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Monday, October 21 • 2:30pm - 3:00pm
Poster Presentation #6-Real-Time Live-Cell Analysis of 3D Tumour Spheroid Invasion

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Cell invasion, a hallmark of malignant cancers, plays a key role in tumour dissemination and metastasis and is responsible for the higher incidence of cancer-related deaths. The ability of tumour cells to form a metastatic tumour primarily involves cell morphological reorganization and degradation of the surrounding extracellular matrix (ECM).Conventional in vitro invasion models, such as the filter based transwell invasion assay (2D) are widely used to assess tumour cell invasion.However, lack of comprehensive cell-ECM interactions may limit their physiological relevance. Here, we provide a simple 3D tumour spheroid invasion model to recapitulate key features of the metastatic phenotype and demonstrate use of live cell imaging to kinetically acquire and quantify 3D single spheroid invasion.



To exemplify the assay platform and assess the metastatic potential of tumour cells in 3D, U87-MG, A172 and HT1080 spheroids were formed for 72 h in Ultra-Low Attachment round bottom 96-well plates and subsequently embedded in Matrigel (4.5 mg/mL). Spheroid invasive properties were measured using novel brightfield image analysis software (IncuCyte® Live-Cell Analysis System) which tracks and quantifies changes in spheroid size (whole spheroid area or invading cell area) over time. To account for variation in size following spheroid formation, whole spheroid area was normalized to spheroid size at t = 0 h and automatically plotted. Cell type specific temporal invasive potential was observed. At 168 h, whole spheroid area of U87-MG spheroids (ratio: 32) increased to approximately 2x and 4x the size of HT1080 (ratio: 18) and A172 (ratio: 8) spheroids respectively. HT1080 spheroids embedded in varying concentrations of Matrigel (1.13 – 4.5 mg/mL) demonstrated that spheroid invasive capacity is Matrigel concentration-dependent. While cell motility was reduced at the highest Matrigel concentration, marked density and elongation of invadopodia was seen. Using available metrics and analysis approaches we could determine the effects of known anti-metastatic compounds on spheroid invasion and spheroid proliferation. Cytochalasin D (300 nM) and PP242 (30 µM) inhibitors caused comparable reduction of U87-MG spheroid size (90% and 80% respectively). However, marked inhibition of invadopodia was only evident with Cytochalasin D. Subtraction of the invading cell area from the whole spheroid area provided a measure of the spheroid body size and revealed the anti-proliferative rather than anti-invasive properties of PP242. A separate but identical study conducted in the absence of Matrigel further supported the anti-proliferative effects of PP242 but not Cytochalasin D.

These data demonstrate the capability to kinetically visualise and quantify 3D single spheroid invasion assays and illustrates the utility of this approach for high throughput real-time anti-metastatic compound profiling. This model can be further developed with the incorporation of stromal cells to address additional aspects of tumour cell invasion.

Speakers
MO

Miniver Oliver

Research Scientist, Sartorius
Research Scientist at Sartorius, working as part of the Applications group for the IncuCyte platform. Expertise indeveloping 3D cellular assays for quantitative live cell analysis.


Monday October 21, 2019 2:30pm - 3:00pm
Sherry Coutu Seminar Suite Foyer