Radiation-Induced Effects on Murine Kidney Tumor Cells: Role in the Interaction of Local Irradiation and Immunotherapy

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THEJOURXAI. OF U R O L O t i Y Copyright 0 1995 by AVERICAN UKOLOUICAL. ASSOCIATION, INC.

Vol. 153, 2029-2033, June 1995 Prinfed in U.S.A.

RADIATION-INDUCED EFFECTS ON MURINE KIDNEY TUMOR CELLS: ROLE IN THE INTERACTION OF LOCAL IRRADIATION AND IMMUNOTHERAPY ELIA YOUNES, GABRIEL P. HAAS", BALAZS DEZSO, ESA ALI, RICHARD L. MAUGHAN, EMILY MONTECILLO, J. EDSON PONTES AND GILDA G. HILLMAN From the Departments of Urology and Radiation Oncology, Wayne State University School of Medicine and Harper Hospital, Detroit, Michigan and the Department of Pathology, Uniuersity of Debrecen Medical School, Debrecen, Hungary

ABSTRACT

Local tumor irradiation enhances the effect of interleukin-2 (IL-2) therapy in the Renca murine renal adenocarcinoma model. To investigate the mechanism(s1 of this interaction, we studied the in vitro a n d i n vivo effects of irradiation on the tumor cells. Tumor cells from in situ irradiated renal tumors had diminished proliferation in vitro. A similar growth inhibition w a s noted following injection of irradiated Renca cells into naive mice, but this effect could be overcome by injecting more cells. Histologic evaluation of tumors derived from irradiated cells revealed a decrease in mitosis a n d an increase in multinucleated giant cells, apoptosis and micronecrosis. The presence of irradiated tumor reduced the growth of nonirradiated tumor cells when both were injected into separate flanks of the same animal, suggesting that irradiated tumor cells m a y trigger a systemic antitumor response. Interleukin-2 therapy given after injection of irradiated t u m o r cells caused a significant increase in leukocytic infiltrates and micronecrosis. O u r findings indicate that radiation directly affects tumor growth and induces a systemic mechanism which could be enhanced by IL-2. KEYWORDS:carcinoma, renal cell; immunotherapy;radiation

High dose interleukin-2 (IL-2) is approved for the treatment of metastatic renal cell carcinoma (RCC). Although durable objective responses, including a few complete responses, have been observed, toxicity has remained the major limiting factor to the widespread applicability of immunotherapy.1~2Current research is directed toward improving results and diminishing toxicity.3 The multimodal treatment of experimental animal tumors with radiation therapy and immunotherapy has been promising.*-G We have reported the synergistic effect of local irradiation of a primary kidney tumor and systemic immunotherapy with IL-2.7.8 This treatment combination induced a significant reduction in the size of the primary tumor and eliminated spontaneous pulmonary metastases. Further studies demonstrated that local irradiation of left lung metastases followed by IL-2 inhibited metastases in both lungs.8.9A recent Phase I clinical trial evaluating local tumor irradiation plus immunotherapy was well tolerated, and objective responses were noted.1° Despite the potential effect of combined radiation and immunotherapy in the preclinical and clinical setting, the mechanism of the interaction is unknown. The data suggest that local tumor irradiation enhances the local and systemic effect of immunotherapy. Radiation may alter the tumor's characteristics or its environment, which could enable the immune system to eliminate the malignancy. In an effort to investigate the potential mechanism(s) responsible for the synergy of radiation therapy and immunotherapy, the current study addresses the effects of in vivo irradiation of Renca tumors on the ability of the tumor cells to divide, induce tumors and affect the growth of nonirradiated tumor in naive animals.

MATERIALS AND METHODS

Animals. Four-to-six week old female Balb/c mice were purchased from Harlan Sprague-Dawley, Inc. (Indianapolis, Indiana). Mice were housed 6 per cage in a pathogen-free animal facility and received free access to standard mouse chow and tap water. Tumor implantation. A poorly differentiated renal adenocarcinoma line, Renca,ll was provided by Dr.Robert Wiltrout, National Cancer Institute, Frederick, Maryland. Renca was cultured in medium (CM) consisting of RPMI 1640 (Gibco Laboratories, Grand Island, New York) supplemented with 10% heat-inactivated newborn serum (NBS, Gibco), 2 mM. glutamine, 100 U/ml. Penicillin G, 100 pg./ml. streptomycin, 0.5 pg./ml. fungizone, 50 pg./ml. gentamicin (Gibco), 1 mM. sodium pyruvate (Sigma Chemical Co., St. Louis, Missouri), 0.1 mM. nonessential amino acids (Gibco) and 10 mM. hepes buffer. Confluent cultures were suspended with versene, washed and resuspended in Hank's balanced salt solution (HBSS) for implantation. For intrarenal injections, mice were anesthetized by intraperitoneal injection of 0.25 cc of lOmg./ml. ketamine (Ketaset, Aveco, Fort Dodge, Iowa). The right kidney was exposed through a right flank incision and injected subcapsularly with lo5Renca cells in 0.05 ml. HBSS using a 30-gauge needle.7 Irradiation. Twenty days after intrarenal Renca injection, local tumor irradiation (LTX) was administered at a dose of 300 cGy to the tumor-bearing kidney in one group of mice whereas a second group of mice received sham radiation (nonLTX). The conditions for irradiation have been previously described.7 Briefly, unanesthetized mice were placed in lucite and acrylic jigs and shielded by 6.4 mm. lead sheets with appropriate openings for LTX. Exposure of the tumorbearing kidney was verified radiographically. Irradiation Accepted for publication December 9, 1994. * Requests for reprints: Department of Urology, Wayne State Un'- was performed with a Siemens Stabilipan x-ray set (Siemens versity School of Medicine, Suite 1017, Harper Professional Buildmg, Medical Systems, Inc., Avon, Connecticut) operated at 4160 John R St., Detroit, Michigan 48201. This study was partially supported by the Elsa U. Pardee Foun- 250kV, 15mA with 1mm. copper filtration at a distance of 47.5 cm. from the target. Dosimetry was carefully monitored, dation and the Soros Foundation's International Program. 2029

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RADIATION EFFECTS ON MURINE KIDNEY TUMOR CELLS

and precautions were taken to minimize backscattering of accompanied by inflammatory cell infiltration)l2,13 was estimated and scored from 0 to 3 + . radiation.? Statistical analysis. Statistical analysis was performed usProcessing of irradiated kidney tumors. Irradiated (LTX) and nonirradiated (nonLTX) renal tumors were resected in ing the one way Anova test for data presented in table 1and sterile conditions either 1hour or 1day after irradiation in the two-tailed t test for independent samples for data preorder to discriminate between immediate and delayed in vivo sented in figure 1 and table 2. effects. Single cell suspensions were prepared using mechanRESULTS ical disaggregation and digestion with 0.4 mg./ml. collagenase type IV (Sigma Chemical Co.) in RPMI 1640 medium Proliferation of irradiated cells. Tumors established in the supplemented with 100 U/ml. Penicillin G, 100 pglml. strep- right kidney were irradiated when they reached a diameter tomycin, 50 pg,/ml. gentamicin and 1OmM. hepes buffer. The of 0.5 to 1 cm. Proliferation of tumor cells obtained 1 hour mixture was filtered through a wire mesh and separated by after irradiation (LTX) was compared with that of cells obFicoll-Hypaque (Histopaque, Sigma) density gradient centrif- tained from nonirradiated tumors (nonLTX) (fig. 1).On day ugation. Tumor cells obtained from the interface were 0, even though the [3H] thymidine uptake was low, a reducwashed twice in HBSS, and viability was determined with tion of 40 to 60% was noted in all LTX cell dilutions compared the exclusion dye eosine. Renca cells isolated 1hour or 1 day with nonLTX cells, indicating a n immediate and direct effect after irradiation were tested for their ability to proliferate in of the radiation on cell division (p <0.05). When incubated for vitro and to induce tumors. 3 days in vitro, a cell growth inhibition of 50 to 63% was still Proliferation assay i n vitro. Cells obtained from irradiated observed at all dilutions (p <0.05). However, following 6 and nonirradiated tumors were plated in 96-well flat-bot- days' incubation, the proliferation of cells in wells containing tomed microplates a t 5000 cells per well and serially diluted 2500 to 5000 cells was comparable to that of nonLTX cells, in 0.2 ml. CM in quadruplicates. Cells were incubated for 1 and a n inhibition of 38 to 63% (p <0.05) was observed only at hour, 3 days, or 6 days at 37C in a 5% CO, incubator, then lower cell concentrations (<1250 cells per well). Thus, the labeled with lpCi [3H] thymidine (Amersham Corp., Arling- long-term assay showed that the radiation effect was overton Heights, Illinois) for 18 hours. Plates were washed and come when high numbers of cells were plated, suggesting incubated with versene t o remove adherent Renca cells, that irradiation affected only a fraction of cells. These data which were subsequently harvested onto a glass fiber filter were reproduced in independent experiments. The LTX cells with a Micromate 196 harvester (Packard Instrument Com- isolated 24 hours after in vivo irradiation proliferated simipany, Meriden, Connecticut). Thymidine incorporation was larly to LTX cells obtained 1hour after irradiation (data not assessed using the p direct Matrix 96 counter (Packard In- shown), suggesting that direct growth inhibitory effect ocstrument). curred immediately after irradiation. Tumorigenicity of irradiated cells. To assess the extent to Tumorigenicity i n pulmonary model. To induce pulmonary metastases, viable Renca cells, from LTX tumors and non- which local tumor irradiation interferes with subsequent tuLTX tumors obtained 1hour or 1day after irradiation, were morigenic potential, pulmonary metastases were induced by injected intravenously into the tail vein at lo5 or 3 X lo5 cells tumor cells obtained 1hour after in vivo irradiation: lo5 LTX per mouse in 0.5cc HBSS using a 30-gauge needle.s Mice cells per mouse induced a lower number of lung metastases were sacrificed on day 25 following Renca injection. Metas- (55% reduction, p <0.05) than cells from nonirradiated tutases developed in the lungs with no evidence of tumor in mors (table 1). However, injection of 3 x lo5 cells from irradiated tumors induced the same number of lung metasother organs.9 The lungs were insufflated with a 15% India ink suspen- tases as lo5 cells from nonirradiated tumors. The same phesion and bleached with Fekete's solution to enumerate the nomenon was observed with cells isolated from renal tumors 24 hours after in vivo irradiation. A greater reduction in lung meta~tases.~.g Tumorigenicity i n subcutaneous model. One day after in metastases (72%, p <0.05) was noted when compared with vivo radiation, lo5 viable LTX or nonLTX Renca cells from data generated from cells obtained 1 hour after irradiation. renal tumors were injected subcutaneously into both flanks This observation may indicate that further cell damage ocof the mice. Cells from nonLTX tumors (group 1) or LTX curs in vivo after irradiation. Effectof irradiated tumor cells on t h g r o w t h of nonirradiated tumors (group 2) were injected into both flanks. In the third group, cells from nonLTX and LTX tumors were injected into tumor. TO assess whether Renca cells obtained from irradithe right and left flanks, respectively, of the same animal. ated renal tumors may reduce the growth of cells from nonTwelve mice were used per group. Half of the mice in each irradiated tumors by an in vivo immune mechanism, mice group received IL-2 therapy 1 day after cell injection (5000 were injected subcutaneously with LTX cells in one flank and Cetus units in 0.5ml. 5% dextrose solution administered in- with nonLTX cells in the other. As controls, nonLTX or LTX traperitoneally twice a day for 5 consecutive days). (1 Cetus unit ICU] is equivalent to 6 international units [IUl). Recombinant human IL-2 (specific activity of 3 x lo6 CU per mg.) TABLE1. Ability of Renca cells obtained from i n vivo irradiated was provided by Chiron Corporation, Emeryville, California. tumors to induce lung metastases Mice were sacrificed on day 23 after Renca injection, and the Time post radiation # Cells Renca cells Pu'monary metastases tumors were measured. mean ? SE reduction ( % I Histology. Serial sections (5pm.) from paraffin-embedded 1 hr 10" NONLTX 150 i17 tumor samples were stained with hematoxylin-eosin (H and LTX 68 i5 55 E ) and Giemsa to evaluate tumor growth pattern, morphol3*105 NONLTX 200 LTX 200 ogy, extent of tumor-infiltrating leukocytes and mitotic rates. 24 hr 105 NONLTX 118'12 The infiltration of the tumor by lymphocytes, macrophages LTX 33 i10 72 and granulocytes was determined based on the characteristic 3 X103 NONLTX 200 morphology of these cells. The average number of cells in LTX 200 mitosis per field was calculated after enumeration of cells Renca cells obtained from irradiated (LTX) and noninadiated (nonLTX) in mitosis in 10 randomly selected high power 1x40) mi- tumors 1 hour and 24 hours after in vivo irradiation were injected intravecroscopic fields. Apoptosis (characterized by sequestrated nously at lo5 or 3 X 10' cells/mouse. Mice were sacrificed on day 25 after Renca cell injection. and pulmonary metastases were enumerated. When the single cells showing chromatin condensations and/or frag- number of lung metastases was higher than 200 a n arbitrary value of 200 was mentations, cytoplasmic shrinkage and shape changes not assigned.

RADIATION EFFECTS ON MURINE KIDNEY TUMOR CELLS

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These tumors had a decreased mitotic rate and invasiveness. The tumors were characterized by an increase in multinucle30000 ated giant cells, apoptotic cells (score 3+) and micronecrosis (table 2, fig. 2,B). To determine whether the decrease in tumor size observed following LTX cell injection correlates cs with the decreased proliferation described earlier (fig. 1,taE, ble l), a group of mice was injected subcutaneously with 0 %fold the number of LTX cells in a separate experiment. c 20000 l d Mice injected with 3 X lo5 cells developed large tumors of 0.42 g. 5 0.08, comparable in size to tumors produced by lo6 u nonLTX cells (0.47 g. 0.14) whereas mice injected with a only lo5 LTX cells had smaller tumors of 0.19 g. -C 0.10. .-c E These data confirm the correlation between the tumor 10000 burden and the ability of the cells to divide. s LTX tumors in mice bearing LTX and nonLTX tumors in Iopposite flanks were comparable in size and frequency to P LTX tumors from group 2 and showed frequent micronecrosis with multifocal scattered lymphocytic infiltration (table 2). 0 NonLTX tumors in this group were reduced by 89%compared 0 1000 2000 3000 4000 5000 with nonLTX cells from mice injected in both flanks (p = 0.001) and showed a marked decrease in the number of cells Cell Number/Well in mitosis (table 2). In fact, in specimens obtained from 6 FIG. 1. Proliferation assay of Renca cells from in vivo irradiated nonLTX tumors in this group, only 2 contained viable cancer tumors. Tumors were resected from kidney 1hour aRer local irradi- whereas the others consisted of connective tissues and reacation of tumor-bearing kidney. Single cell suspensions obtained from irradiated tumors (LTX) and nonirradiated tumors (nonLTX) were tive lymph nodes free of tumor (table 2). Interleukin-2 reduced the size of nonLTX tumors in group tested for proliferation after 1hour (DO), 3 days (D3) and 6 days (D6) incubation in vitro. Data are reported as mean cpm of quadruplicate 4 by 68% compared with group 1 (p = 0.006, table 2). Alexperiments, and error bars represent standard deviation. though IL-2 neither affected the mitotic rate nor caused an increase in apoptotic cells, it increased leukocyte infiltration in both nonLTX and LTX tumors (table 2). In group 6 with cells were injected into both flanks. Subcutaneous tumors nonLTX and LTX cells growing in separate flanks and were resected on day 23, weighed and processed for histopa- treated with IL-2 therapy, variations were noted in the rethology. Large bilateral tumors developed in mice injected in sponse of individual mice to the treatment. Nevertheless a both flanks with lo5 viable cells from nonLTX tumors (group l),(table 2). Sections stained with H and E revealed solid trend showing a decrease in the frequency of tumor growth tumors of pleiomorphic cells with granular cytoplasm and was noted. In this group, the tumors on both sides were only rare giant cells or apoptotic cells (score 1+) (fig. 2 4 ) . significantly smaller than nonLTX cells from group 1 (p < Although surrounded by a pseudocapsule, the tumors invaded 0.05). However, by using the Duncan test for multiple comthe surrounding subcutaneous tissues. Leukocytic infiltra- parisons, no statistical difference was found when comparing tion was not noted in tumor areas free of necrosis (fig. 2,A). the size of the nonLTX + IL-2 tumors (group 6, R) to that of The frequency of mitosis was estimated at 10 cells per field nonLTX tumors (group 3, R) or LTX + IL-2 tumors (group 6, (40X) (table 2). In mice injected in both flanks with cells from L) and LTX tumors (group 3, L). The tumors treated with IL-2 had increased multifocal LTX tumors (group 21, the mean tumor weight was reduced by 65% compared with nonLTX tumors (p = 0.008, table 2). cellular infiltrates of activated (blast-like) lymphocytes, mac-

E

*

5

e.

TABLE2. Irradiated tumor cells affect the growth of nonirradiated tumor cells Group

Treatment

Tumor frequency

nonLTX (R)

616

nonLTX (L)

616

LTX (R)

4/6

1

2

Tumor weightfflank mean (gr) f SE

# cells in

mitosis

0.37 t 0.07

10

0.12 f 0.04

4

LTX (L)

516

nonLTX (R)

2/6

0.03 t 0.02

5

LTX (L)

4/6

0.16

0.08

4

nonLTX (R) +IL-2 nonLTX (L)

4/5 0.11 t 0.04

11

LTX (R)

316

0.16 2 0.06

5

3

4

5

6

+IL2

?

Morphology Pleiomorphic cells with granular cytoplasm, few multinucleated cells, few apoptotic cells and lymphocytes. Increase in multinucleated giant cells, apoptotic cells and micronecrosis. Considerable micronecrosis, multinucleated giant cells and apoptotic cells. Moderate increase in immune cell infiltration in tumors.

2/5

LTX (LI

316

nonLTX ( R ) CIL-2 LTX (L)

316

0.10 f 0.05

6

316

0.08 2 0.04

5

Increase in number and foci of immune cell infiltration in tumors.

Multifocal cellular infiltrates inside tumors, multifocal necrosis. Increase in number and foci of cellular infiltrates associated with micronecrosis.

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RADIATION EFFECTS ON MURINE KIDNEY TUMOR CELLS

FIG.2. Histology of subcutaneous LTX and nonLTX Renca tumors. Hematoxylin and eosin staining of tumor sections from experiment presented in table 2. A, nonLTX tumor (group 1) exhibits pleiomorphic cells with granular cytoplasm and only rare apoptotic cells (arrows) and small lymphocytes (arrowheads).B , LTX tumor (group 2) with multinucleated giant cells (asterisk), apoptotic cells (arrows) and a few small lymphocytes (arrowheads). C , nonLTX tumor following IL-2 therapy (group 6) infiltrated by mononuclear cells, mostly large lymphocytes (arrows).D,LTX tumor following IL-2 therapy (group 6) with a massive infiltration of lymphocytes (arrows) and mononuclear cells surrounding tumor cells (asterisks). (All 425x)

rophages and eosinophils associated with increased micronecrosis, especially in LTX tumors (table 2, fig. 2C,D). DISCUSSION

Early studies of adoptive immunotherapy required pretreatment of the host with either chemotherapy or radiation therapy.14 The precise action of radiation was unknown, but was a n important prerequisite for successful therapy. As chemotherapy became more practical, radiation was abandoned without clarifying the mechanism for the beneficial interaction. I t has been postulated that pretreatment may 1) simply reduce tumor burden or inhibit tumor growth, 2) eliminate suppressor cell populations, 3) alter the tumor environment to make it more accessible to immune surveillance, or 4) increase the antigenicity of the tumor. Cameron e t al.4 demonstrated a synergistic relationship between the sequential application of radiation and immunotherapy, which was subsequently evaluated in the clinical setting.10 Our own studies confirmed that radiation greatly enhances the beneficial effects of immunotherapys and that irradiation of only a portion of the tumor burden will result in equivalent responses both within and outside the field of radiation.” These data suggested that radiation induces a systemic effect. This study was designed to investigate the mechanism of the interaction of radiation and IL-2 therapy. Damage to DNA produced by radiation results in growth rate changes

and the induction of a variety of genes associated with growth control.15 Cells isolated from irradiated Renca tumors had reduced proliferation compared with cells isolated from nonirradiated tumors. The inhibitory effect of radiation was immediate as monitored at 1hour after in vivo irradiation. I n long-term proliferative assays (6 days), the inhibitory effect could be overcome at a high cell concentration, suggesting that irradiation affected only a fraction of the cells. I n vitro irradiation of Renca cells also causes a dose-dependent direct growth inhibitory effect (Younes, E. e t al., submitted). Irradiated Renca cells showed a reduced ability to produce tumors in vivo following intravenous or subcutaneous injection, which was consistent with the decreased proliferation noted in vitro. Proportional increase in the number of irradiated cells compensated for the decrease in tumorigenicity, suggesting t h a t this effect is largely due to growth inhibition by the radiation. The direct growth inhibitory effect of irradiation may be responsible for a local reduction in the tumor burden; however, additional mechanism(s) must account for the systemic effect observed when radiation was combined with IL-2 therapy. We observed that the presence of irradiated tumor cells diminished the growth of nonirradiated tumor cells in the same animal. This important observation suggests that irradiated tumor cells may trigger an in vivo immune mechanism that affects the growth of nonirradiated tumors. Radiation may produce changes in tumor antigens and/or major histo-

RADIATION EFFECTS ON MURINE KIDNEY TUMOR CELLS

compatibility complex (MHC) antigens or in the tumor environment and may render the tumor more susceptible t o recognition and elimination by the immune system activated by immunotherapy. Previous studies have reported MHC class I antigen upregulation by ionizing irradiation in murine B16 melanoma cell@ and a n increased expression of carcinoembryonic antigen and MHC class I antigens in human gastric adenocarcinoma cells.17 We also found that 300 rad irradiation caused an upregulation in H-2Kd class I MHC antigen in the Renca system (Younes, E. et al., submitted). Histologically, tumors from irradiated cells showed a decrease in mitotic cells in correlation with the decreased in vitro proliferation and in vivo tumor growth. The LTX tumors were characterized by a n increase in micronecrosis, multinucleated giant cells and apoptosis, which may result from radiation damage.18 In animals bearing both nonLTX and LTX tumors, a decrease in the mitotic rate of nonLTX tumors consistent with a decrease in tumor growth indicates some systemic effect induced by the irradiated tumor cells on the growth of nonirradiated tumor cells. Interleukin-2 therapy caused a n increase in micronecrosis and infiltrates of blast-like lymphocytes, macrophages and eosinophils, which could be evidence of cytolytic activity.19 This effect was more pronounced in LTX tumors (fig. 2,D). Our studies indicate that in situ tumor irradiation affects the ability of the Renca cells t o divide and grow in vitro and in vivo after subsequent implantation. However, there is also evidence of a systemic effect induced by tumor irradiation. Further studies are underway to clarify whether this systemic mechanism may be the triggering of an immune response induced by radiation-altered tumor cells which can be enhanced by IL-2. Acknowledgement. We thank Dr. Elaine Hockman from the Research Support Laboratory, Wayne State University, for statistical analysis. REFERENCES

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.,