Citation
- Authors: Zhou WH. et al.
- Year: 2022
- Journal: Discov Oncol 13 67
- Applications: in vitro / siRNA / INTERFERin
- Cell types:
- Name: PCI-37B
- Name: PCI-4B
Method
In brief, PCI-4B and PCI-37B cells were seeded the day before transfection to reach 30–50% confluency at the time of transfection. siRNA duplexes were diluted into reduced serum media Opti-MEM®. Then, add transfection reagent INTERFERin® into the siRNA solution, vortex-mixed, and incubated for 10 min at room temperature. Finally, INTERFERin®-siRNA complexes were added in complete fresh medium to incubate cells at 37 ℃. 24 h later, the transfected cells were collected for further experiments. The knockdown efficiency of siRNA was tested by quantitative real-time PCR assay.
Abstract
Purpose: This study aimed to investigate the impact of CC chemokine receptor 7 (CCR7) on the recruitment and polarization of tumor-associated macrophages (TAMs) in oral squamous cell carcinoma (OSCC).
Methods: We analyzed CCR7 expression pattern, clinicopathological significance, and its association with M2 macrophage infiltration in OSCC by bioinformatic methods. Small interfering RNA (siRNA) was utilized to silence CCR7 in OSCC cells. Conditioned media (CM) was harvested from transfected OSCC cells to establish a co-culture model of THP-1 derived macrophages and OSCC cells. Transwell assay and cell adhesion assay were performed to examine the effect of CCR7 on macrophages recruitment and adhesion. Cytoskeleton was labelled by phalloidin to observe macrophage morphological changes. Moreover, phenotypic alteration of macrophages was measured using quantitative real-time PCR (qRT-PCR), flow cytometry, and immunofluorescence (IF) staining. Ultimately, recombinant human CCL19 and CCL21 were added into the medium of THP-1 derived macrophages to explore their effects on polarization in vitro.
Results: In OSCC patients, the overexpression of CCR7 positively correlated with lymph node metastasis and M2 macrophage infiltration. Macrophage not only exhibited enhanced migration, invasion and adhesion abilities, but also appeared more spindle and branched in vitro when treated with CM from OSCC cells. However, these phenomena were abrogated with knockdown of CCR7. We also discovered that inhibition of CCR7 in OSCC cells suppressed TAMs polarization to an M2 phenotype. In addition, recombinant human CCL19 and CCL21 promoted macrophage M2-polarization in vitro.
Conclusion: CCR7 in OSCC cells promoted recruitment and M2-polarization of THP-1 derived macrophages in vitro by regulating production of CCL19 and CCL21.