Clinical trial design overview and sample collection
The clinical trial design follows the standardized protocol outlined in the study “Efficacy and Safety of Surufatinib Combined with Gemcitabine and Albumin-bound Paclitaxel in the Peri-operative Treatment of Pancreatic Cancer” (NCT05908747, https://clinicaltrials.gov/study/NCT05908747).
Sample Collection and Processing: Peripheral blood samples (5–10 mL) were collected using EDTA anticoagulant tubes and centrifuged within 2 h (1,500 × g, 10 min, 4 °C) to separate plasma and peripheral blood mononuclear cells (PBMCs), which were then stored at −80 °C and in liquid nitrogen, respectively. Tumor tissue samples were obtained through surgical biopsy. Freshly resected tumor tissue was digested into a single-cell suspension. Flow cytometry was employed to analyze immune cell composition using the following antibody panels: Myeloid panel: CD45, CD11b, F4/80, CD68, GPR34, MHC-I, MHC-II, CD80, CD86, MRC1 (CD206), MerTK, AXL. Lymphocyte panel: CD45, CD3ε, CD4, CD8α, CD19, NK1.1, Tim-3, PD-1, LAG-3, CTLA-4, GZMB, Ki-67, FOXP3. The gating strategy for identifying cell populations is detailed in Supplementary Fig. 7. Data were acquired on a BD LSRFortessa and analyzed with FlowJo v.10.
scRNA sequencing of patient surgical samples
Fresh tumor samples (~50-100 mg) were dissected into <1 mm³ pieces and incubated with 0.125 mg/mL collagenase IV(Sigma), 0.125 mg/mL Dispase II (Sigma) and Soybean Trypsin Inhibitor (MCE) at 37 °C for 2 h for tissue digestion. Subsequently, cells were filtered through a 70 μm mesh filter and collected. To remove dead cells, SYTOX Blue (Thermo Fisher, Cat.No.S7020) staining was applied to ensure only viable cells were used in subsequent analyses. Single-cell libraries were constructed using the 10x Genomics Chromium Single Cell 3’ GEM, Library & Gel Bead Kit v3.1 (10x Genomics, PN-1000121) targeting 10,000 cells per sample. Sequencing was performed on an Illumina NovaSeq 6000 platform to a median depth of 50,000 reads per cell. Raw sequencing data were initially processed with CellRanger v3.0.2 (10x Genomics) and aligned to the human genome (GRCh38). Downstream analysis was performed using the Seurat package (v5.0.1) in R. for normalization, dimensionality reduction, and clustering. Genes detected in fewer than 20 cells were excluded, and mitochondrial and ribosomal genes were removed. Cells of low quality, defined as having fewer than 200 or more than 6000 genes, or more than 10% mitochondrial gene content, were also filtered out.
scRNA sequencing data underwent quality control (retaining cells with mitochondrial gene percentage < 20% and detected gene counts > 200 and < 5,000), followed by selection of the top 2,000 most highly variable genes for data normalization. Principal component analysis (PCA) was performed using the highly variable gene expression matrix, with the top 30 principal components used for subsequent nonlinear dimensionality reduction and graph-based clustering. Cell clustering was based on shared nearest neighbor graphs (SNN, k. param = 20) and modularity optimization. After batch effect correction, to ensure data quality, the “decontX” function from the celda (v1.26.0) package was used to remove potential contamination from unwanted cell populations. Additionally, to eliminate the influence of homologous doublets, DoubletFinder (v2.0.3) was employed to detect and remove any identified doublets. After testing resolution parameters (0.2–1.2), a resolution of 1 was selected to balance biological rationality and computational efficiency. Cluster results were validated through UMAP visualization, showing good separation between clusters. Differential expression analysis was performed using the Wilcoxon rank-sum test with thresholds of log2FC > 0.5 and adjusted P-value < 0.01. Dimensionality reduction based on the Harmony results was followed by cell clustering using the Seurat functions FindNeighbors and FindClusters. Following clustering, cell type annotation was performed by cross-referencing cluster-specific differentially expressed genes (Wilcoxon rank-sum test, log₂FC > 0.5, adjusted p-value < 0.01) with canonical marker genes: KRT8, KRT18, KRT19 (epithelial cells); CD3D, CD3E, (T cells); CD79A, MS4A1 (B cells); JCHAIN, CD79A (plasma cell); CD14, HLA-DRA, APOE (monocytes/macrophages); FCGR3B (neutrophils); DCN, LUM (fibroblast); CDH5, VWF, PLAVP (endothelial cells); RGS5, PDGFRB (pericyte); CPA3, TPSAB1 (mast cell). A dot plot containing the expression of marker genes for all clusters is provided in Supplementary Fig. 1e. Functional characteristics of the cell types were further validated by their gene expression profiles. To resolve functional heterogeneity within key immune compartments, we performed secondary subclustering and analysis on extracted T cells and macrophages. For each resulting subpopulation, functional annotation was further informed by Gene Ontology (GO) Biological Process and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of their respective marker genes, which corroborated and refined the identity assigned by canonical markers. Characteristic genes for each cell subtype are listed in Table S1.
Gene Set Enrichment Analysis (GSEA) was conducted using the clusterProfiler (v4.10.0) package. Differentially expressed genes were extracted from the differential expression analysis, ranked by log2 fold change, and enriched against Hallmarks, KEGG, and GO gene sets. Enrichment results were visualized using enrichplot, with bar plots showing significantly enriched gene sets and their associated biological functions.
Data were first reduced in dimensionality and clustered based on Seurat clustering results. Pseudotime analysis was then performed using reduce Dimension function (with the DDRTree method) of Monocle 3 (v 0.2.3) followed by orderCells to arrange cells along a differentiation trajectory. The pseudotime analysis results were visualized using UMAP, which displays the position and trajectory of cells during differentiation. The dynamic tracking of cells through different stages of differentiation was used to analyze their differentiation trends.
Intercellular communication analysis was performed using the CellChat (v1.6.1) package. The computeCommunProb function was utilized to calculate the communication probabilities between cell populations, and the resulting signaling pathways were visualized using the netVisual_aggregate function. Based on cell type annotations, further analysis was conducted to explore the interactions and communication networks between different macrophage subpopulations and T cell subpopulations.
Animal Models
Macrophage Gpr34 conditional knockout mouse model (Gpr34ΔLyz2) was constructed by Gpr34flox/flox (Shanghai Model Organisms Center, Inc. Cat.No.NM-CKO-233912) and Lyz2-CreERT mice (B6.129P2-Lyz2tm1(cre)Ifo/J, Jackson Laboratories) were generated by crossing. Cre recombinase is specifically expressed in Lyz2-expressing monocyte-macrophages and induces Gpr34 gene knockout. Three weeks after modeling, tamoxifen (Sigma-Aldrich, Cat. No. T5648) was given to activate Cre recombinase and induce Cre gene deletion. Tamoxifen was dissolved in corn oil (Solarbio, Cat.No.C7030) at a concentration of 20 mg/mL and administered by intraperitoneal injection in a volume of 100 μL/20 g mouse body weight daily for 5 days to ensure effective induction of Cre recombinase activity in macrophages. After the model was established, efficient deletion (>90% reduction in GPR34 protein) in F4/80⁺ tumor-associated macrophages was confirmed by flow cytometry 10 days after the last tamoxifen injection (Supplementary Fig. 8c). Flow cytometry analysis showed that Gpr34 expression was significantly down-regulated in macrophages, confirming the successful construction of Gpr34 CKO model.
Gpr34 global knockout (Gpr34−/−) mice were generated by crossing Gpr34flox/flox mice with Dppa3-Cre mice (C57BL/6Smoc-Dppa3em1(IRES-Cre) Smoc, Shanghai Model Organisms Center, Inc. Cat. No. NM-KI-00040). The Dppa3-Cre transgene activates Cre recombinase in fertilized eggs, enabling global deletion of the Gpr34 gene. Gpr34 gene knockout was detected by next-generation sequencing, and changes in immune cell population were detected by flow cytometry, which confirmed the loss of Gpr34 in immune cells.
KPC spontaneous model: C57BL/6Smoc-Trp53em4(R172H) Krasem4(LSL-G12D) Tg (Pdx1-Cre)Smoc (KrasG12D/+; Trp53R172H/+; Pdx1-Cre, Shanghai Model Organisms Center, Inc. Cat.No.NM-KI-210096) spontaneously developed into pancreatic cancer in a natural background. In these mice, transformation of pancreatic tissue occurs naturally and tumors develop without external intervention. Tumor growth and volume changes were monitored by routine imaging using high-frequency ultrasound (Vevo 2100, VisualSonics). Tumor size and growth trends in the pancreatic region were assessed by ultrasound at early stages of tumor formation. Tumor size was monitored every 7 days to evaluate its growth rate and morphological characteristics. Ultrasound images were processed using VevoLAB software, and tumor volume was calculated using the formula V = 0.5 * (length) * (width2).
OT-1 Mice: C57BL/6-Tg (TcraTcrb)1100Mjb/J mice (Jackson Laboratories, Stock No: 003831) were used as a source of OVA257-264 (SIINFEKL)-specific CD8⁺ T cells.
Mouse CD8+ T cells/ NK cells were depleted using anti-mouse anti-CD8α (Bioxcell, Cat.No.BE0061)/anti-mouse NK1.1 antibody (Bioxcell, Cat.No.BE0036). First, mice were given an intraperitoneal injection of 200 μg. Depletion effects were assessed by looking at surface markers associated with CD8+ T cells /NK cells (Supplementary Fig. 10b, c). After injection, blood and spleen cells were analyzed by flow cytometry to ensure complete depletion of CD8+ T/NK cells. Subsequently, 200 μg/dose was maintained every 5 days. Treatment usually begins one week after intratumoral injection in mice, or when the tumor in KPC mice reaches 150 mm3. The dosage is as follows: Gemcitabine (Selleck, CAT.NO. S4419) 25 mg/kg, twice a week; Nab-paclitaxel (Selleck, E1068, CAT.NO. S4419),10 mg/kg, i.p., once a week; Surufatinib: 50 mg/kg, once every other day, i.p., for a total of 3 times. Anti-CXCL16 (InVivoMAb anti-mouse CXCL16 BioXcell, CAT.NO.BE0450) 5 mg/kg, i.p., 3 times a week. GPR34 antagonist (Compound D2, MCE, CAT. NO. HY-138501), 25 mg/kg, i.p., daily. The treatment cycle for the in-situ injection tumor model is 3 weeks, and the treatment duration for KPC mice is 8 weeks.
Establishment of the Mouse Model of Pancreatitis. Age-matched (8–10 weeks) Gpr34flox/flox or Gpr34ΔLyz2mice pre-treated with tamoxifen as described, received 12 hourly i.p. injections of cerulein (50 µg/kg, Selleck, S9690). Mice were euthanized 48 hours after the first injection for tissue collection and analysis.
Cell line and macrophages isolation
Generation of the KPC Cell Line. KPC mice (KrasG12D/+; Trp53R176H/+; Pdx1-Cre) aged 8–10 weeks were used for tumor isolation. When tumor volume reached approximately 200–300 mm³, tumors were excised under sterile conditions using aseptic techniques. Excised tumors were transferred to sterile glass culture flasks and finely minced into 1–2 mm³ tissue fragments. These fragments were then enzymatically digested in a solution containing collagenase IV (2 mg/mL, Sigma, Cat.No.9001-12-1), soybean trypsin inhibitor (MCE, Cat. No. HY-126388), and DNase I (50 µg/mL, SparkJade, Cat.No.AC1711) at 37 °C for 30 min. The cell suspension was filtered, washed, and plated in DMEM (Gibco, C11995500BT) with 10% FBS (Gibco) and 1% Penicillin/Streptomycin (Solarbio, P7630). After five passages to enrich for epithelial cells, adherent cells were collected.
The established cell lines (designated KPC-0117, used throughout this study) were validated by: a) Morphology: Spindle-shaped epithelial morphology under phase-contrast microscopy. b) Immunophenotype: >95% positive for the epithelial marker CK19 (Cytofix/Cytoperm kit, BD, 554714) by flow cytometry. c) Genotype: Sanger sequencing confirmed the presence of the KrasG12D and Trp53R172H mutations. d) Pathogen Testing: Confirmed negative for mycoplasma (MycoAlert PLUS, Lonza, LT07-710).
For imaging, cells were transduced with lentivirus carrying GFP or firefly luciferase (Luc). For antigen presentation assays, cells were transduced with lentivirus encoding ovalbumin (OVA). Stable pools were selected with puromycin (2 µg/mL).
Isolation and Differentiation of Bone Marrow-Derived Macrophages (BMDMs). BMDMs were isolated from the femur and tibia of 6-week-old C57BL/6, Gpr34−/−, and OT-1 mice following euthanasia. The metaphysis of the bones was removed under sterile conditions, and the bone marrow was flushed out using a 1 mL sterile syringe containing PBS with 2% FBS. The collected bone marrow cells were subjected to erythrocyte lysis and then resuspended in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were seeded into 10 cm² culture dishes and differentiated into macrophages by adding 20 ng/mL monocyte colony-stimulating factor (M-CSF, Proteintech, Cat.No.003831). After five days of culture, non-adherent cells were removed, and the adherent BMDMs were maintained in culture. Prior to co-culture experiments, BMDMs were stimulated with tumor-conditioned medium (TCM) derived from KPC cells for 24 h to mimic the tumor microenvironment and activate macrophages.
Preparation of Tumor-Conditioned Medium (TCM). KPC cells were grown to 70-80% confluence in T75 flasks, washed with PBS, and then cultured in serum-free DMEM for 48 h. The supernatant was collected, centrifuged at 2000 × g for 10 min, filtered through a 0.22 µm filter, and stored at −80 °C. This TCM was used at a 1:1 dilution with fresh complete medium for macrophage stimulation.
Human Cell Lines and Monocyte-Derived Macrophages (hMDMs). Human PDAC cell lines (Panc-1, BxPC-3, AsPC-1) were obtained from the Chinese Academy of Sciences Cell Bank and cultured per supplier instructions. hMDM were isolated from PBMCs obtained from healthy donors. CD14⁺ monocytes were purified using CD14 MicroBeads (Miltenyi Biotec, Cat.No.130-050-201) according to the manufacturer’s protocol. Briefly, PBMCs were incubated with CD14 antibody-conjugated magnetic beads for 20 min at 4 °C with gentle mixing. CD14⁺ cells were then separated from unlabeled cells using magnetic separation (BioLegend, Cat.No.480173). Following isolation, CD14⁺ cells were washed twice with PBS to remove unbound magnetic beads and contaminating cells. The purity of CD14⁺ monocytes was assessed by flow cytometry. Purified CD14⁺ cells were then cultured in RPMI-1640 medium (Gibco, Cat.No. C11875500BT) supplemented with human M-CSF (20 ng/mL, Proteintech, Cat.No. 300-25) and 10% FBS. The culture medium was replaced every two days, and after five days of differentiation, macrophages were used for downstream experiments.
Orthotopic Tumor Model Construction and In vivo Imaging
Pancreatic cancer cells (KPC cells) from KPC mice were transduced with lentivirus carrying luciferase (Luc) plasmids. These stably transduced KPC-Luc cells were then orthotopically injected into the pancreas of recipient mice (Gpr34ΔLyz2 and C57BL/6 strains) to establish an in situ tumor model. In the anesthetized mice, a longitudinal incision was made in the left upper abdominal area to expose the spleen and exteriorize the pancreas. KPC-Luc cells were injected into the pancreas parenchyma (40 μL, 1 × 10⁵ cells). After the surgery, the mice were monitored for 7–10 days, and when the tumors bioluminescence reached approximately 105–106, imaging was performed to monitor tumor growth and metastasis.
Monitoring tumor growth and metastasis: The IVIS Spectrum in vivo imaging system (PerkinElmer) was used for bioluminescence imaging to assess tumor size and location. Mice injected with KPC-Luc cells were given intraperitoneal injections of D-luciferin (150 mg/kg body weight) as a substrate. After 10 min, images were captured using the IVIS Spectrum system. Tumor growth dynamics were quantified by measuring the bioluminescence intensity within the tumor region. During the experiment, tumor growth and the overall health status of the mice were monitored and recorded.
Total body irradiation and bone marrow reconstitution experiments
Total body irradiation was used to ablate blood cells such as hematopoietic stem cells (HSCs) from the bone marrow of mice. The radiation dose was 10 Gy was used to ensure effective destruction of the hematopoietic system. Following irradiation, mice were maintained under standard conditions for 24 hours to allow for the full effect of irradiation on bone marrow depletion. Bone marrow was collected as described previously. The harvested cells were treated with red blood cell lysis buffer (Servicebio, Cat. No. R1010) to remove red blood cells. The cells were then resuspended in DMEM medium and cultured at 37 °C in the presence of 5% CO₂. After bone marrow clearance, Total bone marrow cells were prepared for transplantation. Total bone marrow cells derived from the donor mice were transplanted into recipient mice via tail vein injection. Each recipient mouse received approximately 5 × 106 total bone marrow cells which were resuspended in 200 µL PBS and injected slowly through the tail vein. After injection, mice were placed under standard rearing conditions for recovery. Bone marrow reconstitution was assessed approximately 2 weeks after transplantation. Staining was performed with an anti-mouse CD45 antibody (BD Biosciences, Cat.No.566439) and flow cytometry were used to detect the proportion of CD45+ cells and their immunophenotype in peripheral blood to confirm whether the donor hematopoietic cells were successfully reconstructed. Approximately 2 weeks after transplantation, bone marrow reconstitution was assessed. The peripheral blood was analyzed using flow cytometry to detect the proportion of CD45+ cells and their immune phenotype, confirming whether there was successful reconstitution of hematopoietic cells from the donor.
Tissue Dissociation and Single Cell Suspension Preparation Tissue
Dissociation: Tumors were excised and placed in medium containing PBS and 1% penicillin/streptomycin. The tissue was minced and passed through a 70 µm filter to remove debris to form a single cell suspension. The tissue suspension was then incubated in a digestive enzyme solution containing 0.1% soy trypsin inhibitor and 0.125 mg/mL collagenase IV for 30 min at 37 °C to further digest the tissue and obtain single-cell populations. The digestion was terminated by adding RPMI-1640 medium, and the undigested tissue blocks were removed by centrifugation to obtain the final single-cell suspension.
Preparation of single cell suspensions: Cell suspensions were filtered and centrifuged and washed with PBS. Cell concentrations were determined using a hemocytometer and adjusted as needed to the desired cell density. The cells were resuspended in serum-free RPMI-1640 medium.
Flow Cytometry Analysis
The single cell suspension was resuspended in MACS buffer (1% BSA, 2 mM EDTA, PBS). If it is for detecting the secreted proteins of T cells from PBMCs (such as IFN-γ), the medium should also contain phorbol myristate acetate (PMA, 20 ng/mL; Selleck, Cat. No. S7791) / Ionomycin (1 μg/mL; Selleck, Cat.No.S7074) and Golgi blocker Brefeldin A (50 ng/mL; Selleck, Cat.No.S7046) and incubated for 4 hours at 37 °C with 5% CO₂. To block Fc receptor binding, cells were incubated with anti-mouse CD16/CD32 antibody (BD Biosciences, Cat.No.553141) for 10 minutes on ice. Then, according to experimental requirements, cells were then stained with antibodies against specific surface markers (see in Table S2), followed by two washes to remove unbound antibodies. For analysis of secreted factors and transcription factors, we used a Cytoperm Fixation/Permeabilization Kit (BD Biosciences, Cat.No.554714), and cells were permeabilized and stained according to the manufacturer’s instructions. Subsequently, according to experimental requirements, cells were stained with antibodies against specific transcription factors or intracellular factors (see in Table S2). Finally, the samples were fixed with 1% paraformaldehyde (PFA, Servicebio, Cat. No. G1101). Flow cytometry data were acquired using BD LSRFortessa (BD Biosciences) equipment, and data were processed using FACSDiva software (BD Biosciences). FlowJo v.10.8.1 software was used for data analysis and cell populations were classified according to the fluorescence intensity of specific laser channels. For flow cytometry analysis, appropriate gating strategies were used to exclude debris and doublets. In the analysis of fibroblasts, we excluded EPCAM+, CD31+, and CD45+ cell subsets, gated the Lineage-negative (Lin-) cell population, and used PDPN for positive gating to identify fibroblasts. Myeloid cells were identified by gating on CD45+CD11b+ cells, and lymphocytes were identified by gating on CD45+CD3ε+CD8α+ cells. In specific analyses, we further quantified the immunophenotype, activation status, and cytokine secretion levels of the cell populations by analyzing the corresponding markers.
Macrophage Conditioned Medium (MCM) Preparation
Macrophage-conditioned medium (MCM) was prepared by culturing macrophages under the desired experimental conditions. After the cells were cultured, the medium was replaced, and the macrophages were incubated for an additional 48 hours. The collected medium was filtered through a 0.45 µm filter to remove cell debris, resulting in the macrophage-conditioned medium (MCM). This MCM was used for subsequent co-culture experiments or downstream analyses. To separate MCM from small molecular materials, the supernatant was added to the upper chamber of a 3 kDa molecular weight cutoff protein concentrator (MilliporeSigma, Cat.No.ACK5003PA). The concentrator was placed at 4 °C and centrifuged at 5,000 × g for 1 hour. After centrifugation, approximately 80–90% of the solution was transferred from the upper chamber to the lower chamber. Larger proteins (>3 kDa) were retained in the upper chamber, while small molecules (<3 kDa) were collected in the lower chamber. The concentrated MCM ( > 3 kDa) from the upper chamber was diluted to its original volume with fresh serum-free RPMI 1640 medium, while 10% FBS was added to the MCM ( < 3 kDa) collected in the lower chamber. Both fractions were then used for subsequent experiments as required.
Co-culture of BMDMs and T Cells
BMDMs were pre-stimulated with TCM (50% v/v) for 24 hours. For antigen-specific assays, BMDMs were pulsed with 1 µg/mL SIINFEKL peptide (MCE, HY-P1489) for 2 hours. CD8⁺ T cells were isolated from spleens of OT-1 or C57BL/6 mice using the human CD8+ T Cell Isolation Kit (Negative Isolation) (Miltenyi, Cat. No. 130-104-075). BMDMs and T cells were co-cultured at a 1:5 ratio in RPMI-1640 with 10% FBS. For polyclonal T cell activation, plates were pre-coated with Dynabeads™ mouse T activator CD3/CD28 for T cell activation/expansion kit (Thermo Fisher Scientific, Cat. No. 11452D) according to the instructions provided by the manufacturer. After 48–72 h, cells were harvested for flow cytometric analysis of activation/exhaustion markers and cytokines.
For BMDM and OT-1 CD8+ T cell co-culture, BMDMs from OT-1 mice (8 weeks) were isolated and cultured according to standard methods, and the stimulation reached No. At 5 days, BMDMs were stimulated with OVA peptide (257-264) (SIINFEKL, 1 μg/mL, MCE, HY-P1489) added to TCM for 24 h to induce antigen presentation. After stimulation, medium was replaced. BMDMs were co-cultured with OT-1 mouse-derived CD8+ T cells at a ratio of 1:5 (BMDM:CD8+ T cells) for 24 h. After co-culture, cells were harvested and subjected to surface staining. First, CD8+ T cells were labeled with anti-CD8α antibodies. Then, Tetramer staining was performed using PE-H-2Kb/OVA (SIINFEKL) MHC Tetramer (Creative Biolabs) following the manufacturer’s protocol. For BMDM, Anti-Mouse H-2Kb/SIINFEKL antibody (BD Pharmingen™, 569791) and other indicator antibodies were added. Staining reactions were performed for 30 min on ice. After staining, cells were washed and resuspended in flow cytometry buffer and tested by flow cytometry.
Organoid Construction and In Vitro PBMC Co-culture
Human PDAC tissues were acquired from patients undergoing surgery for pancreatic cancer who consented to donate their samples for research. Briefly, fresh PDAC tissues were collected during surgery operation and immediately washed three times with PBS to remove blood clots, dead tissues and other connective tissues. PDAC tissues were minced and digested with a mix of 5 mL enzymes buffer containing 1 mg/mL collagenase (Sigma-Aldrich, Cat.No.C2799), 2.5 U/mL hyaluronidase (Sigma-Aldrich, Cat.No.H3506) and 0.1 mg/mL DNase (Sigma-Aldrich, Cat. No. DN25) in 37 °C water bath for 15 min. The mixture was then filtered through a 40 μm strainer to obtain single cell suspension. The single cells were embedded in Growth Factor reduced (GFR) Matrigel (Corning, Cat.No.356231) and cultured in complete medium containing Advanced DMEM/F-12 (Gibco, Cat.No.12634-010), 10 mM HEPES pH=7.2–7.5 (Solarbio, Cat.No.H1080), 1 × GlutaMAX Supplement (Invitrogen, Cat.No.35050061), 100 µg/mL Primocin (Invivogen, Cat.No. ant-pm-2), 10 mM Nicotinamide (Sigma-Aldrich, Cat.No.N0636), 1 × Wnt3a-Conditioned Medium, 1 × R-spondin1-Conditioned Medium, 100 ng/mL mNoggin (R&D, Cat.No. 6997-NG-025), 1 × B27 Supplement (Thermo Fisher Scientific, Cat.No.17504044), 100 ng/mL hFGF-10 (Peprotech, Cat.No.100-26), 1.25 mM N-acetylcysteine (Sigma-Aldrich, Cat.No.A9165), 1 µM Prostaglandin E2 (R&D, Cat.No.2296/10), 50 ng/mL hEGF (Peprotech, Cat.No.AF-100-15), 10 µM Rho Kinase inhibitor Y27632 (Sigma-Aldrich), 10 nM hGastrin Ⅰ (Sigma-Aldrich, Cat.No.G9145) and 500 nM A 83-01(R&D, Cat.No.2939-10). PDAC organoids (3 × 103 cells/well) were seeded in 96-well plates. The medium was changed approximately every 3 days and organoids were passaged approximately every 7 days according to their growth conditions.
PBMCs were isolated from the peripheral blood of healthy donor. PBMCs were co-cultured with organoids at a ratio of 5:1 (PBMCs: organoid). CD8+ T cells were enriched from PBMCs using magnetic bead-based isolation to ensure sufficient immune cells in the co-culture system. To activate T cells, Dynabeads Human T-Activator (Gibco, Cat.No.11163) was added to the medium. During co-culture, IL-2 (20 IU/mL, PeproTech, Cat.No.200-02) was added as a stimulant, along with drugs and other reagents. After 36–48 hours of co-culture, organoid morphology, proliferation, and immune responses of the cells were evaluated by immunohistochemistry and flow cytometry. Apoptosis levels were assessed using the caspase 3/7 fluorescence probe (Beyotime, Cat.No.C1073S), and images were captured using both optical and fluorescence microscopy for statistical analysis.
Multi-Immunofluorescence (mIF) Staining of Paraffin and Frozen Sections
Formalin-fixed, paraffin-embedded (FFPE) tissue sections (4 µm) were deparaffinized, rehydrated, and subjected to antigen retrieval in EDTA buffer (pH 9.0) using a pressure cooker. Autofluorescence was quenched with TrueVIEW Autofluorescence Quenching Kit (Vector Labs, SP-8400). Sections were blocked and incubated with primary antibodies overnight at 4 °C (antibody details in Supplementary Table S2). Opal™ 7-Color Manual IHC Kit (Akoya Biosciences, NEL811001KT) was used for tyramide signal amplification with fluorophores Opal 520, 570, 620, 690, and 780. Nuclei were counterstained with DAPI. Slides were scanned using a Vectra Polaris imaging system (Akoya Biosciences) at 20x magnification. Image analysis and cell segmentation were performed using inForm® software (Akoya Biosciences) and QuPath.
Enzyme-Linked Immunosorbent Assay (ELISA)
CXCL16 levels in cell culture supernatants were measured using the Mouse CXCL16 DuoSet ELISA (Biotechwell, Cat. No. EM30142) according to the manufacturer’s instructions. Absorbance was read at 450 nm with correction at 570 nm on a BioTek Synergy MX plate reader.
pHrodo Red Dye and GFP Uptake Assay for Macrophage Phagocytosis Activity
Prior to the experiment, the pHrodo Red AM Ester (Thermo Fisher Scientific, Cat. No. P36600) was equilibrated to room temperature. The working staining solution was prepared as follows: 10 μL of pHrodo Red AM Ester is added to 100 μL of PowerLoad concentrate (provided with the kit), mixed thoroughly, and then diluted with 10 mL of PBS. On day 5 of culture, BMDMs from different mouse strains were treated with TCM and co-cultured with chemotherapy-induced apoptotic tumor cells for 12 h. After removal of apoptotic cells and culture medium, the cells were washed once with PBS. The pHrodo Red AM Ester staining solution is then added, and the cells are incubated at 37 °C with 5% CO₂ for 30 minutes. After incubation, cells are washed with PBS to remove any unbound dye. Stained cells are observed for phagocytosis activity using an integrated fluorescence microscopy imaging system (Ex/Em: 560/585 nm), or the fluorescence signal is quantitatively measured by flow cytometry.
BMDMs were co-cultured with apoptotic KPC-GFP cells (effector: target = 1:5) for 4 hours. After extensive washing to remove non-ingested cells, macrophages were analyzed by flow cytometry for GFP signal and by immunofluorescence microscopy to visualize internalized GFP⁺ material.
Immunofluorescence
Cell coverslips were first immersed in 75% ethanol and washed with PBS, then placed into a 24-well plate, with approximately 2 × 10⁴ cells seeded per well. Cells were cultured for 24 hours. The following day, cells were washed with PBS 5–10 minutes × 3, fixed with 4% paraformaldehyde for 10 minutes, and washed again with PBS 5–10 min × 3. If permeabilization is required, cells were treated with 0.1% Triton X-100 (Solarbio, Cat. No. T8200) for 10 minutes, followed by PBS washing 5–10 min × 3. Then, cells were blocked with 3% BSA for 1 hour at room temperature and washed once with PBS for 10 minutes. Primary antibodies were incubated overnight at 4 °C, protected from light, at a dilution of 1:200 in a humidified chamber. On the third day, cells were brought to room temperature for 1 hour, washed with PBS 5–10 minutes × 3, and then incubated with secondary antibody (1:400, 30 minutes, Thermo fisher scientific, A-11008/ A-11001/ A-21070) protected from light. Cells are washed with PBS 10 minutes × 3. Finally, 20 μL of mounting medium containing DAPI was added to each coverslip. The slides were allowed to dry in the dark before being stored at 4 °C. Images were captured using a fluorescence microscope.
Small interfering RNA (siRNA) transient transfection
After hMDM or BMDM was stimulated with h-mcsf or m-CSF (20 ng/mL) until day 5, siRNA transient transfection was performed. First, siRNA (Shanghai Sangon Inc, see in Table S3) was mixed with Advanced DNA RNA Transfection Reagent (zeta-life, Cat.No.AD600150) in a 1:1 ratio and incubated for 20 minutes at room temperature. The mixture was then added to the macrophage culture medium at a 1:200 ratio (transfection reagent to medium). Transfection was terminated by changing the medium 24 hours after transfection. The transfected cells were used for subsequent experiments.
Cellular RNA extraction and Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from cultured cells using TRIzol reagent (Epizyme, Cat. No. YY101) according to the manufacturer’s instructions. Briefly, cells were collected from culture dishes and gently washed with PBS to remove residual medium. The cells were then lysed in TRIzol reagent to ensure complete disruption and homogenization, with RNA stabilizers included in the lysate to prevent degradation. Chloroform was added to the lysate, followed by thorough mixing and centrifugation at 12,000 × g for 15 minutes at 4 °C to achieve phase separation. The aqueous phase containing RNA was carefully transferred to a fresh RNase-free tube, and an equal volume of isopropanol was added to facilitate RNA precipitation. After incubation at 4 °C for 10 minutes, the RNA was pelleted by centrifugation at 12,000 × g for 10 minutes at 4 °C. The resulting pellet was washed with 75% ethanol, centrifuged, and air-dried before being dissolved in RNase-free water. The concentration and purity of the extracted RNA were assessed using a spectrophotometer.
For cDNA synthesis, a StarScript III All-in-one RT Mix with gDNA Remover (GenStar, Cat.No.A240) was used according to the manufacturer’s protocol. A defined amount of total RNA was reverse-transcribed into cDNA, and inactivation was performed at the end of the reaction. qRT-PCR was performed using a one-step qRT-PCR detection kit (Genstar, Cat. No. A301-10) in a reaction system containing the cDNA template, SYBR Green fluorescent dye, specific primers (see in Supplementary table 2), and qPCR Master Mix. The PCR program consisted of an initial denaturation at 95 °C for 5 minutes, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. The relative expression levels of target genes were analyzed using the ΔΔCt method, with β-actin serving as an internal reference.
Statistical Analysis
Data from at least three independent samples or biological replicates are presented as mean ± standard deviation. Two-tailed Student’s t test was used to compare the data between the two groups. One-way analysis of variance (ANOVA) followed by post-hoc Tukey’s or Dunnett’s tests, or the Kruskal-Wallis H test was used to test P-values for data comparison between more than two groups. For Kaplan-Meier survival analysis, P-values were calculated using the Log-rank (Mantel-Cox) test and multivariate COX regression was used to analyze the influence of various clinical factors to patients’ survival. Use the β test (β regression) to analyze the correlation between the cell proportions in scRNA sequencing data and the clinical treatment effect. Sequencing data were compared relative gene expression using a two-sided Wilcoxon test. *P < 0.05, **P < 0.01, ***P < 0.001.
