Fig. 2: Agnospheres are enriched in stem-like cells that self-sustain their long-term propagation.

a Heatmap showing gene expression levels of selected transcription factors (TFs), quantified by qRT-PCR in agnospheres and tumorspheres derived from metastatic colorectal cancer (mCRC729) or melanoma (mMS321). b Representative immunofluorescent stainings of stem cell markers in agnospheres. Arrowheads: marker co-expression; open arrows: cells with single marker expression (n = 3 independent stainings of agnospheres with similar results were obtained). Scale bar, 50 μm. Inset: magnification of dotted area. Scale bar, 10 μm. c Representative western blot analysis of Polycomb repressor factors, reprogramming and EMT TFs, and epithelial and mesenchymal markers in agnospheres and tumorspheres mCRC729, mCRC0155, and mMS321 (n = 3 independent experiments with similar results were obtained, molecular weights are expressed in kDa). d Heatmap showing the gene set enrichment analysis of the 13 gene sets included in the embryonic stem cell signature²², performed on the transcriptome of human embryonic stem cell lines (hESC), agnospheres, tumorspheres from metastatic cancers, CUP original tissues, and a panel of breast cancer tissues, pooled by grade (the average enrichment score is shown for each grade). Only significant enrichment scores with a false discovery rate < 10% are shown (Supplementary Data 1). NOS: NANOG, OCT4, and SOX2. e In vitro limiting dilution sphere-forming assay. For each agnosphere, plots generated by the ELDA software are shown, reporting the estimated stem cell frequency (percentage of clonogenic cells) with confidence intervals (C.I.; AS901, AS43, AS67, and AS914: n = 3; AS906 and N-AS47: n = 4 independent experiments).