Bevacizumab-induced transient remodeling of the vasculature in neuroblastoma xenografts results in improved delivery and efficacy of systemically administered chemotherapy.

PURPOSE: Dysfunctional tumor vessels can be a significant barrier to effective cancer therapy. However, increasing evidence suggests that vascular endothelial growth factor (VEGF) inhibition can effect transient "normalization" of the tumor vasculature, thereby improving tumor perfusion and, consequently, delivery of systemic chemotherapy. We sought to examine temporal changes in tumor vascular function in response to the anti-VEGF antibody, bevacizumab. EXPERIMENTAL DESIGN: Established orthotopic neuroblastoma xenografts treated with bevacizumab were evaluated at serial time points for treatment-associated changes in intratumoral vascular physiology, penetration of systemically administered chemotherapy, and efficacy of combination therapy. RESULTS: After a single bevacizumab dose, a progressive decrease in tumor microvessel density to <30% of control was observed within 7 days. Assessment of the tumor microenvironment revealed a rapid, sustained decrease in both tumor vessel permeability and tumor interstitial fluid pressure, whereas intratumoral perfusion, as assessed by contrast-enhanced ultrasonography, was improved, although this latter change abated by 1 week. Intratumoral drug delivery mirrored these changes; penetration of chemotherapy was improved by as much as 81% when given 1 to 3 days after bevacizumab, compared with when both drugs were given concomitantly, or 7 days apart. Finally, administering topotecan to tumor-bearing mice 3 days after bevacizumab resulted in greater tumor growth inhibition (36% of control size) than with monotherapy (88% bevacizumab, 54% topotecan) or concomitant administration of the two drugs (44%). CONCLUSIONS: Bevacizumab-mediated VEGF blockade effects alterations in tumor vessel physiology that allow improved delivery and efficacy of chemotherapy, although careful consideration of drug scheduling is required to optimize antitumor activity.

Topotecan central nervous system penetration is altered by a tyrosine kinase inhibitor.

A potential strategy to increase the efficacy of topotecan to treat central nervous system (CNS) malignancies is modulation of the activity of ATP-binding cassette (ABC) transporters at the blood-brain and blood-cerebrospinal fluid barriers to enhance topotecan CNS penetration. This study focused on topotecan penetration into the brain extracellular fluid (ECF) and ventricular cerebrospinal fluid (CSF) in a mouse model and the effect of modulation of ABC transporters at the blood-brain and blood-cerebrospinal fluid barriers by a tyrosine kinase inhibitor (gefitinib). After 4 and 8 mg/kg topotecan i.v., the brain ECF to plasma AUC ratio of unbound topotecan lactone was 0.21 +/- 0.04 and 0.61 +/- 0.16, respectively; the ventricular CSF to plasma AUC ratio was 1.18 +/- 0.10 and 1.30 +/- 0.13, respectively. To study the effect of gefitinib on topotecan CNS penetration, 200 mg/kg gefitinib was administered orally 1 hour before 4 mg/kg topotecan i.v. The brain ECF to plasma AUC ratio of unbound topotecan lactone increased by 1.6-fold to 0.35 +/- 0.04, which was significantly different from the ratio without gefitinib (P < 0.05). The ventricular CSF to plasma AUC ratio significantly decreased to 0.98 +/- 0.05 (P < 0.05). Breast cancer resistance protein 1 (Bcrp1), an efficient topotecan transporter, was detected at the apical aspect of the choroid plexus in FVB mice. In conclusion, topotecan brain ECF penetration was lower compared with ventricular CSF penetration. Gefitinib increased topotecan brain ECF penetration but decreased the ventricular CSF penetration. These results are consistent with the possibility that expression of Bcrp1 and P-glycoprotein at the apical side of the choroid plexus facilitates an influx transport mechanism across the blood-cerebrospinal fluid barrier, resulting in high topotecan CSF penetration.

Immunohistochemical detection of multidrug-resistant protein expression in retinoblastoma treated by primary enucleation.

PURPOSE: To compare the expression of multidrug-resistant proteins in retinoblastoma tumors among eyes treated with primary enucleation. METHODS: A group of 18 patients with unilateral retinoblastoma with advanced intraocular disease was selected for the study. All patients had undergone primary enucleation. A histologic specimen from each patient was retrieved from the pathology archives and a tissue gene microarray was constructed (0.6 x 3-4 mm). Standard immunohistochemical techniques were used to study the tissue microarrays for the expression of the ATP-binding cassette (ABC) transporters: breast cancer resistance protein (BCRP; ABCG2), multidrug-resistant protein 1/P-glycoprotein (MDR1/Pgp; ABCB1), multidrug-resistant-associated protein 1 (MRP1; ABCC1), MRP2 (ABCC2), and MRP4 (ABCC4). RESULTS: Of the 18 specimens retrieved, 16 had adequate tissue for study. MRP1 was expressed in 8 (50%) of 16 tumors, and MRP2 was expressed in 5 (31%) of 16 tumors. MDR1/Pgp was found in 2 (12%) of 16 retinoblastomas. MRP4 and BCRP were not detected in any of the tumors studied. CONCLUSIONS: The results show that multiple ABC transporters were present in a cohort of sporadic patients with unilateral retinoblastoma who underwent primary enucleation. Studies are planned of the expression of ABC transporters in eyes treated by chemotherapy and/or radiation as a comparison with this group.

Tetraspanin CD9 promotes the invasive phenotype of human fibrosarcoma cells via upregulation of matrix metalloproteinase-9.

Tumor cell metastasis, a process which increases the morbidity and mortality of cancer patients, is highly dependent upon matrix metalloproteinase (MMP) production. Small molecule inhibitors of MMPs have proven unsuccessful at reducing tumor cell invasion in vivo. Therefore, finding an alternative approach to regulate MMP is an important endeavor. Tetraspanins, a family of cell surface organizers, play a major role in cell signaling events and have been implicated in regulating metastasis in numerous cancer cell lines. We stably expressed tetraspanin CD9 in an invasive and metastatic human fibrosarcoma cell line (CD9-HT1080) to investigate its role in regulating tumor cell invasiveness. CD9-HT1080 cells displayed a highly invasive phenotype as demonstrated by matrigel invasion assays. Statistically significant increases in MMP-9 production and activity were attributed to CD9 expression and were not due to any changes in other key tetraspanin complex members or MMP regulators. Increased invasion of CD9-HT1080 cells was reversed upon silencing of MMP-9 using a MMP-9 specific siRNA. Furthermore, we determined that the second extracellular loop of CD9 was responsible for the upregulation of MMP-9 production and subsequent cell invasion. We demonstrated for the first time that tetraspanin CD9 controls HT1080 cell invasion via upregulation of an integral member of the MMP family, MMP-9. Collectively, our studies provide mounting evidence that altered expression of CD9 may be a novel approach to regulate tumor cell progression.

Simultaneous determination of cyclophosphamide and carboxyethylphosphoramide mustard in human plasma using online extraction and electrospray tandem mass spectrometry (HTLC-ESI-MS/MS).

A rapid and selective method for simultaneous determination of cyclophosphamide and its metabolite carboxyethylphosphoramide mustard (CEPM) was developed using online sample preparation and separation with tandem mass spectrometric detection. Diluted plasma was injected onto an extraction column (Cyclone MAX 0.5 mm x 50 mm, >30 microm), the sample matrix was washed with an aqueous solution, and retained analytes were transferred to an analytical column (Gemini 3 microm C18 110A, 100 mm x 2.0 mm) using a gradient mobile phase prior to detection by MS/MS. Analytes were detected in an API-3000 LC-MS/MS system using positive multiple-reaction monitoring mode (m/z 261/140 and 293/221 for CTX and CEPM, respectively). Online extraction recoveries were 76% and 72% for cyclophosphamide and CEPM. Within-day and between-day variabilities were <3.0%, and accuracies were between -6.9% and 5.2%. This method has been used to measure plasma cyclophosphamide and CEPM concentrations in an ongoing Phase II study in children with newly diagnosed medulloblastoma.

Interferon beta-mediated vessel stabilization improves delivery and efficacy of systemically administered topotecan in a murine neuroblastoma model.

BACKGROUND: We have recently demonstrated that continuous delivery of interferon beta (IFN-beta) stabilizes solid tumor vasculature and improves tumor perfusion. In this study, we have further investigated the functional consequences of this effect by assessing delivery and efficacy of conventional chemotherapy against neuroblastoma xenografts when used in combination with IFN-beta. METHODS: Mice with established retroperitoneal tumors received adeno-associated virus vector encoding IFN-beta (AAV IFN-beta) or control vector. One week later, at 1 hour before sacrifice, a 1 mg/kg i.v. bolus of topotecan (TPT) was given. Intratumoral levels of TPT were measured by high-performance liquid chromatography and then standardized to plasma levels to determine tumor TPT penetration. Subsequent experiments evaluated the antitumor efficacy of topotecan alone or in combination with AAV IFN-beta. RESULTS: As observed in prior experiments, AAV IFN-beta resulted in a marked increase in tumor vessel association with stabilizing perivascular smooth muscle cells. These more "matured" vessels facilitated improved tumor TPT penetration (51.2% +/- 4.2%) compared with controls (30.8% +/- 4.7%, P = .004). In additional cohorts of mice, this resulted in an improved antitumor effect. Mice with established tumors (301.8 +/- 18.1 mm3) were treated with TPT (1 mg/kg daily for 5 days for 2 consecutive weeks) either alone or in combination with AAV IFN-beta (5 x 10(10) vector particles per mouse). Topotecan monotherapy resulted in a reduction in mean tumor volume of 12% (264.2 +/- 65.8 mm3, P = .66). However, when the same regimen was administered to mice receiving continuous IFN-beta therapy, a 61% (118.9 +/- 42.3 mm3, P = .004) reduction in mean tumor volume was achieved. CONCLUSION: Interferon beta-mediated vessel stabilization resulted in improved intratumoral delivery of systemically administered TPT, enhancing its antitumor efficacy. This approach of altering the tumor vasculature provides a strategy to help overcome solid tumor resistance to traditional cytotoxic agents.