Dendritic cells pulsed with tumor cells killed by high hydrostatic pressure inhibit prostate tumor growth in TRAMP mice

3076 Background: Recent studies have identified molecular events characteristic of immunogenic cell death. These include surface exposure of calreticulin, HSP70 and HSP90, release of intranuclear HMGB1 and secretion of ATP from dying cells. Several chemotherapeutic agents, including anthracyclins, oxaliplatin and bortezomib, and hypericin-based photodynamic therapy have been described to induce the immunogenic cell death in human tumor cells. We investigated the potential of high hydrostatic pressure (HHP) to induce immunogenic cell death in human tumor cells. Methods: Prostate and ovarian cancer cell lines and primary tumor cells were treated by HHP and we analyzed the kinetics of the expression of immunogenic cell death markers. HHP killed tumor cells expressing immunogenic cell death markers were tested for their ability to activate dendritic cells (DCs), to induce tumor specific T cells and regulatory T cells. Results: HHP induced rapid expression of HSP70, HSP90 and calreticulin on the cell surface of all tested cell lines and primary tumor cells. HHP also induced release of HMGB1 and ATP from treated cells. The kinetics of expression was similar to doxorubicin, HHP, however, induced 1.5-2 fold higher expression of HSP70, HSP90 and calreticulin. The interaction of DCs with HHP-treated tumor cells led to the faster rate of phagocytosis, significant upregulation of CD83, CD86 and HLA-DR and release of IL-6, IL-12p70 and TNFα. The ability of HHP-killed tumor cells to promote DCs maturation was cell contact dependent. DCs pulsed with tumor cells killed by HHP induced high numbers of tumor-specific CD4+ and CD8+IFN-g-producing T cells even in the absence of additional maturation stimulus. DCs pulsed with HHP treated tumor cells also induced the lowest number of regulatory T cells among the tested conditions. Cells treated by HHP can by cryopreserved in liquid nitrogen and retain their immunogenic properties upon thawing thus allowing for their convenient use in the manufacturing of cancer immunotherapy products. Conclusions: High hydrostatic pressure is a reliable and very potent inducer of immunogenic cell death in the wide range of human tumor cell lines and primary tumor cells.

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Protease Activated Receptor-1 (PAR-1) Impedes Tumor Progression

Blood ◽

10.1182/blood.v128.22.2560.2560 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2560-2560

Author(s):

Gregory N. Adams ◽

Haley Weston ◽

Leah Rosenfeldt ◽

Malinda Frederick ◽

Joseph S. Palumbo

Keyword(s):

Cell Proliferation ◽

Tumor Growth ◽

Tumor Cell ◽

Tumor Cells ◽

Prostate Tumor ◽

Protease Activated Receptor 1 ◽

Tumor Types ◽

And Control

Abstract Activation of cell signaling by thrombin through Protease Activated Receptor-1 (PAR-1) represents one important interface between blood coagulation and cell activation in response to injury and inflammation. In the context of cancer, PAR-1 has been suggested to promote tumor growth through mechanisms coupled to tumor cell proliferation, tumor cell migration, and the development of a supportive tumor stroma. Consistent with this view, both tumor cells and stromal cells express high levels of PAR-1, and elevated PAR-1 expression has been correlated with a poor prognosis across several tumor types. In the current studies, we tested the hypothesis that PAR-1 is a critical driver of tumorigenesis and tumor growth using murine models of genetically-induced prostate and intestinal tumor growth. To define the role of PAR-1 in prostate tumor progression, we interbred mice expressing the TRAMP transgene (transgenic adenocarcinoma of the mouse prostate; SV40 Large T antigen under the control of a probasin promoter) to PAR-1-deficient mice (PAR-1-/-) in order to generate male TRAMP mice with and without PAR-1 expression for detailed analyses of prostate tumor growth. Surprisingly, prostate tumors harvested from PAR-1-/- mice were significantly larger than those harvested from PAR-1+/+ mice. In order to begin to address the PAR-1 expressing cellular compartments responsible for prostate tumor inhibition, we subcutaneously inoculated immunocompetent C57Bl/6-derived PAR-1-/- and control mice with tumor cells derived from a C57Bl/6 TRAMP mouse. TRAMP-derived tumors grew indistinguishably in PAR-1-/- and control mice, suggesting that stromal-cell associated PAR-1 is dispensable for prostate tumor growth. We next tested the effect of tumor cell-intrinsic inhibition of PAR-1 in TRAMP tumor cells by viral transduction with a construct containing an shRNA against murine PAR-1 in parallel to a non-specific shRNA construct. Diminishing tumor cell-associated PAR-1 expression resulted in significantly more rapid tumor growth in vivo. In order to better define the role of tumor cell-intrinsic PAR-1 we harvested TRAMP tumor cells from a PAR-1 deficient mouse and grew these cells in vitro. We transduced these PAR-1-deficient prostate tumor cells with viral vectors conferring expression of WT murine PAR-1 (PAR-1+), a PAR-1 mutant lacking the thrombin cleavage (R41A mutant) or empty vector (PAR-1-). PAR-1- cells grew robustly and similarly to the parental cells in vitro with a doubling time of approximately 48 hours. Cells expressing the R41A mutant PAR-1 also grew robustly and similarly to PAR-1 deficient cells. However, PAR-1+ cells failed to show any signs of cell proliferation over the span of a 4 day observation period. Furthermore, PAR-1 expression dramatically altered the ability of TRAMP cells to demonstrate signs of cell spreading as measured by the frequency of pseudopodia per cell. As a means of determining the role of PAR-1 in tumorigenesis and tumor growth in another spontaneously occurring setting, we interbred PAR-1-/- mice with APCMin/+ mice genetically predisposed to intestinal adenoma formation due to loss of heterozygosity of the tumor suppressor adenomatous polyposis coli gene. Blinded quantitative histological analyses of the intestinal tracts of PAR-1-/- and PAR-1+/+ APCMin/+ mice revealed that PAR-1 deficiency resulted in a significant 2-fold increase in the number of adenomas observed. Furthermore, the adenomas observed in PAR-1-/- mice were significantly larger based on morphometric analyses of adenoma surface area in histological sections. In sum, these data demonstrate a surprising and unexpected role for PAR-1 in the inhibition of tumor growth in the context of two distinct tumor types. Disclosures No relevant conflicts of interest to declare.

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High hydrostatic pressure affects antigenic pool in tumor cells: Implication for dendritic cell-based cancer immunotherapy

Immunology Letters ◽

10.1016/j.imlet.2017.05.005 ◽

2017 ◽

Vol 187 ◽

pp. 27-34 ◽

Cited By ~ 11

Author(s):

Linda Urbanova ◽

Nada Hradilova ◽

Irena Moserova ◽

Sarka Vosahlikova ◽

Lenka Sadilkova ◽

...

Keyword(s):

Dendritic Cell ◽

Hydrostatic Pressure ◽

Cancer Immunotherapy ◽

Tumor Cells ◽

High Hydrostatic Pressure

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Inhibition of vimentin or β1 integrin reverts morphology of prostate tumor cells grown in laminin-rich extracellular matrix gels and reduces tumor growth in vivo

Molecular Cancer Therapeutics ◽

10.1158/1535-7163.mct-08-0544 ◽

2009 ◽

Vol 8 (3) ◽

pp. 499-508 ◽

Cited By ~ 48

Author(s):

Xueping Zhang ◽

Marcia V. Fournier ◽

Joy L. Ware ◽

Mina J. Bissell ◽

Adly Yacoub ◽

...

Keyword(s):

Extracellular Matrix ◽

Tumor Growth ◽

Tumor Cells ◽

Prostate Tumor ◽

Β1 Integrin ◽

Prostate Tumor Cells

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A combined local treatment with anti-PD-1 antibody and bone marrow derived dendritic cells after x-ray irradiation to subcutaneous melanoma in a murine model.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.e14541 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. e14541-e14541

Author(s):

Yuzi Wang ◽

Lue Sun ◽

Xiaokang Li ◽

Koji Tsuboi

Keyword(s):

Bone Marrow ◽

T Cells ◽

Dendritic Cells ◽

Tumor Growth ◽

Tumor Cells ◽

Combination Treatment ◽

Bone Marrow Cells ◽

Local Treatment ◽

Activated T Cells ◽

X Ray

e14541 Background: In situ dying and just died tumor cells after irradiation give danger signals and release tumor-specific antigens which are supposed to be incorporated into dendritic cells (DCs), and sequentially rendering T-cells activated and proliferated. However, it has been clarified that activated T-cells are killed by PD-L1 ligands on tumor cells which bind to PD-1 receptors on T-cells, consequently suppressing systemic cellular immunological response. To improve local control and prevent metastases after localized radiotherapy, we examined whether the combination of anti-PD-1 antibody and bone marrow derived DCs (BM-DCs) can enhance both the local and systemic antitumor immunoreactions after localized X-ray irradiation in a murine melanoma model. Methods: BM-DCs were induced by using GM-CSF and IL-4 from bone marrow cells taken from the femur and tibia of C57BL/6 mice. Syngeneic B16 melanoma cells implanted subcutaneously at the left thighs of C57BL/6 mice were irradiated with X-ray (8 Gy) 5 days after inoculation. After 1, 3, 5, 7 days from irradiation, induced DCs were injected directly to the tumor site, similarly, after 1, 3, 5 days from irradiation, anti-PD-1 antibody were injected intraperitoneally. To examine the systemic immunoreaction, B16 cells were also inoculated to the right side 4 days after the left side inoculation, and treated with the same protocols only on the left side. The size of tumors was monitored and survival analyses were performed. Results: The induced DCs showed the ability to incorporate antigens and to prime and proliferate T-cells in vitro. The combination treatment of anti-PD-1 antibody, BM-DCs and X-ray irradiation showed a significant delay of tumor growth compared to single or double combination treatments in vivo. In addition, this triple combination treatment significantly inhibited the tumor growth on the other side compared to other treatments. Conclusions: DCs and anti-PD-1 antibody significantly enhanced the antitumor effect of X-ray irradiation and prolonged the survival time. This combination also can induce a strong systemic antitumor immunoreaction which could treat metastatic tumors.

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