Tumor Cell RNA Research Articles

Activating Two-Photon Silica Nanoamplifier-Based CHA and FRET for Accurate Ratiometric Bioimaging of Intracellular MicroRNA.

In situ visualization of microRNA (miRNA) in cancer cells and diseased tissues is essential for advancing our comprehension of the onset and progression of associated diseases. Two-photon (TP) imaging, as an imaging technology with high spatiotemporal resolution, deep tissue penetration, and accurate target quantification, has distinctive advantages over single-photon imaging and has attracted increasing attention. Extensive research has been conducted on two-photon dye-doped silica nanoparticles, which exhibit a large two-photon absorption (TPA) cross-section, high fluorescence quantum yield, and excellent biocompatibility. However, the low abundance of RNA in tumor cells leads to insufficient signal output. Based on functional nucleic acid, a catalyzed hairpin self-assembly (CHA) signal amplification strategy, which has simplicity, robustness, and nonenzymatic characteristics, can achieve the amplification of DNA or RNA signals. Here, a two-photon silica nanoamplifier (TP-SNA) utilizing TP dye-doped silica nanoparticles (SiNPs) and functional nucleic acid was constructed, employing triggering catalyzed hairpin self-assembly and fluorescence resonance energy transfer (FRET) for highly sensitive detection and precise TP imaging of endogenous miRNAs in tumor cells and tissues at varying depths. The TP-SNA demonstrated the capability to detect miR-203 with a detection limit of 33 pM. The maximum two-photon tissue penetration depth of the two-photon nanoamplifier was 210 μm. The two-photon nanoamplifier developed in this study makes full use of the advantages of accurate TP ratiometric bioimaging and the CHA signal amplification strategy, which shows good application value for future transformation into clinical diagnosis.

Non-Coding RNA in Tumor Cells and Tumor-Associated Myeloid Cells-Function and Therapeutic Potential.

The RNA world is wide, and besides mRNA, there is a variety of other RNA types, such as non-coding (nc)RNAs, which harbor various intracellular regulatory functions. This review focuses on small interfering (si)RNA and micro (mi)RNA, which form a complex network regulating mRNA translation and, consequently, gene expression. In fact, these RNAs are critically involved in the function and phenotype of all cells in the human body, including malignant cells. In cancer, the two main targets for therapy are dysregulated cancer cells and dysfunctional immune cells. To exploit the potential of mi- or siRNA therapeutics in cancer therapy, a profound understanding of the regulatory mechanisms of RNAs and following targeted intervention is needed to re-program cancer cells and immune cell functions in vivo. The first part focuses on the function of less well-known RNAs, including siRNA and miRNA, and presents RNA-based technologies. In the second part, the therapeutic potential of these technologies in treating cancer is discussed, with particular attention on manipulating tumor-associated immune cells, especially tumor-associated myeloid cells.

Development of Cell Technologies Based on Dendritic Cells for Immunotherapy of Oncological Diseases.

Immunotherapy using dendritic cell-based vaccination is a natural approach using the capabilities and functions inherent in the patient's immune system to eliminate tumor cells. The development of dendritic cell-based cell technologies evolved as the disorders of dendritic cell differentiation and function in cancer were studied; some of these functions are antigen presentation, priming of cytotoxic T-lymphocytes and induction of antigen-specific immune responses. At the initial stage of technology development, it was necessary to develop protocols for the in vitro generation of functionally mature dendritic cells that were capable of capturing tumor antigens and processing and presenting them in complex with MHC to T-lymphocytes. To achieve this, various forms of tumor-associated antigen delivery systems were tested, including lysates, tumor cell proteins (peptides), and DNA and RNA constructs, and it was shown that the use of DNA and RNA constructs was the most effective method, as it made it possible not only to deliver the most immunogenic epitopes of tumor-associated antigens to dendritic cells, but also to enhance their ability to induce antigen-specific cytotoxic T-lymphocytes. Currently, cell therapy based on dendritic cells is a modern basis for antigen-specific immunotherapy of cancer due to the simplicity of creating DNA and RNA constructs encoding information about both target tumor antigens and regulatory molecules. The potential development of cell technologies based on dendritic cells aims to obtain antigen-specific cytotoxic T-lymphocytes induced by dendritic cells, study their functional activity and develop cell-based therapy.

Deep neural network based tissue deconvolution of circulating tumor cell RNA

Prior research has shown that the deconvolution of cell-free RNA can uncover the tissue origin. The conventional deconvolution approaches rely on constructing a reference tissue-specific gene panel, which cannot capture the inherent variation present in actual data. To address this, we have developed a novel method that utilizes a neural network framework to leverage the entire training dataset. Our approach involved training a model that incorporated 15 distinct tissue types. Through one semi-independent and two complete independent validations, including deconvolution using a semi in silico dataset, deconvolution with a custom normal tissue mixture RNA-seq data, and deconvolution of longitudinal circulating tumor cell RNA-seq (ctcRNA) data from a cancer patient with metastatic tumors, we demonstrate the efficacy and advantages of the deep-learning approach which were exerted by effectively capturing the inherent variability present in the dataset, thus leading to enhanced accuracy. Sensitivity analyses reveal that neural network models are less susceptible to the presence of missing data, making them more suitable for real-world applications. Moreover, by leveraging the concept of organotropism, we applied our approach to trace the migration of circulating tumor cell-derived RNA (ctcRNA) in a cancer patient with metastatic tumors, thereby highlighting the potential clinical significance of early detection of cancer metastasis.

Zwitterionic microgel preservation platform for circulating tumor cells in whole blood specimen

The immediate processing of whole blood specimen is required in circulating tumor cell-based liquid biopsy. Reliable blood specimen stabilization towards preserving circulating tumor cells can enable more extensive geographic sharing for precise rare-cell technology, but remains challenging due to the fragility and rarity of circulating tumor cells. Herein, we establish a zwitterionic magnetic microgel platform to stabilize whole blood specimen for long-term hypothermic preservation of model circulating tumor cells. We show in a cohort study of 20 cancer patients that blood samples can be preserved for up to 7 days without compromising circulating tumor cell viability and RNA integrity, thereby doubling the viable preservation duration. We demonstrate that the 7-day microgel-preserved blood specimen is able to reliably detect cancer-specific transcripts, similar to fresh blood specimens, while there are up/down expression regulation of 1243 genes in model circulating tumor cells that are preserved by commercial protectant. Mechanistically, we find that the zwitterionic microgel assembly counters the cold-induced excessive reactive oxygen species and platelet activation, as well as extracellular matrix loss-induced cell anoikis, to prevent circulating tumor cell loss in the whole blood sample. The present work could prove useful for the development of blood-based noninvasive diagnostics.

Effects of gene expression in 5-FU metabolic pathways in a phase III trial evaluating adjuvant S-1 therapy compared to surgery alone following curative resection for biliary tract cancer (JCOG1202A1).

732 Background: S-1, an oral fluoropyrimidine derivative, is standard adjuvant therapy in Japanese patients with resected biliary tract cancer (BTC), based on the results of JCOG1202, a phase III trial evaluating adjuvant S-1 following curative resection for BTC compared to surgery alone. The efficacy of 5-FU-based therapy is related to the expression of enzymes involved in 5-FU metabolic pathways, such as thymidylate synthase (TS), thymidine phosphorylase (TP), orotate phosphoribosyltransferase (OPRT) and dihydropyrimidine dehydrogenase (DPD). The aim of this study was to evaluate the impact of messenger RNA (mRNA) levels of the four 5-FU metabolic pathway genes on the outcomes of patients enrolled in JCOG1202. Methods: Tumor cell RNA was isolated from formalin-fixed paraffin-embedded primary BTC specimens resected in 264 of 440 randomized patients in JCOG1202. The four 5-FU metabolic pathway genes were measured in 183 patients (surgery alone: n = 94; adjuvant S-1: n = 89) who were randomly divided to training (n = 96) or validation set (n = 87). The endpoints of interest were the predictive values of the four genes for the efficacy of adjuvant S-1 on overall survival and relapse-free survival (RFS), and we here report the results regarding RFS. Cut-off levels for mRNA expression were selected in the training set which minimized the bootstrap p-values (2,000 samples) of an interaction term of treatment (surgery alone or S-1) and mRNA expression in a Cox regression model. Results: There were no obvious differences in each mRNA level and clinical characteristics between surgery alone and adjuvant S-1 groups. RFS tended to be better with adjuvant S-1 (hazard ratio [HR] = 0.790, [95% confidence interval: 0.524-1.192]) compared to surgery alone, which was maintained in the low DPD population (HR = 0.440 [0.216-0.898] in training set and 0.748 [0.334-1.675] in validation set), in the low TP population (HR = 0.709 [0.388-1.296] and 0.602 [0.287-1.262]), and in the high OPRT population (HR = 0.520 [0.152-1.779] and 0.609 [0.161-2.304]). Conclusions: The efficacy of adjuvant S-1 on RFS was representative in BTC patients with intratumoral gene expressions of low DPD, low TP, and high OPRT, which was disappeared in the population with high DPD, high TP, and low OPRT. These biomarkers might be useful to predict therapeutic benefits with S-1 containing chemotherapy for BTC.

Discovery of antitumor effects of leczymes

Sialic-acid binding lectin from bullfrog (Rana catesbeiana) eggs, cSBL, is a cytotoxic ribonuclease (RNase) belonging to the RNase A superfamily. cSBL is cytotoxic to tumor cells, such as malignant pleural mesothelioma by inducing apoptotic cell death caused by the degradation of RNA in tumor cells. In addition, we have reported some data that cSBL could be involved in the endoplasmic reticulum stress pathway, and it was also assumed to cause apoptotic cell death. The most significant property of cSBL is its specificity toward malignant cells. Furthermore, since the antitumor activity of cSBL was confirmed by in vivo experiments using mouse xenograft models, it is expected to be a candidate for clinical chemotherapy. Here, we summarize the history of cSBL, alternatively called "leczyme," with its present and future.

Predictive Immune-Checkpoint Blockade Classifiers Identify Tumors Responding to Inhibition of PD-1 and/or CTLA-4.

Combining anti-PD-1 + anti-CTLA-4 immune-checkpoint blockade (ICB) shows improved patient benefit, but it is associated with severe immune-related adverse events and exceedingly high cost. Therefore, there is a dire need to predict which patients respond to monotherapy and which require combination ICB treatment. In patient-derived melanoma xenografts (PDX), human tumor microenvironment (TME) cells were swiftly replaced by murine cells upon transplantation. Using our XenofilteR deconvolution algorithm we curated human tumor cell RNA reads, which were subsequently subtracted in silico from bulk (tumor cell + TME) patients' melanoma RNA. This produced a purely tumor cell-intrinsic signature ("InTumor") and a signature comprising tumor cell-extrinsic RNA reads ("ExTumor"). We show that whereas the InTumor signature predicts response to anti-PD-1, the ExTumor predicts anti-CTLA-4 benefit. In PDX, InTumorLO, but not InTumorHI, tumors are effectively eliminated by cytotoxic T cells. When used in conjunction, the InTumor and ExTumor signatures identify not only patients who have a substantially higher chance of responding to combination treatment than to either monotherapy, but also those who are likely to benefit little from anti-CTLA-4 on top of anti-PD-1. These signatures may be exploited to distinguish melanoma patients who need combination ICB blockade from those who likely benefit from either monotherapy.

PLASMA EPSTEIN-BARR VIRUS (EBV) DNA AS A BIOMARKER FOR DIAGNOSIS OF SYRIAN EBV-POSITIVE BURKITT’S LYMPHOMA

Epstein-Barr virus - positive Burkitt’s Lymphoma is defined as the presence of Epstein-Barr virus (EBV) in tumor cells, the standard way to detect (EBV) in Burkitt’s Lymphoma is in-situ hybridization (ISH) of EBV-encoded small RNA (EBERs) in tumor cells. The present study aimed to evaluate plasma Epstein-Barr virus (EBV) DNA as a noninvasive biomarker for diagnosis and prognosis of EBV-positive Burkitt’s lymphoma. The study included 40 newly diagnosed patients with Burkitt’s lymphoma, ranging in age from 4 to 60 years, and 55 sex and age-matched controls. Forty formalin-fixed paraffin-embedded blocks of Burkitt’s lymphoma tissue samples were used to investigate the EBV by in-situ hybridization detection of the EBERs. Plasma EBV DNA was quantified by real-time quantitative polymerase chain reaction (PCR) for all Burkitt lymphoma patients prior to therapy and for control. The results showed that (22/40, 55%) of Burkitt lymphoma were positive for histological EBER, whereas plasma EBV DNA was detectable (range from 1.2×104 to 4.7×106 copies/mL) in all EBV-positive Burkitt lymphoma samples (22/22). EBV DNA was undetectable in all cases of EBV-negative Burkitt lymphomas (18/18) and all healthy control (55/55). It is worth mentioning that our results demonstrated that the EBV DNA load was significantly high in the EBV-positive BL patients suffering poor prognostic state. In conclusion: Plasma EBV-DNA can be used as a noninvasive biomarker for diagnosis and prognosis of EBV-positive Burkitt’s lymphoma.

Plasma Epstein-Barr Virus (EBV) DNA as a Biomarker for Diagnosis of Syrian EBV-Positive Burkitt’s Lymphoma

Epstein-Barr Virus -positive Burkitt’s Lymphoma is defined as the presence of Epstein-Barr virus (EBV) in tumor cells, the standard way to detect (EBV) in Burkitt’s Lymphoma is in situ hybridization (ISH) of EBV-encoded small RNA (EBERs) in tumor cells. The present study aimed to evaluate plasma Epstein-Barr virus (EBV) DNA as a noninvasive biomarker for diagnosis and prognosis of EBV-positive Burkitt’s lymphoma. The study included 40 newly diagnosed patients with Burkitt’s lymphoma, ranging in age from 4 to 60 years, and 55 sex and age-matched controls. (40) Formalin-fixed paraffin embedded blocks of Burkitt’s lymphoma tissue samples were used to investigate the EBV by in situ hybridization detection of the EBERs. Plasma EBV DNA was quantified by real-time quantitative polymerase chain reaction (PCR) for all Burkitt lymphoma patients prior to therapy and for control. The results showed that (22/40, 55%) of Burkitt lymphoma were positive for histological EBER, whereas plasma EBV DNA was detectable (range from 1.2 × 104 to 4.7 × 106 copies/mL) in all EBV-positive Burkitt lymphoma samples (22/22). EBV DNA was undetectable in all cases of EBV-negative Burkitt lymphomas (18/18) and all healthy control (55/55). It is worth mentioning that our results demonstrated that the EBV DNA load was significantly high in the EBV-positive BL patients suffering poor prognostic state. In conclusion: Plasma EBV-DNA can be used as a noninvasive biomarker for diagnosis and prognosis of EBV-positive Burkitt’s lymphoma.

Strengths and Challenges of Secretory Ribonucleases as AntiTumor Agents.

Approaches to develop effective drugs to kill cancer cells are mainly focused either on the improvement of the currently used chemotherapeutics or on the development of targeted therapies aimed at the selective destruction of cancer cells by steering specific molecules and/or enhancing the immune response. The former strategy is limited by its genotoxicity and severe side effects, while the second one is not always effective due to tumor cell heterogeneity and variability of targets in cancer cells. Between these two strategies, several approaches target different types of RNA in tumor cells. RNA degradation alters gene expression at different levels inducing cell death. However, unlike DNA targeting, it is a pleotropic but a non-genotoxic process. Among the ways to destroy RNA, we find the use of ribonucleases with antitumor properties. In the last few years, there has been a significant progress in the understanding of the mechanism by which these enzymes kill cancer cells and in the development of more effective variants. All the approaches seek to maintain the requirements of the ribonucleases to be specifically cytotoxic for tumor cells. These requirements start with the competence of the enzymes to interact with the cell membrane, a process that is critical for their internalization and selectivity for tumor cells and continue with the downstream effects mainly relying on changes in the RNA molecular profile, which are not only due to the ribonucleolytic activity of these enzymes. Although the great improvements achieved in the antitumor activity by designing new ribonuclease variants, some drawbacks still need to be addressed. In the present review, we will focus on the known mechanisms used by ribonucleases to kill cancer cells and on recent strategies to solve the shortcomings that they show as antitumor agents, mainly their pharmacokinetics.

Global Transcriptional States of Follicular Lymphoma B Cells Highlight Distinct Groups of Tumor Identity Associated with Somatic Alterations and Tumor Microenvironment

Background: Follicular Lymphoma (FL) is the second most common non-Hodgkin lymphoma and presents with significant clinical, cellular, molecular, and genetic heterogeneity. Despite the name and defining microanatomic location, the genetic and molecular identity and pathogenesis of the FL tumor cell is largely uncharacterized. Prior clinical and molecular classifications of FL have been primarily driven by pathologic classification (Grade 1-3b), genetic classification (M7-FLIPI), or gene expression profiling (IR-1/2). Using a unique cohort of 93 FL tumors, we have explored the transcriptomic signature of purified FL B cells, along with their matched whole tumor, and identified unique molecular subsets which are defined by distinct pathway activation, immune content, and genomic signatures identified through whole exome sequencing (WES). Methods: Frozen tumor biopsies from 93 untreated FL (Grade 1-3b) patients enrolled in the University of Iowa/Mayo Clinic Lymphoma SPORE were used for the study. DNA was isolated from whole tumor cell suspensions and RNA was isolated from both whole tumor and B cell enriched cell suspensions. RNA sequencing (RNAseq) and WES was performed in the Mayo Clinic Genome Analysis Core. RNAseq and WES data were processed using the Mayo Clinic standard pipeline and novel driver genes were identified using 20/20+ driver analysis. Copy number variants were identified using GISTIC 2.0. NMF clustering and single sample gene set testing, for B cell lineage and tumor microenvironment (TME) signatures, was performed in R using the NMF and SingScore packages. Results: NMF consensus clustering of FL B cell RNAseq data identified two distinct subsets, C1 (n=32) and C2 (n=57). Clinically, C1 was associated with being FL grade 3 (p<0.001) and more aggressive therapy (e.g. immunochemotherapy) at diagnosis while other clinical factors were similar between the groups. To determine the biologic underpinnings that drove the NMF analysis, the top NMF metagenes were incorporated (n=389 for C1 and n=293 for C2) for GO analysis. Among those genes were AICDA, IRF4, and EZH2 in C1, and BCL2 in C2. Additionally, we identified significant gene set enrichment with NMF metagenes and calculated differential gene set expression comparing C1, C2, and normal B cells (n=5). Together, these analyses suggest C1 displays significantly (p<0.05) enhanced DNA replication and metabolic activity, whereas C2 displays significantly enhanced cytoskeleton rearrangement, repressed inflammatory cytokine signaling, and NF-kB activity. Based on recent scRNAseq data suggesting that FL derives from distinct germinal center B cells, the B lineage difference between C1 and C2 was explored. Both subgroups were heterogeneous for dark (DZ), intermediate (INT), light (LZ) zones, and plasmablast (PBL) gene signatures. When compared to benign B cell samples, C1 was enriched for a pre-PBL (p<0.01), and C2 for an INT phenotype related to a LZ signature (p<0.01). To define if a genomic signature was associated with C1 and C2, mutations and copy number variants (>5% frequency) were examined. TNFAIP3, TP53, and BCL6 alterations were enriched in C1 samples, whereas C2 associated with alterations in BCL2, KMT2D, CREBBP, REL, and MYC. Finally, B cell clusters were analyzed for TME signatures. C1 samples displayed significant enrichment of macrophage, cytotoxic cell, gamma-delta-Tcell, and endothelial cell TME elements (p<0.05) consistent with an inflamed/hot tumor. In contrast, C2 samples exhibited significantly lower signatures (p<0.05) in all entities except for the fibroblast signature, compared to C1 and normal, consistent with an immune desert phenotype. Conclusion: Our results suggest that B cells from FL patients display two distinct transcriptomic signatures. C1 identifies an immunologically active tumor, driven by TNFAIP3 alterations, with pre-PBL characteristics, DNA replication and repair, inflammatory cytokine secretion/signaling, and hyper-metabolic characteristics. C2 identifies an immunologically quiet tumor, driven by alterations in BCL2 and chromatin modifiers, with an intermediate GC phenotype, repressed cytokine signaling, and active cell cycle progression and cytoskeleton rearrangement. This study improves our understanding of the mechanisms driving FL tumors and motivates further investigation into the relationship between tumor intrinsic factors that may influence the TME. Disclosures Maurer: Morphosys: Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Nanostring: Research Funding; Celgene / BMS: Research Funding. Ansell:ADC Therapeutics: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Regeneron: Research Funding; AI Therapeutics: Research Funding; Takeda: Research Funding; Seattle Genetics: Research Funding; Bristol Myers Squibb: Research Funding. Cerhan:NanoString: Research Funding; BMS/Celgene: Research Funding. Novak:Celgene/BMS: Research Funding.

Abstract 5365: Profiling circulating tumor cell RNA from a large blood screening volume: A pilot study using diagnostic leukapheresis followed by the NanoString low RNA input nCounter assay

Abstract Introduction and objective: The presence of circulating tumor cells (CTC) is associated with poor prognosis in metastatic prostate cancer (mPCa) patients. The aim of this study is to evaluate the feasibility of using diagnostic leukapheresis (LP) followed by gene profiling using the NanoString low RNA input nCounter assay to achieve reliable CTC gene profiling. Methods: Blood samples were collected from 4 mPCa patients and 3 healthy donors. In the LP procedure, different blood elements were separated based on density. The mononuclear cell layer (presumed to contain any CTCs) was collected. The Alere™ CTC AdnaTest (AdnaGen, Langenhagen, Germany), an EpCAM positive selection method for isolation of EpCAM+ CTCs, followed by RT-qPCR for PSA, PSMA, and HOXB13 was used to detect CTCs from LP. The Ct value thresholds for positive CTC signal by RT-qPCR were set based on healthy donor LP subjected to AdnaTest as a negative control (PSA 36.3, PSMA 36.82, HOXB13 40). The cDNA of cells captured by EpCAM+ selection (including putative CTCs) and of the EpCAM+ depleted white blood cells (WBC) were assayed by the nCounter PanCancer Progression Panel to determine expression of 770 selected mRNAs. The NanoString nCounter Low RNA Input Kit with the multiplex 770-gene primer pool was used for the pre-amplification of cDNA and overnight hybridization with the PanCancer Progression panel. To validate the assay, healthy donor LPs were spiked with known numbers of PCa cell line LN95 cells. Results: For mPCa patients, the LP procedure took 136.2±10.6 min, with a screening volume of 10L of peripheral blood. The mean volume of mPCa LP product was 123.64±12.23 ml. The WBC concentration was 69.75±28.2×109/L. All samples were subjected to AdnaTest in triplicate and CTCs were identified in 3 out of 4 patients. The positive Ct values of PSA were 33.86±0.81, 32.14±0.31 and 33.09±0.34; PSMA were 35.55±1.02, 33.55±0.83 and 35.30±0.65; HOXB13 were 36.25±0.34, 37.15±0.22 and 37.09±0.33. To assess profiling of CTCs isolated from LP using the nCounter platform, 100, 500, and 1000 LN95 cells were spiked in healthy LPs and were analyzed with the AdnaTest. Within each spiking group, pre-spiking WBC, captured CTC, post-capture WBC, and LN95 cells in PBS alone were subjected to the Nanostring assay. Eisen Cluster analysis separated pre- and post- WBCs, captured CTCs, LN95 cells into three groups. The gene expression of captured CTCs correlated with those of LN95 cells, and the correlation increased with the spiking cancer cell number (R2=0.45, 0.57, and 0.70, respectively). The gene expression of pre-spiked WBCs were highly correlated with those of post-capture WBCs regardless of spiking cancer cell number (R2=0.83, 0.84 and 0.85). Conclusions: Analyzing CTCs in LP samples by AdnaTest is feasible and offers a promising method for liquid biopsy of mPCa patients. The NanoString low RNA input nCounter assay can provide reliable gene expression profiling of CTC. Citation Format: Liang Dong, Chung-Ying Huang, Changxue Lu, Diane Reyes, Sarah R. Amend, Jun Luo, Kenneth Pienta. Profiling circulating tumor cell RNA from a large blood screening volume: A pilot study using diagnostic leukapheresis followed by the NanoString low RNA input nCounter assay [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 5365.

Fibroblast Growth Factor-14 Acts as Tumor Suppressor in Lung Adenocarcinomas.

Investigation of the molecular dynamics in lung cancer is crucial for the development of new treatment strategies. Fibroblast growth factor (FGF) 14 belongs to the FGF family, which might play a crucial role in cancer progression. We analyzed lung adenocarcinoma (LUAC) patients samples and found that FGF14 was downregulated, correlating with reduced survival and oncogenic mutation status. FGF14 overexpression in lung cancer cell lines resulted in decreased proliferation, colony formation, and migration, as well as increased expression of epithelial markers and a decreased expression of mesenchymal markers, indicating a mesenchymal to epithelial transition in vitro. We verified these findings using small interfering RNA against FGF14 and further confirmed the suppressive effect of FGF14 in a NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ immunodeficient xenograft tumor model. Moreover, FGF14 overexpressing tumor cell RNA sequencing data suggests that genes affected by FGF14 were related to the extracellular matrix, playing a role in proliferation and migration. Notably, newly identified FGF14 target genes, adenosine deaminase RNA specific B1 (ADARB1), collagen and calcium-binding epidermal growth factor domain-containing protein 1 (CCBE1), α1 chain of collagen XI (COL11A1), and mucin 16 (MUC16) expression was negatively correlated with overall survival when FGF14 was downregulated in LUAC. These findings led us to suggest that FGF14 regulates proliferation and migration in LUAC.

Interferon-Induced Transmembrane Protein 1 (IFITM1) Promotes Distant Metastasis of Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is a severe malignancy associated with early and widespread metastasis. To study SCLC metastasis, we previously developed an orthotopic transplantation model using the human SCLC cell line DMS273. In the model, metastatic foci were found in distant tissues such as bone and the adrenal gland, similarly as observed in patients with SCLC. In this study, we evaluated the differentially expressed genes between orthotopic and metastatic tumors in the model. We isolated tumor cells from orthotopic and metastatic sites, and the tumor cell RNA was analyzed using DNA microarray analysis. We found that 19 genes in metastatic tumors were upregulated by more than 4-fold compared with their expression in orthotopic tumors. One of these genes encodes a transmembrane protein, interferon (IFN)-induced transmembrane protein 1 (IFITM1), and immunohistochemical analysis confirmed the higher expression of the protein in metastatic sites than in orthotopic sites. IFITM1 was also detected in some SCLC cell lines and lung tumors from patients with SCLC. The overexpression of IFITM1 in DMS273 cells increased their metastatic formation in the orthotopic model and in an experimental metastasis model. Conversely, the silencing of IFITM1 suppressed metastatic formation by DMS273 cells. We also found that IFITM1 overexpression promoted the metastatic formation of NCI-H69 human SCLC cells. These results demonstrate that IFITM1 promotes distant metastasis in xenograft models of human SCLC.

Abstract PR11: Multimodal analysis of circulating tumor cell RNA, circulating cell-free DNA, and genomic DNA from a single blood sample collected into a PAXgene Blood ccfDNA Tube*

Abstract Background: High demand for liquid biopsy tests in cancer research increases the need for combined approaches that allow multimodal testing from a limited blood volume. Although crosslinking-based fixatives allow for efficient preservation of the circulating cell-free DNA (ccfDNA) profile in whole blood, analysis of mRNA from circulating tumor cells (CTCs) is hampered. Currently, there are no blood collection tubes available that allow RNA analysis from CTCs after prolonged (>48 hours) storage. In this research study, we present a newly developed combined workflow for blood stabilization and subsequent analysis of CTC RNA, ccfDNA, and gDNA after prolonged (≥72 hours) storage. Methods: To establish the multimodal workflow, blood was collected from healthy donors into PAXgene Blood ccfDNA Tubes*, aliquoted and manually spiked with 20 individual LNCaP95 cells/5 mL blood as a CTC model or 20 µL PBS as a control. Blood samples were spiked and stored at 2–8°C for 3, 24, 48, and 72 hours before processing. CTCs were enriched and detected using the AdnaTest ProstateCancerPanel AR-V7† (QIAGEN). The plasma and cellular fraction of the CTC-depleted blood was then used to extract ccfDNA and gDNA, respectively. As a control for ccfDNA and gDNA yield measurements, plasma and the cellular fraction were also generated from whole samples (not CTC-depleted) spiked with 20 LNCaP95 cells/5 mL. The resulting multimodal workflow was tested: 1) in comparison to Streck Cell-Free DNA BCT; 2) to assess the impact of room temperature. Results: Tumor cells spiked into PAXgene Blood ccfDNA Tubes could be enriched and detected at experimental time points 3, 24, 48, and 72 hours after spiking with 100% test sensitivity within 24 hours storage and 91% test sensitivity after 72 hours storage at 2–8°C. In unspiked samples, the PAXgene stabilization solution did not cause false positive signals at any experimental time point. Yields of ccfDNA and gDNA were not statistically significantly affected by CTC capture (p>0.05). RNA from spiked tumor cells was detected in blood samples collected and stored in Streck Cell-Free DNA BCTs for 3 hours, but not at later time points. Storage of PAXgene stabilization solution-treated samples at room temperature is feasible, with 100% test sensitivity for CTC detection within 24 hours storage (similar to storage at 2–8°C) and 80% after 72 hours storage. Conclusions: Blood collected into PAXgene Blood ccfDNA Tubes and stored for up to 72 hours at 2–8°C or at room temperature can be used for multimodal analysis of CTC RNA, ccfDNA, and gDNA from a single blood sample. *For research use only. Not for use in diagnostic procedures. †For molecular biology applications. Not intended for the diagnosis, prevention, or treatment of a disease. This abstract is also being presented as Poster B18. Citation Format: Eric Provencher, Maike Remmel, Siegfried Hauch, Michael Otte, Andrea Ullius, Daniel Groelz, Anna Babayan. Multimodal analysis of circulating tumor cell RNA, circulating cell-free DNA, and genomic DNA from a single blood sample collected into a PAXgene Blood ccfDNA Tube* [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr PR11.

Circulating Tumor Cells and Fibronectin 1 in the Prognosis of Nasopharyngeal Carcinoma.

Objective:Nasopharyngeal carcinoma is highly endemic in Southeast China. Circulating tumor cell is an important biomarker in the prognosis of variety kinds of cancers. Overexpression of fibronectin 1 was observed in variety kinds of malignancies and may contribute to progress and metastasis of the cancers. The current study was aimed to investigate phenotypes of circulating tumor cell in nasopharyngeal carcinoma blood and fibronectin 1 expression in the circulating tumor cell, and their clinical application in predicting nasopharyngeal carcinoma prognosis.Methods:Blood samples were obtained from nasopharyngeal carcinoma patients before and after treatment. CanPatrol circulating tumor cell enrichment and RNA in situ hybridization were applied to identify circulating tumor cell and its phenotypes. Fibronectin 1 messenger RNA expression in the cells of circulating tumors was examined by messenger RNA-in situ hybridization.Results:Circulating tumor cell was not associated with tumor characteristics or lymph node metastasis. Patients with >9 circulating tumor cells or >5 mesenchymal phenotype circulating tumor cell per 5-mL blood had poorer progression-free survival (P < .05). Multivariable analysis demonstrated that 2 or more mesenchymal phenotype circulating tumor cells with high fibronectin 1 messenger RNA expression predicted shorter progression-free survival (P < .05).Conclusions:Circulating tumor cells with high-level fibronectin 1 expression was associated with poor survival in patients with nasopharyngeal carcinoma and could be an independent prognostic factor for nasopharyngeal carcinoma.

RIG-I activation is critical for responsiveness to checkpoint blockade.

Achieving durable clinical responses to immune checkpoint inhibitors remains a challenge. Here, we demonstrate that immunotherapy with anti-CTLA-4 and its combination with anti-PD-1 rely on tumor cell-intrinsic activation of the cytosolic RNA receptor RIG-I. Mechanistically, tumor cell-intrinsic RIG-I signaling induced caspase-3-mediated tumor cell death, cross-presentation of tumor-associated antigen by CD103+ dendritic cells, subsequent expansion of tumor antigen-specific CD8+ T cells, and their accumulation within the tumor tissue. Consistently, therapeutic targeting of RIG-I with 5'- triphosphorylated RNA in both tumor and nonmalignant host cells potently augmented the efficacy of CTLA-4 checkpoint blockade in several preclinical cancer models. In humans, transcriptome analysis of primary melanoma samples revealed a strong association between high expression of DDX58 (the gene encoding RIG-I), T cell receptor and antigen presentation pathway activity, and prolonged overall survival. Moreover, in patients with melanoma treated with anti-CTLA-4 checkpoint blockade, high DDX58 RIG-I transcriptional activity significantly associated with durable clinical responses. Our data thus identify activation of RIG-I signaling in tumors and their microenvironment as a crucial component for checkpoint inhibitor-mediated immunotherapy of cancer.

Circulating tumor cell and cell-free RNA capture and expression analysis identify platelet-associated genes in metastatic lung cancer

BackgroundCirculating tumor cells (CTC) and plasma cell-free RNA (cfRNA) can serve as biomarkers for prognosis and treatment response in lung cancer. One barrier to the selected or routine use of CTCs and plasma cfRNA in precision oncology is the limited quantity of both, and CTCs are only seen in metastatic disease. As capture of CTCs and plasma cfRNA presents an opportunity to monitor and assess malignancies without invasive procedures, we compared two methods for CTC capture and identification, and profiled mRNA from CTCs and plasma cfRNA to identify potential tumor-associated biomarkers.MethodsPeripheral blood was collected from ten patients with small cell lung cancer (SCLC), ten patients with non-small cell lung cancer (NSCLC) and four healthy volunteers. Two methods were used for CTC capture: the standard epithelial cell adhesion molecule (EpCam) CellSearch kit (unicapture) and EpCAM plus HER2, EGFR and MUC-1 specific combined ferrofluid capture (quadcapture). For the quadcapture, anti-cytokeratin 7 (CK7) was additionally used to assist in CTC identification. NanoString analysis was performed on plasma cfRNA and on mRNA from combined ferrofluid isolated CTCs. Expression data was analyzed using STRING and Reactome.ResultsUnicapture detected CTCs in 40% of NSCLC and 60% of SCLC; whereas, quadcapture/CK7 identified CTCs in 20% of NSCLC and 80% of SCLC. Bioinformatic analysis of NanoString data identified high expression of a platelet factor 4 (PF4)-related group of transcripts.ConclusionsQuadcapture ferrofluid reagent did not significantly improve CTC capture efficacy. NanoString analysis based on CTC and plasma cfRNA data highlighted an intriguing PF-4-centric network in patients with metastatic lung cancer.

The expression level of fibulin-2 in the circulating RNA (ctRNA) of epithelial tumor cells of peripheral blood and tumor tissue of patients with metastatic lung cancer.

The Fibulins are a recently discovered family of extracellular matrix proteins. In this study, expression levels of the fibulin-2 (FBLN2) gene and its role in the formation of different metastatic foci were investigated in lung cancer patients. We analyzed 106 lung cancer patients and eight paraffin-embedded tissues, and 27 ethnical-, age- and sex-matched healthy controls for expression levels of the FBLN2 gene. cDNAs obtained from the enriched epithelial cells of peripheral blood lymphocytes and tumor tissues of patients were amplified with specific primers for the target FBLN2 gene and HPRT1 housekeeping gene using quantitative real-time polymerase chain reaction. FBLN2 gene expression levels of the enriched epithelial cells of peripheral blood lymphocytes were found to be decreased approximately twofold in all subsets of patients compared to healthy controls. Our results indicate a significant difference between patient subgroups and controls [F(4.124) = 14.846, p0.05] among patient subgroups: bone metastases versus non-metastatic groups (p = 0.997), bone versus brain metastases (p = 0994), bone metastases versus two primary tumors (p = 0.999), brain metastases versus two primary tumors (p = 0.999), brain metastases versus non-metastatic (p = 0.755), non-metastatic versus two primary tumors (p = 0.996), non-metastatic versus all other metastatic patients (p = 0.731). Moreover, we found a 50-fold upregulation of FBLN2 gene expression in paraffin-embedded tissues compared with the enriched epithelial cells ofperipheral blood lymphocytes of patients. In the study, the enriched epithelial cells of peripheral blood lymphocytes of decreased FBLN2 expression was found to be correlated with metastasis. The fibulin-2 molecules might induce the metastatic potential through interaction with the other molecules in the microenvironment, nevertheless, it is needed further research whether the importance of FBLN2 on lung cancer oncogenesis and as a biomarker for metastatic lung cancer.