Histological Analysis Of Tumors Research Articles

Multiple endocrine neoplasia type 1 with Zollinger-Ellison syndrome: clinicopathological analysis of a Japanese family with focus on menin immunohistochemistry.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by the occurrence of multiple epithelial neuroendocrine tumors (NETs) and non-NETs in various organs. MEN1 encodes a 610-amino acid-long tumor suppressor protein, menin. The optimal treatment for multiple tumors, identification of the most critical tumors for patient prognosis, and menin immunohistochemistry findings remain controversial. Therefore, we aimed to elucidate these issues through a histological analysis of tumors and tumor-like lesions in a Japanese family, comprising a father and his two sons, who had MEN1 with Zollinger-Ellison syndrome (ZES). All family members had a germline alteration in exon 10, c.1714-1715 del TC of MEN1, and exhibited multiple synchronous and metachronous tumors. The patients had pulmonary NETs, hyperparathyroidism, hypergastrinemia, pituitary adenomas, pancreaticoduodenal NETs, adrenocortical adenoma with myelolipoma, nodular goiter of the thyroid, lipomas, and angiofibroma. Most tumors were resected and histologically examined. We compared their clinical courses and tumor histology, and conducted menin immunohistochemistry (IHC). Two patients died of pulmonary NET G2. One patient who underwent pancreaticoduodenectomy was cured of ZES; however, the two other patients who did not undergo pancreaticoduodenectomy suffered persistent ZES despite treatment with octreotide. Menin IHC revealed varying NET intensities, ranging from positive to negative stains. Pancreaticoduodenectomy is the most effective treatment for ZES. Long-term follow-up is essential for pulmonary NET G2 owing to the risk of distant metastasis and/or multiplicity. Moreover, the variability of menin IHC in MEN1-related tumors may indicate the pattern of tumor formation rather than the diagnostic utility of menin in MEN1.

Molecular MR Imaging of Prostate Cancer by Specified Iron Oxide Nanoparticles With PSMA-11 Peptides: A Preclinical Study.

Prostate-specific membrane antigen (PSMA) can provide a prostate cancer (PCa) detection approach in positron emission tomography (PET) using Food and Drug Administration (FDA)-approved PSMA-11 peptide. There are some studies evaluated magnetic-nanoprobes for PSMA detection by MRI, using non-FDA-approved ligands including antibodies or peptides, which are not as specific as PSMA-11. To assess targeted iron oxide nanoparticles (IONPs) by PSMA-11 peptides as a potential specific nano-molecular probes to investigate a PSMA+ PCa-xenograft model by MRI. Prospective. Twenty male C57BL6 nude mice induced subcutaneously PSMA+ LNCaP cell line tumor. 1.5 T, T2-W Fast Spin echo and T2*-W Gradient echo. Coated IONPs with Carboxymethylated-dextran (DNPs) and with bovine serum albumin (BNPs), as well as, targeted DNPs with PSMA-11-HYNIC peptide (TDNPs) and targeted BNPs with PSMA-11-HBED peptide (TBNPs) were injected intravenously with dose 2.8 mg Fe/kg. Coronal T2-W and the T2*-W images were obtained before and 4 hours and 6 hours post-injection. Signal intensity (SI) and relative signal enhancement (RSE) were computed in two- and three-dimensional analyses. Histological analysis of tumors was evaluated, and the Fe distribution within the body based on atomic absorption spectroscopy was calculated. One-way ANOVA followed by Tukey's multiple comparison test, Paired-samples T-test, P < 0.05 was considered significant. A reduction in T2-W SI was achieved as 22 ± 7%, 59 ± 3%, 65 ± 5%, and 78 ± 3% respectively for BNPs, TBNPs, DNPs, and TDNPs 6 hours post-injection. The most difference between targeted and non-targeted groups was observed at 6 hours for PSMA-11-HBED, and at 4 hours for PSMA-11-HYNIC. RSE indicated 88.6 ± 3.1% and 80.7 ± 3.2% enhanced contrast between tumor and muscle region for TBNPs and TDNPs on T2*-W images. Both TBNPs and TDNPs are promising novel nano-molecular probes for PSMA+ PCa tumor detection. The injection dose of non-targeted IONPs can be reduced by using targeted nanoprobes three times for BNPs and two times for DNPs. 1 TECHNICAL EFFICACY: Stage 1.

Evaluation of a photodynamic therapy agent using a canine prostate cancer model.

Male dogs can develop spontaneous prostate cancer, which is similar physiologically to human disease. Recently, Tweedle and coworkers have developed an orthotopic canine prostate model allowing implanted tumors and therapeutic agents to be tested in a more translational large animal model. We used the canine model to evaluate prostate-specific membrane antigen (PSMA)-targeted gold nanoparticles as a theranostic approach for fluorescence (FL) imaging and photodynamic therapy (PDT) of early stage prostate cancer. Dogs (four in total) were immunosuppressed with a cyclosporine-based immunosuppressant regimen and their prostate glands were injected with Ace-1-hPSMA cells using transabdominal ultrasound (US) guidance. Intraprostatic tumors grew in 4-5 weeks and were monitored by ultrasound (US). When tumors reached an appropriate size, dogs were injected intravenously (iv) with PSMA-targeted nano agents (AuNPs-Pc158) and underwent surgery 24 h later to expose the prostate tumors for FL imaging and PDT. Ex vivo FL imaging and histopathological studies were performed to confirm PDT efficacy. All dogs had tumor growth in the prostate gland as revealed by US. Twenty-four hours after injection of PSMA-targeted nano agents (AuNPs-Pc158), the tumors were imaged using a Curadel FL imaging device. While normal prostate tissue had minimal fluorescent signal, the prostate tumors had significantly increased FL. PDT wasactivated by irradiating specific fluorescent tumor areas with laser light (672 nm). PDT bleached the FL signal, while fluorescent signals from the other unexposed tumor tissues were unaffected. Histological analysis of tumors and adjacent prostate revealed that PDT damaged the irradiated areas to a depth of 1-2 mms with the presence of necrosis, hemorrhage, secondary inflammation, and occasional focal thrombosis. The nonirradiated areas showed no visible damages by PDT. We have successfully established a PSMA-expressing canine orthotopic prostate tumor model and used the model to evaluate the PSMA-targeted nano agents (AuNPs-Pc158) in the application of FL imaging and PDT. It was demonstrated that the nano agents allowed visualization of the cancer cells and enabled their destruction when they were irradiated with a specific wavelength of light.

Tumor Radiosensitization by Gene Electrotransfer-Mediated Double Targeting of Tumor Vasculature.

Targeting the tumor vasculature through specific endothelial cell markers involved in different signaling pathways represents a promising tool for tumor radiosensitization. Two prominent targets are endoglin (CD105), a transforming growth factor β co-receptor, and the melanoma cell adhesion molecule (CD1046), present also on many tumors. In our recent in vitro study, we constructed and evaluated a plasmid for simultaneous silencing of these two targets. In the current study, our aim was to explore the therapeutic potential of gene electrotransfer-mediated delivery of this new plasmid in vivo, and to elucidate the effects of combined therapy with tumor irradiation. The antitumor effect was evaluated by determination of tumor growth delay and proportion of tumor free mice in the syngeneic murine mammary adenocarcinoma tumor model TS/A. Histological analysis of tumors (vascularization, proliferation, hypoxia, necrosis, apoptosis and infiltration of immune cells) was performed to evaluate the therapeutic mechanisms. Additionally, potential activation of the immune response was evaluated by determining the induction of DNA sensor STING and selected pro-inflammatory cytokines using qRT-PCR. The results point to a significant radiosensitization and a good therapeutic potential of this gene therapy approach in an otherwise radioresistant and immunologically cold TS/A tumor model, making it a promising novel treatment modality for a wide range of tumors.

Anticancer effects of ABTL0812, a clinical stage drug inducer of autophagy-mediated cancer cell death, in glioblastoma models.

Glioblastoma multiforme (GBM) is the most malignant adult brain tumor. Current standard of care treatments have very limited efficacy, being the patients´ overall survival 14 months and the 2-year survival rate less than 10%. Therefore, the treatment of GBM is an urgent unmet clinical need. The aim of this study was to investigate in vitro and in vivo the potential of ABTL0812, an oral anticancer compound currently in phase II clinical stage, as a novel therapy for GBM. We showed that ABTL0812 inhibits cell proliferation in a wide panel of GBM cell lines and patient-derived glioblastoma stem cells (GSCs) with half maximal inhibitory concentrations (IC50s) ranging from 15.2 µM to 46.9 µM. Additionally, ABTL0812 decreased GSCs neurosphere formation. GBM cells aggressiveness is associated with a trans-differentiation process towards a less differentiated phenotype known as proneural to mesenchymal transition (PMT). ABTL0812 was shown to revert PMT and induce cell differentiation to a less malignant phenotype in GBM cell lines and GSCs, and consequently reduced cell invasion. As previously shown in other cancer types, we demonstrated that the molecular mechanism of action of ABTL0812 in glioblastoma involves the inhibition of Akt/mTORC1 axis by overexpression of TRIB3, and the activation of endoplasmic reticulum (ER) stress/unfolded protein response (UPR). Both actions converge to induce autophagy-mediated cell death. ABTL0812 anticancer efficacy was studied in vivo using subcutaneous and orthotopic intra-brain xenograft tumor models. We demonstrated that ABTL0812 impairs tumor growth and increases disease-free survival and overall survival of mice. Furthermore, the histological analysis of tumors indicated that ABTL0812 decreases angiogenesis. Finally, we investigated the combination of ABTL0812 with the standard of care treatments for GBM radiotherapy and temozolomide in an orthotopic model, detecting that ABTL0812 potentiates the efficacy of both treatments and that the strongest effect is obtained with the triple combination of ABTL0812+radiotherapy+temozolomide. Overall, the present study demonstrated the anticancer efficacy of ABTL0812 as single agent and in combination with the GBM standard of care treatments in models of glioblastoma and supports the clinical investigation of ABTL0812 as a potential novel therapy for this aggressive brain tumor type.

Tumor-Selective Cascade-Amplified Dual-Prodrugs Activation for Synergistic Oxidation-Chemotherapy

Tumor-Selective Cascade-Amplified Dual-Prodrugs Activation for Synergistic Oxidation-Chemotherapy

Morphological characteristics and ecological significance of Volga‐Caspian Basin fish tumors

Aim . To investigate the histological structure of fish tumors in the Volga Delta and the northern part of the Caspian Sea to assess the habitat quality of aquatic organisms. Material and Methods . Neoplasms identified in the following representatives of ichthyofauna were analyzed: black ‐ backed herring, roach, asp, perch, carp, pike, zander, catfish and bream. Biological material was collected during specialized scientific research expeditions to the Volga Delta in April ‐ October 2015 ‐ 2018. Sample analysis were carried out in accordance with standard histological techniques. Results . The research shows that tumors were localized both on the surface of the body and in the internal organs of fish. During the period of investigation individuals examined were diagnosed with the following cancers: adenomectomy, basal cell carcinoma, hepatocellular carcinoma, polypous cancer, rhabdomyoma, fibrosarcoma and follicular, epidermal papilloma. The most common pathology in the populations of semi ‐ migratory, migratory and river fish was a malignant tumor, fibrosarcoma, registered in six of the ten examined ichthyofauna species. Epithelial papilloma was most often observed in catfish. There were no oncological diseases were detected in rudd. Conclusions . Histological analysis of tumors recorded in the Volga ichthyofauna has shown that a number of identified disorders are irreversible and will inevitably lead to the reduction of fish resources as well as generally indicating the environmental problems of the habitat of aquatic organisms.

Electrotransfer of plasmid DNA radiosensitizes B16F10 tumors through activation of immune response.

BackgroundTumor irradiation combined with adjuvant treatments, either vascular targeted or immunomodulatory, is under intense investigation. Gene electrotransfer of therapeutic genes is one of these approaches. The aim of this study was to determine, whether gene electrotransfer of plasmid encoding shRNA for silencing endoglin, with vascular targeted effectiveness, can radiosensitize melanoma B16F10 tumors.Materials and methodsThe murine melanoma B16F10 tumors, growing on the back of C57Bl/6 mice, were treated by triple gene electrotransfer and irradiation. The antitumor effect was evaluated by determination of tumor growth delay and proportion of tumor free mice. Furthermore, histological analysis of tumors (necrosis, apoptosis, proliferation, vascularization, presence of hypoxia and infiltration of immune cells,) was used to evaluate the therapeutic mechanisms.ResultsGene electrotransfer of plasmid silencing endoglin predominantly indicated vascular targeted effects of the therapy, since significant tumor growth delay and 44% of tumor free mice were obtained. In addition, irradiation had minor effects on radioresistant melanoma, with 11% of mice tumor free. The combined treatment resulted in excellent effectiveness with 88% of mice tumor free, with more than half resistant to secondary tumor challenge, which was observed also with the plasmid devoid of the therapeutic gene. Histological analysis of tumors in the combined treatment group, demonstrated similar mode of action of the gene electrotransfer of plasmid encoding shRNA for silencing endoglin and devoid of it, both through the induction of an immune response.ConclusionsThe results of this study indicate that irradiation can in radioresistant melanoma tumors, by release of tumor associated antigens, serve as activator of the immune response, besides directly affecting tumor cells and vasculature. The primed antitumor immune response can be further boosted by gene electrotransfer of plasmid, regardless of presence of the therapeutic gene, which was confirmed by the high radiosensitization, resulting in prolonged tumor growth delay and 89% of tumor free mice that were up to 63% resistant to secondary challenge of tumor. In addition, gene electrotransfer of therapeutic plasmid for silencing endoglin has also a direct effect on tumor vasculature and tumors cells; however in combination with radiotherapy this effect was masked by pronounced immune response.

Evaluation of Efficacy of Radioimmunotherapy with 90Y-Labeled Fully Human Anti-Transferrin Receptor Monoclonal Antibody in Pancreatic Cancer Mouse Models.

ObjectivePancreatic cancer is an aggressive tumor and the prognosis remains poor. Therefore, development of more effective therapy is needed. We previously reported that 89Zr-labeled TSP-A01, an antibody against transferrin receptor (TfR), is highly accumulated in a pancreatic cancer xenograft, but not in major normal organs. In the present study, we evaluated the efficacy of radioimmunotherapy (RIT) with 90Y-TSP-A01 in pancreatic cancer mouse models.MethodsTfR expression in pancreatic cancer cell lines (AsPC-1, BxPC-3, MIAPaCa-2) was evaluated by immunofluorescence staining. 111In-labeled anti-TfR antibodies (TSP-A01, TSP-A02) were evaluated in vitro by cell binding assay with the three cell lines and by competitive inhibition assay with MIAPaCa-2. In vivo biodistribution was evaluated in mice bearing BxPC-3 and MIAPaCa-2 xenografts. Tumor volumes of BxPC-3 and MIAPaCa-2 were sequentially measured after 90Y-TSP-A01 injection and histological analysis of tumors was conducted.ResultsMIAPaCa-2 cells showed the highest TfR expression, followed by AsPC-1 and BxPC-3 cells. 111In-TSP-A01 and 111In-TSP-A02 bound specifically to the three cell lines according to TfR expression. The dissociation constants for TSP-A01, DOTA-TSP-A01, TSP-A02, and DOTA-TSP-A02 were 0.22, 0.28, 0.17, and 0.22 nM, respectively. 111In-TSP-A01 was highly accumulated in tumors, especially in MIAPaCa-2, but this was not true of 111In-TSP-A02. The absorbed dose for 90Y-TSP-A01 was estimated to be 8.3 Gy/MBq to BxPC-3 and 12.4 Gy/MBq to MIAPaCa-2. MIAPaCa-2 tumors treated with 3.7 MBq of 90Y-TSP-A01 had almost completely disappeared around 3 weeks after injection and regrowth was not observed. Growth of BxPC-3 tumors was inhibited by 3.7 MBq of 90Y-TSP-A01, but the tumor size was not reduced.Conclusion 90Y-TSP-A01 treatment achieved an almost complete response in MIAPaCa-2 tumors, whereas it merely inhibited the growth of BxPC-3 tumors. 90Y-TSP-A01 is a promising RIT agent for pancreatic cancer, although further investigation is necessary to improve the efficacy for the radioresistant types like BxPC-3.

Segmental Transarterial Embolization in a Translational Rat Model of Hepatocellular Carcinoma

PurposeTo develop a clinically relevant, minimally invasive technique for transarterial embolization in a translational rat model of hepatocellular carcinoma (HCC). Materials and MethodsOral diethylnitrosamine was administered to 53 male Wistar rats ad libitum for 12 weeks. Tumor induction was monitored using magnetic resonance imaging. Minimally invasive lobar or segmental transarterial embolization was performed through a left common carotid artery approach. Necropsy was performed to evaluate periprocedural mortality. Histologic analysis of tumors that received embolization was performed to assess percent tumor necrosis. ResultsSevere cirrhosis and autochthonous HCCs were characterized in a cohort of rats composed of two groups of rats identically treated with diethylnitrosamine with median survival times of 101 days and 105 days (n = 10/group). A second cohort was used to develop minimally invasive transarterial embolization of HCCs (n = 10). In a third cohort, lobar embolization was successfully performed in 9 of 10 rats and demonstrated a high rate of periprocedural mortality (n = 5). Necropsy performed for periprocedural mortality after lobar embolization demonstrated extensive tissue necrosis within the liver (n = 3) and lungs (n = 2), indicating nontarget embolization as the likely cause of mortality. In a fourth cohort of rats, a segmental embolization technique was successfully applied in 10 of 13 rats. Segmental embolization resulted in a reduction in periprocedural mortality (P = .06) relative to selective embolization and a 19% increase in average tumor necrosis (P = .04). ConclusionsMinimally invasive, segmental embolization mimicking the currently applied clinical approach is feasible in a translational rat model of HCC and offers the critical advantage of reduced nontarget embolization relative to lobar embolization.

Endoglin (CD105) Silencing Mediated by shRNA Under the Control of Endothelin-1 Promoter for Targeted Gene Therapy of Melanoma.

Endoglin (CD105), a transforming growth factor (TGF)-β coreceptor, and endothelin-1, a vasoconstrictor peptide, are both overexpressed in tumor endothelial and melanoma cells. Their targeting is therefore a promising therapeutic approach for melanoma tumors. The aim of our study was to construct a eukaryotic expression plasmid encoding the shRNA molecules against CD105 under the control of endothelin-1 promoter and to evaluate its therapeutic potential both in vitro in murine B16F10-luc melanoma and SVEC4-10 endothelial cells and in vivo in mice bearing highly metastatic B16F10-luc tumors. Plasmid encoding shRNA against CD105 under the control of the constitutive U6 promoter was used as a control. We demonstrated the antiproliferative and antiangiogenic effects of both plasmids in SVEC4-10 cells, as well as a moderate antitumor and pronounced antimetastatic effect in B16F10-luc tumors in vivo. Our results provide evidence that targeting melanoma with shRNA molecules against CD105 under the control of endothelin-1 promoter is a feasible and effective treatment, especially for the reduction of metastatic spread.

Abstract C43: A Sleeping Beauty forward genetic screen identifies novel cancer drivers that cooperate with Pten in lung cancer

Abstract Improved understanding of the genetic drivers of lung cancer will aid in the design of personalized therapy. To this end, we have conducted forward genetic screens in the lungs of mice using the DNA transposon Sleeping Beauty (SB), in order to identify novel cancer driver genes. We conducted the screens on four different backgrounds: wild-type, p19 deficient, p53 dominant negative, and Pten deficient mice. The Pten deficient background was the only screen that resulted in tumors developing at a rate significantly above controls. Experimental mice (SB/Pten-/-) harbored a concatamer of oncogenic transposons, a conditionally activated SB transposase and a floxed Pten gene. A Cre recombinase cDNA under control of the Surfactant Protein C promoter was introduced to simultaneously activate SB and inactivate Pten in alveolar epithelial cells. Two control groups were established. One control group (W/T) did not include the Cre recombinase allele and was essentially wild-type while the second control group (Pten-/-) expressed Cre recombinase but did not contain transposons, causing inactivation of Pten without SB activity. Survival of SB/Pten-/- and Pten-/- mice was similar and both were significantly reduced compared to W/T mice. Tumor penetrance was lowest for W/T mice (4%) followed by Pten-/- mice (11%) and was highest for SB/Pten-/- mice (29%). Histological analysis of tumors indicated the presence of adenomas, adenocarcinomas, and extensive alveolar epithelialization along with bronchiolar epithelial hyperplasia. The latter two findings were present in both SB/Pten-/- and Pten-/- mice. Transposon insertions in 24 tumors from 14 SB/Pten-/- mice were mapped to the mouse genome using linker-mediated PCR followed by amplicon sequencing using Illumina GAIIx. Common insertion sites (CISs) were defined using the TAPDANCE analysis pipeline and over 100 candidate cancer genes were identified. A majority of the CIS genes we discovered have documented mutations in human lung cancers based on recent large-scale genomic analyses. This overlap suggests our mouse data could be used to prioritize research into putative human oncogenes and tumor suppressors. Cullin3 (Cul3) was highly ranked in our CIS list. The protein product of CUL3 is a core scaffolding protein in an E3 ubiquitin ligase complex that regulates ubiquitination of NRF2, IKBKB and other proteins. To determine the role of CUL3 in lung cancer we analyzed expression by western blot in human cancer cell lines and tumors and found CUL3 levels to be lower than normal bronchial epithelial cells. To directly test CUL3's tumor suppressor role we knocked down CUL3 using shRNA in stage II human lung cancer cell lines A549 and H522 either alone or in combination with an shRNA targeting PTEN. Reducing the level of CUL3 in these cell lines resulted in increased proliferation and an increased ability to grow in an anchorage-independent manner. These phenotypes were enhanced when both CUL3 and PTEN were coordinately knocked down. Western blot data indicate PTEN levels increase substantially, while mRNA levels maintain relatively static in CUL3 knockdown cells. In conclusion, we have performed studies that have identified a large number of candidate lung cancer genes and have confirmed a tumor suppressor role for CUL3. We are currently testing the effect of overexpressing CUL3 in both non-transformed and cancerous cell lines, as well as testing more of the candidate genes we have discovered. The results of these studies should lead to new treatment modalities for this deadly cancer. Citation Format: Casey Dorr, Madison Weg, Raha Been, M. Gerard O'Sullivan, Aaron Sarver, David A. Largaespada, Timothy K. Starr. A Sleeping Beauty forward genetic screen identifies novel cancer drivers that cooperate with Pten in lung cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C43.

Reduced pathological angiogenesis and tumor growth in mice lacking GPR4, a proton sensing receptor

The G protein-coupled receptor GPR4 is activated by acidic pH and recent evidence indicates that it is expressed in endothelial cells. In agreement with these reports, we observe a high correlation of GPR4 mRNA expression with endothelial marker genes, and we confirm expression and acidic pH dependent function of GPR4 in primary human vascular endothelial cells. GPR4-deficient mice were generated; these are viable and fertile and show no gross abnormalities. However, these animals show a significantly reduced angiogenic response to VEGF (vascular endothelial growth factor), but not to bFGF (basic fibroblast growth factor), in a growth factor implant model. Accordingly, in two different orthotopic models, tumor growth is strongly reduced in mice lacking GPR4. Histological analysis of tumors indicates reduced tumor cell proliferation as well as altered vessel morphology, length and density. Moreover, GPR4 deficiency results in reduced VEGFR2 (VEGF Receptor 2) levels in endothelial cells, accounting, at least in part, for the observed phenotype. Our data suggest that endothelial cells sense local tissue acidosis via GPR4 and that this signal is required to generate a full angiogenic response to VEGF.

Antitumor Effect of Malaria Parasite Infection in a Murine Lewis Lung Cancer Model through Induction of Innate and Adaptive Immunity

BackgroundLung cancer is the most common malignancy in humans and its high fatality means that no effective treatment is available. Developing new therapeutic strategies for lung cancer is urgently needed. Malaria has been reported to stimulate host immune responses, which are believed to be efficacious for combating some clinical cancers. This study is aimed to provide evidence that malaria parasite infection is therapeutic for lung cancer.Methodology/Principal FindingsAntitumor effect of malaria infection was examined in both subcutaneously and intravenously implanted murine Lewis lung cancer (LLC) model. The results showed that malaria infection inhibited LLC growth and metastasis and prolonged the survival of tumor-bearing mice. Histological analysis of tumors from mice infected with malaria revealed that angiogenesis was inhibited, which correlated with increased terminal deoxynucleotidyl transferase-mediated (TUNEL) staining and decreased Ki-67 expression in tumors. Through natural killer (NK) cell cytotoxicity activity, cytokine assays, enzyme-linked immunospot assay, lymphocyte proliferation, and flow cytometry, we demonstrated that malaria infection provided anti-tumor effects by inducing both a potent anti-tumor innate immune response, including the secretion of IFN-γ and TNF-α and the activation of NK cells as well as adaptive anti-tumor immunity with increasing tumor-specific T-cell proliferation and cytolytic activity of CD8+ T cells. Notably, tumor-bearing mice infected with the parasite developed long-lasting and effective tumor-specific immunity. Consequently, we found that malaria parasite infection could enhance the immune response of lung cancer DNA vaccine pcDNA3.1-hMUC1 and the combination produced a synergistic antitumor effect.Conclusions/SignificanceMalaria infection significantly suppresses LLC growth via induction of innate and adaptive antitumor responses in a mouse model. These data suggest that the malaria parasite may provide a novel strategy or therapeutic vaccine vector for anti-lung cancer immune-based therapy.

Abstract C249: Down-regulation of the HIF pathway enhances anti-tumor effects of the mTOR inhibitor ridaforolimus in the Her2/Neu mouse model of breast cancer

Abstract The Hypoxia-inducible Factor (HIF) pathway is a unique target in cancer because it controls multiple processes involved in tumor progression including neovascularization, tumor cell metabolism, invasion and metastasis. Hypoxia is elevated in a variety of tumor types and associated with resistance to therapeutics resulting in poor prognoses for patients. While a variety of tumor xenograft tumor models have demonstrated sensitivity to HIF pathway inhibition, studies in transgenic mouse models may provide a better understanding of the response as it more closely resembles the disease. Here we evaluate the role of HIF pathway in the MMTV-Neu transgenic (NeuT) breast cancer model and influence of the HIF pathway on response to the mTOR inhibitor ridaforolimus/MK-8669. A HIF regulated neuT model was generated by crossing NeuT transgenic mouse with one expressing a doxycyline-inducible HIF1β shRNA (NeuT-HIF1β) (Taconic-Artemis, Cologne, Germany). HIF1β is the obligate heterodimerization partner for the HIFα subunits and is required for HIF pathway activity. Upon derepression of the HIF1β shRNA by doxycycline treatment, greater than 80% knockdown is observed in most tissues. After 21-days of shRNA induction, tumor growth suppression was observed in the doxycyline-treated NeuT-HIF1β mice, but not NeuT littermate control mice. Growing evidence suggests that in hypoxia mTOR activity is often inhibited by HIF dependent and independent mechanisms. To evaluate the effect of hypoxia on the sensitivity of cells to a mTOR inhibitor, four cell lines (C786; U251; H1915; SKLU1) were treated with ridaforolimus cultured under normoxic (21% oxygen) and hypoxic (0.1% oxygen) conditions, the later oxygen concentration observed in hypoxic region of tumors. Under normoxia, the growth rate for all four cell lines was reduced by ridaforolimus. Under hypoxia, two lines were unaffected (786; U251) by ridaforolimus, while the other (H1915; SKLU1) remained sensitive. Histological analysis of the neuT tumors revealed that PI3K/mTOR and HIF pathways activities are mutually exclusive in these tumors. PI3K/mTOR pathway activity was detected in normoxic regions and HIF pathway activity present only in hypoxic regions. Thus we hypothesized targeting both pathways should achieve a far greater effect than each treatment separately. Treatment of mice with HIF1β knockdown or ridaforolimus resulted in 2-fold reduction in tumor growth and the combination resulted in a 4-fold decrease when both two pathways were targeted together. Histological analysis of these tumors will be discussed. In conclusion these data demonstrate that the effect of ridaforolimus on tumor growth can be improved by combining treatment that reduces the level of HIF pathway activity in tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C249.

Abstract C91: Pharmacodynamic analysis of the Hsp90 inhibitor STA-9090 in a lung cancer xenograft model supports an infrequent dosing schedule in the clinic

Abstract Background: Heat shock protein 90 (Hsp90) is a molecular chaperone that is required for the stability and function of many important signal transduction proteins that regulate the growth of cancer cells. Hsp90 inhibition results in ubiquitination and proteasomal degradation of these client proteins, which include clinically validated drug targets such as BCR-ABL, mutant EGFR, HER2, KIT and VEGFR. STA-9090 is a novel small molecule Hsp90 inhibitor that is currently in multiple Phase 1/2 clinical trials in solid tumor and hematological malignancies. STA-9090 is structurally unrelated to the first-generation anasamycin Hsp90 inhibitors 17-AAG and IPI-504 and inhibits Hsp90 by binding to its N-terminal ATP-binding pocket. Although Hsp90 inhibitors such as STA-9090 induce rapid client protein degradation, cell cycle arrest and apoptosis of cancer cells, it is possible that frequent drug dosing in the clinic may be needed to continuously maintain decreased client protein expression and avoid renewed tumor growth. To investigate this possibility, we conducted in vitro and in vivo studies using the human NCI-H1975 non-small cell lung cancer cell line, which expresses the Hsp90 client protein EGFRL858R/T790M, a mutationally activated and erlotinib-resistant form of the epidermal growth factor receptor. Results: In an in vitro cytotoxicity assay using this cell line, STA-9090 and 17-AAG displayed IC50 values of 10 and 40 nM after 72 hr drug exposure, respectively. These results closely correlated with decreased expression of EGFRL858R/T790M and other Hsp90 client proteins. Unexpectedly, exposure to STA-9090 for only 1 hr still resulted in an IC50 of 670 nM, suggesting that even brief drug exposure in vivo may be sufficient to affect tumor growth. Consistent with this, intravenous dosing of 125 mg/kg STA-9090 on a 1X/week × 3 week schedule (∼80–100% of the highest non-severely toxic dose) induced stable disease in a NCI-H1975 xenograft model, whereas 175 mg/kg 17-AAG resulted in progressive disease, with %T/C values of 15 and 50, respectively. Inhibition of tumor growth was correlated with decreased expression of EGFRL858R/T790M and other client proteins, and importantly, these effects persisted in tumors for 3–6 days after a single drug dose. Similarly, histological analysis of tumors indicated that STA-9090 inhibited cell proliferation by 7-fold and induced apoptosis by 9-fold, with maximal effects being observed at 1–3 days after treatment. Consistent with these observations, STA-9090 accumulated in tumors relative to normal tissues, with a tumor half-life of 58 hr versus 3–5 hr in liver, lung and plasma, and the tumor concentration remained 140-fold higher than the in vitro IC50 (72 hr) even 6 days after a single drug dose. Conclusions: Taken together, these results demonstrate that STA-9090 is a highly potent Hsp90 inhibitor that selectively accumulates in tumors and induces long-lasting client protein degradation, cell cycle arrest, increased apoptosis and tumor growth inhibition in a lung cancer xenograft model. Our results suggest that an infrequent dosing schedule may have clinical activity in cancer patients. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C91.

Avidin chase reduces side effects of radioimmunotherapy in nude mice bearing human colon carcinoma

To evaluate the influence of avidin chase on the side effects of radioimmunotherapy (RIT) in nude mice bearing human colon carcinoma and therapeutic outcome. Purified anti-CEA monoclonal antibody (McAb) was biotinylated with NHS-biotin, and then radiolabeled with 188Re by the direct method. 188Re-labeled biotinylated anti-CEA McAb (188Re-CEA McAb-Bt) was intravenously injected followed by intravenous injection of avidin after 24 h. SPECT imaging and biodistribution study were performed at 28-48 h after the injection of 188Re-CEA McAb-Bt. Three groups of nude mice subcutaneously grafted with human colon carcinoma were treated 7 d after the graft. Mice in the avidin chase group received intravenous injection of 188Re-CEA McAb-Bt (11.1 MBq/20 microg) followed by intravenous injection of cold avidin (80 microg) after 24 h. Mice in the control group (treated group without avidin chase) only received the injection of 188Re-CEA McAb-Bt (11.1 MBq/20 microg), another control group (non-treated group) only received 0.1 mL normal saline solution. Toxicity was evaluated on the basis of change of body weight and peripheral WBC counts, and therapy effects were determined by variation in tumor volume. Histological analysis of tumors was also performed. Avidin chase markedly accelerated the clearance of 188Re-CEA McAb-Bt from the blood and normal tissues. The tumor uptakes of 188Re-CEA McAb-Bt at 28 h were 5.90 and 6.42% ID/g, respectively, in chase group and in non-chase group, while the tumor-to-background (T/NT) ratios were 3.19 and 0.56, respectively. The tumor uptake was slightly decreased by avidin chase, but the T/NT ratios were increased. In treated groups the growth rate of body weight and the number of WBC decreased after injection of 188Re-CEA McAb-Bt, and the WBC counts recovered earlier in the group with avidin chase than in the group without avidin chase. Compared to the non-treated group, treated groups with and without avidin chase showed significant anti-tumor effects. Avidin chase can effectively reduce the side effects of RIT, and improve therapeutic efficacy.

Targeting the tumor vasculature: inhibition of tumor growth by a vascular endothelial growth factor-toxin conjugate.

Tumor-derived vascular endothelial growth factor (VEGF)/ vascular permeability factor (VPF) plays an important role in neovascularization and the development of tumor stroma. Furthermore, VEGF receptors are over-expressed in the endothelial cells of tumor vasculature and almost non-detectable in the vascular endothelium of adjoining normal tissues. The differential expression of receptor offers a selective advantage for targeting cytotoxic toxin polypeptides. We have prepared a vascular targeting reagent by chemically linking recombinant VEGF to a truncated form of diphtheria toxin. The VEGF-toxin conjugate was selectively toxic to endothelial cell lines and inhibited experimental neovascularization of the chick chorioallantoic membrane. In the present study, we examined the effects of VEGF-toxin conjugate on solid tumor growth. Athymic nude mice with established subcutaneous tumors were treated with daily intraperitoneal injections of the VEGF-toxin conjugate or free toxin. When compared with control animals treated with the toxin polypeptide alone, the conjugate-treated animals displayed a significant inhibition of tumor growth. Histological analysis of tumors from conjugate-treated animals revealed hemorrhagic necrosis consistent with a vascular-mediated injury. In contrast, highly vascularized normal tissues from conjugate-treated animals demonstrated no evidence of hemorrhage or tissue injury. The conjugate was well tolerated without apparent toxicities. Our results illustrate the anti-tumor activity of a VEGF-toxin conjugate selectively targeting the tumor neovasculature.