Leukemia Research Vol. 3, No. 1, pp. 23-28. 0145-2126/79/0201-0023 $02.00/0 © Pergamon Press Ltd., 1979. Printed in Great Britain

CHARACTERIZATION OF MALIGNANT CELL POPULATIONS IN MNU-INDUCED LEUKAEMIA OF MICE

PAUL BAINES, T. MICHAEL DEXTER and RAYMOND SCHOFIELD

Paterson Laboratories, Christie Hospital and Holt Radium Institute, Withington, Manchester M20 9BX (Received 7 June 1978. Revised 9 August 1978. Accepted 15 August 1978)

Abstract--A surface marker analysis was found to be of value in classifying leukaemias of poorly differentiated blasts induced by MNU. Thy-1 -t-ve (T cell) lymphoma commonly arose in intact animals with the thymus frequently, though not invariably, enlarged. Thy-1 --ve, Ig--ve leukaemias arose in a low percentage of intact mice and in 8[16 leukaemias of thymectomized mice that were tested. These "markerless" leukaemias remain uncharacterized. Of the remaining leukaemias that developed in thymectomized mice, 4/16 were Ig+ve (or B cell) lymphomas and 4]16 showed low-intensity staining for both Thy-1 and Ig. The mechanism of thymic involvement in MNU-leukaemogenesis is discussed. Leukaemias of intact and thymectomized mice do not appear to arise from a common target cell since Thy-1 +ve leukaemias rarely arose in thymectomized mice given implants of neonatal thymus tissue shortly after MNU-treatment. In fact, MNU-induced thymic lymphoma arises from an intra-thymic target population.

Key words: Murine leukaemia, MNU, surface markers, target cells, T, B lymphomas

INTRODUCTION

LEUKAEMIA developing in mice either spontaneously or as a consequence of treatment with leukaemogenic regimes of irradiation or chemicals, frequently presents as thymic lymphoma. Thymectomy prevents the development of leukaemia in high leukaemia strains and in low leukaemia strains exposed to chronic doses of irradiation or leukaemogenic viruses [7, 9]. This suggests that the thymus plays an essential role in these models of leukaemogenesis. Furthermore, surface marker analyses have demon- strated that the malignant cells of thymic lymphomas frequently possess surface Thy-1 antigen, a character- istic of thymocytes and peripheral T cells [1, 5].

Many chemicals which induce thymic lymphoma in intact mice also induce poorly-differentiated, markerless leukaemia in thymectomized mice [5, 12]. Methylnitrosourea (MNU) is a potent leukaemogen in both intact and thymectomized mice when given as a single intravenous injection [4]. Thymic lym- phomas develop in a high percentage of intact mice within 250 days whilst in approximately 50% of thymectomised recipients, leukaemias involving the spleen and/or mesenteric lymph nodes develop over a period of 250-500 days. A preliminary surface marker analysis has demonstrated that MNU-

Abbreviations: MNU, methyl nitrosourea; Ig, immuno- globulin; PBS, phosphate buffered saline; GPS, Guinea pig serum; NMS, Normal mouse serum; NRS, Normal rabbit serum.

23

induced thymic lymphomas are malignancies of Thy-1 ÷ ve cells and although leukaemias arising in MNU-treated, thymectomized mice were less well characterized [4] it was suggested that T lymphocytes may be the target cells in MNU leukaemogenesis.

The objectives of the current work were: (i) to define more precisely the nature of MNU-susceptible target cells; and (ii) to determine whether or not leukaemias of intact and thymectomized mice arise from a common target cell.

MATERIALS AND METHODS

Mice BDF1 (C57B1/6 x DBA/2J F1), C57B1/6 and DBA/2J

mice were all reared in the Animal Unit, Paterson Laboratories. Only BDF1 female mice were exposed to MNU.

Surgical techniques Thymectomy. Mice were thymectomized at 6 weeks of

age under Nembutal anaesthetic.

Neonatal thymus grafts. Thymuses from 2 day old mice were implanted into adult recipient mice either subcutaneously into the brachial region or under the renal capsule.

Preparation and administration of leukaemogen. MNU was synthesized in the Paterson Laboratories and its purity checked by melting point analysis. For experi- mental use MNU was dissolved in cold (4°C) saline at

24 PAUL BAINES, T. MICHAEL DEXTER a n d RAYMOND SCHOFIELD

2 mg/ml with vigorous shaking, and 50 mg/kg body wt was injected intraveneously into 6-8 week old recipient mice.

Preparation of cell suspensions. Leukaemic tissues, which were grossly enlarged, or normal tissues, were either minced with fine scissors or passed through fine gauze and dispersed in M.E.M. Hepes to give single cell suspensions.

Anti-sera. AKR anti C3H (anti-Thy 1.2): was prepared by the method of Rief and Allen [15]. In both cytotoxicity and fluorescence assay this serum was highly reactive with thymocytes from BDF1 mice (Thy-l.2) but not with t hymocytes from R F M (Thy-l.1) mice; reacted with less than 1 0 ~ of BDF1 marrow cells; was unreactive, after brain absorption, with BDF1 thymocytes or bone marrow cells. Fluorescein conjugated goat anti-mouse IgG (Gam FITC) and rabbit anti-mouse-Ig (Ramg Ig-reactive against IgM, IgG1,2,3, IgA) were supplied by Nordic Ltd. Fluorescein conjugated sheep anti-rabbit IgG was sup- plied by Wellcome Ltd.

Complement. Guinea pig serum, absorbed with mouse liver homogenate, was used as a complement source, at a final dilution of 1:10 in PBS.

Surface marker techniques

(a) Detection of Thy-l.2 by 51Cr release cytotoxicity assay: the method of Raft and Wortis [14] was followed. Briefly, cells labelled with 51Cr were incubated with 1/10 anti-Thy 1 or NMS (controls) for 30rain at 37°C, washed once and incubated with 1/10 complement for a further 30 min at 37°C. The released activity in the supernatant was assayed on a Searle automated (Nuclear Chicago) gamma-counter.

counts released with anti-Thy 1 - -

Cytotoxicity index = Counts released with NMS counts released with Tr i ton- -

Counts released with NMS

(b) Indirect immunofluoresence: both Thy-1 and Ig surface antigens were detected using a modification of the indirect immunofluoresence test [11 ]. To assay Thy-1, cells were incubated at 4°C with a primary layer of anti- Thy-l, at 1/10 dilution, washed 4 times and re-incubated with FITC conjugated goat anti-mouse Ig(Gamlg) at a 1/10 dilution. In control tubes normal mouse serum replaced anti-Thy 1. It is important to note that significant numbers of cells stained in control tubes when cell sus- pensions contained B cells since Gain Ig detects surface Ig directly. Although Thy-1 was routinely assayed by indirect fluorescence (being the most sensitive test avail- able), the cytotoxicity assay was applied to leukaemias comprising large numbers of B cells.

To assay for surface Ig, cells were incubated at 4°C with a primary layer of rabbit anti-mouse Ig (Ram Ig) at a 1/10 dilution, and a second layer of FITC conjugated sheep anti-rabbit IgG at 1/20 dilution. In control tubes normal rabbit serum replaced Ram Ig.

The working dilution of 1/10 for anti Thy-1 and Ram Ig was chosen because, on titration against normal

thymus, spleen and bone-marrow, this dilution was well within the sensitivity limitation of the two assay pro- cedures employed.

X-irradiation. X-irradiation at a dose rate of 40 rad/ min was given using a Siemens stabilipan X-ray machine operating at 390 KV and 12 mA. Mice were housed in Perspex boxes.

Morphology. Air-dried smears of leukaemic tissues were fixed in methanol and stained with benzidene and McNiells tetrachrome.

R E S U L T S

Incidence/induction times o f MNUoinduced leukaemia

As repor ted earl ier [4] t h y m e c t o m y great ly pro- longed the induc t ion t ime a n d reduced the yield of leukaemias developing in M N U - t r e a t e d mice (Fig. 1).

Tissue infiltration in leukaemie mice

Leukaemias o f in tac t mice were c o m m o n l y associated wi th thymic en largement . F requen t ly the spleen, l ymph nodes , a n d liver were en la rged and showed leukaemic infi l t rat ion. Occasional ly, no thymic invo lvement was observed a l though b o t h spleen and lymph nodes were grossly enlarged. Leukaemias of in tac t mice usual ly passaged readi ly to syngeneic recipients. Leukaemic , M N U - t r e a t e d thymec tomized mice usually presen ted wi th enlarged mesenter ic lymph nodes a n d / o r en la rged spleens, which were occasional ly haemorrhag ic . In m a n y

99

9(]

5O

,o_j

i I

J J I I

200 300 500

Doys a f te r MNU

FIG. 1. Cumulative incidence ( ~ ) of leukaemias deve- loping in intact and thymectomized BDF1 mice after i.v.

injection of 50 mg/kg of MNU. O intact mice; • thymectomized mice.

Characterization of MNU-induced leukaemic cells

TABLE 1. M N U - I N D U C E D LEUKAEMIA OF INTACT ~ B D F 1 MICE: SURFACE

MARKERS ON LEUKAEMIC CELLS IN SUSPENSIONS PREPARED FROM GROSSLY

ENLARGED ORGANS OF TERMINAL LEUKAEMIAS

25

No. of Group (organ enlargement mice

in primary leukaemia) tested Surface marker indices mean (range)

Thy-1 Ig

1. Thymic only 3 78 (71-87) 3 (1-8) 2. Thymic disseminated 7 70 (68-91) 7 (0-14) (T+ S-4-LNS) 3. Non-thymic (S+LNS)

(a) 5 80 (63-96) 2 (0-7) (b) 2 9, 25 2, 13

T--Thymus; S--spleen; LNS--lymph nodes.

cases, some months elapsed between injection of cells from grossly enlarged organs of thymectomized mice, and the appearance of leukaemia in secondary hosts.

Leukaemic cell morphology Leukaemias arising in MNU-treated intact or

thymectomized mice commonly comprised large numbers of undifferentiated blasts with variable numbers of lymphocytes. Occasional (3/16) myeloid/ myelomonocytic leukaemias were observed in thymectomized mice.

Surface markers Grossly enlarged, leukaemic organs (usually

thymus, spleen or lymph node) from intact, MNU- treated mice were frequently found to contain large numbers of Thy-1 + v e cells irrespective of whether the thymus was enlarged or not. However, few Thy- l -kve or I g + v e cells were present in 2/7 leukaemias showing no thymic enlargement (Table 1). Grossly enlarged organs (usually spleen or lymph node) from 8/16 leukaemic, thymectomized mice contained less than 2 5 ~ T h y - l + v e or I g + v e cells which indicates substantial proliferation of a

TABLE 2. M N U - I N D U C E D LEUKAEMIA OF THYMECTOMIZED

9 BDFI MICE: SURFACE MARKER VALUES FOR CELLS IN SUSPENSIONS PREPARED FROM GROSSLY ENLARGED ORGANS

OF TERMINAL LEUKAEMIAS

Group

No. of Surface marker indices mean mice (range) tested Thy-1 Ig

1 8 10 (0-25) 8 (0-17) 2 4 8 (3-12) 82 (74-92) 3 4 42 (26-51) 45 (24-63)

Thy- 1 - ve, I g - ve population (Table 2). A further 4/16 leukaemias from thymectomized mice consisted of I g + v e cells which failed to lyse in the presence of anti-Thy-1 + complement. Cells of the remaining 4 leukaemias showed light, diffuse staining for both Thy-1 and Ig giving final indices usually of greater than 20 ~ for each marker.

Thymus implant experiments Implants of neonatal thymus tissue into syngeneic,

thymectomized mice 24h after MNU-treatment failed to reduce the induction time or increase the incidence of leukaemias (Table 3). In one experiment only 2/6 s.c. grafts showed any sign of enlargement and only one of these was T h y - l ÷ v e . Intra-renal thymus implants similarly failed to reduce the induc- tion time of leukaemias.

In a second experiment, thymectomized BDF1 mice received C57B1 neonatal thymus implants 3 weeks before M N U treatment. Leukaemia deve- loped in 7/8 of the recipients within 220 days, and in all these mice the thymus graft was grossly en- larged (Table 4). On transplantation, 3/7 leukaemias progressed in both C57B1 and BDF1 recipients and 4/7 leukaemias progressed in BDF1 but not in C57B1 hosts, which indicates that 3/7 leukaemias were of C57B1 (donor) origin and 4/7 leukaemias were of BDF1 (host) origin. These data strongly suggest that thymic lymphomas only develop where the thymus is exposed directly to MNU.

Effect of irradiation upon MNU-leukaemogenests BDF1 mice were exposed to increasing doses of

irradiation at varying times before M N U treatment, in an attempt to alter the incidence and induction time of thymic lymphoma. However, the incidence and induction time of thymic lymphoma were similar in irradiated or control, non-irradiated groups (Table 5).

26 PAUL BAINES, T. MICHAEL DEXTER and RAYMOND SCHOFIELD

TABLE 3. INCIDENCE AND INDUCTION TIMES OF LEUKAEMIAS DEVELOPING IN THYMECTOMIZED

(Tx) B D F I FEMALE MICE TREATED VClTH M N U AND SUBSEQUENTLY GIVEN NEONATAL THYMUS

GRAFTS SUBCUTANEOUSLY (S.C.) OR INTRA-RENALLY (i.r.)

Observation 50~* Induction Incidence of Enlargement of Protocol period (days) time (days) leukaemia grafted thymus

Tx+s.c. thymus 600 512 6/10 2/6 Tx+i.r. thymus 350 - - 1/11 0/1

* 50 ~ Induction time--time at which 50 ~ of total mice per batch have died with leukaemia.

TABLE 4. INCIDENCE AND ORIGIN OF LEUKAEMIA IN THYMECTOMIZED BDFI FEMALE MICE GIVEN NEONATAL THYMUS IMPLANTS, INTRA-RENALLY, 3 WEEKS

BEFORE MNU INJECTION

No of leukaemias Incidence of 50 ~ Induction showing graft Origin of malignant cells leukaemia time (days) enlargement Donor Host

7/8 145 7/7 3/7 4/7

DISCUSSION

Conventional haematological staining techniques were of limited value in classifying MNU-induced leukaemias since few cells other than poorly dif- ferentiated blasts were present in terminal popula- tions. Surface marker analysis however, permitted further characterization of the cell populations in- volved. In common with other models of induced

TABLE 5. INDUCTION OF LEUKAEMIA IN ~ BDFI MICE TREATED WITH MNU AT VARIOUS TIMES AFTER

X-IRRADIATION

Time of MNU 50% injection after Induction

Irradiation irradiation Incidence of time of dose (rad) (days) leukaemia leukaemia

3OO 6 5/5 111 10 6/6 119 14 5/6 137 22 6/6 132

500 6* 6/6 120 10" 6/6 149 14 6/6 103 22 6/6 110 30 6/6 126

800+bm 6* 3/3 200 10" 4/5 104 30* 5/6 169

300 No MNU 0/6 500 No MNU 0/6 800+bin No MNU 0/6 No Irradiat. MNU 6/6 113

* Groups supported with bone marrow.

leukaemias [1,5], malignant cells from MDU-induced thymic lymphomas were invariably Thy-1 + ve which confirms their T cell nature. Leukaemias of intact mice, which lacked thymus enlargement, were usually T h y - l + v e although occasional Thy-l--ve, Ig--ve leukaemias also arose. Thy-1 --ve, Ig--ve leukaemias were also recorded after a prolonged induction time in 8/16 thymectomized mice that developed the disease after MNU-treatment and the nature of these cells remains undefined. Leukaemic cells of this type may express low amounts of Thy-1 or Ig which are not detected in our assay systems using 1/10 anti-Thy-1 or anti-Ig. However, 3 such "null" leukaemias, which were titrated against increasing concentrations of anti-Thy-1 and anti-Ig, failed to stain or lyse at any serum dilution.

Malignant cells from 4 leukaemias of thymecto- mized mice bore surface Ig but no Thy-1. It is im- probable that this staining represents uptake of immunoglobulin from the serum or uptake of aggregates via Fc receptors since: (a) cells prepared from s.c. grafts of tumour tissue also stained for Ig; and (b) the level of staining was not reduced after culturing the cells in serum-free medium for 1 h. Furthermore, two leukaemias have been successfully cultured in vitro and shown to be capable of syn- thesizing immunoglobulin [13]. The remaining 4 leukaemias that arose in thymectomized mice showed low-intensity staining for both Thy-1 and Ig. It is unclear whether this low-level stain represents actual expression of Thy-1 and Ig or non-specific uptake of serum in vivo or non-specific uptake of serum or aggregates from our test sera.

Characterization of MNU-induced leukaemic cells 27

Thus, removal of the thymus: (a) prolongs the induction time; (b) reduces the incidence; and (c) alters the surface marker characteristics of leukaemias induced by MNU.

The thymus may contain a target population or may furnish an environment suitable for the rapid proliferation of an extra-thymic target-cell popu- lation. I f the latter alternative is correct, then the thymus may act "directly" upon infiltrating cells or "indirectly" upon extra-thymic cells perhaps via "thymic hormone". Thy-1 ÷ v e lymphomas were not always associated with thymus enlargement which could mean that the target cell(s) need not enter the thymus in order to proliferate rapidly. However, it is equally probable that target cells may reside for a brief but critical period in the thymus before migrating and undergoing extensive proliferation in peripheral organs. Whatever the nature of the thymus-target cell interaction, the development of Thy-1 + v e lymphomas requires a prolonged thymic presence since thymectomy as late as 8 weeks after administration of M N U inhibited the early onset of leukaemia in 5/7 mice (unpublished observations).

I f thymectomy does not remove a target popu- lation but instead removes a site essential to the rapid development of T h y - l + v e lymphoma, then it is possible that leukaemias of intact and thy- mectomized mice all derive from a common target cell. It could thus be suggested that in the presence of thymus, many of the 8 Thy- l - -ve , Ig- -ve leukaemias that arose in thymectomized mice would have otherwise developed more rapidly and expressed Thy-1. Furthermore, although 4/16 leukaemias expressed surface Ig, it may be that this represents an alternative method of differentia- tion normally obscured by the onset of thymic lymphoma. However, implants of neonatal thymuses into adult, thymectomized mice which had been treated 24 h earlier with MNU, failed to increase the incidence or reduce the induction time of leukaemias (Table 3). The failure to utilize these grafts indicates that the extra-thymic target popula- tions are either incapable of producing T h y - l + v e lymphoma or, since the thymus graft will take 2-3 weeks to become vascularized, require entry into the thymus during this period. It seems probable that MNU-induced thymic lymphoma usually arises from an intra-thymic target population since malig- nant donor (C57B1) thymocytes developed from thymus grafts implanted into thymectomized, BDF1 mice 3 weeks before M N U treatment (Table 4). In this respect M N U leukaemogenesis bears some

resemblance to the RadLV/radiation-induced lym- phomas of C57B1/Ka mice which appear to develop from an intra-thymic target population [3].

Kaplan [8] has suggested that after irradiation, large numbers of immature thymic blasts (the proposed target cells) are present at a time when leukaemogenic virus, also released by irradiation, is present in quantity. It was thought that injection of exogenous marrow shortly after irradiation, which inhibits the development of leukaemia in C57B1/Ka mice [8], may speed thymic recovery thus shortening the sensitive phase. MNU-lukaemogenesis appears to be largely independent of marked changes in haemopoietic cellularity and kinetics since the incidence and induction times of thymic lymphoma are similar in MNU-treated control mice and in mice given M N U only 6-10 days after 800 rad X-irradiation and supportive marrow cells (Table 5). In addition, injection of marrow cells within 24 h of MNU-treatment failed to inhibit the development of thymic lymphoma (unpublished observations), which suggests that the mechanisms involved in M N U and RadLV]radiation-induced leukaemo- genesis may differ.

That the leukaemic process can be initiated by M N U within 6-10 days of lethal irradiation and reconstitution suggests that the target cell is either radioresistant or is rapidly generated from the donor marrow. Although few donor marrow cells were reported to enter the thymus within 10 days of reconstitution of lethally-irradiated mice [10], more recent data indicate that this may not be so [2]. Radio-resistant thymocytes have been reported [6] and it is possible that M N U acts upon one of these. We are currently establishing radiation chimaeras to resolve this point.

MNU-induced thymic lymphoma may therefore provide an alternative model to the X-irradiation/ RadLV-induced leukaemias of C57B1/Ka mice. Although leukaemias developing in MNU-treated, thymectomized mice have only been partially characterized, the observation that a proport ion of these leukaemias are B-cell lymphomas is of signi- ficance since: (a) this malignancy is not common in the mouse; and (b) B-cell lymphomas are of some value in the study of B lymphocyte development.

Acknowledgements--We wish to thank Dr. A. W. Craig for providing the MNU. This work was supported by grants from the Medical Research Council and Cancer Research Campaign.

28 PAUL BAINES, T. MICHAEL DEXTER and RAYMOND SCHOFIELD

REFERENCES

1. CHAZAN R. & HARAN-GHERA N. (1976) The role of thymus sub-populations in "T" leukaemia development. Cell Immun. 23, 356.

2. DECLEVE A., GERBER G. B., LEONARD A., LAMBERT-COLLIER M., SASSEN A. & MAISIN J. R. (1972) Regeneration of thymus, spleen and bone-marrow in X-irradiated A K R mice. Radiat. Res. 51, 318.

3. DECLEVE A., TRAVIS M., WEISSMAN I. L., LIEBERMAN M. & KAPLAN H. S. (1975) Focal infection and transformation in situ of thymus cell subclasses by a thymotropic murine leukaemia virus. Cancer Res. 35, 5385.

4. DEXTER T. M., SCHOFIELD R., LAJTHA L. G. & MOORE M. (1974) Studies on the mechanisms of chemical leukaemo- genesis. Br. J. Cancer 30, 325.

5. HXAI H., SHISA H., MATSUDAIRA Y. & NlSmZUKA Y. (1973) Theta antigen in N-nitrosobutylurea leukaemogenesis of the mouse. Gann 64, 197.

6. KADISH J. L. & BASCH R. S. (1975) Thymus regeneration after lethal irradiation: evidence for an intra-thymic radioresistant T cell precursor. J. Immun. 114, 425.

7. KAPLAN H. S. (1950) Influence of thymectomy, splenectomy and gonadectomy on incidence of radiation-induced lymphoid turnouts in strain C57 black mice. J. natn. Cancer. Inst. 11, 83.

8. KAPLAN H. S. (1963) The role of radiation on experimental leukaemogenesis. Natn. Cancer Inst. Monogr. 14, 207. 9. METCALF D. (1966) The Thymus: Its Role in Immune Responses, Leukaemia development and Carcinogenesis.

Springer, Berlin. 10. MICKLEM H. S., FORD C. E., EVANS E. P. & GRAY J. (1966) Inter-relationship of myeloid and lymphoid cells:

studies with chromosome-marked cells transfused into lethally irradiated mice. Proc. R. Soc. Ser. B. 165, 78. 11. M6LLER G. (1961) Demonstration of mouse isoantigens at the cellular level by the fluorescent antibody technique.

J. exp. Med. 114, 415. 12. NISHIZUKA Y. & SHISA H. (1972) Topics in Chemical Carcinogenesis. (NAKARA W., TAKAYAMA S., SUGIMARA T. &

ODASHIMA S., Eds.) pp. 493-500. Proc 2rid Int. Syrup. Princell Takamatu Cancer Res. Fund. 13. PAIGE C. J., KINCADE P. W. & RALPH P. (1978) Murine B cell leukaemia with inducible surface immunoglobulin

expression. J. Immun., in press. 14. RAFE M. C. & WORTtS, H. H. (1970) Thymus dependence of 0-bearing cells in the peripheral lymphoid tissue of

mice. Immunology 18, 931. 15. RXEF A. E. & ALLEN J. M. V. (1964) The A K R thymic antigen and its distribution in leukaemias and nervous

tissues. J. exp. Med. 120, 413.