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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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In the absence of CD276 on tumor cells, lower number of macrophages infiltrates into spheroids. Macrophages (MDMs in M-CSF supplemented medium) were genetically edited with Crispr-Cas9 protocol by CD276 specific gRNA (Macrophage^CD276KO). Control cells were treated with scrambled gRNA (Macrophage^WT). Afterwards, cells were rested for 7 days in culture and then cocultured with spheroids for another 4 days. CD11b⁺ MDMs inside the spheroids were analyzed by flow cytometry. (A) A schematic representation of experimental set-up for tumor spheroid culture with macrophages. 5 days after seeding HCT116^WT or HCT116^CD276KO cells, Macrophages^WT or Macrophages^CD276KO were added to the spheroids. 4 days after coculture, a fraction of macrophages infiltrated into the spheroid (yellow), while some macrophages (orange) remain attached in the well, outside of the spheroid. (B) Microscopy images show spheroid-macrophage cocultures for indicated conditions. (C) Histogram displays CD276 expression on Macrophage^WT (black line) and Macrophage^CD276KO (red line) in the spheroids. The graph summarizes the percentage of Macrophage^CD276KO among infiltrated macrophages Spheroid^WT and Spheroid^CD276KOcoculture from four different donors. (D) The count of infiltrated MDMs per spheroid is depicted in the graph. Briefly, 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 MDMs. Macrophage^WT (black dots); Macrophage^CD276KO (red dots). Each dot represents one MDM donor. Statistical analysis was done with paired t-test; *p < 0.05. n.s. = not-significant E) Histograms display the surface expression of indicated markers on Macrophage^WT inside of Spheroid^WT (black line) versus Spheroid^CD276KO (red line) in comparison to not-infiltrated macrophages (dashed line) that were outside of the Spheroid^WT. The data represent one donor from 4 donors MDMs (Macrophage^WT).