Research ArticleClinical Studies

Impact of Extravasated Platelet Activation and Podoplanin-positive Cancer-associated Fibroblasts in Pancreatic Cancer Stroma

TOMOHARU MIYASHITA, HIDEHIRO TAJIMA, RYOSUKE GABATA, MITSUYOSHI OKAZAKI, HIROYUKI SHIMBASHI, YOSHINAO OHBATAKE, KOICHI OKAMOTO, SINICHI NAKANUMA, SEISHO SAKAI, ISAMU MAKINO, JUN KINOSHITA, HIRONORI HAYASHI, KEISHI NAKAMURA, HIROYUKI TAKAMURA, ITASU NINOMIYA, SACHIO FUSHIDA, JOHN W. HARMON and TETSUO OHTA
Anticancer Research October 2019, 39 (10) 5565-5572; DOI: https://doi.org/10.21873/anticanres.13750

Abstract

Background/Aim: The aim of the study was to evaluate the status of extravasated platelet activation (EPA) surrounding podoplanin (PDPN)-positive cancer-associated fibroblasts (CAFs) in pancreatic cancer stroma by neoadjuvant chemotherapy. Patients and Methods: A total of 74 patients were enrolled in this study. We investigated CD42b and PDPN expression in the groups of untreated, gemcitabine (GEM) alone, GEM plus S-1 (GS) and GEM plus nab-paclitaxel (GnP). Results: CD42b expression in surrounding CAFs was observed in 58% patients. CD42b expression was significantly correlated with PDPN expression. CD42b-positive cases were significantly lower in the group treated with GnP than in the untreated group and groups treated with GEM alone or GS. PDPN expression was reduced in the GnP group, as revealed by markedly disorganized collagen and a low density of PDPN-positive fibroblasts. There was a significantly lower CD42b expression and fewer PDPN-positive fibroblasts in the GnP group than in untreated, GEM alone, and GS groups, but there was no significant difference between the latter three groups. Conclusion: There is a significant association between EPA and PDPN-positive CAFs in pancreatic cancer stroma. Our data suggest that the GnP regimen decreases EPA through PDPN-positive CAF depletion.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer, ranked as the 12th most common cancer in the world and the 7th most frequent cause of cancer death worldwide for both sexes in 2012 (1). In the United States, PDAC is anticipated to become the second leading cause of cancer-related death by 2030 (2). In the Asia-Pacific region, such as Armenia, Japan, Kazakhstan, New Zealand, Australia, and Korea, the mortality rate of PDAC is high. Furthermore, in other countries such as China, the death rate due to PDAC is rising (3). Several factors, such as a delay in diagnosis, the aggressiveness of the established tumors, lack of proper therapy, and the development of drug resistance, are contributing to the low survival rate (4). Among them, chemoresistance is a major factor contributing to such a poor prognosis (5, 6). Desmoplasia, in which the stroma can expand to more than 50% of the PDAC tissue and the tumor microenvironment, consists of cancer-associated fibroblasts (CAFs), inflammatory cells, small blood vessels, and extracellular matrix forming a desmoplastic stroma that promotes tumor development and inhibits drug penetration and uptake (7-9). The cellular component of desmoplastic stroma in PDAC is primarily myofibroblasts characterized by αSMA expression (7). CAFs, which are the main effector cells in the desmoplastic reaction, promote tumor proliferation and growth, accelerate invasion and metastasis, induce angiogenesis, promote inflammation and immune destruction, regulate tumor metabolism, and induce chemoresistance. These factors contribute to the acquisition of the major hallmarks of PDAC (10). Therefore, CAFs are major components in the tumor microenvironment and thus represent a molecular target for the treatment of cancer (11). Recently, podoplanin (PDPN; D2-40), a type I transmembrane sialoglycoprotein, has attracted attention as a novel CAF marker and has been reported to be associated with a poor prognosis in several cancers (12). PDPN elicits powerful platelet aggregation and is the endogenous ligand for the platelet C-type lectin receptor CLEC-2 that itself regulates podoplanin signaling (13). Our previous study has demonstrated extravasated platelet activation (EPA) at the invasive front of the tumor in PDAC stroma, which is the epithelial-mesenchymal transition (EMT) portion (14). In addition, our previous study revealed that the gemcitabine plus nab-paclitaxel regimen induces stromal depletion, resulting in fewer myofibroblasts (15). However, the presence of extravasated platelets surrounding CAFs has not been reported so far. Furthermore, the association between EPA and PDPN-positive CAFs has not been fully elucidated. In this study, we investigated the status of EPA surrounding CAFs and the effect of EPA on PDPN-positive CAFs by neoadjuvant therapy.

Patients and Methods

Patients and tissue samples. Between January 2010 and December 2017, 74 patients with PDAC that had been diagnosed radiologically underwent surgery at the Department of Gastroenterological Surgery of Kanazawa University Hospital. Forty-one of these patients had pancreatic head cancer and 33 had cancer of the pancreatic body and tail. They consisted of 48 men and 26 women with an average age of 67.8 years (range=38-84 years). Histological findings, such as histology, tumor size, lymph node metastasis, and stromal volume, were compared (Table I). Fourteen out of the 74 patients had no chemotherapy, eight received gemcitabine (GEM) alone, 24 received GEM plus oral TS-1 (GS), and 28 received GEM plus nab-paclitaxel (n-PTX) (GnP) before undergoing surgical resection. The groups were compared in terms of clinicopathological findings (Table II).

Treatment of the GnP group consisted of 75 mg/m2 n-PTX followed by 1000 mg/m2 GEM administered on days 1, 8, and 15, every 28 days for two cycles. The GS treatment dose was 50 mg/m2 per day (day 1-14) for TS-1 and 1000 mg/m2 for GEM (day 8, 15) every 21 days for two cycles as reported previously (15). The GEM alone group received intravenous GEM at a dose of 1000 mg/m2 on days 1, 8, and 15 for two cycles.

Patients with stable or responding tumors were scheduled for surgical resection at 6-8 weeks after the last chemotherapy dose.

Ethics statement. The study was approved by the Ethics Committee of the Kanazawa University Hospital (UMIN000011062). Written informed consent was obtained from each patient enrolled in the study.

Pathological specimens. Paraffin-embedded tissue samples of the 74 patients with PDAC were obtained from the Institute of Pathology for immunohistochemical analysis. The specimens were previously fixed in 10% formalin and embedded in paraffin. Several 3 μm-thick sections were cut from each tissue block. One sample was stained with H&E and the others were subjected to immunohistochemical staining for PDPN and EPA.

Immunohistochemical examination. For immunohistochemical staining, the Dako Envision system, which uses dextran polymers conjugated to horseradish peroxidase (Dako, Carpinteria, CA, USA), was employed to avoid any endogenous biotin contamination. Sections were deparaffinized in xylene and rehydrated in a graded ethanol series. Endogenous peroxidase was blocked by immersing the sections in 3% H2O2/methanol for 20 min at room temperature. Antigen retrieval was achieved by microwaving the sections for 10 min in 0.001 M citrate buffer (pH 6.7). After blocking endogenous peroxidase, the sections were incubated with Protein Block Serum-Free (Dako) at room temperature for 10 min to block non-specific staining. Subsequently, the sections were incubated for 2 h at room temperature with a 1:100-diluted rabbit monoclonal antibody against CD42b (anti-CD42 rabbit monoclonal antibody, EPR6995; Abcam, Cambridge, CA, USA) or a 1:100-diluted monoclonal mouse antibody against human D2-40 (anti-D2-40 mouse monoclonal antibody, Clone D2-40, Dako). The internal positive control for PDPN staining was lymphatic endothelial cells.

View this table:
Table I.

Patient characteristics and relationship between CD42b expression in surrounding cancer-associated fibroblasts in pancreatic cancer stroma and each parameter.

Peroxidase activity was detected using the enzyme substrate 3-amino-9-ethylcarbazole. Sections were incubated in Tris-buffered saline without the primary antibodies as negative controls. Samples were faintly counterstained with Meyer's hematoxylin.

Evaluation of immunostaining. According to a previously reported definition, spindle-shaped cells in the cancer stroma were identified as fibroblasts (15). The percentage of CD42b-stained cells was recorded in at least five fields at ×400 magnification in randomly selected areas. Cases with >10% stained fibroblasts were defined as positive according to the report by Ishikawa et al. (16). We determined the fibroblast density index by adjusting the number of cells counted to the proportion of tumor fibrosis observed in each specimen. The levels of staining in stroma images were analyzed using Hybrid cell count BZ-H2C software (Keyence, Osaka, Japan) in accordance with the manufacturer's protocol. The digital images were analyzed for the total surface area versus the stained area using Hybrid cell count. Color artifacts and major arteries were excluded manually from the analysis by the investigator. The quantification was performed in five different tumor areas and representative fields for each sample in high power field microscopy. Detected cells were classified depending on the ratio between mean layer intensities of PDPN according to Erkan et al. (15, 17). Results are expressed as the total stained area/total surface area. Two observers who were unaware of the clinical data independently reviewed all pathological sections.

View this table:
Table II.

Patient characteristics according to treatment groups.

Statistical analysis. Differences were analyzed for significance using the Chi-square test, Mann-Whitney rank sum test, or log rank test, as appropriate. Data management and statistical analysis were performed using SPSS version 15 software (SPSS, Chicago, IL, USA). Data values were considered statistically significant when the p-value was less than 0.05.

Results

CD42b expression in surrounding CAFs. We detected expression of CD42b, a platelet-specific marker, in surrounding CAFs. CD42b expression was observed in 43 of 74 (58%) patients (Figure 1A) with particularly strong staining on CAFs (Figure 1B).

Expression of PDPN on CAFs. Tumor tissue samples from patients treated with GEM alone, GS, GnP or non-treated patients were analyzed for PDPN expression. The PDPN expression in patients treated with GnP was less abundant in CAFs around tumor glands. In contrast, PDPN expression in patients with untreated PDAC or treated with GEM alone or GS was found in long, continuous, well-organized, and imbricated CAFs (Figure 1C-F).

Correlation between CD42b and PDPN expression. CD42b expression was significantly correlated with PDPN expression (p=0.0001). In contrast, there was no correlation between CD42b and clinical/histological parameters (Table I).

Figure 1.
Figure 1.

Representative photomicrograph of cancer-associated fibroblasts (CAFs) in PDAC tissue. Immunohistological images of CD42b-positive platelets (black arrows). CD42b expression was observed in surrounding CAFs (A and B). Podoplanin (PDPN) expression according to the treatment group (C-F). Representative photomicrographs of sections stained immunohistologically for PDPN-positive fibroblasts in the area of cancer glands (brown, black arrows). PDPN expression in untreated patients (C) and in those treated with gemcitabine alone (D) or gemcitabine plus S-1 (E) is reflected by long, continuous, well-organized, and overlapping CAFs, whereas in the group treated with gemcitabine plus nab-paclitaxel (F), CAFs were less abundant.

Expression of CD42b in surrounding CAFs according to NAC. CD42b expression surrounding CAFs were observed in 10 (71.4%) of the 14 patients in the control group, five (62.5%) of the eight patients treated with GEM alone, and 19 (79.2%) of the 24 patients in the GS group. In contrast, CD42b expression was found in nine (32.1%) of the 28 patients in the GnP group. CD42b-positive cases were significantly lower in the group treated with GnP than in the untreated group and groups treated with GEM alone or GS (p=0.023, 0.015, and 0.002, respectively). In contrast, there was no difference between the groups in terms of clinicopathological factors (Table II).

Expression of PDPN on CAFs according to NAC. Patients who had been treated with GnP had a lower density of PDPN-positive fibroblasts. The average PDPN density in the group treated with GnP was 1.4±1.6%, whereas it was 3.6±2.3%, 3.5±2.2%, and 4.3±3.2%, respectively, in samples from untreated patients and patients treated with GEM alone or GS. PDPN density was significantly lower in the group treated with GnP than in the untreated, GEM alone- or GS-treated groups (p<0.0001). However, PDPN density did not differ significantly between untreated, GEM alone, and GS groups (p=0.776, 0.473, and 0.392, respectively) (Figure 2).

Figure 2.
Figure 2.

PDPN density according to the treatment group. The median PDPN-positive density index was determined by dividing the proportion of PDPN-positive fibroblasts by stromal content in five random high-power fields. PDPN density was significantly lower in the activated group treated with gemcitabine plus nab-paclitaxel (GnP) than in untreated, gemcitabine alone-, and gemcitabine plus S-1 (GS)-treated groups (*p<0.0001, Mann-Whitney U-test).

Discussion

In this study, we demonstrated the prominence of EPA in surrounding CAFs in 58% of resected pancreatic cancer specimens. Moreover, there was a significant association between EPA and PDPN-positive CAFs in the PDAC stroma. Finally, we also showed that the GnP regimen decreased EPA through PDPN-positive CAF depletion. Therefore, our data provide important insights into the role of EPA in surrounding CAFs and the GnP regimen in PDAC.

Activated myofibroblasts, termed CAFs, are one of the major stromal cell types. With reciprocal crosstalk between cancer cells and fibroblasts, CAFs undergo various morphological and biological transitions in response to tumor progression (18). Furthermore, CAFs have a crucial role in maintaining the tumor microenvironment for cancer cell survival and proliferation. CAFs are essential components of the tumor microenvironment and therefore represent a molecular target for the treatment of cancer (19). In fact, studies have shown that therapeutic targeting of cancer cells alone is insufficient for the treatment of cancer (20). Thus, cancer therapy should co-target cancer cells and cells in their microenvironment such as CAFs.

Despite various proteins suggested as markers for CAFs, including α-smooth muscle actin (SMA) (15), platelet-derived growth factor receptor (PDGFR)α/β, fibroblast activation protein (FAP), PDPN (21), and fibroblast-specific protein (FSP)-1 (22), tumor microenvironment-associated fibroblasts are a heterogeneous population. Thus, the use of α-SMA or vimentin as the only markers will not identify all CAFs. Enhanced expression of PDPN, a type-I transmembrane sialoglycoprotein, has been reported in CAFs, and PDPN expression in CAFs predicts a poor prognosis in several cancers (12). Recently, CLEC-2 was identified as a receptor for the platelet-activating snake venom rhodocytin (23). PDPN is the endogenous ligand for CLEC-2, which is expressed by platelets, dendritic cells, and circulating CD11b+ Gr-1+ myeloid cells (24). The interaction between PDPN and CLEC-2 causes blood platelet degranulation and activation as the result of CLEC-2 oligomerization, thus promoting the formation of further platelet aggregates (25). In the PDAC stroma, CAFs interact with platelets and produce CAF-platelet aggregates. The extravasated platelets promote tumor progression through CAF-induced activation and aggregation. Therefore, CLEC-2 is a physiological target protein of PDPN and may be involved in PDPN-induced platelet aggregation, tumor metastasis, and other cellular responses related to PDPN (25). Thus, the interaction between PDPN in CAFs and CLEC-2 on platelets may be a target to suppress metastasis of PDPN-positive tumors, because platelet-derived factor contributes to the formation of a pre-metastatic niche and promotes tumor growth, metastasis and EMT/invasion which could contribute to extravasation (26).

In our previous study, EPA was observed at the invasive front of the tumor in pancreatic stroma (14). EPA is associated with the first step of EMT, because platelets contain storage pools of growth factors such as platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF), thrombospondin-1 (TSP-1), and plasminogen activator inhibitor-1 (14, 27). In contrast, abundant stromal cells expressing PDGF receptor β are found in dormant tumors. Complex formation between PDGF-β and the TGF-β receptor regulates the differentiation of mesenchymal stem cells into CAFs. However, the source of PDGF-β or TGF-β is unclear. It has been reported that CD44 mediates the adhesion of platelets to hyaluronan (28). Therefore, EPA in surrounding CAFs expressing CD44 may be the origin of PDGF-β or TGF-β, which supports the stemness and drug resistance of malignant tumors (29).

Figure 3.
Figure 3.

Interplay among components of the PDAC microenvironment. Extravasated platelet activation (EPA) plays an important role in the PDAC stroma. Platelet-derived factors, such as TGF-β, VEGF-A, CD40L, cause drug resistance, epithelial–mesenchymal transition, and immune paralysis. Cancer-associated fibroblasts (CAFs) within the PDAC microenvironment are involved in deposition of dense extracellular matrix typical of the desmoplastic reaction. An immunosuppressive inflammatory infiltrate consisting of tumor-associated macrophages (TAMs), regulatory T cells (Tregs), tumor-associated neutrophils (TANs), and myeloid-derived suppressor cells (MDSCs) is recruited to the PDAC microenvironment. These cells play a key role in tumor promotion and in dampening of the cytotoxic T lymphocyte (CTL) response to the tumor.

The lack of effective therapeutic options contributes to the high mortality rate of PDAC. After adjusting for a number of factors, including body mass index, smoking history, and history of diabetes, Risch et al. have reported that regular aspirin use was associated with a 46% decrease in the risk for development of pancreatic cancer. Risk decreased by 8% for each year of aspirin use (30). Streicher et al. have also reported that a daily aspirin regimen, including both low and regular dose aspirin use, may reduce the risk of developing pancreatic cancer (31). Therefore, extravasated platelets may play a central role in the tumor microenvironment in PDAC. Platelets are enriched with a number of growth factors, such as TGF-β, VEGF-A, CD40L, which cause drug resistance, EMT, and immune paralysis (32). CAFs within the PDAC microenvironment are involved in deposition of the dense extracellular matrix typical of the desmoplastic reaction. An immunosuppressive inflammatory infiltrate consisting of tumor-associated macrophages (TAMs), regulatory T cells (Tregs), tumor-associated neutrophils (TANs) and myeloid derived suppressor cells (MDSCs) is recruited to the PDAC microenvironment. These cells play a key role in tumor promotion and in dampening of the cytotoxic T lymphocyte (CTL) response to the tumor (Figure 3). Therefore, it is important to modify the PDAC microenvironment as a new therapeutic approach. Anti-platelet agents inhibit VEGF, TSP1, and TGF-β1 via platelet aggregation (33). Aspirin is an anti-platelet drug whose anticancer activity has been thoroughly investigated both in vitro and in vivo using cancer cell lines, animal models as well as clinical trials (34, 35).

In our previous study, the treatment regimen of GnP induced stromal depletion, resulting in decreased CAF content (15). This study has shown that the GnP regimen was very effective to induce a marked decrease of PDPN-positive CAF content in PDAC compared with untreated specimens or those treated with conventional GEM alone or GS. This study has also found significantly lower CD42b expression surrounding CAFs in the GnP group than in untreated, GEM alone, and GS groups through PDPN-positive CAF depletion. It has been suggested that the potential effect of the GnP regimen on suppression of chemoresistance and metastasis is mediated through alterations in the tumor microenvironment. Conventional anticancer chemotherapy has been historically thought to act through direct killing of tumor cells. Our previous study has shown that induction of MICA/B expression by low dose GEM enhances innate immune functions rather than increasing cytotoxicity and inhibitiing cleavage and release of MIC molecules from the tumor surface in pancreatic cancer (36, 37). Cytotoxic agents such as GEM, PTX, 5-FU, and cisplatin can induce depletion of MDSCs (38). In this way, chemotherapeutic agents stimulate both the innate and adaptive arms of the immune system. Accumulating evidence indicates that the antitumor activities of chemotherapy rely on several off-target effects, especially directed to the host immune system, that cooperate for successful tumor eradication (39).

In conclusion, there was a significant association between EPA and PDPN-positive CAFs in PDAC stroma. The antiplatelet agents and GnP regimen have the potential to alter the tumor microenvironment in PDAC, restricting the progression, invasion, and drug resistance of these aggressive tumor cells.

Acknowledgements

The Authors would like to thank Mitchell Arico from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

The present study was supported by the Japan Society for the Promotion of Science (KAKENHI grant no. 17K10693).

Footnotes

  • Authors' Contributions

    TM and TO made substantial contributions to the design and coordination of the study. TM, RG, MO, HS, YO, SN, IM, HH, HT and HT performed the surgery. TM carried out the immunohistochemistry. TM, KO, JK, SS, KN, IN and SF collected the samples and data. TM and TO analyzed the data. JWH was involved in drafting the manuscript. TO approved the final version of the manuscript. All Authors read and approved the final manuscript.

  • Conflicts of Interest

    The Authors declare they have no financial or other conflicts of interest in relation to the content of this article.

  • Received August 25, 2019.
  • Revision received August 31, 2019.
  • Accepted September 3, 2019.

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Impact of Extravasated Platelet Activation and Podoplanin-positive Cancer-associated Fibroblasts in Pancreatic Cancer Stroma
TOMOHARU MIYASHITA, HIDEHIRO TAJIMA, RYOSUKE GABATA, MITSUYOSHI OKAZAKI, HIROYUKI SHIMBASHI, YOSHINAO OHBATAKE, KOICHI OKAMOTO, SINICHI NAKANUMA, SEISHO SAKAI, ISAMU MAKINO, JUN KINOSHITA, HIRONORI HAYASHI, KEISHI NAKAMURA, HIROYUKI TAKAMURA, ITASU NINOMIYA, SACHIO FUSHIDA, JOHN W. HARMON, TETSUO OHTA
Anticancer Research Oct 2019, 39 (10) 5565-5572; DOI: 10.21873/anticanres.13750
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