Tumour tissues were obtained from baseline biopsies of patients participating in clinical trials for localized prostate cancer (NCT03821246), de novo oligometastatic prostate cancer (NCT03007732) and metastatic mCRPC (NCT03248570). Viable cryopreserved tumour tissue samples were digested in Roswell Park Memorial Institute (RPMI) medium containing Collagenase I and II (0.1 mg ml−1, Thermo Fisher Scientific) and DNase I (Thermo Fisher Scientific), minced and then subjected to 1 h digestion using the gentleMACS system (Miltenyi Biotec). Live cell isolation was done using MACS LS columns (Miltenyi Biotec). The 10x Genomics Chromium Controller was used to generate GEM bead emulsions using the Single Cell 5′ Library & Gel Bead Kit (10x Genomics), followed by cDNA synthesis and amplification, and subsequent library preparation steps using 10x Genomics kits. Library sequencing was done by the University of California, San Francisco (UCSF) Institute for Human Genetics core on a NovaSeq 6000 (Illumina), targeting a median read depth of 150,000 reads per cell for total gene expression libraries and 60,000 reads per cell for CITE-seq libraries. All antibodies were obtained from BioLegend unless otherwise indicated. This work was done with informed consent obtained from all human research participants, and the sample procurement and analysis were approved by the institutional review board committees at UCSF.
The raw data from 10× sequencing were processed using the Cell Ranger pipeline (v.3, Genome build, GRCh38). The raw gene-expression matrices were subjected to processing by CellBender (v.0.1.0)61 to eliminate ambient RNAs. The filtered gene-expression matrices then underwent doublet detection using the package DoubletDetection (https://doi.org/10.5281/zenodo.2678041) with default parameters. The results were analysed through the SCANPY pipeline62. To ensure the retention of high-quality cells, the following filters were applied: first, cells with less than 10% mitochondrial genes were retained; second, the number of detected genes per cell was set between 100 and 2,500 genes; third, genes expressed in at least three cells were kept; and finally, platelets (PF4, unique molecular identifier (UMI) > 0), red blood cells (HBB, UMI > 1) and doublets were removed. The gene-expression matrix was log2-transformed with the addition of 1 and normalized to 10,000 counts per cell, followed by highly variable gene selection using default parameters with the SCANPY function. The resulting matrix was corrected by regressing out total UMI counts and mitochondria percentage, followed by scaling to a mean of 0 and a variance of 1. Principal component analysis was performed using the top 50 principal components, followed by sample-wise batch correction using the SCANPY-implemented Harmony63. Leiden clustering (default resolution = 1.0) and UMAP plotting were performed, with a resolution of 1.0 applied for both T cell and myeloid cell clustering. Differential expression analysis identified the top-ranked genes that were upregulated in each individual cluster relative to the combination of all other cells, as determined by the SCANPY function tl.rank_genes_groups. Annotation of each unbiased population was achieved through manual inspection of the top-ranked genes of each cluster. Analysis of cell density on the UMAP was carried out using the SCANPY function tl.embedding_density, and boxplots were generated to represent cell population frequencies for each cell type. Gene scores were computed using the SCANPY function tl.score_genes with curated gene lists provided. To calculate gene scores at the sample level, scores were computed for each cell and subsequently combined at the sample level by using the median score of cells within a given sample.
FVB/NJ and C57BL/6J male mice (from the Jackson Laboratory) were used in the experiments at 6–10 weeks of age. The STOCK Tg(Spp1-EGFP)PD43Gsat/Mmucd (Spp1-EGFP)64 mouse strain was sourced from the Mutant Mouse Resource & Research Centers at the University of California, Davis. All mice were housed in a pathogen-free facility under standardized environmental conditions, including a controlled 12 h:12 h light:dark cycle, humidity of 30–70% and a temperature range of 20–26 °C. For experiments, a total of 1 × 106 cells (murine prostate cancer cell line MyC-CaP (CRL3255, American Type Culture Collection (ATCC)) or TRAMP-C2 (CRL-2731, ATCC)) were resuspended in sterile PBS and transplanted subcutaneously in the right flank of either FVB/NJ or C57BL/6J mice, respectively. The identities of MyC-CaP and TRAMP-C2 were authenticated using the Mouse Cell STR Profiling Service (137-XV, ATCC), and mycoplasma contamination was tested before each injection using a mycoplasma PCR detection kit (G238, abm). Sample size was determined using preliminary data and previous publications to ensure reproducibility of the experiment. Tumour volume = (L × W × W)/2 (mm3), with length (L) and width (W) being the longest diameter and shortest diameter, respectively. All experimental procedures were approved by the Institutional Animal Care and Use Committee at UCSF.
The model for CRPC was established by subcutaneously engrafting 1 × 106 MyC-CaP cells into the right flank of male FVB/NJ mice 6–10 weeks old. When the tumour size reached 100–200 mm3, each mouse was injected subcutaneously with 1.875 mg degarelix (Firmagon) in 100 μl PBS, followed by a maintenance dose of 0.625 mg degarelix in 100 μl PBS every 28–30 days to induce CRPC. The development of CRPC was defined as a tumour volume that regressed after degarelix treatment and then grew back to 100–200 mm3. Subsequently, the mice were randomized and treated with the indicated antibodies and/or inhibitors.
MyC-CaP and TRAMP-C2 cells were cultured in complete DMEM medium comprising Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS, Omega Scientific) and 1× penicillin/streptomycin (10,000 ml streptomycin sulfate and 10,000 units ml−1 penicillin G). All reagents were obtained from the UCSF Cell Culture Facility, unless otherwise indicated.
Mouse organs were collected and processed as follows. Spleens were mechanically dissociated with FACS wash buffer (PBS supplemented with 2% (v/v) FBS and 0.5 mM EDTA (Teknova)). Tumours were sequentially digested three times with 12 ml of a cocktail of 2 mg ml−1 (w/v) collagenase type IV and 100 Kunitz units per ml DNase I (both from Sigma-Aldrich) for 12 min for each digest. All single-cell suspensions were filtered using 70-μm filters (Fisher Scientific) and subjected to red-blood-cell lysis using ACK Lysing Buffer (Quality Biological). Cells were immunostained by incubating at 4 °C for 30 min with the fluorescently labelled antibodies below (all antibodies were purchased from BioLegend unless otherwise indicated). After staining, cells were washed once or twice in FWB and resuspended in FWB or FWB containing 1 μg ml−1 propidium iodide (PI, BioLegend) to assess viability. All flow cytometric data were acquired using an LSRFortessa X-50 flow cytometer operated with FACSDiva software (BD Biosciences). Post-acquisition data analysis was performed using FlowJo (v.10.10.0, Tree Star). All antibodies used in this study are commercially available and have been validated by the manufacturer or through published literature. On receipt, laboratory testing was conducted with known positive and negative controls to confirm the reliability of each antibody.
For mouse lymphoid staining, we used anti-mouse CD3-Brilliant Ultraviolet 395 (563565, Clone 145-2C11, BD Biosciences, 1:200), CD4-Brilliant Violet 711 (100447, GK1.5, 1:200), CD8-Brilliant Ultraviolet 805 (612898, 53-6.7, BD Biosciences, 1:200), NK-1.1-Alexa Fluor 647 (108719, PK136, 1:200), CD38-PE/Cyanine7 (102717, 90, 1:200), CD39-Brilliant Violet 421 (567105, Y23-1185, BD Biosciences, 1:200), CD45-Brilliant Violet 785 (103149, 30-F11, 1:200), CD279 (PD-1)-PE/Dazzle 594(109115, RMP1-30, 1:200) antibodies were used. For mouse myeloid staining, anti-mouse CD11b-Brilliant Violet 605 (101257, M1/70, 1:200), CD39-Brilliant Violet 421 (567105, Y23-1185, BD Biosciences, 1:200), CD73-PE (12-0731-82, eBioTY/11.8 (TY/11.8), Invitrogen, 1:200), CX3CR1-PE/Cyanine7 (149015, SA011F11, 1:200), F4/80-Alexa Fluor 647 (565853, T45-2342, BD Biosciences, 1:200), I-A/I-E-Alexa Fluor 700 (107621, M5/114.15.2, 1:200), Ly-6G-APC/Cyanine7 (127623, 1A8, 1:200), Podoplanin-PerCP/Cyanine5.5 (127421, 8.1.1, 1:200), Siglec-F-Brilliant Violet 421 or Brilliant Ultraviolet 395 (562681 or 740280, E50-2440, BD Biosciences, 1:200) antibodies. The relevant isotype-matched antibodies (eBRG1, RTK2758, RTK4530 and SHG-1) were used as controls.
For intracellular immunostaining of proteins, single-cell suspensions were labelled with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (L34957, Invitrogen, 1:1,000) and then treated with eBioscience Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), according to the manufacturer’s protocol designed for intracellular (cytoplasmic) proteins. Cells were then stained with fluorescently labelled antibodies against anti-mouse CD3; Brilliant Ultraviolet 395 (563565, 145-2C11, BD Biosciences, 1:200), CD8-Brilliant Ultraviolet 805 (612898, 53-6.7, BD Biosciences, 1:200), CD11b-Brilliant Violet 605 (101257, M1/70, 1:200), CD45-Brilliant Violet 785 (103149, 30-F11, 1:200), IFN-γ-PE/Cy7 (505825, XMG1.2, 1:100), and TNF-α-Brilliant Violet 421 (506327, MP6-XT22, 1:100). The relevant isotype-matched antibodies (RTK2071) were used as negative controls.
Complete RPMI cell culture medium was made up of RPMI 1640 supplemented with 10% FBS (Omega Scientific), 1× β-mercaptoethanol (Gibco, 55 µM), 1× glutamine (29.2 g l−1 l-glutamine, 200 mM), 1 mM sodium pyruvate (11 g l−1 sodium pyruvate), 1× MEM non-essential amino acids, 1× penicillin/streptomycin (10,000 μg ml−1 streptomycin sulfate and 10,000 units ml−1 penicillin G). All reagents were obtained from the UCSF Cell Culture Facility, unless otherwise indicated.
For enrichment of mouse CD8+ T cells, single-cell suspensions of spleens from CRPC-bearing mice were labelled with BD Violet proliferation dye 450 (Fisher Scientific) and subsequently negatively enriched using the MojoSort Mouse CD8 T cell isolation kit, according to the manufacturer’s instructions. For isolation of specific myeloid subsets, single-cell suspensions from CRPC developed in Spp1-EGFP mice were incubated with the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (L34957, Invitrogen, 1:1,000), anti-mouse CD11b-Brilliant Violet 605 (101257, M1/70, 1:200), CX3CR1-PE/Cyanine7 (149015, SA011F11, 1:200), F4/80-Alexa Fluor 647 (565853, T45-2342, BD Biosciences, 1:200), I-A/I-E-Alexa Fluor 700 (107621, M5/114.15.2, 1:200), Ly-6G-APC/Cyanine7 (127623, 1A8, 1:200), Podoplanin-PerCP/Cyanine5.5 (127421, 8.1.1, 1:200) and Siglec-F-Brilliant Violet 421 (562681, BD Biosciences, 1:200) antibodies. After immunostaining, cells were washed twice in FWB and resuspended in FWB containing 1 μg ml−1 propidium iodide to assess viability. The cells of interest were FACS-purified using BD FACSAria Fusion operated with FACSDiva software (BD Biosciences).
To determine whether TAM cells, including Spp1hi-TAMs, CX3CR1hi-TAMs and CD163hi-TAMs, mediate immunosuppression, 1 × 104 CD8+ T cells, labelled with BD Violet Proliferation Dye 450 (BDB562158, Fisher Scientific, 1:1,000) and stimulated with 1 × 104 Dynabeads Mouse T-Activator CD3/CD28 (Gibco) were cultured in the presence or absence of purified myeloid subsets at a 1:1, 5:1 or 10:1 ratio, respectively, in 200 µl complete RMPI medium in round (U)-bottom 96-well plates at 37 °C, 5% CO2 for 3 days. T cell proliferation was assessed by flow cytometry.
To determine whether Spp1hi-TAMs suppress T cell activation, 1 × 104 CD8+ T cells, labelled with BD Violet Proliferation Dye 450 (Fisher Scientific) and activated with 1 × 104 Dynabeads Mouse T-Activator CD3/CD28 (Gibco), were cultured with or without purified Spp1hi-TAMs at a 1:1 ratio in 200 µl complete RMPI medium in round (U)-bottom 96-well plates at 37 °C, 5% CO2 for 3 days. The cells were subsequently restimulated with 1× eBioscience Cell Stimulation Cocktail (plus protein transport inhibitors, Invitrogen) for 5 h. After washing, cells were stained for intracellular immunostaining of proteins. T cell activation was assessed by flow cytometry.
To determine whether Spp1hi-TAM-mediated T cell suppression requires adenosine signalling, 1 × 104 CD8+ T cells, labelled with BD Violet Proliferation Dye 450 (BDB562158, Fisher Scientific, 1:1,000) and activated with 1 × 104 Dynabeads Mouse T-Activator CD3/CD28 (Gibco), were cultured with or without purified Spp1hi-TAMs at a 1:1 ratio in 200 µl complete RMPI medium in the presence of ciforadenant (10 μM, Corvus Pharmaceuticals) or InVivoMAb anti-mouse CD73 (10 μg ml−1; TY/23, BioXCell) in round (U)-bottom 96-well plates at 37 °C, 5% CO2 for 3 days. T cell proliferation was assessed by flow cytometry and compared with cells treated with the equivalent amount of DMSO or isotype-matched control antibodies (2A3, BioXCell).
To determine whether IL-1R signalling is involved in Spp1hi-TAM-mediated T cell suppression, 1 × 104 CD8+ T cells, labelled with BD Violet Proliferation Dye 450 (BDB562158, Fisher Scientific, 1:1,000) and activated with 1 × 104 Dynabeads Mouse T-Activator CD3/CD28 (Gibco), were cultured with or without purified Spp1hi-TAMs at a 1:1 ratio in 200 µl complete RMPI medium in the presence of either purified in vivo GOLD functional grade anti-mouse IL-1R (10 μg ml−1, JAMA-147) or the relevant isotype-matched control antibody (PIP, both from Leinco Technologies) in round (U)-bottom 96-well plates at 37 °C, 5% CO2 for 3 days. T cell proliferation was assessed by flow cytometry.
Spp1hi-TAMs and MDSCs (both 1 × 105 cells) were isolated from the same CRPC and plated in 250 µl complete RMPI medium in flat-bottom 48-well plates at 37 °C, 5% CO2. After 24 h, supernatants were collected and adenosine levels were measured using an adenosine assay kit (Fluoreometric, ab211094, Abcam) following the manufacturer’s protocol. Fluorescence was measured using a GluoMax plate reader (Promega; Ex/Em = 535/587 nm), and the concentration of accumulated extracellular adenosine was calculated by subtracting the baseline adenosine levels from medium without cells.
Transwell assays were performed as previously reported65. In brief, FACS-isolated Spp1hi-TAMs or enriched CD8+ T cells labelled with BD Violet Proliferation Dye 450 (BDB562158, Fisher Scientific, 1:1,000), from CRPC developed in mice or their spleens respectively, were plated into the top or bottom chambers of Transwell plates containing 6.5 mm cell culture inserts with 0.4 µm pore polyester membrane (CLS3379, Corning), as depicted in Extended Data Fig. 6e. In the top chamber of the inserts, 1 × 104 CD8+ T cells alone or those stimulated with 1 × 104 Dynabeads Mouse T-Activator CD3/CD28 (Gibco) were plated, and in the bottom chamber, 6 × 104 Spp1hi-TAMs or 6 × 104 CD8+ T cells with or without anti-CD3/28 stimulation at a 1:1 ratio were plated, each with 100 µl or 600 µl complete RMPI medium, respectively. After 3 days of culture, T cell proliferation in each chamber was assessed by flow cytometry.
To determine whether Spp1hi-TAMs are resistant to CSF1R blockade, mice were randomly divided into two groups when they developed CRPC (tumour volume of 100–200 mm3) and were administered intraperitoneally 1 mg anti-mouse CSF1R (AFS98, BioXCell) or the respective isotype-matched control (2A3, BioXCell) antibodies in 200 μl PBS. A maintenance dose of 0.5 mg in 200 μl PBS was given after 5 days. The myeloid composition was analysed by flow cytometry 2 days after the final injection.
For immune checkpoint inhibition, mice with established CRPC (tumour volume of 100–200 mm3) were randomly divided into four groups and subjected to intraperitoneal injection with these antibodies in 200 μl PBS every 3 days for a total of three injections: 200 μg anti-mouse CTLA-4 (24H2)66 alone; 400 μg anti-mouse PD-1 (17D2)67 alone; a combination of anti-mouse CTLA-4 and PD-1; or the respective IgG2a, κ isotype-matched control. Tumour burden was measured every 2–3 days after the initial injection until it reached 750 mm3, unless otherwise indicated.
To determine whether Spp1hi-TAMs contribute to resistance to ICIs in vivo, mice with developed CRPC (tumour volume of 100–200 mm3) were randomly divided into three groups. They were administered with: a combination of anti-mouse CTLA-4 and PD-1 in 200 μl PBS injected intraperitoneally along with intratumoral injection of 1 × 105 Spp1hi-TAMs purified from digested CRPC (more than 350 mm3) of a mouse from the same cohort in 50 μl PBS; a combination of anti-mouse CTLA-4 and PD-1 in 200 μl PBS injected intraperitoneally along with 50 μl of PBS intratumorally; or the respective isotype-matched control antibody in 200 μl PBS injected intraperitoneally along with 50 μl PBS intratumorally. Antibodies were administered every 3 days for a total of three injections, and Spp1hi-TAMs were adoptively transferred every 5 days for a total of two injections. Tumour growth was measured every 2–3 days after the initial injection until it reached 750 mm3, unless otherwise indicated. The lymphoid composition was analysed by flow cytometry one day after the final injection.
For blockade of adenosine receptors (A2ARs), mice with established CRPC (tumour volume, 100–200 mm3) were randomly divided into two groups. Ciforadenant (10 mg per kg, Corvus Pharmaceuticals) or DMSO vehicle control (Sigma-Aldrich) in 200 μl of injection solution was administered once daily through oral gavage for 10 consecutive days. The injection solution consisted of 10% ciforadenant (or DMSO medium) and 90% corn oil (MedchemExpress). Tumour growth was measured every 2–3 days after the initial injection.
To determine whether A2AR blockade enhances immunotherapy efficacy, mice with established CRPC (tumour volume, 100–200 mm3) were randomly divided into two groups. Ciforadenant (10 mg per kg, Corvus Pharmaceuticals) or DMSO vehicle control (Sigma-Aldrich) in 200 μl of injection solution described above was administered once daily by oral gavage for 10 consecutive days. Simultaneously, mice were injected intraperitoneally with 400 µg anti-mouse PD-1 or the respective isotype-matched control antibodies in 200 µl PBS every 3 days for a total of three injections. Tumour growth was monitored every 2–3 days after the initial injection. The lymphoid and myeloid compositions were analysed by flow cytometry 1–2 h after the eighth injection of ciforadenant (1 day after the final anti-mouse PD-1 antibody injection).
All comparisons within experiments were carried out using age-matched mice (6–10 weeks old) engrafted with the same stock of MyC-CaP throughout the study.
For the single-cell assessment of MyC-CaP, a cohort of FVB/NJ mice bearing MyC-CaP were injected subcutaneously with either degarelix (n = 3) or PBS (n = 3), as described above. HSPC or CRPC tissues were collected on reaching a tumour volume of more than 350 mm3. Tumours were processed and single-cell suspensions were prepared as described above. For the cell-surface protein staining, cells were incubated with CD45.1-Brilliant Violet 510 (A20) for 30 min at 4 °C. After immunostaining, cells were washed twice in FWB and resuspended in FWB containing 1 μg ml−1 propidium iodide for viability assessment. Cells were then sorted into CD45+ and CD45− populations using FACSAria (BD Biosciences). Each population was transferred into separate 75 mm flow-cytometry tubes, centrifuged for 5 min at 250g at 4 °C and the supernatant was discarded. Cells were then resuspended in 100 μl Fc blocking buffer, consisting of 95 μl FWB + 5 μl of 0.5 mg ml−1 anti-mouse CD16/32 antibody (2.4G2, Cytek Biosciences), followed by a 10-minute incubation at 4 °C. CD45+ cells were subsequently stained directly with 2 μl of 0.05 mg ml−1 TotalSeq-C hashtag antibodies 2, 4 and 6 (M1/42, 30-F11) without washing, for 40 min at 4 °C. All sorted populations from each tissue were then pooled to yield a total of 1 × 106 cells and these cells were stained with 100 μl of a cocktail of TotalSeq-C surface antibodies (CD11c (N418), CD163 (S15049I), F4/80 (BM8) and Ly-6G (1A8), each at a concentration of 2.5 μg ml−1) for 30 min at 4 °C. After staining, cells were washed with 1 ml complete RPMI medium and filtered through a 70 µm cell strainer. Cell viability and counting were reassessed and the volumes were adjusted for 10x chromium chip input at a concentration of 1.29 × 106 cells per ml. For scRNA-seq of TRAMP-C2, single-cell suspensions were initially labelled with LIVE/DEAD Fixable Dead Cell Stain Kit (Near-IR; Invitrogen) for 10 min at 4 °C. Subsequently, cells were stained with anti-mouse CD16/32 antibody (2.4G2) and CD45-Brilliant Violet 570 (30-F11) antibodies for 30 min on ice. After immunostaining, cells were washed with FWB and sorted into CD45+ and CD45− populations using a FACSAria2 (BD Biosciences). Each sorted population was transferred into separate 75 mm flow-cytometry tubes, centrifuged for 5 min at 250g at 4 °C and the supernatant was discarded. Cells were then resuspended in 100 μl Fc blocking buffer as above, followed by a 10-minute incubation at 4 °C. Cells were then stained directly with 2 μl of 0.05 mg ml−1 TotalSeq-C hashtag antibodies 1 and 2 (M1/42, 30-F11) for 40 min at 4 °C without washing. Equal proportions of cells labelled with hashtags were pooled together, and three individual reactions, each containing a total of 6 × 104 cells, were washed with 1 ml complete RPMI medium and filtered through a 70 µm cell strainer. After reassessing cell viability and counting, cell concentrations were adjusted to 1.29 × 106 cells per ml for loading into the 10x chromium chip. A 10x Genomics chromium controller was used to create GEM bead emulsions using a Single Cell 5′ Library & Gel Bead Kit (10x Genomics), followed by synthesis and amplification of cDNA and subsequent library preparation steps using 10x Genomics kits. The UCSF Institute for Human Genetics core performed library sequencing on a NovaSeq 6000 (Illumina), targeting a median read depth of 150,000 reads per cell for total gene expression libraries and 60,000 reads per cell for CITE-seq libraries. All antibodies were obtained from BioLegend, unless otherwise indicated.
The raw data obtained from 10× sequencing were processed through the Cell Ranger pipeline (v.5, Genome build GRCm38). Subsequent steps in the analysis were the same as those used in human scRNA-seq until batch correction using Harmony, followed by Leiden clustering (resolution = 1.0) and UMAP plotting. A resolution of 1.0 was applied for myeloid-cell clustering. Differential expression analysis was done to identify the top-ranked genes upregulated in each individual cluster compared with the combination of all other cells. This analysis was done using the SCANPY function tl.rank_genes_groups. Annotation of each population was established through manual examination of the top-ranked genes in each cluster. To analyse cell density on the UMAP, the SCANPY function tl.embedding_density was used. Box plots were generated to visually represent cell population frequencies for each cell type. Gene scores were computed using the SCANPY function tl.score_genes with curated gene lists provided. Gene scores were computed using the SCANPY function tl.score_genes for each cell, using curated gene lists. To calculate similarity scores between myeloid subsets in humans and mice, a pseudo-bulk analysis was done to aggregate gene-expression data from the cellular level to the sample level. Z-scores were computed for each gene on the basis of cells in a given sample, and the mean was determined as the representative value for the sample. We then identified the shared genes in both human and mouse datasets, focusing on the top 50 genes within each subset.
For immunostaining of SPP1hi-TAMs and CD4+ or CD8+ T cells in human tissues, in situ hybridization was done using RNAscope (Advanced Cell Diagnostics, ACDBio) on FFPE sections 4 μm thick from patients with either HSPC or mCRPC (NCT03007732, NCT03248570 and NCT02655822). Tissues were pretreated with target retrieval reagents and protease to improve target recovery according to the RNAscope Multiplex Fluorescent Reagent Kit v.2 assay protocol (323100, ACD Bio). Probes for human SPP1 and CD68 mRNA (420101-C2 and 560591-C4, respectively; ACDBio) were applied at a 1:50 dilution for 2 h at 40 °C. The probes were then hybridized with Opal 7-Color Manual IHC Kit (NEL811001KT, PerkinElmer) for the detection of SPP1 and CD68 transcripts using Opal 650 and Opal 690, respectively, at a dilution of 1:700. Immunofluorescence staining for human CD4 (MA-12259, 4B12, Invitrogen) and CD8 (ab60076, YTC182.20, abcam) was then done at a 1:100 dilution each. Targets were detected using Alexa Fluor 488-conjugated donkey anti-mouse IgG secondary antibody (ab150105, abcam) at a 1:100 dilution and Alexa Fluor 555-conjugated goat anti-rabbit IgG secondary antibody (4050-32, Southern Biotech) at a 1:100 dilution. Tissues were counterstained with 4′,6diamidino-2-phenylindole (DAPI, ACD Bio) and mounted with ProLong Gold Antifade Mountant (P36930, Invitrogen). Slides were imaged at 63× magnification using a Leica SP8 X white-light laser confocal microscope (Leica Microsystems) with multiple regions of interest from each specimen slide randomly selected for analysis. No staining was observed using negative control probes specific for the bacterial dapB gene (321831, ACD Bio) counterstained with Opal dyes, or with secondary antibodies alone on tonsil tissue.
Immunostaining of PD-L1 expression on EpCAM+ cells and CD68+ cells in human tissues was done on FFPE tissue sections 4 µm thick from responders and non-responders in clinical trial NCT02655822. This staining was done using a Ventana DISCOVERY ULTRA automated slide stainer and Ventana DISCOVERY ULTRA reagents (Roche Diagnostics), according to the manufacturer’s instructions (UCSF Protocol 3612), unless otherwise indicated. After deparaffinization, antigen retrieval was performed with cell conditioning 1 solution for 64 min at 97 °C. Primary antibodies for human CD68 (PG-M1; Agilent), PD-L1 and EpCAM (E1L3N and D9S3P, respectively, Cell Signaling Technology) were applied at 1:200, 1:100 and 1:50 dilutions for 32 min, respectively, at 36 °C. Goat Ig Block Ventana (760-6008) was applied for 4 min before the secondary antibodies (OmniMap anti-Ms for the anti-CD68 antibody and OmniMap anti-Rb for the anti-PD-L1 and anti-EpCAM antibodies) were incubated for 12 min. A stripping step between each primary was done with cell conditioning 2 solution at 97 °C for 8 min between primary antibodies. Endogenous peroxidase was inhibited using DISCOVERY Inhibitor RUO Ventana (760-4840) for 12 min. The CD68 was visualized using DISCOVERY Rhodamine 6G Kit Ventana (760-244), PD-L1 was visualized with DISCOVERY Cy5 Kit (760-238) and EpCAM was visualized with DISCOVERY FAM Kit (RUO) (760-243) for 8 min each. Finally, slides were counterstained with spectral DAPI (FP1490, Akoya) for 8 min. Slides were scanned using an AxioScan.Z1 in a whole-slide scanner (Zeiss) with a Plan-Apochromat 20×/0.8 M27 objective lens. Images were captured using an Orca-Flash 4.0 v.2 CMOS camera (Hamamatsu).
Immunostaining of mouse tissues was done on 5-μm acetone-fixed cryosections following standard protocols, as previously described68. Sections were immunostained with the following antibodies: anti-mouse F4/80-Alexa Fluor 647 (565853, T45-2342, BD Biosciences) at a 1:200 dilution, and Spp1-EGFP was amplified using chicken anti-GFP antibody (ab13970, abcam) at a 1:2,000 dilution, followed by donkey anti-chicken IgY(IgG)-DyLight 405 (703-475-155, Jackson ImmunoResearch) at a 1:500 dilution. After staining, slides were washed, stained with DAPI to detect nuclei and mounted with ProLong Gold Antifade Mountant (P36930, Invitrogen). Images were obtained on a Leica DMi8 microscope with a 63×/1.32 oil objective lens and a Leica DFC9000 GTC digital microscope camera, with LAS X software (v.3.5.7.23225). Images were processed using ImageJ (v.2.14.0/1.54 f) for fluorescent channel overlays and uniform exposure adjustment.
Statistical analyses were done using Prism (v.10, GraphPad Software). Normality was determined with the D’Agostino & Pearson or Shapiro–Wilk tests, chosen according to sample size. Statistical significance was determined using two-sided unpaired Student’s t-tests for normally distributed data or the non-parametric Mann–Whitney test, two-sided paired Student’s t-tests, one sample t-tests, ordinary one-way ANOVA with Sidak correction for normally distributed data or the non-parametric Kruskal–Wallis test with Dunn’s correction, ordinary two-way ANOVA with Sidak correction, simple linear regression analyses, Wilcoxon tests with Benjamini–Hochberg correction, or log-rank tests, as indicated in the figure legends.
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
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