Journal: Nature Communications

Article Title: Computational ranking identifies Plexin-B2 in circulating tumor cell clustering with monocytes in breast cancer metastasis

doi: 10.1038/s41467-025-62862-z

Figure Lengend Snippet: a A schematic of an integrated ranking, Rscore, of proteins in breast tumors, cancer cells, and CTCs with tumor specificity and clinical association, using multiple MS proteomic databases The mathematical model of Rscore integrates individual ranks ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${r}_{i}$$\end{document} r i ) of each protein in (1) relative protein abundance, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${r}_{i}$$\end{document} r i (pc), in multiple datasets (patient tumors, CTCs, and cell lines), (2) significance changes, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${r}_{i}$$\end{document} r i (sc) in tumor specificity, including p-value, ratio or fold change, and absolute change, comparing TNBC voxels (laser capture microdissection) to normal adjacent tissues, and (3) clinical association, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${r}_{i}$$\end{document} r i (ca) including p value and hazard ratio, with OS and DMFS among multiple datasets. The significance of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${r}_{i}$$\end{document} r i is multiplied by its constant weight factor ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${c}_{i}$$\end{document} c i ) with the sum divided by n for an integrated Rscore and final top three candidates. Created in BioRender. Tong, F. (2025) https://BioRender.com/nea43wo . b Representative IHC images of PLXNB2 high TNBC tumor and PLXNB2 low normal breast tissue (adjacent to tumors) from a TNBC patient. c KM plot for OS of patients with all breast cancer in the Tang_2018 data set ( n = 108) via Kaplan-Meier plotter, separated by the best cut-off value of PLXNB2 protein expression (4) in primary tumors to define high vs. low within the expression range (0-11). P values were calculated via the Cox-Mantel (log-rank) test. d KM plot for DMFS of patients with ER − breast cancer, divided by median cut-off of PLXNB2 mRNA expression using data from GEO, EGA, and TCGA, n = 218. P values were calculated using a log-rank test. e Schematic depicting the patient blood sample workflow for CTC analysis on CellSearch. f Representative CellSearch images of a homotypic PLXNB2 + CTC-CTC (CD45 − CK + DAPI + ) cluster, a heterotypic PLXNB2 + CTC-WBC (CD45 + CK - DAPI + ) cluster, and a single PLXNB2 - CTC. Scale bar = 5 µm. g Portion (%) of PLXNB2 + CTCs in single CTCs in comparison with homotypic CTC clusters and heterotypic CTC-WBC clusters), respectively, analyzed via CellSearch as shown in ( f ), n = 41 patients. Data are presented as mean values +/- SD, P values reported are from two-sided unpaired t-tests unless specified. Source data are provided as a Source Data file.

Article Snippet: The following overexpression vectors were used: pLV- PLXNB2 - mRBD (Addgene #86240), pLV- PLXNB2-dVTDL (Addgene 86239), pLV- PLXNB2-dECTO (Addgene #86238), PLXNB2 OHu01778C_pcDNA3.1( + ) N-Terminal Flag-Tag (GenScript #SC1626), PLXNB2 OHu01778D_pcDNA3.1 + /C- C-Terminal Flag-Tag (GeneScript #OHu01778D), PLXNB2 Untagged Construct (GeneScript #SC1625).

Techniques: Quantitative Proteomics, Laser Capture Microdissection, Expressing, Comparison

Journal: Nature Communications

Article Title: Computational ranking identifies Plexin-B2 in circulating tumor cell clustering with monocytes in breast cancer metastasis

doi: 10.1038/s41467-025-62862-z

Figure Lengend Snippet: a Schematic showing the experimental workflow of orthotopic implantation and analyses of lung metastases and CTCs. b , c Representative images ( b ) and weight quantification of PLXNB2 Con and KO tumors ( c ) from mice at 10 weeks, n = 8 mice/group. d , e Bioluminescence images (BLI) of mouse lungs ex vivo ( d ) and quantified lung metastasis ( e ) at 10 weeks, n = 8 mice/group. f L2G + CTC counts (single and clusters) detected in PLXNB2 Con and KO mouse blood at 10 weeks of spontaneous metastasis, n = 4 mice/group. g , h Representative images ( g ) and metastatic burden ( h ) in PLXNB2 Con and KO metastatic cells of mouse lungs with H&E staining, scale bar = 250 µm; experiments were repeated with the PB2 KO clone, n = 4 lungs/group. Yellow arrows point to the micro metastases in the lungs. i Schematic representing experimental workflow of dual color implantations of L2T + (red) or L2G + (green) MDA-MB-231 tumor cells with PLXNB2 control (ConT, ConG) or PLXNB2 KO (KOT, KOG); mice were sacrificed after 6 weeks for analyses, n = 4 mice/group. j Representative images of green (ConG/KOG) and red (ConT/KOT) MDA-MB-231 colonies in the lungs of mice, n = 4 mice/group. k Counts of L2T + or L2G + colonies in the lungs of mice after 6 weeks orthopedic, n = 4 mice/group. l Counts of dual color colonies with red and green tumor cells in the lungs, n = 4 lungs/group. m CTC clusters in red/green count as analyzed via flow cytometry in the mice bearing the PLXNB2 Control and KO tumors, n = 8 mice/group. n Top left: Schematic of orthotopic implantation of PLXNB2 Con and KO tumor cells into of NSG mice (200,000 cells/site). Bottom left: KM plot of the mice shows Supplementary survival (2 weeks) in the mice bearing Con vs. KO tumors. Right panels: Photos (top) and bar graphs (bottom) of relatively comparable tumor weight between Con (8-week) and KO groups (10-week), n = 5 mice/group. o – q Bar graphs of blood CTC clusters ( o ), cell cycle phases ( p ), and spontaneous lung metastasis ( q ) between the Con and KO tumors, n = 5 mice/group.Data are presented as mean values +/-SD, with P values reported from two-sided unpaired t-testsunless specified. Source data are provided as a Source Data file.

Article Snippet: The following overexpression vectors were used: pLV- PLXNB2 - mRBD (Addgene #86240), pLV- PLXNB2-dVTDL (Addgene 86239), pLV- PLXNB2-dECTO (Addgene #86238), PLXNB2 OHu01778C_pcDNA3.1( + ) N-Terminal Flag-Tag (GenScript #SC1626), PLXNB2 OHu01778D_pcDNA3.1 + /C- C-Terminal Flag-Tag (GeneScript #OHu01778D), PLXNB2 Untagged Construct (GeneScript #SC1625).

Techniques: Ex Vivo, Staining, Control, Flow Cytometry

Journal: Nature Communications

Article Title: Computational ranking identifies Plexin-B2 in circulating tumor cell clustering with monocytes in breast cancer metastasis

doi: 10.1038/s41467-025-62862-z

Figure Lengend Snippet: a Flow panel showing the gating of PLXNB2 + and PLXNB2 - cells sorted from dissociated primary TN3 L2G + PDX cells for clustering in ( b , c ). b , c Representative images of clustering ( b ) and average cluster area and cluster count curves of sorted PLXNB2 high and low TN3 PDX cells ( c ); n = 5 technical replicates examined over 3 independent experiments. P-value was calculated using two-sided unpaired t-test. d , e Representative images ( d ) and cluster area ( e ) of MDA-MB-231 cells transfected with siRNA control (siCon), PLXNB2 SmartPool siRNA ( siPLXNB2 ), and single siRNA ( siPLXNB2-10 ), n = 5 technical replicates examined over 3 independent experiments. P-value was calculated using two-sided unpaired t-test. f , g Representative images ( f ) and cluster area ( g ) of MDA-MB-468 cells transfected with siCon, si PLXNB2 , and si PLXNB2 -10, n = 5 technical replicates examined over 3 independent experiments. P values were calculated using one-sided ANOVA. h Schematic of PLXNB2 domains: Extracellular (Ecto) domains include SEMA = SEMAphorin domain; IPT = Ig-like fold domain; and PSI = Plexin-SEMAphorin-integrin domain. Intracellular domains include RBD = Rho-binding domain; GAP = GTPase activating protein domain; and VTDL = PDZ-domain binding site (Rho-GEF binding). i Immunoblots of overexpressed PB2 mutants with either full-length (fl) or truncated (tr) depletions: mutated RBD (mRBD) tr, depleted Ecto domain (dECTO) fl and depleted VTDL (dVTDL) tr in MDA-MB-231 PB2 KO cells. j , k Representative images ( j ) and cluster area growth curves ( k ) of MDA-MB-231 PB2 KO clusters with overexpression of PLXNB2 full-length or mutants (mRBD, dECTO, or dVTDL); Con vs. KO + PLXNB2 p = 0.67, Con vs. KO1 p = 0.0002, Con vs. KO2 p = 1.43e-6, KO + PLXNB2 vs. KO+mRBD p = 0.04, KO + PLXNB2 vs. KO+dECTO p = 1.69e-6, KO + PB2 vs. KO+dVTDL p = 0.005, KO + PLXNB2 vs. KO1 p = 0.0002, n = 5 technical replicates examined over 3 independent experiments. P values were calculated using one-sided ANOVA. l – o Bioluminescence images ( l , n ) and quantified BLI signals ( m , o ) of dissected lungs ex vivo after transfections with siCon, siPLXNB2 ( l ), or si PLXNB2 -11 ( n ), n = 4 mice/group ( l , m ) and n = 3 mice/group ( n , o ). P-value between two groups was calculated using two-sided unpaired t-test. Data are presented as mean values ± SD. Source data are provided as a Source Data file.

Article Snippet: The following overexpression vectors were used: pLV- PLXNB2 - mRBD (Addgene #86240), pLV- PLXNB2-dVTDL (Addgene 86239), pLV- PLXNB2-dECTO (Addgene #86238), PLXNB2 OHu01778C_pcDNA3.1( + ) N-Terminal Flag-Tag (GenScript #SC1626), PLXNB2 OHu01778D_pcDNA3.1 + /C- C-Terminal Flag-Tag (GeneScript #OHu01778D), PLXNB2 Untagged Construct (GeneScript #SC1625).

Techniques: Transfection, Control, Binding Assay, Western Blot, Over Expression, Ex Vivo

Journal: Nature Communications

Article Title: Computational ranking identifies Plexin-B2 in circulating tumor cell clustering with monocytes in breast cancer metastasis

doi: 10.1038/s41467-025-62862-z

Figure Lengend Snippet: a Schematic of PLXNB2-SEMA4C intercellular interactions in homotypic tumor cell clustering between two cancer cells. b Immunoblots of PLXNB2 (PB2) and SEMA4C expression in different breast cancer cell lines and PDX models using antibodies that are specific to detect both human and mouse isoforms. c Immunoblots of SEMA4C, CDC42, and PLXNB2 in the protein complex immunoprecipitated by anti-PLXNB2 antibody compared to IgG. d Immunoblot images showing reduced SEMA4C levels after SEMA4C KD in MDA-MB-231 tumor cells (left) and depleted PLXNB2 in PLXNB2 KO1 and KO2 cells (right). e Schematic illustration and immunoblotting of MDA-MB-231 control and PLXNB2 -KO cells transfected with siRNA control or siSEMA4C knockdown, followed by tumor cell clustering assessment in suspension. f , g Representative images ( f ) and average cluster area/size curves ( g ) of MDA-MB-231 control (Con) and PB2 KO (KO) cells transfected with control siRNA (siCon) or SEMA4C siRNAs ( si4C ) for KD, measured by IncuCyte, n = 5 technical replicates examined over 5 independent experiments. For cluster size (area) comparisons with the group of Con+ siCon , Student’s t test p = 0.033 for Con+ si4C , p = 0.021 for KO+ si4C , and p = 0.016 for KO+ siCon . For the cluster number comparisons with the Control group, p < 0.001 for all three pairs above. P value were calculated using one-sided ANOVA. h Principal component analysis clusters of MDA-MB-231 single cells, siCon clusters, and si PLXNB2 clusters analyzed from global proteomics analysis with n = 3 technical replicates/ group over three independent experiments. i Heat map of differentially expressed proteins from global proteomics analysis of siCon single cells (blue), siCon clusters (green), and si PLXNB2 clusters (red), with n = 3 technical replicates/ group over three independent experiments. j Gene ontology biological processes analysis of significantly up-regulated and down-regulated proteins in PLXNB2 control vs. PLXNB2 KD clusters in breast cancer cells analyzed by mass spectrometry. Data are presented as mean values ± SD. P values were calculated using one-sided ANOVA. Source data are provided as a Source Data file.

Article Snippet: The following overexpression vectors were used: pLV- PLXNB2 - mRBD (Addgene #86240), pLV- PLXNB2-dVTDL (Addgene 86239), pLV- PLXNB2-dECTO (Addgene #86238), PLXNB2 OHu01778C_pcDNA3.1( + ) N-Terminal Flag-Tag (GenScript #SC1626), PLXNB2 OHu01778D_pcDNA3.1 + /C- C-Terminal Flag-Tag (GeneScript #OHu01778D), PLXNB2 Untagged Construct (GeneScript #SC1625).

Techniques: Western Blot, Expressing, Immunoprecipitation, Control, Transfection, Knockdown, Suspension, Mass Spectrometry

Journal: Nature Communications

Article Title: Computational ranking identifies Plexin-B2 in circulating tumor cell clustering with monocytes in breast cancer metastasis

doi: 10.1038/s41467-025-62862-z

Figure Lengend Snippet: a Schematic of the interactions between breast cancer cell PLXNB2 and monocyte SEMA4A for heterotypic cluster formation. b Representative images of heterotypic clusters of L2G + MDA-MB-231 control or PLXNB2 KO cells with WBCs from breast cancer patients (Pt). Top: fluorescent images with WBCs from Pt 396 (minimal Cytotox red-stained dead cells). Bottom: brightfield images with WBCs from Pt 431. An average of 13581 and 11376 WBCs is associated with Con and KO clusters, respectively. c Heterotypic cluster area curves as shown in ( b ) ( n = 5 replicates examined over 4 individual experiments). P-value was calculated using two-sided unpaired t-test. d SEMA4A expression on granulocytes, monocytes, and lymphocytes from four patients with advanced breast cancer. P-values were calculated using one-sided ANOVA. e Percent of heterotypic CTC-WBC clusters vs. WBC clusters that express both PB2 and SEMA4A, n = 4 patient samples. P-value was calculated using two-sided unpaired t-test. f , g Immunoblots of SEMA4A ( f ) and CDC42 ( g ) with cell lysates immunoprecipitated with anti-PlexinB2 (αPB2) or IgG isotype. h , i Representative images ( h ) and cluster area curves ( i ) of heterotypic clustering of MDA-MB-231 cells (green) and THP1 monocytes (yellow) mixed at a 1:4 ratio, n = 3 technical replicates examined over 3 individual experiments. P-value was calculated using two-sided unpaired t-test. j , k Representative images ( j ) and cluster size curves ( k ) of heterotypic clustering with L2G + TN1 PDX tumor cells (green, sorted by PLXNB2 high and low) and THP1 (yellow), n = 3 technical replicates examined over 3 individual experiments. P-value was calculated using two-sided unpaired t-test. l-m) Representative images ( l ) and cluster size curves ( m ) of MDA-MB-231 Con or KO cells clustering with THP1 (transfected with siCon or siSEMA4A ), n = 3 technical replicates examined over 3 individual experiments. P values were calculated using one-sided ANOVA.n) BLI images of NSG mice after tail vein injection of pre-clustered MDA-MB-231 cells alone ( PLXNB2 Con or KO), and with THP1 cells ( PLXNB2 Con/THP1, KO/THP1), n = 3 mice/group. P values were calculated using one-sided ANOVA. o BLI flux of lungs, n = 3 mice/group at each time point. Data are presented as mean values ± SD. Source data are provided as a Source Data file.

Article Snippet: The following overexpression vectors were used: pLV- PLXNB2 - mRBD (Addgene #86240), pLV- PLXNB2-dVTDL (Addgene 86239), pLV- PLXNB2-dECTO (Addgene #86238), PLXNB2 OHu01778C_pcDNA3.1( + ) N-Terminal Flag-Tag (GenScript #SC1626), PLXNB2 OHu01778D_pcDNA3.1 + /C- C-Terminal Flag-Tag (GeneScript #OHu01778D), PLXNB2 Untagged Construct (GeneScript #SC1625).

Techniques: Control, Staining, Expressing, Western Blot, Immunoprecipitation, Transfection, Injection

Journal: Nature Communications

Article Title: Computational ranking identifies Plexin-B2 in circulating tumor cell clustering with monocytes in breast cancer metastasis

doi: 10.1038/s41467-025-62862-z

Figure Lengend Snippet: a , b Representative images at 6 h clustering ( a ) and quantification ( b ) of 4T1 cells transfected with siCon and siPlxnb2 during co-clustering with mouse white blood cells over 6 h; n = 5 technical replicates examined over 3 individual experiments, p = 0.008. c Schematic and quantification of % of heterotypic mouse 4T1 CTC clusters with monocytes at 6 h after tail vein injection of the tumor cells transfected with siRNA control (Con) and si Plxnb2 (>80% knockdown efficiencies by flow). 5 × 10 5 4T1 tumor cells were injected into Balb-c mice via the tail vein and cardiac blood was collected at 6 h for CTC analysis via flow cytometry ( n = 5 mice/group). d – i Schematic of orthotopic 4T1 breast tumor implantations (1.5 × 10 6 cells) at the L4/R4 mammary fat pads and following metastasis and CTC analyses ( d ), representative photos of 4T1 tumors (Control and Plxnb2 KO) and ex vivo lung bioluminescence images on Day 9 ( e ), and quantified tumor weight and volume ( f ), lung metastasis (total flux of bioluminescence) ( g ) and CTCs, including single CTCs in live blood cells ( h ) as well as 4T1-WBC heterotypic clusters and CTC-myeloid clusters ( i ) among all white blood cells (WBCs), shown as # of events in 10,000 WBCs, n = 5 mice/group. Tumor burden includes both L4/R4 tumors in each mouse and is used to normalize lung metastasis signals in ( g ). Data are presented as mean values +/-SD, with P values reported from two-sided unpaired t-tests. Source data are provided as a Source Data file.

Article Snippet: The following overexpression vectors were used: pLV- PLXNB2 - mRBD (Addgene #86240), pLV- PLXNB2-dVTDL (Addgene 86239), pLV- PLXNB2-dECTO (Addgene #86238), PLXNB2 OHu01778C_pcDNA3.1( + ) N-Terminal Flag-Tag (GenScript #SC1626), PLXNB2 OHu01778D_pcDNA3.1 + /C- C-Terminal Flag-Tag (GeneScript #OHu01778D), PLXNB2 Untagged Construct (GeneScript #SC1625).

Techniques: Transfection, Injection, Control, Knockdown, Flow Cytometry, Ex Vivo

Journal: bioRxiv

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1101/2024.01.02.573660

Figure Lengend Snippet: (A) Schematic of CRISPR/Cas9-mediated PLXNB2 knockout (KO) with small guide (sg) RNA targeting second coding exon. (B) Western blots show Plexin-B2 expression in different SD2 GSCs, with β-actin as loading control. Note Plexin-B2 precursor at 240 kDa and mature form at 170 kDa. (C) IF images show Plexin-B2 expression in different SD2 GSCs, with Hoechst nuclear counterstain. (D) Left, schematic of atomic force microscopy (AFM) indentation method to probe cell stiffness by cantilever deflection. Middle, AFM indentation curves of different SD2 GSCs; right, box plots of cell stiffness, showing 25– 75% quartiles, median (line), and mean (plus sign). n= 6 cells per group. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (E) Left, depiction of membrane tension measurement with optical tweezers. Middle, force measurements during tether extrusion (shaded box). Right, quantifications of tether extrusion forces. n=5 cells per group. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (F) Left, schematic of FLIM of cell membranes labeled with Flipper-TR membrane dye, with low and high membrane tension associated with shorter and longer lifetimes, respectively. Middle top, representative FLIM images, with lifetime heatmap shown on right. Middle bottom, images show similar fluorescence intensities of Flipper-TR dye in WT and PB2 KO cells. Right top, violin plots show fluorescence lifetime from 3 images per group. Two-sided unpaired t-test. Right bottom, phasor plots of FLIM image data, with arrow indicating a shift to shorter lifetime values for PB2 KO cells. (G) Model of Plexin-B2 regulation of cortical contractility and membrane tension. Phalloidin staining show differences of F-actin network in WT and PB2 KO SD2 GSCs. DAPI for nuclear staining. Arrows point to stress fibers and spread-out contours of the WT GSCs.

Article Snippet: The lentiviral vector for Dox-inducible Plexin-B2 overexpression was generated by inserting human PLXNB2 cDNA into a Dox controlled expression vector (pLenti-CMVtight-PLXNB2 iOE; deposited as Addgene #176849) .

Techniques: CRISPR, Knock-Out, Western Blot, Expressing, Control, Microscopy, Membrane, Labeling, Fluorescence, Staining

Journal: bioRxiv

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1101/2024.01.02.573660

Figure Lengend Snippet: (A) Top, timeline for dextran uptake assay. Bottom, live-cell imaging of WT and PB2 KO SD2 GSCs labeled with SPY-Actin and exposed to dextran-Alexa488. Enlarged images of boxed areas are shown below. Quantification of the areas of dextran + clusters per cell are shown in box plots, with 25–75% quartiles, median (line), and mean (plus sign). n=85 cells for WT, n=44 cells for PB2 KO. Mann–Whitney–Wilcoxon test. (B) Top, live cell confocal plane images of WT and PB2 KO GSCs with side views of z-stacks showing intracellular localization of diffuse dextran-Alexa 488 signals in PB2 KO cells in addition to dextran endosome signals. In contrast, WT cells contained only dextran + endosomes. Bottom, histograms show fluorescence profiles showing bimodal distribution of dextran-Alexa 488 fluorescence intensities in PB2 KO GSCs (blue and brown arrows). n=177 cells for WT, n=161 cells for PB2 KO. Mann–Whitney–Wilcoxon test. (C, D) Left, schematic of myr-palm-GFP or -CFP attached to inner membrane leaflet. Right, live cell fluorescence imaging at 72 hr after transfection shows internalization of myr-palm-GFP or -CPF on endomembranes (arrow) in WT GSCs, in contrast to membrane retention of the probes (arrowhead) in PB2 KO GSCs. (E) Left, schematic of TauSTED super-resolution microscopy of GSCs labeled with MemGlow. Middle, TauSTED live-cell images show reduced endosomes (arrowheads) in PB2 KO cells compared to WT. Right, box plots show areas of MemGlow clusters in each cell. n=26 cells for WT, n=13 cells for PB2 KO. Two-sided unpaired t-test. (F) Working model of regulation of cortical and membrane tension by Plexin-B2, affecting endocytosis and membrane permeability in GSCs.

Article Snippet: The lentiviral vector for Dox-inducible Plexin-B2 overexpression was generated by inserting human PLXNB2 cDNA into a Dox controlled expression vector (pLenti-CMVtight-PLXNB2 iOE; deposited as Addgene #176849) .

Techniques: Live Cell Imaging, Labeling, MANN-WHITNEY, Fluorescence, Membrane, Imaging, Transfection, Super-Resolution Microscopy, Permeability

Journal: bioRxiv

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1101/2024.01.02.573660

Figure Lengend Snippet: (A) Left, schematic of PH(PLCδ1)-GFP PIP2 probe. Right, live-cell imaging at 72 hr post transfection reveals that the PH(PLCδ1)-GFP probes were largely internalized in WT GSCs (arrow), but retained on membrane of PB2 KO GSCs (arrowhead). (B) Left, still images of videography show accumulation of the PH(PLCδ1)-GFP probes (arrow) in front of the nucleus (NucSpot) of migrating WT SD2 GSCs in tunnels, more so in 3 than 8 µm tunnel, but not in PB2 KO cells. Dashed lines delineate cell boundary. Long arrow denotes direction of migration. Right, quantifications of the ratio of PH(PLCδ1)-GFP fluorescence intensity at front vs. rear of GSCs during passage. n=13-16 cells per condition. One-way ANOVA followed by Tukey’s multiple comparison test. Data represent mean ± SEM. (C) Left, schematic of R(+8)-pre-GFP probe for negative surface charge of inner plasma membrane. Right, live-cell imaging at 72 hr post-transfection shows internalization of the probes (arrow) in WT GSCs, in contrast to the predominant membrane localization in PB2 KO GSCs (arrowhead). (D) Left, still images of videography show accumulation of the R(+8)-pre-GFP probes (arrow) at front zone of WT GSCs when traversing the 3 µm tunnel, but not in PB2 KO cells. Right, bar graphs show the ratio of R-pre-GFP fluorescence intensity at rear vs. front of GSCs when passing through tunnels. n=22 cells for WT, n=27 cells for PB2 KO. Mann–Whitney–Wilcoxon test. Data represent mean ± SEM. (E) Diagram illustrating voltage sensitive FluoVolt membrane dye, with fluorescent intensity quenched by voltage-sensitive electron transfer from electron-rich donor mediated by “molecular wire” in plasma membrane. (F) Left, FluoVolt live-cell imaging shows reduced FluoVolt fluorescent intensity in cell membrane of Plexin-B2 KO cells, consistent with higher negative charges of inner membrane. Right, box plots of membrane FluoVolt intensity. n=25 cells for WT, n=27 cells for PB2 KO. Two-sided unpaired t-test. Data represent mean ± SEM. (G) Left, still images from videography show higher FluoVolt fluorescent signals at rear zone (arrowhead) of WT GSCs when traversing tunnels, more so in 3 than 8 µm tunnel, but not in PB2 KO cells. Migration direction is denoted by long arrow. Right, bar graphs show the ratio of FluoVolt intensity at rear vs. front during confined migration. n=15 cells per group. One-way ANOVA followed by Tukey’s multiple comparison test. Data represent mean ± SEM. (H) Live-cell images and quantifications show the effects of constitutive active (CA) RAP1B-V12 or dominant-negative (DN) RAP1B-N17 on FluoVolt intensity in WT or PB2 KO GSCs. n=25 cells per group. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (I) Left, still images capture calcium localization (Fluo4-AM fluorescence, arrowhead) at the rear of WT GSCs when traversing tunnels, more so in 3 than 8 µm tunnel, but not in PB2 KO cells. Migration direction is denoted by long arrow. Right, bar graphs showing Fluo4-AM intensity ratio at rear vs. front in GSC during passage through tunnels. n=15-16 cells. One-way ANOVA followed by Tukey’s multiple comparison test. Data represent mean ± SEM. (J) Left, still images from videography show that calcium chelator BAPTA-AM disrupted the pattern of high FluoVolt signals at the rear of WT GSCs (arrowhead) during confined migration. Right, bar graphs show FluoVolt intensity ratio at rear vs. front of GSCs when traversing tunnels. n=21 cells for WT, n=16 cells for PB2 KO. Two-sided unpaired t-test. Data represent mean ± SEM. (K) Model of Plexin-B2 signaling affecting membrane surface charge and electric field during polarized confined migration, with PIP2 enrichment at cell front and Ca 2+ at rear zone, leading to asymmetry of FluoVolt and R(+8)-pre-GFP.

Article Snippet: The lentiviral vector for Dox-inducible Plexin-B2 overexpression was generated by inserting human PLXNB2 cDNA into a Dox controlled expression vector (pLenti-CMVtight-PLXNB2 iOE; deposited as Addgene #176849) .

Techniques: Live Cell Imaging, Transfection, Membrane, Migration, Fluorescence, Comparison, Clinical Proteomics, MANN-WHITNEY, Dominant Negative Mutation

Journal: bioRxiv

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1101/2024.01.02.573660

Figure Lengend Snippet: (A) Structure model of the extracellular domain of human Plexin-B2 show the locations of lock1 and lock2 mutations predicted to form disulfide bridges that lock the ring structure. (B) Western blots show absence of mature Plexin-B2 (170 kDa) in PB2 KO GSC, and expression of lock mutants in PB2 KO SD2 and SD3 GSCs. β-actin serves as a loading control. (C) Still images from videography show passage of GSCs (nuclei visualized by NucSpot) through microchannels with PB2 wildtype rescue construct but not lock mutants, nor PB2 with deletion of extracellular domain (dECTO). Chevrons point to 3 µm constrictions. (D) Box plots show velocity through constrictions, stalling time at constrictions, and sum of forward and backward movements, with 25–75% quartiles, minimal and maximal values (whiskers), median (line), and mean (cross). For velocity and sum of movements: n=17-20 cells per condition. For stalling time at constriction: n=14-28 cells per condition. One-way ANOVA followed by Dunnett’s multiple comparisons test. (E) Still images from videography show F-actin assembly (SPY-actin, arrowhead) at cell rear and MemGlow + endosomes (arrow) at cell front of SD3 GSCs with Plexin-B2 WT rescue but not mutant rescues when traversing 3 µm constrictions (chevrons). (F) Bar graphs showing fluorescence intensity ratio of SPY-actin and MemGlow at rear vs. front of GSCs during confined migration. n=10-18 cells per condition. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Data represent mean ± SEM. (G) Model of Plexin-B2 signaling and mechano-electrical regulation of membrane tension and membrane surface charge during polarized confined migration. Regionalized enrichment of endocytosis/PIP2 at cell front and F-actin/Ca 2+ at rear zone lead to asymmetry of FluoVolt and R(+8)-pre-GFP membrane probes.

Article Snippet: The lentiviral vector for Dox-inducible Plexin-B2 overexpression was generated by inserting human PLXNB2 cDNA into a Dox controlled expression vector (pLenti-CMVtight-PLXNB2 iOE; deposited as Addgene #176849) .

Techniques: Western Blot, Expressing, Control, Construct, Mutagenesis, Fluorescence, Migration, Membrane