[CANCER RESEARCH 39, 2923-2927, August 1979] 0008-5472/79/0039-0000$02.0O Collagen Synthesis in Capsules Surrounding Dimethylbenzanthracene induced Rat Breast Tumors and the Eftect of Pretreatment with fJ-Aminopropionitrile1 I. Kelman Cohen, Charles W. Moncure, Raphael J. Wltorsch, and Robert F. Dlegelmann2 Departments of Surgery (I. K. C., R. F. Di, Pathology (C. W. M.j, and Physiology (R. J. W.j, MCV/VCU Cancer Center (R. F. DI, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298 ABSTRACT MATERIALSAND METHODS Collagen synthesis is increased over three-fold in capsules surrounding dimethylbenzanthracene-induced rat breast tu mors compared to the tumor parenchyma and over six-fold compared to normal breast connective tissue. Increased col lagen synthesis is independent of the rate of tumor growth and final tumor size. Pretreatment of animals with $-aminopropio nitrile to inhibit collagen cross-linking caused an 82% decrease in tumor formation and a significant reduction in tumor volume (approximately 0.4 cu cm) compared to controls (approxi mately 10 cu cm). The four small tumors that did develop in the lathyritic animals had increased collagen synthesis in the inte nor tumor stroma and reduced collagen synthesis in the tumor capsule. These findings suggest that the collagenous capsule surrounding dimethylbenzanthracene tumors functions as a physical barrier to protect the tumor from the immune system of the host. The apparent antitumor effects of $-aminopropio nitrile may be due to immunopotentiation and/or cytotoxic actions of the drug. INTRODUCTION Most benign and malignant neoplasmsare associated with a collagenousstroma. The variable quantityand quality of this collagenous tissue in and surrounding tumors has never been correlated with tumor virulence. For example, both scirrhous carcinoma and benign fibroadenoma of the breast are com posed of a dense collagenous matrix while other benign and malignant neoplasms may be mainly cellular with a scant col lagenous stroma. As an initialstudy to define some of the basic parametersof collagenmetabolismin malignanttumorand the significanceof a tumor capsule, the DMBA3-induced rat mammary carcinoma model was chosen because it has a well-defined collagen capsule and does not metastasize (8). The objective of this study was to measure collagen and total protein synthesis within the tumor parenchyma compared to tumor capsule and to correlate these findings with the histological appearance of the tumor. Moreover, we sought to determine if alteration in the collagenous stroma by inhibition of collagen cross-linking with BAPN (9) would inhibit capsule formation and allow the DMBA rat breast tumor to metastasize. I This investigation was supported by Grant CAl 7297, awarded by the Na tlonal Cancer Institute, Department of Health, Education, and Welfare. A prelim mary report of a portion of this work was presented (4). 2 To whom requests for reprints should be addressed, at Box 756, Medical College of Virginia, Richmond, Va. 23298. 3 The abbreviations used are: DMBA, dimethylbenzanthracene; BAPN. fl-aml nopropionitrile. Received January 15, 1979; accepted April 24, 1979 Induction of DMBA Rat Breast Tumors by a Multiple Dose RegImen. Breast tumors were induced in female Sprague Dawley rats by jugular i.v. pulse doses of a lipid emulsionof DMBA (2 mg/dose) at 50, 53, and 60 days of age (8). Collagen Synthesis Measurements. Collagen synthesis was measured in vitro (5) by incubating finely minced (0.5 mm) fresh biopsies (100 mg) of normal connective tissue, tumor capsule, and tumor in 3 ml of Krebs-Ringer medium containing 10 @Ci of 5-[3Hjproline (49 Ci/mM; Schwarz/Mann, Orange burg, N. V.). After a 2-hr incubation at 37°,the samples were frozen, sonicated, and homogenized. Unincorporated [3H]proline was removed by repeated 5% tnchloroacetic acid precipitation at 4°,and the radioactive substrate was then washed twice with ethanol:ether(3:1 , v/v) and once with absolute ether to remove the trichloroacetic acid. The precipitated protein was dried slowly to a powder and desiccated under vacuum to a constant dry weight. The dried protein was weighed, dissolved in sodium hydroxide, neutralized, and digested with purified bacterial collagenase as described previously (5, 10). After 90 mmat 37°,the digestion with purified bacterial collagenase was stopped by the addition of an equal volume of 10% trichloroacetic acid:0.5% tannic acid and centnfugation (10). Collagen digestion products in the supernatant fluid and radioactive noncollagen protein in the precipitate were each analyzed by liquid scintillation spectrom etry. All samples were counted in the presence of 1.5 ml of 5% trichloroacetic acid:0.25% tannic acid and were completely dissolved in 10 ml of Triton:Liquifluor(1 :2, v/v) prior to analysis (10). Radioactivity released by collagenase was used as a meas ure of collagen synthesis, and the remaining, undigested pro tein was a measure of noncollagen protein synthesis. Collagen and noncollagen protein synthesis were quantitated per mg tissue dry weight. Measurement of DNA. The procedure of Webb and Levy (1 1) was used for DNA quantitation. AnimalExperiments. Ininitialexperiments, collagenbiosyn thesis was measured in the capsule and parenchyma of estab lished DMBA rat breast tumors and compared to nontumorous breast connective tissue. In subsequent studies, the effect of BAPN on the inductionof DMBA tumors was evaluated in the following treatment groups. Four groups of 12 female Sprague Dawley rats (190 g) were treated as follows:Group 1, 0.5 to 1% fumarate in their diet (control); Group 2, 1 to 2% BAPN: fumarate in their diet (BAPN supplied by Aldrich Chemical Co., Inc., Milwaukee, Wis.); Group 3, 0.5 to 1% fumarate in their diet 2 months prior to tumor initiation; Group 4, 1 to 2% BAPN in their diet 2 months prior to tumor initiation. Animals were AUGUST 1979 2923 Research. on November 7, 2015. © 1979 American Association for Cancer cancerres.aacrjournals.org Downloaded from