Removal of breast cancer cells from bone marrow by in vitro purging with ether lipids and cryopreservation.

Four cell lines from human breast cancer (HTB 19, HTB 133, IZB B, and MCF 7M) were investigated for in vitro growth before and after incubation with the ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) for 4 h and at increasing concentrations (25, 50, 75, and 100 micrograms/ml), as well as with and without cryopreservation. When measured with the human tumor cloning assay in agar or methyl-cellulose the surviving fraction showed an inverse correlation with the concentrations of ET-18-OCH3. After a 4-h exposure with 75 micrograms/ml ET-18-OCH3 at a cell density of 2 x 10(5)/ml the number of colonies of HTB 19 decreased from 75 +/- 10/10(3) cells (100%) to 1 +/- 0/10(3) cells (1%), and after subsequent cryopreservation no remaining colonies were found. In order to simulate the situation in autologous bone marrow transplantation we contaminated normal human bone marrow cells with malignant HTB 19 breast cancer cells at a ratio of 100:1. After incubation with 75 micrograms/ml ET-18-OCH3 for 4 h and subsequent cryopreservation there was a considerable reduction of HTB 19 colonies whereas the majority of normal human hematopoietic progenitors recovered. We conclude that ET-18-OCH3, in combination with cryopreservation, can remove breast cancer cells from bone marrow by up to 1 log and may be useful for purging bone marrow for autologous bone marrow transplantation not only in acute leukemia and non-Hodgkin's lymphoma but also in breast cancer.

Some antagonists of platelet activating factor are cytotoxic for human malignant cell lines.

Nine new platelet activating factor (PAF) antagonists from 4 different chemical classes (thiopyrimidines: SDZ 59-015; thioimidazolines: SDZ 61-813; imidazoisoquinolines: SDZ 62-434, SDZ 62-759, SDZ 63-135, SDZ 63-596; and imidazopiperidines: SDZ 61-638, SDZ 62-293, SDZ 62-694) have been tested for cytostatic/antiproliferative ([3H]thymidine uptake) and cytotoxic (trypan blue dye exclusion) activity in neoplastic human cell lines of different histology in vitro. The antiproliferative activity of 3 of the 9 PAF antagonists (SDZ 61-638, SDZ 61-813, SDZ 62-694) was not stable after freezing and thawing. SDZ 59-015 showed only minor cytotoxic or antiproliferative effects in a dose range of 2-40 microns after 24, 48, and 72 h of incubation. SDZ 62-434 showed varying activity. There were no significant differences between the activities of the other 3 PAF antagonists from the imidazoisoquinoline class, which showed drug concentrations inhibiting 50% of the activity studied (IC50) and drug concentrations yielding a 50% decrease of trypan blue dye exclusion (LC50) of less than or equal to 20 microM at greater than or equal to 48 h, even in the K-562 cell line, which is known to be rather resistant for a variety of cytotoxic drugs related to PAF. SDZ 62-293 showed the best antineoplastic properties with IC50 and LC50 values less than or equal to 10 microM at greater than or equal to 48 h including K-562. SDZ 62-434, SDZ 62-759, SDZ 63-135, SDZ 63-596, and SDZ 62-293 have been further tested in a human tumor cloning assay in 5 cell lines. Colony formation was reproducibly suppressed to less than 30% of the controls only by SDZ 63-135 (less than or equal to 40 microM) and SDZ 62-293 (less than or equal to 20 microM) during continuous exposure. There was no correlation between the IC50 values for the antiproliferative activity of the test compounds and their IC50 values for PAF-induced human platelet aggregation. Furthermore, the antiproliferative activity of the most active compound, SDZ 62-293, could not be antagonized by preincubation with the specific PAF antagonists WEB 2170 or WEB 2086 or PAF itself in noncytotoxic doses.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of interleukin-3 and granulocyte-macrophage colony-stimulating factor on growth of xenotransplanted human tumour cell lines in nude mice.

The clonal growth of cell lines from some human solid tumours can be stimulated by haematopoietic growth factors such as recombinant human (rh) interleukin-3 (IL-3) and rh granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. Among these cell lines are the human colorectal adenocarcinoma cell line HTB 38 and the human small-cell lung cancer cell line HTB 119. Here we report on a series of experiments studying the influence of subcutaneously administered rhIL-3 and rhGM-CSF on the in vivo growth of HTB 38 and HTB 119 cell lines as xenografts in athymic nu/nu BALB/c mice. Beginning 1 day after transplantation of the tumour the cytokines were administered daily for 20 days as a subcutaneous bolus distant from the tumour lesion at dose levels up to 1 mg/m2/day. The cytokines caused no significant and reproducible growth modulation of the tumours in vivo.

Effects of hematopoietic growth factors on malignant nonhematopoietic cells.

We have assayed modulation of clonal growth of cell lines from human solid tumors in vitro by recombinant human interleukin-6 (rhIL-6), rhIL-3, rh granulocyte-macrophage colony-stimulating factor (GM-CSF), rhG-CSF, rhM-CSF, and rh erythropoietin. Effects of hematopoietic growth factors were also tested in the tritiated thymidine uptake assay. No reproducible and significant modulation of clonal growth was found with rhIL-6, rhM-CSF, and rhEPO. The other cytokines showed stimulation of colony formation in some cell lines from colorectal adenocarcinomas and bladder and lung cancers with the following order of activity: rhIL-3 greater than or equal to rhGM-CSF greater than rhG-CSF. Growth stimulation was only found in clonal assays; it was abolished by neutralizing antibodies and was highly dependent on culture conditions. Stimulation could be masked by elevation of serum concentration and there was an inverse correlation between spontaneous plating efficacy of the control cells and growth stimulation by the factor with the highest activity of the colony-stimulating factor at suboptimal growth conditions. Growth inhibition by the cytokines was not observed. We could not establish autocrine loops for the growth modulation by the cytokines in the cell lines tested so far. Furthermore, we xenotransplanted some responsive cell lines into athymic mice and observed their in vivo growth under systemic application of rhIL-3 and rhGM-CSF or vehicle. There was no significant alteration of the tumor growth by these cytokines at plasma levels sufficient for in vitro growth stimulation. In conclusion, tumor growth stimulation by rhGM-CSF and rhIL-3 as potential hazards for their clinical application in cancer patients in conjunction with cytotoxic chemotherapy is unlikely.

Lack of therapeutic effects of platelet activating factor antagonists in WEHI-3B leukemia, human xenotransplanted colorectal and lung cancer and Lewis-lung tumor in vivo.

Four new antagonists of platelet activating factor (PAF) from two different chemical classes (imidazoisoquinolines: SDZ 62-434, SDZ 63-135, SDZ 62-759; imidazopiperidines: SDZ 62-293) were tested for in vivo therapeutic activity in various tumor models including the murine myelomonocytic leukemia WEHl-3B, xenografts of human colon (HTB 38) and lung (HTB 119) cancer cell lines and the murine Lewis-lung tumor. After intraperitoneal (i.p.) injection of 1 x 10(3), 5 x 10(3) and 1 x 10(4) WEHl-3B cells into Balb/c mice, the drugs were given per os (p.o.) or i.p. over 6-14 days. Drug doses were pushed to exceed the lethal dose for 10% of the animals (LD10) and ranged from 1 to 100 mg/kg daily for p.o. treatment and from 1 to 75 mg/kg daily for i.p. treatment. In the xenotransplants and the Lewis-lung tumor experiments, PAF antagonists were given i.p. to nude Balb/c and C57 Black mice after intracutaneous (i.c.) tumor cell inoculation. None of the four compounds induced reproducible prolongation of life span, significant numbers of long term survivors, reduction of tumor size, or delay of tumor growth in any of the therapeutic models. Oral SDZ 62-759 had some activity in experiments in which there was slow WEHl-38 tumor growth in the controls. Toxicity of equivalent drugs doses was higher in the i.p. than in the p.o. schedules.