Project description:The SV-BR-1 cell line was derived from a chest wall lesion of a breast cancer patient. SV-BR-1 cells were stably transfected with CSF2 (encoding GM-CSF), resulting in the SV-BR-1-GM cell line. Following irradiation to prevent cell replication, both SV-BR-1 (Wiseman and Kharazi, The Open Breast Cancer Journal, 2010, 2, 4-11) and SV-BR-1-GM (Wiseman and Kharazi, Breast J. 2006 Sep-Oct;12(5):475-80) cells have been applied as whole-cell therapeutics in clinical trial settings for advanced breast cancer. Molecular profiles of non-irradiated SV-BR-1-GM cells have been established from various manufacturing lots via Illumina HumanHT-12 V4.0 expression beadchip arrays (GPL10558). A key finding from the study is the identification of an immune signature expressed in SV-BR-1-GM cells which includes the MHC class II factors HLA-DMA, HLA-DMB, HLA-DRA, and HLA-DRB3. Since tumor regressions were apparent in clinical trial subjects matching at an HLA-DRB3 allele with SV-BR-1-GM we hypothesize that (partial) HLA matching is needed for maximal tumor-directed clinical responses to occur.
Project description:BACKGROUND: While many authorities theorize that cancer vaccines are too weak to be widely effective, there are quite a few reports with clearly demonstrated, significant clinical benefit in some patients. The literature of cellular cancer immunotherapies shows that 53% (78/147) of phase II studies showed evidence of clinical activity. While not all reached their primary endpoints, 75% (12/16) of phase III studies had positive data. SV-BR-1-GM, derived from a patient with grade 2 (moderately differentiated) breast cancer, is a GM-CSF-secreting breast cancer cell line with properties of antigen-presenting cells we established. METHODS: We report detailed molecular and clinical findings from an open-label phase I, single-arm pilot study in breast (3 subjects) and ovarian (1 subject) cancer with irradiated SV-BR-1-GM cells (ClinicalTrials.gov Identifier NCT00095862). Inoculations of SV-BR-1-GM were preceded by low-dose cyclophosphamide and followed by injections of interferon-alpha2b into the SV-BR-1-GM inoculation sites. We assessed both cellular (delayed-type hypersensitivity (DTH) reactions) and humoral (anti-SV-BR-1 antibody) immune responses and conducted molecular analyses on patient blood cells and SV-BR-1-GM cells. RESULTS: Treatment was generally safe and well tolerated. Immune responses were elicited universally. Overall survival was more than 33 months for three of the four patients. As previously reported, one patient (Subject A002, with grade 2 breast cancer) had regression of metastases in lung, breast and soft tissue within 2 months of treatment initiation. At later relapse, with multiple metastases including several in the brain, rapid tumor response was again seen, including complete regression of CNS metastases. Consistent with a role of Class II HLA in contributing to SV-BR-1-GM’s mechanism of action, Subject A002 allele-matched SV-BR-1-GM at the HLA-DRB1 and HLA-DRB3 loci. Only the HLA-DRB1 alleles were clearly expressed in SV-BR-1-GM cells. However, interferon-gamma (possibly also present in situ) upregulated both HLA-DRB1 and HLA-DRB3 to substantial levels. Gene expression data supports the hypothesis that SV-BR-1-GM cells have retained some of the original breast cancer’s grade 2 character. CONCLUSIONS: We describe a whole-cell immunotherapy regimen with remarkable rapidity of response and a speedy rescue response, including complete resolution of CNS metastases after relapse. Class II HLA matches might be critical for SV-BR-1-GM’s therapeutic potential.
Project description:We report differentially expressed genes by DATS exposure in MCF-10A human epithelial cell line and SK-BR-3 human breast cancer cell line
Project description:Background: Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) is a hematopoietic growth factor and adjuvant in cancer immunotherapy via stimulation of dendritic cells/APCs. However, GM-CSF has yielded inconsistent results and its role regarding in vivo modulation of macrophages remains underexplored. We previously demonstrated that 100ng “high-dose” intratumor (IT) GM-CSF ablated tumor blood vessels and worsened tumor hypoxia after 3 weeks through tumor-associated macrophage (TAM) soluble VEGFR-1 production in PyMT murine breast cancer. Here, we investigate a role for “low-dose” IT GM-CSF on tumor oxygen and the impact on immunotherapy response, TAMs/myeloid cells, and TILs relative to “high-dose”. Methods: We performed IT injections of dose-specific GM-CSF or saline controls and then evaluated phenotypic effects after 3 weeks. We used Electron Paramagnetic Resonance Oximetry to measure and image in vivo tumor oxygen in real-time, and fluorescent immunohistochemistry to assess tumor blood vessels. IT GM-CSF doses were tested in priming PyMT tumors for sensitization to PD1. We performed RNA Sequencing of TAM and CD8 TIL to observe transcriptional changes coupled with flow cytometry of peripheral blood monocytes, tumor myeloid, and TIL populations in immunology cold" PyMT tumors response to dose-optimized GM-CSF. Lastly, we assessed and compared effects of IT GM-CSF on TAM and TIL in an immunologically “hot” 4T1 breast cancer model. Results: 5ng IT GM-CSF significantly increased PyMT tumor oxygen without augmenting tumor growth and promoted tumor vessel health via increased pericyte coverage. Priming of PyMT tumors with 5ng IT GM-CSF (“low-dose”, hypoxia reduced) sensitized “cold” PyMT tumors to PD1, but this synergy was not observed with 100ng (“high-dose”, hypoxia exacerbated). Immunologically, 5ng GM-CSF did not increase monocyte mobilization or alter phenotypic marker expression on TAMs, but reduced hypoxic and inflammatory transcriptional programs in macrophages and CD8 TIL isolated from PyMT tumors. 100ng increased infiltration of myeloid cells and TAMs but these TAMs had reduced MHCII expression, suggesting support of immune-suppressive TAM under hypoxia. Some tumors exhibited increased CD8 polyfunctionality either dose suggesting mild CD8 TIL priming. On the other hand, 100ng in “hot” 4T1 tumors resulted in increased TAM MHCII and other immunostimulatory molecules with moderate increases in CD8 TIL polyfunctionality and exhausted PD1hiTIM3+ phenotype, indicating that GM-CSF may have opposing effects on macrophage modulation based on tumor immunological status.
Project description:Multiple-condition experiment was desinged to be any number of conditions in an experiment without replicate observations for microarray and used to identify genes differentially expressed between different pairs of conditions (treatments).<br> In this study we used breast cancer stable cell lines for overexpressing and silencing annexin A1 (ANXA1), which belongs to a family of -dependent phospholipid binding proteins and are preferentially located on the cytosolic face of the plasma membrane. Cell lines overexpressing ANXA1 (MDA_MB-453/cDNA) were generated by introducing retroviral vectors containing ANXA1 cDNA (pBabe/ANXA1 cDNA) into breast cancer cell line MDA-MB-453 (a low expressor of ANXA1). Breast cancer cell line BT-474, a high expressor of ANXA1, was infected with ANXA1 siRNA-plasmid viruses to knockdown ANAXAI expressor (BT-474/siRNA) where nucleotides corresponding to siRNA were synthesized and ligated into the pLNCX retroviral vector [35,36]. We also used a pLNCX/U6 empty vector to infect BT-474 and obtained an empty vector expressor. Therefore, 5 breast cancer cell lines (MDA_MB-453, MDA_MB-453/cDNA, BT-474, BT-474/siRNA, and BT-474/U6) are attributed to two genotypes: MDA_MB-453 and BT-474. MCE was performed for microarray analysis with these 5 breast cancer cell lines, that is, only one sample was drawn from each breast cancer cell line.
Project description:Myeloid-derived suppressor cells (MDSCs) have emerged as major regulators of immune responses in cancer and other pathological conditions. Multiple factors including cytokines, transcription factors and multiple signaling pathways are involved in MDSC differentiation. Cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin(IL-6) etc could in vitro mediate development of MDSCs.IL-6 with GM-CSF mediated MDSC not only had stronger suppressive function but also the dynamics of their suppressive function was different from GM-CSF alone mediated MDSCs.To found a new regulatory factor (s) in tumor and inflammatory environments, we compared GM-CSF and IL-6 mediated MDSCs with GM-CSF alone mediated MDSCs using lncRNA microarray, miRNA microarrays and protein-coding mRNA microarrays.
Project description:Reactive oxygen species, including RNS, contribute to the control of multiple immune cell functions within the tumor microenvironment (TME). Tumor-infiltrating myeloid cells (TIMs) represent the archetype of tolerogenic cells that actively contribute to dismantle effective immunity against cancer are among the cells most influenced by these molecules. TIMs inhibit T cell functions and promote tumor progression by several mechanisms including the amplification of the oxidative/nitrosative stress within the TME. In tumors, TIM expansion and differentiation is regulated by the granulocyte-macrophage colony-stimulating factor (GM-CSF), which is produced by cancer and immune cells. Nevertheless, the role of GM-CSF in tumors has not yet been fully elucidated. In this study, we show that GM-CSF activity is significantly affected by RNS-triggered post-translational modifications. The nitration of a single tryptophan residue in the sequence of GM-CSF nourishes the expansion of highly immunosuppressive myeloid subsets in tumor-bearing hosts. Importantly, tumors from colorectal cancer patients express higher levels of nitrated tryptophan compared to non-neoplastic tissues. Collectively, our data identify a novel and selective target that can be exploited to remodel the TME and foster protective immunity against cancer.
Project description:Modelling combined virotherapy and immunotherapy:strengthening the antitumour immune response mediated byIL-12 and GM-CSF expression
Adrianne L. Jennera, Chae-Ok Yunb, Arum Yoonb, Adelle C. F. Costercand Peter S. Kimaa
School of Mathematics and Statistics, University of Sydney, Sydney, Australia;bDepartment ofBioengineering, Hanyang University, Seoul, Korea;cSchool of Mathematics and Statistics, University of NewSouth Wales, Sydney, Australia
ABSTRACT
Combined virotherapy and immunotherapy has been emergingas a promising and effective cancer treatment for some time.Intratumoural injections of an oncolytic virus instigate an immunereaction in the host, resulting in an influx of immune cells tothe tumour site. Through combining an oncolytic viral vector withimmunostimulatory cytokines an additional antitumour immuneresponse can be initiated, whereby immune cells induce apoptosisin both uninfected and virus infected tumour cells. We developa mathematical model to reproduce the experimental results fortumour growth under treatment with an oncolytic adenovirus co-expressing the immunostimulatory cytokines interleukin 12 (IL-12)and granulocyte-monocyte colony stimulating factor (GM-CSF). Byexploring heterogeneity in the immune cell stimulation by thetreatment, we find a subset of the parameter space for the immunecell induced apoptosis rate, in which the treatment will be lesseffective in a short time period. Therefore, we believe the bivariatenature of treatment outcome, whereby tumours are either completelyeradicated or grow unbounded, can be explained by heterogeneity inthis immune characteristic. Furthermore, the model highlights theapparent presence of negative feedback in the helper T cell and APCstimulation dynamics, when IL-12 and GM-CSF are co-expressed asopposed to individually expressed by the viral vector.
Project description:Granulocyte-Macrophage colony stimulating factor (GM-CSF) devlops heterogenous myeloid cell populations from bone marrow progenitor cells. In vitro generated bone marrow derived cells are excellent sources for obtaining dendritic cells or macrophages, but it is still not clear about the exact mixed population characteristics of GM-CSF grown cells. We revealed here that GM-CSF grown bone marrow cell derived attaching cells were composed of dendritic cells (GM-BMDC) as well as macrophages (GM-BMM). We compared the transcriptome profiles of these cell populations as well as M-CSF grown bone marrow derived macrophages (M-BMM). We used microarrays to detail the global profile of gene expressions between three populations of CSF-grown bone marrow derived cells: GM-CSF derived dendritic cells (GM-BMDC), GM-CSF derived macrophages (GM-BMM) and M-CSF derived macrophages (M-BMM).