THP-1 Conditioned Medium Research Articles

P165 Guselkumab binding to CD64+ IL-23–producing myeloid cells enhances potency for neutralizing IL-23 signaling

Abstract Background IL-23 is implicated in the pathogenesis of inflammatory bowel disease (IBD) and myeloid cells that express FcγRI (CD64) have been identified as the primary cellular source of IL-23 in inflamed IBD gut tissue. Guselkumab (GUS) and risankizumab (RZB) are monoclonal antibodies (mAbs) specifically directed against the IL-23p19 subunit. GUS is a fully human IgG1 mAb with a native Fc region while RZB is a humanized IgG1 mAb with a mutated Fc region. Here, we evaluated CD64 and IL-23 expression in IBD patient gut biopsies, binding of GUS and RZB to CD64, and the functional consequences of CD64 binding by IL-23p19 subunit mAbs, in in vitro assays. Methods IL23A and FCGR1A (CD64) expression was analyzed from bulk and single-cell RNAseq datasets. Binding of mAbs to IFNγ-primed human monocytes, as well as binding to IL-23–secreting inflammatory monocytes and capture of endogenously secreted IL-23, were assessed by flow cytometry. Internalization of IL-23, GUS, and RZB within CD64+ macrophages was evaluated using live cell confocal imaging. Potency of GUS and RZB for inhibiting IL-23 signaling was determined in a co-culture of THP-1 (a CD64+ monocyte cell line activated to produce IL-23) and an IL-23 reporter cell line (measuring biologically active IL-23). Results Analysis of RNAseq datasets showed that FCGR1A, IL23A, and IL12B were significantly increased in inflamed versus non-inflamed IBD gut biopsies and that IL23A was predominantly expressed by FCGR1A-expressing myeloid cells. In in vitro assays GUS, but not RZB, showed Fc-mediated binding to CD64 on IFNγ-primed monocytes. CD64-bound GUS simultaneously captured IL-23 secreted from the same cells. GUS, but not RZB, bound to the surface of CD64+ macrophages and mediated internalization of IL-23 to low pH intracellular compartments. GUS and RZB demonstrated similar potency for inhibiting signaling by IL-23 present in THP-1–conditioned media. However, in a co-culture of IL-23–producing THP-1 cells with an IL-23–responsive reporter cell line, GUS demonstrated enhanced potency compared to RZB for inhibition of IL-23 signaling. Conclusion Our transcriptomic analysis supported previous observations of CD64+ myeloid cells as a key source of IL-23 production in inflamed IBD gut tissue. GUS binding to CD64 on IL-23–producing cells likely contributed to the enhanced functional potency of GUS compared to RZB for inhibition of IL-23 signaling in the co-culture assay. These in vitro data support a hypothesis for optimal enrichment of GUS in inflamed tissues where CD64+ IL-23–producing myeloid cells are increased and in proximity to IL-23–responsive lymphoid cells, enabling GUS to more potently neutralize IL-23 by targeting IL-23 at its source of production.

Antioxidant, Immunostimulatory, and Anticancer Properties of Hydrolyzed Wheat Bran Mediated through Macrophages Stimulation.

Previous studies demonstrated that enzymatic hydrolysis enhances wheat bran (WB) biological properties. This study evaluated the immunostimulatory effect of a WB hydrolysate (HYD) and a mousse enriched with HYD (MH) before and after in vitro digestion on murine and human macrophages. The antiproliferative activity of the harvested macrophage supernatant on colorectal cancer (CRC) cells was also analyzed. MH showed significantly higher content than control mousse (M) in soluble poly- and oligosaccharides (OLSC), as well as total soluble phenolic compounds (TSPC). Although in vitro gastrointestinal digestion slightly reduced the TSPC bioaccessibility of MH, ferulic acid (FA) levels remained stable. HYD showed the highest antioxidant activity followed by MH, which demonstrated a greater antioxidant activity before and after digestion as compared with M. RAW264.7 and THP-1 cells released the highest amounts of pro-inflammatory cytokines after being treated with 0.5 mg/mL of digested WB samples. Treatment with digested HYD-stimulated RAW264.7 supernatant for 96 h showed the most anticancer effect, and spent medium reduced cancer cell colonies more than direct WB sample treatments. Although a lack of inner mitochondrial membrane potential alteration was found, increased Bax:Bcl-2 ratio and caspase-3 expression suggested activation of the mitochondrial apoptotic pathway when CRC cells were treated with macrophage supernatants. Intracellular reactive oxygen species (ROS) were positively correlated with the cell viability in CRC cells exposed to RAW264.7 supernatants (r = 0.78, p < 0.05) but was not correlated in CRC cells treated with THP-1 conditioned media. Supernatant from WB-stimulated THP-1 cells may be able to stimulate ROS production in HT-29 cells, leading to a decrease of viable cells in a time-dependent manner. Therefore, our present study revealed a novel anti-tumour mechanism of HYD through the stimulation of cytokine production in macrophages and the indirect inhibition of cell proliferation, colony formation, and activation of pro-apoptotic proteins expression in CRC cells.

Alpinumisoflavone ameliorates choroidal neovascularisation and fibrosis in age-related macular degeneration in in vitro and in vivo models

Age-related macular degeneration (AMD) is a major cause of vision loss in the elderly population. Anti-vascular endothelial growth factor (VEGF) antibody therapy is applicable to neovascularisation of AMD; however, the prevention of fibrosis after anti-VEGF monotherapy is an unmet medical need. Subretinal fibrosis causes vision loss in neovascular age-related macular degeneration (nAMD) even with anti-VEGF therapy. We report the anti-fibrotic and anti-neovascularisation effects of alpinumisoflavone (AIF), an isoflavonoid derived from unripe Maclura tricuspidata fruit, in in vitro and in vivo models. For in vitro study, we treated H2O2 or THP-1 conditioned media (TCM) following activation with phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS) in a human retinal pigment epithelial cell line (ARPE-19). Choroidal neovascularisation (CNV) was induced by laser photocoagulation in mice, immediately followed by intravitreal administration of 25 μg AIF. CNV area and fibrosis were measured 7 days after laser photocoagulation. AIF showed anti-fibrosis and anti-neovascularisation effects in both the models. The laser induced CNV area was reduced upon AIF administration in nAMD mouse model. Additionally, AIF decreased the levels of the cleaved form of crystallin alpha B (CRYAB), a chaperone associated with VEGF stabilisation and fibrosis. Our results demonstrate a novel therapeutic application of AIF against neovascularisation and fibrosis in nAMD.

Microfluidic-assisted electrospinning, an alternative to coaxial, as a controlled dual drug release system to treat inflammatory arthritic diseases

Inflammatory arthritic diseases are characterized by a persistent inflammation of the synovial tissues where tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) pro-inflammatory cytokines are over-expressed, leading to progressive musculoskeletal disability. Methotrexate (MTX), a disease-modifying-anti-rheumatic drug (DMARD) commonly applied in their treatment, can be used in combination with biological-DMARDs as anti-TNFα antibody to improve the treatments efficacy. However, their systemic administration comes with severe side-effects and limited therapeutic efficacy due to their off-target distribution and short half-life. To overcome such limitations, encapsulation of clinically relevant concentrations of MTX and anti-TNFα antibody into polycaprolactone (PCL) or poly(vinyl-alcohol) (PVA) microfluidic-assisted or coaxial electrospun fibrous meshes is proposed as local controlled dual drug release systems.Release studies show that microfluidic-assisted electrospinning meshes encapsulating both drugs achieved higher concentrations than coaxials. Biological assays using human articular chondrocytes (hACs) and monocytic cells (THP-1 cell line) demonstrate that fibrous meshes encapsulating the drugs are non-toxic. The systems' efficacy is proved by a significant decrease of TNFα and IL-6 concentrations in conditioned medium of lipopolysaccharide (LPS)-stimulated THP-1 cells, especially in the presence of microfluidic-assisted electrospun meshes, when compared with THP-1 conditioned medium (59.5% and 83.9% less, respectively).Therefore, microfluidic-assisted electrospinning fibrous meshes with encapsulating drugs represent an alternative to coaxial, as a local therapy for inflammatory arthritis diseases.

B-lymphoma moloney murine leukemia virus insertion region 1 promotes epithelial-mesenchymal transition and invasion of colorectal cancer cell HT-29 cells in inflammatory microenvironment

Objective Investigate the effect and mechanism of B-lymphoma Moloney murine leukemia virus insertion region 1 (Bmi-1) on tumor migration and epithelial-mesenchymal transition (EMT) of HT-29 cells in inflammatory microenvironment. Methods Detect the gene and protein levels of Bmi-1, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by qPCR and Western blotingt respectively, and the levels of TNF-α and IL-6 in culture medium supernatant by enzyme linked immunosorbent assay (ELISA). Evaluate the proliferation, migration and invasion in HT-29 cells by MTT assay and Transwell assays. EMT-related markers and nuclear factor-κB (NF-κB) pathway key proteins were detected by Western blotting. Results The mRNA levels of Bmi-1, TNF-α and IL-6 was significantly increased in HT29 cells after THP-1 Conditioned-Medium (THP-1-CM) stimulation (1.86±0.08 vs.1.00±0.05, 2.38±0.13 vs.1.00±0.14, 3.00±0.15 vs.1.00±0.05, P<0.01, respectively), as well as the protein levels[1.68±0.08 vs.1.00±0.05, (73±4) pg/ml vs. (32±4) pg/ml, (140±9) pg/ml vs. (42±9) pg/ml, P<0.01, respectively]. Knockdown of Bmi-1 expression remarkably repressed the secretion of cytokines[ (31±3), (56±8) pg/ml]. THP-1-CM accelerated the protein synthesis of vimentin, N-cadherin and phosphorylation of NF-κB, enhanced invasion and metastasis. Inversely, knockdown of Bmi-1 expression remarkably repressed THP-1-CM-induced EMT and NF-κB activity, but had no effect on cell proliferation in vitro. Conclusion Bmi-1 may be involved in the cytokines secretion of HT-29 cells induced by THP-1-CM, and has effect on migration and invasion through epithelial-mesenchymal transformation, which is related to NF-κB pathway activity. Bmi-1 can be treated as a potential molecular target for metastatic colorectal cancer. Key words: B-lymphoma moloney murine leukemia virus insertion region 1 gene; Tumor inflammatory microenvironment; Metastatic colorectal cancer

Increased infiltration of macrophages to radioresistant lung cancer cells contributes to the development of the additional resistance of tumor cells to the cytotoxic effects of NK cells.

In this study, in order to investigate the effects of increased macrophage infiltration to radioresistant lung tumors in regulating natural killer (NK) cell-mediated immunity, we examined whether the treatment of radioresistant cells with conditioned medium (CM) from phorbol myristate acetate(PMA)/interleukin(IL)-4 treated THP-1 cells (used as a tumor-associated macrophage source) leads to the development of the additional resistance of tumor cells to NK cell cytotoxicity. We found that the susceptibility of THP-1 CM-treated radioresistant cells to NK cell cytotoxicity was decreased compared to the non-treated cells. In addition, it was found that such a decreased susceptibility was associated with increased programmed death receptor ligand1(PD-L1) and decreased natural killer group2D(NKG2D) ligand levels in tumor cells. We further discovered that the THP-1 cells secreted a high level of IL-6, and that blocking IL-6 action by the addition of a neutralizing antibody(Ab) for IL-6 into the THP-1 CM decreased the resistance of THP-1 CM-treated radioresistant cells to NK cell cytotoxicity. Moreover, we discovered that MEK/Erk was the most critical IL-6 downstream signaling pathway in triggering the THP-1 CM effect; thus, the addition of MEK/Erk inhibitor to THP-1 CM enhanced the susceptibility of the THP-1 CM-treated radioresistant cells to NK cell cytotoxicity. On the whole, the findings of this study suggest the existence of a malignant loop characterized by increased macrophage infiltration into radioresistant cells which, in turn, promotes the development of the additional resistance of these cells to NK cell cytotoxicity.

Macrophage inflammatory factors promote epithelial-mesenchymal transition in breast cancer.

The majority of breast cancers (90–95%) arise due to mediators distinct from inherited genetic mutations. One major mediator of breast cancer involves chronic inflammation. M1 macrophages are an integral component of chronic inflammation and the breast cancer tumor microenvironment (TME). Previous studies have demonstrated that up to 50% of the breast tumor comprise of tumor-associated macrophages (TAMs) and increased TAM infiltration has been associated with poor patient prognosis. Furthermore, breast cancer associated deaths are predominantly attributed to invasive cancers and metastasis with epithelial-mesenchymal transition (EMT) being implicated. In this study, we investigated the effects of cellular crosstalk between TAMs and breast cancer using an in vitro model system. M1 polarized THP-1 macrophage conditioned media (CM) was generated and used to evaluate cellular and functional changes of breast cancer lines T47D and MCF-7. We observed that T47D and MCF-7 exhibited a partial EMT phenotype in the presence of activated THP-1 CM. Additionally, MCF-7 displayed a significant increase in migratory and invasive properties. We conclude that M1 secretory factors can promote a partial EMT of epithelial-like breast cancer cells. The targeting of M1 macrophages or their secretory components may inhibit EMT and limit the invasive potential of breast cancer.

Convenience versus Biological Significance: Are PMA-Differentiated THP-1 Cells a Reliable Substitute for Blood-Derived Macrophages When Studying in Vitro Polarization?

Human peripheral-blood monocytes are used as an established in vitro system for generating macrophages. For several reasons, monocytic cell lines such as THP-1 have been considered as a possible alternative. In view of their distinct developmental origins and phenotypic attributes, we set out to assess the extent to which human monocyte-derived macrophages (MDMs) and phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells were overlapping across a variety of responses to activating stimuli. Resting (M0) macrophages were polarized toward M1 or M2 phenotypes by 48-h incubation with LPS (1 μg/ml) and IFN-γ (10 ng/ml) or with IL-4 (20 ng/ml) and IL-13 (5 ng/ml), respectively. At the end of stimulation, MDMs displayed more pronounced changes in marker gene expression than THP-1. Upon assaying an array of 41 cytokines, chemokines and growth factors in conditioned media (CM) using the Luminex technology, secretion of 29 out of the 41 proteins was affected by polarized activation. While in 12 of them THP-1 and MDM showed comparable trends, for the remaining 17 proteins their responses to activating stimuli did markedly differ. Quantitative comparison for selected analytes confirmed this pattern. In terms of phenotypic activation markers, measured by flow cytometry, M1 response was similar but the established MDM M2 marker CD163 was undetectable in THP-1 cells. In a beads-based assay, MDM activation did not induce significant changes, whereas M2 activation of THP-1 decreased phagocytic activity compared to M0 and M1. In further biological activity tests, both MDM and THP-1 CM failed to affect proliferation of mouse myogenic progenitors, whereas they both reduced adipogenic differentiation of mouse fibro-adipogenic progenitor cells (M2 to a lesser extent than M1 and M0). Finally, migration of human umbilical vein endothelial cells was enhanced by CM irrespective of cell type and activation state except for M0 CM from MDMs. In summary, PMA-differentiated THP-1 macrophages did not entirely reproduce the response spectrum of primary MDMs to activating stimuli. We suggest that THP-1 be regarded as a simplified model of human macrophages when investigating relatively straightforward biological processes, such as polarization and its functional implications, but not as an alternative source in more comprehensive immunopharmacology and drug screening programs.

Abstract P5-03-06: Novel targets of breast cancer associated with inflammatory tumor microenvironment

Abstract Breast cancer affects one in eight women in the USA. Considerable progress in the identification of genetic lesions and their modulation has resulted in newer therapies making breast cancer a manageable disease. However, triple negative breast cancer is still difficult to treat and warrants a search for newer targets. To this end, we focused our attention towards the modulation of the breast cancer epithelium by other cell types such as the endothelial cells and the macrophages. The migratory macrophages and the estrogen sensitive migratory endothelial progenitor cells (EPCs) constitute the cellular milieu within the tumor microenvironment which continuously modulates breast cancer epithelium. We analyzed the interactions of the breast cancer cell lines (MCF-7 and MDA-MB-231) with the highly proliferative human umbilical cord derived CD133+/CD34+/VEGFR-2+ EPCs and M1 polarized macrophages (activated THP-1 cell line) in two separate in vitro studies. The readouts were cell proliferation, changes in epithelial to mesenchymal transition (EMT), and cellular differentiation. We observed morphological and cellular growth changes in the EPCs on treatment with conditioned medium (CM) generated from breast cancer cells, consistent with vasculogenesis and in vitro tubulogenesis. Both, MDA-MB-231 and MCF-7 CM, treatments resulted in enhanced EPCs proliferation and differentiation. However, the differentiation patterns were distinct, with MCF-7 CM increasing the number of cell clusters, whereas MDA-MB-231 CM increasing the number of adherent spindle shaped cells. The paracrine interaction was also assessed with M1 polarized macrophages. We observed decreased cell viability in MCF-7 and MDA-MB-231 cells following activated THP-1 CM and exosome treatments. Analysis of exosomes from activated THP-1 indicated an upregulation of 13 miRNAs compared to unactivated THP-1. The miRNA hsa-miR-146a-5p had the highest upregulation (44 fold increase). This specific miRNA has been observed in senescent cell and it inhibits cell proliferation, suggesting a possible mechanism for exosome-associated growth inhibition. The analysis of the paracrine interactive mediators between breast cancer cells, EPCs, and M1 polarized macrophages is likely to yield viable novel clinically translatable therapeutic targets. Citation Format: Tiwari RK, Ben Rahoma G, Tuli N, Bednarczyk R, Maniyar RR, Chakraborty S, Singh S, Mittelman A, Geliebter J. Novel targets of breast cancer associated with inflammatory tumor microenvironment [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-03-06.

In vitro-induced M2 type macrophages induces the resistance of prostate cancer cells to cytotoxic action of NK cells

Previous reports, including our experimental results, showed that macrophages migrate to prostate cancer (PCa) cells. We tested whether the migrated macrophages affect the susceptibility of castration-resistant PCa (CRPC) cells to cytotoxic actions of natural killer (NK) cells. We found treatment of tumor cells with the conditioned media (CM) of the PMA/IL-4 treated THP-1 cells (M2 type macrophages) (THP-1 CM) decreased the susceptibility of tumor cells to NK cell cytotoxicity, as a result of increased programmed death receptor ligand 1 (PD-L1) and decreased NK group 2D (NKG2D) ligands in CRPC cells. Meanwhile, the decreased susceptibility of tumor cells was also detected when NK cells were treated with THP-1 CM and used in NK cell cytotoxicity tests. Therefore, we observed higher resistance of CRPC cells when both tumor and NK cells were treated with THP-1 CM than when tumor cells or NK cells were individually treated. We further discovered that the PMA/IL-4 treated THP-1 cells secrete a high level of IL-6, so blocking the IL-6 action significantly decreased the PD-L1 level while recovering the NKG2D ligands, thus increasing the susceptibility of CRPC cells to NK cell action. Moreover, we discovered that JAK-Stat3 is the most critical IL-6 downstream signaling in triggering the THP-1 CM effect. Consequently, we found the susceptibility of CRPC cells to NK cells was increased when either JAK or Stat 3 inhibitor was added when tumor cells were treated with THP-1 CM, and that the best effect was observed when the JAK inhibitor and PD-L1 Ab were added together.

Abstract TMEM-022: LYSOPHOSPHATIDIC ACID IN OVARIAN CANCER EPITHELIAL–TO–MESENCHYMAL TRANSITION

Abstract Lysophosphatidic acid (LPA) is a bioactive phospholipid found in elevated concentrations in ovarian cancer (OVCA) patients. The objective of this study was to define the mechanism of action by which LPA contributes to ovarian carcinogenesis focusing on epithelial-to-mesenchymal transition (EMT) and role of human macrophages. THP-1 human macrophages and A27801AP human OVCA cells were used and treated with LPA up to 40 µM for 24 h. In THP-1 human macrophages, LPA treatment led to a dose-dependent increase in IL-1β secretion from 333.5 pg/mL (untreated) to 581.9, 661.8 and 1270.1 pg/mL for 10, 20 and 40 µM LPA treatment. We then used the conditioned medium (CM) from THP-1 macrophages to determine the effect on A27801AP OVCA cells. LPA-treated THP-1 CM increased the expression of EMT transcription factors p65, Slug and Snail as compared to non-LPA-treated THP-1 CM and resting OVCA cells. When A27801AP OVCA cells were treated with LPA alone, 20 µM LPA led to the same significant increase in the expression of EMT transcription factors p65, Slug and Snail. In addition, LPA treatment reduced the expression of the epithelial marker E-cadherin while increasing the expression of mesenchymal markers N-cadherin and vimentin which might be associated with increased phosphorylation of extracellular regulated kinase 1/2. The data show the role of LPA in modifying not only OVCA cells but also macrophages that can be found in the tumor microenvironment to promote progression of OVCA by promoting EMT. Citation Format: Vermont P Dia, Ph D. LYSOPHOSPHATIDIC ACID IN OVARIAN CANCER EPITHELIAL–TO–MESENCHYMAL TRANSITION [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-022.

Baicalein reduces angiogenesis in the inflammatory microenvironment via inhibiting the expression of AP-1.

Increasing clinical and experimental studies have demonstrated that refractory chronic inflammation will result in malignant tumor and anti-angiogenic therapy may be an effective way to thwart the progression. Baicalein, one of the major active flavanoids found in Scutellaria baicalensis Georgi, has been exhibited potent anti-inflammation and anti-tumor effects by reducing angiogenesis. However, the exact mechanism of baicalein on endothelial cells in inflammatory microenvironment was not clear yet. Here, we investigated the anti-angiogenic effect of baicalein by incubating human umbilical vein endothelial cells (HUVECs) with THP-1 conditioned medium in vitro. The tube formation of HUVECs and microvessel outgrowth of rat aorta were attenuated, as well as the number of newly formed blood vessels in chicken chorioallantoic membrane (CAM) was reduced by baicalein. This anti-angiogenic effect was mainly on account of the inhibited motility, migration and invasion of HUVECs. In addition, mechanistic studies showed that baicalein could bind to AP-1 directly and the expression of c-Jun and c-Fos in HUVECs was reduced, accompanied by their increased proteasomal degradation. Besides, baicalein suppressed the nuclear translation, heterodimer formation and DNA binding affinity of c-Jun and c-Fos. What's more, the anti-angiogenic effect of baicalein was further confirmed by matrigel plug assay in vivo. Taken together, our study demonstrated that baicalein could exert its anti-angiogenic effect in the inflammation microenvironment via inhibiting the transcriptional activity of AP-1, which suggested that baicalein might be an alternative treatment against refractory chronic inflammation.

Abstract 5105: Exosomes secreted by proinflammatory macrophages modulate breast cancer

Abstract Multiple cell types reside within the tumor microenvironment (TME) including cancer stem cells, stromal cells, and inflammatory cells. Cellular crosstalk is initiated between these cells through a variety of secreted biomolecules such as cytokines, growth factors, and exosomes. The role of inflammation and numerous secretory factors within the TME have been implicated in the development and progression of various cancers including breast. In this study, we wanted to evaluate the effect of macrophages (a major constituent of inflammation) on breast cancer development. Our study focused on macrophage secreted exosomes which are membraneous nanovesicles that contain various miRNA, mRNA, and proteins. We hypothesized that macrophage secreted exosomes will modulate breast cancer phenotype. An in vitro cell culture system was used in this study to examine macrophage exosome treatments on breast cancer cell lines MCF-7 (ER+) and MDA-MB 231 (ER-, PR-, HER2/neu-). Human monocytic THP-1 monocytes were activated with a common phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) and conditioned media (CM) was collected following 48 h. Exosomes were isolated from THP-1 CM via the use of an exosome isolation reagent and standard centrifugation. Breast cancer cells were then treated with THP-1 exosomes and cell morphology, proliferation, MEK/ERK and PI3K/AKT signaling pathways, and cell cycle were evaluated. MCF-7 and MDA-MB 231 exhibited round and spindle-like morphologies as well as a significant decrease in proliferation at 24 h treatments. Furthermore, MDA-MB 231 displayed increased p-ERK, while MCF-7 showed increased p-MEK following Western blot analysis. Both cell lines exhibited decreased p-AKT and p-S6 kinase expression. Cell cycle analysis showed that MCF-7 and MDA-MB 231 arrested at G2/M at 24 h exosome treatments. THP-1 exosome treatments led to a significant increase in MDA-MB 231 senescence as determined by a senescence assay looking at senescence-associated beta-galactosidase. Increased p21 and decreased Cdc2/Cyclin B1 was also observed which indicates a G2/M arrest. We conclude that specific macrophage secreted exosomes may induce breast cancer cell senescence and affect breast cancer progression. Citation Format: Robert Bronislaw Bednarczyk, Neha Y. Tuli, Ghada Ben Rahoma, Rachana Maniyar, Abraham Mittelman, Raj K. Tiwari. Exosomes secreted by proinflammatory macrophages modulate breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5105.

Abstract B04: Tumor-associated macrophages support tumor angiogenesis via CXCL16 signaling in papillary thyroid carcinoma

Abstract Purpose: Tumor-associated macrophages (TAMs) are an important player in tumor microenvironments. We investigated the role of TAMs in angiogenesis of papillary thyroid cancer (PTC). Experimental Design: Human endothelial, HUVEC cells were treated with conditioned medium of PTC (BHP10-3SC)/macrophage (THP-1) co-cultures and cell migration potentials and tube formations were evaluated. Conditioned medium were analyzed by cytokine array. Results: Treatment of BHP10-3SC/THP-1 conditioned medium significantly increased HUVEC cell migrations and tube formation potentials compared to that of BHP10-3SC or THP-1 conditioned mediums. Cytokine array demonstrated that BHP10-3SC/THP-1 conditioned medium contained high level of CXCL16 while it did not expressed in BHP10-3SC alone conditioned medium. Treatment of CXCL16 increased HUVEC cell migrations and tube formation potentials, and blocking CXCL16 signaling using neutralizing antibodies reduced BHP10-3SC/THP-1 conditioned medium effects on HUVEC cell tube formation potentials. Conclusions: TAM angiogenesis in PTC tumor microenvironment, in part, through CXCL16 signaling. Citation Format: Sun Wook Cho, Hyun Jin Sun, Young Shin Song, Young Joo Park. Tumor-associated macrophages support tumor angiogenesis via CXCL16 signaling in papillary thyroid carcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B04.

Abstract 5087: Precise characterization of macrophage secretory exosomes can lead to novel therapeutic approaches

Abstract Multiple cell types interact within the tumor microenvironment (TME) through crosstalk events that are mediated by many biomolecules including cytokines, mRNA, and miRNA-containing exosomes that can influence cancer progression as well as suppression. Inflammation within the TME is a major determinant in tumor progression. Since tumor associated macrophages (TAMs) are a major cellular factor in tumor inflammation, we hypothesized that crosstalk events initiated by macrophage secreted factors such as cytokines and exosomes will modulate breast cancer phenotype. We evaluated the cytokine profile of a human monocytic cell line, THP-1, following stimulation with a phorbol ester TPA (12-O-Tetradecanoylphorbol-13-acetate) in vitro. Activated THP-1 cells exhibited an M1 macrophage phenotype. In addition, we assessed the morphological and cellular growth changes of luminal (T47D, MCF-7) and basal-like (MDA-MB 231) breast cancer cell lines in vitro following treatment with activated THP-1 conditioned medium (CM). Each cell line displayed mesenchymal-like features and decreased cellular growth following activated THP-1 CM treatment. MCF-7 and MDA-MB 231 displayed increased MEK/ERK signaling, while all three cell lines showed increased p-AKT expression. Furthermore, epithelial-like T47D and MCF-7 cells exhibited an increase in EMT transcription factor expression. Moreover, although T47D and MCF-7 did not show drastic changes in E-cadherin expression via western blot analysis, immunofluorescence examination showed a decrease in E-cadherin following activated THP-1 CM treatments emphasizing a possible partial EMT event. Lastly, activated THP-1 exosomes were isolated and used to treat MDA-MB 231 to determine the effects on breast cancer cell proliferation and senescence. MDA-MB 231 exhibited significantly decreased cellular growth and increased senescence following incubation with activated THP-1 exosomes. The variety of secretory factors within the TME as a result of inflammation may contribute to tumor suppression as well as tumor progression via a secretion of cytokines and exosomes. Citation Format: Robert Bronislaw Bednarczyk, Yuki Kitadai, Neha Y. Tuli, Elyse K. Hanly, Ghada Benrahoma, Abraham Mittelman, Raj K. Tiwari. Precise characterization of macrophage secretory exosomes can lead to novel therapeutic approaches. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5087. doi:10.1158/1538-7445.AM2015-5087

Abstract 1171: Proinflammatory chemokine interleukin-8 in the tumor microenvironment modulates breast cancer phenotype

Abstract Cancer growth and progression is influenced by tumor cells, stromal cells, as well as various secreted molecules found within the tumor microenvironment (TME). These secreted molecules participate in various crosstalk events that contribute to cancer development and progression. One such secreted molecule found within the tumor microenvironment is the proinflammatory cytokine interleukin-8 (IL-8). IL-8 influences cancer development by binding to its G-protein coupled receptors CXCR1 and CXCR2 in turn leading to activation of various signal transduction pathways that promotes cell proliferation, survival, angiogenesis, and invasion. We hypothesized that infiltrating macrophages and tumor cells participate in cellular crosstalk through the release of inflammatory cytokines which function to modulate the breast cancer phenotype. Using a cell culture model, conditioned media (CM) generated from activated THP-1 monocytes as well as recombinant IL-8 (rIL-8) were individually tested on the breast cancer cell lines T47D (ER +) and MDA-MB 231 (ER -) in order to evaluate cell proliferation, cell signaling pathways, invasion, and migration. Increased proliferation was observed for both T47D and MDA-MB 231 following treatment with rIL-8 as opposed to an inhibition in proliferation, which was observed following culturing of T47D and MDA-MB 231 with THP-1 CM. It is presumed that THP-1 CM contains both inhibitory and stimulatory factors, which include IL-8. Activation of the MAPK signaling pathway, as observed by phosphorylation of MEK and ERK, was modulated in response to activated THP-1 CM, which may explain the enhanced migration and invasion observed in the breast cancer cells upon treatment with rIL-8. In order to further characterize the crosstalk between THP-1 and breast cancer cells, the inverse experiments were performed by incubating non-activated THP-1 monocytes with CM generated from MDA-MB 231 cells. Interestingly, MDA-MB 231 CM was capable of activating THP-1 monocytes comparable to that of the classical activating agent phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA). Characterization of the MDA-MB 231 CM by cytokine array analysis revealed an abundance of the proinflammatory cytokines IL-6 and IL-8 and although this needs further characterization, we believe that secretion of IL-6 and IL-8 is essential to the crosstalk between breast cancer cells and THP-1 monocytes, which modulates the breast cancer cell phenotype. Citation Format: Robert Bednarczyk, Neha Tuli, Ghada Benrahoma, Rui Kitadai, Robert Suriano, Abraham Mittelman, Raj K. Tiwari. Proinflammatory chemokine interleukin-8 in the tumor microenvironment modulates breast cancer phenotype. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1171. doi:10.1158/1538-7445.AM2014-1171

Abstract 1432: CXCL8 is a secretory inflammatory stimulus of the activated TME that modulates breast cancer phenotype.

Abstract The tumor microenvironment (TME) is a heterogeneous niche consisting of numerous stromal cells, structural components, and secreted molecules from both the malignant and surrounding cells that contribute to tumor growth and progression. Importantly, these diverse types of secreted molecules can initiate crosstalk events between the tumor cells and TME. One such molecule, CXCL8/Interleukin-8 (IL-8), is a proinflammatory chemokine, primarily involved in neutrophil mobilization, which activates multiple cellular signaling pathways via the membrane associated G protein-coupled CXCL8 cognate receptors, CXCR1 and CXCR2. We hypothesized that IL-8, a ubiquitous inflammatory molecule that is secreted by macrophages, activated fibroblasts and mesenchymal cells, can alter breast cancer phenotype. To this end, using a cell culture model we analyzed both estrogen receptor (ER) positive and ER negative breast cancer cell lines T47D and MDA-MB-231 respectively that were found to express the CXCL8 receptors CXCR1 and CXCR2. The cellular source of IL-8 was from conditioned media (CM) from in vitro cultures of primary stromal cells including primary mesenchymal and fibroblast cells, as well as the established monocytic cell lines HL-60 and THP-1. Each cell line secreted adequate amounts of IL-8 as measured by ELISA, however primary mesenchymal cells produced four times that of the fibroblasts and macrophages suggesting that activated mesenchymal cells may be a very potent source of chronic inflammation in the TME. Interestingly, treatment with IL-8 or CM from the phorbol ester treated macrophages THP-1 led to increased proliferation in T47D in vitro however only IL-8 treatment led to proliferation of MDA-MB-231. Recombinant IL-8 as well as CM of THP-1 and mesenchymal cells enhanced migration and invasion of both T47D and MDA-MB-231 cells as evaluated by a modified Boyden chamber assay. Supplementation of the triple negative MDA-MB-231 line with THP-1 CM enhanced its invasive propensity nearly two fold. THP-1 CM treatment also led to the activation of the PI3K/p-Akt and Mek/Erk pathways in the ER− MDA-MB-231 cells but not the ER+ T47D cells suggesting a link between CXCL8 mediated inflammation and constitutive signal transduction pathways that is translatable into enhanced metastatic propensity. Our studies are directed towards the analysis of both chronic and acute inflammation of the TME and we believe IL-8 and its associated intracellular molecular markers may be important preventive and therapeutic targets in triple negative breast cancer. Citation Format: Andrea L. George, Robert Bednarczyk, Shilpi Rajoria, Elyse Hanly, Robert Suriano, Abraham Mittelman, Raj K. Tiwari. CXCL8 is a secretory inflammatory stimulus of the activated TME that modulates breast cancer phenotype. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1432. doi:10.1158/1538-7445.AM2013-1432

The role of myofibroblasts in upregulation of S100A8 and S100A9 and the differentiation of myeloid cells in the colorectal cancer microenvironment

Background/aimS100A8/A9 and myeloid cells in the tumor microenvironment play an important role in cancer invasion and progression, and the effect of tumor-infiltrated myofibroblasts on myeloid cells in the tumor microenvironment is relatively unknown. Accordingly, we investigated the role of myofibroblasts in the upregulation of S100A8/A9 as well as in the differentiation of myeloid cells in the colorectal cancer (CRC) microenvironment. Materials and methodsTo investigate the interactions among cancer cells, myofibroblasts, and inflammatory cells in the microenvironment of CRC, we used 10 CRC cell lines, 18CO cells and THP-1 cells, which were co-cultured with each other or cultured in conditioned media (CM) of other cells. Expression of S100A8/A9 was evaluated via Western blot, immunohistochemical staining and immunofluorescence. The secreted factors from the cell lines were analyzed using cytokine antibody array. Flow cytometry analysis was performed to analyze the differentiation markers of myeloid cells. Results18CO CM induced increased expression of S100A8/A9 in THP-1 cells. Increased expression of S100A8/A9 was noted in inflammatory cells of the peri- and intra-tumoral areas, along with myofibroblasts in colon cancer tissue. S100A8/A9-expressing inflammatory cells also exhibited CD68 expression in colon cancer tissue, and 18CO CM induced differentiation of THP-1 cells into myeloid-derived suppressor cells (MDSCs) or M2 macrophages expressing S100A8/A9. Significant amounts of IL-6 and IL-8 were detected in 18CO CM, compared to those in both controls and THP-1 CM, and tumor-infiltrated myofibroblasts expressed IL-8 in colon cancer tissue. Finally, neutralizing antibodies to IL-6 and IL-8 attenuated 18CO CM-induced increased expression of S100A8/A9. ConclusionsThe upregulation of S100A8/A9 in tumor-infiltrated myeloid cells could be triggered by IL-6 and IL-8 released from myofibroblasts, and myofibroblasts might induce the differentiation of myeloid cells into S100A8/9-expressing MDSCs or M2 macrophages in the CRC microenvironment.

Regulation of human monocyte proMMP-9 production by fetuin, an endogenous TGF-? antagonist

Members of the matrix metalloproteinase family of enzymes degrade specific components of the extracellular matrix. MMP-9 is a type IV/V collagenase necessary for normal osteogenesis and is increased in inflammatory and neoplastic conditions. In such destructive diseases as emphysema and arthritis, and in epithelial cancers, MMP-9 is produced by cells of the monocyte lineage. Fetuin, a prominent serum glycoprotein, binds to and inactivates TGF-beta family members and through this mechanism regulates osteogenesis (Binkert et al., 1999, J Biol Chem 274:28514-28520.). We studied the effects of TGF-beta1 and fetuin on proMMP-9 release by the human monocyte line THP-1. Exogenous TGF-beta1 stimulated proMMP-9 release by THP-1 cells, with half-maximal stimulation at approximately 0.01 ng/ml TGF-beta1. Human fetuin (0.5-2 microM) partially inhibited this stimulation. Human fetuin alone stimulated THP-1 monocyte proMMP-9 release, with half maximal stimulation at approximately 0.25 microM fetuin. Neutralizing antibody specific for TGF-beta1 also stimulated proMMP-9 release, suggesting that endogenously-derived TGF-beta1 has an inhibitory effect. In freshly isolated human peripheral blood monocytes, fetuin stimulated proMMP-9 release with a dose-response curve similar to that observed in THP-1 cells. Human fetuin also activated proMMP-9 present in THP-1 conditioned medium. Taken together, these data suggest that under physiological conditions, fetuin facilitates matrix degradation by monocyte-derived MMP-9, both by opposing the autocrine inhibitory effect of endogenous TGF-beta1 on proMMP-9 release, and by activating proMMP-9 in the pericellular milieu. Conversely, fetuin may limit the stimulation of monocyte proMMP-9 release by high levels of exogenous TGF-beta1. Such modulation could prove important under pathological conditions where TGF-beta1 derived from paracrine sources is abundant, such as in epithelial malignancies.

Interleukin-1β Secreted from Monocytic Cells Induces the Expression of Matrilysin in the Prostatic Cell Line LNCaP

Matrilysin is a matrix metalloprotease that is overexpressed in cancer cells of epithelial origin and in normal tissues during events involving matrix remodeling such as the cycling endometrium. We previously observed that inflamed ductule and acinar epithelia in the prostate also overexpress matrilysin. The presence of infiltrating macrophages in these areas prompted us to determine if factors secreted from monocytes could induce matrilysin expression in a human prostatic cell line. Conditioned media collected from the monocyte cell line THP-1 following lipopolysaccharide treatment substantially induced matrilysin protein and mRNA expression in LNCaP prostate carcinoma cells. Matrilysin expression in LNCaP cells was also induced by recombinant interleukin (IL)-1 (50 pM), but not by equimolar concentrations of recombinant tumor necrosis factor-alpha or IL-6. The matrilysin-inducing activity of THP-1 conditioned medium was completely abrogated by preincubation with a neutralizing antibody to IL-1beta. Transient transfection analyses with a chimeric human matrilysin promoter-chloramphenicol acetyltransferase reporter construct demonstrated that IL-1beta activates transcription through the matrilysin promoter in LNCaP cells. This is the first report of matrilysin induction by an inflammatory cytokine in a cell line of epithelial origin, and the results suggest a potential mechanism for the overexpression of matrilysin in inflamed ducts and glands of the prostate.