Tsuyoshi Enomoto

Histological and prognostic importance of CD44+/CD24+/EpCAM+ expression in clinical pancreatic cancer

CD44+/CD24+/EpCAM+ cells have been reported to be cancer stem cells in pancreatic cancer; however, the histological and clinical importance of these cells has not yet been investigated. Here we clarified the characteristics of CD44+/CD24+/EpCAM+ cells in clinical specimens of pancreatic cancer using immunohistochemical assay. We used surgical specimens of pancreatic ductal adenocarcinoma from 101 patients. In view of tumor heterogeneity, we randomly selected 10 high‐power fields per case, and triple‐positive CD44+/CD24+/EpCAM+ expression was identified using our scoring system. The distribution, histological characteristics, and prognostic importance of CD44+/CD24+/EpCAM+ cells were then analyzed. As a result, the distribution of CD44+/CD24+/EpCAM+ cells varied widely among the 101 cases examined, and CD44+/CD24+/EpCAM+ expression was correlated with poor glandular differentiation and high proliferation. Survival analysis showed that CD44+/CD24+/EpCAM+ expression was not correlated with patient outcome; however, CD44+/CD24+ expression appeared to be correlated with poor prognosis. In conclusion, CD44+/CD24+/EpCAM+ expression overlapped with poorly differentiated cells and possessed high proliferative potential in clinical pancreatic cancer. In particular, the presence of double‐positive CD44+/CD24+ expression seemed to have clinical relevance, associating with poor prognosis.

Anticancer effects of gemcitabine are enhanced by co-administered iRGD peptide in murine pancreatic cancer models that overexpressed neuropilin-1

Background:Impaired drug transport is an important factor that reduces the efficacy of anticancer agents against pancreatic cancer. Here, we report a novel combination chemotherapy using gemcitabine (GEM) and internalised-RGD (iRGD) peptide, which enhances tumour-specific drug penetration by binding neuropilin-1 (NRP1) receptor.Methods:A total of five pancreatic cancer murine models (two cell line-based xenografts (CXs) and three tumour grafts (TGs)) were treated with either GEM (100 mg kg−1, q3d × 4) alone or GEM plus iRGD peptide (8 μmol kg−1). Evaluation of NRP1 expression in xenografts and 48 clinical cancer specimens was performed by immunohistochemistry (IHC).Results:We identified a subset of pancreatic cancer models that showed NRP1 overexpression sensitive to iRGD co-administration. Treatment with GEM plus iRGD peptide resulted in a significant tumour reduction compared with GEM monotherapy in CXs, but not remarkable in TGs. Potential targets of iRGD were characterised as cases showing NRP1 overexpression (IHC-2+/3+), and these accounted for 45.8% of the clinical specimens.Conclusions:Internalised RGD peptide enhances the effects of co-administered drugs in pancreatic cancer models, its efficacy is however only appreciable in those employing cell lines. Therefore, the clinical application needs to be given careful consideration.

Evaluation of serum high-density lipoprotein cholesterol levels as a prognostic factor in gastric cancer patients

Background and Aim:  Although there are some reports of an adverse effect of low serum high‐density lipoprotein cholesterol (HDL‐C) levels on gastrointestinal cancers, the specific correlation between serum HDL‐C levels and gastric cancer remains unknown.

Minimally required heat doses for various tumour sizes in induction heating cancer therapy determined by computer simulation using experimental data

Purpose: Although induction heating cancer therapy (IHCT) using magnetic nanoparticles can be a promising approach to treatment-less multi-nodular cancers, the objective requirement for successful clinical application has not clearly been elucidated. We intended to define objective heat doses suitable for IHCT, especially focusing on the sizes of liver cancer nodules. Materials and methods: Alternating magnetic fields were applied to three human pancreatic cancer cell lines, the intercellular space of those cell pellets were filled with magnetic nanoparticles, and confirmed the cytotoxic effect of IHCT. Subsequently, the temperatures of liver cancer nodules in IHCT were simulated using a computer software program and the required heat dose for various sized tumours were determined. Results: Heating the cancer cells up to 50°C for 10 min was sufficient for complete cell killing and the heat dose of 1.7 W/gtumour is required for 10 mm tumour. Larger tumours require a smaller heat dose, e.g. 20 mm and 40 mm tumours require 0.7 W/gtumour and 0.6 W/gtumour, respectively, whereas smaller tumours require large amounts of heat, e.g. 5 mm and 1 mm tumours require 5.1 W/gtumour and 105 W/gtumour, respectively. Conclusions: Integrating the presently available technologies, including high-quality magnetic nanoparticles (1000 W/gmaterial) and effective drug delivery systems (1–2 mgmaterial/gtumour), treatment of a 10 mm tumour seems possible. Since treatment of smaller tumours less than 5 mm require substantial heat dose, researchers involved in IHCT should target cancer nodules of 10 mm or more, and develop a heat delivery system providing a minimum of 1.7 W/gtumour.

The measurement of small magnetic signals from magnetic nanoparticles attached to the cell surface and surrounding living cells using a general-purpose SQUID magnetometer

Magnetic nanoparticles have recently been widely applied in the bio-medical field. Responding to the demand for a simple and sensitive magnetic assay system for bio-liquid samples, we employed a general-purpose superconducting quantum interference device (SQUID) magnetometer. Strips of filter paper were used as a liquid-specimen sample holder possessing a very small magnetic background signal. An aqueous solution of superparamagnetic iron-oxide nanoparticles (Resovist) was dropped in a tiny blot-like spot in the middle of the filter paper and the magnetization was measured. Magnetic moments of a dilution series of Resovist solutions versus the number of particles provided a linear graph, revealing that the magnetic moment per Resovist particle was 8.25 x 10(-17) emu. 1 x 10(5) cancer cells were incubated with Resovist, and the number of Resovist particles attached to the cell surface and surrounding a living cell was calculated to be 1.02 +/- 0.14 x 10(7) particles/cell. Our system using a commercial SQUID magnetometer should be more than enough to determine the number of magnetic nanoparticles biologically reacting with living cells, contributing to the application of magneto nanomaterials to the life-science field.

Consistent liver metastases in a rat model by portal injection of microencapsulated cancer cells.

Consistent liver metastases in animal models is generally observed only with certain cancer cell lines. With the aim of improving on existing animal models of liver metastases, we hypothesized that cancer cells encased in 300 microm microcapsules, mimicking micrometastatic foci, might be effective seeds of liver metastases. A total of 3,000 microcapsules, containing 700 to 1,500 viable cells/capsule in logarithmic growth phase of three human pancreatic cancer cell lines (SUIT-2, AsPC-1, and BxPC-3), were transplanted in nude rats by portal injection. The rate of liver metastases was 100% (12 of 12), 100% (6 of 6), and 83% (5 of 6) for SUIT-2, AsPC-1, and BxPC-3 microcapsules, respectively. In contrast, the administration of an identical number of single cancer cells (2.1-4.5 x 10(6)) did not lead to liver metastases. Metastases was strictly limited to the liver, was quite stable, and could be proportionately tailored by varying the number of cancer microcapsules administered. Microscopic observation showed that two-thirds of the cancer microcapsules were lodged in the peripheral small (20-50 microm) portal veins, although one-third of the cancer microcapsules were trapped in the central wide (200-400 microm) portal vein. Capsules began to burst at day 3, with recognizable metastases produced at day 7, resulting in overt metastases production at days 28 to 42. The present cancer microcapsule method may be useful for obtaining liver metastases in animal models, especially for cell lines that will not form liver metastases with conventional single cell injection methods and/or for experiments requiring the consistent formation of liver metastases.

Pancreatic neuroendocrine tumor and solid-pseudopapillary neoplasm: Key immunohistochemical profiles for differential diagnosis

AIM To reveal better diagnostic markers for differentiating neuroendocrine tumor (NET) from solid-pseudopapillary neoplasm (SPN), focusing primarily on immunohistochemical analysis. METHODS We reviewed 30 pancreatic surgical specimens of NET (24 cases) and SPN (6 cases). We carried out comprehensive immunohistochemical profiling using 9 markers: Synaptophysin, chromogranin A, pan-cytokeratin, E-cadherin, progesterone receptor, vimentin, α-1-antitrypsin, CD10, and β-catenin. RESULTS E-cadherin staining in NETs, and nuclear labeling of β-catenin in SPNs were the most sensitive and specific markers. Dot-like staining of chromogranin A might indicate the possibility of SPNs rather than NETs. The other six markers were not useful because their expression overlapped widely between NETs and SPNs. Moreover, two cases that had been initially diagnosed as NETs on the basis of their morphological features, demonstrated SPN-like immunohistochemical profiles. Careful diagnosis is crucial as we actually found two confusing cases showing disagreement between the tumor morphology and immunohistochemical profiles. CONCLUSION E-cadherin, chromogranin A, and β-catenin were the most useful markers which should be employed for differentiating between NET and SPN.

Histological advantages of the tumor graft: a murine model involving transplantation of human pancreatic cancer tissue fragments.

Objectives Experimental data based on cell line–derived xenograft models (cell xenograft) seldom reproduce the clinical situation, and therefore we demonstrated here the superiority of a murine model involving transplantation of human pancreatic cancer tissue fragments (tumor graft), focusing on the histological features and drug delivery characteristics. Methods Tumor pieces from 10 pancreatic cancer patients were transplanted into SCID (severe combined immunodeficient) mice. Histological characteristics of tumor grafts, including morphology, desmoplastic reaction, and vascularization, were compared with those of cell xenografts. Drug delivery was evaluated by quantifying the concentrations of injected drug, and the results were compared with its histological features. Results Eight of the 10 transplanted tumors successfully engrafted. Histological comparisons between tumor grafts and cell xenografts revealed the following: the amount of stroma was more (22.9% ± 11.8% vs 10.8% ± 5.4%; P < 0.05), vessel–cancer cell distance was longer (35.3 ± 39.0 vs 3.9 ± 3.1 &mgr;m; P < 0.001), and microvessel density was lower (6.8 ± 1.9 vs 10.8 ± 2.1 vessels/0.4 mm2; P < 0.05) in tumor grafts. Drug concentrations in tumor grafts were lower than those in cell xenografts (3.3 ± 1.2 vs 6.0±0.2 &mgr;g/mL; P = 0.003), and the differences were correlated with the histological differences. Conclusions Pancreatic tumor grafts better reproduce the histological nature of clinical cancer and thus provide a more realistic model that is applicable for pharmacokinetic studies.

Gastric Bronchogenic Cyst Histologically Diagnosed After Laparoscopic Excision: Report of a Case

Abdominal computed tomography of a 71-year-old man revealed a 3-cm mass in gastric cardia. Although the mass was widely attached to the gastric wall, no clear contrast enhancement was observed. Abdominal magnetic resonance imaging revealed the mass to have homogenous high intensity on T2W1 images and isointensity on T1W1 images. On diffusion-weighted imaging, no high intensity was observed. However, the mass had a smooth surface and was widely attached to the gastric wall, consistent with computed tomography findings. A gastric submucosal tumor was suspected. Laparoscopic tumor resection was performed. Histopathologic diagnosis of the mass was a bronchogenic cyst derived from the respiratory primordium originating in the foregut of the primitive intestine. Such cysts are mostly found in the mediastinum or thoracic cavity; their occurrence on the gastric wall is extremely rare. Despite this, we think that bronchogenic cysts should be considered in the differential diagnosis of abdominal unilocular cystic diseases.

Cetuximab delivery and antitumor effects are enhanced by mild hyperthermia in a xenograft mouse model of pancreatic cancer

Even with current promising antitumor antibodies, their antitumor effects on stroma‐rich solid cancers have been insufficient. We used mild hyperthermia with the intent of improving drug delivery by breaking the stromal barrier. Here, we provide preclinical evidence of cetuximab + mild hyperthermia therapy. We used four in vivo pancreatic cancer xenograft mouse models with different stroma amounts (scarce, MIAPaCa‐2; moderate, BxPC‐3; and abundant, Capan‐1 and Ope‐xeno). Cetuximab (1 mg/kg) was given systemically, and the mouse leg tumors were concurrently heated using a water bath method for 30 min at three different temperatures, 25°C (control), 37°C (intra‐abdominal organ level), or 41°C (mild hyperthermia) (n = 4, each group). The evaluated variables were the antitumor effects, represented by tumor volume, and in vivo cetuximab accumulation, indirectly quantified by the immunohistochemical fluorescence intensity value/cell using antibodies against human IgG Fc. At 25°C, the antitumor effects were sufficient, with a cetuximab accumulation value (florescence intensity/cell) of 1632, in the MIAPaCa‐2 model, moderate (1063) in the BxPC‐3 model, and negative in the Capan‐1 and Ope‐xeno models (760, 461). By applying 37°C or 41°C heat, antitumor effects were enhanced shown in decreased tumor volumes. These enhanced effects were accompanied by boosted cetuximab accumulation, which increased by 2.8‐fold (2980, 3015) in the BxPC‐3 model, 2.5‐ or 4.8‐fold (1881, 3615) in the Capan‐1 model, and 3.2‐ or 4.2‐fold (1469, 1922) in the Ope‐xeno model, respectively. Cetuximab was effective in treating even stroma‐rich and k‐ras mutant pancreatic cancer mouse models when the drug delivery was improved by combination with mild hyperthermia.