The role of the tumor microenvironment in tumor cell intravasation and dissemination

Metastasis, a process that requires tumor cell dissemination followed by tumor growth, is the primary cause of death in cancer patients. An essential step of tumor cell dissemination is intravasation, a process by which tumor cells cross the blood vessel endothelium and disseminate to distant sites. Studying this process is of utmost importance given that intravasation in the primary tumor, as well as the secondary and tertiary metastases, is the key step in the systemic spread of tumor cells, and that this process continues even after removal of the primary tumor. High-resolution intravital imaging of the tumor microenvironment of breast carcinoma has revealed that tumor cell intravasation exclusively occurs at doorways, termed “Tumor MicroEnvironment of Metastasis” (TMEM), composed of three different cell types: a Tie2^high/VEGF^high perivascular macrophage, a Mena overexpressing tumor cell, and an endothelial cell, all in direct contact. In this review article, we discuss the interactions between these cell types, the subsequent signaling events which lead to tumor cell intravasation, and the role of invadopodia in supporting tumor cell invasion and dissemination. We end our review by discussing how the knowledge acquired from the use of intravital imaging is now leading to new clinical trials targeting tumor cell dissemination and preventing metastatic progression.     

Variation of cell kinetic parameters in relation to the growth rate of the Ehrlich ascites tumor.

A multicompartmental model of the cell cycle and proliferation kinetics was used to analyse the time-course behavior of the cell cycle time, the growth fraction, and the cell loss rate during Ehrlich ascites tumor growth. The growth rate of Ehrlich ascites tumor cells as the tumor aged was significantly influenced by change in the cell cycle time.     

Requirement of species-specific interactions for the activation of human gamma delta T cells by pamidronate

Human gammadelta T cells bearing Vgamma2Vdelta2-TCR recognize various kinds of small nonpeptide Ags, and activation of them by a nitrogen-containing bisphosphonate Ag, pamidronate, requires Ag presentation by cells other than gammadelta T cells, including many human tumor cells. Present results demonstrated that tumor cell lines of nonhuman origins pulsed with pamidronate failed to activate human gammadelta T cells without exception, whereas most if not all human tumor cell lines could do so. Gammadelta T cells formed stable conjugates with pamidronate-pulsed human tumor cells and both conjugate formation and gammadelta T cell activation were inhibited significantly by anti-LFA-1 mAb, suggesting the requirement of LFA-1-mediated interaction with APC for efficient gammadelta T cell activation. Consistently, ICAM-1(low) tumor cell lines pulsed with pamidronate induced no or only weak activation of gammadelta T cells, whereas similarly treated ICAM-1(high) cell lines could activate them. One of the two ICAM-1(low) tumor cell lines pulsed with pamidronate induced strong gammadelta T cell activation after ICAM-1 gene transfer. However, another ICAM-1(low) human cell line as well as murine tumor cell lines pulsed with pamidronate remained totally defective in gammadelta T cell activation even after expression of human ICAM-1. These results suggested that activation of human gammadelta T cells by nonpeptide Ags required species-specific interactions in addition to LFA-1/ICAM-1-mediated cell adhesion with APC.     

Characterization of mouse tumor cell beta-lipotropin.

Mouse tumor cell beta-lipotropin (beta LPH) and gamma-lipotropin (gamma LPH) were purified from mouse pituitary tumor cell culture medium by ion exchange chromatography and gel filtration. The mouse tumor cell beta LPH was identified by immunoprecipitation with several antisera to beta-endorphin, generation of opioid bioactivity upon brief treatment with trypsin, and its identity with the molecule previously shown to serve as an intermediate in the biosynthesis of beta-endorphin. Mouse tumor cell beta LPH (Mr = 8200 +/- 250) and gamma LPH (Mr = 4600 +/- 200) are significantly smaller than known mammalian beta LPH (Mr = 10,000) and gamma LPH (Mr = 6300) molecules. The beta-endorphin region of mouse tumor cell beta LPH has the same amino acid composition as ovine, bovine, and camel beta-endorphin, and species-specific differences are thus located in the gamma LPH region of the molecule. Mouse tumor cell beta LPH and gamma LPH lack a methionine residue at what had been considered to be a highly conserved site in their beta-melanotropin-like region. A species-specific radioimmunoassay for mouse tumor cell gamma LPH was developed. Rat pituitary beta LPH and gamma LPH were shown to be similar to the corresponding mouse tumor cell molecules in size and lack of methionine in their beta-melanotropin-like segment.     

Increased tumor cell dissemination and cellular senescence in the absence of beta1-integrin function

Integrins are transmembrane receptors that bind extracellular matrix proteins and enable cell adhesion and cytoskeletal organization, as well as transduction of signals into cells, to promote various aspects of cellular behavior, such as proliferation or survival. Integrins participate in many aspects of tumor biology. Here, we have employed the Rip1Tag2 transgenic mouse model of pancreatic beta cell carcinogenesis to investigate the role of beta(1)-integrin in tumor progression. Specific ablation of beta(1)-integrin function in pancreatic beta cells resulted in a defect in sorting between insulin-expressing beta cells and glucagon-expressing alpha cells in islets of Langerhans. Ablation of beta(1)-integrin in beta tumor cells of Rip1Tag2 mice led to the dissemination of tumor cell emboli into lymphatic blood vessels in the absence of ongoing lymphangiogenesis. Yet, disseminating beta(1)-integrin-deficient beta tumor cells did not elicit metastasis. Rather, primary tumor growth was significantly impaired by reduced tumor cell proliferation and the acquisition of cellular senescence by beta(1)-integrin-deficient beta tumor cells. The results indicate a critical role of beta(1)-integrin function in mediating metastatic dissemination and preventing tumor cell senescence.     

Immunity to virus-free syngeneic tumor cell transplantation in the BALB/c mouse after immunization with homologous tumor cells infected with type C virus.

Syngeneic tumor cell lines free of endogenous type C virus or viral antigen antigen expression were derived from spontaneously occurring tumors of the BALB/cCr mouse. Two cell lines free of endogenous type C virus were examined and found to be highly tumorigenic in tumor growth kinetic studies. In vitro inoculation of these cell lines with Rauscher-murine leukemia virus (R-MuLV) resulted in their chronic infection in which 95 to 100% of the cells were scored as virus positive. These infected lines showed a highly significant increase in their immunogenicity as compared to their uninfected controls. Animals in which these virus-positive tumors regressed were then shown to be highly resistant to challenge with the uninfected tumor cell lines as well as to live R-MuLV. This observed resistance to uninfected tumor cell lines could not be induced by immunization of the mouse with uninfected tumor cells and R-MuLV simultaneously at the same injection site, nor could it be induced with lethally irradiated virus-infected tumor cells, subtumorigenic doses of uninfected cells, or inactivated R-MuLV or Gross leukemia virus (G-MuLV). Cell-mediated cytotoxicity studies revealed that spleen cells obtained from animals whose virus-infected tumors regressed were cytotoxic to homologous infected and uninfected tumor cells as well as to other uninfected tumor cell lines syngeneic to the BALB/c mouse. Correlation of in vitro cytotoxicity with in vivo immunity was provided by the Winn assay, by inoculation into susceptible mice of immune and nonimmune spleen cells premixed with uninfected tumor cells. The immune cells were highly effective in preventing this tumor cell transplantation. It was concluded that type-C virus infection of these syngeneic tumor cells resulted in their acquiring strong transplantation antigens that were in part due to the virion, but were at least in part due to alterations of antigens or haptens that are present in a less immunogenic form on the uninfected tumor cell.     

Murine mammary carcinoma exosomes promote tumor growth by suppression of NK cell function

Many tumor cells shed specialized membrane vesicles known as exosomes. In this study, we show that pretreatment of mice with exosomes produced by TS/A or 4T.1 murine mammary tumor cells resulted in accelerated growth of implanted tumor cells in both syngeneic BALB/c mice and nude mice. As implanted TS/A tumor cells grew more rapidly in mice that had been depleted of NK cells, we analyzed the effects of the tumor-derived exosomes on NK cells. The tumor-derived exosomes inhibit NK cell cytotoxic activity ex vivo and in vitro as demonstrated by chromium release assays. The treatment of mice with TS/A tumor exosomes also led to a reduction in the percentages of NK cells, as determined by FACS analysis, in the lungs and spleens. Key features of NK cell activity were inhibited, including release of perforin but not granzyme B, as well as the expression of cyclin D3 and activation of the Jak3-mediated pathways. Human tumor cell lines also were found to produce exosomes that were capable of inhibiting IL-2-stimulated NK cell proliferation. Exosomes produced by dendritic cells or B cells did not. The presentation of tumor Ags by exosomes is under consideration as a cancer vaccine strategy; however, we found that pretreatment of mice with tumor exosomes blunted the protective effect of syngeneic dendritic cells pulsed ex vivo with tumor exosomes. We propose that tumor exosomes contribute to the growth of tumors by blocking IL-2-mediated activation of NK cells and their cytotoxic response to tumor cells.     

Macrophage-mediated cytostasis of neoplastic hemopoietic cells: cytofluorometric analysis of the reversible cell cycle block

Nonactivated mouse peritoneal macrophages inhibit the proliferation of neoplastic hemopoietic cells in vitro. This effect is dependent upon the number of adherent macrophages present in cultures of hemopoietic tumor cells and can be documented by various parameters used as indices of cell proliferation. The two-layer soft agar culture system permits analysis of the regulatory functions of macrophage-derived diffusible substances under conditions of extremely low cell density and where macrophage-tumor cell contact is prevented by the gel matrix. The ability of the underlayers of macrophages to inhibit colony formation by hemopoietic tumor cells indicates that such an effect can be mediated by factor(s) elaborated by macrophages. Evidence that the mode of action of macrophages on tumor cell proliferation is cytostatic rather than cytotoxic is the capability of the macrophages to retard tumor cell growth in a particular phase of the cell cycle with retention of cell viability. Growth inhibition could be demonstrated to be reversible, with tumor cells entering normal cell cycle distributions shortly after being removed from macrophages or macrophage-derived factors. Cytofluorometric analysis of cell cycle inhibition correlated with viable cell counts and mitotic indices and confirms the suitability of this method for studying tumor cell proliferation.     

Possible role of hyaluronectin on cell adhesion in rat histiocytoma.

Distribution of hyaluronectin, a 68-kDa cell surface glycoprotein, has been demonstrated in normal peritoneal, alveolar macrophages as well as in macrophages of the AK-5 tumor cell line. AK-5, a transplantable histiocytic tumor cell line, is a mixture of four different populations and can be grown in both ascites and solid tumors. We are able to demonstrate a differential expression of hyaluronectin on the cell surface of these subpopulations of AK-5 when studied by immunocytochemical staining followed by cytofluorometric analysis. Cell fractions responsible for developing both ascites and solid tumors contain higher amounts of hyaluronectin than fractions which are capable of producing only ascites, suggesting its involvement in solid tumor formation. Furthermore, we established a secretory nature of hyaluronectin as it can be detected in the serum-free medium of AK-5 cells. Since it is localized on the cell surface and secreted into the medium, the cell adhesiveness of hyaluronectin has been examined. Hyaluronectin coating on the plates allowed more cells to attach, which could be specifically blocked by the antibody raised against hyaluronectin, indicating its possible role in cell attachment. The adhesive property of hyaluronectin and its role in tumor formation was further confirmed. The pretreatment of AK-5 cells with hyaluronectin antibody abolished their capacity to grow as solid tumors; however, the cells retained their capacity to grow as ascites tumor. We discuss our observations of hyaluronectin as a cell attachment protein and its specific role on tumor formation.     

Biological impact of mechanical stimuli on tumor metastasis

Increasing evidence suggests tumor cell exposure to mechanical stimuli during the perioperative period as well as throughout the normal disease process may have a discernable impact on tumor metastasis and patient outcome. In vitro studies have demonstrated that transient exposure to increased extracellular pressure and shear forces modulates integrin binding affinity and stimulates cancer cell adhesion through a cytoskeleton- and focal adhesion complex-dependent signaling mechanism. More prolonged exposure to elevated pressures stimulates tumor cell proliferation by a distinct signaling pathway. Whether pressure effects on cell adhesion and proliferation pose biological ramifications in vivo remained unknown. We recently reported that pressure activation of malignant cells does indeed have a biological impact on surgical wound implantation, tumor development, and tumor-free survival in a murine colon tumor model. Moreover, this effect can be disrupted by preoperative administration of colchicine. Taken together with previous work from our laboratory and others, these findings suggest that further

elucidation of the mechanical signaling pathways governing pressure-stimulated tumor cell adhesion and proliferation may identify novel therapeutic targets for the treatment and prevention of tumor metastasis.     

Lymphocyte-induced production of prostaglandin E2 by tumor cells in vitro: Requirements for direct contact and lymphocyte viability

The production of prostaglandin E₂ by tumor cell lines in response to exposure to purified lymphocytes has prompted the suggestion that this phenomenon may represent a defense mechanism whereby tumors may subvert an immune response mounted against them. To further characterize this phenomenon, cell lines derived from carcinogen-induced bladder tumors and embryo fibroblasts in Fischer rats were incubated with purified lymphocytes from peripheral blood, spleen, thymus, and lymph nodes from Fischer rats under a variety of conditions, and the amount of prostaglandin E₂ (PGE₂) production was determined by radioimmunoassay. Increased numbers of blood or splenic lymphocytes were associated with the induction of increased levels of PGE₂ production by the tumor cells. However, no prostaglandin was produced by the tumor cells after exposure to thymus or lymph node lymphocytes. Irradiation of lymphocytes prior to exposure to the tumor cells led to lower levels of PGE₂ production by the tumors, as did sonication of the lymphocyte preparations prior to addition to the tumor monolayers. Separation of lymphocytes from direct contact with the tumor cells resulted in less PGE₂ production by the tumor cell lines; however, when these lymphocytes were later layered onto fresh tumor cell monolayers, PGE₂ production occurred. Results in the present study suggest that direct contact between intact, viable, functionally active lymphocytes and tumor cells is necessary for tumor cell prostaglandin production to occur. Moreover, PGE₂ production only appears to occur in response to exposure to particular populations of lymphocytes, and this may correlate with the number of specific effector or attacker lymphocytes that are present. This specificity of response to effector cell challenge may be important in probing the defense mechanisms tumor cells may have to lymphocyte challenge, as well as in gauging the efficacy of a particular cellular immune response as it may be regulated both by cells involved in effecting this response as well as by the targets in lymphocyte/tumor cell interactions.     

The immunization site of cytokine-secreting tumor cell vaccines influences the trafficking of tumor-specific T lymphocytes and antitumor efficacy against regional tumors

Tumor cells engineered to secrete cytokines, referred to as tumor cell vaccines, can often generate systemic antitumor immunity and, in many cases, cause tumor regression. We compared the efficacy of s.c. immunization or intrahepatic immunization of GM-CSF-expressing tumor cell vaccines on the growth of s.c. or orthotopic liver tumors. A chemically transformed hepatic epithelial cell line, GP7TB, derived from Fischer 344 rats, was used to generate tumor models and tumor cell vaccines. Our results demonstrated that two s.c. injections of an irradiated tumor cell vaccine significantly controlled the growth of s.c. tumors, but was completely ineffective against orthotopic liver tumors. Effector cell infiltration in liver tumors was markedly reduced compared with s.c. tumors. Enhanced apoptosis of some effector cells was observed in the liver tumors compared with the s.c. tumors. Furthermore, the T cells induced by s.c. immunization preferentially migrated to s.c. tumor sites, as demonstrated by adoptive transfer experiments. In contrast, intrahepatic immunization, using parental tumor cells admixed with adenoviruses carrying the GM-CSF gene, yielded significantly better therapeutic effects on the liver tumors than on the s.c. tumors. Adoptive transfer experiments further confirmed that the T cells induced by liver immunization preferentially migrated to the liver tumor sites. Our results demonstrate that distinct T cell populations are induced by different immunization routes. Thus, the homing behavior of T cells depends on the route of immunization and is an important factor determining the efficacy of immunotherapy for regional tumors.     

Quantification of tumor cellularity and mitotic index in invasive ductal carcinoma of the breast

OBJECTIVE: To evaluate the use of stereologically estimated tumor cell counts in the mitotic index as well as to investigate its correlation with the currently used method and test the reproducibility of the method. STUDY DESIGN: The stereologic method described by Simpson et al was used to estimate tumor cellularity in 50 invasive ductal carcinomas. Mitotic counts were also performed, and the mitotic index was calculated by the use of estimated tumor cell counts. Estimated cell counts and the mitotic index calculated were compared statistically with the actual cell counts and the traditional mitotic grades, respectively. Interobserver reproducibility of the method was also tested. RESULTS: Stereologically estimated tumor cell counts had a good correlation with actual cell counts (r = .891, P < .001). Besides, the mitotic indices calculated with tumor cell counts (calculated with both estimated and actual cell counts) in the denominator of the fraction of the mitotic index were in agreement with the currently used method (P < .01 for both). There was no statistically significant difference between the counts of two observers (P = .068). CONCLUSION: The suggested method, considering tumor cellularity as an influencing factor, was practical, reproducible and in agreement with the traditional method. This method should be studied in a large group of patients with follow-up data to determine the threshold values for different grades and determine its prognostic value during the disease course.     

Differential activation of cytotoxic and suppressor T cells against syngeneic tumors in the mouse.

The cytotoxic T cell against a methylcholanthrene-induced sarcoma, S1509a, was induced in syngeneic mice by deliberate immunization with mitomycin C (MMC)-treated live tumor cells. The soluble tumor antigen (STA) extracted from the same tumor by 3 M KCl was, however, unable to induce the cytotoxic T cell upon immunization, although it was able to activate predominantly the suppressor T cell that then specifically suppressed the effect of the cytotoxic T cell against the homologous tumor. The suppressor T cell generated by STA had the same characteristics as those found in tumor-bearing animals: 1) The suppressor T cell has a very strict specificity against individual tumors; 2) The cell expresses cell surface determinants controlled by genes in the I-J subregion of the mouse H-2 complex. The activity of the cytotoxic T cell was completely inhibited by live tumor cells but not by STA, whereas that of the suppressor T cell was neutralized by STA. The results that cytotoxic and suppressor T cells are activated under different conditions, and that the antigenic determinants recognizable by these two cell types are not the same. The soluble extract contains only the determinants recognizable by the suppressor T cell, and the cytotoxic T cell can be activated only by the determinants associated with self antigen present on the surface of live tumor cells.     

Immune mechanism of the antitumor effects generated by bortezomib

Bortezomib, a proteasome inhibitor, is a chemotherapeutic drug that is commonly used to treat a variety of human cancers. The antitumor effects of bortezomib-induced tumor cell immunogenicity have not been fully delineated. In this study, we examined the generation of immune-mediated antitumor effects in response to treatment by bortezomib in a murine ovarian tumor model. We observed that tumor-bearing mice that were treated with bortezomib had CD8(+) T cell-mediated inhibition of tumor growth. Furthermore, the comparison of tumor cell-based vaccines that were produced from tumor cells treated or untreated with bortezomib showed vaccination with drug-treated tumor cell-based vaccines elicited potent tumor-specific CD8(+) T cell immune response with improved therapeutic antitumor effect in tumor-bearing mice. Conversely, the untreated tumor cell-based vaccines led to no appreciable antitumor response. Treatment of tumor cells with bortezomib led to the upregulation of Hsp60 and Hsp90 on the cell surface and promoted their phagocytosis by dendritic cells (DCs). However, cell surface expression of Hsp60, instead of Hsp90, is the more important determinant of whether bortezomib-treated tumor cells can generate tumor-specific CD8(+) T cells. CD11c(+) DCs that were treated with bortezomib in vitro had enhanced phagocytic activities. In addition, CD11c(+) DCs from bortezomib-treated tumor-bearing mice had increased maturation. At lower concentrations, bortezomib had no inhibitory effects on T cell proliferation. Taken together, our data indicate that bortezomib can render tumor cells immunogenic by upregulating the cell surface expression of heat shock protein 60 and heat shock protein 90, as well as improve DC function, which results in potent immune-mediated antitumor effects.     

A novel immunoregulatory axis of NKT cell subsets regulating tumor immunity

There are many mechanisms that regulate and dampen the immune response to cancers, including several types of regulatory T cells. Besides the T reg cell, we have identified another immunoregulatory circuit initiated by NKT cells that produce IL-13 in response to tumor growth and this IL-13 then induces myeloid cells to make TGF-beta that inhibits cytotoxic T cell-mediated tumor immunosurveillance in several mouse tumor models. This finding created a paradox in the role of NKT cells in tumor immunity, in that they can also contribute to protection. We resolve this paradox by the finding that the suppressive NKT cell is a type II NKT cell that lacks the canonical invariant T cell receptor, whereas the protective cell is a type I NKT cell that expresses the invariant receptor. Further, we see that these two subsets of NKT cells counter-regulate each other, defining a new immunoregulatory axis. The balance along this axis may determine the outcome of tumor immunosurveillance as well as influence the efficacy of anti-cancer vaccines and immunotherapy.     

Propagation of mouse mammary tumor cell lines and production of mouse mammary tumor virus in a serum-free medium.

Five different mouse mammary tumor cell lines were propagated in a serum free medium. Evaluation of growth characteristics, including logarithmic growth, cell population increase, protein production and days to confluency, showed serum-free medium comparable to serum-containing medium. Mouse mammary tumor virus expression and production, in C3H and GR tumor cell lines, as determined by virus particle counting and RNA dependent DNA polymerase assays, subsequent to dexamethasone stimulation revealed equivalent to higher levels of virus in serum-free medium as compared to serum-containing medium.     

Specific lysis of human tumor cells by T cells coated with anti-T3 cross-linked to anti-tumor antibody

Heteroaggregates containing anti-T3 cross-linked to anti-target cell antibodies have been shown to cause human T cells to lyse target cells that express antigens recognized by the anti-target cell antibody. In this study, we test targeted human T cells for the ability to lyse human tumor cells as a first step toward the application of this phenomenon to tumor immunotherapy. Several monoclonal anti-human tumor antibodies were assayed for binding to a number of human tumor lines and for the ability to promote specific tumor cell lysis when cross-linked with anti-T3. We found that anti-T3 cross-linked to anti-tumor monoclonal antibodies caused cloned human T cells and fresh peripheral blood T cells to lyse the tumor cells with the same specificity as predicted by the binding studies. Peripheral blood T cells were then tested in the presence of various heteroaggregates for the ability to lyse single cell suspensions prepared from fresh tumor or fresh normal tissue. These studies showed that heteroaggregates containing anti-T3 cross-linked to anti-tumor antibody cause fresh human T cells to specifically lyse fresh tumor cells, but not (with one exception) fresh normal cells.     

Breakdown of the endothelial barrier function in tumor cell transmigration

The ability of tumor cells to metastasize is associated with a poor prognosis for cancer. During the process of metastasis, tumor cells circulating in the blood or lymph vessels can adhere to, and potentially transmigrate through, the endothelium and invade the connective tissue. We studied the effectiveness of the endothelium as a barrier against the invasion of 51 tumor cell lines into a three-dimensional collagen matrix. Only nine tumor cell lines showed attenuated invasion in the presence of an endothelial cell monolayer, whereas 17 cell lines became invasive or showed a significantly increased invasion. Endothelial cells cocultured with invasive tumor cells increased chemokine gene expression of IL-8 and Gro-β. Expression of the IL-8 and Gro-β receptor, CXCR2, was upregulated in invasive tumor cells. Addition of IL-8 or Gro-β increased tumor cell invasiveness by more than twofold. Tumor cell variants selected for high CXCR2 expression were fourfold more invasive in the presence of an endothelial cell layer, whereas CXCR2 siRNA knock-down cells were fivefold less invasive. We demonstrate that Gro-β and IL-8 secreted by endothelial cells, together with CXCR2 receptor expression on invasive tumor cells, contribute to the breakdown of the endothelial barrier by enhancing tumor cell force generation and cytoskeletal remodeling dynamics.