Brief Communication: Invasion and Metastasis of a Xenogeneic Tumor in Nude Mice 1,
2
Henry Maguire, Jr., Henry C. Outzen, R. Philip Custer, and Richmond T. Prehn 3 ABSTRACT-When aliquots of a malignant hamster tumor were transplanted to nude mice and to hamsters, the tumor showed 1) more rapid growth in the hamster, 2) local invasion and metastasis in both species, and 3) occasional spontaneous regression in the nude mouse.-J Natl Cancer Inst 57: 439-442,1976.
RESULTS
In an initial experiment, the tumor was grafted sc by trocar to 11 conventional nude mice and to 6 hamsters. It grew slowly in the mice; at autopsy 8 weeks later, all tumors were less than 1.5 cm in diameter, were bounded by a compression capsule, and had failed to metastasize. In contrast, growth was rapid and aggressive in the hamsters; the tumors averaged 3.7 cm in diameter by 21 days, and most hamsters showed regional lymph node enlargement at that time. All 6 were moribund the following week, and autopsies displayed extensive invasion around the grafted tumor as well as metastases to multiple organs; such findings confirmed prior experience with this tumor in the Syrian hamster (8). Further experiments with both conventional and germfree nude mice and with hamsters supported the finding that the Fabrizio tumor grew more vigorously and consistently in the hamster (table 1). Occasionally, tumors regressed spontaneously in the germfree hosts [#1409, 1410, and 1412 (text-fig. 1) and germfree #2 (table 1)]; regressions were not seen either in the hamsters or in the conventional nudes. Of particular interest were the mice whose tumors exceeded 25 mm in one dimension (fig. 2). Local tumor invasion and metastases were consistent in these animals. The overlying skin was clearly infiltrated by tumor cells (fig. 3) and often extensively ulcerated. Subjacent voluntary muscle was invaded similarly (fig. 4). Metastasis to lymph nodes was found (fig. 6), and 1 mouse had a large mediastinal mass undoubtedly from nodal involvement. Tumor was also noted in the lungs (fig. 5), liver (fig. 10), kidneys (figs. 7, 8), and ovaries (fig. 9). Both hematogenous and lymphatic spread could be demonstrated in addition to extension by contiguity. DISCUSSION
We have shown that a xenogeneic, malignant tumor may establish an invasive and metastatic pattern of growth when directly transplanted to the nude mouse.
MATERIALS AND METHODS
A Syrian hamster tumor was used (8). It had been induced with MCA by one of us (RPC) in 1953 and passed through many hundreds of generations by Dr. Angelina Fabrizio; it is known as the Fabrizio hamster tumor. We obtained a female hamster bearing this tumor from Dr. Fabrizio and banked aliquots of tumor in liquid nitrogen. Its present morphologic characteristics are those of a primitive reticulum cell (histiocytic) sarcoma (fig. 1). Female Syrian hamsters were obtained from the Lakeview Hamstery, Newfield, New Jersey. VOL. 57, No.2, AUGUST 1976
ABBREVIATION USED: MCA=3-methylcholanthrene. Received October 2, 1975; accepted January 26, 1976. Supported by Public Health Service (PHS) grants CA08856 and CA06927 from the National Cancer Institute, by PHS grant RR05539 from the Division of Research Resources, and by an appropriation from the Commonwealth of Pennsylvania. 3 The Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pa. 19111. 1
2
439
J
NATL CANCER INST
Downloaded from http://jnci.oxfordjournals.org/ at The University of British Colombia Library on October 6, 2015
Rygaard and Povlsen (l) made the initial observation that xenogeneic tumors could grow in nude (athymic) mice. They successfully transplanted 7 of9 human colon tumors to the subcutaneous space in these T -ceIl-deficient animals. Other investigators, employing various xenogeneic and allogeneic malignant tissues, confirmed and extended their results. The transplanted neoplastic tissues retain their original histologic characters and karyotypes, even after serial passages through other nude mice (2-4). Marker molecules, such as carcinoembryonic antigen and melanin, often continue to be produced (2, 5). It has been proposed that this model might be useful for predicting the effects of cancer chemotherapeutic drugs and high-energy irradiation on the growth of individual human tumors (6). We successfully transplanted malignant tumors from humans (melanomas, mammary carcinomas, and neuroblastomas), dogs (melanomas and sarcomas), and guinea pigs (sarcomas) into nude mice. The tumor in the recipient mouse often grew to a diameter of several centimeters and could be repeatedly passaged through other nude mice, returned to cell culture, or returned to laboratory animals of the original host strain. Curiously, in autopsies of more than 200 nude mice bearing these xenogeneic tumors, we never encountered a metastasis. This was in accord with the experience of previous investigators (1, 3, 5, 7) apart from Giovanella et al. (2). Further, we consistently found that the xenogeneic tumors in nude mice did not break through their compression pseudocapsules and that they grew as sharply circumscribed "benign" nodules; the hosts died of causes (principally infectious) unrelated to the transplanted tumors. These observations raised the important question of whether the malignant potential of any foreign tumor could be expressed in the environment of the nude mouse. We resolved this issue to our satisfaction by transplanting a highly malignant transmissible tumor of hamsters to the nude mouse and following its growth characteristics.
Conventionally raised and germfree nude mice were from our closed ICR colony (9). In different experiments, a fresh trocar piece or cell suspension of the Fabrizio tumor was inserted sc in the back of each animal. To guarantee that the grafted tissue was malignant, we transplanted aliquots in parallel to nude mice and to hamsters.
440
MAGUIRE, OUTZEN, CUSTER, AND PREHN TABLE 1.---Growth of Fabrizio tumor in hamsters and in nude mice Average tumor diameter in mm at day:
Animals" 0
13
16
19
23
26
14 20 12.5 13.5
20 30 16.5 18.5
24.5 51 23.5 22
27 FDC 28 25
27.5 FDC 27.5
17 6 14 11.5 12.5 9.5
19.5 17.5 18 14.5 20 15.5
21 18.5 18 15.5 20 16.5
18 19 18.5 16 25 20
0 0 0 0 0
2 0 3 6 6
5 0 6 10 12
9 Trace 8 14 13
33
37
22 20 19 15.5 28 21
FDC 25 22.5 23 30 26
25 23.5 25 FDC 26.5
10 8 6 14 13
10 10 10 13 15
-
40
44
10 5 10 15 15
9 3 10 17 17
a On day 0, a suspension of 1.8 x 106 hamster tumor cells in 0.25 ml was injected sc in the backs of hamsters, conventional nude mice, and germfree nude mice. The length +width of tumors (in mm) +2 is recorded. Bold values indicate that the animal was killed that day for histologic examination. FDC=found dead in cage.
50,
r
Mouse No. 40
14031409 ---1410 1412
KILLED
30
KILLED
/
E E 20
i
i
i
i
i
I
I
I
i
.' -t. ,
---- -.~,
10
.\.. O'
o
I
10
'\ ..
'
V._._l",/ 20
30
/
i
i
I
I 40
;
·1··············
.I
.
,,
,
I I
\ 1·....:>--l
50
60
L __ l 70
80
_ 90
DAYS TEXT-FIGURE 1.-5X 106 enzyme-dispersed Fabrizio tumor cells injected sc in backs of 4 germfree nude mice. Average surface diameter (in mm) of each tumor is plotted against time (in days) after inoculation.
The intervention of cell culture was unnecessary. However, a particularly aggressive tumor was required to demonstrate the point. This tumor, an MCA-induced J NATL CANCER INST
and quickly lethal reticulum cell (histiocytic) sarcoma of Syrian hamsters, always grew more rapidly and invasively in hamsters than in nude mice and did not metastasize in the nude mice until the primary graft had become large. Thereafter, local barriers were broken, and there was spread to lymph nodes and/or one or more internal viscera. This host difference in growth rate and spread, not presently understood, might be attributed to metabolic differences between hamster and mouse, to mouse antibody against xenogeneic tumors, or to as yet unidentified factors. Certainly, the result is opposite to the one anticipated, if tumor immunity of the delayed hypersensitivity type is dominant in controlling tumor growth. The spontaneous regressions observed in the germfree mice strongly suggest a host immunologic factor directed against the xenogeneic tumor; such cytotoxic antibodies have been described in the nude mouse (3). The question of why the susceptibility of nude mice raised under conventional conditions (clean but not germfree) is greater than that of germfree nude mice (table 1) is another one to which we have no answer. Possibly, they are prone to infection and their immunologic status thereby is perhaps further depressed . That invasive, metastasizing human tumors when transplanted to nude mice generally fail to invade aggressively and to metastasize probably reflects their lower order of malignancy. REFERENCES (1) RYGAARD], POVLSEN CO: Heterotransplantation of a human ma-
lignant tumor to nude mice. Acta Pathol Microbiol Scand 77:758-760, 1969 (2) GIOVANELLA BC, STEHLIN ]S, WILLIAMS L] ]R: Heterotransplantation of human malignant tumors in "nude" thymusless mice. 11. Malignant tumors induced by injection of cell cultures derived from human solid tumors.] Natl Cancer lnst 52:921-927, 1974 (3) POVLSEN CO, FIALKOW P], KLEIN E, et al: Growth and antigeneic VOL. 57, NO.2, AUGUST 1976
Downloaded from http://jnci.oxfordjournals.org/ at The University of British Colombia Library on October 6, 2015
Hamsters 1 2 3 4 Nude mice (conventional) 1 2 3 4 5 6 Nude mice (germfree) 1 2 3 4 5
XENOGENIC TUMOR METASTASES IN NUDE MICE properties of a biopsy-derived Burkitt's lymphoma in thymusless (nude) mice. Int] Cancer 11:30-39. 1973 (4) VISFELDT ]. POVLSEN CO. RYGAARD]: Chromosome analysis of human tumours following heterotransplantation to the mouse mutant nude. Acta Pathol Microbiol Scand[A] 80:169-176.1972 (5) SORDAT B. FRITSCHE R. MACH ]P. et al: Morphological and functional evaluation of human solid tumours serially transplanted in nude mice. In First International Workshop on Nude Mice (Rygaard ] , Povlsen CO. eds.). Stuttgart. Gustav Fischer. 1974. pp 269-277 (6) POVLSEN CO.]ACOBSEN GK. RYGAARD]: The mouse mutant nude as a model for testing of anticancer agents. In The Laboratory
441
Animal in Drug Testing (Spiegel A. ed.). Stuttgart. Gustav Fischer. 1972. pp 63-72 (7) OZZELLO L. SORDAT B. MERENDA C. et al: Transplantation of a human mammary carcinoma cell line (BT 20) into nude mice.] Nat! Cancer Inst 52:1669-1672.1974 (8) FABRIZIO AM: An induced transmissible sarcoma in hamsters: Eleven-year observation through 288 passages. Cancer Res 25:107-117.1965 (9) EATON G]. OUTZEN HC. CUSTER RP. et al: Husbandry of the nude mouse in conventional and germfree environments. Lab Anim Sci 25:309-314. 1975
Downloaded from http://jnci.oxfordjournals.org/ at The University of British Colombia Library on October 6, 2015
FIGURE I.-Fabrizio hamster tumor growing in hamster. This primitive reticulum cell (histiocytic) sarcoma grows with striking rapidity; many mitotic figures are shown in this field. The fragile cells are pulled apart through fixation shrinkage. whereas the more slowly growing ones in the nude mice tend toward a more syncytial pattern (figs. 3. 6. 9). x 400 FIGURE 2.-Hamster tumor transplant in nude mouse. Massive growth after 63 days. with extensive ulceration of the infiltrated skin. Local invasion can be seen over flank. FIGURE 3.-Skin. Section through a nonulcerated segment shows total replacement of corium by tumor; thin layer of keratotic epidermis persists. x 400 FIGURE 4.-Voluntary muscle. Deep infiltration into body wall is separating and destroying muscle. x 400
VOL. 57, No.2, AUGUST 1976
MAGUIRE. OUT ZEN , CUSTER, AND PREHN
Downloaded from http://jnci.oxfordjournals.org/ at The University of British Colombia Library on October 6, 2015
FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE
442
5.-Lung. Foci of viable tumor cells undoubtedly result from hematogenous dissemination. x 400 6.-Lymph node. Marginal sinuses of node are invaded by bulky sheets of tumor. x 400 7.-Kidney. Large tumor nodule lies in renal cortex. X 400 8.-Kidney. Permeation of tumor along perivascular lymphatic channels. x 400 9.-ovary. Virtually complete displacement of parenchyma by tumor. x 400 1O.-Liver. Tumor emboli in sinusoids. x 400
MAGUIRE, OUTZEN, CUSTER, AND PREHN
2
Henry Maguire, Jr., Henry C. Outzen, R. Philip Custer, and Richmond T. Prehn 3 ABSTRACT-When aliquots of a malignant hamster tumor were transplanted to nude mice and to hamsters, the tumor showed 1) more rapid growth in the hamster, 2) local invasion and metastasis in both species, and 3) occasional spontaneous regression in the nude mouse.-J Natl Cancer Inst 57: 439-442,1976.
RESULTS
In an initial experiment, the tumor was grafted sc by trocar to 11 conventional nude mice and to 6 hamsters. It grew slowly in the mice; at autopsy 8 weeks later, all tumors were less than 1.5 cm in diameter, were bounded by a compression capsule, and had failed to metastasize. In contrast, growth was rapid and aggressive in the hamsters; the tumors averaged 3.7 cm in diameter by 21 days, and most hamsters showed regional lymph node enlargement at that time. All 6 were moribund the following week, and autopsies displayed extensive invasion around the grafted tumor as well as metastases to multiple organs; such findings confirmed prior experience with this tumor in the Syrian hamster (8). Further experiments with both conventional and germfree nude mice and with hamsters supported the finding that the Fabrizio tumor grew more vigorously and consistently in the hamster (table 1). Occasionally, tumors regressed spontaneously in the germfree hosts [#1409, 1410, and 1412 (text-fig. 1) and germfree #2 (table 1)]; regressions were not seen either in the hamsters or in the conventional nudes. Of particular interest were the mice whose tumors exceeded 25 mm in one dimension (fig. 2). Local tumor invasion and metastases were consistent in these animals. The overlying skin was clearly infiltrated by tumor cells (fig. 3) and often extensively ulcerated. Subjacent voluntary muscle was invaded similarly (fig. 4). Metastasis to lymph nodes was found (fig. 6), and 1 mouse had a large mediastinal mass undoubtedly from nodal involvement. Tumor was also noted in the lungs (fig. 5), liver (fig. 10), kidneys (figs. 7, 8), and ovaries (fig. 9). Both hematogenous and lymphatic spread could be demonstrated in addition to extension by contiguity. DISCUSSION
We have shown that a xenogeneic, malignant tumor may establish an invasive and metastatic pattern of growth when directly transplanted to the nude mouse.
MATERIALS AND METHODS
A Syrian hamster tumor was used (8). It had been induced with MCA by one of us (RPC) in 1953 and passed through many hundreds of generations by Dr. Angelina Fabrizio; it is known as the Fabrizio hamster tumor. We obtained a female hamster bearing this tumor from Dr. Fabrizio and banked aliquots of tumor in liquid nitrogen. Its present morphologic characteristics are those of a primitive reticulum cell (histiocytic) sarcoma (fig. 1). Female Syrian hamsters were obtained from the Lakeview Hamstery, Newfield, New Jersey. VOL. 57, No.2, AUGUST 1976
ABBREVIATION USED: MCA=3-methylcholanthrene. Received October 2, 1975; accepted January 26, 1976. Supported by Public Health Service (PHS) grants CA08856 and CA06927 from the National Cancer Institute, by PHS grant RR05539 from the Division of Research Resources, and by an appropriation from the Commonwealth of Pennsylvania. 3 The Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pa. 19111. 1
2
439
J
NATL CANCER INST
Downloaded from http://jnci.oxfordjournals.org/ at The University of British Colombia Library on October 6, 2015
Rygaard and Povlsen (l) made the initial observation that xenogeneic tumors could grow in nude (athymic) mice. They successfully transplanted 7 of9 human colon tumors to the subcutaneous space in these T -ceIl-deficient animals. Other investigators, employing various xenogeneic and allogeneic malignant tissues, confirmed and extended their results. The transplanted neoplastic tissues retain their original histologic characters and karyotypes, even after serial passages through other nude mice (2-4). Marker molecules, such as carcinoembryonic antigen and melanin, often continue to be produced (2, 5). It has been proposed that this model might be useful for predicting the effects of cancer chemotherapeutic drugs and high-energy irradiation on the growth of individual human tumors (6). We successfully transplanted malignant tumors from humans (melanomas, mammary carcinomas, and neuroblastomas), dogs (melanomas and sarcomas), and guinea pigs (sarcomas) into nude mice. The tumor in the recipient mouse often grew to a diameter of several centimeters and could be repeatedly passaged through other nude mice, returned to cell culture, or returned to laboratory animals of the original host strain. Curiously, in autopsies of more than 200 nude mice bearing these xenogeneic tumors, we never encountered a metastasis. This was in accord with the experience of previous investigators (1, 3, 5, 7) apart from Giovanella et al. (2). Further, we consistently found that the xenogeneic tumors in nude mice did not break through their compression pseudocapsules and that they grew as sharply circumscribed "benign" nodules; the hosts died of causes (principally infectious) unrelated to the transplanted tumors. These observations raised the important question of whether the malignant potential of any foreign tumor could be expressed in the environment of the nude mouse. We resolved this issue to our satisfaction by transplanting a highly malignant transmissible tumor of hamsters to the nude mouse and following its growth characteristics.
Conventionally raised and germfree nude mice were from our closed ICR colony (9). In different experiments, a fresh trocar piece or cell suspension of the Fabrizio tumor was inserted sc in the back of each animal. To guarantee that the grafted tissue was malignant, we transplanted aliquots in parallel to nude mice and to hamsters.
440
MAGUIRE, OUTZEN, CUSTER, AND PREHN TABLE 1.---Growth of Fabrizio tumor in hamsters and in nude mice Average tumor diameter in mm at day:
Animals" 0
13
16
19
23
26
14 20 12.5 13.5
20 30 16.5 18.5
24.5 51 23.5 22
27 FDC 28 25
27.5 FDC 27.5
17 6 14 11.5 12.5 9.5
19.5 17.5 18 14.5 20 15.5
21 18.5 18 15.5 20 16.5
18 19 18.5 16 25 20
0 0 0 0 0
2 0 3 6 6
5 0 6 10 12
9 Trace 8 14 13
33
37
22 20 19 15.5 28 21
FDC 25 22.5 23 30 26
25 23.5 25 FDC 26.5
10 8 6 14 13
10 10 10 13 15
-
40
44
10 5 10 15 15
9 3 10 17 17
a On day 0, a suspension of 1.8 x 106 hamster tumor cells in 0.25 ml was injected sc in the backs of hamsters, conventional nude mice, and germfree nude mice. The length +width of tumors (in mm) +2 is recorded. Bold values indicate that the animal was killed that day for histologic examination. FDC=found dead in cage.
50,
r
Mouse No. 40
14031409 ---1410 1412
KILLED
30
KILLED
/
E E 20
i
i
i
i
i
I
I
I
i
.' -t. ,
---- -.~,
10
.\.. O'
o
I
10
'\ ..
'
V._._l",/ 20
30
/
i
i
I
I 40
;
·1··············
.I
.
,,
,
I I
\ 1·....:>--l
50
60
L __ l 70
80
_ 90
DAYS TEXT-FIGURE 1.-5X 106 enzyme-dispersed Fabrizio tumor cells injected sc in backs of 4 germfree nude mice. Average surface diameter (in mm) of each tumor is plotted against time (in days) after inoculation.
The intervention of cell culture was unnecessary. However, a particularly aggressive tumor was required to demonstrate the point. This tumor, an MCA-induced J NATL CANCER INST
and quickly lethal reticulum cell (histiocytic) sarcoma of Syrian hamsters, always grew more rapidly and invasively in hamsters than in nude mice and did not metastasize in the nude mice until the primary graft had become large. Thereafter, local barriers were broken, and there was spread to lymph nodes and/or one or more internal viscera. This host difference in growth rate and spread, not presently understood, might be attributed to metabolic differences between hamster and mouse, to mouse antibody against xenogeneic tumors, or to as yet unidentified factors. Certainly, the result is opposite to the one anticipated, if tumor immunity of the delayed hypersensitivity type is dominant in controlling tumor growth. The spontaneous regressions observed in the germfree mice strongly suggest a host immunologic factor directed against the xenogeneic tumor; such cytotoxic antibodies have been described in the nude mouse (3). The question of why the susceptibility of nude mice raised under conventional conditions (clean but not germfree) is greater than that of germfree nude mice (table 1) is another one to which we have no answer. Possibly, they are prone to infection and their immunologic status thereby is perhaps further depressed . That invasive, metastasizing human tumors when transplanted to nude mice generally fail to invade aggressively and to metastasize probably reflects their lower order of malignancy. REFERENCES (1) RYGAARD], POVLSEN CO: Heterotransplantation of a human ma-
lignant tumor to nude mice. Acta Pathol Microbiol Scand 77:758-760, 1969 (2) GIOVANELLA BC, STEHLIN ]S, WILLIAMS L] ]R: Heterotransplantation of human malignant tumors in "nude" thymusless mice. 11. Malignant tumors induced by injection of cell cultures derived from human solid tumors.] Natl Cancer lnst 52:921-927, 1974 (3) POVLSEN CO, FIALKOW P], KLEIN E, et al: Growth and antigeneic VOL. 57, NO.2, AUGUST 1976
Downloaded from http://jnci.oxfordjournals.org/ at The University of British Colombia Library on October 6, 2015
Hamsters 1 2 3 4 Nude mice (conventional) 1 2 3 4 5 6 Nude mice (germfree) 1 2 3 4 5
XENOGENIC TUMOR METASTASES IN NUDE MICE properties of a biopsy-derived Burkitt's lymphoma in thymusless (nude) mice. Int] Cancer 11:30-39. 1973 (4) VISFELDT ]. POVLSEN CO. RYGAARD]: Chromosome analysis of human tumours following heterotransplantation to the mouse mutant nude. Acta Pathol Microbiol Scand[A] 80:169-176.1972 (5) SORDAT B. FRITSCHE R. MACH ]P. et al: Morphological and functional evaluation of human solid tumours serially transplanted in nude mice. In First International Workshop on Nude Mice (Rygaard ] , Povlsen CO. eds.). Stuttgart. Gustav Fischer. 1974. pp 269-277 (6) POVLSEN CO.]ACOBSEN GK. RYGAARD]: The mouse mutant nude as a model for testing of anticancer agents. In The Laboratory
441
Animal in Drug Testing (Spiegel A. ed.). Stuttgart. Gustav Fischer. 1972. pp 63-72 (7) OZZELLO L. SORDAT B. MERENDA C. et al: Transplantation of a human mammary carcinoma cell line (BT 20) into nude mice.] Nat! Cancer Inst 52:1669-1672.1974 (8) FABRIZIO AM: An induced transmissible sarcoma in hamsters: Eleven-year observation through 288 passages. Cancer Res 25:107-117.1965 (9) EATON G]. OUTZEN HC. CUSTER RP. et al: Husbandry of the nude mouse in conventional and germfree environments. Lab Anim Sci 25:309-314. 1975
Downloaded from http://jnci.oxfordjournals.org/ at The University of British Colombia Library on October 6, 2015
FIGURE I.-Fabrizio hamster tumor growing in hamster. This primitive reticulum cell (histiocytic) sarcoma grows with striking rapidity; many mitotic figures are shown in this field. The fragile cells are pulled apart through fixation shrinkage. whereas the more slowly growing ones in the nude mice tend toward a more syncytial pattern (figs. 3. 6. 9). x 400 FIGURE 2.-Hamster tumor transplant in nude mouse. Massive growth after 63 days. with extensive ulceration of the infiltrated skin. Local invasion can be seen over flank. FIGURE 3.-Skin. Section through a nonulcerated segment shows total replacement of corium by tumor; thin layer of keratotic epidermis persists. x 400 FIGURE 4.-Voluntary muscle. Deep infiltration into body wall is separating and destroying muscle. x 400
VOL. 57, No.2, AUGUST 1976
MAGUIRE. OUT ZEN , CUSTER, AND PREHN
Downloaded from http://jnci.oxfordjournals.org/ at The University of British Colombia Library on October 6, 2015
FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE
442
5.-Lung. Foci of viable tumor cells undoubtedly result from hematogenous dissemination. x 400 6.-Lymph node. Marginal sinuses of node are invaded by bulky sheets of tumor. x 400 7.-Kidney. Large tumor nodule lies in renal cortex. X 400 8.-Kidney. Permeation of tumor along perivascular lymphatic channels. x 400 9.-ovary. Virtually complete displacement of parenchyma by tumor. x 400 1O.-Liver. Tumor emboli in sinusoids. x 400
MAGUIRE, OUTZEN, CUSTER, AND PREHN
