lack of microvessels in well-differentiated regions of

Lack of Microvessels in Well-Differentiated Regions of Human Headand Neck Squamous Cell Carcinoma A253 Associated withFunctional Magnetic Resonance Imaging Detectable Hypoxia,Limited Drug Delivery, and Resistance to Irinotecan Therapy

Arup Bhattacharya,1 Karoly Toth,2

Richard Mazurchuk,1 Joseph A. Spernyak,1

Harry K. Slocum,2 Lakshmi Pendyala,3

Rami Azrak,2 Shousong Cao,2

Farukh A. Durrani,2 and Youcef M. Rustum1

1Departments of Cancer Biology, 2Pharmacology & Therapeutics, and3Medicine, Roswell Park Cancer Institute, Buffalo, New York

ABSTRACTPurpose: Combination chemotherapy with irinotecan

(CPT-11; 50 mg/kg/week � 4 intravenously), followed 24hour later by 5-fluorouracil (50 mg/kg/week � 4 intrave-nously), results in 10 and 100% cure rates of animals bear-ing human head and neck squamous cell carcinoma xe-nografts A253 and FaDu, respectively. A253 consists of 30%well-differentiated and avascular and 70% poorly differen-tiated regions with low microvessel density (10/�400),whereas FaDu is uniformly poorly differentiated with highermicrovessel density (19/�400). Studies were carried out fordetermining the role of well-differentiated and avascularregions in drug resistance in A253 and detection of suchregions with noninvasive functional magnetic resonance(fMR) imaging.

Experimental Design: Tumors were harvested for his-topathologic evaluation and immunohistochemistry (CD31,CD34; differentiation marker: involucrin; hypoxia markers:carbonic anhydrase IX, pimonidazole; vascular endothelialfactor (VEGF) and Ki67) immediately after fMR imagingfollowing the 3rd dose of chemotherapy. High-performanceliquid chromatography determination of intratumoral drugconcentration of 7-ethyl-10-hydroxyl-camptothecin and au-toradiography with 14C-labeled CPT-11 was done 2 hoursafter CPT-11 administration.

Results: Although A253 xenografts showed three timeshigher concentration of 7-ethyl-10-hydroxyl-camptothecin,FaDu was more responsive to therapy. After therapy, A253tumor consisted mostly (�80%) of well-differentiated re-gions (positive for involucrin) lacking microvessels with ahypoxic rim (positive for carbonic anhydrase IX and pi-monidazole) containing few proliferating (Ki67 positive)poorly differentiated cells. Autoradiography revealed thatwell-differentiated A253 tumor regions showed 5-fold lower14C-labeled CPT-11 concentrations compared with poorlydifferentiated areas (P < 0.001). Blood oxygen level depen-dant fMR imaging was able to noninvasively distinguish thehypoxic and well-vascularized regions within the tumors.

Conclusion: Avascular-differentiated regions in squa-mous cell carcinoma offer sanctuary to some hypoxic butviable tumor cells (carbonic anhydrase IX and Ki67 posi-tive) that escape therapy because of limited drug delivery.This study provides direct evidence that because of a specifichistologic structure, avascular, well-differentiated hypoxicregions in tumors exhibit low drug uptake and represent aunique form of drug resistance.

INTRODUCTIONHead and neck squamous cell carcinoma (HNSCC) is the

fifth leading cause of cancer incidence and the sixth leadingcause of cancer-related death in the United States (1). Thesetumors frequently respond to chemotherapeutic agents primarilyused for palliation or as adjuvant therapy in conjunction withsurgery and radiation (2). Irinotecan (CPT-11), a topo I inhibitorin clinical development, is enzymatically converted in vivo bycarboxylesterase 2 in liver and intratumorally to its most activemetabolite, 7-ethyl-10-hydroxyl-camptothecin (SN-38; ref.3–5). The lactone form is active, with SN-38 lactone being 100-to 1,000-fold more cytotoxic than the prodrug in its lactone form(6). The antitumor activity and toxicity of combining CPT-11with 5-fluorouracil (FUra) is highly sequence dependent, with asequence of CPT-11 preceding FUra that results in the mostsignificant increase in therapeutic index (7). Earlier studies fromthis laboratory in which clinically relevant combination ofCPT-11 (50 mg/kg/week �4 intravenously) was used followed24 hours later by FUra (50 mg/kg/week �4 intravenously) onFaDu and A253 tumors growing subcutaneously in nude miceachieved cure rates of 100 and 10%, respectively (8).

Moreover, our preliminary neoangiogenesis studies inthese tumors yielded a surprising and interesting observationabout the morphological distribution of the apparently chaoticmicrovessels that has not been reported previously to the best ofour knowledge. Using CD31 immunostaining specific for mi-crovessels, we observed that well-differentiated regions in A253

Received 7/7/04; revised 8/30/04; accepted 9/9/04.Grant support: in part an Institute Comprehensive Cancer CenterSupport Grant CA 16056 from the National Cancer Institute, Bethesda,Maryland.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.Requests for reprints: Youcef M. Rustum, Department of CancerBiology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buf-falo, NY 14263. Phone: 716-845-2389; Fax: 716-845-7609; E-mail:[email protected].

©2004 American Association for Cancer Research.

8005Vol. 10, 8005–8017, December 1, 2004 Clinical Cancer Research

tumors (with or without keratin pearl formation) had no mi-crovessels. This finding is consistent with the fact that normalsquamous epithelium also lacks microvessels. Because A253originates from the squamous epithelium, it is not surprising thatwell-differentiated tumor regions share this aspect of the normaldifferentiation process. In these regions, multilayered cells arebound tightly together with no space for connective tissuecontaining microvessels. A253 tumors consist of about 30%well-differentiated regions without microvessels and 70%poorly differentiated regions with a low microvessel density(MVD; 10/�400 high power fields). FaDu tumors are uni-formly, poorly differentiated with a higher MVD (19/�400 highpower fields). Because of the potential therapeutic impact ofthese observations in relation to clinical outcomes, this studywas undertaken to determine the role and significance of differ-entiation-linked absence of MVD, and the possible consequencethis might have for drug distribution and therapy resistance. Wehypothesized that avascular and, as a consequence, hypoxicregions within tumors would have limited drug distribution anduptake, and thus these regions could confer resistance to ther-apy. Indeed, poorly differentiated proliferating tumor cells lo-cated in the outer rims of such areas were found to escapetherapy, supporting this premise.

Tumor vasculature is characterized by the following: (a)increased permeability to macromolecules because of large en-dothelial cell gaps or fenestrae (9); (b) incomplete basementmembrane and relative lack of pericyte or smooth muscle asso-ciated with endothelial cells (10); (c) increased vascular tortu-osity (11); (d) vasodilation (12); (e) intermittent or unstable flow(12); and (f) spontaneous hemorrhage and low vascular densitymixed with regions of high angiogenic activity (11). Tumorhypoxia is thus a potential therapeutic hurdle resulting from thisaberrant vasculature. In fact, hypoxia also has been recentlyconsidered a morphogen, inducing terminal differentiation ofcells in well-differentiated squamous cell carcinoma (13). Forthe immunohistochemical detection of hypoxia, carbonic anhy-drase IX (CAIX) has been proposed as an intrinsic, innateconstitutive marker and was found to correlate with loweredoxygen tension (�1%) in tumors (14–16). Pimonidazole hydro-chloride stains hypoxic regions with a partial pressure of oxy-gen � 10 mm Hg. Hypoxia-regulated CAIX has been found torelate to poor vascularization and resistance of HNSCC tochemoradiotherapy in patients (17).

Tumor vascular function (i.e., the ability of vessels totransport oxygen and nutrients) has been noninvasively studiedby mapping changes in the blood oxygen level dependant(BOLD), functional magnetic resonance (fMR) signal intensityin functional vessels in response to a controlled modulation ofblood oxygenation by inhalation of carbogen or other injectiblessystemically administered (19). Initial reports indicate BOLDfMR signal intensity maps correlate with temporal and spatialfluctuations in the measured partial pressure of oxygen values intumor and other tissues (20–22). This study uses the BOLDfMR method as a noninvasive tool for assessing the tumorvascular status: i.e., the net effect of vascular functionality,vessel density, and maturity in individual tumors.

Such an approach is important for several reasons. First,there is a broad spatial and temporal variation in tumor oxygen-ation and perfusion over many tumors as well as within indi-

vidual tumors depending on their stage of development andenvironmental variables (23). And, because noninvasive meth-ods for routine assessment of these factors do not currently exist,optimization of therapeutic efficacy in relation to these variablesis not possible. Second, regional in vivo variability of angio-genic activity cannot be sufficiently assessed by a static, purelymorphological analysis such as MVD measurements of histol-ogy samples. A more robust, easily implemented in vivo assay isneeded. To this end, the feasibility of using BOLD fMR imagingmethods for noninvasive monitoring of hypoxia and blood flowhas been examined herein and validated with detailed his-topathologic evaluation and immunohistologic methods. Spinecho, used for our study, is spatially more precise than gradient-echo, because large vessels downstream from tumors do notcontribute to signal changes (24). Spin echo with a lower mag-nitude of response (a factor of �3) can be a limitation for brainactivation studies but is not a drawback for tumor studies at thehigher field strength of 4.7T (26).

MATERIALS AND METHODSAnimals. Female athymic nude mice (nu/nu, body

weight, 20–25 g), 12 weeks of age, were obtained from HarlanSprague Dawley, Inc. (Indianapolis, IN) and were kept three tofive mice per cage with water and food ad libitum according toa protocol approved by the Institute Animal Care and UseCommittee at Roswell Park Cancer Institute. Non-necrotic tu-mor pieces were transplanted subcutaneously and bilaterally onthe back flanks. Treatments were started when tumor weightsreached �150 to 200 mg.

Tumors. Human HNSCC cell lines A253 (less sensitiveto CPT-11/FUra therapy) and FaDu (sensitive to CPT-11/FUratherapy) were purchased from American Type Culture Collec-tion (Manassas, VA). The cell lines were maintained as amonolayer in RPMI 1640 supplemented with 10% fetal bovineserum (Life Technologies, Inc., Grand Island, NY), and weretested for Mycoplasma every two months with the MycoplasmaT.C. Rapid Detection System (Gen-Probe Inc., San Diego, CA).The xenografts were established initially by implanting subcu-taneously 106 cultured individual cells into mice, and xe-nograpts were passed several generations by transplanting �50mg of non-necrotic tumor tissue. In subsequent experiments,tumors were implanted bilaterally in nude mice (i.e., A253 onthe left and FaDu on the right), so that both tumors developingin a single animal would experience identical whole body en-vironmental factors including drug metabolism. Experimentswith one tumor type per mouse gave identical results in terms ofhistology and response rates when compared with mice bearingboth tumor types. Tumor weight (mg) was approximated as thefollowing: 1⁄2(L� W2)(mm), where L is the longest axis and Wis the shortest axis as measured by digital calipers. Relativetumor volume (100%) was calculated by actual tumor weight(ATW) over initial tumor weight (ITW, day 0) as follows:(ATW/ITW) � 100%. Mice were scanned 24 hours after the 3rdweekly dose of FUra and sacrificed immediately thereafter bycervical dislocation. Tumors were harvested with the outer skinleft intact over the tumor capsule to act as a fiduciary marker forcorrelating BOLD fMR images with histology, whereas the

8006 Differentiated, Avascular Regions and Drug Resistance

tumor capsule facing the interior of the animal was stained withblack ink (Bradley Products Inc., Bloomington, MN).

Patient Samples of HNSCC. We also studied formalin/paraffin sections of six human surgical samples of HNSCC (4well-differentiated and 2 poorly differentiated tumors like A253and FaDu xenografts, respectively) for intratumoral microvesseldistribution using factor VIII (Von Willebrand factor) to con-firm the preclinical results in corresponding human tumors.

Drugs. CPT-11 (Pharmacia, Kalamazoo, MI), FUra(Hoffmann-La Roche, Inc. Nutley, NJ), and 14C-labeled CPT-11(Pharmacia Italia S.p.A, Nerviano, MI, Italy) were dissolved insterile saline before use at a final concentration of 20 and 10mg/mL, respectively. 14C Radiolabel is part of the quinolinediethyl active moiety of CPT-11 that does not get removed withthe cleaved piperidino side chain during conversion to thebiological active form by carboxylesterase intratumorally and inliver.

Drug Doses and Schedules. When tumor weightsreached �150 to 200 mg, the mice were treated with CPT-11(100 mg/kg/week �3 intravenously) followed 24 hours later byFUra (50 mg/kg/week �3 intravenously). The maximum-toler-ated dose for CPT-11 and FUra administered individually is 100mg/kg for the weekly schedule. The combination therapy did notresult in any substantial weight loss or adverse reaction in themice.

Immunohistochemistry. Because detection of mouseendothelial cells by antimouse CD31 monoclonal antibody re-quires the use of zinc fixatives, we developed and optimizedimmunohistochemical methods for involucrin, CAIX, Hy-poxyprobe, and Ki67 in zinc fixatives. This also ensured iden-tical experimental conditions for all of the markers. The stainingcharacteristics of the four markers after zinc fixation, and afterthe traditional 10% neutral buffered formalin fixation, were verysimilar. Before immunostaining, conventional H&E-stained sec-tions were prepared for general histopathologic evaluation andfor comparison with BOLD fMR images. Harvested tumorswere fixed in zinc fixative (BD Biosciences, PharMingen, CA)overnight and then processed for paraffin sections. Immunohis-tochemical staining was carried out on 5-�m sequential sectionson charged slides after conventional deparaffinization andquenching of endogenous peroxidase with 3% H2O2. Casein(0.03%) in PBS with 500 �L/L Tween for 30 minutes at roomtemperature was used to block unspecific binding in each im-munoassay. We carried out all immunohistochemical proce-dures using the automated immunostainer (DAKO, CarpinteriaCA). Antigen retrieval was done with citrate buffer (pH 6) inmicrowave for 4 minutes (Hypoxyprobe and CAIX) or 10minutes (Ki-67, factor VIII), allowed to cool for 15 minutes andthen washed with PBS/500 �L/L Tween. Nuclear counterstain-ing was done with hematoxylin before being dehydrated,cleared, and coverslipped. All washes were done with PBS/500�L/L Tween, and chromogen 3,3�-diaminobenzidine (DAKO)was used for all of the immunostainings for 5 minutes. Anisotype-matched negative control was used on a duplicate slidein place of the primary antibody in the same concentration as theprimary antibody. All histopathologic and immunohistologicanalysis and interpretation was done directly by a board certifiedand experienced pathologist (Dr. Karoly Toth MD, Ph.D.) orunder his supervision.

Involucrin Staining. Involucrin, a marker for keratino-cyte terminal differentiation (like cytokeratin), is expressed inthe suprabasal layers while being absent in the undifferentiatedbasal layer of stratified squamous epithelium. It is a marker fordifferentiation in human squamous cell carcinomas present inthe differentiated cells and absent in the poorly differentiatedtumor cells (27). Epidermis of a human skin biopsy was used asa known positive (suprabasal layers) and negative (basal layers)control to optimize the method. No antigen retrieval was nec-essary. Ten-minute fixation in 10% neutral buffered formalinwas done after deparaffinization and before immunostaining.The primary monoclonal antibody (mouse ascites fluid) anti-involucrin clone SY5 (Sigma, St. Louis, MO) in 16 �g/mLconcentration was applied for 1 hour at room temperature. Thesecondary detection system used was the mouse Envision kit(DAKO).

CD31 and CD34. No antigen retrieval was necessary.The rat antimouse primary antibody CD31 (BD BiosciencesPharMingen) and 10 �g/mL CD34 (clone RAM34, ResearchDiagnostics Inc., Flanders, NJ) was applied for 1 hour at roomtemperature. Biotinylated secondary antibody (antirat, BD Bio-sciences PharMingen) was used for 30 minutes followed by thestreptavidin complex (Zymed Lab Inc., San Francisco, CA) for30 minutes.

Von Willebrand Factor (Factor VIII). Rabbit antihu-man polyclonal antibody (DAKO) in 11.4 �g/mL concentrationwas applied for 1 hour at room temperature. Biotinylated goatantirabbit (Vector Labs, Burlingame, CA) was used for 30minutes, followed by streptavidin complex (Zymed Lab Inc.) for30 minutes.

Carbonic Anhydrase IX (M75). The primary antibodyM75 (20 �g/mL, a gift from Dr. Pastorek, Institute of Virology,Slovak Academy of Sciences, Slovak Republic) was applied for90 minutes at room temperature. Incubation with Elite biotiny-lated secondary antibody (Vector Labs) for 30 minutes wasfollowed by Elite ABC reagent (Vector Labs) for 30 minutes.

Hypoxyprobe. Mice received injection intraperitoneallywith 60 mg/kg of Hypoxyprobe (pimonidazole hydrochloride,NPI, Belmont, MA) 45 minutes before BOLD fMR scanning.Sections were incubated with the primary antibody Hy-poxyprobe-monoclonal antibody (1.4 �g/mL, NPI, Belmont,MA) for 1 hour at room temperature. Incubation with biotiny-lated IgG fragment [F (ab�) 2, Accurate Chemical & ScientificCorporation, Westbury, NY] for 10 minutes was followed by thestreptavidin complex (Zymed Lab Inc.) for 30 minutes.

Vascular Endothelial Growth Factor. Formalin/paraf-fin sections of untreated A253 xenografts were used for vascularendothelial growth factor (VEGF) detection. Polyclonal rabbitantihuman VEGF (0.25 �g/mL A-20, Santa Cruz BiotechnologyInc., Santa Cruz, CA) was applied overnight at 4°C as primaryantibody. Incubation with Elite biotinylated secondary antibodyfor 30 minutes was followed by Elite ABC reagent (VectorLabs) for 30 minutes.

Ki-67. The mouse primary antibody against humanKi-67 (2 �g/�L, clone MB67, Diagnostic Biosystems, Pleasan-ton, CA) was incubated for 1 hour at room temperature. Incu-bation with biotinylated secondary antibody for 30 minutes wasfollowed by the streptavidin-peroxidase complex for 30 minutes

8007Clinical Cancer Research

according to the manufacturer’s instructions (Thermo Shandon,Pittsburg, PA).

Tumor Sample Preparations for High-PerformanceLiquid Chromatography. HNSCCs were implanted bilater-ally in nude mice, so that both A253 and FaDu tumors wouldhave the same drug exposure from circulation after drug treat-ment. Three separate mice with established tumors (�200 mg)were treated with CPT-11 (100 mg/kg), tumors were excised,and blood was collected 2 hours after the treatment. Immedi-ately after the collection, 5 mL of ice-cold methanol and ace-tonitrile (1:1) were added to tumor samples and homogenizedwith a Polytron tissue homogenizer (Brinkmann Instruments,Westbury, NY). After centrifugation of the homogenate, thesupernatant was evaporated to dryness, reconstituted to mobilephase, and subjected to high-performance liquid chromatogra-phy. Protein measurement was done on the pellet with Bradfordprotein assay (28). Plasma was obtained at 4°C immediatelyafter the blood collection and was subjected to the same solventextraction procedure and to high-performance liquid chroma-tography. Three replicates for these experiments were done.

CPT-11 and SN-38 Measurements. We measured thelactone forms of CPT-11 and its active metabolite, SN-38, usinga validated high-performance liquid chromatography methodwith fluorescence detection described by Warner and Burke(29). The separation method was carried out on a Waters Nova-Pak C18 column equipped with �Bondapak C18 guard column,with mobile phase consisting of 20% acetonitrile and 80%triethylamine acetate. The detection was by fluorescence, withexcitation at 370 nm and emission at 510 nm. The limits ofquantitation were 2.5 ng/mL for both. Quality assurance wasmaintained by simultaneously assaying the quality control sam-ples prepared in bulk before assay validation.

Protein Binding Measurements. CPT-11 analogs bindextensively to proteins. The lactone form seems to bind toproteins to a greater extent than the carboxylate form (30, 31).To estimate the actual free (nonprotein bound) drug concentra-tions in the samples, control experiments were analyzed toevaluate the protein binding as described by Stewart et al. (31).This constitutes spiking mouse plasma with known concentra-tions of CPT-11 and SN-38 (100 to 1,000 ng/mL), equilibrationat 25°C and centrifugal ultrafiltration with the Amicon Centri-fuge ultrafiltration system. The ultrafiltrate was processed asdescribed earlier to calculate the nonprotein bound fraction.CPT-11 metabolism to SN-38 lactone was measured in plasmaand tumor tissue samples were taken 2 hours after drug admin-istration. Samples were processed as outlined earlier.

High Resolution Autoradiography. To preserve andmaintain the original tumor in vivo drug distribution, we didautoradiography using frozen sections; thereby, we avoided thenormal embedding procedures that are known to lead to changesin the drug location during processing. 14C-labeled CPT-11(specific activity, 59 mCi/mmol) was mixed with cold CPT-11and given intravenously to achieve a dose of 100 mg/kg, with anactivity of 10 �Ci (specific activity of 0.29 �Ci/mmol/L) tomice bearing HNSCC xenografts. On the basis of the location ofthe 14C label, the radiolabel remains a part of the drug’s activemetabolite (see Drugs section above). After 2 hours, the micewere euthanized by cervical dislocation, and the tumor wasexcised and frozen with cryogel (Instrumedics Inc, Hackensack,

NJ) in liquid nitrogen before being stored in �80°C freezer untilsectioning. Frozen sections (6–8 �) were cut by cryostat(�30°C) and put on amino-acyl silane-treated slides (chargedslides) and stored in �80°C freezer until further processing.They were brought to room temperature in a dark room withonly a red safety light where remaining processing was carriedout. For negative controls, mice with tumors were treated sim-ilarly, with nonlabeled CPT-11, and the same procedure wascarried out. Kodak-NB2 emulsion was placed in a water bath at42°C for 15 minutes. The slides were dipped into a smallamount of emulsion and dried before storing in a black, light-free box at 4°C with desiccant (silica gel). Exposure for durationrequired (7 weeks) was done at 4°C under low humidity con-ditions. For developing, slides at room temperature were im-mersed for 5 minutes in Kodak D19 developer at 22°C. After a30 second wash with distilled water, they were immersed inKodak fixer (1:1) for 4 minutes before washing for 15 minutesand fixing in 10% neutral buffered formalin for 15 minutes. Theconventional H&E counterstaining followed a final wash of 30minutes. Slides were dehydrated, cleared, and coverslipped (32).

Image Analysis of Autoradiographic Slides. For quan-titation of the grains, color microphotographs were taken at�1,000 magnification. Slides were digitized at 1350 dots perinch, and the digital images were converted to gray-scale andfiltered with an unsharp mask (amount 150%, radius 4 pixels,Adobe Photoshop, Adobe Systems Inc., San Jose, CA). We thenanalyzed the resultant images by commercial image analysis andvisualization software (AnalyzePC Version 5, Biomedical Im-aging Resource, Mayo Clinic, Rochester, MN) for grain count-ing using the object counter module.

BOLD fMR Imaging. Mice were anesthetized with 100mg/kg ketamine HCl/10 mg/kg xylazine (intraperitoneally) andimaged with T2*-weighted spin echo-based rapid acquisitionwith refocused echoes (RARE) imaging sequence (Echo time �79.7 ms, Repitition time � 4622.5 ms, number of excitations �2, field of view � 3 cm, 1-mm thick axial slices perpendicularto the spinal axis, with an in-plane spatial resolution of 234 �m).RARE imaging delivers images practically free from artifactswith an appropriate phase-encoding scheme. Biological struc-tures including tumor, with long relaxation times are the onesmost sought after in clinical magnetic resonance imaging, andthus use of a long TR time with a relatively short scan time ispossible with RARE sequences. It can be used to acquire imageswith a very high signal:noise ratio in a reasonable time byaveraging (33). We acquired sequential BOLD fMR images firstwhile the mouse was breathing room air followed later bycarbogen (93% O2 � 7% CO2, for 9 minutes) using a GE CSI4.7T/33 cm imaging spectrometer (GE NMR Instruments, Fre-mont, CA) incorporating AVANCE digital electronics (Biospecplatform with Paravision 2.1 operating system, Bruker Medical,Billerica, MA). For processing fMR data, a customized andinteractive fMR module was developed and interfaced to acommercially available three-dimensional image analysis pack-age (AnalyzePC Version 5, Biomedical Imaging Resource,Mayo Clinic). A functional map was subsequently calculatedpixel-by-pixel for regions visually identified as containingtumor. fMR image intensity changes from pre- and post-carbogen–paired T2-weighted magnetic resonance images werecalculated with the following equation: % signal intensity


change � [(post � pre)/(pre)] � 100, where “pre” refers to theT2-weighted images obtained with the mice breathing room air,and “post” refers to images acquired while breathing carbogen.To serve as a control, functional maps were obtained fromback-to-back scans while the animal breathed room air to ensurepre/post image changes were nonrandom. To aid in visualizationof regions of change, a color lookup table was applied to thefunctional map and an anisotropic diffusion filter (iterations �6, time interval � 0.25) and low-pass filter (kernel 3 � 3 � 1)were applied to reduce systemic noise while preserving grossareas of change from baseline. The colorized functional mapwas then overlaid on a T1-weighted scan to localize areas ofsignal intensity change. In resultant BOLD fMR maps, mini-mum or no change was depicted as “green” whereas regionswith net-positive change from baseline values were depicted as“blue-purple” (50 being the highest positive change), and re-gions with net-negative change are depicted as “yellow-red”(�50 of lookup table being the largest negative change) asillustrated. Regions of different fMR image signal intensitychanges were compared for correlation with MVD count for thatregion.

Statistical Analysis. We analyzed differences betweenthe mean values of the autoradiographic grains and tumor CPT-11/SN-38 lactone levels for significance using unpaired two-tailed t test for independent samples, with Welch correctionapplied. Linear regression and correlation analysis were carriedout for fMR mean signal intensity and MVD with runs test.GraphPad InStat (Version 3.01 for Windows 95, GraphPadSoftware, San Diego, CA) was used to do all analysis. A P valueof �0.05 was considered statistically significant.

RESULTSAntitumor Response in Xenograft. We reported previ-

ously the efficacy of the therapeutic synergy of CPT-11 andFUra (8). Briefly, Fig. 1 summarizes the key findings of the useof CPT-11 (50 mg/kg/week �4 intravenously) followed 24

hours later by FUra (50 mg/kg/week �4 intravenously) on FaDuand A253 tumors growing subcutaneously in nude mice, givinga cure rate of 100% and 10%, respectively. The sequentialcombination with CPT-11 at 100 mg/kg/week, used in thisstudy, gave a lower median tumor volume in A253 as comparedwith the combination using CPT-11 at 50 mg/kg/week. Thehigher dose of CPT-11 in the sequential combination was usedto highlight and confirm the impact of intratumoral tissue ar-chitecture in hampering uniform drug distribution in the lesssensitive xenograft A253.

Detection of Avascular Hypoxia by Immunohistochem-ical Methods in Xenografts. To understand the role ofdifferentiation and avascular hypoxia in drug resistance inHNSCC, the poorly differentiated FaDu (n � 6) and the well-differentiated A253 (n � 6) xenografts were analyzed histolog-ically for differentiation status (H&E, involucrin), MVD withmonoclonal antibodies CD31 and CD34, hypoxia with twomarkers (CAIX and pimonidazole HCl), proliferation markerKi67, and angiogenesis marker VEGF.

Figure 2 summarizes the histologic and immunohistochem-ical characterization of untreated A253 (left) and FaDu (right)xenografts. The representative photomicrographs show the fun-damental differences between the two tumors. Panel A showsthe histologic structure of the well-differentiated HNSCC,A253, containing well- differentiated part with keratin forma-tion (left part) and the characteristic localization of poorlydifferentiated cells at its rim (arrow), whereas the poorly dif-ferentiated tumor cells are present (right side of the panel).Panel B shows the uniformly poorly differentiated HNSCC,FaDu. Panel C clearly shows the complete absence of microves-sels in a large microscopic field of the well-differentiated part ofA253 (arrow), whereas panel D shows a relatively homogene-ous distribution of numerous microvessels in FaDu. FaDu hastwice as many microvessels (19/�400 high power fields) asA253 (10/�400 high power fields), whereas a higher proportionof the total vessels are large in A253. Panels E (brown regions)and G (left, arrow) confirm that the well-differentiated regionsin A253 show positive immunostaining for both hypoxia mark-ers whereas panel G (right, arrow) indicates that such regionsare strongly positive for VEGF. Panels F and H indicate the lackof hypoxia immunostaining almost in the entire FaDu tumor andonly the narrow edge of necrosis at the typical localization(arrows) exhibits hypoxia.

Figure 3 summarizes the remarkable change in intratu-moral tissue architecture over the course of therapy when thedifferentiation marker, involucrin, was used. Panel A showsA253 before therapy with about 30% differentiated regions.Panel B depicts remnant A253 tumor 24 hours after the 3rdsequential treatment with CPT-11 followed 24 hours later byFUra and shows that the remaining small A253 tumor charac-teristically consists of about 80% well-differentiated areas.Panel C shows the presence of poorly differentiated cells (ar-row) in a rim surrounding the differentiated region. Tumor cellsin these locations and nearby are the major source for tumorsurvival and regrowth.

Figure 4 summarizes the histologic and immunohistologicfeatures of A253 (left) and FaDu (right) 24 hours after the 3rdsequential treatment with CPT-11 followed 24 hours later by

Fig. 1 Kinetics of response to the weekly combination of CPT-11 at100 or 50 mg/kg/week intravenously � 4, 24 hours before FUra admin-istration at 50 mg/kg/week intravenously � 4. Arrows indicate CPT-11treatments.


FUra. Panel A shows that the remaining small A253 tumorcharacteristically consists of about 80% well-differentiated ar-eas, with poorly differentiated cells at their rim (arrow). Theuntreated A253 has only about 30% well-differentiated parts.This comparison indicates a remarkable increase in the fractionof the well-differentiated part of A253. Panel B shows theremaining tumor cell nests (arrows) of FaDu in a connectivetissue stroma. Panel C shows again that there are no vessels inthe large well-differentiated areas of A253, as opposed to thehighly vascularized poorly differentiated FaDu (panel D). PanelE shows strong CAIX immunostaining in the poorly differenti-ated cells at the rim of the well-differentiated area, proving thatthe tumor cells within this location remained hypoxic, whereasthe surviving FaDu (panel F) tumor cell nests show no immu-nostaining (arrows), indicating that they are not hypoxic (sur-

rounding mouse stroma shows some unspecific brown staining).Panel G shows positive immunostaining for the other hypoxiamarker in the typical localization of the well-differentiated ar-eas, whereas panel H shows lack of immunostaining in the FaDutumor cells, indicating lack of hypoxia. Panel I visualizes thepresence of Ki67-positive cells among the tumor cells locatedwithin the rim region, indicating that they are surviving andproliferating cells, whereas in FaDu (panel J) the survivingtumor cells (arrows) are Ki67 negative, indicating that they arenot in cell cycle.

We confirmed lack of microvessels in the well-differentiatedparts of HNSCC xenografts before and after therapy usingCD34, an additional marker for endothelial cells. Microvesselswithin the poorly differentiated regions were positive for CD34although weaker when compared with CD31 immunostaining.

Fig. 2 Microphotographs ofthe untreated A253 (left) andFaDu (right) xenografts. A-D,�200; E-H, �100. H&E show-ing well-differentiated A253(A) with an �30% differenti-ated region (arrow indicatingthe rim tumor cells at the edgeof well-differentiated area) anduniformly poorly differentiatedFaDu (B). Microvessel distri-bution by CD31 staining forA253 (C) showing no vesselsin the well-differentiated re-gion (arrow) as compared withthe well-vascularized (brown)FaDu (D). CAIX staining forhypoxia in A253 (E) showslarge positive areas (brown),whereas FaDu (F) is positiveonly in perinecrotic regions(arrow). Pimonidazole HCl,another hypoxia stain, is posi-tive in A253 (G, left) for well-differentiated areas (arrow)whereas in FaDu (H), it is posi-tive only in perinecrotic regions(arrow). G (right) shows strongimmunostaining for VEGF inthe well-differentiated region ofA253 (arrow).


Levels of CPT-11 and SN-38 in Xenografts. Table 1summarizes the plasma and tumor tissue levels of CPT-11 andSN-38. The SN-38 levels in plasma were �9% that of CPT-11.The SN-38 levels were much lower relative to CPT-11 in thetumor. SN-38 in A253 tumor was 1.8% that of CPT-11, and inFaDu it was �0.4%. Although the drug and metabolite levels inplasma were the same for the two tumors, the intratumoral drug

accumulation and conversion, especially for SN-38, differedconsiderably. The CPT-11 levels in A253 and FaDu tumorswere 1.9 and 2.4%, respectively, of the circulating plasma levelsat this 2 hour sampling time, but the SN-38 levels were 0.4 and0.1% of the circulating plasma levels, respectively. The intra-tumoral level of the SN-38 lactone was four times higher inA253 (3.64 ng/mg protein) compared with FaDu (0.93 ng/mgprotein; P � 0.049). Despite a three times higher concentrationof the active metabolite SN-38 in A253, FaDu is more respon-sive to CPT-11 treatment. The in vitro IC50 for SN-38 in A253and FaDu is 0.35 and 0.1 �mol/L, respectively (29). Intratu-moral drug concentration, therefore, does not correspond totreatment response in A253 and, to better understand the role ofintratumoral drug distribution, 14C-labeled CPT-11 autoradiog-raphy was carried out.

Distribution of 14C-labeled CPT-11 in Xenografts byAutoradiography. Figure 5 illustrates the characteristic auto-radiographic distribution of 14C-labeled CPT-11 (black silvergrains) in the xenografts 2 hours after intravenous administra-tion of the drug. Panels A and B show the same A253 tumor,demonstrating that the well-differentiated region (A), includingthe poorly differentiated tumor cells at its rim (arrow), containmany less silver grains, indicating that less 14C-labeled CPT-11is present than in the region with the poorly differentiated cells(B). Panel C, as a control, shows few grains because of baselinebackground radiation (system noise) independent of tissuewhereas panel D shows the grain distribution in the uniformlypoorly differentiated FaDu.

Table 2 summarizes the mean (n � 4) intratumoral graindistribution for both xenografts, after we adjust for backgroundusing image analysis of autoradiographic slides. The rim of cellssurrounding the well-differentiated regions in A253 had a muchlower (22 14 grains/mm2) grain density after the 1st chem-otherapy treatment compared with the poorly differentiated re-gions (103 16 grains/mm2), indicating a significantly lowerdrug distribution (P � 0.001) and, therefore, a lower drugexposure of the cells located within this rim region. The differ-ence was even more significant (rim, 28 15 compared with193 36 grains/mm2 in poorly differentiated regions) after the4th chemotherapy treatment (P � 0.001). The grain distributionin the poorly differentiated regions of A253 after the 4th cycleof chemotherapy showed a significant increase (P � 0.004)when compared with the grain density in a similar region afterthe 1st chemotherapy cycle. This is indicative of effective vas-culature in the remaining poorly differentiated regions of thetumor in A253 during therapy. In FaDu there was no significant(P � 0.0759) increase in the grains during therapy between the1st and 4th cycle. The poorly differentiated well-vascularizedregions in A253 had three times more grains than the poorlydifferentiated regions of FaDu (P � 0.001), consistent with thedifferences seen with total intratumoral SN-38 lactone measure-ments by high-performance liquid chromatography (Table 1).

Noninvasive Characterization of Tumor Hypoxia byBOLD fMR Imaging. BOLD fMR imaging results were com-pared with histopathology and immunohistochemical tumorfindings. Figure 6 summarizes the correlation between well-vascularized, nonhypoxic tumor regions with areas of negativeBOLD fMR signal intensity change (Pearson coefficient r ��0.89, r2 � 0.79, P � 0.001). As an example, these regions are

Fig. 3 Microphotographs of involucrin immunostaining of well-differentiated regions (brown) in A253, which increases from 30% (A)to �80% (B) over the course of therapy. Poorly differentiated cellsincluding those surrounding these regions (C) in a rim (arrow) arenegative for involucrin staining. Tumor cells in these locations andnearby are the major source for tumor survival and regrowth. A and C,�200; B, �100.


illustrated in Fig. 6 (right column) as yellow/red in the resultantBOLD fMR image maps from a FaDu tumor obtained aftertherapy. The higher the number and/or the greater the vesselsize, the greater was the decrease in BOLD fMR signal intensityobserved. Hypoxic regions with poor, little, or no vasculaturewere associated with regions of positive BOLD fMR signalintensity change. Figure 7 (left column) depicts representative

BOLD fMR results obtained from a A253 tumor after therapy.The lookup table used for better visualization shows the colorrange for positive and negative signal intensity change withyellow and red being toward negative change corresponding toMVD whereas blue and purple denotes positive intensitychange. As shown, a significant area within the tumor containsblue/purple pixels as regions with increased BOLD fMR signal

Fig. 4 Microphotographs ofA253 (left) and FaDu (right)xenografts after 3 cycles oftherapy with CPT-11 followedby FUra. All panels, �200.H&E A253 (A) shows an in-crease in well-differentiated re-gions (�80%) whereas FaDu(B) tumor cell nests (arrows)are seen in a connective tissuestroma. CD31 for A253 (C)shows the absence of vesselsfor a large well-differentiatedarea (arrow) with keratin pearls,whereas FaDu (D) is rich inuniformly distributed vessels(brown). CAIX staining in A253(E) is strong particularly inpoorly differentiated rim cells(brown, arrow), whereas FaDu(F) is negative (arrows; mousestroma is false positive). Pi-monidazole HCl staining isstrongly positive (brown, arrow)in the well-differentiated areas ofA253 (G) and negative in FaDu(H). Ki67-positive proliferatingtumor cells (brown nuclei, ar-row) are seen in the rim sur-rounding the well-differentiatedregions in A253 (I), whereas inFaDu (J), tumor cells (arrows)are negative indicating nonpro-liferating and probably dying tu-mor cells.


intensity change associated with hypoxia. The figure legendbelow each fMR image lists MVD results from outlined indi-vidual tumor regions. CD31 results confirm the excellent cor-relation between immunohistochemical data and BOLD fMRimage results. Moreover, Fig. 7 shows the ability and sensitivityof BOLD fMR imaging techniques to be used as a noninvasive“research probe” to identify well-differentiated hypoxic tumorregions and their surrounding rims in our tumor model.

Patient Samples of HNSCC. We detected microvesseldistribution by immunohistochemical method using antihumanfactor VIII to detect intratumoral endothelial cells in formalin/paraffin sections of the surgical specimens (n � 6) of HNSCC.

Smaller and bigger microscopic areas of the well-differen-tiated parts (with or without keratin pearl formation) of tumorsdid not show presence of microvessels. Only the poorly differ-entiated parts of the tumors showed the typical intratumoralchaotic distribution of microvessels. These findings, which useanother endothelial cell marker (factor VIII), confirm a mi-crovessel distribution pattern in human surgical samples similarto that seen in HNSCC xenografts, which use CD31 and CD34endothelial cell markers.

DISCUSSIONWe have used the human HNSCC xenografts A253 and

FaDu in this study to understand intratumoral architecturalchanges during therapy that are linked to resistance of the FDAapproved combination therapy of CPT-11/FUra. Our focus hasbeen on the role of avascular differentiation-linked areas, aunique feature of well-differentiated squamous cell carcinomasuch as A253 and its possible role in facilitating nonuniformintratumoral drug distribution, thereby leading to therapy resist-ance. This study does not focus on hypoxia in any other wayexcept thus far as it is related to such avascular regions and itsdetection using fMR imaging techniques. In vitro IC50 forSN-38 in A253 and FaDu is 0.35 and 0.1 �mol/L, respectively(34). Despite a three times higher concentration of the activemetabolite of CPT-11, SN-38, in A253, FaDu is more respon-sive to CPT-11 treatment in vivo. Intratumoral drug concentra-tion therefore does not correspond to treatment response inA253. FaDu and A253 have been used as a representative modelof poorly and well-differentiated HNSCC, respectively, in thisstudy. Any conclusion and extrapolation from these two modelsonto others should be taken with adequate caution because ofinherent differences between the two tumor types.

The hypoxia markers CAIX and pimonidazole HCl wereused in our study to correlate and compare the BOLD fMRintensity changes. We carried out BOLD fMR imaging to studythe feasibility of using a noninvasive imaging modality to mon-itor intratumoral vessel functionality and tissue oxygenationstatus during therapy in a preclinical mouse model. The ultimategoal is to evaluate the imaging modality for its role as aprognostic predictor of cancer chemotherapy. Our results indi-cate that resultant BOLD fMR signal intensity changes pin-pointed areas of chronic hypoxia as areas of positive intensitychange (blue-purple range in the color look-up table) in both

Fig. 5 Microphotographs ofautoradiographic slides of A253and FaDu xenografts, 2 hoursafter treatment with intrave-nous 14C-labeled CPT-11. Allmagnifications �1,000. A andB show the same A253 tumordemonstrating that the well-differentiated region (A), in-cluding the poorly differenti-ated tumor cells at its rim(arrow), contains many less sil-ver grains and indicates thatless 14C-labeled CPT-11 ispresent than in the region withpoorly differentiated cells (B).C, as a control, shows fewgrains because of backgroundradiation (noise), whereas Dshows the grain distribution inthe uniformly poorly differenti-ated FaDu.

Table 1 Plasma and tumor tissue levels of CPT-11 and SN-38

Plasma/xenograft CPT-11 lactone * SN-38 lactone * P†

Mouse plasma 10,436.53 2872.25 937.87 212.44 0.029A253 197.31 18.56 3.64 1.05 0.437FaDu 251.83 96.19 0.93 0.24 0.049

NOTE. Data are the means of SD of three tumors per experi-ment, three replicates.

* Expressed as ng/ml for plasma and ng/mg protein for the tumortissue.

† Unpaired two-tailed t test Welch corrected.


A253 and FaDu tumors, and this method shows promise fornoninvasive longitudinal monitoring of tumor hypoxia and vas-cular function. The contributing factors to this positive signalintensity change is not well understood although the release ofdiamagnetic oxygen molecules diffusing to the hypoxic regionscould be one contributing factor. These areas colocalized wellwith immunohistologically identified tumor hypoxic regions,whereas areas with negative intensity change (yellow-red re-gions) colocalized with areas rich in vasculature in the corre-

sponding immunohistochemically stained sections. The ob-served BOLD fMR intensity changes were commensurate withthe number and type (small versus large) of vasculature asvisually confirmed with CD31 immunostaining for the corre-sponding regions (Fig. 6). One has to be cautious about extrap-olating the results to other tumor models, because variations inresponse to both carbogen breathing and the magnetic resonancescan protocols have been reported. The potential limitation ofBOLD fMR imaging of tumor is also attributable to confound-ing factors like vessel vasoreactivity, perfusion, and flow. Sig-nificant efforts have been made recently to elicit effectively thepresence and the extent of tumor hypoxia (35, 36). However, invivo assessment of human tumors has been difficult because ofthe lack of consistent and reproducible methods to measureintratumoral oxygen tension. To this end, several single photonemission computed tomography and positron emission tomog-raphy imaging-based hypoxia agents have been developed andare under evaluation (37). These include iodinated azomycinarabinoside for single photon emission computed tomographyand 60Cu(II)-diacetyl-bis (N4-methylthiosemicarbazone and flu-oromisonidazole for positron emission tomography imaging.However, limitations in spatial resolution and imaging charac-teristics have restricted their clinical utility. BOLD fMR imag-ing techniques do not suffer from these limitations and thereforemay have future clinical implications and relevance.

CAIX and Hypoxyprobe immunohistochemical stainingare accepted as reliable markers for chronic hypoxia. Involucrin,a specific marker of differentiation, is oxygen regulated inhuman squamous cell carcinoma and colocalizes with Hy-

Fig. 6 Correlation of MVD inwell-vascularized, non-hypoxictumor regions with areas ofnegative BOLD fMR signal in-tensity change. Pearson coeffi-cient r � �0.89, r2 � 0.79,P � 0.001.

Table 2 Quantitation of the Distribution of 14C-labeled CPT-11*using image analysis of the autoradiographic slides of A253 and

FaDu xenografts

Microscopic fields of samples

Number ofaveragegrains†/

mm2 SD P ‡

A253 rim region after 1st treatment 22 14A253 poorly differentiated region after

1st treatment103 16 �0.001

A253 rim region after 4th treatment 28 15A253 poorly differentiated region after

4th treatment193 36 �0.001

FaDu after 1st treatment 28 15FaDu after 4th treatment 59 13 �0.08

* 2 hours after intravenous administration.† Average number of grains determined by scoring 4 to 7 individ-

ual microscopic fields and accounting for background radiation (24 16 grains/mm2).

‡ Unpaired two-tailed t test.


poxyprobe for some well-differentiated regions (27). CAIXstaining more accurately reflects tissue hypoxia than doeshypoxia-inducible factor-1 alone, although it depends onhypoxia-inducible factor-1 activity and oxygen level (38).CAIX is regulated by cell density and is triggered by an inter-mediate decrease of oxygen tension (�5% and �1%, mildhypoxia), related to increased oxygen consumption (39). SomeCAIX-stained regions in A253 colocalize with Hypoxyprobe(the indicator for true hypoxia with partial pressure of oxygen �10 mm Hg; refs. 40 and 41), whereas all Hypoxyprobe regionswere positive for CAIX. Expression of CAIX, as in A253, is

associated with a tighter intratumoral cellular arrangement andindicates an ability to withstand a hypoxic tumor microenviron-ment. In contrast, FaDu did not show any similarly stainedregions, except the perinecrotic regions. Terminally differenti-ated cells in HNSCC are not the targets of chemotherapy be-cause they do not proliferate. During treatment, in A253, thereduction in the sensitive poorly differentiated proliferating cellsincreases the proportion of well-differentiated regions in theremaining tumor, which are without microvessels and, thus,hypoxic. Cancer chemotherapy relies directly on the ability ofthe tumor neovasculature to deliver the drug to the tumor. The

Fig. 7 Demonstration of mi-croscopic hypoxic regions byBOLD fMR imaging. Redmarked regions in the T1-weighted magnetic resonanceimages (A) and (B) shows theanatomic tumor locations. Amagnetic resonance referencestandard (5 mmol/L CuSO4) isclearly seen as a hyperintensecircle used for orientation andsignal intensity calibration.BOLD fMR signal intensitychanges after 3 weeks of ther-apy from a small (102 mg, 82mm3) A253 tumor (C) indicatethe absence of blood vessels(purple) for a significant por-tion of the tumor as confirmedby CD31 (�200) results (E).These regions consist mainly ofmultiple well-differentiated tu-mor cell nests. In contrast,BOLD fMR signal intensitychanges after 3 weeks of ther-apy from a small (49 mg, 33mm3) FaDu tumor (D) indicatethe presence of blood vessels(red) for a significant portionof the tumor as confirmed byCD31 (�200) results (F). HPF,high power fields.


nonuniform and apparently chaotic arrangement of tumor neo-vasculature becomes more unfavorable for drug delivery toindividual tumor cells in A253 because of the presence ofavascular islands of well-differentiated cells. The outer rimssurrounding these well-differentiated regions have a low partialpressure of oxygen (Hypoxyprobe positive) because of the ves-sel distribution, and they are hypoxic but contain many prolif-erating Ki67-positive tumor cells (Fig. 4, panel I) that do not getsufficient drug delivery, leading to a tumor regrowth. Somepoorly differentiated cells in these regions are nonproliferatingKi67 negative (i.e., in G0 stage of cell cycle that is less sensitiveto CPT-11). Nonuniform drug distribution has been confirmedby high resolution autoradiography (Fig. 5), demonstrating 5- to7-fold less drug in these “rim” regions (Table 2) compared withthe poorly differentiated, well-vascularized regions in the sametumor (P � 0.0002). Studies with simulated percolation pro-cesses to mimic the situation in vivo have produced a highlyvariable intervascular distance that does not allow free diffusionof oxygen (a freely diffusing molecule). Macromolecular drug,such as CPT-11 and FUra with lower diffusivities than oxygen,have little chance of uniform distribution in such a heterogene-ous intratumoral microenvironment, especially in the rim re-gion. Highly heterogeneous and chaotic networks akin to tumorvasculature have areas of higher resistance to blood flow (42).This is particularly the case for A253, where the differentiatedregions are devoid of blood vessels, with their tighter cellulararchitecture as evidenced by CAIX staining. In these regions thetumor cells are very close to each other with no stroma and nomicrovessels between them. The well-differentiated part of thetumor mimics a histologic structure seen in normal squamouscell epithelium of the oral mucosa and epidermis of the skin, inwhich connective tissue and microvessels are absent. A recentstudy (43) with human basal cell skin carcinoma has confirmedthe absence of intratumoral blood vessels, and may explain whybasal cell skin carcinoma does not metastasize, despite itsinvasive character. Our studies with human surgical samplesof HNSCC confirms the presence of similar avascular well-differentiated histologic regions that can result in lower drugdelivery to these specific areas of the tumors.

Immunohistochemical staining for VEGF in A253 showeda relatively stronger staining in the well-differentiated tumorcells (Fig. 2, panel G, right) than in the poorly differentiatedcells. The increased proportion of hypoxic well-differentiatedregions during treatment provides for higher proangiogenic fac-tors, but the tighter cellular architecture of these regions doesnot facilitate penetration by the endothelial cells and thus pre-cludes blood vessel formation. Instead it can lead to highervessel numbers in the poorly differentiated nests of A253 duringtherapy. After 3 weeks of therapy, the well-vascularized poorlydifferentiated regions in A253 had a significantly (P � 0.004)higher drug delivery when compared with similar regions afterthe 1st week of therapy. Such an effect was not seen in FaDuduring therapy. Therefore, the poorly differentiated regions arenot a problem for tumor cell kill during therapy in the well-differentiated tumor A253.

The well-vascularized poorly differentiated compartmentsof A253 during therapy showed a uniform and 3- to 4-foldhigher uptake of the drug compared with FaDu, as representedby the autoradiographic grain distribution. This is consistent

with the results of the 14C-labeled CPT-11 uptake study. Ourstudies indicate that a changing tumor architecture over thecourse of therapy, because of an increasing proportion of dif-ferentiated areas, is linked with unequal drug distribution andresistance in A253. Because of the low drug delivery, avascular-differentiated regions offer sanctuary to some poorly differen-tiated tumor cells within the surrounding outer rim where thecells escape therapy and regrow. Recent data from our labora-tory (44) with another sequential combination, which usesmethyl seleno cysteine as a chemo modulator with CPT-11, hasyielded the same intratumoral architectural changes in resistantA253 xenografts, indicating the likelihood of this as one of thepossible mechanism for well-differentiated squamous cell car-cinoma tumors and tumor cells evading the effects of anticanceragents. Such tumors are also unlikely to respond favorably toantiangiogenic therapy by themselves. In the same study, use ofCPT-11 at double the maximum-tolerated dose (200 mg/kg/week �4 intravenously) was possible with organic seleniumcompounds (decreasing the toxicity of CPT-11; ref. 45) andimproved cure rate from 60 to 80%. This improvement couldpartially be attributable to a better drug distribution to this rimregion through diffusion because of a better drug availability atthe higher drug concentration used, in turn allowing for a bettertumor cell kill.

ACKNOWLEDGMENTSOur grateful thanks to Dr. Jaromir Pastorek of Institute of Virology,

Slovak Academy of Sciences, Slovak Republic, for providing the pri-mary antibody M75 for CAIX staining. We also acknowledge Mary M.Vaughan for excellent assistance with the immunohistochemical meth-ods and with autoradiography.

REFERENCES1. Grenlee RT, Hill-Harmon MB, Murray T, et al. Cancer statistics,2001. CA Cancer J Clin 2001;51:15–36.

2. Murphy BA. New drug therapy for squamous carcinoma of the headand neck. Curr Opin Oncol 1996;8:221–26.

3. Lavelle F, Bissery MC, Andre S, Roquet F, Riou JF. Preclinicalevaluation of CPT- 11 and its active metabolite SN-38. Semin Oncol1996;23:11–20.

4. Rivory LP, Bowles MR, Robert J, Pond SM. Conversion of irinote-can (CPT-11) to its active metabolite, 7-ethyl-10-hydroxycamptothecin(SN-38), by human liver carboxylesterase. Biochem Pharmacol 1996;52:1103–11.

5. Xu G, Zhang W, Ma MK, Mcleod HL. Human carboxylesterase 2 iscommonly expressed in tumor tissue and is correlated with activation ofirinotecan. Clin Cancer Res 2002;8:2605–11.

6. Mathijssen RHJ, Van Alphen RJ, Verweij J, et al. Clinical pharma-cokinetics and metabolism of Irinotecan (CPT-11). Clin Cancer Res2001;7:2182–94.

7. Cao S, Rustum YM. Synergistic antitumor activity of irinotecan incombination with 5-fluorouracil in rats bearing advanced colorectalcancer: role of drug sequence and dose. Cancer Res 2000;60:3717–21.8. Azrak RG, Cao S, Slocum HK, et al. Therapeutic synergy betweenirinotecan and 5-fluorouracil against xenografts bearing human tumors.Clin Cancer Res 2004;10:1121–29.9. Dvorak HF, Nagy JA, Feng D, et al. Vascular permeability factor/vascular endothelial growth factor and the significance of microvascularhyperpermeability in angiogenesis. Curr Top Microbiol Immunol 1999;237:97–132.


10. Benjamin LE, Golijanin D, Itin A, et al. Selective ablation ofimmature blood vessels in established human tumors follows vascularendothelial growth factor withdrawal. J Clin Investig 1998;103:159–65.

11. Dewhirst M. Angiogenesis and blood flow in solid tumors. InTeicher B, editor. Drug Resistance in Oncology. New York: MarcelDekker; 1993. p. 3–24.

12. Braun R, Lanzen J, Dewhirst M. Fourier analysis of fluctuations ofoxygen tension and blood flow in R 3230Ac tumors and muscle of rats.Am J Physiol 1999;277:H551–68.

13. Kaluz S, Kaluzova M, Stanbridge EJ. Expression of the hypoxiamarker carbonic anhydrase IX is critically dependent on SP1 activity.Identification of a novel type of hypoxia-responsive enhancer. CancerRes 2003;63:917–22.

14. Loncaster JA, Harris AL, Davidson SE, et al. Caronic anhydrase(CAIX) expression, a potential new intrinsic marker of hypoxia: corre-lations with tumor oxygen measurements and prognosis in locally ad-vanced carcinoma of the cervix. Cancer Res 2001;61:6394–99.

15. Ivanov S, Liao SY, Ivanova A, et al. Expression of hypoxia-inducible cell-surface transmembrane carbonic anhydrases in humancancer. Am J Pathol 2001;158:905–19.

16. Hockel M, Vaupel P. Tumor hypoxia: definitions and current clin-ical, biologic, and molecular aspects. J Natl Cancer Inst (Bethesda)2001;93:266–76.

17. Koukourakis MI, Giatromanolaki A, Sivridis E, et al. Hypoxia-regulated carbonic anhydrase-9 (CA9) relates to poor vascularizationand resistance of squamous cell head and neck cancer to chemoradio-therapy. Clin Cancer Res 2001;7:3399–403.

18. Abramovitch R, Frenkiel D, Neeman M. Analysis of subcutaneousangiogenesis by gradient echo magnetic resonance imaging. Magn Re-son Med 1998;39:813–24.

19. Griffiths JR, Taylor NJ, Howe FA, et al. The response of humantumors to carbogen breathing monitored by gradient-recalled echomagnetic resonance imaging. Int J Radiat Oncol Biol Phys 1997;39:697–701.

20. Baudelet C, Gallez B. How does blood oxygen level-dependent(BOLD) contrast correlate with oxygen partial pressure (pO2) insidetumors? Magn Reson Med 2002;48:980–6.

21. Al-Hallaq HA, River JN, Zamora M, Oikawa H, Karczmar GS.Correlation of magnetic resonance and oxygen microelectrode measure-ments of carbogen-induced changes in tumor oxygenation. Int J RadiatOncol Biol Phys 1998;41:151–9.

22. Neeman M. Preclinical MRI experience in imaging angiogenesis.Cancer Metastasis Rev 2000;19:39–43.

23. Bernsen HJJA, Rijken PFJW, Oostendorp T, and Van der kogel AJ.Vascularity and perfusion of human gliomas xenografted in the athymicnude mouse. Br J Cancer 1995;71:721–6.

24. Boxerman GL, Hamberg LM, Rosen BR, Weisskoff RM. MRcontrast due to intravascular magnetic susceptibility perturbations.Magn Reson Med 1995;34:555–66.

25. Lee S-P, Cilva AC, Kim S-G. Comparison of diffusion-weightedhigh-resolution CBF and spin-echo BOLD fMRI at 9.4T. Magn ResonMed 2002;47:736–41.

26. Silvennoinen MJ, Clingman CS, Golay X, Kauppinen RA, van ZijlPCM. Comparison of the dependence of blood R2 and R2* on oxygensaturation at 1.5 and 4.7 Tesla. MRM, 2003, 49:47–60.

27. Chou S-C, Azuma Y, Varia MA, Raleigh JA. Evidence that involu-crin, a marker for differentiation, is oxygen regulated in human squa-mous cell carcinomas. Br J Cancer 2004;90:728–35.28. Bradford MM. A Rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dyebinding. Anal Biochem 1976;72:248–54.29. Warner DL, Burke TG. Simple and versatile high-performanceliquid chromatography method for the simultaneous quantitation of thelactone and carboxylate forms of camptothecin anticancer drugs. J Chro-matogr B Biomed Sci Appl 1997;691:161–71.30. Iyer L, Ratain MJ. Clinical Pharmacology of Camptothecins. Can-cer Chemother Pharmacol 1998;42:s31–43.31. Stewart CF, Zamboni WC, Crom WR, Houghton PJ. Disposition ofirinotecan and SN-38 following oral and intravenous irinotecan dosingin mice. Cancer Chemother Pharmacol 1997;40:259–65.32. Rogers A. Techniques of autoradiography, 3rd edition. New York:Elsevier/North-Holland Biomedical Press; 1979.33. Hennig J, Nauerth A, Friedburg H. RARE Imaging: a fast imagingmethod for clinical MRI. Magn Reson Med 1986;3:823–33.34. Hapke G, Yin M-B, Wu J, Frank C, Rustum YM. Phosphorylationof chk1 at serine-345 affected by topoisomerase I poison SN-38. Int JOncol 2002;21:1059–66.35. Brizel DM, Sibley GS, Prosnitz LR, Scher RL, Dewhirst MW.Tumor hypoxia adversely affects the prognosis of carcinoma of the headand neck. Int J Radiat Oncol Biol Phys 1997;38:285–89.36. Brizel DM, Dodge RK, Clough RW, Dewhirst MW. Oxygenationof head and neck cancer: changes during radiotherapy and impact ontreatment outcome. Radiother Oncol 1999;53:113–17.37. Chao KSC, Bosch WR, Mutic S, et al. A novel approach to overcomehypoxic tumor resistance: Cu- ATSM-guided intensity-modulated radi-ation therapy. Int J Radiat Oncol Biol Phys 2001;49:1171–82.38. Brown JM, Le Q-T. Tumor hypoxia is important in radiotherapy,but how should we measure it? Int J Radiat Oncol Biol Phys 2002;54:1299–301.39. Kaluz S, Kaluzova M, Chrastina A, et al. Lowered oxygen tensioninduces expression of the hypoxia marker MN/carbonic anhydrase IX inthe absence of hypoxia-inducible factor 1alpha stabilization: a role forphosphatidylinositol 3�-kinase. Cancer Res 2002;62:4469–77.40. Arteel GE, Thurman RG, Yates JM, Raleigh JA. Evidence thathypoxia markers detect oxygen gradients in liver: pimonidazole andretrograde perfusion of rat liver. Br J Cancer 1995;72:889–95.41. Arteel GE, Thurman RG, Raleigh JA. Reductive metabolism of thehypoxia marker pimonidazole is regulated by oxygen tension independentof the pyridine nucleotide redox state. Eur J Biochem 1998;25:743–50.42. Baish JW, Jain RK. Fractals and cancer. Cancer Res 2000;60:3683–88.43. Chin CWS, Foss AJE, Stevens A, Lowe J. Differences in thevascular patterns of basal and squamous cell skin carcinomas explaintheir differences in clinical behaviour. J Pathol 2003;200:308–13.44. Cao S, Durrani FA, Rustum YM. Selective modulation of thetherapeutic efficacy of anticancer drugs by selenium containing com-pounds against human tumor xenografts. Clin Cancer Res 2004;10:2561–69.45. Rustum YM, Cao S, Durrani FA, Fakih M. Se-(methyl) selenocys-teine (MSC) and seleno-L-methionine (SLM) potentiates the antitumoractivity of irinotecan against human tumor xenografts and protectsagainst drug induced toxicity (abstract 2068). Proc Am Soc Clin Oncol2004;23:144.


lack of microvessels in well-differentiated regions of

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