Heterogeneity of multiorgan metastases of human lung cancer cells genetically engineered to produce cytokines and reversal using chimeric monoclonal antibodies in natural killer cell-depleted severe combined immunodeficient mice

Lung cancer is a major cause of cancer deaths, most of which can be attributed to distant multiorgan metastases. To examine the cellular and molecular mechanisms of lung cancer metastasis to distant organs, we have established novel models of human lung cancer (small cell and non-small cell lung cancer) metastasis in natural killer cell-depleted severe combined immunodeficient (SCID) mice. We investigated whether local production of the cytokines responsible for regulation of macrophage function at tumor growth sites affects the pattern of lung cancer metastasis in distant organs. Several lung cancer cell lines were genetically engineered to produce human macrophage colony-stimulating factor (M-CSF) and monocyte chemoattractant protein-1 (MCP-1), and their metastatic potentials were assessed. Interestingly, M-CSF gene transduction had an antimetastatic effect for the liver and lymph nodes, but not the kidneys. In contrast, MCP-1 gene-modified lung cancer cells and their parent cells had identical metastatic potentials. These findings indicate a possible role for cytokines and suggest that lung cancer has metastatic heterogeneity. Examining ways of controlling human lung cancer metastases, we investigated the antimetastatic effect of chimeric monoclonal antibodies (MAbs) against P-glycoprotein and ganglioside GM2 (MH162 and KM966, respectively). Both MAbs, when given on days 2 and 7, inhibited the development of distant metastases of lung cancer in a dose-dependent fashion. Combined use of anti-P-glycoprotein MAb with M-CSF or MCP-1 gene transduction caused complete inhibition of metastasis of H69/VP cells. The antimetastatic effect of these MAbs in vivo was mainly due to an antibody-dependent cell-mediated cytotoxicity reaction mediated by mouse macrophages. These findings suggest that the mouse-human chimeric MAb in combination with cytokine gene transduction may be useful for the eradication of lung cancer metastases in humans.


Therapeutic efficacy of mouse-human chimeric anti-ganglioside GM2 monoclonal antibody against multiple organ micrometastases of human lung cancer in NK cell-depleted SCID mice

The development of distant metastases to multiple organs is a critical problem in the treatment of human lung cancer. In this study, we evaluated the therapeutic efficacy of a mouse-human chimeric anti-ganglioside GM2 (GM2) monoclonal antibody (MAb), KM966 against metastasis formation of GM2-positive human lung cancer cells inoculated intravenously (i.v.) into natural killer (NK) cell-depleted severe combined immunodeficient (SCID) mice. GM2-positive human small cell lung cancer (SCLC), SBC-3 cells (1 x 10(6)), injected through a tail vein into NK cell-depleted SCID mice, formed large number of metastatic colonies in the liver, kidneys and lymph nodes by 42 days after inoculation (day 42). KM966, but not control MAb, given on days 2 and 7, almost completely inhibited metastasis formation of SBC-3 cells in the liver, kidneys and lymph nodes in a dose-dependent fashion. Moreover, treatment with KM966 at advanced stages of metastasis (even from day 28) significantly suppressed multiple organ metastases of SBC-3 cells. The anti-metastatic effect of KM966 in vivo was mainly due to an antibody-dependent cell-mediated cytotoxicity (ADCC) reaction mediated by macrophages of the SCID mice. Our findings suggest that the mouse-human chimeric anti-GM2 MAb, KM966 may be useful for eradicating multiple organ micrometastases of lung cancer in humans.


Circumvention of multidrug resistance by a quinoline derivative, MS-209, in multidrug-resistant human small-cell lung cancer cells and its synergistic interaction with cyclosporin A or verapamil

To develop a clinically useful approach to circumvent P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in MDR human small-cell lung cancer (SCLC), we examined the ability of a novel quinoline compound, MS-209, to reverse MDR by inhibition of P-gp function in combination with other MDR-reversing drugs using a cytotoxicity assay. We established MDR human SCLC cells by culture in medium with gradually increasing concentrations of adriamycin (ADM). Compared with the parental human SCLC cells, SBC-3, the MDR variant SBC-3 cells obtained (SBC-3/ADM) were highly resistant to various chemotherapeutic agents due to P-gp expression. MS-209 reversed the resistance to ADM and vincristine (VCR) of SBC-3/ADM and H69/VP cells in a dose-dependent manner. Moreover, MS-209 in combination with cyclosporin A (CsA) or verapamil (VER) synergistically enhanced the antitumor effects of ADM and VCR on SBC-3/ADM cells. MS-209 restored ADM incorporation and this effect was enhanced by CsA and VER, suggesting that these synergistic effects were due to competitive inhibition of P-gp function. MS-209 in combination with CsA or VER might increase the efficacy of these chemotherapeutic agents against MDR human SCLC cells.


The role of cyclosporin A on antibody-dependent monocyte-mediated cytotoxicity against human multidrug-resistant cancer cells

A P-glycoprotein (P-gp) inhibitor, cyclosporin A (CsA) was found to enhance the susceptibility of multidrug resistant (MDR) cancer cells to anti-P-gp antibody-dependent cellular cytolysis (ADCC) by monocytes, but the exact mechanism is unknown. In this study, we examined whether CsA enhanced the susceptibility of MDR cells through its inhibitory effect of P-gp function by using anti-ganglioside GM2 (GM2) monoclonal antibody (Ab), KM966, instead of anti-P-gp Ab, MRK16. Monocyte-ADCC induced by both KM966 and MRK16 against P-gp positive human MDR ovarian cancer cells was significantly augmented by addition of CsA. KM966, but not MRK16, induced monocyte-ADCC against P-gp negative human ovarian cancer cells and CsA enhanced this ADCC activity, indicating that suppressive effect of P-gp function by CsA was not essential to the enhancement of ADCC. Moreover, pretreatment of tumor cells with CsA augmented their susceptibility to monocyte-ADCC irrespective of P-gp expression. Interestingly, KM966 or MRK16 induced monocyte-ADCC against various human lung cancer cells expressing either GM2 or P-gp, but CsA did not affect these ADCC. These findings suggest that CsA may enhance the susceptibility to the monocyte-ADCC of ovarian cancer cells, but not of lung cancer cells, irrespective of its suppressive effect of P-gp function.

The Journal of Medical Investigation : JMI


Effect of clarythromycin on the distant metastases of human lung cancer cells in SCID mice

Recently, the use of macrolides is suggested to be therapeutically effective in prolonging the survival of patients with inoperable non-small cell lung cancer. The purpose of this study was to examine therapeutic effects of a macrolide, clarythromycin (CAM) on the metastastic developments of two different human non-small cell lung cancers (squamous cell lung carcinoma RERF-LC-AI, and adenocarcinoma PC-14) in severe combined immunodeficient (SCID) mice depleted or undepleted of natural killer (NK) cells, respectively. CAM, injected subcutaneously at doses of 5 and 10 mg/kg body weight/day from day 7 to 41 after i.v. inoculation of human lung cancer cells, was not effective in inhibiting their distant organ metastases in SCID mice. CAM at concentrations of less than 10 micrograms/ml did not have a direct influence on the proliferation of these tumor cells in vitro. Although CAM alone was not effective in augmenting NK activity, it augmented the IL-2-induced killer (LAK) activity against Daudi cells in vitro. These results suggest that CAM alone may not be enough to control the spread of non-small cell lung cancer in the patient with T cell dysfunction.


Intrapleural instillation of interferon gamma in patients with malignant pleurisy due to lung cancer

The effect of intrapleural instillation of recombinant human interferon gamma (IFN gamma) at increasing doses of (1-12) x 10(6) U was examined in six patients with cytologically positive pleural effusion due to lung cancer. Intrapleural instillation was repeated up to three times. Clinically, no reaccumulation of pleural effusion was observed in one patient and disappearance of lung cancer cells from the pleural effusion was seen in two other patients. No severe side-effects were observed. Considerable levels of IFN gamma remained in the pleural effusion as well as in patients' serum up to 7 days after instillation of 2 x 10(6) U and higher doses. The total cell number showed a transient decrease on day 1 of therapy. Levels of pro-inflammatory cytokines, such as tumor necrosis factor alpha, interleukin(IL)-1 beta and IL-6, in the pleural effusion remained almost stable after IFN gamma instillation. On the other hand, intrapleural IL-1 receptor antagonist levels were remarkably elevated by the instillation of IFN gamma. IL-2- and IL-12-inducible killer activity of pleural mononuclear cells tended to increase slightly. Despite the inability of IFN gamma to control pleural effusion in this treatment schedule, IFN gamma instilled by an intrapleural route had a potential local antitumor activity. Moreover, since IFN gamma persists in pleural effusions for a long time after a single instillation, such a therapy in combination with other fibrogenic biological response modifiers can be promising.


Model of malignant pleural effusion of human lung adenocarcinoma in SCID mice

Malignant pleural effusion (PE) is a frequent problem in lung cancer. In this study, we established a model of malignant PE of human adenocarcinoma cells, PC-14, in SCID mice. Intravenously injected PC-14 cells formed colonies in the lungs as early as week 4 after tumor inoculation, and produced bloody PE in all recipient SCID mice by week 8. Pretreatment of SCID mice with anti-mouse IL-2 receptor beta chain antibody (TM-beta 1) to deplete natural killer (NK) cells markedly promoted the production of bloody PE and metastases to multiple organs, such as the lungs, liver, kidneys, and lymph nodes 4 weeks after tumor inoculation. Histological studies indicated that PC-14 cells formed colonies in the lungs, and then invaded the pleura and spread to the pleural cavity. To establish cell lines with a high potential to produce PE, we harvested PE, expanded the tumor cells in vitro, and injected them into SCID mice again. By four in vivo selection cycles in this way we obtained PC-14-PM4 cells, which produce lung metastases and PE earlier than PC-14 cells. The survival of SCID mice inoculated with PC-14-PM4 cells was significantly shorter than that of mice inoculated with PC-14 cells. The expressions of adhesion molecules, such as CD44, CD49d, ICAM-1, and MHC class I, on PC-14-PM4 cells tended to increase compared with PC-14 cells. These changes of adhesion molecules seem to be one of possible mechanisms involved in higher metastatic potential of PC-14-PM4 cells. PE models with PC-14 and PC-14-PM4 cells should be useful for biological and preclinical studies on malignant PE produced by human lung cancer.


Anti-ganglioside GM2 monoclonal antibody-dependent killing of human lung cancer cells by lymphocytes and monocytes

Ganglioside GM2 (GM2) frequently appears on the cell surface of human cancers of neuroendocrine origin. A mouse-human chimeric monoclonal antibody (mAb), KM966, against GM2 was previously found to promote the lysis of various cancer cells by human blood mononuclear cells (MNC). In this study, we analyzed the effector cells responsible for the chimeric mAb-dependent cell-mediated cytotoxicity (ADCC) against small cell lung cancer (SCLC) cells and examined the enhancing effect of various cytokines on the ADCC activity. The ADCC activity was assessed by 4-h 51Cr release assay. Highly purified lymphocytes (> 99%) and monocytes (> 90%) were separated by centrifugal elutriation from peripheral blood MNC of the same healthy donor. KM966 induced lysis of SCLC cells mediated by both lymphocytes and monocytes to similar extents, in a dose-dependent manner. Pretreatment of lymphocytes with various cytokines [interleukin (IL)-2, IL-12 and interferon-gamma] and that of monocytes with macrophage-colony-stimulating factor significantly augmented the killer activity against SCLC cells in the presence of KM966 mAb. KM966 was also effective for the lysis of non-small cell lung cancer cells in direct proportion to the GM2 expression levels. These findings suggest that combined treatment of KM966 mAb with cytokines may be therapeutically useful for in vivo killing of lung cancer cells expressing GM2 through the ADCC reaction.


Systemic chemotherapy alters interleukin-1 beta and its receptor antagonist production by human alveolar macrophages in lung cancer patients

The purpose of this study was to determine whether cytotoxic chemotherapy influences the number and function of alveolar macrophages (AM) in patients with lung cancer. AM were obtained by bronchoalveolar lavage from 24 patients with lung cancer and 17 control patients. The functional integrity of AM was determined by their ability to produce interleukin-1 beta (IL-1 beta) and interleukin-1 receptor antagonist (IL-1ra) before and after platinum-containing systemic chemotherapy. The productions of IL-1 beta and IL-1ra were quantitated by enzyme immunoassays. The proportions of multinucleated cells among AM were significantly decreased after systemic chemotherapy in lung cancer patients. No significant difference in spontaneous and lipopolysaccharide (LPS)-stimulated IL-1 beta or IL-1ra production by AM was observed between lung cancer patients and control patients. Significant increase of IL-1 beta and significant decrease of IL-1ra production by AM were demonstrated in patients with small cell lung cancer who experienced response to systemic chemotherapy. These results suggest that systemic chemotherapy may influence functional roles of AM in the lung, and consideration of influence of systemic chemotherapy on host functions is important in cancer treatment.


Influence of systemic chemotherapy on anti-P-glycoprotein antibody-dependent cell-mediated cytotoxicity in patients with small cell lung cancer

Anti-P-glycoprotein antibody (MRK-16)-dependent cell-mediated cytotoxicity (ADCC) by blood mononuclear cells (MNC) was examined in patients with small cell lung cancer (SCLC) before and after systemic chemotherapy. The effect of in vitro treatment of MNC with interleukin (IL)-2 and macrophage-colony-stimulating factor (M-CSF) was also examined. The ADCC reaction was assessed by a 6 h 51Cr-release assay using a multidrug-resistant (MDR) SCLC cell line (H69/VP cells). The MRK-16 monoclonal antibody was able to augment spontaneous cytotoxicity by MNC, even in SCLC patients. Pretreatment of MNC with IL-2 significantly augmented their ADCC ability in SCLC patients, while M-CSF had no effect on ADCC activity. After the first cycle of systemic chemotherapy, the ADCC activity tended to decline, but ADCC of MNC pretreated with IL-2 was not affected. The results suggest that anti-P-glycoprotein antibody, in combination with a cytokine such as IL-2, may be therapeutically useful against human SCLC resistant to chemotherapeutic drugs.


高齢者進行・再発非小細胞肺癌に対する Pemetrexed + Bevacizumab 併用療法の有効性と安全性の検討

第62回日本肺癌学会学術集会

第62回日本肺癌学会学術集会