Hepatocellular Carcinoma Is a Natural Target for Adeno-Associated Virus (AAV) 2 Vectors

Although therapeutic options are gradually improving, the overall prognosis for patients with hepatocellular carcinoma (HCC) is still poor. Gene therapy-based strategies are developed to complement the therapeutic armamentarium, both in early and late-stage disease. For efficient delivery of transgenes with antitumor activity, vectors demonstrating preferred tumor tropism are required. Here, we report on the natural tropism of adeno-associated virus (AAV) serotype 2 vectors for HCC. When applied intravenously in transgenic HCC mouse models, similar amounts of vectors were detected in the liver and liver tumor tissue. In contrast, transduction efficiency, as indicated by the level of transgene product, was moderate in the liver but was elevated up to 19-fold in mouse tumor tissue. Preferred transduction of HCC compared to hepatocytes was confirmed in precision-cut liver slices from human patient samples. Our mechanistic studies revealed that this preference is due to the improved intracellular processing of AAV2 vectors in HCC, resulting, for example, in nearly 4-fold more AAV vector episomes that serve as templates for gene transcription. Given this background, AAV2 vectors ought to be considered to strengthen current—or develop novel—strategies for treating HCC.

Identification of Salivary Gland Escape Barriers to Western Equine Encephalitis Virus in the Natural Vector, Culex tarsalis

Herein we describe a previously uninvestigated salivary gland escape barrier (SEB) in Culex tarsalis mosquitoes infected with two different strains of Western equine encephalitis virus (WEEV). The WEEV strains were originally isolated either from mosquitoes (IMP181) or a human patient (McMillan). Both IMP181 and McMillan viruses were fully able to infect the salivary glands of Culex tarsalis after intrathoracic injection as determined by expression of mCherry fluorescent protein. IMP181, however, was better adapted to transmission as measured by virus titer in saliva as well as transmission rates in infected mosquitoes. We used chimeric recombinant WEEV strains to show that inclusion of IMP181-derived structural genes partially circumvents the SEB.

Infrared spectroscopy enables rapid, robust, portable COVID-19 saliva screening based on pathophysiological response to SARS-CoV-2

Fourier-transform infrared (FTIR) spectroscopy provides a (bio)chemical snapshot of the sample, and was recently proposed for COVID-19 saliva screening in proof-of-concept cohort studies. As a step towards translation of this technology, we conducted controlled validation experiments in multiple biological systems. SARS-CoV-2 or UV-inactivated SARS-CoV-2 were used to infect Vero E6 cells in vitro, and K18-hACE2 mice in vivo. Potentially infectious culture supernatant or mouse oral lavage samples were treated with ethanol or Trizol to 75% (v/v) for attenuated total reflectance (ATR)-FTIR spectroscopy, or RT-PCR, respectively. The control condition, UV-inactivated SARS-CoV-2 elicited strong biochemical changes in culture supernatant/oral lavage despite lack of replication determined by RT-PCR or cell culture infectious dose 50%. Crucially, we show that active SARS-CoV-2 infection induced additional FTIR signals over the UV-inactivated SARS-CoV-2 infection, which correspond to innate immune response, aggregated proteins, and RNA. For human patient cohort prediction, we achieved high sensitivity of 93.48% on leave-on-out cross validation (n=104 participants) for predicting COVID-19 positivity using a partial least squares discriminant analysis model, in agreement with recent studies. However, COVID-19 patients negative on follow-up (RT-PCR on day of saliva sampling) were poorly predicted in this model. Importantly, COVID-19 vaccination did not lead to mis-classification of COVID-19 negatives. Meta-analysis revealed SARS-CoV-2 induced increase in Amide II band in all arms of this study and recent studies, indicative of altered β-sheet structures in secreted proteins. In conclusion, ATR-FTIR is a robust, simple, portable method for COVID-19 saliva screening based on detection of pathophysiological responses to SARS-CoV-2.

Human Adenovirus Species D Interactions with Corneal Stromal Cells

Notable among the many communicable agents known to infect the human cornea is the human adenovirus, with less than ten adenoviruses having corneal tropism out of more than 100 known types. The syndrome of epidemic keratoconjunctivitis (EKC), caused principally by human adenovirus, presents acutely with epithelial keratitis, and later with stromal keratitis that can be chronic and recurrent. In this review, we discuss the current state of knowledge regarding the molecular biology of adenovirus infection of corneal stromal cells, among which the fibroblast-like keratocyte is the most predominant, in order to elucidate basic pathophysiologic mechanisms of stromal keratitis in the human patient with EKC.

The cytosolic role of EZH2-IMPDH2 complex in melanoma progression and metastasis via GTP regulation

The enhancer of zeste homolog 2 (EZH2) oncogene is a histone methyltransferase that functions canonically as a catalytic subunit of the polycomb repressive complex 2 (PRC2) to tri-methylate histone H3 at Lys 27 (H3K27me3). Although targeting of EZH2 methyltransferase is a promising therapeutic strategy against cancer, methyltransferase-independent oncogenic functions of EZH2 are also described. Moreover, pharmacological EZH2 methyltransferase inhibition was only variably effective in pre-clinical and clinical studies, suggesting that targeting EZH2 methyltransferase alone may be insufficient. Here, we demonstrate a non-canonical mechanism of EZH2’s oncogenic activity through interactions with inosine monophosphate dehydrogenase 2 (IMPDH2) and downstream promotion of guanosine-5'-triphosphate (GTP) production. Liquid Chromatography-Mass Spectrometry (LC-MS) of EZH2 immunoprecipitates from melanoma cell lines and human patient-derived xenografts (PDXs) revealed EZH2-IMPDH2 interactions that were verified to occur between the N-terminal EED-binding domain of cytosolic EZH2 and the CBS domain of IMPDH2 in a PRC2- and methylation-independent manner. EZH2 silencing reduced cellular GTP, ribosome biogenesis, RhoA-mediated actomyosin contractility and melanoma cell proliferation and invasion by impeding the activity and cytosolic localization of IMPDH2. Guanosine, which replenishes GTP, reversed these effects and thereby promoted invasive and clonogenic cell states even in EZH2 silenced cells. IMPDH2 silencing antagonized the proliferative and invasive effects of EZH2, also in a guanosine-reversible manner. In human melanomas, high cytosolic EZH2 and IMPDH2 expression were associated with nucleolar enlargement, a marker for ribosome biogenesis. We also identified EZH2-IMPDH2 complexes in a range of cancers in which Sappanone A (SA), which inhibits EZH2-IMPDH2 interactions and thereby IMPDH2 tetramerization, was anti-tumorigenic, although notably non-toxic in normal human melanocytes and bone marrow derived blood progenitor cells that lacked observable EZH2-IMPDH2 interactions. These findings illuminate a previously unrecognized, non-canonical, methyltransferase-independent, but GTP-dependent mechanism by which EZH2 regulates tumorigenicity in melanoma and other cancers, opening new avenues for development of anti-EZH2 therapeutics.

TAMI-59. RECIPROCAL IMPACT OF CANCER IMMUNITY AND TUMOR HYPOXIA DURING GLIOBLASTOMA PROGRESSION

Tumor hypoxia is linked to poor outcome for glioblastoma (GBM), a highly malignant brain cancer, but the underlying mechanisms and the environmental factors that initiate tumor hypoxia are poorly understood. We tracked tumor hypoxia in GBM in immunocompetent mice with a hypoxia sensitive fluorescent reporter combined with single cell transcriptomics. We found that hypoxic GBM cells are quiescent, immunosuppressive and display a mesenchymal transition, all of which are linked to malignant potency. We also captured in vivo hypoxia gene signature, which is more represented in recurrent GBM and predicts worse outcome. Interestingly, hypoxic GBM cells is a diverse population, consisted of four subclusters, and enriched for immune pathways. Concordantly, our reporter highlighted a distinct geographic pattern of immune cells in hypoxic regions, with phagocytic tumor-associated macrophages (TAMs) and CD8+ cytotoxic T cells (CTLs) congregated in hypoxic cores confined by hypoxic GBM cells in pseudo-palisading patterns. Mechanistically, this is a dynamic temporospatial process, requiring cytokine CCL8. Remarkably, the sequestered TAMs also experience hypoxia, and they are reprogrammed to express immunotolerant markers by factors released from hypoxic GBM cells. Contrary to the conventional viewpoint that hypoxia arises from rapid tumor expansion outstripping vascular supply, we discovered anticancer immunity as an important driving force of tumor hypoxia; attenuating immune responses by implanting GBM in host mice with immunodeficiency or IL1β deletion greatly decreased GBM hypoxia. Analyses of human patient GBM samples highlighted a connection of mesenchymal subtype, immune response, and tumor hypoxia, all contributing to poor survival. Altogether, our study revealed a reciprocal influence of anti-tumor immunity and tumor hypoxia, which has significant ramifications for prognosis and immunotherapy for GBM.

Spatially resolved transcriptomics reveals the architecture of the tumor-microenvironment interface

During tumor progression, cancer cells come into contact with various non-tumor cell types, but it is unclear how tumors adapt to these new environments. Here, we integrate spatially resolved transcriptomics, single-cell RNA-seq, and single-nucleus RNA-seq to characterize tumor-microenvironment interactions at the tumor boundary. Using a zebrafish model of melanoma, we identify a distinct “interface” cell state where the tumor contacts neighboring tissues. This interface is composed of specialized tumor and microenvironment cells that upregulate a common set of cilia genes, and cilia proteins are enriched only where the tumor contacts the microenvironment. Cilia gene expression is regulated by ETS-family transcription factors, which normally act to suppress cilia genes outside of the interface. A cilia-enriched interface is conserved in human patient samples, suggesting it is a conserved feature of human melanoma. Our results demonstrate the power of spatially resolved transcriptomics in uncovering mechanisms that allow tumors to adapt to new environments.

658 CD8 T cell activation in cancer is comprised of two distinct phases

BackgroundCD8 T cell are a crucial part of the immune response to tumors, with CD8 infiltration predicting disease progression in numerous cancer types. Recently two subsets of CD8 T cells that respond to tumors have been described, a stem-like (TCF1+) CD8 T cell that can give rise to a more cytotoxic terminally differentiated (TD) (TCF1-Tim3+) CD8 T cell. In this study we aimed to understand the origin of stem-like TCF1+ CD8 T cells within tumors.MethodsHuman patient TDLN and tumor samples from kidney and prostate cancer were processed after resection and used for flow cytometry, RNA-seq, TCR-seq and whole genome DNA methylation analysis. We also used a prostate cancer mouse model that expresses the LCMV GP protein (TRAMPC1-LCMV-GP) to track tumor-specific CD8 T cells in both TDLNs and tumors.ResultsWe studied human prostate and kidney cancer tumor-draining lymph nodes (TDLN) and found that CD8 T cells are activated but fail to acquire an effector phenotype within the TDLN. Instead, they share functional, transcriptional, and epigenetic traits with stem-like cells in the tumor. We also found that activated CD8 T cells from TDLNs shared TCR overlap with both CD8 subsets within tumors. This suggests that these activated cells are a precursor to the stem-like CD8 T cells in tumors. To further test this hypothesis, we used our TRAMPC1-LCMV-GP tumor model to study tumor-specific CD8 T cell activation. We found that CD8 T cells are activated in TDLNs but fail to acquire an effector program. These cells then establish the stem-like CD8s within tumor where they require additional co-stimulation from antigen presenting cells to differentiate into TCF1- TD CD8 T cells. This is strikingly different from canonical CD8 T cell activation to acute viruses, where the effector program is acquired immediately. We also showed that human stem-like CD8 T cells require co-stimulation and TCR stimulation to divide and differentiate into terminally differentiated CD8s in-vitro, and DCs from autologous tumors can also induce this differentiation.ConclusionsOverall this work shows a model of CD8 T cell activation in response to tumors that has two distinct phases. The first occurs in the TDLN where CD8 T cells are initially activated, the second occurs in the tumor where CD8 T cells acquire an effector function after additional co-stimulation. This model of T cell differentiation adds to our understanding of basic CD8 T cell biology and has important implications to improve our current immunotherapies.

Type-3 Hyaluronan Synthase Attenuates Tumor Cells Invasion in Human Mammary Parenchymal Tissues

The microenvironment for tumor growth and developing metastasis should be essential. This study demonstrated that the hyaluronic acid synthase 3 (HAS3) protein and its enzymatic product hyaluronic acid (HA) encompassed in the subcutaneous extracellular matrix can attenuate the invasion of human breast tumor cells. Decreased HA levels in subcutaneous Has3-KO mouse tissues promoted orthotopic breast cancer (E0771) cell-derived allograft tumor growth. MDA-MB-231 cells premixed with higher concentration HA attenuate tumor growth in xenografted nude mice. Human patient-derived xenotransplantation (PDX) experiments found that HA selected the highly migratory breast cancer cells with CD44 expression accumulated in the tumor/stroma junction. In conclusion, HAS3 and HA were detected in the stroma breast tissues at a high level attenuates effects for induced breast cancer cell death, and inhibit the cancer cells invasion at the initial stage. However, the highly migratory cancer cells were resistant to the HA-mediated effects with unknown mechanisms.