Correlation between Macrophage Polarization and PD-L1-Related Tumor Microenvironmental Alteration and Metastasis in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with a poor prognosis; nearly 80% patients have regional or distant metastasis when diagnosed. Tumor microenvironment (TME) alteration and epithelial-to-mesenchymal transition (EMT) have been reported to play a key role in cancer metastasis. However, the correlation between TME and EMT was poorly studied in PDAC. This study aims to explore the correlation between EMT markers and TME alteration, mainly including macrophage polarization and PD-L1 expression change, in primary and metastatic PDAC tissues by immunohistochemistry. The results indicated that macrophage polarization was found in metastases with the number of M1 macrophages (CD86+) decreased and M2 (CD163+) increased, though PD-L1 expression did not have a significant alteration. Compared to primary tumors, E-cadherin was significantly lower, while snail was higher, while no difference was found with N-cadherin and ZEB1. Correlation analysis indicated that snail, but not ZEB1, E-cadherin, or N-cadherin, was highly correlated with macrophage polarization. To conclude, the number of CD86+ M1 macrophages was increased while CD163+ M2 macrophages decreased in metastases, with no significant alteration of PD-L1 expression compared to primary tumors. EMT markers—Snail and E-cadherin—but not ZEB1 or N-cadherin, were found to be higher/lower in metastases, which mean that EMT played an important role in PDAC metastasis. Further analysis indicated that snail was highly correlated with M1 to M2 macrophage polarization, which prompted that EMT may be one reason for macrophage polarization induced TME alteration in PDAC metastasis.


Introduction
Pancreatic ductal adenocarcinoma (PDAC) remains an exceedingly aggressive malignancy with increased incidence, and the 5-year survival rate is only around 11% according to the latest statistics [1]. Early metastasis is one of the main reasons for the poor prognosis. Comprehensive therapy performs a necessary position in the treatment of PDAC besides surgical resection. However, there are no extensively accepted targeted drugs, and common chemotherapeutics are still the mainstream treatment with unsatisfed results.
Metastasis is the signal of poor prognosis of malignant tumors including PDAC. Te advent of metastasis means cancer cells have to breach special immune barriers. Tumor microenvironment alteration provides an appropriate condition for cancer metastasis after a complex of regulation. Tumor microenvironment is a complex system composed of cells that evolve with tumor cells and provide support in the process of malignant transformation. Macrophage is an important group in the tumor microenvironment, which can prevent and promote tumor growth. Tumor-associated macrophages (TAMs) have been classifed into tumor-inhibiting (M1) and tumor-promoting (M2) macrophages. Te M1 subtype could be polarized into M2 under diferent conditions. Studies have indicated that macrophage polarization from M1 to M2 is correlated with poor prognosis in various types of cancer [2,3]. However, the relationship between metastasis and macrophage polarization has not been fully elucidated.
Blockade of programmed death-1 (PD-1) or programmed death ligand-1 (PD-L1) has become a new immunotherapy and achieved exciting results in some types of cancer, such as melanoma [4], nonsmall cell lung cancer [5], and hepatocellular carcinoma [6]. However, there were only sporadic case reports and clinical trials, albeit with varying efects in PDAC. Some studies have already pointed out that PD-L1 and PD-1 were diferentially expressed in primary and metastatic sites [7,8]. Unfortunately, this hypothesis has not yet been validated in PDAC, and the potential mechanisms need to be further elucidated. Tus, the efect of immune-checkpoint inhibitors (ICIs) may depend on different tumor microenvironment statuses, and targeting metastatic patients through PD-1/PD-L1 inhibition may be an efective therapeutic strategy under this circumstance.
Epithelial-to-mesenchymal (EMT) is a cellular remodeling process associated with a sequence of biological processes, including tumor growth, invasion, metastasis, and chemotherapy. It has additionally been reported that EMT involves immune suppression, evasion, and tolerance [9]. We hypothesized that the EMT and tumor microenvironment status of the primary tumor and metastatic site of PDAC were diferent. Te result might provide a theoretical foundation for the treatment of metastatic PDAC.

Patients and Specimens.
A total of 50 cases of PDAC diagnosed by pathology from January 2013 to December 2020 in the Pathology Department of the First Afliated Hospital of Dalian Medical University were selected. All patients did not receive any other anticancer treatment, such as chemotherapy or radiotherapy, before surgery. Among the 50 cases, 33 were primary tumors with paired regional metastatic lymph nodes, 9 cases were collected during surgical resection with primary tumors and oligometastases or micrometastases, which were failed to evaluate before surgery but have the probability to get radical resection after exploratory laparotomy and evaluated intraoperatively. Te remaining 8 only have hepatic metastasis through biopsy in order to reach correct diagnosis for future treatment direction. A total of 92 lesions were selected from 42 primary tumor specimens and 50 metastatic tumor specimens. Te clinicopathological parameters of patients with PDAC were collected, including gender, tumor size, degree of tumor diferentiation, neural invasion, vascular invasion, TNM stage, serum CA19-9 level, and serum CA125 level. Te degree of tumor diferentiation was graded according to the grading standard of PDAC in the 5 th edition of the "WHO Classifcation of Tumors of the Digestive System" [10]. Patients with PDAC were clinically staged according to the 8th edition of the American Joint Cancer Society (AJCC) of pancreatic cancer staging [11]. All cases were confrmed and reviewed by two senior pathologists. Tis research was approved by the Medical Ethics Committee of the First Afliated Hospital of Dalian Medical University. All specimens were collected with consent from patients or their relatives.
First, formalin-fxed and parafn-embedded tissues were cut into 4-µm sections. After being deparafnized and rehydrated, the sections were heated with EDTA antigen retrieval solution in a pressure pot for 3 min and cooled at room temperature. Ten, 3% H 2 O 2 solution was used to block endogenous peroxidase activity for 10 minutes. After washing with phosphate bufered solution (PBS), the tissue sections were incubated with specifc antibodies at an appropriate dilution ratio for 1 h at room temperature. After washing with PBS again, the tissue sections were then incubated with horseradish peroxidase-conjugated secondary antibody (PV6000D, Zhongshan Golden Bridge Biotech Co. Ltd) at room temperature for 20 min. Te isotype was used as the control. Te antigen-antibody complexes were visualized using DAB and counterstained with hematoxylin.

Evaluation of Immunochemistry.
All slides were reviewed and evaluated by senior pathologists with no clinicopathological information in a double-blinded manner. Te expression of CD86 was detected on the cell membrane of M1 macrophages. CD163 was located on the cell membrane or in the cytoplasm of M2 macrophages. In this study, we used CD86 and CD163 as markers to assess M1 and M2 macrophages distribution in primary tumors and metastases. CD86 + and CD163 + macrophages were analyzed as follows. Five most representative spots were chosen from ×100 felds per slide. Te number of positive cells was counted under high magnifcation (×400) in the tumor nest and stroma area. Te mean number of M1/M2 macrophages was calculated and recorded. Te medians were calculated, respectively, and those higher than the median were designated as the high expression group, and those lower than the median were designated as the low expression group.
Positive PD-L1 expression was designated when the membrane of tumor cells was stained in comparison with that of the negative control. Te tumor proportion score (TPS) was defned as the number of PD-L1-staining tumor cells divided by the total number of viable tumor cells multiplied by 100. Ten, the positivity of PD-L1 was defned as scores above 0.

Statistical Analysis.
Statistical analysis was performed by SPSS (Version 26, Chicago, USA). Mann-Whitney U test was used to compare the expression of CD86, CD163, PD-L1, E-cadherin, N-cadherin, Snail, and ZEB1 between the primary and metastases of PDAC since they did not ft the normal distribution. Spearman's rank correlation analysis was used to analyze the correlation between EMT (Ecadherin and Snail) and macrophage markers (CD86 and CD163) in metastases of PDAC. Te chi-square test and Fisher's exact test were used to analyze the correlation between the targeted protein expression and clinicopathological parameters in patients with PDAC. P < 0.05 was set as a signifcant diference for all statistical analyses. Table 1. Among the 42 primary tumors of PDAC, 22 cases were male and 20 were female. Te average tumor diameter was 4.1 cm. 20 cases were high to moderate diferentiation and 22 were low diferentiation. 26 cases were validated with vascular invasion (61.9%) and 41 cases with perineural invasion (97.6%). According to the 8 th AJCC TNM stage, the clinical stage was as follows: stage I (0 cases), stage II (26 cases), stage III (7 cases), and stage IV (9 cases). All the cases were defned after surgical resection. Serum CA199 level was elevated in 38 cases (90.5%), serum CA125 level was elevated in 14 cases (33.3%). 8 cases only have metastases from preoperative biopsies and were confrmed as PDAC according to immunohistochemistry and multidisciplinary diagnosis.  Table 3. Tese results demonstrated that the number of M1 macrophages decreased while M2 macrophages increased, which could be regarded as evidence for macrophage polarization during the process of PDAC metastasis. No correlation was observed between the number of CD86/CD163 and clinical parameters in our cohort (Table 4).

Correlation between PD-L1 Expression and Metastasis of
Pancreatic Ductal Adenocarcinoma. According to the tumor proportion score (TPS), the expression of PD-L1 in primary tumors ranged from 0 to 50%, while from 0 to 20% in metastases ( Figure 2). Tere was no signifcant diference between these two groups (P = 0.468) ( Table 5). Correlation analysis indicated that PD-L1 level was highly correlated with TNM stage (P = 0.031) ( Table 6), which demonstrated that the early stage of PDAC had a relatively higher-level PD-L1 score.

Correlation between EMT and Metastasis of Pancreatic
Ductal Adenocarcinoma. E-cadherin, the most commonly used epithelial marker, was signifcantly decreased in metastases (P < 0.001) (Figure 3(a)). Tree mesenchymal markers-N-cadherin, Snail, and ZEB1 were also detected in primary tumors and metastases. Results indicated that ZEB1 was nearly negative in epithelial cells but strongly positive in interstitial cells, which were not used for further analysis (Figure 3(b)). Tere was no signifcant diference of Ncadherinexpression between these two groups (Figure 3(c),         Journal of Oncology 5 P � 0.698). However, Snail expression was signifcantly higher in metastases compared to primary tumors (Figure 3(d), P < 0.001). Te above results indicated that epithelial marker Ecadherin was decreased in metastases, while mesenchymal marker Snail was upregulated, which further validated that EMT was highly associated with cancer metastasis in PDAC. Correlation analysis indicated that N-cadherin was highly related with tumor diferentiation and CA125 level, while Snail was related to clinical TNM stage. Tis was also in accordance with the current results, which show that higher mesenchymal markers are associated with aggressive phenotypes (Table 7).

Correlation between EMT and Macrophage Polarization.
In order to clarify whether EMT was correlated with macrophage polarization, we analyzed the relationship between EMT markers (E-cadherin and Snail) and the number of M2 macrophages infltration in metastases. Te results indicated that, though no clear relationship was found between Ecadherin and CD163 + cells alteration, Snail was signifcantly correlated with M2 macrophage infltration in metastases (Figure 4), which indicated that Snail may play a role in macrophage polarization in PDAC metastasis.

Discussion
One of the reasons for the poor prognosis of PDAC is the early metastasis to regional or distal organs [12]. Tus, investigation of the potential mechanisms for PDAC metastasis should assist help to come throughout the barrier of the cancer therapy. As time progresses and we learn more, researchers now not solely pay interest to the cancer cells themselves, however, additionally the interstitial cells around the cancer cells, such as fbroblasts and immune cells. Tese cells consisted a new environment supporting tumor development, which is known as the tumor microenvironment (TME). In TME, the crosstalk between most cancer cells and interstitial cells could arise many biological processes like tumor growth, metastasis, and therapeutic resistance [13].
According to the latest studies, the immune cells are doubleedged swords with the feature of each tumor promoting and suppression [14]. Te immune cells originally could play an important role in the procession of recognition, initiation of infammation, and antitumor responses in tumorigenesis. Among the immune cells, macrophages play a necessary position in the process of antitumor responses. However, there are two subtypes of macrophages. M1 macrophages have antitumor efects, while M2 macrophages could promote tumor progression by diferent regulatory mechanisms, such as infammation promotion and immune adaptation [15][16][17]. Te transition from M1 to M2 macrophages used to be named as macrophage polarization which has been validated to be associated with tumor invasion and metastasis [18]. Te hypothesis indicated that during the process of metastasis, the TME will change to create an appropriate circumstance for cancer cells colonization. Among these, macrophage polarization is an important event. Our study was consisted with this, compared to primary tumors, the number of M1 macrophages  decreased while M2 macrophages increased through immunohistochemistry validation in clinical samples, which imply macrophage polarization was once correlated with PDAC metastasis. Except tumor-associated macrophages, PD-L1 and PD-1 are additionally hot spots in cancer research. Te ligandreceptor combination could restrict the function of CD8 + T cells. Targeting PD-L1 has become an efective treatment in some solid malignancies [7,19], however, not in PDAC. One of the reasons is that PD-L1 expression is vulnerable in PDAC tissues, however, few studies have investigated its expression in metastases. Schneider et al. validated that PD-L1 expression was associated with the presence of lymph node metastasis in head and neck squamous cell carcinoma, while Wei et al. stated that the expression of PD-L1 in liver metastases was higher than in primary tumors of colorectal cancer [19]. We considered that if PD-L1 was higher in metastases, this might also provide a new theoretical approach for anti-PD-L1 therapy in advanced PDAC. In our study there was no signifcant diference of PD-L1 expression between primary tumors and metastases; however, we discovered PD-L1 is relatively higher in early PDAC patients (stage I and II); this reminded us that whether or not anti-PD-L1 may hardly have impact in late stage of PDAC due to immune suppression, which needs further investigation.
As we discussed above, TME alteration may create an appropriate environment for cancer cells metastasis. Except for TME alteration, cancer cells also adapted themselves to the new microenvironment. EMT, a vital form of cell remodeling, has been reported with cancer metastasis [20], however, whether EMT was once correlated with TME, especially macrophage polarization and PD-L1 expression, was poorly investigated in PDAC. In our study, we unexpectedly found this transformation in metastases, with Ecadherin decreased and Snail increased. We further studied the relationship between TME and EMTand found that, though there was no correlation between EMT markers and PD-L1 expression, the mesenchymal marker Snail was highly related to M2 macrophage infltration in PDAC metastasis. Tese results indicated that cell remodeling and microenvironment alteration are dynamic processes.
Our study has some limitations. First, no matter if it is EMT or tumor-associated macrophages, there are many markers; it is hard to fnish all of them, and the present biomarkers could not represent the whole group, while not only immune cells act as a role in tumorigenesis, TME is a complicated content. Second, the small size of the matched distant metastases cohorts may be another defect due to the difculty of sample collection. Tird, our study was conducted only in clinical samples, without in vivo or in vitro validation.
In summary, macrophage polarization was found in metastases, with the number of CD86 + M1 macrophages reduced and CD163 + M2 macrophage increased. E-cadherin was signifcantly lower in metastases, while mesenchymal marker Snail was higher. Correlation analysis indicated that Snail was highly related to macrophage polarization, which reminded us that TME was may be associated with EMT in PDAC metastasis.

Conclusion
Compared to primary tumors, the number of CD86 + M1 macrophages was decreased, while CD163 + M2 macrophages increased in metastases with no signifcant alteration of PD-L1 expression. EMT markers, Snail and E-cadherin were found to be higher/lower in metastases, whichmeans that EMT played an important role in PDAC metastasis. Further analysis indicated that Snail was highly correlated with M2 macrophage infltration, which prompted that EMT may be one reason for macrophage polarization associated TME alteration in PDAC metastasis.

Data Availability
Te data can be supplied with appropriate request from the corresponding author.

Ethical Approval
Tis study was approved by the Ethics Committee of the First Afliated Hospital of Dalian Medical University of Science and Technology.