bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract PO010: The inhibition of IWS1 phosphorylation promotes genomic instability, the cGAS/STING pathway activation and PD-L1 levels, through the U2AF2 alternative RNA splicing and Sororin expression
Authors: Laliotis Georgios I. , Chavdoula Evangelia , Kaba Abdul , La Ferlita Alessandro , Anastas Vollter , Orlacchio Arturo , Sehgal Lalit , Carbone David P. , Coppola Vincenzo , Tsichlis Philip N.
Published: February 2021

We have previously shown that the loss of IWS1 phosphorylation promotes the alternative RNA splicing of U2 Associated-Factor 2 (U2AF2), resulting in transcripts lacking exon 2 in lung adenocarcinoma cells. This exon encodes part of the U2AF65 Serine-Rich (SR) Domain, which is required for its binding with pre-mRNA Processing factor 19 (Prp19). We have also shown that inhibition of the pathway results in the downregulation of cell cycle division associated 5 (CDCA5), and its protein product Sororin, a phosphorylation target and regulator of ERK and member of the cohesin complex, leading to G2/M phase arrest, sister-chromatid cohesion defects, impaired cell proliferation and tumor growth in mouse xenografts models. Here, we show that the loss of IWS1 results in genomic instability and accumulation of cytosolic dsDNA, an effect rescued by the expression of Sororin. The dsDNA is censored by cyclic GMP-AMP synthetase (cGAS), activating the STING/TBK1 pathway. The latter leads to increased expression of Interferon Regulatory Factor-3 (IRF-3) targets along with PD-L1. More importantly, IWS1 phosphorylation, U2AF2 RNA splicing pattern and Sororin expression negatively correlate with the cGAS/STING pathway and PD-L1 expression in lung adenocarcinoma patients. These results highlight the role of IWS1 phosphorylation-dependent RNA splicing in governing genomic stability, and proposes this axis as a novel drug target for a synergy with PD-L1/PD-1 blockade in lung adenocarcinoma patients.

Citation Format: Georgios I. Laliotis, Evangelia Chavdoula, Abdul Kaba, Alessandro La Ferlita, Vollter Anastas, Arturo Orlacchio, Lalit Sehgal, David P. Carbone, Vincenzo Coppola, Philip N. Tsichlis. The inhibition of IWS1 phosphorylation promotes genomic instability, the cGAS/STING pathway activation and PD-L1 levels, through the U2AF2 alternative RNA splicing and Sororin expression [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO010.
Abstract PO011: Systemic administration of Poly-ICLC promotes T cell tumor infiltration generating antitumor responses
Authors: Sultan Hussein , Wu Juan , Fesenkova Valentyna , Fan Aaron , Addis Diane , Salazar Andres , Celis Esteban
Published: February 2021

Background: Recent advances in immunotherapy have revolutionized cancer treatment. Immune checkpoint blockade and adoptive T cell therapy can induce durable responses in some patients. However, the majority of patients fail to respond. Absence of T cell infiltration to the tumor site in solid tumors is considered as one of the major obstacles for effective immunotherapy. Thus, strategies to enhance T cell trafficking and infiltration into the tumor parenchyma is a major research need.

Methods: Using a combination of in vivo mouse tumor models and in vitro assays we compared the capacity of various formulations and routes of administration of poly-IC (a dsRNA mimic that functions as a pattern recognition receptor ligand) in enhancing T cell tumor infiltration and generating antitumor responses.

Results: Our results showed that poly-ICLC (poly-IC containing poly-lysine and carboxymethylcellulose) enhanced CD8 T cell infiltration into the tumors resulting in the control of tumor growth. These effects relied both on the route of administration as well as on the formulation of poly-IC. Systemic administration (i.v. or i.m.) of poly-ICLC was significantly more effective in inducing CD8 T cell tumor infiltration as compared to intra-tumoral injections. Also, the effects poly-ICLC were substantially more pronounced as compared to unmodified poly-IC. The antitumor effect of poly-ICLC was mediated via MDA-5 and IFN-I, whereas TLR3 stimulation was not required. Interestingly, poly-ICLC stimulated IFN-I responses on tumor vascular endothelial cells (VECs) enhancing the expression of adhesion molecules (VCAM-I), production of IFN-I and T cell recruiting chemokines (CXCL9/CXCL10). Using conditional knockout mice, showed that ablation of IFNab receptors in VECs impaired the antitumor effects of poly-ICLC. IFN-I production upon MDA5 stimulation is required to enhance the secretion of (CXCL9/CXCL10) by VECs indicating that this IFN-I/CXCL9/CXCL10 regulatory axis is crucial for recruiting effector T cells into the tumor parenchyma. The T cell infiltrating effects of poly-ICLC were mainly observed in tumors and not in normal tissues.

Conclusions: Systemic administration of poly-ICLC improves T cell infiltration to solid tumors in an MDA5/IFN-I dependent manner. These findings should have a strong impact on improving T cell-based treatments for solid cancers such as adoptive T cell therapy and antitumor vaccines.

Citation Format: Hussein Sultan, Juan Wu, Valentyna Fesenkova, Aaron Fan, Diane Addis, Andres Salazar, Esteban Celis. Systemic administration of Poly-ICLC promotes T cell tumor infiltration generating antitumor responses [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO011.
Abstract PO012: A subset of monocyte-derived macrophages in glioblastoma multiforme supports antitumor immune responses
Authors: Kim Hyun-Jin
Published: February 2021

Glioblastoma multiforme (GBM) is the most aggressive type of brain tumor and effective therapies are not well established. Lymphopenia and large infiltration of macrophages are major features of GBM. Although tumor-associated macrophages (TAMs) are known to suppress antitumor immune responses and support tumor growth and metastasis, recent studies reported that TAMs are a heterogeneous cell population, which includes both immune activating as well as immunosuppressive TAMs. However, because markers identifying these cells are not established, targeting TAMs is to induce antitumor immune responses is not yet an effective strategy. In this study, we identified a subset of macrophages that promote an anti-tumoral microenvironment in GBM. In single cell transcriptome analysis, we found that macrophages were divided to three populations based on their gene expression. One of these macrophage subsets expressed markers of monocyte-derived macrophages. These cells were a major source of pro-inflammatory chemokines among brain immune cells. In brain tumors without this monocyte-derived macrophage subpopulation, accumulation of CD4 and CD8 T cells and their cytokine production were diminished. Immuno-suppressive cells, such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), and chemokines recruiting these cells were increased. Higher HIF-1α expression in tumors without these macrophages indicate that this subset might resolve hypoxia. Our finding suggests that this macrophage subset can support antitumor immune responses by suppressing a pro-tumoral microenvironment.

Citation Format: Hyun-Jin Kim. A subset of monocyte-derived macrophages in glioblastoma multiforme supports antitumor immune responses [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO012.
Abstract PO013: The mechanism of γδ T cell-mediated antitumor immunity in Glioblasotma multiforme
Authors: Park Jang Hyun , Lee Heung Kyu
Published: February 2021

Glioblastoma multiforme (GBM) is a highly malignant cancer with one of the highest mortality rates. The average survival is less than 2 years after diagnosis even with current therapies, which include chemotherapy and radiation. Furthermore, GBM has shown severe resistance to immunotherapies such as immune checkpoint blockade, which mainly target CD8 T cells. Recently, several studies showed that γδ T cells can be an alternative target of immunotherapy. Unlike conventional ab T cells, γδ T cells recognize stress-induced surface antigens such as NKG2D ligands and phosphoantigens. Because GBM cells highly express NKG2D ligands, γδ T cells could be good target for immunotherapy. However, a basic understanding of γδ T cell-mediated antitumor immunity is lacking. Using single-cell transcriptomic analysis, we analyzed the characteristics of GBM-infiltrating γδ T cells. Because γδ T cells were highly suppressed by the poorly oxygenated microenvironment of brain tumors, we inhibited tumor cell respiration using metformin, a drug used for type 2 diabetes, and found that this sufficiently reinvigorated γδ T cells. Reinvigorated γδ T cells had enhanced expression of NKG2D and multiple cytokines. In conclusion, this study highlights the importance of γδ T cells in GBM and the potential for new druggable pathways through reinvigoration of γδ T cells.

Citation Format: Jang Hyun Park, Heung Kyu Lee. The mechanism of γδ T cell-mediated antitumor immunity in Glioblasotma multiforme [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO013.
Abstract PO015: Tumor-cell-intrinsic epigenetic factors underlie the heterogeneity of immune infiltration and response to immunotherapy in pancreatic cancer
Authors: Li Jinyang , Yuan Salina , Norgard Robert J. , Yan Fangxue , Vonderheide Robert H. , Blanco Andres , Stanger Ben Z.
Published: February 2021

Resistance to immunotherapy is a major problem of current clinical care for cancer patients. While T cell abundance is essential for tumor responsiveness to immunotherapy, factors that dictate T cell infiltration in tumor microenvironments are not fully understood. To understand the tumor-cell-intrinsic factors underlying the heterogeneity of tumor immunity and sensitivity to immunotherapy, we established a new experimental system by generating a library of congenic pancreatic tumor cell clones from a genetic mouse model driven by mutant Kras and p53. These tumor cell clones robustly formed implanted tumors that recapitulated T-cell-inflamed and non-T-cell-inflamed tumor microenvironments, associated with distinct patterns of infiltration by T cells and myeloid cells. We found that the non-T-cell-inflamed phenotype was dominant over the T-cell-inflamed phenotype in the local tumor microenvironment. An integrated transcriptomic and epigenetic analysis revealed that tumor-cell-intrinsic expression of CXCL1, EPHA2, PTGS2, and USP22 as determinants of the non-T-cell-inflamed microenvironment, and ablation of tumor-cell-intrinsic CXCL1, EPHA2, PTGS2, or USP22 promoted T cell infiltration and sensitivity to a combination of chemotherapies, CD40 agonist, and checkpoint blockades. Furthermore, we performed an in vivo CRISPR-based genetic screen to identify tumor cell intrinsic epigenetic regulators of anti-tumor immunity and discovered novel therapeutic opportunities to improve the efficacy of currently developed immunotherapy for pancreatic cancer. These results demonstrated that heterogeneity of tumor immune phenotypes is driven by tumor-cell-intrinsic factors, including epigenetic factors, that can be manipulated to influence the outcome of immunotherapies.

Citation Format: Jinyang Li, Salina Yuan, Robert J. Norgard, Fangxue Yan, Robert H. Vonderheide, Andres Blanco, Ben Z. Stanger. Tumor-cell-intrinsic epigenetic factors underlie the heterogeneity of immune infiltration and response to immunotherapy in pancreatic cancer [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO015.
Abstract PO016: Single-cell analyses characterize circulating anti-tumor CD8 T cells and identify markers for their isolation
Authors: Pauken Kristen E. , Shahid Osmaan , Lagattuta Kaitlyn A. , Mahuron Kelly M. , Luber Jacob M. , Lowe Margaret M. , Huang Linglin , Delaney Conor , Long Jaclyn M. , Fung Megan E. , Newcomer Kathleen , Tsai Katy K. , Chow Melissa , Guinn Samantha , Kuchroo Juhi R. , Burke Kelly P. , Schenkel Jason M. , Rosenblum Michael D. , Daud Adil I. , Sharpe Arlene H. , Singer Meromit
Published: February 2021

The ability to monitor anti-tumor CD8+ T cell responses in the blood has tremendous therapeutic potential. Here, we used paired single-cell RNA sequencing and T cell receptor (TCR) sequencing to detect and characterize “tumor matching” (TM) CD8+ T cells in the blood of mice with MC38 tumors and melanoma patients using the TCR as a molecular barcode. TM cells showed increased activation compared to non-matching T cells in blood, and appeared less exhausted than matching counterparts in tumor. Importantly, PD-1, which has been used to identify putative circulating anti-tumor CD8+ T cells, showed poor sensitivity for identifying TM cells. By leveraging the transcriptome we identified candidate cell surface marker panels for TM cells in mice and melanoma patients, and validated markers in mice. Here, using combinations of markers provided better performance than single markers in identifying TM cells from non-TM cells in the blood. These data demonstrate that the TCR can be used to identify tumor-relevant populations for comprehensive characterization, reveal unique transcriptional properties of TM cells, and develop marker panels for tracking and analysis of these cells.

Citation Format: Kristen E. Pauken, Osmaan Shahid, Kaitlyn A. Lagattuta, Kelly M. Mahuron, Jacob M. Luber, Margaret M. Lowe, Linglin Huang, Conor Delaney, Jaclyn M. Long, Megan E. Fung, Kathleen Newcomer, Katy K. Tsai, Melissa Chow, Samantha Guinn, Juhi R. Kuchroo, Kelly P. Burke, Jason M. Schenkel, Michael D. Rosenblum, Adil I. Daud, Arlene H. Sharpe, Meromit Singer. Single-cell analyses characterize circulating anti-tumor CD8 T cells and identify markers for their isolation [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO016.
Abstract PO032: TIM3 regulation by phosphatidylserine
Authors: Smith Courtney M. , Li Alice , Krishnamurthy Nithya , Lemmon Mark A.
Published: February 2021

Immune responses have been successfully reactivated in cancer by targeting inhibitory receptors such as PD-1 on exhausted T cells to block immune checkpoints. Many patients do not respond, however, and a key challenge in immuno-oncology is to identify and understand new immune-regulatory targets to increase response rates. One other co-inhibitory receptor in T cells is TIM3, which – like PD-1 – is upregulated on exhausted T cells in the tumor microenvironment. TIM3 belongs to a family of phosphatidylserine (PS) receptors with well-documented roles in efferocytosis by phagocytes. However, the role of PS in regulating TIM3 remains unclear. Previous studies have shown that TIM3 can variously enhance or inhibit T cell signaling, though exactly how TIM3 accomplishes this range of activity is unknown. We therefore investigated how TIM3 levels modulate T cell signaling, and how PS in turn influences TIM3 activity. We found that TIM3 promoted T cell activity in a Jurkat T cell model when expressed at high – but not low – levels on the cell surface. We also confirmed the ability of human TIM3 to bind PS with low micromolar affinity using surface plasmon resonance. To investigate how PS affects the ability of TIM3 to promote T cell signaling in this model, we employed several approaches, including adding PS to the cell culture, manipulating TIM3 to reduce or abrogate PS binding (using chimeric receptors and TIM3 mutants), and pharmacological blockade of PS binding using a functional TIM3 antibody. Together, these studies supported a role for PS in regulating TIM3 activity, where decreased or abrogated PS binding prevented TIM3 from promoting T cell signaling. We also sought to understand what changes in T cell receptor (TCR) signaling might account for the TIM3-dependent effects we observed. We found that TIM3 did not impact phosphorylation of downstream components in the TCR signaling cascade. Rather, cells expressing high – but not low – surface levels of TIM3 displayed enhanced phosphorylation of receptor components in the TCR signaling pathway, similar to what has been reported for PD-1. We hypothesize that TIM3 modulates TCR signaling by modifying the localization and/or dwell time of one or more signaling effector proteins (e.g. phosphatases) in a manner dependent on PS engagement. The consequences of this are then determined by surface levels of TIM3. Our results demonstrate that PS does function as a ligand capable of modulating TIM3 activity, and provide new mechanistic insight into how TIM3 modulates TCR signaling.

Citation Format: Courtney M. Smith, Alice Li, Nithya Krishnamurthy, Mark A. Lemmon. TIM3 regulation by phosphatidylserine [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO032.
Abstract PO034: Mechanisms and microbial Influences on CTLA-4 and PD-1-based immunotherapy in the treatment of cancer: A narrative review
Authors: Miller Peter L. , Carson Tiffany L.
Published: February 2021

Background: The relationship between gastrointestinal (GI) bacteria and the response to anti-CTLA-4 and anti-PD-1 immunotherapy in the treatment of cancer can potentially be enhanced to allow patients to maximally respond to these treatments. Insight into the complex interaction between gut microbiota and the human adaptive immune system will help guide future immunotherapeutic cancer treatments to allow a more robust clinical response and fewer adverse effects in patients requiring these drugs. This review highlights these interactions as well as the potential for the creation of “oncomicrobiotics” that would selectively tailor one’s GI bacteria to maximally respond to anti-CTLA-4 and anti-PD-1 treatments.

Main Body: CTLA-4 is an antigen on the surface of T cells which, upon stimulation, leads to inhibition of activated T cells to terminate the immune response. However, many types of tumor cells can upregulate CTLA-4 in the tumor microenvironment, allowing these cells to evade targeting and destruction by the body’s immune system by prematurely inhibiting T cells. Increased representation of Bacteriodes fragilis and Burkholderia cepacia in the GI tract of patients receiving CTLA-4-based immunotherapy led to a stronger therapeutic effect while minimizing adverse side effects such as colitis. In addition, introducing bacteria involved in vitamin B and polyamine transport to the GI tracts of patients treated with anti-CTLA-4 drugs led to increased resistance to colitis while maintaining therapeutic efficacy. PD-1 is another molecule upregulated in many tumor microenvironments which acts in a similar manner to CTLA-4 to tone down the anti-neoplastic actions of T cells. Antibodies to PD-1 have shown promise to help allow the body’s natural immune response to appropriately target and destroy tumor cells. The presence of Bifidobacterium, namely Bifidobacterium breve and longum, in the GI tracts of cancer patients has the potential to create a more robust immune response to anti-PD-1 drugs while limiting medication-induced adverse effects. The development of “oncomicrobiotics” has the potential to help tailor one’s gut microbiota to allow patients to maximally respond to immunotherapy without sacrificing increases in toxicity. These oncomicrobiotics may possibly include antibiotics, probiotics, postbiotics and/or prebiotics. However, many challenges lie ahead in the creation of oncomicrobiotics.

Conclusion: The creation of oncomicrobiotics may allow many patients receiving anti-CTLA-4 and PD-1 immunotherapy to experience prolonged survival and a better quality of life.

Citation Format: Peter L. Miller, Tiffany L. Carson. Mechanisms and microbial Influences on CTLA-4 and PD-1-based immunotherapy in the treatment of cancer: A narrative review [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO034.
Abstract PO040: High-throughput vascularized tumor array for In-vitro natural killer cell cytotoxicity testing
Authors: Song Jiyoung , Choi Hyeri , Park Dohyun , Yu James , Jeon Nooli
Published: February 2021

Colorectal cancer is second leading cause of cancer death. There have been many approaches proposed, such as anti-cancer drugs, chemotherapy and radiotherapy, however, there are still lack of breakthrough therapy designation. Cancer immunotherapies hold great promise, where the mechanism of interaction between immune cell and tumor remains poorly understood. Understanding of immune cell infiltration in solid tumor plays an important role in cancer immunotherapy. This paper describes injection molded plastic chip to mimic complex three-dimensional tumor-vasculature for evaluating immune cell infiltration and their cytotoxicity. Our device facilities simple and high-throughput screening, constituting of 28 wells per device. Using our system, Co-culture conditions that both tumors and blood vessels were adequately benefited were established. Also, we characterized the aberrant vasculature according to the colorectal tumor subtypes and confirmed increased blood vessel the permeability, which is the major feature of aberrant tumor vasculature. Moreover, we evaluated Natural killer(NK) cell infiltration and cytotoxicity according CMS subtypes. NK cell showed higher cytotoxicity to CMS 1 type colorectal cancer compared to the other subtypes. This result confirmed that NK cell is the crucial effector in innate immune system as a first line of defense against pathogens. Therefore, we propose our plastic microfluidic chip as a high-throughput screening platform to evaluate lymphocyte cytotoxicity to various solid tumor including immune infiltration.

Citation Format: Jiyoung Song, Hyeri Choi, Dohyun Park, James Yu, Nooli Jeon. High-throughput vascularized tumor array for In-vitro natural killer cell cytotoxicity testing [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO040.
Abstract PO056: The kinetics of the anti-glioblastoma immune response in immunocompetent mouse models is influenced by neglected factors
Authors: Noffsinger Breanna R. , Witter Alexandra , Shebani Natasha , Xiao Aizhen , Zhong Qing , Harris Tajie , Purow Benjamin
Published: February 2021

Due to the recent rise in immunotherapy research to treat Glioblastoma, immunocompetent mouse models have become increasingly crucial for the study of novel immunotherapies against brain tumors. However, the kinetics of the immune response against the most prevalent immunocompetent GBM models, GL261 and CT2A, have not been well studied, nor have they been compared adequately in the literature. In this study, we compared the immune response in these models using flow cytometry and immunohistochemistry. We investigated several factors that influence the immune response, such as kinetics, tumor size, and transfection status. We hypothesize that these factors influence the immune response enough to warrant consideration while studying new immunotherapeutic approaches for treating GBM. Our results will aid in the preclinical studies of immunotherapy for GBM by allowing scientists to make informed decisions about their chosen models.

Citation Format: Breanna R. Noffsinger, Alexandra Witter, Natasha Shebani, Aizhen Xiao, Qing Zhong, Tajie Harris, Benjamin Purow. The kinetics of the anti-glioblastoma immune response in immunocompetent mouse models is influenced by neglected factors [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO056.
Abstract PO062: Isolation of tumor-infiltrating lymphocytes for higher sensitivity gene expression profiling
Authors: Webb Lindsay M. , Naughton Bartholomew , Yalavarthi Sireesha , Bourin Clotilde , Moisan Jacques , Xu Chunxiao
Published: February 2021

Gene expression profiling is a powerful approach to address the mechanism of action (MOA) of cancer immunotherapy-driven reprogramming of tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment. However, TILs, particularly T and NK cells, are a very rare population within the tumor. Gene expression profiling using Nanostring or RNAseq is often not sensitive enough to detect expression changes in rare populations, and it is difficult to separate gene expression profiles between different cell subsets. Although single-cell RNAseq can distinguish gene expression profiles between different cell subsets, it is very costly and would require many cells to be sequenced before obtaining the rare cell type of interest. Classically, fluorescence-activated cell sorting (FACS) has been used to isolate rare populations, but this method is a long process that can affect cell phenotype and results in dramatic cell loss. Thus, we developed a protocol for rapid and reproducible magnetic bead-based isolation of tumor-infiltrating lymphocytes (TILs) for higher sensitivity MOA studies. CD8 and CD4 TILs were isolated from both MC38 and CT26.KSA tumor models with high purity and sufficient yield for further Nanostring gene expression analysis. As expected, CD8 and CD4 T cell gene expression profiles were dramatically enriched in the isolated TILs compared to the whole tumor. Thus, our protocol for isolating TILs from mouse tumors can be applied broadly to multiple tumor types and provides sufficient yield to perform downstream high-sensitivity gene expression profiling.

Citation Format: Lindsay M. Webb, Bartholomew Naughton, Sireesha Yalavarthi, Clotilde Bourin, Jacques Moisan, Chunxiao Xu. Isolation of tumor-infiltrating lymphocytes for higher sensitivity gene expression profiling [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO062.
Abstract PO077: BT7480, a novel fully synthetic tumor-targeted immune cell agonist (TICA™) induces tumor localized 4-1BB agonism
Authors: Repash Elizabeth M. , Upadhyaya Punit , Lahdenranta Johanna , Kublin Jessica , Ma Jun , Kleyman Marianna , Chen Liuhong , Haines Eric , Battula Sailaja , McDonnell Kevin , Hurov Kristen , Brandish Philip , Keen Nicholas
Published: February 2021

Harnessing costimulatory molecules expressed on T and NK cells namely 4-1BB (CD137/TNFRSF9) is ideal for cancer immunotherapy. Despite promise preclinically, 4-1BB agonistic antibodies, were unable to demarcate hepatoxicity from efficacy in the clinic. Bispecific approaches promote target-mediated clustering of 4-1BB while limiting systemic and liver toxicity. Bicycles® are novel, fully synthetic, constrained bicyclic peptides with high affinity and selectivity to their targets. Their small size (~2kDa) and tunable pharmacokinetic parameters allow Bicycles to have superior tumor penetration and de-risk hepatoxicity concerns due to a renal clearance mechanism. We hypothesized that clustering and activation of 4-1BB could be attained by conjugating a 4-1BB binding Bicycle to a tumor antigen targeting Bicycle promoting a potent tumor-localized immune response. BT7480 is a tumor-targeted immune cell agonist (TICA™) targeting Nectin-4 and agonizing 4-1BB. Nectin-4 (PVRL4) is highly expressed in numerous tumor indications with unmet clinical needs. In an engineered 4-1BB reporter system, we found BT7480 induced potent 4-1BB agonism correlating to target antigen surface expression on the co-cultured tumor cells. Moreover, BT7480 induces cytokine secretion, including interleukin-2 and interferon gamma, in immune cell co-culture in the presence of tumor antigen. This activity is strictly dependent on Nectin-4 expression on tumor cells and the ability of the TICA to bind to both Nectin-4 and 4-1BB. In a MC38 (Nectin-4-expressing) syngeneic mouse model, intermittent dosing of BT7480 led to robust anti-tumor efficacy (22 out of 24 complete responders (CRs)). Importantly, a memory response was established in CR mice as resistance to a re-challenge with MC38 tumors was observed. Additionally, BT7480 led to increased intratumoral T cell infiltration without elevation of liver enzymes in a CT26 (Nectin-4-expressing) syngeneic mouse model. In non-human primates (NHPs), BT7480 exhibits dose linear exposure and is well tolerated up to 10mpk. BT7480 represents a new generation of chemically synthetic tumor antigen targeted 4-1BB agonists with potent efficacy and a favorable safety profile.

Citation Format: Elizabeth M. Repash, Punit Upadhyaya, Johanna Lahdenranta, Jessica Kublin, Jun Ma, Marianna Kleyman, Liuhong Chen, Eric Haines, Sailaja Battula, Kevin McDonnell, Kristen Hurov, Philip Brandish, Nicholas Keen. BT7480, a novel fully synthetic tumor-targeted immune cell agonist (TICA™) induces tumor localized 4-1BB agonism [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO077.
Abstract PO081: Systematic Generation of Allogeneic Immune-targeting Modalities for Glioblastoma
Authors: Salim Sabra K. , Wei Jiarun , Dimitrov Vassil , Chen Katherine , Venugopal Chitra , Vora Parvez , Moffat Jason , Singh Sheila K.
Published: February 2021

Glioblastoma (GBM) is one of the most common brain tumors in adults. Despite a standard-of-care including surgery and chemoradiotherapy, patients only live to a median of 15 months. This may be due to extensive spatiotemporal, intratumoral heterogeneity observed. This is thought to be created by a small population of stem-like cells marked by expression of CD133. CD133 has been shown to correlate with poor patient prognosis, metastases, relapse and worse overall survival in GBM. We thus created an anti-CD133 CAR-T therapy. Our CD133-targeting CAR-T (CART133) has shown great efficacy and in our patient-derived models of GBM. It has also exhibited safety in sparing healthy CD133-expressing cells, particularly, hematopoietic stem and progenitor cells, in our humanized model of hematopoiesis. However, in looking to the clinic, the generation of autologous CAR-Ts may be difficult as it requires sufficient numbers of a patient's own T-cells who may be immunocompromised after first-line therapy. Thus, we propose to generate allogeneic CAR-Ts that target CD133 (AlloCART133) from healthy donor T- cells. AlloCAR-T cells are genetically-edited to abrogate the T-cell receptor (TCR) to avoid graft-versus- host disease, a phenomenon in which immune-mismatch causes donor tissues to attack recipient tissues. These cells can thus be safe for a recipient while also maintaining their anti-tumor activity. AlloCART133 thus presents a clinically-relevant, off-the-shelf therapy for patients with GBM.

Citation Format: Sabra K. Salim, Jiarun Wei, Vassil Dimitrov, Katherine Chen, Chitra Venugopal, Parvez Vora, Jason Moffat, Sheila K. Singh. Systematic Generation of Allogeneic Immune-targeting Modalities for Glioblastoma [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO081.
Abstract PR002: Highly parallel knock-in targeting for genome engineering of cellular therapies
Authors: Roth Theodore L. , Marson Alexander
Published: February 2021

Genetically-engineered immune cell therapies have proven effective to treat some types of cancer, but most tumors still cannot be cured. Forward genetics with CRISPR loss-of-function screens in T cells have identified some mutations to enhance cellular therapies, however these methods do not yet take full advantage of the power of gain-of-function knock-in targeting to engineer cell functions. We developed a robust new platform to assess the functional effects of pools of knock-in constructs in parallel. We used high-efficiency nonviral gene editing to introduce large panels of candidate therapeutic knock-in constructs into a defined genome position in human T cells allowing us to compete the cells against each other and test which constructs enhance T cell function. High-throughput pooled screening of targeted cells identified distinct library members that promoted T cell fitness under various resting, stimulated and immunosuppressive in vitro conditions. Direct competition among adoptively transferred human T cells in immunodeficient mice revealed a subset of constructs that promoted in vivo accumulation of tumor infiltrating lymphocytes. Pooled knock-ins combined with single-cell sequencing also revealed high-dimensional cellular phenotypes induced by each construct ex vivo and in an in vivo tumor microenvironment. Overall, these studies demonstrate the power of pooled knock-in technology to discover and functionally characterize complex synthetic gene programs that can be written into targeted genome sites to generate more effective cellular therapies.

This abstract is also being presented as PO073.

Citation Format: Theodore L. Roth, Alexander Marson. Highly parallel knock-in targeting for genome engineering of cellular therapies [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR002.
Abstract PR006: 27-Hydroxycholesterol acts on myeloid immune cells to induce T cell dysfunction, promoting breast cancer progression
Authors: Ma Liqian , Wang Lawrence , Nelson Adam T. , Han Chaeyeon , He Sisi , Henn Madeline A. , Menon Karan , Chen Joy J. , Baek Amy E. , Vardanyan Anna , Shahoei Sayyed Hamed , Park Sunghee , Shapiro David J. , Nanjappa Som G. , Nelson Erik R.
Published: February 2021

Breast cancer remains one of the leading causes of cancer mortality in the US. Elevated cholesterol is a major risk factor for breast cancer onset and recurrence, while cholesterol-lowering drugs, such as statins, are associated with a good prognosis. Previous work in murine models showed that cholesterol increases breast cancer metastasis, and the pro-metastatic effects of cholesterol were due to its primary metabolite, 27HC. In our prior work, myeloid cells were found to be required for the pro-metastatic effects of 27HC, but their precise contribution remains unclear. Here we report that 27HC impairs T cell expansion and cytotoxic function through its actions on myeloid cells, including macrophages, in an LXR-dependent manner. Many oxysterols and LXR ligands had similar effects on T cell expansion. Moreover, their ability to induce the LXR target gene ABCA1 was associated with their effectiveness in impairing T cell expansion. Interestingly, the enzyme responsible for the synthesis of 27HC, CYP27A1, is highly expressed in myeloid cells, suggesting that 27HC may have important autocrine or paracrine functions in these cells, a hypothesis supported by our finding that breast cancer metastasis was reduced in mice with a myeloid specific knockout of CYP27A1. Pharmacologic inhibition of CYP27A1 reduced metastatic growth and improved the efficacy of checkpoint inhibitor, anti-PD-L1. RNA sequencing of 27HC-treated macrophages and GSEA analysis provide further mechanistic insight, revealing an enrichment of MYC and NOTCH signaling pathways, both of which are documented pathways involved in tumor-associated macrophages. Taken together, our work suggests that targeting the CYP27A1 axis in myeloid cells may present therapeutic benefits and improve the response rate to immune therapies in breast cancer. Delineating the link between LXR and the known mechanisms of tumor-associated macrophages is important to fully understand 27HC-driven cancer metastasis. This work was supported by grants to ERN from the National Cancer Institute of the National Institutes of Health (R01CA234025) and METAvivor.

This abstract is also being presented as PO050.

Citation Format: Liqian Ma, Lawrence Wang, Adam T. Nelson, Chaeyeon Han, Sisi He, Madeline A. Henn, Karan Menon, Joy J. Chen, Amy E. Baek, Anna Vardanyan, Sayyed Hamed Shahoei, Sunghee Park, David J. Shapiro, Som G. Nanjappa, Erik R. Nelson. 27-Hydroxycholesterol acts on myeloid immune cells to induce T cell dysfunction, promoting breast cancer progression [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR006.
Abstract PR008: Armed Myxoma virus demonstrates therapeutic activity in pre-clinical xenograft models
Authors: Torres-Dominguez Lino E. , Franco Lina S. , Abrantes Mario , Walker Benjamin S. , Tacner Zachary , Kien Cassandra , Waters Anna K. , McFadden Grant , Potts Steven J. , Sharp Leslie L.
Published: February 2021

Oncolytic Viruses (OV) selectively replicate in and lyse tumor cells and provide stimulation to the immune system. This represents a promising therapeutic option for cancer patients that do not respond well to treatment with immune checkpoint inhibitors. Myxoma virus (MYXV) is a member of the Poxviridae family of double stranded DNA viruses. The natural host of MYXV is a subset of lagomorphs, but MYXV is able to infect cancer cell lines of humans and other species. The genome of MYXV is relatively large and is amenable to engineering for expression of transgenic proteins making it an excellent oncolytic virus for introduction of immunomodulatory proteins. The current work describes the oncolytic activity, transgene production capability, and in vivo activity of armed myxoma viruses in human cancer models.

This abstract is also being presented as PO090.

Citation Format: Lino E. Torres-Dominguez, Lina S. Franco, Mario Abrantes, Benjamin S. Walker, Zachary Tacner, Cassandra Kien, Anna K. Waters, Grant McFadden, Steven J. Potts, Leslie L. Sharp. Armed Myxoma virus demonstrates therapeutic activity in pre-clinical xenograft models [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR008.
Complex Soft Tissue Defects in Operative Urology: Pedicled Perforator Flaps of the Anterior Lateral Thigh for Tissue Reconstruction of the Lower Trunk
Authors: Jakubietz Rafael , Jakubietz Michael , Vater Adrian , Kocot Arkadius , Kübler Hubert , Kalogirou Charis
Published: February 2021

Background: Complex soft tissue defects involving the lower abdomen, perineum, and groin (LAPG) represent reconstructive challenges following urologic surgery. Consequently, these often require an interdisciplinary approach involving plastic surgery. While pedicled flaps from the lower abdomen are a reliable option, previous placement of colostomies and urinary urostomies renders these flap types unavailable. Here, the pedicled anterolateral thigh perforator (ALT) flap represents a reliable pedicled alternative which can harvest from the thigh. Materials and Methods: A retrospective data analysis of pedicled perforator flaps harvested from the thigh to reconstruct soft tissue defects was conducted. Seven patients treated in the urology department with soft tissue defects following tumor resection, infection, and dehiscence were included. Results: In all patients, the ALT flap was utilized successfully to reconstruct soft tissue defects up to 450 cm2 in size. All flaps survived. In 1 case, prolonged wound healing was observed. All defects were adequately addressed with no recurrence of dehiscence or fistula formation. Conclusions: Pedicled perforator flaps represent a valid option for the soft tissue reconstruction of the lower abdomen and perineum when a pedicled rectus abdominis flap is no longer available. This flap is, therefore, a good option in an interdisciplinary approach to soft tissue reconstruction, especially following urologic surgery, which is predominantly performed in the LAPG region.
Cortisol and Cortisone in Early Childhood in Very-Low-Birthweight Infants and Term-Born Infants
Authors: de Jong Miranda , Cranendonk Anneke , Twisk Jos W.R. , van Weissenbruch Mirjam M.
Published: February 2021

Introduction: Besides programming of the hypothalamic-pituitary-adrenal (HPA) axis, changes in the activity of 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) could contribute to the later metabolic and cardiovascular consequences of preterm birth. Objective: We compared serum cortisol, cortisone, and cortisol/cortisone ratio in early childhood in very-low-birthweight (VLBW) infants and term appropriate for gestational age (AGA) born infants. Methods: We included 41 VLBW infants, participating in the randomized controlled Neonatal Insulin Replacement Therapy in Europe trial, and 64 term AGA-born infants. Cortisol and cortisone were measured in blood samples taken at 6 months and 2 years corrected age (VLBW children) and at 3 months and 1 and 2 years (term children). At 2 years of (corrected) age (HDL) cholesterol, triglycerides, glucose, and insulin were also measured. Results: During the first 2 years of life, cortisol/cortisone ratio is higher in VLBW children compared to term children. In the total group of children, cortisol/cortisone ratio is positively related to triglycerides at 2 years of (corrected) age. In VLBW children, over the first 2 years of life both cortisol and cortisone are higher in the early-insulin group compared to the standard care group. Conclusions: In VLBW infants, lower 11β-HSD2 activity probably contributes to the long-term metabolic and cardiovascular risks. In VLBW infants, early insulin treatment could affect programming of the HPA axis, resulting in higher cortisol and cortisone levels during early childhood.
The Application of Fine Needle Aspiration Biopsy in the Diagnosis of Axillary Masses
Authors: Huang Cong-Gai , Li Meng-Ze , Wang Shao-Hua , Tang Xiao-Qin , Haybaeck Johannes , Yang Zhi-Hui
Published: February 2021

Introduction: We intend to determine the diagnostic power of fine needle aspiration biopsy (FNAB) for differentiation between malignant and benign lesions on axillary masses and draw the physicians’ attention to the benefits of FNAB cytology in the diagnosis of axillary masses. Methods: In this study, 1,328 patients with an axillary mass diagnosed by FNAB were retrospectively reviewed. These cases were registered at the affiliated hospital of Southwest Medical University (China), July 2014 to June 2017. Cytological results were verified either by histopathology following surgical resection or clinical follow-up. Results: Of the 1,328 patients affected by axillary masses, 987 (74.3%) cases were female, and 341 (25.7%) cases were male. The highest incidence of patients was in the age group of 41–50 years (375, 28.2%). There were 1,129 (85.0%) patients with benign lesions and 199 (15.0%) with malignant lesions. Of the 199 malignant lesions cases, 21 cases were lymphomas, 2 cases were accessory breast cancers, and 176 cases were lymph node metastatic tumors. Under lymph node metastases, the most frequent primary tumors were breast cancer (141, 80.1%), followed by lung cancer (21, 11.9%). According to the study, the characters of 1,328 cases showed statistically significant difference (χ2 = 4.534, p = 0.033), and the incidence of females with axillary mass was significantly higher than that of males. There was a statistically significant difference in the distribution of benign and malignant cases in the patient age groups (χ2 = 1.129, p = 0.000), and the incidence of patients of 41–50 years of age was significantly higher than that of other patients. The diagnostic accuracy of FNAB in axillary masses was analyzed with the results of 95.98% of sensitivity, 99.56% of specificity, 97.45% of positive predictive value, and 99.29% of negative predictive value. Conclusion: Our results confirm that FNAB is a valuable initial screening method regarding pathologic diagnosis of axillary mass, in particular with respect to malignancy in 41- to 50-year-old female patients.
The Ordination Prayers in the So-Called Apostolic Tradition
Authors: Bradshaw Paul F.
Published: February 2021

The anonymous church order formerly identified as the Apostolic Tradition and attributed to Hippolytus is now regarded by many scholars as a composite work made up of layers of redaction from around the mid-second to mid-fourth centuries. This essay revises the unsatisfactory attempt to discern such strata in its ordination prayers that was made by Eric Segelberg as long ago as 1975, and argues that their earliest forms are among the oldest material in the so-called Apostolic Tradition, belonging to the first half of the second century.
Who Should Do What for the Wild Animals We Abandoned?
Authors: Fischer Bob
Published: February 2021

When COVID-19 struck, tourists stopped visiting sites where they formally fed animals. As a result, the animals went hungry, with some starving to death. I argue, however, that this doesn’t show that it’s wrong to create such dependency: had we been willing to intervene on behalf of wild animals, there wouldn’t have been any moral issue. Moreover, I argue that we can identify the individuals who most plausibly have some responsibilities to help animals in crisis situations – namely, those who are bound up in caring relationships with those animals. At the same time, though, I don’t think it’s obvious how they should help, and I think there is a serious case to be made for not distinguishing between wild and domestic animals in this context. Given that, euthanization becomes an option that needs to be taken seriously.
Characterization of Anti-Pollution Films by Thickness for Photovoltaic Modules That Can Be Applied in the Field
Authors: Lee Changyeon , Kim Jung Hyun , Park Young , Joung Yeon-Ho , Choi Wonseok
Published: February 2021

In this study, the characteristics of functional coating films were investigated to improve the anti-pollution properties and efficiency of PV (Photovoltaic) module according to the variation of coating film thickness. Functional coating was applied of a glass substrate, which was composed
with the same material as PV module. Brush coating method was used for the coating process. We coated the functional film on the glass substrate 1, 2 and 3 times alternately in the horizontal and vertical directions to change the film thickness, and a hot air fan equipment was used as a heat
treatment process for easy application to existing PV modules. The heat treatment process was performed for 2 minutes after the coating process using a hot air fan equipment. After coating and heat treatment, glass substrates were analyzed for the anti-pollution properties, contact angle,
optical properties and the mechanical properties such as hardness and adhesion. The anti-pollution properties were identified through self-cleaning test. The contact angle of the functional coating film was measured by a contact angle analyzer, and the optical properties were measured via
UV-visible spectroscopy, which can be used as an integrating sphere. The hardness of the functional film was measured by the standard hardness testing method using H-9H, F, HB and B-6B pencil. Also, the adhesion of the functional film was measured by the standard adhesion testing method.