Fig 1: Gene expression defines initiating cell potential and other functional roles in tumor cell clusters.a The UMAP visualization showed the clusters found in the tumor cells from tumor and PDX after integration and reclustering. Cells in different states are labeled. Tr1 and Tr4 are clusters with relatively low sequencing depth. b UMAP visualization of sample-group distribution (Top) and percentages of clusters in each sample group (Bottom). c A heatmap was drawn for 200 most upregulated genes per tumor cluster in each sample group. Cell types in background liver and tumor microenvironment are shown as the reference. Normalized values were used for expression levels. Key signature genes in some clusters are shown on the right. Similarity matrix of tumor clusters was calculated with Pearson correlation based on a combined gene list from (c). Hierarchical clustering was applied for rows and columns. d IHC was conducted in B, T, and PDX samples from HB30, B and T from HB53, and PDX from HB17 to show localization of CCND1, KI67, CD34, GPC3, YAP, and EZH2 to differing regions of tumor (20X, scale bar 50 µM). e Violin plots of gene expression for the corresponding genes in (d) are shown to show the expression patterns in each tumor cluster. Dots are only shown in the violin plot of CD34 due to its relatively sparse expression in the single-cell data. f A heatmap of gene set enrichment scores (-log10Padj) of each cluster by ToppGene enrichment using genes in (c). Tumor-association enrichment terms in Gene Ontology (Biological Process) were selected and grouped into several categories. Hierarchical clustering was applied for rows and columns.
Fig 2: GPC3 targeted therapy performed on OS organoids with different GPC3 expression patterns. (A) Immunohistochemical (×20) demonstration of differential GPC3 expression in OS tissues used for organoid culture. (B) Evaluation of apoptosis (TUNEL) and proliferation (EdU) of GPC3-antibody (GPC3-Ab)-treated OS organoids with different levels of GPC3 expression (×40). (C) The response of OS organoids to GPC3-Ab treatment in a GPC3-related manner
Fig 3: RNAseq comparison of HB samples.a Heat map to demonstrate the expression differences between human background liver, source tumor, and PDX tumor over time (passages F0–F5 demonstrate expansion of tumor through multiple sets of mice). Overexpressed genes in HB tumor are often overexpressed to a greater degree in PDX and genes with lower expression can demonstrate further reduction in PDX. PDX tumor maintains a similar gene expression profile through multiple passages. b Validation of upregulation in genes of interest. qRT-PCR validation of gene expression in human HB background liver (B) compared with tumor (T). Each number—B/T represents matched samples from one patient. GPC3 is overexpressed in T vs. B. YAP1 shows some elevation in T, but expression is variable. SHH is generally overexpressed in T. Beta-catenin (CTNNB1) is variable but generally has moderate elevation in T. AXIN2 and FANCD2 are elevated in many T samples and appear to correlate with PDX tumor-growth success.
Fig 4: Establishment and characterization of OS organoids. (A) Bright-field demonstration of continuous growth of an OS-1 organoid (×40). (B) H&E morphological staining was performed on OS tissues from three cases and the corresponding organoids and bright-field images of the organoids of the matched patients with OS. Tissue, ×20; organoid, ×40. (C) Double immunofluorescent labeling of OS-specific biomarkers, SOX9 and vimentin, on OS tissues and organoids with (OS-1) and without (OS-13) GPC3 expression. Tissue, ×20; organoid, ×40. (D) GPC3 and CD133 double immunofluorescent labeling of paired OS tissue and organoids of OS-1 and OS-13. Tissue, ×20; organoid, ×40
Fig 5: Immunohistochemical profiling of intertumoral and intratumoral GPC3 expression patterns of OS specimens. (A) The specificity results of GPC3 immunohistochemical staining (×20) ascertained using two anti-GPC3 antibodies from Abcam and BioMosaics. (B) The grades of GPC3 immunohistochemical labeling (×20) were scored as -, negative; +, weakly positive; ++, moderately positive; and +++, strongly positive, according to the staining intensities (left). (C) Classification of intertumoral GPC3 immunohistochemical labeling (×20) according to the percentage of GPC3-positive tumor cells: –, all negative; +, focal positive; ++, partially positive; and +++, generally positive (left). IHC data were collected from 61 OS specimens
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