Supplementary Fig. 5 from Spatial Profiling of Circular RNAs in Cancer Reveals High Expression in Muscle and Stromal Cells

Analysis of Ki67 staining on colon adenocarcinoma and adjacent normal tissue sections. Representative Ki67 staining from tumor (left) and adjacent normal (right) tissues from the five patients’ samples subjected to the analysis. Positive staining is visible as brown color. In the tumor tissues, Ki67 is clearly present in the cancer cells and in some cells in the tumor stroma. In the normal tissue sections, the positive staining is found at the bottom of the crypts in the epithelium whereas no staining is detected in the mucosa and muscle sub-compartments.</p

Supplementary Fig. 6 from Spatial Profiling of Circular RNAs in Cancer Reveals High Expression in Muscle and Stromal Cells

Expression levels of MKI67. Measurement of levels of MKI67 by RT-qPCR normalized to MRPL19.</p

Supplementary Table 1 from Spatial Profiling of Circular RNAs in Cancer Reveals High Expression in Muscle and Stromal Cells

List of primers designed for Sanger sequencing across the back splicing junctions.</p

Supplementary Figure 1 from Spatial Profiling of Circular RNAs in Cancer Reveals High Expression in Muscle and Stromal Cells

Evaluation of the most abudant circRNAs from human datasets. Boxplot showing the normalized nCounter counts from the top 50 circRNAs extracted from publicly available human RNAseq datasets (2 normal colon, 1 tumor and paired normal adjacent tissue) (GSE77661) (A) and from the top 20 high-abundance circRNAs according to the data extracted from the three different microarray datasets comparing human tumor vs paired normal tisues (GSE126094, GSE142837 and GSE138589) (B). C, NanoString nCounter analysis of 48 circRNA and 7 mRNA in mock and RNase R treated RNA samples from the Caco-2 cell line. Among the 48 circRNAs only 37 were expressed above cutoff levels in this cell line. D, Sanger sequencing across the backsplicing junction of the 3 circRNAs that haven't been described before. The scattered horizontal lines represent the background threshold. The box represents the interquartile range (Q1 and Q3), the median is marked with horizontral line, and whiskers represent m and maximum values.</p

Supplementary Table 2 from Spatial Profiling of Circular RNAs in Cancer Reveals High Expression in Muscle and Stromal Cells

List of reference genes, tissue markers genes and top circular RNAs for which nanostring nCounter probes were designed.</p

Supplementary Fig. 10 from Spatial Profiling of Circular RNAs in Cancer Reveals High Expression in Muscle and Stromal Cells

Muscle and epithelial marker correlations to circSLC8A1 in prostate cancer patients. Scatter plots comparing the normalized expression levels of circSLC8A1 with levels of the muscle-specific marker, DES, ACTA2 and the epithelial markers KLK3, NKX3.1, and KRT8 for each of the 172 prostatic tumor tissue samples. Linear regression was performed to assess potential correlation and the Pearson correlation coefficient (R). Type refers to tissue sample type, where 0 stands for Adjacent normal, 1 stands for localized prostate cancer and 2 stands for Primary tumor sample from patient with metastatic prostate cancer.</p

Supplementary Fig. 8 from Spatial Profiling of Circular RNAs in Cancer Reveals High Expression in Muscle and Stromal Cells

Single molecule in situ hybridization for circSLC8A1 in bladder adenocarcinoma. Brightfield and fluorescent microscopy images of circSLC8A1 at 200X magnification. A signal from circSLC8A1 can be observed in muscle cells, whereas the cancer cells were negative.</p

Supplementary Fig. 9 from Spatial Profiling of Circular RNAs in Cancer Reveals High Expression in Muscle and Stromal Cells

Single molecule in situ hybridization for circSLC8A1 in prostate adenocarcinoma. Brightfield microscopy image at 200X magnification of DESMIN immunohistochemical staining (left panel). Brightfield and fluorescent microscopy images of circSLC8A1 at 200X magnification. A signal from circSLC8A1 can be observed in muscle cells, whereas the cancer cells were negative (right panel).</p

Supplementary Fig. 3 from Spatial Profiling of Circular RNAs in Cancer Reveals High Expression in Muscle and Stromal Cells

Heatmap and unsupervised hierarchical clustering of normalized nanostring nCounter data from cancer cells, tumor stroma and normal epithelium. A clearer clustering of these sample groups can be observed when the muscularis propria samples are not included in the analysis.</p

Supplementary Fig. 2 from Spatial Profiling of Circular RNAs in Cancer Reveals High Expression in Muscle and Stromal Cells

Tissue markers specificity. Normalized NanoString counts from specific tissue markers used for sub-compartment purity assessment after laser capture microdissection. KRT8, CDX2 and SATB2 are epithelial markers. VIM is an activated stromal marker and ACTA2 and DES are muscle cell markers. Parametric t-tests were performed to compute the p-values using the values from the cancer cells as reference. The box represents the interquartile range (Q1 and Q3), the median is marked with a horizontal line, and whiskers represent minimum and maximum values.</p