Live slow-frozen human tumor tissues viable for 2D, 3D, ex vivo cultures and single-cell RNAseq

Biobanking of surplus human healthy and disease-derived tissues is essential for diagnostics and translational research. An enormous amount of formalin-fixed and paraffin-embedded (FFPE), Tissue-Tek OCT embedded or snap-frozen tissues are preserved in many biobanks worldwide and have been the basis of translational studies. However, their usage is limited to assays that do not require viable cells. The access to intact and viable human material is a prerequisite for translational validation of basic research, for novel therapeutic target discovery, and functional testing. Here we show that surplus tissues from multiple solid human cancers directly slow-frozen after resection can subsequently be used for different types of methods including the establishment of 2D, 3D, and ex vivo cultures as well as single-cell RNA sequencing with similar results when compared to freshly analyzed material

Single-cell Analysis Reveals Inter- and Intratumour Heterogeneity in Metastatic Breast Cancer

Metastasis is the leading cause of cancer-related deaths of breast cancer patients. Some cancer cells in a tumour go through successive steps, referred to as the metastatic cascade, and give rise to metastases at a distant site. We know that the plasticity and heterogeneity of cancer cells play critical roles in metastasis but the precise underlying molecular mechanisms remain elusive. Here we aimed to identify molecular mechanisms of metastasis during colonization, one of the most important yet poorly understood steps of the cascade. We performed single-cell RNA-Seq (scRNA-Seq) on tumours and matched lung macrometastases of patient-derived xenografts of breast cancer. After correcting for confounding factors such as the cell cycle and the percentage of detected genes (PDG), we identified cells in three states in both tumours and metastases. Gene-set enrichment analysis revealed biological processes specific to proliferation and invasion in two states. Our findings suggest that these states are a balance between epithelial-to-mesenchymal (EMT) and mesenchymal-to-epithelial transitions (MET) traits that results in so-called partial EMT phenotypes. Analysis of the top differentially expressed genes (DEGs) between these cell states revealed a common set of partial EMT transcription factors (TFs) controlling gene expression, including ZNF750, OVOL2, TP63, TFAP2C and HEY2. Our data suggest that the TFs related to EMT delineate different cell states in tumours and metastases. The results highlight the marked interpatient heterogeneity of breast cancer but identify common features of single cells from five models of metastatic breast cancer.ISSN:1083-3021ISSN:1573-703

Live slow-frozen human tumor tissues viable for 2D, 3D, ex vivo cultures and single-cell RNAseq

Biobanking of surplus human healthy and disease-derived tissues is essential for diagnostics and translational research. An enormous amount of formalin-fixed and paraffin-embedded (FFPE), Tissue-Tek OCT embedded or snap-frozen tissues are preserved in many biobanks worldwide and have been the basis of translational studies. However, their usage is limited to assays that do not require viable cells. The access to intact and viable human material is a prerequisite for translational validation of basic research, for novel therapeutic target discovery, and functional testing. Here we show that surplus tissues from multiple solid human cancers directly slow-frozen after resection can subsequently be used for different types of methods including the establishment of 2D, 3D, and ex vivo cultures as well as single-cell RNA sequencing with similar results when compared to freshly analyzed material.ISSN:2399-364

A high-throughput drug screen reveals means to differentiate triple-negative breast cancer.

Plasticity delineates cancer subtypes with more or less favourable outcomes. In breast cancer, the subtype triple-negative lacks expression of major differentiation markers, e.g., estrogen receptor α (ERα), and its high cellular plasticity results in greater aggressiveness and poorer prognosis than other subtypes. Whether plasticity itself represents a potential vulnerability of cancer cells is not clear. However, we show here that cancer cell plasticity can be exploited to differentiate triple-negative breast cancer (TNBC). Using a high-throughput imaging-based reporter drug screen with 9 501 compounds, we have identified three polo-like kinase 1 (PLK1) inhibitors as major inducers of ERα protein expression and downstream activity in TNBC cells. PLK1 inhibition upregulates a cell differentiation program characterized by increased DNA damage, mitotic arrest, and ultimately cell death. Furthermore, cells surviving PLK1 inhibition have decreased tumorigenic potential, and targeting PLK1 in already established tumours reduces tumour growth both in cell line- and patient-derived xenograft models. In addition, the upregulation of genes upon PLK1 inhibition correlates with their expression in normal breast tissue and with better overall survival in breast cancer patients. Our results indicate that differentiation therapy based on PLK1 inhibition is a potential alternative strategy to treat TNBC