Distribution of EC in BCa tissues increases from grade 1 to grade 3, manifesting as a risk factor for prognosis. Angiogenesis, defined as the formation of new blood vessels from existing vasculature, plays an important role in tumor growth and metastasis by providing oxygen, nutrients and growth factors to cancer cells. CD31, an existing marker in ECs, is widely used for evaluating microvessels in numerous studies [26]. Anti-CD31 microvessel immunostaining has several advantages over anti-factor VIII, and is a more sensitive method for detecting small, immature microvessels or single EC. This could be of importance in revealing a possible correlation of tumor angiogenesis with metastatic behavior, prognosis, or angiogenic factor overexpression [27]. In the present study, CD31 was used as an EC marker in the BCa sections. As shown in Fig. 1A and B, the number of CD31+ cells gradually increased according to tumor grade (grade 3 vs. grade 1; P = 0.014 < 0.05), indicating that the number of ECs in grade 3 is significantly increased compared with grade 1 in BCa sections. To further understand the association between the distribution of CD31+ cells and the time from tumor diagnosis to relapse, 49 patients were followed up until the tumor relapse period. As expected, the time from tumor diagnosis to relapse is negatively related to the number of CD31+ cells (Fig. 1C). All 49 patients are divided into two groups by the average number of CD31+ cells (average, 5.69). Kaplan-Meier analysis suggested that number of CD31+ cells is an independent risk factor for patients with BCa (Fig. 1D).
Tumor cells cultured in CM manifests with enhanced malignant and proliferation abilities. The role of stromal cells and the tumor microenvironment in general in modulating tumor sensitivity is increasingly becoming a key consideration for the development of active anticancer therapeutics [4]. Tumor-stroma interactions affects tumor cell signaling, survival, proliferation and drug sensitivity via different mechanisms, and among all stromal cells, microvessel endothelial cells are irreplaceable [12]. Consistent with a previous study [28], the present results showed that the number of ECs increased according to tumor progression and is negatively correlated to BCa prognosis. It was hypothesized that ECs may exert direct or indirect effects on BCa cells, aside from metabolism-related roles, such as oxygen supply. To further investigate this hypothesis, CM was used to culture T24 and J82 cells (Fig. 2A). As shown in Fig. 2C, Boyden chamber assay indicated that culture in CM resulted in enhanced malignancy of T24/J82 cells. This also lead to the process of epithelial to mesenchymal transition (EMT) as indicated by western blotting and RT-qPCR results (Fig. 2B).
Additionally, the proliferation abilities were assessed by BrdU incorporation to demonstrate whether culturing T24/J82 cells in CM affected the proliferative ability of tumor cells. As shown in Fig. 2D, comparing with the control, the fluorescence intensity of T24/J82 cells cultured in CM increased by > 5-fold, indicating that CM contribute to tumor cell proliferation in some way.
Tumor cells cultured in CM manifest with enhanced recruitment abilities. Due to the different distribution of ECs in differing grade of BCa sections and the role of CM on educating tumor cell lines, we determined whether there is discrepancy in HUVECs recruited by parental tumor cell and CM-cultured tumor cells. As shown in Fig. 2E, T24/J82 cells cultured in CM manifested with enhanced ability of HUVEC recruitment compared with parental T24/J82 cells. However, the mechanism is still unknown.
CM culture induces elevated expression of CXCL1/5/8 and CXCR2/3 in T24/J82 cells compared with controls. Recent studies showed that the CXC-chemokine family, which are produced by tumor and stromal cells, play important roles in the process of tumor angiogenesis, recruitment and progression [17, 29]. Our previous results showed evidence that the BCa cells cultured in CM showed enhanced malignancy, proliferation and HUVEC recruitment abilities. It was hypothesized that this chemokine family may be involved in this process. To investigate this hypothesis, CXC-chemokines and their receptors were monitored in parental and CM-cultured T24/J82 cells. CM culture resulted in different expression of CXC chemokines in T24 and J82 cells, especially CXCL1, CXCL5 and CXCL8 (Fig. 3A). The corresponding receptors of these chemokines were also monitored, whereby CXCR2 and CXCR3 levels significantly increased compared with parental cell lines (Fig. 3B). This indicated that the CM contributed to the upregulation of those CXC-chemokines /receptors via an unknown mechanism, which was further investigated.
NF-κB signaling in T24/J82 cells is activated by CM culture. Numerous studies showed that the NF-κB pathway played important roles in the progression of cancer, through which cancer progressed into more malignant types [30, 31]. This pathway in cancer cells could be activated by the components of the tumor niche or the interaction between tumor and stromal cells of the niche [32–34]. The present results demonstrate that in the BCa cell lines T24 and J82, CM culture lead to P65 nuclear translocation, which indicated the activation of the NF-κB signaling pathway (Column 2 vs. column 1; Fig. 4A).
Inhibition of the NF-κB signaling pathway in T24/J82 cells attenuates CM culture-induced malignancy, HUVEC recruitment and proliferation, accompanied by EMT reversal. In bladder transitional cancer, EMT is regarded to be one of the important processes in maintaining malignancy and promoting tumor progression [35]. Activation of the NF-κB signaling pathway was reported to be positively related to BCa progression but negatively related to prognosis. Based on this observation, siRNA for knocking down NF-κB, or PDTC, an inhibitor of NF-κB pathway, was used to inhibit this signaling. Both siRNA and PDTC resulted in the attenuation of CM culture-induced NF-κB signaling (Fig. 4A; column 3 vs. column 2; Fig. 4B-D), accompanied by attenuated malignancy (Fig. 5A). In addition, CM culture-induced HUVEC recruitment by T24/J82 cells was also attenuated (Fig. 5B). Inhibition of this signaling significantly inhibited the CM culture-induced proliferation of T24/J82 cells (Fig. 5C). This indicated that the NF-κB signaling pathway is a regulator during CM culture. Additionally, inhibition of NF-κB signaling also lead to the reversal of EMT, including downregulation of matrix metalloproteinase MMP 2, MMP9, N-Cadherin, Vimentin but upregulation of E-Cadherin (Fig. 4B), consistent with reports showing that the NF-κB pathway is a key regulator of EMT.
Inhibition of the NF-κB signaling pathway results in the decreased expression of CXCL1/8 and CXCR2 in CM-cultured tumor cell lines.
Previous results suggested that tumor cells cultured in CM resulted in different expression of CXC-chemokines/receptors, accompanied by the enhanced tumor cell malignancy, proliferation and ability of HUVEC recruitment, and inhibition of the NF-κB pathway attenuated these CM culture-induced phenomena. This result lead us to postulate the roles of the NF-κB signaling pathway on the expression of these CXC-chemokines/receptors. T24/J82 cells were cultured in CM in the presence of PDTC or si-p65, and RT-qPCR was performed to assess the expression of CXC-chemokines/receptors in tumor cells. As shown in Fig. 4A, the nuclear translocation of P65 was reversed by PDTC. RT-qPCR results also indicated that si-p65 induced the downregulation of P65 expression (Fig. 4C) CM culture-induced elevation of CXCL1/8 and its receptor CXCR2 was significantly attenuated in the absence of NF-κB signaling (Fig. 5D); however, CXCL5 and CXCR3 expression was not significantly altered (Fig. 5D).
Activation of the NF-κB signaling pathway in T24/J82 cells by TNF-α mimics the effects induced by CM culture. To determine whether CM culture-induced effects involved the NF-κB signaling pathway, TNF-α, a classical activator of NF-κB signaling [30], was used to activate this pathway. This was followed by monitoring tumor cell malignancy, proliferation and HUVEC recruitment. TNF-α-treated T24/J82 manifested as P65 nuclear translocation, as indicated by immunofluorescence (Fig. 6A), indicating the activation of the NF-κB signaling pathway. As expected, this activation resulted in enhanced malignancy (Fig. 6D), proliferation (Fig. 6F) and ability of HUVEC recruitment (Fig. 6E). In addition, TNF-α induced the elevation of CXCL1/8 and CXCR2 levels (Fig. 6B) and contributed to EMT (Fig. 6C).
Functional inhibition of CXCR2 attenuates CM culture-induced HUVEC recruitment.
It was reported that CXC chemokines played important roles in inflammatory cell recruitment, and the present results also showed that CM culture-induced upregulation of CXCL1/8 in tumor cells was accompanied by enhanced HUVEC recruitment. As aforementioned, binding with the corresponding receptor is the first step for CXCL chemokine to play its role. In addition, CXCR2 is stably expressed on the surface of HUVECs, as shown by our results (data not shown). Therefore, it was hypothesized that enhanced HUVEC recruitment by CM-cultured T24/J82 cells might be mediated by the CXCL1/8-CXCR2 axis. CXCR2 neutralizing antibody (1 µg/ml; Invitrogen; Thermo Fisher Scientific, Inc.) was used to treat tumor cells in the presence of CM. As shown in Fig. 7A, enhanced HUVEC recruitment induced by CM-cultured T24/J82 cells were attenuated. However, this functional inhibition showed no visible effects on tumor cell proliferation (Fig. 7B) and malignancy (Fig. 7C).
CM culture of T24/J82 cells resulted in IκBα degradation and activation of the NF-κB signaling pathway. IκBα is regarded as the key upstream inhibitor of NF-κB signaling, whereby the degradation of IκBα is necessary for NF-κB signaling activation [36]. It was hypothesized that CM-cultured T24/J82 cells might be involved in the degradation or decreased expression of IκBα. Therefore, the expression of this protein in CM-cultured T24/J82 cells was monitored using RT-qPCR and western blotting. Prior to protein extraction, cells were treated with MG-132, an inhibitor of the 26S-dependent protein degradation pathway, for 4 h to prevent spontaneous IκBα degradation. As shown in Fig. 7D (top panel), CM culture induced the attenuation of IκBα, however, this attenuation did not occur at the mRNA level (Fig. 7D; bottom panel). This indicated that CM culture-induced attenuation of IκBα may have been due to degradation instead of decreased expression.