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Single-cell transcriptomic effects of LOX knockdown on tumor cell xenografts implanted on aged mouse breast dECM scaffolds

ABSTRACT: We profiled the single-cell transcriptomic effects of LOX (lysyl oxidase) knockdown on MCF10DCIS.com human breast tumor cells and on CD11b+ tumor-associated myeloid cells in an aged-microenvironment xenograft model. GFP-reporting MCF10DCIS.com cells transduced with either pLKO empty-vector (control) or pLKO-LOX shRNA were seeded onto decellularized/delipidized breast dECM scaffolds derived from aged C57BL/6 mice, cultured for 12 days, and implanted bilaterally into Rag1-/- (B6.129S7-Rag1tm1Mom/J) host mice (right flank = pLKO ctrl, left flank = shLOX, matched within-mouse pairing). Tumors grew for 7 weeks. Three biological replicate pairs (n=3 pLKO + n=3 shLOX, from 3 host mice) were selected for Parse Biosciences Evercode WT v2 single-cell RNA-seq. Each tumor was dissociated and split into a CD11b- tumor-cell fraction and a CD11b+ tumor-associated myeloid fraction, yielding 12 biological samples total (6 tumor + 6 immune). All 12 samples were combinatorially barcoded together and split into 8 sublibraries. Deposited FASTQs are per-biological-sample read groups: after split-pipe v1.0.6p combinatorial demultiplexing, reads were partitioned by assigned sample identity and concatenated across all 8 sublibraries and both sequencing runs, producing one R1/R2 pair per biological sample.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE333415 | GEO | 2026/05/27

REPOSITORIES: GEO

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Project description:Lipoxygenases (LOX) form a heterogeneous family of lipid peroxidizing enzymes capable of converting polyunsaturated fatty acids to their hydroperoxy derivatives. They have been implicated in basic cellular processes such as proliferation and differentiation but are also involved in the pathogenesis of various diseases such as inflammatory disorders and atherosclerosis. The catalytic activity of LOXs increases the intracellular peroxide tone, which is an important parameter for expression regulation of redox-sensitive genes. Previously, we showed that induction of 12/15-expression by interleukin-4 profoundly alters the gene expression pattern of human peripheral monocytes, which led to a profound switch of the cellular phenotype. To explore, which of these changes might be a direct consequence of 12/15-LOX expression we stably transfected a permanent human monocytic cell line (U937) with a 12/15-LOX containing eukaryotic expression plasmid and an empty control vector (mock-control). After a multi-step selection procedure a viable clones (12/15-LOX transfectants and mock-transfectants) were isolated and further cultured. We found that 12/15-LOX expression did not significantly alter basic cellular functions and that the cells, which express the 12/15-LOX at similar levels as IL4-treated human monocytes, did not show major functional deficits when compared with the mock-transfectants. However, more detailed comparison of the gene expression profiles of 12/15-LOX transfected cells and that of corresponding mock-transfectants revealed subtle differences. Among the 22,300 genes present on the chip about one half was not expressed regardless whether 12/15-LOX was present in the cells or not. In 12/15-LOX expressing cells 11,000 genes (50 % of the genes present on the chip) and in the corresponding mock-transfectants 10,600 genes (47 % of the genes present on the chip) were expressed. Among the expressed genes only 900 (7 %) were upregulated more than 2-fold and 1,400 (12 %) were down regulated to a similar extent. Among the regulated gene products we detected several connective tissue matrix proteins (collagen isoforms, lamins, fibulins, fibronectins, aggrecans, claudins), extracellular proteinases (serpins, matrix metalloproteinases), tissue inhibitors of metalloproteinases, growth factors and their receptors, and adhesion molecules. In most cases, we did not see simultaneous up- or down-regulation of all family members but rather selective regulatory response of individual proteins (for instance 5-fold upregulation of collagen XV, alpha –1, but 20-fold downregulation of collagen VII, alpha-1 after 12/15-LOX transfection). These data suggest that induction of 12/15-LOX specifically modifies the gene expression pattern of U937 cells but did not lead to comprehensive alterations. Keywords: other

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Single-cell transcriptomic effects of LOX knockdown on tumor cell xenografts implanted on aged mouse breast dECM scaffolds

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