Journal: bioRxiv

Article Title: Single-cell profiling guided combinatorial immunotherapy for fast-evolving CDK4/6 inhibitor resistant HER2-positive breast cancer

doi: 10.1101/671198

Figure Lengend Snippet: ( A ) Gr1+ MDSCs isolated from Ab+Pal treatment resistant tumors inhibited CD3/CD28 stimulated proliferation of CD4+ and CD8+ T cells in vitro. Left, representative flow cytometry histograms measuring carboxyfluorescein succinimidyl ester (CFSE); right, quantification of T-cell proliferation. P -value by Student’s t-test. ( B ) t-SNE plot of single cell RNA sequencing data from CD45+ TILs, Gr1high Ly6G+cells and Gr1dim Ly6G-cells. Each point represents a single cell. ( C ) Expression of key marker genes used for immune cell-type identification and annotation was overlaid on t-SNE plots as shown in (B). ( D ) mRNA levels of Lcn2 and Mgst1 in sorted T cells (CD3+), macrophages (F4/80+) and MDSCs/IMCs (Gr1+) from APR tumors were quantified by RT-PCR. P -value by one-way ANOVA with Tukey’s test.

Article Snippet: The following pre-conjugated antibodies purchased from Fluidigm were used in this study: CD45-089Y (3089005B, 30-F11); Ly-6G-141Pr (3141008B, 1A8,); CD11c-142Nd (3142003B, N418); CD45R-144Nd (3144011B, RA3-6B2); CD4-145Nd (3145002B, RM4-5); CD11b-148Nd (3148003B, M1/70); CD44-150Nd (3150018B, IM7); CD25-151Eu (3151007B, 3C7); CD3e-152Sm (3152004B, 145-2C11); PD-L1-153Eu (3153016B, 10F.9G2); CTLA-4-154Sm (3154008B, UC10-4B9); PD-1-159Tb (3159024B, 29F.1A12); Ly-6C-162Dy (3162014B, HK1.4); CX3CR1-164Dy (3164023B, SA011F11); NK1.1-165Ho (3165018B, PK136); c-Kit-166Er (3166004B, 2B8); CD8a-168Er (3168003B, 53-6.7); CD86-172Yb (3172016B, GL1); I-A/I-E-209Bi (3209006B, M5/114.15.2).

Techniques: Isolation, In Vitro, Flow Cytometry, RNA Sequencing Assay, Expressing, Marker, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Cancer-driven neutrophil priming couples systemic epithelial regenerative programs with pre-metastatic niche formation

doi: 10.1101/2025.10.20.683483

Figure Lengend Snippet: a) UMAP embedding of lung scRNAseq data from Naïve IgG, Tumour-bearing (TB) (PyMT-FVB) IgG and TB αLy6G mice. Cells are coloured by cluster with cell type identities indicated in the legend. Alveolar type 2 (AT2) clusters are outlined with dashed line. b) PHATE embedding generated from AT2 clusters across Naïve IgG, TB IgG, and TB αLy6G conditions. Cells are coloured by subclusters (0–7). c) Expression of alveolar epithelial progenitor-associated signatures across AT2 subclusters shown in (b). Colour indicates scaled expression level. d) (Left) MELD analysis showing per cell MELD relative likelihoods represented on the PHATE embedding for Naïve IgG vs TB IgG conditions. Red indicates higher relative likelihood associated with TB IgG condition. (Right) Jitterplot shows per-cell TB IgG-associated likelihood across AT2 subclusters. e) (Left) MELD analysis comparing TB IgG and TB αLy6G conditions. Red indicates higher likelihood of association with the TB IgG condition. (Right) Jitter plot shows per-cell likelihoods across clusters. f) UMAP embedding of Intestinal scRNAseq data from Naïve IgG, TB IgG and TB αLy6G conditions. Cells are coloured by cluster with major cell type identities indicated in the legend. Intestinal epithelial clusters are outlined with dashed line. g) PHATE embedding generated from epithelial cell clusters across Naïve IgG, TB IgG and TB αLy6G conditions shown in (f), coloured by cell-type identity. h) Per-cell MELD relative likelihood represented on PHATE embedding comparing TB IgG and Naïve IgG conditions. Red indicates higher relative likelihood associated with TB IgG. i-j) Jitter plots showing per-cell MELD relative likelihoods across intestinal cell-type clusters for (i) TB IgG versus naïve IgG and (j) TB IgG versus TB αLy6G comparisons. Higher likelihood values correspond to TB IgG conditions. k) Quantification of organoid formation efficiency of lung epithelial cells isolated from Naïve IgG, TB IgG and TB αLy6G mice (n=5 mice per group, 3 independent experiments), paired one-way ANOVA. l) Quantification of organoid formation efficiency from intestinal epithelial cells isolated from naïve IgG, TB IgG and TB αLy6G mice (n=3 mice per group, 3 independent experiments), paired one-way ANOVA.

Article Snippet: For neutrophil isolation, cell preparations were first incubated with anti-Ly6G FITC conjugated antibody (1:100) for 15 min on ive, washed and then incubate wth with anti-FITC beads (Miltenyi Biotec).

Techniques: Generated, Expressing, Isolation

Journal: bioRxiv

Article Title: Cancer-driven neutrophil priming couples systemic epithelial regenerative programs with pre-metastatic niche formation

doi: 10.1101/2025.10.20.683483

Figure Lengend Snippet: a) Pathways enriched in mature bone marrow (BM) neutrophils from tumour bearing (TB) versus Naïve mice. Inflammatory and metabolic pathways are coloured in red and blue, respectively; enrichment P values are indicated. b) LC–MS–based metabolic profiling of control or neutrophil-conditioned media. Neutrophils were isolated from lung or spleen of naïve or TB (4T1 Balb/c) mice (n=4 per group) and incubated in Advanced DMEM/F12 medium. Data are shown as mean z-score per metabolite. c-d) Abundance of uridine (c) and uracil (d) in neutrophil-conditioned or control media generated from lung or splenic neutrophils isolated from naïve or TB (4T1 Balb/c) mice, incubated in uridine-containing Plasmax medium (n=4 mice per group), ordinary one-way ANOVA. e) Schematic representation of Uridine phosphorylase 1 (Upp1) enzymatic activity. f) Upp1 mRNA expression in lung and splenic neutrophils from naïve or TB (PyMT-FVB) mice lung (n=5 per group, spleen n=4 per group), two-tailed students t-test. g) (Left) representative image and (Right) quantification of UPP1 immunofluorescence intensity in lung neutrophils (LuNeu) isolated from naïve or tumour bearing (PyMT-FVB) mice. Data are represented a per cell mean intensity (n=4). Welch’s t-test. h) Plasma Uracil abundance from Naïve (Balb/c) IgG, TB (4T1) IgG or TB (4T1) αLy6G mice measured by LC-MS (Naïve IgG and TB IgG n=10, TB αLy6G n=5), ordinary one-way ANOVA. i) Upp1 expression in circulating neutrophils from healthy volunteers (HV) and patients with breast cancer (P). Data are normalized counts from bulk RNA-seq of purified neutrophils (n=14 per group), two-tailed Wilcoxon test. Hormone-receptor status of patients is indicated. j) Plasma uracil concentrations in matched samples from HV and P with breast cancer (n=17 per group), quantified by LC–MS, two-tailed Wilcoxon test.

Article Snippet: For neutrophil isolation, cell preparations were first incubated with anti-Ly6G FITC conjugated antibody (1:100) for 15 min on ive, washed and then incubate wth with anti-FITC beads (Miltenyi Biotec).

Techniques: Liquid Chromatography with Mass Spectroscopy, Control, Isolation, Incubation, Generated, Activity Assay, Expressing, Two Tailed Test, Immunofluorescence, Clinical Proteomics, RNA Sequencing, Purification

Journal: bioRxiv

Article Title: Cancer-driven neutrophil priming couples systemic epithelial regenerative programs with pre-metastatic niche formation

doi: 10.1101/2025.10.20.683483

Figure Lengend Snippet: a) PHATE embedding of bone marrow (BM) scRNAseq data from Naïve WT (C57/Bl6), Tumour-bearing (TB) (PyMT-C57/Bl6) WT and TB UPP1KO (PyMT-C57/Bl6) mice. Cells are coloured by cluster. Neutrophil and neutrophil progenitor clusters are outlined with dashed line. b) Granulopoiesis neutrophil and progenitor clusters from (a) represented by PHATE embedding. Cells are coloured by neutrophil and progenitor subtype as indicated in the legend. c) UPP1 expression across the dataset, shown per cell on the PHATE embedding (left) and as normalized expression across neutrophil subclusters (right) . d) MELD transcriptional perturbation analysis showing per-cell relative likelihoods for TB WT versus TB UPP1KO samples. Red indicates higher likelihood associated with the TB WT condition. e) RNA velocity and transcriptional dynamics across pro-neutrophil (Pro-Neu) cells. (Top) RNA velocity vectors overlaid on PHATE embedding indicating differentiation trajectories within the granulopoiesis dataset; Pro-Neu population marked in red. (Bottom) Predicted transcription (Y-axis) and degradation (X-axis) rates for individual genes in Pro-Neu cells derived from scVelo dynamical modelling. Dashed lines and arrow encircle a group of ‘protected’ transcripts with low degradation rates specific to the TB WT condition. f) Velocity length as a measure of differentiation rate within Pro-Neu population from Naïve WT, TB WT and TB UPP1KO mice. Data represent average velocity length per cell within the Pro-Neu population. Cell numbers are indicated in red, Kruskall-wallace test. g) Predicted degradation rates of eukaryotic translation initiation factor (eIF) transcripts within the stable RNAs in Pro-Neu population from TB mice (highlighted in g). Mean degradation rate for all transcripts is indicated in blue and ‘protected’ transcripts with degradation rate < 0.1 are shown in purple. h-k) In vivo translation rate measured by OP-Puro incorporation in Naïve WT, TB WT and TB UPP1KO mice. OP-Puro fluorescence intensity was quantified by flow cytometry in GMPs (h), (CD115 - Siglech - CD11b + Ly6C + Ly6G + cKit + ) Neutrophil progenitors (i) immature Ly6G low (j) and mature Ly6G high (k) bone marrow neutrophils isolated (Naïve WT n=4, TB WT n=3 TB UPP1KO n=5). Ordinary one-way ANOVA. l) Pathway enrichment analysis in mature neutrophils (M-Neu; from panel b) comparing TB WT and TB UPP1KO mice. Enrichment P values are shown. Inflammatory pathways are indicated in red, metabolic pathways in blue and transcription in black. m) Schematic of GMP colony-formation assay. FACS-sorted GMPs from naïve mice (FVB) were plated one cell per well with or without uracil. After colonies established, single GMP-derived colonies were profiled by FACS for cell number (colony size) and neutrophil/monocyte content (Ly6G+ and Ly6C+ cell count). n) Colony size (left) and Ly6G⁺ cell number per colony (right) from GMP colony-formation assay with or without uracil (n=6 mice. 62 PBS and 66 uracil-treated colonies) Mann-Whitney test. o) Schematic of bone-marrow chimera experiment. CD45.2 recipient mice (C57/Bl6) were reconstituted with either WT or UPP1KO CD45.1 bone marrow (C57/Bl6), generating chimeras with 10% WT CD45.1 and 90% WT or UPP1KO CD45.2 cells. This setup allows profiling of WT CD45.1 cells within a WT or UPP1KO haematopoietic environment. Mire where transplanted with PyMT tumour cells (C57/Bl6). p) Ly6G and CD11b intensity in circulating WT or UPP1KO CD45.2+ (donor) neutrophils and in WT CD45.1+ (recipient) neutrophils from bone marrow chimeric mice. (WT n=24, UPP1KO n=23 mice) Welch’s t-test.

Article Snippet: For neutrophil isolation, cell preparations were first incubated with anti-Ly6G FITC conjugated antibody (1:100) for 15 min on ive, washed and then incubate wth with anti-FITC beads (Miltenyi Biotec).

Techniques: Expressing, Derivative Assay, In Vivo, Fluorescence, Flow Cytometry, Isolation, Colony Assay, Cell Counting, MANN-WHITNEY

Journal: bioRxiv

Article Title: Cancer-driven neutrophil priming couples systemic epithelial regenerative programs with pre-metastatic niche formation

doi: 10.1101/2025.10.20.683483

Figure Lengend Snippet: a) Schematic for DSS induced colitis assessment in tumour bearing mice following neutrophil depletion. Naïve or MMTV-PyMT (FVB) orthotopic mammary tumour bearing animals received DSS drinking water (or control) for 7 days. From day 6 mice receive αLy6G antibody or isotype control (IgG). Colitis assessment was performed on day 11. b) Quantification of DSS-induced colitis severity in naïve IgG, tumour-bearing (TB) IgG and TB αLy6G (1A8) mice based on histological grading of colon sections. Data represent the proportion of colon length exhibiting each severity grade (n=5 mice per group), ordinary two-way ANOVA. c) Schematic of DSS-induced colitis in WT and UPP1KO bone-marrow chimeras. WT or UPP1KO chimeras were either left untreated or given DSS treatment for 7 days. Colitis severity was assessed 4 days after treatment. d) Quantification of DSS-induced colitis severity in naïve WT, naïve UPP1KO, TB WT and TB UPP1KO bone-marrow chimeas based on histological grading of colon sections. Data represent the proportion of colon length exhibiting damage severity (naïve WT n=4, naïve UPP1KO n=5, TB WT n=7 and TB UPP1KO n=7 mice), ordinary two-way ANOVA. e) Schematic for metastasis assessment in tumour-bearing UPP1KO bone-marrow chimeras. WT or UPP1KO BM-chimeras bearing orthotopic MMTV-PyMT tumours were intravenously injected with primary MMTV-PyMT tumour cells 7 days before assessing metastatic efficiency. f) Quantification of lung metastasis based on histological analysis (WT n=5, UPP1KO n=7), Mann-Whitney test. g) Schematic of metastasis assessment in WT (Ela2het, C57/Bl6) and neutrophil-conditional UPP1fox/flox (Ela2Cre-UPP1KO, C57/Bl6) mice. Mice bearing orthotopic MMTV-PyMT tumours were intravenously injected with primary MMTV-PyMT tumour cells (C57/Bl6) 7 days before assessing metastatic efficiency. h) Quantification of lung metastasis based on histological sections from tumour-bearing WT and Ela2CRE-UPP1KO mice. Data represent number of metastatic foci per lung (WT n=11, Ela2CRE-UPP1KO n=12), Mann-Whitney test.

Article Snippet: For neutrophil isolation, cell preparations were first incubated with anti-Ly6G FITC conjugated antibody (1:100) for 15 min on ive, washed and then incubate wth with anti-FITC beads (Miltenyi Biotec).

Techniques: Control, Injection, MANN-WHITNEY

Journal: bioRxiv

Article Title: Cancer-driven neutrophil priming couples systemic epithelial regenerative programs with pre-metastatic niche formation

doi: 10.1101/2025.10.20.683483

Figure Lengend Snippet: a) Schematic of experimental design to assess lung epithelial organoid formation in mice with neutrophil specific UPP1KO TB mice. b) Quantification of organoid formation efficiency of lung epithelial cells isolated from naïve WT, tumour-bearing (TB) WT and TB Ela2CRE-UPP1KO mice (PyMT, C57/Bl6) (n=4 per biological replicates per group, large points, technical replicates, small points). one-way ANOVA performed on mean value of biological replicates. c) (Left) Representative brightfield and immunofluorescence images from ImageStream analysis of circulating neutrophils from tumour-bearing mice. CD41 (FITC, green) marks platelets and Ly6G (APC, red) marks neutrophils. (Right) Quantification of platelet-neutrophil clusters (PNCs) in circulation of a naïve (black) and a tumour-bearing (red) animals, shown as the percentage of PNCs containing increasing platelet numbers. d) Representative IF images of perfused lung from naïve IgG (FVB), TB (PyMT, FVB) IgG an TB (PyMT, FVB) αLy6G mice stained with SPC (AT2 cells, white), MPO (neutrophils, green) and CD42 (platelets, red). e) Quantification of platelet abundance in perfused lung from naïve IgG, TB IgG an TB αLy6G mice from (d), represented as platelets per mm2 of lung tissue. (naïve IgG n=4, TB IgG and TB αLy6G n=5), ordinary one-way ANOVA. f) Percentage of platelet-neutrophil clusters (CD42+ platelets in contact with MPO+ neutrophils) out of total platelets in lungs of naïve IgG, TB IgG an TB αLy6G mice (naïve IgG n=4, TB IgG and TB αLy6G n=5), ordinary one-way ANOVA. g) Percentage of circulating CD41b+ neutrophils out of all neutrophils in tumour-bearing Ela2CRE WT and Ela2CRE-UPP1KO mice (Ela2CRE WT n=7, Ela2CRE-UPP1KO n=6). (PyMT, C57/Bl6). Unpaired t-test. h) Representative IF images of perfused lung from TB Ela2CRE-WT and TB Ela2CRE-UPP1KO mice (PyMT, C57/Bl6), stained for SPC (AT2 cells, white), MPO (neutrophils, green) and CD42 (platelets, red). i) Quantification of platelet-neutrophil clusters represented as number of MPO+ neutrophils contacting CD42+ platelets per mm2 lung tissue. (Ela2CRE WT n=3, Ela2CRE-UPP1KO n=4) unpaired t-test. j) Quantification of platelet number (CD42+) per mm lung tissue in TB Ela2CRE-WT and Ela2CRE-UPP1KO mice from (h) (Ela2CRE WT n=3, Ela2CRE-UPP1KO n=4) unpaired t-test. k) (left) Schematic of experimental design and (right) lung epithelial organoid formation efficiency in presence of ex vivo generated platelet-neutrophil clusters (FVB) (+PNC), or neutrophils alone (+Neu) (n=3 biological replicates per group, large points, technical replicates, small points). Ordinary one-way ANOVA on mean of biological replicates. l) (left) Schematic of experimental design and (right) lung epithelial organoid formation efficiency in presence of platelets (+Platelets) (FVB) (- n=2, +Platelets n=4, biological replicates, large points, technical replicates, small points) Welch’s t test on mean of biological replicates. m-n) Schematic of experimental design (m) and tumour organoid formation efficiency (n) of primary MMTV-PyMT mammary tumour cells (C57/Bl6) cultured with or without lung epithelial cells isolated form naïve Ela2CRE-WT, TB Ela2CRE-WT or TB Ela2CRE-UPP1KO mice (C57/Bl6) (naïve Ela2CRE-WT n=4, TB Ela2CRE-WT n=5 or TB Ela2CRE-UPP1KO n=4, technical replicates shown in small points) Ordinary one-way ANOVA on mean of each n.

Article Snippet: For neutrophil isolation, cell preparations were first incubated with anti-Ly6G FITC conjugated antibody (1:100) for 15 min on ive, washed and then incubate wth with anti-FITC beads (Miltenyi Biotec).

Techniques: Isolation, Immunofluorescence, Staining, Ex Vivo, Generated, Cell Culture