Taken together, these results suggest that a periostin-positive CAF is an early, activated CAF, associated with aggressive tumors, whereas a podoplanin-positive CAF is associated with an immune-related phenotype. These two subpopulations cooperate to define specific tumor microenvironment and patient prognosis, and are of putative interest for future therapeutic stratification of patients.

Taken together, these results suggest that a periostin-positive CAF is an early, activated CAF, associated with aggressive tumors, whereas a podoplanin-positive CAF is associated with an immune-related phenotype. These two subpopulations cooperate to define specific tumor microenvironment and patient prognosis, and are of putative interest for future therapeutic stratification of patients.

Total RNA was extracted from FFPE sections using a high pure FFPE RNA isolation kit (Roche®, Basel, Switzerland) following the manufacturer’s protocol. RNA yield and quality was determined using a NanoDrop™ One spectrophotometer and fragment size was analyzed using an RNA ScreenTape assay run on a 4200 Bioanalyzer (Agilent Technologies®, Santa Clara, CA, USA . DV200 values representing the percentage of RNA fragments above 200 nucleotides in length were estimated, and cases with DV200 more than 30% were included for library preparation.

Total RNA was extracted from FFPE sections using a high pure FFPE RNA isolation kit (Roche®, Basel, Switzerland) following the manufacturer’s protocol. RNA yield and quality was determined using a NanoDrop™ One spectrophotometer and fragment size was analyzed using an RNA ScreenTape assay run on a 4200 Bioanalyzer (Agilent Technologies®, Santa Clara, CA, USA . DV200 values representing the percentage of RNA fragments above 200 nucleotides in length were estimated, and cases with DV200 more than 30% were included for library preparation.

Library preparation was performed using QuantSeq 3’ mRNA-Seq REV (Lexogen® , Vienna, Austria) with an input of 150 ng of total FFPE RNA. The pool was sequenced on a NovaSeq 6000 system flow cell SP (Illumina Inc., San Diego, CA) using a 75-cycle, paired-end protocol providing approximately 10 million reads per sample. Base call files were converted to fastq format using Bcl2Fastq (Illumina®, San Diego, CA). All RNA-seq reads were aligned to the human reference genome (GRCh37, hg19) using STAR (version 2.6.1a_08-27), quantified using FeatureCount and Upper-Quartile normalized.

Library preparation was performed using QuantSeq 3’ mRNA-Seq REV (Lexogen® , Vienna, Austria) with an input of 150 ng of total FFPE RNA. The pool was sequenced on a NovaSeq 6000 system flow cell SP (Illumina Inc., San Diego, CA) using a 75-cycle, paired-end protocol providing approximately 10 million reads per sample. Base call files were converted to fastq format using Bcl2Fastq (Illumina®, San Diego, CA). All RNA-seq reads were aligned to the human reference genome (GRCh37, hg19) using STAR (version 2.6.1a_08-27), quantified using FeatureCount and Upper-Quartile normalized.