Patients and clinical samples
Tumor biopsy samples were collected from 82 patients with advanced PDAC who underwent GEM-based chemotherapy at the Sun Yat-Sen Memorial Hospital and Guangdong Provincial People's Hospital between 2015 and 2021. The obtained tissues were immediately snap-frozen in liquid nitrogen and transferred at −80 °C for further experiments. All samples were histologically confirmed with PDAC. Progression-free survival (PFS) was defined as the time interval beginning the date of chemotherapy to the date of disease progression event occurrence. All patients provided informed consent, and all related procedures were performed with the approval of Ethical Committee of indicated hospitals.
Isolation and culture of stromal fibroblasts
Cancer-associated fibroblasts (CAFs) and Primary normal fibroblasts (NFs) were isolated from pancreatic ductal carcinoma and adjacent normal tissues. The Human Tumor Dissociation Kit (130-095-929, Miltenyi Biotec, German) was used for the generation of single cells from dissociated tissues. Fibroblast populations were isolated by differential velocity adherent technique. Primary CAFs and NFs were culture in Dulbecco’s modified Eagle’s medium (DMEM, GBICO) plus 15% fetal bovine serum (FBS, GBICO) and 1% penicillin-streptomycin at 37°C in humidified air with 5% CO2.
Conditioned medium preparation and co-culture
About 2 × 106 stable transfected CAFs were cultured in a 10 cm cell culture dish for 48 h. The culture medium was collected and centrifuged to removed cell pellets. Panc-1 and MiaPaCa-2 were culture with the conditioned medium (CM) for 2 weeks and then were subjected for cytological experiments.
PDAC cell lines Panc-1 and MiaPaCa-2 were obtained from American Type Culture Collection (ATCC) and cultured at 37°C condition with 5% CO2 and at least 95% humidity atmosphere with Dulbecco’s Modified Eagle Medium (DMEM, Gibco). Medium was added 10% fetal bovine serum (FBS, Gibco).
RNA isolation and quantitative real-time PCR (qRT-PCR)
Total RNA from frozen tissues and cultured cell lines was extracted with TRIzol reagent (Life, USA) and weas reverse transcribed into cDNA using PrimeScript RT Reagent Kit (Takara, Japan). The qRT-PCR analysis was performed with TB Green Premix Ex TaqTM kit (Takara, Japan) on Light Cycler 480 Detection System (Roche, Switzerland), The 2 −ΔΔCt method was used to analyze the relative levels of target gene. All primer sequences for the qRT-PCR assay were listed in supplementary Table S1.
The full length of circFARP1 sequence was cloned into pCD-ciR vector by IGE (Guangzhou, China). shRNA targeting circFARP1, LIF or CAV1 were ordered from IGE. The miR-660-3p mimic and inhibitor were purchased from IGE. The sequences of shRNA are provided in supplementary Table S2.
RNase R digestion and actinomycin D assay
For RNase R digestion assay, total RNA of NFs and CAFs were treated with or without 5 U/μg RNase R (RNR07250, Epicenter Technologies) and incubated at 37℃ for 30 min. For actinomycin D assay, cells were treated with 2μg/mL actinomycin D (Sigma, USA) for 0 h, 4 h, 8 h, 12h and 24 h. And qRT-PCR was used to detected circFARP1 and FARP1 expression levels. The experiments were performed three times.
For drug response of PDAC cells, 4000 of treated pancreatic cells were seeded in 96-well plates per well. The next day, fresh medium containing gemcitabine at a gradient concentration of 0, 0.001, 0.01, 0.1, 1, 10, 100, 1000μM was added into cells and incubated for 72 h. The cells were incubated with 10μl CCK-8 solution (Dojindo, Japan) at 37°C for 2h. Then, the absorbance was measured using a microplate reader (Tecan Trading AG, Switzerland) at 450 nm. The degree of drug response for tumor cells was determined by the half-maximal inhibitive concentration (IC50), which was calculated with software GraphPad Prism 8.0
For cell proliferation, 4000 pretreated cells were seeded in 96-well plates per well and incubated for 4 days. The cell viability was measured daily by reading the absorbance at 450 nm.
EdU labelling assay
5-ethynyl-2’-deoxyuridine (EdU) immunofluorescence assay was performed with BeyoClick™ EdU-555 kit (Beyotime, Guangzhou) according to the manufacturer’s instructions. Briefly, Indicated CAFs were seeded in 96-well plate and incubated with EdU for 3 h. After fixation and permeabilization, cells were stained with anti-EdU reagents and DAPI. Images were acquired by fluorescence microscopy.
Scratch wound healing assay
2×105 indicated CAFs were cultured in 24-well plate to reach confluence. The cells were scratched with a sterile 10 μL pipette tip and incubated with FBS-free culture medium. The degree of cell migration was monitored at 0 and 24 h post-scratching.
Collagen contraction assay
1×105 indicated CAFs were mixed with 200 μl of collagen containing 168.75 μL culture medium, 0.72 μL NaOH, and 31.25 μL Rat Tail Collagen I (Corning) and seeded in 24-well plates. Culture medium was added on top of the gels after polymerization. Plates were scanned 24 h after plating and percentage of contraction was calculated using the formula: Area (well)-Area (gel)/Area (well).
Colony formation assay
500 Panc-1 or MiaPaCa-2 cells with indicated treatments were seeded into 6-well plates and allowed to attach for 24h. After treated with gemcitabine (5μM) for 2 days, the media was replaced with complete media and the cells were cultured for 2 weeks. Then the colonies were fixed in 4% paraformaldehyde for 20 min, followed by staining with 0.1% crystal violet. Colonies were then manually counted. Three different independent experiments were performed.
Sphere formation assay
PANC-1 and MiaPaCa-2 cells were plated in 96-well ultra-low attachment plates (Corning, NY, USA) at a density of 1000 cells per well. Cells were maintained in serum-free DMEM/F-12 supplemented with 20ng/ml human recombinant epidermal growth factor, 20ng/ml basic fibroblast growth factor, and 1× B27 serum substitute; all from Invitrogen, Carlsbad, CA, USA. After incubated at 37°C in 5% CO2 for 2 weeks, spheres with larger than 50μm in diameter were counted.
Annexin V-FITC assay
The Annexin V-FITC Apoptosis Detection Kit was applied to detected cell apoptosis in line with the manufacturer’s instructions. In brief, cells were digested with trypsin and washed by PBS twice. Then cells were dual-stained with PI and Annexin V-FITC, using the Annexin V/FITC kit (Thermo Fisher Scientific, Shanghai, China). The analysis was carried out in the BDTM LSRII flow cytometer (BD Biosciences). and the data were measured with the Cell Quest (BD Bioscience, San Jose, CA, United States) software.
Enzyme-linked immunosorbent assay (ELISA)
Concentration of cytokines were determined by using human LIF ELISA Kit (BMS242, Thermo, USA) according to the manufacturer's instructions. In brief, stable transfected CAFs were cultured with serum-free media when reaching 80% of confluency. After 24 h, 100 µl of indicated CAFs medium was collected and incubated with plates at 37℃ for 90 min. Then detection antibody, streptavidin-HRP and TMB were added in order. The absorbance of each well was measured at 450 nm with SPARK 10 M spectrophotometer (Tecan, Austria).
Protein was extracted from the cells using RIPA lysis buffer (CWBIO, China), followed by subjected to SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% BSA for 1 hour at room temperature. Corresponding primary antibodies including anti-LIF (1:500, ab138002, Abcam), anti-STAT3 (1:1000, 9139, CST), anti-p-STAS3(Tyr705) (1:1000, 9145, CST), anti-CAV1 (1:1000, ab32577, Abcam), anti-SOX2 (1:1000, ab92494, Abcam), anti-ABCC2 (1:1000, ab172630, Abcam), anti-CDA (1:1000, ab222515, Abcam), anti-ZNRF1 (1:1000, ab175125, Abcam), anti-ubiquitin (1:1000, 3936, CST), anti-GAPDH (1:1,000, abs132004, Absin) were added to the membrane at 4℃ overnight. HRP-conjugated secondary antibodies were used. The protein bands were visualized by ECL detection system (Millipore, Germany).
Fluorescence in situ hybridization (FISH)
FISH was performed using a In Situ Hybridization Kit (Gene Pharma, Guangzhou, China) according to the manufacturer's instructions. Cy3-labeled circFARP1 and Cy5-labeled hsa-miR-660-3p probes (Gene Pharma, Guangzhou, China) were hybridized with cells overnight at 37℃. All images were captured by confocal microscopy. The targeted sequences of probes are provided in supplementary Table S2.
RNA in situ hybridization (ISH)
circFARP1 expression in PDAC tissues was detected by ISH analysis using ISH Detection kit (MK1032, BOSTER, China) as manufacture’s instruction. In brief, after deparaffinization, rehydration, and digestion, specimens were incubated with digoxin-labeled circFARP1 probes for 18 h at 40°C. followed with the incubation of anti-digoxin antibody at 37℃ for 2h, BCIP/NBT was used to for the colorimetric detection of circFARP1. The circFARP1 probe sequence was list in Supplementary Table S2.
The staining scores of circFARP1 were determined based on both staining intensity and number of positive cells. Scoring for staining intensity was as follow: 0 (none), 1 (light blue), 2 (bule) and 3 (dark bule). Scoring for ratio of circFARP1 positive cells was as follow: 1 (<25%), 2 (25-50%), 3 (50-75%), 4 (75-100%). The final score was equal to multiply staining intensity and proportion of positively stained cells. The expression of circFARP1 was evaluated by final score, with a cut-off point of <4 versus ≥4.
Histologic sections from formalin-fixed, paraffin-embedded tissues were subjected to antigen retrieval in citrate buffer for 15min, followed by blocking in normal goat serum for 30min. Then tissue sections were incubated with primary antibody as follow: anti-α-SMA (1:200, 67735-1-Ig, proteintech),anti-Ki-67(1:200, ab15580, abcam), anti-CAV1(1:200, ab32577, Abcam), anti-LIF(1:200, ab138002, Abcam) at 4°C overnight. Avidin–biotin peroxidase detection systems with DAB substrate were used to mark the locations of antigens, followed by counterstaining with hematoxylin. Immunohistochemical signal intensity and positively stained field of tissue sections were evaluated and scored independently by two observers.
RNA pull-down assay
The biotinylated probes targeting junction sites of circFARP1 were synthesized by IGE (Guangzhou, China). 1×107 CAF cells were harvested, wash with ice-cold PSB twice, and lysed with co-IP buffer. The supernatant was extracted after centrifugation and incubated with 3 μg biotinylated probes at 4℃ overnight. Streptavidin-coated magnetic beads (Invitrogen, Waltham, MA, USA) were added to the mixture and incubated at room temperature for 1 h. The captured protein or RNA were eluted from the magnetic beads and analyzed by mass spectrometry, western blotting, or qRT-PCR. For mass spectrometry, the bound proteins were subjected SDS-PAGE gel and visualized by Silver Staining Kit (24612, Thermo, USA) as manufacture’s instruction. The different band was cut and analyzed by mass spectrometry. The sequences of probes are shown in in supplementary Table S2.
Luciferase reporter assay
The circFARP1/LIF wild-type or mutant plasmids and miR-660-3p mimic were co-transfected into CAFs cells using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s protocol. Then the transfected Cells were seeded into 96-well plates and luciferase activities were determined by dual-luciferase reporter assay system (Promega, USA) according to the manufacturer's instructions.
Xenograft tumor model experiments
For mouse subcutaneous xenograft model, 5 × 106 Panc-1 cells alone or mixed with stable transfected CAFs at a ratio of 1:1, were injected into the right flank of 4-weeks-old the BALB/c nude mice. The animals of each group (n =5) were assigned to gemcitabine (50 mg/kg, once every four days, intraperitoneally) for 24 days when the tumors reached approximately 3mm in diameter, and tumor size was monitored meantime. All the mice were sacrificed five weeks later. Both maximum (L) and minimum (W) lengths of the tumor were measured using a slide caliper, and the tumor volume was calculated as (LW2)/2.
For mouse PDX models, primary tumor specimens collected from GEM-resistant PDAC patients who underwent surgery were propagated as subcutaneous tumors in 4-week-old NSG mice (F1). Xenografts from F1 mice were cut into small pieces and then implanted into other mice (F2). When tumors grew up to about 1500 mm3, they were excised and cut again into small pieces and transplanted to other mice (F3). The combined treatment of In vivo siRNA/ neutralizing antibodies against LIF and GEM chemotherapy was performed when xenografts volume reached about 200 mm3. In vivo-optimized si-Ctrl (5 nmol, intra-tumor injection), si-circFARP1 (5 nmol, intra-tumor injection) or LIF antibody (10 mg/kg, intra-venous injection). Tumor volume was monitored every week and tumor were further analyzed by IHC and qRT-PCR. The chemotherapy responses were assessed refer to the human clinical evaluation standard. Complete Response (CR) was defined as disappearance of tumor; Partial Response (PR) was defined as at least a 30% reduction of tumor volume; Progressive Disease (PD) was defined as at least a 20% of tumor volume; Stable Disease (SD) was defined as no sufficient to qualify as PR and PD. CR and PR were classified as GEM-sensitive, while SD and PD were classified as GEM-resistant.
Animal experiments were conducted according to guidelines approved by the Animal Experimental Research Ethics Committee of South China University of Technology.
RNA immunoprecipitation (RIP)
RIP was performed by Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s protocol. Briefly, cells were washed twice with ice-cold PBS, followed by cell lysis using RIP lysis buffer. Magnetic beads were washed twice with RIP wash buffer, followed by incubation with 2 μg antibody against CAV1 or rabbit anti-IgG as a negative control for 30 mins at room temperature. Immunoprecipitation was performed by incubating cell lysate with the magnetic bead-antibody complex overnight at 4°C. Then the beads were washed six times with RIP wash buffer. The precipitated RNAs were eluted and further analyzed by qRT-PCR assays.
Cells were lysed in IP lysis buffer and protease inhibitors. For immunoprecipitation, indicated antibody or rabbit anti-IgG as the control was added to the lysates and incubated overnight at 4 °C. Then Dynabeads Protein A /G (10002D/10003D, Invitrogen) was added and then incubated for 3h at 4 °C. The precipitated proteins were analyzed by western blotting.
Total RNA was isolated and purified using TRIzol (Life, USA) following the manufacturer's procedure. After the quality inspection of Agilent 2100 Bioanalyzer (Agilent, USA) and NanoPhotometer (Implen, Germany), ribosomal RNA was removed from 1 μg total RNA. VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina (Vazyme, China) was used for lncRNA library construction following the manufacturer's protocol. Each library was sequenced on an Illumina Novaseq 6000 (Illumina Corporation, USA) in 150PE mode following the vendor's recommended protocol by Guangzhou Huayin Health Medical Group CO.,Ltd. (Guangzhou, China).
All experimental data were expressed as mean ± standard deviation (SD) using GraphPad Prism 8.0. The differences between parametric variables were determined by Student’s t-test or one-way analysis of variance (ANOVA), and nonparametric variables were determined by Mann-Whitney U test. Statistical significance of survival was estimated by Kaplan-Meier analysis and the log-rank test, and correlation analysis was performed by two-sided Pearson’s correlation. Correlation analysis was examined with two-sided Pearson’s correlation. p <0.05 was used as an indicator of statistical significance.