Mitochondrial DNA drives abscopal responses to radiation that are inhibited by autophagy.

Autophagy supports both cellular and organismal homeostasis. However, whether autophagy should be inhibited or activated for cancer therapy remains unclear. Deletion of essential autophagy genes increased the sensitivity of mouse mammary carcinoma cells to radiation therapy in vitro and in vivo (in immunocompetent syngeneic hosts). Autophagy-deficient cells secreted increased amounts of type I interferon (IFN), which could be limited by CGAS or STING knockdown, mitochondrial DNA depletion or mitochondrial outer membrane permeabilization blockage via BCL2 overexpression or BAX deletion. In vivo, irradiated autophagy-incompetent mammary tumors elicited robust immunity, leading to improved control of distant nonirradiated lesions via systemic type I IFN signaling. Finally, a genetic signature of autophagy had negative prognostic value in patients with breast cancer, inversely correlating with mitochondrial abundance, type I IFN signaling and effector immunity. As clinically useful autophagy inhibitors are elusive, our findings suggest that mitochondrial outer membrane permeabilization may represent a valid target for boosting radiation therapy immunogenicity in patients with breast cancer.

Dosing time-dependent difference in the suppressive effect of empagliflozin on the development of mechanical pain hypersensitivity in diabetic mice.

A problem for patients with diabetes is the rise of complications, such as peripheral neuropathy, nephropathy and retinopathy. Among them, peripheral neuropathy, characterized by numbness and/or hypersensitivity to pain in the extremities, is likely to develop in the early stages of diabetes. Empagliflozin (EMPA), a sodium-glucose cotransporter-2 inhibitor, exerts hypoglycemic effects by preventing glucose reabsorption in proximal tubular cells. EMPA can improve cardiovascular and renal outcomes in diabetic patients, but its suppressive effect on the development of diabetic neuropathy remains unclear. In this study, we demonstrated that optimizing the dosing schedule of EMPA suppressed the development of pain hypersensitivity in streptozotocin (STZ)-induced diabetic model mice maintained under standardized light/dark cycle conditions. A single intraperitoneal administration of STZ to mice induced hyperglycemia accompanied by pain hypersensitivity. Although EMPA did not exert anti-hypersensitivity effect on STZ-induced diabetic mice after the establishment of neuropathic pain, the development of pain hypersensitivity in the diabetic mice was significantly suppressed by daily oral administration of EMPA at the beginning of the dark phase. On the other hand, the suppressive effect was not observed when EMPA was administered at the beginning of the light phase. The hypoglycemic effect of EMPA and its stimulatory effect on urinary glucose excretion were also enhanced by the administration of the drug at the beginning of the dark phase. Nocturnal mice consumed their food mainly during the dark phase. Our results support the notion that morning administration of EMPA may be effective in suppressing the development of peripheral neuropathy in diabetic patients. Significance Statement Empagliflozin, a sodium-glucose cotransporter-2 inhibitor suppressed the development of neuropathic pain hypersensitivity in streptozotocin-induced diabetic model mice in a dosing time-dependent manner.

Improving glycemic control by transitioning from the MiniMedTM 640G to 770G in Japanese adults with type 1 diabetes mellitus: a prospective, single-center, observational study.

The effectiveness of a hybrid closed-loop (HCL) system in improving glycemic control is unclear in Japanese individuals. Therefore, we assessed the effect impact of the MiniMed 770G HCL system on glycemic control in this population. This prospective, single-center, 24-week observational study (registration number: UMIN000047394) enrolled 23 individuals with type 1 diabetes mellitus using the Medtronic MiniMed 640G system. The primary endpoint was the improvement in time in the range of 70-180 mg/dL after transitioning to the MiniMed 770G HCL system. We observed an increase in time in range (from 64.1 [55.8-69.5] to 70.9 [67.1-74.4] %, interquartile range 25-75%, p < 0.001) and a decrease in glycated hemoglobin level (from 7.4 [7.0-7.9] to 7.1 [6.8-7.4] %, p = 0.003). There was a significant reduction in time above the range (181-250 mg/dL: 25.8 [20.9-28.6] to 19.5 [17.1-22.1] %, p < 0.001; >251 mg/dL: 8.7 [4.0-13.0] to 4.7 [3.6-9.1] %, p < 0.001). Time below the range remained unchanged (54-69 mg/dL: 1.8 [0.4-2.4] to 2.1 [0.4-3.9] %, p = 0.24; <54 mg/dL: 0.2 [0.0-1.0] to 0.5 [0.1-1.3] %, p = 0.14). In a subgroup of 12 patients with a high HCL implementation rate, the basal insulin infusion decreased immediately after mealtime insulin administration and increased after approximately 120 minutes. The ratings from questionnaires assessing treatment burden, satisfaction, and quality of life remained unchanged. The MiniMed 770G HCL system improved glycemic control and optimized insulin delivery, particularly in patients with high implementation rates.

Cellular senescence in the response of HR+ breast cancer to radiotherapy and CDK4/6 inhibitors.

BACKGROUND: Preclinical evidence from us and others demonstrates that the anticancer effects of cyclin-dependent kinase 4/6 (CDK4/6) inhibitors can be enhanced with focal radiation therapy (RT), but only when RT is delivered prior to (rather than after) CDK4/6 inhibition. Depending on tumor model, cellular senescence (an irreversible proliferative arrest that is associated with the secretion of numerous bioactive factors) has been attributed beneficial or detrimental effects on response to treatment. As both RT and CDK4/6 inhibitors elicit cellular senescence, we hypothesized that a differential accumulation of senescent cells in the tumor microenvironment could explain such an observation, i.e., the inferiority of CDK4/6 inhibition with palbociclib (P) followed by RT (P→RT) as compared to RT followed by palbociclib (RT→P). METHODS: The impact of cellular senescence on the interaction between RT and P was assessed by harnessing female INK-ATTAC mice, which express a dimerizable form of caspase 8 (CASP8) under the promoter of cyclin dependent kinase inhibitor 2A (Cdkn2a, coding for p16Ink4), as host for endogenous mammary tumors induced by the subcutaneous implantation of medroxyprogesterone acetate (MPA, M) pellets combined with the subsequent oral administration of 7,12-dimethylbenz[a]anthracene (DMBA, D). This endogenous mouse model of HR+ mammary carcinogenesis recapitulates key immunobiological aspects of human HR+ breast cancer. Mice bearing M/D-driven tumors were allocated to RT, P or their combination in the optional presence of the CASP8 dimerizer AP20187, and monitored for tumor growth, progression-free survival and overall survival. In parallel, induction of senescence in vitro, in cultured human mammary hormone receptor (HR)+ adenocarcinoma MCF7 cells, triple negative breast carcinoma MDA-MB-231 cells and mouse HR+ mammary carcinoma TS/A cells treated with RT, P or their combination, was determined by colorimetric assessment of senescence-associated ß-galactosidase activity after 3 or 7 days of treatment. RESULTS: In vivo depletion of p16Ink4-expressing (senescent) cells ameliorated the efficacy of P→RT (but not that of RT→P) in the M/D-driven model of HR+ mammary carcinogenesis. Accordingly, P→RT induced higher levels of cellular senescence than R→TP in cultured human and mouse breast cancer cell lines. CONCLUSIONS: Pending validation in other experimental systems, these findings suggest that a program of cellular senescence in malignant cells may explain (at least partially) the inferiority of P→RT versus RT→P in preclinical models of HR+ breast cancer.

Kallikrein-Related Peptidase 12 (KLK12) in Breast Cancer as a Favorable Prognostic Marker.

Kallikrein-related peptides (KLKs) form an evolutionally conserved subgroup of secreted serine proteases that consists of 15 members (KLK1-15). Previous studies have shown that KLKs regulate diverse biological processes, but the clinical significance of KLKs remains largely unclear in human breast cancers. We examined the expression profile of 15 KLK genes in breast carcinomas using microarray data. Next, we immunolocalized KLK12 in 140 breast carcinomas and evaluated its clinical significance. Subsequently, we examined the effects of KLK12 on proliferation and migration in breast cancer cell lines. From microarray analyses, it turned out that KLK12 was the most strongly associated with low-grade malignancy in breast carcinomas among the 15 KLK members. Immunohistochemical KLK12 status was positively associated with ER and PR status, while it was inversely associated with stage, pathological T factor, lymph node metastasis, and distant metastasis. Prognostic analyses demonstrated that KLK12 was a favorable prognostic factor for both disease-free and breast cancer-specific survival of the patients. Furthermore, the knockdown of KLK12 significantly increased cell proliferation activity and cell migration of breast cancer cells. These results suggest that KLK12 has antitumorigenic effects associated with proliferation and migration and immunohistochemical KLK12 status as a potent favorable prognostic factor in breast carcinoma patients.

Microevolution of Pieris butterfly genes involved in host plant adaptation along a host plant community cline.

Herbivorous insects have evolved counteradaptations to overcome the chemical defences of their host plants. Several of these counteradaptations have been elucidated at the molecular level, in particular for insects specialized on cruciferous host plants. While the importance of these counteradaptations for host plant colonization is well established, little is known about their microevolutionary dynamics in the field. In particular, it is not known whether and how host plant diversity shapes diversity in insect counteradaptations. In this study, we examine patterns of host plant use and insect counteradaptation in three Pieris butterfly species across Japan. The larvae of these butterflies express nitrile-specifier protein (NSP) and its paralogue major allergen (MA) in their gut to overcome the highly diversified glucosinolate-myrosinase defence system of their cruciferous host plants. Pieris napi and Pieris melete colonize wild Brassicaceae whereas Pieris rapae typically uses cultivated Brassica as a host, regardless of the local composition of wild crucifers. As expected, NSP and MA diversity was independent of the local composition of wild Brassicaceae in P. rapae. In contrast, NSP diversity correlated with local host plant diversity in both species that preferred wild Brassicaceae. Both P. melete and P. napi revealed two distinct major NSP alleles, which shaped diversity among local populations, albeit with different evolutionary trajectories. In comparison, MA showed no indication for local adaptation. Altogether, MA appeared to be evolutionary more conserved than NSP, suggesting that both genes play different roles in diverting host plant chemical defence.

Thermodynamics for the Self-Assembly of Alkylated Peptides.

Self-assembling peptides form aggregates with various nanostructures such as spheres, sheets, and fibers and have potential applications in nanomedicine and drug delivery. The alkylation of peptides is a promising strategy for controlling the self-assembly of peptides. In this study, we investigated the thermodynamic properties associated with the aggregation of alkyl-chain-modified self-assembling peptides. The tripeptide sequence, KYF, which has been reported to form fibrous aggregates via self-assembly, was modified with various fatty acids at the N-terminus. The fibrous morphology of the aggregates was observed by transmission electron microscopy and atomic force microscopy. Thioflavin T fluorescence and circular dichroism spectroscopy revealed the formation of ß-sheet structures. The critical micelle concentration and its temperature dependence were determined to obtain the thermodynamic parameters for aggregation. The results showed that the aggregation was an entropy-driven process at low temperatures, whereas it was enthalpy-driven at high temperatures. The negative heat capacity changes for aggregation suggested that hydrophobic interactions were the major driving force for self-assembly. Other entropic and enthalpic interactions were also contributed in part to the self-assembly. We individually identified the contributions of the peptide and alkyl chain moiety to the self-assembly. These contributions can be explained by the theoretical values for the self-assembly of each component. The results of this study provide fundamental insights into the design of self-associating peptides.

Validity of Endoscopic Ultrasound Findings of Chronic Pancreatitis: Evaluation from the Viewpoint of Disease Risk Factors.

BACKGROUND: The diagnosis of chronic pancreatitis (CP) using endoscopic ultrasound (EUS) criteria, referred to as the Rosemont classification (RC), has been widely performed. However, the validity of the RC, which was based on expert opinion, is still controversial. If EUS findings are associated with CP, then they should be associated with risk factors for CP. In this study, to verify the appropriateness of the RC and each EUS finding, we performed a retrospective analysis from the viewpoint of risk factors for CP. SUMMARY: Three hundred and forty-four patients were enrolled in this study. Clinical background characteristics that associate with CP were alcohol intake, smoking, history of acute pancreatitis (AP), and age. The correlation between EUS criteria for CP and clinical background was investigated. All EUS findings except the presence of cysts showed significant correlations with one or 2 of the 3 following factors: ethanol (EtOH) intake, smoking status, and history of AP. Results of the univariate and multivariate analyses showed that 3 factors (EtOH intake, smoking, and history of AP) other than age were positively correlated with the RC. Moreover, the risk of progression from normal to consistent CP to indeterminate and suggestive CP was found to increase with increasing EtOH intake. Key Messages: The RC and each EUS finding was validated from the viewpoint of risk factors for CP.

Role of exosomes as a proinflammatory mediator in the development of EBV-associated lymphoma.

Epstein-Barr virus (EBV) causes various diseases in the elderly, including B-cell lymphoma such as Hodgkin's lymphoma and diffuse large B-cell lymphoma. Here, we show that EBV acts in trans on noninfected macrophages in the tumor through exosome secretion and augments the development of lymphomas. In a humanized mouse model, the different formation of lymphoproliferative disease (LPD) between 2 EBV strains (Akata and B95-8) was evident. Furthermore, injection of Akata-derived exosomes affected LPD severity, possibly through the regulation of macrophage phenotype in vivo. Exosomes collected from Akata-lymphoblastoid cell lines reportedly contain EBV-derived noncoding RNAs such as BamHI fragment A rightward transcript (BART) micro-RNAs (miRNAs) and EBV-encoded RNA. We focused on the exosome-mediated delivery of BART miRNAs. In vitro, BART miRNAs could induce the immune regulatory phenotype in macrophages characterized by the gene expressions of interleukin 10, tumor necrosis factor-α, and arginase 1, suggesting the immune regulatory role of BART miRNAs. The expression level of an EBV-encoded miRNA was strongly linked to the clinical outcomes in elderly patients with diffuse large B-cell lymphoma. These results implicate BART miRNAs as 1 of the factors regulating the severity of lymphoproliferative disease and as a diagnostic marker for EBV+ B-cell lymphoma.

Endoscopic ultrasound-guided coil deployment with sclerotherapy for isolated gastric varices: Case series of feasibility, safety, and long-term follow-up.

The purpose of the present study is to report our experience of endoscopic ultrasound-guided coil deployment with sclerotherapy (EUS-CS) for isolated gastric varices (IGV) through a case series. Eight consecutive patients who had risky IGV were prospectively enrolled. EUS-CS was performed according to the following procedures: (i) several coils were first deployed in the IGV under EUS guidance; (ii) contrast medium was subsequently injected without removing the needle; (iii) if the infused contrast medium stayed in the IGV and feeding vein, sclerosant was then injected to obliterate the IGV and feeders. Coil deployment in the IGV was successfully performed in all cases. Sclerosant was injected both into the IGV and feeders in seven patients (87.5%). There was no adverse event during the procedure. During a median follow-up of 57 months, one patient who could not inject the sclerosant into IGV and feeders had an early hemorrhagic recurrence. Our case series showed that EUS-CS could be a feasible and safe procedure for the treatment of IGV.

Development of behavioural automaticity by extended Pavlovian training in an insect.

The effect of repetitive training on learned actions has been a major subject in behavioural neuroscience. Many studies of instrumental conditioning in mammals, including humans, suggested that learned actions early in training are goal-driven and controlled by outcome expectancy, but they become more automatic and insensitive to reduction in the value of the outcome after extended training. It was unknown, however, whether the development of value-insensitive behaviour also occurs by extended training of Pavlovian conditioning in any animals. Here we show that crickets Gryllus bimaculatus that had received minimal training to associate an odour with water (unconditioned stimulus, US) did not exhibit conditioned response (CR) to the odour when they were given water until satiation before the test, but those that had received extended training exhibited CR even when they were satiated with water. Further pharmacological experiments suggested that octopamine neurons, the invertebrate counterparts of noradrenaline neurons, mediate US value signals and control execution of CR after minimal training, but the control diminishes with the progress of training and hence the CR becomes insensitive to US devaluation. The results suggest that repetitive sensory experiences can lead to a change from a goal-driven response to a more automatic one in crickets.

Molecular signatures of selection associated with host plant differences in Pieris butterflies.

Adaptive traits that enable organisms to conquer novel niches and experience subsequent diversification are ecologically and evolutionarily important. The larvae of Pieris butterflies express nitrile-specifier proteins (NSPs), a key innovation for overcoming the glucosinolate (GLS)-myrosinase-based defence system of their Brassicales host plants. Nitrile-specifier proteins are a member of the NSP-like gene family, which includes the major allergen (MA) protein, a paralog of NSP with a GLS-disarming function, and a single domain major allergen (SDMA) protein, whose function is unknown. The arms-race between GLS-based defences and the NSP-like gene family is suggested to mediate diversification in both Pierid butterflies and Brassicales plants. Here, we tested whether the expected strong selection on NSP-like gene family correlates with shifts in host plant spectra among Pierid butterflies. We combined feeding experiments using 25 Brassicaceae plants and five Pieris species with larval transcriptome data to investigate the patterns of selection acting on NSP-like gene family members. Although we observed significantly elevated nonsynonymous to synonymous substitution rate ratios in NSPs on branches associated with changes in patterns of host plant usage, no such pattern was observed in MAs or SDMAs. Furthermore, we found evidence for positive selection of NSP at a phylogenetic branch which reflects different host plant spectra. Our data indicate that the NSP-related gene members have evolved differently: NSPs have accumulated more amino acid changes in response to shifting preferences for host plants, whereas MAs and SDMAs appear to be more conserved. Further detailed functional assays of these genes would provide important insights to understand their role in the chemical arms-race between Pieris butterflies and their Brassicales host plants.

Interspecific Differences in the Larval Performance of Pieris Butterflies (Lepidoptera: Pieridae) Are Associated with Differences in the Glucosinolate Profiles of Host Plants.

The tremendous diversity of plants and herbivores has arisen from a coevolutionary relationship characterized by plant defense and herbivore counter adaptation. Pierid butterfly species feed on Brassicales plants that produce glucosinolates as a chemical deterrent against herbivory. In turn, the larvae of pierids have nitrile specifier proteins (NSPs) that are expressed in their gut and disarm glucosinolates. Pierid butterflies are known to have diversified in response to glucosinolate diversification in Brassicales. Therefore, each pierid species is expected to have a spectrum of host plants characterized by specific glucosinolate profiles. In this study, we tested whether the larval performance of different Pieris species, a genus in Pieridae (Lepidoptera: Pieridae), was associated with plant defense traits of putative host plants. We conducted feeding assays using larvae of three Pieris species and 10 species of the Brassicaceae family possessing different leaf physical traits and glucosinolate profile measurements. The larvae of Pieris rapae responded differently in the feeding assays compared with the other two Pieris species. This difference was associated with differences in glucosinolate profiles but not with variations in physical traits of the host plants. This result suggests that individual Pieris species are adapted to a subset of glucosinolate profiles within the Brassicaceae. Our results support the idea that the host ranges of Pieris species depend on larval responses to glucosinolate diversification in the host species, supporting the hypothesis of coevolution between butterflies and host plants mediated by the chemical arms race.

Endoscope-Assisted Resection of Intramuscular Cavernous Hemangioma Within the Temporal Muscle.

Hemangiomas are benign vascular soft tissue tumors, which most frequently occur in the skin or subcutaneous tissue. Intramuscular hemangiomas typically occur in the trunk and extremities and less frequently in the head and neck. Among these, those occurring in the temporalis muscle are extremely rare. The authors report the case of a 43-year-old Japanese male with a mass in his left temporal fossa. Computed tomography images showed no erosion of the zygomatic bone, and magnetic resonance imaging revealed an ovoid well-marginated mass within the temporal muscle. The lesion was surgically excised with an endoscopy procedure used for minimally invasive lesions and complete removal. Histopathological examination confirmed the diagnosis of intramuscular cavernous hemangioma. The postoperative clinical course was good, with no indications of temporary nerve paralysis. No signs of local recurrence were observed postoperatively. Therefore, a cavernous hemangioma should be suspected when a mass occurs in the temporal region with accompanying radiologic findings suggesting vascular origin. In surgical treatment, the endoscopy-assisted technique is very useful to achieve complete tumor resection and prevent relapse while avoiding serious complications due to surgical procedures.

OLFM4, LY6D and S100A7 as potent markers for distant metastasis in estrogen receptor-positive breast carcinoma.

Metastatic breast cancer is a highly lethal disease, and it is very important to evaluate the biomarkers associated with distant metastasis. However, molecular features of distant metastasis remain largely unknown in breast cancer. Estrogens play an important role in the progression of breast cancer and the majority of stage IV breast carcinomas express estrogen receptor (ER). Therefore, in this study, we examined molecular markers associated with distant metastasis in ER-positive breast carcinoma by microarray and immunohistochemistry. When we examined the gene expression profile of ER-positive stage IV breast carcinoma tissues (n = 7) comparing ER-positive stage I-III cases (n = 11) by microarray analysis, we newly identified OLFM4, LY6D and S100A7, which were closely associated with the distant metastasis. Subsequently, we performed immunohistochemistry for OLFM4, LY6D and S100A7 in 168 ER-positive breast carcinomas. OLFM4, LY6D and S100A7 immunoreactivities were significantly associated with stage, pathological T factor, distant metastasis and Ki67 status in the ER-positive breast carcinomas. Moreover, these immunoreactivities were significantly associated with a worse prognostic factor for distant metastasis-free and breast cancer-specific survival in ER-positive stage I-III breast cancer patients. However, when we performed immunohistochemistry for OLFM4, LY6D and S100A7 in 40 ER-negative breast carcinomas, these immunoreactivities were not generally associated with the clinicopathological factors examined, including distant metastasis and prognosis of patients, in this study. These results suggest that OLFM4, LY6D and S100A7 immunoreactivity are associated with an aggressive phenotype of ER-positive breast carcinoma, and these are potent markers for distant metastasis of ER-positive breast cancer patients.

Antitumor activity of arsenite in combination with tetrandrine against human breast cancer cell line MDA-MB-231 in vitro and in vivo.

BACKGROUND: Triple-negative breast cancer (TNBC) is one of the most difficult subtypes of breast cancer to treat due to its aggressive, metastatic behavior, and a lack of a targeted therapy. Trivalent arsenic derivatives (arsenite, AsIII) with remarkable clinical efficacy in acute promyelocytic leukemia has been demonstrated to exhibit inhibitory effect against breast cancer cells. To provide novel insight into the development of new therapeutic strategies, antitumor activity of AsIII and tetrandrine (Tetra), a Chinese plant-derived alkaloid, against the TNBC cell line MDA-MB-231 in vitro and in vivo was investigated. METHODS: Cytotoxicity was evaluated using cell viability, lactate dehydrogenase leakage and cell cycle assay. Alterations of genes related to cell proliferation and death were analyzed using western blotting. In vivo antitumor activity of AsIII alone or in combination with Tetra was studied using MDA-MB-231 xenografts in nude mice. RESULTS: Synergistic cytotoxic effects of two drugs were observed in the cells. In vivo study also showed that co-administration of AsIII and Tetra significantly reduced tumor volume and weight, directly supporting its in vitro antitumor activity. No deaths and reduction of body-weight were observed after a long-term co-administration, indicating its good tolerability. S-phase arrest associated with the upregulation of FOXO3a, p27 along with decreased Cyclin D1 expression was observed in the cells treated with the combined regimen. A substantial upregulated p21 expression and downregulated phospho-FOXO3a and Cyclin D1 expression was observed in the tumor tissues of mice co-administered with AsIII and Tetra. Autophagy induction was observed in the combination treatment in vitro and in vivo. The addition of wortmannin, a potent autophagy inhibitor, significantly rescued MDA-MB-231 cells from their cytotoxicity of AsIII and Tetra. CONCLUSIONS: S-phase arrest, autophagic and necrotic cell death contribute to the cytocidal effects of the combined regimen of AsIII and Tetra. Considering our previous study showing synergistic cytotoxic effects of the combined regimen in estrogen receptor-positive breast cancer cell line MCF-7, these results suggest that development of the combination regimen of AsIII plus Tetra may offer many benefits to patients with different types of breast cancer.

Tumor-derived spheroids: Relevance to cancer stem cells and clinical applications.

Recently, many types of in vitro 3-D culture systems have been developed to recapitulate the in vivo growth conditions of cancer. The cancer 3-D culture methods aim to preserve the biological characteristics of original tumors better than conventional 2-D monolayer cultures, and include tumor-derived organoids, tumor-derived spheroids, organotypic multicellular spheroids, and multicellular tumor spheroids. The 3-D culture methods differ in terms of cancer cell sources, protocols for cell handling, and the required time intervals. Tumor-derived spheroids are unique because they are purposed for the enrichment of cancer stem cells (CSCs) or cells with stem cell-related characteristics. These spheroids are grown as floating spheres and have been used as surrogate systems to evaluate the CSC-related characteristics of solid tumors in vitro. Because eradication of CSCs is likely to be of clinical importance due to their association with the malignant nature of cancer cells, such as tumorigenicity or chemoresistance, the investigation of tumor-derived spheroids may provide invaluable clues to fight against cancer. Spheroid cultures have been established from cancers including glioma, breast, colon, ovary, and prostate cancers, and their biological and biochemical characteristics have been investigated by many research groups. In addition to the investigation of CSCs, tumor-derived spheroids may prove to be instrumental for a high-throughput screening platform or for the cultivation of CSC-related tumor cells found in the circulation or body fluids.

Cytochrome c1 in ductal carcinoma in situ of breast associated with proliferation and comedo necrosis.

It is well known that comedo necrosis is closely associated with an aggressive phenotype of ductal carcinoma in situ (DCIS) of human breast, but its molecular mechanisms remain largely unclear. Therefore, in this study, we first examined the gene expression profile of comedo DCIS based on microarray data and identified CYC1 as a gene associated with comedo necrosis. Cytochrome c1 (CYC1) is a subunit of complex III in the mitochondrial oxidative phosphorylation that is involved in energy production. However, the significance of CYC1 has not yet been examined in DCIS. We therefore immunolocalized CYC1 in 47 DCIS cases. CYC1 immunoreactivity was detected in 40% of DCIS cases, and the immunohistochemical CYC1 status was significantly associated with tumor size, nuclear grade, comedo necrosis, van Nuys classification, and Ki-67 labeling index. Subsequent in vitro studies indicated that CYC1 was significantly associated with mitochondrial membrane potential in MCF10DCIS.com DCIS cells. Moreover, CYC1 significantly promoted proliferation activity of MCF10DCIS.com cells and the cells transfected with CYC1 siRNA decreased pro-apoptotic caspase 3 activity under hypoxic or anoxic conditions. Considering that the center of DCIS is poorly oxygenated, these results indicate that CYC1 plays important roles in cell proliferation and comedo necrosis through the elevated oxidative phosphorylation activity in human DCIS.

Clinicopathological characteristics and genomic profile of primary sinonasal tract diffuse large B cell lymphoma (DLBCL) reveals gain at 1q31 and RGS1 encoding protein; high RGS1 immunohistochemical expression associates with poor overall survival in DLBCL not otherwise specified (NOS).

AIMS: We aimed to define the clinicopathological characteristics of 29 primary sinonasal diffuse large B cell lymphoma (DLBCLsn ) in a series of 240 cases of DLBCL not otherwise specified [DLBCLall (NOS) ], including DLBCLsn training set (n = 11) and validation set (n = 18), and DLBCLnon-sn (n = 211). METHODS AND RESULTS: In the training set, 82% had a non-germinal center B-cell-like (Hans' Classifier) (non-GCB) phenotype and 18% were Epstein-Barr virus-encoded small RNAs (EBER)+ . The genomic profile showed gains(+) of 1q21.3q31.2 (55%), 10q24.1 (46%), 11q14.1 (46%) and 18q12.1q23 (46%); losses(-) of 6q26q27 (55%) and 9p21.3 (64%); and copy number neutral loss of heterozygosity (LOH) (acquired uniparental disomy, UPD) at 6p25.3p21.31 (36%). This profile is comparable to DLBCLNOS (GSE11318, n = 203.) and closer to non-GCB/activated B-cell-like subtype (ABC). Nevertheless, +1q31, -9p21.3 and -10q11.1q26.2 were more characteristic of DLBCLsn (P < 0.001). Array results were verified successfully by fluorescence in situ hybridization (FISH) on +1q21.3 (CKS1B), -6q26 (PARK2), +8q24.21 (MYC), -9p21.3 (MTAP, CDKN2A/B), -17p13.1 (TP53) and +18q21.33 (BCL2) with 82-91% agreement. Minimal common regions included biologically relevant genes of MNDA (+1q23.1), RGS1 and RGS13 (+1q31.2), FOXP1 (+3p13), PRDM1 (BLIMP1) and PARK2 (-6q21q26), MYC (+8q24.21), CDKN2A (-9p21.3), PTEN (-10q23.31), MDM2 (+12q15), TP53 (-17p13.1) and BCL2 (+18q21.33). Correlation between DNA copy number and protein immunohistochemistry was confirmed for RGS1, RGS13, FOXP1, PARK2 and BCL2. The microenvironment had high infiltration of M2-like tumour associated macrophages (TAMs) and CD8+ T lymphocytes that associated with higher genomic instability. The DLBCLsn validation set confirmed the clinicopathological characteristics, all FISH loci and immunohistochemistry (IHC) for RGS1. RGS1, one of the most frequently altered genes, was analysed by IHC in DLBCLall and high RGS1 expression associated with non-GCB, EBER+ and unfavourable overall survival (hazard ratio = 1.794; P = 0.016). CONCLUSIONS: DLBCLsn has a characteristic genomic profile. High RGS1 IHC expression associates with poor overall survival in DLBCLall (NOS) .