Blockades Adenosine Receptor 2B Suppresses Pancreatic Adenocarcinoma Progression by Inhibiting Leukemia Inhibitory Factor Secretion from Macrophages

doi:10.21203/rs.3.rs-4829619/v1

Download PDF

Research Article

Blockades Adenosine Receptor 2B Suppresses Pancreatic Adenocarcinoma Progression by Inhibiting Leukemia Inhibitory Factor Secretion from Macrophages

https://doi.org/10.21203/rs.3.rs-4829619/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background

Pancreatic adenocarcinoma (PDAC) is a lethal disease with a five-year survival rate of less than 10%. The immunosuppressive tumor microenvironment (TME) was primarily responsible for the poor prognosis in PDAC. M2 Macrophages are a crucial cell population with pro-tumorigenic effects in response to extrinsic signals. Adenosine, a purine nucleoside catabolite of ATP, is one of the standard signals in TME that drives macrophage M2 polarization by activating adenosine receptor (ADOR). Although four types of ADOR have been reported previously, it is still unclear which receptor mediates the main pro-cancer effects in PDAC.

Methods

The conditioned medium (CM) was made by supernatants from ADOR-activation macrophages. The wound healing, trans-well, and CCK-8 assay detected the phenotypic change of pancreatic cancer cell lines PANC-1 and BxPC-3. The transcriptome sequencing was performed to screen the specific cytokine secreted from ADOR-activation macrophages. The ELISA assay was used to verify the cytokine concentration in the supernatants of ADOR-activation macrophages. The Western blot was performed to explore the expression level of proteins related to EMT, cell cycle, and cytokine. The bioinformatics analysis was utilized to find the signaling pathways modulating cytokine secretion. Immunohistochemistry (IHC) was used to calculate the IHC score of the ADOR correlated with the cytokine secretion. The Kaplan-Meier analysis was conducted to predict the prognosis of PDAC patients according to the IHC score of ADOR. The receptor antagonists were used in vivo experiments for mechanism validation.

Results

The CM promoted PANC-1 and BxPC-3 migration, invasion, and proliferation. Leukemia inhibitory factor (LIF) was the specific cytokine contained in the CM with cancer-promoting capacity based on the result of bioinformatics analysis. The activation of ADORA2B elevated the LIF concentration in the macrophage supernatants through the RAF-MEK-ERK signaling pathway. The expression ratio of ADORA2B ranks second among the four types of ADOR in PDAC. The IHC score of ADORA2B in PDAC significantly correlates with overall and disease-free survival in PDAC patients. LIF stimulated PANC-1 and BxPC-3 migration, invasion, and proliferation by connecting with the LIF receptor (LIFR) and activating the JAK-STAT signaling pathway. The ADORA2B and LIFR antagonists decreased the tumor size and number of hepatic metastatic lesions in the pancreatic orthotopic implantation model.

Conclusion

Activation of ADORA2B promotes LIF secretion from macrophages through the RAF-MEK-ERK signaling pathway. Meanwhile, the LIF secreted from macrophages promotes PDAC progression by activating the JAK-STAT signaling pathway.

PDAC was demonstrated as a lethal disease with an overall five-year survival rate that does not exceed 10%[1, 2]. Moreover, PDAC is the second cause of cancer-related death worldwide, reflecting its remarkable biological aggressiveness[3]. So far, radical resection followed by adjuvant chemotherapy has been performed as a standard treatment for PDAC patients [4, 5]. However, nearly 85% of the patients still suffer from tumor recurrence or metastasis in the five years after radical resection[6–13]. The communication between the tumor cell and tumor microenvironment (TME) was indicated to be correlated with PDAC recurrence and metastasis[14–17].

Tumor-associated macrophages, cancer-associated fibroblasts, neutrophils, lymphocytes, and myeloid-derived suppressor cells were the common cell populations in TME[18–20]. Of those, the macrophages were the earliest to infiltrate TME and acquired pro-tumoral phenotypic characteristics in response to TME signals during PDAC development[21, 22]. Therefore, blocking the specific signaling pathway in macrophages may suppress PDAC advancement. The triggering of the ADOR-related signaling pathway enhanced immune suppression and tumor proliferation[23–25]. Meanwhile, there are four types of ADOR (A1, A2A, A2B, A3) distributed on the cell membrane of macrophages[26–28]. The activation of diverse ADOR leads to alternative functional changes in macrophages. As shown in the previous study, ADORA2A and ADORA2B activation promotes the secretion of IL-10 in macrophages[28]. At the same time, ADORA1 and ADORA3 activation is intimately involved in TNF-α production of macrophages[29]. Moreover, adenosine switched macrophages into an M2-like phenotype by activating the ADORA2A receptor[30–32]. However, which receptor mediates the main pro-cancer effects in PDAC is still controversial.

The present study found that ADORA2B activation in macrophages promotes PDAC proliferation and metastasis by secreting LIF through the RAF-MEK-ERK signaling pathway.

Cell line and cell culture

The human pancreatic cancer cell lines PDACN-1 (ATCCID: CRL-1469), BxPC-3 (ATCCID: CRL-1687), and human monocytic cell line Thp-1 (ATCCID: TIB-202) were purchased from Cell Bank. The PANC-1 cell line was cultured in DMEM containing 10% fetal bovine serum (FBS) at 37℃ in humidified air with 5% CO₂. The BxPC-3 and THP-1 cell lines were cultured in RMPI 1640 containing 10% fetal bovine serum (FBS) at 37℃ in humidified air with 5% CO₂. The above cell lines have been fully authenticated and tested free of mycoplasma.

Animal experiments

15 Female BABL/c nude mice aged 4–6 weeks were utilized in the present study. All mice were born in the Huangpu Experimental Animal Center of Sun Yat-sen University Cancer Center under the care of a professional breeder. The mice were randomly assigned into five groups based on their body weight as follows: group1: no drug treatment, group2: treated with ADO, group3: treated with macrophage scavenger and ADO, group4: treated with ADORA2b antagonist and ADO, group5: treated with LIFR antagonist and ADO (n = 3 per group). On the day of surgery, BxPC-3 with luciferase was digested, washed, and resuspended in PBS. Then, the mice were anesthetized by intraperitoneal injection of 350µL mixture of ketamine (100 mg/mL) and xylazine (20 mg/mL). A median abdominal incision (1 cm) was made below the sternum by scissors after disinfection. Consequently, 2.5 × 105 BxPC-3 cells were injected into the head of the pancreas without causing injury and torsion. Finally, the abdominal incision was closed with a 2-layer suture. After ten days of housing, the mice were detected by a fluorescence imager for fluorescence intensity measurements. Dosing was initiated when the average tumor volume in each group was > 100 mm³ based on the results of fluorescence images. It should be emphasized that the mice in the group were given an intraperitoneal injection of clodronate liposomes (macrophages scavenger, 200 µL per mouse) on days − 1 and 0. The mice in the group were given an intraperitoneal injection of ADO (20mg/kg) daily. For group 3, mice were given an intraperitoneal injection of clodronate liposomes (10mg/kg) every three days and ADO (20mg/kg) daily. Mice in the group received PSB-603 (10mg/kg) every three days and ADO (20mg/kg) daily through intraperitoneal injection. In group 5, the mice were treated with EC359 (5 mg/kg) subcutaneously every three days and ADO (20mg/kg) daily by intraperitoneal injection. The mice in the group were treated with an equivalent volume of saline.

Western blot test

The cells cultured in 6 well plates were washed in 1X PBS, followed by rapid lysis on an ice box with 1X RIPA buffer (Cell Signaling Technology). The supernatant of the lysates was discarded after sonication and centrifugation at 4°C. A Nuclear Extraction Kit (Abcam) was used for nuclear lysates per the manufacturer’s protocol. Then, the protein quantification was made by Pierce™ BCA Protein Assay Kit (Thermo Fisher). 20µg of total protein was added to each lane with the loading of SDS-PAGE. After separation on the gel, proteins were transferred to a PVDF membrane. Next, the PVDF membrane was blocked in 5% milk solution and incubated with a primary antibody overnight at 4°C. Consequently, secondary conjugation was performed using HRP-conjugated anti-rabbit or anti-mouse antibodies. The membrane was imaged using the ChemiDoc Imaging System (BioRad) after development with a chemiluminescent substrate. Finally, immunoblots were quantified using ImageJ (NIH, Bethesda, MD) and graphed using Prism Software (GraphPad Software Inc.). Primary antibodies used in the present study: rabbit anti-E-cadherin antibody (WB: dil. 1:1000, Proteintech, 20874-1-AP); rabbit anti-N-cadherin antibody (WB: dil. 1:1000, Proteintech, 22018-1-AP); anti-Vimentin antibody (WB: dil. 1:1000, Proteintech, 10366-1-AP); anti-PKC antibody (WB: dil. 1:1000, Proteintech, 21991-1-AP); anti-Phospho-PKC antibody (WB: dil. 1:1000, Proteintech, 28926-1-AP); anti-MEK antibody (WB: dil. 1:1000, Proteintech, 11049-1-AP); anti-Phospho-MEK antibody (WB: dil. 1:1000, Proteintech, 28930-1-AP); anti-Phospho-ERK antibody (WB: dil. 1:1000, Proteintech, 11257-1-AP); anti-Phospho-ERK antibody (WB: dil. 1:1000, Proteintech, 28733-1-AP); anti-Snial antibody (WB: dil. 1:1000, ABclonal, A11794); anti-β-Actin antibody (WB: dil. 1:20,000, Proteintech, 66,0091-Ig); anti-α-tublin antibody (WB: dil. 1:20,000, Proteintech, 11224-1-AP); (dil., dilution).

Enzyme-linked immunosorbent assay (ELISA)

After the induction of adherents with PMA, the THP-1 cells were cultured in 10% RMPI 1640 medium for 24h. Then, the medium was changed to fresh 0% RMPI 1640, and the cells were continuously incubated with ADO for another 24 hours. Next, conditioned media (CM) was harvested, and cellular components were removed after centrifugation at 4°C. Consequently, the cytokine LIF contained in the CM was detected by commercial ELISA kits (Biosharp) for human LIF in 96-well microtiter plates.

Wound healing assay

The pancreatic cells were cultured in six-well plates until complete confluence.

Next, a cell-free zone was scratched on the cell monolayer with a 200 µ L plastic pipette tip, and the wounded cell layer was washed away by PBS twice. The images of the wound healing process were photographed with an inverted microscope immediately after the wound scratch (0h) and at 24h incubation for cells (24h). Finally, the wound healing ratio was calculated as distance (0h) / distance (24h) × 100% based on the measurement of image J.

Tran-swell assay

Eight thousand cells were seeded in the upper chamber and cultured with 200 µL non-serum culture medium. Then, 600 µL complete medium was added into the lower chamber. After being cultured for 24h, the cells on the upper chamber were fixed with 500 µL paraformaldehyde and stained with crystal violet, followed by photographed cells that penetrated through the membrane under an inverted microscope. Finally, the relative ratio of invasive cells was calculated and analyzed using image J.

CCK8 viability assay

1500 cells/well were seeded in a 96-well plate and cultured for 24h. Then, 100 µL culture medium contained with 10% CCK8 (Beyo-time, Suzhou, China) was added into each well and incubated for another two h. Then, the OD values under 452 nm were measured by a microplate reader (TECAN Spark 10 M, Shengyang, China).

Dual-luciferase reporter assay

A dual luciferase vector psi-check2 was cloned into the binding sites between circRIP2 and miR-1305, miR-1305, and Tgf- β 2. Then, the dual luciferase reporter vector and miR-1305 mimics were co-transfected into 293T cells. Finally, the cell lysates were harvested after 24h incubation, followed by the luciferase measurement with a Dual Luciferase Assay system (Promega, USA).

Macrophage Differentiation

Macrophages used in the present study were derived from THP-1 cells. THP-1 cells were transformed into MΦ macrophages by incubating with 320-nM phorbol 12-myristate 13-acetate (PMA) for 24 h.

Cell coculture model

A 6-well plate with a 0.4 mm trans-well pore membrane was used for cancer cell/macrophage coculture. Firstly, 2.0×10⁵ THP-1 cells were transformed into MΦ macrophages with stimulation of PMA in the upper chamber. Meanwhile, equal PANC-1 and BxPC-3 cells were seeded in the lower chamber. 24h After incubating, fresh medium was replaced in the upper and lower chamber, followed by added of 10µM ADO in the upper chamber. After 24h, the pancreatic cancer cells in the lower chamber were used for further functional experiments or harvested for RNA and protein exploration.

Conditioned medium from Thp-1 macrophages stimulated by ADO enhances the malignant phenotypes of PDAC cells.

To explore the effect of ADOR-activation macrophages on the pancreatic cancer cell lines, we collected the supernatant of Thp-1 macrophages activated with ADO in a concentration gradient. Then, the supernatant was mixed with a fresh medium containing 10% FBS in a 2:1 proportion to prepare the conditioned medium (CM). Subsequently, the CM was used for culturing pancreatic cancer cell lines, and the related phenotypes were tested respectively. The results showed that the migration of pancreatic cancer cell lines PANC-1 and BxPC-3 was significantly increased compared with the control group (Fig. 1A-B). Meanwhile, the invasion of PANC-1 and BxPC-3 was enhanced considerably compared with the control group (Fig. 1C-D). Furthermore, the proliferation of PANC-1 and BxPC-3 was improved compared with the control group (Fig. 1E-F). At last, the western blot results exhibited that the expression of EMT and cell-cycle-related proteins in PANC-1 and BxPC-3 was also significantly elevated compared with the control group (Fig. 1G).

ADO promotes LIF secretion from Thp-1 macrophages.

Next-generation sequencing was performed to screen for potential cytokine secreted from the ADOR-activation macrophages that promote the malignant phenotype of pancreatic cancer cells. At first, the heat and volcano map exhibited 2560 up-regulated genes and 1307 down-regulated genes (Fig. 2A). Moreover, KEGG analysis for the sequencing data showed that the cytokine-cytokine receptor interaction signaling pathway was significantly enriched in ADOR-activation macrophages (Fig. 2B). Meanwhile, the GO analysis indicated that the cytokine activity of ADOR-activation macrophages was also markedly improved (Fig. 2C). Subsequently, cytokine-related genes in ADOR-activation macrophages were screened by taking the intersection from the two gene pools of KEGG and GO enrichment previously described.

Moreover, we removed those genes with primary copy numbers less than ten to ensure the practical value of cytokine used in subsequent studies. Then, the genes were sorted by the value of the fold change. (Fig. 2D). The ranking results indicated that IL23A, CXCL5, IL1A, LIF, and CCL20 were ADOR-activation macrophages' top five most highly expressed cytokine-related genes. However, only IL23A, CXCL5, LIF, and CCL20 exhibited the expression difference between cancer and cancer-adjacent tissue in PDAC (Fig. 2E). Furthermore, only the expression of LIF and IL1A was significantly correlated with the level of ADOR2AB in PDAC (Fig. 2F). Finally, based on the analysis results of the public database, LIF was the only one simultaneously satisfying the two criteria in Fig. 2E-F.

Activation of ADORA2B promotes LIF secretion from Thp-1 macrophages through the RAF-ERK-MEK signaling pathway.

Then, we explored the change of LIF secretion through ELISA assay in Thp-1 macrophages stimulated by ADO. It was revealed that the concentration of LIF in the supernatant of Thp-1 macrophages was significantly elevated after ADO stimulation (Fig. 3A). Meanwhile, the western blot results in Thp-1 macrophages showed that the LIF expression was significantly elevated after ADO stimulation (Fig. 3B). Based on the results of previous research, the four types of adenosine receptors on the surface of Thp-1 macrophages were demonstrated to be connected with the MAPK signaling pathway. Similarly, the KEGG enrichment of the present study also exhibited that the MAPK signaling pathway was remarkedly activated (Fig. 3C). Subsequently, we confirmed the activation of the RAF-MEK-ERK signaling pathway by western blot in Thp-1 macrophages stimulated by ADO (Fig. 3D).

Moreover, combined treatment of ADO, antagonists for ADOR, and RAF were performed on the Thp-1 macrophages. The western blot results indicated that ADO activates the RAF-MEK-ERK signaling pathway and elevates the expression of LIF in Thp-1 macrophages compared to the control group. Meanwhile, the use of antagonists for ADORA2B and RAF could reverse it. On the contrary, using the antagonists for ADORA1, ADORA2A, and ADORA3 had no significant effect on the RAF-MEK-ERK signaling pathway. It also did not influence the expression of LIF in ADOR-activation macrophages. (Fig. 3E). Finally, the results of ELISA also established that only antagonists for ADORA2B and RAF reduced the LLF concentration in the supernatant of Thp-1 macrophages after ADO stimulated. In contrast, the ADORA1, ADORA2A, and ADORA3 antagonists observed no similar inhibitory effect (Fig. 3F).

ADORA2B is overexpressed in PDAC tissues and correlates with poor prognosis.

Data from TCGA was analyzed to explore the oncogenic role of different types of AODR in PDAC. It has been indicated that the expression of ADORA2A, ADORA2B, and ADORA3 in tumor tissue was significantly higher than in normal tissue (Fig. 4A). Meanwhile, the expression ratio of ADORA3 was the highest among the four types of ADOR, followed by ADORA2B, ADORA2A, and ADORA1 (Fig. 4B). However, the TCGA data showed that only the expression levels of ADORA2A and ADORA2B significantly correlated with OS and PFS in PDAC patients (Fig. 4C). Next, IHC staining for ADORA2B was performed on 100 PDAC patients who received radical resection. Moreover, the staining tissue section was further analyzed through the HALO system for H-score (Fig. 4D). Then, a matched-pairs comparison of the ADORA2B H-score between normal and tumor tissue was conducted, exhibiting that ADORA2B expression in tumor tissue was significantly higher than in normal tissue (Fig. 4E). Next, the correlation between the expression level of ADORA2B and OS, PFS was analyzed, indicating that PDAC patients with higher ADORA2B expression level were more likely to have a poor OS and PFS (Fig. 4F).

Macrophages-derived LIF promotes malignant phenotypes of PDAC cells by activating the JAK-STAT signaling pathway.

To verify the promoting effect of LIF, rh-LIF with concentration gradient was used to treat pancreatic cancer cell lines, and the functional change was recorded. The results of phenotypic experiments exhibited that the migration capacity of pancreatic cancer cell lines PANC-1 and BxPC-3 was significantly increased compared with the control group (Fig. 5A-B). Moreover, the invasion of PANC-1 and BxPC-3 were considerably enhanced compared with the control group (Fig. 5C-D). Furthermore, the proliferation of PANC-1 and BxPC-3 was elevated after rh-LIF stimulation (Fig. 5E-F). Subsequently, the western blot for PANC-1 and BxPC-3 exhibited that EMT and cell cycle-related proteins were significantly up-regulated after rh-LIF stimulation (Fig. 4G). Last but not least, the western blot results also revealed that rh-LIF activated the JAK-STAT signaling pathway in PANC-1 and BxPC-3, while the addition of EC359 (antagonist for the LIF receptor) could revise it (Fig. 5H).

The ADORA2B and LIFR antagonists inhibit malignant phenotypes of PDAC cells in the coculture system.

Then, to verify the promoting effect of the ADO/ADORA2B/LIF axis previously described, a combined treatment of ADO, PSB603 (antagonists for ADORA2B), and EC359 (LIFR) was utilized in the co-cultured system consisting of Thp-1 macrophages and pancreatic cancer cells lines PANC-1 and BxPC-3 (Fig. 6A-B). As the results showed, the migration of PANC-1 and BxPC-3 was significantly increased after adding ADO, while this promoting effect could be inhibited by PSB603 and EC359 (Fig. 6C-D). Similarly, the invasion of PANC-1 and BxPC-3 was significantly strengthened after adding ADO, while it could be revised by PSB603 and EC359 (Fig. 6E-F). Moreover, the proliferation of PANC-1 and BxPC-3 has been elevated with the addition of ADO, while the promoting effect could be prevented by PSB603 and EC359 (Fig. 6G-H). Finally, the western blot indicated that the expression of EMT and cell cycle-related proteins in PANC-1 and BxPC-3 were significantly up-regulated after the addition of ADO. At the same time, it could be reversed by PSB603 and EC359 (Fig. 6I).

ADORA2B and LIFR antagonists inhibit the progression and metastasis of pancreatic orthotopic BxPC-3 tumors.

We further explored whether clodronate liposomes (macrophage scavenger), PSB603, and EC359 could inhibit the tumorigenicity of pancreatic orthotopic cancer cells in vivo. The schematic diagram shows the medication regimens (Fig. 7A). After eight weeks of treatment, the vivo bioluminescence imaging was performed and recorded (Fig. 7B). As the results showed, the fluorescence intensity of the tumor in the mice that only received ADO (group 2) was significantly more robust than the no-drug group (group 1). In contrast, the fluorescence intensity of the tumor in mice receiving ADO combined with clodronate liposomes, PSB603, and EC359 (group 3–5) was remarkedly lower than group 2 (Fig. 7B). Then, the mice were sacrificed, followed by the orthotopic tumor and liver destruction (Fig. 7C). Subsequently, the orthotopic tumors were weighed and rinsed in PBS. Meanwhile, the surface metastatic foci in the liver were counted manually. The analysis showed that tumor weight in group 2 was significantly higher than in group 1. Meanwhile, the tumor weight in groups 3–5 was lighter than in group 2 (Fig. 7D). Furthermore, more liver metastatic foci occurred in group 2 compared with group 1. Meanwhile, surface liver metastatic foci in groups 3–5 were notably fewer than in group 2 (Fig. 7E).

PDAC was described as a malignant tumor with a poor prognosis[33, 34]. Currently, the optimal treatment for PDAC is still radical surgery[5, 35]. However, the recurrent risk of PDAC patients was 70–90% within five years after curative resections, even when adjuvant chemotherapy was performed according to the NCCN guidelines[36–38]. The previous research demonstrated that the interaction between the pancreatic cell and macrophages in TME was a crucial reason for PDAC progression and metastasis[39–41]. Furthermore, the activation of ADOR was exhibited to play a significant role in the functional change of macrophages[42–45]. However, the molecular mechanism followed by the activation of ADOR has yet to be investigated thoroughly. Meanwhile, the role of ADOR-activated macrophages in PDAC progression and metastasis has yet to be discussed. In the present study, we identified that activation of ADORA2B stimulated LIF secretion from macrophages by the RAF-MEK-ERK signaling pathway. Furthermore, the LIF derived from macrophages promotes PDAC progression and metastasis.

Purine metabolism is essential in immune escape and tumor progression[46–49]. Moreover, the ADO, which served as the metabolite of AMP decomposition, facilitated tumor immune escape by inhibiting the maturation of CD8 + T lymphocytes and promoting the secretion of IL-10 from macrophages[50–53]. Similarly, in the present study, we confirmed that CM made of supernatant from ADOR-activation macrophages promoted the migration, invasion, and proliferation of two pancreatic cancer cell lines. Meanwhile, the western blot also showed the elevation of EMT and cell cycle proteins in two pancreatic cancer cell lines treated by the CM. Based on these results, we speculated that the specific cytokine secreted from ADOR-activation macrophages enhanced the malignant phenotype of pancreatic cancer cells.

LIF is a highly pleiotropic cytokine, serving as an inhibitory factor for proliferation

of myeloid leukemic cells[54, 55]. Moreover, LIF was also identified to regulate pancreatic cancer cell proliferation, differentiation, and survival[56, 57]. This study performed transcriptomic sequencing on the Thp-1 macrophages stimulated by ADO to clarify the specific cytokines in the CM. After screening two gene pools from KEGG and GO enrichment, the LIF was finally selected. Then, the ELISA assay identified that the concentration of LIF in the supernatant of Thp-1 macrophages was significantly elevated after the ADO treatment. Similarly, the expression level of LIF proteins in Thp-1 macrophages was also considerably up-regulated after ADO stimulation. These results confirmed that LIF was one of the crucial cytokines secreted from ADOR-activation macrophages. Furthermore, based on previous research, most LIF in TME is produced by pancreatic stellate cells[57–59]. Similarly, in the present study, we also found that macrophages are another vital source of LIF in TME.

The ADOR belongs to class A rhodopsin-like G protein-coupled receptor with seven transmembrane domains. Four different types of ADOR have been discovered so far (A1, A2A, A2B, and A3)[42, 60, 61]. Additionally, activation of ADORA1 promotes tumor growth by exerting antiapoptotic effects in breast cancer[62, 63]. Triggers of ADORA2A suppress antitumor immune responses in TME by enhancing immune suppressive activity in regulatory T cells and inhibiting antigen presentation of dendritic cells[64–66]. The role of ADORA3A in regulating cancer progression is still controversial, as this receptor acts differently in various tissue types[67–69]. The ADORA2B can only be activated by high ADO concentrations because of the low affinity to ADO[70, 71]. Hence, the activation of ADORA2B usually happens under extreme external conditions, such as inflammation, injury, hypoxia, or cellular stress[72, 73]. Except for the promoting effect in proliferation, angiogenesis, cell invasion, and metastasis of cancer, the presence of ADORA2B in mast cells, neutrophils, dendritic cells, macrophages, and lymphocytes has also shown critical immunoregulatory roles within the immune suppressive TME[42, 74, 75]. In the present study, we found a novel mechanism in which the activation of ADORA2B promoted LIF secretion in macrophages. Firstly, we found that the triggering of ADORA2B instead of the other three ADO receptors promoted the secretion of LIF in the Thp-1 macrophages by western blot and ELISA assay. Meanwhile, we also identified that the Raf-MEK-ERK signaling pathway was significantly activated after the ADORA2B triggering. Moreover, the Raf-MEK-ERK signaling pathway was verified to be correlated with LIF secretion through rescue experiments with an antagonist for Raf. Based on these findings, we believe that triggering ADORA2B promotes LIF secretion in macrophages by activating the Raf-MEK-ERK signaling pathway.

LIF is the most pleiotropic member of the interleukin-6 family of cytokines, playing a vital role in promoting cancer progression[54, 55]. LIF stimulates the JAK-Tyk family of cytoplasmic tyrosine kinases after binding with LIFR[76, 77]. Then, the downstream signaling of LIFR was further activated. It has been indicated that LIF promotes tumorigenesis and advancement in pancreatic and breast cancer through LIF/LIFR-JAK-STAT3 and LIF/LIFR-AKT-mTOR signaling, respectively[78, 79]. We got a similar conclusion in the present study. At first, the migration, invasion, and proliferation capacity of pancreatic cancer cell lines PANC-1 and BxPC-3 were enhanced after rh-LIF stimulation. Then, the EMT and cell cycle-related proteins were up-regulated by adding rh-LIF. Moreover, we also identified that the JAK-STAT signaling pathway in PANC-1 and BxPC-3 was remarkedly activated after using rh-LIF. At the same time, the JAK-STAT signaling pathway in PANC-1 and BxPC-3 was inhibited by an antagonist for LIFR. Based on these results, we argue that LIF promotes malignant phenotypes of PANC-1 and BxPC-3 by connecting with LIFR and activating the JAK-STAT signaling pathway.

Multiple studies have shown the importance of ADORA2B in cytokine release and tumor progression[53, 80–82]. A recent study utilizing macrophages isolated from ADORA2B KO mice showed that adenosine elicits IL-6 production from macrophages via the ADORA2B[45]. Similarly, based on the results of the present study, we speculated that triggering ADOR2B promotes LIF secretion from macrophages by activating the Raf-MEK-ERK signaling pathway. For instance, LIF was also a cytokine accelerating PDAC progression by connecting with LIFR[83–85]. To verify this series of conclusions, we constructed a cocultured system consisting of Thp-1 macrophages and pancreatic cancer cell lines. The cell functional experiments revealed that the migration, invasion, and proliferation capacity of PANC-1 and BxPC-3 in the cocultured system significantly increased after the addition of ADO. Meanwhile, the antagonist for ADOR2B and LIFR could reverse it. The results from the cocultured system were consistent with our previous conclusion.

Targeted therapy highlights the association between neoplastic characterization and individual therapeutic responses[86]. ADORA2B inhibition with the antagonist PSB1115 decreased tumor metastasis of CD73 + melanoma cells (B16F10, LWT1) and mammary carcinoma cells (TNBC, E0771)[87]. Moreover, it has been reported that the anti-metastatic effect of PSB1115 was independent of immune responses[87]. Furthermore, it is suggested that LIF is a target to limit neural remodeling in pancreatic cancer[57]. Moreover, LIF-blocking antibodies reduced intra-tumoral nerve density[57]. Another study also confirmed that blockade of LIF by neutralizing antibodies represents an attractive approach to improving therapeutic outcomes in KRAS-driven pancreatic cancer[88]. To confirm the antitumor effect of the antagonist for ADORA2B and LIFR, we performed an in vivo validation experiment with orthotopic pancreatic cancer. The results exhibited that ADO promoted cancer progression and metastasis in macrophage-sufficient mice. On the contrary, adding the antagonist for ADOR2B or LIFR would hinder this pro-cancerogenic effect. Based on the results of the animal experiments, we argued that ADORA2B and LIFR may represent therapeutic targets for pancreatic cancer.

However, the present research needs further improvement and optimization. Firstly, we only verified the level of LIF in the supernatant of Thp-1 macrophages after ADO was stimulated. At the same time, the rest of the four possible cytokines filtered out by bioinformatics analysis had never been tested. Then, only the indirect mechanisms of LIF secretion from Thp-1 macrophages after ADOR2B motivation were explored. The direct mechanisms of how ADO connected with ADORA2B and the biological process after ERK was transferred into the nuclear reactor were also worth investigating. Similarly, only the activation of the signaling pathway in the LIFR was verified. The direct mechanisms of LIF-promoted pancreatic development have yet to be explored. Moreover, unlike lentivirus transfection or other genome editing technologies, the antagonist was the only tool used in rescue experiments. Furthermore, credibility would be enhanced if IHC staining was performed to determine the correlation between the expression of LIF and ADOR2B. Finally, there will be more practical value in guiding clinical treatment if antagonists for ADOR2B and LIFR were combined to prevent pancreatic progression.

Ethics approval and consent to participate

The present study complies with the ethical guidelines of the 1975 Declaration of Helsinki, as reflected by a priori approval by the Sun Yat-sen University Cancer Center Ethics Committee. Written informed consent was obtained from all of the patients. For animal studies, the research was approved by the Animal Care and Use Committee of the University of Sun Yat-sen University Cancer Center.

Consent for publication

All authors listed in the article have approved the submit this material to JECCR.

Availability of data and materials

The datasets in the present study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare no conflicts of interest.

Funding

National Natural Science Funds (Nos. 81972299 and 81672390),

National Key Research and Development Plan (No. 2017YFC0910002).

Authors’ contributions

Dailei Qin, Kewei Huang, and Zeihui Yao designed the experiments. Pu Xi, Lingmin Jiang experimented and analyzed the data. Dailei Qin wrote the manuscript. Ran Wei and Shengping Li supervised the study.

Acknowledgments

We thank the State Key Laboratory of Sun Yat-sen University Cancer Center for the experimental instruments and technical support.

Gudjonsson B: Cancer of the pancreas. 50 years of surgery. Cancer 1987, 60(9):2284-2303.
Bray F, Ferlay J, Soerjomataram I, Siegel R, Torre L, Jemal A: Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians 2018, 68(6):394-424.
Rahib L, Smith B, Aizenberg R, Rosenzweig A, Fleshman J, Matrisian L: Projecting cancer incidence and deaths to 2030: the unexpected burden of thyroid, liver, and pancreas cancers in the United States. Cancer research 2014, 74(11):2913-2921.
Kamisawa T, Wood L, Itoi T, Takaori K: Pancreatic cancer. Lancet (London, England) 2016, 388(10039):73-85.
Salem A, Alfi M, Winslow E, Cho C, Weber S: Has survival following pancreaticoduodenectomy for pancreas adenocarcinoma improved over time? Journal of surgical oncology 2015, 112(6):643-649.
Whittington R, Bryer M, Haller D, Solin L, Rosato E: Adjuvant therapy of resected adenocarcinoma of the pancreas. International journal of radiation oncology, biology, physics 1991, 21(5):1137-1143.
Kayahara M, Nagakawa T, Ueno K, Ohta T, Takeda T, Miyazaki I: An evaluation of radical resection for pancreatic cancer based on the mode of recurrence as determined by autopsy and diagnostic imaging. Cancer 1993, 72(7):2118-2123.
Griffin J, Smalley S, Jewell W, Paradelo J, Reymond R, Hassanein R, Evans R: Patterns of failure after curative resection of pancreatic carcinoma. Cancer 1990, 66(1):56-61.
Tepper J, Nardi G, Sutt H: Carcinoma of the pancreas: review of MGH experience from 1963 to 1973. Analysis of surgical failure and implications for radiation therapy. Cancer 1976, 37(3):1519-1524.
Further evidence of effective adjuvant combined radiation and chemotherapy following curative resection of pancreatic cancer. Gastrointestinal Tumor Study Group. Cancer 1987, 59(12):2006-2010.
Neoptolemos J, Stocken D, Friess H, Bassi C, Dunn J, Hickey H, Beger H, Fernandez-Cruz L, Dervenis C, Lacaine F et al: A randomized trial of chemoradiotherapy and chemotherapy after resection of pancreatic cancer. The New England journal of medicine 2004, 350(12):1200-1210.
Conroy T, Desseigne F, Ychou M, Bouché O, Guimbaud R, Bécouarn Y, Adenis A, Raoul J, Gourgou-Bourgade S, de la Fouchardière C et al: FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. The New England journal of medicine 2011, 364(19):1817-1825.
Von Hoff D, Ervin T, Arena F, Chiorean E, Infante J, Moore M, Seay T, Tjulandin S, Ma W, Saleh M et al: Increased survival in pancreatic cancer with nab-paclitaxel plus gemcitabine. The New England journal of medicine 2013, 369(18):1691-1703.
Saka D, Gökalp M, Piyade B, Cevik N, Arik Sever E, Unutmaz D, Ceyhan G, Demir I, Asimgil H: Mechanisms of T-Cell Exhaustion in Pancreatic Cancer. Cancers 2020, 12(8).
Rhim A, Mirek E, Aiello N, Maitra A, Bailey J, McAllister F, Reichert M, Beatty G, Rustgi A, Vonderheide R et al: EMT and dissemination precede pancreatic tumor formation. Cell 2012, 148:349-361.
Farrell A, Joly M, Allen-Petersen B, Worth P, Lanciault C, Sauer D, Link J, Pelz C, Heiser L, Morton J et al: MYC regulates ductal-neuroendocrine lineage plasticity in pancreatic ductal adenocarcinoma associated with poor outcome and chemoresistance. Nature communications 2017, 8(1):1728.
Reichert M, Bakir B, Moreira L, Pitarresi J, Feldmann K, Simon L, Suzuki K, Maddipati R, Rhim A, Schlitter A et al: Regulation of Epithelial Plasticity Determines Metastatic Organotropism in Pancreatic Cancer. Developmental cell 2018, 45(6):696-711.e698.
Feig C, Jones J, Kraman M, Wells R, Deonarine A, Chan D, Connell C, Roberts E, Zhao Q, Caballero O et al: Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer. Proceedings of the National Academy of Sciences of the United States of America 2013, 110(50):20212-20217.
Zhang Y, Yan W, Mathew E, Bednar F, Wan S, Collins M, Evans R, Welling T, Vonderheide R, di Magliano M: CD4+ T lymphocyte ablation prevents pancreatic carcinogenesis in mice. Cancer immunology research 2014, 2(5):423-435.
Stromnes I, Brockenbrough J, Izeradjene K, Carlson M, Cuevas C, Simmons R, Greenberg P, Hingorani S: Targeted depletion of an MDSC subset unmasks pancreatic ductal adenocarcinoma to adaptive immunity. Gut 2014, 63(11):1769-1781.
Clark C, Hingorani S, Mick R, Combs C, Tuveson D, Vonderheide R: Dynamics of the immune reaction to pancreatic cancer from inception to invasion. Cancer research 2007, 67(19):9518-9527.
Beatty G, Eghbali S, Kim R: Deploying Immunotherapy in Pancreatic Cancer: Defining Mechanisms of Response and Resistance. American Society of Clinical Oncology educational book American Society of Clinical Oncology Annual Meeting 2017, 37:267-278.
Seitz L, Jin L, Leleti M, Ashok D, Jeffrey J, Rieger A, Tiessen R, Arold G, Tan J, Powers J et al: Safety, tolerability, and pharmacology of AB928, a novel dual adenosine receptor antagonist, in a randomized, phase 1 study in healthy volunteers. Investigational new drugs 2019, 37(4):711-721.
Beavis P, Divisekera U, Paget C, Chow M, John L, Devaud C, Dwyer K, Stagg J, Smyth M, Darcy P: Blockade of A2A receptors potently suppresses the metastasis of CD73+ tumors. Proceedings of the National Academy of Sciences of the United States of America 2013, 110(36):14711-14716.
Hatfield S, Kjaergaard J, Lukashev D, Schreiber T, Belikoff B, Abbott R, Sethumadhavan S, Philbrook P, Ko K, Cannici R et al: Immunological mechanisms of the antitumor effects of supplemental oxygenation. Science translational medicine 2015, 7(277):277ra230.
Wang J, Wang Y, Chu Y, Li Z, Yu X, Huang Z, Xu J, Zheng L: Tumor-derived adenosine promotes macrophage proliferation in human hepatocellular carcinoma. Journal of hepatology 2021, 74(3):627-637.
Murphy P, Wang J, Bhagwat S, Munger J, Janssen W, Wright T, Elliott M: CD73 regulates anti-inflammatory signaling between apoptotic cells and endotoxin-conditioned tissue macrophages. Cell death and differentiation 2017, 24(3):559-570.
Haskó G, Pacher P: Regulation of macrophage function by adenosine. Arteriosclerosis, thrombosis, and vascular biology 2012, 32(4):865-869.
Sajjadi F, Takabayashi K, Foster A, Domingo R, Firestein G: Inhibition of TNF-alpha expression by adenosine: role of A3 adenosine receptors. Journal of immunology (Baltimore, Md : 1950) 1996, 156(9):3435-3442.
Pinhal-Enfield G, Ramanathan M, Hasko G, Vogel S, Salzman A, Boons G, Leibovich S: An angiogenic switch in macrophages involving synergy between Toll-like receptors 2, 4, 7, and 9 and adenosine A(2A) receptors. The American journal of pathology 2003, 163(2):711-721.
Csóka B, Selmeczy Z, Koscsó B, Németh Z, Pacher P, Murray P, Kepka-Lenhart D, Morris S, Gause W, Leibovich S et al: Adenosine promotes alternative macrophage activation via A2A and A2B receptors. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2012, 26(1):376-386.
Ferrante C, Pinhal-Enfield G, Elson G, Cronstein B, Hasko G, Outram S, Leibovich S: The adenosine-dependent angiogenic switch of macrophages to an M2-like phenotype is independent of interleukin-4 receptor alpha (IL-4Rα) signaling. Inflammation 2013, 36(4):921-931.
Chu L, Goggins M, Fishman E: Diagnosis and Detection of Pancreatic Cancer. Cancer journal (Sudbury, Mass) 2017, 23(6):333-342.
McGuigan A, Kelly P, Turkington R, Jones C, Coleman H, McCain R: Pancreatic cancer: A review of clinical diagnosis, epidemiology, treatment and outcomes. World journal of gastroenterology 2018, 24(43):4846-4861.
Mizrahi J, Surana R, Valle J, Shroff R: Pancreatic cancer. Lancet (London, England) 2020, 395(10242):2008-2020.
Zhang Y, Frampton A, Kyriakides C, Bong J, Habib N, Ahmad R, Jiao L: Loco-recurrence after resection for ductal adenocarcinoma of the pancreas: predictors and implications for adjuvant chemoradiotherapy. Journal of cancer research and clinical oncology 2012, 138(6):1063-1071.
Hernandez J, Morton C, Al-Saadi S, Villadolid D, Cooper J, Bowers C, Rosemurgy A: The natural history of resected pancreatic cancer without adjuvant chemotherapy. The American surgeon 2010, 76(5):480-485.
Van den Broeck A, Sergeant G, Ectors N, Van Steenbergen W, Aerts R, Topal B: Patterns of recurrence after curative resection of pancreatic ductal adenocarcinoma. European journal of surgical oncology : the journal of the European Society of Surgical Oncology and the British Association of Surgical Oncology 2009, 35(6):600-604.
Ware M, El-Rayes B, Lesinski G: Mirage or long-awaited oasis: reinvigorating T-cell responses in pancreatic cancer. Journal for immunotherapy of cancer 2020, 8(2).
De Sanctis F, Solito S, Ugel S, Molon B, Bronte V, Marigo I: MDSCs in cancer: Conceiving new prognostic and therapeutic targets. Biochimica et biophysica acta 2016, 1865(1):35-48.
Biswas S, Mantovani A: Macrophage plasticity and interaction with lymphocyte subsets: cancer as a paradigm. Nature immunology 2010, 11(10):889-896.
Vijayan D, Young A, Teng M, Smyth M: Targeting immunosuppressive adenosine in cancer. Nature reviews Cancer 2017, 17(12):709-724.
undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u: Activation of adenosine A3 receptor alleviates TNF-α-induced inflammation through inhibition of the NF-κB signaling pathway in human colonic epithelial cells. Mediators Inflamm 2014, 2014.
undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u et al: A(3) Adenosine Receptors as Modulators of Inflammation: From Medicinal Chemistry to Therapy. Med Res Rev 2017, 38.
undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u et al: HIF1A up-regulates the ADORA2B receptor on alternatively activated macrophages and contributes to pulmonary fibrosis. FASEB J 2017, 31.
Burnstock G, Di Virgilio F: Purinergic signalling and cancer. Purinergic signalling 2013, 9(4):491-540.
undefined u, undefined u, undefined u, undefined u, undefined u, undefined u: Intercellular crosstalk shapes purinergic metabolism and signaling in cancer cells. Cell Rep 2024, 43.
undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u: Purinergic signalling in cancer therapeutic resistance: From mechanisms to targeting strategies. Drug Resist Updat 2023, 70.
undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u et al: Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion. Elife 2023, 12.
Young A, Ngiow S, Barkauskas D, Sult E, Hay C, Blake S, Huang Q, Liu J, Takeda K, Teng M et al: Co-inhibition of CD73 and A2AR Adenosine Signaling Improves Anti-tumor Immune Responses. Cancer cell 2016, 30(3):391-403.
undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u et al: Systemic oxygenation weakens the hypoxia and hypoxia inducible factor 1α-dependent and extracellular adenosine-mediated tumor protection. J Mol Med (Berl) 2014, 92.
undefined u, undefined u, undefined u, undefined u: Targeting cancer-derived adenosine: new therapeutic approaches. Cancer Discov 2014, 4.
Németh Z, Lutz C, Csóka B, Deitch E, Leibovich S, Gause W, Tone M, Pacher P, Vizi E, Haskó G: Adenosine augments IL-10 production by macrophages through an A2B receptor-mediated posttranscriptional mechanism. Journal of immunology (Baltimore, Md : 1950) 2005, 175(12):8260-8270.
undefined u, undefined u: Leukemia-inhibitory factor-neuroimmune modulator of endocrine function. Endocr Rev 2000, 21.
Nicola N, Babon J: Leukemia inhibitory factor (LIF). Cytokine & growth factor reviews 2015, 26(5):533-544.
Shi Y, Gao W, Lytle N, Huang P, Yuan X, Dann A, Ridinger-Saison M, DelGiorno K, Antal C, Liang G et al: Targeting LIF-mediated paracrine interaction for pancreatic cancer therapy and monitoring. Nature 2019, 569(7754):131-135.
Bressy C, Lac S, Nigri J, Leca J, Roques J, Lavaut M, Secq V, Guillaumond F, Bui T, Pietrasz D et al: LIF Drives Neural Remodeling in Pancreatic Cancer and Offers a New Candidate Biomarker. Cancer research 2018, 78(4):909-921.
Öhlund D, Handly-Santana A, Biffi G, Elyada E, Almeida A, Ponz-Sarvise M, Corbo V, Oni T, Hearn S, Lee E et al: Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer. The Journal of experimental medicine 2017, 214(3):579-596.
Biffi G, Oni T, Spielman B, Hao Y, Elyada E, Park Y, Preall J, Tuveson D: IL1-Induced JAK/STAT Signaling Is Antagonized by TGFβ to Shape CAF Heterogeneity in Pancreatic Ductal Adenocarcinoma. Cancer discovery 2019, 9(2):282-301.
undefined u, undefined u: Role of G-protein-coupled adenosine receptors in downregulation of inflammation and protection from tissue damage. Nature 2002, 414.
undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u et al: THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: Nuclear hormone receptors. Br J Pharmacol 2021.
undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u: RNA interference targeting of A1 receptor-overexpressing breast carcinoma cells leads to diminished rates of cell proliferation and induction of apoptosis. Cancer Biol Ther 2005, 4.
Lin Z, Yin P, Reierstad S, O'Halloran M, Coon V, Pearson E, Mutlu G, Bulun S: Adenosine A1 receptor, a target and regulator of estrogen receptoralpha action, mediates the proliferative effects of estradiol in breast cancer. Oncogene 2010, 29(8):1114-1122.
Novitskiy S, Ryzhov S, Zaynagetdinov R, Goldstein A, Huang Y, Tikhomirov O, Blackburn M, Biaggioni I, Carbone D, Feoktistov I et al: Adenosine receptors in regulation of dendritic cell differentiation and function. Blood 2008, 112(5):1822-1831.
Ohta A, Kini R, Ohta A, Subramanian M, Madasu M, Sitkovsky M: The development and immunosuppressive functions of CD4(+) CD25(+) FoxP3(+) regulatory T cells are under influence of the adenosine-A2A adenosine receptor pathway. Frontiers in immunology 2012, 3:190.
undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u: Adenosine affects expression of membrane molecules, cytokine and chemokine release, and the T-cell stimulatory capacity of human dendritic cells. Blood 2002, 101.
Borea P, Gessi S, Merighi S, Varani K: Adenosine as a Multi-Signalling Guardian Angel in Human Diseases: When, Where and How Does it Exert its Protective Effects? Trends in pharmacological sciences 2016, 37(6):419-434.
undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u: The A3 adenosine receptor: history and perspectives. Pharmacol Rev 2014, 67.
undefined u, undefined u, undefined u: Effects of synthetic A3 adenosine receptor agonists on cell proliferation and viability are receptor independent at micromolar concentrations. J Physiol Biochem 2012, 69.
undefined u, undefined u, undefined u, undefined u, undefined u: Medicinal chemistry and pharmacology of A2B adenosine receptors. Curr Top Med Chem 2003, 3.
undefined u, undefined u, undefined u, undefined u, undefined u: Probing biased/partial agonism at the G protein-coupled A(2B) adenosine receptor. Biochem Pharmacol 2014, 90.
undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u et al: Oxidative stress controls regulatory T cell apoptosis and suppressor activity and. Nat Immunol 2017, 18.
undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, undefined u et al: Adenosine A(2A) receptor ligand recognition and signaling is blocked by A(2B) receptors. Oncotarget 2018, 9.
Yan A, Joachims M, Thompson L, Miller A, Canoll P, Bynoe M: CD73 Promotes Glioblastoma Pathogenesis and Enhances Its Chemoresistance via A Adenosine Receptor Signaling. The Journal of neuroscience : the official journal of the Society for Neuroscience 2019, 39(22):4387-4402.
Antonioli L, Blandizzi C, Pacher P, Haskó G: Immunity, inflammation and cancer: a leading role for adenosine. Nature reviews Cancer 2013, 13(12):842-857.
Zhang C, Liu J, Wang J, Hu W, Feng Z: The emerging role of leukemia inhibitory factor in cancer and therapy. Pharmacology & therapeutics 2021, 221:107754.
Dahéron L, Opitz S, Zaehres H, Lensch M, Andrews P, Itskovitz-Eldor J, Daley G: LIF/STAT3 signaling fails to maintain self-renewal of human embryonic stem cells. Stem cells (Dayton, Ohio) 2004, 22(5):770-778.
Niwa H, Burdon T, Chambers I, Smith A: Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes & development 1998, 12(13):2048-2060.
Li X, Yang Q, Yu H, Wu L, Zhao Y, Zhang C, Yue X, Liu Z, Wu H, Haffty B et al: LIF promotes tumorigenesis and metastasis of breast cancer through the AKT-mTOR pathway. Oncotarget 2014, 5(3):788-801.
Haskó G, Pacher P, Deitch E, Vizi E: Shaping of monocyte and macrophage function by adenosine receptors. Pharmacology & therapeutics 2007, 113(2):264-275.
Gessi S, Fogli E, Sacchetto V, Merighi S, Varani K, Preti D, Leung E, Maclennan S, Borea P: Adenosine modulates HIF-1{alpha}, VEGF, IL-8, and foam cell formation in a human model of hypoxic foam cells. Arteriosclerosis, thrombosis, and vascular biology 2010, 30(1):90-97.
Ernens I, Léonard F, Vausort M, Rolland-Turner M, Devaux Y, Wagner D: Adenosine up-regulates vascular endothelial growth factor in human macrophages. Biochemical and biophysical research communications 2010, 392(3):351-356.
Gearing D, Thut C, VandeBos T, Gimpel S, Delaney P, King J, Price V, Cosman D, Beckmann M: Leukemia inhibitory factor receptor is structurally related to the IL-6 signal transducer, gp130. The EMBO journal 1991, 10(10):2839-2848.
Stahl N, Boulton T, Farruggella T, Ip N, Davis S, Witthuhn B, Quelle F, Silvennoinen O, Barbieri G, Pellegrini S: Association and activation of Jak-Tyk kinases by CNTF-LIF-OSM-IL-6 beta receptor components. Science (New York, NY) 1994, 263(5143):92-95.
Taga T, Kishimoto T: Gp130 and the interleukin-6 family of cytokines. Annual review of immunology 1997, 15:797-819.
Qian Y, Gong Y, Fan Z, Luo G, Huang Q, Deng S, Cheng H, Jin K, Ni Q, Yu X et al: Molecular alterations and targeted therapy in pancreatic ductal adenocarcinoma. Journal of hematology & oncology 2020, 13(1):130.
Mittal D, Sinha D, Barkauskas D, Young A, Kalimutho M, Stannard K, Caramia F, Haibe-Kains B, Stagg J, Khanna K et al: Adenosine 2B Receptor Expression on Cancer Cells Promotes Metastasis. Cancer research 2016, 76(15):4372-4382.
Wang M, Fer N, Galeas J, Collisson E, Kim S, Sharib J, McCormick F: Blockade of leukemia inhibitory factor as a therapeutic approach to KRAS driven pancreatic cancer. Nature communications 2019, 10(1):3055.

Download PDF

Version 1

posted

You are reading this latest preprint version

Blockades Adenosine Receptor 2B Suppresses Pancreatic Adenocarcinoma Progression by Inhibiting Leukemia Inhibitory Factor Secretion from Macrophages

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Methods

Cell line and cell culture

Animal experiments

Western blot test

Enzyme-linked immunosorbent assay (ELISA)

Wound healing assay

Tran-swell assay

CCK8 viability assay

Dual-luciferase reporter assay

Macrophage Differentiation

Cell coculture model

Results

Discussion

Declarations

References

Status:

Version 1