Brian J. Morrison

Vaccination by Genetically Modified Dendritic Cells Expressing a Truncated neu Oncogene Prevents Development of Breast Cancer in Transgenic Mice

Dendritic cells (DCs) are powerful antigen-presenting cells that process antigens and present peptide epitopes in the context of the major histocompatibility complex molecules to generate immune responses. DCs are being studied as potential anticancer vaccines because of their ability to present antigens to naïve T cells and to stimulate the expansion of antigen-specific T-cell populations. We investigated an antitumor vaccination using DCs modified by transfer of a nonsignaling neu oncogene, a homologue of human HER-2/neu, in a transgenic model of breast cancer. BALB-neuT mice develop breast cancers as a consequence of mammary gland-specific expression of an activated neu oncogene. We vaccinated BALB-neuT mice with bone marrow-derived DCs transduced with Ad.Neu, a recombinant adenovirus expressing a truncated neu oncoprotein. The vaccine stimulated the production of specific anti-neu antibodies, enhanced interferon-γ expression by T cells, and prevented or delayed the onset of mammary carcinomas in the mice. Over 65% of vaccinated mice remained tumor free at 28 weeks of age, whereas all of the mice in the control groups developed tumors. When challenged with a neu-expressing breast cancer cell line, vaccinated tumor-free animals had delayed tumor growth compared with controls. The antitumor effect of the vaccine was specific for expression of neu. Studies showed that CD4+ T cells were required in order to generate antitumor immunity. Importantly, the effectiveness of the vaccine was not diminished by preexisting immunity to adenovirus, whereas the protection afforded by vaccination that used direct injection of Ad.Neu was markedly reduced in mice with anti-adenovirus antibody titers. DCs modified by recombinant adenoviruses expressing tumor-associated antigens may provide an effective antitumor vaccination strategy.

Sphere Culture of Murine Lung Cancer Cell Lines Are Enriched with Cancer Initiating Cells

Cancer initiating cells (CICs) represent a unique cell population essential for the maintenance and growth of tumors. Most in vivo studies of CICs utilize human tumor xenografts in immunodeficient mice. These models provide limited information on the interaction of CICs with the host immune system and are of limited value in assessing therapies targeting CICs, especially immune-based therapies. To assess this, a syngeneic cancer model is needed. We examined the sphere-forming capacity of thirteen murine lung cancer cell lines and identified TC-1 and a metastatic subclone of Lewis lung carcinoma (HM-LLC) as cell lines that readily formed and maintained spheres over multiple passages. TC-1 tumorspheres were not enriched for expression of CD133 or CD44, putative CIC markers, nor did they demonstrate Hoechst 33342 side population staining or Aldefluor activity compared to adherent TC-1 cells. However, in tumorsphere culture, these cells exhibited self-renewal and long-term symmetric division capacity and expressed more Oct-4 compared to adherent cells. HM-LLC sphere-derived cells exhibited increased Oct-4, CD133, and CD44 expression, demonstrated a Hoechst 33342 side population and Aldefluor activity compared to adherent cells or a low metastatic subclone of LLC (LM-LLC). In syngeneic mice, HM-LLC sphere-derived cells required fewer cells to initiate tumorigenesis compared to adherent or LM-LLC cells. Similarly TC-1 sphere-derived cells were more tumorigenic than adherent cells in syngeneic mice. In contrast, in immunocompromised mice, less than 500 sphere or adherent TC-1 cells and less than 1,000 sphere or adherent LLC cells were required to initiate a tumor. We suggest that no single phenotypic marker can identify CICs in murine lung cancer cell lines. Tumorsphere culture may provide an alternative approach to identify and enrich for murine lung CICs. Furthermore, we propose that assessing tumorigenicity of murine lung CICs in syngeneic mice better models the interaction of CICs with the host immune system.

Lung cancer-initiating cells: a novel target for cancer therapy

Lung cancer is a major public health problem causing more deaths than any other cancer. A better understanding of the biology of this disease and improvements in treatment are greatly needed. Increasing evidence supports the concept that a rare and specialized population of cancer cells, so-called cancer-initiating cells with stem cell-like characteristics, is responsible for tumor growth, maintenance, and recurrence. Cancer-initiating cells also exhibit characteristics that render them resistant to both radiation and chemotherapy, and therefore they are believed to play a role in treatment failure. This has led to the hypothesis that traditional therapies that indiscriminately kill tumor cells will not be as effective as therapies that selectively target cancer-initiating cells. Investigating putative cancer-initiating cells in lung cancer will greatly benefit the understanding of the origins of this disease and may lead to novel approaches to therapy by suggesting markers for use in either further isolating this population for study or for selectively targeting these cells. This review will discuss (1) lung cancer, (2) stem cells, and the role of cancer-initiating cells in tumorigenesis; (3) markers and functional characteristics associated with lung cancer-initiating cells; and (4) the potential to selectively target this subpopulation of tumor cells.

Genetically Modified Dendritic Cells in Cancer Immunotherapy

Dendritic cells are powerful antigen-presenting cells that can generate primary cytolytic T lymphocyte responses against tumors. Consequently, there has been much interest in their application as antitumor vaccines. A number of dendritic cell-based vaccine trials targeting a variety of tumors have been conducted in different countries; however, the rate of clinical responses remains low. The majority of these studies have administered dendritic cells loaded with synthetic peptide epitopes or tumor lysates. Genetic modification of dendritic cells to express tumor antigens or immunostimulatory molecules through gene transfer or mRNA transfection offers a logical alternative with potential advantages over antigen loading in dendritic cells. In this chapter, we review the current and future prospects for genetically modified dendritic cell vaccines for cancer therapy.

Murine hepatocellular carcinoma derived stem cells reveal epithelial-to-mesenchymal plasticity

AIM To establish a model to enrich and characterize stem-like cells from murine normal liver and hepatocellular carcinoma (HCC) cell lines and to further investigate stem-like cell association with epithelial-to-mesenchymal transition (EMT). METHODS In this study, we utilized a stem cell conditioned serum-free medium to enrich stem-like cells from mouse HCC and normal liver cell lines, Hepa 1-6 and AML12, respectively. We isolated the 3-dimensional spheres and assessed their stemness characteristics by evaluating the RNA levels of stemness genes and a cell surface stem cell marker by quantitative reverse transcriptase-PCR (qRT-PCR). Next, we examined the relationship between stem cells and EMT using qRT-PCR. RESULTS Three-dimensional spheres were enriched by culturing murine HCC and normal hepatocyte cell lines in stem cell conditioned serum-free medium supplemented with epidermal growth factor, basic fibroblast growth factor and heparin sulfate. The 3-dimensional spheres had enhanced stemness markers such as Klf4 and Bmi1 and hepatic cancer stem cell (CSC) marker Cd44 compared to parental cells grown as adherent cultures. We report that epithelial markers E-cadherin and ZO-1 were downregulated, while mesenchymal markers Vimentin and Fibronectin were upregulated in 3-dimensional spheres. The 3-dimensional spheres also exhibited changes in expression of Snai, Zeb and Twist family of EMT transcription factors. CONCLUSION Our novel method successfully enriched stem-like cells which possessed an EMT phenotype. The isolation and characterization of murine hepatic CSCs could establish a precise target for the development of more effective therapies for HCC.

575. Dendritic Cells Modified Using an Adenovirus Expressing Murine IL-12 Inhibits Development of Tumors in a Mouse Colon Cancer Model

DCs are specialized antigen presenting cells that generate immune responses by presenting antigen epitopes in the context of MHC. DCs also secrete cytokines that direct the immune response. Interleukin-12 (IL-12) is important for directing immune system toward a cytolytic T cell response. We studied the effect of vaccination with DCs modified to express the murine IL-12 gene using a recombinant adenovirus. DCs were generated from bone marrow of C57Bl/6 mice in media supplemented with GM-CSF. Recombinant adenoviral vectors, Ad.mIL-12 expressing the p35/p40 subunits of murine IL-12 and a control vector, Ad.null, were generated in 293 cells. DCs were infected with Ad.mIL-12 at various MOIs and IL-12 secretion was measured by ELISA. Splenocytes (Spc) from naive C57Bl/6 mice were mixed with irradiated DCs infected with Ad.mIL-12 (DCAd.mIL-12) or Ad.null (DCAd.null) and in the presence or absence of anti-CD4 or anti-CD8 antibodies. Spc proliferation was assessed by [3H]-thymidine incorporation and IFN-γ production was measured. Groups of C57Bl/6 or IFN-γ −/− knockout C57Bl/6 mice were injected weekly with 1×106 DCAd.mIL-12, DCAd.null or with unmodified DCs for 2 weeks. A week after the last vaccination, mice were injected with 1×105 MC38.KRas murine colon carcinoma cells expressing mutant human K-Ras (G12D). Some groups of DCAd.mIL-12 treated mice received injections of anti-CD4 or anti-CD8 antibodies beginning either two days before initiation of vaccine or before tumor challenge. Tumor and Spc were obtained from each group of mice 21 days after injection of tumor. Tissues were examined for CD4 and CD8 cells, and for IFN-γ expression by in situ hybridization. Spc were co-cultured with irradiated MC38.KRas for 5 days and IFN-γ and IL-4 concentrations were measured by ELISA. After infection with Ad.mIL-12 (MOI = 10 and 30), mIL-12 p70 1628 and 3826 pg/mL/105 cells was measured, respectively. Spc stimulated with DCAd.mIL-12 had 2-fold greater [3H]-thymidine incorporation than controls. Anti-CD4 antibody, but not anti-CD8-antibody abolished this stimulation. DCAd.mIL-12 co-cultured with Spc produced 10-fold higher concentrations of IFN-γ than Spc stimulated with 300 ng of recombinant murine IL-12. When mice were challenged with MC38.KRas cells, inhibition of tumor growth was seen in the DCAd.mIL-12 vaccinated group but not the controls. Sixty percent of DCAd.mIL-12 treated mice were free of tumor at 77 days, while less than 20% of mice in the control groups tumor-free. Infiltration of tumors by CD4+ cells and expression of IFN-γ was seen in DCAd.mIL-12 treated mice. When Spc from mice treated with DCAd.mIL-12, DCAd.null or unmodified DC were mixed with MC38.KRas in vitro, supernatant contained 120, 35, 0.7 ng/ml of IFN-γ and 17.3, 8.9, 42.3 pg/ml of IL-4, respectively. Protection by DCAd.mIL-12 was abolished by depletion of CD4+ cells, but not by depletion of CD8+ cells. DCAd.mIL-12 vaccination was ineffective at inhibiting tumor growth in IFN-γ −/− knockout mice. [Conclusion] DCs modified with Ad.mIL-12 successfully protected C57Bl/6 mice from challenge with MC38KRas tumor cells. CD4+ T cells and secretion of IFN-γ appear to be important for the effectiveness of DCAd.mIL-12 vaccination.