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. 2022 Aug 3;22:845. doi: 10.1186/s12885-022-09941-2

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© The Author(s) 2022

Open AccessThis 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/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 4 — Simultaneous effects of immune checkpoint inhibitors (ICI) on primary large-sized tumors and secondary tumor-bearing mice. (A, B, C, D) Mice bearing MC-C (A) or LMM3 tumor (C) measuring 800 mm³ (1°MC-C or 1°LMM3, respectively) were challenged at day 17 with a secondary tumor implant (B, D) carried out in the contralateral flank (2°MC-C or 2°LMM3, respectively) and, simultaneously, treated with ICI (anti-CTLA-4 + anti-PD-L1). 5 × 10⁵ tumor cells were inoculated for MC-C and LMM3 primary tumors, and 2 × 10⁵ tumor cells for secondary implants. For each tumor, the figure shows a representative experiment (n = 4–6 mice per group) out of two experiments that rendered similar results. Data were expressed as mean (mm³) ± SEM of tumor volume. (E) Representative CFSE flow cytometric histograms of splenic T-cells from MC-C and LMM3 primary and secondary tumors bearing mice. (F, G) Percentage of the proliferation of splenic T cells from MC-C (F) and LMM3 (G) primary and secondary tumors bearing mice. Mice were treated with anti-CTLA-4 + anti-PD-L1 (immunized group), and non-treated mice served as control (control group). Each value represents the mean ± SEM of two assays. Each dose of anti-CTLA-4 and anti-PD-L1 was 100 μg per mouse. For simplicity, groups treated with anti-CTLA-4 alone and anti-PD-L1 alone were omitted. (H, I) Expression of phosphorylated (p)-38 (p38) by Western blotting. Macrophages (3 × 10⁶ cells) were collected surrounding the s.c. primary and secondary MC-C tumors 7 days post-secondary implant. Mice were treated with anti-CTLA-4 + anti-PD-L1 (immunized group), and non-treated mice served as control. A representative experiment is shown. The figure shows levels of p38 in the different groups, normalized with beta-actin densitometric units, representing the mean ± SE of three independent experiments. Statistical comparison between experimental groups and control: * p < 0.02; ** p < 0.01; *** p < 0.001. Immunized primary tumor (I 1°), immunized secondary tumor (I 2°), control primary tumor (C 2°) and control secondary tumor (C 2°).