Stem cells have been isolated by their ability to efflux Hoechst 33342 dye and are referred to as the ''side population'' (SP). In this study, we used flow cytometry and Hoechst 33342 dye efflux assay to isolate and characterize SP cells from six human lung cancer cell lines (H460, H23, HTB-58, A549, H441, and H2170). Nonobese diabetic/severe combined immunodeficiency xenograft experiments showed that SP cells were enriched in tumorinitiating capability compared with non-SP cells. Matrigel invasion assay showed that SP cells also have higher potential for invasiveness. Further characterization of this SP phenotype revealed several stem cell properties. We found evidence for repopulating ability by SP to regenerate a population resembling the original population. SP displayed elevated expression of ABCG2 as well as other ATP-binding cassette transporters and showed resistance to multiple chemotherapeutic drugs. Human telomerase reverse transcriptase expression was higher in the SP, suggesting that this fraction may represent a reservoir with unlimited proliferative potential for generating cancer cells. mRNA levels of minichromosome maintenance (MCM) 7, a member of the MCM family of proteins critical to the DNA replication complex, were lower in SP cells, suggesting that a majority of the SP fraction was in the G 0 quiescent state. Sixteen clinical lung cancer samples also displayed a smaller but persistent SP population. These findings indicate that SP is an enriched source of lung tumor-initiating cells with stem cell properties and may be an important target for effective therapy and a useful tool to investigate the tumorigenic process. [Cancer Res 2007;67(10):4827-33]

To understand the molecular pathways involved in the pathogenesis of squamous cell lung carcinoma, we obtained DNA from 94 microdissected foci from 12 archival surgically resected tumors including histologically normal epithelium (n=13), preneoplastic lesions (n=54), carcinoma is situ (CIS) (n=15) and invasive tumors (n=12). We determined loss of heterozygosity (LOH) at 10 chromosomal regions (3p12, 3p14.2, 3p14.1-21.3, 3p21, 3p22-24, 3p25, 5q22, 9p21, 13q14 RB, and 17p13 TP53) frequently deleted in lung cancer, using 31 polymorphic microsatellite markers, including 24 that spanned the entire 3p arm. Our major ®ndings are as follows: (1) Thirty one percent of histologically normal epithelium and 42% of mildly abnormal (hyperplasia/metaplasia) specimens had clones of cells with allelic loss at one or more regions; (2) There was a progressive increase of the overall LOH frequency within clones with increasing severity of histopathological changes; (3) The earliest and most frequent regions of allelic loss occurred at 3p21, 3p22-24, 3p25 and 9p21; (4) The size of the 3p deletions increased with progressive histologic changes; (5) TP53 allelic loss was present in many histologically advanced lesions (dysplasia and CIS); (6) Analyses of 58 normal and non-invasive foci having any molecular abnormality, indicated that 30 probably arose as independent clonal events, while 28 were potentially of the same clonal origin as the corresponding tumor; (7) Nevertheless, when the allelic losses in the 30 clonally independent lesions and their clonally unrelated tumors were compared the same parental allele was lost in 113 of 125 (90%) of comparisons. The mechanism by which this phenomenon (known as allele speci®c mutations) occurs is unknown; (8) Four patterns of allelic loss in clones were found. Histologically normal or mildly abnormal foci had a negative pattern (no allelic loss) or early pattern of loss while all foci of CIS and invasive tumor had an advanced pattern. However dysplasias demonstrated the entire spectrum of allelic loss patterns, and were the only histologic category having the intermediate pattern. Our ®ndings indicate that multiple, sequentially occurring allele speci®c molecular changes commence in widely dispersed, apparently clonally independent foci, early in the multistage pathogenesis of squamous cell carcinomas of the lung.

In vivo autofluorescence spectra were obtained in 5 patients with carcinoma in situ, 26 patients with invasive tumors, and 1 patient with severe dysplasia. Significant spectral differences were observed between pre-cancerous, cancerous, and normal bronchial tissues. This difference may afford a method to image and/or detect early lung cancer by using tissue autofluorescence alone.

Allelic loss is a hallmark of tumor suppressor gene (TSG) inactivation. We have allelotyped 29 paired lymphoblastoid and lung cancer cell lines derived from 11 patients with small cell (SCLC) and 18 patients with non‐small cell lung carcinomas (NSCLC). Statistical analysis indicated that a threshold of 30% separated non‐random allelic loss from the random genetic deletions of malignancy. We have identified non‐random allelic loss at 42 of 54 (78%) specific chromosomal regions examined, with 22 regions (52%) common between the two major lung cancer histologic types. There were 3 regions (7%) with allelic loss specific for SCLC and 17 regions (41%) specific for NSCLC. Furthermore, there were significant differences in loss of heterozygosity (LOH) frequencies between NSCLC and SCLC at 13 regions on eight chromosome arms (3p, 5q, 6q, 9p, 10q, 11p, 13q, and 19p). Eight homozygous deletions were present in seven cell lines at four regions, 3p12, 3p14.2, 9p21, and 10q23–25. We have also identified novel sites of chromosomal deletions. In particular, there was frequent loss at 11p13 in SCLC and loss at 6p21.3 and 13q12.3 in NSCLC. In this study, we demonstrate that a) non‐random allelic losses in lung cancer involve multiple regions; b) some losses are common to both NSCLC and SCLC subtypes, whereas others are subtype specific; c) there are genetic deletions at novel chromosomal regions; and d) several homozygous deletions have been noted. Our studies demonstrate the usefulness of continuous cell lines for detailed allelotyping, for comparing genetic abnormalities between SCLC and NSCLC, and for identifying homozygous deletions. Genes Chromosomes Cancer 21:308–319, 1998. © 1998 Wiley‐Liss, Inc.

LOH at 9p loci occurs at the earliest stage in the pathogenesis of lung cancer and involves all regions of the respiratory tract. LOH in NSCLC is not random but targets a specific allele in individuals. Studying preneoplastic lesions may help identify intermediate markers for risk assessment and chemoprevention.