Granulopoiesis and Thrombopoiesis in Mice Bearing Transplanted Mammary Cancer1

[CANCER RESEARCH 26, 149-159, January 1966|

L. DELMONTE, A. G. LIEBELT, AND R. A. LIEBELT Department of Anatomy, Baylor University College of Medicine, Houston, Texas

Summary from a transplantable mouse mammary tumor (CE 1460 MA CA) that arose as a spontaneous tumor, associated with a severe Growth of a transplantable mammary cancer in CE mice (CE leukocytosis and extramedullary granulopoiesis (EMG)2, in a fe 1460 MA CA) was paralleled by hyperplasia of the granulocytic male mouse of the CE strain (22). CE 1460 MA CA has been elements of the bone marrow and peripheral leukocytosis (up to carried through more than 100 transplant generations in CE mice 350,000 leukocytes/cu mm) with a lymphoid-myeloid ratio re versal to 1:4 and a shift to the left. About 5-20% of the granulo and through more than 50 transplant generations in (BALB/C x CE) FI hybrid mice (hereafter referred to as Ft hybrids); it has cytic elements in the blood and bone marrow showed morpho logic abnormalities. Hematocrit values decreased 10-15%. shown remarkable stability as regards tumor biology and the tumor-associated hÃ©matologieresponse in both the parent strain Peripheral platelet levels manifested a slight depression in the (CE) and FI hybrid mice. face of bone marrow megakaryocytopenia and splenic mega- During a partial survey of other transplanted mouse tumors in karyocytosis. Extramedullary granulopoiesis, with the presence our laboratories, we observed a fluctuating increase in circulating of mitoses and all stages of development of the granulocyte series platelets but no leukemoid reaction to be associated with another in diffuse and discrete foci, was observed in the liver, spleen, transplantable mammary tumor (BALB/C 2301 MA CA) of lungs, lymph nodes, and adrenals of CE 1460 tumor bearers. The spontaneous origin. The specificity of this response was sug development of foci of extramedullary granulopoiesis, despite the gested by the finding that none of the other tumors studied in the absence of mÃ©tastasesin the bone marrow, suggests that the survey to date evoked a comparable platelet response. former resulted from direct stimulation of both intra- and extra- Although tumor-associated hÃ©matologie changesâ€”particu medullary hemopoietic tissue by a tumor-elaborated, granulo- larly leukemoid reactions and extramedullary hematopoiesisâ€” cyte-specific factor, rather than from a reduction of bone marrow have been reported frequently, and reviewed extensively by space available for granulopoiesis. Dunn (5) and Ebbenhorst-Tengbergen and MÃ¼hlbock(7), it was In BALB/C mice, another transplantable mammary cancer felt that the extraction of a granulocytosis-promoting factor from (BALB/C 2301 MA CA) induced periodic fluctuation of platelets CE 1460 tumor tissue and the consequently implied possibility between base-line and elevated levels and bone marrow mega- that another humoral (thrombocytosis-promoting) factor is karyocytosis but no change in splenic megakaryoctye levels. The elaborated by the BALB/C 2301 tumor were of sufficient interest bone marrow erythroid-myeloid ratio and the peripheral leuko cytes' lymphoid-myeloid ratio remained normal. Hematocrit to warrant a study of the biology of these tumors. Hence, the present report attempts (a) to relate the leukemoid and thrombo values decreased 10-15CÂ¿.No foci of extramedullary granulo cytic responses to tumor characteristics (e.g., growth rate, size, poiesis developed in the host tissues. and tumor viability), changes in blood morphology, and host tis The tumor specificity of the leukemoid and thrombocytic re sue histopathology, and (b) to differentiate between the host sponses was suggested by the host response to transplantation of strain-dependent and the tumor-dependent aspects of the hemo the respective tumors into (BALB/C x CE) FI hybrid mice and by poietic responses to the 2 tumors in the FI hybrid system. the regression of the specific CE 1460 tumor-associated and BALB/C 2301 tumor-associated hÃ©matologiechanges following Materials and Methods tumor excision. HÃ©matologieand histopathologic changes were studied in 98 control and 155 tumor-bearing mice of the CE, BALB/C, and FI Introduction hybrid strains. These included: Group I (control mice), (a) 74 in A recent report from our laboratories (3) described the extrac tact young adult (6-8 weeks old) and 6 intact 3-month-old CE, tion and partial purification of a granulocytosis-promoting factor BALB/C, and FI hybrid mice, and (&) 18 intact sham-operated young adult CE, BALB/C, and FI hybrid mice; Group II (mice 1Supported in part by USPHS Grants SF3CA-17941-01/02, bearing CE 1460 MA CA), 42 young adult, 6 3-month-old, and CA-07788-01, CA-04517-03and by American Cancer Society In 13 12-month-old CE mice and 45 young adult FI hybrid hosts; stitutional Grants ACS-IN-27D-Project No. 12 and ACS-IN-27E- Group III (mice bearing BALB/C 2301 MA CA), 31 young adult Project No. 1. BALB/C and 18 young adult FI hybrid hosts. Mice were kept in Received for publication March 16, 1905; revised September 7, groups of 3 per cage, separated as to sex, and given Purina Lab 1965. Chow and drinking water ad lib.

JANUARY 1966 149

Each host received a s.c. implant (l cu mm) of fresh donor days in 3- and 12-month-old CE mice of both sexes. Macro- tumor tissue delivered with a trocar into the right flank on Day 1. scopically, the tumor was a homogeneous white mass that, at a Of these mice, 105 animals were killed after 2-3 weeks (Group II) diameter of approximately 15 mm, began to manifest superficial or 9-11 weeks (Group III). Another 48 hosts of Groups II and eschar formation and erosion as well as central necrosis and cystic III were subjected to excision of moderately advanced (16-22 fluid accumulation. mm in diameter) tumors, while under light ether anesthesia, and MÃ©tastasesdid not develop in the tissues of young adult CE were observed for periods up to 30 days following both successful tumor bearers, but they occasionally occurred in the form of pin and unsuccessful (i.e., local recurrence or mÃ©tastases,or both) point to 3-mm foci in the lungs and livers of 3- and 12-month-old tumor excision. CE hosts during the usual 3-4 week survival period. MÃ©tastases Morning samples of caudal vein blood were obtained (a) at were also observed in the lungs of several young adult CE mice weekly intervals in intact controls, (6) prior to transplantation, which survived for exceptionally long periods of time (6-12 (c) at weekly intervals thereafter in unoperated tumor-bearing weeks) following tumor implantation. hosts, and (d) prior to operation and at 3-day intervals thereafter Tumor growth was paralleled by a marked rise in total circu in sham-operated controls and tumor-excised hosts. A single lating leukocytes (frequently to 350,000/cu mm and occasionally 13-fil blood sample, diluted 1:100 with 1.3 ml of ammonium to 750,000/cu mm) as well as by slight depression of both hemato- oxalate in an Unopette disposable blood-diluting pipette, sufficed crit (from 45-54% to 40-46%) and platelet levels (from 1.3-2.2 for both total leukocyte and platelet determinations by light million/cu mm to 1.0-1.7 million /cumm) (Chart 1). Circulating microscopy. Heparinized capillary tubes were used for micro- leukocytes showyeda reversal of the lymphoid-myeloid ratio to hematocrit determinations. Wright-Giemsa-stained blood films 1:4 and a shift to the left (5-15% immature granulocytes). and bone marrow imprints were prepared for differential leuko The bone marrow presented severe, predominantly granulocyte counts and studies of blood morphology. cytic hyperplasia with a shift to the left (Fig. 16), as well as pro Intact control mice (Group la) were sacrificed at ages com nounced megakaryocyte depression (Table 1). Megakaryocyte parable to those of the corresponding nonoperated tumor hosts. size remained within normal limits (20-50 fi in diameter). Sham-operated control mice (Group lÃ²)were sacrificed 21 days About 5-20% of the granulocytes in both blood and bone mar following surgery. row were enlarged up to 1.5-fold and showed other morphologic Tissue samples were fixed in 10% neutral formalin, sectioned abnormalities (Fig. lo) commonly seen in tumor-associated at 5 n, and stained routinely with hematoxylin and eosin as well leukemoid reactions (5, 21, 27) as well as in pernicious anemia, as with (PAS)2 plus alcian blue. drug toxicity, and radiation disease states (36). The cytoplasm of Megakaryocytes were counted in 100 high power fields ( X 450) the abnormal granulocytes tended towards extreme basophilia in of 5-/Ã-thickPAS-stained sections of femoral and sternal bone both immature and mature cells and frequently presented ir marrow and of spleenâ€”equivalent to 2.35 X IO5cu n of the fixed regular coarse granulation. The nuclei manifested swelling, hyper- tissue sample. segmentation, clumped and accessory chromatin, and occa Samples of blood, tumor tissue (with and without central sionally abnormal mitotic figures. necrosis), liver, and spleen were taken at autopsy, under aseptic The tumor size-tumor weight curve of the CE 1460 tumor was conditions, from 12 ~Fihybrid mice bearing CE 1460 MA CA and linear in transplanted and biphasic in recurrent (postexcision) cultured both aerobically and anaerobically on blood agar and in tumors (Chart 2). In the latter, it increased in a linear fashion up thioglycolate broth. Whenever necessary, further diagnostic cul to around the 3.5-gm level, then leveled off as the tumor began to ture media were used to identify any microorganisms present. manifest marked eschar formation, erosion, central necrosis, and A preliminary study of 6 other transplantable solid tumors cystic fluid. Occasionally, tumors underwent central erosion with carried in mice of 5 different strains in the Kirschbaum Memorial subsequent peripheral regrowth. Laboratory, Baylor University College of Medicine, was under Circulating leukocytes averaged 65,000-80,000/cu mm/gm of taken for the purpose of relating possible changes occurring in tumor weight and 8,000-12,000/cu mm/mm of tumor diameter the hosts' leukocyte and platelet picture with tumor type, growth (Chart 2). Leukocytosis averaging 100,000 leukocytes/cu mm rate, and size. These tumors included 1 skin cancer (A+ 9966), 3 was associated with a 10-mm tumor; yet macroscopic evidence of mammary cancers [(BALB/C x CE) FI 3206, DBA/2/ff 5199, necrosis was seen only after the tumor had attained a diameter of BALB/C 926)], 1 testicular tumor (CE 8), and 1 lymphoma 15 mm or more. Histologie analysis of CE 1460 tumors during ((BALB/C x CE) F, 1182). Leukocyte and platelet values in recent transplant generations in FI hybrid mice showed no con caudal vein blood were determined as described above. sistent relationship between the presence or absence of micro scopic foci of necrosis in 5.5- to 6.5-mm tumors and the onset of Results the myeloid reaction. Nor was the degree of microscopically evident necrosis in 9.5- to 11.5-mm tumors consistently propor CE Host tional to the degree of the leukemoid reaction observed. CE 1460 MA CA, an adenocarcinoma Type B according to In cases where necrosis advanced to the stage of tumor erosion, Dunn's (6) classification, attained a diameter of approximately the leukemoid reaction became markedly depressed, and the de 25 mm within 14-21 days in 6- to 8-week-old and within 21-28 pression appeared to be reversed only upon renewed growth along the tumor periphery. Cystic-necrotic material (devoid of viable cells) from large CE 1460 tumors, when injected i.p., failed 'The following abbreviations are used: PAS, periodic acid- to evoke significant leukocytosis in young adult CE mice (L. Schiff ; EMG, extramedullary granulopoiesis. Delmonte and R. A. Liebet, unpublished data). The peripheral

150 CANCER RESEARCH VOL. 26

leukocyte, or more specifically the peripheral granulocyte level TABLE 1 apparently was directly proportional to the volume of viable TUMOR SPECIFICITYOF HEMOPOIETICTISSUERESPONSEIN 3 tumor tissue present. STRAINSOFMICE BEARINGCE 1460 MA CA ORBALB/C 2301 Mitotically active foci of EMG developed consistently in the MA CA liver, spleen and lungs and occasionally in the lymph nodes and adrenals of CE mice bearing CE 1460 MA CA (Figs. 1 c-e; 2 CACE(BALB/C1460 MA CA(BALE/2301 MA f-k). EMG was more pronounced in 6- to 8-week-old than in 3- TISSUECE x CE) C x CE) F, and 12-month-old hosts. EMG foci were not usually associated KI hybridBALB/C hybridBALB/C with metastatic proliferation. Granulocytes in EMG foci en compassed both normal and morphologically aberrant forms. Only rarely did erythropoietic and thrombopoietic proliferation occur at sites of EMG. LiverSpleen ++++++Â±+ ++++++Â±-â€”-â€” Hepatic EMG foci occurred mainly within the walls of the larger venous vessels and occasionally among the liver cord cells Lungs AdrenalsLymph nodes+

Â¡esoIs CEHOST^,jf^.^r-""^.Â»â€¢â€¢""'' Megakaryocytes6 20Â§~ Bone marrow 100â€¢ Spleen38%278%38% 330%123% 165%146%126% ii ii 0 Degree of extramedullary granulopoiesis. * Percentage of control (100%) levels.

60 themselves (Fig. 2f,

I The spleen, which doubled or tripled its size within 2-3 weeks 20 of CE 1460 tumor implantation, showed evidence of diffuse EMG that disrupted the splenic architecture and occasionally even 0 displaced the lymphoid elements in germinal centers (Fig. le). Megakaryocytes, easily distinguished from other giant cells by 300 their strong affinity for the PAS stain, increased about 3-fold 250 (Table 1); they occurred mainly in discrete nests of both imma z ture and mature cells and occasionally .singly within the EMG o

BALB/C Host BALB/C 2301 MA CA, an adenoacanthoma according to 7 10 14 17 21 Dunn's (6) classification, had the macroscopic aspect of a homo DAYS POST-TRANSPLANTATION geneous white mass throughout the course of its development, i -M.IS.E. although it occasionally showed eschar formation towards the # 99.7% CONTROL CONFIDENCE LIMITS terminal stage (30-38 mm in diameter). CHART1. Peripheral blood changes accompanying growth of In young adult hosts of both sexes, the tumor developed to a CE 1460 MA CA in CE mice. diameter of approximately 25 mm within 9-11 weeks and at-

JANUARY 1960 151

Â®fflS^&&**f -Â»* â€¢s felÂ» &Ã• 152 CANCER RESEARCH VOL. 26

50 t-3UliÂ»I'Â»> /C HOST..* Lu K 40 TRANSPLANT LU RECURRENCE *-a"f <~30

'20 0a6040 iâ€¢-*.,-.,.*f i i i i i i i i K O IO ti ^ jt ,LJlrâ€¢â€¢â€¢%â€¢â€¢â€¢â€¢*â€¢-â€¢â€¢*.....:..â€¢.â€¢..::.

I 234567 TUMOR WEIGHT (GRAMS ) I-20o300_2503200m i,i i i i i i i i i 50

LU l- 40 LU <~305

o z150Â§ 1 IO 100K5O2.55

200 400 600 800 WBC(THOUSANDS/CU MM)

o ' â€” CD 2.OS| < 4 ='â€¢â€¢Â¿B'&Â¿jÂ¿Â¿Â¿&&Â¿Â¿6%%Â¿Â¿&LWrtfy//1**^ ^v//,~ â€¢ .^-M *J * LÂ°05:-c-P--P?t-BALB O 200 400 600 800 WBC(THOUSANDS/CU MM)

CHART 2. Correlation of leukocytosis, tumor size, and tumor I 3456789 10 II weight in mice bearing transplanted and recurrent CE 1460 MA WEEKS POST-TRANSPLANTATION CA. Upper graph: Ratio of tumor size to tumor weight. Trans I â€¢M.Â±S.E. plant: Y = Ã•.95X+ 15.28, r = 0.782, P < 0.001. Recurrence: â€” â€¢99 7% CONTROL CONFIDENCE LIMITS YÂ¡= 2.63X + 16.7Â«, ri = 0.646, PÂ¡< 0.010. Recurrence: Y2 = CHART 3. Peripheral blood changes accompanying growth of -2.44X + 38.90, r, = 0.812, I\ < 0.025. Interclass: r = 0.739, BALB/C 2301 MA CA in BALB/C mice. P < 0.001. Center graph: Ratio of tumor size to leukocytosis. Transplant: Y = 0.03X + 10.93, r = 0.581, P < 0.001. Recur crit levels were depressed slightly (from 47-60% to 40-48%). rence: Y = 0.04X + 8.42, r = 0.624, P < 0.001. Interclass: r = 0.979, P < 0.001. Lower graph: Ratio of tumor weight to leuko The striking characteristic of this tumor was that platelets cytosis. Transplant: F = 0.007X + 1.55, r = 0.509, P < 0.001. showed periodic fluctuation between baseline (0.8-1.8 million/ Recurrence: Y = 0.005X + 1.77, r = 0.329, P < 0.200. Interclass: cu mm) and elevated levels (to 3.0 million/cu mm), albeit without r = 0.613, P < 0.100. any discernably constant rhythm or correlation with tumor size and weight (Chart 3). Thrombocytosis was evident as early as tained a maximal diameter of 30-38 mm within 13-15 weeks, 2-3 weeks following transplantation of BALB/C 2301 MA CA, although some hosts survived up to 24 weeks. when the tumor measured approximately 3-5 mm in diameter; Neither mÃ©tastasesnorfoci of EMG developed in the tissues of the phenomenon of recurrent thrombocytosis became well es BALB/C hosts bearing BALB/C 2301 MA CA. tablished during the ensuing 9-11 weeks (Chart 3) and persisted During the course of BALB/C 2301 tumor growth, total and thereafter. Individual platelet fluctuations were far more irregu differential leukocyte numbers remained normal, while hemato- lar in the circulation of tumor hosts than in that of control mice

FIG. 1. Leukemoid blood picture and granulopoiesis in tissues of CE 1460 MA CA hosts: a, peripheral blood with aberrant granulocytic forms (X 1000); 6, bone marrow (X 150); c, spleen (X 050); d, lymph node (X 300); e, lymph node (X 750).

JANUARY 1960 153

154 CANCER RESEARCH VOL. 26

HOST;HYBRID CE 50 bJÂ¡IHaSi3020 *"""*'â€”/*..._.** * TRANSPLANT 40 RECURRENCE 10604O

|fr '*^aT^ Til I 30

*" 3bfc.-"^-rw:-g-.-.-.-:-:.-Tr--.III! -ar- â€” "*Â»v â€” - 10

20-F|

(BALB/C X CE) F, 50 ce o 4O _ 250 - 30 roo O 20 "S I50h *<3 IO

mO O 7 14 21 28

__ â€¢Â»_ â€¢. T'"Â«â€¢Â¡a T â€¢>a ^Â¿Â¿- DAYS POST-TRANSPLANTATION CHART5. Growth rate of transplanted and recurrent CK 1400 MA CA in CE and (BALB/C x CE) F, hybrid mice. Upper graph: CE Host, Transplant: Y = 1.24A"- 0.57, r = 0.501, P < 0.001. I Recurrence: Y = 2.13X - 0.12, r = 0.727, P < 0.001. Interclass: Ã®3- "w r = 0.494,P < 0.500.Lower graph: (BALB/C x CE) Fi hybrid host, V.T ^ Transplant: Y = 0.54X + 4.%, r = 0.624, P < 0.001. Recurrence: Â¿i Y = 1.98X - 0.10, r = 0.942, P < 0.001. Interclass: r = 0.143, P < 0.500. OI O morphologic forms and no macro- or microscopically discernible I 3456789 10 II WEEKS POST-TRANSPLANTATION metastatic foci.

!â€¢ M.Â±SE. CE 1460 MACA Â«p. 99.7% CONTROL CONFIDENCE LIMITS BALB/C 2301 MACA (BALB/C x CE) I'\ Hybrid Host

CHART4. Peripheral blood changes accompanying growth of In Fi hybrid mice, both CE 1460 MA CA and BALB/C 2301 CE 1460MA CA and BALB/C 2301 MA CA in (BALB/C x CE) MA CA retained their biologic and specific hÃ©matologiecharac Fi hybrid mice. teristics (Chart 4; Table 1). CE 1460 tumor growth rate in young adult F[ hybrids lagged 1 and periodically attained peak levels in the former that were up to 50% higher than any ever observed in the latter. At 9-11 week behind that observed in young adult CE hosts (Chart 5). This lag, which was significant within 99.9% confidence limits, weeks, tissue megakaryocytc concentrations manifested an in was reproduced in the development of both the leukemoid re crease of about 50% in the bone marrow and 2590 in the spleen (Table 1). The tumor dependence of these changes in the mega- sponse and the platelet depression (Charts 1 and 4), although leukocyte and platelet values as related to tumor size were sta karyocyte-platelet axis has been studied extensively and will be reported elsewhere.3 tistically identical in the parent strain and the FI hybrid. It is of interest that the CE 1460 tumor growth rate in young adult The bone marrow of BALB/C 2301 MA CA hosts presented an Fi hybrids was comparable to that observed in 12-month-okl unchanged erythroid-myeloid ratio. There were no aberrant CE hosts. The incidence of lung and liver mÃ©tastasesin CE 1460 tumor 3L. Delmonte and R. A. Liebelt, Granulopoiesis and Thrombo bearers appeared to be dependent on time rather than on tumor poiesis in Mice Bearing Transplanted Mammary Cancer. II. size or strain. In 3- to 12-week-old CE and in young adult FI Megakaryocyte-Platelet Axis, in manuscript. hybrid hosts bearing advanced tumors (20-25 mm in diameter),

FIG. 2. Leukemoid blood picture and granulopoiesis in tissues of CE 1460MA CA hosts:/, liver (X 150); g, liver (X 475); h, lung (X 200); i, lung (X 600); j, adrenal (X 60); k, adrenal (X 475).

JANUARY 1966 155

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1966 American Association for Cancer Research. L. Delmonte, A. G. Liebelt, and R. A. Liebelt the incidence of mÃ©tastaseswas negligible at 2 weeks, slight at 3 were elevated more than 3-fold in CE 1460 tumor bearers but weeks, and increasingly marked at 4 or more weeks after tumor only about Â§inBALB/C 2301 tumor bearers (Table 1). transplantation. BALB/C 2301 tumor growth rate was identical in young adult Tumor Excision Fi hybrid and BALB/C hosts (Charts 3 and 4). No leukemoid Moderate transitory leukocytosis (25,000-45,000 leukocytes/ reaction occurred in either host strain. The fluctuating platelet cu mm)â€”probablydue toa transi tor y increase in leukopoiesis com elevation was slightly less pronounced in Fi hybrids than in pensating for blood loss at surgeryâ€”followed not only CE 1460 BALB/C hosts (Charts 3 and 4). Unlike CE 1460 MA CA, and BALB/C 2301 tumor excision in hosts of the respective BALB/C 2301 MA CA was already associated with a rise in parent strain (CE and BALB/C) and in FI hybrid hosts, but also platelet levels by the time a 5-mm tumor diameter had been at sham operation in all 3 strains of mice (Table 2). tained in the FI hybrid host. Renormalization of total and differential leukocyte values and Hematocrit changes were slight (10-15% maximum decrease) disappearance of aberrant granulocytic forms occurred in CE and and were comparable for the 2 tumors and all 3 strains of mice Fi hybrid hosts within 3-9 days of excision of CE 1460 MA CA (Charts 1, 3, 4). (Table 2). Excision of a 16- to 20-mm tumor was usually fol The bone marrow and EMG and megakaryocyte picture in FI lowed within 5-21 days by tumor recurrence along the line of hybrids bearing CE 1460 MA CA or BALB/C 2301 MA CA was excision. Recurrence of the leukemoid response was observed as comparable to that of hosts of the respective strain of tumor early as 24-48 hr prior to macroscopically evident tumor re- origin. growth. The growth rate of CE 1460 tumors recurring at the site Splenomegaly in FI hybrids, as well as in the strain of tumor of excision was accelerated by comparison to that of the trans origin, depended on tumor size. Splenic megakaryocyte levels planted tumors (Chart 5).

TABLE 2 PERIPHERALBLOODPICTUREFOLLOWINGSHAMOPERATIONANDTUMOREXCISIONINCE, BALB/C AND(ALB/C x CE) FI HYBRID MICE (Mean Â±S.E.)

POSTSURGERY036912is21\VBC

STRAINCE(BALB/CMOUSE EXCISIONShamTumorShamTumorTumorShamTumorShamTumorShamTumorTumorShamTumorDAYS

mm)10.6Â±2.9185.3Â±7.113.3Â±0.7175.1Â±19.612.0Â±1.59.6Â±1.812.6Â±4.014.8Â±1.835.9Â±2.310.3Â±0.944.0Â±3.924.4Â±2.110.3Â±1.124.8Â±2.014.0Â±1.432.4Â±8.913.0Â±1.125.7Â±3.421.3Â±1.08.9Â±1.330.5Â±2.022.9Â±2.221.5Â±5.319.6Â±1.919.5Â±1.525.0Â±1.518.4Â±2.732.4Â±3.222.8Â±1.420.4Â±1.222.4Â±1.921.3Â±1.524.9Â±2.218.2Â±0.523.0Â±2.916.0Â±1.723.5Â±3.522.4Â±1.518.6Â±0.720.1Â±3.610.8Â±3.241.8Â±6.211.9Â±5.120.2Â±1.811.9Â±1.011.3Â±0.511.8Â±0.611.8Â±2.114.1Â±2.4Platelets(thousands/cu

CANoneCE1460 MA

F,BALB/CCE(BALB/Cx CE)

CABALB/C1460 MA

CANoneBALB/C2301 MA

CANoneCE2301 MA

mm)1.863Â±0.1301.239Â±0.0811.548Â±0.1241.465Â±0.2131.783Â±0.0381.468Â±0.1211.948Â±0.2171.803Â±0.1323.093Â±0.3051.675Â±0.0452.897Â±0.1982.824Â±0.1461.660Â±0.1112.773Â±0.3022.723Â±0.3504.454Â±0.5802.116Â±0.0694.334Â±0.2312.649Â±0.2841.719Â±0.0944.474Â±0.4732.630Â±0.4152.643Â±0.7292.163Â±0.2682.461Â±0.1782.468Â±0.4281.758Â±0.1672.934Â±0.1762.191Â±0.1721.827Â±0.0221.819Â±0.0902.832Â±0.7842.783Â±0.1742.003Â±0.1961.602Â±0.0551.902Â±0.0972.493Â±0.0081.889Â±0.1712.122Â±0.2862.196Â±0.1261.676Â±0.0593.061Â±0.5911.984Â±0.1492.277Â±0.1021.800Â±0.0771.710Â±0.1962.364Â±0.2131.678Â±0.0961.884Â±0.080(millions/eu

CANoneCE1460 MA

F,BALB/CTUMORNoneCEx CE)

CABALB/C1460 MA

CANoneBALB/C2301 MA

2301 MA CATYPE

156 CANCER RESEARCH VOL. 26

With CE 1460 MA CA recurrence after unsuccessful excision, affect the type of hÃ©matologieresponse associated with the the tumor size-tumor weight relationship was initially (up to the tumor: A+ 9966 skin cancer, (BALB/C x CE) FI 3206 mam 3.5-gm level) similar to that observed before excision; then, with mary cancer, CE 8 testicular tumor, and DBA/2/ff 5199 mam the development of a disproportionate weight increase due to mary cancer hosts survived approximately 4-5, 5-6, 6-7, and massive accumulation of cystic fluid, the ratio began to show a 19-20 weeks, respectively; yet all showed normal platelet values. trend towards a linear decrease rather than towards an increase A+ 9966 and (BALB/C x CE) F! 3206 tumor hosts also mani (Chart 2). However, the pre- and postexcision ratios of tumor size fested a leukemoid reaction, whereas CE 8 and DBA/2/ff 5199 to leukocyte number were statistically identical (interclass hosts showed a normal leukocyte picture. BALB/C 962 mam r = 0.979) (Chart 2). Following recurrence, the relationship of mary cancer and (BALB/C x CE) Ft 1882 lymphoma killed their tumor weight to leukocyte number Ix-came random (r = 0.329), respective hosts within approximately 4-5 weeks; hosts bearing although it manifested a trend towards the same ratio as that of both these tumors manifested a trend towards platelet depression the pre-excision group (interclass r = 0.613) (Chart 2). similar to that seen in CE 1460 tumor hosts, whose survival was Following CE 1460 tumor excision, pinpoint to 3-mm mÃ©tas 3-4 weeks, but only BALB/C 962 hosts also manifested Icuko- tases were found frequently in the lungs and liver and occa cytosis. None of the tumors studied evoked a recurrent throm- sionally in the lymph nodes, kidneys, and adrenals, but not in bocytosis like that observed in BALB/C 2301 tumor hosts. femoral or sternal bone marrow of the mice that presented no recurrence at the tumor implantation site. Microscopically evi Discussion dent foci of lung mÃ©tastasesbut no foci or EMG were found upon histologie examination of all CE and FI hybrid hosts with The tumor specificity of the leukemoid and thrombocytic re macroscopic-ally and hematologically diagnosed successful CE sponses associated with CE 1460 MA CA and BALB/C 2301 MA 1460 tumor excision. CA, respectively, was demonstrated by the response of geneti In BALB/C and F! hybrid mice bearing BALB/C 2301 MA cally isologous hosts (Fi hybrid mice) to the transplantation of CA, tumor excision was usually successful, recurrence being rare these genetically unrelated tumors and by the regression of the within the 21-day postsurgical observation period of our experi specific tumor-associated hÃ©matologieresponses following tumor ments. Neither of 2 mice that manifested incipient (palpable) excision. tumor recurrence presented mÃ©tastases. Over the past 50 years, leukocytosis associated with extra- A transitory 2- to 3-fold increase in platelet levels with a peak medullary hematopoiesisâ€”both leukopoiesis and erythropoiesisâ€” at 6 days followed both tumor excision and sham operation in all has been reported repeatedly in tumor-bearing mice (1, 2, 5, 7, 3 strains of mice, persisted for 9-12 days, and manifested a trend 13, 14, 19-21, 26-28), rats (21, 23), hamsters (15), rabbits (8), towards renormalization by 21 days (Table 2). The immediate dogs (8), monkeys (8), and man (4, 9, 10-12, 16-18, 24, 25, reaction, following sham operation or tumor excision, appeared 29-35). Thrombocytosis associated with leukemoid reactions has to be dependent on (i.e., compensatory for) blood loss or trauma, been reported both in man and in tumor-bearing mice manifest or both, at surgery rather than on tumor ablation itself, but it was ing extramedullaiy hematopoiesis (2). A direct correlation be less pronounced after sham operation than after tumor excision. tween splenic megakaryocytosis and peripheral leukocytosis has In all 3 strains of mice, the tumor-dependent platelet changes been reported by Parsons et al. (28) in mice bearing transplanted were totally or partially reversed within 21 days of excision of sarcomas. Most of the tumor-associated leukemoid reactions either CE 1460 MA CA or BALB/C 2301 MA CA. described in the literature have been associated with moderate to extreme concurrent effects on erythropoiesis or thrombopoiesis, Microbiology of CE 1460 MA CA or both, and have resulted in extramedullaiy hemopoiesis involv Although all the mice from which these tissue samples had been ing all cell lines rather than a single one, as is the case for the taken presented a marked leukemoid reaction, only some of the CE 1460 and BALB/C 2301 tumor systems. The tumor-host relationship has been shown to be affected by samples of tumor (67%) and spleen (50%), a single sample of blood (8%), and no sample of liver (0%) from mice bearing ad a variety of factors. Whereas the growth rate of CE 1460 MA CA vanced (20- to 25-mm diameter) CE 1460 tumors manifested was influenced by factors such as host age and genetics, the de evidence of microorganisms in thioglycolate broth or on blood velopment of granulocytosis and EMG foci appeared to be de agar. Among the positive cultures, 4 different Gram-negative pendent upon the volume of viable tumor tissue present and on organismsâ€”Pseudomonas aeruginosa, Aerobacter aerogenes, Pro the hemopoietic mitotic and maturation potential of intra- and teus vulgaris, and Staphylococcus aureusâ€”were identified; a extramedullary host tissue in response to the tumor-elaborated Bacillus (species not identified) also was isolated. The presence stimulus. The occurrence of EMG foci, despite an actual quanti or absence of bacterial contamination had no effect on either the tative increase rather than a reduction of granulocytic tissue in occurrence or the magnitude of the CE 1460 tumor-associated the bone marrow cavities by metastatic or other factors, further leukemoid reaction. supports the concept that the host's granulocytic response is tumor dependent. HÃ©matologieSurveyof Other Transplantable Mouse Tumors Although several investigators including Bateman (2) found The hÃ©matologiestudy of hosts of 5 different strains of mice that tumor age, necrosis, ulcÃ©ration,and eschar formation had a bearing additional tumors of various types of tissues revealed that greater influence on tumor-associated leukemoid reactions in neither the tumor tissue type and growth rate nor the host strain mice than did tumor tissue viability or volume, our data, like and survival time following tumor transplantation appeared to those of Ebbenhorst-Tengbergen and MÃ¼hlbock(7), suggested

JANUARY 1906 157

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1966 American Association for Cancer Research. L. Delmontc, A. G. Liebelt, and R. A. Liebelt that the amount of viable tumor tissue present rather than the Acknowledgments presence of necrotic tissue was the prime factor in the develop ment of the tumor-associated leukemoid reaction. In fact, in the The authors wish to express their appreciation to Miss Kath- aryn Kemp and Mr. William T. Miller for their respective roles CE 1460 tumor system, whenever the ratio of tumor weight to in the survey of the Kirschbaum Memorial Laboratory's trans tumor size increased disproportionately owing to the appearance planted mouse tumors for tumor-associated leukemoid and throm of cystic fluid and necrosis at the core of the tumor, the pe bocytic reactions. ripheral leukocytosis tended to level off despite a continuous in crease in absolute tumor weight. Furthermore, association of ero sion with necrosis, resulting in an even greater loss of viable References tumor tissue, was actually accompanied by regression of the 1. Barnes, W. A., and Sisman, I. E. Myeloid Leukemia and Non- leukocytosis, which recurred only upon renewal of growth at the malignant Extramedullary Myelopoiesis in Mice. Am. J. Can tumor periphery. cer, 37:1-35, 1939. The possibility that tumor-associated microorganisms induce 2. Bateman, J. C. Leukemoid Reactions to Transplanted Mouse the leukemoid reaction has been proposed by Barnes and Sisman Tumors. J. Nati. Cancer Inst., //.-671-87, 1951. (1). Bateman (2) found leukemoid reactions to occur in tumor- 3. Delmonte, L., and Liebelt, R. A. Granulocytosis-promoting bearing mice without demonstrable bacterial infection. In the Extract of Mouse Tumor Tissue: Partial Purification. Science, 148:521-23, 191.5. present study, we found the presence or absence of bacterial con 4. Downey, II., Major, S. C., and Noble, J. F. Leukemoid Blood tamination of CE 1460 tumor or host tissue to have no effect on Pictures of the Myeloid Type. Folia Haematol., 4/.-493-511, the induction or the magnitude of the leukemoid reaction, sug 1930. gesting that there probably is no bacterial pathogenesis of the 5. Dunn, T. B. Normal and Pathologic Anatomy of the Reticu tumor-associated leukocytosis and EMG. However, the possi lar Tissue in Laboratory Mice, with a Classification and Dis bility of viral pathogenesis cannot be excluded, since recent pre cussion of Neoplasms. J. Nati. Cancer Inst., 14:1281-1433, liminary electron microscope studies in our laboratories (L. 1954. Delmonte, T. Smith, and R. A. Liebelt, unpublished data) have 6. . Morphology of Mammary Tumors in Mice. In: F. revealed the presence of virus-like particles in CE 1460 tumor Homburger (ed.), The Physiopathology of Cancer, pp. 38-84, tissue. New York: Paul B. Hoeber, Inc., 1959. 7. Ebbenhorst-Tengbergen, W. J. P. It. van, and MÃ¼hlbock,O. The disparity between the ratios of tumor size to leukocytosis Disorders of the Blood of Mice Bearing Spontaneous or Trans in mice with transplanted and those with recurrent CE 1460 planted Tumors. Brit. J. Cancer, ^2:81-97, 1958. tumors raises an interesting point: Is the disproportionate de 8. Engelbreth-Holm, J. Spontaneous and Experimental Leukae crease in this ratio due to reactivation of residual EMG foci mia in Animals. Edinburgh and London: Oliver and Boyd, that have become dormant following tumor ablation, superim Ltd., 1942. posed on the laying down of new EMG foci during recurrent 9. Fahey, R. J. LTnusual Leukocyte Response in Primary Carci tumor growth? Or has the host become "sensitized" to hemo- noma of the Lung. Cancer, 4/930-35, 1951. poietic stimuli by the original tumor transplant? The phe 10. Hensler, L.: Hohe Leukoytose durch Karinom. Schweiz. nomenon only serves to emphasize the delicate balance of tumor Med. Wochschr., 83:1032-34, 1953. and host factors in tumor-host relationships. 11. Hill, J. M., and Duncan, C. N. Leukemoid Reactions. Am. J. Med. Sci., 20/.-847-57, 1941. Similarly to the CE 1460 tumor-associated leukemoid and 12. Hinshaw, D. B., and Hoxie, H. J. Leukemoid Reactions in EMG reaction, the BALB/C 2301 tumor-associated thrombo- Carcinoma. Calif. Med., 71:300-1, 1949. cytosis and bone marrow megakaryocytosis showed direct de 13. Hueper, W. C. Leukemoid and Leukemic Conditions in White pendence on the presence of viable tumor tissue. However, it Mice with Spontaneous Mammary Carcinoma. Folia Hae appeared that even a small volume of BALB/C 2301 tumor tissue matol., 52:167-78, 1934. sufficed to initiate a platelet releaseâ€”as manifested by the ap 14. â€” â€”. Megakaryocytosis in White Mice with Spontaneous pearance of the 1st platelet peak 2-3 weeks following tumor im Mammary Carcinoma. Am. J. Med. Sci., Ã•SS.-41-48,1934. plantationâ€”but that the bone marrow megakaryocyte build-up 15. Kelsall, M. A. Hematopoiesis in the Spleen of Tumor-bearing Hamsters. J. Nati. Cancer Inst., /0.-625-36, 1949. (hyperplasia) was a more gradual process paralleling tumor 16. Krumbhar, E. B. Leukemoid Blood Pictures in Various Clini volume increase. cal Conditions. Am. J. Med. Sci., Ã•72.-519-33,1320. The specificity of BALB/C 2301 MA CA for evoking a fluctuat 17. Kugelmeier, L. M. LeukÃ¤moide Reaktionen bei Karzinom. ing thrombocytosis is further supported by the lack of consistent Folia Haematol., 53:370-81, 1935. relationship found by both ourselves and Bateman (2) between 18. Lang, F. J. Myeloid Metaplasia. In: H. Downey (ed.), Hand tumor tissue type, size, and growth rate and host strain and sur book of Hematology, pp. 2105-45. New York: Paul B. Hoeber, vival time, following tumor transplantation, on the one hand, and Inc., 1938. the development of tumor-associated thrombocytic (and/or 19. Lappat, E. J., and Cawein, M. A Study of the Leukemoid Re leukocytic) reactions, on the other hand. Further investigation sponse to Transplantable A-280 Tumor in Mice. Cancer Res. is necessary to determine whether a thrombocytosis-promoting 24:302-11, 1964. factor can be extracted from BALB/C 2301 tumor, just as a 20. Lewis, M. R. Myeloid Hyperplasia Brought About in Mice by granulocytosis-promoting factor was extracted from CE 1460 the Growth of Dibenzanthracene Tumors and Its Relation to the Transplantability of the Tumors into Mice of Alien mammary tissue (3). The BALB/C 2301 tumor system appears Strains. Am. J. Cancer, 29:510-16, 1937. to present us with a biologic model from which we can postulate 21. â€¢ . Myeloid Infiltrations Occurring in the Adrenals of the probable presence of a tumor-dependent factor influencing Animals Bearing Certain Tumors. Ibid., 30:95-101, 1937. circulating platelet levels. 22. Liebelt, R. A., Lay, M., and Davis, B. Extramedullary He-

158 CANCER RESEARCH VOL. 26

matopoiesis Associated with a Transplantable Mouse Mam Beitrug zur histopathologischen Bedeutung des makrophagen mary Cancer. Proc. Am. Assoc. Cancer Res., 3:130, 1960 (ab (retikulo-endothelialen) Systems.) Yirchow. Arch. Pathol. stract). Anat. Physiol. Klin. Med., Â£42:138-94,1923. 23. McKucn, C. S., and Selye, H. Histologie Changes in the Adre 30. Plonskier, M. Ueber tumorformige (extramedullaere, hetero- nals of Tumor-bearing Rats. Am. J. Mcd. Sci., /S9.-423-24, tope) subpleurale Knochenmarksherde. Ibid., 277:804-9, 1930. 1935. 31. Saleeby, E. R.: Heterotopia of Bone Marrow without Appar- 24. McFadzcan, A. J. S., Todd, D., and Tsang, K. C. Polycythe- rent Cause. Am. J. Pathol., Ã•.-69-76,1925. mia in Primary Carcinoma of the Liver. Blood, J3:427-35, 32. Sonnenfeld, A. Leukaemische Reaktionen bei Karzinom. Z. 1958. Klin. Med., Ã•Ã•/.-108-1G,1929. 25. Meyer, L. M., and Rotter, S. D.: Leukemoid Reaction (Hy- 33. Starr, G. F., Stroebel, C. F., Jr., and Kearnes, T. P. Poly- perleukocytosis) in Malignancy. Am. J. Clin. Pathol., IS: cythemia with Papilledema and Infratentorial Vascular Tu 218-22, 1942. mors. Ann. Internal Med., 4Â«:978-8(>,1958. 20. Parsons, L. D. Leukemia Coincident with and Transmissible 34. Steigbigcl, N. H., Oppenheim, J. J., Fishman, L. M., and Car by a Spindle-celled Sarcoma in the Mouse. J. Pathol. Bac- bone, P. P. Metastatic Embryonal Carcinoma of the Test is teriol, 40:45-54, 1935. Associated with Elevated Plasma TSH-like Activity and Hy- 27. â€” â€”. Blood Changes in Mice Bearing Experimental Sarco perthyroidism. New Engl. J. Med., 27/:345-49, 1904. mas. Ibid., 43:1-22, 1930. 35. Waldmann, T. A., and Bradley, J. E. Polycythemia Secondary 28. Parsons, L. D., and Warren, F. L. Cellular Changes in the to a Pheoehromocytoma with Production of an Erythropoiesis Spleen and Lymph (Â¡lands of Mice Used for Carcinogenic and Stimulating Factor by the Tumor. Proc. Soc. Exptl. Biol, Related Experiments, with Special Reference to Giant Cells Med., / 08:425-27, 1901. of the Spleen. Ibid., 52:305-21, 1941. 36. Wintrobe, M. M. Clinical Hematology, 1180 pp. Philadelphia: 29. Paunz, T. Ueber die Rundzellhcrde der Nebenniere. (Ein Lea & Febiger, 1901.

JANUARY 1900 159

L. Delmonte, A. G. Liebelt and R. A. Liebelt

Cancer Res 1966;26:149-159.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/26/1/149

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/26/1/149. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.