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. 2021 Jul 21;11:14849. doi: 10.1038/s41598-021-94360-9

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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HCT116^CD276KO cells form tumor spheroids similar to HCT116^WT cells. (A) CD276 expression on HCT116^WT and HCT116^CD276KO cells was analyzed by flow cytometry after Crispr-Cas9 gene editing. (B) The heat-map shows the differentially regulated genes related to cell adhesion and migration in HCT116^CD276KO cells compared to HCT116^WT cells according to global transcriptome analysis. The bar shows the fold change (log2) of the ratio from tpm values (transcripts per kilobase million) of genes in HCT116^CD276KO cells to genes in HCT116^CD276WT cells. (C) Tumor spheroids formed from HCT116^WT and HCT116^CD276KO cells were monitored up to 12 days for shape. Representative microscope images are from 9-day and 12-day culture. (D) Quantification of viable tumor cells per spheroid was assessed by flow cytometry. 5–10 spheroids were pooled into a single sample for each group and before dissociation a defined number of negative compensation flow cytometry beads were added. Ratio of recorded bead count to the initial bead amount was used to back calculate viable tumor cells. The graph summarizes data (Mean + SD) from three independent experiments. (E) WST-1 viability assay treatment of spheroids over time. Each dot is one spheroid.