Regulative role of the CXCL13-CXCR5 axis in the tumor microenvironment

Abstract Chemokines are best known for their abilities of recruiting immune cells and forming lymphoid tissue. Through interactions between chemokines and their receptors, various immune cell subsets are recruited into the tumor microenvironment which is the primary location for tumor cells interacting with responding host cells. In recent decades, a large volume of studies have revealed chemokines’ role in the tumor microenvironment in regulating tumor growth, invasion, and/or metastasis as well as tumor immune response; however, their molecular mechanisms are not well understood. Recently, increasing evidence has reported the importance of the CXCL13-CXCR5 axis in the tumor microenvironment of various human malignancies. Thus, in this review, we will focus on the CXCL13-CXCR5 axis and elaborate on the expression patterns, regulating and corresponding regulatory mechanisms as well as clinical values in a wide range of human cancers.

series of different chemokines, host cells and tumors cells in the tumor microenvironment can recruit and activate different types of immune cell and pathways to regulate anti-tumor and pro-tumor responses. 6,7 Recently, the expression patterns and regulative roles of most chemokines in various human tumor microenvironments have been summarized. 8,9 In particular, CXC-chemokine ligands and their corresponding receptors have been proven to play an important role in angiogenesis, neoplastic transformation, as well as cancer cell migration, invasion, and metastasis. 10,11 However, these reviews completely ignored CXCL13, one of the most important members of the CXC-chemokine family. CXCL13 is a chemotactic protein for B cells, and its receptor, CXCR5, is expressed by a specific subset of T cells. 12 CXCL13 is usually expressed by stromal cells and follicular DC (FDC) in B cell follicles. 13 It is also expressed in lymphoid organs, such as in lymph nodes and the spleen. 13 Studies have proven that CXCL13 can also be secreted by tumor cells, myofibroblasts, CXCL13producing CD4+ follicular helper T cells, bone marrow endothelial cells, and osteoblasts. 9,[12][13][14] Also, CXCL13 can sustain an active immune response by inducing an influx of immune cells to lymph organs and activating B cells to produce immunoglobulins. 15 To date, a large number of studies have reported that CXCL13 and CXCR5 might also play important roles in regulating tumor growth, progression, and metastasis in the tumor microenvironment. [16][17][18][19][20][21] Figure 1 shows the pathway of the CXCL13-CXCR5 axis in promoting tumor immunity and regulating tumor cell growth, invasion, and migration. In this review, we will discuss the regulative role of CXCL13-CXCR5 axis in the tumor microenvironment.

Role of the CXCL13-CXCR5 axis in breast cancer
Through quantified gene analysis based on Graphical Gaussian Models, a Harvard group found that CXCL13 was one of two notable gene hubs (the other is MMP12) in breast cancer (BC). 22 Many studies have reported that, compared with normal breast tissue, the expression level of CXCL13 is significantly higher in both tumor tissues and peripheral blood in BC patients. [23][24][25][26][27][28][29] Panse et al. showed that peripheral serum CXCL13 level increased in patients with metastatic BC but decreased after removing the primary tumors by surgery. 24 Those authors also reported that CXCR5 was positively related to the level of CXCL13. 24 Several studies have suggested that the CXCL13-CXCR5 axis plays a critical role in lymph node metastasis as the tumor expression level of CXCL13 was significantly higher in lymph node (LN) positive BC patients. 16,27,30 Furthermore, vascular invasion, as well as LN involvement, was found to be significantly related to expression of CCR5, CXCL13, and SDF-1. 30 In addition, expression of CXCR5 was significantly higher in patients with stage 3 tumor than those with stage 2 disease. 30 Interestingly, one study reported that CXCL13 expression was higher in younger BC patients and closely associated with negative estrogen. 16 It is difficult to determine the mechanism of action of this chemokine axis based on expression data from clinical specimens. Several studies turned to BC cell lines to investigate the functional activities of CXCL13 and CXCR5, as CXCL13 and CXCR5 are both expressed at high level in common BC cell lines such as MCF-7, MDA-MB-231 ZR-75, and BT-20. 24, 28 Biswas et al. added soluble CXCL13 to BC cell lines and induced expression of matrix metalloproteinase-9 (MMP-9), EMT regulators (Snail, Slug), and mesenchymal markers (Vimentin, Ncadherin) in a CXCR5-dependent manner via RANKL/Src axis. 27 That study further documented that patients with LN involvement co-expressed CXCL13-CXCR5 and displayed significantly higher expression of EMT markers in their primary tumors. 27 Taken together, a collection of studies have reported that high expression of CXCL13 and CXCR5 is associated with adverse prognosis or LN metastasis in BC. 9,12-15 Irshad et al. further proved that neutralizing CXCL13, CCL21, or RANKL was sufficient to decrease lymph node metastasis in BC. 31 Furthermore, these studies suggest that BC cells can secrete CXCL13 and activate its receptor CXCR5 in an autocrine fashion to induce EMT, along with promoting cancer progression and metastasis. 9,12,13 Contrary to the perspective noted above, which argued that the upregulation of CXCL13 axis is a poor prognosticator, a dozen or so studies have supported that this axis is associated with a favorable outcome in BC. For instance, a study reported that in high grade, hormone receptor negative, HER2 (human epidermal growth factor receptor 2)-positive traits in patients with axillary node involvement, high expression of the CXCL13-CXCR5 was associated with improved outcomes. 25 This positive prognostic value of CXCL13 and CXCR5 has also been reported in several other studies. 14,25,[32][33][34][35] Although CXCL13 expression is often lost in aggressive triple-negative breast cancers (TNBC), those patients that retained CXCL13 or those with high expression were found to have superior outcomes. 23,[34][35][36][37] Moreover, studies found that compared with low tumor CXCL13 mRNA expression levels, the high expression levels were strongly related to favorable 5-year distant metastasis-free survival and disease-free survival rate in TNBC sets. 36,38 A four-gene signature (HLF, CXCL13, SULT1E1, and GBP1) was proved to be associated with favorable outcome in TNBC. 39 Recent studies have suggested that the immune modulating activity of CXCL13 could confer its favorable nature in BC. For instance, a novel set of 14 prognostic genes including CXCL13 was proved to be superior to other reported gene signatures in predicting the metastatic outcome of early stage and conservatively managed HRneg and Tneg BC. 34 Importantly, CXCL13 expression has been proved to be the best prognostic predictor in BC no matter the subtype (Her2/neu, triple negative, and so on). 14 Increased CXCL13 secreted by tumor tissues was able to recruit more CXCR5+ T and B cells to the tumor, an activity which could enhance the anti-tumor immunity. 23,24 Indeed, CXCL13 is a known critical factor for triggering development of secondary lymphoid organs. 40 Interestingly, recent studies have proved that CXCL13 is also specifically related to induction of the formation of tertiary lymphoid structures (TLSs) in non-lymphoid tissues 41,42 ; the inhibition of CXCL13 can interfere with formation of TLSs, 43 which are essential for shaping a favorable immune microenvironment to inhibit tumor progress. [44][45][46] Another study detected CXCL13-producing CD4+ follicular helper T (TFHX13) cells in extensively infiltrated tumors, which contributed to organization of LN-like structures. 14 Those TFHX13 may affect recruitment of immune cells to the tumor microenvironment, and also affect TLS formation, thus helping to create a niche in the tumor mass in which effective and durable antitumor immune responses can be generated. 14 In addition, the major source of CXCL13 production was revealed to be CD4 + PD-1 hi CD200 hi TFH cells infiltrating in BC. 14 However, the initial attraction may be attributed to tumor-dependent secretion of CXCL13. 23 A study by Gu-Trantien et al. found that, at the tumor site, TFHX13 cells can potentially initiate TLS formation and thereby generate germinal center B cell responses. In addition, in the BC microenvironment, TFHX13 cell differentiation may play a critical role in converting Treg-mediated immune suppression to de novo activation of adaptive antitumor humoral responses. 19 Pimenta et al. showed that IRF5 (interferon regulatory factor 5) can bind to the promoter of CXCL13 and directly regulate its expression in mammary epithelial tumor cells. In addition, their results revealed that IRF5-induced CXCL13 expression is responsible for recruiting CD19+CXCR5+ B-cell and CD4+CXCR5+ T-cell to the tumor. 23 An in vivo study found that CCX-CKR (ChemoCentryx chemokine receptor), also known as CCR11, a member of atypical chemokine binders, can decrease CCL19/21/25 and CXCL13 protein levels in CCX-CKR-transfected BC xenograft tumor mice models and can significantly inhibit tumor growth and lung metastasis. 47

Role of CXCL13-CXCR5 axis in prostate cancer
In recent decades, scientists discovered that CXCchemokines play significant roles in cell adhesion to endothelium and extracellular matrix in prostate tumor cell lines. 48 Recently, Singh et al. reported significantly higher serum CXCL13 levels in prostate cancer (PCa) patients than those in normal healthy donors and patients with other prostate diseases. 49 Also, those authors found that CXCL13 was positively correlated with serum PSA in PCa patients. 49 However, the result from another group showed that CXCL13 levels were not significantly different between PCa and non-PCa patients. 50 Although they found that CXCL13 gene expression level was higher in PCa tissues compared with adjacent normal tissue, the difference was not statistically significant. 50 An analysis including 137 clinical PCa samples reported significantly higher gene expression and protein levels of CXCL13 in tumor tissues than those in adjacent normal tissues. 18 Conversely, another study reported that CXCL13 was significantly reduced in prostate tumors compared with adjacent normal tissues. 51 The structural and functional features of the prostate-associated lymphoid tissue (PALT) compartment can enable the gland to mount a local immune response against infections and tumors in the course of malignant lesions; its destruction, and quantitative or morphological changes can down-regulate CXCL13 expression. 51,52 Thus, Wedel et al. deduced that CXCL13 and CXCR5 gene expression were gradually decreased in the progression of PCa. 51 In vitro, Fan et al. found that levels of CXCL13 were much higher in LNCaP and CWR22Rv1 than in DU145 and PC3. 18 In terms of the origin of CXCL13 in prostate tissue, some scientists concluded that IL-6, which is highly elevated in PCa patients, can induce CXCL13 production by human bone marrow endothelial cells and osteoblasts. 49 However, in in vivo PCa models, evidence showed that CXCL13 was expressed by tumor-associated myofibroblasts. 53 It was found that androgen deprivation could activate myofibroblasts and induce CXCL13 expression in both prostate tumor and normal prostate through inducing hypoxia, a condition which activates hypoxiainducible factor 1 (HIF-1) and autocrine TGF-β signaling and CTGF. Moreover, after TGF-βR1 signaling was inhibited, myofibroblast activation, B cell infiltration, CXCL13-expressing myofibroblast induction, nuclear translocation of IKKα, and CRPC progression were all prevented. 53 In addition, a study found that androgen stimulation can enhance expression of CXCL13 in androgen receptor (AR)-expressing PCa cells. 18 Knockdown of AR suppressed CXCL13 expression while AR over-expression enhanced CXCL13 expression in both LNCaP and CWR22Rv1 cell lines. AR ChIP-seq results from LNCaP cells revealed that AR regulated expression of CXCL13 by binding to its enhancer sequences. Also, CXCL13 is reported to be involved in AR-induced migration and invasion of PCa cells through its interaction with ARresponsive genes, such as EST-A, Snall, and Cyclin B1. 18 However, for CXCR5, some studies argued that CXCR5 was either reduced significantly in the prostate tumor tissues 51 or expressed identically in normal tissues. 50 While other studies inferred that expression of CXCR5 was significantly higher in prostate tumor than in normal prostate tissues, and that the expression of CXCR5 was positively related to tumor stage and grade. Moreover, compared with normal prostatic epithelial cells, PCa cell lines expressed a higher level of CXCR5. 17,48,54,55 In the case of chemokines, it is noted that mRNA expression patterns do not always mirror those of their encoded proteins. Generally, normal prostate tissues and BPH samples predominately express CXCR5 on the membrane and/or in the cytoplasm. In terms of PCa cases, those with Gleason scores ≤6 also showed predominantly membrane and cytoplasmic CXCR5 expression patterns, but interestingly, those with advanced disease had more nuclear CXCR5 expression patterns. [35][36][37] As attested above, PCa cell lines express different levels of CXCR5. Of interest, the level of CXCR5 expression in a cell line positively correlates with its ability of migration and invasion of extracellular matrix components following interaction with CXCL13. 49,54 In PCa cell lines (LNCaP and PC3), expression of collagenase-1 (MMP-1), collagenase-3 (MMP-13), stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), and stromelysin-3 (MMP-11) were increased after CXCL13 treatment. 36 These data suggest clinical and biological relevance of the CXCL13-CXCR5 pathway and its potential role in PCa cell invasion and migration. Evidence from studies using PC3 cells, an established hormone-refractory PCa cell line with ability of skeletal (osteolytic) metastases and tumor growth, proved that CXCL13 blockade can significantly delay growth of prostate tumor and its spread to bones, as well as osteolysis. This result indicates that the CXCL13-CXCR5 axis supports PCa bone metastasis and growth. 56 Another study also concluded that CXCL13 was secreted by human bone marrow endothelial cells and osteoblasts cells after IL-6 stimulation. 49 The CXCL13 secreted by these stromal cells could attract CXCR5+ PCa cells to bone site, which may partially explain why PCa cells tend to metastasize to bone. Additionally, this group found co-localization of a v β 3 with CXCR5, and clustering of this integrin complex after CXCL13 treatment, which suggest that the CXCR5-CXCL13 axis was also involved in adhesion in PCa cells. 49 Another study disclosed that tumor infiltrating B cells were critical inflammatory cells that promoted the progression to castration-resistant PCa after androgen-deprivation treatment. These B cells can produce LTα:β heterotrimers that stimulate LTβR on PCa cells to induce IKKα nuclear translocation and STAT3 activation, thereby enhancing androgen-independent growth. CXCL13 could be a major signal to recruit these B cells, as a CXCL13 blocking antibody was used to successfully prevent castration-induced B-cell recruitment. 57 In a mouse model, Garg et al. demonstrated that PKCε cooperates with loss of Pten to promote prostate cancer development by individually and synergistically up-regulating the CXCL13 via a non-canonical NF-κB pathway. 58 Chemokine receptors are coupled to heterotrimeric G protein α, β, and γ subunits, thereby subsequently causing Class IA and IB PI3K activation. 55 Gαq/11/Gβ3/Gγ9 in LNCaP and Gαi2/Gβ3/Gγ9 in C4-2B and PC3 cell lines were coupled to CXCR5 and disassociated the following CXCL13 stimulation. Both of these G-proteins can activate the phosphoinositide-3 kinase (PI3K)⁄Akt pathway and the ERK1⁄2 pathway. 59 Generally, in PC3 cells, CXCL13 mainly regulates the PI3K/Akt and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase-1 (JNK) pathways. By means of binding to CXCR5, CXCL13 can activate PI3K/ Akt, Raf/MEK/ERK, Integrin-β3/Src/FAK (focal adhesion kinase), and dedicator of cytokinesis 2 (DOCK2)/Rac/JNK pathways, which regulate advanced PCa cell survival, invasion, and growth. 60 CXCL13 induced cell proliferation in a DOCK2-dependent manner in PC3 cells, but promoted proliferation in a JNK-dependent manner in LNCaP cells regardless of the presence of DOCK2. 59 An earlier study found that PC3 and LNCaP cell lines both expressed Class IA PI3Kp110α and p110β. In addition, LNCaP selectively expressed Class IA p110δ, but PC3 cells expressed Class IB PI3Kp110γ. CXCL13 treatment caused phosphorylation of Class IA PI3Kp85α in LNCaP cells and Class IA PI3Kp85α as well as Class IB PI3Kp101 regulatory subunits in PC3 cells. Thus, LNCaP cells can only use the Gα-Src-FAK-mediated pathway, while PC3 cells have the ability to exploit both Gα-Src-FAK and Gβ/γ-mediated events to promote migration and invasion following CXCL13-CXCR5 interaction. This evidence may also reflect the high metastatic potential of PC3 cells and their ability to spread to bone. 61

Role of CXCL13-CXCR5 axis in gastrointestinal cancers
Prominent overexpression of CXCL13 in liver cancer tissues and serum was observed in patients with hepatocellular carcinoma (HCC). [62][63][64] Moreover, its level was positively associated with serum ALB, ALT, AST, and Child-Pugh scores. 62,63 In patients with a heavy mass burden, advanced and metastatic HCC, serum CXCL13 expression was further increased. 49 Duan et al. reported that the number of infiltrated CXCR5+CD45RA-CD4+ T cells was higher in tumor than in para-tumor tissues, and they found that serum CXCL13 was related to recurrence-free survival after surgery. 64 Li et al. revealed that CXCL13 and Wnt/β-catenin signaling shared a positive feedback loop and that CXCL13 promoted liver cancer by activating Wnt/β-catenin pathway, inducing IL-12, IL-17, and IgG4. 62 Bai et al. showed that CD8+CXCR5+ T cells were abundant in pancreatic cancer patients, especially in the tumor microenvironment, responding to anti-PD-1/anti-TIM-3 blockade by functional upregulation. 65 Moreover, Xing et al. reported that CD8+CXCR5+ T cells could also contribute to antitumor immunity in colorectal cancer. 66 For gastric cancer, no studies have evaluated different expression levels of CXCL13-CXCR5 in tumor tissue and normal tissue. Only one study found that high intratumoral CXCL13 expression contributed to inferior prognosis in patients undergoing gastric cancer resection. Moreover, CXCL13 expression may act as an indicator of response to adjuvant chemotherapy after surgery in patients with T2-4 stage disease. 67 Compared with adjacent normal tissue, CXCL13 and CXCR5 expression were significantly higher in colorectal cancer (CRC) tissues. 68 Furthermore, the expression levels of CXCR5 and CXCL13 were significantly higher in high stage CRC tissues (≥T3). 68 Further evidence proved that high levels of CXCR5 and CXCL13 were also associated with poorer survival. 68 The molecular mechanisms of the CXCL13-CXCR5 axis in gastrointestinal cancer remain elusive. A recent study revealed that CXCL13 promoted growth, invasion, and migration of colon cancer cells through the CXCL13-CXCR5-PI3K/AKT pathway. 69 Likewise, CXCL13 increased MMP-13 expression and secretion. 69 Another study using an endoscopic orthotopic colon cancer model found that tumor growth in CXCR5-/-mice was severely increased, with a similar number of tumorinfiltrating CD4+ T cells to that in tumors of wild-type mice but significantly decreased number of B cells. Vice versa, mice bearing CXCL13-overexpressing MC38 cells had reduced tumor growth compared with wild-type mice, a conclusion that indicated the important role of CXCL13-CXCR5 signaling in recruitment of T and B cells into the tumor microenvironment and anti-tumor immune response in colorectal cancer. 70

Role of CXCL13-CXCR5 axis in other cancers
Head and neck squamous cell carcinomas have been found with high expression of CXCL13 as well as CCR7 and CXCR5 in tumor tissues compared with normal tissues. 71 A high level of expression of CXCL13 was also identified in oral squamous cell carcinoma (OSCC), which was proven to be related to OSCC development and progression. 72,73 An in vitro study identified that expression of both CXCR5 and MMP-9 was upregulated in the OSCC cell line, SCC14a, after treatment with exogenous CXCL13, a result that suggests CXCL13 could enhance CXCR5 receptor expression in an autocrine regulatory manner in OSCC cells. 74 Fortunately, this corresponding response was also observed in a human bone marrow-derived stromal cell line (SAKA-T) and murine preosteoblast cell (MC3T3-E1). 75 By establishing a subcutaneous OSCC model, Pandruvada et al. identified that CXCL13 was also attributed to bone invasion. 74 In models bearing CXCL3 knock-down tumor, the tumor's ability to invade into bone matrix was inhibited and the number of osteoclasts at the tumor-bone interface was also significantly decreased. 74 Researchers uncovered that CXCL13 could significantly enhance the RANKL-RANK signaling pathway, which is an important pathway for promoting osteoclast growth and bone resorption. 75,76 But a small difference of note is that CXCL13 stimulation increased the expression level of NFATc3 in OSCC cells and p-c-Myc in SAKA-T/MC3T3-E1 cells, respectively. And these two proteins both could further induce RANKL expression by targeting the RANKL promoter region and stimulating its activity. 75,76 Lu et al. found that IL-17A could contribute to migration and tumor killing capability of B cells in esophageal squamous cell carcinoma by inducing tumor cells to produce CXCL13, CCL2, and CCL20, which recruit B cells into the tumor microenvironment. 77 Singh et al. reported increased expression levels of CXCR5 and CXCL13 in ovarian cancer cell lines and in clinical samples compared with non-neoplastic ovarian tissues. Moreover, CXCR5 and CXCL13 expression levels were different among different types of cancerous ovarian tissues. 78 Evidence of the role of the CXCL13-CXCR5 axis in terms of lung cancer is limited. 79,80 In a study including a small sample of non-small cell lung cancer (NSCLC) patients, Singh et al. reported that CXCR5 expression was higher in tumor tissues compared with nonneoplastic tissues. 79 Also, they found elevated serum CXCL13 in lung carcinoma patients compared with healthy volunteers. 79 Furthermore, they uncovered that CXCR5 expression was only significantly increased in squamous cell carcinoma (SCC) sets with nodal metastases, not in other subtypes of lung cancer. 79 Ma et al. reported that the frequency of CD4+CXCR5+ T cells was significantly lower in peripheral blood of NSCLC patients than that of healthy controls. 81 They found that CD4+CXCR5+ T cells contributed to antitumor immunity and were related to better outcomes in NSCLC. 81 Wang et al. proved that benzo(a)pyrene (BaP) could induce CXCL13 expression in lung epithelial cells and lung cancer formation in mice, while knockout CXCL13 or CXCR5 could significantly attenuate BaPinduced lung cancer in mice. 82 Similar to breast cancer, intratumoral TLS in lung cancers is also associated with patients' survival. 83 The latest studies concluded that CXCL13 was strongly expressed in the germinal centers by CD21 + follicular dendritic cells in TLS and its higher expression was associated with CXCR5 + T cell recruitment to intratumoral TLS. 84,85 Conclusion The CXCL13-CXCR5 axis plays a critical role in tumor growth, invasion, and ultimately metastasis in a wide range of human malignancies. Understanding its potential mechanisms will likely lead to novel anti-tumor therapies.