Multidimensional pooled shRNA screens in human THP-1 cells identify candidate modulators of macrophage polarization

Surdziel, Ewa; Clay, Ieuan; Nigsch, Florian; Thiemeyer, Anke; Allard, Cyril; Hoffman, Gregory R.; Reece‐Hoyes, John S.; Phadke, Tanushree; Gambert, Romain; Keller, Caroline Gubser; Ludwig, Marie-Gabrielle; Baumgarten, Birgit; Frederiksen, Mathias; Schübeler, Dirk; Seuwen, Klaus; Bouwmeester, Tewis; Fodor, Barna D.

doi:10.1371/journal.pone.0183679

Cited by 39 publications

(53 citation statements)

References 62 publications

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“…To analyze the protein expression of OPN across cell types, cells were seeded in six‐well plates at a density of 100 000 cells/well and grown for 2 days. THP‐1 cells were differentiated into M0 macrophages by adding 60 ng/mL phorbol myristate acetate to the growth medium for 2 days followed by resting in normal growth medium for another day 32 . Cells were washed with ice‐cold PBS two times and then 100 µL radioimmunoprecipitation assay (RIPA) buffer (120 mM NaCl, 50 mM; pH 8.0; Tris 0.5% NP‐40; 1 mM EGTA) containing protease and phosphatase inhibitors (100 µg/mL phenylmethylsulfonyl fluoride/PMSF, 1 mM NaOrVa, 50 µg/mL aprotinin, 50 µg/mL leupeptin, all from Fisher Scientific) was added to the cells for 10 to 15 minutes.…”

Section: Methodssupporting

(Expert classified)

Prostatic osteopontin expression is associated with symptomatic benign prostatic hyperplasia

et al. 2020

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Background: Male lower urinary tract symptoms (LUTS) occur in more than half of men above 50 years of age. LUTS were traditionally attributed to benign prostatic hyperplasia (BPH) and therefore the clinical terminology often uses LUTS and BPH interchangeably. More recently, LUTS were also linked to fibrogenic and inflammatory processes. We tested whether osteopontin (OPN), a proinflammatory and profibrotic molecule, is increased in symptomatic BPH. We also tested whether prostate epithelial and stromal cells secrete OPN in response to proinflammatory stimuli and identified downstream targets of OPN in prostate stromal cells. Methods: Immunohistochemistry was performed on prostate sections obtained from the transition zone of patients who underwent surgery (Holmium laser enucleation of the prostate) to relieve LUTS (surgical BPH, S-BPH) or patients who underwent radical prostatectomy to remove low-grade prostate cancer (incidental BPH, I-BPH). Images of stained tissue sections were captured with a Nuance Multispectral Imaging System and histoscore, as a measure of OPN staining intensity, was determined with inForm software. OPN protein abundance was determined by Western blot analysis. The ability of prostate cells to secrete osteopontin in response to IL-1β and TGF-β1 was determined in stromal (BHPrS-1) and epithelial (NHPrE-1 and BHPrE-1) cells by enzyme-linked immunosorbent assay. Quantitative polymerase chain reaction was used to measure gene expression changes in these cells in response to OPN. Results: OPN immunostaining and protein levels were more abundant in S-BPH than I-BPH. Staining was distributed across all cell types with the highest levels in epithelial cells. Multiple OPN protein variants were identified in immortalized prostate stromal and epithelial cells. TGF-β1 stimulated OPN secretion by NHPrE-1 cells and both IL-1β and TGF-β1 stimulated OPN secretion by BHPrS-1 cells. Interestingly, recombinant OPN increased the mRNA expression of CXCL1, CXCL2, CXCL8, PTGS2, and IL6 in BHPrS-1, but not in epithelial cell lines. DK104310; start-up fund provided by the Department of Urology, Case Western Reserve UniversityConclusions: OPN is more abundant in prostates of men with S-BPH compared to men with I-BPH. OPN secretion is stimulated by proinflammatory cytokines, and OPN acts directly on stromal cells to drive the synthesis of proinflammatory mRNAs.Pharmacological manipulation of prostatic OPN may have the potential to reduce LUTS by inhibiting both inflammatory and fibrotic pathways. K E Y W O R D S chemokine, cytokine, fibrosis, prostatic inflammation Tissue collection was approved by the Institutional Review Board at Vanderbilt University Medical Center (VUMC), and analysis of specimens at Case Western Reserve University and the University of Wisconsin-Madison (UW-M). Deidentified prostate tissues were acquired from the VUMC BPH Tissue and Data Biorepository and their 732 | POPOVICS ET AL.

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Section: Methodssupporting

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Prostatic osteopontin expression is associated with symptomatic benign prostatic hyperplasia

et al. 2020

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“…Increased cell and nucleus size is observed in co-cultured cells, consistent with reported morphological differences between human monocytes and activated macrophages ( Fig 6A) 76 . Expression of M1-like and M2-like macrophage signature genes demonstrate heterogeneity in macrophage phenotype for human co-cultures 77,78 . 24 hours of co-culture significantly upregulated only IL1B, while expression of TNF and PTGS2 significantly decreased in 48 hour co-cultures ( Fig 6B).…”

Section: Figure 4: Metabolic Autofluorescence Imaging Of Mouse Macropmentioning

confidence: 99%

Autofluorescence imaging of 3D tumor-macrophage microscale cultures resolves spatial and temporal dynamics of macrophage metabolism

Heaster

Humayun

et al. 2020

Preprint

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Macrophages within the tumor microenvironment (TME) exhibit a spectrum of pro-tumor and antitumor functions, yet it is unclear how the TME regulates this macrophage heterogeneity. Standard methods to measure macrophage heterogeneity require destructive processing, limiting spatiotemporal studies of function within the live, intact 3D TME. Here, we demonstrate twophoton autofluorescence imaging of NAD(P)H and FAD to non-destructively resolve spatiotemporal metabolic heterogeneity of individual macrophages within 3D microscale TME models. Fluorescence lifetimes and intensities of NAD(P)H and FAD were acquired at 24, 48, and 72 hours post-stimulation for mouse macrophages (RAW 264.7) stimulated with IFN-γ or IL-4 plus IL-13 in 2D culture, validating that autofluorescence measurements capture known metabolic phenotypes. To quantify metabolic dynamics of macrophages within the TME, mouse macrophages or human monocytes (RAW264.7 or THP-1) were cultured alone or with breast cancer cells (mouse PyVMT or primary human IDC) in 3D microfluidic platforms. Human monocytes and mouse macrophages in tumor co-cultures exhibited significantly different FAD mean lifetimes and greater migration than mono-cultures at 24, 48, and 72 hours post-seeding. In co-cultures with primary human cancer cells, actively-migrating monocyte-derived macrophages had greater redox ratios (NAD(P)H/FAD intensity) compared to passively-migrating monocytes at 24 and 48 hours post-seeding, reflecting metabolic heterogeneity in this subpopulation of monocytes. Genetic analyses further confirmed this metabolic heterogeneity. These results establish label-free autofluorescence imaging to quantify dynamic metabolism, polarization, and migration of macrophages at single-cell resolution within 3D microscale models. This combined culture and imaging system provides unique insights into spatiotemporal tumorimmune crosstalk within the 3D TME.

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“…To analyze the protein expression of OPN across cell types, cells were seeded in 6-well plates at a density of 100,000 cells/well and grown for 2 days. THP-1 cells were differentiated into M0 macrophages by adding 60 ng/ml phorbol myristate acetate to the growth medium for 2 days followed by resting in normal growth medium for another day 29 . Cells were washed with ice cold PBS 2 times and then 100 µl RIPA buffer (120mM NaCl, 50 mM pH 8.0 Tris, 0.5% NP-40, 1 mM EGTA) containing protease and phosphatase inhibitors (100 µg/mL PMSF, 1 mM NaOrVa, 50 µg/mL aprotinin, 50µg/mL leupeptin, all from Fisher Scientific) was added to the cells for 10-15 min.…”

Section: Western Blotmentioning

confidence: 99%

“…produced by an alternative translation initiation site 30,37 . To compare OPN variants expressed in prostate cells to those produced by macrophages, we evaluated OPN variants in THP-1 cells monocytes, before and after converting them into M0 macrophages with phorbol 12-myristate 13acetate 29 . OPN variants in undifferentiated THP-1 cells were the same as those detected in prostate stromal and epithelial cells ( Figure 3B).…”

Section: Macrophages Express Various Opn Splice Variants Including Thmentioning

confidence: 99%

Prostatic osteopontin expression is associated with symptomatic benign prostatic hyperplasia

Popovics

Awadallah

Kohrt

et al. 2019

Preprint

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AbstractBackgroundMale lower urinary tract symptoms (LUTS) occur in more than half of men above 50 years of age. LUTS were traditionally attributed to benign prostatic hyperplasia (BPH) and therefore the clinical terminology often use LUTS and BPH interchangeably. More recently, LUTS were also linked to fibrogenic and inflammatory processes. We tested whether osteopontin (OPN), a pro-inflammatory and pro-fibrotic molecule, is increased in symptomatic BPH. We also tested whether prostate epithelial and stromal cells secrete OPN in response to pro-inflammatory stimuli and identified downstream targets of OPN in prostate stromal cells.MethodsImmunohistochemistry was performed on prostate sections obtained from the transition zone (TZ) of patients who underwent surgery (Holmium laser enucleation of the prostate) to relieve LUTS i.e. surgical BPH (S-BPH) or patients who underwent radical prostatectomy to remove low-grade prostate cancer (incidental BPH, I-BPH). Images of stained tissue sections were captured with a Nuance Multispectral Imaging system and histoscore, as a measure of OPN staining intensity, was determined with inForm software. OPN protein abundance was determined by Western blot. The ability of prostate cells to secrete osteopontin in response to IL-1β and TGF-β1 was determined in stromal (BHPrS-1) and epithelial (NHPrE-1 and BHPrE-1) cells by ELISA. qPCR was used to measure gene expression changes in these cells in response to OPN.ResultsOPN immunostaining (p=0.0107) and protein levels were more abundant in S-BPH than I-BPH. Staining was distributed across all cell types with highest levels in epithelial cells. Multiple OPN protein variants were identified in immortalized prostate stromal and epithelial cells. TGF-β1 stimulated OPN secretion by NHPrE-1 cells and both IL-1β and TGF-β1 stimulated OPN secretion by BHPrS-1 cells. Interestingly, recombinant OPN increased the mRNA expression of CXCL1, CXCL2, CXCL8, PTGS2 and IL6 in BHPrS-1, but not in epithelial cell lines.ConclusionsOPN is more abundant in prostates of men with S-BPH compared to men with I-BPH. OPN secretion is stimulated by pro-inflammatory cytokines, and OPN acts directly on stromal cells to drive the synthesis of pro-inflammatory mRNAs. Pharmacological manipulation of prostatic OPN may have the potential to reduce LUTS by inhibiting both inflammatory and fibrotic pathways.

show abstract

Multidimensional pooled shRNA screens in human THP-1 cells identify candidate modulators of macrophage polarization

Cited by 39 publications

References 62 publications

Prostatic osteopontin expression is associated with symptomatic benign prostatic hyperplasia

Prostatic osteopontin expression is associated with symptomatic benign prostatic hyperplasia

Autofluorescence imaging of 3D tumor-macrophage microscale cultures resolves spatial and temporal dynamics of macrophage metabolism

Prostatic osteopontin expression is associated with symptomatic benign prostatic hyperplasia

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