Combined therapy with anti-P-glycoprotein antibody and macrophage colony-stimulating factor gene transduction for multiorgan metastases of multidrug-resistant human small cell lung cancer in NK cell-depleted SCID mice.

Our aim was to determine the antimetastatic potential of anti-P-glycoprotein (P-gp) antibodies (Abs) against multidrug-resistant (MDR) human small cell lung cancer (SCLC) cells expressing P-gp. Human SCLC cells H69 (P-gp negative) and its etoposide-resistant variant H69/YP (P-gp positive) were used. H69 and H69/VP cells injected i.v. metastasized to the liver, kidneys and systemic lymph nodes of NK cell-depleted severe combined immunodeficient (SCID) mice. H69/VP cells, but not H69 cells, were resistant to treatments with vindesine. Treatment with mouse-human chimeric anti-P-gp Ab (MH162) and its mouse counterpart (MRK-16) reduced metastasis of H69/VP cells in various organs and prolonged the survival of tumor-bearing mice, although they were less effective if injected at late times (after 28 days). Treatment with another mouse anti-Pgp Ab, MRK-17, was effective only against liver metastasis. MH162 and MRK-16 efficiently induced Ab-dependent cellular cytotoxicity (ADCC) by peritoneal macrophages against H69/VP cells in vitro, but MRK-17 was less effective, in accordance with their in vivo antimetastatic potential. Gene transfection of macrophage colony-stimulating factor (M-CSF) into H69/VP cells to augment macrophage-mediated ADCC resulted in inhibition of metastasis to the liver and lymph nodes, but not kidneys. Combined treatment with a low dose of MRK-16 completely cured metastasis of M-CSF transfectant, but not of the mock transfectant. Our findings suggest that while anti-P-gp Abs had antimetastatic potential against SCLC cells expressing P-gp, combined treatment with M-CSF gene transduction to augment the therapeutic efficacy of anti-P-gp Abs may be beneficial for eradicating metastatic MDR SCLC in humans.     

Combined therapy of multidrug-resistant human lung cancer with anti-P-glycoprotein antibody and monocyte chemoattractant protein-1 gene transduction: The possibility of immunological overcoming of multidrug resistance

We determined whether transduction of the monocyte chemoattractant protein-I (MCP-I) gene into MDR human lung cancer cells affected their tumorigenicity and sensitivity to antibody-dependent cellular cytotoxicity (ADCC) reaction mediated by the anti-P-glycoprotein (P-gp) monoclonal antibody MRK 16. The human MCP-I gene inerted into an expression vector (BCMGSNeo) was transfected into MDR human small-cell lung cancer (H69/VP) cells. Monocyte chemotactic activity was found in culture supernatants collected from MCP-I-transfected H69/VP cells, but not in supernatants of parent and mock-transfected cells. In an in vitro experiment, recombinant MCP-I did not affected monocytemediated ADCC against H69/VP cells when added to the monocyte culture in either the activation or the effector phase at sufficient concentrations to attract and activate monocytes. Tumorigenicity and growth rates of MCP-I-producing H69/VP cells in nude mice were similar to those of parental cells and mock-transfected cells. However, systemic treatment with MRK16 was move effective in inhibiting the formation of tumors by MCP-I-gene-transfected cells than by mock-transfected cells. Systemic treatment with MRK16 also inhibited the growth of a mixture (1:1) of MCP-I-producing cells and mock-transfected cells. These results suggest that combination therapy with MRK16 and MCP-I gene transduction may be a useful immunological strategy to inhibit the growth of human MDR cancer cells expressng P-gp. Int. J. Cancer 71:170–177, 1997. © 1997 Wiley-Liss, Inc.     

Transduction of the Macrophage Colony-stimulating Factor Gene into Human Multidrug Resistant Cancer Cells: Enhanced Therapeutic Efficacy of Monoclonal Anti-P-glycoprotein Antibody in Nude Mice

To develop a therapeutic modality for overcoming multidrug-resistant (MDR) cancer with anti-MDR1 antibody, we examined the effect of macrophage colony-stimulating factor (M-CSF) gene transfection into MDR AD10 cells on therapy of MDR cancer with anti-MDR1 antihody (MRK17) in nude mice. MDR human ovarian cancer (AD10) cells were transduced with the human M-CSF gene inserted into an expression vector to establish gene-modified cells capable of producing low (ML-AD10), intermediate (MM-AD10) and high (MH-AD10) amounts of M-CSF. Systemic administration of MRK17 resulted in significant dose-dependent inhibition of subcutaneous growth of ML-AD10 tumors. In contrast, systemic administration of recombinant M-CSF in combination with MRK17 did not augment the therapeutic efficacy of MRK17 alone, but rather promoted the growth of the parent AD10 cells. To test the efficacy of in vivo M-CSF gene therapy combined with antibody, we mixed the parent AD10 cells with MH-AD10 cells producing a large amount of M-CSF, and inoculated the mixed cells subcutaneously. Treatment with MRK17 inhibited growth of the mixed cells more than that of the parent cells alone. Thus, combined therapy with anti-MDR1 mAb and M-CSF gene modification of MDR cancer cells may provide a new immunotherapeutic modality for overcoming MDR in humans.     

A new quinoline derivative MS-209 reverses multidrug resistance and inhibits multiorgan metastases by P-glycoprotein-expressing human small cell lung cancer cells.

Development of distant metastases and acquired multidrug resistance (MDR) are major problems in therapy for human small cell lung cancer (SCLC). MS-209 is a novel quinoline compound, which reverses P-glycoprotein (P-gp)-mediated MDR. We previously reported that MS-209 reversed in vitro MDR of human SCLC (SBC-3 / ADM and H69 / VP) cells expressing P-gp. In the present study, we determined the therapeutic effect of MS-209 in combination with chemotherapy against multiorgan metastases of MDR SCLC cells. SBC-3 / ADM cells expressing P-gp were highly resistant to etoposide (VP-16), adriamycin (ADM), and vincristine (VCR) in vitro, compared with parental SBC-3 cells lacking P-gp expression. MS-209 restored chemosensitivity of SBC-3 / ADM cells to VP-16, ADM, and VCR in a dose-dependent manner in vitro. Intravenous injection with SBC-3 or SBC-3 / ADM cells produced metastatic colonies in the liver, kidneys and lymph nodes in natural killer (NK) cell-depleted severe combined immunodeficiency (SCID) mice, though SBC-3 / ADM cells more rapidly produced metastases than did SBC-3 cells. Treatment with VP-16 and ADM reduced metastasis formation by SBC-3 cells, whereas the same treatment did not affect metastasis by SBC-3 / ADM cells. Although MS-209 alone had no effect on metastasis by SBC-3 or SBC-3 / ADM cells, combined use of MS-209 with VP-16 or ADM resulted in marked inhibition of metastasis formation by SBC-3 / ADM cells to multiple organs. These findings suggest that MS-209 reversed the MDR of SBC-3 / ADM cells, but not SBC-3 cells, growing in the various organs, and inhibited metastasis formation in vivo. Therefore, this chemosensitizing agent, MS-209, may be useful for treatment of refractory SCLC patients with multiorgan metastases.     

Novel metastasis model of human lung cancer in SCID mice depleted of NK cells

Metastasis is a critical problem in the treatment of human lung cancer. Thus, a suitable animal model of metastasis of human lung cancer is required for in vivo biological and preclinical studies. In this study, we tried to establish a suitable model for this, using SCID mice. Neither human SCLC H69/VP cells (5 × 10⁶) nor squamous-cell carcinoma RERF-LC-AI cells (1 × 10⁶), injected through a tail vein, formed metastases in untreated SCID mice. Pre-treatment of SCID mice with anti-asialo GM₁ serum resulted in only a few metastases of H69/VP cells, but pre-treatment with anti-mouse IL-2 receptor β chain Ab (TM-β1) resulted in numerous lymph-node metastases 56 days after tumor inoculation. H69/VP-M cells, an in vivo-selected variant line, formed significant numbers of lymph-node metastases even in SCID mice pre-treated with anti-asialo GM₁ serum. SCID mice depleted of NK cells by treatment with TM-β1 showed different patterns of metastasis when inoculated intravenously with the 2 different human lung cancer cell lines (H69/VP and RERF-LC-AI cells): H69/VP cells formed metastases mainly in systemic lymph nodes and the liver, whereas RERF-LC-AI cells formed metastases mainly in the liver and kidneys, with only a few in lymph nodes. A histopathological study showed that the metastatic colonies consisted of cancer cells. The numbers of metastatic colonies formed by the 2 cell lines increased with the number of cells inoculated. TM-β1 treatment of SCID mice efficiently removed NK cells from peripheral blood for at least 6 weeks, whereas, after treatment of the mice with anti-asialo GM₁ serum, NK cells were recovered within 9 days. These findings suggest that NK-cell-depleted SCID mice may be useful as a model in biological and pre-clinical studies on metastasis of human lung cancer. © 1996 Wiley-Liss, Inc.     

A New Quinoline Derivative MS-209 Reverses Multidrug Resistance and Inhibits Multiorgan Metastases by P-glycoprotein-expressing Human Small Cell Lung Cancer Cells

Development of distant metastases and acquired multidrug resistance (MDR) are major problems in therapy for human small cell lung cancer (SCLC). MS-209 is a novel quinoline compound, which reverses P-glycoprotein (P-gp)-mediated MDR. We previously reported that MS-209 reversed in vitro MDR of human SCLC (SBC-3/ADM and H69/VP) cells expressing P-gp. In the present study, we determined the therapeutic effect of MS-209 in combination with chemotherapy against multiorgan metastases of MDR SCLC cells. SBC-3/ADM cells expressing P-gp were highly resistant to etoposide (VP-16), adriamycin (ADM), and vincristine (VCR) in vitro , compared with parental SBC-3 cells lacking P-gp expression. MS-209 restored chemosensitivity of SBC-3/ADM cells to VP-16, ADM, and VCR in a dose-dependent manner in vitro. Intravenous injection with SBC-3 or SBC-3/ADM cells produced metastatic colonies in the liver, kidneys and lymph nodes in natural killer (NK) cell-depleted severe combined immunodeficiency (SCID) mice, though SBC-3/ ADM cells more rapidly produced metastases than did SBC-3 cells. Treatment with VP-16 and ADM reduced metastasis formation by SBC-3 cells, whereas the same treatment did not affect metastasis by SBC-3/ADM cells. Although MS-209 alone had no effect on metastasis by SBC-3 or SBC-3/ADM cells, combined use of MS-209 with VP-16 or ADM resulted in marked inhibition of metastasis formation by SBC-3/ADM cells to multiple organs. These findings suggest that MS-209 reversed the MDR of SBC-3/ADM cells, but not SBC-3 cells, growing in the various organs, and inhibited metastasis formation in vivo. Therefore, this chemosensitizing agent, MS-209, may be useful for treatment of refractory SCLC patients with multiorgan metastases.     

Therapeutic efficacy of a new topoisomerase I and II inhibitor TAS-103, against both P-glycoprotein-expressing and -nonexpressing drug-resistant human small-cell lung cancer

We examined the effect of a novel topoisomerase I and II (topo I and II) inhibitor, TAS-103, on P-glycoprotein (P-gp)-expressing and -nonexpressing drug-resistant human small-cell lung cancer (SCLC) cells in vitro and in vivo. We observed that TAS-103 was effective in inhibiting in vitro proliferation of human SCLC (SBC-3 and H69) cells and their drug-resistant variants SBC-3/ADM or SBC-3/CDDP and H-69/VP, respectively. SBC-3/ADM and H-69/VP expressed high P-gp, whereas SBC-3/CDDP did not. TAS-103 also effectively reduced the tumor growth (more than 50% inhibition) of the parental as well as MDR SCLC cells grown SC in nude mice. Adriamycin (ADM) and cisplatin (CDDP), on the other hand, were effective only against the parental cells, while these drugs failed to inhibit the respective drug-resistant variants in vitro or in vivo. TAS-103 was observed to induce apoptosis dose dependently in the parental as well as drug-resistant SCLC cells as analyzed after 48 h of in vitro treatment, suggesting that the stabilization of cleavable topo I- or II-DNA complexes by topo I and II inhibitors like TAS-103 is followed by apoptosis of the cells. Overall, our study suggests that TAS-103 may have clinical application against drug-resistant human SCLC.     

Monoclonal antibody MRK16 reverses the multidrug resistance of multidrug-resistant transgenic mice.

Using multidrug-resistant (MDR)-transgenic mice, whose bone marrow cells express the human MDR1 gene at a level approximately equal to that found in many human cancers, we determined the efficacy of human-specific anti-P-glycoprotein monoclonal antibody MRK16 in overcoming multidrug resistance in an intact animal. MRK16 alone (2 mg) did not significantly affect the WBC counts of the MDR-transgenic mice, but MRK16, as well as the F(ab')2 fragments of MRK16, led to a dose-dependent circumvention of bone marrow resistance against daunomycin, doxorubicin, vincristine, vinblastine, etoposide, and taxol. This sensitizing effect could not be enhanced by combining MRK16 with low molecular weight chemosensitizing agents such as verapamil, quinine, quinidine, or cyclosporin A. We also investigated the concept of specifically targeting and killing multidrug-resistant cells by using MRK16 coupled to Pseudomonas exotoxin (PE). MRK16-PE resulted in a dose-dependent killing of bone marrow cells in MDR-transgenic mice, whereas no bone marrow toxicity was observed in normal control mice. Administration of excess MRK16 prior to injection of MRK16-PE successfully blocked the effect of MRK16-PE. MOPC-PE, a non-MDR-related control monoclonal antibody conjugate, did not target and kill multidrug-resistant bone marrow cells in MDR-transgenic mice. Thus, these immunological approaches to reversing multidrug resistance appear to be both specific and effective.     

Combination Therapy with Antibody and Interleukin–2 Gene Transfer against Multidrug–resistant Cancer Cells

In the present study, we examined the effect of intcrlcukin–2 (IL–2) gene transfer into multidrug resistance (MDR) cancer cells on the therapeutic efficacy of MRK16. Human MDR ovarian cancer cells, AD10, were transduced with a bicistronic IL–2 retrovirus, Ha–IL2–IRES–Neo. The G418resistant population, IL2–AD10, secreted IL–2 into the culture supernatant and did not form a tumor mass in nude mice. The IL2–AD10 cells were more susceptible to the cytotoxicity of murine spleen cells than AD10 cells in vitro. For examination of the effect of IL–2 gene transfer on the antitumor activity of MRK16 against P–glycoprotein–positlve tumors, IL2–AD10 cells were cotransplanted s.c. with AD10 cells into nude mice in a ratio of 1: 3, and the mice were treated with MRK16 on days 2 and 7. MRK16 markedly inhibited the growth of AD10 cells mixed with IL2 AD10 cells under conditions (0.3–1 jag/body) where it showed only marginal effects on the growth of AD10 tumors. These findings suggest that IL–2 gene transfer potentiates the antitumor activity of MRK16 against MDR tumors.     

Amplification and expression of mdr1 gene in a multidrug resistant variant of small cell lung cancer cell line NCI-H69

Amplification and expression of the mdr1 gene encoding P-glycoprotein have been studied in H69/LX4 a multidrug resistant variant (MDR) of small cell lung cancer (SCLC) cell line NCI-H69. Recently a second independently derived MDR variant of this cell line designated H69/AR was found by others not to show amplification, rearrangement or over-expression of the mdr1 gene. The present study reports that in marked contrast to H69/AR, H69/LX4 shows amplification and expression of the P-glycoprotein gene and raises the possibility that P-glycoprotein hyperexpression may be a clinically relevant component of MDR in some SCLC tumours.     

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

Purpose and methods: 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. Results: 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. Conclusion: MS-209 in combination with CsA or VER might increase the efficacy of these chemotherapeutic agents against MDR human SCLC cells. Received: 10 December 1997 / Accepted: 16 April 1998     

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-mediatedcytotoxicity (ADCC by blood mononuclear cells (MNC was examinedin patients with small cell lung cancer (SCLC) before and aftersystemic chemotherapy. The effect of in vitro treatment of MNCwith interleukin (IL)-2 and macrophage-colony-stimulating factor(M-CSF) was also examined. The ADCC reaction was assessed bya 6 h ⁵¹ Cr-release assay using a multidrug-resistant (MDR)SCLC cell line (H69/VP cells). The MRK-16 monoclonal antibodywas able to augment spontaneous cytotoxicity by MNC, even inSCLC patients. Pretreatment of MNC with IL-2 significantly augmentedtheir ADCC ability in SCLC patients, while M-CSF had no effecton ADCC activity. After the first cycle of systemic chemotherapy,the ADCC activity tended to decline, but ADCC of MNC pretreatedwith IL-2 was not affected. The results suggest that anti-P-glycoproteinantibody, in combination with a cytokine such as IL-2, may betherapeutically useful against human SCLC resistant to chemotherapeuticdrugs.     

Characterization of the human MDR3 P-glycoprotein and its recognition by P-glycoprotein-specific monoclonal antibodies.

We have cloned a human MDR3 complementary DNA, coding for a P-glycoprotein, into a mammalian expression vector and cotransfected it with a selectable marker into drug-sensitive human BRO melanoma cells. With low frequency we obtained stable, MDR3-expressing clones. Immunocytochemical and immunoblotting analysis of these clones using the monoclonal antibody C219 indicated that human MDR3 P-glycoprotein, like human MDR1 P-glycoprotein, was mainly localized in the plasma membrane and probably glycosylated. Although a significant fraction of the cells (5-10%) in one of the MDR3-expressing clones expressed as much P-glycoprotein as a clearly drug-resistant MDR1-transfected clone, we found no resistance against a range of drugs affected by multidrug resistance. The drugs tested included vincristine, colchicine, VP16-213, daunorubicin, doxorubicin, actinomycin D, and gramicidin D. We did not detect enhanced daunorubicin efflux either in any of the MDR3-expressing cells by fluorescence microscopy. Direct selection with vincristine, actinomycin D, gramicidin D, or daunorubicin of BRO cells transfected with expression constructs containing the regular MDR3 complementary DNA, or a complementary DNA representing a major MDR3 splice variant (C(-141)), likewise failed to yield resistant clones. Thus, although human MDR3 P-glycoprotein is highly similar to human MDR1 P-glycoprotein, we found no indications that it can transport drugs. We investigated the cross-reactivity of the monoclonal antibodies C219, C494, JSB-1, HYB-241, and MRK16, recognizing human MDR1 P-glycoprotein, with human MDR3 P-glycoprotein using immunocytochemistry and immunoblotting. Apart from monoclonal antibody C219, none of the monoclonal antibodies showed detectable cross-reactivity with human MDR3 P-glycoprotein. In our hands, monoclonal antibodies MRK16 and HYB-241 were most suitable for sensitive and specific cytochemical detection of human MDR1 P-glycoprotein.     

Combined suicide gene and immunostimulatory gene therapy using AAV-mediated gene transfer to HPV-16 transformed mouse cell: decrease of oncogenicity and induction of protection

To test the effects of combined transduction of a suicide gene and genes coding for various immunostimulatory factors on the oncogenicity and immunogenicity of TC-1 cells (HPV-16 transformed C57BL/6 mouse cells), several bicistronic recombinant adeno-associated viruses (rAAV) were constructed. Each of these constructs carried, and in infected cells expressed, the herpes simplex type 1 thymidine-kinase gene (HSV-TK) and the gene of one of the following immunostimulatory factors: human monocyte chemoattractant protein 1 (MCP-1), mouse B7.1 costimulatory molecule (B7.1), or mouse granulocyte-macrophage colony-stimulating factor (GM-CSF). For control purposes, an rAAV carrying the HSV-TK gene and neomycin resistance gene (neo) and an rAAV containing the lacZ gene were used. All of these constructs proved functional both in mouse TC-1 and human 293T cells. For experiments in mice, TC-1 cells were infected in vitro with the AAV recombinants at an input multiplicity of 50 particles/cell; these cells were then administered to 5-week-old mice. As from day 5, half of the animals were given ganciclovir (GCV) (2.5 mg/day) for 10 days. With a single exception, none of the mice inoculated with cells treated with rAAV expressing HSV-TK + B7.1 or HSV-TK + MCP-1 developed tumour irrespective of GCV treatment. The tumour suppressive effect was less marked in animals inoculated with TC-1 cells infected with rAAV expressing HSV-TK + GM-CSF, and among these it was somewhat more pronounced in GCV-untreated animals. A clear antitumour effect of GCV treatment was only observed in mice inoculated with TC-1 cells transduced with rAAV expressing HSV-TK but no immunostimulatory factor. Mice that remained tumour-free on day 54 were challenged with untreated TC-1 cells. The tumour resistance rates found were related not only to the immunostimulatory gene used for the transduction, but also to GCV treatment. The best protection was recorded in mice pre-inoculated with TC-1 cells transduced with either B7.1 or MCP-1-expressing rAAV and not given GCV.     

Mouse-Human Chimeric Antibody MH171 against the Multidrug Transporter P-Glycoprotein

We have developed a mouse-human chimeric antibody MH171, in which the antigen-recognizing variable regions of the mouse monoclonal antibody MRK17 are joined with the constant regions of human IgG1 antibodies. The MRK17 recognizes specifically the multidrug transporter P-glycoprotein and inhibits the growth of human multidrug resistant (MDR) tumor cells in vitro and in the xenograft nude mouse model system. The established chimeric MH171 antibody forms an apparently intact IgG composed of heavy and light chains covalently assembled via disulfide bonds in sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and is specific to MDR cell lines with a similar affinity to the original mouse MRK17. MH171 also displays strong antibody-dependent cell-mediated cytotoxicity to the target cells in vitro , when human mononuclear cells are used as effector cells. The chimeric antibody against P-glycoprotein, MH171, should be a useful agent in the treatment of human drug-resistant tumors.     

Mouse-human chimeric antibody MH171 against the multidrug transporter P-glycoprotein.

We have developed a mouse-human chimeric antibody MH171, in which the antigen-recognizing variable regions of the mouse monoclonal antibody MRK17 are joined with the constant regions of human IgG1 antibodies. The MRK17 recognizes specifically the multidrug transporter P-glycoprotein and inhibits the growth of human multidrug resistant (MDR) tumor cells in vitro and in the xenograft nude mouse model system. The established chimeric MH171 antibody forms an apparently intact IgG composed of heavy and light chains covalently assembled via disulfide bonds in sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and is specific to MDR cell lines with a similar affinity to the original mouse MRK17. MH171 also displays strong antibody-dependent cell-mediated cytotoxicity to the target cells in vitro, when human mononuclear cells are used as effector cells. The chimeric antibody against P-glycoprotein, MH171, should be a useful agent in the treatment of human drug-resistant tumors.     

逆转录病毒载体介导MDR1基因转染人外周血造血干细胞的研究

目的：多药耐药基因（ＭＤＲ１）的异常扩增和过度表达是肿瘤细胞产生多药抗药性的重要原因之一。本研究试图将ＭＤＲ１导入骨髓或外周血造知干细胞并使入获得耐药表型,以用于减轻大剂量化疗对造血细胞的毒性。方法：应用反转录病毒载体ｐＨａＭＤＲ１／Ａ将ＭＤＲ１阳性。ＰＣＲ法检测转染细胞所 ＣＦＵ－ＧＭ落集中,外源ＭＤＲ１阳性比例为１／４;而未转染细胞所形成的ＣＦＵ－ＧＭ集匀为阴性。集落形成试验证明,转染和非转染