Elsevier

Oral Oncology

Volume 36, Issue 5, September 2000, Pages 474-483
Oral Oncology

Gene expression profiles in squamous cell carcinomas of the oral cavity: use of laser capture microdissection for the construction and analysis of stage-specific cDNA libraries

https://doi.org/10.1016/S1368-8375(00)00039-7Get rights and content

Abstract

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer among men in the developed world affecting the oral cavity, salivary glands, larynx and pharynx. Utilizing tissue from patients with HNSCC, we sought to systematically identify and catalog genes expressed in HNSCC progression. Here, we demonstrate the successful use of laser capture microdissection for procuring pure populations of cells from patient tissue sets comprised of oral squamous cell carcinomas (OSCCs) and matching normal tissue. From the estimated 5000 cells procured for each sample, we were able to extract total RNA (14.7–18.6 ng) of sufficient quality to transcribe GAPDH by reverse transcriptase-polymerase chain reaction (RT-PCR). The RNA was used for the synthesis of blunt-ended, double-strand complementary DNAs (cDNAs) by oligo (dT)-mediated reverse transcription, followed by addition of linkers. Primers specific for these linkers with uracil deglycosylase-compatible ends were used to amplify these cDNAs by PCR and the product was subcloned into the pAMP10 cloning vector. Ninety-six clones from each of six libraries were randomly sequenced and results indicated that 76–96% of the inserts represent either anonymous expressed sequence tags (ESTs) (25–48%), known genes (9–29%) or novel sequences (27–51%), respectively, with very little redundancy. These results demonstrate that high quality, representative cDNA libraries can be generated from microdissected OSCC tissue. Furthermore, these finding suggest the existence of at least 132 novel genes expressed in our cDNA libraries, which may have a role in the pathogenesis of HNSCC, and may represent novel markers for early detection as well as targets for pharmacological intervention in this disease.

Introduction

From the estimated 100,000 genes in the human genome, 4000 of these may be directly related to disease, including cancer [1]. Indeed, altered expression of some of these genes is now thought to be the basis of most neoplasias, either because they are expressed at abnormally high or low levels, or due to their ability to encode aberrant proteins upon mutations in their coding sequence [2]. In this regard, the availability of a catalog of genes expressed in tumor cells may provide a fingerprint of their genetic make up, and comparison with that of their matching cells exhibiting a normal phenotype can help identify genes that either by their presence or absence, can be causal in cancer. It follows that knowing the identity of these genes will not only enhance our understanding of the molecular basis of this disease and its progression, but it will also provide novel means for its early detection and subsequent treatment.

In response to our limited knowledge of the molecular mechanisms of many neoplasias, the Cancer Genome Anatomy Project (CGAP) supported by the National Cancer Institute (NCI), was established with the goal of creating a complete information infrastructure of genes expressed during tumor progression, which is also expected to yield early markers of cancer, thus providing an opportunity to improve our ability to match patients with appropriate treatment strategies. The CGAP initiative involves the generation of complementary DNA (cDNA) libraries from cancer cells, and after random sequencing, expressed genes are then cataloged and compared with those from the corresponding normal tissues. In doing so, CGAP has also become the leading effort in gene discovery. Further success of this approach has been the development of robust databases and easily accessible Web-based analytical tools for comparative use [3], [4], [5].

Squamous cell carcinoma of the head and neck (HNSCC) are neoplastic lesions found predominantly in the oral cavity, including the salivary glands, larynx and pharynx [6]. Despite recent advances in our understanding, prevention, and treatment of other types of neoplasias, HNSCC still remains the sixth most common cancer among men in the developed world [7] and in the United States alone approximately 13,000 deaths occur yearly as a result of this disease [8]. The high morbidity rate for this malignancy can be attributed to many factors, which include lack of suitable markers for early detection, late presentation, insensitivity to available treatment, and our limited understanding of the molecular mechanisms responsible for this disease [9]. In this regard, the identity of those genes that may have a role in the progression of HNSCC has yet to be fully elucidated. Therefore, in an attempt to begin addressing the molecular basis of this cancer, the Head and Neck CGAP (HNCGAP) was established as a cooperative effort between the National Institute of Dental and Craniofacial Research (NIDCR) and the National Cancer Institute's CGAP initiative.

A major scientific challenge in HNSCC is our understanding of the molecular events that drive tumor progression in vivo [10]. This problem is further compounded by the heterogeneity of this tumor type. Thus, gene expression analysis using bulk tissue or tissue areas of interest manually microdissected, might not be representational and of limited value when using this body of information for assessing gene expression profiles in HNSCC. Of interest, the use of laser capture microdissection (LCM) [11], [12], [13] allows the procurement of pure cell populations for RNA isolation, thus providing an appropriate platform for current efforts in defining the nature of those genes expressed in HNSCC, and their potential contribution to neoplasia [14], [15].

In this study we have used HNSCC and their matching normal tissues from patients with oral cancer lesions. We demonstrate the successful use of LCM to procure specific cell populations. Furthermore, we show that 5000 cells are sufficient to extract RNA of high integrity for the synthesis of high-quality representational cDNAs libraries. Furthermore, sequence analysis of randomly selected clones from each library indicates that 76–96% of the inserts represented anonymous expressed sequence tags (ESTs) (25–48%), known genes (9–29%) or novel sequences (27–51%), respectively, and with very little redundancy among libraries. Emerging sequence information suggests the existence of many novel genes, whose function in tumor development can now begin to be evaluated.

Section snippets

Tissue samples and LCM

Biopsies from patients confirmed to have carcinomas of the oral cavity were immediately fixed in 70% ethanol and subsequently embedded in optical cutting temperature (OTC) as described (http://dir.nichd.nih.gov/lcm). Using a cryostat, 8-μm thick tissue sections were cut onto RNAase free glass slides, and prior to LCM, hematoxylin and eosin (H&E)-stained sections were analyzed and confirmed by a board-certified pathologist. The use of LCM (Arcturus Engineering, Mountain View, CA, USA) was

Scheme of experimental procedure

The experimental strategy for this study is illustrated in Fig. 1. Normal and pathological oral squamous epithelium were visualized under the microscope and appropriate cells were microdissected with individual laser shots. Caps containing approximately 5000 cells were processed for RNA and subsequently assessed for quality. The mRNA served as a template for library production. After transformation, clones from each cDNA library were sequence analyzed and prepared for CGAP submission. The

Discussion

Approximately 10% of the total number of genes are suspected to be expressed in a given cell type. Determining their identity is an important first step towards understanding the patterns of gene expression that mediate normal cellular physiology and disease process. In this study, we report the construction of six high-quality cDNA libraries from tissues of oral origin, including normal and malignant epithelium. Previous studies have reported on genes that are expressed in tissue specimens

Acknowledgements

We thank Dr. Vladimir Knezevic for helpful advice on quantification of total RNA using Fluorometer system.

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