Vol. 8, 221 232, January 2002
`
`Clinical Cancer Research 221
`
`Differences in Therapeutic Indexes of Combination Metronomic
`
`Chemotherapy and an Anti-VEGFR—Z Antibody in Multidrug-
`resistant Human Breast Cancer Xenografts1
`
`Giannoula Klement, Ping Huang, Barbara Mayer,
`Shane K. Green, Sban Man, Peter Bohlen,
`Daniel Hicklin, and Robert s. Kerbelz
`Sunnybrook and Women’s College Health Sciences Centre, Molecular
`and Cellular Biology, Toronto, Ontario, M4N 3 M5 Canada [G. K.,
`P. H., B. M., S. K. G., S. M., R. S. K.], and ImClone Systems, Inc.,
`New York, New York 10014 [P. B., D. H.] and Department of
`Medical Biophysics, University of Toronto [R. S. K.]
`
`ABSTRACT
`
`One of the greatest barriers to the treatment of cancer
`with chemotherapeutic drugs is acquisition of drug resist-
`ance. This includes multidrug resistance mediated by P-
`glycoprotein (ng) to multiple lipophilic natural compounds
`such as taxanes, doxorubicin (Adriamycin), and vinblastine.
`The considerable efforts made thus far to reverse this and
`
`other types of drug resistance have had very limited success.
`We report here that a variety of orthotopic human breast
`cancer xenografts selected for high levels of ng and multi-
`drug resistance respond in a significant and durable manner
`to different continuous low-dose (e.g., one-tenth the maxi-
`mum tolerated dose of chemotherapy) chemotherapy regi-
`mens, when used in combination with an antivascular endo-.
`thelial cell growth factor (anti-VEGF) receptor-2 (ilk-1)-
`neutralizing
`antibody
`(DC101). The ng substrates
`paclitaxel (Taxol), Adriamycin, and vinblastine were all ef-
`fective using this type of combination treatment, although
`the chemotherapy protocols showed little or no effect as
`monotherapies. Similar results were also obtained using
`cisplatinum (a non-ng substrate drug) against cisplatinum-
`resistant tumors. Evidence of significant tumor cell death by
`the combination treatment was detected within 3 weeks of
`
`in the
`initiation of therapy by histopathological analysis,
`absence of shrinkage of tumor mass. There were, however,
`marked differences in the cumulative toxicity of long-term
`
`Received 7/26/01; revised 9/18/01; accepted 9/18/01.
`The costs of publication of this article were defrayed in part by the
`payment of page charges. This article must therefore be hereby marked
`advertisement in accordance with 18 U.S.C. Section 1734 solely to
`indicate this fact.
`' Supported by grants to R. S. K. by the National Institutes of Health
`(Grant CA-41233) and the Canadian Institutes of Health Research, and
`by contract fimds from ImClone Systems, New York. G. K. was funded
`for most of this work by the Terry Fox Fellowship of the National
`Cancer Institute of Canada.
`2 To whom requests for reprints should be addressed, at Molecular &
`Cellular Biology Research, S-218 Research Building, Sunnybrook and
`Women’s College Health Sciences Centre, 2075 Bayview Avenue,
`Toronto, Ontario, Canada M4N 3 M5. Phone: (416) 480-5711; Fax:
`(416) 480-5703; E-mail: robert.kerbel@swchsc.on.ca.
`
`regimens of Adriamycin and cisplatinum, where toxicity
`was observed, when compared with the tubulin inhibitors,
`vinblastine and Taxol, where it was not. We conclude that
`vascular-targeting protocols involving frequent adminis-
`tration of very low doses of certain chemotherapeutic
`drugs can provide a stable and safe way to circumvent
`multidrug resistance in established orthotopically grow-
`ing tumors, as long as these are used in combination with
`a second antiangiogenic drug, in this case, anti-VEGFR—Z
`blocking antibodies.
`
`INTRODUCTION
`
`One of the major factors that account for the limited
`advances made in cancer treatment is acquired drug resistance.
`Cancers that respond to chemotherapeutic drugs such as ovarian,
`breast, colon, and non-small cell lung cancer, or to antihormonal
`therapies. e.g., breast and prostate cancer, almost always relapse
`in a resistant form some time later. Information is also emerging
`that even the newest generation of anticancer drugs such as the
`signal
`transduction inhibitors (e.g.,
`the bcr-abl antagonist.
`ST1571) may be compromised by acquired drug resistance (1—
`3). The numerous and diverse genetic instabilities of cancer cells
`are thought to be a major factor in explaining the propensity of
`cancer cells to give rise to such drug-resistant mutant or variant
`subpopulations (4, 5).
`Various strategies to circumvent or reverse acquired resist-
`ance to chemotherapeutic drugs have had little clinically signif-
`icant success thus far, with respect to the treatment of the
`common adult solid malignancies. Examples of such strategies
`include the use of combination chemotherapy, multimodality
`therapies, and the use of drugs such as ng3 antagonists to block
`the function of this particular mediator of multidrug resistance
`to natural
`lipophilic compounds (6). A new and seemingly
`counterintuitive preclinical strategy to combat drug resistance in
`cancer was developed recently (7). which exploits “chemother-
`apeutics as antiangiogenic agents,” i.e.,
`the property of such
`drugs to damage or kill the genetically stable, host endothelial
`cells of a tumor’s newly, formed neovasculaturc (8, 9). It in-
`volves the use of various chemotherapeutic drugs, e.g., cyclo-
`phosphamide (8) or vinblastine (9), given frequently and in a
`chronic manner (with no significant rest periods), at doses lower
`than the MTD. The administration of chemotherapeutic drugs in
`
`3 The abbreviations used are: ng, P-glycoprotein; MTD, maximum
`tolerated dose of chemotherapy; EC, endothelial cell; VEGF, vascular
`endothelial cell growth factor; VEGFR-Z, type 2 receptor for vascular
`endothelial cell growth factor; SCID, severe combined immunodefi-
`cient; HUVEC, human umbilical vein endothelial cell; MDR, multidrug
`resistance.
`Genentech 2083
`
`Hospira v. Genentech
`|PR2017—00737
`
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`Genentech 2083
`Hospira v. Genentech
`IPR2017-00737
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`

`222 Chemotherapy and Anti-VEGFR-Z Antibody in Breast Cancer
`
`this manner has been termed “antiangiogenic” or “metronomic”
`chemotherapy (8, 10).
`A rationale for this type of therapeutic approach is that the
`dividing ECs of newly forming tumor vessels (1]) should be
`sensitive to chemotherapeutic drugs, similar to other types of
`normal dividing cells such as hair follicle, bone marrow, or gut
`mucosa] cells (4). However, because such cells lack the genetic
`instabilities of tumor cells, their ability to mutate and acquire
`resistance properties would be expected to be much more lim-
`ited (4, 5, 12). Indeed, Browder et a1.
`(8) have shown that
`chemotherapeutic drugs given at the MTD can cause EC apo-
`ptosis of tumor-associated vessels in ectopically growing mouse
`tumors, but this damage can be repaired rapidly during the
`prolonged recovery periods necessary for myeloid recovery
`following MTD chemotherapy. Hence, by giving chemotherapy
`more frequently, e.g., weekly or twice weekly, the EC repair
`process can be compromised and the potential antiangiogenic
`effects of chemotherapy enhanced (8).
`These preclinical results may provide an explanation of the
`clinical cases in which patients not responsive to standard MTD
`chemotherapy respond to the same drug at a lower dose, but
`administered more frequently (13—15). In the past such sched-
`ules were used primarily for palliation, because of the less
`severe side effects (14, 15). The availability of oral chemother-
`apeutic drugs (16) makes chemotherapy administered in this
`manner a more practical possibility.
`The long-term antitumor efficacy of antiangiogenic/metro-
`nomic chemotherapy protocols in ectopic syngeneic mouse or
`human tumor xenograft models can be increased, sometimes
`substantially so, by combination with a second,,antiangiogenic
`drug such as D(chloroacetyl-carbamoyl) Fumagillo]
`(8) or
`blocking monoclonal antibodies to VEGFR-2 (9). The rationale
`for this combination is that anti-VEGFR-Z- or anti-VEGF-
`
`targeting drugs (17). agents capable of specific blockade of
`activated EC cell survival mechanisms (18), will selectively
`enhance the damaging or cytotoxic effects of continuous low-
`dose chemotherapy on newly formed blood vessels (9).
`The purpose of the present paper was to address several
`important questions that the previous preclinical studies on
`antiangiogenic/metronomic chemotherapy have raised. First,
`do different classes of chemotherapeutic agents have similar
`potentials in terms of both efficacy and toxicity, when used
`alone or in combination with a second drug such as anti-
`VEGFR-2 antibodies? Would this type of therapeutic ap-
`proach work as well on orthotopically transplanted tumors?
`Finally. would the approach have efficacy on multidrug-
`resistant tumors with high levels of resistance attributable to
`mechanisms such as overexpression of ng? Our results show
`that, indeed, various low-dose antiangiogenic chemotherapy
`regimens are highly effective against orthotopically grown
`multidrug-resistant human breast cancers in SCID mice, but
`, usually only when the drugs are used in combination with the
`antiangiogenic, VEGFR-2-inhibiting antibody. Furthermore,
`at
`least in our study, some drugs [for example, paclitaxel
`(Taxol) and vinblastine] exhibit better therapeutic profiles
`than others
`[cisplatinum or doxorubicin (Adriamycin)]
`mainly because of their lack of cumulative toxicity.
`
`MATERIALS AND METHODS
`Cells and Culture Conditions. Two human breast can-
`
`cer cell lines, MDA-MB—231 and MDA-MB-435, and a number
`of multidrug-resistant, ng-positive variants selected from these
`lines were used in the studies: MVB9, MD22, MPAHS, and
`T01. MVB9 was selected for resistance in vitro to vinblastine
`
`sulfate and maintained in 5 ng/ml vinblastine in culture, MD22
`was selected for resistance to doxorubicin and maintained in 12
`
`mg of doxorubicin/ng, and MPAHS was isolated by transfection
`of MDA-MB-231with mdr—I gene and maintained in 10 ng/ml
`vincristine sulfate and 400 ug of G418. All three were originally
`obtained from Dr. Jeff Lemontt (Genzyme Corporation, Fram-
`ingham, MA). The derivation of these lines is similar to that
`described by Lemontt et al. (19), and expressed stable and high
`levels of cross-resistance to drugs such as colchicine, vinblas-
`tine, vincristine, and Adriamycin“. T0,], a ng-positive multi-
`drug—resistant variant of MDA-MB-435 breast carcinoma, was
`obtained from Dr. Dalia Cohen (Novartis, East Hanover, NJ)
`and was derived by progressive serial in vitro exposure of the
`cell line to increasing concentrations of Taxol; these cells ex-
`press 100-fold resistance to Taxol relative to the parental line
`(20). Finally. a cisplatinum-resistant variant called MDA-
`CDDP—S4 was selected from MDA-MB-231 by serial in viva
`drug exposure, in our laboratory. This variant does not express
`ng. To select this variant, 4 X 106 MDA-MB-23l cells were
`implanted into the mammary fat pads of 8-week-old female
`athymic nude mice, tumors were grown to ~150 mm3, and the
`mice were treated with 5 mg/kg cisplatin every second day three
`times. The tumors were then removed, adapted to culture, and
`grown in vitro for 3 weeks, reimplanted into the mammary fat
`pad of a new group of athymic nude mice, and treated in the
`same manner; the selection process was repeated three more
`times.
`
`lines were expanded as monolayer cultures by
`All cell
`serial passage on tissue culture plates (Nalge Nunc Inter-
`national, Naperville, IL) in DMEM, 5% fetal bovine serum
`(Invitrogen, Carlsbad, CA) with the addition of 22 nM (12
`ng/ml) Adriamycin (Pharmacia Upjohn, Mississauga, Canada)
`for MD22, 12.2 nM (10 ng/ml) vincristine sulfate (Sigma-
`Aldrich Chemical C0,. Canada, Oakville, Ontario, Canada) for
`MPAHS, 6 W (5 ng/ml) vinblastine sulfate (Calbiochem, La
`Jolla, Ca) for MVB9, and 0.1 |.LM Taxol (Abbott Laboratories,
`North Chicago, IL) for TO.1. HUVECs (Clonetics, San Diego,
`CA) were expanded on 1% gelatin-coated tissue culture plates in
`MCDB131 culture medium (JRH Biosciences, Lenexa, KS)
`supplemented with 5 ng/ml bFGF (R & D Systems, Minneap-
`olis, MN), 10 units/ml heparin (Wyeth-Ayerst Laboratories,
`Philadelphia, PA), 10 ng/ml epidermal growth factor (Upstate
`Biotechnology, Lake Placid, NY), and 10% fetal bovine serum.
`In Vitro Determination of Drug Sensitivity. Analysis
`of in vitro drug sensitivity was assessed on cells grown in
`monolayer as well as three-dimensional multicellular spheroids.
`For monolayer analysis, 3,000 cells in 200 H1 of growth medium
`were plated per well
`in 96-well flat-bottomed tissue culture
`plates (Nunc) and incubated at 37°C, 5% CO2 for 24 h prior to
`
`4 J. Lemontt, personal communication.
`
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`Clinical Cancer Research 223
`
`Table 1 Differences in sensitivity of cancer cell lines and l-lUVECs to chemotherapeutic agents
`
`IC50 (nM)
`
`Monolayer
`
`Spheroid
`
`
`Cell population tested
`Agent
`HUVECs
`Vinblastine
`HUVECs
`Adriamycin
`HUVECs
`Cisplatinum
`HUVECs
`Taxol
`MDA-MB-231 parental tumor
`Taxol
`MD22 variant”
`Adriamycin
`MPAHS variant”
`Vinblastine
`MVB9 variant”
`Taxol
`CDDP-S4 variant”
`Cisplatinum
`
`at 24 h
`0.55
`1.65
`4
`0.4
`2
`79.4
`15.7
`27.4
`2581
`
`at 72 h
`at 24 h
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`848
`3,434
`260
`1,610
`>123,301
`1,349
`1,059
`10,084
`
`27,773 2,3 80
`" All of the variants are drug-resistant sublines of MDA-MB-231 human breast carcinoma cell line.
`
`at 72 h
`N/A
`N/A
`N/A
`N/A
`4.5
`41.5
`7.1
`19
`2083
`
`initiation of treatment. For analysis in three-dimensional culture.
`multicellular tumor spheroids were formed using the liquid overlay
`technique, as described previously (21). Ninety-six-wel] round-
`bottomed tissue culture plates (Nunc) were .covered with 2% Poly-
`Hema in 100% ethanol (Aldrich Chemical C0,, Milwaukee, WI) to
`prevent attachment to the tumor cells to the plastic of the dish. The
`formation of spheroids was initiated by a gentle 10-min spin (1,000
`rpm/min) of the freshly detached cells in suspension, followed by
`24—48 h incubation at 37°C, 5% C02. To assess for the inhibitory
`activity of chemotherapeutic agents, tumor cells, grown either as
`spheroids or as a monolayer, were exposed to 0—12 uM (0—10,000
`ng/ml) of either vinblastine sulfate (Calbiochem, San Diego, CA),
`Adriamycin (Pharmacia & Upjohn Inc. Mississauga, Ontario,
`Canada), cisplatinum (Faulding Canada Inc., Vaudreuil, Que—
`bec, Canada), or Taxol (Abbot) for 24, 48, and 72 h. The cells
`were then pulsed with 2 uCi/well of [methyl-3H]thymidine
`(Amersham Life Science, Buckinghamshire, United Kingdom),
`incubated at 37°C for 6 h to allow for incorporation of [3H]
`thymidine into their DNA, frozen, and thawed; then. the DNA
`was harvested onto a filterrnat using a Titertek cell harvester.
`Radioactivity was measured on a Wallac 1205 BetaPlate scin-
`tillation counter (Wallac Oy, Turku, Finland), and proliferation
`was expressed as absolute counts of [3H]thymidine per minute
`or as percentage of untreated control. Each dose concentration
`was done in sextuplicate and repeated twice. Because the most
`significant effect was observed at 24 h for the monolayer culture
`and at 72 h for the Spheroid culture, only these points are
`presented in Table l and Fig. 1.
`In Vitro Confirmation of Drug Resistance. MDA-MB-
`231 and its drug-resistant variants, grown and expanded in
`DMEM with 5% FCS, were washed with PBS twice and incu-
`bated in fresh medium with 5 uM rhodamine chloride (Sigma-
`Aldrich Canada) with or without either 5 uM cyclosporin
`A (Novartis Pharmaceuticals, Canada Inc., Dorval, Quebec,
`Canada) or 30 uM verapamil hydrochloride (Sigma-Aldrich,
`Canada) for 30 min at 37°C. Cells were then trypsinized at room
`temperature, washed, and resuspended in PBS and analyzed for
`intracellular fluorescence by flow cytometry (excitation 488 nm,
`emission 550 nm) with an EPICS Elite V flow cytometer
`(Coulter Electronics, Ltd., Luton. United Kingdom).
`Effect of Antitubulin Agents in Combination with Anti-
`VEGFR—Z Antibody on HUVECs. Passage two of HUVECs
`was seeded on 1% gelatin-coated sterile microscope slides and
`
`allowed to grow to approximately 80% confluence before stain-
`ing. HUVECs treated with medium containing 0.5 or 1 ng/ml
`vinblastine, 25 ug/ml 1C1] (monoclonal antibody against the
`VEGFR-Z/KDR; Ref. 22), or the combination of the two for 4 h
`were stained for B-tubulin as follows. After a thorough wash
`with PBS, the monolayer was covered with ice-cold methanol
`for 10 min at —20°C, rinsed with ice cold acetone twice for 10 s,
`washed twice with PBS and rehydrated in fresh PBS for at least
`30 min prior to labeling with antibody. The anti-B-tubulin/Cy-3
`conjugate (Sigma-Aldrich Chemical Co., St. Louis, MO) was
`then diluted to 1:100 in PBS, 1% BSA and added to the slides
`for 60 min at room temperature. After rinsing the excess anti-
`body off with three 5-min washes with PBS, the tissues were
`mounted, eoverslipped, and evaluated using a Zeiss confocal
`microscope. The same procedure was done using Taxol at 0.6
`nM (data not shown).
`In Vivo Tumor Growth Assessment. Each of the cell
`
`lines was harvested on the day of injection using 1% trypsin-
`EDTA (Invitrogen), and a single-cell suspension of 2 X 106
`cells in 0.05 ml of serum-free growth medium was injected into
`the mammary fat pad of 4—6-week-old CB-17 SCID mice
`(Charles River, St.-Constant, Quebec, Canada). Approximately
`3 weeks later, when most of the tumors had grown to 300 mm3,
`the mice were randomized into groups of five animals. Two
`independent experiments were done for each xenograft, each
`totaling 30 animals in six groups. The treatment groups were as
`follows: group I (Control): 0.2 m1 of PBS (DC101 vehicle) i.p.
`every 3 days and 0.2 m1 injectable saline (vinblastine vehicle)
`i.p. every 3 days; group II: 0.4 ml of 2 mg/ml DC101 antibody
`(800 [Lg/11101156) i.p. every 3 days and 0.2 m1 of injectable saline
`i.p. every 3 days: group III: 0.5 mg/kg (1.5 mg/mz) vinblastine
`sulfate i.p. every 3 days in the case of MPAHS and MVB9,
`1
`mg/kg (3 mg/mz) Adriamycin i.p. every 3 days in the case of
`MD22.
`1 or 2 mg/kg (3 and 6 mg/mz) cisplatinum i.p. every 3
`days in the case of CDDP-S4, and 0.4 ml of PBS i.p. every 3
`days; group IV (ng inhibitor control): cyclosporin A, 10 mg/kg,
`i.p. every 3 days in the case of MPAHS, MDA-MB-231, and
`MD22, or verapamil, 20 mg/kg,
`i.p. every 3 days in case of
`MVB9; group V: chemotherapy as in group 11 combined with
`ng inhibitor in group IV; group VI: chemotherapy as in group
`11 combined with DC101. Body weight and tumor size were
`assessed weekly, and general clinical status of the animals was
`assessed every day. Perpendicular tumor diameters were meas-
`
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`

`224 Chemotherapy and Anti-VEGFR-Z Antibody in Breast Cancer
`
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`
`Adfinmyc'n [nM]
`
`CDDP-S4
`
`MVB9
`..
`
`I
`
`I
`
`120—
`110
`100
`
`= w
`‘s’
`so
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`60
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`
`Fig. I Chemotherapy sensitivity comparison for HUVECs, MVB9 (vinblastine-resistant variant), MD22 (Adriamycin-resistant variant), and
`CDDP-S4 (cisplatinum-resistant variant). HUVECs were grown in monolayer, whereas cancer cells were grown as both spheroids (blue bars) and
`monolayer (red bars). Comparison of the effect of different chemotherapeutic agents on the proliferative capacity of each was made. In HUVECs
`Taxol and vinblastine were inhibitory at the lowest dose concentrations, followed by Adriamycin and cisplatinum. Tumor cell lines were consistently
`more resistant in spheroid culture, but even in monolayer the drug concentrations necessary for inhibition of proliferation were much higher than those
`for endothelial cells. The results represent two independent experiments, done in sextuplicate on two different experimental days (mean : S.E.).
`
`ured using a vemier scale caliper and tumor volume estimated
`using the formula for ellipsoid: (width2 X length)/2. Growth
`curves were analyzed statistically using repeated measures
`ANOVA.
`
`For histological comparison, a separate group of animals
`was treated and sacrificed at 3 weeks of therapy, at which time
`tumors were excised and fixed in 10% (v/v) formalin or cryo-
`preserved in Tissue-Tek O.C.T. compound (Bayer Corp.,
`Elkhart, IN) until processed for histochemical analysis. Animal
`care was in accordance with institutional guidelines.
`Formalin—
`Immunohistochemistry of Tumor Tissues.
`fixed paraffin-embedded sections were cut to S-um sections and
`stained with H&E according to standard protocols.
`
`RESULTS
`
`In Vitro Drug Sensitivity Determination. Prior to un-
`dertaking any in vivo experiments, drug levels at which signif-
`icant toxicity against endothelial, but not tumor cells, might be
`
`achieved were established. A difference in relative chemother-
`
`apeutic drug sensitivities between human neuroblastoma cells
`and activated endothelial cells has been reported previously for
`vinblastine (9), and we asked whether similar differential sen-
`sitivities could be detected with other chemotherapeutic drugs
`and human breast cancer cells. To address this question, we
`subjected monolayer and spheroid cultures to increasing dose
`concentrations of one of four different drugs: Adriamycin, vin-
`blastine, cisplatinum, or Taxol. To optimize growth conditions
`and achieve comparable baseline growth levels in tumor cells
`and HUVECs, tumor cell lines were grown in DMEM with 10%
`bovine serum, but HUVECs were grown on gelatinized plates,
`and in the presence of growth factors. The untreated controls
`showed similar levels of [3H]thymidine incorporation for all
`three cell lines at the baseline drug level, eliminating the con-
`cern that the differences in proliferation may be attributable to
`inherent factors (Fig. l). The chemotherapeutic drug sensitivi-
`ties were found to be remarkably different between HUVECs
`
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`Clinical Cancer Research 225
`
`and the tumor cells tested. Whereas the proliferation of HU-
`VECs was inhibited by dose concentrations <0.5 nM (0.45
`ng/ml) vinblastine, <0.4 nM (0.33 ng/ml) Taxol, <1.7 nM (0.9
`ng/ml) Adriamycin, and <4 nM (1.2 ng/ml) cisplatinum, there
`was no significant growth inhibition of the parental (“drug-
`sensitive”) mammary carcinoma cell line MDA-MB-23l until
`dose concentrations greater than 3000 nM (3,000 ng/ml) were
`reached. This was further enhanced by culturing cancer cells as
`three-dimensional spheroids, which mirrored the tumor growth
`in vivo. The chemotherapeutic resistance of cancer cells grown
`in three-dimensional spheroid-like structures is,
`interestingly,
`further enhanced and enabled the cancer cell to withstand con-
`
`agent 800—>l0,000-fold
`centrations of chemotherapeutic
`higher than those of ECs. In fact, all of the selected drug-
`resistant cell lines were resistant to a degree that would preclude
`clinical relevance, because these dose concentrations are un-
`achievable in patients. For example,
`the growth rate of the
`MDA-MB-23 l-derived, cisplatinum-resistant variant CDDP—S4
`was not affected until dose concentrations of 2,600 nM (770
`ng/ml) were reached in monolayer, and 27,773 nM (8,330 ng/ml)
`in spheroid culture. This in vitro “screen” suggests that actively
`proliferating endothelial cells may be sensitive to a much lower
`range of dose concentrations of chemotherapeutic agents than
`actively proliferating tumor cells. If so,
`there is at
`least a
`theoretical possibility of substantially lowering the present clin-
`ically used MTDs of chemotherapeutic drugs to specifically
`target dividing endothelial cells present in tumors.
`The Effect of Tubulin Inhibitors and Anti-VEGFR-2
`
`Antibodies on HUVECs Grown in Monolayer Culture. De-
`spite the limitations of in vitro culture as a model, including the
`use of (large vein) HUVECS rather than microvascular endothe-
`lial cells, it provides an approach for direct observation of the
`effects of tubulin inhibitors, a monoclonal antibody against the -
`VEGFR-Z, or a combination of the two on endothelial cells. We
`observed no appreciable effect of IMC-lCll, a monoclonal
`neutralizing antibody, against the human VEGFR-2/KDR recep-
`tor, when used alone (Fig. 2). However,
`in combination with
`low-dose concentration vinblastine,
`the effects were striking.
`For example, in combination with 0.5 nM (0.5 ng/ml) vinblas-
`tine, IMC-lCll caused retraction of the cellular membrane and
`full coagulation of the cytoskeleton (Fig. 2). Interestingly, a
`potent effect was observed with the lower dose concentration,
`and doubling the dose concentration to 1 ng/ml provided no
`additional benefits (Fig. 2). This suggests that, at least in com-
`bination with a specific inhibitor of EC survival and other
`functions, lower doses of tubulin inhibitors may produce ade-
`quate anti-endothelial effects.
`Drug Resistance Testing. Before proceeding to study
`the efficacy of low-dose continuous chemotherapy in vivo
`against multidrug-resistant tumors, we verified the functional
`presence of the ng membrane pump. Using rhodamine chloride
`(a ng substrate that is easily detected by flow cytometry), we
`found a clear shift of the fluorescent population to the left (Fig.
`3), confirming the presence of ng-mediated efflux of rhoda-
`mine chloride from the MVB9, MPAHS, MD22, and T01 cells,
`but not, as expected, from the ng-negative parental cell line,
`MDA-MB-231, or the cisplatinum-resistant CDDP—S4 variant.
`As expected, we also found that this drug efflux is reliably
`
`in vitro, by ng reversal agents such as
`least
`inhibited, at
`cyclosporin A or verapamil (data not shown).
`In Vivo Tumor Growth Assessment. Building upon the
`striking in vitro differences in relative chemotherapeutic drug
`sensitivities between the tumor and endothelial cells tested, we
`evaluated the effects of a continuous low-dose regimen of each
`of these agents in inhibiting growth of orthotopic tumor xe-
`nografts in vivo. The cell lines were intentionally selected for
`their high levels of acquired resistance, e.g., up to lOO-fold, to
`negate the possibility that any antitumor effects were due to
`direct antitumor cell activity.
`The first group,
`treated with the neutralizing antibody
`directed against mouse VEGFR-Z/flk-l (DC101) shown previ-
`ously to inhibit the s.c. growth of many different kinds of human
`xenografi in immune-deficient mice (23), displayed the ex-
`pected effectiveness in inhibiting tumor growth (Fig. 4, green
`line, filled circles in all panels). Over the first weeks of therapy
`the effectiveness of DC101 was comparable with that of the
`combination treatment group, such that if this experiment had
`been terminated at 30—60 days, erroneous conclusions might
`have been drawn regarding the apparent equal efficacy of the
`two treatment groups. In most of the long-term experiments, the
`tumor volume curves diverge beyond 60—70'days and the ben-
`efit of adding the chemotherapeutic agent becomes apparent.
`Further confirmation of this delayed divergence effect was seen
`upon analysis of histological specimens at 3 weeks (see below
`and Fig. 5).
`The findings in the second treatment group (chemothera-
`peutic agent alone) showed minimal to no growth inhibition
`(Fig. 4, blue lines, filled squares), and combining the chemo-
`therapeutic agent with a ng pump inhibitor failed to improve
`this lack of an antitumor effect (Fig. 4, mauve diamonds).
`Regardless of the lack of effect of the chemotherapeutic
`agent alone,
`in most instances addition of the anti-flk-l anti-
`body, DC101, produced tumor growth suppressions over and
`above those observed with either agent alone (Fig. 4, red line,
`filled squares in all panels). Most importantly, this combination
`therapy did not result in detectable drug resistance to the treat-
`ment even after >100 days of continuous treatment. With the
`exception of those treated with Adriamycin and cisplatinum, the
`mice remained healthy throughout the course of treatment, and
`in all cases the tumor remnants, when assessed by histology,
`appeared to contain mainly fibrous scar tissue.
`Histopathological Analysis.
`In the multiple in viva pre-
`clinical trials undertaken in our laboratory, we have frequently
`observed that even through significant differences can be seen
`with tumor volume measurements,
`the true degree of tumor
`regression is not always appreciated. However controlled, the
`gross measurement of tumor volume reflects a delayed response,
`and resorption, of the tissues and not necessarily the actual
`degree of the tissue damage inflicted. We have therefore eval-
`uated representative samples of tumor tissues from all groups at
`arbitrary time points and observed, surprisingly. that significant
`differences were apparent as early as 3 weeks post initiation of
`therapy. Care was taken to include reference mammary gland
`tissue in all tumor samples (visible on the left in Fig. 5, aee), to
`avoid concerns about sampling bias. The MPAHS tumor histol-
`ogy, shown in Fig. 5, represents a typical example of the
`histopathology of all of the different tumor subtypes and their .
`
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`226 Chemotherapy and Anti-VEGFR-Z Antibody in Breast Cancer
`
` Fig. 2 Sensitivity
`
`of HUVECs
`grown in monolayer to vinblastine
`alone or in combination with [MC-
`lCIl (a monoclonal antibody to hu-
`man VEGFR-Z/KDR). HUVECs
`grown on glass slides coated with 1%
`gelatin,
`and
`supplemented with
`growth factors, were treated with ei-
`ther 0.5 ng/ml vinblastine or a com-
`bination of vinblastine and 1C11(25
`ug/ml),
`a monoclonal
`antibody
`against
`the VEGFR-Z. The culture
`was then fixed and stained with anti-
`B-tubulin/Cy-3 conjugate. The mild
`polymerization of tubulin evident in
`the cultures treated with 0.5 ng/ml
`vinblastine is visibly enhanced by
`combining the agent with IMC-ICI l,
`and no further escalation of this effect
`is achieved by doubling the vinblas-
`tine dose concentration.
`
`1 nglml Vb]
`
`1 ng/ml Vbl + 25 ug/ml 1C1]
`
`respective changes. No easily appreciated differences were seen
`in mice treated with saline or vinblastine alone. Healthy looking
`cancer cells with high nuclearzcytoplasmic ratio, high mitotic:
`karyorrhetic index, and high invasive potential are the prevalent
`component in tissues from these two groups (Fig. 5, a, f—h,
`k—m). In contrast. cells with pyknotic nuclei and a high degree
`of cytoplasmic blebbing, features consistent with apoptosis,
`predominate in the combination group (Fig. 5e) and are preva-
`
`lent in the DC101 alone group (Fig. 5d). In fact, it appears that
`the degree of tissue necrosis and apoptosis is over and above
`that expected on the basis of tumor size measurements at 3
`weeks (Fig. 4, day 51 of MPAHS panel). Similar differences are
`evident for mitoticzkaryorrhetic indexes. Whereas a typical field
`in the control and vinblastine alone group may manifest many
`mitotic figures (Fig. 5, fand g), these are hard to find in samples
`treated with DC101 (Fig. 51'), and none were found in the
`
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`Clinical Cancer Research 227
`
`MDA231
`Parental cell line
`
`MDA231/MD22
`Adriamycin-Resistant clone
`
`Fig.3 In vitro inhibition of
`MDR of selected cancer cell
`lines. The MDA-MB-23l pa-
`rental cell
`line and its drug-
`resistant variants were grown in
`monolayer,
`incubated with 5
`rig/ml
`rhodamine chloride (a
`fluorescent ng pump sub-
`strate) for 30 min, and assessed
`for
`relative
`fluorescence by
`flow cytometry. The human
`mammary carcinoma parental
`cell lines MDA-MB-23l and its
`cisplatinum-resistant
`variant
`CDDP—S4 readily take up rho-
`damine chloride, shifting the
`curve to the right, and this up-
`take cannot be inhibited by cy-
`closporin A (a ng inhibitor). In
`contrast, its drug-resistant vari-
`ants show less rhodamine chlo-
`ride uptake, or efflux of rhoda-
`mine chloride from the cells,
`and this is, at
`least
`in vitro,
`readily inhibited by cyclosporin
`A or verapamil, suggesting a
`ng-mediated resistance.
`
`Rhod+ ClA
`
`
`
`"‘
`
`m
`
` m-u
`
`MDA23l/MPAHS
`mdr.‘ transfected clone
`
`MDA23l/MVB9
`in vitro selected Vinblastinc