Self-assembling A6K peptide nanotubes as a mercaptoundecahydrododecaborate (BSH) delivery system for boron neutron capture t (BNCT)

Boron neutron capture therapy (BNCT) is a tumor selective therapy, the effectiveness of which depends on sufficient 10B delivery to and accumulation in tumors. In this study, we used self-assembling A6K peptide nanotubes as boron carriers and prepared new boron agents by simple mixing of A6K and BSH. BSH has been used to treat malignant glioma patients in clinical trials and its drug safety and availability have been confirmed; however, its contribution to BNCT efficacy is low. A6K nanotube delivery improved two major limitations of BSH, including absence of intracellular transduction and non-specific drug delivery to tumor tissue. Varying the A6K peptide and BSH mixture ratio produced materials with different morphologies—determined by electron microscopy—and intracellular transduction efficiencies. We investigated the A6K/BSH 1:10 mixture ratio and found high intracellular boron uptake with no toxicity. Microscopy observation showed intracellular localization of A6K/BSH in the perinuclear region and endosome in human glioma cells. The intracellular boron concentration using A6K/BSH was almost 10 times higher than that of BSH. The systematic administration of A6K/BSH via mouse tail vein showed tumor specific accumulation in a mouse brain tumor model with immunohistochemistry and pharmacokinetic study. Neutron irradiation of glioma cells treated with A6K/BSH showed the inhibition of cell proliferation in a colony formation assay. Boron delivery using A6K peptide provides a unique and simple strategy for next generation BNCT drugs

Generation of Vascular Endothelial Cells and Hematopoietic Cells by Blastocyst Complementation.

In the case of organ transplantation accompanied by vascular anastomosis, major histocompatibility complex mismatched vascular endothelial cells become a target for graft rejection. Production of a rejection-free, transplantable organ, therefore, requires simultaneous generation of vascular endothelial cells within the organ. To generate pluripotent stem cell (PSC)-derived vascular endothelial cells, we performed blastocyst complementation with a vascular endothelial growth factor receptor-2 homozygous mutant blastocyst. This mutation is embryonic lethal at embryonic (E) day 8.5-9.5 due to an early defect in endothelial and hematopoietic cells. The Flk-1 homozygous knockout chimeric mice survived to adulthood for over 1 year without any abnormality, and all vascular endothelial cells and hematopoietic cells were derived from the injected PSCs. This approach could be used in conjunction with other gene knockouts which induce organ deficiency to produce a rejection-free, transplantable organ in which all the organ's cells and vasculature are PSC derived

Efficient Drug Delivery of Paclitaxel Glycoside: A Novel Solubility Gradient Encapsulation into Liposomes Coupled with Immunoliposomes Preparation

Although the encapsulation of paclitaxel into liposomes has been extensively studied, its significant hydrophobic and uncharged character has generated substantial difficulties concerning its efficient encapsulation into the inner water core of liposomes. We found that a more hydrophilic paclitaxel molecule, 7-glucosyloxyacetylpaclitaxel, retained tubulin polymerization stabilization activity. The hydrophilic nature of 7-glucosyloxyacetylpaclitaxel allowed its efficient encapsulation into the inner water core of liposomes, which was successfully accomplished using a remote loading method with a solubility gradient between 40% ethylene glycol and Cremophor EL/ethanol in PBS. Trastuzumab was then conjugated onto the surface of liposomes as immunoliposomes to selectively target human epidermal growth factor receptor-2 (HER2)-overexpressing cancer cells. In vitro cytotoxicity assays revealed that the immunoliposomes enhanced the toxicity of 7-glucosyloxyacetylpaclitaxel in HER2-overexpressing cancer cells and showed more rapid suppression of cell growth. The immunoliposomes strongly inhibited the tumor growth of HT-29 cells xenografted in nude mice. Notably, mice survived when treated with the immunoliposomes formulation, even when administered at a lethal dose of 7-glucosyloxyacetylpaclitaxel in vivo. This data successfully demonstrates immunoliposomes as a promising candidate for the efficient delivery of paclitaxel glycoside

A Model of Cancer Stem Cells Derived from Mouse Induced Pluripotent Stem Cells

Cancer stem cells (CSCs) are capable of continuous proliferation and self-renewal and are proposed to play significant roles in oncogenesis, tumor growth, metastasis and cancer recurrence. CSCs are considered derived from normal stem cells affected by the tumor microenvironment although the mechanism of development is not clear yet. In 2007, Yamanaka's group succeeded in generating Nanog mouse induced pluripotent stem (miPS) cells, in which green fluorescent protein (GFP) has been inserted into the 5′-untranslated region of the Nanog gene. Usually, iPS cells, just like embryonic stem cells, are considered to be induced into progenitor cells, which differentiate into various normal phenotypes depending on the normal niche. We hypothesized that CSCs could be derived from Nanog miPS cells in the conditioned culture medium of cancer cell lines, which is a mimic of carcinoma microenvironment. As a result, the Nanog miPS cells treated with the conditioned medium of mouse Lewis lung carcinoma acquired characteristics of CSCs, in that they formed spheroids expressing GFP in suspension culture, and had a high tumorigenicity in Balb/c nude mice exhibiting angiogenesis in vivo. In addition, these iPS-derived CSCs had a capacity of self-renewal and expressed the marker genes, Nanog, Rex1, Eras, Esg1 and Cripto, associated with stem cell properties and an undifferentiated state. Thus we concluded that a model of CSCs was originally developed from miPS cells and proposed the conditioned culture medium of cancer cell lines might perform as niche for producing CSCs. The model of CSCs and the procedure of their establishment will help study the genetic alterations and the secreted factors in the tumor microenvironment which convert miPS cells to CSCs. Furthermore, the identification of potentially bona fide markers of CSCs, which will help the development of novel anti-cancer therapies, might be possible though the CSC model

A cancer stem cell model as the point of origin of cancer-associated fibroblasts in tumor microenvironment

Cancer-associated fibroblasts (CAFs) are one of the most prominent cell types in the stromal compartment of the tumor microenvironment. CAFs support multiple aspects of cancer progression, including tumor initiation, invasion, and metastasis. The heterogeneous nature of the stromal microenvironment is attributed to the multiple sources from which the cells in this compartment originate. The present study provides the first evidence that cancer stem cells (CSCs) are one of the key sources of CAFs in the tumor niche. We generated CSC-like cells by treating mouse induced pluripotent stem cells with conditioned medium from breast cancer cell lines. The resulting cell population expressed both CSC and pluripotency markers, and the sphere-forming CSC-like cells formed subcutaneous tumors in nude mice. Intriguingly, these CSC-like cells always formed heterogeneous populations surrounded by myofibroblast-like cells. Based on this observation, we hypothesized that CSCs could be the source of the CAFs that support tumor maintenance and survival. To address this hypothesis, we induced the differentiation of spheres and purified the myofibroblast-like cells. The resulting cells exhibited a CAF-like phenotype, suggesting that they had differentiated into the subpopulations of cells that support CSC self-renewal. These findings provide novel insights into the dynamic interplay between various microenvironmental factors and CAFs in the CSC niche

HSP‐enriched properties of extracellular vesicles involve survival of metastatic oral cancer cells

Cancer cells often secrete extracellular vesicles (EVs) that carry heat shock proteins (HSPs) with roles in tumor progression. Oral squamous cell carcinoma (OSCC) belongs to head and neck cancers (HNC) whose lymph-node-metastases often lead to poor prognosis. We have examined the EV proteome of OSCC cells and found abundant secretion of HSP90-enriched EVs in lymph-node-metastatic OSCC cells. Double knockdown of HSP90α and HSP90β, using small interfering RNA significantly reduced the survival of the metastatic OSCC cells, although single knockdown of each HSP90 was ineffective. Elevated expression of these HSP90 family members was found to correlate with poor prognosis of HNC cases. Thus, elevated HSP90 levels in secreted vesicles are potential prognostic biomarkers and therapeutic targets in metastatic OSCC

P-NETG2に対しPRRTを施行した4例に関する検討

In June 2021, 177Lu-oxodotreotide, a PRRT, was approved by insurance for unresectable or recurrent NETs. We investigated the efficacy and safety of PRRT in four patients with P-NETs at our hospital. All patients were confirmed to be positive for somatostatin receptors by using octreoscan. PRRT treatment was then performed and retrospectively evaluated for efficacy and safety. PRRT was administered as lutetium oxodotreotide（177Lu） at 7.4 GBq per dose over 30 - minutes for up to 1 - 4 doses at 8 - week intervals. Efficacy was evaluated using RECIST v1. 1. Adverse effects were evaluated by CTCAE（v5.0 JCOG）. The mean patient age was 60±12.0 years, and all patients were male. Three patients had a PS of 0, and one patient had a PS of 1 or higher. Metastatic organs were the liver in four patients and intra-abdominal lymph nodes in one patient, all of whom were Stage IV. Three patients underwent transarterial embolization. 177Lu-DOTATOC PRRT was administered every 8 weeks, and a total of four courses were administered in two patients, three courses in one patient, and one course in one patient. One patient had grade 2 thrombocytopenia after one course, and the second course was administered at a half dose（3.7 GBq）. The overall response rate（ORR） was 25%, with one PR and two SD. The median PFS was 9 months（95% Cl, 8-NA）, and the median overall survival from diagnosis was 119 months（95%Cl, 31 - NA）. Adverse events during PRRT included leukopenia in two patients（one G3, one G2）, lymphopenia in one patient（one G3）, thrombocytopenia in two patients（two G2, one G3）, creatinine increase in one patient（one G2）, and skin rash in one patient. In conclusion, PRRT is expected to be highly effective and safe compared with conventional therapy for neuroendocrine tumors with unresectable or distant metastases

A Novel Combination Cancer Therapy with Iron Chelator Targeting Cancer Stem Cells via Suppressing Stemness

Excess iron causes cancer and is thought to be related to carcinogenesis and cancer progression including stemness, but the details remain unclear. Here, we hypothesized that stemness in cancer is related to iron metabolism and that regulating iron metabolism in cancer stem cells (CSCs) may be a novel therapy. In this study, we used murine induced pluripotent stem cells that expressed specific stem cell genes such as Nanog, Oct3/4, Sox2, Klf4, and c-Myc, and two human cancer cell lines with similar stem cell gene expression. Deferasirox, an orally available iron chelator, suppressed expression of stemness markers and spherogenesis of cells with high stemness status in vitro. Combination therapy had a marked antitumor effect compared with deferasirox or cisplatin alone. Iron metabolism appears important for maintenance of stemness in CSCs. An iron chelator combined with chemotherapy may be a novel approach via suppressing stemness for CSC targeted therapy

Deep Learning of Cancer Stem Cell Morphology Using Conditional Generative Adversarial Networks

Deep-learning workflows of microscopic image analysis are sufficient for handling the contextual variations because they employ biological samples and have numerous tasks. The use of well-defined annotated images is important for the workflow. Cancer stem cells (CSCs) are identified by specific cell markers. These CSCs were extensively characterized by the stem cell (SC)-like gene expression and proliferation mechanisms for the development of tumors. In contrast, the morphological characterization remains elusive. This study aims to investigate the segmentation of CSCs in phase contrast imaging using conditional generative adversarial networks (CGAN). Artificial intelligence (AI) was trained using fluorescence images of the Nanog-Green fluorescence protein, the expression of which was maintained in CSCs, and the phase contrast images. The AI model segmented the CSC region in the phase contrast image of the CSC cultures and tumor model. By selecting images for training, several values for measuring segmentation quality increased. Moreover, nucleus fluorescence overlaid-phase contrast was effective for increasing the values. We show the possibility of mapping CSC morphology to the condition of undifferentiation using deep-learning CGAN workflows