Role of Cellular and Humoral Factors in the Destruction ofNascent Plasma Cell Tumors1'2
Hisashi Yamada, Atsuko Yamada, and Vincent P. Hollander
Research Institute for Skeletomuscular Diseases of the Hospital for Joint Diseases & Medicai Center, and The Mount Sinai School of Medicine,New York, New York 1003S
SUMMARY
Previous work from this laboratory established that chronicadministration of glycoprotein pituitary hormones to mineraloil-injected BALB/c mice almost completely prevented development of the expected plasma cell tumors in the peritoneal cavity. Tumor prevention was accompanied by the intraperitoneal proliferation of mast-like cells (granulated macrophage in the present study) which, in a few mice, containedphagocytosed myeloma cells. In the present study, cytotoxicantibodies reactive to six Unes of transplantable oil-inducedplasma cell tumors were sought in the sera of these mice by afluorochromatic test for immunocytotoxicity in agarose gel ofCelada and Rotman; and phagocytic activity of the peritonealgranulated macrophages from individual mice for four lines ofplasma cell tumor and two primary plasma cell tumors wasstudied by in vitro incubation of these cells. Specific phagocytosis of myeloma cells by the granulated macrophage was demonstrated in certain combinations of tumor lines and macrophage donors. Peritoneal macrophages from mice treated withoil alone failed to ingest myeloma cells. Antibodies cytotoxicto myeloma cells were demonstrated in the sera in certaincombinations between tumor Unes and sera. However, theseantibodies were also found in some mice treated with oilalone. Antibodies to the tumor lines derived from primarytumors of short latent periods were found more frequentlythan to those with a long latent period. The presence of cytotoxic antibodies in the sera of granulated macrophage-bearingmice did not coincide with phagocytosis of tumor cells.
INTRODUCTION
Potter and his associates demonstrated that most BALB/cmice given intraperitoneal mineral oil injection develop plasma cell tumors (10, 11). Previous work from this laboratoryestablished that chronic administration of thyroid-stimulating,
'This work was supported by Grant #P397 from the American Can
cer Society.2Presented in part at the Annual Meeting of the American Association
for Cancer Research, San Francisco, Calif., March 1969 (Proc. Amer.Assoc. Cancer Res., 10: 101, 1969).
Received November 19, 1968; accepted February 14, 1969.
follicle-stimulating, or luteinzing hormone (glycoprotein pituitary hormone, GPH) to oil-injected BALB/c mice almost completely prevented the development of the expected plasma celltumors on the peritoneal surfaces (13). Tumor prevention wasuniformly accompanied by the intraperitoneal proliferation ofa very large cell filled with metachromatic basophilic granules.In prior publications, this cell was referred to as mast or mast-like (7, 13). We now designate this unique cell which distinguishes the peritoneal fluid of GPH-treated mice as granulatedmacrophage for reasons discussed below. In several micetreated with oil plus GPH, these cells had been shown to contain phagocytosed tumor cells when no gross tumor development was subsequently observed. The hypothesis was offeredthat GPH-induced phagocytosis of nascent tumor cells resultedin subsequent immunity. The present study demonstrates thespecific phagocytosis of myeloma cells of BALB/c origin bythe granulated macrophages. Since antibodies are required forthe phagocytosis by macrophages of some tumor cells, theywere sought in sera of animals producing granulated macrophages as a result of GPH treatment. Antibodies cytotoxic formyeloma cells were found in the sera of mice treated withintraperitoneal oil and subcutaneous GPH but also in the seraof mice treated with oil alone.
MATERIALS AND METHODS
Animals. BALB/c mice were obtained from MicrobiologicalAssociates, Bethesda, Md. C57BL/6J mice were purchasedfrom the Jackson Laboratory, Bar Harbor, Maine.
Glycoprotein Pituitary Hormones. Thyroid-stimulating(TSH), follicle-stimulating (FSH), and luteinizing (LH) hormones were of heterlogous origins (bovine, ovine, or porcine).They were gifts from the National Institutes of Health, Bethesda, Md. The origin and batch number used in each experiment was included in the tables.
Plasma Cell Tumors. Plasma cell tumors were induced byintraperitoneal mineral oil injection (11) in BALB/c mice andmaintained in our laboratory by serial subcutaneous transplantation. Six different lines of plasma cell tumors, TPC-3, 8, 12,16, 17, and 18, were used in the tests between the 10th and32nd transplantation generation. Details of these tumors aredescribed in Table 1. In some phagocytosis experiments invitro, tumor cells were obtained from ascites of mice bearingprimary plasma cell tumors. They were designated as ascitesprimary tumor 1 and 2.
Characteristics of transplantable plasma cell tumors."0.5 ml of U.S.P. mineral oil was injected into the peritoneal cavity at 2,
4, and 6 months of age. Latent period was calculated from the first mineraloil injection.
Single Cell Preparations. Single cell suspensions were prepared from solid plasma cell tumors by trypsinization (4). Subcutaneous transplanted tumors were removed aseptically andput through a 60-mesh stainless steel screen. The resulting material was suspended in 0.5% trypsin in Eagle's medium with
1,000 units desoxyribonuclease and incubated at room temperature for 30 minutes. Dead cells and cell debris were digested by this procedure. Viability of the cells varied from75% to 98% as judged by trypan blue (4). From 1 gm tumor,about 5 X IO7 tumor cells were obtained. When many erythro-
cytes were present in the preparation these were destroyed byshort (30 sec) exposure to 0.45% saline.
Cytotoxic Test in Gel. A fluorochromatic test for immuno-cytotoxicity in agarose gel was performed according to Celadaand Rotman (5). Tumor cell suspension (0.1 ml) containing 2X IO6 cells was mixed with 0.6 ml of 0.6% agarose (MannResearch Laboratory, New York, N. Y.) in Eagle's mediumkept at 42°Cand quickly spread on a 5-cm Falcon plastic Petri
dish. Eight wells of 1.5-mm diameter were punched out of theagarose film. For each serum, 2—3Allwere placed in a well andallowed to diffuse. Normal BALB/c serum as negative control,and isoantiserum to BALB/c prepared in C57BL/6J mice aspositive control, were always included in each Petri dish. Normal C57BL/6J serum also served as a negative control. Fluores-
cein diacetate was obtained from Mann Research Laboratory.Immunocytotoxic Electrophoresis. To test the electropho-
retic mobility of cytotoxic activity in the sera, the fluorochromatic test of Celada and Rotman (5) was modified by combining it with electrophoresis. The serum samples were separatedby electrophoresis on cellulose acetate strips (Gelman Instrument Co., Ann Arbor, Mich.) with 0.05 M phosphate buffer(pH 7.4) for 30 minutes at 175 volts. The center portion ofthe strip, measuring 1X3 cm, was cut out and placed onagarose film containing tumor cells. After one hour's incubation at 37°C, the strip was removed and the agarose films in
the Petri dish were treated with complement, followed bytreatment with fluorescein diacetate. The rest of the celluloseacetate strip was stained with Ponceau 2R and served as a
reference profile.In Vitro Phagocytosis of Tumor Cells by Peritoneal Cells.
About 0.05 ml of ascites containing 1-3 X IO6 peritoneal
macrophages was obtained by aspiration with a 1-ml syringe
from the peritoneal cavity of mice treated with mineral oil andGPH or oil alone. The ascites was mixed in 0.7 ml Eagle's
medium-10% normal BALB/c serum and kept in an ice bath;0.1 ml of tumor cell suspension containing 2 X 10s cells was
added. These were then plated in a Falcon culture dish, 5 cmin diameter, with an inside well of 1 ml capacity. A roundcover glass was placed on the bottom of the well. The coverglass was removed after one hour's incubation and stained with
Wright-Giemsa stain. The peritoneal macrophages from individual mice were studied separately.
RESULTS
Tumor Prevention with GPH. Table 2 demonstrates prevention of plasma cell tumors by administration of glycoproteinpituitary hormones. Female BALB/c mice were injected with0.5 ml mineral oil at 2, 4, and 6 months of age. In addition tothe oil-treatment, glycoprotein pituitary hormones were given
subcutaneously, 100 fig/day, five times per week, for 14months, starting at the first dose of oil. The inhibition oftumorigenesis was impressive. Tumor incidence by 17 monthsof age in female BALB/c mice injected with mineral oil aloneis about 70% (13). After 5 months of hormone administration,before the appearance of tumor in the control group, the distinction between the peritoneal fluid of mice treated with oilalone or with oil plus hormone could be made by microscopicexamination with near certainty.
The Granulated Macrophages. Fig. 1 contrasts the peritonealcell cytology of BALB/c mice eight months after the intraperi-
toneal injection of mineral oil with that of mice receiving subcutaneous FSH (100 jug, 5 times per week) in addition to theoil treatment. Macrophages treated with oil alone contain oilglobules. In contrast, the large cell which distinguishes theperitoneal fluid of mice treated with FSH contains no oil butinnumerable basophilic granules. The granules are finer thanthose previously reported (13). The figure serves to define thegranulated macrophage. Similar granulated cells are obtainedfollowing administration of LH or TSH used in this study.Detailed cytology and electron microscopic and cytochemicalcharacterization form the basis for a future report.
Phagocytosis of Myeloma Cells. Preliminary studies established that peritoneal macrophages from mice injected withmineral oil alone failed to ingest any of our established lines of
Table2Number
ofmice3104SexFFFTreatmentFSHLHTSHIncidence of plasma celltumors0021
Tumor prevention with glycoprotein pituitary hormone (GPH) (source ofgranulated macrophages). All mice showed intraperitoneal proliferation ofgranulated macrophages. FSH, follicle-stimulating hormone, NIH-FSH-S3,ovine; LH, luteinizing hormone, NIH-LH-S11, ovine; TSH, thyroid-stimulating hormone, NIH-TSH-S4,ovine. Hormones were given: 100Mg, 5 timesper week for 14 months.
"Tumor incidence at 17 months of age; 70% of female BALB/c miceinjected with mineral oil alone develop plasma cell tumors by this age (13).
transplantai)le myeloma cells although they avidly ingested latex particles. Peritoneal fluid from mice ten months after thefirst injection of mineral oil was incubated with TPC-3, 8, 12,16, 17, and 18 under the condition outlined in the method.No phagocytosis was observed.
Fig. 2 illustrates that granulated macrophages from the peritoneal fluid of mice treated with GPH frequently ingest myeloma cells. Vacuole formation around ingested cells in suchmacrophages was frequent. When normal spleen cells, methyl-cholanthrene-induced fibrosarcoma, and lymphosarcoma P1798cells, all of BALB/c origin, were incubated under the sameconditions, phagocytosis was not seen.
Table 3 illustrates that granulated macrophages from eachanimal tested phagocytosed at least one of the strains of myeloma cell tested. Also, every tumor cell tested was ingested bygranulated macrophages from more than one GPH-treated animal. Ascites primary tumors 1 and 2 were of particular interest. Since GPH treatment prevents almost all tumor formation,it was expected that primary tumor cells should be ingested bythe granulated macrophages of all GPH-treated animals.However, ascites-1 cells proved to be poor subjects forphagocytosis.
Cytotoxic Antibody in Sera. Bennett et al. (3) showed thatstarch-induced macrophages would ingest Meth A sarcoma
cells coated with isoantibody. The observation that a uniquegranulated macrophage present in the peritoneal fluid ofGPH-treated mice ingested only some myeloma cells but notnormal spleen cells, stimulated us to look for a humoral antibody in the sera of hormone-treated mice.
The fluorochromatic method of Celada and Rotman wasideal for the search for such antibodies in the small amounts ofsera available from individual mice. Only a few |Ltlof serum wassufficient for testing against one tumor strain. Results wereunequivocal and completely reproducible. All strains of myeloma cells tested were susceptible to C57BL anti-BALB/c iso-antiserum. Out of 38 normal BALB/c sera from mice of different sex and age, none was cy to toxic for any tumor strain.Normal BALB/c spleen cells were negative against all sera fromoil-injected mice and positive against C57BL anti-BALB/c iso-ant¡scruni.All positive results were complement dependent.
Table 4 shows the results of testing sera from mice injectedwith oil alone or oil plus GPH. Mice treated with oil alonewere tested 8—12months after the first injection and weredivided for testing into mice with and without tumor. Micetreated with oil plus GPH were tested 11—13months after thefirst dose of oil. The table shows that 12-17% of the possiblecombinations between sera and six strains of plasma cell tumors demonstrated cytotoxicity. No apparent difference in
Table 3
Tumor strains(% viability)
Source of macrophages;mice treatedwith:1.2.3.4.5.6.7.8.9.10.FSHLHLHLHLHLHLHTSHTSHTSHTPC-8(96%)_—+__D»_±D++DTPC-12
TPC-17(90%)(98%)_«
±++-H-+
-H-+-H--M-+
D++-t-H-
+++-H--m-
-H-D-t-H-TPC-18
Ascites-1(90%)(86%)+D-
—±D
+__+
_D•H-
-H--
-Ascites-2(90%)±++±DD±-H--H-*-D
Phagocytosis of myeloma cells by granulated macrophages from mice treated with glycoproteinpituitary hormones. FSH, follicle-stimulating hormone, NIH-FSH-S3,ovine; LH, luteinizing hormone,NIH-LH-S11, ovine; TSH, thyroid-stimulating hormone, NW-TSH-S4, ovine. Hormones were givensubcutaneously : 100 /ig, 5 times per week for 14 months.
"—,no phagocytosis; +++,very frequent phagocytosis; + and -H-,intermediate.*D, Discarded because of poor macrophage culture or contamination with intestinal contents. The
granulated macrophages were harvested from the peritoneal cavities of oil-injected mice treated withglycoprotein pituitary hormones. Tumor cell suspensions were obtained from 4 strains of trans-plantable plasma cell tumors and two primary plasma cell tumors.
Table 4
Serumdonors(treatment)Mineral
oil +GPHMineraloil alone withouttumorMineraloil alone with tumorNo.
ofpositivetests16145No.ofcombinationsbetween
seraandtumorstrains968442Incidence
ofpositivetests16.7%16.7%11.9%
Incidence of cytotoxic antibody. GPH, glycoprotein pituitary hormones.
the incidence of cytotoxic antibody was seen in the animalsgiven GPH from those given oil alone.
Immunocytotoxic Electrophoresis. To ascertain the antibody nature of cytotoxic activities, the test described abovewas performed with TPC-8, 17, and 18. Fig. 3 shows a typicalresult. The area of dead cells was located in the gamma-globulin region.
Examination of the data from individual mice treated withGPH allowed additional conclusions. Results of 16 sera frommice treated with GPH studied extensively are shown in Table5. Out of 96 combinations of the 16 sera and 6 strains ofplasma cell tumors, 16 combinations showed cytotoxic activity in the sera. Nine sera out of 16 showed cytotoxicity to atleast one line of tumors. Five of six lines of plasma cell tumorswere susceptible to at least one serum. Six sera reacted withone strain of tumor only. Three others showed multispecificitywith different profiles. TPC-16 was resistant to all of the seratested. However, all six strains were susceptible to C57BL/6Jisoantiserum, indicating the presence of histoincompatibilityantigens on the cells. No relationship was demonstrated between the nature of myeloma proteins produced by the testtumor and their susceptibility to the sera. Some but not all ofthe sera showed precipitin-lines on Ouchterlony's agar plates
against the preparation of hormone injected. This antibodyhad no relationship to cytotoxic activity. To test whether thecytotoxic activities reside in antibodies against myeloma proteins, the sera were incubated with myeloma proteins from theplasma cell tumor tested (100 /zg myeloma protein/50 fu serafor 15 min at room temperature). The cytotoxic activities
were not absorbed. The finding of Klein et al. (8) that anti-myeloma protein antibodies were not cytotoxic to myelomacells is in accordance with this result.
Old et al. (9) demonstrated, in their study on methylcholan-threne-induced sarcoma, that primary neoplasms arising after ashort latent period are more strongly antigenic than tumors oflate development. Whether this was present in our six lines oftumor was studied. The number of sera to which tumor issusceptible is plotted against the latent period of that tumor inChart 1. That tumors with shorter latent periods have strongerantigenecity to the hosts is apparent. This finding suggests thatthe antigens to which the cytotoxic antibodies are reactingexisted in the primary tumors. The finding is difficult to reconcile with viral antigens.
Discrepancy between the Presence of Cytotoxic Activity inthe Sera and Phagocytic Activity of Granulated Macrophage ofthe Individual Mice Treated with GPH. When the cytotoxicactivities of the sera from a mouse were compared to thephagocytic activities of granulated macrophages derived fromthat mouse, apparent discrepancies were observed (Table 6).Some granulated macrophages showed avid phagocytosis formyeloma cells when no cytotoxic activity was detected in thecorresponding sera for these tumor cells. Granulated macrophages from some mice showed poor phagocytic activity for astrain of myeloma cells in spite of the presence of cytotoxicactivity in the sera. When granulated macrophages with highphagocytic activity for myeloma cells were washed three timeswith normal saline, the observed phagocytic activity was notaffected.
Immunocytotoxicity of the sera from GPH-treated mice against 6 strains of plasma cell tumors.GPH, glycoprotein pituitary hormones; TSH, NIH-ISH-B2, bovine, 100 fig 5 times per week; FSH,NIH-FSH-P1,porcine, 100 /lg 5 times per week; LH.NIH-LH-S8, ovine, 100 Jig 5 times per week; n.t.,not tested.
" ++, degree of cytotoxicity; all other spaces were negative.
Chart 1. Antigenicity of plasma cell tumors and their inductionperiods. Cytotoxic activities of 36 sera were tested against six plasmacell tumors, TPC-3, 8, 12, 16, 17, and 18. Sixteen sera were from micetreated with mineral oil and glycoprotein pituitary hormones, 21 werefrom mice treated with mineral oil alone. Plasma cell tumors wereinduced in BALB/c mice by intraperitoneal injection of 0.5 ml mineraloil at 2, 4, and 6 months of age. The latent period was calculated fromthe first mineral oil injection. Correlation coefficience T - —0.853,P < 0.05.
Effect of Isoantiserum on Tumor Ingestion. Since it has beenestablished that C57BL anti-BALB/c sera were uniformly cyto-toxic for all strains of tumor cells tested, the effect of suchsera on the phagocytosis of tumor cells by ordinary macrophages was tested. When 10s tumor cells, 50 ¡Aantiserum, and3 X IO6 starch-induced macrophages from normal C57BLmice were incubated in 0.7 ml Eagle's medium containing10-15% normal BALB/c serum for one hour at 37°C, no
phagocytosis was seen. Addition of complement did not affectthe result. The experiment was repeated more than ten times.These starch-induced macrophages phagocytosed latex particles and, in the presence of isoantiserum, could phagocytosenormal spleen cells from BALB/c mice.
DISCUSSION
These experiments were done to explain the virtually complete prevention of plasma cell tumor development in oil-injected BALB/c mice treated with GPH. Old et al. demon
strated that BCG vaccination delayed development of methyl-cholanthrene-induced sarcomas, presumably by increasing thehost's resistance to the nascent tumors (9). The same mecha
nism might be postulated for the prevention of plasma celltumors by glycoprotein pituitary hormones. The hormonepreparation can be simply acting as a general antigen to stimulate the host. However, prevention is neither specific for allpituitary hormones nor proteins in general. Serum albumin iscompletely without effect (14). Administration of growth hormone markedly accelerates tumorigenesis (14). The hormonepreparations used were of high potency and almost free ofcontaminating hormonal activity. However, they are not freeof contaminating proteins. It is unlikely that the activity isrelated to the biologic activities of the hormones used because they have little in common and do not maintain hormonal activity on continued administration because of theirantigenic properties.
The nature of the granulated macrophage called forth byGPH administration is under study. However, it is clear thatthis cell can ingest some plasma tumor cells without the addition of antisera or complement to the incubation system.Washing of the granulated macrophages failed to remove specific phagocytotic activity toward appropriate myeloma cells.
Bennett et al. (3) showed that some tumor cells are phagocytosed by starch-induced macrophages in the presence of iso-antibody. Macrophages in his system were totally inactive inthe absence of antibody. In the present study many attemptsto demonstrate phagocytosis of myeloma cells by starch-induced macrophages with this system failed. Why are not allstrains of myeloma cells ingested by granulated macrophagessince animals bearing these macrophages in their peritonealfluid fail to develop the disease? If antibody is required forphagocytosis, it could be already present on the surface of thetumor cells. This would explain our inability to demonstratean antibody requirement for phagocytosis. It is possible thatthe gentle trypsinization used in the present study would failto remove tightly bound antibody but might remove less adherent antibody. However, this explanation is inconsistentwith the observation that every strain of tumor cell employed,including two early primary tumors, was ingested by macrophages from some glycoprotein pituitary hormone-treated animal. Two main possibilities emerge to explain the coincidenceof granulated macrophages in the peritoneal fluid of micetreated with oil and glycoprotein pituitary hormone and inhibition of tumorigenesis. It is possible that the macrophages
Table 6
Phagocytosis of myeloma cells bythe peritoneal granulated macrophage
from the individual mice treated with GPH
The incidence of cytotoxic antibodies in the sera" 0/4 6/8 4/7 0/3 2/9
Discrepancy between the presence of cytotoxic antibodies in the sera and phagocytosis of myelomacells by the granulated macrophage from individual mice. (The same BALB/c mice described in Table4. Target tumor cells were TPC-8,12,17 and 18). GPH, glycoprotein pituitary hormones.
"Cytotoxic antibodies in the sera of the same individuals against the particular lines of plasma celltumor tested for phagocytosis (No. of mice with cytotoxic antibody/no, of mice).
may destroy tumor cells without actual phagocytosis. Amos(1) described two types of tumor cell destruction by macrophages when cells were injected into the peritoneal cavity ofan histoincompatible host. LI210 and EL4 cells were vigorously phagocytosed. Rejection of Sarcoma I cells was characterized by close association of macrophages and tumor cells followed by destruction of both cell types but without evidentphagocytosis. Granger and Weiser (6) observed that peritonealmacrophages from animals immunized with histoincompatibletarget cells (Sarcoma I and fibroblasts) can destroy these cellsby contact. During the preparation of anti-BALB/c isoantiseraduring the present study, it was observed that living BALB/cmyeloma cells injected intraperitoneally into C57BL/6J micegave rise to potent anti-BALB/c isoantibody. However, frequent examination of peritoneal smears from recipient micefailed to disclose phagocytosis. Cell destruction of nascent tumor cells might be achieved without phagocytosis, and ingestión of damaged cells might be a secondary, unnecessary partof rejection. It is also possible that an intrinsic specificity lieswithin the granulated macrophage so that it is always able tocope with any myeloma cell capable of forming in that particular host. The granulated macrophage has some specificity formyeloma cells. It fails to ingest several other types of tumorcells or normal BALB/c spleen cells. The specificity is notabsolute since the cells do ingest latex particles. But the specificity toward myeloma cells may represent an immune reaction to the type of nascent clones which were successfullydestroyed. Experiments are planned to test both major possibilities outlined above.
The meaning of cytotoxic antibody in the sera of oil-injectedmice is not clear. It is attractive to think of the activity asspecific for tumor antigens. The cytotoxicity is not directedtoward normal BALB/c spleen cells. The mice with cytotoxicsera received no injection to generate antibodies toward histo-incompatibility antigens. Natural antibodies based on possiblesmall differences in genes within the colony of BALB/c miceseems unlikely. Normal sera of C57BL/6J mice showed nocytotoxicity toward the BALB/c tumor cells. The possibilityof cytotoxic antibodies based on the presence of passengervirus in the mice was considered (2, 12), but the specificity ofcertain sera for specific lines of tumor renders this hypothesiscomplicated since it requires too many passengers. The relation between the susceptibility of a line of tumor and thelatent period of its primary tumor can be best explained bythe presence of these antigens in the primary tumor. The roleof cytotoxic antibodies has not been established. It is notrequired for tumor prevention by glycoprotein pituitary hormones. There is no difference in the number of cytotoxicreactions for different tumor cells between the group receivingoil alone (before or after tumor development) and oil plusGPH. Phagocytosis of tumor cells occurs even when the serum
of the macrophage donor fails to contain the appropriate cytotoxic antibody. Finally, we have not been able to set up anormal macrophage system which ingests tumor cells knownto be coated with anti-BALB/c isoantibody. Our present working hypothesis is that cytotoxic antibodies are specific againstnascent tumors which arise within oil-treated mice. Their role,if any, in the prevention of tumor development is unexplainedby the present study.
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3. Bennett, B., Old, L. J., and Boyse, E. A. Opsonization of Cells byIsoantibody in vitro. Nature, 198: 10-12, 1963.
4. Boyse, E. A. A Method for the Production of Viable Cell Suspension from Solid Tumors. Transplant. Bull., 7: 100-104, 1960.
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7. Hollander, V. P., Takakura, K., and Yamada, H. Endocrine Factorsin the Pathogenesis of Plasma Cell Tumors. Recent Progr. HormoneRes., 25: 81-137, 1968.
8. Klein, E., Klein, G., Nadkarni, J. S., Nadkarni, J. J., Wigzell, H.,and Clifford, P. Surface IgM-kappa Specificity on a BurkittLymphoma Cell In vivo and in Derived Culture Lines. Cancer Res.,28: 1300-1310,1968.
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Fig. 1. A, peritoneal macrophages from BALB/c mouse 8 months after the first intraperitoneal injection of mineral oil. x 1500. B, thegranulated macrophages from the peritoneal fluid of mineral oil-injected mice treated with follicle stimulating hormone (500 /Jg, 5 times per week)for 8 months, x 1500.
Fig. 2. Myeloma cells ingested by granulated macrophages from glycoprotein pituitary hormone-treated mice. Surrounding the ingested tumorcells (darkly stained cells), vacuole formation is seen. Nuclei of macrophages appear pale. Granules of macrophages are not conspicuous in culture.Myeloma cells: TPC-17. Granulated macrophages from a mouse treated with luteinizing hormone. See Table 3 for the conditions of incubation.The arrows indicate phagocytosed tumor cells, x 500.
Fig. 3. Immunocytotoxic electrophoresis. Reference profile (upper): Electrophoresis on cellulose acetate strip stained with Ponceau 2R; thearrow indicates the point of serum application; the cathode is to the right of the strip. Cytotoxic test in gel (lower): The figure was reconstructedfrom a series of microscopic pictures; the arrow indicates the area of dead cells.
1969;29:1420-1427. Cancer Res Hisashi Yamada, Atsuko Yamada and Vincent P. Hollander Nascent Plasma Cell TumorsRole of Cellular and Humoral Factors in the Destruction of
