Isotope-Release Cytotoxicity Assay With the Use of Indium-111: Advantage Over Chromium-51 in Long-Term Assays 1.2 R. H. Wiltrout,
3.4
P. Frost,
5
and G. D. Cummings
ABSTRACT-The adaptation of Indlum-111-oxlne (also known as 8-hydroxyqulnoline) ('''lnOx) chelate for long-term (18-48 hr) isotope-release assays of ceil-mediated cytotoxicity (CMC) and Its advantages over the use of 51Cr are described. Labeling of DBAl2 P815 mastocytoma cells with "'lnOx resulted In the incorporation of as many as a million counts per minute In 10' cells with no reduction In cell viability. '''lnOx labeled both mouse and human tumor cells. '''lnOx, like "Cr, primarily labeled cytoplasmic constituents; up to 80% of the label existed In a releasable form. "'lnOx was quantitatively released from labeled P815 In response to specifically sensitized C57BL/6 lymphocytes. The high labeling efficiency of '''lnOx offered a significant advantage over "Cr In 18to 48-hour assays for CMC by reducing the counting error and thus making the assay more precise. Because of its higher labeling efficiency, '''lnOx can be used in mlcrocytotoxiclty assays. '''lnOx has the added advantage of a lower spontaneous release in culture than 51Cr. This feature of "'lnOx also makes the calculation of specific isotope release more accurate than that achieved with "Cr in long-term cytotoxic assays.-J Nail Cancer Inst 61: 183-188, 1978.
Measurement of CMC by specifically sensitized lymphocytes has been most successfully quantitated by the 5lCr-release assay developed by Brunner et al. (1). This assay equates the level of 5lCr release from labeled target cells with the destruction of the target cells by specifically sensitized lymphoid cells (1-5). Target cell destruction in the model of Cerouini et al. (6) is due to T-cells and is most efficiently measured in short-term, allogeneic cytotoxicity studies (1-6). Because of the magnitude of these allogeneic reactions, they are readil y detectable by 5lCr release in 2-6 hours. However. the measurement of other low-level cytotoxic reactions often employs long-term (18-24 hr) 5lCr-release assays (7, 8). These assays are complicated by several inherent properties of 51Cr. First, cells such as the widely employed P815 mastocytoma are maximally labeled at 50,00080.000 cpm/105 cells. only if 5lCr of high specific activity (200-500 mCi/mg) is used in conjunction with an incubation period of 1 or more hours. If we assume maximum labeling efficiency, labeling with 5lCr results in approximately 800 cpm/103 P8I5 targets and makes 5lCr relatively ineffective in systems that of necessity have few available target and/or effector cells. Second, prolonging the assay time [to take advantage of the purported multihit potential of T-cells (9, 10)] does not effectively increase sensitivity of this assay because of the relatively high spontaneous release of 5lCr from target cells. These deficiencies of 5lCr become more obvious when one attempts to evaluate cytotoxicity against human epithelium-derived tumor cells (11), because these cells label poorly with 51Cr. Thus a radioisotope with high labeling efficiency and low spontaneous release, which can be specifically released by cytotoxic lymphoid cells, VOL. 61. NO.1. JULY 1978
6.7
would be of particular value in this system. This study demonstrates that IIlln, an isotope used in labeling white blood cells (12-16) and platelets (17), has characteristics that make it a useful tumor target cell label. MATERIALS AND METHODS
Animals.-Female BALB/c, DBA/2, and C57BL/6 mice, 6-8 weeks of age, were purchased from The Jackson Laboratory, Bar Harbor, Maine. The mice were allowed food and water ad libitum and were used at 8-12 weeks of age. Tumor cell lines.-Mouse tumor lines were maintained by in vivo passage. DBA/2 P815 mastocytoma and C57BL/6 EL 4 lymphoma were maintained in ascitic form by serial ip passage. Three additional tumors (in BALB/c mice) were used in our radiolabeling studies. SS-I arose as a spontaneous adenoacanthoma, whereas SS-2 and Meth A were both sarcomas induced by 3-methylcholanthrene. All three tumors were stored in dimethyl sulfoxide in liquid nitrogen. Cells were obtained by the injection of 107 thawed tumor cells ip into BALB/c mice. EB-33 is a human prostate carcinoma line originally isolated by Dr. F. H. Schroeder (Dept. of Urology, University of Wiirzburg, Wlirzburg, Federal Republic of Germany). Malme is a human melanoma cell line kindly supplied by Dr. J~rgen Fogh, (Sloan-Kettering Institute for
ABBREVIATIONS llSED: CMC=cell-mediated cytotOXICIty; cpm=counts per minute; FCS=fetal calf serum; (lH)dThd=tritiated thymidine; llIInOx=indium-lll-oxine; PBS=phosphate-buffered saline; P /S=penicillin and streptomycin; RNL=isotope release in presence of normal lymphocytes; RSL=isotope release in presence of sensitized lymphocytes; SR=specific release of isotope; TC=total incorporated counts.
Received October 7, 1977; accepted February 21. 1978. Supported in part by Public Health Service grant CA16426-03 from the National Cancer Institute and by the Harper Hospital Medical Staff Trust Fund. 3 Department of Immunology, Wayne State University School of Medicine. 540 East Canfield Ave .• Detroit. Mich. 48201. • Performed in partial fulfillment of the requirements for a doctor of philosophy degree. S Departments of Medicine and Immunology. Wayne State University School of Medicine; and Department of Medicine, Harper Hospital. 3990 John R St.. Detroit, Mich. 48201. 6 Department of Oncology. Wayne State University School of Medicine. 7 We thank Mr. Julian Smith and Mr. John Kehrl for their technical assistance. We also thank Dr. Myron A. Leon and Dr. Noel R. Rose of the Departments of Immunology and Microbiology, Wayne State University School of Medicine. for reviewing the manuscript. 1
2
183
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NATL CANCER INST
184 Wiltrout, Frost, and Cummings
Cancer Research, Rye, N.Y.). Both of these human cell lines are maintained in vitro (11). Radioisotopes.- 5I Cr (sp act, 200-500 mCi/mg) and [3H]dThd (5 mCi/ml) were purchased from New England Nuclear, Boston, Massachusetts. JIlIn chloride was obtained from Medi-Physics, Inc., Emeryville, California. JIlIn (sp act, 2,400 mCilmg) has a half-life of 67 hours and emits -y-photons at 173 keV (89%) and 247 keV (93%). Labeling procedures.-Cells were labeled with 51Cr by suspension of 5XI06 or 107 viable tumor cells in 0.2 ml RPMI-1640 medium (Grand Island Biological Co., Grand Island, N.Y.) supplemented with 10% FCS (Flow Laboratories, Rockville, Md.). We then added 50-250 J.l.Ci 51Cr and incubated the cells at 37° C for 30-60 minutes. The variations in cell number, 51Cr dose, ap.d incubation time reflect different experiments. Labeling with JIlIn required chelation of the isotope with 8hydroxyquinoline (oxine; Sigma Chemical Co., St. Louis, Mo.) as described by Thakur et al. (15). The chelation, which required less than 1 hour, involved addition of 3 mCi of JIlIn chloride at pH 1-3 to an equal volume of sterile distilled water to which 200 ",I of 0.3 M acetate buffer (pH 5) was added. A solution containing 200 J.l.g of oxine dissolved in absolute ethanol (10 mg/ml; IMC Chemical Group, Terre Haute, Ind.) was added to the buffered isotope. After being mixed, the tube was allowed to stand at room temperature for 15 minutes, when an equal volume of chloroform was added. The solution was mixed and allowed to stand at room temperature for 10 minutes. The water layer was then discarded and the chloroform was evaporated in a boiling water bath. The remaining JllInOx chelate was dissolved in 100 "I absolute ethanol and diluted to 1,500 ",1 with normal saline. In all preparations of the IJlInOx complex, excess oxine was added to saturate any cadmium impurities. Under these conditions, 75-85% of the original gamma activity of the JIlIn chloride was extracted as the chelated JllInOx
JIlIn crossover. which averaged 8-10% of the counts present. Mechanical disruption of labeled P815 mastocytoma and determination of label. localization. -Mastocytoma cells labeled with either 100 ",Ci of 51Cr/108 cells or 100 J.l.Ci JIIInOx/108 cells were washed three times and resuspended to 2XI07 cells/ml in PBS. The cells were then mechanically disrupted by a cell-rupturing pump (18). The cell lysate was centrifuged at 4,000Xg for 30 minutes, and the supernatant was retained and recentrifuged at 20.000Xg for 30 minutes. This procedure allowed for separation of nuclear, cytoplasmic, and membrane components (18). The resulting membrane pellet was washed twice in PBS (at 20,000Xg for 30 min), and the total radioactivity of all supernatants and centrifugates was counted. Assay for CMC.-CMC was measured by a modification of the isotope-release method described by Brunner et al. (1). Three to five C57BL/6 mice were inoculated ip with 3XI07 viable P815 mastocytoma cells. and their spleens. along with spleens from 2 normal, nonimmunized C57BL/6 mice, were excised 10-11 days post immunization. We prepared single-cell suspensions by teasing the spleens in RPMI-1640 medium. The spleen cells were washed three times in medium and resuspended to 107 viable cells/ml in RPMI-1640 medium with 10% FCS and PIS (100 U penicillin/ml and 100 J.l.g streptomycin/ml. Tumor target cells (P815) were prepared by the method of Henney (2). They were labeled with various doses of either 51Cr or IJlInOx and resuspended to 106 viable cells/ml in identical medium as was used for the spleen cells. Lymphocytes and tumor target cells were incubated in duplicate for various periods of time (2-48 hr) at a ratio of effector cells to target cells of 30: I in 5% CO 2 in a total volume of I ml in 12X75-mm loosely capped plastic tubes (Falcon Plastics). Target cell numbers varied from 103 to 105, depending on the experiment. The assays were terminated by centrifugation of the tubes at 1,600 rpm for 6-8
IIlJnOx was prepared, cell labeling was extremely simple and rapid, requiring only 15 minutes. Cells were labeled with various doses of IIIInOx at a concentration of 107 cells/0.5 ml in RPMI-1640 medium with 10% FCS and 20 mM HEPES buffer 7 for 15 minutes at 37° C and washed three times in 15 ml of RPMI-1640 medium. Cell viability.-The viability of cell suspensions was determined by trypan blue exclusion and/or by measurement of [3H]dThd uptake in vitro. Quantitation of [3H]dThd uptake was performed by triplicate platings of 104 tumor cells/well in microplates (Falcon Plastics, Oxnard, Calif.) as follows: Cultures of JIIInOx-labeled cells were pulsed with I ",Ci [3H]dThd/well and incubated for 24 hours, at which time they were harvested on fiber glass discs and counted on a Beckman Model LS-330 liquid scintillation counter. The total [3H]dThd counts/well were corrected by subtraction of
supernatant was removed from each tube for enumeration in a Beckman model 300 gamma counter. By counting all experimental and control tubes at the same time and by incorporating internal standards for each experimental group (not exceeding 50 samples). we automatically corrected for radioactive decay. Normal counting time was I minute per sample. Microcytotoxicity assay.- Microcytotoxicity assays were performed in round-bottom microplates (Falcon Plastics) in a total volume of 0.2 ml medium (RPMI1640 + FCS + PIS). Incubation was for 24 hours at 37° C in 5% CO 2• JIlIn Ox-labeled P815 targets were incubated in triplicate with sensitized or normal C57BL/6 lymphocytes at various effector-to-target ratios. Immediately prior to incubation. the plates were centrifuged at 300 rpm for 3 minutes. At the conclusion of incubation. the plates were centrifuged at 1,600 rpm for 10 minutes, and 100 ",I of the supernatant was removed from each well for counting. Calculation of SR.-The percent SR or spontaneous release was calculated by the following formulas:
complex, which we routinely IIsed for 2 weeks. Once the
7
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N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid. NATL CANCER INST
minutes at room temperature. and one-half of the
VOL. 61. NO. I, JULY 1978
Cell-Mediated Cytotoxicity With Indium-111 Isotope Release 185 , Formula I: Percent SR Formula la: Percent
- RNL = RSL TC _ RNL spontaneous release =
X 100.
RNL - cpm present in supernatant at initiation of assayTC X 100.
The total releasable radioactivity was determined by six cycles of rapid freeze-thawing of 105 labeled target cells in 1.0 ml RPMI-1640 medium with 10% FCS. This value was approximately 80% for both 1I1InOx and 51Cr. We also expressed our data by utilizing the method (formula 2) advocated by Herberman et al. (19). , Formula 2: Percent SR
=
RSL - RNL TC X 100.
Levels of significance were calculated by Student's t-test unless otherwise indicated.
RESULTS
TABLE 1.-Radiolabeling of P815 mastocytoma with 11I1nOxa Dosage of 1I1InOx, /lCi 5 10 20 30 40
Labeling of Different Tumor Cell Types With "'lnOx Six mouse tumor cell lines as well as the two human cell lines, EB-33 and Malme, were labeled with 1I1InOx. Twenty ,",Ci I11InOx was added to 10 7 viable tumor cells. All cell lines labeled well, and all except EL 4 exhibited good viability and low 24-hour spontaneous release (table 2). EL 4, a mouse T-lymphocyte cell line (20), has characteristics similar to those of normal mouse lymphocytes. We have reported similar decreased viabilities and elevations of spontaneous release from mouse spleen lymphocytes labeled with large doses of llllnOx (16). By merely adjusting the labeling dose, one can efficiently label most cell types without reducing the viability. 11IinOx, like 51Cr, is not significantly reused when released from cells (16). In our laboratory, the maximum achievable label with 51Cr for any of the mouse tumor cells shown in table 2 is approximately 60,000 cpm/105 viable cells. The human tumors EB-33 and Malme label at insignificant levels with 51Cr [(J 1); Wiltrout RH, Frost P: Unpublished observation].
Localization of "'lnOx in P815 Mastocytoma Cells labeled with either 51Cr or II1InOx were disrupted, and the localization of label in cellular constituents was determined by differential centrifugation. VOL. 61. NO. I. JULY 1978
1.31 1.82 5.33 6.41 10.54
95.3 94.5 92.7 95.4 88.2
1.53 1.21 1.47 1.17 1.57
[lHJdThd uptake, percent control 114.8 90.7 109.5 87.7 117.2
7 a P815 mastocytoma cells (10 ) were labeled at 37° C for 15 min with increasing amounts of IIIInOx. The labeling efficiency is expressed as cpm/10 1 viable cells as determined by trypan blue exclusion and CH]dThd uptake. b Determined after 24-hr incubation. 4 c [lH]dThd uptake was assessed by pulsing of 10 11IInOx P815 cells with 1 /lCi of [lH]dThd and incubation for 24 hr at 37° C in RPMI-1640 medium with FCS and PIS. Correction of cpm for crossover of IllInOx was made during scintillation counting. Average cross-over is 8-10% of the total counts present.
Labeling of P815 Mastocytoma With "'lnOx Table I demonstrates the labeling efficiency of 111ln_ Ox added to 107 P81S target cells in O.S ml medium. The pattern of 11IInOx labeling observed for P81S was similar to that reponed by Frost et al. (16) for rodent lymphocytes. Table I also demonstrates that the viability of 1I1InOx-labeled P8IS cells remained the same as that of non-labeled cells as assessed by try pan blue exclusion and [3H]dThd uptake. llllnOx will label heatkilled and glutaraldehyde-fixed cells (Wiltrout RH, Frost P: Unpublished observation). This was not a problem to us, since the cell preparations we utilized had viabilities approaching 100%. Neither 11IInOx nor 51Cr labels cell membrane preparations.
Percent vicpml10 1 ability by [lH]dThd viable trypan uptake, cellsX10- 1 cpmxlO-4c blue exclusion b
TABLE 2.-Radiolabeling of mouse or human tumor cells with III/nOxa Cell line P815 mastocytoma
EL 4
SS-I SS-2 Meth A EB-33 Malme
cpm/10 1 viable cellsXlO-l~
Percent viability
Percent spontaneous release
5.33 0.79 3.52 6.91 6.74 2.62 2.88
95.3 61.7 100.0 78.0 88.6 84.2 83.8
14.0 24.6 3.6 5.4 14.1 12.2 8.6
a Tumor cells were labeled with 20 /lCi of 11IInOx in RPMI-1640 medium with 10% FCS and PIS, and the percent viability and spontaneous release were determined after 24 hr of in vitro culture. 1 b Expressed as cpm/10 tumor cells.
Table 3 demonstrates that the intracellular label distribution of both isotopes was similar. The cytoplasm contained 70-80% of either label, with approximately 20% present in panially disrupted cells and sedimentable cell components. Some label remained bound to membrane, but this represented a small portion of the total. The precise binding mechanism of I1IInOx to cell cytoplasmic components is currently unknown. The 80% maximum release of both isotopes was confirmed by freeze-thawing experiments.
Kinetic Analysis of SR and Spontaneous Release of 11'lnOx and 5'Cr From Labeled P815 Target Cells The kinetics of the SR and spontaneous release of isotope from P8lS target cells (as calculated by formula I) are shown in table 4. The higher labeling efficiency of 1I1InOx was reflected in the generally tenfold increase in the cpm released from llllnOx-labeled cells. This increase in cpm offered a significant statistical advantage over 51Cr. The smaller standard deviations for 51Cr at early time points merely reflected the variations between experimental animals. The statistical analyses
J
NATL CANCER INST
186 Wiltrout, Frost, and Cummings TABLE 3.-Comparison o/the localization Total label, cpmx10- 6
Cell fraction Whole cells Pellet' Wash, three timesd Pelletd Total supernatants and centrifugates
IICr
IllInOx
6.35 1.08 4.89 0.11 6.08
25.20 5.20 18.50 1.29 24.99
0/ IIIInOx or IICr within PS15 mastocytoma cells"
Total percent recovered label b IICr IIIInOx 17.8 80.4 1.8 100.00
Percent label recoveryb IICr
20.8 74.0 5.2 100.00
95.7
Status of label
IIIInOx
99.1
Bound, but not to membrane Released into supernatant Bound to membrane fragments
a P815 mastocytoma cells were labeled with IICr (3X10" cells) or IIIInOx (2xlO" cells) at 100 #-ICi/lOB cells. Labeled cells were then mechanically disrupted, and intracellular localization of both isotopes was determined by differential centrifugation. b Dash indicates not determined. C Centrifuged at 4,OOOXg. d Centrifuged at 20,000Xg.
TABLE 4.-SR and spontaneous release of IIIInOx and Iler from
labeled P815 target cells as a function of time"
Time of assay, hr 2 4 6 12 18 24 48
SR, Cpm±sE b• ' '11InOx
IICr
49,909±21,331 121,465±31,690 175,433±27,256 237.052±13,589 230,401±11,246 220.349±11,818 181,369±15,516
9,780±3,259 17,085±3,565 21,835±1,574 22,O46±614 21,O37±334 17.989±446 1l.877±648
Percent spontaneous release RNL IllInOx IICr 1.0 1.7 1.8 3.2 6.5 10.8 25.4
5.7 8.0 9.3 16.3 21.7 30.0 51.1
" Performed by addition of 3X106 sensitized C57BL/6 spleen cells to 10 5 P815 target cells labeled with IIIInOx or 51Cr. Supernatant was harvested at the indicated times. b Values are cpm in 0.5 ml supernatant times 2 and corrected for RNL. , Calculated by formula 1.
discussed below corrected for this biologic variation by the analysis of each animal's responses separately as a function of time. Text-figure I illustrates the SR of both 51Cr and lllInOx as calculated by formulas I and 2. Formula 1 demonstrates that 51Cr was released more rapidly in the presence of sensitized cells than was lllInOx. This difference in release was significant at 6 hours but not by 12 hours. Because spontaneous release was subtracted from the total counts in the denominator of this formula, the true advantages of lllInOx were not revealed. In contrast, formula 2, which does not correct the denominator for spontaneous release, demonstrates the advantage of the use of a low-spontaneous-release isotope such as IllInOx. The difference between the SR for lllInOx when calculated by either formula was negligible until 48 hours. With 51Cr, these differences were clearly apparent at 12 hours (P
3.4
P. Frost,
5
and G. D. Cummings
ABSTRACT-The adaptation of Indlum-111-oxlne (also known as 8-hydroxyqulnoline) ('''lnOx) chelate for long-term (18-48 hr) isotope-release assays of ceil-mediated cytotoxicity (CMC) and Its advantages over the use of 51Cr are described. Labeling of DBAl2 P815 mastocytoma cells with "'lnOx resulted In the incorporation of as many as a million counts per minute In 10' cells with no reduction In cell viability. '''lnOx labeled both mouse and human tumor cells. '''lnOx, like "Cr, primarily labeled cytoplasmic constituents; up to 80% of the label existed In a releasable form. "'lnOx was quantitatively released from labeled P815 In response to specifically sensitized C57BL/6 lymphocytes. The high labeling efficiency of '''lnOx offered a significant advantage over "Cr In 18to 48-hour assays for CMC by reducing the counting error and thus making the assay more precise. Because of its higher labeling efficiency, '''lnOx can be used in mlcrocytotoxiclty assays. '''lnOx has the added advantage of a lower spontaneous release in culture than 51Cr. This feature of "'lnOx also makes the calculation of specific isotope release more accurate than that achieved with "Cr in long-term cytotoxic assays.-J Nail Cancer Inst 61: 183-188, 1978.
Measurement of CMC by specifically sensitized lymphocytes has been most successfully quantitated by the 5lCr-release assay developed by Brunner et al. (1). This assay equates the level of 5lCr release from labeled target cells with the destruction of the target cells by specifically sensitized lymphoid cells (1-5). Target cell destruction in the model of Cerouini et al. (6) is due to T-cells and is most efficiently measured in short-term, allogeneic cytotoxicity studies (1-6). Because of the magnitude of these allogeneic reactions, they are readil y detectable by 5lCr release in 2-6 hours. However. the measurement of other low-level cytotoxic reactions often employs long-term (18-24 hr) 5lCr-release assays (7, 8). These assays are complicated by several inherent properties of 51Cr. First, cells such as the widely employed P815 mastocytoma are maximally labeled at 50,00080.000 cpm/105 cells. only if 5lCr of high specific activity (200-500 mCi/mg) is used in conjunction with an incubation period of 1 or more hours. If we assume maximum labeling efficiency, labeling with 5lCr results in approximately 800 cpm/103 P8I5 targets and makes 5lCr relatively ineffective in systems that of necessity have few available target and/or effector cells. Second, prolonging the assay time [to take advantage of the purported multihit potential of T-cells (9, 10)] does not effectively increase sensitivity of this assay because of the relatively high spontaneous release of 5lCr from target cells. These deficiencies of 5lCr become more obvious when one attempts to evaluate cytotoxicity against human epithelium-derived tumor cells (11), because these cells label poorly with 51Cr. Thus a radioisotope with high labeling efficiency and low spontaneous release, which can be specifically released by cytotoxic lymphoid cells, VOL. 61. NO.1. JULY 1978
6.7
would be of particular value in this system. This study demonstrates that IIlln, an isotope used in labeling white blood cells (12-16) and platelets (17), has characteristics that make it a useful tumor target cell label. MATERIALS AND METHODS
Animals.-Female BALB/c, DBA/2, and C57BL/6 mice, 6-8 weeks of age, were purchased from The Jackson Laboratory, Bar Harbor, Maine. The mice were allowed food and water ad libitum and were used at 8-12 weeks of age. Tumor cell lines.-Mouse tumor lines were maintained by in vivo passage. DBA/2 P815 mastocytoma and C57BL/6 EL 4 lymphoma were maintained in ascitic form by serial ip passage. Three additional tumors (in BALB/c mice) were used in our radiolabeling studies. SS-I arose as a spontaneous adenoacanthoma, whereas SS-2 and Meth A were both sarcomas induced by 3-methylcholanthrene. All three tumors were stored in dimethyl sulfoxide in liquid nitrogen. Cells were obtained by the injection of 107 thawed tumor cells ip into BALB/c mice. EB-33 is a human prostate carcinoma line originally isolated by Dr. F. H. Schroeder (Dept. of Urology, University of Wiirzburg, Wlirzburg, Federal Republic of Germany). Malme is a human melanoma cell line kindly supplied by Dr. J~rgen Fogh, (Sloan-Kettering Institute for
ABBREVIATIONS llSED: CMC=cell-mediated cytotOXICIty; cpm=counts per minute; FCS=fetal calf serum; (lH)dThd=tritiated thymidine; llIInOx=indium-lll-oxine; PBS=phosphate-buffered saline; P /S=penicillin and streptomycin; RNL=isotope release in presence of normal lymphocytes; RSL=isotope release in presence of sensitized lymphocytes; SR=specific release of isotope; TC=total incorporated counts.
Received October 7, 1977; accepted February 21. 1978. Supported in part by Public Health Service grant CA16426-03 from the National Cancer Institute and by the Harper Hospital Medical Staff Trust Fund. 3 Department of Immunology, Wayne State University School of Medicine. 540 East Canfield Ave .• Detroit. Mich. 48201. • Performed in partial fulfillment of the requirements for a doctor of philosophy degree. S Departments of Medicine and Immunology. Wayne State University School of Medicine; and Department of Medicine, Harper Hospital. 3990 John R St.. Detroit, Mich. 48201. 6 Department of Oncology. Wayne State University School of Medicine. 7 We thank Mr. Julian Smith and Mr. John Kehrl for their technical assistance. We also thank Dr. Myron A. Leon and Dr. Noel R. Rose of the Departments of Immunology and Microbiology, Wayne State University School of Medicine. for reviewing the manuscript. 1
2
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184 Wiltrout, Frost, and Cummings
Cancer Research, Rye, N.Y.). Both of these human cell lines are maintained in vitro (11). Radioisotopes.- 5I Cr (sp act, 200-500 mCi/mg) and [3H]dThd (5 mCi/ml) were purchased from New England Nuclear, Boston, Massachusetts. JIlIn chloride was obtained from Medi-Physics, Inc., Emeryville, California. JIlIn (sp act, 2,400 mCilmg) has a half-life of 67 hours and emits -y-photons at 173 keV (89%) and 247 keV (93%). Labeling procedures.-Cells were labeled with 51Cr by suspension of 5XI06 or 107 viable tumor cells in 0.2 ml RPMI-1640 medium (Grand Island Biological Co., Grand Island, N.Y.) supplemented with 10% FCS (Flow Laboratories, Rockville, Md.). We then added 50-250 J.l.Ci 51Cr and incubated the cells at 37° C for 30-60 minutes. The variations in cell number, 51Cr dose, ap.d incubation time reflect different experiments. Labeling with JIlIn required chelation of the isotope with 8hydroxyquinoline (oxine; Sigma Chemical Co., St. Louis, Mo.) as described by Thakur et al. (15). The chelation, which required less than 1 hour, involved addition of 3 mCi of JIlIn chloride at pH 1-3 to an equal volume of sterile distilled water to which 200 ",I of 0.3 M acetate buffer (pH 5) was added. A solution containing 200 J.l.g of oxine dissolved in absolute ethanol (10 mg/ml; IMC Chemical Group, Terre Haute, Ind.) was added to the buffered isotope. After being mixed, the tube was allowed to stand at room temperature for 15 minutes, when an equal volume of chloroform was added. The solution was mixed and allowed to stand at room temperature for 10 minutes. The water layer was then discarded and the chloroform was evaporated in a boiling water bath. The remaining JllInOx chelate was dissolved in 100 "I absolute ethanol and diluted to 1,500 ",1 with normal saline. In all preparations of the IJlInOx complex, excess oxine was added to saturate any cadmium impurities. Under these conditions, 75-85% of the original gamma activity of the JIlIn chloride was extracted as the chelated JllInOx
JIlIn crossover. which averaged 8-10% of the counts present. Mechanical disruption of labeled P815 mastocytoma and determination of label. localization. -Mastocytoma cells labeled with either 100 ",Ci of 51Cr/108 cells or 100 J.l.Ci JIIInOx/108 cells were washed three times and resuspended to 2XI07 cells/ml in PBS. The cells were then mechanically disrupted by a cell-rupturing pump (18). The cell lysate was centrifuged at 4,000Xg for 30 minutes, and the supernatant was retained and recentrifuged at 20.000Xg for 30 minutes. This procedure allowed for separation of nuclear, cytoplasmic, and membrane components (18). The resulting membrane pellet was washed twice in PBS (at 20,000Xg for 30 min), and the total radioactivity of all supernatants and centrifugates was counted. Assay for CMC.-CMC was measured by a modification of the isotope-release method described by Brunner et al. (1). Three to five C57BL/6 mice were inoculated ip with 3XI07 viable P815 mastocytoma cells. and their spleens. along with spleens from 2 normal, nonimmunized C57BL/6 mice, were excised 10-11 days post immunization. We prepared single-cell suspensions by teasing the spleens in RPMI-1640 medium. The spleen cells were washed three times in medium and resuspended to 107 viable cells/ml in RPMI-1640 medium with 10% FCS and PIS (100 U penicillin/ml and 100 J.l.g streptomycin/ml. Tumor target cells (P815) were prepared by the method of Henney (2). They were labeled with various doses of either 51Cr or IJlInOx and resuspended to 106 viable cells/ml in identical medium as was used for the spleen cells. Lymphocytes and tumor target cells were incubated in duplicate for various periods of time (2-48 hr) at a ratio of effector cells to target cells of 30: I in 5% CO 2 in a total volume of I ml in 12X75-mm loosely capped plastic tubes (Falcon Plastics). Target cell numbers varied from 103 to 105, depending on the experiment. The assays were terminated by centrifugation of the tubes at 1,600 rpm for 6-8
IIlJnOx was prepared, cell labeling was extremely simple and rapid, requiring only 15 minutes. Cells were labeled with various doses of IIIInOx at a concentration of 107 cells/0.5 ml in RPMI-1640 medium with 10% FCS and 20 mM HEPES buffer 7 for 15 minutes at 37° C and washed three times in 15 ml of RPMI-1640 medium. Cell viability.-The viability of cell suspensions was determined by trypan blue exclusion and/or by measurement of [3H]dThd uptake in vitro. Quantitation of [3H]dThd uptake was performed by triplicate platings of 104 tumor cells/well in microplates (Falcon Plastics, Oxnard, Calif.) as follows: Cultures of JIIInOx-labeled cells were pulsed with I ",Ci [3H]dThd/well and incubated for 24 hours, at which time they were harvested on fiber glass discs and counted on a Beckman Model LS-330 liquid scintillation counter. The total [3H]dThd counts/well were corrected by subtraction of
supernatant was removed from each tube for enumeration in a Beckman model 300 gamma counter. By counting all experimental and control tubes at the same time and by incorporating internal standards for each experimental group (not exceeding 50 samples). we automatically corrected for radioactive decay. Normal counting time was I minute per sample. Microcytotoxicity assay.- Microcytotoxicity assays were performed in round-bottom microplates (Falcon Plastics) in a total volume of 0.2 ml medium (RPMI1640 + FCS + PIS). Incubation was for 24 hours at 37° C in 5% CO 2• JIlIn Ox-labeled P815 targets were incubated in triplicate with sensitized or normal C57BL/6 lymphocytes at various effector-to-target ratios. Immediately prior to incubation. the plates were centrifuged at 300 rpm for 3 minutes. At the conclusion of incubation. the plates were centrifuged at 1,600 rpm for 10 minutes, and 100 ",I of the supernatant was removed from each well for counting. Calculation of SR.-The percent SR or spontaneous release was calculated by the following formulas:
complex, which we routinely IIsed for 2 weeks. Once the
7
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N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid. NATL CANCER INST
minutes at room temperature. and one-half of the
VOL. 61. NO. I, JULY 1978
Cell-Mediated Cytotoxicity With Indium-111 Isotope Release 185 , Formula I: Percent SR Formula la: Percent
- RNL = RSL TC _ RNL spontaneous release =
X 100.
RNL - cpm present in supernatant at initiation of assayTC X 100.
The total releasable radioactivity was determined by six cycles of rapid freeze-thawing of 105 labeled target cells in 1.0 ml RPMI-1640 medium with 10% FCS. This value was approximately 80% for both 1I1InOx and 51Cr. We also expressed our data by utilizing the method (formula 2) advocated by Herberman et al. (19). , Formula 2: Percent SR
=
RSL - RNL TC X 100.
Levels of significance were calculated by Student's t-test unless otherwise indicated.
RESULTS
TABLE 1.-Radiolabeling of P815 mastocytoma with 11I1nOxa Dosage of 1I1InOx, /lCi 5 10 20 30 40
Labeling of Different Tumor Cell Types With "'lnOx Six mouse tumor cell lines as well as the two human cell lines, EB-33 and Malme, were labeled with 1I1InOx. Twenty ,",Ci I11InOx was added to 10 7 viable tumor cells. All cell lines labeled well, and all except EL 4 exhibited good viability and low 24-hour spontaneous release (table 2). EL 4, a mouse T-lymphocyte cell line (20), has characteristics similar to those of normal mouse lymphocytes. We have reported similar decreased viabilities and elevations of spontaneous release from mouse spleen lymphocytes labeled with large doses of llllnOx (16). By merely adjusting the labeling dose, one can efficiently label most cell types without reducing the viability. 11IinOx, like 51Cr, is not significantly reused when released from cells (16). In our laboratory, the maximum achievable label with 51Cr for any of the mouse tumor cells shown in table 2 is approximately 60,000 cpm/105 viable cells. The human tumors EB-33 and Malme label at insignificant levels with 51Cr [(J 1); Wiltrout RH, Frost P: Unpublished observation].
Localization of "'lnOx in P815 Mastocytoma Cells labeled with either 51Cr or II1InOx were disrupted, and the localization of label in cellular constituents was determined by differential centrifugation. VOL. 61. NO. I. JULY 1978
1.31 1.82 5.33 6.41 10.54
95.3 94.5 92.7 95.4 88.2
1.53 1.21 1.47 1.17 1.57
[lHJdThd uptake, percent control 114.8 90.7 109.5 87.7 117.2
7 a P815 mastocytoma cells (10 ) were labeled at 37° C for 15 min with increasing amounts of IIIInOx. The labeling efficiency is expressed as cpm/10 1 viable cells as determined by trypan blue exclusion and CH]dThd uptake. b Determined after 24-hr incubation. 4 c [lH]dThd uptake was assessed by pulsing of 10 11IInOx P815 cells with 1 /lCi of [lH]dThd and incubation for 24 hr at 37° C in RPMI-1640 medium with FCS and PIS. Correction of cpm for crossover of IllInOx was made during scintillation counting. Average cross-over is 8-10% of the total counts present.
Labeling of P815 Mastocytoma With "'lnOx Table I demonstrates the labeling efficiency of 111ln_ Ox added to 107 P81S target cells in O.S ml medium. The pattern of 11IInOx labeling observed for P81S was similar to that reponed by Frost et al. (16) for rodent lymphocytes. Table I also demonstrates that the viability of 1I1InOx-labeled P8IS cells remained the same as that of non-labeled cells as assessed by try pan blue exclusion and [3H]dThd uptake. llllnOx will label heatkilled and glutaraldehyde-fixed cells (Wiltrout RH, Frost P: Unpublished observation). This was not a problem to us, since the cell preparations we utilized had viabilities approaching 100%. Neither 11IInOx nor 51Cr labels cell membrane preparations.
Percent vicpml10 1 ability by [lH]dThd viable trypan uptake, cellsX10- 1 cpmxlO-4c blue exclusion b
TABLE 2.-Radiolabeling of mouse or human tumor cells with III/nOxa Cell line P815 mastocytoma
EL 4
SS-I SS-2 Meth A EB-33 Malme
cpm/10 1 viable cellsXlO-l~
Percent viability
Percent spontaneous release
5.33 0.79 3.52 6.91 6.74 2.62 2.88
95.3 61.7 100.0 78.0 88.6 84.2 83.8
14.0 24.6 3.6 5.4 14.1 12.2 8.6
a Tumor cells were labeled with 20 /lCi of 11IInOx in RPMI-1640 medium with 10% FCS and PIS, and the percent viability and spontaneous release were determined after 24 hr of in vitro culture. 1 b Expressed as cpm/10 tumor cells.
Table 3 demonstrates that the intracellular label distribution of both isotopes was similar. The cytoplasm contained 70-80% of either label, with approximately 20% present in panially disrupted cells and sedimentable cell components. Some label remained bound to membrane, but this represented a small portion of the total. The precise binding mechanism of I1IInOx to cell cytoplasmic components is currently unknown. The 80% maximum release of both isotopes was confirmed by freeze-thawing experiments.
Kinetic Analysis of SR and Spontaneous Release of 11'lnOx and 5'Cr From Labeled P815 Target Cells The kinetics of the SR and spontaneous release of isotope from P8lS target cells (as calculated by formula I) are shown in table 4. The higher labeling efficiency of 1I1InOx was reflected in the generally tenfold increase in the cpm released from llllnOx-labeled cells. This increase in cpm offered a significant statistical advantage over 51Cr. The smaller standard deviations for 51Cr at early time points merely reflected the variations between experimental animals. The statistical analyses
J
NATL CANCER INST
186 Wiltrout, Frost, and Cummings TABLE 3.-Comparison o/the localization Total label, cpmx10- 6
Cell fraction Whole cells Pellet' Wash, three timesd Pelletd Total supernatants and centrifugates
IICr
IllInOx
6.35 1.08 4.89 0.11 6.08
25.20 5.20 18.50 1.29 24.99
0/ IIIInOx or IICr within PS15 mastocytoma cells"
Total percent recovered label b IICr IIIInOx 17.8 80.4 1.8 100.00
Percent label recoveryb IICr
20.8 74.0 5.2 100.00
95.7
Status of label
IIIInOx
99.1
Bound, but not to membrane Released into supernatant Bound to membrane fragments
a P815 mastocytoma cells were labeled with IICr (3X10" cells) or IIIInOx (2xlO" cells) at 100 #-ICi/lOB cells. Labeled cells were then mechanically disrupted, and intracellular localization of both isotopes was determined by differential centrifugation. b Dash indicates not determined. C Centrifuged at 4,OOOXg. d Centrifuged at 20,000Xg.
TABLE 4.-SR and spontaneous release of IIIInOx and Iler from
labeled P815 target cells as a function of time"
Time of assay, hr 2 4 6 12 18 24 48
SR, Cpm±sE b• ' '11InOx
IICr
49,909±21,331 121,465±31,690 175,433±27,256 237.052±13,589 230,401±11,246 220.349±11,818 181,369±15,516
9,780±3,259 17,085±3,565 21,835±1,574 22,O46±614 21,O37±334 17.989±446 1l.877±648
Percent spontaneous release RNL IllInOx IICr 1.0 1.7 1.8 3.2 6.5 10.8 25.4
5.7 8.0 9.3 16.3 21.7 30.0 51.1
" Performed by addition of 3X106 sensitized C57BL/6 spleen cells to 10 5 P815 target cells labeled with IIIInOx or 51Cr. Supernatant was harvested at the indicated times. b Values are cpm in 0.5 ml supernatant times 2 and corrected for RNL. , Calculated by formula 1.
discussed below corrected for this biologic variation by the analysis of each animal's responses separately as a function of time. Text-figure I illustrates the SR of both 51Cr and lllInOx as calculated by formulas I and 2. Formula 1 demonstrates that 51Cr was released more rapidly in the presence of sensitized cells than was lllInOx. This difference in release was significant at 6 hours but not by 12 hours. Because spontaneous release was subtracted from the total counts in the denominator of this formula, the true advantages of lllInOx were not revealed. In contrast, formula 2, which does not correct the denominator for spontaneous release, demonstrates the advantage of the use of a low-spontaneous-release isotope such as IllInOx. The difference between the SR for lllInOx when calculated by either formula was negligible until 48 hours. With 51Cr, these differences were clearly apparent at 12 hours (P
