Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) BTSCs were subjected to immunoblotting analysis using the antibodies indicated on the blots. wtEGFR and EGFRvIII bands are marked with * and **, respectively. ( b ) Densitometric quantification of galectin1 protein level normalized to tubulin in different BTSC lines is shown. ( c-d ) EGFR / EGFRvIII KD (si EGFR ) and control BTSCs (siCTL) were analyzed by immunoblotting as described in a. ( e-h ) BTSCs were treated with 1 or 5 µM lapatinib and galectin1 expression was assessed by immunoblotting (e-f) and immunostaining (g-h). Nuclei were stained with DAPI. Scale bar = 10 μm. ( i ) BTSCs were subjected to immunoblotting analysis using the antibodies indicated on the blots. ( j ) Pearson correlation analysis of pSTAT3-Y705 and galectin1 protein expression in different BTSCs is shown. ( k-l ) STAT3 KD (si STAT3 ) and siCTL BTSCs were analyzed by immunoblotting as described above. ( m-p ) BTSCs were subjected to immunoblotting or immunostaining following treatment with 25 or 50 µM of the STAT3 inhibitor, S3I-201. Scale bar = 10 μm. ( q-s ) EGFRvIII-expressing BTSCs were subjected to ChIP using an antibody to STAT3 or IgG control followed by qPCR using two different pairs of primers ( LGALS1 -a and LGALS1 -b). OSMR , and HPRT loci were used as positive and negative controls, respectively. ( t-u ) Luciferase reporter assay was performed in BTSC73 following KD of STAT3 using siRNA (t) or treatment with STAT3 inhibitors, 5 µM WP1066 or 50 μM S3I-201 (u). Data are presented as the mean□±□SEM, n ≥ 3. Unpaired two-tailed t -test (q, r and s); one-way ANOVA followed by Dunnett’s test (b) or Tukey’s test (t and u),*p < 0.05, **p < 0.01, ***p < 0.001. See also Figures S1 and S2.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: Western Blot, Expressing, Immunostaining, Staining, Luciferase, Reporter Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a-b ) Cell viability was assessed by CellTiter-Glo assay in LGALS1 CRISPR and CTL BTSCs. ( c ) Population growth curves for LGALS1 CRISPR and CTL BTSC73 are shown. ( d-f ) Cell viability assay (d-e) and population growth curves (f) of BTSC73 treated with 1 or 10 µM OTX008 are shown. ( g ) Representative images of EdU staining in LGALS1 CRISPR and CTL BTSC73 are shown. ( h ) The number of EdU positive cells was quantified using Fiji software. ( i ) EdU incorporation was analyzed by flow cytometry in LGALS1 CRISPR and CTL BTSC73. Representative scatter plots of flow cytometry analyses are shown. Data are presented as the mean□±□SEM, n = 3. Unpaired two-tailed t -test (a, b, c and h); one-way ANOVA followed by Dunnett’s test (d, e and f), **p < 0.01, ***p < 0.001. See also Figures S3 and S4.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: Glo Assay, CRISPR, Viability Assay, Staining, Software, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a-b ) LGALS1 CRISPR or CTL BTSC73 were subcutaneously injected into SCID mice. Representative bioluminescence real-time images tracing tumour growth are shown (a). Graph represents tumour mass (b). ( c-f ) BTSC73 or BTSC147 were injected subcutaneously into SCID mice and treated with 10 mg/kg OTX008. Representative bioluminescence real-time images tracing tumour growth are shown (c, e). Graphs represent tumour mass (d, f). ( g-j ) LGALS1 CRISPR or CTL BTSC73 were intracranially injected into SCID mice. Representative bioluminescence real-time images tracing tumour growth are shown (g). Intensities of luciferase signal were quantified at different time points using Xenogen IVIS software (h). Graph represents quantification of animal weight (i). KM survival plot was graphed to evaluate mice lifespan in each group (j). Data are presented as the mean□±μSEM, n ≥ 4 mice. Unpaired two-tailed t -test (b, d, f, h and i); log-rank test (j), **p < 0.01, ***p < 0.001.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: CRISPR, Injection, Luciferase, Software, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) Volcano plot representing LGALS1 differentially regulated genes is shown. ( b-c ) GSEA analysis demonstrates enrichment for gene sets corresponding to mesenchymal (b) and proneural (c) subtypes of glioblastoma. ( d ) GSEA analysis demonstrates enrichment for gene sets corresponding to mesenchymal-like meta-module (MES1-like) signature. ( e-f ) GSEA analysis demonstrates enrichment for gene sets corresponding to recruitment of NuMA to mitotic centrosomes (e) and mitotic G2−G2/M phases (f). ( g-h ) RNA-seq data was validated by RT-qPCR in BTSC73 and BTSC147. ( i-j ) Cell cycle distribution was assessed by flow cytometry after PI staining in LGALS1 CRISPR BTSCs. Data are presented as the mean□±□SEM, n = 3. One-way ANOVA followed by Dunnett’s test (g and h); unpaired two- tailed t -test (i and j), *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S5.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Flow Cytometry, Staining, CRISPR, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a-d ) LGALS1 CRISPR and CTL EGFRvIII-expressing BTSCs were subjected to LDA (a-b) or ELDA (c-d). ( e-f ) EGFRvIII-expressing LGALS1 CRISPR and CTL BTSCs were subjected to clonogenicity assay performed by culturing one single cell per well. ( g-h ) BTSCs that don’t harbour the EGFRvIII mutation were electroporated with siCTL or si LGALS1 and subjected for ELDA analysis. ( i-p ) EGFRvIII-expressing BTSCs were subjected to LDA (i, j, m and n) or ELDA (k, l, o and p) following the treatment with 1 or 10 µM OTX008. ( q-t ) BTSCs that don’t harbour the EGFRvIII mutation were subjected to LDA (q-r) or ELDA (s-t) following the treatment with 1 or 10 µM OTX008. *p < 0.05, **p < 0.01, ***p < 0.001; unpaired two-tailed t -test (a, b, e and f); one-way ANOVA followed by Dunnett’s test (i, j, m and n), n = 3. Data are presented as the mean□±□SEM. See also Figure S6.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: CRISPR, Expressing, Mutagenesis, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) ELDA was performed following 4 Gy of IR in LGALS1 CRISPR or CTL BTSCs. ( b-c ) LGALS1 CRISPR and CTL BTSC73 were subjected to IR (8□Gy). Apoptosis analysis was performed by flow cytometry 48□h following IR using annexin V and PI double staining. Representative scatter plots of flow cytometry analyses are shown (b). The percentage of cell death (annexin V positive cells) is presented in the histogram (c), n□=□3. ( d ) Schematic diagram of the experimental procedure is shown. BTSC73 were intracranially injected into SCID mice and then treated with OTX008, 4□Gy of IR or a combination of OTX008 and IR. ( e ) Representative bioluminescence real-time images tracing tumour growth are shown, n□=□6 mice. ( f ) Coronal sections of mouse brains were stained with hematoxylin and eosin on day 22 after injection. Representative images of 3 different tumour sections are shown. Scale bar = 1□mm, scale bar (inset) = 0.2 mm. ( g ) Intensities of luciferase signal were quantified at different time points, n = 6 mice. ( h ) KM survival plot was graphed to assess animal lifespan, n□=□6 mice. ( i ) Survival extension of mice bearing BTSC-derived tumours treated with OTX008, IR, or OTX008 + IR relative to those treated with the vehicle control. Data are presented as the mean□±□SEM. One-way ANOVA followed by Tukey’s test (c and i); log-rank test (h), *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: CRISPR, Flow Cytometry, Double Staining, Injection, Staining, Luciferase, Derivative Assay

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) LGALS1 -differentially regulated genes were subjected to enrichment analysis of TF binding motifs using oPOSSUM-3 software. ( b ) Volcano plot representing the HOXA5 target genes among the LGALS1 -differentially-regulated genes is shown. ( c ) BTSCs were analyzed by immunoblotting using the antibodies indicated on the blots. ( d ) Pearson correlation analysis of HOXA5 and galectin1 protein expression is shown. ( e ) KM survival plot describing the association between LGALS1 and HOXA5 expression and the survival of glioblastoma patients is shown. ( f ) Relative positions of HOXA5 ChIP-seq peaks to the adjacent TSS of LGALS1 -differentially regulated genes are shown. The x-axis indicates the distance between peak centers and the TSS of adjacent LGALS1 -differentially regulated genes. The y-axis denotes the expression ratios (log2) of the LGALS1 -differentially regulated gene. Circle size indicates HOXA5 peak height, and color denotes the conservation score of HOXA5 peaks. ( g-h ) HOXA5 KD (si HOXA5 ) and siCTL BTSCs were subjected to RT-qPCR analysis. ( i ) ELDA was performed following 4LGy of IR in si HOXA5 vs. siCTL. ( j - m ) Endogenous Co-IP experiments were performed in different BTSC lines using an anti-HOXA5 antibody, followed by immunoblotting with galectin1 and HOXA5 antibodies. ( n ) Co-IP experiment was performed using anti-FLAG antibody, followed by immunoblotting with anti-FLAG and anti-HOXA5 antibodies. ( o - r ) PLA of galectin1 and HOXA5 were performed in different BTSC lines. Primary antibodies were omitted for the controls. Nuclei were stained with DAPI. Scale bar = 10 μm. ( s ) LGALS1 CRISPR and CTL BTSC73 were subjected to ChIP using an antibody to HOXA5 followed by qPCR for HOXA5 candidate target genes. HBB locus was used as a negative control. ( t-u ) KM survival plot describing the association between LGALS1 and HOXA5 expression and the survival of glioblastoma patients treated with radiotherapy (microarray G4502A Agilent, level 3, n = 489). Data are presented as the meanL±LSEM, n = 3. Log-rank test (e, t and u); one-way ANOVA followed by Dunnett’s test (g and h); unpaired two-tailed t -test (s). *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S7.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: Binding Assay, Software, Western Blot, Expressing, ChIP-sequencing, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Staining, CRISPR, Negative Control, Microarray, Two Tailed Test

Journal: Thrombosis Update

Article Title: Negatively charged phospholipids stimulate factor XI activation by thrombin

doi: 10.1016/j.tru.2020.100022

Figure Lengend Snippet: Fig. 6. Binding of thrombin and FXI to phospholipids. Extruded PS/PC vesicles (40%/60% M/M) were immobilized on a L1 chip. Increasing concentrations of thrombin (0–1000 nM) (A) or FXI (0–100 nM) (B), or a fixed concentration of thrombin (IIa, 500 nM) in combination with thrombin aptamer HD1 or HD22 (500 mM) (C) were perfused over the chip and the binding response was recorded as described under Methods.

Article Snippet: Phospholipid vesicles (PS/PC, 40%/60%M/M) prepared by sonication were extruded through a liposome extruder with a 200-nm filter (Avestin Europe GmbH, Mannheim, Germany) and immobilized on a L1 sensor chip (GE Healthcare) to ~5000 resonance units.

Techniques: Binding Assay, Concentration Assay

Journal: Advanced Science

Article Title: M 6 A‐mediated upregulation of lncRNA TUG1 in liver cancer cells regulates the antitumor response of CD8 + T cells and phagocytosis of macrophages

doi: 10.1002/advs.202400695

Figure Lengend Snippet: Tug1 in tumor cells regulates the antitumor immune response of CD8 + T cells and phagocytosis of macrophages through PD‐L1 and CD47, respectively. A) The correlation of TUG1 with PD‐L1 and CD47 in HCC patients ( n = 371). B) The correlation of METTL3 with PD‐L1 and CD47 in HCC patients ( n = 371). C) The expressions of PD‐L1 and CD47 at the protein and mRNA levels in the control and sh‐METTL3 HepG2 cells. D) The expressions of Pdl1 and Cd47 at the protein and mRNA levels in the control and sh‐Mettl3 Hepa1‐6 cells. E) Representative images of IHC staining and mean intensities of PD‐L1 and CD47 ( n = 5. Magnification: 40 ×). F–H) The frequencies of IFN‐γ, TNF‐α, and GzmB in CD8 + T cells co‐cultured with sh‐NC or sh‐Tug1 Hepa1‐6 cells ( n = 3). I) Representative images from immunofluorescence (IF) staining of peritoneal cavity‐derived macrophages and BMDMs engulfing cancer cells. The white arrows indicate macrophages that phagocytose cancer cells. Macrophages are shown in red (F4/80 + ), cancer cells are shown in green (GFP + ) and nuclei are shown in blue (DAPI). Magnification: 100 ×. J, K) Statistical analysis of phagocytosis by macrophages as detected via IF staining ( n = 3). L, M) Representative plots and statistical analysis of phagocytosis by macrophages derived from the peritoneal cavity and bone marrow as detected using a flow cytometer ( n = 3). Results are represented as the mean ± SEM. Data were statistically analyzed using unpaired two‐tailed Student's t‐test (C‐H, J‐M) or Spearman's correlation analysis (A‐B).*, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Anti‐ Pdl1 antibodies (Clone No.10F.9G2, BioXcell) or IgG isotype control were given intraperitoneally at 200 μg/day every 4 days.

Techniques: Control, Immunohistochemistry, Cell Culture, Immunofluorescence, Staining, Derivative Assay, Flow Cytometry, Two Tailed Test

Journal: Advanced Science

Article Title: M 6 A‐mediated upregulation of lncRNA TUG1 in liver cancer cells regulates the antitumor response of CD8 + T cells and phagocytosis of macrophages

doi: 10.1002/advs.202400695

Figure Lengend Snippet: Tug1 acts as a microRNA sponge to promote PD‐L1 and CD47 expressions, thereby regulating the antitumor immune response of CD8 + T cells and phagocytosis of macrophages. A) The luciferase reporter assays verify the interaction sites of miR‐141 with Tug1 and Pdl1 ( n = 3). B) The expressions of Tug1, miR‐141, and Pdl1 in the control and si‐Tug1‐transfected Hepa1‐6 cells. C) The expression of miR‐141 and Pdl1 in the control and miR‐141 mimic‐transfected Hepa1‐6 cells. D) The production frequency of IFN‐γ in CD8 + T cells co‐cultured with miR‐NC or miR‐141‐overexpressed Hepa1‐6 cells ( n = 3). E) Representative images of tumors, tumor growth curves, and tumor weights from mice bearing miR‐NC and miR‐141‐overexpressed Hepa1‐6 cells bearing mice ( n = 5). F) Immunohistochemistry (IHC) staining and mean intensity of Pdl1 ( n = 5. Magnification: 40 ×). G) The secretion of IFN‐γ in CD8 + T cells from the tumors of mice bearing miR‐NC and miR‐141‐overexpressed Hepa1‐6 cells ( n = 5). H) The luciferase reporter assays verified the interaction sites of miR‐340 with Tug1 and Cd47 ( n = 3). I) The expressions of miR‐340 and Cd47 in the control and si‐Tug1‐transfected Hepa1‐6 cells. J) The expressions of miR‐340 and Cd47 in the control and miR‐340 mimic‐transfected Hepa1‐6 cells. K) Representative plots of flow cytometry and statistical analysis of F4/80 + macrophages phagocytosing miR‐NC or miR‐340‐overexpressed Hepa1‐6 cells ( n = 3). L) Representative images of tumors, tumor growth curves, and tumor weights from mice bearing miR‐NC and miR‐340‐overexpressed Hepa1‐6 cells ( n = 5). M) Immunohistochemistry (IHC) staining of Cd47 ( n = 5. Magnification: 40 ×). N) The frequency of M1‐like macrophages in the tumors obtained from mice bearing miR‐NC and miR‐340‐overexpressed Hepa1‐6 cells ( n = 4). O) The expression of miR‐340 and miR‐141 were upregulated in sh‐Tug1 Hepa1c1c7 cells. P) Pdl1 and Cd47 expression at both mRNAs and protein levels in sh‐Tug1 Hepa1c1c7 cells. Results are represented as the mean ± SEM. Data were statistically analyzed using unpaired two‐tailed Student's t‐test or two‐way ANOVA (E, L). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Anti‐ Pdl1 antibodies (Clone No.10F.9G2, BioXcell) or IgG isotype control were given intraperitoneally at 200 μg/day every 4 days.

Techniques: Luciferase, Control, Transfection, Expressing, Cell Culture, Immunohistochemistry, Flow Cytometry, Two Tailed Test

Journal: Advanced Science

Article Title: M 6 A‐mediated upregulation of lncRNA TUG1 in liver cancer cells regulates the antitumor response of CD8 + T cells and phagocytosis of macrophages

doi: 10.1002/advs.202400695

Figure Lengend Snippet: Tug1 interacts with Ybx1 to transcriptionally regulate Pdl1 and Cd47, thereby regulating the antitumor immune response of CD8 + T cells and phagocytosis of macrophages. A) Hepa1‐6 cells were used for CHIRP assay. The enrichment efficiency of Tug1 probes is confirmed via qPCR and electrophoresis. B) Silver staining of Tug1‐associated proteins from CHIRP. C) Enrichment of Ybx1 protein in the pull‐downs of both “odd” and “even” probes targeting Tug1 relative to LacZ probes. D) RIP‐qPCR shows enrichment of Tug1 after immunoprecipitation of Ybx1. E) The tertiary structure of TUG1 was docked with the YBX1 protein, the intermolecular interaction details were presented. F, G) The correlation of YBX1 with PD‐L1 and CD47 in HCC patients ( n = 371). H) Pdl1 and Cd47 expressions in the control and Ybx1 siRNAs‐transfected Hepa1‐6 cells at protein and mRNA levels. I) Pdl1 and Cd47 expression in sh‐NC and sh‐Ybx1 Hepa1c1c7 cells at protein and mRNA levels. J) CHIP‐qPCR verifies the enrichment of YBX1 in the promoter regions of PD‐L1 and CD47. K) The CHIP‐qPCR results showed a reduction in the recruitment of YBX1 in the promoter regions of PD‐L1 and CD47 when TUG1 is downregulated. L) Representative plots and statistical analysis of macrophages phagocytosing sh‐NC or sh‐Ybx1 Hepa1‐6 cells ( n = 3). M, N) Representative images of tumors, tumor growth curves, and tumor weights from sh‐NC and sh‐Ybx1 Hepa1‐6 cells tumor‐bearing mice ( n = 5). O) The secretion of cytokines in CD8 + T cells from the tumors of sh‐NC and sh‐Ybx1 Hepa1‐6 cell‐bearing mice ( n = 5). P, Q) The immunohistochemistry (IHC) staining and mean intensities of Ybx1, Pdl1 and Cd47. ( n = 5. Magnification: 40 ×). Results are represented as the mean ± SEM. Statistical analysis was performed using the unpaired two‐tailed Student's t‐test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Anti‐ Pdl1 antibodies (Clone No.10F.9G2, BioXcell) or IgG isotype control were given intraperitoneally at 200 μg/day every 4 days.

Techniques: Electrophoresis, Silver Staining, Immunoprecipitation, Control, Transfection, Expressing, ChIP-qPCR, Immunohistochemistry, Two Tailed Test

Journal: Advanced Science

Article Title: M 6 A‐mediated upregulation of lncRNA TUG1 in liver cancer cells regulates the antitumor response of CD8 + T cells and phagocytosis of macrophages

doi: 10.1002/advs.202400695

Figure Lengend Snippet: TUG1, but not TUG1‐ORF, regulates the expressions of PD‐L1 and CD47 in liver cancer cells. A) The expressions of miR‐141 and miR‐340 are upregulated in TUG1‐downregulated HepG2 cells. B) PD‐L1 and CD47 expressions at both the mRNAs and protein levels in TUG1‐downregulated HepG2 cells. C) The expression of miR‐141 and PD‐L1 in the control and miR‐141 over‐expressed HepG2 cells. D) The expressions of miR‐340 and CD47 in the control and miR‐340 over‐expressed HepG2 cells. E) The expressions of miR‐141 and miR‐340 are upregulated in TUG1‐downregulated LM3 cells. F) PD‐L1 and CD47 expressions at both the mRNAs and protein levels in TUG1‐downregulated LM3 cells. G) The expression of miR‐141 and PD‐L1 in the control and miR‐141 over expressed LM3 cells. H) The expressions of miR‐340 and CD47 in the control and miR‐340 over expressed LM3 cells. I) PD‐L1 and CD47 expressions at mRNA levels in YBX1‐downregulated HepG2 cells. J) PD‐L1 and CD47 expressions at mRNA levels in YBX1‐downregulated LM3 cells. K) PD‐L1 and CD47 expressions at protein levels in YBX1‐downregulated HepG2 cells and LM3 cells. L) Pdl1 and Cd47 expressions at both the mRNAs and protein levels in Tug1 full length (Tug1‐FL)‐overexpressed Hepa1‐6 cells. M) The frequencies of IFN‐γ in CD8 + T cells co‐cultured with NC and Tug1‐FL Hepa1‐6 cells ( n = 3). N) Representative plots and statistical analysis of macrophages phagocytosing NC or Tug1‐FL Hepa1‐6 cells ( n = 3). O) Pdl1 and Cd47 expressions at both the mRNAs and protein levels in Tug1‐ORF‐overexpressed Hepa1‐6 cells. P) Representative images of tumors, tumor growth curves, and tumor weights from mice bearing NC and Tug1‐ORF‐overexpressed Hepa1‐6 cells tumor‐bearing mice ( n = 5). Results are represented as the mean ± SEM. Statistical analysis was performed using the unpaired two‐tailed Student's t‐test.*, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Anti‐ Pdl1 antibodies (Clone No.10F.9G2, BioXcell) or IgG isotype control were given intraperitoneally at 200 μg/day every 4 days.

Techniques: Expressing, Control, Cell Culture, Two Tailed Test

Journal: Advanced Science

Article Title: M 6 A‐mediated upregulation of lncRNA TUG1 in liver cancer cells regulates the antitumor response of CD8 + T cells and phagocytosis of macrophages

doi: 10.1002/advs.202400695

Figure Lengend Snippet: TUG1 is a potential biomarker and immunotherapeutic target for liver cancer. A) Expression of TUG1 in 40 pairs of clinical specimens of both normal and tumor tissues was determined by qRT‐PCR. B) Correlative analysis of the relative expression of TUG1 with PD‐L1 in human HCC tumors ( n = 40). C) Correlative analysis of the relative expression of TUG1 with CD47 in human HCC tumors ( n = 40). D) Correlative analysis of the relative expression of TUG1 with YBX1 in human HCC tumors ( n = 40). E) Correlative analysis of the relative expression of YBX1 with PD‐L1 in human HCC tumors ( n = 40). F) Correlative analysis of the relative expression of YBX1 with CD47 in human HCC tumors ( n = 40). G) Schematic of Tug1 siRNAs therapy in combination with an anti‐Pdl1 antibody. H–J) Representative images of tumors, tumor volumes, and tumor weights ( n = 3). K) Schematic of the molecular mechanism of TUG1 in regulating antitumor immune response. Results are represented as the mean ± SEM. Statistical analysis was performed using the unpaired two‐tailed Student's t‐test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Anti‐ Pdl1 antibodies (Clone No.10F.9G2, BioXcell) or IgG isotype control were given intraperitoneally at 200 μg/day every 4 days.

Techniques: Biomarker Discovery, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , Schematic of metastatic LUAD and patient-derived xenograft (PDX) engraftment. b , Box and whisker plots of L1CAM IHC H-score in patient-derived primary tumor and metastasis samples. Primary tumor, n = 15; metastases, n = 54. *** P = 0.0002. c , Box and whisker plots of L1CAM IHC H-score in the primary tumors in ( b ) and in PDXs derived from primary tumor or metastases. Primary tumors, n = 15; primary tumor-derived PDXs, n = 36; metastasis-derived PDXs, n = 70. ns, P = 0.8811; * P = 0.0294 (left), 0.0420 (right). d , Schematic of the oncogenic transformation of AT2 cells into lung adenocarcinoma in the KP GEMM ( left panel ), and box and whisker plots of the percentage of L1CAM + cells in the KP mouse lungs and tumor areas at different time points after lentivirus instillation ( right panel ). Week 0, n = 6; week 14-19, n = 6; week 20-32, n = 15. ns, P = 0.5710; ** P = 0.0032. e , Schematic of the tumor tissue section ( left panel ), and box and whisker plots of the percentage of L1CAM + cells in the tumor center and invasive front ( right panel ). Tumor center, n = 300 cells; invasive front, n = 258 cells. N = 7 image frames. * P = 0.0152. f , L1CAM immunofluorescence (IF) staining of LUAD patient tissue samples at the tumor center (P, papillary) or the invasive front (M, micropapillary). Magnified regions are indicated in red boxes. Scale bar, 20 μm. g , Confocal microscopy image of L1CAM IF staining and Hoechst counterstaining of nuclei in KP tumoroids grown for 5 days. Schematic of tumoroids generated from KP-derived cancer cells ( lower right panel ). Scale bar, 10 μm. h , Percentage of L1CAM + cells in KP primary tumors versus tumoroids. Primary tumor, n = 4 experiments; tumoroids, n = 6 experiments. Mean ± s.e.m. ** P = 0.0095. i , Widefield fluorescence microscopy image of KP tumoroids stained with calcein AM in KP tumor cells sorted by L1CAM expression and grown as tumoroids for 7 days. The magnified region showing a single cell is indicated by a dotted box and that of a tumoroid by a solid box. Scale bar, 400 μm. j , Box and whisker plots of tumoroids formed per 1,000 cells after 7 days in culture. L1CAM − , n = 16; L1CAM + , n = 16. **** P < 0.0001. k , Box and whisker plots of cross-section area per tumoroid in the experiment of panel ( I ). L1CAM - , n = 807; L1CAM + , n = 477. **** P < 0.0001. l , Schematic representation of the generation of L1cam knockout KP LUAD GEMMs. m , H&E staining of KP and KPL1 primary tumors (26 week post-Cre) followed by their histopathological grading using an automated deep neural network. Magnified regions are shown in a red square. Scale bar, 1 mm. n , Fraction of KP and KPL1 tumors (22-26 week post-Cre) based on histopathological grading. Grade 1, green ; Grade 2, orange ; Grade 3, blue ; Grade 4, red . KP, n = 4; KPL1, n = 4. Mean ± S.D. o , H&E staining of KP metastasis in the subcapsular sinus of the lymph node (top panel) or ribcage bone (bottom panel). Invasive front is shown with a dotted yellow line. T, tumor; LN, lymph node. Scale bar, 100 μm. p , Incidence of primary tumor and spontaneous metastases upon viral transduction in KP and KPL1 mice. The organ specificity of spontaneous metastasis is shown as a percentage of the total metastatic tumors. LN, lymph node. KP primary, n = 46; KPL1 primary, n = 30; KP met, n = 42; KPL1 met, n = 16. ns, P = 1; * P = 0.0122. q , Representative images of subcutaneous tumors formed 6 weeks after inoculating 500 KP or KPL1 cells in athymic mice. Scale bar, 2 mm. r , Limiting dilution assay of subcutaneous tumor formation at various doses of KP or KPL1 cells in athymic mice. n = 10 for each condition. s , Fluorescent images of GFP + KP or KPL1 cells seeded in lungs at week 1 after tail vein injection (10 5 cells each) into athymic mice. Scale bar, 10 μm. t , Quantification of KP and KPL1 cells seeded in lungs from the experiment in ( S ). KP, n = 9; KPL1, n = 10. ns, P = 0.3154. u , Representative image of ex vivo lung BLI at week 5 after tail vein injection of single-cell suspension of KP and KPL1 tumoroids (2 x 10 4 cells) into athymic mice. v , Quantification of ex vivo lung BLI signal in the experiment in ( u ). KP, n = 28; KPL1, n = 14. *** P = 0.0006. w , The KM plot showing the overall survival after performing tail vein injections with KP or KPL1 cells. KP, n = 10; KPL1, n = 10. **** P < 0.0001. Statistical significance was assessed using the two-tailed Mann-Whitney test ( b , e , h , j , k , n , t , v ), one-way analysis of variance followed by the Tukey test ( c , d ), two-tailed Fisher’s exact test ( p ) or log-rank (Mantel-Cox) test ( o , w ). Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( b - e , j , k , t , v ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Derivative Assay, Whisker Assay, Transformation Assay, Immunofluorescence, Staining, Confocal Microscopy, Generated, Fluorescence, Microscopy, Expressing, Knock-Out, Transduction, Limiting Dilution Assay, Injection, Ex Vivo, Suspension, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , Representative L1CAM IHC staining in primary tumors, pleural fluids, and metastatic samples from LUAD patients and three tissue microarrays (Array 1, green; Array 2, blue; Array 3, red) containing normal human lung tissue and LUAD PDX (Passage 0 or 1). Scale bar, 100 μm. b , Heatmap of L1CAM IHC staining quantification (H-score) in primary tumor, pleural fluid, metastatic samples, and PDXs from LUAD patients. Each column represents an individual sample. Samples are color-coded according to their site of tissue. c , Maximum projection of a PDX tumoroid fluorescently stained with L1CAM. Scale bar, 10 μm. d , Cross-section of a PDX tumoroid stained with L1CAM IF and DAPI (nuclei). Scale bar, 10 μm. e , Close up of a PDX tumoroid stained with L1CAM IF and DAPI. Scale bar, 5 μm.

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , L1CAM IHC staining in lung and tumor sections in KP mice at baseline and at different time points after lentivirus instillation to induce adenocarcinoma formation. Yellow, week 0; orange, week 14; red, week 24. Scale bar, 50 μm. b , L1CAM IHC staining of the patient tissue sections including normal tissue and tumor. The invasive tumor front is indicated with a yellow line. Scale bar, 200 μm. c , H&E and L1CAM IHC staining of KP primary tumor sections (26 week post-Cre) showing the indicated histological subtypes. Scale bar, 50 μm. d , Fraction of histological subtypes observed in KP primary tumors. n = 203,870 cells. e , Percentage of L1CAM + cells in KP primary tumors with respect to histological subtypes. Lepidic ( n = 26,871); Papillary ( n = 88,381); Micropapillary (red colored, n = 4,116); Acinar ( n = 3,430); Solid ( n = 81,072). **** P < 0.0001. f , Representative GFP (cancer cells) IF and L1CAM IF images of primary tumor and an autochthonous lymph node metastasis from a KP mouse (35 week post-Cre). Scale bar, 20 μm. g , Quantification of L1CAM IF intensity in the experiment ( f ). LN, lymph node; AU, arbitrary unit. n = 24 image frames; N = 3 mice in each condition. **** P < 0.0001. h , H&E staining of KP tumoroids grown in Matrigel over the course of 7 days. Scale bar, 100 μm. I , Brightfield and GFP fluorescence microscopy images of KP tumoroids at day 7. Scale bar, 10 μm. j , Close up of L1CAM IHC staining of patient samples and KP tumors. Scale bar, 20 μm. Statistical significance was assessed using the two-tailed Mann-Whitney test ( g ) or one-way analysis of variance followed by the Tukey test ( e ). Data are shown as mean ± S.D. ( d , e ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Immunohistochemistry, Staining, Fluorescence, Microscopy, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , Representative images of tumor-bearing KP and KPL1 mice analyzed by BLI at week 16 after viral transduction. b , Growth curve of KP and KPL1 primary tumor burden as determined by BLI. KP, n = 37; KPL1, n = 14. ns, P > 0.9999. c , Micro-CT scan images of normal lung and tumor-bearing lungs from KP and KPL1 mice at week 22 after viral transduction. Arrows , tumors; red h , heart. d , Tumor burden comparison between KP and KPL1 mice based on micro-CT scans at week 22. n = 38 KP mice, n = 10 KPL1 mice. ns, P = 0.3192. e , Tumor areas of KP and KPL1 mouse lungs at week 22-30 after viral transduction. n = 4 mice. ns, P = 0.6857. f , Graphical representation of mouse survival prior to reaching the target tumor burden, set at a luciferase bioluminescence flux of 10 7 photons/sec per mouse chest. KP, n = 35; KPL1, n = 14. ns, P = 0.7143. g , Quantification of tumoroids formed per 10,000 cells over 7 days. KP, n = 6; KPL1, n = 6. ns, P = 0.6991. h , Schematic of three different metastasis inoculation routes. i , Representative image of ex vivo lung BLI signals at week 5 after intratracheal delivery of single-cell suspension of KP and KPL1 tumoroids (2 x 10 4 cells) into athymic mice. j , Quantification of ex vivo lung BLI signal in the experiment in panel ( i ). KP, n = 8; KPL1, n = 6. ** P = 0.0027. k , Representative image of ex vivo lung BLI signals at week 3 after intracardiac injection of single-cell suspension of KP and KPL1 tumoroids (5 x 10 4 cells) into athymic mice. l , Quantification of ex vivo lung BLI signals in the experiment in panel ( k ). KP, n = 9; KPL1, n = 5. ** P = 0.0070. m , Representative image of ex vivo kidney BLI signals at week 3 after intracardiac injection of single-cell suspension of KP tumoroids or KPL1 tumoroids (5 x 10 4 cells). n , Quantification of ex vivo kidney BLI signals at week 3 after intracardiac injection of single-cell suspension of KP tumoroids or KPL1 tumoroids. KP, n = 9; KPL1, n = 5. * P = 0.0120. o , Representative image of ex vivo brain BLI signals at week 3 after intracardiac injection of single-cell suspension of KP tumoroids or KPL1 tumoroids (5 x 10 4 cells). p , Quantification of ex vivo brain BLI signals at week 3 after intracardiac injection of single-cell suspension of KP tumoroids or KPL1 tumoroids. KP, n = 9; KPL1, n = 5. * P = 0.0120. q , Representative image of ex vivo liver BLI signals at week 3 after intracardiac injection of single-cell suspension of KP tumoroids or KPL1 tumoroids (5 x 10 4 cells). r , Quantification of ex vivo liver BLI signals at week 3 after intracardiac injection of single-cell suspension of KP tumoroids or KPL1 tumoroids. KP, n = 9; KPL1, n = 5. * P = 0.0120. s , Western immunoblotting analysis of Ru631 PDX cells transduced with two different L1CAM shRNAs. t , H&E staining of lung sections four weeks after injecting Ru631 L1CAM knockdown via tail vein. Scale bar, 50 μm. u , Quantification of Ru631 tumors after tail vein injection. n = 4 mice per condition. * P = 0.0337; ** P = 0.0013. v , Western immunoblotting of L1CAM knockdowns with two different short hairpins in Ru323 PDXs. w , H&E staining of lung sections four weeks after injecting Ru323 with L1CAM knockdown via tail vein. Scale bar, 50 μm. x , Quantification of Ru323 tumors after tail vein injection. n = 5 mice per condition. *P = 0.0123; ** P = 0.0058. Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( d , e , j , l , n , p , r ). Error bar indicates mean ± S.D. ( b ). Statistical significance was assessed using Kolmogorov-Smirnov test ( b ), two-tailed Mann-Whitney test ( d , e , g , j , l , n , p , r ) or one-way analysis of variance followed by the Tukey test ( u , x ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Transduction, Micro-CT, Comparison, Luciferase, Ex Vivo, Suspension, Injection, Western Blot, Staining, Knockdown, Whisker Assay, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , Schematic summary of lung developmental continuum from early to late progenitor stages defined by specific transcription factors. b , Representative 3D maximum intensity projections (MIP) images of SOX2, NKX2-1 and SOX9 IF in the anterior foregut domain in embryonic day E9.5 mouse embryos and the distal lung bud tip in E10.5 mouse embryos. The dashed box demarcates the SOX2/NKX2-1 boundary in E9.5 and NKX2-1/SOX9 boundary in E10.5 as illustrated in the zoomed 2D (Z-Slice) panels on the right. All data were independently validated from replicate samples of at least n = 4 embryos with similar results obtained. c , SOX2, NKX2-1, and SOX9 IF staining in patient-derived primary tumor and metastasis tissue sections. Magnified regions are highlighted in yellow boxes. Scale bar, 50 μm. d , Fraction of cancer cells expressing SOX2, NKX2-1, or SOX9 by IF analysis in patient-derived primary tumor and metastasis tissue sections. Mean ± S.D. n = 6 patient samples each. ns, P = 0.4848 (NKX2-1), 0.3095 (SOX9); ** P = 0.0043. e , SOX2 and SOX9 IHC staining in LUAD patient-derived primary tumor and pleural metastasis samples. Magnified regions are highlighted in red boxes. Scale bar, 100 μm. f , Box and whisker plots of SOX2 and SOX9 IHC H-score in the patient-derived primary tumor and metastasis samples. Primary, n = 15; Metastasis, n = 7. ns, P = 0.1229; ** P = 0.0011. g , Relative median survival of LUAD patients based on the expression of SOX2 , NKX2-1 , and SOX9 in the primary tumor. Gene expression and survival data compiled from GEO, EGA and TCGA. SOX2 (low, n = 706; high, n = 705); NKX2-1 (low, n = 1083; high, n = 1083); SOX9 (low, n = 1083; high, n = 1083). * P = 0.0219; *** P = 0.0003; **** P < 0.0001. h , H&E staining of lung tissue sections harboring metastatic colonies upon tail vein injection of KP tumoroid cells expressing control or Sox2 short hairpins (sh). Magnified regions are indicated in red boxes. Scale bar, 5 mm. i,j , Number per lung ( I ) and percent area ( j ) of metastases in the experiment of panel ( h ). n = 10. **** P < 0.0001. k , UMAP of scRNA-seq data from autochthonous KP tumors collected 32 weeks after viral instillation. n = 4,489 cells. Transcriptionally distinct clusters were computed by Leiden algorithm and numbered. l , Heatmap showing imputed average LUAD developmental marker expression in the clusters from primary tumors ranked left to right according to the average L1cam expression level. m , UMAP of scRNA-seq data from KP tumoroids collected after 7 days. n = 12,962 cells. Transcriptionally distinct clusters were computed by Leiden algorithm and are represented by a different color and a number. n , Heatmap showing imputed average LUAD developmental marker expression in the clusters from 7-day tumoroids ranked left to right according to the average L1cam expression level. Lung developmental progenitor stages represented by the predominant expression of Sox2 ( early developmental stage), Nkx2-1 and Foxa2 ( middle progenitor stage), Hmga2 and Sox9 ( late progenitor stage) are indicated. o , Representative L1CAM and SOX2 IF staining of the invasive front ( dotted yellow line ) within an autochthonous KP tumor (32 week post-Cre) and of KP-derived tumoroids grown for 4 days or 7 days. T , tumor; N , normal tissue. Magnified regions are indicated in red boxes. Scale bar, 50 μm for primary tumor; 20 μm for tumoroids. p , Relative Sox2 mRNA expression of KP tumoroid-derived cells sorted by L1CAM expression. Mean ± S.E.M. n = 3. * P = 0.0258. q , L1CAM and SOX2 IHC staining of representative LUAD PDXs with high or low expression of L1CAM. Scale bar, 50 μm. r , Scatter plot and linear regression ( red line ) of L1CAM versus SOX2 IHC H-score data from LUAD PDXs. Data are log-transformed for visualization. n = 36. s , L1CAM and SOX2 IHC staining in serial sections of patient-derived bone metastasis and liver metastasis tissues. Scale bar, 100 μm. t , Scatter plot and linear regression ( red line ) of L1CAM versus SOX2 IHC H-score data from LUAD patient primary and metastasis samples. Data are log-transformed for visualization. n = 61 samples (primary, 15; metastasis, 46). Spearman correlation was used to calculate the relationship between L1CAM expression and SOX2 expression ( r,t ). Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( f , i,j ). Statistical significance was assessed using the two-tailed Mann-Whitney test ( d , f , g , i , j ) or two-tailed t test after passing the Shapiro-Wilk normality test ( p ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Staining, Derivative Assay, Expressing, Immunohistochemistry, Whisker Assay, Gene Expression, Injection, Control, Marker, Transformation Assay, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , SOX2 and NKX2-1 IF staining of patient-derived LUAD primary tumor tissue section. The invasive front is marked with a dotted yellow line, and the magnified low-grade and high-grade tumor areas are shown in red boxes. Scale bar, 1 mm. b , SOX2 and NKX2-1 IF staining of patient-derived primary tumor tissue section with different tumor grade areas. Invasive front is shown with a dotted yellow line. Scale bar, 20 μm. c , SOX2 and SOX9 IF staining in KP primary tumor and metastasis tissue sections collected simultaneously from the same mouse (35 week post-Cre). Scale bar, 100 μm. d , Western immunoblotting analysis of SOX2 expression after dox-induced Sox2 knockdown in KP tumoroids. e , f , Box and whisker plots of the imputed transcript levels of Sox2 ( e ) and L1cam ( f ) in the scRNA-seq transcriptional clusters from KP primary tumors. g , Scatter plot of L1cam versus Sox2 expression in the scRNA-seq dataset from KP primary tumors. Data from transcriptional cluster 9 are shown in black. H , I , Box and whisker plots of the imputed transcript levels of Sox2 ( h ) and L1cam ( i ) from 7-day tumoroids. Data from the cluster 12 are shown in red. j , Scatter plot of L1cam versus Sox2 expression in the scRNA-seq dataset from 7-day tumoroids. Data from transcriptional cluster 12 are shown in black. k , Proportion of SOX9 + cells detected by IF in lung colonies after injecting a high number (1 x 10 5 ) of KP or KPL1 cells via the tail vein into athymic mice. KP, n = 10; KPL1, n = 5. ** P = 0.0013. l , Relative expression of SOX9 in two independent KP tumoroid lines upon conditional knockdown of L1CAM. Bar graphs, mean ± S.D. n = 3 in each condition. ns, P = 0.8301; * P = 0.0202, 0.0305; ** P = 0.0094. m , Western immunoblotting analysis of L1CAM levels after L1CAM overexpression in KPL1 tumoroids. Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( e , f , h , i , k ). Spearman correlation was used to calculate the relationship between L1CAM expression and SOX2 expression ( g , j ). Statistical significance was assessed using the two-tailed Mann-Whitney test ( k ) or one-way analysis of variance followed by the Tukey test ( l ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Staining, Derivative Assay, Western Blot, Expressing, Knockdown, Whisker Assay, Over Expression, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , SOX2 IF staining of KP and KPL1 primary tumors (32 week post-Cre). Scale bar, 10 μm. b , Percentage of SOX2 + cells in the experiment from panel ( a ). n = 3 mice for each condition. * P = 0.0411. c , d , Relative L1cam mRNA expression and Sox2 mRNA expression in KP and KPL1 tumoroids. KP, n = 4; KPL1, n = 4. * P = 0.0286 ( c , d ). e , IF images of KP and KPL1 tumors in the lung after injecting a high number (1 x 10 5 ) of single cells from KP and KPL1 tumoroids into athymic mice via the tail vein. Magnified regions are highlighted in yellow boxes. Scale bar, 100 μm. f , Proportion of SOX2 + cells detected by IF in lung colonies after injecting a high number (1 x 10 5 ) of KP or KPL1 cells via the tail vein in athymic mice. KP, n = 10; KPL1, n = 5. * P = 0.0400. g , h , Relative expression of L1cam ( g ) and Sox2 ( h ) in two independent KP tumoroid lines upon Dox-dependent conditional knock down of L1cam . n = 4. **** P < 0.0001. i , j , Western immunoblot analysis ( i ) and quantification ( j ) of SOX2 and SOX9 levels in control and L1CAM-overexpressing (OE) KP tumoroids. n = 4. * P = 0.0286. k , l , H&E staining ( k ) and quantification ( l ) of subcutaneous tumors or lung metastases after injection of KPL1 cells with or without L1CAM overexpression into athymic mice, analyzed at 4 weeks after subcutaneous (subQ) injection or 5 weeks after tail vein (TV) injection. Scale bar, 1 mm (SubQ) and 100 μm (TV). n = 5. ns, P > 0.9999; * P = 0.0238. m , GFP (cancer cells) and SOX2 IF staining of control vs L1CAM overexpressing KPL1 cells in lung metastasis after 5 week-post tail vein injection. Magnified regions are highlighted in yellow boxes. Scale bar, 10 μm. n , Fraction of SOX2 + cells in the experiment from panel ( m ). Control, n = 5; L1CAM overexpression, n = 5. ** P = 0.0079. Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( f ). Bar graphs, mean ± S.D. ( c , d , g , h , j ) or ± S.E.M. ( b , l , n ). Statistical significance was assessed using the two-tailed t test after passing the Shapiro-Wilk normality test ( b ), two-tailed Mann-Whitney test ( c , d , f , j , l , n ) or one-way analysis of variance followed by the Tukey test ( g , h ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Staining, Expressing, Knockdown, Western Blot, Control, Injection, Over Expression, Whisker Assay, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , Scatter plot of accumulated enrichment score and negative log-transformed false discovery rate (FDR) of pathways associated with the L1CAM + /SOX2 + tumoroid cell cluster defined by scRNA-seq. The pathway with the highest total enrichment score and the lowest FDR is labeled in red. b , Scatter plot showing the expanded list of individual WNT/PCP-related terms. c , Schematic representations of L1CAM and PCP complex components at a cell-cell junction. d , Heatmap displaying the average expression of core PCP components in the KP tumoroid cell clusters defined in . The clusters are ranked left to right according to the average L1cam expression level. e , L1CAM and CELSR1 IF staining in KP tumoroid-derived cell monolayer. The merged image is magnified for visualization ( red box ). Scale bar, 10 μm. f , L1CAM and CELSR1 IF staining of lung metastasis one week after tail vein injection of H23 LUAD cells into athymic mice. Scale bar, 10 μm. g , Co-immunoprecipitation of L1CAM and the PCP component CELSR1 in H23 LUAD cell lysates. h , Quantification of L1CAM/CELSR1 PLA dots per cell in H23 LUAD cells. Negative PLA control, n = 25 cells; L1CAM/CELSR1 PLA, n = 31 cells. **** P < 0.0001. i , Control and L1CAM-knockout (KO) H23 cell monolayers were subjected to cytokeratin, CELSR1, and SOX2 IF staining. Scale bar, 20 μm. j , Fraction of SOX2 + cells quantified in control versus L1CAM-knockout H23 cell monolayers. Mean ± S.D. Control, n = 43 cells; L1CAM KO, n = 58 cells. * P = 0.0488. k , Western immunoblotting analysis of control and L1CAM-knockdown ( shL1CAM ) PDXs after incubating the cells with 1 μM bafilomycin A for 24 h. l , H&E staining of lung sections of athymic mice after tail-vein inoculation of L1CAM-knockout H23 cells with or without SOX2 overexpression. Scale bar, 20 μm. m , n , Box and whisker plots showing the number ( m ) and percent area ( n ) of metastatic lesions per lung in the experiments of panel ( l ). n = 5 for L1CAM KO-1; 10 for L1CAM KO-2. * P = 0.0317; ** P = 0.0012 in ( m ). ** P = 0.0079; *** P = 0.0002 ( n ). Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( h , m , n ). Statistical significance was assessed using the two-tailed Mann-Whitney test ( h , j , m , n ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Transformation Assay, Labeling, Expressing, Staining, Derivative Assay, Injection, Immunoprecipitation, Control, Knock-Out, Western Blot, Knockdown, Over Expression, Whisker Assay, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , WNT/PCP pathway scoring as the top hit among highly enriched genes in L1CAM + /SOX2 + KP tumoroid cells. Top 3000 ranked differentially expressed genes in cluster #12 versus the rest of cell population in day-7 KP tumoroids were analyzed to determine uniquely enriched biological pathways from the Gene Ontology (GO), Reactome, and PANTHER databases. 30 pathways shared among these three analyses were concatenated into three related processes. 13 of these pathways were from the WNT/PCP-related process and had the lowest accumulated false discovery rate (FDR). Within the WNT/PCP-related processes, the PCP and non-canonical WNT signaling pathways showed the highest enrichment score. b , Heatmap displaying the average expression of core PCP components in the primary tumor clusters defined in . The clusters are ranked left to right according to the average L1cam expression level. c , L1CAM and CELSR1 IF staining in KP tumoroids. The magnified region is indicated by red or white boxes . Scale bar, 10 μm. d , Image analysis workflow for detecting the colocalization of L1CAM and CELSR1 at cell-cell junctions using a steerable filter to extract curvilinear image features. e , Computer vision-driven detection and segmentation of L1CAM and CELSR1 at cell-cell junctions through steerable filtering. The thickness of segmented junctions was dilated for visualization purposes. Scale bar, 1 μm. f , L1CAM and CELSR1 PLA fluorescence in H23 LUAD cells. The PLA signals are shown as red dots with nuclei counterstaining. Scale bar, 10 μm. g , L1CAM western immunoblotting analysis in parental and L1CAM knockout H23 cells. h , Western immunoblotting analysis of SOX2 overexpression in H23 LUAD cells upon CRISPR/Cas9-mediated L1CAM knockout, clones 1 and 2. i. L1CAM and CELSR1 IF staining in HEK293T cells engineered to overexpress L1CAM versus control. Scale bar, 10 μm. j , L1CAM and CELSR1 segmentation analysis at cell-cell junctions in L1CAM overexpressing HEK293T cells. The region of higher magnification is indicated by a red box . k , Quantification of CELSR1 at cell-cell junctions in control or L1CAM overexpressing HEK293T cells. Control, n = 3406 junctions; L1CAM OE, n = 6508 junctions. **** P < 0.0001. l , L1CAM and FZD6 IF staining in control or L1CAM overexpressing HEK293T cells. Scale bar, 10 μm. m , Western immunoblotting analysis of SOX2 overexpression in CHD1 knockout H23 cells. n , Box and whisker plots showing the size of metastatic lesions per lung in SOX2 overexpressing, CHD1 knockout H23 cells. n = 9 for CHD1 KO-1; 10 for CHD1 KO-2. ns, P = 0.2805; * P = 0.0415. Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( k , n ). Statistical significance was assessed using the one-way analysis of variance followed by the Tukey test ( k ) or two-tailed Mann-Whitney test ( n ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Protein-Protein interactions, Expressing, Staining, Fluorescence, Western Blot, Knock-Out, Over Expression, CRISPR, Clone Assay, Control, Whisker Assay, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , FZD6 IF staining with counterstained nuclei in KP tumoroids upon conditional knockdown of L1cam . Scale bar, 10 μm. b , Relative intensity of FZD6 IF quantified in KP tumoroids upon conditional knockdown of L1cam . Left to right, n = 25, 23, 35, 29, 39, 41, 45, 53 tumoroids. * P = 0.0440, *** P = 0.0010. c , Relative Fzd6 mRNA level in upon conditional knockdown of Fzd6 in KP tumoroids. n = 4 experiments. **** P < 0.0001. d , Relative Sox2 mRNA level upon conditional knockdown of FZD6 in KP tumoroids. n = 4 experiments. ** P = 0.0040. e , IF staining for L1CAM, c-Jun(pS73), and SOX2 in KP tumoroids ( upper panels ) and image segmentation to quantify the signal ( bottom panels ). Scale bar, 10 μm. f , Pie chart showing the percent of cells staining positive for c-Jun(pS73) and SOX2 in KP tumoroids. n = 649 cells. g , IF staining for cytokeratin, L1CAM, and c-Jun(pS73) in a patient-derived LUAD lymph node metastasis. Scale bar, 50 μm. h , Pie chart showing the percent of cells staining positive for c-Jun(pS73) and L1CAM in patient-derived LUAD lymph node metastases. n = 2,166 cells from 12 different lymph nodes. i , c-Jun(pS73) IF staining and counterstained nuclei in KP tumoroids upon conditional knockdown of L1CAM. Scale bar, 20 μm. j , Percentage of cells staining positive for c-Jun(pS73) after conditional knockdown of L1cam in KP tumoroids. Left to right, n = 94, 67, 87, 101, 38, 41, 36, 36 tumoroids. **** P < 0.0001. k , Western immunoblotting analysis of L1CAM, S73 phosphorylated and total c-Jun levels in H23 LUAD cells treated with 10 μM (JNK-IN-8) JNK inhibitor (JNKi) for 2 h. l , Sox2 mRNA relative expression level upon incubation of KP tumoroids with 20 μM JNK inhibitor for 24 h. n = 7 for KP tumoroid #1; 4 for KP tumoroid #2. *** P < 0.0006; * P = 0.0286. m , Western immunoblotting analysis of S73 phosphorylated and total c-Jun levels upon the treatments with anisomycin for 6 h. n , Sox2 mRNA relative expression level upon incubation of KP cells with anisomycin for 6 h. n = 3. ** P = 0.0064 (left), 0.0021 (right). The bar graph indicates mean ± S.E.M. ( b - d , j , l , n ). Statistical significance was assessed using the one-way analysis of variance followed by the Tukey test ( b - d , j , n ) or two-tailed Mann-Whitney test ( l ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Staining, Knockdown, Derivative Assay, Western Blot, Expressing, Incubation, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , Venn diagram of transcription factors and chromatin modifiers differentially active in L1CAM + /SOX2 + KP tumoroid cells (scRNA-seq cluster #12, ) based on CHEA and ENCODE databases. b , PLA image of CHD1 and c-Jun in H23 LUAD cells. The PLA signals are shown as red dots overlayed with nuclei staining. Scale bar, 10 μm. c , Quantification of PLA signals per nucleus. Control, n = 63 cells; CHD1/c-Jun, n = 39 cells. **** P < 0.0001. d , Co-immunoprecipitation of CHD1 and c-Jun in KP tumoroids. e , Metaplots of CHD1 only (green), c-Jun only (blue), and CHD1/c-Jun overlap (red) ChIP-seq peak summits relative to peak center of CHD1 (left) or c-Jun (right) in H23 cells. f , Venn diagram showing the overlap between CHD1 and c-Jun genome-wide peaks. g , ChIP-seq analysis of CHD1 and c-Jun binding to the Sox2 locus in H23 cells. Sox2 enhancers ( red ), promoter and gene body ( green ) are indicated. h , ChIP-qPCR analysis of CHD1 binding to the SOX2 promoter in H23 cells with and without JNK inhibitor. n = 3 experiments. *** P = 0.0002. i , Co-immunoprecipitation of CHD1 and the transcriptional elongation factor RTF1 in H23 LUAD cells. j , Western immunoblotting analysis of SOX2 levels upon CHD1 knockout in H23 LUAD cells. k , Western immunoblotting analysis of SOX2 levels upon CHD1 knockdown in KP tumoroids using two different shRNAs. l , H&E staining of lung sections from NSG mice after tail-vein inoculation of H23 cells with CRISPR/Cas9-induced knockouts of CHD1 or L1CAM. Magnified regions are shown ( red boxes ). Scale bar, 100 μm. m , n , Box and whisker plots showing the number ( m ) and percent area ( n ) of metastatic lesions per lung in the experiments of panel ( j ). n = 6 per experimental condition. ** P = 0.0035, 0.0060, 0.0015 from left to right; * P = 0.0301 in ( m ). ** P = 0.0026, 0.0028, 0.0011 from left to right; *** P = 0.0006 in ( n ). o , Model of L1CAM-dependent PCP activation of c-Jun/CHD1 driven SOX2 expression in LUAD progenitors to generate a metastasis-initiating state. Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( c , m , n ). The bar graph indicates mean ± S.E.M. ( h ). Statistical significance was assessed using a one-way analysis of variance followed by the Tukey test ( h , m , n ) or two-tailed Mann-Whitney test ( c ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Staining, Control, Immunoprecipitation, ChIP-sequencing, Genome Wide, Binding Assay, ChIP-qPCR, Western Blot, Knock-Out, Knockdown, CRISPR, Whisker Assay, Activation Assay, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Journal for Immunotherapy of Cancer

Article Title: Cell-intrinsic PD-L1 signaling drives immunosuppression by myeloid-derived suppressor cells through IL-6/Jak/Stat3 in PD-L1-high lung cancer

doi: 10.1136/jitc-2024-010612

Figure Lengend Snippet: IL-6 predicts a poor response to ICI therapy in patients with PD-L1-high NSCLC. ( A, B ) Tumor tissues from 234 patients with NSCLC before ICI therapy were subjected to RNA-seq. The patients were divided into low- CD274 and high- CD274 (PD-L1) groups based on the median value. Differences in pathway enrichment between responders with a partial response (PR) and non-responders with stable disease (SD) or progressive disease (PD) in each group were analyzed using GSEA. ( C ) The heatmap shows the relative mRNA expression levels of the genes in core enrichment sites of the IL-6/Jak/Stat3 gene set. ( D ) Differences in IL6 expression (as presented as z-scores) among patients with PR, SD, and PD or between those with PR and SD+PD in the low- CD274 and high- CD274 groups. ( E ) ICI responsiveness among patients divided into low-expression and high-expression groups for CD274 and IL6 , based on the median value for CD274 and a TPM cut-off of 1.805 for IL6 , as determined using Cutoff Finder software. Differences in the response rate were compared using Pearson’s χ2 test. ( F ) Kaplan-Meier analysis of progression-free survival (PFS) after ICI therapy was performed according to CD274 (left upper), IL6 (left lower), and combined CD274 and IL6 (right) expression. Survival differences were compared using a log-rank test. ( G ) The heatmap shows the relative immune cell abundance of each group. ( H ) Differences in CTL, M2 macrophage, Treg, and MDSC scores between patients with PR and SD+PD in the low- CD274 and high- CD274 groups. Correlations between MDSC and Treg scores and IL-6 expression among patients with PR and SD+PD in the low-PD-L1 and high-PD-L1 groups. ( I ) Correlations between serum IL-6 levels and PD-L1 expression determined by the TPS of PD-L1 (22C3) IHC in the ICI-serum cohort (n=57). ( J ) The proportions of patients with PD and non-PD in the low-serum and high-serum IL-6 groups (cut-off value estimated using Cutoff Finder software) were compared using Pearson’s chi-squared test. ( K ) Kaplan-Meier analysis of PFS and overall survival (OS) after ICI therapy according to serum IL-6 levels. Differences in survival were analyzed using a log-rank test. The data in the histogram are presented as means±SEM. Correlations were calculated using Spearman’s correlation test. *p<0.05, **p<0.01. ADC, adenocarcinoma; ICI, immune checkpoint inhibitor; MNSCLC, non-small-cell lung cancer; DSC, myeloid-derived suppressor cell; SqCC, squamous cell carcinoma.

Article Snippet: Human PD-L1-expressing plasmids (HG10084-UT), PD-L1-Flag-tagged plasmids (HG10084-NF), control vectors (CV020), and human protein tyrosine phosphatase 1B (PTP1B)-Myc-tagged plasmids (HG10304-CM) were purchased from Sino Biological (China). siRNAs targeting human PD-L1 (NM 014143.2) were designed and synthesized by Bioneer (Republic of Korea) with the following sequences: sense 5′-CUG AGA AUC AAC ACA ACA A (dTdT)-3′ and antisense 5′-UUG UUG UGU UGA UUC UCA G (dTdT)-3′.

Techniques: RNA Sequencing, Expressing, Software, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Cell-intrinsic PD-L1 signaling drives immunosuppression by myeloid-derived suppressor cells through IL-6/Jak/Stat3 in PD-L1-high lung cancer

doi: 10.1136/jitc-2024-010612

Figure Lengend Snippet: PD-L1 activates the IL-6/Jak2/Stat3 pathway in lung cancer cells. ( A, B ) RNA-seq was performed in A549 cells transfected with PD-L1 vector or empty vector and in H460 cells transfected with PD-L1 siRNA or scrambled (sc) siRNA. DEGs in PD-L1-overexpressing A549 cells and PD-L1-knockdown H460 cells are displayed in a volcano plot. ( C ) Venn diagram illustrating the numbers of up-regulated genes in PD-L1-overexpressing A549 cells and down-regulated genes in PD-L1-knockdown H460 cells. Of the 359 shared genes, the expression of representative genes as determined in triplicate RNA-seq experiments with both cell lines is displayed in a heatmap. ( D, E ) GSEA was performed in each cell line using DEGs and the Hallmark gene set. ( F ) Expression of representative cytokines and chemokines included in the IL-6/Jak/Stat3 gene set as determined in triplicate RNA-seq experiments with both cell lines (molecules validated by further in vitro analyses are shown in red). ( G ) Cytokine mRNA expression and secretion were measured by qRT-PCR and ELISA, respectively, in PD-L1-overexpressing (PDL1 OE ) A549 cells and PD-L1-knockdown (PDL1 KD ) H460 cells (EV, empty vector; Con, scrambled siRNA). ( H ) Cytokine secretion determined by ELISA in the culture supernatant of patient-derived primary human lung tumor (cancer) cells with low-PD-L1 expression (TPS<1, n=5) and high-PD-L1 expression (TPS≥50, n=3) (left). Primary lung cancer cells with low and high PD-L1 expression were transfected with PD-L1-overexpressing vector and PD-L1-knockdown siRNA, respectively, and cytokine secretion was measured by ELISA (right). ( I, J ) Phosphorylated Stat3 and Jak2 and IL-6 expression were detected using western blotting and immunofluorescence staining in PD-L1-overexpressing A549 and H522 cells and in PD-L1-knockdown H460 and H596 cells (scale bar=20 µm). The data in the histogram are presented as means ±SEM. *p<0.05, **p<0.01, ***p<0.001. DEGs, differentially expressed genes.

Article Snippet: Human PD-L1-expressing plasmids (HG10084-UT), PD-L1-Flag-tagged plasmids (HG10084-NF), control vectors (CV020), and human protein tyrosine phosphatase 1B (PTP1B)-Myc-tagged plasmids (HG10304-CM) were purchased from Sino Biological (China). siRNAs targeting human PD-L1 (NM 014143.2) were designed and synthesized by Bioneer (Republic of Korea) with the following sequences: sense 5′-CUG AGA AUC AAC ACA ACA A (dTdT)-3′ and antisense 5′-UUG UUG UGU UGA UUC UCA G (dTdT)-3′.

Techniques: RNA Sequencing, Transfection, Plasmid Preparation, Knockdown, Expressing, In Vitro, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Derivative Assay, Western Blot, Immunofluorescence, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Cell-intrinsic PD-L1 signaling drives immunosuppression by myeloid-derived suppressor cells through IL-6/Jak/Stat3 in PD-L1-high lung cancer

doi: 10.1136/jitc-2024-010612

Figure Lengend Snippet: PD-L1 binds to p-Stat3 in the nucleus and enhances its efficiency in IL-6 transcription. ( A ) Three-dimensional visualization of nuclear PD-L1. Images corresponding to x-z sections reconstructed along the red lines are displayed at the top of each x-y section. Images corresponding to y-z sections reconstructed along the green solid lines are displayed at the right of each x-y section (scale bar=10 µm). ( B ) Nuclear localization of PD-L1 and p-Y705-Stat3 assessed by western blotting after cellular fractionation into nuclear and non-nuclear fractions, in PD-L1-overexpressing A549 cells and PD-L1-knockdown H460 cells. ( C ) Co-immunoprecipitation assay for p-Y705-Stat3 and PD-L1 using whole cell lysates (upper panels) and nuclear extracts (lower panels) of PD-L1-overexpressing A549 cells and PD-L1-knockdown H460 cells. ( D ) Interaction between p-Y705-Stat3 and PD-L1 in the cytoplasm and nucleus visualized in a Duolink assay of PD-L1-overexpressing A549 cells and PD-L1-knockdown H460 cells (scale bar=10 µm). Red dots indicate the interaction between p-Y705-Stat3 and PD-L1. Fluorescence intensity across dotted lines is depicted in the red lines of the right graphs (DAPI in blue lines). ( E ) A density heatmap of PD-L1 CUT&Tag sequencing data using H460 cells shows PD-L1 enrichment within 3 kb around the transcription start site (TSS) (Rep, replica). ( F ) Integrative genomics viewer (IGV) tracks for IL6 and CXCL1 promoters from the PD-L1 and p-Stat3 CUT&Tag analyses. ( G ) The density heatmap of the p-Stat3 CUT&Tag data shows compromised p-Stat3 enrichment in PD-L1-knockdown H460 cells vs control cells within 3 kb around the TSS. ( H ) IGV tracks for IL6 and CXCL1 promoters from the p-STAT3 CUT&Tag analysis of PD-L1-knockdown H460 cells and control cells. ( I ) Chromatin immunoprecipitation (ChIP) was performed using anti-PD-L1 and anti-Y705-Stat3 antibodies in PD-L1-knockdown H460 cells. The binding of PD-L1 and p-Y705-Stat3 to the IL6 and CXCL1 promoters was then assessed by qPCR (sequential ChIP-qPCR). The data are presented as the mean±SEM of three independent experiments. ***p<0.001.

Article Snippet: Human PD-L1-expressing plasmids (HG10084-UT), PD-L1-Flag-tagged plasmids (HG10084-NF), control vectors (CV020), and human protein tyrosine phosphatase 1B (PTP1B)-Myc-tagged plasmids (HG10304-CM) were purchased from Sino Biological (China). siRNAs targeting human PD-L1 (NM 014143.2) were designed and synthesized by Bioneer (Republic of Korea) with the following sequences: sense 5′-CUG AGA AUC AAC ACA ACA A (dTdT)-3′ and antisense 5′-UUG UUG UGU UGA UUC UCA G (dTdT)-3′.

Techniques: Western Blot, Cell Fractionation, Knockdown, Co-Immunoprecipitation Assay, Fluorescence, Sequencing, Control, Chromatin Immunoprecipitation, Binding Assay, ChIP-qPCR

Journal: Journal for Immunotherapy of Cancer

Article Title: Cell-intrinsic PD-L1 signaling drives immunosuppression by myeloid-derived suppressor cells through IL-6/Jak/Stat3 in PD-L1-high lung cancer

doi: 10.1136/jitc-2024-010612

Figure Lengend Snippet: PD-L1 promotes CXCL1- and IL-6-dependent migration and activation of MDSCs. ( A ) Heatmap showing the expression of 40 MDSC signature genes in TCGA-LUAD (lung adenocarcinoma) and TCGA-LUSC (lung squamous cell carcinoma) according to PD-L1 low vs high expression (cut-off median). ( B ) The correlation between the MDSC score and the expression of each cytokine in TCGA-LUAD and TCGA-LUSC was calculated using Spearman’s correlation test. ( C ) M-MDSCs and PMN-MDSCs infiltrating the tumor tissues of patients with NSCLC were analyzed by flow cytometry, and the correlation between the MDSC population and PD-L1 TPS was calculated using Spearman’s correlation test. ( D–M ) After Ficoll gradient separation, HLA-DR lo CD11b + CD33 + CD14 + cells/HLA-DR lo CD11b + CD33 + CD15 + cells/and CD15 + low density neutrophils were isolated from PBMCs of healthy donors, and a mixture of these cells (“human MDSCs in figure hereafter”) was submitted to in vitro assays. ( D ) A migration assay for human MDSCs was performed using PD-L1-overexpressing and PD-L1-knockdown NSCLC cells in an 8 μm pore Transwell system. ( E ) Primary lung cancer cells with low and high PD-L1 expression were transfected with PD-L1-overexpressing vector and PD-L1-knockdown siRNA, respectively. MDSC migration assays using CM from PD-L1-overexpressing and PD-L1-knockdown primary lung cancer cells were then performed. ( F, G ) Migration assays of MDSCs with PD-L1-overexpressing A549 cells or CM from PD-L1-overexpressing primary lung cancer cells were performed in the presence of the indicated chemokine-neutralizing antibodies (scale bar=1000 µm). ( H ) MDSCs were co-cultured with PD-L1-overexpressing A549 cells in a 0.4 μm pore Transwell system in the presence of the indicated cytokine-neutralizing antibodies (1 µg/mL each). Arg1 , iNOS , and IDO1 expression in MDSCs was then assessed by qRT-PCR. ( I ) MDSCs and PD-L1-overexpressing A549 cells were co-cultured in a 0.4 μm pore Transwell system in the presence of the indicated cytokine-neutralizing antibodies. MDSCs were then co-cultured in a 0.4 μm pore Transwell system with CFSE-labeled anti-CD3/CD28 bead-stimulated CD4 + or CD8 + T-cells; T-cell proliferation was then analyzed by flow cytometry. ( J ) MDSCs were cultured with CM from PD-L1-low (TPS<1) and PD-L1-high (TPS≥50) primary lung cancer cells or CM from PD-L1-overexpressing or PD-L1-knockdown primary lung cancer cells, then analyzed for IDO1 , Arg1 , and iNOS expression by qRT-PCR. ( K, L ) MDSCs were cultured with CM from PD-L1-overexpressing primary lung cancer cells in the presence of IL-6-neutralizing antibodies and then assessed for IDO1 , Arg1 , and iNOS expression by qRT-PCR. iNOS expression in MDSCs was detected by immunofluorescence staining (scale bar=10 µm). ( M ) MDSCs were cultured in CM from PD-L1-overexpressing primary lung cancer cells or co-cultured with PD-L1-overexpressing A549 cells and then co-cultured with CFSE-labeled anti-CD3/CD28 bead-stimulated CD8 + T-cells in the presence of the indicated inhibitors; T-cell proliferation was analyzed by flow cytometry. The data are presented as the mean±SEM of 4–6 independent experiments. *p<0.05, **p<0.01, ***p<0.001. MDSC, myeloid-derived suppressor cell; PMN, polymorphonuclear; TCGA, The Cancer Genome Atlas.

Article Snippet: Human PD-L1-expressing plasmids (HG10084-UT), PD-L1-Flag-tagged plasmids (HG10084-NF), control vectors (CV020), and human protein tyrosine phosphatase 1B (PTP1B)-Myc-tagged plasmids (HG10304-CM) were purchased from Sino Biological (China). siRNAs targeting human PD-L1 (NM 014143.2) were designed and synthesized by Bioneer (Republic of Korea) with the following sequences: sense 5′-CUG AGA AUC AAC ACA ACA A (dTdT)-3′ and antisense 5′-UUG UUG UGU UGA UUC UCA G (dTdT)-3′.

Techniques: Migration, Activation Assay, Expressing, Flow Cytometry, Isolation, In Vitro, Knockdown, Transfection, Plasmid Preparation, Cell Culture, Quantitative RT-PCR, Labeling, Immunofluorescence, Staining, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Cell-intrinsic PD-L1 signaling drives immunosuppression by myeloid-derived suppressor cells through IL-6/Jak/Stat3 in PD-L1-high lung cancer

doi: 10.1136/jitc-2024-010612

Figure Lengend Snippet: PD-L1 promotes tumor growth in vivo via IL-6-induced immune suppression in a PD-1-independent manner. Stable PD-L1-overexpressing LLC cells and control cells were injected subcutaneously into the flanks of C57BL/6 mice. (A−C) For MDSC depletion, mice were intraperitoneally injected with 150 µg of anti-Ly6C/Ly6G (Gr-1) antibody five times, once every 2 days, starting from 1 day before cancer cell injection. Tumor size was measured every 2–3 days using calipers. Tumor volume was measured using the IVIS luminescence imaging system before the mice were euthanized. ( D ) Immune cell populations in the tumors were assessed using flow cytometry. (E−G) Mice were injected intraperitoneally with 200 µg of anti-mouse IL-6 antibodies every 3 days (a total of five times). Tumor size was measured every 2–3 days using calipers. Tumor volumes were measured using the IVIS luminescence imaging system. (H−J) Immune cell populations in tumors were assessed using flow cytometry. ( K ) MDSCs isolated from the mouse tumors were cultured with CFSE-labeled anti-CD3/CD28 bead-stimulated CD8 + T-cells isolated from the spleen of tumor-free C57BL/6 mice. T-cell proliferation was then assessed using flow cytometry. ( L ) IHC staining of CD8 + , GzmB + , and Ly6C/Ly6G + cells in the tumors (scale bar=200 µm). (M−O) Stable PD-L1-overexpressing LLC cells and control cells were injected subcutaneously into the flanks of PDCD1 (PD-1)-knockout C57BL/6 mice. Tumor size was measured once every 2–3 days using calipers. Tumor volumes were measured using the IVIS luminescence imaging system. (P−R) Immune cell populations in tumors were assessed using flow cytometry. The data are presented as the mean±SEM of five independent experiments. *p<0.05, **p<0.01, ***p<0.001. LLC, Lewis lung carcinoma; MDSC, myeloid-derived suppressor cell.

Article Snippet: Human PD-L1-expressing plasmids (HG10084-UT), PD-L1-Flag-tagged plasmids (HG10084-NF), control vectors (CV020), and human protein tyrosine phosphatase 1B (PTP1B)-Myc-tagged plasmids (HG10304-CM) were purchased from Sino Biological (China). siRNAs targeting human PD-L1 (NM 014143.2) were designed and synthesized by Bioneer (Republic of Korea) with the following sequences: sense 5′-CUG AGA AUC AAC ACA ACA A (dTdT)-3′ and antisense 5′-UUG UUG UGU UGA UUC UCA G (dTdT)-3′.

Techniques: In Vivo, Control, Injection, Imaging, Flow Cytometry, Isolation, Cell Culture, Labeling, Immunohistochemistry, Knock-Out, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Cell-intrinsic PD-L1 signaling drives immunosuppression by myeloid-derived suppressor cells through IL-6/Jak/Stat3 in PD-L1-high lung cancer

doi: 10.1136/jitc-2024-010612

Figure Lengend Snippet: IL-6 secreted by PD-L1-overexpressing cells promotes tumor growth in vivo via myeloid cell activation and an immunosuppressive TME. ( A ) Stable PD-L1-overexpressing LLC cells and control cells were cultured for 48 hours. CD11b + Ly6G + and CD11b + Ly6C + myeloid cells isolated from tumor-free mouse spleens were then cultured for 48 hours in CM from PD-L1-overexpressing LLC cells (LLC-PDL1 OE -CM) in the presence or absence of IL-6-neutralizing antibodies and then injected with fresh wild-type LLC cells (at an equivalent ratio of 1:3) into the flanks of mice (EV, empty vector). ( B, C ) Tumor size was measured once every 2–3 days using calipers. Tumor volume was measured using the IVIS Spectrum imaging system, after which the mice were euthanized and tumor weight was determined. (D−F) Immune cell populations in the tumors were assessed using flow cytometry. (G−I) CD11b + Ly6G + and CD11b + Ly6C + myeloid cells isolated from tumor-free mouse spleens were cultured for 48 hours in CM from PD-L1-knockdown LLC cells (LLC-PDL1 KD -CM) and then injected with fresh wild-type LLC cells (at an equivalent ratio of 1:3) into the flanks of mice (Con, control). For CD8 + T-cell depletion, the mice were intraperitoneally injected with 100 µg of anti-CD8 antibody five times, once every 2 days starting from 1 day before cancer cell injection. Tumor size was measured once every 2–3 days using calipers. Tumor size was measured using the IVIS Spectrum imaging system, after which the mice were euthanized and tumor weight was determined. (J−L) CD11b + Ly6G + and CD11b + Ly6C + myeloid cells isolated from tumor-free mouse spleens were cultured for 48 hours in CM from PD-L1-overexpressing LLC cells (LLC-PDL1 OE -CM) and then injected with fresh wild-type LLC cells (at an equivalent ratio of 1:3) into the flanks of mice. For Treg depletion, the mice were intraperitoneally injected with 100 µg of anti-CD25 antibody five times, once every 2 days starting from 1 day before cancer cell injection. Tumor size was measured once every 2–3 days using calipers. Tumor size was measured using the IVIS Spectrum imaging system, after which the mice were euthanized and tumor weight was determined. *p<0.05, **p<0.01, ***p<0.001. LLC, Lewis lung carcinoma.

Article Snippet: Human PD-L1-expressing plasmids (HG10084-UT), PD-L1-Flag-tagged plasmids (HG10084-NF), control vectors (CV020), and human protein tyrosine phosphatase 1B (PTP1B)-Myc-tagged plasmids (HG10304-CM) were purchased from Sino Biological (China). siRNAs targeting human PD-L1 (NM 014143.2) were designed and synthesized by Bioneer (Republic of Korea) with the following sequences: sense 5′-CUG AGA AUC AAC ACA ACA A (dTdT)-3′ and antisense 5′-UUG UUG UGU UGA UUC UCA G (dTdT)-3′.

Techniques: In Vivo, Activation Assay, Control, Cell Culture, Isolation, Injection, Plasmid Preparation, Imaging, Flow Cytometry, Knockdown

Journal: Journal for Immunotherapy of Cancer

Article Title: Cell-intrinsic PD-L1 signaling drives immunosuppression by myeloid-derived suppressor cells through IL-6/Jak/Stat3 in PD-L1-high lung cancer

doi: 10.1136/jitc-2024-010612

Figure Lengend Snippet: Combined blockade of IL-6 and PD-1 efficiently controls tumor growth and elicits antitumor immune response. ( A ) Wild-type LLC or TC-1 cells were injected subcutaneously into the flanks of C57BL/6 mice. For anti-PD-1 immunotherapy, the mice were injected intraperitoneally with 200 µg of anti-PD-1 antibody every 5 days a total of three times. For anti-IL-6 antibody (Ab) treatment, the mice were injected intraperitoneally with 200 µg of anti-mouse IL-6 Ab every 3 days for a total of five times. ( B, C, E, F ) Tumor size was measured every 2–3 days using calipers. Tumor volume was measured using the IVIS Spectrum imaging system, after which the mice were euthanized and tumor weight was determined. ( D, G ) Mice were observed for survival every 2–3 days. Differences in survival were compared using a log-rank test. (H−L) The immune cell populations of LLC tumors were assessed using flow cytometry. ( M ) IHC staining of CD8 + , GzmB + , and Ly6C/Ly6G + cells in LLC tumors (scale bar=200 µm). ( N ) MDSCs isolated from LLC tumors were cultured with CFSE-labeled anti-CD3/CD28 bead-stimulated CD8 + T-cells isolated from tumor-free C57BL/6 mice; the proliferation of CD8 + T-cells was then assessed using flow cytometry. ( O ) CD8 + T-cells isolated from LLC tumors were cultured with CFSE-labeled wild-type LLC cells for 24 hours. The cells were then stained with 7-AAD and Annexin V, and LLC viability was determined by flow cytometry. ( P ) Schematic diagram showing that tumor-cell-intrinsic PD-L1 activates Jak2/Stat3 signaling and contributes to IL-6 production, which drives MDSC-mediated immunosuppression in PD-L1-high lung cancer. Combined therapy targeting PD-1 and IL-6 may thus be effective in tumor control by restoring antitumor immunity. The data are presented as the mean±SEM of five independent experiments. *p<0.05, **p<0.01, ***p<0.001. CFSE, carboxyfluorescein succinimidyl ester; LLC, Lewis lung carcinoma; MDSC, myeloid-derived suppressor cell.

Article Snippet: Human PD-L1-expressing plasmids (HG10084-UT), PD-L1-Flag-tagged plasmids (HG10084-NF), control vectors (CV020), and human protein tyrosine phosphatase 1B (PTP1B)-Myc-tagged plasmids (HG10304-CM) were purchased from Sino Biological (China). siRNAs targeting human PD-L1 (NM 014143.2) were designed and synthesized by Bioneer (Republic of Korea) with the following sequences: sense 5′-CUG AGA AUC AAC ACA ACA A (dTdT)-3′ and antisense 5′-UUG UUG UGU UGA UUC UCA G (dTdT)-3′.

Techniques: Injection, Imaging, Flow Cytometry, Immunohistochemistry, Isolation, Cell Culture, Labeling, Staining, Control, Derivative Assay