Stroma Formation in the Peritoneal Lining' Janicea

[CANCERRESEARCH55, 376-385, January 15, 1995J Pathogenesis of Ascites Tumor Growth: Angiogenesis, Vascular Remodeling, and Stroma Formation in the Peritoneal Lining' JaniceA. Nagy,2EllenS. Morgan,KempT. Herzberg,EleanorJ. Manseau,Ann M. Dvorak,and HaroldF. Dvorak Departments of Pathology. Beth Israel Hospital and Harvard Medical School, Boston, Massachusetts 02215

ABSTRACT In the two accompanying papers (18, 19), we demonstrated that two transplantable murine ascites carcinomas, MOT and TM/St. (13â€”17) In the accompanying papers, we demonstrated that two murine ascites shared several important properties with these same and many other tumors (MOT and TA3/St) Induced peritoneal lining blood vessels to transplantable and autochthonous tumors growing in solid form (1â€”4, become hyperpermeable to plasma proteins, leading to extravasatlon of fibrinogen and Its dotting to cmss-linked fibrin in peritoneal lining tissues 7, 20â€”27).Shared properties included: (a) secretion by tumor cells of (peritoneal wall, mesentery, and diaphragm). In solid tumors, vascular a potent cytokine, VPF3; (b) hyperpermeabiity of tumor blood yes hyperpermeabifity and fibrin deposition lead to the generation of vascu sels; (c) extravasation of plasma proteins, including fibrinogen; and larized connective tissue. In order to determine whether fibrin had similar (d) clotting of a fraction of extravasated fibrinogen to form cross consequences in ascites tumors, the vasculature and stroma of peritoneal linked fibrin deposits in peritoneal lining tissues. lining tissues were analyzed at successive Intervals after i.p. tumor cell Whether resulting from solid tumors (4, 7, 26), wound healing injecflon In both MOT and TA3/St asdtes tumors, the size and number (21, 23), or from direct implantation of fibrin-filled porous chambers of peritoneal lining mlcrovessels increased significantly by 5-8 days. (28), deposition of fibrin in tissues has been shown to stimulate the Subsequently, peritoneal lining vessels Increased in cross-sectional area by ingrowth of new blood vessels and the subsequent development of as much as 15-fold and peritoneal vascular frequency increased by up to fibrous connective tissue stroma. The present study was undertaken to 11-fold. Incorporation of [3H]thymidine by mesenteric blood vessels was negligible In control animals but came to involve 20 and 40% of endothe determine whether fibrin deposited in the peritoneal lining tissues of Dm1cells lining mesenteric vessels in MOT and TA3/St ascites tumor ascites tumor-bearing animals also induced angiogenesis and the bearing mice, respectively. After an early dramatic Increase In cross laying down of new connective tissue stroma. sectional area, peritoneal lining microvessels subsequently underwent a novel form of remodeling to smaller average size as the result of tram vascular bridging by endothellal cell cytoplasmic processes. Thus, both of MATERIALS AND METHODS the ascites tumors Studiedhere induced anglogenesis and stroma similar to that elicited when these same tumors were grown in solid form. How Tumors. TA3/St and MOT tumor cells (1 X 10@)werepassaged weekly in ever, stroma developed more slowly In ascites than in solid tumors and the peritoneal cavities of syngeneic, 5- to 6-week-old A/Jax and C3Heb/FeJ was entirely confined to a compartment(peritoneal lining tissues) that was mice, respectively (18). Sequential study of peritoneal lining tissues was distinct from that (peritoneal cavity) containing the majority of tumor conducted through day 8 after i.p. injection of 10@TA3/Sttumor cells; most cells and ascites fluid. These findings are consistent with the hypothesis animals died by day 9 or 10. In contrast, the majority of animals injected i.p. that vascular hyperpermeability, induced In both solid and ascites tumors with 10@MOT tumor cells survived for >1 month and were followed at by tumor cell-secreted vascular permeability factor, Is a common early intervals for up to 28 days. step in tumor anglogenesis, resulting in fibrinogen extravasatlon, fibrin Vascular Mapping with Microfil. To map the microvasculature of the deposition, and likely other alterations of the extracellular matrix that tissues lining the peritoneal cavity, animals were perfused with Microfil White together stimulate new vessel and fibroblast lngrowth. (MV-112; Canton Bio-Medical products, Inc., Boulder, CO), a silicone rubber compound (27). After the Microfil had hardened, peritoneal lining tissues were dissected free, fixed in formalin, dehydrated in a sequence of ethyl alcohols, INTRODUCTION and cleared in methyl salicylate for examination in a Wild macroscope. Tissue Processing for Microscopy. Tissues lining the peritoneal cavity Experimental tumors growing in solid form commonly initiate the (peritoneal walls, diaphragm, and mesentery) were fixed and processed for generation of vascularized stroma by rendering local blood vessels 1-p.m-thick, Epon-embedded and Giemsa-stained light microscopic sections hyperpermeable to plasma proteins (1â€”5).As a consequence of vas (18, 27). To evaluate blood vessel size and frequency at various stages of cular hyperpermeability, plasma fibrinogen, normally confined to ascites tumor growth, 1-pm Epon sections of randomly selected portions of plasma, extravasates and clots in the extravascular space to form a mesentery were photographed at constant magnification (X675). Color prints fibrin gel; such gels provide a provisional stroma which imposes (4 x 6 inches;Ektar25; EastmanKodak)were prepared,andall microvessels structure on solid tumors and stimulates the ingrowth of new blood (defined as vessels lacking muscle in their walls) were identified, and their vessels and of fibroblasts which synthesize and deposit the matrix lumens were outlined with a photo marking pen. Individual prints were then proteins and proteoglycans of mature tumor stroma (6, 7). In contrast, imaged with a video camera into a computer equipped with IM-series version it has been thought that ascites tumors do not generate a new sup 3.46p software (Analytical Imaging Concepts). Once digitized, measurements of vessel size (diameterand cross-sectionalarea) and density (numberof porting stroma unless they implant on peritoneal surfaces; rather, vessels per unit area) were calculated. they grow as a suspension of malignant cells in a plasma exudate, Identification of Hyperpermeable Blood Vessels and Endotheilal Celia ascites fluid, that is comparable to the interstitial fluid of solid tumors That Had Incorporated [3H]Thymldlne. To identify leaky tumor blood (8-17). vessels, mice received colloidal carbon i.v. 1 h prior to harvest (18). In some experiments,animals were also injected i.v. with [3H]thymidine(25 @Ci/ Received 6/1/94; accepted 11/11/94. mouse; Du Pont New England Nuclear, Boston, MA) 2 h prior to harvest. The costs of publication of this article were defrayed in part by the payment of page Mesenteries were fixed and processed for 1-p.m Epon sections as above. Epon charges. This article must therefore be hereby marked advertisement in accordance with sections were coated with Kodak NTB-2 emulsion for autoradiography. Sec 18 U.S.C. Section 1734 solely to indicate this fact. tions from randomly selected blocks were examined in their entirety at a I This work was supported by NIH research Grants CA-40624 and CA-58845 and under terms of a contract from the National Foundation for Cancer Research and by a magnification of X1000; all blood vessels were identified, and carbon-labeled grant from the BIH Pathology Foundation, Inc. Part Three of a series on â€œPathogenesisof (hyperpermeable) blood vessels and [3H]thymidine incorporating (proliferat Ascites Tumor Growth.â€• 2 To whom requests for reprints should be addressed, at Department of Pathology, Beth Israel Hospital, 330 Brookline Avenue, Boston, MA 02215. 3 The abbreviation used is: VPF, vascular permeability factor. 376

ANGIOGENESIS/STROMAGENERATION IN ASCITES TUMORS

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@@@ .1T@TiiiT L.. . Fig. 1. Peritoneal walls ofmice bearing TA3/St (A and B)or MOT(C-F) ascites tumors. A and B, peritoneal walls of7-day TM/St ascites tumor-bearing mice exhibit micmvascular congestion, microvascular hyperpermeability (carbon leakage, arrowheads), interstitial edema (spreading apart of muscle bundles), and activation of surface mesothelial cells. C and D, peritoneal walls of 16-day MOT ascites tumor-bearing mice illustrate dramatic increase in size and number of superficial microvessels (v) E, another 16-day peritoneal wall to illustrate collagen deposition (c) between muscle bundles in space formerly occupied by fibrin deposits (Fig. 2; Ref. 19). Blood vessels (arrowheads) have reverted to smaller size. F, peritoneal wall at 27 days after i.p. injection of MOT tumor cells shows piling up of tumor cells on the peritoneal surface with superficial blood vessels (arrowheads) and underlying collagen(c)deposition.Eventhemostsuperficialbloodvessels(arrowheads)havereturnedtoa smallersize.Allare1-gunthick,Giemsa-stainedEponsections.AandB, X430(6w', 16 g@m);C,D, and F, x 270; E, X 340.

ing) endothelial cell nuclei were separately counted. More than 5000 individual becoming large, thin-walled channels with persistent hyperpermeabil vessels and a somewhat larger number of endothelial cell nuclei were counted ity to colloidal carbon (Fig. 1, B-D). These vessels became clustered in the course of these studies. superficially in the muscular layer of the peritoneal wall and dia Statistics, Data did not fit a Gaussian distribution, and there were signifi phragm and for the most part were oriented parallel to the surface; cant differences in the SDs of the various test groups; therefore, data were lining endothelial cells appeared activated with enlarged nuclei, in analyzed with nonparametric tests (Kruskal-Walhis and Dunn's multiple com parisons test) as described previously (18, 29). creased basophilic cytoplasm (Fig. 1B), and frequent mitotic figures. Overall vascular proliferation and vessel orientation parallel to the peritoneal surface were readily appreciated by en face macroscopic RESULTS examination of peritoneal walls following perfusion with Microfil Microvascular Changes in the Peritoneal Lining Tissues of (Fig. 2). Mice Bearing Syngeneic MOT and TA3/St Ascites Tumors. In The mesentery underwent changes similar to those of the peritoneal both animal models, the vasculature supplying the peritoneal wall and wall and diaphragm, but the process evolved with a faster tempo. In diaphragm underwent important changes within a few days of i.p. normal mesentery, capillaries were relatively inconspicuous and of tumor cell injection. As noted previously (18, 19), the initial change normally small size (Fig. 3A). However, within a few days of i.p. was microvascular hyperpermeability to circulating macromolecules, injection of tumor cells, preexisting microvessels became greatly followed by extravasation of plasma fibrinogen and its clotting to enlarged and became grouped together in prominent clusters (Fig. 3, cross-linked fibrin. Leaky microvessels were often distended with B and C). Like their counterparts in the peritoneal wall (Fig. 1) and erythrocytes, suggesting sludging of blood flow (Fig. 1). Thereafter, diaphragm, these vessels were congested with erythrocytes and were microvessels underwent a dramatic increase in cross-sectional area, hyperpermeable to colloidal carbon. By 7 days (TM/St) or 10â€”15 377

Fig. 2. Enface macroscopic view of Microfil White-perfused peritoneal walls of a control C3Heb/FeJ mouse (A) and of mice 12 (B) and 16 (C) days after i.p. injection of 1 X 1O@ MOT tumor cells. Note large increase in the number of perfused vessels in ascites tumor-bearing (B and C) as compared with control animals (A). In all instances, most vessels are oriented parallel to the peritoneal surface. All X 8. days (MOT), clusters of enlarged, thin-walled microvessels had in 12 of 59% (Fig. SB). Thereafter, the frequency of carbon-labeled creased in prominence and had become concentrated at the mesenteric vessels declined but remained significantly elevated above control surface (Figs. 3, D-G). Individual vessels bulged into the peritoneal levels as late as day 28. As with the TM/St tumor, labeling of cavity (Fig. 3, D-G; Fig. 4). Projecting vessels commonly retained mesenteric microvascular endothelial cell nuclei with [3H]thymidine broad-based attachments to the underlying mesentery (Fig. 3, D-G; developed more slowly than mesentenc microvascular carbon label Fig. 4A); sometimes, however, projecting vessels with associated ing in MOT ascites tumor-bearing mice; statistically significant thy connective tissue formed long stalks that extended for significantly midine incorporation did not occur before day 8 when 22% of endo greater distances (i.e., 125 @m)into the peritoneal lumen (Fig. 4B). thelial cells counted were labeled (Fig. SB). Thymidine labeling Bulging and protruding peritoneal surface vessels were fragile and declined progressively thereafter, although remaining significantly highly susceptible to injury and bleeding (Fig. 4B). This was espe above control levels at least through day 20. cially true in TM/St-bearing animals whose ascites fluid was com An additional feature of the mesenteric microvasculature in ascites monly bloody by day 8; i.p. hemorrhage was often sufficient to cause tumor-bearing animals was a change in the ratio of endothelial cell serious anemia or death. Even at the height of the response, vascular nuclei encountered per vascular cross section (Fig. 6). This ratio and interstitial changes were confined to fatty-vascular portions of the provides a collective measure of the number and (to a lesser extent) mesentery; portions of mesentery that lacked adipose tissue remained the size of endothelial cell nuclei. In normal control A/Jax and avascular. C3HebfFeJ mice, the value of this ratio was 0.4 Â± 0.1 and As also observed in the peritoneal wall and diaphragm, tumor cells 0.65 Â±0.04, respectively. In TM/St ascites tumor-bearing bearing commonly adhered to the mesenteric surface, individually and in mice, the ratio increased more than 3-fold to a peak on day 8 (just clumps; attachments were distributed over both fatty and nonfatty prior to animal death) of 1.31 Â±0.11. In MOT-bearing mice, the ratio portions of mesentery, in all cases TM/St >> MOT (Fig. 3H). As increased more than 4-fold to a maximum on day 15 of 1.82 Â±0.11; noted previously (18), attached TM/St tumor cells often invaded the thereafter, it declined slightly but remained significantly elevated mesentery, whereas MOT tumor cells only did so rarely. However, through day 28. mesenteries of MOT-bearing mice showed striking, progressive loss MorphometricAnalysisof the Size and Frequencyof Mesen of fat cell lipid content and reversion of fat cells to a more undiffer teric Blood Vessels as a Function of Ascites Tumor Growth. To entiated phenotype with prominent basophilic cytoplasm and acti test our impression that the blood vessels present in peritoneal lining vated nuclei (Fig. 3, E and F). A modest mononuclear cell and/or tissues underwent a striking early increase in cross-sectional (luminal) plasma cell infiltrate was sometimes also present. area and frequency, we performed morphometric analysis on meson Kinetics of Microvascular Hyperpermeability vis-Ã¢-visEndo teries harvested at successive intervals after i.p. injection of either thelial Cell DNA Synthesis. In control mice, hyperpermeable (car MOT or TM/St tumor cells. The data in Table 1 indicate that, in fact, ben-labeled) mesenteric vessels were not observed and only rare the mean luminal cross-sectional areas of blood vessels increased mesentenc vascular endothelial cells incorporated [3H]thymidine significantly within 6â€”7days of tumor cell injection. In TM/St (Fig. 5). However, as early as 2 days after i.p. injection of 106 TM/St bearing animals, mean mesenteric vessel area increased substantially, tumor cells, 2.4% of mesenteric vessels were carbon-labeled, and this rising from control levels of 28 Â±5.3 @am2toa value of 121 Â±15 frequency increased to a maximum of 43% by day 6, falling slightly @.un2at8 days after i.p. tumor cell injection, a 4.3-fold increase; later thereafter until animal death after day 8 (Fig. 5A). Incorporation of values could not be obtained because most animals died shortly after [3H]thymidine by vascular endothelial cells was insignificant in con day 8. In MOT tumor-injected animals, mean cross-sectional vessel trol animals. In TM/St-injected mice, [3Hjthymidine incorporation area increased from 32 Â±5 p.m2 in control animals to 139 Â±26 gun2 was slower to develop than carbon labeling, becoming statistically by day 6 (a 4.3-fold increase) and to 489 Â±48 g.am2by 8.5 days (a significant only on day 6 when 42.1% of endothelial cells were 15.3-fold increase). Thereafter, mean cross-sectional vessel area de labeled. Thereafter, [3Hlthymidine labeling declined somewhat but dined but remained significantly above control values as late as day remained significantly elevated to the time of animal death. 23. Similar patterns of carbon and [3H]thymidine labeling were ob The increase in average vessel size that accompanied ascites tumor tamed in MOT ascites tumor-bearing mice, but the process developed growth was followed by an equally dramatic increase in the hetero more slowly (Fig. SB). Four days after i.p. injection of 1 X 106 MOT geneity of vessel cross-sectional area (Figs. 7 and 8). Thus, 2 days tumor cells, 5.6% of 387 mesenteric vessels counted were carbon after i.p. injection of TM/St tumor cells into A/Jax mice, almost 90% labeled, and this percentage increased progressively to a peak on day of mesenteric vessels had cross-sectional areas of 50 gun2 and only 378

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Fig. 3. Mesenteries of mice bearing MOT (A-F and H) or TM/St (G) ascites tumors. A, at 0â€”2daysafter i.p. injection of 1 X 1O@MOTtumor cells, the fatty, vascular mesentery appeared normal, consisting of numerous lipid-filled adipocytes (fat cells,J) separated by loose connective tissue stroma and small, inconspicuous blood vessels (arrowheads). B, at 6days,scatteredbloodvessels(arrowheads)hadenlargedincross-sectionalareaandwerehyperpermeable(carbonlabelnotwellvisualizedintheseblackandwhitephotomicrographs). There was also slight interstitial edema and a mild mononuclear cell infiltrate. pe, peritoneal cavity. C, at 8 days, vascular changes were more pronounced. Enlarged, thin-walled, hyperpermeable blood vessels were prominently clustered in the fatty mesentery. pc peritoneal cavity. D, at 12 days, enlarged vessels persisted within the fatty mesentery, but many vessels (v) were situated at or just beneath the mesenteric surface, bulging into the peritoneal cavity (pc). E and F, at 15 days, vascular hyperplasia persisted at the mesenteric surface, but vessels (v) were now of smaller size. Occasional enlarged, thin-walled vessels (v) remained more centrally located. Adipocytes U) exhibited a striking reduction of lipid content and appeared smaller with prominent basophilic cytoplasm. G, mesentery from an A/Jax mouse 7 days after i.p. injection of TM/St tumor cells. Enlarged, thin-walled, carbon-labeled (arrowheads) blood vessels (v) were prominent at the mesenteric surface and bulged into the peritoneal cavity (pc); adherent tumor cells commonly exhibited mitotic figures (rn) H, portion of normally thin, fat-free, avascular mesentery (M) with attached tumor cells, 28 days after i.p. injection of MOT tumor cells. A-C and E, X 220; D, F, and H, X 430; G. X 525.

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Fig. 4. Projection of mesenteric blood vessels into the peritoneal cavities of A/Jax mice 8 days after i.p. injection of 1 X 10@TA3/Sttumor cells. A, enlarged, engorged vessel (V)' still enveloped by activated mesothelial cells (meso), bulges into the peritoneal cavity (pc). Some of the carbon that had been injected iv. 1 h prior to harvest decorates the basement membrane (arrowheads), whereas other clumps of carbon particles persist in the vessel lumen between erythrocytes, indicative of sludged blood flow because normally carbon is entirely cleared from the circulation within 15 mm after iv. injection. Several smaller vessels (two of which are labeled, v) also exhibit basement membrane labeling with carbon (arrowheads), indicating hyperpermeabiity. Occasional macrophages(mac)within the peritoneal cavity have ingested carbon.f fat-laden adipocyte;pc, peritoneal cavity. B, projection of a thin-walled, carbon-labeled microvessel into the peritoneal cavity (pc); arrowheads, focal leakage of erythrocytes. Tumor cells and macrophages are attached to the vascular stalk. Othersmallvessels(v)are locatedatthemesentericsurface.A,X 675;B, X 460(bar, 17 @tm).

A B Microvessel density was also expressed as a percentage of total @ TM/St , b MOT r@i, b , ,@ tissue area, a measure that encompasses both microvessel size (cross sectional area) and frequency (Table 1). In TM/St ascites tumor bearing mice, the percentage of mesentery occupied by vessels in @ 0 :. j@ I creased from 0.4 Â±0.1% in control animals to 4.9 Â±1.1% at 8 days, a >12-fold increment. In MOT tumor-bearing mice, the percentage of mesentery occupied by vessels rose from control levels of 0.7 Â±0.4% Ii to a maximum of 18.2 Â±1.8% on day 15.5, a 26-fold increase. o_@LHI @_@LIiLT1@ Microvascular Remodeling. The new large-diameter blood yes 023 4 678 0246 8121520 sels that formed in the mesentery, peritoneal wall, and diaphragm Days Afterip Tumor Cell injection during the course of ascites tumor growth subsequently underwent an Fig. 5. Hyperpermeabiity of mesenteric microvessels to carbon and incorporation of unusual form of remodeling, beginning about days 7â€”8in mice [3H@thymidinebyendothelial cells lining these vessels as a function of time after i.p. bearing either ascites tumor. Remodeling was characterized by the injection of 1 x 1O'@TM/St(A) or MOT (B) tumor cells into A/Jax and C3HebIFeJ mice, respectively. Counts were performed on 1-sm Epon sections. Data are presented as a formationofâ€œbridgesâ€•ofendothelialcellcytoplasmicprocessesthat percentage of carbon-labeled mesenteric vessels and a percentage of mesenteric small vessel endothelial cells that incorporated [3H]thymidine(mean). a, P < 0.05; b, P < 0.01. n = 3 animals at each time point; bars, SE. A B

1.6% of vessels had areas >200 @m2;incontrast, by day 8, more than one-half of the mesenteric microvessels had cross-sectional areas >50 p.m2, and 11% had luminal areas >200 p.m2. The vascular size heterogeneity that developed in MOT ascites tumor-bearing animals was even more striking (Fig. 8). Thus, whereas nearly 90% of vessels in control C3HebIFeJ mice had cross sectional areas of S50 p.m2 and fewer than 2% had areas >200 p.m2. by day 8.5 after injection of MOT tumor cells almost 49% of vessels had areas I >200 p.m2, and 32% had luminal areas >400 p.m2. The number of microvessels per unit area also increased progres sively in the mesenteries of animals bearing ascites tumors (Table 1). I In TM/St-bearing mice, vessel frequency increased from control levels of 131 Â±11 vessels/mm2 to 404 Â±52 vessels/mm2 on day 8, 0 2 3 4 6 7 8 0 1 2 4 6 8121520 a 3.1-fold increase. Vessel frequency also increased in the mesenteries of MOT ascites tumor-bearing mice, rising from 110 Â±13 vessels/ Days After ip TumorCell Injection @2in control mice to 282 Â± 26 vessels/mm2 at 8.5 days, a 2.6-fold Fig. 6. Ratio of numbers of endothelial cell (EC) nuclei to number of microvessel increase. Vessel frequency continued to rise thereafter, achieving a profiles in the mesenteries of ascites tumor-bearing mice at various intervals after i.p. injection of 1 x 10'@TA3/St(A)or MOT (B) tumor cells. Counts were performed on 1-pm peak at 20 days (1223 Â±94 vessels/mm2, an 11.1-fold increase) Epon sections. Data are presented as the mean; bars, SE. b, P < 0.01. n = 3 animals at before falling off slightly on day 23. each time point. 380

Table1 Morphometricanalysisof vesselsize(cross-sectionalarea),frequency(numbersofvessels/mm2),andpercentageoftotaltissuearea occupiedbyvesselsinthe mesenteries ofconzrol and TA3/St or MOT ascites tumor-bearing AIJax or C3HebfFeJ mice size frequency areaâ€•Mean(cross-sectional area)aVessel (no. vessels/mm2)â€•Percentage of vesse1 tissue (%)MedianTA3/St0(control)TumorDaysNo. vesselsTotalarea (@un@)Vessel Â±SEM(psn@)MedianMean Â±SEM MedianMean Â±SEM Â±11 120 5 88 477,474 43Â±9.3 16 242Â±28 240 1.0Â±0.2 0.8 7 175 598,649 76Â±14*** 292Â±25' @4 2.2Â±0,4*** 2.2 4.0MOT0 8158 2461,199,549589,12028Â±5.3 121Â±15@12 49131 404Â±52' 3740.36Â±0.094.9Â±1,[email protected]

(control) Â±5.0 Â±13 90 Â±0.36 2 178 1,216,340 31Â±5.5 14 146Â±17 128 0.42Â±0.06 0.26 4 85 549,625 29 Â±6.6 15 155 Â±7.0 149 0.45 Â±0.10 0.35 6 168 1,062,018 139Â±26*** 44 158Â±28 149 2.14Â±0.44 1.35 8.5 315 1,114,222 489Â±48 191 282Â±26w 272 14.3Â±2.0 11.8 12 329 443,398 117Â±14 44 742Â±62w 731 12,3Â±1,3w 7.8 15.5 843 1,083,395 2O7Â±19*** 74 775Â±58w 862 18.2Â±1.8 16.6 20 624 510,413 75Â±14@@ 30 1223Â±94 1194 9.2Â±1.9 9.5 4,4a 23119 3851,078,134471,58832 61Â±6,9***19 25110 816Â±5O [email protected] 4.9Â±0.6'0.29 @ p

crossed the lumens of enlarged vessels; as a result, single large-bore DISCUSSION vascular channels became divided into two, three, or more channels of smaller diameter (Fig. 9). The vessels that underwent remodeling The data presented here demonstrate that TM/St and MOT ascites remained hyperpermeable, as judged by continued deposition of col tumors elicited a prominent vascularized connective tissue stroma in loidal carbon in their basement membranes (Fig. 9, arrowheads); the tissues lining the peritoneal cavity. This connective tissue stroma however, they remained patent, and intravascular clotting or throm was qualitatively similar to that generated in solid tumors but devel bosis was never observed. oped more slowly and formed a thin superficial rim in peritoneal lining tissues (peritoneal wall, mesentery, and diaphragm), i.e., stroma developed in a compartment separate from the vast majority of tumor cells which were suspended in ascites fluid. That ascites tumors 2d 5d 7d 8d induce angiogenesis and generate connective tissue stroma has not 10@ been generally appreciated; a literature search revealed only six pre Cl) vious citations describing stromal changes of the peritoneal lining in @8o@ ascites tumors (30â€”36).Two of these references are abstracts, and none provided a comprehensive description of ascites tumor stroma or attempted to elucidate pathogenesis. At their respective maxima, new blood vessels occupied 4.9â€”18.2% of total mesenteric area in TM/St and MOT ascites tumors, respec tively; these values are comparable to those (3â€”18%)reported for several murine tumors growing in solid form (reviewed in Ref. 37). Therefore, the angiogenesis that developed in ascites and solid tumors appeared to be of similar magnitude. Furthermore, angiogenesis and I@ ,[email protected]. L.1.@ Li., stroma formation in TM/St and MOT ascites tumors were preceded by events similar to those that preceded angiogenesis and stroma formation in these same tumors grown in solid form (21). In both Cross-sectionalArea (@im2) tumors, secreted VPF rendered blood vessels hyperpermeable, leading Fig. 7. Distribution of cross-sectional areas of mesenteric microvessels at various to extravasation of plasma fibrinogen and its clotting to fibrin; cx intervals up to 8 days after i.p. injection of 1 X 1O@TA3/St tumor cells into A/Jax mice. Vessels were sorted into bins on the basis ofsize (i.e., 0â€”50pm2,50â€”100pm2,100â€”150 travascular fibrin deposits, in turn, provided a provisional stroma that ,i.m2,etc.), and the frequency of each bin was plotted. A total of 632 microvessels was supported the ingrowth of new blood vessels and of fibroblasts which measured. synthesized and deposited structural proteins and proteoglycans (3, 7,

Fig. 8. Distribution of cross-sectional areas of mesenteric microvessels at various intervals up to I 23daysafteri.p.injectionof1 x 106MOTtumor cells into t3Heb/FeJ mice. Vessels were sorted into bins on the basis of size (i.e., 0â€”SOpm2, 50â€”100pm2, 100â€”iSOpm2,etc.), and the fre quency of each bin was plotted. A total of 3046 vessels was analyzed. @ @@@â€˜-@â€œ1!@â€˜@@Ã³!

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Fig. 9. Remodeling of microvessels in mesentenes of mice bearing 12-day MOT (A and B) or 8-day TM/St (C-F) ascites tumoN A and B, thin walled, dilated vessels are concentrated at the mesenteric surface and bulge into the peritoneal cavity (pc). Note multiple thin projections of endothelial cell cytoplasm that cross the vascular lumens (v), subdividing the lumens into multiple smaller channels. The large vessel in B (labeled twice with v) is subdivided into atleast 20 subchannels; there are, in addition, several cytoplasmic projections that, at least in this section plane, terminate blindly and do not bridge across the lumen (arrowheads). :, adherent tumor cells; f adipocyte. C and D, similar mesenteric vessels undergoing remodeling by transluminal bridging in 8-day TA3/St ascites tumor-bearing mice. E and F, tracings ofvascular endotheial cells in C and D, respectively, to illustrate more clearly transluminal bridging by endotheial cell processes. Endothelial cell cytoplasm is stippled; colloidal carbon deposits are indicated as slightly larger black densities. A and B, X 670; C and E, x 790; D and F, x 970.

26, 38). VPF is also a selective endothelial cell mitogen (hence, its metric analysis (Table 1), did not become statistically significant until alternate name, vascular endothelial growth factor or VEGF) and, day 6 (TM/St) or day 8 (MOT) and did not become maximal until day therefore, likely also promoted angiogenesis by stimulating endothe 8 (TM/St) or day 20 (MOT). The likeliest explanation for the delayed hal cell division. Taken together, it would seem that similar mecha kinetics of stroma formation in ascites tumors is that increased mi nisms are involved in the induction of angiogenesis in solid and crovascular permeability, the earliest step we have been able to ascites tumors. identify in the cascade of events leading to angiogenesis, is similarly Despite these similarities, angiogenesis and stroma generation de delayed; i.e., microvascular hyperpermeability began days after i.p. veloped with different kinetics in solid and ascites tumors. When tumor cell injection in contrast to hours after s.c. injection of the same TM/St or MOT cells were injected s.c. or intradermally to form solid number of tumor cells (18). Once microvascular permeability had tumors, angiogenesis was evident within 48â€”72h (21, 27). By con increased, subsequent events such as fibrin deposition and increased trast, following i.p. injection of the same number of tumor cells, vessel number proceeded rapidly as in solid tumors. angiogenesis, as measured by Microfil perfusion (Fig. 2), vascular Our studies uncovered an additional novel property of ascites tumor endothelial cell [3H]thymidine incorporation (Fig. 5), or by morpho growth, namely, that it is accompanied by an early and dramatic 382

Fig. 10. Schematic diagram illustrating the proposed sequence of vascular changes involving peritoneal lining microvessels during ascites tumor growth. Initially, microvessels of small calibre (A) became leaky (carbon deposits indicated between endothelial cell ablumens and underlying basement membrane) and increased in cross-sectional area (B); lining endothelial cells proliferated (Al, endotheial cell mitosis). Subsequently, vessels underwent remodeling by transluminal bridging of endothelial cell processes (C); bridges eventually subdivided enlarged vessels into multiple smaller vessels (D and E')comparable in size to those in (A) prior to i.p. tumor cell injection.

enlargement in the cross-sectional areas of peritoneal microvessels revealed that some laminin and type IV collagen persisted. Taken (Figs. 1, 3, 4, 7, 9; Table 1). In both normal A/Jax and C3Heb/FeJ together, these data are consistent with the possibility that basal mice, the mean cross-sectional area of mesenteric microvessels meas lamina proteolysis is among the earliest events in angiogenesis, lead ured â€”30gun2, corresponding to a mean vessel diameter of 6.2 p.m ing to reduction in basal lamina mass and integrity. Such changes, (assuming a circular cross-sectional profile). However, by 8 days after accompanied by maintenance of a positive pressure gradient across i.p. injection of TM/St or MOT ascites tumor cells, mean mesenteric the vascular wall (intravascular hydrostatic + colloid osmotic pres cross-sectional areas had increased enormously to 121 p.m2 and sure > extravascular hydrostatic + colloid osmotic pressure) would be 489 p.m2, respectively, in TM/St and MOT ascites tumor-bearing expected to cause vessels to dilate by stretching their walls and animals (corresponding to vessel diameters of -â€˜-12.4and 25 p.m. thinning and flattening their lining endothelium. This physical effect respectively). Increased microvascular size was accompanied by a could itself have important biological significance. Others have parallel increase in vessel size heterogeneity (Figs. 7 and 8). In normal shown, for example, that prolonged â€œstretchingâ€•ofthe blood vessel mice of both strains, microvascular cross-sectional area was relatively wall leads to increased vessel size (45) and that flattening of endo homogeneous; i.e., 90% of microvessels had cross-sectional areas thelial cells stimulates endothelial cell growth (49â€”51). It seems of S0p.m2(meandiameterof7.8p.m).However,8.0â€”8.5likely, therefore, that partial proteolysis of vascular basal lamina and days following i.p. tumor cell injection, only 50% of TM/St and consequent mechanical stretching are responsible for the observed <20% of MOT mesenteric microvessels fell into any single 50-p.m2 increase in ascites tumor microvessel cross-section and may simulta size bin, and cross-sectional areas of individual vessels varied from neously stimulate vascular endothelium to divide. <50 to >>2000 p.m2.With the further passage of time, mean vascular The formation of enlarged microvessels early in the course of cross-sectional area returned toward control levels in MOT-bearing ascites tumor angiogenesis may also relate to the fibrin stroma in animals, and vascular cross-sectional areas became less heterogeneous which these vessels develop. In an accompanying paper, we showed (Fig. 8). that fibrinogen extravasates from leaky blood vessels to form a fibrin Others have reported that the blood vessels of solid tumors may gel matrix in the peritoneal lining tissues of mice bearing TM/St or also be increased in size (27, 37, 39â€”44).More particularly, Less et MOT ascites tumors (19). Moreover, the diameters of newly formed a!. (41) reported a mean diameter of 10 p.m for capillary meshwork vessels were maximal in temporal association with maximum fibrin vessels in the rat R3230AC mammary adenocarcinoma, a value sig deposition. Relevant to these findings are studies of Nicosia and nificantly larger than that for capillaries in most normal tissues. Ottinetti (52), who used a three-dimensional culture system to study Similarly, Solesvik et al. (37, 43) found that mean vessel diameters vessel development in vitro. They related the size of newly forming ranged from 14.1 to 29.3 p.m in three solid syngeneic mouse tumors vascular sprouts to the extracellular matrix in which their cultures and in five melanoma xenografts. Therefore, increased average cross sectional area is likely a general property of tumor microvessels, both were embedded. Sprouts that formed in plasma clots or fibrin gels had solid and ascites. significantly larger mean luminal areas than vessels that formed in The mechanisms responsible for microvessel enlargement in ascites either collagen or Matrigel matrices. Taken together, these data sug (or solid) tumors have not been carefully investigated. Increased mean gest that the extracellular matrix influences microvessel morphogen vascular cross-sectional area was accompanied by vascular endothe esis and that fibrin matrices favor the generation of larger-sized hal cell proliferation. Therefore, increased microvessel size cannot vessels. The underlying mechanisms that might be responsible for simply be explained by dilation of pre-existing, normal-sized vessels determining this relationship are presently unknown. as might have resulted, for example, from a vascular response to An additional novelty of the enlarged vessels that form in ascites smooth muscle-relaxing metabolites (e.g., adenosine) released from tumor-bearing animals was their subsequent return toward normal size hypoxic tissues (45). by a peculiar form of vascular remodeling. Remodeling was accom Several groups have suggested that partial proteolytic breakdown of plished by the formation of endothelial cell cytoplasmic bridges which vascular basal lamina occurs as an early step in the cascade of events crossed vascular lumens; as a consequence of such bridging, individ leading to angiogenesis (46, 47). In support of this hypothesis, Palm ual larger vessels were divided into two, three, and often multiple and Paweletz (48) reported â€œexpansionâ€•ofindividual vessels sur smaller components (Figs. 9 and 10). To our knowledge, such changes rounding the rat BSp73 AS adenocarcinoma within 1â€”2days of have not been reported previously in other tumors. However, some transplant. These enlarged vessels, which they termed â€œmotherâ€•yes what analogous events have been described during primary vascular sels because they subsequently served as the source of the more ization of fetal lung (53), in cultured visceral capillary endothelial familiar tumor vessel sprouts, appeared by electron microscopy to cells (54), and, more recently, in the chorioallantoic membrane of the have lost much or all of their basal lamina; however, although basal developing chick, where the process has been referred to as â€œintus lamina ultrastructure was markedly altered, immunocytochemistry susceptive microvascular growthâ€•(55). To the extent that this analogy 383

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1995 American Association for Cancer Research. ANGIOGENESIS/STROMAGENERA11ON IN ASCITE5 TUMORS holds, it represents yet another example of malignancy mimicking 10. Klein,G.,andKlein,[email protected] ontogeny. Aced. Sd., 63: 640â€”661,1956. 11. Goldie, H., and Felix, M. D. Growthcharacteristicsof free tumorcells transferred An instructivefeatureof ascitestumorangiogenesisisthefailed seriallyintheperitonealfluidof themouse.CancerRes.,11: 73â€”81,1951. attempt of new blood vessel sprouts to extend any significant distance 12. Yoshida, T. The Yoshida sarcoma, an ascites tumor. Gann, 40: 1-21, 1949. from the peritoneal walls and mesentery into the lumen of the pen 13. Garrison, R. N., Kaelin, L D., Heuser, L S., and Galloway, R. H. Malignant ascites: clinical and experimental observations. Ann. Surg., 203: 644â€”649,1986. toneal cavity. At the height of the angiogenic response in both TM/St 14. Garrison, R. 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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1995 American Association for Cancer Research. Pathogenesis of Ascites Tumor Growth: Angiogenesis, Vascular Remodeling, and Stroma Formation in the Peritoneal Lining

Janice A. Nagy, Ellen S. Morgan, Kemp T. Herzberg, et al.

Cancer Res 1995;55:376-385.

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