Cellular immunotherapy using irradiated lung cancer cell vaccine co-expressing GM-CSF and IL-18 can induce significant antitumor effects.

BACKGROUND: Although the whole tumor cell vaccine can provide the best source of immunizing antigens, there is still a limitation that most tumors are not naturally immunogenic. Tumor cells genetically modified to secrete immune activating cytokines have been proved to be more immunogenic. IL-18 could augment proliferation of T cells and cytotoxicity of NK cells. GM-CSF could stimulate dendritic cells, macrophages and enhance presentation of tumor antigens. In our study, we used mouse GM-CSF combined with IL-18 to modify Lewis lung cancer LL/2, then investigated whether vaccination could suppress tumor growth and promote survival. METHODS: The Lewis lung cancer LL/2 was transfected with co-expressing mouse GM-CSF and IL-18 plasmid by cationic liposome, then irradiated with a sublethal dose X ray (100 Gy) to prepare vaccines. Mice were subcutaneously immunized with this inactivated vaccine and then inoculated with autologous LL/2 to estimate the antitumor efficacy. RESULTS: The studies reported here showed that LL/2 tumor cell vaccine modified by a co-expressing mouse GM-CSF and IL-18 plasmid could significantly inhibit tumor growth and increased survival of the mice bearing LL/2 tumor whether prophylactic or adoptive immunotherapy in vivo. A significant reduction of proliferation and increase of apoptosis were also observed in the tumor treated with vaccine of co-expressing GM-CSF and IL-18. The potent antitumor effect correlated with higher secretion levels of pro-inflammatory cytokines such as IL-18, GM-CSF, interferon-γ in serum, the proliferation of CD4+ IFN-γ+, CD8+ IFN-γ+ T lymphocytes in spleen and the infiltration of CD4+, CD8+ T in tumor. Furthermore, the mechanism of tumor-specific immune response was further proved by 51Cr cytotoxicity assay in vitro and depletion of CD4, CD8, NK immune cell subsets in vivo. The results suggested that the antitumor mechanism was mainly depended on CD4+, CD8+ T lymphocytes. CONCLUSIONS: These results provide a new insight into therapeutic mechanisms of IL-18 plus GM-CSF modified tumor cell vaccine and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity.

Cytokine changes in response to radio-/chemotherapeutic treatment in head and neck cancer.

BACKGROUND: Radiation and systemic chemotherapy are standard treatment strategies for advanced or metastatic head and neck cancer. However, little is known about the implications and changes in the tumor microenvironment, including the T-helper (TH)1/TH2 balance in response to these treatment regimens. The aim of the current study was to unravel the effects of chemotherapeutic drugs and radiation on cytokine changes. MATERIALS AND METHODS: In this study, the effect of radiation and chemotherapeutic treatment (5-fluorouracil and cisplatin) on eight cell lines was determined. Before and after exposure, cytokine levels in culture supernatants of cell lines were evaluated using the Bio-Plex Assay (Bio-Rad) and the Human TH1/TH2 Cytometric Bead Array (Becton Dickinson). Results were correlated with parallel measurements for cellular proliferation assessed by cytotoxicity assay. RESULTS: Seven out of eight cell lines of primary tumors or metastases demonstrated an enhanced level of the cytokines interleukin (IL)-1ß, IL-6, IL-8, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF) and tumor necrosis factor-α (TNF-α), after sub-lethal radiation doses. Under treatment with low concentrations of 5-fluorouracil and cisplatin, all examined cell lines showed an increasing secretion of the cytokines IL-6 and G-CSF. In contrast, sub-lethal doses of both cytostatic drugs revealed a dose-dependent decrease in secretion IL-1ß. Regarding GM-CSF and TNF-α, we demonstrated an increase in secretion by the primary tumors under low doses of 5-fluorouracil and cisplatin, whereas the metastases showed a sharp drop of GM-CSF and TNF-α secretion. Chemotherapeutic treatment led to no changes of the IL-8 cytokine profile. CONCLUSION: The results suggest complex cytokine changes of the tumor microenvironment and more aberrant expression profiles under treatment with radiation and the chemotherapeutic drugs 5-fluorouracil and cisplatin.

Marrow toxicity of 33P-versus 32P-orthophosphate: implications for therapy of bone pain and bone metastases.

UNLABELLED: Several bone-seeking radiopharmaceuticals, such as 32P-orthophosphate, 89Sr-chloride, 186Re-1,1 hydroxyethylidene diphosphonate (HEDP), and 153Sm-ethylene diamine tetramethylene phosphonic acid (EDTMP), have been used to treat bone pain. The major limiting factor with this modality is bone marrow toxicity, which arises from the penetrating nature of the high-energy beta particles emitted by the radionuclides. It has been hypothesized that marrow toxicity can be reduced while maintaining therapeutic efficacy by using radionuclides that emit short-range beta particles or conversion electrons. In view of the significant clinical experience with 32P-orthophosphate, and the similarity in pain relief afforded by 32P-orthophosphate and 89Sr-chloride, this hypothesis is examined in this study using 32P- and 33P-orthophosphate in a mouse femur model. METHODS: Survival of granulocyte macrophage colony-forming cells (GM-CFCs) in femoral marrow was used as a biologic dosimeter for bone marrow. 32P- and 33P-orthophosphate were administered intravenously, and GM-CFC survival was determined as a function of time after injection and, at the nadir, as a function of injected activity. The kinetics of radioactivity in the marrow, muscle, and femoral bone were also determined. The biologic dosimeter was calibrated by assessing GM-CFC survival at its nadir after chronic irradiation of Swiss Webster mice with exponentially decreasing dose rates of gamma rays (relative biologic effectiveness equivalent to that of beta particles) from a low-dose rate 137Cs irradiator. Dose-rate decrease half-times (Td) (time required for 137Cs gamma ray dose rate to decrease by one half) of 62, 255, and 425 h and infinity were used to simulate the dose rate patterns delivered by the radiopharmaceuticals as dictated by their effective clearance half-times from the mouse femurs. These data were used to experimentally determine the mean absorbed dose to the femoral marrow per unit injected activity. Finally, a theoretical dosimetry model of the mouse femur was developed, and the absorbed doses to the femoral marrow, bone, and endosteum were calculated using the EGS4 Monte Carlo code. RESULTS: When the animals were irradiated with exponentially decreasing dose rates of 137Cs gamma rays, initial dose rates required to achieve 37% survival were 1.9, 0.98, 0.88, and 0.79 cGy/h for dose rate decrease half-times of 62, 255, and 425 h and infinity, respectively. The D37 values were 144 +/- 15, 132 +/- 12, 129 +/- 3, and 133 +/- 10 cGy, respectively, compared with a value of 103 cGy for acute irradiation. When 32P and 33P were administered, the injected activities required to achieve 37% survival were 313 and 2,820 kBq, respectively. Theoretical dosimetry calculations show that 33P offers a 3- to 6-fold therapeutic advantage over 32P, depending on the source and target regions assumed. CONCLUSION: The low-energy beta-particle emitter 33P appears to offer a substantial dosimetric advantage over energetic beta-particle emitters (e.g., 32p, 89Sr, 186Re) for irradiating bone and minimizing marrow toxicity. This suggests that low-energy beta or conversion electron emitters may offer a substantial advantage for alleviation of bone pain as well as for specifically irradiating metastatic disease in bone.

Granulocyte colony-stimulating factor and drugs elevating extracellular adenosine synergize to enhance haematopoietic reconstitution in irradiated mice.

The activation of adenosine receptors has recently been demonstrated to stimulate haematopoiesis. In the present study, we investigated the ability of drugs elevating extracellular adenosine to influence curative effects of granulocyte colony-stimulating factor (G-CSF) in mice exposed to a sublethal dose of 4 Gy of 60Co radiation. Elevation of extracellular adenosine in mice was induced by the combined administration of dipyridamole, a drug inhibiting the cellular uptake of adenosine, and adenosine monophosphate (AMP), an adenosine prodrug. The effects of dipyridamole plus AMP, and G-CSF, administered either alone or in combination, were evaluated. The drugs were injected to mice in a 4-d treatment regimen starting on d 3 after irradiation and the haematopoietic response was evaluated on d 7, 10, 14, 18 and 24 after irradiation. While the effects of G-CSF on the late maturation stages of blood cells, appearing shortly after the completion of the treatment, were not influenced by dipyridamole plus AMP, positive effects of the combination therapy occurred in the post-irradiation recovery phase which is dependent on the repopulation of haematopoietic stem cells. This was indicated by the significant elevation of counts of granulocyte-macrophage progenitor cells (GM-CFC) and granulocytic cells in the bone marrow (d 14), of GM-CFC (d 14), granulocytic and erythroid cells (d 14 and 18) in the spleen, and of neutrophils (d 18), monocytes (d 14 and 18) and platelets (d 18) in the peripheral blood. These effects suggest that the repopulation potential of the combination therapy lies in a common multilineage cell population. The results of this study implicate the promising possibility to enhance the curative effects of G-CSF under conditions of myelosuppressive states induced by radiation exposure.

[Ex vivo study of ultraviolet-B inactivating immunocompetence and hematopoietic potential of cord blood].

OBJECTIVE: To determine the differences of the effect of ultraviolet-B(UVB) on lymphocyte and hematopoietic progenitor cell in cord blood. METHODS: The viability, proliferative response and stimulatory activity in mixed lymphocyte culture (MLC), colony forming unit-granulo-monocyte (CFU-GM) colony growth and percentage of CD34+ cell of cord blood in Hanks medium after 0, 5, 10, 20, 50 mJ/cm2 UVB-irradiation were compared. RESULTS: Like the proliferative response and stimulatory activity, CFU-GM colony also showed UVB-irradiation dose dependent decrease. However, much less colonies were destroyed by UVB irradition less than 10 mJ/cm2. More obvious differences were found when 20%(vol/vol) bovine serum was added to the MNC medium. The percentage of CD34+ cell remained unchanged after UVB exposure. CONCLUSION: A lower dose of UVB exposure can slectively inactivate cord blood lymphocyte and antigen presenting cell while sparing hematopoietic progenitor cells. The data indicates the possibility of utility of UVB irradiation for graft-versus-host disease prophylaxis in cord blood transplantation.

GM-CSF production by epithelial cell line: upregulation by ultraviolet A.

It was demonstrated that UVB increases synthesis and expression of IL-1 alpha and GM-CSF by keratinocytes. Upregulation of GM-CSF by UVB is reported to be mediated by IL-1 alpha. However, regulation of IL-1 alpha and GM-CSF by UVA is not well-known. The purpose of the present study was to evaluate the effects of UVA on IL-1 alpha and GM-CSF production. Here we used a competitive RT-PCR for measuring cytokine gene expression in an epidermal cell line after UVA irradiation. IL-1 alpha and GM-CSF mRNA did not show any change at 1 h and 6 h following exposure to UVA. After UVA irradiation, however, IL-1 alpha mRNA decreased and GM-CSF mRNA increased at 24 h and the level of GM-CSF in culture supernatant increased at 24 h and 48 h. Addition of antihuman IL-1 alpha neutralizing antibody to UVA irradiated cells did not prevent the increase of GM-CSF mRNA expression. These results suggest that UVA radiation may induce GM-CSF production through an IL-1 alpha independent pathway.

Study of irradiation effects on cytokine secretion from retrovirally-transduced tumor cells: a model for tumor vaccination.

We have examined the effects of irradiation on the cytokine secretion from genetically modified human esophageal and gastric carcinomas. Both cell lines were transduced retrovirally to secrete interleukin-2, interleukin-6 and granulocyte-macrophage colony-stimulating factor, respectively. The metabolism of all the transduced cells was partially inhibited by 6 Gy, and greatly inhibited by 20 Gy irradiation. Cytokine productions, however, was not affected by 6 Gy in many cases, and continued to be detected even after 60 Gy irradiation. The analysis of the dose and time of irradiation in each cytokine producer is useful for the designing of tumor vaccine using cytokine gene transfer.

Ionizing radiation-induced IL-1 alpha, IL-6 and GM-CSF production by human lung cancer cells.

A cell line derived from human lung cancer (AOI) was employed in the present study. A panel of cytokines were quantified by ELISA technique following cellular exposure to X-irradiation. Tremendous increase in the levels of both IL-1 alpha and IL-6 were observed, GM-CSF was also detected. A comparison of time kinetics of IL-1 alpha and IL-6 production was made with that of cell cycle progression which was determined by FCM BrdU/DNA bivariate analysis. No cell cycle specific changes were found. The biological implication of radiation-induced cytokine production was discussed.

Experimental radiotherapy and metastasis of human lung cancer xenografts in nude mice.

Using a cell line derived from human lung cancer (AOI), we successfully established human xenografts in KSN nude mice, which showed high incidence of multiple spontaneous metastases. The highest incidence of metastasis in untreated hosts was observed in the spleen followed by the lungs and lymph nodes. The rate of metastasis reached 100% in the mice bearing large sized tumors, when metastasis to any organ or tissue was counted. Experimental radiotherapy caused remarkable redistribution of metastatic foci among different organs. Lung metastasis tended to decrease, while metastasis to the liver and the kidney was increased after radiotherapy. Radiation-induced production of cytokines was speculated to be responsible for such an alteration of metastasis pattern.

Effects of gamma-irradiation on the M-CSF-promoter linked to a chloramphenicol aminoacyl transferase reporter gene expressed in a clonal murine bone marrow stromal cell line.

The effects of cytokines produced by bone marrow stromal cells on closely associated hematopoietic cells constitute a major component of the physiology of the hematopoietic microenvironment. A major cytokine produced by marrow stromal cells is macrophage colony-stimulating factor (M-CSF). To determine the effect of gamma-irradiation on the M-CSF promoter in bone marrow stromal cells, we selected a clonal cell line from the C3H/HeJ mouse marrow stromal cell line D2XRII and stably transfected a reporter construct containing the murine M-CSF-promoter linked to a chloramphenicol aminoacyl transferase (CAT) gene. CAT activity was measured at serial time points after gamma-irradiation in vitro to doses between 500 and 10,000 cGy at a dose rate of 116 cGy/min. D2XRII marrow stromal cells treated with phorbol myristate acetate (40 micrograms/ml, four h), demonstrated a significant two-fold increase in CAT activity. In contrast, CAT activity measured immediately, 24 h, 72 h or 1 week after gamma-irradiation, showed no significant increase or decrease in CAT activity. An increase in CAT activity was detected 48 h after irradiation with cells that received 5,000 cGy. Thus, single fraction gamma-irradiation of plateau phase bone marrow stromal cells did not decrease M-CSF-promoter activity. These results are consistent with prior experimental data demonstrating stable levels of release of M-CSF protein following gamma-irradiation of bone marrow stromal cells and imply that the stability of transcription of the gene for this important cytokine is protected from irradiation.