Dynamics of inflammatory signals within the tumor microenvironment

Abstract

Tumor stroma, or tumor microenvironment (TME), has been in the spotlight during recent years for its role in tumor development, growth, and metastasis. It consists of a myriad of elements, including tumor-associated macrophages, cancer-associated fibroblasts, a deregulated extracellular matrix, endothelial cells, and vascular vessels. The release of proinflammatory molecules, due to the inflamed microenvironment, such as cytokines and chemokines is found to play a pivotal role in progression of cancer and response to therapy. This review discusses the major key players and important chemical inflammatory signals released in the TME. Furthermore, the latest breakthroughs in cytokine-mediated crosstalk between immune cells and cancer cells have been highlighted. In addition, recent updates on alterations in cytokine signaling between chronic inflammation and malignant TME have also been reviewed.

Key Words: Inflammatory signals; Tumor microenvironment; Cytokines; Interleukins; Transforming growth factor

Core Tip: Tumor microenvironment consisting of the cancer related fibroblasts can inhibit the tumor cell killing activity of cytotoxic T-cells. This activity is promoted by secretion of a variety of immunosuppressive cytokines, such as interleukins (ILs) and transforming growth factor beta (TGF-β). IL-1 has been found to be associated with tumor growth and its dissemination in the body. Similarly, other cytokines and inflammatory proteins such as IL-6, IL-33, TGF-β, nuclear factor kappa-B, IL-6, signal transducer and activator of transcription 3 pathway, interferons and toll-like receptors have been linked to tumor invasion and immunosuppression, creating conditions that facilitate tumor growth and metastasis.

INTRODUCTION

Inflammation is the body’s natural defense against pathogens, helping protect the human body from illness. Since inflammation occurs across all cellular levels, mechanisms involved in its activation are ubiquitously expressed in all cells[1]. To achieve this goal, the immune cells and signaling molecules generate various communication agents to coordinate the defense against harmful invaders[2]. Once the danger is eliminated, the inflammation response diminishes, enabling the restoration of balance and stability within the body whereas the secretion of the cytokines and chemokines subsides. This is an acute inflammation episode where the duration is short-lived and concise. The process is usually followed by tissue repair involving a myriad of components in this process. However, there are instances when this equilibrium is not achieved. The inflammatory response involves a continuous and prolonged reactive process within the body, sustaining the immune system's active state. While inflammation is a vital mechanism to counter anomalies, persistent or chronic inflammation can lead to disruptions in the tissue, leading to irreversible damage[2].

While the link between chronic inflammation and cancer is well-established, it is important to elaborate on the mechanisms underlying this connection to fully understand its role in promoting tumorigenesis. Chronic inflammation creates a microenvironment that is conducive to the development and progression of cancer through several pathways. Prolonged inflammation leads to continuous tissue damage and regeneration, which increases the likelihood of DNA mutations during cell replication. Furthermore, inflammatory cells produce reactive oxygen species (ROS) and nitrogen species (RNS) that can directly damage DNA, proteins, and lipids, contributing to genomic instability[3,4].

Additionally, chronic inflammation often involves the sustained production of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-1β, which can activate signaling pathways like nuclear factor kappa-B (NF-кB) and signal transducer and activator of transcription (STAT) 3. These pathways are known to promote cell survival, proliferation, angiogenesis, and metastasis, thereby facilitating the transformation of normal cells into malignant ones[5]. Thus, a more comprehensive exploration of these mechanisms is essential for a deeper understanding of how chronic inflammation contributes to cancer development.

Genetic mutations and alterations within a cell lead to the development of a tumor. The immune system is also responsible for identifying and eliminating transformed cells, a process known as immunosurveillance. However, as cancer progresses, the interplay between cancer cells and the surrounding tissue initiates a series of complex changes in the functions of various cell types and signaling molecules, ultimately leading to the establishment of an immunosuppressive milieu (Table 1). The immunosuppressive environment is primarily driven by the recruitment and activation of specific immune cells such as regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs). These cells produce a range of immunosuppressive cytokines, including transforming growth factor beta (TGF-β), IL-10, and vascular endothelial growth factor (VEGF), which inhibit the activity of cytotoxic T cells and natural killer (NK) cells, the primary effectors of anti-tumor immunity[6,7]. Additionally, cancer cells themselves often overexpress immune checkpoint molecules such as programmed death-ligand 1 (PD-L1), which interact with programmed death-1 (PD-1) receptors on T cells, leading to their functional exhaustion and preventing them from attacking the tumor[8].

Table 1 Players in the immunosuppressive milieu and their roles.

Immune cells/cytokines	Role in tumorigenesis
Regulatory T cells	Suppress anti-tumor immune responses, contributing to immune tolerance of tumor cells
Myeloid-derived suppressor cells	Inhibit T-cell function, promote tumor progression by creating a pro-tumoral environment
Tumor-associated macrophages	Support tumor growth and metastasis through cytokine release and immune suppression
Transforming growth factor beta	Inhibits cytotoxic T cells and natural killer cells; promotes extracellular matrix remodeling and metastasis
Interleukin-10	Inhibits anti-tumor immunity and contributes to an immunosuppressive microenvironment
Vascular endothelial growth factor	Promotes angiogenesis, providing nutrients and oxygen to tumors
Programmed death-ligand 1	Interacts with programmed death-1 on T cells, leading to immune exhaustion and preventing T cells from attacking tumors

This immunosuppressive milieu not only allows cancer cells to evade the immune response but also modifies the tissue environment in a way that supports tumor growth and metastasis. For instance, the immunosuppressive cytokines like IL-10 and TGF-β also contribute to the remodeling of the extracellular matrix (ECM), making the tissue environment more conducive to cancer cell invasion and metastasis[9]. Additionally, these cytokines can promote angiogenesis, which is the formation of new blood vessels that supply the growing tumor with nutrients and oxygen, further facilitating its progression[10]. Within this immunosuppressive environment, cancer cells can evade immunoediting process of immune responses and manipulate the activities of inflammatory signals and cytokines, driving them towards a pro-tumoral trajectory[11] as depicted in Figure 1.

Figure 1 Immunosuppressive tumor microenvironment development and progression. The flow from tumor development to progression involves several key steps. Genetic mutations lead to tumor formation. The immune system attempts immunosurveillance, identifying and eliminating transformed cells. When immunosurveillance fails, cancer cells survive and continue to grow. This failure creates an immunosuppressive microenvironment, driven by the recruitment of immune cells such as regulatory T cells, myeloid-derived suppressor cells, and tumor-associated macrophages. These immune cells release immunosuppressive cytokines (transforming growth factor beta, interleukin-10, vascular endothelial growth factor), which inhibit cytotoxic T cells and natural killer cells. As a result, tumor growth and metastasis are facilitated, including processes like angiogenesis and extracellular matrix remodeling. Ultimately, the tumor evades immune responses and progresses further. ECM: Extracellular matrix; IL: Interleukin; MDSCs: Myeloid-derived suppressor cells; NK: Natural killer; TAMs: Tumor-associated macrophages; TGF-β: Transforming growth factor beta; Tregs: Regulatory T cells; VEGF: Vascular endothelial growth factor.

FORMATION OF THE TUMOR MICROENVIRONMENT

The tumor microenvironment (TME) includes all the non-cancerous host cells within the tumor, such as fibroblasts, endothelial cells, neurons, adipocytes, and both adaptive and innate immune cells. It also consists of non-cellular components (Figure 2), including the ECM and soluble substances like chemokines, cytokines, growth factors, and extracellular vesicles[12].

Figure 2 Complex cellular and non-cellular components within the TME. CAFs: Cancer associated fibroblasts; EMT: Epithelial mesenchymal transition; IL: Interleukin; M2: Type 2 macrophages; NF-кB: Nuclear factor kappa-B; NO: Nitrous oxide; ROS: Reactive oxygen species; STAT3: Signal transducer and activator of transcription 3; TAMs: Tumor associated macrophages; TNF-α: Tumor necrosis factor-alpha; TGF-β: Transforming growth factor beta; VEGF: Vascular endothelial growth factor.

Immunoediting occurs within the TME, which is defined as the milieu of various cellular and non-cellular elements that interact with cancer cells with the main objective to drive and support tumor growth[13]. Stromal cells in the TME include cancer associated fibroblasts (CAFs) and endothelial cells[14].

CAFs are known to secrete a variety of immunosuppressive cytokines, such as TGF-β and IL-6, which can inhibit the activity of cytotoxic T cells and promote the recruitment of Tregs. This creates an environment that dampens anti-tumor immune responses. Additionally, CAFs contribute to the remodeling of the ECM, which not only facilitates tumor invasion but also provides a physical barrier that limits the infiltration of immune cells into the TME. Recent studies have demonstrated that CAF-induced ECM remodeling can enhance the desmoplastic reaction, leading to increased tissue stiffness and further promoting immune evasion by cancer cells[15,16]. Endothelial cells also play a role by expressing immune checkpoint molecules like PD-L1, which can directly inhibit T cell function, thus contributing to a more tumor-permissive environment (Table 2).

Table 2 Summary of the roles of cancer-associated fibroblasts and tumor-associated macrophages in tumor growth and immunosuppression.

Key players	Roles and mechanisms in tumor microenvironment	Impact on tumor progression
Cancer-associated fibroblasts	Secretion of immunosuppressive cytokines: Transforming growth factor beta, IL-6. Extracellular matrix remodeling: Facilitates tumor invasion and forms physical barrier to immune cells. Promotion of regulatory T cell recruitment: Inhibits cytotoxic T cells	Immune evasion: Dampens anti-tumor immune response. Enhanced tumor invasion: Promotes desmoplastic reaction and tissue stiffness. Physical barrier: Limits immune cell infiltration
Tumor-associated macrophages	Predominantly type 2 macrophages phenotype macrophages: Secrete vascular endothelial growth factor and IL-10. Angiogenesis: Promotes new blood vessel formation. Immune suppression: Dampens immune response	Tumor progression: Supports angiogenesis, providing nutrients to tumors. Immune suppression: Contributes to immune evasion and poor prognosis
Endothelial cells	Expression of immune checkpoint molecules: Programmed death-ligand 1, which inhibits T cell function	Tumor-permissive environment: Contributes to immune evasion and progression

IL: Interleukin.

The notable cellular components related to inflammatory and immune response such as NK cells, regulatory T cells, TAMs, MDSCs mediated by chemokines released and matrix metalloproteinases (MMPs) are present within the TME[13,17]. TAMs are associated with poor prognosis in many cancers. In glioma related tumors, there is a consensus that blood-borne monocytes and macrophages contribute to suppressing immune surveillance functions[18]. These immune cells and signals are believed to sustain a chronic state of inflammation by the constant and unregulated release of inflammatory molecules and cytokines into the TME. Other features that contribute to dynamics and tumorigenesis within the TME include ECM remodeling, hypoxia, and acidosis[13].

ECM REMODELING

The ECM is a complex three-dimensional molecular framework that surrounds and supports cells within tissues. This elaborate structure is made up of a variety of macromolecules, including proteins such as collagens, proteoglycans, and matricellular proteins, as well as glycosaminoglycans like hyaluronan[19]. The ECM plays a crucial role in various developmental stages, ranging from embryogenesis to adult development, tissue repair, and the maintenance of tissue and organ homeostasis. After being synthesized in the cytoplasm, ECM components are secreted into the extracellular space, where they undergo further modifications to form their final molecules[20,21]. ECM remodeling under normal physiological conditions is a highly regulated and intricate process, involving various proteins that contribute to maintaining homeostasis. During tumorigenesis, the ECM also undergoes remodeling, with multiple studies suggesting that it can both promote tumorigenesis and exhibit antitumorigenic effects[22-24]. Increased ECM synthesis is associated with the development of chemoresistance in various cancers[25-27]. Tumor progression is marked by significant remodeling of the surrounding ECM, leading to the formation of a tumor-specific ECM that is often enriched in collagen and displays increased rigidity. The biochemical diversity and spatial organization of ECM components play a crucial role in regulating the migration, maturation, and activation of immune cells[28]. For tumor cells to migrate and metastasize, they require space and the secretion of factors that facilitate invasion. Tumor cells downregulate ECM deposition by releasing colony-stimulating factor 1[29-31]. This reduction in ECM synthesis promotes enhanced tumor cell invasion and migration. Tumorigenesis leads to alterations of the ECM in terms of structure and composition, with such alterations being pro-tumorigenic[32-34].

Various factors can drive ECM remodeling, including the roles of CAFs, lysyl oxidase, MMPs, and other stromal cells or proteins. TAMs play a crucial role in orchestrating ECM remodeling. They are involved in a complex process of degrading existing ECM components while simultaneously stimulating fibroblasts and promoting the synthesis of new proteins[35]. The importance of TAMs' specialized function in breast cancer is highlighted by their secretion of MMP11, which plays a pivotal role in promoting the migration of human epidermal growth factor receptor 2-positive breast cancer cells. This process, mediated by the chemokine ligand 2 and chemokine receptor 2 signaling pathway, underscores the targeted and selective actions of TAMs across different cancer subtypes[36]. TAMs with elevated B7-H3 expression further contribute to the promotion of regulators involved in ECM remodeling and angiogenesis, such as MMP2, VEGF-A, and TGF-β. These activities aid in ECM degradation and the formation of new vascular networks, both of which are critical for tumor metastasis[37]. Neutrophils contribute to ECM remodeling through the formation of neutrophil extracellular traps. Neutrophil elastase and MMP9 play key roles in targeting laminin. The remodeling of laminin creates a new epitope, which then activates integrin α3β1 signaling in cancer cells. This activation is a crucial event in reawakening dormant cancer cells, setting off a series of biological processes that ultimately drive tumor growth and metastasis[38]. Cathepsin G is another key component of neutrophil serine proteases, released by activated neutrophils via azurophil granules[39]. Dendritic cells (DCs) also contribute to ECM remodeling. The interaction between the ECM and bone marrow-derived DCs displays unique characteristics in the context of lung cancer. The mechanism of monocyte-derived DCs involves the expression and activation of heparanase, a heparan sulfate-degrading enzyme, by monocytes and early immature DCs. Heparanase accumulates in membrane extensions during DC maturation, facilitating ECM degradation[40]. The involvement of NK cells in ECM remodeling occurs through the expression of heparanase. Upon activation, NK cells upregulate both the transcription and protein levels of the heparanase gene. The enzymatic activity of heparanase enables NK cells to degrade the ECM, a critical step for cell invasion and migration through basement membranes[41].

CHRONIC INFLAMMATION WITHIN THE TME

To understand the relationship between chronic inflammation and tumor progression, we need to know the triggers associated with chronic inflammation, including the mediators involved in activating and sustaining a chronic inflammation state[42,43]. Further, we need to understand how these mediators orchestrate and blend in the TME triggering further immunosuppression and immune evasion. Recognizing the complexities of immunoediting is essential for grasping how cancer can adapt and survive despite immune system attacks. This insight is fundamental for developing new therapeutic strategies that can interrupt the cycle of immune evasion and suppression, making it possible to enhance immune responses against tumors and potentially leading to innovative treatments and better patient outcomes. Understanding these processes will also help identify new biomarkers for early cancer detection and guide the development of personalized immunotherapies.

The balance between antineoplastic immune system and oncogenic inflammation is so intricate that not only depend on immune cells and mesenchymal cells activation, but also strongly related to the pivotal cytokines, chemokines and growth factor[17].

Chronic inflammation has been recognized as a connecting factor to contribute to several cancer types such as liver cancer[44], colorectal cancer, pancreatic cancer, breast cancer, and lung cancer[45-49]. Inflammatory processes can play a significant role in cancer progression by causing immune suppression, facilitating tissue remodeling, inducing DNA damage, and promoting cell proliferation[50]. Additionally, pro-inflammatory cytokines—TNF-α, IL-6, and IL-1β—activate critical pathways like NF-κB and STAT3, promoting cell survival, proliferation, angiogenesis, and metastasis[5]. Expanding on these pathways with specific examples provides a more robust understanding of inflammation-driven cancer development. Chronic inflammation can disrupt cellular activity by interfering with various cell signaling pathways, such as the Janus kinase (JAK)/STAT, phosphoinositide 3-kinase/protein kinase B, and mitogen-activated protein kinase pathways[3,5]. Chronic inflammation can suppress the immune response that is responsible for identifying and eliminating tumor cells. Cytokines produced by inflammatory cells can impair the function of immune cells, making it easier for cancer cells to proliferate and spread[51]. In addition, numerous recent studies have highlighted several shared features between cancer and chronic inflammation, such as lymphocyte infiltration, involvement of mast cells, and angiogenesis[3,52]. Chronic inflammation leads to the constant release of pro-inflammatory cytokines, such as TNF-α, IL-1 and IL-6, and chemokines, which support tumor cell survival, proliferation, angiogenesis, and invasion. It also triggers the release of growth factors like TGF-β and platelet-derived growth factor, which drive tissue remodeling, fibrosis, and angiogenesis, ultimately promoting tumor growth. This prolonged environment rich in pro-inflammatory cytokines and growth factors fosters tumor development and progression by enhancing tumor cell survival, proliferation, invasion, and angiogenesis[53]. Several studies have depicted the association of microbial infections with the development of different types of cancers. Infection of the gastric mucosa with Helicobacter pylori is recognized as the primary risk factor for both intestinal and diffuse gastric cancer[49,54], Epstein-barr virus infection is linked to various types of lymphoma and nasopharyngeal carcinoma[55], and is responsible for about 8.4% of gastric cancers[56], hepatitis B virus and hepatitis C virus are known to cause hepatocellular carcinoma[57]. Chronic inflammation sustained over decades, along with co-factors originating from microbes, plays a role in the development of cancer. Both viruses and bacteria can interact with host cells through interferons and toll-like receptors (TLRs), promoting chronic inflammation. For example, activation of TLR4 by bacterial lipopolysaccharides triggers NF-κB signaling, which leads to the release of cytokines and immune system modulation (Figure 3).

Figure 3 Molecular pathways of chronic inflammation leading to tumorigenesis. Genetic mutations trigger tumor development. The immune system attempts to perform immunosurveillance by identifying and eliminating transformed cells, but this fails as cancer progresses. During tumorigenesis and chronic inflammation, the pathogens such as bacteria and viruses interact with toll-like receptors triggering nuclear factor kappa-B signaling produces proinflammatory cytokines such as tumor necrosis factor-alpha, interleukin (IL)-1, IL-6 and other chemokines. IL-1 production mediates recruitment of inflammatory cells and synthesis of reactive oxygen species leading to genetic mutations and DNA damage. Additionally, chronic inflammation aids in epithelial-to-mesenchymal transition that enhances the tumor development. EMT: Epithelial-to-mesenchymal transition; IL: Interleukin; NF-кB: Nuclear factor kappa-B; ROS: reactive oxygen species; TNF-α: Tumor necrosis factor-alpha.

Chronic inflammation can promote the development of cancer by increasing DNA damage, oxidative stress, and other intracellular stressors that can harm cells and lead to cancer-causing mutations[3]. Additionally, chronic inflammation may aid in the spread of cancer cells to other parts of the body during metastasis by triggering epithelial-to-mesenchymal transition (EMT) and enhancing the infiltration and migration of cancer cells[5,58]. Within cellular signaling pathways, NF-κB acts as a transcription factor, regulating cell growth, lifespan, metastasis, angiogenesis, and drug resistance. It plays a crucial role in both chronic inflammation and cancer by driving tumor progression. Various plant-derived polyphenols can influence the NF-κB pathway by preventing its translocation to the nucleus through the inhibition of kinase phosphorylation[59,60]. In both inflammatory conditions and cancer, activator protein-1 and NF-κB regulate several pro-inflammatory cytokines, including TNF-α, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1, and TGF-β[61].

CYTOKINES AND THEIR INTERACTIONS

Interleukins, a subset of cytokines, are generated by leukocytes and various other cells in the body. They play a critical role in immune responses by engaging in cellular signaling. Within the TME, the intricate network of cellular receptors and signaling pathways provides an ideal platform for the multifaceted involvement of interleukins in cancer progression[11].

IL-1

The interactions between a tumor and its microenvironment through the immune system are critical for tumor development and progression[62]. The immune component of the TME plays an important role in contributing to therapeutic resistance for some tumors, despite the promising effects they register in vitro. This necessitates the importance of investigating the inflammatory and immune signals within the tumor niche[18].

The IL-1 superfamily is composed of several subfamilies such as IL-18, IL-33, IL-36, IL-37, and IL-38[63,64] that are pivotal in mediating an inflammatory response. IL-1 has been also found to be associated with the development of cancers such as colorectal cancer[65], and lung cancer[66].

IL-1, particularly its subtypes IL-1α and IL-1β, is predominantly produced by myeloid and epithelial cells during chronic inflammation episodes. This cytokine family is instrumental in promoting the production of RNS and ROS, both of which are known carcinogenic mediators. IL-1 also facilitates the recruitment of innate immune cells and the release of other pro-inflammatory cytokines, such as IL-6. A crucial factor in the action of IL-1 is NF-κB, which accumulates in the nucleus and amplifies the inflammatory response[11].

IL-1 must bind to its receptor, IL-1R, via agonistic molecules like IL-1α and IL-1β to initiate and amplify local inflammation[67]. However, IL-1β is more closely associated with cancer progression than IL-1α. While IL-1α is typically membrane-bound and acts locally, IL-1β is secreted in a mature form and can influence the TME more profoundly[68].

The presence of IL-1β in the TME has been linked to tumor invasion and immunosuppression, creating conditions that facilitate tumor growth and metastasis. This is why elevated levels of IL-1β often correlate with poor prognosis in cancer patients[60]. The quantity of IL-1β present in the TME appears to be a critical factor; higher concentrations are generally associated with more aggressive disease and worse outcomes. In contrast, IL-1α does not have the same effect on tumor progression and prognosis[63]. The reasons for this difference lie in the distinct mechanisms of action of these cytokines. IL-1α remains primarily at the site of production and does not promote the same systemic inflammatory response or immunosuppressive environment that IL-1β does. The activation of mature IL-1β is heavily influenced by the NF-κB pathway. NF-κB's role in the inflammatory response is to upregulate various genes involved in immune response and inflammation, including those coding for IL-1β. The activation of NF-κB and subsequent production of IL-1β can create a feedback loop that exacerbates inflammation and promotes tumor progression. Recent research using cryo-electron microscopy has shown that the conditions within the TME, such as hypoxia and acidic pH, are conducive to the activation and transit of mature IL-1β, further supporting its role in promoting a pro-tumorigenic environment[64,67].

IL-17, a cytokine with proinflammatory properties, is produced by T cells, specifically Th17 cells. Its contribution is well-documented in autoimmune conditions and the invasiveness of tumors[63]. Much like IL-1, IL-17 is a pleiotropic cytokine, and both inflammatory signals have numerous cellular targets within the TME. Investigating the interplay between these two cytokines holds promise for gaining insights into their involvement in the development of cancer. It was demonstrated that IL-1 affects Th17 cells to produce IL-17[63].

IL-6 and STAT3 pathway

IL-6 is a pleotropic cytokine involved in many biological mechanisms such as hematopoiesis, immune regulation, and inflammation[69,70]. The dysregulation of IL-6 is linked to the development of chronic low-grade inflammation and cancer which is evident through the high abundance of this cytokine within the TME of many cancers including breast cancer[70,71]. Moreover, IL-6 itself is an autocrine and paracrine growth factor as well as a survival factor for different forms of cancers, and it is secreted by multiple immune and non-immune cells within the TME[72]. For instance, a recent study on renal cell carcinoma demonstrated that elevated levels of IL-6 in the TME are linked to a poor prognosis[73]. Furthermore, many studies have demonstrated the role of IL-6 in various health conditions and diseases, including plasmacytoma and myeloma[62].

IL-6 family functions as a ligand and exerts its role by binding to a corresponding receptor forming a dimerized receptor complex[74]. This leads to the activation of JAK associated with this receptor. JAK1 and JAK2 stimulates the phosphorylation and activation of STAT3[72]. STAT3 is involved in multiple cellular functions utilizing canonical cytokine signaling[75]. Once STAT3 is activated, it translocates from cytoplasm to nucleus and target gene promoters to regulate gene transcription[76]. This activation is tightly controlled under normal physiological state; however, it becomes dysregulated during cancer development[14] resulting in altered cell biology[77].

STAT3 is also involved in the cross talk among the different constituents of the TME, by increasing the expression of TGF-β and by interacting with other signaling pathways such as NF-кB. The increased and persistent activation of STAT3 results in amplified production of IL-6. This feedback loop will lead to the activation and production of a greater number of cytokines and growth factors supporting further development of cancer[74]. IL-6/STAT3 downstream signaling is the most crucial where in colorectal cancer, this signaling pathway is induced within the TME to promote tumor growth[76] through facilitating proliferation, migration, EMT, and angiogenesis[62]. EMT is defined as the loss of cellular polarity and cell-to-cell adhesion[78]. By modulating some of the transcription factors involved in EMT process, STAT3 can influence the phenotype of the mesenchymal cells and promote the ability of these cells to migrate and invade, thus positively impacting cancer progression[74].

STAT3 inhibits DC differentiation, maturation and antigen presenting abilities leading to inhibition of tumor immunity. Consequently, IL-6/STAT3 signaling reduces intracellular major histocompatibility complex (MHC) II levels in DC[62]. This is important because MHC-II is critical for antigen presentation to CD4⁺ T lymphocytes, whose role in antitumor immunity is becoming increasingly appreciated[79]. IL-6/STAT3 pathway is also involved in the conversion of Treg cells to Th17 cells. Th17 cells are favored for inflammation and autoimmunity, while Treg cells suppress these responses, and thus Treg cell functions are suppressed in several autoimmune diseases[62].

STAT3 along with the abundantly present IL-6 and IL-10 within the TME, also play a key role in TAMs type 2 macrophages (M2) differentiation[76]. As previously discussed, M2 cells support tumor progression and metastasis by inhibiting tumor immunity through IL-10, arginase-1 and TGF-β[62]. M2 phenotype is responsible for the abundance of IL-6 secretion within the TME[76] and Th17 cells play a role in M2 macrophages and MDSCs differentiation which suppress tumor immunity[62].

IL-10

The anti-inflammatory cytokine IL-10 is secreted by cancer cells and several immune cells of myeloid and lymphoid lineage that plays a crucial role in regulating carcinogenesis. Research has shown that IL-10 secreted by tumor cells activates tumor-infiltrating macrophages[80-82].

IL-10 is a versatile immune-suppressive cytokine that plays roles in immune regulation and angiogenesis of gastric cancer[83,84]. It promotes tumor cell survival, proliferation, and metastasis by modulating antitumor immunity[85]. Additionally, IL-10 inhibits the activity of key effector immune cells, such as powerful antitumor cytotoxic NK cells and CD8 T cells[86]. A recent study has demonstrated that neutralizing the effects of IL-10 triggers a tumor-specific cytotoxic immune response[87]. Additional research by Naing et al[88] revealed that PEGylated IL-10 stimulates a CD8 T-cell-mediated immune response in cancer patients by boosting their proliferation, tumor infiltration, and production of interferon-gamma (IFN-γ) and granzyme B. Moreover, combining PEGylated IL-10 with immune checkpoint blockade further promotes the intra-tumoral expansion of CD8 T cells[88].

Therapeutic blockade of IL-10 has been shown to enhance the antitumor immune response mediated by CD8 T cells and NK cells[89]. A study conducted by Hu et al[90] showed that IL-10 secreted by B cells suppresses effector CD4 and CD8 T-cell-mediated immunity in gastric carcinoma patients by reducing the production of IFN-γ, TNF-α, and IL-17, while also converting these effector T cells into IL-10-producing T cells. In contrast, Zhang et al[91] demonstrated that IL-10 secreted by TAMs creates an immune-evasive microenvironment, leading to poor prognosis and reduced therapeutic response to adjuvant chemotherapy in gastric cancer patients. Recent studies have extensively characterized the immune-modulatory effects of IL-10 on antitumor NK cells. In pancreatic ductal adenocarcinoma patients, CD16hiCD57hi NK cells produce elevated levels of IL-10, which suppresses their cytotoxic activity and IFN-γ expression[92]. These findings suggest that IL-10 exerts a range of suppressive effects on antitumor immune cells, which are essential for the clearance of tumor cells.

IL-33

According to Jou et al[93], IL-33 has been recently implicated in association with the development of colorectal cancer. The role of IL-33, which is a member of IL-1 family, is to act as an alarm to mediate an immune response when needed[94]. By binding to the suppressor of tumorigenicity (ST) 2, which is a transmembrane receptor, IL-33 can activate intracellular signaling and mediates its proinflammatory action[95] such as activating different cytokines or polarizing into the corresponding phenotype in different pathological conditions[96]. It also functions as a regulator of gene expressions in cells by binding to chromosomes when it translocates to the nucleus[18,94]. Thus, it can effectively function as both a nuclear factor as well as an extracellular cytokine when released from cells exposed to stress[97]. However, this alarmin cytokine is currently being investigated for its role in tumorigenesis[18]. Depending on the nature of the inflammatory environment, tumor and cellular context, expression levels and bioactivities, IL-33 may play a dual role as a pro-tumorigenic or anti-tumorigenic cytokine within the TME[96]. High level of IL-33 within TME is attributed to the complex interactions between CAFs and TAMs. The abundant presence of these cells within the TME of different tumors strengthened the assumption that these cellular collaborations play a pivotal role in tumor development[11,98,99]. This cytokine is also responsible for amplifying TGF-β signaling. IL-33 is also secreted by tumor-initiating cells, which are cells that have transformed to acquire tumorigenic capacity[11]. It was also reported in a recent study that a specific protein of IL-33 (intracellular IL-33; icIL-33) mediates the activation of suppressors of mother against decapentaplegic (SMAD) which is essential for cancer development during an episode of chronic inflammation[94].

Understanding the nuclear function of IL-33 can help decrease the pro-tumorigenic environment by stopping the regulation of other chemokines that function in recruiting and activating innate immune cells within the cancer niche[18].

The high level of IL-33 as well as the invasion of TAMs and monocytes were investigated in the progression of glioma within the glioblastoma microenvironment[18]. Increased secretion of IL-33 induces the M1 to M2 polarization in TAMs through the IL-33/ST2 signaling pathway, which has been proved to be involved in helping tumor growth[74,94]. The M2 type macrophages are referred to as the alternatively activated macrophages and they are pro-tumorigenic[18]. This specific phenotype of macrophages seems to tolerate and help tumor growth rather than resisting it[74]. M1 type macrophages are referred to as classically activated macrophages and are known predominantly for their anti-tumorigenic function[18]. However, this notion about M1 phenotype is debatable as some scientists have shown that TNF-α derived from M1 type macrophages is associated with accumulating ROSs within tumor cells and is directly involved in damaging proto-oncogenes and anti-oncogenes[17].

The M1 to M2 transition is accompanied by a dramatic > 200-fold increase in MMP9, a critical factor in metastasis. A recent study identified the IL-33-ST2-NF-κB-MMP9-laminin signaling pathway as pivotal in orchestrating tumor stroma-mediated metastasis[100]. Importantly, it was revealed that the genetic deletion of IL-33, ST2, or MMP9 in mouse and human fibroblast-rich pancreatic cancers effectively blocked metastasis, and pharmacological inhibition of NF-κB and MMP9 yielded similar results. Additionally, the deletion of IL-33, ST2, or MMP9 restored laminin levels, a vital basement membrane component linked to tumor micro-vessels. These results underscore the importance of the IL-33-NF-κB-MMP9-laminin axis in CAF-TAM-mediated metastasis and suggest that targeting this axis offers a promising therapeutic avenue for cancer treatment[101-104].

According to Pisani et al[105], the outcome of the IL-33/ST2 signaling pathway depends on the ST2-expressing cell types involved in epithelial cells, various chemokines are produced, while in TH2 cells, cytokines such as IL-4, IL-5, and IL-13 are released. Thus, the cellular components of the TME can either be influenced to be protumorigenic or antitumorigenic depending on the activation of different immune cells[105].

TGF-β

TGF-β is one of the most significant key signal transducers found in the TME. It is postulated as the main immune checkpoint in some cancers for its role in immune suppression and EMT. This transition is considered a significant occurrence in some cancers[106,107]. TGF-β exists in three prototypic isoforms which is a cytokine involved in a myriad of physiological and pathological conditions leading to the transition from non-cancerous disease state to the cancer in the gastrointestinal tract, liver and pancreas[108,109]. It functions in regulating cell growth and differentiation, wound healing and tissue repair, apoptosis, cell motility, ECM production, angiogenesis, and cellular immune responses[106,110].

For TGF-β to be functional, it needs to interact with type II receptor TGF-BRII. When this ligand is formed, then type II receptor TGF-BRII binds and phosphorylates type I receptor TGF-BRI. These two transmembrane receptors activation stimulate several downstream signaling pathways including transcription factors SMADs, which are later involved in altered transcription of TGF-β responsive gene[111]. This disruption in signaling is speculated to be the reason for its pathogenesis and protumor activity[107].

“TGF-β paradox” stipulates that mutations in genes encoding for TGF-β binding proteins reduce secretion in ECM, yielding elevated levels of the cytokine. This thought can be closely related to multiple scripts discussing function of TGF-β as an inhibitor of epithelial cell tumorigenesis should be alleviated, however, tumor cells produce TGF-β in larger quantities than normal epithelial cells do. TGF-β has a tumor suppressive activity at early stages of cancer, however, as tumor progresses, the cytokine alters the signaling pathway to create a more tumor favorable environment[107]. A hyperactive activity of STAT3 within the TME is also speculated to cause an increase in the expression of TGF-β[14].

Recently, it was also noted that the regulation of TGF-β signaling is mediated by CAFs and TNF-α. The latter was shown to deregulate the receptors specific for TGF-β as well as modifying the normal fibroblasts to exhibit CAF characteristics[112].

The clinical implication indicates that the selective blockade of the TGF-β isoform using anti-glycoprotein A repetitions predominant: TGF-β1 monoclonal antibodies can overcome resistance to PD-1/PD-L1 blockade in cancer patients[113]. In patients with esophageal squamous cell carcinoma, TGF-β secreted by MDSCs enhances PD-1 expression on CD8 T cells, leading to resistance to PD-1/PD-L1 blockade. A combination of PD-1/PD-L1 and TGF-β blockade restored tumor-antigen-specific CD8 T cells. Similarly, TGF-β blockade combined with immune checkpoint blockade promotes Th1 polarization and boosts the clonal expansion of CD8 T cells, improving survival in patients with metastatic castration-resistant prostate cancer[114]. A similar study demonstrated that dual blockade of TGF-β1 and GM-CSF enhances chemotherapy efficacy in pancreatic cancer patients by preventing the polarization of TAMs into the immune-suppressive M2 phenotype and stimulating the response of CD8 T cells[115]. An alternative study has shown that the combined blockade of PD-L1 and TGF-β increases the intra-tumoral infiltration of CD8⁺ T cells and improves survival in mice with colon and pancreatic adenocarcinoma[116].

TUMOR ASSOCIATED NEUTROPHILS AND METALLOPROTEINASES

One specific protease secreted by tumor associated neutrophils is the MMP9, which has beneficial functions in tissue remodeling, wound healing, and mobilization of tissue-bound growth factors and cytokines. However, it was found to be of negative impacts in TME where it is stipulated that it correlates with collagen deposition accompanying pancreatic cancer, with lymph node metastasis in breast cancer and with regional vessel invasion by giant cell tumor of bone[117]. Some of these factors play a role in tumor development by gene alteration like the NF-κB. This transcription factor is responsible for promoting the inflammatory environment by regulating the gene expression of inflammatory cytokines[118].

NF-κB

NF-кB is a transcription factor that induces the expression of genes encoding the release of cytokines and chemokines and other pro-inflammatory molecules. Due to its importance in regulating adaptive and innate immunity, any irregularities in the signaling pathway would lead to the development of pathological processes related to immune system like chronic inflammatory diseases[2].

NF-кB as well as other transcription factors are known to be regulators of inflammation and cancer[74]. The signaling pathway of NF-кB is activated during inflammation, and it proved to be of importance as a mediator in cancer development by inflammation-induced carcinogenesis and anti-tumor immunity[14]. The transformation of malignant cells triggers the immune response to activate NF-кB which can neutralize and eliminate malignant cells, where this process resembles acute inflammation, and it is associated with the release of cytotoxic immune cells[1]. TLRs which will be discussed later were also found to be involved in NF-кB activation[119]. Patients who are immunodeficient were categorized to have higher risks of cancer development, highlighting the importance of immune surveillance in fighting tumorigenesis[1].

Interestingly, the knockdown of intercellular IL-33 in cholangiocarcinoma cells upregulated the expression of NF-kB and IL-6, which resulted in increased cell proliferation and invasion[94]. However, when cancer cells overtake the ability of the immune system to resist, this phase is characterized by “chronic” inflammation features. Interestingly, it was noted that NF-кB values remain high, suggesting that patients with chronic inflammation are within the same category of immunodeficient patients. The persistent presence of NF-кB results in the upregulation of anti-apoptotic genes which provide a cell survival mechanism[1].

The continuous state of activation of NF-кB along with the presence of STAT3 induces the release of pro-inflammatory cytokines that can regulate the immune response[1,74]. These cytokines are TNF-α, IL-1, IL-6, IL-8, and VEGF. On the other hand, an increased cytokine release from the TME results in elevated NF-кB and STAT3 activity[1]. Since these growth factors are involved in linking inflammation to cancer because they are activated in both tumor cells and tumor-related myeloid cells, they further enhance tumor invasion and resistance to therapy[1,74]. This has also been postulated in another review by Hirano et al[62] where it was mentioned that the persistent and collaborative activation of NF-кB and STAT3 in non-immune cells leads to a positive feedback loop of NF-κB activation by the IL-6/STAT3 axis since Il-6 is considered an NF-кB target.

Release of NF-кB and STAT3 within the TME favors the production of M2 phenotype macrophages which are the predominant TAMs in the TME[1]. To accomplish its role, and further instill immunosuppression, the activity of NF-κB is enhanced by TLR4 and lipopolysaccharide[119]. TGF-β also participates in promoting the immune suppression of MDSCs, which would help in promoting angiogenesis leading to an influx of oxygen and nutrients into the tumor cells and aiding in tumor growth and metastasis[17].

INTERFERONS AND TOLL LIKE RECEPTORS

Interferons are cytokines involved in innate and adaptive immunity and were proved to play an important role in survival and death of cancer cells[120]. TLRs on the other hand, which are stimulated in innate immunity act as receptors for activated biomolecules which in turn induce the production of interferons and other cytokines[119]. TLR4, which was the first mammalian TLR identified, is ubiquitously expressed by immunocytes, epithelial cells, and other stromal cells as well as many cancer cells[119]. As mentioned earlier, these cells are essential components within the multicellular complex of TME. One of the recently published articles pointed out that IFN-γ, which is produced by tumor reactive T-cells, is highly involved in the influx of large number of cytokines to the TME. This in turn proves that IFN-γ has a bystander activity by acting distantly on the TME through the sustained and prolonged signaling which is mediated by tumor reactive T-cells[120].

IMMUNOGENIC CELL DEATH

Immunogenic cell death (ICD) role is to activate the immune system to eliminate any residual cancer cells that survive conventional cancer treatments[121] and is currently being investigated as a means for targeting cancer cells in combination with apoptosis[122] which is immunologically silent form of cell death[123]. Necroptosis, on the other hand, generates second messengers that act on immune cells in the TME, alerting them of danger[123]. Even though IL-6 is involved in eliciting an immune response, chronic activation of IL-6 and STAT3 can possibly weaken the generation of an immune response and an adaptive immune response. In the context of ICD, IL-6 shapes a microenvironment that is less capable of generating an effective immune response to acute inflammatory stimuli. Thus IL-6 may impair anti-cancer immunity through the control of a mechanistic switch between primarily cytotoxic cell death and immune-mediated clearance of tumor cells after genotoxic chemotherapy treatment[69].

CONCLUSION

Future trends for better understanding the complexity of signaling pathways within the TME are focused on utilizing this sum of information to halt and treat different tumors. To accomplish this, TME models must be created in vitro to screen for potential therapy. Until now, the two-dimensional monolayer cell culture technique has not proved efficient in properly demonstrating and reflecting the true heterogeneity of the TME[124]. This has made it challenging to find accuracy in answering questions related to the complex intricate network of cellular and non-cellular signals. Ultimately this also has led to the scarcity of information on how administered therapeutics function within the cancer microenvironment. Three-dimensional systems for tissue culture are gaining spotlight for their ability in the provision of a holistic approach to decipher these ambiguities[124]. Microfluidic platforms are systems used to accomplish this goal, where its level of multiplexing plays a pivotal role in unraveling the mystery of TME[125]. Several models are described with the main purpose of collectively culturing different cell lines within the same medium allowing to study the biochemical complexity of cross-talks, differentiation, migration, and metastasis taking place within the TME[125] as well as the pH, oxygen, nutrients and promigratory factors[126]. Advantages of such systems include the vast operability of analysis performed as the in vivo features of heterogenous cell populations within the TME niche are elucidating the cell-cell and cell-ECM interactions[126]. It was also noted from previous literature that TME changes between the primary tumors and the secondary metastasizing tumors and that cancer cells adapt their migration and behavior accordingly. Studying this peculiar and elusive mechanism can be accomplished by the utilization of different microfluidics platforms[126].

One of the latest breakthroughs is the involvement of cytokines with the propagation of exosomes within the TME[127]. It was noted that cytokines tagged to exosomes from cancer patients hold a different composition to those from healthy individuals[128]. This process facilitates the take-up of these exosomes by receptors specific to these cytokines, playing an integral role in metastasis[127]. The nature of exosomes as extracellular vesicles allows them to regulate progression or inhibition of cancer depending on their intracellular components[129]. Combining this with the cytokine tag can have pronounced therapeutic goals. As more emerging evidence and breakthroughs of the TME are being discussed, cytokines are being highlighted for their role in pro-tumorigenic inflammation. Further studies on the signaling pathways and regulatory functions of these inflammatory signals have the potential to pave the way for more targeted cancer immunotherapies.