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PDF file, 1193K, Figure S1. Snail+ tumor cells frequently metastasize into bone marrow. Figure S2. Differentiation activity of the specific subpopulations isolated from the Snail+ tumor-stimulated BMCs. Figure S3. The Snail+ tumor-induced MSCs promote tumor metastatic seeding into bone. Figure S4. Increase of fstl1 and its receptor dip2a mRNA expressions in Snail+ tumor cells. Figure S5. FSTL1-knockdown suppresses metastatic seeding of human tumor cells into bone. Figure S6. FSTL1-induced cancer bone metastasis mechanism.

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Kouichi Murakami,1 Emi Inayama,1 Yukiko Itoh,1 Shoji Tuchiya,1 Masashi Iwasaki,2 Nobuko Tamura,1 Toshihiko Suzuki,3 Noriko Iwai,1 Junji Utino,4 Motoyuki Masai,4 Daiji Nagayama,5,6 Kohji Shirai4,6 1Seijinaki Mihama Narita Clinic, Narita City, Chiba, 286-0041, Japan; 2Seijinkai Mihama Katori Clinic, Katori City, Chiba, 287-0041, Japan; 3Seijinkai Mihama Sakura Clinic, Sakura City, Chiba, 285-0841, Japan; 4Seijinkai Mihama Hospital, Chiba City, Chiba, 261-0013, Japan; 5Nagayama Clinic, Oyama City, Tochigi, 323-0032, Japan; 6Sakura Hospital, School of Medicine Toho University, Sakura City, Chiba, 285-8741, JapanCorrespondence: Daiji NagayamaNagayama Clinic, 2-12-22, Tenjin-Cho, Oyama City, Tochigi, 323-0032, JapanTel/Fax +81-285-22-0219Email deverlast96071@gmail.comAim: Mortality rate of maintenance hemodialysis patients is known to be high. Cardio-ankle vascular index (CAVI) is an index reflecting the proper stiffness of the arterial tree from the origin of the aorta to the ankle. We aimed to clarify the utility of CAVI as a predictor of mortality in hemodialysis patients. The roles of age and nutritional conditions on survival were also examined.Methods: We followed 242 patients undergoing hemodialysis for 6 consecutive years. Data from 209 patients (mean age was 60 ± 11 years) excluding those with ankle-brachial index < 0.90 were then analyzed. CAVI and heart to ankle pulse wave velocity (haPWV) were measured using Vasera 1500.Results: Thirty-eight hemodialysis patients who died during the 6-year period had higher age, cardiothoracic ratio (CTR), CAVI, and haPWV, and lower diastolic blood pressure, albumin, phosphate, and calcium phosphate product. The Kaplan–Meier curves for cumulative survival among the tertile groups showed that the mortality rate was higher in the highest tertile (T3) compared to T1/T2 for both CAVI and haPWV. Receiver operating characteristic (ROC) analysis revealed that CAVI had better discriminatory power for all-cause mortality compared to haPWV. In the Cox-proportional hazards analyses, 1 SD increase in both parameters contributed independently to all-cause mortality [CAVI: HR 1.595 (95% CI 1.108– 2.297), haPWV: HR 1.695 (95% CI 1.185– 2.425)], as well as age and CTR. Both parameters above the cut-offs estimated in the ROC analysis (CAVI ≥ 9.2, haPWV ≥ 8.9) also had independent contributions to mortality.Conclusion: Through the 6 consecutive years of follow-up in 209 HD patients, increased CAVI might represent a major modifiable risk factor for all-cause mortality. Further research is needed to examine whether CAVI-lowering interventions contribute to improved prognosis.Keywords: hemodialysis patient, mortality, cardio-ankle vascular index, phosphorus

The number of limit cycles for three dimensional Lotka–Volterra competitive systems is an open problem. Recently, we have presented a concrete example with three limit cycles in Zeeman’s class 27 [6]. In this paper, we present a concrete example with three limit cycles which belongs to Zeeman’s class 29. We explicitly give the critical parameter values such that the interior equilibrium is an exact unstable weak focus of order two. Also we verify that the system is permanent. This implies that there can exist three limit cycles around the interior equilibrium under suitable perturbations. We actually generate multiple limit cycles, and confirm them by numerical simulation. In addition, we present some other examples with three limit cycles in Zeeman’s class 27.

The number of limit cycles for three dimensional Lotka–Volterra systems is an open problem. Recently, Yu et al. (2016) constructed some examples with the possibility of the existence of four limit cycles. Unfortunately, multiple limit cycles are not visible by numerical simulations, because all of them are very close to the interior equilibrium and extremely small. We present a concrete example with multiple limit cycles for three dimensional Lotka–Volterra systems which we can confirm them by numerical simulations. First we prepare the modified formula to compute coefficients of the normal form for the generalized Hopf bifurcation. Applying this formula to three dimensional Lotka–Volterra competitive systems with the aid of the computer algebra system, we derive the critical parameter values explicitly such that the interior equilibrium is exactly an unstable weak focus. Also we show that the heteroclinic cycle on the boundary of R + 3 is repelling. This implies that there exists a stable limit cycle by the Poincare–Bendixson theorem. Then, adding some suitable perturbations to parameters, we generate additional two limit cycles near the interior equilibrium by the generalized Hopf bifurcation. Finally we confirm that there exist three limit cycles by numerical simulations.

Thymine DNA glycosylase (TDG) is a base excision repair (BER) enzyme, which is implicated in correction of deamination-induced DNA mismatches, the DNA demethylation process and regulation of gene expression. Because of these pivotal roles associated, it is crucial to elucidate how the TDG functions are appropriately regulated in vivo. Here, we present evidence that the TDG protein undergoes degradation upon various types of DNA damage, including ultraviolet light (UV). The UV-induced degradation of TDG was dependent on proficiency in nucleotide excision repair and on CRL4CDT2 -mediated ubiquitination that requires a physical interaction between TDG and DNA polymerase clamp PCNA. Using the Tdg-deficient mouse embryonic fibroblasts, we found that ectopic expression of TDG compromised cellular survival after UV irradiation and repair of UV-induced DNA lesions. These negative effects on cellular UV responses were alleviated by introducing mutations in TDG that impaired its BER function. The expression of TDG induced a large-scale alteration in the gene expression profile independently of its DNA glycosylase activity, whereas a subset of genes was affected by the catalytic activity of TDG. Our results indicate the presence of BER-dependent and BER-independent functions of TDG, which are involved in regulation of cellular DNA damage responses and gene expression patterns.

FSCE focused on resist materials and coordinate correction technology for FPD mask and carried out research on underlying technology. The results we obtained are reported.

Purposes: We previously demonstrated that Snail expression in tumor cells promotes bone metastasis not only by enhancing motility and bone polarity of tumor cells, but also by hindering anti-tumor immunity via increase of immunoregulatory cells including CD4(+)Foxp3(+) Tregs, CD11c(+)MHC II(-/low)PDL1(+) DCregs and CD45(-)ALCAM(+) mesenchymal stem cells (MSCs), and the consequent CD8(low)Tim3(+)PD1(+) exhausted/impaired T cells. In this study, we examined whether immune checkpoint inhibitors targeting CTLA4, PD1 or PDL1 would rescue from the bone metastasis-associated immune dysfunction using a bone metastasis tumor model that we previously established. Results: C57BL/6 mice are implanted with snail-transduced B16-F10 melanoma cells both subcutaneously and intravenously, and were intraperitoneally injected with anti-CTLA4, anti-PD1, or anti-PDL1 mAb, or the isotype IgG as a control (10 mg/kg) on days 5 and 8 after tumor implantation. Treatment with any immune checkpoint inhibitors significantly inhibited increase of CD4(+)Foxp3(+) Tregs followed by increase of tumor-specific CD8(+) T cells in the subcutaneous tumors, resulting in significant suppression of the tumor growth as compared to that of the control group. Unexpectedly, however, bone metastasis was rather promoted in these treated mice. Nevertheless, anti-PDL1-treated mice survived in good condition for a long time, although anti-CTLA4 or anti-PD1-treated emaciated mice died earlier than the control mice. In the mice receiving anti-CTLA4 or anti-PD1 mAb, although CD4(+)Foxp3(+) Tregs systemically decreased, other immunoregulatory cells such as CD45(-)ALCAM(+) MSCs, CD11b(+)Gr1(+) MDSCs and CD8(+)Foxp3(+) Tregs, and impaired CD8(low)Tim3(+)PD1(+) T cells systemically increased more than those in the control group. In addition, tumor cells isolated from the bone marrow (BM) formed a lot of larger sphere colonies in the matrigel culture than those of the control group, and were resistant to chemotherapeutics and CTLs, indicating cancer stem-like features. In contrast, in the anti-PDL1-treated mice, tumor-specific CD8(+) T cells were systemically induced, although immunoregulatory or impaired cells still increased. Interestingly, the BM-isolated tumor cells were quite different from those of other groups: high adhesive and chemo/CTL-sensitive, suggesting tumor transition into a less malignant epithelial type. The molecular mechanisms underlying these effects are now being investigated in detail. Conclusions: These results suggest that anti-PDL1 mAb is effective in treating bone metastasis by modulating both host immunity and tumor cells. Our study, at least using this tumor model, also gives a caution in the application of anti-CTLA4 and anti-PD1 mAb for cancer patients with bone metastasis. Citation Format: Chie Kudo-Saito, Takafumi Fuwa, Kouichi Murakami. Differential efficacy of immune checkpoint inhibitors on bone metastasis and its associated immune dysfunction. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 250. doi:10.1158/1538-7445.AM2015-250

Purposes: We have been investigating the interplay between cancer cells and host immunity, and previously demonstrated that Snail expression in tumor cells accelerates metastasis not only by enhancing tumor motility but also by inducing immune dysfunction through increase of a variety of immunoregulatory cells using murine tumor models implanted with snail-transduced B16-F10 melanoma (F10-snail+). We additionally found that Snail+ tumor cells frequently metastasize into bone marrow (BM) of the mice, and pluripotent CD45-ALCAM+ mesenchymal stem cells (MSCs) consequently increased in the mice. In this study, we attempted to characterize the Snail+ tumor-induced CD45-ALCAM+ MSCs (sMSCs). Results: The sMSCs were prepared by stimulating BMCs with F10-snail+ tumor supernatant for 5-7 days, or by sorting CD45-ALCAM+ cells from BM or spleen of the F10-snail+ tumor cells 2-3 weeks after implantation. When the mixture of the sMSCs and F10-mock tumor cells was subcutaneously injected in C57BL/6 mice, tumor growth was aggressively promoted, and spontaneous tumor dissemination in BM indicating de novo bone metastasis was observed, accompanied by increase of sMSCs in BM. In the tumor milieu and spleen of these mice, functionally impaired CD8(low) T cells and immunosuppressive Treg-inducible CD11b+Gr1+ MDSCs predominantly increased. Indeed, CD8(low) T cells were generated in the culture with splenic T cells and sMSCs, and MDSCs were expanded in the culture with BM cells and sMSCs. We identified key molecules responsible for the sMSC-induced events: ALCAM, ANGPT2 and FSTL1, which significantly increased only in the sMSC but not F10-mock-derived MSCs and normal MSCs. FSTL1 inhibited bone metastasis and consequent increase of sMSCs, CD8(low) T cells and MDSCs. Blocking ALCAM with mAb inhibited both sMSC expansion and CD8(low) T-cell induction. Blocking ANGPT2 inhibited expansion of MDSCs and Tregs rather than angiogenesis. Blocking FSTL1, which is a key trigger to initiate these cascades, in combination with elimination of other sMSC-associated factors significantly induced anti-tumor immune responses, resulting in complete inhibition of tumor growth and metastasis. Conclusions: These results suggest that sMSCs play a central role in the cancer EMT-induced immune dysfunction leading to tumor progression. Snail is reported to regulate acquisition of stem-like properties in tumor cells. However, how cancer stem cells would modulate the BM microenvironment and alter anti-tumor immune responses also remains unclear. Targeting sMSCs and its associated molecules may be a promising strategy for abrogating even cancer stem cells through reprogramming and activating anti-tumor immune responses appropriately. Citation Format: Chie Kudo-Saito, Takafumi Fuwa, Kouichi Murakami. Immune dysfunction cascades caused by ALCAM+ mesenchymal stem cells toward tumor progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 183. doi:10.1158/1538-7445.AM2014-183