Origin of the vasculature supporting growth of primary patient tumor xenografts.

BACKGROUND: Studies of primary patient tumor xenografts grown in immunodeficient mice have shown that these tumors histologically and genetically closely resemble the original tumors. These patient xenograft models are becoming widely used for therapeutic efficacy studies. Because many therapies are directed at tumor stromal components and because the tumor microenvironment also is known to influence the response of a tumor to therapy, it is important to understand the nature of the stroma and, in particular, the vascular supply of patient xenografts. METHODS: Patient tumor xenografts were established by implanting undisrupted pieces of patient tumors in SCID mice. For this study, formalin fixed, paraffin embedded specimens from several types of solid tumors were selected and, using species-specific antibodies which react with formalin fixed antigens, we analyzed the species origin of the stroma and blood vessels that supported tumor growth in these models. Additionally, we investigated the kinetics of the vascularization process in a colon tumor and a mesothelioma xenograft. In mice bearing a head and neck xenograft, a perfusion study was performed to compare the functionality of the human and mouse tumor vessels. RESULTS: In patient tumors which successfully engrafted, the human stroma and vessels which were engrafted as part of the original tumor did not survive and were no longer detectable at the time of first passage (15-25 weeks). Uniformly, the stroma and vessels supporting the growth of these tumors were of murine origin. The results of the kinetic studies showed that the loss of the human vessels and vascularization by host vessels occurred more rapidly in a colon tumor (by 3 weeks) than in a mesothelioma (by 9 weeks). Finally, the perfusion studies revealed that while mouse vessels in the periphery of the tumor were perfused, those in the central regions were rarely perfused. No vessels of human origin were detected in this model. CONCLUSIONS: In the tumors we investigated, we found no evidence that the human stromal cells and vessels contained in the original implant either survived or contributed in any substantive way to the growth of these xenografts.

AGE-RAGE in multiple sclerosis brain.

This pilot study used immunohistochemical techniques to investigate the advanced glycation end-product (AGE) Nepsilon-(carboxymethyl)lysine (CML) and its receptor (RAGE) in the brains of multiple sclerosis (MS) patients, comparing them with the brains of patients with Alzheimer's disease (AD) (positive controls) and with age-matched control subjects (negative controls). Postmortem slides derived from the hippocampi of MS patients, AD patients, and controls were stained with monoclonal antibodies for CML and human RAGE. Results showed increased AGE and RAGE immunostaining in the hippocampi of MS patients, similar to AD patients.

Hypoxia-inducible factor-1α protein expression is associated with poor survival in normal karyotype adult acute myeloid leukemia.

We examined the predictive impact of HIF-1α protein expression on clinical outcome of 84 normal karyotype acute myeloid leukemia (NK-AML) patients (median age 66.5 years) at our institute. Thirty percent of NK-AML cells expressed cytoplasmic HIF-1α. In univariate analysis, low HIF-1α (≤ 5%, n = 66) was associated with improved event-free survival (p = 0.0453, HR = 0.22). Multivariate analysis incorporating age, complete remission, FLT3-ITD mutation, and marrow blast percentage demonstrated that HIF-1α was independently associated with poorer overall and event-free survival. HIF-1α expression correlated with VEGF-C but not VEGF-A, marrow angiogenesis, FLT3 ITD or NPM1 mutations. These results support HIF-1α as an outcome marker for NK-AML.

Double immunohistochemical staining method for HIF-1alpha and its regulators PHD2 and PHD3 in formalin-fixed paraffin-embedded tissues.

Hypoxia-inducible factor (HIF-1alpha) is expressed in the nuclei of tumor cells under hypoxic conditions, and is regulated, in part, by cytoplasmic prolyl hydroxylases (PHDs). As HIF-1alpha is selectively expressed in tumor cells, inhibitors are being developed for cancer therapy. Although methods for the detection of HIF-1alpha and PHDs are available, an immunohistochemical double staining method for these markers in individual tumor cells is not available. For method development a human squamous cell carcinoma (SCC) xenograft, A253, was used as a known positive control tissue for HIF-1alpha in well-differentiated areas without microvessels. This laboratory showed that tumor cells in these areas are strongly positive for hypoxia markers. Another human, poorly differentiated SCC xenograft, FaDu, without hypoxic areas, was used as a negative control. PHD2 and 3 immunostaining was optimized individually using the human kidney. To optimize HIF-1alpha detection the pressure cooker time for antigen retrieval, concentration of the primary antibody, amplification reagent, and DAB development time were decreased. Casein blocking further decreased the background. Double staining resulted in brown nuclei for HIF-1alpha (DAB), and pink cytoplasmic staining for PHD2, 3 (fast red). The isotype-matched controls were negative. Normal human tissues had no detectable HIF-1alpha, but expressed PHD2, 3. The potential use of this new and improved method was confirmed by analyzing 15 surgical biopsies of oropharyngeal SCC of which 6 were positive for HIF-1alpha. This new method defined the optimal conditions for detection of HIF-1alpha and PHDs in individual tumor cells and could have a diagnostic and therapeutic potential.

Se-methylselenocysteine sensitizes hypoxic tumor cells to irinotecan by targeting hypoxia-inducible factor 1alpha.

PURPOSE: Hypoxic tumor cells overexpressing hypoxia-inducible factor 1alpha (HIF-1alpha) are generally resistant to chemo/radiotherapy. We have reported that Se-methylselenocysteine (MSC) therapeutically enhances the efficacy and selectivity of irinotecan against human tumor xenografts. The aim of this study was to delineate the mechanism responsible for the observed efficacy targeting on HIF-1alpha and its transcriptionally regulated genes VEGF and CAIX. METHODS: We investigated the mechanism of HIF-1alpha inhibition by MSC and its critical role in the therapeutic outcome by generating HIF-1alpha stable knockdown (KD) human head and neck squamous cell carcinoma, FaDu by transfecting HIF-1alpha short hairpin RNA. RESULTS: While cytotoxic efficacy in combination with methylselenic acid (MSA) with SN-38 (active metabolites of MSC and irinotecan) could not be confirmed in vitro against normoxic tumor cells, the hypoxic tumor cells were more sensitive to the combination. Reduction in HIF-1alpha either by MSA or shRNA knockdown resulted in significant increase in cytotoxicity of SN38 in vitro against hypoxic, but not the normoxic tumor cells. Similarly, in vivo, either MSC in combination with irinotecan treatment of parental xenografts or HIF-1alpha KD tumors treated with irinotecan alone resulted in comparable therapeutic response and increase in the long-term survival of mice bearing FaDu xenografts. CONCLUSIONS: Our results show that HIF-1alpha is a critical target for MSC and its inhibition was associated with enhanced antitumor activity of irinotecan. Inhibition of HIF-1alpha appeared to be mediated through stabilization of PHD2, 3 and downregulation of ROS by MSC. Thus, our findings support the development of MSC as a HIF-1alpha inhibitor in combination chemotherapy.

Inactivation of LGI1 expression accompanies early stage hyperplasia of prostate epithelium in the TRAMP murine model of prostate cancer.

The LGI1 gene has been implicated in tumor cell invasion through regulation of the ERK pathway. To determine whether human prostate cancer cells (PC3, 22RV, Du145) are similarly affected by exposure to LGI1, we conducted scratch wound assays and demonstrated that the secreted LGI1 protein can reduce cell motility, an essential component of invasion and metastasis. These studies have now been extended to an in vivo mouse model of prostate cancer. Using a BAC transgenic mouse expressing a GFP reporter gene under the control of cis regulatory elements, we demonstrated that LGI1 is highly expressed in the normal prostate epithelium. To determine whether loss of LGI1 expression is associated with development and progression of murine prostate cancer, we bred the GFP reporter BAC transgenic mice with TRAMP mice which undergo early hyperplasia and progressive stages of prostate cancer. In the F1 animals, although the surrounding normal prostate epithelium expressed high levels of LGI1 in the double transgenic mice, the LGI1 gene had been inactivated even at the earliest stages of hyperplasia. This observation supports the suggestion that inactivation of LGI1 in certain cell types is related to tumor progression. Taken together these results suggest that LGI1 may be an important molecule for the arrest of prostate cancer cell invasion and possibly as a biomarker for early detection of prostate hyperplasia.

The Vps33a gene regulates behavior and cerebellar Purkinje cell number.

A mutation in the Vps33a gene causes Hermansky-Pudlak Syndrome (HPS)-like-symptoms in the buff (bf) mouse mutant. The encoded product, Vps33a, is a member of the Sec1 and Class C multi-protein complex that regulates vesicle trafficking to specialized lysosome-related organelles. As Sec1 signaling pathways have been implicated in pre-synaptic function, we examined brain size, cerebellar cell number and the behavioral phenotype of bf mutants. Standardized behavioral tests (SHIRPA protocols) demonstrated significant motor deficits (e.g., grip strength, righting reflex and touch escape) in bf mutants, worsening with age. Histological examination of brain revealed significant Purkinje cell loss that was confirmed with staining for calbindin, a calcium binding protein enriched in Purkinje cells. This pathologic finding was progressive, as older bf mutants (13-14 months) showed a greater attrition of neurons, with their cerebella appearing to be particularly reduced (approximately 30%) in size relative to those of age-matched-control cohorts. These studies suggest that loss of Purkinje neurons is the most obvious neurological atrophy in the bf mutant, a structural change that generates motor coordination deficits and impaired postural phenotypes. It is conceivable therefore that death of cerebellar cells may also be a clinical feature of HPS patients, a pathological event which has not been reported in the literature. In general, the bf mutant may be a potentially new and useful model for understanding Purkinje cell development and function.

Tumor vascular maturation and improved drug delivery induced by methylselenocysteine leads to therapeutic synergy with anticancer drugs.

PURPOSE: Our previously reported therapeutic synergy between naturally occurring seleno-amino acid methylselenocysteine (MSC) and anticancer drugs could not be shown in vitro. Studies were carried out to investigate the potential role of MSC-induced tumor vascular maturation and increased drug delivery in the observed therapeutic synergy in vivo. EXPERIMENTAL DESIGN: Mice bearing s.c. FaDu human head and neck squamous cell carcinoma xenografts were treated with MSC (0.2 mg/d x 14 days orally). Changes in microvessel density (CD31), vascular maturation (CD31/alpha-smooth muscle actin), perfusion (Hoechst 33342/DiOC7), and permeability (dynamic contrast-enhanced magnetic resonance imaging) were determined at the end of the 14-day treatment period. Additionally, the effect of MSC on drug delivery was investigated by determining intratumoral concentration of doxorubicin using high-performance liquid chromatography and fluorescence microscopy. RESULTS: Double immunostaining of tumor sections revealed a marked reduction ( approximately 40%) in microvessel density accompanying tumor growth inhibition following MSC treatment along with a concomitant increase in the vascular maturation index ( approximately 30% > control) indicative of increased pericyte coverage of microvessels. Hoechst 33342/DiOC7 staining showed improved vessel functionality, and dynamic contrast-enhanced magnetic resonance imaging using the intravascular contrast agent, albumin-GdDTPA, revealed a significant reduction in vascular permeability following MSC treatment. Consistent with these observations, a 4-fold increase in intratumoral doxorubicin levels was observed with MSC pretreatment compared with administration of doxorubicin alone. CONCLUSION: These results show, for the first time, the antiangiogenic effects of MSC results in tumor growth inhibition, vascular maturation in vivo, and enhanced anticancer drug delivery that are associated with the observed therapeutic synergy in vivo.

Defining the expression pattern of the LGI1 gene in BAC transgenic mice.

The LGI1 gene has been implicated in the development of epilepsy and the invasion phenotype of glial cells. Controversy over the specific tissue expression pattern of this gene has stemmed from conflicting reports generated using immunohistochemistry and the polymerase chain reaction. LGI1 is one of a four-member family of secreted proteins with high homology and here we demonstrate, using GFP-tagged constructs from the four LGI1family members, that commonly used antibodies against LGI1 cross-react with different family members. With the uncertainty surrounding the use of commercially available antibodies to truly establish the expression pattern of LGI1, we generated transgenic mice carrying the LGI1-containing BAC, RP23-127G7, which had been modified to express the GFP reporter gene under the control of the endogenous regulatory elements required for LGI1 expression. Three founder mice were generated, and immunohistochemistry was used to determine the tissue-specific pattern of expression. In the brain, distinct regions of glial and neuronal cell expression were identified, as well as the choriod plexus, which is largely pia-derived. In addition, strong expression levels were identified in glandular regions of the prostate, individual tubules in the kidney, sympathetic ganglia in the kidney, sebaceous glands in the skin, the islets of Langerhans, the endometrium, and the ovary and testes. All other major organs analyzed were negative. The pattern of reporter gene expression was identical in three individual founder mice, arguing against a position effect altering expression profile due to the integration site of the BAC.

Down-regulation of WAVE3, a metastasis promoter gene, inhibits invasion and metastasis of breast cancer cells.

The expression of WAVE3, an actin-cytoskeleton and reorganization protein, is elevated in malignant human breast cancer, yet the role of WAVE3 in promoting tumor progression remains undefined. We have recently shown that knockdown of WAVE3 expression in human breast adenocarcinoma MDA-MB-231 cells using small interfering RNA resulted in a significant reduction of cell motility, migration, and invasion, which correlated with a reduction in the levels of active p38 mitogen-activated protein kinase. Here, we investigated the effect of stable suppression of WAVE3 by short hairpin RNA on tumor growth and metastasis in xenograft models. Breast cancer MDA-MB-231 cells expressing short hairpin RNA to WAVE3 (shWAVE3) showed a significant reduction in Matrigel invasion and formation of lung colonies after tail-vein injection in SCID mice. In the orthotopic model, we observed a reduction in growth rate of the primary tumors, as well as in the metastases to the lungs. We also show that suppression of p38 mitogen-activated protein kinase activity by dominant-negative p38 results in comparable phenotypes to the knockdown of WAVE3. These studies provide direct evidence that the WAVE3-p38 pathway contributes to breast cancer progression and metastasis.

SSeCKS metastasis-suppressing activity in MatLyLu prostate cancer cells correlates with vascular endothelial growth factor inhibition.

SSeCKS, a Src-suppressed protein kinase C substrate with metastasis suppressor activity, is the rodent orthologue of human gravin/AKAP12, a scaffolding protein for protein kinase A and protein kinase C. We show here that the tetracycline-regulated reexpression of SSeCKS in MatLyLu (MLL) prostate cancer cells suppressed formation of macroscopic lung metastases in both spontaneous and experimental models of in vivo metastasis while having minimal inhibitory effects on the growth of primary-site s.c. tumors. SSeCKS decreased angiogenesis in vitro and in vivo by suppressing vascular endothelial growth factor (VEGF) expression in MLL tumor cells as well as in stromal cells. The forced reexpression of VEGF(165) and VEGF(121) isoforms was sufficient to reverse aspects of SSeCKS metastasis-suppressor activity in both the experimental and spontaneous models. SSeCKS reexpression in MLL cells resulted in the down-regulation of proangiogenic genes, such as osteopontin, tenascin C, KGF, angiopoietin, HIF-1alpha, and PDGFRbeta, and the up-regulation of antiangiogenic genes, such as vasostatin and collagen 18a1, a precursor of endostatin. These results suggest that SSeCKS suppresses formation of metastatic lesions by inhibiting VEGF expression and by inducing soluble antiangiogenic factors.

Temporal and spatial expression of TACC1 in the mouse and human.

TACC1 is the founding member of the evolutionarily conserved transforming acidic coiled coil genes. These genes play a role in normal development and tumorigenesis through interactions with multiple complexes involved in transcription, translation, and centrosomal dynamics. Despite its importance, detailed examination of the expression of TACC1 and splice variants has not previously been performed. In this study, the spatiotemporal distribution of the Tacc1 protein was examined immunohistochemically in cross-sections of mouse embryonic tissues. We also report the distribution of currently known/predicted TACC1 splice variants in adult humans. These results indicate that Tacc1 is regulated in a dynamic manner during embryogenesis. In adult humans, ubiquitous expression of at least one TACC1 splice variant is noted, although specific combinations of variants are evident in individual differentiated tissues. An important observation is that in the in vivo three-dimensional tissue architecture of the growing organism, both the human and mouse TACC1 protein can be localized to different subcellular compartments in a cell- and tissue-specific manner. This indicates that exploration of TACC1 function must take into account the temporal expression of specific splice variants that may perform different cell-type and tissue-specific functions. Furthermore, this analysis will provide the groundwork from which future Tacc1 knockout strategies can be designed and properly interpreted.

Aberrations of TACC1 and TACC3 are associated with ovarian cancer.

BACKGROUND: Dysregulation of the human Transforming Acidic Coiled Coil (TACC) genes is thought to be important in the development and progression of multiple myeloma, breast and gastric cancer. Recent, large-scale genomic analysis and Serial Analysis of Gene Expression data suggest that TACC1 and TACC3 may also be involved in the etiology of ovarian tumors from both familial and sporadic cases. Therefore, the aim of this study was to determine the occurrence of alterations of these TACCs in ovarian cancer. METHODS: Detection and scoring of TACC1 and TACC3 expression was performed by immunohistochemical analysis of the T-BO-1 tissue/tumor microarray slide from the Cooperative Human Tissue Network, Tissue Array Research Program (TARP) of the National Cancer Institute, National Institutes of Health, Bethesda, MD, USA. Tumors were categorized as either positive (greater than 10% of cells staining) or negative. Statistical analysis was performed using Fisher's exact test and p < 0.05 (single comparisons), and p < 0.02 (multiple comparisons) were considered to be significant. Transgenomics WAVE high performance liquid chromatography (dHPLC) was used to pre-screen the TACC3 gene in constitutional DNA from ovarian cancer patients and their unaffected relatives from 76 families from the Gilda Radner Familial Ovarian Cancer Registry. All variant patterns were then sequenced. RESULTS: This study demonstrated absence of at least one or both TACC proteins in 78.5% (51/65) of ovarian tumors tested, with TACC3 loss observed in 67.7% of tumors. The distribution pattern of expression of the two TACC proteins was different, with TACC3 loss being more common in serous papillary carcinoma compared with clear cell carcinomas, while TACC1 staining was less frequent in endometroid than in serous papillary tumor cores. In addition, we identified two constitutional mutations in the TACC3 gene in patients with ovarian cancer from the Gilda Radner Familial Ovarian Cancer Registry. These patients had previously tested negative for mutations in known ovarian cancer predisposing genes. CONCLUSION: When combined, our data suggest that aberrations of TACC genes, and TACC3 in particular, underlie a significant proportion of ovarian cancers. Thus, TACC3 could be a hitherto unknown endogenous factor that contributes to ovarian tumorigenesis.

Isomers of conjugated linoleic acid differ in their effects on angiogenesis and survival of mouse mammary adipose vasculature.

Dietary conjugated linoleic acid (CLA) is a cancer chemopreventive agent that has been shown to inhibit angiogenesis in vivo and in vitro, and to decrease vascular endothelial growth factor (VEGF) and Flk-1 concentrations in the mouse mammary gland. To determine which isomer mediates the antiangiogenic effects of CLA in vivo, the effects of diets supplemented with 5 or 10 g/kg c9,t11- or t10,c12-CLA isomers were compared in CD2F1Cr mice. Both isomers inhibited functional vascularization of a matrigel pellet in vivo and decreased serum VEGF concentrations; the t10,c12 isomer also decreased the proangiogenic hormone leptin (P < 0.05). Additionally, the t10,c12 isomer, but not c9,t11-CLA, rapidly induced apoptosis of the white and brown adipocytes as well as the preexisting supporting vasculature of the mammary fat pad (P < 0.05). Independent of this isomer-specific adipose apoptotic effect, both isomers induced a rapid and reversible decrease in the diameter of the unilocular adipocytes (P < 0.05). The ability of both CLA isomers to inhibit angiogenesis in vivo may contribute to their ability to inhibit carcinogenesis. Moreover, we propose that each CLA isomer uniquely modifies the mammary stromal "soil" in a manner that is useful for chemoprevention of breast cancer.

Successful treatment of bowenoid papulosis in a 9-year-old girl with vertically acquired human immunodeficiency virus.

A 9-year-old black girl with vertically acquired human immunodeficiency virus (HIV) and no history of condyloma acuminata presented with a 4-year history of enlarging and spreading dark brown flat papules in the perineum. Some of the lesions were confluent and extended from the clitoris to the labia majora and posteriorly to the buttocks and perianal region. A biopsy of one of the lesions showed bowenoid features. Our patient had a normal Pap smear, but vaginal and cervical biopsy specimens revealed human papillomavirus type 16. Therapy with topical imiquimod cream every other day was started, but little improvement was noted after 2 months. Application of 25% podophyllin every 4 to 8 weeks was added, and improvement was noted within 1 month. After 1 year of treatment, the patient had complete resolution of all lesions, and she has had no further appearance of lesions. Our case emphasizes the need for increased awareness of the potential for development of bowenoid papulosis in HIV-positive children as well as the successful treatment of our patient with topical therapy alone.

Inhibition of angiogenesis by the cancer chemopreventive agent conjugated linoleic acid.

Dietary conjugated linoleic acid (CLA) has been shown previously to inhibit rat mammary carcinogenesis. In addition to direct effects on mammary epithelial cells,including decreased proliferation and induction of apoptosis, CLA may exert its effects indirectly by inhibiting the differentiation of mammary stromal cells to an endothelial cell type. Specifically, CLA was found to decrease the ability of mammary stromal cells to form complex anastomosing microcapillary networks in vitro on Engelbreth-Holm-Swarm-derived reconstituted basement membrane. This suggested that CLA might inhibit angiogenesis in vivo. To test this possibility, CD2/F(1) mice were placed on synthetic diets containing 0, 1, or 2% CLA for 6 weeks, before angiogenic challenge by s.c. injection with an angiogenic gel substrate (Matrigel pellet assay). After 7 days, the pellets from animals fed the control diet were infiltrated by abundant branching networks of blood vessels with patent lumen-containing RBCs. In contrast, pellets from the CLA-fed animals contained fewer infiltrating cells, which formed limited branching cellular networks, the majority of which had collapsed lumen and no RBCs. Both levels of dietary CLA showed similar effects, with the number of RBC-containing vessels per 20x field decreased to a third of that seen in control. Dietary CLA decreased serum levels of vascular endothelial growth factor (VEGF) and whole mammary gland levels of VEGF and its receptor Flk-1. Both cis-9, trans-11 and trans-10, cis-12 CLA isomers were effective in inhibiting angiogenesis in vitro in a dose-dependent fashion. The ability of CLA to inhibit angiogenesis may contribute to its efficacy as a chemopreventive agent.