Tumor imaging with multicolor fluorescent protein expression

Imaging with fluorescent proteins has been revolutionary and has led to the new field of in vivo cell biology. Many new applications of this technology have been developed. Green fluorescent protein (GFP)-labeled or red fluorescent protein (RFP)-labeled HT-1080 human fibrosarcoma cells were used to determine clonality of metastasis by imaging of metastatic colonies after mixed implantation of the red and green fluorescent cells. Resulting pure red or pure green colonies were scored as clonal, whereas mixed yellow colonies were scored as nonclonal. Dual-color fluorescent cancer cells expressing GFP in the nucleus and RFP in the cytoplasm were engineered. The dual-color cancer cells enable real-time nuclear–cytoplasmic dynamics to be visualized in living cells in vivo, including mitosis and apoptosis. The nuclear and cytoplasmic behavior of dual-color cancer cells in real time in blood vessels was observed as they trafficked by various means or extravasated in an abdominal skin flap. Dual-color cancer cells were also visualized trafficking through lymphatic vessels where they were imaged via a skin flap. Seeding and arresting of single dual-color cancer cells in the lung, accumulation of cancer-cell emboli, cancer-cell viability, and metastatic colony formation were imaged in real time in an open-chest nude mouse model using assisted ventilation. Novel treatment was evaluated in these imageable models. UVC irradiation killed approximately 70% of the dual-color cancer cells in a nude mouse model. An RFP-expressing glioma was transplanted to the spinal cord of transgenic nude mice expressing nestin-driven green fluorescent protein (ND-GFP). In ND-GFP mice, GFP is expressed in nascent blood vessels and neural stem cells. ND-GFP cells staining positively for neuronal class III-β-tubulin or CD31 surrounded the tumor, suggesting that the tumor stimulated both neurogenesis and angiogenesis. The tumor caused paralysis and also metastasized to the brain. The Salmonella typhimurium A1-R tumor-targeting bacterial strain was administered in the orthotopic spinal cord glioma model. The treated animals had a significant increase in survival and decrease in paralysis. S. typhimurium A1-R was effective against primary bone tumor and lung metastasis expressing RFP in a nude mouse model. S. typhimurium A1-R was effective against both axillary lymph and popliteal lymph node metastases of human dual-color pancreatic cancer and fibrosarcoma cells, respectively, as well as lung metastasis of the fibrosarcoma in nude mice. Imaging with fluorescent proteins will reveal mechanisms of cancer progression and provide visual targets for novel therapeutics.     

Color coding cancer cells with fluorescent proteins to visualize in vivo cellular interaction in metastatic colonies

We report here in vivo visualization of cancer cell interaction using stable sublines of green fluorescent protein (GFP)- and red fluorescent protein (RFP)-expressing HT-1080 human fibrosarcoma cells. These color-coded fibrosarcoma cell lines show similar cell proliferation and lung metastasis potential. The color-coded cells were mixed at a ratio of 1:1 and were injected into the tail vein of severe combined immunodeficient (SCID) mice. The resulting experimental lung metastases were simultaneously imaged and their ratio was determined by color-pixel analysis. Fluorescence color coding of cancer cells enables visualization of the interaction of cancer cells and can be used to distinguish cancer cells of any genotype or phenotype.     

Comparison of 'spontaneous' and 'experimental' metastasis using rat 13762 mammary adenocarcinoma metastatic cell clones

Rat 13762NF mammary adenocarcinoma cloned cell lines were assayed at different in vitro passage numbers and compared for their abilities to form 'spontaneous' metastases by subcutaneous injection of cells and 'experimental' metastases by intravenous injection of cells. Tumor cell clones were established from locally growing tumor and spontaneous lung metastases, and these clones were found to possess heterogeneous metastatic potentials in both metastasis assays. The rank order of clonal metastatic potentials based on either the average number of lung tumor colonies or the average total lung tumor volume was generally equivalent for 'spontaneous' and 'experimental' metastases, but some differences were noted. Ranking of 'spontaneous' metastasis by average total lung tumor volumes more closely resembled the rank order of 'experimental' metastasis than by the average number of spontaneous metastases. The results demonstrated that in the 13762NF mammary adenocarcinoma system (i) there is heterogeneity in tumor cell clonal metastatic potential using either 'spontaneous' or 'experimental' assays; (ii) these two assay methods yield generally the same rank order of metastatic potential; (iii) the metastatic potential of each of the tumor cell clones drifts with time (passage number) in cell culture, and (iv) ranking by average tumor burden calculated from total lung tumor volumes may yield a better estimate of metastatic potential than ranking by the average number of lung tumor colonies.     

Viable circulating metastatic cells produced in orthotopic but not ectopic prostate cancer models

Elucidation of the mechanisms of hormone-independent metastatic prostate cancer remains a significant and highly relevant challenge. We report here that hormone-refractory human prostate carcinoma growing orthotopically efficiently deliver viable metastatic cells in the host circulation. This is in contrast to the ectopic tumors of the same lineage, which do not deliver live cells into the circulation. To investigate the malignant potential of viable circulating carcinoma cells, we developed a novel dual-color orthotopic coimplantation model of human prostate cancer metastasis in nude mice. This model is comprised of coinjection of an equivalent mixture of isolated and cultured circulating green fluorescent protein-expressing clones and parental red fluorescent protein-expressing human prostate carcinoma cells. In the dual-color model, the selected green fluorescent protein-labeled viable circulating cells have an increased metastatic propensity relative to the red fluorescent protein-labeled parental cells. The identification and isolation of highly malignant viable circulating human prostate carcinoma cells from orthotopic but not ectopic models will enable important new insights into the metastatic process including the role of the tumor microenvironment.     

Imaging the inhibition by anti-β1 integrin antibody of lung seeding of single osteosarcoma cells in live mice

Integrins play a role in tumor growth and metastasis. However, the effect of integrin inhibition has not been visualized on single cancer cells in vivo. In this study, we used a powerful subcellular in vivo imaging model to demonstrate how an anti-integrin antibody affects seeding and growth of osteosarcoma cells on the lung. The 143B human osteosarcoma cell line, expressing red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) in the nucleus, was established. Such double-labeled cells enable imaging of apoptosis and mitosis and other nuclear-cytoplasmic dynamics. Using the double-labeled osteosarcoma cells, single cancer-cell seeding in the lung after i.v. injection of osteosarcoma cells was imaged. The anti-β1 integrin monoclonal antibody, AIIB2, greatly inhibited the seeding of cancer cells on the lung (experimental metastasis) while a control antibody had no effect. To image the efficacy of the anti-integrin antibody on spontaneous metastasis, mice with orthotopically-growing 143B-RFP cells in the tibia were also treated with AIIB2 or control anti-rat IgG1 antibody. After 3 weeks treatment, mice were sacrificed and primary tumors and lung metastases were evaluated with fluorescence imaging. AIIB2 significantly inhibited spontaneous lung metastasis but not primary tumor growth, possibly due to inhibition of lung seeding of the cancer cells as imaged in the experimental metastasis study. AIIB2 treatment also increased survival of mice with orthotopically growing 143B-RFP.     

Intrapulmonary spread of established B16 melanoma lung metastases and lung colonies

Spontaneous metastasis of subcutaneous B16 melanoma transplants proceeds in two distinct stages: initially to the lungs, and secondarily, following tumor removal, from established lung metastases to extrapulmonary systemic sites. Coincident with extrapulmonary metastasis, there is a dramatic amplification of visible lung metastases, with death generally resulting from extensive lung metastasis. The progression of lung metastasis, and lung colonization initiated by intravenous injection of tumor cells, was investigated using B16 melanoma clone G3.12. Analysis of the growth of invisible metastases in organ culture explants of lung revealed that tumors continually disseminated relatively small numbers of lung metastases after reaching a size of about 6 mm in diameter. However, most terminal-stage lung metastases, along with all extrapulmonary metastases, apparently arise from a secondary spread of tumor cells from tumor-derived lung metastases 1-2 mm in size. Individual lung colonies, initiated with G3.12 cells bound to single microbeads, also disseminated large numbers of secondary lung colonies, as well as extrapulmonary colonies, at a 1- to 2-mm size. The mechanism for intrapulmonary spread of secondary metastases and colonies is unclear, but the consequence appears to be a secondary stage of intrapulmonary and extrapulmonary metastasis with selection for tumor cells with rapid growth rates.     

Lectin-binding patterns in transplantable mouse mammary tumors and their metastases.

Lectin binding was assessed in a transplantable pregnancy-dependent mouse mammary tumor line (TPDMT-4), its autonomous sublines (T4-0196 and T4-01165) and their artificial metastases (lung colonies), using the avidin-biotin-peroxidase technique. Soybean agglutinin (SBA) and peanut agglitinin (PNA) bound to the luminal surfaces of TPDMT-4 tumor cells, while dolicos biflorus agglutinin (DBA) showed no binding. In T4-0196 and T4-01165 tumors as well as their lung metastases, SBA and PNA binding was mixed and both positive and negative cells were detected, indicating that these lectins were not associated with the metastatic phenotype. Although the T4-0196 and T4-01165 sublines had a mixture of DBA-positive and DBA-negative cells, all the metastatic T4-0196 subclones contained only DBA-positive cells and all the metastatic T4-01165 subclones had DBA-negative cells. Thus DBA-positive, and DBA-negative subclones had respectively metastasized to the lungs from these autonomous sublines, implying that the carbohydrate moieties detected by DBA were not associated with metastatic potential but that the lung metastases were clonal in origin.     

Evidence that intravenously derived murine pulmonary melanoma metastases can originate from the expansion of a single tumor cell

The purpose of these studies was to determine whether hematogenous clonal pulmonary melanoma metastases originate from the expansion of a single cell and if so, by extrapolation, metastasis can be considered a cloning process. Three different cell lines of murine K-1735 melanoma with different metastatic properties and unique karyotypes were injected i.v. into syngeneic C3H/HeN mice as multicell aggregates of individual cell lines or combinations of cell lines. Resultant solitary lung metastases were isolated in culture as individual lines and then karyotyped. Even when heterogeneous clumps of tumor cells were injected, the individual metastases exhibited a karyotype unique to one metastatic cell type. Furthermore, when cellular aggregates were composed of metastatic cells admixed with cells that were tumorigenic but nonmetastatic, the resultant metastases exhibited only the karyotype of the metastatic cells. This finding suggests that the presence of metastatic cells did not change the inability of nonmetastatic cells to proliferate in a distant organ. Collectively, the results indicate that the resultant metastases were of clonal origin owing to the expansion of a single metastatic tumor cell in the lung parenchyma.     

High frequency of clonally related tumors in cases of multiple synchronous lung cancers as revealed by molecular diagnosis.

In patients with multiple synchronous lung tumors, discrimination of multicentric lung cancers from intrapulmonary metastasis is important for treatment decision, but this is sometimes difficult. The aim of this study was to retrospectively distinguish multicentric lung cancers from intrapulmonary metastases in 14 such cases by loss of heterozygosity (LOH) and p53 mutational status. DNA was extracted from microdissected tumor cells in paraffin-embedded archival tissue, and 3p14.2, 3p21, 3p25, 9p21, and 18q21.1 were investigated for LOH. Exons 5-8 of the p53 gene were examined for mutations by the PCR, followed by single-strand conformation polymorphism analysis and DNA sequencing. For cases with the same LOH pattern, we calculated a clonality index, the probability of the given LOH pattern when these tumors were hypothesized to be independent in origin. Eleven of 14 cases (79%) were thus diagnosed as having pulmonary metastasis and only one case as having genuinely multicentric lung cancers. Two cases presented difficulty in diagnosis. In several cases, the LOH patterns conflicted with p53 mutation patterns, suggesting that clonal evolution is directly affected by certain genetic changes. The combination of p53 with LOH helped increase both the sensitivity and specificity of the assay.     

Follistatin suppresses the production of experimental multiple-organ metastasis by small cell lung cancer cells in natural killer cell-depleted SCID mice.

PURPOSE: Follistatin (FST), an inhibitor of activin, regulates a variety of biological functions, including cell proliferation, differentiation, and apoptosis. However, the role of FST in cancer metastasis is still unknown. Previous research established a multiple-organ metastasis model of human small cell lung cancer in natural killer cell-depleted SCID mice. In this model, i.v. inoculated tumor cells produced metastatic colonies in multiple organs including the lung, liver, and bone. The purpose of this study is to determine the role of FST in multiple-organ metastasis using this model. EXPERIMENTAL DESIGN: A human FST gene was transfected into the small cell lung cancer cell lines SBC-3 and SBC-5 and established transfectants secreting biologically active FST. The metastatic potential of the transfectants was evaluated using the metastasis model. RESULTS: FST-gene transfection did not affect the cell proliferation, motility, invasion, or adhesion to endothelial cells in vitro. I.v. inoculated SBC-3 or SBC-5 cells produced metastatic colonies into multiple organs, including the lung, liver, and bone in the natural killer cell-depleted SCID mice. FST transfectants produced significantly fewer metastatic colonies in these organs when compared with their parental cells or vector control clones. Immunohistochemical analyses of the liver metastases revealed that the number of proliferating tumor cells and the tumor-associated microvessel density were significantly less in the lesions produced by FST transfectants. CONCLUSIONS: These results suggest that FST plays a critical role in the production of multiple-organ metastasis, predominantly by inhibiting the angiogenesis. This is the first report to show the role of FST in metastases.     

Morpho-functional study of vascular fluorochrome delivery to lung and liver metastases of Lewis lung carcinoma (3LL).

The growth of 3LL liver and lung metastases related to its vascular organization was studied by morphological and functional methods, using the Hoechst 33342 fluorescent DNA staining technique. Experimental liver and lung metastases were produced in syngeneic C57BL/6 mice by injection of 3LL tumor cells into a lateral tail vein or into the spleen, respectively. The resulting neoplasms were composed of large cells arranged in sheets with a thin irregularly distributed stroma. Scattered blood vessels with an open or closed lumen were observed within the tumor. Functional study of H33342 diffusion showed a single and reticular fluorescent pattern in liver metastases. In contrast, in lung metastases the fluorochrome diffusion revealed two different fluorescent patterns related to the location of the metastasis. Thus, parenchymal and subpleural metastasis presented a fluorescent pattern similar to that observed in the liver whereas metastases located around blood vessels and conducting airways never displayed fluorescence. In summary, our results suggest that the target metastatic organ and/or intra-organ location modulates the characteristics of metabolic exchange of the tumor cells in relation to the vascular organization of the metastatic focus.     

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 × 10⁶), 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.Int. J. Cancer 78:480–485, 1998. © 1998 Wiley-Liss, Inc.     

Hypoxia enhances metastatic efficiency in HT1080 fibrosarcoma cells by increasing cell survival in lungs, not cell adhesion and invasion

This study examined possible mechanisms for hypoxia-increased metastasis in a green fluorescent protein-labeled human fibrosarcoma cell line (HT1080). The efficiency of the lung arrest of tumor cells, which can be dependent on the adhesive potential of the tumor cells, was assessed by measuring the level of integrin alpha3beta1 protein and by adhesion assays, whereas the extravasation potential was examined by an invasion assay. These properties were not changed by exposure to hypoxia, indicating that lung arrest and extravasation are unlikely to play a major role in the effect of hypoxia on metastasis in this model. The main effect of hypoxic exposure was found to be increased survival after lung arrest as determined by clonogenic assay of tumor cells recovered from mouse lungs after i.v. injection. Concomitantly, apoptosis was identified as responsible for the death of lung-arrested cells, suggesting the involvement of an altered apoptotic response following hypoxic exposure of these cells. Consistent with this finding, we found that the effect of hypoxia on both increased metastasis and survival of arrested cells was inhibited by treatment with farnesylthiosalicylic acid. However, this effect was not due to down-regulation of hypoxia-inducible factor-1alpha, a mechanism of action of this drug reported by previous studies. Further detailed studies of the mechanisms of action of the drug are needed.     

Direct evidence of the importance of stromal urokinase plasminogen activator (uPA) in the growth of an experimental human breast cancer using a combined uPA gene-disrupted and immunodeficient xenograft model

Several studies have indicated an interaction between tumor cells and infiltrating stromal cells regarding the urokinase plasminogen activation (uPA) system. By developing combined uPA gene-disrupted and immunodeficient mice, we have studied the role of stromal uPA for the growth of the MDA-MB-435 BAG human tumor xenograft. Subcutaneous tumor growth and lung metastasis were compared between wild-type immunodeficient mice and mice with the combined deficiencies. Tumor growth was evaluated by volume measurements and plasma beta-galactosidase activity and metastasis was evaluated by counting lung surface metastases. Although no differences appeared in primary tumor take between the two groups of mice, a significant difference was observed in primary tumor growth, with tumors in uPA-/- mice growing significantly more slowly. In addition, a nonsignificant trend toward fewer lung metastases in uPA-/- mice was observed. The present data points to a critical role of stromal-derived uPA in the primary tumor growth of MDA-MB-435 BAG xenografts, whereas only a trend toward fewer lung metastases in uPA gene-disrupted mice was found.     

Orthotopic Transplant Mouse Models with Green Fluorescent Protein-expressing Cancer Cells to Visualize Metastasis and Angiogenesis

There have been major efforts in metastasis research in recent years, especially on the role of angiogenesis in the metastatic process. Much of the information in this area has been obtained from model systems that are not representative of clinical cancer. The technique of surgical orthotopic implantation (SOI) has allowed the development of clinically relevant metastatic models of human cancer in immunodeficient rodents such as the nude and SCID mouse. In order to allow direct visualization of the metastatic process, we took advantage of the green fluorescent protein (GFP) of the jellyfish, Aequorea victoria. A series of cancer cell lines have been stably transfected with vectors containing humanized GFP cDNA. To utilize GFP expression for metastasis studies, fragments of subcutaneously growing tumor, which were comprised of GFP-expressing cells, were implanted by SOI in nude mice. Subsequent metastases were visualized in systemic organs by GFP fluorescence in the lung, liver, bones, brain and other organs down to the single-cell level. With this fluorescent tool, we detected and visualized for the first time tumor cells at the microscopic level in fresh viable tissue in their normal host organs even in the live animal. Angiogenesis is readily visualized in the transplanted GFP-expressing tumors in real time in situ in the live animal using simple laparotomy and fluorescent techniques. The results with the GFP-transfected tumor cells, combined with the use of SOI, demonstrate a fundamental advance to visualize and study cancer metastasis and the role of angiogenesis and other factors in the metastatic process. This revised version was published online in July 2006 with corrections to the Cover Date.     

A critical step in metastasis: in vivo analysis of intravasation at the primary tumor

Detailed evaluation of all steps in tumor cell metastasis is critical for evaluating the cell mechanisms controlling metastasis. Using green fluorescent protein transfectants of metastatic (MTLn3) and nonmetastatic (MTC) cell lines derived from the rat mammary adenocarcinoma 13762 NF, we have measured tumor cell density in the blood, individual tumor cells in the lungs, and lung metastases. Correlation of blood burden with lung metastases indicates that entry into the circulation is a critical step for metastasis. To examine cell behavior during intravasation, we have used green fluorescent protein technology to view these cells in time lapse images within a single optical section using a confocal microscope. In vivo imaging of the primary tumors of MTLn3 and MTC cells indicates that both metastatic and nonmetastatic cells are motile and show protrusive activity. However, metastatic cells show greater orientation toward blood vessels and larger numbers of host cells within the primary tumor, whereas nonmetastatic cells fragment when interacting with vessels. These results demonstrate that a major difference in intravasation between metastatic and nonmetastatic cells is detected in the primary tumor and illustrate the value of a direct visualization of cell properties in vivo for dissection of the metastatic process.     

Inhibition of pulmonary and skeletal metastasis by a transforming growth factor-beta type I receptor kinase inhibitor

Transforming growth factor-beta (TGF-beta) signaling has been shown to promote invasion and metastasis in various models of human cancers. In this study, we investigated the efficacy of a TGF-beta type I receptor kinase inhibitor (TbetaRI-I) to limit early systemic metastases in an orthotopic xenograft model of lung metastasis and in an intracardiac injection model of experimental bone and lung metastasis using human breast carcinoma MDA-MB-435-F-L cells, a highly metastatic variant of human breast cancer MDA-MB-435 cells, expressing the enhanced green fluorescent protein (EGFP). Treatment of the cells with the TbetaRI-I had no effect on their growth but blocked TGF-beta-stimulated expression of integrin alpha(v)beta(3) and cell migration in vitro. Systemic administration of the TbetaRI-I via i.p. injection effectively reduced the number and size of the lung metastasis in both orthotopic xenograft and experimental metastasis models with no effects on primary tumor growth rate compared with controls. TbetaRI-I treatment also reduced the incidence of widespread early skeletal metastases in the femur, tibia, mandible, and spine detected by whole-body EGFP fluorescence imaging. Tumor burden in femora and tibiae was also reduced after TbetaRI-I treatment as detected by histomorphometry analysis compared with the placebo controls. Our results indicate for the first time that abrogation of TGF-beta signaling by systemic administration of the TbetaRI-I can inhibit both early lung and bone metastasis in animal model systems and suggest antimetastatic therapeutic potential of the TbetaRI-I.     

In vivo color-coded imaging of the interaction of colon cancer cells and splenocytes in the formation of liver metastases

The role of host cells in tumor progression and metastasis is critical. Intrasplenic injection of tumor cells has long been known as an effective method of developing liver metastases in nude mice, whereas portal vein (PV) injection of tumor cells can result in rapid death of the tumor cells. Host cells were thought to play a role in these phenomena. We report here that after splenic injection of tumor cells, splenocytes cotraffic with the tumor cells to the liver and facilitate metastatic colony formation. Human colon cancer cells that express green fluorescent protein (GFP) linked to histone H2B in the nucleus and red fluorescent protein (RFP) in the cytoplasm (HCT-116-GFP-RFP) were injected in either the PV or spleen of nude mice and imaged at the subcellular level in vivo. Extensive clasmocytosis (destruction of the cytoplasm) of the cancer cells occurred within 6 hours after PV injection and essentially all the cancer cells died. In contrast, splenic injection of these tumor cells resulted in the aggressive formation of liver and distant metastasis. GFP spleen cells were found in the liver metastases that resulted from intrasplenic injection of the tumor cells in transgenic nude mice ubiquitously expressing GFP. When GFP spleen cells and the RFP cancer cells were coinjected in the PV, liver metastasis resulted that contained GFP spleen cells. These results suggest a novel tumor-host interaction that enables efficient formation of liver metastasis via intrasplenic injection.     

Combined therapy with a new bisphosphonate, minodronate (YM529), and chemotherapy for multiple organ metastases of small cell lung cancer cells in severe combined immunodeficient mice.

PURPOSE: Lung cancer in the advanced stage frequently metastasizes to multiple organs, including the liver, lungs, lymph nodes, and bone. Bisphosphonates have been widely used to treat osteolytic bone metastasis in the past years; however, many studies have implicated that a single use of bisphosphonates could not prolong the survival of patients. In the present study, using a multiple-organ metastasis model of human lung cancer cells, we examined the effect of combined therapy with a new bisphosphonate (YM529) and etoposide (VP-16). EXPERIMENTAL DESIGN: Human small cell lung cancer (SBC-5) cells i.v. inoculated into natural killer cell-depleted severe combined immunodeficient mice metastasized to multiple organs, including the lungs, liver, kidneys, lymph nodes, and bone. SBC-5-bearing mice were treated with YM529 and/or VP-16 and sacrificed 5 weeks after tumor cell inoculation. Bone metastasis was assessed by X-ray photographs, and visceral metastasis was evaluated macroscopically. The number of osteoclasts in the bone lesions was examined by tartrate-resistant acid phosphatase staining. RESULTS: Monotherapy with YM529 suppressed the production of bone metastases, but not visceral metastasis. Histological analyses revealed that the number of osteoclasts in bone lesions was lower in YM526-treated mice, compared with control mice. VP-16 inhibited both bone metastasis and visceral (lung and liver) metastasis. However, neither YM529 alone nor VP-16 alone significantly prolonged the survival of SBC-5-bearing mice. Combined use of YM529 and VP-16 further inhibited the production of bone metastasis and significantly prolonged survival. CONCLUSIONS: Combined therapy with bisphosphonate and chemotherapy may be useful for small cell lung cancer patients with multiple organ metastases including bone metastasis.     

Comparison of cancer-cell seeding, viability and deformation in the lung, muscle and liver, visualized by subcellular real-time imaging in the live mouse

The comparison of cancer cell seeding, deformation and viability in the lung, muscle and liver of nude mice in real-time is reported here. The mice were intubated to support ventilation with positive end-respiratory pressure (PEEP) for imaging on the lung. Human fibrosarcoma cells with green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm (dual-color HT-1080 cells) were injected into the tail vein for lung imaging, the portal vein for liver imaging or the abdominal aorta for muscle imaging which was performed with an Olympus OV100 Small Animal Imaging System. The length of the cytoplasm and nuclei in 20 seeded cancer cells were measured. A large number of cells initially arrested in the lung capillaries and many cells formed aggregates. The cell number decreased rapidly at 6 and 24 h. There was no significant difference in cancer cell survival when immunocompetent C57BL/6 mice were used in place of the nude mice, suggesting that T cell reaction is not very important in the first 24 h after seeding of cancer cells in the lung. In the lung and liver, little cancer cell deformation occurred. In contrast in the muscle, the cytoplasm and nuclei of the seeded cells were highly deformed and many fragmented cells were observed. The rate of cancer cell death was highest in the lung and lowest in the muscle. In each organ, single disseminated cells tended to die earlier than aggregated cells. The results of this study suggest that the early steps of metastasis are different in the lung, liver and muscle.