Extended Data Fig. 4: Chemosensor genetic deletion re-educate TAMs.
From: Chemosensor receptors are lipid-detecting regulators of macrophage function in cancer
a) Experimental scheme of OLFR644 and VMN2R29 genetic deletion on primary bone marrow derived macrophages. b) FACS analysis of LGP and NT macrophages exposed to Pten-/-; Trp53-/- media. NT= non- targeting guides. (CD206-MHCII + LGP n = 5, NT n = 5; CD206BrightMHCII- LGP n = 6, NT n = 9; ARG LGP n = 6, NT n = 9; CD39 LGP n = 6, NT n = 8). c) RT-qPCR gene expression analysis on LGP or chemosensor KO macrophages. Bar graphs show the fold change of treated cells versus untreated for each condition. (CD206: LGP n = 4, OLFR229-/- n = 3, OLFR644-/- n = 4, OLFR192-/- n = 4, VMN1R87-/- n = 3, VMN2R29-/- n = 4; MHCII: LGP n = 4, OLFR229-/- n = 4, OLFR644-/- n = 3, OLFR192-/- n = 4, VMN1R87-/- n = 4, VMN2R29-/- n = 3). d-e) FACS analysis on macrophages in absence (LGP n = 4) or presence of OLFR229 deletion (n = 3), OLFR192 deletion (n = 3) and VMN1R87 deletion (n = 3). Events are gated on F4/80 + CD11b+ cells. Bar graphs show the ratio between conditions: (d) % of CD206-MHCII+ and % of CD206brightMHCII- (e) % of ARG1+ and % of CD39+ cells. (d) and (e) one-way ANOVA test with Tukey’s multiple comparisons test was used. f) Bulk mRNA-seq was performed on non-conditioned LGP, Olfr644-/-, and Vmn2r29-/- Macs and LGP, Olfr644-/-, and Vmn2r29-/- Macs exposed to conditioned media from Pten-/-; Trp53-/-cells. The balloon plot shows predicted upstream regulators identified using Ingenuity Pathway Analysis (IPA) by comparing macrophages exposed to conditioned medium and their respective untreated controls. In the LGP condition, genes were ranked based on their pvalue and the top 25 genes were selected. Each balloon represents an upstream regulator, with size indicating the activation Z-score and color reflecting its predicted activation state (red = activated, blue = inhibited). g) RT-qPCR gene expression analysis on Hif1a downstream genes performed on LGP or chemosensor- KO macrophages. Bar graphs show the fold change of treated cells versus untreated for each condition. h) Scratch assay: graph and curves showing the distance (um) covered by tumor cells over time after exposure to supernatant from untreated macrophages. When not specified, statistical analyses were performed using two-tailed unpaired Student’s t test. Values are presented as mean ± SEM. Schematic in a created using BioRender.com.
