Dissection of paracrine/autocrine interplay in lung tumor microenvironment mimicking cancer cell-monocyte co-culture models reveals proteins that promote inflammation and metastasis

Abstract

Abstract Background Tumor cell-monocyte interactions play crucial roles in shaping up the pro-tumorigenic phenotype and functional output of tumor-associated macrophages. Within the tumor microenvironment, such heterotypic cell–cell interactions are known to occur via secretory proteins. Secretory proteins establish a diabolic liaison between tumor cells and monocytes, leading to their recruitment, subsequent polarization and consequent tumor progression. Methods We co-cultured model lung adenocarcinoma cell line A549 with model monocytes, THP-1 to delineate the interactions between them. The levels of prototypical pro-inflammatory cytokines like TNF-𝛼, IL-6 and anti-inflammatory cytokines like IL-10 were measured by ELISA. Migration, invasion and attachment independence of lung cancer cells was assessed by wound healing, transwell invasion and colony formation assays respectively. The status of EMT was evaluated by immunofluorescence. Identification of secretory proteins differentially expressed in monocultures and co-culture was carried out using SILAC LC–MS/MS. Various insilico tools like Cytoscape, Reacfoam, CHAT and Kaplan–Meier plotter were utilized for association studies, pathway analysis, functional classification, cancer hallmark relevance and predicting the prognostic potential of the candidate secretory proteins respectively. Results Co-culture of A549 and THP-1 cells in 1:10 ratio showed early release of prototypical pro-inflammatory cytokines TNF-𝛼 and IL-6, however anti-inflammatory cytokine, IL-10 was observed to be released at the highest time point. The conditioned medium obtained from this co-culture ratio promoted the migration, invasion and colony formation as well as the EMT of A549 cells. Co-culturing of A549 with THP-1 cells modulated the secretion of proteins involved in cell proliferation, migration, invasion, EMT, inflammation, angiogenesis and inhibition of apoptosis. Among these proteins Versican, Tetranectin, IGFBP2, TUBB4B, C2 and IFI30 were found to correlate with the inflammatory and pro-metastatic milieu observed in our experimental setup. Furthermore, dysregulated expression of these proteins was found to be associated with poor prognosis and negative disease outcomes in lung adenocarcinoma compared to other cancer types. Pharmacological interventions targeting these proteins may serve as useful therapeutic approaches in lung adenocarcinoma. Conclusion In this study, we have demonstrated that the lung cancer cell-monocyte cross-talk modulates the secretion of IFI30, RNH1, CLEC3B, VCAN, IGFBP2, C2 and TUBB4B favoring tumor growth and metastasis.

Keywords

• Tumor microenvironment
• Tumor associated macrophages
• Inflammation
• Secretome
• Metastasis