Journal: Cancer Science

Article Title: EGFR inhibition in EGFR ‐mutant lung cancer cells perturbs innate immune signaling pathways in the tumor microenvironment

doi: 10.1111/cas.15701

Figure Lengend Snippet: CD24 expression is induced in EGFR‐mutant NSCLC cells upon EGFR‐TKI treatment. (A) Differential gene expression between EGFR‐TKI‐treated versus ‐untreated EGFR‐mutant NSCLC cells in GSE75308 (left) and GSE57156 (right) were analyzed. (B) EGFR‐mutant PC9 and H1975 cells, and EGFR‐wild‐type RERF‐LC‐Ad1 and H522 cells were treated with osimertinib for 72 h in vitro. CD24 expression was analyzed by flow cytometry. Gray: isotype control; dotted line: untreated; line: treated. Data are representative of four independent experiments. (C) Numbers of CD24 molecules expressed on cells with or without EGFR‐TKI treatment in vitro. Data are presented as mean ± SEM from four independent experiments. Numbers of the receptors were calculated using the BD QuantiBrite kit as described in Materials and Methods. (D) CD24 gene expression in tumor cells with or without EGFR‐TKI treatment in vitro were analyzed by qRT‐PCR. Data are presented as mean ± SEM of technical replicates from at least two independent experiments. (E) EGFR‐mutant PC9 and EGFR‐wild‐type H522 cells were treated either with osimertinib, gefitinib, or afatinib for 72 h in vitro. CD24 expression was analyzed by flow cytometry. Data are representative of two independent experiments. * q < 0.01.

Article Snippet: The lung cancer cell line, PC9, was obtained from the European Collection of Authenticated Cell Cultures.

Techniques: Expressing, Mutagenesis, Gene Expression, In Vitro, Flow Cytometry, Control, Quantitative RT-PCR

Journal: Cancer Science

Article Title: EGFR inhibition in EGFR ‐mutant lung cancer cells perturbs innate immune signaling pathways in the tumor microenvironment

doi: 10.1111/cas.15701

Figure Lengend Snippet: CD24 induction in EGFR‐mutant NSCLC cells is a target of antibody‐dependent cellular phagocytosis. (A) The fraction of immune cell types in the tumor microenvironment of NSCLC from the TCGA‐LUAD dataset (B) Arbitrary gene expression levels of SIGLEC‐10 and MRC1 across macrophages in the tumor microenvironment of NSCLC (C) Siglec‐10 expression was analyzed by flow cytometry. Gray: isotype control, line: Siglec‐10. Human monocyte‐derived macrophages were differentiated according to the protocols shown in Supplementary Table . Data are a representative of at least two independent experiments from three healthy donors. (D) M‐CSF differentiated macrophages and EGFR‐TKI‐treated PC9 cells were incubated for 9 h with anti‐CD24 antibodies or isotype control antibodies. Cytochalasin D was used to inhibit macrophage phagocytosis as described in Materials and Methods. Data are presented as the mean ± SEM of technical replicates from at least two independent experiments. *** p < 0.001.

Article Snippet: The lung cancer cell line, PC9, was obtained from the European Collection of Authenticated Cell Cultures.

Techniques: Mutagenesis, Gene Expression, Expressing, Flow Cytometry, Control, Derivative Assay, Incubation

Journal: Cancer Science

Article Title: EGFR inhibition in EGFR ‐mutant lung cancer cells perturbs innate immune signaling pathways in the tumor microenvironment

doi: 10.1111/cas.15701

Figure Lengend Snippet: EGFR‐TKIs accelerate the release of the cell‐free DNA activating type I IFN response in a STING‐dependent manner. (A) EGFR‐mutant tumor cells were treated with either osimertinib (EGFR‐TKI) or paclitaxel. Data are presented as the mean ± SEM of technical triplicates from at least two independent experiments. (B) cfDNA concentrations in the supernatant were measured as described in Materials and Methods. Data are presented as the mean ± SEM of technical triplicates from at least two independent experiments. (C) THP‐1‐Dual and THP‐1‐Dual STING‐knockout (KO) cells were cultured either with LPS (500 ng/ml), IFNa2a (10,000 U/ml), tumor‐derived cfDNA (PC9 and H1975 cfDNA)/lipofectamine, or lipofectamine alone. IRF (left) and NF‐κB (right) activities were analyzed as described in Materials and Methods. (D) PMA‐differentiated THP‐1 monocytes were pretreated either with lipofectamine, poly(dA:dT) (100 ng/ml), tumor‐derived cfDNA (PC9) (100 ng/ml)/lipofectamine, or IFNa2a (20,000 U/ml) followed by IFN‐γ stimulation (100 ng/ml). (E) Expression levels of CXCL9 and CXCL10 were analyzed using flow cytometry. * p < 0.05, ** p < 0.01, **** p < 0.0001, * q < 0.01. ns, not significant.

Article Snippet: The lung cancer cell line, PC9, was obtained from the European Collection of Authenticated Cell Cultures.

Techniques: Mutagenesis, Knock-Out, Cell Culture, Derivative Assay, Expressing, Flow Cytometry

Journal:

Article Title: Tumorigenic Role of Orphan Nuclear Receptor NR0B1 in Lung Adenocarcinoma

doi: 10.2353/ajpath.2009.090010

Figure Lengend Snippet: High NR0B1 expression in SP of A549. A: Dot blot analysis of A549 cells stained with Hoechst 33342 dye in the absence (left) or presence (right) of verapamil. SP and MP cells were boxed. B: Quantitative real-time RT-PCR was performed with mRNA obtained from SP and MP of A549. The amount of NR0B1 mRNA was normalized for the amount of GAPDH mRNA. The values represent the mean ± SE of three experiments. *P < 0.01 by the Student’s t-test.

Article Snippet: The human lung adenocarcinoma cell lines A549 and PC-14 were purchased from the American Type Culture Collection (Rockville, MD) and the Riken Bioresource Center (Tsukuba, Japan), respectively.

Techniques: Expressing, Dot Blot, Staining, Quantitative RT-PCR

Journal:

Article Title: Tumorigenic Role of Orphan Nuclear Receptor NR0B1 in Lung Adenocarcinoma

doi: 10.2353/ajpath.2009.090010

Figure Lengend Snippet: Expression of apoptosis- and invasion-related genes in NR0B1-knocked down A549 cells. A: Semiquantitative RT-PCR. B: Real-time quantitative RT-PCR to examine the expression level of Bcl-2 and MMP-2. The value is shown as the mean ± SE. *P < 0.01 by the Student’s t-test.

Article Snippet: The human lung adenocarcinoma cell lines A549 and PC-14 were purchased from the American Type Culture Collection (Rockville, MD) and the Riken Bioresource Center (Tsukuba, Japan), respectively.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Cancer Immunology, Immunotherapy

Article Title: The expression of PD-1 ligand 1 on macrophages and its clinical impacts and mechanisms in lung adenocarcinoma

doi: 10.1007/s00262-022-03187-4

Figure Lengend Snippet: Effect of anti-cancer compounds in cell lines. A schematic diagram of the cell cultures and methods ( A ). A549 and PC9 cells were cultured with dimethyl sulfoxide (DMSO), paclitaxel (PTX), docetaxel (DOC), carboplatin (CBDCA), and pemetrexed (PMET) for 24 h at the same concentration (40 µM), and the mRNA expression of GM-CSF was evaluated by real-time polymerase chain reaction ( B ). The cells were subsequently cultured for another day, and the concentration of GM-CSF in the medium was tested by ELISA ( C ). Macrophages were stimulated with the CM of control PC9 cells or PEMT-treated PC9 cells, and surface PD-L1 expression was detected by fluorescence-activated cell sorting ( D ). Macrophages were stimulated with the CM of PEMT-treated PC9 cells with control immunoglobulin G or anti-GM-CSF antibody (20 µg/mL) (E). *: statistically significant (n = 3 to 4 each), p value < 0.05

Article Snippet: A549 and PC9 were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan).

Techniques: Cell Culture, Concentration Assay, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Control, Fluorescence, FACS

Journal: Antioxidants

Article Title: H 2 S-Synthesizing Enzymes Are Putative Determinants in Lung Cancer Management toward Personalized Medicine

doi: 10.3390/antiox13010051

Figure Lengend Snippet: NSCLC cell lines present cysteine reliance on H 2 S production. ( A ) NSCLC cell lines showed similar basal levels of H 2 S production. To verify if H 2 S production was directly dependent on cysteine degradation, cells were exposed to AOAA/PAG treatment. ( B ) NSCLC cells cultured in the presence of the H 2 S donor NaHS alone and in combination with cysteine showed that H 2 S levels decreased when exposed to AOAA and PAG. Upon cysteine treatment, PC-9 showed a tendency to decrease the average levels of H 2 S in control conditions and compensated production of H 2 S upon exposure to inhibitors. ( C ) Upon cysteine supplementation, NSCLC cell lines tended to decrease H 2 S production at T = 0 h but compensated the CBS and CSE inhibition, maintaining (H292 and PC-9) or increasing (A549) H 2 S levels upon AOAA and PAG exposure. Analysis of H 2 S at T = 0 h indicated that A549 and H292 cell lines presented decreased levels of H 2 S in the presence of cysteine with and without glycolysis inhibition with BPA, while the PC-9 cell line presented no significant changes in all conditions. ( D ) Analysis of H 2 S levels showed that all cell lines presented a similar basal ability to produce H 2 S. Maximum score ( E ) or the average levels ( F ) of ATP production indicated that A549 cells increased the production of ATP upon cysteine supplementation independently of the presence of inhibitors, while H292 and PC-9 were able to maintain ATP levels in all experimental conditions. Maximum ATP score and average levels indicated that A549 cells increased the levels of ATP upon BPA and/or cysteine supplementation, and H292 and PC-9 cells maintained the ATP levels in all culture conditions independently of glycolysis inhibition. All data were normalized to the control condition and are represented as mean ± SD. * p < 0.5, ** p < 0.01, **** p < 0.0001.

Article Snippet: Human adenocarcinoma cell line A549 (CCL-185™), mucoepidermoid carcinoma cell line H292 (CRL-1848™), tubular renal cell line HK2 (CRL-2190™), and keratinocyte cell line HaCaT (PCS-200-011™) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and adenocarcinoma cell line PC-9 (90071810) was obtained from European Collection of Authenticated Cell Cultures (ECACC, Porton Down, Salisbury, UK).

Techniques: Cell Culture, Inhibition

Journal: Antioxidants

Article Title: H 2 S-Synthesizing Enzymes Are Putative Determinants in Lung Cancer Management toward Personalized Medicine

doi: 10.3390/antiox13010051

Figure Lengend Snippet: NSCLC cells express distinct expression patterns of enzymes and transporters involved in cysteine metabolism. ( A ) mRNA expression level analysis showed that H292 cells expressed noteworthy levels of CBS and SLC7A11 compared to A549 cells, while PC-9 cells showed low overall expression of CTH , MPST , SLC1A1, and SLC7A11 . ( B ) Immunofluorescence analysis showed that protein expression followed mRNA expression patterns, reporting similar differences. ( C ) mRNA expression level analysis further showed that H292 cells highly expressed GOT2 , while PC-9 showed high expression levels of GOT2 and CDO1 compared to A549 cells. All mRNA expression level data are relative to HPRT1 and represented as mean ± SD. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human adenocarcinoma cell line A549 (CCL-185™), mucoepidermoid carcinoma cell line H292 (CRL-1848™), tubular renal cell line HK2 (CRL-2190™), and keratinocyte cell line HaCaT (PCS-200-011™) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and adenocarcinoma cell line PC-9 (90071810) was obtained from European Collection of Authenticated Cell Cultures (ECACC, Porton Down, Salisbury, UK).

Techniques: Expressing, Immunofluorescence

Journal: Antioxidants

Article Title: H 2 S-Synthesizing Enzymes Are Putative Determinants in Lung Cancer Management toward Personalized Medicine

doi: 10.3390/antiox13010051

Figure Lengend Snippet: Distinct molecular backgrounds induce individual metabolic patterns in NSCLC cells. ( A – C ) 1 H-Nuclear magnetic resonance (NMR) of the NSCLC panel studied indicated alterations between A549, H292, and PC-9 regarding levels of intracellular amino acids and peptides, sugars and organic acids, and other metabolites. ( D – F ) NMR of the NSCLC panel studied further indicated alterations between A549, H292, and PC-9 regarding levels of extracellular amino acids and peptides and sugars and organic acids. Data are represented as mean ± SD. * p < 0.5, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ( G , H ) PCA showed no differences in the endometabolome ( G ), but the exometabolome ( H ) indicated that A549 and PC-9 cells present distinct metabolic profiles, and H292 cells had common metabolic patterns with A549 and PC-9 cells. ( I ) PLS-DA analysis of the exometabolome allowed the discrimination of the three cell lines. From the PLS-DA analysis of the metabolites present in the cell media, it was possible to discriminate PC-9 from the other cell lines in the first component, while A549 could be discriminated in the second component (upper panel), Q 2 = 0.873. ( J ) The loading plot (lower panel) showing the metabolites important for the discrimination, colored by VIP in the first component. 2-Oxoisocaproate increased in A549, while aspartate, lactate, phosphocholine, and glycerophosphocholine increased in PC-9, making them important for discrimination between these cell lines. ( K ) From the PLS-DA analysis of the metabolites present in the cell media, it was possible to discriminate between A549 and PC-9 cell lines (upper panel), Q 2 = 0.975. ( L ) The loading plot (lower panel) showing the metabolites important for the discrimination, colored by VIP in the first component. ( M ) Metabolites significantly different between PC-9 and A549. Volcano plot (fold change >2 and p value < 0.05) of the intracellular metabolites between A549 and PC-9. Arginine, proline, and nicotinurate were significantly increased in A549 and guanosine in PC-9. ( N ) Nicotinurate was only present in A549 cells. Box plot of nicotinurate intracellular concentrations in the three cell lines (ANOVA analysis p value = 0.00028097 × 10 4 .

Article Snippet: Human adenocarcinoma cell line A549 (CCL-185™), mucoepidermoid carcinoma cell line H292 (CRL-1848™), tubular renal cell line HK2 (CRL-2190™), and keratinocyte cell line HaCaT (PCS-200-011™) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and adenocarcinoma cell line PC-9 (90071810) was obtained from European Collection of Authenticated Cell Cultures (ECACC, Porton Down, Salisbury, UK).

Techniques: Nuclear Magnetic Resonance

Journal: Antioxidants

Article Title: H 2 S-Synthesizing Enzymes Are Putative Determinants in Lung Cancer Management toward Personalized Medicine

doi: 10.3390/antiox13010051

Figure Lengend Snippet: NSCLC cell lines are chemoresistant, and SeChry@PURE G4 -FA induces decreased cell viability in NSCLC cells, with specificity toward tumor cells rather than nontumoral cells. ( A ) NSCLC cell lines exposed to the most commonly used therapy regimens showed overall resistance to these drugs, except for cisplatin. ( B ) EC 50 curves and values for SeChry, Sechry@PURE G4- FA, and PURE G4- FA in NSCLC indicated a higher sensitivity of A549 and H292 to the treatment than PC-9. Empty nanoparticles (PURE G4. FA) did not induce relevant toxicity on cell viability. ( C ) EC 50 curves and values for SeChry, Sechry@PURE G4- FA, and PURE G4- FA in noncancer cell lines (HaCaT and HK2) indicated no effect on cell viability. Empty nanoparticles (PURE G4. FA) did not induce relevant toxicity on cell viability. ( D ) Detection of FR-α by immunofluorescence indicated high protein levels in NSCLC but not in nontumoral cell lines. FR- α is labeled in green, and nuclei were counterstained with DAPI (blue). Magnification 400×, scale 20 µm. *** p < 0.001, **** p < 0.0001.

Article Snippet: Human adenocarcinoma cell line A549 (CCL-185™), mucoepidermoid carcinoma cell line H292 (CRL-1848™), tubular renal cell line HK2 (CRL-2190™), and keratinocyte cell line HaCaT (PCS-200-011™) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and adenocarcinoma cell line PC-9 (90071810) was obtained from European Collection of Authenticated Cell Cultures (ECACC, Porton Down, Salisbury, UK).

Techniques: Immunofluorescence, Labeling