LBC Cells Research Articles

Abstract 3354: Capturing tumor heterogeneity to personalize therapy in young patients with hormone receptor positive breast cancer

Abstract Background: Breast cancer (BC) in young women (Y) is a distinct entity in terms of prognosis. Although triple negative and HER2 positive subtypes are enriched in Y patients, the hormone receptor (HR) positive subtype (luminal, LBC) remains the most common. A thorough understanding of LBC biology in Y women has not been reached yet, also hampered by lack of appropriate models that faithfully recapitulate these tumors in molecular and preclinical studies. Methods: To investigate molecular determinants of progression and therapeutic resistance, we collected a large panel of fresh specimens (N=226) from Y (< 40 years) and nY (>55 years) LBC patients, characterized by targeted sequencing and used to generate Patient-Derived Organoids (PDO) and Xenografts (PDX). A selected panel of Y and nY LBC patients (N=96), sharing similar clinical-pathological features, was used for whole exome sequencing (WES) and transcriptomic analyses. Results: We generated stable LBC PDO (N=32, 13 from Y patients), with a success rate of 50% and stable LBC PDX (N=15, 11 from Y patients), with a success rate 17%. Our targeted panel of NGS, covering most mutated genes in LBC at high coverage (1000x), revealed a significantly higher number of Y patients displaying mutations in PIK3CA and in PIK3CA+TP53. However, these data were not observable in WES analyses (200x), likely because many of those mutations displayed a VAF < 5% (20% of PIK3CA and 30% of TP53). The transcriptomic analysis showed an enrichment in signatures of aggressiveness in the Y LBC subgroup. The EGF family member AREG (Amphiregulin) was the most enriched gene in tumors from pre- versus post-menopausal patients. Following validation in a larger panel of LBC, both at RNA and protein level, we tested the impact of AREG in LBC cell lines. AREG expression was timely induced by growth factor and hormonal stimulation. Furthermore, stimulation using amphiregulin induced the activation of EGFR and its downstream pathway, creating a positive feedback loop. Relevant to the Y LBC phenotype, a consistent and time dependent induction of AREG transcription was induced following estrogen and progesterone stimulation, indicating that, in LBC, AREG might act as a transcriptional target of ESR1. Finally, high levels of amphiregulin reduced response to tamoxifen and CDK4/6 inhibitors in LBC cells and PDOs. Conclusions and Perspectives: Our study provides valuable molecular and biological insights into LBC in Y women. We identified distinct genetic and transcriptomic profiles, including PIK3CA and TP53 mutations and AREG overexpression, which may be exploited to plan more targeted and efficient treatments. The establishment of a living biobank of PDO and PDX from LBC patients of Y age will enable preclinical trials, facilitating discovery of biomarkers and therapeutic strategies tailored to the unique biology of Y LBC. Citation Format: Ilenia Segatto, Maria Chiara Mattevi, Alessandra Dall'Acqua, Lorena Musco, Federica Ruggiero, Gabriele Di Giustino, Andrea Favero, Gian Luca Rampioni Vinciguerra, Nicole Crestan, Luca Pellizzari, Riccardo Spizzo, Riccardo Bomben, Filippo Vit, Andrea Vecchione, Lorenzo Gerratana, Fabio Puglisi, Samuele Massarut, Tiziana Perin, Gustavo Baldassarre, Barbara Belletti. Capturing tumor heterogeneity to personalize therapy in young patients with hormone receptor positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3354.

Gene expression comparison between primary aggressive luminal breast cancer and paired axillary and sentinel lymph node metastasis.

e13060 Background: More aggressive and therapy-resistant hormone receptor (HR)-positive breast cancer (hereafter luminal breast cancer, LBC) remains a great challenge. Few studies examine the genomics of axillary lymph node (ALN) metastasis in LBC. The aim of this study was to characterize and compare the mRNA expression patterns of primary breast cancers and paired ALN metastases. Methods: Aggressive pT1-2N2-3M0 LBC patients aged over 18 years who were treated at Shandong Provincial Hospital Affiliated to Shandong First Medical University and had available matched primary and metastatic tumor samples for gene expression analyses were retrospectively recruited. Bulk mRNA expression levels were determined by RNA sequencing (Illumina NovaSeq 6000 system) and calculated using featureCounts. Differential gene expression analysis was performed using DESeq2. All analyses were carried out with differentially expressed genes (DEGs) with an FDR <0.05. Changes of intrinsic subtypes from primary to metastatic disease were assessed. Results: Ten LBC patients with paired tumor samples were included. Compared with the primary tumor, ALN metastatic tumor had 1509 statistically significant DEGs, including 743 upregulated genes and 766 downregulated genes. Notably, there was an upregulation of genes related to immune response-activating signal transduction, T cell receptor signaling pathway, B cell receptor signaling pathway, and cytokine-cytokine receptor interaction in the ALN metastasis. The downregulation of genes were mainly enriched in the PI3K-Akt signaling pathway, ECM-receptor interaction, Wnt signaling pathway, and TGF-beta signaling pathway. Intrinsic subtype distribution in primary tumors was different from those in ALN metastatic tumors, such as Luminal A (30% vs. 50%), Luminal B (10% vs. 20%), HER2-enriched (20% vs. 10%), Basal-like (10% vs. 10%), and Normal-like (30% vs. 10%). These results suggest that LBC is a highly heterogeneous disease. Conclusions: In aggressive pT1-2N2-3M0 LBC, ALN metastases have a distinct gene expression profile and show upregulation of immune-related genes ( CR2, BLK, PAX5, CD19, FCRL1, TNFRSF13B). Genes associated with signaling pathways are involved in the development of breast cancer ( FST, TNN, LAMC3, SFRP2, FGF18), and are downregulated when compared to autologous primary cancer. These pathways are potential targeted pathways in the treatment of breast cancer. LBC cells metastatic to lymph nodes undergo a change to metastasize and survive in the new microenvironment, which may provide insights into the metastatic process. Besides, a switch towards more aggressive intrinsic subtype from primary to advanced disease is also observed.

Analysis of Resistance and Surface Recombination Velocities by Contact Coverage for Optimizing Electrical Loss in c-Si Local Back Contact.

Recently, the importance of solar cell research has emerged due to emerging social issues such as environmental pollution problems and rising oil prices. Accordingly, each company is studying to make solar cell of high efficiency. In order to fabricate high-efficiency solar cells, the two major techniques have to be applied on the rear. One is complete passivation of the surface using a thermal oxide and the other one is the part that comes in contact with the electrode doped partially LBSF (Local BSF) formation. In this paper, LBC technology which is usually applied for high efficiency crystalline silicon solar cell, applied to mass productive solar cell to achieve high open circuit voltage and short circuit current with low surface recombination from rear side. Thermal SiO2/SiN(x) double layer which has superior thermal stability is formed on rear surface as passivation layer, then 1% of the whole rear surface area is locally contacted with aluminum. Finally, the cell has been fired at high temperature and the cell process has complete. The fabricated LBC cells conversion efficiency was 18.0% with 625 mV of open-circuit voltage (V(oc)), 37.58 mA/cm2 of current density (J(sc)), 76.3% of fillfactor (FF) at 5% contact coverage, respectively.

EXOSOMES DERIVED FROM THE ASCITIC FLUID OF MICE BEARING A T-CELL LYMPHOMA EXPRESS TUMOR ASSOCIATED ANTIGENS

EXOSOMES DERIVED FROM THE ASCITIC FLUID OF MICE BEARING A T-CELL LYMPHOMA EXPRESS TUMOR ASSOCIATED ANTIGENS

EXOSOMES DISPLAYING HIGH EXPRESSION OF CD24 AND HSP90 INDUCED CELLULAR AND HUMORAL IMMUNE RESPONSE IN VIVO

Event Abstract Back to Event EXOSOMES DISPLAYING HIGH EXPRESSION OF CD24 AND HSP90 INDUCED CELLULAR AND HUMORAL IMMUNE RESPONSE IN VIVO Claudia Mongini1*, Leticia Hershlik1, Paula Di Sciullo1, Florencia Menay1, María J. Gravisaco2, Alejandrina Vendrell1, Claudia Waldner1 and Julieta De Toro1 1 National Research Council of Argentina (CONICET), Argentina 2 Instituto Nacional de Tecnología Agropecuaria (INTA), Argentina Exosomes have emerged as a new approach for the diagnosis and therapy of cancer. The use of these nano-vesicles as modulators of the immune system is being studied presenting them as readily available and stable tumor antigens. The objective of this work was to characterize exosomes derived from the murine T-cell lymphoma LBC and their immunogenic properties. The expression of proteins of immunological relevance, such as MHC I, CD8, CD24 and Hsp90 on exosomes membrane was demonstrated. We demonstrated surface expression of Hsp90 and intralumen Hsp70, indicative of a different protein expression pattern with the cells from which they derive. An over-expression of tumor associated antigens on LBC exosomes when compared to LBC cell lysates or even to intact LBC cells, corresponding to proteins of 51 kDa on LBC-derived exosomes that could not be detected in LBC cells, was found. These tumor antigens were recognized not only by memory lymphocytes but also induced a primary immune response in vitro and a humoral and cellular immune response in vivo, as INF gamma secreted LBC-sensitized splenocytes and specific antibodies were identified in the sera of mice immunized with LBC exosomes. In conclusion, the association of tumor-associated antigens and other molecules of immunologic interest such as MHC, Hsp or costimulatory molecules on tumor exosomes might determine the their immune properties. All these findings confirm exosomes as promising defined acellular tumor antigens for the development of an antitumor vaccine for the immunotherapy of cancer. Keywords: Exosomes, T-cell lymphoma, tumor vaccines, CD24, HSP90 Heat-Shock Proteins Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Translational immunology and immune intervention Citation: Mongini C, Hershlik L, Di Sciullo P, Menay F, Gravisaco MJ, Vendrell A, Waldner C and De Toro J (2013). EXOSOMES DISPLAYING HIGH EXPRESSION OF CD24 AND HSP90 INDUCED CELLULAR AND HUMORAL IMMUNE RESPONSE IN VIVO. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01161 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 31 Jul 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Claudia Mongini, National Research Council of Argentina (CONICET), Buenos Aires, Buenos Aires, 1121, Argentina, monginic@fmed.uba.ar Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Claudia Mongini Leticia Hershlik Paula Di Sciullo Florencia Menay María J Gravisaco Alejandrina Vendrell Claudia Waldner Julieta De Toro Google Claudia Mongini Leticia Hershlik Paula Di Sciullo Florencia Menay María J Gravisaco Alejandrina Vendrell Claudia Waldner Julieta De Toro Google Scholar Claudia Mongini Leticia Hershlik Paula Di Sciullo Florencia Menay María J Gravisaco Alejandrina Vendrell Claudia Waldner Julieta De Toro PubMed Claudia Mongini Leticia Hershlik Paula Di Sciullo Florencia Menay María J Gravisaco Alejandrina Vendrell Claudia Waldner Julieta De Toro Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Imiquimod enhances anti-lymphoma response in a tumor cell lysate vaccine and inhibits tumor cells growth.

Event Abstract Back to Event Imiquimod enhances anti-lymphoma response in a tumor cell lysate vaccine and inhibits tumor cells growth. Claudia Mongini1*, Julieta De Toro1, Leticia Hershlik1, María J. Gravisaco2, Claudia Waldner1 and Paula Di Sciullo1 1 National Research Council Of Argentina, Argentina 2 Instituto Nacional de Tecnología Agropecuaria (INTA), Argentina Our aim was to study the immunomodulating and proapoptotic properties of the TLR7 agonist Imiquimod to be used as adjuvant in a tumor vaccine. Imiquimod showed an antiproliferative effect on LBC T-cell lymphoma in vitro at 48 and 72 h with doses between 0.5 and 200 μg/ml, inducing apoptosis in a dose dependent manner. BALB/c mice inoculated intraperitoneally twice with LBC cells lysates with or without 100 μg/mouse of Imiquimod and challenged one week later with LBC cells intraperitoneally, showed a significant increase in survival compared to un-immunized controls (LBC-lysate<0.05, LBC-lysate + IMQ<0.0005) and to mice receiving Imiquimod (p<0.0002; Logrank).Tumor incidence was only 5±5% in LBC-lysate+IMQ group whereas in LBC-lysate group was 52.5±2.5%. Although the percentages of specific cytotoxicity were above normal in both immunized groups (p<0.05, ANOVA), LBC-lysate+IMQ immunization induced the highest levels. A significant enhance in peritoneal cavity CD8+, CD4+ T and B cells (p<0.0001, ANOVA) with an increase IFNγ in supernatant from peritoneal cavity cells and splenocytes was demonstrated in all vaccinated groups compared to normal (p<0.001, ANOVA). Our vaccine also generated circulating antibodies in all immunized mice that rejected the tumor up to 75 days after challenge. Finally, mice that rejected the first tumor challenge were re-challenged with LBC tumor with an increased in survival in all immunized groups over the control (p<0.01, Logrank) with rejection rates close to 100%. We demonstrated that Imiquimod not only modulates immune response efficiently to reject tumor generating a long-lasting memory, but also is capable of inducing tumor apoptosis. Keywords: imiquimod, T-cell lymphoma, TLR7 agonist, Apoptosis, tumor cell lysate vaccine Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Translational immunology and immune intervention Citation: Mongini C, De Toro J, Hershlik L, Gravisaco MJ, Waldner C and Di Sciullo P (2013). Imiquimod enhances anti-lymphoma response in a tumor cell lysate vaccine and inhibits tumor cells growth.. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01162 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 31 Jul 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Claudia Mongini, National Research Council Of Argentina, Buenos Aires, Buenos Aires, 1121, Argentina, cmongini@yahoo.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Claudia Mongini Julieta De Toro Leticia Hershlik María J Gravisaco Claudia Waldner Paula Di Sciullo Google Claudia Mongini Julieta De Toro Leticia Hershlik María J Gravisaco Claudia Waldner Paula Di Sciullo Google Scholar Claudia Mongini Julieta De Toro Leticia Hershlik María J Gravisaco Claudia Waldner Paula Di Sciullo PubMed Claudia Mongini Julieta De Toro Leticia Hershlik María J Gravisaco Claudia Waldner Paula Di Sciullo Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Lineage-Specific Functions of LKB1 in CML and B Lymphoid Blast Crisis

Abstract 34 Background:The serine-threonine liver kinase B1 (LKB1, also called STK11) acts as negative regulator of aerobic glycoloysis, a metabolic pathway that is typically used in cancer cells (commonly referred to as ‘Warburg effect'). LKB1, together with AMPKs, shifts the metabolic balance from aerobic glycolysis to oxidative phosphorylation and thereby reverses the metabolic program of cancer cells and functions as tumor suppressor. Recently, it has been shown that LKB1 plays a critical role in the maintenance of quiescence and metabolic homeostasis of hematopoietic stem cells (HSCs). Hypothesis:In the present study, we focused on the roles of LKB1 in BCR-ABL1-driven leukemias including CML and B lymphoid blast crisis/Ph+ ALL (LBC). While LKB1 is widely seen as tumor suppressor in solid tumors, we found that high expression levels of LKB1 at diagnosis predict poor clinical outcome in patients with high risk acute lymphoblastic leukemia (n=207; COG P9906 trial; p=0.0204). In addition, high levels of LKB1 expression correlate with positive minimal residual disease (MRD+, p=0.0323) status in patients. These findings were unexpected and seem to contradict the common notion of LKB1 as a tumor suppressor. Results:To study the function of LKB1 in CML and B lymphoid blast crisis/Ph+ ALL (LBC), we developed a mouse model for inducible ablation of Lkb1 in BCR-ABL1-transformed hematopoietic stem and progenitor cells (CML-like) and B cell progenitors (LBC). To this end, Lkb1-fl/fl bone marrow hematopoietic stem and progenitor cells and B cell precursor cells were transformed with BCR-ABL1 and transduced with tamoxifen-inducible Cre. Unexpectedly, Cre-mediated deletion of Lkb1 had opposite effects in CML and LBC. While Lkb1-deletion in CML results in an initial proliferative burst of the leukemia cells, the vast majority of B cell lineage LBC cells undergo rapid cell cycle arrest. These findings are consistent with changes of cell cycle checkpoint proteins in response to Lkb1 deletion in CML and B cell lineage LBC. While Lkb1 deletion in CML cells results in downregulation of Arf, p53 and p27 levels, Lkb1 deletion in B lineage LBC cells resulted in upregulation of Arf and p27. In addition, Lkb1 deletion in CML resulted in inactivation of AMPK, a known substrate of LKB1, as well as enhanced activation of mTORC1. By contrast, while deletion of Lkb1 in B cell lineage LBC cells resulted in inactivation of AMPK as shown by reduced phosphorylation of AMPKα T172, there was reduction in mTORC1 activity based on diminished levels of phospho-p70 S6 kinase and S6 following LKB1 deletion.The effects of LKB1 on sensitivity of BCR-ABL1 CML and B lineage LBC cells to Imatinib were also examined. Lkb1-deficient ALL cells became more sensitive to Imatinib treatment. On the other hand, initial Lkb1 deletion rendered CML cells more resistant to Imatinib treatment. When primary patient-derived Ph+ ALL cells (n = 3) were treated with Imatinib, upregulation of phospho-LKB1 (S428) was observed. Finally, LKB1 was also shown to regulate energy homeostasis in CML and B cell lineage LBC in different manners, as measured by monitoring ATP and lactate production. Conclusions:Here we show that Lkb1 plays critical roles in mediating proliferation and cell growth in BCR-ABL1-driven leukemias. While LKB1 is widely seen as a tumor suppressor that limits aerobic glycolysis in cancer cells according to the Warburg effect, our findings demonstrate that LKB1 has lineage-specific functions in BCR-ABL1 driven leukemias. While LKB1 function in CML resembles its tumor suppressor function in solid tumors, LKB1 is critical for survival and proliferation on B cell lineage CML blast crisis and Ph+ ALL. The finding of a divergent role of Lkb1 in CML and B cell lineage LBC/Ph+ ALL is relevant because small molecule inhibitors of AMPK and mTORC1 are currently under development for the treatment of BCR-ABL1-driven leukemias. Disclosures:No relevant conflicts of interest to declare.

Beneficial activity of Enterococcus faecalis CECT7121 in the anti-lymphoma protective response

To study the anti-tumour effects of Enterococcus faecalis CECT7121 on LBC cells, an aggressive murine T-cell lymphoma that kills the host in 18 days when is intraperitoneally (i.p.) administrated. In vitro studies have shown that LBC cell proliferation was inhibited by Ent. faecalis CECT7121 stimulus in a dose-dependent manner, inducing apoptosis. The production of ceramide was involved in the latter effect. To undertake in vivo studies, syngeneic BALB/c mice pre-treated i.p. with Ent. faecalis CECT7121 (2.5 × 10(8 ) CFU) were challenged i.p. with LBC cells (1.0 × 10(6) cells) the day after. On day 30 post-inoculation of LBC cells, 70% of Ent. faecalis CECT7121 pre-treated mice survived, whereas no survivals were recorded in the control group. A group of surviving mice was re-challenged with LBC cells, and 89% of them survived. Upon stimulation with irradiated LBC cells, spleen cell proliferation, high IFNγ, IL-12 and IL-10 levels were observed in surviving animals. Enterococcus faecalis CECT7121 affected multiple factors of the tumour establishment by the following methods: down-regulating the LBC cell proliferation and inducing apoptosis in these cells; and enhancing the immune response that protects animals from lymphoma challenge and re-challenge. This study demonstrate that Ent. faecalis CECT7121 has potential as a probiotic that could facilitate the development of novel complements to therapeutic strategies against oncological diseases.

Complete rejection of a T-cell lymphoma due to synergism of T-cell receptor costimulatory molecules, CD80, CD40L, and CD40

The equal importance of the qualitative and quantitative characteristics of antigen presentation as well as the set of costimulatory signals provided by antigen presenting cells to T-cells in determining the outcome of T-cell responses at the time of antigen recognition is now clear. Moreover, an important function in innate mechanisms has been recently attributed to costimulatory molecules demonstrating their relevant role in different stages of immune response. In this paper, we demonstrated the ability of CD40L (CD154) and CD80 costimulatory molecules expression in a T-cell lymphoma to induce both T-cell dependent and independent immune responses leading to an important anti-tumor effect. CD40 expression by LBC cells enhanced only T-cell dependent anti-tumor immune response resulting in tumor rejection. Furthermore, this work represents the first report to describe complete tumor rejection after co-inoculation of lymphoma cells transfected with CD40L and CD80 in either presence or absence of CD40 expressing lymphoma cells. In addition, this synergistic effect resulted in long lasting immunity to parental tumor cells. Co-inoculation of tumor cells each genetically modified to express a different costimulatory molecule circumvents the need to co-transfect genetically unstable tumor cells and represents an option for those weakly or non-immunogenic tumors where either treatment alone proved to be inefficient. This strategy represents a promising approach for inducing anti-tumor immunity and provides a new rational design of cancer therapies.

Transgene expression enhancement in T-lymphoma cell lines

In transfection protocols, the expression levels of the transgene is important to define, still is difficult to obtain in certain cell lines such as those derived from T-lymphoma cells. In this study we evaluate transgene expression kinetics in the presence and absence of two well known transcription activators such as phorbol-12-myristate13-acetate (PMA) and Ionomicin (IO). Three murine T lymphoma cell lines (LBC, EL4 and BW5147) were transfected by electroporation using green fluorescent protein (GFP) as a reporter gene and analyzed by flow cytometry. Addition of PMA/IO resulted in a significant increase of the Mean Fluorescence Intensity but not in GFP-positive cell percentages, either in transient or stable transfected LBC and EL4 cells. Remarkable, BW5147 cells showed low GFP induction with a significant increment only in stable transfected cells. Our results demonstrated that CMV promoter activity can be enhanced in transfected lymphoma cells by PMA/IO suggesting that transgene expression levels can be optimized by means of the use of transcription activators.

IL-2, IL-10, IL-15 and TNF are key regulators of murine T-cell lymphoma growth

We studied the role of IL-2, IL-15, IL-10, TNF and IL-2 receptor complexes (IL-2R) produced constitutively by a T-cell lymphoma line (LBC) on their own proliferation. The constitutive expression of surface alpha, beta and gamma chains IL-2R was detected in tumor cells by flow cytometry. Using reverse-transcription PCR, mRNA for IL-2, IL-15, IL-10 and TNF were found to be present in LBC. In addition, tumor cells were found to constitutively express intracellular IL-2, IL-15, IL-10 and TNF. Despite the production of these cytokines by tumor cells, specific neutralising antibodies did not inhibit LBC proliferation; surprisingly, anti-IL-15 increased LBC cell growth. We also demonstrated that recombinant IL-2 or IL-15 enhanced LBC cell proliferation. Our data suggest that endogenous IL-2 and IL-15 may trigger the proliferation of lymphoma LBC cells, and so their growth could be regulated, at least partly, by IL-2/IL-15/IL-2R system. In addition, IL-10 and TNF, immunosuppressor and pro-metastatic cytokines, respectively, may promote the in vivo growth of the tumor. The fact that leukaemia-lymphoma cells produce simultaneously both IL-2 and IL-15 should be taken into consideration in the design of immunotherapy protocols directed to IL-2R.

Induction of apoptosis in murine lymphoma cells by cyclosporin A

The aim of this study was to investigate if CsA could induce apoptosis in the murine T-lymphoma cell line LBC, whose growth is inhibited by this immunosuppressive drug. CsA induced programmed cell death in LBC cells with typical features of apoptosis demonstrated by exposure of phosphatidyl serine residues on the cell membrane, the decrease of cell DNA content, chromatin condensation, and nuclear fragmentation. Apoptosis was evident within 12 h after CsA incubation, with a maximal effect at 48 h, in a time and dose-dependent fashion. In addition, the role of apoptosis inhibitors (Bcl-2 and Bcl-x) and the apoptosis inducer (Bax) in CsA induced-apoptosis was evaluated. The expression of Bcl-2 and Bax proteins were high in LBC cells and following CsA treatment the expression of these proteins as well as Bcl-XL decreased. In this work we demonstrated that cell growth inhibition following CsA treatment in LBC was paralleled by the induction of apoptosis thus providing an interesting animal model to identify the mechanism participating in the regulation of apoptotic genes by CsA in T-cell neoplasms and to assess preclinical in vivo trials of T-cell lymphoma-related disorders.

Characterization of the immunophenotype and the metastatic properties of a murine T-lymphoma cell line. Unexpected expression of cytoplasmatic CD4.

We report the first characterization of a mouse T-lymphoma cell line that surprisingly expresses cytoplasmatic (cy) yCD4. Phenotypically, LBC cells are CD5+, CD8+, CD16+, CD24+, CD25+, CD2-/dim, CD3-/dim, TCRbeta-/dim, TCRgammadelta, CD154 , CD40-, and CD45R. Coexpress cyTCRbeta, cyCD3, cyCD4, and yet lack surface CD4 expression. Transplantation of LBC cells into mice resulted in an aggressive T-lymphoblastic lymphoma that infiltrated lymph nodes, thymus, spleen, liver, ovary, and uterus but not peripheral blood or bone marrow. LBC cells display a modal chromosome number of 39 and a near-diploid karyotype. Based on the characterization data, we demonstrated that the LBC cell line was derived from an early T-cell lymphocyte precursor. We propose that the malignant cell transformation of LBC cells could coincide with the transition stage from late double-negative, DN3 (CD4- CD8 CD44-/low, CD25+) or DN4 (CD4-low, CD8-/low, CD44-, CD25-) to double-positive (DP: CD4+CD8+) stage of T-cell development. LBC cells provide a T-lymphoblastic lymphoma model derived from a malignant early T-lymphocyte that can be potentially useful as a model to study both cellular regulation and differentiation of T-cells. In addition, LBC tumor provides a short latency neoplasm to study cellular regulation and to perform preclinical trials of lymphoma-relatel clisorders.

Replication of rat coronaviruses in intestinal cell line, RCN-9, derived from F344 rats.

To examine the susceptibility of the epithelial cell line to rat coronavirus (RCV), we inoculated sialodacryoadenitis virus and Parker's RCV into five cell lines; JTC-19, rat L2, LLC, RCN-9 and LBC cells originating in the lungs, intestines and mammary tumors of rodents. Both RCVs were replicated in LBC and RCN-9 cells, but not in the others. The infectivity titers of both RCVs grown in RCN-9 cells were significantly higher than those in LBC cells in every passage (2.5-3.9 log rate). Both RCVs replicated in LBC cells showed higher tropism to RCN-9 cells than to LBC cells, suggesting that RCN-9 cells are more suitable for the replication of RCVs than LBC cells. The RCN-9 cell line would be useful for the investigation of RCV infection in rodents.

Induction of anti-tumour immunity in syngeneic mice by a leukaemic cell line.

Induction of anti-tumour immunity in syngeneic mice by LBC cell line derived from a non-immunogenic T cell leukaemia was studied. The immunization of BALB/c mice with LBC irradiated cells induced in them anti-tumour spleen cells, cytotoxic T lymphocytes and anti-LBC antibodies. The anti-LBC antibodies reacted with components of 14, 16 and 27 kDa present on LB tumour cells, LBC cell line and normal thymocytes, but not with normal lymph node cells. Furthermore, immunization of the autologous hosts with LBC cells partially protected them against subsequent challenge with the original LB leukaemic cells. These findings demonstrate that culture conditions induced modifications in the antigenic properties of the leukaemic cells, allowing LBC cells to stimulate an immune response directed against components expressed at early stages during T cell maturation. These results also suggest that the immune response is responsible for the prolongation of the survival time of the mice inoculated with the parental leukaemic cells.

Isolation and characterization of sialodacryoadenitis virus (coronavirus) from rats by established cell line LBC.

The outbreak of sialoadenitis occurred in a laboratory rat colony and the causative agent was isolated from the affected salivary glands of diseased rats using the established cell line LBC. The isolate readily multiplied, producing clear cytopathic effects with syncytium formation, and it was identified virologically and serologically as rat sialodacryoadenitis virus. In attempts to isolate the virus by primary rat kidney (PRK) cells and suckling mice as well as LBC cells, the LBC cells showed higher susceptibility for the virus growth as compared with PRK cells or the brain of suckling mice. The isolation rate of virus was 100% (5/5) in LBC, 40% (2/5) in PRK cells and 60% (3/5) in suckling mice. After four passages in the LBC cells, the virus did not produce disease in adult rats, while the mouse brain-passaged virus did.

Axial heterogeneity of potassium transport across hamster thick ascending limb of Henle's loop.

Functional significance of morphological heterogeneities along the thick ascending limb of Henle's loop of hamsters was explored by the in vitro microperfusion technique with special reference to K+ transport. The transmission electron microscopic study confirmed that there are two types of cells, with smooth surface (S-cell) and rough surface (R-cell), respectively, and that the former is abundant in the medullary thick ascending limb (MTAL), whereas the latter is in the cortical portion (CTAL). The electrophysiological study revealed that in both segments there are two cell populations, one having high basolateral and low apical membrane K+ conductances (HBC) and the other having low basolateral and high apical K+ conductances (LBC). Random cell puncture revealed that the ratios of HBC/LBC were 24/7 (77%/23%) in the MTAL and 7/22 (24%/76%) in the CTAL, suggesting that HBC corresponds to S-cell, whereas LBC corresponds to R-cell. Net K+ transport was determined in two segments by measuring K+ concentration in the collected and perfused fluid by ultramicroflame photometry. In all six tubules of MTAL, net K+ flux had a direction to reabsorption with a mean of 4.87 +/- 0.46 pmol.min-1.mm-1. In marked contrast, in all six tubules of CTAL, we observed K+ secretion with a mean of -3.81 +/- 0.49 pmol.min-1.mm-1. The transmural voltage was positive in both segments and was significantly higher in the CTAL (7.8 +/- 0.5 mV) than in the MTAL (2.5 +/- 0.2 mV). From these observations, we conclude that the S-cell corresponding to the HBC cell reabsorbs K+, whereas the R-cell corresponding to the LBC cell secrets K+.(ABSTRACT TRUNCATED AT 250 WORDS)

LBC細胞で作製したラット唾液腺涙腺炎ウイルスによる血清抗体の検出

The TG strain of sialodacryoadenitis (SDA) virus propagated in LBC cell culture (TGr/LBC) reacted strongly with anti-TGr rat serum in complement fixation (CF) tests, showing much higher titers with anti-TGr rat serum than mouse hepatitis virus MHV-NuU antigen. The antigenicity was not affected after ether treatment while infectivity was lost. The TGr/LBC antigen might be useful in seromonitoring for SDA infection in rats.

Plaque Assay and Propagation in Rat Cell Line LBC Cells of Rat Coronavirus and 5 Strains of Sialodacryoadenitis Virus

Various factors influencing the plaque formation of rat coronavirus (RCV) and sialodacryoadenitis virus (SDAV) in LBC cell monolayers were studied to develop the practical method for plaque assay. By this method, 4 Japanese isolates of SDAV also produced clear plaques. In one-step growth experiments of these viruses, newly formed virus was first recognized within 7.5 h postinfection and showed subsequently a rapid exponential increase. The virus was released rapidly from the infected cells. By indirect immunofluorescence virus specific antigen was detected as perinuclear granules in the cytoplasm of the cells at 5-6 h postinfection, and all the cells revealed fluorescence at 12 h postinfection.

Replication of sialodacryoadenitis virus in mouse L-2 cells

SummarySialodacryoadenitis (SDA) is a naturally-occurring infection of the laboratory rat raused by the coronavirus, sialodacryoadenitis virus (SDAV). The study of SDAV has been limited because there is no widely available continuous cell line for the propagation of high titers of the virus. The purpose of this study, therefore, was to compare the ability of SDAV to replicate in the permanent cell lines, LBC, of rat origin, and the mouse cell lines. L-929 and L-2. Following 2 to 6 repeated passages of SDAV in LBC cells, the virus could be readily propagated in LBC and L-2 cells, but not in L-929 cells. Similarly, SDAV adapted to replicate directly in L-2 cells could be readily propagated in LBC, but not L-929 cells. In LBC and L-2 cells, cytopathic effect (CPE), viral antigen, viral particles, and virus infectivity could be demonstrated. Titers of up to 108.0 infectious viral particles/0.25 ml of culture fluid were obtained at 48 hours in L-2 cells. Titers in LBC cells were one to two logs lower. When susceptible rats were inoculated with eighth passage L-2 cell-adapted virus, they developed typical lesions of SDA. Virus could be recovered from infected tissues and propagated in L-2 cells on first passage. The ability to propagate SDAV to high titers in the widely available L-2 cell line should promote the study of this virus and facilitate its comparison with other murine coronaviruses.