Graham F. Barnard

Characterization of a side population of cancer cells from human gastrointestinal system.

A subset of stem cells, termed “side population” (SP) cells, has been identified and characterized in several mammalian tissues and cell lines. However, SP cells have never been identified or isolated from gastrointestinal cancers. We used flow cytometry and the DNA‐binding dye Hoechst 33342 to isolate SP cells from various human gastrointestinal system cancer cell lines. Fifteen of sixteen cancer cell lines from the gastrointestinal system contained 0.3%–2.2% SP cells. Next, we used an oligonucleotide microarray to analyze differentially expressed genes between SP and non‐SP cells of hepatoma HuH7. The expression of GATA6, which is associated with embryonic development and hepatocytic differentiation, was significantly upregulated in HuH7 SP cells. The expression of ABCG2, ABCB1, and CEACAM6, which are associated with chemoresistance, was also significantly increased in SP cells. In addition, some epithelial markers and mesenchymal markers were overexpressed in SP cells. Reverse transcription‐polymerase chain reaction and immunocytochemical staining validated these results and suggested a multilineage potential for HuH7 SP cells. In hepatoma HuH7 and colorectal SW480 cell lines, SP cells showed evidence for self‐renewal, generating both SP and non‐SP cells. Finally, chemoresistance to anticancer agents, including doxorubicin, 5‐fluorouracil, and gemcitabine, were compared between HuH7 SP and non‐SP cells using an ATP bioluminescence assay. The HuH7 SP cells expressed a higher resistance to doxorubicin, 5‐fluorouracil, and gemcitabine compared with non‐SP cells. These findings demonstrate that cancers of the gastrointestinal system do contain SP cells that show some characteristics of so‐called stem cells.

CD13 is a therapeutic target in human liver cancer stem cells

Cancer stem cells (CSCs) are generally dormant or slowly cycling tumor cells that have the ability to reconstitute tumors. They are thought to be involved in tumor resistance to chemo/radiation therapy and tumor relapse and progression. However, neither their existence nor their identity within many cancers has been well defined. Here, we have demonstrated that CD13 is a marker for semiquiescent CSCs in human liver cancer cell lines and clinical samples and that targeting these cells might provide a way to treat this disease. CD13+ cells predominated in the G0 phase of the cell cycle and typically formed cellular clusters in cancer foci. Following treatment, these cells survived and were enriched along the fibrous capsule where liver cancers usually relapse. Mechanistically, CD13 reduced ROS-induced DNA damage after genotoxic chemo/radiation stress and protected cells from apoptosis. In mouse xenograft models, combination of a CD13 inhibitor and the genotoxic chemotherapeutic fluorouracil (5-FU) drastically reduced tumor volume compared with either agent alone. 5-FU inhibited CD90+ proliferating CSCs, some of which produce CD13+ semiquiescent CSCs, while CD13 inhibition suppressed the self-renewing and tumor-initiating ability of dormant CSCs. Therefore, combining a CD13 inhibitor with a ROS-inducing chemo/radiation therapy may improve the treatment of liver cancer.

Molecular detection of circulating solid carcinoma cells in the peripheral blood: the concept of early systemic disease

Detection of the mRNA of selected genes by reverse transcriptase‐polymerase chain reaction (RT‐PCR) is a sensitive and powerful tool for detecting cancer cells in bone‐marrow or peripheral‐blood samples. In this study, we determined whether carcinoembryonic antigen (CEA) mRNA is detectable in the peripheral blood of patients with gastrointestinal or breast cancer. In addition, we studied selected patients undergoing surgical procedures to assess whether tumor manipulation during operation enhances cancer‐cell dissemination. Peripheral blood from 55 patients with gastrointestinal or breast cancer and from 22 control cases was analysed for CEA mRNA using RT‐PCR. For 15 selected cases undergoing curative surgery for cancer, samples were also obtained during and after surgery. The lower limit of detection was 1 to 10 CEA‐positive cells diluted among 1 × 107 blood mononuclear cells. The test was positive for 20 of the 55 patients with cancer (36%). None of the 22 control samples were positive. An increase in positivity was observed with increasing stage of disease; however, even some patients with early‐stage cancer showed positive results. In addition, CEA mRNA could be detected in the peripheral blood during operation in 3 of 13 patients whose pre‐operative CEA mRNA in the peripheral blood had been negative. These findings suggest that, (1) RT‐PCR amplification of CEA mRNA is an efficient means of detecting circulating solid cancer cells in the peripheral blood, although long‐term clinical studies should be done to evaluate its usefulness; (2) not only breast cancer but also gastrointestinal cancer might be better regarded as a systemic disease even in early stages of carcinoma; and (3) surgical manipulation can provoke cancer‐cell dissemination. ©1996 Wiley‐Liss, Inc.

Clinical significance of molecular detection of carcinoma cells in lymph nodes and peripheral blood by reverse transcription-polymerase chain reaction in patients with gastrointestinal or breast carcinomas.

PURPOSE This study evaluates the clinical significance of detection of carcinoembryonic antigen (CEA) mRNA in the dissected lymph nodes and peripheral blood samples of patients with gastrointestinal or breast carcinomas. PATIENTS AND METHODS A total of 406 lymph nodes obtained from 65 patients were analyzed by both histologic and molecular examination of CEA-specific reverse transcriptase-polymerase chain reaction (RT-PCR). Peripheral blood samples from another 102 patients were also analyzed by CEA-specific RT-PCR. Patients were followed up prospectively for 24 +/- 12 months. RESULTS Of 406 lymph nodes, the positive detection rate increased from 20% by histologic examination to 60% by RT-PCR examination. The recurrence rate was 40% in 15 cases showing positive results in both examinations, 14% in 29 cases showing histologically negative but RT-PCR positive results, and none in 21 cases showing negative results in both examinations. The positive detection rate for CEA mRNA in peripheral blood samples increased with advancing stage of disease. With respect to 62 curatively operated cases, CEA mRNA was detected in 12 cases. Four of these 12 cases developed metastatic disease after surgery whereas none of 50 cases negative by RT-PCR developed metastasis. CONCLUSION It has been shown that RT-PCR is a powerful tool to detect CEA mRNA in the lymph nodes or the peripheral blood. This is potentially very useful to determine high-risk patients for metastasis. Serial analysis is warranted to assess the long-term significance of this method and its therapeutic and prognostic implications.

Plastin3 Is a Novel Marker for Circulating Tumor Cells Undergoing the Epithelial–Mesenchymal Transition and Is Associated with Colorectal Cancer Prognosis

Circulating tumor cells (CTC) in blood have attracted attention both as potential seeds for metastasis and as biomarkers. However, most CTC detection systems might miss epithelial-mesenchymal transition (EMT)-induced metastatic cells because detection is based on epithelial markers. First, to discover novel markers capable of detecting CTCs in which EMT has not been repressed, microarray analysis of 132 colorectal cancers (CRC) from Japanese patients was conducted, and 2,969 genes were detected that were overexpressed relative to normal colon mucosa. From the detected genes, we selected those that were overexpressed CRC with distant metastasis. Then, we analyzed the CRC metastasis-specific genes (n = 22) to determine whether they were expressed in normal circulation. As a result, PLS3 was discovered as a CTC marker that was expressed in metastatic CRC cells but not in normal circulation. Using fluorescent immunocytochemistry, we validated that PLS3 was expressed in EMT-induced CTC in peripheral blood from patients with CRC with distant metastasis. PLS3-expressing cells were detected in the peripheral blood of approximately one-third of an independent set of 711 Japanese patients with CRC. Multivariate analysis showed that PLS3-positive CTC was independently associated with prognosis in the training set (n = 381) and the validation set [n = 330; HR = 2.17; 95% confidence interval (CI) = 1.38-3.40 and HR = 3.92; 95% CI = 2.27-6.85]. The association between PLS3-positive CTC and prognosis was particularly strong in patients with Dukes B (HR = 4.07; 95% CI = 1.50-11.57) and Dukes C (HR = 2.57; 95% CI = 1.42-4.63). PLS3 is a novel marker for metastatic CRC cells, and it possesses significant prognostic value.

Somatic Mutations of Epidermal Growth Factor Receptor in Colorectal Carcinoma

Purpose: Somatic mutations of the epidermal growth factor receptor (EGFR) gene may predict the sensitivity of non–small cell lung carcinoma to gefitinib. However, no mutations have been reported for colorectal carcinoma. We therefore analyzed EGFR mutations in colorectal adenocarcinomas by the combined use of laser microdissection and sequencing of genomic DNA. Experimental Design: We examined 11 representative colorectal adenocarcinoma cell lines and 33 clinical samples of colorectal carcinoma. In the clinical cases, we carefully dissected only carcinoma cells from frozen sections by laser microdissection. After DNA extraction and PCR, we examined EGFR mutations by sequencing genomic DNA. Results: None of 11 colorectal carcinoma cell lines exhibited somatic mutations, but 4 of 33 clinical tumors (12%) exhibited mutations in the EGFR kinase domain. This may be the first report of somatic mutations in colorectal adenocarcinoma. Conclusions: Our findings suggest that a distinct minority of colorectal adenocarcinomas exhibit somatic mutations of EGFR, and these tumors may be susceptible to gefitinib treatment.

The significance of carbonic anhydrase expression in human colorectal cancer

BACKGROUND Carbonic anhydrase isoenzymes I and II are present in normal colorectal mucosa. This study aimed to determine if carbonic anhydrase is present in colorectal cancer and what is its potential clinical significance. METHODS The messenger RNA (mRNA) and protein expression of carbonic anhydrase isoenzyme I were analyzed in fresh specimens of colorectal adenocarcinoma by Northern and Western blots, respectively. The immunohistochemical expression was subsequently studied in a larger number of formalin-fixed surgical specimens. RESULTS All of 30 normal colon samples had a strong RNA hybridization signal. Only 4 of 39 paired colorectal cancer and none of 9 normal liver samples had detectable levels of carbonic anhydrase mRNA. Isoenzyme I protein expression showed similar results. In a separate group of patients, immunohistochemical studies showed that 16 of 96 colorectal tumors had positive staining cells. All positive tumors were well or moderately differentiated carcinomas (P < 0.05). When analyzed retrospectively, immunoreactive cases were more likely to be in a group with a good outcome (P < 0.01) and to lack vascular invasion (P < 0.01) than negative cases. CONCLUSIONS The majority of colorectal cancers do not express carbonic anhydrase isoenzyme I. The presence of any isoenzyme I-positive immunoreactive cancer cells may be associated with a more favorable outcome in colorectal cancer.

Regional Expression of CXCL12/CXCR4 in Liver and Hepatocellular Carcinoma and Cell-cycle Variation during in vitro Differentiation

The CXCL12/CXCR4 system may be important in carcinoma. Expression of the a‐chemokine SDF‐lα (stromal cell derived factor‐lα)/CXCL12 mRNA is reduced in many carcinomas, yet its tissue protein expression may guide metastasis. Here we first compare the mRNA and protein expression of CXCL12 and its receptor CXCR4 in human liver, hepatocellular carcinoma, and malignant cell lines, and then assess cell cycle variation in CXCR4 expression. CXCR4 mRNA was present in most normal human tissues and malignant cell lines; it was only marginally reduced in hepatomas, while CXCL12 was markedly reduced, P<0.0001. Immuno‐histochemical staining of adjacent non‐malignant liver showed regional CXCR4 cytoplasmic and cell‐surface staining, limited to those hepatocytes around the central vein, a distribution resembling that of CXCL12. CXCL12 protein was not present in hepatocellular carcinoma cells in vivo, nor was cytoplasmic CXCR4 staining; nuclear CXCR4 protein expression in some malignant hepatocytes and CXCR4 staining of capillary endothelial cells around tumor cells were noted. In some malignant cell lines that had no CXCL12 on northern blots CXCL12 was weakly detectable by RT‐PCR or protein staining in the cytoplasm of a few cells. With a view to future manipulation of CXCL12/CXCR4 expression and growth we noted that in HT‐29 cells CXCR4 protein expression was less on confluent than on non‐confluent cells and varied during the cell cycle. Higher expression was associated most closely with the percentage of cells in the S‐phase and inversely with the percentage of cells in the G1‐phase. Treatment of HT‐29 cells with butyrate reduced CXCR4 cell surface expression and reduced the percentage of cells in S‐phase. In summary, CXCL12 protein expression parallels its mRNA, being markedly reduced in malignant cell lines and hepatomas; in liver, the regional distributions of CXCL12 and cytoplasmic CXCR4 are similar; finally, in HT‐29, CXCR4 expression correlates with the S‐phase of the cell cycle and is reduced during butyrate‐induced differentiation.

Clinicopathologic and Biological Significance of Kallikrein 6 Overexpression in Human Gastric Cancer

Purpose: Human kallikrein genes (KLK) have been reported to be involved in human malignancies and several KLKs are promising biomarkers of prostate, ovarian, testicular, and breast cancers. Herein, we investigated the clinicopathologic and biological significance of KLK6 gene expression in human gastric cancer. Patients and Methods: Using real-time reverse transcription-PCR, we analyzed the KLK6 expression status with respect to various clinicopathologic variables in 66 patients with gastric cancer. In addition, we established a KLK6 stably suppressed gastric cancer cell line (MKN28) using small interfering RNA–mediated gene silencing, and investigated its effects on the cell proliferation rate, cell cycle, and invasiveness. Results: The KLK6 gene expression in cancerous tissue (0.37 ± 0.53) was significantly (P < 0.000001) higher than that in noncancerous tissue (0.026 ± 0.060). Elevated KLK6 expression was significantly associated with lymphatic invasion (P = 0.03). Furthermore, patients with a high KLK6 expression had a significantly poorer survival rate than those with a low KLK6 expression (P = 0.03). Therefore, we showed that KLK6 gene silencing with KLK6 small interfering RNA effectively suppressed the cell proliferation rate (P = 0.002), cell population in the S phase (P < 0.01), and invasiveness (P < 0.01) in comparison to mock-transfected cells. Conclusions: The KLK6 gene is markedly overexpressed in gastric cancer tissue and its expression status may be a powerful prognostic indicator for patients with gastric cancer. Our findings also suggest that KLK6 may possibly be a novel target for gastric cancer therapy by gene-silencing procedures.

Interleukin-8 and SDF1-α mRNA expression in colonic biopsies from patients with inflammatory bowel disease

OBJECTIVES:Interleukin-8 (IL-8) as an α-chemokine recruits and activates neutrophils, which are abundant in the intestinal lesions of ulcerative colitis (UC) and Crohns disease (CD). Stromal cell-derived factor 1 (SDF1-α) is a new chemokine that is chemotactic to neutrophils. The aims of this study were to assess the relative expression of SDF1-α and IL-8 mRNA in different colonic regions and patients with inflammatory bowel disease with varied degrees of inflammation in the colon.METHODS:Colon biopsy samples were obtained from 19 patients with UC, 12 with CD, and 5 with irritable bowel syndrome (IBS) who underwent colonoscopy. Levels of IL-8 and SDF1-α mRNA expression were measured semiquantitatively by reverse-transcription and polymerase chain reaction amplification. The cytokine mRNA levels were corrected for glyceraldelyde-3-phosphate dehydrogenase mRNA expression.RESULTS:IL-8 mRNA expression was significantly correlated with SDF1-α expression in normal biopsies from IBS patients (r = 0.58, p < 0.01). There was no significant difference in cytokine mRNA expression (IL-8 or SDF1-α) across different regions of the colon or rectum in uninflamed normal biopsies. The IL-8 mRNA expression ratios in UC (mean ± SD, 1.03 ± 0.52) and CD (0.90 ± 0.38) patients were significantly higher than in IBS (0.52 ± 0.17) (p < 0.01, p < 0.05, respectively). The SDF1-α mRNA expression ratio in UC (0.30 ± 0.52) was higher than in both CD (0.21 ± 0.10) and IBS patients (0.22 ± 0.11) (p < 0.01, <0.05, respectively). A statistically significant correlation was found between the IL-8 mRNA expression and the colonic inflammation in UC patients (r = 0.44, p < 0.05) but not for SDF1-α expression in UC patients.CONCLUSIONS:IL-8 but not SDF1-α mRNA expression was associated with inflammation in UC. This suggests that IL-8 may play a more important role in inflammatory bowel disease than does SDF1-α.