Vascular anatomy and organ-specific tumor growth as critical factors in the development of metastases and their distribution among organs

We have examined with 19 tumor cell lines the discrete roles that vascular anatomy and tumor-cell-organ-affinity play in the development of metastases and their distribution among organs. Spontaneous metastases of B16-G3.26 melanoma cells from a primary tumor growing in the foot pad of mice, or experimental metastases 21 days after intravenous tumor-cell injection resulted in tumor colonies only in the lungs. In contrast, when the lung microvasculature was bypassed, and the same cells given by systemic intra-arterial (s.i.a.) injection, large tumor colonies developed selectively in the ovaries, adrenal glands and bones, but rarely in the lungs. When animals injected i.v. were allowed to live with lung metastases for a long period of time, small tumor colonies began to develop in extra-pulmonary organs with a distribution identical to that seen after s.i.a. injection. Seven murine tumor cell lines (previously characterized by their ability to colonize primarily the lungs after i.v. injection) and 7 of the 8 studied human tumor cell lines colonized different specific extra-pulmonary organs after s.i.a. injection, frequently producing metastatic syndromes commonly described in patients with cancer, but rarely seen in animal models of metastasis. These results suggest that metastatic cells, even those capable of colonizing specific organs, do not freely circulate in the blood stream and lodge in specific tissues. In contrast, the cells must establish a vascular route of access to the target organ, e.g., through the systemic circulation from metastatic tumors in the lungs. Two cell lines considered to be tumorigenic but non-metastatic failed to colonize the lungs or extra-pulmonary organs after i.v. injection, but readily colonized specific organs after s.i.a. injection. Thus, tumor cells considered to be non-metastatic may be indeed metastatic if they are provided with vascular access to an organ more congenial to their growth requirements.     

Persistence of solitary mammary carcinoma cells in a secondary site: a possible contributor to dormancy

Tumors can recur years after treatment, and breast cancer is especially noted for long periods of dormancy. The status of the cancer during this period is poorly understood. As a model to study mechanisms of dormancy, we used murine D2.0R mammary carcinoma cells, which are poorly metastatic but form occasional metastases in liver and other organs after long latency. Highly metastatic D2A1 cells provided a positive, metastatic control. Our goals were to learn how the cell lines differ in survival kinetics in a secondary site and to seek evidence for the source of D2.0R dormancy. In spontaneous metastasis assays from mammary fat pad injections, we found evidence for dormancy because of a persistence of large numbers of solitary cells in the liver. To quantify the fate of cells after arrival in liver, experimental metastasis assays were used. To permit identification of cells that had not divided, cells were labeled before injection with fluorescent nanospheres, which were diluted to undetectable levels by cell division. Cancer cells were injected i.v. to target them to the liver and coinjected with reference microspheres to monitor cell survival. Dormancy was defined as retention of nanosphere fluorescence in vivo, as well as negative staining for the proliferation marker Ki67. A large proportion of D2.0R cells persisted as solitary dormant cells. No metastases formed, but viable cells could be recovered from the liver 11 weeks after injection. Large numbers of solitary, dormant, Ki67-negative D2A1 cells were also detected against a background of progressively growing metastases. Thus, this study identified a possible contributor to tumor dormancy: solitary, dormant cells that persist in tissue. If such cells are present in patients, they could contribute to tumor recurrence and would not be susceptible to current therapeutic strategies targeting proliferating cells.     

Ineffectiveness of Doxorubicin Treatment on Solitary Dormant Mammary Carcinoma Cells or Late-developing Metastases

Breast cancer is noted for long periods of tumor dormancy and metastases can occur many years after treatment. Adjuvant chemotherapy is used to prevent metastatic recurrence but is not always successful. As a model for studying mechanisms of dormancy, we have used two murine mammary carcinoma cell lines: D2.0R/R cells, which are poorly metastatic but form metastases in some mice after long latency times, and D2A1/R cells, which form more numerous metastases much earlier. Previously we identified a surprisingly large population of dormant but viable solitary cells, which persisted in an undivided state for up to 11 weeks after injection of D2.0R/R cells. Dormant cells were also detected for D2A1/R cells, in a background of growing metastases. Here we used this model to test the hypothesis that dormant tumor cells would not be killed by cytotoxic chemotherapy that targets actively dividing cells, and that the late development of metastases from D2.0R/R cells would not be inhibited by chemotherapy that effectively inhibited D2A1/R metastases. We injected mice with D2A1/R or D2.0R/R cells via a mesenteric vein to target liver. We developed a doxorubicin (DXR) treatment protocol that effectively reduced the metastatic tumor burden from D2A1/R cells at 3 weeks. However, this treatment did not reduce the numbers of solitary dormant cells in mice injected with either D2A1/R or D2.0R/R cells. Furthermore, DXR did not reduce the metastatic tumor burden after an 11-week latency period in mice injected with D2.0R/R cells. Thus, apparently effective chemotherapy may spare non-dividing cancer cells, and these cells may give rise to metastases at a later date. This study has important clinical implications for patients being treated with cytotoxic chemotherapy.     

Intranasal therapy with an adenoviral vector containing the murine interleukin-12 gene eradicates osteosarcoma lung metastases.

The purpose of these studies was to determine the effect of adenovirus-mediated interleukin-12 (IL-12) gene transfer on the growth and development of osteosarcoma (OS) lung metastases in nude mice. A nude mouse model was produced by repetitive cycling of human SAOS OS cells through the lung. The resultant SAOS-LM6 cell line produced microscopic lung metastases by 5-6 weeks after i.v. injection of the tumor cells, with visible lung metastases present 8 weeks after injection. Transfection of SAOS-LM6 cells with a plasmid containing the murine IL-12 gene resulted in a decrease in metastatic potential. Animals injected with IL-12-transfected clones had fewer metastases compared with mice injected with SAOS-LM6 cells transfected with a control plasmid. Furthermore, nasal delivery of an adenoviral vector containing the murine IL-12 gene resulted in the inhibition of pulmonary metastases. Together, these data indicate that IL-12 may be an effective agent against OS and that nasal delivery may offer a unique way to deliver the gene to the local tumor environment, potentially decreasing systemic toxic effects.     

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.     

Evaluation of systemic chemotherapy with magnetic liposomal doxorubicin and a dipole external electromagnet

The development of an active drug delivery system is an attractive approach to increase the targetability of anticancer agents. In the present study, we examined the efficiency of systemic chemotherapy with small magnetic liposomes containing doxorubicin (magnetic DOX liposomes) and an externally applied electromagnetic force in osteosarcoma-bearing hamsters. Syrian male hamsters inoculated with osteosarcoma, OS515, in the limb were studied 7 days after inoculation. The efficiency of this system was evaluated by measuring the tissue distribution and tumor-suppressing effects of DOX on primary tumor growth and lung metastases. A DC dipole electromagnet was used, and the hamster's tumor-bearing limb was placed between 2 poles after the i.v. administration of liposomes. The dose of DOX and the magnetic field strength were fixed at 5 mg/kg and 0.4 T, respectively. Administration of magnetic DOX liposomes followed by 60 min application of magnetic field produced a 3- to 4-fold higher maximum DOX concentration in the tumor. This newly designed systemic chemotherapy significantly suppressed primary tumor growth for at least 2 weeks, though other DOX treatments also suppressed compared to control. Histologic examination confirmed a greater antitumor effect of this systemic chemotherapy compared to standard methods. In addition, this approach significantly suppressed lung metastases measured at 3 weeks posttreatment. These results suggest that this systemic chemotherapy can effectively reduce primary tumor growth and suppress lung metastasis due to increased targeting of DOX. Such targeted drug delivery for anticancer agents would provide clinical advantages compared to current methods.     

Dietary genistein reduces metastasis in a postsurgical orthotopic breast cancer model

Metastatic spread, not primary tumor burden, is the leading cause of breast cancer deaths. For patient prognosis to improve, new systemic adjuvant therapies that are capable of effectively inhibiting the outgrowth of seeded tumor cells after surgical treatment of the primary breast tumor are needed. To facilitate the preclinical development of such therapies, relevant animal models of breast cancer metastasis that can mimic the postsurgical adjuvant setting are required. Here we developed a preclinical xenograft model of breast cancer metastasis where the primary tumor was removed by surgical resection before systemic adjuvant treatment. We used this model to assess the antimetastatic effect of postsurgical dietary intervention with the soy isoflavone genistein. The anticancer activity of genistein has been established in vitro and in vivo, however, few studies have tested the potential of genistein as an antimetastatic therapy. Using our model, we tested the efficacy of adjuvant treatment with genistein to inhibit the outgrowth of metastases postsurgery. To establish primary tumors, human breast carcinoma cells, MDA-MB-435/HAL, were implanted into the mammary fat pad of female nude mice. Primary tumors were left to grow for 5 weeks before being surgically removed. Mice were then randomized into two diet groups: control soy-free diet versus genistein-supplemented diet. Five weeks later, metastatic burden was assessed. Genistein reduced the percent metastatic burden in the lungs by 10-fold. These results indicate that dietary intervention following cancer surgery can affect the outgrowth of seeded tumor cells. The availability of well-characterized, clinically relevant animal models for studying factors that regulate metastatic outgrowth postsurgery will provide an important tool for developing new systemic adjuvant therapies.     

Mesenchymal stem cells as a vehicle for targeted delivery of CRAds to lung metastases of breast carcinoma

Purpose Alternative and complementary therapeutic strategies need to be developed for metastatic breast cancer. Virotherapy is a novel therapeutic approach for the treatment of cancer in which the replicating virus itself is the anticancer agent. However, the success of virotherapy has been limited due to inefficient virus delivery to the tumor site. The present study addresses the utility of human mesenchymal stem cells (hMSCs) as intermediate carriers for conditionally replicating adenoviruses (CRAds) to target metastatic breast cancer in vivo. Experimental design HMSC were transduced with CRAds. We used a SCID mouse xenograft model to examine the effects of systemically injected CRAd loaded hMSC or CRAd alone on the growth of MDA-MB-231 derived pulmonary metastases (experimental metastases model) in vivo and on overall survival. Results Intravenous injection of CRAd loaded hMSCs into mice with established MDA-MB-231 pulmonary metastatic disease homed to the tumor site and led to extended mouse survival compared to mice treated with CRAd alone. Conclusion Injected hMSCs transduced with CRAds suppressed the growth of pulmonary metastases, presumably through viral amplification in the hMSCs. Thus, hMSCs may be an effective platform for the targeted delivery of CRAds to distant cancer sites such as metastatic breast cancer.     

Promotion of experimental liver metastasis by tumor necrosis factor

Models for experimental metastasis were established to investigate the influence of rmTNF on tumor-colony formation in the liver. Highly metastatic lymphoma tumor cells were either injected i.v. or inoculated s.c. to form spontaneous metastases. In both systems, administration of rmTNF to the animals led to significant enhancement of the number of liver metastases in comparison with control groups. The number of metastatic tumor-cell colonies at an early stage of metastasis was increased, as well as the number of surface metastases in a late stage. Consequently, TNF-treated animals revealed a higher mortality. The optimal time for TNF to exert this metastasis-enhancing effect was found to be 7 days after tumor inoculation, In vitro adhesion of the lymphoma tumor cells to a mouse endothelioma cell line was strongly inhibited by monoclonal antibodies interfering with the interaction of VCAM-1 with VLA-4. These results support and extend earlier results with a fibrosarcoma lung colonization model. In addition, they show that stimulation of the immune system in tumor-bearing hosts activates tumor-promoting pathways, in addition to having possible beneficial effects.     

Simultaneous radiolabel, genetic tagging and proliferation assays to study the organ distribution and fate of metastatic cells

We compared the suitability of 3 techniques to study tumor cell survival in the lungs of mice and proliferation into metastases. Genetic tagging of tumor cells with the bacterial beta-galactosidase marker gene lacZ, radiolabeling of tumor cells with [I-125]IdUrd, and S-phase labeling of cells with bromodeoxyuridine (BrdUrd) were used simultaneously to track the fate of highly metastatic K-1735 X-21 melanoma cells injected into syngeneic C3H/HeN mice. The melanoma cells were transfected with a plasmid containing lacZ and neomycin resistance genes. After growth in selective medium, the cells were incubated in medium containing [I-125]IdUrd and then injected i.v. into mice. Lungs isolated at various times after i.v. injection were processed for staining with X-gal, radioactive monitoring, and immunohistochemical staining with a monoclonal antibody against BrdUrd. At early time points, the presence of lacZ-positive cells directly correlated with radioactivity associated with viable cells. However, the expression of the beta-galactosidase was only stable for 1 week, and by 3 weeks after injection, large metastases contained only a few lacZ-positive cells. The combination of lacZ tagging with BrdUrd proliferation assay accurately identified dividing tumor cells in micrometastases. The simultaneous use of these 3 techniques allowed us to conclude that quantitative analysis of tumor cell survival is best accomplished by radioactive labeling of cells, whereas the use of lacZ-tagged cells allowed for studies of localization. Analysis of tumor cell proliferation requires the use of both lacZ tagging and immunohistochemistry using anti-BrdUrd antibodies. Since the process of metastasis consists of a series of distinct steps, each technique presents its own advantages and limitations.     

Promotion of pulmonary metastasis in mice by bleomycin-induced endothelial injury

The passage of circulating tumor cells across the vascular wall is an important step in the evolution of cancer metastases. Since tumor cells attach preferentially to subendothelial matrix at sites of endothelial injury and retraction in vitro, we have used an established in vivo model of pulmonary endothelial damage to examine the effects of endothelial injury on the localization and metastasis of circulating tumor cells in vivo. C57BL/6 mice were given a single i.v. dose of bleomycin (120 mg/kg) or multiple i.p. injections (10 mg/kg, twice weekly for 6 wk). Five days after the single injection or 4 days after the last i.p. injection, 2 X 10(5) [131I] iododeoxyuridine-labeled fibrosarcoma cells or unlabeled cells were injected i.v. Two to 8 times as many labeled cells were found in the lungs of bleomycin-treated animals after 24 h. Two and 3 wk after injecting unlabeled fibrosarcoma cells, 1.4 to 5 times more metastatic lung colonies were counted in bleomycin-treated animals than in controls. Morphometric analysis of histological sections demonstrated that the percentage of lung area occupied by tumor in bleomycin-treated animals was between 4 and 16 times that of controls. Analysis of bronchoalveolar lavage fluids demonstrated 5-fold increases of total protein content and leakage of previously injected 125l-labeled albumin, indicating increased endothelial permeability. Electron microscopic examination of lungs of bleomycin-treated mice demonstrated endothelial retraction with exposure of the underlying basement membrane. Electron microscopy of [3H]thymidine-labeled tumor cells, located by autoradiography, demonstrated their attachment to exposed basal lamina. Data from these experiments in vivo support the hypothesis that endothelial damage can facilitate the metastasis of circulating tumor cells.     

Effective elimination of lung metastases induced by tumor cells treated with hydrostatic pressure and N-acetyl-L-cysteine

In previous studies, we have demonstrated that application of high hydrostatic pressure (P) to tumor cells in the presence of a slow-reacting membrane-impermeable cross-linker (CL), 2'-3'-adenosine dialdehyde, can rearrange cell surface proteins into immunogenic clusters. Here, we present evidence indicating that subsequent reduction of surface protein disulfides with N-acetyl-L-cysteine (NAC) further augments the immunogenic potential of PCL-modified tumor cells both in vitro and in vivo. Immunotherapy with PCL+NAC-modified 3LL-D122 Lewis lung carcinoma cells plus i.v. delivery of NAC in mice bearing established lung metastases provoked an antitumor response capable of eradicating the metastatic nodules as demonstrated by restoration of normal lung weight and histology. In addition, immunization with PCL+NAC-modified tumor cells gave rise to a strong delayed-type hypersensitivity recall response against parental D122 cells. We propose that this novel two-prong strategy, based on local immunization with autologous PCL+NAC-modified tumor cells and systemic boosting with NAC, could provide a practical, effective immunotherapeutic regimen for the treatment of human cancer.     

The role of polymorphonuclear leukocytes (PMN) on the growth and metastatic potential of 13762NF mammary adenocarcinoma cells

Circulating neutrophil (PMN) levels can increase in rats bearing subcutaneously growing clones of the 13762NF mammary adenocarcinoma and the level of increase correlates with the metastatic potential of the clone. In rats with poorly metastatic MTC tumors, numbers of circulating PMN did not rise, whereas PMN levels rose 50-fold in rats bearing highly metastatic MTLn3, 12-fold in rats with weakly metastatic MTLn2, and 14-fold in those with moderately metastatic MTF7 tumors. Neutrophilia was caused partly by tumor size, but metastatic potential was a stronger determinant, suggesting that PMNs may play a role in the metastatic process. To determine whether circulating PMNs indeed contribute to cellular metastatic potential, we examined effects of PMN on various aspects of the metastatic process. Experimental metastasis assays involving i.v. co-injections of PMNs yielded a dose-dependent increase in extrapulmonary metastases for MTLn3, but no change in lung colonization potential for any of the clones examined. The change in the metastatic profile was not due to any modification in in vivo distribution of i.v. injected tumor cells or in adhesion to endothelial monolayers in vitro. PMNs also had no effect on in vitro DNA, RNA or protein synthesis and were not cytolytic (E:T 100:1). However, PMNs collected from high-passage MTLn3 tumor-bearing rats had a 50% increase in heparanase and type-IV collagenolytic activity as compared to unstimulated PMNs isolated from normal rats. These results indicate that polymorphonuclear cells may contribute to the metastatic potential of highly metastatic clones from the 13762NF mammary adenocarcinoma cells by assisting in the degradation of basement membrane during extravasation.     

TMTP1, a novel tumor-homing peptide specifically targeting metastasis

PURPOSE: Tumor metastasis continues to be the major obstacle to cancer therapy and the leading cause of cancer-related death. Methods used to detect metastasis, especially occult metastases, have received a great deal attention. In this study, a novel selective peptide was assessed for its specific binding to metastasis. METHODS: The FliTrx bacterial peptide display system, an alternative to phage peptide display, was used to identify a 5-amino acid peptide termed TMTP1 (NVVRQ), which binds to the highly metastatic prostate cancer cell line PC-3M-1E8. The synthetic TMTP1 was tested in vitro for its binding specificity and affinity to highly metastatic cancer cells. The tumor targeting assays were done in vivo by i.v. injection of FITC-conjugated TMTP1 into tumor-bearing mice. RESULTS: TMTP1 specifically bound to a series of highly metastatic tumor cells, including prostate cancer PC-3M-1E8, breast cancer MDA-MB-435S, lung cancer PG-BE1, and gastric cancer MKN-45sci, in vitro and in vivo but not to the poorly metastatic or nonmetastatic cell line, including prostate cancer PC-3M-2B4, breast cancer MCF-7, lung cancer PG-LH7, or murine fibroblast cell NIH/3T3. FITC-TMTP1 strongly and specifically targeted the metastasis foci in tumor-bearing mice 24 h after i.v. peptide injection. Moreover, the occult metastases were specifically detected by FITC-TMTP1. CONCLUSION: Our results suggest that TMTP1 is a potential strategy for the development of new diagnostic tracers or alternative anticancer agents for tumor metastasis.     

Syngeneic in vivo passage of the murine BW 5147 lymphoma results in the expression of a stable metastatic phenotype

BW 5147 lymphoma cells are non-invasive tumor cells which do not generate experimental metastases following i.v. inoculation. In contrast, s.c. and intra-splenic (i.s.) growth of BW cells resulted in widespread colonization of liver and spleen. Cells derived from either s.c. tumors or metastatic lesions did generate metastases after i.v. administration. The capacity of these tumor-derived BW cells to disseminate via blood-borne cells was irreversible and stable, indicating that one in vivo passage of BW cells results in the generation of new, metastatic BW variants. Concomitantly, these variants exhibited an inherent invasive potential as manifested by their capacity to infiltrate in vitro monolayers of hepatocytes and fibroblasts. The BW variants expressed new membrane markers such as H-2 antigens, the Lyt 1.2 T-cell differentiation antigen and the MTH antigen (a newly defined membrane antigen expressed predominantly on murine metastatic T-cell lymphomas and mature T lymphocytes). This phenomenon was observed with both cloned and uncloned BW populations, suggesting that an inductive rather than a selective mechanism accounts for the transition of BW cells towards a more malignant phenotype. These observations confirm the concept that local factors at the growth site of a tumor might influence the metastatic behavior of that tumor, possibly via induction of silent differentiation programs.     

Growth and metastasis of tumor cells isolated from a human renal cell carcinoma implanted into different organs of nude mice

The purpose of this study was to determine whether the methods for isolating tumor cells from a human renal cell carcinoma (HRCC) influence the biological behavior of the cancer cells. Renal cell carcinoma obtained from a surgical specimen was dissociated by enzymatic treatment and cells were plated into culture dishes or injected s.c. into the kidney of BALB/c nude mice. The resultant kidney tumor produced liver metastasis and ascites. All tumors growing in nude mice (s.c., kidney, liver, ascites) were also established in culture. The human origin of all five lines was ascertained by karyotypic and isoenzyme analyses. Cells from all lines were injected, s.c., i.p., i.v., intrasplenically, and beneath the renal capsule of nude mice. All the lines were tumorigenic after s.c. or renal subcapsule injection, although the rate of tumor growth varied among the five lines. The metastatic behavior of the HRCC cells was influenced by both the nature of the tumor cells and the route of injection into nude mice. In general, cells derived from the liver metastasis produced more metastases in nude mice than other lines. The lines established in culture from the primary HRCC and the ascites were poorly metastatic. Even with highly metastatic cells, i.v. injection did not yield significant metastasis, but the injection of cells into the renal subcapsule resulted in extensive metastasis to the lungs and in all peritoneal organs. These results indicate that nude mice can be used for the isolation of populations of HRCC cells with different growth and metastatic potential and that, of the organ sites tested, the renal subcapsule is the most advantageous site for implantation of HRCC cells.     

Genetic evidence for progressive selection and overgrowth of primary tumors by metastatic cell subpopulations

We have exploited random insertions of transfected DNA as unique clonotypic markers to follow cell lineages during primary and metastatic tumor growth of a mouse mammary adenocarcinoma, SP1. Southern analysis was undertaken of primary solid tumors and metastases obtained after injection of a pooled population of individual SP1 transfectants, or reconstituted mixtures of genetically marked metastatic and unmarked nonmetastatic cells. Here we provide evidence for the reproducible selection and eventual overgrowth of primary tumors by genotypically distinct metastatic clones, thereby illustrating that late-state, advanced primary tumors can evolve to become biologically similar, or even identical, to distant metastases. The selective growth advantage of metastatic cells within primary tumors was shown to occur despite the fact that tumors generated by both metastatic and nonmetastatic SP1 cell populations grew at comparable growth rates when injected and analyzed separately. The extent of the local growth advantage manifested by individual metastatic clones varied considerably, from 5- to 50-fold. Clonal overgrowth was also observed whether the tumor cells were injected ectopically, or orthotopically (i.e., into the mammary fat). This type of experimental approach should provide new insights into the dynamics of tumor progression and metastasis, the lineage relationship of primary tumors to metastases, the influence of clonal interactions on tumor behavior, and the physiological changes which are causative of malignant disease.     

Adoptive immune therapy in mice bearing poorly immunogenic metastases, using T lymphocytes stimulated in vitro against highly immunogenic mutant sublines

MDW3, a highly immunogenic and non-tumorigenic (tum-) mutant of the poorly immunogenic metastatic murine tumor called MDAY-D2, has been employed in an immune therapy scheme for the treatment of widespread established visceral MDAY-D2 metastases in syngeneic mice. MDW3 was selected from a mutagenized population of MDAY-D2 cells for the ability to grow in the presence of toxic concentrations of wheat-germ agglutinin (WGA) in vitro. The mutant expresses a common tumor-associated antigen (TAA) present on MDAY-D2 as well as a new antigen whose presence enhances the anti-TAA cell-mediated immune response in vivo and in mixed lymphocyte tumor cultures (MLTC) in vitro. For immune therapy, spleen cells from DBA/2 mice which had rejected an inoculum of MDW3 cells were restimulated in MLTC and injected i.v. into MDAY-D2 tumor-bearing mice. Two protocols were used. In the first, mice were given an i.v. injection of 10(3) MDAY-D2 cells ("artificial metastasis") and subsequently treated with 400 R whole-body irradiation and MDW3-stimulated T cells. Such mice had a 75% long-term survival rate, whereas 400 R alone, or no treatment, resulted in 25% and 0% long-term survivors, respectively. In the second protocol, treatment of mice bearing a 12-day-old subcutaneous MDAY-D2 tumor by surgical removal of the solid tumor, 400 R whole-body irradiation, and systemic administration of MDW3-stimulated spleen cells, resulted in a 75-100% survival rate, whereas omitting any part of the treatment resulted in 0-50% survival rates. The treatment increased splenic anti-TAA CTL activity, and the mice acquired immunity against the new antigen on MDW3, suggesting that the injected lymphocytes were proliferating in the host. The optimal combination of resection, whole-body irradiation and passive infusion of MDW3-stimulated spleen cells was ineffective when used on mice bearing a tumor-antigen-loss variant of MDAY-D2, suggesting that success of our immune therapy protocol required specific recognition of the tumor-associated antigen of MDAY-D2.     

Expression of a novel factor in human breast cancer cells with metastatic potential.

Clinical and experimental evidence suggests that tumor cells shed into the circulation from solid cancers are ineffective in forming distant metastasis unless the cells are able to respond to growth conditions offered by the secondary organs. To identify the phenotypic properties that are specific for such growth response, we injected carcinoma cells, which had been recovered from bone marrow micrometastases in a breast cancer patient who was clinically devoid of overt metastatic disease and established in culture, into the systemic circulation of immunodeficient rats. The animals developed metastases in the central nervous system, and metastatic tumor cells were isolated with immunomagnetic beads coated with an antibody that was reactive with human cells. The segregated cell population was compared with the injected cells by means of differential display analysis, and two candidate fragments were identified as up-regulated in the fully metastatic cells. The first was an intracellular effector molecule involved in tyrosine kinase signaling, known to mediate nerve growth factor-dependent promotion of cell survival. The second was a novel gene product (termed candidate of metastasis-1), presumably encoding a DNA-binding protein of helix-turn-helix type. Constitutive expression of candidate of metastasis-1 seemed to distinguish breast cancer cells with metastatic potential from cells without metastatic potential. Hence, our experimental approach identified factors that may mediate the growth response of tumor cells upon establishment in a secondary organ and, thereby, contribute to the metastatic phenotype.     

CXC chemokine receptor-4 antagonist blocks both growth of primary tumor and metastasis of head and neck cancer in xenograft mouse models

Squamous cell carcinoma of the head and neck (SCCHN) metastasizes to the lymph nodes and lungs. We have generated previously an orthotopic mouse model for head and neck metastasis and did in vivo selection of SCCHN cells through four rounds of serial metastases. A subpopulation of 686LN cells with high metastatic potential (686LN-Ms) was isolated. When the highly metastatic cells were compared with their low metastatic parental cells (686LN-Ps), we found that CXC chemokine receptor-4 (CXCR4) mRNA levels were significantly higher in the 686LN-Ms cells than the 686LN-Ps cells. Interestingly, the metastatic subclones had lost epithelial morphology and acquired mesenchymal features, which were maintained during cell expansion in vitro. This was featured by decreased E-cadherin and involucrin and increased vimentin and integrin beta(1). These results imply that CXCR4 and epithelial-mesenchymal transition markers can be potential biomarkers to identify the subpopulation of cells with high metastatic potential. Using the orthotopic SCCHN animal model, we showed that anti-CXCR4 treatment suppressed primary tumor growth by inhibiting tumor angiogenesis and prevented lung metastasis. Because the reduction of metastasis seen in the treated group could have resulted from 2-fold reduction in primary tumor size compared with that in the control group, we examined the effects of the CXCR4 antagonist in an experimental metastatic animal model in which 686LN-Ms cells were i.v. injected. 686LN-Ms cells failed to metastasize in the CXCR4 antagonist-treated group, whereas they metastasized to the lungs in the control group. Our data indicate that CXCR4 is an important target to inhibit tumor progression in SCCHN.