Daniel Lazar

Characterization of circulating tumor cell aggregates identified in patients with epithelial tumors

Circulating tumor cells (CTCs) have been implicated as a population of cells that may seed metastasis and venous thromboembolism (VTE), two major causes of mortality in cancer patients. Thus far, existing CTC detection technologies have been unable to reproducibly detect CTC aggregates in order to address what contribution CTC aggregates may make to metastasis or VTE. We report here an enrichment-free immunofluorescence detection method that can reproducibly detect and enumerate homotypic CTC aggregates in patient samples. We identified CTC aggregates in 43% of 86 patient samples. The fraction of CTC aggregation was investigated in blood draws from 24 breast, 14 non-small cell lung, 18 pancreatic, 15 prostate stage IV cancer patients and 15 normal blood donors. Both single CTCs and CTC aggregates were measured to determine whether differences exist in the physical characteristics of these two populations. Cells contained in CTC aggregates had less area and length, on average, than single CTCs. Nuclear to cytoplasmic ratios between single CTCs and CTC aggregates were similar. This detection method may assist future studies in determining which population of cells is more physically likely to contribute to metastasis and VTE.

Cytomorphology of circulating colorectal tumor cells:a small case series.

Several methodologies exist to enumerate circulating tumor cells (CTCs) from the blood of cancer patients; however, most methodologies lack high-resolution imaging, and thus, little is known about the cytomorphologic features of these cells. In this study of metastatic colorectal cancer patients, we used immunofluorescent staining with fiber-optic array scanning technology to identify CTCs, with subsequent Wright-Giemsa and Papanicolau staining. The CTCs were compared to the corresponding primary and metastatic tumors. The colorectal CTCs showed marked intrapatient pleomorphism. In comparison to the corresponding tissue biopsies, cells from all sites showed similar pleomorphism, demonstrating that colorectal CTCs retain the pleomorphism present in regions of solid growth. They also often retain particular cytomorphologic features present in the patients primary and/or metastatic tumor tissue. This study provides an initial analysis of the cytomorphologic features of circulating colon cancer cells, providing a foundation for further investigation into the significance and metastatic potential of CTCs.

Potent and Targeted Activation of Latent HIV-1 Using the CRISPR/dCas9 Activator Complex

HIV-1 provirus integration results in a persistent latently infected reservoir that is recalcitrant to combined antiretroviral therapy (cART) with lifelong treatment being the only option. The “shock and kill” strategy aims to eradicate latent HIV by reactivating proviral gene expression in the context of cART treatment. Gene-specific transcriptional activation can be achieved using the RNA-guided CRISPR-Cas9 system comprising single guide RNAs (sgRNAs) with a nuclease-deficient Cas9 mutant (dCas9) fused to the VP64 transactivation domain (dCas9-VP64). We engineered this system to target 23 sites within the long terminal repeat promoter of HIV-1 and identified a “hotspot” for activation within the viral enhancer sequence. Activating sgRNAs transcriptionally modulated the latent proviral genome across multiple different in vitro latency cell models including T cells comprising a clonally integrated mCherry-IRES-Tat (LChIT) latency system. We detected consistent and effective activation of latent virus mediated by activator sgRNAs, whereas latency reversal agents produced variable activation responses. Transcriptomic analysis revealed dCas9-VP64/sgRNAs to be highly specific, while the well-characterized chemical activator TNFα induced widespread gene dysregulation. CRISPR-mediated gene activation represents a novel system which provides enhanced efficiency and specificity in a targeted latency reactivation strategy and represents a promising approach to a “functional cure” of HIV/AIDS.

Cytometric comparisons between circulating tumor cells from prostate cancer patients and the prostate tumor derived LNCaP cell line

Many important experiments in cancer research are initiated with cell line data analysis due to the ease of accessibility and utilization. Recently, the ability to capture and characterize circulating tumor cells (CTCs) has become more prevalent in the research setting. This ability to detect, isolate and analyze CTCs allows us to directly compare specific protein expression levels found in patient CTCs to cell lines. In this study, we use immunocytochemistry to compare the protein expression levels of total cytokeratin (CK) and androgen receptor (AR) in CTCs and cell lines from patients with prostate cancer to determine what translational insights might be gained through the use of cell line data. A non-enrichment CTC detection assay enables us to compare cytometric features and relative expression levels of CK and AR by indirect immunofluorescence from prostate cancer patients against the prostate cancer cell line LNCaP. We measured physical characteristics of these two groups and observed significant differences in cell size, fluorescence intensity and nuclear to cytoplasmic ratio. We hope that these experiments will initiate a foundation to allow cell line data to be compared against characteristics of primary cells from patients.

The emerging role of long non-coding RNAs in HIV infection.

The discovery of long non-coding RNAs (lncRNAs) and the elucidation of the mechanisms by which they affect different disease states are providing researchers with a better understanding of a wide array of disease pathways. Moreover, lncRNAs are presenting themselves as both unique diagnostic biomarkers as well as novel targets against which to develop new therapeutics. Here we will explore the intricate network of non-coding RNAs associated with infection by the human immunodeficiency virus (HIV). Non-coding RNAs derived from both the human host as well as those from HIV itself are emerging as important regulatory elements. We discuss here the various mechanisms through which both small and long non-coding RNAs impact viral replication, pathogenesis and disease progression. Given the lack of an effective vaccine or cure for HIV and the scale of the current pandemic, a deeper understanding of the complex interplay between non-coding RNAs and HIV will support the development of innovative strategies for the treatment of HIV/acquired immunodeficiency disease (AIDS).

693. Potent and Targeted Activation of Latent HIV-1 Using Multiplexed Guide RNAs and the CRISPR/dCas9 Activator Complex

Cells latently infected with HIV provide a reservoir that promotes new viral infection indefinitely, thus making it exceedingly difficult for antiretroviral therapy (ART) to completely ablate the infection. A powerful strategy to eradicate latent HIV reservoirs has been to reactivate proviral gene expression using histone deacetylase (HDAC) inhibitors followed by ART: an approach called “shock and kill”. However, this approach suffers from lack of efficacy and specificity. Gene specific transcriptional activation can be achieved using the RNA-guided CRISPR-Cas9 system comprising a nuclease deficient mutant of Cas9 (dCas9) fused to a C-terminal VP64 or VP160 acidic transactivation domain. We initially identified 18 sites for targeting of small guide RNAs (sgRNAs) in the U3 region, upstream of the canonical HIV transcriptional start site TSS (-450 to 0 bp). Activating sgRNA candidates were identified and verified by transient transfection in TZM-Bl cells and also in HEK293 cells with Subtype B and C LTR-driven luciferase reporters. Several candidate sgRNA individually induced a 10-fold increase in gene expression, with a “hotspot” region between -200 and -250 bp of the TSS. However, when combined in multiplex fashion, with four conserved gRNAs expressed off four independent Pol III promoters in a single vector, a synergistic enhancement of HIV transcription of ~30 fold was observed. Given the complexity and multifaceted mechanisms of latency, current cell line models are unable to completely recapitulate the conditions of endogenous HIV reservoirs. As an initial proof-of-concept that the CRISPR/Cas9 system can be used to purge the latent HIV proviral genome, we used a CEM T cell-based reporter system, comprising a single copy of HIV, that encodes mCherry-IRES-Tat from the full-length HIV LTR (LChIT). The LChIT system enables a Tat-mediated positive feedback loop that drives expression of Tat and mCherry allowing for exquisite sensitivity to alterations in viral gene expression, either directly or indirectly. CEM-LChIT clone 3.2 expressed mCherry in 50% of the population (a bimodal state). In this model, nucleofected sgRNAs and dCas9-VP64 led to a 200-fold activation of gene expression; a result higher than treatment controls using a combination of HDAC inhibitor trichostatin A (TSA) and TNFa. In conclusion, the potential for RNA-guided gene activation systems to provide maximum efficiency and specificity in a targeted reactivation strategy augurs well for the future of the “shock and kill” approach as an impending cure for HIV.

Abstract 3619: The absence of cleaved caspase-3 in circulating tumor cells detected using a non-enrichment-based assay

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: The role of cleaved caspase-3 as an integral player in the apoptotic process of mammalian cells has prompted the investigation of an assay to detect cleaved caspase-3 in circulating tumor cells (CTCs) as one way to assess CTC viability. Methods: Using a previously developed non-enrichment based assay, nucleated blood cells and CTCs are adhered to a single slide. An antibody for cleaved caspase-3 was incorporated into the assay in addition to the existing antibodies for cytokeratin (to identify tumor cells) and CD45 (to identify white blood cells), as well as a nuclear stain (DAPI). The modified assay was initially tested on cultured MCF 10a cells spiked into normal donor blood. Before spiking, apoptosis was induced in these cells using staurosporine. Following validation of the assay in cell lines, the assay was performed using blood samples from 9 different breast cancer patients, as well as 3 different normal donor samples. Results: CTCs were detected in the patient samples as single cells, as well as clusters. The results of the assay showed that only 1 out of 896 detected CTCs was positive for cleaved caspase-3 (0.11%). Of these CTCs, 134 of them were found to be in clusters, and all 134 were negative for cleaved caspase-3, indicating that whether in clusters or as single cells, the vast majority of CTCs detected with this assay are not undergoing irreversible apoptotic processes via cleaved caspase-3. The results also showed that 0.40% of white blood cells in healthy donors expressed cleaved caspase-3, compared to 0.51% of the white cells in cancer patients, indicating that healthy donors and cancer patients express cleaved caspase-3 in their white blood cells at nearly the same rate. Conclusions: The morphologic characterization used in this platform to select healthy-appearing cells identifies a population of circulating tumor cells with virtually no evidence of caspase-related ongoing apoptosis, a finding that supports the morphologic impression of viability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3619. doi:1538-7445.AM2012-3619

The probacterial effect of type I interferon signaling requires its own negative regulator USP18

USP18 acts as a key effector molecule downstream of IFN-I signaling to promote primary and secondary bacterial infections. Turning the tables on interferon An early step in the host response to viral infection involves a burst of synthesis of type I interferons that allow cells to quickly fight back against the offending viruses. Shaabani et al. investigated how the same interferon-stimulated genes (ISGs) that usually help against viruses surprisingly dampen the host’s ability to resist many bacterial infections. Deletion of a single ISG known as Usp18 in mouse dendritic cells was sufficient to enhance host control of infections with two strains of Gram-positive bacteria. Normal induction of USP18 after infection impaired antibacterial responses mediated by tumor necrosis factor. These findings spotlight USP18 as a new potential target for therapeutics aimed at improving control of serious bacterial infections. Type I interferon (IFN-I) signaling paradoxically impairs host immune responses during many primary and secondary bacterial infections. Lack of IFN-I receptor reduces bacterial replication and/or bacterial persistence during infection with several bacteria. However, the mechanisms that mediate the adverse IFN-I effect are incompletely understood. Here, we show that Usp18, an interferon-stimulated gene that negatively regulates IFN-I signaling, is primarily responsible for the deleterious effect of IFN-I signaling during infection of mice with Listeria monocytogenes or Staphylococcus aureus. Mechanistically, USP18 promoted bacterial replication by inhibiting antibacterial tumor necrosis factor–α (TNF-α) signaling. Deleting IFNAR1 or USP18 in CD11c-Cre+ cells similarly reduced bacterial titers in multiple organs and enhanced survival. Our results demonstrate that inhibiting USP18 function can promote control of primary and secondary bacterial infection by enhancing the antibacterial effect of TNF-α, which correlates with induction of reactive oxygen species (ROS). These findings suggest that USP18 could be targeted therapeutically in patients to ameliorate disease caused by serious bacterial infections.

62. Aptamer-siRNA Conjugate Directed Transcriptional Gene Silencing in HIV-1 Infected T Cells

The primary standard of care against human immunodeficiency virus (HIV) -1 is highly active antiretroviral therapy which is plagued by toxicity and patient compliance. As such, alternative therapies are sought to provide sustained viral inhibition with reduced treatment intervention. One new potential approach, termed transcriptional gene silencing (TGS), involves the use of small non-coding RNAs (sncRNAs) to inhibit gene expression via the induction of concomitant silent-state epigenetic marks on histones and DNA. We previously demonstrated the ability to silence HIV-1 via TGS by targeting sites within the 5’ LTR promoter [1, 2]. However, the lack of efficient, systemic delivery of these RNAs to infected cells hinders successful clinical progression. To address this hurdle, we describe here the development of chimeric molecules based on the previously reported dual function of the gp120 (A-1) aptamer conjugated to 27-mer Dicer-substrate anti-HIV siRNA (dsiRNA) to deliver these sncRNAs to virally infected cells. The efficacy of such a system had been previously demonstrated both in vitro and in vivo to target selectively gp120-expressing cells and to deliver siRNAs for the inhibition of HIV-1 through post-transcriptional gene silencing (PTGS) [3]. To validate the ability for nuclear delivery of sncRNAs, we demonstrated, by fluorescent confocal microscopy, 15% nuclear delivery for our novel chimera-delivered dsiRNAs in a gp120 expressing CHO cells. Using nuclear-cytoplasmic fractionation of HIV-infected PBMCs, dsiRNAs targeting the susceptible regions within the 5’ LTR showed preferential nuclear localization when compared to a scrambled siRNAs. Additionally, two 800 nM doses at day 5 and 7 post-infection (p.i.) induced 10-fold suppression of viral p24 levels as measured at day 12 p.i. in CEM cells. The LTR-362 27-mer aptamer chimera proved more potent than A-1 tat/rev 27mer chimera, a positive control that inhibits HIV via PTGS. To validate the inhibition occurred via TGS, 5’-Azacytidine and trichostatin A were added at day 5 p.i, and resulted in transcriptional derepression by the A-1 LTR-263 27-mer, but not the A-1 Tat/Rev 27mer. Nuclear run-on analysis provided further confirmation of TGS inhibition. This is the first study to demonstrate RNA-induced TGS of an existing viral infection in both human PBMC and CEM cell lines following application of aptamer-siRNA chimeras.

119. HIV-1 Proviral DNA Excision and Deactivation Using a CRISPR/Cas9 “Nickase” Targeted to the 5’ and 3’ LTR

Permanent integration of the HIV-1 provirus in the host genome ensures a persistent supply of latently infected HIV cells capable of reactivation. This latent reservoir is recalcitrant to antiretroviral therapy (ART) making lifelong treatment the only option for patients. Therapies aimed at targeting the latent virus offer a promising approach to a “functional cure” of HIV/AIDS. Several gene editing technologies, including ZFNs, TALENs, Tre-recombinases and RNA-guided CRISPR/Cas9 tools have been used to disrupt the HIV-1 genome and suppress viral gene expression and replication in latently-infected cells. While CRISPR/Cas9 represents a powerful and facile DNA editing technology, toxicities associated with the Cas9 nuclease activity remains a concern. Here we explore the use of a “nickase” Cas9 (nCas9), which has a mutated RuvC I catalytic domain, and requires the combination of two adjacent small guide RNAs (sgRNAs) for on-target editing and specificity. We initially identified 9 sgRNA sequences targeted to conserved sites within the HIV TAR element, found on both the 5’ and 3’ LTRs. Using integrated and episomal LTR-driven luciferase reporters, we identified functional sgRNA “nickase pairs” capable of inducing an 80% reduction in gene expression, as well as excision of the proviral DNA. Using HIV-1 latency models, plasmids expressing nCas9 and the sgRNA pairs were nucleofected into HIV-1 integrated cell lines J1.1, CHME5 and U1 and treated with the histone deacetylase inhibitor trichostatin A (TSA) to activate infection. All treated cells showed significant reduction (up to 80% for J1.1) in infectious viral output. Cleavage was further verified using ChIP assays to detect DNA damage response proteins recruited to the targeted locus in all cell lines. Lastly, using a custom algorithm for detecting off-target cleavage sites (https://cm.jefferson.edu/Off-Spotter/), we identified 8 sites for each sgRNA within the “nickase pair”. No off-target editing was observed at these loci following high throughput NGS analysis. In conclusion, we showed that CRISPR/Cas technology represents a powerful tool at eradicating and/or deactivating the integrated HIV-1 provirus and that using the Cas9 nickase is just as effective as wild-type Cas9 but with the added advantage of providing a significant additional layer of safety for future in vivo applications.