DOI: 10.5061/dryad.4b8gthtqb
title: Single-cell and spatial RNA sequencing identify divergent microenvironments and progression signatures in early- versus late-onset prostate cancer

description: The clinical and pathological outcomes differ between early- (EOPC;
 diagnosed in men ≤ 55 years of age) and late-onset prostate cancer (LOPC),
 potentially attributed to the changes in hormone levels and immune
 activities associated with ageing. Exploring the heterogeneity therein
 holds the potential for developing age-specific precision interventions.
 Here, through single-cell and spatial transcriptomic analyses of prostate
 cancer tissues, we identified that an androgen response-related
 transcriptional meta-program (AR-MP) might underlie the age-related
 heterogeneity of tumour cells and microenvironment. APOE+
 tumour-associated macrophages infiltrated AR-MP-activated tumour cells in
 EOPC, potentially facilitating tumour progression and immunosuppression.
 By contrast, inflammatory cancer-associated fibroblasts in LOPC could
 downregulate the AR-MP of tumour cells, promote epithelial-to-mesenchymal
 transition and induce pre-existing castration resistance, which effects
 could also be caused by smoking. This study provides insight into
 precision treatments tailored to diverse age groups, emphasizing
 interventions that include targeting AR and tumour-associated macrophages
 in young patients but anchoring epithelial-to-mesenchymal transition and
 inflammatory cancer-associated fibroblasts in old counterparts.
description: The tumour samples used in this study for scRNA-seq and ST-seq
 were consecutively collected from treatment-naïve patients with aggressive
 PCa [ISUP grade group ≥ 4, or tumour stage T3/T4, or tumour in nodes (N1),
 or metastasis present (M1)] who underwent prostate biopsy or radical
 prostatectomy (RP), which consisted 9 EOPC and 11 LOPC patients in
 total. Sequencing Protocol:
 <em>scRNA-seq (discovery cohort):</em> The fresh
 tumour tissues were stored in the GEXSCOPE® Tissue Preservation Solution
 and transported on ice, which were then washed (Hanks Balanced Salt
 Solution), minced and digested (GEXSCOPE® Tissue Dissociation Solution),
 centrifuged and resuspended. GEXSCOPE® red blood cell lysis buffer was
 used to remove the red blood cells. Single-cell suspensions were converted
 to barcoded scRNA-seq libraries using the Chromium Single Cell Library,
 Gel Bead & Multiplex Kit (10x Genomics), following the
 manufacturer’s instructions. Eventually, libraries were prepared using 10x
 Genomics Library Kits and sequenced on Illumina Nova6000 with a pair-end
 150 bp (PE150) reading strategy. <em>scRNA-seq
 (validation cohort):</em> After washed, minced, digested, and
 removed for red blood cells, the single-cell suspensions were subjected to
 SeekOne® DD Single Cell 3’ library preparation kit (SeekGene) to prepare
 barcoded scRNA-seq libraries. Purified libraries were sequenced on
 Illumina Nova6000 with PE150 reading strategy.
 <em>ST-seq for frozen fresh sample: </em>The fresh
 samples from EOPC-4, Rep-EOPC4, and Rep-LOPC1 were snap frozen in optimum
 cutting temperature (OCT) compound (SAKURA, Cat#: 4583) and stored at −80
 °C, and then placed on the Visium Spatial Gene Expression slide (10x
 Genomics). Methoxylated sections were performed according to Visium
 Spatial Gene Expression for OCT (10x Genomics), incubated at −20°C for 30
 min, then stained with hematoxylin and eosin (H&E), and finally
 stained for imaging. The Visium Spatial Gene Expression Slide &
 Reagent Kit (Cat:1000184,10x Genomics) was used to construct sequencing
 libraries according to the Visium Spatial Gene Expression User Guide
 (CG000408, 10x Genomics), which were sequenced using Illumina Nova6000
 with PE150 under a sequencing depth of at least 50,000 reads per
 spot. <em>ST-seq for Formalin-fixed,
 paraffin-embedded (FFPE) sample:</em> An additional tumour tissue
 from LOPC-3 was prepared as a FFPE sample and subjected to Visium
 CytAssist Spatial Gene Expression for FFPE (PN-1000520, 10× Genomics).
 Deparaffinization, hematoxylin, and eosin staining and decrosslinking were
 performed after overnight drying, followed by probe hybridization and
 probe connection. Then, RNA from the tissue section was digested to
 release the probes, which were captured by the capture sequences on the
 Visium CytAssist Spatial Gene Expression slide (10x Genomics) using the
 Visium CytAssist instrument. The probe extension and library construction
 steps were then conducted following the standard Visium for FFPE workflow
 outside of the instrument, which was finally sequenced on Illumina
 Nova6000 with a sequencing depth of at least 100,000 reads per spot with
 PE150. Data processing to generate matrix:
 Raw reads of scRNA-seq were processed to generate gene expression
 profiles using <em>Cell Ranger</em><em>
 </em>(v.3.0.3, 10x Genomics). Reads from the 10x library were mapped
 to GRCh38 with ensemble version 92 gene annotation. ST-seq data were
 processed by <em>Space Ranger</em> (v.2.1.0, 10x Genomics) to
 generate RNA profiles and the result matrix files.
description: # Single-cell and spatial RNA sequencing identify divergent
 microenvironments and progression signatures in early- versus late-onset
 prostate cancer
 [https://doi.org/10.5061/dryad.4b8gthtqb](https://doi.org/10.5061/dryad.4b8gthtqb) ## Description of the data and file structure This dataset consists of matrices of single-cell RNA sequencing from 20 prostate cancer tissues, and of spatial transcriptomic sequencing from 4 prostate cancer tissues, emphasising the differences in tumour microenvironment associated with age-of-onset. ### Files and variables #### File: PCSC003\_SC\_matrix.tar.gz **Description:** 10X Genomics-typed matrix files, which are generated from CellRanger pipeline:  matrix.mtx.gz, features.tsv.gz and barcodes.tsv.gz. #### File: PCSC005\_SC\_matrix.tar.gz **Description:** 10X Genomics-typed matrix files, which are generated from CellRanger pipeline:  matrix.mtx.gz, features.tsv.gz and barcodes.tsv.gz. #### File: PCSC002\_SC\_matrix.tar.gz **Description:** 10X Genomics-typed matrix files, which are generated from CellRanger pipeline:  matrix.mtx.gz, features.tsv.gz and barcodes.tsv.gz. #### File: PCSC007\_SC\_matrix.tar.gz **Description:** 10X Genomics-typed matrix files, which are generated from CellRanger pipeline:  matrix.mtx.gz, features.tsv.gz and barcodes.tsv.gz. #### File: PCSC008\_Spatial\_matrix.tar.gz **Description:** 10X Genomics-typed matrix files, which are generated from SpaceRanger pipeline:  filtered_feature_bc_matrix.h5. #### File: PCSC008\_SC\_matrix.tar.gz **Description:** 10X Genomics-typed matrix files, which are generated from CellRanger pipeline:  matrix.mtx.gz, features.tsv.gz and barcodes.tsv.gz. #### File: PCSC009\_SC\_matrix.tar.gz **Description:** 10X Genomics-typed matrix files, which are generated from CellRanger pipeline:  matrix.mtx.gz, features.tsv.gz and barcodes.tsv.gz. #### File: PCSC010\_Spatial\_matrix.tar.gz **Description:** 10X Genomics-typed matrix files, which are generated from SpaceRanger pipeline:  filtered_feature_bc_matrix.h5. #### File: PCSC010\_SC\_matrix.tar.gz **Description:** 10X Genomics-typed matrix files, which are generated from CellRanger pipeline:  matrix.mtx.gz, features.tsv.gz and barcodes.tsv.gz. #### File: PCSC012\_SC\_matrix.tar.gz **Description:** 10X Genomics-typed matrix files, which are generated from CellRanger pipeline:  matrix.mtx.gz, features.tsv.gz and barcodes.tsv.gz. #### File: PCSC013\_SC\_matrix.tar.gz **Description:** 10X Genomics-typed matrix files, which are generated from CellRanger pipeline:  matrix.mtx.gz, features.tsv.gz and barcodes.tsv.gz. #### File: PCSC015\_SC\_matrix.tar.gz **Description:** 10X Genomics-typed matrix files, which are generated from CellRanger pipeline:  matrix.mtx.gz, features.tsv.gz and barcodes.tsv.gz. #### File: Validate-PCSC003\_Spatial\_matrix.tar.gz **Description:** 10X Genomics-typed matrix files, which are generated from SpaceRanger pipeline:  filtered_feature_bc_matrix.h5. #### File: Validate-PCSC001\_SingleCell\_matrix.tar.gz **Description:** 10X Genomics-typed matrix files, which are generated from CellRanger pipeline:  matrix.mtx.gz, features.tsv.gz and barcodes.tsv.gz. #### File: Validate-PCSC003\_SingleCell\_matrix.tar.gz **Description:** 10X Genomics-typed matrix files, which are generated from CellRanger pipeline:  matrix.mtx.gz, features.tsv.gz and barcodes.tsv.gz. #### File: Validate-PCSC002\_SingleCell\_matrix.tar.gz **Description:** 10X Genomics-typed matrix files, which are generated from CellRanger pipeline:  matrix.mtx.gz, features.tsv.gz and barcodes.tsv.gz. #### File: Validate-PCSC004\_SingleCell\_matrix.tar.gz **Description:** 10X Genomics-typed matrix files, which are generated from CellRanger pipeline:  matrix.mtx.gz, features.tsv.gz and barcodes.tsv.gz. #### File: Validate-PCSC007\_Spatial\_matrix.tar.gz **Description:** 10X Genomics-typed matrix files, which are generated from SpaceRanger pipeline:  filtered_feature_bc_matrix.h5. #### File: Validate-PCSC005\_SingleCell\_matrix.tar.gz **Description:** 10X Genomics-typed matrix files, which are generated from CellRanger pipeline:  matrix.mtx.gz, features.tsv.gz and barcodes.tsv.gz. #### File: Validate-PCSC006\_SingleCell\_matrix.tar.gz **Description:** 10X Genomics-typed matrix files, which are generated from CellRanger pipeline:  matrix.mtx.gz, features.tsv.gz and barcodes.tsv.gz. #### File: Validate-PCSC008\_SingleCell\_matrix.tar.gz **Description:** 10X Genomics-typed matrix files, which are generated from CellRanger pipeline:  matrix.mtx.gz, features.tsv.gz and barcodes.tsv.gz. #### File: Validate-PCSC007\_SingleCell\_matrix.tar.gz **Description:** 10X Genomics-typed matrix files, which are generated from CellRanger pipeline:  matrix.mtx.gz, features.tsv.gz and barcodes.tsv.gz. #### File: Validate-PCSC009\_SingleCell\_matrix.tar.gz **Description:** 10X Genomics-typed matrix files, which are generated from CellRanger pipeline:  matrix.mtx.gz, features.tsv.gz and barcodes.tsv.gz. #### File: Validate-PCSC010\_SingleCell\_matrix.tar.gz **Description:** 10X Genomics-typed matrix files, which are generated from CellRanger pipeline:  matrix.mtx.gz, features.tsv.gz and barcodes.tsv.gz. ## Code/software Seurat pipeline based on R software. The single-cell RNA sequencing matrix (each for three files including "barcodes.tsv.gz", "features.tsv.gz", "matrix.mtx.gz") should be read with "Read10X()" and the spatial transcriptomic data (each for a "filtered_feature_bc_matrix.h5" file) should be read with "Read10X_h5()".