Heterogeneity of Soluble Suppressor Factors in Rat Malignant Ascites

. A study was undertaken to enumerate and partially characterize soluble factors generated by tumor-bearing animals capable of suppressing PHA-induced splenocyte proliferation. Sprague-Dawley rats were induced to form malignant ascites by the intraperitoneal injection of the Walker 256 carcinoma. Intact ascites suppressed splenocyte proliferation by 96%. Molecular sieving of the ascites by means of ultrafiltration (10-kilodalton particle cutoff) revealed suppressor activity to reside in both the ultrafiltrate and reténtate. Further enumeration of suppressor factors was achieved by preparative polyacrylamide gel electrophoresis of the ascites ultrafiltrate and reténtate. Five discrete bands of suppressor activity were resolved in the ultrafiltrate, three of which were heat-labile. Three discrete bands of suppressor activity were resolved in the reténtate, none of which were heat-labile. This study underscores the complexity and heterogeneity of soluble factors elab­ orated in a cancer-bearing animal. acrylamide slacking gel and a 7-cm 7% acrylamide separating gel in a solution of 50 mM Tris-glycine at pi 1 9.6. A current of 4 mA/gel was utilized to perform electrophoresis which was run at 4 C. The gel were cut into 2-mm slices and two adjacent slices were incubated in 200 pi RPMI for 24 h at 37 C. The eluents were assayed for immunosuppressive activity in the PHA-stimulated splcnocyte proliferation assay. The position of the gel slice was identified by its RF value determined by its position in the gel relative to the marker dye. bromophenyl


Introduction
The negative consequences of the immunosuppres sion associated with neoplasia are substantial.In addi tion to the loss of host defenses against growth and spread of tumor, there are also the problems of oppor tunistic infection, and intolerance to anticancer therapy.This immunosuppression has been demon strated to be due, at least in part, to the presence of serum suppressor factor(s).Sera from patients with a variety of malignancies inhibit mitogen-stimulated proliferation in vitro of lymphocytes obtained from healthy donors [1,2].Furthermore placing unrespon sive lymphocytes from cancer patients into normal serum will restore responsiveness to mitogenic sti mulation [3].Thus, one of the defects in immune function related to cancer may be a reversible inhibi tion of lymphocyte function mediated by serum sup pressor factors.
Despite the importance of the problem of immuno suppression in neoplasia, this area is still not well understood.One of the reasons for this lack of general understanding is the difficulty inherent in human ex perimentation.Serial sampling of functioning immune tissue from a cancer patient is difficult to justify.In addition, therapeutic manipulation with chemothera peutic agents, radiation, and surgery may perturb the natural host immune response complicating inter pretation of experimental data [4][5][6][7].In order to cir cumvent these problems we have investigated an ani mal model capable of generating large quantities of soluble suppressor factors [8].
Sprague-Dawley rats injected intraperitoneally with the Walker 256 carcinoma develop a malignant ascites.We demonstrated the cell-free component of this ascites to be capable of inhibiting mitogeninduced proliferation of normal rat splenocytes.In vitro studies indicated that the source of this material was both lymphoid and nonlymphoid tissues of tum or-bearing animals.Preliminary characterization of suppressor factor(s) in this ascites revealed activity to be heat-stable and of low molecular weight (less than 10.000 daltons).Thin-layer chromatography revealed the presence of prostaglandins E2 and F2a.The ability of prostaglandins to inhibit mitogen-induced blastogenesis has been previously documented [9].The present study incorporates a more detailed examina tion of the experimental malignant ascites and demon strates that numerous soluble factors capable of sup pressing lymphocyte function are present in the ascites of these tumor-bearing animals.

Materials and Methods
Solid Walker 256carcinoma (Mason Research Institute.Worchester.Mass.) grown intramuscularly was minced with iris scissors in a sterile Petri dish containing RPMI 1640 tissue culture medium.Adult male Sprague-Dawley rats (Harlan Spraguc-Dawley.Mad ison.Wise.) were injected intraperitoneally with 1 ml of a suspen sion of finely minced tumor in RPMI.Ten to 14 days after inocula tion ascites was harvested by transabdominal needle aspiration from halothanc-ancsthetized rats.The ascites was allowed to stand at room temperature for 1 h to permit clot formation.The fluid component was separated by centrifugation at 1,500 rpm for 10 min.The supernatant was removed, aliquoted, and stored at -3 5 C.
A microplalc assay measuring [H3]thymidinc incorporation by PHA-stimulated rat splcnocytcs was employed.The details of this assay have been described previously [8].Briefly, splcnocytcs from a normal spleen obtained from a healthy donor were suspended in RPMI 1640 supplemented with 10% fetal calf serum and the cell concentration adjusted to 5 x 10ft viable cells/ml.One hundred and eighty microliters of the splcnocyte suspension were placed in each well of the microlitcr test plate.Twenty microliters of the experi mental material were added to each well.Proliferation was induced by adding 2.5-5.0 mg/ml of PllA (Wellcome Research Labs, Beck enham, England).Normal rat serum and ascites in various stages of fractionation were also assayed with and without the addition of PH A. Experimental samples were run in sextuplicate, except for assays conducted with material obtained from polyacrylamide gels.Material sufficient only for a single determination was eluted from the gel slices.The plates were incubated for 72 h at 37 C in an atmosphere of 95% Ch 5% CO2 .Following the 72-hour incuba tion.10 pi of [H-'Jthymidine (IOOgCi/ml.Amersham.Arlington Heights, 111.) were added to each well.Following a second incuba tion of 18 h, the wells were aspirated and the contents deposited onto filter paper, dried at 95 C, placed in scintillation vials with 2 ml of scintillation cocktail, and counted in a liquid scintillation counter for 1 min.For samples run in sextuplicate, a mean and standard deviation were calculated.Percent inhibition of DNA synthesis was then calculated by the following formula: eounts/min experimental sample counls/min control Ascites was first subjected to centrifugation at 30.000 rpm for 1 h at 4 C to remove fine particulate matter.The supernatant was then passed through a PM-10 Diaflo® ultrafiltration membrane (Amicon Corporation, Lexington, Mass.).The ultrafiltrate was collected.The reténtate was further cleansed of low molecular weight moieties by adding RPMI 1640 tissue culture medium 3 times to the Amicon chamber and subjecting the reténtate to re peated ultrafiltration.The ultrafiltrate and PM-10 reténtate were aliquoted and stored at -35 C.
Crude ascites, ultrafiltrate, and the reténtate following the ul trafiltration were subjected to heating at 100 "C for 40 min.This resulted in the formation of a precipitate which was removed by centrifugation at 1,500 rpm for 10 min.The supernatant was collect ed, aliquoted, and frozen at -35 C.
Polyacrylamide gel electrophoresis (PAGE) was performed by the technique of Davis [10].A 30-gl sample in 20% sucrose was placed on a 0.5 x 8.0 cm gel column made up of a 1-cm 3% acrylamide slacking gel and a 7-cm 7% acrylamide separating gel in a solution of 50 mM Tris-glycine at pi 1 9.6.A current of 4 mA/gel was utilized to perform electrophoresis which was run at 4 C.The gel were cut into 2-mm slices and two adjacent slices were incubated in 200 pi RPMI for 24 h at 37 C.The eluents were assayed for immunosuppressive activity in the PHA-stimulated splcnocyte proliferation assay.The position of the gel slice was identified by its RF value determined by its position in the gel relative to the marker dye.bromophenyl blue.

Results
The suppressor activities of ascites and its com ponents separated by ultrafiltration are summarized in table I. Mitogen-induced lymphocyte proliferation was consistently inhibited in the presence of crude ascites.Soluble suppressors present in crude ascites were separated into two fractions utilizing ultrafiltra tion.The low molecular weight moieties present in the ultrafiltrate produced a 71% inhibition when assayed with PHA-stimulated splenocytes.Heat treatment of the ultrafiltrate abolished the suppressor activity and resulted in an enhancing effect.Substances present in the retained material following ultrafiltration were found to suppress PHA-stimulated splenocytes by 95%.Heat treatment of the PM-10 reténtate resulted in a loss of suppressor activity.These results were observed with a minimum of two experiments and were consistent throughout the study.
Examination of eluted material from polyacryl amide gel slices in the blastogenesis assay proved to be capable of resolving ascites and its components into bands of suppressor activity.Preliminary work re vealed that the most consistent results could be ob tained when material eluted from the gel slices was further diluted to a 50% concentration with RPMI 1640 tissue culture medium.Hence, all results record ASCITES (Rf value) ed in this study relating to the PAGE work are derived from material assayed at the 50% concentration.A definition of significant suppression was chosen to be greater than 50% inhibition of blastogenesis when the eluted material was assayed at a 50% concentration.This definition of suppression was arrived at since normal rat serum or its components rarely suppressed splenocyte proliferation to this degree.
Ascites eluted from PAGE gels and assayed for suppressor activity was generally suppressive through-  out the gel.This contrasted with the PAGE assay of normal rat serum, which demonstrated rare suppres sor activity at the 50% concentration (fig. 1, 2).The ultrafiltrate eluted from PAGE gels and assayed for suppressor activity revealed five discrete areas of sup pression (fig.3).Following heating bands A, B, and C were no longer observed (fig.4).Band D persisted after heating and band E was diminished.In addition, the areas previously occupied by suppressor bands A, B, and C were found to be strongly enhancing.
The reteníate eluted from PAGE gels and assayed for suppressor activity revealed three broad bands of suppression (fig.5).Heat treatment of the reteníate did not substantially affect the bands of suppression, but did result in the observation of enhancement of blastogenesis in the areas which were previously not enhancing (fig.6).Although the general trends in suppression were preserved following heating, minor changes were noted in the region of bands G and H.

Discussion
The present studies were undertaken to further examine the characteristics of suppressor factor(s) in the ascites of rats bearing the Walker 256 carcinoma.The initial observation that suppressor factors were multiple occured when substances suppressing splenocyte proliferation appeared in both the retén tate and ultrafiltrate following ultrafiltration of malig nant ascites.These suppressor factors were further enumerated with PAGE into five bands of suppressor activity present in the ultrafiltrate and three bands of suppressor activity were resolved in that portion of ascites retained by the ultrafilter.
Heat treatment of the ultrafiltered ascites resulted in a decreased suppression of splenocyte proliferation by the PM-10 reteníate and enhancement of prolifera tion by the ultrafiltrate.These studies suggest that some of the suppressor factors are heat-labile.This finding was demonstrated more clearly by examining heat-treated components of ascites following PAGE in the splenocyte proliferation assay.It was observed that the three slowly migrating bands of suppressor activity has been replaced by activity which enhanced splenocyte proliferation.This effect is likely the result of heat dénaturation of low molecular weight peptide suppressor substances.Yet, the two rapidly migrating bands of suppressor activity were generally intact.These heat-stable bands may be accounted for by prostaglandins, described by us previously to be present in this model.The large molecular weight moieties which suppressed splenocyte proliferation did not appear to be significantly altered by heating.These large molecular weight substances may consist of complex molecules such as glycolipids or lipopoly saccharides which would not be expected to be altered by heating.
The description of multiple soluble suppressors in this manuscript bears close resemblance to the de scription of an immunosuppressor peptide isolated from human plasma [11].The cited reference describes a low molecular weight substance with an estimated molecular weight of 4-6 kilodaltons (KD) which is noncovalently bound to a large carrier protein.This concept would fit with our observations of both low and high molecular weight suppressor substances.This previous investigation also demonstrated multi ple low molecular weight peptides following paper electrophoretic analysis.Our work confirms that numerous low molecular weight substances are present and has demonstrated that each substance has suppressor activity.
A variety of suppressor molecules obtained from malignant ascites have been demonstrated previously.Hess et al. [12] described a substance with a molecular weight between 50 and 100 KD obtained from pa tients with ovarian neoplasms capable of inhibiting PHA-induced blastogenesis.However, a step in puri fication was dialysis which would have allowed low molecular weight substances to pass undetected.Sheid and Boyce [13] also examined ascitic fluid from women with ovarian cancer and described a low mo lecular weight substance which they attributed to macrophage origin.They were able to discern 6 moi eties following electrophoresis of lyophilized material.The authors were not certain whether the numerous substances were a result of a complex mixture or were a result of changes in a single molecule brought about by the method of preparation.A third description of an ascites-derived suppressor factor involves a mole cule less than 2 KD capable of inhibiting natural killer cell-mediated cell lysis [14], The authors of this article likewise conclude that this material is of host origin.However, this investigation did not reveal the presence of any large molecular weight suppressor substances.
This study has established that a number of heterogeneous suppressor substances are elaborated in a cancer-bearing animal.This observation would appear to inject a sense of complexity in the area of soluble factors capable of inhibiting cellular immune function.Additional work in our laboratory has re stored some order to this area.By examining blood samples from tumor-bearing animals over time we were able to document an initial enhancing effect of serum on in vitro lymphocyte function followed by a suppressor effect [15].We were able to separate these effects on the basis of molecular weight with the vast majority of suppressor activity residing in the ultrafil trate.
An issue not addressed in this study is the specific ity of these suppressor substances to cancer.Acute-phase reactants present in the sera of animals and humans afflicted with both benign and malignant dis eases have the capacity to suppress immune function [16][17][18], These acute-phase reactants may account for a number of these suppressor factors demonstrated in this project.This issue could be resolved by applying the methodology utilized in this study in benign and malignant diseases resulting in immune suppression and enumerating the suppressor factors elaborated in each disease condition.In this manner suppressor factors uniquely related to cancer would be sought.The potential clinical utility of specific cancer-asso ciated suppressor factors in the diagnosis and possible treatment of cancer warrants further study in the characterization of suppressor factors related to can cer.

Fig. 1 .Fig. 2 .
Fig. 1.Examination of suppressor activity following PAGE separation of intact malignant ascites demonstrated significant sup pression of splcnocyte proliferation by material obtained from the entire gel.Each point represents the activity from material eluted from two adjacent slices.Note that almost all points fall below the 50% inhibition level.

Fig. 3 .
Fig. 3. Suppressor activity of material passing through the ultrafiltcr after PAGE separation.Five (A-E) relatively discrete bands of suppressor activity could be observed.

Fig. 4 .Fig. 5 .Fig. 6 .
Fig. 4. Suppressor activity of material passing through the ul trafiller, then heated (100 C x 40 min) and subjected to PAGE separation.Suppressor bands A, B. and C appeared to be heatlabile, whereas regions D and E persisted relatively unchanged.

Table ( .
Suppression of PHA-stimulated splcnocyte proliferation by malignant ascites following ultrafiltration and heat dénaturation " Assayed at 50% concentration.h Negative suppression indicated enhancement of blastogenesis.^ x 100.