
==== Front
Mol Biol Rep
Mol Biol Rep
Molecular Biology Reports
0301-4851
1573-4978
Springer Netherlands Dordrecht

9928
10.1007/s11033-024-09928-1
Original Article
Cancer-associated fibroblast-derived exosome Leptin promotes malignant biological lineage in pancreatic ductal adenocarcinoma by regulating ABL2 via miR-224-3p
Zhang Li
Chen Yesheng
Dai Yihe
Mou Weicheng
Deng Pan
Jin Yan
Xu Jing
Jin Yun colourcloud@126.com

grid.414918.1 Department of Hepatopancreatobiliary Surgery, The First People’s Hospital of Yunnan Province, the Affiliated Hospital of Kunming University of Science and Technology, Kunming, Yunnan 650034 China
19 9 2024
19 9 2024
2024
51 1 99517 5 2024
10 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Background

Cancer-associated fibroblasts, as a major component of the tumor microenvironment, have been shown to exhibit protumorigenic effects in pancreatic ductal adenocarcinoma. Moreover, cancer-associated fibroblasts-derived exosomes have been reported to promote tumor development, but exact mechanisms have not been elucidated. The purpose of this study was to investigate the processes by which exosomes generated from cancer-associated fibroblasts promote tumor growth.

Methods

twenty-one patients with pancreatic ductal adenocarcinoma who evaluated preoperatively as potentially surgically resectable without distant metastasis and pathologically examined postoperatively as pancreatic ductal cell carcinoma were included. We determined the expression of Leptin as well as downstream proteins at the clinical and cellular levels. Cancer-associated fibroblast-derived exosomes were characterised by nanoparticle transmission electron microscopy and tracking analysis. To ascertain the mechanism mediating the action of exosomal Leptin in pancreatic ductal adenocarcinoma, we performed CCK-8 assay, colony formation assays, transwell and wound healing assays in PSN1 cells to evaluate cell proliferation, migration and invasion. Western blotting was used to detect the level of Leptin, ABL2 and exosome markers. qRT-PCR was employed to evaluate miR-224-3p. Cancer-associated fibroblasts markers and exosome uptake were verified by immunofluorescence.

Results

Western blotting assays show that Leptin is present inside tissues and cancer-associated fibroblasts in pancreatic ductal adenocarcinoma. Cancer-associated fibroblasts stimulated PSN1 cells growth, migration and invasion in vitro by secreting the exosomal Leptin. Exosomal Leptin could regulate miR-224-3p, which targets negative regulation of ABL2. Inhibiting Leptin significantly limited PSN1 cells growth, migration and invasion. In vitro analyses revealed that miR-224-3p mimics mitigate the inhibitory effect of cancer-associated fibroblasts knockdown of Leptin on PSN1 cells development, but overexpression of ABL2 partly abolished the tumor-promoting phenotype of miR-224-3p mimics.

Conclusion

Our results revealed that cancer-associated fibroblasts mediate pancreatic ductal adenocarcinoma development by regulating the miR-224-3p/ABL2 molecular axis through the secretion of the exosomal Leptin.

Keywords

Cancer-associated fibroblasts (CAFs)
Pancreatic cancer
Pancreatic ductal adenocarcinoma (PDAC)
Exosomes
miR-224-3p
Leptin
ABL2
The Yunnan Science and Technology Department and Kunming Medical University Special Fund202101AY070001-234 The Yunnan Digestive Endoscopy Clinical Medical Center Foundation for Health Commission of Yunnan Province of (ZX2019-01-02)2022LCZXKF-XH02 The Yunnan Fundamental Research Projects202401AT070053 issue-copyright-statement© Springer Nature B.V. 2024
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pmcIntroduction

There were 511,000 new cases of pancreatic cancer and 467,000 deaths in 2022. The disease is among the poorest in terms of prognosis and hence the disease ranks as the sixth leading cause of cancer mortality in both sexes combined [1]. Pancreatic ductal adenocarcinoma (PDAC) is the predominant kind of pancreatic cancer, accounting for over 90% of cases [2–4]. The mechanism of PDAC development has not been fully elucidated, and studies at the molecular pathological level have revealed that the dense mesenchymal components surrounding the tumor parenchyma comprise the tumor microenvironment (TME) that supports tumor cell growth, proliferation, infiltration, and metastasis [5,6]. Stromal cells that have been activated in the tumor microenvironment are known as cancer-associated fibroblasts (CAFs), and they are essential to the growth of cancer.

It has been found that activated CAFs can promote tumor development through various pathways, including paracrine, direct contact, immune response, and promotion of extracellular matrix (ECM) [7,8]. There is growing evidence that exosomes mediate intercellular signaling [9]. For instance, esophageal squamous cell carcinoma proliferates and migrates more readily when exposed to exosomal SHH generated from CAFs [10]. Uncertainty surrounds the mechanism by which tumor-derived exosomes drive Cancer Progress.

Leptin is an adipokine that involved in the control of immunological response, neuroendocrine function, energy homeostasis, and glucose metabolism [11]. Previous studies have shown that Leptin is involved in regulating the process of cancer development and progression [12,13]. Accumulating evidence suggested cancer cell-derived exosomes are closely related to cancer malignant behavior [14]. According to a recent study, Leptin is substantially expressed in exosomes generated from gallbladder cancer (GBC) cells, and Leptin transfer from GBC cell-derived exosomes improves the invasion and migration of GBC cells [15]. Additionally, it has high expression as an oncogene in a variety of tumor cells and organs, including breast cancer, ovarian cancer, and gastric cancer [16–18]. However, the mechanism by which exosomal Leptin promotes tumor invasion and metastasis has not yet been elucidated.

ABL2 (ABL Proto-Oncogene 2, ABL2) is a member of the Abelson family of non-receptor complex amino acid kinases, and ABL2 regulates cell proliferation, polarity, and migration by participating in actin reorganization. Studies have shown that ABL2 is lowly expressed in various tumor cells, such as nasopharyngeal carcinoma and hepatocellular carcinoma [19,20]. In addition, Lu et al. found that ABL2 was also down-regulated in PDAC cells and that overexpression of ABL2 inhibited pancreatic cancer development [21]. However, further studies are needed to elucidate the role of ABL2 in pancreatic cancer.

Comparing pancreatic cancer tissues to paraneoplastic tissues, our findings revealed a drop in ABL2 levels and an increase in Leptin and miR-224-3p levels. In addition, PSN1 cells treated with exosomal Leptin showed increased proliferation, migration, and invasion. Subsequently, we assessed the association between Leptin and miR-224-3p in order to ascertain the fundamental process by which CAFs cells facilitate the migration and invasion of PSN1 cells through exosomal Leptin. In conclusion, our results revealed that CAFs cells mediate PDAC development by regulating the miR-224-3p/ABL2 molecular axis through the secretion of the exosomal Leptin.

Materials and methods

Patients and samples

We collected 21 specimens of cancerous and paracancerous tissues from patients diagnosed with PC in the First People’s Hospital of Yunnan Province. These patients underwent radical surgical resection at the Pancreas Center of the First People’s Hospital of Yunnan Province between 2021 and 2023. Pathological confirmation was performed for pancreatic ductal epithelial cell carcinoma. These samples were collected after surgery. The samples were kept at -80 °C after being quickly frozen in liquid nitrogen. The eighth edition of the American Joint Committee on Cancer’s pancreatic cancer staging system was used to determine the clinical TNM of the patient’s stages. The study design was authorized by the First People’s Hospital of Yunnan Province Research Ethics Committee prior to tissue collection (KHLL2022-KY145) in October 2022. Written informed consent was provided by every patient.

Inclusion criteria: A total of 30 specimens and paracancerous tissues (0.5 cm x 0.5 cm x 0.5 cm) of pancreatic ductal cell carcinoma that were evaluated preoperatively as potentially surgically resectable without distant metastasis and pathologically examined postoperatively as pancreatic ductal cell carcinoma were collected.

Exclusion criteria: Specimens and paracancerous tissues (0.5 cm x 0.5 cm x 0.5 cm) were collected for preoperative evaluation of possible pancreatic ductal cell carcinoma that was surgically resectable without distant metastases and postoperative pathologic examination of non-pancreatic ductal cell carcinoma.

Cell culture

In patients undergoing malignant tissue resection, pancreatic cancer tissues were used to isolate CAFs, and paired neighboring normal areas were used to isolate normal fibroblasts (NFs). PSN1 cells (human pancreatic cancer cells) were obtained from. CAFs cells, NFs cells and PSN1 cells were cultured in Dulbecco’s modified eagle medium (DMEM) containing 10% fetal bovine serum. All cells tested negative for mycoplasma. CAFs cells were cultured in a medium containing serum free of exosomes and were washed three times with PBS (cat#C3593-0500, Viva Cell BIOSCIENCES, Shanghai, China) prior to the collection of supernatants for exosome extraction for 48 h.

Immunofluorescence

The slices were incubated with a fluorescent secondary antibody for an hour at room temperature in a humid box in order to perform immunofluorescence. DAPI (cat#CM07245, Proteintech, Wuhan, China) was used as a counterstain for the cell nuclei [22]. Images were acquired on an inverted fluorescence microscope (Nikon, Tokyo, Japan). The following primary antibodies were used: rabbit polyclonal anti-FSP1 (cat#20886-1-AP), anti-α-SMA (cat#14395-1-AP) antibody and anti-FAP (cat#11779-1-AP) (Proteintech, Wuhan, China).

Cell co-culture

CAFs or NFs (1 × 105 cells) were inoculated into the 0.4 μm Transwell chamber (cat#CLS3470-48EA, Corning, Beijing, China). Meanwhile, PSN1 cells (1 × 104 cells) were inoculated into plate. After that, transwell chambers containing CAFs cells were placed into plates containing PSN1 cells and incubated for 24 h [23].

Small-molecule, CM and exosomes treatments

To confirm that the substances affecting PSN1 cells are of extracellular origin, we collected the corresponding CM (cell medium) and mixed it 1:1 with fresh medium for the treatment of the cells [24]. For small-molecule treatment, cells were treated with the 10µM GW4869 (cat#HY-19363, MCE, Shanghai, China) to inhibit exosomes secretion. For exosomes treatment, the extracted exosomes are added to the corresponding cells to be treated.

Isolation of exosomes

Before gathering the cell supernatant, CAFs cells were cultured in exosome-free FBS medium for 48 h after three PBS washes. Subsequently, the cell supernatants underwent a 5-minute 500 × g centrifugation to separate the cells, and a 10-minute 3000×g centrifugation was performed to eliminate any remaining cellular debris. After adding the supernatant to an equal volume of PBS that had been pre-cooled, Exosome Concentration Solution was added. The mixture was then centrifuged at 10,000×g for 60 min at 4 °C. To evaluate the protein content, RNA isolation, and in vitro investigations, the precipitate was collected as exosomes and resuspended in 100 µL of PBS in RNase-free tubes. The precipitate was then refrigerated at -80 °C for Transmission Electron Microscopy.

Transmission electron microscopy of exosomes

After we extracted exosomes using the kit, 5 µl of exosome suspension was added to a Formvar-carbon sample-carrying copper mesh. A good field of view was chosen for further examination. Electron micrographs were taken at 100 kV using a JEOL JEM-1230 Transmission Electron Microscopy.

Exosome uptake assay

Using the PKH67 Green Fluorescence Kit (cat#UR52303, Umibio, Shanghai, China), we tagged exosomes. A 0.22 μm filter was used to filter 10 µg of exosomes that had been resuspended in 500 µL PBS for the exosome uptake experiment. To 30% confluent PSN1 cells on confocal Petri dishes, exosomes were introduced, and the mixture was cultured for a whole day. Using an inverted fluorescent microscope (Nikon, Tokyo, Japan), pictures were captured [25].

CCK8 assay

Using the cell counting kit-8 (cat#PF00004, Proteintech, Wuhan, China), the vitality of the cells was assessed. In short, the cells underwent a 24-hour treatment with exosomes before being planted at a density of 1 × 105 cells per well in 96-well plates. Afterward, the cells were analyzed for cell viability overnight. Next, 10 µL CCK-8 solution was added to the well and the plates were incubated at 37 °C for 3 h. Finally, the absorbance in each well was measured at 450 nm using a microplate auto-reader (Thermo, USA).

Quantitative reverse transcriptase-PCR (qRT-PCR)

Total RNA was isolated from tissues, cells, and exosomes using a total RNA extraction kit (cat#R1200, Solarbio, Beijing, China). After that, RNA was reverse transcribed using the manufacturer’s instructions to create complementary DNA. For miR-224-3p, we used the Enhanced miRNA First Strand Synthesis Kit (cat#PC4801, Aidlab Biotechnologies, Beijing, China) to reverse transcribe the RNA to generate complementary DNA and the Enhanced miRNA Fluorescence Quantitative PCR Detection Kit (cat#PC4901, Aidlab Biotechnologies, Beijing, China) for qRT-PCR. PCR was performed on an ABI 7500 Rapid Real-Time RCR system (Applied Biosystems, USA). In order to standardize the data, U6 served as miRNA’s internal control. Finally, we quantified gene expression relative to each other using the 2-ΔΔCt technique. The primer pairs used in this study were: U6-forward 5′-ATGGACTATCATATGCTTACCGTA-3′ and U6-reverse 5′-GAGCAGGGTCCGAGGT-3′.

miR-224-3p-forward 5′-CCCTAAAATGGTGCCCTAGTG-3′ and miR-224-3p-reverse 5′-TCCTCCTCTCCTTCCTTCTC-3′.

Western blotting and antibodies

Using RIPA lysis buffer (cat#R0010, Solarbio, Beijing, China) containing PMSF (cat#P0100, Solarbio, Beijing, China), proteins from tissues, cells, and exosomes were isolated. Following separation on SDS-PAGE, 10–20 µg of proteins were transferred to a PVDF membrane (cat#IPVH00010, MerckMillipore, Germany). After blocking the membranes for an hour with 5% fat-free milk (cat#L8590, Solarbio, Beijing, China) in a Tris-buffered solution containing 0.1% Tween-20 (TBST), the primary antibody was then incubated at 4 °C for the entire night. The following primary antibodies were used: rabbit anti-Leptin (cat#ET1704-44, HUABIO, Hangzhou, China; RRID: AB_2934245), rabbit anti-ABL2 (cat#ET1702-07HUABIO, Hangzhou, China; RRID: AB_3068595), rabbit anti-β-actin (cat#81115-1-RR, Proteintech, Wuhan, China; RRID: AB_2923704), rabbit anti-CD63 (cat#AF5117, Affinity Biosciences, Jiangsu, China; RRID: AB_2837603), and rabbit anti-TSG101 (cat#ET1701-59, HUABIO, Hangzhou, China; RRID: RRID: AB_3068599) were the primary antibodies used. Subsequently, the membranes were treated for one hour with HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (cat#SA00001-2, Proteintech, Wuhan, China, RRID: AB_2722564) secondary anti-rabbit IgG antibodies. Finally, An enhanced chemiluminescence kit (cat#PK10001, Proteintech, Wuhan, China) was used to observe the bands, and the ImageJ software was used for quantitation.

Transfection

The manufacturer’s instructions for transfection were followed, and HANBIO (Shanghai, China) provided the pcDNA-ABL2, sh-Leptin, and miR-224-3p mimics that we used. CAFs were transfected with sh-Leptin, and PSN1 cells were transfected with pcDNA-ABL2 and miR-224-3p mimics. Further analyses and experiments were performed 48 h after transfection.

PSN1 cells per well were grown in 2 mL of medium with 10% FBS in 6-well plates (cat#CLS3428, Corning, Beijing, China). Ensure that the fusion rate is 30–40% when infected with the virus the next day. Replaced with DMEM medium containing 8ug/ml Polybrene, then added virus. Observe cell status 12–24 h after viral infection. After 48 h of virus infection of cells, observe the cells under a fluorescence microscope to determine the lentivirus infection. The efficiency of lentivirus infection in PSN1 cells is determined.

Colony formation assay

Initially, 200 PSN1 cells per well were grown for 4 weeks in 2 mL of medium with 15% FBS in 6-well plates (cat#CLS3428, Corning, Beijing, China). After that, these colonies were fixed and stained for 20 min with 4% paraformaldehyde (PFA) and two minutes at room temperature with crystal violet staining solution. Rinsed under running water and allowed to air dry in a cool, room temperature environment. Finally, they were observed and photographed under an inverted microscope (Nikon, Tokyo, Japan).

Transwell migration assay

We used a 12-well 8.0 μm Transwell chamber (cat#CLS3428, Corning, Beijing, China) to determine cell migration. Firstly, incubate in an incubator for 24 h to detect PSN1 cells migration from the upper to the lower layers. The chambers were fixed for 15 min at room temperature in a 4% paraformaldehyde solution. After cleaning the cells on the chamber’s upper surface, the chamber was let to sit at room temperature for 30 min in a crystal violet staining solution. The chambers were taken out after staining, cleaned under running water, and allowed to air dry at room temperature in a cold environment. Finally, the cells were observed and imaged under an inverted microscope (Nikon, Tokyo, Japan).

Wound healing assay

6-well plates were used for the wound healing assays (cat#CLS3516-50EA, Corning, Beijing, China). After that, PSN1 cells were cultivated to 95% confluence in 6-well plates after being inoculated. After making a scratch with 100 l sterile tips, the area was cleaned with PBS to remove any floating cells, and the cells were cultured in a medium without serum. Lastly, pictures were taken with an inverted microscope at 24 and 72 h (Nikon, Tokyo, Japan). Mobility was analyzed using the “Image J” software using the following formula: wound healing capacity (%) = (initial scratch distance - final scratch distance)/initial scratch distance × 100%.

Statistical analysis

All statistical analyses were performed with the GraphPad Prism software (version 8.0.2). The data is shown as mean ± SD. Student’s t-test was used to analyze differences between two groups, while one-way ANOVA was used to compare differences between three groups, and the Sidak test was used as post hoc. p-values less than 0.05 were considered statistically significant. “*, **, and ***” indicate P < 0.05; **, P < 0.01; ***, P < 0.001, respectively.

Results

Leptin, mir-224-3p and ABL2 expression in pancreatic ductal adenocarcinoma

To determine the expression of Leptin, miR-224-3p, and ABL2 in PDCA, we extracted RNA and protein from clinical samples, respectively, and Western blotting detected the expression of Leptin and ABL2. RT-qPCR detected miR-224-3p expression. The results showed that Leptin expression levels were significantly increased and ABL2 expression was decreased (**P < 0.01) in PDCA tissues compared with paracancerous tissues (Fig. 1A). We used immunofluorescence to label α-SMA, FSP-1, and FAP in CAFs and NFs(Fig. 1B). miR-224-3p expression was significantly increased (**P < 0.01) in PDCA tissues (Fig. 1C). The findings showed that PDCA tissues had reduced ABL2 levels while increasing the expression of Leptin and miR-224-3p.

Fig. 1 Leptin, miR-224-3p and ABL2 expression in PDCA. A Western blot analysis of Leptin and ABL2 in tumor tissues and their adjacent normal tissues. B α-SMA, FSP-1, and FAP immunofluorescence staining in CAFs and NFs. C RT-qPCR analysis showed that miR-224-3p expressed in tumor tissues and their adjacent normal tissues. The data is shown as mean ± SD. Analyses were performed by Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001

Cancer-associated fibroblasts promote PSN1 cells proliferation, invasion and migration

We co-cultured PSN1 cells with CAFs or NFs to determine the function of CAFs. When compared to PSN1 cells alone and NFs co-cultured groups, the CCK-8 assay showed that the viability of PSN1 cells co-cultured with CAFs was significantly up-regulated (**P < 0.01) (Fig. 2A). Colony formation assay showed a significant increase in proliferative capacity with CAFs co-cultured with PSN1 cells compared with the PSN1 cells alone and NFs co-cultured groups (Fig. 2B). Transwell and wound healing assays showed increased migration and motility in the CAFs co-culture compared to the group cultured with PSN1 cells alone (**P < 0.01) and the NFs co-culture group (*P < 0.05) (Fig. 2C, D). Our results indicate that CAFs increase PSN1 cells cell viability and promote proliferation, invasion, and migration.

Fig. 2 CAFs promote PSN1 cells proliferation, invasion and migration. A CCK8 assay was performed in PSN1 cells. B Colony formation assay was performed in PSN1 cells. C The transwell assay was used to assess PSN1 cells’ capacity for invasion. D Assays for wound healing were used to evaluate PSN1 cells’ capacity for migration. The data is shown as mean ± SD. Analyses were performed by one-way ANOVA, and the Sidak test was used as post hoc. *p < 0.05; **p < 0.01; ***p < 0.001

Cancer-associated fibroblasts promote PSN1 cells proliferation, invasion, and migration by secreting exosomes

The exosome inhibitor GW4869 was used to determine that CAFs promote PSN1 cells proliferation, invasion, and migration by secreting exosomes. CAFs-conditioned medium (CM) was collected for the treatment of PSN1 cells. PSN1 cells cell viability was detected by CCK-8, cell proliferation by Colony formation assay, cell invasion by Transwell assay, and PSN1 cells cell migration by scratch assay. The results showed that PSN1 cells cell viability was significantly upregulated (**P < 0.01) in PSN1 cells co-cultured with CAFs-conditioned medium compared to PSN1 cells co-cultured with CAFs-conditioned medium with the exosome inhibitor GW4869 (Fig. 3A). Colony formation, transwell (**P < 0.01), and scratch assay assays (**P < 0.01) showed a significant increase in cell proliferation, invasion, and migration (Fig. 3B-D). It suggests that the promotional effect of CAFs on PSN1 cells cell viability, proliferation, invasion, and migration may act through CAFs exosomes.

Fig. 3 CAFs promote PSN1 cells proliferation, invasion, and migration by secreting exosomes. A CCK8 assay was performed in PSN1 cells. B Colony formation assay was performed in PSN1 cells. C Transwell assay was performed to evaluate the invasive ability of PSN1 cells. D Assays for wound healing were used to evaluate PSN1 cells’ capacity for migration. The data is shown as mean ± SD. Analyses were performed by one-way ANOVA, and the Sidak test was used as post hoc. *p < 0.05; **p < 0.01; ***p < 0.001

Cancer-associated fibroblasts exosomes promote PSN1 cells proliferation, invasion, and migration via leptin

The isolated CAFs exosomes were detected by transmission electron microscopy (Fig. 4A). Detection of exosome marker proteins CD63 and TSG101 by Western blotting. The isolated exosomes expressed TSG101 and CD63, according to Western blotting data (Fig. 4B). To incubate PSN1 cells, exosomes from CAFs were tagged with PKH67. The findings demonstrated that PSN1 cells ingested exosomes generated from CAFs (Fig. 4C).

Next, we determined Leptin expression in NFs and CAFs. The results showed that Leptin expression was significantly increased (**P < 0.01) in CAFs cells compared to NFs (Fig. 4D). To determine Leptin levels in exosomes, we isolated exosomes from the supernatants of NFs and CAFs. Western blotting results showed significantly higher expression (**P < 0.01) of exosomal Leptin secreted by CAFs compared with exosomes from NFs (Fig. 4E). We constructed stable Leptin (***P < 0.001) knockdown CAFs (Fig. 4F). We then ascertained the expression of Leptin in CAFs-exo. The expression of Leptin was significantly reduced (**P < 0.01) in exosomes produced from Leptin knockdown CAFs (Fig. 4G). This showed a favorable correlation between changes in Leptin expression in CAFs and Leptin expression in exosomes produced from CAFs.

Fig. 4 CAFs exosomes promote PSN1 cells proliferation, invasion, and migration via Leptin. A Transmission Electron Microscopy of exosomes produced from CAFs. B Analysis of CD63, TSG101 and HSP70 using Western blotting in CAFs-exo and CAFs-CM. C Exosomes tagged with PKH67 from CAFs co-cultured with PSN1 cells (green-stained exosomes, blue-stained nuclei). D Examining Leptin in NFs and CAFs using Western blot. E Western blotting study of CAF exosomes containing Leptin. F Western blot analysis of Leptin in CAFs and CAFs + sh-Leptin. G Western blot analysis of Leptin in exosomes from CAFs and CAFs + sh-Leptin. The data is shown as mean ± SD. Analyses were performed by Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001

PSN1 cells were then given the CAFs + sh-Leptin-exo treatment. Expression of Leptin was detected in PSN1 cells using Western blotting. CCK-8 results showed that cell viability was significantly reduced (**P < 0.01) in CAFs + sh-Leptin-exo treated PSN1 cells compared to CAFs-exo treated PSN1 cells (Fig. 5A). The findings demonstrated that PSN1 cells treated with CAFs + sh-Leptin-exo had significantly lower levels (**P < 0.01) of Leptin expression than CAFs-exo. (Fig. 5B). The proliferative capacity of CAFs + sh-Leptin-exo treated PSN1 cells was much lower than that of CAFs-exo treated PSN1 cells, as demonstrated by the Colony creation data (Fig. 5C). Transwell (**P < 0.01) and scratch assays (**P < 0.01) showed that the migration and invasion ability of CAFs + sh-Leptin-exo treated PSN1 cells were significantly reduced compared to CAFs-exo treated PSN1 (Fig. 5D, E).

Fig. 5 CAFs exosomes promote PSN1 proliferation, invasion and migration via Leptin. A CCK8 assay was performed in PSN1 cells. B Western blot analysis of Leptin in PSN1. C Colony formation assay was performed in PSN1 cells. D The Transwell assay was used to assess PSN1 cells’ capacity for invasion. E Assays for wound healing were used to evaluate PSN1 cells’ capacity for migration. The data is shown as mean ± SD. Analyses were performed by Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001

Mir-224-3p mitigates the inhibitory effect of cancer-associated fibroblasts knockdown of leptin on PSN1 cells development

PSN1 cells transfected with miR-224-3p mimics was treated with CAFs + sh-Leptin-exo. Our results showed that the viability of PSN1 cells transfected with miR-224-3p mimics was significantly higher (**P < 0.01) than that of PSN1 cells not transfected with miR-224-3p mimics after CAFs-sh-Leptin exosome treatment (Fig. 6A). The expression level of miR-224-3p was significantly up-regulated (**P < 0.01) in the miR-224-3p mimic-transfected PSN1 cells (Fig. 6B). The colony formation results showed that the proliferative capacity of PSN1 cells transfected with miR-224-3p mimics was significantly increased compared to PSN1 cells (Fig. 6C). Transwell (**P < 0.01) and wound healing assays (**P < 0.01) showed that PSN1 cell migration and invasion were significantly increased in PSN1 cells transfected with miR-224-3p mimics (Fig. 6D, E). It indicated that miR-224-3p could alleviate the inhibitory effects of CAFs transfected with sh-Leptin on PSN1 cell viability, cell proliferation, invasion, and migration.

Fig. 6 miR-224-3p mitigates the inhibitory effect of CAFs knockdown of Leptin on PSN1 cells development. A CCK8 assay was performed in PSN1 cells. B RT-qPCR analysis of miR-224-3p. C Colony formation assay was performed in PSN1 cells. D The Transwell assay was used to assess PSN1 cells’ capacity for invasion. E Assays for wound healing were used to evaluate PSN1 cells’ capacity for migration. The data is shown as mean ± SD. Analyses were performed by Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001

Cancer-associated fibroblasts exosomal leptin promotes PSN1 cells by inhibiting ABL2 expression through upregulation of miR-224-3p

To determine the role of ABL2 in cell development in PSN1 cells, we simultaneously overexpressed ABL2 in PSN1 cells transfected with miR-224-3p mimics. The CCK8 results demonstrated that, in comparison to PSN1 cells transfected with miR-224-3p mimics, the cell viability of PSN1 cells concurrently transfected with pcDNA-ABL2 and miR-224-3p mimics was considerably increased (**P < 0.01) following CAFs-sh-Leptin exosome treatment (Fig. 7A). Western blotting detection of ABL2 expression levels indicated that ABL2 expression was significantly increased (*P < 0.05, **P < 0.01) in PSN1 cells transfected with both miR-224-3p mimics and pcDNA-ABL2 compared to PSN1 cells transfected with miR-224-3p mimics (Fig. 7B). The results of colony formation demonstrated that PSN1 cells transfected with pcDNA-ABL2 in addition to miR-224-3p mimics had a much lower proliferative ability than PSN1 cells transfected with miR-224-3p mimics alone (Fig. 7C). Cell migration and invasion were dramatically reduced in PSN1 cells transfected with both pcDNA-ABL2 and miR-224-3p mimics, according to transwell (**P < 0.01) and wound healing (**P < 0.01) experiments (Fig. 7D, E). Our results indicate that CAFs exosomal Leptin promotes the development of PSN1 cells by reducing ABL2 through upregulation of miR-224-3p (Fig. 8).

Fig. 7 CAFs exosomal Leptin promotes PSN1 cells by inhibiting ABL2 expression through upregulation of miR-224-3p. A PSN1 cells were used for the CCK8 test. B Western blot examination of PSN1’s ABL2. C PSN1 cells were used for the colony formation experiment. D The Transwell assay was used to assess PSN1 cells’ capacity for invasion. E Assays for wound healing were conducted to evaluate PSN1 cells’ capacity for migration. The data is shown as mean ± SD. Analyses were performed by one-way ANOVA, and the Sidak test was used as post hoc. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 8 Diagram showing how the Leptin/miR-224-3p/ABL2 axis controls the invasion and migration of pancreatic cancer cells originating from CAF. Exosomes carry CAF-derived Leptin, which enters pancreatic cancer cells and uses miR-224-3p to suppress ABL2 expression, therefore fostering the growth of cancerous biological lineages

Discussion

The TEM plays an important role in tumorigenesis and development. Tumor-associated fibroblasts are cancer-activated fibroblasts with myofibroblast-like properties in tumor tissues [26]. The phenotypic malignant transition of CAFs is closely related to tumor evolution, and CAFs are the main tissue component of the TEM [27]. Growing data highlights the significance of cell-to-cell communication in the TEM, which is essential for the growth, invasion, and metastasis of tumors. A previous research indicates that CAFs-secreted exosomes play a role in tumor development, invasion, and metastasis [28]. For example, exosomal SOX21-AS1 is secreted by PDAC cells, and HUVECs absorb it, promoting angiogenesis [29]. The overexpression of CD9 on the surface of CAF-derived EVs including ANXA6-EVs was shown to boost PDAC aggressive behavior in the work by Nigri et al. [30]. This leads to increased EMT, activation of the MAPK pathway, and PDAC proliferation. Leptin is an oncogenic adipokine that activates many pathways to mediate obesity-associated PDAC. By boosting proliferation and glucose uptake through leptin receptor-dependent activation of the AKT pathway, it encourages the growth of tumors 31. However, further research is necessary to fully understand the underlying mechanism of CAFs exosomes in PDAC, as it is currently unknown.

In our study, CAFs were isolated and extracted from tissues of PDCA patients, and after co-culturing CAFs with PDCA, we found that the co-culture system significantly promoted cell proliferation and invasion, but the specific regulation was not clear. Therefore, investigating the pertinent molecular regulation mechanisms of CAFs during invasion and metastasis in pancreatic cancer cells is extremely important from a research and therapeutic standpoint. According to studies, CAFs can participate in interactions between tumor cells by secreting a range of growth factors, cytokines, and chemokines in the form of exosomes. These interactions can induce tumor angiogenesis, cell proliferation, and the epithelial-mesenchymal transition, all of which can support tumor growth, invasion, and metastasis [32].

Our results indicated that CAFs may regulate pancreatic cancer development by secreting exosomes. Then, we treated PDAC using CAFs medium supernatant and CAFs medium supernatant with exosome release inhibitors, respectively, and the results showed that compared with PDAC with CAFs medium supernatant with exosome inhibitors, PDAC cells treated with CAFs medium supernatant showed a significant increase in cell proliferation and invasive ability. Further studies on exosomes revealed that Leptin was significantly highly expressed in CAFs exosomes. Leptin is involved in regulating the process of cancer development and progression. Nevertheless, the underlying mechanism of exosomal Leptin in PDAC metastasis remains unclear. However, the regulatory mechanism of Leptin in tumorigenesis and development needs to be further explored [33].

MicroRNAs (miRNAs) have been documented to play critical roles in cancer [34,35]. It has been suggested that Leptin may regulate tumor development through miRNAs [36]. Peng et al. found that Leptin promotes epithelial-mesenchymal transition and vascular regeneration in cholangiocarcinoma cells through the miR-122/PKM2 axis [37]. Our group found that miR-224-3p was significantly highly expressed in the co-culture system by RNA-seq experiments in the previous period and that miR-224-3p was significantly down-regulated in CAFs cells transfected with sh-Leptin and then co-cultured. Furthermore, our investigation reported that Leptin may promote the development of PDAC by upregulating miR-224-3p.

In order to explore the downstream proteins of miR-224-3p, we screened the genes with targeting relationships using miR-224-3p and and found that ABL2 was down-regulated in PDAC cells by detection. Our experiments revealed that miR-224-3p promotes PDCA by targeting ABL2.

This study has certain limitations. Our results proved that CAFs cells promote miR-224-3p levels by secreting Leptin, up-regulating Leptin expression in PSN1 cells, targeting down-regulation of ABL2, and promoting malignant biological behaviors such as proliferation, invasion, and migration of PSN1 cells, leading to pancreatic cancer progression.

Conclusion

In conclusion, this study indicates that Leptin in exosomes derived from CAFs promoted the proliferation and metastasis in PDAC. By controlling the miR-224-3p/ABL2 molecular axis, exosomal Leptin acts as a biomarker of PDAC and offers fresh insight into the development of exosome-based therapy in PDAC.

Author contributions

Yun Jin designed the study and revised the manuscript; Li Zhang analyzed the data and drafted the manuscript; Yesheng Chen performed cell culture, cell co-culture, small-molecule, isolation of exosomes and exosome uptake assay; Yihe Dai performed Immunofluorescence, Transfection and CCK8 assay; Weicheng Mou performed qRT-PCR, CM and Exo treatments and Wound healing assay; Pan Deng performed Western blotting; Yan Jin performed Colony formation assay; Jing Xu performed Transwell migration assay. All authors read and approved the final manuscript.

Funding

This work was supported by the Yunnan Science and Technology Department and Kunming Medical University Special Fund (Grant No. 202101AY070001-234) ,the Yunnan Digestive Endoscopy Clinical Medical Center Foundation for Health Commission of Yunnan Province (No.2022LCZXKF-XH02) of (ZX2019-01-02) and the Yunnan Fundamental Research Projects (grant NO. 202401AT070053).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethical approval

Prior to tissue collection, the study performed was approved by the First People’s Hospital of Yunnan Province Research Ethics Committee (No.KHLL2022-KY145) in October 2022.

Competing interests

The authors declare no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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