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.

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.

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.

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.

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.

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.

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.

Selective inhibition of CCR2 expressing lymphomyeloid cells in experimental autoimmune encephalomyelitis by a GM-CSF-MCP1 fusokine.

We describe the generation of a fusion cytokine consisting of GM-CSF in tandem with N-terminal-truncated MCP-1 (6-76), hereafter GMME1. Treatment of activated T cells with recombinant GMME1 protein leads to proinflammatory cytokine reduction and apoptosis via a CCR2-restricted pathway. Similarly, cell death is triggered in macrophages cultured with GMME1, while an inhibition of Ab production from plasma cells is observed. Treatment of CD4 T cells derived from experimental autoimmune encephalomyelitis mice with GMME1 leads to p38 hyperphosphorylation, inhibition of p44/42, AKT and STAT3 phosphorylation, and caspase-3 activation. GMME1 administration to experimental autoimmune encephalomyelitis mice suppresses symptomatic disease and correlates with decreased levels of inflammatory cytokines including IL-17, MOG-specific Ab titers, and blockade of CD4 and CD8 T cell infiltration in spinal cords. We propose that GMME1 defines a new class of agents for the treatment of autoimmune ailments by selectively targeting lymphomyeloid cells expressing CCR2.

An engineered GM-CSF-CCL2 fusokine is a potent inhibitor of CCR2-driven inflammation as demonstrated in a murine model of inflammatory arthritis.

CCR2 is a chemokine receptor widely expressed by lymphomyeloid cells involved in maladaptive autoimmune ailments. Therefore CCR2 is of great interest as a biological target for immune suppression due to its direct implication in autoimmune diseases such as rheumatoid arthritis. We have generated a novel fusion protein using GM-CSF and an N-terminal truncated version of MCP-1/CCL2 (6-76, GMME1) and investigated its utility as a CCR2-specific immune suppressor. Using BRET studies, we found that distinct to CCL2, GMME1 binding to CCR2 led to altered conformational changes in the CCR2 homodimer and did not induce the recruitment of beta-arrestin 2 to the receptor. However, CCR2-dependent calcium mobilization, BAX induction and caspase-3 activation followed by cell death was observed. Using Th17 cells harvested from DBA/1 mice ill with bovine collagen-induced arthritis, we demonstrate that GMME1 is capable of blocking their production of IL-17 in vitro. Upon its delivery to mice symptomatic with inflammatory arthritis, a robust clinical recovery occurred with decreased paw thickness to normal levels and a significant reduction in anti-collagen Ab titer and rheumatoid factor titer, as well as reduction of proinflammatory cytokines levels both intraarticular and systemic. Our data demonstrate that GMME1 is a powerful synthetic suppressor cytokine that coopts CCR2-dependent cellular signaling and blunts the effects of CCR2-expressing lymphomyeloid cells causative of autoimmune arthritis.

Mesenchymal stromal cells ameliorate experimental autoimmune encephalomyelitis by inhibiting CD4 Th17 T cells in a CC chemokine ligand 2-dependent manner.

The administration of ex vivo culture-expanded mesenchymal stromal cells (MSCs) has been shown to reverse symptomatic neuroinflammation observed in experimental autoimmune encephalomyelitis (EAE). The mechanism by which this therapeutic effect occurs remains unknown. In an effort to decipher MSC mode of action, we found that MSC conditioned medium inhibits EAE-derived CD4 T cell activation by suppressing STAT3 phosphorylation via MSC-derived CCL2. Further analysis demonstrates that the effect is dependent on MSC-driven matrix metalloproteinase proteolytic processing of CCL2 to an antagonistic derivative. We also show that antagonistic CCL2 suppresses phosphorylation of AKT and leads to a reciprocal increased phosphorylation of ERK associated with an up-regulation of B7.H1 in CD4 T cells derived from EAE mice. CD4 T cell infiltration of the spinal cord of MSC-treated group was robustly decreased along with reduced plasma levels of IL-17 and TNF-alpha levels and in vitro from restimulated splenocytes. The key role of MSC-derived CCL2 was confirmed by the observed loss of function of CCL2(-/-) MSCs in EAE mice. In summary, this is the first report of MSCs modulating EAE biology via the paracrine conversion of CCL2 from agonist to antagonist of CD4 Th17 cell function.

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.

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.

Mesenchymal stromal cell-derived CCL2 suppresses plasma cell immunoglobulin production via STAT3 inactivation and PAX5 induction.

We demonstrate that the secretome of mesenchymal stromal cells (MSCs) suppresses plasma cell (PC) immunoglobulin (Ig) production, induces plasmablast proliferation, and leads to interleukin-10-mediated blockade in vitro. We found that these effects are the result of MSC-derived CC chemokine ligands CCL2 and CCL7. More specifically, MSCs further processed these CC chemokines by the activity of matrix metalloproteinases (MMPs), leading to the generation of proteolytically processed antagonistic CCL2 variant. Neutralizing CCL2 or inhibiting MMP enzymatic activity abolished the PC-suppressive effect of MSCs. We also observed that MMP-processed CCL2 suppresses signal transducer and activator of transcription 3 (STAT3) activation in PC. As a result, the transcription factor PAX5 is induced, thus explaining the inhibition of Ig synthesis. The absence of inhibitory effects by MSC on the humoral response of CCR2(-/-) mice to xenoantigen suggests that MMP-cleaved CCL2/CCR2 interaction as well as downstream phosphatase activity is necessary for antagonistic effect. We tested syngeneic MSCs in hemophilic B6 mice with predeveloped antihuman factor VIII (hFVIII) antibodies and demonstrated a robust decrease in hFVIII-specific IgG levels. Thus, MSCs may play a role in modulating Ig production by PCs via MMP processing of CCL2 and may represent an appealing cell therapy approach for pathologic humoral responses.

Allogeneic mesenchymal stem cells for treatment of experimental autoimmune encephalomyelitis.

The use of allogeneic "universal donor" mesenchymal stromal cells (MSCs) may be a substantial clinical convenience for treatment of autoimmune ailments such as multiple sclerosis. We therefore tested whether allogeneic MSCs can be exploited for treatment of experimental autoimmune encephalomyelitis (EAE) in mice with otherwise intact immune system. Administration of allogeneic Balb/c-derived MSCs to C57Bl/6 mice with pre-established EAE led to a significant decrease in disease score over time comparable to that achieved with syngeneic MSCs, and was correlated with a significant blunting of immune cell infiltration to the spinal cord and reduced circulating levels of interferon-gamma (IFN-gamma) and interleukin-17. Pretreatment of allogeneic MSCs with IFN-gamma increased the expression levels of CCL2 as well as major histocompatibility complex I (MHCI) and MHCII, but also led to complete loss of suppressive activity in vivo that correlated with immune rejection. In conclusion, allogeneic MSCs can suppress the manifestations of EAE, yet retain the potential for alloimmunization.

Mesenchymal stromal cells engineered to express erythropoietin induce anti-erythropoietin antibodies and anemia in allorecipients.

Autologous bone marrow mesenchymal stromal cells (MSCs) have been successfully used for the delivery of erythropoietin (EPO) in murine models of anemia and myocardial infarction. For clinical applications where a transient effect would be adequate, such as myocardial infarction, the use of EPO-engineered universal donor allogeneic MSCs would be a substantial convenience. We thus investigated whether MSCs from C57BL/6 mice would permit robust transient EPO delivery in normal BALB/c allorecipients. Implantation of MSCs overexpressing murine EPO led to increases in hematocrit in syngeneic and allogeneic mice, but the latter eventually developed severe anemia due to acquired neutralizing anti-EPO antibodies. As MSCs constitutively produce the CCL2 chemokine which may behave as an adjuvant to the anti-EPO immune response, experiments were performed using EPO-engineered MSCs derived from CCL2(-/-) mice and similar results were obtained. In conclusion, MHC-mismatched MSCs can break the tolerance to autoantigens and lead to the development of pathogenic autoantibodies.

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.

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.

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.

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.