Potent antitumor effects of CD154 transduced tumor cells in experimental bladder cancer

PURPOSE: Current intravesical immunotherapy for bladder cancer with bacillus Calmette-Guerin instillations is standard treatment for patients with high risk superficial tumors but relapses are common. We evaluated the tumor vaccine concept in murine bladder cancer by comparing tumor cell transduction with genes coding for the immunostimulatory molecules CD154, interleukin (IL)-12 and CD80 to design a novel vaccination strategy. MATERIALS AND METHODS: Adenoviral vectors were used to transduce murine bladder cancer MB-49 cells with genes coding for CD154, IL-12 and CD80. Parental or transduced MB-49 cells were injected subcutaneously into syngeneic mice. The effects of transgene expression on tumorigenicity and the generation of protective immunological memory against challenge with parental tumor were studied. RESULTS: All 76 animals injected with parental MB-49 cells had tumors within 8 to 12 days. Tumor cell expression of CD154 combined with IL-12 completely inhibited tumor outgrowth with all 21 mice tumor-free and CD154 transduction alone was almost as effective with 33 of 35 tumor-free. IL-12 production by tumor cells delayed tumor outgrowth and 4 of 10 mice remained tumor-free. Over expression of CD80 had no effect on tumorigenicity. CD154 expressing tumors were rapidly infiltrated with large numbers of CD4+ and CD8+ T cells. Mice vaccinated 4 times with adenoviral CD154 transduced MB-49 cells were completely protected against challenge with parental tumor. Co-injection of CD154 modified cells with parental MB-49 cells retarded tumor growth. CONCLUSIONS: Our experimental results suggest that the potent antitumor effects of CD154 gene transduction should be considered for immunostimulatory gene therapy for bladder cancer.     

A Tat Fusion Protein–Based Tumor Vaccine for Breast Cancer

Background We recently reported that dendritic cells (DCs) transduced with a fusion protein between Her2/neu and the protein transduction domain Tat (DC-Tat-extracellular domain [ECD]) induced Her2/neu-specific CD8⁺ T cells in vitro. This study tested the in vivo efficacy of DC-Tat-ECD in a murine breast cancer model.Methods FVB/N mice received one or two weekly intraperitoneal immunizations with syngeneic DC-Tat-ECD followed by a tumor challenge with syngeneic neu⁺ breast cancer cells, and tumor development was monitored. To test for Her2/neu specificity, CD4⁺ and CD8⁺ cells were isolated through magnetic bead separation and analyzed for specific interferon γ release.Results Intraperitoneally injected DCs migrated to secondary lymphoid organs, as evidenced by small-animal positron emission tomography studies. Immunized mice developed palpable tumors significantly later than control mice injected with DC-Tat-empty (P = .001 and P < .05 for two immunizations and for one immunization, respectively) or mice that received no DCs (P = .001 and P < .05). Similarly, immunized mice had smaller resulting tumors than mice injected with DC-Tat-empty (P < .05 and P < .01) or untreated mice (P < .001 and P < .001). Significantly more tumor-specific CD8⁺ splenocytes were found in twice-immunized mice than in untreated animals (P < .001). Similarly, a T-helper type 1 CD4⁺ T-cell response was observed.Conclusions Protein-transduced DCs may be effective vaccines for the treatment of cancer.Presented at the 57th Annual Cancer Symposium of the Society of Surgical Oncology, New York, New York, March 18–21, 2004.Published by Springer Science+Business Media, Inc. © 2005 The Society of Surgical Oncology, Inc.     

Cortical and Subventricular Zone Glioblastoma-Derived Stem-Like Cells Display Different Molecular Profiles and Differential In Vitro and In Vivo Properties

Background

Cellular self-renewal capacity in glioblastomas is heterogeneous, with only stem-like cells having this property. These cells generate a specific tumor phenotype, but no link with tumor location or molecular characteristics has ever been made.

Methods

Two cells lines, established from cell-dissociated glioblastomas and A2B5+ magnetic cell sorting, were used to decipher the mechanisms of cell migration in glioblastomas. GBM6 was derived from a glioblastoma close to the subventricular zone, whereas GBM9 was derived from a cortical glioblastoma and contained a high number of CD133⁺ cells.

Results

Orthotopic injections in both the subventricular zone and the cortex of nude mice showed that GBM6 and GBM9 cells had a differential pattern of migration that mirrored that of adult and fetal normal neural stem cells, respectively. GBM6 demonstrated higher tumorigenicity than GBM9, and whichever cell line was injected, subventricular zone-implanted tumors were larger than cortical ones. In vitro, GBM6 and GBM9 displayed high autorenewal and proliferation rates, and their expression profiles and genomic status showed that they had distinctive molecular signatures: GBM6 was classified as a mesenchymal glioblastoma and GBM9 as a proneural glioblastoma.

Conclusions

Altogether, our findings suggest that tumor location in addition to molecular signature influence tumor growth and migration pattern.

     

Studies on naïve CD4+CD25+T cells inhibition of naïve CD4+CD25- T cells in mixed lymphocyte cultures

BackgroundNaïve CD4⁺CD25⁺T cells suppress immune responses in a non-antigen specific manner. The effects of naïve CD4⁺CD25⁺T cells in suppressing alloimmune responses as assayed in the mixed lymphocyte culture (MLC) is poorly understood.MethodThe alloreactivity of naïve CD4⁺CD25⁺, CD4⁺CD25⁻ and unfractionated CD4⁺T cells from DA rats was compared in MLC with MHC incompatible stimulator cells. The response of Lewis and PVG cells to semi-allogeneic (Lewis × PVG)F₁ cells and fully allogeneic stimulators were compared. Potential mechanisms of suppression were examined by blocking T cell cytokines, produced by activated CD4⁺CD25⁺T cells.ResultsProliferation of CD4⁺CD25⁻T cells was significantly greater than unfractionated CD4⁺T cells to both allogeneic and syngeneic stimulator cells. CD4⁺CD25⁺T cells had no response to syngeneic stimulators and very low proliferative responses to alloantigen due to the Foxp3⁻ cells. Admixing CD4⁺CD25⁺T cells with CD4⁺CD25⁻T cells at a ratio of 1:10 reduced the proliferation to that of unfractionated CD4⁺T cells. At a ratio of 1:1 proliferation was nearly totally suppressed, IL-2, IL-4 and IL-5 mRNA induction was reduced but IFN-γ, IL-10, TGF-β and inducible nitric oxide (iNOS) mRNA induction was spared. The inhibition by CD4⁺CD25⁺T cells was not due to their consumption of IL-2 nor to anti-CD25mAb that had been used to enrich the cells being releases and blocking the IL-2 receptor on CD4⁺CD25⁻T cells that had been activated by alloantigen and induced to express CD25. Blocking IFN-γ, IL-10, TGF-β, IL-5 or iNOS did not prevent CD4⁺CD25⁺T cell's inhibition of CD4⁺CD25⁻T cell proliferation. Blocking IFN-γ or iNOS enhanced CD4⁺CD25⁻T cell proliferation only in the absence of CD4⁺CD25⁺T cells. Depletion of CD4⁺CD25⁺T cells enhanced responses to syngeneic stimulator cells, but this anti-self suppression did not regulate the response to alloantigen on semi-allogeneic stimulators.ConclusionsTwo independent mechanisms that control proliferation of CD4⁺CD25⁻T cells in MLC were identified that naive CD4⁺CD25⁺T cells mediated by cell to cell contact and not release of cytokines produced in the cultures, and that CD4⁺CD25⁻T cells producing IFN-γ to induce iNOS.     

Interleukin-2 transfected prostate cancer cells generate a local antitumor effect in vivo

In an effort to stimulate host-mediated antitumor response against prostate cancer in an animal model, highly malignant Dunning MAT-LyLu rat prostate carcinoma cells were transfected with the interleukin-2 (IL-2) cDNA, resulting in their ability to secrete large amounts of biologically active IL-2. Although parental cells form lethal tumors when injected subcutaneously into syngeneic hosts at doses of ≥5,000, injections of IL-2 secreting cells initially formed tumors and regressed completely in each of over 200 animals at all doses tested (10⁴-8 × 10⁷ cells). Mixtures of parental and IL-2 transfected cells were similarly rejected, demonstrating the non-cell autonomous nature of the response. Histological analysis of regressing tumors revealed a vigorous, predominantly lymphocytic and macrophage infiltrate at day 2 and marked tumor necrosis by day 6. Immunohistochemical staining of infiltrating lymphocytes at this latter time point demonstrated numerous T cells bearing either CD4 or CD8 surface markers, suggesting these cells as possibly mediating the tumor rejection. The ability of athymic mice to reject the IL-2 secreting tumor cells, however, suggests a non-T-cell-mediated mechanism. Although splenic natural killer (NK) activity is increased following injection of IL2 secreting tumor cells, this activity appears to be unnecessary for tumor elimination since syngeneic animals injected with asialo-GM1 antiserum to decrease NK activity also rejected IL-2 transfected cells, albeit slightly less effectively than untreated animals. Immunization of animals with subcutaneous injections of IL-2 transfected cells protected animals against a subsequent challenge of 10⁴ wild-type cells 1 to 2 weeks later in 19 of 51 cases; however, immunization did not confer protection against larger doses of parental tumor. These studies indicate that high local concentrations of IL-2 stimulate the elimination of large local burdens of prostate cancer in this model system, and this elimination results in a weak, but detectable systemic immune response against wild-type prostate cancer cells. © 1994 Wiley-Liss, Inc.     

Incomplete tumour control following DNA vaccination against rat gliomas expressing a model antigen

Background

Vaccination against tumour-associated antigens is one approach to elicit anti-tumour responses. We investigated the effect of polynucleotide (DNA) vaccination using a model antigen (E. coli lacZ) in a syngeneic gliosarcoma model (9L).

Methods

Fisher 344 rats were vaccinated thrice by intramuscular injection of a lacZ-encoding or a control plasmid in weekly intervals. One week after the last vaccination, lacZ-expressing 9L cells were implanted into the striatum.

Results

After 3 weeks, in lacZ-vaccinated animals the tumours were significantly smaller than in control-vaccinated animals. In cytotoxic T cell assays lysis rates of >50 % could only be observed in a few of the lacZ-vaccinated animals. This response was directed against lacZ-expressing and parental 9L cells but not against syngeneic MADB 106 adenocarcinoma cells. In Elispot assays interferon-γ production was observed upon stimulation with 9LlacZ and 9L wild-type but not MADB 106 cells. This response was higher for lacZ-immunized animals. All animals revealed dense infiltrates with CD8+ lymphocytes and, to a lesser extent, with NK cells. CD25-staining indicated cells possibly associated with the maintenance of peripheral tolerance to self-antigens. All tumours were densely infiltrated by microglia consisting mostly of ramified cells. Only focal accumulation of macrophage-like cells expressing ED1, a marker for phagocytic activity, was observed.

Conclusion

Prophylactic DNA vaccination resulted in effective but incomplete suppression of brain tumour formation. Mechanisms other than cytotoxic T cell responses as measured in the generally used in vitro assays appear to play a role in tumour suppression.     

CD133<Superscript>+</Superscript>CD44<Superscript>+</Superscript> Population Efficiently Enriches Colon Cancer Initiating Cells

Background  Previous reports have demonstrated that CD133⁺ cells or CD44⁺ cells might be cancer initiating cells (CIC) of colon cancer. However, the association between the two cell types is unclear. In this study, we evaluated the tumorigenicity of each population of human colon cancer divided by CD133 and CD44 using non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. Methods  Using the colon cancer cell lines HT29 and Caco2 we evaluated the change of expression status of CD133 or CD44 by a treatment with sodium butyrate (NaBT) that can induce cellular differentiation. Next, we prepared ten clinical samples of colon cancer and analyzed the expression and tumorigenicity of CD133 and CD44. Results  With NaBT treatment, CD44 expression was greatly downregulated in both HT29 and Caco2 (HT29: nontreatment versus treatment; 77.8% versus 0.6%, Caco2: 14.0% versus 0.4%, respectively), more than CD133 expression (HT29: nontreatment versus treatment; 90.1% versus 67.7%, Caco2: 98.9% versus 76.3%, respectively). In clinical samples, the percentages of CD133⁺ cells and CD44⁺ cells varied from 0.3% to 82.0% (mean 35.5%), and from 11.5% to 58.4% (mean 30.0%), respectively. Subcutaneous injection of CD133⁺ or CD44⁺ cells made a tumor in all mice (3/3 and 4/4, respectively). The combined analysis of CD133 and CD44 revealed that only the CD133⁺CD44⁺ population had the ability to produce a tumor (3/3). Conclusion  The findings demonstrate that, at present, the CD133⁺CD44⁺ population may be the best to identify tumor initiating cells of human colon cancer. Naotsugu Haraguchi: Awardee of Research Resident Fellowship from the Foundation for Promotion of Cancer Research (Japan) for the 3^rd Term Comprehensive 10- Year Strategy for Cancer Control.     

Development of Functional Human Immune System With the Transplantations of Human Fetal Liver/Thymus Tissues and Expanded Hematopoietic Stem Cells in RAG2−/−γc−/− MICE

BackgroundThere is an increasing need for suitable animal models for the study of the human immune system and disease. The purpose of this study was to develop a practical in vivo model of human immune cell repopulation using ex vivo expanded human fetal liver-derived CD34⁺ hematopoietic stem cells and subrenally coimplanted fetal liver/thymus tissues.MethodsFreshly isolated fetal liver-derived CD34⁺ hematopoietic stem cells were frozen until injected and ex vivo expanded with various cytokines for 7 days. After fetal liver/thymus tissues were subrenally coimplanted into preirradiated Rag2^?/?γ_c^?/? mice, frozen and ex vivo expanded CD34⁺ cells were injected intravenously. The peripheral blood of the mice was monitored for the detection of human cell engraftment using flow cytometry. Then we confirmed human T-cell function by in vitro function assays.ResultsAfter fetal liver/thymus tissues were coimplanted into the irradiated Rag2^?/?γ_c^?/? mice, with frozen and ex vivo expanded CD34⁺ hematopoietic stem cells, human cell engraftments were determined using hCD45 and multilineage markers. The cultured cells with the cytokine combination of stem cell factor, thrombopoietin, Flk2/Flk3 ligand (FL), and interleukin-3 showed stable and long-term engraftment compared to other combinations. The ex vivo expanded human fetal liver-derived CD34⁺ hematopoietic stem cells, under our culture conditions, accomplished a large volume of expanded cells that were sustained, demonstrating self-renewal of the evaluated markers, which may have indicated long- term repopulation activity.ConclusionThe results of this study demonstrated a practical mouse model of expanded human immune cells especially T cells in Rag2^?/?γ_c^?/? mice.     

Cellular mechanisms of interleukin-12-mediated neuroblastoma regression

Background/Purpose: Interleukin-12 (IL-12) is a proinflammatory cytokine with potent antitumor effects. Previous studies from the authors laboratory showed regression of established neuroblastoma in mice vaccinated with IL-12 transduced dendritic cells (DC). Although regression was associated with intense T cell infiltration, the precise role of T cells is unknown. The purpose of this work is to study the cellular mechanisms in IL-12[ndash ]mediated tumor regression. Methods: Three groups of mice (n = 12) received subcutaneous inoculation with 1 [times ] 10⁶ murine neuroblastoma cells (TBJ). Anti-CD4 (T helper), anti-CD8 (T cytotoxic), or antiasialo-GM1 (natural killer) antibodies were injected intravenously at 3-day intervals to deplete various immune effector cell populations. Mice in each depletion group and the control (nondepleted) group were injected intratumorally on day 7 with 1 [times ] 10⁶ DC IL-12[ndash ]transduced DC. Tumors were harvested for morphometry and immunohistochemistry at 21 days. Results: CD4 depletion had no effect on tumor growth in either control or IL-12[ndash ]vaccinated animals. In contrast, CD8-depleted animals treated with IL-12[ndash ]transduced DC underwent initial regression followed by progressive tumor growth (P [lt ] .01). These tumors were smaller in size at the same time-point. However, NK cell depletion (antiasialo GM1) completely abrogated the antitumor effects of IL-12[ndash ]transduced DC, leading to progressive tumor growth from the outset. There was no difference between the control and treated animals in this group. Conclusions: Contrary to our hypothesis that IL-12 DC primarily function to stimulate a T cell[ndash ]mediated response, these data suggest that NK cells are essential for the initial antitumor response of animals treated with IL-12[ndash ]transduced DC. CD8+ T cells appear to be necessary effector cells for complete rejection of tumor and possibly memory. NK cells are responsible for the early immune response. Furthermore, CD4+ (T helper) cells did not play any role in IL-12[ndash ]induced regression. These results imply that for DC to generate an effective antitumor response against neuroblastoma both acquired and innate effector cells are required. J Pediatr Surg 38:199-204.     

Immunisation with an allogeneic peptide promotes the induction of antigen-specific MHC II(pos) CD4+ rat T cells demonstrating immunostimulatory properties

BackgroundThe phenomenon of T cell stimulation by MHC class II expressing (MHC II^pos) CD4⁺ T cells has been intensively investigated for T cell clones but, so far, not for native T cells. The extensive use of T cell clones may explain the inconsistent outcomes of T cell-mediated antigen-presentation. Therefore, we used freshly isolated primed rat CD4⁺ T cells induced by immunisation with an allogeneic peptide P1, which is involved in allograft rejection.MethodsMHC II^pos and MHC II^neg CD4⁺ T cells were isolated from popliteal lymph nodes of P1-immunised Lewis rats and were purified by combining depletion and positive selection steps. Purified MHC II^pos CD4⁺ T cells and MHC II^neg CD4⁺ T cells (10⁵ cells per well each) were autostimulated or restimulated with P1-loaded (33 μg/ml peptide P1) and subsequently irradiated (with 20 Gy) autologous DC.ResultsSeven days after immunisation, a small population of MHC II^pos CD4⁺ T cells was detectable (approximately 8.0% of total lymph node cells), as well as a large population of MHC II^neg CD4⁺ T cells (up to 45%). Antigen-specific proliferation was observed for both T cell populations but only P1-loaded MHC II^pos CD4⁺ T cells presented antigen presenting cell (APC) function for P1-primed T cells. Their inability to activate unprimed T cells may be due to impaired surface expression of costimulatory molecules (CD80 and CD86).ConclusionImmunisation with the allogeneic peptide antigen P1 induced antigen-specific MHC II^pos CD4⁺ rat T cells demonstrating perfect APC function for primed T cells in vitro.     

Expression and prognostic significance of cancer stem cell markers CD24 and CD44 in urothelial bladder cancer xenografts and patients undergoing radical cystectomy

ObjectivesTo evaluate CD24/CD44/CD47 cancer stem cell marker expressions in bladder cancer (BCa) and provide data on their prognostic significance for clinical outcome in patients undergoing radical cystectomy (RC).Material and methodsPrimary BCa tissue was used for xenograft studies. A tissue microarray was prepared using specimens from a cohort of 132 patients. All patients underwent RC for urothelial BCa between 2001 and 2010. Expression of CD24, CD44, and CD47 was examined in primary samples and xenografts by fluorescence-activated cell sorting. Populations of CD24^low- and CD24^high- expressing cells were sorted and evaluated for tumorigenicity in vivo. Tissue microarray was analyzed for CD24/CD44 staining intensity and tumor-specific vs. stromal cell staining. Associations with BCa survival, BCa stage, and lymph node status were evaluated by univariate and multivariate analyses.ResultsCD24 and CD44/CD47 expressions mark distinct cell populations within the normal urothelium as well as in BCa. CD24^high/low expression was not sufficient to characterize CD24 as a BCa-initiating marker in in vivo primary xenotransplants. CD24 and CD44 expressions correlated with lower cancer-specific survival in patients. However, multivariate analyses of CD24 or CD44 did not demonstrate significantly increased hazards for cancer-specific death if analyzed together with stage, grade, and nodal status of patients.ConclusionsCancer stem cell markers CD24/CD44/CD47 are differentially expressed in cells of urothelial BCa in patients undergoing RC and influence cancer-specific survival of patients. Further evaluation of CD24/CD44/CD47 protein expression could be of high therapeutic value in BCa. However, both CD24 and CD44 expressions cannot be regarded as independent prognostic parameters for patients undergoing RC.     

Effective enrichment of prostate cancer stem cells from spheres in a suspension culture system

BackgroundStem-like prostate cancer cells are also called prostate cancer stem cells (PrCSCs). These rare cells are supposed to be highly tumorigenic and to be involved in maintenance of tumor homeostasis and mediation of tumor metastasis. Methods for sorting PrCSCs are mainly based on sorting cells with the marker (CD133⁺/CD44⁺) or side population cells. However, CD133⁺/CD44⁺ cells or side population cells are very rare or even undetectable. The scarcity of approaches for isolation and purification of PrCSCs is the main obstacle to studying PrCSCs.MethodsIn the present study, suspension culture was used for enrichment of PrCSCs. And PrCSCs were verified by side population technology, drug sensitivity assays, and the molecular marker analysis of prostate cancer stem cell.ResultsPC3 cells survived and formed spheres in nonadherent suspension culture. The percentage of CD44⁺/CD133⁺ cells was 18-fold higher in the nonadherent sphere-forming cell population than in the adherent PC3 cell population (13.94% vs. 0.77%, respectively). This side population was increased to 3.1% in the nonadherent population but undetectable in adherent population. Resistance to cisplatin was higher in the nonadherent cells than adherent cells.ConclusionSuspension culture can be used to enrich for PrCSCs. This approach will aid prostate stem cell biology research and facilitate identification of novel therapeutic agents for prostate cancer.     

小鼠CD154真核表达质粒的构建及其对膀胱癌细胞体内成瘤性的影响

目的 构建小鼠CD154真核表达质粒，观察其转染膀胱癌细胞后对癌细胞体内成瘤性的影响。方法 逆转录-聚合酶链反应（RT-PCR）法扩增T739小鼠CD154 cDNA，重组到pcD-NA3．1／Zeo（＋）构成重组质粒。经阳离子脂质体转染小鼠膀胱移行细胞癌细胞BTT739，将转染后的细胞接种T739小鼠，观察肿瘤在体内的生长情况。结果 RT-PCR产物进行凝胶电泳可见0．8kb处的目的基因条带，序列测定结果正确；CD154重组质粒转染BTT739细胞后能够在肿瘤细胞内表达；转染CD154的肿瘤细胞在小鼠体内不能成瘤。结论 成功构建小鼠CD154真核表达质粒，将其转染肿瘤细胞后能抑制肿瘤细胞的体内成瘤性。     

Biological and Genetic Characteristics of Tumor-Initiating Cells in Colon Cancer

Background Human prominin-1 (PROM1, CD133) was used as a marker to detect stem cells (progenitor cells) and cancer stem cells (tumor-initiating cells) in various tissues. The purpose of this study was to investigate the biological and genetic characteristics of tumor-initiating cells in colon cancer with both in vitro and in vivo analyses. Methods The CD133 expression of 12 colon cancer cell lines was evaluated. CD133⁺ cells were isolated by flow cytometry and examined for in vivo tumor formation, in vitro proliferation, colony formation, and invasion ability. Additionally, we used microarray analysis to compare gene expression profiles between CD133⁺ and CD133^– isolated cells. Results CD133⁺ cells were found in 5 of 12 colon cancer cell lines. Isolated CD133⁺ cells from the HT29 colon cancer cell line exhibited a higher tumorigenic potential than CD133^– cells in the in vivo tumor formation assay. Furthermore, it was shown that CD133⁺ cells are more proliferative and have higher colony-forming and invasive abilities than CD133^– cells in vitro. Microarray analysis found differential gene expression correlating with CD133 expression. Conclusions It was confirmed that CD133⁺ cells in colon cancer are useful markers for the detection of tumor-initiating cells. Intimate biological and genetic features of CD133⁺ cells in colon cancer cell lines were also revealed. The biological characteristics of CD133⁺ cells and differentially expressed genes in these cells will help elucidate more details of tumor-initiating cells in colon cancer. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users     

Dendritic cells (DC) loaded with apoptotic, allogeneic melanoma cells reduce tumor progression and improve survival in a murine immunotherapy model

Introduction: DC loaded with apoptotic, syngeneic melanoma cells slow tumor growth and improve survival in a murine immunotherapy model. The use of allogeneic cells as a source of tumor antigen could increase the clinical applicability of this therapy, so we investigated the efficacy of DC loaded with apoptotic, allogeneic cells in this model. Methods: DC were cultured from bone marrow from C-57/BL-6 mice with GMCSF (10 ng/ml), IL-4 (5 ng/ml). Syngeneic B-16 or allogeneic K-1735 melanoma cells were irradiated (20 Gy) to induce apoptosis and cultured with DC at a 1:1 ratio for 48 hr to make DC/Apo vaccine. C-57/BL-6 mice (n = 6) were injected with 2.5 x 10⁵ B-16 SQ and treated with 1 x 10⁶ DC/Apo vaccine cells SQ or saline control weekly x4 starting 5 days after tumor implantation. In other experiments, splenocytes were harvested from control and immunized tumor-bearing animals at day 30 and stimulated weekly x2 with IFN-gamma treated, irradiated B-16 cells. Cytotoxic T lymphocyte (CTL) assays were performed with B-16 targets for 48 hr and % specific lysis calculated. Results: Summarized in table 1. Conclusions: Mice treated with DC loaded with apoptotic, syngeneic melanoma cells (DC/ApoB-16) or apoptotic, allogeneic melanoma cells (DC/ApoK-1735) had delayed tumor progression and improved survival compared to controls. CTL responses to B-16 cells were seen in both treatment groups, and there was no difference in tumor size, survival, or CTL responses among the treatment groups. We conclude that DC loaded with apoptotic allogeneic melanoma cells may be a practical and effective approach to polyvalent immunotherapy of melanoma. We are currently designing a clinical trial to evaluate the efficacy of this treatment in patients with high-risk disease.     

Prognostic value of peripheral blood T lymphocyte subsets in clear cell renal cell carcinoma

BackgroundTo date, few studies have evaluated the role of peripheral blood T lymphocyte subsets in patients with clear cell renal cell carcinoma (ccRCC). Here we measured the levels of peripheral blood T lymphocyte subsets and evaluated its prognostic value in ccRCC.MethodsData from 122 patients with RCC from January 2018 to January 2020 were collected. Preoperative peripheral blood T lymphocyte subsets and medical records were analyzed. Kaplan-Meier cures and log rank test were used for analyzing overall survival (OS). Univariate and multivariate survival analyses were underwent by performing the Cox proportional hazards models. Correlations were tested by Pearson’s correlation analysis.ResultsOf 122 patients, a total of 80 ccRCC patients was enrolled. Patients with low CD3⁺ T cells and low CD4⁺/CD8⁺ ratio displayed a worse OS than patients with high CD3⁺ T cells and high CD4⁺/CD8⁺ ratio (P=0.029 and 0.002, respectively). Multivariate analyses showed CD3⁺ T cells and CD4⁺/CD8⁺ ratio were independent predictive factors for the OS (HR: 0.295, 95% CI, 0.091–0.956; P=0.042 and HR: 0.244, 95% CI, 0.065–0.920; P=0.037, respectively). Moreover, NLR negatively correlated with both levels of CD3⁺ T cells and CD4⁺/CD8⁺ ratio (P<0.001, r=−0.398 and P=0.012, r=−0.280, respectively).ConclusionsThe findings of our study suggest that preoperative CD3⁺ T cells and CD4⁺/CD8⁺ ratio in peripheral blood are independent predictors for patients with ccRCC.     

Sequential administration of GM-CSF and IL-2 surface-modified MB49 cells vaccines against the metastatic bladder cancer

ObjectivesMany strategies are pursued to enhance tumor vaccine immune response, including the utilization of cytokines. We have developed a novel protein-anchor technology to immobilize cytokines on tumor cell surface. Here we reported the preparation of tumor cell vaccines by immobilizing GM-CSF or IL-2 on MB49 bladder cancer cells and evaluated their antitumor efficacy (administrated alone or sequentially) in a metastatic mouse model.Materials and methodsSA-mGM-CSF or SA-hIL-2 surface-modified MB49 cells were prepared as vaccine. Mice were treated with MB49 cell vaccines (administrated alone or sequentially). Survival time, tumor growth, flow cytometry, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and cytotoxic T lymphocytes (CTL) assay were used to evaluate the antitumor efficiency of the vaccines in the pulmonary metastatic model of bladder cancer.ResultsGM-CSF vaccine induced more mature dendritic cells in the mice spleen. Combination with subsequent IL-2 vaccine significantly increased CD4⁺, CD8⁺, and IFN-γ⁺CD8⁺ T but not CD4⁺Foxp3⁺ T cell population and induced the highest production of IFN-γ, IL-12, but not IL-10. Furthermore, the splenocytes from the sequentially combined vaccines group showed the most potent cytotoxicity on MB49 cells. Finally, the sequentially combined vaccines evidently extended the survival time of mice (the median survival time of PBS, ethanol-fixed, anchored GM-CSF, anchored IL-2, and anchored GM-CSF + anchored IL-2 groups were 34, 37, 45, 47, and 59 days, respectively) and effectively protected the mice against a second MB49 cells but not RM-1 cells challenge.ConclusionsThis study demonstrated that sequential administration of GM-CSF and IL-2 surface-modified MB49 cells vaccines could effectively induce specific antitumor immune response.     

A novel xenograft model of human hepatocellular carcinoma in immunocompetent mice based on the microcarrier-6

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignant tumors that threaten human health; thus, the establishment of an animal model with clinical features similar to human hepatocellular carcinoma is of important practical significance.MethodsTaking advantage of the novel microcarrier-6, human HCC cells were injected into immunocompetent mice to establish a novel human HCC patient-derived xenograft (PDX) model. Primary HCC cells were isolated from fresh hepatocellular carcinoma tissues, which were subsequently co-cultured with microcarrier-6 to construct a three-dimensional tumor cell culture model in vitro. The HCC-microcarrier complex was implanted into mice by subcutaneous inoculation, and the tumor formation time, tumor formation rate, and pathological manifestation were recorded. Changes of immune parameters in mice were detected by flow cytometry.ResultsThe success rate was 60% (6/10) in the establishment of hepatocellular carcinoma PDX mouse model, and the total tumor formation rate of the tumor-forming model is 90–100%. H&E staining and immunohistochemical experiments indicate that the model well retained the characteristics of the primary tumor. Interestingly, M2 macrophages in tumor-bearing mice increased significantly, and the levels of CD4⁺ T cells were significantly reduced.ConclusionsThrough the application of the microcarrier-6 in immunocompetent mice, we successfully established a novel human HCC PDX model, which can be used to better study and further elucidate the occurrence and pathogenic mechanism of HCC, improve the predictability of toxicity and drug sensitivity in HCC.     

Transplant long-surviving induced by CD40-CD40 ligand costimulation blockade is dependent on IFN-γ through its effect on CD4(+)CD25(+) regulatory T cells

BackgroundIFN-γ was documented to be commonly associated with acute rejection. In the present study, we investigated the role of IFN-γ in the transplant long-surviving induced by blocking CD40–CD40 ligand (CD40–CD40L) costimulation and its mechanisms.MethodsIFN-γ expression in cardiac allografts and spleens from syngeneic and allogeneic recipients with or without anti-CD40L monoclonal antibody (MR-1) treatment was examined by real-time RT-PCR. The grafts survival time in Wild type (IFN-γ^+/+) and IFN-γ deficient (IFN-γ^?/?) recipients was investigated. Mixed lymphocyte reaction (MLR) of CD4⁺ T cells and cytotoxic T lymphocyte (CTL) assay of CD8⁺ T cells were also studied. FoxP3 expression in allografts and spleens from IFN-γ^+/+ or IFN-γ^?/? recipients with MR-1 treatment was examined. Furthermore, FoxP3, IL-10 and CTLA-4 expressions and the suppressive capability of CD4⁺CD25⁺ regulatory T cells were examined.ResultsRejected allografts showed significantly higher IFN-γ expression than long-surviving allografts. Allograft survival was not prolonged in nonimmunosuppressed IFN-γ^?/? mice. Administration of MR-1 induced long-term survival in 90.1% of IFN-γ^+/+ recipients (98 ± 6.6 days) but failed to do so in IFN-γ^?/? group (16.2 ± 4.0 days). IFN-γ^?/? recipients facilitated the proliferation and CTL generation of T cells. The allografts and spleens from IFN-γ^+/+ recipients contained higher FoxP3 expression than IFN-γ^?/? recipients. Moreover, CD4⁺CD25⁺ T cells from IFN-γ^+/+ recipients displayed a higher FoxP3 and IL-10 expression and suppressive capability.ConclusionIFN-γ plays an important role in the long-surviving induced by blocking CD40–CD40L through inhibiting the function of activated T cells and increasing suppressive capability of CD4⁺CD25⁺ regulatory T cells.     

Tumor-Associated Antigen Expressing Listeria monocytogenes Induces Effective Primary and Memory T-Cell Responses Against Hepatic Colorectal Cancer Metastases

Purpose

Despite advances in therapy for the treatment of metastatic colorectal cancer, many patients die of hepatic disease. Current immunotherapeutic strategies are likely limited by inhibitory signals from the tumor. To successfully eliminate tumor deposits within an organ, an appropriate immunologic milieu to amplify antitumor responses must be developed.

Methods

We used a murine model utilizing the CT26 colon cancer cell line to analyze primary and memory tumor-specific T-cell responses induced by an attenuated actin A and internalin B deleted immunodominant tumor-associated antigen expressing strain of Listeria monocytogenes for the treatment of metastatic colorectal cancer.

Results

Treatment of mice bearing established hepatic metastases with this L. monocytogenes strain led to the generation of a strong initial tumor-specific cytotoxic CD8⁺ T-cell response that successfully treated 90% of animals. Tumor antigen-specific central and effector memory T cells were also generated and protected against tumor rechallenge. These cell populations, when measured before and after tumor rechallenge, showed a marked expansion of antigen-specific effector CD8⁺ effector memory T cells. This strain of L. monocytogenes was able to down-modulate the expression of the immune checkpoint molecule, PD-1, within the tumor microenvironment but had variable effects on CTLA-4 expression.

Conclusions

This L. monocytogenes strain generated a highly effective antitumor T-cell response, providing a basis for the development of this vaccine platform in patients with liver metastases.