Tumor-associated macrophages induced spheroid formation by CCL18-ZEB1-M-CSF feedback loop to promote transcoelomic metastasis of ovarian cancer

Abstract

BackgroundOvarian cancer (OvCa)-tumor-associated macrophages (TAMs) spheroids are abundantly present within ascites of high malignant patients. This study investigated the mutual interaction of OvCa cells and TAMs in the spheroids.MethodsThree-dimensional coculture system and transwell coculture system were created to mimic the OvCa and TAMs in spheroids and in disassociated state. Transwell-migration assay and scratch wound healing assay were used to measure the invasive and migratory capacity. Western blot, quantitative reverse transcription-PCR and immunostaining were used to measure the mesenchymal and epithelial markers. Flow cytometry was used to assess the polarization of TAMs. Also, the differential gene expression profile of OvCa cells and OvCa cells from spheroids were tested by RNA-sequence. Finally, the ovarian mice models were constructed by intraperitoneal injection of ID8 or OvCa-TAMs spheroids.ResultsOur results indicated that the formation of OvCa-TAMs spheroids was positive related to the malignancy of OvCa cells. M2-TAMs induced the epithelial-mesenchymal transition of OvCa cells by releasing chemokine (C-C motif) ligand 18 (CCL18) in the spheroids. While, CCL18 induced macrophage colony-stimulating factor (M-CSF) transcription in OvCa cells through zinc finger E-box-binding homeobox 1 (ZEB1). This study further indicated that M-CSF secreted by OvCa cells drived the polarization of M2-TAMs. Therefore, a CCL18-ZEB1-M-CSF interacting loop between OvCa cells and TAMs in the spheroids was identified. Moreover, with blocking the expression of ZEB1 in the OvCa cell, the formation of OvCa-TAMs spheroids was impeded. In the ovarian mice models, the formation of OvCa-TAMs spheroids in the ascites was promoted by overexpressing of ZEB1 in OvCa cells, which resulted in faster and earlier transcoelomic metastasis.ConclusionThese findings suggested that the formation of OvCa-TAMs spheroids resulted in aggressive phenotype of OvCa cells, as a specific feedback loop CCL18-ZEB1-M-CSF in it. Inhibition of ZEB1 reduced OvCa-TAMs spheroids in the ascites, impeding the transcoelomic metastasis and improving the outcome of ovarian patients.

Cancer-Associated Fibroblasts: Mechanisms of Tumor Progression and Novel Therapeutic Targets

“…When such shedding occurs, tumor cells initially lose their original ECM and/or cell-to-cell attachments, creating an elevated risk for anoikis and subsequent apoptosis [ 83 ]. As a protective mechanism, disseminated OVCA cells cluster in the peritoneal fluid where they form heterotypic aggregates or spheroid-like structures composed of tumor cells and modified stromal cells including CAFs, TAMs, adipocytes and mesothelial cells [ 84 , 85 ]. These multicellular aggregates provide a unique opportunity to assess the contribution of the component cell types to the acquisition of a progressively malignant phenotype.…”

Spheroid Formation and Peritoneal Metastasis in Ovarian Cancer: The Role of Stromal and Immune Components

“…In malignant ascites, TAMs float separately or are located in the center of spheroids surrounded by tumor cells; they possess M2 polarization by the abundant expression of CD163 and CD206 [ 83 , 84 , 85 ]. Additionally, fewer CD163+ TAMs can be found outside of spheroids in a free-floating state [ 66 ].…”

“…In ovarian cancer patients, the five-year overall survival rate was significantly lower in OC patients with high percentages of CD68+ TAMs (>14.5%) in spheroids compared with low percentages (<14.5%) of CD68-positive cells [ 84 ]. EGF-secreted TAMs also increased the invasive and migratory capacity of SKOV3 cells isolated from in vitro 3D spheroids, generated by TAMs and tumor cells [ 85 ]. Spheroid formation was facilitated by the CCL18-ZEB1-M-CSF axis.…”

“…The morphology of SKOV3 cells isolated from spheroids resembled that of mesenchymal cells with increased expression of the mesenchymal markers (ZEB1, SNAIL and TWIST) and decreased E-cadherin expression, compared to SKOV3 cells in a transwell system ( Figure 2 ). In vivo, spheroids containing ZEB1-overexpressed OC cells and TAMs were shown to be critical for transcoelomic metastasis [ 85 ].…”

“…Numerous soluble tumor cell-derived and TAM-derived factors in ascites facilitate tumor progression [ 36 , 84 , 85 , 90 ]. A transcriptomic analysis revealed several signaling networks providing tumor cell-TAMs interactions in ascites.…”