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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, soluble PAI-1 is increased, while uPA is decreased. (A&B) Spheroids formed from HCT116^WT or HCT116^CD276KO were cocultured with Macrophages^WT or Macrophages^CD276KO (M-CSF) for 4 days and supernatants were collected. (A) Proteome Profiler Cytokine Arrays from equal volume of supernatants from Spheroid^WT or Spheroid^CD276KO cocultured with the same Macrophages^WT are presented here. (B) The graphs summarize the concentration of PAI-1 and uPA in the supernatants performed with relevant ELISA for indicated samples. (C) Spheroids were cultured without macrophages for the same period (5 days after initial seeding + 4 days after medium exchange). To collect enough supernatant, 5 wells from each group were pooled into one sample. PAI-1 and uPA concentrations were analyzed with ELISA. The data is from three independent experiments. (D-F) Spheroids formed from HCT116^WT or HCT116^CD276KO cells were cocultured with macrophages (7 days cultured in M-CSF supplemented medium, no gene editing) for 4 days. Quantifications for CD11b⁺ infiltrated macrophages were done by flow cytometry. 6–10 spheroids from each group were pooled into one sample, and before dissociating the spheroids, a defined number of beads (negative compensation beads for flow cytometry) was added. The ratio of recorded bead counts to initial bead amount was used to back calculate MDMs per spheroid. (D) Spheroids and macrophages were cultured in the absence or presence of recombinant uPA protein with indicated concentration. Six macrophage donors in total were tested. (E–F) Spheroid^WT cocultured with macrophages were treated with 20 µg/ml αCD276 antibody or isotype control. (E) Graph depicts the count of infiltrated macrophages per spheroid from six different donors. (F) The graph summarizes the concentration of PAI-1 in the supernatants from indicated samples (G) CD276 expression on macrophages and tumor cells treated with αCD276 antibody or isotype controls were analyzed. Histograms represent experiment with one macrophage donor and tumor cells in the same sample, and the graphs summarize the data from six donors and corresponding tumor cells. Dotted line indicates isotype treated controls, black line αCD276 antibody treated samples. Statistical analysis was done with paired t-test; **p < 0.01; ***p < 0.001.