BAG3‐positive pancreatic stellate cells promote migration and invasion of pancreatic ductal adenocarcinoma

Abstract BAG3 is constitutively expressed in multiple types of cancer cells and its high expression is associated with tumour progression and poor prognosis of PDAC. However, little is known about the role of BAG3 in the regulation of stromal microenvironment of PDAC. The current study demonstrated that beside PDAC tumour cells, BAG3 was also expressed in some activated stroma cells in PDAC tissue, as well as in activated PSCs. In addition, the current study demonstrated that BAG3 expression in PSCs was involved in maintenance of PSCs activation and promotion of PDACs invasion via releasing multiple cytokines. The current study demonstrated that BAG3‐positive PSCs promoted invasion of PDACs via IL‐8, MCP1, TGF‐β2 and IGFBP2 in a paracrine manner. Furthermore, BAG3 sustained PSCs activation through IL‐6, TGF‐β2 and IGFBP2 in an autocrine manner. Thereby, the current study provides a new insight into the involvement of BAG3 in remodelling of stromal microenvironment favourable for malignant progression of PDAC, indicating that BAG3 might serve as a potential target for anti‐fibrosis of PDAC.

extracellular matrix (ECM). 9,10 These complex and heterogeneous stromal components constitute a sophisticated microenvironment that facilitates tumour growth and metastasis. Complex interactions between stromal cells and pancreatic cancer cells exert influences upon each other. On one hand, tumour cells secrete pro-inflammatory soluble factors such as TGF-β1, PDGF, TNF-α and IL-1/6, which recruit and activate PSCs/CAFs. On the other hand, activated PSC/ CAFs secrete large amounts of extracellular matrix (ECM) proteins and signalling factors to remodel tumour microenvironment-assisting malignant progression of PDAC. 11 Based on the key role of tumour stroma, a number of stromal-targeting strategies in PDAC have been developed. However, so far none of the stromal-ablation therapeutic strategies have improved patient survival and some of them even had the adverse effect, [12][13][14] suggesting that more studies are needed to further decipher the complexity of PDAC tumour-stromal interactions.
Bcl2-associated athanogene (BAG) 3 belongs to BAG family of cochaperones that interact with the ATPase domain of the heat shock protein 70 (Hsp70) via the carboxyl terminal BAG domain. 15 Besides, BAG3 has multiple domains such as WW domain, proline-rich (PxxP) domain and IPV (Ile-Pro-Val) motifs, providing the structural basis for interactions with other partners. By interacting with different partners, BAG3 protein participates in modulating a variety of biological processes including anti-apoptosis, autophagy, cytoskeleton organization and cell motility. BAG3 is constitutively expressed in many cancer tissues, including pancreatic ductal adenocarcinoma cells (PDACs), 16 melanomas, 17 colorectal carcinomas 18 and thyroid carcinomas, 19 contributing to tumour growth, invasiveness and resistance to therapy. More recent literature shows that BAG3 can be secreted by pancreatic cancer cells. 20,21 The secreted BAG3 can bind and activate stromal macrophages to promote pancreatic cancer cells growth in turn. However, involvement of BAG3 in remodelling of stromal microenvironment in PDAC is not fully studied.
In the current study, we observe that conditioned media from BAG3-overexpression PSCs facilitate migration and invasion of PDACs and promote proliferation and migration of PSCs.
Furthermore, we demonstrate that ectopic expression of BAG3 in PSCs remodels stromal microenvironment of PDACs through mediating secretion of some cytokines/chemokines. These cytokines/ chemokines exert an influence on PDACs and PSCs in a paracrine and autocrine manner respectively. Thereby, we provide a new insight into the involvement of BAG3 in interaction between PDACs and PSCs, indicating that BAG3 might serve as a potential target for anti-fibrosis of PDAC.

| Patients and tissue samples
In this study, we enroled 30 patients with PDAC who had undergone pancreatic surgery at Liaoning Cancer Hospital & Institute between July 2016 and July 2018. Eligible patients were the participants diagnosed pathologically with PDAC by two qualified pathologists according to the WHO classification. Those who accepted radiotherapy, chemotherapy or other treatments before surgery were excluded from this study. All tissue specimens were processed in formalin fixation for 24 hours and then embedded in paraffin. The protocol was authorized by the Ethics Committee of China Medical University and the informed consent was obtained from each participant.

| Immunohistochemistry
Briefly, 4-micrometre sections were cut from Paraffin-embedded tissue blocks, mounted on poly-L-lysine-coated slides, deparaffinized and hydrated. After being boiled in citric acid buffer for 90 seconds and blocked by hydrogen peroxide and normal goat serum, sections were incubated with anti-BAG3 antibody (GeneTex) and anti-α-SMA antibody (Abcam) for 2 hours at 25°C. Subsequent to incubation with secondary antibodies for one hour, sections were detected using the Streptavidin-Peroxidase complex (

| Collection of conditioned media
HPanSteC cells (5 × 10 6 cells) were cultured in T-175 flasks in Stellate Cell Medium. On the next day, the cells were washed two to three times with PBS until no suspended dead cells were left.
Then the cells were cultured in Stellate Cell Medium containing 1% FCS for additional 72 hours. Thereafter, the supernatants were collected, aliquoted into 1.5-mL tubes and stored at −80°C until usage.

| Growth and migration assays using real-time cellular analysis (RTCA)
Real-time monitoring of cell proliferation and migration were performed with the xCELLigence system (ACEA Biosciences, San

| Migration and invasion assays by transwell
Cell migration/invasion assays were measured in Corning 3422 transwell permeable support chambers with 8-mm pore filter inserts in 24well plates (Corning Incorporated Life Sciences, Munich, Germany). The invasion assay shared the same procedures, except that the filter inserts were pre-coated with Matrigel at a 1:4 dilution in DMEM. Briefly, 600 µL DMEM containing 10% FBS was added to the lower chamber.
A total of 100 µL cells in serum-free DMEM were seeded into the top chamber (3 × 104 cells/well). After incubation for 24 hours, the cells on the upper surface were removed using a cotton swab. The migrated/ invaded cells on the lower surfaces of inserts were fixed in methanol and stained with crystal violet. The migrated/invaded cells were counted in 10 representative microscopic fields and photographed.  (B) HPanSteC cells were treated with TGF-β1 (1, 2, 5 and 10 ng/mL) for 48 hours and the protein levels of BAG3 and α-SMA were analysed by Western blot. (C) HPanSteC cells were treated with the indicated concentrations of TGF-β1 for 24 h, the BAG3 mRNA level was detected by RT-qPCR. (D and E) HPanSteC cells were stimulated with PDGF and IL6, mRNA level and protein level of BAG3 were analysed by RT-qPCR and Western blotting respectively. (F) HPanSteC cells were infected with lentivirus containing shRNAs against BAG3 (shBAG3) for 48 h, then treated with 10 ng/mL TGF-β1 for additional 24 h. Western blotting was performed to detect the protein levels of BAG3 and α-SMA. *P < 0.01. Error bars indicate means ± SD Briefly, the membranes were incubated in conditioned medium overnight at 4°C after half an hour of incubation with blocking buffer.

| Human cytokine antibody array
Membranes were washed three times with wash buffer Ⅰ and Ⅱ respectively, followed by incubation with biotinylated antibodies mixture overnight at 4°C. Then, membranes were incubated with horseradish peroxidase-conjugated streptavidin overnight at 4°C after washing. Finally, after full washing, the membranes were detected using detection buffer and scanned with an imaging system (Tanon-4200; Tanon Science & Technology Co., Ltd).

| Enzyme-linked immunosorbent assays
In order to quantify the content of some cytokines/chemokines in supernatant of HPanSteC cells, enzyme-linked immunosorbent assays (ELISA) were conducted according to the provided instructions.
ELISA kits for all factors were purchased from RayBiotech.

| RNA isolation and Quantitative reverse transcriptase PCR
RNA from cultured cells was isolated using RNeasy® Mini Kit (Qiagen), followed by cDNA synthesis using GoScript TM Reverse Transcription System (Promega). Quantitative reverse transcriptase PCR was performed with GoTaq® qPCR Master Mix (Promega) on the ABI prism 7000 sequence detection system (Applied Biosystems, Eugene, OR). The results for each sample were normalized to the 18SrRNA. All assays were conducted at least three times.

| Western blot analysis
Cells were lysed by RIPA lysis buffer (Thermo Fisher) supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Protein amount was determined using the BCA protein assay kit (Thermo Fisher).
Twenty micrograms of total protein was separated on 10% SDS-PAGE and transferred to PVDF membrane (Millipore). The membranes were blocked in 5% skimmed milk in Tris-buffered saline buffer with 1% Tween-20 (TBST) for 1 hour at room temperature, followed by incubation with primary antibodies overnight at 4°C.
Subsequently, the membranes were subjected to HRP-conjugated

| Statistical analysis
SPSS (16.0) software (SPSS, Chicago, IL) was adopted for statistical analysis. All results were presented as the mean ± standard deviation. Data were analysed by Student's t-test. All tests were twotailed and P < 0.05 was considered statistically significant.

| BAG3 expression is up-regulated in activated PSCs
Immunohistochemistry staining demonstrated that BAG3 was expressed in the stoma of some PDAC tissues, accompanied by positive expression of alpha smooth muscle actin (α-SMA) ( Figure 1A). α-SMA is one of the critical hallmarks of activated BAG3 mRNA ( Figure 1D) and protein ( Figure 1E) expression levels.
To study the potential involvement of BAG3 up-regulation in activation of PSCs, BAG3 was knocked down using two distinct shR-NAs against BAG3 (shBAG3) ( Figure 1F). Importantly, knockdown of BAG3 significantly decreased induction of α-SMA expression by TGF-β1 ( Figure 1F). These data suggested that except for tumour cells, BAG3 was also highly expressed in some activated PSCs in PDAC tissues.

| Ectopic BAG3 overexpression promotes proliferation and migration of PSCs
To observe the potential effect of BAG3 on PSCs, HPanSteC cells were infected with lentivirus vectors harbouring BAG3 gene.

Western blot analyses indicated that BAG3 elevation in PSCs led
to a significant increase in α-SMA (Figure 2A). RTCA ( Figure 2B) and Edu incorporation ( Figure 2C) demonstrated that up-regulation of BAG3 promoted proliferation of HPanSteC cells. Moreover, BAG3 elevation facilitated migration of HPanSteC cells, as assessed by transwell migration assay ( Figure 2D) and RTCA analysis ( Figure 2E). These data indicated that BAG3 overexpression per se could activate PSCs and promote growth and migration of PSCs.

| Conditional media from PSCS with BAG3 overexpression facilitates migration and invasion of PDACS and proliferation and migration of PSCS themselves
Activated PSCs can exert profound impact on PDACs through secretion of various pro-inflammatory cytokines/growth factors.
Therefore, the influence of BAG3 elevation in PSCs on PDACs was then explored. Edu incorporation assays found that prolifera- by Edu incorporation and transwell migration assay respectively.
These results implied us that BAG3-positive PSCs might release some factors that promote PDACs invasion and sustain PSCs activation. and IGFBP2 mRNA levels, while had no effect on MCP1, CXCL6 or TGF-β2 mRNA expression ( Figure 4D). The results suggested that complicated mechanisms might underlie altering secretory profile of PSCs by BAG3 up-regulation.

| Implication of IL-6, TGF-β2 and IGFBP2 in maintenance of HPanSteC activation by BAG3 in an autocrine manner
To explore the potential involvement of secretory factors in sus- HPanSteC cells (Figure 5A-B). As CXCL6 release was decreased in HPanSteC cells with BAG3 overexpression ( Figure 4A,C), recombinant CXCL6 (reCXCL6) was then included in the culture media.
Compared with bovine serum albumin (BSA), reCXCL6 did not alter migration of HPanSteC cells ( Figure 5C-D). Edu incorporation experiments demonstrated that proliferation of BAG3-overexpressed HPanSteC cells was inhibited by addition of IL-6 and IGFBP2 antibodies, while other antibodies had no obvious effects ( Figure 5E). Neither reCXCL6 affected proliferation of BAG3-overexpressed HPanSteC cells ( Figure 5F). These data indicated that BAG3 might promote release of IL-6, TGF-β2 and IGFBP2 by PSCs to sustain their own activation.

| D ISCUSS I ON
BAG3 is up-regulated in cancer cells and its overexpression is correlated with tumour progression and poor prognosis of PDAC. 22,23 Recent study has demonstrated that BAG3 can be secreted by PDAC cells and secreted BAG3 promotes pancreatic ductal adenocarcinoma proliferation via activating stromal macrophages in tumour microenvironment. 20 Previous reports have mainly highlighted the oncogenic role of BAG3 highly expressed by cancer cells themselves.
The current study demonstrates that BAG3 produced by stromal cells plays a different role, focusing on tumour microenvironment.
In this study, we observed that beside in PDACs, BAG3 was also significantly expressed in the stroma of some PDAC tissues. In vitro, we found that BAG3 was hardly expressed in quiescent PSCs, while de novo expression of BAG3 was marked induced once PSCs were activated by TGF-β1, PDGF and IL-6. Importantly, knockdown of BAG3 decreased the extent of PSCs activation induced by TGF-β1, indicating that BAG3 at least partially implicated in PSCs activation.
Furthermore, ectopic expression of BAG3 through lentivirus infection could activate PSCs, evidenced by elevated α-SMA expression.
Besides, ectopic BAG3 overexpression directly stimulated proliferation and migration of PSCs. These data suggest that BAG3-positive PSCs might play a potential role in remodelling of tumour microenvironment in PDAC.
It is well known that tumour microenvironment plays a pivotal role in cancer progression, metastasis and chemotherapy resistance. 24,25 The pancreatic tumour microenvironment is comprised of various cells including stellate cells, fibroblast, immune cells, as well as blood vessels, extracellular matrix proteins. 26 The interaction between tumour microenvironment and PDAC cells has extensively been demonstrated. 4 PSCs, one of important cellular components in stroma of PDAC, exist in two main forms: quiescent and activated. 27 Quiescent PSCs have key function in regulation of extracellular matrix turnover and in maintenance of normal tissue architecture. 27  Alternatively, we detected the secretion levels of the above cytokines and growth factors; we cannot exclude the possibility that BAG3 might regulate release of cytokines and growth factors by regulating cytoskeleton. A previous study reported that BAG3 could regulate insulin secretion. 39 The exact mechanisms by which BAG3 regulates secretion of multiple key protein factors in PSCs need further investigation.
In conclusion, the current study demonstrated that BAG3 ex-

CO N FLI C T O F I NTE R E S T
The authors declared there are no competing financial interests.