Tumor Pericytes Research Articles

Abstract 930: Combined inhibition of pan-RAF and VEGFR-2 mediates antitumor activity in KRAS mutant non-small cell lung cancer (NSCLC) through enhanced inhibition of tumor angiogenesis and growth

Abstract Lung cancer is the leading cause of cancer death worldwide. MAPK activation via KRAS mutation is present in up to 30% of lung cancer patients. NSCLC patients with KRAS mutation is associated with poor prognosis and represents an unmet medical need. LY3009120, a pan-RAF and RAF dimer inhibitor which is in phase I clinical trial, was previously demonstrated to have anti-tumor activities in BRAF or RAS mutant tumor cells in vitro and in vivo. Ramucirumab, a fully-human antagonist monoclonal antibody to human VEGFR-2 was recently approved as an anti-angiogenic treatment for several cancer indications including second-line NSCLC. Combination strategies in cancer including targeting both tumor cells and the surrounding stroma cells have been shown to be effective in various disease subtypes. In this study, the combination effect of LSN3074753 (a surrogate and an analogue of LY3009120) with VEGFR-2 inhibitor DC101 (a monoclonal antibody specific for murine VEGFR-2 and a surrogate for ramucirumab) were evaluated in KRAS mutant NSCLC models, including NCI-H2122 (G-12C), A549 (G-12S) and NCI-H441 (G-12V). LSN3074753 treatment alone resulted in 66.9% and 82.4% tumor growth inhibition in H2122 and A549 xenograft tumors, respectively; and 41.4% tumor regression in H441 xenograft tumors. DC101 treatment alone resulted in 64.5%, 75% and 102.2% tumor growth inhibition in H2122, A549 and H441, respectively. The combination of LSN3074753 and DC101 led to more significant tumor growth inhibition (87.2% of tumor growth inhibition for H2122, p<0.001) or tumor regression (16.3% and 51.5% tumor regression for A549 and H441, respectively, p<0.001) when compared to single agent treatment. The molecular mechanism was further investigated in H2122 and H441 tumor xenografts in terms of MAPK signaling and its gene signatures, tumor vascularization, cell proliferation and apoptosis. Treatment with LSN3074753 or DC101 alone reduced the tumor vessels and pericytes, but the combination treatment showed better effects. Similarly, the analysis of multiple cell cycle markers indicated that the combination treatment inhibited cell proliferation more than the single agent alone. Moreover, the combination effect of LSN3074753 with ramucirumab was also investigated in vitro, including endothelial cell sprouting, cord formation and tumor cell driven cord formation assays to further understand the molecular mechanisms underlying the combination benefit on different tumor compartments including tumor and stroma cells. Overall, combined inhibition of pan-RAF and VEGFR-2 enhanced both antiangiogenesis and antitumor effects, and the data support that combining a panRAF inhibitor with ramucirumab represents a promising strategy for the treatment of KRAS mutant NSCLC. Citation Format: Wenjuan Wu, Julie Stewart, Constance King, Bonita Jones, Robert Flack, Susan Pratt, Randi Berryman, Michelle Swearingen, Diane Bodenmiller, Xi Lin, Mark Uhlik, Beverly Falcon, Anthony Fischl, Jason Manro, Ramon Tiu, Sudhakar Chintharlapalli, Bronislaw Pytowski, Shripad V. Bhagwat, Sean Buchanan, Sheng-Bin Peng. Combined inhibition of pan-RAF and VEGFR-2 mediates antitumor activity in KRAS mutant non-small cell lung cancer (NSCLC) through enhanced inhibition of tumor angiogenesis and growth. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 930.

Abstract 3330: Tumor angiogenesis and cancer stem cells in serous adenocarcinoma of the ovary

Abstract Angiogenesis is required for tumor growth and metastasis. The conventional view of tumour angiogenesis is that tumours get their blood supply from the neighbouring normal stroma. However, recently the origin of tumour endothelial cells or pericytes in part has been shown to be derived from cancer stem cells (CSC) in glioma. The spread of ovarian cancer (SOC) is different and the origin of endothelial cells in the tumor is not known. Using spheroids as an in vitro model (which is enriched for CSC), we have evaluated the role of CSC in primary malignant cells (PMCs) from patients with serous adenocarcinoma of ovary (n = 30) cultured under endothelial conditions. The expression of endothelial markers (CD31, CD105, and CLEC14a) was evaluated by flow cytometry. Further, the ability of CSC to express endothelial markers under appropriate growth conditions was also evaluated with Bevacizumab (Avastin) or Cediranib which antagonize VEGF or its receptor (VEGFR2) respectively. In addition, functional assays such as uptake of Dil labelled acetylated low density lipoprotein (Dil-ac-LDL) and expression of endothelial nitric oxide synthase (e-NOS) was performed to assess the endothelial phenotype. The localization of tumour blood vessels and CSC in primary ovarian tumors was examined by double immunohistochemistry of CD31/CD105/CLEC14a and ALDH1a1. Primary malignant cells (n = 30) grown in endothelial growth medium (EGM) showed significantly higher expression of CD105 (mean 20.8%, p = 0.001) and CLEC14a (mean 5.3%, p = 0.012) and a co-expression of CD105/CLEC14a (mean 1.6%, p = 0.012) than that of control (mean, 12.5%, 2.5% and 0.95% respectively). The co-expression of ALDH1a1 with endothelial markers, CD31, (mean, 1.58%), CD105, (mean 0.7%), CLEC14a (mean 0.44%) in primary malignant cells (n = 5), denovo, suggest that there is a small proportion of cells which are in transit to form endothelial cells. When the primary malignant cells were grown as spheroids in endothelial conditions in the presence or absence of Avastin (1 μg/μl), there was reduction in the expression of CD105 (mean, 12.5% (EGM) and 7.9% (Avastin), P = 0.056) and CLEC14a (mean. 5.5% (EGM) and 1.5% (Avastin), P = 0.04). However, when the spheroids were cultured in presence of cediranib (10 nM), the expression of CD105 was not reduced (Mean, 2.53% (EGM) and 4% (cediranib). The cells grown in endothelial conditions showed uptake of Dil-ac-LDL (n = 3) and expressed e-NOS (n = 3), confirming their endothelial phenotype. The double immunostaining with ALDH1a1 and CD31/CD105 demonstrated that the blood vessels were in proximity to ALDH1A1+ cells in primary serous adenocarcinoma of the ovary (n = 5). These results suggest that a proportion of endothelial cells (probably 20%) could be derived from CSC in serous ovarian carcinoma and the VEGF pathway has an important role. This property of CSC to contribute to tumour angiogenesis can be inhibited. Citation Format: Syama Krishna Priya, Rohit P. Nagare, V S. Sneha, C Sidhanth, S Bindhya, P Manasa, Trivadi S. Ganesan. Tumor angiogenesis and cancer stem cells in serous adenocarcinoma of the ovary. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3330.

Role of Tumor Pericytes in the Recruitment of Myeloid-Derived Suppressor Cells.

Pericytes are members of the tumor stroma; however, little is known about their origin, function, or interaction with other tumor components. Emerging evidence suggest that pericytes may regulate leukocyte transmigration. Myeloid-derived suppressor cells (MDSC) are immature myeloid cells with powerful inhibitory effects on T-cell-mediated antitumor reactivity. We generated subcutaneous tumors in a genetic mouse model of pericyte deficiency (the pdgfb (ret/ret) mouse) and littermate control mice (n = 6-25). Gene expression profiles from 253 breast cancer patients (stage I-III) were evaluated for clinic-pathological parameters and survival using Cox proportional hazard ratios (HRs) and 95% confidence intervals (CIs) based on a two-sided Wald test. We report that pericyte deficiency leads to increased transmigration of Gr1(+)/CD11b(+) cells in experimentally induced tumors. Pericyte deficiency produced defective tumor vasculature, resulting in a more hypoxic microenvironment promoting IL-6 upregulation in the malignant cells. Silencing IL-6 expression in tumor cells attenuated the observed differences in MDSC transmigration. Restoring the pericyte coverage in tumors abrogated the increased MDSC trafficking to pericyte-deficient tumors. MDSC accumulation in tumors led to increases in tumor growth and in circulating malignant cells. Finally, gene expression analysis from human breast cancer patients revealed increased expression of the human MDSC markers CD33 and S100A9 with concomitant decreased expression of pericyte genes and was associated with poor prognosis (HR = 1.88, 95% CI = 1.08 to 3.25, P = .03). Our data uncovers a novel paracrine interaction between tumor pericytes and inflammatory cells and delineates the cellular events resulting in the recruitment of MDSC to tumors. Furthermore, we propose for the first time a role for tumor pericytes in modulating the expression of immune mediators in malignant cells by promoting a hypoxic microenvironment.

A novel population of local pericyte precursor cells in tumor stroma that require Notch signaling for differentiation

Pericytes are perivascular support cells, the origin of which in tumor tissue is not clear. Recently, we identified a Tie1+ precursor cell that differentiates into vascular smooth muscle, in a Notch-dependent manner. To understand the involvement of Notch in the ontogeny of tumor pericytes we used a novel flow immunophenotyping strategy to define CD146+/CD45−/CD31−/lo pericytes in the tumor stroma. This strategy combined with ex vivo co-culture experiments identified a novel pericyte progenitor cell population defined as Sca1hi/CD146−/CD45−/CD31−. The differentiation of these progenitor cells was stimulated by co-culture with endothelial cells. Overexpression of the Notch ligand Jagged1 in endothelial cells further stimulated the differentiation of Sca1hi/CD146−/CD45−/CD31− cells into pericytes, while inhibition of Notch signaling with a γ-secretase inhibitor reduced this differentiation. However, Notch inhibition specifically in Tie1-expressing cells did not change the abundance of pericytes in tumors, suggesting that the pericyte precursor is distinct from the vascular smooth muscle cell precursor. Transplant experiments showed that the bone marrow contributes minimally to tumor pericytes. Immunophenotyping revealed that Sca1hi/CD146−/CD45−/CD31− cells have greater potential to differentiate into pericytes and have increased expression of classic mesenchymal stem cell markers (CD13, CD44, Nt5e and Thy-1) compared to Sca1−/lo/CD146−/CD45−/CD31− cells. Our results suggest that a local Sca1hi/CD146−/CD45−/CD31− pericyte progenitor resides in the tumor microenvironment and requires Notch signaling for differentiation into mature pericytes.

Endogenous brain pericytes are widely activated and contribute to mouse glioma microvasculature.

Glioblastoma multiforme (GBM) is the most common brain tumor in adults. It presents an extremely challenging clinical problem, and treatment very frequently fails due to the infiltrative growth, facilitated by extensive angiogenesis and neovascularization. Pericytes constitute an important part of the GBM microvasculature. The contribution of endogenous brain pericytes to the tumor vasculature in GBM is, however, unclear. In this study, we determine the site of activation and the extent of contribution of endogenous brain pericytes to the GBM vasculature. GL261 mouse glioma was orthotopically implanted in mice expressing green fluorescent protein (GFP) under the pericyte marker regulator of G protein signaling 5 (RGS5). Host pericytes were not only activated within the glioma, but also in cortical areas overlying the tumor, the ipsilateral subventricular zone and within the hemisphere contralateral to the tumor. The host-derived activated pericytes that infiltrated the glioma were mainly localized to the tumor vessel wall. Infiltrating GFP positive pericytes co-expressed the pericyte markers platelet-derived growth factor receptor-β (PDGFR-β) and neuron-glial antigen 2. Interestingly, more than half of all PDGFR-β positive pericytes within the tumor were contributed by the host brain. We did not find any evidence that RGS5 positive pericytes adopt another phenotype within glioma in this paradigm. We conclude that endogenous pericytes become activated in widespread areas of the brain in response to an orthotopic mouse glioma. Host pericytes are recruited into the tumor and constitute a major part of the tumor pericyte population.

Recruitment and retention: factors that affect pericyte migration

Pericytes are critical for vascular morphogenesis and contribute to several pathologies, including cancer development and progression. The mechanisms governing pericyte migration and differentiation are complex and have not been fully established. Current literature suggests that platelet-derived growth factor/platelet-derived growth factor receptor-β, sphingosine 1-phosphate/endothelial differentiation gene-1, angiopoietin-1/tyrosine kinase with immunoglobulin-like and EGF-like domains 2, angiopoietin-2/tyrosine kinase with immunoglobulin-like and EGF-like domains 2, transforming growth factor β/activin receptor-like kinase 1, transforming growth factor β/activin receptor-like kinase 5, Semaphorin-3A/Neuropilin, and matrix metalloproteinase activity regulate the recruitment of pericytes to nascent vessels. Interestingly, many of these pathways are directly affected by secreted protein acidic and rich in cysteine (SPARC). Here, we summarize the function of these factors in pericyte migration and discuss if and how SPARC might influence these activities and thus provide an additional layer of control for the recruitment of vascular support cells. Additionally, the consequences of targeted inhibition of pericytes in tumors and the current understanding of pericyte recruitment in pathological environments are discussed.

Tumor Angiogenesis: A New Source of Pericytes

Glioblastoma stem cells have been reported to directly contribute to the tumor vasculature by endothelial cell differentiation. Interestingly, a recent study demonstrates that glioblastoma stem cells preferentially differentiate into vascular pericytes to support vasculature function and tumor growth.

330. Tumor Pericytes: A Novel Target for Immunotherapy in Renal Cell Carcinoma

330. Tumor Pericytes: A Novel Target for Immunotherapy in Renal Cell Carcinoma

Abstract 1421: Down-regulation of neuronal repressor gene REST inhibits tumor growth in Ewing's sarcoma

Abstract Ewing's sarcoma is a peripheral primitive neuroectodermal tumors (PNET). The abnormal differentiation of neuronal stem cells may relate to tumorigenesis of Ewing's sarcoma. The RE1-silencing transcription factor (REST) is a neuronal repressor gene which regulates neuronal stem cell differentiation. The aim of this study is to investigate the role of REST in Ewing's sarcoma. Our results demonstrated that three human Ewing's sarcoma cell lines TC71, A4573 and SK-ES expressed high levels of REST. Six of seven tumor samples from patients with Ewing's sarcoma were shown REST positive. Transfection of EWS-FLI-1 into mesenchymal stem cells up-regulated REST expression. Down-regulation of EWS-FLI-1 using si-RNA inhibited REST expression in TC71 cells, suggesting EWS-FLI-1 fusion protein regulates REST expression in Ewing's sarcoma. The siRNA-REST stable transfected cell line was established to down-regulate REST expression in TC71 Ewing's sarcoma cells. In vivo experiments indicated that Ewing's sarcoma tumor growth was significantly inhibited in the mice injected with si-REST-TC71 cells compared to the mice injected with si-control cells. Down-regulation of REST inhibited cell proliferation and induced apoptosis in TC71 tumors. The immunohistochemical staining indicated that the tumor vessels and pericyte marker expression were reduced in si-REST tumor tissues. Inhibition of REST decreased the early neural differentiation marker MSX-1 expression and up-regulated the terminal neural differentiation marker β-Tubulin III (Tuj) expression. These data suggesting that neuronal repressor gene REST plays an important role in tumor development of Ewing's sarcoma. Inhibition of REST suppressed tumor growth and altered neuronal differentiation marker expression in Ewing's sarcoma. REST is a potential target for treatment of Ewing's sarcoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1421. doi:1538-7445.AM2012-1421

Influence of Morphine on Pericyte-Endothelial Interaction: Implications for Antiangiogenic Therapy

Morphine stimulates tumor angiogenesis and cancer progression in mice. We examined if morphine influences endothelial-pericyte interaction via platelet-derived growth factor-BB (PDGF-BB) and PDGF receptor-β (PDGFR-β). Clinically relevant doses of morphine stimulated PDGF-BB secretion from human umbilical vein endothelial cells and activated PDGFR-β and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) phosphorylation in human pericytes. These in vitro effects of morphine were translated into promotion of tumor angiogenesis in a transgenic mice model of breast cancer when treated with clinically used dose of morphine. Increased vessel-associated immunoreactivity of desmin and PDGFR-β was observed on pericytes in tumors of morphine-treated mice. These data suggest that morphine potentiates endothelial-pericyte interaction via PDGF-BB/PDGFR-β signaling and promotes tumor angiogenesis, pericyte recruitment, and coverage of tumor vessels. We speculate that morphine may impair the effectiveness of antiangiogenic therapy by influencing vascular pericyte coverage.

EGFR Inhibition Fails to Suppress Vascular Proliferation and Tumor Growth in a Ewing's Sarcoma Model

Expression of epidermal growth factor receptor (EGFR), a potent regulator of cellular homeostasis, is associated with aggressive tumor behavior. The mechanism by which EGFR inhibition functions is unclear, with controversial results demonstrating an effect on the tumor cells, endothelial cells, or pericytes. EGFR activation has been linked to the expression of vascular endothelial growth factor (VEGF), a known mitogen of angiogenesis, but the relationship between these factors and their effect on tumor vessel development is vague. We hypothesized that using an EGFR inhibitor on a human Ewing's sarcoma model would inhibit tumor growth by suppressing vessel proliferation. A cell proliferation assay was performed on the Ewing's sarcoma (SK-NEP-1) cell line. Tumor cells were implanted intrarenally in athymic mice. Animals received daily gavage with vehicle or gefitinib 1 wk following implantation. Mice (n = 12/cohort) were euthanized 6 wk following implantation. Remaining mice were maintained without treatment for 2 wk. Vascular changes were assessed by angiography and immunohistochemically. EGFR and vascular endothelial growth factor (VEGF) expression were quantified using quantitative polymerase chain reaction (qPCR). Gefitinib suppressed in vitro cell growth with an IC(50) = 1.36 μM. Minimal tumor growth suppression was noted at 6 wk (6.01 ± 1.2 g in control versus 4.61 ± 0.9 g treated, P = 0.36). After cessation of gefitinib, tumor growth was increased in both groups (7.37 ± 1.62 g versus 6.77 ± 1.53 g, P = 0.79). Microvessel density was unchanged despite EGFR inhibition (161,000 ± 16,000 pixels versus 135,000 ± 18,000 pixels, P = 0.31). At 6 wk, the vascular maturity index was similar in both groups (3.63 ± 1.12 versus 4.09 ± 1.71, P = 0.83). A downward trend in EGFR expression (49% of control) and an upward trend in VEGF levels (50% of control) occurred in the treated group. EGFR expression was suppressed in cultured cells and xenograft tumors. Despite a cytotoxic effect on cell lines, gefitinib had little effect on tumor growth. No effects on the tumor vasculature were noted in the setting of EGFR suppression, suggesting that angiogenesis induced by SK-NEP-1 cells is refractory to EGFR inhibition. Interestingly, the resulting increase in VEGF expression following EGFR blockade, provides an alternative pro-angiogenic pathway promoting tumor survival.

Abstract 4271: Combination of VEGF Trap (Aflibercept) with docetaxel, but not with gemcitabine, is efficacious in tumors resistant to anti-VEGF therapy

Abstract Increased anti-tumor efficacy is often observed when VEGF blockade is combined with certain chemotherapy agents. However, the mechanism by which combination treatment with certain agents, but not others, results in enhanced anti-tumor effects is poorly understood. In this study, we compared the anti-tumor effects of VEGF Trap in combination with either docetaxel or gemcitabine on HT1080, a human fibrosarcoma tumor line that is resistant to anti-VEGF treatment. Immunodeficient SCID mice bearing subcutaneous HT1080 tumors (size ∼150 mm3) were treated with either control protein plus vehicle, VEGF Trap, chemotherapy agent (docetaxel or gemcitabine), or the combination. Following three treatments administered every 3-4 days, tumors were harvested either one day after the final dose or allowed to grow and harvested when the average size of the treatment group reached ∼500mm3. We found that combination treatment with VEGF Trap plus docetaxel was significantly more effective compared to single agents, while there was no added benefit of combining VEGF Trap with gemcitabine. Although treatment with chemotherapy agents alone and in combination with VEGF Trap both initially regressed tumor size, only the combination with docetaxel was effective at delaying long-term tumor re-growth. Immunohistochemical analysis of tumors showed that both combination regimens produced a dramatic decrease in the number of proliferating tumor cells. However, only VEGF Trap and docetaxel combination treatment resulted in pruned tumor vessels with less branching, whereas VEGF Trap plus gemcitabine had minimal effect on tumor vessel morphology. Furthermore, VEGF trap plus docetaxel produced an increase in TUNEL-positive cells associated with the tumor vasculature and a marked decrease in tumor pericytes. These results suggest that in tumors resistant to VEGF blockade, taxanes (docetaxel) can sensitize the vasculature to anti-VEGF treatment (aflibercept), thus resulting in enhanced effects on the tumor vessels and improved anti-tumor efficacy. Further understanding of the mechanistic basis of different combination regimens may allow improvement of current strategies to treat tumors resistant to antiangiogenic therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4271. doi:10.1158/1538-7445.AM2011-4271

Abstract 3466: Tumor pericytes: A novel target for immune therapy in renal cell carcinoma

Abstract Tumor vasculature is classified by leaky, chaotic blood vessels consisting of several components including vascular endothelial cells (VEC) and mural cells (smooth muscle cells and pericytes), as well as immune regulatory cells, stem cells and vascular progenitors. Irregular tumor vasculature limits immune effector cell access to the tumor, and may in part be responsible for the modest success observed in many current anti-cancer immunotherapies. Certain drugs targeting tyrosine kinase receptors expressed on tumor stromal cells induce a Type-1 proinflammatory skewing of the tumor microenvironment (TME) with a coordinate reduction in the number of immune regulatory cell populations. A transient normalization of the vasculature may also occur in association with enhanced leukocyte recruitment. We have shown that vaccination against tumor-associated stromal antigens leads to the activation of VCAM-1 expression on tumor vascular endothelial cells and the enhanced recruitment of protective Type-1 T cells into the TME. Currently little is known regarding pericyte biology and their behavior in the TME, with no existing treatments specifically targeting pericytes. We hypothesize that pericytes residing in the TME differentially express antigens that distinguish them from pericytes in healthy tissue, and vaccination specifically targeting tumor pericyte antigens will result in vascular normalization and improved delivery of protective Type-1 T cells into the TME. To address these hypotheses, we examined mRNA expression of pericytes and VEC that have been flow-sorted from human renal cell carcinoma (RCC) tumors and patient-matched normal adjacent kidney (NAK) tissue. We have identified antigens that are differentially expressed by pericytes and/or by VEC in the TME versus NAK (such as RGS5). We are currently investigating the therapeutic efficacy of genetic (lentiviral) vaccines encoding tumor-associated stromal antigens in murine tumor models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3466. doi:10.1158/1538-7445.AM2011-3466

Intratumoral IL-12 Gene Therapy Results in the Crosspriming of Tc1 Cells Reactive Against Tumor-associated Stromal Antigens

HLA-A2 transgenic mice bearing established HLA-A2(neg) B16 melanomas were effectively treated by intratumoral (i.t.) injection of syngeneic dendritic cells (DCs) transduced to express high levels of interleukin (IL)-12, resulting in CD8(+) T cell-dependent antitumor protection. In this model, HLA-A2-restricted CD8(+) T cells do not directly recognize tumor cells and therapeutic benefit was associated with the crosspriming of HLA-A2-restricted type-1 CD8(+) T cells reactive against antigens expressed by stromal cells [i.e., pericytes and vascular endothelial cells (VEC)]. IL-12 gene therapy-induced CD8(+) T cells directly recognized HLA-A2(+) pericytes and VEC flow-sorted from B16 tumor lesions based on interferon (IFN)-γ secretion and translocation of the lytic granule-associated molecule CD107 to the T cell surface after coculture with these target cells. In contrast, these CD8(+) T effector cells failed to recognize pericytes/VEC isolated from the kidneys of tumor-bearing HHD mice. The tumor-associated stromal antigen (TASA)-derived peptides studied are evolutionarily conserved and could be recognized by CD8(+) T cells harvested from the blood of HLA-A2(+) normal donors or melanoma patients after in vitro stimulation. These TASA and their derivative peptides may prove useful in vaccine formulations against solid cancers, as well as, in the immune monitoring of HLA-A2(+) cancer patients receiving therapeutic interventions, such as IL-12 gene therapy.

Abstract 388: Epithelial to mesenchymal transition is associated with acquired resistance to sunitinib in patients with renal cell carcinoma: evidence from a xenograft study

Abstract Purpose: Tyrosine kinase inhibitors (TKIs) targeting angiogenesis via inhibition of the vascular endothelial growth factor (VEGF) pathway have changed the medical management of metastatic renal cell carcinoma. While the treatment with TKIs has demonstrated clinical benefit these drugs will eventually fail patients. The potential mechanisms of resistance to TKIs are poorly understood. Experimental Design: To address this question we obtained an excisional biopsy of a skin metastasis from a patient with clear cell renal carcinoma who initially had a response on sunitinib and eventually progressed on therapy. Tumor pieces were grafted subcutaneously in athymic nude mice. Established xenografts were treated with sunitinib. Tumor size, microvascular density and pericyte coverage were determined. Plasma as well as tissue levels for sunitinib were assessed. A tumor derived cell line was established and assessed in vitro for a potential direct antitumor effects of sunitinib. Results: To our surprise, xenografts from the patient who progressed on sunitinib regained sensitivity to the drug. At a dose of 40 mg/kg sunitinib caused regression of the subcutaneous tumors. Histology showed a marked reduction in microvascular density and pericyte dysfunction. More interestingly, histological examination of the original skin metastasis revealed evidence of epithelial-to-mesenchymal-transition while the xenografts showed reversion to the clear cell phenotype. In vitro studies showed no inhibitory effect on tumor cell growth at pharmacologically relevant concentrations. Conclusions: The histological examination in this xenograft study suggests that reversible epithelial-to mesenchymal-transition may be associated with acquired tumor resistance to TKIs in patients with clear cell renal carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 388.

Targeting fibroblast activation protein inhibits tumor stromagenesis and growth in mice

Membrane-bound proteases have recently emerged as critical mediators of tumorigenesis, angiogenesis, and metastasis. However, the mechanisms by which they regulate these processes remain unknown. As the cell surface serine protease fibroblast activation protein (FAP) is selectively expressed on tumor-associated fibroblasts and pericytes in epithelial tumors, we set out to investigate the role of FAP in mouse models of epithelial-derived solid tumors. In this study, we demonstrate that genetic deletion and pharmacologic inhibition of FAP inhibited tumor growth in both an endogenous mouse model of lung cancer driven by the K-rasG12D mutant and a mouse model of colon cancer, in which CT26 mouse colon cancer cells were transplanted into immune competent syngeneic mice. Interestingly, growth of only the K-rasG12D-driven lung tumors was also attenuated by inhibition of the closely related protease dipeptidyl peptidase IV (DPPIV). Our results indicate that FAP depletion inhibits tumor cell proliferation indirectly, increases accumulation of collagen, decreases myofibroblast content, and decreases blood vessel density in tumors. These data provide proof of principle that targeting stromal cell-mediated modifications of the tumor microenvironment may be an effective approach to treating epithelial-derived solid tumors.

Endosialin Protein Expression and Therapeutic Target Potential in Human Solid Tumors: Sarcoma versus Carcinoma

Endosialin/CD248/tumor endothelial marker 1 is expressed in stromal cells, endothelial cells, and pericytes in various tumors; however, few studies have focused on expression in malignant cells. We studied expression of endosialin in clinical specimens, cell culture, and animal models and designed an anti-endosialin therapeutic prototype. Fifty human tumor cell lines and 6 normal cell types in culture were assayed by reverse transcription-PCR and/or flow cytometry for endosialin. Cell surface protein was found on 7 sarcoma lines, 1 neuroblastoma, and 4 normal cell types in culture. A fully human anti-endosialin antibody bound to human A-673 Ewing's sarcoma cells and SK-N-AS neuroblastoma cells but not HT-1080 cells. Exposure of cells to an anti-human IgG conjugated to saporin resulted in growth inhibition only of endosialin-expressing cells. Endosialin expression was assessed by immunohistochemistry in 250 clinical specimens of human cancer including 20 cancer subtypes. Endosialin is frequently found in human cancers. Endosialin expression is mainly a perivascular feature in carcinomas, with some expression in stromal cells. In sarcomas, endosialin is expressed by malignant cells, perivascular cells, and stromal cells. Development and characterization of experimental models for studying endosialin biology in sarcomas and evaluating anti-endosialin therapies is presented. Findings suggest that an anti-endosialin immunotoxin might be a promising therapeutic approach for endosialin-positive neoplasia, especially synovial sarcoma, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, and osteosarcoma. Thus, a diagnostic/therapeutic targeted therapeutic approach to treatment of endosialin-expressing tumors may be possible.

144 CDx2 Promotes Tumor Pericyte Content and Vascular Function in Human Colon Cancer Xenografts

144 CDx2 Promotes Tumor Pericyte Content and Vascular Function in Human Colon Cancer Xenografts

Sequential Loss of Tumor Vessel Pericytes and Endothelial Cells after Inhibition of Platelet-Derived Growth Factor B by Selective Aptamer AX102

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.

Overexpression of PDGF-BB decreases colorectal and pancreatic cancer growth by increasing tumor pericyte content

We hypothesized that overexpression of PDGF-BB in colorectal cancer (CRC) and pancreatic cancer cells would result in increased pericyte coverage of ECs in vivo, rendering the tumor vasculature more resistant to antiangiogenic therapy. We stably transfected the cDNA for the PDGF-B into HT-29 human CRC and FG human pancreatic cancer cells. Surprisingly, when HT-29 or FG parental and transfected cells were injected into mice (subcutaneously and orthotopically), we observed marked inhibition of tumor growth in the PDGF-BB-overexpressing clones. In the PDGF-BB-overexpressing tumors, we observed an increase in pericyte coverage of ECs. Treatment of PDGF-BB-overexpressing tumors with imatinib mesylate (PDGFR inhibitor) resulted in increased growth and decreased total pericyte content compared with those in untreated PDGF-BB-overexpressing tumors. In vitro studies demonstrated the ability of VSMCs to inhibit EC proliferation by approximately 50%. These data show that increasing the pericyte content of the tumor microenvironment inhibits the growth of angiogenesis-dependent tumors. Single-agent therapy targeting PDGF receptor must be used with caution in tumors when PDGFR is not the target on the tumor cell itself.