Optimized Directed Virus Evolution to Accelerate the Generation of Oncolytic Coxsackievirus B3 Adapted to Resistant Colorectal Cancer Cells

Figures

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  Figure 1. Adaptation of PD-H to MC38 cells. (A) Virus growth kinetics. MC38 cells were infected with PD-H at MOI 0.1. Virus titer was determined by plaque assay 24 h, 48 h, and 72 h p.i. Shown are mean values ± SEM (n = 3). (B) Cell viability of MC38 cells after infection with PD-H. MC38 cells were infected with PD-H at the indicated MOI. Cell viability was measured by XTT assay after 24 h, 48 h, and 72 h. Cell viability was normalized to untreated cells (=100%). Shown are mean values ± SEM (n = 3). (C) Scheme of the serial passaging of PD-H on MC38 cells. For P1, cells were infected with PD-H at MOI 0.1 and incubated for 72 h. For further passaging to P10, the stated amount of a 1:10 dilution was added to fresh MC38 cells and incubated for the indicated time. (D) Development of viral titers during serial passaging of PD-H on MC38 cells. The virus titers were determined by plaque assay on HeLa cells. (E) Changes in the ability of PD-H to lyse MC38 cells during passaging. MC38 cells were infected with PD-H, P-2, P-4, P-5, P-6, P-8, and P-10 at MOI 1. Cell viability was measured by XTT assay 48 h after infection. Shown are mean values ± SEM (n = 3). Significance, * p < 0.05; ** p < 0.01;…

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  Figure 2. Sequence analysis of P-10 and comparison of P-10 with other CVB3 isolates. (A) Analysis of mutations that occurred in P-10. Shown are eight electropherograms of the sanger sequencing of viral RNA isolated from PD-H and P-10. Nucleotide substitutions are highlighted in yellow boxes. For seven mutations, a double signal in sanger sequencing was detected. (B) Scheme of the genome of PD-H. The location of the mutations, the mutated nucleotides, and the resulting amino acid substitutions detected in P-10 are indicated. (C) Comparison of the nucleotide and amino acid substitutions found in PD-10 with other CVB3 strains. Only a partial cDNA is available for the RD strain; no comparison was carried out for the mutations in 2A and 3A protein.

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  Figure 3. Replication and oncolytic activity of PD-MC38. (A) Virus growth kinetics. MC38 cells were infected with PD-H, P-10, and PD-MC38 at MOI 0.1. Virus titer was determined by plaque assay. Shown are mean values ± SEM (n = 3). There was no significant difference between P-10 and PD-MC38. Significance of PD-MC38 compared to PD-H, ** p < 0.01; *** p < 0.001. (B) Cell viability of MC38 cells after infection. MC38 cells were infected with PD-H, P-10, and PD-MC38 at the indicated MOI. Cell viability was measured by XTT assay 48 h later. Cell viability was normalized to untreated cells (=100%). Shown are mean values ± SEM (n = 3). Significance, * p < 0.05; ** p < 0.01; *** p < 0.001. (C) Representative images of viral plaques (white dots) of PD-H, P-10, and PD-MC38 on HeLa cell monolayers. (D) Graphical representation of plaque size of PD-H, P-10, and PD-MC38 shown under C. Shown are mean values ± SEM for n = 50 plaques.

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  Figure 4. Comparison of oncolytic efficiency of PD-MC38 and PD-H in different colorectal cancer cell lines. The colorectal cancer cell lines DLD-1, Colo320, Colo205, CaCo-2, Colon-26 and CT-26Luc were infected with PD-H and PD-MC38 at the indicated MOI; cell viability was measured 48 h after infection with XTT assay. Shown are mean values ± SEM (n = 3). Significance, * p < 0.05; ** p < 0.01; **** p < 0.0001.

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  Figure 5. Schematic procedure and time schedule of the generation of tumor cell-specific adapted oncolytic CVB3 strain PD-H.