[CANCER RESEARCH 47, 1762-1766, April 1, 1987)

Intravesical Bacillus Calmette-GuérìnTherapy for Murine Bladder Tumors:

Initiation of the Response by Fibronectin-mediated Attachment of BacillusCalmette-Guérìn1

Timothy L. RatlifF,2 James O. Palmer, Janet A. McGarr,3 and Eric J. Brown

Washington University School of Medicine at Jewish Hospital of St. Louis, Division of Urology [T. L. RJ, Jewish Hospital of St. Louis, Department of Pathologyfj. O. P.], and Washington University School of Medicine, Departments of Medicine and Microbiology and Immunology [J. A. M., E. J. B.J, St. Louis, Missouri 63110

ABSTRACT

Intravesical Bacillus Calmette-Guérìn(BCG) is considered to be one

of the most effective treatments for superficial bladder cancer. Althoughthe mechanisms by which BCG inhibits tumor growth are not known,previous studies have shown that systemic immunization to BCG and thelocal expression of the immune response in the bladder are associatedwith a favorable response to BCG therapy. We have investigated theconditions required for the initiation of an ¡mmunological response afterthe intravesical instillation of BCG. Initial histológica! studies showedthat BCG attached to the bladder wall only in areas where the urotheliumwas damaged by electrocautery and suggested that attachment was associated with the fibrin clot. Quantitative studies verified the histológica!observations. Minimal BCG attachment (mean <102 colony forming

units) was observed in normal bladders in contrast with a mean of 1.42x IO4colony forming units/bladder in bladders damaged by electrocautery

(10 separate experiments). BCG attachment to the bladder wall wasdurable since organisms were observed in bladders 48 h after instillation.To investinate the proteins to which BCG attached, we tested the bindingof BCG to extracellular matrix and inflammatory proteins which comprisea significant portion of the fibrin clot. BCG bound in vitro to coverslipscoated in vivo with extracellular matrix proteins but did not bind tocontrol albumin-coated coverslips. BCG also bound to coverslips coated

with purified plasma fibronectin but not to coverslips coated with otherpurified extracellular matrix proteins including laminin, fibrinogen, andtype IV collagen. BCG attachment to coverslips coated with eitherextracellular matrix proteins or purified fibronectin was inhibited byantibodies specific for fibronectin. Moreover, BCG attachment to cauterized bladders in vivo was inhibited by antifibronectin antibodies. Theseresults demonstrate that fibronectin mediates the attachment of BCG tosurfaces and suggest that it is the primary component mediating attachment within the bladder. Moreover, the data suggest that the BCG-

fibronectin interaction may be a requisite first step for the initiation ofthe antitumor activity in intravesical BCG for bladder cancer.

INTRODUCTION

Treatment of superficial transitional cell carcinoma of thebladder with intravesical BCG4 alone has been shown to be aneffective treatment for superficial bladder cancer (1-5). In theclinical studies, viable BCG organisms are instilled into thebladder via a catheter once each week for a minimum of 6consecutive weeks. Such administration eliminates existing tumors and prevents subsequent tumor recurrence. Systemic sen-sitization to BCG, granulomatous inflammation, and in somecases systemic BCG infections have been observed to occur

Received 10/14/86; revised 1/5/87; accepted 1/7/87.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by USPHS Grant CA/AM37926 from the NationalCancer Institute through the National Bladder Cancer Project and by a grantfrom the Monsanto Corporation.

2To whom requests for reprints should be addressed, at Department ofSurgery, The Jewish Hospital of St. Louis, 216 South Kingshighway. St. Louis,MO 63110. Recipient of USPHS Grant CA/AM37926.

3Recipient of American Cancer Society Grant PRTF-66.4The abbreviations used are: BCG, Bacillus Calmette-Guérìn;CFU, colony

forming units; FN, fibronectin; PPD, purified protein derivative; ECM, extracellular matrix; PBS, phosphate buffered saline.

concomitant with antitumor activity (1,3,5). The mechanism(s)by which BCG mediates antitumor activity is not known.

A systemic immune response is often associated with intravesical BCG therapy. A previous report showed that 44% ofpatients treated with 6 weeks of intravesical BCG convertedfrom PPD skin test negative to positive (5). In addition, granulomatous inflammation in bladder biopsies was observed in65% of these patients (5). Other investigators also have reportedboth PPD conversion and granulomatous inflammation in thebladder after intravesical BCG therapy (1, 3). Some investigators (2, 5) have suggested that the development of either adelayed type hypersensitivity to PPD or granulomatous inflammation of the bladder correlate with a favorable antitumorresponse, although these observations remain controversial (6).

Studies in an animal bladder tumor model also demonstratedthat intravesical BCG alone inhibited bladder tumor growthand resulted in the development of delayed type hypersensitivityto PPD (7). Moreover, adoptive transfer studies in athymicmice showed that BCG inhibited bladder tumor growth only inmice receiving splenocytes containing T-lymphocytes, suggesting that T-lymphocytes were required for BCG-mediated anti-tumor activity (8). Thus, both clinical and animal studies suggest that ¡mmunological responsiveness to BCG and the localexpressions of immunity within the bladder may be requiredfor antitumor activity.

A requisite first step in the initiation of an immunologicalresponse is introduction of the antigen to the immune system.The mechanisms by which BCG gains exposure to the immunesystem after intravesical instillation are not known. Since asignificant correlation between the development of immunological responsiveness to BCG and a therapeutic response has beendemonstrated, a clear understanding of the molecular mechanising) involved in the presentation of BCG to immunologicalcells, and the subsequent induction of a local and systemicimmune response may provide a basis for enhancing therapeuticresults.

We have begun studies to determine the mechanism(s) bywhich the immunological and antitumor responses to intravesical BCG is initiated. The results reported herein suggest thatthe first step in the intravesical immunization process and thesubsequent antitumor response is FN-mediated attachment ofBCG to the bladder wall.

MATERIALS AND METHODS

Bacteria. BCG were obtained from Armand Frappier, Quebec, Canada as a lyophilized preparation containing IO7 CFU/mg. Before use,

BCG were resuspended in 0.1 M PBS, pH 7.4 to the desired concentration.

In Vivo Adherence of BCG. Intravesical BCG instillation was performed as previously described (7). Briefly, mice (OH/HEN, CharlesRiver) were anesthetized with Nembutal, i.p. (0.05 mg/g animalweight). Anesthetized mice were secured on a grounded plate and anelectrode consisting of an Amplatz curved wire guide (Cook, Inc.,

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ATTACHMENT OF BCG TO FN-COATED SURFACES

Bloomington, IN), insulated by a 24-gauge Teflon sheath (Viera, Division of Travenol Laboratory, Inc.) was inserted through the urethrainto the bladder. The electrode was attached to a Bovie electrocauteryunit. While the tip of the electrode was inside the bladder, the cauteryapparatus was activated for 4 s at the lowest coagulation setting. Aftercauterization, the wire was removed from the catheter and 10' CPU of

BCG in 0.1 ml PBS were instilled into the bladder, and the catheterwas removed. Controls included cauterized mice with instillation ofPBS and noncauterized mice with instillation of BCG. Bladders weresurgically removed 30 min after BCG instillation, washed extensivelyin PBS, and examined histologically or used to quantitate adherentBCG (see below).

BCG Quant ¡talion.Washed bladders were placed in 6 ml of collagen-äse(l mg/ml), minced with scissors, and digested in a spinner flask for40 min at 37'C. BCG CPU were quantitated as described in Section

620.44 of the Federal Register (9). The supernatant was diluted inYouman's medium in serial 1:3 dilutions, and the mixture was solidifiedwith 0.5% agar. Dilutions resulting in 10-50 colonies after 3 weeks ofincubation at 37'C were used for quantitating results.

Coating Coverslips in Vivo with ECM and Inflammatory Proteins.Coverslips (0.5 x 1.0 cm) were placed in polypropylene tubes (trimmedto approximately 8x15 mm) made porous by puncturing approximately50 times with a sharp object. The open end of the tubes was sealed withsilicone type A medical adhesive (Stortz, St. Louis, MO). Tubes containing the coverslips were implanted s.c. in anesthetized mice. Thecoverslips were harvested 3-4 weeks later (ECM-coated foreign bodyimplants), and the supernatant was cultured on blood agar plates. Onlysterile preparations were used for adherence studies.

Adherence of Purified ECM Proteins to Coverslips. Coverslips (0.5 x1.0 cm) were cleaned with 0.1 N HC1, rinsed, and incubated for l h at37'C in 1.0 ml 0.15 M NaCl (normal saline) containing 120 /ig/ml of

the respective purified protein. After incubation the coverslips werewashed with normal saline and used in adherence assays. Controlcoverslips were coated with either bovine serum albumin or humanserum albumin (Sigma Chemical Co.) in an identical manner.

Radiolabeling of Bacteria. BCG (120 mg of the manufacturer's lyoph-ilized preparation) were cultured for 7 days in 120 ml Youman'smedium at 37'C in 5% CO2. Bacteria were washed in PBS and resus-

pended in 10 ml of a 1:10 dilution of heart infusion broth (Difco) aspreviously described (10). "P ("P¡;Amersham) was added to a finalconcentration of 20 /iC'i/ml. The bacteria were incubated for l h at37°C,washed 3 times in PBS supplemented with 0.1% human serumalbumin, and resuspended to 10" CFU/ml in PBS.

Staphylococcus aureus Woods strain (ATCC 10832) were cultured inRPMI 1640. Log phase cells were washed, resuspended in 10 mlMedium 199 containing 100 /iCi ('H)thymidine, and incubated for 3 hat 37'C. Bacteria were washed and resuspended to 10" CFU/ml in PBS.

Coverslip Adherence Assay. Coverslips were submerged in 1.0 ml ofbacterial suspension and incubated for l h at 37*C. Coverslips were

removed, washed extensively in normal saline, and bound bacteria werequantitated by either staining for acid fast bacilli or liquid scintillationcounting for radiolabeled bacteria. Radioisotope assays were performedin triplicate while acid fast-staining assays were performed in duplicate.Adherent bacteria stained by the acid fast method were counted in aminimum of 10 separate fields for each coverslip, and adherence isreported as the mean number of bacteria per oil immersion field.

Extracellular Matrix Proteins. Human plasma FN was punned aspreviously described (11). Briefly, the 10% polyethylene giycol 3350precipitate from EDTA, benzamidine, and phenylmethylsulfonyl fluoride-treated plasma was resuspended in a buffer of 150 mM NaCl, 50mM KH2PO«/K2HPO4, and 10 mM EDTA, pH 7.4. This plasmafraction was then adsorbed by passage over Sepharose 4B (PharmaciaFine Chemicals, Piscataway, NJ), and the FN was purified by elutionfrom gelatin-Sepharose with 1 M arginine. Further purification wasobtained by adsorption to, and elution from, arginine-Sepharose. Allbuffers used for chromatography and elution contained 5 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, and/or 25 /¿Mp-nitro-phenyl p-amidino benzoate to inhibit residual serine proteases. Thepurified FN showed a single line on immunoelectrophoresis versus anti-whole human serum, and a single major band at M, 440,000 on sodium

dodecyl sulfate-polyacrylamide gel electrophoresis. Upon the reductionof disulfide bonds, sodium dodecyl sulfate-polyacrylamide gel electro

phoresis revealed a closely spaced doublet, as has been reported previously for human plasma FN (12). Antibodies raised against this FNpreparation in rabbits and goats gave a monospecific response onimmunoelectrophoresis and Ouchterlony double diffusion againstwhole human plasma.

Type IV collagen and laminin were purified as previously described(13) and were the kind gift of Dr. Hynda Kleinman (NIH). Fibrinogenwas purchased from Sigma Chemical Co. (Catalog No. F4753).

Inhibition of BCG Binding to Cauterized Bladders by Anti-FN. Micewere anesthetized, catheterized, and the bladder was cauterized asdescribed above. Goat anti-FN antibody and normal goat serum wereinstilled into respective bladders for 30 min. Serum was removed andBCG was instilled and quantitated as described above. Three mice wereincluded in each experimental group. The results were reproducible in2 separate experiments, and are reponed as a single representativeexperiment.

Table 1 Effect of bladder urolhelial damage on BCG adherence

Experiment no.

1234S6 7 89 10

Catheter onlyCautery

0.18°

9.407.0

01.7

04.4

00.21

00.42

0.6731

082

042

00.42

" Bacterial attachment is reported as the number of CPU x 10'.

6.0

5.0

4.0

30

2.0

1.0

0.5 24

Hours Post-BCG48

Instillation

Fig. 1. Time-course analysis of the presence of BCG in cauterized bladders.Each lime point included 3 mice and the data were reproducible in 2 separateexperiments.

CoverslipCoating

BSA°

ECM6

ECM

ECM

Coversliptreatment beforeBCG addition

NONE

NONEC

TRYPSIN

ANTIFIBRONECTINANTIBODY

K) 20 30

BCG/OIL IMMERSION FIELD

Fig. 2. Effect of trypsin and anti-FN antibody on the attachment of BCG tocoverslips coated in vivo with extracellular matrix proteins. * Coverslips coatedwith bovine serum albumin (OSA) as described in "Materials and Methods."*Coverslips implanted s.c. and processed as described in "Materials and Methods." ' In this experiment coverslip pretreatment with preimmune goat serum

was not used as a control. In numerous other experiments preimmune goat serumhad no effect on BCG attachment while antifibronectin antibodies inhibitedattachment (see Figs. 4 and 6).

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ATTACHMENT OF BCG TO FN-COATED SURFACES

FISRONECTIN(ug/ml)

0°

30

o°o°o°ooooo°oc>oooooooooool—t

100 ZOO

BCG/OIL IMMERSION FIELD

Fig. 3. Effect of coating coverslips with varying concentrations of purified FNon BCG binding. The results were reproducible in 3 experiments. °Controlcoverslip coated with bovine serum albumin as described in "Materials andMethods."

CoverslipCoatingBSA"

FNbCoverslip

treatment beforeBCGadditionNONE

CONTROL0SERUMÌ9:

FN

FN

FN

ANTIFIBRONECTINANTIBODY (1:100)

ANTIFIBRONECTINANTIBODY (1:500)

ANTIFIBRONECTINANTIBODY(I:25OO) üoJ°g°iliiiiili°ili°l

100 200 300

BCG/OIL IMMERSION FIELD

Fig. 4. Effect of anti-FN antibody on the binding of BCG to coverslips coatedwith purified FN. 'Coverslips coated with bovine serum albumin (BSA) asdescribed in "Materials and Methods." 'Coverslips incubated with 120 fig/mlpurified FN overnight. ' Preimmune goat serum.

s-o30O»0»oCL

20KOl

"o.oBCG.

T«-iilIIS.

AUREUSTp<00051-p<005p<O25

^,p<005N!-I^—i-ia

o30

Õuo20

è1IIO

*oX0.

O

Fig. S. Binding capacity of BCG and S. aureus to individual purified extracellular matrix proteins. Statistical analysis by Student's t test. /' values indicatesignificance of binding compared with human serum albumin (//.S.-l) controls.

RESULTS

In Vivo Adherence of BCG. Histological studies were performed to evaluate BCG retention and attachment after intravesical instillation. The results showed that acid fast bacilliwere present only in areas where the urothelium was damagedby electrocautery (data not shown).

Quantitative studies were performed to confirm the subjective histological data. Table 1 shows the effects of urothe-

10CMQ

8.0X&E

6.0o25

«(Ao

4.0^

oo•

2.0

u.0-JL1.

.CauteryCautery + Cautery +

Normal Goat Anti-FNSerum Antibody

Fig. 6. Effect of anti-FN antibody on the in vivo attachment of BCG tocauterized mouse bladders.

Hal damage on the intravesical attachment of BCG. Electrocau-tery consistently resulted in elevated BCG colony counts. BCGwas detected in only 2 of 10 experiments in which the bladderurothelium lacked intentional damage and no adherent BCGwere detected in the other 8 experiments. In all experimentscolony counts for bladders damaged by electrocautery exceedednoncauterized bladders. Statistical evaluation of the datashowed that the CPU of BCG observed in cauterized bladderswas significantly (P< 0.001; Student's t test) greater than those

observed in noncauterized bladders. At 95% confidence limits,8-132 times more BCG bound to cauterized than to noncauterized bladders.

Because the in vivo adherence assays were performed 30 minafter BCG instillation, the stability of the attachment of BCGto the bladder was not known. Further studies were performedto determine whether BCG attachment was transient or longlasting. Bladders were removed and CPU were quantitated atvarious times after instillation (Fig. 1). The results show thatBCG were detectable in bladders through 48 h, demonstratingthat the attachment of BCG to the bladder was durable.

Attachment of BCG to Coverslips Coated in Vivo with ECMand Inflammatory Proteins. Since the histological data suggestedthat BCG attachment was associated with the fibrin clot, weinitiated in vitro studies to evaluate the mechanisms of BCGbinding. Poreign body implants (coverslips placed in porouspolypropylene tubes and implanted s.c. for 3 weeks) which areknown to be coated with ECM proteins were tested for theirability to bind BCG as described for Staphylococcus aureus(l4).Coverslips coated with bovine serum albumin were used ascontrols. The data show that BCG attached to ECM-coatedforeign body implants but not bovine serum albumin-coatedcoverslips (Fig. 2). Pretreatment of ECM-coated foreign bodyimplants with trypsin (500 ¿ig/mlfor l h at 37°C)eliminated

BCG binding (Fig. 2). Moreover, pretreatment of ECM-coatedforeign body implants with polyclonal antifibronectin antibodies also abrogated BCG binding (Fig. 2). These data suggestthat fibronectin is necessary for BCG attachment to ECM-coated foreign body implants and that it could be a mediator ofthe in vivo attachment of BCG to the bladder.

In Vitro BCG Attachment to Coverslips Coated with PurifiedECM Proteins. Fig. 3 shows that BCG attached to surfacescoated with purified FN in a dose-dependent manner demonstrating that FN alone is sufficient for mediating BCG adherence. As expected, FN-mediated attachment of BCG was inhib-

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ATTACHMENT OF BCG TO FN-COATED SURFACES

ited by anti-FN antibody in a concentration-dependent manner

(Fig. 4).The ability of BCG to attach to other purified ECM proteins

including laminiti, type IV collagen, and flbrinogen also wastested (Fig. 5). BCG bound only to FN in contrast to S. aureuswhich bound to all ECM tested, as has been previously reported(15, 16).

Effect of Anti-FN Antibodies on BCG Attachment to Cauterized Bladders. The in vitro binding studies to the respectivepurified ECM proteins suggested that FN was both necessaryand sufficient for BCG attachment to the coverslips coated invivo with ECM and inflammatory proteins, and further suggested that FN might mediate the in vivo attachment of BCGto the bladder wall. To test the relevance of these observationsfor the binding of BCG to cauterized bladders, the effects ofanti-FN antibodies on the in vivo attachment of BCG wasstudied. The results show that pretreatment of cauterized bladders with anti-FN antibodies inhibited the in vivo attachmentof BCG but preimmune goat IgG did not (Fig. 6). Takentogether these in vivo and in vitro studies suggest that FNmediates binding of BCG to the bladder wall.

DISCUSSION

Fibronectins are a family of glycoproteins found in solubleform in plasma and other body fluids and in an insoluble formin connective tissue and basement membranes (17). Plasmafibronectin has a molecular weight of approximately 440,000and is composed of two homologous but non-identical disul fide-linked polypeptide chains (17). Fibronectins are major participants in adhesion of both eukaryotic and prokaryotic cells toother cells and substrates, and in the regulation of cell locomotion and morphology (17).

The role of FN in mediating attachment of BCG to thebladder wall is supported by several studies reported herein. Invitro studies demonstrated that FN was important in the attachment of BCG to coverslips coated in vivo with ECM proteins since the attachment was inhibited by anti-FN antibodies.Further studies showed that FN alone was sufficient for theattachment of BCG but other ECM proteins were not. Finally,anti-FN antibodies inhibited the in vivo attachment of BCG tocauterized bladders by >95%, suggesting that FN is the primarymediator of BCG attachment to the bladder wall.

It is well established that FN binds to collagen and fibrin andis a major constituent of healing wounds (18). Pode et al. (19)have analyzed the distribution of FN in normal human bladdermucosa. FN was absent from the apical surface of epithelialcells but was observed at the basement membrane and in thesubmucosa. The FN distribution suggests that FN-mediatedbinding of BCG can occur only in the presence of a disruptedurothelium. This hypothesis is supported by our in vivo bindingstudies in which BCG attached primarily to damaged urothelialsurfaces. Either extracellular matrix FN or plasma FN incorporated into the fibrin clot could contribute to the binding ofBCG to the damaged urothelium. The relative binding roles ofFN from these two sources remain to be determined.

The attachment of BCG to the bladder wall via FN may beimportant to the antitumor activity of BCG for several reasons.It may function in a passive manner as an attachment matrixthat mediates retention of BCG, and thus allows induction ofan immune response to the organism as with other foreigninvaders. The expression of this anti-BCG immune responsewithin the bladder may then eliminate the tumors. FN alsocould play a more active role in the induction of the immune

response and expression of antitumor activity. Since FN maybe opsonic (20), it may direct BCG to macrophages for processing and presentation to the immune system. Furthermore,by entering the macrophages complexed to BCG, FN maybecome a target of the immune response as an "altered self"

antigen. Since FN is important for tumor cell attachment withinthe bladder,5 an immunologie-ai response to FN could result in

the elimination of the tumor cells.This is the first report of a mycobacterial-FN interaction.

Other bacteria including staphylococci and streptococci havebeen shown to bind to FN while Gram-negative bacteria suchas Escherichia coli do not (21-26). The molecular interactionsof S. aureus and streptococci with FN have been investigated.While S. aureus binds FN via a cell surface protein, streptococciapparently interact with FN via exposed glycolipids (21-26).Although the nature of the binding molecule on BCG has notbeen determined, preliminary experiments in our laboratoryshow that the binding of BCG to FN-coated surfaces is inhibitedby pretreatment of BCG with either protease or detergent.5 Themolecular details of the FN-binding molecule on BCG arecurrently under investigation.

The FN-BCG interaction may have implications beyond itsapparent effects on immunotherapy for bladder cancer. SinceFN can bind to phagocytes as well as BCG and has been shownto enhance phagocytosis (27), it may modify ingestion of BCGby macrophages by either acting as an opsonin or by directeffects on the macrophage. Such modulation of the BCG-macrophage interaction could alter BCG uptake, intracellularreplication, and/or antigen presentation. Thus, the interactionmay play a role in the host's defense against the intracellular

organisms which in itself may be a subject of potential clinicalrelevance.

In summary, our studies demonstrate that FN mediates attachment of BCG to surfaces in vitro and suggest an importanteffector role in BCG attachment to epithelial surfaces in vivo.The data suggest that FN-mediated attachment of BCG to thebladder wall is a requisite first step in the initiation of theantitumor response. Furthermore, this observation may haveimportant implications for mycobacterial colonization and invasion in general.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Hynda Kieinman for the kindgift of laminin and collagen type IV, and Dr. John Rüsselfor his helpfuldiscussions of this work. We also thank Julie Duello Ritchey for hertechnical assistance and Regina Wigger for her secretarial assistance.

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1987;47:1762-1766. Cancer Res   Timothy L. Ratliff, James O. Palmer, Janet A. McGarr, et al.  

Bacillus Calmette-GuérinFibronectin-mediated Attachment of Bladder Tumors: Initiation of the Response by

Therapy for MurineBacillus Calmette-GuérinIntravesical

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