[CANCER RESEARCH48, 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 Urushizaki
`DepartmentofInternal Medicine (Section 4), Sapporo Medical! College, South-1, West-16, Chuo-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, througha sight glass consisting of two stain-
`less steel plates (11), one containing a drilled hole 13 mm in diameter
`which was covered by a clear glass plate affixed to the outside surface.
`On day0, the sight glass was affixed with surgical thread to a raised
`portion of the dorsum as shown in Fig. 1 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 (1 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 tumorvasculature 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 Antitumor 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 (1 x 10°-1 x 10* units/0.1 ml) was administered i.v. with or
`withoutprior 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(6) axes, as a? X 6/2. 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 yl 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 sight 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 (1-3) and is known to exert a strong antitumori-
`genic effect against tumorcells both in vivo (1, 4) and in vitro
`(5). Its direct effect on tumorcells has been demonstrated in
`vitro and undoubtedlyplaysa part in its antitumoreffect. Many
`aspects of the overall antitumor mechanism nevertheless remain
`unclear. One of these is the possible involvementof reactions
`affecting the tumor blood vessels, which is suggested by the
`hemorrhagic necrosis which is usually observed in the course
`of transplanted tumorregression following administration of
`TNFin 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-
`coagulantfactor (8, 9). No direct evidence of their (6-9) occur-
`rence in vivo has yet been reported.
`Thepresent study is a more direct investigation of the effect
`of TNF on tumorvasculature. To allow visual observation of
`changes in the tumorvasculature underthe 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 antitumoreffect of TNF.
`
`MATERIALS AND METHODS
`
`Materials. Human recombinant TNF (2.37 x 10° units/mgprotein)
`(10) was provided by Asahi Chemical Industry Co., Ltd. Heparin
`(Midori Juji, Osaka, Japan) was used as anticoagulant. BALB/c mice
`(female, 5 weeks of age; Clea Japan Co., Ltd.) were used.
`Cell Culture. CPAEcells (bovine endothelial cells of the pulmonary
`artery, L-M cells (mouse tumorigenic fibroblast) and Meth-A cells
`(mouse fibrosarcoma) were used. CPEA and L-Mcells 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, tumornecrosis factor; Meth-A, methyl-
`cholanthrene-induced A-cells; MEM, minimal essential medium; i.d., intrader-
`mal(ly); IDso, 50% inhibitory dose.
`
`Effect of TNF on Tumor Vasculature. Stereomicroscopic ob-
`servation of the newly formed blood vessels of the Meth-A
`tumorunderthe sight glass showed hemorrhage from capillary
`vessels at 1 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 nonein 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 aloneand those given both TNF and heparin (Table
`1).
`CPAE Cell Susceptibility. TNF showed dose-dependent cy-
`totoxicity against CPAEcells (Fig. 4) but the IDso of 1.6 x 10*
`2179
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`TOXIC EFFECT OF TNF ON TUMOR VASCULATURE
`
`units/ml was muchhigherthan the IDspo 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-Acells 24 h after administration of TNF (1 X 10* units/
`mouse) or the TNFdiluent as control were excised. Examina-
`tion by optical microscope showed no difference between the
`excised tissue of the TNF group andthatof the control group
`(Fig. 5).
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`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 tumorcells 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 tumorcell lines
`as shown bythe IDso of 1.6 x 10‘ units/ml in the 48-h assay.
`Theobservation ofin vitro cytotoxicity against vascular cells,
`however, does not necessarily imply significant alteration of
`tumorblood vessels by TNF. As shown in Fig. 6, the clearance
`of TNF from the bloodis relatively rapid. In this experiment,
`the TNFblood level 30 min after i.v. administration of 1 < 10*
`units/mouse was 4485 units/ml, and the concentration half-life
`
`
`
`Fig. 2. Effect of TNF on tumorvasculature. Meth-A cells (1 x 10° cells/mouse) were injected into the region underthe drilled hole ofsight glass. On day 9, TNF
`(1 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. a, tumorvasculature at 24hafter i.v. injection of the TNF diluent; 6-d, tumor vasculature at 1, 5, or 24 h, respectively, after i.v. injection of
`TNF.
`
`2180
`
`Fa ® =
`
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`TOXIC EFFECT OF TNF ON TUMOR VASCULATURE
`
`
`1
`eB OOH s Kade
`ra = oe eae
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`"ie
`Fig. 3. Histology of the Meth-A tumors resected from mice treated with the diluent solution of TNF(a), heparin (6), TNF(c), and TNF-heparin (d). Meth-A cells
`(1 x 10° cells/mouse) were inoculated i.d. on day 0, and on day 6 TNF(3 x 10° units/0.1 ml) was administered i.v. with (a) or without(c) prior injection of heparin
`(3 units/0.1 ml) via the caudal vein. The TNFdiluent (0.1% gelatin-phosphate buffered saline, pH 7.4, 0.1 ml) (a) or heparin (3 units/0.1 ml!) (6) was administered
`i.v. as control. The histology of the tumor vessels was examined 4 h after each administration. Arrow, thrombus formation.
`
`ae ee
`
`uf
`
`Table 1 Effect ofheparin on antitumor effect of TNF against Meth-A fibrosarcoma cells
`Meth-A cells (1 < 10° cells/mouse) were inoculated i.d. and on day 6, TNF (1 x 10° - 1 x 10* units/mouse) and heparin (3 units/mouse) were administered i.v.;
`0.1% gelatin-phosphate buffered saline, pH 7.4, the diluent solution of TNF, was administered as control. Necrotic response was judged on day 8, with grades as
`described in “Materials and Methods.” Cured ratio was judged on day 97.
`
`
`TNF(units/___Netroticresponse(m)Mean tumorgrowth,
`mouse)
`-
`+
`++
`+++
`days 6-8
`Cured ratio
`1x 10
`0
`0
`5
`2
`-11
`6/7
`3x 10
`0
`2
`5
`0
`+4
`5/7
`1x10?
`0
`6
`1
`0
`+9
`4/7
`Control
`4
`3
`0
`0
`+71
`0/7
`
`Experiment 1 (TNF)
`
`Experiment 2 (TNF + heparin)
`
`1 x 10%
`3x10
`1x 10°
`Control
`
`0
`0
`0
`6
`
`0
`0
`5
`1
`
`5
`7
`2
`0
`
`2
`0
`0
`0
`
`-38
`+21
`+8
`+53
`
`5/7
`7/7
`4/7
`0/7
`
`
`
`
`
`Cellsurvival(%) 8
`
`was approximately 27 min. Tumorvasculature, 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 tumorvasculature. These included hemorrhaging at 1-
`2 h, congestion at 4-6 h, and complete loss of circulation in
`the blood vessels of tumors in mice after TNF administration.
`Noneofthese effects was observed under the same conditions
`in the mice that received no TNF.
`Histological examination of vascular tissue from the Meth-
`140.
`40°
`40?
`40"
`A tumors following TNF administration showed thrombus
`Concentration of TNF (U/mé)
`formationat4h and shereafier. by N
`th
`and St
`(8)
`d
`. Stern(8)anLee . i ord with reports by Nawroth and
`
`
`
`
`
`
`
`
`
`survival(3)aemetsaredby dheuptabausayaferincabetioneecals.Coll
`Bevilacquaet al. (9) which show that TNF promotes thrombus
`M cells with 1-1 x 10° units (U)/ml of TNFfor 48 h.
`formation by acting on vascular endothelial cells and stimulat-
`2181
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`CPAE
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`of
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`TOXIC EFFECT OF TNF ON TUMOR VASCULATURE
`
`=
`
`
`"a
`=
`eee.
`
`
`Fig. 5. Histology of the skin vessels (a, 5), aorta (c, d), and vena cavainferior (e, f) in BALB/c mice treated
`with or without TNF. Meth-A cells (1 < 10° units/
`mouse) were inoculated i.d., and TNF (1 x 10* units/mouse)(a, c, e) or the TNF diluent as control (6, d, f) was administered i.v. on day 9 following transplanting.
`Histology was examined 24 h after TNFinjection. H & E, x 100.
`
`ae.
`
`a
`
`ing the production of procoagulant factor in vitro. Thrombus
`formation is apparently not essential to the antitumoreffect,
`however, since no lowering of tumor growthinhibition, 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
`Wewish 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 CPAEcells
`suggest that the endothelial cells of normal vasculature are far
`less susceptible to TNF than are those of newly formed tumor
`vasculature.
`
`ACKNOWLEDGMENTS
`
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`TOXIC EFFECT OF TNF ON TUMOR VASCULATURE
`
`%clearance 8
`
`°
`
`1
`
`2
`
`3
`
`4
`
`time after TNF injection (h)
`Fig. 6. Clearance of TNF in BALB/c mouse. TNF(1 X 10‘ units/mouse) was
`injected into the caudal vein, and TNF activity of serum was measured by dye
`uptake assay using L-M cells as target. Bars, SD.
`
`REFERENCES
`
`1. Carswell, E. A., Old, L. J., Kassel, R. L., Green, S., Fiore, N., and William-
`son, B. An endotoxin induced serum factor that causes necrosis of tumors.
`Proc. Natl. Acad. Sci. USA, 72: 3666-3670, 1975.
`2. Matthews, N. Tumor necrosis factor from the rabbit. II. Production by
`monocytes. Br. J. Cancer, 38: 310-315, 1978.
`3. Niitsu, Y., Watanabe, N., Sone, H., Neda, H., Yamauchi, N., and Urushizaki,
`I. Mechanism of the cytotoxic effect of tumor necrosis factor. Gann, 76:
`1193-1197, 1985.
`4. Watanabe, N., Niitsu, Y., Sone, H., Neda, H., Yamauchi, N., and Urushizaki,
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`

`Cancer Research
`
`The Journal of Cancer Research (1916-1930)
`
`| The American Journal of Cancer (1931-1940)
`
`AAGR American Association
`for Cancer Research
`
`Toxic Effect of Tumor Necrosis Factor on Tumor Vasculaturein
`Mice
`
`Naoki Watanabe, Yoshiro Niitsu, Hiroshi Umeno, et al.
`
`Cancer Res 1988;48:2179-2183.
`
`Updated version
`
`Access the most recentversion ofthis article at:
`http://cancerres.aacrjournals.org/content/48/8/2179
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`
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