Using microarray technology, we analyzed 12,000 genes for regulation by TNF-alpha and the synthetic glucocorticoid, dexamethasone, in the human lung epithelial cell line, A549. Only one gene was induced by both agents, the cellular inhibitor of apoptosis 2 (c-IAP2), which was induced 17-fold and 5-fold by TNF-alpha at 2 h and 24 h, respectively, and increased 14-fold and 9-fold by dexamethasone at 2 h and 24 h, respectively. The combination of the two agents together led to an additive increase (34-fold) at 2 h and a more than additive effect (36-fold) at 24 h. The human c-IAP2 promoter contains two nuclear factor (NF)-kappaB sites that have been shown to be required for transcriptional activation by TNF-alpha. To test whether glucocorticoids regulate the c-IAP2 gene at the level of the promoter, a reporter vector containing 947 bases upstream of the start site of transcription of the human c-IAP2 promoter was linked to luciferase [IAP(-947-+54)-LUC] and transfected into A549 cells. Dexamethasone and TNF-alpha each induced reporter activity, whereas the combination of the two agents led to greater induction of luciferase than either one alone. Truncation of the promoter region containing a putative glucocorticoid response element (GRE) at -515 [IAP(-395-+54)-LUC] or mutation of the GRE in the context of the natural promoter [IAP(-947-+54mutGRE)-LUC] resulted in a loss of dexamethasone-mediated induction of reporter activity. Although the functional NF-kappaB sites were retained in the truncated and mutant c-IAP2 promoter constructs, dexamethasone did not inhibit the TNF-alpha induction of luciferase activity, indicating that GR repression through the NF-kappaB sites did not occur. Regulation of the c-IAP2 gene is therefore unique, as GR and NF-kappaB signaling pathways are usually mutually antagonistic, not cooperative. Treatment of A549 cells with TNF-alpha and/or dexamethasone had no effect on cell death, but the two agents were able to inhibit interferon-gamma/anti-FAS antibody-mediated apoptosis. In human glioblastoma A172 cells, TNF-alpha and dexamethasone together elicited a greater than additive increase in c-IAP2 mRNA levels and also inhibited anti-FAS antibody-mediated A172 cell apoptosis. In contrast, in human CEM-C7 leukemic T cells, whereas TNF-alpha and dexamethasone treatment also led to an increase in c-IAP2 mRNA, the two agents were able to induce apoptosis on their own. However, TNF-alpha and dexamethasone were also able to blunt anti-FAS-induced apoptosis in the T cells. These data indicate that the induction of the antiapoptotic protein, c-IAP2, by glucocorticoids and TNF-alpha correlates with the ability of these agents to inhibit apoptosis in a variety of cell types.

Selective targeting of up-regulated integrins on tumor cells is a novel antiangiogenesis strategy for treating solid tumors. CNTO 95 is a fully human anti-A v integrin monoclonal antibody and has shown antitumor activity when used as a single agent in preclinical studies. We previously showed that radiation combined with an integrin A v B 3 antagonist cRGD peptide increased the therapeutic efficacy of radiation in preclinical tumor models. We hypothesized that the combination of radiation and CNTO 95 would synergistically enhance the efficacy of radiation therapy. The in vitro studies showed that CNTO 95 radiosensitized and induced apoptosis in M21 cells in vitronectin-coated dishes. In mice bearing established human cancer xenograft tumors, CNTO 95 alone had only a moderate effect on tumor growth. The combined therapy of CNTO 95 and fractionated radiation significantly inhibited tumor growth and produced the longer tumor growth delay time in multiple tumor models. Maintenance dosing of CNTO 95 following irradiation contributed to efficacy and was important for continued inhibition of tumor regrowth. Immunohistochemistry studies showed that the combined use of CNTO 95 and radiation reduced the A v integrin and vascular endothelial growth factor receptor expression and the microvessel density and increased apoptosis in tumor cells and the tumor microenvironment. CNTO 95 alone and in combination with radiation did not produce any obvious signs of systemic toxicity. These results show that CNTO 95 can potentiate the efficacy of fractionated radiation therapy in a variety of human cancer xenograft tumor types in nude mice. These findings are very promising and may have high translational relevance for the treatment of patients with solid tumors. [Mol Cancer Ther 2008;7(6):1569 -78]

In vivo models that reproduce human disease are needed for the evaluation of acute myeloid leukemia (AML) disease progression and the development of effective treatments for the disease. The NSG mouse strain is used to model human AML due to the absence of mature B and T lymphocytes and lack of functional NK cells. Successful systemic engraftment of human AML cells in these mice may provide similar disease manifestation observed in patient disease, thus allowing evaluation of therapeutic candidates. The Kasumi-3 cell line from ATCC was established from the blast cells of a myeloperoxidase-negative acute leukemia patient and expresses CD7, CD4, CD13, CD33, CD34, HLA-DR and c-Kit cell surface markers which are compatible with acute myelocytic leukemia, subtype M0 (AML-M0). The prognosis of patients with AML-M0 is poor in comparison to other classifications of AML due to the association of these leukemia cells as being extremely immature and either undifferentiated or minimally differentiated. We have developed a systemic Kasumi-3 human AML model, characterizing tumor cell engraftment over time, following intravenous implantation of Kasumi-3 AML cells in NSG mice. Kasumi-3 cells were implanted at either 1e6 or 10e6 cells per NSG mouse and tumor burden throughout disease progression was assessed by monitoring the engraftment of these cells in the peripheral blood, bone marrow and spleen by FACS analysis. The engraftment of huCD45+ cells was used as a marker for determining the percentage of human tumor cell burden in the mice. Results indicated that within 28 days post intravenous implantation of Kasumi-3 cells at either concentration, huCD45+ cells were present in the peripheral circulation, bone marrow and spleens of NSG mice, and the tumor cell engraftment percentages strongly correlated with the number of implanted Kasumi-3 AML cells. Both the 1e6 and 10e6 cell implant concentrations exhibited unidirectional changes in the percentage of huCD45+ cells engrafting in the peripheral blood, bone marrow, and spleen at all time points throughout the experiment. After analyzing the percentages of human tumor cell engraftment in the NSG mice, the 10e6 Kasumi-3 cell implant concentration showed tumor cell engraftment of 35% in the blood, 67% in the bone marrow and 16% in the spleen on Day 28 and provided the most appropriate environment for the in vivo onset and evaluation of AML tumor burden. The 1e6 Kasumi-3 concentration had a lower percentage of systemic tumor cell engraftment at Day 28 when compared to the percentage of huCD45+ cell engraftment with 10e6 cells. The higher implant concentration showed established tumor burden in the peripheral blood and spleen 14 days sooner than the 1e6 cell dose, which would allow for the observation of efficacy and treatment effects in this model sooner than the 1e6 cell concentration. The establishment of this human AML model in NSG mice can serve as an in vivo tool for the evaluation of putative therapeutics for AML. Citation Format: Diana Chin, Christine Pietsch, Francis McCabe, Susan Chippari, Elizabeth Kaiser, Rebecca Hanson, Mariusz Lubomirski. Development of a systemic Kasumi-3 acute myeloid leukemia model in NSG mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 90. doi:10.1158/1538-7445.AM2014-90

Immunocompromised mice engrafted with human hematopoietic stem cells (HSC) or peripheral blood mononuclear cells (PBMC) can be promising models of human immune system compartments, useful for studying diseases such as diabetes, lupus, GVHD, and cancer. We have utilized the PBMC-humanized mouse as a platform for xenograft models. To investigate the effect of human antibody (Ab) treatment on immune cells and xenografts using this platform, we examined the infiltrate of human immune cells in A431 xenografts with treatment of an effector function-enhanced anti-tissue factor VIIA (TFVIIA) Ab. Female NSG mice were intravenously engrafted with human PBMC, and subcutaneously implanted two weeks later with A431 human SCC cells. One week post tumor cell implant and thereafter twice weekly, mice received treatment with anti-TFVIIA Ab. Peripheral blood, bone marrow and spleen were analyzed by FACS and terminal tumor samples were analyzed by FACS and immunohistochemistry (IHC). FACS analysis of blood from Ab treated and untreated tumor-bearing mice collected from baseline to termination, showed an overall increase in the frequency of human CD45+, CD3+, CD8+, and CD4+ cells, while the frequency of CD56+ cells remained stable. In comparison, non-tumor bearing mice had increased CD56+ cells and decreased CD3+ cells. FACS analysis of tumors showed a robust increase in the frequency of CD56+ cells and a decrease in the frequency of CD3+ cells with treatment of anti-TF Ab. With treatment, the frequency of CD8+ cells was markedly lower and the frequency of CD4+ cells unchanged. Histological analysis was performed on A431 FFPE xenografts from both the treated and non-treated groups. Hematoxylin and eosin (H&E) staining of the xenografts confirmed the presence of inflammatory cells in the tumors and also showed greater PBMC infiltration of the tumors in the presence of the anti-TFVIIA Ab. Xenografts treated with anti-TFVIIA Ab exhibited greater infiltration of human CD3+, CD8+, and CD57+ cells. There was an increase in cleaved caspase-3 (CC3)+ cells in Ab-treated xenografts, indicating an increase in apoptosis with anti-TFVIIA Ab treatment. In conclusion, anti-TFVIIA Ab treatment causes changes in immune cell populations in xenografts. While the mechanism is unknown, one possibility is that NK migration into the tumor coupled with increased apoptosis with treatment indicates ADCC activity by the NK cells. This would signify some preservation of immune cell function in the PBMC engraftment model, and would enhance studying the effects of experimental antibodies in a more humanized setting. Citation Format: Hillary Millar-Quinn, Brenda Hertzog, Rebecca Hanson, Jeffrey Nemeth, John Alvarez. Human immune cell infiltration of tumors in a PBMC-humanized NSG mouse xenograft model changes with treatment of an anti-tissue factor antibody. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1655. doi:10.1158/1538-7445.AM2014-1655