Angiogenesis inhibitors are receiving increased attention as cancer therapeutics, but little is known of the cellular effects of these inhibitors on tumor vessels. We sought to determine whether two agents, AG013736 and VEGF-Trap, that inhibit vascular endothelial growth factor (VEGF) signaling, merely stop angiogenesis or cause regression of existing tumor vessels. Here, we report that treatment with these inhibitors caused robust and early changes in endothelial cells, pericytes, and basement membrane of vessels in spontaneous islet-cell tumors of RIP-Tag2 transgenic mice and in subcutaneously implanted Lewis lung carcinomas. Strikingly, within 24 hours, endothelial fenestrations in RIP-Tag2 tumors disappeared, vascular sprouting was suppressed, and patency and blood flow ceased in some vessels. By 7 days, vascular density decreased more than 70%, and VEGFR-2 and VEGFR-3 expression was reduced in surviving endothelial cells.

Inhibitors of VEGF signaling can block angiogenesis and reduce tumor vascularity, but little is known about the reversibility of these changes after treatment ends. In the present study, regrowth of blood vessels in spontaneous RIP-Tag2 tumors and implanted Lewis lung carcinomas in mice was assessed after inhibition of VEGF receptor signaling by AG-013736 or AG-028262 for 7 days. Both agents caused loss of 50%-60% of tumor vasculature. Empty sleeves of basement membrane were left behind. Pericytes also survived but had less α-SMA immunoreactivity. One day after drug withdrawal, endothelial sprouts grew into empty sleeves of basement membrane. Vessel patency and connection to the bloodstream followed close behind. By 7 days, tumors were fully revascularized, and the pericyte phenotype returned to baseline. Importantly, the regrown vasculature regressed as much during a second treatment as it did in the first. Inhibition of MMPs or targeting of type IV collagen cryptic sites by antibody HUIV26 did not eliminate the sleeves or slow revascularization. These results suggest that empty sleeves of basement membrane and accompanying pericytes provide a scaffold for rapid revascularization of tumors after removal of anti-VEGF therapy and highlight their importance as potential targets in cancer therapy.

Purpose: Axitinib (AG-013736) is a potent and selective inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases 1to 3 that is in clinical development for the treatment of solid tumors. We provide a comprehensive description of its in vitro characteristics and activities, in vivo antiangiogenesis, and antitumor efficacy and translational pharmacology data. Experimental Design: The potency, kinase selectivity, pharmacologic activity, and antitumor efficacy of axitinib were assessed in various nonclinical models. Results: Axitinib inhibits cellular autophosphorylation of VEGF receptors (VEGFR) with picomolar IC 50 values. Counterscreening across multiple kinase and protein panels shows it is selective for VEGFRs. Axitinib blocks VEGF-mediated endothelial cell survival, tube formation, and downstream signaling through endothelial nitric oxide synthase, Akt and extracellular signal-regulated kinase. Following twice daily oral administration, axitinib produces consistent and dose-dependent antitumor efficacy that is associated with blocking VEGFR-2 phosphorylation, vascular permeability, angiogenesis, and concomitant induction of tumor cell apoptosis. Axitinib in combination with chemotherapeutic or targeted agents enhances antitumor efficacy in many tumor models compared with single agent alone. Dose scheduling studies in a human pancreatic tumor xenograft model show that simultaneous administration of axitinib and gemcitabine without prolonged dose interruption or truncation of axitinib produces the greatest antitumor efficacy. The efficacious drug concentrations predicted in nonclinical studies are consistent with the range achieved in the clinic. Although axitinib inhibits platelet-derived growth factor receptors and KIT with nanomolar in vitro potencies, based on pharmacokinetic/pharmacodynamic analysis, axitinib acts primarily as a VEGFR tyrosine kinase inhibitor at the current clinical exposure. Conclusions: The selectivity, potency for VEGFRs, and robust nonclinical activity may afford broad opportunities for axitinib to improve cancer therapy.

Purpose Patients with advanced, incurable thyroid cancer not amenable to surgery or radioactive iodine (131I) therapy have few satisfactory therapeutic options. This multi-institutional study assessed the activity and safety of axitinib, an oral, potent, and selective inhibitor of vascular endothelial growth factor receptors (VEGFR) 1, 2, and 3 in patients with advanced thyroid cancer. Patients and Methods Patients with thyroid cancer of any histology that was resistant or not appropriate for 131I were enrolled onto a single-arm phase II trial to receive axitinib orally (starting dose, 5 mg twice daily). Objective response rate (ORR) by Response Evaluation Criteria in Solid Tumors was the primary end point. Secondary end points included duration of response, progression-free survival (PFS), overall survival, safety, and modulation of soluble (s) VEGFR. Results Sixty patients were enrolled. Partial responses were observed in 18 patients, yielding an ORR of 30% (95% CI, 18.9 to 43.2). Stable disease lasting ≥ 16 weeks was reported in another 23 patients (38%). Objective responses were noted in all histologic subtypes. Median PFS was 18.1 months (95% CI, 12.1 to not estimable). Axitinib was generally well tolerated, with the most common grade ≥ 3 treatment-related adverse event being hypertension (n = 7; 12%). Eight patients (13%) discontinued treatment because of adverse events. Axitinib selectively decreased sVEGFR-2 and sVEGFR-3 plasma concentrations versus sKIT, demonstrating its targeting of VEGFR. Conclusion Axitinib is a selective inhibitor of VEGFR with compelling antitumor activity in all histologic subtypes of advanced thyroid cancer.

004). MMP-2 is expressed by neuroblastoma tumor cells and stromal cells, whereas MMP-9 is exclusively expressed by stromal cells, particularly vascular cells.To examine the contribution of MMP-9 to tumor angiogenesis, we generated RAG1/MMP-9 double-deficient mice. We observed a significant inhibition of angiogenesis in the immunodeficient RAG1/MMP-9 double-deficient mice orthotopically implanted with tumor cells (P ‫؍‬ 0.043) or implanted s.c. with a mixture of tumor cells and Matrigel (P < 0.001). Using an FITC-labeled lectin, we demonstrated an inhibition in the architecture of the tumor vasculature in MMP-9-deficient mice, resulting in fewer and smaller blood vessels. These changes were associated with a 48% decrease in pericytes present along microvessels. Taken together, the data demonstrate that in neuroblastoma, stromally derived MMP-9 contributes to angiogenesis by promoting blood vessel morphogenesis and pericyte recruitment.

Purpose: Antigenic overlap among circulating endothelial cells (CEC) and progenitors (CEP), platelets, and other blood cells led to the need to develop a reliable standardized method for CEC and CEP quantification. These cells are emerging as promising preclinical/clinical tools to define optimal biological doses of antiangiogenic therapies and to help stratify patients in clinical trials. Experimental Design: We report the experimental validation of a novel flow cytometry method that precisely dissects CEC/CEP from platelets and other cell populations and provides information about CEC/CEP viability.+ CECs, investigated by electron microscopy, were found to be bona fide endothelial cells by the presence of Weibel-Palade bodies. More than 75% of the circulating mRNAs of the endothelial-specific gene,VE-cadherin, found in the blood were present in the sorted population. CECs were 140 F 171/mL in healthy subjects (n = 37) and 951 F1,876/mL in cancer patients (n = 78; P < 0.0001). The fraction of apoptotic/necrotic CECs was 77 F 14% in healthy subjects and 43 F 23% in cancer patients (P < 0.0001). CEPs were 181 F 167/mL in healthy donors and 429 F 507/mL in patients (P = 0.00019). Coefficients of variation were 4 F 4% (intrareader), 17 F 4% (interreader), and 17 F 7% (variability over 0-72 h), respectively. Parallel samples were frozen by a standardized protocol. After thawing, coefficients of variation were 12 F 8% (intrareader), 16 F 10% (interreader), and 26 F 16% (variability over 0-14 days of frozen storage), respectively. Conclusions: This procedure enumerates a truly endothelial cell population with limited intrareader and interreader variability. It appears possible to freeze samples for large-scale CEC enumeration during clinical trials.This approach could be enlarged to investigate other angiogenic cell populations as well.

Purpose: There is an unmet need for noninvasive markers to measure the biological effects of targeted agents, particularly those inhibiting the vascular endothelial growth factor (VEGF) receptor (VEGFR) pathway, and identify patients most likely to benefit from treatment. In this study, we investigated potential blood-based biomarkers for SU11248 (sunitinib malate), a multitargeted tyrosine kinase inhibitor, in patients with metastatic imatinib-refractory gastrointestinal stromal tumors. Experimental Design: Patients (n = 73) enrolled in a phase I/II trial received SU11248 daily for 14 or 28 days followed by 14 days without treatment per cycle. Clinical benefit was defined as progression-free survival of &gt;6 months. We assessed plasma markers, including VEGF and soluble VEGFR-2 (sVEGFR-2), and two cellular populations bearing VEGF receptors: monocytes and, in a subset of patients, mature circulating endothelial cells (CEC). Results: Compared to patients with progressive disease, patients with clinical benefit had significantly greater increases in CECs (0.52 versus −−0.01 CEC/μL/d, P = 0.03) and smaller decreases in monocyte levels (47% versus 60%, P = 0.007) during cycle 1. VEGF increased by 2.2-fold and sVEGFR-2 decreased 25% during the first 2 weeks of treatment. Neither plasma marker correlated with clinical outcome although a modest inverse correlation was observed between sVEGFR-2 changes and plasma drug levels. Monocytes, VEGF, and sVEGFR-2 all rebounded towards baseline off treatment. Conclusions: Monocytes, VEGF, and sVEGFR-2 were consistently modulated by treatment, suggesting that they may serve as pharmacodynamic markers for SU11248. Changes in CECs and monocytes, but not the plasma markers, differed between the patients with clinical benefit and those with progressive disease. These end points merit further investigation in future trials to determine their utility as markers of SU11248 activity and clinical benefit in gastrointestinal stromal tumors and other tumor types.

We studied AG3340, a potent metalloproteinase (MMP) inhibitor with pM affinities for inhibiting gelatinases (MMP-2 and -9), MT-MMP-1 (MMP-14), and collagenase-3 (MMP-13) in many tumor models. AG3340 produced dose-dependent pharmacokinetics and was well tolerated after intraperitoneal (i.p.) and oral dosing in mice. Across human tumor models, AG3340 produced profound tumor growth delays when dosing began early or late after tumor implantation, although all established tumor types did not respond to AG3340. A dose-response relationship was explored in three models: COLO-320DM colon, MV522 lung, and MDA-MB-435 breast. Dose-dependent inhibitions of tumor growth (over 12.5-200 mg/kg given twice daily, b.i.d.) were observed in the colon and lung models; and in a third (breast), maximal inhibitions were produced by the lowest dose of AG3340 (50 mg/kg, b.i.d.) that was tested. In another model, AG3340 (100 mg/kg, once daily, i.p.) markedly inhibited U87 glioma growth and increased animal survival. AG3340 also inhibited tumor growth and increased the survival of nude mice bearing androgen-independent PC-3 prostatic tumors. In a sixth model, KKLS gastric, AG3340 did not inhibit tumor growth but potentiated the efficacy of Taxol. Importantly, AG3340 markedly decreased tumor angiogenesis (as assessed by CD-31 staining) and cell proliferation (as assessed by bromodeoxyuridine incorporation), and increased tumor necrosis and apoptosis (as assessed by hematoxylin and eosin and TUNEL staining). These effects were model dependent, but angiogenesis was commonly inhibited. AG3340 had a superior therapeutic index to the cytotoxic agents, carboplatin and Taxol, in the MV522 lung cancer model. In combination, AG3340 enhanced the efficacy of these cytotoxic agents without altering drug tolerance. Additionally, AG3340 decreased the number of murine melanoma (B16-F10) lesions arising in the lung in an intravenous metastasis model when given in combination with carboplatin or Taxol. These studies directly support the use of AG3340 in front-line combination chemotherapy in ongoing clinical trials in patients with advanced malignancies of the lung and prostate.