Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A ) Schematic of integrating computational rankings of adhesion proteins into a four-way Venn diagram, based on mass spectrometry proteomic abundance in primary breast tumors (N=122) and TNBC cell lines, altered protein expression in TNBC tumor vs. normal tissue voxels, and breast cancer cell-secreted EVs; to identify novel candidates associated with patient outcomes. B ) Representative IHC images of human PB2 + TNBC tumor and PB2 - normal breast tissue (adjacent to tumor) from a patient with TNBC. C ) KM plot for overall survival of all patients with breast cancer, divided by best cut-off of high vs. low PB2 protein expression in primary tumors, N=108. D ) KM plot for distant metastasis-free survival (DMFS) of patients with ER - breast cancer, divided by median cut-off of high vs. low PB2 mRNA expression, N=218. E ) Schematic depicting the patient blood sample analysis workflow for CTC analysis on CellSearch. F ) Representative CellSearch images of a homotypic PB2 + CTC-CTC (CD45 - CK + DAPI + ) cluster, a heterotypic PB2 + CTC-WBC (CD45 + CK - DAPI + ) cluster, and a single PB2 - CTC, stained after anti-EpCAM bead enrichment. Scale bar = 5 µm. G ) Portion of CellSearch-analyzed PB2 + CTCs (%) in single CTCs, homotypic CTC clusters, and heterotypic CTC-WBC clusters, as shown in (F), N=10. Data are presented as mean values +/- SD, p-values reported are from two-sided unpaired t-tests.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

Techniques: Mass Spectrometry, Expressing, Staining

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A ) Average spectral counts-based abundance ranking of 398 identified adhesion/surface proteins across 122 treatment-naïve primary breast patient samples (N=122) ( https://doi.org/10.1016/j.cell.2020.10.036 ), highlighting key proteins previously identified to be significant in CTCs (CD44, ICAM1, CD81 in green) and a novel candidate PB2 at a higher rank (red). B ) Average peptide spectrum match (PSM)-based abundance ranking of 252 identified adhesion/surface proteins in MDA-MB-231 triple negative breast cancer (TNBC) cell lines (N=3) ( https://doi.org/10.1021/acs.jproteome.1c00293 ). C ) Categories of MS-identified extracellular vesicle proteins (total 1603 peptides) secreted by normal and cancer cells. D ) Heat map of MS-identified differentially expressed proteins between EVs derived from breast cancer cell lines vs. EVs from immortalized normal epithelial cells (unpaired t test P<0.05). PB2 is indicated by a red box. E ) Table showing the top 11 proteins significantly up-regulated in breast tumor tissue vs. normal tissue and whether or not they significantly correlate to poor prognosis in breast cancer. F ) Average intensity expression (in millions) of PB2 in EVs of normal non-tumor epithelial cells compared to that of metastatic breast cancer cells. Data are reported as mean +/- SD, p-value is from a two-sided unpaired t-test.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

Techniques: Derivative Assay, Expressing

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A ) KM plot for distant metastasis-free survival (DMFS) of grade 3 breast cancer with high vs. low PB2 mRNA expression, N=836 (data from KMPlotter). B ) Tissue microarray of advanced breast cancer patient samples subjected to IHC staining for PB2 (N=86). The first 11 cases from the B06 cohort of patients (STU00203283), as highlighted by the green box on the TMA (see Suppl. Excel 3), had simultaneous analyses for the number of CTCs in the blood. C & D) Annotation of PB2 status of TMA patients stratified by specific molecular subtype of breast cancer (C) and tabular annotation of the cases (D). E ) PB2 status of first 11 patients from the B06 cohort in correlation with CTC status in blood.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

Techniques: Expressing, Microarray, Immunohistochemistry

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A ) Schematic showing workflow for flow cytometry analysis of blood cells from patients with breast cancer. B ) Representative flow cytometry gating of single and clustered blood cells, by PB2 +/- and CD45 +/- status, from the B06 cohort of advanced breast cancer patients. C ) Average percentage of cells expressing PB2 in CD45 - single and clustered CTCs from flow analysis, including all EpCAM +/- CTCs, N=17 patients, data reported as mean values +/- SD, p-value from a two-sided unpaired t-test. D ) Percentage of PB2 + CD45 - single and CD45 - clustered CTCs from advanced breast cancer patients at the time of blood draw, N=17 patients, data stratified by individual patient. E ) Table showing total number and proportion of CTC single cells or CTC clusters that are PB2 + from breast cancer patients as reported by flow analysis, N=23.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

Techniques: Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A) Flow panel showing the gating of PB2 + and PB2 - cells sorted from dissociated primary TN3 L2G + PDX cells for clustering in (B-C). B & C) Representative images at 0 and 6 h of clustering (B) and cluster count curves of sorted PB2 high and PB2 low TN3 PDX cells (C), measured by IncuCyte imaging system; N=3 experiments with at least 5 technical replicates each. D & E) Representative images (D) and cluster area growth curves (E) of MDA-MB-231 cells treated with control (siCon), PB2 KD via SmartPool siRNA (siPB2), and single target siRNA (siPB2-10), N=3 experiments with at least 5 technical replicates each. F & G) Representative images (F) and cluster area growth curves (G) of MDA-MB-468 cells treated with siCon, PB2 KD via siPB2 SmartPool (siPB2), and siPB2-10. H ) Schematic of PB2 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 PB2 full-length or mutants (mRBD, dECTO, or dVTDL) over 6 h as measured via IncuCyte; Con vs. KO+PB2 p=0.67, Con vs. KO1 p=0.0002, Con vs. KO2 p=1.43e-6, KO+PB2 vs. KO+mRBD p=0.04, KO+PB2 vs. KO+dECTO p=1.69e-6, KO+PB2 vs. KO+dVTDL p=0.005, KO+PB2 vs. KO1 p=0.0002. L-O) IVIS bioluminescence images (L, N) of NSG mice on days 0 and 4, and quantified signals of metastasis (M, O) of dissected lungs ex vivo on day 4 after tail vein injection of MDA-MB- 231 cells transfected with siCon, siPB2 (L), or siPB-11 (N), N=4 (L-M) and 3 (N-O) biological replicates. Data are presented as mean values +/- SD, p-values reported are from two-sided unpaired t-tests.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

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

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A ) Left panel: Western blot showing MDA-MB-231 KD efficiency of PB2 using SmartPool (siPB2) siRNA as well as single siRNAs (si09, si10, si11), N=3 experiments; Right panel: Proliferation of MDA-MB-231 cells measured as percent confluence using Incucyte. Breast cancer cells plated after double transfection with either SmartPool siRNA (siPB2) or single siRNA KD of PB2 (siPB2-11), N=3 biological replicates with at least 3 technical replicates each, data are presented as mean +/- SD, p-value at 84 h reported as two-sided unpaired t-test. B ) Flow analysis showing MDA-MB-468 KD of PB2 using SmartPool (siPB2) siRNA as well as single siRNA (siPB2-10), N=3 experiments. C ) Flow panel showing KD efficiency of SKBR3 breast cancer cells after double transfection with PB2 SmartPool siRNA. D ) Representative images of SKBR3 cell clusters after 4 h as taken by the Incucyte Live Cell Imager. E ) Quantification of clusters of SKBR3 cells with PB2 KD after 4 h, average cluster area measured by IncuCyte, with N=4 experiments. Data are presented as mean values +/- SD, p- value is from a two-sided unpaired t-test. F ) Flow panel showing KD efficiency of 4T1 mouse breast cancer cells after double transfection with PB2 SmartPool siRNA. G ) Representative images of 4T1 cell clusters after 6 h as taken by the Incucyte Live Cell Imager. H ) Quantification of clustering assay of 4T1 cells with PB2 KD after 6 h, average cluster area measured by Incucyte, with N=3 experiments. Data are presented as mean values +/- SD, p- value is from a two-sided unpaired t-test. I ) Flow panel showing KD efficiency of HS578T breast cancer cells after double transfection with PB2 SmartPool siRNA and siPB2-10 single PB2 siRNA. J ) Representative images of HS578T cell clusters after 4 h as taken by the Incucyte Live Cell Imager. K ) Quantification of clustering assay of HS578T cells with PB2 KD after 4 h, average cluster area measured by Incucyte, with N=3 experiments. Data are presented as mean values +/- SD, p-value is from a two-sided unpaired t-test.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

Techniques: Western Blot, Transfection

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A ) Immunoblots of full-length (FL) and truncated (Tr) PB2 in MDA-MB-231 cells with two pooled clones of CRISPR/Cas9-mediated KO, KO1 and KO2, with PB2-specific bands at 250 kDa and 75 kDa, N=3 experiments; B-D) Flow cytometry analysis of MDA-MB-231 Cas9 control cells for PB2, KO1, and KO2. E) Proliferation assay of MDA-MB-231 control and KO cells over 60+ h measured as percent confluence by IncuCyte, N=3 experiments, data reported as mean +/- SD, p-value is from a two-sided unpaired t-test. F-G) Representative images (F) and cluster size quantification (G) of MDA-MB-231 PB2 control, KO1, and KO2 cell clusters after 6 h as taken by the IncuCyte Live Cell Imager; N=3 experiments with at least 3 technical replicates each, data are presented as mean values +/- SD, p-value is from a two-sided unpaired t-test. H ) Flow cytometry analysis of PB2 overexpression in MDA-MB-231 PB2 KO cells. I & J) Representative images (I) of average mammosphere counts of 10 cells or more (J) of MDA-MB-231 WT cells with scramble (siCon), PB2 SmartPool (siPB2), and PB2 single (siPB2-10) siRNA KD after 7 days; N=3 experiments with at least 5 technical replicates each, data are presented as mean values +/- SD, p-values are from a two-sided unpaired t-test. K & L) Mammosphere images (K) and counts (L) of MDA-MB-231 KO2 cells with overexpression of PB2 full-length (PB2 OE) and a PB2 mutant with depleted extracellular domain (dECTO) for 10 days, N=4 replicates (bottom). Mammospheres larger than 100 microns across were counted. Data are presented as mean +/-SD, p-value is from a two-sided unpaired t-test.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

Techniques: Western Blot, Clone Assay, CRISPR, Flow Cytometry, Control, Proliferation Assay, Over Expression, Mutagenesis

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A ) Abundance ranking of PB2 and Sema4 family members based on average spectral counts among 398 identified adhesion/surface proteins across 122 treatment-naïve primary breast patient samples ( https://doi.org/10.1016/j.cell.2020.10.036 ); the canonical binding ligands of PB2, Sema4C, Sema4D, and Sema4A, are shown by yellow dots. B ) KM plot of distant metastasis-free survival (DMFS) of breast cancer patients based on mRNA expression of Sema4C, p=0.0051. C ) KM plot of overall survival (OS) of breast cancer patients based on mRNA expression of Sema4C, p=0.049.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

Techniques: Binding Assay, Expressing

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A ) Schematic of PB2-Sema4C intercellular interactions in homotypic tumor cell clustering between two cancer cells. B ) Immunoblots of 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 PB2 in the protein complex immunoprecipitated by anti-PB2 antibody compared to IgG. D ) Immunoblot image with reduced Sema4C after Sema4C KD in MDA-MB-231 tumor cells. E & F) Representative images (E) and average cluster growth area curves (F) of MDA-MB-231 control and PB2 KO cells in co-culture with Con or Sema4C KD cells, measured by IncuCyte, N=3 experiments with at least 5 technical replicates each, PB2 + Sema4C + vs. PB2 + Sema4 - p=1.86e-9, PB2 + Sema4C + vs. PB2 - Sema4C - p=6.81e-10, PB2 - Sema4C + vs. PB2 - Sema4C - p=0.22, PB2 + Sema4C - vs. PB2 - Sema4C - p=0.47. G ) Principal component analysis clusters of MDA-MB-231 single cells, siCon clusters, and siPB2 clusters analyzed from global mass spectrometry with N=3 replicates per group. H ) Heat map of differentially expressed proteins from global mass spectrometry analysis of siCon single cells (blue) N=3, siCon clusters (green) N=3, and siPB2 clusters (red) N=3 I ) Gene ontology biological processes analysis of significantly up-regulated and down-regulated proteins in PB2 control vs. PB2 KD clusters in breast cancer cells analyzed by mass spectrometry. Data are presented as mean values +/- SD, p-values reported are from two-sided unpaired t-tests.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

Techniques: Western Blot, Expressing, Immunoprecipitation, Control, Co-Culture Assay, Mass Spectrometry

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A ) Table showing fold change in the top hits from GO analysis of biological processes for proteins that were significantly up-regulated in PB2 KD compared to control cells by global mass spectrometry analysis of clustered cells. B ) GO biological processes analysis of altered proteins in Groups C and F in , with clustering-specific up-regulation and down-regulation, respectively, and reversed in a siPB2- dependent manner. C ) Western blot validation of top hits from Group B (see ) of global mass spectrometry analysis of siPB2 single cells vs. clusters. D & E) Representative images (D) and average cluster area (E) of MDA-MB-231 control vs. MCM7 KD cells as taken by the IncuCyte. N=3 experiments with at least 3 technical replicates each, data are presented as mean values +/- SD, p-value is from a two-sided unpaired t-test.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

Techniques: Control, Mass Spectrometry, Western Blot, Biomarker Discovery

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A ) Schematic of the interactions between breast cancer cell PB2 and monocyte Sema4A for heterotypic cluster formation. B ) Representative images at 4 h co-culture of ex vivo heterotypic clusters of L2G + MDA-MB- 231 control or PB2 KO cells with WBCs from breast cancer patients (Pt). Top row: fluorescent images with WBCs from Pt 396 (minimal cytotox red-stained dead cells). Bottom row: brightfield images with WBCs from Pt 431. C ) Area curves of heterotypic tumor cell clusters co-cultured with WBCs from patients (N=4) as shown in (B), measured by Incucyte imaging. Data are reported as mean +/-SD, with p-values from two-sided unpaired t-tests. D ) Abundance ranking of 96 identified adhesion/surface proteins in human monocytes (N=3), based on mass spectrometry (MS) quantitative analysis ( https://doi.org/10.1038/s41598-020-61356-w ). E ) Sema4A expression on granulocytes, monocytes, and lymphocytes from patients with advanced breast cancer (N=4). F ) Percent of heterotypic CD45 + EpCAM + CTC-WBC clusters vs. CD45 + EpCAM - WBC clusters that express both PB2 and Sema4A, as analyzed by flow analysis, N=4 patients. G ) PB2 immunoprecipitation of MDA-MB-231 tumor-monocyte (THP1) clusters or THP1 monocytes only, blotted for Sema4A and CDC42. H & I) Representative images at 4 h (H) and cluster area curves (I) of heterotypic clustering with MDA-MB-231 tumor cells (green) and THP1 monocytes (red) mixed at a 1:4 ratio, reported as mean +/-SD, N=3 with at least 3 technical replicates each. J & K) Representative images at 6 h (J) and cluster size curves (K) of heterotypic clustering with L2G + TN1 PDX tumor cells (green, sorted by PB2 high and PB2 low ) and THP1 monocytes (red), N=2 experiments with at least 3 technical replicates each. L & M) Representative images at 6 h (L) and cluster size curves (M) of MDA-MB-231 WT (Con) or PB2 KO cells cocultured with THP1 monocytes (sorted by Sema4A+/- expression), N=3 experiments with at least 5 technical replicates each. Data are presented as mean values +/- SD, p-values reported are from two-sided unpaired t-tests.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

Techniques: Co-Culture Assay, Ex Vivo, Control, Staining, Cell Culture, Imaging, Mass Spectrometry, Expressing, Immunoprecipitation

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A ) Quantification of normalized cytotoxic red dye-labeled dead cell events as measured by IncuCyte at 12 h during the MDA-MB-231 breast cancer cell co-culture with WBCs isolated from breast cancer patients, N=4. Data reported as mean +/-SD, p-value is from a two-sided unpaired t-test. B ) Sema4A expression in human PBMCs, analyzed via single-cell sequencing of the Human Protein Atlas. C ) Representative flow plots of Sema4A expression in monocytes, granulocytes, and lymphocytes derived from the blood of patients with breast cancer. D ) Representative gating strategy of flow cytometry analysis of advanced stage breast cancer patient WBCs stained for EpCAM, CD45, PB2, and Sema4A to identify PB2 + Sema4A + heterotypic CTC clusters. E ) Quantification of double positive PB2 + Sema4A + clusters (CD45 +/- ) in advanced stage breast cancer PBMCs, N=4 patients, data are presented as mean +/-SD, p-value is from a two-sided unpaired t-test. F ) Flow cytometry analysis of THP1 monocyte cell line for PB2 and Sema4A expression, N=3. G ) Average Cytotox Red counts in heterotypic THP1 co-culture system as counted by IncuCyte, N=3, data are presented as mean +/-SD, p-value is from an unpaired two-sided t-test.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

Techniques: Labeling, Co-Culture Assay, Isolation, Expressing, Sequencing, Derivative Assay, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A) Schematic showing the experimental workflow of orthotopic implantation of tumor cells into mouse mammary fat pads and subsequent analyses of lung metastases and blood CTCs. B-C) Representative images (B) and weight quantification of PB2 Con and KO tumors (C) upon harvest from mice at 10 weeks (N=8). D-E) Bioluminescence images of mouse lungs ex vivo (D) and normalized lung metastasis burden (E) at 10 weeks, with bioluminescence counts normalized to cumulative tumor weight, N=5 biological replicates. F-G) Representative images (F) and metastatic burden (G) in control and PB2 KO metastatic cells of mouse lungs with H&E staining at 10 weeks after orthotopic implantation, scale bar = 250 µm; experiments were repeated with the PB KO2 clone. H ) L2G + CTC counts (single and clusters) detected in control and PB2 KO mouse blood via flow cytometry at 10 weeks of spontaneous metastasis, N=4. I ) IVIS bioluminescence images of NSG mice after tail vein injection of pre-clustered MDA- MB-231 cells alone (PB2 Con or KO), and with THP1 cells (PB2 Con/THP1, KO/THP1) on day 0 (D0), 12 h, day 4 (D4). N=3 biological replicates. J-K) Mean photon counts/s of ROI (region of interest) of lungs ex vivo after harvesting lungs at 12 h (J) and day 4 (K), N=3 biological replicates per time point. Data are presented as mean values +/-SD, with p-values reported from two-sided unpaired t-tests.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: PB2 PE (human, R&D Systems #FAB53291P), PB2 APC (human, R&D Systems #FAB53291A), PB2 FITC (mouse, R&D Systems #FAB6836G), Sema4A PE (human/mouse, BioLegend #148404).

Techniques: Ex Vivo, Control, Staining, Flow Cytometry, Injection