Human samples. Paired human lung tumour and macroscopically healthy lung resection specimens were obtained from patients with lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) at Sotiria Chest Hospital. The study was approved by the ethical committee of the Hospital and informed consent was obtained from all patients. LUADs and LUSCs were clinically scored and staged according to the International Union against Cancer (UICC) TNM staging system. The clinical and pathological characteristics of all patients included in this study are summarized (Supplementary Table 1).
Mice. Col1a2-cre/ERT,-ALPP mice (Jackson Laboratories ,ID #029235), Tg (Col6a1-cre)1Gkl (ColVI-Cre) mice (MGI:3775430), B6.129X1-Twist2tm1.1(cre)Dor/J (Twist2-Cre) mice (Jackson Laboratories ,ID # 008712), mTmG mice (Jackson Laboratories ,ID #007576), IL12 p35 knockout (Jackson Laboratories ,ID #002691), IL12 p40 knockout (Jackson Laboratories ,ID # 002693) were provided by G. Kollias, (BSRC Al. Fleming), OTII mice (Jackson Laboratories ,ID #004194) by V. Andreakos (BRFAA), IFNgR (Jackson Laboratories ,ID #003288) and IFNg knockout mice (Jackson Laboratories ,ID #002287) from J.P. Gorvel (CIML). B6.129X1-H2-Ab1tm1Koni/J (I-AB-flox, ID #013181) and B6.SJL-Ptprca (CD45.1, ID #002014) mice were purchased from The Jackson Laboratories). For MHCII deletion in fibroblasts the Col1a2-cre mice were crossed to I-AB-flox mice that possess a loxP-flanked neo cassette upstream of exon 1, and a single loxP site downstream of exon 1 of the H2-Ab1 locus. For T cell responses against ovalbumin-expressing cancer cells, OTII mice that express the mouse alpha-chain and beta-chain T cell receptor that pairs with the CD4 co-receptor and is specific for the chicken ovalbumin 323-339 peptide in the context of I-Ab, were crossed to Rag1 knockout mice and mice that express the CD45.1 (Ly5.1 PTP) alloantigen. For reporter studies Col1a2-cre, ColVI-Cre and Twist2-Cre were crossed to mTmG mice.
Cell lines. The Lewis Lung Carcinoma cell line (LLC), obtained from American Type Collection Cultures (Manassas, VA), the C57BL/6-derived urethane-induced lung adenocarcinoma CULA cell line, provided by G. Stathopoulos (Helmholtz Zentrum München) and the C57BL/6-derivered melanoma cell line B16F10, provided by V. Kostourou (BSRC Al. Fleming), were transduced with zsGreen and/or OVAmCherry lentiviral vectors. Cells were maintained in DMEM, containing 10% heat-inactivated FBS, 1% L-glutamine and 1% penicillin/streptomycin (Gibco). All the cells were tested negative for the presence of mycoplasma contamination using a PCR-based technology. Cell lines were not authenticated.
Sample preparation and staining. Human and murine tissue specimens were perfused with PBS and cut into pieces. Minced samples were immediately processed or cryopreserved using BioCool (SP Scientific) in Recovery Cell Culture Freezing Medium (Gibco, Cat. No.12648-010) enriched with 10% DMSO. Human and mouse tumour and lung tissue fragments were enzymatically digested in 10% FBS/HBSS (Gibco) using Collagenase IV (Sigma-Aldrich, 1 mg/ml, Cat. No.C7657), Dispase II (Roche, 1 mg/ml, Cat. No.SCM133) and Dnase I (Sigma-Aldrich, 0.09 mg/ml, Cat. No DN25) for 1 hour at 37°C with agitation. Murine lymph nodes were digested 10% FBS/HBSS using Collagenase P (Sigma-Aldrich, 1 mg/ml, Cat. No. 11 249 002 001), Dispase II (Roche, 1 mg/ml,) and Dnase I (Sigma-Aldrich, 0.09 mg/ml,) for 1 hour at 37°C with agitation. Human tumor cells were passed through a 100um cell strainer and murine tumor cells were passed through a 70um cell strainer. Murine splenocytes were mechanically dissociated by passing through a 40um cell strainer. Cells were washed with FACS buffer (2%FBS/PBS/1.5mM EDTA), centrifuged and re-suspended in FACS buffer. Non-specific binding was blocked by incubating cells with human anti-Fc Receptor antibodies (TrueStain, Biolegend, Cat. No. 101320) or anti-mouse CD16/32 Fc block (Biolegend, Cat. No. 101310).
Staining of human tissue was performed with the following antibodies (all from Biolegend, unless otherwise stated): CD45-PE, (Cat. No. 304007), CD31-PE, (Cat. No. 303105), EpCAM-PE, (Cat. No. 324205), FAP-Alexa700 (R&D Biosystems, Cat. No. MAB3715), Podoplanin APC-Fire 750, (Cat. No. 337023), CD140a (PDGFRa)-PE-Cy7, (Cat. No. 323507), CD152-PE (CTLA-4, Cat. No. 369603), CD40-BV421, (Cat. No. 334331), CD80-BV510, (Cat. No. 305233), CD86-APC, (BD,Cat. No. 555660), CD45-PE, (Cat. No. 304007), CD8-PERCP-Cy5.5, (Cat. No. 301031), HLA-DR-BV785 (Cat. No. 307641), HLA-DR-DP-DQ-APC (Cat. No. 361713), CD44-APC (Cat. No. 103011), CD14-PE (Cat. No. 301805), CD15-PE (Cat. No. 301905), CD16-PE (Cat. No. 302007), CD19-PE (Cat. No. 302207), CD34-PE (Cat. No. 343505), CD36-PE (Cat. No. 336205), CD56-PE (Cat. No. 304605), CD123-PE (Cat. No. 306005) and CD253-PE (Cat. No. 308215) for 30 minutes at 4°C.
Murine cells were stained with the following antibodies (all from Biolegend, unless otherwise stated): CD45-APC-CY7 (Cat. No. 103115), CD3-PE-CY7 (eBioscience, Cat. No. 25-0031-82), B220-PERCP (eBioscience, Cat. No. 45-0452-82), CD8-PERCP (Cat. No. 126610), CD4-FITC (BD Pharmigen, Cat. No. 553651), CD45-Alexa700 (Cat. No. 123128), CD31-Alexa700 (Cat. No. 102443), EpCAM-Alexa700 (Cat. No. 334331), CD140a (PDGFRa)-PE (Cat. No. 334331) or BV785 (Cat. No. 118239), Podoplanin-PE Cy7 (Cat. No. 127412), MHCII-APC-Cy7 (Cat. No.107627), CD45.1-Alexa700 (Cat. No. 110723), CD11b (BD Pharmigen, Cat. No.557397), CD11c (BD Pharmigen, Cat. No. 553802), CD19 (Cat. No. 115507), B220 (BD Pharmigen, Cat. No.553089), CD49b (Cat. No. 103506), CD105 (Cat. No. 110723), MHCII (Cat. No. 107607) and TER119 (Cat. No. 116207). Tetramers SIINFEKL-PE and SIINFEKL-APC were provided by NIH.
For intracellular staining, cells were fixed and permeabilised using the Intracellular Fixation & Permeabilization Buffer Set (eBioscience, Cat. No. 88-8823-88), followed by staining for human samples with PAN-Cytokeratin-PE (Sigma-Aldrich, Cat. No. SAB4700668), Vimentin-Alexa Fluor 674 (Abcam, Cat. No. ab194719), a-Sma-FITC (Sigma, Cat. No. F3777), and Alexa Fluor 647-anti-mouse (Invitrogen, Cat. No.A21235) secondary antibody. For murine samples, also a-Sma-FITC (Sigma, Cat. No. F3777) and Vimentin-Alexa Fluor 674 (Abcam, Cat. No. ab194719) were used.
For intracellular staining of phosphorylated proteins, cells were fixed and permeabilised using the Intracellular Fixation & Permeabilization Buffer Set (eBioscience, Cat. No. 88-8823-88) as per manufaturers’ recommendations for detection of intracellular phosphorylated proteins, followed by staining with pmTor-PE-Cy7 (eBiosciences, Cat. No. 25-9718-42).
Sytox-Green viability dye (ThermoScientific, Cat. No.S7020), Zombie NIR (Biolegend, Cat. No. 423105), Zombie Violet (Biolegend, Cat. No. 423113) or BD Horizon, Fixable, Viabilility, Stain 700 (BD Pharmigen, Cat. No. 564997) were used to exclude dead cells.
For Annexin V/ PI stain up to 5x104 cells were resuspended in Binding Buffer (0.01M Hepes, pH 7.4, 0.14M NaCl, and 2.5 mM CaCl2) and stained with Annexin V FITC (Cat. No. 640906) for 30 minutes at 4°C. Cells were washed and resuspended in propidium iodide (PI)-Binding Buffer solution (0.1mg/ml, Sigma, Cat. No. P4170).
Functional ex-vivo assays. For human immunological assays, CAFs and CD4+ T cells were harvested from the same tumour fragment and dispersed into single-cell suspensions as stated above. CAFs were sorted as FSC-AHighCD45-CD31-EPCAM-FAP+PDGFRa+ cells. To preserve functional TCRs, intratumoral CD4+ T cells were sorted as FSC-ALowSSC-ALowCD45+CD14-CD15-CD16-CD19-CD34-CD36-CD56-CD123-CD8-CTLA-4- cells. CAFs were co-cultured overnight with CD4+ T cells at a 1:1 ratio in the presence of pan-HLA antibody (5μg/ml, Biolegend, Cat. No.361702) or GC1q R antibody (10μg/ml, Abcam, Cat. No. ab24733) or isotypic controls (mouse IgG2a (Biolegend, Cat. No.361702) and mouse IgG1 (Abcam, Cat. No. ab170190) respectively). Cells were co-cultured in complete RPMI (Gibco) medium.
Classical Ficoll-Pague (StemCell, Cat. No. 07861) density gradient centrifugation was followed for PBMCs isolation from human peripheral blood. Untouched CD4+ T cells were negatively selected from PBMCs using anti-PE MicroBeads (Miltenyi Biotech) after incubation with the following PE-conjugated antibodies: CD14, CD15, CD16, CD19, CD34, CD36, CD56, CD123, CD253 and CD8 according to manufacturer’s instructions. Human T Cell Activation/Expansion Kit (Miltenyi Biotech) was utilized for activation of CD4+ T cells according to manufacturer’s protocol. T cells were cultured in Click’s medium supplemented with 10% heat-inactivated human serum (Sigma-Aldrich, H4522), 1% L-glutamine and 1% penicillin/streptomycin, 1% sodium pyruvate, 1% MEM non-essential amino acids, 2mM HEPES, β-mercaptoethanol and 50U/ml huIL-2 (Peprotech, Cat. No. 200-02). For selected experiments T cells were cultured in serum-depleted medium with or without native human C1q protein (100ug/ml, Abcam, ab96363).
For murine 3D CAF cultures, CAFs sorted as FSC-AHighCD45-CD31-EPCAM-PDPN+PDGFRa+ cells were resuspended in liquified Matrigel (354254, Corning) at 4oC. Approximately 30.000cells/well were seeded in a 25ul drop of Matrigel in the center of wells of a 48-well tissue culture plate. The matrix was allowed to set for 15 minutes at 37oC before adding 400ul complete RPMI into each well. At day 5 of culture, RPMI medium was replaced with fresh RPMI supplemented with 30% healthy lung or tumor lung homogenate plus/minus anti-IFN-γ (10ug/ml, Biolegend, Cat. No. 505705). To obtain tissue homogenates, tumors or healthy lungs were excised, weighed and homogenized in PBS (30% weight/volume) on ice. Samples were centrifuged at 13000rpm for 10 min at 4oC and supernatants were added in wells containing matrigel encapsulated cells.
In vivo studies. Gender and age-matched, over 8-week-old mice were used for all studies. All mice were housed under standard special pathogen-free conditions at BSRC Alexander Fleming. All animal procedures were approved by the Veterinary Administration Bureau, Prefecture of Athens, Greece under compliance to the national law and the EU Directives and performed in accordance with the guidance of the Institutional Animal Care and Use Committee of BSRC Al. Fleming.
For the lung cancer model, mice were anesthetized via i.p. injection with xylazine and ketamine. Cancer cells (2 × 105) resuspended in 50ul DMEM (Gibco) and enriched with 20% growth-factor reduced extracellular matrix (Matrigel, BD Biosciences) were intrapleurally injected in the lung parenchyma of mice using a 29G needle (BD Biosciences). For the metastatic cancer model, mice were injected i.v. (intravenously) with 4 × 105 B16F10 cells in 100μl DMEM medium. Tamoxifen (50mg/kg, Sigma T5648) dissolved in corn oil (Sigma C8267) was given at Col1a2-cre mice i.p. once per day for five consecutive days prior to inoculation of tumor cells.
Monoclonal antibody aCD4/GK1.5 (ATCC/ TIB-207) was administered intraperitonealy (i.p.) 2 days before tumor implantation and continuing 3 times per week for the duration of the study (150ug/mouse). FTY720 (Cayman Chemicals, USA) was administered i.p. for 5 consecutive days, starting on day 7 after tumor cell transplantation at 20mg per mouse.
For the adoptive T cell transfer experiments, splenocytes from OT-II/Rag1/CD45.1 mice were cultured in a-CD28 coated plates (Biolegend, Cat. No. 102115, 1ug/ml) with complete RPMI (10%FBS, 1% Pen/Strep, 50uM b-mercaptoethanol) supplemented with 1ug/ml OVA Peptide (323-339) (Genscript, Cat. No. RP10610) (DAY0). Untouched CD45.1 OTII cells were magnetically sorted from cultured splenocytes on DAY2 using anti-PE MicroBeads (Miltenyi Biotech) after incubation with the following PE-conjugated antibodies: CD11b, CD11c, CD19, B220, CD49b, CD105, MHCII and TER119, according to manufacturer’s instructions. T cells were cultured in the aforementioned medium supplemented with 1ug/ml OVA Peptide (323-339) and 50U/ml mIL-2 (Peprotech, Cat. No. 212-12). On DAY4 OTII cells were transduced with C1qbp lentiviral particles (Lenti ORF, C1qbp, Origene, NM_007573) versus Lenti-ORF Control Particles at a multiplicity of infection (MOI) of 2. The transduction was performed for 24 hours in medium supplemented with 8ug/ml polybrene (Sigma-Aldrich). Two days after lentiviral transduction cells were placed in puromycin selection for 2 weeks (2ug/ml, Sigma P8833). On DAY22 100.000 T cells per mouse were injected intravenously in LLC-OVA tumor-bearing mice.
RNA extraction and qPCR. For assessment of human C1q expression, total RNA was extracted from apCAFs, nonapCAFs, blood and intratumoral CD4+T cells that were isolated as stated above, using Single Cell RNA Purification Kit (Norgen Biotek) according to manufacturer’s instructions. Superscript II reverse-transcriptase (Thermo Fisher Scientific) was used for cDNA synthesis and SYBR Green (Thermo Fisher Scientific) for qPCR performed on the CFX96 Touch™ Real-Time PCR Detection System from Bio-Rad. Transcript levels of C1q were determined relative to U6 reference gene, using the ΔΔCt method. The following primers sets were used: human C1qb Fw: TAAAAGGAGAGAAAGGGCTTCCAGGG and Rv: TGGCCTTGTAGTCTCCCGATTCACC and human U6, Fw: 5'-CTCGCTTCGGCAGCACA-3' and Rv: 5'-AACGCTTCACGAATTTGCGT-3’.
Flow cytometry. FACS analysis or sorting was performed using FACSCANTO II (BD Biosciences) or BD FACSARIA III (BD Biosciences) and data were analyzed using Flowjo software. For calculation of absolute numbers of tumor cells (burden) or immune cells, counting beads (123Count eBeads, ThermoScientific) were used.
Immunohistochemistry/ Immunofluorescence. For mouse studies, lungs were excised from healthy and tumor bearing mice and fixed in 4% freshly prepared paraformaldehyde for 14-18 hours at 4oC. After fixation and incubation in 30% sucrose, samples were embedded in O.C.T (VWR Cat. 361603E), then sectioned (10μm thick) onto SuperFrost Plus™ microscope slides (Thermo Scientific™). Slides were washed using PBS followed by 1 hour incubation in blocking solution (1% BSA, 0.1% Saponin, PBS). Sections were incubated with primary antibodies Anti-Mouse MHC-II 1:250 (EBiosciences 14-5321-81) and Anti-Mouse Podoplanin eFluor® 660 1:500 (EBiosciences 50-5381-82) diluted in BSA 1%/ PBS overnight at 4oC. After rinsing with 0,1% Saponin/PBS, sections were incubated with secondary antibody for MHC-II (Goat anti-Rat IgG (H+L) Alexa Fluor 594™) for 1 hour at room temperature. Nuclei were stained with DAPI and slides were mounted using Fluoroshield™ mounting media (Sigma F6182). Images were acquired with a TCS SP8X White Light Laser confocal system (Leica).
For IHC and IF stainings of human formalin-fixed paraffin-embedded (FFPE) tissues, 5μm thick sections on charged glass slides were deparaffinized in xylene and rehydrated with ethanol. Sections were incubated for antigen retrieval (Tris EDTA, pH9) for 20 minutes in the microwave and let cool down for 20 minutes at room temperature. For IHC, endogenous peroxidase was blocked by applying UltraVision Hydrogen Peroxide Block (ThermoScientific) for 15 min. Nonspecific protein-binding sites were blocked with UltraVision Protein Block (ThermoScientific) for 5 minutes. Sections were stained with anti-FAP (R&D AF3715) 1:100 or anti-HLA-DR+DP+DQ (Abcam ab7856) 1:200 overnight at 4 oC. Immunodetection was performed using UltraVision Quanto Detection System HRP Polymer DAB (ThermoScientific) according to the manufacturer’s instructions. 3,3′diaminobenzidine Quanto Chromogen (ThermoScientific) was used as chromogen. Slides were counterstained with hematoxylin. For IF, nonspecific protein-binding sites were blocked with 2% BSA/ 2% NGS. Then sections were incubated with anti-FAP (R&D AF3715) 1:100, anti-HLA DR+DP+DQ (Abcam ab7856) 1:200 and anti-CD4 (Abcam ab133616) 1:100 overnight at 4 oC. After rinsing, sections were incubated with secondary antibodies (Goat anti-Rabbit IgG (H+L) Cross-Adsorbed ReadyProbes™ Secondary Antibody, Alexa Fluor 594 Catalog # R37117, Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 Catalog # A-11029 and Donkey anti-Sheep IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 Catalog # A-21448). Nuclei were stained with DAPI and slides were mounted using Fluoroshield™ mounting media (Sigma F6182). Images were acquired with a TCS SP8X White Light Laser confocal system (Leica).
Immunofluorescence in T cells for the expression and localization of C1qbp were conducted in sorted intratumoral T cells. After sorting T cells were washed twice and cytospun at 400g for 4 minutes. After cytospin slides let dry for 5-10 min and then cells were fixed for 15 minutes using 4% PFA. After fixation cells were washed twice with PBS 1x and permeabilized for 10 minutes using 0.1% Triton-X/ PBS. Blocking was performed using 2% BSA/ 2% NGS in 0.1% Triton-X 100/ PBS. Cells were stained overnight at 4oC using Recombinant Anti-GC1q R antibody [EPR23238-6] (Abcam ab270033).
Image acquisition and analysis. Images were acquired using a TCS SP8X confocal system (Leica). We selected a fluorophore panel which allowed for simultaneous visualization of three targets and a nuclear stain (DAPI). During acquisition fluorophores were excited with 405nm (UV Laser), 488 nm (Argon), 594 nm and 647nm (White Light Laser). For images shown in Figure 1 analysis was performed using Leica LASX. Tile scanning was performed in slides stained for pan-MHCII, CD4, FAP and nuclei were stained using DAPI. Autostitching using 10% overlap was followed by analysis. Imaris 9.6 was used for subsequent image manipulations. After creating a colocalization channel between pan-MHCII and FAP all channels were used to define “primary objects” (surfaces - spots) used to analyze the image (distances, cell number, size, XY positioning etc.). Shortest distance calculation and object identification tools were used for data acquisition and image analysis. Cell density analysis was performed by identifying each "object " respectively in fields of 250.000 μm^2. Data exported from Imaris 9.6 including XY location of CD4+, MHCII+FAP+ and MHCII+FAP- objects were used for density plot creation using custom R scripts.
Identification of human MHCII fibroblasts in scRNA sequencing datasets. Processed data were collected from publicly available paired human lung tumour and healthy lung scRNA-seq datasets (E-MTAB-6149 and E-MTAB-6653). The log2cpm values of the 22180 ensembl gene IDs were used. The transition from the raw sequence read data to the gene expression table can be found in the original publication (24). Processed cells annotated as fibroblasts were included in the analysis. Patient #1 was excluded from the analysis because of the small number of Fibroblasts (9 cells). Patient #2 was excluded from the analysis because of the small number of healthy fibroblasts and dendritic cells (5 and 11 cells respectively). Only fibroblasts of patients #3, #4 and #5 were analyzed (n=643, 155 and 486, respectively). To identify MHCII+ and MHCII- fibroblasts, cells annotated as cross-presenting dendritic cells in the same dataset were used as positive control for MHCII expression. The 9 MHCII genes (HLA-DRA,HLA-DRB5, HLA-DRB1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DPA1, HLA-DPB1) were used for re-clustering fibroblasts and dendritic cells with kmeans function of cluster R package with k=3 (tuned for k=3:10), supported by mean Silhouette information scoring, calculated by silhouette function of cluster R package. This resulted in the splitting of the cells into 3 groups (low, middle and high MHCII expression). Fibroblasts of middle and high groups were considered as MHCII+ fibroblasts (206 cells), while those of low group as MHCII- fibroblasts. Gene IDs that had zero expression in all fibroblasts were removed from the analysis (16877 IDs were kept). 15487 out of them were successfully translated into gene names using the biomaRt R package. After the characterization of MHCII+ and MHCII- fibroblasts, genes with non-zero expression values in more than 25% of each group were kept (1699 genes for apCAFs and 1388 genes for non-apCAFs). The union of the both lists was considered as the list of the genes that are expressed in at least one group. This resulted in 1785 genes.
Differential expression and pathway analysis of human datasets. To identify the differential expressed genes between the MHCII+ and MHCII- we used the FindAllMarkers function of the Seurat R package, giving as input non-scaled read values of the two clusters. We kept cells that had at least 10 features, and removed ribosomal and mitochondrial genes. This resulted to 79 statistical significant up regulated genes (adjusted p-value <0.05, 67 with logFC>1) and 55 statistical significant downregulated (adjusted p-value <0.05, 48 with logFC< -1). The 67 up and the 55 down-regulated were submitted to DAVID for GO term enrichment analysis. DAVID analysis was performed as previously described (Huang et al., 2009) using the οfficial gene names (hg19) as background. Gene ontology using DAVID (6.8) (Huang et al., 2009) was performed for terms classified as biological processes. An enrichment map was created in Cytoscape (v3.7.2) (Shannon et al., 2003) using all significantly enriched GO terms (FDR < 0.1) with a gene set size between 10-1000. Overlap was used as a metric and the cutoff was set to 0.5. Clustering was performed using AutoAnnotate (v1.3.2) (Kucera et al., 2016) with overlap used as edge weight values.
Bulk mouse RNA sequencing data acquisition, processing and analysis. Intratumoral CD45+CD3+CD4+ T cells were FACS sorted in RL buffer. Total RNA was isolated by using NucleoSpin RNA (Macherey-Nagel). RNA integrity was assessed on an Agilent Bioanalyzer RNA 6000 Pico Chip, library was constructed using NEB Next Ultra RNA Library Prep Kit. The sequencing platform Ion Torrent PROTON and 3′-UTR sequencing strategy was used. Quality of FASTQ files, obtained after Ion Proton sequencing, was assessed using FastQC, following the software recommendations. Quality of FASTQ files was assessed using FastQC, following the software recommendations. Alignment of sequencing reads to the reference genome was performed using the software HISAT2 (version 2.1.0) with the genome reference version mm10. Bam files containing reads that were uniquely aligned were summarized to read counts table using the GenomicRanges package through MetaseqR2 pipeline and default settings for 3’UTR data. The resulting gene counts table was subjected to differential expression analysis (DEA) with metaseqr2 function, using DESeq2 algorithm for the normalization and statistical testing. Genes with less than 5 counts in 75% of the samples were excluded from downstream analysis. DEA thresholds were set for FDR equal to 0.01 and for logFC +-1, returning 365 down-regulated and 19 up-regulated genes. The 365 down-regulated genes were submitted to DAVID for GO term enrichment analysis. DAVID analysis was performed as previously described (Huang et al., 2009) using the official gene names (mm10) as background. Gene ontology using DAVID (6.8) (Huang et al., 2009) was performed for terms classified as biological processes, as described earlier.
MHCII+ CAFs and MHCII- CAFs from tumor-bearing mice were FACS sorted as FSC-AhighCD45-CD31-EPCAM-mCherry-PDPL+PDGFRa+MHCII+ and FSC-AhighCD45-CD31-EPCAM-mCherry-PDPL+PDGFRa+MHCII- cells respectively. Total RNA was isolated by using Single Cell RNA Purification Kit (Norgen Biotek) according to manufacturer’s instructions. RNA integrity was assessed on an Agilent Bioanalyzer RNA 6000 Pico Chip, library was constructed using NEB Next Ultra RNA Library Prep Kit. The sequencing platform Illumina Novaseq 6000 and Pair-end 150 strategy were used. Quality of FASTQ files was assessed using FastQC, following the software recommendations. Alignment of sequencing reads to the reference genome was performed using a two way alignment procedure of initial mapping with STAR (version 2.7.3) and remapping of the unmapped reads with bowtie2 (version 184.108.40.206) software packages with the genome reference version mm10. The raw bam files were summarized to read counts table using the GenomicRanges through MetaseqR2 R package pipeline, and the resulting gene counts table was subjected to differential expression analysis (DEA) using the R package DESeq2 again through MetaseqR2 pipeline. DEA thresholds set for p-value equal to 0.05 and for logFC 1.5 returned 2216 down-regulated and 164 up-regulated genes. The 418 down-regulated genes with logFC >3 were submitted to DAVID for GO term enrichment analysis, as described earlier.
Gene set enrichment analysis of CAFs for alveolar genes. Gene set enrichment analysis was performed with GSEA (v4.1.0) software. The normalized count table produced by DESeq2 was used for the GSEA according to software recommendations on the standard GSEA run. For human CAFs, the gene set included marker genes of an alveolar cluster that was identified in the same dataset (24). For murine CAFs, we used a gene set that included marker genes of a murine alveolar cluster that was identified in another dataset (54). The rest of the analysis was performed with the default thresholds.
Cross-species homology analysis. Upregulated genes in MHCII+ versus MHCII- fibroblasts were identified independently for each species, as above, and normalized by dividing the average expression of the gene plus a regularization constant (10e-4) by the average of the cluster averages plus a regularization constant (10e-4). After selecting genes with conserved gene symbols, the normalized expression matrices were log-normalized and their correlation calculated by Pearson correlation distance.
Statistics and reproducibility. For the G-squared tests of independence the g2Test_univariate function of Rfast R package was used. The p-value was calculated only for the upper tail through the pchisq function of the same package. The Pearson correlation values were calculated with cor base R function.
Visualization. PCA was performed using the prcomp base R function, with centering but no scaling. MDS plot were drawn in R with ggplot2. Box plots were generated using the ggplot2 R package and default parameters. Violin plots were generated using the geom_violin function of ggplot2 R package, with default parameters. MA plots and GO terms dot plot were generated using the geom_point function of ggplot2 R package. Plots were generated using the ggplot2 with dropping. Heatmaps were generated using the pheatmap function of pheatmap R package.