Chemotherapy-induced metastasis: molecular mechanisms and clinical therapies

Abstract

Chemotherapy, the most widely accepted treatment for malignant tumors, is dependent on cell death induced by various drugs including antimetabolites, alkylating agents, mitotic spindle inhibitors, antitumor antibiotics, and hormonal anticancer drugs. In addition to causing side effects due to non-selective cytotoxicity, chemotherapeutic drugs can initiate and promote metastasis, which greatly reduces their clinical efficacy. The knowledge of how they induce metastasis is essential for developing strategies that improve the outcomes of chemotherapy. Herein, we summarize the recent findings on chemotherapy-induced metastasis and discuss the underlying mechanisms including tumor-initiating cell expansion, the epithelial-mesenchymal transition, extracellular vesicle involvement, and tumor microenvironment alterations. In addition, the use of combination treatments to overcome chemotherapy-induced metastasis is also elaborated.

Introduction

Chemotherapy aims to improve the survival of cancer patients by eliminating tumor cells. It is the most widely accepted treatment for malignant tumors and uses various drugs to induce tumor cell death. Such drugs include antimetabolites, alkylating agents, mitotic spindle inhibitors, antitumor antibiotics, and hormonal anticancer drugs. However, accumulating evidence indicates that chemotherapeutic drugs can cause biological deterioration. Metastasis is the most prominent “by-effect” in patients receiving chemotherapy (Table 1). It has been demonstrated that cyclophosphamide induces the metastasis of fibrosarcoma in vivo [1]. Concordantly, other chemotherapeutic agents, such as paclitaxel, doxorubicin, gemcitabine, 5-fluorouracil (5-FU), sunitinib, and tamoxifen, have been reported to initiate metastasis [2–5]. Hence, preventing or reversing the pro-metastatic effect induced by chemotherapy is of great significance in clinical cancer treatment.

Table 1.

The pro-metastatic effects of chemotherapeutic agents.

Classification	Chemotherapeutic agents	Cancer type	Pro-metastatic mechanism	Reference
Taxanes	Paclitaxel	Cancer	Increase invadopodia number, screen out aggressive phenotype	[11]
		Ovarian cancer, breast cancer	Dimerize and activate TLR4, activate the NF-κB pathway	[101, 103]
		Lewis lung carcinoma (mouse model)	Induce bone marrow-derived cells secrete more matrix metalloproteinase 9 (MMP9)	[50]
		Ovarian carcinoma, small cell lung carcinoma, and glioma (mouse model)	Trigger angiogenic factors VEGF and proteinases and mediate macrophage infiltration into tumors	[54]
		Breast cancer	Stimulate monocytosis to systemically elevate monocyte chemoattractant proteins. Increase EZH2 expression, secrete exosomes enriched in miR-378a-3p and miR-378d	[24]
		Ovarian cancer	Activate NF-κB, stimulate growth, invasion, and drug resistance	[2]
	Docetaxel	Breast cancer	Elevate miR-9–5p, miR-195–5p, and miR-203a-3p in circulating extracellular vesicles (EVs) and decrease ONECUT2 expression, promote the expansion of tumor-initiating cells (TICs), increase cancer stemness	[46]
Alkylating agents	Cyclophosphamide	Ovarian carcinoma, small cell lung carcinoma, and glioma (mouse model)	Trigger angiogenic factors VEGF and proteinases and mediate macrophage infiltration into tumors	[54]
		Breast cancer	Increase vascular permeability and the level of MMP2	[49]
	Cisplatin	Ovarian cancer	Activate NF-κB, stimulate growth, invasion, and drug resistance	[2]
			Increase ERK2 phosphorylation	[12]
	Carboplatin	Triple-negative breast cancer	Regulate p38 MAPK signaling pathway through HIF-1, enrich breast cancer stem cells	[21]
		Breast cancer	Directly induct the expression of glutathione S-transferase omega 1 (GSTO1), activate hypoxia-inducible factors and Ca2+ release	[23]
	Oxaliplatin	Colon carcinoma	Promote epithelial-mesenchymal transition (EMT)	[2]
		Colon cancer	Stimulate the NF-κB signaling pathway	[119]
		Colorectal cancer	Induce cancer-associated fibroblasts (CAFs) secreting exosomes to enhance miR-92a-3p, activate the Wnt/β-catenin pathway and inhibit mitochondrial apoptosis	[44]
Antitumor antibiotics	Doxorubicin/Adriamycin	Breast cancer	Stimulate monocytosis to systemically elevate monocyte chemoattractant proteins Increase EZH2 expression, secrete exosomes enriched in miR-378a-3p and miR-378d	[24]
		Colon cancer	Stimulate the NF-κB signaling pathway	[115–119]
		Breast cancer	Upregulate Tie2 by inhibiting the expression of SIRT7, promote EMT	[58]
	Anthracyclines	Breast cancer	Secrete exosomes that suppress chemosensitivity by promoting the expansion of TICs	[46]
Antimetabolites	Gemcitabine	Pancreatic tumor	Promote EMT, activation of the c-Met tyrosine kinase	[3]
		Pancreatic cancer	Generate reactive oxygen species (ROS), affect NF-κB and HIF-1α, activate ERK1/2 and Akt signaling pathways	[120]
		Hepatocellular carcinoma	Activate the EGFR-pSTAT3 pathway, upregulate HAb18G/CD147 protein expression	[55]
	5-Fluorouracil (5-FU)	Colon cancer	Stimulate the NF-κB signaling pathway	[119]
		Colorectal cancer	Induce CAFs secreting exosomes to enhance miR-92a-3p, activate the Wnt/β-catenin pathway and inhibit mitochondrial apoptosis	[44]
Receptor tyrosine kinase (RTK) inhibitor	Sunitinib	Renal cell carcinoma	Decrease mir-452-5p and regulate SMAD4/SMAD7 pathway	[111]
Humoral Antiestrogens	Tamoxifen	Breast cancer	Increase cell motility and invasion, promote EMT	[28, 29]

Tumor metastasis involves several important steps including cell migration, local invasion, entry into the circulation, arrest at secondary sites, extravasation, and colonization [6, 7]. Accumulating evidence reveals that chemotherapy can induce metastasis at multiple steps. It may directly affect tumor cells or cause changes in the tumor microenvironment (TME). It kills cancer cells while simultaneously screens out those with more invasive and metastatic phenotypes from the primary tumors, such as cancer stem cells (CSCs) or stem-like tumor-initiating cells (TICs). It also induces epithelial-mesenchymal transition (EMT) and extracellular vesicles (EVs) in tumor cells, which confers chemoresistance and metastasis [5]. Besides, chemotherapy increases vascular permeability and the release of vascular endothelial growth factor (VEGF), facilitating tumor cell intravasation and dissemination. It can also damage the vascular endothelium and impair the host’s natural antitumor defenses, enabling increased tumor cell extravasation [1, 8]. Moreover, chemotherapy can contribute to a favorable TME for tumor cells metastasis by promoting the mutual participation of cancer-associated fibroblasts (CAFs), altered extracellular matrix (ECM), and pro-metastatic cytokines released by tumor cells and stromal cells, in parallel with changes of immune composition of the TME at the pro-metastatic niche or metastatic site. Hence, understanding the mechanisms underlying chemotherapy-induced metastasis merits great significance for effective cancer treatment in the clinic.

In this review, we summarize the pro-metastatic effects induced by chemotherapeutic agents as well as the underlying molecular mechanisms. The use of combination treatments to overcome chemotherapy-induced metastasis, as based on preclinical studies, is also discussed. Our review provides helpful information for cancer researchers and clinical strategies for combating the by-effect of chemotherapy.

Chemotherapy enhances the metastatic potential of tumor cells

In addition to killing activities, chemotherapeutic drugs also have metastasis-promoting activities; these include screening out cells with highly metastatic phenotypes, expanding cancer stem cell populations, and activating the EMT.

Chemotherapy screens out cancer cells with invasive and metastatic phenotypes

Anti-cancer drugs often fail to completely eradicate cancer cells and enable the emergence of drug-resistant cells and cells with aggressive (e.g., invasive and metastatic) phenotypes, ultimately leading to tumor regrowth and metastasis [9]. Invadopodia are important convergence points and the hallmarks of cancer cells that undergo systemic dissemination and metastasis [10]. The chemotherapeutic agent paclitaxel increases the number of invadopodia and, consequently, the invasive behavior of cancer cells [11]. High basal levels of phosphorylated extracellular signal-related kinases 1 and 2 (ERK1/2) render tumor cells resistant to cisplatin [12], resulting in metastasis [13]. Chemotherapy also induces aggressive phenotypes via metastatic regulators, such as mammalian-enabled protein (MENA), an actin-regulatory protein that promotes the invasion and migration of breast cancer cells in vivo. MENA^Calc (also known as MENA^INV-high) [14] is a marker of invasive and metastatic tumor cells and is associated with poor prognosis in breast cancer patients [15, 16]. MENA^Calc-positive tumor cells tend to enter blood vessels, which facilitates tumor dissemination to distant organs in the host body [17].

Chemotherapy induces the expansion of cancer stem cell populations

Chemotherapeutic drugs can cause expansion of TICs or elevate cancer stemness-related genes, contributing to cell stemness and malignancy. TIC expansion is essential for tumor therapeutic resistance and metastasis [18]. Enhancer of zeste homolog 2 (EZH2) promotes TIC expansion in breast [19] and other [20] cancers [18]. Paclitaxel and carboplatin enrich breast cancer stem cell populations via hypoxia-inducible factor-1-dependent regulation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway [21]. In a previous study, the number of circulating tumor cells increased by more than 1000-fold in breast cancer patients treated with paclitaxel [22]. Moreover, chemotherapy can induce the expression of stemness-related genes through various mechanisms that may be dependent or independent of EV secretion. Independent mechanisms include the direct induction of glutathione S-transferase omega 1 (GSTO1), activation of hypoxia-inducible factors, stimulation of Ca²⁺ release in cancer cells [23], or the stimulation of monocytosis, which systemically elevates monocyte chemoattractant proteins in cancer cells exposed to drugs [24]. The mechanisms underlying the dependence of chemotherapy-induced stemness on EV secretion are summarized in the following paragraphs.

Chemotherapy induces the EMT in cancer cells

The EMT is a fundamental process in embryonic development and tissue repair, but confers malignant properties to carcinoma cells, including invasive behavior, cancer stem cell activity, and DNA instability; as such, it plays an important role in cancer progression, metastasis, and drug resistance [25]. As cells undergo the EMT, they shed epithelial characteristics (e.g., adhesion to the basement membrane and adjacent cells) and assume the invasive mesenchymal phenotype, which allows them to leave the tissue parenchyma and enter the systemic circulation [26]. Hallmarks of the EMT include reduced abundance of the epithelial proteins zona occludens 1, occludin, and cytokeratin and increased expression of the mesenchymal markers vimentin, fibronectin, fibroblast specific protein 1, α-smooth muscle actin (α-SMA), and N-cadherin [27].

Chemotherapy serves as a stressor for cancer, which leads to DNA damage, cell fragmentation, and upregulation of genes controlling the EMT [3]. When β-catenin is tyrosine-phosphorylated by highly active endogenous epidermal growth factor receptors (EGFRs) in tamoxifen-resistant breast cancer cells, it dissociates from E-cadherin, and the subsequent loss of cell-cell adhesion increases cell motility and invasiveness [28, 29]. Induction of the EMT by chronic paclitaxel treatment enhances the entry of ovarian carcinoma cells into the peritoneal cavity; this process is accompanied by upregulation of the transcription factors Snail and Twist, which bind directly to the E-boxes on the E-cadherin promoter to repress its transcription in ovarian carcinoma cells [5]. Decreased expression of E-cadherin has also been observed in oxaliplatin-resistant colon carcinoma cells undergoing the EMT [2]. Gemcitabine-resistant pancreatic cancer cells undergo morphological and molecular alterations consistent with the EMT, including the near restriction of β-catenin to the nucleus where it can promote transcription, and the translocation of E-cadherin from the plasma membrane to the cytoplasm and its consequent inability to function as an adhesion molecule. An association between activation of the c-Met tyrosine kinase and chemoresistance was also suggested [3].

Signal transducer and activator of transcription 3 (STAT3) induces EMT-mediated metastasis in various cancers and affects responses to chemotherapeutic drugs [30]. For instance, in ovarian tumors, miRNA-10b-induced STAT3 signaling and the EMT trigger cisplatin-resistance, as indicated by E-cadherin downregulation and vimentin upregulation [31]. Mechanistically, cisplatin resistance resulted from miRNA-10b-mediated upregulation of CHRF, a lncRNA that is overexpressed in many drug-resistant diseases. Thus, CHRF-miR-10b signaling is a promising therapeutic target for sensitizing drug-resistant ovarian cancer cells [31]. STAT3 also mediates gemcitabine resistance in pancreatic cancer cells; in these cells, binding of the pleiotropic lipid molecule sphingosine-1-phosphate to sphingosine 1-phosphate receptor 1 (S1PR1) stimulates STAT3 signaling. FTY720, a functional antagonist of S1PR1, either alone or in combination with gemcitabine, reduces S1PR1 expression and STAT3 phosphorylation (i.e., activation) and consequently decreases vimentin and N-cadherin expression and increases E-cadherin expression [32].

Chemotherapy elicits the production of exosomes and extracellular vesicles in cancer cells

Exosomes are microvesicles composed of a lipid bilayer and various bioactive molecules, including DNA, miRNAs, proteins, and lipids; they are secreted by various cells to modulate angiogenesis, invasion, and metastasis [33]. Cell-secreted exosomes function as vital intermediators between cancer cells and stromal cells by transferring genetic message-associated content to the TME [34]. They also promote vascular permeability, premetastatic niche formation, and chemotherapy resistance [35–37]. Stromal cells enhance the malignant phenotype of cancer cells by secreting tumor-promoting exosomes [38]. Numerous studies over the last decade have shown that exosomes and extracellular vesicles produced in response to cytotoxic chemotherapeutic drugs contribute to metastatic processes. Malignant-activated platelets secrete exosomes, conferring to angiogenesis, invasion, and metastasis in lung cancer [39]. Exosomes secreted by cancer-associated fibroblasts (CAFs) enhance the proliferation of pancreatic cells and induce gemcitabine resistance by increasing the expression of Snail [40]. CD81-positive exosomes derived from CAFs activate the Wnt-planar signal pathway and promote migration and lung metastasis of breast cancer [41].

Exosomes promote drug resistance and cancer metastasis through multiple mechanisms. First, they sequester cytotoxic drugs, thus reducing their effective concentration at the target site [24]. For instance, ABCG is overexpressed in drug-resistant MCF-7 breast cancer cells and is also contained in released exosomes, which promotes drug accumulation in exosomes and drug release [42]. Second, they mediate the communication between cancer cells and stromal cells in the TME [43]. For example, CAFs promote stemness, EMT, metastasis, and 5-FU/oxaliplatin resistance in colorectal cancer (CRC) cells by secreting exosomes to enhance miR-92a-3p in CRC cells. Specifically, increased expression of miR-92a-3p activates the Wnt/β-catenin pathway and inhibits mitochondrial apoptosis by directly inhibiting interaction between F-Box and WD Repeat Domain Containing 7 (FBXW7) and Modulator of apoptosis protein1 (MOAP1), thereby contributing to cancer progression [44]. Third, they convey mRNAs, miRNAs, lncRNAs, and proteins from drug-resistant tumor cells to additional tumor cells [45]. Doxorubicin and paclitaxel increase EZH2 expression, which activates STAT3, allowing it to bind to the promoter regions of miR-378a-3p and miR-378d, and the secretion of exosomes enriched with these miRNAs influences multiple signaling pathways including the WNT/β-catenin and Notch pathways [24]. Fourth, they promote cancer stemness by activating TICs or transporting miRNAs. Taxanes and anthracyclines stimulate cancer cells to secrete exosomes that suppress chemosensitivity by promoting TIC expansion during neoadjuvant chemotherapy. In mice bearing xenograft mammary tumors, docetaxel elevates miR-9-5p, miR-195-p, and miR-203a-3p levels in circulating extracellular vesicles and decreases the expression of one-cut domain family member 2 (ONECUT2), enhancing cell stemness [46].

Chemotherapy affects the hematological system

Chemotherapy-induced vascular alterations include increased blood vessel permeability, vascular endothelial growth factor (VEGF) production, and angiogenesis, all of which facilitate the dissemination of tumor cells to distant organs in the body.

Chemotherapy increases vascular permeability

Neoadjuvant paclitaxel treatment increases vascular permeability and induces blood vessel leakage [47]. Tie2, a tyrosine kinase receptor, is expressed in endothelial cells and a subset of macrophages that promote tumor angiogenesis and lymphangiogenesis, as well as cancer cell intravasation and metastasis. Chemotherapeutic drugs mobilize bone marrow-derived mesenchymal and endothelial progenitor CD11b⁺ myeloid cells, including those expressing Tie2. Tie2⁺ cells transform into Tie2^Hi macrophages, which associate with newly constructed tumor blood vessels [48]. Rebastinib inhibits the VEGF-A-dependent increase in blood vessel permeability and tumor cell intravasation and dissemination induced by Tie2^Hi macrophages. By increasing vascular permeability and matrix metalloproteinase (MMP)-2 levels, cyclophosphamide remodels the basement membrane to promote breast cancer metastasis to the lungs [49]. Similarly, MMP-9 levels are higher in the conditioned medium of bone marrow-derived cells from paclitaxel-treated vs. control mice [50]. Paclitaxel increases vascular permeability [51], as does bleomycin [52].

Chemotherapy promotes angiogenesis

Angiogenesis is essential for the malignant progression and metastasis of both solid and non-solid tumors [53]. VEGF promotes tumor angiogenesis by stimulating endothelial cell proliferation and migration, inhibiting apoptosis, remodeling the extracellular matrix (ECM), and increasing vascular permeability. Cyclophosphamide and paclitaxel upregulate the levels of VEGF and proteinases [54]. Short-term high-dose gemcitabine treatment activates the EGFR-STAT3 axis, upregulates HAb18G/CD147 expression, and induces the secretion of MMPs, thereby promoting invasion and metastasis [55]. Notably, gemcitabine enhances the invasiveness of pancreatic cells [56, 57]. Adriamycin indirectly upregulates Tie2 by inhibiting the expression of sirtuin 7 in breast cancer cells in vitro; consequent upregulation of the vascular endothelial marker CD31 promotes tumor angiogenesis and metastasis in vivo [24, 58].

Chemotherapy activates platelets, contributing to cell adhesion

In addition to increasing vascular permeability, cyclophosphamide enables the adherence of cancer cells to blood vessels; it does so by increasing the levels of cell adhesion molecules [59–61]. Moreover, cyclophosphamide activates platelets, which aggregate on disseminated cancer cells in the bloodstream to facilitate cancer cell adherence to the vascular walls [61, 62]. Platelets also become active in response to chemotherapy-induced stimulation of plasminogen receptors and thrombin upregulation and promote metastasis by shielding cancer cells from shear forces generated by blood flow as they travel to distant sites [63]. Therefore, platelet-based drug delivery systems can be used to target cancer cells in the clinic [64].

Chemotherapy alters the TME

The TME is comprised of cellular and molecular components, including normal epithelial cells, fibroblasts, stromal cells, blood vessels, resident and infiltrating immune cells, and the ECM [65]. It is the context in which the tumor originates, grows, and eventually disperses to normal tissues. Chemotherapeutic drugs induce a series of changes in both immune and non-immune cells and the ECM in the TME, providing a favorable condition for tumor metastasis. The TME of metastasis (TMEM) is composed of perivascular Tie2^Hi/VEGF^Hi macrophages in contact with MENA-overexpressing tumor and endothelial cells, all of which are potential therapeutic targets [14].

Effect of chemotherapy on immune cells

Most immune cells in the TME are myeloid-derived macrophages, which are essential mediators of the innate immune response [66]. M1 macrophages are thought to be anti-tumorigenic, whereas M2-polarized macrophages elicit several pro-tumorigenic events including angiogenesis, lymphangiogenesis, immune suppression, hypoxia induction, tumor cell proliferation, and metastasis [67]. The TME influences macrophage recruitment and polarization, which are directly modulated by cancer cells [68].

Emerging evidence indicates that chemotherapy induces macrophage infiltration and polarization. Chemotherapy recruits M2-polarized macrophages to tumors where they release VEGF to promote tumor neovascularization, lymphangiogenesis, and the subsequent spread of tumor cells through blood and lymphatic vessels [69]. In a rat xenograft model, cyclophosphamide increased vascularization and primary and metastatic tumor growth by stimulating macrophage infiltration and the production of angiogenic factors including VEGF and proteinases [54]. Inflammatory factors released into the TME following M2 macrophage polarization contribute to the migration and invasion of tumor cells and include proteases, eicosanoids, cytokines, and chemokines. Cytokines include macrophage migratory inhibitory factor (MIF), tumor necrosis factor (TNF-α), interleukin-6 (IL-6), IL-17, IL-12, IL-23, IL-10, and transforming growth factor-β (TGF-β) [70]. With the treatment of paclitaxel, macrophage-derived cathepsins promote the progression of pancreatic neuroendocrine and mammary tumors [71, 72]. In addition, polarization of M2 macrophage activates pro-survival signaling pathways, providing cancer metastasis. For instance, macrophages and monocytes impede immune-mediated clearance or directly interact with tumor cells to promote extravasation and survival of disseminated tumor cells [73]. In pancreatic tumors, tumor-infiltrating macrophages and inflammatory monocytes enhance TIC activity by activating STAT3. Inactivation of myeloid cell receptors, colony-stimulating factor 1 receptors (CSF1R), or C-C motif chemokine receptors (CCR2) decreases the number of TICs, enhances antitumor T cell responses, inhibits metastasis, and improves chemotherapeutic efficacy [74].

In addition to regulating macrophage activity in the TME, chemotherapy also prevents the production of perforin and granule enzymes by natural killer cells and CD8⁺ T cells, and the resultant local immune imbalance and immunosuppression facilitates tumor metastasis. T cell-inflamed malignancies are characterized by large numbers of CD8⁺ T cells. However, these cells are frequently depleted in cancer patients [75]. Moreover, in the T cell-inflamed microenvironment, immunological inhibitory pathways triggered by interferon (IFN) are generally upregulated and regulatory T cells are abundant [76, 77]. Treatment of mice with gemcitabine plus anti-programmed cell death ligand 1 (PD-L1) antibody significantly increases the overall number of CD8⁺ T cells and IFN-γ expression in the TME [78].

Effect of chemotherapy on non-immune cells

Chemotherapeutic drugs induce tumor and stromal cells to recruit cancer-associated fibroblasts (CAFs) by secreting a variety of cytokines, including transforming factor β (TGF-β), epidermal growth factor, platelet-derived growth factor (PDGF), fibroblast growth factor 2, and C-X-C motif chemokine ligand (CXCL) 12 [79, 80]. Originating from a variety of cell types, CAFs are characterized by overexpression of α-SMA, fibroblast activation protein, fibroblast-specific protein 1, PDGF receptor-α/β, and vimentin [81]. Depending on the circumstances, CAFs are quiescent [82, 83] or function as mesenchymal stem cells [84, 85] or adipocytes, particularly white adipocytes [86, 87].

CAFs promote tumor growth [40], angiogenesis [88], migration [89], invasion [90], inflammation [91], and metastasis [44], along with ECM remodeling and even chemoresistance [92]. Specifically, they induce the EMT in non-small cell lung cancers (NSCLCs) through the TGF-β-interleukin (IL)-6 axis [93]. CAFs are also intimately involved in immune regulation. As examples, they hinder the recruitment of immune effector cells (e.g., CD8⁺ T cells) to tumors by secreting cytokines [80] and alter the proportions of M2 macrophages, regulatory T cells, myeloid-derived suppressor cells (MDSCs), and other immunosuppressive cells. The presence of podoplanin-positive CAFs in surgically resected primary lung adenocarcinomas predicts a shorter progression-free survival period in patients with recurrence who received platinum-based chemotherapy [94].

Effect of chemotherapy on the ECM of tumor tissue

The metastatic sites of tumors are not randomly selected by cancer cells. Rather, they preferentially seed in organs that selectively support metastatic colonization [95]. Numerous events are thought to prime secondary organ sites before the arrival of cancer cells and formation of the metastatic niche; such events include changes in cell and ECM components [95]. Hematopoietic progenitors and myeloid cells home to pre-metastatic sites, where they cluster and create a structural niche for cancer cell seeding [95]. During metastasis, changes in fibronectin, collagen, versican, periostin, and tenascin C expression occur in the ECM [96, 97]. Hence, ECM remodeling plays a crucial role in tumor metastasis. Paclitaxel increases the expression and activity of ECM remodeling enzymes in CD8⁺ T cells, priming the lung microenvironment for the seeding of metastatic cancer cells [98].

Effect of chemotherapy on inflammatory responses

Cancer-related inflammation is an essential process in cancer progression and metastasis. It promotes metastasis by increasing the secretion of cytokines and other pro-metastatic factors such as tumor necrosis factor-α and Toll-like receptor (TLR) 2, both of which are necessary for the metastasis of Lewis lung carcinoma [99]. Inflammation-related signaling pathways, such as the nuclear factor-κB (NF-κB), SMAD4/SMAD7, TLR4, ERK1/2, and AKT signaling pathways, are frequently activated during chemotherapy.

NF-κB activity is dysregulated in chemotherapy-induced metastasis. Paclitaxel aberrantly activates NF-κB by inducing the dimerization and consequent activation of TLR4 [100, 101], TLR4 is a natural lipopolysaccharide receptor that is highly upregulated in malignant epithelial cells [100, 102, 103]. Gene targets of NF-κB include those encoding numerous inflammatory, migratory, and pro-survival proteins such as VEGF-A, cyclooxygenase-2 (COX-2), IL-6, IL-8, MMP-9, X-linked inhibitor of apoptosis protein, and Bcl-2 [104–106]. The expression of TLR4 and MyD88 in ovarian cancer cells may facilitate the transmission of environmental stimuli for NF-κB activation [107]. Overactivation of NF-κB results in resistance to paclitaxel and cisplatin in ovarian cancer [107–109]. In a breast cancer mouse model, a low dose of paclitaxel stimulated the phosphorylation of IκB kinase and the p65 subunit of NF-κB, whereas the NF-κB inhibitor, IκB, was significantly downregulated [110]. p65 increases miR-452-5p expression by directly binding to the miR-452-5p promoter in renal cell carcinoma; miR-452-5p directly binds to SMAD4 and, via the SMAD4/SMAD7 pathway, ultimately enhances cell invasion and metastasis [111]. Neutrophil extracellular traps (NETs) are net-like structures composed of DNA-histone complexes and proteins released by activated tumor-associated neutrophils in the TME [112], which can be induced by phorbol 12-myristate 13-acetate and activate NF-κB by increasing the interaction of NF-κB essential modifier with IκB kinase α/β [113]. NETs also activate the NF-κB/NLRP3 pathway by downregulating MIR503HG expression to promote the EMT and NSCLC metastasis [114]. Multiple agents have been investigated for targeting the formation of NETs or other associated steps to alleviate cancer progression and metastasis in vitro and in vivo. Doxorubicin [115], 5-fluoracil (5-FU) [116–118], and oxaliplatin [119] can also stimulate the NF-κB signaling pathway. Gemcitabine causes reactive oxygen species (ROS) generation in pancreatic cancer patients, which induces NF-κB and HIF-1α by activating the ERK1/2 and AKT signaling pathways [120].

Host cells in the TME are the main source of inflammation-induced pro-metastatic factors, such as TGF-β, MMP-9, chemokine C-C motif ligand-2 (CCL2), and IL-6; the host cells include bone marrow-derived progenitor cells of monocyte or endothelial origin, macrophages, endothelial cells, and CAFs [121, 122]. TGF-β is a cytokine with antitumorigenic and strong anti-inflammatory properties in normal cells and tumor-promoting properties in cancer cells. Its production by cancer cells and CAFs promotes the accumulation of stromal collagen in the ECM. TGF-β increases blood vessel formation at lower concentrations and suppresses angiogenesis at higher concentrations. It also directly regulates endothelial cell growth and migration, the activation state of the endothelium, and the differentiation of regulatory T cells [123], M2 macrophages, tumor-associated macrophages (TAMs) [124], and tumor-associated neutrophils [125]. Calcitriol upregulates TGF-β expression in host cells (e.g., macrophages, endothelial cells, and CAFs) in the TME, thus enhancing the probability of cancer metastasis [126]. Doxorubicin induces the release of urine diphosphate into the TME, activates highly expressed P2Y6 in tumors, and facilitates cancer cell migration. Urine diphosphate activates the MAPK and NF-κB signaling pathways by increasing the expression and enzymatic activity of MMP-9 [127].

CCL2 is an inflammatory chemokine recruited by immune cells in the TME. It promotes cancer progression, and co-culture of stromal fibroblasts with breast cancer cells increases its production by the fibroblasts in a STAT3-dependent manner [128]. In ovarian cancer, its inclusion in a combined paclitaxel and carboplatin regimen increases anti-tumor efficacy compared with paclitaxel plus carboplatin alone [129]. IL-6, which is mainly secreted by CAFs, not only promotes the metastasis of NSCLCs by upregulating T cell immunoglobulin mucin receptor 4 (TIM-4) via NF-κB [130], but also accelerates the growth and invasion of cancer cells via STAT3 [131].

Recent research suggests that autocrine CXCL10/C-X-C motif receptor 3 (CXCR3) signaling is crucial for tumor motility and metastasis. Cancer cells spread to distant organs in the early stages of metastasis; thus gut inflammation could upset the equilibrium in the organs housing the dormant metastatic cells. In a previous study, high IP-10 (CXCL10) expression levels correlated with worse survival and higher tumor grade in patients with stage IV breast cancer. Moreover, ligand-bound CXCR3 drove the growth of metastatic breast cancer cells in an ex vivo metastatic liver microphysiological model in a dose-dependent manner [132].

Clinical treatment of chemotherapy-induced metastasis

Chemotherapy-induced metastasis is a major obstacle to the survival of cancer patients. Like chemotherapy resistance, it involves numerous molecular mechanisms, including those responsible for tumor invasion and metastasis, as well as tumor growth and cell fate [133]. Several strategies (single or combination) that limit the progression of malignant tumors can be implemented to overcome chemotherapy-induced metastasis; these include radiotherapy, immunotherapy, and administration of small molecule inhibitors.

Radiotherapy

Radiotherapy has several benefits in limiting tumor metastasis, including interfering with tumor immunity and altering the TME. Low doses of radiotherapy (≤2 Gy) reprogram TAMs, resulting in an anti-metastatic phenotype and consequent normalization of the tumor vasculature (including a reduction in the number of CD31⁺ cells in blood vessels) and upregulation of vascular cell adhesion protein-1 (VCAM-1) in the tumor endothelium [134]. In response to ionizing radiation, chemokines recruit effector T cells to attack tumor tissues [135]. In a xenograft mouse model, hypofractionated high-dose radiotherapy (i.e., >7 Gy on more than 5 consecutive days) enhanced major histocompatibility complex 1 antigen presentation [136] by increasing the local production of type I IFNs [137] and IFN-γ [138]. It is noted that single-dose radiotherapy has limitations; it can cause inflammation, cycling hypoxia, immunomodulation, revascularization, CAF-coordinated ECM remodeling, and fibrosis in the TME [139]. Combining radiotherapy with immunotherapy or rational medications will be beneficial.

Small molecule inhibitors

Owing to their low molecular weights, small molecule inhibitors have been used to target numerous cancer-related processes, including migration, invasion, and metastasis [140]. In the context of metastasis, promising results have been obtained for its effects against tumor-associated cells, cancer stem cell markers, and several cytokines and pro-inflammatory mediators. For instance, in preclinical models of breast cancer, inhibition of Tie2-expressing macrophages suppressed TMEM-mediated vascular permeability, thereby inhibiting the intravasation of cancer cells and their dissemination to premetastatic niches [48, 69]. Moreover, activation of tissue macrophages by liposome-encapsulated immunomodulators has tumoricidal effects [141].

Small molecule inhibitors can also be used to target the cytokines and pro-inflammatory mediators. For instance, the dual COX-2 and soluble epoxide hydrolase inhibitor PTUPB prevents chemotherapy-induced cytokine storms [142]. In line with the premise that chemotherapeutic agents are stressors, repression of the stress-inducible gene ATF3 by small molecule inhibitors inhibits metastasis [143]. Co-administration of paclitaxel and MMP-9 inhibitors targeting MMP-9-producing bone marrow-derived cells reduces chemotherapy-driven EMT and metastatic dissemination [50].

Combination therapy

In recent clinical trials, multiple combination therapies have generated encouraging results in terms of restricting tumor metastasis (Table 2). Such combinations include radiotherapy and immunotherapy [144]. Tumor irradiation and monoclonal antibodies targeting key immune checkpoints [CD40, CD137, programmed cell death protein 1 (PD-1), and cytotoxic T-lymphocyte antigen-4 (CTLA-4)] have shown promising synergistic efficacy in patients with metastatic tumors [145–147].

Table 2.

Current preclinical and clinical treatments of chemotherapy-induced metastasis.

Compound	Combination	Mechanism/Target	Types of cancer	Phase	NCT https://clinicaltrials.gov/
Immunotherapy in combination with radiotherapy
APX005M	mFOLFOX and radiation therapy 5 Gy  5 days	CD40	Advanced rectal adenocarcinoma	Phase II	NCT04130854 (Active, not recruiting)
Avelumab	Stereotactic ablative radiotherapy (SAR)	PD-1/PD-L1	Metastatic Non-small Cell Lung Cancer (NSCLC)	Early Phase I	NCT03158883 (completed)
Nivolumab/Pembrolizumab	Radiation therapy	PD-1/PD-L1	Metastatic Cancer	/	NCT03453892 (completed)
Ipilimumab	Radiation therapy	CTLA-4	Melanoma	Phase II	NCT01449279 (completed)
Ipilimumab	Radiation therapy	CTLA-4	Metastatic Cancer	/	NCT03453892 (completed)
Photothermal therapy (PTT)
AuroLase Therapy	/	/	Head and Neck Cancer	/	NCT00848042 (completed)
Signaling pathway inhibitors in combination with immunotherapy
SAR-439459	Cemiplimab	TGF-β and PD-L1	Advanced Malignant Solid Neoplasm Metastatic Malignant Solid Neoplasm Unresectable Malignant Solid Neoplasm	Phase I	NCT04729725 (Active, not recruiting)
M7824 (MSB0011359C)	/	Bifunctional fusion protein targeting PD-L1 and TGF-β, reduces the number of MDSCs	Solid tumors	Phase I	NCT02517398 (Completed)
Pembrolizumab (MK-3475)	Regorafenib (Stivarga, BAY73-4506)	PD-1/PD-L1 and multi-target	Hepatocellular Carcinoma	Phase II	NCT04696055 (Active, not recruiting)
Signaling pathway inhibitors in combination with chemotherapy
Bevacizumab	Docetaxel	VEGF	Recurrent Breast Cancer Stage IV Breast Cancer	Phase II	NCT00055861 (Completed)
Lenvatinib (E7080/MK-7902)	Docetaxel, Pembrolizumab (MK-3475)	PD-1/PD-L1 and multi-target (including VEGF)	Non-Small Cell Lung Cancer	Phase III	NCT03976375 (Active, not recruiting)
Imiquimod	Abraxane	TLR7 agonist	Recurrent Breast Cancer Skin Metastases Stage IV Breast Cancer	Phase II	NCT00821964 (Completed)
Immunotherapy in combination with chemotherapy
Durvalumab Tremelimumab	Platinum-Based Drug	CTLA-4 and PD-1/PD-L1	Metastatic Lung Cancer	Phase II	NCT03057106 (Active, not recruiting)
Ipilimumab	Paclitaxel and carboplatin	CTLA-4	Non-Small Cell Lung Carcinoma	Phase II	NCT00527735 (Completed)
Anti-PD-1/PD-L1 antibodies	Taxane-platinum	PD-1/PD-L1	Non-Small Cell Lung Carcinoma	Phase III	NCT03875092 (Active, not recruiting) NCT02367794 (Completed)
Pembrolizumab/Vibostolimab coformulation	Docetaxel	Dual inhibition on TIGIT and PD-1/PD-L1	Metastatic Non-Small Cell Lung Cancer	Phase II	NCT04725188 (Active, not recruiting)
Durvalumab	Carboplatin Paclitaxel Stereotactic body radiation therapy (SBRT)	PD-1/PD-L1, CTLA-4, radiotherapy	Non-small Cell Lung Cancer Stage IV Oligometastasis	Phase II	NCT03965468 (Active, not recruiting)
Neutrophil targeted therapy in combination with chemotherapy
Galunisertib	Lomustine	TGF-β pathway	Glioblastoma	Phase II	NCT01582269 (Active, not recruiting)
Reparixin	Paclitaxel	CXCR1/CXCR2	Breast cancer	Phase II	NCT02370238 (Completed)
Napabucasin (BBI608)	FOLFIRI	STAT3	Colorectal Cancer	Phase III	NCT02753127 (Completed)
Plerixafor	/	CXCR4	Pancreatic Adenocarcinoma Metastatic Ovarian Serous Adenocarcinoma Colorectal Cancer Metastatic	Phase I	NCT02179970 (Completed)

Photothermal therapy (PTT) is a recently developed anti-metastasis treatment with great potential owing to its unique advantages of high specificity and minimal invasiveness [148]. In mice bearing colon or head and neck carcinomas, a single round of PTT combined with a sub-therapeutic dose of doxorubicin elicited robust anti-tumor immune responses and eliminated local as well as untreated distant tumors [149]. Hence, PTT plus chemotherapy is a promising strategy for treating chemotherapy-induced metastasis.

Combination therapies targeting the signaling pathways that promote chemotherapy-induced metastasis have shown favorable results. In a mouse model of liver metastasis, paclitaxel and a decoy cis-element oligo-deoxyribonucleic acid against the NF-κB-binding site effectively induced cancer cell apoptosis in the metastasis and significantly prolonged survival, without side effects [150]. TGF-β promotes tumor growth, invasion, and metastasis when overexpressed in advanced tumors and thus is an excellent target for metastasis treatment. Monoclonal TGF-β-neutralizing antibodies, soluble TGF-β receptor type-2 (TβRII) and TβRIII functioning as ligand traps, antisense-mediated therapy, and small molecule inhibitors of TGF-β receptor kinases are promising candidates for TGF-β inhibition [151]. In pancreatic tumor mouse models, TGF-β inhibition combined with nal-IRI plus 5-fluorouracil/leucovorin suppresses invasion and prolongs survival [152]. M7824 (MSB0011359C), a bifunctional fusion protein that targets both PD-L1 and TGF-β reduces the number of MDSCs and decreases TMEM formation in advanced cancers [153]. Simultaneous inhibition of two molecular targets by a single agent is a potentially efficacious strategy.

Newly developed combinations of targeted drugs can be used to hinder tumor metastasis. Small-cell lung cancer (SCLC) is a highly aggressive neuroendocrine cancer with high metastatic potential [154, 155]. By targeting the histone variant H3.3, darinaparsin (ZIO-101, S-dimethylarsino-glutathione), an organic derivative of arsenic trioxide, induces apoptosis and increases the sensitivity of SCLCs to poly (ADP-ribose) polymerase inhibitors [156].

Currently, the inhibition of immune regulatory checkpoints, especially the CTLA-4 and the PD-1/PD-L1 axis, is at the forefront of cancer immunotherapy. Ipilimumab, a CTLA-4 inhibitor, in combination with paclitaxel and carboplatin in the first-line treatment of NSCLC modestly improved progression-free survival and overall survival [157, 158]. Moreover, a phase III trials (KEYNOTE-407 and IMpower131) reported that anti-PD-1/PD-L1 antibodies in combination with taxane-platinum improves the therapeutic efficacy for advanced squamous NSCLC [159]. Given the significant role of neutrophils in metastasis, neutrophil-targeted therapy has recently become a hotspot for cancer intervention. Chemotherapy increases growth arrest-specific 6 (Gas6) expression in circulating neutrophils and leads to AXL receptor activation in tumor cells; recombinant Gas6 plus chemotherapy disrupts neutrophil infiltration and Gas6/AXL signaling and thus inhibits metastatic pancreatic cancer growth [160].

Newly developed drug delivery systems have improved the efficacy of anti-tumor agents. Tactical nanomissile therapy has been proposed as an effective way to eradicate tumor cells in neoadjuvant chemotherapy. It consists of three functional components: a guidance system (anti-PD-1, anti-PD-L1), ammunition (mitoxantrone), and projectile bodies with both adjuvant effects and tumor intracellular azoreductase responsiveness (tertiary amine-modified azobenzene derivatives). MPAL has been shown significant effect against pulmonary metastasis, which increases the infiltration of antitumor CD8⁺ T lymphocytes, and prevents postsurgical tumor metastasis and recurrence [161]. In addition, nanoparticles have emerged as a valuable tool for delivering anti-cancer agents to target sites. Recently, a tumor microenvironment-based mPEG-PLGA nanoparticle loaded with baicalein (PMs-Ba) has been developed, exhibiting a high drug-loading rate and stability, excellent biocompatibility, and low toxicity. This nanoparticle has been found to remodel the TME, leading to a substantial improvement in the antitumor effectiveness of doxorubicin (adriamycin)-loaded mPEG-PLGA [162].

Conclusion

Chemotherapy slows cancer progression and improves patient outcomes, which is the most commonly used therapy in the clinic. However, a major challenge associated with chemotherapy is its potential to induce malignancy, particularly metastasis. Recent research has focused on investigating the molecular mechanisms and potential cellular markers that could serve as prognostic markers for patients undergoing specific chemotherapy treatments (Fig. 1). Further elucidating the role of chemotherapy in metastasis is required for guiding future clinical treatment, helping the large population of cancer patients.

Fig. 1. Mechanism of chemotherapy-induced metastasis.

Chemotherapy screens out cancer cells with more invasive and metastatic phenotypes, along with stem-like tumor-initiating cell (TIC) expansion, from the primary tumor. The remaining tumor cells undergo epithelial-mesenchymal transition (EMT) or elicit extracellular vesicles (EVs) to gain the ability to disseminate to distant organs. Moreover, chemotherapy-induced increased vascular permeability and vascular endothelial growth factor (VEGF) release facilitate the intravasation and dissemination of cancer cells. Additionally, chemotherapy alters the tumor microenvironment (TME) and immune composition in pro-metastatic niches or metastatic sites, including macrophage polarization and T-cell failure. Cancer-associated fibroblasts (CAFs), modulated extracellular matrix (ECM), and pro-metastatic cytokines released by tumor and stromal cells create a favorable microenvironment for tumor cells to form secondary metastatic sites.

Competing interests

The authors declare no competing interests.