SUPPLEMENTARY FILE

SUPPLEMENTARY MATERIALS AND METHODS

Bioinformatics analysis

Four Gene Expression Omnibus (GEO) datasets (GSE15471, GSE16515, GSE28735, and

GSE43288) were used in this study to determine the expression of neurotransmitter receptors, especially GABRP, EDNRA and F2R. The expression microarray data of these datasets are all available at https://www.ncbi.nlm.nih.gov/gds/. The gene expression data for pancreatic adenocarcinoma (PAAD) was downloaded from TCGA, which were processed by Broad

Institute’s TCGA workgroup. And our reproduction abided by the rules of the TCGA request.

The RNA-sequencing expression data contain log2-transformed RNA-seq by expectation maximization (RSEM) values were summarized at gene level. For gene set enrichment analysis (GSEA) of TCGA data, genes expressed in less than 80% of the samples were removed. Three gene sets related to tumour growth and metastasis deposited in the GSEA

MSigDB resource were used to identify the difference between the high-GABRP group and low-GABRP group. We used median expression as cutoff. Gene expression higher than median was designated “High”, while lower than median was designated “Low”. GSEA was performed on the Broad Institute Platform and statistical significance (false discovery rate,

FDR) was set at 0.25. The GABRP-correlated genes were analyzed by online available software R2 (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi). For annotating the biological roles of these GABRP-correlated genes, Cytoscape software and the ClueGo Apps

(http://www.cytoscape.org/) were used. ClueGO presents enriched pathways within a network interconnected based on the k score. The node size shows the term significance after

Bonferroni correction. Terms found in the GO interval of 3 to 8, with at least three genes from the initial list representing a minimum of 4%, were selected. Fusion was applied to reduce the redundancy.

Cell culture

Pancreatic ductal adenocarcinoma cell lines (AsPC-1, BxPC-3, Capan-1, Capan-2, CFPAC-1,

HPAC, MiaPaCa-2, PANC-1, and SW1990) were all preserved in Shanghai Cancer Institute,

Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University and maintained at 37°C in a 5% carbon dioxide incubator with a humidified atmosphere. Panc-02 cell line was a gift by

Prof. Jing Xue. Cells were cultured in Dulbecco’s modified Eagle’s media (DMEM), DMEM/F12 or Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 100 Units/mL of penicillin, and 100 μg/mL of streptomycin as suggested by the American Type Culture Collection (ATCC, Manassas, VA, USA).

siRNA transfection

Exponentially growing untreated BxPC-3 and PANC-1 cells were plated in 60 mm dishes for

24 h before transfection. When cells were grown at 60-70% confluence, 50 ng of specific siRNA targeting the mRNA of GABRP or KCNN4, or scrambled siRNA targeting no known gene sequence accompanied with Lipofectamine® RNAiMAX reagent (ThermoFisher

Scientific, #13778030) were added to conduct siRNA transfection according to the manufacturer’s protocol. After treatment for at least 48 h, the interference efficiency was certified by western blotting and the cells were harvested or processed for further assays. The siRNA oligonucleotides were synthesized from GenePharma (Shanghai, China) and the detailed sequences of the siRNA used in this study were shown as follows: si-CXCR2-1,

GGAGAGUGACAGCUUUGAAdTdT; si-CXCR2-2, GCCAUGGACUCCUCAAGAUdTdT; si-CCR6-1, GCAUUUCUCCAGGCUAUUUdTdT; si-CCR6-2,

CCAUUGUACAGGCGACUAAdTdT; si-KCNN4-1, CCUGUUCCUGGUUAAAUGCdTdT; si-KCNN4-2, GCCGUGCGUGCAGGAUUUAdTdT.

Construction of lentivirus constructs

The shRNA constructs against GABRP and scrambled sequences were purchased from

GenePharma (Shanghai, China). Lentiviral particles were generated using a three plasmid system (pPACKH1-GAG, pPACKH1-REV, and pVSV-G). Lentivirus packaging was performed in 293T cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Cells were infected with 1 × 106 recombinant lentivirus-transducing units in the presence of 6 μg/ml polybrene (Sigma-Aldrich, H9268, USA).

Twenty-four hours after infection, cells were treated with 2 μg/ml puromycin (Gibco, A1113802,

USA) for 7 days to eliminate the uninfected cells and thus yield mass populations of puromycin-resistant cells expressing the shRNAs. The knockdown efficiency of GABRP was detected by western blotting.

Western blotting analysis

Cell lysates were collected from 10 cm plates using a total protein extraction buffer (Beyotime, Shanghai, China) with phosphatase and proteinase inhibitors (1 mM EDTA, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 100 mM NaF, 10 mg/ml leupeptin, 10 units/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride) and protein concentration was measured using a BCA Protein Assay Kit (Pierce Biotechnology, USA). Protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Millipore, Danvers, MA). Nonspecific binding of the membranes was blocked by 5% non-fat dried milk in Tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature. Then the membrane was probed with one of the following primary antibodies: GABRP (1: 1000, Abcam, ab26055), KCNN4 (1:1000, Proteintech, 23271-1-AP),

Caveolin-1 (1: 1000, Abcam, ab2910), B-adaptin (1:1000, Proteintech, 15690-1-AP), Lamin B1

(1: 1000, Cell Signaling Technology, #13435), α-Tubulin (1:1000, Abcam, ab176560), and

β-actin (1:1000, Abcam, ab5644), at 4°C overnight. After washing in TBST for three times, the membranes were incubated with species-specific secondary antibodies (ThermoFisher

Scientific, USA) and bound secondary antibodies were detected by Odyssey imaging system

(LI-COR Biosciences, Lincoln, NE, USA).

Immunofluorescence

For tissue immunofluorescence analysis, paraffin sections (5 μm) of human tumours were deparaffinized, rehydrated with graded ethanol, and subjected to antigen retrieval. For cell immunofluorescence analysis, the cells were grown on chamber slides, fixed with 4% paraformaldehyde and permeabilized with phosphate-buffered saline (PBS) containing 0.1%

Triton X-100. Both tissue and cell samples were blocked with 10% donkey serum (Sigma-Aldrich, D9663, USA) for 1 h at room temperature, followed by incubation with primary antibodies at 4°C overnight. These slides were then washed three times with PBS, and incubated with secondary antibodies for 30 min at room temperature. Nuclei were visualized using 4ʹ,6-diamidino-2-phenylindole (DAPI) staining. Digital images were acquired with fluorescence or confocal microscopes equipped with a digital camera (Nikon, Japan). The detailed information for primary and secondary antibodies in this assay was provided in

Supplementary Table 3.

Immunohistochemistry

Immunohistochemical analysis (IHC) was performed as previously reported 1. Scoring was conducted based on the percentage of positive-staining cells: 0-5% scored 0, 6-35% scored 1,

36-70% scored 2, and more than 70% scored 3; and staining intensity: no staining scored 0, weakly staining scored 1, moderately staining scored 2 and strongly staining scored 3. The final score was calculated using the percentage score multiply staining intensity score as follows: “-” for a score of 0-1, “+” for a score of 2-3,“++” for a score of 4-6 and “+++” for a score of > 6. Low expression was defined as a total score < 4 and high expression with a total score ≥ 4. These scores were determined independently by two senior pathologists in a blinded manner. Specifically, 16 dots in the tissue microarray were lost during dewaxing or staining process, and finally 295 dots were remained for analysis. Specific antibody used in this assay were: PCNA (1:5,000, Cell Signaling Technology, #13110), MMP9 (1:200,

Proteintech, 10375-1-AP), GABRP (1:200, Abcam, ab26055), CCL20 (1:200, Proteintech,

26527-1-AP), CXCL5 (1:500, Abcam, ab9802), and F4/80 (1:250, Cell Signaling Technology, #70076S). The scoring was determined independently by two senior pathologists in a blinded manner.

Quantitative real-time PCR

Cells were washed once with PBS, and total RNA was extracted using Trizol reagent (Takara,

Japan) and reversely transcribed through PrimeScript RT-PCR kit (Takara, Japan) according to the manufacturer’s instructions. Real-time quantitative PCR analysis was performed with

SYBR Premix Ex Taq (Takara, Japan) on a 7500 Real-time PCR system (Applied Biosystems,

USA) at the recommended thermal cycling settings: one initial cycle at 95°C for 10 min followed by 40 cycles of 5 sec at 95°C and 31 sec at 60°C. Relative mRNA expression was calculated using the 2(−ΔΔCt) method and normalized to β-actin mRNA levels. Sequences of the primers used to amplify genes are indicated in Supplementary Table 4.

Plate clone formation assay

Single-cell suspension was plated at a density of 1,000 cells per plate (6-well plate). The culture medium was replaced every three days. Two weeks later, the formed clones were then fixed and stained with 0.1% crystal violet/40% methanol and colonies with more than fifty cells were counted under a microscope.

Cell invasion assay

In brief, Matrigel was diluted in cold DMEM at 1:6 and then 100 μl diluted Matrigel was added to the upper wells of the Transwell chambers. The Matrigel was reconstituted with medium for 1 h at 37°C before the addition of cells. Equal number of the treated viable cells (10,000 cells) resuspended in 100 μl serum-free medium were seeded onto the upper wells of the Transwell chambers. The lower chamber was supplemented with 700μl medium containing 5% FBS.

Forty-eight hours later, the cells that had not invaded were removed from the upper surface of the filters using cotton swabs and the migrated cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The invaded cells were counted under a light microscope in six random fields.

Cell apoptosis assay

Serum starvation-induced apoptosis in PDAC cells was detected by the Caspase-3/7 activity kit (Promega, USA). After serum starvation for 48 h, the cell number and caspase-3/7 activity in indicated groups were monitored using CellTiter-Blue (Promega, G8081) and Apo-ONE

Caspase-3/7 assay (Promega, G7790), respectively. Relative Caspase-3/7activity was calculated as the ratio of Apo-ONE/CellTiter-Blue signals.

In vivo tumour growth and metastasis assays

Athymic male nu/nu mice ages 6 to 8 weeks were kept on a 12-hr day/night cycle with free access to food and water. For subcutaneous xenograft study, a total of 2 × 106 sh-Ctrl or sh-GABRP BxPC-3 cells in 200 μl Hanks buffered saline were injected subcutaneously in the lower back. Four weeks later, mice were sacrificed and the tumour was isolated and tumour weight was measured. Tumour volumes were calculated: volume = length × width2/2. For experimental lung metastasis assay, a total of 1 × 106 sh-Ctrl or sh-GABRP BxPC-3 cells in 100 μl Hanks buffered saline were intravenously injected and lung colonization was quantified after 4 weeks.

Macrophage depletion assay

For macrophage depletion assay, all mice were housed and maintained under specific pathogen-free conditions and used in accordance with institutional guidelines. The animal studies and the process were approved by Ethics Committee of Ren Ji Hospital, School of

Medicine, Shanghai Jiao Tong University, China. Clodronate liposomes and PBS liposomes were purchased from Dr Nico van Rooijen (Vrije Universiteit, Amsterdam, Netherlands). In brief, C57BL/6J mice were injected intraperitoneally with clodronate liposomes (1.4 mg/20 g body weight) or with an equivalent volume of PBS liposomes twice per week until week 5. At week 2, C57BL/6J mice were injected orthotopically with 2 × 106 ov-vector and ov-Gabrp

Panc-02 cells. Finally, a bioluminescent imaging (BLI) analysis was used to measure tumour burden using an IVIS Spectrum system (PerkinElmer, Waltham, MA, USA).

In situ proximity ligation assay

In situ proximity ligation assay (PLA, Olink Bioscience, DUO92007) was performed as previously reported 11. Briefly, BxPC-3 and PANC-1 cells were fixed with 4% formaldehyde for

30 min at room temperature, followed by permeabilization with 0.05% Triton-X100 for 5 min.

After incubation with DuoLink blocking buffer for 30 min at 37°C, cells were incubated with primary antibodies against GABRP and KCNN4 from two different species at 4°C overnight.

The next day, cells were washed with a suitable wash buffer and incubated with species-specific PLA probes in a pre-heated humidity chamber for 1 h at 37°C. The ligation solution, consisting of two additional oligonucleotides and ligase, was added to join the hybridized oligonucleotides to form a closed circle. In the presence of fluorescently labeled oligonucleotides and polymerase, a rolling-circle amplification reaction was conducted to generate a concatemeric product. Finally, cells were counterstained with DAPI to locate the nuclei.

Co-immunoprecipitation assay

The protein G Dynabeads (Invitrogen, USA) were pre-cleaned and incubated with anti-GABRP antibody (Abcam, ab26055), anti-KCNN4 antibody (GeneTex, GTX54786) or control IgG for

15-30 min in PBS at room temperature. After washed with PBS for three times, the beads were incubated with total cell extracts with gentle shaking for 30 min at room temperature. The beads were washed and resuspended in 40 μl of 1 × loading buffer and boiled at 70°C for 10 min. The proteins were separated by SDS-PAGE (8% SDS) and transferred to a nitrocellulose membrane for immunoblot detection with anti-KCNN4 antibody or anti-GABRP antibody.

Chemokine chip assay

To generate conditional medium (CM), 5 × 106 sh-Ctrl or sh-GABRP BxPC-3 cells were seeded on 10-mm plates. The next day culture media were replaced with RPMI-1640 free of

FBS. Four-eight hours later, CM was collected and subjected to chemokine antibody array.

Human chemokine antibody array kit C1 (Raybiotech, AAH-CHE-1-2) was used according to the manufacturer’s instructions. Detailed antibody array information can be found in https://www.raybiotech.com/files/manual/Antibody-Array/AAH-CHE-1.pdf. Briefly, the arrays were blocked with blocking buffer, incubated with 1 ml of undiluted conditional medium and incubate at 4°C overnight, followed by incubation with biotin-conjugated antibodies cocktail for

2 h at room temperature. After washing for three times, the membranes were incubated with

HRP-Streptavidin for 2 h at room temperature, and incubated with chemiluminescent substrate and exposed to x-ray film for 15 minutes before development. Quantitative array analysis was performed using Array Vision Evaluation 8.0 (GE Healthcare Life Science).

ELISA analysis of CXCL5 and CCL20

Cells were washed once with PBS and incubated for 24 h with medium free of FBS. Cell supernatants were collected, cleared by centrifugation and used immediately. The amount of supernatants used was normalized to cell number. The CXCL5 and CCL20 ELISA kits were purchased from Sigma-Aldrich (Shanghai, China) and used according to the manufacturer’s instructions.

Chemotaxis assays

Human peripheral blood mononuclear cells (PBMCs) were isolated from the heparinized venous blood of healthy donors (Shuheng Jiang) by standard density gradient centrifugation and resuspended in RPMI medium. The blood donors acquired verbal consent, volunteered to contribute blood to accomplish these experiments, knew that the risk was minimal at donation and blood was not used for other experiments. Monocytes were then purified from PBMCs by negative depletion of lymphocytes, dendritic cells, erythrocytes, granulocytes, and macrophages. For the migration assay, THP-1 cells or human monocytes (2 × 104) were plated in the upper compartment of Transwell chambers, and the lower chamber contained

RPMI 1640 medium with indicated treatment. To inhibit the stimulatory effects of CXCL5 or

CCL20, cell culture supernatants and recombinant CXCL5 or CCL20 (R&D Systems) were treated with 5 μg/mL anti-CXCL5 antibody (R&D Systems) and 5 μg/mL anti-CCL20 antibody

(R&D Systems) for 30 min before addition to THP-1 cells or human monocytes. After a 12-h incubation period for THP-1 cells and a 24-h incubation period for human monocytes, the migrated cells were stained with 2 μM Calcein-AM for 15 min, followed by photographing under a fluorescence microscope.

Ca2+ measurements

Intracellular free Ca2+ concentration was studied in live BxPC-3 and PANC-1 cells using

FLUO-3 AM (Invitrogen, USA). Cells were loaded with FLUO-3 AM (5 mmol/l) for 45 min at

37°C in serum-free medium and washed for 30 min in PBS at 37°C to allow cleavage of the acetoxymethyl esters. In association with FLUO-3, Hoechst 33342 (1.0 mmol/l, Dojindo

Molecular Technologies, Japan) nuclear staining was applied for 10 min. Fluorescence was measured at 505-535 nm with excitation at 488 nm.

Electrophysiology

Macroscopic currents were recorded from BxPC-3 cells in the whole-cell configuration by an

EPC-10 amplifier controlled by PatchMaster software (HEKA Electronics). Patch pipettes were prepared from borosilicate glass (Warner) and had a resistance of 2–5 MΩ when filled with pipette solution containing: 140 mM K-aspartate, 4.3 mM CaCl2, 2.06 mM MgCl2, 5 mM EGTA,

10 mM HEPES, pH 7.2. Free Ca2+ concentration was calculated to 1 μM using Maxchelator

2+ program (Stanford University). For experiments with 0 μM free [Ca ]i in pipette solution, CaCl2 was omitted and EGTA was increased to 10 mM. Standard bath solution contained: 150 mM

Na-aspartate, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM Glucose, 10 mM HEPES, pH 7.4.

Currents were elicited by 1 s-ramps from −100 to 100 mV every 5 s. Series resistance was compensated by 60-70%. Activation of KCNN4 K+ currents by fast perfusion of intracellular

Ca2+ was performed by automated planar patch-clamp instrument (Nanion) as described previously (Han et al., PMID:27005320). Automated whole-cell electrophysiological measurements were conducted according to Nanion’s standard procedure with 8-channel

Patchliner (Nanion) equipped with an EPC-10 quadro patch-clamp amplifier (HEKA

Electronics). Single-use borosilicate glass chips with medium resistance (1.8 to 3 MΩ, NPC-16,

Nanion) were used for all recordings. The PatchControlHT (Nanion) and PatchMaster (HEKA

Electronics) were used for cell capture, seal formation, whole-cell access, and subsequent recording in the voltage-clamp configuration. All electrophysiological experiments were performed at room temperature.

Isolation of lipid rafts

The protocol for lipid raft isolation is available at www.biodynamics.co.jp.

Luciferase reporter assay

BxPC-3 and PANC-1 cells were plated at 60-70% confluence in 24-well plates and 24 h later transiently transfected with 1 μg total DNA using FuGENE 6 transfection reagent (Roche

Applied Science). The pGMNF-κB-Luc, pGMNFAT-Luc, pGMCRE-Luc vectors (Genomeditech,

Shanghai, China) were transfected into cells with one of the following expression vectors: si-GABRP, si-KCNN4, or scrambled siRNA according to the manufacturer’s instruction.

Co-transfection of Renilla luciferase under the control of the SV40 early enhancer/promoter region (pSV40-RL, Promega) was used to normalize for transfection efficiency. After 24 h, the cells were collected and subjected to a Dual Luciferase Reporter Assay System (E1910,

Promega, USA). Firefly luciferase activity was normalized to Renilla luciferase activity for each sample. All transfections were performed at least two times, in triplicate. The results are shown as means ± SD.

References

1. Jiang SH, Li J, Dong FY, et al. Increased Serotonin Signaling Contributes to the Warburg

Effect in Pancreatic Tumour Cells under Metabolic Stress and Promotes Growth of Pancreatic

Tumours in Mice. Gastroenterology. 2017 Jul;153(1):277-291.e19.

SUPPLEMENTARY FIGURES

Supplementary Figure 1 GABRP expression in human and mouse PDAC tissues. (A)

Kaplan-Meier analysis (log-rank test) of the overall survival of PDAC patients based on

EDNRA and F2R expression. Data were derived from TCGA cohort. (B) Immunofluorescence staining for CK19 and GABRP in mouse PanINs and PDAC tissues. PanIN tissues were collected from KC mice and PDAC tissues were collected from KPC mice. Scale bar, 50 μm.

(C) Immunofluorescence staining for CK19 and GABRP in human PDAC tissues. Scale bar,

50 μm. (D) Western blotting analysis of nuclear/cytoplasmic/membrane GABRP expression in

PDAC cells.

Supplementary Figure 2 GABRP contributes to macrophage recruitment. (A, B) Transwell experiment was used to demonstrate the effect of conditioned medium from sh-Ctrl and sh-GABRP cells on migration of PMA-stimulated THP-1 cells and monocytes (n = 6). Scale bar:

200 μm. (C, D) Transwell experiment was used to verify the effect of conditioned media from different pancreatic cancer cells on migration of PMA-stimulated THP-1 cells and monocytes

(n = 6). Scale bar: 50 μm. **p < 0.01, ***p < 0.001.

Supplementary Figure 3 GABRP correlates with macrophage infiltration in PDAC. (A, B)

Immunohistochemistry was performed in 81 cases of pancreatic cancer tissue microarray to analyze the relationship between the expression of GABRP and the number of CD68+ (A) and

CD163+ (B) macrophages. Scale bar: 50 μm. (C) Immunohistochemical analysis of F4/80+ macrophages from sh-Ctrl and sh-GABRP subcutaneous xenograft tissues and lung metastasis tissues. Scale bar: 50 μm. **p < 0.01, ***p < 0.001.

Supplementary Figure 4 GABRP overexpression upregulates CXCL5 and CCL20 in PDAC cells. (A) ELISA analysis of the CXCL5 and CCL20 level in the CM from pancreatic cancer cells (n = 3). (B) Western blotting analysis of GABRP overexpression efficiency in HPAC and

SW1990 cells. (C) ELISA analysis of the effect of GABRP overexpression on CXCL5 and

CCL20 level in HPAC and SW1990 cells (n = 3). **p < 0.01, ***p < 0.001.

Supplementary Figure 5 CXCR2 and CCR6 signaling are crucial for macrophage recruitment induced by CM from PDAC cells. (A) Expression profile of CXCR2 and CCR6 in THP-1 cells and monocytes as well as human PDAC tissues was analyzed by co-immunofluorescence with

CD68. (B, C) Real-time qPCR analysis showed the knockdown efficiency of CXCR2 and

CCR6 in THP-1 and monocytes. (D) Effects of CXCR2 or CCR6 knockdown on the migratory ability of PMA-primed THP-1 cells and human monocytes (n = 6). *p < 0.05, **p < 0.01, ***p <

0.001.

Supplementary Figure 6 Effect of GABA signaling on intracellular Ca2+ signaling, PDAC cell proliferation and invasion. (A) IHC for the expression of GABA in human PDAC tissues (n = 10);

2 representative images with both tumour and nerve tissues were shown. Scale bar: 50 μm. (B)

FLUO-3 AM staining was used to measure the effect of GABA stimulation on the concentration

2+ of free cytosolic calcium ([Ca ]i) in PDAC cells (n = 3). Scale bar: 30 μm. (C) CCK-8 assay

was used to determine the effect of GABA and two GABAA receptor agonists (propofol and pregnenolone) on cell viability under 1% and 10% serum culture conditions (n = 3). (D)

Transwell experiment was used to demonstrate the effect of GABA, propofol and pregnenolone on cell invasion (n = 6). (E) Real-time qPCR was used to verify the effect of

BAPTA on the mRNA expression levels of CXCL5 and CCL20 (n = 3). *p < 0.05, **p < 0.01.

Supplementary Figure 7 Measurement of KCNN4 expression and its activity in PDAC cells.

(A) The expression of potassium channels in PDAC. (B) Correlation analysis of potassium channel and GABRP, red column indicates positive correlation and blue column indicates negative correlation. (C) Whole-cell patch clamp recordings of BxPC-3 cells with 1 μM free

Ca2+ in pipette solution. Cells were exposed to KCa3.1 inhibitor TRAM-34 (5 μM). (D)

Whole-cell patch clamp recordings of PANC-1 cells with 0/1 μM free Ca2+ in pipette solution.

Cells were exposed to KCa3.1 inhibitor TRAM-34 (5 μM).

Supplementary Figure 8 Effects of KCNN4 on intracellular calcium signaling and chemokine expression in PDAC cells. (A) The effects of KCNN4 knockdown on the free cytosolic calcium

2+ ([Ca ]i) concentration were measured using FLUO-3 AM staining (n = 3). Scale bar: 30 μm. (B)

Real-time qPCR analysis of the effect of KCNN4 knockdown on the CXCL5 and CCL20 mRNA level in BxPC-3 and PANC-1 cells (n = 3). (C) ELISA analysis of the CXCL5 and CCL20 level in the CM from si-Ctrl and si-KCNN4 BxPC-3 and PANC-1 cells (n = 3). (D) Real-time qPCR analysis of the effect of 1-EBIO treatment on the CXCL5 and CCL20 mRNA level in BxPC-3 and PANC-1 cells (n = 3). (E) Real-time PCR analysis of the effect of TRAM-34 treatment on the CXCL5 and CCL20 mRNA level in BxPC-3 and PANC-1 cells (n = 3). Values in graphs are mean ± SD. *p < 0.05, **p < 0.01.

Supplementary Figure 9 MβCD inhibits intracellular calcium signaling and chemokine expression in PDAC cells. (A) Membrane cholesterol content in PDAC cells after MβCD treatment (5 mM) was determined using the Amplex Red Cholesterol Assay Kit (n = 5). (B) The

2+ effects of MβCD on the free cytosolic calcium ([Ca ]i) concentration in BxPC-3 and PANC-1 cells were measured using FLUO-3 AM staining (n = 3). Scale bar: 50 μm. (C) Real-time qPCR analysis of the effect of MβCD treatment on the CXCL5 and CCL20 mRNA level in BxPC-3 and

PANC-1 cells (n = 3). Values in graphs are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Supplementary Figure 10 Loss of GABRP, loss of KCNN4, and treatment with MβCD all inhibit cell proliferation and invasion and promote cell apoptosis. (A) Plate clone formation assay was used to verify the proliferation ability of PDAC cells upon GABRP/KCNN4 interference or MβCD treatment. (B) Caspase-3/7 activity kit was used to verify the apoptotic

PDAC cells upon GABRP/KCNN4 interference or MβCD treatment (n = 3). (C) Transwell experiment was used to verify the invasion ability of of PDAC cells upon GABRP/KCNN4 interference or MβCD treatment. (n = 6). *p < 0.05, **p < 0.01.

Supplementary Figure 11 Correlation between GABRP/KCNN4 axis and chemokine expression in PDAC. (A) Immunohistochemistry was used to analyze the protein expression of

GABRP, CXCL5 and CCL20 in GABRP-silenced or Gabrp-overexpressed tumour tissues.

Scale bar: 25 μm. (B) Correlation between GABRP level and chemokine expression in TCGA

PDAC cohort. (C) Correlation between KCNN4 level and chemokine expression in TCGA

PDAC cohort. Values in graphs are mean ± SD. *p < 0.05, **p < 0.01.

SUPPLEMENTARY TABLES

Supplementary table 1 Neurotransmitter receptors analyzed in this study Neurotransmitter Neurotransmitter receptors Glutamate GRIA1, GRIA2, GRIA3, GRIA4, GRIK1, GRIK2, GRIK3, GRIK4, GRIK5, GRID1, GRID2, GRIN1, GRIN2A, GRIN2B, GRIN2C, GRIN2D, GRIN3A, GRIN3B, GRM1, GRM2, GRM3, GRM4, GRM5, GRM6, GRM7, GRM8 Gamma aminobutyric GABRA1, GABRA2, GABRA3, GABRA4, GABRA5, GABRA6, GABRB1, GABRB2, acid (GABA) GABRB3, GABRD, GABRE, GABRG1, GABRG2, GABRG3, GABRP, GABRQ, GABRR1, GABRR2, GABRR3, GABBR1, GABBR2 Glycine GLRA1, GLRA2, GLRA3, GLRB Histamine HRH1, HRH2, HRH3, HRH4 Acetylcholine CHRM1, CHRM2, CHRM3, CHRM4, CHRM5, CHRNA1, CHRNA2, CHRNA3, CHRNA4, CHRNA5, CHRNA6, CHRNA7, CHRNA9, CHRNA10, CHRNB1, CHRNB2, CHRNB3, CHRNB4, CHRND, CHRNE, CHRNG Dopamine DRD1, DRD2, DRD3, DRD4, DRD5 Epinephrine (E) ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, Norepinephrine (NE) ADRB3 Serotonin (5-HT) HTR1A, HTR1B, HTR1D, HTR1F, HTR2A, HTR2B, HTR2C HTR3A, HTR3B, HTR3C, HTR3D, HTR3E, HTR4, HTR5A, HTR6, HTR7 Neuro-active ligand TAAR, TACR1, TAKR, NPY1R, NPY2R, NPY4R, NPY5R, NPY6R, NPYNR, GPR83, OPRM1, OPRK1, OPRD1, OPRL1, AVPR1A, AVPR1B, AVPR2, CCKAR, CCKBR, CCKLR, OXTR, ATGR1, ATGR2, APLNR, NMBR, GRPR, BRS3, BDKRB1, BDKRB2, C3AR1, C5AR1, FPR1, FPRL, EDNRA, EDNRB, GALR1, GALR2, GALR3, GALRN, GHSR, KISS1R, MC1R, MC2R, MC3R, MC4R, MC5R, MLNR, NMUR1, NMUR2, NPFFR1, NPFFR2, NPBWR1, NPBWR2, NTSR1, NTSR2, HCRTR1, HCRTR2, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TACR2, TACR3, OPRM1, OPRK1, OPRD1, OPRL1, AVPR1A, AVPR1B, AVPR2, UTS2R, F2R, F2RL1, F2RL2, F2RL3, PARD3, PRLHR, MCHR1, MCHR2, FSHR, LHCGR, PTGDR, PTGER1, PTGER2, PTGER3, PTGER4, PTGFR, PTGIR, TBXA2R, ADORA1, ADORA2A, ADORA2B, ADORA3, P2RY2, P2RY1, P2RY4, P2RY6, P2RY8, P2RY10, P2RY11, P2RY13, P2RY14, LPAR4, LPAR6, GPR35, CNR1, CNR2, PTAFR, GNRHR, TRHR, MTNR1A, MTNR1B, GPR50, LPAR1, LPAR2, LPAR3, LPAR4, SIPR1, SIPR2, SIPR3, SIPR4, SIPR5, LTB4R1, LTB4R2, MAS1, RXFP1, RXFP2, RXFP3, RXFP4, CYSLTR1, CYSLTR2, CALCR, CALCRL, CRHR1, CRHR2, GIPR, GCGR, GLP1R, GLP2R, GHRGR, PTHR1, PTHR2, ADCYAP1R1, SCTR, VIPR1

Supplementary table 2 The top 300 GABRP-correlated genes in TCGA cohorts HUGO R-value P-value HUGO R-value P-value

GJB4 0.646 1.26E-20 DAPP1 0.553 6.11E-14 MMP7 0.631 1.96E-19 GRHL2 0.553 5.86E-14 TMPRSS4 0.621 1.33E-18 SERPINB8 0.553 5.72E-14 LOC284578 0.611 7.11E-18 ALPK1 0.552 7.29E-14 GCNT3 0.608 1.26E-17 IL18 0.551 7.94E-14 LY75 0.608 1.26E-17 PLSCR1 0.551 8.12E-14 RHBDL2 0.606 1.68E-17 ANXA2P2 0.55 8.52E-14 ADGRG6 0.602 3.71E-17 ZNF267 0.549 1.06E-13 ADGRF1 0.601 3.93E-17 MUC4 0.548 1.22E-13 SH3RF2 0.601 4.25E-17 RASEF 0.548 1.23E-13 AIM1 0.598 7.02E-17 KRT7 0.547 1.40E-13 SAMD9L 0.595 1.13E-16 F3 0.546 1.54E-13 CEACAM6 0.589 2.71E-16 PARP14 0.546 1.48E-13 GBP2 0.589 2.93E-16 XAF1 0.546 1.56E-13 MYO1E 0.588 3.51E-16 POU2F3 0.545 1.68E-13 PDZK1IP1 0.586 4.56E-16 ADAM28 0.543 2.43E-13 ITGB6 0.585 5.15E-16 KYNU 0.543 2.44E-13 CAPZA1 0.582 8.96E-16 MOB1A 0.543 2.38E-13 CTTNBP2NL 0.581 1.06E-15 TNFSF10 0.543 2.35E-13 PROM1 0.581 1.05E-15 TACSTD2 0.542 2.53E-13 GJB3 0.58 1.15E-15 BCL2L15 0.541 2.92E-13 PIGR 0.58 1.17E-15 MYOF 0.541 2.97E-13 GPRC5A 0.579 1.27E-15 SERPINB5 0.541 2.92E-13 SNAP23 0.578 1.49E-15 TGFA 0.541 2.88E-13 LIPH 0.576 2.07E-15 LMO4 0.54 3.26E-13 FAM83B 0.575 2.30E-15 CORO2A 0.539 3.88E-13 TCN1 0.574 2.94E-15 RBPMS 0.539 3.98E-13 KLF5 0.573 3.46E-15 ARHGAP26 0.537 5.05E-13 NMI 0.572 3.61E-15 CARD6 0.537 5.08E-13 CLDN1 0.571 4.50E-15 PRSS8 0.537 5.27E-13 TPM1 0.57 5.13E-15 RASSF9 0.536 5.89E-13 DOCK5 0.566 9.42E-15 TFPI 0.536 5.78E-13 PLA2R1 0.566 9.37E-15 ABCA13 0.535 6.42E-13 CASP8 0.565 1.13E-14 EPS8 0.534 7.70E-13 ANXA3 0.563 1.47E-14 BIRC3 0.533 8.35E-13 FGD6 0.562 1.65E-14 TMEM133 0.533 8.34E-13 ARHGAP42 0.558 2.93E-14 LINC01559 0.531 1.17E-12 IL1RN 0.558 2.98E-14 TRIM34 0.531 1.09E-12 TRIM29 0.558 2.93E-14 TRIM5 0.531 1.13E-12 AHR 0.556 3.71E-14 ERN2 0.53 1.20E-12 HNMT 0.556 3.98E-14 INPP4B 0.53 1.22E-12 TLR3 0.555 4.77E-14 MYEOV 0.53 1.20E-12 SFN 0.53 1.32E-12 GNAI3 0.511 1.23E-11 ST6GALNAC1 0.53 1.34E-12 MUC16 0.51 1.51E-11 LCN2 0.528 1.58E-12 SLC6A20 0.51 1.37E-11 LYPD6 0.528 1.66E-12 GBP1 0.508 1.85E-11 SLC6A14 0.528 1.61E-12 IGSF3 0.508 1.93E-11 PLEKHG1 0.527 1.79E-12 ANO6 0.507 2.09E-11 RNF213 0.526 2.16E-12 TNFRSF10A 0.507 1.98E-11 C1orf106 0.525 2.35E-12 PLEKHS1 0.506 2.26E-11 EIF2AK2 0.525 2.30E-12 IYD 0.506 2.21E-11 LGALS3 0.525 2.24E-12 SFTA2 0.506 2.25E-11 RTP4 0.525 2.48E-12 ANXA2 0.505 2.57E-11 GNG12 0.523 3.12E-12 KRT17 0.505 2.53E-11 TMEM92 0.523 3.20E-12 PVRL4 0.505 2.60E-11 ANXA2P1 0.522 3.55E-12 ENC1 0.504 3.06E-11 MST1R 0.522 3.39E-12 OASL 0.504 2.79E-11 CHMP2B 0.521 3.74E-12 OSBPL3 0.504 3.01E-11 POF1B 0.521 3.73E-12 PQLC3 0.504 2.78E-11 SP100 0.521 3.73E-12 STK38 0.504 2.93E-11 CAP1 0.519 5.27E-12 TMC5 0.504 3.01E-11 CFLAR 0.519 4.88E-12 TRIM38 0.504 2.83E-11 LGALS9 0.519 4.93E-12 ZFP36L2 0.504 2.93E-11 PLBD1 0.519 4.87E-12 SPPL2A 0.503 3.26E-11 RAP1A 0.518 5.81E-12 CTSE 0.502 3.73E-11 RHOF 0.518 5.73E-12 SP110 0.502 3.65E-11 GALM 0.517 6.36E-12 FRRS1 0.501 4.15E-11 KITLG 0.517 6.10E-12 HKDC1 0.501 4.02E-11 NRAS 0.517 6.54E-12 MBOAT1 0.501 4.07E-11 ZBP1 0.517 6.11E-12 SEMA3C 0.501 4.22E-11 ZFP36L1 0.517 6.64E-12 SPATS2L 0.501 4.02E-11 ABCC3 0.516 7.30E-12 ALDH1A3 0.5 4.74E-11 TRIM31 0.516 7.03E-12 BNIP2 0.5 4.44E-11 ANKRD22 0.515 8.12E-12 EDN1 0.499 5.03E-11 SAMD9 0.515 8.44E-12 LRRK1 0.499 4.84E-11 IFI44 0.514 9.55E-12 PROM2 0.499 4.93E-11 TRIM21 0.514 8.66E-12 RARRES3 0.499 4.88E-11 MET 0.513 1.01E-11 SLFN13 0.499 4.96E-11 STAT6 0.513 9.79E-12 UCA1 0.499 4.94E-11 TPM4 0.513 1.06E-11 VRK2 0.499 4.85E-11 CCDC68 0.512 1.12E-11 APOBEC3C 0.498 5.81E-11 LYPD6B 0.512 1.14E-11 TES 0.498 5.76E-11 SP1 0.512 1.17E-11 ALOX5 0.497 6.14E-11 ACSL5 0.511 1.28E-11 KCNN4 0.497 6.62E-11 DKK1 0.511 1.23E-11 MUC5B 0.497 6.16E-11 NOTCH2 0.497 6.36E-11 BCAS1 0.487 1.87E-10 PSME4 0.497 6.04E-11 CDK2 0.487 1.78E-10 TGIF1 0.497 6.62E-11 CMPK1 0.487 1.88E-10 TJP3 0.497 6.29E-11 DHCR24 0.487 1.89E-10 VDR 0.497 6.36E-11 OSMR 0.487 1.77E-10 CST1 0.496 7.14E-11 TRIM14 0.487 1.77E-10 BAZ1A 0.495 8.21E-11 PTPN22 0.486 1.95E-10 BMPR1B 0.495 8.11E-11 AQP5 0.485 2.19E-10 CDH3 0.495 8.21E-11 CAPN5 0.485 2.32E-10 OAS2 0.495 7.87E-11 CYP2C18 0.485 2.22E-10 BCL2L14 0.493 9.48E-11 GPX8 0.485 2.17E-10 LGALS4 0.493 9.84E-11 NCK1 0.485 2.34E-10 RALB 0.493 9.61E-11 PTGFRN 0.485 2.18E-10 CHST4 0.492 1.10E-10 SDR16C5 0.485 2.21E-10 PLS3 0.492 1.10E-10 TMPRSS3 0.485 2.31E-10 PTP4A2 0.492 1.10E-10 TP53I3 0.485 2.29E-10 SP140L 0.492 1.07E-10 TSPAN6 0.485 2.18E-10 CASP4 0.491 1.21E-10 FOSL2 0.483 2.92E-10 FHL2 0.491 1.29E-10 FUT3 0.483 2.94E-10 FRMD4B 0.491 1.24E-10 ITGAV 0.483 2.92E-10 IGF2BP2 0.491 1.29E-10 ITGB4 0.483 2.69E-10 LPP 0.491 1.23E-10 SLC25A24 0.483 2.84E-10 MPZL2 0.491 1.28E-10 YAP1 0.483 2.70E-10 CD58 0.49 1.35E-10 APOL6 0.482 3.00E-10 DCLRE1B 0.49 1.33E-10 CST4 0.482 3.15E-10 FAM111B 0.49 1.38E-10 EML4 0.482 3.05E-10 TMOD3 0.49 1.42E-10 NFE2L3 0.482 3.28E-10 TPRG1 0.49 1.32E-10 PLA2G10 0.482 3.21E-10 VTCN1 0.49 1.37E-10 TRIM22 0.482 3.18E-10 ARL14 0.489 1.57E-10 UBE2L6 0.482 2.98E-10 CTSS 0.489 1.51E-10 ANO1 0.481 3.58E-10 GJB2 0.489 1.52E-10 CASP10 0.481 3.58E-10 IFIH1 0.489 1.46E-10 CTNNA1 0.481 3.45E-10 PGM2 0.489 1.45E-10 FUT2 0.481 3.36E-10 SLC30A7 0.489 1.57E-10 GALNT5 0.481 3.57E-10 GBP4 0.488 1.64E-10 GJB5 0.481 3.60E-10 MITF 0.488 1.68E-10 KIAA1217 0.481 3.58E-10 NCEH1 0.488 1.62E-10 TEAD3 0.481 3.58E-10 PDLIM5 0.488 1.62E-10 ACTR3 0.48 3.86E-10 RASAL1 0.488 1.70E-10 LAMB3 0.48 3.81E-10 S100P 0.488 1.67E-10 MACC1 0.48 3.99E-10 SLC5A1 0.488 1.64E-10 OAS3 0.48 3.94E-10 ATP10B 0.487 1.76E-10 PDGFC 0.479 4.26E-10 PICALM 0.479 4.44E-10 PPAP2C 0.475 6.13E-10 ACOT9 0.478 4.79E-10 RIPK2 0.475 6.38E-10 GIMAP2 0.478 4.78E-10 SLC35A3 0.475 6.37E-10 KRT19 0.478 4.61E-10 HSD17B2 0.474 6.94E-10 PCSK5 0.478 4.60E-10 MYD88 0.474 6.92E-10 TMEM173 0.478 4.64E-10 RASSF3 0.474 7.26E-10 ANXA4 0.477 5.35E-10 FAM3D 0.473 8.14E-10 B2M 0.477 5.35E-10 IRAK2 0.473 7.87E-10 CAMKK2 0.477 5.06E-10 PTAFR 0.473 7.67E-10 DPYD 0.477 5.29E-10 SLC16A4 0.473 7.64E-10 LAMP3 0.477 5.34E-10 ZNFX1 0.473 7.67E-10 TSPAN8 0.477 5.32E-10 ARAP2 0.472 8.38E-10 WNT2 0.477 5.25E-10 GALNT4 0.472 8.28E-10 DEPDC1B 0.476 5.87E-10 SSPN 0.472 8.64E-10 ETV6 0.476 5.68E-10 UGT1A10 0.472 8.83E-10 IL1RAP 0.476 5.81E-10 ATL3 0.471 9.62E-10 MFAP5 0.476 5.90E-10 NAALADL2 0.471 9.04E-10 STIL 0.476 5.94E-10 PDCD10 0.471 9.04E-10 C8orf4 0.475 6.28E-10 RUNX1 0.471 9.31E-10 LACTB 0.475 6.30E-10 C3orf52 0.47 1.07E-09 NFE2L2 0.475 6.58E-10 IFIT2 0.47 1.02E-09

Supplementary table 3 The antibodies used in co-immunofluorescence experiment

Group Antigen Primary antibodies Secondary antibodiese

#1 GABRP 1: 200, Abcam, ab26055 donkey anti-rabbit Alexa Fluor 488 (1:400, #711-545-152)

CK19 1: 200, Proteintech, 60187-1-Ig donkey anti-mouse Alexa Fluor 594 (1:400, #715-585-150)

#2 CXCR2 1: 200, Proteintech, 20634-1-AP donkey anti-rabbit Alexa Fluor 488 (1:400, #711-545-152)

CD68 1: 200, Abcam, ab955 donkey anti-mouse Alexa Fluor 594 (1:400, #715-585-150)

#3 CCR6 1: 500, Abcam, ab78429 donkey anti-rabbit Alexa Fluor 488 (1:400, #711-545-152)

CD68 1: 200, Abcam, ab955 donkey anti-mouse Alexa Fluor 594 (1:400, #715-585-150)

All the secondary antibodies were purchased from Jackson ImmunoResearch.

Supplementary table 4 The sequences of primers used in this study

Gene Forward primer (5’-3’) Reverse primer (5’-3’)

GABRP CAGCGGCTGGTGTTTGAAG GAGGCGGATGAGCCTGTTTC

CXCL5 AGCTGCGTTGCGTTTGTTTAC TGGCGAACACTTGCAGATTAC

CCL20 TGCTGTACCAAGAGTTTGCTC CGCACACAGACAACTTTTTCTTT

Cxcl5 TCCAGCTCGCCATTCATGC TTGCGGCTATGACTGAGGAAG

Ccl20 GCCTCTCGTACATACAGACGC CCAGTTCTGCTTTGGATCAGC

CXCR2 CCTGTCTTACTTTTCCGAAGGAC TTGCTGTATTGTTGCCCATGT

CCR6 GGCTATTTGTACCGATTGCCT GATGCCTTTTAGCAACTTGCAC

ACTB CATGTACGTTGCTATCCAGGC CTCCTTAATGTCACGCACGAT

Actb GGCTGTATTCCCCTCCATCG CCAGTTGGTAACAATGCCATGT