Functional Tumor Vasculature Research Articles

Depletion of slow-cycling PDGFRα+ADAM12+ mesenchymal cells promotes antitumor immunity by restricting macrophage efferocytosis

The capacity to survive and thrive in conditions of limited resources and high inflammation is a major driver of tumor malignancy. Here we identified slow-cycling ADAM12+PDGFRα+ mesenchymal stromal cells (MSCs) induced at the tumor margins in mouse models of melanoma, pancreatic cancer and prostate cancer. Using inducible lineage tracing and transcriptomics, we demonstrated that metabolically altered ADAM12+ MSCs induced pathological angiogenesis and immunosuppression by promoting macrophage efferocytosis and polarization through overexpression of genes such as Gas6, Lgals3 and Csf1. Genetic depletion of ADAM12+ cells restored a functional tumor vasculature, reduced hypoxia and acidosis and normalized CAFs, inducing infiltration of effector T cells and growth inhibition of melanomas and pancreatic neuroendocrine cancer, in a process dependent on TGF-β. In human cancer, ADAM12 stratifies patients with high levels of hypoxia and innate resistance mechanisms, as well as factors associated with a poor prognosis and drug resistance such as AXL. Altogether, our data show that depletion of tumor-induced slow-cycling PDGFRα+ MSCs through ADAM12 restores antitumor immunity.

SYST-27 INTERROGATION OF THE TUMOR IMMUNE MICROENVIRONMENT REVEALS DISTINCT IMMUNOSUPPRESSIVE LANDSCAPES IN GLIOBLASTOMA

Abstract High-resolution sequencing technologies have revealed significant inter-and intra-tumoral molecular heterogeneity in glioblastoma (GBM), leading to an urgent need for novel targeted therapies. Our research using representative murine GBM models and various experimental techniques has shown that classical GBMs (e.g., EGFRvIII+, TIMElow) and mesenchymal GBMs (e.g., NF1ko/PTENko/p53ko, TIMEhigh) are both immunosuppressive but in distinct ways. TIMElow GBMs have a more impaired and less functional tumor vasculature, resulting in low infiltration of T cells with anergic features. TIMEhigh GBMs have a more intact BBB revealing overall more adaptive and innate immune cell infiltrates, but with highly dysfunctional T cells and immunosuppressive TME. Congruently, GSEA analysis of both human and murine GBM scRNA-seq data demonstrated enriched immunosuppressive signatures in the myeloid cell populations of TIMEhigh GBM compared to TIMElow GBM. Our findings have also revealed potential adaptive and intrinsic resistant mechanisms to antiangiogenic immunotherapy in TIMElow and TIMEhigh GBMs, respectively, with TIMElow GBMs exhibiting a transitory response to the treatment, while TIMEhigh GBMs being intrinsically resistant due to their intact tumor vasculature and reliance on dysfunctional T cells. We then tested the efficacy of different immunotherapies, including dendritic cell vaccination, a CD40 agonist, and a myeloid cell PI3K activity inhibitor in TIMElow and TIMEhigh tumors. Surprisingly, our results revealed that the response to these treatments can be either beneficial or detrimental and may depend on factors such as tumor antigenicity and TIME subtype. Overall, our study provides new insights into the intricate immune landscape of GBMs, emphasizes the importance of considering tumor subtypes as they may lead to potential unintended consequences of immunotherapies.

Role of the Tyrosine Phosphatase SHP-2 in Mediating Adrenomedullin Proangiogenic Activity in Solid Tumors.

VE-cadherin is an essential adhesion molecule in endothelial adherens junctions, and the integrity of these complexes is thought to be regulated by VE-cadherin tyrosine phosphorylation. We have previously shown that adrenomedullin (AM) blockade correlates with elevated levels of phosphorylated VE-cadherin (pVE-cadherinY731) in endothelial cells, associated with impaired barrier function and a persistent increase in vascular endothelial cell permeability. However, the mechanism underlying this effect is unknown. In this article, we demonstrate that the AM-mediated dephosphorylation of pVE-cadherinY731 takes place through activation of the tyrosine phosphatase SHP-2, as judged by the rise of its active fraction phosphorylated at tyrosine 542 (pSHP-2Y542) in HUVECs and glioblastoma-derived-endothelial cells. Both pre-incubation of HUVECs with SHP-2 inhibitors NSC-87877 and SHP099 and SHP-2 silencing hindered AM-induced dephosphorylation of pVE-cadherinY731 in a dose dependent-manner, showing the role of SHP-2 in the regulation of endothelial cell contacts. Furthermore, SHP-2 inhibition impaired AM-induced HUVECs differentiation into cord-like structures in vitro and impeded AM-induced neovascularization in in vivo Matrigel plugs bioassays. Subcutaneously transplanted U87-glioma tumor xenograft mice treated with AM-receptors-blocking antibodies showed a decrease in pSHP-2Y542 associated with VE-cadherin in nascent tumor vasculature when compared to control IgG-treated xenografts.Our findings show that AM acts on VE-cadherin dynamics through pSHP-2Y542 to finally modulate cell-cell junctions in the angiogenesis process, thereby promoting a stable and functional tumor vasculature.

ABT-165, a Dual Variable Domain Immunoglobulin (DVD-Ig) Targeting DLL4 and VEGF, Demonstrates Superior Efficacy and Favorable Safety Profiles in Preclinical Models.

Antiangiogenic therapy is a clinically validated modality in cancer treatment. To date, all approved antiangiogenic drugs primarily inhibit the VEGF pathway. Delta-like ligand 4 (DLL4) has been identified as a potential drug target in VEGF-independent angiogenesis and tumor-initiating cell (TIC) survival. A dual-specific biologic targeting both VEGF and DLL4 could be an attractive strategy to improve the effectiveness of anti-VEGF therapy. ABT-165 was uniquely engineered using a proprietary dual-variable domain immunoglobulin (DVD-Ig) technology based on its ability to bind and inhibit both DLL4 and VEGF. In vivo, ABT-165 induced significant tumor growth inhibition compared with either parental antibody treatment alone, due, in part, to the disruption of functional tumor vasculature. In combination with chemotherapy agents, ABT-165 also induced greater antitumor response and outperformed anti-VEGF treatment. ABT-165 displayed nonlinear pharmacokinetic profiles in cynomolgus monkeys, with an apparent terminal half-life > 5 days at a target saturation dose. In a GLP monkey toxicity study, ABT-165 was well-tolerated at doses up to 200 mg/kg with non-adverse treatment-related histopathology findings limited to the liver and thymus. In summary, ABT-165 represents a novel antiangiogenic strategy that potently inhibits both DLL4 and VEGF, demonstrating favorable in vivo efficacy, pharmacokinetic, and safety profiles in preclinical models. Given these preclinical attributes, ABT-165 has progressed to a phase I study. Mol Cancer Ther; 17(5); 1039-50. ©2018 AACR.

Abstract LB-151: The novel tubulin-binding, tumor checkpoint controller BAL101553 has differential effects on tumor vascularization with IV and oral dosing and provides superior anti-tumor activity in combination with bevacizumab

Abstract Background: BAL101553 is a highly soluble prodrug of BAL27862, a novel, small molecule, microtubule-depolymerizer inducing tumor cell death through the spindle assembly checkpoint. BAL101553 has anti-cancer activity in diverse tumor models refractory to standard therapies, and is currently undergoing Phase 1/2a evaluation in advanced cancer patients with IV (weekly) and oral (daily) dosing schedules. Here, the vascular disruption activity of BAL101553 was evaluated in the context of administration route and implications for combinations with VEGF-targeted therapies. Methods: MTD IV (day[d] 1) and oral (d1 & d1 - 4) dosing regimens were tested in H460 NSCLC xenografts. Tumors were analyzed by IHC for proliferation (Ki67) and vascularization (CD31, lectin perfusion). Drug combinations were performed in Pgp-overexpressing, colon carcinoma SW480 xenografts using BAL101553 IV 1xweek (21.3 mg/kg) or oral daily (15 mg/kg) and bevacizumab IP d1 & 4 (5 mg/kg). Results: H460 tumor-bearing mice received once weekly (IV 25 mg/kg; oral 60 mg/kg; high Cmax regimens) or daily (oral 25 mg/kg; low Cmax, high AUC) BAL101553 regimens which elicited equipotent anti-tumor effects (ΔT/Cs = 26-34%). Although all treatments elicited a similar anti-proliferative response (% reduction of mean Ki67 signal+/-SD=10+/-1.2; p<0.05 vs. controls), tumor vessel density was most profoundly reduced by high Cmax regimens. Interestingly, daily-oral treatment (low Cmax, high AUC) had reduced tumor anti-vascular activity, while presenting equivalent anti-proliferative and anti-tumor efficacy. In vitro assays against tumor cell lines showed that sub-cytotoxic BAL27862 concentrations reduced HIF-1α protein levels in normoxic and hypoxic conditions, diminishing VEGF-expression and secretion, indicating direct interference of the drug on hypoxic adaptation and endothelial tube formation. Based on these data, combination of the anti-VEGF antibody bevacizumab with BAL101553 was assessed in the paclitaxel refractory SW480 tumor model, using MTD BAL101553 dosing (IV weekly & oral daily). All monotherapies induced an anti-tumor response (ΔT/Cs bevacizumab: 35%; IV BAL101553 22%; oral BAL101553: 53%). However, bevacizumab combined with IV or oral BAL101553 was superior to single agents (ΔT/C: 6% and 17%, resp., p<0.05 vs. control) with IV combination associated with the least functional tumor vasculature. Conclusions: BAL101553 targets tumor cell proliferation, tumor hypoxic adaptation and vascularization. The latter is Cmax-driven and can be attenuated by daily oral dosing, providing an alternative pharmacodynamic endpoint with implications for clinical treatment strategies. IV and orally administered BAL101553 both elicit superior anti-tumor responses in combination with anti-VEGF therapy. Citation Format: Ashish Sharma, Felix Bachmann, Angela Broggini-Tenzer, Matthias Guckenberger, Heidi Lane, Martin N. Pruschy. The novel tubulin-binding, tumor checkpoint controller BAL101553 has differential effects on tumor vascularization with IV and oral dosing and provides superior anti-tumor activity in combination with bevacizumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-151. doi:10.1158/1538-7445.AM2017-LB-151

Assessment of tumor hypoxia in BEV resistant GBM using FMISO 18F-PET and MRI.

2045 Background: Glioblastoma (GBM) is the most common malignant brain tumor in adults, and is characterized by poor survival and marked resistance to treatment. Hypoxic volume is directly correlated with poor outcome in GBM, with structural and functional tumor vasculature changes regarded as a primary driver of tumor hypoxia. As part of an ongoing clinical trial with a hypoxia activated prodrug, we sought to explore the correlation between abnormal tumor vasculature and hypoxia in bevacizumab (BEV) resistant GBM. Methods: MRI and 18F-FMISO PET scans were acquired at study entry. The MRI protocols include standard anatomical sequences as well as Dynamic Susceptibility Contrast (DSC/perfusion) imaging. Enhancing tumor ROIs were determined from Delta T1 maps, non-enhancing tumor ROIs were obtained from FLAIR images, ratio between enhancing and non-enhancing volumes were calculated, values from standardized rCBV maps were extracted from corresponding enhancing ROIs. Decay correction was applied to18F-FMISO images and converted to SUV units. After registration to MR images, cerebellar regions were used as surrogates for blood activity. The FMISO images were then normalized to generate tissue to blood ratio (TB). A threshold of TB > 1.2 was used in discriminating the hypoxia volume (HV). Correlation between parameters was assessed using Pearson correlation. Results: 7 patients from University of Texas Health Science center and 7 patients from Dana-Farber Cancer Institute had evaluable MR and PET imaging. We compared MRI parameters (enhancing and non-enhancing tumor volumes, srCBV) with hypoxia (HV and maxSUV). There was a positive correlation between HV and enhancing volume (R = 0.732, p = 0.0029) as well as between HV and ratio (R = 0.644, p = 0.013. The correlation between other pairs were not statistically significant (HV vs srCBV: p = 0.862, T1 vs maxSUV p = 0.492, srCBV vs maxSUV: p = 0.393, ratio vs maxSUV = 0.178). Conclusions: The hypoxic volume following bevacizumab failure correlates with both the volume of enhancement and the fraction of enhancement within the mass. The clinical implications of this is being assessed in an ongoing phase 2 study. Clinical trial information: NCT02342379.

Optimized time-resolved imaging of contrast kinetics (TRICKS) in dynamic contrast-enhanced MRI after peptide receptor radionuclide therapy in small animal tumor models.

Anti-tumor efficacy of targeted peptide-receptor radionuclide therapy (PRRT) relies on several factors, including functional tumor vasculature. Little is known about the effect of PRRT on tumor vasculature. With dynamic contrast-enhanced (DCE-) MRI, functional vasculature is imaged and quantified using contrast agents. In small animals DCE-MRI is a challenging application. We optimized a clinical sequence for fast hemodynamic acquisitions, time-resolved imaging of contrast kinetics (TRICKS), to obtain DCE-MRI images at both high spatial and high temporal resolution in mice and rats. Using TRICKS, functional vasculature was measured prior to PRRT and longitudinally to investigate the effect of treatment on tumor vascular characteristics. Nude mice bearing H69 tumor xenografts and rats bearing syngeneic CA20948 tumors were used to study perfusion following PRRT administration with (177) lutetium octreotate. Both semi-quantitative and quantitative parameters were calculated. Treatment efficacy was measured by tumor-size reduction. Optimized TRICKS enabled MRI at 0.032 mm(3) voxel size with a temporal resolution of less than 5 s and large volume coverage, a substantial improvement over routine pre-clinical DCE-MRI studies. Tumor response to therapy was reflected in changes in tumor perfusion/permeability parameters. The H69 tumor model showed pronounced changes in DCE-derived parameters following PRRT. The rat CA20948 tumor model showed more heterogeneity in both treatment outcome and perfusion parameters. TRICKS enabled the acquisition of DCE-MRI at both high temporal resolution (Tres ) and spatial resolutions relevant for small animal tumor models. With the high Tres enabled by TRICKS, accurate pharmacokinetic data modeling was feasible. DCE-MRI parameters revealed changes over time and showed a clear relationship between tumor size and Ktrans .

Highlights of This Issue

Dysregulation of Notch signaling promotes cancer growth through several mechanisms, including the maintenance of tumorigenic cells and functional tumor vasculature. Yen and colleagues report the development and characterization of a dual antagonist antibody that inhibits both Notch2 and Notch3 (OMP-59R5, tarextumab). OMP-59R5 inhibited tumor growth, decreased cancer stem cell frequency, and modulated the function of tumor vasculature in a variety of patient-derived xenografts. In pancreatic cancer, efficacy was associated with higher levels of Notch3 expression, providing a potential biomarker for patient selection. Tarextumab is currently advancing in Phase 2 clinical testing for treatment of pancreatic and small cell lung cancers.The microRNA miR-27a directly regulates dihydropyrimidine dehydrogenase (DPD), the key enzyme in fluoropyrimidine catabolism. To investigate if miR-27a may influence toxicity risk in fluoropyrimidine-based chemotherapy, Amstutz and colleagues assessed the association of genetic polymorphisms in MIR27A with early-onset fluoropyrimidine toxicity. The common MIR27A variant rs895819—previously shown to affect miR-27a levels—was strongly associated with severe early-onset toxicity in patients also carrying risk variants in the DPD gene. This suggests that miR-27a and polymorphisms affecting its expression may serve as novel, complementary markers for further patient risk stratification in DPD risk variant carriers to improve the specificity of genetic testing.Chemotherapy targets rapidly proliferating cells, thus sparing slowly-dividing hypoxic cells that can contribute to repopulation in between courses of treatment. Hypoxia-activated prodrugs (TH-302) selectively kill hypoxic cells and may inhibit tumor repopulation and, in combination with chemotherapy, may increase tumor control. Saggar and colleagues investigated changes in reoxygenation within the tumor microenvironment by administrating two sequential markers of hypoxia following treatment with doxorubicin/docetaxel +/− TH-302. It was found that (i) chemotherapy induces reoxygenation of previously hypoxic cells and leads to tumour repopulation, and (ii) TH-302 reduces this effect. This supports the potential of TH-302 to increase therapeutic effects of chemotherapy.EGFR blockade with the antibody cetuximab is effective in patients with wt-RAS metastatic colorectal cancer (CRC). Unfortunately, responses are transient and patients invariably develop acquired resistance. Arena and colleagues analyzed the genetic profile of clinical samples and preclinical models of acquired resistance to cetuximab. They identified five novel point mutations in the EGFR ectodomain, which confer resistance to cetuximab in CRC patients and cells. Functional analysis of cellular models revealed that a subset of the EGFR mutations retains sensitivity to panitumumab. This knowledge is being incorporated in clinical trials aimed at overcoming acquired resistance to cetuximab in colorectal cancer patients.

The effect of chemotherapeutic agents on tumor vasculature in subcutaneous and orthotopic human tumor xenografts.

BackgroundThe growth of solid tumors and their regrowth after treatment is dependent upon functional tumor vasculature. Some chemotherapeutic agents have shown anti-angiogenic properties but there are limited studies of the effect of chemotherapy on tumor vasculature. Here we investigate the effect of paclitaxel, 5-fluorouracil (5-FU) and doxorubicin on tumor vasculature in subcutaneous and orthotopic xenografts in mice.MethodsThe vascular density and percentage of functional blood vessels were evaluated in subcutaneous A431 human vulvar cancer xenografts, and in subcutaneous and orthotopic MCF-7 human breast cancer xenografts, following single doses of paclitaxel, 5-FU or doxorubicin.ResultsThere was no significant difference in total (CD31+) blood vessels between untreated ectopic and orthotopic MCF-7 tumors, but there was a significantly lower proportion of functional blood vessels in orthotopic tumors. After paclitaxel treatment, there was a decrease in functional tumor vasculature in A431 subcutaneous xenografts, followed by a subsequent rebound. There was a significant decrease in total vascular density on day 12 in A431 tumors following 5-FU or doxorubicin treatment, but no change in the percentage of functional vessels. An increase in functional blood vessels or percentage of functional vasculature was noted in MCF-7 subcutaneous and orthotopic xenografts following chemotherapy treatment.ConclusionsThere are differences in the vasculature and microenvironment of ectopic and orthotopic xenografts in mice. Anti-tumor effects of chemotherapy may be due, in part, to effects on tumor vasculature and may vary in different tumor models.

The effect and mechanism of M402 on gemcitabine uptake into pancreatic tumors.

215 Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive desmoplasia, which has been attributed to high interstitial pressure and poor drug delivery. In nonclinical studies, M402 affected multiple growth factors, adhesion molecules, and chemokines, inhibiting tumor progression, metastasis, and angiogenesis by clearing heparin-binding factors from the tumor microenvironment. We hypothesized that M402 could modulate tumor-stroma interactions in an orthotopic pancreatic cancer model (rich in desmoplasia), decreasing the fibrotic response and in turn increasing tumor perfusion and drug delivery. Methods: Capan-2 human PDAC cells were injected into the pancreata of nude mice. M402 (40 mg/kg/day, s.c.) or saline began Week 5. Gemcitabine (GEM; 30 mg/kg, i.p. biweekly) started Week 7. At different time points, primary tumors were analyzed for mRNA arrays, immunohistochemistry (ECM components, CD31), and functional tumor vasculature. GEM tumor uptake was evaluated by quantifying the incorporation of dFdC into tumor DNA by LC-MS/MS. M402 tumor uptake was visualized using HyLite-750-labeled drug in a Xenogen system. Results: M402 readily targeted pancreatic tumors with long residence time. Alone and particularly in combination with GEM, M402 significantly reduced tumor size and fibrosis, which was accompanied by down-regulation of SHH, PDGF, and TGF-β signaling in stromal cells. Tumors treated with M402 or M402+GEM showed perfusion and microvessel penetration into the tumor centers while vehicle or GEM treated tumors exhibited limited tortuous vessels surrounding the tumor rim. This translated into increased GEM incorporation into the tumor DNA when mice were pre-treated with M402 vs. saline (10.9±5.5 vs. 6.7±1.8 pg of dFdC/µg dG, respectively; p=0.037). Conclusions: M402 appears to inhibit stromal activation within the tumor microenvironment. Reduction in desmoplasia led to improved tumor perfusion, delivery of GEM, and tumor shrinkage. These and other promising nonclinical data support the rationale for the ongoing Phase 1/2 study evaluating the safety and tolerability of M402 in combination with nab-paclitaxel and GEM in patients with metastatic pancreatic cancer.

Abstract 4459: Evaluating imaging biomarkers of acquired resistance to targeted EGFR therapy in xenograft models of human squamous cell carcinoma of the head and neck (SCCHN).

Abstract Overexpression of EGFR has been identified as a negative prognostic factor in squamous cell carcinoma of the head and neck (SCCHN). Patients with SCCHN who respond to EGFR-targeted tyrosine kinase inhibitors (TKIs) eventually develop acquired resistance. Colocalization of EGFR expression and tumor hypoxia in SCCHN is associated with poor outcome, suggesting a role for hypoxia in drug resistance. Non-invasive imaging strategies to accurately identify and monitor patients whose tumors have become resistant to EGFR-TKI therapy would have clinical value. As part of a pre-clinical multiparametric imaging study, we have investigated EGFR-TKI resistance using intrinsic susceptibility magnetic resonance imaging (IS-MRI), which relies on the dependence of the MRI transverse relaxation rate R2* on the ratio of oxy- to deoxyhemoglobin in blood. Given its relationship to blood oxygen saturation and pO2 in and around blood vessels, R2* and hyperoxia-induced ΔR2* are being investigated as imaging biomarkers of tumor oxygenation. IS-MRI was performed on size-matched xenografts derived from the EGFR TKI sensitive SCCHN cell line CAL27 and an isogenic subline resistant to multiple TKIs (gefitinib, erlotinib, and afatinib). Tumor-bearing mice were administered the hypoxia marker pimonidazole, and baseline R2* quantified from resistant (n=8) or sensitive (n=7) tumors whilst the host breathed air. Gas delivery was then switched to 100% O2, and tumour R2* measured again. Finally, mice were administered the perfusion marker Hoechst 33342. The extent of functional tumor vasculature and hypoxia were quantified ex vivo using fluorescence microscopy. Resistant tumors revealed regions of fast R2* (functional, deoxygenated vasculature) restricted primarily to the tumor periphery, while sensitive tumors exhibited a more heterogeneous distribution of fast R2* throughout. There was no significant difference in baseline R2* between the two cohorts (64 ± 4s-1 and 76 ± 5s-1, p=0.07). Hyperoxia resulted in a significantly smaller ΔR2* in the resistant tumors (-2.4 ± 1.4s-1) compared to sensitive tumors (-9.1 ± 2s-1, p<0.05). Resistant tumors had significantly lower Hoechst 33342 uptake (9.5 ± 1.3%, p<0.01) and increased pimonidazole adducts (21 ± 2%, p<0.01) compared to sensitive tumors (19.2 ± 3% and 10.4 ± 2% respectively). Tumor regions of fast R2* and hyperoxic-induced ΔR2* were spatially associated with Hoechst 33342 uptake. Tumors with resistance to EGFR-TKIs exhibited a reduced hemodynamic MRI response that was associated with decreased vessel perfusion and increased tumor hypoxia. IS-MRI informs on phenotypic differences in tumor blood vessel distribution, functionality and oxygenation associated with EGFR-TKI resistance and sensitivity, affording useful non-invasive imaging biomarkers for investigating EGFR drug resistance and tumor hypoxia. Citation Format: Lauren CJ Baker, Carol Box, Arti Sikka, Gary Box, Suzanne A. Eccles, Simon P. Robinson. Evaluating imaging biomarkers of acquired resistance to targeted EGFR therapy in xenograft models of human squamous cell carcinoma of the head and neck (SCCHN). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4459. doi:10.1158/1538-7445.AM2013-4459

Abstract 5293: In vitro modeling of tumor-derived endothelial cell derivation

Abstract Background: Tumor growth beyond a few millimeters in size requires the development of a neovasculature. In addition to providing a portal for the systemic dissemination of metastatic cells, this allows the tumor bed to overcome the severe hypoxia, acidosis, and nutrient deprivation that otherwise limits tumor growth. Early research presumed the development of genetically and functionally normal tumor vasculature from normal host tissue. Anti-angiogenic agents were thus developed to inhibit tumor blood supply with the hope that the presumed normal tumor vascular endothelial cells (EC's), would be less inclined to develop drug resistance. Thus far, however, the experience with these agents has been one of only incremental improvement in patient survival. We have previously demonstrated that tumor cells can differentiate, in vivo, into an EC phenotype and form functional tumor vasculature. Formation of these “tumor derived endothelial cells” (TDEC's) offers a partial explanation for the disappointing clinical results of the angiogenesis inhibitors and suggests a new, complementary, therapeutic target. Objectives: To develop an in vitro system that would recapitulate the tumor cell to EC transition seen in vivo. We hypothesize that manipulation of culture conditions to mimic the tumor microenvironment would result in a switch to an EC phenotype. Design/Methods: A variety of human cancer cell lines (H460 lung cancer, PC3 prostate cancer, OVCAR-8 ovarian cancer) were cultured in 1% O2 in low glucose DMEM supplemented with EC-specific growth factors. The cells were assayed for EC-specific proteins and transcripts with western blotting, immunofluorescence, flow cytometry, and rtPCR. Functionality was quantified by the uptake of acetylated-LDL and the ability to vascularize tumor xenografts. Results: Human cancer cell lines exposed to hypoxia and EC-specific growth factors increase expression of a variety of EC-specific proteins including von Willebrand's factor, ESAM, and VE-Cadherin, while down regulating the epithelial specific cytokeratins. Inclusion of a twenty percent minority of these in vitro-derived TDEC's in a tumor inoculum results in an increase in both tumor growth rate and vascular density. These in vitro-derived TDEC's are also found within the tumor neo-vasculature. Conclusions: We have developed culture conditions that can, within a span of only five days, induce many of the same phenotypic and functional characteristics of the TDEC's isolated from tumor xenografts grown for nearly two months. Furthermore, these cells are able to act as functional endothelial cells in vivo. Potential uses for this system include the ability to study the mechanistic underpinnings of the tumor cell to TDEC transition and to identify pharmacologic inhibitors of this transition. 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 5293. doi:1538-7445.AM2012-5293

Quantitative Immunohistochemistry for Evaluating the Distribution of Ki67 and Other Biomarkers in Tumor Sections and Use of the Method to Study Repopulation in Xenografts after Treatment with Paclitaxel

Surviving cells may repopulate tumors between courses of chemotherapy, thereby reducing the effectiveness of treatment. Using a novel quantitative method, we characterize the influence of the tumor microenvironment on repopulation of surviving cells in human tumor xenografts after paclitaxel treatment and evaluate the potential of gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, to inhibit repopulation. High-EGFR-expressing A431 xenografts and low-EGFR-expressing MCF-7 xenografts were treated with paclitaxel or gefitinib. Time-dependent changes in cell proliferation (Ki67) and apoptosis (cleaved caspase 3) in relation to total and functional tumor blood vessels (recognized by CD31 and a flow marker), and regions of hypoxia (recognized by EF5) were quantified using fluorescence microscopy. Decrease in functional tumor vasculature and in cell proliferation and increase in apoptosis were observed in A431 xenografts after treatment with either paclitaxel or gefitinib. There was a rebound in functional vasculature and cell proliferation ≈ 12 days after treatment with paclitaxel, and repopulation was observed from tumor cells close to regions of hypoxia. Cell proliferation increased ≈ 5 days after the last dose of gefitinib. There were minimal effects of paclitaxel or gefitinib on cell proliferation, cell death, or tumor vasculature in MCF-7 xenografts. Repopulation in A431 xenografts after treatment with paclitaxel was associated with changes in functional tumor vasculature. Gefitinib decreased cell proliferation in EGFR-overexpressing tumor xenografts, suggesting its potential to inhibit repopulation when used in sequence with chemotherapy.

Abstract 5317: Non-invasive dynamic bioluminescence imaging of tumor functional vascular changes during antiangiogenic therapy in conscious mice

Abstract Bioluminescence imaging has been used for several years in oncology research to quantify tumor growth and metastatic spread rather than functional physiological effects. Two recent publications have highlighted the use of non-invasive dynamic bioluminescence imaging (DBLI) in isofluran anesthetized mice to assess vascular changes in subcutaneous xenografts (1) and orthotopic implanted tumors (2) post-treatment. We have previously reported that anesthesia impairs bioluminescence signal intensity (3) and may in turn profoundly affect interpretation of compound activity based on BLI (4). Thus, we decided to investigate DBLI for non-invasive, longitudinal and quantitative monitoring of functional vascular changes (tumor perfusion, blood volume, vascular flow) in conscious mice during antiangiogenic therapy. Subcutaneous xenografts (HCT116 colorectal, U87MG glioblastoma and SJSA-1 osteosarcoma) were treated with a blocking antibody to vascular endothelial growth factor receptor 2 (DC101) and repeatedly examined at bi-weekly intervals. Our results have clearly demonstrated that in all tested models, tumor growth was significantly inhibited (T/C: 0.33, 0.52 and 0.53 for HCT116, U87MG and SJSA-1, respectively) after treatment with DC101 when compared to control. DBLI analysis revealed an initial increase in vascular flow (Day 2 to 3) followed by a gradual decrease after a 2-wk therapy, suggesting early and long-term effects on functional tumor vasculature. Notably, this pattern was more pronounced in highly vascularized (SJSA-1) when compared with less well vascularized (HCT116) tumors. Lastly, very similar decreased microvessel density (MVD) was noted by histological immuno-fluorescence staining following chronic treatment with DC101 in all three tumor models. In conclusion, DBLI measurements in conscious tumor bearing mice offer an optimal and reliable approach for non-invasive functional tumor vessel assessment without the confounding effects of anesthesia. It can be foreseen that with future development of more efficient light-emitting enzyme/substrate systems and the improvement of detection devices it will be possible to increase the sensitivity of this technique allowing the measurement of weaker bioluminescence signals and extending spatial resolution.

Abstract LB-304: Bone marrow-derived endothelial cells migrate to tumor sites and contribute to functional tumor vasculature

Abstract Tumor expansion is dependent on neovascularization, a process that requires sustained new vessel formation. Although the critical role of angiogenesis by endothelial sprouting in this process, controversy still prevails on the whether vasculogenesis, involving bone marrow (BM)-derived endothelial cells, does contribute to this process. The objective of this study was to investigate the contributions of BM-derived endothelial cells to tumor vasculature, in a melanoma model using chimeric GFP mice. Methodology: First, wild-type C57BL/6 mice were was exposed to a single dose of 10 Gy X-irradition and rescued by transplantation of BM cells from C57BL/6-GFP mice. At d30 post-transplant, the animals received a subcutaneous infusion of melanoma cells stably transfected with the luciferase gene (B16/F10-Luc+). After 20 days, the tumors were harvested, and tumor stroma characterized using flow cytometry, bioluminescence-based image processing and confocal microscopy, to define the specific contribution of BM-derived cells. Results: Mice transplanted with donor GFP+ cells showed significant BM chimerism (90.9 ± 0.87%) demonstrating successful engraftment of donor BM stem/progenitor cells. Analyses of tumor specimens showed the presence of donor cells in the tumor sites (3.5±1.7%) in all animals, showing that BM-derived cells were effectively recruited to the developing melanoma. Interestingly, these cells represent endothelial cells (CD31+ cells; mean=X%) and myeloid-lineage cells (CD11b+ cells; mean=80%), but also tumor-infiltrating lymphocytes (CD8+ T cells, mean = 13.31; CD4+ T-cells, mean=2.1). Examination of the tumor endothelium by confocal microscopy clearly showed the presence of donor CD31/GFP cells in the vessel wall, namely in vessels lacking other mural components (as pericytes). Conclusions: Taken together, this study demonstrates that BM-derived cells are actively recruited to tumor sites, contributing to the tumor stroma and the formation of blood vessels in the developing tumor. We are performing studies to elucidate the molecular mechanisms involved in mobilization of of BM-derived precursor cells to the tumor microenvironment, and whether disruption of this vasculogeneic process significant impairs tumor development. 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 LB-304. doi:10.1158/1538-7445.AM2011-LB-304

Vascular and Metabolic Response to Bevacizumab-Containing Regimens in Two Patients With Colorectal Liver Metastases Measured by Dynamic Contrast-Enhanced MRI and Dynamic 18F-FDG-PET

Early monitoring of response to treatment is one of the cornerstones of personalized treatment. As new and often expensive targeted therapies, which are tumoristatic rather than tumoricidal, become available, new demands are posed on response assessment. Bevacizumab, an antiangiogenic agent causing normalization of the tumor microvasculature, potentiates the effect of cytotoxic agents on colorectal liver metastases. It is known that assessment of glucose metabolism by (dynamic) positron emission tomography using [(18)F]-2-fluoro-2-deoxy-D-glucose ((18)F-FDG PET) can be used as an early surrogate endpoint to determine treatment efficacy. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) can be used to quantify functional tumor vasculature (permeability, vascular surface area). Here, we describe the response of colorectal liver metastases to cytotoxic regimens including bevacizumab using both (18)F-FDG PET and DCE-MRI in 2 cases. In both cases, a large reduction in glucose metabolic rate and functional tumor vasculature are observed after 3 treatment cycles.

VEGF121b and VEGF165b are weakly angiogenic isoforms of VEGF-A

BackgroundDifferent isoforms of VEGF-A (mainly VEGF121, VEGF165 and VEGF189) have been shown to display particular angiogenic properties in the generation of a functional tumor vasculature. Recently, a novel class of VEGF-A isoforms, designated as VEGFxxxb, generated through alternative splicing, have been described. Previous studies have suggested that these isoforms may inhibit angiogenesis. In the present work we have produced recombinant VEGF121/165b proteins in the yeast Pichia pastoris and constructed vectors to overexpress these isoforms and assess their angiogenic potential.ResultsRecombinant VEGF121/165b proteins generated either in yeasts or mammalian cells activated VEGFR2 and its downstream effector ERK1/2, although to a lesser extent than VEGF165. Furthermore, treatment of endothelial cells with VEGF121/165b increased cell proliferation compared to untreated cells, although such stimulation was lower than that induced by VEGF165. Moreover, in vivo angiogenesis assays confirmed angiogenesis stimulation by VEGF121/165b isoforms. A549 and PC-3 cells overexpressing VEGF121b or VEGF165b (or carrying the PCDNA3.1 empty vector, as control) and xenotransplanted into nude mice showed increased tumor volume and angiogenesis compared to controls. To assess whether the VEGFxxxb isoforms are differentially expressed in tumors compared to healthy tissues, immunohistochemical analysis was conducted on a breast cancer tissue microarray. A significant increase (p < 0.05) in both VEGFxxxb and total VEGF-A protein expression in infiltrating ductal carcinomas compared to normal breasts was observed. A positive significant correlation (r = 0.404, p = 0.033) between VEGFxxxb and total VEGF-A was found.ConclusionsOur results demonstrate that VEGF121/165b are not anti-angiogenic, but weakly angiogenic isoforms of VEGF-A. In addition, VEGFxxxb isoforms are up-regulated in breast cancer in comparison with non malignant breast tissues. These results are to be taken into account when considering a possible use of VEGF121/165b-based therapies in patients.

Inhibition of vasculogenesis, but not angiogenesis, prevents the recurrence of glioblastoma after irradiation in mice

Despite the high doses of radiation delivered in the treatment of patients with glioblastoma multiforme (GBM), the tumors invariably recur within the irradiation field, resulting in a low cure rate. Understanding the mechanism of such recurrence is therefore important. Here we have shown in an intracranial GBM xenograft model that irradiation induces recruitment of bone marrow-derived cells (BMDCs) into the tumors, restoring the radiation-damaged vasculature by vasculogenesis and thereby allowing the growth of surviving tumor cells. BMDC influx was initiated by induction of HIF-1 in the irradiated tumors, and blocking this influx prevented tumor recurrence. Previous studies have indicated that BMDCs are recruited to tumors in part through the interaction between the HIF-1-dependent stromal cell-derived factor-1 (SDF-1) and its receptor, CXCR4. Pharmacologic inhibition of HIF-1 or of the SDF-1/CXCR4 interaction prevented the influx of BMDCs, primarily CD11b+ myelomonocytes, and the postirradiation development of functional tumor vasculature, resulting in abrogation of tumor regrowth. Similar results were found using neutralizing antibodies against CXCR4. Our data therefore suggest a novel approach for the treatment of GBM: in addition to radiotherapy, the vasculogenesis pathway needs to be blocked, and this can be accomplished using the clinically approved drug AMD3100, a small molecule inhibitor of SDF-1/CXCR4 interactions.

Platelet-Derived Growth Factor-B Normalizes Micromorphology and Vessel Function in Vascular Endothelial Growth Factor-A-Induced Squamous Cell Carcinomas

Vascular endothelial growth factor (VEGF), which is a key regulator of angiogenesis, often induces formation of immature vessels with increased permeability and reduced vessel functionality. Here, we demonstrate that de novo expression of murine (m)VEGF-164 induces malignant and invasive tumor growth of HaCaT keratinocytes. However, the mVEGF-164-induced tumors are ulcerated with a disorganized epithelium that is interrupted by lacunae with limited basement membrane and endothelial cell coverage. Vessel maturation is strongly impaired. Tumor and vessel micromorphology are markedly improved by the combined expression of human platelet-derived growth factor (hPDGF)-B and mVEGF-164. Although tumor size and malignancy are comparable with either mVEGF-164 alone or combined human PDGF-B and mVEGF-164 expression, combined hPDGF-B and mVEGF-164 expression leads to a more solid and compact tumor tissue with a mature functional tumor vasculature and a higher microvessel density, as demonstrated histologically and by dynamic contrast-enhanced magnetic resonance imaging. Treatment of the hPDGF-B- and mVEGF-164-expressing tumors with imatinib mesylate to block PDGF-B signaling reverses this effect. In addition, tumor cell invasion of mVEGF-164 transfectants and mVEGF-164 plus hPDGF-B transfectants in vivo is associated with a marked induction of tumor-derived matrix metalloproteinase-1 and stromal matrix metalloproteinase-9 and -13, as was confirmed in three-dimensional organotypic co-cultures with fibroblasts in vitro. These data clearly demonstrate the need for a concerted action of different growth factors in the establishment of solid tumors with functional vasculature and emphasize the need for a multifactorial therapy.

Embryonic stem cell tumor model reveals role of vascular endothelial receptor tyrosine phosphatase in regulating Tie2 pathway in tumor angiogenesis

Inhibiting angiogenesis has become an effective approach for treating cancer and other diseases. However, our understanding of signaling pathways in tumor angiogenesis has been limited by the embryonic lethality of many gene knockouts. To overcome this limitation, we used the plasticity of embryonic stem (ES) cells to develop a unique approach to study tumor angiogenesis. Murine ES cells can be readily manipulated genetically; in addition, ES cells implanted subcutaneously in mice develop into tumors that contain a variety of cell types (teratomas). We show that ES cells differentiate into bona fide endothelial cells within the teratoma, and that these ES-derived endothelial cells form part of the functional tumor vasculature. Using this powerful and flexible system, the Angiopoietin/Tie2 system is shown to have a key role in the regulation of tumor vessel size. Endothelial differentiation in the ES teratoma model allows gene-targeting methods to be used in the study of tumor angiogenesis.