The frozen skin material (BCC, peritumoral skin and buttock skin) including both the dermal and epidermal part, that was frozen until qRT-PCR analysis, was thawed in RNA later (Sigma, St. Louis MO, USA) and minced into 1 mm pieces. The method was performed as described previously [
8]. All analyses were run as triplicates. The primer-probes used were: GAPDH (HS02758991_g1), IL6 (HS00985639_m1), CD3 (HS00174158_m1), CXCL12 (HS03676656_mH), CXCR4 (HS00607978_s1), CCL11 (HS_00237013_m1), CCL17 (HS00171074_m1), CCL18 (HS00268113_m1), CCL22 (HS01574247_m1), CCL25 (HS00608373_m1), PDGFRβ (HS01019589_m1), FAP-α (HS00990806_m1), Collagen 11A (HS01097664_m1) and P4HA2 (HS00990001_m1). The data obtained from CC17, CCL18, and CCL22 analyses have previously been published together with data on T-regs [
8]. Obtained data were analysed by the ∆∆CT method [
9,
10] with GAPDH as housekeeping reference gene.
Immunohistochemistry and immunofluorescence
For immunohistochemical staining, 10 μm sections were cut from the embedded tissue blocks on a Microm HM560 cryostat and mounted on glass slides for immunohistochemical and immunofluorescent stainings. For 3,3′-Diaminobenzidine (DAB) (Dako, Glostrup, Denmark) stainings, the sections were fixed using acetone at -20o C for 10 min. Endogenous peroxidase activity was quenched using 0.3% H2O2 (Merck, Millipore, Darmstadt, Germany) in PBS for 15 min at room temperature (RT) in the dark. The sections were blocked in 10% horse serum (Gibco, Fisher Scientific, New Zealand) and 1% bovine serum albumin (BSA) (Sigma, St. Louis MO, USA) in PBS for 1 h at RT and followed by incubation with the primary antibody (rabbit anti-FAP-α (1:100, LS-C313051, LSBio, Seattle, USA), mouse anti- CXCL12 (1:40, MAB350, R & D systems, Oxon, UK), rabbit anti-Collagen 11A (1:100, ab64883, Abcam, Cambridge, UK), rabbit anti-CXCR4 (1:400, AHP442, AbD Serotec, Oxford, UK), rabbit anti-IL6 (1:600, ab6672, Abcam), rabbit anti-PDGFRβ (1:100, LS-C312148, LSBio), mouse anti-CCL17 (1:80, LS-C198166, LSBio), mouse anti-CCL22 (1:100, MAB336, R & D systems) or rabbit anti-P4HA2 (1:80, LS-C91131, LSBio)) diluted in 1%BSA in PBS over night at 4 °C. Incubation with secondary antibody (goat anti-rabbit HRP (1:200, P0448, Dako), goat anti-mouse HRP (1:200, P0447, Dako)) diluted in 10% horse serum and 1% BSA in PBS for 40 min at RT was performed. DAB was added to visualise the staining and the sections were incubated with haematoxylin for 1 min followed by coverslipping with glycergel mounting medium (Dako).
For immunofluorescence (IF) stainings, sections were fixed and blocked as described above (peroxidase quenching step was omitted) followed by incubation with combinations of two primary antibodies (rabbit anti-FAP-α (1:200, LS-C313051, LSBio), mouse anti-CCL17 (1:80, LS-C198166, LSBio) PCGFR-β (1:100, LS-C312148, LSBio), CXCL12 (1:40, MAB350, R & D systems, Oxon, UK), CCL22 (1:100, MAB336, R & D systems)) diluted in blocking solution overnight at 4 °C. Incubation with secondary antibodies (goat anti-rabbit Alexa568 (1:500, A11036, Life Technologies) and/or goat anti-mouse Alexa488 (1:500, A11001, Life Technologies, Thermo Fischer Scientific)) diluted in 1% BSA in PBS was then performed for 1 h at RT in the dark. This was followed by incubation with 0.5 μg/ml 4′,6-Diamino-2-phenylindole (DAPI) (Sigma) and coverslipping with glycergel mounting medium.
Transcriptome sequencing
Large RNAs were extracted and isolated from the samples using the NucleoSpin® miRNA kit [
11] following the protocol for purification of small and large RNA in separate fractions but skipping the DNA digestion step. The frozen tissue samples were cut into smaller pieces on dry ice, transferred to lysis buffer containing two stainless steel beads of 2–3 mm in diameter, and homogenised using the TissueLyser II (Quiagen, Hilden, Germany) prior to extraction. For each sample, 5 μl extract were DNase treated for 30 min at 37° with 1 μl TURBO DNase enzyme (ThermoFisher,
www.thermofisher.com) in a total volume of 50 μl. The RNA was subsequently purified using the RNeasy MinElute Cleanup Kit (Quiagen). Libraries were prepared from 100 ng of RNA, using the ScriptSeq v2 RNA-Seq Library Preparation kit (Epicentre, Illumina,
www.illumina.com), following the manufacturer’s guidelines, and with 12 cycles of PCR amplification. Paired-end sequencing of 100 base pairs was performed on the Illumina HiSeq 2000 platform, yielding more than 380 million paired-end sequence reads (Additional file
1: Table S1). Removal of adapters, quality trimming and merging of paired reads was carried out using open-source AdapterRemoval software [
12]. Reads were mapped onto the human genome (assembly version hg19) using the RNA-seq aligner RNAstar [
13]. Potential PCR duplicate reads were discarded using the rmdup function in samtools [
14]. Whereas approximately 97% of all reads mapped to the human genome, duplicate reads constituted a significant fraction leaving only around 48 million unique reads (Additional file
1: Table S1). From this mapping, transcript abundance was estimated running FLUX CAPACITOR [
15] provided with the GENCODE gene annotation [
16]. The raw number of reads assigned to each transcript by FLUX CAPACITOR was used as input for EdgeR (Version 3.2, Bioconductor) [
17].