Figure 1.
JIMT1 cells form mass-shaped structures in 3D.
A. Time series of JIMT1 cells grown as either a Matrigel (MG) or polyHEMA (PH) culture. B. Zoom-in representative images of mass-shaped JIMT1 structures in Matrigel and polyHEMA. C. Growth rate comparison of 2D, Matrigel, and polyHEMA cultures. The growth rate was calculated by dividing the CTG (CellTiter-Glo) read by the starting cell number and expressed as % of 2D. Pictures were taken with IncuCyte.
Figure 2.
Drug screening shows differences between the culture models and responses to individual drugs vary greatly.
Cells cultured in 2D, Matrigel (MG4+7d, MG7d), or polyHEMA (PH4+7d, PH7d) were treated with colchicine, methotrexate, helenine or API-2 with the indicated concentrations for 7 days. The cells were treated either directly up on plating for 7 days (2D7d, MG7d, PH7d) or after 4 day pre-growth at 3D (MG4+7d, PH4+7d). CellTiter-Glo (CTG) was used as a cell viability measure; data are an average of three biological replicates. Error bars are STDEV, * indicate statistically (t-test) significant changes compared to the corresponding 2D7d concentration, * < 0.05, ** < 0.01, *** < 0.001.
Figure 3.
JIMT1 cells are more sensitive to drugs in Matrigel.
A. Average drug response of the JIMT1 cells in different cell culture models. The average responses of the 63 drugs are shown individually for the three highest concentrations (high (0.34-20 µM), medium (0.034-2 µM), and low (0.0034-0.2 µM) and across the concentrations. Response, p-value, and significance (** < 0.01, * < 0.05) compared to 2D are shown above the bars and average across the concentrations is shown above the line. Drug annotations and concentrations used for each drug are shown in File S1. B. Multiple linear regression analysis of individual drug screens comparing general drug effects to 2D. T-value shows the response difference to 2D (33.46 or 26.06). ***< 0.001, **<0.01, *<0.05.
Figure 4.
Gene expression of JIMT1 cells grown in Matrigel is closest to xenografts.
A. Dendrogram showing hierarchical clustering of genome-wide gene expression data. au = approximately unbiased p-value (%) in red, bp = bootstrap probability value (%) in blue, edge = cluster number in gray. Data shown are the average of two biological RNA replicates. Correlation of the replicates is shown in File S6 B. Venn diagram of ≥2-fold upregulated genes compared to 2D7d shows the number of shared genes in every combination of culture models. A list of common genes for each category is shown in File S4.
Figure 5.
HER2 signaling is altered in xenograft.
The most significantly altered upstream regulator in xenograft compared to 2D according to IPA; the HER2 pathway with arrows pointing to the top five changing genes shown in Table 1. Genes in red were upregulated in xenografts, and genes in green were downregulated. The 200 most upregulated and the 200 most downregulated genes in the xenografts compared to the 2D cultures were subjected to IPA analysis using the log-fold differences in expression as comparison values.
Figure 6.
Interferon pathway is activated in polyHEMA cultures.
A. The canonical pathway that changed the most in PH4d and in PH7d compared to 2D according to IPA; the interferon pathway with arrows pointing to the top five changing genes in PH4d is shown in Table 2. Genes in red were upregulated in PH4d. Two hundred of the most up- and downregulated genes in PH4d/PH7d compared to the 2D cultures were subjected to IPA analysis. B. The gene expression results were validated by measuring interferon-alpha and beta activity from JIMT1 cells by transfecting them with a Cignal ISRE Reporter dual-luciferase assay kit (Qiagen). The kit measures induction of the STAT1 and STAT2 components of the JAK/STAT-signal transduction pathways as a readout for interferon activity. The cells were grown in 2D, MG, or PH for 3 days before reporter plasmids and transfection reagents were added for 24 hours. *** p-value < 0.001.