Fig 1: Human tumors coexpress LIF and IL-1a, which synergize in mouse models to potentiate EMH. (A–C) In mice with 10 days of LIF overexpression lentivirus or empty vector control lentivirus and 24 hours after treatment with 200 ng IL-1a IV, PMNs in the PB as a percent of total leukocytes (A, n = 7–8), KSL cells as a fraction of total splenic CD45+ cells (B, n = 7–8), GMP cells as a fraction of total splenic CD45+ cells (C, n = 7–8). (D–F) RNA-seq expression of IL1A, LIF, and CSF3 expression in tumor compared to normal tissue for breast (D, 292–1,099), colonic (E, n = 288–349), and pancreatic (F, n = 171–178). (G) Our proposed model of parallel mechanisms for tumor-associated EMH mediated in part by indirect inflammatory changes to HSPCs by tumor-derived IL-1a through local HSPC TNFa expression and direct proliferative effects on splenic ABS cells from tumor-derived LIF. Processed data for this figure can be found in S1 Data. EMH, extramedullary hematopoiesis; GMP, granulocyte–monocyte precursor; HSPC, hematopoietic stem and progenitor cell; IV, intravenous; KSL, Kit+/Sca-1+/Lineage-; LIF, leukemia inhibitory factor; PB, peripheral blood; PMN, polymorphonuclear neutrophil; RNA-seq, RNA-sequencing.
Fig 2: TCM prolongs the expression of pluripotency features in ESCs and EBs. (A) Alkaline phosphatase (AP) staining as pluripotency marker in ESCs grown for 5 days in BM or TCM. The number of AP-positive colonies is significantly higher in TCM conditions. Data are from three independent biological replicates. Statistical analysis was by multiple unpaired Student’s t-tests with Holm-Šídák multiple comparisons test. ** p < 0.01. (B) Heatmap of normalized Log2-transformed RNA-seq read counts of 10 selected, key pluripotency genes that are differentially expressed between D4 EBs grown in BM vs. TCM. The majority of these genes retains higher expression levels in TCM conditions, with the most pronounced differences (>8-fold) observed for Sox2, Esrrb and Nr0b1. (C) Gene set enrichment analysis using enrichR [26] of the 416 genes with =8-fold higher expression in TCM- vs. BM-grown EBs identifies a high enrichment for genes regulated by pluripotency factors OCT4, SOX2 and NANOG in the TF-target interaction database TRRUST [25]. (D) Wiggle plots of gene expression data across the indicated pluripotency gene loci of D4 EBs grown in either BM or TCM. Sox2, Esrrb and Nr0b1 expression remains far higher in EBs cultured in TCM. ChIP-seq binding sites for the same three factors in ESCs were integrated from previous studies [29,30] and are indicated by the dark blue horizontal bars. (E) RT-qPCR expression data of EMT marker genes in EBs at D4 of differentiation. E-Cadherin (Cdh1), an epithelial marker, remains more highly expressed in TCM- compared to BM-grown EBs, whereas mesenchymal markers are reduced. Data of six independent biological replicates are normalized to BM conditions and are displayed as mean ± SEM. Statistical analysis was by unpaired two-tailed Student’s t-test. *** p < 0.001 and **** p < 0.0001. (F) Expression of Lif as the most pertinent pluripotency-promoting factor in two independent TSC lines across a 6-day differentiation time course. Data of two independent biological replicates is normalized to TSC (stem) conditions and are displayed as mean ± SEM. (G) LIF protein abundance determined by ELISA and displayed as relative fold-change normalized to total protein levels. Values are displayed as mean ± SEM. Statistical analysis was by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05.
Fig 3: Tumor-produced LIF induces EMH.(A) LIF concentration from serum of mice with or without 21 days of PyMT-B6 tumor (n = 11). (B) LIF concentration from base media, 1956, LLC, or PyMT-B6-conditioned media (n = 4, significance assigned by one-way ANOVA). (C–E) In mice with 10 days of LIF overexpression or empty vector control, fraction of PMNs in the PB as a percent of total leukocytes (C, n = 11–12), KSL cells per spleen (D, n = 7–8), GMP cells per spleen (E, n = 7–8). (F) Twenty-eight days after subcutaneous injection of 2.5 × 105 PyMT-B6 ?G-CSF parental cells or ?G-CSF ?LIF cells, KSL cells per spleen (F, n = 12). Processed data for this figure can be found in S1 Data. EMH, extramedullary hematopoiesis; GMP, granulocyte–monocyte precursor; KSL, Kit+/Sca-1+/Lineage-; LIF, leukemia inhibitory factor; LLC, Lewis lung carcinoma; PB, peripheral blood; PMN, polymorphonuclear neutrophil; PyMT, polyomavirus middle T antigen.
Fig 4: LIF directly expands the splenic niche.(A) Violin plot of expression of Lifr in reanalyzed scRNA-seq data from Tikhonova and colleagues of BM niche cell types (0 –HSC, 1 –endothelium (EC), 2 –proliferating CD45+, 3 –ABS cell, 4 –GMP, 5 –CLP, 6 –Sca-1+ EC, 7 –B cell progenitor, 8 –osteoblast, 9 –RBC progenitor). (B) In LIFRflox mice with LIF overexpression and Cdh-Cre+ or Cdh5-Cre-, KSL cells per spleen (n = 10–11, contains male mice). (C–F) In day 12 postpartum LIFRflox mice with PDGFRa-Cre+ or PDGFRa-Cre- littermates, KSL cells as a fraction of total splenic CD45+ cells (C, n = 6–9, contains male mice), KSL cells as a fraction of total BM CD45+ cells (D, n = 6–9, contains male mice), KSL cells per spleen (E, n = 6–9, contains male mice), KSL cells per leg (F, n = 6–9, contains male mice). (G) Twenty-one days after injection of 2 × 106 1956 tumor cells, 5 × 105 LCC tumor cells, or 5 × 105 PyMT-B6 tumor cells, injected subcutaneously compared to control animals injected with PBS, total ABS cells per spleen (n = 4, 1 independent experiment, significance assigned by one-way ANOVA). (H) Representative RT-qPCR expression data of Mki67 from splenic ABS cells treated for 72 hours with 20 ng/mL LIF (n = 6). (I) Fraction of splenic PDGFRa+ cell that are Ki67+ by immunofluorescence with 7 days of LIF overexpression or empty vector lentivirus control (n = 8). (J, K) Enrichment of MSCs (J) and stromal scoring (K) as calculated by xCell from RNA-seq data of human tumors split by top and bottom quartile of LIF expression (n = 416–417). Processed data for this figure can be found in S1 Data. BM, bone marrow; CLP, common lymphoid progenitor; EC, endothelial cell; GMP, granulocyte–monocyte precursor; HSC, hematopoietic stem cell; KSL, Kit+/Sca-1+/Lineage-; LLC, Lewis lung carcinoma; LIF, leukemia inhibitory factor; MSC, mesenchymal stromal cell; PBS, phosphate buffered saline; PyMT, polyomavirus middle T antigen; RBC, red blood cell; RT-qPCR, reverse transcription quantitative PCR; scRNA-seq, single-cell RNA-sequencing.
Supplier Page from Abcam for Mouse LIF ELISA Kit