High-speed live cell interferometry for screening bioprinted organoids

There is increasing interest in leveraging tumor organoids for high-throughput drug screenings to investigate tumor biology and identify therapeutic leads. However, functional precision medicine platforms are limited by the difficulties of creating, scaling, and analyzing physiological disease models. Most systems use manually seeded organoids and take advantage of destructive endpoint assays to rapidly characterize response to treatment. These approaches fail to capture transitory changes and intra-sample heterogeneity that underlies much of the clinically observed resistance to therapy. We therefore developed bioprinted tumor organoids linked to real-time growth pattern quantitation via high-speed live cell interferometry (HSLCI). We demonstrate that bioprinting gives rise to 3D organoid structures that preserve histology and gene expression. These are suitable for imaging with HSLCI, enabling accurate parallel mass measurements for thousands of bioprinted organoids. In drug screening experiments, HSLCI rapidly identifies organoids transiently or persistently sensitive or resistant to specific therapies. We show that our approach can provide detailed, actionable information to guide rapid therapy selection.

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High-speed synthetic aperture microscopy for live cell imaging

Optics Letters ◽

10.1364/ol.36.000148 ◽

2011 ◽

Vol 36 (2) ◽

pp. 148 ◽

Cited By ~ 76

Author(s):

Moonseok Kim ◽

Youngwoon Choi ◽

Christopher Fang-Yen ◽

Yongjin Sung ◽

Ramachandra R. Dasari ◽

...

Keyword(s):

Live Cell Imaging ◽

High Speed ◽

Cell Imaging ◽

Live Cell ◽

Synthetic Aperture ◽

Synthetic Aperture Microscopy

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Cost-Effective High-Speed, Three-Dimensional Live-Cell Imaging of HIV-1 Transfer at the T Cell Virological Synapse

SSRN Electronic Journal ◽

10.2139/ssrn.3956819 ◽

2021 ◽

Author(s):

Alice Sandmeyer ◽

Lili Wang ◽

Wolfgang Hübner ◽

Marcel Müller ◽

Benjamin Chen ◽

...

Keyword(s):

T Cell ◽

Live Cell Imaging ◽

High Speed ◽

Cell Imaging ◽

Three Dimensional ◽

Cost Effective ◽

Live Cell ◽

Virological Synapse ◽

Hiv 1

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Cerebrospinal Fluid Access Devices in Pediatric Diffuse Midline Glioma Patients: Literature Review and Evaluation of Institutional Practices

Neurosurgery ◽

10.1093/neuros/nyz310_650 ◽

2019 ◽

Vol 66 (Supplement_1) ◽

Author(s):

Daphne Li ◽

Wendy Stellpflug ◽

Amanda Muhs Saratsis

Keyword(s):

Cerebrospinal Fluid ◽

Liquid Biopsy ◽

Tumor Biology ◽

Significant Proportion ◽

Circulating Tumor Dna ◽

Response To Treatment ◽

Institutional Practices ◽

Medline Search ◽

Tumor Dna ◽

Diffuse Midline Glioma

Abstract INTRODUCTION Diffuse midline gliomas (DMG) are the number one cause of cancer death in children. H3K27M mutations occur in 80% of DMG, with distinct tumor biology and poorer response to treatment. H3K27M is detectable in cerebrospinal fluid (CSF) circulating tumor DNA (ctDNA), depending on CSF tumor proximity, and correlates with tumor volume and treatment response. Ventricular access devices (VAD) for serial CSF sampling (liquid biopsy) could therefore play a significant role in DMG management. Here, we set to characterize VAD placement practices in pediatric DMG. METHODS A retrospective review of patients <21 yr treated for DMG at our institution was performed (1984-2019). A MEDLINE search was conducted to identify reports of VAD placement in DMG. Full-text English reports of patients = 21 yr with VAD outcomes were analyzed. RESULTS A total of 106 DMG patients at our institution were identified. In total 49% had brainstem disease (n = 52). A total of 46.23% (n = 49) had VADs: 32.65% transient (ETV n = 5, EVD n = 11), 67.35% permanent (reservoir n = 7, shunt n = 26). A total of 17 had ETV at biopsy, 7 with concurrent reservoir placement. Of 10 ETV patients without initial reservoir, 5 ultimately underwent permanent VAD placement (reservoir n = 1, shunt n = 4). A total of 9 patients received EVDs at tumor surgery, 8 required EVD for acute hydrocephalus (HCP), with 6 converted to shunts. A total of 15 shunts were placed at tumor diagnosis: 4 required revision (27%). A total of 14 articles describing 240 DMG patients cited HCP in 22%-100%, with VAD placement in 22%-63%, and shunt-induced extraneural metastases in 7. Ventricular chemotherapy via indwelling reservoirs (481 patients) was associated with 29 infectious and 50 noninfectious complications. Standardized reservoir access procedures decreased infection rates. CONCLUSION VAD placement is clinically indicated in a significant proportion of pediatric DMG patients, with low morbidity. Ventricular CSF is superior to lumbar for ctDNA sequencing and quantification. VAD placement should therefore be considered to facilitate liquid biopsy in DMG.

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High-speed Live-cell Super-resolution Microscopy with Stochastically Switching Fluorophores

CLEO: 2013 ◽

10.1364/cleo_si.2013.ctu3n.1 ◽

2013 ◽

Author(s):

Fang Huang ◽

Tobias M. P. Hartwich ◽

Felix E. Rivera-Molina ◽

Yu Lin ◽

Jordan R. Myers ◽

...

Keyword(s):

High Speed ◽

Super Resolution ◽

Live Cell ◽

Super Resolution Microscopy

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Survival of advanced melanoma patients with normal LDH treated with oblimersen, temozolomide, and nab-paclitaxel

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.9080 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. 9080-9080

Author(s):

A. C. Pavlick ◽

P. Ott ◽

J. Escalon ◽

K. Madden ◽

E. Yepes ◽

...

Keyword(s):

Survival Data ◽

Caspase 3 ◽

Subcutaneous Tissue ◽

Tumor Biology ◽

Metastatic Tumor ◽

Advanced Melanoma ◽

Response To Treatment ◽

Antisense Therapy ◽

Melanoma Patients ◽

Ecog Ps

9080 Background: Oblimersen (OBL), temozolomide (TMZ), and abraxane (ABX) act synergistically in preclinical studies with melanoma cell lines. Bcl-2 antisense therapy in combination with dacarbazine was encouraging in advanced melanoma patients(pts) with normal LDH. Methods: Chemotherapy-naïve advanced melanoma pts (ECOG PS ≤ 2, baseline LDH ≤1.1 × ULN, measurable disease per RECIST) were enrolled on a phase I/II protocol. The treatment regimen consisted of 56-day cycles of OBL (7 mg/kg/d continuous IV infusion, d 1–7 and 22–28), TMZ (75 mg/m2/d, d 1–42), and ABX (175 mg/m2 in Cohort 1, 260 mg/m2 in Cohort 2, d 7 and 28). Immunohistochemical (IHC) staining for Bcl-2, Bcl-XL, BAK and caspase 3 was performed in pre- and post-therapy tumor samples. Serum shed collagen cryptic epitope levels were monitored. Results: 18 pts were treated (Cohort 1 = 14 pts [1–6 cycles];Cohort 2 = 4 pts [2–3 cycles]). Median age was 58 years (range: 34–78). Disease sites included liver (6), other visceral sites (10), skin, subcutaneous tissue, and lymph nodes (2). The overall survival (OS) was 14.7 months and showed a trend towards superiority when compared to both arms of the prior oblimersen trial (DTIC, OS 9.7 months, p = 0.078 and DTIC-OBL, OS 11.4 months, p = 0.31) in pts with the same LDH cut-off (Bedikian et al. JCO. 2006). 50% of pts survived > 1 year. One CR lasted 25+ mo, five PR (>50% tumor reduction) lasted > 2 cycles, and 7 SD lasted > 3 cycles. Five PD after 1 cycle were seen. One ocular melanoma pt survived 15 mo despite PD. Shed cryptic epitopes correlated with clinical response versus disease progression. Alteration of the tumor biology based on phenotypic changes in Bcl-2, Bcl-xL, BAK and caspase 3 correlated with response to treatment. Conclusions: Our data suggest that the combination of OBL, TMZ, and ABX is synergistic in advanced melanoma pts with normal LDH, possibly translating into improved OS compared to prior regimens with dacarbazine ± OBL. Biomarker studies support the rationale that Bcl-2 antisense therapy specifically impacts apoptotic signaling pathways in melanoma cells from metastatic tumor. The survival data in the limited number of pts enrolled in cohort 1 and 2 of this trial are encouraging; further exploration with this combination is underway using 1-hour infusions of OBL. No significant financial relationships to disclose.

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Advances in High-Speed Structured Illumination Microscopy

Frontiers in Physics ◽

10.3389/fphy.2021.672555 ◽

2021 ◽

Vol 9 ◽

Author(s):

Tianyu Zhao ◽

Zhaojun Wang ◽

Tongsheng Chen ◽

Ming Lei ◽

Baoli Yao ◽

...

Keyword(s):

Live Cell Imaging ◽

High Speed ◽

Cell Imaging ◽

Super Resolution ◽

Live Cell ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Light Power ◽

Recent Developments ◽

Super Resolution Microscopy

Super-resolution microscopy surpasses the diffraction limit to enable the observation of the fine details in sub-cellular structures and their dynamics in diverse biological processes within living cells. Structured illumination microscopy (SIM) uses a relatively low illumination light power compared with other super-resolution microscopies and has great potential to meet the demands of live-cell imaging. However, the imaging acquisition and reconstruction speeds limit its further applications. In this article, recent developments all targeted at improving the overall speed of SIM are reviewed. These comprise both hardware and software improvements, which include a reduction in the number of raw images, GPU acceleration, deep learning and the spatial domain reconstruction. We also discuss the application of these developments in live-cell imaging.

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AutoCLEM: An Automated Workflow for Correlative Live-Cell Fluorescence Microscopy and Cryo-Electron Tomography

Scientific Reports ◽

10.1038/s41598-019-55766-8 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 4

Author(s):

Xiaofeng Fu ◽

Jiying Ning ◽

Zhou Zhong ◽

Zandrea Ambrose ◽

Simon Charles Watkins ◽

...

Keyword(s):

Electron Microscopy ◽

High Speed ◽

Electron Tomography ◽

Light And Electron Microscopy ◽

Live Cell ◽

Host Cells ◽

Spatiotemporal Information ◽

Fluorescence Light ◽

Correlation Process ◽

Electron Imaging

AbstractCorrelative light and electron microscopy (CLEM) combines the strengths of both light and electron imaging modalities and enables linking of biological spatiotemporal information from live-cell fluorescence light microscopy (fLM) to high-resolution cellular ultra-structures from cryo-electron microscopy and tomography (cryoEM/ET). This has been previously achieved by using fLM signals to localize the regions of interest under cryogenic conditions. The correlation process, however, is often tedious and time-consuming with low throughput and limited accuracy, because multiple correlation steps at different length scales are largely carried out manually. Here, we present an experimental workflow, AutoCLEM, which overcomes the existing limitations and improves the performance and throughput of CLEM methods, and associated software. The AutoCLEM system encompasses a high-speed confocal live-cell imaging module to acquire an automated fLM grid atlas that is linked to the cryoEM grid atlas, followed by cryofLM imaging after freezing. The fLM coordinates of the targeted areas are automatically converted to cryoEM/ET and refined using fluorescent fiducial beads. This AutoCLEM workflow significantly accelerates the correlation efficiency between live-cell fluorescence imaging and cryoEM/ET structural analysis, as demonstrated by visualizing human immunodeficiency virus type 1 (HIV-1) interacting with host cells.

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The Gastrointestinal Tumor Microenvironment: An Updated Biological and Clinical Perspective

Journal of Oncology ◽

10.1155/2019/6240505 ◽

2019 ◽

Vol 2019 ◽

pp. 1-22 ◽

Cited By ~ 1

Author(s):

Silvia Batista ◽

Ana C. Gregório ◽

Andreia Hanada Otake ◽

Nuno Couto ◽

Bruno Costa-Silva

Keyword(s):

Extracellular Matrix ◽

Immune Modulation ◽

Tumor Biology ◽

Tumor Development ◽

Therapeutic Strategies ◽

Clinical Perspective ◽

Gastrointestinal Cancers ◽

Gastrointestinal Tumor ◽

Resistance To Therapy ◽

Potential Benefits

Gastrointestinal cancers are still responsible for high numbers of cancer-related deaths despite advances in therapy. Tumor-associated cells play a key role in tumor biology, by supporting or halting tumor development through the production of extracellular matrix, growth factors, cytokines, and extracellular vesicles. Here, we review the roles of these tumor-associated cells in the initiation, angiogenesis, immune modulation, and resistance to therapy of gastrointestinal cancers. We also discuss novel diagnostic and therapeutic strategies directed at tumor-associated cells and their potential benefits for the survival of these patients.

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