All Publications

  • Detection of effusion tumor cells under different storage and processing conditions. Cancer cytopathology Libert, D. M., Zhu, Y., Wang, A., Allard, G. M., Cheng-Yi Lowe, A. 2024


    Circulating tumor cells (CTCs) shed into blood provide prognostic and/or predictive information. Previously, the authors established an assay to detect carcinoma cells from pleural fluid, termed effusion tumor cells (ETCs), by employing an immunofluorescence-based CTC-identification platform (RareCyte) on air-dried unstained ThinPrep (TP) slides. To facilitate clinical integration, they evaluated different slide processing and storage conditions, hypothesizing that alternative comparable conditions for ETC detection exist.The authors enumerated ETCs on RareCyte, using morphology and mean fluorescence intensity (MFI) cutoffs of >100 arbitrary units (a.u.) for epithelial cellular adhesion molecule (EpCAM) and <100 a.u. for CD45. They analyzed malignant pleural fluid from three patients under seven processing and/or staining conditions, three patients after short-term storage under three conditions, and seven samples following long-term storage at -80°C. MFI values of 4',6-diamidino-2-phenylindol, cytokeratin, CD45, and EpCAM were compared.ETCs were detected in all conditions. Among the different processing conditions tested, the ethanol-fixed, unstained TP was most similar to the previously established air-dried, unstained TP protocol. All smears and Pap-stained TPs had significantly different marker MFIs from the established condition. After short-term storage, the established condition showed comparable results, but ethanol-fixed and Pap-stained slides showed significant differences. ETCs were detectable after long-term storage at -80°C in comparable numbers to freshly prepared slides, but most marker MFIs were significantly different.It is possible to detect ETCs under different processing and storage conditions, lending promise to the application of this method in broader settings. Because of decreased immunofluorescence-signature distinctions between cells, morphology may need to play a larger role.

    View details for DOI 10.1002/cncy.22803

    View details for PubMedID 38373107

  • Comprehensive epithelial biomarker analysis of malignant mesothelioma: EpCAM positivity is a potential diagnostic pitfall. Cancer cytopathology Zhu, Y., Moore, S., Wang, A., George, E., Allard, G. M., Libert, D. M., Lowe, A. C. 2023


    Epithelial cell adhesion molecule (EpCAM) is frequently used to distinguish carcinoma from background mesothelial cells during cytologic examination of body cavity fluids. Previously, the authors identified one malignant mesothelioma case with strong and diffuse membranous EpCAM staining, making it indistinguishable from carcinoma.In this study, the authors evaluated all available effusion specimens from patients with malignant mesothelioma, including the above-mentioned index case, obtained at Stanford Health Care, from 2011 to 2021 (N = 17) as well as control cases (N = 5). Analyses included an immunohistochemistry (IHC) assay for EpCAM and claudin-4, a multiplexed immunofluorescent (IF) assay for EpCAM, and an RNA in situ hybridization assay targeting EpCAM.The authors detected EpCAM positivity of variable intensity and percentage in four malignant mesothelioma cases (23.5%; although only two showed positivity for the epithelial-specific IHC marker MOC31 in ≥40% of cells) and claudin-4 negativity in all cases, with two cases displaying focal and weak claudin-4 staining in <1% of cells. Multiplexed IF staining on the cases with EpCAM IHC positivity showed strong, membranous EpCAM staining in one of four cases. RNA in situ hybridization also was used to assess the correlation between EpCAM positivity by IHC/IF and RNA expression levels. Strong EpCAM RNA expression was detected in the three malignant mesothelioma cases.The current findings revealed that a subset of epithelioid malignant mesothelioma cases mimic or exhibit the immunophenotypic features of carcinoma when evaluating for EpCAM only. Additional biomarker testing, such as claudin-4, may help avoid this potential pitfall to yield accurate diagnoses.

    View details for DOI 10.1002/cncy.22706

    View details for PubMedID 37069606

  • Single-cell Retrieval from Clinical Cytology Slides Under Morphologic Guidance Facilitates Future Comprehensive Genomic Profiling from Paucicellular Samples Zhu, Y., Aragon, A., Wang, A., Gonzalez-pena, V., Gawad, C., Lowe, A. ELSEVIER SCIENCE INC. 2023: S388
  • Detecting Effusion Tumor Cells (ETCs) Under Different Storage and Processing Conditions Libert, D., Zhu, Y., Wang, A., Lowe, A. ELSEVIER SCIENCE INC. 2023: S328-S329
  • Structure-Function Relationship for a Divergent Atg8 Protein Required for a Nonautophagic Function in Apicomplexan Parasites. mBio Walczak, M., Meister, T. R., Nguyen, H. M., Zhu, Y., Besteiro, S., Yeh, E. 2023: e0364221


    Atg8 family proteins are highly conserved eukaryotic proteins with diverse autophagy and nonautophagic functions in eukaryotes. While the structural features required for conserved autophagy functions of Atg8 are well established, little is known about the molecular changes that facilitated acquisition of divergent, nonautophagic functions of Atg8. The malaria parasite Plasmodium falciparum offers a unique opportunity to study nonautophagic functions of Atg8 family proteins because it encodes a single Atg8 homolog whose only essential function is in the inheritance of an unusual secondary plastid called the apicoplast. Here, we used functional complementation to investigate the structure-function relationship for this divergent Atg8 protein. We showed that the LC3-interacting region (LIR) docking site (LDS), the major interaction interface of the Atg8 protein family, is required for P. falciparum Atg8 (PfAtg8) apicoplast localization and function, likely via Atg8 lipidation. On the other hand, another region previously implicated in canonical Atg8 interactions, the N-terminal helix, is not required for apicoplast-specific PfAtg8 function. Finally, our investigations at the cellular level demonstrate that the unique apicomplexan-specific loop, previously implicated in interaction with membrane conjugation machinery in recombinant protein-based in vitro assays, is not required for membrane conjugation nor for the apicoplast-specific effector function of Atg8 in both P. falciparum and related Apicomplexa member Toxoplasma gondii. These results suggest that the effector function of apicomplexan Atg8 is mediated by structural features distinct from those previously identified for macroautophagy and selective autophagy functions. IMPORTANCE The most extensively studied role of Atg8 proteins is in autophagy. However, it is clear that they have other nonautophagic functions critical to cell function and disease pathogenesis that are so far understudied compared to their canonical role in autophagy. Mammalian cells contain multiple Atg8 paralogs that have diverse, specialized functions. Gaining molecular insight into their nonautophagic functions is difficult because of redundancy between the homologs and their role in both autophagy and nonautophagic pathways. Malaria parasites such as Plasmodium falciparum are a unique system to study a novel, nonautophagic function of Atg8 separate from its role in autophagy: they have only one Atg8 protein whose only essential function is in the inheritance of the apicoplast, a unique secondary plastid organelle. Insights into the molecular basis of PfAtg8's function in apicoplast biogenesis will have important implications for the evolution of diverse nonautophagic functions of the Atg8 protein family.

    View details for DOI 10.1128/mbio.03642-21

    View details for PubMedID 36625582

  • Immunofluorescent and molecular characterization of effusion tumor cells reveal cancer site‐of‐origin and disease‐driving mutations Cancer Cytopathology Zhu, Y., Wang, A., Allard, G. M., Nordberg, J. J., Nair, R. V., Kunder, C. A., Lowe, A. C. 2022

    View details for DOI 10.1002/cncy.22610

  • Identification and characterization of effusion tumor cells (ETCs) from remnant pleural effusion specimens. Cancer cytopathology Zhu, Y., Allard, G. M., Ericson, N. G., George, T. C., Kunder, C. A., Lowe, A. C. 2021


    Cancer is a leading cause of death worldwide, and patients may have advanced disease when diagnosed. Targeted therapies guided by molecular subtyping of cancer can benefit patients significantly. Pleural effusions are frequently observed in patients with metastatic cancer and are routinely removed for therapeutic purposes; however, effusion specimens have not been recognized as typical substrates for clinical molecular testing because of frequent low tumor cellularity.Excess remnant pleural effusion samples (N = 25) from 21 patients with and without suspected malignancy were collected at Stanford Health Care between December 2019 and November 2020. Samples were processed into ThinPrep slides and underwent novel effusion tumor cell (ETC) analysis. The ETC results were compared with the original clinical diagnoses for accuracy. A subset of confirmed ETCs was further isolated and processed for molecular profiling to identify cancer driver mutations. All samples were obtained with Institutional Review Board approval.The authors established novel quantitative standards to identify ETCs and detected epithelial malignancy with 89.5% sensitivity and 100% specificity in the pleural effusion samples. Molecular profiling of confirmed ETCs (pools of 5 cells evaluated) revealed key pathogenic mutations consistent with clinical molecular findings.In this study, the authors developed a novel ETC-testing assay that detected epithelial malignancies in pleural effusions with high sensitivity and specificity. Molecular profiling of 5 ETCs showed promising concordance with the clinical molecular findings. To promote cancer subtyping and guide treatment, this ETC-testing assay will need to be validated in larger patient cohorts to facilitate integration into cytologic workflow.

    View details for DOI 10.1002/cncy.22483

    View details for PubMedID 34171181

  • Multiplexed fluorescence in situ hybridization-based detection of circulating tumor cells: A novel liquid-based technology to facilitate accurate and early identification of non-small cell lung cancer patients. Cancer cytopathology Zhu, Y., Lowe, A. C. 2020

    View details for DOI 10.1002/cncy.22277

    View details for PubMedID 32320525

  • An in vitro Microscopy-based Assay for Microtubule-binding and Microtubule-crosslinking by Budding Yeast Microtubule-associated Protein BIO-PROTOCOL Zhu, Y., Tan, W., Lee, W. 2018; 8 (23)


    In this protocol, we describe a simple microscopy-based method to assess the interaction of a microtubule-associated protein (MAP) with microtubules. The interaction between MAP and microtubules is typically assessed by a co-sedimentation assay, which measures the amount of MAP that co-pellets with microtubules by centrifugation, followed by SDS-PAGE analysis of the supernatant and pellet fractions. However, MAPs that form large oligomers tend to pellet on their own during the centrifugation step, making it difficult to assess co-sedimentation. Here we describe a microscopy-based assay that measures microtubule binding by direct visualization using fluorescently-labeled MAP, solving the limitations of the co-sedimentation assay. Additionally, we recently reported quantification of microtubule bundling by measuring the thickness of individual microtubule structures observed in the microscopy-based assay, making the protocol more advantageous than the traditional microtubule co-pelleting assay.

    View details for DOI 10.21769/BioProtoc.3110

    View details for Web of Science ID 000458028500014

    View details for PubMedID 30733975

    View details for PubMedCentralID PMC6363367

  • Microtubule cross-linking activity of She1 ensures spindle stability for spindle positioning JOURNAL OF CELL BIOLOGY Zhu, Y., An, X., Tomaszewski, A., Hepler, P. K., Lee, W. 2017; 216 (9): 2759–75


    Dynein mediates spindle positioning in budding yeast by pulling on astral microtubules (MTs) from the cell cortex. The MT-associated protein She1 regulates dynein activity along astral MTs and directs spindle movements toward the bud cell. In addition to localizing to astral MTs, She1 also targets to the spindle, but its role on the spindle remains unknown. Using function-separating alleles, live-cell spindle assays, and in vitro biochemical analyses, we show that She1 is required for the maintenance of metaphase spindle stability. She1 binds and cross-links MTs via a C-terminal MT-binding site. She1 can also self-assemble into ring-shaped oligomers. In cells, She1 stabilizes interpolar MTs, preventing spindle deformations during movement, and we show that this activity is regulated by Ipl1/Aurora B phosphorylation during cell cycle progression. Our data reveal how She1 ensures spindle integrity during spindle movement across the bud neck and suggest a potential link between regulation of spindle integrity and dynein pathway activity.

    View details for DOI 10.1083/jcb.201701094

    View details for Web of Science ID 000409075500019

    View details for PubMedID 28794129

    View details for PubMedCentralID PMC5584168

  • The role of plus TIPs in directional tip expansion MOLECULAR MICROBIOLOGY Zhu, Y., Lee, W. 2014; 94 (3): 486–89


    Aspergillus nidulans is an ideal model to study nuclear migration and intracellular transport by dynein and kinesin owing to its long neuron-like hyphae, conserved transport mechanisms, and powerful genetics. In this organism, as in other filamentous fungi, microtubules have been implicated in patterning cell shape through polarized tip growth - the hallmark mode of growth that generates the elongated hyphae. Exactly how microtubules regulate tip growth is incompletely understood and remains a fascinating question for various cell types, such as pollen tubes and root hairs. Zeng et al. (2014) describe important new findings in A. nidulans regarding the role of EBA, the master regulator of microtubule plus end-tracking proteins, in specifying microtubule dynamics required for directional tip growth at the hyphal tip.

    View details for DOI 10.1111/mmi.12791

    View details for Web of Science ID 000344465000002

    View details for PubMedID 25213368

    View details for PubMedCentralID PMC4213288