importance of timing in cyclophosphamide therapy of mopc ... · importance of timing in...

[CANCER RESEARCH 40, 2135-2141. July 1980]0008-5472/80/0040-0000$02.00

Importance of Timing in Cyclophosphamide Therapy of MOPC-315Tumor-bearing Mice1

James C. D. Hengst,2 Margalit B. Mokyr, and Sheldon Dray3

Department ol Microbiology and Immunology, University of Illinois at the Medical Center, Chicago, Illinois 606 i 2

ABSTRACT

The timing of cyclophosphamide (CY) administration aftertumor inoculation was found to be critical for successful therapy of MOPC-315 tumor-bearing mice. Following inoculationwith 3.5 x 106 viable tumor cells, a single i.p. injection of CY

(15 mg/kg) into mice bearing 10- to 25-mm (Days 8 to 14)

tumors cured most mice, whereas injection into mice bearingnonpalpable (Day 4) tumors cured only a few of the mice. Thetime interval between tumor inoculation and CY administrationrather than the tumor size was critical for successful therapysince mice bearing nonpalpable tumors 12 to 13 days postin-oculation with 105 viable tumor cells were cured by CY therapy.

Furthermore, CY therapy of mice bearing large (19-mm) tumors

was not curative for mice that had been treated previouslywhen their tumors were nonpalpable. A curative injection of CYinto mice bearing large tumors resulted in an augmented abilityof their spleen cells to mount a cytotoxic antitumor responseupon in vitro immunization with mitomycin C-treated s'timulator

tumor cells. This was not further augmented by depletion ofglass-adherent cells and was accompanied by a decrease inthe percentage of cells bearing surface MOPC-315 myelomaprotein in the spleen. The level of antitumor cytotoxicity exhibited by in w'fro-immunized spleen cells from CY-treated mice

was equivalent to that exhibited by in wfro-immunized spleencells from untreated tumor-bearing mice that prior to in vitroimmunization were depleted of glass-adherent cells. Sincedepletion of glass-adherent cells from tumor bearer spleen

cells prior to in vitro immunization was shown to result ingreater augmentation of antitumor cytotoxicity than that obtained by depletion of tumor cells (25), the present data suggestthat in addition to the drug's tumoricidal activity, it also elim

inates other suppressor elements in the spleen. Mice cured oftumors following CY therapy exhibited a high degree of anti-tumor immunity as judged in vivo by their ability to reject alarge tumor challenge and in vitro by the ability of their spleencells to mount a "secondary type" antitumor response upon in

vitro immunization.

INTRODUCTION

A number of reports demonstrate the necessity for an activeantitumor response for the eradication of established tumorsby CY4 therapy (7, 19, 26, 28). Several mechanisms for syn-

' Supported in part by NIH Grant ROI CA-26480.2 In partial fulfillment of the requirements for a Ph.D. in Microbiology and

Immunology in the Graduate College of the University of Illinois at the MedicalCenter.

3 To whom requests for reprints should be addressed, at P. O. Box 6998,

Chicago, III. 60680.4 The abbreviations used are: CY, cyclophosphamide; MEM, minimal essential

medium; TNP-SRBC. trinitrophenylated sheep red blood cells.Received September 13, 1979; accepted March 19, 1980.

ergism between chemotherapy and host antitumor immunityhave been suggested. Thus, the drug might (a) reduce tumorburden to a level whereby existing host antitumor immunity caneliminate residual tumor cells (28), (o) slow tumor growth longenough to allow development of a potent antitumor response(7), (c) render residual tumor cells more susceptible to immunelysis (3), and (d) potentiate cytotoxic immunity by eliminationof suppressor elements that interfere with an effective antitumorimmune response (12).

Progression of tumor growth can be attributed to eitherinefficient in vivo sensitization or the appearance of suppressorelements that interfere with an effective antitumor response.Such suppressor elements were first described in the serum oftumor-bearing hosts and included blocking antibodies, freeantigen shed by tumor cells, or antigen-antibody complexes (1,16). More recently, evidence has accumulated regarding theappearance of 0-positive (11) as well as 0-negative (13) sup

pressor lymphoid cells during progressive tumor growth. Wehave shown that in comparison to normal spleen cells, spleencells from mice bearing large MOPC-315 tumors contain met-

astatic tumor cells as well as an increased percentage ofmacrophages and upon in vitro immunization exhibit reducedlevels of antitumor cytotoxicity (22-24). Both cell types wereimplicated as suppressor cells in the MOPC-315 tumor system

since depletion of tumor cells from tumor bearer spleen cellsprior to in vitro immunization resulted in less augmentation ofantitumor cytotoxicity than that obtained by depletion of glass-adherent cells which included the removal of most tumor cellsand macrophages (25) and possibly suppressor T-cells (10).

In the present study, we evaluated the importance of timingCY therapy for its effectiveness in curing mice bearing MOPC-315 tumors. In addition, we evaluated whether augmentationin the antitumor potential of spleen cells observed following CYtreatment of mice bearing large tumors can be attributed to theelimination of suppressor elements.

MATERIALS AND METHODS

Spleen Cell Suspensions. Spleen cell suspensions wereprepared from spleens of normal female BALB/c mice (8 to 12weeks old; Laboratory Supply Co., Indianapolis, Ind.) or fromBALB/c mice bearing various sizes of s.c. MOPC-315 tumors.In any individual experiment performed, the spleens used ineach group were obtained from at least 3 but usually 5 to 7mice. Single-cell suspensions were prepared by mechanical

disruption between glass slides as previously described (24),and the viability as determined by trypan blue dye exclusion(0.4%) always exceeded 95%.

Target Cells. Target cells used in the 51Cr release assay

included MOPC-315 plasmacytoma, EL4 leukemia, and normalBALB/c spleen cells. MOPC-315 was maintained by serial s.c.inoculation in syngeneic BALB/c mice. Single-cell suspensions

JULY 1980 2135

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J. C. D. Hengst et al.

were prepared by mechanical disruption (24), and the viabilityas determined by trypan blue dye exclusion (0.4%) alwaysexceeded 85%. EL4 was maintained in ascitic form by weeklyi.p. injection into C57BL/6 mice (8 to 12 weeks old; CharlesRiver Breeding Labs., Inc., Wilmington, Mass.). Normal BALB/c target cells were blast cells induced by stimulation withconcanavalin A (5 fig/ml) for 48 to 72 hr as previously described (22).

Chemotherapy. CY (Cytoxan, Mead Johnson & Co., Evans-

ville, Ind.) was dissolved in sterile distilled water (to a concentration of 20 mg/ml) and further diluted in MEM (Grand IslandBiological Co., Grand Island, N. Y.). Various doses of the drugranging from 5 to 300 mg/kg were injected i.p. into MOPC-315 tumor-bearing mice. Mice were examined 3 times weekly

for the presence of tumors, and mice that had no detectabletumors 60 days after therapy were considered to be cured.

In Vitro Immunization. The in vitro method for generatingantitumor cytotoxicity in lymphoid cells was described previously (6, 24). Briefly, responder spleen cells (75 x 106) were

cultured with or without mitomycin C-treated MOPC-315 stimulator tumor cells (2.5 x 106) at 37Â°in a humidified atmosphere

of 5% CO2 in air for 5 days unless otherwise specified.Antitumor Cytotoxicity Assay. The cell-mediated lysis was

determined as we have previously described (24) utilizing the3.5-hr in vitro 5'Cr release assay (5). The percentage of specific51Cr release was assessed by the following formula:

T â€”C. of specific 51Cr release = â€”â€”â€”x 100

M â€”C

where T is the percentage of release with test spleen cells; Cis the percentage of spontaneous release by the target tumorcells alone which ranged between 14 and 18%; and M is thepercentage of maximum 5'Cr release obtained by 3 cycles of

freezing and thawing, which ranged between 78 and 81%.Each experiment was performed 2 to 4 times. We have observed, as have others (6, 21), that the level of antitumorcytotoxicity obtained with spleen cells immunized in vitro underidentical conditions may vary substantially from one experimentto another. Still, the effect of treating tumor-bearing mice with

CY was consistent. Therefore, in order to obtain a more accurate comparison of the levels of antitumor cytotoxicity exhibitedby in wfro-immunized spleen cells at different times following

CY therapy, the level of antitumor cytotoxicity exhibited byexperimental groups was corrected with respect to that ofparallel cultures of in wfro-immunized normal spleen cells. The

following formula was used:

% of corrected 5'Cr release = â€”x Ã‘N

where E is the percentage of specific release with test spleencells; N is the percentage of specific release with normal spleencells immunized in parallel; and Ã‘ is the average percentage ofspecific release with all the in wfro-immunized normal groupsin the serial study. The level of antitumor cytotoxicity is presented as the mean 51Cr release of triplicate samples Â±theS.E. Variations in the 51Cr release between individual samples

rarely exceed 3% of the mean. All points that differed by 5%release or more from the level exhibited by in wiro-immunizednormal spleen cells were judged to be significantly different bythe Student's f test (p = 0.05).

Enumeration of Cells Rosettable with TNP-SRBC. SinceMOPC-315 tumor cells possess high-affinity surface IgA with

specificity for nitrophenyl compounds, we enumerated tumorcells in tumor bearer spleen cells by utilizing their ability torosette with TNP-SRBC. Trinitrophenol was coupled to sheep

RBC by the method of Rittenberg and Pratt (29) as modified byHannestad ef al. (15). The rosetting procedure used was essentially the method described by Hannestad et al. (15). Thepercentage of rosettes in spleen cell suspensions was determined by counting triplicate samples of 300 spleen cells.

Depletion of Cells Adherent to Glass Wool. Tumor bearerspleen cells (8 x 108) suspended in 30 ml warmed MEM

supplemented with 20% fetal calf serum were applied to individual 50-ml sterile glass wool columns. Columns were incubated at 37Â°for 45 min with occasional rotation. Nonadherent

spleen cells were eluted in 100 ml of warmed MEM. The yieldof cells recovered from the columns was about 15 to 25%.

Determination of the Percentage of Macrophages. Thepercentage of macrophages in spleen cell suspensions wasdetermined by morphological criteria by counting at least 2500but usually 5000 nucleated cells on Wright-stained smears.

Enumeration of macrophages by functional criteria was doneby determining the percentage of cells that ingested latexbeads (Difco Laboratories, Detroit, Mich.) according to themethod of Rosenstreich ef al. (30). More than 90% of the cellsidentified as macrophages by morphological criteria phagocy-

tized latex beads (22).

RESULTS

Determination of the Minimum Dose of CY Required forthe Cure of BALB/c Mice Bearing Large MOPC-315 Tumors.Mice bearing 20-mm tumors were given a single i.p. injection

of various doses of CY ranging from 5 to 300 mg/kg (Table 1).Treatment of mice with 5 mg/kg cured only 17% of the mice,whereas treatment of mice with 15 to 200 mg/kg cured mostmice. Treatment of mice with 300 mg/kg resulted in toxicity,and although some reduction in tumor size was observedfollowing CY treatment, 67% of the mice died. Therefore, infurther experiments, we have treated tumor-bearing mice with

CY (15 mg/kg) which was determined to be the lowest curativedose.

Effectiveness of CY Therapy for Mice Bearing VariousSizes of MOPC-315 Tumors. Initially, mice bearing eithernonpalpable (Day 4 tumor bearers) or large (19-mm, Days 10to 14) s.c. MOPC-315 tumors were treated with a single i.p.injection of CY (15 mg/kg) (Table 2). Inoculation of 3.5 x 106

Table 1Effectiveness of various doses of CY tor therapy of mice bearing large3 MOPC-

315 tumors

Dose(mg/kg)05152550100200300Effect

oftherapyCured

(%)017100929210010033No.ofcured/total0/122/1212/1211/1211/1212/1212/124/12

Tumors s.c. with diameters of 20.2 Â±0.3 mm.

2136 CANCER RESEARCH VOL. 40



Importance of Timing CY Therapy

viable MOPC-315 tumor cells into normal BALB/c mice re

sulted in progressively growing tumors that killed the mice inabout 21 days. Treatment of mice bearing nonpalpable tumorscured 12% of the mice and extended the survival of theremainder by 12 days, whereas treatment of mice bearing largetumors cured 81 % of the mice and extended the survival of theremainder by 21 days. Large tumors regressed within 10 to 14days after CY treatment and were nondetectable during the 60days posttherapy. Next, the effectiveness of a single i.p. injection of CY (15 mg/kg) for curing mice bearing various sizes ofs.c. MOPC-315 tumors was evaluated (Table 3). As above,

treatment of mice bearing nonpalpable (Day 4) tumors curedonly relatively few mice (7%). Treatment was progressivelymore curative when administered to mice bearing increasinglylarger tumors such that treatment of mice bearing 5-mm tumorscured 47% of the mice, whereas treatment of mice bearing 25-

mm tumors cured 92%. Thus, CY therapy with the selecteddose was more successful in curing mice bearing larger MOPC-315 tumors than those bearing smaller tumors.

Effect of Time Interval between Tumor Inoculation and CYAdministration on the Success of Therapy. Experiments wereperformed to determine whether the increasing effectivenessof CY therapy with progression of tumor growth could beattributed to differences in the time of drug therapy after tumorinoculation or whether the effectiveness could be attributed tothe tumor size at the time of therapy (Table 4). BALB/c micewere inoculated with various numbers of MOPC-315 tumorcells ranging from 1 x 105 to 3.5 x 106, and 12 to 13 days

later when the resulting tumor sizes ranged from nonpalpableto 24 mm, they received a single i.p. injection of CY (15 mg/kg). Whereas CY therapy of mice bearing nonpalpable tumors4 days after tumor inoculation was essentially noncurative,therapy of mice bearing nonpalpable tumors 12 to 13 daysafter inoculation with 106 tumor cells cured most mice (7%

versus 87%). Furthermore, CY therapy given to mice bearing11-, 17-, or 24-mm tumors 12 to 13 days post-tumor inocula-

Table 2Effect of a single injection of CY (15 mg/kg) i.p. on the survival of mice bearingnonpalpable or large MOPC-315 tumors resulting from the prior inoculation of

3.5 x )06 viable tumor cells s.c.

Time oftherapyDays

post-tumor inocula

tionUntreated

410-14Size

of tumors(mm Â±S.E.)Nonpalpable

19.1 Â±2.4Cured

(%)0

1281Effect

oftherapyNo.

of cured/total0/25

3/2550/62Survival

time ofmice not cured(days Â±S.E.)21.3

Â±1.033.4 Â±2.042.7 Â±2.7

Table 3Effectiveness of CY therapy3 for mice bearing various sizes of MOPC-315

tumors

Effect of therapysize ot tumors at tnerapy

(mm Â±S.E.)Tumor

bearercontrolsNonpalpable"4.9

Â±0.29.9Â±0.217.2Â±0.225.2Â±0.3Cured

(%)0747737792No. ofcured/total0/151/157/1511/1510/1312/13

tion was equally curative (87, 87, and 100% cured, respectively). Thus, the effectiveness of CY therapy can be attributedto the time of CY administration post-tumor inoculation ratherthan to the tumor size at the time of therapy.

Effectiveness of CY Therapy for Mice Bearing LargeMOPC-315 Tumors That Were Not Cured by Previous Ther

apy when They Had Nonpalpable Tumors. Since CY therapywas relatively curative for mice bearing 10- to 25-mm tumorsbut not for mice bearing nonpalpable (Day 4) tumors, experiments were performed to determine whether mice not curedby CY when they had nonpalpable tumors would be cured bya second injection of CY (15 mg/kg) when their tumors reached19 mm (Table 5). As above, CY treatment of mice bearing 19-

mm tumors cured most mice (76%), whereas treatment of micebearing 19-mm tumors that had received CY treatment when

they had nonpalpable tumors cured only 7% of the mice. Thus,therapy of mice bearing large MOPC-315 tumors with CY is

curative only if the mice were not treated with the drug at earlystages after tumor inoculation.

Effect of CY Therapy of Mice Bearing Large MOPC-315Tumors on the in Vitro Antitumor Potential of Their SpleenCells. Mice bearing 20-mm tumors were treated with CY, and14 days later when the tumors had completely regressed,spleen cells were obtained. The spleen cells were immunizedin vitro with mitomycin C-treated tumor cells for 5 days and

subsequently assayed for in vitro antitumor cytotoxicity. Thelevel of cytotoxicity obtained was compared to that exhibitedby in w'fro-immunized spleen cells from normal mice or fromuntreated mice bearing 20-mm tumors (Table 6). In w'fro-im

munized spleen cells from untreated tumor-bearing mice ex

hibited a lower level of antitumor cytotoxicity than that exhibitedby in w'fro-immunized spleen cells from normal mice (32%

versus 48%) [in confirmation of our previous studies (21-

Table 4Effect of the time interval between tumor inoculation and CY therapy1 on the

survival of mice bearing various sizes of s.c. tumors

Time oftherapyNo.

of tumorcells inoculated"1

x 1055 x 10s1 x 10s

3.5 x1063.5x 10sCays

post-tumor inocula

tion12-13

12-1312-1312-13

4Sizes

of tumors(mm Â±S.E.)Nonpalpable

10.8 Â±0.517.1 Â±0.523.9 Â±0.4NonpalpableEffect

oftherapyCured

(%)87

8787

1007No.

ofcured/total13/15

13/1513/1515/15

1/15

CY (15 mg/kg) i.p.Mice in untreated control groups of 15 mice/group all developed lethal

tumors following inoculation with 105or more viable tumor cells s.c.

Table 5Effectiveness of CY therapy" for mice bearing large MOPC-315 tumors not

cured by previous therapy when they had nonpalpable tumors

Effect of therapy

Experimental groups Cured (%) No. of cured/total

Untreated tumorbearersTreatedlarge-tumorbearers"Treatednonpalpable-tumorbearers0Double-treated

tumor bearers0076770/1519/251/151/15

CY (15 mg/kg) i.p.' Mice treated 4 days after inoculation with tumor cells.

CY(15 mg/kg) i.p.Mice treated once when tumors were 19.4 Â±0.6 mm.

c Mice treated once 4 days after inoculation with tumor cells.d Mice were treated 4 days after inoculation with tumor cells, and those not

cured were treated again when tumors reached 19.3 Â±0.4 mm.

JULY 1980 2137




Table 6Effect of CY therapy of mice bearing large MOPC-315 tumors on the in vitro

antitumor potential of their spleen cells

In wfro-immunizedspleen cells from

Antitumor cytotoxicity(% of specific 5'Cr release Â±S.E.)a

Normal miceUntreated tumor bearers6Treated tumor bearers0

47.9 Â±0.931.6 Â±0.393.9 Â±0.3

At an effector/target cell ratio of 100/1.6 Mice bearing s.c. tumors with diameters of 19.8 Â±0.3 mm.c Mice bearing s.c. tumors with diameters of 19.8 Â±0.3 mm received injections

of CY (15 mg/kg) Â¡.p.14 days prior to in vitro immunization.

23)]. CY treatment of tumor-bearing mice enabled their spleen

cells to develop upon in vitro immunization a much higher levelof antitumor cytotoxicity than that exhibited by either in wfro-immunized spleen cells from untreated tumor-bearing mice orin wfro-immunized spleen cells from normal mice (94% versus32% and 48%, respectively). Thus, CY treatment of micebearing large tumors results in augmented antitumor potentialof their spleen cells.

Determination of the Optimal Duration of in Vitro Immunization for the Development of in Vitro Antitumor Cytotoxicityin Spleen Cells from Mice That Rejected Large Tumorsfollowing CY Treatment. Mice bearing 19-mm tumors were

treated with CY, and 21 days later when their tumors hadcompletely regressed ("cured" mice), spleen cells were ob

tained. These spleen cells or spleen cells from normal micewere evaluated for antitumor cytotoxicity prior to, or 1 to 7days after, the initiation of in vitro culturing in the presence orabsence of stimulator tumor cells (Chart 1). Freshly preparedspleen cells from normal mice were noncytotoxic, whereasthose from "cured" mice exhibited some cytotoxicity (0%

versus 7%) which was maintained during the 7 days of culturingin the absence of stimujator tumor cells. In vitro immunizationof normal spleen cells resulted in the appearance of antitumorcytotoxicity on the fourth day of culture (31%) which reacheda peak on the fifth day and was nondetectable by the seventhday (<5%) [as we had previously shown (23)]. In vitro immunization of spleen cells from "cured" mice resulted in a sub

stantial augmentation of antitumor cytotoxicity (as compared tothat exhibited by unimmunized spleen cells) as early as thesecond day of in vitro immunization (25% versus 8%) whichreached maximal levels on the fourth day (100%) and wasmaintained at high levels through the seventh day (85 to 100%).On Days 2 through 7 of the immunization process, the levels ofantitumor cytotoxicity exhibited by in wfro-immunized spleencells from "cured" mice when tested at an effector/target cell

ratio of 100/1 or 20/1 were much higher than those exhibitedby in wfro-immunized normal spleen cells tested at 100/1.

Thus, spleen cells from mice that following CY therapy rejectedlarge tumors respond to in vitro stimulation with tumor cells ina manner similar to a secondary response. It is also clear fromthese data that the optimal length of in vitro immunization forthe development of antitumor cytotoxicity with spleen cellsfrom normal mice is 5 days and with spleen cells from "cured"

mice is 4 to 7 days. Therefore, in further experiments, in vitroimmunization was performed for 5 days to allow a bettercomparison of the levels of antitumor cytotoxicity exhibited byin w'fro-immunized spleen cells from CY-treated mice with thoseexhibited by in w'fro-immunized normal spleen cells.

Effect of CY Therapy of Mice Bearing Large Tumors on

the Presence of Suppressor Elements in the Spleens. Experiments were performed to determine how soon after CY therapyof tumor-bearing mice their spleen cells exhibit augmentedantitumor potential and whether this augmented potential isdue to elimination or inactivation of suppressor elements.Spleen cells were obtained from normal mice, mice bearing24-mm tumors, or mice that had 24-mm tumors when treatedwith CY and their spleens removed 2, 4, or 7 days posttreat-

ment (Table 7). Spleen cells were examined for the percentageof macrophages and tumor cells to determine whether theaugmented cytotoxic potential correlates with a decrease inthe percentage of such cells. In addition, we evaluated theeffect of removing glass-adherent cells from the spleens ofuntreated or CY-treated tumor-bearing mice on the level of

antitumor cytotoxicity obtained upon in vitro immunization.Identification of tumor cells was done utilizing their ability toform rosettes with TNP-SRBC. Normal spleen cells which con

tained 3.6% macrophages and 2.8% rosettable cells exhibiteda substantial level of antitumor cytotoxicity upon in vitro immunization (81 %). Spleen cells from untreated tumor-bearing

mice which contained a higher percentage of macrophages(11%) and rosettable cells (11%) exhibited lower levels of

Days of in Vitro Immunization

Chart 1. The levels of antitumor cytotoxicity exhibited by spleen cells fromnormal mice or CY-cured mice upon culturing in the presence or absence ofstimulator tumor cells. O, unimmunized spleen cells from normal mice at aneffector/target cell ratio of 100/1 ; â€¢,immunized spleen cells from normal miceat the same ratio; A, unimmunized spleen cells from CY-cured mice; A, immunizedspleen cells from similar mice; â€¢immunized spleen cells from CY-cured mice atan effector/target cell ratio of 20/1.

Table 7Effect of CY therapya for mice bearing 24-mm tumors on the percentage of

rosettable cells and macrophages in the spleen and also on the level ofantitumor cytotoxicity obtained with unfractionated or glass-nonadherent spleen

cells upon in vitro immunization

Antitumor cytotoxicity (% of corrected 5'Cr release Â±S.E.)6

Dayspost-

therapy0

247%

of rosettablecells Â±S.E.10.6

Â±0.42.6 Â±0.43.2 Â±0.43.0 Â±0.6%

of macrophages10.8

Â±0.911.4 Â±0.9

9.6 Â±0.67.4 Â±0.1Unfractionated

spleencells23.5

Â±0.659.9 Â±0.585.7 Â±0.7

100.3 Â±1.6Glass-nonad

herent spleencells91.4

Â±1.157.4 Â±1.185.7 Â±0.760.7 Â±1.8

CY (15 mg/kg) Â¡.p.' At an effector/target cell ratio of 100/1.





antitumor cytotoxicity upon in vitro immunization (24%). Asearly as 2 days post-CY treatment, the percentage of rosettable

cells dropped to background levels (2.6%) while the percentage of macrophages was virtually unchanged (11 %), and uponin vitro immunization of the spleen cells, augmented levels ofantitumor cytotoxicity were obtained (60% versus 24%). Between Days 2 and 7 post-CY treatment, further augmentation

of the antitumor potential of tumor bearer spleen cells wasobserved, yet a drop in the percentage of macrophages wasfirst noted on Day 7 (7% versus 11 %).

Removal of suppressor elements from the spleens of MOPC-315 tumor-bearing mice was achieved in our previous studiesby depletion of glass-adherent cells (22). Here, depletion ofglass-adherent cells prior to in vitro immunization resulted in ahigher level of antitumor cytotoxicity than that exhibited byunfractionated spleen cells when spleen cells from untreatedbut not CY-treated tumor-bearing mice were used. Moreover,when glass-adherent cells were depleted from the spleens of

mice treated with CY 7 days prior to in vitro immunization, thelevel of antitumor cytotoxicity was lower than that exhibited bythe unfractionated in wfro-immunized spleen cells (61 % versus

100%). Thus, CY treatment results in the elimination of theglass-adherent suppressor elements that operate in the spleensof MOPC-315 tumor-bearing mice.

in Vitro Cytotoxicity of in V/tro-lmmunized or Unimmunized

Spleen Cells from Mice Cured of Large Tumors following CYTherapy when Evaluated against MOPC-315, EL4, or NormalBALB/c Target Cells. Mice that following CY therapy rejected20-mm tumors were used as spleen cell donors 21 days afterthe initiation of treatment. The spleen cells were cultured invitro in the presence or absence of stimulator tumor cells andsubsequently were tested for their cytotoxicity against MOPC-

315, EL4 of C57BL/6 origin, or normal BALB/c target cells(Table 8). The normal BALB/c target cells used were blastcells induced by stimulation with concanavalin A and weresusceptible to lysis by C57BL/6 spleen cells immunizedagainst MOPC-315 but resistant to lysis by either C57BL/6 or

BALB/c spleen cells immunized with EL4 (data not shown).Unimmunized spleen cells from "cured" mice exhibited appre

ciable levels of cytotoxicity against MOPC-315 (15%) but no

cytotoxicity against EL4 or normal BALB/c target cells (lessthan 3%). In vitro immunization of spleen cells from "cured"

mice resulted in augmented anti-MOPC-315 cytotoxicity (95%)

and virtually no cytotoxicity against EL4 or normal BALB/cblast cells (1 % or 4%, respectively).

Ability of Mice "Cured" by CY of Large MOPC-315 Tumors

to Reject a Lethal Challenge with Viable MOPC-315 Tumor

Table 8In vitro cytotoxicity against MOPC-315. EL4. or normal BALB/c target cellsexhibited by in vitro-immunized or unimmunized spleen cells from mice that

following CY therapy rejected large MOPC-315 tumors

Source of

Cytotoxicity (% of specific 51Cr release Â±S.E.)a

spieencellsTreated

mice0

Treatedmice0btimuiator

tumorcellsNone

MOPC-315MOPC-31515.3

Â±1.3

94.8 Â±2.8EL40.5

Â±0.7

0.6 Â±0.7Normal01.0

Â±0.3

3.7 Â±1.1

Cells. Mice that following CY treatment rejected 21 -mm tumorswere challenged 21 to 54 days later with 3.5 x 106 viable

MOPC-315 tumor cells (Table 9). The challenge dose is 350-

fold greater than the minimum dose required for the appearance of lethal tumors in all mice receiving injections (4). Noneof the mice that were "cured" of a large tumor by CY developed

tumors following challenge with viable tumor cells. Thus, animals that following CY treatment were "cured" of MOPC-315

tumors exhibit an augmented potential to mount an in vivo anti-MOPC-31 5 tumor response.

DISCUSSION

In the present study, we have shown that the timing of CYadministration to MOPC-315 tumor-bearing mice is critical forsuccessful therapy. Following inoculation with 3.5 x 106 viable

tumor cells, a single i.p. injection of CY (15 mg/kg) into micebearing nonpalpable (Day 4) tumors extended their survival butcured only a few of the mice, whereas injection of the samedose of CY into mice bearing 10- to 25-mm (Days 8 to 14)

tumors cured most of the mice and extended the survival of theremainder. The time interval between tumor inoculation and CYadministration, rather than the size of the tumor, was found tobe critical for successful therapy since mice bearing nonpalpable tumors 12 to 13 days postinoculation with 105 viable

tumor cells were cured by CY (15 mg/kg). In addition, CYtherapy of mice bearing large tumors was not successful if themice had been treated previously with CY when their tumorswere nonpalpable. A curative injection of CY into mice bearinglarge tumors resulted in an augmented ability of their spleencells to mount a cytotoxic antitumor response upon in vitroimmunization. This was not further augmented by depletion ofglass-adherent cells and was accompanied by a decrease inthe percentage of cells bearing surface MOPC-315 myeloma

protein in the spleen. The level of antitumor cytotoxicity exhibited by in w'fro-immunized spleen cells from CY-treated mice

was equivalent to that exhibited by spleen cells from untreatedtumor-bearing mice that were depleted of glass-adherent cells

prior to in vitro immunization. Following tumor regression, miceexhibited a high degree of antitumor immunity in vivo by theability to reject a large tumor challenge and in vitro by theability of their spleen cells to mount a "secondary type" anti-

tumor response upon in vitro immunization.A single injection of at least 15 mg of CY per kg was required

to cure mice bearing large MOPC-31 5 tumors. This remarkable

sensitivity to relatively low doses of CY is not confined to theMOPC-315 plasmacytoma since the MOPC-104E (18), the AdjPC-5 (27), and the Adj PC-6 (9) plasmacytomas are also

extremely sensitive to CY therapy.CY therapy of mice shortly after tumor inoculation was less

curative than was therapy of mice at a later time. It seems

Table 9Ability of mice "cured"a by CY of 21-mm tumors to reject a lethal challenge

with 3.5 x JO6 viable MOPC-315 tumor cells'1

Mice Survival (%) No. of survivors/total

At an effector/target cell ratio of 100/1.b As normal target cells, concanavalin A-induced blast cells were used.

Mice bearing s.c. tumors with diameters of 20.1 Â± 0.5 mm were giveninjections of CY (15 mg/kg) i.p. 19 days prior to in vitro immunization.

Normal

"Cured" mice 100

0/15

15/15a Challenged 21 to 54 days after CY therapy of 15 mg/kg i.p.

Three hundred fifty-fold greater than the minimum tumor dose required for

the appearance of lethal tumors in all mice inoculated.

JULY 1980 2139




unlikely that tumor regression in mice bearing large tumors iscaused by a direct tumoricidal action of the drug alone. Analternative and more probable explanation is that at late stagesof tumor growth, tumor bearers have mounted an antitumorresponse that facilitates the effectiveness of CY therapy. Aswell, the possibility that the ineffectiveness of CY therapy formice bearing nonpalpable tumors is due to the presence of CY-

resistant suppressor cell(s) or weak antitumor potential shouldbe considered. The requirement for an active antitumor response to facilitate the action of CY has been reported byothers (7, 8, 19, 26, 28). CY was more effective when administered to tumor-bearing animals that exhibited some antitumor

immunity due to previous immunization than to unimmunizedtumor-bearing counterparts (7, 26). Furthermore, CY treatmentof tumor bearers that were immunosuppressed [by anti-thy-mocyte serum (14, 28), X-irradiation (19, 28), or high doses of

the drug (20)] has been reported to be less effective thansimilar treatment of immunocompetent tumor bearers.

We have previously shown that in w'fro-immunized spleen

cells from mice bearing large MOPC-315 tumors exhibited

much lower levels of in vitro antitumor cytotoxicity than did inwfro-immunized spleen cells from normal mice (22-24). We

have suggested (25) that this reduced ability of tumor bearerspleen cells to respond in vitro to stimulator tumor cells may beattributed to the action of: (a) metastatic tumor cells in thespleen capable of inhibiting both the generation and expression(by cold target inhibition) of antitumor cytotoxicity; (o) an increased percentage of splenic macrophages capable of inhibiting the generation of antitumor cytotoxicity; and (c) possiblysuppressor T-lymphocytes. Removal of such cells from MOPC-

315 tumor bearer spleen cells was achieved by fractionation ofthe spleen cells on glass wool prior to in vitro immunization (22,23, 25). Thus, in the MOPC-315 tumor system, the elimination

of such cells appears to be required for CY therapy to actsynergistically with a cytotoxic antitumor response. Indeed, CYtherapy of mice bearing large tumors resulted in a rapid disappearance of metastatic tumor cells in the spleen which wasaccompanied by augmentation of antitumor potential that couldnot be further augmented by fractionation of the spleen cellson glass wool. These data indicate that glass-adherent cells

had been eliminated during the course of CY therapy.Therapy of mice bearing large MOPC-315 tumors with CY

was curative only if the mice were not treated with the drug atearly stages after tumor inoculation. Preliminary data5 suggest

that one effect of the administration of CY to mice bearing Day4, nonpalpable tumors may be the selection of CY-resistanttumor cells, and this might explain the ineffectiveness of thesecond dose of CY when the tumors are large. This question iscurrently under further investigation.

The importance of the time of CY administration relative toimmunization with allogeneic (2) or syngeneic (12) tumor cellson the strength of the antitumor response obtained was evaluated by other investigators. Administration of CY prior toimmunization resulted in the elimination of antigen-nonspecificsuppressor cells and led to the development of stronger anti-

tumor responses than those exhibited by untreated, immunizedcounterparts (12). On the other hand, administration of CY toanimals 2 to 6 days after immunization led to elimination ofantigen-stimulated prekiller or killer cells and resulted in

' J. C. D. Hengst. M. B. Mokyr, and S. Dray, unpublished data.

weaker antitumor responses than those exhibited by immunized, untreated counterparts (2, 12). In these studies, theeffect of administering CY to animals later than 6 days afterimmunization was not evaluated. Such experiments should beperformed since it is possible that administration of CY toimmunized animals at a stage when the immune response isbeing turned off and when suppressor cells are actively proliferating will results in potentiation of this response due toelimination of suppressor cells.

In our previous studies, the reduced ability of in wfro-immunized spleen cells from MOPC-315 tumor-bearing mice to

mediate antitumor cytotoxicity correlated with the appearanceof metastatic tumor cells and an increase in the percentage ofmacrophages in the spleen (25). Since we have found thatdepletion of tumor cells from tumor bearer spleen cells prior toin vitro immunization resulted in less augmentation in the levelof antitumor cytotoxicity than that obtained by depletion ofglass-adherent cells, which included the removal of most mac

rophages and tumor cells, both cells were implicated as suppressor cells in the generation of antitumor cytotoxicity in theMOPC-315 tumor system (25). Further evidence for the contribution of the tumor cell to the reduced levels of antitumorcytotoxicity of in w'fro-immunized tumor bearer spleen cells is

illustrated here by the correlation between the rapid disappearance of cells bearing MOPC-315 myeloma protein from

the spleen and rapid augmentation of antitumor potential inspleen cells from tumor-bearing mice following CY therapy. In

addition, we have previously shown that addition of viabletumor cells to the in vitro immunization culture of normal spleencells inhibits the generation of antitumor cytotoxicity (25).However, the macrophage may not be involved in suppressionsince (a) the percentage of macrophages began to decline onlyon Day 7 post-CY treatment when the antitumor potential oftumor bearer spleen cells is almost fully developed, and (b)depletion of glass-adherent cells from the spleens of micetreated 2 days prior to the initiation of in vitro immunization didnot augment the antitumor cytotoxicity beyond that obtainedwith the unfractionated, in w'fro-immunized cells. Nevertheless,

the possibility that the macrophage requires the presence ofviable tumor cells to be suppressive cannot be ruled out assuggested by Kolb ef al. (17) for the inhibition of primaryantibody responses by suppressor macrophages, the appearance and expression of which are induced by plasmacytomacells. This is supported by preliminary data5 that suggest that

addition of tumor bearer macrophages but not of normal macrophages can inhibit the generation of antitumor cytotoxicityby normal spleen cells upon in vitro immunization. In addition,the possibility that glass-adherent suppressor T-lymphocytesoperate in the MOPC-315 tumor system should be considered

(10). Thus, the effectiveness of CY therapy for curing micebearing large MOPC-315 tumors appears to be due, at least in

part, to elimination or inactivation of tumor cells and macrophages. Metastatic tumor cells are apparently eliminated by atumoricidal action of the drug, and macrophages and perhapsother cells are probably rendered inactive by either a toxiceffect of the drug or their dependence on viable tumor cells foractivity.

We have shown that a drug given at the "wrong" time did

not cure mice with relatively small (nonpalpable) tumors andleaves them resistant to further treatment by the drug when thetumor enlarges. Yet, the same dose of drug given at the ' 'right' '





time cures mice with relatively large tumors and leaves themimmune to further tumor challenge. This phenomenon apparently has an immunological basis in that the observationscorrelated with the ability of spleen cells from these mice tomount an in vitro cytotoxic immune response to the tumor. Inview of these findings, it seems of immediate interest to investigate how timing of drug therapy affects the immunologicalresponse and cure rates in other tumor systems.

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JULY 1980 2141



1980;40:2135-2141. Cancer Res James C. D. Hengst, Margalit B. Mokyr and Sheldon Dray MOPC-315 Tumor-bearing MiceImportance of Timing in Cyclophosphamide Therapy of

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