(CANCER RESEARCH 48. 6826-6831. December 1. 1988] Regulation of Tumor-induced Myelopoiesis and the Associated Immune Suppressor Cells in Mice Bearing Metastatic Lewis Lung Carcinoma by Prostaglandin E21

M. Rita I. Young,2 Melvin E. Young, and Kimberly Kim

Department of Research Services, Edward J. Hiñes,Jr., Veterans Administration Hospital, Hiñes,Illinois 60141 [M. R. I. Y., M. E. Y., K. K.J, and Department of Pathology, Loyola University Stritch School of Medicine, Maywood, Illinois 60153 [M. R. I. Y.J

ABSTRACT In our studies, we have shown that the decline in immune The in vitro and in vivo effects of prostaglandin I^ ( l'( ;I-'.,)and of its competence of mice with progressively growing metastatic LLC-C3 tumor cells was associated with the appearance of a stable analogue, 16,16-dimethyl-PGE2 (dmPGE2), on myelopoiesis and sequence of immune suppressor cell populations (5, 16, 17, 24, on the myelopoiesis-associated immune suppressor cell activity of mice 25). These suppressor cells were inhibitory to the activities of bearing metastatic variant Lewis lung carcinoma (LLC-C3) tumors are T-lymphocytes and natural killer cells, both of which are known assessed. In vitro studies showed a reduced susceptibility of bone marrow to be important components of the host anti-tumor defense myeloid progenitor cells (CFC) from LLC-C3 tumor bearers versus normal mice to the growth-inhibitory effects of PGE2. When added to system. First the immune suppression was mediated by PGE2- cocultures of bone marrow cells with LLC-C3 supernatant*, PGE2 less producing adherent splenic and peritoneal macrophages (16, ened the frequency of CEC and slightly reduced the generation of bone 17, 24). However, as tumors became large (>3 g), the suppres- marrow immune suppressor cells. In vivo studies showed that 4 daily sive activity of mature adherent macrophages declined as did injections of dmPGE2 into LLC-C3 tumor-bearing mice caused some their production of PGE2. The decline of the PGE2-mediated reduction in femoral bone marrow CEC and had an insignificant effect on immune suppressor mechanism coincided with an increased bone marrow suppressor cell activity. In contrast to the relative insensi- stimulation of myelopoiesis and the appearance of a population tivity of bone marrow cells of tumor bearers to the effects of PGE2, in of immature bone marrow-derived immune suppressor cells (5, vitro studies showed that CFC formation by spleen cells of tumor bearers 25). The myelopoiesis stimulation resulted in splenomegaly and was readily inhibited by PGE2. Likewise, in vivo studies showed that spleen cells of dmPGEj-treated LLC-C3-bearing mice had a reduction in in increased numbers of progenitor cells in both the bone marrow and spleen. The myelopoiesis-associated suppressor cellularity, CEC, and the level of spontaneous proliferation; a reduction in suppressor cell activity; and an increase in blastogenesis. Thus, short- cells resembled immature cells of the monocyte lineage, and term dmPGE2 treatment of LLC-C3-bearing mice limited the tumor- their presence was associated with an increased frequency of induced splenic myelopoiesis and reduced the associated splenic immune monocytic cells in the spleen and peripheral blood (5). suppressor cell activity. The regulation of myelopoiesis is normally controlled by positive mediators such as the M-CSF, GM-CSF, G-CSF, and interleukin 3 (26-28), and negative regulators such as acidic INTRODUCTION isoferritins, interferons, and prostaglandins (29-34). Adminis tration of PGE2 to mice or the addition of macrophage-derived The progressive growth of tumors in patients and experimen tal animals results in an aberration in the regulation of both PGE to in vitro bone marrow cultures resulted in a reduction myelopoiesis and immunity. For example, growth in mice of a in myeloid CFC (35, 36). The PGE can also modulate expres variety of tumor types, including TS/A mammary adenocarci- sion of la antigen on stem and myeloid progenitor cells and, in noma, LLC,3 or 4T00.1 plasmacytoma, resulted in splenomeg turn, regulate the sensitivity of these cells to inhibition by acidic aly and an increase in myeloid progenitor cells (CFC) (1-10). isoferritins (37-39). This tumor stimulation of myelopoiesis was associated with In the current study, a known negative regulator of myelo tumor cell production of CSFs. Tumor stimulation of myelo poiesis, PGE2, was used to minimize the myelopoiesis stimu poiesis in mice bearing BMT-11 fibrosarcomas enhanced the lation which was prominent in mice bearing LLC-C3 tumors. By doing so, we were able to minimize the myelopoiesis-asso ability of injected B16 melanoma tumor cells to colonize and to metastasize (11). ciated immune suppressor activity of spleen cells and, conse quently, augment the T-lymphocyte immune competence of the Tumor growth in a host frequently results in reduced immune competence and an increased appearance of immune suppres- LLC-C3 tumor bearers. sive cells (12-17). Increased suppressor T-lymphocyte activity was apparent in lymph nodes adjoining melanotic tissue and in MATERIALS AND METHODS the spleens of mice bearing a variety of tumor types (18-20). Medium. The medium used was RPMI-1640 containing 100 units/ Suppressive activity of monocytes and macrophages became ml of penicillin, 100 ^g/ml of 4-(2-hydroxyethyl)-l-piperazineethane- more prominent during progressive growth of X5563 or suifonic acid buffer solution, 5 x 10~5M 2-mercaptoethanol, 2 HIML- MOPC-315 plasmacytomas, methylcholanthrene-induced fi glutamine, and 10% endotoxin-free FBS (Hyclone Laboratories, Logan, brosarcomas, and of LLC tumors (17, 21-24). UT). Mice. Six- to 8-wk-old male C57BL/6 mice were used for all studies. Received 6/23/88; revised 8/15/88; accepted 8/18/88. The costs of publication of this article were defrayed in part by the payment The mice were obtained from Cumberland View Farms (Clinton, TN) of page charges. This article must therefore be hereby marked advertisement in and then housed at the HiñesV. A. animal research facility. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. LLC-C3 Tumor Cells (40). Cloned metastatic variant cells of the ' This study was supported in part of the Medical Research Services, HiñesV. Lewis lung carcinoma, LLC-C3, were used in all studies. These cells A. Hospital, Hiñes,IL, and by the National Cancer Institutes of Health Grant ÇA4S080. do not produce immune suppressive factors and produce only minimal 2To whom requests for reprints should be addressed, at Pathology Research levels of PGE2. However, the LLC-C3 cells secreted CSF activities (151-Z2), HiñesV. A. Hospital, Hiñes,IL 60141. which stimulate proliferation of monocytic and monocytic-granulocytic 3The abbreviations used are: LLC, Lewis lung carcinoma; CSF, colony- bone marrow CFC (5). In the present studies, the LLC-C3 tumor cells stimulating factors; PGE2, prostaglandin E2; FBS, fetal bovine serum; dmPGE2, were implanted into mice by dorsal s.c. injection of 5 x IO5cells. 16.16-dimethyl-PGE2; PWMSCS, pokeweed mitogen-stimulated spleen cell su pernatant; ConA, concanavalin A. Culture of Bone Marrow Cells with LLC-C3 Supernatant. Normal 6826 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1988 American Association for Cancer Research. PGEj, MYELOPOIESIS, AND SUPPRESSOR CELLS

bone marrow cells were suspended to 5 x 106/ml in medium only or the frequency of CFC contributed to an increase in the number medium containing 20% supernatant from 24-h cultures of 1 x 106/ml of CFC per femur or per spleen. For example, the number of of LLC-C3 cells. In some of these bone marrow cultures, PGE2 was CFC per femur of tumor bearers was increased to 40.2 ±1.9 x added to yield 1 ng/ml or 0.1 ng/ml. After 3 days of culture, both the IO3as compared to 15.6 ±2.0 x IO3 for normals. The number adherent and nonadherent cells were collected and used in CFC and of CFC per spleen of tumor bearers was increased to 25.3 ± suppressor cell assays. 4.4 x IO3 as compared to 2.2 ±1.5 x IO3 for normals. The Treatment of Mice with dmPGE2. At 3 wk after LLC-C3 tumor implantation, when tumors were approximately 20 mm in diameter, addition of PGE2 to the cultures of bone marrow or spleen cells normal and tumor-bearing mice were treated daily for 4 days by i.p. resulted in a dose-dependent reduction in the CFC formation. injection of diluent or 10 /¿gofdmPGE2 (Upjohn Diagnostics, Kala- This was evident for cells obtained from either normal mice or mazoo, MI). On the day following the last dmPGE2 treatment, mice LLC-C3 bearers. There was, however, a reduced susceptibility were sacrificed, and their spleen cells and femoral bone marrow cells of the bone marrow cells from tumor bearers versus normals to were used. Studies were conducted with at least 5 mice/group. CFC Assay (41). Bone marrow cells (7.5 x 10") or spleen cells (7.5 the inhibitory effects of PGE2. A 50% inhibition of CFC for x 10*) from individual mice were placed into each 35 x 10-mm tissue mation by bone marrow cells of normal mice was achieved with culture dish in 1 ml of semisolid supplemented RPMI-1640 medium less than 1 ng/ml of PGE2, while the 50% inhibitory PGE2 dose was approximately 460 ng/ml for bone marrow cells of containing 20% FBS and 0.3% agar (Bacto Agar; Difco Laboratories, LLC-C3-bearing mice. In contrast to the relative insensitivity Detroit, MI). PWMSCS was used as a source of CSF (42) at a final concentration of 10%. This preparation of murine CSF, which was free of bone marrow cells of tumor bearers to the inhibitory effects of known inhibitory molecules, stimulated colonies containing granu- of PGE2, CFC formation by spleen cells of tumor bearers was locytes (approximately 10%), macrophages (approximately 30%), and more readily inhibited by PGE2 with the 50% inhibitory PGE2 granulocytes plus macrophages (approximately 50%). In some assays, dose being approximately 22 ng/ml. various concentrations (100 pg/ml to 1 Mg/ml) of PGE2 were added to In Vitro Effects of PGE2 on the Frequency of Progenitor Cells the dishes. The colonies (>50 cells) were counted after 6 days of culture. and on the Generation of Suppressor Activity in Cultures of Bone T-Lymphocyte Blastogenesis. The T-lymphocyte activity was assessed Marrow Cells with LLC-C3 Supernatant. Our previous studies by culturing 2 x 10s spleen cells for 3 days in microtiter plates with showed that LLC-C3 tumor cells secrete CSFs and that the Con-A. For the last 18 h of culture. 1 ¿¿Ciof[3H]thymidine was added to each well. The amount of [3H]thymidine incorporated by the spleen LLC-C3 supernatants induced the appearance of suppressor cells was measured in a liquid scintillation counter. All assays were cells from normal bone marrow cells (5). Culturing bone mar conducted in triplicate on mice from groups of at least 5 mice each. row cells for 3 days in medium containing 20% LLC-C3 super Each assay was repeated at least 3 times. natant resulted in the persistence of bone marrow progenitor Suppressor Cell Assay. The presence of immune suppressor cells was cells which subsequently were able to grow into colonies in the measured by the capacity to suppress the T-lymphocyte blastogenic soft agar CFC assay (Table 2), and in the generation of immune response of normal spleen cells to Con-A. Prior to being added to suppressor cells which were inhibitory to T-lymphocyte activa responder spleen cells, the suppressor cells were irradiated, washed, and added at different ratios to 2 x IO5normal responder spleen cells tion (Table 3). The immune suppressor cells which were induced by culture with LLC-C3 supernatants mediate their suppressive and 4 fig/ml of Con-A. activities through a prostaglandin-independent mechanism Analysis of Data. The Student t test was used to determine the significance of the differences between values. which was not influenced by the addition of the prostaglandin synthesis inhibitor, indomethacin. For example, in one study the tumor supernatant-cultured bone marrow cells inhibited RESULTS normal spleen cell blastogenesis to Con-A by 82% in the absence of indomethacin and by 78% in the presence of indomethacin. In Vitro Effect of PGE2 Formation by Bone Marrow and In contrast to the effects of culturing bone marrow cells with Spleen Cells of LLC-C3 Tumor-bearing Mice. The frequency of tumor supernatant, culturing bone marrow cells with only me CFC in the bone marrow and spleen cells of mice bearing large dium resulted in a loss of progenitor cells and in marginal levels metastatic LLC-C3 tumors was increased to 1.6-fold and 5.5- of suppressor cell activity. The addition of PGE2 to bone fold, respectively, that of normals (Table 1). This increase in marrow cells cultured with either LLC-C3 supernatant or with

Table 1 In vitro effect ofPGE2 on colony formation by bone marrow and spleen cells from LLC-C3-bearing mice Into each 35-mm dish were added various amounts of PGE2 and either 7.5 x IO4 bone marrow cells or 7.5 x 10* spleen cells from normal or LLC-C3 tumor- bearing mice with 10% (v/v) PWMSCS as a source of CSF. The percentage of the number of colonies which formed in the absence of added PGE2 and the significance of this difference are also shown. fromAdded Colonies/dish by cells mice%-0 bearers%-0 PGE2Cell (ng/ml)Bonesource PGE2"(100)7641281514(100)8777928842PNS<0.001<0.001<0.001<0.001NSNSNSNS<0.001CFC131PGE2(100)112101997633(100)9773553822PNSNSNS<0.02<0.001NS<0.05<0.01<0.01<0.001 00.11101001000Spleenmarrow ±9*62 ±15147 ±1433 ±17132 ±222 ±18130 ±712±711 ±1599 ±1144 ±717± ±393

00.11101001000CFC811I5±413±616 ±1191 ±468 ±1051 ±615±27±1Control ±935 ±721 ±6LLC-C3 * %-0 PGE2, % of the number of colonies which formed in the absence of added PGEZ; NS, not significant. * Mean ±SD. 6827 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1988 American Association for Cancer Research. PGE2, MYELOPOIESIS, AND SUPPRESSOR CELLS

Table 2 In vitro reduction by PGE2 of the number of progenitor cells in cultures of bone marrow cells with LLC-C3 supernatant frequency of bone marrow CFC and not due to a reduction in Normal bone marrow cells were cultured for 3 days with medium or with 20% femoral cellularity which was not significantly altered by either LLC-C3 supernatant in medium with either no added PGE2 or with O.I ng/ml or the presence of the tumor or by the treatment with dmPGE2. 1.0 ng/ml of PGE2. These cells were then collected and used in a CFC assay by adding 7.5 x IO4cells to 35-mm dishes with 10% (v/v) PWMSCS as a source of In contrast to the bone marrow, both the spleen cellularity and the frequency of CFC of LLC-C3-bearing mice were in CSF. creased resulting in over a 20-fold increase in the number of effect,(%)"-27 assayFreshCells in CFC CFC per spleen. Treatment of the LLC-C3 bearers with dm- BM*BM ±8.4r18.3 PGE2 resulted in a 56% decrease in CFC per spleen which was due to both a decrease in the frequency of CFC and in the precultured with Medium ±1.5 number of nucleated cells in the spleen. Treatment of normal + 0.1 ng/ml of PGE2 13.3 ±0.6 mice with dmPGE2 also decreased the number of cells per PGE2BM+ 1.0 ng/ml of 9.0 ±2.659.0 -51-25 spleen but caused a slight increase in the frequency of splenic precultured with CFC. Consequently, dmPGE2 treatment of normal mice had LLC-C3 supernatant ±5.2 no effect on the number of CFC per spleen. + 0.1 ng/ml of PGE2 44.3 ±4.2 In a separate study, the effect of dmPGE2 treatment on the + 1.0 ng/ml of PGE2CFC/dish83.7 30.3 ±5.7PGE2 -49 spleen cell incorporation of [3H]thymidine during the 18 h °Values for the number of CFC formed by PGE2-precultured cells which are following removal from tumor-bearing or normal mice was significantly different (P < 0.05) from the CFC values for cells precultured in the absence of added !'(rl are expressed as the percentage of change due to PGE2. measured and was shown to agree with the above results of the ft BM, bone marrow. CFC assay. In the absence of any stimulants, the [3H]thymidine ' Mean ±SD from at least 3 dishes per group. incorporated (cpm) by spleen cells of placebo-treated normal and LLC-C3-bearing mice was 13,189 ±2,551 and 120,100 ± 5,482, respectively. The [3H]thymidine incorporated by spleen medium alone caused a reduction in the frequency of CFC cells of dmPGE2-treated normal and LLC-C3-bearing mice was (Table 2). The addition of PGE2 to these bone marrow cell reduced, respectively, to 9,571 ±2,719 (not significantly differ cultures also caused a slight reduction in the generation of ent) and to 81,457 ±11,622 (P < 0.01 ). suppressor cell activity by the LLC-C3 supernatant and in the Effect of Treating Normal and LLC-C3-bearing Mice with less prominent suppressor cell activity which was present after dmPGE2 on Their Spleen Cell Blastogenic Response. The effect culture with only medium (Table 3). of treating normal and tumor-bearing mice with dmPGE2 on Bone Marrow and Spleen Cellularity and Number of CFC their spleen cell blastogenic response to various doses of Con- following Treatment of LLC-C3-bearing Mice with dmPGE2. A was measured (Table 5). Treatment of normal mice with The effect of treating LLC-C3 tumor-bearing mice with dm- dmPGE2 had no effect on blastogenesis in response to 4 /ug/ml PGE2 on the myelopoiesis parameters of the femoral bone of Con-A and inhibited blastogenesis in response to 2 or 1 ¿tg/ marrow and spleens was evaluated (Table 4). The frequency of ml of Con-A by 24% and 26%, respectively (P< 0.05). Placebo- bone marrow CFC and the number of CFC/femur in LLC-C3 treated LLC-C3 bearers were minimally able to respond to bearers were increased to 1.9-fold and 1.7-fold, respectively, Con-A with their blastogenesis being reduced by over 60% as that of normal mice. Treatment of LLC-C3-bearing mice with compared to that of normal mice. In contrast to the immune dmPGEi decreased the frequency of bone marrow CFC by 32% suppressive effect of dmPGE2 treatment in normal mice, treat and the number of CFC per femur by 38%. Similarly, treatment ment of tumor-bearing mice with dmPGE2 resulted in a signif of normal mice with dmPGE2 decreased the frequency of bone icant restoration of their Con-A responsiveness. For example, marrow CFC by 26% and the number of CFC per femur by dmPGE2 treatment of tumor bearers restored their responsive 24%. The reduction in CFC/femur in dmPGE2-treated tumor- ness to 4 jig/ml of Con-A from 37% to 75% of the response by bearing and normal mice was due to the reduction in the normals.

Table 3 In vitro inhibition of the generation of bone marrow suppressor cells by culture with PGEj Normal bone marrow cells were cultured for 3 days with medium or with 20% LLC-C3 supernatant in medium with either no added PGE2 or with O.I ng/ml or 1.0 ng/ml of n .1 The ability of these cells to suppress normal spleen cell blastogenesis to Con-A was measured and compared to that for freshly isolated bone marrow cells. Splenic Con-A response by spleen cells With added BM cells at ratio of* Added BM°cells Alone 1:0.5 1:0.25 None 132,647 ±3,260'

Fresh BM 121,797 ±6,153(8)'' 140,333 ±13,040

BM precultured with Medium only 78,302 ±10,640(41) 118,810 ±15,743 + 0.1 ng/ml of PGE; 81,607 ±3,218 (38) 111,680 ±12,871 (16) + 1.0 ng/ml of PGE¡ 104,143 ±6,780*(21) 141,220 ±4,640'

BM precultured with LLC-C3 supernatant 27,208 ±1,524(79) 40,992 ±4,868 (69) + 0.1 ng/ml of PGEi 54,982 ±5,288' (59) 69,982 ±3,143'(47) + 1.0 ng/ml of PGEj 51,590 ±6,039'(61) 78,091 ±4,601'(41) " BM, bone marrow. " Ratio of responder normal spleen cells to added suppressor cells. ' Mean ±SD of at least triplicate cultures (cpm). 11Numbers in parentheses, significant (P < 0.05) levels of suppressor cell activity expressed as the percentage of suppression of the normal spleen cell response. ' Significant (/' < 0.05) reduction in suppressor activity of PGEi-cultured cells as compared to that of cells cultured in the absence of added I'<. I

6828 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1988 American Association for Cancer Research. PGE¡,MYELOPOIESIS, AND SUPPRESSOR CELLS Table 4 Effect afin vivo administration ofdmPGEi to LLC-Ci-bearing mice on the myelopoietic parameters of their femoral bone marron1 and spleens Bone marrow cells (7.5 x IO4)or spleen cells (7.5 x 10') from individual mice were seeded in triplicale into 35-mm dishes with 10% (v/v) PWMSCS. After 6 days, the number of CFC per dish was determined. In Experiments 3 to 5, the numbers of nucleated cells in the femoral bone marrow or in the spleen of individual mice were also determined and the numbers of CFC per organ were then calculated. 1-5CFC/dish

dmPGE2 x IO7 x IO3 treatmentBoneCells Mice (% ofnorm)85.3 (% ofnorm)2.0 (% ofnorm)22.9 marrow Normals ±3.0° ±0.32.2 + 2.2 63.4 + 2.4* (74)' ±0.4 17.4 ±0.7*(76) bearersSpleen LLC-C3 158.9 + 7.9(186) 1.8 ±0.3 39.1 ±1.0(171) 108.7(127)13.8 ±8.1* 1.7±0.111.2± 24.3 ±2.8*1.4

Normals ±1.317.5+ 1.8 ±0.1 1.7*(127) 7.6 ±1.3*(67) 1.6 ±0.4 LLC-C3 bearersExperiments 95.9 ±7.4 (693) 27.2 ±3.2 (242) 31.5 + 5.6(2,176) 68.3 ±2.2*(494)ExperimentsCells/organ,14.6 ±3.4*(130)3-5CFC/organ,14.0 ±3.0*(968) °Mean ±SEM. * Values for dmPGE2-treated mice is significantly different (P < 0.05) from the value for placebo-treated mice. ' Numbers in parentheses, values which are significantly different (P < 0.05) from those for normal placebo-treated mice expressed as the percentage of the normal value.

Table 5 Effect ofdmPGEi treatment ofLLC-Ci tumor bearers on their Con-A blastogenic response with"Placebo183, response of mice treated Con-A Spleen cell (¡.•j.milsource4 change*-3+99-24 value'NS' Normals 160 ±6,424'' 77,387 ±10,811 bearers2 LLC-C3 (37/227,33068,368 ±12,906 136,730 +5,096(75)173,037+ <0.01<0.05

Normals ±36,471 10,315(76) bearers1 LLC-C3 1.354(20)196,46346,459 ± 136,373 +9,682(60)1 +194-26 <0.001<0.01

Normals ±6,958 44,623 ±11,537(74) LLC-C3 bearersCon-A 18,584 ±2,936 (9)dmPGEj1 77,162 ±8,364 (39)%of +315P <0.001 °The Con-A blastogenic response of individual control and LLC-C3 tumor-bearing mice which had been treated with placebo or with dmPGEj was measured. Each treatment group contained 8 mice. * The percentage of change in Con-A blastogenesis due to dmPGE; treatment of mice. ' Level of significance in the difference between the response of dmPGE2-treated mice as compared to the response of placebo-treated mice. d Mean ±SEM (cpm). ' NS, not significant. ^Numbers in parentheses, values which are significantly different (P < 0.05) from those for normal placebo-treated mice expressed as the percentage of the normal value.

Table 6 Effect ofdmPGE2 treatment ofLLC-Ci tumor bearers on their suppressor cell activity Con-A blastogenic response by spleen cells" '' ' withMediumWith added cells of mice treated

supernatant)NoneCells Source Ratio* Alone (% of ofsupernatant)1 19,501''BM N/A' N/A 148.613 ±

Normals 100,721 ±10,441 (32)' 55,456 ±13,21y :0.5 104,966 ±9,800 (29) 164,345+ 14.85/ bearersSpleen LLC-C3 :1 74,657 + 8,740(50) 83,803 + 8,919(44) :0.5±8,787(33):1 100,071 114.258+10,970(23)150,096

Normals 157, 170 ±7,237 10,149 :0.5 146,626 ±9,249 137,376 ±3,046 LLC-C3 bearers:1 :1 87,079 ±7,454 (41) 133,212 ±11,826^ :0.5 112,133 + 5,270(25)dmPGE2(% 139.054±±11,20/ " The effect of bone marrow and spleen cells from placebo- or dmPGEj-treated normal or LLC-C3-bearing mice on the Con-A responses of normal spleen cells was assessed. Each treatment group contained 8 mice. * Ratio of responder normal spleen cells to added suppressor cells. ' N/A, not available; BM, bone marrow. **Mean ±SEM of 3 experiments. ' Significant (P < 0.05) levels of suppressor cell activity expressed as the percentage of suppression of the normal spleen cell response by the added cells. * Value for dmPGE2-treated mice is significantly (P < 0.05) different from the value for placebo-treated mice.

Effect of dmPGEz Treatment of Normal and LLC-C3-bearing dmPGEi caused a complete elimination of suppressor activity Mice on the Suppressor Activity of Their Bone Marrow and of their bone marrow cells. In contrast, dmPGE2 treatment of Spleen Cells. Studies were conducted to determine if dmPGEj tumor-bearing mice tended to only minimally lessen their bone treatment of normal and LLC-C3-bearing mice would minimize marrow suppressor activity (not statistically significant). the levels of immune suppressor activity of their bone marrow The effects of dmPGE2 administration to tumor bearers on and spleen cells (Table 6). While bone marrow cells of normal the suppressor activity of their spleen cells differed from the mice had some suppressor cell activity, causing 32% suppres results described above for bone marrow cells. No suppressive sion of normal spleen cell blastogenesis, bone marrow cells of activity was present in spleen cells prepared from either placebo- LLC-C3-bearing mice had more suppressor activity and sup treated or dmPGE2-treated normal mice. In contrast, spleen pressed blastogenesis by 50%. Treatment of normal mice with cells of LLC-C3-bearing mice suppressed normal spleen cell 6829 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1988 American Association for Cancer Research. PGE2, MYELOPOIESIS, AND SUPPRESSOR CELLS blastogenesis by 41%. This suppressive activity was abolished tumor-bearer progenitor cells to PGE2 by assessing either their by treatment of LLC-C3-bearing mice with dmPGE2. Similar intracellular levels of cyclic cAMP or the modulation of their results were observed at both of the ratios at which suppressor expression of la-associated antigen. cells were added to normal splenic responder cells. The conclusion of our present studies is that short-term treatment of tumor bearers with PGE2 is efficacious in the spleen in limiting tumor-induced myelopoiesis stimulation and DISCUSSION the presence of myelopoiesis-associated immune suppressor Progressive growth of tumors results in the appearance of cells. immune suppressor cells which limit the antitumor effector mechanisms of the host (12, 16-21). Our studies have shown ACKNOWLEDGMENTS that growth of LLC-C3 tumors profoundly suppresses host immune competence. This immunosuppression is mediated by We thank Dr. Hal E. Broxmeyer of the Indiana University School host suppressor mechanisms, since the tumor itself does not of Medicine and Dr. Carleton C. Stewart of the Roswell Park Memorial produce immune suppressive factors such as PGE2. However, Institute for their encouragement and advice in the preparation of this the LLC-C3 tumor cells do secrete CSFs. Our studies (5, 17, manuscript. 24, 25) have suggested the following cascade of tumor-induced host suppressor mechanisms which overlap with mechanisms REFERENCES that regulate myelopoiesis. During the initial and intermediate periods of LLC-C3 growth, mature adherent macrophages me 1. Hardy, C. L., and Balducci, L. Review: hemopoietic alterations of cancer. Am. J. Med. Sci., 290: 196-205, 1985. diate immune suppression by producing increased amounts of 2. Hardy, C. L., and Balducci, L. Early hematopoietic events during tumor PGE2. So long as the macrophages produce elevated levels of growth in mice. J. Nati. Cancer Inst., 76: 535-540, 1986. PGE2, they also block myelopoietic stimulation by the tumor- 3. Nicoletti, G., Brambilla, P., De Giovanni, C., Lollini, P.-L., Del Re, B., Marocchi, A., Mocarelli, P., Prodi, G., and Nanni, P. Colony-stimulating derived CSF. During the later phases of tumor growth, the activity from the new metastatic TS/A cell line and its high- and low- macrophages lose their suppressive activities and produce only metastatic clonal derivatives. Br. J. Cancer, 52: 215-222, 1985. 4. Kovacs, C. J., Emma, D. A., Evans, M. J., Johnke, R. M., and Scarantino, nominal amounts of PGE2. When macrophage PGE2 produc C. W. Haemopoietic modulation in tumour-bearing animals: enhanced pro tion declines, the myelopoietic stimulation by the tumor-derived genitor-cell production in femoral marrow. Cell Tissue Kinet., 18: 235-246, CSF becomes evident. Consequently, the myelopoiesis-associ- 1985. 5. Young, M. R., Newby, M., and Wepsic, H. T. Hematopoiesis and suppressor ated immune suppressor cells also became apparent. As PGE2 bone marrow cells in mice bearing large metastatic Lewis lung carcinoma is a potent negative regulator of myelopoiesis (31-39), it was tumors. Cancer Res., 47: 100-105, 1987. used in vitro and in vivo in the presently described studies to 6. Merchav, S., Apte, R. N., Tatarsky, I., and Ber, R. Effect of plasmacytoma cells on the production of granulocyte-macrophage colony-stimulating activ restrict the appearance of tumor-induced myelopoiesis and the ity (GM-CSA) in the spleen of tumor-bearing mice. Exp. Hematol. (Copenh.), associated bone marrow-derived suppressor cells. 15:995-1000, 1987. 7. Balducci, L., and Hardy, C. High proliferation of granulocyte-macrophage The results of in vitro studies showed that PGE2 could be progenitors in tumor-bearing mice. Cancer Res., 43:4643-4647, 1983. effective at inhibiting the tumor-induced myelopoiesis in order 8. Ishikawa, M., Hosokawa, M., Oh-hara, N., Nino, Y., and Kobayashi, H. to inhibit the appearance of the myelopoiesis-associated im Marked granulocytosis in C57BL/6 mice bearing a transplanted KMT 11 fibrosarcoma. J. Nati. Cancer Inst., 78: 567-571, 1987. mune suppressor cells. The doses at which these effects could 9. Ikeda, K., Motoyoshi, K., Ishizaka, Y., Hatake, K., Kajigaya, S., Saito, M., be observed, 1 ng/ml and 100 pg/ml, are physiologically perti and Muirá,Y. Human colony-stimulating activity-producing tumor: produc nent, as the plasma PGE2 levels of normal mice and of mice tion of a very low mouse-active colony-stimulating activity and induction of marked granulocytosis in mice. Cancer Res., 45:4144-4149, 1985. bearing large tumors are typically between 530 and 600 pg/ml 10. Lilly, M. B., Devlin, P. E., Devlin, J. J., and Rado, T. A. Production of (Ref. 17 and data not shown). These in vitro studies supported granulocyte colony-stimulating factor by a human melanoma cell line. Exp. the use of PGE2 in LLC-C3 tumor bearers at a time when Hematol. (Copenh.), /5: 966-971, 1987. 11. Ishikawa, M., Koga, Y., Hosokawa, M., and Kobayashi, H. Augmentation of tumor stimulation of myelopoiesis and the associated appear B16 melanoma lung colony formation in C57BL/6 mice having marked ance of bone marrow-derived suppressor cells were prominent. granulocytosis. Int. J. Cancer, 3 7:919-924, 1986. In the in vivo studies, treatment of tumor bearers with dmPGE2 12. Mukherji, B., Nashed, A. L., Guha, A., and Ergin, M. T. Regulation of cellular immune response against autologous human melanoma. II. Mecha was restricted to 4 daily injections to (a) minimize any direct nism of induction and specificity of suppression. J. Immunol., 136: 1893- immune suppressive effects of dmPGE2, and to (b) avoid any 1898, 1986. 13. Elliott, L., Brooks, W., and Roszman, T. Role of interleukin-2 (IL-2) and changes in tumor size that would interfere in the assessment of IL-2 receptor expression in the proliferative defect observed in mitogen- PGE2 effects on myelopoiesis and suppressor cells. Short-term stimulated lymphocytes from patients with gliomas. J. Nati. Cancer Inst., treatment of tumor bearers with dmPGE2 resulted in some 78:919-922, 1987. 14. Lotzova, E., Savary, C. A., and Herberman, R. B. Induction of NK activity reduction in femoral CFC and had an insignificant effect on against fresh human leukemia in culture with interleukin 2. J. Immunol., the presence of immune suppressor bone marrow cells. How 138: 2718-2727, 1987. ever, the dmPGE2 treatment of tumor bearers caused a marked 15. Ridgway, D., Wolff, L. J., and Neerhout, R. C. Enhanced lymphocyte response to PHA among leukemia patients taking oral lithium carbonate. reduction in splenic myelopoiesis and a reduction in splenic Cancer Invest., 4: 513-517, 1986. immune suppressor cells. The capacity of the dmPGE2 treat 16. Young, M. R., Endicott, R. A., Duffle, G. P., and Wepsic, H. T. Suppressor alveolar macrophages in mice bearing Lewis lung carcinoma tumors. J. ments in diminish splenic myelopoiesis and suppressor cell Leukocyte Biol., 42:682-688, 1987. activity prompts further studies to determine whether these 17. Young, M. R., Wheeler, E., and Newby, M. Macrophage-mediated suppres sion of natural killer cell activity in mice bearing Lewis lung carcinoma. J. effects would be expressed as a measurable reduction in tumor Nati. Cancer Inst., 76: 745-750, 1986. growth. These in vivo results were consistent with the results of 18. Hoon, D. S. B., Bowker, R. J., and Cochran, A. J. Suppressor cell activity in in vitro studies showing that bone marrow cells of tumor bearers melanoma-draining lymph nodes. Cancer Res., 47:1529-1533, 1987. 19. Bear, H. D. Tumor-specific suppressor T-cells which inhibit the in vitro were less sensitive to the myelosuppressive effects of PGE2 than generation of cytolytic T-cells from immune and early tumor-bearing host were either bone marrow cells from normal mice or spleen cells spleens. Cancer Res., 46: 1805-1812, 1986. from tumor-bearing mice. It would be of interest to quantitate 20. North, R. J. Radiation-induced immunologically mediated regression of an established tumor as an example of successful therapeutic immunomanipu- PGE2 receptors on progenitor cells of tumor bearers versus of lation. J. Exp. Med., 164: 1652-1666, 1986. control mice or to compare the levels of responsiveness of 21. Fuji, T., Igarashi, T., and Kishimoto, S. Significance of suppressor macro- 6830 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1988 American Association for Cancer Research. PCE2, MYELOPOIESIS. AND SUPPRESSOR CELLS

phages for immunosurveillance of tumor-bearing mice. J. Nati. Cancer Inst., human marrow granulocyte-macrophage progenitor cells by prostaglandin E 78: 509-517, 1987. and recombinant interferon-alpha, -beta, and -gamma and an effect mediated 22. Garner, R. E., Malick, A. P., Yurochko, A. D., and Elgert, K. D. Shifts in by tumor necrosis factor. J. Immunol., 140: 479-484. 1988. macrophage (Mo) surface phenotypes during tumor growth: association of 33. Boorman. G. A., Luster, M. I., Dean, J. H., and Luebke, R. W. Effect of Mac-2* and Mac-3* Mo with immunosuppressive activity. Cell. Immunul.. indomethacin on the bone marrow and immune system of the mouse. J. Clin. 108: 255-268, 1987. Lab. Immunol., 7: 117-126, 1982. 23. Ye, Q.-W.. Mokyr, M. B., Pyle. J. M.. and Dray. S. Suppression of antitumor 34. Nikcevich. D. A., Young, M. R.. Ellis. N. K., Newby, M., and Wepsic, H. T. immunity by macrophages in spleens of mice bearing a large MOPC-315 Stimulation of hematopoiesis in untreated and cyclophosphamide treated tumor. Cancer Immunol. Immunother., 16: 162-169, 1984. mice by the inhibition of prostaglandin synthesis. J. Immunopharmacol., S: 24. Young, M. R., and Newby, M. Differential induction of suppressor macro 289-313, 1986. phages by cloned Lewis lung carcinoma variants. J. Nati. Cancer Inst., 77: 35. Gentile. P. S., and Pelus, L. M. In vivo modulation of myelopoiesis by 1255-1260, 1986. prostaglandin E2. II. Inhibition of granulocyte-monocyte progenitor cell 25. Young, M. R., Young, M. E., and Wepsic, H. T. The effect of recombinant (CFU-GM) cycle rate. Exp. Hematol. (Copenh.), 15: 119-126, 1984. murine interferon-gamma on the hematopoietic and immunological param 36. Kurland, J. I.. Bockman, R. S., Broxmeyer, H. E., and Moore, M. A. S. eters of mice bearing metastatic Lewis lung carcinoma tumors. Exp. Hematol. 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Relationships between morphol normal donors and patients with leukemia: synergism of tumor necrosis ogy, dissemination, migration, and prostaglandin E2 secretion by cloned factor and interferon-gamma. J. Immunol.. 136:4487-4495, 1986. variants of Lewis lung carcinoma. Cancer Res., 45: 3918-3923, 1985. 30. Broxmeyer, H. E., Juliano, L., Waheed, A., and Shadduck, R. K. Release 41. Broxmeyer, H. E., Cooper, S., Rubin, B. Y., and Taylor, M. W. The from mouse macrophages of acidic isoferritins that suppress hematopoietic synergistic influence of human interferon-gamma and interferon-alpha on progenitor cells is induced by purified L cell colony stimulating factor and suppression of hematopoietic progenitor cells is additive with the enhanced suppressed by human lactofemn. J. Immunol., 135: 3224-3231, 1985. sensitivity of these cells to inhibition by interferons at low oxygen tension in 31. Kurland, J., and Moore, M. A. S. Modulation of hemopoiesis by prostaglan- vitro. J. 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6831 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1988 American Association for Cancer Research. Regulation of Tumor-induced Myelopoiesis and the Associated Immune Suppressor Cells in Mice Bearing Metastatic Lewis Lung Carcinoma by Prostaglandin E2

M. Rita I. Young, Melvin E. Young and Kimberly Kim

Cancer Res 1988;48:6826-6831.

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