This article is mentioned in:

Introduction

Pancreatic ductal adenocarcinoma (PDAC), globally recognized as the ‘king of cancers’, is projected to become the second deadliest cancer in the United States by 2026, but its cause remains elusive (1,2). This lethal disease is typically diagnosed at an advanced metastatic stage owing to a lack of early clinical manifestations (3). The mortality rate of patients with PDAC closely mirrors the morbidity rate, with a brief disease course and a survival period typically <1 year (4). Early screening techniques such as endoscopic ultrasound, magnetic resonance/magnetic resonance pancreaticobiliary imaging and PDAC detection with artificial intelligence can accurately detect and classify pancreatic lesions using non-contrast CT (5,6). However, early diagnosis and screening for pancreatic cancer remain extremely limited in most countries (7).

Yachida et al (8) proposed a model for the genetic evolution of PDAC, dividing its development into stages of precursor lesion pancreatic intraepithelial neoplasia (PanIN), invasive carcinoma to a metastatic mass and metastatic transmission to death. In the past few years, single-cell RNA sequencing (scRNA-seq) has revealed that the tumor microenvironment (TME) of PDAC is a complex ecosystem with intricate cellular composition and heterogeneity among populations (9). The TME is characterized by an abundance of extracellular matrix (ECM), including cell components such as fibroblasts and immune cells (10). Moreover, the progression from early to late PDAC is characterized by a decrease in the number of cancer-associated fibroblasts (CAFs), which are composed of fibroblasts and stellate cells, and a progressive increase in the proportion of immune cells (11). Throughout these stages, a transition from a proinflammatory microenvironment to a highly fibrotic and immunosuppressive TME was observed (Fig. 1) (9,12). Therefore, fibrosis and immunity are indispensable factors in the progression of PanIN to PDAC. Activated pancreatic stellate cells (aPSCs), which constitute ~50% of the TME, dominate dense stroma composed of PDAC fibroblasts (13). These aPSCs are present at all stages of pancreatic cancer, including early PanIN lesions, where they accelerate the progression of high-grade PanIN lesions, thereby exhibiting tumor-promoting properties (14). Interleukins and related cytokines are a means of communication between innate and adaptive immune cells and non-immune cells and tissues (15). Members of the interleukin family have emerged as novel protein markers for pancreatic cancer risk in the China Kadoorie Biobank Chronic Disease Prospective Project study (16).

Figure 1. Changes in the TME of pancreatic cancer progression. (A) Pre-invasive PanIN lesions develop from normal ductal epithelia, through PanIN stages 1, 2 and 3, to stage 3 development process. (B) Quiescence and differentiation of aPSC. qPSC markers include Nrxn2, Prelp, Serping1, ADIRF and FABP4, myCAF markers include Acta2, Tagln, Tpm2, Col12a1 and Tpm1, iCAF markers include IL-6 and Pdgfra, and apCAF markers include H2-Ab1, CD74, Hla-dra and slpi. (C) Components of the PDAC tumor microenvironment. qPSC, quiescent pancreatic stellate cell; aPSC, activated pancreatic stellate cell; Mø, macrophages; M2 ø, type 2 macrophages; MDSC, myeloid-derived suppressor cell; Treg, regulatory cell; Nrxn2, neurexin 2; Prelp, proline/arginine-rich end leucine-rich repeat protein; Serping1, serpin peptidase inhibitor, clade G (C1 inhibitor), member 1; ADIRF, adipogenesis regulatory factor; FABP4, fatty acid binding protein 4; Acta2, actin α2, smooth muscle; Tagln, transgelin; Tpm1/2, tropomyosin 1/2; Col12a1, collagen type XII α1; Pdgfra, platelet-derived growth factor receptor α; H2-Ab1, histocompatibility 2, class II antigen A, β1; CD74, leukocyte differentiation antigen 74; Hla-dra, human leukocyte antigen-dr α; slpi, secretory leukocyte protease inhibitor; PanIN, pancreatic intraepithelial neoplasia; PDAC, pancreatic ductal adenocarcinoma.

The present review focuses on the effects of aPSCs in the interleukin-1 (IL-1) family, interleukin-6 (IL-6) family, interleukin-10 (IL-10) family and interleukin-17A (IL-17A) cytokines on pancreatic cancer development (Fig. 2). In addition to the IL-1 family, the IL-6 family and IL-10 family of cytokines are involved in the survival of patients with PDAC (Fig. 3) highlighting their potential as critical targets for early cancer screening and diagnosis. These interleukin family cytokines are prospective candidates in the field of fibrosis as well as immune regulation and have considerable potential to improve the early developmental profile of PDAC (15–17). However, most other interleukin family members have only been evaluated in preclinical or clinical trials for immune regulatory signatures (Table I).

Figure 2. Crosstalk between pancreatic stellate cells and the IL-1 family, IL-6 family, IL-10 family and IL-17A of cytokines in PDAC. aPSC, activated pancreatic stellate cell; NK, natural killer; ILC, innate lymphoid cell; Th1/2, helper T1/2; CD4+ T cell, CD4+ T helper cell; CD8+ T cell, CD8+ T helper cell; IL-18Rα, interleukin-18 receptor α; IL-18Rβ, interleukin-18 receptor β; IL-1RAcP, interleukin-1 receptor accessory protein; Tyk2, tyrosine kinase 2; PDAC, pancreatic ductal adenocarcinoma.

Figure 3. Survival of IL-1 family, IL-6 family and IL-10 family cytokines in patients with pancreatic ductal adenocarcinoma. Overall survival for patients with high (top quartile) and low (bottom quartile) level expression of (A) IL-1, (B) IL-6, (C) IL-10, (D) IL-11, (E) IL-18, (F) IL-18BP, (G) IL-20 and (H) IL-33. HR, hazard ratio.

Table I. Preclinical or clinical trials of IL family members on PanIN/PDAC in the previous five years.

Table I.

Preclinical or clinical trials of IL family members on PanIN/PDAC in the previous five years.

A, IL-2 family
Cytokine	Cotreatment	Sample size	Study model	Abstract	(Refs.)/Phase (0-III)/Clinical Trials gov identifier
IL-2	NA	21/11	Human	IL-2RG is overexpressed in PanIN	(126,127)
				RNA sequences and in PDAC tissues
	PD-L1 + IFNγ	NA	Mouse	Targeting cytokine therapy to the	(128)
				PDAC microenvironment using
				specific VHHs
	NA	48	Human	Peripheral blood and tissue assessment	(129)
				highlights differential tumor-
				circulatory gradients of IL-2 with
				prognostic significance in resectable
				PDAC
	Gemcitabine	8	Human	ALT-803 combination with gemcita-	Phase Ib/II
	Nab-paclitaxel			bine and nab-paclitaxel in patients	NCT02559674
	ALT-803			with advanced PDAC
	Cyclophospha-	11	Human	TCR-T cell therapy on advanced	Phase I
	mide +			pancreatic cancer	NCT05438667
	fludarabine
	Neoantigen	180	Human	Autologous T cells to express TCRs	Phase Ib/II
	specific TCR-			in subjects with solid tumors	NCT05194735
	T cell drug
	product
Salmonella-	FOLFIRINOX	60	Human	Saltikva for metastatic pancreatic	Phase II
IL2	or gemcitabine/			cancer	NCT04589234
	abraxane
IL-9	NA	25	Human	Circulatory IL-9 is high in healthy	(60)
				individuals, and IL-9 in the PDAC
				matrix is associated with improved
				survival
IL-15	Behavioral:	30	Human	Secondary outcome measures included	NCT05483075
	Health care			a change in the number of circulating
	provider-guided			CD8+ T cells expressing IL-15Ra
	exercise training
	program
IL-15	ETBX-011	3	Human	ETBX-011 vaccine in combination	Phase I/II
(ALT-803)				with ALT-803 in patients with CEA-	NCT03127098
				expressing cancer
	NANT	80/3/	Human	Combination immunotherapy in	Phase I/II
	pancreatic	173		patients with pancreatic cancer who	NCT03329248
	cancer vaccine			have progressed on or after standard-	NCT03136406
				of-care therapy	NCT03586869
IL-21	IL-26	19	Human	DW-MRI diagnostic PDAC immune	(130)
				cell infiltration, increased ADC and
				aggressive tumor disease
IL-21/	NA	221	Human	IL-21 receptor/ligand interaction is	(131)
IL-21R				linked to disease progression in PDAC
B, IL-3 family
IL-3	CTLA-4	NA	Mouse	Anti-CTLA-4 synergizes with	(132)
				dendritic cell-targeted vaccine to
				promote IL-3-dependent CD4+ T cell
				infiltration into murine PDAC
C, IL-12 family
IL-12	IL-18	NA	Mouse	IFNγ produced by IL-12 and IL-18	(133)
				treatment to control early tumor
				growth
Ad5-yCD/	5-FC+	12	Human	IL-12 therapy with standard	Phase I
mutTKSR	[18F]-FHBG			chemotherapy in metastatic pancreatic	NCT03281382
39rep-				cancer
hIL12
Human-IL-	Nivolumab	51	Human	VG161 combination with Nivolumab	Phase I/II
12/15/PDL				in patients with advanced PDAC	NCT05162118
1B
IL-23	TGF-ß	NA	Human	IL-23 and TGF-β diminish macro-	(134)
			and	phage associated metastasis in PDAC
			mouse
IL-35	NA	NA	Human	IL-35 drives STAT3-dependent CD8+	(135)
			and	T-cell exclusion and immunotherapy
			mouse	resistance in PDAC
D, Other families
Anti-IL-8	Nivolumab	76	Human	Neoadjuvant and adjuvant immuno-	Phase II
antibody;				therapy for patients with resectable	NCT02451982
HuMax-IL-				PDAC
8 (BMS-
986253)
IL-13	NA	NA	Human	Presence of IL-13 in pancreatic PanIN	(136)
			and	alters macrophage populations and
			mouse	mediates pancreatic fibrosis and
				tumorigenesis
IL-13Rα1	NA	NA	Human	IL-13Rα1 deficiency induces PDAC	(137)
				apoptosis and promotes EMT
IL-13Rα2	NA	236	Human	Novel marker of IL-13Rα2 in PNI and	(138)
				its association with poor prognosis in
				PDAC
IL-34	NA	NA	Human	CTC in patients with PDAC raises	(139)
				IL-34 levels and may affect myeloid
				differentiation and/or present antigens for cell activation

[i] ALT-803, ETBX-011, GI-4000, capecitabine, cyclophosphamide, nab-paclitaxel, oxaliplatin and SBRT are NANT pancreatic cancer vaccines. VHH are single-domain antibodies against PD-L1 fused with IL-2 and IFN-γ. [¹⁸F]-FHBG is a HSV-1 TK substrate that involves PET imaging to quantify the intensity, persistence and biodistribution of HSV-1 TK gene expression in the pancreas. VG161 is a human-IL12/15/PDL1B oncolytic HSV-1 injection. NA, not applicable; 5-FC, 5-fluorocytosine; DW-MRI, diffusion-weighted magnetic resonance imaging; ADC, apparent diffusion coefficient; TCR, T-cell receptor; PNI, perineural invasion; CTC, circulating tumor cell; TCR-T, T cell receptor-engineered T; IFNγ, interferon-γ; CTLA-4, cytotoxic T-lymphocyte antigen-4; EMT, epithelial-mesenchymal transition; CEA, carcinoembryonic antigen; PDAC, pancreatic ductal adenocarcinoma; PanIN, pancreatic intraepithelial neoplasia.

Previous and current understanding of pancreatic stellate cells (PSCs) in PDAC

In 1998, PSCs from healthy human and rat pancreases were successfully isolated and cultured for the first time (18). Most of the initial understanding of PSCs was based on their similarity with hepatic stellate cells, but in-depth exploration has led to a more comprehensive understanding of the morphology and location of PSCs, as well as their unique characteristics of being able to store lipid droplets and vitamin A (19). Transcriptome analysis of human and mouse PSCs revealed two distinct clusters of PSCs including ‘activate’ [enrichment of ECM genes, collagen type 1 α1 (COL1A1) and fibronectin 1] and ‘quiescent’ (enrichment of adipose genes, adipogenesis regulatory factor and fatty acid binding protein 4) (20). Under physiological conditions, PSCs are in a quiescent state and are the only pancreatic cells that store vitamin A (21). PSC activation is characterized by a spindle-like shape in vitro and the disappearance of vitamin A lipid droplets, although the mechanism of the disappearance or absence of vitamin A in PDAC progression has yet to be elucidated (22). In the aPSC state, there are α-smooth mucle actin (SMA) and collagen fibers, ECM deposition, and the release of epithelial-mesenchymal transition (EMT)-associated soluble inflammatory factors (23). Simultaneously, aPSCs begin to proliferate and manifest the proinflammatory phenotype, releasing chemokines, cytokines and growth factors that recruit other inflammatory factors into the pancreas, perpetuating the inflammatory response (24). aPSCs can also promote an immunosuppressive microenvironment in mouse models of pancreatic cancer (25). Sustained aPSCs can create a TME conducive to cancer cell growth (Fig. 1C).

PSC-derived CAFs in human and mouse PDAC were analyzed by scRNA-seq technology and classified into myofibroblast CAFs (myCAFs), inflammatory CAFs (iCAFs) and antigen-presenting CAFs (apCAFs) based on positional and functional characteristics (26,27). These three heterogeneous CAF subsets function in mutual conversion (26,27). However, apCAFs are rarely found in patients with PDAC (9). MyCAFs dominate connective tissue proliferation and form a physical protective barrier outside the cells of pancreatic cancers, protecting them from drug intervention and immune recognition (28). ECM depletion has been proposed to remove the fibrous barrier, but it has not been successful in the treatment of pancreatic cancer (29). The depletion of SMA+ myCAFs led to a reduction in the tumor stroma in mice (30). This accelerates PanIN and PDAC formation and development and reduces survival (30). The second CAF subgroup includes iCAFs. In a broad sense, myCAFs appear to be involved in EMT and ECM remodeling, whereas iCAFs appear to be associated with inflammation and ECM deposition (31). Mouse pancreatic tumor scRNA-seq revealed that the IL-1/JAK/STAT3 and TGFβ/Smad3 signaling are key pathways that regulate iCAF and MyCAF heterogeneity and function (32). Furthermore, myCAFs and iCAFs are considered to play opposing roles in cancer (33). The third subpopulation of CAFs is apCAFs, which express a wide range of fibroblast markers including COL1A1, COL1A2, decorin and podoplanin as well as major histocompatibility complex class II (MHC II)-related genes that are limited to antigen-presenting cells (APC) of the immune system and present model antigens to CD4+ T cells in an antigen-dependent manner (26). The source and nature of the antigen presented by apCAFs are not known, which is an important mystery in the study of the interaction between CAFs and tumor-infiltrating T cells.

PSC-derived CAFs are classified as cancer-promoting CAFs (pCAFs) or cancer-restraining CAFs (rCAFs) based on their role in fighting cancer (26,34). Fibroblasts that maintain homeostasis and innately suppress tumorigenesis are collectively referred to as rCAFs (35). However, the nature and characteristic markers of rCAFs are unknown. Meflin is highly expressed in quiescent PSCs, oncogenic meflin-tagged rCAFs appear around cells in PanIN, meflin expression decreases during PDAC progression, and α-SMA expression increases, leading to phenomena such as pCAFs (34). pCAFs secrete matrix components such as collagen, fibronectin and proteoglycans that are involved in EMT, invasion, metastasis and tumor angiogenesis and have been extensively studied (35). Currently, using pharmacological methods to synthesize the unnatural retinoid Am80, clinical trials on the conversion of pCAFs to rCAFs for the treatment of pancreatic cancer have been initiated (clinical trial NCT05064618). Recently, several vitamin analogs that downregulate α-SMA, reduce the movement and activation of PSCs, restore PSCs to quiescence, and lead to increased apoptosis in neighboring cancer cells in combination with chemotherapy drugs have entered clinical trials in patients with PDAC (https://clinicaltrials.gov/; Table II). However, PSC-derived CAF subpopulations are diverse (26,27,34). Thus, interfering with PSC subpopulations is one of the more challenging therapeutic strategies for pancreatic cancer.

Table II. Ongoing clinical trials for combined vitamin analogues for PDAC.

Table II.

Ongoing clinical trials for combined vitamin analogues for PDAC.

Target	Candidate drug and combination regimen	Trial design	Sample size	Primary outcome	Phase (O-III)/Clinical Trials gov identifier
VB	FOLFIRINOX regimen:	Single-center,	60	Percentage of patients that were	NA
	FOL+ 5-FU + Irinotecan	single-arm,		subject to surgical resection	NCT05262452
	(eloxatin) + OX-	investigator-		(4 months or 6 months after the
	oxaliplatin (eloxatin)	initiated, open		start of the combined treatment)
		labeled
VC	Intravenous melphalan +	Single-arm	10	Evaluate the safety of the	Phase I
	BCNU + low-dose I.V.			investigational treatment on	NCT04150042
	ethanol + vitamin B12b +			pancreatic cancer in patients
	vitamin C			who were carriers for BRCA1
				or BRCA2
	Gemcitabine and Nab-	Parallel	65	Overall survival after treatment	Phase II
	paclitaxel + pharma-	assignment			NCT02905578
	cological ascorbate
	High dose vitamin C +	Open, prospective,	30	Progression-free survival	Phase II
	metformin	single-arm, multi-		(up to 12 weeks)	NCT04033107
		cohort
VD	Paricalcitol +	Single group	21	Response evaluation in solid	Phase II
	hydroxychloroquine	assignment		tumors	NCT04524702
	gemcitabine + Nab-
	paclitaxel
VDR	Nivolumab + albumin-	Single-arm, open-	10	Complete response rate	Phase II
	bound paclitaxel +	label			NCT02754726
	paricalcitol + cisplatin +
	gemcitabine
	Gemcitabine + Nab-	Parallel	112	Assess adverse events and	Phase I and II
	paclitaxel + two	assignment		overall survival	NCT03520790
	formulations (iv or
	oral or placebo) of
	paricalcitol
	High-dose oral vitamin	Randomized,	60	Blood level of vitamin D3	Phase III
	D3 supplementation	open-label,		(60-day postoperative	NCT03300921
	(prior to PDAC surgery)	parallel		mortality is a secondary	NCT03472833
		assignments		outcome)
Vitamin	ARACOMPLEX®	Randomized,	234	Change in quality of life	NCT05360745
complexes	or placebo	parallel assignment

[i] NA, not applicable; PDAC, pancreatic ductal adenocarcinoma; VB/C/D, vitamin B/C/D; vitamin VDR, vitamin D receptor; FOL, folinic acid (leucovorin; vitamin B derivative); 5-FU, 5-fluorouracil; ARACOMPLEX^®, food supplement that contains maca extract, vitamin complexes and ions.

Interleukin family synergizes the role of the PSC in fibrosis and immune regulation in PanIN and PDAC

Spatial transcriptomics and scRNA-seq have revealed distinct transcriptomic features of PanIN fibroblasts and macrophages in healthy adult organ donors and patients with PDAC (36). Furthermore, macrophages are observed in close proximity to PSCs, and aPSCs drive anti-inflammatory M2 type macrophages to produce an immunosuppressive environment for pancreatic cancer, which may explain the aberrant interactions between PSCs and immune cells (37,38). scRNA-seq analysis conducted on PDAC samples before and after chemotherapy revealed that chemotherapy might enhance resistance to immunotherapy (9). However, T cells and macrophages are the primary populations shaping the immune landscape in the TME (39). Baron et al (20) conducted scRNA-seq on four cases of PDAC and found that PSC, in addition to expressing genes associated with the ECM, were also closely associated with high interleukin expression. Interleukins are cytokines with immunomodulatory functions that serve as a means of communication between cells and tissues (20). Interleukins cultivate an environment conducive to cancer growth and are critical for tumor immunotherapy and targeting (15).

IL-1 family

The IL-1 family was originally used to generalize the products of macrophage-induced inflammation. A total of seven agonist cytokines (IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β and IL-36γ), three receptor antagonists (IL-1Ra, IL-36Ra and IL-38) and one anti-inflammatory cytokine (IL-37) currently belong to the IL-1 family (40,41). With the notable exception of IL-1Ra, each family member is produced as a precursor (40). Most of these precursors, except for IL-1α and IL-33, which are biologically inactive until they are cleaved and mature, do not activate their receptors (40). The antagonistic function of the IL-1 family and its own proinflammatory effects confer a wide range of biological activities, including proinflammatory and anti-inflammatory activities (42). The receptor structure contains one or three extracellular immunoglobulin structural domains and a transmembrane structural domain (42). Apart from type 2 IL-1 receptor (IL-1R2), other receptors share a conserved intracellular Toll/IL-1 receptor signaling domain, which is the structural basis for the IL-1 response to immunology (43). Therefore, the regulation of these signals is critical for the regulation of the immune system. In summary, the IL-1 family is a powerful toolbox.

IL-1

IL-1α and IL-1β were the first cytokines in the IL-1 family to be discovered (44). The expression of iCAF marker genes [IL-1α, IL-6, leukemia inhibitory factor, CXC motif chemokine ligand 1 (CXCL1) and granulocyte colony-stimulating factor 3] were significantly upregulated after IL-α and IL-1β induction in PSCs, whereas the expression of myCAF marker genes (actin α2, smooth muscle and connective tissue growth factor) were reduced (32). In particular, the mature form of IL-α enhances crosstalk between CAFs and PDAC cells, resulting in a chronic inflammatory tumor environment, characterized by the expression of the inflammatory factors IL-6 and CXCL8, but no downstream mechanism has been reported (32,45). Schwann cells enhance the proliferation and migration of PDAC cells by promoting the switch of CAFs to malignant subtypes of iCAFs via IL-1α (46). These preclinical studies have encouraged early phase I clinical trials. The maximum established tolerated dose of XB2001 (an IL-1α antagonist), in combination with irinotecan liposome injection (trade name ‘ONIVYDE’) plus 5-FU/LV (+ folinic acid), is being implemented in a PDAC clinical trial (clinical trial NCT04825288). IL-1β promotes immunosuppression by regulating PSC activation and the secretory phenotype in the PanIN microenvironment (47). Inflammatory loops are induced between tumor cells and IL-1β-expressing macrophages, a subset of macrophages elicited by synergy between prostaglandin E2 (PGE2) and tumor necrosis factor (TNF) (48). The PGE2-IL-1β axis may enable preventive or therapeutic strategies to reprogram immune dynamics in pancreatic cancer (48). However, IL-1β is associated with poor outcomes after neoadjuvant therapy of PDAC (49). IL-1 is essential for the formation of both iCAFs and myCAFs but it does not serve as a successful target for specific inhibition of mesothelial cell transition to apCAFs (49). Simultaneously, IL-1 inhibits the expression of MHC II and APC-related genes in CAFs, while IL-1R2 receptors, which are highly expressed by regulatory T cells (Tregs) in the TME, decoy IL-1 to block IL-1 CAF signaling and enhance the ability to deliver APCs, thereby increasing the number of Tregs infiltrating tumors (50). IL-1Ra binds to and blocks proinflammatory IL-1α and IL-1β (42). The IL-1Ra protein signature significantly distinguishes benign pancreatic tissue from PDAC (51). Given the wealth of preclinical studies of IL-1 signaling in iCAFs, anakinra, a human recombinant IL-1Ra, is currently being clinically evaluated in combination with standard chemotherapy (clinical trial NCT02550327).

Interleukin-18 (IL-18)

IL-18 is mainly secreted by macrophages, dendritic cells and epithelial cells, and can stimulate a variety of cell types and has numerous biological functions (52). IL-18 exists in an inactive form within cells (53). Structurally, IL-18-like IL-1β is an important effector molecule downstream of the NLRP3 and NLRP1 inflammasomes (53). IL-18 mediates the MyD88-NFκΒ signaling pathway in combination with its heterodimeric receptor (IL-18Rα/βR), is activated by the rapid release of caspase-1 excised precursor peptides from the inflammasome during inflammation and is considered a proinflammatory cytokine (53,54). Preliminary studies have revealed that IL-18 plays an instrumental role in the development of the pancreas from the acute to the chronic disease stage, while activating the PSC to promote fibrosis in the pancreas (55,56). Concurrently, elevated levels of IL-1β and IL-18 proteins in chronic pancreatitis (CP) have been attributed to the direct involvement of the NLRP3 inflammasome in PSC activation both in vivo and in vitro (57,58). The subsequent promotion of pancreatic fibrosis is mediated by pathogen-associated molecular patterns (57,58).

In the search for specific cytokines in the TME, the application of scRNA-seq technology to tumor-infiltrating lymphocytes revealed specifically high expression of IL-18 and its receptor (59). High expression of IL-18 in the stroma is associated with poor prognosis in patients (60). One function of IL-18 is to stimulate the production of IFNγ by natural killer (NK) cells and Th1 cells and synergize with IL-12 to enhance cytotoxicity against tumor cells (53,54). Therefore, IL-18 has been used in tumor immunotherapy, however, this approach has failed in phase II clinical trials (61). This raises the question of why IL-18 is ineffective against solid tumors. First, the Cancer Genome Atlas (TCGA) database and tissue microarrays revealed that IL-18 binding protein (IL-18BP) is more widely distributed than IL-18R in various solid tumor tissues and sera. Second, IL-18BP binds IL-18 with high affinity (1.1 pM), prevents it from binding to the receptor and reduces the IFNγ-secreting activity of IL-18 (62,63). Thus, IL-18BP is a major barrier to IL-18 immunotherapy (62). The next question was whether bypassing IL-18BP would exert an antitumor effect. A mutant decoy-resistant IL-18 (DR-18) variant, which combines with IL-18Rα but not with IL-18BP, has been screened by directed evolutionary means for its full mobilization of a variety of immune cells, including CD8+ T cells, NK cells and intratumor cell-like T cells (60). DR-18 exhibits good antitumor activity, and its efficacy alone is superior to that of anti-PD-1 monotherapy (60). In 2022, Simcha Therapeutics (60) commenced a phase I clinical trial on the safety and bioactivity of ST-067 (DR-18) in multiple solid tumor types based on this basic study. In a second study, IL-37 was shown to have high homology with IL-18 (61). IL-37 binds to IL-18Rα after maturation via caspase-1 cleavage but binds less efficiently than does IL-18 (62). However, IL-37 can act as a binder for IL-18BP and antagonize the binding of IL-18/IL-18Rα to IL-18Rβ, whereas the low-affinity dimer IL-18/IL-18Rα needs to bind to IL-18Rβ for cell signaling, thus inhibiting IL-18 innate immunity and reducing IFNγ expression (61,64). IL-18 is a promising alternative therapeutic target for pancreatitis, PanIN and pancreatic cancer.

Interleukin-33 (IL-33)

IL-33 induces a type 2 immune response (65). Following sustained stimulation by damage signals, IL-33 is rapidly released from the nucleus of cells distributed in the pretumor period into the extracellular space, where it binds to the surface receptor tumorigenicity 2 (ST2) of innate lymphoid cells 2 (ILC2s) to activate the Th2 immune response (66). PSCs were the predominant cell type in CP fibroblastic tissue (43.8% of the total cells), and human PSCs were further characterized upon activation with an immune-activated genome enriched for IL-33 and IL-11 (20). IL-33-mediated activation of ILC2s directly contributes to PSCs proliferation and activation, ultimately leading to CP fibrosis (25). The IL-33-ST2 signaling axis in cancer cells promotes type 2 immune reactions, induces an immunosuppressive microenvironment and accelerates PDAC progression in the context of chronic inflammation accompanied by fibrosis or intratumoral fungi (67,68). Conversely, genetic deletion of IL-33, ST2 or MMP-9 has been shown to significantly block tumor metastasis in mouse and human fibroblast-rich PDAC (69). Moreover, blockade of PD-1 signaling by IL-33-activated ILC2s has a direct antitumor effect, and modulation of the IL-33-PD-1 axis demonstrates a more favorable prognosis in individuals with PDAC tumors (70). Furthermore, a recent study demonstrated that IL-6+ CAFs secrete IL-33 to induce CXCL3 secretion via macrophages. Notably, CXCL3 engages with its receptor CXCR2 on CAFs to convert them into myCAFs, and this IL-33-CXCL3-CXCR2 loop eventually promotes PDAC metastasis (71).

IL-33, the most responsive chromatin-activated tumor-forming effector, cooperates with mutant KRAS to generate a specific transcriptional program for tumor formation that contributes to epigenetic remodeling of early neoplasia and tumor transformation (72). Despite being expressed in only a fraction of KRAS-mutant pancreatic epithelial cells, IL-33 resulted in marked changes in the premalignant pancreatic cellular state and subsequently prevented the transition of the plasticized progenitor-like state to the PanIN populations (73). Among the downstream targets of oncogenic genes, IL-33 is the most altered cytokine (72,73). In conclusion, IL-33 is a vital target allele in damaged pancreatic tissue, acting as an immunotherapeutic enhancer in the PDAC stage while exerting opposite proinflammatory and fibrotic effects in precancerous tissue (72). IL-33 directly facilitates TGFβ-triggered differentiation of immunosuppressive Treg cells and IFNγ production in other solid tumors, thereby reducing immunotherapy efficacy (74). IL-33 is specifically elevated in human PDACs and is positively associated with tumor immunity in human patients with PDAC (71). However, the combined clinical effects of IL-33 require further investigation.

IL-6 family

The IL-6 family comprises cytokines with a similar structure and signaling mechanism as the subunit glycoprotein 130 kDa (GP130) (75). GP130 dimers are recruited through conjugation to the non-signaling α receptor (IL-6R) to form an IL-6/IL-6R/GP130 hexamer that initiates the intracellular signaling chain (75). During PanIN-PDAC progression, resident PSCs in the ECM secrete large amounts of inflammatory cytokines IL-6 and IL-11 (76). Bazedoxifene has been shown to have effective antitumor effects on pancreatic cancer via inhibition of the IL-6 (GP130/STAT3) pathway (clinical trial NCT04812808). Specifically, the JAK/STAT pathway activates JAK proteins in cells and phosphorylates the transcription factor STAT3, which translocates to the nucleus to regulate target gene expression (77). Although JAK/STAT signaling is the primary pathway for downstream activation of the IL-6 family of cytokines, the mitogen-activated protein kinase (MAPK) pathway can also undergo activation (78). IL-6 collaborates with the oncogene KRAS to activate the reactive oxygen species detoxification program downstream of the MAPK/ERK signaling pathway (79). For instance, a synergistic therapeutic combination with the CAF inhibitor nintedanib enhances PDAC chimeric antigen receptor-NK-mediated cytotoxicity via a reduction in CAF-released IL-6 (80). Clinically high levels of IL-6 in patients with PDAC are typically associated with large tumor volumes and distant metastases (81). Moreover, patients with unresectable and systemic metastatic PDAC have high IL-6 production (82). Thus, high levels of IL-6 indicate an accurate prognosis for adverse outcomes (82). In addition, the serum marker IL-6 is superior to C-reactive protein, carcinoembryonic antigen and carbohydrate antigen 19-9 for the diagnosis and prognosis of patients with PDAC (83). These phenomena clearly demonstrate that IL-6 is the intrinsic mechanism of PDAC development, recapitulating most of the hallmarks of cancer. However, it should be noted that IL-6 can be secreted from other compartments, such as immune cells and can affect PDAC growth and progression (31). Therefore, systemic depletion of IL-6 affects CAF-independent pathways in PDAC.

The IL-6 series of cytokines and their downstream mediators contributes to the initiation of PDAC. IL-6 release by aPSCs leads to the conversion of immature myeloid cells into myeloid-derived suppressor cells (MDSCs), which then inhibit the action of CD4+ T cells, CD8+ T cells and NK cells, thus forming an immunosuppressive environment for PDAC (22). Moreover, Nagathihalli et al (84) demonstrated that IL-6 secreted by PSCs causes marked fibrosis and catheterization of pancreatic tissue and that compromising the IL-6/Stat3 axis inhibits PanIN carcinogenesis. Several clinical trials targeting the IL-6/JAK/STAT3 pathway have been conducted (77). The chimeric mouse-human antibody siltuximab is the most widely developed anti-IL-6 clinical drug, but the highly heterogeneous nature of KRAS-mutated PDAC tumors and their autocrine IL-6 status may result in the clinical ineffectiveness of siltuximab against these tumors (85). Tocilizumab is an antibody against IL-6R that inhibits IL-6 signaling to significantly reduce the growth and recurrence of primary cancer (86). An early phase clinical trial on the safety and efficacy of tocilizumab in patients with PDAC is ongoing (clinical trial NCT02767557). Ruxolitinib is a clinically useful oral inhibitor of JAK and has been shown to inhibit tumor growth in several preclinical studies in mouse models of pancreatic cancer (87). However, ruxolitinib did not improve the survival rate of patients with advanced/metastatic pancreatic disease (87). The use of STAT3 as a possible inhibitor is challenging owing to its lack of enzyme activity (88). Nevertheless, a synthetic STAT3 inhibitor compound, AZD9150, is now in a phase 2 clinical trial (NCT02983578), but its clinical outcome is not yet known. Currently, IL-6 is a critical player in all stages of PDAC and is a potential therapeutic target.

IL-10 family

The IL-10 family is classified based on structural similarity, common receptor use and downstream signaling. This family consists of nine members including IL-10 and IL-20 subfamily members IL-19, IL-20, IL-22, IL-24, IL-26, IL-28A, IL-28B and IL-29, which are categorized as type III interferons (IFNs) (89,90). All IL-10 family members preferentially bind to Janus kinase 1 and tyrosine kinase 2, and mediate differentiation through the downstream signaling JAK/STAT transcription factor pathway (89). IL-10 and IL-22 are the most widely studied family members in the pancreas (91,92).

IL-10

IL-10 cytokines are produced primarily by macrophages and T cell subsets (Th2 and Treg) in immune cells, and have been linked to the pathogenesis and development of autoimmune diseases and cancers (93). IL-10 predominates in inflammatory activity and wound recovery, releasing regenerative anti-inflammatory factors that suppress inflammation and promote favorable matrix remodeling and repair to alleviate organ impairment (94). IL-10 [source bone marrow, (BM)] knockout transplanted mice compared with wild-type mice exhibit substantially more fibrosis, inflammatory cell infiltration and BM-derived myofibroblasts, which emphasizes the crucial role of IL-10 in pancreatitis (95). However, whether IL-10 can be recovered by aPSCs requires further investigation (95).

The effect of IL-10 multipotency as an immunotherapeutic strategy has been investigated. A cetuximab-based IL-10 fusion protein exhibits powerful antitumor activity by blocking dendritic cell-mediated apoptosis of tumor-infiltrating CD8+ T cells (96). Conversely, IL-10 levels in the blood of patients with PDAC were 35-fold greater than the systemic concentrations, which may be associated with NK cells immune escape, a cytotoxic CD16hiCD57hi NK phenotype and impeded expression of cytotoxic T lymphocytes and IFNγ (91). PEGylated IL-10 treatment restored tumor-specific CD8+ T cell reactions and diminished tumor proliferation (97). However, a phase 3 trial of single pegilodecakin (PEGylated human IL-10) tumor immunotherapy revealed no evidence of improved survival in patients with PDAC (clinical trial NCT02923921). This may be the reason why IL-10 research in the field of pancreatic fibrosis has ended abruptly in recent years. However, it has also been shown that genetically engineered macrophages producing an IL-10-blocking antibody (αIL-10) can increase cancer cell death in human gastrointestinal tumors (98). A trial with a combination treatment is anticipated based on successful preclinical outcomes.

Interleukin-22 (IL-22)

IL-22 is mainly derived from CD4+ T cells (Th1, Th17 and Th22), NK cells and innate lymphoid cells, and plays an instrumental role in host defense, tissue repair and gastrointestinal tumor formation (99). The pancreas is one of the major targets of IL-22, as IL-22R is stably expressed in pancreatic epithelial cells and fibroblasts (100). IL-22 activates STAT-3 in coordination with the inflammatory response and affects the production of Reg survival genes, vascular endothelial growth factor (VEGF) and antiapoptotic protein Bcl-X (101,102). It also promotes repair and renewal signaling in impaired pancreatic vesicle cells, effectively protecting mice from induced acute pancreatitis or CP (103). However, IL-22 has not yet been investigated clinically (103,104). Conversely, with sustained increases in IL-22 expression and signaling imbalances, this protective effect can be easily manipulated into carcinogenesis (92). CD4+ T cell-derived IL-22 amplifies liver metastasis by promoting angiogenesis (105). The absence of PanIN and PDAC in IL-22 knockout mice confirms the importance of IL-22 in ductal differentiation (100). Moreover, STAT inhibitors prevent IL-22-induced ductal hyperplasia of the alveoli, the expression of stem-associated transcription factors and tumor regeneration during EMT (100). The aryl hydrocarbon receptor (AHR) in cigarette smoke dramatically increased pancreatic fibrosis in a mouse pancreatitis model (106). AHR is an area for the convergence of numerous cellular and environmental pathways, and the gut microbiota regulates IL-22 transcription and oncogenic IL-10 expression through AHR via tryptophan metabolites (107). Homologous IL-20RB includes immune-related genes in a prognostic model of pancreatic cancer, and IL-20 antagonists inhibit PD-L1 expression and prolong survival in PDAC (108,109). The IL-10 family is still a viable target in clinical trials, but it is possible that the collapse of clinical trials on IL-10 has put research projects in this category in jeopardy.

Interleukin-17 (IL-17) family

The IL-17 family comprises six subtypes of multifunctional cytokines (IL-17A to IL-17F) that perform diverse functions despite their amino acid sequence homology (110). Th17 cells preferentially produce IL-17A, IL-17F, IL-21 and IL-2 (110). IL-17A is active in epithelial cells and fibroblasts and is a characteristic hallmark of the proinflammatory cytokine Th17 cells, which participate in autoimmune, inflammatory and tumor pathogenesis, whereas IL-17F is mainly involved in mucosal host defense mechanisms (110,111). IL-17A signal-mediated tissue remodeling MMP may lead to ECM destruction and tissue lesions and is also capable of downregulating tissue inhibitors of metalloproteinases (111). IL-1β, TNFα, IL-6, IL-10 and IL-2 are typically induced following IL-17A stimulation of macrophages (112). The inflammatory and cancer paracrine factor regenerating islet-derived 3β stimulates IL-17RA, promotes cell proliferation, reduces susceptibility to apoptosis through coupling of the gp130-JAK2-pSTAT3 signaling pathway and initiates PanIN onset (113). IL-17A binds to IL-17RA/RC complexes to secrete cytokines to recruit neutrophils, which inhibits CD8+ T cells in the PDAC TME (114). Intestinal IL-17-IL-17RA signaling regulates microbes to promote barrier immunity and drive distant pancreatic tumor proliferation (115). Subsequently, IL-17A participates in the evolution of PanIN and promotes PDAC via the induction of iCAFs, which results in poor prognosis for patients (116,117). Ablation of IL-17A limits the immunosuppression of T cells to alter cytokine release by tumor fibroblasts (118). Thus, the inhibition of IL-17A may be a novel combination treatment (118). To assess the role of IL-17 therapy in tumor initiation and early tumor progression, an anti-IL-17 antibody was injected weekly to ensure inhibition of the IL-17A/IL-17RA axis throughout tumor development (119). However, there was no prolongation of overall survival in this curative model, which indicates that IL-17A may have a variable effect on PanIN and PDAC progression (119). Blocking the IL-17A/RA axis with antibodies alone is ineffective in preventing the development and progression of pancreatic cancer (119). Therefore, IL-17A is not suitable as primary monotherapy for pancreatic cancer in clinical practice. IL-17A is also a double-edged sword in the immune system (120). During tumorigenesis, IL-17A recruits MDSCs to suppress antitumor immunity, but it also activates STAT3 to induce IL-6 to promote tumor growth in vivo and upregulates oncogenes to promote tumor survival and angiogenesis (121). Additional studies are required to clarify the role of IL-17A in the progression of PanIN and PDAC.

Summary and overall conclusions

In conclusion, the interaction of PDAC with its TME components is becoming increasingly important. However, long-term fibrosis evolution and tumor immune cell imbalance result in an egregious TME (11). Currently, depletion of the ECM to remove the fibrous barrier has been proposed, but this approach has been found to be ineffective for PDAC treatment (122). Therefore, ameliorating the accumulation of the ECM in PDAC and targeting the ECM for immune cell modulation may have unexpected outcomes. Despite well-developed theories related to fibrosis or immune modulation in the pancreas, several questions remain to be addressed to translate mechanistic insights into new and effective therapeutic interventions. First, even with comprehensive combined systematic screening of fibrosis and immunomodulation with PSC and interleukins for PanIN and PDAC, there are limitations in extracting only gross information from the available study data. Utilizing scRNA-seq data for in-depth exploration of the mechanisms of PSC to CAF conversion remains a priority. Second, CAF-forming fibrosis interacts with immune cells in various ways, with the mode of action depending in part on the type of CAF under investigation. As markers for different CAF subgroups are not common to all CAF regulatory factors, personalized regimens are needed for the treatment of different stages of PDAC. Moreover, interleukin families affect the development of PDAC to varying extents, but a standard for interleukins to cause PDAC fibrosis directly by inducing PSC activation is lacking. Consequently, IL-1, IL-6, IL-10 and IL-17A are promising targets for the treatment of PDAC. Targeting the PanIN-PDAC process for immunity and fibrosis treatment is complex and requires further rigorous testing and validation of targets. The complex interactions between these elements and the development of specific therapeutic strategies require further elucidation.

Future research directions must focus on the stroma and immune cells in the TME of PDAC, both of which play critical roles in establishing structural and functional barriers to protect PDAC from external attack. Most studies investigating the interactions between PDAC and its ecotope have relied on traditional two-dimensional cell cultures, which may not accurately represent the complex three-dimensional microenvironment of PDAC in vivo and could be utilized in coculture modeling or organoid culture (123). Organoid technology has revolutionized in the field of precision medicine for treating PDAC (124). Concurrently, the emergence of high-resolution scRNA-seq technology provides an in-depth characterization of malignant cell types and increases the understanding of the heterogeneity and plasticity of PDAC in response to homeostatic and therapeutic perturbations (125).

In conclusion, pancreatic fibrosis and immunomodulation have been identified as key drivers in the development of PDAC and are major impediments to the efficacy of therapeutics for PDAC. With increasing research on PDAC-related signaling, it is anticipated that combination therapy regimens will be more successful, providing a more authoritative basis for drug development and clinical treatment, and thus improving the survival of patients with PDAC.

Acknowledgements

Not applicable.

Funding

The present study was supported by The National Natural Science Foundation of China (grant no. 82372686) and The Guangzhou Medical University Research Capacity Enhancement Program (grant no. 2024SRP192).

Availability of data and materials

The survival data of patients with pancreatic cancer generated in the present study may be found in the gene expression profiling interactive analysis 2 (GEPIA 2) using data from The Cancer Genome Atlas (http://gepia2.cancer-pku.cn/#survival).

Authors' contributions

HL and DL wrote the manuscript; KL acquired the data, YW and GZ analyzed and interpreted the data included in the review. KL, YW and GZ also contributed to the study design. LQ and KX conceived the original idea, corrected and finalized the manuscript and contributed to critical discussion. All authors read and approved the final version of the manuscript and checked and confirmed the authenticity of all the raw data.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References