Organotypic slice cultures of pancreatic ductal adenocarcinoma preserve the tumor microenvironment and provide a platform for drug response

doi:10.1016/j.pan.2018.09.009

Pancreatology

Volume 18, Issue 8, December 2018, Pages 913-927

https://doi.org/10.1016/j.pan.2018.09.009 Get rights and content

Abstract

Background

/Objective: The conventional models currently used to evaluate various anti-tumor therapeutic agents are not sufficient for representing human pancreatic ductal adenocarcinoma (PDA), which has a unique tumor microenvironment. We aimed to produce an organotypic slice culture model from human PDA that resembles the in vivo situation and to evaluate the responses of PDA slices to established cytotoxic drugs.

Methods

PDA tissues were obtained from 10 patients who underwent pancreatic resection. The tissues were sliced by a vibratome, and the tumor slices were then cultured. The viability of tumor slices during slice culture was evaluated using H&E and immunohistochemical staining, and stromal cells were demonstrated. The effects of cytotoxic drugs on PDA cell lines and slices were analyzed.

Results

Tumor slices maintained their surface areas and tissue viability for at least five days during culture. Preserved proliferation and apoptosis in tumor slices were observed by the expression of Ki-67 and cleaved caspase-3. Stromal cells including macrophages (CD68⁺ and CD163⁺), T cells (CD3⁺, CD8⁺, and FOXP3⁺), and myeloid cells (CD11b⁺) were present throughout the culture period. Staurosporine, gemcitabine, and cisplatin treatment of PDA cell lines and tumor slices exerted proportional cytotoxic effects in terms of MTT viability, tumor cell number, and Ki-67 and cleaved caspase-3 expression.

Conclusions

Organotypic human PDA slice cultures preserved their viability and tumor microenvironment for at least five days during slice culture. PDA slice culture appears to be a feasible preclinical test model to assess the response to anti-tumor agents.

Introduction

Pancreatic ductal adenocarcinoma (PDA) represents a therapeutic challenge due to its lack of responsiveness to current treatments and its poor prognosis. Unfortunately, the five-year survival rate of patients with pancreatic cancer has not significantly improved over the last two decades and remains as low as under 10% (NIH surveillance, epidemiology, and end results program) which stems from difficulties in early detection, adjacency to major vessels, and poor response rates to cytotoxic drugs. Although traditional cytotoxic drug therapy and radiotherapy do not have satisfying results, FOLFIRINOX and nab-paclitaxel have recently shown improved survival and response rates in randomized control studies [1,2]. In addition, newly developed target agents and immunologic drugs have raised the expectation of a treatment response to PDA. For the evaluation of such drugs, appropriate ex vivo models are needed to accurately represent the complex and heterogeneous in vivo tumor biology and pathology of tumors. However, there has not yet been a proper ex vivo platform to predict an individual's response to drugs.

Current ex vivo tumor models including primary cell lines, xenografts, or genetically engineered mice (GEM) have been actively used to test tumor biology and drug response. However, these models are inherently limited by an absence of stroma and low heterogeneity. PDA characteristically has abundant stroma that is considered to play a major role in tumor growth and drug resistance [3,4]. On the other hand, organotypic slice cultures preserve the stromal microenvironment as well as tumor cells, which leads to the maintenance of the tumor-stroma interaction. Recently, organotypic slice cultures have been developed and studied in several tumors, such as head, neck, gastric, and breast tumors [[5], [6], [7], [8]]. In addition to preserving the tumor-stroma interaction, organotypic slice culture models allow the rapid assessment of drugs requiring only a few days and may serve to find drugs tailored to individual patients. Because different tumor entities display distinct growth and culture characteristics, careful consideration of slice preparation and culture conditions suitable for PDA is needed. A recent study that included JH Chang as a coauthor established a PDA slice culture model and demonstrated its usefulness for immune microenvironment research [9]. We aimed to reproduce this precision-cut organotypic slice culture model from human PDA and to evaluate the responses of PDA slices to established cytotoxic drugs compared to the response of PDA cell lines.

Section snippets

Patients and cell lines

A total of 10 pancreatic tumor specimens were collected during surgery from patients who underwent surgery for pancreatic cancer. Details regarding the patients and surgery information are described in Table 1. Six patients underwent Whipple's operation, two underwent pylorus-preserving pancreaticoduodenectomy (PPPD), one underwent distal pancreatectomy, and one underwent radical antegrade modular pancreato-splenectomy (RAMPS). The final stages of pancreatic cancer according to the 8th edition

Gross morphologies and surface areas of tumor slices were preserved during slice culture

The gross morphology of each PDA slice showed a rectangular shape, and this shape did not change during culture (Fig. 1A). The size of the tumor slices did not grossly change for the first few days of culture but tended to gradually decrease. The surface areas of the tumor slices were compared based on their culture day, as shown in Fig. 1B. The mean slice surface areas (n = 10) on each culture day compared that on day 0 were 89.6% (day 1), 79.0% (day 3), 68.6% (day 5), 55.2% (day 7), and 47.0%

Discussion

Conventional evaluation models for anti-tumor therapeutic agents, such as primary cell lines and tumor xenografts, are limited in their representations of human PDA. Primary cell lines are derived from fast-growing tumor cells during cultivation and do not contain stroma, which result in the absences of tumor-stroma interactions and tumor heterogeneity. Although the mouse tumor xenograft model retains the tumor microenvironment, it is within the murine stroma and the mouse is an

Conflicts of interest

The authors declare no competing financial interest.

Acknowledgments

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2016R1D1A1B03931040) and supported by the Institute of Clinical Medicine Research of Bucheon St. Mary's Hospital (Research Fund, BCMC17LH03). We acknowledge Dr. Xiuyun Jiang and Prof. Venu G. Pillarisetty of the Department of Surgery, University of Washington for supporting the concept and design of our experiment.

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Organotypic slice cultures of pancreatic ductal adenocarcinoma preserve the tumor microenvironment and provide a platform for drug response

Abstract

Background

Methods

Results

Conclusions

Introduction

Section snippets

Patients and cell lines

Gross morphologies and surface areas of tumor slices were preserved during slice culture

Discussion

Conflicts of interest

Acknowledgments

FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer

N Engl J Med

Increased survival in pancreatic cancer with nab-paclitaxel plus gemcitabine

N Engl J Med

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Cell Cycle

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N Engl J Med

Slice cultures from head and neck squamous cell carcinoma: a novel test system for drug susceptibility and mechanisms of resistance

Br J Canc

Organotypic slice cultures of human gastric and esophagogastric junction cancer

Canc Med

Tumor slice culture system to assess drug response of primary breast cancer

BMC Canc

The practicalities of using tissue slices as preclinical organotypic breast cancer models

J Clin Pathol

Long-lived pancreatic ductal adenocarcinoma slice cultures enable precise study of the immune microenvironment

OncoImmunology

Ex-vivo evaluation of gene therapy vectors in human pancreatic (cancer) tissue slices

World J Gastroenterol