Tatsuya Manabe

Tumor–stromal interactions with direct cell contacts enhance proliferation of human pancreatic carcinoma cells

Pancreatic ductal adenocarcinoma is often characterized by an abundant desmoplastic stroma that is partially induced by activated pancreatic stellate cells (PSCs). Indirect co‐culture has often been used to investigate the effects of cancer–stromal interactions on the proliferation of cancer cells, but the effects of cell–cell adhesion and juxtacrine signaling between cancer and stromal cells cannot be evaluated using this method. This study aimed to establish a simplified direct co‐culture system that could be used to quantify populations of cancer cells in co‐culture with PSCs, and to evaluate the effects of direct cell contact on the proliferation of cancer cells. We established three green fluorescent protein (GFP)‐expressing pancreatic cancer cell lines and were able to quantify them with high reliability and reproducibility, even when co‐cultured directly with PSCs, using a color plate reader. We assessed the differential effects of direct and indirect co‐culture with PSCs on the proliferation of cancer cells, and found that the proliferation of GFP‐expressing pancreatic cancer cell lines was dramatically enhanced by direct co‐culture with PSCs, compared with the indirect co‐culture system. We also found that direct co‐culture of cancer cells and PSCs activated the Notch signaling pathway in both cell types. Direct cell contact between cancer cells and PSCs plays an important role in the control of cancer cell proliferation, and is essential to the understanding of tumor–stromal interactions. (Cancer Sci 2009; 100: 2309–2317)

α-Smooth muscle actin expressing stroma promotes an aggressive tumor biology in pancreatic ductal adenocarcinoma

Objectives: Pancreatic ductal adenocarcinoma (PDAC) is often characterized by a prominent desmoplastic stroma that is induced partially by &agr;-smooth muscle actin (SMA)-expressing activated pancreatic stellate cells (PSCs). This study aimed to investigate the significance of &agr;-SMA expression in PDAC and the correlation between &agr;-SMA mRNA levels and the patient prognosis. Methods: We obtained formalin-fixed, paraffin-embedded tissue samples from 109 patients with PDAC, who underwent pancreatectomy at our institution from 1992 to 2007. We measured &agr;-SMA mRNA levels by quantitative real-time reverse transcription-polymerase chain reaction and investigated the association of &agr;-SMA mRNA expression with clinicopathologic parameters and survival time. We also assessed the influence of activated PSCs on malignant behaviors of pancreatic cancer cells using in vitro experiments. Results: &agr;-SMA immunoreactivity was detected exclusively in the stroma of PDAC. The group with high &agr;-SMA expression showed a significantly shorter survival, as shown by univariate analysis (P = 0.005) and multivariate analysis (P < 0.0001). &agr;-SMA-expressing activated PSCs enhanced the invasiveness, proliferation, and colony formation of pancreatic cancer cells. Conclusions: Quantitative analysis of &agr;-SMA mRNA expression using formalin-fixed, paraffin-embedded tissue samples was useful to predict the prognosis of patients with PDAC. Activated PSCs may regulate the malignant behavior of pancreatic cancer cells. Abbreviations: PDAC - pancreatic ductal adenocarcinoma, PSCs - pancreatic stellate cells, &agr;-SMA - &agr;-smooth muscle actin, FFPE - formalin-fixed paraffin-embedded, qRT-PCR - quantitative real-time reverse transcription-polymerase chain reaction, ECM - extracellular matrix, GFP - green fluorescent protein, CM - conditioned media, UICC - Union Internationale Contre le Cancer and the American Joint Committee on Cancer, COL1 - collagen type I, AC - adjuvant chemotherapy

Predicting the chemosensitivity of pancreatic cancer cells by quantifying the expression levels of genes associated with the metabolism of gemcitabine and 5-fluorouracil

Gemcitabine (GEM) is the standard treatment for advanced/metastatic pancreatic cancer. However, there is a substantial subset of patients in whom the efficacy of GEM, when used as a single agent, is inadequate. Recently, the 5-fluorouracil (5-FU) prodrugs capecitabine and S-1 have been used as an alternative, either alone or in combination with GEM. The aim of the present study was to investigate the expression pattern of genes that render pancreatic cancer cells sensitive to GEM and 5-FU, and to identify markers for individualized chemotherapy, even in patients who have developed resistance. We investigated the correlation between the expression of genes associated with the metabolism of GEM and 5-FU, and sensitivity to these drugs in 15 human pancreatic cancer cell lines. We also established GEM- and 5-FU-resistant pancreatic cancer cell lines to investigate changes in the expression levels of these genes and the effects of one drug on cells resistant to the other. We found no correlation between pancreatic cancer cell sensitivity to either GEM- or 5-FU. GEM-resistant cells did not become resistant to 5-FU and vice versa. High expression of RRM1 (P=0.048) and TS x DPD (P=0.035) correlated significantly with sensitivity to GEM and 5-FU, respectively. 5-FU-resistant cells expressed significantly higher levels of TP than parental cells (P<0.05). In conclusion, pancreatic cancer cells showed no cross-resistance to GEM and 5-FU. Quantitative analyses of RRM1, TP, DPD and TS mRNA levels in pancreatic cancer cells may be useful for predicting their sensitivity to GEM and 5-FU.

High EGFR mRNA expression is a prognostic factor for reduced survival in pancreatic cancer after gemcitabine-based adjuvant chemotherapy

Pancreatic ductal adenocarcinoma (PDAC) still presents a major therapeutic challenge and a phase III clinical trial has revealed that the combination of gemcitabine and a human epidermal growth factor receptor type I (HER1/EGFR) targeting agent presented a significant benefit compared to treatment with gemcitabine alone. The aim of this study was to investigate EGFR mRNA expression in resected PDAC tissues and its correlation with patient prognosis. We obtained formalin-fixed paraffin-embedded (FFPE) tissue samples from 88 patients with PDAC who underwent pancreatectomy, and measured EGFR mRNA levels by quantitative real-time reverse transcription-polymerase chain reaction. The high-level EGFR group had significantly shorter disease-free-survival (p=0.029) and overall-survival (p=0.014) as shown by univariate analyses, although these did not reach statistical significance, as shown by multivariate analyses. However, we found that high EGFR expression was an independent prognostic factor in patients receiving gemcitabine-based adjuvant chemotherapy (p=0.023). Furthermore, we measured EGFR mRNA levels in 20 endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) cytological specimens. Altered EGFR levels were distinguishable in microdissected neoplastic cells from EUS-FNA cytological specimens compared to those in whole cell pellets. In conclusion, quantitative analysis of EGFR mRNA expression using FFPE tissue samples and microdissected neoplastic cells from EUS-FNA cytological specimens could be useful in predicting prognosis and sensitivity to gemcitabine in PDAC patients.

Tumor deposit is a poor prognostic indicator for patients who have stage II and III colorectal cancer with fewer than 4 lymph node metastases but not for those with 4 or more.

BACKGROUND: Extranodal tumor deposits are involved in TNM classification. However, it is uncertain whether a tumor deposit is a regular lymph node metastasis, and its prognostic significance in patients with stage II or III colorectal cancer remains to be established. OBJECTIVE: This study aimed to determine the prognostic significance of tumor deposits for stage II and III colorectal cancer. DESIGN: This study is a retrospective review of clinicopathological data. SETTING: This study was conducted at a tertiary care hospital/referral center in Japan. PATIENTS: We reviewed the clinical course of 171 stage II and 173 stage III consecutive patients between January 1999 and December 2006. MAIN OUTCOME MEASURES: We examined the clinicopathological features of colorectal cancers with tumor deposits and calculated overall survival and recurrence-free survival of the patients according to the status of tumor deposits. The primary outcome was the impact of tumor deposits on patient survival. RESULTS: Thirty-five (10.2%) patients with colorectal cancers had tumor deposits in the pericolic and/or mesocolic region. Survival rates among the patients with tumor deposits were significantly lower than those without (5-year overall survival: 58.4% vs 81.0%, p < 0.0001; 5-year recurrence-free survival: 47.1% vs 73.4%, p < 0.0001). Tumor deposit was an independent prognostic factor for patients with colorectal cancer in multivariate analysis (overall survival: HR, 2.30; 95% CI, 1.26–4.04; p = 0.04; recurrence-free survival: HR, 2.42; 95% CI, 1.04–4.90; p = 0.04). Tumor deposit was an independent prognostic factor in N0 and N1 colorectal cancer, whereas N2 cancer had poor survival outcome regardless of tumor deposit. LIMITATIONS: Our study was a single-institution retrospective study, and the numbers of patients were relatively small to draw firm conclusions. CONCLUSION: Tumor deposit may be an independent adverse prognostic factor for stage II and III N1 colorectal cancer.

Extra-pancreatic invasion induces lipolytic and fibrotic changes in the adipose microenvironment, with released fatty acids enhancing the invasiveness of pancreatic cancer cells

Pancreatic cancer progression involves components of the tumor microenvironment, including stellate cells, immune cells, endothelial cells, and the extracellular matrix. Although peripancreatic fat is the main stromal component involved in extra-pancreatic invasion, its roles in local invasion and metastasis of pancreatic cancer remain unclear. This study investigated the role of adipose tissue in pancreatic cancer progression using genetically engineered mice (Pdx1-Cre; LSL-KrasG12D; Trp53R172H/+) and an in vitro model of organotypic fat invasion. Mice fed a high fat diet had significantly larger primary pancreatic tumors and a significantly higher rate of distant organ metastasis than mice fed a standard diet. In the organotypic fat invasion model, pancreatic cancer cell clusters were smaller and more elongated in shape and showed increased fibrosis. Adipose tissue-derived conditioned medium enhanced pancreatic cancer cell invasiveness and gemcitabine resistance, as well as inducing morphologic changes in cancer cells and increasing the numbers of lipid droplets in their cytoplasm. The concentrations of oleic, palmitoleic, and linoleic acids were higher in adipose tissue-derived conditioned medium than in normal medium, with these fatty acids significantly enhancing the migration of cancer cells. Mature adipocytes were smaller and the concentration of fatty acids in the medium higher when these cells were co-cultured with cancer cells. These findings indicate that lipolytic and fibrotic changes in peripancreatic adipose tissue enhance local invasiveness and metastasis via adipocyte-released fatty acids. Inhibition of fatty acid uptake by cancer cells may be a novel therapy targeting interactions between cancer and stromal cells.

Suppression of CD51 in pancreatic stellate cells inhibits tumor growth by reducing stroma and altering tumor-stromal interaction in pancreatic cancer

Pancreatic stellate cells (PSCs) enhance the malignant behavior of pancreatic cancer by interacting with cancer cells and producing extracellular matrix (ECM). To date, several stroma-targeted therapies for pancreatic cancer have been attempted, but these therapies are still not in practical use. Integrins expressed in stromal cells are involved in fibrosis of several organs, as well as promoting tumor malignancy. We investigated whether CD51, also known as integrin αV, expressed in PSCs was associated with stromal formation of pancreatic cancer and enhancement of tumor malignancy. We also assessed the effects of suppression of CD51 in PSCs on pancreatic cancer. Immunohistochemistry for CD51 in resected pancreatic cancer tissues showed that high expression of CD51 in the tumor stroma was associated with lymph node metastasis (P=0.025), positive pathologic margin (P=0.025), and shorter patient survival times (P=0.043). Lentivirus-mediated short hairpin RNA knockdown of CD51 decreased the proliferation and migration of PSCs. Quantitative real-time polymerase chain reaction showed that expression levels of genes related with ECM and tumor-stromal interactions were decreased by CD51 knockdown in PSCs. In a co-implantation model of pancreatic cancer cells and PSCs, tumor growth in vivo was inhibited by CD51 knockdown in PSCs (P<0.05). We also found reduced tumor stroma and decreased proliferation of cancer cells in implanted cancer tissues with CD51-silenced PSCs (P<0.05). Our results showed that CD51 expression in pancreatic cancer stroma is associated with enhanced tumor malignancy and that CD51 may be a potential therapeutic target for pancreatic cancer.

Calpain inhibitor calpeptin suppresses pancreatic cancer by disrupting cancer–stromal interactions in a mouse xenograft model

Desmoplasia contributes to the aggressive behavior of pancreatic cancer. However, recent clinical trials testing several antifibrotic agents on pancreatic cancer have not shown clear efficacy. Therefore, further investigation of desmoplasia‐targeting antifibrotic agents by another mechanism is needed. Calpeptin, an inhibitor of calpains, suppressed fibroblast function and inhibited fibrosis. In this study, we investigated the anticancer effects of calpeptin on pancreatic cancer. We investigated whether calpeptin inhibited tumor progression using a mouse xenograft model. We used quantitative RT‐PCR to evaluate the expression of calpain‐1 and calpain‐2 mRNA in pancreatic cancer cells (PCCs) and pancreatic stellate cells (PSCs). We also undertook functional assays, including proliferation, migration, and invasion, to evaluate the inhibitory effects of calpeptin on PCCs and PSCs. Quantitative RT‐PCR indicated that PCCs and PSCs expressed calpain‐2 mRNA. Calpeptin reduced tumor volume (P = 0.0473) and tumor weight (P = 0.0471) and inhibited the tumor desmoplastic reaction (P < 0.001) in xenograft tumors in nude mice. Calpeptin also inhibited the biologic functions of PCCs and PSCs including proliferation (P = 0.017), migration (P = 0.027), and invasion (P = 0.035) in vitro. Furthermore, calpeptin reduced the migration of PCCs and PSCs by disrupting the cancer–stromal interaction (P = 0.0002). Our findings indicate that calpeptin is a promising antitumor agent for pancreatic cancer, due not only to its suppressive effect on PCCs and PSCs but also its disruption of the cancer–stromal interaction.

Galanin plays an important role in cancer invasiveness and is associated with poor prognosis in stage II colorectal cancer

Reliable predictors of tumor recurrence for patients with stage II colorectal cancer (CRC) are needed to select patients who should receive adjuvant chemotherapy. Although galanin (GAL) is expressed in several malignant tumors and is associated with cell proliferation and tumor growth, the prognostic value of GAL expression in CRC is poorly understood. We compared GAL expression between 56 patients with stage II and III CRC who developed tumor recurrences and 56 patients who did not. The clinical and prognostic significance of GAL expression was examined using our data and independent public datasets. We also analyzed the influence of GAL expression on the proliferation and invasive activity of CRC cells. Higher expression of GAL was associated with tumor recurrence among the CRC patients (P<0.001). Stage II CRC patients who presented with high expression levels of GAL had significantly poorer prognosis than those with low expression levels of GAL [5-year overall survival: hazard ratio (HR), 7.31; 95% confidence interval (CI), 2.38–24.04; P<0.001; 5-year recurrence-free survival: HR, 3.99; 95% CI, 1.61–9.44; P=0.004], but there was no association between GAL expression and survival in stage III CRC patients. These findings were supported by analysis of two public datasets. Functionally, siRNA-mediated silencing of GAL resulted in a significant decrease in the proliferative and invasive activities of CRC cells. In conclusion, high expression of GAL is associated with poor prognosis of stage II CRC patients and GAL expression may be related to the aggressive behavior of CRC.

TM4SF1 as a prognostic marker of pancreatic ductal adenocarcinoma is involved in migration and invasion of cancer cells.

The cell surface protein Transmembrane 4 L6 family member 1 (TM4SF1) has been detected in various tumors, and its expression on tumor cells is implicated in cancer cell metastasis and patient prognosis. The role of TM4SF1 in malignant tumors remains poorly understood, particularly in pancreatic cancer. We performed immunohistochemical staining to analyze the expression of TM4SF1 in resected pancreatic tissues and investigated the correlation between TM4SF1 expression and prognosis. The function of TM4SF1 in the invasion and migration of pancreatic cancer cells was analyzed in vitro using an RNA interference technique. In pancreatic cancer tissues, TM4SF1 expression was detected in cancer cells, and patients with high tumor levels of TM4SF1 showed longer survival times than those with low TM4SF1 levels (P=0.0332). In vitro, reduced TM4SF1 expression enhanced the migration (P<0.05) and invasion (P<0.05) of pancreatic cancer cells partially via decreased E-cadherin expression. TM4SF1 protein levels were also reduced after TGF-β1-induced epithelial-mesenchymal transition (EMT).TM4SF1 expression is associated with better prognosis in pancreatic cancer. Loss of TM4SF1 contributes to the invasion and migration of pancreatic cancer cells.