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[CANCER RESEARCH 40, 1501-1505, May
1980]0008-5472/80/0040-OOOOS02.00
Tumor-immunotherapeutic Efficacy of Serratia marcescens
Polyribosomes1
Richard W. Urban2, Bruce S. Edwards, and William Segal
Animal Tumor Program. Department of Animal. Dairy and Veterinary
Science, UMC 56, Utah State University, Logan, Utah 84321 [R. W.
U.¡:Department of HumanOncology. University of Wisconsin Clinical
Cancer Center, Madison. Wisconsin 53706 [B. S. E.J; and Department
of Environmental, Population and OrganismicBiology, University of
Colorado, Boulder, Colorado 80309 [W. S.¡
ABSTRACT
The ability of polyribosomes, obtained from several
bacterialspecies, to suppress the development of cutaneous
SaD2fibrosarcomas in DBA/2 mice was evaluated. Suppression oftumor
appearance depended upon the tumor load at the timeof treatment,
dose of polyribosomes, and species source ofpolyribosomes, with
Serratia marcescens being superior toEscherichia coli,
Streptococcus pneumoniae, Mycobacteriumbow's (Pasteur),
Mycobacterium smegmatis, and Propionibac-
terium acnes (formerly Corynebacterium parvum). A
singleinjection of 5 or 50 fig of Serratia polyribosomes at the
tumorsite 72 hr after the intradermal administration of 1.5 x
103
SaD2 cells resulted in 66 to 95% survival. All untreated
animalsexpired within 50 days. Tumor suppression occurred at
bothflank and footpad sites. Presensitization with polyribosomesand
incorporation of polyribosomes into adjuvant were notrequired for
the tumor-suppressive effect. Treatment of Serratia
polyribosomes with RNase or pronase reduced the number
ofsurvivors. Endotoxin was not detectable with the Limulus ame-
bocyte lysate assay.
INTRODUCTION
Several bacterial species belonging to the CMN3 group have
proven to be active immunopotentiators in the suppression
oftumors in animals and humans. BCG, a live attenuated strain
ofMycobacterium bow's, has been most intensively investigated
and has been found to be therapeutic in many animal
tumorsystems, as well as in malignant melanoma and acute
myelog-enous leukemia in humans (5-7, 10, 25). However, there
are
several serious limitations to therapy using whole
bacterialcells, especially the variability of the tumor response
and thehigh incidence of clinical complications including
deaths.
In several laboratories, investigators have managed to extract
fractions from bacterial cells which are immunotherapeu-
tically active and which reduce the harmful side effects
seenwith whole cells. The methanol-extracted residue of BCG
(24),
the mycobacterial RNA fraction of Millman era/. (16), and theP.
acnes cytoplasmic fraction of Millman et al. (15) seem themost
promising in this respect.
Youmans and Youmans (27-32) established several years
ago that ribosomal vaccines prepared from
Mycobacteriumtuberculosis confer protection against homologous
challenge.Since then, several investigators have demonstrated that
ribosomal vaccines prepared from a great variety of
organismsprotect animals against homologous infection (2, 4, 8, 9,
13)or homologous and heterologous infections (1, 17, 23). The
1 Supported by American Cancer Society Grants IN-103B and
IN-103C.2 To whom requests for reprints should be addressed.3 The
abbreviations used are: CMN, corynebacteria, mycobacteria,
nocardia;
BCG, Bacillus Calmette-GuerinReceived November 18. 1979;
accepted January 31, 1980.
emphasis of this work has been directed toward the treatmentof
infectious disease, and little effort has been made to
evaluatethese fractions in the treatment of tumors. Millman et al.
(14,16) did, however, extend the use of mycobacterial RNA vaccines
to the treatment of tumors.
The mechanism of the action of the ribosomal fraction
differsaccording to the ribosome source. Although the majority
ofthese vaccines require adjuvant for activity, ribosomal
vaccinesprepared from Staphylococcus aureus (26) and Neisseria
men-
ingitidis (22) do not. In addition, vaccines prepared from
M.tuberculosis (11) and Salmonella typhimurium (20) appear toinduce
a cell-mediated response, whereas those prepared from
Streptococcus pneumoniae and Streptococcus pyogenes mediate a
humoral response (21). Ribosomal vaccines thereforedeserve
investigation as immunotherapeutic agents, sincethere is a
potential for manipulating the immune response andsince such
vaccines are generally considered to be nontoxic.
This study represents an extension of the use of
ribosomalvaccines to the treatment of tumors through the use of the
asyet untried polyribosome fraction. Polyribosomes from selectedCMN
bacteria, as well as from unrelated organisms not commonly used as
tumor immunotherapeutic agents, were examined to avoid the a priori
assumption of CMN antitumor superiority.
MATERIALS AND METHODS
Mice. Five-week-old male DBA/2 mice (The Jackson Labo
ratory, Bar Harbor, Maine) were housed under standard conditions
and maintained on a Purina mouse chow diet and waterad libitum.
Tumor. Male DBA/2 mice carrying nonmetastatic SaD2 fibrosarcomas
(originating as a spontaneous tumor in DBA/2mice) were obtained
from The Jackson Laboratory. Tumorswere aseptically excised and
mechanically dissociated in balanced salt solution (0.49% NaCI,
0.75% KCI, 0.048% CaCI2,0.3% MgCI2-6H2O, and 0.17% sodium acetate
in water) usingsterile forceps. Cells were quantitated in a
Neubauer hemocy-
tometer, and viabilities were determined by the trypan
blueexclusion method. Tumor stocks were maintained by passagingat
biweekly intervals in nonimmunosuppressed mice. For passage, 2 x
106 tumor cells in 0.2 ml balanced salt solution were
injected at cutaneous flank sites.Tumor Suppression. Mice were
examined 3 times/week for
the development of tumors. Those mice that did not developtumors
were maintained for a minimum of 100 days followingtreatment.
Tumor Kinetics. A generation time for the SaD2 cells
wasapproximated by the method of Attia and Weiss (3).
Tumor Immunity. Mice successfully rejecting a primary tumor
transplant were challenged 30 days later at a contralateralsite
with 1.5 x 103 SaD2 cells. Absence of tumor development
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R. W. Urban et al.
at the challenge site indicated the development of systemictumor
immunity. A challenge control group received the samenumber of
tumor cells at the same site.
RNase Treatment. RNase (Sigma Chemical Co., St. Louis,Mo.; 0.5
mg/100 mg RNA dissolved in 10 mw Tris/HCI, pH7.6) was added to a
fresh polysome preparation and incubatedat 4°for 30 min prior to
injection.
Pronase Treatment. Crystalline pronase (Sigma; 0.05 mg/ml
polysome suspension) was added, and the mixture wasincubated at
4°for 30 min prior to injection.
Endotoxin Assay. The Limulus Amebocyte Lysate Pyrogentstandard
assay kit was from Mallinckrodt Chemical Works, St.Louis, Mo.
Cultures. Eschehchia coli B, S. pneumonÃ-as,Serratia mar-
cescens, and MycobacterÃ-um smegmatis were obtained
fromdepartmental stocks. BCG (Pasteur) and Propionibactehumacnes
were provided by the National Jewish Hospital (Denver).Stock
cultures were maintained in appropriate media and transferred at
regular intervals.
Bacterial cultures for polyribosome extraction were raised
inliquid media shaker cultures at 37°and harvested during mid-
log phase. E. coli, S. marcescens, and S. pneumoniae weregrown
as spheroplasts or protoplasts in hypertonic nutrientbroth (Difco
Laboratories, Inc., Detroit, Mich.) containing penicillin G
potassium (E. R. Squibb and Sons, Princeton, N.J.;200 units/ml). P.
acnes was grown as a stationary culture inthioglycollate broth
(Difco). Mycobacteria were grown in 7H11medium containing 4.97 g of
Middlebrook 7H9 broth, 5.0 ml ofglycerol, 1.0 g of casein
hydrolysate, and 5.0 ml of a 10%solution of Tween 80 (Sigma) per
liter.
Cell Lysis. Prior to harvesting, all cultures were
rapidlychilled to 0°in acetone-dry ice. From this point on, all
procedures were carried out at 0-4°.
E. coli. Pneumococcal and Serratia cultures were osmoti-
cally lysed by suspension in 8 to 10 ml ribosome buffer (10
rriMMgSCvSO rriM NH4CI-10 mw Tris-HCI, pH 7.6) containing
0.15% sodium deoxycholate, DNase (Sigma), 10 mg/ml, and1.0 mg
lysozyme (Sigma). P. acnes and the mycobacterialspecies were
suspended in 8 to 10 ml ribosome buffer andmechanically disrupted
by passage through a prechilled pressure cell (American Instrument
Co., Silver Spring, Md.) at10,000 psi (P. acnes) or 15,000 psi
(mycobacteria).
Polysomes. Cell lysates were allowed to clear for 15 to 20min
after which cellular debris was removed by centrifugationat 30,000
x g for 15 min in a Sorval RC-2B centrifuge fittedwith a SS-34
head. The resulting supernatant was immediatelylayered on
discontinuous gradients consisting of 3 ml of 15%sucrose and 5 ml
of 30% sucrose for pelleting. Sucrose gradients were made with
ribosome buffer as diluent. Discontinuous gradients were run at
108,000 x g for 66 min at 0-4°
with a Beckman 50 rotor in a Beckman Model L ultracentrifuge.No
protease or RNase inhibitors were utilized in this study.
Polysome dosages were based on RNA content determinedby the
Warburg nomograph (Calbiochem, Los Angeles, Calif.)following
absorbance determinations at 260 and 280 nM.
Treatment. Tumor cells (1.5 x 103) in balanced salt solution
were injected intradermally at shaved left flank (0.05 to 0.1
ml)or left footpad sites (0.02 to 0.04 ml). A given polysome
dosagewas administered in 0.05 to 0.1 ml ribosome buffer at 24,
48,or 72 hr after the injection of tumor cells. Both injections
wereadministered at the same site.
Statistics. Survival data were arranged in a 2 x 2 contingency
table, and x2 analysis was carried out using the Yates
correction factor for small sample size. Where indicated,
pol-ysome-induced tumor suppression was analyzed through theuse of
a functional, negative, exponential transformation (0 =0.01 ; t =
100 days). A detailed discussion of the method and
criteria for the application of transformations is given by
Liddell(12).
RESULTS
Tumor kinetic studies (data not presented) demonstratedthat the
time interval required to develop a palpable 1.0-mmmass (106 cells)
was directly related to the number of tumorcells injected in the
range of 102 to 5 x 105 cells. All animals
receiving injections of tumor cells within this range, at
flanksites, developed tumors and expired within 50 days.
Tumormetastasis did not occur.
Tumor volumes increased exponentially and doubled every1.1 days.
Following the injection of 1.5 x 103 SaD2 cells at
flank sites, 61 % (36 of 59 animals examined) developed a 1.0-mm
mass in 9 to 12 days. This corresponded to a doublingtime of
approximately 1.3 days. The mean tumor appearancetime for all
animals was 12 days (range, 9 to 21 days). Theslight difference in
tumor appearance in the remaining animalswas probably due to an
extended lag phase prior to the onsetof tumor growth.
Equivalent doses (in terms of nucleic acid content) of poly-
ribosomes from different bacteria were administered
intradermally at the sites of syngeneic SaD2 fibrosarcoma
transplantsin age-matched (7- to 12-week-old) male DBA/2J mice,
and
the immunotherapeutic effectiveness was evaluated in terms ofthe
time at which a palpable (approximately 1 mm diameter)tumor
appeared and in terms of long-term survival (100 days).
The polyribosomes from S. marcescens were significantly (p<
0.01 to p < 0.001 ) superior to all the others tested (Table
1).BCG, E. coli, S. pneumoniae, P. acnes, and M. smegmatiswere
consecutively less effective in delaying tumor appearance(Chart 1)
and in promoting long-term survival, given the limita
tions of the specific conditions of culture, the specific
strains,and the specific doses of each of these species.
When polyribosomes from S. marcescens were tested ingraded doses
against 3-day-old fibrosarcoma transplants, abimodal dose-response
relationship was observed. Significant
numbers of survivors were obtained only after the administration
of 5 or 50 jug Serratia polysemes at the tumor site. Althoughthe
50-(ug dose was superior to the 5-^ig dose in delaying
tumorappearance, equivalent numbers of long-term survivors
wereobserved in both groups in individual experiments. A
100-jugdose of Serratia polysomes did not delay tumor
appearancesignificantly; however, it did suppress the rate of tumor
growth(controls, 31.8 mm diameter, versus treated, 16.6 mm
diameter, at 32 days posttreatment). Other doses tested (1.25,
2.5,10, and 20 /¿g)were ineffective in delaying tumor
appearanceand in producing significant numbers of tumor-free
animals.Polysomes prepared from the other organisms examined
wereineffective at these doses.
Successful prevention of tumor appearance depended onthe tumor
load (Table 2). All mice receiving greater than 2 x103 SaD2 cells
or treatment later than 72 hr after the receipt of1.5 x 103 tumor
cells developed and succumbed to their
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Tumor Immunotherapy by S. marcescens Polysomes
Table 1Tumor-suppressive efficacy of various sources of
polyribosomes
Treatment3SourceSerratiaE.
coli"BCG*P.
acnes"M.smegmatis"S.pneumoniaeeBufferonly'No
treatment'Dosage
(/ig)1.252.55.01020501001.252.55.010205.05.05.05.0S/N"0/100/1020/304/103/108/100/100/100/104/201/200/201/200/200/101/100/1000/100%of
survivors00066.7"40308000020505001000p
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R. W. Urban et al.
doses, the observed dose response could represent stimulationof
2 different effector cell populations. This possibility is
currently being investigated in our laboratory. The results
givenhere indicate that S. marcescens polyribosomes are
effectiveagainst residual tumor loads; however, work in progress
demonstrates consistent regression of established tumors (5
mmdiameter) by Serratia polysomes, with the response being
dosedependent.
The presence of endotoxin (a potent lipopolysaccharidefound in
the cell walls of gram-negative bacteria) in our prep
arations was not unequivocally ruled out. Treatment of
Serratiapolysomes with RNase or pronase reduced the number
ofsurvivors. Diarrhea, hair ruffling, ulcération of injection
sites,and conjunctivitis have not been observed in
non-tumor-bearing or tumor-bearing mice following single
intradermal injec
tions of Serratia polysomes in doses ranging from 1.25 jug to1.0
mg. Single intradermal Serratia polysome injections (0.4 to1.0 mg)
in guinea pigs result in very mild localized inflammatoryreactions
with little or no erythema. The response is maximalat 24 hr and
absent by 48 hr in non-tumor-bearing animals.Mice, guinea pigs, and
rats respond to endotoxin with notemperature change or a
hypothermie response. Thus far, allanimals treated with Serratia
polysomes have demonstrated amoderate hyperthermic response (2-3°)
which consistently
begins in 30 min following treatment. The duration of
hyper-thermia appears to depend upon the presence or absence
oftumor and the tumor load at the time of treatment. In
addition,Limulus amebocyte lysate assays of Serratia polysome
preparations are consistently negative; however, endotoxin could
bepresent at concentrations of less than 5 ng/15 /ig of
polyribo-somal RNA. Such a low concentration of endotoxin, if
present,could not account for the observed tumor-suppressive
effect.In addition, it is well established that endotoxin is not
effectivein the treatment of residual tumor loads.
The mechanism of the immune response could not be determined
from the data presented here. Reduction in the numberof survivors
obtained, when polysomes were treated with pronase or RNase,
suggested a requirement that these aggregatesbe intact in order to
achieve an optimal response. Polysomeprofiles differed
significantly among each of the organismsexamined, and it is
conceivable that the size of a particularaggregate or the degree of
aggregation was an importantparameter in determining the level of
induction. The aggregatenature of polyribosomes was also probably
responsible for thedevelopment of an effective response in the
absence of complete or incomplete Freund's adjuvant. Such adjuvants
are
usually required for effective use of most ribosomal vaccines.By
virtue of their RNA content and large molecular size,
polyribosomes may stimulate the release and/or production
ofInterferon which then could activate resident macrophages
asnonspecific killers (18, 19). Interferon production is
typicallymaximal within 24 hr of the inducing stimulators.
The bimodal response could be explained if 2 different
cellpopulations are stimulated. Polysome-based immunotherapymay
then be more specific than other forms of immunopoten-tiation.
The predictable nature of the antitumor response, high levelsof
long-term survival, and absence of secondary complicationssuggest
that polyribosomes may offer a new and more effectiveapproach to
the experimental study of tumor immunotherapy.
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1980;40:1501-1505. Cancer Res Richard W. Urban, Bruce S. Edwards
and William Segal Polyribosomes
Serratia marcescensTumor-immunotherapeutic Efficacy of
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