Murray A. Stackhouse

Treatment of pancreatic cancer xenografts with Erbitux (IMC-C225) anti-EGFR antibody, gemcitabine, and radiation.

PURPOSE To investigate treatment of human pancreatic cancer cell lines and xenografts with combinations of Erbitux (IMC-C225) anti-epidermal growth factor receptor (EGFR) antibody, gemcitabine, and radiation. METHODS AND MATERIALS BxPC-3 and MiaPaCa-2 human pancreatic carcinoma cells were treated in vitro for 24 h with IMC-C225 (5 microg/mL), then exposed to epidermal growth factor (EGF) (10 mM) for 5 min. Immunoblots were screened for EGFR expression and the ability of IMC-C225 to block EGF-induced tyrosine phosphorylation of EGFR. Cells were treated with IMC-C225 (5 microg/mL) on Day 0, the IC(50) dose of gemcitabine on Day 1 for 24 h, followed by 3 Gy 60Co irradiation on Day 2, or the combination of each agent. For cell proliferation, cells were counted on Day 4, and for apoptosis, cells were stained with annexin V-FITC and propidium iodide, then analyzed by FACS. Cells were treated with the same single or multiple treatments and analyzed in a clonogenic cell survival assay. The effect of IMC-C225, gemcitabine, and radiation on the growth of BxPC-3 and MiaPaCa-2 tumor xenografts was determined. Athymic nude mice bearing established s.c. tumor xenografts of 6-8 mm diameter received 6 weeks of treatment with IMC-C225 (1 mg every 3 days x 6) alone or in combination with gemcitabine (120 mg/kg i.v. every 6 days x 6), and 6 weekly fractions of 3 Gy radiation on the days after gemcitabine administration. Tumor growth was measured with Vernier calipers. RESULTS BxPC-3 and MiaPaCa-2 cell lines expressed low levels of EGFR. IMC-C225 inhibited EGF-induced tyrosine phosphorylation of the EGF receptor on both cell lines. Treatment of cells with a combination of IMC-C225 + gemcitabine + radiation produced the highest induction of apoptosis and inhibition of proliferation in vitro. Combination treatment with IMC-C225, gemcitabine, and radiation produced 100% complete regression of MiaPaCa-2 tumors for more than 250 days, and the greatest growth inhibition of BxPC-3 tumors compared to any single or dual treatments. CONCLUSIONS The IMC-C225 therapy in combination with gemcitabine chemotherapy and radiation therapy demonstrated statistically significantly greater efficacy over the single and double combination therapies. This form of multimodality treatment shows potential clinical application in the treatment of pancreatic cancer in humans.

An adenovirus encoding proapoptotic Bax synergistically radiosensitizes malignant glioma

PURPOSE We explore the utility of the adenovirus-mediated delivery of proapoptotic Bax for enhancing the cytotoxicity of radiotherapy (RT) in RT-refractory glioma cells. MATERIALS AND METHODS Cell lines D54 MG and U87 MG (p53 wild-type), and U251 MG and U373 MG (p53 mutant), and patient-derived astrocytes were evaluated. Cells were irradiated and infected with an inducible adenovirus encoding Bax. Cell proliferation, colony formation assay, quantification of early apoptotic alteration in the plasma membrane by fluorescence-activated cell sorter using annexin V, and nuclear staining with H33258 were used to evaluate apoptosis. The capacity of the combined treatment to induce regression of subcutaneous D54 MG tumors was tested in nude mice. A dose of 5 Gy was administered every other day, four times, for a total dose of 20 Gy. One day after each irradiation, tumors were injected with 1 x 10(9) plaque-forming units (PFU). RESULTS Apoptotic death was enhanced by the combination of Ad/Bax and RT. In D54 MG, levels of apoptosis after RT alone, Ad/Bax alone, or the combination were, respectively, 12.3%, 32.1%, and 78.5%. In contrast, treatment of astrocytes did not significantly induce apoptosis. A colony-formation assay showed a 2-log inhibition with respect to controls after combined treatment, irrespective of the endogenous levels of p53. The other apoptosis assays also showed the defining characteristics of apoptosis in the combination group. Remarkably, combined treatment induced regression of tumors in mice. CONCLUSIONS Ad/Bax synergistically radiosensitizes glioma, with a seemingly favorable therapeutic index.

Specific membrane receptor gene expression targeted with radiolabeled peptide employing the erbB-2 and DF3 promoter elements in adenoviral vectors

Radioimmunotherapy is limited by a variety of factors, including poor tumor penetration of monoclonal antibodies and low levels of intratumoral antigen expression. To address these limitations, a gene therapy strategy was devised to genetically induce tumor cells to express enhanced levels of membrane receptors with high affinity for a radiolabeled peptide. We designated this approach as genetic radioisotope targeting strategy. To this end, an adenoviral vector (AdCMVGRPr) encoding the murine gastrin-releasing peptide receptor (GRPr) was used to achieve a high level of binding of radiolabeled bombesin (BBN). To achieve genetic induction of membrane GRPr specifically to tumor cells, we constructed two adenoviral vectors encoding the GRPr gene under the control of the tumor-specific regulatory elements, DF3 (AdDF3GRPr) or erbB-2 (AderbGRPr). We investigated the binding of [125I]BBN to the GRPr following infection with AdDF3GRPr and AderbGRPr in a panel of human breast, pancreatic, and cholangiocarcinoma tumor cell lines. [125I]BBN binding and GRPr expression increased with increasing multiplicities of infection of AdCMVGRPr in all of the cell lines tested. Breast cancer cell lines expressing erbB-2 showed significant GRPr expression using AderbGRPr. A similar result was observed in breast and cholangiocarcinoma cells infected with AdDF3GRPr expressing MUC1 as detected by immunohistochemistry but was not seen in the pancreatic cell lines tested. Thus, adenoviral vectors with tissue-specific promoter elements can be used to achieve a selective expression of membrane receptors that can be targeted with a radiolabeled peptide. The use of such a transcriptional targeting approach may restrict gene expression to tumors and limit the radiation dose deposited in normal tissues in vivo.

An ionizing radiation-sensitive CHO mutant cell line: irs-20. IV. Genetic complementation, V(D)J recombination and the scid phenotype.

The genetic defect responsible for hypersensitivity of Chinese hamster ovary (CHO) irs-20 cells to ionizing radiation was found to be recessive in nature and could be complemented to produce wild-type radiosensitivity in irs-20/human hybrids. The radiosensitivities of six hybrid clones were determined based on their colony-forming ability under continuous irradiation at 6 cGy/h. A parallel cytogenetic analysis revealed a concordance between the presence or absence of human chromosome 8 and the resistant or sensitive phenotype. Confirming evidence was obtained using human chromosome 8-specific PCR primers. Positive amplification was obtained in hybrids with wild-type radiosensitivity, while no amplification was obtained in sensitive hybrids. Complementation analysis between radiosensitive CHO irs-20 and murine scid cell lines was carried out to determine whether the defects leading to their ionizing radiation hypersensitivity could be corrected by genetic complementation in the hybrids. Complementation did not occur. A transient V(D)J recombination assay after the introduction of the RAG1 and RAG2 genes indicated that the V(D)J recombination ability of the CHO irs-20 cells was about 10% of that for the CHO wild-type cells for signal join formation with an 80% joining fidelity and only 3% of the parental level for coding join formation. These data show that murine scid and irs-20 mutant hamster cells fall into the same complementation group and show similar defects in V(D)J recombination.

Radiosensitization mediated by a transfected anti-erbB-2 single-chain antibody in vitro and in vivo.

PURPOSE The erbB-2 receptor is overexpressed in several human cancers, including ovarian, prostate, and breast. We have developed plasmid and adenoviral vectors expressing an anti-erbB-2 single chain antibody (sFv), directed to the endoplasmic reticulum (ER) of target cells, that is cytotoxic to tumor cells overexpressing erbB-2 through induction of apoptosis. The anti-erbB-2 sFv also sensitizes erbB-2 overexpressing cells to the cytotoxic effects of cisplatin. On this basis, we hypothesized that human ovarian cancer cells expressing anti-erbB-2 sFv with downregulated erbB-2 product, p185erbB-2, also would be sensitized to ionizing radiation. Therefore, we designed experiments to test the ability of the anti-erbB-2 sFv to radiosensitize human ovarian cancer cells in vitro and in vivo. METHODS AND MATERIALS To test our hypothesis, we established subcutaneous (s.c.) tumors in the flanks of nude mice with SKOV3.ip1 human ovarian cancer cells and SKOV3 cells stably expressing the ER directed anti-erbB-2 sFv (SKOV3/pGT21). The tumors were treated with 10 Gy 60Co, or received no radiation. We then determined the regression rate, delay in regrowth, and time to tumor doubling of the tumors treated with radiation in the transfected group and controls. In addition, SKOV3.ip1 and SKOV3/pGT21 tumors were dissected from the irradiated animals and assayed for differences in p185erbB-2 expression at 12 weeks after irradiation by immunohistochemistry. Further, in vitro clonogenic survival assays were performed on the parental SKOV3.ip1 and SKOV3/pGT21 cell lines. RESULTS A statistical analysis of the combined data was done for two in vivo experiments. The analysis of the combined data showed that animals with irradiated tumor SKOV3/pGT21 had a significantly higher regression rate (p = 0.0055), longer delay in regrowth (p = 0.0001) and time to tumor doubling (p = 0.0004), than those animals with tumor SKOV3.ip1 that received radiation. We observed a similar significant effect for the same parameters in the unirradiated tumor SKOV3/pGT21 compared to unirradiated tumor SKOV3.ip1. Immunohistochemical analysis of the SKOV3/pGT21 tumor cells demonstrated focal accumulation of p185erbB-2 in scattered clumps of cells and less p185erbB-2 membrane expression than cells of SKOV3.ip1 tumors. However, SKOV3.ip1 and SKOV3/pGT21 cells had similar in vitro sensitivity to radiation. CONCLUSIONS These data support the hypothesis that tumors with reduced p185erbB-2 expression mediated by the anti-erbB-2 sFv are rendered more susceptible in vivo to the cytotoxic effects of ionizing radiation than tumors that maintain their normal expression of p185erbB-2. However, a similar effect was not observed with the same tumor cells in vitro. Thus, as has been described by others (1, 2), in vitro and in vivo results do not always correlate. Therefore, appropriate assays to assess clinical relevance need to be determined for each particular system studied.

Combined cytosine deaminase expression, 5-fluorocytosine exposure, and radiotherapy increases cytotoxicity to cholangiocarcinoma cells☆☆☆

Cholangiocarcinoma is a malignancy that is resistant to current therapy. We applied the toxin gene therapy strategy of cytosine deaminase conversion of the nontoxic prodrug 5-fluorocytosine to 5-fluorouracil combined with radiotherapy to cholangiocarcinoma. The transduction efficiency of SK-ChA-1 cholangiocarcinoma cells was determined by fluorescence-activated cell-sorting analysis following infection with recombinant adenovirus AdCMVLacZ, which encodes the gene for β-galactosidase. To evaluate cytosine deaminase-mediated conversion of 5-fluorocytosine to 5-fluorouracil and subsequent cytotoxicity, SK-ChA-1 cells were infected with the recombinant adenovirus AdCMSXD, which encodes cytosine deaminase, and exposed to S-fluorocytosine for 6 to 8 days. Additive cytotoxicity of radiation therapy was evaluated by cobalt-60 exposure following AdCMVVCD infection and S-fluorocytosine treatment. SK-Cl&l cells were transduced (98.4%) by AdCMVLacZ at 100 plaque-forming units per cell. Following infection with AdCMIVCD and exposure to 5 to 100 μ,g/ml of 5-fluorocytosine, 20% to 64% of SK-&A-l cells were killed. A combination of radiation and cytosine deaminase/5-fluorocytosine therapy resulted in enhanced cell killing (83.5% to 91.5%). Cholangiocarcinoma cells were transduced by recombinant adenoviral vectors and were killed by cytosine deaminase-mediated production of 5-fluorouracil. Enhanced cytotoxicity was seen with the addition of external beam radiation. These results provide a foundation for multimodality therapy for human cholangiocarcinoma that combines gene therapy technology with radiation therapy.

Vitamin E Phosphate Nucleoside Prodrugs: A Platform for Intracellular Delivery of Monophosphorylated Nucleosides

Vitamin E phosphate (VEP) nucleoside prodrugs are designed to bypass two mechanisms of tumor resistance to therapeutic nucleosides: nucleoside transport and kinase downregulation. Certain isoforms of vitamin E (VE) have shown activity against solid and hematologic tumors and result in chemosensitization. Because gemcitabine is one of the most common chemotherapeutics for the treatment of cancer, it was used to demonstrate the constructs utility. Four different VE isoforms were conjugated with gemcitabine at the 5′ position. Two of these were δ-tocopherol-monophosphate (MP) gemcitabine (NUC050) and δ-tocotrienol-MP gemcitabine (NUC052). NUC050 was shown to be able to deliver gemcitabine-MP intracellularly by a nucleoside transport independent mechanism. Its half-life administered IV in mice was 3.9 h. In a mouse xenograft model of non-small cell lung cancer (NSCLC) NCI-H460, NUC050 at a dose of 40 mg/kg IV qwk × 4 resulted in significant inhibition to tumor growth on days 11–31 (p < 0.05) compared to saline control (SC). Median survival was 33 days (NUC050) vs. 25.5 days (SC) ((hazard ratio) HR = 0.24, p = 0.017). Further, NUC050 significantly inhibited tumor growth compared to historic data with gemcitabine at 135 mg/kg IV q5d × 3 on days 14–41 (p < 0.05). NUC052 was administered at a dose of 40 mg/kg IV qwk × 2 followed by 50 mg/kg qwk × 2. NUC052 resulted in inhibition to tumor growth on days 14–27 (p < 0.05) and median survival was 34 days (HR = 0.27, p = 0.033). NUC050 and NUC052 have been shown to be safe and effective in a mouse xenograft of NSCLC.

Abstract 2778: Tumor target vs. tissue of tumor origin: cluster analysis of genomic profile of 42 human tumor in vitro and in vivo models.

Traditionally, drug development has relied upon testing cancer drug candidates in cell lines. Active drugs are then tested in human tumor xenograft models, usually selected based upon the cell lines in which the drug showed activity. The majority of drugs fail at this stage as they do not show activity in the xenograft models chosen. We performed Affymetrix genomic analysis on 42 human tumor xenograft models and the original cell lines from which they were established. The genomic profiles obtained underwent Unsupervised Hierarchical Cluster Analysis to ascertain which cell lines and xenograft models had similar genomic profiles and which did not. The analysis showed that only 24 of 42 human tumor xenograft models clustered side-by-side with the cell line from which they were established. All 6 human leukemia/lymphoma xenograft models clustered very well with the cell lines from which they were established, and they clustered perfectly according to histological class. Five out of six human colon tumor xenograft models clustered well with the cell lines from which they were established and according to histotype. Of the 18 xenograft/cell line pairs that did not cluster side-by-side, 10 pairs remained in the same general cluster, whereas the partners of 8 other pairs were dispersed across different major clusters. Ovarian, breast, melanoma, and pancreatic human tumor xenograft models did not cluster according to histotype. Our data may explain why some drugs that show in vitro activity in some cell lines are not active in other cell lines of the same histological type, and also why some drugs that show activity in vitro then fail in xenograft models. In our laboratory, the PANC-1 cell line is very often chosen as a model of pancreatic cancer. A drug showing activity in the PANC-1 cell line would next be tested in other in vitro models of pancreatic cancer (e.g., MIA PaCa-2, CFPAC-1, and BxPC-3). However, none of these other pancreatic models have a similar genetic profile to PANC-1. Based upon our data, the cell line showing most similarity to the PANC-1 cell line is the breast cancer cell line MDA-MB-231. It is our suggestion that a drug showing activity in the PANC-1 cell line should be tested in other cell lines showing similar genetic profiles, not in cell lines based on histotype. Another example from our analysis is the LOX-IMV1 melanoma cell line. Not only does this cell line not cluster with its corresponding LOX-IMV1 xenograft model, it clusters most closely with the NCI/ADR-RES ovarian cell line. In summary, the genomic profiles of approximately 57% of the tumor xenograft models analyzed closely associate with the cell line from which they were established. Some of the tumor xenograft models show very little similarity to the cell lines from which they were established. Additionally, many of the models (both xenografts and cell lines), do not cluster according to their tissue of origin. Citation Format: Michael J. Roberts, Michael S. Koratich, Murray Stackhouse, Richard D. May, Andrew D. Penman, Tommie A. Gamble, Kristy L. Berry, Joseph F. Murphy, Robert J. Rooney, Yulia Y. Maxuitenko. Tumor target vs. tissue of tumor origin: cluster analysis of genomic profile of 42 human tumor in vitro and in vivo models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2778. doi:10.1158/1538-7445.AM2013-2778

Abstract 3114: Effects of tumor-stromal interactions on gene expression in panel of mouse tumor models

Tumor growth is not determined solely by the tumor cells but is governed by interactions between tumor cells and host stromal cells, including endothelial cell activation and fibroblastic stroma response. Tumor stroma profoundly influences many steps of tumor progression. In many human cancers, such as breast, prostate, and colon, the stroma comprises the majority of the tumor mass, as a hallmark of the clinical feature called desmoplasia. Numerous studies have showed that tumor-stromal cell interactions play crucial roles in supporting cancer progression and in promoting anticancer drug resistance by alternating gene expression profiles in both tumor and stromal cells through network tumor-stromal interactions in the tumor microenvironments. It has been challenging to obtain separate gene profiles for tumor and stroma with human tumor samples as both tumor and stromal cells share the same genome. In xenograft mouse tumor models, human tumor cells are supported by mouse host stromal microenvironment. Therefore, effects of tumor stromal integrations on gene expression can be profiled separately by taking advantage of this heterogeneous genetic makeup. To understand the underlying biological process of stroma in cancer and select relevant in vitro and in vivo model systems for various targeted anticancer drug discovery and development projects, we selected a panel of 30 commonly used xenograft tumor mouse models that are derived from human tumor cell lines of various cancer histotypes and conducted analyses of differential gene expression in both human cancer cells and mouse host stromal cells before and after their interactions in vivo by using quantitative PCR with mouse and human specific primers. Growing the human tumors as a continuous in vivo passage subcutaneously in immunodeficient mice permits stroma infiltration over a long time. We have examined a number of anticancer target genes involved in different signaling pathways, such as tumor angiogenesis, apoptosis and survival (Akt/mTOR signaling pathway). Our study results have demonstrated that tumor stromal interactions significantly regulate expression levels of various genes important in tumor progression and development of resistance to treatment in both tumor and stromal cells. Tumor animal models play a critical role in translating the bench science to the bedside medical care of cancer patients. Decisions for moving new anticancer agents into costly clinical investigations are mostly based on the preclinical results using xenograft mouse models. The results of this gene profiling approach could provide tools for studying tumor microenvironment and tumor stromal interactions in vivo to advance anticancer drug development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3114. doi:10.1158/1538-7445.AM2011-3114