Increase In Tumor-associated Macrophages Research Articles

CD47-mediated immune evasion in early-stage lung cancer progression

Alveolar and interstitial macrophages play crucial roles in eradicating pathogens and transformed cells in the lungs. The immune checkpoint CD47, found on normal and malignant cells, interacts with the SIRPα ligand on macrophages, inhibiting phagocytosis, antigen presentation, and promoting immune evasion. In this study, we demonstrated that CD47 is not only a transmembrane protein, but that it is also highly concentrated in extracellular vesicles from lung cancer cell lines and patient plasma. Abundant CD47 was observed in the cytoplasm of lung cancer cells, aligning with our finding that it was packed into extracellular vesicles for physiological and pathological functions. In our clinical cohort, extracellular vesicle CD47 was significantly higher in the patients with early-stage lung cancer, emphasizing innate immunity inactivation in early tumor progression. To validate our hypothesis, we established an orthotopic xenograft model mimicking lung cancer development, which showed increased serum soluble CD47 and elevated IL-10/TNF-α ratio, indicating an immune-suppressive tumor microenvironment. CD47 expression led to reduced tumor-infiltrating macrophages during progression, while there was a post-xenograft increase in tumor-associated macrophages. In conclusion, CD47 is pivotal in early lung cancer progression, with soluble CD47 emerging as a key pathological effector.

Abstract 6358: Arming tumor-associated macrophages to inhibit cancer progression

Abstract Integrin αvβ3 is a marker of cancer progression in a range of epithelial cancers. In the 1990s, a humanized anti-αvβ3 antibody (Etaracizumab) was developed to target αvβ3+ cancer cells via NK cell-mediated cytotoxicity. While it demonstrated safety and some efficacy in clinical trials, our recent study of the immune microenvironment in αvβ3+ human epithelial tumors revealed increased tumor-associated macrophages (TAMs) but minimal NK cell accumulation. Accordingly, we re-engineered Etaracizumab to favor TAM engagement over NK cells and compared the efficacy of this antibody (ABT101) to that of Etaracizumab in various αvβ3+ epithelial cancer models. While ABT101 and Etaracizumab exhibited identical affinity for αvβ3, ABT 101 showed superior anti-tumor activity in vivo. Notably, depleting NK cells in mice had no impact on ABT101’s effectiveness, while macrophage depletion completely abolished its anti-tumor activity. Interestingly, tumors in mice treated with ABT101 displayed a significant increase in TAMs. Here we define an “antigen-effector cell matching” strategy that allows for maximal anti-tumor activity for various drug resistant epithelial cancers. ABT101 will enter clinical trials for patients with drug resistant lung cancer in the first quarter of 2024. Citation Format: Joshua P. Reddy, Ziqi Yu, Ryan M. Shepard, Tami Von Schalscha, Stephen J. McCormack, Sara M. Weis, David A. Cheresh, Hiromi I. Wettersten. Arming tumor-associated macrophages to inhibit cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6358.

Abstract A150: Effects of radioimmunotherapy on human and canine osteosarcoma microenvironment

Abstract Introduction: Osteosarcoma (OS) is a type of primary bone cancer that predominantly affects children and adolescents with an unacceptably high mortality rate of 30% within 5 years of diagnosis. Though several treatment options including radiotherapy, chemotherapy, and amputation surgery exist, these are either ineffective or very invasive. Also, no significant advancements in treatment options have occurred in the last 30 years. The incidence of OS among companion dogs is much higher than in humans and phenotypically OS is very similar between humans and canines. We are using a comparative oncology approach to OS treatment by developing radioimmunotherapy (RIT) which targets the insulin-like growth factor receptor type 2 (IGF2R) on OS cells. IGF2R is abundant in human and canine OS and has shown promise a novel target for RIT of OS using a comparative oncology approach. It is therefore important to investigate the mechanistic underpinnings of RIT efficacy by investigating its interaction with OS microenvironment to develop improved RIT regimens for OS. Materials and Methods: Severe combined immunodeficiency (SCID) female mice bearing human and canine patient-derived OS xenografts were treated with RIT utilizing IF3, a novel full-length human mAb targeting IGF2R obtained by phage-display technology. The IF3 antibody was radiolabeled with an alpha emitter 225Actinium (225Ac) or a beta emitter 177Lutetim (177Lu) radionuclides. The tumors were removed at 1, 3 and 7 days post-RIT and the effects of RIT on IGF2R-positive tumor cells, OS stem cells, and immune components of the tumor microenvironment such as natural killer (NK) cells and tumor-associated macrophages (TAMs) were assessed using quantitative immunohistochemistry (IHC). Results: In the untreated tumors IHC detected the presence of IGF2R-positive tumor cells, OS stem cells, and immune components of the tumor microenvironment such as NK cells and TAMs, in both human and canine OS tumors obtained from mouse models. Quantitative analysis of IHC results in the tumors post-RIT demonstrated a time-dependent decrease in IGF2R-positive OS tumor cells and OS stem cells and an increase in TAMs and NK cells. Conclusions: Deciphering the mechanisms of RIT interaction with tumor microenvironment can assist in the design of the clinical trials in companion dogs with OS, which will be followed by the trials in children with this cancer. Also, it supports combining RIT with immunotherapy targeting OS stem cells and other immune components of tumor microenvironments such as NK cells and TAMS, which can result in improved patient outcomes. Citation Format: Sabeena Giri, Kevin J.H. Allen, Maruti Uppalapati, Ekaterina Dadachova. Effects of radioimmunotherapy on human and canine osteosarcoma microenvironment [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A150.

Ketogenic Diet Promotes Epithelial Ovarian Cancer Progression and Alters Tumor Gene Expression in-vivo (104)

<b>Objectives:</b> The ketogenic diet (KD) is hypothesized to impact tumor progression indirectly through altering the immune system as well as directly at the cellular level. Our studies were designed to assess changes in tumor resident immune cells and tumor gene expression in relation to epithelial ovarian cancer (EOC) progression in KD-fed mice. <b>Methods:</b> ID8 EOC cells, syngeneic with C57Bl/6J mouse strain, transfected with luciferase (ID8-Luc), were injected and monitored for tumor development via imaging with an <i>in vivo</i> Imaging System (IVIS) intraperitoneal D-luciferin. Tumor-bearing mice were fed a KD (10% protein, 0% carbohydrates, 90% fat), high fat diet (HFD) (10% protein, 15% carbohydrates, 75% fat), or control diet (CD) (10% protein, 77% carbohydrates, and 13% fat) (<i>n</i>=10 mice per group). EOC tumor growth was monitored weekly, and tumor burden was determined based on total flux recorded in photons/second measured. At the endpoint, tumors were collected and processed for flow cytometry and RNAseq analysis. Single-cell dissociated tumors (Papain System) were stained with an immune targeted antibody panel and analyzed on the BD Cytek Aurora. RNA was isolated (Qiagen), and sequencing was performed. Statistical tests included analysis of variance (ANOVA), permutational multivariate analysis (PERMANOVA), linear regression, and Wilcoxon test; a p-value of <0.05 was considered statistically significant. <b>Results:</b> Throughout the study, KD-fed mice exhibited a statistically significant increase in tumor progression. We observed a 9.1-fold increase in tumor volume from baseline in the KD-fed group compared to HFD and CD-fed groups (9.1 vs 2.0 vs 3.1-fold, respectively, p<0.001). At the endpoint, tumor-associated macrophages (TAM) were significantly enriched and activated lymphoid cells (NK cells) were significantly diminished in the KD group (p<0.001). There was a trend toward increased M2:M1 polarization in KD-fed mice. However, there was no significant difference in Treg cells or activated CD8+ cells among diet groups. EOC tumors in KD-fed mice exhibited a significant decrease in differential gene expression on RNA seq compared to CD- and HFD-fed tumors, with no significant difference between CD- and HFD-fed groups at the gene expression level. Gene set enrichment analysis (GSEA) demonstrated that EOC tumors from KD-fed mice had enhanced angiogenesis, inflammatory response, and epithelial to mesenchymal transition (EMT) phenotype, as well as altered lipid metabolism. Specifically, an increase in lipid catalytic activity and catabolism and decrease in lipid synthesis were noted among KD-fed mice versus HFD-fed mice. <b>Conclusions:</b> KD induced tumor progression in our murine EOC model compared to HFD and CD. KD was associated with an increase in TAM and a decrease in activated NK cells consistent with a protumorigenic immune environment. KD-fed mice exhibited markedly increased alterations in angiogenesis, inflammation, and EMT pathways with enhanced lipid catabolic activity. Further testing of KD in human EOC cells is needed to clarify the <i>in-vivo</i> effect of KD on EOC growth.Fig. 1

Abstract PO089: Comparison of Two oHSV Vectors for the Treatment of Glioblastoma

Abstract Glioblastoma multiforme (GBM) is the most common human brain cancer. Despite a well-established standard of care, the 5-year mortality rate of GBM patients is 95%, highlighting the need for innovative therapeutic interventions. A variety of oncolytic viruses, including those derived from herpes simplex virus (oHSV), have been designed for GBM therapy, but early-phase clinical trials have reported few complete responses without evidence of durable anti-tumor immunity. Potential reasons for the lack of efficacy are limited vector potency (i.e., virulence) and the presence of a highly immunosuppressive tumor microenvironment (TME) comprised of few activated lymphocytes, large numbers of immunosuppressive myeloid cells (macrophages, myeloid derived suppressor cells [MDSCs], microglia), and an agglomerate of immunosuppressive cytokines (IL-10, VEGF, MIF, etc.). Herein we explore these obstacles by comparing the anti-tumor activity of two different oHSV designs and by arming oHSV with an immunomodulatory payload designed to encourage the development of anti-tumor immunity. We first compared the activities of an HSV-1 KOS strain derivative designated KG4:T124 or colloquially, 2A5B, and an F strain derivative designated rQNestin34.5v.1 (a similar oHSV, rQNestin34.5v.2, is currently in a phase I clinical trial for GBM). We evaluated viral entry, replication capacity, and cytotoxicity using both murine and human glioma cell lines in vitro. Viral entry was relatively similar between both vectors, but rQNestin34.5v.1 replicated more effectively and generally induced greater viral mediated cytotoxicity. In syngeneic mice, rQNestin34.5v.1 reduced orthotopic GL261N4 tumor burden compared to 2A5B, but neither virus impeded disease progression in the more pernicious CT2A model. Evaluation of either the GL261N4 or CT2A TME revealed that both viruses induced a high influx of tumor associated macrophages, a transient polymorphonuclear (PMN) cell population, but did not alter the frequency of monocytic MDSCs. As a strategy to enhance oHSV efficacy, we sought to differentiate the highly immunosuppressive MDSC population into pro-inflammatory macrophages using oHSV armed with IL-12, a cytokine that has been shown to provide this activity. We show that oHSV:IL-12 induced a significant increase in tumor-associated macrophages and increased the survival of animals bearing either tumor type. Although we observed a low percentage of complete responses with the presence of anti-tumor CTLs demonstrated by tumor rechallenge experiments, IL-12 alone was not sufficient to significantly improve overall long-term animal survival, suggesting that additional arming genes should be considered in combination with IL-12. Citation Format: Joseph W. Jackson, Bonnie L. Hall, Lisa Bailey, Christophe Quéva, E. Antonio Chiocca, Justus B. Cohen, Joseph C. Glorioso. Comparison of Two oHSV Vectors for the Treatment of Glioblastoma [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO089.

Myeloid cell-targeted STAT3 inhibition sensitizes head and neck cancers to radiotherapy and T cell-mediated immunity.

The tumor microenvironment affects the outcome of radiotherapy against head and neck squamous cell carcinoma (HNSCC). We recently found that tolerogenic myeloid cells accumulate in the circulation of HNSCC patients undergoing radiotherapy. Here, we analyzed tumor-containing lymph node biopsies collected from these patients. After 2 weeks of radiotherapy, we found an increase in tumor-associated macrophages (TAMs) with activated STAT3, while CD8+ T cells were reduced as detected using multiplex IHC. Gene expression profiling indicated upregulation of M2 macrophage-related genes (CD163, CD206), immunosuppressive mediators (ARG1, LIF, TGFB1), and Th2 cytokines (IL4, IL5) in irradiated tumors. We next validated STAT3 as a potential target in human HNSCC-associated TAMs, using UM-SCC1 xenotransplants in humanized mice. Local injections of myeloid cell-targeted STAT3 antisense oligonucleotide (CpG-STAT3ASO) activated human DCs/macrophages and promoted CD8+ T cell recruitment, thereby arresting UM-SCC1 tumor growth. Furthermore, CpG-STAT3ASO synergized with tumor irradiation against syngeneic HPV+ mEERL and HPV- MOC2 HNSCC tumors in mice, triggering tumor regression and/or extending animal survival. The antitumor immune responses were CD8+ and CD4+ T cell dependent and associated with the activation of antigen-presenting cells (DCs/M1 macrophages) and increased CD8+ to regulatory T cell ratio. Our observations suggest that targeted inhibition of STAT3 in tumor-associated myeloid cells augments the efficacy of radiotherapy against HNSCC.

Abstract A105: PARP inhibition modulates the infiltration, phenotype, and function of tumor-associated macrophages (TAMs) in BRCA-associated breast cancer and can be augmented by harnessing the antitumor potential of TAMs

Abstract Patients with BRCA-associated triple-negative breast cancer (TNBC) have few effective treatment options. PARP inhibitors are promising, and we recently showed they induce an influx of white blood cells, including CD8+ T cells and macrophages into the tumor. The influx of CD8+ cells, mediated by activation of the STING pathway in tumor cells, contributes substantially to efficacy of PARP inhibition in mice. Strikingly, in these studies the greatest infiltration of immune cells into the tumor was macrophages. Given that objective responses to PARP inhibition have been observed in clinical trials but the benefits are transitory, we hypothesized that this was due to a suppressive tumor microenvironment, driven by tumor macrophages. To better understand the molecular basis of resistance to PARP inhibitors, we used high-dimensional single-cell immune profiling on human TNBC. We observed a ≥10-fold increase in TAMs in BRCA-associated TNBC compared to BRCA-wild-type TNBC. Using a preclinical model of BRCA1-deficient triple-negative breast cancer, we found that PARP inhibitors not only further increased TAM abundance but also induced functional and phenotypic changes associated with STING pathway activation, antigen presentation, and chemokine and cytokine signaling. PARP inhibitors increased the frequency of TAMs expressing costimulatory molecules CD80 and CD86 as well as the activation and maturation marker CD40, which are indicative of an antitumor phenotype. We also identified a novel negative feedback mechanism that limits the functionality of the anti-tumor TAMs and is consistent with induction of an immune-suppressive macrophage population. Utilizing transcriptomic, proteomic, and metabolic profiling of ex vivo cultured human myeloid cells, we identified multiple biologic processes associated with PARP inhibition, showing that these drugs directly affect macrophage states and phenotypes. Remarkably, in the preclinical BRCA1-deficient TNBC model, the novel combination of PARP inhibition with macrophage modulation significantly extended remissions obtained with PARP inhibitor therapy only, and this advantage persisted when treatment was discontinued, suggestive of a durable reprogramming of the tumor microenvironment. Moreover, CD8+ cells were required for the extension of PARP inhibitor-induced remissions, suggesting that targeting macrophages lifted the constraints imposed by protumor macrophages on CD8+ T cell-mediated tumor cell killing. We identify mechanisms related to macrophage and T-cell activation that increase PFS and provide evidence that TAMs may serve as targets for new therapeutic interventions designed to overcome PARP inhibitor resistance in BRCA-associated TNBC. Citation Format: Anita K. Mehta, Emily M. Cheney, Jessica A. Castrillon, Jia-Ren Lin, Mateus de Oliveira Taveira, Christina A. Hartl, Nathan T. Johnson, William M. Oldham, Marian Kalocsay, Sarah A. Boswell, Olmo Sonzogni, Constantia Pantelidou, Brett P. Gross, Shawn Johnson, Deborah A. Dillon, Sandro Santagata, Judy E. Garber, Nadine Tung, Elizabeth A. Mittendorf, Gerburg M. Wulf, Geoffrey I. Shapiro, Peter K. Sorger, Jennifer L. Guerriero. PARP inhibition modulates the infiltration, phenotype, and function of tumor-associated macrophages (TAMs) in BRCA-associated breast cancer and can be augmented by harnessing the antitumor potential of TAMs [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A105.

Abstract P5-04-01: PARP inhibition modulates the infiltration, phenotype and function of tumor-associated macrophages (TAMs) in BRCA-associated breast cancer and can be augmented by harnessing the anti-tumor potential of TAMs

Abstract Patients with BRCA-associated triple negative breast cancer (TNBC) have few effective treatment options. PARP inhibitors are promising, and we recently showed they induce an influx of white blood cells, including CD8+ T-cells and macrophages into the tumor. The influx of CD8+ cells, mediated by activation of the STING pathway in tumor cells, contributes substantially to efficacy of PARP inhibition in mice. Strikingly, in these studies, the greatest infiltration of immune cells into the tumor was macrophages. Given objective responses to PARP inhibition have been observed in clinical trials but the benefits are transitory, we hypothesized that this was presumably due to a suppressive tumor microenvironment, driven by tumor macrophages. To better understand the molecular basis of resistance to PARP inhibitors, we used high dimensional single-cell immune profiling on human TNBC. We observed a ≥10-fold increase in TAMs in BRCA-associated TNBC compared to BRCA-wildtype TNBC. Using a pre-clinical model of BRCA1-deficient triple-negative breast cancer, we found that PARP inhibitors not only further increased TAM abundance but also induced functional and phenotypic changes associated with STING pathway activation, antigen presentation, and chemokine and cytokine signaling. PARP inhibitors increased the frequency of TAMs expressing co-stimulatory molecules CD80 and CD86 as well as the activation and maturation marker CD40, which are indicative of an anti-tumor phenotype. We also identified a novel negative feedback mechanism which limits the functionality of the anti-tumor TAMs, and is consistent with induction of an immune suppressive macrophage population. Utilizing transcriptomic, proteomic and metabolic profiling of ex vivo cultured human myeloid cells, we identified multiple biological processes associate with PARP inhibition, showing that these drugs directly affect macrophage states and phenotypes. Remarkably, in the pre-clinical BRCA1-deficient TNBC model, the novel combination of PARP inhibition with macrophage modulation significantly extended remissions obtained with PARP inhibitor therapy only, and this advantage persisted when treatment was discontinued, suggestive of a durable reprogramming of the tumor microenvironment. Moreover, CD8+ cells were required for the extension of PARP inhibitor-induced remissions, suggesting that targeting macrophages lifted the constraints imposed by pro-tumor macrophages on CD8+ T cell-mediated tumor cell killing. We identify mechanisms related to macrophage and T-cell activation that increase PFS and provide evidence that TAMs may serve as targets for new therapeutic interventions designed to overcome PARP inhibitor resistance in BRCA-associated TNBC. Citation Format: Jennifer L Guerriero, Anita K Mehta, Emily M Cheney, Jessica A. Castrillon, Jia-Ren Lin, Mateus de Oliveira Taveira, Olmo Sonzogni, Constantia Pantelidou, Christina A Hartl, William M Oldham, Nathan T Johnson, Sarah A Boswell, Marian Kalocsay, Matthew J Berberich, Sholin Mei, Dan Wang, Shawn Johnson, Brett Gross, Deborah A Dillon, Mikel Lipschitz, Evisa Gjini, Scott Rodig, Sandro Santagata, Judy E Garber, Nadine Tung, Peter Sorger, Geoffrey I Shapiro, Gerburg M Wulf, Elizabeth A Mittendorf. PARP inhibition modulates the infiltration, phenotype and function of tumor-associated macrophages (TAMs) in BRCA-associated breast cancer and can be augmented by harnessing the anti-tumor potential of TAMs [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-01.

Abstract 5031: Regulatory T cell ablation accelerates early stage breast cancer progression

Abstract Breast cancer is the most common malignancy in women worldwide. Early detection of pre-invasive breast tumor has dramatically improved due to the routine screening for women. Ductal carcinoma in situ is a non-obligate precursor of breast cancer, and it only progresses to invasive breast cancer in about 40% of patients. We have previously shown that transient ablation of regulatory T (Treg) cells in established breast tumors results in significant reduction of primary and lung metastatic tumor growth. In this study, we aim to investigate the specific role of Treg cells at the pre-malignant stages. Using a spontaneous, stage-wise MMTV-driven polyoma middle T (PyMT) model of murine mammary carcinogenesis, crossed to Foxp3DTR knock-in mice where injection of diphtheria toxin leads to a complete ablation of Treg cells for around 7 days, we found that ablation of Treg cells at the pre-invasive tumor stage resulted in a significant increase in the number of mammary glands developing tumors, as well as tumor growth compared to control mice. Histological examination of tissues revealed that the area of the incipient tumors was larger in Treg cell-ablated mice, and presented a more advanced tumor stage. In addition, Treg cell ablation resulted in a pronounced mammary inflammatory infiltrate, including an increase in tumor-associated macrophages and a soluble cytokine network conducive to tumor progression. Interestingly, Treg cell ablation increased the percentage of cancer stem/progenitor cells in the mammary compartment. Overall, our study demonstrates that unlike their role in established cancers, Treg cells ablation promotes breast cancer progression at early stages. Citation Format: Wei Du, Leandro Martinez, Valentina Robila, Nicholas Clark, Michael Idowu, Paula Bos, Paula Bos. Regulatory T cell ablation accelerates early stage breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5031.

Abstract 4486: Androgen-deprivation therapy promotes immune suppression in a murine model of prostate cancer

Abstract Most prostate cancer (PCa) deaths are due to castration resistant PCa (CRPC), following failure of androgen deprivation therapy (ADT). ADT is the standard of care for patients with advanced PCa but nearly universal progression to CRPC occurs 2-3 years after ADT is initiated. Immunotherapy with checkpoint inhibitors has not been effective in most prostate cancers, perhaps because such cancers lack functional CD8 T-cells. This may be caused by infiltration of myeloid cell populations into the tumor immune cell microenvironment (TIME). Recently, we found that in a PTEN-deficient mouse PCa model, castration induces an immunosuppressive state within the tumor that is concurrent with tumor recurrence. The response to castration/ADT is tri-phasic: a pro-apoptotic regression phase when tumor shrinks, followed by selection for a residual population of resistant tumor cells and finally recurrent growth as CRPC. Using PCa cell lines to model the first two phases of the response to ADT, we have shown that ADT induces apoptosis, thereby enriching for an ADT-resistant stem/progenitor population that we propose is the in vivo source of TNF. Mechanistically, in our model system the response to ADT is driven by the soluble mediators TNF and CCL2, which facilitate communication within the TIME. Specifically, a TNF-CCL2-CCR2 paracrine loop is induced between prostate cancer cells and non-tumor cells in the microenvironment: TNF produced by tumor cells acts on myofibroblasts to induce CCL2 production, which in turn recruits CCR2+ tumor-associated macrophages (TAMs). To investigate the ADT response within the TIME in an in vivo model of prostate cancer, we employed a prostate-specific PTEN-deficient mouse model (PbCre4 x PTENf/f). Castration caused the tumors to regress, consistent with initial phase of the response that is seen in the human disease. At late times post-castration (5-6 weeks), corresponding to the selection phase, we observed a coordinate increase in the stem/progenitor tumor cell population, as well as TNF and CCL2, within the TIME. Immunohistochemical staining of tumors 5 weeks post-castration revealed an increase in TAMs, and a decrease in CD8 T cells, consistent with an immuno-suppressive or immuno-evasive state. This phenotype was reversed by a soluble receptor that binds TNF (etanercept). Thus, following ADT, TNF derived from an ADT-resistant stem/progenitor epithelial tumor cell population promotes an immunosuppressive state via CCL2 in the TIME. Analysis of public human PCa data sets show the transcripts for TNF, as well as gene signatures for stem/progenitor tumor cells and M2 TAMs are increased in CRPC, consistent with our hypothesis that ADT drives the development of myeloid immuno-suppressive state via a TNF-CCL2-CCR2 axis. Our results set the stage for the future development of immunotherapies that could improve the efficacy of ADT. Citation Format: John J. Krolewski, Kai Sha, Kent L. Nastiuk. Androgen-deprivation therapy promotes immune suppression in a murine model of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4486.

Aryl hydrocarbon receptor acts as a tumor suppressor in a syngeneic MC38 colon carcinoma tumor model.

Background: Aryl hydrocarbon receptor (AHR), commonly known as an environmental sensor involved in the metabolism and elimination of xenobiotic substances, is also an important modulator in the development and functioning of the immune system. AHR expression is varied in the T cell subsets with the highest expression in T-helper 17 and T regulatory cells. It has been reported that AHR can act as a tumor promoter or a tumor suppressor, depending on the tumor type.Methods: In an effort to understand the role played by AHR in tumor growth, the MC38 syngeneic colon carcinoma tumor model was used on C57BL/6 or ahr knockout (KO, -/-) mice with or without AHR antagonist (CH223191) treatment. Tumor sizes were measured, and biomarkers were quantified in tumor microenvironment and draining lymph nodes using flow cytometry. Enzyme-linked immunosorbent assay was used to determine the amount of cytokines in tumors.Results: In ahr deficient mice, MC38 tumors progress more rapidly than in wild-type mice, accompanied by an increase in tumor-associated macrophages and M2 macrophages and a decrease in CD8a positive cytotoxic lymphocytes. Analysis of cytokines in the tumor microenvironment reveals a pro-inflammatory phenotype. Similar changes were observed by pharmacologic blockade of the receptor using CH223191.Conclusion: AHR acts as a tumor suppressor in mice implanted with MC38 colon carcinoma cells as evidenced by either a blockade or deficiency of AHR.

Abstract A084: Towards combining androgen deprivation and immunotherapy to prevent progression to castration-resistant prostate cancer

Abstract Most prostate cancer (PCa) deaths are due to castration-resistant PCa (CRPC), following failure of androgen-deprivation therapy (ADT). ADT is the standard of care for patients with advanced PCa. However, nearly universal progression to castration-resistant prostate cancer (CRPC) occurs 2-3 years after ADT is initiated. Although there have been recent improvements in the treatment of CRPC, even the most promising therapies are still not curative. One approach to this problem is to improve the initial treatment of advanced prostate cancers, by combining complementary therapies with ADT, to prevent progression of such advanced cancers to CRPC. Immunotherapy with checkpoint inhibitors (CPIs) has not been effective in prostate cancers, perhaps because such cancers are “cold” (lacking cytolytic CD8 T-cells). Some cold tumors may be caused by infiltration of myeloid cell populations (tumor associated macrophages and myeloid-derived suppressor cells) into the tumor immune cell microenvironment (TIME). Recently, we found that in a PTEN-deficient mouse PCa model, castration induces an immunosuppressive state within the tumor that is concurrent with tumor recurrence. The response to castration/ADT is tri-phasic: a pro-apoptotic regression phase when tumor shrinks, followed by selection for a residual population of resistant tumor cells and finally recurrent growth as CRPC. Using PCa cell lines to model the first two phases of the response to ADT, we have shown that ADT induces apoptosis, thereby enriching for an ADT-resistant stem/progenitor population that we propose is the in vivo source of TNF. Mechanistically, in our model system the response to ADT is driven by the soluble mediators TNF and CCL2, which facilitate communication within the TIME. Specifically, a TNF-CCL2-CCR2 paracrine loop is induced between prostate cancer cells and non-tumor cells in the microenvironment: TNF produced by tumor cells acts on myofibroblasts to induce CCL2 production, which in turn recruits CCR2+ tumor-associated macrophages (TAMs). To investigate the ADT response within the TIME in an in vivo model of prostate cancer, we employed a prostate-specific PTEN-deficient mouse model (PbCre4 x PTENf/f). Castration caused the tumors to regress, consistent with initial phase of the response that is seen in the human disease. At late times post-castration (5-6 weeks), corresponding to the selection phase, we observed a coordinate increase in the stem/progenitor tumor cell population, as well as TNF and CCL2, within the TIME. Immunohistochemical staining of tumors 5 weeks post-castration revealed an increase in TAMs, and a decrease in CD8 T-cells, consistent with an immunosuppressive or immunoevasive state. This phenotype was reversed by a soluble receptor that binds TNF (etanercept). We also observed increased myeloid-derived suppressor cells (MDSC). Thus, following ADT, TNF derived from an ADT-resistant stem/progenitor epithelial tumor cell population promotes an immunosuppressive state via CCL2 in the TIME. Analysis of public human PCa data sets shows TNF and stem/progenitor marker expression are both increased in CRPC, consistent with our hypothesis that ADT drives the development of an immunosuppressive state via a TNF-CCL2-CCR2 axis. Our results set the stage for the future development of immunotherapies that could improve the efficacy of ADT. Citation Format: John J. Krolewski, Kai Sha, Michalis Mastri, Dean Tang, Kevin Eng, Kent L. Nastiuk. Towards combining androgen deprivation and immunotherapy to prevent progression to castration-resistant prostate cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A084.

Quantitative Imaging of Tumor-Associated Macrophages and Their Response to Therapy Using 64Cu-Labeled Macrin.

Tumor-associated macrophages (TAMs) are widely implicated in cancer progression, and TAM levels can influence drug responses, particularly to immunotherapy and nanomedicines. However, it has been difficult to quantify total TAM numbers and their dynamic spatiotemporal distribution in a non-invasive and translationally relevant manner. Here, we address this need by developing a pharmacokinetically optimized, 64Cu-labeled polyglucose nanoparticle (Macrin) for quantitative positron emission tomography (PET) imaging of macrophages in tumors. By combining PET with high-resolution in vivo confocal microscopy and ex vivo imaging of optically cleared tissue, we found that Macrin was taken up by macrophages with >90% selectivity. Uptake correlated with the content of macrophages in both healthy tissue and tumors ( R2 > 0.9) and showed striking heterogeneity in the TAM content of an orthotopic and immunocompetent mouse model of lung carcinoma. In a proof-of-principle application, we imaged Macrin to monitor the macrophage response to neo-adjuvant therapy, using a panel of chemotherapeutic and γ-irradiation regimens. Multiple treatments elicited 180-650% increase in TAMs. Imaging identified especially TAM-rich tumors thought to exhibit enhanced permeability and retention of nanotherapeutics. Indeed, these TAM-rich tumors accumulated >700% higher amounts of a model poly(d,l-lactic- co-glycolic acid)- b-polyethylene glycol (PLGA-PEG) therapeutic nanoparticle compared to TAM-deficient tumors, suggesting that imaging may guide patient selection into nanomedicine trials. In an orthotopic breast cancer model, chemoradiation enhanced TAM and Macrin accumulation in tumors, which corresponded to the improved delivery and efficacy of two model nanotherapies, PEGylated liposomal doxorubicin and a TAM-targeted nanoformulation of the toll-like receptor 7/8 agonist resiquimod (R848). Thus, Macrin imaging offers a selective and translational means to quantify TAMs and inform therapeutic decisions.

Macrophage Depletion in Elderly Mice Improves Response to Tumor Immunotherapy, Increases Anti-tumor T Cell Activity and Reduces Treatment-Induced Cachexia.

Most cancers emerge in the elderly, including lung cancer and mesothelioma, yet the elderly remain an underrepresented population in pre-clinical cancer studies and clinical trials. The immune system plays a critical role in the effectiveness of many anti-cancer therapies in young hosts via tumor-specific T cells. However, immunosuppressive macrophages can constitute up to 50% of the tumor burden and impair anti-tumor T cell activity. Altered macrophage phenotype and function during aging may further impact anti-tumor T cell responses. Yet, the impact of macrophages on anti-tumor T cell responses and immunotherapy in the elderly is unknown. Therefore, we examined macrophages and their interaction with T cells in young (3 months) and elderly (20–24 months) AE17 mesothelioma-bearing female C57BL/6J mice during tumor growth. Mesothelioma tumors grew faster in elderly compared with young mice, and this corresponded with an increase in tumor-associated macrophages. During healthy aging, macrophages increase in bone marrow and spleens suggesting that these sites have an increased potential to supply cancer-promoting macrophages. Interestingly, in tumor-bearing mice, bone marrow macrophages increased proliferation whilst splenic macrophages had reduced proliferation in elderly compared with young mice, and macrophage depletion using the F4/80 antibody slowed tumor growth in young and elderly mice. We also examined responses to treatment with intra-tumoral IL-2/anti-CD40 antibody immunotherapy and found it was less effective in elderly (38% tumor regression) compared to young mice (90% regression). Tumor-bearing elderly mice decreased in vivo anti-tumor cytotoxic T cell activity in tumor draining lymph nodes and spleens. Depletion of macrophages using F4/80 antibody in elderly, but not young mice, improved IL-2/anti-CD40 immunotherapy up to 78% tumor regression. Macrophage depletion also increased in vivo anti-tumor T cell activity in elderly, but not young mice. All the tumor-bearing elderly (but not young) mice had decreased body weight (i.e., exhibited cachexia), which was greatly exacerbated by immunotherapy; whereas macrophage depletion prevented this immunotherapy-induced cachexia. These studies strongly indicate that age-related changes in macrophages play a key role in driving cancer cachexia in the elderly, particularly during immunotherapy, and sabotage elderly anti-tumor immune responses.

Imaging and Treatment of Primary Tumors and Metastases by Targeted Macrophage Therapy, Using Radiation Therapy-Directed Encapsulated Nanoparticles

Liposome-protamine-hyaluronic acid nanoparticles (LPH-NPs; 173 ± 7.2 nm in diameter) encapsulated in oxygen-rich microcapsules (3.6 ± 0.8 μm in diameter) that release their contents upon exposure to radiation were used in two radiotherapy sessions. We investigated primary tumor and metastasis imaging and macrophage-based therapy through silencing of “don’t eat me” signals and an increase in tumor-associated macrophages (TAMs) of M1 phenotype. For the first radiotherapy session, LPH-NPs, containing 5% iopamiron, and 250 nmol anti-CD47 small interfering RNA (siRNA) were mixed with 1.0 mL oxygen-rich solution (2200 ppm O2) containing 4.0% alginate, 3.0% hyaluronate, 1 mg ascorbate, and 1 μg/mL CD-40 and sprayed into 0.5 mmol/L FeCl2 supplemented with 1 μg/mL anti-vascular endothelial growth factor receptor (VEGFR)-1/2 antibody (Ab). The resultant microcapsules (1 × 1010) were intravenously (i.v.) administered into C57BL/6 mice with a primary MC38 tumor in the left hind leg and lung metastases, and their accumulation in the tumor was monitored by computed tomography (CT). Subsequently, the primary tumors and metastases were treated with 10 or 20 Gy 60Co γ-radiation. For the second session, microcapsules were generated by mixing LPH-NPs containing 200 μg anti-CD40 monoclonal Ab (moAb) and 900,000 IU interleukin (IL)-2, with the same oxygen-rich cocktail used in session one, and were sprayed into 0.5 mmol/L FeCl2 supplemented with 1 μg/mL anti-P-selectin Ab. They were then i.v. administered into the mice and allowed to interact with P-selectin for 9 h prior to a second radiotherapy with 10 or 20 Gy 60Co γ-radiation. In session one, the anti-VEGFR-1/2 microcapsules accumulated around the primary and metastatic tumors as detected by CT, and in response to the irradiation, they released anti-CD 47 siRNA LPH-NPs and oxygen-rich water, which silenced “don’t eat me” signals and increased tumor oxygen concentration, respectively. In session two, the microcapsules accumulated around the primary tumor through a P-selectin antigen-antibody reaction, and in response to the irradiation, they released anti-CD 40 moAb/IL-2 LPH-NPs and oxygen-rich water. Additionally, the radiation recruited TAMs in the tumors. The released anti-CD 40 moAb/IL-2 switched the phenotype of the recruited TAMs from M2 to M1, and the increased oxygen concentration due to the repeatedly released oxygen-rich water inhibited TAM repolarization from M1 to M2, leading to an increase in TAMs of M1 phenotype, which then attacked the tumors for which the “don’t eat me” signals were already silenced in session one. These treatments significantly increased the antitumor effect (EF 1.8) and reduced metastasis by 82.6%. Our CT-detectable microcapsules exhibited targeted macrophage therapy, improving tumor diagnosis and treatment.

Abstract 574: Phosphatidylserine targeting antibody in combination with tumor radiation and immune checkpoint blockade promotes anti-tumor activity in mouse B16 melanoma

Abstract Phosphatidylserine (PS) is a phospholipid that is exposed on surface of apoptotic cells, tumor cells and tumor endothelium. PS has been shown to promote immunosuppressive signals in the tumor microenvironment. Antibodies that target PS have been shown to reactivate anti-tumor immunity by polarizing tumor associated macrophages into a pro-inflammatory M1 phenotype, reducing the number of MDSCs in tumors and promoting the maturation of dendritic cells into functional APCs. In a mouse B16 melanoma model, targeting PS in combination with immune checkpoint blockade promoted greater anti-tumor activity than either agent alone. This combination was shown to enhance CD4+ and CD8+ T cell infiltration and activation in the tumors of treated animals. Radiation therapy (RT) is an effective focal treatment of primary solid tumors, but is less effective in treating metastatic solid tumors as a monotherapy. There is evidence that RT induces immunogenic tumor cell death and enhances tumor-specific T cell infiltration in treated tumors. The abscopal effect, a phenomenon in which tumor regression occurs outside the site of RT, has been observed in both preclinical and clinical trials when RT is combined with immunotherapy. In this study, we show that irradiation treatment of B16 melanoma causes an increase in PS expression on the surface of viable tumor and immune infiltrates. We subsequently examined the effects of combining RT with an antibody that targets PS (mch1N11) and immune checkpoint blockade (anti-PD-1) in B16 melanoma. We found that treatment with mch1N11 synergizes with RT to improve anti-tumor activity and overall survival in tumor bearing mice. In addition, the triple combination of mch1N11, RT and anti-PD-1 treatment displayed even greater anti-tumor and survival benefit. Analysis of local immune responses in the tumors of treated animals revealed an increase in tumor-associated macrophages with a shift towards a pro-inflammatory M1 phenotype after treatment with RT and mch1N11. In addition, analysis of the systemic immune responses in the spleen and tumor draining lymph nodes revealed an increase in CD8 T cell activation, effector cytokine production and differentiation into effector memory cells in the triple combination. This finding highlights the potential of combining these three agents to improve outcome in patients with advanced-stage melanoma and other cancers and may inform the design of clinical studies combining PS-targeting antibodies with RT and/or checkpoint blockade. Citation Format: Sadna Budhu, Olivier De Henau, Roberta Zappasodi, Rachel Giese, Luis F. Campesato, Christopher Barker, Bruce Freimark, Jeff Hutchins, Jedd D. Wolchok, Taha Merghoub. Phosphatidylserine targeting antibody in combination with tumor radiation and immune checkpoint blockade promotes anti-tumor activity in mouse B16 melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 574. doi:10.1158/1538-7445.AM2017-574

Telomere profiles and tumor-associated macrophages with different immune signatures affect prognosis in glioblastoma

Telomere maintenance is a hallmark of cancer and likely to be targeted in future treatments. In glioblastoma established methods of identifying telomerase and alternative lengthening of telomeres leave a significant proportion of tumors with no defined telomere maintenance mechanism. This study investigated the composition of these tumors using RNA-Seq. Glioblastomas with an indeterminate telomere maintenance mechanism had an increased immune signature compared with alternative lengthening of telomeres and telomerase-positive tumors. Immunohistochemistry for CD163 confirmed that the majority (80%) of tumors with an indeterminate telomere maintenance mechanism had a high presence of tumor-associated macrophages. The RNA-Seq and immunostaining data separated tumors with no defined telomere maintenance mechanism into three subgroups: alternative lengthening of telomeres like tumors with a high presence of tumor-associated macrophages and telomerase like tumors with a high presence of tumor-associated macrophages. The third subgroup had no increase in tumor-associated macrophages and may represent a distinct category. The presence of tumor-associated macrophages conferred a worse prognosis with reduced patient survival times (alternative lengthening of telomeres with and without macrophages P=0.0004, and telomerase with and without macrophages P=0.013). The immune signatures obtained from RNA-Seq were significantly different between telomere maintenance mechanisms. Alternative lengthening of telomeres like tumors with macrophages had increased expression of interferon-induced proteins with tetratricopeptide repeats (IFIT1–3). Telomerase-positive tumors with macrophages had increased expression of macrophage receptor with collagenous structure (MARCO), CXCL12 and sushi-repeat containing protein x-linked 2 (SRPX2). Telomerase-positive tumors with macrophages were also associated with a reduced frequency of total/near total resections (44% vs >76% for all other subtypes, P=0.014). In summary, different immune signatures are found among telomere maintenance mechanism-based subgroups in glioblastoma. The reduced extent of surgical resection of telomerase-positive tumors with macrophages suggests that some tumor-associated macrophages are more unfavorable.

Abstract B121: Low dose, metronomic cyclophosphamide therapy sensitizes tumors to CpG-1826 immunotherapy in a preclinical glioma model

Abstract Metronomic chemotherapy, in which cytotoxic drugs are delivered at low doses at regular, intermittent intervals, has shown promising clinical results in several cancers and offers the benefit of reduced patient toxicity compared to conventional maximum tolerated dose chemotherapy. We previously showed that cyclophosphamide (CPA) treatment on a 6-day repeating metronomic schedule regresses implanted gliomas by an immune-mediated mechanism. Here, we examine whether this immune-stimulatory metronomic chemotherapy can be enhanced by combination with TLR9-stimulating CpG-1826 immunotherapy in GL261 gliomas implanted in immunocompetent BL6 mice. CpG-1826 treatment activated an anti-tumor immune response in ~50% of mice, associated with a strong increase in tumor-associated macrophages. CpG-1826 was ineffective against GL261 tumors implanted in T cell- and B cell-deficient scid mice, suggesting that adaptive immunity is essential. Combination of CpG-1826 with metronomic CPA sensitized GL261 gliomas in both CpG-1826-responsive and CpG-1826-non-responsive mice, increasing infiltration of tumor-associated macrophages, dendritic cells, and T cells and increasing anti-tumor activity. Further, the combination treatment elicited long-term regression and immune memory in a subset of glioma-bearing mice. To identify potential mechanisms for this enhanced response to the combination therapy, we assayed a number of immune-related genes in the treated tumors, including feedback inhibitory immunosuppressive T cell factors that are induced by metronomic CPA treatment. Combination of CpG-1826 with metronomic CPA failed to induce several of these factors, suggesting that TLR9-based immunotherapy can enhance the anti-tumor activity of metronomic CPA by interdicting feedback immune inhibition mechanisms. Grant support: CA49248 (to DJW). Citation Format: Marie Jordan, David J. Waxman. Low dose, metronomic cyclophosphamide therapy sensitizes tumors to CpG-1826 immunotherapy in a preclinical glioma model. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B121.

Increase in tumor-associated macrophages after antiangiogenic therapy is associated with poor survival among patients with recurrent glioblastoma

Antiangiogenic therapy is associated with increased radiographic responses in glioblastomas, but tumors invariably recur. Because tumor-associated macrophages have been shown to mediate escape from antiangiogenic therapy in preclinical models, we examined the role of macrophages in patients with recurrent glioblastoma. We compared autopsy brain specimens from 20 patients with recurrent glioblastoma who received antiangiogenic treatment and chemoradiation with 8 patients who received chemotherapy and/or radiotherapy without antiangiogenic therapy or no treatment. Tumor-associated macrophages were morphologically and phenotypically analyzed using flow cytometry and immunohistochemistry for CD68, CD14, CD163, and CD11b expression. Flow cytometry showed an increase in macrophages in the antiangiogenic-treated patients. Immunohistochemical analysis demonstrated an increase in CD68+ macrophages in the tumor bulk (P < .01) and infiltrative areas (P = .02) in antiangiogenic-treated patients. We also observed an increase in CD11b+ cells in the tumor bulk (P < .01) and an increase in CD163+ macrophages in infiltrative tumor (P = .02). Of note, an increased number of CD11b+ cells in bulk and infiltrative tumors (P = .05 and P = .05, respectively) correlated with poor overall survival among patients who first received antiangiogenic therapy at recurrence. In summary, recurrent glioblastomas showed an increased infiltration in myeloid populations in the tumor bulk and in the infiltrative regions after antiangiogenic therapy. Higher numbers of CD11b+ cells correlated with poor survival among these patients. These data suggest that tumor-associated macrophages may participate in escape from antiangiogenic therapy and may represent a potential biomarker of resistance and a potential therapeutic target in recurrent glioblastoma.

Abstract LB-339: Increase in tumor-associated macrophages (TAMs) after antiangiogenic therapy is associated with poor survival in recurrent glioblastoma (GBM) patients.

Abstract Antiangiogenic therapy is associated with increased radiographic responses in glioblastomas (GBMs), but tumors invariably recur. Because tumor-associated macrophages (TAMs) have been shown to mediate escape from antiangiogenic therapy in preclinical models, we examined the role of TAMs in recurrent glioblastoma (rGBM) patients. We compared autopsy brain specimens from 20 rGBM patients who received antiangiogenic treatment and chemoradiation (AAT+) to 8 patients who received chemotherapy and/or radiotherapy without antiangiogenic therapy, or no treatment (AAT-). TAMs were morphologically and phenotypically analyzed using flow cytometry and immunohistochemistry (IHC) for CD68, CD14, CD163, and CD11b expression. Flow cytometry showed an increase in TAMs in the AAT+ patients. IHC analysis demonstrated an increase in CD68+ TAMs in the tumor bulk (p&amp;lt;0.01) and infiltrative areas (p=0.02) in AAT+ patients. We also observed an increase in CD11b+ cells in the tumor bulk (p&amp;lt;0.01) and an increase in CD163+ TAMs in infiltrative tumor (p=0.02). Of note, an increased number of CD11b+ cells in bulk and infiltrative tumor (p=0.05 and p=0.05, respectively) correlated with poor overall survival in patients who first received antiangiogenic therapy at recurrence. In summary, rGBMs showed an increased infiltration in myeloid populations in the tumor bulk and in the infiltrative regions after antiangiogenic therapy. Higher numbers of CD11b+ cells correlated with poor survival in rGBM patients. These data suggest that TAMs may participate in escape from antiangiogenic therapy and may represent a potential biomarker of resistance and a potential therapeutic target in rGBM. Citation Format: Christine Lu-Emerson, Matija Snuderl, Nathaniel D. Kirkpatrick, Jermaine Goveia, Jennie Taylor, Christian Davidson, Yuhui Huang, Lars Riedemann, S. Percy Ivy, G. Dan Duda, Marek Ancukiewicz3, Scott R. Plotkin, Andrew Chi, Elizabeth R. Gerstner, April F. Eichler, Jorg Dietrich, Anat O. Stemmer-Rachamimov, Tracy T. Batchelor, Rakesh K. Jain. Increase in tumor-associated macrophages (TAMs) after antiangiogenic therapy is associated with poor survival in recurrent glioblastoma (GBM) patients. [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 LB-339. doi:10.1158/1538-7445.AM2013-LB-339