[CANCER RESEARCH 48. 2179-2183. April 15. 1988]
`
`Toxic Effect of Tumor Necrosis Factor on Tumor Vasculature in Mice
`
`Naoki Watanabe, Yoshiro Niitsu, Hiroshi Umeno, Hiroshi Kuriyama, Hiroshi Neda, Naofumi Yamauchi,
`Masahiro Maeda, and Ichiro Urushimki
`Department ofInternal Medicine (Section 4). Sapporo Medical College, South-I, West-l6, CInw-ku, Sapporo, Japan
`
`bations were performed under the same conditions except as otherwise
`noted. Meth-A cells were passaged i.p. in mice.
`Observation of Tumor Vasculature. Observations were made under a
`stereoscopic microscope, through a sight glass consisting of two stain-
`less steel plates (1 1), one containing a drilled hole 13 mm in diameter
`which was covered by a clear glass plate affixed to the outside surface.
`On day 0, the sight glass was affixed with surgical thread to a raised
`portion of the dorsum as shown in Fig. l with an excised dermal area
`facing the drilled hole, and Meth-A cells (1 x 10‘ cells/0.1 ml Eagle's
`MEM) were then injected into the region under the drilled hole. On
`day 9, TNF (l x 10‘ units/0.1 ml 0.1% gelatin-phosphate buffered
`saline, pH 7.4) or the TNF diluent as control was administered i.v.,
`and the tumor vasculature under the sight glass was observed 1, 2, 4,
`6, and 24 h later with the mouse under secobarbital anesthesia (0.6 mg,
`i.p.; Yoshitomi Pharmaceutical). Prophylactic penicillin (0.8 mg; Toy-
`ama Chemical Industry Co.) was administered on days 0, 1, 2, 4, and
`6.
`
`Evaluation of Antimmor Effect. Meth-A cells (1 x 10‘ cells/0.1 ml
`Eagle’s MEM) were injected i.d. in each mouse on day 0, and on day 6
`TNF (l x 10’-1 X 10‘ units/0.1 ml) was administered iv. with or
`without prior injection of heparin (3 units/0.1 ml) via the caudal vein.
`Control mice each received TNF diluent (0.1% gelatin-phosphate buff-
`ered saline, pH 7.4, 0.1 ml) instead of TNF. Tumor weight was
`estimated from measurements on days 6 and 8 of the minor (a) and
`major (b) axes, as a2 X b/2. Necrotic response was graded on day 8 as
`+++ (necrosis over entire tumor), ++ (necrosis over 50% or more of
`tumor), + (necrosis over less than 50% of tumor), and —- (no apparent
`necrosis). Occurrence of complete cure was judged on day 97. Tumor
`sections were obtained from 8 mice 4 h after TNF administration with
`and without heparin and periodically thereafter, embedded in paraffin,
`and subjected to histological examination by optical microscope after
`hematoxylin and eosin staining.
`Cytotoxic Assay. One hundred ul of CPAE or L—M cells (1 x 10’
`cells/ml) and 100 pl of TNF at various concentrations were added to
`the wells of a 96-well microculture plate and incubated for 48 h.
`Cytotoxicity was then assessed by dye uptake method (12-14).
`
`RESULTS
`
`ABSTRACT
`
`Stereoscopic observation via an implanted swat glass in mice bearing
`transplanted methylcholanthrene-induced A-cells showed tumorivascular
`hemorrhage at 1—2 h after tumor necrosis factor (TNF) administration,
`congestion at 4-6 h, and hemorrhage, congestion, and blood circulation
`blockage at 24 h.
`Histological examination after TNF administration to mice bearing
`similar methylcholanethrene-induced A-cell transplants showed throm-
`bus formation in the tumor vasculature at 4 h and thereafter. Suppression
`of this thrombus formation with heparin had no apparent influence on
`the necrotic response, tumor growth inhibition or complete cure rate
`following TNF administration to mice bearing the methylcholanethrene-
`induced A-cell tumors. The results suggest that direct toxicity of TNF
`on tumor vasculature is a factor in the overall antitumor mechanism of
`TNF.
`
`INTRODUCTION
`
`TNF' is an anticancer cytokine derived from monocytes and
`macrophages (l-3) and is known to exert a strong antitumori-
`genic effect against tumor cells both in vivo (1, 4) and in vitro
`(5). Its direct effect on tumor cells has been demonstrated in
`vitra and undoubtedly plays a part in its antitumor effect. Many
`aspects of the overall antitumor mechanism nevertheless remain
`unclear. One of these is the possible involvement of reactions
`affecting the tumor blood vessels, which is suggested by the
`hemorrhagic necrosis which is usually observed in the course
`of transplanted tumor regression following administration of
`TNF in mice (1). In vitro studies have provided evidence of
`such reactions. TNF reportedly exerts a cytotoxic effect on
`endothelial cells from bovine arteries (6) and human umbilical
`veins (7), and promotes thrombus formation by acting on
`vascular endothelial cells and stimulating production of a pro-
`coagulant factor (8, 9). No direct evidence of their (6-9) occur-
`rence in vivo has yet been reported.
`The present study is a more direct investigation of the effect
`of TNF on tumor vasculature. To allow visual observation of
`changes in the tumor vasculature under the influence of TNF,
`we implanted a sight glass in mice before their inoculation with
`Meth-A cells. We also investigated the influence of thrombus
`suppression with heparin on the antitumor effect of TNF.
`
`MATERIALS AND METHODS
`
`Materials. Human recombinant TNF (2.37 x 10‘ units/mg protein)
`(10) was provided by Asahi Chemical Industry Co., Ltd. Heparin
`(Midori Juji, Osaka, Japan) was used as anticoagulant. BALD/c mice
`(female, 5 weeks of age; Clea Japan Co., Ltd.) were used.
`Cell (hilture. CPAE cells (bovine endothelial cells of the pulmonary
`artery, L—M cells (mouse tumorigenic fibroblast) and Meth-A cells
`(mouse fibrosarcoma) were used. CPEA and L-M cells were maintained
`in Eagle’s MEM (Nissui Pharmaceutical) containing 10% fetal bovine
`serum (Flow Laboratories) in a 5% CO; incubator at 37'C. All incu-
`
`Received 9/ 14/87; revised 12/28/87; accepted 1/22/88.
`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.
`' The abbreviations used are: TNF. tumor necrosis factor. Meth-A, methyl-
`cholanthrene-induced A-cells; MEM. minimal essential medium; i.d., inn-ader-
`mal(ly); ID”, 50% inhibitory dose.
`
`Effect of TNF on Tumor Vasculature. Stereomicroscopic ob-
`servation of the newly formed blood vessels of the Meth-A
`tumor under the sight glass showed hemorrhage from capillary
`vessels at l and 2 h after TNF administration, both hemorrhage
`from capillary vessels and congestion of nutrient vessels at 4
`and 6 h, and congestion of entire vasculature and complete loss
`of blood circulation due to blocking at 24 h (Fig. 2). None of
`these effects was observed in the control group. Histological
`examination of the Meth-A tumors resected from mice 4 h after
`administration of TNF revealed extensive thrombus formation
`in the tumor vessels of those receiving TNF alone (Fig. 3c,
`arrow), but none in those receiving both TNF and heparin (Fig.
`3d).
`Influence of Thrombus Suppression on TNF Antitumor Effect.
`Despite the histologically observed suppression of thrombus
`formation by heparin in combination with TNF, no significant
`difference in Meth-A tumor growth inhibition, necrotic re-
`sponse, or complete cure rate was observed between the mice
`given TNF alone and those given both TNF and heparin (Table
`l).
`CPAE Cell Susceptibility. TNF showed dose-dependent cy-
`totoxicity against CPAE cells (Fig. 4) but the ID” of 1.6 x [0‘
`2179
`
`Downloaded from cancerres.aacrjournals.org on April 23, 2018. © 1988 American Association for Cancer Research.
`|PR2018-00685
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Celgene Ex. 2032, Page 1
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`

`
`
`
`
`
`
`
`
`
`

`
`

`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`IPR2018-00685
`Celgene Ex. 2032, Page 1
`
`

`

`TOXIC EFFECT OF TNF 0N TUMOR VASCULATURE
`
`units/ml was much higher than the ID” of 1.0 unit/ml observed
`for L-M cells.
`Effect of TNF on Normal Blood Vessels. The skin vessels,
`pulmonary aorta, and abdominal vena cava of mice bearing
`
`Meth-A cells 24 h after administration of TNF (l x 10‘ units/
`mouse) or the TNF diluent as control were excised. Examina-
`tion by optical microscope showed no difference between the
`excised tissue of the TNF group and that of the control group
`(Fig. 5).
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`DISCUSSION
`
`Various reports have suggested that reactions affecting the
`tumor vasculature may be involved in the overall antitumor
`mechanism of TNF, in addition to its proven direct cytotoxicity
`against tumor cells and activities mediated by neutrophils (15)
`or macrophages (16). This is based on observation of cytotoxic
`effects by TNF on vascular endothelial cells derived from bovine
`aorta (6) and from human umbilical cord (7). In the present
`study with CPAE cells, a cytotoxic effect was similarly observed.
`The effect was dose dependent in the range of 1-1 x 10’
`units/ml. The level of cytotoxicity against these cells was never-
`theless much lower than that against various tumor cell lines
`as shown by the ID“. of 1.6 x 10‘ units/ml in the 48-h assay.
`The observation of in vitro cytotoxicity against vascular cells,
`however, does not necessarily imply significant alteration of
`tumor blood vessels by TNF. As shown in Fig. 6, the clearance
`of TNF from the blood is relatively rapid. In this experiment,
`the TNF blood level 30 min after i.v. administration of 1 x 10‘
`units/mouse was 4485 units/ml, and the concentration half-life
`
`
`
`Fig. 2. Effect of TNF on tumor vasculature. Meth—A cells (I X 10‘ cells/mouse) were injected into the region under the drilled hole of sight glass. On day 9, TNF
`(I x I0‘ units/0.1 ml 0.1% gelatin-phosphate buifered saline. pH 7.4) or the TNF diluent as control was administered i.v. and the tumor vasculature under the sight
`glass was observed. a. tumor vasculature at 24 h after i.v. injection of the TNF diluent; b—d, tumor vasculature at l. 5, or 24 h. respectively. after u. injection of
`TNF.
`
`2180
`
`.
`
`z-
`
`! Tar 3-.
`
`_
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Downloaded from cancerres.aacrjournals.org on April 23, 2018. © 1988 American Association for Cancer Research.
`|PR2018—00685
`
`Celgene Ex. 2032, Page 2
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`

`
`
`
`
`
`
`
`
`
`

`
`

`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`IPR2018-00685
`Celgene Ex. 2032, Page 2
`
`

`

`TOXIC EFFECT OF TNF ON TUMOR VASCUIATURE
`
`in." 11'";
`" vii" ‘I:
`I
`
`..a5". as?"
`
`€3.12! \.
`
`- r
`'r-
`
`
`30
`s
`gig I
`
`
`
`
`A tuft-ital. mitt-flan." _ .' Ain‘t“). Ir
`.
`‘3.
`i
`'.
`Fig. 3. Histology of the Meth-A Inmors resected from mice treated with the diluent solution ofTNF (a), heparin (b), TNF (c). and TNF-Ileparin (d). Meth-A cells
`I I x 10“ ct-Ilsjmouse) were inoculated in. on day (I. and on day 6 TM" (3 x 10' units/0.1 ml) was administered i.v. with (d) or without (c) prior injection of heparin
`(3 units/0.1 ml) via the caudal vein. The TNF diluent (0.1% gelatin-phosphate buffered saline, pH 7.4, 0.1 ml) (0) or heparin (3 units/0.1 ml) (11) was administered
`i.v. as control. The histology of the tumor vessels was examined 4 h after each administration. Arrow, thrombus formation.
`
`'
`
`Table l Eflect ofheparin on ontl'tuntor eflecr of INF against Moth-A ftbmaarooma ceILr
`Math-A cells (I x 10‘ cells/mouse) were inoculated id. and on day 6, TNF (l x 10’ - l x 10‘ units/mouse) and heparin (3 units/mouse) were administered i.v.;
`0.1% gelatin-phosphate buttered saline. pH 7.4, the diluent solution of TNF, was administered a control. Necrotic response was judged on day 8, with grades as
`described in “Materials and Methods." Cured ratio was judged on day 97.
`
`Experiment 1 (TNF)
`
`Experiment 2 (TNF + heparin)
`
`TNF (units/ & Mean tumor growth,
`mouse)
`-
`+
`++
`+++
`days 6-8
`l x 10‘
`0
`0
`S
`2
`-11
`3 x 10’
`o
`2
`s
`0
`+4
`I x 10’
`0
`6
`I
`0
`+9
`Control
`4
`3
`0
`0
`+71
`
`1 x 10‘
`3 x 10’
`1 x 10’
`Control
`
`0
`0
`0
`6
`
`0
`0
`S
`1
`
`S
`7
`2
`0
`
`2
`0
`0
`0
`
`-38
`+21
`+8
`+53
`
`Cured ratio
`6/7
`5/7
`4/7
`0/7
`
`5/7
`7/7
`4/7
`0/7
`
`
`
`
`
`was approximately 27 min. Tumor vasculature, moreover, is
`known to be structurally different from that of normal blood
`vessels (17, 18).
`The sight glass observations in the present study provided
`clear and direct evidence of toxic effects by TNF on newly
`formed tumor vasculature. These included hemorrhaging at l-
`2 h, congestion at 4-6 h, and complete loss of circulation in
`the blood vessels of tumors in mice after TNF administration.
`None of these eifects was observed under the same conditions
`in the mice that received no TNF.
`Histological examination of vascular tissue from the Meth-
`A tumors following TNF administration showed thrombus
`105
`102
`10
`1
`formation at 4 h and ,thereafter'
`Conceniration of TNF (U /1)
`This '5 In accord mm reports by Nawroth and Stern (8) and
`Fig. 4. Cytotoxic activity of TNF against CPAE cells and L-M cells. Cell
`survival (95) was measured by dye uptake assay after incubation of CPAE and L- Bevilacqua 9’ al- (9) WhiCh Show that TNF Prommes thrombus
`M cells with 1—1 x 10’ units (U)/ml ofTNF for 48 ll.
`formation by acting on vascular endothelial cells and stimulat-
`2181
`
`/T
`
`CPAE
`
`L'"
`
`Cellsurvival(96) 8
`
`°
`
`0
`
`104
`
`105
`
`Downloaded from cancerres.aacrjournals.org on April 23, 2018. © 1988 American Association for Cancer Research.
`|PR2018-00685
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Celgene Ex. 2032, Page 3
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`

`
`
`
`
`
`
`
`
`
`

`
`

`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`IPR2018-00685
`Celgene Ex. 2032, Page 3
`
`

`

`TOXIC EFFECT OF TNF ON TUMOR VASCULATURE
`
`
`
`
`Fig. 5. Histology of the skin vessels (a, b), aorta (c, d), and vena cava inferior (e, f) in BALB/c mice treated with or without TNF. Meth-A cells (l x 10‘ units/
`mouse) were inoculated i.d.. and TNF (l x [0‘ units/mouse) (a, c. e) or the TNF diluent as control (b, d. I) was administered i.v. on day 9 following transplanting.
`Histology was examined 24 h alter TNF iniection. H & E, x 100.
`
`L .
`
`ing the production of procoagulant factor in vitra. Thrombus
`formation is apparently not essential to the antitumor effect,
`however, since no lowering of tumor growth inhibition, necrotic
`response, or cure rate occurred under complete thrombus
`suppression with heparin.
`It may further be noted that, at least in the dose range used
`in this study, TNF apparently had no toxic effect on normal
`blood vessels.
`Histological examination of the skin vessels, pulmonary
`aorta, and abdominal vena cava from mice in which tumor
`We wish to thank Orville M. Stever for his help in preparation of
`this manuscript.
`necrosis or complete cure had been observed showed no abnor-
`2182
`
`mality in any of the vascular endothelial cells. These results as
`well as those of the in vitro investigation with CPAE cells
`suggest that the endothelial cells of normal vasculature are far
`less susceptible to TNF than are those of newly formed tumor
`vasculature.
`
`ACKNOWLEDGMENTS
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Downloaded from cancerres.aacrjournals.org on April 23, 2018. © 1988 American Association for Cancer Research.
`|PR2018-00685
`
`Celgene Ex. 2032, Page 4
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`

`
`
`
`
`
`
`
`
`
`

`
`

`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`IPR2018-00685
`Celgene Ex. 2032, Page 4
`
`

`

`TOXIC EFFECT OF TNF 0N TUMOR VASCLMTURE
`
`9bclearance 8
`
`100
`
`0
`
`1
`
`2
`
`3
`
`4
`
`time after TNF Injection (h)
`Fig. 6. Clearance of TNF in BALD/c mouse. TNF (I X 10‘ units/mouse) was
`iniectedintothecaudal vein,and'1'Nl-‘activityofsensmwasmeasuredbydye
`uptake assay using L-M cells as target. Bars, SD.
`
`REFERENCES
`
`1. Carswell. E. A., Old. L. J.. Kassel, R. L., Green. 8., Flore, N., and William-
`son, B. An cndotoxin induced scrum factor that causes necrosis of tumors.
`Proc. Natl. Acad. Sci. USA 72: 3666-3670. 1975.
`2. Matthews. N. Tumor necrosis factor from the rabbit. 11. Production by
`monocytes. Br. J. Cancer. 38: 310—315, 1978.
`3. Niitsu. Y., Watanabe, M, Sone, H., Neda. 11., Yamsuchi. N., and Urushinlri,
`1. Mechanism of the cytotoxic effect of tumor necrosis factor. Gann. 76:
`1193—1197, 1985.
`4. Watanabe, N., Niitsu, Y., Sone, H.. Neda. 11.. Yamauchi, N., and Urushinki,
`l. Inhibitory effect of tumor necrosis serum on the metastasis of 13-16 mouse
`melanoma cells. Oahu, 76: 989-994. 1985.
`S. Watanabe. N., Niitsu. Y., Neda, H., Sone, H.. Yamauchi, N.. Umetau, '11.
`
`and Urusbinki. 1. Aatitumor effect of motor necrosis factor against various
`primarilyculhued humancancercells. Gann, 76:1115—1119,l985.
`Kull, F. C., Jr.. Jacobs, 8., .lr.. and Cuatreeass, P. Cellular receptor for n’1-
`labeled tumor necrosis factor: specific binding. affinity labeling and relation-
`ship to sensitivity. Proc. Natl. Acad. Sci. USA, 82: 5756—5760. 1985.
`Sato. M. Goto, T., Haranalta, IL, Satomi, N., Nariuehi, l-l.. Mano-Hiram,
`Y..sndSawasaki.Y. Actiousoftumornecrosisfactoronculturedvsscular
`endothel'nl cells: morphologic modulation. growth inhibition. and cytotox-
`icity. .1. Natl. Canceling” 76: 1113-1121, 1986.
`Nswroth. P. P.. and Stern. D. M. Modulation ofendothelial cell hemostatic
`properties by tumor necrosis factor. .1. Exp. Med.. 163.- 740—745. 1986.
`Bevilacqna, M. It, Rober, J. 8., Maieau. G. 11., Piers. W., Cotran. 11., and
`Gimbrone. M. A.. Jr. Recombinant tumor necrosis factor induces procoag-
`ulant activity in cultured human vascular endothelium: characterintion and
`comparison with the actions of interleukin 1. Proc. Natl. Acad. Sci. USA.
`83: 4533-4537, 1986.
`Shirai. '11, Yamguchi, H., Ito. H., Todd, C. W.. and Wallace, R. B. Cloning
`andexpresn'oninircbericltiacoliofthegeneforhumantumornecrosis
`factor. Noam (land), 313: 803-806, 1985.
`Hot-i, 1L, Suzuki, M., Abe, 1.. and Saito, S. Increased tumor tissue pressure
`inassociationviththegrowthofrsttumors. Gann. 77:65-73, I986.
`Yamanhi, 8.. Ouishi. F... Enami, 1L, Natori, 1L, Kohaae. M., Sakamoto, H.,
`TanouchLM.,andHayashi.H.Proposalofstandardizedmethodsand
`reference for assaying recombinant human tumor necrosis factor. Jpn. J.
`Med. Sci. Biol.. 39.- 105-118, 1986.
`Watanabe. N., Niitsu, Y.. Umeno. H., Sone, H., Neda. H., Yamauchi, N.,
`Maeda. M., and Urushinki, 1. Synergistic cytotoxic and antitumor effects of
`recombinant humantumornecrosisfactorandhyperthermia.CancerRes.,
`48: 650-653, 1988.
`Niitsu, Y., Watanabe, N.. Umeno. H.. Sone, H., Neda, H., Yarnauchi, N.,
`Maeda, M., and Urushinki, l. Synergistic effects ofrecombinant human
`tumor necros‘nl‘actorand hyperthermiaon in vinocytotoxicityand artificial
`metastasis. Cam Rec, 48: 654-657. 1988.
`Shalby, M. 11., Palladino, M. A., Jr., Hirabayashi, S. E, Eessalv, T. 15.,
`lewis, G. D.. Shepard. H. M., and Aggarrval. B. 3. Receptor binding and
`activation of polymorphonuclear neutrophils by tumor necrosis factor-alpha.
`J. Leukocyte Biol, 41: 196-220, 1987.
`Hori. IL. Ehrke. M. J., Mace. IL. Maccubbin. 0.. Doyle. M. 1.. Otsulra. Y..
`andMihich.E. Efl‘ectofrecombinanthumantumornecrosisfactoronthe
`inducdonofmurinemacrophagemmoricidslactivity.€anoerkee.47:2793—
`2798. 1987.
`VogeLAWJnu'atumorvascularchangeswithincteasedsizeofamammary
`adenocarcinoma. New method and results. J. Natl. Cancer inst. 35: 571-
`578, I965.
`Yamaura, H., and Sato, H. Quantitative studies on the developing vascular
`system of rat hepatoma. J. Natl. Cancer Inst., 53: 1229-1240. 1974.
`
`10.
`
`11.
`
`12.
`
`13.
`
`I4.
`
`15.
`
`16.
`
`17.
`
`18.
`
`2183
`
`Downloaded from cancerres.aacrjournals.org on April 23, 2018. © 1988 American Association for Cancer Research.
`|PR2018-00685
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Celgene Ex. 2032, Page 5
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`

`
`
`
`
`
`
`
`
`
`

`
`

`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`IPR2018-00685
`Celgene Ex. 2032, Page 5
`
`

`

`Cancer Research
`
`ThejoumalofCancer Research( ‘1915—4930) | TheAmerIcanJ ournalofCancerl 1931-4940)
`
`for Cancer Research
`MGR American Association
`
`Toxic Effect of Tumor Necrosis Factor on Tumor Vasculature in
`
`Mice
`
`Naoki Watanabe, Yoshiro Niitsu, Hiroshi Umeno, et al.
`
`Cancer Res 1988;48:2179-2183.
`
`Updated version
`
`Access the most recent version of this article at:
`http://cancerres.aacrjournals.org/content/48/8/2179
`
`*
`F
`4
`9
`<
`=
`5
`:
`<
`E
`D
`7
`@
`?
`5
`A
`7
`C
`=
`B
`?
`9
`7
`3
`5
`A
`7
`@
`>
`=
`<
`;
`4
`:
`4
`9
`4
`8
`7
`6
`5
`4
`3
`7
`?
`5
`2
`/
`1
`0
`/
`.
`-
`,
`+
`*
`)
`"
`
`&
`(
`!
`'
`
`&
`%
`"
`$
`#
`"
`"
`!
`
`%
`9
`4
`<
`F
`U
`7
`9
`A
`4
`?
`@
`9
`Z
`5
`:
`5
`7
`?
`A
`<
`Y
`9
`:
`<
`U
`<
`A
`9
`?
`5
`<
`@
`9
`?
`?
`U
`U
`X
`7
`E
`<
`R
`Q
`P
`O
`N
`L
`M
`I
`L
`K
`J
`I
`H
`G
`!
`'
`
`&
`T
`"
`T
`"
`$
`T
`9
`:
`<
`9
`:
`5
`U
`T
`W
`A
`5
`*
`?
`F
`4
`:
`A
`B
`5
`V
`A
`U
`4
`4
`*
`?
`<
`A
`A
`<
`U
`:
`4
`U
`T
`T
`%
`S
`9
`9
`@
`Rightslink site.
``
`9
`<
`7
`<
`A
`<
`F
`4
`9
`_
`9
`5
`9
`@
`7
`?
`4
`A
`9
`7
`U
`F
`<
`5
`A
`V
`5
`B
`A
`:
`4
`F
`*
`W
`:
`B
`S
`5
`A
`<
`U
`<
`7
`Y
`<
`Z
`A
`<
`<
`<
`E
`4
`7
`F
`(
`4
`F
`A
`9
`?
`[
`\
`]
`J
`P
`^
`J
`^
`L
`N
`K
`O
`A
`7
`?
`h
`A
`9
`B
`;
`5
`:
`5
`5
`<
`5
`A
`_
`<
`A
`A
`S
`:
`9
`5
`Z
`9
`@
`7
`?
`4
`9
`7
`U
`F
`<
`A
`5
`?
`;
`?
`U
`A
`7
`<
`9
`5
`9
`@
`<
`V
`B
`A
`:
`4
`F
`=
`U
`5
`9
`4
`U
`9
`9
`@
`<
`X
`X
`i
`j
`k
`B
`;
`F
`7
`U
`4
`9
`7
`5
`:
`?
`L
`N
`R
`R
`I
`J
`e
`H
`P
`K
`O
`g
`E
`4
`B
`;
`?
`f
`*
`S
`4
`4
`U
`A
`*
`5
`A
`W
`<
`S
`4
`A
`9
`<
`:
`9
`9
`c
`a
`b
`d
`H
`P
`O
`O
`N
`P
`K
`Q
`R
`5
`:
`5
`5
`h
`S
`?
`U
`A
`A
`7
`<
`F
`5
`n
`B
`<
`E
`B
`A
`4
`A
`<
`<
`?
`9
`S
`7
`?
`?
`9
`A
`<
`(
`?
`4
`F
`A
`4
`9
`5
`Z
`9
`@
`7
`9
`7
`F
`<
`=
`B
`?
`<
`9
`@
`7
`?
`F
`7
`:
`6
`P
`l
`L
`O
`Q
`R
`O
`N
`]
`P
`O
`@
`9
`9
`S
`%
`T
`T
`U
`4
`:
`U
`<
`A
`A
`<
`?
`*
`4
`4
`U
`A
`V
`5
`B
`A
`:
`4
`F
`?
`*
`5
`A
`W
`T
`U
`5
`:
`9
`<
`:
`9
`T
`$
`"
`T
`"
`T
`&
`
`'
`!
`*
`i
`6
`:
`U
`7
`F
`m
`9
`7
`5
`j
`<
`n
`B
`<
`?
`9
`k
`<
`A
`E
`?
`?
`7
`5
`:
`?
`m
`o
`@
`7
`U
`@
`o
`7
`F
`F
`9
`4
`6
`<
`p
`5
`B
`9
`5
`9
`@
`<
`i
`5
`S
`p
`A
`7
`W
`@
`9
`i
`F
`<
`4
`A
`4
`:
`U
`<
`i
`<
`:
`<
`A
`q
`?
`r
`i
`i
`i
`s
`j
`?
`F
`*
`7
`6
`?
`W
`@
`9
`7
`:
`7
`9
`<
`
`E-mail alerts
`
`Sign up to receive free email-alerts related to this article orjournal.
`
`Reprints and
`Subscriptions
`
`To order reprints of this article or to subscribe to the journal, contact the AACR Publications
`Department at pubs@aacr.org.
`
`Permissions
`
`To request permission to re-use all or part of this article, use this link
`http://cancerres.aacrjournals.org/content/48/8/2179.
`Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
`
`Downloaded from cancerres. aacrjournals. org on April 23, 2018. © 1988 American Association for Cancer Research.
`|PR2018-00685
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Celgene Ex. 2032, Page 6
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`

`
`
`
`
`
`
`
`
`
`

`
`

`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`IPR2018-00685
`Celgene Ex. 2032, Page 6
`
`