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{"title":"Abstract B174: Co-expression of stimulators and inhibitors of T-cell activation in melanoma","authors":"R. Maniyar, R. Freund, A. Malhotra, Sanjukta Chakraborty, J. Geliebter, M. Wallack, R. Tiwari","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B174","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B174","url":null,"abstract":"Melanoma, one of the most aggressive skin cancers, has steadily been on the rise over the last three decades. With limited treatment options, the recent impetus in immuno-oncology drugs, especially checkpoint inhibitors, has changed the treatment landscape. While anti-CTLA4 and anti-PD1 demonstrated success in clinic, the search for additional targets is needed to enable potent comprehensive curative combination therapies. Our laboratory has characterized and screened five primary patient-derived melanoma cell lines, MEL-2, MEL-V, 3MM, KFM, and GLM-2. While immunomodulatory molecules are canonically present on antigen presenting cells and T-cells, increasing evidence suggests that they are not expressed in isolation. In an effort to identify immunomodulatory molecules expressed on our primary melanoma cells, a comprehensive RT-PCR screen of 29 co-inhibitory and co-stimulatory molecules was carried out. Several molecules including CD160, CD226, TIM1, HVEM, and BTLA were seen to be differentially expressed in melanoma cells compared to normal melanocytes at the mRNA level. Western blots and immunocytochemistry validated the differential expression of these molecules at the protein level. 50-80% of melanoma cases are positive for the BRAFV600E mutation, and are treated with a small molecule inhibitor of the mutated BRAF, vemurafenib (PLX4032). Treatment of these cells with PLX4032 led to an upregulation of transcription factors MITF and AP-1, as well as immunomodulatory molecules, CD160, CD226, TIM1, HVEM, and BTLA, a phenomenon seen only in cells positive for the BRAFV600E lesion. MITF and AP-1, owing to the binding sites present in the promoter regions of these molecules, can drive their expression upon treatment with PLX4032. These additional immune-regulatory molecules of T-cell activation and/or immune tolerance mechanisms are potential targets for a combination therapy with PLX4032 in melanoma patients positive for the BRAFV600E genetic lesion. Our future directions aim to elucidate the role of these molecules on the tumor cells and devise an effective combination with small-molecule inhibitors and immunotherapies. Citation Format: Rachana Maniyar, Robert Freund, Aryan Malhotra, Sanjukta Chakraborty, Jan Geliebter, Marc Wallack, Raj K. Tiwari. Co-expression of stimulators and inhibitors of T-cell activation in melanoma [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B174.","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"1 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"130222538","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B168: A Radiosensitivity gene signature and PD-L1 status predict clinical outcome of patients with glioblastoma multiforme in The Cancer Genome Atlas Dataset","authors":"I. Kim, B. Jang","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B168","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B168","url":null,"abstract":"Background: A combination of radiotherapy and immune checkpoint blockade, such as programmed death-1 (PD-1) or programmed death-ligand 1 (PD-L1) blockade, is being actively tested in clinical trial settings. Here, we tried to identify a subset of patients that could potentially benefit from this strategy using The Cancer Genome Atlas (TCGA) dataset for glioblastoma (GBM). Methods: A total of 399 cases were clustered into radiosensitive (RS) versus radioresistant (RR) groups based on radiosensitivity gene signature and were further stratified into PD-L1 high versus PD-L1 low groups according to the median expression of CD274 mRNA. Differential and integrated analyses with expression and methylation data were performed. CIBERSORT was used to enumerate the immune repertoire that resulted from transcriptome profiles. Results: We identified a subset of GBM patients, the “PD-L1 high-RR group” that had a worse clinical outcome compared to the other groups. In this group, differentially expressed genes (DEG) were highly enriched for an immune response and mapped into activation of PI3K-AKT and MAPK signaling pathways. Through integration of DEG and differentially methylated regions, kinase MAP3K8, which is involved in T-cell receptor signaling, was found to be upregulated, while BAI1, a factor that inhibits angiogenesis, was silenced. CIBERSORT showed that a higher infiltration of the immune repertoire, which included M2 macrophages and regulatory T-cells, contributed to an immunosuppressive tumor microenvironment. Conclusion: Looking at the results collectively, the “PD-L1-high-RR group” could potentially benefit from radiotherapy combined with PD-1/PD-L1 blockade and angiogenesis inhibition. Citation Format: Inah Kim, Bum Sup Jang. A Radiosensitivity gene signature and PD-L1 status predict clinical outcome of patients with glioblastoma multiforme in The Cancer Genome Atlas Dataset [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B168.","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"28 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"130388756","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B178: Development and preclinical efficacy characterization of a systemically administered multiple Toll-like receptor (TLR) agonist for antitumor immunotherapy","authors":"M. J. Newman","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B178","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B178","url":null,"abstract":"Background: The first cancer immunotherapy was developed in the 1890s by Dr. William Coley, who observed tumor regressions after injecting cancer patients with a heat-killed mixture of Gram-positive and negative pathogenic bacteria. Dr. Coley determined that the Gram-negative bacteria were the principal contributor to antitumor activity and that the product would likely be most effective when administered intravenously (i.v.), but was too toxic in this setting, limiting use to intramuscular and intratumoral administration. Coley’s toxins, as it was known, was credited with curing hundreds of late-stage cancer patients over 70 years. However, lack of knowledge regarding mechanism of action made it difficult to optimize or standardize, producing high variability. The use of multiple, local administration routes also likely contributed to variability in response, leading the FDA to refuse to grandfather in the product in 1963. We now know the mechanism of action of Coley’s toxins and the source of the i.v. toxicity. Gram-negative bacteria contain immune system danger signals, including multiple TLR agonists (activating TLRs 2, 4, 5 and 9), which directly and indirectly, via induction of cytokine and chemokine secretion, participate in the activation of most of the cellular mediators of innate and adaptive immune responses. Lipopolysaccharide (LPS), which activates TLR4, has been identified as a major contributor to both the antitumor activity and i.v. toxicity of Gram-negative bacteria. Decoy’s hypothesis is that significant reduction without complete elimination of LPS activity, in conjunction with killing and stabilization of nonpathogenic, Gram-negative bacteria, may produce a multiple TLR product that can safely and effectively induce antitumor immune responses via i.v. administration. Methods and Results: Nonpathogenic, Gram-negative E. coli were treated with polymyxin B and glutaraldehyde under conditions to kill and stabilize the cells, producing >90% reduction in LPS endotoxin activity and pyrogenicity. Endotoxin activity and pyrogenicity were quantified using Limulus amebocyte lysate (LAL) and in vivo rabbit assays. Bacterial integrity was assessed by electron and light microscopy. Antitumor activity was determined using standard syngeneic and xenograft tumor models. Decoy-treated bacteria exhibited a 3-fold reduction in acute in vivo toxicity relative to untreated bacteria. Surprisingly, induction of antitumor cytokine secretion by murine and human peripheral blood mononuclear cells (PBMCs) was not compromised, relative to untreated bacteria. Treatment with Decoy bacteria (i.v.) produced significant single-agent antitumor activity against orthotopic murine colorectal carcinoma and metastatic murine pancreatic carcinoma. Synergistic combination activity, including eradication of established tumors, with a therapeutic index of up to 10-fold, was observed in combination with IL-2 or low-dose cyclophosphamide (LDC) in murine colorectal carcino","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"41 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"133707469","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B162: Developmental programming of long-term immunity of CD8 T-cells by perinatal glucocorticoids","authors":"J. Hong, B. Vaidyanathan, J. Cho, R. Medzhitov","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B162","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B162","url":null,"abstract":"Stress has been associated with various types of diseases including cancer. It was suggested that compromised antitumor immunity is often responsible for tumor progression during stress, which is caused by immunosuppressive glucocorticoids (GC), stress hormone, and sympathetic nervous system activation. Perinatal period is critical for immunity as the first major contact with the environment is made and this interaction can shape the immune system development. Epidemiologic studies found that early-life exposure to specific environment may have life-long impact on immunity, affecting the development of immune-related diseases such as inflammatory diseases, metabolic diseases, allergic diseases as well as cancer. Nevertheless, it is still largely unknown whether developmental programming of immunity exists due to the lack of mechanistic understanding on this subject. Since most of the environmental factors that are reported to cause later-life development of diseases are associated with stress, we adopted a model to directly test the effect of stress hormone. We introduced the in vivo mouse model of perinatal glucocorticoid receptor (GR) activation via dexamethasone (DEX) treatment in drinking water perinatally (embryonic day 7.5-postnatal day 1). Then, we analyzed the T-cell immunity and tumor susceptibility when the offspring became mature. We found that perinatal exposure of DEX permanently repressed the CD8 T-cell response. Consistently, accelerated tumor growth and decreased intra-tumor CD8 T-cell response were observed with perinatal DEX exposure in B16-F10 melanoma model. In OT-I T-cell receptor transgenic model, OT-I CD8 T-cells, from the mice with perinatal DEX exposure, showed suppressed ovalbumin (OVA) antigen-specific immune response as well as repressed antitumor immune response against OVA expressing E.G7 lymphoma. Furthermore, we found CD8 T-cell population expressing glucocorticoid-induced TNF receptor-related protein (GITR) was decreased with perinatal DEX exposure. Finally, we observed that endogenous corticosterone level as well as GR signaling pathway in T-cell was reduced with perinatal DEX exposure. We also identified that GR in T-cell was required for adequate CD8 T-cell activation and survival, suggesting that reduction of endogenous GC level with perinatal DEX was responsible for suppressed CD8 T-cell function. These results showed that perinatal GC exposure persistently programed the threshold for hypothalamus-pituitary-adrenal axis for regulating endogenous GC level, and that reduced systemic GC level elicited repressed CD8 T-cell activation and survival, leading to tumor susceptibility. Citation Format: Jun Young Hong, Bharat Vaidyanathan, Jen Young Cho, Ruslan Medzhitov. Developmental programming of long-term immunity of CD8 T-cells by perinatal glucocorticoids [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 20","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"9 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"133952323","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B177: Analysis of immunogenic cell death (ICD) induced in multiple myeloma cells","authors":"M. Matsushita, Sho Kashiwazaki, Satoshi Kamiko, Ryousuke Uozaki, D. Ichikawa, Y. Hattori","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B177","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B177","url":null,"abstract":"Background: Development of novel drugs, such as immunomodulatory drugs (IMiDs), proteasome inhibitors (PIs), or monoclonal antibodies, has prolonged survival of multiple myeloma (MM) patients. However, high-risk patients harboring del 17, t(14;17), or t(11;14) still have poor prognosis. Therefore, clarification of the drugs that could potentiate the effect of these treatments is necessary. Several drugs have been reported to cause cell surface expression of calreticulin (CRT), release of high mobility group box 1 (HMGB 1) and ATP, activating an immune response, which is called immunogenic cell death (ICD). However, there is almost no information on ICD in MM. The purpose of this study is to investigate whether anti-MM drugs can induce not only direct cell-killing effect but also immunomodulatory effects in high-risk MM cells. Methods: High-risk myeloma cell line, MUM24 was treated with dexamethasone, melphalan, lenalidomide, bortezomib, carfilzomib, and panobinostat. Expression of cell surface CRT and HMGB1 release were detected using flow cytometry and Western blotting, respectively. We then co-cultured DiD-labeled monocyte-derived dendritic cells with GFP-induced MUM24 treated with each drug and evaluated phagocytosis of DCs using fluorescence microscope. Results: After treatment with drugs, bortezomib and carfilzomib induced higher expression of CRT and release of HMGB1 in MUM24 compared with other drugs. We also observed that DCs could engulf MUM24 cells treated with carfilzomib. Conclusion: Our results suggest that PIs, such as bortezomib or carfilzomib, could not only kill MM cells but also induce ICD. These effects are expected to improve the prognosis of MM patients by evoking strong immune response against MM cells. Citation Format: Maiko Matsushita, Sho Kashiwazaki, Satoshi Kamiko, Ryo Uozaki, Daiju Ichikawa, Yutaka Hattori. Analysis of immunogenic cell death (ICD) induced in multiple myeloma cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B177.","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"92 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"133130842","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B191: The impact of postoperative myeloid-derived suppressor cells on prognosis of gastric cancer patients","authors":"S. Urakawa, H. Wada, M. Mori, Y. Doki","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B191","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B191","url":null,"abstract":"Background and Aim: Myeloid cells such as myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs) and neutrophils have a suppressive role in the antitumor immunity. In gastric cancer (GC) patients, it has been reported that these myeloid cells in peripheral blood or tumor tissue were associated with their prognosis and increased after surgical treatments. Some reported that the tumor progression and frequent recurrence were caused by increased MDSC after surgical stress in a mouse model. However, in human GC patients, it is still unclear whether such an increase of myeloid cells after gastrectomy is related to their poor prognosis. In this study, we investigate the relationship between the increase of myeloid cells after surgery and prognosis in gastric cancer patients. Materials and Methods: For analysis of relationship between increased myeloid cells after surgery and patients’ prognosis, general archival data were obtained from 278 pStgae2-3 GC patients who received curative resection between January 2007 and December 2014. For analysis of relationship between perioperative changes of MDSCs and patients’ clinicopathologic data, peripheral blood was obtained from 75 GC patients who underwent gastrectomy between April 2016 and December 2017, and MDSCs, CD8, CD4 T-cells, and regulatory T-cells (Tregs) were analyzed by flow cytometry. For analysis of the immunosuppressive function, MDSCs were purified by FACS Aria II and the IFNγ secretion assay were performed using activated autologous T-cells as responder cells. Result and Discussion: In the analysis with 278 GC patients, short RFS was significantly correlated with increased number of monocytes after surgery (#monocytes) (P=0.0073), but not increased number of neutrophils (#neutrophils) (P=0.26). A multivariate analysis demonstrated that #monocytes (HR 1.49; 95% CI 1.01-2.21), pT (HR 2.14; 95% CI 1.26-3.83), pN (HR 2.98; 95% CI 1.81-5.18), postoperative complications (HR 1.69; 95% CI 1.03-2.68) were independently associated with RFS. In the analysis with 75 GC patients, preoperative % M-MDSCs (CD11b+ CD33+ HLA-DR- in CD14+ PBMCs) was positively associated with pathologic stages. After gastrectomy, % M-MDSCs dramatically increased in most patients. On the other hand, % Foxp3+ CD25+ in CD4+ increased but not significantly, while % CD4 and %CD8 in CD3+ were stable. By the suppression assay, smaller number of IFNγ-secreting responder cells were observed when they were co-cultured with M-MDSCs than with CD11b+ CD33+HLA-DR+ cells. Because the increase in the number of M-MDSCs (#M-MDSCs) was positively correlated with #monocytes (r2=0.57 P Citation Format: Shinya Urakawa, Hisashi Wada, Masaki Mori, Yuichiro Doki. The impact of postoperative myeloid-derived suppressor cells on prognosis of gastric cancer patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New Yor","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"58 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"114993177","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B181: Adenovirus, Semliki Forest virus and vaccinia virus-induced immunogenic cell death augments oncolytic virus immunotherapy","authors":"Mohanraj Ramachandran, M. Essand","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B181","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B181","url":null,"abstract":"Oncolytic viruses represent new immunotherapy agents with therapeutic effects consisting of direct oncolysis of cancer cells and anticancer immune responses induced upon oncolysis. However, the exact pathways of cell death and the subsequent immune response initiations after oncolysis are not fully elucidated. We have examined the cell death pathways and immune-stimulatory pathways involved in adenovirus (Ad), Semliki Forest virus (SFV) and vaccinia virus (VV) infection. The three viruses activate distinct but different pathways but all of them induced immunogenic cell death (ICD), which is depicted by elevated cell surface exposure of calreticulin (CRT) and extracellular secretion of ATP. Oncolysis-mediated ICD grants dying cells to be phagocytosed and it promotes DC maturation and activation with a Th1-polarized cytokine secretion profile. As a proof of concept, we validated that when DCs are incubated with Ad- or SFV-, but not VV-infected tumor cells expressing a surrogate antigen (CMVpp65), they can efficiently process and cross-present peptides of this antigen to CD8+ T-cells, as shown by specific IFN-γ production. Mice immunized with SFV- or VV-infected murine glioblastoma cells (GL261) elicit a strong and specific T-cell response against GL261. This adaptive immune response can control tumor growth and prolong mice survival after GL261 rechallenge. Citation Format: Mohanraj Ramachandran, Magnus Essand. Adenovirus, Semliki Forest virus and vaccinia virus-induced immunogenic cell death augments oncolytic virus immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B181.","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"41 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"116013806","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B194: Quantifying the interaction between macrophages and deformable microparticles with cell-like mechanical properties","authors":"D. Vorselen, Yifan Wang, M. Footer, W. Cai, J. Theriot","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B194","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B194","url":null,"abstract":"Macrophages are able to phagocytose vastly different targets, ranging from bacteria to apoptotic and cancer cells. Especially the difference in rigidity between such targets is striking, with bacteria being ~1000 fold stiffer than human cells. Phagocytosis can also be strongly affected by target rigidity and is seemingly less efficient for softer targets. However, mechanistic studies have largely focused on the uptake of stiff particles, and it is currently poorly understood how macrophages adapt the phagocytic mechanism for efficient uptake of soft targets. Here, we developed deformable hydrogel microparticles to study phagocytosis in unprecedented detail, using targets that more accurately mimic cellular physical properties. We used extrusion through Shirasu Porous glass (SPG) membranes to create emulsions with uniformly sized droplets containing acrylamide, bis(acrylamide) and acrylic acid. Subsequent polymerization yields monodisperse (CV 10 Pa) directly from the deformed particle shape. We observed highly localized force exertion by the phagocytes and 4 implied mechanically distinct steps in the phagocytic process. Initially, we observe outward-directed pushing forces from the phagocytic cup base. During subsequent pseudopod extension the majority of the deformation is localized in a ring that is initially irregular, but becomes uniform during cup closure. Surprisingly, strong localized punches at the cup base occur in these stages. After cup closure, we observe some of the strongest forces, seemingly pushing the engulfed target into the cell. Our novel approach gives us unprecedented detail on the mechanical interaction between phagocyte and target in phagocytosis. The presented method here is expected to find broad applications in the study of the immune system in vitro and in vivo. Citation Format: Daan Vorselen, Yifan Wang, Matt Footer, Wei Cai, Julie Theriot. Quantifying the interaction between macrophages and deformable microparticles with cell-like mechanical properties [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B194.","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"16 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"123685870","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B180: Effects of EMT process under MHC class I and TAP1 gene expression related to antigen presentation","authors":"P. R. L. Pires, P. Xavier, H. Fukumasu","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B180","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B180","url":null,"abstract":"Metastasis is a process that involves tumor cell migration from a primary tumor and its invasion to other tissues, which in turn are believed to be driven by a process called epithelial-mesenchymal transition (EMT). EMT process in cancer cells implies increase of malignance and metastatic potential by molecular and phenotypic modifications. MHC class I (MHC-I) is a protein complex used by cells for antigen presentation, specifically to T-cell CD8. It is believed that MHC-I gene expression may fluctuate along different cancer types, biologic processes and molecular modifications which cancer cells may suffer, and it could directly impact on system immunologic responses and T-cell-dependent immunotherapy treatment. This project aimed to evaluate MHC-I and TAP1 gene expression in nonmetastatic origin and metastatic origin cells under the effect of tumor growth factor beta (TGFb), a factor that induces EMT process. Three nonmetastatic cell lineages (A549, H1703, H23) and three metastatic cell lineages (H1792, H2023, H2030) of pulmonary carcinoma were cultured under controlled conditions in RPMI culture media supplemented with 10% of bovine fetal serum (BFS), 1% of antibiotics (penicillin and streptomycin), 2% of glutamine in incubator at 37oC and air atmosphere containing 5% of CO2. Cell cultures were supplemented or not with TGFb (4µg/mL) for 5 days to induce EMT process. After 5 days, cells were evaluated for acquiring mesenchymal cell morphology and MHC-I and TAP1 gene expression (relative quantification). Pool of cells was used to obtain RNA using RNAesy Extraction Kit (QIAGEN®) followed by RT-PCR reaction using High Capacity RNA-to-cDNA Kit (Applied Biosystems®) to obtain cDNA. Relative gene expression was analyzed using Real Time PCR using Fast SYBR™ Green Master Mix (Applied Biosystems®). Except by H23 cell, TGFb incubation showed to be effective and seems to induce EMT process within 5 days culture for both cell types. The cells acquired mesenchymal morphology characteristics such as elongation and increased size. It was also possible to identify a significant gene expression pattern for MHC-I and TAP1 gene expression between control and TGFb groups. Both MHC-I and TAP1 gene expression were shown to be upregulated in the majority of non-metastatic origin cells (A549, H1703, H23; p Citation Format: Pedro Ratto Lisboa Pires, Pedro L.P. Xavier, Heidge Fukumasu. Effects of EMT process under MHC class I and TAP1 gene expression related to antigen presentation [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B180.","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"58 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"128438597","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B185: Epithelial cancer cell-expressed genes contribute to clinically relevant immune-based classifications of breast cancer","authors":"J. Shepherd, C. Perou","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B185","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B185","url":null,"abstract":"Introduction: With the development of immunotherapies for breast cancer therapy, reliable methods to evaluate the extent and type of immune involvement present in tumors and investigation of its effect on patient prognosis and treatment are needed. Identifying tumor-specific features that affect immune involvement will be a key to understand tumor-immune involvement. Therefore, we evaluated expression of immune-related mRNA signatures in TCGA breast cancer data to identify distinct immune-related tumor subsets and asociated prognostic values. We also evaluated immune cell features, including B cell and T-cell receptor richness and diversity, as well as epithelial tumor cell-specific features, including somatic mutations, copy number alterations and differential RNA expression between identified groups. Methods: More than 130 published immune related gene signatures were evaluated in 1095 breast tumors and 97 normal mammary samples. Groups were identified by consensus based hierarchical clustering of the immune signatures, using the proportion of ambiguous clustering to select the optimal number of groups. An ElasticNet model trained on TCGA data was applied to two other breast tumor datasets to predict immune group classification. RNA-sequencing (RNAseq) data from 70 breast cancer cell lines and from human tumor xenografts passaged in immune-compromised mice and processed through a human specific sequencing pipeline provided in vitro and in vivo sources of epithelial cancer cell expression with limited stromal content that was used to filter TCGA bulk RNAseq data for epithelial expressed genes. Results: We identified three distinct immune groups present in breast cancer: immune-low, immune-normal, and immune-high. The immune-high group is characterized by high T-cell scores, including both cytotoxic and regulatory T-cell signatures, and increased B cell and macrophage signatures. The immune-normal set shows signs of normal epithelia and low proliferation. The immune-low group has very low immune cell signatures. Intrinsic breast cancer subtypes (Basal, luminal A, Luminal B, Her2 and Normal-like) are present in each of the immune groups; however, enrichment of basal tumors in immune-high, luminal tumors in the immune-low, and normal mammary, normal-like tumors and luminal A tumors in the immune-normal group demonstrate an interaction between intrinsic tumor type and immune involvement. Immune groups are prognostic in TCGA, with the immune-high group having improved recurrence-free survival. Two more breast tumor datasets confirmed improved survival for basal tumors in the immune-high group relative to the immune-low tumors. Total mutation burden, unique somatic mutations, and copy number alterations did not show significant changes between immune-low and –high groups, whereas RNA expression differs between groups. Selecting for genes with evidence of expression by epithelial breast cancer cells identified over 8,000 genes differentially expressed ","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"4 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"121597783","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}