Single cell-based automated quantification of therapy responses of invasive cancer spheroids in organotypic 3D culture

Veelken, Cornelia; Bakker, Gert-Jan; Drell, David W.; Friedl, Peter

doi:10.1016/j.ymeth.2017.07.015

Cited by 30 publications

(18 citation statements)

References 29 publications

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“…However, adaptation and adoption of these models for HT-HC screens has been hampered by lack of standardization of both spheroid and matrix components and challenges associated with scaling. Other commonly cited shortcomings include: (1) time consuming nature of spheroid creation 10 , 22 ; (2) lack of user control of spheroid size, shape, and cell density 25 , 26 ; (3) reproducibility issues and questionable relevance of 3D matrix component 27 , 28 ; and (4) lack of rapid, reproducible methods for spheroid embedment 26 .…”

Section: Introductionmentioning

confidence: 99%

Development of a Novel 3D Tumor-tissue Invasion Model for High-throughput, High-content Phenotypic Drug Screening

Puls

Tan

Husain

et al. 2018

Sci Rep

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While much progress has been made in the war on cancer, highly invasive cancers such as pancreatic cancer remain difficult to treat and anti-cancer clinical trial success rates remain low. One shortcoming of the drug development process that underlies these problems is the lack of predictive, pathophysiologically relevant preclinical models of invasive tumor phenotypes. While present-day 3D spheroid invasion models more accurately recreate tumor invasion than traditional 2D models, their shortcomings include poor reproducibility and inability to interface with automated, high-throughput systems. To address this gap, a novel 3D tumor-tissue invasion model which supports rapid, reproducible setup and user-definition of tumor and surrounding tissue compartments was developed. High-cell density tumor compartments were created using a custom-designed fabrication system and standardized oligomeric type I collagen to define and modulate ECM physical properties. Pancreatic cancer cell lines used within this model showed expected differential invasive phenotypes. Low-passage, patient-derived pancreatic cancer cells and cancer-associated fibroblasts were used to increase model pathophysiologic relevance, yielding fibroblast-mediated tumor invasion and matrix alignment. Additionally, a proof-of-concept multiplex drug screening assay was applied to highlight this model’s ability to interface with automated imaging systems and showcase its potential as a predictive tool for high-throughput, high-content drug screening.

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Section: Introductionmentioning

confidence: 99%

Development of a Novel 3D Tumor-tissue Invasion Model for High-throughput, High-content Phenotypic Drug Screening

Puls

Tan

Husain

et al. 2018

Sci Rep

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“…3D spheroid culture: 3D spheroid culture was established as described 46 . Shortly, HT1080 fibrosarcoma cells from subconfluent culture were detached with 2mM EDTA (1 mM) and spheroids containing 1000 cells were formed with the hanging drop method.…”

Section: Attenuation Length Analysismentioning

confidence: 99%

Intravital Deep-Tumor Single-Beam 2-, 3- and 4-Photon Microscopy

Bakker

Weischer

Heidelin

et al. 2020

Preprint

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Three-photon excitation has recently been introduced to perform intravital microscopy in deep, previously inaccessible layers of the brain. The applicability of deep-tissue three-photon excitation in more heterogeneously structured, dense tissue types remains, however, unclear. Here we show that in tumors and bone, high-pulse-energy low-duty-cycle infrared excitation near 1300 and 1700 nm enables two- up to fourfold increased tissue penetration compared to conventional 2-photon excitation. Using a single laser line, simultaneous 2-, 3- and 4-photon processes are effectively induced, enabling the simultaneous detection of blue to far-red fluorescence together with second and third harmonic generation. This enables subcellular resolution at power densities in the focus that are not phototoxic to live cells and without color aberration. Thus, infrared high-pulse-energy low-duty-cycle excitation advances deep intravital microscopy in strongly scattering tissue and, in a single scan, delivers rich multi-parameter datasets from cells and complex organ structures.

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“…To avoid the possible concerns of cells touching the glass surface beneath the matrigel dam during invasion, a possible mixed 2.5-D 33 and 3-D cell invasion ( Figure 1C), we employed a full 3-D spheroid cell invasion assay (Figure 2A) 34 . Interestingly in such assays, pure cancer cells expressing cadherin-11 did not invade much out of the spheroids into the surrounding 3-D matrices.…”

Section: Cancer Cells Hijack Fibroblast Cell Bodies For Invasion Thromentioning

confidence: 99%

“…Cells were pre-labeled by using the Vybrant™ Multicolor Cell-Labeling Kit (DiO, DiI, DiD Solutions) (Invitrogen, Carlsbad, CA). Each spheroid was made by resuspending 8,000 cells in 15 µl media with 0.24% methyl cellulose (4,000 cpi, Sigma-Aldrich, St. Louis, MO) by the hanging drop method 34 . Each spheroid was then embedded in 400 µl extracellular matrix (ECM) with 6 mg/ml matrigel (Cultrex® BME, Trevigen, Gaithersburg, MD) and 1.4 mg/ml collagen (Corning® Collagen I, Corning, NY).…”

Section: -D Spheroid Cell Invasion Assay and Microfluidic Devicesmentioning

confidence: 99%

Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

Kang

Fan

et al. 2019

Preprint

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Current prevailing knowledge on EMT (epithelial mesenchymal transition) deems epithelial cells acquire the characters of mesenchymal cells to be capable of invading and metastasizing on their own.One of the signature events of EMT is called "cadherin switch", e.g. the epithelial E-cadherin switching to the mesenchymal Cadherin-11. Here, we report the critical events after EMT that cancer cells utilize cadherin-11 to hijack the endogenous cadherin-11 positive fibroblasts. Numerous 3-D cell invasion assays with high-content live cell imaging methods reveal that cadherin-11 positive cancer cells adhere to and migrate back and forth dynamically on the cell bodies of fibroblasts. By adhering to fibroblasts for co-invasion through 3-D matrices, cancer cells acquire higher invasion speed and velocity, as well as significantly elevated invasion persistence, which are exclusive characteristics of fibroblast invasion.Silencing cadherin-11 in cancer cells or in fibroblasts, or in both, significantly decouples such physical co-invasion. Additional bioinformatics studies and PDX (patient derived xenograft) studies link such cadherin-11 mediated cancer hijacking fibroblasts to the clinical cancer progression in human such as triple-negative breast cancer patients. Further animal studies confirm cadherin-11 mediates cancer hijacking fibroblasts in vivo and promotes significant solid tumor progression and distant metastasis.Moreover, overexpression of cadherin-11 strikingly protects 4T1-luc cells from implant rejection against firefly luciferase in immunocompetent mice. Overall, our findings report and characterize the critical post-EMT event of cancer hijacking fibroblasts in cancer progression and suggest cadherin-11 can be a therapeutic target for solid tumors with stroma. Our studies hence provide significant updates on the "EMT" theory that EMT cancer cells can hijack fibroblasts to achieve full mesenchymal behaviors in vivo for efficient homing, growth, metastasis and evasion of immune surveillance. Our studies also reveal that cadherin-11 is the key molecule that helps link cancer cells to stromal fibroblasts in the "Seed & Soil" theory.

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Single cell-based automated quantification of therapy responses of invasive cancer spheroids in organotypic 3D culture

Cited by 30 publications

References 29 publications

Development of a Novel 3D Tumor-tissue Invasion Model for High-throughput, High-content Phenotypic Drug Screening

Development of a Novel 3D Tumor-tissue Invasion Model for High-throughput, High-content Phenotypic Drug Screening

Intravital Deep-Tumor Single-Beam 2-, 3- and 4-Photon Microscopy

Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

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