tbuckley/cabot-search huggingface dataset:
Abstract 5115: Cancer-associated fibroblasts under the influence of melanoma cells might stimulate the lymphatic vessel formation
Authors: Shusaku Maeda, Masakazu Yashiro, Hisashi Motomura, Takaharu Hatano, Heishiro Fujikawa
Journal: Cancer Research (2020)

Abstract Introduction: Cancer-associated fibroblasts (CAFs) in tumor microenvironment have an interaction with tumor cells and play an important role for the progression and metastasis. Tumor lymphangiogenesis may be controlled by not only tumor cells but also tumor microenvironment, and some reports suggested that CAFs may promote tumor lymphangiogenesis. In malignant melanoma, lymph node metastasis is important prognostic factor. However, no study about the interaction among CAFs, tumor cells, and human lymphatic endothelial cells (HLECs) was found. In this study, we evaluated the effect of CAFs on lymphatic vessel formation in malignant melanoma. Materials and methods: We used CAFs established from a primary malignant melanoma, HLECs, and two melanoma cell lines (MMAc, COLO679). CAFs and melanoma cell lines were cultured with DMEM. Serum-free conditioned medium (CM) was obtained from CAFs (CAF-CM) and two melanoma cells (MMAc-CM, COLO679-CM). Also, CM from CAFs cultured with MMAc-CM (MMAcCAF-CM) and COLO679-CM (COLO679CAF-CM) was obtained. HLECs were cultured by endothelial growth medium (EGM) in combination with 50% of each CM. Control group was cultured with 50% EGM and 50% of serum-free endothelial basal medium. The effect of each CM on the proliferation of HLECs was examined by CCK assay and tube formation assay. Tube formation was assessed by counting total tube length, number of tubes and junctions at 12 hours of incubation. Result: Both MMAcCAF-CM and COLO679CAF-CM significantly increased the proliferation of HLECs in compared with the control or CAF-CM alone, after 24, 48 and 72 hours of incubation. In tube formation assay, both tube length and number of tubes formed by HLECs were significantly longer and larger in MMAcCAF-CM and MMAc-CM group than control group. The tube length, number of tubes and junctions treated by MMAcCAF-CM or COLO679CAF-CM were longer and larger than in control and CAF-CM group. Conclusion: CAFs under the influence of melanoma cells might have a more progressive effect on HLECs, resulting in promotion of lymphangiogenesis. Citation Format: Shusaku Maeda, Masakazu Yashiro, Hisashi Motomura, Takaharu Hatano, Heishiro Fujikawa. Cancer-associated fibroblasts under the influence of melanoma cells might stimulate the lymphatic vessel formation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5115.
Abstract 1596: Profiling immune cell populations in the tumor microenvironment with complementary capillary-based and single-cell western assays
Authors: Charles Haitjema, Rainer Grant, Lindsay Wentz, Stefan Luhowskyj, Jeff Cooper, Annegret Boge, Kathy Brumbaugh, Kelly Gardner, Christopher Heger
Journal: Cancer Research (2020)

Abstract The tumor microenvironment (TME) is a complex mixture of cancerous and non-cancerous cells, including immune cells like T-cells, macrophages, and neutrophils. The TME plays a key role in tumorigenesis and metastasis, and it has recently been recognized that it can dramatically shape a response to therapy. Thus, there is a pressing need to accurately identify and quantify the variety of cell types in any given TME. However, studying the TME presents major challenges. For example, the heterogeneity of the environment requires sensitive and high-resolution techniques to parse subpopulations of different cell types. This challenge is compounded by the severely limited sample size that can be obtained from donor tissues. To address these challenges, we use an in-capillary immunoassay with small sample sizes (3 µL) to identify immune cells commonly found in the TME. We also leverage single-cell western to uncover trends in population heterogeneity. Human peripheral blood mononuclear cells (PBMCs) were differentiated into dendritic cells (DCs) and regulatory T cells (Tregs), and natural killer (NK) cells were expanded from isolated NK cells. These samples were then analyzed by in-capillary immunoassay and single-cell western. These analyses revealed the identification and characterization of cell types, at both the single-cell and population level, based on the differential expression of protein biomarkers. Specifically, in-capillary immunoassay analysis identified mature populations by CD209 and CD83 expression for DCs, a CD56+/CD3- phenotype for NK cells, and CD25 and Foxp3 expression for Tregs. Analysis of single cells provided further detail within these populations, for example, we observed FoxP3low and FoxP3high subpopulations in Tregs, and an unexpectedly large (81%) CD56-/CD3- subpopulation in undifferentiated PBMCs, suggesting the presence of other cell subtypes. We anticipate that the small sample size, automation, single-cell resolution, and multiplexing ability of these assays collectively will enable a more efficient and deeper characterization of the TME not possible with traditional immunoassays like western blot and flow cytometry. Citation Format: Charles Haitjema, Rainer Grant, Lindsay Wentz, Stefan Luhowskyj, Jeff Cooper, Annegret Boge, Kathy Brumbaugh, Kelly Gardner, Christopher Heger. Profiling immune cell populations in the tumor microenvironment with complementary capillary-based and single-cell western assays [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1596.
Abstract 2579: Kras expression level regulates heparanase activity
Authors: Donghong Ju, Mary A. Kosir
Journal: Cancer Research (2020)

Abstract Introduction: Sarcomas are locally and systemically aggressive, significantly interacting with extracellular matrix. The purpose of this study is to quantify the expression of heparanase by K-ras expressing sarcoma cells. Methods: Balb/c3T3 and K234 (Ki-ras-transformed Balb/c 3T3) cells (ATCC) were cultured using DMEM with 5% FBS. Heparanase assays were performed with the heparan degrading enzyme assay kit (cat# MK412; Takara) on cell lysate for each cell line according manufacturer protocol. Dhmaracon on-Target Plus SMARTpool siRNA was used to knock down Kras. Total RNA was isolated with Qiagen RNeasy kit, cDNA was made using Invitrogen SuperScript™ III First-Strand Synthesis System, real time RT-PCR was done by use SYBR green Master Mix and Applied Biosystems® 7500 system, mRNA level for HPSE and PPBP was measured by RTPCR. The UCSC genome browser was used to show HPSE sequence and primer location. Results: The heparan degrading activity of lysate from K234 cells was 20-30 times higher than from Balb/c3T3 cells. However, nine pairs of primers for mHPSE RT-PCR results show that mHPSE mRNA is expressed differently in Balb/c3T3 and K234 cell with K234 cell mHPSE mRNA level is lower than in Balb/c 3T3 cells. The mRNA sequence is the same in these two cell lines, however. Using siRNA knock down of greater than 80% Kras from K234 and Balb/c3T3 cells, the heparanase activity of lysate decreased by 78% in K234 cells, and 25% in Balb/c3T3 cells. Kras knock down also changed cell morphology in both cell lines, and changed the mRNA levels of HPSE, PPBP and some EMT markers. Conclusion: Kras overexpressed in K234 cells is associated with increase in heparanase activity, and knock down of Kras decreases heparanase activity. However, heparanase activity does not correlate with HPSE (heparanase 1) expression level, so there is likely another mechanism(s) for Kras expression to regulate heparanase activity. Citation Format: Donghong Ju, Mary A. Kosir. Kras expression level regulates heparanase activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2579.
Abstract 4311: <i>PPIB</i> expression associates with tumor progression and unfavorable disease-free survival of non-small cell lung cancer
Authors: Jeongwon Kim, Joon-Yong Chung, Stephen M. Hewitt, Ilseon Hwang, Joon Seon Song, Chang-Min Choi, Eunho Cho, Kwon-Ho Song, Tae Woo Kim, Kris Ylaya
Journal: Cancer Research (2020)

Abstract Background: PPIB (Peptidylprolyl isomerase B) is involved in cellular transcriptional regulation, immune response, chemotaxis, and proliferation. Recent studies have shown that PPIB is linked to tumor progression and chemoresistance. The role of PPIB in non-small cell lung cancer (NSCLC) has not been explored. We aimed to investigate the significance of PPIB in NSCLC and its mechanism of action. Methods: The role of PPIB was assessed in 5 human NSCLC cell lines. PPIB expression was examined using RNA in situ hybridization (RNA-ISH) on tissue microarrays consisting of 544 NSCLC cases and Ki-67 expression was evaluated by immunohistochemical assays (IHC). RNA-ISH and IHC scores were evaluated by digital image analysis and used to compare patients' prognostic outcomes. Results: PPIB overexpression promoted proliferation, colony formation and migration of NSCLC tumor cells. We also confirmed the oncogenic properties of PPIB expression in PPIB transfected human bronchogenic epithelial cells. In NSCLC tumor samples, high PPIB expression correlated with lymph node metastasis and recurrence (p = 0.018 and 0.026, respectively), while Ki-67 overexpression correlated with advanced tumor stage (p &amp;lt; 0.001). Patients with high PPIB expression assessed by RNA-ISH (PPIBhigh), exhibited significantly shorter disease-free survival (DFS) time than those who were PPIBlow [HR (95% CI) = 1.53 (1.11-2.10), p = 0.008]. Multivariate analysis revealed that combined Ki-67high/PPIBhigh expression was an independent predictor of shorter DFS [HR (95% CI) = 1.71 (1.26-2.31), p &amp;lt; 0.001]. Conclusions: These findings suggest that PPIB plays a role in NSCLC tumor growth and is associated with unfavorable disease-free survival. PPIB may have value as a prognostic marker and a potential target molecule for NSCLC treatment. Citation Format: Jeongwon Kim, Joon-Yong Chung, Stephen M. Hewitt, Ilseon Hwang, Joon Seon Song, Chang-Min Choi, Eunho Cho, Kwon-Ho Song, Tae Woo Kim, Kris Ylaya. PPIB expression associates with tumor progression and unfavorable disease-free survival of non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4311.
Abstract 2253: Lymphangiogenesis augments the anti-tumor efficacy of radiotherapy in mouse melanoma
Authors: Nikolaos Mitrousis, Maria Stella Sasso, Ralph R. Weichselbaum, Jeffrey A. Hubbell, Melody A. Swartz
Journal: Cancer Research (2020)

Abstract Radiotherapy (RT) constitutes a significant component of cancer therapy in the clinic, with approximately 50% of all cancer patients receiving treatment over the course of their disease. In addition to acting as a DNA-damaging agent, RT leads to the release of tumor antigens, damage-associated molecular patterns, and cytokines, a process termed immunogenic cell death. This phenomenon triggers the initiation of anti-tumor immune responses, which have been shown to be essential for the complete therapeutic response to RT. Despite its diverse mechanisms of action and broad applicability, RT does not typically lead to curative responses and combinations with other treatment modalities such as immunotherapy are being explored to improve efficacy. Our laboratory and others have previously established that tumor-associated lymphatics can actively modulate anti-tumor immune responses and potentiate immunotherapy. Here, we asked whether lymphangiogenesis influences the immune response and anti-tumor efficacy elicited by RT. We employed two variants of the aggressive B16F10 melanoma model: a variant that was transduced to overexpress the lymphangiogenic factor VEGF-C (B16-VEGF-C), and a mock-transduced (B16-ctrl) variant. Tumor-bearing animals were treated by ablative doses of localized RT and monitored for tumor growth and survival, while anti-tumor immune responses were assessed by flow cytometry and ELISA. We observed that the therapeutic efficacy of RT was greater in B16-VEGF-C melanomas compared to B16-ctrl, as evidenced by a significant inhibition of tumor growth and extended animal survival. In line with this finding, B16-VEGF-C melanomas exhibited a significantly higher CD8+ T cell infiltration and activation upon ex vivo restimulation after RT. These responses were partially inhibited by a function-blocking antibody of the main VEGF-C receptor, VEGFR3. To gain further insight, we assessed the tumor infiltration and activation status of antigen-presenting cells. Consistent with the observed T cell response, we found that both dendritic cells and macrophages exhibited a greater expression of co-stimulatory molecules in B16-VEGF-C melanomas after RT. Together, our findings demonstrate that lymhangiogenesis may be a key contributing factor underlying tumor responsiveness to RT. Citation Format: Nikolaos Mitrousis, Maria Stella Sasso, Ralph R. Weichselbaum, Jeffrey A. Hubbell, Melody A. Swartz. Lymphangiogenesis augments the anti-tumor efficacy of radiotherapy in mouse melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2253.
Abstract 4992: In vitro exhausted T cell assay for immuno-oncology candidates
Authors: Amin Osmani, Juliette Lamy, Thibaut Janss, Séverine Giltaire, Sofie Pattijn, Jana Schockaert
Journal: Cancer Research (2020)

Abstract The need to better understand the tumour microenvironment (TME) dictates the characterization of the cell types involved, the roles they play and how they respond to treatment. To develop better cancer immunotherapies, in vitro primary immune cell bioassays offer an early assessment of their effects on the various players of the TME. Here we describe the development of an exhausted T cell assay and demonstrate its potential in identifying candidates that can reverse exhaustion. Exhausted T cells were first discovered in LCMV virus infections where chronic stimulation due to remaining virus antigens leads to T cells that stop responding to stimulation and lose their functionality (cytokine secretion and cytotoxic activity). The same phenotype was afterwards found in the tumor microenvironment where a lot of T cells were found to be “anergic”. Reversing their loss of functionality as a therapeutic strategy has made the characterization of these exhausted T cells a priority. Their phenotype is currently defined by an increase in the expression of a specific set of cell surface markers such as PD-1, TIM-3 and LAG-3. As exhaustion builds, the expression of these marker increases while T cell functionality decreases. The loss of functionality is defined by a reduction in cytokine secretion, proliferation and cytotoxic activity in the presence of an antigen. To evaluate this phenotype in more depth, transcription factors can also be monitored. For example, BLIMP-1 and BATF, which play a role in PD-1 expression, are shown to increase following exhaustion. Using primary immune cells, in vitro bioassays were developed to better screen the potential effect of new therapeutics on exhausted T cells. Their ability to reverse the exhausted phenotype and facilitate the anti-tumour immune response can as a result be assessed early in the drug development process. These experiments help advance our understanding of the tumour microenvironment and optimize the therapeutic effects of new drugs, design better clinical trials and ultimately discover relevant biomarkers. Citation Format: Amin Osmani, Juliette Lamy, Thibaut Janss, Séverine Giltaire, Sofie Pattijn, Jana Schockaert. In vitro exhausted T cell assay for immuno-oncology candidates [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4992.
Abstract 773: Tumor-derived Extracellular Vesicles (EVs) expressing TGFb as potential biomarkers for anti-TGFb antibody activity and drug activity in liquid biopsy
Authors: Manoel Nunes, Camille Bernard, Gaelle Muzard, Wilson Dos-Santos-Bele, Jack Pollard, Alexei Protopopov, Colette Dib
Journal: Cancer Research (2020)

Abstract The field of liquid biopsies is of enormous interest in cancer using a noninvasive process. There are currently three major approaches: Circulating Cell Tumors (CTCs), circulating cell free DNA (cfDNA) and Extracellular Vesicles (EVs) such as exosomes, microvesicles and apoptotic bodies. The EVs are cell-derived small membrane vesicles secreted by cells that convey biological messages, either by surface-to-surface interaction or by shuttling bioactive molecules to a recipient cell's cytoplasm. The EVs released by cells harbor the cargos of their original cells and could be a good tool to follow drug activity. We are developing anti-TGFβ antibody SAR439459 for the treatment of cancer (NCT03192345), and in this vitro study we explore the effect of anti-TGFβ antibody in EVs carrying TGFβ. The cargo of EVs were analyzed by an Elisa test for both latent and active free TGFβ and by miRNA expression. Cell lines were selected based on their expression of TGFβ (A549, DU45, PC3, MCF7 and Caco2), treated for 72h and compared with respective untreated controls. The EVs were isolated from the supernatant and purified. Nanoparticle Tracking Analysis was used to quantify and determine size distribution of EVs. The expression of active free and latent TGFβ was done using Elisa tests. TGFβ antibody-coated magnetic beads were used to capture EVs, and miRNA expression sequencing was done using the Illumina NextSeq. Anti-TGFβ treatment reduced notably the expression of latent TGFβ in the supernatant of cultured tumor cells; the active free TGFβ was very low expressed or not detected in the supernatants. EVs express both latent and active free TGFβ; the anti-TGFβ antibody treatment significantly reduced the active free TGFβ and altered miRNA expression in EVs. Finally, using TGFβ Antibody-coated magnetic beads, we demonstrate that intact EVs express TGFβ at their surface. In conclusion, these results suggest that EVs express TGFβ at their surface and carry the active free TGFβ. Finally, the expression of active free TGFβ in EVs is decreased with anti-TGFβ antibody, suggesting that it might be a biomarker for monitoring anti-TGFβ antibody activity in patients by liquid biopsies. Citation Format: Manoel Nunes, Camille Bernard, Gaelle Muzard, Wilson Dos-Santos-Bele, Jack Pollard, Alexei Protopopov, Colette Dib. Tumor-derived Extracellular Vesicles (EVs) expressing TGFb as potential biomarkers for anti-TGFb antibody activity and drug activity in liquid biopsy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 773.
Abstract 3415: Mutant BRAF small molecule inhibition enhances oncolytic herpes virus immunotherapy through increased immune cell recruitment and activation in melanoma
Authors: Galabina Bozhanova, Victoria Jennings, Malin Pedersen, Joan Kyula, Emmanuel Patin, Jehanne Hassan, Masahiro Ono, Kevin Harrington, Alan Melcher
Journal: Cancer Research (2020)

Abstract Small molecule inhibitors of the MAPK pathway have demonstrated rapid responses in melanoma patients, but their use is hindered by the frequent development of acquired resistance. Oncolytic viruses are a novel class of immunotherapeutics which selectively replicate inside cancer cells, causing lysis and subsequent activation of anti-tumor immunity. BRAF and MEK inhibitors have previously been shown to synergise with oncolytic viruses to kill melanoma cells, but the consequences of these interactions on immune responses have not been assessed. Here, we show that the combination of PLX4720 (BRAFV600E inhibitor) and intratumoral oncolytic herpes virus HSV1716 led to a significant tumor reduction and improved survival over single-agent treatment in a murine BRAFV600E melanoma model. The combination led to a significant increase of PD-L1+ neutrophils and monocytes at an early time point following therapy administration, suggesting an inflammatory switch in the tumor microenvironment. Importantly, the combination led to a significant increase in cytotoxic CD8+ cells at a later time point, which was accompanied by an increase in CD25+ Foxp3+ T-regulatory (Treg) cells. Addition of an anti-CD25 antibody to the PLX4720/HSV1716 doublet led to complete eradication of melanoma tumors in vivo. Current efforts are focused on deciphering T-cell dynamics during this therapy using a novel technology, Timer of cell kinetics and activity (Tocky). Tocky uses a fluorescent Timer protein which spontaneously changes its emission spectrum from blue to red, reporting real-time activation of the T-cell receptor and subsequent activation of immune cells in vivo. Using Tocky, we are identifying specific immune subsets that are stimulated following therapy and the real-time dynamics of antigen recognition and subsequent cell activation. These findings form the basis of future testing of drug-virus combinations in an effort to identify potent cytotoxic, as well as immuno-modulatory, strategies and their detailed mechanism of action. Citation Format: Galabina Bozhanova, Victoria Jennings, Malin Pedersen, Joan Kyula, Emmanuel Patin, Jehanne Hassan, Masahiro Ono, Kevin Harrington, Alan Melcher. Mutant BRAF small molecule inhibition enhances oncolytic herpes virus immunotherapy through increased immune cell recruitment and activation in melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3415.