Advances in Primary Cell Culture of Pancreatic Cancer

Acknowledgments

I would like to thank the following colleagues: Prof. Ugo Boggi, General and Transplants Surgery, University of Pisa, Italy; Prof. Daniela Campani, Surgical Pathology, University of Pisa, Italy; Prof. Marco Del Chiaro, General Surgery, CLINTEC Karolinska, Sweden; Prof. Irene Esposito, Surgical Pathology, Munich University, Germany; Dr Elisa Giovannetti, Cancer Center Amsterdam, VU University, Amsterdam, The Netherlands; Dr Luca Emanuele Pollina, Surgical Pathology, University of Pisa, Italy; Prof. Franco Mosca, General and Transplants Surgery, University of Pisa, Italy; and, Dr Claudio Ricci, Laboratory of Immunology, University of Verona, Italy. The author would like to thank also Fondazione Umberto Veronesi, Milano, Italy. The author was awarded the research grant “Young Investigator Program Year 2013”.

Introduction

Pancreatic cancer is a lethal disease, and despite the low incidence, it is in fact a major cause of cancer-related deaths in industrialized countries. At the time of diagnosis, 75–85% of patients present with advanced tumors and are not amenable to surgical resection with curative intent . Conservative therapeutic strategies, such as chemotherapy and/or radiotherapy, have not shown to improve the prognosis of pancreatic cancer that is not operable . Improvement of the surgical technique in high-volume centers has reduced the intraoperative mortality below 5%, and consequently increased the number of resections with curative intent. However, the local recurrence rate remains very high and, although in subgroups of patients there has been reported actual survival of more than 20% five years post diagnosis, the real long-term survivors are rare (<2%) . The reasons for this aggressive nature of the disease are not fully known, but they seem to have to be sought in the particular biological structure that characterizes cancer of the pancreas. In the last two decades, scientific and technological progress in the field of molecular biology have made it possible to elucidate many of the genetic and epigenetic mechanisms underlying this disease, with the hope that they will lead to the development of better diagnostic and therapeutic modalities. Nevertheless, the lack of known risk factors and the absence of symptoms in the early stages of the disease make the implementation of strategies for primary or secondary prevention very unlikely.

Epidemiology

Pancreatic cancer has the highest incidence in industrialized countries such as the United States, Japan, and Europe, where rates are higher in the northern than the Mediterranean regions. The African and Asian countries are characterized by a low incidence (1–2 cases/10 ⁵ habitants/year) . In Italy there are regional variations; in fact, it has been reported that the incidence is around 1–2 cases/10 ⁵ habitants/year, with an average of 8.4 new 1–2 cases/10 ⁵ habitants/year (National Cancer Registry). Epidemiological studies have shown a constant growth in the overall incidence rate and age-standardized incidence rate until the eighties, followed by a plateau phase. The mortality rate has been shown to coincide with that of incidence . Males are more commonly affected than females, with a ratio ranging from 1.4:1 to 2.9:1 in Brazil and France, respectively. However, in recent decades there has been an increase in the number of female patients suffering from pancreatic cancer, probably because of increased cigarette smoking in women . Cigarette smoking is a clear and well-established risk factor (from two to six times higher) for pancreatic cancer, as demonstrated by some epidemiological studies. However, there is no clear evidence linking cigar or pipe smoking or chewing tobacco with the disease. The consumption of alcohol, coffee, or tea showed no clear association with the development of the disease either. In contrast, medical conditions such as chronic pancreatitis, cystic fibrosis, and diabetes do show strong correlation with the disease. Speaking of chronic pancreatitis, for example, a multicenter study conducted by Lowenfels showed a 14.4- to 16.5-fold increase in the incidence of pancreatic carcinoma compared to the general population , with the risk increasing even higher over the years from the onset of the disease. Individuals with hereditary chronic pancreatitis, which is characterized by an early onset, can achieve a 75% cumulative risk of developing pancreatic cancer if they have inherited the disease from the male branch of the family . Although most cases of pancreatic cancer are sporadic, the disease also occurs in the context of hereditary syndromes, such as dysplastic nevus syndrome, Lynch syndrome type II (colon cancer hereditary nonpolyposis), breast-ovarian cancer syndrome, ataxia-telangiectasia, Peutz-Jeghers syndrome, and the aforementioned hereditary pancreatitis . Furthermore, the possibility of an autosomal dominant inheritance of the disease in the absence of an apparent genetic disorder may have other hereditary links to it. For example, through linkage analysis, change at locus 4q32-34 has recently been identified to associate with the disease significantly, even though no specific gene has been identified responsible . Overall, it is estimated that up to 10% of pancreatic cancer cases are transmitted with an autosomal dominant pattern of inheritance .

The Molecular Genetics of PDAC

In the last decade, with the advancement of molecular biology tools and the development of transgenic animal models, researchers have been able to identify some fundamental genetic alterations underlying the development of pancreatic cancer. Four main events are believed to be critical for the pathogenesis and/or progression of ductal adenocarcinoma:

Activating point mutations of the K-Ras
Inactivation of the tumor suppressor gene TP53
Inactivation of the tumor suppressor gene p16
Inactivation of the tumor suppressor gene Dpc4 (deleted in pancreatic cancer 4).

K-Ras

K-Ras mutations are more frequent in pancreatic cancer than in any other type of human neoplasia: >80% of carcinomas of the pancreas have activating mutations in the first or second base of codon 12 . The Ras family proteins have a function of transmitting growth signals within the cell: they are capable of binding GTP molecules and transform them into GDP (GTPase activity) once the signal is transmitted. Point mutations at codons 12, 13, or 61 lead to loss of the GTPase activity. As a consequence, the ras protein remains in the active state to continuously transmit growth signals. K-Ras mutations appear to be an early event in pancreatic carcinogenesis, as shown by numerous experimental data. In fact, the same type of mutation has been found in tumors and in the lesions associated with them ; also, animal models of pancreatic carcinogenesis have revealed a high incidence of ras mutations in the early stages of neoplastic transformation. Finally, K-Ras mutations have been identified in preneoplastic ductal lesions found in the pancreas of a patient with a family history of pancreatic cancer .

p53

The alterations of the p53 tumor suppressor gene are common in human cancers. This gene encodes a nuclear protein with a short half-life. It acts as a transcription factor to exert a negative regulation of cell growth and proliferation by inducing apoptosis in the presence of genomic damage that is unrepairable. Loss of heterozygosity at the p53 locus occurs in almost 90% of pancreatic tumors, while in 50–75% of cases there is complete loss of function of the protein due to alterations involving the inactivation of the remaining copy of the gene . Of all the p53 gene alterations, missense point mutations are the most frequent ones; frameshift mutations may also occur predominantly represented by intragenic microdeletions, which occur in pancreatic cancer with a frequency significantly higher (up to 30%) compared to other human cancers. The majority of p53 mutations, with exceptions frequently represented by microdeletions and more rarely by nonsense mutations, lead to the synthesis of a mutant protein with increased half-life, which can be easily detected by immunohistochemistry staining .

p16

P16INK4a/CDKN2/MTS1 gene is located on chromosome 9p21 and encodes a protein that binds the cyclin-dependent kinase 4 (Cdk4) to prevent its interaction with cyclin D1. The cyclin D1-Cdk4 interaction regulates the transition from G1 phase to S phase of the cell cycle. In the absence of inhibition by p16 , it leads to continuous activation and therefore uncontrolled cell growth. Three mechanisms are responsible for the loss of function of p16 in almost all cases of carcinomas of the pancreas: (1) homozygous deletions due to loss of both alleles; (2) loss of one allele and mutation in the other allele resulting in altered function (loss of heterozygosity); and (3) methylation of cytosine nucleotides in the promoter region to suppress the expression of the gene .

Dpc4

Dpc4 (Smad4) is a tumor suppressor gene located on the long arm of chromosome 18 and encodes a transcription factor that participates in the cascade mediated by signal transduction-dependent growth factor TGF. It is frequently altered in pancreatic cancer. The loss of its function was observed in 50% of pancreatic carcinomas and is due to two mechanisms: (1) loss of heterozygosity; and (2) homozygous deletion. Immunohistochemistry staining allows one to obtain a very sensitive and specific assessment of the expression level of Dpc4 . Recently, it was shown that the loss of expression of Smad4 is associated with a poorer prognosis of carcinoma of the pancreas.

Other Genes

Alterations of other genes, mainly tumor suppressors, have also been reported in pancreatic cancer. Mutations of the gene APC (adenomatous polyposis coli) are rare in ductal adenocarcinoma but are reported more frequently in solid pseudopapillary pancreatic tumors, acinar carcinoma, and ampullary cancer . DCC (deleted in colorectal cancer) is a tumor suppressor gene that encodes a protein with receptor functions involved in cell migration and apoptosis. It is located on the long arm of chromosome 18 near the gene Dpc4 , leading to an underestimation of its involvement in the molecular pathogenesis of pancreatic cancer because the consequence of the chromosomal deletions discussed in this topic has been mainly attributed to the loss of the Dpc4 locus. However, it has recently been shown that there are some pancreatic carcinomas in which there is a real loss to the locus DCC, while the locus Dpc4 remains intact. BRCA2 is another tumor suppressor gene involved in the pathogenesis of some familial forms of pancreatic carcinoma .

General Considerations

As previously described, at the time of diagnosis pancreatic cancer appears to be an incurable disease in almost all cases, since the rate of incidence of this disease is almost coinciding with the rate of mortality. The cases that are defined as long-term survivors, disease free after five years, make up a very small cohort of patients, estimated at around 2%. This is due to the absence of early diagnosis of pancreatic cancer. The radiodiagnostic methods that are the current ones for diagnosis are not able to detect the presence of this cancer in its early stages, so at the time of diagnosis, this cancer is already in advanced stages and in some cases not operable . Pancreatic surgery has made great strides in substantially reducing the operative mortality and improving morbidity of patients with unresectable tumors, but the absence of an effective drug therapy seems to be the problem . Until now, the gold standard in the treatment of pancreatic cancer has been the use of gemcitabine in adjuvant setting chemotherapy in favor of other drugs such as 5-fluorouracil or platinum derivatives, both of which are less tolerable in therapy .

Our goal in the presented study was to obtain an in vitro model that closely mimics pancreatic ductal adenocarcinoma (PDAC) patients; the purpose was to measure the levels of expression of the molecular determinants involved in the metabolism of gemcitabine and relate them to the survival of patients treated with this drug in monochemotherapy . To eliminate the interference of the abundant desmoplastic component, the tumor epithelial component was isolated using the laser microdissection (LMD) technology. To complete the study of expression in vivo and in vitro, primary cell cultures of pancreatic cancer have been set up .

The purpose of this chapter is to describe the techniques for the isolation of the epithelial component of pancreatic cancer. These methods are:

The realization of primary cultures of PDAC
The LMD of normal and cancerous pancreatic tissues.

The LMD and cell cultures focus on removing the “stroma”, which can mask the true expression at the mRNA level of tumor cells. With microdissection you can study the molecular signature in a static manner for the reason that the tissue is locked in a precise biological moment. Cell cultures offer the opportunity to study the characteristics of the tumor in a dynamic way. Study of the molecular biology of the epithelial component of the tumor, desmoplastic excluding the component, can lead to the realization of a valid system for the study of ductal carcinoma of the pancreas.

Cell Culture of PDAC

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