Targeted Delivery of Zoledronate to Tumor-Associated Macrophages for Cancer Immunotherapy.

Tumor-associated macrophages (TAMs) are recruited from circulatory monocytes by tumor-derived factors, which differentiate into macrophages residing in the tumor microenvironment. TAMs play critical roles in promoting angiogenesis, invasion, metastasis and immune escape, and the direct depletion of TAMs is a promising strategy for tumor immunotherapy. In this study, we developed lipid-coated calcium zoledronate nanoparticles (CaZol@pMNPs) containing conjugated mannose, which were sterically shielded with an extracellular pH-sensitive material. The NPs specifically targeted TAMs and induced their apoptosis in vitro and in vivo. In a S180 tumor-bearing mouse model, CaZol@pMNPs effectively depleted TAMs, markedly decreased angiogenesis, reduced immune suppression, and eventually restrained tumor growth without eliciting systemic effects. The collective data indicate the potential of the direct depletion of TAMs using CaZol@pMNPs for cancer immunotherapy.

Construction of random tumor transcriptome expression library for creating and selecting novel tumor antigens.

Novel tumor antigens are necessary for the development of efficient tumor vaccines for overcoming the immunotolerance and immunosuppression induced by tumors. Here, we developed a novel strategy to create tumor antigens by construction of random tumor transcriptome expression library (RTTEL). The complementary DNA (cDNA) from S180 sarcoma was used as template for arbitrarily amplifying gene fragments with random primers by PCR, then ligated to the C-terminal of HSP65 in a plasmid pET28a-HSP for constructing RTTEL in Escherichia coli. A novel antigen of A5 was selected from RTTEL with the strongest immunotherapeutic effects on S180 sarcoma. Adoptive immunotherapy with anti-A5 sera also inhibited tumor growth, further confirming the key antitumor roles of A5-specific antibodies in mice. A5 contains a sequence similar to protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1). The antisera of A5 were verified to cross-react with PCMT1 by Western blotting assay and vice versa. Both anti-A5 sera and anti-PCMT1 sera could induce antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity toward S180 cells by in vitro assay. Further assay with fluorescent staining showed that PCMT1 is detectable on the surface of S180 cells. Summary, the strategy to construct RTTEL is potential for creating and screening novel tumor antigens to develop efficient tumor vaccines. By RTTEL, we successfully created a protein antigen of A5 with significant immunotherapeutic effects on S180 sarcoma by induction of antibodies targeting for PCMT1.

Selective depletion of tumor neovasculature by microbubble destruction with appropriate ultrasound pressure.

Low-intensity ultrasound-microbubble (LIUS-MB) treatment is a promising antivascular therapy for tumors. We sought to determine whether LIUS-MB treatment with an appropriate ultrasound pressure could achieve substantial and persistent cessation of tumor perfusion without having significant effects on normal tissue. Further, we investigated the mechanisms underlying this treatment. Murine S-180 sarcomas, thigh muscles, and skin tissue from 60 tumor-bearing mice were subjected to sham therapy, an ultrasound application combined with microbubbles in four different ultrasound pressures (0.5, 1.5, 3.0, 5.0 MPa), or ultrasound at 5.0 MPa alone. Subsequently, contrast-enhanced ultrasonic imaging and histological studies were performed. Tumor microvessels, tumor cell necrosis, apoptosis, tumor growth, and survival were evaluated in 85 mice after treatment with the selected ultrasound pressure. We found that twenty-four hours after LIUS-MB treatment at 3.0 MPa, blood perfusion and microvessel density of the tumor had substantially decreased by 84 ± 8% and 84%, respectively (p < 0.01). Similar reductions were not observed in the muscle or skin. Additionally, an extreme reduction in the number of immature vessels was observed in the tumor (reduced by 90%, p < 0.01), while the decrease in mature vessels was not significant. Further, LIUS-MB treatment at 3.0 MPa promoted tumor cell necrosis and apoptosis, delayed tumor growth, and increased the survival rate of tumor-bearing mice (p < 0.01). These findings indicate that LIUS-MB treatment with an appropriate ultrasound pressure could selectively and persistently reduce tumor perfusion by depleting the neovasculature. Therefore, LIUS-MB treatment offers great promise for clinical applications in antivascular therapy for solid tumors.

Involvement of reactive oxygen species in the enhancement of membrane lipid peroxidation by sonodynamic therapy with functionalized fullerenes.

BACKGROUND/AIM: Sonodynamic cancer therapy is based on the preferential uptake and/or retention of a sonosensitizing drug (sonosensitizer) in tumor tissues and subsequent activation of the drug by ultrasound irradiation. In the present study, we investigated the participation of lipid peroxidation in the mechanism of the sonodynamically-induced antitumor effect with functionalized fullerenes, such as polyhydroxy fullerene (PHF. MATERIALS AND METHODS: Ultrasonically-induced cell damage and lipid peroxidation with PHF were compared in the same in vitro insonation setup. Sarcoma 180 cells suspended in PBS were exposed to 2 MHz ultrasound in the presence and absence of PHF. Cell viability was determined by the Trypan Blue exclusion test. Lipid peroxidation in cell membranes was estimated by measuring the amount of malondialdehyde as the thiobarbituric acid-reactive-substances. RESULTS: Significant enhancement of the rates of both ultrasonically-induced cell damage and lipid peroxidation was observed in the presence of PHF, both of which were positively correlated with PHF. The enhancement of cell damage and lipid peroxidation with PHF was suppressed by reactive oxygen scavengers such as histidine and tryptophan. CONCLUSION: The good correlation observed in the presence of PHF suggests that membrane lipid peroxidation is one of the important intermediary events in sonodynamically-induced cellular damage. The inhibitory effects of histidine and tryptophan also provide evidence that singlet oxygen plays an important role in PHF-mediated sonosensitization of membranes and that this moiety may be an important mediator of cell destruction in sonodynamic therapy associated with PHF and ultrasound.

Therapeutic effect of the treatment for colorectal cancer with adenoviral vectors mediated estrogen receptor ß gene therapy combined with thermotherapy.

INTRODUCTION: Our preliminary study on repressing colorectal tumors by recombinant adenoviruses (Ads) delivering the human ERß gene (Ad-ERß) has achieved positive result. METHODS: In this study, hydrophobic fluorescent dyes ICG-Der-01 was entrapped into the N-succinyl-N'-octyl chitosan (SOC) micelles to form the near infrared absorbing dyes SOC-ICG-Der-01 and SOC-ICG-Der-01 mediated near infrared laser (SOC-ICG-Der-01/NIR) thermotherapy was combined with Ad-ERß gene therapy to regress colon cancer in vivo. RESULTS: Firstly, the antitumor efficacies of SOC-ICG-Der-01/NIR thermotherapy were investigated on S180 ascites tumor-bearing mice. Results indicated that, the average tumor volume of SOC-ICG-Der-01/NIR group was the smallest among the three treatment groups. Then, thermotherapy with SOC-ICG-Der-01/NIR combined with Ad-ERß gene therapy to treat HCT-116 colon cancer xenograft model was investigated. Further results demonstrated that, SOC-ICG-Der-01/NIR thermotherapy showed the significantly inhibitory efficiency compared with control group and Ad-ERß enhanced the therapeutic effect of SOC-ICG-Der-01/NIR. CONCLUSION: These findings demonstrated that combined administration of Ad-ERß with SOC-ICG-Der-01/NIR thermotherapy represents a promising colon cancer therapeutic strategy.

Sonodynamic antitumor effect of a novel sonosensitizer on S180 solid tumor.

PURPOSE: The purpose of this study was to evaluate the sonodynamically induced antitumor effect of a novel sonosensitizer (DVDMS) in mice bearing sarcoma 180 solid tumors. METHODS: In order to determine the optimum timing of ultrasound exposure after administration of DVDMS, a three-dimensional optical imaging system (IVIS spectrum) was used to observe the biodistribution of DVDMS in S180 tumor. The antitumor effects were estimated by the tumor inhibition ratio (volume and weight) after sonodynamic therapy. RESULTS: The experiments suggested that DVDMS has a preferential localization in tumors, but a low accumulation in most normal tissues. A significant synergistic effect of ultrasound combined with DVDMS was obtained when the load power indicated 4 W and DVDMS dose was above 2 mg/kg. At day 14 after DVDMS-SDT, the tumor volume inhibition ratio was 56.27%. In addition, the tumor weight inhibition ratio after the synergistic treatment was 55.37%, which was obviously stronger than ultrasound treatment alone (23.85%) and DVDMS alone (23.15%). Moreover, no metastasis occurred to the tumors in the SDT-treated mice compared with the control group. CONCLUSIONS: DVDMS is a potential sensitizer for sonodynamic cancer therapy. The antitumor effect of ultrasound could be enhanced in the presence of DVDMS, which might be involved in a sonochemical mechanism.

Immunotherapy of cancer via mediation of cytotoxic T lymphocytes by methionine enkephalin (MENK).

The aim of this study was to investigate the immunological mechanisms by which synthetic methionine enkephalin (MENK) exerts therapeutic effects on tumor growth. Our findings in vivo or in vitro show that MENK treatment either in vivo or in vitro could up-regulate the percentages of CD8+T cells, induce markers of activated T cells, increased cytotoxic activity against mouse S180 tumor cells and increase secretion of IFNγ. In addition, the adoptively transferred CD8+T cells, after either in vitro or in vivo treatment with MENK, result in significantly increased survival of S180 tumor-bearing mice and significant shrinkage in tumor growth. Opioid receptors are detected on normal CD8+T cells and exposure to MENK leads to increased expression of opioid receptors. Interaction between MENK and the opioid receptors on CD8+T cells appears to be essential for the activation of CTL, since the addition of naltrexone (NTX), an opioid receptor antagonist, significantly inhibits all of the effects of MENK. The evidence obtained indicates that the MENK-induced T cell signaling is associated with a significant up-regulation of Ca2+ influx into the cytoplasm and the translocation of NFAT2 into nucleus, and these signaling effects are also inhibited by naltrexone.

Suppression of tumor growth by systemic delivery of anti-VEGF siRNA with cell-penetrating peptide-modified MPEG-PCL nanomicelles.

Small interfering RNAs (siRNAs) have potential applications for many diseases, such as cancer, since siRNAs can specifically silence disease-associated genes. However, effective siRNA carriers need to be developed to overcome the low siRNA stability in vivo, to form stable complexes and to facilitate intracellular uptake. In this study, to develop a carrier for systemic siRNA delivery, we prepared methoxy poly(ethylene glycol) (MPEG)/polycaprolactone (PCL) diblock copolymers conjugated with a cell-penetrating peptide, Tat, via a disulfide linkage, and evaluated their ability as an siRNA carrier. The particle size of MPEG-PCL-SS-Tat/siRNA complexes was approximately 100-200 nm. The cellular uptake ability after transfection with FAM-siRNA with MPEG-PCL-SS-Tat was significantly higher than that with FAM-siRNA only. MPEG-PCL-SS-Tat did not induce substantial cytotoxicity. Intravenous injection of MPEG-PCL-SS-Tat/anti-vascular endothelial growth factor (VEGF) siRNA (siVEGF) complexes achieved a high anti-tumor effect in tumor-bearing mice. These results suggest that MPEG-PCL-SS-Tat is a potentially effective siRNA carrier for silencing genes in vitro and in vivo.

Influence of lipid components on gene delivery by polycation liposomes: Transfection efficiency, intracellular kinetics and in vivo tumor inhibition.

Transfection efficiency of non-viral gene vectors is influenced by many factors, including chemical makeup, cellular uptake pathway and intracellular delivery. To investigate the effect of lipid saturation on transfection efficiency of polycation liposomes (PCLs), a soybean phospholipids (SPL), egg phospholipids (EPL) and hydrogenated soybean phosphatidylcholine (HSPC) series was used to prepare PCLs. Testing these PCLs in a luciferase assay indicated that with increasing saturation (SPL

Gold nanorod mediated plasmonic photothermal therapy: a tool to enhance macromolecular delivery.

Plasmonic photothermal therapy (PPTT) with gold nanostructures has been used to generate significant heat within tumors to ablate vasculature. Here we report the use of gold nanorod (GNR) mediated PPTT to induce moderate hyperthermia as a tool to enhance the delivery of macromolecules. GNRs were injected intravenously in a mouse sarcoma (S-180) tumor model. After 24h Evans blue dye (EBD) was injected and the right tumor was radiated with a laser diode for 10 min. EBD content in the right and left tumors were extracted in formamide, measured spectrophotometrically and expressed as a thermal enhancement ratio (TER). Enhanced delivery of EBD was observed (up to 1.8-fold) when tumor temperatures reached 43°C or 46°C. No statistical difference was observed between tumors at these two temperatures, though significant hemorrhage was observed in tumors and surrounding areas receiving the higher thermal dose (46°C). These results indicate that tumor directed PPTT may be used to induce moderate hyperthermia and therefore selectively increase the delivery of macromolecules with therapeutic anticancer drugs.

Sonodynamically-induced antitumor effect of mono-l-aspartyl chlorin e6 (NPe6).

BACKGROUND: The sonodynamically-induced in vitro and in vivo antitumor effects of mono-l-aspartyl chlorin e6 (NPe6) was investigated. MATERIALS AND METHODS: Both in vitro and in vivo antitumor effects were tested in combination with ultrasound at 2 MHz. RESULTS: The rate of ultrasonically-induced damage on isolated sarcoma 180 cells in air-saturated suspension was enhanced two-fold with 80 µM NPe6. The co-administration of 25 mg/kg NPe6 followed by ultrasonic exposure at 2 MHz suppressed the growth of implanted colon 26 cell tumors at an intensity at which ultrasound alone showed only a slight antitumor effect. CONCLUSION: These in vitro and in vivo results suggest that NPe6 is a potential sensitizer for sonodynamic tumor treatment. The enhancement of cell damage by NPe6 was significantly inhibited by histidine, which may suggest reactive oxygen species plays a primary role in sonodynamically-induced antitumor effect.

Sonodynamically induced cell damage using rose bengal derivative.

AIM: The ultrasonically induced effect of a tumor accumulative derivative of rose bengal (RB) on isolated tumor cells was investigated to clarify whether the RB derivative (RBD) maintains the sonosensitizing ability of RB. MATERIALS AND METHODS: Sarcoma 180 cells were suspended in air-saturated phosphate-buffered saline and were exposed to ultrasound in standing wave mode for up to 60 s in the presence and absence of RBD or RB. The viability of the cells was determined by the ability to exclude trypan blue. RESULTS: The ultrasonically induced cell-damaging rate with 100 µM RBD was one order of magnitude higher than that with the same concentration of RB. This increase was significantly inhibited by the active oxygen scavengers histidine, tryptophan and N-acetyl-L-cysteine. CONCLUSION: Chemical modification of RB to RBD for tumor accumulation significantly increased the sonodynamically induced antitumor effect of RB.

Comparison of pharmacokinetics, intracellular localizations and sonodynamic efficacy of endogenous and exogenous protoporphyrin IX in sarcoma 180 cells.

PURPOSE: The aim of the present study was to investigate the differences in pharmacokinetics, sub-cellular localizations and sonodynamic efficacy between endogenous and exogenous protoporphyrin IX (endo-PpIX and exo-PpIX) in sarcoma 180 (S180) cells. MATERIALS AND METHODS: The 5-aminolevulinic acid (ALA)-derived endo-PpIX and exo-PpIX pharmacokinetic profiles were determined by the fluorescence intensity of cell extracts with a spectrophotometer based on a standard curve. The changes in their sub-cellular localization patterns over a prolonged incubation time were evaluated by laser scanning confocal microscopy. The cytotoxic effects of 5-ALA-mediated sonodynamic therapy (ALA-SDT) and exogenous PpIX-mediated sonodynamic therapy (PpIX-SDT) were also evaluated by the MTT assay. RESULTS: The exo-PpIX showed dose-dependent pharmacokinetics in which a plateau of intra- and extracellular content was observed 45min after administration. However, the amount of ALA-derived endogenous intracellular PpIX, as well as extracellular PpIX in the same samples, showed linear accumulation with incubation time, which was independent of ALA concentration. Fluorescent imaging revealed that the exo-PpIX mainly accumulated at the plasma membrane in the early stage, whereas the ALA-derived PpIX initially localized in the mitochondria. Cells displayed sonodynamic damage by the synthesized endo-PpIX after addition of 1mM ALA for 12h, but the cytotoxicity induced by the equivalent amount of exo-PpIX was much more significant with increasing ultrasound intensities. CONCLUSIONS: Our findings suggest that endo- and exo-PpIX in S180 cells differ not only in pharmacokinetics but also in sub-cellular localizations, which may affect their sonodynamic efficacy and mechanisms of inducing cell death.

Suppressed CD31 expression in sarcoma-180 tumors after injection with Toxoplasma gondii lysate antigen in BALB/c mice.

The anti-tumorigenic effects of Toxoplasma gondii (RH) antigens were studied in a murine sarcoma-180 tumor model. To determine the anti-tumor effects, the reduction in tumor size and expression of CD31 (an angiogenesis marker in the tumor tissue) were examined after injection of BALB/c mice with T. gondii lysate antigen (TLA) or formalin-fixed, proliferation-inhibited, T. gondii tachyzoites. Tumors were successfully produced by an intradermal injection of sarcoma-180 cells with plain Matrigel in the mid-backs of mice. After injection with TLA or formalin-fixed T. gondii tachyzoites, the increase in tumor size and weight nearly stopped while tumor growth continued in control mice that were injected with PBS. CD31 expression in TLA-treated or formalin-fixed T. gondii-injected mice was lower than the control mice. Accordingly, the present study shows that the treatment of mice with formalin-fixed T. gondii or TLA in the murine sarcoma-180 tumor model results in a decrease of both tumor size and CD31 expression.

Effective in vitro and in vivo gene delivery by the combination of liposomal bubbles (bubble liposomes) and ultrasound exposure.

Gene delivery with a physical mechanism using ultrasound (US) and nano/microbubbles is expected as an ideal system in terms of delivering plasmid DNA noninvasively into a specific target site. We developed novel liposomal bubbles (Bubble liposomes (BLs)) containing the lipid nanobubbles of perfluoropropane which were utilized for contrast enhancement in ultrasonography. BLs were smaller in diameter than conventional microbubbles and induced cavitation upon exposure ultrasound. In addition, when coupled with US exposure, BLs could deliver plasmid DNA into various types of cells in vitro and in vivo. The transfection efficiency with BLs and US was higher than that with conventional lipofection method. Therefore, the combination of BLs and US might be an efficient and novel nonviral gene delivery system.

Infiltration by macrophages and lymphocytes in transplantable mouse sarcoma after irradiation with high-intensity focused ultrasound.

BACKGROUND: High-intensity focused ultrasound (HIFU) therapy offers great promise for the treatment of cancer. The histological changes, including the antitumor immunological response, after HIFU treatment was examined in soft tissue sarcoma. MATERIALS AND METHODS: Sarcoma 180 cells were injected subcutaneously in mice. Approximately 2 weeks after the injection, the tumor was irradiated by a single shot of HIFU. The tumor diameter was measured and the survival rate was observed after treatment. The tumors were resected, and stained with TUNEL stain, tartrate-resistant acid phosphatase (TRAP) stain to detect tumor-associated macrophages, and immunohistochemical stains for CD4 and CD8. RESULTS: The tumor size in the HIFU group was significantly smaller than the control and survival rate was significantly higher. The numbers of TUNEL-, TRAP-, CD4- and CD8-positive cells infiltrating the tumor were significantly higher in the HIFU group. CONCLUSION: HIFU, even when administered as a single shot, induces apoptosis of tumor cells and intratumoral infiltration of macrophages and lymphocytes.

Substance P and beta-endorphin mediate electro-acupuncture induced analgesia in mouse cancer pain model.

BACKGROUND: Opioid analgesics are generally used to combat the pain associated with cancerous conditions. These agents not only inhibit respiratory function and cause constipation, but also induce other significant side effects such as addiction and tolerance, all of which further contribute to a reduced quality of life for cancer patients. Thus, in the present study, the effects of electro-acupuncture treatment (EA) on mechanical allodynia were examined in a cancer pain mouse model. METHODS: In order to produce a neuropathic cancer pain model, S-180 sarcoma cells were inoculated around the sciatic nerve of left legs of Balb/c mice. Magnetic Resonance Imaging (MRI) scanning confirmed the mass of S-180 cancer cells embedded around the sciatic nerve. Mechanical allodynia was most consistently induced in the mouse sarcoma cell line S-180 (2 x 10(6)sarcoma cells)-treated group compared to all the other groups studied. EA stimulation (2 Hz) was administered daily to ST36 (Zusanli) of S-180 bearing mice for 30 min for 9 days after S-180 inoculation. RESULTS: EA treatment significantly prolonged paw withdrawal latency from 5 days after inoculation. It also shortened the cumulative lifting duration from 7 days after inoculation, compared to the tumor control. Also, the overexpression of pain peptide substance P in the dorsal horn of the spinal cord was significantly decreased in the EA-treated group compared to the tumor control on Day 9 post inoculation. Furthermore, EA treatment effectively increased the concentration of beta-endorphin in blood and brain samples of the mice to a greater extent than that of the tumor control as well as the normal group. The concentration of beta-endorphin for EA treatment group increased by 51.457% in the blood and 12.6% in the brain respectively, compared to the tumor control group. CONCLUSION: The findings of this study suggest that a S-180 cancer pain model is useful as a consistent and short time animal model. It also indicated that EA treatment could be used as an alternative therapeutic method for cancer pain due to a consequent decrease in substance P and increase in beta-endorphin levels.

The sonodynamic antitumor effect of methylene blue on sarcoma180 cells in vitro.

BACKGROUND: Sonodynamic therapy (SDT) is a promising methodology for cancer treatment. Methylene blue (MB) is a phenothiazine dye that is widely used in clinical practice and can be administered intravenously. MATERIALS AND METHODS: The sonodynamic antitumor effect of 1, 10 and 100 microM MB on sarcoma180 (S180) cells was investigated in vitro. RESULTS: After ultrasound (US) exposure at 0.24 W/cm(2) for 30 seconds, survival rates of S180 cells in the presence of 10 and 100 microM MB were significantly lower than that of the control group. These effects were significantly inhibited by the addition of D-mannitol, but not by L-histidine or superoxide dismutase. Microvilli loss and blebbing on the surface of S180 cells were observed in the presence of 100 muM MB after US exposure. CONCLUSION: MB has a sonodynamic antitumor effect on S180 cells in vitro and the hydroxyl radical appears to be the principal mediator of this effect.

Bioeffects of low-energy continuous ultrasound on isolated sarcoma 180 cells.

BACKGROUND: The aim of this study was to investigate the mechanism underlying bioeffects of low-intensity continuous ultrasound on isolated sarcoma 180 (S180) cells and cellular responses to these effects. METHODS: After sonication, several structural and functional parameters were examined to elucidate ultrasound-induced cell damage. RESULTS: Instant disruption of the cell membrane might be caused by acoustic cavitation, producing mechanical and chemical effects that acted simultaneously on S180 cells; this could be reflected by immediate (morphological) changes such as membrane permeability, membrane fluidity, lipid peroxidation and the generation of hydroxyl radicals in culture medium. Our results of the delayed effects also indicated S180 cells were sensitive to ultrasound-induced apoptosis, and the rate of apoptosis rose gradually with a prolonged incubation time. The presence of apoptotic cells was identified by a distinct morphological form characterized by membrane blebbing, cell shrinkage, chromatin condensation and DNA fragmentation. Moreover, delayed cytotoxicity was accompanied by an increase in intracellular reactive oxygen species (ROS) and a decrease in the mitochondrial membrane potential, and the two events presented obviously a negative correlation. CONCLUSION: ROS secondarily generated from damaged mitochondria may play a role in the induction of apoptosis.

[Effect of sequential intratumoral injection of xenogeneic antigens for immunotherapy in immunized mice bearing S180 tumor].

OBJECTIVE: To investigate the therapeutic effect of sequential intratumoral injection of xenogeneic antigens in immunized tumor-bearing mice. METHODS: Sequential intratumoral injection of the xenoantigens was performed in immunized mice bearing S180 tumor. The tumor size changes were observed, and the tumor-infiltrating lymphocytes (TIL) including CD3+CD4+T, CD3+CD8+T, and CD3+CD4+CD25+T lymphocytes were counted with flow cytometry. The concentrations of IL-2 and TNF-alpha in the tumor was measured using ELISA. RESULTS: No significant difference was found in the number of CD3+T lymphocytes in the TILs between different groups. After the immunotherapy, the percentages of CD3+CD4+T, CD3+CD8+T and CD3+CD4+CD25+T lymphocytes were 54%, 22% and 2.91%, respectively, with the CD4+/CD8+ ratio of 2.49, significantly different from that in the control group (P<0.05). The concentrations of IL-2 and TNF-alpha were 100.61 pg/ml and 54.114 pg/ml, respectively, significantly different from those in the control group (P<0.05). CONCLUSION: Sequential intratumoral injection of heteragenetic antigena can significantly increase the amount of effector cells and cytokines in the micro-environment of the tumor, and decrease the expression of T regulatory.