Chemokines network in bone metastasis: Vital regulators of seeding and soiling

Abstract

Chemokines are well equipped with chemo-attractive signals that can regulate cancer cell trafficking to specific organ sites. Currently, updated concepts have revealed the diverse role of chemokines in the biology of cancer initiation and progression. Genomic instabilities and alterations drive tumor heterogeneity, providing more options for the selection and metastatic progression to cancer cells. Tumor heterogeneity and acquired drug resistance are the main obstacles in managing cancer therapy and the primary root cause of metastasis. Studies emphasize that multiple chemokine/receptor axis are involved in cancer cell-mediated organ-specific distant metastasis. One of the persuasive mechanisms for heterogeneity and subsequent events is sturdily interlinked with the crosstalk between chemokines and their receptors on cancer cells and tissue-specific microenvironment. Among different metastatic niches, skeletal metastasis is frequently observed in the late stages of prostate, breast, and lung cancer and significantly reduces the survival of cancer patients. Therefore, it is crucial to elucidate the role of chemokines and their receptors in metastasis and bone remodeling. Here, we review the potential chemokine/receptor axis in tumorigenesis, tumor heterogeneity, metastasis, and vicious cycle in bone microenvironment.

1. Introduction

Chemokines were initially discovered as chemoattractant cytokines that could regulate leukocyte trafficking to distant organ sites [1]. Cancer cells express chemokine receptors and migrate towards organ-specific sites according to their chemokine gradient [2]. The non-random migration of cancer cells is regulated by several factors, including the organ’s anatomical location, blood circulation pattern, tumor-intrinsic factors, and the interaction between tumor cells and the microenvironment of the metastatic sites [3]. Therefore, it is logical to anticipate that chemokines are the key metastatic defining factors. The movement and accumulation of immune cells at the tumor site start after the attachment of chemokines to the specific receptors and participate in the regulation of immune surveillance, invasion, angiogenesis, and metastasis [4–6].

Previously, the major role of chemokines was predicted as an essential regulator of immune cell trafficking. However, the concepts are currently updated with crucial roles of chemokines in the biology of non-immune cell-mediated pathological conditions, including cancer initiation and progression (Fig. 1) [1]. Chemokines can regulate the expression of many growth and angiogenic factors in the tumor microenvironment [7], and additionally, they can direct the cancer cell movement required for metastasis [8]. Genome sequencing analysis revealed that tumor cells have diverse genetic profiles due to the frequent somatic alterations with high mutation frequencies in proto-oncogenes, tumor suppressor genes, and some other crucial genes associated with the poor prognosis of cancer patients [9]. Individual genetic makeup and genomic instability foster phenotypic and genetic diversity in cancer cells, contributing to tumor heterogeneity and become more heterogeneous over time. This tumor heterogeneity drives the resistance and metastatic capability of tumor cells [10]. Chemokines and their receptors facilitate the communication between disseminated tumor cells with the microenvironment of distant metastatic organ sites for organ-specific transmigration, seeding, and colonization [11]. Bone metastasis is one of the inclusive examples of organ-specific metastasis, where cancer cells imbalance the bone homeostasis and start a vicious cycle followed by dysregulated bone remodeling. Bone is the most common site for prostate, breast, and lung cancer metastasis [12]. It is important to note that it can rarely be cured once cancer metastasizes to the bones. Consequently, it reduces the life expectancy of cancer patients and often causes limb dysfunction, pathological fractures, spinal cord compression, and severe pain [13–17]. Bone metastatic cells are the malignant tumor cells of the extraosseous organ or tissue that metastasize to the bone via the lymphatic system, which can either be dormant or continue to grow and start a vicious cycle in the bone microenvironment [18,19]. Bone remodeling starts with the expression of the receptor activator of nuclear factor-kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) inside the bone marrow compartment [20]. It initiates the differentiation of osteoclast precursors to osteoclast. The ratio of osteoblast (bone formation) and osteoclast (bone resorption) follow a tight regulation which is crucial to maintain the consistency of bone mass [21]. An imbalance in bone homeostasis leads to pathophysiological conditions, including osteoporosis, heterotopic ossification, and bone fragility [22]. Therefore, it is necessary to gain in-depth knowledge to improve the expectancy and quality of cancer patient’s life. Here, we would like to focus on chemokines networks/axis in different types of cancer, tumor heterogeneity, drug resistance, and bone metastasis, followed by a vicious cycle of bone remodeling.

Fig. 1. Major chemokines/receptor axis involved in malignancy:

This illustration explains the multifaceted roles of chemokine ligands and their receptors in various malignancies. Many chemokines can bind to multiple receptors, and a single receptor can bind to multiple chemokines. Most of the chemokine axis facilitate proliferation, invasion, migration, and bone metastasis. Interaction and activation of different axis have been shown in different colors with major consequences in breast, prostate, and lung tumorigenesis. Other types of cancer cells have different profiles of chemokine-receptor expression, but CXCR4 is most common among breast, prostate and lung cancers.

2. Chemokines/cytokines: types and functions

Chemokines are a small family of chemoattractant cytokines (small molecules of polypeptides with a molecular weight of 8–10 kDa). They are produced by various immunocytes, tumor cells, and normal tissue of different organs in response to infections and are involved in tumor growth and progression [23,24]. Chemokines bind to the specific receptors, particularly the seven-transmembrane G-protein-coupled receptor of target cells, and control numerous biological and pathological processes. A single chemokine can bind with multiple receptors and vice versa [25]. Approximately 50 chemokines have been detected so far and based on the number and location of N-terminal cysteine molecules; chemokines can be classified into four subfamilies: CC, CXC, C, and CX3C. The CC subfamily has a broad chemotactic spectrum/chemokines effect on various immunocytes (monocytes, basophils, eosinophils, T lymphocytes, dendritic cells) and cancer cells. It represents the most significant subgroup with 28 members (CCL1 to CCL28). The CXC chemokines subfamily comprises 16 members (CXCL1 to CXCL16) and, based on EβLR motifs (glutamic acid, leucine, and arginine), can be further divided into two subgroups ELR^+ve and ELR^−ve [26]. This CXC subfamily of chemokines plays a direct/indirect role in either promotion (ELR^+ve) or Inhibition (ELR^−ve) of angiogenesis [27]. Furthermore, the C subfamily of chemokines contains only two members, including XCL1 and XCL2, while CX3CL1 is the only known member in the CX3C subfamily chemokines [28]. To date, more than 20 chemokine receptors have been identified and classified according to the different chemokine subfamily or ligands, i.e., CC receptor (CCR), CXC receptor (CXCR), C receptor (CR), and CX3C receptor (CX3CR) [29]. The expression of chemokine receptors is not limited to macrophages, neutrophils, or inflammatory cells, but some endothelial and tumor-derived epithelial cells also express these receptors [30]. In addition to the above chemokine receptors, some other kinds of chemokine receptors are also expressed on stromal cells called atypical chemokine receptors (ACKR). These receptors do not participate either in cell migration or activation, but they can hamper the availability and function of chemokines by scavenging them either by limiting their spatial availability or remove them from in vivo sites [31]. Though they are not participating in migration and cell activation, they are essential molecules in health and numerous diseases. Four groups of ACKR have been identified, including ACKR1, ACKR2, ACKR3, and ACKR4 [32].

Similar to chemokines, cytokines are a diverse family of low molecular weight proteins involved in cellular signaling pathways [33]. Generally, cytokines act as immuno-modulators in all manners, including autocrine, paracrine, and endocrine signaling [34]. Based on their origin and involvement, cytokines are broadly categorized in chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors (TNF) [35]. Cytokines play crucial roles in the prevention of diseases, specifically in host immune responses to the infection and cancer [36]. It acts through cell surface receptors and can modulate humoral and cell-based immune responses [37]. Specific cytokines can be produced by more than one type of cells [26] and are released in response to different cellular stresses, including infection, inflammation, and carcinogen-induced injury [36]. The vital function of cytokines is to stimulate a host response to diminish cellular stress and subsequent damages [38]. Host reaction to cellular stress can impact tumor initiation and progression. Based on function, cytokines can be categorized into five major groups, including interleukins (IL), tumor necrosis factors (TNF), interferons (IFN), and colony-stimulating factors (CSF). Interleukins are the primary group in cytokines containing 12 members IL-1 to IL-12 [39]. In addition, cytokines can be further classified into the following three groups based on the nature of immune responses: adaptive immunity; pro- and anti-inflammatory signaling. In adaptive immunity, common γ chain receptor ligands, common β chain (CD131) receptor ligands, shared IL-2β chain (CD122) and shared receptors are usually involved in adaptive immunity while IL-1, IL-6, TNFα, Type I, II and III IFN cytokines are involved in pro-inflammatory signaling, whereas IL-10 and IL-12 cytokines participate in anti-inflammatory signaling pathway [35].

3. Chemokines helter-skelter in cancer

In the present scenario, it is well established that chemokines and their receptors participate in cancer progression. However, the regulatory mechanisms related to chemokines/receptors are still unclear [40]. After rigorous studies, researchers came to know that chemokines and their receptors are intensively involved in the process of metastasis [41, 42]. Metastasis is very dynamic and selective towards the specific organ, and different molecular axis is involved in various cancers, including breast, prostate, and lung cancer (Figs. 1 & 2). In addition to the role of chemokines in cancer, cytokines also participate in tumorigenesis, angiogenesis, metastatic niche formation, and bone remodeling.

Fig. 2. Chemokines-mediated tumorigenesis and bone tropism in lung, breast, and prostate cancers:

Chemokines directly affect the tumor microenvironment during the cancer progression. Involvement of chemokines in the different stages of cancer metastasis, such as angiogenesis, extravasation, and colonization to the distant organ or bone site (sequentially illustrated from left to right). Among all, the CXCL12-CXCR4 axis is significantly involved in the entire metastatic process. In contrast, vascular endothelial growth factor (VEGF) and CXCL8 majority participate in angiogenesis, while CCL2 and CCL5 drive metastasis and extravasation.

Statistically, in the year 2020, 19.3 million new cancer cases and 10 million cancer deaths were recorded worldwide. Briefly, female breast cancer (BC) 11.7 % (6.9 % deaths), lung cancer (LC) 11.4 % (18 % deaths), and prostate cancer (PCa) 7.3 % (3.8 % deaths) were the most common types [43]. However, in 2021, 1,898,160 new cancer cases are registered in the United States, out of which 608,570 cancer deaths are projected [44]. According to the American cancer society data, about 350,000 cancer patients die each year in the United States from bone metastasis [45]. The prevalence of bone metastasis among all metastatic cancer was 88.74 % in the PCa, 53.71 % in the BC, and 34.56 % in LC [45]. These three-carcinomas accounts for more than 80 % of patients with metastatic bone disease. Most cancer cells predominantly metastasize to axial skeletal including spine (87 %), ribs (77 %), pelvis (63 %), and humeri and femora (53 %), as well as the skull (35 %) [46]. In this section, we focus on the role of chemokines in the initiation and progression of various cancers which includes breast, prostate and lung and their metastatic potential.

3.1. Chemokines/receptors role in breast cancer and metastasis

Recent studies have elucidated the role of chemokine receptor CCR1 in human breast invasive ductal carcinoma is dependent on epidermal growth factor (EGF) stimulation. For EGF mediated stimulation, a signal transducer and activator of transcription 3 (STAT3) is required to activate the promoter of CCR1 via AKT-mTOR-STAT3 signaling and mediates invasion and metastasis of BC [47]. However, CCR2 chemokine receptors are potentially involved in the progression of early-stage BC and correlate with their invasive potential. The over-expression of CCR2 can enhance the invasion and viability of AUW225 BC cells [48]. Chemokine CCL2 can also induce and recruit the CCR2 expressing macrophages and promote metastasis in BC cells [49]. Specifically, CCR2/CCL2 axis mediates the p42/44 mitogen-activated protein kinase (MAPK) and SMAD3 pathways for cancer cell growth, invasion, and metastasis [50]. CCR4 is expressed differentially in human BC cell lines, and CCR4 over-expression is highly associated with metastasis. The expression of CCR4 is positively correlated to human epidermal growth factor receptor 2 (HER2), which results in tumor relapse and metastasis to lymph nodes, lungs, and bones [51]. Furthermore, BC metastasis can be modulated by the activation of CCL5 and is dependent on the interaction with CCR5 [52]. Zhang et al. delineated the role of CCL5 in the metastasis and primary tumor load in the MMTV-PYMT transgenic BC mouse model, where CCL5/CCR3 signaling promotes pulmonary metastasis and tumor burden by inducing Th2 polarization of CD4⁺ T cells [53]. It has been evidenced that the CCR7 is highly expressed in triple-negative BC cells. In addition to that, ligands (CCL19 and CCL21) of CCR7 are also highly expressed in the lymph nodes of BC patients. At the same time, the rate of proliferation and metastasis is significantly reduced in the absence of CCR7 in BC cells [54,55]. Later, the triple-negative BC metastatic mouse model revealed that the knockout of CCR7 and CCR4 reduced the metastasis of 4T1 cells to the lymph node [56].

Similarly, the CXC family of chemokines and their receptors are also involved in signal transduction for BC metastasis. The involvement of CXCR1 was identified in the proliferation of cancer stem cells (CSCs) of HER-2-negative BC, and Inhibition of CXCR1 with allosteric inhibitor reparixin reduces the CSC population in the xenograft mouse model [57]. Likewise, Interleukin-8 (IL-8) can play a crucial role in the regulation of BC metastasis. Nannuru et al. studied the role of IL-8 and CXCR2 in the progression, migration, invasion, and metastasis of breast mesenchymal stem cells, bone metastasis, and tumor microenvironment [58,59]. Several clinical studies showed a high correlation between CXCR2 and cyclooxygenase 2 (COX2), promoting BC metastasis and chemoresistance via down-regulating E-cadherin and β-catenin in cancer tissues [60]. At the same time, tumor-associated macrophages (TAMs) and the CXCL1/CXCR2 axis promote BC metastasis via NF-κB/SRY-related HMG-box (SOX4) activation [61]. During tumor progression or malignant transformation, chemokines can trigger several signaling pathways. In the 4T1 mouse model of BC, secretion of CXCL10 is gradually increased and is involved in the regulation of cancer cell growth via NF-kB signaling [62]. Chemokine receptor CXCR4 is intensively involved in the tumorigenesis of BC. Previous studies revealed that CXCL12/CXCR4 axis fosters the metastasis of BC cells by natural selection. Moreover, CXCR4 participates in drug resistance by interfering with the recruitment of transcription factors p53 and death receptor 5 (DR5) or TRAIL receptor 2 (TRAILR2) [63,64].

Likewise, over-expression of CXCR6 in BC mediates cancer cell invasion and metastasis, whereas silencing CXCR6 using shRNA reduces cancer-promoting abilities. CXCR6/ERK1/2/RhoA/cofilin/F-actin pathway is involved in BC tumorigenesis and metastasis [65]. Furthermore, the indirect contribution of CXCR7 has been revealed in the regulation of CXCR4 expression on BC cells. CXCR7 acts as a scavenging receptor for CXCL12 and also upregulates the CXCR4 expression and develops the BC metastasis in CXCR7^ΔEND/ΔEND mice [66,67]. Additionally, CXCR7/CXCL12 axis strongly supports BC cell survival with mesenchymal stromal stem cell-derived factors in the tumor microenvironment [68]. In vitro experiment shows that tamoxifen treatment increases the external secretion of CXCL16, which prompts the ability of migration and invasion of MCF-7 BC cells mediated by G-protein coupled receptors 30 [69]. In addition to the direct action on tumor metastasis, chemokines and their receptors influence the infiltration and recruitment of immune cells to create favorable conditions for metastasis. For example, the atypical chemokine receptor 2 (ACKR2) constrains the expression of CCR2 and prevents the infiltration of natural killer cells into the tumor site resulting in facilitating metastasis [70]. Atypical chemokines and their receptors may also participate as a component in the regulatory network for tumorigenesis [71]. Due to the frequent and critical involvement of chemokines and their receptors in breast carcinoma, rigorous attention has been paid to understand the complex phenomenon, including tumor heterogeneity, resistance, recurrence, and metastasis in the tumor microenvironment.

3.2. Chemokines/receptors role in prostate cancer and metastasis

In PCa progression and metastasis, several chemokines are extensively involved. The binding of CCL5 with CCR1 enhances the invasiveness of taxane-resistant PCa cells, via ERK and Rac activation and secretion of MMPs 2 and 9 [72]. Furthermore, the role of CXCR1 in PCa has been revealed by silencing its expression using RNA interference mechanisms. It was shown that CXCR1 is involved in tumor growth and makes androgen-independent, and IL-8 mediated growth of PCa cells [73]. In advanced PCa, blocking of CXCR2 deteriorates aggressiveness and induces senescence. Further, CXCR2 plays an essential role in infiltrating tumor-associated macrophages (TAMs) in the tumor microenvironment [74]. Shen et al. delineated the potential role of CXCR3 in the proliferation and invasion of PCa cells. Abnormal expression of CXCR3 elevates the expression of phospholipase C (PLCβ), matrix metalloproteinase (MMP-1), and MMP-3 in vitro, indicating the role of CXCR3 in PCa cell proliferation, invasion, and metastasis through activation of the PCLβ signaling pathway [75]. Recently, novel crosstalk between CXCR4 and phosphatidylinositol 4-kinase IIIα (PI4KIIIα) has been well established in PCa cells. Furthermore, SILAC-based quantitative proteomic analysis of PCa cells identified the PI4KIIIα and SAC1 phosphatase as potential regulators for CXCR4 expression [76]. Likewise, CXCR5 over-expression and its interaction with CXCL13 in PCa positively correlate with progression, migration, and invasion. Aberrant expression of oncogenic PKCε, a member of the PKC family, and loss of the tumor suppressor PTEN are involved in PCa cells migration and tumorigenesis [77]. Elevated expression of CXCR6 is also associated with PCa invasiveness and tumor growth. However, the CXCR6-CXCL16 axis promotes drug resistance and acts as a counter-defense mechanism [78]. The expression of some angiogenic factors such as IL-8 or VEGF is induced by CXCR6 over-expression. The CXCR6/CXCL16/AKT/mammalian target of rapamycin (mTOR) circuit is mainly involved in PCa tumorigenesis and angiogenesis [79]. One of the pioneer’s studies addressed the role of CXCR7 with CXCL12 in PCa. CXCR7 functions as a chemokine receptor for CXCL12 and, similar to CXCR6, regulates the IL-8 or VEGF expression and activates the AKT pathways resulting in cancer invasion and angiogenesis [80]. In castration-resistance PCa (CRPC), CXCR7 over-expression activates the MAPK pathways that develop drug resistance even for second-generation antiandrogen (enzalutamide) therapy. Further, elevated CXCR7 levels activate ligand-independent MAPK/ERK signaling in enzalutamide resistance [81]. Lastly, CX3CR1 and its cognate ligand CXCL1/fractalkine regulate the PCa cell adhesion, migration, and survival. CX3CR1 expression also enhances the perineural invasion, dissemination, and metastasis of PCa [84]. CX3CR1 receptors are involved in the activation of endothelium results in tumor cell adhesion and neural tropism. Direct correlation of this chemokine receptor with neuronal invasiveness and bone metastasis was supported by over-expression of CX3CR1 in human pancreatic and BC cells. In contrast to the normal prostate gland epithelial tissue, the CX3CR1 over-expressed in PCa malignancy.

3.3. Chemokines/receptors role in lung cancer and metastasis

Likewise, the role of chemokines is also associated with lung tumorigenesis. In a CCR1 knockdown study using RNA-mediated interference, the invasiveness of human non-small cell LC (NSCLC) has been significantly suppressed with reduced expression of matrix metalloproteinase-9 (MMP-9) [82]. However, another study suggests crosstalk between tumor-associated macrophages and cancer cells via CCR2/CCL2 and CX3CR1/CX3CL1 axis are fundamental mechanisms for driving LC. Macrophage depletion using clodronate and genetic ablation of CCR2 and CX3CR1 reduced tumor growth and metastasis in vivo [83]. Similarly, inhibition of CCR4 by miR-532–5p induces apoptosis and reduces the invasiveness, and the metastatic capability of LC cells confirming the role of CCR4 in LC tumorigenesis [84]. However, CCL5/CCR5 axis is also reported in LC metastasis in association with immune cell infiltration and poor prognosis. The expression of CCL5 is inversely correlated with miR-147a level (downregulated) in NSCLC, and upregulation suppressed the growth and metastasis [85,86]. Furthermore, CXCR1 interferes with the therapeutic outcome in EGFR tyrosine kinase inhibitors (TKIs) treatment, first-line treatment for advanced NSCLC via JAK/STAT signaling pathway [87]. However, in a previous study, IL-8 and CXCR1 mediate mitogenic function in autocrine/paracrine signaling [88]. Elevated expression of CXCR2 is observed in both LC Stroma and tumor cells, and inhibition of CXCR2 using a selective inhibitor SB225002 promoted apoptosis and increased drug sensitivity corroborate the role of CXCR2 in drug resistance [89]. A very few reports elucidate the involvement of CXCR3 in lung carcinoma. Expression of IL-7 promotes CXCR3/CXCL9/CXCL10 axis-dependent T cell antitumor reactivity in LC [90]. CXCR4 receptor, specifically with CXCL12, is intensively intricate with tumorigenesis, angiogenesis, and metastatic processes in NSCLC. Additionally, CXCR4/STAT3/Slug signaling is operated to maintain radio-resistance in NSCLCs [91]. Whereas CXCR6 and CXCL16 are expressed together in human LC and involved in the regulation of viability and invasion of LC cells. Downregulation of CXCR6 or neutralization of CXCL16 with antibody retards the viability and invasiveness of LC cells in vitro [92]. The expression of CXCR7 is upregulated in LC tissue and enhances cell migration and polarization in vitro. In the xenograft mouse model, CXCR7 accelerates tumor growth and metastasis to the different organs, such as the liver and/or bone marrow [93]. Furthermore, Liu et al. investigated the role of the CX3CR1/CX3CL1 axis in six different human LC cell lines. Activation of this axis significantly potentiates the migration and invasion of LC via Src/focal adhesion kinase (FAK) signaling pathway [94].

3.4. Cytokines and immune cells in carcinogenesis

Several researchers have examined the tumor susceptibility against chemical carcinogenesis in immuno-deficient mice to delineate the involvement of cytokines and immune cells in carcinogenesis. In a series of findings, growth differentiated factor 15 (GDF15, a divergent member of the transforming growth factor β superfamily) and its cognate receptor, glial cell line-derived neurotrophic factor (GDNF) family receptor α-like (GFRAL) has been elucidated in obesity. GDF15 signaling requires the interaction of GFRAL with a coreceptor RET to induce weight loss in mice and nonhuman primates [95–99]. Most recently, a pathophysiological role of GDF15 has been elaborated in various tumorigenesis, metastasis, and cachexia [100–107]. Another study demonstrates that impairments in the function of IFN-γ enhanced the susceptibility of tumor formation with a short latency period against polycyclic hydrocarbon methylcholanthrene [108–110]. IFN-γ mediates pleiotropic effects in the innate and adaptive response against infection [111]. Two cytokines IL-12, and IL-23, that stimulate the production of IFN-γ and lack of p40 subunit from these interleukins, enhanced susceptibility of tumor growth in response to chemical carcinogens [112]. With this, mice lacking Stat1 and IFN-γ, and its receptor is also prone to develop tumors against chemical carcinogens [113]. Mice devoid of Natural Killer T (NKT) cells are at risk of developing tumors with chemical carcinogens because NKT cells are one of the critical sources for IFN-γ production [114]. IFN-γ can upregulate the expression of MHC class I expression during cancer development and elicit cell-mediated immunity [115,116].

Furthermore, cell-mediated immunity (recognition and rejection) against tumors from immuno-deficient hosts into post-transplanted wild-type animals reveal to be more sensitive towards carcinogens [117]. Recently, B and T cells deficient mice manifested as increased susceptibility for tumorigenesis after methylcholanthrene (a highly carcinogenic polycyclic aromatic hydrocarbon) treatment [118]. Bone tissues constitute a persistent site for metastasis in numerous malignancies, including BC, PCa, and LC. Cytokines such as IL-6, VEGF, RANKL, and TGFβ have been crucial in establishing a premetastatic niche, remodeling the bone microenvironment, and viable macro-metastasis [119,120]. It is not essential that all circulating tumor cells survive in the new tissue microenvironment during metastasis. Often, many cells undergo a dormancy state, while others remain as nonviable micro-metastasis [121,122]. Further, VEGF secretion initiated micro-metastasis formation followed by bone metastasis, inducing neovascularization and providing nutrient supply [123]. Additionally, VEGFR-1-positive bone marrow progenitors have been strongly correlated with the initiation of tumor premetastatic niche formation. Indeed, activation of the VEGF/VEGFR axis is crucial for establishing cancer metastasis [124]. Furthermore, angiogenesis and inflammation provide molecular links for the initiation and promotion of cancer. However, the function of angiogenesis for tumor promotion is well established [125]. Next, to understand the involvement of host immunity in tumor suppression, several investigators assessed the tumor incidence in immuno-deficient or knockdown/knockout animal models. Earlier in 1984, it was observed that CD8T cells could recognize the specific antigen of tumor cells, followed by cloning the first definitive tumor antigens [126,127]. Ever since, it became clear that the immune system of humans can detect the antigenic profile of tumors [128,129]. Other limitations are related to the infiltration of cytotoxic T cells into the tumor tissues, and T-cell infiltration into tumors correlates with an improved prognosis for the patient [130,131]. Some advanced therapies have been developed to overcome the limitations of infiltration of cytotoxic T cells, including antigen-specific vaccines or by adoptive transfer of tumor-specific T cells. Even though significant expansions of circulating tumor-specific T cells do not provide long-term benefits [132], importantly, it has been shown that induced or transferred T cells are very proficient and sensitive to eliminating tumor cells in vitro but frequently fail to do so in in vivo settings. In lymphoma-bearing mice, knockout of perforin and double knockout of perforin/interferon enhanced the metastasis rate results in decreased longevity of mice [133]. Likewise, the development of B cell lymphoma is persistent in fas-fas-ligand defective mice, and tumor induction in immuno-deficient mice compared to wild-type mice is very frequent. Similarly, the development of adenocarcinomas of the breast, lung, and colon in double knockout mice (Rag2/Stat1 and Ifn-α/Trp53) is very frequent as compare to wild-type mice [134].

Two different major pathways have been suggested in support of tumor proliferation and endurance of cancer cells by chemokines, including activation of mitogen-activated protein kinase (MAP kinase) and extracellular signal-regulated kinase (ERK) signaling pathway [135]. Chemokines can also promote the tumor cell growth directly by inducing the expression of specific growth-stimulating genes, e.g., cyclin D1, oncogene FOS, and human heparin-binding epidermal growth factor (HB-EGF), and negatively regulates the expression of anti-apoptotic genes Bcl-2 and MDM2 [54,136]. Furthermore, chemokines and their receptors also participate indirectly in tumor growth, invasion, and immuno-suppression by stimulating the infiltration of leukocytes at the tumor sites to promote the secretion of IL-10 and TGF-β [137].

4. Chemokines in bone microenvironment versus vicious cycle and remodeling

The bone microenvironment is tightly regulated and maintains the homeostasis between diverse populations of resident cells and their functions. Bone is a very dynamic connective tissue that provides a physiological scaffold for the skeleton of the human body. Bone is a dwelling place for bone marrow which acts as a production house for postnatal hematopoiesis and a reservoir for minerals and energy (Fig. 3) [138–140]. In many types of malignancy, including BC, PCa, and LC, bone is a preferred metastatic site for homing cancer cells. Tumor cells, invasion, and colonization to the bone site alter the homeostasis of bone formation, and bone resorption leads to bone remodeling and, finally, the consequences of bone destruction. This osteolytic process is driven by several mediators such as RANKL and other factors which facilitate the expansion of metastasizing cancer cells in this niche [141,142]. Several chemokine family members were found to participate actively in bone remodeling in standard and malignant conditions [12,141–144].

Fig. 3. Bone homeostasis versus vicious cycle of bone metastasis:

Contribution of chemokines/cytokines in seeding and soiling of cancer cells in the bone microenvironment. (A) Normal bone homeostasis has been depicted on the left side, where a balance between bone formation and resorption has been shown with well-regulated RANK, RANKI, and OPG Systems. (B) Metastasis of cancer cells releases several factors such as IL-6, PTH-rP, MCSF, VEGF, MCP-1/CCL2, TGF-β, and RANKL in the bone microenvironment modulate the activities of osteoclasts and osteoblasts or bone homeostasis. In addition, bone cells also release pro-survival factors such as TGF-β, VEGF, IGFs to facilitate the growth of cancer cells with the bone microenvironment. This altered modeling starts a vicious cycle that promotes tumor cell growth and alteration in bone quality and bone remodeling process. Chemokines that regulate the osteolytic and osteoblastic bone remodeling are depicted on the right lower side.

4.1. Bone microenvironment and remodeling

Bone is metabolically active tissue and maintains homeostasis between bone formation and resorption. The basic multicellular unit is inhabited in the bone microenvironment and contributes to maintaining bone homeostasis. This multicellular unit comprises osteoblasts, osteoclasts, bone lining cells, and osteocytes (Fig. 3A). Osteoblasts participate in bone formation while osteoclasts reabsorb the bone. The function of bone lining cells is poorly defined, but these cells cover most of the endosteal bone surface [145]. Osteocytes are composed of almost 90 % of all bone cells and can regulate osteoclast and osteoblast activity. The osteocyte is a source of soluble factors and plays a role in both phosphate metabolism and calcium availability [146]. Mesenchymal origin cells, including osteoblasts, pre-adipocytes, chondrocytes, express osteocalcin, and adiponectin to varying stages of differentiation [147–150]. Additionally, osteoblastic monocyte chemoattractant protein-1 (MCP-1) or CCL2 mediates the anabolic and catabolic effect of parathyroid hormone in bone [151,152]. Chondrocytes, adipocytes, hematopoietic stem cells, mesenchymal stem cells (MSCs), fibroblasts, CXCL12-abimdant reticulocytes (CAR) cells, endothelial cells, pericytes, and nerve cells are also residing in the bone microenvironment [153–155]. Bone matrix which provides a healthy support to bone residing cells and play a crucial role in bone homeostasis, by regulating the level of growth factors, inorganic salts, and organic components [156,157]. Recently, it is revealed that the bone marrow endothelium, adipocytes, and immune cells infiltration also contribute to bone homeostasis. Aging and several diseases including cancer can imbalance the delicate bone homeostasis and create a structural defect and change the bone “soil” [158]. Different types of cells are involved in the formation and resorption of bone tissue. The primary cells which participate in bone formation are osteoblasts. These cells secrete extracellular matrix proteins, including type I collagen, osteopontin, osteocalcin, and alkaline phosphatase. The skeletal lineages towards the specification of osteoblast can be differentiated into three different stages: osteoprogenitor, pre-osteoblast, and osteoblast [159–161]. Furthermore, the differentiation of osteoblast requires a multitude of steps from stem cell differentiation to mature osteoblast. Two different kinds of stem cells, i.e., mesenchymal stem cells (MSCs) and skeletal stem cells (SSCs), differentiate into osteoblast individually. These cells are involved in bone tissue formation and mineralization [159]. Still, the relationship between these two stem cell populations has not been precisely determined. Another category of cells, including osteoclasts and osteocytes, regulate bone resorption. Osteoclasts are derived from the pre-osteoclasts cells that differentiate to form macrophages and monocytes [162]. Briefly, osteoclasts are large and multi-nucleated cells and are mainly involved in the resorption of bone. These cells are originated from hemopoietic lineage and differentiated into osteoclast with the interaction of macrophage-colony stimulating factor (M-CSF) and RANKL. M-CSF and RANKL are required for the proliferation and differentiation of osteoclast precursors, respectively, and RANKL deficiency hinders the bone resorption in mice [163]. Together with this, we emphasize the role of chemokines and cytokines in bone metastasis and vicious cycle of remodeling.

4.2. Chemokines and bone metastasis

Several studies have been done to elucidate the role of chemokines in bone metastasis. The CCR2 receptor mediated CCL2/CCR2 axis significantly contributes to the growth of PCa cells in the bone [164,165]. CCL2 mediated expansion of bone metastasis is facilitated by CCR2^+ve stromal cells, specifically in the case of PCa and BC bone metastasis. Chemotaxis of PCa cells is motivated by CCL2, suggesting the possible involvement of CCL2 in bone-specific migration of PCa cells [166]. Together with CCL2, RANKL, a receptor activator of NF-κB ligand, was reported to involve in the process of bone resorption by osteoclastogenesis and homing of cancer cells in PCa and BC bone metastasis, respectively [165,167,168]. However, another study suggests that the up regulation of CCL2/CCR2 accompanies PCa progression, metastasis, and relapse [169]. Additionally, PCa cells trigger the destructive cascade of osteoblastic CCL2 production in the bone microenvironment for metastatic progression. Further, CCL2 mediated destructive cascade (osteoclastogenesis) has been shown by injecting PCa cells in the tibia of mice [74]. The perturbation of bone marrow by bone marrow-suppressive chemotherapeutic drug cyclophosphamide mediates a transient increase in myeloid cells and myelogenic cytokines with CCL2, IL-6, and VEGF-A create a receptive microenvironment for PCa bone metastasis. Interestingly, anti-CCL2 antibody treatment significantly attenuates the tumor growth in the bone metastatic site [170]. The addition of CCL2 induces the expression of CCR4 together with CCL22 in PCa cells, and the CCR4/CCL17/CCL22 axis enhances the invasion and migration potential via phosphorylation of Akt [171]. Likewise, a comparative study of gene expression between PCa cell lines and tumors in immune-competent mice evidenced that the activation of the CCR5 signaling axis and blocking with CCR5 antagonist (maraviroc) reduces the bone metastatic potential of v-Src oncogene–transformed metastatic PCa cell lines [172]. CXCL10 and its receptor CXCR3 encourage BC bone metastasis and osteoclast activation, results in an increased rate of bone resorption in cancer patients. CXCL12/CXCR4 axis regulates cell migration and is a critical mediator of PCa bone metastasis. Another chemokine receptor, CX3CR1, is utilized by BC cells for extravasation from blood circulation, seeding, and colonization at skeletal metastatic site [173]. The abundant CX3CR1 expression is necessary for the adhesion of the tumor cells to endothelium, whereas mutated CX3CR1 mice show adhesion deficiency and abrogate tumor cell recruitment to the bone [75]. Due to the same axis in differentiated osteoblast, the CXCL1/CX3CR1 axis is intensively involved in PCa bone tropism. In an in vitro study, PCa cells migrate towards a medium conditioned by osteoblasts, which secrete the soluble form of the chemokine [174]. It is also elucidated that androgens facilitate the extravasation of CX3CRl^+ve PCa cells towards the CXCL1 concentration gradient in the bone marrow [175]. The CX3CR1-CXCL1 axis activates EGFR-dependent SRC/FAK pathway and induces the migration of PCa cells towards the bone tissue [176]. In a comparative study with one hundred LC patients (between with and without bone metastasis), the CX3CL1–CX3CR1 axis activation is positively correlated with bone metastasis [177].

4.3. Chemokines and vicious bone cycle

The arrival of cancer cells (seeding) can induce chaos in the bone microenvironment by disrupting normal homeostasis (Fig. 3A) and start a vicious cycle. Several metastasized tumor cells die in the bone microenvironment, and some of them can be in a dormant state for years. In contrast, some tumor cells silently start to change the bone microenvironment as preparing the soil for proliferation. The vicious cycle develops either osteolytic (abnormal bone resorption by osteoclastogenesis) or osteoblastic (ossification by osteoblastogenesis) remodeling [178]. Osteolytic lesions provide suitable niches for tumor cells to communicate with nearby cells (e.g., osteoclast progenitor cells, pe-osteoclasts, mesenchymal cells, and pre-osteoblast cells). Several factors, including chemokines and multistep processes, are involved in starting a vicious cycle, and because of that, bone metastasis is frequently observed in the late stages of cancer. But once cancer cells metastasize to the bone, they become incurable [141]. Metastasized tumor cells produce multiple chemokines and eventually start osteolytic or osteoblastic bone remodeling followed by a vicious cycle, as indicated in Fig 3B. Pathologically activated osteolytic lesions and numerous growth factors from damaged bone matrix accelerate vicious cycle and release large amounts of calcium ions resulting in the maturation of osteoclasts and bone resorption [179].

Additionally, bone is highly vascularized with the network of sinusoidal endothelial cells pervade through the bone marrow and cortical surface lined up with the cortical surface forming arteries and arterioles. Understanding the network interplay is critical as bone provides an avenue for tumor cell migration and homing. In the bone marrow, sinusoidal and arterial endothelial cells are the significant populations. Single-cell RNA sequencing (scRNAseq) data also revealed a complex transcriptional heterogeneity among bone endothelial populations reacting deferentially (differences in vessel size and function) in response to different microenvironmental stimuli. Moreover, the bone microenvironment harboring different populations with MSCs facilitates a favorable niche for tumor cell seeding and colonization [180,181]. Diverse populations of MSCs with an elevated leptin receptor (Lepr) and CXCL12 are identified with scRNAseq studies. These cells are also known as Cxcl12-abundant reticular (CAR) cells and are characterized by their distinct morphology of processes wrapping around blood vessels, specifically in the perivascular region in the bone [182]. According to the transcriptional profile, these CAR cells can be primed towards adipogenic or osteogenic lineage with elevated expression of respective markers and designated as adipo-CARs and osteo-CARs [149,150,183]. Several other studies have also enlightened the heterogeneity of the MSCs and assisted in recognizing the specific cell populations involved in bone metastasis and remodeling (Fig. 3). Briefly, studies reported that different tumor cells have an abnormal expression of some specific chemokine receptors. One of the initial studies delineated the role of chemokines in organ-specific metastasis of breast carcinoma. They describe the role of CCR7 and CXCR4 that regulate the invasion and organ-specific metastasis in lung, liver, and bone and the preferred sites positive for CCL21 and CXCL12 (respective ligands for CCR7 and CXCR4) [184]. However, BC cells’ pervasive tendency to metastasize in lungs, bones, and the brain also depends on vascular anatomy. In the case of colorectal cancer, the primary determinant for metastasis is vascular anatomy which dictates metastatic tendency to the liver. Some organs, including the lung, brain, liver, lymph nodes, and bone marrow, are more prone to cancer metastasis, while others such as the kidneys, pancreas, and skin are less prone to metastasis [185].

Further studies showed that a non-metastatic B16 melanoma and lung metastatic cell line lacking with CCR7 expression was directed to metastasized for lymph nodes after CCR7 over-expression [186,187]. Similarly, CXCR4 expression on B16 melanoma cells induced metastasis to the lung [188]. Metastatic cancer cells derived from different cancers can hijack the chemokine receptor system to facilitate cellular migration and triggers bone metastasis at distant sites. However, various cancers acquire another mechanistic axis for bone metastasis in which CXCL12/CXCR4 are very common [185,189]. Additionally, various resident cells of specific organs, e.g., lung cells, secrete CXCL12 and CCL21, directing BC (CXCR4) and melanoma cells (CCR7) to the lung [184]. CCR9/CCL25 is a specific axis with melanoma tumors that shows a rare metastasis to the intestine [190,191].

In this context, under normal physiological conditions, CXCL8 can regulate the production of RANKL via osteoblasts and increase the rate of bone resorption with osteoclast promotion [142,192–194]. Therefore, metastasizing cancer cells acquired the ability to express CXCL8 can induce osteoclastogenesis followed by bone resorption. Indeed, several studies demonstrated that BC cells promoted osteoclastogenesis as indicated by increased generation of TRAP + cells out of peripheral blood mononuclear cells and inhibition of CXCL8 or its receptors down-regulate the process [190,193,195]. A similar in vivo study showed CXCL8 produced in CXCL8-transgenic mice upregulated the osteolysis, whereas antibodies against CXCL8 prevented bone damage and elevated the survival of mice [193]. Additionally, in BC, semaphorin D-mediated induction of IL-8 and LIX/CXCL5 increases osteoclast numbers, and shRNA silencing of semaphorin D reduces the levels of metastasis [194]. In numerous studies, analysis of plasma from BC patients identified a significant correlation between increased CXCL8 levels and a high degree of bone resorption as well as bone metastasis, supporting key roles for CXCL8 in pro-osteoclastogenic activity [193]. In addition to CXCL8, CCL2 and its receptor CCR2 are involved in the physiological bone remodeling process [196]. Activation of CCR2/CCL2 axis supports colonization of BC cells at bone metastatic site by promotion of osteoclast differentiation. Moreover, it was found that MAPK11 (p38β) activation in BC cells has given rise to elevated CCL2 production, which then contributed to increased bone resorption [193, 197,198]. Axis related to the CXCL12-CXCR4 pair, CCL5, and CCL3, and their shared receptors CCR1 and CCR5 are more complex, regulating bone remodeling in cancer. CXCL12-CXCR4 axis is of particular interest because CXCL12 was found to promote bone resorption under different pathophysiological conditions. In parallel, it is a leading factor in driving tumor cell homing to the bones in a massive number of malignancies [142,199]. Due to these multifaceted roles of CXCL12, it is expected that the CXCL12-CXCR4 pair is involved in regulating osteoclastogenesis and osteolysis in bone homing and needs to be extensively addressed in the future studies. In summary, chemokines/cytokines’ contribution in tumorigenesis and metastasis mainly depends on the metastatic tissue sites and crosstalk between chemokines and their respective receptors within the local microenvironment. However, current experimental evidence links specific chemokine/receptor axis to the metastatic niche formation, which promotes tumor growth in the prostate, breast, and lungs and is followed by tissue-specific metastasis.

5. Chemokines microcosmos versus tumor heterogeneity and resistance

High levels of heterogeneity in a tumor can modulate the aggressiveness, drug tolerance and can expand pre-existing sub-clonal drug-tolerant populations. Additionally, chemotherapeutic resistance is often entwined with cancer stem cells and followed by metastasis. Tumor heterogeneity potentiates cancer cells to evade apoptosis, drug resistance, and subsequently metastasis processes. The capability of tumor cells to initiate the heterogeneous population and appropriate account for metastasis reflecting heterogeneity along with cancer stem cells (CSCs) and assessment of heterogeneity is essential to improve the drug efficacy and therapeutic outcome [200]. One of the influential mechanisms for heterogeneity and subsequent events are interlinked with the secretion of proinflammatory cytokines/chemokines by tumor cells and tumor microenvironment termed as cytokine storm [201]. Chronic inflammation is associated with several types of cancers, including helicobacter-associated gastric cancer, chronic colitis-associated colon cancer, and smoking-induced LC [202,203]. Plausible explanations for this paradigm are multifold. During chronic inflammatory responses to the infection or damage, the compensatory cell proliferation induced to regenerate lost or damaged tissues might lead to an increased chance of multiple mutations and a proliferative advantage for the cells carrying those mutations. Maeda et al. showed the correlation between cell loss and the subsequent regeneration in mouse chemical carcinogenesis models [204]. In addition, free radical oxygen species released by the inflammatory cells may increase the mutation rate in the proliferating epithelial stem cells. Genetic, as well as phenotypic tumor heterogeneity, seems like a big challenge in cancer management. Epithelial to mesenchymal transition (EMT) and cancer stemness are two interlinked driving non-genetic phenotypic plasticity [205]. The microenvironment of cancer cells also remarkably supports CSCs with the secretion of chemokines and cytokines, including IL-6, stromal-derived factor-1, and IL-8, which promote drug resistance and metastasis [206,207]. Prompt microenvironment from cytokines and growth factors also participate crucially to determine the fate of CSCs in non-solid tumors [208]. Currently, the concept about CSCs is that they do not need undifferentiated stem cells for their origin. CSCs can be initiated from reprogrammed somatic cells and can achieve advanced properties, including cellular plasticity, resistance to apoptosis, immune evasion, and co-option of other tumor and stromal cells [209]. Suggestively, in addition to cellular motility, the role of chemokines and their receptors are deeply interlinked with the maintenance of CSCs and metastasis [210].

5.1. Chemokines and tumor heterogeneity

Two major axis of chemokines-receptors are involved in tumor heterogeneity, i.e., CXCL12/CXCR4 and CXCR1/CXCR2. In a recent study, the CXCR4-CXCL12 chemokines/receptor axis is one of the key regulators in maintaining CSCs. This axis is activated in the CD44⁺/CD133⁺ prostate progenitor population and supports clonal growth and malignancy. Further, using a xenograft mouse model, it was observed that as compared to monotherapy, a combination of Taxotere and AMD3100 (an antagonist of CXCR4) significantly eradicated PCa stem-like cells [211]. Similarly, the role of the intracellular chemokine CXCL12γ has been revealed in CSCs induction. Over-expression of CXCL12γ induced CSCs in PCa cells through CXCR4-mediated PKCα/NFκB signaling and promotes prostate tumor growth, metastasis, and chemoresistance in vivo [212]. Additional evidence supporting cancer stemness is linked with CXCR4/CXCL12 axis in carcinoma-associated fibroblasts (CAFs) derived from BC patients. CAFs promote the proliferation of CD44⁺CD24⁻ cells through their ability to secrete stromal cell-derived factor 1 (SDF-1), which may be mediated to SDF-1/CXCR4 signaling resulting in high spheroid formation with an enriched population of CSCs [213] and supported that CXCR4 expression enhances BC cells’ ability to form tumor mammospheres [214]. Later, Kong et al. reported that the over-expression of SDF-1 activates NF-kB pathways and induces the EMT and CSCs in a luminal-A subtype of breast carcinoma [215]. It was previously observed that the expression of CXCR4 is associated with poor prognosis in esophageal cancer patients and its role in an early metastatic spread in lymph nodes and bone marrow [216].

CXCR1/CXCR2 is another important chemokine receptor axis playing a role in tumor heterogeneity and maintains the stemness of CSCs in association with CXCL1 and CXCL8. Isolation and characterization of CSCs in 33 cell lines derived from normal and malignant mammary tissue identified the importance of CXCR1/IL-8 role in tumor heterogeneity, stem cells maintenance, and metastasis [217]. The selective depletion of the aldehyde dehydrogenase-positive BC CSCs population by blocking CXCR1 receptors induces apoptosis in the BC population. It reduces metastasis in vivo via FASL/FAS signaling pathways [218]. Another study demonstrated that the EGFR/HER2-dependent mechanism of the mammospheres-promoting effect of CXCL8 and blockade of CXCR1/2 together with specific inhibitors potentially reduced breast CSCs activity [219]. Further, administration of CXCL1 significantly abolishes the effects of XIAOPI (an inhibitor of pre-metastatic niche formation) in BC migration, invasion, stem cells subpopulations, EMT, or mammospheres formation abilities [220,221]. Similarly, over-expression of CXCL8 self-renews pancreatic CSCs through the CXCR1 and IL-8/CXCR1 axis and is associated with cancer stem cell-like properties that correlate with clinical prognosis in human pancreatic cancer cases [222]. Different studies also support the role of other chemokines, including CCL2, CCL5, CCR5, and CXCR7/ACKR3, in tumor heterogeneity and CSCs maintenance in BC [223–226].

5.2. Chemokines and drug resistance

Drug resistance remains a multifaceted obstacle to cure most cancers at present. Increasing evidence suggests that the tumor microenvironment plays a cardinal role in drug resistance [227]. Previously, researchers have highlighted the role of chemokines and cytokines in the mechanisms of cancer drug resistance and cancer progression. Secretion of inflammatory mediators such as cytokines and chemokines from the tumor microenvironment components or tumor cells directly influences cancer cell proliferation and metastasis-related to chemoresistance and poor prognosis [228]. These messenger molecules can be secreted by both the tumor microenvironment and cancer cells themselves and exert chemoresistance through autocrine/paracrine signaling by inhibiting apoptosis, increasing the proliferation and efflux of chemotherapeutic drugs. Furthermore, recent studies have described how specific cytokines secreted by cancer stromal cells confer resistance to chemotherapeutic treatments [228]. To better understand the mechanism of cancer drug resistance and to predict treatment outcomes, correlations between global chemokine/cytokine profiles and cancer drug resistance must be established. Here we discuss the recent discoveries in this field of therapy and chemoresistance concerning chemokines and cytokines and their implications for the future development of effective outcomes in BC, PCa, and LC and their organ-specific metastasis [229]. The chemoresistance role of chemokines and cytokines is well established in BC, PCa, and LC. For instance, the IL-6 and IL-8 level in tamoxifen-resistant BC cells increases compared to parental cells [229]. CAFs are the crucial components of TME that can reprogram extracellular matrix (ECM) and are an important regulator of chemotherapy resistance. It has been reported that CAFs treated with cisplatin conferred chemoresistance to LC cells by upregulating IL-11 in CAF cells, protects cisplatin-mediated apoptosis. Mechanistically, IL-11 induces the STAT3 pathway, increases anti-apoptotic protein Bcl-2 and survivin expression in cancer cells, and provides resistance to cancer cells [230]. IL-6 is also recognized as a key regulator of immunosuppression in patients with advanced cancer [231]. Several evidences propounded crosstalk of IL-6 with multidrug resistance (MDR) in cancers, suggesting that IL-6 is instrumental in the chemotherapeutic response of the drug. IL-6 promotes MDR expression through JAK/STAT3, PI3K/AKT, and RAS-MAPK pathways. Activation of these pathways induces the expression of genes involved in proliferation and survival, promoting drug resistance [232]. Chemokine and cytokine recruit and activate MSCs at tumor sites. Consequently, MSCs secrete more chemokines and cytokines, which participate in tumor growth and metastasis [233]. In a BC study, MSCs promote proliferation of MCF-7 cells and decrease cisplatin response by releasing IL-6, activating the STAT3 survival pathway, and reducing apoptosis, and explored a novel drug resistance mechanism of cisplatin [234]. Siltuximab, a chimeric, anti-IL-6 monoclonal antibody, harbors potential therapeutic benefit in castration-resistant PCa (CRPC) patients. In combination with docetaxel, siltuximab is safe and shows preliminary efficacy in patients with CRPC [235]. As discussed previously, cancer stem cells play a cardinal role in disease recurrence, metastasis, and drug resistance. CXCR1, a receptor of CXCL8, is present on stem cells in BC that renders them resistant to drug response. Interestingly, reparexin, an allosteric inhibitor of CXCR1/2, reduces the CSCs population in the human BC xenograft mice model, thus, induces chemo-sensitization [236]. In a clinical trial, reparexin drug was safe in 20 patients with no serious adverse effects and CSCs markers were found to be decreased by ≥ 20 % in BC patients [57]. In another Phase1b clinical trial, the dose, safety, and pharmacokinetic profile of paclitaxel plus reparixin were determined in metastatic BC patients [237].

Like BC, chemoresistance was frequently observed in LC. In NSCLC, CCL5 secreted from CAFs induces chemoresistance in tumor cells via upregulating lncRNA HOTAIR expression and high levels of Bcl2 protein, which ultimately lead to cisplatin resistance in NSCLC [238]. Effector T-regulatory (Treg) cells predominantly express CCR4 in cancer tissues [239]. Although mTOR is essential for the immunosuppressive function of myeloid-derived-suppressor cells (MDSCs) [240], consistent rapalog (mTOR inhibitor) treatment promotes FoxP3+Tregs expansion in vivo that counterbalance with effector antitumor CD8 T-cell immunity. Consistently, Tregs Inhibition by using a small molecule antagonist of CCR4, a receptor highly expressed on rapalog-induced T regs, improved the antitumor efficacy of rapalogs and further restoring tumor-specific CD8 T-cell functions [241]. Tregs expansion in vivo limits the efficacy of the bi-therapy by altering the antitumor CD8 T-cell responses. A CCR4 antagonist prevents Tregs induction which considerably improved the efficacy of the bi-therapy by enhancing CD8 T cells-mediated antitumor immunity [242]. Also, the transcriptomic analysis showed marked downregulation of CXCR2 and BCL-2 in taxane-treated patients vs. taxane-naive patients as well as in in vitro settings. Furthermore, they determined that in vivo preclinical data analysis reveals taxane-platinum combinations are highly synergistic and sensitizes metastatic castration resistance PCa (mCRPC) tumors to cisplatin [243]. Over-expression of CXCR4 in NSCLC promotes cisplatin resistance via CXCR4-mediated CYP1B1 upregulation [244]. Similarly, CXCR2 upregulation renders NSCLC cells resistant to cisplatin and gemcitabine by activating JAK2/STAT3 signaling pathway [245]. In a separate study, drugs (cisplatin, carboplatin, and oxaliplatin) showed the same effect on tumor growth in immune-deficient mice. However, oxaliplatin exhibited a better response in wild-type mice with Lewis lung carcinoma (LLC), showing a high level of CXCL9, CXCL10, and CXCL11 in the tumor microenvironment, facilitate the recruitment of T cells and NK cells. Therefore, the combination of oxaliplatin with anti-PDL1 or anti-NKG2D antibodies results in a better therapeutic effect. Combining the three drugs could effectively inhibit the growth of LLC tumors and improve the survival of tumor-bearing mice [246]. Anti-PDL-1 or CCR1 inhibitor alone moderately inhibits the primary tumor growth and lung metastasis. However, the combination of CCR1 inhibitor and anti-PDL1 antibody significantly reduces the tumor burden compared to single agents. In addition, CCR1 expressing CD11b + Ly6Ghi Ly6Chi MDSCs were found in a xenograft BC model and further reduced significantly with CCR1 blockade through CCX9588 [247]. KRAS/LKB1 is a driver mutation to develop primary resistance to immune checkpoint inhibitors (ICIs) in NSCLC. Combination therapy with anti-PDL-1 and all-trans-retinoic acid (ATRA) improved local and systemic T-cell proliferation and generated tumor-specific immunity. This study also implicated ELR + CXC chemokine-mediated enrichment of G-MDSCs as a potential mediator of immunosuppression in LKB1-deficient NSCLC. It provides a rationale for using ATRA in combination with anti-PDL-1 therapy in patients with LKB1-deficient NSCLC refractory to ICIs [248].

Additionally, bone marrow contains candidate components that could contribute to the emergence of drug resistance, including soluble factors, such as interleukins. Moreover, chemokines are known to be essential for hematopoietic cdl homing within the bone marrow. Stromal cell-derived factor-1 (SDF-1 or CXCL12) is constitutively expressed by bone marrow stromal cells, which can be considered as a primary source of chemokine in adults [249–251]. Price et al. demonstrated that dormant and proliferating BC cells occupy the niche in the bone marrow. Dormant cells reside in the E-selectin and CXCL12-rich vascular regions. CXCL12/CXCR4 axis renders dormant cancer cells resistant in the bone microenvironment [252]. In a preclinical model of BC, CXCR4/CXCL12 axis in M2 perivascular TAMs was implicated in disease relapse after chemotherapy. Interestingly a similar M2 population was noticed in BC patients with disease relapse and bone metastasis after chemotherapy [253]. Metastatic bone tumors express a high level of CCR5 and targeting CCR5 by maraviroc in PCa bone metastasis could be a promising treatment option [172]. Since, several studies indicate the substantial correlation of cytokines/chemokines with tumor heterogeneity, drug resistance, and metastasis. Specifically, tumor heterogeneity and drug resistance are the primary root cause of metastasis. Moreover, after acquiring these abilities (heterogeneity and drug resistance), chemokines and cytokines act as torchbearers for specified organotropism.

6. Major clinical trials related to chemokines in bone metastasis

Several clinical trials have been started based on the preclinical evidence related to the modulation of the different axis of chemokine receptors/ligands in PCa, BC, and LC patients with bone metastasis (Table 1) [254–256]. Three clinical trials have been started with bone metastatic cancer patients viz., 1). Evaluation of the safety and blocking efficacy of CCL2 in advanced stages of patients (http://www.clinicaltrials.gov: NCT00992186). In the initial clinical trial, CNTO 888, an antibody that blocks CCL2, was used for metastatic castration-resistant PCa patients. Blocking CCL2 was not very efficient and probably because of compensatory mechanisms [254]. 2.) MLN1202, a monoclonal antibody against CCR2, has been used as anti-metastatic therapy for patients with bone metastasis in the phase II trial. Bone metastatic patients administered anti-CCR2 monoclonal antibody MLN1202 IV over 1 h on days 1,15, and 29 showed a decrease in urine n-telopeptide, suggesting a positive effect. (http://www.clinicaltrials.gov: NCT03550508). To date, there is no availability of any anti-metastatic clinical regimen that demands additional criteria to investigate different factors and etiology for heterogeneity, stemness, and organotropic metastasis of cancer cells and microenvironment. Moreover, future studies will be decisive in targeting specific cancer axis of chemokine-chemokine receptors to manage therapeutic options for cancer and metastasis.

Table 1.

Major clinical trials related to the chemokine’s regulation in bone metastasis.

Clinical trial number	Title	Status	Condition or disease	Interventions/treatment	Sponsors	Phases
NCT03554317	COMbination of Bipolar Androgen Therapy and Nivolumab (COMBAT-CRPC)	Active, not recruiting	Castration-resistant Prostate Cancer Metastatic Prostate Cancer Prostate Cancer	Drug: Testosterone cypionate Drug: Nivolumab	Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins	Phase 2
NCT00992186	A Study of the Safety and Efficacy of Single-agent Carlumab (an Anti-Chemokine Ligand 2 [CCL2]) in Participants with Metastatic Castrate-Resistant Prostate Cancer	Completed	Prostate Cancer	Drug: Carlumab
NCT03599453	Chemokine Modulation Therapy and Pembrolizumab in Treating Participants with Metastatic Triple-Negative Breast Cancer	Active, not recruiting	Triple -Negative Breast Cancer	Drug: Celecoxib Biological: Recombinant Interferon Alfa-2b Drug: Rintatolimod Biological: Pembrolizumab	Roswell Park Cancer Institute	Early Phase 1
NCT04081389	Chemokine Modulation Therapy and Standard Chemotherapy Before Surgery for the Treatment of Early-Stage Triple Negative Breast Cancer	Recruiting	Triple-Negative Breast Carcinoma	Drug: Celecoxib Drug: Cyclophosphamide Drug: Doxorubicin Drug: Doxorubicin Hydrochloride Drug: Paclitaxel Biological: Recombinant Interferon Alfa-2b Drug: Rintatolimod	Roswell Park Cancer Institute	Phase 1
NCT01433172	Combination Immunotherapy of GM.CD40 L Vaccine with CCL21 in Lung Cancer	Completed	Lung Cancer Adenocarcinoma	Biological: Phase I - GM.CD40 L.CCL21 Vaccinations Biological: Phase II - GM.CD40 L cells Vaccinations Biological: Phase II - GM.CD40 L.CCL21 Vaccinations	H. Lee Moffitt Cancer Center and Research Institute	Phase 1 Phase 2
NCT05060796	Study of CXCR5 Modified EGFR Targeted CAR-T Cells for Advanced NSCLC	Recruiting	Non-Small Cell Lung Cancer	Biological: CXCR5 modified EGFR Chimeric Antigen Receptor Autologous T cells	Second Affiliated Hospital of Guangzhou Medical University	Early Phase 1
NCT04153799	Study of CXCR5 Modified EGFE Chimeric Antigen Receptor Autologous T Cells in EGFR- Positive Patients with Advanced Non-small Cell Lung Cancer	Recruiting	Non-Small Cell Lung Cancer	Biological: CXCR5 modified EGFR Chimeric Antigen Receptor Autologous T cells	Sun Yat-sen University	Phase 1
NCT01015560	S0916, MLN1202 in Treating Patients with Bone Metastases	Completed	Metastatic Cancer Unspecified Adult Solid Tumor, Protocol Specific	Drug: anti-CCR2 monoclonal antibody MLN1202	Southwest Oncology Group	Phase 2
NCT03550508	Safety, Tolerability and PK/PD of JMT103 in Patients with Bone Metastases from Tumors (JMT103)	Unknown	Bone Metastases	Biological: Anti-RANKL Monoclonal Antibody	Shanghai JMT-Bio Inc.	Phase 1
NCT02688686	Safety and Efficacy of DC-GK in Patients with Advanced Non-Small-Cell Lung Cancer with Bone Metastases	Unknown	Non-Small-Cell Lung Cancer with Bone Metastases	Biological: genetically modified dendritic cells + cytokine-induced killer	Affiliated Hospital to Academy of Military Medical Sciences	Phase 1 Phase 2

7. Concluding remarks and future perspective

Conclusively, besides their traditional roles as a chemoattractant, chemokines play crucial roles in tumor initiation, progression, and metastasis. Several chemokines and chemokine receptors axis govern primary tumor lesions to bone metastasis and devastating skeletal remodeling. As reviewed in this manuscript, tumor cells in the bone microenvironment, distinct chemokine/chemokine receptor pairs/axis control cancer cell survival, proliferation, dormancy, and initiation of a vicious cycle. Further, we also focused on a massive amount of literature supporting chemokines’ vital role in bone remodeling. However, some axis can be dissected for specific cancer and bone metastasis despite the enormous complexity of the chemokines system and functional redundancy. It is important to note that numerous mice models devoid of specific chemokines/receptors display a distinct impairment of their skeletal remodeling status. Since incurability and high mortality rate (of bone metastatic cancer patients) demand to explore the most critical chemokine receptor axis playing a role in bone metastasis and remodeling, altogether, inhibition of metastasis or activation of dormant cancer cells into the bone and delineating specific axis in different cancers would be crucial for cancer therapeutics. The establishment of such kinds of chemokines/receptors axis could open new avenues in skeletal metastasis regulation and improvement of the quality of life of bone metastatic cancer patients.

In the last decade of cancer research, prevention or eradication of existing metastasis has been developed into the most critical scientific assignments. However, inhibition of metastasis by targeting tumor cells along with tumor microenvironment or specific host cells remains to be achieved. A classical concept related to cancer prevention or therapy is very much focused on tumor cells alone, but in modern notions, the tumor microenvironment is also under important consideration. The concept of pharmacological modulation of chemokines and their receptors to reduce chemokines regulated metastasis is under extensive investigation. Despite of the remarkable success, various knots/obstacles related to chemokines interplay remain to be solved. (A) The secretion of chemokines and the expression of their receptors are highly complex because of multi-ligand and multi-receptors interplay. (B) The expression of receptors and chemokines secretion can be either on tumor cells or at the metastatic site. (C) Modulating chemokines signaling may lead to alteration in chemokines profile, and harmful consequences can arise.

Abbreviations:

RANK

receptor activator of nuclear factor kappa B

RANKL

receptor activator of nuclear factor kappa B -ligand

OPG

osteoprotegerin

IL-6

Interleukin 6

PTH-rP

parathyroid hormone-related peptide

MCSF

macrophage colony-stimulating factor

VEGF

vascular endothelial growth factor

MCP-1/CCL2

monocyte chemoattractant protein-1

TGF-β

transforming growth factor-beta

IGFs

insulin-like growth factors

Data availability

Not applicable, all information in this review can be found in the reference list.