Recent evidence points towards dissemination strategies that proceed in parallel with the development of the primary tumor [25,26]. those cells before they wake up. Abstract (1) Background: metastatic relapse following a prolonged period of disease-free survival is a common cause of mortality for many cancer patients. Disseminated dormant cancer cells (DDCCs) lie below the radar before waking up years, or even decades, after the removal of the primary tumor. This implies that they are able to survive in a latent state in a foreign environment for an extended period of time supported by intrinsic and extrinsic factors still to be elucidated. (2) Methods: we employed a coculture of DDCCs with lung epithelial cells together with RNA sequencing analysis to understand the overlap in gene transcription between in vivo and cocultured DDCCs. (3) Results: we found a significant overlap between the processes activated in DDCCs from lungs and in the coculture, as well as in alveolar type I cells in vivo and in coculture. We identified the transcription factor EB (TFEB)-lysosomal axis as a relevant process activated in DDCCs Pexidartinib (PLX3397) upon dissemination to the lung and confirmed the results in our lung coculture. Interestingly, breast cancer patients with a higher expression of TFEB targets show increased likelihood of developing relapses. (4) Conclusions: we propose that lysosomal accumulation following TFEB activation is an important feature of breast cancer DDCCs that might be exploited for future therapeutic interventions. sample. Read-quality trimming and adaptor removal were carried out using Trimmomatic (version 0.36). The RSEM package (version 1.3.30) , in conjunction with the STAR alignment algorithm (version 2.5.2a) , was used for the mapping and subsequent gene-level counting of the sequenced reads with respect to the Ensembl mouse GRCm.38.89 version transcriptome. The normalization of raw count data and differential expression analysis was performed with the DESeq2 package (version 1.18.1)  within the R programming environment (version 3.4.3) . Differentially expressed genes were defined as those showing statistically significant differences (False Discovery Rate (FDR) 0.05). Differential gene lists ranked by the Wald statistic were used to look for pathways and selected gene sets using the Broads Gene Set Enrichment Analysis (GSEA) software (version 2.1.0) with gene sets from MSigDB (version 6)  and additional published and custom datasets (Table S1). Spearmans rank correlation was used to compare the normalized enrichment scores by comparisons with different Oaz1 experiments to determine which pathways were similarly enriched. Scatterplots (generated using the R base graphics package) shows the correlation between the Walds statistic (gene level differences from DESeq2) or the normalized enrichment score (NES)(pathway level differences from GSEA) when comparing D2.0R lung-disseminated_vs_monoculture and coculture_vs_monoculture comparisons. Survival analysis. Kaplan-Meier were generated with KM Plotter online tool (https://kmplot.com/analysis/ (accessed on 27 February 2021)) which calculates log-rank value. Options used: Use mean Pexidartinib (PLX3397) expression of selected gene, Autoselect best cutoff, User selected probe set and Derive ER status from gene expression data. 2.5. Growth Assays Resazurin staining. AT1-like cells were seeded in 96-well plates (2.176 104/well in quadruplicate) in parallel with standards in the linear range of detection. 24 h after seeding, 4 nM Bafilomycin A1 (Sigma-Aldrich, St. Louis, MO, Pexidartinib (PLX3397) USA, B1793) was added with fresh medium. After 4 days of treatments, cells were washed with PBS and incubated with 100 M Resazurin (Sigma-Aldrich, St. Louis, MO, USA, R7017) in culture medium at 37 C for 2 h. Absorbance at 544/590 nm was measured on live cells by checking that the signal was in the temporal linear range. An absolute cell number was then calculated based on the standard curve, after background subtraction (medium without cells). Cell number quantification with Operetta system. Cells were seeded in 96-well plates 5 104 cells/well in quadruplicate). Then, 24 h after seeding, culture medium was renewed by adding 1, 2 or 4 nM Bafilomycin A1. After 4 days of treatments, cells were fixed for 10 min in PFA 4% at room temperature, washed in sterile PBS and incubated for 15 Pexidartinib (PLX3397) min with Hoechst (Life technologies, Carlsbad, CA, USA, H1399, 1 ug/mL). The cells were automatically counted using the Operetta high-content imaging system based on nuclear counterstaining. Live-cell analysis was performed using Harmony high content imaging and analysis software. 2.6. Reporter Assay At day 1, D2.0R cells were transfected with TFEB transcriptional reporter Pexidartinib (PLX3397) plasmid (RAGD promoter cloned upstream of luciferase gene, a gift from Prof. Graziano Martello, University of Padua, Italy) , together with a plasmid with constitutive expression of Renilla luciferase to normalize for transfection efficiency  with Lipofectamine 3000.
Recent evidence points towards dissemination strategies that proceed in parallel with the development of the primary tumor [25,26]