Immunotherapy of cancer is attractive because of its potential for specificity and limited side effects. The efficacy of this approach may be improved by providing adjuvant signals and an inflammatory environment for immune cell activation. We evaluated antitumor immune responses in mice after treatment of OVA-expressing B16-F0 tumors with single (15 Gy) or fractionated (5 × 3 Gy) doses of localized ionizing radiation. Irradiated mice had cells with greater capability to present tumor Ags and specific T cells that secreted IFN-γ upon peptide stimulation within tumor-draining lymph nodes than nonirradiated mice. Immune activation in tumor-draining lymph nodes correlated with an increase in the number of CD45+ cells infiltrating single dose irradiated tumors compared with nonirradiated mice. Similarly, irradiated mice had increased numbers of tumor-infiltrating lymphocytes that secreted IFN-γ and lysed tumor cell targets. Peptide-specific IFN-γ responses were directed against both the class I and class II MHC-restricted OVA peptides OVA257–264 and OVA323–339, respectively, as well as the endogenous class I MHC-restricted B16 tumor peptide tyrosinase-related protein 2180–188. Adoptive transfer studies indicated that the increased numbers of tumor Ag-specific immune cells within irradiated tumors were most likely due to enhanced trafficking of these cells to the tumor site. Together these results suggest that localized radiation can increase both the generation of antitumor immune effector cells and their trafficking to the tumor site.

Posttranscriptional gene regulation by microRNAs (miRNAs) is important for many aspects of development, homeostasis, and disease. Here, we show that reduction of endothelial miRNAs by cell-specific inactivation of Dicer, the terminal endonuclease responsible for the generation of miRNAs, reduces postnatal angiogenic response to a variety of stimuli, including exogenous VEGF, tumors, limb ischemia, and wound healing. Furthermore, VEGF regulated the expression of several miRNAs, including the upregulation of components of the c-Myc oncogenic cluster miR-17-92. Transfection of endothelial cells with components of the miR-17-92 cluster, induced by VEGF treatment, rescued the induced expression of thrombospondin-1 and the defect in endothelial cell proliferation and morphogenesis initiated by the loss of Dicer. Thus, endothelial miRNAs regulate postnatal angiogenesis and VEGF induces the expression of miRNAs implicated in the regulation of an integrated angiogenic response.endothelium ͉ VEGF M icroRNAs (miRNAs) are short (Ϸ22 nt) noncoding RNAs derived from long primary transcripts through sequential processing by the enzymes Drosha and Dicer. Dicer-generated miRNAs are incorporated into the RNA-induced silencing complex that mediates miRNA-dependent translational suppression or in some instances cleavage of respective mRNA targets or translational activation (1, 2). The significance of miRNAs in mammalian biology has been dissected by Dicer gene disruption in mice. Mutant and disrupted Dicer alleles caused embryonic lethality associated with a loss of pluripotent stem cells (3) and defective blood vessel formation (4). Tissue-specific inactivation of Dicer has led to the conclusion that Dicer is essential for several processes, for example, limb, lung, and skin morphogenesis, the maintenance of hair follicles, T cell development/ differentiation, and neuronal survival (5-11).The growth of blood vessels is essential for organ growth and tissue repair. During adulthood, most blood vessels remain quiescent to fulfill their main function of conducting nutritive blood flow to organs; however, during pathological events such as tissue ischemia, inflammation, and tumor progression, endothelial cells (ECs) become activated and angiogenesis ensues to provide conduits for blood flow (12). An imbalance in the growth of blood vessels contributes to the pathogenesis of numerous disorders (13), and the growth of vessels is a complex process, requiring a finely tuned balance between numerous stimulatory and inhibitory signals (14). VEGF has been identified as a central mediator of angiogenesis (15). We (16) and others (17) have recently shown that reduction of miRNA levels via Dicer silencing strongly impacts EC functions in vitro, suggesting a critical role for miRNAs in angiogenesis. The role of Dicer-regulated miRNAs in ovarian angiogenesis is suggested by data obtained in mice expressing a global hypomorphic Dicer1 allele, where female mice are infertile because of corpus luteum insufficiency and defective ovarian angiogenesis...

Alterations to the tumor microenvironment following localized irradiation may influence the effectiveness of subsequent immunotherapy. The objective of this study was to determine how IFN-γ influences the inflammatory response within this dynamic environment following radiotherapy. B16/OVA melanoma cells were implanted into C57BL/6 (wild-type (WT)) and IFN-γ-deficient (IFN-γ−/−) mice. Seven days after implantation, mice received 15 Gy of localized tumor irradiation and were assessed 7 days later. Irradiation up-regulated the expression of VCAM-1 on the vasculature of tumors grown in WT but not in IFN-γ−/− mice. Levels of the IFN-γ-inducible chemokines MIG and IFN-γ-inducible protein 10 were decreased in irradiated tumors from IFN-γ−/− mice compared with WT. In addition to inducing molecular cues necessary for T cell infiltration, surface MHC class I expression is also up-regulated in response to IFN-γ produced after irradiation. The role of IFN-γ signaling in tumor cells on class I expression was tested using B16/OVA cells engineered to overexpress a dominant negative mutant IFN-γ receptor (B16/OVA/DNM). Following implantation and treatment, expression of surface class I on tumor cells in vivo was increased in B16/OVA, but not in B16/OVA/DNM tumors, suggesting IFN-γ acts directly on tumor cells to induce class I up-regulation. These increases in MHC class I expression correlated with greater levels of activated STAT1. Thus, IFN-γ is instrumental in creating a tumor microenvironment conducive for T cell infiltration and tumor cell target recognition.

Controlling metastases remains a critical problem in cancer biology. Within the peritoneal cavity, omental tissue is a common site for metastatic disease arising from intraperitoneal tumors; however, it is unknown why this tissue is so favorable for metastatic tumor growth. Using five different tumor cell lines in three different strains of mice, we found that the omentum was a major site of metastases growth for intraperitoneal tumors. Furthermore, initial attachment and subsequent growth were limited to specific sites within the omentum, consisting of organized aggregates of immune cells. These immune aggregates contained a complex network of capillaries exhibiting a high vascular density, which appear to contribute to the survival of metastatic cells. We found that the vasculature within these aggregates contained CD105؉ vessels and vascular sprouts, both indicators of active angiogenesis. A subset of mesothelial cells situated atop the immune aggregates was found to be hypoxic, and a similar proportion was observed to secrete vascular endothelial growth factor-A. These data provide a physiological mechanism by which metastatic tumor cells preferentially grow at sites rich in proangiogenic vessels, apparently stimulated by angiogenic factors produced by mesothelial cells. These sites provide metastatic cells with a microenvironment highly conducive to survival and subsequent growth.

The need for an intact immune system for cancer radiation therapy to be effective suggests that radiation not only acts directly on the tumor but also indirectly, through activation of host immune components. Recent studies demonstrated that endogenous type I interferons (type I IFNs) play a role in radiation-mediated anti-tumor immunity by enhancing the ability of dendritic cells (DCs) to cross-prime CD8+ T cells. However, it is still unclear to what extent endogenous type I IFNs contribute to the recruitment and function of CD8+ T cells. Little is also known about the effects of type I IFNs on myeloid cells. In the current study, we demonstrate that type I and type II IFN (IFN-γ) are both required for the increased production of CXCL10 (IP-10) chemokine by myeloid cells within the tumor after radiation treatment. Radiation-induced intratumoral IP-10 levels in turn correlate with tumor-infiltrating CD8+ T cell numbers. Moreover, type I IFNs promote potent tumor-reactive CD8+ T cells by directly affecting the phenotype, effector molecule production and enhancing cytolytic activity. Using a unique inducible expression system to increase local levels of IFN-α exogenously, we show here that the capacity of radiation therapy to result in tumor control can be enhanced. Our pre-clinical approach to study the effects of local increase in IFN-α levels can be used to further optimize the combination therapy strategy in terms of dosing and scheduling, which may lead to better clinical outcome.