In vivo enhancement of tumor radioresponse by C225 antiepidermal growth factor receptor antibody.

Overexpression of epidermal growth factor receptor (EGFR) has been correlated with tumor resistance to cytotoxic agents, including radiation (T. Akimoto et al., Clin. Cancer Res., 5: 2884-2890, 1999), and thus is a candidate target for anticancer treatment. This study investigated whether treatment with C225 anti-EGFR antibody would improve tumor response to radiotherapy. Nude mice bearing 8-mm-diameter A431 tumor xenografts in the hind leg were treated with C225 antibody, 18 Gy of single-dose local tumor irradiation, or both. C225 was given i.p. at a dose of 1 mg/mouse 6 h before irradiation or 6 h before and 3 and 6 days after irradiation. Delay in tumor growth was the treatment end point. C225 dramatically improved the efficacy of local tumor irradiation, particularly when multiple injections of C225 were administered. Tumor radioresponse was enhanced by a factor of 1.59 by a single dose and by a factor of 3.62 by a doses of C225. Histological analyses of tumors revealed that C225 caused a striking increase in central tumor necrosis associated with hemorrhage and vascular thrombosis when combined with radiotherapy. In addition, C225 induced heavy tumor infiltration with granulocytes, increased tumor cell terminal differentiation, and inhibited tumor angiogenesis. We conclude that C225 anti-EGFR antibody enhances tumor radioresponse by multiple mechanisms that may involve direct and indirect actions on tumor cell survival.     

Recombinant human granulocyte colony-stimulating factor induces granulocytosis in vivo

Recombinant human granulocyte colony-stimulating factor (G-CSF) was prepared and its granulopoietic effects on mice were examined. When mice were injected intraperitoneally with the G-CSF daily for 14 days, marked granulocytosis occurred in the mice. The number of progenitor cells (CFU-C) was remarkably increased in the spleen. The results suggest that G-CSF plays a central role in granulocyte production in vivo.     

Opposite effect of tumor necrosis factor alpha on granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor-dependent growth of normal and leukemic hemopoietic progenitors.

The effect of recombinant human tumor necrosis factor alpha (TNF-alpha) on normal and chronic myeloid leukemia granulocyte-macrophage progenitors (CFU-GM) growing in semisolid agar cultures in the presence of recombinant granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor was studied. Granulocyte-macrophage colony-stimulating factor-dependent growth of normal and chronic myeloid leukemia bone marrow CFU-GM was greatly enhanced by TNF-alpha at doses of 0.1 to 100 units/ml. Growth enhancement included neutrophil, eosinophil, and monocyte-macrophage colonies and clusters at 7 and 14 days of culture. Since similar results were achieved with highly enriched progenitor cell populations, devoid of accessory cells, an indirect effect on CFU-GM growth through the release by accessory cells of other cytokines upon TNF-alpha stimulation was thus ruled out. By contrast, the same doses of TNF-alpha inhibited the growth of normal CFU-GM in granulocyte colony-stimulating factor-dependent cultures. Taken together, our findings indicate that the final effect of TNF-alpha on normal bone marrow granulocyte-macrophage progenitor growth is dependent on the specific growth factor interacting with it, and that both normal and chronic myeloid leukemia CFU-GM are equally responsive to the combined effects of TNF-alpha and a given colony-stimulating factor.     

Recombinant human TNF-alpha stimulates the secretion of granulocyte colony-stimulating factor in vivo.

Tumor necrosis factor (TNF) is a macrophage-derived cytokine that causes hemorrhagic necrosis of several human tumors in vitro. It has a wide range of biologic effects including stimulation of secretion of both granulocyte colony-stimulating factor (G-CSF) and granulocyte/macrophage colony-stimulating factor (GM-CSF) by normal adult lung fibroblasts in culture. No in vivo data are available on the effect of exogenously administered TNF on cytokine production. In the studies reported here, we show that G-CSF accumulates in the serum in vivo in response to recombinant TNF (rTNF) administration. At the peak of the response circulating levels of 2-6 ng/ml of biologically active G-CSF are detectable. Surprisingly, circulating levels of GM-CSF, interleukin-3 as well as a number of other cytokines were not detectable within the limits of the assays. The results indicate that the levels of GM-CSF or interleukin-3 are minimally 100-fold lower than the peak levels of G-CSF. These data illustrate the complex interplay that cytokines have in vivo. Understanding these interactions in humans is crucial to the correct use of this new class of agents in the clinic.     

Clonal heterogeneity of a human functioning tumor which produces granulocyte colony-stimulating factor.

Two different clonal strains have been isolated from a human squamous cell carcinoma which produces granulocyte colony-stimulating factor (G-CSF). The clones differed in morphology, growth characteristics, karyotype, production of G-CSF, histology of the tumors produced by inoculating the cultured cells, and in the development of granulocytosis in host mice transplanted with the cultured cells. A well-differentiated squamous cell carcinoma was developed by inoculation with clone 1 cells into athymic nude mice. No CSF activity was detected in the cultured medium of the cells. The host mice with the transplanted cells showed no increase in peripheral blood neutrophils. Clone 2, which formed a poorly-differentiated squamous cell carcinoma in nude mice, produced a large amount of CSF in vitro and developed a marked neutrophilia in host nude mice. Clone 1 cells were more sensitive to bleomycin than were clone 2 cells in vitro. The results suggested the G-CSF producing tumor to be heterologous and the cells with different functional properties, including G-CSF production, sensitivity to bleomycin, and keratinization, to pre-exist in the parental cell population.     

In vitro and in vivo analysis of the effects of recombinant human granulocyte colony-stimulating factor in patients

Twelve patients with small cell lung cancer were treated with recombinant human granulocyte colony-stimulating factor, rhG-CSF, given by continuous infusion at doses ranging from 1 to 40 micrograms kg-1 day-1. Patients received the rhG-CSF before the start of intensive chemotherapy and after alternate cycles of chemotherapy. Several in vitro assays were performed using peripheral blood neutrophils and marrow progenitor cells collected from patients prior to and after infusion of the growth factor. Peripheral blood neutrophils were tested for mobility and phagocytic activity. In addition, in vitro clonogenic assays of marrow haemopoietic progenitor cells and analysis of bone marrow trephines and aspirates were carried out. We found that rhG-CSF in vivo has at least two main effects: (a) an early fall in peripheral neutrophils, within the first hour, followed by a rapid influx of mature neutrophils into the circulatory pool; (b) stimulation of proliferation and differentiation of neutrophil precursors in the bone marrow. Neutrophils released into the circulation were normal in tests of their mobility and phagocytic activity.     

Neutrophils contribute to the measles virus-induced antitumor effect: enhancement by granulocyte macrophage colony-stimulating factor expression

To investigate the contribution of a neutrophil response to the oncolytic effect of replicating attenuated measles virus (MV), MVs expressing murine granulocyte macrophage colony-stimulating factor (GM-CSF) were generated. The growth characteristics and kinetics of GM-CSF production of these viruses were characterized in vitro. Their biological effects were characterized in mice transgenic for the MV receptor CD46. The oncolytic efficacy of MV GM-CSF was then compared with that of a parental MV and a control, UV-irradiated MV using a human lymphoid tumor model in immunodeficient mice. Intratumoral injection of MV resulted in significant tumor regression or slowing of progression compared with injection of the control. Injection of MV GM-CSF further enhanced the oncolytic effect. In additional experiments, the cellular response to MV, MV GM-CSF, recombinant murine GM-CSF alone, or untreated tumors was quantified. The predominant response was an influx of neutrophils. Intratumoral natural killer cells and macrophages were not detected. The magnitude of the neutrophil response correlated well with tumor regression. Our studies suggest that therapy with replicating MV stimulates a strong neutrophil antitumor response, which can be cytokine-enhanced to improve oncolysis.     

Bullous pyoderma gangrenosum after granulocyte colony-stimulating factor treatment.

The hematopoietic growth factors are under investigation for the treatment of patients with chemotherapy-induced bone marrow suppression. One such trial at the University of California, Los Angeles involves chemotherapy with or without granulocyte colony-stimulating factor (G-CSF) in patients with small cell lung cancer. The authors report a case of a patient who had bullous pyoderma gangrenosum at the site of previous eczema during treatment with G-CSF. The lesions resolved promptly when the drug was discontinued. Other investigators have recently reported inflammatory complications of G-CSF and granulocyte-macrophage colony-stimulating factor (GM-CSF) but this is the first case report of biopsy-proven neutrophilic dermatosis associated with administration of a hematopoietic growth factor. Patients should be monitored for development of inflammatory processes during G-CSF therapy and this therapy should be given with caution to those patients with existing inflammatory conditions.     

Distribution of human granulocyte colony-stimulating factor receptors on hematopoietic and nonhematopoietic tumor cell lines.

Introduction of two tyrosine residues into human granulocyte colony-stimulating factor (G-CSF) allowed us to obtain radioiodinated material efficiently. By using this material, specific receptors for human G-CSF were analyzed. It was found that human G-CSF receptors were expressed on several myelocytic and monocytic leukemia cell lines. Furthermore, they were also expressed on two choriocarcinoma cell lines. However, the human G-CSF receptors were not detected on other nonhematopoietic tumor cell lines examined, indicating that expression of G-CSF receptors is strictly limited as compared with receptors for granulocyte/macrophage colony-stimulating factor (GM-CSF), which are widely distributed on nonhematopoietic tumors.     

In vitro growth and clinical response of leukemia cells to macrophage colony-stimulating factor (M-CSF) and granulocyte colony-stimulating factor (G-CSF) in acute leukemia

The in vitro growth and clinical response of leukemia cells to macrophage colony-stimulating factor (M-CSF) and granulocyte colony-stimulating factor (G-CSF) were studied in 24 patients with acute leukemia. Among cases of acute myelogenous leukemia, a positive response to M-CSF (stimulation index > or = 2.5) was seen in 27.3% of the cases, and a significantly higher response rate (81.8%) was seen following G-CSF treatment of leukemia cells in vitro. In cases of acute monocytic leukemia, M-CSF showed a higher stimulating index than that observed for non-monocytic leukemia. G-CSF was administered in 19 cases and M-CSF in 5 cases after chemotherapy, and none of the patients showed leukemia cell proliferation in vivo. There was no correlation between in vitro test results and clinical response of leukemia cells to the CSFs administered.     

Effect of surgery on granulocyte/macrophage colony-stimulating factor.

Eighteen surgical patients were studied to determine the effect of anesthesia (general or spinal) and surgery on serum and urinary colony-stimulating factor(s) (CSF). CSF is a leukopoietin that stimulates proliferation of macrophages and granulocytes from bone marrow precursor cells. CSF was assayed by adding aliquots of serum or urine to semisolid agar cultures of bone marrow cells and scoring the number of colonies developing after 7 days of incubation. In all but 1 case, a 3- to 30-fold increase in the 24 hour excretion of urinary CSF was seen on the day of surgery and the first postoperative day. CSF urinary excretion began declining toward normal by the second postoperative day. A parallel increase in granulocyte count, a delayed rise in monocytes, and a decline in absolute lymphocyte counts were also observed. The data suggest that immediately postoperatively there may be a strong stimulus to granulocyte/macrophage proliferation. More rapidly proliferating cells would be anticipated to have increased vulnerability to toxicity from cell cycle-active chemotherapeutic agents.     

Production of granulocyte colony-stimulating factor by acute myelomonocytic leukemia cells

Two patients with acute myelomonocytic leukemia (AMMoL) were tested to determine whether or not their cells produced granulocyte colony-stimulating factor (G-CSF). Using the NFS-60 bioassay method, relatively high levels of G-CSF were demonstrated in the serum pre-treated with acid-dialysis and in the media conditioned by leukemia cells. Northern blot analysis for G-CSF using cDNA as a probe revealed that G-CSF production was increased at the mRNA level in the cells. No rearrangement at the DNA level, however, was observed by genomic Southern analysis. Our data indicate that the leukemic cells in AMMoL probably have the capacity to synthesize and secrete G-CSF.     

Effect of human recombinant granulocyte/macrophage colony-stimulating factor and native granulocyte colony-stimulating factor on clonogenic leukemic blast cells

The effects of human recombinant granulocyte/macrophage colony-stimulating factor (GM-CSF) and human native purified granulocyte colony-stimulating factor (G-CSF) on the growth of clonogenic leukemic blast cells from eight Japanese patients with acute myeloblastic leukemia were studied, using an in vitro leukemic blast colony assay. The results showed that GM-CSF stimulated leukemic blast colony formation in all cases examined, whereas G-CSF stimulated colony formation in four of the eight cases. The maximum stimulating activity of GM-CSF on the growth of clonogenic leukemic blast cells was higher than that of G-CSF in the majority of cases, while sometimes GM-CSF and G-CSF worked synergistically. Thus, the clonogenic leukemic blast cell populations seemed to be heterogeneous with respect to their in vitro response to growth regulators.     

Granulocyte colony-stimulating factor receptors on human acute leukemia: biphenotypic leukemic cells possess granulocyte colony-stimulating factor receptors.

Granulocyte colony-stimulating factor (G-CSF) receptors on the gated leukemic blast cells from newly diagnosed patients with acute leukemia or crisis of chronic myelogenous leukemia were investigated using flow cytometric detection. Surface marker analysis and cytochemical studies were conducted simultaneously to characterize the blast cells. Among 24 leukemia cases examined, G-CSF receptor-positive blast cells were detected in all 11 cases of acute myeloblastic leukemia even though the percentage range of positive cells was widely variable. On the other hand, they were not detected on the blast cells from patients with peroxidase-negative acute lymphoblastic leukemia with no myeloid surface antigens. However, G-CSF receptors were demonstrated in significant amounts on blast cells from 5 of 8 cases of peroxidase-negative acute leukemia expressing both myeloid and lymphoid surface antigens (biphenotypic leukemia). The percentage of blast cells positive for G-CSF receptors was significantly smaller in biphenotypic cases [33 +/- 14% (SD)] than in acute myeloblastic leukemia cases [65 +/- 22%] (P less than 0.01). The percentage expression of CD13 antigen by blast cells was significantly related to their percentage positivity for G-CSF receptors (rs = 0.50, P less than 0.05). These findings indicate that the distribution of flow cytometrically detectable G-CSF receptors on leukemic cells possessing myeloid characteristics may be related to the maturation process.     

Effect of co-administration of granulocyte colony-stimulating factor on interferon therapy.

The effect of co-administration of granulocyte colony-stimulating factor (G-CSF), as an antineutropenia agent, on interferon therapy was examined in a mouse model, in anticipation of an enhancement of interferon efficacy, because neutrophils induced by G-CSF are thought to act as antitumor effectors. G-CSF was intraperitoneally co-administered with human interferon alpha A/D (IFN) on Day 6 to Day 10 after intradermal inoculation of Meth A fibrosarcoma. Although the co-administration of G-CSF could protect against neutropenia and leukopenia induced by IFN, it did not enhance the regression of tumor, and rather reduced the prolongation of survival time and the long-term survival incidence of IFN therapy. The subsequent in vitro study showed that the antiproliferative activity of peripheral blood leukocytes from Meth A-bearing mice given both IFN and G-CSF was much weaker than that of mice given IFN alone. Whether the observed nullifying effect of G-CSF on IFN therapy is also the case with tumors other than Meth A is open to further study.     

Therapy of advanced prostate cancer with granulocyte macrophage colony-stimulating factor.

Granulocyte macrophage colony-stimulating factor is a pleiotropic cytokine capable of inducing systemic immune responses against experimental and human tumors. To evaluate the efficacy of GM-CSF treatment in patients with hormone-refractory prostate cancer, we conducted sequential Phase II studies in 36 men with progressive disease after androgen deprivation and antiandrogen withdrawal. In a first cohort of patients (n = 23), GM-CSF was administered s.c. at a dose of 250 microg/m2 daily for 14 days of a 28-day treatment period. After we observed oscillating prostate-specific antigen (PSA) responses in several patients in this first cohort, a second trial was performed in which patients (n = 13) received maintenance GM-CSF (250 microg/m2 three times weekly) after the first 14 days of daily GM-CSF. All patients were treated until disease progression. Response was assessed by evaluation of serial changes in serum PSA and sequential imaging studies. In cohort I, 10 of 22 patients (45%) had a PSA versus time plot with a sawtooth pattern, with PSA declining during GM-CSF therapy and climbing during the off-therapy period; 5 patients had at least two consecutive declines in PSA, with a median response duration of 3.5 months. All but one patient in cohort II experienced a decline in PSA (median decline, 32%), but a PSA decline greater than 50% and sustained for more than 6 weeks was seen in only one patient, who had a >99% decline in PSA and an improvement in bone scan lasting for 14+ months. Changes in PSA levels could not be attributed to direct or indirect effects of GM-CSF on the PSA assay or down-regulation of PSA expression by GM-CSF. Toxicity was very mild, consisting primarily of transient constitutional symptoms and injection site reactions. These data suggest that GM-CSF may have antitumor activity in advanced prostate cancer, and the use of GM-CSF may be a confounding variable when PSA responses are used as an end point in clinical trials evaluating new regimens for the treatment of advanced prostate cancer.     

Intratumoral vaccination with vaccinia-expressed tumor antigen and granulocyte macrophage colony-stimulating factor overcomes immunological ignorance to tumor antigen

Using a murine transitional cell carcinoma tumor model, MB49, which naturally expresses the male antigen HY, we evaluated whether tumor ignorance as determined by lack of a systemic immune response could be overcome by immunization with vaccinia expressed tumor antigen and granulocyte macrophage colony-stimulating factor. Systemic tumor ignorance of MB49 was demonstrated by the lack of a splenic HY-specific CTL response in MB49-bearing female mice. In contrast, we demonstrated HY-specific CTL priming in the draining lymph nodes. MB49-bearing female B6 mice were immunized with VVHY+VVGMCSF intratumorally or in the contralateral flank. Intratumoral VVHY, VVGMCSF, and keyhole limpet hemocyanin (to produce CD4 help) generated splenic HY-specific CD8 CTLs, whereas immunization with the combination in the contralateral flank or single agents given intratumorally failed to yield a splenic response. Purified male T cells injected intratumorally, as a source of HY antigen, also generated a HY-specific response, whereas contralateral immunizations did not. These finding expand the understanding of tumor immunological ignorance and support intratumoral vaccination as a strategy for immunotherapy of established tumors.     

Identification of a genetic locus modulating splenomegaly induced by granulocyte colony-stimulating factor in mice.

Clinically detectable splenomegaly and splenic rupture are uncommon but potentially life-threatening consequences of G-CSF administration. Increased spleen size in mice injected with G-CSF is a complex genetic trait amenable to investigation in experimental inter-strain crosses by quantitative trait analysis. A quantitative trait locus (QTL) with highly significant linkage (LOD 7.9) for splenomegaly was identified within a 22 centimorgan (cM) region on chromosome 1. Inheritance of a C57BL/6 haplotype in this region was associated with a greater spleen weight. The relevance of this locus was confirmed by analysing the responses of mice congenic for the distal 12 cM of this region (C57BL/6 and C57BL/6.SJL-Ptprc(a) Pep3(b)). Consistent with the QTL effect, mice lacking C57BL/6 alleles in this region had reduced splenomegaly induced by G-CSF. Intriguingly, peripheral blood neutrophilia and progenitor cell mobilisation responses to G-CSF were also significantly influenced.