[CANCER RESEARCH56. 5647-5652, December 15, 19961

ABSTRACT

Photodynamic therapy (PDT) treatment of murine EMT6 mammarysarcoma using Photofrin (10 mg/kg) and light (110 ,J/cm2)cured all theselesions growing in syngeneic BALB/c mice. In contrast, the same treatment produced initial ablation but no long-term cures of EMT6 tumorsgrowing in either scid or nude mice, the immunodeficient strains sharingthe same genetic background with BALB/c mice. No difference was detected in either the level ofPhotofrin accumulated per g oftumor tissue orthe extent of tumor cell killing during the first 24 h after PDT of EMT6tumors growing in BALB/c or scid mice. The assumption that the difference in tumor cures could be ascribed to the absence of functional lymphoid cells in sdd and nude mice was supported by the results of experimentsinvolving the adoptive T-cell transfer into acid mice or bone marrowtransfer betweenBALB/cand scid mice.The adoptive transfer of splenicvirgin T lymphocytes from BALB/C mice into acid mice performed 9 days

before PDT of EMT6 tumors growing in the recipients was successful indelaying the recurrence of treated tumors. Adoptive transfer done immediately after PDT or 7 days after PDT had no obvious benefit. Even better

improvement and a high cure rate of PDT-treated tumors was obtainedwith scM mice reconstituted with BALB/C bone marrow. In contrast, amarked drop in tumor cure rate was observed with BALB/c mice reconstituted with acid bone marrow. These results suggest that the activity ofhost lymphoid populations was essential for preventing the recurrence ofEMT6 tumors following the PDT treatment used in this study. Thecontribution of PDT-induced immune reaction may, therefore, be ofcritical importance for the cure with at least some tumors.

INTRODUCTION

One of the established strategies for the control of solid cancersinvolves the induction of inflammation within these lesions by eithernonspecific agents such as bacterial vaccines (1, 2) or specific immune agents such as cytokines interleukin 4, interleukin 7, and granulocyte colony-stimulating factor (3). Such inflammation can lead tothe induction of specific immunity against cancer cells and result inthe regression of local as well as metastasized tumors (1, 2, 4, 5). Incontrast, inflammation of normal tissues generally has no significanteffect on tumor growth in the same host. The presence of nonviablecancerous cells damaged by the inflammatory insult and an associatedinflux of inflammatory cells generates conditions for the evolution ofan inflammation-primed immune development cascade (3, 6). Theingestion of dead and damaged tumor cells by macrophages and other“professional―antigen-presenting cells may stimulate the recognitionof tumor epitopes by CD4@ T cells, leading, in turn, to the generationand expansion of tumor-specific CTLs.

Recently, it has become evident that PDT,3 a newly establishedmodality for cancer treatment, exerts a very strong local inflammatoryreaction at the treated tumor site (7, 8). This is characterized by the

Received 6/28/96; accepted 10/28/96.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 in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I Supported by Grant MT-l2l65 from the Medical Research Council of Canada.

2 To whom requests for reprints should be addressed. Phone: (604) 877-6010, ext.

3044; Fax: (604) 875-6857.3 The abbreviations used are: PDT, photodynamic therapy; scid, severe combined

immunodeficient.

release of various cytokines and other inflammatory mediators (7, 9)as well as by a massive accumulation of nonspecific effector cells(i.e., neutrophils, mast cells, and monocytes/macrophages) in PDTtreated tumors (8). Functional activation of these inflammatory cells isindicated by their elevated tumoricidal activity (8). The objective ofthe present study was to test the hypothesis that, in agreement with theeffects of other inflammatory insults on cancerous tissue, this event inPDT-treated tumors also leads to the induction of tumor-specificimmunity mediated by host lymphoid populations. The role of lymph

oid cells in the curative effect of PDT was studied using a murinetumor model, EMT6, growing in syngeneic immunocompetent orimmunodeficient mice.

MATERIALS AND METHODS

Tumor Model and Mice. The EMT6 mammarysarcoma(10) was maintamed in syngeneic BALB/cJ mice by biweekly i.m. passage (11). For exper

iments, this tumor was implanted s.c. by injecting 6—10 X l0@ EMT6 cells into

a dorsal site. In addition to BALB/c mice, the tumor was implanted into scidmice (BALB/cByJ-scid.TO) and nu/nu mice (BALB/cBy-nu). Both of these

immunodeficient mouse strains, hereafter referred to as scid and nude mice,share a common genetic background with BALB/c mice. The rate of EMT6

tumor growth was similar in all these strains. Only female mice were used inthe experiments. Their age was 7—9weeks at the time of PDT treatment, exceptfor those used in the experiments involving bone marrow transfer (as specifiedbelow).

PDT. The mice were administeredPhotofrin(10 mg/kg iv. in most cases)6 days after tumor inoculation, which was followed by light treatment(630 ±10 nm, 110 J/cm2, 130 mW/cm2) 24 h later. The largest tumor diameterat the time of treatment was 5-7 mm, and the thickness never exceeded 3.5mm. The monodirectional light beam was delivered from a tunable light source(model A5000 with a 1-kW xenon bulb, manufactured by Photon Technology

International, Inc.) using a 5-mm core diameter liquid light guide 2000A(Luminex, Munich, Germany).

The end point in most experiments was tumor cure/recurrence. Individualtreatment groups consisted of 8—12mice. The mice were observed for up to 90

days after PDT for signs of tumor regrowth; no sign of tumor at the end of thistime interval qualified as the cure. In some experiments, the mice weresacrificed at 24 h after Photofrin administration or at different times following

photodynamic light treatment. The excised tumors were either digested and

analyzed for Photofnn concentration by a fluorometric assay (12) or enzymatically dissociated into a single-cell suspension. In the latter case, the tumor cellyield was determined, and aliquots of cell suspension were plated into Petri

dishes with DMEM (Sigma Chemical Co., St. Louis, MO) supplemented with

[0% fetal bovine serum (HyClone Laboratories, Inc., Logan, UT). Tumor cellcolonies were stained and counted 7—8 days later. The clonogenicity wascalculated based on the plating efficiency and cell yield normalized to thevalues obtained with tumors from untreated animals (12). These values werenot changed in the control groups receiving Photofrmnonly.

For the fluorometric assay, the tumors were minced and completely digestedin ScintiGest (Fisher Scientific Co., Fair Lawn, NJ). Photofrin concentration in

tumor tissue lysates was determined from fluorescence intensities (405 nmexcitation, 625 emission) using standard calibration curves (12).

Adoptive Transfer of T Lymphocytes and Bone Marrow Transplantation. For the adoptive transfer studies, spleens taken from donor animals werecarefully teased apart to yield splenocyte suspensions without enzymaticdigestion. After removal of erythrocytes by lysis in ice-cold ammoniumchloride buffer (13), T lymphocytes were separated from other splenocytes by

5647

The Role of Host Lymphoid Populations in the Response of Mouse EMT6 Tumor toPhotodynamic Therapy1

Miaden Korbelik,2 Gorazd Krosl, Jana Krosl, and Graeme J. Dougherty

Cancer imaging Department(M. K., G. K.] and Terry Fox Laboratory If. K.. G. J. D.], British Columbia Cancer Agency. British Columbia Cancer Research Centre, 601 West10th Avenue, Vancouver, British Columbia, Canada V5Z ILl

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ROLE OF LYMPHOCYTES IN TUMOR RESPONSE TO PDT

a standard purification procedure using nylon wool fiber (Polysciences Inc.,

Warrington, PA). Immediately thereafter, these cells were transferred into

recipient mice by injecting 1 X l0@cells/mouse into the tail vein. Examinationby flow cytometry (described below) verified that the purity of T-cell populations eluted through nylon wool columns was >85%.

For reconstitution of the hemopoietic system by hone marrow transplantatiOn, bone marrow was collected from femurs of donor mice and injected

immediately into lethally irradiated mice (1 X l0@ cells/recipient, iv.). Lethal

whole-body ‘y-radiation(Shepherd Irradiator, cesium 137 unit) was used todestroy the bone marrow of the recipient and facilitate the reconstitution of thehemopoietic system from the transplanted bone marrow. scid mice received 4.5Gy (0.128 Gy/min), and BALB/c mice were irradiated to 7 Gy (0.94 Gy/min).

Flow Cytometry. The purityof the spleen-derivedT-cell populationsusedfor adoptive transfer as well as the lymphocyte levels in the blood of mice thatreceived T cells or bone marrow transplant were analyzed by standard flowcytometry techniques. Blood samples (0.1 ml) were taken from tail veins ofmice at different times after adoptive T-cell transfer or bone marrow recon

stitution. After lysis of erythrocytes, aliquots of WBC suspensions were

stained with monoclonal antibodies specific for mouse leukocyte antigens that

were conjugated with FITC or phycoerythrin. In addition to identifying Tlymphocytes by their common marker, CD3@,helper T cells were distinguishedby positive staining with anti-CD4, cytotoxic/suppressor T cells by stainingwith anti-CD8, and B lymphocytes by staining with anti-B220. All monoclonal

antibodies were from PharMingen (San Diego, CA). For staining (30 mm at

0°C),the antibodies were diluted in HBSS containing 1% fetal bovine serumand 0.05% sodium azide. Flow cytometry analysis was performed on a FACScan (Becton Dickinson, Mountain View, CA) or Coulter Epics Elite ESP(Coulter Electronics, Hialeah, FL).

RESULTS

Tumor Cell Killing and Photofrin Accumulation. The directkilling of EMT6 cells by PDT treatment of tumors growing in

BALB/c or scid mice was examined by excising tumors immediatelyafter the termination of photodynamic light treatment and plating cellsdissociated from these tumors for the colony formation assay (Fig.Ia). Two PDT treatments based on different Photofrin doses wereused in these experiments. As will be shown later, even the lower PDTdose was sufficient to attain a 100% cure of EMT6 tumor growing inBALB/c mice. No significant difference in cell killing was noted forthe EMT6 tumor growing in BALB/c compared to the same tumorgrowing in scid mice. Very little, if any, increase in tumor cell deathwas reached with the higher compared to the lower Photofrmn dose.

In additional experiments, this type of analysis was expanded to thedetermination of the indirect lethal effect of PDT by delaying tumorexcision for up to 24 h posttreatment (Fig. lb). The PDT dose usedresulted in the killing of 60—70% of tumor cells when assayedimmediately after light treatment. In accordance with previous studiesusing the same tumor model (14), the survival of tumor cells decreased markedly with the time elapsed between the light treatmentand tumor excision. The extent of tumor cell killing at 0, 12, and 24 hpost-PDT was not dependent on the host (BALB/c or scid mice); i.e.,there was no significant difference in the survival of cells in PDTtreated tumors growing in these two different mouse strains.

In addition, no significant difference was detected in the accumulation of Photofrin in EMT6 tumors growing in either BALB/c or scidmice. Based on the standard fluorometric assay, the Photofrin levelsdetermined 24 h after photosensitizer administration (25 mgfkg, i.v.)were 37.2 ±6.1 and 35.3 ±4.2 j.@gper g of tumor tissue (±SD) forthe tumors growing in BALB/c and scid mice, respectively.

PDT Response of Tumors Growing in BALB/C,scid, and NudeMice. The photodynamictreatmentcombininga Photofrindose of 10mg/kg and a light dose of 110 i/cm2 was curative for all EMT6 tumorsgrowing in BALB/c mice (Fig. 2). The same PDT treatment of EMT6tumors growing in scid mice produced a similar initial response, with

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—.— EMT6[BALB/C]

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Fig. 1. The survival of EMT6 tumor cells after PDT treatment of the tumor growing ineither BALB/c or scid mice. a, Photofrmn(10 or 25 mg/kg, iv.) was administered totumor-bearing mice 24 h before the light treatment at 110J/cm2.The tumors were excisedimmediately after the termination of light delivery and dissociated into suspensions ofsingle cells, which were plated for the colony formation assay. b. tumor-bearing mice weregiven Photofrin (10 mg/kg, iv.), which was followed by light exposure (55 J/cm2) 24 hlater. At indicated times after light treatment, the tumors were excised and cells plated forcolony formation. Bars, SD.

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—EMT6[BALB/c]EJ EMT6[scid]

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all the tumors becoming impalpable within 1—2days after lightdelivery. However, in scid mice, all these tumors regrew between 14and 21 days after the treatment. Even doubling the PDT dose byincreasing the light exposure to 220 i/cm2 was not sufficient toproduce any cures of the EMT6 tumor growing in scid mice, althoughthe average tumor recurrence time was delayed by approximately 5days.

The same type of comparison for the EMT6 tumor growing ineither BALB/c or nude mice produced very similar results (Fig. 3).

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Fig. 2. The response of EMT6 tumors growing in BALB/c or acidmice to PDT. Tumor-bearing mice received Photofrmn (10 mg/kg. iv.),followed 24 h later by the light treatment ofeither 110or 220 J/cm2.Themice were observed afterward for signs of tumor growth. Tumor-freemice at 90 days post-PDT were considered cured. Each treatment groupcontainedeightmice.

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Again, the PDT treatment used resulted in a complete initial ablationof tumors in both groups and cured all the EMT6 tumors growing inBALB/c mice, but none of the tumors growing in nude mice. Most ofthetumorsin thelattergrouprecurredby day10post-PDT,i.e.,somewhat earlier than those in scid mice (Fig. 2).

Modulation of Tumor Response by Adoptive Transfer of TCells. In the next series of experiments, attempts were made todetermine whether the response of EMT6 tumors growing in scidmice to PDT can be improved by restoring the T-lymphocyte populations, which are drastically reduced or nonexistent in these hosts.For this purpose, spleens were taken from nontreated BALB/c mice,and T cells were separated from other splenocytes by elution throughnylon wool columns and transferred into scid mice. Flow cytometryanalysis of blood samples taken from the recipient mice 1—3weeksafter T-ceil transfer confirmed the presence of CD4@ and CD8@ cellsat levels that were in most cases normal or near-normal for BALB/cmice. Accumulation of T cells in the spleens of these mice was alsoobvious. As expected, the number of CD4@ cells in the blood ofnontreated scid mice was found to be extremely low (0—2%),andCD8@ cells were practically nonexistent.

Adoptive T-cell transfer was performed at different times relative tothe PDT treatment of EMT6 tumors growing in the recipient mice.The PDT dose was 110 i/cm2 (10 mg/kg Photofrmn).There was noindication of an improvement in the tumor response to PDT (compared to that with scid mice that have not received T lymphocytes)when adoptive T-cell transfer was done either immediately after PDTor at 7 days post-PDT (Fig. 4a). However, the recurrence of EMT6

100.

tumors was delayed for 1 week in the scid mice that received the Tcells 9 days before PDT (i.e., 2 days before the tumor implant; Fig.4b). The average time of tumor recurrence in these mice compared tothe mice not receiving the adoptive T-cell transfer (26 and 18 days,respectively) was statistically different (P < 0.01, Student's z test).The transfer to scid mice of nonselected BALB/c splenocytes 9 daysbefore PDT resulted in a similar benefit to the tumor response. In thiscase, the data even suggest a low level of tumor cures, but statistically(log-rank test) there was no significant difference in cures between theresults obtained with the transfer ofcomplete splenocytes and selectedT cells. The transfer of splenocytes or T cells under the experimentalprotocol used in this study (2 days before the tumor implant) had noobvious effect on the growth rate of EMT6 tumors in the absence ofPDT treatment. The volumes at 7 days postimplantation for tumorsgrowing in control scid mice, T cell recipients, and recipients ofnonselected splenocytes were 63 ±11, 60 ±12, and 56 ±7 mm3(mean ±SD), respectively.

The Effect of PDT foUowing Bone Marrow Transfer. The roleof immune cell populations in the response of EMT6 tumors to PDTwas also studied by performing different combinations of bone marrow transfer. This included the transfer of bone marrow from BALB/cto scid mice and from scid to BALB/c mice, as well as the scid-to-scidtransfer in a control group of mice. Two protocols were tested for theBALB/c bone marrow transfer to scid mice. The difference betweenprotocols 1 and 2 was the age of the scid mice at the time theyreceived the bone marrow, which was 7 and 3 weeks, respectively.Flow cytometry analysis confirmed the presence of mature T and B

BALB/c

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Fig. 3. The response of EMT6 tumors growing in BALB/c or nudemice to PDT. Tumor-bearing mice received PDT treatment of 10 mg/kgPhotofrmn combined with 110 Jkm2 light, with other details as describedin Fig. 2. Each treatment group consisted of 10 mice.

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lymphocytes in the blood of scid mice 8 weeks after they received theBALB/c bone marrow. The blood levels ofCD4@, CD8@, and B220@cells in these mice were similar or even higher than the average levelsin nontreated BALB/c mice. A similar analysis performed at 6 weeksafter bone marrow transfer suggested that the reconstitution of thehemopoietic system was incomplete at that time.

The attempt to replace the hemopoiesis in BALB/c mice with thescid bone marrow-based hemopoiesis was not completely successful.Flow cytometry analysis indicated the presence of CD4@ cells (range,

21—34%)and CD8@ cells (5—8%)but only marginal levels of Blymphocytes (0—2%of B220@ cells) in the blood of these mice. Ourexperience suggests that, at least with BALB/c mice, it may not bepossible to achieve by radiation a 100% destruction of hemopoieticcells of the recipient in a successful bone marrow transplantationexperiment. These mice received whole-body -y-irradiation (7 Gy) thatwould be lethal to them if they were not rescued by the bone marrowtransfer (as confirmed with control mice treated with the radiationonly). Despite this, some irradiated bone marrow cells have obviouslysurvived and eventually reconstituted the T-lymphoid populations inthese mice.

The EMT6 tumors inoculated in mice that received a bone marrowtransplant 8 weeks earlier showed no apparent difference in the

growth rate in these hosts compared to the growth rate in control scidor BALB/c mice. When they reached the appropriate size, thesetumors were treated by PDT, again using the dose of 110 i/cm2(Photofrin 10 mg/kg). The results (Fig. 5) demonstrate the restorationof the curative effect of PDT in the scid mice that received BALB/cbone marrow with the cure rates of 63% with protocol 1 and 80% with

— no transfer

- . . . immed. post POT

—— 7dayspostPDT

Fig. 4. The effect of adoptive T-cell transfer on PDT-mediatedcontrol of EMT6 tumors growing in acid mice. The mice receivedBALB/c spleen-derived T lymphocytes (1 X l07/mouse) eitherimmediately after PDT or 7 days post PDT (a), or at 9 days beforePDT (2 days before the tumor implant; b). In the latter case, anothergroup of acid mice received the nonselected spleen cells fromBALB/c mice. The PDT light dose was I 10 i/cm2. Other detailswere as described in Fig. 2.

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protocol 2. On the other hand, the PDT-based cure rate of EMT6tumors growing in BALB/c mice that had their hemopoietic systemreconstituted with the scid bone marrow was reduced drasticallycompared to the effects seen in normal BALB/c hosts (Figs. 2 and 3).Also shown in Fig. 5 is the PDT response of EMT6 tumors growingin scid mice reconstituted with the scid bone marrow. This transplanthas not changed the immunodeficient status of the recipients, and, asexpected, the PDT treatment was not curative in these mice.

DISCUSSION

A striking difference was demonstrated between the response ofEMT6 tumor growing in immunocompetent BALB/c mice and immunodeficient scid or nude mice (Figs. 2 and 3). The PDT treatmentused cured all the tumors growing in BALB/c mice, whereas itproduced no cure of tumors growing in either scid or nude mice. It isalso shown that the photosensitizer Photofrin accumulated at similarlevels in EMT6 tumors growing in either BALB/c or scid mice andthat the extent of tumor cell killing between 0 and 24 h after PDT wasnot different with these two host strains. This suggests that thedifference in tumor cures originated in the lack of activity of lymphoidcells in severely immunocompromised scid and nude mouse strains.Nude mice, which are characterized by the congenital absence of athymus, have severely reduced numbers of mature T cells, whereasthe levels of B lymphocytes and natural killer cells are normal (15).scid mice have a defect in rejoining of double-strand DNA breaks(required in the V(D)J recombination during normal maturation of Tand B cells) due to a mutation in a gene encoding for a protein

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[presumably a phosphatidylinositol kinases (PIK)-related kinase] thatparticipates in this process (16). Consequently, scid mice have nomature T and B lymphocytes except for the occasional presence ofvery low levels of CD4@ cells that are of oligoclonal origin. It isimportant to note that the myeloid populations and their activity inscid and nude mice are normal. Thus, the PDT-induced acute inflammatory response mediated by myeloid cells appears not to be affectedin these mice.

The contribution of lymphoid populations to the cure of EMT6tumor by PDT was demonstrated further by the adoptive transfer ofTcells to the scid mice and different combinations of bone marrowtransplantation. The adoptive transfer of virgin T lymphocytes ornonselected splenocytes from BALB/c mice to scid mice that servedas the hosts of PDT-treated EMT6 tumors delayed the recurrence ofthese lesions when it was performed at 9 days before PDT. Even morecomplete restoration of the sensitivity of EMT6 tumors to PDT wasobserved with scid mice that had their hemopoietic system reconstituted with the BALB/c bone marrow. Another finding that supportsthe importance of host lymphoid populations in the response of theEMT6 tumor to PDT is the loss of the curative effect in BALB/c micereconstituted with scid bone marrow.

Interestingly, reconstituted bone marrow from scid mice introducedinto other scid mice appears to have produced a delay in tumorregrowth after PDT in a small percentage of animals. The effects ofthe conditioning regimen as well as the transplant procedure used(involving, e.g., protracted cycles of cytokine release) may haveimpacted on the functional activity of macrophages and other immunecell types present within scid mice. This is an aspect that requiresadditional investigation.

The abnormalities in lymphoid tissues and sites in adult scid micemay have hindered the orderly reconstitution of lymphoid populationsafter the T-cell or bone marrow transfer, and this would prevent acomplete “transformation―of scid mice into immune equivalents ofBALB/c mice. The thymus in scid mice is severely hypoplastic andovergrown with fibrocollagenous stroma, whereas lymph nodes arealso very small (17). In addition, histological abnormalities are discernible in the spleen, where lymphoid follicles and germinal centersare replaced by nodular structures consisting mainly of stromal cells.This consideration prompted us to include in the study the transfer ofBALB/c bone marrow into scid mice that were only 3 weeks old(protocol 2 in Fig. 5). The average cure rate for PDT-treated EMT6tumors growing in these mice 9 weeks later was superior to thatobtained when adult scid mice were used for the bone marrow transfer(protocol 1) and close to the fully curative effect seen with BALB/cmice. This supports the assumption that the restoration of the sensi

Fig. 5. The effect of bone marrow transfer on PDT-mediated controlof EMT6 tumors growing in the recipient mice. Bone marrow fromdonor mice was transferred into lethally ‘y-irradiatedrecipient mice. TheEMT6 tumors implanted in the recipients 8 weeks later were treated withPDT using the same dose as in Fig. 4. The age of the acid mice thatreceived BALB/c bone man@ow[scid(BALB/c)] was either 7 weeks(protocol 1;#1) or 3 weeks (protocol 2; #2) at the time of transfer. Alsoshown are the results with BALB/c mice reconstituted with the scid bonemarrow [BAL.B/c(scid)land for a control group of scid mice that weretransplanted acid bone marrow [scid(scid)l. Treatment groups consistedof 10—12mice. Other details were as described in Fig. 2. ‘a

I I I I

30 90

tivity to PDT of EMT6 tumors is affected by how successfully the

lymphoid sites in these animals were reconstituted.Timing was shown to be important for the optimal benefit of the

adoptive transfer of T cells into scid mice to be realized (Fig. 4). Theactivation and expansion of transferred T lymphocytes requires timeand a suitable environment for optimal homing of these cells. Anadditional factor with the timing of T-cell transfer may be the need toavoid the correlation with the immunosuppressive effect induced byPDT. Nearly complete depletion of peritoneal lymphocytes and a lossof responsiveness of splenic lymphocytes to concanavalin A andlipopolysaccharide mitogens was observed in mice after PDT treatment to the exposed musculoperitoneal layer (abdominal skin removed; Ref. 18). The systemic immunosuppression was reported topersist for several weeks after such treatment protocol with Photofrinbased PDT (19). This appears to be mediated by adoptively transferable suppressor cells that are not antigen-specific and was shown notto be reversed by adoptively transferred virgin splenic lymphoid cells(20). However, the immunosuppressive effect seems less severe afterPDT treatment protocols of s.c.-growing EMT6 tumors such as thoseused in this study,4 and even milder after cutaneous PDT exposure(19).

Additional research is required to elucidate specific roles of distinctpopulations of lymphoid cells in PDT-induced immune reaction.

Perhaps other models of immunodeficient mice (e.g., CD4 and CD8knockout mutants), in which the problem with the reconstitution oflymphoid populations encountered with scid mice could be avoided,may prove useful for this purpose.

Most likely, PDT-induced immune activation does not substantiallycontribute to the initial ablation of treated lesions but may be ofcritical importance in the eradication of remaining foci of cancerouscells under the reduced tumor burden. This, as suggested by the results

of the present study, appears to be an essential requirement forpreventing the recurrence of PDT-treated EMT6 tumors. A relevantfactor in the development of a PDT-induced immune reaction may bethe immunogenicity of treated tumors. The EMT6 tumor is stronglyimmunogenic, and this may facilitate the recognition of tumor antigens following PDT by lymphoid cells. However, current investigations in our laboratory indicate that lymphoid populations may alsoplay a role in the response to PDT of tumors that are less immunogenic (21). The induction by PDT of tumor-specific immunity againsta weakly immunogenic murine fibrosarcoma was described by Cantiet al. (22).

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4 M. Korbelik, unpublished results.

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In summary, this study demonstrates that host lymphoid populations can participate in the antitumor effect of PDT, and that thecontribution of immune mechanisms may be of critical importance forthe cure with at least some tumors. Additional investigation is warranted to define the exact nature of the underlying mechanismsresponsible for this effect and to help determine how it might beexploited to enhance the antitumor effect of PDT in the clinic. Animportant implication for preclinical research is that the response toPDT of human and other tumors (even those of poor immunogenicity)xenografted in immunodeficient mice may not adequately reflect theresponse of these lesions in original hosts.

In ongoing studies, we are continuing to exploit the difference insensitivity to PDT of tumors growing in syngeneic immunocompetentand immunodeficient mice for further characterization of PDT-induced tumor immunity (21).

ACKNOWLEDGMENTS

The photosensitizer Photofrmn(porfimer sodium) used in this study wasprovided by QLT Phototherapeutics Inc. (Vancouver, British Columbia, Canada). We appreciate the excellent technical assistance provided by SandyLynde.

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1996;56:5647-5652. Cancer Res   Mladen Korbelik, Gorazd Krosl, Jana Krosl, et al.   Mouse EMT6 Tumor to Photodynamic TherapyThe Role of Host Lymphoid Populations in the Response of

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