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Results for “Targeted expression”

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At least 433 records · Page 24Linked to original sources

PubMed2005

Src-like adaptor protein down-regulates T cell receptor (TCR)-CD3 expression by targeting TCRzeta for degradation.

Src-like adaptor protein (SLAP) down-regulates expression of the T cell receptor (TCR)-CD3 complex during a specific stage of thymocyte development when the TCR repertoire is selected. Consequently, SLAP-/- thymocytes display alterations in thymocyte development. Here, we have studied the mechanism of SLAP function. We demonstrate that SLAP-deficient thymocytes have increased TCRzeta chain expression as a result of a defect in TCRzeta degradation. Failure to degrade TCRzeta leads to an increased pool of fully assembled TCR-CD3 complexes that are capable of recycling back to the cell surface. We also provide evidence that SLAP functions in a pathway that requires the phosphorylated TCRzeta chain and the Src family kinase Lck, but not ZAP-70 (zeta-associated protein of 70 kD). These studies reveal a unique mechanism by which SLAP contributes to the regulation of TCR expression during a distinct stage of thymocyte development.

Animals↗

PubMed1997

Induced PAI-1 mRNA expression and targeted protein accumulation are early G1 events in serum-stimulated rat kidney cells.

Expression of plasminogen activator inhibitor type-1 (PAI-1), a member of the SERPIN gene family that functions to regulate the plasmin-based pericellular proteolytic cascade, is growth state-regulated in normal rat kidney (NRK) cells (Ryan and Higgins, 1990, J. Cell. Physiol., 155:376-384; Ryan et al., 1996, Biochem. J., 314:1041-1046). Comparative analysis of arrest states induced in NRK cells upon exposure to serum-deficient (0.5% FBS) or serum-free culture conditions served to define the kinetics of PAI-1 gene expression and fate of de novo-synthesized PAI-1 protein. While cells rendered quiescent in serum-free or serum-deficient media were equivalent with regard to the time course of PAI-1 mRNA induction, the level of expressed transcripts (27-fold vs. 12-fold) and accumulated saponin fraction PAI-1 protein (12-fold vs. 6-fold) were consistently greater in cells recruited into exponential growth phase from a serum-free as compared to a serum-deficient arrest condition. Relative PAI-1 mRNA abundance increased within 1-2 hr post-serum addition, was maximal at 4 hr, and declined rapidly thereafter; this time course of expression coupled with placement of entry into DNA synthetic phase at approximately 12 hr after stimulation indicates that PAI-1 induction is an early-to-mid G1 phase event. Induced PAI-1 protein was evident immunocytochemically within 2 hr of serum stimulation as a peripheral "rim" of accumulated protein restricted to the cellular ventral surface at the plane of the substrate. No PAI-1 was detected between individual cells suggesting that this protein may be targeted directly to the undersurface region. By 6 hr post-stimulation, the rim of PAI-1 deposition increased in intensity and broadened to occupy approximately 30 to 50% of the total undersurface area. Double-label immunocytochemistry indicated that accumulated PAI-1 was deposited in close proximity to, but not actually within, vinculin-containing focal contact structures. Potential functionality of induced PAI-1 expression to either the initiation or maintenance of the serum-stimulated phenotype was assessed using antibodies to PAI-1. The IgG fractions of two different antisera which neutralize the ability of PAI-1 to complex with and thereby inhibit the catalytic activity of urokinase plasminogen activator significantly reduced (by 25-35%) the incidence of cells displaying the serum-stimulated phenotype; antibodies that bind PAI-1 but do not block PAI-1 inhibitory activity were without effect. In view of the vagaries of antibody accessibility and in situ neutralizing activity (particularly in a region as structurally complex as the focal contact), these data may actually underestimate the importance of PAI-1 in maintaining the activated phenotype.

Animals↗

PubMed2000

T-cell killing of heterogenous tumor or viral targets with bispecific chimeric immune receptors.

We have previously described several novel chimeric immune receptors (CIRs) that redirect human T cells to kill malignant or HIV-infected cells. These CIRs comprise a cancer- or virus-specific ligand or single-chain antibody fused to the signaling domain of the T-cell receptor CD3-zeta subunit. Binding of the ligand- or antibody-based CIR to the target antigen (Ag) triggers T-cell-mediated cytolysis of the tumor- or virus-infected cell independent of target cell major histocompatibility complex class I expression. A new type of CIR was developed to mediate the lysis of cells that expressed one or more distinct viral or tumor Ags; three bispecific CIRs (BCIRs) were generated that recognized the carcinoembryonic Ag (CEA) and TAG-72 tumor Ags or, alternatively, distinct epitopes in the HIV envelope (HIVenv). T cells expressing the antitumoral Ag BCIR lysed both CEA- and TAG-72-expressing targets and did not kill Ag-negative targets or target cells expressing other members of the CEA family. Similarly, T cells expressing the anti-HIVenv BCIR lysed target cells expressing both the wild-type HIVenv and a mutant HIVenv that lacked the epitopes recognized by the monospecific CIRs. This approach permits the generation of T cells with a broader spectrum of activity capable of killing virus-infected cells and malignant cells and reduces the potential of progression of disease due to Ag loss variants.

Amino Acid Sequence↗

PubMed2002

Targeting gene expression in the preimplantation mouse embryo using morpholino antisense oligonucleotides.

Morpholino antisense oligonucleotides act by blocking translation of their target gene products and are effective tools for down-regulating gene expression. The current study was conducted to define treatment conditions for the use of morpholino oligonucleotides (MOs) in mammalian preimplantation embryos, and to employ MOs to target genes and study gene function in the early embryo. For the first time, ethoxylated polyethylenimine (EPEI), Lipofectin or Lysolecithin delivery agents were employed in combination with a fluorescent control MO and an alpha-catenin specific MO, to down-regulate gene expression during murine preimplantation development. Experiments applied to both two- and eight-cell stage murine preimplantation embryos contrasted the efficacy of MO concentrations of 1, 2, 5, 10, and 20 microM and treatment delivery times of 3, 6, 24, and 48 hr. Continuous treatment of two-cell embryos with Lipofectin and 20 microM alpha-catenin MO for 48 hr resulted in a significant (P < 0.05) reduction in development to the blastocyst stage and was accompanied by a marked reduction in alpha-catenin protein. These results indicate that morpholino antisense oligonucleotides are effective tools for down-regulating gene expression during mammalian preimplantation development.

Animals↗

PubMed2003

Down-regulation of Notch target gene expression by Suppressor of deltex.

In Drosophila, Suppressor of deltex (Su(dx)) mutations display a wing vein gap phenotype resembling that of Notch gain of function alleles. The Su(dx) protein may therefore act as a negative regulator of Notch but its activity on actual Notch signalling levels has not been demonstrated. Here we show that Su(dx) does regulate the level of Notch signalling in vivo, upstream of Notch target genes and in different developmental contexts, including a previously unknown role in leg joint formation. Overexpression of Su(dx) was capable of blocking both the endogenous activity of Notch and the ectopic Notch signalling induced by the overexpression of Deltex, an intracellular Notch binding protein. In addition, using the conditional phenotype of the Su(dx)(sp) allele, we show that loss of Su(dx) activity is rapidly followed by an up-regulation of E(spl)mbeta expression, the immediate target of Notch signal activation during wing vein development. While Su(dx) adult wing vein phenotypes are quite mild, only affecting the distal tips of the veins, we show that the initial consequence of loss of Su(dx) activity is more severe than previously thought. Using a time-course experiment we show that the phenotype is buffered by feedback regulation illustrating how signalling networks can make development robust to perturbation.

Alleles↗

PubMed1990

Expression of a chimeric VIP gene is targeted to the intestine in transgenic mice.

We showed previously that a gene construction that consisted of 5.2 kb of 5' flanking sequence, the first exon, and part of the first intron of the human gene encoding vasoactive intestinal peptide (VIP) fused to the reporter gene chloramphenicol acetyltransferase (CAT) fully mimicked the diverse behavior of the endogenous VIP gene when transfected into subclones of the human neuroblastoma cell line SK-N-SH (Waschek et al., 1988). To determine if the same sequences were sufficient to target expression of a reporter to VIP-producing tissues in the mouse, we initiated a pilot study in which we generated four transgenic mice or mouse lines that contained the VIPCAT fusion gene. Detectable levels of CAT were found in the ileum of either founder or offspring of each of the transgenic mouse lines. In all other tissues tested, CAT activity was either below the level of detection or the transgene was not expressed, with the exception of one mouse in which ectopic expression in the cerebellum was observed. The results indicate that the VIP sequences utilized were sufficient to direct expression of the transgene to the intestine, but not necessarily to other sites of VIP expression. To investigate what specific DNA sequences might confer VIP expression in the intestine and other sites, we analyzed further the VIP gene in SK-N-SH subclones using VIP/luciferase fusion gene constructions. A 0.6 kb DNA fragment located between 4.0 kb and 4.6 kb upstream from the VIP transcriptional start site was found to impart a high level of expression in one subclone and an increased degree of phorbol ester induction in another. These and other data indicate that multiple transcriptional elements control VIP expression in neuroblastoma cells and are candidates as mediators of VIP gene expression in the intact animal.

Animals↗

PubMed2000

Induction of human Cdc37 in prostate cancer correlates with the ability of targeted Cdc37 expression to promote prostatic hyperplasia.

The Cdc37 gene encodes a 50 kDa protein which targets intrinsically unstable oncoprotein kinases such as Cdk4, Raf-1, and src to the molecular chaperone Hsp90. This activity is thought to play an important role in the establishment of signaling pathways controlling cell proliferation. The budding yeast Cdc37 homolog is required for cell division and mammalian Cdc37 is expressed in proliferative zones during embryonic development and in adult tissues, consistent with a positive role in proliferation. Here we report that human prostatic tumors, neoplasias and certain pre-malignant lesions display increased Cdc37 expression, suggesting an important and early role for Cdc37 in prostatic transformation. To test the consequences of increased Cdc37 levels, transgenic mice expressing Cdc37 in the prostate were generated. These mice displayed a wide range of growth-related abnormalities including prostatic epithelial cell hyperplasia and dysplasia. These data suggest that the expression of Cdc37 may promote inappropriate proliferation and may be an important early step in the development of human prostate cancer.

Animals↗

PubMed1996

A novel population of CD4+CD56+ myelin-reactive T cells lyses target cells expressing CD56/neural cell adhesion molecule.

CD56 is a member of the neural cell adhesion molecule family expressed on cells of the central nervous system and also on NK cells. Previous studies suggest the involvement of CD56 in effector-to-target cell conjugation mediated by NK cells. It was shown recently that CD56 is also expressed by subpopulations of CD8+ and CD4+ T cells. The present study describes the functional characteristics of CD4+CD56+ T cell lines established from blood of multiple sclerosis patients by stimulation with myelin basic protein (MBP). CD4+CD56+, MBP-specific T cell lines were able to lyse MBP-pulsed target cells in an HLA class II-restricted fashion. At the same time, they mediated MHC-unrestricted lysis of CD56+ target cells such as CD56+ lymphoid or glial tumor cells, but not of the typical NK target, K562. A number of experimental results including separation of CD4+CD56+ T cells into CD56 high and low expressing populations, cold target inhibition, as well as killing of CD56-transfected cells indicate that homotypic CD56 interactions are involved in the MHC-unrestricted lysis. CD56 interactions are not sufficient but are required for effector/target interaction. Our findings raise the possibility that CD4+CD56+ T cells sharing properties of both typical Ag-specific Th0-like T cells and NK cells might be involved in damage of tissues expressing CD56/neural cell adhesion molecule, such as the central nervous system. Thus, we provide evidence for a novel mechanism that could lead to organ-specific autoreactivity.

Biomarkers↗

PubMed2006

Small interfering RNA expression vector targeting hypoxia-inducible factor 1 alpha inhibits tumor growth in hepatobiliary and pancreatic cancers.

Hepatobiliary and pancreatic carcinomas are hypovascular tumors that can proliferate under hypoxic conditions. Recent reports have demonstrated that hypoxia-inducible factor 1 alpha (HIF1alpha) plays an important role in the survival of these cancers. Given these findings, the inhibition of the HIF1alpha pathway might prove to be a powerful tool in the treatment of these cancers. To inhibit HIF1alpha expression, we used small interference RNA (siRNA) expression vectors in this study. The transient transfection of siRNA expression vectors significantly reduced both HIF1alpha mRNA levels (13% of control) and protein levels (41% of control) and significantly inhibited the growth of cancer cell lines (P<0.05). VEGF, Glut1, and aldorase A expressions were also significantly reduced by transfection with these vectors (P<0.05), and we found that these vectors induced apoptosis but not cell cycle arrest. In a subcutaneous tumor model using nude mice, transfected MIA PaCa-2 cells, stably expressing siRNAs, barely formed tumors compared to control (P<0.05). This study thus demonstrates the usefulness of siRNA expression vector in targeting HIF1alpha and points to a potential clinical role in the treatment of pancreatic and hepatobiliary carcinomas.

Animals↗

PubMed1995

Targeting gene expression to the vascular wall in transgenic mice using the murine preproendothelin-1 promoter.

To develop a system for overexpressing genes in the vascular wall, we created transgenic mice using the reporter gene luciferase and the murine preproendothelin-1 promoter. In vitro analysis suggested that the murine 5'-flanking region contained endothelial-specific elements in a 5.9-kb fragment. Five transgenic mice colonies established from independent founders all exhibited the highest level of luciferase activity in the aorta with up to 8,540 light units per microgram of protein. Immunohistochemistry with anti-luciferase antisera revealed high levels of expression in the endothelial cells of both large and small arteries and lower levels of expression in veins and capillaries. Significant expression was also seen in arterial smooth muscle cells and in select epithelial surfaces which is consistent with the known distribution of endothelin-1 in mammals. The further demonstrate the targeting capability of this system, we overexpressed the lipid-peroxidating enzyme, human 15-lipoxygenase, in the vessel wall of transgenic mice. As with luciferase, expression of active enzyme and immunohistochemical localization in vascular cells were documented in transgenic animals. Hence, this new system can be used to direct expression of molecules to the vascular wall for the purpose of examining the biological significance of either overexpression or inhibition of select proteins.

Animals↗

PubMed2006

Ketogenic HMGCS2 Is a c-Myc target gene expressed in differentiated cells of human colonic epithelium and down-regulated in colon cancer.

HMGCS2, the gene that regulates ketone body production, is expressed in liver and several extrahepatic tissues, such as the colon. In CaCo-2 colonic epithelial cells, the expression of this gene increases with cell differentiation. Accordingly, immunohistochemistry with specific antibodies shows that HMGCS2 is expressed mainly in differentiated cells of human colonic epithelium. Here, we used a chromatin immunoprecipitation assay to study the molecular mechanism responsible for this expression pattern. The assay revealed that HMGCS2 is a direct target of c-Myc, which represses HMGCS2 transcriptional activity. c-Myc transrepression is mediated by blockade of the transactivating activity of Miz-1, which occurs mainly through a Sp1-binding site in the proximal promoter of the gene. Accordingly, the expression of human HMGCS2 is down-regulated in 90% of Myc-dependent colon and rectum tumors. HMGCS2 protein expression is down-regulated preferentially in moderately and poorly differentiated carcinomas. In addition, it is also down-regulated in 80% of small intestine Myc-independent tumors. Based on these findings, we propose that ketogenesis is an undesirable metabolic characteristic of the proliferating cell, which is down-regulated through c-Myc-mediated repression of the key metabolic gene HMGCS2.

Acetylation↗

PubMed2006

Targeting iCre expression to murine progesterone receptor cell-lineages using bacterial artificial chromosome transgenesis.

Gene-targeting in embryonic stem cells has been the dominant genetic approach when engineering mouse models to query the physiologic importance of the progesterone receptor (PR). Although these models have been instrumental in disclosing the in vivo significance of the progesterone signaling pathway, generation of such mice exacts considerable expenditure of time, effort, and expense. Considering the growing list of new PR mouse models that are urgently required to address the next questions in progestin biology, bacterial artificial chromosome (BAC) recombineering in conjunction with transgenesis was evaluated as an alternative method to accelerate the creation of these models in the future. Using this approach, we describe the generation of three PR-BAC(iCre) transgenic lines in which improved Cre recombinase (iCre) was targeted in-frame, downstream, and under the control of the PR promoter contained within a BAC transgene. Crossing with the ROSA26R revealed that the PR-BAC(iCre) transgenic expresses active iCre only in cell-lineages that express the PR. The specificity of the PR-BAC(iCre) transgene not only underscores the importance of BAC-mediated transgenesis as a quick, easy, and affordable method by which to engineer the next generation of PR mouse models, but also provides a unique opportunity to investigate transcriptional control of PR expression as well as PR structure-function relationships in vivo.

Animals↗

PubMed1998

Expression of target genes by coinfection with replication-deficient viral vectors.

An in vivo transcription system was developed by coinfection of cells with replication-deficient viral vectors. Recombinant baculovirus (AcT7HCVLuc) and fowlpox virus (FPVT7HCVLuc) carrying a cDNA of the hepatitis C virus (HCV) minigene encoding the HCV 5' untranslated region (UTR), a luciferase gene and the 3' UTR, including the 98 nt extra sequence, under the control of the T7 promoter were constructed. The HCV minigene was synthesized in various cells by coinfection with one of these two viruses and recombinant baculovirus (AcCAT7) or adenovirus (AdexCAT7) expressing T7 RNA polymerase under the control of a mammalian promoter. Only a low level of luciferase expression was obtained in cells coinfected with AcT7HCVLuc and either AcCAT7 or AdexCAT7. In contrast, high-level luciferase expression was detected when the same cells were coinfected with FPVT7HCVLuc and either AcCAT7 or AdexCAT7. We further constructed a recombinant fowlpox virus with its HCV minigene extended to contain the whole HCV core protein region. Significantly high levels of expression of HCV core protein were detected in MT-2, COS7 and Vero cells by coinfection with the recombinant fowlpox virus and AdexCAT7. A coinfection system consisting of recombinant fowlpox virus and AdexCAT7 was established for high level of expression of a target gene in various cells.

Adenoviridae↗

PubMed2006

Human eosinophils have an intact Smad signaling pathway leading to a major transforming growth factor-beta target gene expression.

BACKGROUND: There is a paradoxical finding that eosinophils are frequently accumulated at the sites of allergic inflammation where transforming growth factor (TGF)-beta, a negative regulator of eosinophil survival, is upregulated; however, eosinophil accumulation is persistent. We thus hypothesized that eosinophils might have aberrant TGF-beta signaling and be unresponsive to TGF-beta. To test the hypothesis, we examined the expression and function of Smad proteins, which are central mediators for TGF-beta signaling, in human eosinophils. METHODS: Eosinophils were isolated from the peripheral blood of normal donors, and the expression and activation of endogenous Smad proteins were examined by reverse transcription polymerase chain reaction and Western blotting. The Smad function in the transcription of the major TGF-beta target gene Smad7 was investigated using a dominant negative form of Smad3. The effect of TGF-beta on eosinophil survival was then evaluated by a cell viability assay using normal and asthmatic eosinophils. RESULTS: Human eosinophils expressed mRNAs and proteins of TGF-beta typeI and type II receptors, Smad2, Smad3 and Smad4. TGF-beta induced the phosphorylation of Smad2 in eosinophils, which was blocked by SB431542, an inhibitor of TGF-beta type I receptor kinase. A dominant negative Smad3 protein suppressed TGF-beta-induced Smad7 mRNA expression in eosinophils. Finally, TGF-beta prevented granulocyte macrophage colony-stimulating factor- or interferon-gamma-mediated survival of eosinophils obtained from asthmatic patients as well as normal subjects. CONCLUSION: Human eosinophils have an intact Smad signaling pathway leading to a major TGF-beta target gene expression. Thus, eosinophils might become resistant to TGF-beta only in in vivo circumstances.

Activin Receptors, Type I↗

PubMed2000

A carboxyl-terminal region important for the expression and targeting of the skeletal muscle dihydropyridine receptor.

We have used the yeast two-hybrid technique and expression of truncated/mutated dihydropyridine receptors (DHPRs) to investigate whether the carboxyl tail of the DHPR is involved in targeting to junctions between the sarcolemma and sarcoplasmic reticulum in skeletal muscle. The carboxyl tail was extremely reactive in yeast two-hybrid library screens, with the reactivity residing in amino acids 1621-1647 and abolished by a point mutation (V1642D). Dysgenic myotubes were injected with cDNA encoding green fluorescent protein fused to the amino terminus of DHPRs truncated after either residue 1620 (Delta1621-1873) or residue 1542 (Delta1543-1873) or of full-length DHPRs with the V1642D mutation (V1642D). For either Delta1621-1873 or V1642D, the restoration of excitation-contraction coupling was reduced approximately 40%, and the number of functional DHPRs in the sarcolemma was reduced approximately 30%, compared with the wild-type DHPR. The restoration of excitation-contraction coupling and surface expression was more drastically reduced (by approximately 90 and approximately 55%, respectively) for Delta1543-1873. Fluorescence microscopy revealed that Delta1621-1873 and V1642D were concentrated in a longitudinally restricted region near the injected nucleus, whereas wild-type DHPRs were present relatively uniformly along the length of a myotube. The intensity of fluorescence was greatly reduced for Delta1543-1873, indicating a low level of protein expression. Thus, residues 1543-1647 appear to play a role in the biosynthetic processing, transport, and/or anchoring of DHPRs, with residues 1543-1620 being particularly important for expression.

Animals↗

PubMedDate not supplied

Trastuzumab-polyethylenimine-polyethylene glycol conjugates for targeting Her2-expressing tumors.

In this study, we describe the synthesis and characterization of a conjugate consisting of poly(ethylene glycol 2,000 Da)(10)-graft-poly(ethylene imine 25 kDa) (PEG-PEI) covalently coupled to Trastuzumab (Herceptin) via N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) for specific gene delivery to Her2-expressing cell lines. The efficiency of DNA condensation was studied using an ethidium bromide exclusion assay and demonstrated negligible differences compared to PEG-PEI. Conjugate complex sizes were determined by dynamic light scattering to be in the range 130-180 nm. zeta potentials at different N/P ratios were close to neutral. Flow cytometry and confocal microscopy revealed efficient binding and uptake of Trastuzumab-PEI-PEG complexes using Her2-positive SK-BR-3 cells. In contrast, binding and uptake into Her2-negative OVCAR-3 cells was negligible. In good correlation with these findings, reporter gene expression using targeted complexes in SK-BR-3 cells was up to sevenfold higher than that of unmodified PEG-PEI complexes. With the use OVCAR-3 cells, no significant difference in expression efficiencies could be observed between conjugate and PEG-PEI complexes. Inhibition experiments with free Trastuzumab showed a significant decrease in reporter gene expression using SK-BR-3 cells but no decrease using OVCAR-3 cells, strongly supporting a specific Her2-receptor-mediated uptake mechanism. Our results suggest that Trastuzumab-PEI-PEG might be a promising new bioconjugate for targeted gene transfer to Her2-positive tumor cells in vivo.

Antibodies, Monoclonal↗

PubMed1999

Expression and targeting of the apoptosis inhibitor, survivin, in human melanoma.

The newly described apoptosis inhibitor survivin is expressed in many human cancers and appears to play a critical part in both apoptosis regulation and cell cycle progression. Its potential role in malignant melanoma is unknown. In a panel of 30 malignant melanomas, survivin was strongly expressed in all cases (15 of 15) of metastatic malignant melanomas and 13 of 15 cases of invasive malignant melanomas by immunohistochemistry. In invasive malignant melanomas, survivin was also expressed in the in-situ component of the lesion. Survivin expression was found in all cases (11 of 11) of nevi, but not in melanocytes in sections of normal skin. The apoptosis inhibitor bcl-2 was expressed in 26 of 30 cases, but generally at lower levels than that of infiltrating lymphocytes. The mitotic index, as assessed by MIB-1 staining, was consistently higher in metastatic than invasive malignant melanomas. Assessment of apoptotic index by in situ end-labeling revealed extremely low rates of apoptosis in most malignant melanomas. Survivin expression by western blotting was detected in four human metastatic malignant melanoma cell lines but not in cultured normal human melanocytes. Transfection of both YUSAC-2 and LOX malignant melanoma cells with green fluorescence protein-conjugated survivin anti-sense or green fluorescence protein-conjugated survivin dominant negative mutant (Cys84Ala) [corrected] resulted in increased apoptosis in the absence of other genotoxic stimuli. Two-color flow cytometry confirmed that YUSAC-2 cells transfected with survivin anti-sense expressed less endogenous survivin and exhibited an increased fraction of cells with sub-G1 DNA content. These data demonstrate that apoptosis inhibition by survivin may participate in the onset and progression of malignant melanomas, and suggest that therapeutic targeting of survivin may be beneficial in patients with recurrent or metastatic disease.

Adult↗

PubMed2003

Expression and targeting of human fibroblast activation protein in a human skin/severe combined immunodeficient mouse breast cancer xenograft model.

Antigens and receptors that are highly expressed on tumor stromal cells, such as fibroblast activation protein (FAP), are attractive targets for antibody-based therapies because the supporting stroma and vessel network is essential for a solid neoplasm to grow beyond a size of 1-2 mm. The in vivo characterization of antibodies targeting human stromal or vessel antigens is hindered by the lack of an appropriate mouse model system because xenografts in standard mouse models express stromal and vessels elements of murine origin. This limitation may be overcome by the development of a human skin/mouse chimeric model, which is established by transplanting human foreskin on to the lateral flank of severe combined immunodeficient mice. The subsequent inoculation of breast carcinoma MCF-7 cells within the dermis of the transplanted human skin resulted in the production of xenografts expressing stromal and vessel elements of human origin. Widespread expression of human FAP-positive reactive stromal fibroblasts within xenografts was seen up to 2 months posttransplantation and postinjection of cells. Human blood vessel antigen expression also persisted at 2 months posttransplantation and postinjection of cells with murine vessels coexisting with the human vascular supply. The model was subsequently used to evaluate the biodistribution properties of an iodine-131-labeled humanized anti-FAP monoclonal antibody (BIBH-7). The results showed high specific targeting of the stromal compartment of the xenograft, indicating that the model provides a useful and novel approach for the in vivo assessment of the immunotherapeutic potential of molecules targeting human stroma and angiogenic systems.

Animals↗