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Hypoxia is linked to epithelial-mesenchymal transition (EMT) and tumor progression in numerous carcinomas. Responses to hypoxia are thought to operate via hypoxia-inducible factors (HIFs), but the importance of co-factors that regulate HIF signaling within tumors is not well understood. Here, we elucidate a signaling pathway that physically and functionally couples tyrosine phosphorylation of β-catenin to HIF1α signaling and HIF1α-mediated tumor EMT. Primary human lung adenocarcinomas accumulate pY654-β-catenin and HIF1α. All pY654-β-catenin, and only the tyrosine phosphorylated form, was found complexed with HIF1α and active Src, both within the human tumors and in lung tumor cell lines exposed to hypoxia. Phosphorylation of Y654, generated by hypoxia mediated, reactive oxygen species (ROS)-dependent Src kinase activation, was required for β-catenin to interact with HIF1α and Src, to promote HIF1α transcriptional activity, and for hypoxia-induced EMT. Mice bearing hypoxic pancreatic islet adenomas, generated by treatment with anti-vascular endothelial growth factor antibodies, accumulate HIF1α/pY654-β-catenin complexes and develop an invasive phenotype. Concurrent administration of the ROS inhibitor N-acetylcysteine abrogated β-catenin/HIF pathway activity and restored adenoma architecture. Collectively, the findings implicate accumulation of pY654-β-catenin specifically complexed to HIF1α and Src kinase as critically involved in HIF1α signaling and tumor invasion. The findings also suggest that targeting ROS-dependent aspects of the pY654-β-catenin/ HIF1α pathway may attenuate untoward biological effects of anti-angiogenic agents and tumor hypoxia.

Objectives: The purpose of our study was to assess the chondrogenic potential and the MR signal effects of GadofluorineM-Cy labeled matrix associated stem cell implants (MASI) in pig knee specimen. Materials and Methods: Human mesenchymal stem cells (hMSCs

Thalidomide, which inhibits angiogenesis in certain tumor types, reduced extravasation of a macromolecular contrast medium (MMCM) in a human breast cancer model as assayed by MMCM-enhanced dynamic magnetic resonance imaging (MRI) and fluorescence microscopy in the same tumors. After a 1-week, three-dose course of thalidomide, the mean MRI-assayed endothelial transfer coefficient, K(PS), decreased significantly (p < 0.05) from 19.4 +/- 9.1 to 6.3 +/- 9.1 microl/min.100 cm(3). Correspondingly, microscopic measurements of extravasated MMCM, expressed as fractional area of streptavidin staining, were significantly (p < 0.05) lower in thalidomide-treated tumors (18.6 +/- 11.9%) than in control saline-treated tumors (50.2 +/- 2.3%). On a tumor-by-tumor basis, post-treatment K(PS) values correlated significantly (r(2) = 0.55, p < 0.05) with microscopic measures of MMCM extravasation. However, no significant differences were observed between saline- and thalidomide-treated tumors with respect to rate of growth, vascular richness, or amount of VEGF-containing cells. Because of its sensitivity to the detection of changes in vascular leakage in tumors, this MMCM-enhanced MRI assay could prove useful for monitoring the effects of thalidomide on an individual patient basis. The significant correlation between MRI and fluorescence microscopic measures of MMCM extravasation supports the utility of the non-invasive MRI approach for assessing the action of thalidomide on tumor blood vessels. [ABSTRACT FROM AUTHOR]

Neoantigens are peptides derived from non-synonymous mutations presented by human leukocyte antigens (HLAs), which are recognized by antitumour T cells 1-14 . The large HLA allele diversity and limiting clinical samples have restricted the study of the landscape of neoantigen-targeted T cell responses in patients over their treatment course. Here we applied recently developed technologies 15-17 to capture neoantigen-specific T cells from blood and tumours from patients with metastatic melanoma with or without response to anti-programmed death receptor 1 (PD-1) immunotherapy. We generated personalized libraries of neoantigen-HLA capture reagents to single-cell isolate the T cells and clone their T cell receptors (neoTCRs). Multiple T cells with different neoTCR sequences (T cell clonotypes) recognized a limited number of mutations in samples from seven patients with long-lasting clinical responses. These neoTCR clonotypes were recurrently detected over time in the blood and tumour. Samples from four patients with no response to anti-PD-1 also demonstrated neoantigen-specific T cell responses in the blood and tumour to a restricted number of mutations with lower TCR polyclonality and were not recurrently detected in sequential samples. Reconstitution of the neoTCRs in donor T cells using non-viral CRISPR-Cas9 gene editing demonstrated specific recognition and cytotoxicity to patient-matched melanoma cell lines. Thus, effective anti-PD-1 immunotherapy is associated with the presence of polyclonal CD8 + T cells in the tumour and blood specific for a limited number of immunodominant mutations, which are recurrently recognized over time., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells 1-3 . Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRβ). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated., (© 2022. The Author(s).)

Oncolytic viruses pose many questions in their use in cancer therapy. In this study, we assessed the potential of mpJX-594 (mouse-prototype JX-594), a replication-competent vaccinia virus administered by intravenous injection, to target the tumor vasculature, produce immune activation and tumor cell killing more widespread than the infection, and suppress invasion and metastasis. These actions were examined in RIP-Tag2 transgenic mice with pancreatic neuroendocrine tumors that developed spontaneously and progressed as in humans. mpJX-594 initially infected tumor vascular endothelial cells, leading to vascular pruning and prolonged leakage in tumors but not in normal organs; parallel effects were observed in U87 gliomas. Viral infection spread to tumor cells, where tumor cell killing was much more widespread than the infection. Widespread tumor cell killing at 5 days was prevented by depletion of CD8 + T lymphocytes and did not require GM-CSF, as mpJX-594 variants that expressed human, mouse, or no GM-CSF produced equivalent amounts of killing. The antivascular, antitumor, and antimetastatic effects of mpJX-594 were amplified by concurrent or sequential administration of sunitinib, a multitargeted receptor tyrosine kinase inhibitor. These effects were not mimicked by selective inhibition of VEGFR2 despite equivalent vascular pruning, but were accompanied by suppression of regulatory T cells and greater influx of activated CD8 + T cells. Together, our results showed that mpJX-594 targets tumor blood vessels, spreads secondarily to tumor cells, and produces widespread CD8 + T-cell-dependent tumor cell killing in primary tumors and metastases, and that these effects can be amplified by coadministration of sunitinib. Significance: These findings reveal multiple unrecognized features of the antitumor properties of oncolytic vaccinia viruses, all of which can be amplified by the multitargeted kinase inhibitor sunitinib. Cancer Res; 78(4); 922-37. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Poxvirus-based active immunotherapies mediate anti-tumor efficacy by triggering broad and durable Th1 dominated T cell responses against the tumor. While monotherapy significantly delays tumor growth, it often does not lead to complete tumor regression. It was hypothesized that the induced robust infiltration of IFNγ-producing T cells into the tumor could provoke an adaptive immune evasive response by the tumor through the upregulation of PD-L1 expression. In therapeutic CT26-HER-2 tumor models, MVA-BN-HER2 poxvirus immunotherapy resulted in significant tumor growth delay accompanied by a robust, tumor-infiltrating T cell response that was characterized by low to mid-levels of PD-1 expression on T cells. As hypothesized, this response was countered by significantly increased PD-L1 expression on the tumor and, unexpectedly, also on infiltrating T cells. Synergistic benefit of anti-tumor therapy was observed when MVA-BN-HER2 immunotherapy was combined with PD-1 immune checkpoint blockade. Interestingly, PD-1 blockade stimulated a second immune checkpoint molecule, LAG-3, to be expressed on T cells. Combining MVA-BN-HER2 immunotherapy with dual PD-1 plus LAG-3 blockade resulted in comprehensive tumor regression in all mice treated with the triple combination therapy. Subsequent rejection of tumors lacking the HER-2 antigen by treatment-responsive mice without further therapy six months after the original challenge demonstrated long lasting memory and suggested that effective T cell immunity to novel, non-targeted tumor antigens (antigen spread) had occurred. These data support the clinical investigation of this triple therapy regimen, especially in cancer patients harboring PD-L1neg/low tumors unlikely to benefit from immune checkpoint blockade alone.

About 43 million individuals in the U.S. encounter cartilage injuries due to trauma or osteoarthritis, leading to joint pain and functional disability. Matrix-associated stem cell implants (MASI) represent a promising approach for repair of cartilage defects. However, limited survival of MASI creates a significant bottleneck for successful cartilage regeneration outcomes and functional reconstitution. We report an approach for noninvasive detection of stem cell apoptosis with magnetic resonance imaging (MRI), based on a caspase-3-sensitive nanoaggregation MRI probe (C-SNAM). C-SNAM self-assembles into nanoparticles after hydrolysis by caspase-3, leading to 90% amplification of (1)H MR signal and prolonged in vivo retention. Following intra-articular injection, C-SNAM causes significant MR signal enhancement in apoptotic MASI compared to viable MASI. Our results indicate that C-SNAM functions as an imaging probe for stem cell apoptosis in MASI. This concept could be applied to a broad range of cell transplants and target sites.

Alternative pathways to the VEGF, such as hepatocyte growth factor or HGF/c-met, are emerging as key players in tumor angiogenesis and resistance to anti-VEGF therapies. The aim of this study was to assess the effects of a combination strategy targeting the VEGF and c-met pathways in clear cell renal cell carcinoma (ccRCC) models. Male SCID mice (8/group) were implanted with 786-O tumor pieces and treated with either a selective VEGF receptor tyrosine kinase inhibitor, axitinib (36 mg/kg, 2×/day); a c-met inhibitor, crizotinib (25 mg/kg, 1×/day); or combination. We further tested this drug combination in a human ccRCC patient-derived xenograft, RP-R-01, in both VEGF-targeted therapy-sensitive and -resistant models. To evaluate the resistant phenotype, we established an RP-R-01 sunitinib-resistant model by continuous sunitinib treatment (60 mg/kg, 1×/day) of RP-R-01-bearing mice. Treatment with single-agent crizotinib reduced tumor vascularization but failed to inhibit tumor growth in either model, despite also a significant increase of c-met expression and phosphorylation in the sunitinib-resistant tumors. In contrast, axitinib treatment was effective in inhibiting angiogenesis and tumor growth in both models, with its antitumor effect significantly increased by the combined treatment with crizotinib, independently from c-met expression. Combination treatment also induced prolonged survival and significant tumor growth inhibition in the 786-O human RCC model. Overall, our results support the rationale for the clinical testing of combined VEGF and HGF/c-met pathway blockade in the treatment of ccRCC, both in first- and second-line setting., (©2014 American Association for Cancer Research.)

Purpose: To determine whether intravenous ferumoxytol can be used to effectively label mesenchymal stem cells (MSCs) in vivo and can be used for tracking of stem cell transplants., Materials and Methods: This study was approved by the institutional animal care and use committee. Sprague-Dawley rats (6-8 weeks old) were injected with ferumoxytol 48 hours prior to extraction of MSCs from bone marrow. Ferumoxytol uptake by these MSCs was evaluated with fluorescence, confocal, and electron microscopy and compared with results of traditional ex vivo-labeling procedures. The in vivo-labeled cells were subsequently transplanted in osteochondral defects of 14 knees of seven athymic rats and were evaluated with magnetic resonance (MR) imaging up to 4 weeks after transplantation. T2 relaxation times of in vivo-labeled MSC transplants and unlabeled control transplants were compared by using t tests. MR data were correlated with histopathologic results., Results: In vivo-labeled MSCs demonstrated significantly higher ferumoxytol uptake compared with ex vivo-labeled cells. With electron microscopy, iron oxide nanoparticles were localized in secondary lysosomes. In vivo-labeled cells demonstrated significant T2 shortening effects in vitro and in vivo when they were compared with unlabeled control cells (T2 in vivo, 15.4 vs 24.4 msec; P < .05) and could be tracked in osteochondral defects for 4 weeks. Histologic examination confirmed the presence of iron in labeled transplants and defect remodeling., Conclusion: Intravenous ferumoxytol can be used to effectively label MSCs in vivo and can be used for tracking of stem cell transplants with MR imaging. This method eliminates risks of contamination and biologic alteration of MSCs associated with ex vivo-labeling procedures., (© RSNA, 2013.)

Inhibition of VEGF signaling can promote lymph node metastasis in preclinical models, but the mechanism is not fully understood, and successful methods of prevention have not been found. Signaling of hepatocyte growth factor (HGF) and its receptor c-Met can promote the growth of lymphatics and metastasis of some tumors. We sought to explore the contributions of c-Met signaling to lymph node metastasis after inhibition of VEGF signaling. In particular, we examined whether c-Met is upregulated in lymphatics in or near pancreatic neuroendocrine tumors in RIP-Tag2 transgenic mice and whether lymph node metastasis can be reduced by concurrent inhibition of VEGF and c-Met signaling. Inhibition of VEGF signaling by anti-VEGF antibody or sunitinib in mice from the age of 14 to 17 weeks was accompanied by more intratumoral lymphatics, more tumor cells inside lymphatics, and more lymph node metastases. Under these conditions, lymphatic endothelial cells, like tumor cells, had strong immunoreactivity for c-Met and phospho-c-Met. c-Met blockade by the selective inhibitor, PF-04217903, significantly reduced metastasis to local lymph nodes. Together, these results indicate that inhibition of VEGF signaling in RIP-Tag2 mice upregulates c-Met expression in lymphatic endothelial cells, increases the number of intratumoral lymphatics and number of tumor cells within lymphatics, and promotes metastasis to local lymph nodes. Prevention of lymph node metastasis by PF-04217903 in this setting implicates c-Met signaling in tumor cell spread to lymph nodes., (©2013 AACR.)

Selective inhibition of vascular endothelial growth factor (VEGF) increases the efficacy of chemotherapy and has beneficial effects on multiple advanced cancers, but response is often limited and the disease eventually progresses. Changes in the tumour microenvironment--hypoxia among them--that result from vascular pruning, suppressed angiogenesis and other consequences of VEGF inhibition can promote escape and tumour progression. New therapeutic approaches that target pathways that are involved in the escape mechanisms add the benefits of blocking tumour progression to those of slowing tumour growth by inhibiting angiogenesis.

The purpose of this study was to (1) compare three different techniques for ferumoxide labeling of mesenchymal stem cells (MSCs), (2) evaluate if ferumoxide labeling allows in vivo tracking of matrix-associated stem cell implants (MASIs) in an animal model, and (3) compare the magnetic resonance imaging (MRI) characteristics of ferumoxide-labeled viable and apoptotic MSCs. MSCs labeled with ferumoxide by simple incubation, protamine transfection, or Lipofectin transfection were evaluated with MRI and histopathology. Ferumoxide-labeled and unlabeled viable and apoptotic MSCs in osteochondral defects of rat knee joints were evaluated over 12 weeks with MRI. Signal to noise ratios (SNRs) of viable and apoptotic labeled MASIs were tested for significant differences using t-tests. A simple incubation labeling protocol demonstrated the best compromise between significant magnetic resonance signal effects and preserved cell viability and potential for immediate clinical translation. Labeled viable and apoptotic MASIs did not show significant differences in SNR. Labeled viable but not apoptotic MSCs demonstrated an increasing area of T2 signal loss over time, which correlated to stem cell proliferation at the transplantation site. Histopathology confirmed successful engraftment of viable MSCs. The engraftment of iron oxide-labeled MASIs by simple incubation can be monitored over several weeks with MRI. Viable and apoptotic MASIs can be distinguished via imaging signs of cell proliferation at the transplantation site.

Purpose: To correlate dynamic MRI assays of macromolecular endothelial permeability with microscopic area-density measurements of vascular endothelial growth factor (VEGF) in tumors., Methods and Material: This study compared tumor xenografts from two different human cancer cell lines, MDA-MB-231 tumors (n=5), and MDA-MB-435 (n=8), reported to express respectively higher and lower levels of VEGF. Dynamic MRI was enhanced by a prototype macromolecular contrast medium (MMCM), albumin-(Gd-DTPA)35. Quantitative estimates of tumor microvascular permeability (K(PS); μl/min × 100 cm(3)), obtained using a two-compartment kinetic model, were correlated with immunohistochemical measurements of VEGF in each tumor., Results: Mean K(PS) was 2.4 times greater in MDA-MB-231 tumors (K(PS)=58 ± 30.9 μl/min × 100 cm(3)) than in MDA-MB-435 tumors (K(PS)=24 ± 8.4 μl/min × 100 cm(3)) (p<0.05). Correspondingly, the area-density of VEGF in MDA-MB-231 tumors was 2.6 times greater (27.3 ± 2.2%, p<0.05) than in MDA-MB-435 cancers (10.5 ± 0.5%, p<0.05). Considering all tumors without regard to cell type, a significant positive correlation (r=0.67, p<0.05) was observed between MRI-estimated endothelial permeability and VEGF immunoreactivity., Conclusion: Correlation of MRI assays of endothelial permeability to a MMCM and VEGF immunoreactivity of tumors support the hypothesis that VEGF is a major contributor to increased macromolecular permeability in cancers. When applied clinically, the MMCM-enhanced MRI approach could help to optimize the appropriate application of VEGF-inhibiting therapy on an individual patient basis., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Unlabelled: Invasion and metastasis increase after the inhibition of VEGF signaling in some preclinical tumor models. In the present study we asked whether selective VEGF inhibition is sufficient to increase invasion and metastasis and whether selective c-Met inhibition is sufficient to block this effect. Treatment of pancreatic neuroendocrine tumors in RIP-Tag2 mice with a neutralizing anti-VEGF antibody reduced tumor burden but increased tumor hypoxia, hypoxia-inducible factor-1α, and c-Met activation and also increased invasion and metastasis. However, invasion and metastasis were reduced by concurrent inhibition of c-Met by PF-04217903 or PF-02341066 (crizotinib). A similar benefit was found in orthotopic Panc-1 pancreatic carcinomas treated with sunitinib plus PF-04217903 and in RIP-Tag2 tumors treated with XL184 (cabozantinib), which simultaneously blocks VEGF and c-Met signaling. These findings document that invasion and metastasis are promoted by selective inhibition of VEGF signaling and can be reduced by the concurrent inhibition of c-Met., Significance: This report examines the mechanism of increased tumor aggressiveness after anti-VEGF therapy and presents evidence for roles of vascular pruning, hypoxia, and c-Met activation. The results show that simultaneous inhibition of c-Met and VEGF signaling not only slows tumor growth but also reduces invasion and metastasis., (©2012 AACR.)

Objectives: The purpose of our study was to assess the chondrogenic potential and the MR signal effects of GadofluorineM-Cy labeled matrix associated stem cell implants (MASI) in pig knee specimen., Materials and Methods: Human mesenchymal stem cells (hMSCs) were labeled with the micelle-based contrast agent GadofluorineM-Cy. Ferucarbotran-labeled hMSCs, non-labeled hMSCs and scaffold only served as controls. Chondrogenic differentiation was induced and gene expression and histologic evaluation were performed. The proportions of spindle-shaped vs. round cells of chondrogenic pellets were compared between experimental groups using the Fisher's exact test. Labeled and unlabeled hMSCs and chondrocytes in scaffolds were implanted into cartilage defects of porcine femoral condyles and underwent MR imaging with T1- and T2-weighted SE and GE sequences. Contrast-to-noise ratios (CNR) between implants and adjacent cartilage were determined and analyzed for significant differences between different experimental groups using the Kruskal-Wallis test. Significance was assigned for p<0.017, considering a Bonferroni correction for multiple comparisons., Results: Collagen type II gene expression levels were not significantly different between different groups (p>0.017). However, hMSC differentiation into chondrocytes was superior for unlabeled and GadofluorineM-Cy-labeled cells compared with Ferucarbotran-labeled cells, as evidenced by a significantly higher proportion of spindle cells in chondrogenic pellets (p<0.05). GadofluorineM-Cy-labeled hMSCs and chondrocytes showed a positive signal effect on T1-weighted images and a negative signal effect on T2-weighted images while Ferucarbotran-labeled cells provided a negative signal effect on all sequences. CNR data for both GadofluorineM-Cy-labeled and Ferucarbotran-labeled hMSCs were significantly different compared to unlabeled control cells on T1-weighted SE and T2*-weighted MR images (p<0.017)., Conclusion: hMSCs can be labeled by simple incubation with GadofluorineM-Cy. The labeled cells provide significant MR signal effects and less impaired chondrogenesis compared to Ferucarbotran-labeled hMSCs. Thus, GadoflurineM-Cy might represent an alternative MR cell marker to Ferucarbotran, which is not distributed any more in Europe or North America.

Angiogenesis inhibitors that block VEGF receptor (VEGFR) signaling slow the growth of many types of tumors, but eventually the disease progresses. Multiple strategies are being explored to improve efficacy by concurrent inhibition of other functionally relevant receptor tyrosine kinases (RTK). XL880 (foretinib, GSK1363089) and XL184 (cabozantinib) are small-molecule inhibitors that potently block multiple RTKs, including VEGFR and the receptor of hepatocyte growth factor c-Met, which can drive tumor invasion and metastasis. This study compared the cellular effects of XL880 and XL184 with those of an RTK inhibitor (XL999) that blocks VEGFR but not c-Met. Treatment of RIP-Tag2 mice with XL999 resulted in 43% reduction in vascularity of spontaneous pancreatic islet tumors over 7 days, but treatment with XL880 or XL184 eliminated approximately 80% of the tumor vasculature, reduced pericytes and empty basement membrane sleeves, caused widespread intratumoral hypoxia and tumor cell apoptosis, and slowed regrowth of the tumor vasculature after drug withdrawal. Importantly, XL880 and XL184 also decreased invasiveness of primary tumors and reduced metastasis. Overall, these findings indicate that inhibition of c-Met and functionally related kinases amplifies the effects of VEGFR blockade and leads to rapid, robust, and progressive regression of tumor vasculature, increased intratumoral hypoxia and apoptosis, and reduced tumor invasiveness and metastasis., (©2011 AACR.)

Inhibition of platelet-derived growth factor-B (PDGF-B) has multiple effects on tumors, including loss of pericytes, regression of some vessels, normalization of other vessels, and reduction of interstitial pressure. PDGF-B inhibition also increases the efficacy of cancer therapeutics, but the role on tumor vessel efficiency and drug delivery is unclear. We sought to determine whether inhibition of PDGF-B signaling can increase delivery and efficacy of cyclophosphamide in Lewis lung carcinomas or RIP-Tag2 tumors. PDGF-B blockade in Lewis lung carcinoma tumors by the DNA aptamer AX102 for 14 days increased the number of perfused tumor vessels marked by lectin in the bloodstream by 50%. AX102 also increased the width of sleeves of viable tumor cells around blood vessels by 66%, increased tumor cell proliferation by 90%, and increased intratumoral delivery of Hoechst 33342 by 78%. A low dose of cyclophosphamide (20 mg/kg) reduced tumor cell proliferation by 31% when combined with AX102 but not when given alone. Synergy of cyclophosphamide and AX102 on tumor cell proliferation also was found in RIP-Tag2 tumors. Similarly, the PDGF receptor signaling inhibitor imatinib increased delivery of cyclophosphamide and reduced tumor burden in RIP-Tag2 mice, without evidence of tumor cell sensitization to chemotherapy. Together, these findings indicate that inhibition of PDGF-B signaling promotes the delivery and efficacy of chemotherapeutic agents by increasing the efficiency of tumor blood vessels., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Purpose: This study aims to determine the effect of human mesenchymal stem cell (hMSC) labeling with the fluorescent dye DiD and the iron oxide nanoparticle ferucarbotran on chondrogenesis., Procedures: hMSCs were labeled with DiD alone or with DiD and ferucarbotran (DiD/ferucarbotran). hMSCs underwent confocal microscopy, optical imaging (OI), and magnetic resonance (MR) imaging. Chondrogenesis was induced by transforming growth factor-b and confirmed by histopathology and glycosaminoglycan (GAG) production. Data of labeled and unlabeled hMSCs were compared with a t test., Results: Cellular uptake of DiD and ferucarbotran was confirmed with confocal microscopy. DiD labeling caused a significant fluorescence on OI, and ferucarbotran labeling caused a significant T2* effect on MR images. Compared to nonlabeled controls, progenies of labeled MSCs exhibited similar chondrocyte morphology after chondrogenic differentiation, but the labeled cells demonstrated significantly reduced GAG production (p < 0.05)., Conclusion: DiD and DiD/ferucarbotran labeling of hMSC does not interfere with cell viability or morphologic differentiation into chondrocytes, but labeled cells exhibit significantly less GAG production compared to unlabeled cells.

Objective: To compare magnetic resonance (MR) signal characteristics of contrast agent-labeled apoptotic and viable human mesenchymal stem cells (hMSCs) in matrix-associated stem cell implants., Methods: hMSCs were labeled with Food and Drug Administration-approved ferumoxides nanoparticles. One group (A) remained untreated whereas a second group (B) underwent mitomycin C-induced apoptosis induction. Viability of group A and apoptosis of group B was confirmed by caspase-assays and terminal dUTP nick-end labeling (TUNEL) stains. Labeled viable hMSCs, unlabeled viable hMSCs, labeled apoptotic hMSCs, and unlabeled apoptotic hMSCs (n = 7 samples each) in an agarose scaffold were implanted into cartilage defects of porcine patellae specimens and underwent MR imaging at 7 T, using T1-weighted spin-echo sequences, T2-weighted spin-echo sequences, and T2*-weighted gradient-echo sequences. Signal-to-noise ratios (SNR) of the implants were calculated and compared between different experimental groups using linear mixed regression models., Results: Ferumoxides-labeled hMSCs provided a strong negative T2 and T2*-enhancement. Corresponding SNR data of labeled hMSCs were significantly lower compared with unlabeled controls (P < 0.05). Apoptosis induction resulted in a significant signal decline of ferumoxides-labeled hMSC transplants on short echo time T2-weighted spinecho sequences. SNR data of labeled apoptotic hMSCs were significantly lower compared with labeled viable hMSCs (P < 0.05)., Conclusion: Apoptosis of transplanted ferumoxides-labeled stem cells in cartilage defects can be visualized noninvasively by a significant signal decline on T2-weighted MR images. The described MR signal characteristics may serve as a noninvasive outcome measure for the assessment of matrix-associated stem cell implants in clinical practice. Additional studies are needed to further enhance the observed differences between viable and apoptotic cells, for example, by further optimizing the applied MR pulse sequence parameters or intracellular contrast agent concentration.

Purpose: To determine the effects of MRI-assayed vascular leakiness on the delivery of macromolecular therapeutics to tumors., Materials and Methods: MDA-MB 435 tumors, subcutaneously implanted into nude rats were treated with a single dose of bevacizumab at levels of 0.1 mg (n = 5) or 1.0 mg (n = 10) or received saline (control animals, n = 8). After 24 hours, albumin-(Gd-DTPA) (30)-enhanced MRI was performed. Just prior to MRI, the cytotoxic drug vinorelbine was administered intravenously. Upon completion of the MR experiment, tumor vinorelbine concentrations were quantified by high performance liquid chromatography (HPLC). Vascular leakiness (K (PS)) was calculated based on the MRI data using a pharmacokinetic model., Results: K (PS) was calculated as 3.70 +/- 1.12 (control tumors), 1.95 +/- 0.70 (0.1 mg group) and 0.75 +/- 0.46 microl min (-1)cm (-3) (1.0 mg group). K (PS) was significantly higher in the control group compared to the 1.0 mg bevacizumab group. Vinorelbine concentrations were measured as 409.4 +/- 109.7 (control tumors), 387.5 +/- 47.5 (0.1 mg group) and 250.7 +/- 71.9 (1.0 mg group). These differences were not significant. A moderate and significant correlation was found between K (PS) and Vinorelbine concentrations in tumors (r = 0.49, p < 0.05)., Conclusion: MRI-assayed K (PS) based on dynamic MRI enhanced by albumin-(Gd-DTPA) (30) correlated significantly with vinorelbine accumulation in experimental xenograft tumors under angiogenesis inhibition. Thus, the MRI technique applied in our study could potentially help to predict accumulation of macromolecular cytotoxic drugs and to optimize individual therapeutic regimes in tumors.

The objective of this work is to establish an optical imaging technique that would enable monitoring of the integration of mesenchymal stem cells (MSC) in arthritic joints. Our approach is based on first developing a labeling technique of MSC with the fluorescent dye DiD followed by tracking the cell migration kinetics from the spatial distribution of the DiD fluorescence in optical images (OI). The experimental approach involves first the in vitro OI of MSC labeled with DiD accompanied by fluorescence microscopy measurements to establish localization of the signal within the cells. Thereafter, DiD-labeled MSC were injected into polyarthritic, athymic rats and the signal localization within the experimental animals was monitored over several days. The experimental results indicate that DiD integrated into the cell membrane. DiD-labeled MSC localization in the arthritic ankle joints was observed with OI indicating that this method can be applied to monitor MSC in arthritic joints.

The purpose of this study was to define the feasibility of dynamic contrast-enhanced magnetic resonance imaging (MRI) to estimate the vascular density and leakiness of spontaneous islet cell tumors in RIP-Tag2 transgenic mice. Dynamic T(1)-weighted spoiled gradient echo (SPGR) imaging at 2.0 T was performed in 17 RIP-Tag2 mice using a prototype blood pool macromolecular contrast medium (MMCM), albumin-(Gd-DTPA)(35). Kinetic analysis of the dynamic enhancement responses based on a two-compartment model was used to estimate fractional plasma volume (fPV) and the coefficient of endothelial permeability (K(PS)) for each tumor. The MRI estimate of fPV was correlated on a tumor-by-tumor basis with corresponding microscopic measurements of vascular density. The fPV assays by MMCM-enhanced imaging ranged from 2.4%-14.1% of tissue volume. Individual tumor fPV values correlated significantly (r = 0.79, P < 0.001) with the corresponding microscopic estimates of vascularity consisting of the combined area densities of lectin-perfused microvessels plus erythrocyte-stained blood lakes. A biotinylated derivative of the albumin-based MMCM confirmed extravasation of the contrast agent from some tumor blood vessels and accumulation in 25% of blood lakes. The K(PS) values ranged from 0 (no detectable leak) to 0.356 mL/min/100 cm(3). Dynamic MMCM-enhanced MRI is feasible in RIP-Tag2 pancreatic tumors, yielding estimates of vascular permeability and microscopically validated measurements of vascular richness.

Human mesenchymal stem cells (hMSCs) were labeled with Ferucarbotran by simple incubation and cultured for up to 14 d. Iron content was determined by spectrometry and the intracellular localization of the contrast agent uptake was studied by electron and confocal microscopy. At various time points after labeling, ranging from 1 to 14 d, samples with viable or lysed labeled hMSCs, as well as nonlabeled controls, underwent MRI. Spin-echo (SE) and gradient-echo (GE) sequences with multiple TRs and TEs were used at 1.5T and 3T on a clinical scanner. Spectrometry showed an initial iron oxide uptake of 7.08 pg per cell. Microscopy studies revealed lysosomal compartmentalization. Contrast agent effects of hMSCs were persistent for up to 14 d after labeling. A marked difference in the T(2) effect of compartmentalized iron oxides compared to free iron oxides was found on T(2)-weighted sequences, but not on T(2)*-weighted sequences. The observed differences may be explained by the loss of compartmentalization of iron oxide particles, the uniformity of distribution, and the subsequent increase in dephasing of protons on SE images. These results show that viable cells with compartmentalized iron oxides may-in principle-be distinguished from lysed cells or released iron oxides., ((c) 2009 Wiley-Liss, Inc.)

For in vivo applications of magnetically labeled stem cells, biological effects of the labeling procedure have to be precluded. This study evaluates the effect of different ferucarbotran cell labeling protocols on chondrogenic differentiation of human mesenchymal stem cells (hMSC) as well as their implications for MR imaging. hMSC were labeled with ferucarbotran using various protocols: cells were labeled with 100 microg Fe/ml for 4 and 18 h and additional samples were cultured for 6 or 12 days after the 18 h labeling. Supplementary samples were labeled by transfection with protamine sulfate. Iron uptake was quantified by ICP-spectrometry and labeled cells were investigated by transmission electron microscopy and by immunostaining for ferucarbotran. The differentiation potential of labeled cells was compared with unlabeled controls by staining with Alcian blue and Hematoxylin and Eosin, then quantified by measurements of glucosaminoglycans (GAG). Contrast agent effect at 3 T was investigated on days 1 and 14 of chondrogenic differentiation by measuring signal-to-noise ratios on T(2)-SE and T(2)*-GE sequences. Iron uptake was significant for all labeling protocols (p < 0.05). The uptake was highest after transfection with protamine sulfate (25.65 +/- 3.96 pg/cell) and lowest at an incubation time of 4 h without transfection (3.21 +/- 0.21 pg/cell). While chondrogenic differentiation was decreased using all labeling protocols, the decrease in GAG synthesis was not significant after labeling for 4 h without transfection. After labeling by simple incubation, chondrogenesis was found to be dose-dependent. MR imaging showed markedly lower SNR values of all labeled cells compared with the unlabeled controls. This contrast agent effect persisted for 14 days and the duration of differentiation. Magnetic labeling of hMSC with ferucarbotran inhibits chondrogenesis in a dose-dependent manner when using simple incubation techniques. When decreasing the incubation time to 4 h, inhibition of chondrogenesis was not significant., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

Vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and their receptors are important targets in cancer therapy based on angiogenesis inhibition. However, it is unclear whether inhibition of VEGF and PDGF together is more effective than inhibition of either one alone. Here, we used two contrasting tumor models to compare the effects of inhibiting VEGF or PDGF alone, by adenovirally generated soluble receptors, to the effects of inhibiting both together. In RIP-Tag2 tumors, VEGF and PDGF inhibition together reduced tumor vascularity and abundance of pericytes. However, VEGF inhibition reduced tumor vascularity without decreasing pericyte density, and PDGF inhibition reduced pericytes without reducing tumor vascularity. By contrast, in Lewis lung carcinomas (LLC), inhibition of VEGF or PDGF reduced blood vessels and pericytes to the same extent as did inhibition of both together. Similar results were obtained using tyrosine kinase inhibitors AG-013736 and imatinib. In LLC, VEGF expression was largely restricted to pericytes and PDGF was largely restricted to endothelial cells, but, in RIP-Tag2 tumors, expression of both growth factors was more widespread and significantly greater than in LLC. These findings suggest that inhibition of PDGF in LLC reduced pericytes, and then tumor vessels regressed because pericytes were the main source of VEGF. The vasculature of RIP-Tag2 tumors, in which most VEGF is from tumor cells, was more resistant to PDGF inhibition. The findings emphasize the interdependence of pericytes and endothelial cells in tumors and the importance of tumor phenotype in determining the cellular effects of VEGF and PDGF inhibitors on tumor vessels.

The simultaneous targeting of both endothelial cells and pericytes via inhibition of VEGF receptor (VEGFR) and PDGFbeta receptor (PDGFRbeta) signaling, respectively, has been proposed to enhance the efficacy of antiangiogenic tumor therapy. Clinical and preclinical modeling of combined VEGFR and PDGFRbeta signaling inhibition, however, has used small molecule kinase inhibitors with inherently broad substrate specificities, precluding detailed examination of this hypothesis. Here, adenoviral expression of a soluble VEGFR2/Flk1 ectodomain (Ad Flk1-Fc) in combination with a soluble ectodomain of PDGFRbeta (Ad sPDGFRbeta) allowed highly selective inhibition of these pathways. The activity of Ad sPDGFRbeta was validated in vitro against PDGF-BB and in vivo with near-complete blockade of pericyte recruitment in the angiogenic corpus luteum, resulting in prominent hemorrhage, thus demonstrating an essential function for PDGF signaling during ovarian angiogenesis. Combination therapy with Ad PDGFRbeta and submaximal doses of Ad Flk1-Fc produced modest additive antitumor effects; however, no additivity was observed with maximal VEGF inhibition in numerous s.c. models. Notably, VEGF inhibition via Ad Flk1-Fc was sufficient to strongly suppress tumor endothelial and pericyte content as well as intratumoral PDGF-B mRNA, obscuring additive Ad sPDGFRbeta effects on pericytes or tumor volume. These studies using highly specific soluble receptors suggest that additivity between VEGFR and PDGFRbeta inhibition depends on the strength of VEGF blockade and appears minimal under conditions of maximal VEGF antagonism.

Objectives: To evaluate the potential of quantitative assays of vascular characteristics based on dynamic contrast-enhanced magnetic resonance imaging (MRI) using a macromolecular contrast medium (MMCM) to search for and measure effects of dimethyl sulfoxide (DMSO) on cancer vasculature with microscopic correlations., Material and Methods: Saline-treated control (n = 8) and DMSO-treated (n = 7) human breast cancer xenografts (MDA-MB-435) in rats were imaged dynamically by MMCM-enhanced MRI using albumin-(Gd-DTPA)27-(biotin)11 (molecular weight approximately 90 kDa), before and after a 1-week, 3-dose treatment course. After the posttreatment MRI examinations, tumors were perfused with lectin and fixative and subsequently stained with RECA-1 and streptavidin for quantitative fluorescent microscopy. Quantitative MRI estimates of cancer microvessel permeability (KPS; microL/min.100 cm3) and fractional plasma volume (fPV; %) were based on a 2-compartment kinetic model. Fluorescent microscopy yielded estimates of MMCM extravasation and vascular density that were compared to the MRI results., Results: DMSO decreased cancer vascular endothelial permeability significantly (P < 0.05) from tumor KPSday0 = 19.3 +/- 8.8 microL/min.100 cm3 to KPSday7 = 0 microL/min.100 cm3). K values in the saline-treated tumors did not change significantly. The amount of extravasated albumin-Gd-(DTPA)27-(biotin)11, as assayed by a fluorescently labeled streptavidin stain that strongly binds to the biotin tag on the MMCM, was significantly (P < 0.05) lower in the DMSO-treated cancers than in the control cancers (57.7% +/- 5.5% vs. 34.2% +/- 4.9%). Tumor vascular richness as reflected by the MRI-assayed fPV and by the RECA-1 and lectin-stained microscopy did not change significantly with DMSO or saline treatment., Conclusion: Reductions in cancer microvascular leakiness induced by a 7-day course of DMSO could be detected and measured by dynamic MMCM-enhanced MRI and were confirmed by microscopic measurements of the leaked macromolecular agents in the same cancers. Results support the robustness of an MMCM-enhanced MRI approach to the characterization of cancers and providing first evidence for an in vivo effect of DMSO on cancer blood vessels.

Purpose: To prospectively evaluate in rats the acute change in tumor vascular leakiness (K(PS)) assayed at magnetic resonance (MR) imaging after a single dose of the angiogenesis inhibitor bevacizumab as a predictive biomarker of tumor growth response after a prolonged treatment course., Materials and Methods: Institutional animal care and use committee approval was obtained. Seventeen female rats with implanted human breast cancers underwent dynamic albumin-(Gd-DTPA)(30)-enhanced MR imaging followed by an initial dose of bevacizumab or saline (as a control). Treatment was continued every 3rd day, for a total of four doses at five possible dose levels: 0 mg bevacizumab (n = 4 [control rats]), 0.1 mg bevacizumab (n = 3), 0.25 mg bevacizumab (n = 2), 0.5 mg bevacizumab (n = 5), and 1.0 mg bevacizumab (n = 3). A second MR imaging examination was performed 24 hours after the initial dose to enable calculation of the acute change in MR imaging-assayed leakiness, or Delta K(PS). This acute change in K(PS) at MR imaging was correlated with tumor growth response for each cancer at the completion of the 11-day treatment course. For statistical analyses, an unpaired two-tailed t test, analysis of variance, and linear regression analyses were used., Results: The MR imaging-assayed change in tumor microvascular leakiness, tested as a potential biomarker, correlated strongly with tumor growth rate (R(2) = 0.74, P < .001). K(PS) and tumor growth decreased significantly in all bevacizumab-treated cancers compared with these values in control group cancers (P < .05)., Conclusion: The MR imaging-assayed acute change in vascular leakiness after a single dose of bevacizumab was an early, measurable predictive biomarker of tumor angiogenesis treatment response., ((c) RSNA, 2008.)

Inhibition of platelet derived growth factor (PDGF) can increase the efficacy of other cancer therapeutics, but the cellular mechanism is incompletely understood. We examined the cellular effects on tumor vasculature of a novel DNA oligonucleotide aptamer (AX102) that selectively binds PDGF-B. Treatment with AX102 led to progressive reduction of pericytes, identified by PDGF receptor beta, NG2, desmin, or alpha-smooth muscle actin immunoreactivity, in Lewis lung carcinomas. The decrease ranged from 35% at 2 days, 63% at 7 days, to 85% at 28 days. Most tumor vessels that lacked pericytes at 7 days subsequently regressed. Overall tumor vascularity decreased 79% over 28 days, without a corresponding decrease in tumor size. Regression of pericytes and endothelial cells led to empty basement membrane sleeves, which were visible at 7 days, but only 54% remained at 28 days. PDGF-B inhibition had a less pronounced effect on pancreatic islet tumors in RIP-Tag2 transgenic mice, where pericytes decreased 47%, vascularity decreased 38%, and basement membrane sleeves decreased 21% over 28 days. Taken together, these findings show that inhibition of PDGF-B signaling can lead to regression of tumor vessels, but the magnitude is tumor specific and does not necessarily retard tumor growth. Loss of pericytes in tumors is an expected direct consequence of PDGF-B blockade, but reduced tumor vascularity is likely to be secondary to pericyte regression.

The purpose of this study was to label human monocytes with Gadofluorine M by simple incubation for subsequent cell depiction at 1.5 and 3 T. Gadofluorine M displays a high r(1) relaxivity and is spontaneously phagocytosed by macrophages. Human monocytes were incubated with Gadofluorine M-Cy at varying concentrations and incubation times and underwent MR imaging at 1.5 and 3 T at increasing time intervals after the labeling procedure. R1-relaxation rates and r1 relaxivities of the labeled cells and non-labeled controls were determined. Cellular contrast agent uptake was examined by fluorescence microscopy and quantified by ICP-AES. Efficient cell labeling was achieved after incubation of the cells with 25 mM Gd Gadofluorine M for 12 h, resulting in a maximal uptake of 0.3 fmol Gd/cell without impairment of cell viability. Fluorescence microscopy confirmed internalization of the fluorescent contrast agent by monocytes. The r1 relaxivity of the labeled cells was 137 mM(-1)s(-1) at 1.5 T and 80.46 mM(-1)s(-1) at 3 T. Imaging studies showed stable labeling for at least 7 days. Human monocytes can be effectively labeled for MR imaging with Gadofluorine M. Potential in vivo cell-tracking applications include targeting of inflammatory processes with Gadofluorine-labeled leukocytes or monitoring of stem cell therapies for the treatment of arthritis.

Unlike during development, blood vessels in the adult are generally thought not to require VEGF for normal function. However, VEGF is a survival factor for many tumor vessels, and there are clues that some normal blood vessels may also depend on VEGF. In this study, we sought to identify which, if any, vascular beds in adult mice depend on VEGF for survival. Mice were treated with a small-molecule VEGF receptor (VEGFR) tyrosine kinase inhibitor or soluble VEGFRs for 1-3 wk. Blood vessels were assessed using immunohistochemistry or scanning or transmission electron microscopy. In a study of 17 normal organs after VEGF inhibition, we found significant capillary regression in pancreatic islets, thyroid, adrenal cortex, pituitary, choroid plexus, small-intestinal villi, and epididymal adipose tissue. The amount of regression was dose dependent and varied from organ to organ, with a maximum of 68% in thyroid, but was less in normal organs than in tumors in RIP-Tag2-transgenic mice or in Lewis lung carcinoma. VEGF-dependent capillaries were fenestrated, expressed high levels of both VEGFR-2 and VEGFR-3, and had normal pericyte coverage. Surviving capillaries in affected organs had fewer fenestrations and less VEGFR expression. All mice appeared healthy, but distinct physiological changes, including more efficient blood glucose handling, accompanied some regimens of VEGF inhibition. Strikingly, most capillaries in the thyroid grew back within 2 wk after cessation of treatment for 1 wk. Our findings of VEGF dependency of normal fenestrated capillaries and rapid regrowth after regression demonstrate the plasticity of the adult microvasculature.

The vasculature of the embryo requires vascular endothelial growth factor (VEGF) during development, but most adult blood vessels lose VEGF dependence. However, some capillaries in the respiratory tract and selected other organs of adult mice regress after VEGF inhibition. The present study sought to identify the sequence of events and the fate of endothelial cells, pericytes, and vascular basement membrane during capillary regression in mouse tracheas after VEGF signaling was blocked with a VEGF-receptor tyrosine kinase inhibitor AG-013736 or soluble receptor construct (VEGF Trap or soluble adenoviral VEGFR-1). Within 1 day, patency was lost and fibrin accumulated in some tracheal capillaries. Apoptotic endothelial cells marked by activated caspase-3 were present in capillaries without blood flow. VEGF inhibition was accompanied by a 19% decrease in tracheal capillaries over 7 days and 30% over 21 days. During this period, desmin/NG2-immunoreactive pericytes moved away from regressing capillaries onto surviving vessels. Empty sleeves of basement membrane, left behind by regressing endothelial cells, persisted for about 2 wk and served as a scaffold for vascular regrowth after treatment ended. The amount of regrowth was limited by the number of surviving basement membrane sleeves. These findings demonstrate that, after inhibition of VEGF signaling, some normal capillaries regress in a systematic sequence of events initiated by a cessation of blood flow and followed by apoptosis of endothelial cells, migration of pericytes away from regressing vessels, and formation of empty basement membrane sleeves that can facilitate capillary regrowth.

The cytokine/extracellular matrix protein osteopontin (OPN/Eta-1) is an important component of cellular immunity and inflammation. It also acts as a survival, cell-adhesive, and chemotactic factor for endothelial cells. Here, subtractive suppression hybridization showed that serum-deprived murine aortic endothelial (MAE) cells transfected with the angiogenic fibroblast growth factor-2 (FGF2) overexpress OPN compared with parental cells. This was confirmed by Northern blotting and Western blot analysis of the conditioned media in different clones of endothelial cells overexpressing FGF2 and in endothelial cells treated with the recombinant growth factor. In vivo, FGF2 caused OPN expression in newly formed endothelium of the chick embryo chorioallantoic membrane (CAM) and of murine s.c. Matrigel plug implants. Recombinant OPN (rOPN), the fusion protein GST-OPN, and the deletion mutant GST-DeltaRGD-OPN were angiogenic in the CAM assay. Angiogenesis was also triggered by OPN-transfected MAE cells grafted onto the CAM. OPN-driven neovascularization was independent from endothelial alpha(v)beta(3) integrin engagement and was always paralleled by the appearance of a massive mononuclear cell infiltrate. Accordingly, rOPN, GST-OPN, GST-DeltaRGD-OPN, and the conditioned medium of OPN-overexpressing MAE cells were chemotactic for isolated human monocytes. Also, rOPN triggered a proangiogenic phenotype in human monocytes by inducing the expression of the angiogenic cytokines TNF-alpha and IL-8. OPN-mediated recruitment of proangiogenic monocytes may represent a mechanism of amplification of FGF2-induced neovascularization during inflammation, wound healing, and tumor growth.

Tumors express more than a single angiogenic growth factor. To investigate the relative impact of fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) on tumor growth and neovascularization, we generated tumor cell transfectants differing for VEGF and/or FGF-2 expression. Human endometrial adenocarcinoma HEC-1-B-derived Tet-FGF-2 cells that express FGF-2 under the control of the tetracycline-responsive promoter (Tet-off system) were further transfected with a VEGF(121) anti-sense (AS-VEGF) cDNA. Next, Tet-FGF-2 and AS-VEGF/Tet-FGF-2 cells were transplanted subcutaneously in nude mice that received tetracycline or not in the drinking water. Simultaneous expression of FGF-2 and VEGF in Tet-FGF-2 cells resulted in fast-growing lesions characterized by high blood vessel density, patency and permeability, and limited necrosis. Blood vessels were highly heterogeneous in size and frequently associated with pericytes. Inhibition of FGF-2 production by tetracycline caused a significant decrease in tumor burden paralleled by a decrease in blood vessel density and size. AS-VEGF expression resulted in a similar reduction in blood vessel density associated with a significant decrease in pericyte organization, vascular patency, and permeability. The consequent decrease in tumor burden was paralleled by increased tumor hypoxia and necrosis. A limited additional inhibitory effect was exerted by simultaneous down-regulation of FGF-2 and VEGF expression. These findings demonstrate that FGF-2 and VEGF stimulate vascularization synergistically but with distinctive effects on vessel functionality and tumor survival. Blockade of either one of the two growth factors results in a decrease in blood vessel density and, consequently, in tumor burden. However, inhibition of the expression of VEGF, but not of FGF-2, affects also vessel maturation and functionality, leading to tumor hypoxia and necrosis. Our experimental model represents an unique tool to investigate anti-neoplastic therapies in different angiogenic environments.

Fibroblast growth factor-2 (FGF2) exerts paracrine and autocrine functions on endothelial cells. FGF2-overexpressing murine aortic endothelial cells (FGF2-T-MAE cells) induce opportunistic hemangioendothelioma-like tumors when inoculated in immunodeficient mice. To evaluate the impact of FGF2-mediated activation on gene expression profile in transformed endothelial cells, we performed subtractive suppression hybridization analysis between FGF2-T-MAE cells and parental MAE cells. The two cell populations were compared for differential gene expression also by gene macroarray hybridization with 32P-labeled cDNAs. The two approaches allowed the identification of 27 transcripts whose expression was upregulated by FGF2 in endothelial cells. With the exception of one unknown gene, the differentially expressed transcripts encoded for proteins involved in the modulation of cell cycle, differentiation, and cell adhesion. Among them, the stress-inducible genes A170, GADD45 and GADD153 are upregulated by FGF2 transfection or recombinant growth factor treatment. Their expression was also induced in vascular tumors originated by parental or FGF2-transfected MAE cells in nude mice. This study extends the number of genes involved in tumor angiogenesis and/or endothelial cell transformation, a finding with possible implications for the discovery of novel targets for angiostatic therapy.