Talicia Savage

Expression of NF-κB p50 in Tumor Stroma Limits the Control of Tumors by Radiation Therapy

Radiation therapy aims to kill cancer cells with a minimum of normal tissue toxicity. Dying cancer cells have been proposed to be a source of tumor antigens and may release endogenous immune adjuvants into the tumor environment. For these reasons, radiation therapy may be an effective modality to initiate new anti-tumor adaptive immune responses that can target residual disease and distant metastases. However, tumors engender an environment dominated by M2 differentiated tumor macrophages that support tumor invasion, metastases and escape from immune control. In this study, we demonstrate that following radiation therapy of tumors in mice, there is an influx of tumor macrophages that ultimately polarize towards immune suppression. We demonstrate using in vitro models that this polarization is mediated by transcriptional regulation by NFκB p50, and that in mice lacking NFκB p50, radiation therapy is more effective. We propose that despite the opportunity for increased antigen-specific adaptive immune responses, the intrinsic processes of repair following radiation therapy may limit the ability to control residual disease.

Optimizing Timing of Immunotherapy Improves Control of Tumors by Hypofractionated Radiation Therapy

The anecdotal reports of promising results seen with immunotherapy and radiation in advanced malignancies have prompted several trials combining immunotherapy and radiation. However, the ideal timing of immunotherapy with radiation has not been clarified. Tumor bearing mice were treated with 20Gy radiation delivered only to the tumor combined with either anti-CTLA4 antibody or anti-OX40 agonist antibody. Immunotherapy was delivered at a single timepoint around radiation. Surprisingly, the optimal timing of these therapies varied. Anti-CTLA4 was most effective when given prior to radiation therapy, in part due to regulatory T cell depletion. Administration of anti-OX40 agonist antibody was optimal when delivered one day following radiation during the post-radiation window of increased antigen presentation. Combination treatment of anti-CTLA4, radiation, and anti-OX40 using the ideal timing in a transplanted spontaneous mammary tumor model demonstrated tumor cures. These data demonstrate that the combination of immunotherapy and radiation results in improved therapeutic efficacy, and that the ideal timing of administration with radiation is dependent on the mechanism of action of the immunotherapy utilized.

The Peripheral Myeloid Expansion Driven by Murine Cancer Progression Is Reversed by Radiation Therapy of the Tumor

Expansion of myeloid-lineage leukocytes in tumor-bearing mice has been proposed as a cause of systemic immunosuppression. We demonstrate that radiation therapy of tumors leads to a decline in myeloid cell numbers in the blood and a decrease in spleen size. The frequency of myeloid cells does not decline to the level seen in tumor-free mice: we demonstrate that metastatic disease can prevent myeloid cell numbers from returning to baseline, and that tumor recurrence from residual disease correlates with re-expansion of myeloid lineage cells. Radiation therapy results in increased proliferation of T cells in the spleen and while T cell responses to foreign antigens are not altered by tumor burden or myeloid cell expansion, responses to tumor-associated antigens are increased after radiation therapy. These data demonstrate that myeloid cell numbers are directly linked to primary tumor burden, that this population contracts following radiation therapy, and that radiation therapy may open a therapeutic window for immunotherapy of residual disease.

Expression of Arginase I in Myeloid Cells Limits Control of Residual Disease after Radiation Therapy of Tumors in Mice

An accumulating body of evidence demonstrates that radiation therapy can generate adaptive immune responses that contribute to tumor control. However, in the absence of additional immune therapy, the adaptive immune response is insufficient to prevent tumor recurrence or affect distant disease. It has been shown in multiple models that tumor-infiltrating myeloid cells exhibit alternative activation phenotypes and are able to suppress adaptive immune responses, and recent data suggests that the myeloid response in tumors treated with cytotoxic therapy limits tumor control. We hypothesized that tumor myeloid cells inhibit the adaptive immune response after radiation therapy through expression of the enzyme arginase I. Using a myeloid cell-specific deletion of arginase I in mice, we demonstrate an improved tumor control after radiation therapy. However, tumors still recurred despite the conditional knockdown of arginase I. Since multiple alternative factors may combine to inhibit adaptive immunity, we propose that targeting macrophage differentiation may be a more effective strategy than targeting individual suppressive pathways.

TGFβ inhibition prior to hypofractionated radiation enhances efficacy in preclinical models

Young and colleagues demonstrate in syngeneic mouse models of colorectal and pancreatic cancers that TGFβ inhibition with the oral, small-molecule inhibitor SM16 enhanced adaptive immunity in the tumor microenvironments and significantly improved the efficacy of subsequent radiotherapy. The immune infiltrate in colorectal cancer has been correlated with outcome, such that individuals with higher infiltrations of T cells have increased survival independent of the disease stage. For patients with lower immune infiltrates, overall survival is limited. Because the patients with colorectal cancer studied have received conventional cancer therapies, these data may indicate that the pretreatment tumor environment increases the efficacy of treatments such as chemotherapy, surgery, and radiotherapy. This study was designed to test the hypothesis that an improved immune environment in the tumor at the time of treatment will increase the efficacy of radiotherapy. We demonstrate that inhibition of TGFβ using the orally available small-molecule inhibitor SM16 improved the immune environment of tumors in mice and significantly improved the efficacy of subsequent radiotherapy. This effect was not due to changes in radiosensitivity, epithelial–mesenchymal transition, or changes in vascular function in the tumor; rather, this effect was dependent on adaptive immunity and resulted in long-term protective immunity in cured mice. These data demonstrate that immunotherapy is an option to improve the immune status of patients with poor tumor infiltrates and that pretreatment improves the efficacy of radiotherapy. Cancer Immunol Res; 2(10); 1011–22. ©2014 AACR.

Mertk on tumor macrophages is a therapeutic target to prevent tumor recurrence following radiation therapy

Radiation therapy provides a means to kill large numbers of cancer cells in a controlled location resulting in the release of tumor-specific antigens and endogenous adjuvants. However, by activating pathways involved in apoptotic cell recognition and phagocytosis, irradiated cancer cells engender suppressive phenotypes in macrophages. We demonstrate that the macrophage-specific phagocytic receptor, Mertk is upregulated in macrophages in the tumor following radiation therapy. Ligation of Mertk on macrophages results in anti-inflammatory cytokine responses via NF-kB p50 upregulation, which in turn limits tumor control following radiation therapy. We demonstrate that in immunogenic tumors, loss of Mertk is sufficient to permit tumor cure following radiation therapy. However, in poorly immunogenic tumors, TGFb inhibition is also required to result in tumor cure following radiation therapy. These data demonstrate that Mertk is a highly specific target whose absence permits tumor control in combination with radiation therapy.

Circulating and intratumoral macrophages in patients with hepatocellular carcinoma: correlation with therapeutic approach

BACKGROUND Hepatocellular carcinoma arises in an environment of chronic injury, and wound-healing responses may vary by treatment. METHODS Peripheral blood myeloid populations were quantified in 39 patients with hepatocellular carcinoma treated with surgical or endoluminal therapy. Macrophages were quantified in tissue when available. RESULTS There was a similar expansion of myeloid populations after operative procedures compared with endoluminal treatments. Immunostaining for CD68 revealed no significant differences in the number of macrophages within benign versus malignant tumors and when tumors were compared with nontumor liver. Cytotoxic CD8+ T cells were rare within tumors compared with the surrounding liver (P < .0001). Progression-free survival was reduced in patients with preoperative peripheral blood monocyte expansion (P < .05). CONCLUSIONS These data provide preliminary evidence of poor prognostic significance of elevated peripheral blood monocyte counts. We propose that the inflammatory environment of hepatocellular carcinoma may represent a consistent feature to both predict and alter the course of disease.

Preparative immunotherapy with anti-OX40 and anti-CTLA4 improves the response to chemotherapy

Meeting abstracts Recent studies have reported that decreased T cell infiltrate alone, or co-ordinate with increased macrophage infiltrate, correlate with decreased survival in a range of cancers, including patients with pancreatic cancer. Importantly, in mouse models of pancreatic cancer,

Abstract 1788: Converting radiation-mediated tumor control to tumor cure by manipulating the macrophage response to dying cells

High-dose radiation has the ability to produce large-scale cancer cell death at the tumor site over a short time frame with the potential to provide both antigen and the immunological adjuvants required for effective adaptive immune responses. However, both clinical and preclinical evidence suggests that in the absence of additional immune intervention, adaptive immune responses to distant tumors following radiation therapy are rare and ineffective. In vitro and in vivo, dying cells are efficiently immunosuppressive. Mer is a member of a subgroup of receptor tyrosine kinases and its dominant ligand is Gas6, a protein S-related gene that has been shown to bind exposed phosphatidylserine (PS) on apoptotic cells. The Mer-Gas6-PS interaction results in phagocytosis of the apoptotic cell, and mice with inactivated Mer are defective in their handling of apoptotic cells. We hypothesized that Mer expression on tumor macrophages acted as a receptor for cancer cells killed by radiation therapy and resulted in differentiation of these macrophages into suppressive phenotypes. We demonstrate here that blocking the Mer interaction with irradiated cancer cells blocks suppressive differentiation of tumor macrophages, and Mer blockade combined with TGFβ blockade restored classical proinflammatory macrophage function in the presence of irradiated cancer cells. We demonstrate that Mer ligation results in accumulation of NFkB p50 in macrophages that is required for the switch from anti-inflammatory to pro-inflammatory cytokine secretion, but does not change or interfere with classical polarization as measured by arginase and iNOS expression. Using CT-guided radiation to treat tumors in immune competent wild-type or Mer-/- mice, we demonstrate that loss of Mer is not sufficient to change tumor growth and response to radiation therapy, but when combined with TGFβ blockade using an orally bioavailable small molecule results in significantly extended survival and tumor cures. This does not occur in the absence of radiation therapy, demonstrating that radiation-induced cell death is a necessary trigger for tumor cure in mice lacking Mer and TGFβ signaling. These data demonstrate that targeting specific suppressive pathways involved in the response to dying cells permits tumor clearance by cytotoxic therapy. Citation Format: Marka R. Crittenden, Jason R. Baird, Talicia Savage, Benjamin Cottam, David Friedman, Kristina Young, Pippa Newell, Cynthia Nguyen, Andrew M. Jackson, Michael J. Gough. Converting radiation-mediated tumor control to tumor cure by manipulating the macrophage response to dying cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1788. doi:10.1158/1538-7445.AM2015-1788

Immune consequences of CT-guided radiation therapy of mouse mammary tumors

Transgenic tumor models provide the closest animal approximation of human cancer development and progression, and a challenging treatment model. The orthotopic location of these tumors presents challenges in testing radiation therapies because conventional radiation models using patient linear accelerators or untargeted units with selective shielding are difficult or impractical in transgenic tumor models. We have tested CT-guided radiation therapy using a Small Animal Radiation Research Platform to treat transgenic MMTV-PyMT mammary tumors. Image guiding permits close targeting of tumors with no measurable toxicities at single tumor doses tested up to 20Gy. We demonstrate that tumor treatment results in a dose-dependent control of mammary tumors. Histology illustrates destruction of invasive carcinoma in the mammary gland with remaining tissue features of premalignant disease due to ongoing transgene-driven tumorigenic progression in other mammary cells. We demonstrate progressive expansion of CD11b+Gr1+ myeloid cells detectable in the blood of mice that correlates with tumor progression, and that focal treatment of mammary tumors with radiation therapy reverses this myeloid expansion without affecting systemic T cell numbers. The result is an improved systemic myeloid cell: T cell ratio in treated mice, which has been associated with improved immune function in many animal models of cancer. This platform provides an approach to study hypofractionated radiation therapy in authentic animal models of tumor progression without causing systemic lymphopenia and permits experiments investigating the interaction between radiation therapy and endogenous immune responses in transgenic and orthotopic tumor models.