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Supplementary file containing Supplementary Methods (pertaining to reagents, imaging, immunoblotting, cellular studies, microarray data, NF-κB ELISA, PCR, cell culture, histology, cytology, flow cytometry, constructs, and cellular Assays), 4 Supplementary Tables (pertaining to number of experimental mice, PCR primers, antibodies, and lentiviral shRNA pools), and 7 Supplementary Figures (described in the main and Supplementary text).

Although oncogenic activation of NFκB has been identified in various tumors, the NFκB–activating kinases (inhibitor of NFκB kinases, IKK) responsible for this are elusive. In this study, we determined the role of IKKα and IKKβ in KRAS-mutant lung adenocarcinomas induced by the carcinogen urethane and by respiratory epithelial expression of oncogenic KRASG12D. Using NFκB reporter mice and conditional deletions of IKKα and IKKβ, we identified two distinct early and late activation phases of NFκB during chemical and genetic lung adenocarcinoma development, which were characterized by nuclear translocation of RelB, IκBβ, and IKKα in tumor-initiated cells. IKKα was a cardinal tumor promoter in chemical and genetic KRAS-mutant lung adenocarcinoma, and respiratory epithelial IKKα-deficient mice were markedly protected from the disease. IKKα specifically cooperated with mutant KRAS for tumor induction in a cell-autonomous fashion, providing mutant cells with a survival advantage in vitro and in vivo. IKKα was highly expressed in human lung adenocarcinoma, and a heat shock protein 90 inhibitor that blocks IKK function delivered superior effects against KRAS-mutant lung adenocarcinoma compared with a specific IKKβ inhibitor. These results demonstrate an actionable requirement for IKKα in KRAS-mutant lung adenocarcinoma, marking the kinase as a therapeutic target against this disease.Significance: These findings report a novel requirement for IKKα in mutant KRAS lung tumor formation, with potential therapeutic applications. Cancer Res; 78(11); 2939–51. ©2018 AACR.

Supplementary Methods

Figure S2. Deficiency in IL-5 does not affect infiltration of lungs with T and B lymphocytes and NK cells during initial stages of metastatic load of LLC cells.

Figure S1: Myeloid Ikkβ is required for anti-tumorigenic immunity. Figure S2. Myeloid cell depletion alters tumor immunity. Figure S3. Ikkβ loss alters macrophage phagocytosis capacity. Figure S4. Myeloid Ikkβ deletion reduced the cytotoxicity of T cells Figure S5. Cytokines play a major role in orchestrating the cellular make-up of the tumor microenvironment.

Supplementary Figure 1 from The Nuclear Factor-κB Pathway Controls the Progression of Prostate Cancer to Androgen-Independent Growth

Typically, the initial response of a prostate cancer patient to androgen ablation therapy is regression of the disease. However, the tumor will progress to an “androgen-independent” stage that results in renewed growth and spread of the cancer. Both nuclear factor-κB (NF-κB) expression and neuroendocrine differentiation predict poor prognosis, but their precise contribution to prostate cancer progression is unknown. This report shows that secretory proteins from neuroendocrine cells will activate the NF-κB pathway in LNCaP cells, resulting in increased levels of active androgen receptor (AR). By blocking NF-κB signaling in vitro, AR activation is inhibited. In addition, the continuous activation of NF-κB signaling in vivo by the absence of the IκBα inhibitor prevents regression of the prostate after castration by sustaining high levels of nuclear AR and maintaining differentiated function and continued proliferation of the epithelium. Furthermore, the NF-κB pathway was activated in the ARR2PB-myc-PAI (Hi-myc) mouse prostate by cross-breeding into a IκBα+/− haploid insufficient line. After castration, the mouse prostate cancer continued to proliferate. These results indicate that activation of NF-κB is sufficient to maintain androgen-independent growth of prostate and prostate cancer by regulating AR action. Thus, the NF-κB pathway may be a potential target for therapy against androgen-independent prostate cancer. [Cancer Res 2008;68(16):6762–9]

Figure S1. Lung eosinophils (Eos), identified as CD45+/CD11c-/ F4/80+/siglec-F+ cells using flow cytometry are A) negative for CD68, B) express high levels of CCR3, and C) express low levels of Gr1 compared to the CD45+/Gr1hi neutrophils (PMN).

In many patients with prostate cancer, the cancer will be recurrent and eventually progress to lethal metastatic disease after primary treatment, such as surgery or radiation therapy. Therefore, it would be beneficial to better predict which patients with early-stage prostate cancer would progress or recur after primary definitive treatment. In addition, many studies indicate that activation of NF-κB signaling correlates with prostate cancer progression; however, the precise underlying mechanism is not fully understood. Our studies show that activation of NF-κB signaling via deletion of one allele of its inhibitor, IκBα, did not induce prostatic tumorigenesis in our mouse model. However, activation of NF-κB signaling did increase the rate of tumor progression in the Hi-Myc mouse prostate cancer model when compared with Hi-Myc alone. Using the nonmalignant NF-κB–activated androgen-depleted mouse prostate, a NF-κB–activated recurrence predictor 21 (NARP21) gene signature was generated. The NARP21 signature successfully predicted disease-specific survival and distant metastases-free survival in patients with prostate cancer. This transgenic mouse model–derived gene signature provides a useful and unique molecular profile for human prostate cancer prognosis, which could be used on a prostatic biopsy to predict indolent versus aggressive behavior of the cancer after surgery. Cancer Res; 74(10); 2763–72. ©2014 AACR.

PDF file - 453KB, Supplementary Figure 1. NF-kappaB signaling is continuously activated in the prostate of IkappaBalpha+/- mouse. Supplementary Figure 2. NF-kappaB signaling activated in the prostate of Myc/IkappaBalpha bigenic mouse. Supplementary Figure 3. Continuous activation of NF-kappaB signaling promotes PCa progression in the Hi-Myc transgenic mouse. Supplementary Figure 4. The NF24 gene signature predicts significant differences in the overall cancer-specific survival of the PCa patients. Supplemental Figure 5. Molecular network analysis using Ingenuity Pathway Analysis (IPA). Supplemental Figure 6. NF-kappaB signaling is activated/inactivated in PCa cells by infecting with IKK2-EE or IKK2-KD retroviral vectors. Supplemental Figure 7. Activation of NF-kappaB signaling increases JNK phosphorylation in PCa cells. Supplemental Figure 8. Blocking JNK signaling inhibits NF-kappaB induced invasive ability efficiently in PCa cells. Tables 1 and 2. Matched human homolog genes. Supplementary Table 3. Stratification of 77 PCa patients.

Tumor-associated macrophages (TAMs) represent more than 50% of the immune cells in breast tumors, even in immune dysregulated, ‘cold’ tumors. TAMs that express an M2-like phenotype promote tumor growth and create an immunosuppressive tumor microenvironment (TME). High levels of M2-like TAMs correlate with a poor prognosis. However, macrophages display phenotypic plasticity and can be repolarized from one phenotype to another through signaling molecules, such as cytokines. Macrophages that express an M1-like phenotype are inflammatory and can induce anti-tumor immunity. Administration of pro-inflammatory cytokines reprogram M2 TAMs toward an M1-like phenotype that can reduce tumor growth and stimulate an inflammatory response within the TME. However, systemic delivery of these inflammatory cytokines generates toxic, dose-limiting off target effects. This work seeks to develop a localized delivery approach that enables TAM polarization toward M1 programs while abrogating systemic toxicities. We have developed an injectable alginate cryogel (hydrogel fabricated at -20oC) to act as localized delivery depot for inflammatory cytokines and macrophage-specific chemokines to repolarize TAMs. To delay the release of our chemoattractant relative to our repolarizing cytokines, we are developing a second generation cryogel, loaded with free chemokines and poly(lactic-co-glycolic acid) (PLGA) nanoparticles encapsulated with cytokines to delay their release. With our first generation cryogel (without nanoparticles), we used qRT-PCR, flow cytometry, Luminex panels, and single cell RNAseq to characterize the impact of treatment. For in vitro studies, bone marrow derived macrophages were extracted from FVB mice, while TAMs and tumor explants were extracted from the spontaneous mammary tumors of PyMT mice. It was found that our cryogel significantly increased M1 markers and decreased M2 markers in all three in vitro models.Peritumoral injection of the cryogel system into FVB female mice with orthotopic mammary tumors resulted in significantly suppressed tumor growth, an increase in T cell infiltration, and an increase in the M1:M2 ratio of TAMs. This activated immune response and elevated presence of T cells suggests that localized chemokine/cytokine treatment ‘primes’ the TME, potentially making it more susceptible to immunotherapies that rely on T cells, such as immune checkpoint blockade.We have concluded that our first-generation gel demonstrates the potential of priming the TME to slow tumor growth. Characterization of our second generation, nano-in-cryogel system is currently underway. Based on mathematical modeling, we hypothesize that the nano-in-cryogel will induce a more pronounced repolarization effect by allowing the chemoattractant to induce M2 TAM migration prior to the release of the inflammatory, reprogramming cytokines from the PLGA nanoparticles. Citation Format: Sydney Ros Henriques, Ori Z. Chalom, Evan B. Glass, Sohini Roy, Abigail E. Manning, Benjamin C. Hacker, Marjan Rafat, Laura C. Kennedy, Fiona E. Yull, Young J. Kim, Todd D. Giorgio. Tumor-associated macrophage reprogramming by novel nano-in-cryogel biomaterials for breast cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2719.

Neural circuits between lesions are one mechanism through which local inflammation spreads to remote positions. Here, we show the inflammatory signal on one side of the joint is spread to the other side via sensory neuron–interneuron crosstalk, with ATP at the core. Surgical ablation or pharmacological inhibition of this neural pathway prevented inflammation development on the other side. Mechanistic analysis showed that ATP serves as both a neurotransmitter and an inflammation enhancer, thus acting as an intermediary between the local inflammation and neural pathway that induces inflammation on the other side. These results suggest blockade of this neural pathway, which is named the remote inflammation gateway reflex, may have therapeutic value for inflammatory diseases, particularly those, such as rheumatoid arthritis, in which inflammation spreads to remote positions.

Background New treatment options for ovarian cancer are urgently required. Tumor-associated macrophages (TAMs) are an attractive target for therapy; repolarizing TAMs from M2 (pro-tumor) to M1 (anti-tumor) phenotypes represents an important therapeutic goal. We have previously shown that upregulated NF-kappaB (NF-κB) signaling in macrophages promotes M1 polarization, but effects in the context of ovarian cancer are unknown. Therefore, we aimed to investigate the therapeutic potential of increasing macrophage NF-κB activity in immunocompetent mouse models of ovarian cancer. Methods We have generated a transgenic mouse model, termed IKFM, which allows doxycycline-inducible overexpression of a constitutively active form of IKK2 (cIKK2) specifically within macrophages. The IKFM model was used to evaluate effects of increasing macrophage NF-κB activity in syngeneic murine TBR5 and ID8-Luc models of ovarian cancer in two temporal windows: 1) in established tumors, and 2) during tumor implantation and early tumor growth. Tumor weight, ascites volume, ascites supernatant and cells, and solid tumor were collected at sacrifice. Populations of macrophages and T cells within solid tumor and/or ascites were analyzed by immunofluorescent staining and qPCR, and soluble factors in ascitic fluid were analyzed by ELISA. Comparisons of control versus IKFM groups were performed by 2-tailed Mann-Whitney test, and a P-value Results Increased expression of the cIKK2 transgene in TAMs from IKFM mice was confirmed at the mRNA and protein levels. Tumors from IKFM mice, regardless of the timing of doxycycline (dox) administration, demonstrated greater necrosis and immune infiltration than control tumors. Analysis of IKFM ascites and tumors showed sustained shifts in macrophage populations away from the M2 and towards the anti-tumor M1 phenotype. There were also increased tumor-infiltrating CD3+/CD8+ T cells in IKFM mice, accompanied by higher levels of CXCL9, a T cell activating factor secreted by macrophages, in IKFM ascitic fluid. Conclusions In syngeneic ovarian cancer models, increased canonical NF-κB signaling in macrophages promoted anti-tumor TAM phenotypes and increased cytotoxic T cell infiltration, which was sufficient to limit tumor progression. This may present a novel translational approach for ovarian cancer treatment, with the potential to increase responses to T cell-directed therapy in future studies.

NF-κB is a reduction-oxidation–sensitive transcription factor that plays a key role in regulating the immune response. In these studies, we intended to investigate the role of mitochondrial-derived reactive oxygen species in regulating NF-κB activation by studying transgenic mice that overexpress mitochondrial-targeted human catalase (mCAT). We treated wild-type (WT) and mCAT mice with intratracheal instillation of Escherichia coli LPS and found that mCAT mice had exaggerated NF-κB activation in the lungs, increased neutrophilic alveolitis, and greater lung inflammation/injury compared with WT mice. Additional studies using bone marrow chimeras revealed that this hyperinflammatory phenotype was mediated by immune/inflammatory cells. Mechanistic studies using bone marrow–derived macrophages (BMDMs) showed that LPS treatment induced a sustained increase in NF-κB activation and expression of NF-κB–dependent inflammatory mediators in mCAT BMDMs compared with WT BMDMs. Further investigations showed that cytoplasmic, but not mitochondrial, hydrogen peroxide levels were reduced in LPS-treated mCAT BMDMs. However, mCAT macrophages exhibited increased glycolytic and oxidative metabolism, coupled with increased ATP production and an increased intracellular NADH/NAD+ ratio compared with BMDMs from WT mice. Treatment of BMDMs with lactate increased the intracellular NADH/NAD+ ratio and upregulated NF-κB activation after LPS treatment, whereas treatment with a potent inhibitor of the mitochondrial pyruvate carrier (UK5099) decreased the NADH/NAD+ ratio and reduced NF-κB activation. Taken together, these findings point to an increased availability of reducing equivalents in the form of NADH as an important mechanism by which metabolic activity modulates inflammatory signaling through the NF-κB pathway.

Kirsten rat sarcoma virus (KRAS)-mutant cancers are frequent, metastatic, lethal, and largely undruggable. While interleukin (IL)-1β and nuclear factor (NF)-κB inhibition hold promise against cancer, untargeted treatments are not effective. Here, we show that human KRAS-mutant cancers are addicted to IL-1β via inflammatory versican signaling to macrophage inhibitor of NF-κB kinase (IKK) β. Human pan-cancer and experimental NF-κB reporter, transcriptome, and proteome screens reveal that KRAS-mutant tumors trigger macrophage IKKβ activation and IL-1β release via secretory versican. Tumor-specific versican silencing and macrophage-restricted IKKβ deletion prevents myeloid NF-κB activation and metastasis. Versican and IKKβ are mutually addicted and/or overexpressed in human cancers and possess diagnostic and prognostic power. Non-oncogene KRAS/IL-1β addiction is abolished by IL-1β and TLR1/2 inhibition, indicating cardinal and actionable roles for versican and IKKβ in metastasis.

Background Current cancer immunotherapies have made tremendous impacts but generally lack high response rates, especially in ovarian cancer. New therapies are needed to provide increased benefits. One understudied approach is to target the large population of immunosuppressive tumor-associated macrophages (TAMs). Using inducible transgenic mice, we recently reported that upregulating nuclear factor-kappaB (NF-κB) signaling in TAMs promotes the M1, anti-tumor phenotype and limits ovarian cancer progression. We also developed a mannose-decorated polymeric nanoparticle system (MnNPs) to preferentially deliver siRNA payloads to M2, pro-tumor macrophages in vitro. In this study, we tested a translational strategy to repolarize ovarian TAMs via MnNPs loaded with siRNA targeting the inhibitor of NF-κB alpha (IκBα) using mouse models of ovarian cancer. Methods We evaluated treatment with MnNPs loaded with IκBα siRNA (IκBα-MnNPs) or scrambled siRNA in syngeneic ovarian cancer models. ID8 tumors in C57Bl/6 mice were used to evaluate consecutive-day treatment of late-stage disease while TBR5 tumors in FVB mice were used to evaluate repetitive treatments in a faster-developing disease model. MnNPs were evaluated for biodistribution and therapeutic efficacy in both models. Results Stimulation of NF-κB activity and repolarization to an M1 phenotype via IκBα-MnNP treatment was confirmed using cultured luciferase-reporter macrophages. Delivery of MnNPs with fluorescent payloads (Cy5-MnNPs) to macrophages in the solid tumors and ascites was confirmed in both tumor models. A three consecutive-day treatment of IκBα-MnNPs in the ID8 model validated a shift towards M1 macrophage polarization in vivo. A clear therapeutic effect was observed with biweekly treatments over 2-3 weeks in the TBR5 model where significantly reduced tumor burden was accompanied by changes in immune cell composition, indicative of reduced immunosuppressive tumor microenvironment. No evidence of toxicity associated with MnNP treatment was observed in either model. Conclusions In mouse models of ovarian cancer, MnNPs were preferentially associated with macrophages in ascites fluid and solid tumors. Evidence of macrophage repolarization, increased inflammatory cues, and reduced tumor burden in IκBα-MnNP-treated mice indicate beneficial outcomes in models of established disease. We have provided evidence of a targeted, TAM-directed approach to increase anti-tumor immunity in ovarian cancer with strong translational potential for future clinical studies.

The mechanisms tumor cells use to hijack the immune system are largely uncharted. Here we used bioluminescent nuclear factor (NF)-κB reporter mice and macrophages to discover that metastatic tumors trigger NF-κB activation in host macrophages, dependent on mutant KRAS signaling and delivered via secretory versican. Versican activates NF-κB in tumor-associated macrophages via inhibitor of NF-κB kinase (IKK) β, resulting in release of interleukin (IL)-1β into the tumor microenvironment. Versican silencing in cancer cells or conditional IKKβ deletion in macrophages prevents myeloid NF-κB activation and metastasis. Versican is overexpressed and/or mutated in human cancers and metastatic effusions with KRAS mutations, predicts poor survival, can aid in the development of diagnostic platforms for pleural metastasis, and is druggable via toll-like receptor (TLR) 1/2 inhibition. The data indicate a cardinal role for tumor-derived versican in establishing cross-talk with macrophage IKKβ during metastasis and may foster the development of new therapies and diagnostic tools.

Introduction While individual-level measures of socioeconomic status have been well-studied in relation to ovarian cancer survival, no studies to date have examined both state and national-level Area Deprivation Indices (ADIs), which incorporate neighborhood affluence and resources. Methods We abstracted clinical data from medical records for ovarian cancer cases from the Vanderbilt University Medical Center and obtained ADIs from the Neighborhood Atlas®. Associations with clinical characteristics were assessed with Spearman correlations and Kruskal-Wallis tests; associations with progression-free survival (PFS) and overall survival (OS) were assessed with Cox proportional-hazards regression. Results Among 184 cases, state and national ADIs were highly correlated, but not related to any cancer characteristics. In multivariable adjusted regression models, both were significantly associated with OS; each decile increase in state or national ADI corresponded to a 9 % or 10 % greater risk of death, respectively. Conclusions Increasing area-level deprivation may negatively impact ovarian cancer survival.

Objectives: Identifying new strategies to improve response to poly ADP-ribose polymerase inhibitors (PARPi) in women with homologous recombination (HR)-proficient ovarian cancer is a key clinical challenge. The nuclear orphan receptor NR4A1 has pro-tumor effects in ovarian cancer cells through transcriptional regulation of key genes promoting growth, survival and HR DNA repair. NR4A1 is also expressed in immune cells in the tumor microenvironment and may play a role in inhibiting re-polarization of pro-tumor M2-like tumor-associated macrophages (TAMs) to a more effective anti-tumor M1-like TAM phenotype. Our group has shown that the NF-kappaB (NF-ĸB) pathway is a key regulator of TAM phenotype, with upregulated NF-ĸB signaling promoting M1-like functions. Our goal therefore was to determine whether inhibition of NR4A1 can improve responses to PARPi both by increased tumor DNA damage and apoptosis, and by promoting an anti-tumor M1 phenotype in TAMs by modulating NF-ĸB activity. Methods: Cultured HR-proficient ID8 mouse ovarian cancer cells, mouse ovarian TAMs and mouse bone marrow-derived macrophages were treated with the established NR4A1 antagonist C-DIM-pPHOH (C-DIM) and/or the PARPi olaparib (OLA) for 24-72 hours. C57BL/6 mice with established intra-peritoneal ID8 tumors were treated with vehicle, C-DIM, OLA or combined C-DIM/OLA for 3 weeks. Standard techniques were used to determine cell growth (Sulforhodamine B assay), HR efficiency (BRCA1 and RAD51 foci formation), NF-ĸB activity (nuclear p65, luciferase reporter activity), and expression of markers of DNA damage (pH2AX), apoptosis (cleaved PARP), proliferation (PCNA), and macrophage M1 (CCL3, iNOS) and M2 (CD206, arginase-1) polarization. Statistical significance (p Download : Download high-res image (260KB) Download : Download full-size image Results: In ID8 cells, co-treatment with C-DIM and OLA significantly reduced cell growth and HR efficiency, and increased DNA-damage-induced apoptosis, compared to OLA treatment alone. NR4A1 was highly expressed in macrophages, and C-DIM increased NF-ĸB activity and induced M1 polarization alone and when combined with olaparib. Similar effects of combined treatment were observed in vivo. Compared to mice treated with OLA alone, there was significantly reduced ascites volume and omental tumor mass in C-DIM/OLA mice at the time of sacrifice. Combined treatment also reduced proliferation and increased DNA damage and apoptosis in harvested tumors, and increased M1 polarization of TAMs in ascites fluid, compared to OLA mice. Conclusions: NR4A1 inhibition sensitizes ovarian tumors to PARPi though direct effects on tumors by targeting the HR pathway, and by modulating TAM function towards an anti-tumor phenotype. These findings have important implications for expanding the use of PARPi in women with HR-proficient ovarian tumors, which would benefit a substantial number of women with this devastating disease.

Background The canonical and non-canonical nuclear factor-kappaB (NF-κB) signaling pathways have key roles in cancer, but studies have previously evaluated only the association of canonical transcription factors and ovarian cancer survival. Although a number of in vitro and in vivo studies have demonstrated mechanisms by which non-canonical NF-κB signaling potentially contributes to ovarian cancer progression, a prognostic association has yet to be shown in the clinical context. Methods We assayed p65 and p52 (major components of the canonical and non-canonical NF-κB pathways) by immunohistochemistry in epithelial ovarian tumor samples; nuclear and cytoplasmic staining were semi-quantified by H-scores and dichotomized at median values. Associations of p65 and p52 with progression-free survival (PFS) and overall survival (OS) were quantified by Hazard Ratios (HR) from proportional-hazards regression. Results Among 196 cases, median p52 and p65 H-scores were higher in high-grade serous cancers. Multivariable regression models indicated that higher p52 was associated with higher hazards of disease progression (cytoplasmic HR: 1.54; nuclear HR: 1.67) and death (cytoplasmic HR: 1.53; nuclear HR: 1.49), while higher nuclear p65 was associated with only a higher hazard of disease progression (HR: 1.40) in unadjusted models. When cytoplasmic and nuclear staining were combined, p52 remained significantly associated with increased hazards of disease progression (HR: 1.91, p = 0.004) and death (HR: 1.70, p = 0.021), even after adjustment for p65 and in analyses among only high-grade serous tumors. Conclusions This is the first study to demonstrate that p52, a major component of non-canonical NF-κB signaling, may be an independent prognostic factor for epithelial ovarian cancer, particularly high-grade serous ovarian cancer. Approaches to inhibit non-canonical NF-κB signaling should be explored as novel ovarian cancer therapies are needed.

Tumor associated macrophages (TAMs) often exist in high densities in breast tumors and are associated with adverse clinical outcomes. TAM repolarization as a strategy to shift the breast cancer microenvironment from immunosuppressive to immunostimulatory is an active area of research. The canonical and non-canonical NfkappaB (NFkB) signaling pathways represent potential points of intervention to influence TAM phenotypes. We have found that upregulation of the canonical NFkB signaling pathway specifically in macrophages can be used to repolarize TAMs toward an M1, anti-tumor phenotype. Using our doxycycline inducible mouse model termed IKFM, we are exploring the effects of macrophage specific upregulation of NFkB signaling on different stages of tumor growth and metastasis. In studies using the IKFM mouse model, we found that doxycycline-induced NFkB activation in macrophages in established orthotopic mammary tumors has therapeutic effects on tumor outcomes. Compared to control mice, we found that IKFM mice have reduced tumor burden in the lung, indicating prevention of metastasis. Ongoing studies are focused on understanding how macrophage specific activation of NFkB influences the metastatic niches of lung and bone; two of the most common sites of breast cancer metastasis. To move our findings towards clinical translation, we have developed an M2-macrophage targeted nanoparticle that activates NFkB signaling by delivering an siRNA against IkappaBalpha, an inhibitor of the pathway. The M2 targeted nanoparticle is currently being tested in murine models of breast cancer metastasis to both lung and bone to translate our findings from the IKFM mouse model to a pharmacological manipulation. Citation Format: Kennady K. Bullock, Dominique Parker, Evan B. Glass, Alyssa Hoover, Alyssa Merkel, Richard Maynard, Andrew Wilson, Todd Giorgio, Julie A. Rhoades, Fiona E. Yull. Modulation of NFkappaB signaling influences tumor associated macrophage phenotypes in murine models of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2728.

"Smart", dual pH-responsive, and endosomolytic polymeric nanoparticles have demonstrated great potential for localized drug delivery, especially for siRNA delivery to the cytoplasm of cells. However, targeted delivery to a specific cell phenotype requires an additional level of functionality. Copper-catalyzed azide-alkyne cycloaddition (CuAAC) is a highly selective bioconjugation reaction that can be performed in conjunction with other polymerization techniques without adversely affecting reaction kinetics, but there exists some concern for residual copper causing cytotoxicity. To alleviate these concerns, we evaluated conjugation efficiency, residual copper content, and cell viability in relation to copper catalyst concentration. Our results demonstrated an optimal range for minimizing cytotoxicity while maintaining high levels of conjugation efficiency, and these conditions produced polymers with increased targeting to M2-polarized macrophages, as well as successful delivery of therapeutic siRNA that reprogrammed the macrophages to a proinflammatory phenotype.

Ryan A Ortega,1–3 Whitney Barham,3 Kavya Sharman,4 Oleg Tikhomirov,3 Todd D Giorgio,1–3 Fiona E Yull3 1Department of Biomedical Engineering, Vanderbilt University, 2Vanderbilt Institute for Nanoscale Science and Engineering, 3Department of Cancer Biology, Vanderbilt-Ingram Cancer Center, 4Department of Neuroscience, Vanderbilt University, Nashville, TN, USA Abstract: Tumor-associated macrophages (TAMs) are critically important in the context of solid tumor progression. Counterintuitively, these host immune cells can often support tumor cells along the path from primary tumor to metastatic colonization and growth. Thus, the ability to transform protumor TAMs into antitumor, immune-reactive macrophages would have significant therapeutic potential. However, in order to achieve these effects, two major hurdles would need to be overcome: development of a methodology to specifically target macrophages and increased knowledge of the optimal targets for cell-signaling modulation. This study addresses both of these obstacles and furthers the development of a therapeutic agent based on this strategy. Using ex vivo macrophages in culture, the efficacy of mannosylated nanoparticles to deliver small interfering RNA specifically to TAMs and modify signaling pathways is characterized. Then, selective small interfering RNA delivery is tested for the ability to inhibit gene targets within the canonical or alternative nuclear factor-kappaB pathways and result in antitumor phenotypes. Results confirm that the mannosylated nanoparticle approach can be used to modulate signaling within macrophages. We also identify appropriate gene targets in critical regulatory pathways. These findings represent an important advance toward the development of a novel cancer therapy that would minimize side effects because of the targeted nature of the intervention and that has rapid translational potential. Keywords: nanotechnology, targeted nanoparticles, cancer immunology, RNAi

Although numerous studies have demonstrated a critical role for canonical NF-κB signaling in inflammation and disease, the function of the noncanonical NF-κB pathway remains ill-defined. In lung tissue from patients with acute respiratory distress syndrome, we identified increased expression of the noncanonical pathway component p100/p52. To investigate the effects of p52 expression in vivo, we generated a novel transgenic mouse model with inducible expression of p52 in Clara cell secretory protein–expressing airway epithelial cells. Although p52 overexpression alone did not cause significant inflammation, p52 overexpression caused increased lung inflammation, injury, and mortality following intratracheal delivery of Escherichia coli LPS. No differences in cytokine/chemokine expression were measured between p52-overexpressing mice and controls, but increased apoptosis of Clara cell secretory protein–positive airway epithelial cells was observed in transgenic mice after LPS stimulation. In vitro studies in lung epithelial cells showed that p52 overexpression reduced cell survival and increased the expression of several proapoptotic genes during cellular stress. Collectively, these studies demonstrate a novel role for p52 in cell survival/apoptosis of airway epithelial cells and implicate noncanonical NF-κB signaling in the pathogenesis of acute respiratory distress syndrome.

Objective: Mediated by a family of transcription factors, the canonical and noncanonical nuclear factor-kappaB (NF-kB) signaling pathways have key roles in inflammation, innate immunity, and cancer development and progression. As prior ovarian cancer studies have evaluated only canonical NF-kB transcription factors in relation to survival, this study was undertaken to evaluate both canonical and noncanonical NF-kB pathway components in tumor samples from epithelial ovarian cancer cases from the Vanderbilt Tissue Repository for Ovarian Cancer (TROC). Methods: We abstracted clinical data from electronic medical records and assayed p65 (representing the canonical NF-kB pathway) and p52 (representing the noncanonical NF-kB pathway) by immunohistochemistry for 197 TROC samples. Nuclear and cytoplasmic staining in tumor cells was semiquantified by H-scores and dichotomized at median values. Associations with progression-free survival (PFS) and overall survival (OS) were quantified by hazard ratios (HR) and 95% confidence intervals (CI) using Cox proportional-hazards regression. Results: Median cytoplasmic and nuclear p65 and p52 H-scores were significantly higher among high-grade and serous cases; median p52 H-scores were also significantly higher among late-stage cases and patients treated with chemotherapy (all Wilcoxon rank sum p Conclusions: This is the first study to demonstrate that p52, representing noncanonical NF-kB signaling, may be an independent prognostic factor for epithelial ovarian cancer. Approaches to inhibit noncanonical NF-kB signaling should be explored as novel therapies for ovarian cancer are needed. Citation Format: Demetra Hufnagel, Andrew J. Wilson, Jamie Saxon, Dineo Khabele, Timothy Blackwell, Marta A. Crispens, Fiona Yull, Alicia Beeghly-Fadiel. Noncanonical NF-kappaB signaling is associated with poor ovarian cancer prognosis [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A15.

Ovarian cancer, a rare but fatal disease, has been a challenging area in the field of gynecological cancer. Ovarian cancer is characterized by peritoneal metastasis, which is facilitated by a cross-talk between tumor cells and other cells in the tumor microenvironment (TME). In epithelial ovarian cancer, tumor-associated macrophages (TAMs) constitute over 50% of cells in the peritoneal TME and malignant ascites, and are potential targets for therapy. Here, we review the bipolar nature of TAMs and the evolving strategies to target TAMs in ovarian cancer.

While many studies have demonstrated that canonical NF-κB signaling is a central pathway in lung tumorigenesis, the role of non-canonical NF-κB signaling in lung cancer remains undefined. We observed frequent nuclear accumulation of the non-canonical NF-κB component p100/p52 in human lung adenocarcinoma. To investigate the impact of non-canonical NF-κB signaling on lung carcinogenesis, we employed transgenic mice with doxycycline-inducible expression of p52 in airway epithelial cells. p52 over-expression led to increased tumor number and progression after injection of the carcinogen urethane. Gene expression analysis of lungs from transgenic mice combined with in vitro studies suggested that p52 promotes proliferation of lung epithelial cells through regulation of cell cycle-associated genes. Using gene expression and patient information from The Cancer Genome Atlas (TCGA) database, we found that expression of p52-associated genes was increased in lung adenocarcinomas and correlated with reduced survival, even in early stage disease. Analysis of p52-associated gene expression in additional human lung adenocarcinoma datasets corroborated these findings. Together, these studies implicate the non-canonical NF-κB component p52 in lung carcinogenesis and suggest modulation of p52 activity and/or downstream mediators as new therapeutic targets.

Although oncogenic activation of NFκB has been identified in various tumors, the NFκB–activating kinases (inhibitor of NFκB kinases, IKK) responsible for this are elusive. In this study, we determined the role of IKKα and IKKβ in KRAS-mutant lung adenocarcinomas induced by the carcinogen urethane and by respiratory epithelial expression of oncogenic KRASG12D. Using NFκB reporter mice and conditional deletions of IKKα and IKKβ, we identified two distinct early and late activation phases of NFκB during chemical and genetic lung adenocarcinoma development, which were characterized by nuclear translocation of RelB, IκBβ, and IKKα in tumor-initiated cells. IKKα was a cardinal tumor promoter in chemical and genetic KRAS-mutant lung adenocarcinoma, and respiratory epithelial IKKα-deficient mice were markedly protected from the disease. IKKα specifically cooperated with mutant KRAS for tumor induction in a cell-autonomous fashion, providing mutant cells with a survival advantage in vitro and in vivo. IKKα was highly expressed in human lung adenocarcinoma, and a heat shock protein 90 inhibitor that blocks IKK function delivered superior effects against KRAS-mutant lung adenocarcinoma compared with a specific IKKβ inhibitor. These results demonstrate an actionable requirement for IKKα in KRAS-mutant lung adenocarcinoma, marking the kinase as a therapeutic target against this disease. Significance: These findings report a novel requirement for IKKα in mutant KRAS lung tumor formation, with potential therapeutic applications. Cancer Res; 78(11); 2939–51. ©2018 AACR.

Characterization of markers that identify activated macrophages could advance understanding of inflammatory lung diseases and facilitate development of novel methodologies for monitoring disease activity. We investigated whether folate receptor β (FRβ) expression could be used to identify and quantify activated macrophages in the lungs during acute inflammation induced by Escherichia coli LPS. We found that FRβ expression was markedly increased in lung macrophages at 48 hours after intratracheal LPS. In vivo molecular imaging with a fluorescent probe (cyanine 5 polyethylene glycol folate) showed that the fluorescence signal over the chest peaked at 48 hours after intratracheal LPS and was markedly attenuated after depletion of macrophages. Using flow cytometry, we identified the cells responsible for uptake of cyanine 5-conjugated folate as FRβ(+) interstitial macrophages and pulmonary monocytes, which coexpressed markers associated with an M1 proinflammatory macrophage phenotype. These findings were confirmed using a second model of acute lung inflammation generated by inducible transgenic expression of an NF-κB activator in airway epithelium. Using CC chemokine receptor 2-deficient mice, we found that FRβ(+) macrophage/monocyte recruitment was dependent on the monocyte chemotactic protein-1/CC chemokine receptor 2 pathway. Together, our results demonstrate that folate-based molecular imaging can be used as a noninvasive approach to detect classically activated monocytes/macrophages recruited to the lungs during acute inflammation.

Objective Macrophage accumulation in adipose tissue (AT) during obesity contributes to inflammation and insulin resistance. Recruitment of monocytes to obese AT has been the most studied mechanism explaining this accumulation. However, recent evidence suggests that recruitment-independent mechanisms may also regulate pro-inflammatory AT macrophage (ATM) numbers. The role of increased ATM survival during obesity has yet to be explored. Results We demonstrate that activation of apoptotic pathways is significantly reduced in ATMs from diet-induced and genetically obese mice. Concurrently, pro-survival Bcl-2 family member protein levels and localization to the mitochondria is elevated in ATMs from obese mice. This increased pro-survival signaling was associated with elevated activation of the transcription factor, NF-κB, and increased expression of its pro-survival target genes. Finally, an obesogenic milieu increased ATM viability only when NF-κB signaling pathways were functional. Conclusions Our data demonstrate that obesity promotes survival of inflammatory ATMs, possibly through an NF-κB-regulated mechanism., Graphical abstract, Highlights • Macrophage apoptosis is decreased in obese adipose tissue. • ATMs from obese mice display increased mitochondrial localization of Bcl-2. • The pro-survival targets of NF-κB are increased in ATMs from obese mice. • NF-κB activation in ATMs during metabolic stimulation increases their survival. • Decreased ATM apoptosis contributes to macrophage accumulation in obesity.

Although the lung is the most common metastatic site for cancer cells, biologic mechanisms regulating lung metastasis are not fully understood. Using heterotopic and intravenous injection models of lung metastasis in mice, we found that IL5, a cytokine involved in allergic and infectious diseases, facilitates metastatic colonization through recruitment of sentinel eosinophils and regulation of other inflammatory/immune cells in the microenvironment of the distal lung. Genetic IL5 deficiency offered marked protection of the lungs from metastasis of different types of tumor cells, including lung cancer, melanoma, and colon cancer. IL5 neutralization protected subjects from metastasis, whereas IL5 reconstitution or adoptive transfer of eosinophils into IL5-deficient mice exerted prometastatic effects. However, IL5 deficiency did not affect the growth of the primary tumor or the size of metastatic lesions. Mechanistic investigations revealed that eosinophils produce CCL22, which recruits regulatory T cells to the lungs. During early stages of metastasis, Treg created a protumorigenic microenvironment, potentially by suppressing IFNγ-producing natural killer cells and M1-polarized macrophages. Together, our results establish a network of allergic inflammatory circuitry that can be co-opted by metastatic cancer cells to facilitate lung colonization, suggesting interventions to target this pathway may offer therapeutic benefits to prevent or treat lung metastasis. Cancer Res; 75(8); 1624–34. ©2015 AACR.

Objective Introduction of a high-fat diet to mice results in a period of voracious feeding, known as hyperphagia, before homeostatic mechanisms prevail to restore energy intake to an isocaloric level. Acute high-fat diet hyperphagia induces astrocyte activation in the rodent hypothalamus, suggesting a potential role of these cells in the homeostatic response to the diet. The objective of this study was to determine physiologic role of astrocytes in the acute homeostatic response to high-fat feeding. Methods We bred a transgenic mouse model with doxycycline-inducible inhibition of NFkappaB (NFκB) signaling in astrocytes to determine the effect of loss of NFκB-mediated astrocyte activation on acute high-fat hyperphagia. ELISA was used to measure the levels of markers of astrocyte activation, glial-fibrillary acidic protein (GFAP) and S100B, in the medial basal hypothalamus. Results Inhibition of NFκB signaling in astrocytes prevented acute high-fat diet-induced astrocyte activation and resulted in a 15% increase in caloric intake (P

Myeloid cells are capable of promoting or eradicating tumor cells and the nodal functions that contribute to their different roles are still obscure. Here, we show that mice with myeloid-specific genetic loss of the NF-κB pathway regulatory kinase IKKβ exhibit more rapid growth of cutaneous and lung melanoma tumors. In a BRAFV600E/PTEN−/− allograft model, IKKβ loss in macrophages reduced recruitment of myeloid cells into the tumor, lowered expression of MHC class II molecules, and enhanced production of the chemokine CCL11, thereby negatively regulating dendritic-cell maturation. Elevated serum and tissue levels of CCL11 mediated suppression of dendritic-cell differentiation/maturation within the tumor microenvironment, skewing it toward a Th2 immune response and impairing CD8+ T cell–mediated tumor cell lysis. Depleting macrophages or CD8+ T cells in mice with wild-type IKKβ myeloid cells enhanced tumor growth, where the myeloid cell response was used to mediate antitumor immunity against melanoma tumors (with less dependency on a CD8+ T-cell response). In contrast, myeloid cells deficient in IKKβ were compromised in tumor cell lysis, based on their reduced ability to phagocytize and digest tumor cells. Thus, mice with continuous IKKβ signaling in myeloid-lineage cells (IKKβCA) exhibited enhanced antitumor immunity and reduced melanoma outgrowth. Collectively, our results illuminate new mechanisms through which NF-κB signaling in myeloid cells promotes innate tumor surveillance. Cancer Res; 74(24); 7274–84. ©2014 AACR.

In many patients with prostate cancer, the cancer will be recurrent and eventually progress to lethal metastatic disease after primary treatment, such as surgery or radiation therapy. Therefore, it would be beneficial to better predict which patients with early-stage prostate cancer would progress or recur after primary definitive treatment. In addition, many studies indicate that activation of NF-κB signaling correlates with prostate cancer progression; however, the precise underlying mechanism is not fully understood. Our studies show that activation of NF-κB signaling via deletion of one allele of its inhibitor, IκBα, did not induce prostatic tumorigenesis in our mouse model. However, activation of NF-κB signaling did increase the rate of tumor progression in the Hi-Myc mouse prostate cancer model when compared with Hi-Myc alone. Using the nonmalignant NF-κB–activated androgen-depleted mouse prostate, a NF-κB–activated recurrence predictor 21 (NARP21) gene signature was generated. The NARP21 signature successfully predicted disease-specific survival and distant metastases-free survival in patients with prostate cancer. This transgenic mouse model–derived gene signature provides a useful and unique molecular profile for human prostate cancer prognosis, which could be used on a prostatic biopsy to predict indolent versus aggressive behavior of the cancer after surgery. Cancer Res; 74(10); 2763–72. ©2014 AACR.

Pancreatic cancer occurs in the setting of a profound fibrotic microenvironment that often dwarfs the actual tumor. Although pancreatic fibrosis has been well studied in chronic pancreatitis, its development in pancreatic cancer is much less well understood. This article describes the dynamic remodeling that occurs from pancreatic precursors (pancreatic intraepithelial neoplasias (PanINs)) to pancreatic ductal adenocarcinoma, highlighting similarities and differences between benign and malignant disease. Although collagen matrix is a commonality throughout this process, early stage PanINs are virtually free of periostin while late stage PanIN and pancreatic cancer are surrounded by an increasing abundance of this extracellular matrix protein. Myofibroblasts also become increasingly abundant during progression from PanIN to cancer. From the earliest stages of fibrogenesis, macrophages are associated with this ongoing process. In vitro co-culture indicates there is cross-regulation between macrophages and pancreatic stellate cells (PaSCs), precursors to at least some of the fibrotic cell populations. When quiescent PaSCs were co-cultured with macrophage cell lines, the stellate cells became activated and the macrophages increased cytokine production. In summary, fibrosis in pancreatic cancer involves a complex interplay of cells and matrices that regulate not only the tumor epithelium but the composition of the microenvironment itself.

BACKGROUND: Ovarian cancer is the most lethal gynecologic malignancy. While women with BRCA deficient tumors show sensitivity to PARP inhibitors (PARPi), new treatment options are urgently needed for patients with PARPi-resistant tumors. An emerging strategy to improve PARPi response is combination therapy with epigenetic drugs. A recently recognized epigenetic drug target in ovarian cancer is the bromodomain and extraterminal (BET) family of proteins. BET proteins such as BRD4 promote oncogenic transcription of genes promoting cell growth, survival and DNA repair. One example is the well-established link between inflammation and cancer, nuclear factor-kappaB (NF-κB). In syngeneic mouse ovarian cancer models, M2-like pro-tumor macrophages are a prominent component of the ovarian cancer tumor microenvironment (TME). NF-κB inhibition can reduce the M2 macrophage population; however, more sustained treatment with a systemic NF-κB inhibitor leads to more ascites and reduced survival time. Thus, BET inhibitors (BETi) have the potential to induce transcriptional reprogramming in tumors and macrophages that may be beneficial or harmful depending on context. OBJECTIVE: To determine the cellular and molecular effects of combining BETi and PARPi in mouse ovarian cancer cells and peritoneal macrophages. METHODS: Cultured ID8 mouse ovarian cancer cells, PMJ2-PC mouse peritoneal macrophages and immortalized bone marrow-derived macrophages were treated with vehicle, the PARPi olaparib, the BETi JQ1 or the JQ1/olaparib combination for 24-72h. Sulforhodamine B (SRB) assays assessed cell growth in vitro. C57BL/6 mice injected intra-peritoneally (IP) with ID8 cells were treated with JQ1 (30 days, 50mg/kg by IP injection) with volume of ascites fluid, weight of harvested tumor, and number of tumor implants assessed. Markers of apoptosis (cleaved PARP or cleaved caspase-3), and DNA damage (pH2AX) were measured by immunohistochemistry or western blot. NF-κB activity was measured by luciferase assays of a NF-κB reporter plasmid. Expression of M1 (CCL3) and M2 (CD206, arginase-1) macrophage markers was measured by quantitative real-time RT-PCR (QPCR). RESULTS: In culture, JQ1 treatment sensitized ID8 ovarian cancer cells to olaparib-induced growth inhibition, DNA damage and apoptosis. However, despite modest stimulatory effects on DNA damage and apoptosis of long-term JQ1 treatment in ID8 tumors in vivo, JQ1 unexpectedly increased ascites formation without reducing overall tumor burden. In macrophages, there were also contrasting effects between JQ1 treatment in vitro and in vivo. JQ1 alone or combined with olaparib reduced NF-κB activity in cultured macrophage cell lines, and increased expression of CCL3, and reduced CD206 and arginase-1 expression. In contrast, increased ascites due to JQ1 treatment in vivo was accompanied by a pronounced M2 macrophage shift. CONCLUSIONS: Sustained inhibition of NF-κB activity in macrophages and potentially other cells in the ovarian TME could have overall deleterious effects on tumor progression. Our results strongly suggest that the therapeutic effects of BETi in ovarian cancer, and any therapeutic agent with the potential to alter cell function in the tumor microenvironment, need to be more thoroughly tested in the context of functional immune system before being translated to patients. This is particularly relevant since BETi are currently being tested in human patients in early clinical trials. Citation Format: Andrew J. Wilson, Alyssa Hoover, Whitney Harris, Esther Liu, Dineo Khabele, Fiona E. Yull. BROMODOMAIN INHIBITION IN OVARIAN CANCER AND THE TUMOR MICROENVIRONMENT [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr TMIM-085.

It is recognized that modulation of macrophage phenotypes within the tumor microenvironment has the potential to achieve anti-tumor outcomes. However, formulating a strategy to achieve the benefits of such an approach in a cell-specific manner while minimizing off-target or uncontrolled deleterious side effects remains a challenge. We hypothesize that modulation of Nuclear Factor Kappa-B (NF-kappaB) signaling within macrophages in the proximal tumor microenvironment represents an effective approach. However, there remains controversy whether NF-kappaB signaling in macrophages induces pro- or anti-tumor outcomes. Understanding the mechanisms by which this signaling pathway defines predominant macrophage characteristics is therefore critical in order to use strategic interventions to obtain therapeutic benefits. Our goal is to gain insight into how to modulate NF-kappaB signaling in macrophages to generate anti-tumor phenotypes and to use this information to develop new treatments. We use immortalized bone marrow-derived macrophages or ex vivo macrophages in cell culture approaches to investigate impacts of modulation of NF-kappaB signaling on macrophage phenotypes. We have three methods to modulate signaling: doxycycline inducible transgenic mice, liposomal delivery of the bacterial cell mimic MTP-PE, and optimized polymeric nanoparticle-mediated delivery of siRNA targeting the NF-kappaB inhibitor. In addition to our in vitro assays, we also employ in vivo murine models of ovarian and breast tumor progression. This multi-faceted approach is providing insights into how altering NF-kappaB signaling in macrophages impacts their interactions with tumor cells and other cells in the tumor microenvironment. Our data suggests that high levels of NF-kappaB signaling in macrophages induce both direct tumor cell-killing and immune-stimulating responses. These studies are designed to better understand the mechanisms by which NF-kappaB regulates macrophage functions to inform development of a novel macrophage-based immunotherapy that will be effective across a wide spectrum of solid tumors and metastatic disease. Citation Format: Evan B. Glass, Alyssa Hoover, Whitney Harris, Zahra Mirafzali, Todd D. Giorgio, Andrew Wilson, Fiona Yull. Understanding the effects of NF-kappaB signaling on macrophage phenotype to inform therapeutic approaches [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 4138.

Synovial sarcoma is an aggressive soft-tissue malignancy of children and young adults, with no effective systemic therapies. Its specific oncogene, SYT–SSX (SS18–SSX), drives sarcoma initiation and development. The exact mechanism of SYT–SSX oncogenic function remains unknown. In an SYT–SSX2 transgenic model, we show that a constitutive Wnt/β-catenin signal is aberrantly activated by SYT–SSX2, and inhibition of Wnt signaling through the genetic loss of β-catenin blocks synovial sarcoma tumor formation. In a combination of cell-based and synovial sarcoma tumor xenograft models, we show that inhibition of the Wnt cascade through coreceptor blockade and the use of small-molecule CK1α activators arrests synovial sarcoma tumor growth. We find that upregulation of the Wnt/β-catenin cascade by SYT-SSX2 correlates with its nuclear reprogramming function. These studies reveal the central role of Wnt/β-catenin signaling in SYT–SSX2-induced sarcoma genesis, and open new venues for the development of effective synovial sarcoma curative agents. Significance: Synovial sarcoma is an aggressive soft-tissue cancer that afflicts children and young adults, and for which there is no effective treatment. The current studies provide critical insight into our understanding of the pathogenesis of SYT–SSX-dependent synovial sarcoma and pave the way for the development of effective therapeutic agents for the treatment of the disease in humans. Cancer Discov; 3(11); 1286–1301. ©2013 AACR. This article is highlighted in the In This Issue feature, p. 1207

High salt diet induces renal medullary cyclooxygenase 2 (COX2) expression. Selective blockade of renal medullary COX2 activity in rats causes salt-sensitive hypertension, suggesting a role for renal medullary COX2 in maintaining systemic sodium balance. The present study characterized the cellular location of COX2 induction in the kidney of mice following high salt diet and examined the role of NFκB in mediating this COX2 induction in response to increased dietary salt. High salt diet (8 % NaCl) for 3 days markedly increased renal medullary COX2 expression in C57Bl/6 J mice. Co-immunofluorescence using a COX2 antibody and antibodies against aquaporin-2, ClC-K, aquaporin-1, and CD31 showed that high salt diet-induced COX2 was selectively expressed in renal medullary interstitial cells. By using NFκB reporter transgenic mice, we observed a sevenfold increase of luciferase activity in the renal medulla of the NFκB-luciferase reporter mice following high salt diet, and a robust induction of enhanced green fluorescent protein (EGFP) expression mainly in renal medullary interstitial cells of the NFκB-EGFP reporter mice following high salt diet. Treating high salt diet-fed C57Bl/6 J mice with selective IκB kinase inhibitor IMD-0354 (8 mg/kg bw) substantially suppressed COX2 induction in renal medulla, and also significantly reduced urinary prostaglandin E2 (PGE2). These data therefore suggest that renal medullary interstitial cell NFκB plays an important role in mediating renal medullary COX2 expression and promoting renal PGE2 synthesis in response to increased dietary sodium.

Although reactive oxygen species (ROS) produced by NADPH oxidase are known to regulate inflammatory responses, the impact of ROS on intracellular signaling pathways is incompletely understood. In these studies, we treated wild-type (WT) and p47phox-deficient mice with LPS to investigate mechanisms by which NADPH oxidase regulates signaling through the NF-κB pathway. After intratracheal instillation of LPS, ROS generation was impaired in p47phox−/− mice, whereas these mice had increased neutrophilic alveolitis and greater lung injury compared with WT controls. In mice interbred with transgenic NF-κB reporters (HIV-long terminal repeat/luciferase [HLL]), we found exaggerated LPS-induced NF-κB activation and increased expression of proinflammatory cytokines in lungs of p47phox−/−/HLL mice compared with controls. Both lung macrophages and bone marrow–derived macrophages (BMDMs) isolated from p47phox−/−/HLL mice showed enhanced LPS-stimulated NF-κB activity compared with controls. Although nuclear translocation of NF-κB proteins was similar between genotypes, EMSAs under nonreducing conditions showed increased DNA binding in p47phox−/−/HLL BMDMs, suggesting that ROS production reduces NF-κB binding to DNA without affecting nuclear translocation. Increased intracellular reduced glutathione/glutathione disulfide ratio and greater nuclear redox factor 1 (Ref-1) levels were present in p47phox−/−/HLL compared with WT BMDMs, pointing to NADPH oxidase modulating intracellular redox status in macrophages. Treatment with the Ref-1–specific inhibitor E3330 or hydrogen peroxide inhibited LPS-induced NF-κB activation in p47phox−/−/HLL BMDMs but not in WT/HLL cells. Consistent with these findings, small interfering RNA against Ref-1 selectively reduced NF-κB activity in LPS-treated p47phox−/−/HLL BMDMs. Together, these results indicate that NADPH oxidase limits LPS-induced NF-κB transcriptional activity through regulation of intracellular redox state.

Macrophages represent an important therapeutic target, because their activity has been implicated in the progression of debilitating diseases such as cancer and atherosclerosis. In this work, we designed and characterized pH-responsive polymeric micelles that were mannosylated using “click” chemistry to achieve CD206 (mannose receptor)-targeted siRNA delivery. CD206 is primarily expressed on macrophages and dendritic cells and upregulated in tumor-associated macrophages, a potentially useful target for cancer therapy. The mannosylated nanoparticles improved the delivery of siRNA into primary macrophages by 4-fold relative to the delivery of a nontargeted version of the same carrier (p < 0.01). Further, treatment for 24 h with the mannose-targeted siRNA carriers achieved 87 ± 10% knockdown of a model gene in primary macrophages, a cell type that is typically difficult to transfect. Finally, these nanoparticles were also avidly recognized and internalized by human macrophages and facilitated the delivery of...

Introduction. Since the NF-κB pathway regulates both inflammation and host defense, it is uncertain whether interventions targeting NF-κB would be beneficial in sepsis. Based on the kinetics of the innate immune response, we postulated that selective NF-κB inhibition during a defined time period after the onset of sepsis would reduce acute lung injury without compromising bacterial host defense.Methods. Mice underwent cecal ligation and puncture (CLP). An NF-κB inhibitor, BMS-345541 (50 µg/g mice), was administered by peroral gavage beginning 2 hours after CLP and repeated at 6 hour intervals for 2 additional doses.Results. Mice treated with BMS-345541 after CLP showed reduced neutrophilic alveolitis and lower levels of KC in bronchoalveolar lavage fluid compared to mice treated with CLP+vehicle. In addition, mice treated with CLP+BMS had minimal histological evidence of lung injury and normal wet-dry ratios, indicating protection from acute lung injury. Treatment with the NF-κB inhibitor did not affect the ability of cultured macrophages to phagocytose bacteria and did not alter bacterial colony counts in blood, lung tissue, or peritoneal fluid at 24 hours after CLP. While BMS-345541 treatment did not alter mortality after CLP, our results showed a trend towards improved survival.Conclusion. Transiently blocking NF-κB activity after the onset of CLP-induced sepsis can effectively reduce acute lung injury in mice without compromising bacterial host defense or survival after CLP.

The tumor microenvironment (TME) consists of cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that can promote neoplastic transformation, support tumor growth and invasion, protect the tumor from host immunity, foster therapeutic resistance, and provide niches for dormant metastases to thrive. An American Association for Cancer Research (AACR) special conference held on November 3–6, 2011, addressed five emerging concepts in our understanding of the TME: its dynamic evolution, how it is educated by tumor cells, pathways of communication between stromal and tumor cells, immunomodulatory roles of the lymphatic system, and contribution of the intestinal microbiota. These discussions raised critical questions on how to include the analysis of the TME in personalized cancer diagnosis and treatment. Cancer Res; 72(10); 2473–80. ©2012 AACR.

SummaryAlthough it is believed that neural activation can affect immune responses, very little is known about the neuroimmune interactions involved, especially the regulators of immune traffic across the blood-brain barrier which occurs in neuroimmune diseases such as multiple sclerosis (MS). Using a mouse model of MS, experimental autoimmune encephalomyelitis, we show that autoreactive T cells access the central nervous system via the fifth lumbar spinal cord. This location is defined by IL-6 amplifier-dependent upregulation of the chemokine CCL20 in associated dorsal blood vessels, which in turn depends on gravity-induced activation of sensory neurons by the soleus muscle in the leg. Impairing soleus muscle contraction by tail suspension is sufficient to reduce localized chemokine expression and block entry of pathogenic T cells at the fifth lumbar cord, suggesting that regional neuroimmune interactions may offer therapeutic targets for a variety of neurological diseases.

The mechanisms by which chronic inflammatory lung diseases, particularly chronic obstructive pulmonary disease, confer enhanced risk for lung cancer are not well-defined. To investigate whether nuclear factor (NF)-κB, a key mediator of immune and inflammatory responses, provides an interface between persistent lung inflammation and carcinogenesis, we utilized tetracycline-inducible transgenic mice expressing constitutively active IκB kinase β in airway epithelium (IKTA (IKKβ trans-activated) mice). Intraperitoneal injection of ethyl carbamate (urethane), or 3-methylcholanthrene (MCA) and butylated hydroxytoluene (BHT) was used to induce lung tumorigenesis. Doxycycline-treated IKTA mice developed chronic airway inflammation and markedly increased numbers of lung tumors in response to urethane, even when transgene expression (and therefore epithelial NF-κB activation) was begun after exposure to carcinogen. Studies using a separate tumor initiator/promoter model (MCA+BHT) indicated that NF-κB functions as an independent tumor promoter. Enhanced tumor formation in IKTA mice was preceded by increased proliferation and reduced apoptosis of alveolar epithelium, resulting in increased formation of premalignant lesions. Investigation of inflammatory cells in lungs of IKTA mice revealed a substantial increase in macrophages and lymphocytes, including functional CD4+/CD25+/FoxP3+ regulatory T lymphocytes (Tregs). Importantly, Treg depletion using repetitive injections of anti-CD25 antibodies limited excessive tumor formation in IKTA mice. At 6 weeks following urethane injection, antibody-mediated Treg depletion in IKTA mice reduced the number of premalignant lesions in the lungs in association with an increase in CD8 lymphocytes. Thus, persistent NF-κB signaling in airway epithelium facilitates carcinogenesis by sculpting the immune/inflammatory environment in the lungs.

Bronchopulmonary dysplasia is a common pulmonary complication of extreme prematurity. Arrested lung development leads to bronchopulmonary dysplasia, but the molecular pathways that cause this arrest are unclear. Lung injury and inflammation increase disease risk, but the cellular site of the inflammatory response and the potential role of localized inflammatory signaling in inhibiting lung morphogenesis are not known. In this study, we show that tissue macrophages present in the fetal mouse lung mediate the inflammatory response to LPS and that macrophage activation inhibits airway morphogenesis. Macrophage depletion or targeted inactivation of the NF-κB signaling pathway protected airway branching in cultured lung explants from the effects of LPS. Macrophages also appear to be the primary cellular site of IL-1β production following LPS exposure. Conversely, targeted NF-κB activation in transgenic macrophages was sufficient to inhibit airway morphogenesis. Macrophage activation in vivo inhibited expression of multiple genes critical for normal lung development, leading to thickened lung interstitium, reduced airway branching, and perinatal death. We propose that fetal lung macrophage activation contributes to bronchopulmonary dysplasia by generating a localized inflammatory response that disrupts developmental signals critical for lung formation.

IL-5 is a T helper 2 cytokine important in the trafficking and survival of eosinophils. Because eosinophils can be found in malignant pleural effusions (MPE) from mice and humans, we asked whether IL-5 is involved in the pathogenesis of MPE.To determine the role of IL-5 in MPE formation.The effects of IL-5 on experimental MPE induced in C57BL/6 mice by intrapleural injection of syngeneic lung (Lewis lung cancer [LLC]) or colon (MC38) adenocarcinoma cells were determined using wild-type (il5(+/+)) and IL-5-deficient (il5⁻(/)⁻) mice, exogenous administration of recombinant mouse (rm) IL-5, and in vivo antibody-mediated neutralization of endogenous IL-5. The direct effects of rmIL-5 on LLC cell proliferation and gene expression in vitro were determined by substrate reduction and microarray.Eosinophils and IL-5 were present in human and mouse MPE, but the cytokine was not detected in mouse (LLC) or human (A549) lung and mouse colon (MC38) adenocarcinoma-conditioned medium, suggesting production by host cells in MPE. Compared with il5(+/+) mice, il5⁻(/)⁻ mice showed markedly diminished MPE formation in response to both LLC and MC38 cells. Exogenous IL-5 promoted MPE formation in il5(+/+) and il5⁻(/)⁻ mice, whereas anti-IL-5 antibody treatment limited experimental MPE in il5(+/+) mice. Exogenous IL-5 had no effects on LLC cell proliferation and gene expression; however, IL-5 was found to be responsible for recruitment of eosinophils and tumor-promoting myeloid suppressor cells to MPE in vivo.Host-derived IL-5 promotes experimental MPE and may be involved in the pathogenesis of human MPE.

The transcription factor nuclear factor kappa B (NF-κB) is activated in human breast cancer tissues and cell lines. However, it is unclear whether NF-κB activation is a consequence of tumor formation or a contributor to tumor development. We developed a doxycycline (dox)-inducible mouse model, termed DNMP, to inhibit NF-κB activity specifically within the mammary epithelium during tumor development in the polyoma middle T oncogene (PyVT) mouse mammary tumor model. DNMP females and PyVT littermate controls were treated with dox from 4 to 12 weeks of age. We observed an increase in tumor latency and a decrease in final tumor burden in DNMP mice compared with PyVT controls. A similar effect with treatment from 8 to 12 weeks indicates that outcome is independent of effects on postnatal virgin ductal development. In both cases, DNMP mice were less likely to develop lung metastases than controls. Treatment from 8 to 9 weeks was sufficient to impact primary tumor formation. Inhibition of NF-κB increases apoptosis in hyperplastic stages of tumor development and decreases proliferation at least in part by reducing Cyclin D1 expression. To test the therapeutic potential of NF-κB inhibition, we generated palpable tumors by orthotopic injection of PyVT cells and then treated systemically with the NF-κB inhibitor thymoquinone (TQ). TQ treatment resulted in a reduction in tumor volume and weight as compared with vehicle-treated control. These data indicate that epithelial NF-κB is an active contributor to tumor progression and demonstrate that inhibition of NF-κB could have a significant therapeutic impact even at later stages of mammary tumor progression.

While it is known that nuclear factor κ-B (NF-κB) signaling within macrophages regulates expression of downstream targets that can mediate either pro- or antitumor functions, the mechanisms that define predominant characteristics are not well understood. As macrophages are present in the tumor microenvironment of diverse tumor types, their characteristics have the potential to either support or hinder tumor growth and metastatic spread. Therefore, a method to shift the balance of tumor associated macrophages from pro- to antitumor functions could potentially target tumor cells directly and also recruit and activate other immune cells, resulting in significant therapeutic benefit. Our studies are designed to gain an understanding of how to use modulation of NF-κB signaling as the “pinch point” to generate antitumor phenotypes and determine how to achieve this modulation in a patient. We are using immortalized bone marrow-derived macrophages or ex vivo tumor-associated macrophages in cell culture approaches and inducible transgenic mice in in vivo approaches to investigate the mechanisms by which NF-κB defines macrophage characteristics. Interactions with tumor cells are also being investigated using co-culture and murine models. Our data suggests that high levels of NFκ-B activity in macrophages induce both direct tumor cell killing and immune stimulating responses. We are testing two different translational approaches. The first is liposomal muramyl tripeptide ethanolamine that acts as an immune stimulant by increasing NF-κB activity in macrophages. The second is preferential delivery of siRNA against the inhibitor of NFκ-B (IκB-alpha) to tumor-associated macrophages using polymeric nanoparticles. We hope to better understand the mechanisms by which NFκ-B regulates macrophage functions to inform development of a novel macrophage-based immunotherapy that could be effective across a wide spectrum of solid tumors and metastatic disease. Citation Format: Esther Liu, Alyssa A. Hoover, Whitney R. Harris, Whitney Barham, Oleg Tikhomirov, Dineo Khabele, Zahra Mirafzahli, Todd D. Giorgio, Andrew J. Wilson, Fiona E. Yull. Modulation of NFκ-B signaling to optimize antitumor characteristics in macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4711.