Background
Pancreatic cancer shows rapid growth and metastasis and is one of the most fatal human cancers. Over half of patients are diagnosed at a stage where metastases have developed, and the overall 5-year survival rate for the pancreatic patients with metastases is only 10% [
1,
2]. Only 15–20% of patients have resectable disease at the time of diagnosis [
3]. The majority of patients have local and distant micrometastases at the time of surgery. Therefore, disease recurrence following operation is very high. Hattangadi et al. reported that the peritoneum (55%) and the liver (53%) were the most common sites of recurrence [
4].
Adjuvant therapies such as radiotherapy and chemotherapy (or a combination) help improve the prognosis of pancreatic cancer. However, their effectiveness is limited. Gemcitabine [
5] or S-1 [
6] is the most common adjuvant chemotherapy for pancreatic cancer after surgery. Recent studies showed that in patients with advanced disease, combining gemcitabine with other systemic agents can improve patient outcome compared with standard gemcitabine monotherapy [
7]. For example, the combined therapy with gemcitabine plus albumin-bound paclitaxel reached 8.7 months of median overall survival in phase III trial [
2]. Although the prognosis of this disease has been improved, these results were not acceptable for the patients in clinical. Thus, the identification of novel and effective strategies is critical to improve the prognosis for pancreatic cancer patients.
Mesothelin is a 40-kDa glycosylphosphatidyl inositol-linked protein that is expressed in mesothelial cells lining the peritoneum among the human normal tissues [
8]. Mesothelin is highly overexpressed in various types of cancers, such as mesothelioma [
8], ovarian [
8] and pancreatic cancer [
9]. Recent studies have demonstrated a role for mesothelin in cell survival, cell migration, cell invasion and tumor progression [
10]. The expression of mesothelin is also related to epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs) [
11]. We previously reported the significance of mesothelin expression in the poor prognosis of gastrointestinal cancer patients [
12‐
17]. Because of the expression pattern, mesothelin has recently been targeted for immunotherapy [
18].
Amatuximab is an investigational chimeric high-affinity monoclonal IgG1/k antibody that targets mesothelin [
19]. In vitro, Amatuximab elicits antibody-dependent cellular cytotoxicity against mesothelin-expressing tumor cell lines and inhibits heterotypic cell adhesion of mesothelin-positive tumor cells to CA125-expressing tumor cells. Based on its safety in a Phase I study and preclinical studies showing synergy with chemotherapy [
20], Amatuximab was combined with gemcitabine in a Phase II study (NCT00570713) in patients with previously untreated unresectable stage 3 or 4 pancreatic cancer. However, the study did not demonstrate the efficacy of combined therapy compared with either Amatuximab or chemotherapy alone. The trial included patients with unresectable bulky pancreatic cancer, and we suspect that Amatuximab could not be delivered to pancreatic tumor cells in these patients.
We previously reported that Amatuximab monotherapy reduced the peritoneal mass volume in peritonitis model mouse of human pancreatic cancer cells and observed the appearance of sherbet-like aggregates in this model [
21]. We demonstrated that the combined therapy using Amatuximab and gemcitabine reduced the peritoneal mass and eliminated the sherbet-like aggregates. However, the molecular mechanism of this phenomenon is not yet understood.
The aim of this study is to clarify the role and potential mechanism of mesothelin receptor blockage by Amatuximab in human pancreatic cell lines both in vitro and in vivo.
Methods
Cell culture
The human pancreatic adenocarcinoma AsPC-1 and Capan-2 cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin. Panc-1 and MIA Paca-2 cell lines were cultured in DMEM medium supplemented with 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin. The cells were cultured and maintained as previously described [
21]. We obtained AsPC-1 cells (ATCC® CRL-1682™) in 2010, MIA Paca-2 (ATCC® CRL-1420™) cells and Panc-1 (ATCC® CRL-1469™) cells in 2016 and Capan-2 cells (ATCC® HTB-80™) in 2019. All cell lines were obtained from ATCC (Manassas, VA, USA). After obtaining cells from ATCC, cells were frozen at low passage (p3-p5), and then thawed and used at passage 10 or lower. These cell lines were tested for mycoplasma contamination at regular intervals. we did not authenticate these cell lines.
Reagents
Amatuximab (also known as MORAb-009) was obtained from MORPHOTEK INC (Exton, PA, USA). Gemcitabine hydrochloride (Gemzar) was purchased from Eli Lilly (Indianapolis, IN, USA).
Proliferation assay (cell counting method)
Pancreatic cancer cell lines were seeded at a density of 1.5 × 105 cells per 2 mL medium in a 35 mm plate, and Amatuximab was added at various concentrations (0, 0.01, 1 or 100 μg/mL). After 48 h, cells were harvested by Trypsin/EDTA (0.05%) (Thermo Fisher Scientific Inc., Waltham, MA, USA). Next, 50 μL of the cell suspension were mixed with 50 μL of 0.4% trypan blue (#207–17,081, Wako, Tokyo, Japan) by gentle pipetting and then 20 μL of the mix were loaded into a chamber in a hemocytometer. Counts were performed in triplicate by one analyst under a 40× objective according to standard methodology using BIOREVO (#BZ-9000, KEYENCE, Osaka, Japan).
Invasion assay
In vitro invasion assays were performed using the BD Bio-Coat Matrigel invasion assay system (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. Briefly, cells were seeded into Matrigel-precoated Transwell chambers consisting of polycarbonate membranes with 8.0 μm pores. The Transwell chambers were then placed into 24-well plates, into which basal medium only or basal medium containing 100 μg/mL Amatuximab or control IgG was added. We decided the concentration of Amatuximab or control IgG from our data in proliferation assay and the past literature, which showed the strong blockage of adhesion between mesothelin and CA125 in this concentration of Amatuximab [
19]. After incubating cells for 24 h, the upper surface of the Transwell chambers was wiped with a cotton swab and the invading cells were fixed and stained using the Diff-Quick cell staining kit (Dade Behring, Inc., Newark, DE, USA). We counted the number of invading cells in five random microscopic fields (200× magnification) using BIOREVO (#BZ-9000, KEYENCE, Osaka, Japan).
Migration assay
In vitro migration assays were performed using the BD Bio-Coat migration assay system (BD Biosciences) according to the manufacturer’s instructions and the methodology for the invasion assays.
Cell growth inhibition assay
Pancreatic cancer cells were seeded at a density of 1.5 × 10
5 cells per 35 mm dish in culture medium containing 10% FBS with 1 μM gemcitabine and 100 μg/ml Amatuximab or control IgG. After 48 h, the numbers of viable cells were counted as described in the previous section. The concentration of gemcitabine was determined based on previous studies [
22] and our preliminary experiments. Counts were performed in triplicate by one analyst under a 40× objective according to standard methodology using BIOREVO (#BZ-9000, KEYENCE, Osaka, Japan).
Western blot analysis
Cells were seeded at a density of 1.5 × 10
6 cells in 2 ml medium in a 100 mm dish. Amatuximab (100 μg/ml) or control IgG was added to medium at the time of seeding and cells were cultured for 24 or 48 h. After cells were harvested, western blot analysis was performed as previously described [
21]. The cells were homogenized at 4 °C in lysis buffer (0.1% SDS, 1% Igepal CA-630, 0.5% sodium deoxycholate) and a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Cell lysates (20–50 μg) were resolved by electrophoresis on polyacrylamide gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking the membranes in 5% non-fat dry milk or 3% bovine serum albumin in Tris-buffered saline for 1 h at room temp, the blots were hybridized overnight at 4 °C with primary antibodies. After hybridization with secondary antibodies conjugated with HRP (Cell Signaling Technology, Danvers, MA, USA), immunocomplexes were visualized using an enhanced chemiluminescence kit (GE Healthcare, Chalfont St. Giles, UK). Densitometric analysis of western blots was performed using a ChemiDoc XRS Plus system with Image Lab Software (Bio-Rad, Hercules, CA, USA). Primary antibodies are described in Supplemental Table
1.
FACS analysis
We harvested, washed and resuspended the cells in ice-cold PBS. Primary antibody was added at 2 μg/ml and incubated for 1 h on ice. Cells were then washed with PBS, incubated with Alexa Fluor 488 goat-anti-mouse secondary antibody (Invitrogen, Carlsbad, CA, USA) diluted to 1 μg/ml, washed as above and analyzed on BD FACS Canto™ II flow cytometer (BD Biosciences).
Animals
Six-week-old female BALB/c Slc-nu/nu mice (body weight 14-19 g) were purchased from CLEA Japan, INC. (Tokyo, Japan). Mice were maintained under specific pathogen-free conditions in laminar-flow benches and were allowed to adapt to the environment for 1 or 2 weeks before experiments. All procedures involving animals and their care were approved by the Institutional Animal Care and Use Committee of National University Corporation Hokkaido University and were conducted under National University Corporation Hokkaido University Regulations on Animal Experimentation.
Generation of peritoneal model mouse using AsPC-1 cells
We generated the peritoneal model mouse as described previously [
21]. Briefly, 5 × 10
6 AsPC-1 cells were injected to the abdominal cavity of 7-week-old nude mice. The mice were divided to two groups randomly. Amatuximab (200 mg/kg) (
n = 6) or isotype control IgG (200 mg/kg) (
n = 4), so totally using 10 mice, was administered by injection into the abdominal cavity every 2 days for 22 days. Two groups were maintained in the same type of cage and the environment. The mice were euthanized by isoflurane inhalation followed by cervical dislocation on day 22 and peritoneal masses and sherbet-like aggregations were harvested. No expected or unexpected adverse events happened. The resected specimens were fixed with formalin for 6 h immediately after harvest and embedded in paraffin, and then sliced sequentially at a thickness of 3 μm. We established the humane endpoints that when the model mouse got poor physical condition during the experiment, we planned to euthanize by isoflurane inhalation followed by cervical dislocation. No animals were adapted to the criteria. We set no specific criteria used for including and excluding animals during the analysis. The first author understood the group allocation totally through the experiment. These protocols were prepared before the study.
Immunohistochemistry
We performed the immunohistochemistry using the EnVision+ System-HRP (Dako Japan, Tokyo), in the same method as described previously [
21]. Briefly, the sections were mounted on glass slides, deparaffinized, and rehydrated through several graded ethanol. We retrieved the antigens in Dako EnVision FLEX Target Retrieval Solution low pH using Dako PT Link for 20 min at 97 °C according to the manufacturer’s instructions (Dako, Japan). After we blocked the endogenous peroxidase activity with 0.03% hydrogen peroxide, the tissue sections were incubated with primary antibodies at room temp for 30 min and then we reacted the samples with a dextran polymer reagent combined with secondary antibodies and peroxidase for 30 min at room temperature. Specific antigen-antibody reactions were visualized with diaminobenzidine chromogen applied for 10 min. After that, these slides were stained with hematoxylin, dehydrated and mounted. The primary antibodies are described in Supplemental Table
1. The percentage of stain-positive cells and strength of staining were evaluated in the analysis. The representative images were taken using OLYMPUS equipment described below (microscope BX63–44-FLD-2, Camera DP80, software cellSens, OLYMPUS, Tokyo, Japan).
Quantitative RT-PCR
Total RNA from tissues and cells was isolated using TRIzol reagent (Invitrogen). Relative levels of E-cadherin, ALDH1, CD44 and c-MET mRNA were examined using SYBR Green Real-time Quantitative RT-PCR (qRT-PCR) (LightCycler 480 Roche, Switzerland) and were normalized to levels of GAPDH mRNA. We described the primers in Supplemental Table
2. Relative expression was calculated using the 2
-ΔΔCT method. All qRT-PCR analyses were carried out in triplicate, and the data are presented as means±standard of the means.
Statistical analysis
Data are shown as mean ± SD. Significance of differences was determined by Student’s t-test, one-way ANOVA, two-way ANOVA or Mann-Whitney U-test. P < 0.05 was considered statistically significant. All statistical procedures were performed using JMP Pro 10 (SAS Institute, Japan).
Discussion
In the present study, we demonstrated significant effects of Amatuximab by mesothelin blockage in pancreatic cancer cells. Our in vitro results showed that Amatuximab treatment suppressed the invasiveness and gemcitabine sensitivity of AsPC-1 and Capan-2 pancreatic cancer cells. Amatuximab also induced the downregulation of CSC-related proteins such as pMET in these cell lines.
A full understanding of the biological functions of mesothelin is lacking given that mesothelin knockout mice do not show any developmental phenotype [
23]. Recent reports indicate that mesothelin may play an important role in cell adherence, cell survival/proliferation, tumor progression and chemoresistance [
24]. In pancreatic cancer, Stat3 plays a pivotal role in oncogenic transformation [
25,
26], cell survival, proliferation [
25,
27] and resistance to apoptosis [
28]. Stat3 is also aberrantly activated in a subset of pancreatic tumor tissues and cell lines [
27]. Bharadwaj et al. showed that MSLN upregulation induces the activation of Stat3 in pancreatic cancer cells [
29]. Furthermore, the authors showed that mesothelin induced an NFκB/Akt-dependent anti-apoptotic pathway that can protect pancreatic cancer cells from TNF-α-induced apoptosis, and this was a probable mechanism of pancreatic cancer cell survival in midst of inflammation and inflammatory mediators. Other groups found that mesothelin could confer resistance to cytotoxic drug-induced apoptosis via the ERK signaling pathway [
30,
31]. Some mesothelin monoclonal antibodies were reported as unable to inhibit cancer cell proliferation because the majority of these antibodies target N terminal region I rather than a key signaling domain in mesothelin. Our results showed that Amatuximab did not inhibit cell proliferation, but the agent suppressed the invasiveness and chemoresistance in AsPC-1 and Capan-2 pancreatic cancer cells. We could observe the slight changes of the molecule expression level in the pathway described above in AsPC-1 only. These results might be caused by the difference of the expression level of mesothelin, that of the expression pattern of MMP7, or that of the mutation pattern of p53 between AsPC-1 cells and Capan-2 cells [
32,
33]. These factors might lead to the dissociated results.
CSCs are a population of undifferentiated tumorigenic cells that are responsible for tumor initiation, tumor maintenance and tumor cell spreading to distant organ sites [
34]. These cells exhibit unlimited proliferation potential, self-renewal ability and the capacity for the generation of a progeny of differentiated cells which constitute the major tumor population. CSCs can be characterized by chemoresistance [
35], multipotency, tumorigenicity [
36], stem gene expression [
37] and aldehyde dehydrogenase activity [
38]. Several studies showed that chemotherapy treatment in pancreatic cancer cell lines led to an increased number of CSCs [
39,
40]. CSCs are also thought to be related to the invasiveness of cancer cells [
41]. Ortensi et al. reported that brain tumor cells enriched for stem cell markers displayed greater migratory and invasive potential compared with stem cell marker-negative tumor cells. We can identify the CSCs by the expression of CSC-specific cell surface markers, such as CD133, CD44, c-MET and ALDH1 [
42]. He et al. showed that mesothelin regulates EMT and CSC traits. Knockdown of mesothelin upregulates epithelial and adhesion molecules on one side and on the other side that downregulates mesenchymal and CSC regulatory genes that relieves self-renewal, proliferation, dissemination and metastasis of cancer cells. Overexpression of mesothelin in normal cells stimulates anchorage-independent growth, migration and invasion [
11]. In our study, the mesothelin blockage by Amatuximab directly suppressed the expression level of pMET and led to suppression of malignant features of AsPC-1 and Capan-2 pancreatic cancer cells. To the best of our knowledge, this is the first study to establish a relationship between mesothelin blockage and CSC markers.
MET (also known as c-MET) is a receptor of the tyrosine kinase family that acts as a proto-oncogene and is stimulated by hepatocyte growth factor to mediate motility, invasion, and metastasis [
43]. The intracellular signaling cascades activated by MET include the RAS-MAPK and PI3K-Akt pathways, NFκB and Wnt/GSK-3β/β-Catenin signaling [
44]. The levels of c-MET are increased in pancreatic carcinoma where c-MET signaling induces growth and invasion and some authors have reported c-MET as a stem cell marker in pancreatic tissue [
44]. In our results, we concluded that MET was the important factor in the effects of Amatuximab by mesothelin blockage in pancreatic cancer cells.
Although the molecular changes of AsPC-1 show the limited effects of Amatuximab in vitro study, drastic changes observed in vivo study. Using the peritoneal dissemination model, we demonstrated that several proteins related to proliferation, cancer stemness and chemoresistance were suppressed in the cancer cell clusters (which we named sherbet-like aggregates) that were blocked to attach to peritoneum by mesothelin blockage. We previously demonstrated that the sherbet-like aggregates were sensitive to gemcitabine [
21]. Marjanovic presented a plastic CSC theory as a model of tumor heterogeneity [
45]. The classical CSC theory proposes that tumor heterogeneity would arise when cancer cells within a given tumor reside in different states of stemness or differentiation. Critical to this theory is the notion that CSC-to-non-CSC conversion is a unidirectional process. The plastic CSC theory describes an evolving model in which bidirectional conversions exist between non-CSCs and CSCs that are controlled by extrinsic features, such as extracellular matrix or blood vessels, and intrinsic features, such as genetic or epigenetic changes. This model implies that non-CSCs can continually create CSC populations throughout tumorigenesis. In other words, this theory suggests that CSCs are kept in differentiated states by their surroundings, and their malignancy is suppressed in this condition. Stankevicius et al. reported the importance of microenvironment, including scaffold, for the cancer cell stemness in human colorectal cancer cells [
46]. The authors demonstrated that the expressions of several CSC markers were increased in 3D cultures considered as scaffold compared with those in 2D monolayer cultures. Our results suggested that not only the direct effects of Amatuximab but also the environmental changes induced by Amatuximab could control the stemness of pancreatic cancer cells expressing high mesothelin, and these resulted in an improvement of sensitivity for gemcitabine in mesothelin-expressing pancreatic cancer cells.
There are several limitations in the present study. First, we did not demonstrate the relationship between the expression of mesothelin and CSCs. We need to examine the expression changes of CSC molecules using the cells those expression of mesothelin were genetically controlled. Second, we could not confirm the definite role of p-MET downregulation in the antitumor activity of Amatuximab directly in this study. To confirm that, we should carry out the experiment such as invasion assay, migration assay and gemcitabine sensitivity experiments using AsPC-1 cells or Capan-2 cells those expression of p-MET is controlled genetically or using inhibitor for p-MET. Third, we carried out our experiments using whole pancreatic cancer cells, not dividing cells to CSCs or non-CSCs. We need to determine the specific effects of Amatuximab on CSC population separated by sorting technique for further investigation in the future. Fourth, our in vitro data could not show the relationship between the inhibition of invasion/migration and enhancement of antitumor activity of gemcitabine in mesothelin-high pancreatic cancer cell lines. The setting of the in vitro experiments could not mimic the condition of ex vivo study completely. 3D culture might be suitable for the investigation of cancer stemness and of our ex vivo study. Shishido reported the relationship between the ovarian cancer cells and peritoneal cells in the co-culture experiments [
47]. The ovarian cancer cells showed the stem like features more strongly in co-culture condition than in monoculture. Co-culture study might be useful in our investigation of CSCs. Fifth, our data could not reveal how Amatuximab caused the generations of sherbet like aggregates. Some previous reports showed that CA125 was expressed in the peritoneum in the mouse [
48] and that Amatuximab inhibited the interaction of mesothelin-CA125 [
49]. Although we tried to reveal the direct evidence showing that Amatuximab inhibited the adhesion of cancer cells to peritoneum, we could not confirm the mechanism in this study. Additionally, we examined the in vivo experiments using only AsPC-1 cell lines. To validate the efficacy of Amatuximab for pancreatic cancer cells and the relationship between effect of Amatuximab and expression of mesothelin, we need to perform the experiment using other several pancreatic cancer cell lines. Our findings are limited to pancreatic cancer cell lines. Further research is needed in other types of cancer cells that highly express mesothelin. Finally, we did not demonstrate the relationship between the microenvironment and the phenomenon in the previous study. We need to reveal these mechanisms by establishing the experimental system in those the microenvironment was controlled.
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