Recent evidence suggests that the targeting of membrane transporters specifically activated in cancer stem cells (CSCs) is an important strategy for cancer therapy. The objectives of the present study were to investigate the ion channel expression profiles in digestive CSCs. Cells strongly expressing CSC markers, such as ALDH1A1 and CD44, were separated from the human esophageal squamous cell carcinoma, gastric cancer, and pancreatic cancer cell lines using fluorescence-activated cell sorting, and CSCs were identified based on tumorsphere formation. Messenger RNA levels of CSC markers were higher in CSCs than in non-CSCs. These CSCs also exhibited resistance to anticancer agents. The microarray analysis revealed that the expression of transient receptor potential vanilloid 2 (TRPV2), voltage-gated calcium channels (VGCCs), and voltage-gated potassium channels (VGKCs) were upregulated in esophageal, gastric, and pancreatic CSCs, respectively, compared with non-CSCs. The TRPV2 inhibitor tranilast, VGCCs inhibitors amlodipine and verapamil, and VGKC inhibitor 4-aminopyridine exhibited greater cytotoxicity in CSCs compared with non-CSCs, and their inhibitory effects were also confirmed in a xenograft model in nude mice. Taking these results, phase I/II study to investigate clinical safety and efficacy of neoadjuvant combination chemotherapy of tranilast in advanced esophageal squamous cell carcinoma (TNAC study) is ongoing. These researches identified a role of ion channels in the persistence of CSCs and suggested that their inhibitors may have potential as a therapeutic agent for digestive cancers.

We have developed autologous NKT cell-targeted immunotherapy for lung cancer and head and neck cancer at Chiba University. We induce α-galactosylceramide(αGalCer)-pulsed antigen-presenting cells(APCs)from patients' peripheral blood mononuclear cells(PBMCs)in vitro and give them back to the patients. We transferred them intravenously to patients with lung cancer and demonstrated the potential to improve survival time. For patients with head and neck cancer, we transferred them via the nasal submucosa with ex vivo expanded autologous NKT cells. We demonstrated an increased response rate compared with αGalCer-pulsed APCs alone. This suggested that combination therapy of αGalCer-pulsed APCs and NKT cells can increase the response rate. However, NKT cells circulate at less than 0.1% in human PBMCs. Producing enough autologous NKT cells for adoptive immunotherapy is tough. Furthermore, the immunologic function of patient-derived NKT cells can vary among patients. Because stable cell production in number and nature is essential to show effective treatment results, the development of NKT cell-targeted immunotherapy is moving forward using allogeneic NKT cells worldwide. In this circumstance, we, RIKEN and Chiba University, have been developing allogeneic induced pluripotent stem cell(iPS cell)- derived NKT cell therapy. The phase Ⅰ clinical trial of iPS cell-derived NKT cell therapy for head and neck cancer is ongoing.

The COVID-19 pandemic has cast a shadow over transfusion medicine based on the blood donation system. However, managing alloimmune platelet transfusion refractoriness (allo-PTR) has already been difficult. As a first step toward resolving this issue using induced pluripotent stem cell-derived platelet products (iPSC-PLTs), a clinical trial of autologous products (iPLAT1) was conducted in a patient with allo-PTR caused by anti-HPA-1a antibodies who had no compatible donor, and safety was confirmed. To produce iPSC-PLTs, a master cell bank (MCB) of expandable megakaryocyte lines (imMKCLs) is established from iPSCs. From this MCB, iPSC-PLTs are manufactured using a newly developed turbulent-type bioreactor and various compounds. Their quality, safety, and efficacy are confirmed by extensive preclinical studies. Based on the findings of the iPLAT1 study, a clinical trial of allo-transfusion of HLA homozygous iPSC-PLTs is currently ongoing and HLA class I-deficient O-type universal iPSC-PLTs are also being developed. iPSC-PLTs are expected to solve various problems, including allo-PTR in platelet transfusion, and greatly contribute to the advancement of transfusion medicine.

Our laboratory previously established spinal motor neurons (MN) from induced-pluripotent stem cells (iPSCs) prepared from both sporadic and familial ALS patients, and successfully recapitulated disease-specific pathophysiological processes. We next searched for effective drugs capable of slowing the progression of ALS using a drug library of 1232 existing compounds and discovered that ropinirole hydrochloride prevented MN death. In December 2018, we started an investigator-initiated clinical trial testing ropinirole hydrochloride extended-release tablets in ALS patients. This is an on-going phase I/IIa randomized, double-blind, placebo-controlled, single-center, open-label continuation clinical trial (UMIN000034954). The primary aim is to assess the safety and tolerability of ropinirole hydrochloride in patients with ALS. We will also perform an efficacy evaluation using patient-derived iPSCs/MN. Major inclusion criteria were as follows: 1) 'clinically possible and laboratory-supported ALS', 'clinically probable ALS' or 'clinically definite ALS', according to the criteria for the diagnosis of ALS (El Escorial revised) and within 60 months after disease onset; 2) change in ALSFRS-R score of -2 to -5 points during the 12-week run-in period. Finally, 15 patients will be assigned to the active drug and 5 patients to the placebo. Our trial will be a touchstone trial for iPSC-based drug development strategies.

We performed drug screening using motor neurons derived from disease-specific induced pluripotent stem cells (iPSCs) for amyotrophic lateral sclerosis (ALS) and found that ropinirole hydrochloride prevented motor neuron death. We have started a randomized clinical trial testing ropinirole hydrochloride in ALS patients in December 2018. This is a phase I/IIa randomized, double-blind, placebo-controlled, single-center, open-label continuation clinical trial. The primary aim is to assess the safety and tolerability of ropinirole hydrochloride in patients with ALS. Secondary aims include the following effectiveness evaluations: ALSFRS-R, quantitative muscle strength by a hand-held dynamometer, muscle volume by CT scan, forced vital capacity, physical activity by an activity tracker, survival, ALSAQ40 scale, and a Zarit Caregiver Burden Interview. Moreover, we will perform an efficacy evaluation using subjects-derived iPSCs/motor neurons and assess plasma/CSF biomarkers (TDP-43, and ALS-related RNA/micro RNA) as exploratory research questions. Ropinirole hydrochloride potentially targets multiple mechanisms of ALS pathology (i.e., oxidative stress, mitochondrial dysfunction, and abnormal aggregation of TDP-43/FUS protein, which is representative of the ALS phenotype), with promising preclinical study results based on iPSC research. The availability of the drug suggests that rapid translation to daily clinical use might be possible. Our trial will provide reliable and important data for further potential trials. The results will appear in March 2021.

Sequential high-dose chemotherapy with in vivo rituximab-purged stem cell autografting was designed for previously untreated mantle cell lymphoma (MCL). The response rate, disease-free survival (DFS), overall survival (OS) and toxicity were investigated in this trial. Between November 2001 and August 2008, five patients younger than 65 years of age with MCL at diagnosis were enrolled in this study. Initial chemotherapy consisted of 3 cycles of CHOP regimen followed by four courses of high-dose chemotherapy. During the in vivo purging phase, the patient was administered high-dose cyclophosphamide and cytarabine, and then each administration was followed by two infusions of rituximab. Molecular monitoring of minimal residual disease was performed by assessing DNA samples from bone marrow and autografted cells using PCR amplification of the bcl-1/IgH rearrangement. The complete response rate was 100%, and the 3-year OS and DFS were 100% and 100%, respectively. PCR analysis of autografted cells from four evaluable patients, 75% lymphoma-negative harvests were achieved following in vivo purging. One patient relapsed 3.2 years after treatment. The principal toxicity in the study was hematologic but there were no treatment-related deaths. Intensive high-dose sequential chemotherapy with in vivo purged stem cell support can achieve long-term disease-free survival for MCL.

A feasibility study on high-dose therapy with autologous peripheral blood stem cell transplantation (HDT/PBSCT) was performed in Japanese patients with multiple myeloma (MM). Twenty evaluable patients younger than 65 years old with stage II/III MM were enrolled in this study. Three courses of VAD were used as initial chemotherapy. High-dose etoposide or cyclophosphamide followed by G-CSF was used for PBSCH, and 1.2-89.3 (median 23.4) x 106/kg of CD34+ cells were collected. Single (11 patients) or tandem (9 patients) HDT with melphalan (MEL) 200 mg/m2 or MEL 140 mg/m2 plus TBI 10 Gy were performed. The incidence of grade 4 toxicity (COG) was 10% and treatment-related mortality was 5%. Complete response and tumor reduction of more than 75% were obtained in 4 (21%) and 16 (84%) out of 19 patients, respectively. The actuarial 3-year overall survival (OS) and event-free survival (EFS) after PBSCT/HDT were 65.6% and 22.0%, respectively. The median EFS duration was 18 months. These preliminary results indicated that HDT/PBSCT is feasible for Japanese MM patients. A prospective randomized clinical trial will be required to assess the efficacy.

The aim of this study was to evaluate the phase I aspects of super high-dose chemotherapy for advanced liver cancer with combined use of PIHP and PBSCT. In 4 patients with HCC and 1 patient with colonic liver metastases, peripheral blood stem cells (PBSC) were mobilized by either i.v. infusion of ETP (180 mg/m2/day for 3 days) or i.a. infusion of doxorubicin (100-120 mg/m2). In 3 (60%) of 5 patients, PBSC for PBSCT were harvested in effective quantities (CFU-GM > or = 2 x 10(5)/kg or CD 34 positive cells > or = 2 x 10(6)/kg). In these 3 patients, a repeated PIHP with either doxorubicin, 5-fluorouracil, or CDDP was carried out 4 weeks after the 1st PIHP. Thereafter, auto-PBSCT was intravenously administered 2 days after PIHP. In the repeated PIHP treatments, although anti-cancer drugs were administered at doses equivalent to or even higher than those administered at the 1st PIHP, bone marrow suppressions were transient and well tolerated. Fatal complications were not observed in any patients. These results indicate that with the combined use of PIHP and PBSCT, high-dose regional chemotherapy of the liver can be safely repeated without systemic toxicities.

Chemotherapy plus granulocyte colony-stimulating factor (G-CSF) induced mobilization of peripheral blood stem cells (PBSC) was performed in patients with limited stage small-cell lung cancer. Chemotherapy consisted of cisplatin/etoposide or cisplatin/adriamycin/etoposide. The amounts of CD34 positive cells and granulocyte-macrophage colony forming units (CFU-GM) collected during 2-3 courses of apheresis were 3.1 +/- 2.9 x 10(6)/kg (n = 10) and 3.1 +/- 1.5 x 10(5)/kg (n = 8), respectively. Adequate amounts of PBSC were also harvested even in patients treated with concurrent chemoradiotherapy. Eight patients were successfully treated with high-dose chemotherapy consisting of ifosfamide, carboplatin and etoposide with PBSC transfusion. The patients'-bone marrow reconstruction was rapid and no treatment-related death was observed.

The efficacy and safety of high-dose lenograstim on CD34 positive (CD34+) cell mobilization into peripheral blood were investigated in 18 healthy male volunteers. The volunteers were divided into 3 lenograstim dose groups of 6 subjects each. Lenograstim was administered at a dose of 2, 5, or 10 micrograms/kg/day, b.i.d. by subcutaneous injection for a total of 5 days. The median peak number of CD34+ cells/microliter of blood was 16.3, 53.9, and 96.6 in the 2, 5, and 10 micrograms/kg/day groups, respectively. A positive correlation was observed between the peak CD34+ level and dose of lenograstim (P = 0.002). The percentage of volunteers achieving more than 50 CD34+ cells/microliter of blood was significantly higher in the 10 micrograms/kg/day group (83.3%, P = 0.010) than in the 2 micrograms/kg/day group (0%). On the subject of safety, at least 1 adverse drug reaction (ADR) was observed in each of the volunteers, and a total of 12, 33, and 45 ADRs were observed in the 2, 5, and 10 micrograms/kg/day groups, respectively. A dose-dependent increase in the number of ADRs was also observed, including an elevation of LDH (P < 0.001), bone pain (P < 0.001), and fatigue (P = 0.008). However, no volunteers required symptomatic treatment or discontinuation of lenograstim. We concluded that administration of lenograstim at a dose of 10 micrograms/kg/day for 5 days is highly effective for CD34+ cell mobilization into peripheral blood and tolerable in healthy volunteers.

We examined the clinical results of super high-dose chemotherapy with peripheral blood stem cell transplantation (PBSCT) in 14 patients with poor-risk advanced germ cell tumors. The mean number of nadir white blood cells was 205 +/- 126/microliter; the mean period of number of white blood cells fewer than 1,000/microliter was at 8-10 days (mean +/- SD; 9.2 +/- 0.92). The nadir number of blood platelet cells was 1.7 +/- 0.70 x 10(4)/microliter; the mean period of number of platelet cells fewer than 5 x 10(4)/microliter was at 12.6 +/- 2.17 days. Of 10 patients treated with super high-dose chemotherapy with PBSCT as induction therapy, 8 patients (80%) showed that the serum tumor marker returned within the normal range after super high-dose chemotherapy. Of 8 patients, 7 underwent resection of the residual tumor. Surgical or pathological CR was obtained in 5 of these 7 patients, 4 patients of whom were alive with no evidence of disease 29 to 49 months after initial consultation: the other patient died with recurrence 20 months after initial visit. On the other hand, super high-dose chemotherapy with PBSCT was performed for one patient as consolidation, and for 3 patients with recurrence. Of these 4 patients, one died from disease 6 months after detection of recurrence. The other 3 patients were alive with no evidence of disease at 7-37 months after initial visit. The 1- and 3-year disease-free survival rates were 88% and 72%, respectively. In conclusion, super high-dose chemotherapy with PBSCT can be done safely and could be useful for patients with poor-risk germ cell tumor.

We conducted a multicenter early phase II study to evaluate the feasibility and therapeutic efficacy of high-dose chemotherapy supported by autologous peripheral blood stem cell transplantation (auto-PBSCT) for the treatment of intermediate grade non-Hodgkin's lymphoma (IG-NHL). High-dose etoposide or cyclophosphamide followed by G-CSF was used for PBSC mobilization, and a sufficient number of CD34+ cells (> 1 x 10(6)/kg) were collected. Out of 81 enrolled patients, 50 received high-dose chemotherapy with auto-PBSCT; Hematologic recovery after transplantation was rapid. The incidence of grade III/IV toxicity (Bearman) was about 6%; treatment-related mortality was 6% (3/50). The Disease-free survival rate for the patients in complete remission who received high-dose chemotherapy with auto-PBSCT was better than that for the patients who were treated with conventional chemotherapy (57% vs 35%). These preliminary results indicated that high-dose chemotherapy with auto-PBSCT is feasible and effective. A prospective randomized phase III clinical trial will be required to assess the efficacy of high-dose chemotherapy with auto-PBSCT as a post-remission therapy for IG-NHL.

We performed a multicenter, an early phase II clinical trial to evaluate the feasibility, safety and efficacy of myeloablative therapy supported by autologous peripheral blood stem cell transplantation (auto-PBSCT) for the treatment of acute myelogenous leukemia (AML) in first remission. A total of 105 patients were enrolled in the study, and 56 patients in first complete remission received auto-PBSCT. The median age was 44 years. Of the 56 patients, 34 (60.7%) had M2 or M3 AML by the French-American-British Classification system. The median concentration of infused CD34+ cells was 2.3 x 10(6)/kg by recipient body weight. Median days to reach an absolute neutrophil count > 500/microliter and a platelet count > 20000/microliter were 14 and 16, respectively. The median disease-free survival rate was estimated to be 62.0% at a median follow-up time of 534 days. Although the study enrolled a small number of patients and the follow-up period was relatively short, the preliminary results were encouraging and indicated that myeloablative chemotherapy with auto-PBSCT is feasible and can be performed safely as a post-remission therapy for AML. A prospective randomized clinical trial of auto-PBSCT versus standard chemotherapy alone will be necessary to assess the efficacy of high-dose therapy facilitated by auto-PBSCT as a post-remission therapy for AML.

We studied the possibility of performing peripheral blood stem cell (PBSC) harvests during the course of ABVD therapy by adding G-CSF to the treatment regimen. Six patients with high-risk Hodgkin's disease (HD) (5 untreated cases with bulky mass and 1 relapsed case) received G-CSF (5 micrograms/kg) subcutaneously from day 8 to day 13 of their first course of ABVD treatment; the numbers of CD34+ cells and CFU-GM were monitored. PBSC harvests were performed on day 12 and day 13 of subsequent ABVD plus G-CSF treatment courses. For all patients tested, we were able to harvest CFU-GM (3.78 +/- 1.19 x 10(5) colonies/kg) for peripheral blood stem cell transplants (PBSCT) by performing 2 to 4 cycles. of apheresis, without any modification to the original ABVD protocol. These findings suggest that ABVD plus G-CSF therapy is a strong candidate for the treatment of patients with high-risk HD who may undergo autologous PBSCT.

Of 36 patients with malignant tumors who had been subjected to peripheral blood stem cell harvests (PBSCHs), 22 had undergone peripheral blood stem cell transplants (PBSCTs) since 1993. Flow cytometry recorded higher CD34+ cell yields in the PBSCHs of those patients with high white blood cell (WBC) counts as well as those who had been under intensive chemotherapy. Also, higher CD34+ cell yields were recorded in patients whose peripheral blood WBCs recovered more rapidly from their nadir state. WBC counts recovered rapidly in patients who received transfusions of at least 2.0 x 10(6) CD34+ cells/kg. However, patients with acute non-lymphocytic leukemia (ANLL) demonstrated a delayed recovery in their platelet counts following PBSCT. The mean disease-free survival rate and mean disease-free period were 60% and 12.8 months for the 5 patients with ANLL; and 100% and 11.3 months for the 4 patients with acute lymphocytic leukemia. These findings suggest PBSCT is a safe and effective treatment for patients with malignant tumors following high-dose chemotherapy, and can be performed in a private general hospital.

Placental and umbilical cord blood, as an alternative course of haematopoietic stem cells for bone marrow reconstitution, have recently been showed to yield successful sibling-donor cord blood grafts children. The advantages of using cord blood are related to the high number of haematopoietic progenitors in circulation at birth. In our study there was a remarkable heterogeneity of volume, number of nucleated cells and the number of progenitors from sample to sample. Seven out of 40 samples of more than 61 ml blood volume contained 1.8 (+/- 1.0) x 10(5) CFU-GM or 6.0 (+/- 4.8) x 10(8) nucleated cells. 10 ml of whole blood was necessary for laboratory tests including ABO blood type, screening of infectious markers, HLA typing, as well as frozen sera and cells for the possible future tests. Collected cord blood of more than 70ml in volume may have sufficient numbers of CFU-GM for engraftment to pediatric patients weighing about 20 kg. A cord blood bank project is now going on by collecting blood with informed consent of the mother in cooperation with obstetrics and gynecology staff.

In vitro chemosensitivity of established cell lines from human prostate cancer (8 PC 93, 19 PC 93, DU 145 and PC 3) to various anticancer drugs was examined by clonogenic assay. The drugs used were aclarubicin (ACR), adriamycin (ADM), carboquone (CQ), vincristine (VCR), ifosfamide (IFM), peplomycin (PEP) and cis-platinum (CDDP). To compare antitumor activity of different drugs, the predicted anticancer activity (PAA) was calculated from the 50% inhibition doses and the peak plasma concentration of the clinically used dose. High antitumor activity of drug was considered if PAA > or = 1 was observed. The chemosensitivities were: CQ > ADM > CDDP > VCR > PEP > IFM > ACR in the clonogenic assay. 19 PC 93 and DU 145 were sensitive to CQ, ADM and CDDP, but 8 PC 93 and PC 3 were sensitive to CQ, ADM, CDDP and VCR. Thus, a new combination chemotherapy with CDDP, ADM and CQ (PAQ therapy) was used clinically. PAQ therapy was given to sixteen patients with stage D advanced prostate cancer. Of these patients, 14 were undergoing relapse from antiandrogen therapy and 2 had hormone-resistant prostate cancer. The mean interval from the start of the prior treatment to relapse of the cancer was 18 months. The effectiveness of the new therapy was judged according to the response criteria for prostate cancer treatment of Japan. Three patients showed partial response (PR), 9 were stable disease (Stable) and 4 showed progressive disease (PD). The mean response duration in the patients with PR and with Stable was 11.6 months. The survival length of the responders (PR + Stable) was significantly longer than that of the nonresponders (PD) (p < 0.001). The side effect of PAQ therapy was lower than the moderate degree. Therefore, we considered PAQ therapy to be one of the clinical trials for the treatment of advanced prostate cancer.

Ideally, in preoperative intra-arterial infusion chemotherapy (regarding advanced breast cancer) is to obtain the most significant effects concerning histological features. Intra-arterial infusion chemotherapy using epirubicin (EPI), used in conjunction with daily doses of 1,200 mg medroxyprogesterone acetate (MPA), have recently been performed. This procedure has shown remarkable histological effects in the metastatic lymph nodes as well as in the primary lesions. These results were especially remarkable in the patients who were administered MPA (daily) two weeks prior to EPI infusion. These patients showed a complete disappearance of tumor cells. The results were interesting in view of the mechanism of action. Intra-arterial infusion chemotherapy combined with MPA may also be valuable in treating metastatic liver tumors and recurrent lesions in the regional lymph nodes such as supraclavicular tumors. A high response rate was obtained in the chemo-endocrine therapy when combined with MPA as a pretreatment. Thus, MPA may be expected to be available as a systemic therapy, too. In the future, a new development in the field of intra-arterial infusion chemotherapy may be achieved by utilizing a combination of angiogenesis inhibitors and peripheral blood stem cell transplantation.