Masahisa Ohkuma

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.

CD133 + CD44 + Population Efficiently Enriches Colon Cancer Initiating Cells

BackgroundPrevious reports have demonstrated that CD133+ cells or CD44+ cells might be cancer initiating cells (CIC) of colon cancer. However, the association between the two cell types is unclear. In this study, we evaluated the tumorigenicity of each population of human colon cancer divided by CD133 and CD44 using non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice.MethodsUsing the colon cancer cell lines HT29 and Caco2 we evaluated the change of expression status of CD133 or CD44 by a treatment with sodium butyrate (NaBT) that can induce cellular differentiation. Next, we prepared ten clinical samples of colon cancer and analyzed the expression and tumorigenicity of CD133 and CD44.ResultsWith NaBT treatment, CD44 expression was greatly downregulated in both HT29 and Caco2 (HT29: nontreatment versus treatment; 77.8% versus 0.6%, Caco2: 14.0% versus 0.4%, respectively), more than CD133 expression (HT29: nontreatment versus treatment; 90.1% versus 67.7%, Caco2: 98.9% versus 76.3%, respectively). In clinical samples, the percentages of CD133+ cells and CD44+ cells varied from 0.3% to 82.0% (mean 35.5%), and from 11.5% to 58.4% (mean 30.0%), respectively. Subcutaneous injection of CD133+ or CD44+ cells made a tumor in all mice (3/3 and 4/4, respectively). The combined analysis of CD133 and CD44 revealed that only the CD133+CD44+ population had the ability to produce a tumor (3/3).ConclusionThe findings demonstrate that, at present, the CD133+CD44+ population may be the best to identify tumor initiating cells of human colon cancer.

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.

Increased CD13 expression reduces reactive oxygen species, promoting survival of liver cancer stem cells via an epithelial–mesenchymal transition-like phenomenon

BackgroundRecently, it has been reported that a small population of cancer stem cells (CSCs) play a role in resistance to chemotherapy and radiation therapy. We reported that CD13+ liver CSCs survive in hypoxic lesions after chemotherapy, presumably through increased expression of CD13/Aminopeptidase N, which is a scavenger enzyme in the reactive oxygen species (ROS) metabolic pathway. On the other hand, the concept of epithelial–mesenchymal transition (EMT) was indicated by a recent study showing an increased plasticity linked to the cellular “stemness” of CSCs.MethodsTo study the relationship between CSCs and EMT, we examined biological characteristics of liver cancer cell lines with EMT by exposing transforming growth factor-β (TGF-β).ResultsWe showed that a TGF-β-induced EMT-like phenomenon is associated with increased CD13 expression in liver cancer cells. This phenomenon prevents further increases in the ROS level as well as the induction of apoptosis, promoting the survival of CD13+ CSCs, whereas inhibition of CD13 stimulates apoptosis. Immunohistochemical analysis also indicated that after chemotherapy, CD13 was coexpressed with N-cadherin in surviving cancer cells within fibrous capsules. We have demonstrated that CD13 expression plays a role in supporting the survival of CSCs and that there is an EMT-associated reduction in ROS elevation.ConclusionsThis novel and consistent linkage between functional CSC markers and the EMT phenomenon suggests a bona fide candidate for targeted therapy for EMT-mediated invasion and metastasis of liver cancer.

Absence of CD71 Transferrin Receptor Characterizes Human Gastric Adenosquamous Carcinoma Stem Cells

BackgroundAlthough the importance of cancer stem cells (CSCs) in overcoming resistance to therapy and metastasis has recently been reported, the role of CSCs in gastric cancer remains to be elucidated.MethodsMKN-1 cells were used to study markers of CSCs in gastric adenosquamous carcinoma, as these cells are suitable for determining multidifferentiation ability. Changes in expression of CD44, CD49f, CD133, and CD71 following 5-fluorouracil (5-FU) treatment were assessed.ResultsAfter 5-FU treatment, only the CD71− fraction was significantly increased. Investigation of CD71 indicated that the CD71− cell fraction was present in the G1/G0 cell cycle phase and showed high resistance to the anticancer agent 5-FU. Limiting dilution and serial transplantation assays revealed the CD71− cell fraction to have higher tumorigenicity than the CD71+ cell fraction. The CD71− cell fraction showed multipotency to adenocarcinoma and squamous cell carcinoma. A three-dimensional (3D) invasion assay and immunohistochemical analysis showed CD71− cells to be highly invasive and to exist in the invasive fronts of cancer foci.ConclusionThe present study suggests that use of CD71− as a marker for adenosquamous carcinoma may provide a useful model for studying CSCs.

Thymidine phosphorylase mRNA expression may be a predictor of response to post‑operative adjuvant chemotherapy with S‑1 in patients with stage III colorectal cancer

The aim of the present study was to investigate markers in surgically resected specimens of colorectal cancer that can be used to predict the response to chemotherapy. The mRNA expression levels of enzymes involved in 5-fluorouracil (5-FU) metabolism and folate metabolism were measured in formalin-fixed, paraffin-embedded tumor sections obtained from the primary tumors of 54 patients with resected stage II or III colorectal cancer who received S-1 for one year. The 5-FU metabolizing enzymes studied were thymidylate synthase, dihydropyrimidine dehydrogenase and thymidine phosphorylase (TP). The folate metabolizing enzymes studied were folypolyglutamate synthetase, γ-glutamyl hydrolase and dihydrofolate reductase. The associations between the mRNA expression levels of these enzymes and clinical variables were investigated. Tumors were classified as exhibiting high or low expression as compared with the median mRNA expression level of each metabolizing enzyme defined as the cutoff value. The associations between the high and low expression levels of each enzyme and disease-free survival (DFS) were analyzed with the use of Kaplan-Meier curves and the log-rank test. DFS was not significantly associated with the relative mRNA expression level of any metabolizing enzyme in the study group as a whole, but there was a trend toward longer DFS in patients with high TP expression (P=0.066). In patients with stage III colorectal cancer, high TP expression was associated with significantly improved outcomes compared with low TP expression (P=0.039). These results indicate that the mRNA expression of TP, a metabolizing enzyme of 5-FU, is a significant predictor of response to post-operative chemotherapy with S-1 in patients with stage III colorectal cancer.

A comparison of laparoscopic energy devices on charges in thermal power after application to porcine mesentery.

Introduction: Advances in energy devices have played a major role in the rapid expansion of laparoscopic surgery. However, complications due to these energy devices are occasionally reported, and if the characteristics of these devices are not well understood, serious complications may occur. This study evaluated various typical energy devices and measured temperature rises in the adjacent tissue and in the devices themselves. Equipment and Methods: We used the following 7 types of energy devices: AutoSonix (AU), SonoSurg (SS), Harmonic Scalpel (HS), LigaSure Atlas (LA), LigaSure Dolphin Tip (LD), monopolar diathermy (Mono), and bipolar scissors (Bi). Laparoscopy was performed under general anesthesia in pigs, and the mesentery was dissected using each energy device. Tissue temperature at a distance of 1 mm from the energy device blade before and after dissection was measured. Temperature of the device blade both before and after dissection, time required for dissection, and interval until the temperature fell to 100°C, 75°C, and 50°C were documented. Results: Temperature of the surrounding tissue using each device rose the most with the Mono (50.5±8.0°C) and the least with the HS in full mode (6.2±0.7°C). Device temperature itself rose the highest with the AU in full mode (318.2±49.6°C), and the least with the Bi (61.9±4.8°C). All ultrasonic coagulation and cutting devices (AU, SS, and HS) had device temperatures increase up to ≥100°C, and even at 8 seconds after completing dissection, temperatures remained at ≥100°C. Conclusions: Because the adjacent tissue temperature peaked with the Mono, cautious use near the intestine and blood vessels is necessary. In addition, the active blades of all ultrasonic coagulation and cutting devices, regardless of model, developed high temperatures exceeding 100°C. Therefore, an adequate cooling period after using these devices is therefore necessary between applications.

Human colorectal CD24+ cancer stem cells are susceptible to epithelial-mesenchymal transition

Conventional cancer chemotherapy preferentially destroys non-stem cancer cells within a tumor, and a subpopulation of cancer stem cells (CSCs) is more resistant and survives, leading to relapses and metastasis. Howeve, recent studies suggest that CD24 and susceptibility to epithelial-mesenchymal transition (EMT) can serve as markers of CSCs. We report that CD24(+) cells are susceptible to induction of EMT, a phenotype important for cancer metastasis. We studied the responsiveness of CSC markers to TGF-β , an effective EMT inducer. The data on CD24 demonstrated that CD24(+) cells are susceptible to EMT, a phenotype important for cancer metastasis in two colorectal cancer cell lines, the CaR-1 and CCK81. CD24(+) cells expressed Notch 1 in response to exposure to TGF-β in culture and showed higher tumorigenic activity compared to controls. This evidence shows that CD24(+) cells are susceptible to EMT induction and to cancer progression and is indicative of the candidacy of CD24 as a therapeutic target in CSC.

Standardization of surgical procedures to reduce risk of anastomotic leakage, reoperation, and surgical site infection in colorectal cancer surgery: a retrospective cohort study of 1189 patients

PurposeAnastomotic leakage (AL) and surgical site infection (SSI) are prevalent complications of colorectal surgery. To lower this risk, we standardized our surgical procedures in 2012, with a preferential use of laparoscopic approach (LS) for both colon and rectal surgery, combined with triangulating anastomosis (TA) for colon surgery and defunctioning ileostomy (DI) for low anterior resection. Our aim was to evaluate the outcomes of our standardized procedures.MethodsThe incidence rate of AL (primary outcome) and of reoperation and SSI (secondary outcome) was compared before (early period, n = 648) and after (late period, n = 541) standardization, through a retrospective analysis.ResultsThe incidence rate of AL (6.6 versus 1.8%; P = 0.001), reoperation (3.5 versus 0.7%; P = 0.0012), and SSI (7.7 versus 4.6%; P = 0.029) was lower in late than in the early period. For colon cancer, TA and LS reduced the risk of AL (2.1 versus 0.3%, P = 0.020, for TA, and 3.2 versus 0.4%, P = 0.0027, for LS) and reoperation (2.9 versus 0.3%, P = 0.003, for TA, and 2.5 versus 0.2%, P = 0.0040, for LS). For rectal cancer, the incidence of all adverse outcomes (AL, reoperation, and SSI) was lower in cases treated by LS. However, the incidence of AL was lower in the late than in early period (P = 0.002) and with LS (P = 0.002). On multivariate analysis, late period and LS were independent factors of a lower risk of adverse outcomes.ConclusionsOur surgical standardization seems to be effective in lowering the risks of AL, reoperation, and SSI after colorectal cancer surgery.

Preoperative peripheral blood neutrophil count predicts long-term outcomes following hepatic resection for colorectal liver metastases

Preoperative systemic inflammatory response is associated with a poor long-term prognosis following resection surgery for malignant tumors. Several markers of systemic inflammation have been reported to be associated with the outcome; however, they have not currently been fully investigated. Therefore, the association between preoperative peripheral blood neutrophil count and oncological outcome following hepatic resection for colorectal liver metastasis (CRLM) was retrospectively investigated. The present study comprised 89 patients who had undergone hepatic resection for CRLM between January 2000 and March 2010. The association between preoperative peripheral blood neutrophil count and disease-free survival, in addition to overall survival, was investigated. In multivariate analysis, the presence of neoadjuvant chemotherapy (P=0.015), bilobar distribution (P=0.015) and neutrophil count ≥3,500/µl (P=0.025) were independent and significant predictors of poor disease-free survival, while significant predictors of poor overall survival consisted of >4 lymph node metastases (P=0.001), neo-adjuvant chemotherapy (P=0.003), bilobar distribution (P=0.039) and neutrophil count ≥3,500/µl (P=0.040). Additionally, tumor diameter (P=0.021) and monocyte count (P<0.0001) were observed to be significantly greater in the elevated neutrophil count group. In conclusion, preoperative peripheral blood neutrophil count may be an independent and significant indicator of poor long-term outcomes in patients with CRLM following hepatic resection.