CXCL9: evidence and contradictions for its role in tumor progression

Abstract Chemokines are a group of low molecular weight peptides. Their major function is the recruitment of leukocytes to inflammation sites, but they also play a key role in tumor growth, angiogenesis, and metastasis. In the last few years, accumulated experimental evidence supports that monokine induced by interferon (IFN)‐gamma (CXCL9), a member of CXC chemokine family and known to attract CXCR3‐ (CXCR3‐A and CXCR3‐B) T lymphocytes, is involved in the pathogenesis of a variety of physiologic diseases during their initiation and their maintenance. This review for the first time presents the most comprehensive summary for the role of CXCL9 in different types of tumors, and demonstrates its contradictory role of CXCL9 in tumor progression. Altogether, this is a useful resource for researchers investigating therapeutic opportunities for cancer.


Introduction
The development of malignant tumors is dependent on the tumor microenvironment, where chemokines and their receptors are important participants [1]. Chemokine families are defined as small (8-15 kD) proteins that induce chemotaxis, tissue extravasation and, in some instances, modulate the functional properties of different leukocytes. Chemokines can be subdivided into four classes according to the number and spacing of two conserved N-terminal cysteine residues, consisting of the C, C-C, C-X-C, and C-X3-C families [2]. Chemokine CXCL9 is a member of the CXC family and has an important role in the chemotaxis of immune cells. It is secreted by various cell types including immune cells (T lymphocytes, NK cells [3], dendritic cells [4], macrophages [5], eosinophils [6], etc.), and non-immune cells (hepatic stellate cells [7], preadipocytes, thyrocytes [8], endothelial cell, tumor cells, and fibroblasts [9], etc). CXCL9 has a versatile and controversial role in tumors, and accumulating evidence suggests that CXCL9 is closely associated with the prognosis of tumor patients. Here, the role of CXCL9 in cancer development was reviewed, as well as the molecular mechanisms leading to aberrant expression of CXCL9 in cancer and the potential clinical applications of CXCL9 in diagnosis, prognosis, and cancer treatment.

Regulation of CXCL9
The transcriptional regulation of CXCL9 is a multistep process involving many transcription factors (Fig. 1), of which signal transducer and activator of transcription (STAT1) and nuclear factor κB (NF-κB) are two most well-characterized members [10]. Both the gene mutation ORIGINAL RESEARCH CXCL9: evidence and contradictions for its role in tumor progression of STAT1 [11] and the blocking of the Janus-activated kinase (JAK)/STAT-1 pathway [12,13] can reduce CXCL9 expression induced by IFN-γ. Moreover, CXCL9 expression can be suppressed by reducing the levels of components of the STAT1-IRF-1(IRF-1, Interferon regulatory factor) transcriptional activation pathway by Porphyromonas gingivalis that leads to the immune function decline [14]. Lipopolysaccharide (LPS) and D-galactosamine could induce the phosphorylation of STAT1 and enhance the transcription of CXCL9 leading to the enhancement of liver inflammation, and even liver apoptosis and injury [15]. In this process, VSL#3 (a mixture of eight different probiotic bacteria) can specifically reduce the phosphorylation of STAT-1 [15,16]. STAT-1 can also be activated by IL-27 accompanied with IFN-γ. Then CXCL9 induced by STAT-1 finally affects the liver inflammation [17]. In addition, our previous work by Xia et al. identified that HBx protein can induce the CXCL9 transcription by activating NF-κB that binds to its promoter, and CXCL9 promotes the migration of leukocytes in liver with HBV infection [18]. Unlike STAT1, phosphorylation of NF-κB could not be suppressed by VSL#3 [16]. STAT1 and NF-κB can cooperatively regulate the expression of CXCL9, and this transcriptional synergy could cause the enhanced recruitment of RNA polymerase II complex to the promoter via simultaneous interaction of CBP with STAT1 and NF-κB [10].
Besides STAT1 and NF-κB, myeloid transcription factor PU.1 (PU.1) is also involved in regulating the CXCL9 gene transcription [19,20]. PU.1 is a cell-specific nuclear transcription factor and a key transcription activator of the gene encoding CXCL9 in response to IFN-γ. In microglia of the central nervous system (CNS), both STAT1 and PU.1 constitutively bind to the CXCL9 gene promoter. However, only STAT1 binds to the CXCL9 gene promoter in astrocytes, and this binding can be counteracted by the ectopic expression of PU.1 [19]. Besides, studies also showed that IRF-8 can bind to the CXCL9 gene promoter induced by IFN-γ and this binding was accompanied by decreasing PU.1 binding [20]. In the B-cell lymphoma/ leukemia, multiple myeloma oncogene 1 (MUM1) can upregulate the expression of CXCL9 by activating its promoter in cooperation with PU.1, leading to poor prognosis of B-cell malignancy [21].
Early growth response-1 (Egr-1), a zinc-finger transcription factor, also correlates with the expression of CXCL9 in the invading macrophages and accumulation of NK cells in Lewis lung carcinoma [22]. Nevertheless, this phenomenon is not observed for all types of tumors and whether Egr-1 directly regulates CXCL9 or other Egr family members take part in this process is not clearly understood.
CXCL9 is also associated with human hepatic fibrosis and anti-fibrosis in mice [23,24]. Fos-related antigen 1 (Fra-1) represses CXCL9 expression by direct promoter binding in hepatocytes and affects the fibrotic response to some extent [25].

CXCL9 as a Tumor Suppressor
A summary of the increasing evidence about the effects of CXCL9 on tumor suppression is exhibited in Table 1.

Lung cancer
There is no precise staging system that predicts the prognosis of early-stage non-small-cell lung cancer (NSCLC). Addison et al. found that high protein levels of ELR-(Glu-Leu-Arg) chemokine CXCL9 existed in 90 human NSCLC tissues. Moreover, they showed that either recombinant human cytokine CXCL9 (rhCXCL9) or gene transfer of CXCL9 inhibited tumor-derived angiogenesis, suppressing tumor growth and metastasis, which counteracted the angiogenic role of ELR+ chemokine (such as IL-8 and epithelial neutrophil activating protein 78) partly [36]. Metodieva et al. also showed a high expression level of CXCL9 in 12 NSCLC patients [37]. However, a study by Kowalczuk et al. also showed that CXCL9 expression was low in 109 NSCLC tumor tissues, but it could not influence both overall and disease-free survival [38]. In addition, other studies have also demonstrated that Egr-1 deficiency [22], IL-7 [39], CCL21 [40], and myeloid-derived suppressor cells depletion [41] reduced tumor burden by upregulating CXCL9 and CXCL10 expression, which played an anti-angiogenic role and attracted tumor macrophages, CD4 and CD8+ T lymphocytes, and NK cells. Also, IFNγ, interleukin-12 (IL-12), and granulocyte macrophage colony-stimulating factor were involved in the reduction of tumor burden caused by CCL21 [40]. NSCLC, Non-small-cell lung cancer; CTCL, Cutaneous T-cell lymphoma; UC-Ca, ulcerative colitis-associated cancer; RCC, Renal cell carcinoma; TCs, Tumor cells; GC, Gastric carcinoma. 1 The response as "Good" means good prognosis of cancer patients, good response to tumor therapy, or reduction of tumor burden.

Breast cancer
It is recognized that CXCL9 is significantly associated with lymphocytes infiltration and chemotherapy response in human breast cancer (BC) patients [42][43][44] [47].

Lymphoma
It is reported that CXCL9 plays a significant role in lymphoma because of its chemotaxis on immunocytes. Tensen et al. showed that CXCL9 and CXCL10 mRNA, not IL-8, were highly expressed in 11 patients with cutaneous T-cell lymphoma (CTCL), and correlated with increased CD4+ T cells infiltration, not CD8+ T cells [48]. CXCL9 was also reported to be significantly elevated in nine early CTCL compared to the normal skin or 13 advanced CTCL skin [49]. Przewoznik et al. showed that the overexpression of CXCL9 and CXCL10 revealed a significant correlation with increased NK cells and their migration in late B-cell lymphoma stages, which were prerequisites for the potential tumor therapy of adoptive NK-cell transfer [50]. In Addition, IL-12 and Th1-derived IFN-γ exerted antitumor effects through the inhibitory effects of endogenous CXCL9 and CXCL10 on tumor vasculature in human Burkitt's lymphoma [51] and in B-cell lymphoma [52], respectively.

Colorectal cancer
Colorectal cancer (CRC) is one of the most prevalent tumor types worldwide. Tumor-infiltrating T cells are crucial for anti-tumor immunity [53]. Wu  were selectively increased, followed by accumulation of CXCR3+ conventional T cells, in Treg-cell-depleted tumors, which indicated that targeting Treg cells and upregulating CXCL9 and CXCL10 might be a potential immunotherapy [57].

Other tumors
In renal cell carcinoma, endogenous CXCL9 was closely implicated in the antitumor effects that were produced by IL-2 [64] and IL-12 (also producing CXCL10) [65] by inhibiting tumor angiogenesis and infiltration of CD8+ T lymphocytes, respectively. In ovarian cancer, synergistic effect of tumor-associated IL-17 and Th17 cells [66], as well as IL-18-primed "helper" NK cells [67], induced the production of endogenous CXCL9 and CXCL10 that were directly correlated with tumorinfiltrating CD8+ T cells. The former also attracted the NK cells, and the latter also induced CCL5 that was involved in the T-cells infiltration [66]. In gastric carcinoma, endogenous CXCL9 could promote antitumor immunity exerted by T cells, and CXCL10, occasionally, also participated in this process [68,69]. In 20 Ewing sarcoma patients, Berghuis reported that CXCL9, CXCL10, and CCL5 that is highly expressed by tumor and stromal cells were correlated positively with accumulated CD8+ T cells. However, CXCL9 was not related to patient survival rate [70]. Moreover, CXCL9 was significantly increased in cutaneous tumor patients, and played a critical role in CXCR3+ T cells (including CD4+ and CD8+ T cells) and NK-mediated tumor immune-surveillance and suppression [71,72]. Some other IFN-regulated proteins (MxA, IDO) were also involved in the recruitment [71].

CXCL9 as a Tumor Promoter
An increasing body of evidences has been demonstrated that CXCL9 acts as a tumor promoter in multiple types of cancer (summarized in Table 2).

Hepatocellular carcinoma
Hepatocellular carcinoma (HCC) causes the third most common cancer mortality due to early metastasis and recurrence [73]. Ding et al. in our team showed that CXCL9 promoted the migration and invasion abilities of CD133+ liver cancer cells, and its receptor isoform CXCR3-A upregulated invasion abilities via activation of p-ERK1/2-MMP2/MMP9 pathway induced by rhCXCL9 [74]. Lan et al. in our team also demonstrated that rhCXCL9 enhanced the invasion ability of hepatocellular carcinoma through the upregulation of phosphatidylinositol-3, 4, 5-trisphosphate RAC Exchanger 2 (PREX2) [75]. In addition, Liu et al. found that there was pronounced expression of CXCL9, CXCL10, and CXCL11, induced by IL-17, at the HCC invading edge in 40 patient tissues, which were correlated with the recruitment of CXCR3+ B cells. CXCR3+ B cells could trigger the polarization of protumorigenic M2b macrophages in an IgG-dependent manner and were positively associated with early recurrence in HCC patients [76].

Lung cancer
Although CXCL9 is considered as an anti-tumor factor according to some reports, it could also promote the tumor development as follows. Shiels et al. conducted two independent nested case-control studies (the discovery study and the replication study). The results showed that CXCL9 was associated with lung cancer risk in the replication study, and remained associated with it more than 6 years prior to diagnosis in pooled analyses. Their research suggested that CXCL9 has an etiologic role in lung cancer [77]. On the contrary, Nakanishi et al. showed that in 40 lung cancer patients, although CXCL9 was highly upregulated in tumor tissues, no significant relationship between CXCL9 expression and DFS or lower risk of postsurgical recurrence was observed [78].

Breast cancer
Metastasis of breast cancer (BC) remains a challenge for the therapeutic management. Ejaeidi et al. showed that the levels of CXCL9, CXCL10, and CXCL11 were markedly high in 40 HR+ (hormone receptor) metastatic BC patients' sera when compared to HR-patients and healthy controls. These three chemokines in sera, especially CXCL10, played an important role in BC development through activation of survivin, βcatenin, mitogen-activated protein kinase phosphatase 1 (MKP-1), and matrix metalloproteinase-1 (MMP-1) [79]. Ruiz-Garcia et al. found that among 120 BC patients, the serum concentration of CXCL9 was higher in cancer patients compared with normal volunteers, and the difference between ER-negative BC samples and normal volunteers was statistically significant. However, it was not different between ER-positive BC and normal volunteers. When a cutoff of 1000 pg/mL for CXCL9 was selected, the sensitivity of ER-negative BC diagnosis was 27% and specificity was 90%, and the positive predictive value was 76%. In addition, when CXCL9 combined with fibronectin 1 (Fibronectin 1 > 200 pg/mL as positive; 150 < fibronectin 1 < 200 pg/mL and CXCL9 concentrations >1000 pg/mL as positive), the sensitivity for BC diagnosis was 53% and specificity was 97%. The positive predictive value was up to 96% [80].
Lymphoma CXCL9 could also be of great importance for enhancing the understanding of metastasis of lymphoma. Mir et al. demonstrated that in 209 follicular lymphoma patients receiving chemotherapy, elevated serum levels of CXCL9 predicted a shorter median event-free survival (EFS). When a separate study of 183 patients was combined in a metaanalysis, the results still showed that CXCL9 was still significantly associated with shorter EFS [81]. Venetz et al. found that in primary CNS lymphoma, perivascular CXCL9 correlated with CD8+, CD4+, and Foxp3+ T cells infiltration, and formed heterocomplexes with CXCL12 in vivo. Moreover, rhCXCL9 enhanced rhCXCL12-induced migration of CXCR3+/CXCR4+/CD8+ T cells and malignant B cells in vitro. These results highlighted the importance of regions expressing chemokines in tumor development [82]. Maruoka et al. demonstrated that CXCL9 and CXCL10 (5000 pg/mL and 500 pg/mL as the cutoff levels, respectively) were significantly useful for the early diagnosis and therapeutic outcomes of Lymphoma-associated hemophagocytic syndrome (LAHS). They also could distinguish LAHS from sepsis and, furthermore, severe from moderate/mild LAHS, and B-cell-type from T/NK cell-type LAHS [83]. Many other studies have delineated that CXCL9 was probably involved in tumor-associated macrophages polarization [84], tumor cell motility [85], and the survival of H-RS cells [86].

Melanoma
Amatschek et al. found that low concentration of rhCXCL9 was able to induce melanoma cell migration, conversely at high concentration. And rhCXCL9 enhanced the breakdown of endothelial cells monolayer during transendothelial migration of tumor cells [87]. Moreover, RhCXCL9 facilitated the intracellular actin polymerization, cell adhesions (phosphorylation of focal adhesion kinase and paxillin), and cell survival. RhCXCL9 could also increase the intracellular calcium concentration, an early biochemical events in response to chemokines. Endogenous CXCL9 and CXCL10, upregulated by complete Freund's adjuvant in draining lymph nodes, promoted the metastasis of tumor cells to the lymph nodes [88]. Jehs et al. reported that uveal melanoma cell lines that were cocultured with activated T cells resulted in an upregulation of chemokines in the supernatant, such as CXCL9, CXCL10, CXCL11, CCL2, CCL5, and VEGF. In turn, these cytokines could generate a tumor-promoting inflammatory microenvironment [89].

Head and neck cancer
The head and neck cancer (HNC) is the seventh most common malignancy worldwide, of which squamous cell carcinoma (SCC) is the most common type [90]. In a research study that included sera from 205 nasopharyngeal carcinoma (NPC) patients and 231 healthy individuals,  [91]. Chang et al. found that CXCL9 expression was significantly higher in 50 tumor samples than the normal samples, as well as in the sera of 181 oral cavity SCC patients than the 231 healthy individuals. Serum CXCL9 levels were correlated with pT status, pathological overall stages, tumor depths, and positive bone invasion. Moreover, the results also indicated that higher CXCL9 serum levels predicted a poor prognosis of patient's overall survival and DFS [92].

Other cancers
In cervical cancer, Zhi et al. detected that serum CXCL9 was significantly increased at invasive International Federation of Gynecology and Obstetrics stages II, and III in a total of 1057 women, as compared to the noninvasive stage. This suggested that CXCL9 was involved in the development of cervical cancer [93]. In chronic lymphocytic leukemia, Yan et al. reported that high serum levels of chemokines CL1 (CXCL9, CXCL10, CXCL11, CCL3, CCL4, CCL19, IL-5, IL-12, and IFN-γ) correlated with shorter overall survival, suggesting that high levels of CXCL9 might predict a poor prognosis [94]. In prostate cancer (PCa), Hu et al. reported that PCa cells could secrete CXCL9 more than the normal cells, and CD4+ T cells recruited by endogeneous CXCL9, consequently, promoted PCa metastasis via modulation of FGF11/ miRNA-541/AR/MMP9 signaling [95]. Liu et al. showed that recombinant mouse and human CXCL9 and CXCL10 facilitated proliferation of murine and human gliomaspheres, suggesting that they may promote tumorigenesis [96]. In addition, studies also demonstrate that CXCL9 is highly upregulated in gliolastoma [97] and primary pediatric CNS germ cell tumor (germinoma type) [98].

CXCL9 in cancer therapy
Accumulating evidence indicates that manipulation of the tumor microenvironment, which involves CXCL9, could enhance the therapeutic efficacy of strategies via tumorspecific T cells (summarized in Table 3).
The Role of CXCL9 in Tumor Q. Ding et al. through reduction of T cells infiltration [99]. A further evaluation of genetic immunotherapy by Sharma et al. provided evidence that adenovirus vector expressing interleukin (IL)-7 (DC-AdIL-7) reduced the tumor burden via the increase of IFN-γ and IL-12 as well as the antiangiogenic endogenous chemokines CXCL10 and CXCL9, and the decrease of immunosuppressive cytokines TGF-β and VEGF [100]. Additionally, Zhang et al. showed that the combination regime of plasmid-borne CXCL9 gene therapy plus low-dose cisplatin augmented the antitumor efficacy by enhancing the tumor anti-angiogenesis and apoptosis or CTL activity. These effects were shown in Lewis lung carcinoma (LL/2c) murine models, as well as in the colon carcinoma (CT26) murine models [101].

Breast cancer
Several evidences demonstrate the close relationship between CXCL9 and medicine in BC. The cyclooxygenase-2 (COX-2) and its pro-inflammatory products, prostaglandin E2 (PGE2), are strongly implicated in a range of human cancers including BC in a lymphocyte-dependent manner [102]. Markosyan et al. found that compared to wild-type mice, ErbB2-transgenic mice, deficient in mammary epithelial cells COX-2, showed enhanced immune surveillance by recruiting more CD4+ Th cells and CD8+ CTL, which were coincident with intratumoral CXCL9 influx, a key T-cell chemoattractant [103]. Bronger et al. discovered that there existed inverse correlation between endogenous CXCL9 concentration and COX overexpression in BC tissues. CXCL9 and CXCL10 could be inhibited by PGE2, and induced by unselective COX inhibitors. However, the expected increase of CXCL9 was only observed at low concentrations of COX-2-specific inhibitor celecoxib, and decreased by high concentrations [42]. Specht et al. discovered that CXCL9, and intersectin 2 (ITSN2) in BC tissues were significantly associated with prolonged DFS in 70 patients with cyclophosphamide, methotrexate, and 5-fluorouracile (CMF)-chemotherapy. Furthermore, the results by multivariate Cox analysis showed that the CXCL9/ITSN2 or CXCL9/FLJ22028 (Hypothetical protein FLJ22028 (NM_024854)) ratios could be independent predictive factors of DFS [44]. Hannesdóttir et al. showed that the lapatinib and doxorubicin enhanced the expression of Stat1dependent endogenous chemokines CXCL9, CXCL10, and CXCL11 (importance for attracting CD8+ T cells), and reduced tumor-associated macrophages (TAMs), consequently augmenting the antitumor immune response [104].

Melanoma
The incidence of melanoma has increased significantly, but there are no effective treatments yet [105]. Szabo et al. reported that combination of all-transretinoic acid (ATRA) and polyinosinic:polycytidylic acid (polyI:C) in melanoma cell lines upregulated the expression level of IL-1β, IL-6, IFN-β, CXCL10, CXCL9, CXCL8, and CXCL1 more than treatments with either ATRA or polyI:C separately, which was mediated by toll-like receptor 3 and MDA5. In this study, they found CXCL9 and CXCL10 to attract activated T lymphocytes [106]. Bedognetti et al. found that CXCL9, CXCL10, CXCL11, and CCL5 were all highly expressed in metastatic melanoma patients, and this was associated with responsiveness to adoptive therapy and IL-2 treatment [60].
Head and neck cancer IL-12 has been known as an effective cytokine for cancer treatment. Using the SCCVII tumor model, Li et al. found that intratumoral and intramuscular injection of IL-12 gene by electroporation upregulated CXCL9 expression by 15-fold and 6-fold, respectively. They also detected that CXCL9 gene injection significantly promoted the infiltration of CD4+ T cells in the tumors, not CD8+ T cells, indicating that CXCL9 played a crucial role in the IL-12 antitumor efficacy [107]. Dorsey et al. showed that intratumoral IFN-α DNA electroporation caused 50% of tumor eradication rate and more than doubled the survival time when compared with the controls, of which upregulated CXCL9 and CXCL10 had a pivotal role through inhibiting angiogenesis [108].

Other cancers
In chronic lymphocytic leukemia, Gustafsson et al. detected that tumor-loaded αtype 1-polarized dendritic cells cocktail (IL-1β/TNF-α/IFN-α/IFN-γ/poly-I:C;αDC1) showed a better tumor therapeutic efficiency by producing more NK cell-, NKT cell, and CD8 + T cell-recruiting chemokines CXCL9, CXCL10, and CXCL11 in the culture supernatants, as compared with the "standard" cocktail (IL-1β/TNF-α/ IL-6/PGE2;PGE2DCs), indicating the key role of the three chemokines in cancer treatment [109]. In renal cell carcinoma (RCC), it was reported that high-dose IL-2 treatment in 20 patients elevated the plasma levels of CXCR3 ligands (CXCL9, CXCL10, and CXCL11), sequentially, forming an angiostatic environment [110]. In external genital carcinoma, Soong et al. found that imiquimod, a toll-like receptor 7 agonist, induced local expression of CXCL9 and CXCL10 leading to CXCR3+ CD8+ CTL accumulation in the cervicovaginal tract, and enhanced potent antitumor efficacy in the orthotropic cervical cancer model when combined with intramuscular CRT/E7 vaccination [111]. In Sarcoma, Pardee et al. identified that OX40 ligand-Fc fusion protein (OX40L-Fc), a novel tumor Q. Ding et al. The Role of CXCL9 in Tumor necrosis factor receptor family member, promoted antitumor activity in most mice by rendering the tumor microenvironment permissive to type 1 T-cell infiltration and increase of vascular cell adhesion molecule (VCAM-1) and CXCL9 expression, upregulated by CD31+ vascular endothelial cells [112]. In cutaneous melanoma, Hong et al. discovered that in temozolomide-treated mice CCL5, CXCL9, and CXCL10 were significantly upregulated, which was followed by T-cell infiltration, enhanced tumor control, and this prolonged overall survival [113].

Concluding remarks
Chemokines play a divergent role in controlling the growth and metastasis of malignant tumors. Certain chemokines enhance nonspecific or specific host immunity against tumor implantation, while others could promote tumor growth, metastasis, or neovascularization in tumor tissues. So far, doubts about the role of CXCL9 still exist in tumors, even in the same type of tumor. CXCL9 could promote cancer metastasis via enhanced migration and invasion of tumor cells [74], and breaking of the endothelial cells monolayer [87]. However, as a tumor suppressor, it mainly recruited tumor-infiltrating CD8+ T cells and NK cells [62], and inhibited tumor angiogenesis [36]. However, in the section discussing "CXCL9 in cancer therapy" in this review, all the research conducted so far has suggested that high expression level of CXCL9 might be an important target for anti-cancer therapies. In addition, CXCL9 has been identified as a candidate biomarker in breast cancer [80] and nasopharyngeal carcinoma [91]. Therefore, a joint effort among researchers could provide a pave the path to further understand the role of CXCL9 in therapeutic purposes. The complex role of CXCL9 in tumor might be due to the following reasons: First, CXCL9 plays an important role in tumor immunity. CXCL9 could recruit not only CTL, inhibiting tumor development, but also other host immune cells, such as regulatory T cells (Tregs), tumor-associated macrophages, and MDSC, which mediate immune tolerance in tumors [42,43,46,47]. Role of CXCL9 might be dependent on the cancer immune stage, as discussed by Cancer Immunoediting theory that immunity efficacy was different at different stages [114]. Second, the contradictory role of CXCL9 might be associated with its receptor's splice variants CXCR3-A and CXCR3-B, as they always showed a counteracting role in tumor progression. CXCL9/CXCR3-A could promote tumor migration and invasion via PI3K [33], MAPK [74], pathways and so on, but CXCL9/CXCR3-B could inhibit endothelial cells proliferation [115] and tumor angiogenesis [36], which might be mediated by VEGF/VEGFR2 (KDR) and its downstream phospholipase Cγ, p-JNK, and p-ERK [24]. Lastly, the different functions of CXCL9 were closely related with the many cell types (stated above) secreting it, and its concentration in the tumor microenvironment. CXCL9 will draw more researchers' interests and attention in the future because of its contradictory and key effects on tumor initiation and development. Nevertheless, a more detailed characterization and mechanism of the role of CXCL9 in tumor biology is desperately required, as it may improve cancer treatment and possibly lead to clinical applications in cancer prognosis, diagnosis, and therapy.