Studies on the interaction between interleukin 6 and human malignant nonhematopoietic cell lines.

We have previously reported on stimulation of clonal growth of cell lines from human solid tumors by recombinant human interleukin 3, recombinant human granulocyte-macrophage colony-stimulating factor, and recombinant human granulocyte colony-stimulating factor (W. E. Berdel et al., Blood, 73: 80-83, 1989; Exp. Hematol., 16: 510, 1988). Within an extensive screening program of hematopoietic growth factor activity on malignant cells, the effects of recombinant human interleukin 6 (rhIL-6) were tested on the growth (tritiated thymidine uptake and human tumor cloning assay) of 26 different human cell lines derived from a wide range of solid tumors (head and neck, 4; lung, 1; pancreatic, 1; gastric, 1; colorectal, 3; renal, 3; bladder, 1; prostate, 1; breast, 2; ovary, 2; choriocarcinoma, 1; sarcoma, 2; glioblastoma, 2; neuroblastoma, 2). rhIL-6 (dose range up to 10(4) IU/ml) caused no reproducible enhancement or inhibition of tritiated thymidine uptake by tumor cell lines from nonhematopoietic origin. Furthermore, 19 of the tumor cell lines were clonogenic in a capillary modification of the human tumor cloning assay. No reproducible stimulation of clonal growth by rhIL-6 was observed in any of the cells tested. Particularly, there was no sensitivity of those cell lines for rhIL-6, which were previously shown to be sensitive for recombinant human interleukin 3 and recombinant human granulocyte-macrophage colony-stimulating factor in this assay. On the other hand, there were no significant growth-inhibitory effects of rhIL-6 on the cell lines tested in this study. Further experiments showed no influence of neutralizing monoclonal anti-hIL-6 antibody on the growth of 3 kidney carcinoma cell lines, making autocrine growth-modulating loops for IL-6 in these lines unlikely. In conclusion, no major interactions between hIL-6 and the growth of the human malignant cell lines from nonhematopoietic origin tested were detected in this study.

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)

Recombinant human stem cell factor stimulates growth of a human glioblastoma cell line expressing c-kit protooncogene.

The growth of a panel of eight different human glioblastoma cell lines was examined in a human tumor cloning assay in agar, a tritiated thymidine uptake assay, and by counting cell numbers, in cultures performed in the absence or presence of increasing concentrations (1 to 100 ng/ml) of recombinant human stem cell factor (SCF). Growth of 7 of 8 cell lines was not significantly and reproducibly affected by recombinant human SCF. However, growth of the CRL 1620 cell line could be stimulated up to 5-fold by the cytokine. In contrast to the other cell lines investigated, CRL 1620 expressed the c-kit protooncogene assessed on the mRNA and protein level. Furthermore, SCF-induced proliferation of CRL 1620 cells was sensitive to the tyrosine kinase inhibitor erbstatin. Our data suggest that SCF can be operative in growth modulation of malignant cells outside the hematopoietic system, and this finding should be further studied for its possible clinical implications.

Nerve growth factor stimulates clonal growth of human lung cancer cell lines and a human glioblastoma cell line expressing high-affinity nerve growth factor binding sites involving tyrosine kinase signaling.

The growth of a panel of 22 different human tumor, leukemia, and lymphoma cell lines was examined in a human tumor cloning assay in agar or methylcellulose and a tritiated thymidine uptake assay. The cultures were performed in the absence or presence of increasing concentrations (0.5-500 ng/ml) of nerve growth factor (NGF). The growth of 17 of the 22 cell lines was not significantly and reproducibly affected by NGF. There was minor (1.2-fold) but reproducible stimulation of clonal growth in one glioblastoma cell line (86-HG-39) by NGF, but in this cell line NGF induced no growth modulation in a tritiated thymidine uptake assay. However, clonal growth of another glioblastoma cell line (87-HG-31) and all three lung cancer cell lines tested (HTB 119, HTB 120, CCL 185) could be stimulated up to 3-fold by NGF with a dose-response relationship for the growth factor. Growth stimulation by NGF could be completely reversed by neutralizing anti-NGF antibody and by the tyrosine kinase inhibitor genistein. Evaluation of secondary plating efficiency revealed the stimulation of colony formation as representing self-renewal and not terminal differentiation. Reverse transcriptase-PCR experiments in the five responding cell lines showed expression of both low-affinity NGF receptor (glycoprotein 75) and c-trk transcripts on the mRNA level. Of the five responding cell lines, only 86-HG-39, the cell line with the lowest responsiveness, revealed low-affinity NGF receptor on the protein level; the other four cell lines with high responsiveness, including the three lung cancer cell lines, expressed no low-affinity NGF receptor as shown by fluorescence-activated cell sorter analysis and immunoprecipitation using the ME 20.4 antibody. Immunoprecipitation using anti-trk antibodies was negative in all five responding cell lines. However, binding studies with iodinated NGF showed only low-affinity binding on the 86-HG-39 cell line and only high-affinity binding on the high-responder cell lines CCL 185 and 87-HG-31. In summary, our data suggest that NGF can be operative in stimulation of clonal growth of malignant tumor cells. High-affinity but not low-affinity binding sites mediate signal transduction for clonal growth and signaling involves tyrosine kinase activity.

Recombinant human interleukin 4 has antiproliferative activity on human tumor cell lines derived from epithelial and nonepithelial histologies.

Interleukin 4, a T cell-derived 20-kDa glycoprotein, plays an important role in regulating the immune response of B cells, T cells, and macrophages against infections and malignant cells. For this reason recombinant human interleukin 4 (rhIL-4) has entered early clinical trials in cancer patients. In the present study we report that rhIL-4 has an antiproliferative effect on five of nine cell lines derived from human colon tumors, head and neck tumors, and glioblastomas as measured by a decrease of colony formation in human tumor cloning assays. All of the cell lines with in vitro responsiveness express at least 100 high-affinity receptors for human interleukin 4 per cell on their cell surface, whereas the nonresponsive tumor cell lines lack expression of high-affinity receptors for human interleukin 4 on their cell surface. In the next series of experiments we have xenotransplanted some of the responsive cell lines into athymic nude mice. Subsequently, the animals were treated s.c. twice daily with 0.5 mg/m2 rhIL-4 or control vehicle for at least 12 days. There was a clear growth inhibition of these xenotransplanted tumors in the mice treated with rhIL-4. Histology of the tumors in both groups revealed no marked infiltration with murine hematopoietic and lymphocytic cells as evaluated by staining with a rat anti-mouse CD45 antibody. We conclude that rhIL-4 has a direct therapeutic activity on the growth of some human epithelial and nonepithelial tumor cell lines which, along with its regulatory function on hematolymphopoietic cells, makes this cytokine an interesting candidate for experimental tumor therapy.

Inhibition of proliferation and clonal growth of human breast cancer cells by interleukin 13.

We tested the influence of recombinant human interleukin (rhIL)-l3 and rhIL-4 on clonal growth of human breast cancer cell lines. rhIL-13 and rhIL-4 inhibited clonal growth of three of nine lines to approximately 50% of controls (ED50, 0.5 ng/ml). rhIl-13 reduced [3H]thymidine incorporation in all three cell lines: two showing a minor (84% and 83% of controls) and one showing a major response (25% of control). Both cytokines markedly reduced serum-induced G(0/1) exit (approximately 25% versus 60%). 125I-labeled interleukin (IL) 13 binding assays revealed high-affinity binding sites for IL-13 on two of the three responding cell lines (KD approximately 60 pM). (Y124D)IL-4 effectively antagonized all effects of rhIl-13 and rhIL-4, arguing for shared receptor components between them. However, neither rhIl-4 nor (Y124D) IL-4 could displace 125I-labeled IL-13 from binding, although unlabeled rhIL-13 effectively did so. Using reverse transcription-PCR, we studied the expression of the common gamma chain (gammac) in responding cell lines, putatively being shared between IL-4 receptor and IL-13 receptor; none of the three cell lines express gammac. In conclusion, we demonstrate antiproliferative effects of IL-4 and IL-13 on carcinoma cells which express IL-13 binding sites without participation of gammac.

Differential cytotoxicity of an ether lipid on lymphoma and bone marrow cells and its role in purging malignant lymphoid cells from remission bone marrow contaminated with tumor cells.

Four human clonogenic malignant lymphoid cell lines (CEM, Su-DHL-4, Li-A, and Raji) as well as normal human bone marrow stem cell progenitor cells were investigated for clonal in vitro growth before and after incubation with the ether lipid ET-18-OCH3 for various times (1, 4, and 18 h) and at increasing concentrations of the drug (25, 50, 75, and 100 micrograms/ml). The clonal growth of the malignant lymphoid cell lines was inversely correlated with concentrations and times of drug incubation. The antineoplastic effect of ET-18-OCH3 was further amplified by subsequent cryopreservation. In a situation of 4-h exposure to less than or equal to 50 micrograms/ml ET-18-OCH3 and subsequent cryopreservation, in which greater than 50% of the normal human bone marrow progenitor cells survived, 1-3 logs of the malignant lymphoblastoid cells were killed, indicating a potential value of this drug for bone marrow purging in lymphoid malignancy. In order to simulate the situation of autologous bone marrow transplantation (ABMT) in complete remission of the disease, we contaminated normal human bone marrow cells with malignant CEM or Su-DHL-4 lymphoid cells at a ratio of 100:1. Results show that 4 h of incubation with 75 micrograms/ml ET-18-OCH3 and subsequent cryopreservation can eliminate 2-3 logs of clonogenic cells of the malignant lymphoblastoid cell lines under conditions that allow recovery of greater than 50% of the normal human hematopoietic progenitors.

Recombinant human stem cell factor does exert minor stimulation of growth in small cell lung cancer and melanoma cell lines.

We have previously reported on the stimulation of clonal growth of a glioblastoma cell line by rhSCF (Berdel et al., Cancer Res 1992, 52, 3498-3502). Within an extensive screening programme of haematopoietic growth factor activity on malignant cells, the effects of rhSCF were further tested on the growth of 29 different human cell lines derived from a wide range of solid tumours, among them six lung cancers and five melanomas. RhSCF (0, 1, 10, 100 ng/ml) was tested in a human tumour cloning assay (HTCA) which reliably detects growth modulation of tumour cells by cytokines. Additionally, a tritiated thymidine uptake test was used. Growth of 27 of the 29 cell lines tested was not affected by rhSCF. However, growth of the small cell lung cancer (SCLC) cell line HTB 120 was slightly stimulated (1.5 fold that of controls), and that of the melanoma cell line MeWo was stimulated up to 1.3-fold. This activity was eliminated dose-dependently by the tyrosine kinase inhibitor, genistein. We further analysed the cell lines for expression of the proto-oncogene C-KIT and its ligand SCF. All melanoma and lung cancer cell lines expressed SCF as assessed at the mRNA level. Northern blotting also revealed clear C-KIT mRNA expression in three melanoma (HAS, MeWo, SK-MEL-28), one NSCLC (HTB 53), and four SCLC cell lines (HTB 119, HTB 120, HTB 171, HTB 175). Furthermore, C-KIT protein expression was detected by flow cytometric analysis on the cell surface of MeWo, HTB 119 and HTB 120 cells. Our data indicate that SCF can be operative in growth modulation of non-haematopoietic malignant cells, especially SCLC and melanoma. However, our extensive screening of SCF/tumour cell interaction shows that this interaction is rare and makes potential hazards, such as tumour stimulation upon clinical use of rhSCF in conjunction with chemotherapy in cancer patients, unlikely for the majority of other tumour histologies.

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.

Generation of progenitor-cell precursors in long-term bone-marrow cultures after marrow purging with ether lipids.

Alkyl-lysophospholipid derivates (ALP) are currently being tested as bone marrow (BM) purging agents prior to autologous BM transplantation in different malignancies. We evaluated the toxicity of the ALP ET-18-OCH3 (ET-18; Edelfosine, 1-0-octadecyl-2-0-methyl-rac-glycero-3-phosphocholine) towards early hematopoietic precursors by testing progenitor regeneration of non-purged and ET-18-purged BM (75 mu g and 125 mu g ET-18/ml/2x10(7) BM cells) in autologous long-term bone marrow cultures (LTBMC) from 3 different patients in complete remission. LTBMC feeder layers were irradiated with 875 rad for complete elimination of hematopoietic progenitors and recharged with cryopreserved purged and non-purged BM. In weekly intervals, adherent layer and supernatant LTBMC cells were completely removed and evaluated in colony forming unit (CFU)-assays. We have seen sufficient CFU-regeneration out of ET-18-purged BM up to 8 weeks of LTBMC (>40 CFU/flask). Total CFU-counts from LTBMC with purged BM were slightly reduced compared to non-purged control. High dose purging with 125 mu g ET-18/ml partly inhibited initial CFU-proliferation, but demonstrated elevated CFU-counts after 4 and 8 weeks of LTBMC compared to control. In conclusion, in our LTBMC series ET-18-purging yielded tolerable toxicity towards committed BM-progenitors, but no remarkable decline of early hematopoietic precursors regenerating CFU-progenitors for up to 8 weeks of culture.

Macrophage inflammatory protein (mip)-1-alpha stem-cell inhibitor (sci) does not affect clonal growth of human solid tumor-cell lines in-vitro.

MIP-1alpha is a member of a family of proinflammatory cytokines produced by activated macrophages which has been shown to be a negative regulator of early hematopoietic stem cell progenitors. We report on results testing recombinant human (rh) MIP-1alpha on the clonal growth of different human nonhematopoietic tumor cell lines in vitro. Cell lines tested included the following histologies: 7 glioblastomas, 1 neuroblastoma, 2 head and neck carcinomas, 4 lung carcinomas, 3 colorectal carcinomas, 1 gastric carcinoma, 1 pancreatic carcinoma, 1 breast carcinoma, 1 prostate carcinoma, 1 choriocarcinoma, 1 ovary carcinoma, 1 osteosarcoma, and 3 melanomas. MIP-1alpha (0, 2, 20, 200 ng/ml) was tested in human tumor cloning assays (HTCA) in agar-containing capillaries (HTCAcap) and in mixtures of methylcellulose and agar (HTCAmix), representing assay systems with different plating efficiencies (PE). Tumor cells were continuously exposed to the cytokine for the complete assay period. Clonal growth of none of the cell lines was significantly and reproducibly stimulated or inhibited by MIP-1alpha.

Interleukin-1 receptor antagonist inhibits growth modulation of human tumor-cell lines by interleukin-1 in-vitro.

In this study we report that recombinant human (rh) interleukin-1alpha (IL-1alpha) has direct and dose-dependent growth-modulating effects on human tumor cell lines in vitro as measured by a human tumor cloning assay (HTCA). Colony formation of the melanoma cell line A 375 was inhibited by rhIL-1alpha, whereas colony formation of the glioblastoma cell line HTB 14 was enhanced by this cytokine. Both growth-modulating effects were dose-dependent, however with some saturation. Subsequently, we have tested the activity of recombinant human IL-1 receptor antagonist (rhIL-1ra) on the tumor growth modulation by rhIL-1alpha. Tumor cells were incubated with increasing concentrations of rhIL-1ra and then added to the cultures containing rHIL-1alpha. Concentrations of rhIL-1ra were chosen to achieve a range between 0.01 and 100 ng/ml which includes a 1000-fold molar excess over IL-1alpha. The receptor antagonist was able to block both the inhibition and the stimulation of clonal growth of the respective tumor cell line by rhIL-1alpha. Furthermore, there was a direct dose dependent relationship revealing higher IL-1 antagonism of rhIL-1ra at higher concentrations with maximum efficacy at 1000-molar excess concentrations over IL-1alpha. In addition, rhIL-1ra alone did not reveal major modulation of the growth of A 375, but significantly decreased colony formation of HTB 14. We conclude that rhIL-1ra can counteract modulation of clonal growth of human tumor cells by IL-1alpha in vitro. Since our report provides first evidence that the stimulation of clonal tumor cell growth by IL-1alpha can be blocked by rhIL-1ra, this member of the IL-1 cytokine network should be further studied as a possible candidate for experimental cancer treatment.

Interleukin-4 inhibits growth of a human lung-tumor cell-line in-vitro and has therapeutic activity against xenografts of this cell-line in-vivo.

In the present study we report that recombinant human (rh) interleukin 4 (IL-4) has direct, dose-dependent antiproliferative effects on the human lung cancer cell line CCL 185 in vitro as measured by a human tumor cloning assay (HTCA). The biological response of the tumor cells to rhIL-4 is correlated with expression of specific binding sites for human (h) IL-4 on the cell surface. Furthermore, we have xenotransplanted CCL 185 into BALB/c nu/nu mice. Subsequently, the mice were treated for 17 days with twice 0.5 mg/m2 rhIL-4 or control vehicle subcutaneously per day. Treatment with rhIL-4 yielded a significant inhibition of tumor growth versus control. Histology of the tumors in both groups showed no marked infiltration of the tumors with murine hematopoietic and lymphocytic cells consistent with the species specificity of IL-4. We conclude that rhIL-4 has direct antiproliferative effects on the growth of a human lung tumor cell line in vitro and in vivo which together with its regulatory effects on various effector cell populations makes this cytokine a possible candidate for experimental cancer treatment.

Urokinase-type plasminogen-activator (upa), a protease with cytokine-like activity in human hl-60 leukemic-cell line.

HL-60, a cell line originating from a human myeloid leukemia, expresses urokinase-type plasminogen activator (uPA) on the mRNA level and secretes the protein into the culture supernatant. Additionally, uPA receptors (uPA-R) could be detected in HL-60 on both the mRNA and the protein level, whereas the lymphoblastic cell line Raji studied in parallel was uPA-R negative. The cell lines were further studied in their clonal growth in methylcellulose under the influence of rhuPA (rhpro-uPA). The growth of Raji cells was not influenced by rhuPA. Colony and cluster formation of HL-60 was not reproducibly affected by rhuPA in concentrations between 1-100 ng/ml. In some experiments however, there were higher numbers of colonies in the HL-60 cultures incubated with rhuPA which was due to a cluster-to-colony shift. Furthermore, the HL-60 colonies in the rhuPA incubated plates always showed morphological alterations including an adherent basis indicating functional differentiation. This assumption was further supported by the observation that the secondary plating efficiency (PE2) of HL-60 cells taken from single colonies of uPA-incubated plates decreased significantly when compared with PE2 of cells from colonies grown without the presence of uPA. In conclusion, the intact uPA molecule functions like an autocrine cytokine for a human leukemic cell line, which in addition to its effects in tumor invasion makes it an interesting target molecule for further studies on tumor suppression.

Fresh peripheral blood mononuclear cell preparations are a better starting material than bone marrow after cryopreservation for immunomagnetic harvesting of CD34(+) hematopoietic cells.

Immunomagnetic separation using anti-CD34 monoclonal antibodies and paramagnetic microspheres has been used to enrich hematopoietic stem cells from human bone marrow, whole cord blood, or mobilized peripheral blood mononuclear cell collections. This method has been reported to achieve high separation purity of CD34+ cells in small scale experiments with fresh material. The aim of the present study was to compare the efficacy of the CD34+ cell selection technique, when thawed bone marrow or fresh peripheral blood mononuclear cells were enriched. Starting with thawed bone marrow containing 2.9% CD34+ cells the final product purity was 67.7% with a 6% CD34+ cell yield (enrichment factor 25.7), and a 85-fold CFU-GM enrichment. Using fresh mobilized peripheral blood mononuclear cells the released cells contained 77.6% CD34+ cells with a 47% yield (enrichment 86.5-fold), and a 46-fold CFU-GM enrichment. These results indicate that CD34+ cells can be selected from cryopreserved bone marrow using immunomagnetic procedures. However, fresh leukapheresis products seem to be a much better material for a positive immunomagnetic stem cell selection technique in terms of purity, yield and enrichment.

Chemopurging of peripheral blood-derived progenitor cells by alkyl-lysophospholipid and its effect on haematopoietic rescue after high-dose therapy.

One reason for relapse after high-dose tumor therapy with subsequent autologous stem cell transplantation is tumor cell contamination of the graft. Removal of tumor cells from bone marrow grafts by chemopurging with the ether lipid edelfosine has been established as an effective and simple method. When compared with bone marrow derived grafts, progenitor cells from peripheral blood have considerably reduced the haematological recovery times. However, this advantage is put at risk by the nonspecific haematotoxic activity of the purging agent. We therefore compared the in vitro recovery of peripheral blood derived progenitor cells (PBPC) from either non-purged (n = 41) or purged (75 micrograms/ml of ether lipid for 4 h at 37 degrees C, n = 48) leukapheresis products. The recovery of CFU-GM after cryopreservation was 63 +/- 4% without and 48 +/- 3% with purging (P = 0.007). After high-dose therapy, patients (n = 37) received similar amounts of either non-purged (n = 17) or purged (n = 20) autologous PBPC. The median haematological recovery times (non-purged vs purged) to > 500 WBC/microlitres were 9.0 vs 8.5 days after transplantation, to > 2000 PMN/microlitres 10.5 vs 10.0 days, and to > 50,000 PLT/microlitres 15.5 vs 14.0 days. All differences were statistically not significant. We conclude that ether lipid purging of PBPC leads to a significant, however tolerable loss of progenitor cells in vitro, and that haematological recovery times after high-dose therapy are identically short, provided similar amounts of PBPC are reinfused.

The efficiency of tumor cell purging using immunomagnetic CD34+ cell separation systems.

Immunomagnetic separation with anti-CD34 monoclonal antibodies and paramagnetic microbeads has been used to enrich hematopoietic stem cells from human bone marrow (BM) or mobilized peripheral blood mononuclear cells (PBMNC). The introduction of this technique also constitutes a new principle of tumor cell purging. The efficiency in terms of purging tumor cells from PBMNC was evaluated in seven different experiments. Mobilized (chemotherapy and G-CSF) PBMNC were collected from patients with solid tumors (n = 6) and multiple myeloma (n = 1) by leukapheresis using an automated MNC separation system and contaminated with 1% (n = 5) or 10% (n = 2) tumor cells from different epithelial cell lines being CD34-negative. The cell mixture was sensitized with anti-CD34 (9C5) antibodies and sheep anti-mouse IgG1 paramagnetic microspheres and enriched for CD34+ cells using an Isolex 50 magnetic separator. Purify of CD34+ cells was studied by flow cytometry (FACScan) and tumor cell depletion was evaluated by comparative human tumor cloning assays (HTCA) containing methylcellulose and agar. We achieved a median purity of CD34+ cells of 85.9% (range 69.8-92.9%) and a median yield of 48.1% (range 21.0-85.2%). From these data in each case the estimated log depletion of tumor cells was calculated and compared with the experimentally achieved (HTCA) log depletion (log delta depletion = log experimental depletion--log calculated depletion). In our experiments we achieved a median depletion of 2.75 log (range 1.55-3.69 log). When corrected for CD34+ cell yield of each experiment we observed a median 'yield corrected depletion' of 2.38 log (range 1.48-3.15 log). The following delta depletion values were obtained: +0.32 log (HTB 129, breast), +0.21 log (HTB 26, breast), +0.04 log (HTB 26) for experiments with higher experimental depletion, and -0.23 log (HTB 26), -0.9 log (HTB 26, PBMNC from patient with multiple myeloma), -0.82 log (HTB 131, breast) and -1.66 log (HTB 131) for lower depletion efficacy than calculated. These data suggest that depletion may depend on specific cell surface characteristics of tumor cells. Moreover, plasma factors (eg paraprotein) may also have some impact. In summary, the Isolex 50 provides a high purity of CD34+ cells and depletion of tumor cells was efficient. However, calculated and experimental purging efficiencies are not necessarily identical.

Peripheral blood progenitor cell mobilization with Dexa-Beam/G-CSF, ether lipid purging, and autologous transplantation after high-dose CBV treatment: a safe and effective regimen in patients with poor risk malignant lymphomas.

High-dose chemotherapy followed by autologous peripheral blood progenitor cell transplantation (PBPCT) is increasingly applied in patients with relapsed, poor risk malignant lymphomas. Different strategies for progenitor cell mobilization using cytoreductive chemotherapy, hematopoietic growth factors, or both have been described. We studied the safety and efficacy of a modified DexaBEAM regimen (dexamethasone, BCNU [carmustine], etoposide, ara-C, melphalan) followed by granulocyte-colony stimulating factor (G-CSF) that was administered in order to minimize any residual disease and to obtain a sufficient amount of progenitor cells in the autografts. Until now, 16 patients at poor risk (8 with Hodgkin's disease, 8 with non-Hodgkin's lymphoma) entered the study. All the 12 patients with measurable disease at study entry responded to DexaBEAM. Median time of subsequent leukopenia (leukocytes < 1.000/microL) was 6 days (range 5-8 days). Peak numbers of CD34+ hematopoietic progenitor cells appeared in the peripheral blood after a median of 20 days (range 18-22 days) after onset of therapy. At that time, peripheral mononuclear cells were collected for autografting. Thereafter, the leukapheresis products were frozen until the day of transplantation, either unpurged in the case of Hodgkin's disease or purged with the ether lipid edelfosine in cases of non-Hodgkin's lymphoma. After high-dose chemotherapy with the CBV regimen (cyclophosphamide, BCNU, etoposide) the patients received their autografts, followed again by G-CSF treatment. A stable hematopoietic recovery was reached with granulocytes > 2.000/muL within 11 days (range 8-17 days), and platelets > 50.000/microL within 15 days (range 10-31 days), respectively, without significant differences between the purged and unpurged transplants. After a median follow-up of 28 months (range 1-40 months) 7 patients are alive without signs of recurrent disease, while 1 patient has died due to acute treatment related toxicity. Three patients had refractory disease, and 5 have relapsed of whom 4 have died. In summary, the DexaBEAM/G-CSF/CBV strategy appears to be safe and effective for salvage treatment in patients with poor risk malignant lymphomas.

Antiproliferative effect of human interleukin-4 in human cancer cell lines: studies on the mechanism.

Interleukin-4 (IL-4) plays an important role in activating the immune system against malignant cells. The human interleukin-4 receptor (hIL-4R) is not only expressed by hematopoietic cells but also on a large number of tissue specimens which include colon, breast and lung carcinomas. In this study we report that rhIL-4 has an antiproliferative effect on 2 out of 3 non-small cell lung carcinoma (NSCLC) cell lines in vitro as measured by human tumor cloning assays (HTCA). In comparison, rhIL-4 had no effect on the growth of small cell lung carcinoma cell lines (SCLC) in vitro. The response towards the cytokine is correlated with expression of at least 1500 high affinity receptors/cell for hIL-4 on the responsive cell lines. Xenotransplanting the human lung tumor cell lines into nude mice followed by 12 days of systemic treatment of the mice with rhIL-4 revealed a significant growth retardation of the IL-4R positive NSCLC cell lines when compared with the controls, whereas the growth of the IL-4R negative SCLC cell lines was unaffected also in vivo. Studies of possible mechanisms involved in the antiproliferative effect of rhIL-4 showed that rhIL-4 does not induce apoptosis or modulation of the transcription factor c myc in the responsive NSCLC cell lines. Additionally, the expression of the epidermal growth factor receptor (EGFR), which is discussed as mediating autocrine/paracrine growth stimulation of NSCLC, is unaffected by rhIL-4. However, we have observed that rhIL-4 inhibited G1-S-phase cell cycle progression. We conclude that rhIL-4 has an antiproliferative effect on the growth of some NSCLC in vitro and in vivo. The mechanisms involved remain to be further elucidated.

Studies on the role of recombinant human erythropoietin in the growth regulation of human nonhematopoietic tumor cells in vitro.

Recombinant human (rh) erythropoietin (EPO) is attracting increasing interest as an agent for treating cancer-related anemia. Thus, we have tested the effects of rhEPO on the clonal growth of 22 different cell lines derived from a wide range of human solid tumors (head and neck 3, lung 2, breast 2, stomach 1, colorectal 3, hepatocellular 1, pancreas 1, ovary 1, choriocarcinoma 1, osteogenic sarcoma 1, glioblastoma 2, neuroblastoma 1, prostate 1, renal 2) in vitro. RhEPO (dose range 0.01-100 U/ml) caused no significant and reproducible stimulation of clonal growth as measured by a capillary modification of the human tumor cloning assay in agar in any of the cell lines tested. In particular, there was no sensitivity for rhEPO of those cell lines which were shown to be responsive to interleukin-3 and GM-CSF. On the other hand, there were no growth inhibitory effects of rhEPO on the cell lines of this study. Finally, neutralizing anti-human EPO antibody had no effect on the clonal growth of two kidney carcinoma cell lines, making autocrine growth regulation by hEPO in these lines unlikely.