Cytokine Interaction With Cancer-Associated Fibroblasts in Esophageal Cancer

TGF-β

TGF-β is an anti-inflammatory cytokine involved in numerous cellular functions. ⁵¹ The cytokine exists in three different isoforms (TGF-β1, TGF-β2, and TGF-β3) that have various contributions to EC. ⁴⁷ TGF-β was first identified with epidermal growth factor (EGF) as a factor that pushed fibroblasts into different phenotypes. ⁵² The cytokine plays a major role in general cell signaling with a strong impact on SMAD transcriptional regulation.^51,53 The prevalence of SMAD signaling throughout cell types paired with the pleiotropic nature of TGF-β allows for manipulation of the pathway by EC cells.^51,53 In fact, since its discovery in the 1980s, TGF-β has been strongly implicated in the initiation as well as the progression of many cancer types, including EC.^52,54,55

TGF-β levels remain abundant throughout the EC TME. Under normal conditions, TGF-β displays tumor-suppressing properties, even inducing apoptosis in EC. ⁵⁰ Upon subsequent alterations of the TGF-β pathway, cancer cells are able to highjack the machinery to inhibit the cytokine’s tumor-suppressing abilities. ⁵¹ Cancer cells are able to utilize TGF-β to activate pathways involved in weakening the immune response to EC and building an immune suppressive microenvironment. ⁵³ One major method of immune suppression by cancer cells is to convert resident fibroblasts into a phenotype more conducive to cancer progression. ⁵⁴ The creation of CAFs allows for direct feedback with the cancer cells as well as further conversion of other local fibroblasts. ⁵⁴ Following fibroblast activation, TGF-β is secreted by CAFs to assist in EC cancer cell progression and further development.^51,54

Secretions of TGF-β have been widely implicated in many physiological processes that support tumor initiation, progression, and metastasis. As previously mentioned, TGF-β allows cancer cells to signal to resident fibroblasts for conversion to a cancer-associated phenotype; however, TGF-β also has the ability to act on other stromal elements to induce cancer-favoring processes. ⁵⁴ In EAC, clinical data has shown an association between TGF-β and tumor stage, with lower cytokine levels associated with favorable patient survival. ⁴⁷ The TGF-β pathway has been observed to assist in the remodeling of EC extracellular matrix components via upregulation of matrix metalloproteinase 9 (MMP9), thus creating a habitat hospitable for tumor progression and metastasis.^54,56 Furthermore, TGF-β is linked to EMT, which has been shown to transform epithelial cells to mesenchymal stem cells.^57,58 The mesenchymal stem cells create an environment that is more evasive to the immune system, thus promoting invasion and metastasis in EC.^53,57,58 In ESCC, TGF-β has also been shown to impact EMT via upregulation of MALAT1 and XlAP.^59,60 Additionally, EMT has been linked to decreased patient survival rates and increased metastasis in EC.^58,60 More recent data has also suggested a role of TGF-β on upregulation of PD-1 in CD8⁺ T-cells, which could decrease the efficacy of therapeutics targeting the PD-1 or PD-L1.

Tumor Necrosis Factor-α (TNF-α)

First discovered in the 1970s, TNF-α has long been associated with many cancers including EC. ⁶¹ The pleiotropic cytokine binds to two different receptors, TNFR-1 and TNFR-2, and plays an important role in various oncogenic pathways such as NF-kB and MAPK.^62,63 TNF-α can be secreted by both cancer cells, including EC, as a method to activate fibroblasts and conversely, through CAFs as means to convert other resident fibroblasts and support cancer cell proliferation.^14,64 In stark contrast with its tumor progressive functions, TNF-α has also been shown to kill cancer cells at high concentrations. ⁶¹ While promising, the increased cytokine concentration produces toxicity in patients but yields lower side effects when administered locally.^61,62

Although TNF-α implores a variety of functions, more recent studies have shifted focus to the cytokine’s involvement in the mediation of cancer-related processes. TNF-α has been shown to be involved in cisplatin resistance in ESCC via induction of EMT by increased superoxide dismutase mitochondrial (SOD-2) production. ⁶⁵ TNF-α also increased in vitro proliferation of human ESCC cells. ⁶⁵ TNF-α signaling has been associated with EMT activation in other cancer types by similar mechanisms, as well.^66,67 TNF-α also plays a complex role in the progression of BE to EAC through multiple mechanisms.^68,69 Previous studies suggest that the loss of glutathione peroxidase 7 (GPX7) is attributed to an upregulation of TNF-α, thus producing NF-kB induced tumorigenesis. ⁶⁸ More recently, TNF-α has been implicated in promoting the expression of other CAF secretions such as IL-8 that impact the progression of BE to EAC. ⁶⁹

Interleukin-6

First discovered in 1986, IL-6 is another pleiotropic cytokine that plays an important role in several processes from metabolism to tissue and cell maintenance.^70-72 The 25 kD cytokine has since become the focal point of extensive efforts to halt the progression of numerous cancers, including esophageal.^70,73 IL-6 is able to have such vast impacts on various cancer types via its role in numerous oncogenic pathways including STAT3, NF-kB, PI3K, and MAPK.^71,74 In addition, IL-6 is also enlisted in creating a tumor niche that employs an abundant number of hallmarks necessary for EC and other cancers, specifically angiogenesis and metastasis.^73,75,76

While noted throughout most cancer types, IL-6 is extremely prevalent in EC, evoking an impactful role in tumor progression. ⁷⁷ In vivo experiments elucidated the importance of IL-6 on carcinogenesis via injection of co-culture of CAFs and EC cells in mice, demonstrating its ability to provide a unique, intercellular feedback system between a variety of components in the TME. ⁷⁸ This communication between stromal components and cancer cells allows for alterations to the immune landscape of the TME. Through increased secretion by CAFs, IL-6 promoted decreased CD8⁺ and increased FoxP3+ tumor infiltrating lymphocyte levels within intratumoral tissue which have been implicated clinically in immune system suppression and reduction in overall patient survival. ⁷⁹ In EC, IL-6 has been further implicated in immune suppression for its role in impairing NK cells functionality. ⁸⁰ The cytokine is able to render the NK cells ineffective in EC by promoting the expression of CD39, as demonstrated via blocking of the effect by the receptor antagonist tocilizumab. ⁸⁰

While IL-6 has also been cited in numerous forms of cancer progression, the protein plays a major role in resistance to various treatments. IL-6 has been directly linked to decreased efficacy of cisplatin for treating ESCC patients.^81,82 These findings were verified by utilizing siRNA targeting IL-6 to display the restored sensitivity to cisplatin. ⁸¹ The cytokine yields further prognostic value with respect to patients undergoing esophagectomies where IL-6 rates have been associated with poor response to radiotherapy and chemotherapeutics. ⁸³ Furthermore, the IL-6/CXCR7 axis has been shown to activate the STAT3/NF-kB pathway in EC and induce EMT, leading to further chemoresistance and decreased patient survival. ⁸⁴

Interleukin-8

Interleukin-8, also referred to as CXCL8 and neutrophil-activating factor, was the first chemokine identified in humans and is produced in a plethora of cell types from neutrophils to EC CAFs.^49,85,86 Regulation of this proinflammatory cytokine is controlled via its two receptors, CXCR1 and CXCR2, which assist with cancer hallmarks like angiogenesis in EC. ⁸⁷ In EC, CXCL8 expression yields activation of several different pathways such as Akt1/RelA, STAT3, and NF-kB.^88-90 IL-8 also has wide-reaching effects on EC progression via interactions with other cytokines such as IL-6 and has since been noted as a possible biomarker for ESCC.^91,92

Although IL-8 has been contemplated for immune targeting, the cytokine continues to pose obstacles as it resists many current treatment methods while aiding in carcinogenesis. The presence of IL-8 greatly impacts the recruitment of certain myeloid-derived suppressor cells (MDSC), which cause increased EC tumor progression. ⁹³ A clinical study observed that recurrence-free survival rates fell in ESCC patients expressing the CXCL8/CXCR2 system. Those same patients also experienced resistance to chemotherapy and radiation. ⁹⁴ Similar results were observed in a later study demonstrating that IL-8 assists EC cells in resisting radiation therapy. ⁹⁵ IL-8 is able to manipulate miR-27b-5p expression to aid in the upregulation of proliferating cell nuclear antigen (PCNA), which was the main contributor to EC radio-resistance. ⁹⁵

Hepatocyte Growth Factor (HGF)

Initially observed in the primary culture of rat liver cells, HGF, also known as scatter factor, was discovered in 1984.^96,97 The pleiotropic cytokine is made up of two subunits (alpha and beta), weighs 84 kD and has immense implications in EC and other diseases.^96,98 The role of HGF on EC development and progression is accentuated by the wide range of stromal elements that secret the cytokine, with CAFs producing large quantities.^96,97 HGF is able to promote tumorigenesis in EC through binding of mesenchymal–epithelial transition (MET) receptor tyrosine kinase and subsequent activation of its pathway. ⁹⁹

In addition to EC, the activation of the HGF/MET pathway has been involved in many cancer types. HGF has been shown to increase proliferation in certain ESCC cells while also recruiting VEGF to assist in promoting angiogenesis. ¹⁰⁰ Further studies noted increased HGF levels in over half ESCC histology samples while linking the cytokine’s expression to tumor size and stage. ¹⁰¹ Additionally, heightened degrees of HGF in ESCC patient plasma were associated with advanced disease stage and metastasis. ¹⁰² Upregulation of the cytokine’s receptor has also been observed in esophageal adenocarcinoma as well as its precursor, Barrett’s esophagus. ¹⁰³ Recent findings demonstrated another relationship between HGF and EAC with decreased patient survival rates as cytokine frequencies were elevated. ¹⁰⁴

Other Relevant Signaling Proteins

Although CAFs are able to manipulate the microenvironment of EC via secretion or response to the aforementioned cytokines, recent studies have begun unveiling other signaling proteins involved in EC tumorigenesis. For instance, CXCL12, also known as stromal cell-derived factor-1, is a chemokine that, via upregulation of twist1, is produced and secreted at high volumes by CAFs in EC.^45,105 Subsequently, CXCL12 is able to bind its receptor, CXCR4, to induce EMT and promote EC metastasis.^45,105 CCL2 is another chemokine that has been implicated in tumor progression of ESCC. ¹⁰⁶ CCL2 secretions appear to have some association with FAP expression in CAFs. Knockdowns of FAP in subpopulations of EC CAFs resulted in a reduction of CCL2 secretions. ¹⁰⁶ IL-1 is a prevalent cytokine in esophageal carcinogenesis with secretions producing an environment conducive to treatment resistance in ESCC.^45,107 In addition to its role in ESCC, increased levels of IL-1 were shown to induce a BE phenotype in mouse models. ¹⁰⁸ Furthermore, previous studies demonstrated a significant increase of IL-1 expression in both BE and EAC ¹⁰⁹ . CXCL10, also referred to as interferon-γ inducible protein-10 (IP10), is an anti-inflammatory chemokine where increased rates correlated to improved patient survival in ESCC; however, others observed raised levels of the chemokine associated with advanced stages of EAC.^49,110 Similarly, IL-33 is another cytokine that is upregulated by CAFs and a contributor to metastasis in EC.^45,111

Treatments Targeting Cancer Cells that are Impacted by Interactions with CAFs

Human epidermal growth factor receptor 2, HER2, is a key target of numerous therapies available for EC treatment.^123,124The first FDA approved HER2-specific treatment was the monoclonal antibody trastuzumab that demonstrated improved patient survival in combination with chemotherapy and the immune checkpoint inhibitor.^123,124 Its approval has led to the development of targeted therapies focused on the tyrosine receptor kinase, including pertuzumab and zanidatamab.^123,125 While the results are encouraging, subsequent studies in breast cancer have demonstrated trastuzumab resistance via CAF secretion of FGF5, although the phenomenon has yet to be observed in EC. ¹²⁶ Additionally, side effects such as cardiotoxicity have been observed in various patient populations including those with EC. ¹²⁴

Another receptor tyrosine kinase with profound opportunities for targeting EC is epidermal growth factor receptor (EGFR).^123,125,127 Approved for the treatment of colorectal cancer, cetuximab is a chimeric monoclonal antibody that demonstrated promising results in initial trials with EC.^123,125 Nimotuzumab is another anti-EGFR therapy, which has shown positive results in EC when paired with radiation.^125,128 Recently, lapatinib, a small molecule inhibitor, is gaining traction in EC, which shows dual inhibition of EGFR and HER2. ¹²³ Although promising, recent in vivo studies in lung cancer highlight concerns over therapeutic resistance of tyrosine kinase-specific therapies as cancer cells have been shown to utilize lactate to direct CAF secretion of HGF, hindering therapies such as lapatinib and other immune checkpoint inhibitors (ICIs). ¹²⁹ The findings have yet to be replicated in EC, but questions persist on the responsiveness of EC to the treatments due to inconsistent results in clinical trial. ¹²³

Targeting the interaction between MET and its ligand HGF is another avenue that researchers have explored to combat blockades against the immune system.^123,130-132 Overexpression of c-MET has been demonstrated in both ESCC and EAC while HGF is a known secretion of CAFs.^97,125 Current therapies targeting the pathway have shown promise in obstructing cancer signaling; however, EC patient survival rates have not been significantly impacted. ¹²³ One MET inhibitor gaining attention is AMG337, and while still early in clinical trials, displays anti-cancer properties. ¹²³ That said, CAF-induced therapeutic resistance poses roadblocks with prolonged treatment efficacy. ¹²⁹ Although still unobserved in EC, CAFs have also been implicated in gradual resistance to MET inhibitors in lung cancer. ¹²⁹ While various targeted agents have shown potential, therapeutic efficacy and adverse effects on the heart have led some researchers to seek alternative approaches. ¹³²

A subsequent target that has drawn scientist’s attention is the PD-1/PD-L1 interaction. Programmed death 1 (PD-1) and its ligand (PD-L1) are responsible for the suppression of T-cell response, allowing for evasion of the immune system in esophageal and other cancer types.^118,120 EC cells express PD-L1 on their surface while immune cells like macrophages display PD-1. ¹²³ The interaction between ligand and receptor leads immune cells to perceive the EC cells as harmless, allowing them to escape immune destruction.^123,125,133 Immune checkpoint inhibitors targeting this interaction have shown potential in clinical trials with pembrolizumab, a monoclonal antibody blocking PD-1, demonstrating promising results as improved overall survival when administered alongside chemotherapeutics.^120,123,133 On the other hand, recent studies have shown certain subtypes of CAFs impact the effectiveness of pembrolizumab in melanoma treatment. ¹³⁴ Though yet to be observed in EC, CAF clusters were significantly elevated in patients unresponsive to pembrolizumab. ¹³⁴ Conversely, other immunotherapies targeting the ligand-receptor, such as nivolumab and camrelizumab, have shown promise in treating EC.^123,125

Treatments Directly Targeting CAFs

More recent research has since shifted to disrupting the TME and even utilizing stromal elements, such as CAFs, to assist in killing cancer. ¹³⁵ Various methodologies have been hypothesized for directly targeting CAFs in other cancers, which remains a challenge given their heterogeneity and lack of definitive markers.^64,118,135 Recent clinical trials have attempted to utilize antibodies in order to target FAP-α directly in CAFs (https://clinicaltrials.gov/ct2/show/NCT02209727?cond=sibrotuzumab&draw=2&rank). ¹³⁶ For instance, sibrotuzumab, an anti-FAP-α antibody, was discontinued after failing to produce favorable outcomes. ¹³⁶ In fact, the antibody actually decreased patient survival; however, subsequent immunotherapies that target FAP-α, such as RO6874281, have been proposed in combination with existing therapeutics in hopes of improving treatment efficacy in EC (https://clinicaltrials.gov/ct2/show/NCT03386721). ¹³⁶ Alternatively, as single-cell analyses become more widely available, numerous studies have begun seeking subpopulation-specific therapies in other cancer types.^64,137 One such study targeted CAFs in breast cancer by utilizing pirfenidone and observed favorable results in declining CAF viability; however, the anti-fibrotic drug alone did not impact metastasis as initially hoped. ¹³⁸ Another approach has been to interfere with CAFs ability to perform autophagy. ¹³⁹ Disrupting the function was shown to decrease levels of proinflammatory cytokines, increase responsiveness to chemoradiation, and decrease cell proliferation. ¹³⁹ Other techniques to obstruct CAFs that have been investigated seek to revert the cell type back to its quiescent phase.^13,137 This reversion has been observed experimentally through the usage of distinct agents like all-trans retinoic acid (ATRA), vitamin D, and vitamin A.^135,137 While these cutting-edge studies continue trying to reprogram and repurpose CAFs in other cancers, work remains in order to elucidate whether these findings can produce similar results EC.