CX3CL1 in the red bone marrow promotes renal cell carcinoma to metastasize to the spine by involving the Src-related pathway

by Selleckchem | Oct 30 2023

Spinal metastasis (SM) frequently occurs in renal cell carcinoma (RCC) patients. Our preliminary work showed that CX3CL1 plays a positive role in SM. The objective of the present study was to verify whether CX3CL1 activates the downstream pathway by binding to CX3CR1 in RCC cells, ultimately promoting RCC to metastasize to the spine. The expression of CX3CL1 and CX3CR1 in tissue samples was detected by immunohistochemistry and western blotting. ELISA was used to quantify the concentration of CX3CL1 in the serum. The expression level of CX3CR1 in RCC cell lines was also detected. The CellTiter-Glo assay and flow cytometry were used to analyze cell viability and apoptosis of RCC cells. Transwell and wound healing assay were used to analyze the effect of CX3CL1 on the invasion and migration ability of RCC cells. Specific inhibitors were used to interfere with key molecules in the signaling pathway to further explore the signal transduction in RCC cells after CX3CL1 stimulation. The expression of CX3CR1 in SM from RCC was higher than that in limb bone metastases. Among the five RCC cell lines, 786O cells expressed the highest level of CX3CR1. CX3CL1 neither inhibited the proliferation of 786O cells nor promoted the apoptosis of 786O cells. However, it promoted the migration and invasion of RCC cells. After CX3CL1 stimulation, Src and Focal adhesion kinase (FAK) phosphorylation levels increased in RCC cells. Bosutinib and PF-00562271 inhibited Src/FAK phosphorylation and cell motility and invasion triggered by CX3CL1 stimulation. CX3CL1 in the red bone marrow of spinal cancellous bone enhances migration and invasion abilities of RCC cells, thereby promoting RCC metastasize to the spine. The migration and invasion of RCC cells activated by CX3CL1 are at least partially dependent on Src/FAK activation.

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The study aimed to investigate the role of CX3CL1 (also known as fractalkine) in promoting spinal metastasis (SM) in renal cell carcinoma (RCC). The researchers conducted various experiments to examine the expression of CX3CL1 and its receptor CX3CR1, as well as the downstream signaling pathway activated by CX3CL1 in RCC cells.

First, the expression of CX3CL1 and CX3CR1 was assessed in tissue samples using immunohistochemistry and western blotting techniques. The results revealed higher expression levels of CX3CR1 in SM samples from RCC patients compared to limb bone metastases. Additionally, the researchers quantified the concentration of CX3CL1 in the serum using an enzyme-linked immunosorbent assay (ELISA).

Next, the expression level of CX3CR1 was examined in RCC cell lines. Among the five cell lines tested, 786O cells exhibited the highest expression of CX3CR1.

To determine the functional effects of CX3CL1 in RCC cells, the researchers performed several assays. They utilized the CellTiter-Glo assay to evaluate cell viability and flow cytometry to analyze apoptosis of RCC cells. The results indicated that CX3CL1 neither inhibited the proliferation nor promoted the apoptosis of 786O cells.

However, CX3CL1 did enhance the migration and invasion abilities of RCC cells. This was assessed through Transwell and wound healing assays, which demonstrated that RCC cells treated with CX3CL1 showed increased migration and invasion capacities compared to control cells.

To gain insights into the underlying molecular mechanisms, the researchers explored the signaling pathway activated by CX3CL1 in RCC cells. They found that after CX3CL1 stimulation, phosphorylation levels of Src (a non-receptor tyrosine kinase) and Focal adhesion kinase (FAK) increased in RCC cells. Src and FAK are known to play important roles in cell motility and invasion.

To further investigate the involvement of Src and FAK in mediating the effects of CX3CL1, the researchers used specific inhibitors (Bosutinib and PF-00562271) to interfere with these molecules in the signaling pathway. The inhibition of Src/FAK phosphorylation resulted in reduced cell motility and invasion triggered by CX3CL1 stimulation.

In conclusion, this study suggests that CX3CL1, through its binding to CX3CR1, enhances the migration and invasion abilities of RCC cells, potentially promoting RCC metastasis to the spine. Furthermore, the migration and invasion of RCC cells induced by CX3CL1 are at least partially dependent on Src/FAK activation.