Antibodies and reagents
All information of antibodies and reagents were listed in Supplementary Table 6.
Cell culture and transfection
ESCC cell lines-KYSE410 and KYSE510 were provided by Dr. Yutaka Shimada (Kyoto University). Primary CAFs were purchased from CHI Scientific Inc. All cells were cultured in RPMI-1640 medium contained with 10% heat-inactivated FBS (Gibco) and 1% penicillin/streptomycin in a 37 °C humidified incubator under 5% CO2.
The siRNA-based approach was applied to generate CCTα or CCTβ-knockdown cells. CCTα or CCTβ siRNAs were transfected into primary CAFs using Lipofectamine 2000 reagent. For plasmid stable transfection, pcDNA 3.1-Flag plasmid contained AKT2 S128A or CCTα S315/319/323A mutant was transfected into CAFs. Subsequently, positive clones were selected for further experiments. Tansfection efficacy was evaluated using immunoblotting. Sequences of siRNAs and mutant plasmids were listed in Supplementary Table 7.
For evaluation of CAFs-mediated FAK inhibitor resistance in ESCC treatment, CAFs (3 × 105/well) were plated in the upper chamber of transwell apparatus (0.4 μm insert, 24-well), and ESCC cells (1 × 105/well) were cultured in the lower chamber. Defactinib (0-10 μM) or/and other inhibitors, including miltefosine (25 μM), ruxolitinib, fedratinib (10 μM), or S3I-201 (20 μM), were treated for 4 days, and then the upper chamber was discarded. The growth rate of ESCC cells was assessed using MTS assay (Progema). The IC50 value of defactinib was listed in presented Figures. Other MTS assays of indicated ESCC cells and CAFs were performed in 96-well plates. The exact protocols of MTS assay was according to our previous studies.
EZCellTM cell invasion assay kit (Biovision) was applied to evaluate CAFs-mediated defactinib resistance in the anti-invasion of ESCC cells. Briefly, indicated ESCC cells (2 × 104/well) were plated in the upper chamber of transwell apparatus (8 μm insert), and indicated CAFs (6 × 104/well), PC (16:0/20:4) and glycerophosphocholine (10 μM) were added to the lower chamber. Defactinib (10 μM) or/and other inhibitors, including miltefosine (25 μM), ruxolitinib, fedratinib (10 μM), or S3I-201 (20 μM), were treated for 24 hours, and then remove the non-invasive cells from the top chamber using a cotton swab. The invasive cells in top chamber were incubated with cell dye and dissociation solutions for 1 hour at 37 °C in CO2 incubator. Read the plate at Ex/Em (530/590 nm) to obtain the invasion rate of indicated ESCC cells.
Concentration of phosphoatidylcholine (PC) in condition media (CM) and plasma of ESCC patients
PC assay kit (Sigma-Aldrich) was used to measure the levels of PC in the CM from indicated ESCC cells and CAFs, or ESCC patients’ plasma. Supernatants of indicated cells were collected, and centrifuged at 3000 rpm for 10 minutes to obtain CM. Then, 50 μL sample buffer (44 μL CM, 2 μL PC hydrolysis enzyme, 2 μL PC development mix, and 2 μL fluorescent peroxidase substrate) was combined with 50 μL reaction mix, and incubated at room temperature for 30 minutes. Then, OD value was measured spectrophotometrically at 570 nm. The plasma (50 μL/sample) was directly subjected to PCs assay.
Intracellular concentration of Ca2+
CAFs were collected and suspended in 500 μL calcium assay buffer and put on ice for quickly pipetting. Then, intracellular concentration of Ca2+ was evaluated using Ca2+ detection kit (Nanjing Jiancheng Bioengineering Institute). Briefly, 50 μL sample were incubated with 500 μL MTB reagent, 1mL alkaline solution, and 50 μL protein clarifier for 5 minutes. OD value was measured spectrophotometrically at 610 nm.
Human phospho-STAT3 Tyr705 and total STAT3 ELISA kit (Raybiotech) was used to evaluate the activity of intratumoral STAT3. The protocols were according to manufacture’s instruction and our previous studies.
Immunoprecipitation (IP) and immunoblotting (IB) analysis
For IP assay, indicated cells were washed with PBS, lysed in NP40 buffer supplemented with protease and phosphatase inhibitors for 30 minutes, and then centrifuged at 12000 g for 20 min at 4℃. Supernatants were collected to incubated with indicated primary antibodies (approximately 10 μg antibody/sample) and protein A/G sepharose beads (Thermo Fisher) on a rotator at 4℃ overnight. Then, samples were centrifuged at 4°C for 5 minutes at 3000 g, supernatants were discarded, and pellets were washed with 800 μL cold NP40 buffer for 3 times. Finally, beads were collected, and 60 μL loading buffer was added to the beads. The beads were bathed in metal for 5 minutes, and supernatants were subjected to IB assay.
For IB assay, proteins were separated using sodium dodecyl sulfate (SDS)-PAGE, and transferred onto a nitrocellulose (NC) membrane. After blocking with PBS buffer solution containing 0.1% Tween-20 and 5% nonfat milk for 1 hour, the membranes were incubated with indicated primary antibodies at 4°C overnight. PBST was used to wash NC membranes for three times. The membranes were incubated with secondary antibodies for 1 hour, and then washed an additional three times with PBST and detected by chemiluminescence (Thermo Fisher).
Female BALB/c-nu mice (purchased from Beijing Vital River Laboratory) with 3-4 weeks of age were used in present assay. All animal procedures were approved by Institutional Review Board of Peking University Cancer Hospital & Institute.
KYSE410 or KYSE510 cells were subcutaneously co-injected with indicated CAFs into the flank of mice. When the tumor reached around 100 mm3, defactinib (25mg /kg/day, p.o) alone or in the presence of ruxolitinib (10 mg/kg/day, p.o.), or fedratinib (10 mg/kg/day, p.o.), or S3I-201 (25 mg/kg/day, p.o.) for consecutive 3 weeks (n=5/group). Tumor volume was evaluated using our reported formula. Human Ki67, CD31, or LYVE-1 ELISA kits (Raybiotech) were applied to measure the proliferation, angiogenesis, or lymph-angiogenesis abilities of indicated ESCC tumors. The experimental protocols were according to manufacturer’s instructions.
For evaluation of lymph node metastasis of ESCC cells, KYSE410 or KYSE510 cells were subcutaneously co-injected with indicated CAFs into the footpads of mice (n=5/group). The agents used in this assay was consistent with the model that subcutaneous tumor cells inoculation. Treatment was started for week 2 and this experiment was sustained for 5 weeks. Lymph node volume was evaluated by our reported formula .
ESCC tissues and IHC staining
All procedures and experiments of ESCC tissues were approved by the institutional Review Board of Peking University Cancer Hospital. The protocols of IHC staining and the calculation of staining index were according to our previous studies [12,26]. The dilution of primary antibodies was as follow: FAK (1:100), pFAK Tyr397 (1:100), pAKT2 Ser128 (1:100), pCCTα Ser315/319/323 (1:500), pSTAT3 Tyr705 (1:4000), or αSMA (1:1500).
Protein extraction and tryptic digestion
CAFs samples were washed with three times with phosphate buffer saline (PBS), and then lysed in SDS lysis buffer supplemented with PMSF and phosphatase inhibitors (Cat # 4906837001, Roche). Samples were sonicated with 3 min (1 s on and 1 s off, 80 W power) on ice and centrifuged at 12, 000 g for 10 min to remove insoluble debris. The supernatants were collected, and the protein concentration was measured using BCA protein assay (Cat # 23225, ThermoScientific). Extracted proteins were reduced in 5 mM DTT at 55 °C for 30 min and then alkylated in 10 mM iodoacetamide at room temperature for 15 min in darkness. Six times of the volume of precooled acetone was added to above system to precipitate the proteins, and place samples at -20 °C overnight, then centrifuged at 8, 000 g for 10 min to collect the precipitate. Samples underwent trypsin digestion (enzyme-to-substrate ratio of 1:50 at 37 °C overnight) and lyophilized.
The enrichment of phosphorylated peptides
For iTRAQ labeling, the lyophilized samples were resuspended in 60 μL 100 mM TEAB solution and then transferred into 1.5 mL Eppendorf tube for labeling. 70 μL isopropanol was added to iTRAQ reagent vial (Cat # 4381663, ABSCIEX) at room temperature, and then vortexed for mixing, centrifuged. This step was repeated 1 time. 100 μL iTRAQ label reagent was added to samples for mixing. Tubes were incubated at room temperature for 2 h. Finally, 200 μL HPLC water was added to each sample and incubated for 30 min to stop reaction. The labeled peptides samples were lyophilized and stored at -80 °C.
For phosphopeptides enrichment, the high-selectTM Fe-NTA phosphopeptide enrichment kit was used (Cat # A32992, ThermoFisher). Briefly, the labeling peptides were incubated with 200 μL binding/washing buffer in spin columns and incubated for 30 min. Then, placed the columns into microcentrifuge tubes for centrifuging at 1000 g for 30 s, and discarded the flowthrough. Bound columns were washed by 200 μL binding/washing buffer, and then centrifuged at 1000 g for 30 s. This step was repeated for three times. Bound peptides were washed with 200 μL HPLC water to remove the unphosphorylated peptides. The remaining peptides were added with 100 μL elution buffer, resolved on columns, and then centrifuged at 1, 000 g for 30 s. The incubation reaction was repeated. The final enriched phosphopeptides were immediately vacuum concentrated for further use.
Peptides samples were analyzed on an EASY-nLC 1200 system (ThermoFisher) coupled with a TimsTOF pro mass spectrometry (Bruker). Peptides were re-dissolved in mobile phase A (H2O-FA; 99.9: 0.1, v/v) and immediately loaded onto an 25 cm long, C18 analytical column (75 μm inner diameter; Cat #AUR2-25075C18A; Ionopticks).
Peptides were separated at an fluent flow rate of 400 nL/min. Peptide separation was achieved with a 90 min gradient (0-66 min, 2 to 21% of buffer B; 66-73 min, 21 to 42% of buffer B; 73-82 min, 42 to 95 % of buffer B; 82-90 min, 95% of buffer B). The eluted phosphopeptides were ionized and detected by the TimsTOF pro mass spectrometry. Mass range was 100-1700 m/z. Ion mobility was 0.6-1.6. Collision energy was 20-59 eV. The temperature of drying gas was 180 °C. Capillary voltage was 1.5 KV.
MS database searching
MS raw files generated by LC-MS/MS were searched using Max Quant (version 220.127.116.11.). Protease was Trypsin/P. Up to 2 missed cleavages were allowed. iTRAQ (N-term, K) and carbamidomethyl (C) were considered as fixed modifications. Variable modifications were oxidation (M), acetyl (Protein N-term), and phospho (S/T/Y). The cutoff of false discovery rate (FDR) by using a target-decoy strategy was 1% for peptides.
Identification of variant peptides
MaxQuant search data were analyzed by “R” package (version 2.15) software. MaxQuant output files were log2-transformed, normalized and adjusted in “R” package. Protein, peptide, and phosphorylation-site identifications were filtered by removing entries corresponding to reverse database identifications, potential contaminants and those identified by site alone. The screening conditions were as follows: the phosphorylation sites with expression value ≥ 50% in any groups were retained. Phosphorylation sites with missing value ≤ 50% were filled with the mean value of the same group, screen localization probability ≥ 0.75, Delta score ≥ 8, and converse data via median normalization and log2-transformation to obtain highly reliable phosphorylation sites. Based on obtained highly reliable phosphorylation sites, the differences between two groups was calculated (Difference screening condition: fold change ≥ 2), and the MOMO (http://meme-suite.org/tools/momo) software was applied to analyze the motif of phosphorylation sites to obtain the specific substrates. Then, Uniprot, KEGG/GO databases were used to annotate the function of phospho-proteins. Method of KEGG/GO functional enrichment analysis was as follow: human proteins were used as the background, and the proteins corresponding to the differential phosphorylation sites were screened from background. The Fisher’s exact test (as implemented in R software) were used to evaluate the different pathways corresponding to phospho-proteins between control and treated groups. Pathways with P value ≤ 0.05 were considered to be statistically differential pathways.
1 mL of CM were transferred to a 5 mL Eppendorf tube, and then lyophilized. 400 μL mixture of methanol and water (1/4, vol/vol) were added to each sample. Samples were vortexed for 30 s, sonicated for 3 min, and then added with 10 μL internal standard (2-chloro-L-phenylalanine; 0.3 mg/mL, dissolved in methanol). Mixed samples were place at -20 °C for 2 h, and then centrifuged at 13, 000 g for 10 min at 4 °C. 150 μL supernatants from each sample were collected using crystal syringes (filtered through 0.22 μm microfilters) and transferred to LC vials, which were stored at -80 °C for further LC-MS assay.
Metabolic analyses were performed on an UPLC Ultimate 3000 system (Dionex) coupled to a A-Exactive mass spectrometer (ThermoFisher). The chromatographic separation was conducted using ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm; Waters) with temperature was set at 45 °C. The mobile phases A and B were consisted of water and acetonitrile both containing 0.1 % formic acid, and run at a flow rate of 0.35 mL/min. The linear gradient was as follows: 0.01 min, 5% of phase B; 2 min, 5% of phase B; 4 min, 30 % of phase B; 8 min, 50 % of phase B; 10 min, 80 % of phase B; 14-15 min, 100 % of phase B; 15.1-18 min, 5 % of phase B. The mass spectrometer was performed in a full-scan mode ranging from 100-1000 m/z, running at a 70, 000 resolution in both positive and negative modes simultaneously.
Data extraction and analysis
The acquired raw data were analyzed using the Progenesis QI (version 2.3; Nonlinear Dynamics), and the applied parameters were as follows: precursor tolerance was set 5 ppm, product tolerance was set 10 ppm, and product ion threshold was set 5 %. The metabolite identification was conducted using EMDB database (http://www.emdataresource.org/mission.html). The extracted data were further reduced by removing any peaks with missing value (ion intensity =0) in more than 50 % samples, and the selected score is >36 (full score is 60). Results of score lower than 36 were deleted. The positive and negative data were combined to obtain a combined data which were imported into “R” package (version 2.15) software. Unsupervised principle component analysis (PCA) and PLS-DA and OPLS-DA were combined to discriminate the metabolite profile between groups. The variable importance in projection (VIP) value (from OPLS-DA) > 1.00 and P value (Student’s test) < 0.05 of each metabolite were used as the combined cut-offs of the statistical significance.
Untargeted lipidomic analysis
After lyophilized, CM lipids were extracted by 400 μL mixture of chloroform and methanol (2/1, vol/vol) in the presence of 20 μL internal standard (Lyso PC-17:0, 0.1 mg/mL), and then vortexed for 30 s, sonicated for 3 min, plated for 30 min at -20 °C. Subsequently, samples were centrifuged at 12, 000 g for 10 min at 4 °C, and volatilized. The residue of lipids were re-dissolved with 200 μL isopropanol and methanol (1/1, vol/vol), vortexed for 30 s, sonicated for 3 min, and then centrifuged at 12, 000 g for 10 min at 4 °C. 150 μL supernatants were collected for further study.
Lipidomic analyses were conducted using an UPLC Ultimate 3000 system (Dionex) coupled to a A-Exactive mass spectrometer (ThermoFisher). The chromatographic separation was conducted using ACQUITY UPLC BEH C8 column (100 mm × 2.1 mm, 1.7 μm; Waters) with temperature was set at 55 °C. The mobile phase A was consisted of acetonitrile and water (6/4, vol/vol) containing 10 mM ammonium acetate. The mobile phase B was consisted of isopropanol and acetonitrile (9/1, vol/vol) containing 10 mM ammonium acetate. The flow rate was set at 0.26 mL/min. The linear gradient was as follows: 0.00 min, 32 % of phase B; 1.5 min, 32 % of phase B; 15.5 min, 85 % of phase B; 15.6 and 18.0 min, 97 % of phase B; 18.1 and 21.0 min, 32 % of phase B. The mass spectrometer was performed in a full-scan mode ranging from 100-1500 m/z, running at a 70, 000 resolution in both positive and negative modes simultaneously.
Data extraction and analysis
The raw data were extracted and analyzed using the Lipid Research (version 4.1.16，ThermoFisher). Following analysis methods and protocols were according to metabolomics analysis.
The lipid was extracted from 100 μL plasma using 300 μL choroform-methanol (2:1, v/v, supplementing with 0.1 mM BHT) contained 20 μL known amounts of 74 isotope-labeled internal mix standards (identify 1, 000 lipids), vortex for 30 seconds and ultrasonic extraction for 10 minutes, and then hold 30 minutes at -20 °C, centrifuge 10 minutes (13, 000 rpm) at 4 °C, 200 μL chloroform layers were transferred into a centrifuge tube. Residues were reextracted according to above condition. Combine the chloroform layers together and lyophilize in a centrifugal vacuum evaporator at 4 °C. Samples were reconstituted in isopropanol-methanol (1:1, v/v), vortex for 30 seconds, ultrasonic extraction for 3 minutes, centrifuge 10 minutes (13, 000 rpm) at 4 °C, and then stored at -20 °C. Finally, 150 μL supernates were subjected to LC/MS analysis.
LC-MS analysis was conducted using an ExionLCTM system (ABSCIEX) coupled to Qtrap 6500 plus system (ABSCIEX) equipped with an IonDriveTM Turbo V source. The chromatographic separation was conducted using ACQUITY UPLC BEH C8 column (100 mm × 2.1 mm, 1.7 μm; Waters) with temperature was set at 55 °C. The mobile phase A was consisted of acetonitrile and water (6/4, vol/vol) containing 0.1 % formic acid and 10 mM ammonium formate. The mobile phase B was consisted of isopropanol and acetonitrile (9/1, vol/vol) containing 0.1 % formic acid and 10 mM ammonium formate. The flow rate was set at 0.35 mL/min. The sample injection volume was 5 μL. The linear gradient was as follows: 0.00 min, 0 % of phase B; 1.5 min, 0 % of phase B; 5.0 min, 55 % of phase B; 15.0 min, 90 % of phase B; 16.0 and 18.0 min, 100 % of phase B; 18.1 and 20.0 min, 0 % of phase B. The MS analysis was conducted in the negative/positive-ion mode working in the time-scheduled MRM method to high-throughoutly screen more than 1000 lipids. The source condtion is as follow: curtain gas is 35 psi, the ion spray (IS) voltage is -4500 V/+5500 V, source temperature is 350 °C, and Gas 1 and Gas 2 is 40 psi and 45 psi.
Data extraction and analysis
The acquired raw data were analyzed using the MRMPROBS software  to perform automated batch processing, including peak extraction, alignment, identification, peak area integral. Relevant parameters are set as follows: smoothing level is 2, minimum peak width is 5, minimum peak height is 500, retention time tolerance is 0.2 min, and supplemented by manual correction. The obtained qualitative and quantitative tables were quantitatively analyzed using response factor method. Furthermore, integral peak area of metabolites was brought into following formula: Lipid concentration = Target lipid peak area (A1)/Peak area of lipid internal standard corresponding to target lipid (A2) × The internal standard concentration value of the corresponding lipid internal standard in the sample (C) × The constant volume (V)/Weighed sample mass or volume (N). The concentration of lipids in plasma from different tumor stages of ESCC patients (30 cases) were divided into two groups, including Stage Ⅰ and stage Ⅱ and Ⅲ, and the difference between this two groups was analyzed using Mann-Whitney U test. Variables are expressed as the median with interquartile range (IQR) in Supplementary Table 7.
The phosphoproteomic data have been deposited in https://www.iprox.cn/page/home.html, and the accession number was: PXD032254. The metabolomics related data have been deposited in https://www.ebi.ac.uk/metabolights/, and the accession numbers were: MTBLS4489 and MTBLS4502.
All data are expressed as the mean ± SD, and statistical analyses are performed by Graphpad 7.0 software. Unpaired Student’s t-test (two-tailed) was applied to compare the difference between two groups. For analysis of clinical samples, Chi-square test was used to evaluate the correlation between two factor. Kaplan-Meier method was employed to establish the survival curves of ESCC patients. The statistical analysis of phosphoproteomics, metabolomics, lipidomics, or pseudo-targeted Lipidomics was listed in respective “Materials and methods” section. P-value < 0.05 was considered statistically significant.