Multi-tumor analysis of cancer-stroma interactomes of patient-derived xenografts unveils the unique homeostatic process in renal cell carcinomas.

The patient-derived xenograft (PDX) model is a versatile tool used to study the tumor microenvironment (TME). However, limited studies have described multi-tumor PDX screening strategies to detect hub regulators during cancer-stroma interaction. Transcriptomes of cancer (human) and stroma (mouse) components of 70 PDX samples comprising 9 distinctive tumor types were analyzed in this study. PDX models recapitulated the original tumors' features, including tumor composition and putative signaling. Particularly, kidney renal clear cell carcinoma (KIRC) stood out, with altered hypoxia-related pathways and a high proportion of endothelial cells in the TME. Furthermore, an integrated analysis conducted to predict paracrine effectors in the KIRC cancer-to-stroma communication detected well-established soluble factors responsible for the hypoxia-related reaction and the so-far unestablished soluble factor, apelin (APLN). Subsequent experiments also supported the potential role of APLN in KIRC tumor progression. Therefore, this paper hereby provides an analytical workflow to find hub regulators in cancer-stroma interactions.

Plasma membrane expression of ZNF185 is a prognostic factor in pancreatic ductal carcinoma.

Pancreatic ductal carcinoma (PDC) is one of the major causes of cancer-associated mortality globally due to its high potential for distant metastasis. To understand hematogenous metastasis, the molecular expression profiles of weak metastatic PDC cell subline BxPC-3 and highly liver-metastatic cell subline LM-BxPC-3 were compared, and zinc finger protein 185 (ZNF185) was identified as a molecule that is upregulated in LM-BxPC-3 cells. The aim of the present study was to evaluate the clinicopathological significance of ZNF185 in PDC. Using immunohistochemistry, ZNF185 expression was investigated in 182 patients with PDC, in association with numerous clinicopathological variables. The expression profile of ZNF185 was also characterized using xenograft models. In contrast to parent BxPC-3 cells in subcutaneous transplanted tumor foci, which only expressed ZNF185 on their plasma membrane (m)ZNF185, LM-BxPC-3 cells in liver-metastatic foci that were formed subsequent to transplantation all expressed cytoplasmic (c)ZNF185. Additionally, 51% of the cells at the periphery of the tumor foci expressed mZNF185. Expression of cZNF185, and of mZNF185 and cZNF185 combined was identified in 93 and 39% of clinical patients with PDC, respectively. Patients with mZNF185-negative and -positive PDC exhibited a median survival time of 30.2 months and 21.3 months, respectively. Multivariate analysis indicated that the expression of mZNF185 is closely associated with a shorter overall survival time. Increased marked venous invasion was more prevalent in patients who were mZNF185-positive, as compared with patients who were mZNF185-negative. These data suggest that the expression of mZNF185 is an independent and unfavorable prognosticator in patients with PDC. The results suggested that the amount and subcellular location of ZNF185 are correlated with the position of the cancer cells expressing it within the nests. Additionally, the subcellular location of ZNF185 may be important to its biological function.

Efficient production of recombinant adeno-associated viral vector, serotype DJ/8, carrying the GFP gene.

The purpose of this study was to establish an efficient method for the preparation of an adeno-associated viral (AAV), serotype DJ/8, carrying the GFP gene (AAV-DJ/8-GFP). We compared the yields of AAV-DJ/8 vector, which were produced by three different combination methods, consisting of two plasmid DNA transfection methods (lipofectamine and calcium phosphate co-precipitation; CaPi) and two virus DNA purification methods (iodixanol and cesium chloride; CsCl). The results showed that the highest yield of AAV-DJ/8-GFP vector was accomplished with the combination method of lipofectamine transfection and iodixanol purification. The viral protein expression levels and the transduction efficacy in HEK293 and CHO cells were not different among four different combination methods for AAV-DJ/8-GFP vectors. We confirmed that the AAV-DJ/8-GFP vector could transduce to human and murine hepatocyte-derived cell lines. These results show that AAV-DJ/8-GFP, purified by the combination of lipofectamine and iodixanol, produces an efficient yield without altering the characteristics of protein expression and AAV gene transduction.

Study on AAV-mediated gene therapy for diabetes in humanized liver mouse to predict efficacy in humans.

Most in vivo studies on the conversion to insulin-producing cells with AAV carrying PDX1 gene are performed in rodents. However, there is little information regarding Adeno-associated virus (AAV) carrying PDX1 gene transduced to human liver in vivo because accidental death caused by unpredicted factors cannot be denied, such as the hypoglycemic agent troglitazone with hepatic failure. Here we aim to confirm insulin secretion from human liver transduced with AAV carrying PDX1 gene in vivo and any secondary effect using a humanized liver mouse. As the results, AAV2-PG succeeded to improve the hyperglycemia of STZ-induced diabetic humanized liver mice. Then, the analysis of humanized liver mice revealed that the AAV2-PG was more transducible to humanized liver area than to mouse liver area. In conclusion, the humanized liver mouse model could be used to examine AAV transduction of human hepatocytes in vivo and better predict clinical transduction efficiency than nonhumanized mice.

Establishment of patient-derived cancer xenografts in immunodeficient NOG mice.

Viable and stable human cancer cell lines and animal models combined with adequate clinical information are essential for future advances in cancer research and patient care. Conventional in vitro cancer cell lines are commonly available; however, they lack detailed information on the patient from which they originate, including disease phenotype and drug sensitivity. Patient-derived xenografts (PDX) with clinical information (so-called 'cancer xenopatients') are a promising advance that may accelerate the development of anticancer therapies. We established 61 PDX lines from 116 surgically removed tumor tissues inoculated subcutaneously into NOG mice (53% success rate). PDX lines were established from various types of epithelial tumors and also from sarcomas, including gastrointestinal stromal tumors and Ewing/PNET sarcomas. The metastatic tumors yielded PDX lines more effectively (65%) than the primary tumors (27%, P<0.001). In our PDX models, morphological characteristics, gene expression profiles, and genetic alteration patterns were all well preserved. In eight cases (7%), the transplantable xenografts for several generations were composed of large monotonous nonepithelial cells of human origin, revealed to be Epstein-Barr virus infection-associated lympho-proliferative lesions. Despite this, PDX linked with clinical information offer many advantages for preclinical studies investigating new anticancer drugs. The fast and efficient establishment of individual PDX may also contribute to future personalized anticancer therapies.

Zinc finger protein 185 is a liver metastasis-associated factor in colon cancer patients.

LIM domain proteins are involved in several fundamental biological processes, including cell lineage specification, cytoskeleton organization and organ development. Zinc finger protein 185 (ZNF185) is one of the LIM domain proteins considered to be involved in the regulation of cellular differentiation and/or proliferation. However, the detailed functions and properties of ZNF185 in the multistep process of cancer biology have not yet been elucidated. In this study, we analyzed the association between ZNF185 and the clinicopathological characteristics of colon cancer, such as patient age and gender, histological type, lymphatic and venous involvement, T and N status, liver metastasis and stage. ZNF185 protein expression was immunohistochemically analyzed and ZNF185 was detected in the cancer cells of 78 of the 87 colon cancer patients. The correlation between ZNF185 and histological type was significant (P=0.010, G-test). ZNF185 expression was also significantly correlated with liver metastasis (P=0.030, G-test). A multivariate analysis using the Cox proportional hazards model was performed among cause-specific survival rate, ZNF185 expression and clinicopathological characteristics. Histological type, liver metastasis and ZNF185 expression were found to be independent prognostic indicators (P=0.028, P<0.0001 and P=0.036, respectively). Therefore, ZNF185 expression was found to be an independent indicator of liver metastasis and prognosis in patients with colon cancer.

A new in vivo model to analyze hepatic metastasis of the human colon cancer cell line HCT116 in NOD/Shi-scid/IL-2Rγ(null) (NOG) mice by (18)F-FDG PET/CT.

Clinically, (18)F-fluorodeoxyglucose positron emission tomography/computed tomography ((18)F-FDG-PET/CT) is useful in the evaluation of various types of human cancers. While PET analysis has been established to evaluate subcutaneous lesions of human cancers in mice, its applications for internal lesions are still being developed. We are currently evaluating new PET approaches for the effective evaluation of in vivo metastatic lesions in the internal organs of small experimental animals. In this study, we analyzed in vivo hepatic metastases of human colonic cancer in immunodeficient mice (NOD/Shi-scid/IL-2Rγ(null), NOG) using PET imaging. This new PET approach has been proposed for the evaluation of in vivo metastatic lesions in internal organs. The human colon cancer line HCT116 (1.0x10(5) and 1.0x10(6) cells/mouse) was transplanted by intrasplenic injection. (18)F-FDG-PET/CT scans were performed 2 weeks after transplantation. After PET/CT scans, histopathological examinations were performed. PET/CT analysis disclosed multiple metastatic foci and increased standardized uptake values (SUV) of FDG in the livers of NOG mice (control, SUVmean 0.450±0.033, SUVmax 0.635±0.017; 1.0x10(5) cells, 0.853±0.087, 1.254±0.237; 1.0x10(6) cells, 1.211±0.108, 1.701±0.158). There were significant differences in FDG uptakes between the three groups (ANOVA, P=0.017 in SUVmean; P=0.044 in SUVmax, n=2). We clearly and quantitatively detected images of hepatic metastasis in the livers of NOG mice by (18)F-FDG-PET/CT in vivo. PET/CT analysis of internal organ lesions of human cancerous xenografts is a new reliable experimental system to simulate metastases. This model system is useful for analyzing metastatic mechanisms and for developing new novel drugs targeting hepatic metastases of cancer.

Overexpression of the miR-34 family suppresses invasive growth of malignant melanoma with the wild-type p53 gene.

Malignant melanoma is the most aggressive neoplasm, with severe metastatic potential. microRNAs represent a class of endogenously expressed, small non-coding RNAs that regulate gene expression. As a consequence, the translation of these mRNAs is inhibited or they are destabilized resulting in downregulation of the encoded protein. The microRNA-34 (miR-34) family, which comprises three processed microRNAs (miR-34a/b/c) was identified as the mediator of tumor suppression by p53. Many reports suggest that the miR-34s contribute to the inhibition of invasion or metastasis in various tumor types. In this study, we evaluated the expression of the miR-34 family in four human melanoma cell lines (A375, G361, C32TG and SK-MEL-24) which have the wild-type p53 gene using real-time reverse transcription PCR. We also examined their generative and invasive characteristics using the cell proliferation assay and the invasion/migration assay, respectively. All four melanoma cell lines showed significant expression of miR-34s - A375: miR-34a 0.6176, miR-34b 0.7625, miR-34c 0.7877; G361: 7.6424, 16.4127, 22.0332; C32TG: 2.1630, 2.1091, 8.4425; SK-MEL-24: 0.3621, 2.5659, 8.5907. The cell doubling times of these cell lines in h:min were as follows: A375 23:23, G361 68:24, C32TG 47:22 and SK-MEL-24 67:03. The in vitro generation times of G361 and SK-MEL-24, which showed increased expression of miR-34c, were significantly shorter than A375 with decreased expression of miR-34c (p=0.0063, ANOVA). Invasion (%) of the four cell lines was as follows: A375 44.0%, G361 22.4%, C32TG 13.8% and SK-MEL-24 45.0%. In vitro invasiveness of G361 and C32TG, which showed increased expression of miR-34a, was significantly suppressed (p= 0.005, ANOVA). These results suggest that overexpression of miR-34a and c suppresses invasive and generative potentials, respectively, in human malignant melanoma.

Reduced CD73 expression and its association with altered purine nucleotide metabolism in colorectal cancer cells robustly causing liver metastases.

Liver metastases of colorectal cancers significantly affect the prognoses of patients. To further understand the biological aspects of the metastatic phenotypes, we established the highly liver-metastatic human colorectal cancer cell subline SW48LM2. The subline was established through the serial intrasplenic transfer of cells derived from poor but visible hepatic tumor foci formed by parental SW48 cells and transferred to NOD/SCID/IL-2Rγc(null) mice. The growth, both under monolayer culture conditions and during the formation of subcutaneous tumors, was similar between the two cell lines, although there were morphological differences in the in vitro spheroid formation. Of 41 molecules reportedly associated positively or negatively with tumor progression, four were overexpressed and four were underexpressed in SW48LM2 cells. Notably, this liver-metastatic cell subline exhibited a strongly reduced expression of the ecto-5'-nucleotidase CD73 as well as an altered metabolism of purine nucleotides. Previous studies showed a positive correlation between CD73 expression and metastatic cancer phenotypes. A reduced CD73 expression in tumor cells, however, may contribute to obtaining insight into the mechanisms of liver metastases.

Alternative splicing variant of vascular endothelial growth factor-A is a critical prognostic factor in non-small cell lung cancer.

Lung cancer is one of the most common malignant diseases in the world, and its prognosis is generally poor. Cancer and metastasis involve numerous biological steps, including angiogenesis in both the primary and metastatic sites. Although various molecules that are involved in both tumor neovascularization (angiogenesis) and invasion have been identified, little is known about how these molecules interact in cancerous microenvironments. We previously reported that the gene expressions of some factors associated with vascularization correlated with the prognosis of non-small cell lung cancer (NSCLC). In this study, we performed multivariate analysis of the mRNA levels of 10 selected genes [VEGF-A, VEGF121, VEGF165, VEGF189, S100A4, E-cadherin, Thrombospondin (TSP)-1, TSP-2, matrix metalloproteinase (MMP)-2, and MMP-9] in 130 NSCLC specimens using the real-time quantitative reverse transcription-polymerase chain reaction. Spearman's rank correlation test was used to determine the co-expression patterns. The analysis demonstrated highly significant co-expressions (P<0.0001) among the VEGF isoforms (VEGF-A, VEGF121, VEGF165, and VEGF189). We also analyzed the correlations among the prognosis, gene expressions, clinical factors (age and gender), and pathological features (histological types, TNM status, stages, lymphatic involvement, and venous involvement) using the Cox proportional hazards model. Multivariate analyses showed that only VEGF189 expression was an independent prognostic indicator (P=0.0252). The alternative splicing variant VEGF189, the cell binding isoform, plays a leading role in the progression of NSCLC.

Cancerous, but not stromal, thrombospondin-2 contributes prognosis in pulmonary adenocarcinoma.

Thrombospondin (TSP)-2 is known to be an endogenous negative regulator of vascularization in human cancer. However, it is unclear whether TSP-2 expression is related to neovascularization and prognosis in non-small cell lung cancer. In this study, we quantitatively examined the expression of TSP-2 mRNA by real-time reverse transcription-polymerase chain reaction (RT-PCR) in 102 pulmonary adenocarcinomas. All 102 carcinoma specimens expressed TSP-2 mRNA. The expression of TSP-2 mRNA in carcinoma was significantly higher than normal lung tissues (p<0.0001, Kruskal-Wallis test). Sizes of tumors were significantly correlated with TSP-2 gene expression (p=0.0179, Kruskal-Wallis test). The TSP-2 expression levels of the stage II/III pulmonary carcinomas were significantly increased as compared to those of stage I (p=0.0136, Kruskal-Wallis test). Thirty-five patients with high TSP-2 mRNA expression showed poor prognosis in survival (p=0.0139, log-rank test). We examined TSP-2 protein localizations in the pulmonary adenocarcinoma overexpressing TSP-2 mRNA. The TSP-2 localizations were categorized in two patterns: cancerous TSP-2 expression pattern (TSP-2 expression in the cancerous cells) and non-cancerous TSP-2 expression pattern (TSP-2 expression in the stromal lymphoid cells). Pulmonary adenocarcinoma patients with cancerous TSP-2 expression pattern showed good prognosis (p=0.0322; Fisher's probability exact test). Pulmonary adenocarcinoma patients with non-cancerous TSP-2 expression pattern showed poor prognosis (p=0.0220; Fisher's probability exact test). Non-cancerous TSP-2 expressions may reflect secondary reactions in the cancerous stroma. The stromal TSP-2 expression is not enough to suppress growth of pulmonary adenocarcinoma, while the cancerous TSP-2 expression directly inhibits growth of the carcinoma.

Thrombospondin 2 inhibits metastasis of human malignant melanoma through microenvironment-modification in NOD/SCID/gammaCnull (NOG) mice.

Thrombospondin (TSP) 2 interacts with matrix metalloproteinases (MMPs) and matrix serine proteases such as plasminogen activator (PA). Malignant melanoma is an aggressive human neoplasm showing aggressive metastatic features. We examined the effects of TSP2 gene introduction in the human malignant melanoma cell line A375. We established three clones transfected with human TSP2 (A375/TSP2). The in vitro invasiveness was remarkably suppressed (42-61%) in the TSP2-transfectants, while growth properties were preserved. The A375/TSP2 showed significantly decreased liver metastatic potential (liver weight: 3.88+/-0.30 g in A375/TSP2, 7.07+/-0.67 g in vector-transfectant (A375/V), p<0.01, Mann-Whitney U test) in super immuno-deficient mice (NOD/SCID/gammacnull, NOG). The PA inhibitor-1 (PAI-1) and PAI-2 mRNAs were significantly overexpressed in A375/TSP2. The increased activities of PAI-1 and PAI-2 were confirmed by reverse zymography. The vascularity of metastatic lesions was significantly decreased in A375/TSP2 (vascular density: 0.62+/-0.15% in A375/TSP2, 4.96+/-0.61% in A375/V, p<0.01, Welch test). These results suggest that TSP2 suppresses hematogenous metastasis through microenvironment-modification including PAI up-regulation and anti-vascularization in human malignant melanoma.

Thrombospondin-2 inhibits tumor cell invasion through the modulation of MMP-9 and uPA in pancreatic cancer cells.

The extracellular matrix protein thrombospondin (TSP) plays an important role in a variety of biological processes, including cell-cell and cell-matrix interactions. The biological role of TSP-2 in invasion and metastasis is poorly understood, while it is known that TSP-1 regulates a proteolytic cascade that allows tumor cells to invade and metastasize. In this study, we examined the role of TSP-2 in tumor cell invasion and its association with proteolytic proteins, matrix metalloproteinase (MMP) and the plasminogen/plasmin system, including urokinase-type plasminogen activator (uPA), in the human pancreatic cancer cell line PANC-1. PANC-1 cells expressed a low level of TSP-2, but significant levels of TSP-1. We isolated three clones of PANC-1 transformants stably overexpressing human TSP-2 (PANC-T). PANC-T highly expressed the TSP-2 gene and protein, while TSP-1 expression was not altered. In vitro invasion assays demonstrated that the invasiveness of PANC-T clones was significantly suppressed (p<0.05; Welch test). Zymography revealed that restoration of TSP-2 synthesis in the PANC-T clones significantly inhibited MMP-9 activity (p<0.05; Welch test). uPA activity in the PANC-T clones was significantly suppressed (p<0.05; Welch test). We concluded that restoration of TSP-2 can inhibit cell invasion through the down-regulation of MMP-9 and uPA activity in pancreatic cancer cell lines. Thus, TSP-2 may be a potent inhibitor of metastasis in pancreatic cancer.

[A case of follicular lymphoma with onset of chylothorax].

We report a case of follicular lymphoma with chylothorax. A 45-year-old man visited our hospital complaining of shortness of breath and abdominal distension. Chest X-rays showed bilateral pleural effusion, and an abdominal CT scan revealed a large intraperitoneal tumor around the abdominal aorta and pancreas. Bilateral cervical and inguinal lymph nodes were swollen. Biochemical study of the pleural fluid revealed the presence of chylomicrons, and an inguinal lymph node biopsy led to a follicular lymphoma diagnosis. The patient achieved complete remission, with disappearance of pleural effusion, following 8 cycles of chemothreapy (R-CHOP). Cases of malignant lymphoma with chylothorax are rarely reported in Japan, but should be taken into account when examining cases of non-traumatic chylothorax.

Pilonidal sinus in Japan maritime self-defense force at Yokosuka.

Pilonidal sinus is known to be common among soldiers but not among Asian individuals. Few reports have discussed the occurrence of pilonidal sinus in the navy. We analyzed the incidence and etiological factors of 12 cases (11 patients, with 1 recurrence) of pilonidal sinus diagnosed between 1998 and 2004 in Japan Self-Defense Force Hospital Yokosuka. All patients were male, and all lesions were in the sacral area. The patients' average age and body mass index were 29.1 years and 27.4, respectively. The surgical procedures used were primary closures (10 cases, 83.3%) and flaps (two cases, 16.7%), and the number of postoperative hospital days averaged 11.8. Pilonidal sinus was not found to be significantly common among Japan Self-Defense Force personnel in general (7 of 11 patients with pilonidal sinus; not significant); however, it was significantly more common among the crew members of Japan Self-Defense Force ships (seven of seven patients; p < 0.05), which suggests that the on-board lifestyle contributes to the occurrence of pilonidal sinus.

A case of chronic expanding haematoma on the finger.

We describe a patient with a chronic expanding haematoma developing in the right middle finger. Preoperative ultrasonography showed a cystic-like lesion upon the digital flexor tendon. Pathologic examination revealed a chronic organising haematoma surrounded by fibrous granulation tissue. Neither endothelial nor neoplastic cells were identified. We diagnosed this lesion as a chronic expanding haematoma (CEH) based upon the clinical course and pathological findings. The cause of our case was hypothesised to be repeated strain from the handle of heavy bag.