抗EpCAM单克隆抗体治疗肝母细胞瘤的动物实验研究
|
摘要
目的观察抗上皮细胞粘附分子(epithelial cell adhesion molecule,EpCAM)单克隆抗体靶向治疗人肝母细胞瘤(hepatoblastoma,HB)荷瘤鼠模型的疗效。方法体外培养HB HuH-6细胞,采用免疫组化检测Hu H-6细胞中EpCAM的表达。将10只裸鼠皮下接种HuH-6细胞,待建立稳定的HuH-6细胞荷瘤鼠模型后,将荷瘤鼠等额随机分为两组:实验组经鼠尾静脉注射抗EpCAM单克隆抗体,剂量30 ug/kg,每天1次;对照组经鼠尾静脉注射等量生理盐水;两组均给药3周。停药1周后取荷瘤鼠肿瘤组织进行病理检查,测算肿瘤的重量及体积,并计算抑瘤率。结果 HuH-6细胞传代培养成功,免疫组化染色可见其细胞膜EpCAM呈阳性表达。裸鼠接种Hu H-6细胞6周后可形成肿瘤体,病理检查证实为HB,成功建立HB荷瘤鼠模型。实验组肿瘤重量及体积分别为(35. 6±2. 97) mg和(1. 12±0. 09) cm~3,对照组肿瘤重量及体积分别为(45. 8±4. 59) mg和(1. 37±0. 77) cm~3,差异均有统计学意义(P <0. 05)。实验组肿瘤组织EpCAM表达阳性率(28. 3±5. 4)%,对照组肿瘤组织EpCAM表达阳性率(39. 7±4. 4)%,差异有统计学意义(P <0. 05)。结论动物实验结果表明,抗Ep CAM单克隆抗体能够抑制HB荷瘤裸鼠肿瘤的生长,为HB靶向治疗提供了一定的实验基础和理论依据。
Objective To explore the efficacy of anti-EpCAM( epithelial cell adhesion molecule) monoclonal antibody for hepatoblastoma( HB) in a nude mice model. Methods HB cell line of Hu H-6 was cultured and the expression of EpCAM in Hu H-6 examined. After Hu H-6 cell was inoculated subcutaneously,all mice were randomly divided into treatment and control groups. In treatment group,anti-EpCAM monoclonal antibody was injected into mice by tail vein( 30 ug/kg qd daily) for 3 weeks. In control group,the same volume of saline was applied. The expression of EpCAM from implanted tumor mass was detected by immunohistochemistry. Weight and volume of implanted tumor were measured and then tumor inhibition rate was calculated 1 week later. Results Hu H-6 cell was successfully sub-cultured and the expression of EpCAM was positive on cell membrane. After 6-week cell implantation,mass was confirmed as HB by histopathological examination. Compared with control group,anti-EpCAM monoclonal antibody could significantly inhibit tumor growth in control group. Tumor weight and volume were smaller in treatment group than those in control group [tumor weight:( 35. 6 ± 2. 97) vs.( 45. 8 ± 4. 59) mg( P < 0. 05); tumor size:( 1. 12 ± 0. 09) vs.( 1. 37 ± 0. 77) cm~3( P <0. 05) ]. The positive rate of EpCAM expression was( 28. 3 ± 5. 4) % in tumor tissue of experimental group and( 39. 7 ± 4. 4) % in tumor tissue of control group. The positive rate of EpCAM expression was lower in tumor tissue of experimental group than that of control group. And the difference was statistically significant( P < 0.05). Conclusion Anti-EpCAM monoclonal antibody can inhibit the growth of HB tumor cells in a murine model. And it serves as a potential monoclonal targeting therapy for pediatric HB.
Objective To explore the efficacy of anti-EpCAM( epithelial cell adhesion molecule) monoclonal antibody for hepatoblastoma( HB) in a nude mice model. Methods HB cell line of Hu H-6 was cultured and the expression of EpCAM in Hu H-6 examined. After Hu H-6 cell was inoculated subcutaneously,all mice were randomly divided into treatment and control groups. In treatment group,anti-EpCAM monoclonal antibody was injected into mice by tail vein( 30 ug/kg qd daily) for 3 weeks. In control group,the same volume of saline was applied. The expression of EpCAM from implanted tumor mass was detected by immunohistochemistry. Weight and volume of implanted tumor were measured and then tumor inhibition rate was calculated 1 week later. Results Hu H-6 cell was successfully sub-cultured and the expression of EpCAM was positive on cell membrane. After 6-week cell implantation,mass was confirmed as HB by histopathological examination. Compared with control group,anti-EpCAM monoclonal antibody could significantly inhibit tumor growth in control group. Tumor weight and volume were smaller in treatment group than those in control group [tumor weight:( 35. 6 ± 2. 97) vs.( 45. 8 ± 4. 59) mg( P < 0. 05); tumor size:( 1. 12 ± 0. 09) vs.( 1. 37 ± 0. 77) cm~3( P <0. 05) ]. The positive rate of EpCAM expression was( 28. 3 ± 5. 4) % in tumor tissue of experimental group and( 39. 7 ± 4. 4) % in tumor tissue of control group. The positive rate of EpCAM expression was lower in tumor tissue of experimental group than that of control group. And the difference was statistically significant( P < 0.05). Conclusion Anti-EpCAM monoclonal antibody can inhibit the growth of HB tumor cells in a murine model. And it serves as a potential monoclonal targeting therapy for pediatric HB.
引文
1 Meyers RL,Czauderna P,Otte JB.Surgical treatment of hepatoblastoma[J].Pediatr Blood Cancer,2012,59(5):800-808.DOI:10.1002/pbc.24220.
2董岿然.我国儿童胚胎性恶性实体肿瘤的研究和治疗进展[J].临床小儿外科杂志,2017,16(5):417-421.DOI:10.3969/j.issn.1671-6353.2017.05.001.Dong KR.Diagnosis and treatment of embryonic malignant tumors in children in China[J].J Clin Ped Sur,2017,16(5):417-421.DOI:10.3969/j.issn.1671-6353.2017.05.001.
3 Karski EE,Dvorak CC,Leung W,et al.Treatment of hepatoblastoma with high-dose chemotherapy and stem cell rescue:the pediatric blood and marrow transplant consortium experience and review of the literature[J].J Pediatr Hematol Oncol,2014,36(5):362-368.DOI:10.1097/MPH.0000000000000130.
4 Hishiki T,Matsunaga T,Sasaki F,et al.Outcome of hepatoblastomas treated using the Japanese Study Group for Pediatric Liver Tumor(JPLT)protocol-2:report from the JPLT[J].Pediatr Surg Int,2011,27(1):1-8.DOI:10.1007/s00383-010-2708-0.
5 O'Neill AF,Towbin AJ,Krailo MD,et al.Characterization of Pulmonary Metastases in Children With Hepatoblastoma Treated on Children's Oncology Group Protocol AHEP0731(The Treatment of Children With All Stages of Hepatoblastoma):A Report From the Children's Oncology Group[J].JClin Oncol,2017,35(30):3465-3473.DOI:10.1200/JCO.2017.73.5654.
6 Patriarca C,Macchi RM,Marschner AK,et al.Epithelial cell adhesion molecule expression(c D326)in cancer:a short review[J].Cancer Treat Rev,2012,38(1):68-75.DOI:10.1016/j.ctrv.2011.04.002.
7 Gires O,Stoecklein NH.Dynamic Ep CAM expression on circulating and disseminating tumor cells:causes and consequences[J].Cell Mol Life Sci,2014,71(22):4393-402.DOI:10.1007/s00018-014-1693-1.
8 Wang MH,Sun R,Zhou XM,et al.Epithelial cell adhesion molecule overexpression regulates epithelial-mesenchymal transition,stemness and metastasis of nasopharyngeal carcinoma cells via the PTEN/AKT/m TOR pathway[J].Cell Death Dis,2018,9(1):2.DOI:10.1038/s41419-017-0013-8.
9史淑君,康权.儿童离体或半离体肝切除自体肝移植术-不可切除性肝脏肿瘤的新治疗方式[J].临床小儿外科杂志,2017,16(6):537-541.DOI:10.3969/j.issn.1671-6353.2017.06.004.Shi SJ,Kang Q.Ex vivo liver tumor resection techniques and liver autotransplantation:another resource for otherwise unresectable malignancy in children[J].J Clin Ped Sur,2017,16(6):537-541.DOI:10.3969/j.issn.1671-6353.2017.06.004.
10 Ismail H,Broniszczak D,Kaliciński P,et a1.Changing treatment and outcome of children with hepatoblastoma:analysis of a single center experience over the last 20 years[J].J Pediatr Surg,2012,47(7):1331-1339.DOI:10.1016/j.jpedsurg.2011.11.073.
11 Armeanu-Ebinger S,Hoh A,Wenz J,et al.Targeting EpCAM(CD326)for immunotherapy in hepatoblastoma[J].Oncoimmunology,2013,2(1):e22620.DOI:10.4161/onci.22620.
12 Martowicz A,Seeber A,Untergasser G,et al.The role of EpCAM in physiology and pathology of the epithelium[J].Histol Histopathol,2016,31(4):349-355.DOI:10.14670/HH-11-678.
13 Kurtz JE,Dufour P.Adecatumumab:an anti-EpCAM monoclonal antibody,from the bench to the bedside[J].Expert Opin Biol Ther,2010,10(6):951-958.DOI:10.1517/14712598.2010.482098.
14 Yun WJ,Shin E,Lee K,et al.Clinicopathologic implication of hepatic progenitor cell marker expression in hepatoblastoma[J].Pathol Res Pract,2013,209(9):568-573.DOI:10.1016/j.prp.2013.06.015.
15 Browne M,Stellmach V,Cornwell M,et al.Gene transfer of pigment epithelium-derived factor suppresses tumor growth and angiogenesis in a hepatoblastoma xenograft model[J].Pediatric Research,2006,60(3):282-287.DOI:10.1203/01.pdr.0000232789.86632.91.
16 Naundorf S,Preithner S,Mayer P,et al.In vitro and in vivoactivity of MT201,a fully human monoclonal antibody for pancarcinoma treatment[J].International Journal of Cancer,2002,100(1):101-110.DOI:10.1002/ijc.10443.
17 Segota E,Bukowski RM.The promis of targeted therapy:cancer drugs become more specific[J].Clevel Clin J Med,2004,71(7):551-560.
18 Munz M,Baeuerle PA,Gires O.The emerging role of EpCAM in cancer and stem cell signaling[J].Cancer Res,2009,69(14):5627-5629.DOI:10.1158/0008-5472.CAN-09-0654.
19 Punt CJ,Nagy A,Douillard JY,et al.Edrecolomab alone or in combination with fluorouracil and folinic acid in the adjuvant treatment of stage III colon cancer:a randomised study[J].Lancet,2002,360(9334):671-677.DOI:10.1016/S0140-6736(02)09836-7.
20高茜,王永生.基于肿瘤相关抗原Ep-CAM的免疫治疗现状[J].山东医药,2008,48(14):146-147.DOI:10.3969/j.issn.1002-266X.2008.14.108.Gao X,Wang YG.Current status of immunotherapy based on tumor associated antigen Ep-CAM[J].Shandong Medical Journal,2008,48(14):146-147.DOI:10.3969/j.issn.1002-266X.2008.14.108.
21 Schmidt M,Rüttinger D,Sebastian M,et al.Phase IB study of the Ep CAM antibody adecatumumab combined with docetaxel in patients with Ep CAM-positive relapsed or refractory advanced-stage breast cancer[J].Ann Oncol,2012,23(9):2306-2313.DOI:10.1093/annonc/mdr625.
22 Richter CE,Cocco E,Bellone S,et al.High-grade,chemotherapy-resistant ovarian carcinomas overexpress epithelial cell adhesion molecule(Ep CAM)and are highly sensitive to immunotherapy with MT201,a fully human monoclonal anti-EpCAM antibody[J].Am J Obstet Gynecol,2010,203(6):582 e1-7.DOI:10.1016/j.ajog.2010.07.041.
23 Marschner N,Rüttinger D,Zugmaier G,et al.Phase II study of the human anti-epithelial cell adhesion molecule antibody adecatumumab in prostate cancer patients with increasing serum levels of prostate-specific antigen after radical prostatectomy[J].Urol Int,2010,85(4):386-395.DOI:10.1159/000318055.
24 Brischwein K,Schlereth B,Guller B,et al.MT110:a novel bispecific single-chain antibody construct with high efficacy in eradicating established tumors[J].Mol Immunol,2006,43(8):1129-1143.DOI:10.1016/j.molimm.2005.07.034.
25 Fiedler WM,Ritter B,Seggewiss R,et al.Phase I safety and pharmacology study of the Ep CAM/CD3-bispecific Bi TEantibody MT110 in patients with metastatic colorectal,gastric,or lung cancer[J].J Clin Oncol,2010,28(15S):2573-2573.DOI:10.1200/jco.2010.28.15_suppl.2573.
26 Str9hlein M,Lordick F,Rüttinger D,et al.Peritoneal carcinomatosis immunotherapy with the trifunctional anti-EpCAMx anti-CD3 antibody catumaxomab in patients with colon,gastric,or pancreatic cancer:Long-term results after a 2-year follow-up[J].J Clin Oncol,2009,27(15):302-323.
27 Parsons S,Murawa PX,Koralewski P,et al.Intraperitoneal treatment of malignant ascites due to epithelial tumors with catumaxomab:A phase II/III study[J].J Clin Oncol,2008,26(15S):3000.DOI:10.1200/jco.2008.26.15_suppl.3000.
28 Heiss MM,Str9hlein MA,Bokemeyer C,et al.The role of relative lymphocyte count as a biomarker for the effect of catumaxomab on survival in malignant ascites patients:results from a phase II/III study[J].Clin Cancer Res,2014,20(12):3348-3357.DOI:10.1158/1078-0432.CCR-13-2351.
29 Sulpice L.Epithelial cell adhesion molecule is a prognosis marker for intrahepatic cholangiocarcinoma[J].J Surg Res,2014,192(1):117-23.DOI:10.1016/j.jss.2014.05.017.
2董岿然.我国儿童胚胎性恶性实体肿瘤的研究和治疗进展[J].临床小儿外科杂志,2017,16(5):417-421.DOI:10.3969/j.issn.1671-6353.2017.05.001.Dong KR.Diagnosis and treatment of embryonic malignant tumors in children in China[J].J Clin Ped Sur,2017,16(5):417-421.DOI:10.3969/j.issn.1671-6353.2017.05.001.
3 Karski EE,Dvorak CC,Leung W,et al.Treatment of hepatoblastoma with high-dose chemotherapy and stem cell rescue:the pediatric blood and marrow transplant consortium experience and review of the literature[J].J Pediatr Hematol Oncol,2014,36(5):362-368.DOI:10.1097/MPH.0000000000000130.
4 Hishiki T,Matsunaga T,Sasaki F,et al.Outcome of hepatoblastomas treated using the Japanese Study Group for Pediatric Liver Tumor(JPLT)protocol-2:report from the JPLT[J].Pediatr Surg Int,2011,27(1):1-8.DOI:10.1007/s00383-010-2708-0.
5 O'Neill AF,Towbin AJ,Krailo MD,et al.Characterization of Pulmonary Metastases in Children With Hepatoblastoma Treated on Children's Oncology Group Protocol AHEP0731(The Treatment of Children With All Stages of Hepatoblastoma):A Report From the Children's Oncology Group[J].JClin Oncol,2017,35(30):3465-3473.DOI:10.1200/JCO.2017.73.5654.
6 Patriarca C,Macchi RM,Marschner AK,et al.Epithelial cell adhesion molecule expression(c D326)in cancer:a short review[J].Cancer Treat Rev,2012,38(1):68-75.DOI:10.1016/j.ctrv.2011.04.002.
7 Gires O,Stoecklein NH.Dynamic Ep CAM expression on circulating and disseminating tumor cells:causes and consequences[J].Cell Mol Life Sci,2014,71(22):4393-402.DOI:10.1007/s00018-014-1693-1.
8 Wang MH,Sun R,Zhou XM,et al.Epithelial cell adhesion molecule overexpression regulates epithelial-mesenchymal transition,stemness and metastasis of nasopharyngeal carcinoma cells via the PTEN/AKT/m TOR pathway[J].Cell Death Dis,2018,9(1):2.DOI:10.1038/s41419-017-0013-8.
9史淑君,康权.儿童离体或半离体肝切除自体肝移植术-不可切除性肝脏肿瘤的新治疗方式[J].临床小儿外科杂志,2017,16(6):537-541.DOI:10.3969/j.issn.1671-6353.2017.06.004.Shi SJ,Kang Q.Ex vivo liver tumor resection techniques and liver autotransplantation:another resource for otherwise unresectable malignancy in children[J].J Clin Ped Sur,2017,16(6):537-541.DOI:10.3969/j.issn.1671-6353.2017.06.004.
10 Ismail H,Broniszczak D,Kaliciński P,et a1.Changing treatment and outcome of children with hepatoblastoma:analysis of a single center experience over the last 20 years[J].J Pediatr Surg,2012,47(7):1331-1339.DOI:10.1016/j.jpedsurg.2011.11.073.
11 Armeanu-Ebinger S,Hoh A,Wenz J,et al.Targeting EpCAM(CD326)for immunotherapy in hepatoblastoma[J].Oncoimmunology,2013,2(1):e22620.DOI:10.4161/onci.22620.
12 Martowicz A,Seeber A,Untergasser G,et al.The role of EpCAM in physiology and pathology of the epithelium[J].Histol Histopathol,2016,31(4):349-355.DOI:10.14670/HH-11-678.
13 Kurtz JE,Dufour P.Adecatumumab:an anti-EpCAM monoclonal antibody,from the bench to the bedside[J].Expert Opin Biol Ther,2010,10(6):951-958.DOI:10.1517/14712598.2010.482098.
14 Yun WJ,Shin E,Lee K,et al.Clinicopathologic implication of hepatic progenitor cell marker expression in hepatoblastoma[J].Pathol Res Pract,2013,209(9):568-573.DOI:10.1016/j.prp.2013.06.015.
15 Browne M,Stellmach V,Cornwell M,et al.Gene transfer of pigment epithelium-derived factor suppresses tumor growth and angiogenesis in a hepatoblastoma xenograft model[J].Pediatric Research,2006,60(3):282-287.DOI:10.1203/01.pdr.0000232789.86632.91.
16 Naundorf S,Preithner S,Mayer P,et al.In vitro and in vivoactivity of MT201,a fully human monoclonal antibody for pancarcinoma treatment[J].International Journal of Cancer,2002,100(1):101-110.DOI:10.1002/ijc.10443.
17 Segota E,Bukowski RM.The promis of targeted therapy:cancer drugs become more specific[J].Clevel Clin J Med,2004,71(7):551-560.
18 Munz M,Baeuerle PA,Gires O.The emerging role of EpCAM in cancer and stem cell signaling[J].Cancer Res,2009,69(14):5627-5629.DOI:10.1158/0008-5472.CAN-09-0654.
19 Punt CJ,Nagy A,Douillard JY,et al.Edrecolomab alone or in combination with fluorouracil and folinic acid in the adjuvant treatment of stage III colon cancer:a randomised study[J].Lancet,2002,360(9334):671-677.DOI:10.1016/S0140-6736(02)09836-7.
20高茜,王永生.基于肿瘤相关抗原Ep-CAM的免疫治疗现状[J].山东医药,2008,48(14):146-147.DOI:10.3969/j.issn.1002-266X.2008.14.108.Gao X,Wang YG.Current status of immunotherapy based on tumor associated antigen Ep-CAM[J].Shandong Medical Journal,2008,48(14):146-147.DOI:10.3969/j.issn.1002-266X.2008.14.108.
21 Schmidt M,Rüttinger D,Sebastian M,et al.Phase IB study of the Ep CAM antibody adecatumumab combined with docetaxel in patients with Ep CAM-positive relapsed or refractory advanced-stage breast cancer[J].Ann Oncol,2012,23(9):2306-2313.DOI:10.1093/annonc/mdr625.
22 Richter CE,Cocco E,Bellone S,et al.High-grade,chemotherapy-resistant ovarian carcinomas overexpress epithelial cell adhesion molecule(Ep CAM)and are highly sensitive to immunotherapy with MT201,a fully human monoclonal anti-EpCAM antibody[J].Am J Obstet Gynecol,2010,203(6):582 e1-7.DOI:10.1016/j.ajog.2010.07.041.
23 Marschner N,Rüttinger D,Zugmaier G,et al.Phase II study of the human anti-epithelial cell adhesion molecule antibody adecatumumab in prostate cancer patients with increasing serum levels of prostate-specific antigen after radical prostatectomy[J].Urol Int,2010,85(4):386-395.DOI:10.1159/000318055.
24 Brischwein K,Schlereth B,Guller B,et al.MT110:a novel bispecific single-chain antibody construct with high efficacy in eradicating established tumors[J].Mol Immunol,2006,43(8):1129-1143.DOI:10.1016/j.molimm.2005.07.034.
25 Fiedler WM,Ritter B,Seggewiss R,et al.Phase I safety and pharmacology study of the Ep CAM/CD3-bispecific Bi TEantibody MT110 in patients with metastatic colorectal,gastric,or lung cancer[J].J Clin Oncol,2010,28(15S):2573-2573.DOI:10.1200/jco.2010.28.15_suppl.2573.
26 Str9hlein M,Lordick F,Rüttinger D,et al.Peritoneal carcinomatosis immunotherapy with the trifunctional anti-EpCAMx anti-CD3 antibody catumaxomab in patients with colon,gastric,or pancreatic cancer:Long-term results after a 2-year follow-up[J].J Clin Oncol,2009,27(15):302-323.
27 Parsons S,Murawa PX,Koralewski P,et al.Intraperitoneal treatment of malignant ascites due to epithelial tumors with catumaxomab:A phase II/III study[J].J Clin Oncol,2008,26(15S):3000.DOI:10.1200/jco.2008.26.15_suppl.3000.
28 Heiss MM,Str9hlein MA,Bokemeyer C,et al.The role of relative lymphocyte count as a biomarker for the effect of catumaxomab on survival in malignant ascites patients:results from a phase II/III study[J].Clin Cancer Res,2014,20(12):3348-3357.DOI:10.1158/1078-0432.CCR-13-2351.
29 Sulpice L.Epithelial cell adhesion molecule is a prognosis marker for intrahepatic cholangiocarcinoma[J].J Surg Res,2014,192(1):117-23.DOI:10.1016/j.jss.2014.05.017.