A phenotype of IGFBP-3 knockout mice revealed by dextran sulfate-induced colitis.

BACKGROUND AND AIM: Insulin-like growth factor-1 (IGF-1) bioactivity has been shown to be attenuated by insulin-like growth factor binding protein-3 (IGFBP-3), one of six IGF-binding proteins. While prior work revealed no major phenotype associated with IGFBP-3 knockout mice, we explored the possibility that a phenotype could be revealed under specific conditions of gastrointestinal stress. METHODS: The dextran sodium sulfate (DSS) murine model of ulcerative colitis was used for this study. RESULTS: Insulin-like growth factor binding protein-3 knockout mice had significantly reduced colitis on exposure to DSS as measured by lower levels of pro-inflammatory cytokines IL-6 (P < 0.0001), TNF-α (P = 0.0035), and IL-1ß (P = 0.0112), reduced weight loss (P < 0.0001), reduced myeloperoxidase activity (P = 0.0025), and maintenance of colorectal length (P < 0.05), all relative to wild-type mice exposed to DSS. IGFBP-3 knockout mice also exhibited increased colon epithelial cell proliferation (P < 0.0001) following DSS exposure. Semi-quantitative immunohistochemistry showed greater IGF-1 receptor activation in colon epithelial cells of IGFBP-3 knockout mice compared with control mice following DSS exposure. CONCLUSION: Our data demonstrate that IGFBP-3 influences severity of DSS-induced colitis. The observations suggest that in the absence of IGFBP-3, enhanced IGF bioactivity leads to increased epithelial proliferation and mucosal barrier repair, thereby lessening inflammation.

Germ line knockout of IGFBP-3 reveals influences of the gene on mammary gland neoplasia.

Insulin-like growth factor binding protein-3 (IGFBP-3) is an important carrier protein for insulin-like growth factors (IGFs) in the circulation. IGFBP-3 antagonizes the growth-promoting and anti-apoptotic activities of IGFs in experimental systems, but in certain contexts can increase IGF bioactivity, probably by increasing its half-life. The goal of this study was to investigate the role of IGFBP-3 in breast carcinogenesis and breast cancer metastasis. In the first part of the study, we exposed IGFBP-3 knockout and wild-type female mice to dimethylbenz[a]anthracene (DMBA) and followed them for appearance of primary tumors for up to 13 months. In the second part, mice of each genotype received an IV injection of 4T1 mammary carcinoma cells and then lung nodules were counted. Our results show that IGFBP-3 knockout mice developed breast tumors significantly earlier than the wild-type (13.9 ± 1.1 versus 22.5 ± 3.3 weeks, respectively, P = 0.0144), suggesting tumor suppression activity of IGFBP-3. In tumors of IGFBP-3 knockout mice, levels of phospho-AKT(Ser473) were increased compared to wild-type mice. The lung metastasis assay showed significantly more and larger lung nodules in IGFBP-3 knockout mice than in wild-type mice. While we observed increased levels of IGFBP-5 protein in the IGFBP-3 knockout mice, our findings suggest that this was not sufficient to completely compensate for the absence of IGFBP-3. Even though knockout of IGFBP-3 is associated with only a subtle phenotype under control conditions, our results reveal that loss of this gene has measurable effects on breast carcinogenesis and breast cancer metastasis.

Serine deprivation enhances antineoplastic activity of biguanides.

Metformin, a biguanide widely used in the treatment of type II diabetes, clearly exhibits antineoplastic activity in experimental models and has been reported to reduce cancer incidence in diabetics. There are ongoing clinical trials to evaluate its antitumor properties, which may relate to its fundamental activity as an inhibitor of oxidative phosphorylation. Here, we show that serine withdrawal increases the antineoplastic effects of phenformin (a potent biguanide structurally related to metformin). Serine synthesis was not inhibited by biguanides. Instead, metabolic studies indicated a requirement for serine to allow cells to compensate for biguanide-induced decrease in oxidative phosphorylation by upregulating glycolysis. Furthermore, serine deprivation modified the impact of metformin on the relative abundance of metabolites within the citric acid cycle. In mice, a serine-deficient diet reduced serine levels in tumors and significantly enhanced the tumor growth-inhibitory actions of biguanide treatment. Our results define a dietary manipulation that can enhance the efficacy of biguanides as antineoplastic agents that target cancer cell energy metabolism.

Quantification of binding of IGF-1 to BI 836845, a candidate therapeutic antibody against IGF-1 and IGF-2, and effects of this antibody on IGF-1:IGFBP-3 complexes in vitro and in male C57BL/6 mice.

IGF-1 and IGF-2 are potent mitogens acting through the IGF-1 receptor (IGF-1R). The importance of the IGF system in neoplasia has been demonstrated in several models, and IGF-1 signaling has become a target for drug development. The drug candidate BI 836845 is a fully human IgG1 ligand-neutralizing antibody that cross-reacts with IGF-1 and IGF-2. It has been shown to reduce both IGF-1R phosphorylation and cellular proliferation in preclinical studies. In rodent studies, administration of BI 836845 leads to large increases in total IGF-1 concentration in serum, despite reduced serum IGF-1 activity as measured by a kinase activation assay. Despite the fact that anti-IGF-ligand antibodies have entered clinical trials, their effect on IGF-binding proteins has not been described. In this report, we developed a novel technique to measure ligand-BI 836845 binding, and we apply it to a mouse model in various contexts. We show that although large increases in total serum IGF-1 levels are observed, the vast majority of ligand is present as a complex with BI 836845, and total serum IGF-binding protein-3 levels are decreased. Finally, we show that BI 836845 treatment induces an increase in GH levels, a finding consistent with attempted compensation at the level of the pituitary. Our results reveal complexities in the physiologic sequelae of BI 836845 administration that have implications for determination of optimal dosing regimens and for development of pharmacodynamic endpoints for clinical trials.

Treatment with insulin analog X10 and IGF-1 increases growth of colon cancer allografts.

Obesity and type 2 diabetes are associated with an increased risk for development of certain forms of cancer, including colon cancer. The publication of highly controversial epidemiological studies in 2009 raised the possibility that use of the insulin analog glargine increases this risk further. However, it is not clear how mitogenic effects of insulin and insulin analogs measured in vitro correlate with tumor growth-promoting effects in vivo. The aim of this study was to examine possible growth-promoting effects of native human insulin, insulin X10 and IGF-1, which are considered positive controls in vitro, in a short-term animal model of an obesity- and diabetes-relevant cancer. We characterized insulin and IGF-1 receptor expression and the response to treatment with insulin, X10 and IGF-1 in the murine colon cancer cell line (MC38 cells) in vitro and in vivo. Furthermore, we examined pharmacokinetics and pharmacodynamics and monitored growth of MC38 cell allografts in mice with diet-induced obesity treated with human insulin, X10 and IGF-1. Treatment with X10 and IGF-1 significantly increased growth of MC38 cell allografts in mice with diet-induced obesity and we can therefore conclude that supra-pharmacological doses of the insulin analog X10, which is super-mitogenic in vitro and increased the incidence of mammary tumors in female rats in a 12-month toxicity study, also increase growth of tumor allografts in a short-term animal model.

Inducible IL10(+) suppressor B cells inhibit CNS inflammation and T helper 17 polarization.

Granulocyte-macrophage colony-stimulating factor (GMCSF) and MCP3 (aka CCL7) exert complementary, nonoverlapping, proimmune effects on responsive lymphoid and myeloid cells. We hypothesized that a synthetic cytokine linking GMCSF to MCP3 (hereafter GMME3) as part of a single polypeptide would acquire novel, therapeutically desirable immunomodulatory properties. We demonstrate that GMME3 has enhanced CC-chemokine receptor (CCR)-mediated intracellular Ca(++) mobilization with selective effects on the CD21(hi)CD24(hi) CD1.d(hi) subset of splenic B cells inducing substantial interleukin 10 (IL10) production. We demonstrate that B(GMME3) exert their suppressive effect through an IL10-mediated inhibition of antigen presentation. More importantly, B(GMME3) inhibit the reactivation of encephalomyelitis (EAE)-derived or TGFß/IL6 differentiated Th17 cells by altering their polarization toward a Th1 or Th2 phenotype. The secretion of interferon-γ (IFNγ) and IL4 in turn inhibits IL17 production. The adoptive transfer of B(GMME3), but not IL10(-/-) B(GMME3) cells, to mice symptomatic with experimental autoimmune encephalitis significantly improves their disease score and inhibits lymphoid infiltration into the central nervous system (CNS). We propose that designed CCR modulators such as GMME3, allows for conversion of naive B-cells to a novel suppressor phenotype allowing for the personalized cell therapy of autoimmune ailments.

Stimulation of MC38 tumor growth by insulin analog X10 involves the serine synthesis pathway.

Recent evidence suggests that type II diabetes is associated with increased risk and/or aggressive behavior of several cancers, including those arising from the colon. Concerns have been raised that endogenous hyperinsulinemia and/or exogenous insulin and insulin analogs might stimulate proliferation of neoplastic cells. However, the mechanisms underlying possible growth-promoting effects of insulin and insulin analogs in cancer cells in vivo, such as changes in gene expression, are incompletely described. We observed that administration of the insulin analog X10 significantly increased tumor growth and proliferation in a murine colon cancer model (MC38 cell allografts). Insulin and X10 altered gene expression in MC38 tumors in a similar fashion, but X10 was more potent in terms of the number of genes influenced and the magnitude of changes in gene expression. Many of the affected genes were annotated to metabolism, nutrient uptake, and protein synthesis. Strikingly, expression of genes encoding enzymes in the serine synthesis pathway, recently shown to be critical for neoplastic proliferation, was increased following treatment with insulin and X10. Using stable isotopic tracers and mass spectrometry, we confirmed that insulin and X10 increased glucose contribution to serine synthesis in MC38 cells. The data demonstrate that the tumor growth-promoting effects of insulin and X10 are associated with changes in expression of genes involved in cellular energy metabolism and reveal previously unrecognized effects of insulin and X10 on serine synthesis.

Adoptive transfer of mesenchymal stromal cells accelerates intestinal epithelium recovery of irradiated mice in an interleukin-6-dependent manner.

BACKGROUND AIMS: Apoptosis of radiosensitive cells in the bone marrow and gut is a serious, at times life-threatening, complication arising from radiation exposure. METHODS: We investigated whether adoptive transfer of allogeneic bone marrow-derived mesenchymal stromal cells (MSC) could exert cytoprotective and life-sparing effects in a mouse model of sublethal total body irradiation (TBI). RESULTS: We demonstrated that a single intraperitoneal injection of C57Bl/6 MSC given to major histocompatibility complex (MHC)-mismatched Balb/c mice within 24 h of sublethal TBI significantly reduced mortality in a dose-dependent manner. Histologic analysis and Ki67 immunostaining of jejunum sections collected 3 and 6 days post-TBI indicated that MSC protected the gastrointestinal epithelium from TBI-induced damage and significantly accelerated recovery of the gut by stimulating proliferation of the crypt cell pool. Using interleukin-6(-/-) (IL-6) MSC, we demonstrated that IL-6 expressed by MSC played a role in gastrointestinal epithelium regeneration. CONCLUSIONS: Our results suggest that allogeneic MHC-mismatched MSC may be exploited to reduce gastrointestinal complications and mortality arising from ionizing radiation exposure.

B effector cells activated by a chimeric protein consisting of IL-2 and the ectodomain of TGF-ß receptor II induce potent antitumor immunity.

We have previously shown that interleukin (IL)-2 receptor-expressing lymphoid cells stimulated with a chimeric protein linking IL-2 to the ectodomain of TGF-ß receptor II (also known as FIST) become resistant to TGF-ß-mediated suppression and produce significant amounts of proinflammatory cytokines. In this study, we have characterized the antigen presentation properties of FIST-stimulated B cells (hereafter inducible B effector cells, iBEC). FIST converts naïve splenic B cells to B effector cells characterized by potent antigen presentation properties and production of TNFα and IFNγ. iBECs display hyperphosphorylation of STAT3 and STAT5 downstream of the IL-2 receptor and upregulation of T-bet expression. iBECs maintain B-cell identity based on the expression of PAX5 and CD19 and overexpress Smad7, which confers resistance to TGF-ß-mediated suppression of B-cell activation. iBEC antitumor immunity was determined by a mouse model of lymphoma-expressing ovalbumin (E.G7-OVA) as a specific tumor antigen. OVA-pulsed iBECs function as antigen-presenting cells (APC) in vitro by inducing the activation of OVA-specific CD4(+) and CD8(+) T cells, respectively, and in vivo by conferring complete protective immunity against E.G7-OVA tumor challenge. In addition, OVA-pulsed iBECs promote tumor regression in immunocompetent C57Bl/6 mice bearing E.G7-OVA tumors. In conclusion, iBECs represent an entirely novel B cell-derived APC for immune therapy of cancer.

Metformin reduces endogenous reactive oxygen species and associated DNA damage.

Pharmacoepidemiologic studies provide evidence that use of metformin, a drug commonly prescribed for type II diabetes, is associated with a substantial reduction in cancer risk. Experimental models show that metformin inhibits the growth of certain neoplasms by cell autonomous mechanisms such as activation of AMP kinase with secondary inhibition of protein synthesis or by an indirect mechanism involving reduction in gluconeogenesis leading to a decline in insulin levels and reduced proliferation of insulin-responsive cancers. Here, we show that metformin attenuates paraquat-induced elevations in reactive oxygen species (ROS), and related DNA damage and mutations, but has no effect on similar changes induced by H(2)0(2), indicating a reduction in endogenous ROS production. Importantly, metformin also inhibited Ras-induced ROS production and DNA damage. Our results reveal previously unrecognized inhibitory effects of metformin on ROS production and somatic cell mutation, providing a novel mechanism for the reduction in cancer risk reported to be associated with exposure to this drug.

A MCP1 fusokine with CCR2-specific tumoricidal activity.

BACKGROUND: The CCL2 chemokine is involved in promoting cancer angiogenesis, proliferation and metastasis by malignancies that express CCR2 receptor. Thus the CCL2/CCR2 axis is an attractive molecular target for anticancer drug development. METHODS: We have generated a novel fusion protein using GMCSF and an N-terminal truncated version of MCP1/CCL2 (6-76) [hereafter GMME1] and investigated its utility as a CCR2-specific tumoricidal agent. RESULTS: We found that distinct to full length CCL2 or its N-truncated derivative (CCL2 5-76), GMME1 bound to CCR2 on mouse lymphoma EG7, human multiple myeloma cell line U266, or murine and human medulloblastoma cell lines, and led to their death by apoptosis. We demonstrated that GMME1 specifically blocked CCR2-associated STAT3 phosphorylation and up-regulated pro-apoptotic BAX. Furthermore, GMME1 significantly inhibited EG7 tumor growth in C57BL/6 mice, and induced apoptosis of primary myeloma cells from patients. CONCLUSION: Our data demonstrate that GMME1 is a fusokine with a potent, CCR2 receptor-mediated pro-apoptotic effect on tumor cells and could be exploited as a novel biological therapy for CCR2+ malignancies including lymphoid and central nervous system malignancies.

IGF1/insulin receptor kinase inhibition by BMS-536924 is better tolerated than alloxan-induced hypoinsulinemia and more effective than metformin in the treatment of experimental insulin-responsive breast cancer.

Epidemiologic and experimental evidence suggest that a subset of breast cancer is insulin responsive, but it is unclear whether safe and effective therapies that target the insulin receptor (IR), which is homologous to oncogenes of the tyrosine kinase class, can be developed. We demonstrate that both pharmacologic inhibition of IR family tyrosine kinase activity and insulin deficiency have anti-neoplastic activity in a model of insulin-responsive breast cancer. Unexpectedly, in contrast to insulin deficiency, pharmacologic IR family inhibition does not lead to significant hyperglycemia and is well tolerated. We show that pharmacokinetic factors explain the tolerability of receptor inhibition relative to insulin deficiency, as the small molecule receptor kinase inhibitor BMS-536924 does not accumulate in muscle at levels sufficient to block insulin-stimulated glucose uptake. Metformin, which lowers insulin levels only in settings of hyperinsulinemia, had minimal activity in this normoinsulinemic model. These findings highlight the importance of tissue-specific drug accumulation as a determinant of efficacy and toxicity of tyrosine kinase inhibitors and suggest that therapeutic targeting of the IR family for cancer treatment is practical.

Metformin abolishes increased tumor (18)F-2-fluoro-2-deoxy-D-glucose uptake associated with a high energy diet.

Insulin regulates glucose uptake by normal tissues. Although there is evidence that certain cancers are growth-stimulated by insulin, the possibility that insulin influences tumor glucose uptake as assessed by ( 18) F-2-Fluoro-2-Deoxy-d-Glucose Positron Emission Tomography (FDG-PET) has not been studied in detail. We present a model of diet-induced hyperinsulinemia associated with increased insulin receptor activation in neoplastic tissue and with increased tumor FDG-PET image intensity. Metformin abolished the diet-induced increases in serum insulin level, tumor insulin receptor activation and tumor FDG uptake associated with the high energy diet but had no effect on these measurements in mice on a control diet. These findings provide the first functional imaging correlate of the well-known adverse effect of caloric excess on cancer outcome. They demonstrate that, for a subset of neoplasms, diet and insulin are variables that affect tumor FDG uptake and have implications for design of clinical trials of metformin as an antineoplastic agent.

Erythropoietin gene-enhanced marrow mesenchymal stromal cells decrease cisplatin-induced kidney injury and improve survival of allogeneic mice.

Bone marrow-derived mesenchymal stromal cells (MSCs) are promising for regenerative medicine applications, such as for renoprotection and repair in acute kidney injury (AKI). Erythropoietin (Epo) can also exert cytoprotective effects on various tissues including the kidney. We hypothesized that MSCs gene-enhanced to secrete Epo may produce a significant beneficial effect in AKI. Mouse Epo-secreting MSCs were generated, tested in vitro, and then implanted by intraperitoneal injection in allogeneic mice previously administered cisplatin to induce AKI. Epo-MSCs significantly improved survival of implanted mice as compared to controls (67% survival versus 33% with Vehicle only). Also, Epo-MSCs led to significantly better kidney function as shown by lower levels of blood urea nitrogen (72 ± 9.5 mg/dl versus 131 ± 9.20 mg/dl) and creatinine (74 ± 17 µmol/l versus 148±19.4 µmol/l). Recipient mice also showed significantly decreased amylase and alanine aminotransferase blood concentrations. Kidney sections revealed significantly less apoptotic cells and more proliferating cells. Furthermore, PCR revealed the presence of implanted cells in recipient kidneys, with Epo-MSCs leading to significantly increased expression of Epo and of phosphorylated-Akt (Ser473) (P-Akt) in these kidneys. In conclusion, our study demonstrates that Epo gene-enhanced MSCs exert significant tissue protective effects in allogeneic mice with AKI, and supports the potential use of gene-enhanced cells as universal donors in acute injury.

Novel TGF-beta antagonist inhibits tumor growth and angiogenesis by inducing IL-2 receptor-driven STAT1 activation.

Carcinoma derived TGF-ß acts as a potent pro-oncogenic factor and suppresses antitumor immunity. To antagonize TGF-ß-mediated effects in tandem with a proinflammatory immune stimulus, we generated a chimeric protein borne of the fusion of IL-2 and the soluble extracellular domain of TGF-ßR II (FIST). FIST acts as a decoy receptor trapping active TGF-ß in solution and interacts with IL-2-responsive lymphoid cells, inducing a distinctive hyperactivation of STAT1 downstream of IL-2R, which in turn promotes SMAD7 overexpression. Consequently, FIST-stimulated lymphoid cells are resistant to TGF-ß-mediated suppression and produce significant amounts of proinflammatory cytokines. STAT1 hyperactivation further induces significant secretion of angiostatic CXCL10. Moreover, FIST upregulates T-bet expression in NK cells promoting a potent Th1-mediated antitumor response. As a result, FIST stimulation completely inhibits pancreatic cancer (PANC02) and melanoma (B16) tumor growth in immunocompetent C57BL/6 mice. In addition, melanoma cells expressing FIST fail to form tumors in CD8(-/-), CD4(-/-), B cell-deficient (µMT), and beige mice, but not in NOD-SCID and Rag2/γc knockout mice, consistent with the pivotal role of FIST-responsive, cancer-killing NK cells in vivo. In summary, FIST constitutes a novel strategy of treating cancer that targets both the host's angiogenic and innate immune response to malignant cells.

A dendritic cell population generated by a fusion of GM-CSF and IL-21 induces tumor-antigen-specific immunity.

We have previously shown that the fusion of GM-CSF and IL-21 (GIFT-21) possesses a potent immune stimulatory effect on myeloid cells. In this study, we define the effect of GIFT-21 on naive murine monocytes (GIFT-21 dendritic cells [DCs]), which express increased levels of Gr-1, CD45R, MHC class I, CD80, CD86, and CXCR4 and suppress CD11c and MHC class II. Compared with conventional dendritic cells, GIFT-21 DCs produced substantially more CCL2, IL-6, TNF-α, and IFN-α and induced significantly greater production of IFN-γ by CD8(+) T cells in MHC class I-restricted Ag presentation assays. B16 melanoma and D2F2 Neu breast cancer growth was inhibited in mice treated with Ag-naive GIFT-21 DCs. This effect was lost in CD8(-/-) and CCR2(-/-) mice and when mice were treated with ß(2)-microglobulin-deficient GIFT-21 DCs, indicating that GIFT-21 DCs migrated to and sampled from the tumors to present tumor Ags to CCL2 recruited CD8(+) T cells via MHC class I. We propose that autologous GIFT-21 DCs may serve as a cell therapy platform for the treatment of cancer.

Mesenchymal stromal cells expressing ErbB-2/neu elicit protective antibreast tumor immunity in vivo, which is paradoxically suppressed by IFN-gamma and tumor necrosis factor-alpha priming.

It is unknown whether mesenchymal stromal cells (MSC) can regulate immune responses targeting tumor autoantigens of low immunogenicity. We tested here whether immunization with MSC could break immune tolerance towards the ErbB-2/HER-2/neu tumor antigen and the effects of priming with IFN-γ and tumor necrosis factor-α (TNF-α) on this process. BALB/c- and C57BL/6-derived MSC were lentivirally transduced to express a kinase-inactive rat neu mutant (MSC/Neu). Immunization of BALB/c mice with nontreated or IFN-γ-primed allogeneic or syngeneic MSC/Neu induced similar levels of anti-neu antibody titers; however, only syngeneic MSC/Neu induced protective neu-specific CD8(+) T cell responses. Compared to immunization with nontreated or IFN-γ-primed syngeneic MSC/Neu, the number of circulating neu-specific CD8(+) T cells and titers of anti-neu antibodies were observed to be decreased after immunizations with IFN-γ- plus TNF-α-primed MSC/Neu. In addition, syngeneic MSC/Neu seemed more efficient than IFN-γ-primed MSC/Neu at inducing a protective therapeutic antitumor immune response resulting in the regression of transplanted neu-expressing mammary tumor cells. In vitro antigen-presenting cell assays performed with paraformaldehyde-fixed or live MSC showed that priming with IFN-γ plus TNF-α, compared to priming with IFN-γ alone, increased antigen presentation as well as the production of immunosuppressive factors. These data suggest that whereas MSC could effectively serve as antigen-presenting cells to induce immune responses aimed at tumor autoantigens, these functions are critically regulated by IFN-γ and TNF-α.

Human marrow-derived mesenchymal stromal cells decrease cisplatin renotoxicity in vitro and in vivo and enhance survival of mice post-intraperitoneal injection.

Acute kidney injury (AKI) can occur from the toxic side-effects of chemotherapeutic agents such as cisplatin. Bone marrow-derived mesenchymal stromal cells (MSCs) have demonstrated wide therapeutic potential often due to beneficial factors they secrete. The goal of this investigation was to evaluate in vitro the effect of human MSCs (hMSCs) secretome on cisplatin-treated human kidney cells, and in vivo the consequence of hMSCs intraperitoneal (ip) implantation in mice with AKI. Our results revealed that hMSCs-conditioned media improved survival of HK-2 human proximal tubular cells exposed to cisplatin in vitro. This enhanced survival was linked to increased expression of phosphorylated Akt (Ser473) and was reduced by a VEGF-neutralizing antibody. In vivo testing of these hMSCs established that ip administration in NOD-SCID mice decreased cisplatin-induced kidney function impairment, as demonstrated by lower blood urea nitrogen levels and higher survival. In addition, blood phosphorous and amylase levels were also significantly decreased. Moreover, hMSCs reduced the plasma levels of several inflammatory cytokines/chemokines. Immunohistochemical examination of kidneys showed less apoptotic and more proliferating cells. Furthermore, PCR indicated the presence of hMSCs in mouse kidneys, which also showed enhanced expression of phosphorylated Akt. In conclusion, our study reveals that hMSCs can exert prosurvival effects on renal cells in vitro and in vivo, suggests a paracrine contribution for kidney protective abilities of hMSCs delivered ip, and supports their clinical potential in AKI.

A fusion of GMCSF and IL-21 initiates hypersignaling through the IL-21Ralpha chain with immune activating and tumoricidal effects in vivo.

We hypothesized that fusing granulocyte-macrophage colony-stimulation factor (GMCSF) and interleukin (IL)-21 as a single bifunctional cytokine (hereafter GIFT-21) would lead to synergistic anticancer immune effects because of their respective roles in mediating inflammation. Mechanistic analysis of GIFT-21 found that it leads to IL-21Ralpha-dependent STAT3 hyperactivation while also contemporaneously behaving as a dominant-negative inhibitor of GMCSF-driven STAT5 activation. GIFT-21's aberrant interactions with its cognate receptors on macrophages resulted in production of 30-fold greater amounts of IL-6, TNF-alpha, and MCP-1 when compared to controls. Furthermore, GIFT-21 treatment of primary B and T lymphocytes leads to STAT1-dependent apoptosis of IL-21Ralpha(+) lymphocytes. B16 melanoma cells gene-enhanced to produce GIFT-21 were immune rejected by syngeneic C57Bl/6 mice comparable to the effect of IL-21 alone. However, a significant GIFT-21-driven survival advantage was seen when NOD-SCID mice were implanted with GIFT-21-secreting B16 cells, consistent with a meaningful role of macrophages in tumor rejection. Because GIFT-21 leads to apoptosis of IL-21Ralpha(+) lymphocytes, we tested its cytolytic effect on IL-21Ralpha(+) EL-4 lymphoma tumors implanted in C57Bl/6 mice and could demonstrate a significant increase in survival. These data indicate that GIFT-21 is a novel IL-21Ralpha agonist that co-opts IL-21Ralpha-dependent signaling in a manner permissive for targeted cancer immunotherapy.

Differential roles of Trk and p75 neurotrophin receptors in tumorigenesis and chemoresistance ex vivo and in vivo.

The neurotrophin receptors TrkA (NGF receptor) and TrkC (NT-3 receptor) have been shown to be important in staging disease and predicting progression and drug response for various neoplasias such as neuroblastoma, medulloblastoma and prostate cancer. Less is known about the role of the p75 neurotrophin receptor in cancer, but it influences metastatic potential in glioblastoma. To determine the effect of each neurotrophin receptor or co-receptor expression in tumorigenesis, we examined PC12 pheochromocytomas. PC12 wild type (TrkA(+), p75(++)) were compared to three PC12-derived cell lines expressing varying levels of TrkA or TrkC and/or p75. Growth rates, tumorigenic potential ex vivo and in vivo, and chemotherapeutic drug response profiles differed depending on the neurotrophin receptor phenotype. The ability of neurotrophins to rescue cells from doxorubicin or cisplatin induced cell death also varied depending on phenotype. Thus, unique neurotrophin receptor tumor profiles may determine tumor aggressiveness and chemoresistance. This work may help to develop tailored therapies for specific tumor phenotypes by combining traditional chemotherapy with neurotrophin receptor modulators.