Chromogranin A Restricts Drug Penetration and...

Therapeutics, Targets, and Chemical Biology

Chromogranin A Restricts Drug Penetration and Limits theAbility of NGR-TNF to Enhance Chemotherapeutic Efficacy

Eleonora Dondossola, Anna Maria Gasparri, Barbara Colombo, Angelina Sacchi,Flavio Curnis, and Angelo Corti

AbstractNGR-TNF is a derivative of TNF-a that targets tumor blood vessels and enhances penetration of chemother-

apeutic drugs. Because of this property, NGR-TNF is being tested in combination with chemotherapy in variousphase II and III clinical trials. Here we report that chromogranin A (CgA), a protein present in variable amountsin the blood of normal subjects and cancer patients, inhibits the synergism of NGR-TNF with doxorubicin andmelphalan inmousemodels of lymphoma andmelanoma. Pathophysiologically relevant levels of circulating CgAblocked NGR-TNF–induced drug penetration by enhancing endothelial barrier function and reducing drugextravasation in tumors. Mechanistic investigations done in endothelial cell monolayers in vitro showed thatCgA inhibited phosphorylation of p38MAP kinase, disassembly of VE-cadherin–dependent adherence junctions,paracellular macromolecule transport, and NGR-TNF–induced drug permeability. In this system, the N-terminalfragment of CgA known as vasostatin-1 also inhibited drug penetration and NGR-TNF synergism. Together, ourresults suggest that increased levels of circulating CgA and its fragments, as it may occur in certain cancerpatients with nonneuroendocrine tumors, may reduce drug delivery to tumor cells particularly as inducedby NGR-TNF. Measuring CgA and its fragments may assist the selection of patients that can respond better toNGR-TNF/chemotherapy combinations in clinical trials. Cancer Res; 71(17); 5881–90. �2011 AACR.

Introduction

The efficacy of chemotherapy in cancer patients is oftenlimited by inadequate and uneven diffusion of drugs in tumortissues (1). This phenomenon is related to abnormal vascu-lature and altered composition of tumor tissues, which maycause irregular blood flow, heterogeneous permeability,increased interstitial fluid pressure and, consequently, unevendrug penetration in tumors (2, 3).We have previously shown that a neovasculature-homing

TNF-aderivative, calledNGR-TNF, canbe exploited to improvedrug penetration in neoplastic tissues (3, 4). NGR-TNF is acytokine–peptide fusion protein, originally developed by ourgroup, consisting ofTNF fused to theCNGRCGpeptide (3). Thispeptide is a ligand of a CD13 form overexpressed in the tumorneovasculature (5–7). Experiments in mouse models haveshown that the administration of ultra-low doses of NGR-TNF (picograms) increases the penetration of doxorubicin,melphalan, cisplatin, gemcitabine, and paclitaxel into solidtumors (4, 8). NGR-TNF can also induce, at later time points,

vascular damage and inflammatory/immune responses (4, 8).Phase I and II clinical studies with low-dose NGR-TNF, aloneand in combination with chemotherapy, showed manage-able toxicity profile and evidence of disease control, parti-cularly in subpopulations of patients with hepatocellularcarcinoma and pleural mesothelioma (9–14). A randomizeddouble-blind phase III study of NGR-TNF (in combinationwith chemotherapy or supportive care) started in 2010 inpatients with malignant pleural mesothelioma (http://www.clinicaltrialsfeeds.org/clinical-trials/show/NCT01098266). Theidentification of factors that promote tumor resistance totargeted-TNF/chemotherapy combination could be, therefore,pharmacologically and clinically relevant.

We have previously shown that chromogranin A (CgA), aprotein released in circulation by many endocrine and neu-roendocrine cells, neurons and granulocytes, is an importantmodulator of the endothelial barrier function and a potentinhibitor of TNF-induced vascular leakage in the liver (15, 16).Structure–activity studies have shown that a bioactive site islocated in the N-terminal region of CgA (15, 17). A fragment ofCgA spanning the N-terminal residues 1–78, called vasostatin-1 (VS-1), can inhibit a series of effects exerted by TNF onendothelial cells, including phosphorylation of p38 MAPkinase [p38-MAPK (mitogen-activated protein kinase)], redis-tribution of VE-cadherin, formation of gaps, paracellulartransport of macromolecules (15, 18). VS-1 can also inhibitVEGF- and thrombin-induced endothelial cell permeability(19). Thus, CgA and its N-terminal fragment VS-1 are efficientinhibitors of endothelial cell activation and permeabilitycaused by TNF and by other stimuli.

Authors' Affiliation: Division of Molecular Oncology and IIT NetworkResearch Unit ofMolecular Neuroscience, San Raffaele Scientific Institute,Milan, Italy

Corresponding Author: Angelo Corti, Division of Molecular Oncology,San Raffaele Scientific Institute, via Olgettina 58, 20132 Milan, Italy.Phone: 39-02-26434802; Fax: 39-02-26434786;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-1273

�2011 American Association for Cancer Research.

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Increased levels of circulating CgA have been detected inpatients with neuroendocrine tumors, in subpopulations ofpatients with prostate, breast or non–small cell lung cancer(NSCLC), and in patients with heart failure, renal failure,hypertension, rheumatoid arthritis, sepsis, hepatocellularcarcinoma, liver cirrhosis, chronic hepatitis, pancreatitis,inflammatory bowel disease, or atrophic gastritis (20–31).Furthermore, increased levels of CgA have been detected alsoin patients treatedwithprotonpump inhibitors, a class of drugscommonly used to treat acid peptic disorders (32, 33). Thus,CgA levels higher than normal values can occur not only inpatients with neuroendocrine tumors or with tumors that mayundergo neuroendocrine differentiation but also in patientswith nonneuroendocrine tumors for a variety of reasons (30).

Given these premises and considering the protective effectsof CgA on TNF-induced alteration of the endothelial barrierfunction, we have hypothesized that abnormal levels of cir-culating CgA might inhibit the synergism between NGR-TNFand chemotherapy. To address this hypothesis, we haveinvestigated the effect of CgA and VS-1 on the antitumoractivity of NGR-TNF/chemotherapy against murine lym-phoma and melanoma, that is, 2 models of nonneuroendo-crine tumors. The rationale for this choice relies on the factthat these models have been largely exploited for preclinicalstudies on NGR-TNF combined with various chemotherapeu-tic drugs and that this drug combination is currently inves-tigated in patients with nonneuroendocrine tumors (3, 4, 8).We show that both proteins, at levels similar to those found inpatients, can markedly reduce the response of tumors to NGR-TNF combined with melphalan or with doxorubicin, by redu-cing the transport and the penetration of these drugs intoneoplastic tissues.

Materials and Methods

Drugs and reagentsMelphalan (Alkeran) was obtained from Glaxo-Wellcome;

doxorubicin (Adriblastina) was purchased from Pharmacia-Upjohn. Murine NGR-TNF (consisting of TNF fused with theC-terminus of CNGRCG) were prepared by recombinant DNAtechnology and purified from E. coli cell extracts as described(3).

VS-1, CgA, and anti-CgA antibodiesSer-Thr-Ala-CgA1–78 (VS-1), human CgA (hCgA), and mur-

ine CgA (mCgA) were prepared by recombinant DNA tech-nology and expression in E. coli cells as described (34).

Anti-human CgA monoclonal antibody (mAb) 5A8 (epitopeCgA53-57), B4E11 (epitope CgA68-71, all IgG1k) were pro-duced and characterized as described previously (35, 36).

In vivo studiesStudies on animal models were approved by the Ethical

Committee of the San Raffaele Scientific Institute and doneaccording to the prescribed guidelines. C57BL/6 mice (CharlesRiver Laboratories), weighing 16 to 18 g, were challenged withsubcutaneous injection in the left flank of 1 � 105 RMA orB16-F1 living cells. Six to 10 days later, mice were treated with

VS-1 (3 mg) or CgA (0.2 mg or 1 mg) 60, 30, and 0 minutes beforeadministration of NGR-TNF (0.1 ng). Two hours later, micewere given melphalan or doxorubicin solutions (90 or 80 mg,respectively). All drugs were administered intraperitoneally(i.p.), dilutedwith 0.9% sodium chloride, containing 100 mg/mLendotoxin-free HSA (Farma-Biagini SpA), except for doxoru-bicin and melphalan, which were diluted in 0.9% sodiumchloride alone. Tumor growth wasmonitored daily by measur-ing the tumors with callipers as previously described (4).Animals were killed before the tumors reached 1.0 to 1.5 cmin diameter.

In vitro endothelial permeability assayThe endothelial permeability assay was done using a trans-

well system (6.5-mm insert, 0.4-mm pore size filters; Corning),essentially as previously described, using 40 kDa fluoresceinisothiocyanate (FITC)–dextran conjugate (1 mg/mL; Sigma),as a tracer. The FITC–dextran conjugate present in the lowerchamber of the transwell system at various time points wasanalyzed using a fluorimeter (excitation, 495 nm; emission,520 nm). The same assays were done using doxorubicin(200 mg/mL) in place of FITC–dextran (excitation, 471 nm;emission, 556 nm).

Results

CgA and VS-1 inhibit the synergistic activity of NGR-TNFand chemotherapy in tumor-bearing mice

To investigate the effect of CgA and VS-1 on the synergismbetween NGR-TNF and chemotherapeutic drugs, we treatedRMA lymphoma–bearing mice with CgA or VS-1 (1 or 3 mg,respectively, 3 times in 1 hour), then with NGR-TNF (0.1 ng),and finally with doxorubicin (80 mg, 2 hours after NGR-TNF;Fig. 1A). The combination of doxorubicin with NGR-TNF, butnot doxorubicin alone, delayed tumor growth, as previouslyshown (4). This effect was completely inhibited by CgA or VS-1(Fig. 1B). We obtained similar results using a model based onB16-F1 melanoma–bearing mice and melphalan. Also in thiscase, both CgA and VS-1 inhibited the antitumor effect exertedby NGR-TNF in combination with melphalan (Fig. 1C). Theseresults suggest that CgA can inhibit the synergism betweenNGR-TNF and chemotherapy and that its N-terminal domaincontains a bioactive site.

Notably, CgA and VS-1 reached concentrations of about5 nmol/L after 1 to 2 hours, that is, at the time at whichNGR-TNF is expected to exert its action (Fig. 1D). Notably,these levels of CgA are in the range of those observed in certaincancer patients with nonneuroendocrine tumors (31, 37).

CgA and VS-1 do not inhibit the cytotoxic activity ofdoxorubicin and melphalan alone and in combinationwith NGR-TNF

The mechanism of action of CgA and VS-1 was theninvestigated. Neither CgA nor VS-1 affected the cytotoxicactivity of doxorubicin and melphalan, alone and in combina-tion with NGR-TNF, against human umbilical vein endothelialcells (HUVEC) and RMA cells (Fig. 2). These resultssuggest that the inhibition of the antitumor activity of

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Figure 1. CgA and VS-1 inhibit thesynergistic antitumor effects betweenNGR-TNF and chemotherapeutic drugs.A, tumor-bearing mice were treated, i.p.,with CgA (1 or 0.2 mg) or VS-1 (3 mg),NGR-TNF (0.1 ng) and doxorubicin (80 mg)or melphalan (90 mg) at the indicated time.B and C, effect of CgA or VS-1 on thesynergistic activity of NGR-TNF andchemotherapy on tumor growth (n ¼ 5mice per experiment, mean � SE).D, serum levels of human CgA and VS-1as measured by ELISA (mean � SE, n ¼ 5mice per experiment). **, P < 0.01;***, P < 0.001 by t test (2-tailed).

Chromogranin A and NGR-TNF/Chemotherapy

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NGR-TNF/chemotherapy by CgA or VS-1 was not related toinhibition of their cytotoxic activity against endothelial ortumor cells. More likely, it was related to a decreased drugdelivery to tumor cells.

CgA and VS-1 inhibit the penetration of doxorubicinin RMA tumors induced by NGR-TNF

To investigate whether CgA and VS-1 could affect thedelivery of chemotherapeutic drugs to tumor cells in vivo,we exploited the fact that doxorubicin is a fluorescent com-pound and that tumor cell fluorescence is an indication of theamount of doxorubicin that penetrate tumor cells (4). Tumor-bearing mice were treated with doxorubicin and, 2 hours later,tumor were excised, disaggregated, and analysed by fluores-cence-activated cell sorting (FACS). Preadministration of NGR-TNF to mice (2 hours before doxorubicin) increased the tumorcell fluorescence (i.e., the penetration of doxorubicin in tumorcells), as observed previously (4). However, pretreatment withCgAandVS-1 completely inhibited this effect (Fig. 3A). Notably,VS-1 significantly reduced also the spontaneous penetration ofdoxorubicin in tumor cells (Fig. 3A). These results support the

hypothesis that CgA and VS-1 can inhibit the delivery ofdoxorubicin to RMA cells in vivo.

Induction of endogenous CgA with omeprazoleinhibits the penetration of doxorubicin in RMAtumors induced by NGR-TNF

To assess the role of endogenous CgA, we investigated theeffect of omeprazole, a drug known to induce the release ofCgA in circulation in patients, on doxorubicin penetration inRMA tumors. Preliminary experiments showed that a singleadministration of omeprazole to mice (1 mg, i.v.) can increasethe circulating levels of murine CgA from 0.9 � 0.08 to1.48 nmol/L � 0.079 (n ¼ 5, mean � SE) by ELISA, peaking2 hours after injection (Fig. 3B, left). Furthermore, repeatedinjection of omeprazole (1 mg/day, for 5 days) induced anincrease in circulating murine CgA, reaching levels of1.75 nmol/L at day 4. The CgA returned to normal values 4 daysafter discontinuation of omeprazole administration. Repeatedadministration of omeprazole significantly decreased thepenetration of doxorubicin induced by NGR-TNF in RMAtumors (Fig. 3B, right). No significant effects were observed

Figure 2. CgA and VS-1 do notinhibit the cytotoxic activity ofNGR-TNF in combination withchemotherapeutic drugs in vitro.A and B, effect of CgA, VS-1,NGR-TNF, and chemotherapeuticdrugs on HUVEC cell viability.HUVEC cells were treated for 48hours with the indicated drugs,alone or in combination. Viablecells were stainedwithMTT. C andD, the same procedure wasapplied for RMA cell cytotoxicityassays (mean � SE of 2 assays,each conducted in quadruplicate).

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when omeprazole was administered in combination with mAb5A8, a neutralizing anti-CgA antibody (Fig. 3B, right), suggest-ing that the effect of omeprazole was mediated by CgA.These results suggest that even a moderate increase of

endogenous CgA is sufficient to significantly reduce the pene-tration of doxorubicin induced by NGR-TNF in tumor cells.

CgA and VS-1 do not affect RMA cell membranepermeability

The observation that CgA and VS-1 can reduce the deliveryof doxorubicin to RMA tumor cells in vivo prompted us toinvestigate whether these polypeptides could affect the trans-port of drugs through RMA cell membranes. To this aim, we

Figure 3. CgA and VS-1 inhibit theNGR-TNF�induced penetration ofdoxorubicin in RMA tumor cellsin vivo. A, effect of exogenousCgA and VS-1 on the NGR-TNF�induced penetration ofdoxorubicin in RMA tumor. Micebearing RMA tumors (diameter0.5–1 cm) were treated with VS-1(3 mg), CgA (1 mg), NGR-TNF(0.1 ng), or doxorubicin (360 mg) asindicated. Mice were then killed.Tumors were excised, weighed,disaggregated, and filteredthrough 70-mm filters. Then, theamount of doxorubicin thatpenetrated tumor cells wasanalysed by FACS.Representative FACS analysisand fluorescence positive tumorcells are shown (mean � SE,cumulative data of 2 (left) or 3(right) independent experiments,n ¼ 5–6 mice per group perexperiment). B, left: effect of singleor repeated administrations ofomeprazole (Ome) on CgA serumlevels (mean � SE; n ¼ 8 mice).Right: effect of omeprazole andanti-CgA mAb 5A8 on the NGR-TNF–induced penetration ofdoxorubicin in RMA tumors. Micewere injected with omeprazole(1 mg per mouse) and mAb (10 mgper mouse) as indicated. The % offluorescence-positive cellsrecovered from RMA tumors isshown (mean � SE, n ¼ 6 miceper group). *, P < 0.05; **, P < 0.01;and ***, P < 0.001 by t test.






preincubated RMA cells in vitro with different amounts ofNGR-TNF, CgA, or VS-1 (1 hour) and then with differentamounts of doxorubicin (1 hour). Finally, we analyzed thefluorescence intensity of cells by FACS. As expected, increas-ing concentrations of doxorubicin increased the fluorescenceintensity of cells, indicating that doxorubicin could penetratethe cells in a dose-dependent manner under these conditions(Fig. 4, left panel). Neither NGR-TNF nor CgA/VS-1 couldaffect the fluorescence intensity of doxorubicin-treated cells(Fig. 4, right panels). These results argue against the hypoth-esis that these polypeptides can affect the transport of dox-orubicin through cell membranes.

CgA inhibits the NGR-TNF�induced transport ofmolecules from the vascular compartment to thetumor cell microenvironment

We then investigated whether CgA could inhibit the NGR-TNF�induced transport of drugs from blood to the tumormicroenvironment. To this aim, we treated RMA-bearing micewith CgA, alone or in combination with NGR-TNF, followed 2hours later by Patent Blue VF, a hydrophilic nontoxic dye thatdoes not penetrate into cells and that can be easily quantified.After 5 minutes, the mice were killed and perfused with asaline solution. The amount of Patent Blue VF present intumor tissue homogenates was then quantified spectropho-tometrically. CgA inhibited the NGR-TNF–induced penetra-tion of Patent Blue VF in tumor tissues (Fig. 5). These resultssupport the hypothesis that CgA can indeed affect the trans-port of molecules from the vascular compartment to thetumor cell microenvironment induced by NGR-TNF.

CgA and VS-1 inhibit NGR-TNF�induced endothelialbarrier alteration

The positive effect of TNF on endothelial permeability andvascular leakage is a well-known phenomenon (38). This effectcan be efficiently inhibited by CgA and VS-1 (15). Keeping thisin mind, we tested the hypothesis that NGR-TNF can promotedrug extravasation by affecting the endothelial permeabilityand that CgA and VS-1 can inhibit this effect by protecting theendothelial barrier integrity. To this aim we analyzed, first, theeffect of NGR-TNF on the paracellular transport of FITC–dextran (a macromolecular tracer) through endothelialcell monolayers cultured in transwell systems. As expected,

NGR-TNF could increase the flux of FITC–dextran fromthe upper to the lower chamber of the transwell system.CgA (4–20 nmol/L) efficiently inhibited this effect (Fig. 6A,top panel). This result supports the concept that this proteincan inhibit the paracellular transport of molecules induced byNGR-TNF through endothelial cells.

Figure 4. CgA and VS-1 do not affect the penetration of doxorubicin in RMA tumor cells in vitro. RMA cells were incubated with different amounts ofCgA, VS-1, or NGR-TNF in complete medium for 1 hour and then with various amounts of doxorubicin (1 hour). Fluorescence intensity of cells (as measuredby FACS) is shown (mean � SE, cumulative results of 3 independent experiments, each conducted in duplicate).

Figure 5. CgA inhibits the NGR-TNF�induced penetration of PatentBlue VF in tumor. RMA tumor–bearing mice (diameter, 0.5–1 cm) weretreated with CgA (1 mg), NGR-TNF (0.1 ng), and, 2 hours later, with PatentBlue VF (12.5 mg/mL, 0.1 mL, i.v.). After 5 minutes, mice were killed andperfused. Tumors were excised, homogenized, and centrifuged. Thesupernatants were mixed with trichloroacetic acid [10% (v/v), finalconcentration] and cleared by centrifugation. The absorbance at 405 nmwas then measured using a spectrophotometer (n ¼ 4 mice per group,box-plots with median, interquartile, and min/max values). *, P < 0.05by t test (2-tailed).

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To assess weather CgA and VS-1 could also inhibit thetransport of drugs induced by NGR-TNF, we carried out asimilar experiment using doxorubicin in place of FITC-dextran. Both CgA and VS-1 could inhibit the flux of doxor-ubicin from the upper chamber to the lower chamber inducedby NGR-TNF (Fig. 6A, bottom panels). These data support thehypothesis that these polypeptides can affect drug delivery totumor cells in vivo by inhibiting the alteration of the endothe-lial barrier function induced by NGR-TNF.

CgA inhibits the NGR-TNF�induced disassemblyof VE-cadherin–dependent adherence junctionsThe molecular mechanism underlying the inhibitory effect

exerted by CgA on the NGR-TNF�induced permeability wasthen investigated. First, we analyzed whether NGR-TNF couldinduce the disassembly of VE-cadherin, a protein critical foradherence junctions, in confluent HUVEC cells. Immunofluor-escence staining of VE-cadherin showed a homogeneous dis-tribution of this molecule at cell–cell contacts (Fig. 6B).Incubation with NGR-TNF induced the disassembly of VE-cad-herin and caused the formation of intercellular gaps. This effectwas inhibitedbyCgA(Fig. 6B).These results suggest thatCgAcan

inhibit the permeabilizing activity of NGR-TNF by preventingVE-cadherin–dependent adherence junction disassembly.

CgA inhibits the phosphorylation of p38-MAPKinduced by NGR-TNF in endothelial cells

TNF is known to induce the phosphorylation of p38-MAPK,a signaling mechanism that can alter VE-cadherin expressionand endothelial permeability (39). To assess whether NGR-TNF and CgA regulate this signaling pathway in a positive andnegative manner, respectively, we analysed the effect of theseproteins on p38-MAPK phosphorylation in HUVEC cells atdifferent time points. Western blot analysis of total andphosphorylated p38-MAPK showed that NGR-TNF can inducethe phosphorylation of this enzyme in a time-dependentmanner and that CgA can inhibit this effect in a dose-dependent manner (Fig. 6C). CgA alone did not affectp38-MAPK phosphorylation (data not shown).

Discussion

The results of this study show that pretreatment of micebearing RMA lymphomas or B16-F1 melanomas with doses of

Figure 6. CgA inhibits the NGR-TNF�induced permeability, gap formation, VE–cadherin delocalization, and p38-MAPK phosphorylation in endothelial cellmonolayers in vitro. A, effect of NGR-TNF and CgA on the flux of FITC–dextran (40 kDa) and doxorubicin through endothelial cell monolayers (HUVEC).Experiments were carried out as described in "Materials and Methods" (n ¼ 4, mean � SE). *, P < 0.05; **, P < 0.01; and ***, P < 0.001 by t test (2-tailed).B, effect of CgA (2.5 mg/mL) on VE–cadherin delocalization and gap formation (arrows) induced by NGR-TNF (5 ng/mL) in confluent endothelial cells.VE–cadherin was detected by immunofluorescence analysis with a rabbit anti–VE-cadherin antibody and goat anti-rabbit Alexa Fluor 546 conjugate (red).Nuclei were stained with DAPI (blue; representative picture of 2 experiments each conducted in duplicate). Bar, 10 mm. C, effect of CgA on NGR-TNF�inducedphosphorylation of p38 MAPK in endothelial cells. Endothelial cells were incubated with NGR-TNF and CgA and analyzed by Western blot with anti�p38-MAPK and anti�phosphorylated p38-MAPK antibodies after 0, 5, and 20 minutes (representative results of 3 experiments).






CgA that enhance its circulating levels 4 to 5 times above thenormal values completely inhibits the synergism of NGR-TNFwith doxorubicin and melphalan. This inhibitory activity isunlikely related to inhibition of the cytotoxic activity of thisdrug combinations, as no effect was observed in cytotoxicityassays with cultured endothelial or tumor cells. More likely,the inhibitory activity of CgA was related to a reduction of thedelivery of chemotherapeutic drugs to tumor cells. Thishypothesis is supported by the results of FACS analysis oftumor cells recovered from tumor-bearing mice treated withdoxorubicin (which is a fluorescent compound) showing thatthe penetration of drugs into neoplastic cells induced by NGR-TNF in vivo can be almost completely inhibited by CgA.

How does NGR-TNF and CgA increase and decrease, respec-tively, the penetration of drugs into tumor cells? Drug deliveryto neoplastic cells in solid tumors requires, in principle, atleast 4 steps: the drug must (a) enter the tumor blood vessels(step 1), (b) cross the endothelial barrier (step 2), (c) migratethrough the tumor interstitium (step 3), and (d) cross themembrane of the target cells (step 4; see Fig. 7 for a schematicrepresentation). The results obtained with cultured cells showthat neither NGR-TNF nor CgA can affect the penetration ofdoxorubicin into RMA cells, arguing against a crucial role ofthese molecules on step 4. The effect on other steps are likelymore important. Interestingly, the results of in vivo assaysdone with RMA tumor–bearing mice injected with Patent BlueVF (a hydrophilic dye that does not cross the cell membrane)show that NGR-TNF can increase the accumulation of dye inthe tumor microenvironment and that CgA can inhibit thiseffect. Because tumors were perfused before excision (toremove the dye present in the intravascular compartment),these findings suggest that the dye was located in the extra-

vascular compartment of the tumor microenvironment. Thus,NGR-TNF enhances the drug extravasation steps (steps 2 and3), whereas CgA can inhibit this effect. Accordingly, the resultsof endothelial permeability assays showed that CgA caninhibit the NGR-TNF�induced flux of FITC-dextran throughendothelial cell monolayers. Given that FITC-dextran is a goodtracer for the paracellular transport of soluble compoundsthrough the endothelial barrier, these findings suggest thatNGR-TNF can reduce the barrier function of endothelial cellswhereas CgA inhibits this effect (step 2). Notably, CgA couldalso inhibit the NGR-TNF�enhanced flux of doxorubicinthrough endothelial cell monolayers. Thus, whereas the altera-tion of the endothelial barrier function by NGR-TNF mightaccount for the improved delivery of chemotherapeutic drugsto tumor cells, the protective activity exerted by CgA on theendothelial barrier integrity might account for the inhibitionof drug penetration and therapeutic activity observed in vivo.Notably, all the in vitro and in vivo effects observed with CgAwere observed also with its N-terminal fragment (VS-1). Thisobservation suggests that the biologically active site of CgA islocated in its N-terminal domain. A schematic representationof these concepts is shown in Figure 7.

How do NGR-TNF, CgA, and VS-1 affect endothelial cellpermeability? The results of in vitro studies show that NGR-TNF, similar to TNF, can alter the barrier function ofendothelial cell monolayers by causing a redistribution ofVE-cadherin, a component of adherence junctions criticalfor the maintenance of endothelial barrier integrity (40) andthat both CgA and VS-1 can inhibit this effect. The changesof VE-cadherin expression induced by TNF involvep38-MAPK phosphorylation (39). We observed that alsoNGR-TNF, similar to TNF, can induce p38-MAPK phosphor-ylation in endothelial cells. Again, CgA could inhibit thiseffect. Notably, CgA did not inhibit the cytotoxic activity ofNGR-TNF in assays done in the presence of actinomycin D, atranscription inhibitor (data not shown). This observationsuggests that CgA does not inhibit the interaction ofNGR-TNF with TNF membrane receptors. More likely,CgA inhibits the NGR-TNF–induced phosphorylation ofp38-MAPK by interfering with downstream signaling path-ways triggered by TNF receptor activation.

Are the doses of CgA used in our experimental modelsphysiologically relevant? Administration of 1 mg of CgA, i.p.,generated circulating levels of about 3 to 5 nmol/L at the timeof NGR-TNF administration in our murine models. This dosewas selected to achieve circulating levels similar to thoseobserved in subpopulation of patients with nonneuroendo-crine tumors (11, 12, 31, 37). For example, although serum CgAin the blood of normal subjects is about 1 nmol/L (21),increased levels of CgA, up to 4 to 8 nmol/L have beendetected in the sera of a subgroup of patients with NSCLC,despite the lack of neuroendocrine differentiation, withimportant prognostic implication (37). Considering that thecombination of NGR-TNF and chemotherapy is currentlyunder investigation in patients with NSCLC, hepatocellularcarcinoma, or other nonneuroendocrine tumors (9–14), theresults showing inhibitory activity of CgA in animal models ofnonneuroendocrine tumors might be clinically relevant.

Figure 7. Hypothetical mechanism of the effects exerted by NGR-TNFand CgA on the penetration of drugs in tumors. To reach cancer cellsdrugs must (1) enter tumor vasculature, (2) cross the vessel wall,(3) diffuse through the interstitium, and (4) penetrate into the cancer cells.NGR-TNF exerts synergistic effects with chemotherapy by altering theendothelial barrier function and promoting drug penetration in tumors(step 2). CgA and VS-1 inhibit NGR-TNF/chemotherapy synergism byprotecting the endothelial barrier integrity.

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A growing body of evidence suggest that administration ofproton pump inhibitors, which are drugs commonly used inthe treatment of acid peptic disorders, can enhance 2 to 3times, and in certain patients even up to 10 times, thecirculating levels of CgA, depending on the time of adminis-tration (32, 33). These drugs, by decreasing gastric acidity,induce the release of gastrin that, in turn, can induce enter-ochromaffin cell-like cell hyperplasia and CgA secretion (41,42). Accordingly, we found that daily treatment of mice withomeprazole can increase 2-fold the circulating levels of CgAand that 4 day discontinuation of therapy is necessary toreturn to base line. Interestingly, we observed that admini-stration of omeprazole to mice can inhibit the penetration ofdoxorubicin in tumors and that this effect can be inhibited byadministration of a CgA-neutralizing antibody. Thus, a 2-foldenhancement of CgA levels was sufficient to inhibit thepenetration of doxorubicin induced by NGR-TNF in tumors.Considering the wide use of proton pump inhibitors inpatients, also these findings may have important clinicalimplications.This study has a limitation that must be highlighted:

although our models are suitable to investigate the role ofcirculating CgA in nonneuroendocrine tumors, they arenot necessarily suitable to address the role of tumorderived/locally produced CgA that may occur in patients withneuroendocrine tumors. Indeed, in the latter case the tumorvasculature is exposed to much higher levels of CgA (possibly>1 mmol/L) compared with those of nonneuroendocrinetumors, owing to the fact that the source of CgA is the tumoritself. Because the dose–response curves of CgA and VS-1 invarious biological assays are "bell shaped" with loss of activityat micromolar concentrations (36, 43, 44), higher levels of CgAin the tumor microenvironment could be, paradoxically, lessactive than low levels of circulating CgA. Furthermore, dif-ferent proteolytic processing may occur to CgA released byneuroendocrine tumor cells and by normal cells (45). Thus,further studies with suitable neuroendocrine tumor modelsare necessary to address this point.Although many studies showed that CgA is a good marker

for diagnosis and prognosis of neuroendocrine tumors, evenfor monitoring their responses to chemotherapy (29, 30, 46),only few works have investigated the capability of baseline

circulating CgA to predict the therapeutic response. Interest-ingly, in 3 studies carried out on SCLC, NSCLC, and prostatecancer, the response rates to chemotherapy inversely corre-lated with baseline CgA (37, 47, 48). However, another study onprostate cancer, a tumor that may undergo neuroendocrinedifferentiation, showed higher response rates in patientswith high baseline CgA (49). Moreover, in a recent study, alarge proportion of patients with metastatic pancreatic endo-crine carcinomas experienced a major biochemical responsedespite an elevated serum CgA before therapy (50). Theseconflicting results further underline the need of new studies toelucidate the role of CgA in the response to chemotherapy ofneuroendocrine tumors or of tumors that may undergo neu-roendocrine differentiation.

In conclusion, the results of this study suggest that CgA caninhibit the NGR-TNF/chemotherapy synergism in murinemodels of nonneuroendocrine tumors by inhibitingNGR-TNF–induced changes of vascular permeability and drugpenetration in neoplastic tissues. These results could stimu-late further work aimed at assessing whether circulating CgAand VS-1 levels can predict the response to NGR-TNF/chemotherapy in patients. Furthermore, the finding thatomeprazole-induced CgA can inhibit the penetration of dox-orubicin in tumor tissues may suggest that discontinuation ofadministration of proton pump inhibitors to patients and/orits replacement with H2 receptor antagonists (a class of drugsthat do not increase CgA levels; ref. 33) might increase theresponse rate to this therapy in patients with documentedelevation of circulating CgA or VS-1.

Disclosure of Potential Conflicts of Interest

Angelo Corti is the inventor of a patent on NGR-TNF.

Grant Support

This work was supported by Associazione Italiana per la Ricerca sul Cancro,Alleanza Contro il Cancro (ACC), and FIRB (Italy).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 13, 2011; revised July 7, 2011; accepted July 7, 2011;published OnlineFirst July 28, 2011.

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Dondossola et al.





2011;71:5881-5890. Published OnlineFirst July 28, 2011.Cancer Res Eleonora Dondossola, Anna Maria Gasparri, Barbara Colombo, et al. of NGR-TNF to Enhance Chemotherapeutic EfficacyChromogranin A Restricts Drug Penetration and Limits the Ability

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