Protumor M2 Phenotype Research Articles

Effects of macrophage polarization on gold nanoparticle-assisted plasmonic photothermal therapy.

Tumor associated macrophages (TAM) are key pathogenic factors in neoplastic diseases. They are known to have plasticity and can polarize into two opposing phenotypes, including the tumoricidal M1 and the protumoral M2 phenotypes with high prevalence of M2-phentoypes in patients with poor prognosis. Strategies for targeting M2-TAM may consequently increase the efficacy of therapeutic strategies for cancer treatment. Gold nanorod-assisted plasmonic photothermal therapy (PPTT) has emerged as a promising treatment for cancer but the effects of macrophage polarization parameters in the performance of this new treatment modality is still unknown. Herein, human monocytic THP-1 cells were polarized into two opposite phenotypic macrophages (M1-TAM and M2-TAM) and their response to PPTT was examined. M2-TAM exhibits a three-fold increase in AuNP uptake compared to M1-TAM. Laser irradiation results in selective killing of pro-tumoral M2-TAM after treatment with AuNPs with limited effects on anti-tumoral M1-TAM. A positive correlation between the expression of CD206 marker and the AuNP uptake may indicate the role of CD206 in facilitating AuNP uptake. Our findings also suggest that the differences in AuNP avidity and uptake between the M1-TAM and M2-TAM phenotypes may be the rationale behind the effectiveness of PPTT in the treatment of solid tumors.

Multifunctional ginsenoside Rg3-based liposomes for glioma targeting therapy

Liposomes have been widely used for targeted drug delivery. However, nonselective distribution, low blood-brain barrier penetration, and the disadvantages of cholesterol greatly limit the application of conventional liposomes in the treatment of brain tumors. In the present study, we aimed to develop a multifunctional ginsenoside Rg3-based liposomal system (Rg3-LPs). Compared to cholesterol liposomes (C-LPs), Rg3-LPs not only significantly improved cellular uptake and penetration across glioma spheroids in vitro, but also remarkably enhanced active glioma targeting and intratumoral diffusion capability in vivo. Paclitaxel-loaded Rg3-LPs (Rg3-PTX-LPs) exhibited a substantially stronger anti-proliferation effect on C6 glioma cells than paclitaxel-loaded C-LPs and re-educated tumor-associated macrophages from the protumor M2 phenotype to the antitumor M1 phenotype in vivo. Rg3-PTX-LPs significantly prolonged median survival time of intracranial C6-bearing mice/rats by activating the immune microenvironment in glioma, facilitating T-cell immune responses with expansion of the CD8+ T-cell population, increasing the M1/M2 ratio, and decreasing regulatory T and myeloid-derived suppressor cells. Together, the results demonstrated that ginsenoside Rg3 is a good alternative for cholesterol in drug delivery liposomes and has a synergistic effect with loaded anticancer drugs. Rg3-PTX-LPs can serve as a multifunctional potential drug for the treatment of glioma.

Hepatocellular carcinoma-derived high mobility group box 1 triggers M2 macrophage polarization via a TLR2/NOX2/autophagy axis

In many human cancers, including hepatocellular carcinoma (HCC), high density of infiltrating tumor-associated macrophages (TAM) is associated with poor prognosis. Most TAMs express a M2 phenotype subsequently supporting tumor growth. How tumor cells polarize these TAMs to a pro-tumor M2 phenotype is still poorly understood. Our previous studies have revealed that a Toll-like receptor 2 (TLR2)-dependent autophagy triggered by hepatoma-derived factors down-regulates NF-κB p65 and drives M2 macrophage differentiation. However, the underlying mechanisms and potential hepatoma-derived TLR2 ligands are not clear. Here, we provide evidence to reveal that NADPH oxidase 2 (NOX2)-dependent reactive oxygen species (ROS) generation is crucial for HCC-induced autophagy, NF-κB p65 down-regulation and M2 phenotype polarization in primary macrophages. This NOX2-generated ROS production in abolished in TLR2-deficient macrophages. HCC-derived or recombinant high-mobility group box 1 (HMGB1) is able to trigger this TLR2-mediated M2 macrophage polarization. Blockage of HMGB1 and ROS by inhibitors, ethyl pyruvate and N-acetylcysteine amide, respectively, significantly reduces both M2 macrophage accumulation and liver nodule formation in HCC-bearing mice. Our findings uncover a HMGB1/TLR2/NOX2/autophagy axis to trigger M2 macrophage polarization in HCC that can be considered as a novel therapeutic target for treating HCC.

М3 макрофаги останавливают деление клеток предстательной железы больного раком простаты

В развитии рака предстательной железы (РПЖ) макрофаги играют важную роль. Многие опухоли выделяют противовоспалительные цитокины, которые перепрограммируют М1 фенотип макрофагов на проопухолевый М2 фенотип. М2 макрофаги подавляют противоопухолевый иммунитет, способствуют делению и метастазированию опухолевых клеток. Предыдущие исследования позволили нам обосновать предположение, что деление опухолевых клеток РПЖ человека может быть ограничено особым М3 фенотипом макрофагов. Фенотип М3, в отличие от М1 фенотипа, реагирует на противовоспалительные цитокины увеличением продукции провоспалительных противоопухолевых цитокинов, что способствует сохранению их противоопухолевых свойств в зоне опухоли. Цель исследования - проверка гипотезы о способности М3 макрофагов останавливать деление клеток предстательной железы больного РПЖ. Методика. В работе использовали макрофаги мышей, выделенные из перитонеального лаважа и макрофаги человека, полученные из моноцитов крови больных РПЖ. Фенотип М3 макрофагов получали добавлением в среду культивирования IFN-γ, ингибиторов STAT3, STAT6 и SMAD3 с последующей стимуляцией липополисахаридом. Результаты. Показано, что М3 макрофаги мышей и человека ограничивали деление клеток предстательной железы больных РПЖ в условиях 2D (на плоскости) культивирования на 43% и 93%, соответственно. При 3D (в объеме) культивировании М3 макрофаги мышей не ограничивали, а М3 макрофаги человека лишь незначительно ограничивали деление клеток предстательной железы у больных РПЖ. Заключение. Результаты работы делают обоснованными дальнейшие исследования и разработку клинической версии биотехнологии лечения рака предстательной железы с использованием М3 макрофагов.Macrophages play an important role in the development of prostate cancer (PCa). Many tumors, including PCa, secrete anti-inflammatory cytokines that reprogram the M1 macrophage phenotype into the pro-tumor M2 phenotype. M2 macrophages suppress antitumor immunity and promote division and metastasis of tumor cells. We hypothesized that the division of human PCa cells may be restricted by a specific M3 macrophage phenotype. The M3 phenotype, in contrast to the M1 phenotype, responds to anti-inflammatory cytokines by increasing the production of inflammatory anti-tumor cytokines and retains its anti-tumor properties in the tumor area. The aim of the study was to test the hypothesis on the ability of M3 macrophages to stop division of prostate cells from patients with PCa. Methods. This study used murine macrophages isolated from the peritoneal lavage and human macrophages obtained from blood monocytes of patients with PCa. The M3 macrophage phenotype was obtained by adding IFN-γ, STAT3, STAT6, and SMAD3 inhibitors to the cultural medium followed by lipopolysaccharide (LPS) stimulation. Results. Murine and human M3 macrophages restricted the division of patients’ PCa cells in the conditions of 2D cultivation by 43% and 93%, respectively. In 3D cultivation, murine M3 macrophages did not restrict whereas human M3 macrophages only slightly limited the division of prostate cells from PCa patients. The results of the study warrant further research and development of a clinical biotechnology for PCa treatment with reprogrammed M3 macrophages.

Detection of pancreatic ductal adenocarcinoma with galectin-9 serum levels

Pancreatic ductal adenocarcinoma (PDAC) responds poorly to checkpoint blockade, such as anti-CTLA-4 and anti-PD-1. Galectin-9, a β-galactoside-binding lectin, promotes immune suppression through T-cell inhibition, and programming of tolerogenic macrophages. Of all cancers tested, PDAC showed the highest expression of LGALS9 (galectin-9) mRNA. We analyzed formalin-fixed and paraffin-embedded specimens from 83 patients with PDAC stained for galectin-9. Using flow cytometry, we determined galectin-9 expression on immune cells from tumor and matched blood samples from 12 patients with resectable PDAC. Furthermore, we analyzed galectin-9 serum levels by enzyme-linked immunosorbent assay using serum samples from 70 patients with PDAC, from 36 individuals with benign pancreatic disease, and from 28 healthy controls. Galectin-9 was highly expressed in human PDAC compared with normal pancreas and present on both tumor and immune cells. Tumor-infiltrating immune cells, especially CD3+ T cells, showed upregulation of galectin-9 compared with immune cells from matched blood. Blood γδ T cells from PDAC patients had higher galectin-9 expression than γδ T cells from healthy individuals. Galectin-9 polarized macrophages toward a protumoral M2 phenotype leading to suppressed T-cell cytokine secretion. Furthermore, serum concentration of galectin-9 was able to discriminate PDAC from benign pancreatic disease and healthy individuals, and was prognostic for stage IV patients. Galectin-9 is a new biomarker for the detection of PDAC.

1999P - Impact of tumor reoxygenation by nanoparticles on tumor associated macrophages (TAMs)

BackgroundGlioblastoma (GB), a highly hypoxic brain tumor (Bekaert et al. 2017), is characterized by a massive macrophage (MΦ) infiltration (Lapa et al. 2015). Hypoxia triggers a shift to a pro-tumoral M2 phenotype in GB (Leblond et al. 2016). Thus, strategies aiming to reduce hypoxia could promote an anti-tumoral M1 phenotype. Among these reoxygenation strategies, we recently developed a new approach with zeolites nanoparticles. These zeolites are able to carry hyperoxic/hypercapnic gases and release them according to a hypoxic gradient. We have demonstrated that the charge balancing cation changes affinity to the gases but also the ability to track zeolite with MRI. Our objective is to study the reoxygenation efficacy of zeolites specifically in the GB and to evaluate their impact on tumor associated MΦ with in vitro and in vivo studies. MethodsFaujasite zeolites (FAU, ∼20nm of diameter) were used and modified by ion exchange with various cations (Fe, Gd, Cu, Ag). GB model was obtained by orthotopic glioblastoma cells implantation (U251) in nude rats (ONCOModels/Unicaen). 7T MRI (Bruker/Cyceron) was used to follow zeolites after intravenous injection and for oxygen measurement. Murine bone marrow derived MΦ were prepared and polarized to M1 and M2 using LPS/IFNg or IL4 as previously described (Leblond et al. 2016). Zeolites were added in MΦ medium and their impact on MΦ were evaluated by crystal violet dye assay, flow cytometry (Plateau ICORE/Unicaen) and polarization assays. ResultsOur results show that zeolites are able to accumulate and release the carried gases specifically in the brain tumor leading to tumor reoxygenation. Regarding the effect on MΦ, our preliminary results show, in vitro, the safety of as-prepared zeolites or Fe, Gd or Cu dopped zeolites on M0, M1 and M2 MΦ cultures. Similarly, no alteration of the cell cycle was observed. As a positive control of cell death, the presence of Ag-dopped zeolites dramatically decreased M0 and M1 MΦ viability. ConclusionsZeolites can deliver oxygen to the brain tumor and may improve the effectiveness of conventional treatments. Zeolites do not exhibit toxicity on primary cultures of MΦ. Additional studies are underway to evaluate the effect of zeolites on the polarization of MΦ, both in vitro and in vivo. Legal entity responsible for the studyThe authors. FundingRégion Normandie, CNRS, Université de Caen Normandie, Ministère de l’Enseignement Supérieur et de la Recherche, European Union-Fonds Européen de Développement Régional (FEDER), HABIONOR European project, co-funded by the Normandy County Council, the French State in the framework of the interregional development Contract “Vallée de la Seine” 2015-2020, ARCHADE, Fédération pour la Recherche sur le Cerveau (FRC) et INCa (INCA-11699). DisclosureAll authors have declared no conflicts of interest.

Tumor-associated macrophage infiltration in meningioma.

BackgroundMeningioma, a most common brain tumor, has a high rate of recurrence. Tumor-associated macrophages (TAMs) are the most abundant immune cell type in meningioma. TAMs display functional phenotypic diversity and may establish either an inflammatory and anti-tumoral or an immunosuppressive and pro-tumoral microenvironment. TAM subtypes present in meningioma and potential contribution to growth and recurrence is unknown.MethodsImmunofluorescence staining was used to quantify M1 and M2 TAM populations in tissues obtained from 30 meningioma patients. Associations between M1 and M2 cells, M1:M2 cell ratio to tumor characteristics, WHO grade, recurrence, size, location, peri-tumoral edema, and patient demographics such as age and sex were examined.ResultsTAM cells accounted for ~18% of all cells in meningioma tissues. More than 80% of infiltrating TAMs were found to be of pro-tumoral M2 phenotype and correlated to tumor size (P = .0409). M1:M2 cell ratio was significantly decreased in WHO grade II, compared to grade I tumors (P = .009). Furthermore, a 2.3-fold difference in M1:M2 ratio between primary (0.14) and recurrent (0.06) tumors was observed (n = 18 and 12 respectively, P = .044).ConclusionThis study is the first to confirm existence of pro-tumoral M2 TAMs in the meningioma microenvironment, emphasizing its potential role in tumor growth and recurrence.

Abstract P2-02-08: Slit2 induced anti-tumor activity may be mediated through metabolism driven immunomodulation

Abstract Metabolism of immune cells plays an important role in regulating tumor growth by modulating the anti-tumor M1 phenotype or pro-tumor M2 phenotype in macrophages. However, the role of bone marrow derived macrophages (BMDM) and their metabolic profile in promoting tumor growth is unknown. Slit2 is an anti-tumor molecule that is suppressed in breast cancer; however, the mechanism by which Slit2 mediates its function is not fully elucidated. We hypothesize that Slit2 mediated metabolic reprogramming of BMDMs favors anti-tumor M1 phenotype in these macrophages, which in turn reduces tumor growth. Here we assessed cellular metabolism in BMDMs from the MMTV-PyMT mouse, a model showing spontaneous tumor development. Shortly, age matched female mice with palpable tumors and control mice without tumors were treated with recombinant Slit2 (Slit2) or PBS intraperitoneally every third for 2 weeks (n=4 mice per group). Tumor volume was measured in each mouse before and at the end of treatment. Next, mice were euthanized and bone marrow was flushed from both the tibia and femor for culturing in vitro in the presence of macrophage chemotactic factor rich conditioned media. Rate of glycolysis was described based on extracellular acidification rate (ECAR) as measured by the Seahorse Bioanalyzer® under glycostress conditions. Lactate dehydrogenase (LDH) activity, which is linked with breast cancer progression and pro-tumor macrophage phenotype in experimental models, was also assayed using a commercially available kit. Finally, to elucidate potential pathways involved in Slit2 induced metabolic change, we assessed the expression of several proteins and factors involved in cellular metabolism. Firstly, we observed that PyMT mice treated with Slit2 showed lower tumor volume compared to mice treated with PBS confirming Slit2 anti-tumor activity. Hematoxylin & Eosin staining of tumor sections from these mice also showed better tissue structure, with a higher cytoplasm to nuclear ratio in Slit2 treated PyMT mice compared to PBS treated mice. Furthermore, BMDMs from PyMT mice show lower aerobic glycolysis with higher lactate dehydrogenase activity (LDH) compared to control mice. Moreover, treatment with Slit2 appeared to lower LDH activity and trended to increase glycolysis in the BMDMs isolated from Slit2 treated PyMT compared to PBS treated PyMT. Expression analyses using quantitative PCR showed a 2-4 fold decrease in PGC-1α and CPT-2 in Slit2 treated BMDMs, indicating a reduction in fatty acid oxidation in these cells. This coupled with a 3 fold increase in IL-6 expression, and 2-3 fold decrease in arginase and IL-10 expression in tumor tissue suggest a potential shift from pro-tumor M2 to anti-tumor M1 phenotype. In spite of these preliminary trends, changes in metabolism and associated signals may be clearer in isolated, enriched populations of macrophages alone. Nevertheless, our findings suggest that Slit2 reduces tumor growth by affecting immune cell metabolism. Furthermore, these studies provide novel evidence of the potential immunomodulatory effects of Slit2 on macrophages. This may lead to development of Slit2 as a novel non-invasive therapeutic strategy against highly aggressive and metastatic cancers associated with high mortality and low quality of life. Citation Format: Kaul K, Ahirwar DK, Benej M, Denko N, Ganju R. Slit2 induced anti-tumor activity may be mediated through metabolism driven immunomodulation [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-02-08.

Pooled Safety Analysis and Efficacy of Tenalisib (RP6530), a PI3Kδ/γ Inhibitor, in Patients with Relapsed/Refractory Lymphoid Malignancies

IntroductionTenalisib (RP6530) is a next generation, oral, selective, PI3Kδ/g inhibitor with nanomolar inhibitory potency. Besides its apoptotic and anti-proliferative activity, Tenalisib modulates the tumor microenvironment resulting in reprogramming of tumor associated-macrophages (TAMs) from a protumor M2 phenotype to an antitumor M1 phenotype and a marked reduction of angiogenesis in pre-clinical models. Tenalisib has demonstrated activity in patients with relapsed/refractory lymphoid malignancies (Carmelo, ASH 2016 and Oki, ASCO 2018).Since there are concerns over long-term safety of PI3K δ or PI3K dual δ/g inhibitors with respect to immune-mediated toxicities (e.g. transaminitis, colitis and pneumonitis), cytopenias, and infections, a pooled safety analysis across two Phase I studies in patients treated with Tenalisib was performed.MethodsSafety data was pooled from two Phase I Tenalisib monotherapy trials (NCT02017613 and NCT02567656) with similar key eligibility criteria. Patients had R/R lymphoid malignancies with ≥1 prior therapy. Responses were evaluated in lymphoid malignancies using IWG criteria (Cheson et al., 2007) and in CTCL using the modified Severity Weighted Assessment Tool (mSWAT). Adverse events were graded according to CTCAE v4.03.ResultsA total of 93 patients were included in the analysis. Among these patients, 34% were PTCL, 32% CTCL, 16% HL, 6% DLBCL and 12% were other lymphomas. Patients received a median of 5 prior therapies. 53 % of patients received Tenalisib for ≤ 3 months, 21% for 3-6 months and 26% for > 6 months.The overall incidence of related AEs and ≥G3 AEs were 58% and 29% respectively (Table 1). Very few AEs were seen with exposures >6 months and mainly included single cases of diarrhea, anemia, edema, and abdominal pain. There were no incidences of late onset toxicities such as colitis and pneumonitis and most of the AEs happened during the initial three months of therapy. No treatment discontinuations due to AE's were seen in patients exposed to > 6 months of treatment.Efficacy response assessments of the 66 evaluable patients demonstrated an ORR of 45% in TCL (44% in PTCL (8/18, 3 CR, 4 PR) & 45% in CTCL (9/20, 9 PR)), 29% (4/14; 1CR; 3PR) in HL, and 13% (1/6; 1CR) in DLBCL.ConclusionIn this pooled safety analysis with long term follow-up, Tenalisib exhibited an improved safety profile when compared to other investigational/marketed PI3K inhibitors. The incidence of transaminitis was low and occurred within the first two to three cycles of therapy. In particular, there were no occurrences of pneumonitis or colitis in patients that had been on treatment for > 3 months and beyond. Incidence of neutropenia/thrombocytopenia and infections was limited. Tenalisib can therefore be safely combined with a diverse array of other agents active in lymphoid malignancies. Tenalisib is currently being studied in combination with Pembrolizumab and Romidepsin and as a monotherapy in a Phase II trial in indolent NHL. [Display omitted] DisclosuresHaverkos:Viracta Therapeutics: Membership on an entity's Board of Directors or advisory committees. Ramchandren:Pharmacyclics LLC an AbbVie Company: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy; Merck: Research Funding; Janssen: Consultancy, Research Funding. Devata:Affimed: Research Funding. Routhu:Rhizen Pharmaceuticals SA: Employment. Barde:Rhizen Pharmaceuticals SA: Employment. Nair:Rhizen Pharmaceuticals SA: Employment. Carlo-Stella:Boehringher Ingelheim Italia: Consultancy; Sanofi: Consultancy; MSD Italia: Speakers Bureau; Rhizen Pharmaceuticals: Research Funding; AstraZeneca: Speakers Bureau; ADC Therapeutics: Research Funding, Speakers Bureau; Janssen: Speakers Bureau; Genenta Science: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Amgen: Speakers Bureau.

A vascular disrupting agent overcomes tumor multidrug resistance by skewing macrophage polarity toward the M1 phenotype

Multidrug resistance (MDR) mediated by ATP-binding cassette (ABC) transporters is the major obstacle for chemotherapeutic success. Although attempts have been made to circumvent ABC transporter-mediated MDR in past decades, there is still no effective agent in clinic. Here, we identified a vascular disrupting agent, Z-GP-DAVLBH, that significantly inhibited the growth of multidrug-resistant human hepatoma HepG2/ADM and human breast cancer MCF-7/ADR tumor xenografts, although these cells were insensitive to Z-GP-DAVLBH in vitro. Z-GP-DAVLBH increased the secretion of granulocyte-macrophage colony-stimulating factor in tumor tissues and serum of tumor-bearing mice to skew tumor-associated macrophages from the pro-tumor M2 phenotype to the antitumor M1 phenotype, thereby contributing to the induction of HepG2/ADM and MCF-7/ADR cell apoptosis. Our findings shed new light on the underlying mechanisms of VDAs in the treatment of drug-resistant tumors and provide strong evidence that Z-GP-DAVLBH should be a promising agent for overcoming MDR.

Reprogramming Tumor-Associated Macrophages To Reverse EGFRT790M Resistance by Dual-Targeting Codelivery of Gefitinib/Vorinostat.

Gefitinib is a first-line therapy in the EGFR-mutated nonsmall cell lung cancer (NSCLC). However, the development of drug resistance is almost unavoidable, thus leading to an unsustainable regimen. EGFRT790M mutation is the major cause responsible for the molecular-targeting therapy failure in NSCLC. Although the recently approved osimertinib is effective for the EGFRT790M-positive NSCLC, the osimertinib-resistant EGFR mutation is rapidly developed, too. In this study, we proposed a tumor-associated macrophage (TAM) reprogramming strategy for overcoming the EGFRT790M-associated drug resistance via a dual-targeting codelivery system of gefitinib/vorinostat that acted on both TAM with overexpression of mannose receptors and the HER-2 positive NSCLC cells. The trastuzumab-modified, mannosylated liposomal system was able to repolarize the protumor M2 phenotype to the antitumor M1 and cause the elevating ROS in the cancer cells, consequently modulating the intracellular redox balance via ROS/NOX3/MsrA axis. The suppressed MsrA facilitated the EGFRT790M degradation through 790M oxidation by ROS, thus resensitizing the EGFRT790M-positive cells to gefitinib. The dual-targeting codelivery and TAM-reprogramming strategies provided a potential method for rescuing the EGFRT790M-caused resistance to tyrosine kinase inhibitor treatment.

PKCζ in prostate cancer cells represses the recruitment and M2 polarization of macrophages in the prostate cancer microenvironment

Tumor-associated macrophages are key regulators of the complex interplay between tumor and tumor microenvironment. M2 Macrophages, one type of tumor-associated macrophages, are involved in prostate cancer growth and progression. Protein kinase C zeta has been shown to suppress prostate cancer cell growth, invasion, and metastasis as a tumor suppressor; however, its role in chemotaxis and activation of tumor-associated macrophages remains unclear. Here, we investigated the role of protein kinase C zeta of prostate cancer cells in regulation of macrophage chemotaxis and M2 phenotype activation. Immunohistochemistry was performed to analyze the expression of protein kinase C zeta and the number of CD206+ M2 macrophages in human prostate tissue. Macrophage chemotaxis and polarization were examined using Transwell migration assays and a co-culture system. Quantitative real-time polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay were used to detect M2 markers, protein kinase C zeta, interleukin-4, and interleukin-10 expression. We found the expression of protein kinase C zeta increased in prostate cancer tissues, especially in the early stage, and was negatively associated with tumor grade and the number of CD206+ macrophages. Inhibition of protein kinase C zeta expression in prostate cancer cells promoted chemotaxis of peripheral macrophages and acquisition of M2 phenotypic features. These results were further supported by the finding that silencing of endogenous protein kinase C zeta promoted the expression of prostate cancer cell-derived interleukin-4 and interleukin-10. These results suggest that protein kinase C zeta plays an important role in reducing infiltration of tumor-associated macrophages and activation of a pro-tumor M2 phenotype, which may constitute an important mechanism by which protein kinase C zeta represses cancer progression.

Role of Cyclooxygenase-2 on Intermittent Hypoxia-Induced Lung Tumor Malignancy in a Mouse Model of Sleep Apnea

An adverse role for obstructive sleep apnea (OSA) in cancer epidemiology and outcomes has recently emerged from clinical and animal studies. In animals, intermittent hypoxia (IH) mimicking OSA promotes tumor malignancy both directly and via host immune alterations. We hypothesized that IH could potentiate cancer aggressiveness through activation of the cyclooxygenase-2 (COX-2) pathway and the concomitant increases in prostaglandin E2 (PGE2). The contribution of the COX-2 in IH-induced enhanced tumor malignancy was assessed using celecoxib as a COX-2 specific inhibitor in a murine model of OSA bearing Lewis lung carcinoma (LLC1) tumors. Exposures to IH accelerated tumor progression with a tumor associated macrophages (TAMs) shift towards a pro-tumoral M2 phenotype. Treatment with celecoxib prevented IH-induced adverse tumor outcomes by inhibiting IH-induced M2 polarization of TAMs. Furthermore, TAMs isolated from IH-exposed mice treated with celecoxib reduced the proliferation of LLC1 naïve cells, while the opposite occurred with placebo-treated IH-exposed mice. Finally, in vitro IH exposures of murine macrophages and LLC1 cells showed that both cell types increased PGE2 release in response to IH. These results suggest a crucial role for the COX-2 signaling pathway in the IH-exacerbated malignant processes, and designate macrophages and lung adenocarcinoma cells, as potential sources of PGE2.

Selective upregulation of TNFα expression in classically-activated human monocyte-derived macrophages (M1) through pharmacological interference with V-ATPase

Pharmacological interference with vacuolar-type H(+)-ATPase (V-ATPase), a proton-translocating enzyme involved in protein transport and pH regulation of cell organelles, is considered a potential strategy for cancer therapy. Macrophages are critically involved in tumor progression and may occur as pro-tumoral M2 phenotype, whereas classically-activated M1 can inhibit tumor development for example by releasing tumor-suppressing molecules, including tumor necrosis factor (TNF)α. Here, we show that targeting V-ATPase by selective inhibitors such as archazolid upregulates the expression and secretion of TNFα in lipopolysaccharide (LPS)- or LPS/interferon (INF)γ-activated M1-like macrophages derived from human blood monocytes. In contrast, archazolid failed to elevate TNFα production from uncommitted (M0) or interleukin (IL)-4-treated M2-like macrophages. Secretion of other relevant cytokines (i.e., IL-1β, IL-6, IL-10) or chemokines (i.e. IL-8 and monocyte chemotactic protein-1) from M1 was not affected by archazolid. Though V-ATPase inhibitors elevated the lysosomal pH in M1 comparable to chloroquine or ammonium chloride, the latter agents suppressed TNFα secretion. Archazolid selectively increased TNFα mRNA levels, which was abolished by dexamethasone. Interestingly, archazolid enhanced the phosphorylation and nuclear translocation of the p65 subunit of NFκB and stimulated phosphorylation of SAPK/JNK. In a microfluidically-supported human tumor biochip model, archazolid-treated M1 significantly reduced tumor cell viability. Together, our data show that V-ATPase inhibition selectively upregulates TNFα production in classically-activated macrophages along with NFκB and SAPK/JNK activation. Such increased TNFα release caused by V-ATPase inhibitors may contribute to tumor suppression in addition to direct targeting cancer cells.

Onionin A, a sulfur-containing compound isolated from onions, impairs tumor development and lung metastasis by inhibiting the protumoral and immunosuppressive functions of myeloid cells.

Recent studies have demonstrated that myeloid lineage cells, such as macrophages and myeloid suppressor cells (MDSCs), are major components exhibiting protumoral functions in the setting of tumor progression. Tumor-associated macrophages polarized to the protumoral M2 phenotype promote tumor proliferation and are considered to be a therapeutic target in patients with malignant tumors. We identified a new candidate compound, called onionin A (ONA) isolated from onions, that inhibits macrophage polarization into the M2 phenotype, as well as the immunosuppressive activity of MDSCs and tumor proliferation, by suppressing signal transducer and activator of transcription-3 (Stat3) activation. Furthermore, ONA administration was found to significantly suppress subcutaneous tumor development and lung metastasis in tumor-bearing mice. ONA administration also inhibited Stat3 activation and increased the number of infiltrating lymphocytes in tumor tissues, and an ex vivo analysis showed that the immunosuppressive effect of MDSCs in tumor-bearing mice is impaired by ONA. Moreover, ONA regulated tumor proliferation by inhibiting cell-cell interactions between macrophages and tumor cells, and ONA administration enhanced the antitumor effects of cisplatin in the tumor-bearing mice. These findings demonstrate that ONA may be a potential new tool for antitumor therapy and also for tumor prevention.