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a) Schematic of experiment. Mixed subcutaneous tumours were engrafted with BFP KRAS G12C cells plus 0.2% Luc-eGFP KRAS G12D cells. Mice were treated for six days with the RAS G12C(ON) inhibitor RMC-4998 (100 mg/kg; G12Ci / Ci) with or without the SHP2 inhibitor RMC-4550 (30 mg/kg; SHP2i / Si) or a-PD-1 (10 mg/kg). After treatment flow cytometry of tumours and tumour-draining lymph nodes (tdLN) was performed. For all subsequent plots, each dot represents one individual tumour; bar graphs indicate mean ± SD; One-way ANOVA Kruskal-Wallis test comparing vehicle to each of the treated conditions; only significant comparisons are shown. b) Fraction of KRAS G12C (left) and KRAS G12D (right) mutant tumour cells out of all life cells. c) Fraction of macrophages (MPs) out of all immune cells (CD45 + ). d) Macrophage polarisation: Fraction of Arg1 high /MHC-II low (left) and Arg1 low/MHC-ll high (right) MPs out of all MPs. e) Fraction of CD8 + T cells out of CD45 + cells. f) Fraction of NK cells out of CD45 + cells. g) CD8 + T cells to Treg (CD4 + <t>Foxp3</t> + T cells) ratio by treatment group. h) Fraction of effector memory (Tem; CD62L - /CD44 + ; left) and TIM3 + /Lag3 + (right) CD8 + T cells out of CD45 + cells. i) In tumour draining lymph node (tdLN): fraction of Tem (CD62L - /CD44 + ) CD8 + T cells out of all CD8 + T cells.
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a) Schematic of experiment. Mixed subcutaneous tumours were engrafted with BFP KRAS G12C cells plus 0.2% Luc-eGFP KRAS G12D cells. Mice were treated for six days with the RAS G12C(ON) inhibitor RMC-4998 (100 mg/kg; G12Ci / Ci) with or without the SHP2 inhibitor RMC-4550 (30 mg/kg; SHP2i / Si) or a-PD-1 (10 mg/kg). After treatment flow cytometry of tumours and tumour-draining lymph nodes (tdLN) was performed. For all subsequent plots, each dot represents one individual tumour; bar graphs indicate mean ± SD; One-way ANOVA Kruskal-Wallis test comparing vehicle to each of the treated conditions; only significant comparisons are shown. b) Fraction of KRAS G12C (left) and KRAS G12D (right) mutant tumour cells out of all life cells. c) Fraction of macrophages (MPs) out of all immune cells (CD45 + ). d) Macrophage polarisation: Fraction of Arg1 high /MHC-II low (left) and Arg1 low/MHC-ll high (right) MPs out of all MPs. e) Fraction of CD8 + T cells out of CD45 + cells. f) Fraction of NK cells out of CD45 + cells. g) CD8 + T cells to Treg (CD4 + <t>Foxp3</t> + T cells) ratio by treatment group. h) Fraction of effector memory (Tem; CD62L - /CD44 + ; left) and TIM3 + /Lag3 + (right) CD8 + T cells out of CD45 + cells. i) In tumour draining lymph node (tdLN): fraction of Tem (CD62L - /CD44 + ) CD8 + T cells out of all CD8 + T cells.
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a) Schematic of experiment. Mixed subcutaneous tumours were engrafted with BFP KRAS G12C cells plus 0.2% Luc-eGFP KRAS G12D cells. Mice were treated for six days with the RAS G12C(ON) inhibitor RMC-4998 (100 mg/kg; G12Ci / Ci) with or without the SHP2 inhibitor RMC-4550 (30 mg/kg; SHP2i / Si) or a-PD-1 (10 mg/kg). After treatment flow cytometry of tumours and tumour-draining lymph nodes (tdLN) was performed. For all subsequent plots, each dot represents one individual tumour; bar graphs indicate mean ± SD; One-way ANOVA Kruskal-Wallis test comparing vehicle to each of the treated conditions; only significant comparisons are shown. b) Fraction of KRAS G12C (left) and KRAS G12D (right) mutant tumour cells out of all life cells. c) Fraction of macrophages (MPs) out of all immune cells (CD45 + ). d) Macrophage polarisation: Fraction of Arg1 high /MHC-II low (left) and Arg1 low/MHC-ll high (right) MPs out of all MPs. e) Fraction of CD8 + T cells out of CD45 + cells. f) Fraction of NK cells out of CD45 + cells. g) CD8 + T cells to Treg (CD4 + <t>Foxp3</t> + T cells) ratio by treatment group. h) Fraction of effector memory (Tem; CD62L - /CD44 + ; left) and TIM3 + /Lag3 + (right) CD8 + T cells out of CD45 + cells. i) In tumour draining lymph node (tdLN): fraction of Tem (CD62L - /CD44 + ) CD8 + T cells out of all CD8 + T cells.
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pfa fixation solution  (Thermo Fisher)
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a) Schematic of experiment. Mixed subcutaneous tumours were engrafted with BFP KRAS G12C cells plus 0.2% Luc-eGFP KRAS G12D cells. Mice were treated for six days with the RAS G12C(ON) inhibitor RMC-4998 (100 mg/kg; G12Ci / Ci) with or without the SHP2 inhibitor RMC-4550 (30 mg/kg; SHP2i / Si) or a-PD-1 (10 mg/kg). After treatment flow cytometry of tumours and tumour-draining lymph nodes (tdLN) was performed. For all subsequent plots, each dot represents one individual tumour; bar graphs indicate mean ± SD; One-way ANOVA Kruskal-Wallis test comparing vehicle to each of the treated conditions; only significant comparisons are shown. b) Fraction of KRAS G12C (left) and KRAS G12D (right) mutant tumour cells out of all life cells. c) Fraction of macrophages (MPs) out of all immune cells (CD45 + ). d) Macrophage polarisation: Fraction of Arg1 high /MHC-II low (left) and Arg1 low/MHC-ll high (right) MPs out of all MPs. e) Fraction of CD8 + T cells out of CD45 + cells. f) Fraction of NK cells out of CD45 + cells. g) CD8 + T cells to Treg (CD4 + <t>Foxp3</t> + T cells) ratio by treatment group. h) Fraction of effector memory (Tem; CD62L - /CD44 + ; left) and TIM3 + /Lag3 + (right) CD8 + T cells out of CD45 + cells. i) In tumour draining lymph node (tdLN): fraction of Tem (CD62L - /CD44 + ) CD8 + T cells out of all CD8 + T cells.
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a) Schematic of experiment. Mixed subcutaneous tumours were engrafted with BFP KRAS G12C cells plus 0.2% Luc-eGFP KRAS G12D cells. Mice were treated for six days with the RAS G12C(ON) inhibitor RMC-4998 (100 mg/kg; G12Ci / Ci) with or without the SHP2 inhibitor RMC-4550 (30 mg/kg; SHP2i / Si) or a-PD-1 (10 mg/kg). After treatment flow cytometry of tumours and tumour-draining lymph nodes (tdLN) was performed. For all subsequent plots, each dot represents one individual tumour; bar graphs indicate mean ± SD; One-way ANOVA Kruskal-Wallis test comparing vehicle to each of the treated conditions; only significant comparisons are shown. b) Fraction of KRAS G12C (left) and KRAS G12D (right) mutant tumour cells out of all life cells. c) Fraction of macrophages (MPs) out of all immune cells (CD45 + ). d) Macrophage polarisation: Fraction of Arg1 high /MHC-II low (left) and Arg1 low/MHC-ll high (right) MPs out of all MPs. e) Fraction of CD8 + T cells out of CD45 + cells. f) Fraction of NK cells out of CD45 + cells. g) CD8 + T cells to Treg (CD4 + Foxp3 + T cells) ratio by treatment group. h) Fraction of effector memory (Tem; CD62L - /CD44 + ; left) and TIM3 + /Lag3 + (right) CD8 + T cells out of CD45 + cells. i) In tumour draining lymph node (tdLN): fraction of Tem (CD62L - /CD44 + ) CD8 + T cells out of all CD8 + T cells.

Journal: bioRxiv

Article Title: Leveraging death of drug-sensitive cancer cells to promote immune-mediated bystander killing of subclones of drug-resistant tumour cells

doi: 10.1101/2025.10.27.684825

Figure Lengend Snippet: a) Schematic of experiment. Mixed subcutaneous tumours were engrafted with BFP KRAS G12C cells plus 0.2% Luc-eGFP KRAS G12D cells. Mice were treated for six days with the RAS G12C(ON) inhibitor RMC-4998 (100 mg/kg; G12Ci / Ci) with or without the SHP2 inhibitor RMC-4550 (30 mg/kg; SHP2i / Si) or a-PD-1 (10 mg/kg). After treatment flow cytometry of tumours and tumour-draining lymph nodes (tdLN) was performed. For all subsequent plots, each dot represents one individual tumour; bar graphs indicate mean ± SD; One-way ANOVA Kruskal-Wallis test comparing vehicle to each of the treated conditions; only significant comparisons are shown. b) Fraction of KRAS G12C (left) and KRAS G12D (right) mutant tumour cells out of all life cells. c) Fraction of macrophages (MPs) out of all immune cells (CD45 + ). d) Macrophage polarisation: Fraction of Arg1 high /MHC-II low (left) and Arg1 low/MHC-ll high (right) MPs out of all MPs. e) Fraction of CD8 + T cells out of CD45 + cells. f) Fraction of NK cells out of CD45 + cells. g) CD8 + T cells to Treg (CD4 + Foxp3 + T cells) ratio by treatment group. h) Fraction of effector memory (Tem; CD62L - /CD44 + ; left) and TIM3 + /Lag3 + (right) CD8 + T cells out of CD45 + cells. i) In tumour draining lymph node (tdLN): fraction of Tem (CD62L - /CD44 + ) CD8 + T cells out of all CD8 + T cells.

Article Snippet: Subsequently, extracellular master mix, prepared with the antibodies listed in Table S6 diluted in Foxp3 Fixation/Permeabilization Buffer (Invitrogen, 00-5523-00), was added and samples were incubated for 30 min at 4°C.

Techniques: Flow Cytometry, Mutagenesis