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Characterization of a Transplan Table, Canine, Immature Mast Cell Tumor1

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Characterization of a Transplan Table, Canine, Immature Mast Cell Tumor1 (CANCER RESEARCH 32, 1434-1441, July 1972] Characterization of a Transplan table, Canine, Immature Mast Cell Tumor1 C. A. Bowles, W. T. Kerber, S. R. S. Rangan,2 R. Kwapien, W. Woods, and E. M. Jensen Hazleton Laboratories, Vienna, Virginia 22180 ¡C.A. B., W. T. K., S. R. S. R., R. K., E. M. J.f, and National Cancer Institute, NIH, Bethesda, Maryland 20014 ¡W.W.] SUMMARY This report presents detailed information about this tumor system. Results of serial passage, a histopathological A transplantable canine tumor has been established and description, results of tissue culture studies, and electron carried through 10 serial passages in newborn beagles without microscopic observations of the original and transplanted immunosuppression of the recipient dogs. Histopathologically, tumor are presented. the tumor appeared as a reticulum cell sarcoma, but ultrastructural studies by electron microscopy have shown the tumor to be an immature mast cell sarcoma. Histamine was MATERIALS AND METHODS demonstrated in the tumor cell extracts. Tumors were induced Spontaneous Tumor Donor. The original donor was a in dogs, no more than 10 days old, with IO6 cells. A tissue 7-year-old neutered female purebred beagle with an immature culture-propagated cell line was established from a second in mast cell neoplasm. The disease was characterized by vivo passage dog, and this has been found to induce tumors generalized weakness, anorexia, mild anemia, and enlargement after over 1 year of in vitro cultivation. Attempts to pass the of both superficial inguinal lymph nodes and the right tumor in vivo with cell-free extracts have not been successful, popliteal lymph node. The white blood cell count was and no type C or other identifiable virus particles have been 13,900/cu mm. Neoplastic cells were not observed in the observed in the tumor cells by electron microscopy. peripheral blood smear. The superficial inguinal lymph nodes were surgically removed and prepared for inoculation. Eleven INTRODUCTION days after surgery, the dog was killed and necropsied. The anterior mediastinal lymph nodes, deep inguinal lymph nodes, and spleen were markedly enlarged. The thymus was Unequivocal evidence that a virus is the causative agent of normal and skin lesions were not observed. A small perforated cancer in dogs has not been demonstrated, despite extensive ulcer, loosely covered by the omentum, was present in the efforts in recent years. Most of this work has centered around proximal duodenum. studies of transplantable malignant lymphoreticular tissue, Experimental Animals. Newborn dogs used for subpassage although transplants of nonlymphoid tumors have been study were obtained from a randomly bred colony of purebred reported also (1,11,15). beagles (Hazleton Research Animals, Cumberland, Va.). The Lymphosarcomas have been successfully transplanted with pups were derived by cesarean section and hand raised in cellular preparations through as many as 15 serial passages in sterile isolators except for a few that were whelped naturally irradiated, neonatal dogs (3, 6, 8, 9). Some of these and raised by the bitch. Pups in isolators were deprived of lymphosarcomas (3, 6, 9) were thought to be transmitted in early passage, but neoplasms could not be induced by cell-free colostrum by feeding of a sterile commercial liquid diet (Orphalac; Riviana Foods, Inc., Topeka, Kan.) during the first preparations of the tumor material and no evidence of virus 6 weeks after birth. The pups were inoculated i.p., usually particles was shown by electron microscopy. Lombard et al. (7) and Post et al. (12) successfully induced within 72 hr after birth, and examined at least twice weekly mast cell leukemias in nonirradiated, neonatal beagle dogs for evidence of tumor formation. Immunosuppression or through as many as 15 serial passages. Demonstration of virus irradiation generally was not used. particles in the tumor tissue was not clearly established, but Dogs with tumors were killed in the terminal stage of the the induction of neoplasms with filtered tumor extracts disease, and selected tissue was removed for subsequent suggests a viral etiology for this disease. passage and pathological evaluation. Those dogs that did not develop tumors were removed from the isolators between 5 In a preliminary report (2), we described a transplantable, and 6 weeks of age and placed in standard dog cages in an canine, immature mast cell tumor which was carried through 10 in vivo passages without irradiation of the recipient dogs. isolated area within the facility. They were maintained in this area without vaccination for up to 12 months. Inocula Preparation. The inguinal lymph node of the donor 1This study was conducted under Contract NIH-69-2079 within the dog was minced, washed with balanced salt solution, and Special Virus Cancer Program of the National Cancer Institute, NIH, trypsinized according to standard procedures. Cells were USPHS. suspended in Eagle's minimum essential medium with 'Present address: Tulane University, Delta Regional Primate Re search Center, Covington, Louisiana 70433. penicillin (100 units/ml) and streptomycin (100jug/ml) so that Received January 13, 1972; accepted March 23, 1972. the final solution contained approximately 2X IO8 cells/ml. 1434 CANCER RESEARCH VOL. 32 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1972 American Association for Cancer Research. A Canine Immature Mast Cell Tumor Cells derived from ascitic fluid of dogs with transplanted Ascitic fluid cells obtained from 2nd- and 5th-passage dogs tumor or from floating cells propagated in tissue culture were were prepared with a tissue grinder as 20% extracts in 0.9% washed with balanced salt solution and resuspended in Earle's NaCl solution. The extracts were clarified by centrifugation at base minimal essential medium. Cell-free inocula consisted of 10,000 X g for 20 min. A portion of the clarified extract was 10 or 20% extracts of tumor tissue or tumor concentrates heat inactivated by boiling for 15 min. This material was prepared by the method of Moloney (10). centrifuged, and the supernatant was collected. All samples, Histológica! Methods. Necropsy examination was performed boiled or unboiled, were administered i.v. on all animals. Tissues were fixed in 10% neutral buffered Tissue Culture. Cells for in vitro propagation were processed formalin or Zenker's solution and were prepared for from solid tumor tissue by standard trypsinization procedures. Cells from ascitic fluid were processed without trypsin. Sterile microscopic examination by embedding in paraffin. Sections 32-oz prescription bottles containing 40 to 50 ml of Hanks' were cut at 3 to 6 m/¿routinely and stained with hematoxylin and eosin. Selected tissues were stained with base minimum essential medium, supplemented with 20% fetal May-Grunwald-Giemsa, 0.5% toluidine blue 0 or Luna's stain bovine serum, penicillin (100 units/ml), and streptomycin for mast cells, Gridley's reticulum stain, and periodic (100pig/ml), were seeded with 5 X IO6 cells and incubated at 37°.The culture grew as a mixture of monolayer and floating acid-Schiff reaction. Electron Microscopy. Tumor nodules and ascitic fluid cells cells. Media changes were made twice each week at which time for electron microscopy were obtained by biopsy or at floating cells in the supernatant fluid were transferred to fresh necropsy. Cells from the various subpassage levels of tissue 32-oz bottles. A floating culture was thus established and was culture cell line 32043 were also processed. fed by withdrawal of half of the supernatant fluid and cells Tissues 1 to 2 cu mm in size were processed as described and replacement with an equal volume of fresh media twice elsewhere (13). Ascitic fluid and tissue culture cells were each week. initially pelleted by low-speed centrifugation, and the pellets were processed similar to the solid tissues. Thin sections were cut on a Porter-Blum MT-1 ultramicrotome and were double RESULTS stained with alcoholic uranyl acetate, then with lead citrate. Sections were examined in a Philips 200 electron microscope. Transplant Studies Pharmacodynamic Study. For determination of the pharmacological activity of the tumor cells, 2 adult beagles Transplanted Tumor Dogs. Summarized in Table 1 are the (each weighing about 10 kg) were anesthetized by i.v. injection results of 10 serial passages representing 36 of 47 dogs of pentobarbital sodium, 30 mg/kg. The dogs were prepared inoculated by the i.p. or i.m. routes with whole-cell for measurement of respiration by endotracheal cannulation preparations. The table also shows the cell inocula, latent and for arterial blood pressure by a cannulated femoral artery. periods, and the times to death of pups at various passage Both values were recorded on a Sanborn polygraph. levels. These in vivo passages are designated as Series A. A Responses to appropriate doses of epinephrine (1 /ug/kg), separate in vivo passage designated Series B, which is described acetylcholine (5/ag/kg), norepinephrine (1.5jug/kg), and later, was initiated with tissue culture cells from a 2nd-passage histamine (2 /ug/kg), as standard reference agents, were dog (32043) of Series A. obtained. The standard reference agents were prepared in 0.9% Latent period and death time of early-passage, Series A dogs NaCl solution and administered i.v. in total volumes less than ranged from 1 to 2 months, and neoplasms were characterized 1.0 ml. Diphenhydramine hydrochloride was used as an by massive tumor growth. In later passages, the induction and antagonist of histamine. death time were greatly reduced, and less extensive gross Table 1 Summary of Series A in vivo passages of the immature mast cell tumor in newborn beagles Inoculum Mean time (days) to Passage12345678910No.ofcellsl.OX RouteLymph 10'7.0 i.p.Ascitesnode X10sl.OX i.p.Ascites 10'1.8X i.p.Ascites 10sl.OX i.p.Ascites 10'2.9 i.p.AscitesLeg X10"l.OX 10s1.5 tumorPooled X10'l.OX tumors0AscitesAscites.m..p.•P-•P-•P-Results"6/64/108/8l/5b6/64/52/2l/ld2/22/2Induction663530291313151069Death83443748282823352318 10'7.5 X 10'Source 0 No.
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