Transplantability of Naturally Occurring Benign and ... · benign from malignant tumors (1, 7,11). Descriptive terminology for neoplasms has evolved since the 19th century (6, 25,
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[CANCER RESEARCH 44, 2608-2615, June 1984]
Transplantability of Naturally Occurring Benign and Malignant Neoplasmsand Age-associated Nonneoplastic Lesions of the Aging F344 Rat asBiological Evidence for the Histological Diagnosis of Neoplasms1
Jerrold M. Ward2 and Peter H. Lynch
Tumor Pathology and Pathogenesis Section, Laboratory of Comparative Carcinogenesis, Division of Cancer Etiology, National Cancer Institute, NIH,Frederick, Maryland 21701
ABSTRACT
Portions of 162 naturally occurring neoplasms and 26 nonneo-
plastic lesions from 93 aged male or female F344/NO rats wereimplanted into the left inguinal mammary fat pads of weanlingsyngeneic recipients. As controls, 95 normal tissues were implanted to the right inguinal fat pad. Transplant recipients weremaintained for up to 1 year. Essentially, all types of naturallyoccurring benign and malignant tumors were successfully transplanted, i.e., grew progressively forming nodules and masses.For the transplants, the latency period preceding palpablegrowth, tumor growth rate, invasiveness, metastatic rate, andtime to death were associated with the degree of histologicalmalignancy of the primary tumor. The tumors which were themost malignant based on these criteria included large granularlymphocyte leukemia, sarcomas, and carcinomas. Fibromas,mammary fibroadenomas, and papillomas were easily transplanted but were not invasive. Endocrine tumors generally werethe slowest-growing tumors. This study provides evidence that
successful tumor transplantation is only evidence of neoplasiaand does not distinguish whether a primary tumor is benign ormalignant.
INTRODUCTION
Tumor transplantation has been utilized to study the autonomous growth of neoplasms and to differentiate neoplastic fromnonneoplastic lesions (4, 7, 13, 14, 23, 24, 35). Transplantedtumors grow progressively and may kill the host. Some investigators believe transplantation of a tumor is the ultimate proof ofmalignancy (4, 23, 24, 35), while others provide evidence thattransplantation is proof of neoplasia but does not distinguishbenign from malignant tumors (1, 7,11).
Descriptive terminology for neoplasms has evolved since the19th century (6, 25, 34, 35). Benign tumors, which grow autonomously and progressively, must be distinguished from hyper-
plastic lesions which are reaction to injury or which may precedeneoplasia and are reversible. Benign tumors are histologicallywell differentiated, are delineated from adjacent normal tissue,and do not invade or metastasize (6, 7, 18, 20, 34). Clinically,such tumors may be removed surgically with an excellent prognosis but if not removed may cause death, usually by mechanicalinterference with adjacent normal tissues. Malignant tumors mayarise de novo, from benign neoplasia or from hyperplastic lesions(7, 23, 26, 31, 32). They are defined as tumors with histological
1This project has been supported in part by USPHS Contracts N01-CO-21910
to Program Resources, Inc., and N01-CO-23912 to LJtton Bionetics, Inc.2To whom requests for reprints should be addressed.
Received November 15, 1983; accepted February 27, 1984.
characteristics of poorly to well-differentiated tumor cells; inva
In F344 rats, tumors are seen which occur spontaneously andfit the description of "benign" tumors given above (3, 8, 10, 26,
27, 31 ). These include fibromas in dermal areas of the mammaryfat pads, mammary fibroadenomas, certain endocrine tumors,cutaneous papillomas, and uterine stromal polyps. Several isolated reports have indicated the transplantability of at least somehistologically benign tumors in rodents including mammary fibroadenomas (11, 24) and skin papillomas (1). There are nostudies on the transplantability of representative naturally occurring tumors of all sizes and the association of transplantationwith histological appearance and biological behavior in the primary host. The study described herein attempts to define transplantation characteristics of histologically benign and malignanttumors occurring naturally in the aging F344 rat in an effort toprovide a biological basis for histological tumor diagnosis.
MATERIALS AND METHODS
Ninety-three male and female retired breeder F344/NCr rats were
obtained from the National Cancer Institute Animal Genetics and Production Branch and Animal Program, Frederick, MD, and maintained 3/cagein polycarbamate cages with hardwood bedding until 20 to 38 monthsold. They were fed Wayne Sterilizable Lab-Blox (Allied Mills, Inc., Chi
cago, IL) and water ad libitum. They were sacrificed at various ages, anda selective necropsy was performed on each rat.
Two- to 3-mm-square portions of grossly visible tumors, nonneoplastic
lesions, or grossly normal tissues were transplanted into the inguinalmammary fat pad of normal weanling (4- to 5-week-old) male or female
F344/NCr according to the method of Williams er al. (15, 16, 32, 33).Ketamine hydrochloride (Ketaset; Bristol Laboratories, Syracuse, NY)anesthesia was used at a dosage level of 50 mg/kg i.m. A small pocketwas prepared in the left or right inguinal fat pad; one tissue implant wasplaced in each pocket. The pocket was closed with a purse string sutureof 4-0 surgical silk to enclose the implant and provide a gross and
histological marker of the implant site. The skin was closed with stainlesssteel clips that were removed 1 week later. Usually, tumor tissue wasplaced in the left inguinal fat pad, and a normal portion of the sametissue was placed in the right inguinal fat pad of the same rat. Eachtumor was implanted in only one rat. Transplant sites were palpated,and rats were weighed weekly. When tumors appeared, they weremeasured weekly and their progression was recorded. Rats died or weresacrificed only when moribund or when the transplant became largeenough to interfere with the rat's normal functions. All surviving rats
were sacrificed 1 year after transplantation. Selected tumors werepassed into second- and third-generation recipients.
All donor rats and transplant recipients were necropsied, and selectedtissues fixed in 10% neutral buffered formalin or Bouin's solution. Por
tions of primary and transplanted tumors, regional lymph nodes adjacentto the primary tumor or tumor transplant, and lungs were fixed fromeach rat. For diagnosis of LGL3 leukemia in donor or recipient rats, spleen
and liver were fixed from the majority of the rats. Tissues were embeddedin paraffin, sectioned at 4 to 6 /¿m,and stained with hematoxylin andeosin. Selected tumors were stained by one of the following methods:periodic acid-Schiff; Masson's trichrome; or Wilder's reticulin. Tumors
from donor rats were classified by conventional histological criteria.Primary benign neoplasms were well differentiated and well circumscribed and did not invade adjacent normal tissues. Benign tumors ofrats, however, usually have no capsule. These tumors grow by expansion. Primary malignant neoplasms were usually less differentiated orhad areas of various degrees of differentiation, had more mitotic figuresthan did benign tumors, and invaded adjacent normal tissues to varyingdegrees. Some malignant neoplasms metastasized to distant tissues.
For immunoperoxidase localization of a cell surface antigen reactingwith monoclonal OX-8 antibody, the avidin-biotin-peroxidase complexmethod was used (12, 22, 29). The mouse monoclonal OX-8 antibodywas obtained from Sera-Lab, Sussex, United Kingdom, and the Vectas-
tain ABC Kit was used (Vector Laboratories, Inc., Burlingame, CA).
Histological Appearances of Transplanted Tumors. In general, the histological appearance of the first passage of each
3The abbreviations used are: LGL, large granular lymphocyte; NK, natural killer
cell.
tumor was identical or similar to that of the primary tumor.Histologically malignant primary neoplasms including sarcomasand carcinomas usually were invasive when transplanted. LGLleukemias disseminated quickly from the transplant site (22, 30).The site usually was only slightly enlarged while the spleen, liver,lungs, and other organs were severely infiltrated by leukemiccells. Only 4 transplants other than LGL leukemias metastasizedto the lungs. These included one each of the following tumortypes: fibrosarcoma; carcinoma of the auditory sebaceousglands; pheochromocytoma; and mesothelioma.
Many transplant recipients died of LGL leukemia. Aged F344/NCr rats (2 to 3 years) have a 65% incidence of LGL leukemia(28). The aged rat donors usually had typical lesions of fulminating LGL leukemia (30, 31). Some donor rats had evidence of theearly stage of LGL leukemia (12, 31) and a few donor ratsappeared histologically normal without evidence of early or lateLGL leukemia. The stroma of some primary tumors which weretransplanted into weanling recipients was composed of bloodvessels or connective tissue containing LGL leukemia cells, asshown by the OX-8 cell surface antigen marker (22, 29, 30) (Fig.
1). Some primary epithelial and endocrine tumors were transplanted with leukemia cells growing within the transplant in therecipient rat (Fig. 2). In some rats that died with leukemia, themammary fat pad site of tumor implantation contained foci ofresting or degenerative nonleukemic tumor cells or small epithelial or endocrine tumors in an early stage of growth. Characteristicresting tumors were composed of an island of cells similar tothose in the primary tumor but which were atrophied, degenerative, or well differentiated (Fig. 3). Early tumors appear to arisefrom these resting cells (Fig. 4). These small implants had fewreactive lymphocytes and scant connective tissue stroma orcapsule. Histologically, transplanted tumors were nodules ormasses, sometimes with areas of connective tissue capsuleformation. They usually were identical in morphology to theprimary tumor (Figs. 5 to 10). Some tumors had variable patternswhich were different than those of the primary tumor. Theprimary pulmonary adenocarcinoma (composed primarily of alveolar type II cells by ultrastructural examination) had foci ofsquamous metaplasia while the transplant did not. The 2 skinpapilloma transplants formed cystic tumors with papillary, acan-
thotic, and normal squamous lining epithelium (Figs. 5 and 6).Benign tumors were not invasive (Fig. 7), but a few formedmultiple lobules or nodules (Fig. 8). Transplanted tumors of mixedepithelial and mesenchymal elements such as the uterine stremaipolyp maintained both their epithelial and their stromal components. The mesothelioma formed solid sheets of poorly differentiated cells.
Hepatocellular neoplasms were difficult to transplant, in partbecause the majority of recipients died from LGL leukemia. In 4recipients, foci of hepatocytes with chronic inflammatory cellswere seen within the implant sites up to 1 year later.
Nonneoplastic Lesions and Normal Tissues. Nonneoplasticlesions did not form tumors at implant sites except for theappearance of a transplantable transitional cell carcinoma froma kidney implant with lesions of aging nephropathy (Table 4). Notumor was seen in the donor kidney. The common nodularlesions of epidermal inclusion cyst, abdominal fat necrosis, andherniated hepatic nodule (at diaphragm) did not form tumors.Normal tissues did not produce tumors (Table 4), but 26 of 92implants (effective number) yielded nonneoplastic lesions composed of tissues resembling the implanted tissue (Figs. 11 and
"Rats living more than 250 days after transplantation or developinga tumor transplant prior to 250 days even if developing leukemia."Numbers in parentheses, range.cNumbers in parentheses, number of tumors."NA, not applicable (no tumors found, or leukemiatransplants did not grow to 20 mm at transplant site).
Table 2Transplantationcharacteristics of naturally occurring skin and adnexalgland tumors in F344ratsTumor
"Rats living more than 250 days after transplantation or developinga tumor transplant prior to 250 days even if developing leukemia.6Numbers in parentheses, range.cNumbers in parentheses, number of tumors.
Table 3Summaryof the transplantability of tumors of F344 rats
Mean latentperiod (days to
No. tumor takes/no. palpabletrans-Histotogicaltumor type transplanted plant)
12). These tissues were often degenerative, inactive, or atrophied.
Suture granulomas were usually seen at the implant site forup to 1 year after surgery. These lesions were used as markersto help find the implants, which were usually nearby.
DISCUSSION
Our study showed that histologically malignant naturally occurring neoplasms were transplantable at a higher rate and witha shorter latent period than were benign tumors. Benign tumors,
"Rats living more than 250 days after transplantation or developing a tumor
transplant prior to 250 days even if developing leukemia.
however, were consistently transplantable from most tissueswith the exception of the liver. Our results agree with conceptspromoted by Foulds (7) and others; i.e., progressive growth ontransplantation is evidence of neoplasia but does not provemalignancy as has been proposed by some pathologists (4, 17,19, 24, 35). The histological appearance of the primary tumorsin our study correlated well with the transplantability, latentperiod, growth rate, histology, and invasiveness of the transplants in tumor recipients. Similar findings have been reported inliver tumor transplant studies (15, 16, 21, 32, 33). The highsuccess rate in our experiment is related to the use of an inbredstrain. Spontaneous tumors have been shown to be easily transplanted in syngeneic hosts and evoke little immune response (5,10,13,36).
The LGL leukemia occurs in high incidence in F344 rats (8,26, 28, 30) and less commonly in other rat strains. Some caseshave leukemic cells with high NK activity (22, 30). Similar diseases have recently been found in humans (9). It is tempting tospeculate that the LGL leukemia cells had NK activity againstthe spontaneous tumors in the donor F344 rats. Thus, whentumors were implanted into recipient rats, some LGL leukemiacells were also implanted from those donor rats with LGL leukemia, explaining the appearance of leukemia in recipient rats. Itmay also be possible that LGL leukemia cells were present indonor tumors because of the vascularity of the primary tumors,since only 20 to 30% of primary LGL leukemias have high NKactivity as measured in vitro (22, 30). We found no evidence forNK activity against natural primary tumors in aged F344 ratswith LGL leukemia (27). Since we did not perform a completepathology survey of all tissues of donor rats, we cannot evaluatethe possibility that transplanted tumors stimulated the development of leukemia in recipient rats. The vast majority of recipientrats which developed leukemia received tumors or tissues fromdonors which had lesions of leukemia.
ACKNOWLEDGMENTS
The authors are grateful for the aid of Drs. Amos Palmer, Donald Fish, andDraginja Djurickovic, Fred Argilan, Bill Danner, Robert Payne, Tom Tyler, Lee Dove,Lam/ Ostby, Gary Best, Kevin Beali, Jeannie Huntzberry, and Marcha Gamber.
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Fig. 1. Portion of primary preputial gland adenoma showing infiltration of LGL leukemia cells which are reactive with OX-8 monoclonalantibodies (dark cells). Avidin-biotin-peroxidasecomplex immunocytochemicaltechnique, hematoxylin, x 130.
Fig.2. Portion of transplanted pituitary adenoma (pale areas),with outgrowth of LGL leukemia(dark cells). H & E, x 80.Fig.3. Plaqueat fat pad implant site from testicular interstitial cell adenoma.Tumor cells are inactive and nondividing.H & E, x 80.Fig.4. Portion of nodule at implant site showing growth of large neoplastiainterstitial cells and smaller degenerativeinactive implantedcells. H & E, x 220.
Fig. 5. Primary papilloma in aged F344 rat which was successfully transplanted to weanling recipient. H & E, x 27.Fig.6. Transplanted papilloma in the mammary fat pad. The tumor is cystic and contains papillary areas resembling papillomaand squamous areas resembling the
histological appearanceof the epidermal inclusioncyst. H & E, x 27.Fig. 7. Portion of transplanted fibroma. Note absence of invasion. H & E, x 80.Fig.8. Transplanted uterine stromal polyp showing polypoid tumors and the absenceof invasion into fat pad. H & E, x 38.
Primary preputial gland adenoma in aged F344 rat. H & E, x 130.
I1 2^•' '^:^^5^1S?4'. «üÃTTj../- <
Fig. 9. Primary preputial gland adenoma in aged F344 rat. H & E, x 130.
Fig. 10. Transplanted preputial gland adenoma in recipient showing granularity of tumor cells and localized growth without invasion. Note similarity to primary tumorFig. 9. H & E. x 330.
Fig. 11. Island of hepatocytes and lymphocytes in mammary fat pad. Primary tumor was a hepatocellular adenoma. H & E, x 130.
Fig. 12. Thyroid follicles in fat pad after implantation of normal thyroid gland. Suture granuloma at lower portion of figure. H & E, x 54.
1984;44:2608-2615. Cancer Res Jerrold M. Ward and Peter H. Lynch Diagnosis of NeoplasmsAging F344 Rat as Biological Evidence for the HistologicalNeoplasms and Age-associated Nonneoplastic Lesions of the Transplantability of Naturally Occurring Benign and Malignant