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1. PD-1 suppresses the maintenance of cell couples between cytotoxic T cells and target tumor cells within the tumor

   https://doi.org/10.1126/scisignal.aau4518  

   Ambler, Rachel ; Edmunds, Grace L. ; Tan, Sin Lih ; Cirillo, Silvia ; Pernes, Jane I. ; Ruan, Xiongtao ; Huete-Carrasco, Jorge ; Wong, Carissa C. W. ; Lu, Jiahe ; Ward, Juma ; et al ( September 2020 , Science Signaling)

   The killing of tumor cells by CD8⁺T cells is suppressed by the tumor microenvironment, and increased expression of inhibitory receptors, including programmed cell death protein-1 (PD-1), is associated with tumor-mediated suppression of T cells. To find cellular defects triggered by tumor exposure and associated PD-1 signaling, we established an ex vivo imaging approach to investigate the response of antigen-specific, activated effector CD8⁺tumor-infiltrating lymphocytes (TILs) after interaction with target tumor cells. Although TIL–tumor cell couples readily formed, couple stability deteriorated within minutes. This was associated with impaired F-actin clearing from the center of the cellular interface, reduced Ca²⁺signaling, increased TIL locomotion, and impaired tumor cell killing. The interaction of CD8⁺T lymphocytes with tumor cell spheroids in vitro induced a similar phenotype, supporting a critical role of direct T cell–tumor cell contact. Diminished engagement of PD-1 within the tumor, but not acute ex vivo blockade, partially restored cell couple maintenance and killing. PD-1 thus contributes to the suppression of TIL function by inducing a state of impaired subcellular organization.

    

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2. New Labyrinth Microfluidic Device Detects Circulating Tumor Cells Expressing Cancer Stem Cell Marker and Circulating Tumor Microemboli in Hepatocellular Carcinoma

   https://doi.org/10.1038/s41598-019-54960-y  

   Wan, Shanshan ; Kim, Tae Hyun ; Smith, Kaylee J. ; Delaney, Ryan ; Park, G-Su ; Guo, Hui ; Lin, Eric ; Plegue, Thomas ; Kuo, Ning ; Steffes, John ; et al ( December 2019 , Scientific Reports)

   Hepatocellular Carcinoma (HCC) is one of the most lethal cancers with a high mortality and recurrence rate. Circulating tumor cell (CTC) detection offers various opportunities to advance early detection and monitoring of HCC tumors which is crucial for improving patient outcome. We developed and optimized a novel Labyrinth microfluidic device to efficiently isolate CTCs from peripheral blood of HCC patients. CTCs were identified in 88.1% of the HCC patients over different tumor stages. The CTC positivity rate was significantly higher in patients with more advanced HCC stages. In addition, 71.4% of the HCC patients demonstrated CTCs positive for cancer stem cell marker, CD44, suggesting that the major population of CTCs could possess stemness properties to facilitate tumor cell survival and dissemination. Furthermore, 55% of the patients had the presence of circulating tumor microemboli (CTM) which also correlated with advanced HCC stage, indicating the association of CTM with tumor progression. Our results show effective CTC capture from HCC patients, presenting a new method for future noninvasive screening and surveillance strategies. Importantly, the detection of CTCs with stemness markers and CTM provides unique insights into the biology of CTCs and their mechanisms influencing metastasis, recurrence and therapeutic resistance.

    

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3. Autofluorescence imaging identifies tumor cell‐cycle status on a single‐cell level

   https://doi.org/10.1002/jbio.201600276  

   Heaster, Tiffany M. ; Walsh, Alex J. ; Zhao, Yue ; Hiebert, Scott W. ; Skala, Melissa C. ( May 2017 , Journal of Biophotonics)

   The goal of this study is to validate fluorescence intensity and lifetime imaging of metabolic co‐enzymes NAD(P)H and FAD (optical metabolic imaging, or OMI) as a method to quantify cell‐cycle status of tumor cells. Heterogeneity in tumor cell‐cycle status (e. g. proliferation, quiescence, apoptosis) increases drug resistance and tumor recurrence. Cell‐cycle status is closely linked to cellular metabolism. Thus, this study applies cell‐level metabolic imaging to distinguish proliferating, quiescent, and apoptotic populations. Two‐photon microscopy and time‐correlated single photon counting are used to measure optical redox ratio (NAD(P)H fluorescence intensity divided by FAD intensity), NAD(P)H and FAD fluorescence lifetime parameters. Redox ratio, NAD(P)H and FAD lifetime parameters alone exhibit significant differences (p<0.05) between population means. To improve separation between populations, linear combination models derived from partial least squares ‐ discriminant analysis (PLS‐DA) are used to exploit all measurements together. Leave‐one‐out cross validation of the model yielded high classification accuracies (92.4 and 90.1 % for two and three populations, respectively). OMI and PLS‐DA also identifies each sub‐population within heterogeneous samples. These results establish single‐cell analysis with OMI and PLS‐DA as a label‐free method to distinguish cell‐cycle status within intact samples. This approach could be used to incorporate cell‐level tumor heterogeneity in cancer drug development.magnified image

    

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4. Development and initial clinical testing of a multiplexed circulating tumor cell assay in patients with clear cell renal cell carcinoma

   https://doi.org/10.1002/1878-0261.12931  

   Bade, Rory M. ; Schehr, Jennifer L. ; Emamekhoo, Hamid ; Gibbs, Benjamin K. ; Rodems, Tamara S. ; Mannino, Matthew C. ; Desotelle, Joshua A. ; Heninger, Erika ; Stahlfeld, Charlotte N. ; Sperger, Jamie M. ; et al ( March 2021 , Molecular Oncology)

   Although therapeutic options for patients with advanced renal cell carcinoma (RCC) have increased in the past decade, no biomarkers are yet available for patient stratification or evaluation of therapy resistance. Given the dynamic and heterogeneous nature of clear cell RCC (ccRCC), tumor biopsies provide limited clinical utility, but liquid biopsies could overcome these limitations. Prior liquid biopsy approaches have lacked clinically relevant detection rates for patients with ccRCC. This study employed ccRCC‐specific markers, CAIX and CAXII, to identify circulating tumor cells (CTC) from patients with metastatic ccRCC. Distinct subtypes of ccRCC CTCs were evaluated for PD‐L1 and HLA‐I expression and correlated with patient response to therapy. CTC enumeration and expression of PD‐L1 and HLA‐I correlated with disease progression and treatment response, respectively. Longitudinal evaluation of a subset of patients demonstrated potential for CTC enumeration to serve as a pharmacodynamic biomarker. Further evaluation of phenotypic heterogeneity among CTCs is needed to better understand the clinical utility of this new biomarker.

    

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5. Fundamentals of integrated ferrohydrodynamic cell separation in circulating tumor cell isolation

   https://doi.org/10.1039/D1LC00119A  

   Liu, Yang ; Zhao, Wujun ; Cheng, Rui ; Harris, Bryana N. ; Murrow, Jonathan R. ; Hodgson, Jamie ; Egan, Mary ; Bankey, Anastacia ; Nikolinakos, Petros G. ; Laver, Travis ; et al ( May 2021 , Lab on a Chip)

   Methods to separate circulating tumor cells (CTCs) from blood samples were intensively researched in order to understand the metastatic process and develop corresponding clinical assays. However current methods faced challenges that stemmed from CTCs' heterogeneity in their biological markers and physical morphologies. To this end, we developed integrated ferrohydrodynamic cell separation (iFCS), a scheme that separated CTCs independent of their surface antigen expression and physical characteristics. iFCS integrated both diamagnetophoresis of CTCs and magnetophoresis of blood cells together via a magnetic liquid medium, ferrofluid, whose magnetization could be tuned by adjusting its magnetic volume concentration. In this paper, we presented the fundamental theory of iFCS and its specific application in CTC separation. Governing equations of iFCS were developed to guide its optimization process. Three critical parameters that affected iFCS's cell separation performance were determined and validated theoretically and experimentally. These parameters included the sample flow rate, the volumetric concentration of magnetic materials in the ferrofluid, and the gradient of the magnetic flux density. We determined these optimized parameters in an iFCS device that led to a high recovery CTC separation in both spiked and clinical samples. 

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