biotinylated anti-cd45 and anti-cd16/32 antibodies  (Thermo Fisher)

Journal: Nature Communications

Article Title: Tumor cell-based liquid biopsy using high-throughput microfluidic enrichment of entire leukapheresis product

doi: 10.1038/s41467-024-55140-x

Figure Lengend Snippet: The leukapheresis product (leukopak) is collected from a patient during a 1 to 2 h procedure that samples 3 to 6 L of blood volume. Following the addition of biotinylated antibodies to tag WBCs, a microfluidic debulking chip is used to remove unbound antibodies, RBCs, platelets, and excess plasma. Streptavidin-conjugated magnetic beads are added to tag WBCs, which are then separated from untagged CTCs using an ultrahigh-throughput microfluidic magnetic sorter. Enriched CTCs are imaged using immunofluorescent staining for lineage or tumor markers, subjected to RNA-based quantitation of specific transcripts (ddPCR), or cultured ex vivo. Single CTCs may be isolated for RNAseq or DNA analyses, including mutational profiling and CNV analyses. Together, the two chips comprise the LP CTC-iChip platform. Illustration by Nicole Wolf, MS, ©2024. Printed with permission.

Article Snippet: The WBC depletion cocktail included three antibodies: biotinylated anti-human CD45 (Thermo Fisher Scientific, clone HI30, IgG1, 0.25 μg/million cells), biotinylated anti-human CD16 (BD Biosciences, clone 3G8, IgG1, 0.05 μg/million cells), and biotinylated anti-human CD66b (Novus Biologicals, clone 80H3, IgG1, 0.025 μg/million cells).

Techniques: Clinical Proteomics, Magnetic Beads, Staining, Quantitation Assay, Cell Culture, Ex Vivo, Isolation

Journal: Nature Communications

Article Title: Tumor cell-based liquid biopsy using high-throughput microfluidic enrichment of entire leukapheresis product

doi: 10.1038/s41467-024-55140-x

Figure Lengend Snippet: A – D Immunofluorescence images of representative CTCs enriched from leukopak samples from patients with metastatic prostate cancer (GU-1 and GU-2, n = 2), triple-negative breast cancer (TNBC-1, n = 1), hepatocellular carcinoma (HCC-1 and HCC-2, n = 2), and uveal melanoma (UM-1 and UM-2, n = 2). Bulk cell populations are stained with DAPI nuclear marker (blue), the relevant tumor markers grouped within a single color (green) (see individual epitopes below), and with antibodies against the WBC markers CD45, CD16, CD66b (red). Tumor markers: A GU-1 and GU-2 (EpCAM, pan CK, CK19), B TNBC-1 (EpCAM, pan CK, CK19), C HCC-1 and HCC-2 (EpCAM, pan CK, CK19, ASGR1, GPC3), D UM-1 and UM-2 (Sox10, Melan-A, NG2). E Representative contaminating WBCs. F Table listing blood volumes processed and yield of CTCs obtained from patient-derived leukopaks (median 2799 CTCs per leukopak). G Droplet digital RNA-PCR (ddPCR) analysis of 0.5 to 1% of the bulk CTC products from all seven cases, quantifying expression of previously curated RNA signatures that denote either tissue lineage or cancer-specific transcripts within the background of normal blood cells. Of note, GU-1 CTCs express multiple neuroendocrine genes (CHGA, SYP, and DLL3), consistent with immunohistochemistry staining for synaptophysin and chromogranin A in a resected adrenal metastasis from this patient. Bar graphs showing expression of wild-type androgen receptor (AR-wt) and AR variant 7 (AR-V7) for GU-1 and GU-2 are shown in accompanying Supplementary Fig. . Healthy donor blood is shown as negative control, with positive controls drawn from either cultured prostate cancer cell lines (LNCaP, 22Rv1, and PC3), cultured breast CTCs (BRx-142 ), cultured liver cancer cells (HepG2), or cultured melanoma CTCs (Mel-167 ), respectively. (H) Measured whole cell and nucleus diameters of individual CTCs ( n = 5543), compared with WBCs ( n = 223). Substantial overlap in size is evident between CTC and WBC populations. I Variation across individual CTCs from cases GU-1, GU-2, TNBC-1, HCC-1, HCC-2, and UM-1 in their intensity of staining for the combined lineage markers (see above). Source data are provided as a Source Data file.

Article Snippet: The WBC depletion cocktail included three antibodies: biotinylated anti-human CD45 (Thermo Fisher Scientific, clone HI30, IgG1, 0.25 μg/million cells), biotinylated anti-human CD16 (BD Biosciences, clone 3G8, IgG1, 0.05 μg/million cells), and biotinylated anti-human CD66b (Novus Biologicals, clone 80H3, IgG1, 0.025 μg/million cells).

Techniques: Immunofluorescence, Staining, Marker, Derivative Assay, Expressing, Immunohistochemistry, Variant Assay, Negative Control, Cell Culture

Journal: Nature Communications

Article Title: Tumor cell-based liquid biopsy using high-throughput microfluidic enrichment of entire leukapheresis product

doi: 10.1038/s41467-024-55140-x

Figure Lengend Snippet: A Schematic of single-cell isolation from LP CTC-iChip-enriched leukopak samples using Fluorescence Activated Cell Sorting (FACS). The CTC-enriched LP CTC-iChip product is free of magnetic-conjugated antibodies against WBC markers CD45, CD16 and CD66b. For FACS single-cell isolation, two-color separation is achieved using an AF488-conjugated antibody cocktail against EpCAM and PSMA (for patients GU-1 and GU-2; green) and a PE-Cy7-conjugated antibody cocktail against CD45, CD16, CD66b (labeling contaminating low-expressing WBCs that escaped LP CTC-iChip depletion; red). Individual cells (CTCs and WBCs) are then subjected to paired single-cell whole-genome sequencing and single-cell RNA-seq. Figure was created using BioRender (Agreement number: DF26ZQNZ7S). B Two-step FACS-sorting strategy using an initial ultra-yield bulk sorting to remove dead cells and debris, followed by single-cell plate sorting. A representative single-cell sorting profile is shown here, isolating candidate CTCs that are positive cells for either EpCAM (epithelial marker) or PSMA (prostate lineage marker); cells of uncertain identity that are negative for both EpCAM and PSMA, as well as the WBC markers CD45, CD16 and CD66b (Double-negative; DN); and white blood cells that are positive for CD45, CD16 and CD66b. C Representative DNA copy-number variation (CNV) analysis in individual CTCs, compared with diploid WBCs from the same patient (X chromosome haploid in male patient). Cancer cell-associated aneuploidy was confirmed by CNV in 36/39 single CTC candidates (EpCAM- and/or PSMA-positive) and in 7/10 single DN cells from patient GU-1 and in 76/109 single EpCAM- and/or PSMA-positive CTCs and in 24/52 DN cells from patient GU-2. Ginkgo was used for DNA copy-number analysis from single-cell whole-genome sequencing data. D Normalized heatmap shows clustering of single-cell CNVs across the genome in CTCs ( n = 43) from patient GU-1. All CTCs show shared core chromosomal alterations, while WBCs are diploid. Variability score (VS) quantified DNA and assay quality (low VS corresponds to high quality). Source data are provided as a Source Data file.

Article Snippet: The WBC depletion cocktail included three antibodies: biotinylated anti-human CD45 (Thermo Fisher Scientific, clone HI30, IgG1, 0.25 μg/million cells), biotinylated anti-human CD16 (BD Biosciences, clone 3G8, IgG1, 0.05 μg/million cells), and biotinylated anti-human CD66b (Novus Biologicals, clone 80H3, IgG1, 0.025 μg/million cells).

Techniques: Single-cell Isolation, Fluorescence, FACS, Labeling, Expressing, Sequencing, RNA Sequencing, Marker