Everolimus RAD001 and anti angiogenic cyclophosphamide show long term gastric cancer growth in vivo

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Cancer Biology & Therapy

ISSN: 1538-4047 (Print) 1555-8576 (Online) Journal homepage: https://www.360docs.net/doc/0015105909.html,/loi/kcbt20

Everolimus (RAD001) and anti-angiogenic

cyclophosphamide show long-term control of gastric cancer growth in vivo

Daniel Cejka, Matthias Preusser, Adelheid Woehrer, Wolfgang Sieghart,

Sabine Strommer, Johannes Werzowa, Thorsten Fuereder & Volker Wacheck

To cite this article: Daniel Cejka, Matthias Preusser, Adelheid Woehrer, Wolfgang Sieghart,Sabine Strommer, Johannes Werzowa, Thorsten Fuereder & Volker Wacheck (2008) Everolimus (RAD001) and anti-angiogenic cyclophosphamide show long-term control of gastric cancer growth in vivo , Cancer Biology & Therapy, 7:9, 1377-1385, DOI: 10.4161/cbt.7.9.6416To link to this article:

https://www.360docs.net/doc/0015105909.html,/10.4161/cbt.7.9.6416

Published online: 01 Sep 2008.

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[Cancer Biology & Therapy 7:9, 1377-1385; September 2008]; ?2008 Landes Bioscience

Metronomic dosing of cytotoxic drugs such as cyclophosphamide has shown anti-angiogenic activity, most likely by inducing hypoxia in tumors. Hypoxia leads to activation of escape mechanisms allowing tumor cell survival. This potentially limits the activity of anti-angiogenic strategies. We hypothesized that mTORC1 inhibi-tion by everolimus (RAD001) leads to suppression of HIF-1α and VEGF resulting in synergistic anti-tumor activity in combination with anti-angiogenically dosed cyclophosphamide.

In vitro, effects of everolimus on mTORC1 signaling, prolif-eration, cell cycle, HIF-1α expression and VEGF secretion were evaluated in two gastric cancer cell lines. In vivo, anti-tumor activity of everolimus in combination with metronomic cyclophosphamide was studied in a NCI-N87 human gastric cancer SCID mouse xeno-graft model. Expression of Ki-67 and HIF-1α, activated caspase 3, microvascular density (MVD) and tumor necrotic area assessed.Everolimus decreased proliferation and attenuated production of HIF-1α as well as VEGF in gastric cancer cells in vitro. In vivo, everolimus significantly inhibited tumor growth. This anti-tumor activity was linked to a significant increase in tumor necrotic area (p < 0.02) and trends for decreased proliferation, increased apop-tosis, decreased HIF-1α and lower tumor MVD (p = n.s.). The combination of everolimus and cyclophosphamide resulted in a striking and highly significant long-term tumor growth control compared to monotherapy (p < 0.001), which was associated with a sharp increase in central tumor necrosis (p < 0.001).

In conclusion, the combination of everolimus and metro-nomic cyclophosphamide showed synergistic anti-tumor activity.

Depriving cancer cells by everolimus of factors necessary for their survival under hypoxia induced by anti-angiogenic chemo-therapy appears to be a promising approach for treatment of gastric cancer.

Introduction

The mammalian target of rapamycin (mTOR) pathway is a central sensor for nutrient and energy availability. It is modulated by various growth factor signaling pathways and negatively regulated by the tumor suppressor PTEN.1 The mTOR protein forms with the adaptor protein raptor the mTOR complex 1 (mTORC1) and stimulates the translational machinery by activating the ribosomal protein S6 kinase 1 (S6K1) and inhibiting the initiation factor 4E binding proteins (4E-BP1-3). Subsequent activation of eIF-4E results in translation of multiple malignancy-associated proteins, including cyclin D1 and VEGF .2 In gastric cancer, mTOR activity and components of the mTOR signaling network including PTEN, 4E-BP1 and eIF-4E, have been reported to be deregulated and are linked with tumor progression, metastasis or poor survival.3-7

The macrolide antibiotic rapamycin binds to the 12-kDa immu-nophilin FK506-binding protein (FKBP12) and inhibits the serine/threonine kinase activity of mTORC1. Inhibition of mTORC1 induces cell cycle arrest, apoptosis or autophagy depending on cancer cell type. In addition to its direct effect on tumor cells, rapamycin has anti-angiogenic activity by lowering VEGF secretion, impairing endothelial cell proliferation or inducing tumor vessel thrombosis.8-12

Clinically, the rapamycin derivatives everolimus (RAD001), temsirolimus (CCI-779) and AP23573 are currently being evaluated in phase I-III trials against a variety of tumor entities.13 However, monotherapy with mTORC1 inhibitors often shows only modest therapeutic activity.14

Besides molecular targeting of cancer by novel compounds, recent advances in the understanding of additional mechanism of action of well known cytotoxics have prompted attention. Of note, some

Research Paper

Everolimus (RAD001) and anti-angiogenic cyclophosphamide show long-term control of gastric cancer growth in vivo

Daniel Cejka,1 Matthias Preusser,2 Adelheid Woehrer,3 Wolfgang Sieghart,1 Sabine Strommer,1 Johannes Werzowa,1 Thorsten Fuereder 1 and Volker Wacheck 1,*

1Section of Experimental Oncology/Molecular Pharmacology; Department of Clinical Pharmacology; 2Department of Internal Medicine I; 3Institute of Neurology; Medical

University Vienna; Vienna Austria

Abbreviations: 4E-BP , 4E binding proteins; eIF-4E, eukaryotic initiation factor 4E; ELISA, enzyme-linked immunosorbent assay; FKBP12, FK506-binding protein 12; GIST, gastrointestinal stromal tumor; HIF-1α, hypoxia inducible factor 1 alpha; Ki-67, antigen identified by monoclonal antibody 67, a proliferation marker; mTOR, mammalian target of rapamycin; mTORC1, mammalian target of rapamycin complex 1; MVD, microvascular density; RECIST, response evaluation criteria in solid tumors; SCID, severe combined immunodeficient; SDS, sodium dodecyl sulfate; VEGF , vascular endothelial growth factor

Key words: mTORC1, RAD001, HIF-1α, metronomic, hypoxia

*Correspondence to: Volker Wacheck; Department of Clinical Pharmacology; Medical University Vienna; Waehringer Guertel 18-20; Vienna 1090 Austria; Tel.: 43.1.40400.2989; Fax: 43.1.40400.2998; Email: Volker.Wacheck@ meduniwien.ac.at

Submitted: 04/05/08; Revised: 05/30/08; Accepted: 06/02/08Previously published online as a Cancer Biology & Therapy E-publication: https://www.360docs.net/doc/0015105909.html,/journals/cbt/article/https://www.360docs.net/doc/0015105909.html,

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ytotoxics also act as anti-angiogenic agents if given at appropriate dosing regimens such as cyclophosphamide at metronomic (i.e., daily, low-dose) administration.15 Metronomic cyclophosphamide directly damages endothelial cells, thereby inducing hypoxia in tumors.16-18 Cancer cell hypoxia activates escape mechanisms like stabilization of transcription factor HIF-1α leading to production of pro-angiogenic molecules.19 Expression of HIF-1α has been identified as unfavor-able prognostic factor in a variety of solid tumors, including gastric cancer.20 Similar observations have been made for expression of HIF-1α target genes in gastric cancer specimens.21-24 Preclinical studies indicate that blocking HIF-1α activity leads to profound inhibition of tumor growth and angiogenesis in gastric cancer models in vivo.25,26 Interestingly, translation of HIF-1α has been reported to be sensitive to mTORC1 inhibition by rapamycin.27-30

In this study it was hypothesized that depriving cancer cells of “hypoxia escape mechanisms” such as HIF-1α expression by RAD001 would render tumors more sensitive to anti-angiogenic chemotherapy. Thus, the combination of RAD001 with anti-angio-genic cyclophosphamide chemotherapy was assessed for anti-tumor activity and angiogenesis biomarkers in a human gastric cancer SCID mouse xenograft model.

Results

Gastric cancer cells are sensitive to mTORC1 inhibition in vitro. We first assessed the anti-proliferative activity of RAD001 in vitro. RAD001 displayed a dose dependent effect on NCI-N87 and MKN-45 gastric cancer cells. At a concentration of approximately 20 nM RAD001 cell numbers were decreased by 34% and 30% for NCI-N87 and MKN45 cells, respectively (p < 0.01 for both; Fig. 1A and B). Cell cycle analysis indicated that 20 nM RAD001 induced an accumulation in G 1 phase in both gastric cancer cell lines. Compared to control groups, percentages of RAD001-treated cells in G 1 phase were 54% vs. 67% for NCI-N87 and 45% vs. 52% for MKN45 (p < 0.01 for both). RAD001 did not induce cell death of gastric cancer cells (not shown).

RAD001 inhibits phosphorylation of downstream targets of mTORC1 in gastric cancer cells. In order to investigate the effect of RAD001 on downstream targets of mTORC1, the phosphorylation status of S6K1 (Thr421/Ser424) and 4E-BP1 (Ser65) in NCI-N87 and MKN45 cells was evaluated (Fig. 2A). Incubation of gastric cancer cells with 20 nM RAD001 resulted in markedly decreased phosphorylation of both S6K1 isoforms (p70, p85) and 4E-BP1.mTORC1 inhibition decreases HIF-1α and VEGF-A levels in NCI-N87 and MKN45 cells. Incubation of cells with 20 nM of RAD001 resulted in suppression of HIF-1α protein expression (Fig. 2B). Additionally, secretion of VEGF-A was reduced significantly compared to controls (p < 0.02 for NCI-N87 and p < 0.01 for MKN45; Fig. 2C). Both findings support the working hypothesis that inhibition of mTORC1 by RAD001 deprives gastric cancer cells of proteins involved in escape from tumor hypoxia.

Synergistic tumor growth control exerted by RAD001 in combi-nation with metronomic cyclophosphamide. T reatment with the mTORC1 inhibitor RAD001 resulted in a profound single agent activity (Fig. 3A). RAD001 significantly delayed xenograft growth resulting in more than 75% smaller xenografts relative to control group (p < 0.001). T umor growth was substantially impaired by RAD001 with a more than three-fold prolongation of time-to-endpoint

(TTE = 121 days). Metronomic cyclophosphamide showed only

minor anti-tumor activity on gastric cancer xenografts (T/C 73%; p = 0.07). Time to endpoint (TTE) was 32 days for the control group and 36 days for the metronomic cyclophosphamide group, respectively.The combination of RAD001 with metronomic cyclophosph-amide showed outstanding long-term tumor growth control. After a month of treatment, tumor volume in the combination group was similar to RAD001 monotherapy (T/C 23% for RAD monotherapy and T/C 20% for combination treatment). However, sustained long-term tumor growth control was achieved only by the combination of RAD001 and metronomic cyclophosphamide. Even after 121 days of treatment, TTE was not reached (tumor volume 452 mm 3 +/- 198). At this time point, tumors in the combination arm were more than 50% smaller than the tumors in the RAD-monotherapy group (RAD vs RAD + mCy, p < 0.001).

To assess the impact on mTORC1 signaling, phosphorylated S6K1 was evaluated as biological marker for RAD001 activity (Fig. 3B and C). Corresponding to the effects observed in vitro, treatment with RAD001 resulted in markedly decreased phosphorylation status of S6K1 in vivo.

As a result of anti-angiogenic therapy tumor xenografts may display necrotic central areas surrounded by viable tumor tissue.34 Possibly, anti-angiogenic agents may increase the proportion of tumor necrotic area. Thus, we compared the necrotic index of tumor xenografts as a biomarker for anti-angiogenic activity among treatment groups (Fig. 4A). Remarkably, the necrotic index in the RAD001 monotherapy group increased more than six-fold compared to control (RAD vs. Ctrl, p < 0.001), leaving a narrow rim of viable cancer cells in the tumor periphery. The metronomic cyclophosphamide group showed an increase by 50% of necrotic index compared to control tumors (p < 0.02). Strikingly, tumors in the combination therapy arm showed a more than eleven-fold increase in necrotic index compared to control (RAD + mCy vs. Ctrl, p < 0.001).

To gain more insight into the biology underlying the anti-tumor activity of treatments, excised tumor xenografts were investigated in more detail. By immunohistochemistry (IHC) only minor differences between groups were detected regarding tumor cell proliferation, tumor cell apoptosis, HIF-1α expression and tumor vascularization (Fig. 4B–E), none of which reached statistical significance (p = n.s. between groups for all parameters). Figure 5 shows representative tissue sections of tumor xenografts.

Discussion

Inhibitors of mTORC1 show great promise to improve thera-peutic regimens of solid tumors, but the ideal setting for the use of mTORC1 inhibitors has yet to be defined.13 Another promising target for anti-cancer therapy is the tumor vasculature. Appropriate dosing of the cytotoxic drug cyclophosphamide was shown to have anti-angiogenic activity by directly damaging endothelial cells and induction of tumor hypoxia.16,18

It was the hypothesis of this study that deprivation of “hypoxia escape mechanisms” (like HIF-1α induction) in gastric cancer cells by mTORC1 inhibition may synergize with anti-angiogenic cyclo-phosphamide.

Importantly, our studies showed that only the combination of RAD001 with metronomic cyclophosphamide led to potent, long-lasting tumor growth control in vivo. In line with previous reports,

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RAD001 single agent treatment showed strong anti-tumor effects in the gastric cancer xenograft model.3 However, tumor growth rate in the RAD001 monotherapy arm accelerated over time (approx. from day 50) until terminal disease was reached. In contrast, tumor growth rate in the combination group was constant, suggesting a loss of anti-tumor activity of RAD001 over time as well as an additive mode of action for metronomic cyclophosphamide in combination with RAD001.

How can these findings be explained? RAD001 inhibited mTORC1 in vivo as it decreased phosphorylation of S6K, a commonly used marker of mTORC1 activity.1 As a pharmacodynamic response to this inhibition, we observed an increase in central necrosis after

long-term treatment with RAD001. Interestingly, in pilot studies short-term treatment with RAD001 resulted in a decrease of central necrosis (data not shown). It might be reasonable to speculate, that short-term administration of RAD001 decreases tumor metabolism by limiting tumor oxygen—and nutrient supply, effecting low cell turn-over and resulting in reduced tumor necrosis. However, if RAD001 treatment is prolonged this oxygen and nutrient shortage might finally induce cancer cell necrosis in vulnerable regions of the xenograft such as the tumor center which is surrounded by a small rim of viable tumor cells (as shown in Fig. 5E).

Most notably, tumors in the RAD001 + metronomic cyclophosph-amide treatment arm showed a more pronounced increase in tumor

Figure 1. mTORC1 inhibition attenuates gastric cancer cell growth by inducing accumulation in G 1 phase without inducing cell death in vitro. Proliferation of NCI-N87 (A) and MKN45 (B) cells exposed to increasing concentrations of RAD001 (2.5–160 nM) or solvent control for 72 h. n = 4 for both cell lines.

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necrosis relative to RAD001, suggesting potentiation of anti-angiogenic activity by combination therapy. This finding supports our hypothesis that the combi-nation of RAD001 with anti-angiogenic cyclophosphamide results in superior anti-tumor activity by inhibiting angiogenesis in regions prone to hypoxia. The inhibition may be the effect of both, direct suppres-sion of endothelial cell function as well as suppression of the hypoxia-induced, pro-angiogenic escape mechanisms.

Following the notion of deprived “hypoxia escape mechanisms” by mTORC1 inhibition we noticed that RAD001 not only suppressed HIF-1α protein expression in vitro but also lowered median HIF-1α expression levels by 48% in RAD001 treated tumors in vivo. In contrast, metronomic cyclo-phosphamide induced median HIF-1α expression by 58% in vivo, whereas levels of HIF-1α in the RAD001 + metronomic cyclophosphamide group were similar to control tumors (-8% compared to control). These findings

support our hypothesis that in vivo induction of HIF-1α by metronomic cyclophosphamide might be blunted by RAD001 and might therefore provide a biological rationale for the observed superior anti-tumor activity.

However, it needs to be emphasized that HIF-1α expression was found to be heterogeneously distributed in vivo,

and tumors in all therapy arms were focally positive for HIF-1α. Intra-group variability of HIF-1α expression was high, a finding which also has been reported for HIF-1α immunostaining of surgical specimens in clinical studies, and differences in HIF-1α expression between treatment groups did not reach statistical significance.35

Regarding tumor vascularisation RAD001 + metronomic cyclo-phosphamide showed only minor, statistically not significant decrease of MVD of gastric cancer xenografts. Moreover, no significant differ-ences in tumor cell proliferation could be detected. This might be attributable, at least in part, to the longitudinal design of this study. Cross-sectional evaluation of immunohistochemical endpoints might lead to statistically significant results. Furthermore, in the

clinical setting immunohistochemical studies of MVD or tumor cell proliferation are hampered by considerable heterogeneity of tumor architecture and the invasive nature of tumor biopsies. However, these finding suggest that evaluation of cancer cell proliferation or tumor MVD might be of limited usefulness to monitor mTORC1-inhibitor therapy.

In line, tumor volume measurements of gastric cancer xeno-grafts treated with RAD001, metronomic chemotherapy or both continued to increase in size despite a sharp increase in tumor necrosis. This finding is of clinical importance as monitoring of therapeutic activity of mTORC1-inhibitors or metronomic chemotherapy by commonly used RECIST criteria might not be adequate.36 T umors may increase in diameter while showing tumor response as central tumor necrosis. Alternative assessment methods,

such as detecting radiographic changes in tumor density which have been recently introduced by Choi and colleagues for GIST might be preferable for monitoring mTORC1 inhibitor activity.37 Furthermore, since treatment with anti-angiogenic treatment combinations allows survival and growth of a viable tumor rim, additional therapeutic modalities targeting the tumor rim should be considered in further studies.

In conclusion, our data suggest that gastric cancer is sensitive to mTORC1 inhibition by RAD001, resulting in impaired tumor growth and induction of central tumor necrosis. The combination of RAD001 with metronomic cyclophosphamide shows durable tumor growth control in the gastric cancer xenograft model studied. Given the anti-tumor activity and the acceptable toxicity profile of both

agents, the combination of mTORC1 inhibitors with metronomic cyclophosphamide may constitute a cost-effective long-term treat-ment option for gastric cancer patients in the future, particularly after tumor debulking therapy.38,39

Figure 2. Effects of RAD001 on mTORC1-pathway signaling, HIF-1α expression and VEGF-A secretion in vitro. (A) mTORC1-pathway signaling. NCI-N87 and MKN45 gastric cancer cell lines were exposed to 20

nM RAD001 for 24 h followed by Western blotting. mTORC1 inhibition by RAD001 markedly decreased

phosphorylation status of S6K1 and 4E-BP1 at indicated residues. Of note, phosphorylation status of AKT (Ser473) was not altered by RAD001 treatment. (B) RAD001 reduces HIF-1α protein expression. Gastric cancer cell lines were pre-treated with 20 nM RAD001 for 1 h followed by incubation with 150 μM CoCl 2

for additional 24 h to allow HIF-1α protein accumulation. Protein extracts were analyzed by Western blot-ting. (C) VEGF-A levels are decreased by RAD001. VEGF-A levels were detected by ELISA in cell culture

supernatant. Bars indicate percentage of VEGF-A levels compared to controls (n = 4).1380

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Materials and Methods

Cell lines. NCI-N87 human gastric cancer cells (intestinal type) were purchased from ATCC. MKN45 human gastric cancer cells (diffuse type) were kindly provided by Prof. H. Yokozaki, 1st Department of Pathology, Hiroshima University School of Medicine. Both cell lines were cultured in RPMI 1640 medium supplemented with 10% FCS, L-glutamine and an antibiotic mixture containing penicillin, streptomycin and amphotericin B (all Gibco Invitrogen) in a fully humidified 5% CO 2, 95% ambient air atmosphere at 37°https://www.360docs.net/doc/0015105909.html,pounds. RAD001 (everolimus) was kindly provided by The Novartis Institutes for BioMedical Research Basel, Oncology,

Switzerland. For in vitro studies, a 1 mM RAD001 stock solution was prepared in DMSO and stored at -20°C. Aliquots were diluted in cell culture medium and used immediately. Controls were treated with appropriate concentrations of DMSO. CoCl 2 (Sigma Aldrich, Austria) was prepared as 1 M stock in H 2O. For in vivo experiments, formulated RAD001 was supplied as oral microemulsion. Aliquots were stored at -20°C and diluted with 5% glucose solution before administration. Microemulsion without RAD001 was given as carrier control. Cyclophosphamide (Ebewe, Austria) was diluted in normal saline (0.9% w/v).

Proliferation assay. Gastric cancer cells (10,000 cells re-suspended in 1 ml medium) were seeded in 24-well plates. 24 h after seeding,

Figure 3. Long-term treatment of RAD001 in combination with metronomic cyclophosphamide exerts potent tumor growth control in vivo. (A) Tumor xeno-graft volume mean +/- SEM. NCI-N87 xenografts were treated with placebo (Ctrl; black diamonds), metronomic cyclophosphamide (mCy; open triangles), RAD001 (RAD; open squares), or combination of both (RAD + mCy; black squares). The combination of RAD001 with metronomic cyclophosphamide shows significantly improved anti-tumor activity compared to RAD001 monotherapy (RAD001 vs. RAD + mCy; p < 0.05 on day 55 to p < 0.001 on day 121). (B) Representative Western blot from xenograft tumors showing markedly reduced phosphorylation of S6K1 (Thr421/Ser424) by RAD001. Animals were treated with control, RAD001, metronomic cyclophosphamide (mCy) or combination of both as indicated. (C) Densitometric measurements of signals from phosphorylation-specific Western blots of p85 (left) and p70 (right) S6K-isoforms from tumor xenografts (median values). Placebo-treated tumors (Ctrl) served as reference (i.e., 100%).

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Figure 4. Histological analysis of gastric cancer xenografts. All parameters are presented relative to control (Ctrl) tumors (arbitrary units; Ctrl = 1). (A) Tumor necrotic index. RAD001 as well as metronomic cyclophosphamide induce significant increases in tumor necrosis (RAD001 vs. Ctrl, p < 0.02 and mCy vs. Ctrl, p < 0.001). The combination of both treatment induces an eleven-fold increase in tumor necrosis compared to control (RAD + mCy vs. Ctrl, p < 0.001). (B) Tumor cell proliferation (Ki-67). Decreases in tumor cell proliferation induced by mCy and mCy + RAD were not statistically significant. (C) Microvascular density (MVD; Factor VIII). Combination of RAD001 with metronomic cyclophosphamide showed a trend for reduction of MVD compared to placebo control (p = n.s.). (D) Expression of HIF-1α. RAD001-containing treatment groups showed a trend for decreased HIF-1α protein levels (p = n.s.). (E) Apoptotic cell death (activated caspase 3). No statistically significant increases in apoptotic cell death were found in treatment groups compared to control tumors.

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0.5 ml medium containing RAD001 was added to yield desired concentrations of RAD001. At the time points indicated, gastric cancer cells were harvested and counted with a Coulter Z1 electronic cell counter (Beckman Coulter, UK).

Western blot. For in vitro studies, 200,000 cells were seeded in 6-well plates in 4 ml of culture medium. The day after seeding, 1 ml of RAD001 solution was added to yield a final concentration of 20 nM. Cells were incubated for additional 24 h and protein was subse-quently extracted. For HIF-1α detection, cells were pre-treated with 20 nM RAD001 for 1 h followed by addition of CoCl 2 solution to a final concentration of 150 μM and incubated for additional 24 h until protein extraction. Snap frozen tumor xenografts were pulverized with a MM 200 mixer mill (Retsch, Germany). Protein was extracted as previously described.31 A total of 10 μg protein per lane were loaded on a 10% SDS-polyacrylamide-gel and electrophoretically separated followed by blotting on nitrocellulose membranes (Schleicher & Schuell, Germany). After blocking for 1 h membranes were incubated with primary antibodies diluted in blocking solution at 4°C over night. Antibodies were directed against PTEN (dilution 1:2,000), phos-pho-AKT Ser473 (dilution 1:2,000), phospho-S6 Kinase Thr421/

Ser424 (dilution 1:5,000), phospho-4E-BP1 Ser65 (dilution 1:2,000), all polyclonal rabbit (Cell Signaling T echnology, USA); HIF-1α (mono-clonal mouse, clone 54; dilution 1:2,000, BD Biosciences, USA) and Actin (purified from rabbit serum, dilution 1:10,000; Sigma, USA). Primary antibodies were detected by alkaline phosphatase-conjugated secondary anti-rabbit or anti-mouse antibodies (T ropix; 1:5,000) and visualized by chemiluminescence using CSPD (T ropix) substrate. T o obtain numeric data on changes in phosphorylation status of proteins, band intensity was semiquantitatively measured on digitalized films using T otalLab Software (Phoretix Inc.).

ELISA. For detection of human VEGF-A, cell culture supernatant from wells designated for Western blot experiments was harvested and detected with a Quantikine human VEGF Immunoassay kit (R&D Systems, USA) according to manufacturer’s recommenda-tions. Absorption was measured with a Victor II multilabel plate reader (Wallac, Finland). After subtraction of blanks, secreted VEGF-A levels were calculated as percentage of treatment groups relative to controls.

Flow cytometry. For in vitro experiments, cells were treated as described above. Cells were washed with DPBS (Cambrex) once.

Figure 5. Representative tissue sections of NCI-N87 xenografts. Representative IHC staining of Ki-67 expression (A), activated caspase 3 (B), HIF-1α (C) and Factor VIII (D) are shown. (E) shows tissue sections of complete tumor xenografts showing varying extents of tumor necrosis (left: control; right: long-term RAD001).

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Cells were re-suspended in DPBS, fixed with ice-cold ethanol-DPBS (70:30) solution and stored at 4°C. Following centrifugation, cells were incubated with RNase A (Sigma-Aldrich; 125 μg/ml) and propidium iodide (Sigma-Aldrich; 10 μg/ml) at 37°C for 30 minutes and analyzed immediately by flow cytometry. For cell cycle distribu-tion analysis, debris and aggregates were excluded by appropriate gating. Cancer cell death was quantified by placing subG 1-markers in ungated FL2A-histograms. Cell culture supernatant was included for analysis.

T umor xenograft model. Pathogen-free, 4–6 week old, female CB-17 SCID mice (Charles River Laboratories, Germany) were housed under sterile conditions. The study was approved by the local animal welfare committee and was performed in accordance with the local regulations.

The combination of RAD001 (5 mg/kg solved in microemul-sion qd p.o.) and anti-angiogenic therapy by metronomic dosing of cyclophosphamide (20 mg/kg qd p.o.) was studied.32 Mice (n = 6/group) were inoculated with NCI-N87 cells bilaterally. Animals were assigned randomly to one of the following treatment groups:(1) carrier control (Ctrl): saline + microemulsion p.o.;

(2) metronomic cyclophosphamide (mCy): cyclophosphamide + microemulsion p.o.;

(3) RAD001 (RAD): RAD001 + saline p.o.;

(4) RAD001 + metronomic cyclophosphamide (RAD + mCy).T umor volume was assessed biweekly by caliper measurements and calculated according to the approximation formula: volume (mm 3) = 4/3 π x (long diameter 2 x short diameter)/2. T reatment was initiated when tumor volumes reached approximately 150 mm 3 and continued until tumor volume reached 1,000 mm 3 as the predefined surrogate endpoint for terminal disease. The time to endpoint (TTE) was defined as days from initiation of treatment until tumor volume exceeding 1,000 mm 3. The treatment-to-control ratio (T/C%) is the tumor volume of the respective treatment group given as percentage of control group tumor volume at the time of control group sacrifice.Immunohistochemistry (IHC). Specimens were fixed in 4.5% phosphate-buffered formaldehyde solution, embedded in paraffin and cut at a thickness of 3–5 μm. Sections were deparaffinized and heated in 0.01 M citrate buffer (pH 6.0) for 30 minutes (for anti-Ki-67 immunostaining), 20 minutes (for anti-caspase-3 immu-nostaining), or 10 minutes (for anti-Factor-VIII immunostaining) in a microwave oven at 600 Watts.

We used the following antibodies for immunostainings: anti-Ki-67 (monoclonal mouse, clone MIB-1, Dako, Denmark; dilution 1:50), anti- HIF-1α (monoclonal mouse, clone 54, T ransduction laboratories, UK; dilution 1:30), anti-Factor-VIII (polyclonal rabbit, Dako, dilution 1:1,000), anti-caspase-3 (monoclonal rabbit, clone 5A1, Cell signaling, dilution 1:100). Detection of immunostaining was performed using the Envision kit (Dako) and diaminobenzidine was used as chromogen. For all IHC staining, control of antibody specificity included omission or substitution of the primary anti-bodies by non-specific, isotype-matched antibodies or substitution of the primary antiserum by normal rabbit serum. Photographs of negative controls are shown in Supplemental Figure S1.

For quantitative assessment of Ki-67 and caspase-3 immunola-beling, a total of 500 tumor cells were evaluated in each specimen in fields showing the highest density of immunopositive cells. For assessment of MVD, anti-Factor-VIII immunostained tissue sections

with the highest density of distinctly highlighted microvessels (“hot-spot”) were manually counted at 200-fold magnification within an examination area of 0.25 mm 2.33 For evaluation of anti-HIF-1α immunostaining slides were semiquantitatively analyzed using an Axioplan 2 imaging microscope with Zeiss plan-Neofluar 20x/0.5 numeric aperture objective and Vision Axiovision 3.1 software (Carl Zeiss Vision, Mannheim, Germany).

Results of IHC studies were interpreted by a trained pathologist blinded regarding treatment groups.

For assessment of necrotic area, necrotic area was identified by morphology and measured using Photoshop CS2 software (Adobe Inc., USA) and calculated as percentage of total area of complete cross sections of tumors. To correct for different tumor size, a necrotic index (NI) was calculated as tumor necrotic area/tumor volume.Statistical analysis. For comparisons between two groups, data sets were analyzed by the Mann-Whitney-U test. For multiple compari-sons, the Kruskal-Wallis test was followed by pairwise comparisons using the Mann-Whitney-U test and Bonferroni-Holm correction for multiple testing. p values <0.05 were considered statistically significant. Data is given as percentage of control values unless indi-cated otherwise. Boxplots show median values, interquartile ranges and 95% confidence intervals, respectively. Outliers are indicated as circles or asterisks. Statistical analysis was performed using SPSS 10.0 software (SPSS Inc., USA).

Acknowledgements

D.C. received research funding from the Hochschuljubil?umsstif tung der Stadt Wien.

We thank Ondina Cejka for technical support regarding compila-tion and layout of figures.

Note

Supplementary materials can be found at:

https://www.360docs.net/doc/0015105909.html,/supplement/CejkaCBT7-9-Sup.pdf

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