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Supplementary Figure S1-S7

Primary central nervous system lymphoma (PCNSL) is an isolated type of lymphoma of the central nervous system and has a dismal prognosis despite intensive chemotherapy. Recent genomic analyses have identified highly recurrent mutations of MYD88 and CD79B in immunocompetent PCNSL, whereas LMP1 activation is commonly observed in Epstein–Barr virus (EBV)-positive PCNSL. However, a lack of clinically representative preclinical models has hampered our understanding of the pathogenic mechanisms by which genetic aberrations drive PCNSL disease phenotypes. Here, we establish a panel of 12 orthotopic, patient-derived xenograft (PDX) models from both immunocompetent and EBV-positive PCNSL and secondary CNSL biopsy specimens. PDXs faithfully retained their phenotypic, metabolic, and genetic features, with 100% concordance of MYD88 and CD79B mutations present in PCNSL in immunocompetent patients. These models revealed a convergent functional dependency upon a deregulated RelA/p65-hexokinase 2 signaling axis, codriven by either mutated MYD88/CD79B or LMP1 with Pin1 overactivation in immunocompetent PCNSL and EBV-positive PCNSL, respectively. Notably, distinct molecular alterations used by immunocompetent and EBV-positive PCNSL converged to deregulate RelA/p65 expression and to drive glycolysis, which is critical for intracerebral tumor progression and FDG-PET imaging characteristics. Genetic and pharmacologic inhibition of this key signaling axis potently suppressed PCNSL growth in vitro and in vivo. These patient-derived models offer a platform for predicting clinical chemotherapeutics efficacy and provide critical insights into PCNSL pathogenic mechanisms, accelerating therapeutic discovery for this aggressive disease.Significance:A set of clinically relevant CNSL xenografts identifies a hyperactive RelA/p65-hexokinase 2 signaling axis as a driver of progression and potential therapeutic target for treatment and provides a foundational preclinical platform.

Supplementary Table S1-S12

BackgroundMeningiomas are often embolized preoperatively to reduce intraoperative blood loss and facilitate tumor resection. However, the procedure is controversial and its effects have not yet been reported. We evaluated preoperative embolization for meningiomas and its effect on postoperative outcome and recurrence.MethodsWe retrospectively reviewed the medical records of 186 patients with WHO grade I meningiomas who underwent surgical treatment at our hospital between January 2010 and December 2020. We used propensity score matching to generate embolization and no-embolization groups (42 patients each) to examine embolization effects.ResultsPreoperative embolization was performed in 71 patients (38.2%). In the propensity-matched analysis, the embolization group showed favorable recurrence-free survival (RFS) (mean 49.4 vs 24.1 months; Wilcoxon p=0.049). The embolization group had significantly less intraoperative blood loss (178±203 mL vs 221±165 mL; p=0.009) and shorter operation time (5.6±2.0 hours vs 6.8±2.8 hours; p=0.036). There were no significant differences in Simpson grade IV resection (33.3% vs 28.6%; p=0.637) or overall perioperative complications (21.4% vs 11.9%; p=0.241). Tumor embolization prolonged RFS in a subanalysis of cases who experienced recurrence (n=39) among the overall cases before variable control (mean RFS 33.2 vs 16.0 months; log-rank p=0.003).ConclusionsAfter controlling for variables, preoperative embolization for meningioma did not improve the Simpson grade or patient outcomes. However, it might have effects outside of surgical outcomes by prolonging RFS without increasing complications.

Primary central nervous system lymphoma (PCNSL) is an isolated type of lymphoma of the central nervous system and has a dismal prognosis despite intensive chemotherapy. Recent genomic analyses have identified highly recurrent mutations of MYD88 and CD79B in immunocompetent PCNSL, whereas LMP1 activation is commonly observed in Epstein–Barr virus (EBV)-positive PCNSL. However, a lack of clinically representative preclinical models has hampered our understanding of the pathogenic mechanisms by which genetic aberrations drive PCNSL disease phenotypes. Here, we establish a panel of 12 orthotopic, patient-derived xenograft (PDX) models from both immunocompetent and EBV-positive PCNSL and secondary CNSL biopsy specimens. PDXs faithfully retained their phenotypic, metabolic, and genetic features, with 100% concordance of MYD88 and CD79B mutations present in PCNSL in immunocompetent patients. These models revealed a convergent functional dependency upon a deregulated RelA/p65-hexokinase 2 signaling axis, codriven by either mutated MYD88/CD79B or LMP1 with Pin1 overactivation in immunocompetent PCNSL and EBV-positive PCNSL, respectively. Notably, distinct molecular alterations used by immunocompetent and EBV-positive PCNSL converged to deregulate RelA/p65 expression and to drive glycolysis, which is critical for intracerebral tumor progression and FDG-PET imaging characteristics. Genetic and pharmacologic inhibition of this key signaling axis potently suppressed PCNSL growth in vitro and in vivo. These patient-derived models offer a platform for predicting clinical chemotherapeutics efficacy and provide critical insights into PCNSL pathogenic mechanisms, accelerating therapeutic discovery for this aggressive disease. Significance: A set of clinically relevant CNSL xenografts identifies a hyperactive RelA/p65-hexokinase 2 signaling axis as a driver of progression and potential therapeutic target for treatment and provides a foundational preclinical platform.

We previously identified a novel nanomagnetic particle, N,N'-bis(salicylidene)ethylenediamine iron [Fe(Salen)]. Fe(Salen) not only shows antitumor effects but also magnetic properties. We found that Fe(Salen) can be used for magnet-guided drug delivery and visualization of accumulated drug by magnetic resonance imaging (MRI) because of its magnetism. In addition, Fe(Salen) can generate heat by itself when exposed to an alternating current magnetic field (AMF), resulting in a hyperthermia effect. Herein, we partly elucidated the antitumor mechanism of Fe(Salen) and carried out an i.v. repeated dose toxicity study to decide the therapeutic amount. Furthermore, we evaluated the antitumor effect of selective intra-arterial injection or i.v. injection of Fe(Salen) by catheter and the hyperthermia effect of Fe(Salen) when exposed to AMF in vivo. We used a rabbit model grafted with VX2 cells (rabbit squamous cell carcinoma) on the right leg. Intra-arterial injection of Fe(Salen) showed a greater antitumor effect than did i.v. injection. The combination of Fe(Salen) intra-arterial injection and AMF exposure showed a greater antitumor effect than did either Fe(Salen) or methotrexate (MTX) without AMF exposure, suggesting that AMF exposure greatly enhanced the antitumor effect of Fe(Salen) by arterial injection by catheter. This is the first report that the effectiveness of Fe(Salen) was evaluated in the point of administration route; that is, selective intra-arterial injection by catheter. Taken together, these results indicate a new administration route; that is, selective arterial injection of Fe(Salen) by catheter, and the development of a new strategy of simultaneous hyperthermia-chemotherapy in the future.

We previously reported the efficacy of anti‐cancer therapy with hyperthermia using an alternating magnetic field (AMF) and a magnetic compound. In the course of the study, unexpectedly, we found that an AMF enhances the cytotoxicity of Compound C, an activated protein kinase (AMPK) inhibitor, although this compound is not magnetic. Therefore, we examined the cellular mechanism of AMF‐induced cytotoxicity of Compound C in cultured human glioblastoma (GB) cells. An AMF (280 kHz, 250 Arms) for 30 minutes significantly enhanced the cytotoxicity of Compound C and promoted apoptosis towards several human GB cell lines in vitro. The AMF also increased Compound C‐induced cell‐cycle arrest of GB cells at the G2 phase and, thus, inhibited cell proliferation. The AMF increased Compound C‐induced reactive oxygen species production. Furthermore, the AMF decreased ERK phosphorylation in the presence of Compound C and suppressed the protective autophagy induced by this compound. The application of an AMF in cancer chemotherapy may be a simple and promising method, which might reduce the doses of drugs used in future cancer treatment and, therefore, the associated side effects.

The potential of positron emission tomography/computed tomography using 62Cu-diacetyl-bis (N4-methylthiosemicarbazone) (62Cu-ATSM PET/CT), which was originally developed as a hypoxic tracer, to predict therapeutic resistance and prognosis has been reported in various cancers. Our purpose was to investigate prognostic value of 62Cu-ATSM PET/CT in patients with glioma, compared to PET/CT using 2-deoxy-2-[18F]fluoro-d-glucose (18F-FDG). 56 patients with glioma of World Health Organization grade 2–4 were enrolled. All participants had undergone both 62Cu-ATSM PET/CT and 18F-FDG PET/CT within mean 33.5 days prior to treatment. Maximum standardized uptake value and tumor/background ratio were calculated within areas of increased radiotracer uptake. The prognostic significance for progression-free survival and overall survival were assessed by log-rank test and Cox’s proportional hazards model. Disease progression and death were confirmed in 37 and 27 patients in follow-up periods, respectively. In univariate analysis, there was significant difference of both progression-free survival and overall survival in age, tumor grade, history of chemoradiotherapy, maximum standardized uptake value and tumor/background ratio calculated using 62Cu-ATSM PET/CT. Multivariate analysis revealed that maximum standardized uptake value calculated using 62Cu-ATSM PET/CT was an independent predictor of both progression-free survival and overall survival (p

Cortical plasticity is fundamental to motor recovery following cortical perturbation. However, it is still unclear how this plasticity is induced at a functional circuit level. Here, we investigated motor recovery and underlying neural plasticity upon optogenetic suppression of a cortical area for eye movement. Using a visually-guided eye movement task in mice, we suppressed a portion of the secondary motor cortex (MOs) that encodes contraversive eye movement. Optogenetic unilateral suppression severely impaired contraversive movement on the first day. However, on subsequent days the suppression became inefficient and capability for the movement was restored. Longitudinal two-photon calcium imaging revealed that the regained capability was accompanied by an increased number of neurons encoding for ipsiversive movement in the unsuppressed contralateral MOs. Additional suppression of the contralateral MOs impaired the recovered movement again, indicating a compensatory mechanism. Our findings demonstrate that repeated optogenetic suppression leads to functional recovery mediated by the contralateral hemisphere.

OBJECTIVEErythropoietin (EPO) is a clinically available hematopoietic cytokine. The aim of this study was to evaluate the effect of EPO on a rat model of cervical cord compression myelopathy and to explore the possibility of its use as a pharmacological treatment.METHODSTo produce the chronic cervical cord compression model, thin polyurethane sheets were implanted under the C5-C6 laminae of rats and gradually expanded due to water absorption. In this model, motor functions significantly declined from 7 weeks after surgery. Based on the result, EPO administration was started 8 weeks after surgery. Motor function as seen with rotarod performance and grip strength was measured 16 weeks after surgery, and then motor neurons were stained with H-E and NeuN staining, and counted. Apoptotic cell death was assessed with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining. To assess transfer of EPO into spinal cord tissue, the EPO level in spinal cord tissue was measured with an enzyme-linked immunosorbent assay for each group after subcutaneous injection of EPO.RESULTSHigh-dose EPO (5000 IU/kg) administered from 8 weeks after surgery markedly restored and maintained motor function in the Compression groups (P < 0.01). EPO significantly prevented loss of motor neurons in the anterior horn (P < 0.05) and significantly decreased the number of TUNEL-positive apoptotic cells (P < 0.05). The EPO level in spinal cord tissue was significantly higher in the High-dose EPO group than other groups.CONCLUSIONSEPO improves motor function in rats with progressive chronic compression myelopathy. EPO protects anterior horn motor neurons and inhibits neuronal cell apoptosis in spinal cord compression. The neuroprotective effects can be produced through transfer of EPO into spinal cord tissue. These findings suggest that EPO has high potential as a treatment for developing compression myelopathy.

The potential of positron emission tomography/computed tomography using56 patients with glioma of World Health Organization grade 2-4 were enrolled. All participants had undergone bothDisease progression and death were confirmed in 37 and 27 patients in follow-up periods, respectively. In univariate analysis, there was significant difference of both progression-free survival and overall survival in age, tumor grade, history of chemoradiotherapy, maximum standardized uptake value and tumor/background ratio calculated using

BACKGROUND AND PURPOSE: (62)Cu-diacetyl-bis(N(4)-methylthiosemicarbazone) was developed as a hypoxic radiotracer in PET. We compared imaging features among MR imaging and (62)Cu-diacetyl-bis(N(4)-methylthiosemicarbazone)-PET, FDG-PET, and L-methyl-[(11)C]methionine)-PET in gliomas. MATERIALS AND METHODS: We enrolled 23 patients who underwent (62)Cu-diacetyl-bis(N(4)-methylthiosemicarbazone)-PET and FDG-PET and 19 (82.6%) who underwent L-methyl-[(11)C]methionine)–PET, with all 23 patients undergoing surgery and their diagnosis being then confirmed by histologic examination as a glioma. Semiquantitative and volumetric analysis were used for the comparison. RESULTS: There were 10 newly diagnosed glioblastoma multiforme and 13 nonglioblastoma multiforme (grades II and III), including 4 recurrences without any adjuvant treatment. The maximum standardized uptake value and tumor/background ratios of (62)Cu-diacetyl-bis(N(4)-methylthiosemicarbazone), as well as L-methyl-[(11)C]methionine, were significantly higher in glioblastoma multiforme than in nonglioblastoma multiforme (P = .03 and P = .03, respectively); no significant differences were observed on FDG. At a tumor/background ratio cutoff threshold of 1.9, (62)Cu-diacetyl-bis(N(4)-methylthiosemicarbazone) was most predictive of glioblastoma multiforme, with 90.0% sensitivity and 76.9% specificity. The positive and negative predictive values, respectively, for glioblastoma multiforme were 75.0% and 85.7% on (62)Cu-diacetyl-bis(N(4)-methylthiosemicarbazone), 83.3% and 60.0% on L-methyl-[(11)C]methionine, and 72.7% and 75.0% on MR imaging. In glioblastoma multiforme, volumetric analysis demonstrated that (62)Cu-diacetyl-bis(N(4)-methylthiosemicarbazone) uptake had significant correlations with FDG (r = 0.68, P = .03) and L-methyl-[(11)C]methionine (r = 0.87, P = .03). However, the (62)Cu-diacetyl-bis(N(4)-methylthiosemicarbazone)–active region was heterogeneously distributed in 50.0% (5/10) of FDG-active and 0% (0/6) of L-methyl-[(11)C]methionine)–active regions. CONCLUSIONS: (62)Cu-diacetyl-bis(N(4)-methylthiosemicarbazone) may be a practical radiotracer in the prediction of glioblastoma multiforme. In addition to FDG-PET, L-methyl-[(11)C]methionine)–PET, and MR imaging, (62)Cu-diacetyl-bis(N(4)-methylthiosemicarbazone)-PET may provide intratumoral hypoxic information useful in establishing targeted therapeutic strategies for patients with glioblastoma multiforme.

Melanoma has an extremely poor prognosis due to its rapidly progressive and highly metastatic nature. Several therapeutic drugs have recently become available, but are effective only against melanoma with specific BRAF gene mutation. Thus, there is a need to identify other target molecules. We show here that Transient receptor potential, canonical 3 (TRPC3) is widely expressed in human melanoma. We found that pharmacological inhibition of TRPC3 with a pyrazole compound, Pyr3, decreased melanoma cell proliferation and migration. Similar inhibition was observed when the TRPC3 gene was silenced with short-hairpin RNA (shRNA). Pyr3 induced dephosphorylation of signal transducer and activator of transcription (STAT) 5 and Akt. Administration of Pyr3 (0.05 mg/kg) to mice implanted with human melanoma cells (C8161) significantly inhibited tumor growth. Our findings indicate that TRPC3 plays an important role in melanoma growth, and may be a novel target for treating melanoma in patients.

Object The authors retrospectively studied the mechanism of cyst formation and enlargement after Gamma Knife surgery (GKS) for arteriovenous malformations (AVMs). Methods Eighteen patients in whom cyst formation developed following GKS for AVM were retrospectively identified among 775 patients who underwent GKS for AVM at Yokohama Rosai Hospital. The study group was composed of 12 male and 6 female patients ranging in age from 17 to 47 years. Results Chronic encapsulated expanding hematoma was associated with the cyst in 5 patients. The AVM nidus volume at the time of GKS ranged from 1.9 to 36 cm3, and the prescription radiation dose was 18–25 Gy. Complete obliteration of the AVM nidus was obtained in 13 patients and partial obliteration in 5 patients. Cyst formation was detected between 2.6 and 15 years after GKS. Craniotomy was performed in 10 patients, including 2 patients in whom the incompletely obliterated nidus was removed at the same time, and an Ommaya reservoir was placed in 2 patients. Spontaneous regression of the cyst was observed in 1 patient. Serial MR imaging was performed in the other patients because the size of the cyst was stable or the lesion was asymptomatic. Histological examination of the cyst wall revealed linear hemosiderin deposits with gliosis. The nodular lesion, which was enhanced on MR images, contained granulation tissue with chronic hemorrhage from newly developed capillary vessels. Conclusions Cysts developing after GKS for AVM enlarge mainly due to repeated minor hemorrhages from a reddish nodular angiomatous lesion that develops within an adjacent brain area. Thus, the optimal treatment is wide opening of the cyst with removal of the associated angiomatous lesion by craniotomy.

We previously reported that μ-oxo N,N’-bis(salicylidene)ethylenediamine iron [Fe(Salen)], a magnetic organic compound, has direct anti-tumor activity, and generates heat in an alternating magnetic field (AMF). We showed that Fe(Salen) nanoparticles are useful for combined hyperthermia-chemotherapy of tongue cancer. Here, we have examined the effect of Fe(Salen) on human glioblastoma (GB). Fe(Salen) showed in vitro anti-tumor activity towards several human GB cell lines. It inhibited cell proliferation, and its apoptosis-inducing activity was greater than that of clinically used drugs. Fe(Salen) also showed in vivo anti-tumor activity in the mouse brain. We evaluated the drug distribution and systemic side effects of intracerebrally injected Fe(Salen) nanoparticles in rats. Further, to examine whether hyperthermia, which was induced by exposing Fe(Salen) nanoparticles to AMF, enhanced the intrinsic anti-tumor effect of Fe(Salen), we used a mouse model grafted with U251 cells on the left leg. Fe(Salen), BCNU, or normal saline was injected into the tumor in the presence or absence of AMF exposure. The combination of Fe(Salen) injection and AMF exposure showed a greater anti-tumor effect than did either Fe(Salen) or BCNU alone. Our results indicate that hyperthermia and chemotherapy with single-drug nanoparticles could be done for GB treatment.

Background: It has been well known that melanoma has a poor prognosis due to its rapid progression and high metastatic ability before. Although a few new drugs came out into clinical field in very recent years, these drugs are restricted to use for the patients who have BRAF or MEK gene mutation. Therefore we still have to search new target points that are not dependent on these gene mutations. TRP channels are activated by changes of temperature or membrane voltage, resulting in activation of intracellular responses. Here, we investigate whether TRPC3 regulates cell proliferation and migration of human melanoma in vitro and in vivo. Material: C8161 cells, a BRAF wild type human melanoma cell line, were mainly used in this study. Cell proliferation was assessed by 2,3, -bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt (XTT) assay. Cell cycle was analyzed by fluorescence-activated cells sorting. In order to examine the role of TRPC3 in human melanoma, Short heparin RNA (shRNA) transductions with lentivirus encoding either TRPC3 or scramble was performed according to the protocols of the manufacture. Secretion level of matrix metalloproteinase (MMP) 9 was assessed in the presence of Pyr3, TRPC selective inhibitor or not by gelatin zymography. We transplanted C8161 cells into the side chest of female Balb-c nude mice at the level of intradermal depth. Immediately after tumors were formed, Pyr3 or DMSO (control) was injected intradermaly around the tumor every day. We measured each diameters of tumor every two days, and calculated volume and regression rate. Result: mRNA and protein of TRPC3 were expressed in multiple human melanoma cell lines and primary tissue. Proliferation rate of TRPC3 knocked down C8161 decreases in 48 hours (p Conclusion: TRPC3 regulates proliferation and migration, and thus tumor growth and metastasis of melanoma, suggesting that TRPC3 could be a novel target for treating BRAF wild type human melanomas. Note: This abstract was not presented at the meeting. Citation Format: Kayoko Oda, Masanari Umemura, Mayumi Katsumata, Haruki Aoyama, Ayako Makino, Makoto Ohtake, Itaru Sato, Yukie Yamaguchi, Yoji Nagashima, Michiko Aihara, Yoshihiro Ishikawa, Akane Nagasako. Transient receptor potential cation channel 3 (TRPC3) regulates tumor proliferation and migration of BRAF wild type human malignant melanoma. [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 4371. doi:10.1158/1538-7445.AM2015-4371

Background: Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumor in humans. In spite of advances in treatment strategies, survival rate of GBM remains poor. We have recently reported a novel anti-cancer compound with intrinsic magnetism(EI236). In addition to its anti-cancer effect, EI236 generates a large amount of heat upon an alternating magnetic field (AMF) application because of its magnetism, i.e., hyperthermic effect. In this study, we have examined the effect of EI236 on human GBM in vitroand vivo. Materials and Methods: Human GBM cell lines, U251MG, YKG-1, and U87, were used in this study. Cell proliferation was assessed by methyl thiazolyl tetrazorium (MTT) assays in the presence of EI236, in comparison to temozolomide (TMZ) and calmustine (BCNU), i.e. the clinical standard drugs for GBM. Apoptosis was analyzed by fluorescence activated cells sorting. In addition, we have examined whether EI236 inhibited the tumor growth with/without AMF application in animal models. Tumors were induced by implantation of U251MG cells into the hip of female Balb-c mice. Seven days after the implantation of GBM cells, EI236, BCNU or normal saline (control) was injected respectively into the tumors and mice were exposed to an AMF application(330.8A, 280kHz) for 30 minutes. Therefore, we calculated the tumor volume and the regression rate of tumors twice a week. Result: EI236 inhibited the proliferation of all cell lines and increased apoptosis in a dose dependent manner. Moreover, we found that EI236 had much greater anti-cancer effect than the clinical standard drugs. Mice bearing GBM cancer cells in their hip were then subjected to a double combination therapy, consisting of local injection of EI236 and hyperthermic treatment by AMF application. Tumor sizes in both drug stimulation groups were reduced, especially that in EI236 with AMF application group was highly reduced, while that in control group was increased. Conclusion: EI236 has greater anti-cancer effect than TMZ and BCNU, i.e. the clinical standard drugs. Besides this strong anti-cancer effect, EI236 has hyperthermic effect upon an AMF application because of heat generation. Accordingly, EI236 may enable us to develop novel strategies in GBM treatment, i.e. hyperthermic-chemotherapy with a single-drug compound. Note: This abstract was not presented at the meeting. Citation Format: Makoto Ohtake, Masanari Umemura, Itaru Sato, Kayoko Oda, Akane Nagasako, Ayako Makino, Haruki Aoyama, Mayumi Katsumata, Haruki Eguchi, Nobutaka Kawahara, Yoshihiro Ishikawa. Simultaneous hyperthermic-chemotherapy for glioblastoma using a single anti-cancer compound with intrinsic magnetism. [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 4548. doi:10.1158/1538-7445.AM2015-4548

Background: We previously reported the generation of a novel paclitaxel derivative with intrinsic magnetism. Similarly, we have synthesized a novel derivative of methotrexate, a conventional drug for cancer and rheumatic diseases, with intrinsic magnetism (M-MTX). MTX is widely used in clinical treatments, however various severe side effects such as renal, hepatic, and pulmonary toxicities or bone marrow suppression have been reported. This is a single methotrexate compound with intrinsic magnetism and is not a methotrexate encapsulated in micelle with magnetic particles. The magnetic property contributes to unique features. 1) It can be attracted by a magnet, resulting in reduction of the side effects. 2) It can be visualized by magnetic resonance imaging (MRI), suggesting that we can identify the localization of drug and quantify the amount of this drug. In this study, we have examined whether M-MTX has both the magnetic and the anti-cancer property with similar efficacy to commercial available methotrexate. Materials & Methods: The magnetic property of M-MTX was measured by Electron Spin Resonance (ESR) and Superconducting Quantum Interference Device (SQUID). MCF7, breast cancer cells were obtained from RIKEN Bioresource center in this study. Cell proliferation was assessed by a commercially available kit, XTT Cell Proliferation Assay Kit (ATCC). Apoptotic cells were stained with APC Annexin V and 7-AAD, and measured by fluorescence activated cell sorting (FACS), to evaluate early and late apoptosis. The intracellular ROS level was then measured using a fluorescent dye, 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Sigma, Japan) Results: M-MTX was easily attracted by a neodium magnet. Plots of magnetization versus magnetic field revealed that the magnetized methotrexate exhibits spontaneous magnetization in SQUID. ESR also showed that M-MTX has an intrinsic magnetism. Furthermore, M-MTX inhibited cell proliferation and induced cellular apoptosis in MCF7 cell lines in a dose-dependent manner. M-MTX also increased reactive oxygen species (ROS) generation, suggesting that M-MTX might retained the original anti-cancer property. Conclusion: M-MTX may provide us a new strategy for cancer therapy, i.e., chemotherapy with magnetic drug delivery with a single agent. These results also suggested that various conventional anti-cancer drugs might be similarly magnetized, leading to novel drug developments in future cancer chemotherapy and other treatments. Note: This abstract was not presented at the meeting. Citation Format: MASANARI UMEMURA, Mayumi Katsumata, Itaru Sato, Akane Nagasako, Haruki Aoyama, Ayako Makino, Makoto Ohtake, Kayoko Oda, Kosuke Matsuo, Haruki Eguchi, Yoshihiro Ishikawa. Methotrexate derivative with intrinsic magnetism. [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 4398. doi:10.1158/1538-7445.AM2015-4398

Background: We previously reported the identification of a novel nano-organic compound, EI236, an anti-cancer agent with intrinsic magnetic property. In addition to anti-cancer effect, its ferromagnetic property contributes to unique features.1) It can be attracted by a magnet. 2) It can be visualized by magnetic resonance imaging (MRI). Hereby, we have identified the key mechanism that contributes to magnetism by X-ray crystallographic analysis, and succeeded in generating a novel paclitaxel with intrinsic magnetism; this is a single paclitaxel compound, and is not a paclitaxel encapsulated in micelle with magnetic particles. Our aim is to examine its effect on triple negative-breast cancer (TNBC) cells. Material and Method: The magnetization of the magnetized paclitaxel was measured with a superconducting quantum interference device (SQUID) (Quantum Design MPMS7 system). Breast cancer cells, MDA-MB-453 (TNBC) and MCF7 (Non-TNBC), were obtained from RIKEN Bioresource center. Cell proliferation assay was performed using a commercially available kit, XTT Cell Proliferation Assay Kit. Apoptotic cells were stained with Annexin V, allophycocyanin conjugate and 7-amino-actinomycin D, and measured by fluorescence activated cell sorting (FACS), to evaluate early and late apoptosis. Cell cycle analysis was performed using The Cycletest™ Plus DNA Reagent Kit and assessed using FACS. Results: Plots of magnetization versus magnetic field revealed that the magnetized paclitaxel exhibits magnetic property in SQUID. Magnetized paclitaxel was easily attracted by a commercial bar magnet. Magnetized paclitaxel exhibits greater anti-cancer effect than original paclitaxel in TNBC and Non-TNBC cells in a dose-dependent manner. Magnetized paclitaxel induced apoptosis and G2/M arrest in cell cycle analysis in a dose-dependent manner, suggesting that magnetized paclitaxel retained the original anti-cancer property. In MRI T2 -weighted imaging, signal intensity was changed in a concentration-dependent manner with magnetized paclitaxel, but not with commercial available paclitaxel. Conclusion: These results suggested that various conventional anti-cancer drugs might be similarly magnetized, leading to novel drug development in future cancer chemotherapy. Citation Format: Masanari Umemura, Ayako Makino, Itaru Sato, Xianfeng Feng, Kayoko Oda, Makoto Ohtake, Satoshi Izuka, Maki Iwai, Kosuke Matsuo, Haruki Eguchi, Yoshihiro Ishikawa. A novel treatment for triple-negative breast cancer using intrinsic magnetized paclitaxel. [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 5399. doi:10.1158/1538-7445.AM2014-5399