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Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115 [C. S. M., N. S. M., D. C., N. M., S. P. T., T. H., K. C. A.]; Departments of Medicine [C. S. M., N. S. M., D. C., N. M., S. P. T., T. H., K. C. A.] and Pathology [R. T. B.], Harvard Medical School, Boston, Massachusetts 02115; Cross Cancer Institute, University of Alberta, Edmonton, Alberta, T6G 1Z2 Canada [C. A. M., L. P.]; and Department of Surgery, University of California at San Diego, San Diego, California 92111 [R. M. H.]; and AntiCancer, Inc., San Diego, California 92111 [R. M. H.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To address these limitations of conventional in vivo models for hematological malignancies, we developed an in vivo model of MM where MM cells stably transfected with construct for the GFP of the jellyfish Aequorea Victoria are injected i.v. in SCID/NOD mice: the subsequent development of diffuse MM lesions can be monitored by whole-body fluorescence imaging, which detects the fluorescence emitted by the GFP+ MM cells when illuminated with near-infrared light. Because of the marked visual contrast generated between fluorescent GFP+ MM cells and nonfluorescent GFP- normal tissues of the host, we were able to visualize noninvasively and monitor serially, the anatomical distribution of MM lesions in the intact mouse, including s.c. tissues, bone, and BM, as well as visceral sites of tumor infiltration.
This first GFP-based in vivo model of a hematological malignancy represents a new avenue of research distinct from previous applications of fluorescence imaging in solid tumor models (5, 6, 7, 8, 9, 10, 11, 12, 13) . Herein, this GFP model recapitulates the diffuse bone disease of MM, and importantly, the anatomical distribution of GFP+ lesions, confirmed by flow cytometry and extensive histopathological analyses, is consistent with the distribution of tumors in MM patients and, in particular, their preferential development in sites of the axial skeleton. Therefore, this study not only provides a clinically relevant in vivo model for more accurate preclinical evaluations of investigational therapies against MM but also generates a reproducible framework to characterize the interactions of MM cells with the BM milieu in vivo, with particular emphasis on the preferential targeting by MM cells of distinct areas of the skeleton. Because the BM microenvironment is now emerging as a key aspect of the pathophysiology of other hematological malignancies and not just MM, our study constitutes a framework for development of similar models for those diseases as well.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MTT Colorimetric Survival Assay.
RPMI-8226/S cells transfected with GFP or control (neo) vector were incubated with 250 ng/ml doxorubicin (Sigma Chemical Co., St. Louis, MO) for 48 h, and cell survival was assessed by MTT colorimetric survival assay (14)
.
SCID/NOD Mice and MM Cell Injection.
A cohort of 85 male (6–8-week old) immunodeficient SCID/NOD mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and were housed and monitored in the Animal Research Facility of the Dana-Farber Cancer Institute. All experimental procedures and protocols had been approved by the Institutional Animal Care and Use Committee. Mice were 
-irradiated (300 rads) using a 137Cs -irradiator source. After 24 h, 75 mice received tail vein injections of 5 x 106 RPMI-8226/S-GFP+ cells suspended in a total volume of 100 µl PBS/mouse, whereas a control cohort of 10 mice received equal volume of PBS i.v. injections. Mice were monitored daily for changes in their body weight, signs of infection, and paralysis and thrice weekly by fluorescence imaging. In accordance with institutional guidelines, mice were sacrificed by CO2 inhalation in the event of paralysis or major compromise in their quality of life.
Whole-Body Real-Time Fluorescence Imaging.
The development of fluorescent MM lesions in the skeleton and extraskeletal sites was periodically monitored by whole-body fluorescence imaging using the LT-9500 fluorescent light box (Lightools Research, Encinitas, CA). Fluorescence excitation of GFP tumors was produced through a 440 ± 20-nm interference filter (excitation filter) using slit fiber optics for animal illumination. Fluorescence was observed through a 520-nm long pass filter (viewing filter). Fluorescence imaging results were digitally captured on a FujiFilm FinePix 6800Z digital camera (FujiFilm USA, Edison, NJ) and analyzed with Adobe PhotoShop 7.0 and Image Pro Plus v.4.0 software (Media Cybernetics, Silver Spring, MD). Before fluorescence imaging, cutaneous hair of the mice was removed with Nair (Carter-Wallace, New York, NY).
Fluorescence Imaging-Guided Necropsy.
During necropsy of sacrificed mice, fluorescence imaging-guided macroscopic inspection of internals organs was performed after generation of skin flaps (to evaluate whether the autofluorescence of GFP+ tumors is attenuated by the skin), as well as during the preparation of the spine, lung, liver, and spleen after the dissection of each specimen but before additional processing for histopathological analysis. Samples from the spine, skull, pelvis, extremities, thoracic cage, s.c. tissue, or any other organ that contained fluorescing lesions (as well as control samples obtained from sites adjacent to fluorescent lesions or from unaffected contralateral sites) were processed for conventional histopathological examination.
Flow-Cytometric Analysis of s.c. Plasmacytomas and BM Aspirates.
s.c. plasmacytomas from GFP+-injected mice (or s.c. tissue from control mice) were removed during necropsy and kept in PBS until processing for preparation of single-cell suspensions. BM aspirates from both femoral bones were obtained from all GFP+-injected and control mice by flushing PBS into the marrow cavity of femoral bones and were processed for RBC lysis. All samples were then analyzed with flow cytometry, using RPMI-8226/S-GFP+ cells growing in monolayer cultures as positive controls and neo (vector)-transfected RPMI-8226/S cells as negative controls. Counterstaining of GFP+ MM cells was performed with phycoerythrin-conjugated antihuman CD38 monoclonal antibody (Beckman Coulter, Miami, FL). All flow cytometric analysis (single color-flow cytometry for assessment of GFP+ and dual color cytometry for GFP+ status and CD38+ expression) was performed according to previously described protocols (14, 15, 16)
and analyzed on an Epics-XL-MCL flow cytometer (Beckman Coulter).
Statistical Analysis.
The overall survival of mice was defined as the time between i.v. injection of tumor cells and sacrifice or death. Kaplan-Meier survival analysis was performed as described previously (17)
. To confirm that differences in frequency of development of tumor lesions in individual organs are statistically significant, we calculated the 95% CI of each frequency as described previously (18)
. A difference in frequencies of tumor involvement for two organs or tissues was considered significant (P < 0.05) if the 95% CI of two compared frequencies of tumor involvement had no overlap (18)
.
Fluorescent Imaging Cross-Sections.
After acquisition of external fluorescent images, 15 of the 80 mice of our cohort were kept at -80°C for 12–24 h, then sliced in cross-section at 
10-mm thickness using disposable microtome blades (Model 818; Leica) The sections were observed both under fluorescence microscopy or fluorescence light box imaging, simulating the acquisition of tomographic images to confirm the tumor localization determined by external visualization using whole-body fluorescence imaging.
Histolopathological Analysis.
Immediately after fluorescence imaging-guided necropsy, specimens from tissues with fluorescent lesions, as well as representative samples of nonfluorescent areas of several organs (lungs, liver, spleen, and so on) were fixed in 10% buffered formalin; skeletal tissues were decalcified with 14% EDTA and embedded in paraffin by previously described standard techniques (19
, 20)
. Soft tissues were fixed in 10% buffered formalin and embedded in paraffin without prior decalcification. Nonconsecutive sections were cut longitudinally using a standard microtome through the sagittal plane of the lumbar vertebrae and through blocks of liver, spleen, lung, kidney, intestines, s.c. tissue, and other specimens in each animal. The sections were then placed on poly-L-lysine-coated glass slides and stained with H&E (19
, 20)
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Whole-Body Radiographic Analysis.
After sacrifice, whole body radiographs were obtained with a Faxitron radiographic inspection unit (Field Emission Corporation, Inc., McMinnville, OR) as described previously (21)
. After tissue dissection, removal of the skin, and fixation in 10% buffered formalin, similar radiographs were also taken of the limbs, spine, and calvaria.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. GFP+ cells retained bright GFP fluorescence after numerous passages in the absence of selective agents, whereas their responsiveness to conventional anti-MM drugs, e.g., doxorubicin, did not significantly differ from control RPMI-8266/S cells (Fig. 1B)
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. Table 1
summarizes the anatomical distribution (with 95% CIs of site-specific frequencies) of MM lesions in a cohort of 75 SCID/NOD mice that received injected with RPMI-8226/S-GFP+ cells. The overwhelming majority of mice (74 of 75 mice, 98.7%, 95% CI: 92.9–100) developed skeletal lesions. Specifically, 72 of 75 mice (96.0%, 95% CI: 88.9–99.7) developed lesions in their spine, primarily lumbar (57 of 75 mice, 76.0% of mice, 95% CI: 65.2–84.2), and thoracic (39 of 75 mice, 52.0%, 95% CI: 40.8–63.0) vertebrae, whereas in a great majority of cases (64 of 75 mice, 85.3%), MM lesions were detected in the skull. Extraskeletal lesions were also formed, including s.c. plasmacytomas in 50 of 75 mice (66.7%, 95% CI: 55.4–76.3), and plasmacytomas in soft tissues of the posterior cervical area in 49 of 75 mice (65.3%, 95% CI: 54.0–75.1). Interestingly, however, visceral MM lesions in lung, liver, spleen, or kidney developed only rarely (<5% of mice). The development of spinal lesions was associated with hind limb paralysis (78.8% of mice), after a median of 29 days (range, 22–43 days), prompting sacrifice of mice per protocol (Fig. 3)
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Analysis of in Vivo GFP+ Expression.
s.c. plasmacytomas from GFP+ cell-injected mice or s.c. tissue samples from control mice were removed immediately after sacrifice and kept in cold 1x PBS. Single-cell suspensions were prepared and analyzed by flow cytometry to compare their GFP-derived fluorescence with RPMI-8226/S-GFP+ cells cultured in vitro as positive control versus non-GFP-expressing RPMI-8226/S cells as negative control. After several weeks of growth in vivo, RPMI-8226/S-GFP+ cells explanted from s.c. plasmacytomas did not significantly differ in their GFP+ expression from GFP-transfected cells cultured in vitro during the same period of time (Fig. 4A)
, consistent with previous reports of stable long-term fluorescence of tumor cell lines transfected with GFP vectors (5, 6, 7, 8, 9, 10, 11, 12, 13)
. Importantly, tumor cells explanted from these s.c. plasmacytomas were subsequently cultured in vitro for several months, confirming their viability, which is consistent with prior studies that GFP-fluorescent lesions represent viable cells (5, 6, 7, 8, 9, 10, 11, 12, 13)
rather than accumulations of cellular debris or cells internalizing fluorescent protein from dead GFP+ cells. Moreover, GFP protein irreversibly loses its fluorescence in the event of cell death (R. M. Hoffman, unpublished observations).
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. Additional flow cytometric analyses confirmed cell surface expression of human CD38, which is expressed by RPMI-8226/S cells on fluorescent cells from affected bones (Fig. 4C)
, providing additional support of specific homing of GFP+ MM cells to the BM. Interestingly, flow cytometric analyses of BM aspirates from femurs with bilateral involvement of GFP+ lesions demonstrated variability in the extent of BM infiltration by GFP+ MM cells, reflecting the heterogeneity of BM infiltration with MM cells noted clinically (Fig. 5)
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depicts representative H&E staining of histological sections of bone, confirming homing of malignant plasma cells to BM at distinct skeletal sites.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3 years, which has not significantly improved over the last three decades (22)
. Recent preclinical and early clinical studies indicate that new therapies, which target not only MM cells but also their interactions with the host BM microenvironment (14, 15, 16
, 23, 24, 25, 26, 27)
, may improve outcome of MM patients. Nonetheless, MM is still considered an invariably fatal disease, highlighting the urgent need for accelerated bench-to-bedside translation of investigational therapies. This necessitates the development of preclinical in vivo models that accurately simulate the clinical presentation of the disease in patients and, in particular, the homing of MM cells to the BM, a critical process in the pathophysiology of MM (28)
, and other hematological malignancies. Unfortunately, currently available animal models for MM are not optimal because (a) conventional models of s.c. implanted malignant cells are not consistent with the diffuse systemic nature of hematological malignancies and, in particular, do not reflect the close interaction of MM cells with the local BM milieu; and (b) diffuse tumors developing after i.v. injection cannot be readily detected and serially followed-up by conventional imaging modalities, e.g., radiographic examination is often not informative of soft tissue lesions and generally is not sufficiently sensitive to monitor tumor-associated skeletal disease (29)
because in rodents, which are much smaller than the human body, tumors are also much smaller and often fall below the threshold for radiographic detection (30)
. More sensitive imaging modalities, e.g., computerized tomography or magnetic resonance imaging, currently have very high cost, which prohibits serial measurements required for large-scale screening studies of new anticancer therapies. Furthermore, the comprehensive histopathological analyses of the entire body of experimental animals might help define the distribution of tumor cells in various organs and tissues but can only be performed after necropsy and not serially during the course of administration of an antitumor regimen. The current study fulfills the major prerequisites for a clinically relevant in vivo model of MM, namely the development of diffuse bone lesions, with anatomical distribution and resulting manifestations (e.g., paralysis) consistent with the clinical picture of the disease in human patients and the ability to perform, with high sensitivity and specificity, noninvasive, real-time monitoring of the precise localization of tumor lesions. Indeed, MM cells, malignant counterparts of postgerminal center B-cells (28) , are considered home to the BM after the venous and subsequently the arterial circulation. In that respect, our model of SCID/NOD mice injected i.v. with MM cells stably expressing GFP constitutes an orthotopic model and allows these cells to reach their preferred sites of homing, i.e., the bone and form diffuse skeletal lesions, primarily in the axial skeleton, in a manner consistent with the clinical presentation of the disease. The significant visual contrast generated by the fluorescence of GFP+ tumors versus adjacent nonfluorescent healthy tissues allows for detailed analysis by whole-body fluorescence imaging of total number and size of even small tumors, which might otherwise escape detection. The use, during necropsy of sacrificed mice, of fluorescence imaging-guided sampling of tissue specimens with fluorescent lesions, and subsequent histopathological analyses confirmed, in this study, the very high sensitivity and specificity of whole-body fluorescent imaging in this GFP+ model. The selective noninvasive macroscopic visualization of tumor burden without exogenous use of contrast agents, radioactivity, fluorescent antibodies, or other preparative procedures that might limit the application of this model or confound interpretation of its results (13) , allows for minimal alterations in tumor cell behavior in vivo (31) . Furthermore, collection of whole-body fluorescence imaging data does not require (in contrast to other imaging modalities) lengthy exposures of immobilized animals and was performed with only short-acting anesthetics (e.g., isofluorane), which did not adversely impact on the survival of mice, in this study.
Our study also indicates that in mice with diffuse MM lesions, the overall survival and quality of life are not affected by the total tumor burden in a strictly proportional manner. Indeed, vertebral bone lesions (which can be of a relatively small size in comparison to the overall tumor burden) were the cause of paralysis and sacrifice in the overwhelming majority of mice, whereas lesions in other less critical skeletal or extraskeletal (e.g., s.c. tissues) areas with greater local tumor involvement did not have the same impact on the course of the disease. These findings suggest that systemic markers of total tumor burden (e.g., serum or urine levels of monoclonal immunoglobulins in MM or other plasma cell dyscrasias), although informative of its overall changes during the course of the disease, may not reflect how individual tumor lesions at various organs can differentially affect the survival and quality of life of mice. Importantly, the critical role of the local bone microenvironment in conferring drug resistance not only to MM cell but also to cells from other osteotropic malignancies (3 , 32) indicates that systemic markers of total tumor burden may not reliably reflect how tumor cells homing in different organs may differentially respond to therapy because of differential interactions within the distinct local microenvironments of the various host tissues.
Because of these differential, site-dependent interactions of tumor cells with the local microenvironment, it is important for in vivo animal models of systemic malignancies such as MM to mimic the patterns of organ involvement, clinical course, and manifestations of the disease in patients. In this study, the SCID/NOD model of diffuse GFP+ MM lesions simulated the clinical behavior of MM more closely than conventional models of ectopic s.c. implantation of human MM cells (6) because >95% of mice injected with GFP+ MM cells developed bone lesions at one or more sites in the spine, and at least one tumor site in the axial skeleton was noted in 98% of SCID/NOD mice, which mirrors the osteotropic behavior of MM in patients (30) . Importantly, GFP+ MM lesions targeted mainly the axial skeleton, which is consistent with the actual pattern of anatomical distribution of MM bone disease observed clinically (30) . The clinical relevance of this model is additionally highlighted by the high incidence of clinical manifestations directly attributed to bone lesions, including the development of paralysis secondary to vertebral MM involvement.
Because of its clinical relevance, this GFP+ MM mouse model provides a valuable experimental setting to preclinically evaluate the in vivo antitumor activity of investigational agents. Indeed, recent studies of our group have applied this model not only to evaluate the antimyeloma activity of new therapeutic approaches such as the heat shock protein 90 inhibitors (33) but also to elucidate the in vivo molecular sequelae of such therapies and confirm that they are consistent with their in vivo mechanism(s) of action. This is possible because GFP+ MM cells can be purified on the basis of their fluorescence using flow cytometry-based cell sorting from nonfluorescent normal host cells and subsequently analyzed by conventional or high-throughput molecular studies of gene expression or proteomic profile (33) . Of note, in ongoing studies from our group using other GFP-expressing human MM cell lines (e.g., MM-1S, MM-1R, OPM-1) we have also found formation of MM bone lesions in SCID/NOD mice, similarly to the current results with RPMI-8226/S-GFP cells.5 Although the precise anatomical pattern of lesions generated by each line can depend on its individual molecular characteristics (and can potentially differ among different sublines or subclones of the same line if these exhibit different osteotropic potential), the tendency of MM cells for establishment of bone lesions is not limited to RPMI-8226-derived cells. Furthermore, these findings indicate that fluorescence imaging of diffuse MM bone lesions is applicable for a broader spectrum of MM cell lines. In addition, whole-body fluorescence imaging for detection of MM cells can also be incorporated in the setting of other of other in vivo MM models such as syngeneic mouse models of MM (e.g., by GFP-expressing 5T2 mouse MM cells injected in C57BL/KaLwRij mice; Ref. 34 , 35 ), as well as in the SCID-hu model of MM (36 , 37) . In this latter model, direct injection of human MM cells in human bone grafts implanted in SCID mice leads to osteolytic lesions in the injected graft, as well as to a secondary (metastatic) bone lesion in contralateral human bone grafts not injected with MM cells. These models, which have provided important insight in the pathophysiology of the osteolytic lesions of MM, may be complemented by injection of GFP+ human MM cells to allow for better monitoring of the MM tumor burden (and its response to therapy) in the bone grafts and to facilitate the characterization of interactions between MM cells and the BM microenvironment.
Furthermore, because of its clinical relevance, this in vivo model is not only appropriate for preclinical evaluation of potential new therapies but also for addressing important questions regarding the pathophysiology of MM. Of particular biological significance is the marked osteotropism of MM cells and their heterogeneous but not random skeletal distribution. The degree of BM infiltration by GFP+ MM cells, as assessed by flow cytometry of BM aspirates, was not uniform but heterogeneous and analogous to the variable percentage of BM infiltration by malignant plasma cells in different skeletal sites in the same patients noted clinically. Furthermore, detailed statistical analyses, including calculation of 95% CIs for the frequencies of tumor involvement in individual organ sites confirmed that the distribution of tumor lesions was not random and involved preferential bone involvement in the bones and, primarily, in the axial skeleton, which is characterized by the presence of trabecular bone, a major site of MM bone involvement in human patients. The precise etiology for this site-specific pattern of tumor formation is under investigation using this GFP+ MM model. It conceivable that the mechanism underlying this site specificity is multifactorial and could include heterogeneous expression of cytokines, growth factors, and/or adhesion molecules in different parts of the skeleton. Because MM cell proliferation and survival is significantly enhanced by BM-derived cytokines such as insulin-like growth factors (38) and interleukin 6 (32) or by binding of MM cells to the ECM or stromal cells in the BM (2) , it is possible that skeletal areas with most frequent establishment of MM lesions represent areas with, e.g., higher local production of proliferative/antiapoptotic factors for MM cells, enhanced activity of BMSCs, and/or expression of specific ECM components. Interestingly, axial skeletal areas most frequently targeted by MM lesions also harbor active red BM (39) , a principal site for adult hematopoiesis: the very active local network of BMSCs in those sites may not offer support via cytokine- or cell adhesion-mediated pathways not only for normal hematopoietic lineages but also for locally seeded tumor cells. The nonrandom distribution of MM lesions could also be due, at least in part, to well-documented organ- or tissue-specific molecular heterogeneity of the vascular tree (40 , 41) . Indeed, in vivo screening of phage-displayed peptide libraries has identified peptide sequences capable of homing selectively to the vasculature of specific organs (42) . Such organ- or tissue-specific vascular markers may not only facilitate organ-specific blood-tissue exchange of biomolecules or selective homing of normal cells, e.g., migration of neutrophils to inflamed tissues, lymphocyte homing to lymphoid tissues (43, 44, 45) , but may also function as receptors for metastasizing malignant cells (46, 47, 48) , thus determining, e.g., the osteotropism of MM cells. Our study’s finding of more frequent bone lesions in the axial skeleton raise the hypothesis that the concept of heterogeneous molecular signature of the vasculature may apply not only to different tissues but maybe to different parts of the same organ or tissue, e.g., in axial skeleton versus other bone regions. Determination of such axial skeleton-specific endothelial markers could serve to design new therapeutic strategies specifically delivering antitumor agents in sites of predominant MM cell homing as in previous studies of selective vascular targeting of metastatic disease (49 , 50) .
Although GFP+ MM cells predominantly homed to the bones, extraskeletal tumors were also observed in our model. Some of these plasmacytomas, e.g., paraspinal soft tissue GFP+ lesions, resulted from direct extension of plasma cell infiltration from adjacent bone lesions. Other extraskeletal tumors such as s.c. plasmacytomas may reflect the derivation of RPMI-8226/S cells from circulating malignant plasma cells of a patient with advanced MM (plasma cell leukemia; Ref. 51 ). At this stage of the disease, MM cells have acquired additional genetic abnormalities, allowing them to survive in the absence of interaction with the BM microenvironment. Importantly, however, the striking osteotropic behavior of these cells, with bone involvement in almost all mice of our study, indicates that the BM milieu can still play an important role in promoting the proliferation and survival of even very aggressive MM cells.
This study, which represents a first application of whole-body fluorescence imaging in vivo models for the study of MM may also be applied to evaluate the development of diffuse lesions in other hematological malignancies, provides the context for additional characterization of disease pathogenesis and, importantly, establishes a clinically relevant framework for preclinical testing of new therapeutic strategies.
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FOOTNOTES |
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1 Supported by the Multiple Myeloma Research Foundation (to C. S. M., N. M.), Lauri Strauss Leukemia Foundation (to C. S. M., N. M.), International Waldenstrom’s Macroglobulinemia Foundation (to C. S. M.), National Institutes of Health Grants RO-1 50947 and PO-1 78378, and Doris Duke Distinguished Clinical Research Scientist Award (to K. C. A.). C. S. M. is a Special Fellow of the Leukemia and Lymphoma Society. 
2 To whom requests for reprints should be addressed, at Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston MA 02115. Phone: (617) 632-1962; Fax: (617) 812-7701; E-mail: Constantine_Mitsiades{at}dfci.harvard.edu and Kenneth_Anderson{at}dfci.harvard.edu
3 Both authors contributed equally to this work.
4 The abbreviations used are: MM, multiple myeloma; BM, bone marrow; BMSC, BM stromal cell; ECM, extracellular matrix; SCID/NOD, severe combined immunodeficient/nonobese diabetic; GFP, green fluorescent protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CI, confidence interval.
5 C. S. Mitsiades et al., unpublished observation.
Received 3/16/03. Revised 6/ 2/03. Accepted 7/ 1/03.
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) versus control RPMI-8226/S transfected with empty (neo) vector (
). B, after a 48-h incubation with doxorubicin (250 ng/ml), MTT colorimetric assay indicates that there is no difference in the survival of RPMI-8226/S control cells versus RPMI-8226/S-GFP+ cells.
) in G418 (500 µg/ml) selection medium for the entire duration of the in vivo experiment is compared with non-GFP-transfected RPMI-8226/S cells (
