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Multilayer spheroid contractility assay. (a) Schematic representation of spheroid formation in a 96-well U-bottom well plate. The image on the right shows a spheroid formed at Day 1 post cell seeding. (b) Schematic representation of the process of spheroid encapsulation. Different layers can be distinguished: Layer 1 (collagen and beads), Layer 2 (collagen, beads, and spheroid), Layer 3 (fibrin and stromal cells). Stromal cells considered in the study are -ECs: Human Umbilical Vein Endothelial Cells, NFs: Normal Human Lung Fibroblasts, CAFs: Human Lung Cancer Associated Fibroblasts. The image on the right is a representative image of an A549 spheroid embedded with beads in collagen hydrogel at t = 0 h. (c) Experimental timeline. The image on the right is a representative image of an A549
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The remarkable contractility and force generation ability exhibited by cancer cells empower them to overcome the resistance and steric hindrance presented by a three-dimensional, interconnected matrix. Cancer cells disseminate by actively remodelling and deforming their extracellular matrix (ECM). The process of tumour growth and its ECM remodellin...
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... schematic in Fig. 1 was constructed using BioRender (biorender. com). Fig. 1 b shows the spheroid encapsulation process in collagen gel, followed by stromal cell embedding. The hydrogel solutions were kept on ice during the entire cell seeding process. Layer 1 in the 4-well dish (Nunc, 10507591, Fisher Scientific) was prepared by dispensing approximately ...
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... schematic in Fig. 1 was constructed using BioRender (biorender. com). Fig. 1 b shows the spheroid encapsulation process in collagen gel, followed by stromal cell embedding. The hydrogel solutions were kept on ice during the entire cell seeding process. Layer 1 in the 4-well dish (Nunc, 10507591, Fisher Scientific) was prepared by dispensing approximately 200 μl/well of unpolymerised collagen hydrogel solution ...
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... study the effect of stromal cells on biophysical parameters associated with cancer invasion, we modified a previously described spheroid contractility assay [6] to incorporate the stromal cell compartment. Briefly, multicellular spheroids were formed by suspending cancer cells in a U-shaped 96-well plate (Fig. 1 a). The spheroids, similar in size and aspect ( Supplementary Fig. 1), were mixed with 4 μm fluorescent fiducial marker beads in the unpolymerised collagen solution and poured onto a pre-coated collagen layer (also called layer 1) in a 24-well plate to polymerise. It was ensured that the spheroids were located near the centre of the well ...
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... multicellular spheroids were formed by suspending cancer cells in a U-shaped 96-well plate (Fig. 1 a). The spheroids, similar in size and aspect ( Supplementary Fig. 1), were mixed with 4 μm fluorescent fiducial marker beads in the unpolymerised collagen solution and poured onto a pre-coated collagen layer (also called layer 1) in a 24-well plate to polymerise. It was ensured that the spheroids were located near the centre of the well and the distance amongst each was relatively high (>3 mm) to avoid any edge-effects and spheroid-spheroid interactions, respectively, thus reducing variability in data collection. ...
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... cells appear to modulate the rate of cell dissemination by enhancing the invasiveness of cancer cells through stromal cellgenerated signals. In addition, we also observed a transition in the invasion pattern of cancer spheroids from collective to single cell invasion in the presence of stromal cells (Supplementary video 1, 2). ...
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... the trend of the collagen displacement pattern (observed with fiducial beads) was largely consistent with the radial alignment of collagen fibrils in the control. The angular distribution was also evaluated in terms of their kurtosis coefficients (degree of peakedness of a distribution) as shown in Fig. 4 c and Supplementary Fig. 10. For non-metastatic cells (Control: 2.74 ± 0.15, ECs: 2.37 ± 0.11, NFs: 2.33 ± 0.11, CAFs: 2.38 ± 0.09) and for metastatic cells (Control: 2.81 ± 0.11, ECs: 2.65 ± 0.14, NFs: 2.30 ± 0.11, CAFs: 2.45 ± 0.11), the coefficients were calculated. ...
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... growth cell culture media was collected after 24 h of the experiment and sent for the evaluation of a defined list of growth factors, cytokines, and chemokines. Fig. 4 d compares the fold change of the various cytokines/chemokines with the controls (Supplementary Fig. 11). We observe certain similarities in the overproduction of pro-inflammatory cytokines (such as IL-2, IL-6, IL-8, MCP-1, G-CSF) in the presence of stromal cells in both metastatic and non-metastatic spheroids. ...
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... further supplemented the media in the control case with IL-6 (10 ng/ml, Bio-Techne) to see if it elicits similar response to CAFs case. We observed a significant increase in the invasion area in both metastatic and non-metastatic cell lines, however, the corresponding radial displacements although lesser than the control were not significantly different in both cell lines (Supplementary Fig. 12). This maybe a result of the compounding effect of a number of soluble factors that results in the observation of the negative correlation between collagen deformation and spheroid invasion ability. ...
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... the stromal cells were not in physical contact with the cancer spheroids to alter the collagen matrix [38], we speculate that the upregulation of MCP-1 in the stromal cell cases suggests the induction of MMP production by cancer cells that enable the cancer cells to break down the collagen matrix to invade [39,40]. We identified a preliminary role of MMP-2 and MMP-9 activity in SK-MES-1 and A549 spheroids respectively, in all stromal cell cases ( Supplementary Fig. 13). Further quantification of MMPs and their corresponding inhibitors will give important insights into understanding their role in identifying the relationship between cancer cell invasion and collagen deformation. ...
