Metformin reshapes the tumor microenvironment and enhances prognosis in invasive pancreatic ductal adenocarcinoma with diabetes mellitus

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is an increasingly prevalent malignancy and ranking as the 7th leading cause of cancer death worldwide (1). Diabetes mellitus (DM) is a well-established risk factor for pancreatic cancer, as well as for various other malignancies. Long-standing diabetes (>3 years) has been associated with a 1.5- to 2.4-fold increased risk of pancreatic cancer (2,3). Conversely, accumulating evidence suggests that pancreatic cancer itself can induce β-cell dysfunction, peripheral insulin resistance, and subsequent diabetes. These metabolic alterations, often presenting as new-onset diabetes, may serve as an early diagnostic marker for pancreatic cancer (4), underscoring the close interplay between pancreatic cancer and diabetes.

Despite advancements in surgical and systemic therapies, the prognosis of PDAC remains poor, with a 5-year postoperative survival rate of only 20–40% (5). Among these patients, those with DM have been reported to have an especially poor prognosis. In recent years, metformin, a widely used first-line treatment for type 2 DM, has attracted increasing attention for its potential anti-cancer properties (6). Epidemiological studies have shown that metformin reduces the incidence of various malignancies, including PDAC, in patients with type 2 DM (7-10). Moreover, a number of clinical studies have suggested that metformin use may improve the survival of cancer patients, although the magnitude of this effect appears to vary by cancer type (11,12). In the context of PDAC, several retrospective studies have reported an association between metformin use and improved survival outcomes (13-16). However, conflicting results have also been reported, likely due to heterogeneity in study design, treatment regimens, and patient selection criteria (17,18).

In preclinical models, metformin has been shown to suppress tumor growth and prolong survival in multiple murine models of PDAC (19-21). While metformin’s anti-tumor mechanisms are not yet fully elucidated, previous studies have demonstrated that it can directly inhibit tumor cell proliferation and survival via both AMP-activated protein kinase (AMPK)-dependent and AMPK-independent pathways (22,23). More recently, growing evidence suggests that metformin’s anti-tumor efficacy may be mediated, at least in part, by modulation of the host immune response, thereby enhancing anti-tumor immunity (24,25).

However, the impact of oral metformin intake on prognosis and the tumor immune microenvironment (TIME), particularly its effects on intratumoral T lymphocytes, remains incompletely understood in PDAC patients. To address this gap, we investigated retrospectively survival outcomes and T cell infiltration patterns in pancreatic cancer patients with diabetes who underwent curative resection at our institution, comparing those who received metformin with those who did not. We present this article in accordance with the REMARK reporting checklist (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-457/rc).

Methods

Patients

This retrospective study included 143 patients who underwent surgery for invasive PDAC at Jichi Medical University Hospital between April 2010 and March 2022. Patients with distant metastases or multiple organ resections were excluded. Clinical data—including surgical procedures, medical history, medication use, preoperative test results, pathological findings, and outcomes—were retrieved from electronic medical records. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethical Committee of Jichi Medical University Hospital (approval No. Clinic A21-064). Owing to the anonymous nature of the data, the requirement for written informed consent was waived.

Reagents

The mAbs targeting CD3 (clone SP7: MA5-14524) were obtained from Invitrogen (Thermo Fisher Scientific, MA, USA), the mAbs targeting CD8α (clone 1G2B10) from Proteintech (Rosemont, USA) and additional reagents for immunohistochemistry (IHC) such as Signal Enhancer HIKARI, antibody dilution buffer and blocking Solution One Histo from Nacalai Tesque (Kyoto, Japan).

Histopathology, multiple IHC, and image processing

Surgical specimens were fixed in formalin, embedded in paraffin, and sectioned into 4-µm-thick slices for hematoxylin and eosin (HE) staining and IHC. The pathological response in tumors which received neoadjuvant treatment was determined according to College of American Pathologists Classification (26). Multiplex immunostaining and image processing were performed as previously described (27). Briefly, after deparaffinization, tissue sections were stained with hematoxylin for 1 minute and scanned using the OlyVIA^® SLIDEVIEW VS200 (Evident Corporation, Tokyo, Japan). Endogenous peroxidase activity was blocked by immersing the sections in a 0.3% hydrogen peroxide solution for 30 minutes. Antigen retrieval and hematoxylin removal were achieved by immersing the sections in 10 mM sodium citrate buffer and heating them in a microwave oven for 10 minutes. To prevent non-specific binding, a blocking agent (Blocking One Histo, Nacalai Tesque, Kyoto, Japan) was applied.

The sections were then incubated with a primary antibody against CD3 (1:150 dilution) at room temperature for 60 minutes. Following washing, the sections were treated with a rabbit-specific kit from the Histofine Simple Stain PO (M) (Nichirei Bioscience Inc., Tokyo, Japan). T cells were visualized using the ImmPACT^® AMEC Red Substrate Kit (Vector Laboratories, Newark, CA, USA), enclosed in VectaMount AQ aqueous mounting medium (Vector Laboratories, Newark, CA, USA), and subsequently scanned. The sections were then dehydrated in an alcohol gradient for 2 minutes to remove the 3-amino-9-ethylcarbazole (AEC) reaction product. After rehydration, they were incubated in 10 mM sodium citrate buffer in a microwave oven for 10 minutes to remove the primary antibody. Subsequently, the sections were stained with a primary antibody against CD8α (1:2,000 dilution).

Digitized images were iteratively co-registered using the CellProfiler version 2.1.1 pipeline “Alignment_Batch.cppipe” (General Public License version 2.0) to ensure precise pixel-level alignment. For visualization, co-registered images were converted into pseudo-colored single-marker images and combined using ImageJ Fiji (Bethesda, Maryland, USA, National Institutes of Health). Tumor-infiltrating T cell density was quantified by measuring the number of CD3-positive and CD8-positive T cells per square millimeter at the tumor margin, based on the average of three randomly selected areas.

Statistical analyses

Statistical analyses of clinicopathological data were performed using EZR (28). Differences in clinical and pathological factors were evaluated using the Mann-Whitney U test or Fisher’s exact test. Recurrence-free survival (RFS) and overall survival (OS) were assessed using the Kaplan-Meier method, with significance determined by the log-rank test. Statistical analysis of IHC data was conducted using GraphPad Prism 9. A P value <0.05 was considered statistically significant for all analyses.

Results

Patient characteristics

Among the 143 patients who underwent curative resection for PDAC, 65 (45.5%) were diagnosed with DM at the time of surgery (Table 1); 65 patients (45.5%) received neoadjuvant therapy prior to surgery. For patients with resectable PDAC, radiotherapy combined with S-1 was administered in 27 cases prior to 2019. Since 2019, two courses of gemcitabine plus S-1 (GS) therapy have been employed in 32 cases, in accordance with the results of the Prep-02/JSAP05 study (29). Patients with borderline resectable PDAC received either gemcitabine plus nab-paclitaxel (GnP) or FOLFIRINOX (GnP: 5 cases, FOLFIRINOX: 1 case). Adjuvant chemotherapy, consisting of oral S-1 within 10 weeks or gemcitabine administration, was provided to 115 patients (80.4%). The proportion of patients receiving adjuvant chemotherapy was significantly lower in the diabetic group compared to the non-diabetic group (72.3% vs. 87.1%, P=0.03). However, no significant differences were observed in other clinical characteristics between the two groups. Pathological factors were analyzed separately based on receipt of neoadjuvant therapy, and no significant differences were identified between diabetic and non-diabetic patients in either subgroup.

The impact of metformin intake on patient outcome

Among the 65 diabetic patients, there was a trend toward shorter RFS and OS compared to non-diabetic patients; however, these differences did not reach statistical significance (Figure 1). Within the diabetic cohort, 17 patients (26.1%) were receiving metformin at the time of surgery (Table 2). No significant differences in clinical characteristics were observed between the metformin group and the remaining 48 diabetic patients not receiving metformin.

Figure 1 The impact of diabetes on the survival of PDAC patients. (A) Recurrence-free and (B) overall survival curve; red curve: patients with diabetes, blue curve: patients without diabetes mellitus. DM, diabetes mellitus; HR, hazard ratio; OS, overall survival; PDAC, pancreatic ductal adenocarcinoma; RFS, recurrence-free survival.

Pathological analysis was stratified based on whether patients had undergone neoadjuvant therapy. Among patients who underwent upfront surgery, no significant differences were noted between metformin users and non-users. However, among the 33 patients who received neoadjuvant therapy, the metformin group exhibited a lower incidence of perineural invasion compared to non-users (35.2% vs. 47.9%, P=0.050). Additionally, the metformin group showed a trend toward a higher pathological response rate to neoadjuvant chemotherapy, although this difference did not reach statistical significance (23.5% vs. 8.3%, P=0.17).

In diabetic patients receiving metformin demonstrated improved survival outcomes, with significantly better RFS [hazard ratio (HR) =0.46, P=0.03] and OS (HR =0.30, P=0.01) (Figure 2). This survival benefit was particularly evident in patients who underwent upfront surgery (RFS: HR =0.29, P=0.009; OS: HR =0.2,8 P=0.02). In contrast, no significant differences were observed among those who received neoadjuvant therapy, including neoadjuvant chemoradiotherapy (NACRT) and NAC-GS (Figure 3).

Figure 2 The impact of metformin on the survival of PDAC patients with diabetes. (A) Recurrence-free and (B) overall survival curve; red curve: non-metformin group, blue curve: metformin group. HR, hazard ratio; OS, overall survival; PDAC, pancreatic ductal adenocarcinoma; RFS, recurrence-free survival.

Figure 3 The impact of metformin on the survival was analyzed in the patients who received upfront surgery and those who receive neoadjuvant therapy, respectively. (A) Recurrence-free and (B) overall survival in patients who received upfront surgery; red curve: non-metformin group, blue curve: metformin group. (C) Recurrence-free and (D) overall survival in patients who received neoadjuvant therapy. HR, hazard ratio; OS, overall survival; RFS, recurrence-free survival.

In univariate analysis, metformin use was associated with improved RFS (HR =0.42, P=0.03) and remained an independent prognostic factor in multivariate analysis (HR =0.40, P=0.02), along with lymph node metastasis and adjuvant chemotherapy. Similarly, metformin use was a prognostic factor for OS in univariate analysis (HR =0.237, P=0.02) and remained an independent factor in multivariate analysis [HR =0.206, 95% confidence interval (CI): 0.054–0.776, P=0.02], together with age >71 years, lymph node metastasis, and adjuvant chemotherapy (Table 3).

The impact of metformin on the TIME

We analyzed the density of CD3(+) and/or CD8(+) T cells, as well as the CD8/CD3 ratio within tumor tissues, using IHC. This analysis was conducted in 17 patients from the metformin group and 17 propensity score-matched patients from the non-metformin group to minimize the influence of confounding factors (Table 4). Representative images of HE and multiplex IHC are shown in Figure 4A-4F. In these images, CD3-positive and CD8-positive T cells are stained red and green, respectively. Consequently, CD3(+)CD8(−) T cells appear red, while CD3(+)CD8(+) T cells appear yellow. These T cells were predominantly localized around the tumor periphery.

Figure 4 The impact of metformin on the intra-tumoral infiltration of T cells. Representative images of HE staining (A,B) and multiplex immunohistochemical staining (C-F) of tumor infiltrating CD3⁺ (red) and CD8⁺ (green) T cells at the invasive front of PDAC. (A,C,E) Metformin group, (B,D,F) non-metformin group. The density of CD3-positive cells (G) and CD8-positive cells (H), and the percentage of CD8-positive cells to CD3-positive cells were compared between metformin group (red squared dot) and non-metformin group (blue round dot) (I). Those were compared in those who received upfront surgery (J-L) and in those who received neoadjuvant therapy (M-O), respectively. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant. HE, hematoxylin and eosin; PDAC, pancreatic ductal adenocarcinoma.

The density of total CD3(+) T cells was significantly higher in the metformin group compared to the non-metformin group {median =1,087 [453–1,783] vs. 667 [391–987] cells/mm², P<0.001}. Furthermore, the density of CD3(+)CD8(+) T cells was more markedly elevated in the metformin group {median =538 [279–1,079] vs. 295 [195–481] cells/mm², P<0.001}, accompanied by a significantly increased CD8/CD3 ratio (58.8% vs. 45.1%, P<0.001) (Figure 4G-4I). These elevations in metformin group were kept when confined to upfront resected PDAC {CD3(+) T cells: median =1,074 [736.3–1,438] vs. 558.3 [390.7–870] cells/mm², P=0.003, CD3(+)CD8(+) T cells: median =581.2 [485.7–904] vs. 247.7 [184.3–380] cells/mm², P<0.001, CD8/CD3 ratio: 61.7% (47.4–73.7%) vs. 43.4% (35.9–51.6%), P=0.003} (Figure 4J-4L). In those who underwent neoadjuvant therapy, significant differences were not observed on the density of CD3(+) T cells {median =1087 [452.7–1,783] vs. 695.3 [392–987.3] cells/mm², P=0.15}, while the density of CD3(+) CD8(+) T cells and CD3/CD8 ratio were higher in metformin group {CD3(+) CD8(+) T cells: median =434.3 [279.3–1,080] vs. 309.7 [195.7–481.7] cells/mm², P=0.013, CD8/CD3 ratio: 60.4% (38.3–64.0%) vs. 46.0% (31.0–58.9%), P=0.01} (Figure 4M-4O).

Subsequently, the impact of metformin intake on the intratumoral myeloid cells was analyzed. Representative images are shown in Figure 5A-5L. Metformin intake significantly suppressed the intratumoral infiltration of neutrophils as a whole {Figure 5M, median =126.7 [32–1,008] vs. 417.3 [61.3–1,561] cells/mm², P=0.02} and when confined to those who received neoadjuvant therapy {Figure 5N, median =90.7 [32–568] vs. 417.3 [77.3–1,561] cells/mm², P=0.03}. This trend was seen in patients who underwent upfront surgery, although it was not statistically significant {Figure 5O, median =244.7 [42.7–1,009] vs. 727.3 [61.3–1,215] cells/mm², P=0.18}. Intratumoral neutrophils sparsely formed MPO(+)citH3(+) neutrophil extracellular traps (NETs). Metformin intake did not have significant impact on NETs formation (Figure 5P-5R), intratumoral infiltration of CD68(+) macrophage (Figure 5P) or that of CD163(+) M2 macrophage (Figure 5S-5V), regardless of neoadjuvant therapy.

Figure 5 The impact of metformin on the intra-tumoral infiltration of myeloid cells. Representative images of HE staining (A,B,G,H) and multiplex immunohistochemical staining of neutrophils [C-F; hematoxylin (blue), citH3 (red), and MPO (green)], and macrophages [I-L; hematoxylin (blue), CD163 (red), CD68 (green)] at the invasive front of PDAC. (A,C,E,G,I,K) Metformin group, (B,D,F,H,J,L) non-metformin group. The density of MPO-positive cells (M-O) and citH3-positive cells (P-R) were compared between metformin group (red squared dot) and non-metformin group (blue round dot). Those were compared in those who received neoadjuvant therapy (N,Q) and in those who did not (O,R), respectively. The density of CD68-positive cells (S) and CD163-positive cells (T-V) were compared between metformin group and non-metformin group. CD163-positive cells were compared in those who received neoadjuvant therapy (U) and in those who did not (V), respectively. *, P<0.05; ns, not significant. HE, hematoxylin and eosin; MPO, myeloperoxidase; NETs, neutrophil extracellular traps; PDAC, pancreatic ductal adenocarcinoma.

Discussion

Epidemiological studies have suggested a positive association between metformin use and improved survival in diabetic patients with PDAC, although outcomes appear to be influenced by tumor stage, treatment modality, concomitant medications, and other confounding factors (23). In the present cohort, we evaluated the clinical outcomes of diabetic patients who underwent curative resection for PDAC and found that both RFS and OS were significantly improved in the metformin group compared to the non-metformin group. Multivariate analysis further identified metformin use as an independent prognostic factor for favorable outcomes. These findings are consistent with those of Jang et al., who reported that among 764 patients undergoing surgical resection for PDAC, metformin users exhibited improved cancer-specific survival in a dose-dependent manner (14). Collectively, these data strongly suggest that metformin use may reduce mortality in patients with resectable pancreatic cancer and pre-existing diabetes.

More importantly, immunohistochemical analysis demonstrated that the number of tumor-infiltrating lymphocytes (TILs), particularly CD8(+) T cells, was significantly higher in tumors from patients in the metformin group than in those from non-users. Recent studies have highlighted metformin’s immunomodulatory effects in both preclinical and clinical settings. For example, Eikawa et al. demonstrated that metformin enhances the infiltration and cytotoxic activity of CD8(+) T cells in murine tumor models (30). Finisguerra et al. reported that metformin prevents CD8(+) T cell apoptosis in hypoxic and immunosuppressive tumor microenvironments, potentiating the efficacy of chemotherapy when combined with cyclophosphamide (31). Crist et al. further showed that metformin enhances natural killer (NK) cell-mediated cytotoxicity in patients with head and neck squamous cell carcinoma (32). Additionally, increased CD8(+) TIL density has been observed in metformin-treated patients with various malignancies, including head and neck squamous cell carcinoma (33), non-small cell lung cell tumors (34), colorectal cancer (35) and breast cancer (36). These findings are in concordance with our observations and support the hypothesis that metformin may help reprogram the immunosuppressive TIME into an immune-active state, thereby contributing to improved clinical outcomes in PDAC.

Intriguingly, the survival benefit of metformin was observed exclusively in patients who underwent upfront surgery, whereas no significant effect was noted in those who received neoadjuvant therapy. In parallel, the density of CD8(+) TILs was less pronounced in tumors from patients in the neoadjuvant group. These findings align with a recent study by van Eijck et al., who reported that metformin significantly improved survival in 82 patients with PDAC who underwent upfront surgery, but not in 66 patients treated with gemcitabine-based NACRT (37). Their immuno-profiling of resected tumors revealed that metformin-treated tumors downregulated genes associated with pro-tumoral immunity only in the upfront surgery cohort. Our findings are totally consistent with theirs and suggest that the clinical benefit of metformin may be mediated by its ability to modulate the immune microenvironment, which may be altered or masked following neoadjuvant therapy.

This study has several limitations, including a relatively small sample size of metformin users, the retrospective design, and the single-institution setting. Despite these limitations, our findings are in line with previous reports and support the hypothesis that modulation of immune cell infiltration within the tumor microenvironment is a key mechanism by which metformin improves outcomes in PDAC. Further prospective studies are warranted to validate these findings and to elucidate the broader impact of metformin on host anti-tumor immunity, including its effects on T cell exhaustion and myeloid cell differentiation within the TIME.

Conclusions

Metformin may enhance the infiltration of cytotoxic CD8(+) T cells into the tumor microenvironment and improve the prognosis of diabetic patients with PDAC, particularly in those undergoing upfront surgical resection.

Acknowledgments

We thank J. Shinohara, H. Hatakeyama, N. Nishiaki, and I. Nieda for their technical and clerical contributions.

Footnote

Reporting Checklist: The authors have completed the REMARK reporting checklist. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-457/rc

Data Sharing Statement: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-457/dss

Peer Review File: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-457/prf

Funding: This study was supported by the Japan Society for the Promotion of Science (No. 23K24411).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-457/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethical Committee of Jichi Medical University Hospital (approval No. Clinic A21-064). Owing to the anonymous nature of the data, the requirement for written informed consent was waived.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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