Characterization of two highly metastatic variants of Lewis lung carcinoma with different organ specificities

The biological properties and metastasis of two sublines of the Lewis lung carcinoma (3LL) which have maintained a stable pattern of organ-selective metastasis have been studied. Subline M-3LL, a lung-specific variant which originated from a lung metastasis of the parent line, metastasized only to the lung following injection of 10(4)-10(6) tumor cells i.v. or s.c. Lymphatic metastases of this tumor were rarely detected. Subline H-3LL which was developed from a rare, spontaneous hepatic metastasis of the parent line metastasized primarily to the liver, but pulmonary metastases have also been observed. While it grew at local s.c. sites, this tumor metastasized to the regional lymph nodes draining the tumor site, as determined by histology and by bioassay of the lymph nodes following their grafting into new recipient animals. Histologically, the two lines were indistinguishable with the exception of a higher incidence of giant cells detected in tissue sections and culture monolayers of the liver-colonizing variant H-3LL. Ten clones derived from each of the variant lines were expanded in vitro and inoculated i.v. While none of the ten clones derived from line M-3LL gave rise to extrapulmonary metastasis, nine of ten clones derived from Tumor H-3LL gave rise to hepatic metastasis. Highly metastatic clones selected from each tumor were subsequently used to study the patterns of distribution and arrest of radiolabeled tumor cells following their inoculation i.v. No correlation could be found between the initial distribution of the radiolabeled tumor cells and the organ selectivity eventually noted in the site of the metastases.     

Increased metastatic capacity of Lewis lung tumor cells by in vivo selection procedure

Lewis lung tumor cells from liver metastases originally obtained from an intrasplenic tumor, and lung metastases obtained from an intramuscular transplant, were repeatedly passaged in the corresponding transplantation sites (spleen, intramuscular). Cells from liver metastases injected into the spleen gained an increased metastatic capacity. The same phenomenon was observed with lung metastatic cells injected intramuscularly, but to a lesser degree. In all passages metastases occurred only in the organs receiving the venous blood from the primary site. The enhanced metastasis formation may be a result of a selection of tumor cells resistant to host cytotoxic cells and/or of selection of tumor cells 'seeding' successfully in target organs.     

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.     

Inhibitory effects of heparin plus cortisone acetate on endothelial cell growth both in cultures and in tumor masses

A combination of heparin and cortisone acetate significantly inhibited both embryonic angiogenesis and the tumor growth of Lewis lung carcinoma (3LL) transplanted into C57BL/6 mice, although each of these agents used alone affected neither angiogenesis nor tumor growth. On the other hand, this combination neither decreased the number of metastatic foci in the lung nor prolonged the survival time of mice with 3LL. All tumor-bearing mice died of hemothorax due to pulmonary metastases. Cortisone acetate by itself increased metastasis, and addition of heparin did not affect accelerated metastasis. Because an antiangiogenic activity appears independent of metastasis acceleration by cortisone acetate, the use of steroids other than cortisone acetate having no metastasis-promotion effect should be required for an antiangiogenic tumor therapy in the presence of heparin. Heparin plus cortisone acetate prevented the DNA synthesis of cultured vascular endothelial cells but not that of cultured 3LL cells. Additionally, oral administration of this combination decreased the [3H]thymidine labeling of endothelial cells of tumor blood vessels prior to the suppression of tumor growth. The specific inhibition of the growth of endothelial cells by heparin plus cortisone acetate was revealed in both the in vitro and the in vivo tests.     

The role of the intravascular microenvironment in spontaneous metastasis development

Metastasis is primarily responsible for the morbidity and mortality of cancer. Improved therapeutic outcomes and prognosis depend on improved understanding of mechanisms regulating the establishment of early metastasis. In this study, use of green fluorescent protein (GFP)‐expressing PC‐3 orthotopic model of human prostate cancer and two complementary fluorescence in vivo imaging systems (Olympus OV100 and VisEn FMT) allowed for the first time real‐time characterization of cancer cell–endothelium interactions during spontaneous metastatic colonization of the liver and lung in live mice. We observed that prior to the detection of extra‐vascular metastases, GFP‐expressing PC‐3 cancer cells resided initially inside the blood vessels of the liver and the lung, where they proliferated and expressed Ki‐67 and exhibited matrix metalloprotenases (MMP) activity. Thus, the intravascular cancer cells produced their own microenvironment, where they could continue to proliferate. Extravasation occurred earlier in the lung than in the liver. Our results demonstrate that the intravascular microenvironment is a critical staging area for the development of metastasis that later can invade the parenchyma. Intravascular tumor cells may represent a therapeutic target to inhibit the development of extravascular metastases. Therefore, this imageable model of intravascular metastasis may be used for evaluation of novel anti‐metastatic agents.     

GROWTH OF A LOCAL TUMOR EXERTS A SPECIFIC INHIBITORY EFFECT ON PROGRESSION OF LUNG METASTASES

The effects of local tumor growth on the progression of lung metastasis of two mouse tumors of C57BL origin—the 3LL Lewis lung carcinoma and the B-16 melanoma—were studied. Excision of primary footpad grafts of 3LL or B-16 resulted in a significant increase in incidence and size of lung metastases. The incidence, but not the size, of lung metastases was found to be a function of size of the local tumor at time of excision. Reinoculation of tumor cells in the left footpad following excision of tumor from the right footpad suppressed the acceleration of growth of lung metastases and their increased incidence. Thus, the progression of the local tumor exerts an inhibitory effect on lung metastasis. The extent of inhibition of accelerated metastatic development by reinoculated tumor cells is a function of size of the cell inoculum or, in fact, of mass of the local tumor. The inhibitory effect of the local tumor on lung metastasis seems to be tumor-specific. 3LL metastases were not inhibited by local B-16 or EL4, neither was B-16 metastasis inhibited by 3LL. The specificity of interactions between the local tumor growth and its metastases may suggest the participation of immunological mechanisms in the control of lung metastasis. The involvement of the lymphoid system in the control of metastatic progression is also supported by the observation that total-body X-irradiation was associated with an increase in lung metastasis and that splenomegaly was observed in mice bearing the footpad tumor. Its excision resulted in the prevention of splenomegaly but reinoculation of the tumor, leading to metastatic suppression, was again associated with splenomegaly.     

Differences in cell surface antigens of tumor metastases and those of the local tumor.

Tests were made to determine whether cell surface tumor antigens of metastases differed from the tumor antigens of the cell population of the local tumor growth. C57BL/6 mouse spleen lymphocytes sensitized against monolayers of the local growth of the 3LL Lewis lung carcinoma (L-3LL) in the presence of syngeneic serum generated lymphocytes that were cytotoxic to L-3LL but significantly less cytotoxic to target cells derived from lung metastases (M-3LL). Lymphocytes sensitized against M-3LL were significantly more cytotoxic against M-3LL than against L-3LL cells. Anti-M-3LL cytotoxic lymphocytes but not anti-L-3LL, admixed with either L-3LL cells or M-3LL tumor cells, when injected into syngeneic recipients reduced lung metastasis significantly. Results indicated that cells with high metastatic capacity and distinct antigenic properties exist within the tumor cell population and that immunoselection might be involved in the production of lung metastases.     

癌瘤转移规律的探讨

在 4 0 0例恶性肿瘤尸解中 ,32 1例 (80 3% )癌 ,79例肉瘤 ,其中 6 5例淋巴瘤 ,14例 (3 5 % )为软组织及骨肿瘤 ,肿瘤转移到肝及肺最常见。有 16 3例转移到肺及肝 ,各占 4 0 5 %。肝的转移瘤依次主要来自乳腺、大肠、卵巢、胃及非何杰金淋巴瘤 (NHL)。肺的转移瘤主要来自乳腺、肝、NHL ,胃及卵巢。淋巴结转移主要累及颈部、纵隔及主动脉周围淋巴结。广泛转移的肿瘤是肺癌、胃癌、乳腺癌和淋巴瘤。尸检材料显示 ,宫颈癌、膀胱癌、咽癌及睾丸肿瘤主要是局部侵犯 ,转移并不广泛。本研究显示 ,恶性肿瘤的扩散与转移基本有下列方式 :直接侵袭 :很多肿瘤可能直接累及周围组织。例如胃肠癌 ,癌细胞沿着肌组织间隙进入临近器官。淋巴管扩散 :任何器官或组织的肿瘤细胞可能进入淋巴管而转移到局部或远处淋巴结 ,它是癌的主要转移方式。血道转移 :远处转移在尸检材料中很常见 ,最常累及的器官是肝和肺 ,特别是软组织肉瘤 ,但晚期癌也很常见血道转移。种植性转移 :这也是很常见的转移方式 ,特别是胃癌、大肠癌、卵巢癌等。本研究也显示与癌瘤手术标本对比研究、肿瘤转移与临床分期、肿瘤部位、组织学类型及分化程度等有密切关系     

Temporal progression of metastasis in lung: cell survival, dormancy, and location dependence of metastatic inefficiency

Cancer metastasis is an inefficient process. The steps in metastasis responsible for this inefficiency and how metastatic inefficiency can vary in different locations within an organ remain poorly understood. B16F10 cells were injected to target mouse lung, and at sequential times thereafter we quantified in lung the time course of: (a) overall cell survival and metastatic development; and (b) local cell survival and growth with respect to the lung surface and specific interior structures. We found high rates of initial survival of cells trapped in the lung circulation, extravasation into lung tissue, and subsequent survival of extravasated solitary cells (74% at day 3) before metastasis formation. However, at the time of initial replication of metastatic cells a major loss of cells occurred. Although only a small proportion of injected cells started to form metastases, most of these developed into macroscopic tumors. Solitary cells found at later times were dormant. Thus, overall metastatic inefficiency was largely due to postextravasation events affecting solitary cells. Regionally within the lung, cells and metastases were randomly distributed to day 4, but by day 10 preferential tumor growth was found along the lung surface and around arterial and venous vessels. Thus, trapping and early growth of injected cells was unaffected by location within the lung, whereas subsequent metastatic growth was enhanced in specific microenvironments. This study: (a) quantifies early temporal and spatial progression of metastasis in lung; (b) documents persistence of solitary dormant cells; and (c) shows that metastatic inefficiency depends on the initiation of growth in a subset of extravasated cells, whereas continued growth of metastases occurs preferentially in specific tissue environments.     

Intrabronchial implantation of the Lewis lung tumor cell does not favor tumorigenicity and metastasis

The purpose of these studies was to determine whether the orthotopic implantation of the Lewis lung tumor favors tumorigenicity and production of metastasis as compared with implantation at an ectopic site. Different numbers of viable cells were implanted subcutaneously (footpad), intrabronchially, or intrathoracically into groups of syngeneic mice. Tumorigenicity, production of distant metastases and survival were recorded. Tumor cells implanted subcutaneously produced fast-growing local tumors and numerous spontaneous lung metastases. Tumor cells implanted intrabronchially produced slow-growing tumors that did not produce distant metastasis. Since radiolabeled cells implanted into different sites exhibited similar patterns of survival, the differences in behavior of 3LL cells in the skin or lung were due to other factors. We conclude that for the Lewis lung carcinoma, orthotopic implantation does not favor increased tumorigenicity and metastatic potential.     

Inhibition of metastasis of lewis lung carcinoma by urinary trypsin inhibitor in experimental and spontaneous metastasis models

A purified human urinary trypsin inhibitor (UTI) and its related synthetic peptides were examined to determine whether they could inhibit production of experimental and spontaneous lung metastases by murine Lewis lung carcinoma (3LL) cells. Three peptides, peptide 1, peptide 2 and peptide 3, representing the amino acid sequences within the UTI molecule, were synthesized. UTI and peptide 2 inhibited human leukocyte elastase (HLE). UTI and peptide 3 specifically inhibited human and murine plasmin activity. Peptide 1 had essentially no inhibitory activity. In an in vivo spontaneous metastasis model, multiple s.c. injections of UTI or peptide 3 for 7 days immediately after s.c. tumor cell inoculation significantly inhibited the formation of lung metastasis in CS7BL/6 mice in a dose-dependent manner. UTI reduced lung tumor colonization more effectively than peptide 3. Peptides 1 and 2, however, did not affect the formation of lung metastasis. Inhibition of lung metastasis was not due to direct anti-tumor effects of UTI and peptide 3. In an in vivo experimental metastasis assay, multiple s.c. injections of UTI for 7 days after i.v. tumor cell inoculation inhibited metastatic lung tumor colonization, while peptide 3 did not affect metastasis. Peptides 1 and 2 did not affect the formation of lung metastasis. When examined with an in vitro assay system using a modified Boyden chamber, UTI and peptide 3 suppressed the invasion of tumor cells through Matrigel. UTI and peptide 3 inhibited neither cell proliferation nor the binding of tumor cells to Matrigel and showed no significant suppression of chemotactic migration of tumor cells to fibronectin. Our results suggest that UTI efficiently regulates the mechanism involved in not only the entry into vascular circulation of tumor cells (intravasation, though, at least in part, inhibition of the proteolytic enzyme plasmin) but also the extravasation step of the metastatic process.     

Embryonic endothelial progenitor cells armed with a suicide gene target hypoxic lung metastases after intravenous delivery

We show that mouse embryonic endothelial progenitor cells (eEPCs) home preferentially to hypoxic lung metastases when administered intravenously. This specificity is inversely related to the degree of perfusion and vascular density in the metastasis and directly related to local levels of hypoxia and VEGF. Ex vivo expanded eEPCs that were genetically modified with a suicide gene specifically and efficiently eradicated lung metastases with scant patent blood vessels. eEPCs do not express MHC I proteins, are resistant to natural killer cell-mediated cytolysis, and can contribute to tumor vessel formation also in nonsyngeneic mice. These results indicate that eEPCs can be used in an allogeneic setting to treat hypoxic metastases that are known to be resistant to conventional therapeutic regimes.     

Activity of a new vascular targeting agent,ZD6126, in pulmonary metastases by human lung adenocarcinoma in nude mice

ZD6126 (ANG453) is a novel vascular targeting agent that selectively disrupts the cytoskeleton of endothelial cells in tumor. In mouse s.c. xenograft models, ZD6126 was found to induce selective occlusion of tumor blood vessels, cessation of tumor blood flow, and death of tumor cells because of the starvation of oxygen and nutrition. Here, we investigated whether ZD6126 inhibited the metastatic formation of human non-small cell lung cancer cells. PC14PE6 (adenocarcinoma) and H226 (squamous cell carcinoma) cells were injected into the tail vein of nude mice, and lung metastases were estimated. ZD6126 treatment involved either a single dose on 24 h before killing or daily doses from day 14 until the end of the experiment. Single treatment with i.p. injection of 200 mg/kg ZD6126 caused bleeding and necrotic changes in the tumor by 24 h. Histological analysis revealed that apoptotic tumor cells were markedly increased in the ZD6126-treated group. Moreover, ZD6126 induced the apoptosis of CD31-positive vascular endothelial cells in tumors but not in the normal lung parenchyma. When mice were treated daily with 100 mg/kg ZD6126 from day 14 until the end of the experiment, the lung weight was significantly less in the ZD6126-treated group than that of the control group, despite no difference in the number of metastatic nodules. These data suggest that ZD6126 could demonstrate its antitumor activity against both already established and early phase of lung cancer metastasis by causing the selective apoptosis of tumor endothelial cells and destruction of the tumor vasculature.     

Decrease of metastatic ability after selection for intravasating ability in Lewis lung carcinoma (3LL) cell line.

The release of cancer cells from the primary site and penetration into blood vessels are obligatory preliminary steps for metastasis. To investigate the mechanism of such steps we isolated variant cells (designated as Int-3LL) possessing enhanced intravasating ability from Lewis lung carcinoma (3LL) cells by in vivo selection. In spite of the enhanced intravasating ability of Int-3LL, the spontaneous and experimental metastatic abilities of Int-3LL decreased significantly compared to parent cells. Such a cell line has never been reported so far. The matched pair of cell lines described in this report provides a useful system for investigating the primary steps of metastasis.     

Expression of variant CD44, exon 6 in patients with metastatic pulmonary tumor

High expression ratios of CD44 variant 6 (CD44 V6) in patients with metastatic pulmonary tumor were found in those with primary lesions of cancer of the colon, uterus, larynx, liver and osteosarcoma. It was clarified that patients showing expression of CD44 variant 6 likely revealed pulmonary metastasis at earlier time following operations of primary cancer (p<0.05). CD44 V6, an adhesion molecule, was a factor to participate in pulmonary metastases from various organ cancers. No significant correlation was observed in survival between patients with CD44 V6 positive versus negative tumors, except laryngeal tumor after resection of primary or metastatic lung tumor. CD44 V6 related to its invasive and further metastatic functions in metastatic lung tumor. We suggest that cancer cells expressing the CD44 molecule especially V6 may adhere to vascular endothelium and hyaluronic acid in the lung. And cancer cells without this molecule liberated from the primary focuses hardly adhere to the pulmonary tissues supposedly resulting in delayed metastases and proliferations in the pulmonary tissues.     

Identification of a surface antigen, common to murine lung and Lewis lung carcinoma, by monoclonal antibody

Lewis lung carcinoma (3LL) is a spontaneous murine lung carcinoma which preferentially metastasizes into the lung. Monoclonal antibodies (MAb) against 3LL cells were prepared by fusing spleen cells from rats immunized with 3LL and the P3-NSI/I-Ag4 (NSI) plasmacytoma. Two hybridomas were selected which secreted antibodies capable of preferential binding to 3LL cells and not to other malignant cells. Immunohistochemical staining of sections prepared from various mouse organs with one of the MAbs (22.2), revealed exclusive staining of the lung tissue. The data suggest that a lung-specific, 3 LL-associated antigen, is detected by the 22.2 MAb on 3LL cells. The 22.2 antigen is a surface molecule, and is stable following trypsin treatment and fixation by glutaraldehyde or picric acid. Cells that were prepared from a subcutaneous tumor or from lung metastases express high amounts of 22.2 antigen compared to cells of the culture 3LL line (3LLC). Upon reinoculation of 3LLC cells into mice there was a gradual increase in 22.2 antigen expression. It was possible to select from the heterogeneous 3LLC cell population several clones which express high amounts of antigen. These clones differed also in their metastatic patterns. However, the metastatic capacity of these clones did not correlate with expression of the 22.2 antigen. Thus, it appears that expression of the 22.2 antigen is not a necessary condition for formation of lung metastases by 3LL.     

Imaging of cancer invasion and metastasis using green fluorescent protein

The use of green fluorescent protein to fluorescently tag tumour cells has allowed investigators to open the “black box” of metastasis in order to visualise the behaviour of tumour cells in living tissues. Analysis of cells leaving the primary tumour indicates that highly metastatic cells are able to polarise more effectively towards blood vessels while poorly metastatic cells fragment more often when interacting with blood. In addition, there appear to be greater numbers of host immune system cells interacting with metastatic tumours. After arresting in target organs such as the lungs or liver, most tumour cells become dormant or apoptose. A small fraction of the arrested cells form metastases. In some target organs, migration of tumour cells may enhance the ability to form metastases.     

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.     

Establishment and Characterization of a New Spontaneous Metastasis Model of Human Gastric Carcinoma in Nude Mice

A poorly differentiated medullary carcinoma of human stomach, designated HY–1, was successfully transplanted to nude mice by either the subcutaneous or intramuscular route for five generations. The transplanted tumor showed spontaneous lung metastases in nearly 100% of KSN and Balb/c female nude mice. There were over 20 visible lung metastatic nodules in KSN and Balb/c nude mice bearing tumors for over SO days. Immunostaining of type IV collagen and electron microscopy revealed that tumor cells were often in direct contact with basement membrane (BM) of tumor blood vessels in the primary tumor tissue. At the site of contact between tumor cells and vascular BM, focal disappearance of the BM, disruption of endothelial cells and entry of tumor cell clusters into vascular lumen were observed. Immunostaining of 72 kDa gelatinase/type IV collagenase demonstrated that tumor cells expressed this enzyme in their cytoplasm. These results suggest that spontaneous metastasis of this tumor may be partly due to a marked tendency to vascular invasion involving the following sequential events: tumor cell contact with vascular BM, BM degradation possibly by 72 kDa gelatinases and endothelial disruption. This model could be a useful tool for understanding the mechanism of hematogenous metastasis of human gastric cancer.     

Micronodular transformation as a novel mechanism of VEGF-A-induced metastasis

How and why tumors metastasize is still a matter of debate. The assumption is that mutations render tumor cells with a metastatic phenotype, enabling entrance in and transport through lymph or blood vessels. Distant outgrowth is thought to occur only in a suitable microenvironment (the seed and soil hypothesis). However, the anatomical location of most metastases in cancer patients suggests entrapment of tumor cells in the first microcapillary bed that is encountered. We here investigated how vascular endothelial growth factor-A (VEGF-A) attributes to the metastatic process. We describe here that VEGF-A enhances spontaneous metastasis by inducing intravasation of heterogeneous tumor cell clusters, surrounded by vessel wall elements, via an invasion-independent mechanism. These tumor clusters generate metastatic tissue embolisms in pulmonary arteries. Treatment of tumor-bearing mice with the antiangiogenic compound ZD6474 prevented the development of this metastatic phenotype. This work shows that tumors with high constitutive VEGF-A expression metastasize via the formation of tumor emboli and provides an alternative rationale for anti-VEGF-A therapy, namely to inhibit metastasis formation.