DNA barcoding and gene expression recording reveal the presence of cancer cells with unique properties during tumor progression.

Tumors comprise diverse cancer cell populations with specific capabilities for adaptation to the tumor microenvironment, resistance to anticancer treatments, and metastatic dissemination. However, whether these populations are pre-existing in cancer cells or stochastically appear during tumor growth remains unclear. Here, we show the heterogeneous behaviors of cancer cells regarding response to anticancer drug treatments, formation of lung metastases, and expression of transcription factors related to cancer stem-like cells using a DNA barcoding and gene expression recording system. B16F10 cells maintained clonal diversity after treatment with HVJ-E, a UV-irradiated Sendai virus, and the anticancer drug dacarbazine. PBS treatment of the primary tumor and intravenous injection of B16F10 cells resulted in metastases formed from clones of multiple cell lineages. Conversely, BL6 and 4T1 cells developed spontaneous lung metastases by a small number of clones. Notably, an identical clone of 4T1 cells developed lung metastases in different mice, suggesting the existence of cells with high metastatic potential. Cas9-based transcription recording analysis in a human prostate cancer cell line revealed that specific cells express POU5F1 in response to an anticancer drug and sphere formation. Our findings provide insights into the diversity of cancer cells during tumor progression.

[Delayed diagnosis of hydroa vacciniforme-like lymphoproliferative disorder in a patient with skin rashes].

Chronic active Epstein-Barr virus (CAEBV) infection is characterized by persistent EBV infection and can lead to fatal conditions such as hemophagocytic syndrome and malignant lymphoma through the clonal expansion of EBV-infected T or natural killer (NK) cells. Hydroa vacciniforme lymphoproliferative disorder (HV) and hypersensitivity to mosquito bites (HMB) have been identified as skin diseases in EBV-associated T- or NK-cell lymphoproliferative diseases. We present the case of a 33-year-old man. The patient had frequent episodes of a facial rash for three years before he visited our hospital, he visited several dermatologists but did not receive a diagnosis of HV. He was referred to the hematology department of our hospital for assessment of atypical lymphocytes in peripheral blood. Based on routine blood and bone marrow test we were unable to diagnose HV. However, when the patient's liver function deteriorated six months later, we considered the possibility of HV after reevaluating the skin rash. After performing EBV-related tests, we were able to definitively diagnose CAEBV with HV. It is crucial to be able to connect clinical observations to EBV-related tests when diagnosing CAEBV. Hematologists must be knowledgeable of the EBV-associated skin conditions of HV and HMB.

[Effects of ordinances prohibiting smoking in private homes with children following smoke-free household rules in Tokyo].

Objectives　Exposure to secondhand smoke is harmful to children's health. Therefore, the Tokyo metropolitan area implemented an ordinance on April 1st, 2018 that prohibits smoking in private homes when children are present. To date, the effect of this ordinance has not been studied. In this study, we evaluated the change in the percentage of residents in the Tokyo metropolitan area who have smoke-free household rules using difference-in-difference (DID) analysis.Methods　A one year, follow-up, longitudinal internet survey of the general Japanese population was conducted from 2018 to 2019 (Japan Society and New Tobacco Internet Survey, JASTIS). DID analysis was conducted using the percentage of residents who have smoke-free household rules in Tokyo metropolitan area and control groups in 2018 and 2019.Results　We utilized three control groups (Control 1: residents in Japan except from Tokyo metropolitan area; Control 2: residents in Japan except from Kanto region; Control 3: residents in prefectures which have ordinance-designated city) for DID analysis. Covariate-adjusted DID estimates for each control group were -1.0 percentage points (Control 1, 95% Confidence Interval (CI)=-5.8, 3.9), -1.0 percentage points (Control 2, 95% CI=-5.9, 4.0), and -1.0 percentage points (Control 3, 95% CI=-5.9, 3.9) indicating that there was no significant difference for all control groups. Moreover, no significant difference was observed when respondents' answers were analyzed and stratified according to sex, age, household income, housing tenure, smoking status, education, or marital status.Conclusions　DID analysis revealed no significant change in the percentage of residents of the Tokyo metropolitan area who have smoke-free household rules after the implementation of the ordinance. This study will be useful for local governments when planning and promoting more effective smoke-free ordinances.

Self-assembling DNA hydrogel-based delivery of immunoinhibitory nucleic acids to immune cells.

Immunoinhibitory oligodeoxynucleotides (INH-ODNs) are promising inhibitors of Toll-like receptor 9 (TLR9) activation. To efficiently deliver INH-ODNs to TLR9-positive cells, we designed a Takumi-shaped DNA (Takumi) consisting of two partially complementary ODNs as the main component of a DNA hydrogel. Polyacrylamide gel electrophoresis showed that Takumi-containing INH-ODNs (iTakumi) and iTakumi-based DNA hydrogel (iTakumiGel) were successfully generated. Their activity was examined in murine macrophage-like RAW264.7 cells and DC2.4 dendritic cells by measuring tumor necrosis factor-α and interleukin-6 release after the addition of a TLR9 ligand (CpG ODN). Cytokine release was efficiently inhibited by the iTakumiGel. Flow cytometry analysis and confocal microscopy showed that cellular uptake of INH-ODN was greatly increased by the iTakumiGel. These results indicate that a Takumi-based DNA hydrogel is useful for the delivery of INH-ODNs to immune cells to inhibit TLR9-mediated hyperinduction of proinflammatory cytokines. From the Clinical Editor: Toll-like receptor 9 activation has been reported to be associated with many autoimmune diseases. DNA inhibition using oligodeoxynucleotides is one of the potential treatments. In this article, the authors described hydrogel-based platform for the delivery of the inhibitory oligodeoxynucleotides for enhanced efficacy. The positive findings could indicate a way for the future.

Ebp2 and Brx1 function cooperatively in 60S ribosomal subunit assembly in Saccharomyces cerevisiae.

The yeast protein Ebp2 is required for early steps in production of 60S ribosomal subunits. To search for cofactors with which Ebp2 functions, or substrates on which it acts, we screened for mutants that were synthetically lethal (sl) with the ebp2-14 mutation. Four different mutant alleles of the 60S ribosomal subunit assembly factor Brx1 were found. To investigate defects of the double mutant, we constructed strains conditional for the ebp2-14 brx1- synthetic lethal phenotype. These ebp2-14 brx1 mutants were defective in processing of 27S pre-rRNA and production of 60S subunits, under conditions where each single mutant was not. Ebp2 and Brx1 exhibit a strong two-hybrid interaction, which is eliminated by some combinations of brx1 and ebp2 mutations. In one such mutant, Ebp2 and Brx1 can still associate with pre-ribosomes, but subunit maturation is perturbed. Depletion of either Ebp2 or Brx1 revealed that Brx1 requires Ebp2 for its stable association with pre-ribosomes, but Ebp2 does not depend on the presence of Brx1 to enter pre-ribosomes. These results suggest that assembly of 60S ribosomal subunits requires cooperation of Ebp2 with Brx1, together with other molecules present in pre-ribosomes, potentially including several found in assembly subcomplexes with Brx1 and Ebp2.

Efficient delivery of immunostimulatory DNA to mouse and human immune cells through the construction of polypod-like structured DNA.

Investigation of mouse macrophage-like RAW264.7 cells showed that the immunostimulatory activity of CpG DNA is increased by formation of polypod-like structured DNA (polypodna), an assembly consisting of three or more oligodeoxynucleotides. To apply CpG polypodna to immunotherapy, its activity was examined in murine dendritic DC2.4 cells, splenic macrophages, and bone marrow-derived dendritic cells (BMDCs). In all cell types, increasing the pod number increased the cellular uptake of DNA and cytokine release. No significant release of cytokines was observed in macrophages lacking Toll-like receptor 9. Similar results were obtained after intradermal injection of polypodna. The polypodna preparations produced significantly higher amounts of interferon α in human peripheral blood mononuclear cells (PBMCs) compared with single-stranded DNA. The conditioned medium of hexapodna-treated human PBMCs effectively inhibited the activity of a hepatitis C virus subgenomic replicon reporter system. These results indicate that polypodna preparations are useful as an immunostimulator. FROM THE CLINICAL EDITOR: This study demonstrates the utility of polypoid-like structured DNA (polypodna) preparations as potent immunostimulators in a murine model.

Tyrosine Kinase Inhibitor-induced Cerebrovascular Occlusion Presenting with Moyamoya Disease-like Stenosis of the Circle of Willis.

Vascular occlusive events are notable adverse effects of tyrosine kinase inhibitors (TKIs), which are promising treatments for chronic myeloid leukemia (CML). We herein report the case of a patient with CML who developed cerebrovascular occlusion of the circle of Willis during TKI treatment. Our patient did not meet the diagnostic criteria for moyamoya disease due to the insignificant development of moyamoya vessels. The lack of moyamoya vessel development may be explained by the suppression of tyrosine kinases that are responsible for angiogenesis. Cerebrovascular occlusion of the circle of Willis, without significant development of moyamoya vessels, may be an important phenotype of TKI-associated vasculopathy.

Multilineage Lymphoblastic Lymphoma as an Initial Presentation of Mixed Phenotype Acute Leukemia.

Mixed phenotype acute leukemia (MPAL) is characterized by leukemic blasts that express markers of multiple lineages. Compared with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), MPAL is considered to have a poor treatment outcome. We report a case of MPAL T/myeloid not otherwise specified that was initially presented as multilineage lymphoblastic lymphoma and subsequently developed into leukemic MPAL. An acute lymphoblastic leukemia-based treatment regimen was ineffective, but azacitidine and venetoclax therapy resulted in hematological complete remission. Our case suggests that multilineage lymphoblastic lymphoma should be considered to be the same disease as MPAL, albeit with different clinical presentations. Optimal treatment for MPAL has not been established yet, but azacitidine and venetoclax therapy may be a potential approach.

Combined encapsulation of a tumor antigen and immune cells using a self-assembling immunostimulatory DNA hydrogel to enhance antigen-specific tumor immunity.

Our previous study demonstrated that the incorporation of a tumor antigen into a self-assembling DNA hydrogel, comprised of a DNA containing un-methylated cytosine-phosphate-guanine (CpG) dinucleotides (CpG DNA), efficiently induced antigen-specific tumor immunity after intra-tumoral injection into tumor-bearing mice. We hypothesized that the additional incorporation of immune cells, the target for the antigen and immunostimulatory CpG DNA, would increase the antitumor response. To prove this, immune cells were also encapsulated into the CpG DNA hydrogel and delivered along with the antigen. Mouse dendritic DC2.4 cells maintained their form even after incorporation into the DNA hydrogel. The incorporation of mouse macrophage-like J774.1 cells and RAW264.7 cells into CpG DNA hydrogel did not significantly affect their viability. J774.1, RAW264.7, DC2.4, and mouse bone marrow-derived dendritic cells (BMDCs) were efficiently activated when incorporated into the CpG DNA hydrogel. The CpG DNA hydrogel incorporated with both the tumor antigen and BMDCs effectively induced antigen-specific immune responses, and retarded tumor growth following intradermal administration before and after tumor inoculation without severe local and systemic adverse events. These data indicate that the combined delivery of a tumor antigen and immune cells using an immunostimulatory CpG DNA hydrogel is effective in inducing antigen-specific antitumor immunity.

Retardation of Antigen Release from DNA Hydrogel Using Cholesterol-Modified DNA for Increased Antigen-Specific Immune Response.

Our previous study indicates that cationization of an antigen is effective for sustained release of both immunostimulatory DNA containing unmethylated cytosine-phosphate-guanine (CpG) dinucleotides, or CpG DNA, and antigen from a DNA hydrogel. Another approach to sustained antigen release would increase the applicability and versatility of the system. In this study, a hydrophobic interaction-based sustained release system of ovalbumin (OVA), a model antigen, from immunostimulatory CpG DNA hydrogel is developed by the use of cholesterol-modified DNA and urea-denatured OVA (udOVA). Cholesterol-modified DNA forms a hydrogel, Dgel(chol), and induces IL-6 mRNA expression in mouse skin after intradermal injection, as DNA without cholesterol does. Cholesterol-modified DNA associated with OVA and denaturation of OVA using urea increases the interaction. The release of udOVA from Dgel(chol) is significantly slower than that from DNA hydrogel with no cholesterol, Dgel. Moreover, intratumoral injections of udOVA/Dgel(chol) significantly inhibit the growth of EG7-OVA tumors in mice. These results indicate that sustained release of antigen from Dgel can be achieved by the combination of urea denaturation and cholesterol modification, and retardation of antigen release is effective to induce antigen-specific cancer immunity.

Improved sustained release of antigen from immunostimulatory DNA hydrogel by electrostatic interaction with chitosan.

Immunostimulatory DNA hydrogel (sDNA hydrogel) containing unmethylated cytosine-phosphate-guanine (CpG) sequences has been demonstrated to be a useful antigen delivery system, which can effectively induce an antigen-specific immune response through stimulation of the innate immune system. However, relatively rapid release of antigens from the sDNA hydrogel limits its potential. To enhance the potency of the sDNA hydrogel via improvement of its sustained release property, we selected chitosan, a biocompatible cationic polymer which electrostatically interacts with DNA, and mixed it with the sDNA hydrogel. Compared to unmixed sDNA hydrogel, sDNA hydrogel mixed with chitosan (Chitosan-sDNA hydrogel) was more stable, tougher, had more bound water, released a model antigen ovalbumin (OVA) more slowly in vitro, and provided longer retention of OVA at the injection site after intradermal injection into mice. Intradermal immunization of mice with the OVA-loaded Chitosan-sDNA hydrogel resulted in the induction of a higher level of OVA-specific IgG in serum compared with OVA-loaded sDNA hydrogel with no chitosan. These results indicate that the Chitosan-sDNA hydrogel is an improved sustained release formulation for efficient induction of antigen-specific immune responses.

DNA nanotechnology-based composite-type gold nanoparticle-immunostimulatory DNA hydrogel for tumor photothermal immunotherapy.

Success of tumor photothermal immunotherapy requires a system that induces heat stress in cancer cells and enhances strong anti-tumor immune responses. Here, we designed a composite-type immunostimulatory DNA hydrogel consisting of a hexapod-like structured DNA (hexapodna) with CpG sequences and gold nanoparticles. Mixing of the properly designed hexapodna and oligodeoxynucleotide-modified gold nanoparticles resulted in the formation of composite-type gold nanoparticle-DNA hydrogels. Laser irradiation of the hydrogel resulted in the release of hexapodna, which efficiently stimulated immune cells to release proinflammatory cytokines. Then, EG7-OVA tumor-bearing mice received an intratumoral injection of a gold nanoparticle-DNA hydrogel, followed by laser irradiation at 780 nm. This treatment increased the local temperature and the mRNA expression of heat shock protein 70 in the tumor tissue, increased tumor-associated antigen-specific IgG levels in the serum, and induced tumor-associated antigen-specific interferon-γ production from splenocytes. Moreover, the treatment significantly retarded the tumor growth and extended the survival of the tumor-bearing mice.

Nasal delivery of Japanese cedar pollen Cryj1 by using self-gelling immunostimulatory DNA for effective induction of immune responses in mice.

To develop an immunotherapeutic vaccine for treatment of allergic rhinitis, we developed a controlled release formulation of Cryj1, a major Japanese cedar pollen allergen, with immunostimulatory potency. Two sets of hexapod-like structured DNA (hexapodna) were prepared using six oligodeoxynucleotides (ODNs) each, including ODNs with an unmethylated cytosine-phosphate-guanine (CpG) sequence (CpG motif), to obtain an immunostimulatory DNA hydrogel (sDNA hydrogel). A non-immunostimulatory DNA hydrogel (nsDNA hydrogel) was also prepared using ODNs with no CpG motifs. The sDNA hydrogel was more effective than its components or the nsDNA hydrogel for production of interleukin (IL)-12 after addition to murine macrophage-like RAW264.7 cells or after intranasal administration to mice. Then, a Cryj1-loaded sDNA hydrogel (Cryj1/sDNA hydrogel) formulation was prepared by mixing solutions containing both Cryj1 and hexapodna. Cryj1 was slowly released from the sDNA hydrogel in phosphate-buffed saline. After intranasal administration of the fluorescein isothiocyanate (FITC)-labeled Cryj1/sDNA hydrogel in mice, FITC-Cryj1 was retained in the nasal cavity for a longer period than FITC-Cryj1 mixed with hexapodna in solution. Intranasal immunization of mice with the Cryj1/sDNA hydrogel resulted in high levels of Cryj1-specific IgG in nasal lavage fluid (NFL), IL-12 and interferon-γ release from spleen cells after re-stimulation with Cryj1 when compared with intranasal immunization with the other formulations examined. These results indicate that the self-gelling immunostimulatory DNA hydrogel is an effective formulation for controlled induction of allergen-specific immune responses.

Injectable, self-gelling, biodegradable, and immunomodulatory DNA hydrogel for antigen delivery.

DNA nanotechnology-based nanosystems and macrosystems have attracted much attention in the biomedical research field. The nature of DNA endows these systems with biodegradable, biocompatible, and immunomodulatory properties. Here, we present an injectable hydrogel system that consists only of chemically synthesized short DNA strands, water, and salts. Several preparations of polypod-like structured DNA, or polypodna, were designed, including tri-, tetra-, penta- and hexapodna, as the building blocks of self-gelling DNA hydrogel. Under physiological conditions, properly designed polypodna preparations formed a hydrogel. The analysis of the modulus data of the hydrogel consisting of two sets of hexapodna preparations showed that this injectable hydrogel was reorganized at a time scale of 0.25s. Then, DNA hydrogel containing unmethylated cytosine-phosphate-guanine (CpG) dinucleotides was used to stimulate innate immunity through Toll-like receptor 9, the receptor for CpG DNA. Gel formation significantly increased the activity of immunostimulatory CpG DNA, retarded the clearance after intradermal injection into mice, and increased the immune responses to ovalbumin (OVA) incorporated into the hydrogel as a model antigen. OVA/CpG DNA hydrogel induced much less local or systemic adverse reactions than OVA injected with complete Freund's adjuvant or alum. GpC DNA hydrogel containing no CpG sequences was less effective, indicating the importance of immunomodulation by CpG DNA hydrogel. Thus, we have created an efficient system for sustained delivery of antigens or other bioactive compounds.