Pancreatic cancer is considered as a chemoresistant tumor because of its abundant cancer stroma which contains cancer-associated fibroblasts (CAFs). Nab-paclitaxel (Nab-PTX, cytoskeletal anticancer drug) is expected as a key drug for pancreatic cancer. The patients of pancreatic cancer treated with Nab-Paclitaxel show remarkable tumor reduction in size. We analyzed total 20 surgically resected cases of pancreatic ductal adenocarcinoma; ten cases treated with Nab-PTX before surgical operation (treated group) and ten cases without neoadjuvant chemotherapy (untreated group). The treated group showed high density of aniline blue (AnB) positive collagen bundles in the stroma and low density of α-smooth muscle actin (α-SMA) positive CAFs. On the other hand, the untreated group exhibited low density of AnB positive collagen bundles and high density of CAFs. We speculated that the Nab-PTX neoadjuvant chemotherapy plays in roles of stromal collagenous fibrosis and inactivation of CAFs.

Keywords: Pancreatic ductal adenocarcinoma, Neoadjuvant chemotherapy, Nab-Paclitaxel, Cancer associated fibroblast

In this study we investigated why Nab-PTX has prominent anticancer effects, analyzing histopathological specimens of pancreatic cancer stroma which were performed neoadjuvant chemotherapy with Nab-PTX.

MATERIALS AND METHODS

We investigated total 20 surgically resected cases of pancreatic ductal adenocarcinomas; ten cases treated with Nab-PTX before surgical operation (treated group) and ten cases without neoadjuvant chemotherapy (untreated group). Numbers of the cases were limited because the Nab-PTX treatment has recently established as the neoadjuvant chemotherapy for pancreatic cancer.

We evaluated the cancer stromal phenotypes using Heidenhain’s AZAN trichrome stain (AZAN stain) [10,11] and immunohistochemical α-smooth muscle actin (α-SMA) stain. Collagen bundles in the cancer stroma are stained with aniline blue (AnB) of AZAN stain, while CAFs in the cancer stroma are immunohistochemically positive for α-SMA. We used imaging software “Image J” to analyze fibrosis of cancer stroma, i.e., quantification of collagen bundles (AnB) and CAFs (α-SMA). We selected three invasive foci which represented the most intense fibrosis, and took photographs with x40 objective lens for all cases. Then, we analyzed binarized/extracted images of collagen bundles (AnB) and CAFs (α-SMA) using the software. The images were represented as pixels on the computer screen. We calculated the average of three areas for all the cases.

RESULTS

Pancreatic ductal adenocarcinoma was characterized by an invasive growth pattern with abundant fibrous stroma in both treated/untreated groups (Figure 1). However, the treated/untreated groups revealed different distribution pattern of AnB positive collagen bundles and α-SMA positive CAFs. The treated group showed the very high density of collagen bundles and the low density of CAFs. On the other hand, the untreated group exhibited the low density of collagen bundles and high density of CAFs.

Table 1 is the results of Image J analyzation. Student t-test proved that the treated group significantly increased AnB positive collagen bundle area (P<0.01) and significantly decreased α-SMA positive CAF area (P<0.01).

 DISCUSSION AND CONCLUSION

In the present study, we demonstrated the Nab-PTX neoadjuvant chemotherapy induced the unique fibrous stroma of pancreatic ductal adenocarcinoma, i.e., dense AnB positive fibrous stroma with limited numbers of CAFs. We think the dense collagenous fibrosis in the stroma contributes to tumor shrinkage in an anticancer effective manner. We have speculated that the pancreatic ductal adenocarcinomas in the untreated group continuously induced active CAFs in the stroma, and made use of the CAFs for the chemoresistance [5,6]. However, the treated group with Nab-PTX rapidly induced AnB positive collagen bundles in the stroma, and then decreased numbers of CAFs.

Our previous studies reported that the transcriptional factors associated epithelial-mesenchymal transition (EMT) contributed pancreatic cancer invasiveness/aggressiveness, and were thought to be related with CAFs [12,13]. The Nab-PTX neoadjuvant chemotherapy plays in roles of stromal collagenous fibrosis and inactivation of CAFs. However, details of molecular mechanisms of Nab-PTX are still unknown. We try to analyze the molecular mechanisms of collagenous fibrosis as the Nab-PTX anticancer effects in the near future.

1.       Jemal A, Siegel R, Xu J, Ward E (2010) Cancer statistics. CA Cancer J Clin 60: 277-300.

2.       Bosman FT, Carneiro F, Hruban RH, Theise ND (2010) WHO classification of tumors of the digestive system. 4th Edn. International Agency for Research on Cancer (IARC).

3.       Tsutsumi S, Morohashi S, Kudo Y, Akasaka H, Ogasawara H, et al. (2011) L1 cell adhesion molecule (L1CAM) expression at the cancer invasive front is a novel prognostic marker of pancreatic ductal adenocarcinoma. J Surg Oncol 103: 669-673.

4.       Sakuraba S, Morohashi S, Yoshizawa T, Tsutsumi S, Kimura N, et al. (2015) MUC5AC-negative phenotype is correlated with poor patient prognosis of pancreas head ductal carcinoma. Hirosaki Med J 66: 28-37.

5.       McCarroll JA, Naim S, Sharbeen G, Russia N, Lee J, et al. (2014) Role of pancreatic stellate cells in chemoresistance in pancreatic cancer. Front Physiol 5: 141.

6.       Schober M, Jesenofsky R, Faissner R, Weidenauer C, Hagmann W, et al. (2014) Desmoplasia and chemoresistance in pancreatic cancer. Cancers (Basel) 6: 2137-2154.

7.       Zalatnai A, Molnar J (2007) Molecular background of chemoresistance in pancreatic cancer. In Vivo 21: 339-347.

8.       Miyashita T, Tajima H, Makino I, Okazaki M, Yamaguchi T, et al. (2018) Neoadjuvant chemotherapy with gemcitabine plus Nab-paclitaxel reduces the number of cancer-associated fibroblasts through depletion of pancreatic stroma. Anticancer Res 38: 337-343.

9.       Alvarez R1, Musteanu M, Garcia-Garcia E, Lopez-Casas PP, Megias D, et al. (2013) Stromal disrupting effects of nab-paclitaxel in pancreatic cancer. Br J Cancer 109: 926-933.

10.    Ohno S, Tachibana M, Fujii T, Ueda S, Kubota H, et al. (2002) Role of stromal collagen in immunomodulation and prognosis of advanced gastric carcinoma. Int J Cancer 97: 770-774.

11.    Heidenhain M (1915) Uber die Mallorysche Bindegewebsf¨arbung mit Karmin und Azokarmin als Vorfarben. Z Wissenschaft Mikrosk Mikrosk Techn 32: 361-372.

12.    Wu Y, Kijima H (2018). BHLH transcription factors DEC1 and DEC2: From structure to various diseases. Biomed Res J 2: 28-33.

13.    Wu Y, Sato F, Yamada T, Bhawal UK, Kawamoto T, et al. (2012) The BHLH transcription factor DEC1 plays an important role in the epithelial-mesenchymal transition of pancreatic cancer. Int J Oncol 41: 1337-1346.