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Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Radiation is the standard of care for many cancers and has conventionally been exploited for its direct cytotoxic effect on tumors or palliative effects in patients. Interestingly, numerous classes of genes have been reported to be modulated after irradiation of both murine and human tissues (16) and has led to our interest in the use of radiation therapy of tumors in combination with active immunotherapy approaches. Recent preclinical work from our laboratory has shown that irradiation of murine tumors enhanced their ability to be killed by specific CTLs in a Fas-dependent mechanism (6) .
Although human colon, lung, and prostate are among the most prevalent cancers, very few studies, typically limited to one or two cell lines, have focused on the phenotypic changes in response to irradiation (17, 18, 19, 20, 21, 22) . In addition, most of these studies examined changes in only one or two surface molecules. Less than one third of the cell lines used in this study have been examined by others in similar phenotypic assays (16) . In fact, to our knowledge, none have reported the functional significance, as it relates to CTLs, of phenotypic changes observed after irradiation of human colon, lung, or prostate carcinoma.
Here, we examine the effects of sublethal radiation on molecules that have each been implicated in enhancing T-cell–mediated tumor cell killing through diverse mechanisms: functional Fas, the cell adhesion/costimulatory molecule ICAM-1, MHC class I, and the human carcinoma-associated antigens CEA and MUC-1. Results of this study also examine whether changes induced by irradiation of tumor cells could improve CTL-mediated tumor killing. Lastly, microarray analysis of colorectal tumor cells also revealed changes in numerous additional genes that could serve as additional potential targets for immunotherapy.
Overall, these studies show, for the first time (1) , a comparison of the effects of nonlethal radiation on surface molecule expression in a large panel of human colon, lung, and prostate carcinoma cell lines (2) , the use of radiation to functionally enhance Ag-specific CTL-mediated killing in several human cell lines, and (3) the use of microarray to identify additional potential immune targets enhanced by irradiation. Thus, these findings have potentially important implications on the combined use of local tumor irradiation and cancer immunotherapies in the clinic.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tumor Irradiation.
Human tumor cells were harvested while in log-growth phase. Tumor cells, in suspension, were placed on ice and irradiated (0 to 20 Gy) by a cesium-137 source (Gammacell-1000; AECL/Nordion, Kanata, Ontario, Canada) at a dose rate of 0.74 Gy/min. Control samples were also placed on ice but not irradiated. Both irradiated and nonirradiated cells were then washed in fresh media and seeded in 75-cm2 tissue culture flasks. After 72 hours, cells were harvested for surface molecule analysis by flow cytometry.
Flow Cytometric Analysis.
Cell surface staining of tumor cells was done with the following primary-labeled monoclonal antibodies: CD95-FITC, CD54-PE, CD66-FITC, COL-1-FITC (23)
, CD227-FITC, HLA-ABC-PE, and the appropriate isotype matched controls. 7-amino actinomycin D staining was used as a measure of cell death following the manufacturer’s instructions. All antibodies, with the exception of COL-1, were purchased from BD PharMingen (San Diego, CA). Stained cells were acquired on a FACScan flow cytometer using CellQuest software (BD PharMingen). Isotype control staining was <5% for all samples analyzed. Dead cells were excluded from the analysis based on scatter profile.
Functional Fas Assay.
Human tumor cells were nonirradiated (0 Gy) or irradiated (10 and 20 Gy) and recultured. After 72 hours, cells were harvested and counted. A total of 2 x 106 tumor cells was labeled with 75 µCi 111Indium Oxine (Amersham Health, Silver Spring, MD) for 30 minutes at 37°C. Cells were washed and subsequently incubated for 18 hours with varying concentrations of anti-Fas antibody, clone CH11 (MBL, Watertown, MA). Control cells were incubated with IgM isotype control antibody (BD PharMingen), and Jurkat cells were used as a positive control for Fas-mediated cytotoxicity. Functional Fas was determined as described previously (6)
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CTL Cell Line.
The A2-restricted, CEA-specific, CD8+ CTL line, designated V8T, recognizes the CEA peptide epitope YLSGANLNL (CAP-1; refs. 24
, 25
).
Cytotoxicity Assays.
V8T cells were used on day 4 of the restimulation cycle after Ficoll purification. Irradiated (10 Gy) and nonirradiated (0 Gy) human tumor cells were cultured for 72 hours and subsequently used as targets in a standard cytotoxicity assay using indium-111. A total of 2 x 103 radiolabeled tumor cells were incubated with 6 x 104 V8T CTLs (E:T of 30:1) for 18 hours at 37°C with 5% CO2. Targets and CTLs were suspended in complete medium supplemented with 10% human AB serum in 96-well U-bottomed plates (Costar, Cambridge, MA). After incubation, supernatants were collected and assayed as above.
RNA Isolation.
WiDr (human colon carcinoma) cells in suspension were irradiated (20 Gy) or nonirradiated (0 Gy) on ice and reseeded in 150-cm2 tissue culture flasks. After 8 and 24 hours, cells were harvested from flasks, and total RNA was extracted and purified from 5 x 107 cells with the RNeasy midi kit (Qiagen, Inc., Valencia, CA) according to the manufacturer’s instructions.
cDNA Microarray.
Dual-color microarray hybridizations were performed as follows: RNA isolated from irradiated (20 Gy) WiDr cells was compared with RNA isolated from nonirradiated WiDr cells at two different time points after irradiation (8 and 24 hours). Analysis was carried out as previously described (26)
. Genes were considered to be up-regulated or down-regulated, after irradiation, if their normalized intensity ratio was 
2 or 
0.5 (2-fold cutoff) and 3 or 0.33 (3-fold cutoff). Hierarchical clustering (27)
was applied to those genes that had a normalized intensity ratio 3 or 0.33. Pearson correlation was used as distance matrix.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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10% or if the mean fluorescence intensity doubled after irradiation. To confirm that increased cell surface levels of proteins reflected an increase in overall levels rather than increased movement to the cell surface, WiDr and HCT116 cells were subjected to 0 and 20 Gy of radiation, and protein extracts were made after 72 hours. CEA protein levels were quantified by enzyme linked immunosorbant assay. The detection limit for CEA protein was 1 ng/mL. For both cell lines, the CEA level was undetectable before radiation. After radiation, for WiDr cells the CEA level was 90 ng/106 cells, and for HCT116, the CEA level was 85 ng/106 cells.
The results obtained using 11 additional human colorectal tumor cell lines, 7 human lung cell lines, and 4 human prostate cell lines are summarized in Fig. 1
. In total, 21 of 23 (91%) human tumor cell lines responded to low-dose irradiation by up-regulating one or more surface molecules (Fig. 1A–C)
. Of the colorectal cells lines tested, 12 of 12 up-regulated at least one of the monitored surface proteins (Fig. 1A)
. CEA was up-regulated most often, and an increase in this molecule was observed in 83% of these lines. In the lung tumor lines, CEA and ICAM-1 were the molecules increased most often after radiation (Fig. 1B)
; two lung cancer cell lines, HLF-a and Calu-1, showed no up-regulation after radiation. Taken as a whole, prostate cell lines were the least altered after irradiation. Although each prostate line did respond by up-regulating at least one protein, no single line responded by increasing more than three proteins, as was observed in both colon and lung carcinomas. In addition, none of the prostate carcinomas showed an increase in surface MHC class I expression. Only one cell line, colon carcinoma WiDr, responded to irradiation by up-regulating all five of the monitored surface molecules. Overall, ICAM-1 and CEA, up-regulated 61 and 69%, respectively, were the most commonly up-regulated molecules. Twenty-six percent up-regulated functional Fas, 74% (17 of 23) up-regulated a TAA (either CEA or MUC-1), and 35% up-regulated MHC class I (Fig. 1)
. In addition, no clear distinctions, in response to radiation, could be made between lines derived from primary tumor sites and those derived from metastatic sites.
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as Fas functional and nonfunctional examples, respectively. Fas-sensitive Jurkat cells were used as a positive control; the antibody efficiently mediated lysis of 45% of these cells in an 18-hour assay. Overall, 6 of 10 cell lines that up-regulated surface Fas after irradiation underwent enhanced lysis (ranged from 35 to 65%) in response to anti-Fas stimulation when compared with their nonirradiated counterparts (Fig. 1
, hatched boxes). Interestingly, Fas up-regulation following radiation did not always result in increased Fas-mediated lysis. Specifically, A549 and LNCaP cells both up-regulated Fas to similar levels (Fig. 2)
; however, only A549 cells were killed upon surface Fas cross-linking. No lysis was observed when irradiated cells were incubated in the presence of an isotype-matched control IgM antibody (Fig. 2
, open symbols). Functional Fas was subsequently confirmed, in selected cell lines, by active caspase-3 detection after Fas receptor cross-linking (data not shown).
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. LS 174T is a CEA-positive, HLA-A2–negative tumor that greatly up-regulated surface Fas in response to radiation. This cell line, however, was not killed by the HLA-A2–restricted, CEA-specific CTLs used in these experiments and was included as a negative control (Fig. 3B)
. Enhanced CTL killing was not always linked to Fas up-regulation in this study. For example, two of the five cell lines (Caco-2 and SW1463) showed significantly enhanced killing after irradiation, although they did not up-regulate surface Fas after irradiation (Fig. 3)
. Furthermore, SW620 up-regulated surface Fas that was not functional, yet these cells demonstrated the most significant enhancement of killing by CTLs. In fact, of the five molecules observed, MHC class I was the only other molecule observed to increase after irradiation of this cell line. Although it is possible that the five molecules examined could contribute to enhanced CTL killing of tumor targets, it is also likely that many other proteins could contribute to these observations.
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Additional analysis focused on those genes that exhibited a change in expression of at least 3-fold, which corresponded to 22 genes up-regulated at 8 hours after irradiation and 90 genes differentially expressed at 24 hours after irradiation. A hierarchical clustering has placed these 90 genes into two groups (Fig. 4, A and B)
. Cluster A contains 84 genes up-regulated after irradiation, and cluster B contains the only six genes whose expression was repressed at 24 hours after irradiation. The set of 90 genes that was differentially expressed at 24 hours included very few genes whose expression was also modified at a shorter time after irradiation, suggesting that many of those 90 genes belong to a category of late-responsive genes because their RNA levels were only modified at the later time point. Exceptions were CDKN1A, CTBS, and FSLT3 genes that were also induced at least 3-fold at 8 hours after irradiation.
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. Also shown in the table is the normalized relative intensity ratio for each individual gene. Of a total of 109 genes differentially expressed at 8 or 24 hours, 10 genes encoded proteins that participate in the control of cell cycle and cell proliferation. Among them was CDKN1A (p21), which is p53 dependent and was previously described as one of the acute-response genes in irradiation responses (30)
. p21 was detected early and was 12-fold up-regulated at 8 hours after irradiation and maintained its high level of expression in irradiated cells at 24 hours after irradiation. A group of seven genes encoding for apoptosis-related proteins was also differentially expressed after irradiation, among them the apoptosis inhibitors Bcl-2 and MCL-1 (a Bcl-2–related gene) were both 3.3-fold repressed at 24 hours postirradiation. Also in this group were PTPN6 (Sph-1), a negative regulator of signaling that is downstream of a wide range of receptors, including cytokine receptors, growth factor receptors, and receptor complexes of the immune system (31)
, and PLAB/MIC-1, a member of the transforming growth factor ß superfamily that can block cell proliferation and induce apoptosis (32)
. As would be expected, a set of genes that participate in the DNA repair process was also up-regulated at 24 hours postirradiation, such as DDB2 that encodes a p53-inducible damage-specific DNA binding protein. Similarly, oxidative stress genes involved in p53-mediated apoptosis, such as ferredoxin reductase, which generates oxidative stress in mitochondria (33)
, were also differentially expressed 24 hours after irradiation. Additionally, many genes whose expression was modified in response to radiation belonged to the category of receptors, membrane transporters, and adhesion molecules, such as CD40, a member of the tumor necrosis factor receptor superfamily that is known for mediating costimulatory signals to the T cells, as well as for its ability to modulate the viability of various tumor cells (34)
. Several adhesion molecule-encoding genes were also up-regulated in irradiated WiDr cells, such us LMAN2, ICAM-1, and Ninjurin-1. Included in this cell adhesion category is Mesothelin, whose gene expression was increased by 2.5-fold and therefore missed the 3-fold cutoff used in Table 1
. This gene is of particular interest because it has recently been identified as a potential target for immune-mediated attack of carcinoma cells (35
, 36)
. The up-regulation of ICAM-1 gene expression was in accordance with the observations at the protein level observed previously (Fig. 1)
. Two genes observed as up-regulated by flow cytometry, CEA, and HLA-B were also both up-regulated by microarray analysis (CEA, 1.4-fold; HLA-B, 1.7 fold), however, not to 3-fold levels reported in Table 1
. No change in MUC-1 mRNA expression was detectable by microarray. The gene chips used in this assay did not include Fas cDNA. This discrepancy, however, could be due to factors such as RNA turnover and/or stability or the fact that the RNA was analyzed only at 8 and 24 hours after irradiation, whereas the flow cytometry was carried out at 72 hours after irradiation.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous studies with human tumor lines have shown that doses of radiation, lower than those that cause direct damage, may up-regulate MHC class I (40, 41, 42) , tumor-associated molecules (21 , 43) , adhesion molecules (3 , 44) , and death receptors (45 , 46) . Few, however, have focused on the phenotypic changes in colorectal (17, 18, 19, 20) , lung (21) , or prostate (22) human tumor cells’ response to irradiation. These studies have typically been limited to one or two cell lines. Specifically, previous phenotypic studies on human colon carcinoma cells’ response to radiation in vitro have been limited to three cell lines. In addition, each of these studies typically reported on a single surface molecule per cell line (20 , 47) . In a more recent study, LOVO colon carcinoma cells were observed by protein microarray analysis after receiving 4 to 8 Gy of radiation (19) . CEA protein levels were decreased 4 hours after irradiation but were subsequently shown to increase 24 hours after irradiation. We extended our observations to include 12 colon carcinomas in a single study 72 hours after irradiation. Findings reported here confirm and extend those of Sheard et al. (18) , who demonstrated HCT116 up-regulation of surface Fas 24 hours after irradiation, and the lack thereof in HT-29. In contrast, however, increased MUC-1 expression was not observed by us in HT-29 cells as observed by Kang et al. (20) . These investigators reported increased MUC-1 expression by flow cytometry with an antibody to the glycosylated form of the protein. This difference in results could be attributed to the monoclonal antibodies used for detection.
In vitro studies of human lung carcinoma cells’ response to irradiation are more limited. Matsumoto et al. (21) reported CEA expression on GLL-1 cells increased after irradiation at a single dose of 10 Gy, with the peak of CEA expression observed on day 4 after irradiation. One additional study reported enhancement in the expression of MHC class I in cultured human lung A549 cells after low-dose radiation. We examined phenotypic changes in response to radiation in a total of seven lung carcinomas and found that ICAM-1 and CEA were up-regulated most often in response to irradiation.
Phenotypic studies of this kind are currently the most limited for prostate carcinomas. A single study examined the effects of radiation in human prostate carcinoma cells.
In a very recent study, Shimada et al. (22) reported that surface Fas expression increased 24 hours after 5 Gy of irradiation. We observed the same modulation of Fas for this cell line and extended observations to three additional prostate carcinomas, as well as examining other surface markers. Although this carcinoma type was the least responsive to tumor irradiation, all four lines did up-regulate at least one surface molecule.
Overall, the data reported here are the first to show a comparison of the effects of nonlethal radiation on surface Fas, ICAM-1, CEA, MUC-1, and MHC class I expression in a large panel of human colon, lung, and prostate tumor cell lines. In contrast to previous observations, here, we extended our phenotypic observations to include, in a single study, five surface molecules and 23 cell lines, representing three categories of cancer. As it has been previously observed that surface molecule up-regulation after irradiation is durable and can be maintained for long periods of time (6) , we examined phenotypic changes 72 hours after irradiation. Although the number and magnitude of molecules up-regulated in response to sublethal radiation varied between cell lines, an overwhelming majority (91%) of human tumor lines demonstrated changes in surface molecule expression in response to irradiation. Perhaps not surprisingly, of the 21 that responded by surface molecule up-regulation, no consistent pattern of up-regulation was observed across the group.
Also reported here for the first time is the use of radiation to functionally enhance Ag-specific CTL-mediated killing in numerous human colon cell lines. We observed enhanced Ag-specific killing of human tumor cells by CTL after tumor irradiation. Five of five irradiated CEA+/A2+ colon tumor cells lines were killed better by CEA-specific HLA-A2–restricted CD8+ CTL than their nonirradiated counterparts. In addition, we additionally demonstrated that we could observe a significant enhancement in killing by CTLs, regardless of the surface molecule expression pattern observed in the five molecules monitored. However, in contrast to in vivo and in vitro observations in a single murine tumor cell line (6)
, we show that enhanced sensitivity to CTL attack was not solely dependent on Fas because it did not always correlate with functional Fas up-regulation or high expression levels. In fact, one of the tumor cell lines exhibiting significantly greater lysis by CTL (SW620) expressed surface Fas that was not functional after irradiation. In addition, two cell lines that did not up-regulate Fas after irradiation were subsequently killed better by CTLs. Whether the enhanced killing by CTLs, in these instances, reflects increased presentation of TAAs by MHC class I molecules or increased ICAM expression, resulting in enhanced cell adhesion, is unknown. The enhanced killing could even be a result of other accessory molecules not observed in this study. Observations in this study were extended beyond surface staining and enhanced killing by CTLs to include microarray analysis of a colon carcinoma cell line. To our knowledge, this is the first time a cell line has been examined so extensively after radiation. RNA from the colon carcinoma cell line WiDr was used in these analyses at 8 and 24 hours after irradiation. Using this broad-based approach, we were able to identify additional molecules modulated by radiation that could potentially be targets for immunotherapy or cancer vaccine strategies (Table 2)
. For instance, numerous up-regulated molecules, such as Ninjurin-1, PLAU, mesothelin, glypican, EphA2, CYP1A1, or HSRTS-ß, could serve as potential tumor-associated molecules and targets for tumor-specific CTLs. CD40 up-regulation could serve to enhance costimulatory signals to tumor-specific T cells and as a result stimulate better T-cell killing. The antiapoptotic protein bcl-2 was down-regulated upon tumor irradiation. This alteration could increase cell susceptibility to apoptotic signals given to the tumor, whereas the increase in kallikrein 10 could complement this process by slowing tumor growth. Of particular interest is mesothelin, which has recently been identified as a potential target for immune mediated attack of carcinoma (35
, 36)
. Future studies will explore the functional significance that some of these genes may play in enhanced tumor immunogenicity and sensitization to T-cell attack.
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It is estimated that during their illnesses, 50 to 60% of patients with cancer will undergo radiation therapy, either for cure or palliation (49)
. Standard of care doses of radiation vary depending upon the cancer being treated, ranging from 45 to 50 Gy for residual prostate tumors to 56 to 60 Gy for lung cancer, both delivered fractionated over a period of weeks (49)
. However, it is unclear what dose of radiation is given to each tumor cell, and the result may be that some tumor cells are killed while others are phenotypically modified. Although a single, sublethal, low dose of radiation (20Gy) was used in this study, previous studies suggest that similar protein level increases are observed at higher doses of radiation (6
, 40)
. In addition, fractionation, as radiation is often given in the clinic, achieved similar response rates both in vitro and in vivo similar to that of a bolus dose in a preclinical model (6
, 47)
. These results suggest that radiation doses need not necessarily be lowered below those typically used for patient care to benefit from radiation immune enhancing strategies. Results also suggest that radiation could be beneficial for treating tumors along with vaccines for which radiation is currently not the standard of care. Overall, results of this study suggest that nonlethal doses of radiation can be used to make human tumors more amenable to immune system recognition and attack and form the rational basis for the combinatorial use of cancer and vaccines and local tumor irradiation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Requests for reprints: Jeffrey Schlom, Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, NIH, 10 Center Drive, Building 10, Room 8B09, MSC 1750, Bethesda, MD 20892-1750. Phone: (301) 496-4343; Fax: (301) 496-2756; E-mail: js141c{at}nih.gov
Received 4/30/04. Revised 7/ 7/04. Accepted 8/24/04.
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REFERENCES |
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. Increased surface Fas after irradiation, which was not functional as defined by antibody cross-linking assay, is shown as shaded boxes. Protein expression that remained unchanged after irradiation is indicated by 
. MHC-I, MHC class I; Met, metastasis; 1°, primary.
) or irradiated with 10 Gy (
) and recultured for 72 hours (A and B). Cells were then labeled with 111In and were co-incubated with HLA-A2–restricted CEA-specific CTLs for 18 hours at an E:T ratio of 30:1. LS 174T (B) is a CEA-positive, HLA-A2–negative cell line and is shown as a negative control. All other cell lines shown were both CEA and HLA-A2 positive. * denotes statistical significance. C, numbers indicate % positive and those in parentheses indicate mean fluorescent intensity of cells expressing molecule on cell surface after receiving 0 Gy/10 Gy of radiation, respectively. Numbers in bold represent molecules classified as up-regulated after irradiation.