DOI: 10.5061/dryad.kkwh70shs
title: Data from: Engineering a spatiotemporal macrophage circuit via STING phase separation to override immune suppression in pancreatic cancer

description: Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal
 malignancies, largely due to its highly immunosuppressive tumor
 microenvironment (TME), which fuels metastasis and resistance to
 immunotherapy. Through comprehensive analysis of single-cell RNA
 sequencing (scRNA-seq) datasets, we identified multiple heterogeneous
 tumor-associated macrophage (TAMs) subpopulations as key regulators of
 PDAC progression, which co-express MRC1 and exert their effects by
 actively suppressing anti-tumor immune responses. To overcome this
 barrier, we developed a spatiotemporal macrophage reprogramming platform
 that leverages STING phase separation to reprogram TAM plasticity and
 reshape the immune landscape. This system, PMMB, integrates a CSF-1R
 inhibitor and a STING agonist within a macrophage-mimetic nanostructure,
 enabling sequential, controlled reprogramming of TAMs. By leveraging STING
 phase separation, PMMB stabilizes TAMs in an anti-tumor CD80⁺ phenotype
 while preventing excessive inflammation, achieving durable immune
 activation. In preclinical models, PMMB not only suppresses both primary
 and metastatic PDAC but also enhances CD8⁺ T cells infiltration,
 reinvigorates anti-PD-1 therapy responses, and mitigates immune
 exhaustion. These findings establish spatiotemporal macrophage circuit
 engineering via STING phase separation as a novel cross-scale strategy to
 override PDAC’s immune barriers and drive next-generation
 macrophage-targeted immunotherapy. This study paves the way for rationally
 designed, precision macrophage modulation strategies in solid tumors.
description: <strong>Single-cell RNA sequencing of PDAC tumors in
 mice</strong> To create a subcutaneous xenograft
 PDAC model, 5×106 KPC cells were administered via injection into the right
 flank of male C57BL/6 mice. PMMB was injected into the tail vein of mice
 on days 0, 3, 6, 9, and 12. On Day 14, the tumors from the mice were
 collected for scRNA-seq. Tumors were enzymatically
 dissociated into single-cell suspensions. After removal of dead cells,
 viable cells (&gt;80%) were resuspended in PBS with 0.5% BSA and
 processed for scRNA-seq. Single-cell libraries were generated using a
 droplet-based platform according to the manufacturer’s protocol, including
 reverse transcription, cDNA amplification, and library indexing.
 Libraries were sequenced on an Illumina NovaSeq platform,
 yielding ~200,000 reads per cell with a median detection of ~2,000–2,500
 genes. Raw reads were filtered with fastp (4), aligned to the mouse mm10
 genome, and processed to generate gene-barcode matrices for downstream
 analysis in Seurat. <strong>Bulk RNA
 sequencing</strong> Total RNA was isolated from
 PMMB- and PBS-treated MRC1⁺ macrophages (n = 3 per group) and PDAC mouse
 tumor tissues using TRIzol reagent (Invitrogen) following
 the manufacturer’s instructions. RNA quality was confirmed using NanoDrop
 and Agilent Bioanalyzer (RIN &gt; 7.0, OD260/280 &gt; 1.8, total
 RNA &gt; 1 μg). Libraries were constructed with the Illumina Stranded
 mRNA Prep Kit, including polyA selection, RNA fragmentation, cDNA
 synthesis, adapter ligation, and PCR amplification. Sequencing was
 performed on an Illumina NovaSeq 6000 platform (paired-end, 150
 bp). Raw reads were aligned to the mouse genome (mm10)
 or human genome (GRCh38) using STAR, and gene counts were generated with
 featureCounts. Differential expression analysis was performed with DESeq2
 (|log₂ fold change| &gt; 1, adjusted p &lt; 0.05). DEGs were
 further subjected to GO, KEGG, and GSEA analyses to evaluate
 immune-related pathways, TAM-associated functions, and STING
 signaling.
description: # Data from: Engineering a spatiotemporal macrophage circuit via STING
 phase separation to override immune suppression in pancreatic cancer
 Dataset DOI:
 [10.5061/dryad.kkwh70shs](https://doi.org/10.5061/dryad.kkwh70shs) ##
 Description of the data and file structure #### File: scRNA-Seq.zip
 **Description:** This compressed folder contains raw and processed
 single-cell RNA sequencing data used in the study. Six data files are
 included, representing two experimental groups (control and treatment),
 with three biological replicates per group: PBS-1, PBS-2, PBS-3: Control
 group (phosphate-buffered saline–treated samples) PMMB-1, PMMB-2, PMMB-3:
 Experimental group (treated samples) In each subfolder of this dataset,
 you will find three essential files that together form the Gene Expression
 Matrix for a single sample. Here is a description of what each file
 contains: 1\. ‘barcodes.tsv.gz’：A list of all unique cell barcodes
 identified in the sample. Each line represents a single cell (or droplet
 that contained a cell). The barcode is a short DNA sequence that is unique
 to each cell's RNA molecules, allowing the sequencing data to be
 assigned back to its cell of origin. 2\. ‘features.tsv.gz’：A list of all
 genes (features) that were measured in the experiment. Each line
 corresponds to a gene and typically includes three pieces of information:
 Gene ID，Gene Symbol，Feature Type. 3\. ‘matrix.mtx.gz’：A sparse matrix
 containing the actual expression counts. This file stores the number of
 RNA transcripts (UMI counts) for each gene in each cell. It uses a
 "sparse matrix" format to efficiently store data, as the
 majority of genes in a given cell have a count of zero. The file structure
 is: First line: Header with the total number of genes, cells, and non-zero
 entries. Subsequent lines: ‘[row] [column] [count]’, where the row
 corresponds to a gene in ‘features.tsv’, the column corresponds to a cell
 in ‘barcodes.tsv’, and the count is the UMI count for that gene in that
 cell. #### File: Bulk_RNA-Seq.zip **Description:** This compressed folder
 contains the bulk RNA sequencing (RNA-seq) data used in this study. Two
 data tables are included: Cell sample data table Animal sample data table
 Each table includes two experimental groups (control and treatment), with
 three biological replicates per group: PBS-1, PBS-2, PBS-3: Control group
 (PBS-treated samples) PMMB-1, PMMB-2, PMMB-3: Treatment group (treated
 samples)