THE BIOLOGICAL CHARACTERISTICS OF ADENOVIRUS-MEDIATED IL-18 GENE-MODIFIED MURINE COLORECTAL ADENOCARCINOMA CELL IN VIVO AND IN VITRO

Interleukin 18 (IL-18) has multiple biological activities, such as promoting the generation of Th1 cytokines and GM-CSF, activating NK cells and CTL, which contributes to its anti-tumor activity.[1(5] So IL-18 might have promising application in the immunotherapy and gene therapy of cancer. To confirm the anti-tumor activity of IL-18, we constructed the recombinant adenovirus encoding IL-18 gene, observed preliminarily the biological characteristics of IL-18-modified murine colorectal …     

Gene transfer of NK4, an angiogenesis inhibitor,induces CT26 tumor regression via tumor‐specific T lymphocyte activation

Hepatocyte growth factor (HGF) has been shown to be involved in malignant behaviors, such as invasion and metastasis, in different tumors. Hence, HGF could be a target molecule for control of the malignant potential of cancer. NK4 is a competitive antagonist for HGF and exerts an antitumor activity, not only by HGF antagonism but also by antiangiogenesis. Here, we studied the participation of cellular immunity in CT26 tumor regression by NK4 gene transfer. In vivo experiments showed that NK4‐induced inhibition of subcutaneous tumor growth (as demonstrated in immunocompetent BALB/c mice) was weakened in T lymphocyte‐deficient nude mice. In addition, the immunocompetent BALB/c mice that had shown complete regression of CT26‐NK4 tumors generated an immune memory against repeated challenge with the same tumor antigen. Immunohistochemistry of tumor‐infiltrating lymphocytes showed that the ratio of CD8/CD4 in CT26‐NK4 tumors was significantly higher than that in control tumors. Also, the presence of tumor‐specific cytotoxic T lymphocytes (CTL) was demonstrated by cytotoxicity assays. Depletion of CD8+ T lymphocytes markedly abrogated the antitumor activity of NK4. However, NK4 had no direct effect on the in vitro cellular immune system. Taken together, these data indicate that NK4 expression by gene transfer, at the tumor site, triggers tumor‐specific CTL activation, resulting in complete CT26 tumor regression in vivo. This action was considered to be due to apoptosis induced by NK4's potent antiangiogenic and HGF antagonistic effects. © 2009 UICC     

Interleukin-18 Synergism with Interleukin-2 in Cytotoxicity and NKG2D Expression of Human Natural Killer Cells

Natural killer (NK) cells play an important role in anti-tumor immunity. Interleukin (IL)-18 is animmunoregulatory cytokine that induces potent NK cell-dependent anti-tumor responses when administratedwith other cytokines. In this study, we explored the effects of combining IL-18 and IL-2 on NK cytotoxicity aswell as expression levels of the NK cell receptor NKG2D in vitro. Freshly isolated PBMCs were incubated for48 h with IL-18 and IL-2, then CD107a expression on CD3-CD56+ NK cells was determined by three-colour flowcytometry to evaluate the cytotoxicity of NK cells against human erythroleukemia K562 cells and human coloncarcinoma HT29 cells. Flow cytometric analysis was also employed to determine NKG2D expression on NK cells.The combined use of IL-18 and IL-2 significantly increased CD107a expression on NK cells compared with usingIL-18 or IL-2 alone, suggesting that the combination of these two cytokines exerted synergistic enhancement ofNK cytotoxicity. IL-18 also enhanced NKG2D expression on NK cells when administered with IL-2. In addition,blockade of NKG2D signaling with NKG2D-blocking antibody attenuated the up-regulatory effect of combiningIL-18 and IL-2 on NK cytolysis. Our data revealed that IL-18 synergized with IL-2 to dramatically enhance thecytolytic activity of human NK cells in a NKG2D-dependent manner. The results appear encouraging for theuse of combined IL-18 and IL-2 in tumor immunotherapy.     

Tumor cell lysate-pulsed dendritic cells induce a T cell response against colon cancer in vitro and in vivo

To investigate whether tumor cell lysate-pulsed (TP) dendritic cells (DCs) induce cytotoxic T lymphocyte (CTL) activity against colon cancer in vitro and in vivo. Hematopoietic progenitor cells were magnetically isolated from BALB/c mice bone marrow cells. These cells were cultured with cytokines GM-CSF, IL-4, and TNFα to induce their maturation. They were analyzed by morphological observation and phenotype analysis. DCs were pulsed with tumor cell lysate obtained by rapid freezing and thawing at a 1:3 DC:tumor cell ratio. CTL activity and interferon gamma (IFNγ) secretion was evaluated ex vivo. In order to determine whether or not vaccination with CT26 TP DCs induce the therapeutic potential in the established colon tumor model, CT26 colon tumor cells were implanted subcutaneously (s.c.) in the midflank of naïve BALB/c mice. Tumor-bearing mice were injected with vaccination with CT26 TP DCs on days 3 and 10. Tumor growth was assessed every 2–3 days. Finally, CTL activity and IFNγ secretion were evaluated in immunized mice. Hematopoietic progenitor cells from mice bone marrow cells cultured with cytokines for 8 days showed the character of typical mature DCs. Morphologically, these cells were large with oval or irregularly shaped nuclei and with many small dendrites. Phenotypically, FACS analysis showed that they expressed high levels of MHC II, CD11b, CD80, and CD86 antigen, and were negative for CD8α. However, immature DCs cultured with cytokines for 5 days did not have typical DCs phenotypic markers. Ex vivo primed T cells with CT26 TP DCs were able to induce effective CTL activity against CT26 tumor cells, but not B16 tumor cells (E:T = 100:1, 60.36 ± 7.11% specific lysis in CT26 group vs. 17.36 ± 4.10% specific lysis in B16 group), and produced higher levels of IFNγ when stimulated with CT26 tumor cells but not when stimulated with B16 tumor cells (1210.33 ± 72.15 pg/ml in CT26 group vs. 182.25 ± 25.51 pg/ml in B16 group, P < 0.01). Vaccination with CT26 TP DCs could induce anti-tumor immunity against CT26 colon tumor in murine therapeutic models (tumor volume on day 19: CT26 TP DCs 342 ± 55 mm³ vs. the other control groups, P < 0.05). In addition, all splenic CD3⁺ T cells obtained from mice vaccinated with CT26 TP DCs produced high levels of IFNγ and shown specific cytotoxic activity against CT26 tumor cells, but no cytotoxic activity when stimulated with B16 tumor cells. Tumor cell lysate-pulsed DCs can induce tumor-specific CTL activity against colon cancer in vitro and in vivo.     

腺病毒介导IL-2基因转染的瘤苗体内抗肿瘤作用

目的 :探讨重组腺病毒介导的 IL- 2基因转染的瘤苗的体内抗肿瘤作用及其免疫学机制。方法 :应用腺病毒介导的鼠 IL- 2基因转染 CT2 6小鼠结肠癌细胞 ,灭活后用作瘤苗治疗荷瘤小鼠 ,观察皮下肿瘤生长及其存活期。采用乳酸脱氢酶释放法检测荷瘤小鼠脾细胞 CTL、L AK、NK细胞的杀伤活性。结果 :鼠 IL- 2基因转染瘤苗治疗能显著抑制荷瘤小鼠皮下肿瘤生长并明显延长其存活期 (P<0 .0 1)。体内免疫功能检测表明 ,鼠 IL- 2基因转染疫苗治疗组小鼠脾细胞 CTL 活性、L AK活性和 NK活性显著高于对照组 (P<0 .0 1)。结论 :腺病毒介导鼠 IL- 2基因转染的瘤苗体内具有较强的抗肿瘤效应 ,其机制可能是提高了荷瘤小鼠特异性和非特异性抗肿瘤免疫反应     

Cryo-thermal therapy inducing MI macrophage polarization created CXCL10 and IL-6-rich pro-inflammatory environment for CD4+ T cell-mediated anti-tumor immunity

Purpose: We previously developed a novel cryo-thermal therapy to treat malignant mammary carcinoma and melanoma in a mouse model; long-term survival and CD4⁺ T cell orchestrating anti-tumor immune memory response were achieved. Moreover, cryo-thermal-induced CD4⁺ T cell differentiation into Th1 and CD4⁺CTL sub-lineages, in which M1 macrophage polarization played a direct, important role. In particular, cryo-thermal therapy triggered M1 macrophage polarization with up-regulated expression of C–X–C motif ligand 10 (CXCL10) and Interleukin 6 (IL-6). But whether CXCL10 and IL-6 contribute to CD4⁺ T cell-mediated anti-tumor immunity remains unclear. In this study, the role of cryo-thermal-induced CXCL10 and IL-6 in anti-tumor immunity was determined.

Methods: The level of CXCL10 and IL-6 in spleen and serum was determined by RT-PCR and ELISA on day 14 after cryo-thermal therapy. Splenic dendritic cells (DCs) and macrophages were isolated from cryo-thermal-treated mice on day 5 and 14, and the level of CXCL10 and IL-6 in macrophages and DCs was determined by ELISA. The transwell migration assay was performed to study immune cell migration. In vivo neutralization of CXCL10 or IL-6 was performed to investigate the phenotypic changes of immune cells.

Results: Cryo-thermal therapy induced M1 macrophage polarization with up-regulation of CXCL10 and IL-6 expression in spleen. CXCL10 and IL-6 promoted DCs migration and maturation, and subsequently promoted CD4⁺ T cell migration and differentiation into Th1 and CD4⁺ CTL, moreover, reduced myeloid-derived suppressor cells (MDSCs) accumulation.

Conclusions: Cryo-thermal-induced CXCL10 and IL-6 created acute inflammatory environment to initiate a systemically cascading innate and adaptive anti-tumor immunity, which was more permissive for tumor eradication.     

IL-18基因修饰增强肿瘤细胞裂解物致敏的树突状细胞的抗肿瘤效应

目的:观察经肿瘤细胞裂解物致敏的白细胞介素18(IL-18)基因修饰的树突状细胞(DC)体内诱导的抗肿瘤免疫应答反应.方法:体外培养的小鼠骨髓树突状细胞经IL-18重组腺病毒感染后(IL18-DC),再经Hepal-6肝癌细胞裂解物冲击致敏后通过皮下注射用于荷瘤小鼠的治疗.用ELISA检测细胞因子,4 h51Cr释放法检测NK细胞活性及CTL杀伤活性.结果:致敏IL18-DC组体内诱导NK细胞活性与未致敏IL18-DC组无明显差别(P＞0.05),但明显高于致敏DC组和DC组(均P＜0.01);致敏IL18-DC组体内诱导特异性CTL杀伤活性明显高于IL18-DC组、致敏DC组和DC组(均P＜0.01);致敏IL18-DC组免疫治疗作用明显优于未致敏IL18-DC组、致敏DC组和DC组(均P＜0.01).结论:肿瘤细胞裂解物致敏的IL-18基因修饰的DC疫苗进行体内免疫治疗,能诱导出显著的抗肿瘤免疫反应,为DC介导的肿瘤基因治疗开辟了新的途径.     

Spleen-derived dendritic cells engineered to enhance interleukin-12 production elicit therapeutic antitumor immune responses

The major goal in cancer immunotherapy is the induction of tumor-specific T lymphocytes capable of killing tumor cells. As both dendritic cells (DCs) and interleukin-12 (IL-12) can play immunostimulatory roles in vivo, the use of a combination of these has become a promising approach. In the present study, we used a murine tumor model to examine whether spleen-derived DCs transduced with the IL-12 gene could elicit tumor-specific immune responses. BALB/c mice injected peritumorally with adenovirus-mediated IL-12 gene-transduced antigen-unpulsed DCs inhibited the growth of day 5-established subcutaneous CT26 tumors. Splenocytes from treated mice responded specifically to parental tumor cells and showed increased production of interferon gamma (IFN-gamma) and antitumor cytotoxic T-lymphocyte (CTL) activity. Increased numbers of both CD4(+) and CD8(+) T cells were detected in the treated tumors. The inhibition of tumor growth was significantly greater in mice injected with IL-12 gene-transduced DCs than in those injected with IL-12 gene-transduced fibroblasts or the IL-12 gene-encoding adenovirus itself. Taken together, these results indicate that DCs transduced with the IL-12 gene by a recombinant adenovirus are effective in inducing tumor-specific Th1 and CTL responses that inhibit the growth of established subcutaneous tumors.     

A phase-I clinical study of autologous tumor cells plus interleukin-2-gene-transfected allogeneic fibroblasts as a vaccine in patients with cancer

Tumor cells transfected to express immunostimulatory cytokines, or admixed with similarly modified bystander cells, are able to induce immune responses against unmodified tumor cells in animal models. For treatment of human patients, a vaccine composed of autologous tumor cells and IL-2-secreting allogeneic fibroblasts was developed. Autologous tumor cells were isolated from biopsy specimens. A clone (KMST6.14) of an immortalized human fibroblast line that stably secreted 5290 IU IL-2 per 10⁶ cells and per 24 hr was obtained by cationic lipofection with an expression construct for human IL-2 and Neo^r. Fifteen patients with refractory malignant tumors received 3–4 injections of irradiated KMST6.14 and autologous tumor cells in a phase-I clinical trial. Increasing transient inflammatory responses without systemic toxicity developed at vaccination sites and after injections with irradiated tumor cells only (p < 0.05). These sites contained a dense infiltrate of CD3⁺ T cells with numbers of CD4⁺ helper cells exceeding those of CD8⁺ cytotoxic T cells (CTL). CD8⁺ T-cell lines isolated from vaccination sites of 2 malignant melanoma patients but not of renal-cell carcinoma patients exhibited a dominant lytic activity against autologous tumor cells in vitro. CD8⁺ T-cell clones established from the vaccination site of 1 of 2 renal-cell carcinoma patients preferentially lysed autologous and partially matched allogeneic renal-cell carcinoma cells. In conclusion, a vaccine composed of IL-2 gene-transfected allogeneic fibroblasts and autologous tumor cells is able to enhance specific anti-tumor T-cell responses in vivo without major side-effects. Malignant melanoma and renal-cell carcinoma appear to be promising entities for testing of similar approaches in future therapeutic trials. Int. J. Cancer, 70:269–277, 1997. © 1997 Wiley-Liss, Inc.     

EZH2 identifies the precursors of human natural killer cells with trained immunity

Objective:Trained immunity of natural killer (NK) cells has shown great potential in the treatment of cancers by eliciting enhanced effector responses to restimulation by cytokines or cancer cells for long time periods after preactivation. However, the human NK cells responsible for the generation and maintenance of trained immunity are largely unknown. We hypothesized that heterogeneous human NK cells would respond differentially to stimulation with a combination of IL-12, IL-15, and IL-18, and that an NK cell subset might exist that is mainly responsible for the induction of trained immunity. On the basis of our hypothesis, we aimed to identify the subset from which cytokine-trained human NK cells originate and to explore possible regulatory targets for drug intervention.Methods:Flow cytometry assays were performed to analyze the functions of cytokine-trained NK cells and examine cell division and protein expression in NK cell subsets. Single-cell RNA sequencing (scRNA-seq) plus TotalSeq™ technology was used to track the heterogeneity of NK cells during the induction of trained immunity.Results:Traditional developmental markers for peripheral NK cells were unable to identify the precursors of human NK cells with trained immunity. Therefore, we used scRNA-seq plus TotalSeq™ technology to track the heterogeneity of NK cells during the induction of trained immunity and identified a unique cluster of CD57⁻NKG2A⁺EZH2⁺IFNG⁺MKI67⁺IL12R⁺IL15R⁺IL18R⁺ NK cells. Enrichment and pseudotime trajectory analyses suggested that this cluster of NK cells contained the precursor of trained NK cells. We then used flow cytometry to further investigate the role of EZH2 in trained NK precursors and found that CD57⁻NKG2A⁺EZH2⁺ NK cells had faster cell cycles and an enhanced trained phenotype, and EZH2 inhibition significantly impaired the induction of trained immunity in NK cells. These results suggested that EZH2 is a unique epigenetic marker of precursors of human NK cells with trained immunity.Conclusions:Our work revealed human NK heterogeneity in the induction of trained immunity, identified the precursor subset for trained NK cells, and demonstrated the critical role of EZH2 in the induction of trained immunity in human NK cells.     

Experimental Studies of Immunotoxicity of a Photosensitizing Agent (Photofrin II) in Mice

Summary

Immunotoxicity studies have been performed on the photosensitizing agent Photofrin II (PHFR), a porphyrin derivative used in photodynamic therapy. Hybrid CD2F1 (H-2^d/H-2^d) or inbred C57B1/6 (H-2^b) male mice were injected with graded doses of the agent (from 1.2 to 12 mg/Kg ip) on day -5, -3 and -1 before assays. The animals, or spleen cells collected from them on day 0 with respect to PHFR treatment, were tested for: a) competence of producing GVHD upon cell transfer into allogeneic, immunosuppressed recipients; b) graft response against challenge with allogeneic lymphoma cells; c) delayed-type hypersensitivity (DTH) against sheep red blood cells; d) in vitro response to mitogens; e) NK cell activity; f) in vitro generation of alloreactive cytotoxic T lymphocytes (CTL); g) resistance against the challenge of a sublethal dose of Pseudomonas aeruginosa. Moreover the LD50 of the drug given ip has been determined in male CD2F1 mice.

The results show that PHFR, even at the highest doses used, does not affect most of the immunological parameters studied, except for a marginal inhibition of CTL generation and increment in proliferative responses to Con A or LPS. These data along with parallel studies performed by our group on human models in vitro, showing increased susceptibility of PHFR-treated tumors to NK or LAK effector cells, point out that PHFR, in the absence of systemic photoactivation, is essentially non-immunotoxic in vivo and could render tumor cells more susceptible to natural immunity.     

Immune Changes in Patients with Colorectal Cancer Treated by Adjuvant Therapy with Monoclonal Antibody 17-1 A: A Pilot Study

Abstract

We investigated the efficacy of Mo17-1A to improve important immunological parameters in vivo and in vitro in colorectal cancer (CRC) patients receiving no second-line treatment. Eighteen CRC patients stage Dukes’ C were treated post-operatively with 5 doses of Mo17-1A. Peripheral blood mononuclear cells (PBMC) were analyzed for proliferative responses and cytolytic activity. Serum levels of cytokines were determined during treatment. Enhancement in the proliferative activity of patients' T cells, improvement in their lymphocytes' killing ability of natural killer (NK) and lymphokine-activated killer (LAK) sensitive tumor targets, and an in vivo increase in serum levels of interleukin (IL)-2, IL-12, IL-15, interferon (IFN)-γ and granulocyte-macrophage colony-stimulating factor (GM-CSF) were noted. We conclude that treatment of CRC patients with Mo17-1A partially restores deficient cellular immune responses and the secretion of cytokines with immuno-enhancing properties. The development of novel immunotherapeutic protocols using combinations of Mo17-1A with stimuli that enhance the lytic capacity of effector cells (e.g. cytokines), may improve clinical responses in these types of patients.     

Tumor-derived transforming growth factor-β1 and interleukin-6 are chemotactic for lymphokine-activated killer cells

Adherent lymphokine-activated killer (A-LAK) cells are purified IL-2 activated natural killer (NK) cells with potent anti-tumor cytotoxic activity. They have been used in the adoptive immunotherapy of metastatic canters. However, it has been shown that intravenously transferred LAK cells have a poor horning capacity to tumor sites. For the present study, the effects of tumor-derived factors on the in vitro migratory capacity of A-LAK cells was investigated. In a micropore migration assay the conditioned medium from 3LL Lewis lung carcinoma cell cultures was found to exert a strong chemotactic, but not chemokinetic effect on A-LAK cells. This effect was partially inhibited by neutralizing antibodies against the cytokines TGF-β1 and IL-6. A combination of the 2 antibodies completely suppressed the chemotactic activity of tumor-cell-conditioned medium. Purified TGF-β1 and recombinant IL-6 were chemotactic for A-LAK cells. Biological activities of both cytokines were detectable in the tumor-cell-conditioned medium. The in vivo relevance of these findings, with respect to tissue infiltration of NK cells and LAK cells in inflammation or cancer, remains to be elucidated.     

Adenovirus-mediated gene transfer of interleukin-23 shows prophylactic but not therapeutic antitumor effects

A novel cytokine interleukin (IL)-23 bears a structural and functional resemblance to IL-12. A recombinant adenovirus expressing IL-23N220L (recombinant replication-defective adenovirus (rAd)/IL-23N220L) that selectively secrets IL-23 was constructed and compared with rAd/IL-12N220L in terms of immunological and antitumor effects. In a prophylactic setting, vaccination with rAd/ovalbumin (OVA) and rAd/IL-23N220L enhanced OVA-specific CD8(+) T-cell responses that were closely associated with complete protection against the subsequent challenge of OVA-expressing E.G7 thymoma. However, in a therapeutic setting, the intratumoral injection of rAd/IL-23N220L showed only marginal antitumor activity against several established tumors such as E.G7, CT26 and B16F10. Interestingly, whereas IL-23 still induced tumor-specific CD8(+) T-cell responses, it could not activate natural killer (NK) cells in vitro and in vivo. In addition, the adoptive transfer of activated NK cells partially restored the therapeutic antitumor effect of IL-23, indicating that NK cells are one of the crucial factors responsible for the regression of established tumors. Taken together, we demonstrated that adenovirus-mediated gene transfer of IL-23 induces a potent prophylactic, but not a therapeutic, antitumor effect.     

Systemic administration of interleukin-12 can restore the anti-tumor potential of B16 melanoma-draining lymph node cells impaired at a late tumor-bearing state

We investigated the effect of the systemic administration of interleukin (IL)-12 on the anti-tumor potential of tumor-draining lymph nodes (LNs). Tumor-draining LN cells on day 10 after s.c. inoculation of B16 melanoma showed a significant anti-tumor effect against established pulmonary metastases after in vitro expansion, whereas those on either day 20 or 30 exhibited an impaired anti-tumor potential. However, i.p. injections of IL-12 (0.5 μg) on days 18, 20 and 22 significantly increased the total number of tumor-draining LN cells on day 24 and, furthermore, restored their anti-tumor potential after in vitro expansion. In addition, i.p. injection of IL-12 (0.1 μg) on days 20, 22, 24, 26 and 28 significantly suppressed the growth of s.c.-inoculated B16 melanoma and finally cured the tumors in 6 of 12 mice (50%), whereas dissection of the tumor-draining LNs on day 18, prior to the IL-12 treatment, decreased both the IL-12-induced anti-tumor effect and the percentage of cured mice (8.3%). Cured mice acquired a specific protective immunity. Collectively, our results indicate that the systemic administration of IL-12 can restore the immunotherapeutic potential of tumor-draining LNs, which was impaired at the late tumor-bearing state, and that the IL-12-induced systemic anti-tumor activity is preceded by the restoration of an anti-tumor response in tumor-draining LNs. Int. J. Cancer 75:400–405, 1998. © 1998 Wiley-Liss, Inc.     

Suppression of Anti-tumor CD4+ T Cell Responsiveness in the Tumor-bearing State and Its Recovery in in vitro Culture Free of Tumor Burden

We investigated whether the responsiveness of anti-tumor CD4⁺ T cells suppressed in the tumor-bearing state is reversed in conditions free of tumor burden. Spleen cells from BALB/c mice bearing a syngeneic tumor (CSA1M) 1–3 wk after inoculation with CSA1M cells produced interleukin-2 (IL-2) and IL-4 upon in vitro culture without addition of exogenous tumor antigens. This lymphokine production was achieved through collaboration between anti-CSA1M CD4⁺ T cells and antigen-presenting cells (APC) that had been pulsed with CSA1M tumor antigens in vivo in the tumor-bearing state. However, spleen cells from late (8–10 wk) tumor-bearing stages produced reduced levels of lymphokine production despite the presence of comparable proportions of CD4⁺ T cells. Because APC in these cell populations exhibited enhanced capacities to present tumor antigens, reduced responsiveness was ascribed to the dysfunction of CD4⁺ T cells themselves. When spleen cells from early tumor-bearing mice were preincubated for 1–2 days and recultured in fresh medium, the magnitude of lymphokine production by these cells was not changed. In contrast, the same protocol of preincubation and reculture for cells from late tumor-bearing mice resulted in the recovery of anti-tumor lymphokine-producing capacity. The recovered capacity was comparable to or slightly higher than that expressed by cells from early tumor-bearing stages. Since the CD4⁺ T cell content did not significantly differ before and after preincubation, enhanced lymphokine production was due to the recovered responsiveness of anti-tumor CD4⁺ helper T cells. The recovery of anti-tumor responsiveness was also induced in vivo by tumor removal at the late tumor-bearing stage: spleen cells from mice 2–4 wk after tumor resection efficiently produced IL-2 and IL-4. These results indicate that the immunodysfunction of anti-tumor CD4⁺ T cells induced in the tumor-bearing state is reversible because release from tumor burden either by preincubation in vitro or by tumor removal in vivo results in almost complete recovery of the potent anti-tumor responsiveness initially expressed.     

Effects of hMIP-1beta gene modification on in vivo tumorigenicity and vaccine efficacy of tumor cells

OBJECTIVE To explore the effects of human macrophage inflammatory protein-1 beta (hMIP-1beta) modification on the in vivo tumorigenicity and vaccine efficacy of tumor cells. METHODS: Murine colorectal adenocarcinoma CT26 cells were transfected with a recombinant adenovirus carring the hMIP-1beta gene (AdhMIP-1beta). The efficacy of gene transfection was tested by X-gal staining. The hMIP-1beta level in the supernatant of hMIP-1beta gene-modified CT26 cells was assayed by ELISA, and the chemotactic activity for CD4+ T cells, CD8+ T cells, NK cells and immature dendritic cells (imDCs) were assayed by a transwell chamber. The changes of growth characteristics and in vivo tumorigenicity of hMIP-1beta gene-modified CT26 cells were also assessed. BALB/c mice were immunized with hMIP-1beta gene-modified CT26 tumor vaccine and the antitumor effect was evaluated. RESULTS: hMIP-1beta gene could be transfected into CT26 cells by AdhMIP-1beta with an efficiency over 95%. The level of hMIP-1beta in the culture supernatant of hMIP-1beta gene-modified CT26 cells was 980 pg/ml and the supernatant displayed ramarkable chemotactic activity to CD4+ T cells, CD8+ T cells, NK cells and imDCs compared with LacZ gene-modified CT26 cells and control. When the hMIP-1beta gene-modifited CT26 cells were subcutaneously inoculated in BALB/c mice, the tumorigencity was delayed and suppressed, and overt necrosis and lymphocyte infiltration were observed in the tumor tissue, but not in those inoculated with LacZ gene-modified CT26 cells or parental CT26 cells. The mice immunized with hMIP-1beta gene-modified CT26 tumor vaccine could induce tumor specific CTL activity and nonspecific NK activity, and exhibited resistance to later challenge with wild-type CT26 cells. CONCLUSION: hMIP-1beta gene-modified CT26 cells exhibit decreased tumorigenicity, and hMIP-1beta gene-modified tumor vaccine may induce a powerful specific and nonspecific antitumor response. The data suggested that hMIP-1beta gene-modified tumor vaccine may play a potent role in prevention of metastasis and recurrence of malignant tumors.     

Significance of the expression of green fluorescent protein on detection of glioma invasion in vivo

Objective: To investigate the invasion and metastasis of glioma in vivo by xenotransplanted tumor established by implanting C6 glioma cells transfected with green fluorescent protein (GFP) gene in vitro into the brain of SD rats. Methods: C6 cells were transfected with a plasmid vector (pEGEP-N3) containing the GFP gene. Stable GFP-expressing clones were isolated and performed examination by flow cytometry and electron microscope. GFP-expressing cells were stereotactically injected into the brain parenchyma of SD rats to establish xenotransplanted tumor. Four weeks later rats were killed and continuous brain sections respectively were examined by HE staining, immunohistochemistry method and fluorescence microscopy for detection of tumor cell invasion. Xenotransplanted tumor was primarily cultured to determine the storage of exotic GFP gene in vivo. Results: There were not obvious changes in cell cycle and ultrastructure for the cells transfected with GFP gene. C6 cells transfected with GFP gene maintained stable high-level GFP expression in the central nervous system during their growth in vivo. GFP fluorescence clearly demarcated the primary tumor margin and readily allowed for the detection of distant invasion on the single-cell level, which was evidently superior to HE and immunohistochemistry staining. There was not GFP gene loss of transfected cells in vivo. Conclusions: It is suggested that C6 cells transfected with GFP gene can be visualized by fluorescent microscopy after intracranial implantation. This model is an excellent experimental animal model in research on invasion of glioma. Foundation item: This work was supported by a grant from the National Natural science foundation of China (No. 39970752). Biography: LI Xia (1975–), master of neurosurgery, Xijing Hospital, the Fourth Military Medical University, majors in gene diagnosis and gene therapy of brain malignant tumor.     

Effects of Diheptyldiselenide (DDS) on Human Tumor Cell Lines and on Peripheral Blood Mononuclear Cells

Summary

In vitro effects of graded concentrations of diheptyldiselenide (DDS) on human tumor cell proliferation, and on the proliferative responses and immunological functions of peripheral blood mononuclear cells (MNC) were investigated. The agent significantly decreased tumor cell proliferation in a dose and time dependent manner. Proliferative responses of MNC to phytohemagglutinin (PHA) and interleukin-2 (IL-2) were also significantly depressed when MNCs were exposed to DDS at the onset of the culture. Pretreatment of MNCs with DDS (250 uM for 18 h) led to a significant increase in NK activity only in MNC samples showing very limited baseline NK function. On the other hand, generation of LAK cells was significantly inhibited by DDS. However, when the agent was added to the effector and target cell mixture during the 4 h ⁵¹Cr release cytotoxicity assay, no influence was found on NK and LAK-mediated target cell lysis. These studies show that high concentrations of DDS inhibit tumor cell proliferation and could also impair certain proliferative-dependent immune functions, without directly affecting cell-mediated cytolytic activity of effector cells.