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Abstract
Introduction: TGF-β is an important target for many cancer therapies under development. In addition to suppressing anti-tumor immunity, it has pleiotropic direct pro- and anti-tumor effects. The actions of increased endogenous TGF-β production remain unclear, and may affect the outcomes of anti-TGF-β cancer therapy. We hypothesize that tumor-derived TGF-β (td-TGF-β) plays an important role in maintaining tumor remission by controlling tumor proliferation in vivo, and that decreasing td-TGF-β in the tumor microenvironment will result in tumor progression. The aim of this study was to examine the effect of TGF-β in the tumor microenvironment on the balance between its anti-proliferative and immunosuppressive effects.
Methods: A murine BALB/c spontaneous colon adenocarcinoma cell line (CT26) was genetically engineered to produce increased active TGF-β (CT26-TGF-β), a dominant-negative soluble TGF-β receptor (CT26-TGF-β-R), or the empty neomycin cassette as control (CT26-neo). In vitro proliferation rates were measured. For in vivo studies, the three cell lines were injected into syngeneic BALB/c mice, and tumor growth was measured over time. Immunodeficient BALB/c nude mice were used to investigate the role of T and B cells.
Results: In vitro, CT26-TGF-β-R and CT26-TGF-β cells showed increased and suppressed proliferation, respectively, compared to control (CT26-neo), confirming TGF-β has direct anti-tumor effects. In vivo, we found that CT26-TGF-β-R cells displayed slower growth compared to control, likely secondary to reduced suppression of anti-tumor immunity, as this effect was ablated in immunodeficient BALB/c nude mice. However, CT26-TGF-β cells (excess TGF-β) exhibited rapid early growth compared to control, but later failed to progress. The same pattern was shown in immunodeficient BALB/c nude mice, suggesting the effect on tumor growth is direct, with minimal immune system involvement. There was minimal effect on systemic antitumor immunity as determined by peripheral antigen-specific splenocyte type 1 cytokine production and tumor growth rate of CT26-neo on the contralateral flank of the same mice.
Conclusion: Although TGF-β has opposing effects on tumor growth, this study showed that excessive td-TGF-β in the tumor microenvironment renders the tumor non-proliferative. Depleting excess td-TGF-β may release this endogenous tumor suppressive mechanism, thus triggering the progression of the tumor. Therefore, our findings support cautions against using anti-TGF-β strategies in treating cancer, as this may tip the balance of anti-immunity vs. anti-tumor effects of TGF-β, leading to tumor progression instead of remission.
36. Murphy E.A. Interferon-gamma is crucial for surviving a Brucella abortus infection in both resistant C57BL/6 and susceptible BALB/c mice / E.A. Murphy, J. Sathiyaseelan, M.A. Parent, B. Zou, C.L. Baldwin // Immunology. – 2001. – V. 103. – I. 4. – P. 511-518.
ABSTRACT
Brucella abortus is an intracellular bacterial pathogen that causes chronic infections in humans and a number of agriculturally important species of animals. It has been shown that BALB/c mice are more susceptible to infections with virulent strains of Brucella abortus than C57BL/6 or C57BL/10 strains. In experiments described here, gene knock-out mice were utilized to elucidate some of the salient components of resistance. Resistant C57BL/6 mice with gene deletions or disruptions in the interferon-γ (IFN-γ), perforin or β2-microglobulin genes had decreased abilities to control intracellular infections with B. abortus strain 2308 during the first week after infection. However, only the IFN-γ knock-out mice had a sustained inability to control infections and this resulted in death of the mice at approximately 6 weeks post-infection. These mice had a continual increase in the number of bacterial colony-forming units (CFU) in their spleens until death. When BALB/c mice with the disrupted IFN-γ gene were infected they had more splenic CFU at one week post-infection than control mice but the increase was not statistically significant and by 3 weeks they did not have more CFU than control mice. Moreover, the number of splenic bacteria did not increase in the BALB/c IFN-γ knock-out mice between 6 and 10·5 weeks, although they died at 10·5 weeks, the time by which normal BALB/c mice were clearing the infection. Death in both strains of IFN-γ gene disrupted mice coincided with symptoms of cachexia and macrophages comprised ≥75% of the splenic leucocytes.
37. Huang G.M. Gambogic acid induces apoptosis and inhibits colorectal tumor growth via mitochondrial pathways / G.M. Huang, Y. Sun, X. Ge, X. Wan, C.B. Li // World J Gastroenterol. – 2015. – V. 21. – I. 20. – P. 6194-6205.
Abstract_AIM'>Abstract
AIM: To investigate the effect of gambogic acid (GA) on apoptosis in the HT-29 human colon cancer cell line.
METHODS: H-29 cells were used for in vitro experiments in this study. Relative cell viability was assessed using MTT assays. Cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling and Hoechst 33342 staining, and quantified by flow cytometry. Cellular ultrastructure was observed by transmission electron microscopy. Real-time PCR and Western blot analyses were used to evaluate gene and protein expression levels. For in vivo experiments, BALB/c nude mice received subcutaneous injections of HT-29 cells in the right armpit. When well-established xenografts were palpable with a tumor size of 75 mm3, mice were randomly assigned to a vehicle (negative) control, positive control or GA treatment group (n = 6 each). The animals in the treatment group received one of three dosages of GA (in saline; 5, 10 or 20 mg/kg) via the caudal vein twice weekly, whereas animals in the negative and positive control groups were given equal volumes of 0.9% saline or 10 mg/kg docetaxel, respectively, via the caudal vein once weekly.
RESULTS: The cell viability assay showed that GA inhibited proliferation of HT-29 cells in a dose- and time-dependent manner after treatment with GA (0.00, 0.31, 0.62, 1.25, 2.50, 5.00 or 10.00 μmol/L) for 24, 48 or 72 h. After 48 h, the percentage of apoptotic cells in cells treated with 0.00, 1.25, 2.50 and 5.00 μmol/L GA was 1.4% ± 0.3%, 9.8% ± 1.2%, 25.7% ± 3.3% and 49.3% ± 5.8%, respectively. Ultrastructural analysis of HT-29 cells treated for 48 h with 2.5μmol/L GA revealed apoptotic bodies and condensed and fragmented nuclei. Levels of caspase-8, -9 and -3 mRNAs were significantly increased after treatment with GA (1.25, 2.50 or 5.00 μmol/L) for 48 h (P < 0.05 for all). Protein levels of apoptosis-related factors Fas, FasL, FADD, cytochrome c, and Apaf-1 were increased in GA-treated cells, whereas levels of pro-caspase-8, -9 and -3 were significantly decreased (P < 0.05 for all). Furthermore, GA significantly and dose-dependently inhibited the growth of HT-29 tumors in a mouse xenograft model (P < 0.05).
CONCLUSION: GA inhibits HT-29 proliferation via induction of apoptosis. The anti-cancer effects are likely mediated by death receptor (extrinsic) and mitochondrial (intrinsic) pathways.
38. Sigalov A.B. A novel ligand-independent peptide inhibitor of TREM-1 suppresses tumor growth in human lung cancer xenografts and prolongs survival of mice with lipopolysaccharide-induced septic shock / A.B. Sigalov // Int Immunopharmacol. – 2014. – V. 21. – I. 1. – P. 208-219.
Abstract
Triggering receptor expressed on myeloid cells-1 (TREM-1) amplifies the inflammatory response and plays a role in cancer and sepsis. Inhibition of TREM-1 by short hairpin RNA (shRNA) in macrophages suppresses cancer cell invasion in vitro. In the clinical setting, high levels of TREM-1 expression on tumor-associated macrophages are associated with cancer recurrence and poor survival of patients with non-small cell lung cancer (NSCLC). TREM-1 upregulation on peritoneal neutrophils has been found in human sepsis patients and in mice with experimental lipopolysaccharide (LPS)-induced septic shock. However, the precise function of TREM-1 and the nature of its ligand are not yet known. In this study, we used the signaling chain homooligomerization (SCHOOL) model of immune signaling to design a novel, ligand-independent peptide-based TREM-1 inhibitor and demonstrated that this peptide specifically silences TREM-1 signaling in vitro and in vivo. Utilizing two human lung tumor xenograft nude mouse models (H292 and A549) and mice with LPS-induced sepsis, we show for the first time that blockade of TREM-1 function using non-toxic and non-immunogenic SCHOOL peptide inhibitors: 1) delays tumor growth in xenograft models of human NSCLC, 2) prolongs survival of mice with LPS-induced septic shock, and 3) substantially decreases cytokine production in vitro and in vivo. In addition, targeted delivery of SCHOOL peptides to macrophages utilizing lipoprotein-mimicking nanoparticles significantly increased peptide half-life and dosage efficacy. Together, the results suggest that ligand-independent modulation of TREM-1 function using small synthetic peptides might be a suitable treatment for sepsis and NSCLC and possibly other types of inflammation-associated disorders.
39. Shibahara I. OX40 ligand expressed in glioblastoma modulates adaptive immunity depending on the microenvironment: a clue for successful immunotherapy / R. Saito, R. Zhang et al. // Mol Cancer. – 2015. – V. 14. – P. 41. – DOI: 10.1186/s12943-015-0307-3.
Abstract
Background: Glioblastoma is the most malignant human brain tumor and has a dismal prognosis; however, some patients show long-term survival. The interaction between the costimulatory molecule OX40 and its ligand OX40L generates key signals for T-cell activation. The augmentation of this interaction enhances antitumor immunity. In this present study, we explored whether OX40 signaling is responsible for antitumor adaptive immunity against glioblastoma and also established therapeutic antiglioma vaccination therapy.
Methods: Tumor specimens were obtained from patients with primary glioblastoma (n = 110) and grade III glioma (n = 34). Quantitative polymerase chain reaction (PCR), flow cytometry, and immunohistochemistry were used to analyze OX40L expression in human glioblastoma specimens. Functional consequences of OX40 signaling were studied using glioblastoma cell lines, mouse models of glioma, and T cells isolated from human subjects and mice. Cytokine production assay with mouse regulatory T cells was conducted under hypoxic conditions (1.5% O2).
Results: OX40L mRNA was expressed in glioblastoma specimens and higher levels were associated with prolonged progression-free survival of patients with glioblastoma, who had undergone gross total resection. In this regard, OX40L protein was expressed in A172 human glioblastoma cells and its expression was induced under hypoxia, which mimics the microenvironment of glioblastoma. Notably, human CD4 T cells were activated when cocultured in anti-CD3-coated plates with A172 cells expressing OX40L, as judged by the increased production of interferon-γ. To confirm the survival advantage of OX40L expression, we then used mouse glioma models. Mice bearing glioma cells forced to express OX40L did not die during the observed period after intracranial transplantation, whereas all mice bearing glioma cells lacking OX40L died. Such a survival benefit of OX40L was not detected in nude mice with an impaired immune system. Moreover, compared with systemic intraperitoneal injection, the subcutaneous injection of the OX40 agonist antibody together with glioma cell lysates elicited stronger antitumor immunity and prolonged the survival of mice bearing glioma or glioma-initiating cell-like cells. Finally, OX40 triggering activated regulatory T cells cultured under hypoxia led to the induction of the immunosuppressive cytokine IL10.
Conclusion: Glioblastoma directs immunostimulation or immunosuppression through OX40 signaling, depending on its microenvironment.
40. Miura K. Molecular and genetic characterization of partial masculinization in embryonic ovaries grafted into male nude mice / K. Miura, K. Harikae, M. Nakaguchi et al. // PLoS One. – 2019. – V. 14. -I. 3: e0212367. - DOI: 10.1371/journal.pone.0212367.
Abstract
In most of mammalian embryos, gonadal sex differentiation occurs inside the maternal uterus before birth. In several fetal ovarian grafting experiments using male host mice, an experimental switch from the maternal intrauterine to male-host environment gradually induces partial masculinization of the grafted ovaries even under the wild-type genotype. However, either host-derived factors causing or molecular basis underlying this masculinization of the fetal ovaries are not clear. Here, we demonstrate that ectopic appearance of SOX9-positive Sertoli cell-like cells in grafted ovaries was mediated by the testosterone derived from the male host. Neither Sox8 nor Amh activity in the ovarian tissues is essential for such ectopic appearance of SOX9-positive cells. The transcriptome analyses of the grafted ovaries during this masculinization process showed early downregulation of pro-ovarian genes such as Irx3, Nr0b1/Dax1, Emx2, and Fez1/Lzts1 by days 7–10 post-transplantation, and subsequent upregulation of several pro-testis genes, such as Bhlhe40, Egr1/2, Nr4a2, and Zc3h12c by day 20, leading to a partial sex reversal with altered expression profiles in one-third of the total numbers of the sex-dimorphic pre-granulosa and Sertoli cell-specific genes at 12.5 dpc. Our data imply that the paternal testosterone exposure is partially responsible for the sex-reversal expression profiles of certain pro-ovarian and pro-testis genes in the fetal ovaries in a temporally dependent manner.
41. Uematsu K. Osteoclastogenic Potential of Tissue-Engineered Periosteal Sheet: Effects of Culture Media on the Ability to Recruit Osteoclast Precursors / K. Uematsu, T. Ushiki, H. Ishiguro et al. // Int J Mol Sci. – 2021. – V. 22. – I. 4. – P. 2169. - DOI: 10.3390/ijms22042169.
Abstract
Cell culture media influence the characteristics of human osteogenic periosteal sheets. We have previously found that a stem cell medium facilitates growth and collagen matrix formation in vitro and osteogenesis in vivo. However, it has not yet been demonstrated which culture medium is superior for osteoclastogenesis, a prerequisite for reconstruction of normal bone metabolic basis. To address this question, we compared chemotaxis and osteoclastogenesis in tissue-engineered periosteal sheets (TPSs) prepared with two types of culture media. Periosteal tissues obtained from adult volunteers were expanded with the conventional Medium 199 or with the stem cell medium, MesenPRO. Hematopoietic enhanced-green-fluorescent-protein (EGFP)-nude mice were prepared by γ-irradiation of Balb/c nu/nu mice and subsequent transplantation of bone marrow cells from CAG-EGFP C57BL/6 mice. TPSs were implanted subcutaneously into the chimeric mice and retrieved after intervals for immunohistopathological examination. EGFP+ cells were similarly recruited to the implantation site in both the TPSs prepared, whereas the distribution of CD11b+ cells was significantly lower in the TPS prepared with the stem cell medium. Instead, osteoclastogenesis was higher in the TPS prepared with the stem cell medium than in the one prepared with the conventional medium. These findings suggest that the stem cell medium is preferable for the preparation of more functional TPSs.
42. Bucana C.D. Different patterns of macrophage infiltration into allogeneic-murine and xenogeneic-human neoplasms growing in nude mice / C.D. Bucana, A. Fabra, R. Sanchez, I.J. Fidler // Am J Pathol. – 1992. – V. 141. – I. 5. – P. 1225-1236.
Abstract
This study determined the distribution pattern of tumor-associated macrophages (TAM) in murine and human neoplasms growing subcutaneously in nude mice. Seven different human neoplasms (cancers of the breast, kidney, colon, prostate, lung, and skin, and a melanoma) and five different murine neoplasms (carcinomas of the lung, colon, and kidney, melanoma, and fibrosarcoma) were injected into nude mice. The murine tumors also were injected into syngeneic mice. Tumor-associated macrophages in small and large tumors were studied immunohistochemically by the use of several antibodies, including the macrophage-specific F4/80. The pattern of TAM distribution differed between mouse and human tumors. Regardless of histologic classification, TAM were uniformly distributed throughout all the murine neoplasms growing in syngeneic or nude mice. In the human neoplasms, TAM were found on the periphery of the lesions and in association with fibrous septae. The distribution of TAM in murine and human tumors was associated with a pattern of vascularization as determined by antibodies to basement membrane collagen type IV. Because the pattern of TAM distribution in neoplasms influences their antitumor activity, the data question the validity of the nude mouse model for the study of macrophage infiltration into human neoplasms.
48. Keyaki A. Effector mechanisms of syngeneic anti-tumour responses in mice. II. Cytotoxic T lymphocytes mediate neutralization and rejection of radiation-induced leukaemia RL male 1 in the nude mouse system / A. Keyaki, K. Kuribayashi, S. Sakaguchi et al. // Immunology. – 1985. – V. 56. – I. 1. – P. 141-151.
Abstract
We demonstrated the efficacy of a long-term cultured cytotoxic T-lymphocyte line, CTLL-D4, on tumour growth inhibition using athymic nude mice as recipients. CTLL-D4, specific for a unique surface determinant on a radiation-induced leukaemia RL male 1 of BALB/c origin, was obtained from the limiting dilution culture of MLTC cells performed between spleen cells of a CB6F1-nu/+ mouse immunized in vivo and RL male 1 stimulator cells, and cultured for several months in the absence of added TCGF as described in our preceding paper (Kuribayashi, 1985). The specific inhibition of tumour growth by CTLL-D4 was demonstrated both in Winn-type neutralization assay and in systemic transfer experiments. A subcutaneous inoculation of the mixture of CTLL-D4 and RL male 1 cells resulted in the complete inhibition of tumour growth, even at the effector to tumour cell ratio of 1:1, whereas non-cytolytic D4f, which was self-Ia antigen(s)-reactive, composed entirely of Lyt-1+23- T cells and derived originally from CTLL-D4 but completely lost its cytotoxic activity during culture with the irradiated syngeneic feeder cells alone, had no inhibitory effect at all. In the adoptive transfer studies, the subcutaneously established tumours were rejected by the single i.v. transfer of 2 X 10(7) CTLL-D4 cells into CB6F1-nu/nu mice. However, D4f was ineffective again in this systemic transfer system. When the effect of CTLL-D4 cells on tumour rejection in vivo was compared to that of non-cultured spleen cells hyperimmunized with RL male 1 cells, the former exhibited more rapid rejection in nude mice after i.v. transfer than the latter did, suggesting that CTLL-D4 cells also attack the tumour cells much more effectively as effectors in vivo. Thus, it is conceivable that CTLs are mainly involved in tumour rejection in this adoptive transfer system using RL male 1 tumour cells and athymic nude mice.
49. Gulig P.A. Analysis of host cells associated with the Spv-mediated increased intracellular growth rate of Salmonella typhimurium in mice / P.A. Guling, T.J. Doyle, J.A. Hughes, H. Matsui // Infect Immun. – 1998. – V. 66. – I. 6. – P. 2471-2485.
ABSTRACT
The 90-kb virulence plasmid of Salmonella typhimurium encodes five spv genes which increase the growth rate of the bacteria within host cells within the first week of systemic infection of mice (P. A. Gulig and T. J. Doyle, Infect. Immun. 61:504–511, 1993). The presently described study was aimed at identifying the host cells associated with Spv-mediated virulence by manipulating the mouse host and the salmonellae. To test the effects of T cells and B cells on the Spv phenotype, salmonellae were orally inoculated into nude and SCID BALB/c mice. Relative to normal BALB/c mice, nude and SCID BALB/c mice were unaffected for splenic infection with either the Spv+ or Spv− S. typhimurium strains at 5 days postinoculation. When mice were pretreated with cyclophosphamide to induce granulocytopenia, there was a variable increase in total salmonella infection, but the relative splenic CFU of Spv+ versus Spv− S. typhimurium was not changed after oral inoculation. In contrast, depletion of macrophages from mice by treatment with cyclophosphamide plus liposomes containing dichloromethylene diphosphate resulted in equivalent virulence of Spv+ and Spv− salmonellae. To examine if the spv genes affected the growth of salmonellae in nonphagocytic cells, an invA::aphT mutation was transduced into Spv+ and Spv− S. typhimurium strains. InvA− Spv+ salmonellae were not significantly affected for splenic infection after subcutaneous inoculation compared with the wild-type strain, and InvA− Spv− salmonellae were only slightly attenuated relative to InvA+ Spv− salmonellae. Invasion-defective salmonellae still exhibited the Spv phenotype. Therefore, infection of nonphagocytes is not involved with the Spv virulence function. Taken together, these data demonstrate that macrophages are essential for suppressing the infection by Spv− S. typhimurium, by serving as the primary host cell for Spv-mediated intracellular replication and possibly by inhibiting the replication of salmonellae within other macrophages.
50. Lagrange P.H. Local immune response to Mycobacterium lepraemurium in C3H and C57Bl/6 mice / P.H. Lagrange, B. Hurtrel // Clin Exp Immunol. – 1979. – V. 38. – I. 3. – P. 461-474.
Abstract
Subcutaneous footpad inoculation of living M. lepraemurium (L.MLM) induced, in high responder C57Bl/6 mice, a local granulomatous reaction associated with the production of effector cells which stopped the multiplication of bacilli in the draining popliteal node with the concurrent development of 24--48 hr delayed type hypersensitivity (DTH). The thymus-dependent local reaction did not occur after the injection of heat-killed M. lepraemurium (HK.MLM) or after the inoculation of L.MLM in nude mice. However, HK.MLM injection interfered with the onset of the local reaction and enhanced acid-fast bacteria (AFB) counts in the draining node. In low responder C3H mice, L.MLM produced a local and delayed footpad swelling but no restriction of bacilli multiplication in the draining lymph node was observed. This unresponsiveness was not due to an overloading of the inoculum dose since doses ranging from 3 x 10(4) to 3 x 10(7) MLM did not produce any granulomatous local reaction as in C57Bl/6 mice. The injection of dead bacilli in the contralateral footpad of subcutaneously (s.c.) infected C3H mice revealed Arthus-like and 18--24 hr delayed reactions. When 10(6) L.MLM per mouse were injected intravenously (i.v.), systemic infection, measured in the spleen, was found to be less restricted in C57Bl/6 than in C3H mice. Moreover, in C57Bl/6 mice low doses of L.MLM injected i.v. delayed the local reaction at first, then enhanced footpad swelling and AFB counts in the draining nodes, indicating some acquired defect of peripheral immunity. When a high dose of L.MLM (2 x 10(8)/mouse) was injected i.v., C57Bl/6 mice died sooner than C3H mice, indicating certain discrepancies between local resistance and systemic susceptibility.