. Orthotopic liver xenotransplantation was performed with modified two-cuff technique. The survival time and liver function of recipients, morphological and pathological changes in rat livers were investigated. RESULTS: There was no piebald like change in the recipient livers in all experiment groups. The survival time of recipients in all experiment groups was longer than that in control group (p<0.05). Moreover, the survival time in the RS+CVF group was markedly longer than that in the RS group (p<0.01) and CVF group (p<0.05). The serum ALT level in all experiment groups were lower than that in the control group (p<0.05). Furthermore, the ALT level in the RS+CVF group was significantly lower than that in the CVF group (p<0.05) and RS group (p<0.01). The histological damages were significantly improved when compared with the control group, and the histological damages in the RS+CVF group were milder than those in the remaining groups (p<0.05) CONCLUSION: Pre-perfusion of donor liver with recipient serum and cobra venom factor treatment can exert synergistic suppressive effects on the hyperacute rejection following liver xenotransplantation.

OBJETIVO: Investigar a supressão sinérgica da pré-perfusão do doador de fígado com soro do receptor (SR) e tratamento com fator veneno de cobra (FVC) na rejeição hiperaguda (RHA) após o xenotransplante de fígado. MÉTODOS: Foram utilizados Cobaias (GP, n=24) e ratos Sprague-Dawley (SD, n=24). Antes do transplante foram coletadas amostras de soro dos ratos SD e usados para a preparação dos complementos inativados. Cobaias GP e ratos SD foram randomicamente distribuídos em quatro grupos (n=6), respectivamente: grupo RS, grupo FVC, grupo SR+FVC e grupo controle. Xenotransplante ortotópico do fígado foi realizado com a técnica de dois cuffs modificados. Foram investigados o de tempo de sobrevida, a função hepática dos receptores e alterações morfopatológicas em fígados de ratos. RESULTADOS: Não houve alteração na coloração do parênquima dos fígados nos receptores. O tempo de sobrevida dos receptores em todos os grupos experimentais foi mais longo do que o grupo controle (p<0,05). Além disso, o tempo de sobrevida do grupo SR+ FVC foi marcadamente maior do que o grupo SR (p<0,01) e o grupo FVC (p<0,05). O nível sérico ALT foi menor em todos os grupos experimentais do que o grupo controle (p<0,05). O nível de ALT no grupo SR+ FVC foi significantemente menor do que no grupo FVC (p<0,05) e o grupo SR (p<0,01). As alterações histológicas foram significantemente melhoradas quando comparado com o grupo controle, e os danos histológicos no grupo SR+ FVC foram mais moderados do que nos grupos restantes (p<0,05). CONCLUSÃO: Pré-perfusão do fígado doador com soro do receptor e fator veneno de cobra pode exercer efeito supressor sinérgico da rejeição hiperaguda após xenotransplante de fígado.

VHL-regulated MiR-204 suppresses tumor growth through inhibition of LC3B-mediated autophagy in renal clear cell carcinoma.

The von Hippel-Lindau tumor-suppressor gene (VHL) is lost in most clear cell renal cell carcinomas (ccRCC). Here, using human ccRCC specimens, VHL-deficient cells, and xenograft models, we show that miR-204 is a VHL-regulated tumor suppressor acting by inhibiting macroautophagy, with MAP1LC3B (LC3B) as a direct and functional target. Of note, higher tumor grade of human ccRCC was correlated with a concomitant decrease in miR-204 and increase in LC3B levels, indicating that LC3B-mediated macroautophagy is necessary for RCC progression. VHL, in addition to inducing endogenous miR-204, triggered the expression of LC3C, an HIF-regulated LC3B paralog, that suppressed tumor growth. These data reveal a function of VHL as a tumor-suppressing regulator of autophagic programs.

Real-time visualization and quantitation of vascular permeability in vivo: implications for drug delivery.

The leaky, heterogeneous vasculature of human tumors prevents the even distribution of systemic drugs within cancer tissues. However, techniques for studying vascular delivery systems in vivo often require complex mammalian models and time-consuming, surgical protocols. The developing chicken embryo is a well-established model for human cancer that is easily accessible for tumor imaging. To assess this model for the in vivo analysis of tumor permeability, human tumors were grown on the chorioallantoic membrane (CAM), a thin vascular membrane which overlays the growing chick embryo. The real-time movement of small fluorescent dextrans through the tumor vasculature and surrounding tissues were used to measure vascular leak within tumor xenografts. Dextran extravasation within tumor sites was selectively enhanced an interleukin-2 (IL-2) peptide fragment or vascular endothelial growth factor (VEGF). VEGF treatment increased vascular leak in the tumor core relative to surrounding normal tissue and increased doxorubicin uptake in human tumor xenografts. This new system easily visualizes vascular permeability changes in vivo and suggests that vascular permeability may be manipulated to improve chemotherapeutic targeting to tumors.

Effect of cryopreservation and transplantation on the expression of kit ligand and anti-Mullerian hormone in human ovarian tissue.

BACKGROUND: Although cryopreservation and transplantation of ovarian tissue represent a promising alternative to safeguard fertility in cancer patients, low recovery rates of oocytes aspirated from antral follicles and a significant number of empty follicles have been observed in women with transplanted frozen-thawed ovarian tissue. In order to understand how freezing and/or grafting may affect follicular development, the follicular expression of kit ligand (KL) and anti-Müllerian hormone (AMH), two key factors activating and inhibiting follicle growth, were assessed after long-term grafting in severe combined immunodeficient (SCID) mice. METHODS: Ovarian biopsies from eight patients were used for fresh and frozen-thawed tissue xenografting in 13 SCID mice for a period of 28 weeks, including 2 weeks of gonadotrophin stimulation. KL, AMH and proliferating cell nuclear antigen (PCNA) immunostaining were quantified before and after grafting in the two treatment groups (fresh and frozen-thawed grafted ovarian tissue). RESULTS: Lower expression of KL was found in primordial and primary follicles after grafting of both fresh and frozen-thawed tissue. Consistent expression of AMH was found in most growing follicles at a similar rate in both graft types. In fresh and frozen-thawed grafts, 13-14% of primordial follicles were PCNA-positive, indicating a similar maintenance of quiescent follicles despite follicle activation. CONCLUSIONS: Grafting and/or gonadotrophin stimulation appear to affect the follicular expression of KL, which may alter oocyte quality. AMH expression in growing follicles after ovarian tissue transplantation may be one of the factors contributing to the preservation of resting follicles in 28-week-old grafts.

Subcutaneous passage increases cell aggressiveness in a xenograft model of diffuse large B cell lymphoma.

Xenograft models of human diffuse large B cell lymphoma (DLBCL) are widely used to test new drugs against this neoplasia. Most of them, however, are subcutaneous xenografts that do not show a disseminated disease as it is found in the human neoplasia. In this paper, we aimed to develop a disseminated xenograft model of DLBCL by performing a subcutaneous passage of DLBCL cells before their intravenous injection in mice. WSU-DLCL-2 (WSU) cells were injected into both flanks of NOD/SCID mice. The subcutaneous tumours were disaggregated and a cell suspension (WSU-SC) was obtained. Two groups of 10 NOD/SCID mice were intravenously injected with WSU-SC or WSU cells. All mice injected with WSU-SC cells developed lymphoma in 32-47 days and showed lymph node and bone marrow infiltration. WSU-SC cells showed a significantly higher engraftment rate and faster dissemination than WSU cells after intravenous injection in mice. When molecularly compared, WSU-SC cells showed higher expression levels of FAK, p130Cas and phosphorylated AKT than WSU cells. The subcutaneous passage enhanced the engraftment and the metastatic capacity of WSU cells, allowing the generation of a rapid and disseminated DLBCL xenograft model. The aggressive behaviour of WSU-SC cells was associated with increased p130Cas and FAK expression and AKT activation.

Immune response to bovine pericardium implanted into α1,3-galactosyltransferase knockout mice: feasibility as an animal model for testing efficacy of anticalcification treatments of xenografts.

OBJECTIVES: Glutaraldehyde (GA)-fixed xenografts are prone to calcification after implantation in humans and there is evidence that immune reaction to the Galα1,3-Galß1,4GlcNAc-R (α-Gal) antigen may play a part in this process. The objectives of this study were to evaluate the immune response of α1,3-galactosyltransferase knockout (α-Gal KO) mice to bovine pericardium and to evaluate the effect of various anticalcification treatments on bovine pericardium using mouse subcutaneous implantation model. METHODS: Bovine pericardial tissues were divided into eight groups according to the method of anticalcification treatments. Prepared tissues were subcutaneously implanted into the α-Gal KO and wild-type mice for 2 months, and anti-α-Gal antibodies were measured at 2 weeks and 2 months after implantation. Explanted tissues were examined by immunohistochemistry and calcium contents of the explanted tissues were measured. RESULTS: Titres of IgM and IgG antibodies in the α-Gal KO mice increased significantly according to the duration of implantation, whereas titres of IgM and IgG antibodies in the wild-type mice increased until 2 weeks after implantation without further increase thereafter. Titres of IgG antibodies measured at 2 months after implantation were significantly higher in the α-Gal KO mice than in the wild-type mice. Immunohistochemistry revealed macrophages surrounding the pericardial tissues irrespective of the mouse type into which the tissues implanted, whereas T-cells could only be observed in the tissues implanted into the α-Gal KO mice. Except the high-concentration GA-treated group, calcium contents of anticalcification-treated groups were all significantly lower or tended to be lower than that of the control group, irrespective of the mouse type. Calcium contents of the control group were significantly higher in the α-Gal KO mice than in the wild-type mice. CONCLUSIONS: Bovine pericardium implanted into the α-Gal KO mice caused significant increase in anti-α-Gal antibodies, showed some histologic evidences of chronic rejection and revealed a potential toward more calcification. These findings suggest a possible role of immune response in calcification of xenografts. High-concentration GA fixation alone did not prove to be an effective anticalcification treatment in mouse subcutaneous implantation model. α-Gal KO mouse subcutaneous implantation model might be a feasible animal model for testing efficacy of anticalcification treatments incorporating immunologic approach.

Genetically-engineered pig-to-baboon liver xenotransplantation: histopathology of xenografts and native organs.

Orthotopic liver transplantation was carried out in baboons using wild-type (WT, n = 1) or genetically-engineered pigs (α1,3-galactosyltransferase gene-knockout, GTKO), n = 1; GTKO pigs transgenic for human CD46, n = 7) and a clinically-acceptable immunosuppressive regimen. Biopsies were obtained from the WT pig liver pre-Tx and at 30 min, 1, 2, 3, 4 and 5 h post-transplantation. Biopsies of genetically-engineered livers were obtained pre-Tx, 2 h after reperfusion and at necropsy (4-7 days after transplantation). Tissues were examined by light, confocal, and electron microscopy. All major native organs were also examined. The WT pig liver underwent hyperacute rejection. After genetically-engineered pig liver transplantation, hyperacute rejection did not occur. Survival was limited to 4-7 days due to repeated spontaneous bleeding in the liver and native organs (as a result of profound thrombocytopenia) which necessitated euthanasia. At 2 h, graft histology was largely normal. At necropsy, genetically-engineered pig livers showed hemorrhagic necrosis, platelet aggregation, platelet-fibrin thrombi, monocyte/macrophage margination mainly in liver sinusoids, and vascular endothelial cell hypertrophy, confirmed by confocal and electron microscopy. Immunohistochemistry showed minimal deposition of IgM, and almost absence of IgG, C3, C4d, C5b-9, and of a cellular infiltrate, suggesting that neither antibody- nor cell-mediated rejection played a major role.

Mapping of homozygous deletions in verified esophageal adenocarcinoma cell lines and xenografts.

Human esophageal adenocarcinoma (EAC) cell lines and xenografts are powerful tools in the search for genetic alterations because these models are composed of pure human cancer cell populations without admixture of normal human cells. In particular detection of homozygous deletions (HDs) is easier using these pure populations of cancer cells. Identification of HDs could potentially lead to the subsequent identification of new tumor suppressor genes (TSGs) involved in esophageal adenocarcinogenesis. Genome wide single nucleotide polymorphism (SNP) arrays were used to identify HDs in 10 verified EAC cell lines and nine EAC xenografts. In total, 61 HDs (range 1-6 per sample) were detected and confirmed by polymerase chain reaction. Besides HDs observed in common fragile genomic regions (n = 26), and gene deserts (n = 8), 27 HDs were located in gene-containing regions. HDs were noted for known TSGs, including CDKN2A, SMAD4 and CDH3/CDH1. Twenty-two new chromosomal regions were detected harboring potentially new TSGs involved in EAC carcinogenesis. Two of these regions of homozygous loss, encompassing the ITGAV and RUNX1 gene, were detected in multiple samples indicating a potential role in the carcinogenesis of EAC. To exclude culturing artifacts, these last two deletions were confirmed by fluorescent in situ hybridization in the primary tumors of which the involved cell lines and xenografts were derived. In summary, in this report we describe the identification of HDs in a series of verified EAC cell lines and xenografts. The deletions documented here are a step forward identifying the key genes involved in EAC development.

The inhibition of Endostar on the angiogenesis and growth of gastrointestinal stromal tumor xenograft.

To investigate whether Endostar can inhibit the angiogenesis and growth of gastrointestinal stromal tumor (GIST) xenografts in nude mice and the feasibility of antiangiogenesis as a treatment modality for GIST. Twenty Balb/c-nu/nu mice burdened with GIST were randomly divided into two groups. Endostar (2 mg/kg) was injected around the tumor once per day for 10 days in the experimental group and with normal saline (NS) (0.1 ml) in the control group. The tumor bulk was measured every 5 days until 5 days after the end of the injections. The inhibition tumor rate (ITR) was calculated. Tumor bulk, microvascular density (MVD), rate of bcl-2-positive expression, and AI were assessed in the two groups. Tumor volumes were compared before and after treatment in the experimental group. The difference in tumor bulk between the two groups was not statistically significant before treatment (P = 0.628), but at the end of test, the difference was significant (P < 0.0001), and in the test group, the tumor bulk was also decreased significantly after treatment (P < 0.0001). The ITR was 86.5%. All xenografts showed CD117-positive staining. MVD and bcl-2-positive rate were lower in the experimental group than in the control group (P = 0.020 and P = 0.023, respectively). AI increased significantly in the experimental group compared with the control group (P = 0.020). Endostar can reduce angiogenesis,promote cell apoptosis, and inhibit the growth of a GIST xenograft. It is possible that Endostar will be used as an effective drug for GIST in the future.

Thrombocytopenia after pig-to-baboon liver xenotransplantation: where do platelets go?

BACKGROUND: In baboons with orthotopic pig liver xenografts, profound thrombocytopenia was observed within 1 h after reperfusion. Assessment of the fate of platelets may shed light on the underlying mechanisms leading to thrombocytopenia and may allow preventive therapies to be introduced. METHODS: Platelet-white blood cell (WBC) aggregation was studied in two baboons that received orthotopic liver xenografts from α1,3-galactosyltransferase gene-knockout pigs transgenic for human CD46 (GTKO/CD46). Percentages of CD42a-positive platelet aggregates with WBC-subtypes were determined by flow cytometry, and absolute numbers (per mm(3) ) were calculated. Platelet aggregates in the liver xenografts were identified by immunofluorescence and electron microscopy. Mean platelet volume (MPV) was determined before and after transplantation. RESULTS: After pig liver reperfusion, profound thrombocytopenia was associated with aggregation of platelets with WBC-subtypes. Increasing aggregation of platelets with WBC-subtypes was detected throughout the post-transplant period until the recipient was euthanized. Significant negative correlation was found between platelet counts in the blood and aggregation of platelets with monocytes (P < 0.01) and neutrophils (P < 0.01), but not with lymphocytes. MPV remained within the normal range. Two hours after reperfusion, platelet and fibrin deposition were already detected in the liver xenografts by immunofluorescence and by electron microscopy. CONCLUSIONS: Following liver xenotransplantation, the early disappearance of platelets from the circulation was at least in part due to their aggregation with circulating WBC, which may augment their deposition in the liver xenograft and native lungs. Prevention of platelet aggregation with monocytes and neutrophils is likely beneficial in reducing their subsequent sequestration in the liver xenograft and native organs.

Xenoreactive CD4+ memory T cells resist inhibition by anti-CD44 mAb and reject islet grafts via a Th2-dependent pathway.

BACKGROUND: Memory T cells are a significant barrier to the induction of transplant tolerance. Our previous study demonstrated that multiple applications of anti-CD44 monoclonal antibody (mAb) could significantly inhibit CD4(+) memory T cells from mediating rejection of cardiac allografts. Now, we sought to explore the effect and mechanism of anti-CD44 mAb on the rejection of islet allografts and xenografts mediated by CD4(+) memory T cells. METHODS: In this study, we first engrafted skin grafts of C57BL/6 (B6) mice or Dark Agouti (DA) rats onto BALB/c mice to induce donor-reactive memory T cells. We adoptively transferred purified CD4(+) memory T cells to BALB/c origin nude mice and then transplanted islet allografts and xenografts to produce the Allo-Tx and Xeno-Tx models, respectively. We subsequently administered multiple anti-CD44 mAb and observed changes in the survival times of the islet grafts. RESULTS: In the Allo-Tx model, the mean survival time (MST) of the grafts was 7.7 days in the isotype group, and 20.3 days in the anti-CD44 group. In the Xeno-Tx model, the MST of the grafts was 7.2 days in the isotype group and 8.2 days in the anti-CD44 group. Compared with the isotype group, CD4(+) T cells on the grafts in the anti-CD44 group were significantly decreased in both the Allo-Tx and Xeno-Tx models, but the proportion of CD4(+) memory T cells in the spleens and draining lymph nodes of the recipient nude mice in the anti-CD44 group was significantly decreased in the Allo-Tx model, while it was increased in the Xeno-Tx model. The production of donor-specific IgG antibody in the anti-CD44 group did not vary in the Allo-Tx model, while it was markedly elevated in the Xeno-Tx model. Furthermore, the expression of interferon gamma in the anti-CD44 group was markedly decreased in both the Allo-Tx and Xeno-Tx models, while the expression of IL-4 in the anti-CD44 group was significantly increased only in the Xeno-Tx model. CONCLUSION: Multiple applications of the anti-CD44 mAb could significantly inhibit donor-reactive CD4(+) memory T cells from rejecting grafts via a Th1-dependent pathway, but xenoreactive CD4(+) memory T cells can avoid the effects of anti-CD44 mAb to reject islet xenografts via a Th2-dependent pathway.

Transplanted neurally modified bone marrow-derived mesenchymal stem cells promote tissue protection and locomotor recovery in spinal cord injured rats.

BACKGROUND: Stem cell-based therapy for repair and replacement of lost neural cells is a promising treatment for central nervous system (CNS) diseases. Bone marrow (BM)-derived mesenchymal stem cells (MSCs) can differentiate into neural phenotypes and be isolated and expanded for autotransplantation with no risk of rejection. OBJECTIVE: The authors examined whether transplanted neurally induced human MSCs (NI hMSCs), developed by a new procedure, can survive, differentiate, and promote tissue protection and functional recovery in injured spinal cord (ISC) rats. METHODS: Neural induction was achieved by exposing cells simultaneously to inhibitors of DNA methylation, histone deacetylation, and pharmacological agents that increased cAMP levels. Three groups of adult female Sprague-Dawley rats were injected immediately rostral and caudal to the midline lesion with phosphate-buffered saline, MSCs, or NI hMSCs, 1 week after a spinal cord impact injury at T-8. Functional outcome was measured using the Basso Beattie Bresnahan (BBB) locomotor rating scale and thermal sensitivity test on a weekly basis up to 12 weeks postinjury. Graft integration and anatomy of spinal cord was assessed by stereological, histochemical, and immunohistochemical techniques. RESULTS: The transplanted NI hMSCs survived, differentiated, and significantly improved locomotor recovery of ISC rats. Transplantation also reduced the volume of lesion cavity and white matter loss. CONCLUSION: This method of hMSC modification may provide an alternative source of autologous adult stem cells for CNS repair.