Fig 2: Patient-derived organoid (PDOs) propagation, characterization, and bidirectional interactions with stromal cells in welled plates.a Schematic describing the workflow used to generate PDOs from biopsy samples via fine-needle biopsy (FNB). PDOs (generated from the biopsy sample of one patient) formed sphere-shaped cell clusters in 3D Matrigel culture. Scale bar, 100 µm. b Characterization of the isolated PDOs by H&E and EpCAM staining. Scale bar, 100 µm. c PDOs cocultured with PSCs and U937 monocytes showed an increased average diameter (±SEM, n = 3) over a 6 day culture period in welled plates. Scale bar, 100 µm. d Coculture of the organoids with PSCs significantly increased collagen deposition (n = 3 wells, expression quantified from ten fields). Scale bar, 20 µm. E Bidirectional increase in the proliferation of neoplastic epithelial cells (PDO and MIA PaCa-2) and U937 monocytes in a transwell culture assessed by a cell-counting kit assay (n = 5, CCK8). *p < 0.05, **p < 0.01, ***p < 0.001

Fig 3: Software-based determination of EpCAM and Slug expression intensities in HNSCC tissue microarrays. (A) Tissue microarray samples stained for the expression of EpCAM were analyzed with the Definiens Developer XD2 (Definiens AG) to identify nuclei and the remaining membrane and cytoplasmic areas (middle panel). EpCAM staining intensity was thereafter determined in the membrane/cytoplasm as no (0, white), weak (1, yellow), intermediate (2, orange), to high expression (3, brown). (B) Tissue microarray samples stained for the expression of Slug were analyzed with the Definiens Developer XD2 (Definiens AG) to identify nuclei and Slug staining intensities were determined as (0, blue), weak (1, yellow), intermediate (2, red), to high expression (3, brown).

Fig 4: ELTD1 Facilitates EMT of GC Cells. (A) The Pearson correlation between ELTD1 and epithelial markers (CDH1, EPCAM), mesenchymal markers (VIM, CDH2) was analyzed using TCGA STAD data. (B) The Pearson correlation between ELTD1 and EMT transcription factors (ZEB1, ZEB2, SLUG, TWIST2) was analyzed using TCGA STAD data. (C) Cytoskeleton of indicated cells is visualized by staining F-actin with rhodamine-phalloidin. Scale bars, 5 µm. (D) qRT-PCR was used to detect the expression of EMT markers in ELTD1-interfered and control cells. (E) Western blot assay was used to detect the expression of EMT markers, ZEB1, and MMP2 in ELTD1-interfered and control cells.

Fig 5: CSCs isolated from standard breast and ovarian cancer cell lines express higher protein levels of HDAC1 and HDAC7, and have increased HDAC enzymatic activity. (a) The higher sphere forming capacity of CD44high/CD166high CSCs (black bars) compared to CD44low/CD166low nsTC cells (white bars) is observed in multiple breast (SUM159, MDA-231, and MCF7) and ovarian (SKOV3 and OV90) cell lines. The data are presented as the mean of sphere counts from triplicate wells, the error bars represent standard deviation of the mean (P<0.01). The results are representative of at least three independent experiments. (b) The higher HDAC enzyme activity is observed CD44high/CD166high CSCs (black bars) compared to CD44low/CD166low nsTCs (white bars) in multiple breast (SUM159, MDA-231, and MCF7) and ovarian (SKOV3 and OV90) and colon (HT29) cancer cell lines. Results are representative of at least three independent experiments. The error bars represent standard deviation of the mean (P<0.01). (c) HDAC1 and HDAC7 protein expression is significantly higher in CD44high/CD166high CSCs (+/+) from multiple breast (BPLER, MDA-MB-231, MCF7, SUM159) and ovarian (SKOV3) cancer cell lines compared to nsTC (-/-, CD44low/CD166low). Merged image of two Western blots run with whole-cell lysates, gel#1(BPLER, MDA-MB-231, MCF7, SUM159) and gel#2(SKOV3). ß-Actin represents loading control. (d) HDAC7high (red) and CD44high (green) co-expression in ovarian cancer cell line SKOV3, demonstrated with double IF staining. Scale bar 25 µM. (e) Double IF staining identifies co-expression of CSC-markers (CD44, CD326, CD166, ALDH1, CD29) with HDAC7 in standard breast cell lines (MDA-MB-231, MCF7, SUM159), and primary human ovarian (OCI-P5x and OCI-E1p) and breast cancer cells (BCI-1009 and BCI-1133). (Y) represents consistent positive correlation of HDAC7 and CSC marker expression in surveyed lines. (y) represents positive, but inconsistent correlation. (N) represents lack of correlation. (n/a), not analyzed. (n/e) CSC marker not expressed in cell line. IF staining was repeated a minimum of two times for each line. See Supplementary Figure 10 for additional markers and cell lines.