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Complement Activation Drives a Metastatic Phenotype of Ovarian Cancer Cells Co-Cultured with Adipocytes

ABSTRACT: Background: The preferred site of metastasis in ovarian cancer (OC) is the fat-rich omentum. The interaction between cancer cells and adipocytes induces transcriptomic changes driving an invasive and pro-metastatic phenotype. Here we studied the effects of the OC cell-adipocyte crosstalk by using direct co-culture with immortalized human visceral pre-adipocytes and OC cells. Methods: OVCAR5 or OVCAR8 OC cells expressing either GFP or RFP were co-cultured with matured immortalized visceral pre-adipocyte (VNPAD). After direct co-culture, OC cells were FACS-sorted and cell proliferation, invasiveness, resistance to cisplatin were assessed. RNA-sequencing determined transcriptomic changes induced by co-culture. Immunohistochemistry (IHC) and ELISA measured C3 expression in metastatic tumors and in malignant ascites. Results: Direct co-culture with VNPAD led to increased proliferation, invasiveness and resistance to cisplatin of OC cells compared to monoculture. RNA-sequencing revealed 205 differentially expressed genes (DEGs) common to both VNPAD co-cultured OVCAR5 and OVCAR8 cells and enriched pathways such as PI3K/AKT and complement activation. Co-culture induced lipid transfer into OC cells promoted upregulation of complement C3 and C5 units, as measured at mRNA and protein levels. C3 or C5 inhibition reverted the invasive phenotype of OC cells and C3 knockdown reduced tumor progression in a xenograft model. Increased C3 expression was detected in metastatic vs. primary ovarian tumors (p<0.0001) and increased C3 secretion was observed in ascites from women with BMI > 25 (p = 0.01). C3 upregulation in OC cells was driven through activation of stress response pathway regulated by ATF4. Conclusions: Co-culture of adipocytes and cancer cells drives an invasive and chemo-resistant phenotype. These changes are induced by transfer of lipids from adipocytes to cancer cells causing activation of the complement proteins C3, C5, and of the integrated stress response pathway.

ORGANISM(S): Homo sapiens

PROVIDER: GSE271466 | GEO | 2025/04/03

REPOSITORIES: GEO

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Project description:Systemic inflammation like in sepsis is still lacking specific diagnostic markers and effective therapeutics. The first line of defense against intruding pathogens and endogenous damage signals is pattern recognition by e.g., complement and Toll-like receptors (TLR). Combined inhibition of a key complement component (C3 and C5) and TLR-co-receptor CD14 has been shown to attenuate certain systemic inflammatory responses. Using DNA microarray and gene annotation analyses, we aimed to decipher the effect of combined inhibition of C3 and CD14 on the transcriptional response to bacterial challenge in human whole blood. Importantly, combined inhibition reversed the transcriptional changes of 70% of the 2335 genes which significantly responded to heat-inactivated Escherichia coli by on average 80%. Single inhibition was less efficient (p<0.001) but revealed a suppressive effect of C3 on 21% of the responding genes which was partially counteracted by CD14. Furthermore, CD14 dependency of the Escherichia coli-induced response was increased in C5-deficient compared to C5-sufficient blood. The observed crucial distinct and synergistic roles for complement and CD14 on the transcriptional level correspond to their broad impact on the inflammatory response in human blood, and their combined inhibition may become inevitable in the early treatment of acute systemic inflammation. A microarray study was performed on whole blood from two healthy donors, a C5-deficient patient (C5D) (with and without C5-reconstitution) incubated for 120 min at 37oC with (i) PBS, (ii) E. coli or (iii) E. coli and inhibitors of complement (C3 or C5aR) and/or CD14 or inhibitor controls. The experiments were performed as biological replicates on two different days (healthy donor and C5D). Due to lack of a second C5-deficient patient, microarray analyses were prerformed twice (technical replicate) for C5D and day 1. Expression data in presence of PBS were used as reference for those in pressence of E. coli. Expression data in presence of E. coli were used as reference for those in pressence of E. coli and inhibitors. Expression data from 0 min incubation with PBS represent the initial state (T0). Whole blood was lysed in Applied Biosystems AB6100 total RNA chemistry lysis buffer and stored at -80oC prior to RNA extraction.

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Complement Activation Drives a Metastatic Phenotype of Ovarian Cancer Cells Co-Cultured with Adipocytes

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