{'fraction_non_alphanumeric': 0.039183029453015425, 'fraction_numerical': 0.03348527349228612, 'mean_word_length': 5.094551282051282, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Distinct immune signatures in directly treated and distant tumors result from TLR adjuvants and focal ablation

Both adjuvants and focal ablation can alter the local innate immune system and trigger a highly effective systemic response. Our goal is to determine the impact of these treatments on directly treated and distant disease and the mechanisms for the enhanced response obtained by combinatorial treatments. Methods: We combined RNA-sequencing, flow cytometry and TCR-sequencing to dissect the impact of immunotherapy and of immunotherapy combined with ablation on local and systemic immune components. Results: With administration of a toll-like receptor agonist agonist (CpG) alone or CpG combined with same-site ablation, we found dramatic differences between the local and distant tumor environments, where the directly treated tumors were skewed to high expression of F4/80, Cd11b and Tnf and the distant tumors to enhanced Cd11c, Cd3 and Ifng. When ablation was added to immunotherapy, 100% (n=20/20) of directly treated tumors and 90% (n=18/20) of distant tumors were responsive. Comparing the combined ablation-immunotherapy treatment to immunotherapy alone, we find three major mechanistic differences. First, while ablation alone enhanced intratumoral antigen cross-presentation (up to ~8% of CD45+ cells), systemic cross-presentation of tumor antigen remained low. Combining same-site ablation with CpG amplified cross-presentation in the draining lymph node (~16% of CD45+ cells) compared to the ablation-only (~0.1% of CD45+ cells) and immunotherapy-only cohorts (~10% of CD45+ cells). Macrophages and DCs process and present this antigen to CD8+ T-cells, increasing the number of unique T-cell receptor rearrangements in distant tumors. Second, type I interferon (IFN) release from tumor cells increased with the ablation-immunotherapy treatment as compared with ablation or immunotherapy alone. Type I IFN release is synergistic with toll-like receptor activation in enhancing cytokine and chemokine expression. Expression of genes associated with T-cell activation and stimulation (Eomes, Prf1 and Icos) was 27, 56 and 89-fold higher with ablation-immunotherapy treatment as compared to the no-treatment controls (and 12, 32 and 60-fold higher for immunotherapy-only treatment as compared to the no-treatment controls). Third, we found that the ablation-immunotherapy treatment polarized macrophages and dendritic cells towards a CD169 subset systemically, where CD169+ macrophages are an IFN-enhanced subpopulation associated with dead-cell antigen presentation. Conclusion: While the local and distant responses are distinct, CpG combined with ablative focal therapy drives a highly effective systemic immune response.

on either a FACScan or LSRII flow cytometer (BD Biosciences). All data were analyzed using FlowJo v10 software (TreeStar).

IFN-β and IFN-γ Stimulation of Ifi27l2a in vitro
NDL tumor cells were plated in 6-well tissue-culture treated plates at a concentration of 5 x 10 5 cells/well. The next day, media was changed to complete media (Gibco, #11995 containing 10% FBS and 1% penicillinstreptomycin) supplemented with either IFN-γ (#575302, BioLegend) or IFN-β1 (#581302, BioLegend) at 500 or 1000 U/mL in a final volume of 2 mL per well. Cells were incubated continuously for 24 h in a 37°C humidified chamber containing 5% CO2, after which the cells were collected, counted and snap frozen in liquid nitrogen for submission to the UC Davis Real-Time PCR Research and Diagnostics Core Facility for quantification of Ifi27l2a expression via qPCR.
RNA Isolation for RNA-seq NDL tumors were submitted to the UC Davis Comprehensive Cancer Center's Genomics Shared Resource (GSR) for isolation of total cellular RNA and subsequent RNA-seq analysis. Total cellular RNA was isolated from snap-frozen NDL tumor pieces using the TRIzol Reagent (Invitrogen) and a modified protocol that incorporates an additional extraction with phenol/chloroform/isoamyl alcohol (25:24:1, pH 4.3) followed by an additional clean-up with an RNeasy spin column (Qiagen). RNA concentration and purity were assessed with a NanoDrop 2000 Spectrophotometer (Thermo Scientific) and quality assessments were made using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).

Cell Isolation and nucleic acid preparation for real-time quantitative PCR
Mice were treated as described in the "in vivo studies" section and euthanized on day 38. NDL tumors were harvested and processed to single cell suspensions through mechanical and enzymatic disruption, followed by magnetic bead isolation of CD8 + T-cells and NDL tumor cells. CD8 + T-cells were isolated from total cell samples using the negative-selection EasySep™ Mouse CD8 + T-Cell Isolation Kit (Stemcell Technologies). NDL tumor cells were isolated from total cell samples using the negative-selection Tumor Cell Isolation Kit (Miltenyi Biotech). Isolated cell pellets were resuspended in 200 µl phosphate buffered saline (PBS) and then mixed with 200 µl of Buffer VXL + 20 µl Proteinase K. Two stainless steel grinding beads (4 mm diameter, SpexCertiprep, Metuchen, NJ) were added and the cells homogenized in a GenoGrinder2000 (SpexCertiprep) for 2.5 min at 1750 strokes per minute. Lysate was incubated for 10 minutes at 56°C. 200 µl of lysate was removed and used for total nucleic acid (TNA) extraction and the remaining saved as backup. TNA extraction was performed on a semi-automated extraction system (QIAcube, Qiagen) according to manufacturer's instructions. TNA was eluted in 100 µl of diethylpyrocarbonate-treated water.
Directional RNA-seq library preparation and next-generation sequencing Indexed RNA-seq libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit (New England BioLabs, Ipswich, MA), according to the manufacturer's standard protocol. Briefly, poly-adenylated mRNA was purified from total RNA (100 ng) and ribosomal RNA removed by binding to magnetic oligo(dT)25 beads, which was followed by RNA fragmentation. Double-stranded cDNA was then generated by randomprimed first-strand synthesis and subsequent second strand synthesis in the presence of dUTP for strand marking (1, 2). The double-stranded cDNA was then end repaired, 3'-dA tailed, and an Illumina-compatible NEBNext adaptor was ligated. Uridine excision was performed with the USER (Uracil-Specific Excision Reagent) enzyme, and the libraries were then indexed and enriched by high-fidelity PCR amplification (15 cycles) with Q5 High-Fidelity DNA Polymerase and NEBNext multiplex primers. Subsequently, libraries were combined for multiplex sequencing on an Illumina HiSeq 4000 System (150-bp, paired-end, ~25-30 x10 6 reads per sample).

NGS Data Analysis
RNA-seq data was analyzed using a STAR-StringTie-Cufflinks pipeline. De-multiplexed raw sequence reads (FASTQ format) were mapped to the reference mouse genome assembly (GENCODE, GRCm38, release 05/2017) using STAR (Spliced Transcripts Alignment to a Reference) software with a 2-pass alignment approach that uses initial alignment to detect novel junctions and insert them into the genome index, followed by a second pass to re-align reads using both annotated (GENCODE, Release M14, GRCm38.p5) and novel (detected in the first pass) junctions (3). Mapped reads were then passed onto StringTie for transcript assembly (4). Subsequently, gene-and transcript-level expression were comprehensively quantified with Cufflinks tools (e.g., Cuffquant, Cuffnorm) to yield normalized expression as FPKM (fragments per kilobase of transcript per million fragments mapped reads) and test for differential expression (Cuffdiff).

NGS Data Processing
Cuffnorm files were imported to MATLAB for processing and analysis. Z-scores were calculated using raw FPKM values across all presented samples for a single gene. Principal component analysis was performed on the log2 transformed FPKM values using the singular value decomposition algorithm. Hierarchical clustering was performed using Euclidean pairwise distances and unweighted average cluster distances (UPGMA). Gene set enrichment analysis was performed on the set of all differentially expressed genes for each CuffDiff analysis (i.e., genes with a differential expression at p<0.01) using DAVID's Functional Annotation Tool. Specifically, the GO Biological Processes and the KEGG Pathways were investigated (5).

RT-reaction and real-time TaqMan PCR
Real-time PCR was performed through the UC Davis Real-Time PCR Research and Diagnostics Core Facility. TaqMan Gene Expression Assays were ordered from Applied Biosystems (Carlsbad, CA) for murine Ki67 (Mm01278617_m1), Slc2a1 (Mm0044148_m1), Wnt7b (Mm01301717_m1), Ifi27l2a (Mm01329883_gH), Psrc1 (Mm00498358_m1), Pdcd1 (Mm01285676_m1), and the reference genes Ubc (Mm02525934_g1, for normalization in CD8 + T-cell samples) and Actb (Mm00607939_s1, for normalization in tumor-cell samples). The 5´ end of the TaqMan MGB probes are labeled with FAM reporter dye, the 3´ end with quencher dye NFQMGB (Non-Fluorescent Quencher Minor Grove Binding). The Quantitect Reverse transcription kit (Qiagen) was used for cDNA synthesis following the manufacturer's directions with the following modifications. Ten µl of RNA were digested with 1 µl of gDNA WipeOut Buffer by incubation at 42°C for five minutes and then briefly centrifuged. Genomic DNA contamination was tested by using 1 µl of digested RNA and running the Real-time PCR housekeeping gene. Then 0.5 µl of Quantitect Reverse Transcriptase, 2 µl Quantitect RT buffer, 0.5 µl RT Primer Mix, 0.5 µl 20 pmol Random Primers (Invitrogen) were added and brought up to a final volume of 20 µl and incubated at 42°C for 40 minutes. The samples were inactivated at 95°C for 3 minutes, chilled, and 80 µl of water was added (6). Each qPCR reaction contained 20x primer and probes for the respective TaqMan system and commercially available PCR mastermix (TaqMan Universal PCR Mastermix, Applied Biosystems) containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, 2.5 mM deoxynucleotide triphosphates, 0.625 U AmpliTaq Gold DNA polymerase per reaction, 0.25 U AmpErase UNG per reaction and 5 µl of the diluted cDNA sample in a final volume of 12 µl. The samples were placed in 384 well plates, in singlet, and amplified in an automated real-time PCR System (ABI PRISM 7900 HTA FAST, ABI). ABI's standard amplification conditions were used: 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 s at 95°C and 60 s at 60°C. Fluorescent signals were collected during the annealing temperature and Cq values extracted with a threshold of .2 and baseline values of 3-15 (6).

Relative quantitation of gene transcription
Final quantitation was done using the comparative Cq method (User Bulletin #2, Applied Biosystems) and is reported as relative transcription or the n-fold difference relative to a calibrator cDNA. In brief, the reference gene was used to normalize the Cq values of the target genes (ΔCq). The ΔCq was calibrated against the average of the control group within each target gene. The linear amount of target molecules relative to the calibrator was calculated by 2 -ΔΔCq . Therefore, all gene transcription is expressed as an n-fold difference relative to the calibrator.       Tlr1  Tlr2  Tlr4  Tlr6  Tlr7  Tlr8  Tlr9   Myd88  Irak4  Rac1   Tnf  Il12a  Il12b   Tnfrsf1a Traf5 Ripk1 Ccl3 Ccl4 Bcl3 Nfkbia Socs3