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.

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.

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.

Sonodynamic effects of lomefloxacin derivatives conjugated with methoxy polyethylene glycol on sarcoma 180 cells.

BACKGROUND: Sonodynamic therapy (SDT) is a promising methodology for cancer treatment. Lomefloxacin hydrochloride (LFLX) has been reported to have sonodymamic antitumor effects. MATERIALS AND METHODS: We synthesized LFLX derivatives conjugated with methoxy polyethylene glycol (PEGylated LFLXs) and investigated their ultrasonically induced antitumor effects. RESULTS: After ultrasound exposure at 2.0 MHz for 30 s, the survival rates of Sarcoma 180 cells in the presence of lower molecular weight PEGylated LFLXs (200 microM) were significantly lower than those of the control and the LFLX at 1.5 and 2.0 W/cm2. This enhancement was significantly inhibited by the addition of L-histidine, but not by D-mannitol or superoxide dismutase. There was no apparent cell damage in the presence of high molecular weight PEGylated LFLX even at 3.0 W/cm2. CONCLUSION: These findings indicate that the sonodynamic antitumor effects of lower molecular weight PEGylated LFLXs are better than those of LFLX.

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.

The tumoricidal effect of sonodynamic therapy (SDT) on S-180 sarcoma in mice.

There are increasing data showing that sonodynamic therapy (SDT), which refers to a synergistic effect of drugs and ultrasound, is a promising new modality for cancer treatment. However, few clinical data on SDT have been published. One reason is the lack of suitable drugs for clinical SDT use. Recently a new sonosensitizing agent has been developed by SonneMed, LLC, referred to as SF1. In this study the effect of SDT with SF1 on S-180 sarcoma in mice was examined. The tumor bearing mice were allocated to the following groups: (1) sham-treatment (control, C); (2) ultrasound treatment (only ultrasound treatment, 1.2 mW/cm2 , without SF1, U); (3) SF1 treatment (SF1 20 mg/kg intraperitoneal [ip] without ultrasound treatment, S); and (4) SF1 + ultrasound treatment (SU). Following treatment, tumor volume was monitored. Tumor growth inhibition was seen only in group SU, and with increasing ultrasound intensity, the inhibitory effect was enhanced. Tumor growth inhibition was also visible even when covered by a barrier of bone. Pathological slices showed coagulated necrosis or metamorphic tissue with inflammatory reaction in the tumor taken from 2 to 36 hours after SDT. These data revealed that SDT with SF1 did inhibit growth of mouse S-180 sarcoma and the inhibitory effect was sound intensity dependent. SDT also induced some inflammation while it destroyed the tumor, indicative of a "vaccine" effect. SF1 shows great promise for clinical use in the future.

[Electrochemotherapy for tumor and mechanism analysis].

Electrochemotherapy was instituted for sarcoma, and the tumor inhibitory ratio, curing ratio, vascular endothelial growth factor, microvessel density and mechanism were measured and analyzed. The results indicate that the curing ratio of electrochemotherapy for sarcoma is 84.6%. The present research provides experimental evidence for the security, mechanism and feasibility of electrochemotherapy in clinical practice.

Comparison between sonodynamic effect with protoporphyrin IX and hematoporphyrin on sarcoma 180.

PURPOSE: The comparison between sonodynamic antitumor effect with protoporphyrin IX (PPIX) and hematoporphyrin (Hp) at a concentration of 5 mg/kg on Sarcoma 180 (S180) cells was studied in vivo, and the potential cell damage mechanism was also investigated. METHODS: The sonodynamically induced anti-tumor effect of PPIX was studied in mice bearing S180 solid tumors. In order to determine the optimum timing of ultrasound exposure after administration of PPIX, the PPIX concentrations in plasma, skin, muscle and tumor were determined by the fluorescence intensity of tissue extractions with a fluorescence spectrophotometer based on the standard curve. Anti-tumor effects were estimated by measuring the tumor size and the tumor weight. Additionally, the morphological changes of S180 cells were evaluated by transmission electron microscope (TEM) observation immediately after sonodynamic therapy (SDT) treatment. RESULTS: A time of 24 h after the intravenous administration of PPIX was chosen as the best time for ultrasound exposure. The antitumor effect induced by PPIX mediated sonodynamic therapy (PPIX-SDT) was in a dose dependent manner when ultrasound intensity was at or above the inertial cavitation threshold (5 W/cm(2)). A significant tumor growth delay was observed both in PPIX mediated sonodynamic therapy and in Hp mediated sonodynamic therapy treatments (Hp-SDT), and the tumor weight inhibition ratios after the synergistic treatments were 42.82 +/- 0.03 and 35.22 +/- 0.03%, respectively, this difference was significant at P < 0.05. While ultrasound alone (5 W/cm(2)) showed a slight tumor growth inhibitory effect compared with the control group, and PPIX or Hp alone showed almost no significant effect. Furthermore, TEM observation indicated cell damage was more serious in PPIX-SDT treatment group than in Hp-SDT treatment group. After sonication, the cell ultra-structure such as cell membrane destruction, mitochondria swelling, chromatin condensation might be important factors that inhibited the tumor growth and even induced cell death. CONCLUSIONS: The comparative results suggested that PPIX as a sonosensitizer might have more potential cytotoxicity than Hp when irradiated with ultrasound, and the ultra-structural changes may account for cell destruction induced by sonodynamic therapy in our experiment mode.

Antitumor effect of acridine orange under ultrasonic irradiation in vitro.

BACKGROUND: Sonodynamic synergistic antitumor therapy is a promising new methodology for cancer treatment. MATERIALS AND METHODS: To determine the antitumor effects of ultrasound (US) in the presence of acridine orange (AO), 1.0 microg/ml solution of AO was tested as a sonodynamic compound against sarcoma 180 cells in vitro. RESULTS: After US irradiation at 2.0 W/cm2 for 60 sec, the survival rate of tumor cells in the presence of AO was significantly lower than in its absence (p < 0.001). In the AO-treated group, the tumor cells were mostly fragmented. When D-mannitol or L-histidine was used along with the AO, the survival rate of tumor cells after irradiation was significantly higher than that when AO alone was applied. CONCLUSION: AO could exhibit useful antitumor activity under US irradiation, and the generation of singlet oxygen and hydroxyl radicals is involved in the processes of tumor cell damage.

Sonodynamic antitumor effect of protoporphyrin IX disodium salt on S180 solid tumor.

BACKGROUND: The sonodynamically induced antitumor effect of protoporphyrin IX (PPIX) disodium salt was studied in mice bearing sarcoma 180 solid tumors. METHODS: In order to determine the optimum timing of ultrasound exposure after administration of PPIX, the PPIX concentrations in plasma, skin, muscle and tumor were estimated by measuring the fluorescence intensity of tissue extractions with a fluorescence photometer based on the standard curve. Antitumor effects were estimated by measuring tumor size and calculating the average survival time of tumor-bearing mice after sonodynamic therapy; additionally, the morphological changes of sarcoma 180 cells were evaluated by transmission electron microscope observation in vivo. RESULTS: Our experiments suggested a time of 24 h after the administration of PPIX to be best for ultrasound exposure. At an ultrasound intensity >or=5 W/cm(2) and a PPIX dose >or=5 mg/kg, a significant synergistic effect of ultrasound combined with PPIX was observed, reducing tumor volume and increasing average animal survival time; this synergistic effect was obviously stronger than ultrasound treatment alone, while PPIX alone showed no significant effect. Transmission electron microscope observation indicated that changes in cell ultrastructure, such as cell membrane destruction, mitochondria swelling and chromatin condensation, were important factors that inhibited tumor growth and even induced cell death. CONCLUSION: The results implied that the antitumor effect of ultrasound could be enhanced in the presence of PPIX which might be involved in a sonochemical mechanism.

[The killing effect of focused ultrsound activating protoporphyrin IX on S180 cells].

The killing effect on S180 cells was studied using the combination of protoporphyrin IX and focused ultrasound at the frequency of 2.2 MHz and different intensities. Cell viability was determined by trypan blue exclusion test, morphology changes were evaluated by means of scanning electron microscopy and transmission electron microscopy after ultrasonic exposure. The results indicated that protoporphyrin IX(PPIX) alone showed no significant effect on S180 cells when compared with that of control group. Ultrasound alone and ultrasound combined with PPIX groups showed some anti-tumor effect, which became more noticeable as the ultrasound intensity and PPIX concentration increased, and when the concentration of PPIX increased to 120 microM, the ultrasound combined with PPIX exerted a more significant anti-tumor effect than did the ultrasound alone in the same experiment.

Antitumor of a boiled scallop extract.

A fraction from a boiled scallop extract modified by a sonication procedure exhibited marked antitumor activity when it was injected intradermally into either ICR or C3H/He mice that had been given sc implants of sarcoma 180 and Ehrlich carcinoma. The active moiety was heat-stable and noncytotoxic. In contrast, preparations from raw scallop showed lower antitumor activity and were heat-labile.

Effects of systemic acidification of mice with Sarcoma 180.

The effects of dietary-induced acidosis on the growth and rates of complete regression of Sarcoma 180 in mice have been studied. The experiments here reported have demonstrated that mineral acidification of laboratory food produces a late decrease in tumor growth and significantly increases the rates of complete tumor regression. Blood acid-base studies also demonstrate the effects of these diets in altering the acid-base balance, and seemingly, this is independent of starvation and/or ketosis. The relationships of such in vivo acid-base metabolic changes to the control of tumor metabolism are briefly discussed. A therapeutic potential for this preliminary approach is considered.

Gene therapeutic exploration: retrovirus-mediated soluble vascular endothelial growth factor receptor-2 (sFLK-1) inhibits the tumorigenicity of S180, MCF-7, and B16 cells in vivo.

Inhibition of tumor angiogenesis is an anticancer strategy in which neovasculature is targeted because tumor progression relies on neovascularization. The soluble, truncated form of vascular endothelial growth factor receptor-2 (VEGFR-2), sFLK-1, is a well-known inhibitor of endothelial cells. This kind of soluble receptor retains its high-affinity binding to VEGF, but cannot work with the receptor tyrosine transphosphorylation and activation of downstream signal transduction to induce endothelial proliferation due to the lack of the tyrosine kinase domain. Therefore, we tried to use this sFLK-1 as an inhibitor for malignant tumor gene therapy. In this study we transferred a soluble VEGFR-2 (sFLK-1) from embryo mouse liver by RT-PCR to PA317 cells through retroviral vector (pLXSN) and obtained stable expression. NIH3T3 cells were used for measuring the virus titer. The virus titer in this experiment was 2 x 10(7) CFU/ml. After 7 days of preparing subcutaneous tumor models bearing S180, MCF-7, and B16 cells in mice, respectively, 2 x 10(7) PFU of recombinant retroviruses were injected locally into the tumors the treatment groups. After treatment, the tumor size and weight were significantly smaller than that of control (p < 0.05). After autopsy, the metastasic focus numbers in the treatment groups were also less than control groups. We also measured VEGFR-2 expression in tumor tissues by Western blot to check if sFLK-1 had been integrated into the cells of tumor tissues. Expression in the treatment groups was significantly greater than that of control groups (p < 0.001). Microvessel density (MVD) and proliferative cell nuclear antigen (PCNA) were investigated to determine whether the Re-sFLK-1 fragment had the ability to inhibit tumor angiogenesis and proliferation in mice bearing S180 and MCF-7 cells. The results showed that MVD and PCNA in th e treatment groups werelower than in control groups. There were significant difference between treatment groups and control groups (p < 0.0001). The results indicated that retroviral-mediated sFLK-1 gene therapy in animal tumor models has significant therapeutic effect.

Cellular toxic effects of combination of low dose of cisplatin and interstitial injection of 32P glass microspheres on mouse solid tumor S180.

BACKGROUND: Although 32P-glass microspheres (32P-GMS) have been used in internal radiotherapy for malignant tumors, it has been one of the key obstacles to improve the effect of radiotherapy. We investigated the cellular and hypersensitive effect of combined use of low dose of cisplatin and interstitial injection of 32P-GMS on mouse solid tumor S180. METHODS: The mice with solid tumor S180 were randomly divided into four groups (controls, cisplatin therapy, 32P-GMS therapy and combination therapy). The specimens of the mice were sectioned two weeks after treatment and weighed. The death rate of tumor cells and the inhibition rate of tumor were calculated respectively. The cell cycle and apoptosis rate were evaluated with flow-cytometry. The ultrastructural changes of the four groups were observed by a transmission electron microscope. The data were analyzed by the chi-square test. RESULTS: The growth of tumor was slower in the combination therapy group than in the simple therapy groups by macrography. The inhibition rate and the death rate of tumor cells of the combination therapy group were significantly higher than those of the control group and the other two simple therapy groups (P < 0.05). More cell damages were displayed in the combination therapy group than in the other groups under the light and electronic microscope. CONCLUSION: Low-dose cisplatin combined with interstitial injection of 32P glass microspheres could be used as an effective hypersensitive regimen for the internal radiotherapy of mouse solid tumor S180.

Response of S 180 murine tumor to bleomycin in combination with radiation and hyperthermia using micronucleus assay: a multimodality approach for therapeutic augmentation.

Response of a transplantable tumor, S180, grown intradermally in inbred Balb/c mice, was assessed by using micronucleus assay after treating the solid tumors with bleomycin (BLM), radiation (RT) and hyperthermia (HT) vis-a-vis multimodality approach. The frequency of micronuclei (MN) though did not vary greatly during the one week of observation in untreated tumors, it significantly increased in the drug and RT groups at 24 hr post-treatment. However, MN frequency was non-significant in the HT group from the control. A drug dose dependent linear increase in the frequency of MN induction was evident in 10, 15 and 20 mg/kg body weight BLM alone treated groups. Combination of radiation with BLM or HT further increased the MN counts in the bimodality groups. But, MN induction at 24 hr post-treatment in the trimodality group (BLM + RT + HT) was non-significant from that of the bimodality treatments. However, the tumors treated with trimodality treatment presented severe tumor necrosis, indicating increased cell loss, and resulting in immediate tumor regression. In all the bi-modality groups MN counts though declined 3 or 5 days post-treatment, the values remained significantly higher than the control, on day 7 post-treatment. Micronucleus assay could be used as a predictive parameter for the assessment of post-irradiation tumor regression response. However, the tumor response assessment with MN assay alone may not be sufficient and the role of other parameters, such as apoptosis and necrosis, in immediate tumor regression, especially radiosensitive/thermosensitive tumors can not be ignored while taking multimodality approach into consideration for cancer therapy.

[Electrochemotherapy and its mechanism for sarcoma of KM mice].

In this paper are analyzed the effect of electrochemotherapy and its mechanism. The favorable parameter of electric pulses (EP) was studied in vitro using the S-180 cells exposed to various EP. After the tumor model was copied, the tumor-bearing mice were randomly divided into four groups: control, chemotherapy, electrotherapy, and electrochemotherapy. The tumor inhibitory ratio, the cure ratio and the level of oxygen free radicals (OFR) were determined. The inhibitory ratio and cure ratio of electrochemotherapy group were significantly higher than those in the chemotherapy, electrotherapy and control groups (P < 0.05). The injury of OFR was decreased while the immunological competence was increased. The mechanism of electrochemotherapy may be related with the enhancement of cell membranepermeability, the depression of drug resistance, the improvement of immunological competence, and so on.