Tumor-bearing Nude Mouse Model Research Articles

Induction of CTLs by DCs pulsed with K-ras mutant peptide on the surface of nanoparticles in the treatment of pancreatic cancer

The aim of this study was to investigate the role of specific cytotoxic T lymphocytes (CTLs) activated by dendritic cells (DCs) presenting cationic nanoparticles with the K-ras (12-Val) mutant peptide in the killing of different pancreatic cancer cell lines invivo and invitro. Peripheral blood DCs were induced by rhGM-CSF and IL-4 and cultured. DCs were sensitized by whole antigen of PANC-1 with expression of K-ras mutant, K-ras mutant peptide (K-ras+peptide) and cationic nanoparticles with K-ras mutant peptide (K-ras+peptide-CNP), respectively. Cell surface markers were measured by flow cytometry. Lymphocyte proliferation was detected by the 3H-TdR test, and IL-12 and IFN‑γ secretion was detected by ELISA. 125I-UdR was used to measure the killing effect of CTLs. The antitumor activity of CTLs in tumor-bearing nude mouse models prepared with PANC-1 and SW1990 cells was evaluated. Results showed that, compared with K-ras+peptide, low concentrations of K-ras+peptide-CNP were effectively presented by DCs (P<0.05). CTLs induced by DCs pulsed with whole tumor antigen had a significantly greater killing effect (P<0.05) on PANC-1 and SW1990 pancreatic cancer cells compared with K-ras+peptide- and K-ras+peptide-CNP-induced CTLs. CTLs induced by DCs pulsed with K-ras+peptide and K-ras+peptide- CNP had a specific killing effect (P<0.05) on PANC-1 cells and no effect (P>0.05) on SW1990 cells. In conclusion, cationic nanoparticles with the K-ras (12-Val) mutant peptide can be effectively presented by DCs at a low concentration. CTLs induced by K-ras+peptide-CNP had specific killing activity for the pancreatic cancer cell line with the K-ras mutant and significantly inhibited tumor growth and increased the survival time of tumor-bearing nude mice. Although this study confirmed that whole cell antigen induced a good antitumor immune response, the possibility of immune tolerance and autoimmunity which has been previously proven contribute to the difficulty in the application of this DC vaccine.

PEG-liposomal oxaliplatin potentialization of antitumor efficiency in a nude mouse tumor-xenograft model of colorectal carcinoma

The non-selectivity of chemotherapeutics between normal tissue and pathological sites poses a challenge for the treatment strategy for advanced colorectal carcinoma. To obtain sufficient antitumor activity, optimization of the therapeutic regimen is of great importance. We investigated PEG-liposomal oxaliplatin potentialization of antitumor efficiency in a nude mouse tumor-xenograft model of colorectal carcinoma. A tumor-bearing nude mouse model, intravenous injections of (Dio)-labeled PEG-liposomes via tail vein and fluorescence imaging with in vivo imaging system were employed. Mice were treated with free L-oHP, PEG-liposomal L-oHP via the tail vein, followed by analysis of the accumulation of L-oHP in tumor tissues by high-performance liquid chromatography (HPLC), observation of the tumor volume and the survival rate. Apoptosis and proliferation of tumors were detected by TUNEL assay and immunohistochemistry. The mRNA and protein levels of Bcl-2, Bax, caspase-3 (P17) and Ki-67 were determined by RT-PCR and Western blotting. Fluorescence imaging with in vivo imaging showed PEG-liposome targeting in tumor tissues. After intravenous injections of PEG-liposomal oxaliplatin, tumor tissue maximum accumulation of L-oHP was 9.37 ± 0.79 µg/g at 24 h; The tumor volume was significantly suppressed, and mice showed longer survival, compared with the free oxaliplatin group. Apoptosis increased, but proliferation decreased in tumor tissues. The mRNA expression of Bcl-2 and Ki-67 was down-regulated, while Bax and caspase-3 expression was up-regulated. Protein expression of Bcl-2 was down-regulated, while Bax and P17 expression was up-regulated. The results indicate that PEG-liposomal oxaliplatin can improve antitumor efficiency in a nude mouse tumor-xenograft model of colorectal carcinoma.

Evaluation of the efficacy, toxicity and safety of vinorelbine incorporated in a lipid emulsion

To reduce the severe adverse effects of vinorelbine (VRB) with the aim of improving patient compliance, a parenteral vinorelbine-loaded lipid emulsion (VLE) has been developed. The objective of the present study was to get insight into the preclinical antitumor efficacy, toxicity and safety of VLE, and compare this with that of the commercial product, Navelbine ® i.v. (VS). Comparable antitumor efficacy of VLE and VS was observed in tumor-bearing nude mouse models inoculated with A549 human lung cancer, hepatoma solidity (Heps) G2 cancer and BCAP-37 human breast cancer cells. The median lethal dose (LD 50) in mice was 29.3 mg/kg (male) and 32.1 mg/kg (female) for VLE, while the corresponding value was 30.5 mg/kg (male and female) for VS. In the long-term toxicity study, VLE significantly reduced the decreases in RBC, HC, WBC and WBC differential count (DC) levels. Lesions in spleen, thymus, lymph nodes, bone marrow, testis, ovary and injection site induced by VS were much more severe compared with VLE. VLE also exhibited less local venous irritation than VS, as well as no hemolysis or hypersensitivity. Consequently, these observations clearly indicate that the lipid emulsion could be a useful potential parenteral carrier for VRB with equivalent efficacy and lower toxicity.

Abstract 3569: Photodynamic activity of the glucoconjugated silicon(IV) phthalocyanine SiPcGlu

Abstract Photodynamic therapy involves the use of a non-toxic photosensitizer which exhibits killing effect upon activation by light. It has been used successfully in treating age-related macular degeneration, actinic keratosis and various cancers like lung, bladder and esophageal cancers. The success of photodynamic therapy depends on the properties of the photosensitizer and there is a continuous search for better photosensitizers with desirable photophysical and biochemical effects. In the last few years, we have synthesized a series of silicon(IV) phthalocyanines with different axial substituents and demonstrated their photocytotoxic effects in the in vitro heptocellular carcinoma HepG2 cells. Among them the glucoconjugated silicon(IV) phthalocyanine SiPcGlu is most promising with IC50 value down to nanomolar range in the cell culture system. In the present investigation, the in vivo effect of SiPcGlu was studied in the HepG2 tumor-bearing nude mice model. The biodistribution of SiPcGlu was followed to evaluate its tumor-selectivity and the tumor volume was measured after photodynamic therapy. The level of intrinsic toxicity induced was also investigated by examining the level of plasma enzyme markers. The distribution and clearance results showed that the concentration of SiPcGlu in the tumor was relatively higher at 24 hr post-injection when compared with all the other tissues examined. One percent of initial dose per gram tissue could be found in the tumor while the corresponding values for the other tissues ranged from 0.8% (lung) to 2.5% (skin). Thus, a drug-light interval of 24 hr was selected in the photodynamic therapy experiment. A laser light dose of 30 J/cm2 (bandwidth 675 ± 3 nm) was spotted onto the tumor 24 hr after intravenous injection of the drug (one μmol per kg body weight). Significant tumor regression was observed for the experimental group of mice. Fifteen days after photodynamic treatment, the volume of the tumor was less than 20% of the initial volume while mice in the two control groups, viz. dark control in which the mice received drug but no irradiation and the light control group in which the mice received irradiation but no drug, did not show any tumor regression. The photodynamic treatment did not cause any apparent toxicity to the animals as the plasma levels of the three enzyme markers, alanine transaminase, aspartate transaminase and creatine kinase, were similar in all the three groups of mice. To conclude, the present results suggest that SiPcGlu is a good candidate for further evaluation on its anti-tumor potential. (This project is supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, Project No. CUHK 4569/05M.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3569.

Establishment of subcutaneously transplanted and metastatic neuroblastoma models in nude mice

To establish a tumor-bearing nude mouse model of human neuroblastoma in order to study the mechanisms of neuroblastoma invasion and metastasis, and to investigate potential therapeutic modalities in the experimental animal models. A human neuroblastoma cell line was cultured in vitro. 1 x 10(7) cells undergoing exponential growth were collected in 0.1 ml of suspension and subcutaneously inoculated into the right flank next to the forelimb in nude mice. The biological characteristics of the developed tumors were observed, and histopathological and DNA microarray analyses were performed. The expressions of NSE in the subcutaneous tumor, metastatic tumor and the primary neuroblastoma tumor tissues from a pediatric patient were analyzed by immunohistochemistry. Tumors successfully grew in 36 out of 48 injected mice, with a total tumor-formation rate of 75.0%. Metastasis occurred in 10 cases, and the metastatic rate was 20.8%. Tumors in five injected mice grew locally without metastasis. These tumors had large volume and the tumor weight reached up to half of the body weight of the host animal. Four mice exhibited systemic metastasis without tumor growth at the primary inoculation site. There were six mice with locally growing tumor accompanied by metastasis. We have successfully established a human neuroblastoma xenograft model in nude mice with high tumor growth and metastatic rates. This model depicting the natural cell growth, local infiltration and distant metastasis characteristics of human neuroblastoma, providing an ideal animal model for in vivo studies of neuroblastoma. In addition, the results of this study indicate the heterogeneous nature of neuroblastoma, it may play an important role in metastasis of this tumor.

Tumor Uptake of the RGD Dimeric Probe 99mTc-G3-2P4-RGD2 is Correlated with Integrin αvβ3 Expressed on both Tumor Cells and Neovasculature

Integrin αvβ3 has been well-documented as one of the key players in the process of tumor angiogenesis. Radiolabeled RGD (Arg-Gly-Asp) peptides that specifically target integrin αvβ3 have great potential for tumor early detection and noninvasively monitoring the status of tumor angiogenesis. We developed a cyclic RGD dimeric probe (99m)Tc-HYNIC-Gly3-E[PEG4-c(RGDfK)]2 ((99m)Tc-G3-2P4-RGD2) (using tricine and TPPTS as the coligands, TPPTS = trisodium triphenylphosphine-3,3',3''-trisulfonate), and investigated whether it could be used to noninvasively visualize and quantify integrin αvβ3 expression in vivo. HYNIC-Gly3-E[PEG4-c(RGDfK)]2 was synthesized and labeled with (99m)Tc. The biodistribution and planar γ-imaging studies of (99m)Tc-G3-2P4-RGD2 were performed in both U87MG (human integrin αvβ3 positive/murine integrin αvβ3 positive) and HT-29 (human integrin αvβ3 negligible /murine integrin αvβ3 positive) tumor-bearing nude mouse models. The correlation of (99m)Tc-G3-2P4-RGD2 tumor uptake values (measured by ex vivo biodistribution) with expression levels of human integrin αvβ3 or murine integrin αvβ3 (measured by Western blot) were determined in U87MG and HT-29 tumor models, respectively. (99m)Tc-G3-2P4-RGD2 exhibited increased receptor binding affinity and in vivo tumor uptake as compared with previously reported RGD dimeric tracer (99m)Tc-RGD2 (without Gly3 and PEG4 spacers). The tumor uptake of (99m)Tc-G3-2P4-RGD2 was related to the expression levels of both human integrin αvβ3 (expressed on tumor cells) and murine integrin αvβ3 (expressed on newborn tumor vasculature). Our results demonstrate that (99m)Tc-G3-2P4-RGD2 is a useful agent for integrin αvβ3 imaging. The relationship between (99m)Tc-G3-2P4-RGD2 uptake and integrin αvβ3 expression level as determined by this study would provide useful information for clinical translation of RGD probes.

Antitumor effect of RNA interference on non-small-cell lung cancer in vivo

To investigate whether chemically synthesized double-stranded RNA (dsRNA) targeting epidermal growth factor receptor (EGFR) can induce gene silencing in non-small cell lung cancer (NSCLC) cells in vivo. The NSCLC cell line SPC-A1 was transfected with EGFR sequence-specific dsRNA formulated with Lipofectamine 2000. SPC-A1 cells (1 x 10(7)/ml) in 200 microl were injected s.c. into the left flank area of the athymic nude mice to establish a tumor-bearing nude mouse model. To calculate the tumor growth inhibition rate by measuring the diameter and the weight of the tumor. Immunohistochemistry and Western blot were used to monitor the reduction of EGFR protein production. Real-time RT-PCR was used to detect the silencing of the EGFR mRNA level. The EGFR sequence specific dsRNA (dsRNA-EGFR) significantly inhibited the tumor growth in vivo. The tumor growth inhibition rate was 75.0%. The dsRNA-EGFR sequence specifically silenced EGFR with 53.6% of down-regulation of EGFR protein production and 32.3% of silencing of EGFR mRNA level. dsRNA-EGFR show a blockbuster effect in down-regulation of EGFR mRNA level and protein production, and inhibition of tumor growth in vivo.

Silencing of signal transducer and activator of transcription 3 expression by RNA interference suppresses growth of human hepatocellular carcinoma in tumor-bearing nude mice

To explore the effect of silencing of signal transducer and activator of transcription 3 (STAT3) expression by RNA interference (RNAi) on growth of human hepatocellular carcinoma (HCC) in tumor-bearing nude mice in vivo. To construct the recombinant plasmid of pSilencer 3.0-H1-STAT3-siRNA-GFP (pSH1-siRNA-STAT3) and establish the tumor-bearing nude mouse model of the HCC cell line SMMC7721, we used intratumoral injection together with electroblotting to transfect the recombinant plasmid pSH1-siRNA-STAT3 into the transplanted tumor. The weight of the nude mice and tumor volumes were recorded. STAT3 gene transcription was detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Level of protein expression and location of STAT3 were determined by Western blotting and immunohistochemical staining. STAT3-related genes such as survivin, c-myc, VEGF, p53 and caspase3 mRNA and protein expression were detected in tumor tissues at the same time. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used to detect apoptosis of tumor cells. The weight of the treated nude mice increased, and the tumor volume decreased markedly compared with those of the mock-treated and negative control groups (P < 0.01). The results of RT-PCR and Western blotting showed that mRNA and protein levels of STAT3 declined markedly in the treated group. The change in STAT3-related gene expression in tumor tissues at the mRNA and protein level also varied, the expression of survivin, VEGF and c-myc were obviously reduced, and expression of p53 and caspase3 increased (P < 0.01). Most of the tumor tissue cells in the treated group developed apoptosis that was detected by TUNEL assay. Silencing of STAT3 expression by RNAi significantly inhibits expression of STAT3 mRNA and protein, and suppresses growth of human HCC in tumor-bearing nude mice. The mechanism may be related to down-regulation of survivin, VEGF and c-myc and up-regulation of p53 and caspase3 expression. Accordingly, the STAT3 gene may act as an important and effective target in gene therapy of HCC.

Impact of Human Neutralizing Antibodies on Antitumor Efficacy of an Oncolytic Adenovirus in a Murine Model

The purpose of this study was to assess the impact of anti-adenovirus neutralizing antibodies (AdNAbs) on the distribution, tolerability, and efficacy of intravenously administered oncolytic adenovirus. A translational model was developed to evaluate the impact of humoral immunity on intravenous administration of oncolytic adenovirus in humans. Initially, severe combined immunodeficient (SCID)/beige mice were passively immunized with various amounts of human sera to establish a condition of preexisting humoral immunity similar to humans. A replication-deficient adenovirus encoding beta-galactosidase (rAd-betagal) was injected intravenously into these mice. An AdNAb titer that mitigated galactosidase transgene expression was determined. A xenograft tumor-bearing nude mouse model was developed to assess how a similar in vivo titer would impact the activity of 01/PEME, an oncolytic adenovirus, after intravenous administration. In SCID/beige mice, there was a dose dependence between AdNAbs and galactosidase transgene expression; 90% of transgene expression was inhibited when the titer was 80. A similar titer reconstituted in the nude mice with human serum, as was done in the SCID/beige mice, did not abrogate the antitumor efficacy of the replicating adenovirus after intravenous administration. Viral DNA increased in tumors over time. In intravenous administration, preexisting AdNAb titer of 80 significantly attenuated the activity of a 2.5 x 10(12) particles per kilogram dose of nonreplicating adenovirus; the same titer had no affect on the activity of an equivalent dose of replicating adenovirus. Our results suggest that a majority of patients with preexisting adenovirus immunity would be candidates for intravenous administration of oncolytic adenovirus.

Enhanced photodynamic antitumor effect on gastric cancer by a novel photosensitive stealth liposome

Lipophilic photosensitizers hold potential for cancer photodynamic therapy. We sought to develop a novel photosensitive stealth liposome (PSSL) which incorporating a lipophilic photosensitizer into its lipid bilayer and to examine its photoactivity. We prepared PSSL composed of lipophilic chlorin e6 (Ce6) ester, dilauroylphosphatidylcholine, dioleoylphosphatidylethanolamine and distearoyl-phosphoethanolamine- N-[poly (ethylene glycol) 2000] and evaluated its photodynamic effect against gastric cancer cell lines and tumor-bearing nude mice models. In gastric cancer cell lines, LC 80 of PSSL was a maximum of 53 times as low as that of Ce6 sodium salt (Ce6-Na). PSSL completely destroyed all tumors in animal models and tumor recurrence levels were minimal (1.5±0.9%). PSSL achieved greater photodynamic effects in gastric cancer cell lines and in murine models than Ce6-Na. PSSL holds promise for photodynamic therapy for gastric cancer.

Structure-activity of novel rhodacyanine dyes as antitumor agents.

We have previously reported that rhodacyanine dyes, such as 1 and 2, exhibited a potent inhibitory effect on the growth of several tumor cells and that 4-oxothiazolidine (rhodanine) was an essential moiety for antitumor activity. On the basis of our foregoing work, two types of rhodacyanine dyes, which categorized into class I and II depending on the methine length, were synthesized and evaluated as a novel antitumor agent. Attention was particularly focused on the structure-activity study of two heteroaromatic rings. In class I, where the A rings were conjugated to rhodanine via two methine groups, compounds 1, 20, 23, and 24 were found to be efficacious in tumor-bearing nude mice model study, but they did not have the chemical properties (stability, solubility) suitable for clinical use. In contrast, in class II, where the A rings were directly conjugated to rhodanine, compounds 13 and 25, which possessed a benzothiazole moiety for the A ring, exhibited the favorable biological and chemical properties. Therefore, we decided to have a benzothiazole moiety as the A ring and introduce various heterocyclic groups for the B ring. As a result, the pyridinium ring was selected as the optimal moiety for the B ring (compound 13). Further, the variation of counteranion had a profound effect on solubility in water without influence on antitumor activity. Chloride anion was selected as the favorable anion with respect to synthetic method as well as solubility in water. Our study finally led us to the identification of compound 3 (MKT 077, 1-ethyl-2-[[3-ethyl-5-(methylbenzothiazolin-2-ylidene)-4-oxothi azolidin-2 -ylidene]methyl]pyridinium chloride) as the candidate for clinical trials and is currently subjected to further investigation as a potent antitumor agent in phase I clinical trial for the treatment of solid tumors.

Synthesis and evaluation of novel rhodacyanine dyes that exhibit antitumor activity.

Rhodacyanine dyes and several analogous delocalized lipophilic cations (DLCs) were synthesized and evaluated as novel antitumor agents. Rhodacyanine dye consists of two heteroaromatic rings such as thiazoles at both termini of the conjugate systems and 4-oxothiazolidine (rhodanine) in the middle of it. Compounds with such a unique double-conjugate structure were found to inhibit the growth of several tumor cell lines, such as colon carcinoma CX-1, and to exhibit relatively low toxicity against normal kidney cell line CV-1 (e.g., IC50(CX-1) = 50 nM, IC50(CV-1) = 17.3 microM; selectivity index = 346 for compound 5). These compounds were also found to be efficacious in the tumor-bearing nude mice model (e.g., against human melanoma LOX; T/C (%) = 168 for compound 5). Structural modifications on rhodacyanine, including deletion of a heteroaromatic ring involved in the merocyanine conjugate system and replacement of rhodanine with a structurally related moiety such as 4-oxoimidazolidine or 4-oxo-1,3-dithiolane, resulted in a loss of the selectivity and/or the activity. Our current structure-activity studies imply that the double-conjugate system with a rhodanine moiety is essential for the selective activity of rhodacyanine dyes, and we find this class of compounds as unique antitumor agents candidates.