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. 2023 Jul 28;72(12):2307–2320. doi: 10.1136/gutjnl-2022-329147

Figure 5.

Figure 5

Anti-MS4A4A mAb treatment delays CRC progression. (A–B) Mouse bone marrow-derived macrophages were treated with MC38-CM or MC38-CM+anti-MS4A4A mAb (10 µg/mL). FACS analyses of iNOS and CD206 expression by the macrophages (n=3). (C–D) Mouse bone marrow-derived macrophages (M) were cocultured with mouse splenocytes (S) and MC38 tumour cells (T) at a 1:1:1 ratio. The cells were treated with 10 µg/mL anti-MS4A4A mAb for 2 days. FACS analyses of Ki67 and IFN-γ expression by the CD8+ T-cells (n=3). (E–O) Tumour growth in mice injected subcutaneously (s.c.) with murine MC38 colon cancer cells treated with anti-MS4A4A mAb (n=5/group) (E) Schematic showing the treatment plan. (F–G) Tumour growth. (H–K) FACS analysis of specific molecule expression on tumour-infiltrating CD8+ T-cells and TAMs from MC38 tumour-bearing mice. (L) IHC staining with CD206-specific antibodies to detect CD206+ macrophage infiltration in subcutaneously transplanted MC38 tumours. The number of CD206-positive cells per high-power field (HPF) was counted in subcutaneous tumour sections from each group of mice. Five random HPFs were selected for analysis on each slide. (M–O) Relative expression of the indicated genes determined by qRT-PCR. Data depict the mean±SEM (n=3) and are representative of three independent experiments. (P–Q) Murine CT26 colon cancer cells were transplanted subcutaneously (s.c.) into BALB/c mice and treated with an isotype control or anti-MS4A4A mAb (n=3/group). t-SNE analysis of CyTOF data for immune cells from the isotype-treated and anti-MS4A4A mAb-treated CT26 tumours. (R) Histogram showing the quantification of tumour-infiltrating immune cells. (S) CyTOF analysis to compare the differences in the expression of specific molecules in TAMs within CT26 tumours between the control antibody-treated and MS4A4A mAb-treated groups. (T) CyTOF analysis to compare the differences in the expression of specific molecules in CD8+ T-cells within CT26 tumours between the control antibody-treated and MS4A4A mAb-treated groups. CM, conditioned medium; CRC, colorectal cancer; CyTOF, mass cytometry by time of flight; FACS, flow cytometry; FSC, forward scatter; IHC, immunohistochemistry; i.p., intraperitoneal injection; mAb, monoclonal antibody; qRT-PCR, quantitative real-time PCR; TAMs, tumour-associated macrophages; t-SNE, t-distributed stochastic neighbour embedding.