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[CANCER RESEARCH 41, 3995-4000, October 1981]0008-5472/81 /0041-OOOOS02.00
Biological Behavior of Human Malignant Tumors Grown in the Nude Mouse1
Andreas P. Kyriazis,2 Aikaterini A. Kyriazis, William B. McCombs, III, and James A. Kereiakes
Departments of Pathology [A. P. K.] and Radiology ¡J.C. K.], University of Cincinnati Medical Center, Cincinnati, Ohio 45267, and the Scott and White Clinic,Temple, Texas 76501 [W. B. M.¡
ABSTRACT
The biological behavior of a number of nude mouse-grown
human epithelial tumors of different histogenetic backgroundwas studied. They included three transitional cell carcinomasand a mucin-producing carcinoma of the urinary bladder, two
pancreatic adenocarcinomas, a colon adenocarcinoma, abreast ductal carcinoma, and an epidermoid carcinoma. Mi-
croinvasion was a constant finding in all tumors examined. Allbut one of the tumors studied showed macroinvasion andmétastases.Tumors transplanted s.c. in the anterior aspect ofthe lateral thoracic wall metastasized primarily to the regionaland mediastinal lymph nodes. In certain cases, systemic métastases were observed affecting the submaxillary, contralat-
eral axillary, and inguinal lymph nodes. Lung métastasesoccurred through the lymphatic and hematogenous routes. It isconcluded that the nude mouse-grown human tumors investi
gated in the present study show overt malignant behaviorrecapitulating the biological characteristics of the tumor oforigin.
INTRODUCTION
The lack of a functional thymus-dependent immune system
in nude mice has made possible the transplantation of manysolid human tumors into these animals. Furthermore, a numberof reports have indicated that transplanted human tumors preserve the morphological, biological, and biochemical characteristics of the tumor of origin (3, 6,16, 20, 24, 27). In addition,several studies have shown a correlation between human tumorxenograft response to chemotherapy and clinical results withthe same drugs in cancer patients (2, 7, 9, 10, 11, 17, 18, 21 ).These observations have stressed the potential value of thenude mouse system in cancer research. Unfortunately, questions as to the validity of the nude mouse system may be raisedbased primarily on reports on the biological behavior of varioustransplanted tumors. With only a few exceptions (6, 13, 25,26), 2 aspects of the problem have been overemphasizedduring the past decade, lack of invading capabilities and absence of métastases (4, 15, 19, 20, 24, 27). Particularlypuzzling have been reports by leading investigators on theabsence of métastases. This is clearly exemplified by Ry-gaard's statement "why do tumors in nude mice not metasta-size but rather behave like benign tumors" (23). Assuming that
the validity of the nude mouse is based on the assumption thattransplanted tumors should behave in a manner closely simulating that of the primary neoplastic growth, a benign behavioras it might be indicated by lack of invasion and métastasesmay
' This work was supported by Grant CA-26693 from the National Cancer
Institute through the National Bladder Cancer Project.2 To whom requests for reprints should be addressed, at Department of
Pathology, University of Cincinnati Medical Center, MSB, R-1253, Cincinnati,Ohio 45267.
Received January 15, 1981 ; accepted July 1, 1981.
question its usefulness and narrow its applicability in cancerresearch.
This paper addresses these questions and summarizes ourpersonal experience with regard to the biological behavior ofa number of nude mouse-grown human tumors of different
histogenetic backgrounds.
MATERIALS AND METHODS
For the purpose of the present study, 4- to 6-week-old femaleBALB/c nude mice were purchased from Sprague-Dawley, Madison,Wis. All mice were maintained in a pathogen-free environment as
described previously (5). Human epithelial tumor cell lines grown intissue culture and human tumors removed at surgery and directlytransplanted into nude mice were used. The following tumor cell lineswere used in the present study. Capan-1, a pancreatic carcinoma, and
RT-4, a transitional cell carcinoma of the bladder, were provided byDr. J. Fogh, Sloan Kettering Institute for Cancer Research, Rye, N. Y.Lines SW-480, a colon carcinoma, and SW-780 and SW-800, both
derived from transitional cell carcinomas of the bladder, were providedby Dr. W. McCombs, Scott and White Clinic, Temple, Texas. Cell lineHEP-2 was obtained from the American Tissue Culture Collection,
Rockville, Md.
Of the primary transplants, tumor line 13678, a bladder adenocarcinoma, was kindly provided by Drs. D. Paulson and R. Bonar, DukeUniversity. BrCa, a breast carcinoma, and PaCa, a pancreatic adenocarcinoma, were established in nude mice from primary transplants.All tested tumors were established in the nude mouse before theinitiation of the experimental procedure. Mice received s.c. through askin incision in the anterior aspect of the lateral thoracic region a smallpiece of tumor measuring approximately 0.3 x 0.4 cm. Tumor growthwas determined weekly by using the formula length x (width)2 x 0.4
(1 ). The study was conducted on mice serving as control in a broadspectrum of experiments. All mice remained healthy throughout theentire observation period, free of hepatitis or wasting disease. Theaverage tumor size at the time of animal sacrifice was calculated to be1875 ± 182 (S.E.). Most of the tumors were studied in successivepassages ranging from 4 to 10.
Animals were killed by cervical dislocation. Autopsy was performedon all animals. Tumors, regional and distant lymph nodes, and representative sections from various organs were fixed in 10% bufferedformalin solution. Removal and processing of lungs was done asdescribed previously (13).
For histological examination, tissue paraffin blocks were cut 5 jumthick and stained with hematoxylin and eosin. Occasional sectionswere stained with periodic acid-Schiff and reticulin. From both lymph
nodes and lungs, serial sections were examined.
RESULTS
On the basis of light microscopy, local involvement by tumorwas distinguished in 2 forms: a microinvasive form whereneoplastic cells infiltrated the surrounding soft tissues to adistance not exceeding 200 firn from the tumor pseudocapsule(Fig. 1) and a macroinvasive form with the tumor havingreached the pectoral and intercostal muscles (Figs. 2 to 4). Inboth cases, groups of neoplastic cells infiltrated the surround-
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A. P. Kyriazis et al.
ing tumor soft tissues in a multifocal pattern. Both micro- and
macroinvasion were typical findings in all tumor lines examined,the only exception being the bladder carcinoma RT-4 which
demonstrated only the microinvasive pattern. In the majority ofcases, tumors exhibiting macroinvasion had invaded the locallymphatic channels which appeared dilated and filled withgroups of neoplastic cells (Fig. 3). Lymph node involvementwas characterized by the presence of neoplastic cells in theperipheral sinus with extension into the paracortical areas andthe medullary parts (Figs. 5 and 6). Nodal capsular invasionand tumor extension in the surrounding tissue were also observed. Of the various lymph nodes, métastases were seenprimarily in the homolateral axillary and less frequently themediastinal groups. On many occasions, bladder tumors SW-780 and SW-800 and pancreatic carcinomas had metastasizedto the controlateral axillary and submaxillary lymph nodes (Fig.8) and diaphragm (Fig. 7), and in a small number of cases,involvement of the homolateral inguinal lymph nodes wasnoted. In the lungs, métastaseswere found mainly in areasadjacent to medium and small size bronchi and bronchioles(Figs. 10 to 13). Their presence within lymphatics, alveolarcapillaries, and branches of the pulmonary artery with involvement of the surrounding lung parenchyma indicated that distantmétastasesoccurred by both the lymphatic and hematogenousroutes. These findings were consistently observed regardlessof the number of tumor passages in the nude mouse. It shouldbe mentioned that without exception métastasesshowed morphological characteristics identical to the tumor of origin. Table1 summarizes the observations with regard to the infiltratingand metastasizing capabilities of the tumors studied. Criteriafor invasion and métastaseswere the same as described in thetext.
DISCUSSION
There are 3 factors taken under consideration in determiningthe malignant behavior of a neoplastic growth in humans:histological grading; invasion; and métastases.Invasion of thetumor bed, the adjacent tissues, and less frequently vessels,primarily lymphatics, is a common observation in most humantumors. Métastases, however, is an inconstant finding. Although it is encountered in the majority of cancers, there areoccasions where the most extensive search, even at the autopsy table, fails to reveal metastatic lesions in tumors otherwise characterized as having a rather aggressive behavior.This negative finding, however, does not rule out the presenceof métastasessince it is known that metastatic foci not seen atthe time of the patient's treatment may become apparent yearslater ultimately contributing to the patient's death. It should be
kept in mind that between the time of the inception of neoplasiato the stage of metastatic disease there is an interval of monthsor years. Detectable métastasesfor most tumors represent alate event. Therefore, their presence depends largely on thetiming the neoplastic growth is taken under consideration.
Another characteristic is the variability in growth rates seenin tumors of the same histogenetic background, covering awide spectrum from the very slow to the very fast growth. Aclear example of this is the transitional cell carcinoma of theurinary bladder. It may take years for a Stage I tumor toprogress to Stage II or III, or the transition may be rapid (8).Transplants taken from such tumors should biologically behave
TatHelBiological behavior of various human tumors grown s.c. in nude mice
Local invasion Métastases
Tumor Histological typeLymph
Micro Macro nodes Lungs
Capan-1 Moderately well-differentiatedpancreatic adenocarcinoma(metastasizing)9
PaCa Well-differentiated pancreaticadenocarcinoma(metastasizing)
PT-4 Transitional-cell carcinoma ofbladder. Grade I
SW-800 Transitional-cell carcinoma ofbladder, poorlydifferentiated (infiltrating)
SW-780 Transitional-cell carcinoma ofbladder, Grade II to III(infiltrating)
SW-480 Poorly differentiated colonadenocarcinoma (notavailable)
Hep-2 Epidermoid laryngealcarcinoma, poorlydifferentiated6 (not
available)BrCa Breast ductal adenocarcinoma
(metastasizing)13678 Mucinous adenocarcinoma of
bladder (infiltrating)NE
' Information available on tumor behavior in the host patient at the time of
tumor biopsy.'"'This cell line is considered in HeLa contamination.
c ±,inconstant finding; NE, not examined.
in a manner similar to the primary growth with regard to growthrate, invasion, and metastatic capabilities. A deviation from thisrule may weaken rather than strengthen the nude mouse-human tumor xenograft model.
From the presented data, it becomes evident that the studiedhuman tumors grown in the nude mouse exhibited 3 consistently observed interrelated behavioral characteristics; namely,local invasion, vascular invasion, and a tendency to metasta-size. These findings were independent of the number of tumorpassages, an observation confirming a previous report on thissubject (25). Of all tumors examined, only the RT-4 failed toshow the degree of aggressiveness characteristic of most ofthe tumors of the present study. This is not surprising in viewof the origin of the tumor. Here, we deal with a Stage I, GradeI bladder carcinoma grown in the nude mouse and recapitulating in an exact manner the behavior of the same tumor in thehuman.
It is worth noting that examination of serial lung sectionsdemonstrated without exception the presence of isolatedgroups of neoplastic cells in pulmonary capillaries with earlyinvolvement of the adjacent lung parenchyma (Figs. 9 to 11).This observation was viewed as an indication of the positivemetastatic potential of the transplanted tumors regardless ofthe absence of recognizable large tumor formations. Failure todetect the latter may be related to various host factors, e.g.,mouse strain (22), health status of animals (12), site of transplantation (13, 14), tumor size, and growth rate, and thebiological characteristics of the original neoplastic growth fromwhich the transplanted tumor originated. Furthermore, sincemétastasesare not synchronous, those taking place late in thelife of the animal may not have the time required to reach thestage of detectability, being only microscopically seen as smallaggregates or neoplastic cells within lymphatic channels and
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Biology of Human Tumors in Nude Mouse
pulmonary vessels.In conclusion, a large number of human tumors grown in
nude mice, as the ones described in the present study, preserve their malignant behavior as evidenced by their capabilityto infiltrate adjacent tissues, invade lymphatic and blood vessels, and metastasize. Absence of métastases should beviewed within the context of tumor-host relationship and tumor
biology rather than as evidence of benign tumor behavior. It isimperative that, in attempting to interpret the behavior of transplanted human tumors in the nude mouse, both the individualbiological characteristics of the primary neoplasm and thevarious host factors as described above should always betaken under consideration. When all these factors are considered, it becomes evident that human tumors grown in the nudemouse mimic to a great extent the behavior of the tumor oforigin.
ACKNOWLEDGMENTS
We thank Indu Malhotra and Barbara Holman for technical assistance, TinaLink for the photography work, and Joyce Turner for typing the manuscript.
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Fig. 1. Typical picture of microinvasion. Pancreatic carcinoma capan-1 has invaded the tumor pseudocapsule (arrows) and the fibroadipose tissue surroundingthe tumor, x 160.
Fig. 2. Macroinvasion of bladder carcinoma SW-780. Groups of neoplastic cells involving extensively soft tissues at a considerable distance from the originaltumor, x 100.
Fig. 3. Macroinvasion in bladder carcinoma SW-780. Invasion of the skin covering the tumor and presence of neoplastic cells in dilated lymphatics (arrows').
x 100.Fig. 4. Macroinvasion in bladder carcinoma SW-780 extending to the pectoral and intercostal muscles, x 100.
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Figs. 5 to 13. Local and distant métastases.Fig. 5. A well-differentiated pancreatic adenocarcinoma having metastasized to the regional axillary lymph nodes, x 100.
Fig. 6. Massive involvement of mediastinal lymph nodes by bladder carcinoma SW-780. x 100.
Fig. 7. Pancreatic adenocarcinoma Capan-1 with métastasesto the diaphragm, x 160.
Fig. 8. Bladder carcinoma SW-800 with métastasesto submaxillary lymph nodes and salivary gland, x 100.
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Fig. 9. Early presence of single neoplastia cells from bladder carcinoma SW-780 in pulmonary alveolar capillaries (4 weeks posttransplantation), x 400.
Fig. 10. Same tumor type as in Fig. 9. A more advanced stage characterized by the presence of a small group of neoplastic cells infiltrating the lung parenchymaadjacent to an alveolar bronchiole (6 weeks posttransplantation), x 250.
Fig. 11. Same tumor type as in Fig. 9. Neoplastic cells, at a later stage, aggregate to form a microscopically well-recognizable tumor nodule replacing the lungtissue (10 weeks posttransplantation), x 250.
Fig. 12. Bladder tumor SW-780 involving the bronchial wall and peribronchial space. Presence of tumor within lymphatics adjacent to the bronchial wall are seen,x 160.
Fig. 13. Pancreatic adenocarcinoma with lung métastasesadjacent to small bronchioles, x 160.
4000
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1981;41:3995-4000. Cancer Res Andreas P. Kyriazis, Aikaterini A. Kyriazis, William B. McCombs III, et al. Nude MouseBiological Behavior of Human Malignant Tumors Grown in the
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