Expression Of CD137L In Colorectal Cancer Tissues And Its Biological Function

Background and objectivesCD137/CD137L(4-1BB/4-1BBL) are a pair of important and powerful T-cell costimulatory molecules which are involved in the bidirectional signal transduction between activate T cells and antigen presenting cells(APC) and have a special significance for the maintainance of specific cellular immune response and immune memory. The CD137 molecule is a member of the TNF receptor gene superfamily which is mainly expressed on activated T cells. Its ligand, CD137L, is expressed on APCs, such as monocytes, splenic dendritic cells, activated B cells and macrophages. Through the ligation of CD137 and CD137L, they can provide a co-stimulatory signal to T cells and cooperate with CD28/B7 to activate T cells. And in the absence of CD28/B7 signal, CD137/CD137L can provide costimulatory signals independently and play an important role in the activation of CD28- T cells. CD137 can not only activate T cells, but also protect T cells from the activation-induced cell death.More and more researchers began to concern the reverse signal transduction by CD137L. The interaction of CD137 with CD137L on monocytes stimulates cell proliferation and production of TNF, IL-6, IL-8 and IL-12. CD137L can induce the expression of ICAM-1 and significantly promote the migratory activity of monocytes in vitro and in vivo. Drenkard found that CD137 can be strongly expressed by blood vessel walls at sites of inflammation and the expression of CD137 is induced by proinflammatory cytokines in endothelial cells that can induce migration of monocytes in vitro and in vivo. CD137L enhances the rate of apoptosis in monocytes as well as proliferation. However, signaling through the CD137L induces proliferation of monocytes, which overcompensate the loss of cells through apoptosis. CD137L can enhance proliferation and Ig synthesis of preactivated B cells. However, constitutive expression of the CD137L on APC causes the elimination of peripheral B cells. It has been shown that cross-linking of CD137 on monocytes causes them to induce apoptosis in B cells. CD137L is also expressed on activated T cells and transducts an apotosis signal independent of CD95.CD137L is constitutively expressed on Raji, Daudi, Colo 205, HCT 116, HT 29, LX 1, PC3, SKBR and human tumor tissue cells. Cross-linking of the CD137L induces release of IL-8 from tumor cells, which means CD137L on tumor cells surface is biofunctional. HCT 116 cells and human T cells were co-cultured in 96-well plates in the presence of soluble anti-CD3 and this resulted in the release of IFN-y into the culture medium. And there appears to be a dose-dependent relationship among the density of CD137L expressed on tumor cells, the number of cells, and the levels of IFN-y production. CD137 and CD137L are constitutive expressed on human T and B leukemia cell lines, such as CCRF-CEM, Jurkat, and Raji. In vitro experiments found that both tumor CD137 and CD137L molecules signaled in T and B leukemia cells inducing proliferation and prolonging survival. In addition, CD137/CD137L system ligation opposed the anticancer drug cytotoxic effects, reducing the apoptotic DNA fragmentation and stimulating proliferation of doxorubicin-escaped leukemia cells. A recent study assessed the expression of CD137 and CD137L by immunohistochemistry in frozen sections of 12 human normal tissues,15 benign tumors of epithelial or mesenchymal origin(adenoma and leiomyoma) and 36 malignant tumors of epithelial origin (squamous cell carcinoma and adenocarcinoma). The expression of CD137 and CD137L was observed only in human benign (2/15,3/15) or malignant tumors (15/36,21/36), but not in normal tissues (0/12,0/12). CD 137 was expressed on the vessel walls within tumor tissues, whereas CD137L was expressed on tumor cells. The expression of CD 137 and CD137L was more common in malignant tumors, especially in moderate or low-differentiated tumors.CD137L is shed from the cell surface due to the activity of matrix metalloproteinases (MMP) and its secretion can be inhibited by matrix metalloproteinase inhibitor (MMPI). Salih found that the serum level of soluble CD137L (sCD137L) is very low in healthy donors (3.2pg/ml). However, sCD137L is present at high levels in the sera of patients with various hematological diseases. The mean serum levels of sCD137L are 57pg/ml,210pg/ml, and 682pg/ml in 31 patients with non-Hodgkin lymphoma (NHL),52 patients with myelodysplastic syndrome (MDS) and 20 patients with acute myeloic leukemia (AML). The levels of sCD137L in the sera were markedly higher than those found necessary in vitro to effectively stimulate T cells. This indicates that the in vivo release from cells and/or dispersion to a distal site is likely to have physiological significance. Further study found that high levels of sCD137L correlate with statistical significance to rapid progression of disease. Statistically significant higher median levels of sCD137L are present in patients with undifferentiated AML (M1/M2,1470 pg/ml), poor cytogenetic risk (288 pg/ml) and higher levels of BM-blasts(186 pg/ml) compared with patients with monocytoid AML (M4/M5,89 pg/ml), intermediate cytogenetic risk (59 pg/ml) and lower levels of BM-blasts(14 pg/ml) respectively. Furthermore, in AML patients sCD137L levels correlate significantly with the probabilities to achieve complete remission (CR), stay in CR or with progress of the disease.It is believed that tumor cells should not express costimulatory molecules or express them at a low level in tumor genesis and progression. CD137L is expressed on several human carcinoma cell lines, such as Colo 205, HT 29, and HCT 116 and so on. And CD137L on carcinoma cell surface is biofunctional. It can induce tumor cell proliferation and prolonging survival, enhance the secretion of IL-8 and contribute to drug resistance. CD137L is expressed on carcinoma cell, while CD137 is expressed on the vessel walls. So we presume that CD137/CD137L could play an important role in tumor microenvironment and have a special significance for tumor genesis and progression via bidirectional signal transduction. To discuss the possible role of CD137L in colorectal cancer and pave the way for finding out a new biomarker and therapy target in CRC, we evaluate the expression of CD137L in CRC tissue by real-time RT-PCR and immunohistochemistry. And we stimulate the CD137L on Colo 205 with immobilized CD137-Fc and analyze the cell proliferation by CCK-8, detect the secretion of IL-8 by ELISA, assess the invasion ability by invasive chamber, analyze the MMP-9 production by gelatin zymogram and analyze the expressions of Akt, phospho-Akt, p38, phospho-p38, ERK and phospho-ERK by western blotting.Methods1. A series of 54 colorectal cancer patients who were treated surgically between Jury 2007 and April 2008 were entered in this study. We collected the tumor tissues and adjacent noncancerous tissues. Tumor tissues were all adenocarcinoma diagnosed by clinical pathologists. The tissue samples were collected within 30m when cutted off and dipped into RNAlater(?) at 4℃ overnight and then stored at -80℃for the analysis of CD137L mRNA expression by real-time RT-PCR.2. The tumor tissues and adjacent noncancerous tissues were collected and embedded in OTC and wrapped with aluminum foil. Then the tissues were quickly frozen by dipped into liquid nitrogen and stored at -80℃for the analysis of CD 137L protein expression by immunohistochemistry.3. Expression of CD137L was assessed by two independent pathologists who were blinded to the patient's outcome. The results of immunohistochemistry were estimated by semi quantitative analysis. The immunostaining score was a product of the percentage of positively stained cells and the staining intensity. The percentage of positively stained cells scored "0"(<5% of cells stained), "1"(5-10% of cells stained), "2"(11-50% of cells stained) or "3"(>50% of cells stained). The staining intensity scored "0" (negatively stained), "1" (weakly stained), "2" (moderately stained) and "3" (strongly stained). Both percentage of positively stained cells and staining intensity were decided in a double-blind manner. The final expression score was the product by percentage of positively stained cells score and staining intensity score. We defined the final expression score <2 as weak expression and≥2 as high expression.4. We detected the expression of CD137L on the surface of Colo 205 preserved in our lab. To probe into the possible role that CD137L may play in the microenvironment of colorectal cancer, we observed the biological effects that CD137L might have on Colo 205.5. Colo 205 was cultured in the presence of immobilized CD137-Fc or IgG1 Fc and the cell proliferation was detected by CCK-8 at 24h,48h and 72h.6. Colo 205 was cultured in the presence of immobilized CD137-Fc or IgG1 Fc and the secretion of IL-8 was analyzed by ELISA at 48h.7. CD137-induced cell invasion was studied in vitro. We loaded matrigel with CD137-Fc or Fc protein and coated it onto the upper chamber of transwell. Colo 205 was cultured in the upper chamber for 48h and then the cells on the upper surface of the filters were removed by swabbing softly with a cotton swab. The cells that had migrated to the reverse side were fixed with methanol and stained with Giemsa, and counted in 5 random fields under a microscope at×200 magnification. We estimated the effects of CD137L on invasive ability of Colo 205 by the mean migrated cell number per field.8. Colo 205 was cultured in the presence of immobilized CD137-Fc or IgG1 Fc and the production of MMP-9 was measured by gelatin zymogram at 48h.9. Colo 205 was cultured in the presence of immobilized CD137-Fc or IgG1 Fc and the expressions of Akt, phospho-Akt, p38, phospho-p38, ERK and phospho-ERK were analyzed by western blotting at 12h.Results1. CD137L mRNA expression was analyzed by real-time RT-PCR. The relative expression of CD137L mRNA in tumor tissues is 0.035±0.013 which is significantly higher than the expression in adjacent tissues (0.008±0.005, P=0.000). CD137L mRNA expression in colorectal cancer tissues shows no significant differences among different age, sex, tumor location, Duke's classification, and lymph node metastasis.2. CD137L protein expression was analyzed by frozen section and immunohistochenmistry. We found that only 2 samples in 18 adjacent tissues show "+" expression and the other 16 samples show negative expression. The expression rate of CD137L protein in tumor tissues is 88.9% (48/54):11.1% (6/54) "-",18.5% (10/54) "+", 37.0%(20/54) "++",33.3% (18/54) "+++"3. Among 54 CRC patients, there were 29 males and 23 females. The expression of CD137L in the tumor tissues of 21 males and 17 females is high and the high expression rate is 72.4% and 68.0% respectively which shows no significant difference(χ2=0.125, P=0.772, two tailed). There were 27 patients who were older than 60 years old and 27 patients younger than 60 years old. The high expression case number of older and younger patients is 17 and 21 respectively and the high expression rate is 63.0% and 77.8% respectively which shows no significant difference(χ2= 1.421, P=0.372, two tailed).28 patients were lymph node metastasis positive and 26 were lymph node metastasis negative. The high expression case number of lymph node metastasis positive and negative patients is 18 and 20 respectively and the high expression rate is 64.3% and 77.0% respectively which shows no significant difference(χ2=1.033, P=0.379, two tailed). There were 38 colon cancer patients and 16 rectum and sigmoid colon cancer patients. The high expression case number of lymph node metastasis positive and negative patients is 28 and 10 respectively and the high expression rate is 73.7% and 62.5% respectively which shows no significant difference(χ2=0.675, P=0.517, two tailed).4. Among 54 CRC patients, there are 1 high expression case in Dukes A,11 high expression cases in Dukes B,13 high expression cases in Dukes C and 13 high expression cases in Dukes D. The high expression rate is 16.7%,81.2%,91.6% and 65.0% respectively. And the high expression rate shows significant differences in different Dukes classification (χ2=12.094, P=0.007).5. Colo 205 was cultured in the presence of immobilized CD137-Fc or IgG1 Fc and the cell proliferation was detected by CCK-8 at 24h,48h and 72h. There is significant difference among different culture groups (F=231.548,P=0.000) and different culture time (F=98.579,P=0.000). However, there is no interaction effect between culture condition and culture time by multifactorial analysis (F=0.726, P=0.585). Multiple comparison (LSD) results indicate that the cell proliferation of CD137-Fc group is significantly increased compared with IgG1 Fc group and blank control group at 24h,48h and 72h. The difference between IgG1 Fc group and blank control group has no statistical significance.6. Colo 205 was cultured in the presence of immobilized CD137-Fc or IgG1 Fc and the concentration of IL-8 was analyzed by ELISA at 48h. There is significant difference among different culture groups (F=68.585, P=0.000). The IL-8 concentration in the culture supernatant of CD137-Fc group is 195.3±24.3ng/ml, which is significantly higher than that of IgG1 Fc group and blank control group (P=0.000,0.000). The IL-8 concentration in the culture supernatant of IgG, Fc group is 52.7±11.4ng/ml, and the concentration of blank control group is 54.7±12.5ng/ml. The difference between IgG1 Fc group and blank control group has no statistical meaning (P=0.891).7. We loaded matrigel with CD137-Fc or Fc protein and coated it onto the upper chamber of transwell. Colo 205 was cultured in the upper chamber for 48h and then the cells migrated to the reverse side were fixed, stained with Giemsa, and counted in 5 random fields under a microscope at x200 magnification. There is significant difference among different culture groups (F=47.988, P=0.000). The invasion assay showed that the migrated cell number of CD137/Fc group is 105±10 per field, which is significantly higher than that of IgGi Fc group and blank control group (P=0.000,0.000). The migrated cell number of IgG1 Fc group is 57±3 per field, and the concentration of blank control group is 60±6 per field. The difference between IgG1 Fc group and blank control group has no statistical meaning (P=0.602).8. Colo 205 was cultured in the presence of immobilized CD137-Fc or IgG1 Fc and the production of MMP-9 was measured by gelatin zymogram at 48h. There is significant difference among different culture groups (F=14.593,P=0.005). The MMP-9 expression in the culture supernatant of CD137-Fc group is 81.33±6.11, which is significantly higher than that of IgG1 Fc group and blank control group (P=0.004,0.003). The MMP-9 expression in the culture supernatant of IgG1 Fc group is 58.00±6.25, and the expression of blank control group is 59.00±5.57. The difference between IgG1 Fc group and blank control group has no statistical meaning (P=0.845).9. Colo 205 was cultured in the presence of immobilized CD137-Fc or IgG1 Fc. The expressions of Akt, phospho-Akt, p38, phospho-p38, ERK and phospho-ERK were analyzed by western blotting at 12h. The expression of Akt shows no significant differences (F=0.128, P=0.882). The expression of phospho-Akt shows significant differences (F=34.403, P=0.001). The expression of phospho-Akt in CD137-Fc group is significantly higher than that of IgG1 Fc group and blank control group (P=0.000,0.000). The expression of phospho-Akt shows no significant difference between IgG1 Fc group and blank control group (P=0.505). The expression of p38 shows no significant differences in all groups (F=0.282, P=0.764). The expression of phospho-p38 shows significant differences (F=22.110, P=0.002). The expression of phospho-p38 in CD137-Fc group is significantly higher than that of IgG1 Fc group and blank control group (P=0.001,0.001). The expression of phospho-p38 shows no significant difference between IgG1 Fc group and blank control group (P=0.964). The expressions of ERK and phospho-ERK show no significant differences in all groups (F=1.845,1.288, P=0.237,0.342).Conclusions1. CD137L mRNA and protein are highly expressed in colorectal cancer tissues. CD137L protein is expressed on tumor cell surface and is significantly higher in Dukes B and Dukes C colorectal cancer tissues. The result indicates that CD137L could play an important role in the genesis and progression of colorectal cancer.2. CD137L protein is positively expressed in most of tumor tissue, while negatively expressed in most of adjacent tissues, suggesting that CD137L has sort of clinical value to be a diagnostic tumor biomarker for colorectal cancer.3. CD137L can significantly enhance the cell proliferation, IL-8 secretion, MMP-9 expression and invasive ability and induce Akt and p38 MAPK activation of Colo 205. Our study indicated that CD137L may be an important molecule which is involved in the genesis, progression and metastasis of colorectal cancer.Innovative points1. We found that the CD137L mRNA and protein were highly expressed in colorectal cancer tissues by real-time RT-PCR and IHC. CD137L protein is expressed on tumor cell surface and is significantly higher in Dukes B and Dukes C colorectal cancer tissues, weak in Dukes D colorectal cancer tissues and weaker in Dukes A colorectal cancer tissues.2. By activating the CD137L by immobilized CD137-Fc in vitro, we found that CD137L can significantly enhance the cell proliferation, IL-8 secretion, MMP-9 expression and invasive ability and induce Akt and p38 MAPK activation of Colo 205, and preliminarily reveal the biological effects of CD137L on Colo 205 and the possible mechanisms.