RESUMEN
BACKGROUND: Cancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment and significantly contribute to immune evasion. We investigated the effects of CAFs on the immune function of CD4+ and CD8+ T cells in non-small cell lung cancer (NSCLC). METHODS: We isolated CAFs and normal fibroblasts (NFs) from tumors and normal lung tissues of NSCLC patients, respectively. CAFs were co-cultured with activated T cells to evaluate their immune regulatory function. We investigated the effect of CAF conditioned medium (CAF-CM) on the cytotoxicity of T cells. CAFs were also co-cultured with activated peripheral blood mononuclear cells and further incubated with cyclooxygenase- 2 (COX2) inhibitors to investigate the potential role of COX2 in immune evasion. RESULTS: CAFs and NFs were isolated from the lung tissues (n=8) and lymph nodes (n=3) of NSCLC patients. Immune suppressive markers, such as COX2 and programmed death-ligand 1 (PD-L1), were increased in CAFs after co-culture with activated T cells. Interestingly, CAFs promoted the expression of programmed death-1 in CD4+ and CD8+ T cells, and strongly inhibited T cell proliferation in allogenic and autologous pairs of CAFs and T cells. CAF-CM decreased the cytotoxicity of T cells. COX2 inhibitors partially restored the proliferation of CD4+ and CD8+ T cells, and downregulated the expression of COX2, prostaglandin E synthase, prostaglandin E2, and PD-L1 in CAFs. CONCLUSION: CAFs promote immune evasion by suppressing the function of CD4+ and CD8+ T cells via their effects on COX2 and PD-L1 in NSCLC. The immunosuppressive function of CAFs could be alleviated by COX2 inhibitors.
RESUMEN
Extracellular vesicles (EVs) have emerged as novel biomarkers and therapeutic material. However, the small size (~200 nm) of EVs makes efficient separation challenging. Here, a physical/chemical stress-free separation of EVs based on diffusion through a nanoporous membrane chip is presented. A polycarbonate membrane with 200 nm pores, positioned between two chambers, functions as the size-selective filter. Using the chip, EVs from cell culture media and human serum were separated. The separated EVs were analyzed by nanoparticle tracking analysis (NTA), scanning electron microscopy, and immunoblotting. The experimental results proved the selective separation of EVs in cell culture media and human serum. Moreover, the diffusion-based separation showed a high yield of EVs in human serum compared to ultracentrifuge-based separation. The EV recovery rate analyzed from NTA data was 42% for cell culture media samples. We expect the developed method to be a potential tool for EV separation for diagnosis and therapy because it does not require complicated processes such as immune, chemical reaction, and external force and is scalable by increasing the nanoporous membrane size.