Identification of Breast Cancer Stem Cells Using a Newly Developed Long-acting Fluorescence Probe, C5S-A, Targeting ALDH1A1.

BACKGROUND/AIM: Aldehyde dehydrogenase (ALDH) 1A1 is a well-known marker for cancer stem cells (CSCs), characterized by self-renewal capacity and multidrug resistance in breast cancer. We developed a near-infrared turn-on fluorescence probe for ALDH1A1, C5S-A, which is suitable for observing and analyzing viable cells. Here, we demonstrated the utility of C5S-A in CSC research using breast cancer cell lines. MATERIALS AND METHODS: To evaluate concordance between C5S-A and conventional stem cell markers, breast cancer cells sorted for ALDEFLUOR-positive cells and for CD44+/CD24- cell populations were stained with C5S-A. Tumorigenicity of C5S-A-positive cells was examined by mammosphere formation assay and subcutaneous transplantation to immunodeficient mice. Additionally, to determine how long fluorescence from a single staining remained observable, we cultured breast cancer cells for 5 days after C5S-A staining. We then evaluated whether C5S-A-positive cells possessed resistance to cytotoxic drugs by chronological imaging. RESULTS: C5S-A staining showed good concordance with conventional breast CSC markers, and good utility for research into CSC characteristics in breast cancer cell lines, including tumorigenesis. Additionally, C5S-A was observable for more than 3 days with a single staining. Using this property, we then confirmed that C5S-A-positive cells possessed resistance to cytotoxic drugs, which is one of the characteristics of CSCs. CONCLUSION: We showed that C5S-A is suitable for CSC research using breast cancer cell lines, and confirmed its utility in observing cells over time.

Safety and immunogenicity of the COVID-19 vaccine BNT162b2 in patients undergoing chemotherapy for solid cancer.

BACKGROUND: Although COVID-19 severity in cancer patients is high, the safety and immunogenicity of the BNT162b2 mRNA COVID-19 vaccine in patients undergoing chemotherapy for solid cancers in Japan have not been reported. METHODS: We investigated the safety and immunogenicity of BNT162b2 in 41 patients undergoing chemotherapy for solid cancers and in healthy volunteers who received 2 doses of BNT162b2. We evaluated serum IgG antibody titers for S1 protein by ELISA at pre-vaccination, prior to the second dose and 14 days after the second vaccination in 24 cancer patients undergoing cytotoxic chemotherapy (CC group), 17 cancer patients undergoing immune checkpoint inhibitor therapy (ICI group) and 12 age-matched healthy volunteers (HV group). Additionally, inflammatory cytokine levels were compared between the HV and ICI groups at pre and the next day of each vaccination. RESULTS: Anti-S1 antibody levels were significantly lower in the ICI and CC groups than in the HV group after the second dose (median optimal density: 0.241 [0.063-1.205] and 0.161 [0.07-0.857] vs 0.644 [0.259-1.498], p = 0.0024 and p < 0.0001, respectively). Adverse effect profile did not differ among the three groups, and no serious adverse event occurred. There were no differences in vaccine-induced inflammatory cytokines between the HV and ICI groups. CONCLUSION: Although there were no significant differences in adverse events in three groups, antibody titers were significantly lower in the ICI and CC groups than in the HV group. Further protection strategies should be considered in cancer patients undergoing CC or ICI.

Microsatellite instability-high colorectal cancer patient-derived xenograft models for cancer immunity research.

CONTEXT: There is an increasing demand for appropriate preclinical mice models for evaluating the efficacy of cancer immunotherapies. AIMS: Therefore, we established a humanized patient-derived xenograft (PDX) model using microsatellite instability-high (MSI-H) colorectal cancer (CRC) tissues and patient-derived peripheral blood mononuclear cells (PBMCs). SUBJECTS AND METHODS: The CRC tissues of patients scheduled for surgery were tested for MSI status, and CRC tumors were transplanted into NOD/LtSz-scid/IL-2Rg-/-(NSG) mice to establish MSI-H PDX models. PDX tumors were compared to the original patient tumors in terms of histological and genetic characteristics. To humanize the immune system of MSI-H PDX models, patient PBMCs were injected through the tail vein. RESULTS: PDX models were established from two patients with MSI-H CRC; one patient had a germline mutation in MLH1 (c.1990-2A > G), and the other patient had MLH1 promoter hypermethylation. PDX with the germline mutation was histologically similar to the patient tumor, and retained the genetic characteristics, including MSI-H, deficient mismatch repair (dMMR), and MLH1 mutation. In contrast, the histological features of the other PDX from a tumor with MLH1 promoter hypermethylation were clearly different from those of the original tumor, and MLH1 promoter hypermethylation and MSI-H/dMMR were lost in the PDX. When T cells from the same patient with MLH1 mutation were injected into the PDX through the tail vein, they were detected in the PDX tumor. CONCLUSIONS: The MSI-H tumor with an MMR mutation is suitable for MSI-H PDX model generation. The PBMC humanized MSI-H PDX has the potential to be used as an efficient model for cancer immunotherapy research.

Serum Soluble Interleukin-2 Receptor as a Potential Biomarker for Immune-related Adverse Events.

BACKGROUND/AIM: Biomarkers for immune-related adverse events (irAEs) induced by immune checkpoint inhibitors (ICIs) are required. We encountered a patient whose skin irAE fluctuated in parallel with serum soluble interleukin-2 receptor (sIL-2R). PATIENTS AND METHODS: We examined 15 patients with cancer who received ICIs. Serum sIL-2R levels before and during ICI treatment were measured. The sIL-2R levels of preserved serum samples from another five patients who developed grade 3 irAEs were measured. RESULTS: Twelve patients showed no significant changes in sIL-2R levels during ICI treatment. Baseline serum sIL-2R levels in three patients increased beyond the normal range before the second cycle. These three patients had grade ≥2 irAEs at the second cycle treatment visit, supporting our hypothesis. Furthermore, at diagnosis of irAEs, the sIL-2R levels of all preserved samples from patients with grade 3 irAEs were significantly elevated. CONCLUSION: Serum sIL-2R is a promising biomarker for the diagnosis of irAEs.

The effects of interleukin-6 signal blockade on immune system, reproductive and skeletal development in juvenile mice.

Interleukin-6 (IL-6) is involved in the pathogenesis of multiple disorders, including juvenile autoimmune diseases. IL-6 participates in a broad spectrum of physiological events, and the IL-6 receptor (IL-6R) is widely distributed across multiple organs. The interrelationship of development phases in juveniles together with organs involved in IL-6 signaling called for evaluations of anti-IL-6R antibody induced effects in a juvenile mouse model to assess the safety of such an approach in human juvenile arthritis. Here we show that naive mice in which IL-6 signals have been transiently blocked during the juvenile period develop normally. The fatal immunogenic reactions recorded earlier by repeated administration of the chosen rat anti-mouse IL-6R antibody, MR16-1, to mice were avoided successfully by application of a high loading dose followed by lower maintenance doses, with the support of modeling data. The high loading-dose regimen enabled us to conduct assessments without any major interference due to immunogenicity. Transient and complete inhibition of IL-6 signals from postnatal days 22 to 79 in mice exhibited no biologically important changes in sexual maturation or development of immune and skeletal systems. Although tendencies toward reductions of peripheral blood T-cell counts were observed, normal levels of antigen-specific IgG/IgM antibody productions indicating sufficient immunological functions were confirmed. Our results demonstrate that blockage of IL-6R by the neutralizing antibody does not affect juvenile development. This may be in part due to the generation or existence of compensatory pathways in the whole body system.

The effects of interleukin-6 signal blockade on fertility, embryo-fetal development, and immunization in vivo.

Possible effects of interleukin-6 (IL-6) on reproductive performance, embryonal development, parturition, and postnatal development have been suggested based on protein/mRNA expression level of IL-6 in related organs, but less is known about functions of IL-6 signals in these areas. Following two different approaches have been employed to investigate the role of IL-6 signals in fertility and pre-/postnatal development: administration of a rat anti-mouse IL-6 receptor antibody, MR16-1, to mice as a neutralizing antibody system, and B6.129S2-Il6(tm1Kopf)/J (IL-6 knockout [KO]) mice as a KO system. By intravenously dosing 50 mg/kg of MR16-1 every 3 days, animals in male and female fertility studies and dams in a pre-/postnatal development study exhibited plasma MR16-1 concentrations much higher than the effective plasma concentration, indicating that MR16-1 exposure was sufficient to completely block IL-6 signals. The concentration of MR16-1 in the plasma of fetuses exceeded that in the plasma of pregnant animals, and MR16-1 concentration in milk was about one-fourth of that in plasma. Both the transient IL-6 signal blockade by MR16-1, and the constitutive IL-6 signal inhibition using IL-6 KO mice in a combined fertility and pre-/postnatal development study, revealed no biologically important effects on fertility, early embryonic development to implantation, or pre-/postnatal development, including IgG/IgM production by keyhole limpet hemocyanin sensitization. These results indicate that IL-6 signals have no unique, noncompensable roles in reproduction and development in the whole body system, although contributions of IL-6 in the signaling network appear to exist, as suggested by previously published investigations.

Collaborative work on evaluation of ovarian toxicity. 10) Two- or four-week repeated dose studies and fertility study of di-(2-ethylhexyl) phthalate (DEHP) in female rats.

The main purpose of this collaborative work is to determine the optimal administration period required to detect toxic effects in evaluation of ovarian morphological changes in repeated-dose toxicity studies. To assess the morphological and functional changes induced in the ovaries by di-(2-ethylhexyl) phthalate (DEHP), two repeated-dose toxicity studies (repeated dose for 2 or 4 weeks) of DEHP administrated to female rats at dose levels of 0, 300, 1,000 and 3,000 mg/kg were conducted in collaboration with a female fertility study at the same dosages from 2 weeks prior to mating to Day 7 of pregnancy. Histopathology of the ovaries in both repeated-dose toxicity studies showed vacuolation of stromal cells in the groups receiving 300 mg/kg or more and an increase of large atretic follicles in groups at 1,000 mg/kg or more. In the 4-week study, a decrease in new corpora lutea was observed in the 3,000 mg/kg group. In the female fertility study, the 3,000 mg/kg group showed prolongation of the mean estrous cycle and irregular estrous cycles. Cesarean section revealed a decrease of pregnancy rate in the 3,000 mg/kg group. No effects on fertility or early embryonic development were found in groups at 1,000 mg/kg or less. These findings indicate that histopathological changes in the ovary are important endpoints for the evaluation of drugs which induce ovarian damage. In conclusion, for a repeated-dose toxicity study, a 2-week administration period is sufficient to detect ovarian toxicity caused by DEHP.

Application of an indwelling vascular access port for intravenous administration in a repeated and intermittent dose toxicity study in rats.

Totally implantable catheter animal models are considered useful for pharmacological and toxicological studies. In this report, we assessed the feasibility of using an indwelling vascular access port (VAP) in rats for long-term evaluation of repeated and intermittent dose toxicity studies. In Experiment 1, the VAP devices were implanted in male and female rats and a saline solution administered intravenously via the posterior vena cava for 2 weeks (4 ml/kg, 2 ml/min, 5 times/week, 10 times total). General conditions, body weight and blood chemistry showed no toxicological changes compared with the rats in the non-implanted, non-treated group. Hematology changes such as transient increases in peripheral blood reticulocytes and eosinophils were noted post-implantation. In pathology, proliferation of the endothelium at the site of VAP implantation and perivascular inflammatory cell infiltration including eosinophils in lung were noted at the end of the treatment period. Moreover, we found that the lumbar area is more suitable for VAP implantation than the back of neck for young, still growing rats. Experiment 2 included a 1-month intravenous intermittent dose (4 ml/kg, 2 ml/min, 1 time/week, 5 times total) toxicity study in VAP-implanted rats followed by a 1-month recovery period conducted under Good Laboratory Practice (GLP) regulations. The results suggested that an animal model with implanted VAP is useful for intermittent intravenous dosing of drugs. Moreover, VAP implantation in animals is expected to be extrapolated to use VAP in humans in clinical studies.

Contribution of intestinal calcium absorption to 1,25-dihydroxyvitamin D3-induced calcium action in the total parenteral nutrition rat.

To investigate the contribution of intestinal calcium (Ca) absorption to 1,25-dihydroxyvitamin D(3) (1,25(OH) (2)D(3))-induced Ca action, we assessed parameters related to Ca metabolism after a single dosing of 1,25(OH)(2)D (3) in the total parenteral nutrition (TPN) solution or 5% D-mannitol (MAN) solution treatment with rats. Animals were divided into 6 groups (vehicle, 100 microg/kg p.o. and 25 microg/kg i.v.; n=8) in Experiment 1 and 8 groups (vehicle, 1, 10 and 100 microg/kg p.o.; n=6) in Experiment 2 at TPN or MAN solution treatment. In both experiments, the parameters related to Ca metabolisms, urinary Ca and urinary deoxypyridinoline on 0-24 hr or serum Ca, osteocalcin and parathyroid hormone at 24 hr were measured after 1,25(OH)(2)D (3) dosing. 1,25(OH)(2)D(3)-related increased urinary Ca or serum Ca were observed in both experiments. Decrease rates in change of urinary Ca in TPN solution treatment rats were 36.3% (100 microg/kg p.o.) or 47.1% (25 microg/kg i.v.) of MAN solution treatment rats in Experiment 1, and 29.0% (1 microg/kg), 56.2% (10 microg/kg) or 35.3% (100 microg/kg) of MAN solution treatment rats in Experiment 2. Decrease rates in change of serum Ca at 72 hr in TPN solution treatment rats were 57.3% (100 microg/kg p.o.) or 44.5% (25 microg/kg i.v.) of MAN solution treatment rats in Experiment 1, and were 57.0% (100 microg/kg) of MAN solution treatment rats in Experiment 2. There were no differences in the change of serum Ca in the 1,25(OH)(2)D(3) 1 or 10-microg/kg group in Experiment 2. Our results suggest that differences in urinary Ca or serum Ca between MAN solution treatment rats and TPN solution treatment rats express the contribution of intestinal Ca absorption to 1,25(OH)(2)D(3)-induced Ca action in the conditions of the study.

Total parenteral nutrition using continuous intravenous infusion via the posterior vena cava in rats.

This study was conducted to evaluate the feasibility of total parenteral nutrition (TPN) in rats using continuous intravenous infusion (tail cuff method) via the posterior vena cava. A catheter was inserted into the posterior vena cava from the femoral vein of 10 females (Experiment 1) and 16 females (Experiment 2). The depth of the inserted catheter from the femoral vein was set at 4.5 cm for Experiment 1 and was set at 6 cm for Experiment 2. The test animals were divided into two groups in each experiment: a 5% D-Mannitol (MAN) group and a TPN group. In Experiment 1, TPN rats showed macroscopic lesions (edema in peritoneum, increased collateral vasculature, induration in perivenous tissue, and thrombus) at the tip of the catheter. The diameter of the posterior vena cava (2.86 +/- 0.16 mm, mean +/- S.D.) was significantly greater than that of the anterior vena cava (2.45 +/- 0.22 mm) in 10 rats of Experiment 1. In Experiment 2, TPN rats showed no abnormalities at necropsy. Our findings suggest that TPN administered via the posterior vena cava in Sprague-Dawley rats requires the catheter to be inserted to a depth of 6 cm from the femoral vein. We hypothesize that this is because it is inserted to the level of the renal vein branch where the diameter of the posterior vena cava may be greatest.