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

Figure 3.

Figure 3

In vivo inhibition of macrophage MS4A4A delays CRC progression. (A–J) Tumour growth in mice injected subcutaneously (s.c.) with the MC38 or CT26 cell line and treated with siRNA (siMs4a4a) in vivo (n=5/group). (A) Schematic showing the treatment plan. (B) Tumour growth in MC38 tumour-bearing C57BL/6 mice. (C) Schematic showing the treatment plan. (D) Tumour growth in CT26 tumour-bearing BALB/c mice. (E–H) FACS analysis of specific molecule expression on tumour-infiltrating CD8+ T cells and TAMs from CT26 tumour-bearing BALB/c mice. (I) IHC staining with CD206-specific antibodies to detect CD206+ macrophage infiltration in subcutaneously transplanted MC38 or CT26 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. (J) Relative expression of the indicated genes determined by qRT-PCR. (K–L) C57BL/6 mice were implanted with MC38 cells and received siNC plus PBS liposome (PL), siMs4a4a plus PL, siNC plus clodronate liposome (CL) or siMs4a4a plus CL treatment (n=5/group). (K) Schematic showing the treatment plan. (L) Left: Representative images of tumours in mice from different treatment groups. Right: Tumour growth. (M–N) BALB/c mice were implanted with CT26 cells and received siNC plus PL, siMs4a4a plus PL, siNC plus CL or siMs4a4a plus CL treatment (n=5/group). (M) Schematic showing the treatment plan. (N) Left: Representative images of tumours in mice from different treatment groups. Right: Tumour growth. (O–Q) C57BL/6 mice were implanted with MC38 cells and received siNC plus IgG antibody, siMs4a4a plus IgG antibody, siNC plus anti-CD8 antibody or siMs4a4a plus anti-CD8 antibody treatment (n=5/group). (O) Schematic showing the treatment plan. (P) Left: Representative images of tumours in mice from different treatment groups. Right: Tumour growth. (Q) FACS analysis of the depletion efficiency for CD8+ T-cells in MC38 tumours. (R–T) BALB/c mice were implanted with CT26 cells and received siNC plus IgG antibody, siMs4a4a plus IgG antibody, siNC plus anti-CD8 antibody or siMs4a4a plus anti-CD8 antibody treatment (n=5/group). (R) Schematic showing the treatment plan. (S) Left: Representative images of tumours in mice from different treatment groups. Right: Tumour growth. (T) FACS analysis of the depletion efficiency for CD8+ T-cells in CT26 tumours. CRC, colorectal cancer; FACS, flow cytometry; FSC, forward scatter; IHC, immunohistochemistry; i.p., intraperitoneal injection; i.t., intratumoral injection; mAb, monoclonal antibody; PBS, phosphate buffer solution; qRT-PCR, quantitative real-time PCR; siMs4a4a, MS4A4A-specific siRNA; siNC, control siRNA; siRNA, small interfering RNA; TAMs, tumour-associated macrophages;