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
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.
References (28)
- et al.
The Cancer Genome Anatomy Projectbuilding an annotated gene index
Trends Genet
(2000) - et al.
Laser-capture microdissectionopening the microscopic frontier to molecular analysis
Trends Genet
(1998) - et al.
Genome-wide analysis of oral cancer — early results from the Cancer Genome Anatomy Project
Oral Oncology
(2000) - et al.
The origin and function of tumor-associated macrophages (see comments)
Immunol Today
(1992) - et al.
Chemotactic factors, passive invasion and metastasis of cancer cells (letter; comment)
Immunol Today
(1992) - et al.
Human monocyte chemotactic protein-3 (MCP-3)molecular cloning of the cDNA and comparison with other chemokines
Biochem Biophys Res Commun
(1993) - et al.
The human MCP-3 gene (SCYA7)cloning, sequence analysis, and assignment to the C-C chemokine gene cluster on chromosome 17q11.2-q12
Genomics
(1994) - et al.
The small proline-rich proteins constitute a multigene family of differentially regulated cornified cell envelope precursor proteins
J Invest Dermatol
(1995) - et al.
Differential expression of human cornifin alpha and beta in squamous differentiating epithelial tissues and several skin lesions
J Invest Dermatol
(1997) - et al.
Cytokeratin expression in squamous cell carcinomas of the tongue and alveolar mucosa
Eur J Cancer B Oral Oncol
(1996)
Use of microgenomic technology for analysis of alterations in DNA copy number and gene expression in malignant melanoma
Clin Exp Immunol.
Oncogenes, cancer and imaging
J Nucl Med.
Cited by (64)
Animal models in oral cancer research
2006, Oral OncologyGene expression profile in oral squamous cell carcinoma: A pilot study
2005, Journal of Oral and Maxillofacial SurgeryIL-1/IL-1R Signaling in Head and Neck Cancer
2021, Frontiers in Oral HealthThe role of icIL-1RA in keratinocyte senescence and development of the senescence-associated secretory phenotype
2021, Journal of Cell Science
- 1
Equal contribution by the authors.