Loycano, M. | bioRxiv Science

bioRxiv2024

Single-Cell Proteomic and Transcriptomic Characterization of Drug-Resistant Prostate Cancer Cells Reveals Molecular Signatures Associated with Morphological Changes

This study delves into the proteomic intricacies of drug-resistant cells (DRCs) within prostate cancer, which are known for their pivotal roles in therapeutic resistance, relapse, and metastasis. Utilizing single-cell proteomics (SCP) with an optimized high-throughput Data Independent Acquisition (DIA) approach with the throughput of 60 sample per day, we characterized the proteomic landscape of DRCs in comparison to parental PC3 cells. This optimized DIA method allowed for robust and reproducible protein quantification at the single-cell level, enabling the identification and quantification of over 1,300 proteins per cell on average. Distinct proteomic sub-clusters within the DRC population were identified, closely linked to variations in cell size. The study uncovered novel protein signatures, including the regulation of proteins critical for cell adhesion and metabolic processes, as well as the upregulation of surface proteins and transcription factors pivotal for cancer progression. Furthermore, by integrating SCP and single-cell RNA-seq (scRNA-seq) data, we identified six upregulated and ten downregulated genes consistently altered in drug-treated cells across both SCP and scRNA-seq platforms. These findings underscore the heterogeneity of DRCs and their unique molecular signatures, providing valuable insights into their biological behavior and potential therapeutic targets.

cancer biology↗

bioRxiv2024

Comparison of Different Approaches to Single Cell RNA Sequencing of Cancer Associated Fibroblasts

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with a poor prognosis. PDAC has a high propensity for metastasis, particularly to the lungs and liver. Cancer associated fibroblasts (CAFs) represent a major stromal component of PDAC with both tumor-promoting and restraining properties. Of note, CAFs play a significant role in the creation of an immunosuppressive tumor microenvironment (TME) and the metastasis of PDAC. Studies have demonstrated functional heterogeneity among different subpopulations of CAFs, highlighting the need to identify specific subpopulations when targeting CAFs. MethodsThe orthotopic model was used for both KPC-4545 and KPC-3403 cell lines, which were derived from the primary tumors of KPC mice with liver metastases and lung metastases only, respectively. In brief, 2x106 KPC cells were injected subcutaneously into the flanks of synergic female C57BI6 mice. Tumors were harvested and cut into 2-3 mm3 pieces before being implanted into the pancreas of new 6-8-week-old syngeneic female C57Bl/6 mice. Murine orthotopic tumors were dissected, mechanically and enzymatically processed with Miltenyi Tumor Dissociation Kit (Miltenyi Biotec) thirteen days after tumor implantation. Samples were filtered with a 100 {micro}m strainer, washed with T cell media, and centrifuged twice. Two different samples underwent single cell RNA-sequencing (scRNA-seq) for each cell line: an unenriched sample, which represents all cells following dissociation of the tumor, and a CAF-enriched sample. To further obtain the CAF-enriched sample, cells were then stained with CD45-AF657 (BioLegend clone 30-F11, 1:20), CD31-AF647 (BioLegend clone 390, 1:20), EPCAM-AF647 (BioLegend, clone G8.8, 1:20), and TER119-AF647 (BioLegend clone TER-119 1:20) for 30 minutes on ice. After two washes, CD45-, CD31-, EPCAM-, and TER119-negative cells, representing the CAF-enriched fraction, were obtained via cell sorting. scRNA-seq of both the unenriched and CAF-enriched fractions were performed using 10X Chromium microfluidic chips and data was analyzed using CellRanger v6.1.1, mm10 transcriptome reference, and 10X Loupe Browser. ResultsWe found that scRNA-seq of the unenriched whole tumor showed only one cluster of CAFs for both cells lines, making it difficult for studying CAF heterogeneity. Enriching for CAFs prior to scRNA-seq allowed for better capture of CAFs and provided more granularity on CAF heterogeneity for both KPC-4545 and KPC-3403. ConclusionsWhile enrichment provides more information on CAF heterogeneity, the process results in the loss of other cells within the TME. The need to capture CAF heterogeneity while studying cell-cell interaction between CAFs and other cells within the TME and identifying how distinct CAF populations respond differently to treatment warrants the use of other methods such as single-nuclear RNA-seq.

cancer biology↗