use of a Sorvall JB-4 rotary microtome. Bone mar- row cellularity was estimated on H&E-stained sec- tions with the use of a calibrated intraocular grid on.
Carcinoma, Pulmonary Metastases, Neutrophilic Leulocytosis VX2
Possible Animal Model
of
and
Tumor-Associated Granulocytosis
A. J. HOUGH, Jr., MD, W. C. HUBBARD, PhD, and J. A. OATES, MD
From the Laboratory Service, Veterans Administration Medical Center, Nashville, Tennessee, and the Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee
In New Zealand white rabbits bearing the transplantable VX2 carcinoma intramuscularly in the hind limbs uniformly a granulocytosis syndrome developed, characterized by peripheral blood neutrophilia and marked granulocytic bone marrow hyperplasia, whereas the syndrome did not always develop in animals bearing the tumor intraabdominally. The relationship between the extent of pulmonary metastases and granulocytosis/bone marrow hyperplasia was significant (P < 0.005). This positive correlation (correlation coefficient = 0.85) existed for both animals bearing intraabdominal tumors and animals bearing intramus-
cular tumors. The granulocytic syndrome occasionally occurred in rabbits with mild degrees of pulmonary metastatic involvement, but never with total absence of pulmonary metastases. Hypercalcemia occurred prior to the appearance of pulmonary metastases and was not essential to the development of granulocytosis. These data suggest that granulocytosis in VX2 carcinomabearing rabbits is dependent on the presence of tumor in the lungs and is not directly related to implantation site. This tumor-bearing animal model may be suitable for studies of tumor-associated granulocytosis in vivo. (Am J Pathol 1983, 112:231-237)
THE VX2 CARCINOMA-bearing rabbit has been employed extensively as an animal model of malignancy-associated hypercalcemia.1-12 In addition to becoming hypercalcemic, in rabbits bearing the VX2 carcinoma in hind limb musculature granulocytosis develops, accompanied by bone marrow hyperplasia.'9'3 Granulocytic syndromes have been described in animals with other tumors 14-7 and in human patients with certain carcinomas that involve the lung either primarily or secondarily.18-25 The granulocytic syndrome associated with the VX2 carcinoma in rabbits is of interest not only because of possible analogies to human tumor-associated granulocytosis21 but also because of the potential role of prostaglandins of the E series in modulation of granulocyte production.22-25 The incidence of VX2 carcinoma-associated granulocytosis and hypercalcemia varies with respect to the primary site of implantation of the tumor.2413 PGE2 production in vivo does not significantly vary in rabbits bearing the VX2 carcinoma intramuscularly and those bearing the tumor intraabdominally. Since granulocytosis
does not occur in all rabbits bearing the tumor, we undertook this study to examine the relationship between the site of tumor growth and the development of the granulocytic syndrome.
Materials and Methods Animals Female New Zealand white rabbits weighing from 2.5 to 3 kg, maintained on a standard laboratory diet Supported by the Veterans Administration and by NIGMS-1543 1. Dr. Oates is a Joe and Morris Werthan Professor of Investigative Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee. Dr. Hubbard is with the Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee. Accepted for publication March 30, 1983. Address reprint requests to Dr. A. J. Hough, Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR 72203.
0002-9440/83/0805-0231$01.15 ©) American Association of Pathologists
231
232
HOUGH ET AL
AJP * August 1983
were given transplants of either 0.5 g of partially hemogenized VX2 carcinoma in both hind limbs or 1.0 g in the peritoneal cavity, as described previously.4 Control animals of similar age, weight, and sex were employed. A total of 9 control, 10 leg-tumorbearing, and 26 abdominal-tumor-bearing rabbits were studied. Leukocyte and Platelet Counting Blood samples for leukocyte and platelet counts were obtained by cardiac puncture. Leukocyte and platelet counts were performed on Coulter S and F particle counters, respectively. Leukocyte differential counts were performed manually by counting 200 consecutive leukocytes on Wright-stained, air-dried smears.
Quantitation of Disease Control and tumor-bearing rabbits were subjected to autopsy at the conclusion of each experiment. The extent of tumor involvement of the liver, diaphragm, and mediastinum was estimated grossly and rated on a scale of 1 to 4. The lungs were immediately fixed in neutral 4% formaldehyde. The whole lungs were deWV
_W
hydrated, embedded in paraffin, and sectioned at 6 , in the midcoronal plane on a Lipshaw 80-A sliding microtome. Whole-mount lung sections stained with hematoxylin and eosin (H&E) were examined, and the total percentage of surface involved by tumor was estimated with the use of a 0.5-cm ruled grid overlay. Less than 20% involvement was considered 1 +; 2140%o involvement, 2+; 41-60% involvement, 3+; and >6007o involvement, 4+. The humeri, radii, femurs, and tibias of each animal were dissected free of soft tissues, bisected in the midsagittal plane, and fixed in Technicon Zenker's solution for 6-10 hours. After decalcification in 507o vol/vol formic acid, the bones were dehydrated, embedded with paraffin, and sectioned at 5 , with the use of a Sorvall JB-4 rotary microtome. Bone marrow cellularity was estimated on H&E-stained sections with the use of a calibrated intraocular grid on 10 consecutive medium power fields (x 160) in the diaphysis of each of the bones removed. We used periodic acid-Schiff and hematoxylin-stained sections to evaluate incidence of granulocytic precursors in relation to total marrow cellularity. Marrow cellularity was defined as the percentage of marrow occupied by hematopoietic tissue. We averaged the means of the scores of each bone provide the bone
_- --N
V
M&
.
4l
9 4
2 A
Sn Figure 1 -Bone marrow of the midfemoral shaft in a control rabbit. The marrow section demonstrates approximately 40% cellularity and is composed of an admixture of cellular types. (H&E, x900)
VX2 CARCINOMA
Vol. 112 * No. 2
233
Figure 2- Bone marrow of the midfemoral shaft in a rabbit bearing VX2 carcinoma in the hind limb musculature. The marrow section demonstrates greater than 80% cellularity (4 +) and an increase in granulocyte precursors characterized by reniform and segmented nuclei. (H&E, x 900)
marrow cellularity index for each animal. Mean cellularity was graded at 1 +, 80%. Control rabbits uniformly had mean cellularity of 40-50% by this method.
Statistical Analysis Statistical analysis of data obtained was performed with the Student t test, the F test, and linear regression analysis.
Results Hematopoietic Changes In rabbits with hind limb transplants a severe granulocytosis of peripheral blood developed, as compared with control animals. White blood cell counts in tumor-bearing animals ranged up to 38,000/cu mm, with from 90% to 98% neutrophilic granulocytes on differential counts. Control animals had normal total leukocyte counts with 50-65% granulocytes. The mean percentage of juvenile (unsegmented) neutrophilic granulocytes did not differ significantly between the control and tumor-bearing groups. In arithmetic terms, the marked increase in
peripheral white blood count was due almost entirely to mature granulocytes. No elevation in eosinophils, basophils, or monocytes was noted in smears; nor was a corresponding increase in platelet counts noted by automated particle counting. In addition, there was an incidence in the mean bone marrow cellularity index (BMCI) from 1.1 ± 0.06 (SE) in control (Figure 1) to 3.8 ± 0.13 (SE) in rabbits bearing hind limb tumors by 3 weeks of tumor growth (Figure 2). The increase in bone marrow cellularity was almost entirely due to granulocytic precursors, unsegmented neutrophils, and mature neutrophilic granulocytes (Figure 2), and in this respect resembled the peripheral blood smears in the tumor-bearing rabbits. In contrast to the constant occurrence of granulocytosis after hind limb transplants, abdominal transplants caused only a portion of the rabbits to develop the granulocytic syndrome, in spite of the rapid growth of the tumor in the abdomen. Some rabbits with abdominal transplants did not evince granulocytosis, while in others a syndrome developed identical to that of the animals with hind limb transplants. Tumors at hind limb and intraabdominal sites often contained extensive necrosis, but bacterial cultures and smears from the necrotic areas were uni-
234
xw-
HOUGH ET AL
AJP * August 1983
EJ ABDOMINAL TUMOR
50
z HINDLIMB TUMOR
40
PERCENT OF 30 TOTAL 20
I
-
-
10
7I "I 11
K
with pulmonary
metastases without pulmonary metastases
30,000 WBC per
F
cu mm
Kl. . F' .11t Q L--j 0 1+ 2+ 3+ 4+ EXTENT OF PULMONARY METASTASES Figure 3-Distribution of rabbits with varying extents of pulmonary
20,000 _
S.E.
10,000 _-
metastasis. In rabbits with hind limb transplants more severe pulmonary involvement consistently developed.
r-f .
formly negative. Granulocytosis was noted in both peripheral blood and bone marrow in animals without, as well as animals with, tumor necrosis. Pulmonary Metastases In these particular studies all of the animals with hind limb transplants had pulmonary metastatic involvement by 3 weeks of tumor growth. When metastases were quantitated in midcoronal lung sections, 60% of the animals with hind limb transplants showed extensive pulmonary metastatic burden (34+). Although 81 07o of the animals with abdominal transplants developed some degree of pulmonary metastasis, only 15% showed extensive pulmonary metastatic burden (3-4+), as seen in Figure 3. The overall comparison of control, leg-tumor-bearing and abdomen-tumor-bearing groups is summarized in Table 1. Of rabbits with intraabdominally implanted tumors, only those with pulmonary metastases developed granulocytosis of the peripheral blood (Figure 4) and an increase in bone marrow cellularity (Figure 5), while animals without pulmonary metastases maintained normal values for these parameters. This difference was significant (P < 0.005) for both
CONTROL ABDOMEN
LEG
Figure 4-The effect of the variation of primary VX2 tumor transplant sites on white blood cell (WBC) per cubic millimeter. No significant difference in WBC per cum mm is present between abdominal and hind limb transplants when pulmonary metastases are present. The WBC counts of rabbits with abdominal transplants without pulmonary metastases are not significantly different from those of control rabbits.
white blood count and bone marrow cellularity index. Combined data from both tumor-bearing groups can be seen in Table 2. Further examination of the relationship of the pulmonary metastatic burden to the peripheral white blood count and bone marrow cellularity index dis-
E -
with pulmonary metastose s J without pulmonary meta stases
BONE MARROW CELLULARITY INDEX + S.E.
Table 1 -Comparison of Control, Leg Tumor, and Abdomen Tumor Groups Control Leg tumor Abdomen tumor Number Pulmonary metastatic index Bone marrow index Initial WBC Week 3 WBC A WBC *
9
10
26
-
2.6 (0.26)* 3.8 (0.13)*
1.5 (0.22) 2.8 (0.19)
1.1 (0.06) 6.9 (0.55) 7.2 (0.68) 0.3 (0.18)
Standard error of the mean.
7.7 (0.36)* 21.2 (1.46) 13.6 (1.36)
7.8 (0.37) 21.4 (1.70) 13.2 (1.76)
CONTROL ABDOMEN
LEG
Figure 5-The effect of the variation of primary VX2 tumor transplant sites on bone marrow cellularity index (BMCI). Values in abdominaltumor-bearing rabbits with pulmonary metastases were similar to those of animals with leg transplants with pulmonary metastases. Animals with abdominal transplants without pulmonary metastases had values similar to those of control animals.
VX2 CARCINOMA
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Table 2-Effect of the Degree of Pulmonary Metastasis on Bone Marrow Cellularity and the WBC WBC count (10-3) Pulmonary Bone marrow metastatic index Week 3 Number index Initial 0
5
9 11 9 2
1+
2+ 3+ 4+ *
1.3 (0.05)' 2.5 (0.20) 3.5 (0.15)
3.8 (0.13) 4.0 (-)
6.7 (1.03) 8.4 8.0 7.6 7.8
(0.38) (0.38) (0.60) (-)
9.8 17.8 24.5 25.6 30.1
(0.21) (1.38) (1.94) (1.91) (-)
235
A 3.1 9.4 16.5 18.0 22.3
(1.05) (1.71) (2.0) (1.71) (-)
Standard error of the mean.
closed a positive correlation between the extent of pulmonary metastasis and the white blood count (Figure 6) as well as the bone marrow cellularity index. The increments in these hematologic parameters were proportionally greater from 0 to 1 + and 1 + to 2+ pulmonary metastatic involvement than from 2 + to 3 +. Further demonstration of the relationship between pulmonary metastases and bone marrow cellularity is seen in Figure 7, where a linear relationship (correlation coefficient = 0.85) existed for both hind limb- and abdominal-tumor-bearing rabbits. Animals with slight and moderate degrees of pulmonary metastatic spread (1 + and 2 +) had more variable bone marrow pictures than those with more extensive pulmonary involvement (3 + and 4 + ). Occasionally, the syndrome appeared in animals with less than 20%o pulmonary involvement, but never in animals without any pulmonary metastases.
Extrapulmonary Metastases Autopsies revealed that in addition to pulmonary metastatic involvement noted above, many animals developed varying degrees of diaphragmatic involve-
30,000-
WBC per
cu mm + S.E.
1 + 0 21 3+ EXTENT OF PULMONARY
METASTASES Figure 6-Relationship of the degree of pulmonary metastasis to the WBC per cubic millimeter. P < 0.01 (F test).
ment. This was predominantly observed in intraabdominal tumor transplantation and, when present without pulmonary metastases, was not associated with granulocytosis. Hepatic metastases were identified in 25% of rabbits given intraabdominal trans-
plants and 5% of animals given implants intramuscularly. Granulocytosis was not associated with hepatic involvement. In these studies, only one rabbit developed renal metastasis. There was no evidence of bony metastases of the tumor in any of the animals. Discussion The present studies show a statistically significant relationship between metastatic pulmonary tumor and granulocytosis in the VX2 carcinoma-bearing rabbit. The exact nature of the stimulus leading to the development of the granulocytic leukemoid syndrome in rabbits is not known but does not appear to be related to secondary infection of the tumor. One area of speculation is that pulmonary tumor deposits may secrete and release mediators of the granulocytic leukemoid syndrome directly into the systemic circulation. However, granulocytosis associated with other animal tumors and in man does not appear dependent on pulmonary metastases. The relationship between pulmonary tumor involvement and granulocytosis in man has not been systematically evaluated. The incidence of granulocytosis associated with certain carcinomas in man has been noted, but the pathophysiology is unclear.19 26"27 Colony-stimulating activity has been isolated from some human carcinomas in vitro.28 The relationship between prostaglandins of the E series and other substances affecting granulopoiesis has been extensively investigated in vitro.2225 Comparable studies in vivo have not yet been reported. Chronically administered indomethacin prevents bone destruction in VX2tumor-bearing rabbits.5 29 Whether prostaglandin synthesis inhibitors can modify the granulocytosis syndrome as well as hypercalcemia has not been established. Irrespective of the exact role of prostaglandins in the complex events leading to the granu-
236
AJP * August
HOUGH ET AL
1983
AX
4+ A - Hindlimb Tumor X- Abdominol Tumor
r=0.85 y= 0.94X- 1.06 3+
y x
-
A^ /A x AA
PULMONARY METASTATIC X
INDEX INDEX 2+
1+
X
-
-
xx x
XX ~ AX ~ xx ~~~~XA&
XAX
lvv YY s { lo
1+
Figure7-Linearregressionanalysisof the bone marrow cellularity index (X axis) versus pulmonary metastatic index (Y axis). r = 0.85.
I
4+ 3+ 2+ BONE MARROW CELLULARITY INDEX
locytic leukemoid syndrome in vivo, we suggest that the VX2 carcinoma-bearing rabbit may be a suitable animal model of tumor-associated neutrophilic
9.
granulocytosis. References 1. Voelkel EF, Tashjian AH Jr, Franklin R, Wasserman E, Levine L: Hypercalcemia and tumor-prostaglandins: The VX2 carcinoma in the rabbit. Metabolism 1975, 24:973-986 2. Young DM, Fioravanti JL, Prieur DJ, Ward JM: Hypercalcemic VX2 carcinoma in rabbits: A clinicopathologic study. Lab Invest 1976, 35:30-46 3. Wilson JR, Merrick H, Vogel SD, Woodward ER: Hyperparathyroid-like state in rabbits with the VX2 carcinoma: Further studies. Am Surg 1965, 31:145-152 4. Hubbard WC, Hough AJ, Johnson RM, Oates JA: The site of tumor transplantation affects the development of hypercalcemia in rabbits. Prostaglandins 1980, 19:881-889 5. Seyberth HW, Hubbard WC, Oelz 0, Sweetman BJ, Watson JT, Oates JA: Prostaglandin-mediatedhypercalcemia in the VX2 carcinoma-bearing rabbit. Prostaglandins 1977, 14:319-331 6. Wolfe HJ, Bitman WR, Voelkel EF, Griffiths JH, Tashjian AH Jr: Systemic effects of the VX2 carcinoma on the osseous skeleton: A quantitative study of trabecular bone. Lab Invest 1978, 38:208-219 7. Hough AJ Jr, Seyberth HW, Oates JA, Hartmann WH: Changes in bone and bone marrow of rabbits bearing the VX2 carcinoma. Am J Pathol 1977, 87: 537-552 8. Tashjian AH Jr, Voelkel EF, Levine L: Plasma concentrations of 13,14-dihydro-i5-keto prostaglandin E2 in rabbits bearing the VX2 carcinoma: effects of hydro-
10.
11.
12.
13. 14.
15. 16. 17.
cortisone and indomethacin. Prostaglandins 1977, 14:309-317 Hubbard WC, Hough A, Brash AR, Johnson RM, Oates JA: The VX2 carcinoma: Humoral effects and arachidonic acid metabolism, Advances in Prostaglandin Thromboxane Research. Edited by B Samuelsson, P Ramwell, R Paoletti. New York, Raven Press, 1980, pp 525-527 Hubbard WC, Hough AJ, Brash AR, Watson JT, Oates JA: Metabolism of linoleic and arachidonic acids in VX2 carcinoma tissue: Identification of monohydroxyoctadecadienoic acids and monohydroxyeicosatetraenoic acids. Prostaglandins 1980, 431-447 Klein DC, Raisz LG: Prostaglandins: simulators of bone resorption in tissue culture. Endocrinology 1970, 86:1436-1440 Holtrop ME, Raisz LG, King GJ: The response of osteoclasts to prostaglandin and osteoclast activating factor as measured by ultrastructural morphometry, Proceedings, Mechanisms of Localized Bone Loss. Edited by Horton JE, Tarpley TE, Davis WF. Special Supplement to Calcified Tissue Abstracts, 1978, p 13 Hough AJ, Hubbard WC, Oates JA: Pathogenic studies of VX2 carcinoma-associated granulocytosis (Abstract). Lab Invest 1980, 42:27 Osipova TV, Bukhman VM, Belyanchikova NI, SvetMoldavski GY: Hematopoietic tissue reactions associated with growth of syngeneic transplantable tumors in mice. Byulleten Eksperimental'noi Biolgii Meditsiny 1978, 85:346-349 Lappat EJ, Cawein M: A study of the leukemoid response to transplantable A-280 tumor in mice. Cancer Res 1964, 24:302-311 Grezes F, Salomon JC: Myoepithelioma with a leukemoid reaction in BALB/c mice. J Natl Cancer Inst 1972, 197:1527-1538 Delmonte L, Liebelt AG, Liebelt RA: Granulopoiesis and thrombopoiesis in mice bearing transplanted mammary cancer. Cancer Res 1966, 26:149-159
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18. Asano S, Urabe A, Sato N, Kondo Y, Ueyama Y, Chiba S, Ohsawa N, Kosaka K: Demonstration of granulopoietic factor(s) in plasma of nude mice transplanted with a human lung cancer and in tumor tissue. Blood 1977, 49:845-852 19. Sato N, Asano S, Ueyama Y, Mori M, Okabe T, Kondo Y, Ohsawa N, Kosaka K: Granulocytosis and colony-stimulating activity (CSA) produced by a human squamous cell carcinoma. Cancer 1979, 43:605610 20. Johnson RL, Donnell RM: Diffuse granulocytic infiltration of giant cell carcinoma of the lung: A distinctive histologic finding with clinical significance (Abstr). Lab Invest 1979, 40:262 21. Robinson WA: Granulocytosis in neoplasia. Ann NY Acad Sci 1974, 230:212-218 22. Chan HSL, Saunders EF, Freedman MH: Modulation of human hematopoiesis by prostaglandins and lithium. J Lab Clin Med 1980, 95:125-132 23. Kurland JI, Broxmeyer HE, Pelus LM, Bockman RS, Moore MAS: Role of monocyte-macrophage-derived colony-stimulating factor and prostaglandin E in the positive and negative feedback control of myeloid stem cell proliferation. Blood 1978, 52:388-407
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24. Motomura S, Dexter RM: The effect of prostaglandin E1 on hemopoiesis in long-term bone marrow cultures. Exp Hemat 1980, 8:298-303 25. Brennan JK, Lichtman MA, Dipersio JF, Abboud CN: Chemical Mediators of granulopoiesis: A review. Exp Hematol 1980, 8:441-464 26. Rubins J, Waken CJ: Hypoglycemia and leukemoid reaction with hypernephroma. NY State J Med 1977, 77:406-409 27. Gibson EL, Herbert V, Robinson WA: Granulocyte colony stimulating activity and vitamin B12 binding proteins in human urine. Br J Haematol 1974, 28:191197 28. Kimura N, Yoshiyuki N, Yanase T: A high level of colony-stimulating activity in a lung cancer patient with extensive leucocytosis, and the establishment of a CSA producing cell line (KONT). Scand J Hematol 1982, 28:417-424 29. Galasko CSB, Rawlins R, Bennett A: Timing of indomethacin in the control of prostaglandins, osteoclasts and bone destruction by VX2 carcinoma in rabbit. Br J Cancer 1979, 40:360-364
