The human tumor colony-forming assay was used to compare chemosensitivity among tumor cells within a primary tumor, between primary tumor and its metastasis, and between different metastases. The results indicate that the reported discrepancies of in vitro and in vivo results in clinical trials with TCFA for predicting of resistance or sensitivity to cytostatic drugs may be due to therapeutic heterogeneity among tumor colony-forming units within a primary tumors and between primary tumor and metastases, and that the results from a metastatic lesion may have more profound implications in planning treatment of other metastatic lesions of the same patient.

The experimental and clinical studies were carried out to alleviate the bone marrow suppression by antineoplastic drugs. Faster recovery of granulopoiesis was observed by pretreating recipients with RBC hypertransfusion and/or OK-432 (Picibanil, biological products of beta-streptococci.) In experimental mice, higher granulocyte counts could be maintained with hypertransfusion in the peripheral blood, and the recovery of granulopoietic series and CFU-S of bone marrow cells were found to be more rapid after cyclophosphamide administration. OK-432 also resulted in higher peripheral granulocyte, whereas the total nucleated cell counts and CFU-S were decreased in the bone marrow, suggesting sparing bone marrow granulocyte reserve and its migration to the peripheral blood. The mechanism of higher granulocyte count after hypertransfusion was not clearly explained but it was considered that erythroid suppression caused colateral flow of multipotential stem cells to granulopoiesis. The effect of both combinations was unexpectedly less significant in the recovery of granulopoiesis, but it was thought that the optimal time interval should be sought between pretreatment and the administration of anti-neoplastic agents. The clinical use of hyper transfusion and OK-432 also proved the alleviation of granulocytopenia, and rapid granulocyte recovery at the time of consolidation therapy among children with AML.

This experiment was carried out, in order to investigate the effect of antibiotics and natural mycotoxin on the hematopoietic stem cells at the normal and inflammatory condition. Adjuvant-treated rats (Aj-rats) are considered as a model of human rheumatoid arthritis. We measured the CFU-C and CFU-E of bone marrow of normal and Aj-rats which were injected with large (1.0 g/kg X 3) and small doses (0.5 g/kg X 3) of ampicillin (ABPC), cefazolin (CEZ), chloramphenicol (CP) and fusarenon-X (F-X). In Aj-rats the number of CFU-C was 1.5 times higher and CFU-E 60% less than normal. Injection of large doses of ABPC enhanced markedly the numbers of CFU-C in Aj-rats and suppressed slightly CFU-E in normal rats. Large doses of CEZ inclined to increase CFU-C and decreased CFU-E in normal and Aj-rats. Injection of small doses of CP tended to increase CFU-C and to decrease CFU-E, and large doses of CP to suppress both CFU-C and CFU-E levels in normal or Aj-rats. F-X, natural mycotoxin suppressed markedly both CFU-C and CFU-E levels of normal rats, and slightly the CFU-E in Aj-rats. These results suggest that one should pay attention to the fact that some doses of antibiotics or natural mycotoxin might be harmful on the bone marrow hematopoietic stem cells.

The chemotherapeutic susceptibility of normal human granulocyte-macrophage progenitor cells (CFU-C) and established human acute myelogenous leukemia (AML) cells (HL-60) in co-culture were determined. Nucleated marrow cells (2 X 10(5)) and HL-60 cells (5 X 10(3)) were mixed in 0.33% agar containing McCoy's 5a medium, 10% fetal calf serum, 0.1 ml of human placenta-conditioned medium and various concentrations of vincristine, cytosine arabinoside or daunorubicin to a total of 1.1 ml. They were incubated in 5% humidified CO2 at 37 degrees C for 8 to 10 days. CFU-C and HL-60 colonies were differentiated morphologically. In the absence of chemotherapeutic agents, the CFU-C colony formation was inhibited with the increasing number of HL-60 cells. CFU-C is equivalent or less sensitive than the leukemic cells in separate culture, but in co-culture it becomes more susceptible to vincristine and daunorubicin. These data indicate AML cells exert inhibitory effects on normal marrow CFU-C. In such state, normal hematopoietic cells become more susceptible to certain chemotherapeutic agents.

One of the problems in administration of anticancer drugs is a decrease of peripheral blood leucocytes which causes reduction of defense mechanism against various infections. In this study, for the purpose of protecting leucopenia after administration of anticancer drugs, an effect of cepharanthine which is a kind of alkaroid materials was examined in normal and tumor-bearing mice. Peripheral leucocytes of both normal and tumor-bearing mice receiving anticancer drugs gradually decreased during first two weeks, and in third and forth week they decreased rapidly. On the other hand, leucopenia was improved by simultaneous administration of anticancer drugs and cepharanthin; however, a complete repairment was hardly expected when anticancer drugs were continuously administered. A significant recovery of leucocytes was observed in mice receiving cepharanthin compared to those receiving only anticancer drugs. In any process of decreasing and recovering from peripheral leucocytes, proportions of differential counts of leucocytes were approximately identical. On the colony forming experiment for granulocytic stem cell (CFU-C) using bone marrow cells of normal, anticancer drugs-treated, and anticancer drugs plus cepharanthin-treated mice, it has been suggested that cepharanthin might have some influencing activity on hematopoietic cells or cell-mediated activity on hematopoiesis only in vivo.

In male dd-mice aged at 0, 20, 35, 70, and 150 days, erythropoiesis in the splenic red pulp was examined histologically, particularly using semithin plastic sections. In the red pulp, a majority of hemopoietic cells consist of erythroblasts. In early life until 20 days, erythroblasts constituted 91 to 96% of all the hemopoietic cells, and they then decreased gradually in proportion, although they composed 71% even at 150 days of age. The total number of erythroblasts contained in the red pulp increased in early life until it reached maximal at 35 days. At 150 days of age, erythroblasts were a half of those at 35 days in number. In semithin sections, erythroblasts were classified into two types: large erythroblasts and small erythroblasts. During life, large erythroblasts were most numerous at birth, constituting 13% of all the erythroblasts even at 150 days of age. In the red pulp, hemopoietic cells other than erythroids, such as lymphocytes, granulocytes, and megakaryocytes, were contained in relatively small numbers. Lymphocytes increased gradually in number with advancing age. In adults, macrophages were often positive in ferruginous reaction. Such macrophages were not seen in mice younger than 35 days of age.

Many hybridoma monoclonal antibodies to lymphocytes of T cell and B cell lineage and of both T and B cell lineage and stem cells were newly developed and characterized. Lymphocyte surface antigens that appear at the specific stages of T and B cell differentiation were clearly detected by these monoclonal antibodies. These antigens were also expressed on the cell surface of lymphatic leukemias and malignant lymphomas, suggesting the differentiation stages from which the tumor was derived. Leukemias and malignant lymphomas were diagnosed and classified objectively with these monoclonal antibodies. Immunohistochemical approach with the antibodies was very helpful for diagnosis of malignant lymphomas. Since the cell surface phenotypes are often related to biological behavior of the cell, the classification was expected to reflect the pathologic expression of tumor cells, and to have relationship with the clinical course of the patients with lymphoid malignancies. In fact, it has been suggested that the classification of leukemia and lymphoma based on the antigens defined by the monoclonal antibodies is related to the clinical features and prognosis of the patients.

Colony forming ability of solid tumor cells was studied in a tumor colony assay (human tumor stem cell assay). In 50 cases of solid tumors, cloning efficiencies of 5 X 10(5) cells plated were as follows: breast cancer 12/12 (100%), colon cancer 10/11 (91%), ovarian cancer 9/9 (100%), sarcomas 7/9 (78%), gastric cancer 3/6 (50%), endometrial cancer 2/2 and pancreatic cancer 1/1. An overall cloning efficiency was 88% (44/50) and this rate is higher than those reported in literatures. Ovarian cancer showed the highest plating efficiency of 0.07% (number of colonies/number of cells plated X 100%) in various solid tumors tested. Subsequently, plating efficiencies of colon and breast cancer were 0.03 and 0.01%, respectively. In the cases of sarcomas and gastric cancer, low plating efficiencies were seen (0.008%, 0.003%). The overall rate succeeded colony growth of solid tumors was somewhat higher in enzymatically treated tumor cells, that is, cloning efficiencies in mechanical and enzymatic methods were 85 and 90%, respectively. The enzymatic disaggregation is an advantageous method in gastric cancer and sarcomas. Various solid tumors can be formed colonies in soft agar and chemosensitivity test using in vitro colony assay is expected in solid tumors.

The sensitivity of leukemic progenitor cells (L-CFU) to cytosine arabinoside (Ara-C) and daunomycin (DM) in vitro was studied using PHA -LCM two step assay for L-CFU. Continuous exposure of leukemic and normal bone marrow cells to DM as well as Ara-C in vitro appeared to be more effective than pulse exposure because colony formation was suppressed by a dose dependent fashion. The relationship between in vitro sensitivity to DM and Ara-C and that of in vivo to chemotherapy was investigated in 17 untreated and 5 relapsed patients with acute nonlymphocytic leukemia. The sensitivity of L-CFU to the two chemotherapeutic agents varied from patient to patient. These studies indicated a clear-cut relationship between in vitro drug sensitivity and in vivo response to patients whose L-CFU were sensitive to both agents and entered complete remission, whereas patients whose L-CFU were insensitive to one or both drugs in vitro failed to enter remission. This assay system appears to be useful in predicting response of patients to chemotherapy and in selecting the most effective drugs for an individual patient use.