Standardized xeno- and serum-free culture platform enables large-scale expansion of high-quality mesenchymal stem/stromal cells from perinatal and adult tissue sources.

BACKGROUND AIMS: Mesenchymal stem/stromal cells (MSCs) are of interest for the treatment of graft-versus-host disease, autoimmune diseases, osteoarthritis and neurological and cardiovascular diseases. Increasing numbers of clinical trials emphasize the need for standardized manufacturing of these cells. However, many challenges related to diverse isolation and expansion protocols and differences in cell tissue sources exist. As a result, the cell products used in numerous trials vary greatly in characteristics and potency. METHODS: The authors have established a standardized culture platform using xeno- and serum-free commercial media for expansion of MSCs derived from umbilical cord (UC), bone marrow and adipose-derived (AD) and examined their functional characteristics. RESULTS: MSCs from the tested sources stably expanded in vitro and retained their biomarker expression and normal karyotype at early and later passages and after cryopreservation. MSCs were capable of colony formation and successfully differentiated into osteogenic, adipogenic and chondrogenic lineages. Pilot expansion of UC-MSCs and AD-MSCs to clinical scale revealed that the cells met the required quality standard for therapeutic applications. CONCLUSIONS: The authors' data suggest that xeno- and serum-free culture conditions are suitable for large-scale expansion and enable comparative study of MSCs of different origins. This is of importance for therapeutic purposes, especially because of the numerous variations in pre-clinical and clinical protocols for MSC-based products.

Inactivation of Wnt signaling by a human antibody that recognizes the heparan sulfate chains of glypican-3 for liver cancer therapy.

UNLABELLED: Wnt signaling is important for cancer pathogenesis and is often up-regulated in hepatocellular carcinoma (HCC). Heparan sulfate proteoglycans (HSPGs) function as coreceptors or modulators of Wnt activation. Glypican-3 (GPC3) is an HSPG that is highly expressed in HCC, where it can attract Wnt proteins to the cell surface and promote cell proliferation. Thus, GPC3 has emerged as a candidate therapeutic target in liver cancer. While monoclonal antibodies to GPC3 are currently being evaluated in preclinical and clinical studies, none have shown an effect on Wnt signaling. Here, we first document the expression of Wnt3a, multiple Wnt receptors, and GPC3 in several HCC cell lines, and demonstrate that GPC3 enhanced the activity of Wnt3a/ß-catenin signaling in these cells. Then we report the identification of HS20, a human monoclonal antibody against GPC3, which preferentially recognized the heparan sulfate chains of GPC3, both the sulfated and nonsulfated portions. HS20 disrupted the interaction of Wnt3a and GPC3 and blocked Wnt3a/ß-catenin signaling. Moreover, HS20 inhibited Wnt3a-dependent cell proliferation in vitro and HCC xenograft growth in nude mice. In addition, HS20 had no detectable undesired toxicity in mice. Taken together, our results show that a monoclonal antibody primarily targeting the heparin sulfate chains of GPC3 inhibited Wnt/ß-catenin signaling in HCC cells and had potent antitumor activity in vivo. CONCLUSION: An antibody directed against the heparan sulfate of a proteoglycan shows efficacy in blocking Wnt signaling and HCC growth, suggesting a novel strategy for liver cancer therapy.

Glypican-3 targeted human heavy chain antibody as a drug carrier for hepatocellular carcinoma therapy.

Glypican-3 (GPC3) represents an attractive target for hepatocellular carcinoma (HCC) therapy because it is highly expressed in HCC but not in adult normal tissue. Recently, high affinity anti-GPC3 antibodies have been developed; however, full antibodies may not penetrate evenly into tumor parenchyma, reducing their effectiveness. In this study, we compared a whole IgG antibody, anti-GPC3 YP7, with an anti-GPC3 human heavy chain antibody, HN3, with regard to their relative therapeutic effects. Both YP7 and HN3 bound to GPC3-positive A431/G1 cells and were internalized by the cells by in vitro evaluation with (125)I- and (111)In-radiolabeling antibodies. In vivo biodistribution and tumor accumulation was performed with (111)In-labeled antibodies, and intratumoral microdistribution was evaluated using fluorescently labeled antibodies (IR700). HN3 showed similar high tumor accumulation but superior homogeneity within the tumor compared with YP7. Using the same IR700 conjugated antibodies photoimmunotherapy (PIT) was performed in vitro and in a tumor-bearing mouse model in vivo. PIT with IR700-HN3 and IR700-YP7 demonstrated that comparable results could be achieved despite of low reaccumulation 24 h after the first NIR light exposure. These results indicated that a heavy-chain antibody, HN3, showed more favorable characteristics than YP7, a conventional IgG, as a therapeutic antibody platform for designing molecularly targeted agents against HCC.

The molecular basis of hepcidin-resistant hereditary hemochromatosis.

The interaction between the hormone hepcidin and the iron exporter ferroportin (Fpn) regulates plasma iron concentrations. Hepcidin binds to Fpn and induces its internalization and degradation, resulting in decreased iron efflux from cells into plasma. Fpn mutations in N144, Y64N, and C326 residue cause autosomal dominant disease with parenchymal iron overload, apparently due to the resistance of mutant Fpn to hepcidin-mediated internalization. To define the mechanism of resistance, we generated human Fpn constructs bearing the pathogenic mutations. The mutants localized to the cell surface and exported iron normally, but were partially or completely resistant to hepcidin-mediated internalization and continued to export iron despite the presence of hepcidin. The primary defect with exofacial C326 substitutions was the loss of hepcidin binding, which resulted in the most severe phenotype. The thiol form of C326 was essential for interaction with hepcidin, suggesting that C326-SH homology is located in or near the binding site of hepcidin. In contrast, N144 and Y64 residues were not required for hepcidin binding, but their mutations impaired the subsequent internalization of the ligand-receptor complex. Our observations explain why the mutations in C326 Fpn residue produce a severe form of hemochromatosis with iron overload at an early age.

New high affinity monoclonal antibodies recognize non-overlapping epitopes on mesothelin for monitoring and treating mesothelioma.

Mesothelin is an emerging cell surface target in mesothelioma and other solid tumors. Most antibody drug candidates recognize highly immunogenic Region I (296-390) on mesothelin. Here, we report a group of high-affinity non-Region I rabbit monoclonal antibodies. These antibodies do not compete for mesothelin binding with the immunotoxin SS1P that binds Region I of mesothelin. One pair of antibodies (YP218 and YP223) is suitable to detect soluble mesothelin in a sandwich ELISA with high sensitivity. The new assay can also be used to measure serum mesothelin concentration in mesothelioma patients, indicating its potential use for monitoring patients treated with current antibody therapies targeting Region I. The antibodies are highly specific and sensitive in immunostaining of mesothelioma. To explore their use in tumor therapy, we have generated the immunotoxins based on the Fv of these antibodies. One immunotoxin (YP218 Fv-PE38) exhibits potent anti-tumor cytotoxicity towards primary mesothelioma cell lines in vitro and an NCI-H226 xenograft tumor in mice. Furthermore, we have engineered a humanized YP218 Fv that retains full binding affinity for mesothelin-expressing cancer cells. In conclusion, with their unique binding properties, these antibodies may be promising candidates for monitoring and treating mesothelioma and other mesothelin-expressing cancers.

Photoimmunotherapy of hepatocellular carcinoma-targeting Glypican-3 combined with nanosized albumin-bound paclitaxel.

AIM: Effectiveness of Glypican-3 (GPC3)-targeted photoimmunotherapy (PIT) combined with the nanoparticle albumin-bound paclitaxel (nab-paclitaxel) for hepatocellular carcinoma was evaluated. MATERIALS & METHODS: GPC3 expressing A431/G1 cells were incubated with a phthalocyanine-derivative, IRDye700DX (IR700), conjugated to an anti-GPC3 antibody, IR700-YP7 and exposed to near-infrared light. Therapeutic experiments combining GPC3-targeted PIT with nab-paclitaxel were performed in A431/G1 tumor-bearing mice. RESULTS: IR700-YP7 bound to A431/G1 cells and induced rapid target-specific necrotic cell death by near-infrared light exposure in vitro. IR700-YP7 accumulated in A431/G1 tumors. Tumor growth was inhibited by PIT compared with nontreated control. Additionally, PIT dramatically increased nab-paclitaxel delivery and enhanced the therapeutic effect. CONCLUSION: PIT targeting GPC3 combined with nab-paclitaxel is a promising method for treating hepatocellular carcinoma.

Cytomegalovirus and Epstein-Barr virus in breast cancer.

Findings of polymerase chain reaction (PCR) studies of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) and breast cancer vary, making it difficult to determine whether either, both, or neither virus is causally associated with breast cancer. We investigated CMV and EBV in paired samples of breast cancer and normal breast tissue from 70 women using quantitative PCR. A serum sample from each woman was tested for CMV and EBV IgG. To place our results in context, we reviewed the existing literature and performed a meta-analysis of our results together with previous PCR studies of EBV, CMV, and breast cancer. Of the serology samples, 67 of 70 (96%) were EBV IgG positive and 49 of 70 (70%) were CMV IgG positive. QPCR detected EBV in 24 (34%) of the tumour and 9 (13%) of the paired normal specimens and CMV in 0 (0%) of the tumour and 2 (3%) of the paired normal specimens. Our findings, together with earlier results summarised in the meta-analysis, suggest several possibilities: variable findings may be due to limitations of molecular analyses; 'hit and run' oncogenesis may lead to inconsistent results; one or both viruses has a role at a later stage in breast cancer development; infection with multiple viruses increases breast cancer risk; or neither virus has a role. Future studies should focus on ways to investigate these possibilities, and should include comparisons of breast cancer tissue samples with appropriate normal tissue samples.

Identification of one-electron reductases that activate both the hypoxia prodrug SN30000 and diagnostic probe EF5.

SN30000 is a second-generation benzotriazine-N-oxide hypoxia-activated prodrug scheduled for clinical trial. Previously we showed that covalent binding of the hypoxia probe EF5 predicts metabolic activation of SN30000 in a panel of cancer cell lines under anoxia, suggesting that they are activated by the same reductases. However the identity of these reductases is unknown. Here, we test whether forced expression of nine oxidoreductases with known or suspected roles in bioreductive prodrug metabolism (AKR1C3, CYB5R3, FDXR, MTRR, NDOR1, NOS2A, NQO1, NQO2 and POR) enhances oxic or anoxic reduction of SN30000 and EF5 by HCT116 cells. Covalent binding of (14)C-EF5 and reduction of SN30000 to its 1-oxide and nor-oxide metabolites was highly selective for anoxia in all lines, with significantly elevated anoxic metabolism of both compounds in lines over-expressing POR, MTRR, NOS2A or NDOR1. There was a strong correlation between EF5 binding and SN30000 metabolism under anoxia across the cell lines (R(2)=0.84, p=0.0001). Antiproliferative potency of SN30000 under anoxia was increased most strongly by overexpression of MTRR and POR. Transcript abundance in human tumours, evaluated using public domain mRNA expression data, was highest for MTRR, followed by POR, NOS2A and NDOR1, with little variation between tumour types. Immunostaining of tissue microarrays demonstrated variable MTRR protein expression across 517 human cancers with most displaying low expression. In conclusion, we have identified four diflavin reductases (POR, MTRR, NOS2A and NDOR1) capable of reducing both SN30000 and EF5, further supporting use of 2-nitroimidazole probes to predict the ability of hypoxic cells to activate SN30000.

A human single-domain antibody elicits potent antitumor activity by targeting an epitope in mesothelin close to the cancer cell surface.

Monoclonal antibodies against mesothelin are being evaluated for the treatment of mesothelioma and multiple forms of cancers, and show great promise for clinical development for solid cancers. Antibodies against mesothelin have been shown to act via immunotoxin-based inhibition of tumor growth and induction of antibody-dependent cell-mediated cytotoxicity (ADCC). However, complement-dependent cytotoxicity (CDC), considered an important additional mechanism of therapeutic antibodies against tumors, is inactive for such antibodies. Here, we used phage display antibody engineering technology and synthetic peptide screening to identify SD1, a human single-domain antibody to mesothelin. SD1 recognizes a conformational epitope at the C-terminal end (residues 539-588) of mesothelin close to the cell surface. To investigate SD1 as a potential therapeutic agent, we generated a recombinant human Fc (SD1-hFc) fusion protein. Interestingly, the SD1-hFc protein exhibits strong CDC activity, in addition to ADCC, against mesothelin-expressing tumor cells. Furthermore, it causes growth inhibition of human tumor xenografts in nude mice as a single agent. SD1 is the first human single-domain antibody targeting mesothelin-expressing tumors, shows potential as a cancer therapeutic candidate, and may improve current antibody therapy targeting mesothelin-expressing tumors.

Changes in global gene expression associated with 3D structure of tumors: an ex vivo matrix-free mesothelioma spheroid model.

Tumor microenvironments present significant barriers to anti-tumor agents. Molecules involved in multicellular tumor microenvironments, however, are difficult to study ex vivo. Here, we generated a matrix-free tumor spheroid model using the NCI-H226 mesothelioma cell line and compared the gene expression profiles of spheroids and monolayers using microarray analysis. Microarray analysis revealed that 142 probe sets were differentially expressed between tumor spheroids and monolayers. Gene ontology analysis revealed that upregulated genes were primarily related to immune response, wound response, lymphocyte stimulation and response to cytokine stimulation, whereas downregulated genes were primarily associated with apoptosis. Among the 142 genes, 27 are located in the membrane and related to biologic processes of cellular movement, cell-to-cell signaling, cellular growth and proliferation and morphology. Western blot analysis validated elevation of MMP2, BAFF/BLyS/TNFSF13B, RANTES/CCL5 and TNFAIP6/TSG-6 protein expression in spheroids as compared to monolayers. Thus, we have reported the first large scale comparison of the transcriptional profiles using an ex vivo matrix-free spheroid model to identify genes specific to the three-dimensional biological structure of tumors. The method described here can be used for gene expression profiling of tumors other than mesothelioma.

High-affinity monoclonal antibodies to cell surface tumor antigen glypican-3 generated through a combination of peptide immunization and flow cytometry screening.

Isolating high-affinity antibodies against native tumor antigens on the cell surface is not straightforward using standard hybridoma procedures. Here, we describe a combination method of synthetic peptide immunization and high-throughput flow cytometry screening to efficiently isolate hybridomas for cell binding. Using this method, we identified high-affinity monoclonal antibodies specific for the native form of glypcian-3 (GPC3), a target heterogeneously expressed in hepatocellular carcinoma (HCC) and other cancers. We isolated a panel of monoclonal antibodies (YP6, YP7, YP8, YP9 and YP9.1) for cell surface binding. The antibodies were used to characterize GPC3 protein expression in human liver cancer cell lines and tissues by flow cytometry, immunoblotting and immunohistochemistry. The best antibody (YP7) bound cell surface-associated GPC3 with equilibrium dissociation constant, KD = 0.3 nmol/L and was highly specific for HCC, not normal tissues or other forms of primary liver cancers (such as cholangiocarcinoma). Interestingly, the new antibody was highly sensitive in that it detected GPC3 in low expression ovarian clear cell carcinoma and melanoma cells. The YP7 antibody exhibited significant HCC xenograft tumor growth inhibition in nude mice. These results describe an improved method for producing high-affinity monoclonal antibodies to cell surface tumor antigens and represent a general approach to isolate therapeutic antibodies against cancer. The new high-affinity antibodies described here have significant potential for GPC3-expressing cancer diagnostics and therapy.

Rapid generation of in vitro multicellular spheroids for the study of monoclonal antibody therapy.

Tumor microenvironments present significant barriers to penetration by antibodies and immunoconjugates and are difficult to study in vitro. Cells cultured as monolayers typically exhibit less resistance to therapy than those grown in vivo. Therefore, it is important to develop an alternative research model that better represents in vivo tumors. We have developed a protocol to produce multicellular spheroids, a simple and more relevant model of in vivo tumors that allows for further investigations of the microenvironmental effects on drug penetration and tumor cell killing. The protocol is used to produce in vitro three-dimensional tumor spheroids from established human cancer cell lines and primary cancer cells isolated from patients without the use of any extracellular components. To study the ability of tumor-targeting immunoconjugates to penetrate these tumor spheroids in vitro, we have used an immunotoxin targeting mesothelin, a surface protein expressed in malignant mesotheliomas. This method for producing consistent, reproducible 3D spheroids may allow for improved testing of novel monoclonal antibodies and other agents for their ability to penetrate solid tumors for cancer therapy.

The development and characterization of a human mesothelioma in vitro 3D model to investigate immunotoxin therapy.

BACKGROUND: Tumor microenvironments present significant barriers to penetration by antibodies and immunoconjugates. Tumor microenvironments, however, are difficult to study in vitro. Cells cultured as monolayers exhibit less resistance to therapy than those grown in vivo and an alternative research model more representative of the in vivo tumor is more desirable. SS1P is an immunotoxin composed of the Fv portion of a mesothelin-specific antibody fused to a bacterial toxin that is presently undergoing clinical trials in mesothelioma. METHODOLOGY/PRINCIPAL FINDINGS: Here, we examined how the tumor microenvironment affects the penetration and killing activity of SS1P in a new three-dimensional (3D) spheroid model cultured in vitro using the human mesothelioma cell line (NCI-H226) and two primary cell lines isolated from the ascites of malignant mesothelioma patients. Mesothelioma cells grown as monolayers or as spheroids expressed comparable levels of mesothelin; however, spheroids were at least 100 times less affected by SS1P. To understand this disparity in cytotoxicity, we made fluorescence-labeled SS1P molecules and used confocal microscopy to examine the time course of SS1P penetration within spheroids. The penetration was limited after 4 hours. Interestingly, we found a significant increase in the number of tight junctions in the core area of spheroids by electron microscopy. Expression of E-Cadherin, a protein involved in the assembly and sealing of tight junctions and highly expressed in malignant mesothelioma, was found significantly increased in spheroids as compared to monolayers. Moreover, we found that siRNA silencing and antibody inhibition targeting E-Cadherin could enhance SS1P immunotoxin therapy in vitro. CONCLUSION/SIGNIFICANCE: This work is one of the first to investigate immunotoxins in 3D tumor spheroids in vitro. This initial description of an in vitro tumor model may offer a simple and more representative model of in vivo tumors and will allow for further investigations of the microenvironmental effects on drug penetration and tumor cell killing. We believe that the methods developed here may apply to the studies of other tumor-targeting antibodies and immunoconjugates in vitro.

Mesothelin as a potential therapeutic target in human cholangiocarcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) are the two most common primary liver cancers, yet there have been no significant advances in effective therapeutics. Mesothelin has been reported as a new therapeutic target in various types of cancer. Here, we investigated the expression of mesothelin in liver cancer and its potential role as a novel therapeutic target for immunotherapy. METHODS: HCC and CCA specimens were examined by immunohistochemistry for mesothelin expression. Protein expression was assessed by immunoblotting and flow cytometry. The SS1P immunotoxin targeting mesothelin was evaluated in the well-established CCA cell lines HuCCT1, HuH-28, KMBC, KMCH, Mz-ChA-1 and OZ. RESULTS: We showed strong immunochemical mesothelin staining in 33% of the surgically resected CCA specimens and 3 of 6 CCA cell lines (OZ, KMBC and KMCH). No mesothelin staining was found in HCC or normal liver tissue. Mesothelin was primarily localized to the cellular plasma membrane and the mature form (molecular weight, ~40 kDa) was expressed at a high level in CCA tissues. Moreover, 22% of CCA specimens had a high mesothelin expression level which was comparable to the CCA cell line models. Interestingly, SS1P showed very high and specific growth inhibition when added to mesothelin-expressing CCA cells with IC(50) values ranging from 0.5 to 11 ng/mL. CONCLUSIONS: Mesothelin is overexpressed in one-third of CCA tissues. SS1P targeting mesothelin reveals a remarkable single agent activity against CCA in vitro. These findings indicate a potential for SS1P in the immunotherapeutic treatment of CCA.

Blunted hepcidin response to oral iron challenge in HFE-related hemochromatosis.

Inadequate hepcidin synthesis leads to iron overload in HFE-related hemochromatosis. We explored the regulation of hepcidin by iron in 88 hemochromatosis patients (61 C282Y/C282Y, 27 C282Y/H63D) and 23 healthy controls by analyzing urinary hepcidin before and 24 hours after a 65-mg oral iron dose. Thirty-four patients were studied at diagnosis and had iron overload, and 54 patients were iron depleted. At diagnosis, hepcidin values in C282Y homozygotes were similar to controls, whereas values in C282Y/H63D heterozygotes were higher (P = .02). However, the hepcidin/ferritin ratio was decreased in both homozygotes (P < .001) and heterozygotes (P = .017), confirming the inadequate hepcidin production for the iron load with both genotypes. In iron-depleted patients of both genotypes studied at a time remote from phlebotomy, basal hepcidin was still lower than in controls (P < .001 for C282Y/C282Y and P = .002 for heterozygotes). After an iron challenge, mean urinary hepcidin excretion increased in controls (P = .001) but not patients, irrespective of genotype and iron status. Significant hepcidin increase ( > or = 10 ng/mg creatinine) was observed in 74% of controls, 15% of homozygotes, and 32% of heterozygotes. The hepcidin response to oral iron is blunted in HFE-related hemochromatosis and not improved after iron depletion. The findings support the involvement of HFE in iron sensing and subsequent regulation of hepcidin.