Eradication of osteosarcoma lung metastases following intranasal interleukin-12 gene therapy using a nonviral polyethylenimine vector

The use of adenoviral vectors for therapeutic delivery of genes via pulmonary application poses several problems in terms of immune responses. The purpose of this study was to determine whether polyethylenimine (PEI), a polycationic DNA carrier, can be used to deliver the IL-12 gene into the lungs of mice having microscopic osteosarcoma (OS) lung metastases. Incubation of SAOS-LM6 cells in vitro with PEI containing the murine IL-12 (mIL-12) gene (PEI:IL-12) resulted in expression of both the p35 and p40 subunits of IL-12 mRNA and production of mIL-12 protein. Using our newly developed OS nude mouse model, we demonstrated that treatment of mice using intranasal PEI:IL-12 resulted in significant IL-12 mRNA expression in the lung but not the liver. Furthermore, plasma IL-12 was undetectable after up to 4 weeks of intranasal PEI:IL-12 therapy given twice weekly. No IL-12 expression was seen following intranasal PEI therapy alone. The number of lung metastases in animals that received intranasal PEI:IL-12 twice weekly for 4 weeks starting 6 weeks after tumor inoculation was significantly decreased (median, 11; range, 0-47) compared with those that received PEI alone (median, 89; range, 2 to >200; P=.012). Also, the size of the nodules was significantly smaller in the PEI:IL-12-treated animals, with 90% measuring < or =0.5 mm in diameter compared with 56% in the PEI-alone group. Animals that received PEI alone also had numerous large nodules (3-6 mm) throughout the lungs. Intranasal therapy is a noninvasive way to administer agents and has the advantage of targeting the pulmonary region, resulting in higher concentrations in the tumor area. Additionally, delivery of IL-12 to the lung via the airway using PEI may avoid systemic toxicity. Because OS metastasizes almost exclusively to the lung, this may be a novel approach to the treatment of pulmonary OS metastases.     

Aerosol gene therapy with PEI: IL-12 eradicates osteosarcoma lung metastases.

PURPOSE: We determined whether polyethylenimine (PEI), a polycationic DNA carrier, can be used to deliver the interleukin (IL) 12 gene by aerosol to treat established osteosarcoma (OS) lung metastases in a nude mouse model. EXPERIMENTAL DESIGN: Tumor response was assessed using our OS lung metastases model. Treatment with aerosolized PEI containing the murine IL-12 gene (PEI:IL-12; 600 microl PEI and 2 mg IL-12) was given twice weekly for 5-6 weeks. RESULTS: Aerosol therapy for 2 weeks resulted in high expression of both the p35 and p40 subunits of IL-12 in the lungs but not in the livers of mice. Peak IL-12 mRNA expression was seen 24 h after a single aerosol PEI:IL-12 treatment. This expression gradually decreased with continued detection for up to 7 days. IL-12 protein was not detectable in plasma even after 6 weeks of aerosol therapy. The number of lung metastases in mice treated with aerosol PEI:IL-12 was decreased significantly (median, 0; range, 0-33) compared with mice that received PEI alone (median, 37.5; range, 11-125; P = 0.002). Nodule size was also significantly smaller in the aerosol PEI:IL-12 group with 87% of the nodules measuring 1 mm. Weekly aerosol PEI:IL-12 therapy was as effective as twice weekly therapy. CONCLUSIONS: Aerosol therapy resulted in selective gene expression and protein production in the tumor area. Aerosol PEI:IL-12 may avoid the systemic toxicities described previously in patients treated with i.v. IL-12. Because OS metastasizes almost exclusively to the lung, aerosol PEI:IL-12 therapy may provide a therapeutic option, which may be especially valuable.     

Growth suppression of established human osteosarcoma lung metastases in mice by aerosol gene therapy with PEI-p53 complexes

Lung metastases are a frequent complication of osteosarcoma and a treatment that would reduce the severity of this complication would be of great benefit to patients. We have used a formulation consisting of polyethyleneimine (PEI) and a p53 gene administered in aerosol to treat established lung micrometastases as a model of human osteosarcoma in nude mice. The SAOS-LM6 cell line, a metastatic derivative of the p53 null SAOS-2 line, expresses high levels of p53 protein after in vitro transfection with PEI-p53 complexes as determined by ELISA, and transfection with both p53wt and the p53 variant, p53-CD(1-366) in vitro, results in a marked inhibition of SAOS-LM6 cell proliferation. Aerosol delivery of plasmid DNA containing either the p53 gene or a p53-CD(1-366) variant gene formulated with PEI to mice resulted in highly significant reductions in the numbers and size of tumors (P<.001), the total number of tumor foci in the lungs (P<.001) and the size of individual tumor nodules in treated animals compared to untreated, PEI only-treated and PEI-CAT-treated control animals. The different tissues examined did not reveal any signs of toxicity or inflammation after repeated exposure to PEI-DNA. The aerosol delivery of PEI-based formulations of p53 or synthetic p53 variant genes represents a promising new strategy for the treatment of established human osteosarcoma lung metastases. The noninvasive nature of aerosol delivery coupled with low toxicity also make this therapeutic approach potentially appropriate for combination therapy with either radio- or chemotherapy.     

IL-12基因联合吡柔比星治疗裸鼠膀胱癌移植瘤的实验

目的 探讨联合应用白细胞介素12(IL-12)质粒和吡柔比星（THP）对人膀胱癌裸鼠移植瘤生长的抑制作用。方法 将人EJ膀胱癌细胞株裸鼠腋下接种,成瘤后设4组（每组6只）：联合组于骨骼肌注射IL-12真核表达质粒pcAGGS-mIL-12和腹腔注射THP;IL-12组骨骼肌注射pcAGGS-mIL-12;THP组腹腔注射THP;对照组肌肉及腹腔注射0.9%氯化钠溶液,以上注射每周2次,共4次。观察各组裸鼠肿瘤大小变化。4周后处死荷瘤鼠。免疫组织化学法检测肿瘤内VEGF 表达。ELISA法测定血液及肿瘤组织中IL-12和γ-干扰素(IFN-γ)的表达。结果 联合组肿瘤生长明显抑制,瘤重及体积明显小于其他组。ELISA法表明IL-12组和联合组 IL-12和IFN-γ都高表达。免疫组织化学法表明联合组VEGF基因表达量明显低于其他组（P<0.05）。结论 联合应用IL-12真核表达质粒pCAGGS-mIL-12和THP,对人膀胱癌裸鼠移植瘤的抑制作用明显强于单独应用IL-12和THP。     

Interleukin (IL)-12 and IL-12 gene transfer up-regulate Fas expression in human osteosarcoma and breast cancer cells

Expression of Fas (CD95, APO-1), a cell surface receptor capable of inducing ligand-mediated apoptosis, is involved in tissue homeostasis and elimination of targeted cells by natural killer and T cells. Corruption of this pathway, such as reduced Fas expression, can allow tumor cells to escape elimination and promote metastatic potential. In this study, the status of Fas expression has been examined in the parental SAOS human osteosarcoma cells that do not metastasize and in selected variants that cause lung metastases in 16 weeks (LM2) or 8 weeks (LM6) after i.v. injection into nude mice. Fas expression correlated with the metastatic potentials of the three cell lines. Northern and fluorescence-activated cell-sorting analyses indicated that LM6 cells expressed Fas at a lower level than seen in the parental cells. Infection of the LM6 cells with an adenoviral vector containing the murine interleukin (IL)-12 gene (AD:mIL-12) or treatment with recombinant murine IL-12 resulted in a dose-dependent up-regulation of FAS: The up-regulation of Fas by IL-12 was also demonstrated in human etoposide-resistant MDA-MB-231 breast cancer cells. [(3)H]Thymidine growth inhibition studies indicated that the cell surface Fas induced after IL-12 exposure was functional and able to mediate cell death on cross-linking with anti-FAS: We also demonstrate that this effect is independent of IFN-gamma. Whereas these cell lines are sensitive to IFN-gamma, incubation with IFN-gamma does not increase susceptibility to Fas-mediated cell death, nor do these cells produce IFN-gamma with or without IL-12 treatment. We hypothesize that expression of Fas may play a role in the elimination of metastatic tumor cells in the lung, an organ in which Fas ligand is expressed. The antitumor activity of IL-12 may be secondary in part to its ability to up-regulate Fas expression on tumor cells, which subsequently increases immune-mediated destruction of osteosarcoma cells.     

TGFβ1质粒DNA直接瘤内注射地小鼠肿瘤生长的影响

目的探讨裸ＤＮＡ质粒ＰＭＡＭｎｅｏ－ＴＧＦβ１小人直接注射后的表达及其对肿瘤生长的影响。方法 肺腺癌细胞株ＬＭ３小鼠背部皮下接种,两周后瘤。瘤体内多点多次直接注入质粒ＤＮＡＰＭＡＭｎｅｏＴＧＦβ１,并设空质粒和生理盐水注射组为对照,观察肿瘤生长情况。于第８周将小鼠处死,取新鲜的铰瘤组织提取ＲＮＡ,行Ｎｏｒｔｈｅｒｎ杂交,并病理制片观察肿瘤组织结构的变化。结果ＴＧＦβ基因治疗组肿瘤生长速度增快,但各     

Effect of interleukin-1-beta on metastasis formation in different tumor systems

Experiments were done to determine the effect of interleukin-1-beta (IL-1 beta) on metastasis formation in different tumor systems. Intravenous administration of 1 microgram of human recombinant IL-1 beta given 1 hour before tumor cell injection augmented lung colony formation (experimental metastases) by the human A375 melanoma variants, the human HT-29M colon carcinoma, the SN12-K1 renal carcinoma in nude mice, the murine B16 melanoma variants, and the murine UV-2237M fibrosarcoma in syngeneic recipients. The same treatment did not induce lung colony formation by a human rectal carcinoma (HCC-P2988) or by a murine reticulum cell sarcoma (M5076), both of which are not metastatic to the lung. Spontaneous metastases were studied in C57BL/6 mice bearing the B16-BL6 melanoma (metastatic to the lung) in their footpad and the M5076 reticulum cell sarcoma (metastatic to the liver) subcutaneously. Daily intraperitoneal treatment with 1 microgram of IL-1 beta increased lung and liver metastases. These findings indicate that treatment of mice with IL-1 beta can increase the number of artificial or spontaneous metastases and that this effect is not limited to a single tumor type or to a specific organ.     

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.     

Interleukin 1-induced augmentation of experimental metastases from a human melanoma in nude mice

This study has examined the effect of the cytokine interleukin 1 (IL-1) on metastasis formation by the human melanoma A375M in nude mice. We have found that human recombinant IL-1 beta (a single injection greater than 0.01 micrograms per mouse i.v. given before tumor cells) induced an augmentation of experimental lung metastases from the A375M tumor cells in nude mice. This effect was rapidly induced and reversible within 24 h after IL-1 injection. A similar effect was induced by human recombinant IL-1 alpha and human recombinant tumor necrosis factor, but not by human recombinant interleukin 6. 5-[125I]odo-2'-deoxyuridine-radiolabeled A375M tumor cells injected i.v. remained at a higher level in the lungs of nude mice receiving IL-1 than in control mice. In addition, IL-1 injected 1 h, but not 24 h, after tumor cells enhanced lung colonization as well, thus suggesting an effect of IL-1 on the vascular transit of tumor cells. These findings may explain the observation of enhanced secondary localization of tumor cells at inflammatory sites and suggest that modulation of secondary spread should be carefully considered when assessing the ability of this cytokine to complement cytoreductive therapies.     

Immunotherapy of disseminated fibrosarcoma in mice using IL-2-producing tumor cells: studies on its mechanism and specificity

Genetically modified, IL-2-producing tumor cells have been shown to regress in vivo and immunize mice against subsequent challenge with parental tumor. We investigated whether IL-2-producing tumor cells may serve as immunotherapy of established tumors in mice. MCA 205 and MCA 203, weakly immunogenic murine sarcomas of B6 origin, were transfected with the pBMGNeo-mIL-2 vector containing the murine IL-2 cDNA. Mice receiving intraperitoneal injections of the parental sarcoma cells developed ascites and died within 4 weeks. The intraperitoneal injection of IL-2-producing tumor cells significantly prolonged survival and, moreover, significantly reduced the number of established pulmonary metastases. The specificity of this effect was indicated by the unaltered course of disease in mice that were injected with unrelated IL-2-producing tumor cells. FACS analysis of peritoneal cells obtained from treated mice showed a predominance of Vbeta3-positive cells. In a 4 h 51Cr release assay, these Vbeta3-positive cells exhibited tumor-specific cytotoxicity and also nonspecific effector cells are shown to be involved in tumor rejection.     

肿瘤靶向性载体介导的IL-12 基因增强裸鼠抗骨肉瘤免疫

 目的 观察以转铁蛋白-多聚乙烯亚胺(Transferrin-polyethylenimine，Tf-PEI)为靶向性载体，体内转染小鼠白细胞介素12(murine interleukin-12，mIL-12)基因，治疗裸鼠骨肉瘤模型的疗效。方法 以Tf-PEI为载体，体外转染mIL-12基因入人骨肉瘤细胞，并以游离转铁蛋白竞争性拮抗Tf-PEI，观察基因表达情况。将Tf-PEI包裹mIL-12基因直接注入荷瘤裸鼠的肿瘤局部，检测此基因在肿瘤细胞中的表达情况和小鼠脾脏自然杀伤细胞(NK)活性。结果 Tf-PEI可以靶向性的将mIL-12基因导入人骨肉瘤细胞。在Tf-PEI／DNA治疗组小鼠的肿瘤局部，mIL-12蛋白水平明显升高，小鼠脾细胞NK活性增强。结论 Tf-PEI是一种高效率的肿瘤靶向性基因转染载体，它可以成功的将mIL-12基因导入裸鼠骨肉瘤模型，mIL-12基因治疗可提高机体的抗肿瘤免疫应答。     

Correlation of growth capacity of cells in hard agarose with successful transfection by the activated c-Ha-ras oncogene and in vivo proliferative capacity at metastatic sites

The purpose of this study was to determine whether the degree of anchorage-independent growth of rodent or human cells in increasing concentrations of agarose correlated with successful transfection of the cells with an activated c-Ha-ras oncogene and tumorigenicity in nude mice. NIH 3T3 cells, C3H 10T1/2 fibroblasts, four clones of the murine K-1735 melanoma with different metastatic capacities and the TE85 human osteogenic sarcoma line were transfected with plasmids containing the 6.6-kilobase BamHI fragment of the mutant human c-Ha-ras gene and the neo gene, which confers resistance to neomycin (pSV2-neoEJ). Cells transfected with pSV2-neo, a plasmid containing the neo gene, served as controls. Cells from parental or transfected lines (selected by Geneticin) were plated into medium containing 0.3%, 0.6% 0.9%, or 1.2% agarose. These cells were also injected subcutaneously and intravenously into nude mice. The production of tumor cell colonies in dense agarose (greater than or equal to 0.6%) correlated with successful transfection with pSV2-neoEJ and production of experimental metastases in the lung of nude mice. We conclude that the degree of anchorage-independent growth of cells predicts successful transfection with activated c-Ha-ras oncogene and tumorigenic behavior in vivo. Thus this technique may be useful for the detection of cells transfected with transforming oncogenes.     

Conversion of a poorly differentiated human adenocarcinoma to ascites form with invasion and metastasis in nude mice

Cells (1 X 10(7)/0.5 ml) from a Borrmann type III poorly differentiated adenocarcinoma of the human stomach were injected ip into nude mice. The injection resulted in ascites carcinoma with invasion (carcinomatous peritonitis) and liver metastasis. The inoculum was obtained from subcutaneous tumors at passage 9 in nude mice that had received serial transplants from the patient with Borrmann type III poorly differentiated adenocarcinoma of the stomach. Serial transfers of 1.5 X 10(6) dispersed cancer cells/0.5 ml into the peritoneal cavity of nude mice converted this adenocarcinoma to an ascites form. Hemorrhagic ascites accumulated within 3 weeks at the first passage and 4-6 weeks in serial passages. Carcinomas peritonitis occurred consistently and was observed in the diaphragm, mesenteries, omentum, and pancreas; metastases were seen in the liver and spleen. Subsequently, iv injection of ascites at passage 3 (6 X 10(5) cells/0.2 ml) into nude mice produced metastatic lesions in the lung and the heart. The histology of the invasive and metastatic lesions in the nude mice was similar to that of the original tumor in the patient with stomach carcinoma.     

Specific immunotherapy against occult cancer metastases

We investigated the efficacy of a preparation containing High Five (H5) insect cells infected with recombinant baculovirus encoding the murine interferon-beta gene (H5BVIFN-beta) against established primary tumors and occult lung metastases. Injection of live or lyophilized H5BVIFN-beta into established subcutaneous tumors of the highly metastatic murine UV-2237m fibrosarcoma or K-1735M2 melanoma in syngeneic mice eradicated both primary tumors and preexisting lung metastases. The therapeutic effects of H5BVIFN-beta were not observed in nude mice and were diminished in syngeneic mice depleted of CD4(+) and CD8(+) T cells. Immunohistochemical staining showed that tumors injected with H5BVIFN-beta were densely infiltrated by CD4(+) and CD8(+) T cells in mice with normal CD4/CD8 complement. These data demonstrate that, unlike most immunologic approaches in which prophylaxis can be achieved but eradication of established tumor is rare, lyophilized preparations of H5BVIFN-beta can serve as a novel immunotherapy against both primary tumors and their occult metastases.     

Fibrosarcoma cells transduced with the IL-6 gene exhibited reduced tumorigenicity, increased immunogenicity, and decreased metastatic potential.

Murine fibrosarcoma cell lines transduced with retroviral vectors containing the murine interleukin 6 (IL-6) gene constitutively secreted IL-6. When injected s.c. into normal mice these IL-6-secreting tumors exhibited reduced tumorigenicity. This reduced tumorigenicity was not seen in nude or irradiated mice, implicating a T-cell-dependent, radiosensitive host response activated by the cytokine. Subcutaneous IL-6-secreting tumor did not retard the growth of distant deposits of wild-type tumor in the same host. However, animals rejecting IL-6-secreting tumors exhibited resistance to later challenge with wild-type tumor. When injected i.v. in an experimental metastasis model the IL-6-secreting tumors failed to or were extremely inefficient in giving rise to pulmonary nodules; this was observed in both normal and immunoincompetent mice, implicating a second, nonimmune mechanism affecting the growth of the tumor modified to secrete IL-6.     

In vivo immunologic selection of class I major histocompatibility complex gene deletion variants from the B16-BL6 melanoma

The mechanism by which tumor allografts escape host immunologic attack was investigated. B16-BL6 cells (the bladder 6 subline of the B16 melanoma) (H-2b) were transfected with a gene (Dd) encoding an allogeneic class I major histocompatibility complex antigen. Clones that expressed Dd antigen were injected into the footpads of nonimmune syngeneic mice, syngeneic immune mice, and nude mice. Under conditions of immunologic selection a clone that contained multiple copies of the transfected gene formed variants that lacked the transfected gene. Primary tumors and pulmonary metastases of immunized mice and pulmonary metastases of nonimmunized mice had lost the Dd gene and, in most cases, all of the associated plasmid. In contrast, in immunodeficient nude mice, primary tumors and pulmonary metastases retained the Dd gene and the associated plasmid. Deletion of genes encoding cell surface antigens may be one of the mechanisms by which allogeneic tumors escape immunologic attack.     

Hiwi Knockdown Inhibits the Growth of Lung Cancer in Nude Mice

Hiwi, a human homologue of the Piwi family, plays an important role in stem cell self-renewal and isoverexpressed in various human tumors. This study aimed to determine whether an RNA interference-basedstrategy to suppress Hiwi expression could inhibit tumor growth in a xenograft mouse model. A rare population ofSSCloAldebr cells was isolated and identified as lung cancer stem cells in our previous study. Plasmids containingU6 promoter-driven shRNAs against Hiwi or control plasmids were successfully established. The xenograft tumormodel was generated by subcutaneously inoculating with lung cancer stem cell SSCloAldebr cells. After the tumorsize reached about 8 mm in diameter, shRNA plasmids were injected into the mice via the tail vein three timesa week for two weeks, then xenograft tumor growth was assessed. In nude mice, intravenously delivery of HiwishRNA plasmids significantly inhibited tumor growth compared to treatment with control scrambled shRNAplasmids or the vehicle PBS. No mice died during the experiment and no adverse events were observed in miceadministered the plasmids. Moreover, delivery of Hiwi shRNA plasmids resulted in a significant suppressedexpression of Hiwi and ALDH-1 in xenograft tumor samples, based on immunohistochemical analysis. Thus,shRNA-mediated Hiwi gene silencing in lung cancer stem cells by an effective in vivo gene delivery strategyappeared to be an effective therapeutic approach for lung cancer, and may provide some useful clues for RNAigene therapy in solid cancers.     

Rapid, in vivo, evaluation of antiangiogenic and antineoplastic gene products by nonviral transfection of tumor cells

Using a nonviral, electroporation-based gene transfection approach, we demonstrate the efficient and consistent transfection of two poorly immunogenic tumor cell lines: B16F10 melanoma and renal carcinoma (RENCA). Three genes, IL-12, angiostatin (AS), and an endostatin:angiostatin fusion protein (ES:AS) were subcloned into a DNA plasmid containing EBNA1-OriP, which was then transfected into B16F10 and RENCA cells. Significant levels of protein were secreted into the culture supernatants of transfected cells in vitro. Transfected tumor cells were injected subcutaneously into mice. All the three transgenes were capable of significantly delaying and reducing the formation of primary B16F10 and RENCA tumors, as well as B16F10 lung metastases. By day 11 post-injection, all control mice that received either mock-transfected or empty vector DNA-transfected B16F10 tumor cells had developed large primary tumors. In contrast, mice that received IL-12-transfected B16F10 cells did not develop appreciable tumors until day 17, and these were significantly smaller than controls. Similar results were observed for the RENCA model, in which only one of the IL-12 mice had developed tumors out to day 31. Expression of AS or ES:AS also significantly delayed and reduced primary tumors. Overall, ES:AS was more effective than AS alone. Furthermore, 25% of the AS mice and 33% of the ES:AS mice remained tumor-free at day 17, by which point all control mice had significant tumors. Mouse survival rates also correlated with the extent of tumor burden. Importantly, no lung metastases were detected in the lungs of mice that had received either AS or ES:AS-transfected B16F10 tumor cells and significantly fewer metastases were found in the IL-12 group. The consistency of our transfection results highlight the feasibility of directly electroporating tumor cells as a means to screen, identify, and validate in vivo potentially novel antiangiogenic and/or antineoplastic genes.     

The effect of TGFβ1 on murine tumor growth following direct intratumoral injection of plasmid DNA

In order to investigate TGFβ1 gene expression and its effect on murine tumor growth following direct intratumoral injection of naked plasmid DNA encoding human TGFβ1, LM₃ murine lung adenocarcinoma cells were inoculated subcutaneously to T₇₃₉ mice and grew to tumor nodules in 2 weeks. Multiple direct intratumoral injection of plasmid DNA, PMAMneo- TGFβ1, were given and compared with saline or vector plasmid administration groups. The growth of tumor was observed till the 8th week when the mice were killed for Northern blot analysis and histopathological study of tumoral tissue. The results showed that the growth of tumor was boosted in the TGFβ1 gene treated mice as compared with the control groups, whereas there was no significant difference in the metastatic behavior. Northern blot showed efficient expression of TGFβ1 mRNA in the treated group. The present study indicated that TGFβ1 may stimulate tumor growthin vivo through certain mechanisms. And direct intra-tumoral injection of nude plasmid DNA may be a promising gene transfer strategyin vivo. This work was supported by the National Natural Science Foundation of China (No. 39370761).     

Attenuated Salmonella typhimurium containing interleukin-2 decreases MC-38 hepatic metastases: a novel anti-tumor agent

Currently, there is no long-term effective treatment for unresectable hepatic malignancies. Salmonella sp. are known to naturally track to the liver during active infection. To develop a biological vector for delivery of Interleukin-2 (IL-2) to the liver for anti-tumor purposes, the avirulent and highly immunogenic chi 4550 strain of Salmonella typhimurium was used as a vector for IL-2. The gene for human IL-2 was cloned into plasmid pYA292 (renamed pIL-2) and inserted into the attenuated Salmonella typhimurium and renamed [chi 4550 (pIL-2)]. This transformant was found to produced biologically active IL-2 demonstrated by NK cell activation in a 4 hour chromium release cytotoxicity assay. To determine anti-tumor potential, MCA-38 murine adenocarcinoma cells were injected intrasplenically into C57BL/6 mice to produce hepatic metastases and metastases were subsequently enumerated after 12 days. Statistical significance was determined by ANOVA with Fisher's test for significance. Hepatic metastases enumerated by blinded observers revealed that the mean number of metastases was 106.4 in control mice, 103.7 in mice gavage fed attenuated salmonella without IL-2 [chi 4550(pYA292)], and 44.3 in mice fed the chi 4550(pIL2); (ANOVA: p < 0.01). Culture of livers and spleens in mice administered a single gavage dose of salmonella demonstrated persistent colonization for up to 4 weeks. No observable toxicity was seen to either IL-2 or salmonella. These studies demonstrate that the chi 4550(pIL2) is a novel form of in vivo biotherapy which produces biologically active IL-2 and employs the oral route of administration to stimulate an immune response against malignancy in the liver.