Adenovirus-mediated TNF-alpha gene transfer induces significant tumor regression in mice

Recombinant adenovirus vectors are highly efficient at in vitro and in vivo gene delivery. The in vitro infection of a mouse colon adenocarcinoma cell line MCA-26 with the adenovirus AdV-LacZ can reach a maximal 75% of infectivity at an MOI of 1000. Intratumoral injection of AdV-LacZ (2X10(9) pfu) resulted in substantial gene transfer in nearly 70% of MCA-26 tumors. After the in vitro infection of AdV-TNF-alpha, infected MCA-26 cells showed significant secretion of TNF-alpha (45 ng/ml/10(6) cells) in tissue culture. The secretion peaks at day 2 and is diminished at day 4 following the viral infection. Infected MCA-26 tumor cells secreting TNF-alpha significantly reduced their tumorigenicity in syngeneic BALB/c mice. In mice bearing small tumors, intratumoral injection of 2X10(9) pfu of AdV-TNF-alpha virus with a repeated booster treatment resulted in complete regression of three tumors and significant diminution of the other two with a mean tumor-weight of 0.16 g; this is in contrast to 0.85 and 1.62 g for tumors injected with the control AdV-pLpA and PBS respectively (p < 0.01). Mice with complete tumor regression further developed protective immunity against the second challenge of MCA-26 inoculation. In mice bearing large tumors, this treatment also caused significant inhibition of tumor growth with a mean tumor weight of 0.65 g vis-a-vis 3.05 g for tumors injected with the control AdV-pLpA. On the contrary, in mice bearing large tumors, the treatment of tumors with pCI-TNF-alpha delivered by the gene gun did not induce significant tumor inhibition. These results indicate that the adenoviral delivery of TNF-alpha gene is more efficient than the particle-mediated gene gun device, and that adenovirus-mediated cytokine gene therapy may be a useful approach in the clinical management of human solid tumors.     

Adenovirus-mediated interferon alpha gene transfer induces regional direct cytotoxicity and possible systemic immunity against pancreatic cancer

We previously demonstrated a characteristically high sensitivity of pancreatic cancer cells to interferon alpha (IFN-alpha) gene transfer, which induced a more prominent growth suppression and cell death in pancreatic cancer cells than in other types of cancers and normal cells. The IFN-alpha protein can exhibit both direct cytotoxicity and indirect immunological antitumour activity. Here, we dissected and examined the two mechanisms, taking advantage of the fact that IFN-alpha did not show any cross-species activity in its in vivo effect. When a human IFN-alpha adenovirus was injected into subcutaneous xenografts of human pancreatic cancer cells in nude mice, tumour growth was significantly suppressed due to cell death in an adenoviral dose-dependent manner. The IFN-alpha protein concentration was markedly increased in the injected subcutaneous tumour, but leakage of the potent cytokine into the systemic blood circulation was minimal. When a mouse IFN-alpha adenovirus was injected into the same subcutaneous tumour system, all mice showed significant tumour inhibition, an effect that was dependent on the indirect antitumour activities of IFN-alpha, notably a stimulation of natural killer cells. Moreover, in this case, tumour regression was observed not only for the injected subcutaneous tumours but also for the untreated tumours at distant sites. This study suggested that a local IFN-alpha gene therapy is a promising therapeutic strategy for pancreatic cancer, due to its dual mechanisms of antitumour activities and lack of significant toxicity.     

Anti-tumor immunity generated by tumor cells engineered to express B7-1 via retroviral or adenoviral gene transfer

We engineered B7-1 retroviral and adenoviral gene transfer systems and studied them in four immunogenic tumor models. M-MSV tumor cells, but not K-Balb, 38.2 and 205 tumor cells, when expressing B7-1 by retroviral transduction were rejected and conferred protection against a tumor challenge. Transient expression of B7-1 after transduction with adenoviruses was less efficient. We observed enhanced cytotoxic T-lymphocyte activity accompanied by increased secretion of IL-6, IFNgamma and GM-CSF. GM-CSF secretion correlated with tumor rejection. Enhanced IFNgamma but unchanged IL-4 secretion suggested a T-helper 1-mediated anti-tumor immune response.     

Vaccination of tumor cells transfected with the B7-1 (CD80) gene induces the anti-metastatic effect and tumor immunity in mice

The present study demonstrates that the transfection of B7-1 or its variant MB7-2 genes into MHC class I⁺ tumor cells (B16-BL6 or K1735-M2 melanoma) resulted in the remarkable reduction of lung metastasis caused by i.v. injection into immunocompetent syngeneic mice. However, i.v. injection of the transfectants into T cell-deficient nude mice did not affect reduction of lung tumor colonies as compared with parental wild-type tumors, suggesting that such an inhibitory effect was closely associated with T cell-mediated responses. The reduced metastasis of B7⁺ tumor cells consequently led to the significant prolongation of survival. Expression of B7 on tumor cells did not influence the tumorigenicity in vivo and tumor cell invasion into basement membrane Matrigel in vitro. We also found that immunization of X-irradiated B7 transfectants was effective as a tumor vaccine for preventing lung metastasis caused by i.v. injection of B7⁻ parental B16-BL6 cells but not against other syngeneic 3LL tumors. Thus, the B7-mediated anti-metastatic effect was tumor-specific. Vaccinations of irradiated B7⁺ tumor cells before and after surgical excision of the s.c. inoculated primary B7⁻ tumors on day 21 achieved effectively the prevention of spontaneous lung metastasis. Our report that vaccination of irradiated B7⁺ tumor cells led to a therapeutic effect in an established tumor metastasis model clearly expands and confirms previous related observations. © 1996 Wiley-Liss, Inc.     

Suppression of tumor growth and pulmonary metastasis in murine osteosarcoma using gene therapy

We evaluated the effect of gene therapy in the murine osteosarcoma cell line, LM8, which preferentially metastasizes to the lungs. LM8 cells were transduced with the gene for a herpes simplex virus thymidine kinase (HSV-tk) or Escherichia coli beta-galactosidase (lacZ). We investigated the cytotoxicity of LM8 cells bearing an HSV-tk gene after treatment with ganciclovir (GCV). LM8 cells bearing an HSV-tk gene were more sensitive than non-transduced cells. The remarkable inhibition of tumor growth and pulmonary metastases was confirmed in vivo. Our findings indicated that GCV kills tumor cells transduced with HSV-tk in vitro and in vivo.     

FasL和B7-1联合基因修饰的肺癌细胞诱导抗肺癌主动免疫

目的:探讨FasL和B7-1联合基因转移是否具有协同的抗肿瘤效应.方法:采用重组腺病毒载体将FasL和B7-1基因导入人肺癌细胞A-549,G418阳性克隆筛选,流式细胞分析,RT-PCR显示FasL和B7-1的表达,并且通过Hoechst33342染色和DNA片段分析检测肺癌细胞的凋亡;将携带FasL和B7-1基因的肺癌细胞(命名为A-549/FB-11)接种于C57BL/6小鼠背部皮下,观测其致瘤性能;A-549/FB-11致敏的小鼠对野生型瘤细胞是否具有免疫保护作用;用A-549和A-549/FB-11细胞分别经腹腔免疫小鼠,得到腹腔浸润淋巴细胞及致敏脾细胞,MTT法检测其体外杀伤实验.结果:FasL和B7-1基因在肺癌细胞中获得高表达并可诱导肺癌细胞的凋亡,双基因转染的肺癌细胞的致瘤性明显下降; A-549/FB-11 诱导的CTL (cytotoxic T lymphocyte)对A-549的杀伤活性显著高于野生型A-549诱导的CTL对相同靶细胞的杀伤活性,P<0.05;A-549 /FB- 11诱导的CTL对A-549/FB-11的杀伤率显著高于对野生型A-549的杀伤率,P<0.05.结论:FasL促进肺癌细胞凋亡,B7-1促进抗肺癌CTL的增殖、活化,在效应阶段两基因发挥着重要的协同作用.     

Adenovirus-mediated N5 gene transfer inhibits tumor growth and metastasis of human carcinoma in nude mice

The therapeutic effectiveness of cancer therapy often relies on induction of apoptotic cell death. Gene-therapy-mediated induction of apoptosis, therefore, may provide an effective means to kill cancer cells. The N5 gene encodes a death-domain-containing protein (p84N5) that can trigger atypical apoptosis from within the nucleus, suggesting it may be a candidate for use as a gene therapy for cancer. In the present study, we test the potential utility of a recombinant adenovirus designed to express the N5 gene(AdN5) for the treatment of a variety of human cancers using in vitro and animal models. In vitro, adenoviral-mediated N5 gene transfer inhibits the growth of five different tumor cell lines, but not a normal diploid fibroblast cell line. Adenoviral-mediated N5 gene transfer also reduces the growth and metastasis of primary human tumors in subcutaneous and orthotopic xenograft mouse models. Reduction in tumor cell growth in vitro and in vivo correlates with increased expression of p84N5 and induction of apoptosis. The relative sensitivity of different human cancer cells to AdN5 or Adp53 varies, suggesting that AdN5 may be effective in tumors relatively resistant to p53 gene therapy. We conclude that N5 has potential utility for the gene therapy of cancer.     

In vivo immunogenicity of osteosarcoma cells that express B7-1a,an alternatively spliced form of B7-1

BACKGROUND: B7 family members play a central costimulatory role in T cell activation. We have previously identified B7-1a, an alternatively spliced form of B7-1. The function of B7-1a in induction of anti-tumor immunity remains uncertain. MATERIALS AND METHODS: The cDNAs of murine B7-1, B7-1a and B7-2 were introduced into a murine osteosarcoma cell line, LM8. The ability of B7 transfectants to elicit in vivo anti-tumor immunity was comparatively analyzed with respect to tumorigenecity, pulmonary metastasis and survival time. RESULTS: LM8 cells expressing B7-1, B7-1a, or B7-2 all elicited immunological responses in immunocompetent C3H/He mice. Notably, the anti-tumor effects were most obvious in mice inoculated with B7-1a-transfected LM8 cells. Such a difference among B7-transfectants became indistinguishable in immunodeficient nude mice. CONCLUSION: These findings indicate that B7-1a serves as a more efficacious costimulatory molecule than B7-1 or B7-2 in the induction and maintenance of anti-tumor immune responses against a poorly immunogenic osteosarcoma cell line.     

The immune anti-tumor effects of GM-CSF and B7-1 gene transfection are enhanced by surgical debulking of tumor

Malignant mesothelioma (MM) is a solid tumor largely unresponsive to conventional therapies. Immunological gene therapy shows promise in murine models and human clinical trials; however, the role of surgery in combination with gene therapy has not been widely studied. The aim of this study was to determine if debulking surgery improved the effectiveness of gene therapy in a murine MM model. Mice were subcutaneously inoculated with the MM cell line, AC29, at two different sites, 4 days apart, to allow a surgical and distal site tumor to develop. Once tumors were established, the surgical site tumor was debulked and vaccination of syngeneic tumor transfectants encoding genes for IL-4, IL-2, GM-CSF, B7-1 or allogeneic MHC molecules commenced at a site away from both tumors, and tumor growth was measured. Neither debulking surgery nor gene therapy alone delayed tumor growth. However, there was a clear delay of tumor growth when debulking surgery was combined with vaccination of tumor transfectants expressing B7-1 or high levels of GM-CSF. Combinations of these two transfectants did not lead to a synergistic effect. This study demonstrates that debulking surgery can augment the immunostimulatory effects of immunological gene therapy and can delay tumor growth. This has implications for the future design of human gene therapy trials for solid tumors such as MM.     

Overexpression of cadherins suppresses pulmonary metastasis of osteosarcoma in vivo

Osteosarcoma by nature shows aggressive pulmonary metastasis; however, the underlying molecular mechanisms remain unclear. We previously showed that N-cadherin and cadherin-11 (OB-cadherin), which are highly expressed in normal osteoblasts, are anomalously expressed in human osteosarcoma (Kashima et al., Am J Pathol 1999;155:1549-55). In the present study, we examined the role of cadherins in osteosarcoma metastasis using the mouse osteosarcoma cell line Dunn and its highly metastatic subline LM8. Oligonucleotide array and RT-PCR analyses demonstrated that Dunn and LM8 cells did not express appreciable levels of several members of the cadherin family, and Western blot analysis confirmed that Dunn and LM8 cells did not express P-cadherin, E-cadherin, N-cadherin or cadherin-11 protein. We therefore investigated the functional consequences of cadherin overexpression on cell migration and in vivo metastatic potential of LM8 cells. Several LM8 clones were isolated which expressed exogenous N-cadherin and cadherin-11 localized to the cell membrane and able to bind to beta-catenin. Overexpression of N-cadherin or cadherin-11 in LM8 cells did not affect cell proliferation but caused an inhibitory effect on cell migration in vitro. In vivo analysis showed that N-cadherin- and cadherin-11-overexpressing cells exhibited a marked reduction in their ability to form pulmonary metastases, with significant decreases in lung weight and the number and weight of metastatic lesions, as well as the size and weight of primary lesions at the s.c.-inoculated site. These observations demonstrate that disruption of N-cadherin- and cadherin-11-mediated cell-cell adhesion is critical in the pulmonary metastasis of osteosarcoma.     

Adenovirus-mediated gene transfer of P16INK4/CDKN2 into bax-negative colon cancer cells induces apoptosis and tumor regression in vivo

The tumor-suppressor gene p16INK4/CDKN2 (p16) is a cyclin-dependent kinase (cdk) inhibitor and important cell cycle regulator. Here, we show that adenovirus-mediated gene transfer of p16 (AdCMV.p16) into colon cancer cells induces uncoupling of S phase and mitosis and subsequently apoptosis. Flow cytometric analysis revealed that cells infected with AdCMV.p16 showed an initial G2-like arrest followed by S phase without intervening mitosis (DNA >4N). Using microscopic analysis, deformed polyploid cells were detectable only in cells infected with AdCMV.p16 but not in control-infected cells. Subsequently, AdCMV.p16-infected polyploid cells underwent apoptosis, as assessed by AnnexinV staining and DNA fragmentation, suggesting that cell cycle dysregulation is upstream of the onset of apoptosis. Treatment of mice with subcutaneously transplanted tumors of colorectal cancer cells with AdCMV.p16 but not AdCMV.p53 resulted in significantly reduced tumor volume and prolonged survival. Using an orthotopic model of liver metastasis, we observed both reduced local tumor growth and secondary intrahepatic metastasis after AdCMV.p16 treatment. Importantly, induction of apoptosis in vitro and reduction of tumor growth in vivo by p16 was p53- as well as bax-independent because identical results were obtained using cancer cells, either wild type or mutant for p53 or bax. The studies suggest that an AdCMV.p16-based treatment may be especially effective in patients with bax-negative colon cancer where overexpression of p53 appears not to be of therapeutic value.     

B7-1基因转染骨肉瘤细胞诱导抗骨肉瘤主动免疫的实验研究

目的:探讨B7-1基因转染骨肉瘤细胞能否诱导抗骨肉瘤主动免疫作用。方法:利用脂质体将B7-1真核表达载体pcDNA3-B7-1和空载体pcDNA3分别导入骨肉瘤细胞株LM8中,G418筛选出阳性克隆(分别命名为LM8/B7-1和LM8/pcDNA3),通过RT-PCR、Western blot和流式细胞仪检测B7-1基因与蛋白的表达;通过软琼脂克隆形成实验观察转基因细胞株LM8/B7-1的体外增殖能力;用LM8、LM8/B7-1和LM8/pcDNA3分别经腹腔免疫小鼠,得到腹腔浸润淋巴细胞和致敏脾细胞,MTT法检测其体外杀伤活力;将LM8、LM8/B7-1和LM8/pcDNA3细胞分别接种到C3H雄性小鼠前腋下,观察其致瘤能力;观察LM8/B7-1致敏的小鼠对骨肉瘤细胞LM8是否具有免疫保护作用。结果:B7-1基因在转基因细胞LM8/B7-1中能得到高表达;LM8/B7-1体外增殖能力与LM8和LM8/pcDNA3无明显差异(P>0.05);LM8/B7-1诱导的CTL的杀伤活力显著高于LM8和LM8/pcDNA3诱导的CTL对相同靶细胞的杀伤活力,LM8/B7-1诱导的CTL对LM8/B7-1的杀伤活力也明显高于对LM8和LM8/pcDNA3的杀伤活力(P<0.05);LM8/B7-1致瘤能力较LM8和LM8/pcDNA3细胞明显下降(P<0.01);LM8/B7-1致敏的小鼠对骨肉瘤细胞LM8具有免疫保护作用。结论:B7-1基因转染骨肉瘤细胞能诱导抗骨肉瘤主动免疫作用,为利用B7-1基因进行骨肉瘤免疫基因治疗提供了实验依据。     

Adenovirus-mediated retinoblastoma 94 gene transfer induces human pancreatic tumor regression in a mouse xenograft model.

PURPOSE: Gene transfer of a truncated variant of the retinoblastoma (RB) gene encoding a M(r) 94000 protein that lacks the NH(2)-terminal 112 amino acid residues, termed RB94, has been shown to inhibit proliferation of several human tumor cell types. We have assessed its therapeutic effectiveness on pancreatic cancer, one of the most aggressive and therapy-resistant types of cancer. For this purpose, preclinical studies aimed to evaluate the therapeutic potential of RB94 gene transfer in pancreatic cancer were carried out. EXPERIMENTAL DESIGN: We have compared the antiproliferative effects of adenovirus-mediated gene transfer of RBwt and RB94 at the in vitro and in vivo levels in three RB-positive human pancreatic tumor cell lines: (a). NP-9; (b). NP-18; and (c). NP-31. We have also examined their effects on cell cycle and their capacity to induce apoptosis. RESULTS: In vitro results indicate that RB94 gene transfer has stronger antiproliferative effects compared with RBwt. RB94 transduction correlated with accumulation at the S-G(2) phase of the cell cycle in the three cell lines tested and induction of apoptosis in two of them. In vivo studies show significant decreases in the growth rate of tumors treated with Ad-RB94 when compared with those treated with Ad-RBwt. Moreover, terminal deoxynucleotidyl transferase-mediated nick end labeling analyses of Ad-RB94-treated tumor sections revealed that only RB94 is able to significantly induce apoptosis. CONCLUSIONS: RB94 gene expression has antiproliferative effects also in human pancreatic tumor cells, being more effective than wild-type RB in preventing tumor growth.     

Concurrent induction of T-cell activation and apoptosis of osteosarcoma cells by adenovirus-mediated B7-1/Fas chimeric gene transfer

To establish an effective B7-based gene therapy against osteosarcoma, we transferred B7-1/Fas chimeric gene adenovirally into poorly immunogenic osteosarcoma cells. We found that adenovirus-mediated rat B7-1/Fas gene transfer induced (i) expression of rat B7-1/Fas chimeric molecules in osteosarcoma cells, (ii) activation of murine T cells, (iii) apoptosis of murine osteosarcoma cells in the presence of anti-rat B7-1 mAb in vitro, and (iv) therapeutic effects more prominently than B7-1 gene transfer on the development of pulmonary metastasis and survival of mice. These findings collectively support the therapeutic value of adenovirus-mediated B7-1/Fas gene transfer on poorly immunogenic osteosarcoma, which is resistant to a treatment protocol using transduction of B7-1 alone.     

Adenovirus-mediated REIC/Dkk-3 gene transfer inhibits tumor growth and metastasis in an orthotopic prostate cancer model

We had previously reported that REIC/Dkk-3, a member of the Dickkopf (Dkk) gene family, works as a tumor suppressor. In this study, we evaluated the therapeutic effects of an intratumoral injection with adenoviral vector encoding REIC/Dkk-3 gene (Ad-REIC) using an orthotopic mouse prostate cancer model of RM-9 cells. We also investigated the in vivo anti-metastatic effect and in vitro anti-invasion effect of Ad-REIC gene delivery. We demonstrated that the Ad-REIC treatment inhibited prostate cancer growth and lymph node metastasis, and prolonged mice survival in the model. These therapeutic responses were consistent with the intratumoral apoptosis induction and in vitro suppression of cell invasion/migration with reduced matrix metalloprotease-2 activity. We thus concluded that in situ Ad-REIC/Dkk-3 gene transfer may be a promising therapeutic intervention modality for the treatment of prostate cancer.     

Adenovirus-mediated interleukin-18 mutant in vivo gene transfer inhibits tumor growth through the induction of T cell immunity and activation of natural killer cell cytotoxicity

We report here that gene transfer using recombinant adenoviruses encoding interleukin (IL)-18 mutants induces potent antitumor activity in vivo. The precursor form of IL-18 (ProIL-18) is processed by caspase-1 to produce bioactive IL-18, but its cleavage by caspase-3 (CPP32) produces an inactive form. To prepare IL-18 molecules with an effective antitumor activity, a murine IL-18 mutant with the signal sequence of murine granulocyte-macrophage (GM)- colony stimulating factor (CSF) at the 5'-end of mature IL-18 cDNA (GMmIL-18) and human IL-18 mutant with the prepro leader sequence of trypsin (PPT), which is not cleaved by caspase-3 (PPThIL-18CPP32-), respectively, were constructed. Adenovirus vectors carrying GMmIL-18 or PPThIL-18CPP32- produced bioactive IL-18. Ad.GMmIL-18 had a more potent antitumor effect than Ad.mProIL-18 encoding immature IL-18 in renal cell adenocarcinoma (Renca) tumor-bearing mice. Tumor-specific cytotoxic T lymphocytes, the induction of Th1 cytokines, and an augmented natural killer (NK) cell activity were detected in Renca tumor-bearing mice treated with Ad.GMmIL-18. An immunohistological analysis revealed that CD4+ and CD8+ T cells abundantly infiltrated into tumors of mice treated with Ad.GMmIL-18. Huh-7 human hepatoma tumor growth in nude mice with a defect of T cell function was significantly inhibited by Ad.PPThIL-18CPP32- compared with Ad.hProIL-18 encoding immature IL-18. Nude mice treated with Ad.PPThIL-18CPP32- contained NK cells with increased cytotoxicity. The results suggest that the release of mature IL-18 in tumors is required for achieving an antitumor effect including tumor-specific cellular immunity and augmented NK cell-mediated cytotoxicity. These optimally designed IL-18 mutants could be useful for improving the antitumor effectiveness of wild-type IL-18.     

Adoptive transfer via immune T-lymphocytes of effective anti-tumor immunity against a malignant rat glioma in the brain

The aim of the present study was to develop an animal model to test the therapeutic potential of purified CD4 and CD8 T-lymphocytes against the intracerebrally implanted rat glioma cell line TZ363. Peripheral immunization of donor rats was performed by subcutaneous injection of viable TZ363 tumor cells while control animals received buffer injection. Donor splenic T-lymphocytes were prepared 14 days later and enriched by immune-bead MACS sorting. FACScan analysis revealed that of the pooled and sorted cells 91% of the tumor immune group were T-lympocytes and from the control animals 96%. The purified immune CD4/CD8 T-lymphocytes (1.2 to 5x10(7) cells) were injected intraperitoneally into 12 adult rats (three groups; each four animals), which were challenged five days later by an intracerebral injection of 5x10(4) TZ363 glioma cells. Four rats received 1.4x10(7) T-cells from control animals. While 3 of 4 animals developed a brain tumor and died in the control group, all animals, which received 5x10(7) immune T-cells survived the intracerebral tumor challenge. In the other groups survival rate depended on the amount of T-cells given. All other rats were sacrificed 32 days after intracerebral grafting. No tumor was found in these animals. Our data demonstrate that an anti-tumor T-cell response can be raised against the malignant rat glioma TZ363 and that purified CD4 and CD8 T-lymphocytes from tumor immunized donors can transfer protective immunity across the blood-brain barrier into recipient rats which are tumor challenged intracerebrally.