Suppression of in vivo tumor growth by the transfection of the interleukin-5 gene into colon tumor cells

To investigate the influence of tumor producing interleukin-5 (IL-5) on growth kinetics of tumors, we transduced the murine IL-5 gene into murine colon C26 tumor cells. Two IL-5-secreting clones, low-level IL-5 producer C26-8B and high-level IL-5 producer C26-6F, were established. Both tumors, C26-6F and C26-8B, grew more slowly than the mock C26 tumor, although the in vitro growth rate of these IL-5 transfectants was much the same as that of the mock C26 cells. There was a significantly decreased number of colonies in the lung of mice given C26-6F or C26-8B tumors i.v. than in mice given mock C26 tumors i.v. Moreover, in mice given C26-6F cells i.v., a smaller number of tumor colonies in the lung was observed, as compared to the case with C26-6B cells. While the growth rate of C26-8B tumors in mice treated with anti-IL-5 mAb was more rapid than that seen in control mAb-treated mice, growth of C26-6F tumors in anti-IL-5-mAb-treated mice was slightly more rapid compared to findings in control mAb-treated mice. The isotypematched mAb did not alter the in vitro growth of mock-C26 cells or of the IL-5-gene-modified C26 cells. Growth of IL-5-secreting C26 tumors transplanted in nude mice was also inhibited. These results suggest that tumor-producing IL-5 inhibits growth of colon tumors mediated through T-cell-independent protective mechanisms of the host.     

Suppression of in vivo tumor growth by the transfection of the interleukin-5 gene into colon tumor cells

To investigate the influence of tumor producing interleukin-5 (IL-5) on growth kinetics of tumors, we transduced the murine IL-5 gene into murine colon C26 tumor cells. Two IL-5-secreting clones, low-level IL-5 producer C26-8B and high-level IL-5 producer C26-6F, were established. Both tumors, C26-6F and C26-8B, grew more slowly than the mock C26 tumor, although the in vitro growth rate of these IL-5 transfectants was much the same as that of the mock C26 cells. There was a significantly decreased number of colonies in the lung of mice given C26-6F or C26-8B tumors i.v. than in mice given mock C26 tumors i.v. Moreover, in mice given C26-6F cells i.v., a smaller number of tumor colonies in the lung was observed, as compared to the case with C26-6B cells. While the growth rate of C26-8B tumors in mice treated with anti-IL-5 mAb was more rapid than that seen in control mAb-treated mice, growth of C26-6F tumors in anti-IL-5-mAb-treated mice was slightly more rapid compared to findings in control mAb-treated mice. The isotypematched mAb did not alter the in vitro growth of mock-C26 cells or of the IL-5-gene-modified C26 cells. Growth of IL-5-secreting C26 tumors transplanted in nude mice was also inhibited. These results suggest that tumor-producing IL-5 inhibits growth of colon tumors mediated through T-cell-independent protective mechanisms of the host.     

Analysis of changes in DNA copy number in radiation-induced thymic lymphomas of susceptible C57BL/6, resistant C3H and hybrid F1 Mice

Radiation-induced thymic lymphoma in mice is a useful model for studying both the mechanism of radiation carcinogenesis and genetic susceptibility to tumor development. Using array-comparative genomic hybridization, we analyzed genome-wide changes in DNA copy numbers in radiation-induced thymic lymphomas that had developed in susceptible C57BL/6 and resistant C3H mice and their hybrids, C3B6F1 and B6C3F1 mice. Besides aberrations at known relevant genetic loci including Ikaros and Bcl11b and trisomy of chromosome 15, we identified strain-associated genomic imbalances on chromosomes 5, 10 and 16 and strain-unassociated trisomy of chromosome 14 as frequent aberrations. In addition, biallelic rearrangements at Tcrb were detected more frequently in tumors from C57BL/6 mice than in those from C3H mice, suggesting aberrant V(D)J recombination and a possible link with tumor susceptibility. The frequency and spectrum of these copy-number changes in lymphomas from C3B6F1 and B6C3F1 mice were similar to those in C57BL/6 mice. Furthermore, the loss of heterozygosity analyses of tumors in F(1) mice indicated that allelic losses at Ikaros and Bcl11b were caused primarily by multilocus deletions, whereas those at the Cdkn2a/Cdkn2b and Pten loci were due mainly to uniparental disomy. These findings provide important clues to both the mechanisms for accumulation of aberrations during radiation-induced lymphomagenesis and the different susceptibilities of C57BL/6 and C3H mice.     

Chloroprene: overview of studies under consideration for the development of an IRIS assessment

Beta-chloroprene (C(4)H(5)Cl, chloroprene, 2-chloro-1,3-butadiene, CASRN 126-99-8) is a volatile, flammable liquid monomer utilized primarily in the manufacture of neoprene (polychloroprene) elastomer used in belts, hoses, gloves, wire coatings, and tubing. Absorption into the body occurs primarily via the respiratory system and may occur via the gastrointestinal tract or the skin. Once absorbed, chloroprene is widely distributed as evidenced by effects in several target organs including nose and lung, liver, and skin. Chloroprene metabolism is believed to include cytochrome P450 oxidation to a monoepoxide, hydrolysis by epoxide hydrolases, and glutathione conjugation. Similar to 1,3-butadiene, the epoxide is considered to be the toxic moiety, and species differences in metabolic capacity may influence the severity of effects as well as what tissues are affected. EPA has not previously developed an assessment of chloroprene's potential for human health effects. Existing human epidemiological studies offer little data on noncancer effects, and the associations of exposure with increased cancer (liver and lung) mortality reported are inconclusive. Recent epidemiological studies (submitted for publication) could offer information that may impact chloroprene's health assessment. Multiple-site tumors have been reported in rats and mice exposed to chloroprene by inhalation; nevertheless, there are marked differences in strain sensitivities (i.e., tumors in F344 rats versus no tumors in Wistar rats). Recently developed physiologically based toxicokinetic models may allow for the resolution of species and tissue differences and sensitivities as well as exposure-dose-response relationships relevant to humans. (This presentation does not necessarily reflect EPA policy.).     

Genetic analysis of Raf1, Mdm2, c-Myc, Cdc25a and Cdc25b proto-oncogenes in 2',3'-dideoxycytidine- and 1,3-butadiene-induced lymphomas in B6C3F1 mice

We have previously identified activation of ras proto-oncogenes and inactivation of tumor suppressor genes including p53, p16(INK4a) and p15(INK4b) in 2',3'-dideoxycytidine (ddC)- and/or 1,3-butadiene (BD)-induced lymphomas derived from B6C3F1 (C57BL/6xC3H/He) mice, indicating that alterations of ras signaling pathway, p53 and pRb growth control pathways are important in the development of these chemically induced lymphomas. However, there is still a subset of tumors that displayed no changes in these genes. Thus, we investigated whether the Raf1, Mdm2, c-Myc, Cdc25a and Cdc25b proto-oncogenes, which are implicated in the ras or p53 or pRb pathways, are alternative oncogenic target genes. Analyses of gross genomic alterations by Southern blots failed to reveal rearrangement or amplification in any of the tumors examined. Frequent point mutations on the substrate binding domain of the Raf1 gene has been reported in 1-ethyl-1-nitrosourea (ENU)-induced murine lymphomas and lung tumors, along with a conspicuous lack of ras mutations [U. Naumann, I. Eisenmann-Tappe, U.R. Rapp, The role of raf kinases in development and growth of tumors, Recent Results Cancer Res., 143 (1997) 237-244]. To investigate whether Raf1 mutation is involved in our set of tumor especially those without ras mutations, the PCR-based single-strand conformation analyses (SSCA) and direct DNA sequencing were employed. No mutations but four genetic polymorphisms between C57BL/6 and C3H/He were found, with two of them reported as point mutations previously (op. cit.). The polymorphisms were utilized for allelic loss study of Raf1 locus. Losses of heterozygosity were found in six of 31 BD-induced lymphomas. These results indicate that genetic alterations of c-Myc, Cdc25, Raf1 and Mdm2 proto-oncogenes may not be involved in the development of ddC- and BD-induced lymphomas and the inactivation of tumor suppressor gene(s) located close to Raf1 gene might be important in the development of a subset of BD-induced lymphomas.     

Inhibition of metastasis of syngeneic murine melanoma in vivo and vasculogenesis in vitro by monoclonal antibody C11C1 targeted to domain 5 of high molecular weight kininogen

Metastasis of malignant tumors is a major cause of morbidity and mortality. Inhibition of tumor growth in distant organs is of clinical importance. We have demonstrated that C11C1, a murine monoclonal antibody to the light chain region of high molecular weight kininogen (HK), reduces growth of murine multiple myeloma in normal mice and human colon cancer in nude mice. C11C1 inhibits angiogenesis by reducing tumor microvascular density by blocking binding of HK to endothelial cells. We now evaluate the anti-metastatic effect of C11C1 on C57BL/6 mouse lung metastatic model using B16F10 melanoma cells. The tail veins of mice were injected with 0.5 × 10⁶ cells of melanoma B16F10. One group received C11C1 and the other received saline (control) intraperitoneally. When mice were killed at 28 days, 6 of 10 control mice had detectable metastatic pulmonary nodules which stained positive with an antibody against S-100 protein, a tumor antigen present in malignant melanoma cells. In the C11C1 groups, none of the mice showed metastatic foci in their lungs. We showed that C11C1 inhibits endothelial cell tube formation in a 3-D collagen fibrinogen gel model by inhibiting the rate of cleavage of HK by plasma kallikrein without changing the binding affinity for HK. These studies demonstrate that a monoclonal antibody to HK has the potential to prevent metastasis with minimal side effects.     

Cell-mediated immune responses to syngeneic tumors. I. Identification of two distinct CTL effector pathways which differ in antigen specificity, genetic regulation, and cell surface phenotype

It has been demonstrated previously that draining lymph nodes (DLN) from tumor-immunized mice contain a population of lymphoid cells that are capable of differentiating into functional antitumor cytotoxic T lymphocytes (CTL) during in vitro culture. In the present studies, it was observed that DLN cells from either C57BL/10 (B10) or C3H mice that had been footpad-immunized with syngeneic tumor cells differentiated into CTL during a 4-day in vitro culture in the absence of added antigen. The specificity patterns of the CTL thus generated, however, were quite different in the two strains. DLN from B10 mice immunized with ultraviolet light-induced fibrosarcoma cells of B10 origin differentiated into CTL which were only capable of lysing target cells from the tumor used for immunization. Thus, the antitumor CTL which differentiate from B10 DLN appeared to be specific for the tumor-specific antigen (TSA) expressed by these tumor cells. In contrast, DLN from C3H mice immunized with a syngeneic ultraviolet light-induced fibrosarcoma differentiated into CTL which effectively lysed not only target cells from the immunizing tumor, but several other fibrosarcomas of both B10 and C3H origin, and which did not lyse normal nontumor targets. These C3H effectors thus appeared to be specific for a tumor-associated antigen (TAA) which is widely shared by a number of tumors. Cold target-blocking studies demonstrated that the CTL generated by C3H DLN cells contained a subpopulation of TSA-specific cells in addition to cross-reactive TAA-specific effectors. (B6 X C3H)F1 (B6C3F1) mice generated cross-reactive TAA-specific CTL in response to in vivo challenge with either B10 or C3H tumors, indicating that the ability to generate a TAA-specific CTL response behaves as a dominant trait of the responding mouse strain and not as a function of the tumor used for immunization. TSA-specific CTL and cross-reactive TAA-specific CTL were distinguishable on the basis of their cell surface phenotypes, because the TSA-specific CTL generated by B10 DLN cells were Thy-1.2+ Lyt-2.2+, whereas TAA-specific B6C3F1 CTL were Thy-1.2+ Lyt-2.2-; alloantigen-specific CTL generated from the same B6C3F1 lymph nodes were Thy-1.2+ Lyt-2.2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultraviolet light induces tumors with both unique and host-associated antigenic specificities

Murine tumors induced by ultraviolet light (UV) are immunogenic in syngeneic and semi-syngeneic hosts, evoking antibody of several different specificities. Cytotoxic antibody specific for the immunizing syngeneic tumor (tumor-specific antigen) comprises the early response and a minor portion of the later response of C3H and C3H.SW mice. It is the primary specificity to which C57BL/6 and C57BL/10 hosts respond. The major portion of the antibody produced by C3H and C3H.SW against syngeneic tumors cross-reacts strongly with other tumors, both UV and chemically induced, arising in C3H and C3H.SW but not in B6.H2k, C57BL/6, C57BL/10, or (C3H X B6)F1. Normal adult cells or embryonic fibroblasts do not cross-react with the antisera. These results are interpreted as evidence for the involvement of a host component (non-MHC) in this tumor-associated antigen (TAA). (C3H X B6)F1 and (C3H X BALB/c)F1 hosts respond to C3H tumors with antibody with cross-reactive specificities identical to those of the C3H and C3H.SW hosts. thus detecting the TAA(C3H) specificity. (C3H X B6)F1 hosts respond to syngeneic F1 tumors, however, with a totally cross-reactive antibody that is interpreted as evidence for the existence of a common antigen in addition to the evident immune response control. An undetected TAA (B6) specificity in the (C3H X B6)F1 tumors is speculatively proposed.     

Anti-tumor effects of allogeneic bone marrow transplantation in (NZB X NZW)F1 hybrids with spontaneous lymphosarcoma

Untreated female (NZB X NZW)F1 hybrid mice (B/W F1) were found to develop lymphosarcoma spontaneously as they aged. Tumor incidence was evaluated in B/W F1 mice immunosuppressed with total lymphoid irradiation (TLI) and in TLI-conditioned B/W F1 mice reconstituted with 3 X 10(7) BALB/c bone marrow (BM) cells. BALB/C leads to B/W F1 chimerism (79 to 89% BALB/c-type cells) was confirmed by typing peripheral blood lymphocytes with specific alloantisera and complement by using a microcytotoxicity assay. Chimeras showed no clinical signs of graft-vs-host disease (GVHD). TLI-treated mice seemed to show a slightly accelerated onset of lymphosarcoma as compared with untreated controls, but the difference was not significant (p = 0.08). BALB/c leads to B/W F1 chimeras reconstituted at 1 to 3 mo of age (25 mice) developed no tumors for an observation period of 18 mo after transplantation. In contrast, tumors developed in 24/130 of age-matched controls, and in 13/57 of TLI-treated nonreconstituted age-matched B/W F1 mice. Tumor incidence in BALB/c leads to B/W F1 chimeras transplanted at an older age (9 to 11 mo) was similar to that observed in age-matched TLI-treated B/W F1 mice and age-matched untreated controls. The data suggests that the high naturally occurring incidence of lymphosarcoma could be reversed by reconstituting TLI-treated mice with BM cells (p = 0.027). Thus, allogeneic BM transplantation may exert potent graft-vs-tumor effects (GVT) when tumor susceptible hosts are reconstituted at an early age, whereas GVT is relatively ineffective at an advanced age, which probably correlates with an advanced stage of tumor development. Allogeneic BM transplantation should be additionally explored as a potential clinical tool for eradication of certain solid tumors in adjunct to high-dose radiochemotherapy, inasmuch as GVT seems to be independent of GVHD.     

Growth inhibition of an orthotopic glioblastoma in immunocompetent mice by cationic lipid-DNA complexes

The effect of a cationic liposome non-coding plasmid DNA complex on the growth of an intracerebral glioblastoma in an immunocompetent syngeneic mouse strain was evaluated. Previous studies of extraneural tumors in mice have demonstrated that such complexes containing plasmid DNA are capable of stimulating a potent Th-1 cytokine immune-mediated response with a dramatic inhibition of tumor growth. A DOTIM-cholesterol cationic liposome complexed to non-coding plasmid DNA (EV-CLDC) was administered intravenously (i.v.) at weekly intervals to 6-week-old male mice of the B6D2F1 strain at either 3, 10 or 17 days post-inoculation (DPI) of 4C8 glioblastoma cells. Tumor growth was monitored by volumetric image analysis obtained from sequential weekly magnetic resonance imaging studies of the brain. Experiments were terminated between 30 to 38 DPI. Terminal tumor volumes calculated from histological sections directly correlated with tumor volumes from corresponding MR images. The EV-CLDC administered at 3 DPI resulted in a statistically significant (P<0.0001) sustained inhibition of tumor growth compared with tumors in mice administered only individual components of the EV-CLDC. The EV-CLDC similarly inhibited growth of longer established glioblastomas. Histopathologic evaluation of terminal tumors did not find any hemorrhage, edema or necrosis in either the EV-CLDC-treated or control tumors. The results indicate that an i.v.-administered EV-CLDC can significantly inhibit the growth of a brain tumor in immunocompetent syngeneic mice.     

miR-200 Inhibits lung adenocarcinoma cell invasion and metastasis by targeting Flt1/VEGFR1

The microRNA-200 (miR-200) family is part of a gene expression signature that predicts poor prognosis in lung cancer patients. In a mouse model of K-ras/p53-mutant lung adenocarcinoma, miR-200 levels are suppressed in metastasis-prone tumor cells, and forced miR-200 expression inhibits tumor growth and metastasis, but the miR-200 target genes that drive lung tumorigenesis have not been fully elucidated. Here, we scanned the genome for putative miR-200 binding sites and found them in the 3'-untranslated region (3'-UTR) of 35 genes that are amplified in human cancer. Mining of a database of resected human lung adenocarcinomas revealed that the levels of one of these genes, Flt1/VEGFR1, correlate inversely with duration of survival. Forced miR-200 expression suppressed Flt1 levels in metastasis-prone lung adenocarcinoma cells derived from K-ras/p53-mutant mice, and negatively regulated the Flt1 3'-UTR in reporter assays. Cancer-associated fibroblasts (CAFs) isolated from murine lung adenocarcinomas secreted abundant VEGF and enhanced tumor cell invasion in coculture studies. CAF-induced tumor cell invasion was abrogated by VEGF neutralization or Flt1 knockdown in tumor cells. Flt1 knockdown decreased the growth and metastasis of tumor cells in syngeneic mice. We conclude that miR-200 suppresses lung tumorigenesis by targeting Flt1.     

Normal tissue alloantigens and genetic control of susceptibility to tumors: microcytotoxicity studies on resistant C3Hf and susceptible (A X C3Hf) F1 mice inoculated with transplacentally induced C3Hf lung tumor.

The immune response of mice to a transplacentally induced lung tumor was investigated with the microcytotoxicity (MC) assay. The tumor, originally induced in C3Hf mice, does not grow readily when transplanted to normal syngeneic C3Hf recipients. It grows readily, however, in (A C3Hf)F1 hybrids and in strain C3H mice, which express in their normal lung tissue a component which constitutes a strong lung tumor-associated transplantation antigen (TATA) in C3Hf mice. Both lung tumor-immunized C3Hf and tumor-bearing (A X C3Hf)F1 and C3H mice possessed lymphoid cells reactive against cultured lung tumor cells in the MC assay. Reactivity was also observed against cells cultured from normal lungs of (A X C3Hf)F1 and C3H mice, but not against cells similarly cultured from C3Hf of C57BL/6 mice. Anti-tumor MC was inhibited by serum-blocking factors present in some but not all tumor-bearing and tumor-immunized mice. The MC assay and detection by it of serum-blocking factors does not distinguish the effective anti-C3Hf lung tumor immune response of immunized C3Hf mice from the ineffective immune response of tumor-bearing (A X C3Hf)F1 and C3H mice. Furthermore, in lung tumor-bearing mice cells reactive in the MC assay may be directed against a normal tissue antigen rather than a tumor-associated antigen.     

K-ras mutations in mouse lung tumors of extreme age: independent of paternal preconceptional exposure to chromium(III) but significantly more frequent in carcinomas than adenomas

Preconceptional exposure of male NIH Swiss mice to chromium(III) chloride resulted in increased incidence of neoplastic and non-neoplastic changes in their progeny, including lung tumors in females [Toxicol. Appl. Pharmacol. 158 (1999) 161-176]. Since mutations in the K-ras protooncogene are frequent, early changes in mouse lung tumors, we investigated possible mutational activation of this gene as a mechanism for preconceptional carcinogenesis by chromium(III). These offspring had lived until natural death at advanced ages (average 816+/-175 days for controls, 904+/-164 for progeny of chromium-treated fathers). Mutations of K-ras, analyzed by single-strand conformation polymorphism and sequencing, were, in codon 12, wild type GGT (glycine), to GAT (aspartic acid); to GTT (valine); and to CGT (arginine); and in codon 61, wild-type CAA (glutamine), to CGA (arginine). K-ras mutation frequencies in lung tumors were very similar in control progeny (4/14) and in progeny of chromium-treated fathers (5/15). Thus, germline mutation or tendency to spontaneous mutation in K-ras does not seem to be part of the mechanism of preconceptional carcinogenesis here. However, an additional interesting observation was that K-ras mutations were much more frequent in lung carcinomas (8/16) than in adenomas (1/13) (P=0.02), for all progeny combined. This was not related to age of the tumor-bearing mice or the size of the tumors. K-ras mutations may contribute to malignant tumor progression during aging, of possible relevance to the putative association of such mutations with poor prognosis of human lung adenocarcinomas.     

Protection against metastasis by immunization with an allogeneic lymphocyte antigen

Q5 antigens are expressed on the surface of various experimental murine tumor cells. They share partially common antigenicity with Qa-2 alloantigens expressed on normal lymphocytes. For that reason we tested the immunoprotection by anti-Qa-2 immunization of mice against a Q5+ tumor. Nerve fibrosarcoma (NSFA) tumor, which specifically develops metastasis in the lung, has been reported to be poorly immunogenic. However, expression of the Q5 antigen was evident on the surface of NFSA cells. After immunizing (C3H/He x B6.K1)F1 (Qa-2-) mice with B6 (Qa-2+) lymphocytes, the protection against the proliferation of the semi-syngeneic NFSA tumor was examined First, immunization of normal mice induced resistance to NFSA cell transplants. Second, when the tumor cells were transplanted to the hind foot of a mouse and the resulting tumor was removed by amputating the leg, the mice were protected against the development of lung metastasis after immunization by intraperitoneal inoculation of B6 cells 3 days after tumor removal. Immunization with attenuated NFSA cells in this system failed to protect the mice from lung metastasis. On the other hand, inoculation of the mice with B6 cells without removal of the original tumor on the foot showed little effect on the progression of the tumor. Thus, cytotoxic T lymphocytes (CTL), which seemed to be present in an inactive form in the mice from which the tumor had not been removed, were induced in the mice after the removal of the major tumor followed by immunization with B6 lymphocytes. The induction of CTL by the immunization was suppressed in mice bearing large tumors. Cells stimulated by the tumor antigen seemed to be involved in the suppression. It was also shown that the Q5 antigen is the direct recognition target of the CTL since the activity of Q5-specific CTL clones in lysing tumor cells was inhibited by a monoclonal antibody specific for the Q5 antigen. In contrast to immunization with attenuated tumor cells, our novel allogeneic lymphocyte immunization procedure offers high CTL activation, by-passing the induction of T cell unresponsiveness.     

Protection against metastasis by immunization with an allogeneic lymphocyte antigen

Q5 antigens are expressed on the surface of various experimental murine tumor cells. They share partially common antigenicity with Qa-2 alloantigens expressed on normal lymphocytes. For that reason we tested the immunoprotection by anti-Qa-2 immunization of mice against a Q5+ tumor. Nerve fibrosarcoma (NSFA) tumor, which specifically develops metastasis in the lung, has been reported to be poorly immunogenic. However, expression of the Q5 antigen was evident on the surface of NFSA cells. After immunizing (C3H/He x B6.K1)F1 (Qa-2-) mice with B6 (Qa-2+) lymphocytes, the protection against the proliferation of the semi-syngeneic NFSA tumor was examined First, immunization of normal mice induced resistance to NFSA cell transplants. Second, when the tumor cells were transplanted to the hind foot of a mouse and the resulting tumor was removed by amputating the leg, the mice were protected against the development of lung metastasis after immunization by intraperitoneal inoculation of B6 cells 3 days after tumor removal. Immunization with attenuated NFSA cells in this system failed to protect the mice from lung metastasis. On the other hand, inoculation of the mice with B6 cells without removal of the original tumor on the foot showed little effect on the progression of the tumor. Thus, cytotoxic T lymphocytes (CTL), which seemed to be present in an inactive form in the mice from which the tumor had not been removed, were induced in the mice after the removal of the major tumor followed by immunization with B6 lymphocytes. The induction of CTL by the immunization was suppressed in mice bearing large tumors. Cells stimulated by the tumor antigen seemed to be involved in the suppression. It was also shown that the Q5 antigen is the direct recognition target of the CTL since the activity of Q5-specific CTL clones in lysing tumor cells was inhibited by a monoclonal antibody specific for the Q5 antigen. In contrast to immunization with attenuated tumor cells, our novel allogeneic lymphocyte immunization procedure offers high CTL activation, by-passing the induction of T cell unresponsiveness.     

B16/F10 tumors in aged 3D collagen in vitro simulate tumor growth and gene expression in aged mice in vivo

Although the incidence of cancer rises with age, tumor growth is often slowed in older hosts. The B16/F10 melanoma cell line is commonly used in murine models of age-related tumor growth suppression. We wished to determine if the growth pattern and gene expression of B16/10 tumors grown in aged mice could be simulated in 3D collagen matrices derived from aged mice. Outcome measures were tumor size in vitro and gene expression of the key growth regulatory molecules: growth hormone receptor (GHR), IL-10Rβ, IL-4Rα, and IL-6. B16/F10 tumors were grown in 20–25-mo-old C57/BL6 male mice. Tumor sizes ranged from 30 to 4,910 mg in vivo. Tumors from a subset of mice were removed after euthanasia, and equivalent amounts of each tumor were placed in aged 3D collagen and grown for 5 d. Tumor sizes in aged 3D collagen correlated highly with their original tumor size in vivo. Gene expression changes noted in vivo were also maintained during tumor growth in aged 3D collagen in vitro. The relative expression of GHR was increased, IL-10Rβ was unchanged, and IL-4Rα and IL-6 were decreased in the larger tumors relative to the smaller tumors in vitro, in a pattern similar to that noted in vivo. We propose that 3D matrices from aged mice provide an in vitro model of tumor growth that correlates highly with tumor size and expression of key regulatory molecules in vivo.     

Dual roles of sialyl Lewis X oligosaccharides in tumor metastasis and rejection by natural killer cells

Aberrant expression of cell surface carbohydrates such as sialyl Lewis X is associated with tumor formation and metastasis. In order to determine the roles of sialyl Lewis X in tumor metastasis, mouse melanoma B16-F1 cells were stably transfected with alpha1, 3-fucosyltransferase III to express sialyl Lewis X structures. The transfected B16-F1 cells, B16-FTIII, were separated by cell sorting into three different groups based on the expression levels of sialyl Lewis X. When these transfected cells were injected into tail veins of C57BL/6 mice, B16-FTIII.M cells expressing moderate amounts of sialyl Lewis X in poly-N-acetyllactosamines produced large numbers of lung tumor nodules. Surprisingly, B16-FTIII.H cells expressing the highest amount of sialyl Lewis X in shorter N-glycans died in lung blood vessels, producing as few lung nodules as B16-FTIII.N cells which lack sialyl Lewis X. In contrast, B16-FIII.H cells formed more tumors in beige mice and NK cell-depleted C57BL/6 mice than did B16-FTIII.M cells. B16-FTIII.H cells bound to E-selectin better than did B16-FTIII.M cells, but both cells grew at the same rate. These results indicate that excessive expression of sialyl Lewis X in tumor cells leads to rejection by NK cells rather than tumor formation facilitated by attachment to endothelial cells.     

Loss of the putative tumor suppressor band 4.1B/Dal1 gene is dispensable for normal development and does not predispose to cancer

The band 4.1 proteins are cytoskeletal proteins, harboring a conserved FERM domain highly homologous to the N-terminal FERM domain of ezrin, radixin, moesin, and merlin. Recently, a truncated form of the 4.1B protein, termed Dal-1, was identified in a screen as down regulated in adenocarcinoma of the lung and was mapped to chromosome 18p11.3, which is lost in 38% of primary non-small cell lung carcinoma tumors. Analysis of several meningiomas has shown that Dal-1 expression was lost in 76% of the tumors. To further elucidate the function of the 4.1B/Dal-1 gene in development and tumorigenesis we generated mice deficient for this allele. The 4.1B/Dal-1 null mice develop normally and are fertile. Rates of cellular proliferation and apoptosis in brain, mammary, and lung tissues from the 4.1B/Dal-1 null mice were indistinguishable from those seen with wild-type mice. Aging studies indicate that these mice do not have a propensity to develop tumors. Analysis of fibroblasts from these mice demonstrated that the growth characteristics and kinetics of these cells were not different from those of cells from the wild-type mice. These findings indicate that the 4.1B gene is not required for normal development and that 4.1B/Dal-1 does not function as a tumor suppressor gene.