联合自杀基因与IL-2基因转移疗法的抗肿瘤效果及其抗肿瘤免疫应答的诱导作用

单用自杀基因疗法或单用细胞因子基因疗法抗肿瘤效果不理想,本研究中我们观察了大肠杆菌胞嘧啶脱胺酶(CD)基因与白细胞介素2(IL-2)基因联合转移对荷瘤小鼠的治疗效果及其对抗肿瘤免疫的诱导作用。复制荷瘤小鼠模型后在荷瘤部位注射表达CD基因的重组腺病毒(AdCD)及表达小鼠IL-2基因的重组腺病毒(AdIL2),并连续10天、每天1次腹腔注射5氟胞嘧啶(5FC)对荷瘤小鼠进行治疗。结果表明,AdCD/5FC/AdIL2联合基因治疗能显著抑制荷瘤小鼠皮下肿瘤的生长,并明显延长其生存期(P<0.01)。联合基因治疗组小鼠肿瘤细胞发生明显的坏死,瘤内及瘤周有大量的炎性细胞浸润,瘤内CD4~ 和CD8~ T细胞明显增加,脾细胞NK和CIL杀伤活性明显高于单用AdCD/5FC、对照病毒AdLacZ/5FC或PBS组。实验结果表明,联合应用自杀基因与细胞因子基因治疗可更有效诱导机体的抗肿瘤免疫反应,从而更显著地抑制荷瘤小鼠肿瘤的生长。     

腺病毒介导的胞嘧啶脱氨酶自杀基因联合IL-2基因疗法的肿瘤治疗作用及免疫机理分析

目的以腺病毒作为载体，将大肠杆菌胞嘧啶脱氨酶（ＣＤ）基因与小鼠ＩＬ－２基因联合转移，研究其体内抗肿瘤作用及免疫机理。方法小鼠皮下接种黑色素瘤Ｂ１６Ｆ１０细胞后３天，肿瘤局部注射表达ＩＬ－２的重组腺病毒ＡｄＩＬ－２和表达ＣＤ的重组腺病毒ＡｄＣＤ，然后连续１０天给予５－氟胞嘧啶（５－Ｆｃ）３００ｍｇ／ｋｇ进行治疗。结果联合治疗组荷瘤小鼠皮下肿瘤结节的生长明显受到抑制，小鼠存活期明显长于ＡｄＩＬ－２、ＡｄＣＤ／５－Ｆｃ、ＡｄｌａｃＺ／５－Ｆｃ或ＰＢＳ组。经联合治疗后，小鼠脾细胞的ＮＫ活性和ＣＴＬ杀伤活性明显增强；肿瘤瘤体内ＣＤ４、ＣＤ８细胞浸润增加；肿瘤细胞表达Ｈ－２Ｋｂ和Ｂ７－１分子明显增加。结论联合应用自杀基因和ＩＬ－２基因治疗，一方面可以明显抑制荷瘤小鼠肿瘤生长，另一方面可以提高机体对肿瘤细胞免疫应答，增加机体的抗肿瘤作用，是肿瘤基因治疗中一条行之有效的途径。     

Dose of adenoviral vectors expressing interleukin-2 plays an important role in combined gene therapy with cytosine deaminase/5-fluorocytosine: preclinical consideration.

Using a syngeneic murine model, we investigated the therapeutic efficacy of combined gene therapy using adenoviral vectors expressing murine interleukin-2 (AdmIL-2) and Escherichia coli cytosine deaminase (AdCD). In a subcutaneous tumor model, tumor-bearing mice were treated with an intratumoral injection of adenoviral vectors and received an intraperitoneal administration of 5-fluorocytosine (5-FC). Only the mice treated with AdCD (2 x 10(8) pfu) and an intermediate dose of AdmIL-2 (1 x 10(6) pfu) survived significantly longer than mice treated with AdCD alone (P < 0.01). Moreover, 40% of these treated mice obtained complete remission from tumor-bearing status. The cytotoxicity of splenocytes obtained from the treated mice was related to the survival period. Tumor-specific cytotoxic T lymphocyte assay showed that the cell-mediated cytotoxic response was specific for parental tumor cells. In a hepatic metastasis model, mice treated with an intravenous administration of both AdCD (2 x 10(8) pfu) and an intermediate dose of AdmIL-2 (1 x 10(6) pfu) demonstrated the most significant reduction of metastatic foci and the longest survival following a 5-FC administration. These results suggest that gene therapy combined with AdmIL-2 and AdCD may be a promising strategy for clinical application and, in addition, that translation of combined gene therapy from murine models into the clinical setting will require careful attention to the variables of cytokine expression levels in the design of clinical trials and in the evaluation of treatment efficacy.     

Dose of Adenoviral Vectors Expressing Interleukin-2 Plays an Important Role in Combined Gene Therapy with Cytosine Deaminase/5–Fluorocytosine: Preclinical Consideration

Using a syngeneic murine model, we investigated the therapeutic efficacy of combined gene therapy using adenoviral vectors expressing murine interleukin-2 (AdmIL-2) and Escherichia coli cytosine deaminase (AdCD). In a subcutaneous tumor model, tumor-bearing mice were treated with an intratumoral injection of adenoviral vectors and received an intraperitoneal administration of 5–fluorocytosine (5–FC). Only the mice treated with AdCD (2×10⁸ pfu) and an intermediate dose of AdmIL-2 (1×10⁶ pfu) survived significantly longer than mice treated with AdCD alone ( P <0.01). Moreover, 40% of these treated mice obtained complete remission from tumor-bearing status. The cytotoxicity of splenocytes obtained from the treated mice was related to the survival period. Tumor-specific cytotoxic T lymphocyte assay showed that the cell-mediated cytotoxic response was specific for parental tumor cells. In a hepatic metastasis model, mice treated with an intravenous administration of both AdCD (2×l0⁸ pfu) and an intermediate dose of AdmIL-2 (1×10⁶ pfu) demonstrated the most significant reduction of metastatic foci and the longest survival following a 5–FC administration. These results suggest that gene therapy combined with AdmIL-2 and AdCD may be a promising strategy for clinical application and, in addition, that translation of combined gene therapy from murine models into the clinical setting will require careful attention to the variables of cytokine expression levels in the design of clinical trials and in the evaluation of treatment efficacy.     

腺病毒介导的大肠杆菌CD自杀基因体内外对肿瘤杀伤作用的研究

编委会 Editorial Board名誉主编 Honorary Editor-in-chief吴孟超 Wu Meng chao(Second Military Medical University,Shanghai 200433学术顾问 Academic Advisers巴德年 Ba Denian(Chinese Academy of Medical Sciences,Beijing 100730)刘新垣 Liu Xinyuan(Shanghai Institute of Biochemistry,Chinese Academy of Sciences,Shanghai 200031)吴旻 WuMin(Cancer Institute,Chinese Academy of Medical Sciences,Beijing 100021)汤钊猷 Tang Zhaoyou(Shanghai Medical University,Shanghai 200032)主编 Editor-in-chief张友会 Zhang Youhui(Cancer Institute,Chinese Academy of Medical Sciences,Beijing,100021)副主编 Associate Editor-in-chief崔正言 Cul Zhenyan(Department of Immunology,Shandong Academy of Medical Sciences,Jinan 250001)钱振超 Qian Zhenchao(Department of Patho-physiology,Dalian Medical University,Dalian 116027)何球藻 He Qiuzao(Department of Immunology,Shanghai Medical University,Shanghai 200032)董志伟 Dong Zhiwei(Cancer Institute,Chinese Academy of Medical Sciences,Beijing 100021)常务副主编 Managing Editor-in-chief     

Adenovirus-mediated combined P16 gene and GM-CSF gene therapy for the treatment of established tumor and induction of antitumor immunity

Antitumor effects of combined transfer of P16 and cytokine genes were investigated in this study. The adenovirus harboring the P16 gene (AdP16) and murine granulocyte-macrophage colony-stimulating factor gene (AdGM-CSF) were utilized for the treatment of established tumors. The mice were inoculated subcutaneously with Renca cells and, 6 days later, received an intratumoral injection of AdP16 in the presence or absence of AdGM-CSF. The results demonstrated that tumor-bearing mice treated with AdP16 in combination with AdGM-CSF showed more potent inhibition of tumor growth and survived much longer than did mice treated with AdP16, AdGM-CSF, adenovirus expressing beta-galactosidase, or phosphate-buffered saline alone (P<.01). The tumor mass showed obvious necrosis and inflammatory cell infiltration, and more CD(4)(+) and CD(8)(+) T cells infiltrating the tumor after combined therapy. After combined therapy, the expression of MHC-1 (H-2K(d)) and Fas molecules on freshly isolated tumor cells increased greatly. The activity of specific cytotoxic T lymphocytes was also found to be induced more significantly after the combined therapy (P<.01). Our results demonstrated that combined therapy with P16 and GM-CSF genes can inhibit the growth of established tumors in mice significantly and induce antitumor immunity of the host efficiently.     

腺病毒介导的胞嘧啶脱氨酶自杀基因联合IL—2基因疗法的肿瘤治 …

以腺病毒作为载体,将大肠杆菌胞嘧啶脱氨酶基因与小鼠ＩＬ－２基因联合转移,研究其体内抗肿瘤作用及免疫机理。方法小鼠皮下接种黑色素瘤Ｂ１６Ｆ１０细胞后３天,肿瘤局部 注射表达ＩＬ－２的重要腺病毒ＡｄＩＬ－２和表达的ＣＤ的重组腺病毒ＡｄＣＤ,然后连续０天给予５－氟胞嘧啶３００ｍｇ／ｋｇ进行治疗。     

CD自杀基因联合淋巴细胞趋化因子基因疗法的肿瘤治疗作用及免疫机理研究

本研究以腺病毒作为载体,将大肠杆菌胞嘧啶脱氨酶(CD)基因与小鼠淋巴细胞趋化因子(Ltn)基因体内联合转染,观察了其抗肿瘤效应并分析了免疫机理.小鼠皮下接种结肠腺癌CT26细胞后3天,肿瘤局部注射表达Ltn的重组腺病毒AdLtn和表达CD的重组腺病毒AdCD,然后连续10天给予5一氟胞嘧啶(5-FC)300mg/kg进行治疗,结果表明,联合治疗组荷瘤小鼠皮下肿瘤结节的生长受到明显抑制,小鼠存活期明显长于单用AdLtn治疗组或单用AdCD/5-FC治疗组.经联合治疗后小鼠脾细胞的NK活性和对(37结肠腺癌细胞的CTL杀伤活性明显增强.瘤体细胞FACS分析结果表明,经联合基因治疗后,肿瘤组织CD4~ 、CD8~ 细胞浸润增加,结肠腺癌细胞表达H-2Kd和B7-1分子明显增加.提示经CD自杀基因和Ltn基因联合治疗后,肿瘤细胞免疫原性增加.本研究结果表明联合应用自杀基因和Ltn基因治疗可以提高机体对肿瘤细胞免疫的应答,增加机体的抗肿瘤作用,是肿瘤基因治疗中一条新的途径.     

p16基因重组质粒的构建及其对人肺癌细胞的抑制作用

表达大肠杆菌胞嘧啶脱胺酶(CD)基因的重组腺病毒AdCD体外转染小鼠黑色素瘤细胞B16F10,结果显示转染了CD基因的B16F10细胞对5-氟胞嘧啶(5FC)的敏感性显著提高.将经AdCD/5FC系统处理的B16F10细胞上清倍比稀释后.加至野生型B16F10细胞中,发现当上清仅占6.25%时即可对野生型B16F10细胞发挥明显的杀伤作用,提示AdCD/5FC介导的旁观者效应可能是通过5FC经CD酶代谢产生的毒性产物扩散而实现的.本实验还观察了CD基因体内转染后的杀伤效果,荷瘤小鼠经注射AdCD并连续10天给予5FC治疗后,与PBS、对照病毒AdLacZ/5FC治疗小鼠比较,小鼠肿瘤生长明显受到抑制,小鼠存活期明显延长.     

氟胞嘧啶/胞嘧啶脱氨酶自杀基因疗法结合热休克蛋白-多肽复合物治疗小鼠胃癌

目的；研究氟胞嘧啶／胞嘧啶脱氨酶（5－FC／CD）基因疗法与热休克蛋白－多肽复合物（FSP70－PC）免疫疗法联合抗肿瘤效果。方法；将携带CD基因的重组腺病毒注射到小鼠MFC瘤体内，腹腔注射5－FC，同时皮下接种HSP70－PC。结果；经联合治疗后，70％荷瘤小鼠肿瘤体答缩小，消退，小鼠存活期延长，细胞毒T淋巴细胞（CTL）杀伤活性增高，CD4^ 及CD^8 T细胞浸润明显。结论：5－FC／CD基因疗法结合HSP－PC免疫疗法抗小鼠MFC瘤作用显著，具有临床应用前景。     

大肠杆菌CD自杀基因转染诱导肿细胞凋亡及旁观者效应的研究

以重组腺病毒AdCD为载体将大肠杆菌胞嘧啶脱氨酶(CD)基因体外传染小鼠红白血病细胞FBL3,结果显示,转染了CD基因的FBL3细胞对5-氟胞嘧啶(5-FC)的敏感性显著提高,进一步研究发现,AdCD/5-FC系统可以诱导肿瘤细胞发生凋亡;将经AdCD/5-FC处理过的FBL3细胞上清倍比稀释后,加入到野生型FBL3细胞中,发现当上清仅占6.25％时,即对野生型FBL3细胞发挥明显的杀伤作用,提示旁观者效应在AdCD介导的细胞毒作用中起着重要的作用。本实验还观察了CD基因体内转染后的杀伤效果,荷瘤小鼠局部注射AdCD并连续10天给予5-FC(300mg/kg)治疗后,小鼠皮下肿瘤结节的生长受到明显抑制。     

5-FC/CD 自杀基因疗法结合热休克蛋白-多肽复合物瘤苗治疗小鼠黑色素瘤的研究

 目的　研究 5 -氟尿嘧啶 /胞嘧啶脱氨酶 (5 FC/ CD)基因疗法与热休克蛋白 -多肽复合物 (HSP- PC)瘤苗免疫疗法联合抗肿瘤效果。方法　将携带 CD基因的重组腺病毒注射到小鼠黑色素瘤体内 ,腹腔注射 5 - FC,同时皮下接种 HSP70 - PC。结果　经联合治疗后 ,70 %荷瘤小鼠肿瘤体积缩小、消退 ,小鼠存活期延长 ,肿瘤组织明显坏死 ,炎症细胞、CD+4 及 CD+8T细胞浸润明显。结论5 - FC/ CD基因疗法结合 HSP- PC瘤苗免疫疗法抗小鼠黑色素瘤作用显著 ,具有临床应用前景。     

肺腺癌组织特异性自杀基因治疗实验研究

目的 探讨肺腺癌组织特异性自杀基因治疗的安全性及有效性。方法 采用病毒感染法，将癌胚抗原(CEA)基因启动子所驱动的CD基因的组织特异性逆转录病毒载体(G1CEACDNa)，导入分泌CEA的肺腺癌细胞系A549细胞．研究裸鼠体内抑瘤效果；应用重组逆转录病毒裸鼠体内治疗A549肿瘤，观察G1CEACDNa／5-氟胞嘧啶(5-FC)对A549细胞致瘤裸鼠的治疗作用及毒副反应。结果 (1)将转基因的A549细胞和未转基因的A549细胞接种至裸鼠皮下．两者成瘤性无明显差异；(2)在转基因细胞致瘤裸鼠实验中，5-FC对转CEA启动子调控自杀基因的肿瘤生长具有明显的抑制作用；(3)将G1CEACDNa重组逆转录病毒上清直接注射到裸鼠成瘤部位．然后腹腔内注射5-FC同样获得明显的抑瘤效果；(4)与直接注射5-FU相比，组织特异性自杀基因治疗对骨髓的抑制明显降低。结论 组织特异性自杀基因治疗可能成为肿瘤治疗个体化的重要方法之一。     

SCF或（和）GM—CSF体内基因转染对“自杀基因”疗法抗肿瘤作用的增？…

目的 通过增强体内抗原提呈功能提高“自杀基因”疗法疗效,探讨其相关免疫学机理。方法 采用腺病毒介导的ＳＣＦ,ＧＭ－ＣＳＦ基因体内转染联合ＣＤ／５ＦＣ“自杀基因”疗法治疗小鼠的ＣＴ２６结肠腺癌,观察肿瘤的生长和荷瘤小鼠的存活期,不同方法所诱导的ＣＴＬ杀伤活性及肿瘤局部的细胞因子表达。结果 一次性低剂量腺病毒介导的ｍＧＭ－ＣＳＦ或（和）ｍＳＣＦ基因的体内转染,能增强“自杀基因”疗法的疗效,在肿瘤局部出     

腺病毒介导的CD/5FC自杀基因疗法对小鼠结肠腺癌的治疗作用及相关免疫机理的研究

大肠杆菌胞嘧啶脱氨酶CD可以将前体药物5FC代谢为毒性产物5FU,具有抗肿瘤作用.本课题我们观察了腺病毒介导的CD/5FC自杀基因疗法对小鼠结肠癌生长的治疗作用并探讨其作用机理.结果表明,首先,CD/5FC自杀基因疗法小鼠结肠癌CT26体内外生长均具有显著的抑制作用,4O%的荷瘤小鼠经过治疗后长期存活;其次,我们还研究了自杀基因疗法治疗肿瘤时可能涉及的免疫机理.结果发现在以CD/5FC系统治疗结肠腺癌小鼠过程中能在一定程度上诱导出机体的抗肿瘤免疫反应,包括脾脏CT6L活性的增高、肿瘤局部浸润免疫细胞表达,DEC-205、B7-2、I-A~(b,d)分子增加等.本实验结果提示虽然CD/5FC系统可以诱导机体产生一定的抗肿瘤免疫反应,但其程度不够显著.     

Gene therapy of metastatic colon carcinoma: regression of multiple hepatic metastases by adenoviral expression of bacterial cytosine deaminase

Colon carcinoma accounts for 20% of deaths due to malignancies in the Western world. Once metastases occur, therapeutic options are limited, with an approximate 5-year survival of only 5%. To investigate the potential of new gene therapeutic approaches, a hepatic micrometastasis model of colon carcinoma in BALB/c mice was established. Inoculation of syngeneic MCA26 colon carcinoma cells into the spleens of 18- to 20-week-old mice resulted in the formation of multiple hepatic metastases. Selective transduction of developing hepatic metastases was demonstrated using a beta-galactosidase-expressing recombinant adenovirus. Cytosine deaminase (CD) can metabolize 5-fluorocytosine into the chemotherapeutic reagent 5-fluorouracil (5FU). The antitumoral potential of this suicide gene therapy approach was explored by systemic application of a recombinant replication-deficient adenovirus encoding for the bacterial CD gene under the control of the cytomegalovirus promoter (Ad.CMV-CD). Injection into the tail vein of tumor-bearing mice resulted in delayed tumor growth with significant reduction in hepatic metastases. The potential of this experimental approach for possible future clinical applications was evaluated by investigating adenoviral transduction efficiency, 5FU sensitivity, and 5-fluorocytosine-dependent Ad.CMV-CD toxicity in a variety of human colon cancer cell lines. Although the murine cell lines MCA26 and CC36 were highly sensitive to 5FU, the human colon cancer cell lines showed a 1-100 times higher resistance to 5FU. Specific Ad.CMV-CD toxicity correlates with 5FU toxicity. Transduction efficiency in human colon carcinoma cell lines was shown to be 10-1700 times higher compared with murine cell lines, thus compensating for 5FU resistance. In conclusion, suicide gene therapy using CD may be promising as an adjuvant treatment regimen for hepatic micrometastases of human colon carcinoma.     

Interleukin-12 gene therapy of a weakly immunogenic mouse mammary carcinoma results in reduction of spontaneous lung metastases via a T-cell-independent mechanism

In our previous studies using gene gun-mediated delivery of interleukin 12 (IL-12) cDNA in vivo, we observed T-cell-mediated regression of established murine tumors and demonstrated the induction of systemic immunity in test animals. In this study, we further characterized the antitumoral and anti-metastatic effect of this gene therapy approach by employing two murine metastatic mammary tumor models: the immunogenic TS/A adenocarcinoma and the weakly immunogenic 4T1 adenocarcinoma. In the TS/A model, gene transfer into the skin overlying an established intradermal tumor with an IL-12 cDNA expression vector resulted in complete tumor regression in 50% of mice followed by the development of immunological memory. In contrast, the growth of the intradermal 4T1 tumors was not affected by the IL-12 gene therapy protocol. However, this treatment resulted in a substantial reduction of spontaneous metastases in the lungs of 4T1 tumor-bearing mice and significantly prolonged their survival time. T cells were not required for this anti-metastatic effect, because it was also observed in nude mice and in mice depleted of CD4+ and CD8+ T cells. Tumor-draining lymph node cells obtained from 4T1 tumor-bearing mice treated with IL-12 cDNA exhibited increased natural killer (NK) activity and produced enhanced levels of interferon-gamma (IFN-gamma) compared with similar mice treated with luciferase cDNA. In addition, in vivo depletion of NK cells or neutralization of IFN-gamma resulted in partial suppression of the anti-metastatic effect of IL-12 gene therapy, suggesting the involvement of both NK cells and IFN-gamma in this effect.     

Expression of Escherichia coli uracil phosphoribosyltransferase gene in murine colon carcinoma cells augments the antitumoral effect of 5-fluorouracil and induces protective immunity

Uracil phosphoribosyltransferase (UPRT) of Escherichia coli origin can convert 5-fluorouracil (5-FU), a chemotherapeutic agent widely used for solid tumors, to an active intermediate, 5-fluorouridine-5'-monophosphate, as mammalian orotate phosphoribosyltransferase does. To examine whether the E. coli UPRT gene expressed in tumor cells can confer increased sensitivity to 5-FU, we retrovirally transduced Colon 26 cells, a murine colon carcinoma cell line, with the UPRT gene (Colon 26/UPRT cells) and tested the in vivo antitumoral effect of 5-FU in syngeneic immunocompetent mice. After 5-FU administration, tumors of Colon 26/UPRT cells regressed, whereas those of wild-type cells were unaffected. The mice that once eliminated Colon 26/UPRT tumors after 5-FU treatment rejected wild-type cells that were subsequently inoculated but not irrelevant syngeneic tumor cells. This suicide gene/prodrug system was less efficient in nude mice, suggesting that mature alphabeta T cells play a role in the antitumoral effect. The cytotoxicity mediated by the bystander effect was marginal in this system, contrary to the herpes simplex virus-thymidine kinase gene/ganciclovir system. Therefore, expression of the UPRT gene in tumor cells followed by 5-FU administration is a possible strategy for cancer gene therapy, but potentiation of the bystander effect is required for its therapeutic application.     

Murine model of cancer gene therapy

In order to examine how the adenovirus-mediated gene therapy induce anti-adenoviral immunity, we set up experiments for a murine cancer model. We first administered adenovirus carrying LacZ (AxCALacZ) gene to DBA/2 mice intraperitoneally, then two weeks after administration of the virus, P815 tumor cells infected with adenovirus carrying LacZ gene were inoculated intraperitoneally to the AxCALacZ immune mice. The mice rejected the P815 tumor infected with AxCALacZ and survived for long periods. The peritoneal T cells in adenovirus immune mice had a strong killing effect of adenovirus-infected P815 cells. Further, parental P815 tumor cells were inoculated into the mice, which rejected the P815 tumor infected with AxCALacZ. All mice rejected P815 tumor cells and survived for long periods. These mice developed P815 specific tumor immunity, which was confirmed by a cytotoxicity assay.     

Antitumoral vaccination with granulocyte-macrophage colony-stimulating factor or interleukin-12-expressing DHD/K12 colon adenocarcinoma cells

Immunomodulating gene therapy for the treatment of malignant diseases is under extensive investigation. In this study, we induced an antitumoral immune response with murine interleukin-12 (mIL-12) and murine granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor cells in a model of peritoneal carcinomatosis. Intraperitoneal injection of DHD/K12 tumoral cells engineered to produce IL-12 or GM-CSF did not generate any tumors, whereas untransduced DHD/K12 cells gave rise to peritoneal carcinomatosis. IL-12-expressing DHD/K12 cells also protected against tumors derived from coinjected parental cells. To test whether cytokine-producing cells could elicit a memory antitumoral immune response, animals received a challenge with parental DHD/K12 cells 35 days after the injection of proliferating or irradiated DHD/K12 engineered cells. Under our experimental conditions, irradiated tumor cells did not generate any antitumoral immunity. In contrast, tumor development was delayed and survival increased in the animals vaccinated with cytokine-secreting proliferating cells. A specific cytotoxic T-lymphocyte response against DHD/K12 parental cells was observed after vaccination with GM-CSF-expressing cells. Our results demonstrated that intraperitoneal vaccination with IL-12- or GM-CSF-expressing adenocarcinoma cells induced a systemic immune antitumoral response that may be useful as an adjuvant therapy after surgical resection of colorectal cancer.