bluuebunny/crossref_metadata_2025_split huggingface dataset:
Influence of DNA damage repair defects on tumor immunogenicity
Authors: Samstein Robert 
Published: January 2023

Abstract
In recent years, immune checkpoint inhibitors (ICI) have dramatically altered the treatment landscape for patients with advanced stage and metastatic cancers. Durable benefit, however, is limited to subsets of histologies and patients, highlighting the importance of identifying predictive biomarkers to select for patients who may benefit. DNA damage repair and response (DDR) deficiency including mismatch repair deficiency and resulting increased tumor neoantigens have been associated with improved response. We hypothesize that a similar concept may extend to other defects in DDR pathways, the most common being homologous recombination (HR). In ongoing work, we have implicated mutations in the DDR pathways including the HR pathway with improved survival after ICI in a large retrospective cohort. However, divergent responses were noted depending on the individual HR gene mutated. Syngeneic murine models were used to study individual DDR genes using CRISPR-Cas9 mutagenesis and their role in altering the tumor microenvironment and response to immune checkpoint blockade in isogenic model systems. We report differential effects of mutations in genes of the homologous recombination (HR) pathway on response after ICI administration in mouse and human tumors, and show that truncating mutations in BRCA2 are associated with superior response to ICI compared to BRCA1-deficient tumors. Single cell RNA-sequencing of CD45+ cells sorted from tumors reveals distinct immune landscapes. These data have significant implications for elucidating the genetic and microenvironmental determinants of response to immunotherapy in the setting of DDR deficiency and ultimately expand the therapeutic window of immunotherapy.
Metastatic colorectal cancer cells harness nonsense-mediated mRNA decay for immune evasion
Authors: Wu Xingxin , Tan Tao , Xu Qiang 
Published: January 2023

Abstract
Immunosuppression microenvironment allows primary tumor growth, while tumor-inherent factors that bring about immune evasion during metastasis remain elusive. Here, we observed a stronger nonsense-mediated mRNA decay (NMD) activity with a higher expression of up-frameshift protein 1 (UPF1) in colorectal cancer (CRC) metastasis than in matched primary cancer cells. In metastatic CRC SW620 cells, the higher UPF1 expression was found to arise from the increased stability of UPF1 by ubiquitin specific peptidase 10 (USP10)-mediated deubiquitination. In contrast, in primary CRC SW480 cells, autophagy promotes proteolysis of ubiquitinated UPF1 and inhibits NMD activity. Interestingly, in SW620 cells NMD selectively controlled a cluster of immune-related genes including ICAM1. Furthermore, NMD inhibition exposed SW620 cells but not SW480 cells to natural killer (NK) cell-mediated cytolysis that depended on ICAM1.

Conclusion
NMD activity can be recommissioned in CRC metastasis cells to escape from NK-dependent immune surveillance, and inhibiting NMD to boost anti-tumor immunity could be a novel strategy to treat CRC metastasis.
Isolation of Tumor-Infiltrating Leukocytes from Mouse Tumors
Authors: Poon Grace FT , Antignano Frann , Boyd Lyz , Ennis Siobhan , Deng Joe , Kokaji Andy I , Woodside Steven M , Eaves Allen C , Louis Sharon A 
Published: January 2023

Abstract
Cell-based immunotherapy is being evaluated in various types of cancer and it is one of the most rapidly growing and promising areas of cancer research. Tumor-infiltrating leukocytes (TILs) consist of highly diverse leukocyte subsets with major roles in cancer immune surveillance. Due to their relatively low frequency, tumor heterogeneity, and the abundance of tissue debris in tumor samples, it is difficult to isolate or analyze TILs with sensitivity and precision. To address this challenge, we have developed a simple method for isolating CD45+ TILs from mouse tumors. Performance was evaluated in three commonly used mouse models, namely the B16 melanoma, CT26 colon carcinoma, and 4T1 mammary tumor models. Solid tumors were induced by subcutaneous implantation of B16, CT26.WT, and 4T1 cancer cell lines into syngeneic recipients. Starting with a single-cell suspension, TILs from tumor samples were labeled with an antibody complex that links CD45+ cells to magnetic particles, then separated using an EasySep™ magnet. Using this method, TILs were enriched from 17.5 +/− 5.8% to 90.1 +/− 6.0% (n = 13) from B16 tumors, 27.9 +/− 9.4% to 74.2 +/− 12.3 % (n = 9) from CT26 tumors, and 39.5 +/− 8.0% to 87.5 +/− 3.7% (n = 6) from 4T1 tumors. The protocol can be easily modified to achieve higher purity or recovery as required by adjusting the addition volume of the antibody complex or particles. Importantly, major immune subsets including T cells, B cells, and myeloid cells are recovered after isolation. The EasySep™ Mouse CD45 TIL Isolation Kit allows researchers to isolate leukocytes from tumors with ease, improving the TIL downstream workflow. Furthering our understanding of TILs will be essential for developing effective immunotherapeutic strategies.
Modulating the cross-talk between the immune and nervous systems to treat rectal cancer
Authors: Uccello Taylor Paige , Mills Bradley N. , Repasky Elizabeth A. , Lord Edith M. , Linehan David C. , Gerber Scott A. 
Published: January 2023

Abstract
Cancer patients with perineural invasion (PNI) have reduced overall survival, enhanced rates of local recurrence, and increased metastasis. Nerves secrete catecholamines (CA) that bind beta 2 adrenergic receptors (B2AR) on various cells, including immune cells. Signaling through this neuronal-immune axis drives immune cells towards a suppressive phenotype. Therefore, the sympathetic peripheral nervous system is considered to be a source of immunosuppression in the tumor microenvironment (TME). The impact that various therapies, such as radiation therapy (RT), have on innervation and CA secretion is unknown. We hypothesize that proinflammatory cytokines induced by RT signal nerves to secrete CAs that propagate tumor growth. We have shown via qPCR and flow cytometry that tumor cell lines exposed to RT in vitro upregulate B2AR transcription and protein level, respectively. Consequently, tumor lines exposed to B2AR agonists are more radioresistant by colony forming assays. To further explore the role of the nervous system in enhancing tumor growth, we have developed an orthotopic model of rectal cancer. Using this model, we visualized PNI by immunohistochemistry. We also detected positive B2AR expression on immune, tumor, and stromal cells excised from the TME. Upon treating mice bearing orthotopic tumors with a B2AR agonist, we observed increased metastatic burden, accompanied by reduced overall survival compared to untreated controls. Moving forward, we will inhibit immunosuppressive neuronal signaling in the TME in combination with RT. Our goal is to elucidate the immunosuppressive role of the nervous system in rectal cancer and use this information to identify a novel RT adjuvant that potentiates a robust antitumor response.
The changes of immunologic properties and their prognostic implications: the paired analysis of initial and recurrent head and neck squamous-cell carcinoma
Authors: So Yoon Kyoung , Cheong Taek Yoon , Byeon Sun-ju , Chung Man Ki 
Published: January 2023

Abstract

Purpose
The immunologic properties of tumors can change during the clinical course. We aimed to compare the expression of PD-L1 and the infiltration of CD8+ tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment between initial and recurrent head and neck squamous-cell carcinomas (HNSCCs). We also evaluated whether the changes of those immunologic properties in recurrent HNSCCs affect the oncologic outcome.

Methods
We included 42 patients who had been treated for both initial and recurrent HNSCCs. Pathologic specimens from initial and salvage surgery were obtained for each patient, and IHC staining of CD3, CD8, PD-1, and PD-L1 were done. Also, neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) were collected. The change of each immunologic profile was expressed as the recurrent-to-initial ratio (R/I ratio).

Results
The change of CD3+, CD8+, PD-1, and PD-L1 positive cells varied widely. For more than half the patients, those values decreased in recurrent tumors. The median R/I ratio was 0.5 for CD3+ TILs, 0.6 for CD8+ TILs, 0.5 for PD-1 and 0.4 for PD-L1. In contrast, NLR and PLR increased in recurrent tumors for more than half the patients. The median R/I ratio of NLR and PLR was 1.6 and 1.4, respectively. In multivariate analysis, increased CD8 (R/I ratio &gt;1) was the independent prognostic factor for better OS (hazard ratio 0.228; 95% CI 0.067 – 0.777; p= 0.018).

Conclusion
The change of immunologic properties along with the recurrence of HNSCC varied widely from patient to patient. Generally, the intratumoral biomarkers decreased, while the systemic inflammatory markers increased. The increased CD8+ TILs in recurrent HNSCCs was the significant prognostic factor for better overall survival.
The role of osteosarcoma cell-derived exosomes in the promotion of lung metastasis
Authors: Mathias Alissa B , Palmer Eric P , Kruh-Garcia Nicole , Regan Daniel P 
Published: January 2023

Abstract
Osteosarcoma (OS) is the most common primary tumor of bone and typically occurs in children and young adults. 30–40% of OS patients develop lung metastases after initial diagnosis, with only 20% of these patients surviving 4 years post-relapse. For over 30 years, there have been no improvements in the treatment of OS, and more importantly, no diagnostic tools that help identify those patients at high risk for lung metastases. Recent data has shown that in other tumor types, tumor-secreted extracellular vesicles (EVs) deliver biological cargo to distant sites, resulting in priming of non-malignant host cells for promotion of metastasis. The aim of this project is to understand how OS EVs influence the response of lung fibroblasts (LFs) and alveolar macrophages (AMs), and if their uptake of EVs results in pre-metastatic priming that can be detected via AM transcriptomic changes.

Hypothesis
LFs are primary targets of OS EVs, which will increase LF IL-6 production and cause paracrine STAT3 activation of AM’s into an M2 tumor-promoting phenotype.

Results
Flow cytometry and confocal microscopy demonstrated in vitro uptake of OS EVs by LF cells. In vitro ‘education’ of LFs with OS EVs induced significant production of IL-6, IL-8, and CCL2, with the greatest mean difference between EV-educated and naïve LFs observed with IL-6 secretion.

Conclusion
This data suggest that OS EVs efficiently prime LFs to induce IL-6 secretion, and this response may be associated with promotion of OS lung metastasis. Further data is being collected to determine the role of IL-6 in OS cell survival and lung metastatic colonization. RNA sequencing of AMs will be used to characterize phenotype which could serve as an effective cellular biomarker to predict lung metastasis.
Characterization of the genomic and immunological diversity of malignant human brain tumors through multi-sector analysis
Authors: Schaettler Max , Richters Megan , Griffith Malachi , Dunn Gavin 
Published: January 2023

Abstract
While the degree of spatial genomic heterogeneity within primary glioblastoma (GBM) has been previously investigated, the extent of this heterogeneity in other malignant brain tumors and its connection to the local immune microenvironment remains unknown. To address this, we performed whole-exome, RNA, and TCR sequencing on multiple spatially distinct sectors from a cohort of malignant brain tumors comprised of both brain metastases and primary and recurrent GBMs. Our results suggest a striking difference in which a majority of mutations and predicted neoantigens are shared between spatially distinct regions of metastatic brain tumors in contrast to the spatial heterogeneity observed within both primary and recurrent GBMs. Additionally, despite substantial differences in immunotherapy responsiveness between brain metastases and GBM, we can detect significantly expanded T-cell clonotypes within both tumor types with some clonal frequencies exceeding 10% of intratumoral T-cells. Similar to the observed distribution of variants and neoantigens, the expanded clonotypes are more shared among spatially distinct sectors in metastatic brain tumors than in primary or recurrent GBMs. Interestingly, the frequencies of most of these enriched T-cell clones are significantly diminished within expanded tumor infiltrating lymphocyte (TIL) cultures, advising caution in the use of expanded TIL for the detection of neoantigen-specific responses. These results provide novel insight into the immunogenomic landscape of malignant brain tumors with implications for our understanding of tumor-immune interactions and the development of immunotherapies.
Neoantigens derived from differential mutations of invasive ductal carcinomas with axillary lymph node metastasis
Authors: Bagriacik Emin Umit , Ermis Ilker , Ekinci Ozgur , Girgin Cansu , Oruklu Nihan , Bayram Orhan 
Published: January 2023

Abstract
Invasive ductal carcinomas (IDCs) are the most common breast cancer type. It was reported that almost 80% of breast cancers are IDCs. IDCs can be treated by immunotherapy using neoantigens. Therefore, common neoantigens that are specific for ductal carcinomas should be determined. We aimed to determine differential expression of neoantigens for IDCs in the presence or absence of axillary lymph node metastasis. DNA was isolated from paraffin-embedded (FFPE) tissue specimens by using GeneRead DNA FFPE Kit. QIAseq Targeted DNA panel for human breast cancer was used for sequencing of isolated tumor DNA. Next generation sequencing was performed using a Illumina MiSeq. Total exons of 93 genes were studied. A bioinformatic analysis was conducted by a platform-specific pipeline software (Qiagen Clinical Insight-AnalyseTM and Clinical Insight-InterpretTM). We found significant variations in several genes for individuals with or without axillary lymph node metastasis. In particular, variations in CDKN2A and PIK3R1genes were quite interesting to determine individual neoantigens of ductal carcinomas as potential immunotherapy targets. Currently, these findings are still under further investigations. Based on these findings, we concluded that NGS analysis is a very powerful technology in determination of specific neoantigens of individuals with ductal carcinomas. Also NGS can be used to discover new genes related to axillary lymph node metastasis for ductal carcinomas. Supported by Gazi University BAP.
Immune profiling of the tumor microenvironment (TME) using multiplexed ion beam imaging (MIBI)
Authors: Ptacek Jason , Johnson Robert W. , Palma Joann P , Tarolli Jay G. , Sigal Yari , Finck Rachel , Aksoy Murat , Zhang Yi , Finn Jessica 
Published: January 2023

Abstract
Understanding the cell types present in the tumor microenvironment and their activation state is at the forefront of immunotherapy research. To address this, MIBI has been developed to simultaneously image more than 40 markers at single cell resolution. Staining of 10 NSCLC formalin-fixed paraffin embedded tissue sections was performed with a panel of 20 metal labeled antibodies stained together. The tissue was imaged at subcellular resolution using an ion beam and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Masses of detected species were assigned to target biomolecules given the unique label of each antibody and multi-step processing and segmentation were performed to create images of the TME. Each tumor sample was imaged across 10 regions of interest (ROIs) to assess heterogeneity of the TME. The resulting single cell segmentation enabled quantitative analyses of both marker expression and the spatial relationships between cells of different types. At the highest level, cells were classified as immune (CD45) and tumor cells (keratin) based on measured intensities of marker expression. Co-expression of markers were used to classify B cells, macrophages, and T cells subsets. Markers associated with immune suppression such as PD-L1 were also quantified. Cell types and their frequency were compared within the 10 ROIs collected per sample as well as between samples. Finally, distances between tumor and the closest immune cell were measured as a means for describing the spatial organization of the TME, which has been linked to patient survival. In conclusion, MIBI offers high-parameter capability, at a sensitivity and resolution uniquely suited to understanding the complex tumor immune landscape.
A novel genetic screen to identify genes that enhance T cell trafficking into solid tumors
Authors: Rogers Laura M , Dupuy Adam , Weiner George 
Published: January 2023

Abstract
T cell mediated cancer immunotherapies, including anti-PD-1 and CAR-T, are becoming standard of care for a variety of cancer types. However, patients with tumors that lack T cell infiltration generally do not respond well to immunotherapy. One potential strategy to overcome immunotherapy resistance is to enhance the ability of T cells to traffic into the tumor. The objective of this study was to develop an in vivo genetic screen in endogenous T cells using the Sleeping Beauty (SB) mutagenesis system to identify candidate genes that drive intratumoral T cell accumulation.
Mutations in one candidate gene, Aak1, were identified in intratumoral T cells of multiple tumor models, including those treated with anti-PD-1. Insertions were highly clustered, suggesting that a gain-of-function Aak1 mutation may be favorable for driving T cells into the tumor. This gain-of-function is likely dominant-negative, as inhibition of Aak1 kinase activity enhanced intratumoral T cell infiltration. Surprisingly, inhibition of Aak1 kinase activity also abolished the therapeutic effect of anti-PD-1 in mouse models of lymphoma and melanoma.
Our data demonstrates that our SB mutagenesis approach can be used to identify target genes that could be therapeutically modified to enhance T cell infiltration into tumors. Our innovative method can be adapted to other immune cell types. At least one candidate gene we identified (Aak1) deserves further evaluation as a potential therapeutic target, though a better understanding of the antagonistic relationship between anti-PD-1 action and Aak1 kinase activity is needed. Immediate next steps also include assessing the impact of candidate gene modification in CAR-T cells on their ability to traffic into solid tumors.
High parameter immune cell quantification in a melanoma-derived tumor model by Chipcytometry
Authors: Gamber Kevin , Detmers Jan , Schupp Jonathan , Tuttenberg Andrea , Christians Arne 
Published: January 2023

Abstract
Immune evasion is a hallmark of cancer and the presence and interaction of immune cells within a tumor and its microenvironment can have a great impact on disease progression and prognosis. Unravelling the complex interplay of neoplastic and immune cells requires both phenotypic and spatial analysis of each involved cell population in the tissue.
The aim of this study is to compare 3 methods of biomarker analysis in tissues, flow cytometry, immunohistochemistry (IHC) and Chipcytometry, in their ability to detect immune cells in a tumor model.
NOD-Scid tg HLA-A2.1 mice were implanted with human Ma-Mel-19 melanoma cells. After 21 days, the mice were injected with human PBMC at the tumor site. Local treatment with Tasquinimod was performed three times once a week. Tumors, including the neighbouring PBMC injection site were excised on day 48 and immune cell content was analysed by Chipcytometry, flow cytometry and IHC. All three methods were successful in detecting human immune cells in the samples consisting mostly of T-cells (CD45+CD3+). However, flow cytometry and IHC were unable to provide either accurate localization or phenotyping data, respectively. With Chipcytometry we were able to specifically quantify tumor-infiltrating immune cells. In treated samples immune cell count (110/mm^2) and composition (72% T-cells) was significantly higher than in the untreated samples (54/mm^2, 49% T-cells). We show that Chipcytometry is able to produce “flow-like” quantitative data, while also retaining spatial information. This makes Chipcytometry a powerful tool for the study of immunological processes in tumors.
High-throughput screening for rare antigen-reactive TCRs using natively-paired TCRαβ expression libraries generated from millions diverse primary T cells
Authors: Spindler Matthew J , Nelson Ayla L , Heather James M , Wagner Ellen K , Adler Adam S , Johnson David S 
Published: January 2023

Abstract
A major goal of T cell research is to profile the repertoire of antigen-reactive TCRs targeting specific peptide:MHCs (pMHC). Single cell sorting of pMHC-binding T cells using multimers and ex vivo antigen expansion are the current gold standards for identifying antigen-reactive primary T cells. However, functional validation of the TCRs identified by these approaches requires resource intensive cloning of each individual TCRαβ pair. Additionally, primary T cells, especially tumor infiltrating lymphocytes (TILs), are a limited resource, which restricts the number of antigens that can be screened.
To address this, we developed a microfluidic approach to capture and functionally express natively-paired TCRαβ libraries from millions of single T cells. Unlike DNA barcoding approaches that mark single cells by adding a sequence tag, we physically link the TCRα-TCRβ chains to generate sequencing and full-length expression libraries which we introduce into Jurkat cells for functional testing.
Using these methods, we captured over 2.9 million TCRαβ clonotypes from six healthy PBMC donors and over 0.5 million from expanded melanoma TIL samples. We applied pMHC binding and cellular activation screens to identify and validate 14 TCRs reactive to common viral and tumor associated antigens with starting library frequencies of &lt;0.0001 – 0.3% and functional avidities ranging from 0.3 – 592nM. We found that including the cellular activation screen reduced our false-positive TCR discovery rate from 65% for pMHC binding alone to 18%. We are further developing these cellular activation enrichment approaches and applying them to broadly profile anti-tumor TCR reactivity from TILs.