Pancreatic cancer (PC) ranks the third leading cause of cancer-related death in the developed countries and is expected to be the second-leading cause of cancer-related mortality by 2040 (1, 2). Interest in PC has increased owing to its increasing annual incidence and high mortality (3). Currently, surgery is the main curative treatment for patients with PC. However, less than 20% of patients are suitable for operation at diagnosis (4). Furthermore, even in cases where surgery is suitable, about 75% of them will experience recurrence within 2 years (5), and the 5-year overall survival (OS) rate remains only less than 10% (6). For patients with recurrent or advanced tumors, other treatments, including chemotherapy, radiotherapy, and immune therapies, have shown limited efficacy. Therefore, it is necessary to determine and implement the new treatment strategies that benefit more patients.

Aminooctylamine (ANO1) is a calcium-activated chloride channel protein (7) which serves the functions such as smooth muscle contraction, the epithelial ion transport and glandular secretion (8, 9). In studies to date that have shown that ANO1 is overexpressed in multiple diseases and tumor types and contributes to the process of carcinogenesis (10). For example, in esophageal squamous cell carcinoma, both mRNA and protein levels of ANO1 were upregulated and correlated with an unfavorable clinical prognosis (11). Additionally, ANO1 is highly expressed in breast, ovarian, and hepatocellular carcinomas and promotes the malignant biological behavior of cancer cells (12–14). Although one research showed that ANO1 was overexpressed in PC (15), the prognostic value and specific role of ANO1 in PC progression remain unclear, and its association with the tumor microenvironment (TME) has not been explored.

TME play a crucial role in regulating cancer cells progression. This topic has received increasing appreciation recently. The pancreatic TME contains multiple fibroblasts and heterogeneous immune cell populations with inhibitory effects (16). Cancer-associated fibroblasts (CAFs) are a heterogeneous group consisting of myofibroblast CAFs (myCAFs), inflammatory CAFs (iCAFs), and antigen-presenting CAFs (17). MyCAFs are located near tumor cells and is considered to be the cause of the extracellular matrix deposition. iCAFs tend to dominate the space between islands of PC cells and release inflammatory cytokines such as interleukin-6 (IL-6) (18). Fibroblast activation protein (FAP) and alpha smooth muscle actin (α-SMA) are markers of myCAFs and iCAFs, respectively. Recent studies have shown that targeted FAP-positive CAFs may inhibit tumor growth (19–21). In addition, the absence of FAP in the stromal cells delayed the progression of PC disease in mice (19). α-SMA-positive CAFs have tumor-inhibiting effects in a genetically engineered mouse model of PC and contribute to the differentiate of tumor-infiltrating T cells (22). Positive α-SMA was associated with increased OS, whereas positive FAP was associated with decreased OS (23). Tumor-infiltrating lymphocytes (TILs) represent the immune response to tumor progression, and they are another important component of TME (24). CD8 and FOXP3 play important but opposite roles in cancer and are two specific subgroups of TILs (21). CD8-positive TILs are key immune cells in tumor immunity that kill cancer cells by triggering apoptosis, while foxp3 positive TILs are regulatory T cells (Tregs) with immunosuppressive functions that inhibit the proliferation and activation of CD8-positive TILs (25). A recent study found that IL-6 secreted by α-SMA positive CAF conferred chemotherapy resistance and negatively regulated T cells in the TME (26). In addition, growth factors and inflammatory cytokines secreted by CAFs activate oncogenic pathways in cancer cells. Interestingly, IL-6 and epidermal growth factor promote the expression of ANO1 by signal transducer and transcriptional activator 3 and phosphatidylinositol 3 kinase/protein kinase B signaling (27). This suggests that ANO1 levels can be regulated in cancer cells by cytokines, which may be the molecular mechanism by which ANO1 is involved in inflammation and tumor immunity. However, whether ANO1 regulates TIL and CAF populations in PC is unclear and requires further investigation.

Therefore, the aims of this study were to probe into the prognostic value of ANO1 in PC patients and explore the relationship between ANO1 expression and TIL and CAF populations.

Multiple studies have identified the effects of ANO1 on tumor cells in various types of cancer,however, its effects on PC and the TME of PC have rarely been studied. Our results indicate that ANO1 positivity is an independent risk factor for PC. Further, ANO1 expression correlated positively with accumulation of FAP-positive CAFs. ANO1 high expression was significantly correlated with a lower number of CD8-positive TILs and possibly correlated with a high number of FOXP3-positive TILs. Because increased FAP-positive CAFs and decreased CD8-positive TILs are associated with an immunosuppressive environment in PC, ANO1 may be a key regulatory factor of the TME in PC. A bioinformatics analysis of the TCGA dataset showed the same results. Furthermore, the possible mechanism by which ANO1 affects the TME was investigated, and the results indicated a paracrine mechanism was involved. To the best of our knowledge, this study is the first to identify the prognostic role of ANO1 and its effect on the TME in PC.

The most specific hallmark of PC is remarkable desmoplasia/fibrosis, which accounts for 80% of the tumors and infiltrating immunosuppressive cells (28). CAFs, including activated pancreatic stellate cells, and their deposition in the extracellular matrix influence tumor progression, metastasis, therapeutic resistance, and angiogenesis (29). CAFs play a key role in tumor promotion through paracrine signaling and direct physical interactions (30). Recent lineage-tracing studies on pancreatic stellate cells have shown that native pancreatic fibroblast populations differ in their ability to expand during carcinogenesis (31, 32). CAFs with opposing functions in PC progression have been identified through single-cell RNA sequencing, multiple immunostaining, and various genetic mouse models (21). myCAFs and iCAFs play the most important functional roles and express FAP and α-SMA, respectively. A previous study showed that depletion of FAP-positive CAFs leads to increase survival rate, while the depletion of α-SMA-positive CAFs, which leads to decrease survival rate (16). PCs tend to have little CD8-positive T cell infiltration but are readily infiltrated by multiple types of immunosuppressive cells, such as FOXP3-positive Tregs, myeloid suppressor cells, and macrophages (33). previous studies have shown that increased numbers of CD8-positive TILs are associated with better survivalin patients with colorectal and ovarian cancer, whereas an increase in the number of Tregs is linked with poor survival in patients with PC (24, 34). Notably, CAFs have a clear relationship with immune cells. FAP-positive CAFs and α-SMA-positive CAFs can regulate both tumor-related pathways and Treg accumulation. Inhibition of α-SMA-positive CAFs can reduce the Teff/Treg ratio and promote tumor growth (35). Conversely, inhibition of FAP-positive CAFs decreases CD11b-positive myeloid cells and reduces PC growth (20, 36). Previous studies have shown that the general functional contribution of α-SMA-positive CAFs is tumor suppression, which is partly mediated by its production of type I collagen and IL-6 (37). However, this IL-6 production does not promote the progression of PC, and we observed an approximately 50% decrease in IL-6 in the TME (16). FAP-positive CAFs inhibit T cell infiltration into the tumor through CXCL12, and depletion of these fibroblasts or targeting of CXCL12 increases the sensitivity to immune checkpoint blockade of PC in preclinical models (38). Interestingly, although CD4-positive T cell ablation enables CD8-positive T cell-mediated deterioration of pancreatic tumors in mice, consumption of FOXP3-positive Tregs accelerates tumorigenesis as a result of compensatory myeloid infiltration (39). Our study found that ANO1 expression positively correlated with FAP-positive CAFs and negatively correlated with CD8-positive TILs, suggesting that ANO1 may be a key regulator of the tumor immune microenvironment.

Ion channel dysfunction is an emerging field in cancer research, and the differential expression of several types of ion channels in cancer have been reported (15, 26, 40). As a calcium-activated chloride channel, ANO1 can increase vascular smooth muscle contractility and can be a promising target for treating hypertension (41). The expression of ANO1 is influenced by multiple molecular mechanisms (42). Recent studies have revealed that ANO1 expression is controlled by 11q13 gene amplification and that ANO1 may play a cell-specific role through its interconnected protein network, phosphorylation of different kinases, and signaling pathways (11). We found that ANO1 is highly expressed in various malignancies and is associated with poor survival outcomes, which highlight its potential as a biomarker for the detection of certain malignancies. ANO1 not only inhibits tumor apoptosis but also promotes tumor immune escape. Recent researches have revealed that ANO1 overexpression promotes the progression of lung cancer, head and neck squamous cell carcinoma, and breast cancer through the epidermal growth factor receptor signaling pathway (43, 44). Specifically, in breast cancer cells, ANO1, epidermal growth factor receptor, and signal transducer and transcriptional activator 3 form a positive feedback pathway that promotes cancer cell proliferation and growth (45). ANO1 also activates the Ras-Raf-MEK-ERK1/2 signaling pathway, contributing to the development of head and neck squamous cell carcinoma and colon cancer (45). In esophageal squamous cell carcinoma, ANO1 regulates the tumor growth factor-B signaling pathway and promotes proliferation, migration, and invasion (46). In addition, ANO1 promotes metastasis of liver cancer and ovarian cancer by activating the phosphatidylinositol 3 kinase/protein kinase B signaling pathway (13, 47). However, the relationship between ANO1 and the TME has attracted increasing attention. Research have revealed that ANO1 is participate in immune escape in digestive system-related tumors (48). In one study, ANO1-expressing esophageal squamous cell carcinoma cells activated NFκB signaling in fibroblasts, stimulating CCL1 production, and subsequently enhance invasion (49). In gastrointestinal stromal tumors, the expression of ANO1 was significantly negatively correlated with infiltration of plasma cells and memory-activating CD4-positive T cells, suggesting that ANO1 may be participate in the functional suppression of T cells (50). ANO1 also conduces to immunosuppression of TME and induces acquired resistance to anti-PD-1 immunotherapy, while knockdown or inhibition of ANO1 enhances immunotherapy efficacy and overcomes resistance to immunotherapy. From the mechanism, ANO1 inhibits ferroptosis of cancer in a phosphatidylinositol 3 kinase/protein kinase B signaling-dependent manner, promotes tumor progression by promoting tumor growth factor-B release, and promotes the recruitment of CAFs, thereby anti-tumor immunity mediated by CD8-positive T cells is weakened and resistance to immunotherapy is generated (51). However, the role of ANO1 in PC has rarely been investigated. We speculated that ANO1 might regulate the TME through regulating the infiltration of FAP-positive CAFs and CD8-positive TILs in a paracrine manner. Through bioinformatics analysis of the TCGA dataset, we found that ANO1 may regulate the TME of PC through cytokine signaling, interleukin signaling, and cytokine-cytokine receptor interaction signaling pathways; however, the specific mechanisms remain to be further studied.

Our study has certain limitations. First. this was a single-center study, and the sample size limited the validity of our results. Therefore, a larger number of cases and mechanistic studies are required to validate our results. Second, to our knowledge, this is the first research to report that ANO1 is positively associated with CD8-positive TILs and FAP-positive CAFs in PC. However, our analysis used only immunohistochemistry, Therefore, this phenomenon needs to be further confirmed in vivo and in vitro.

In conclusion, ANO1 is an important prognostic factor for patients with PC after radical resection. ANO1 may induce an immunosuppressive tumor microenvironment in PC cells in a paracrine manner; therefore, ANO1 may be a new therapeutic target in PC.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving humans were approved by Institutional Review Committee of the Affiliated Hospital of North Sichuan Medical College (number: 2023ER041-1). The studies were conducted in accordance with the local legislation and institutional requirements. The human samples used in this study were acquired from primarily isolated as part of your previous study for which ethical approval was obtained. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and institutional requirements.

GZ: Conceptualization, Data curation, Methodology, Writing – original draft. ZS: Data curation, Formal analysis, Methodology, Writing – original draft. JY: Writing – original draft. JL: Writing – original draft. PY: Writing – original draft. BW: Writing – original draft. DD: Writing – original draft. SY: Writing – original draft. YL: Writing – original draft. DR: Writing – original draft. YH: Writing – review & editing. CL: Writing – original draft, Writing – review & editing.