Reduction In Tumor Blood Flow Research Articles

Abstract 3182: CAR T cells as micropharmacies to induce locoregional tumor vascular infarction by antigen-specific delivery of tissue factor to the tumor microenvironment

Abstract CAR T cell therapy of solid tumors is challenged by the heterogeneity of target expression and by mechanical and immune-modulatory barriers in the tumor microenvironment (TME). To combine CAR-retargeted T cell effector functions with a second therapeutic mode of action, we designed an innovative cell-based combination strategy. CAR-engineered antitumor effector T cells are used as micropharmacies to produce and deliver a pro-coagulatory fusion protein, tTF-NGR, in the TME to induce locoregional tumor vascular infarction for combined T-cell mediated and hypoxic tumor cell death. tTF-NGR is a CD13-targeted tissue factor variant with coagulation activity upon relocalization into the phospholipid membranes of the CD13-expressing vascular endothelial cells that invade tumor tissues. Consequent thrombosis in tumor blood vessels induces tumor infarction, growth retardation and regression in preclinical in vitro and in vivo studies and selective reduction of tumor blood flow in a clinical phase I study. Human T cells were co-transduced by retroviral one-vector gene transfer to express genes encoding for a GD2-specific CAR and for tTF-NGR, the latter in an antigen-dependent CAR-mediated manner. The engineered T cells exerted potent GD2 antigen-specific effector functions, including secretion of IFN-γ and TNF-α, upregulation of CD107 and tumor cell lysis, comparable to control CAR T cells producing mutant tTF-NGR lacking pro-coagulatory function. They secreted recombinant tTF-NGR in a strictly antigen-dependent manner upon coincubation with the anti-idiotype antibody ganglidiomab, which selectively engages the extracellular scFv of the CAR, or with GD2-positive tumor cells, shown by ELISA. tTF-NGR produced by human T cells effectively activates the extrinsic coagulation cascade, thus it retains its pro-coagulatory activity. In a murine Ewing sarcoma xenograft model which expresses the CAR target GD2on tumor cells along with CD13 on tumor vascular endothelial cells, GD2-specific CAR T cells with inducible tTF-NGR had noticeably superior therapeutic activity compared with control cells excreting mutant tTF-NGR. Mechanistic evidence hints at hypoxia-induced higher CAR T cell cytolytic activity. We conclude that combined CAR-mediated T cell targeting of cancer cells with CD13-targeted vascular infarction of the TME in a one-vector engineering strategy is a promising approach to overcome limitations of both strategies for effective targeting and eradication of solid cancers. Citation Format: Bianca Altvater, Sareetha Kailayangiri, Christian Spurny, Maike Flügge, Jutta Meltzer, Lea Greune, Christian Schwöppe, Caroline Brand, Christoph Schliemann, Wolfgang Hartmann, Hinrich Abken, Axel Schambach, Nicole Farwick, Wolfgang E. Berdel, Claudia Rossig. CAR T cells as micropharmacies to induce locoregional tumor vascular infarction by antigen-specific delivery of tissue factor to the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3182.

Effect of Ultrasound-Stimulated Microbubbles and Hyperthermia on Tumor Vasculature of Breast Cancer Xenograft.

The objective of the present study was to investigate the treatment effects of ultrasound-stimulated microbubbles (USMB) and hyperthermia (HT) on breast tumor vasculature. Tumor-bearing mice with breast cancer xenografts (MDA-MB-231), were exposed to different treatment conditions consisting of control (no treatment), USMB alone, HT alone, USMB with HT exposures of 10 and 50minutes. Quantitative 3D Doppler ultrasound and photoacoustic imaging were used to detect tumor blood flow and oxygen saturation, respectively. In addition, histopathological analysis including TUNEL staining for cell death, and CD31 staining for the vessel count, was performed to complement the results of power Doppler and photoacoustic imaging. Results demonstrated a decrease in tumor blood flow as well as oxygenation level following 50minutes HT treatment either alone or combined with USMB. In contrast, 10minutes HT alone or combined with USMB had minimal effects on blood flow and tumor oxygenation level. Treatment with HT for 50 minutes caused drops in tumor oxygenation, which were not evident with USMB treatment alone. Additionally, results revealed an increase in cell death after 10minutes HT with or without USMB and a decrease in vessel count compared to control. Unlike previous studies which demonstrated synergistic treatment effects combining USMB with other modalities such as radiation or chemotherapy, USMB and HT effects were not synergistic in the present study. The results here demonstrated HT and USMB both alone or together resulted in a significant reduction in tumor blood flow, tumor oxygenation, and vessel count with observed increases in cell death response.

Tumor Blood Flow Is a Predictor of Radiotherapy Response in Patients With Nasopharyngeal Carcinoma.

PurposeThe aim of this study was to evaluate tumor blood flow (TBF) as a predictor of radiotherapy response for nasopharyngeal carcinoma (NPC).Materials and MethodA total of 134 patients were divided into two groups, the complete response (CR) group and the partial response (PR) group based on RECIST 1.1 recommendations. The statistical difference was evaluated for pre- and mid- or post-treatment TBF and changes of TBF for tumors and metastatic lymph nodes between CR and PR, respectively. The receiver operation characteristic (ROC) curve was utilized to evaluate the accuracy of TBF in predicting the response of radiation therapy. The association between TBF and SUVmax was also investigated.ResultsThe reduction of TBF in CR was significantly lower than that in PR for primary tumors (P <0.001) and metastatic lymph nodes (P <0.001). The multivariate logistic regression analysis indicated that the reduction of TBF is an independent predictor of the response of radiation therapy for primary tumors (P <0.001) and metastatic lymph nodes (P <0.001). The accuracy of TBF reduction in predicting the response of radiation therapy was 0.817 in primary tumors and 0.924 in metastatic lymph nodes, respectively. No significant correlation was observed between the TBF values and SUVmax of primary tumors (r = -0.008, P = 0.954) and metastasis lymph nodes (r = -0.061, P = 0.652).ConclusionThis study suggests that the reduction of TBF is a promising parameter for evaluating the response of radiation therapy.

Efficacy of endovascular intratumoral embolization for meningioma: assessment using dynamic susceptibility contrast-enhanced perfusion-weighted imaging

BackgroundIn preoperative embolization for intracranial meningioma, endovascular intratumoral embolization is considered to be more effective for the reduction of tumorous vascularity than proximal feeder occlusion. In this study, we aimed...

First-In-Class CD13-Targeted Tissue Factor tTF-NGR in Patients with Recurrent or Refractory Malignant Tumors: Results of a Phase I Dose-Escalation Study.

Background: Aminopeptidase N (CD13) is present on tumor vasculature cells and some tumor cells. Truncated tissue factor (tTF) with a C-terminal NGR-peptide (tTF-NGR) binds to CD13 and causes tumor vascular thrombosis with infarction. Methods: We treated 17 patients with advanced cancer beyond standard therapies in a phase I study with tTF-NGR (1-h infusion, central venous access, 5 consecutive days, and rest periods of 2 weeks). The study allowed intraindividual dose escalations between cycles and established Maximum Tolerated Dose (MTD) and Dose-Limiting Toxicity (DLT) by verification cohorts. Results: MTD was 3 mg/m2 tTF-NGR/day × 5, q day 22. DLT was an isolated and reversible elevation of high sensitivity (hs) Troponin T hs without clinical sequelae. Three thromboembolic events (grade 2), tTF-NGR-related besides other relevant risk factors, were reversible upon anticoagulation. Imaging by contrast-enhanced ultrasound (CEUS) and dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) showed major tumor-specific reduction of blood flow in all measurable lesions as proof of principle for the mode of action of tTF-NGR. There were no responses as defined by Response Evaluation Criteria in Solid Tumors (RECIST), although some lesions showed intratumoral hemorrhage and necrosis after tTF-NGR application. Pharmacokinetic analysis showed a t1/2(terminal) of 8 to 9 h without accumulation in daily administrations. Conclusion: tTF-NGR is safely applicable with this regimen. Imaging showed selective reduction of tumor blood flow and intratumoral hemorrhage and necrosis.

PO-263 Assessment of the accumulation of hyaluronan in the tumour microenvironment (TME) of solid tumours

IntroductionHyaluronan (HA), a primary component of the extracellular matrix, can accumulate in a variety of solid malignancies, where histological analyses of patient tumour tissues demonstrated that HA levels may correlate with patient outcomes. HA accumulation in the TME may be associated with poor prognosis. Preclinical studies demonstrated elevated TME HA levels increased tumour interstitial pressure, reduced tumour blood flow, and resulted in poor penetration of anticancer agents and immune cells. A PEGylated recombinant human hyaluronidase, pegvorhyaluronidase alfa (PEGPH20), depolymerizes HA, increases tumour vascular perfusion, and may increase access and anti-tumour efficacy of cytotoxic and immunotherapies.Material and methodsPegvorhyaluronidase alfa is being evaluated in clinical studies for pancreatic, breast, lung, cholangiocarcinoma, and gastric cancers. To broaden the spectrum of possible tumour types that may benefit from pegvyorhyaluronidase alfa, we assessed the prevalence and accumulation of HA in 3633 human histological samples in 22 solid tumour types. HA levels were measured using tissue microarrays (TMAs) stained with a proprietary HA probe, HTI–601, a recombinant immunoadhesin containing a modified HA–specific link domain from TNF-Stimulated Gene 6 protein (TSG–6) and the Fc portion of human IgG1. The resulting HA probe was biotinylated for use in an affinity-based histochemistry method. HA staining was evaluated with research-grade histological image analysis software. HA levels associated with distinct tumour compartments (stroma or tumour cells) were assessed. HA accumulation associated with immune infiltrates present in the TMA cores was also evaluated.Results and discussionsDistribution of HA accumulation across compartments fell into 4 main arrangements:- For desmoplastic tumour indications (pancreatic ductal adenocarcinoma, cholangiocarcinoma), the stroma was associated with high percentage of surface area stained with HTI-601 relative to the tumour cells;- Comparable distribution of HA staining in both tumour cells and stroma (gall bladder and glioblastoma);- Sub-clusters of HA staining within a specific tumour type (NSCLC non-squamous);- Distribution bias towards increased or decreased HA intensity staining (head and neck excluding nasopharyngeal cavities and renal cell carcinoma, respectively).ConclusionUnderstanding HA prevalence and accumulation in solid tumours may potentially identify malignancies that may benefit from pegvorhyaluronidase alfa therapy targeting HA.

Nuclear survivin expression correlates with endoglin-assessed microvascularisation in laryngeal carcinoma

AimsSurvivin—a member of the family of inhibitor of apoptosis proteins that control cell division, apoptosis and metastasis—is overexpressed in virtually all human cancers, including laryngeal squamous cell carcinoma (LSCC). Recent...

Fosbretabulin tromethamine in the treatment of thyroid cancer

ABSTRACTIntroduction: Anaplastic thyroid carcinoma [ATC] is a rare, highly aggressive neoplasm with a disease-specific mortality of nearly 100%. Available treatment options include surgery, radiotherapy, chemotherapy or their combination and, more often recently, participation in clinical trials, as recommended by all available guidelines. However, despite of many attempts with the use of new targeted drugs, to date no remarkable progress in the improvement in patients’ survival has been made. Fosbretabulin (combretastatin A4 phosphate, CA4P) is a vascular disrupting agent, and a new drug that targets tumor neovasculature leading to an acute, reversible reduction in tumor blood flow and tumor central necrosis. Its activity against ATC was evaluated in the FACT trial.Areas covered: Published results of preclinical, phase I and II studies, and other reports evaluating the mechanism of action, efficacy and safety of fosbretabulin alone or combination with other drugs in ATC are evaluated.Expert Opinion: Disappointing results of previous randomized studies, referred to ATC treatment, force us to draw conclusions. Among the reasons leading to the failure of FACT study was an insufficient number of enrolled patients. Since available guidelines emphasized the necessity to treat ATC under clinical trials, inclusion criteria should be reconsidered to make the recruitment process much easier and faster in future.

NG2 proteoglycan as a pericyte target for anticancer therapy by tumor vessel infarction with retargeted tissue factor.

tTF-TAA and tTF-LTL are fusion proteins consisting of the extracellular domain of tissue factor (TF) and the peptides TAASGVRSMH and LTLRWVGLMS, respectively. These peptides represent ligands of NG2, a surface proteoglycan expressed on angiogenic pericytes and some tumor cells. Here we have expressed the model compound tTF-NGR, tTF-TAA, and tTF-LTL with different lengths in the TF domain in E. coli and used these fusion proteins for functional studies in anticancer therapy. We aimed to retarget TF to tumor vessels leading to tumor vessel infarction with two barriers of selectivity, a) the leaky endothelial lining in tumor vessels with the target NG2 being expressed on pericytes on the abluminal side of the endothelial cell barrier and b) the preferential expression of NG2 on angiogenic vessels such as in tumors. Chromatography-purified tTF-TAA showed identical Factor X (FX)-activating procoagulatory activity as the model compound tTF-NGR with Km values of approx. 0.15 nM in Michaelis-Menten kinetics. The procoagulatory activity of tTF-LTL varied with the chosen length of the TF part of the fusion protein. Flow cytometry revealed specific binding of tTF-TAA to NG2-expressing pericytes and tumor cells with low affinity and dissociation KD in the high nM range. In vivo and ex vivo fluorescence imaging of tumor xenograft-carrying animals and of the explanted tumors showed reduction of tumor blood flow upon tTF-TAA application. Therapeutic experiments showed a reproducible antitumor activity of tTF-TAA against NG2-expressing A549-tumor xenografts, however, with a rather small therapeutic window (active/toxic dose in mg/kg body weight).

Abstract 296: Synergistic actions of oncolytic vaccinia virus and sunitinib on pancreatic neuroendocrine tumors in RIP-Tag2 mice

Abstract Pexa-Vec (JX-594) is an engineered oncolytic vaccinia virus that lacks thymidine kinase and expresses human granulocyte-macrophage colony stimulating factor. In addition to having oncolytic effects on tumor cells and promoting an immune response, Pexa-Vec has been found to cause rapid reduction of tumor blood flow in preclinical models and in clinical trials. The angiogenesis inhibitor sunitinib reduces tumor vascularity and can augment the activity of vesicular stomatitis virus by suppressing two interferon-induced anti-viral host proteins, RNA-dependent protein kinase and ribonuclease L. We explored the interaction of mpJX-594, a mouse-adapted version of Pexa-Vec, and sunitinib. The mouse-adapted version, mpJX-594, was engineered from Western Reserve strain vaccinia virus by deletion of thymidine kinase and addition of human GM-CSF, yellow fluorescent protein, and luciferase genes. The effects of mpJX-594 or sunitinib given individually on pancreatic neuroendocrine tumors in RIP-Tag2 mice were compared to those of the two agents given together. Treatment with mpJX-594 alone reduced tumor vascularity and induced widespread apoptosis in the tumors. After treatment with sunitinib alone, tumor vascularity was decreased, but apoptosis was much less than after mpJX-594 alone. When mpJX-594 was given together with sunitinib, the amount of viral infection in tumors, assessed by vaccinia immunoreactivity, was much more than after mpJX-594 alone. Vaccinia staining was detected in most of the tumor blood vessels and in large patches of tumor cells. The reduction of tumor vascularity and amount of apoptosis were greater after both agents than after either of them given alone. To determine whether mpJX-594 increased the efficacy of sunitinib in tumors or vice versa, we examined the effects of sequential administration, where mpJX-594 or sunitinib was given first and then followed 5 days later by the other. In both cases, the reduction in tumor vascularity after sequential treatment was greater than after either agent given alone. When sunitinib was given first, tumor vascularity was less throughout tumors, and the surviving vessels appeared more normal. By comparison, when mpJX-594 was given before sunitinib, the tumor vasculature was greatly reduced in some regions and less so in others, and the remaining blood vessels were abnormal. Apoptotic cells were much more numerous and widespread when mpJX-594 preceded sunitinib than after either agent given alone. In contrast, when sunitinib was given first, tumor apoptosis was greater than after sunitinib alone but not after mpJX-594 alone. Together these findings indicate that mpJX-594 amplifies the anti-vascular and anti-tumor effects of sunitinib and vice versa in RIP-Tag2 pancreatic neuroendocrine tumors, regardless of the sequence of administration, but the magnitude of the changes differs according to the sequence of administration. Citation Format: Corry E. McDonald, Barbara Sennino, Brian J. Schriver, John C. Bell, David H. Kirn, Caroline J. Breitbach, Donald M. McDonald. Synergistic actions of oncolytic vaccinia virus and sunitinib on pancreatic neuroendocrine tumors in RIP-Tag2 mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 296. doi:10.1158/1538-7445.AM2015-296

Anti-S1P Antibody as a Novel Therapeutic Strategy for VEGFR TKI-Resistant Renal Cancer.

VEGFR2 tyrosine kinase inhibition (TKI) is a valuable treatment approach for patients with metastatic renal cell carcinoma (RCC). However, resistance to treatment is inevitable. Identification of novel targets could lead to better treatment for patients with TKI-naïve or -resistant RCC. In this study, we performed transcriptome analysis of VEGFR TKI-resistant tumors in a murine model and discovered that the SPHK-S1P pathway is upregulated at the time of resistance. We tested sphingosine-1-phosphate (S1P) pathway inhibition using an anti-S1P mAb (sphingomab), in two mouse xenograft models of RCC, and assessed tumor SPHK expression and S1P plasma levels in patients with metastatic RCC. Resistant tumors expressed several hypoxia-regulated genes. The SPHK1 pathway was among the most highly upregulated pathways that accompanied resistance to VEGFR TKI therapy. SPHK1 was expressed in human RCC, and the product of SPHK1 activity, S1P, was elevated in patients with metastatic RCC, suggesting that human RCC behavior could, in part, be due to overproduction of S1P. Sphingomab neutralization of extracellular S1P slowed tumor growth in both mouse models. Mice bearing tumors that had developed resistance to sunitinib treatment also exhibited tumor growth suppression with sphingomab. Sphingomab treatment led to a reduction in tumor blood flow as measured by MRI. Our findings suggest that S1P inhibition may be a novel therapeutic strategy in patients with treatment-naïve RCC and also in the setting of resistance to VEGFR TKI therapy.

Evaluation of the "steal" phenomenon on the efficacy of hypoxia activated prodrug TH-302 in pancreatic cancer.

Pancreatic ductal adenocarcinomas are desmoplastic and hypoxic, both of which are associated with poor prognosis. Hypoxia-activated prodrugs (HAPs) are specifically activated in hypoxic environments to release cytotoxic or cytostatic effectors. TH-302 is a HAP that is currently being evaluated in a Phase III clinical trial in pancreatic cancer. Using animal models, we show that tumor hypoxia can be exacerbated using a vasodilator, hydralazine, improving TH-302 efficacy. Hydralazine reduces tumor blood flow through the “steal” phenomenon, in which atonal immature tumor vasculature fails to dilate in coordination with normal vasculature. We show that MIA PaCa-2 tumors exhibit a “steal” effect in response to hydralazine, resulting in decreased tumor blood flow and subsequent tumor pH reduction. The effect is not observed in SU.86.86 tumors with mature tumor vasculature, as measured by CD31 and smooth muscle actin (SMA) immunohistochemistry staining. Combination therapy of hydralazine and TH-302 resulted in a reduction in MIA PaCa-2 tumor volume growth after 18 days of treatment. These studies support a combination mechanism of action for TH-302 with a vasodilator that transiently increases tumor hypoxia.

Abstract 3000: Angiogenic recovery under chronic exposure to sunitinib is associated with vasculogenic mimicry in renal cell carcinoma

Abstract Background. Sunitinib inhibits VEGFR/PDGFR signaling in cancer cells and reduces tumor blood flow, leading to tumor starvation, hypoxia, and necrosis. The aim of this study was to investigate mechanisms associated with adaptive resistance to sunitinib in vitro and in vivo in renal cancer models (RCC). Materials and Methods: CAKI1 (VHL+) and 786-0 (VHL-) RCC cells were exposed in vitro to stepwise increasing doses of sunitinib for &amp;gt;6 months. In vitro, vascular mimicry was assessed by pseudo-tube formation assay using Matrigel. Pseudo-tube networks were quantified by “angiogenesis analyzer” for Image J, assessing the meshes number. CAKI1 or 786-0 cells were engrafted in nude mice and treated at 60 mg/kg/day of sunitinib until progression. Results. We confirmed that the VHL pathway was functional in CAKI1 but not in 786-0 cells. In vivo, sunitinib significantly delay tumor growth with a median time-to-tumor progression of 50 days versus 17 days (p&amp;lt;0.001) in CAKI and over 60 days versus 22 days (p&amp;lt;0.01) in 786-0 xenogratfs. In CAKI1 and 786-0, quantitative immunohistochemistry and fluorescence analyses at the time of sunitinib progression showed patterns of angiogenesis recovery with tumor blood vessel sprouting associated with reduction of hypoxic and necrotic areas. In these sunitinib-resistant tumors, cancer cells displayed stem cell and mesenchymal differentiation markers such as CD133 vimentin and CXCR4. Vessels sprouting under sunitinib exposure contained CD10/CD31/von Willebrand co-stained cells embedded in their walls suggesting vascular mimicry. We further investigated whether cancer cell differentiation into endothelial cell was related to chemical inhibition of VEGFR/PDGFR by sunitinib. We first characterized CAKI and 786 cells for VEGFR and PDGFR expression, showing that CAKI expressed high level of PDGFR, whereas 786 expressed low levels of VEGFR and PDGFR. In normally oxygenated culture conditions, 786-0 cells formed spontaneously more meshes than CAKI1 cells (22 versus 0.33, p&amp;lt;0.05). Interestingly, chronic exposure of CAKI1 cells to sunitinib led to a 32-fold increase in the number of meshes (10.6 versus 0.33, p&amp;lt;0.01), which could be related to the inhibition of the PDGFR pathway. Conclusions. Our data showed that chronic exposure to subtoxic doses of sunitinib might force cancer stem cells to differentiate into endothelial-like cells in vitro and in vivo independently of the VHL status of tumor cells. This phenomenon known as vasculogenic mimicry is associated with poor prognosis in renal cell carcinoma. VHL-mutated 786-0 cells appeared more prompt to form vessels and can spontaneously form meshes in normoxic condition in vitro. Interestingly, this plastic capacity of forming vessels in normoxic condition can be acquired in wild-type VHL CAKI1 when treated chronically with subtoxic doses of sunitinib. Citation Format: Annemilai Tijeras-Raballand, Célia Dos Santos, Maria Serova, Matthieu Martinet, Sandrine Faivre, Armand de Gramont, Eric Raymond. Angiogenic recovery under chronic exposure to sunitinib is associated with vasculogenic mimicry in renal cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3000. doi:10.1158/1538-7445.AM2014-3000

Phase I Safety, Pharmacokinetic and Pharmacodynamic Evaluation of the Vascular Disrupting Agent Ombrabulin (AVE8062) in Patients with Advanced Solid Tumors

The vascular disrupting agent ombrabulin rapidly reduces tumor blood flow and causes necrosis in vivo. A phase I dose-escalation study was designed to determine the recommended phase II dose (RP2D) of single-agent ombrabulin administered once every three weeks in patients with advanced solid malignancies. Ombrabulin (30-minute infusion) was escalated from 6 to 60 mg/m2, with RP2D cohort expansion. Safety, tumor response, pharmacokinetics, and pharmacodynamic biomarkers were evaluated. Eleven dose levels were evaluated in 105 patients. Two patients had dose-limiting toxicities in cycle 1 during escalation: grade 3 abdominal pain at 50 mg/m2, grade 3 tumor pain/grade 3 hypertension at 60 mg/m2, and the RP2D was 50 mg/m2 (39 patients). Common toxicities were headache, asthenia, abdominal pain, nausea, diarrhea, transient hypertension, anemia, and lymphopenia. No clinically significant QTc prolongations or left ventricular ejection fraction (LVEF) decreases occurred. Ombrabulin was rapidly converted to its active metabolite RPR258063 (half-life 17 minutes and 8.7 hours, respectively), both having dose-proportional exposure. Weak inhibition of CYP2C19-mediated metabolism occurred at the clinical doses used and there was no effect on CYP1A2 and CYP3A4. A patient with rectal cancer had a partial response and eight patients had stable disease lasting four months or more. Circulating endothelial cells (CEC), VEGF, and matrix metalloproteinase (MMP)-9 levels increased significantly six to 10 hours postinfusion in a subset of patients. The recommended schedule for single-agent ombrabulin is 50 mg/m2 every 3 weeks. CECs, VEGF, and MMP-9 are potential biomarkers of ombrabulin activity.

Tumor-derived hydrogen sulfide, produced by cystathionine-β-synthase, stimulates bioenergetics, cell proliferation, and angiogenesis in colon cancer

The physiological functions of hydrogen sulfide (H2S) include vasorelaxation, stimulation of cellular bioenergetics, and promotion of angiogenesis. Analysis of human colon cancer biopsies and patient-matched normal margin mucosa revealed the selective up-regulation of the H2S-producing enzyme cystathionine-β-synthase (CBS) in colon cancer, resulting in an increased rate of H2S production. Similarly, colon cancer-derived epithelial cell lines (HCT116, HT-29, LoVo) exhibited selective CBS up-regulation and increased H2S production, compared with the nonmalignant colonic mucosa cells, NCM356. CBS localized to the cytosol, as well as the mitochondrial outer membrane. ShRNA-mediated silencing of CBS or its pharmacological inhibition with aminooxyacetic acid reduced HCT116 cell proliferation, migration, and invasion; reduced endothelial cell migration in tumor/endothelial cell cocultures; and suppressed mitochondrial function (oxygen consumption, ATP turnover, and respiratory reserve capacity), as well as glycolysis. Treatment of nude mice with aminooxyacetic acid attenuated the growth of patient-derived colon cancer xenografts and reduced tumor blood flow. Similarly, CBS silencing of the tumor cells decreased xenograft growth and suppressed neovessel density, suggesting a role for endogenous H2S in tumor angiogenesis. In contrast to CBS, silencing of cystathionine-γ-lyase (the expression of which was unchanged in colon cancer) did not affect tumor growth or bioenergetics. In conclusion, H2S produced from CBS serves to (i) maintain colon cancer cellular bioenergetics, thereby supporting tumor growth and proliferation, and (ii) promote angiogenesis and vasorelaxation, consequently providing the tumor with blood and nutritients. The current findings identify CBS-derived H2S as a tumor growth factor and anticancer drug target.

Phase II randomized study of dalantercept in combination with axitinib compared to axitinib alone as second-line treatment in patients with metastatic renal cell carcinoma.

TPS4595 Background: The treatment of metastatic renal cell cancer (mRCC) with therapies targeting the vascular endothelial growth factor (VEGF) pathway delays disease progression; however, overcoming tumor resistance to these agents remains a therapeutic challenge. Activin receptor-like kinase 1(ALK1) is a type 1 receptor in the TGF-ß superfamily and is selectively expressed on activated endothelial cells. While VEGF drives the proliferative stage of angiogenesis, ALK1 is primarily involved in the maturation phase. Dalantercept is a human ALK1-Fc receptor fusion protein that binds to bone morphogenetic proteins (BMP) 9 and 10 (ligands for ALK1) and acts as a ligand trap. Preclinically, dalantercept showed delayed tumor growth in solid tumor models, including RCC models alone and in combination with sunitinib. In RCC models, the addition of dalantercept to sunitinib enhanced the reduction in tumor blood flow compared to sunitinib alone. Dalantercept showed anti-tumor activity in a completed phase 1 study in 37 pts. with advanced solid tumors. Based on this promising data, we hypothesize that ALK1 inhibition may be synergistic with axitinib, a VEGFR tyrosine kinase inhibitor (TKI), in pts. with mRCC. Methods: A two-part, multi-center, open label phase 2 study to evaluate safety, tolerability, efficacy, pharmacokinetics (PK), and pharmacodynamics (PD) of dalantercept plus axitinib as second line therapy is ongoing. In Part 1, dose escalation using a 3+3 design will assess the safety and PK of dalantercept SC every 3 weeks plus axitinib 5 mg PO BID until disease progression or unacceptable toxicity. Once the maximum tolerated dose (MTD) has been determined, up to 20 pts. may be enrolled in an expansion cohort to establish safety for the recommended dose for Part 2. Part 2 will include 112 pts. randomized 1:1 to dalantercept plus axitinib vs. axitinib alone. Key eligibility criteria are one prior TKI in the first-line setting, ECOG &lt;/= 1, and measurable disease. The primary efficacy endpoint is PFS. Secondary endpoints are OS, TTP, ORR, DOR, DCR, and PD biomarkers on archived tumor and serum specimens including BMP9/10 and ALK1 expression. Clinical trial information: NCT01727336.

Post‐therapeutic needle biopsy in patients with hepatocellular carcinoma is a useful tool to evaluate response to proton irradiation

Proton beam therapy is safe and more effective than conventional radiation therapy for the local control of nodular hepatocellular carcinoma (HCC). However, evaluating therapeutic response by imaging is not accurate during the early post-irradiation period. Therefore, we examined whether the histopathological study of biopsy specimens obtained at 3 weeks after irradiation can be used to more accurately assess therapeutic response. Fifteen HCC lesions from 13 patients were treated with proton beam irradiation. Tissue biopsy samples were obtained using abdominal ultrasound-guided percutaneous fine-needle aspiration from the center of the tumor before, 3 weeks after and 1 year post-proton therapy. The specimens were examined after staining with hematoxylin-eosin (HE) and a MIB-1 antibody. MIB-1 labeling indices (LI) before treatment were 13.0 ± 8.5% (mean ± SD; range, 0.6-27.0), whereas those 3 weeks after proton therapy were significantly reduced to 3.2 ± 2.4% (range, 0.6-8.9) (P < 0.05). Although the tumor size was reduced, we did not observe a reduction in tumor blood flow by dynamic computed tomography or degenerative changes by HE. All lesions that displayed reduced MIB-1 LI at 3 weeks post-proton treatment were ultimately diagnosed as complete response at 1 year after treatment. In contrast, one case with increased MIB-1 LI at 3 weeks had significant tumor size progression at 1 year post-treatment. The percutaneous fine-needle aspiration biopsy of HCC is a safe and useful tool that can be used to evaluate the response to proton irradiation. In particular, MIB-1 LI may provide additional information to assess the therapeutic response of HCC during the early post-irradiated period.

Abstract 2841: Widespread endothelial cell infection and tumor cell apoptosis after intravenous injection of oncolytic vaccinia virus JX-594 into RIP-Tag2 mice.

Abstract Replication-competent oncolytic viruses are being developed as a promising strategy for treating certain types of cancer. JX-594 is an oncolytic vaccinia virus that lacks thymidine kinase and expresses human granulocyte-macrophage colony stimulating factor (hGM-CSF). Replication of JX-594 is promoted by EGFR/Ras pathway signaling in cancer cells. In addition to having direct oncolytic effects on tumor cells and recruiting an immune response, JX-594 has been found to cause rapid reduction of tumor blood flow in preclinical models and in clinical trials. To understand the mechanism of this vascular effect, we injected mouse-adapted JX-594 vaccinia virus (mJX-594, on Western Reserve backbone and expressing hGM-CSF) intravenously into RIP-Tag2 transgenic mice and examined the pancreatic neuroendocrine tumors at 6 hours or 1, 2, or 5 days after injection. Dot-like vaccinia immunoreactivity was widespread in endothelial cells of tumor vessels at 6 hours. Scattered endothelial cells had intense staining. Vaccinia immunoreactivity in tumor blood vessels was still widespread at 1 day, but at 5 days, most tumor vessels were narrowed, some appeared fragmented, and tumor vascularity was reduced by 40%. Normalization of tumor vessels, as observed after inhibition of VEGF signaling, was not found at any time examined. Intratumoral hypoxia was evident as diffuse regions of pimonidazole staining at 6 hours. Pimonidazole staining increased from 1 to 5 days, and was especially strong in regions of vascular pruning. Extravascular vaccinia immunoreactivity was located in scattered cells at 1 day and increased with time. At 5 days, most tumors had large patches of vaccinia-positive cells. This staining was not present in the surrounding normal acinar pancreas. Apoptotic cells stained for activated caspase-3 were scattered throughout tumors at 6 hours. Most of these apoptotic cells were endothelial cells. Apoptotic tumor cells were sparse at 6 hours, but at 5 days apoptotic cells were 23-fold more numerous than at baseline and even more widespread than vaccinia immunoreactivity, and tumors tended to be smaller than corresponding controls. These findings are consistent with oncolytic virus mJX-594 causing rapid infection of tumor vascular endothelial cells in RIP-Tag2 mice. Endothelial cell infection is followed by regression of tumor blood vessels, exaggeration of intratumoral hypoxia, and oncolytic actions on tumor cells that lead to widespread tumor cell apoptosis. Citation Format: Barbara Sennino, Brian J. Schriver, Caroline J. Breitbach, Naomi De Silva, John C. Bell, David H. Kirn, Donald M. McDonald. Widespread endothelial cell infection and tumor cell apoptosis after intravenous injection of oncolytic vaccinia virus JX-594 into RIP-Tag2 mice. [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 2841. doi:10.1158/1538-7445.AM2013-2841

Comparison of Optical and Power Doppler Ultrasound Imaging for Non-Invasive Evaluation of Arsenic Trioxide as a Vascular Disrupting Agent in Tumors

Small animal imaging provides diverse methods for evaluating tumor growth and acute response to therapy. This study compared the utility of non-invasive optical and ultrasound imaging to monitor growth of three diverse human tumor xenografts (brain U87-luc-mCherry, mammary MCF7-luc-mCherry, and prostate PC3-luc) growing in nude mice. Bioluminescence imaging (BLI), fluorescence imaging (FLI), and Power Doppler ultrasound (PD US) were then applied to examine acute vascular disruption following administration of arsenic trioxide (ATO).During initial tumor growth, strong correlations were found between manual caliper measured tumor volume and FLI intensity, BLI intensity following luciferin injection, and traditional B-mode US. Administration of ATO to established U87 tumors caused significant vascular shutdown within 2 hrs at all doses in the range 5 to 10 mg/kg in a dose dependant manner, as revealed by depressed bioluminescent light emission. At lower doses substantial recovery was seen within 4 hrs. At 8 mg/kg there was >85% reduction in tumor vascular perfusion, which remained depressed after 6 hrs, but showed some recovery after 24 hrs. Similar response was observed in MCF7 and PC3 tumors. Dynamic BLI and PD US each showed similar duration and percent reductions in tumor blood flow, but FLI showed no significant changes during the first 24 hrs.The results provide further evidence for comparable utility of optical and ultrasound imaging for monitoring tumor growth, More specifically, they confirm the utility of BLI and ultrasound imaging as facile assays of the vascular disruption in solid tumors based on ATO as a model agent.

Abstract 295: The Role of 20-HETE in the Control of Prostate Cancer Tumor Vasculature

20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P450-derived arachidonic acid metabolite endowed with vasoconstrictor. Recent reports documented that 20-HETE synthesis inhibitors decrease the growth of glioma and renal cell carcimona, and that urinary 20-HETE levels are significantly elevated in prostate cancer patients. These reports led us to investigate the role of 20-HETE in the regulation of blood flow to prostate cancer tumors. First, we compared 20-HETE levels in prostate cancer cell line-LNCaP and normal prostate epithelial cells. Cultured LNCaP cells produced high levels of 20-HETE (566±139 pg/mg protein), whereas 20-HETE production was undetectable in normal prostate epithelial cells. Second, subcutaneous tumors were induced in balb/c nude mice by injection of LNCaP cells, followed by assessment of 20-HETE in both tumors and isolated tumor vessels. We found that LNCaP cell-derived tumors had high levels of 20-HETE (425±97 pg/mg protein), while the level in isolated tumor vessels was undetectable. Third, we used laser-doppler flowmetry to evaluate whether tumor-derived 20-HETE influences tumor blood flow and vessel diameter in vivo. Topical application of the 20-HETE antagonist 20-6,15-HEDE (1 mM) to tumor vessels in vivo increased blood flow by 1.81±0.34-fold and vessel diameter by 1.286±0.133-fold, which responses were reversed by topical application of 20-HETE in a dose-dependent manner. We conclude that LNCaP cell-derived tumors produce 20-HETE which, in turn, promotes vasoconstriction leading to reduction of tumor blood flow.