br Of great interest and importance continuous monitoring in
Of great interest and importance, continuous monitoring indicated that the serum IL-8 levels in the chemoresistant patients increased gradually during NAC (P = 0.023), and it decreased dramatically post radical surgery (446.71 ± 111.07 pg/ml vs 33.74 ± 12.27 pg/ml, P = 0.037); however, it decreased slightly in chemosensitive patients (P = 0.070) (Fig. 1B). The serum IL-6 level remained nearly unchanged in these two groups (P > 0.05) (Fig. 1C). Thus, it may be considered that the serum IL-8, rather than IL-6, in gastric cancer patients is in-volved in chemoresistance, and high IL-8 level may be produced by tumor tissues.
3.2. IL-8 is highly expressed in CAFs in the tumor microenvironment of gastric cancer
To determine whether IL-8 was expressed in tumor tissues in these patients and which SRS 16-86 IL-8 located in, we performed im-munohistochemistry assays. As shown in Fig. 2, IL-8 was highly ex-pressed in gastric tumor tissues in the chemoresistant patients rather than in the chemosensitive patients. Cytologically, IL-8 exclusively lo-cated in the fibroblasts in the tumors, which expressed α-SMA, namely IL-8 was highly expressed in CAFs.
Furthermore, we obtained the primary cultured CAFs from gastric cancer tissues and normal fibroblasts (NFs) from the corresponding noncancerous mucosa tissues in these patients, and we evaluated the secretion of IL-8 in these CAFs and NFs. Fig. 3A showed that α-SMA was overexpressed in CAFs by comparison with NFs (P = 0.018), indicating these primary cultured fibroblasts were activated. Higher level of IL-8 was detected in the cell supernatant of CAFs rather than NFs (P = 0.012).
It has been well established that cancer cell supernatant may acti-vate fibroblasts. Thus, we used the conditioned media from human gastric cancer cell lines, AGS and MGC-803, to co-culture human sto-mach fibroblast line Hs738. As shown in Fig. 3B, both AGS supernatant and MGC-803 supernatant upregulated the α-SMA expression in Hs738 (P < 0.05), namely gastric cancer cell supernatant activates fibro-blasts. Importantly, the activated Hs738 produced more IL-8 (Fig. 3C, P < 0.05). Thus, it can be inferred that IL-8 secreted by CAFs was in-volved in human gastric cancer chemoresistance.
3.3. IL-8 mediates chemoresistance to cispatin in human gastric cancer cells via NF-κB activation and ABCB1 up-regulation
IL-8 is one of proinflammatory cytokine, and we assayed the ex-pression of nuclear factor kappa B (NF-κB) pathway-associated proteins. We found that the expression of PI3K, p-AKT, p-IKb and p-p65 was enhanced after IL-8 treatment (Fig. 4B), indicating IL-8 mediates che-moresistance via NF-κB activation. Importantly, ATP-binding cassette subfamily B member 1 (ABCB1) expression was increased accordingly. It has been well established that ABCB1 acts as drug pump to reduce the intracellular drug concentration, and leads to chemoresistance in sev-eral kinds of human cells . We further examined the expression of ABCB1 and p-p65 in human gastric cancer tissues, and showed that ABCB1 and p-p65 were overexpressed in tumor tissues of chemoresis-tant patients rather than tissues of chemosensitive patients (P = 0.046 and P = 0.033, respectively) (Fig. 4C). Thus, it can be concluded that CAFs derived IL-8 promotes chemoresistance in human gastric cancer via NF-κB activation and ABCB1 up-regulation.
Recurrence and metastasis after radical gastrectomy results in dismal prognosis in advanced gastric cancer patients, and cancer's growing chemoresistance continues to be a challenge for the overall survival of patients with advanced gastric cancer undergoing che-motherapy [13,27,28]. Resistance to chemotherapy involves several mechanisms concerning both genetic characteristics of cancer cells and the interaction with their tumor microenvironment (TME), which consists of the extracellular matrix (ECM), growth factors, cytokines, and a variety of cell types, such as CAFs, immune cells, endothelial cells, and inflammatory cells . CAFs are the main cell type in TME, and originate from normal fibroblasts, mesenchymal stem cells (MSCs), and even the perivascular cells in the vicinity of tumor sites during tumourgenesis [19,30]. CAFs specifically play crucial roles in pro-moting tumor development and progression of many cancer types, in-cluding gastric cancer [21,27,31,32]. However, the exact molecular mechanism underlying the interaction between gastric cancer cells and CAFs remains poorly understood .
Alpha-smooth muscle actin (α-SMA), fibroblast-specific protein-1 (FSP-1), and fibroblast-activated protein (FAP) are specific markers of CAFs, and α-SMA was used in our study. Intensive researches have revealed that CAFs mediates chemoresistance through producing IL-6 [17,20,21,33], IL-11 , Lumican , and vascular endothelial growth factor (VEGF) , and that the expression of tumor en-dothelial marker 1 (TEM1)  or CXCL12  in CAFs is correlated with a poor prognosis in patients with gastric cancer. Our study in-vestigated the clinicopathological significance of IL-6 and IL-8 in ad-vanced gastric cancer patients, and IL-8 derived from CAFs was re-vealed to be associated with gastric cancer chemoresistance, which may be due to the organ specificity of fobroblasts. We speculated that a set of factors are involved in the mechanism of CAFs in chemoresistance, and that its roles in chemoresistance may be tumor-specific, which needs to be further investigated.