Fig. 1

A Clinical features of case 1: primary breast cancer and relative brain metastases. B Contrast-enhanced brain computed tomography scan showing a solid and enhanced large lesion (3.9 cm) in the cerebellum with perilesional oedema. C Representative staining results from haematoxylin–eosin (HE) and immunohistochemistry for ER, PR, HER2 and Ki-67 in the metastatic lesion from patient 1. Scale bar = 200–500 µM. ER, oestrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor. D Representative bright-field microscopy image of organoids generated from metastatic lesions. Magnification: 20X. E Summary of the mutational profile of case 1 brain metastases and related organoids. VAF: variant allele frequency. F All expressed gene distributions by RNA-seq analysis of metastatic lesions (METs) and related organoids (PDOs). RNAseq data counts were normalized as transcripts per million (TPM). G Preranked gene set enrichment analysis (GSEA; https://www.gsea-msigdb.org) was performed on all expressed genes in the metastatic lesion (MET) and related organoid (PDO) samples. GSEA was run in preranked mode using classic as a metric and 1000 permutations selecting the curated gene sets of the Molecular Signatures Database (MsigDB) derived from the Hallmark and KEGG collections. As a ranking metric, the z scores of the genes were evaluated in each sample. Gene set enrichment was assessed through the normalized enrichment score (NES). The size of the circles reflects the percentage of genes in the core enrichment of the pathway. H Representative bright-field microscopy images of organoids generated from brain metastatic lesions carrying PIK3CA mutations treated with 10 µM alpelisib for 7 days. Magnification: 10X. The number of organoids and relative area for each condition are plotted in the bar graphs. I Flow cytometry of Helix NP blue-stained organoids generated from brain metastatic lesions carrying PIK3CA mutations after 7 days of treatment with 10 µM alpelisib. J Representative bright-field microscopy images of organoids generated from brain metastatic lesions with wild-type PIK3CA treated with 10 µM alpelisib for 7 days. Magnification: 10X. The number of organoids and relative area for each condition are plotted in the bar graphs. K Flow cytometry of Helix NP blue-stained organoids generated from brain metastatic lesions with wild-type PIK3CA after 7 days of treatment with 10 µM alpelisib