Figure 1. Fate mapping of monocyte-derived or tissue-resident macrophages in PDA.

(A) In vivo monocyte tracking approach in CCR2^CreER R26^TdTomato mice orthotopically implanted with KPC2a tumor cells and treated with tamoxifen. See also Figure S1.

(B) Gating strategy for Tomato⁺ MHCII^hi and MHCII^lo macrophages.

(C) Representative CD86 and Arg1 histograms gated on Tomato⁺ MHCII^hi or MHCII^lo TAMs.

(D) Frequency and number of Tomato⁺ TAMs. Each dot is an independent mouse. Data are mean ± SEM; n = 3–4 mice per group; **p < 0.005; one-way ANOVA with Tukey’s posttest.

(E) Proportion of intratumoral Tomato⁺ cells that are macrophages (CD64⁺F4/80⁺) or monocytes (F4/80⁻Ly6C⁺). Each dot is an independent mouse. Data are mean ± SEM; n = 3–4 mice per group; ****p < 0.0001; one-way ANOVA with Tukey’s posttest.

(F) Proportion of intratumoral Tomato⁺ MHCII^hi or MHCII^lo macrophages. Each dot is an independent mouse. Data are mean ± SEM; n = 3–4 mice per group; **p < 0.005; one-way ANOVA with Tukey’s posttest.

(G) In vivo monocyte tracking approach in CCR2^CreER R26^TdTomato mice implanted with KPC2a tumor cells and treated with tamoxifen twice.

(H) Representative plot gated on intratumoral F4/80⁺CD64⁺ macrophages on day 7. Proportion of total Tomato⁺ macrophages. Each dot is an independent mouse. Data are mean ± SEM; n = 4 mice per group; ****p < 0.0001; Student’s t test.

(I) Representative plots gated on day 7 and proportion of Tomato⁺ or Tomato⁻ intratumoral MHCII^hi or MHCII^lo macrophages on day 7. Each dot is an independent mouse. Data are mean ± SEM; n = 4 mice per group; ***p < 0.001; Student’s t test.

(J) Representative plots gated on day 7 and proportion of Tomato⁺ and Tomato⁻ MHCII^loCD206⁺FRβ⁺ macrophages on day 7. Each dot is an independent mouse. Data are mean ± SEM; n = 4 mice per group; ***p < 0.001; Student’s t test.

(K) PD-L1 staining and mean fluorescence intensity (MFI) of Tomato⁺ and Tomato⁻ macrophages at day 7. Each dot is an independent mouse. Data are mean ±SEM; n = 4 mice per group; *p < 0.05; Student’s t test.

(L) Representative histograms and frequency of Ki67⁺ macrophages on day 7. Each dot is an independent mouse. Data are mean ± SEM; n = 4 mice per group; **p < 0.005; Student’s t test.

(M) Embryonic fate-mapping approach in CX3CR1^CreER R26^TdTomato. Briefly, pregnant mothers were gavaged with tamoxifen on embryonic day 14.5 and then progeny were implanted with tumors. See also Figure S2.

(N) Representative plot gated on intratumoral F4/80⁺CD64⁺ macrophages on day 7 from (M). Proportion of each cell subtype that is Tomato⁺ is shown. Each dot is an independent mouse. Data are mean ± SEM; n = 3 mice per group; **p < 0.005, ****p < 0.0001; one-way ANOVA with Tukey’s post test.

(O) Representative plots gated on day 7 and proportion of Tomato⁺ and Tomato⁻ MHCII^loCD206⁺FRβ⁺ tumor macrophages. Each dot is an independent mouse. Data are mean ± SEM; n = 3 mice per group; **p < 0.005; Student’s t test.

(P) Experimental schematic to label the initial wave of recruited monocytes (purple) or subsequent waves of recruited monocytes (green).

(Q) Proportion of each monocyte wave that gives rise to MHCII^hi or MHCII^lo macrophages. Each dot is an independent mouse. Data are mean ± SEM; n = 3–4 mice per group; **p < 0.005; Student’s t test.

(R) Proportion of each monocyte wave that gives rise to CD206⁺ macrophages. Each dot is an independent mouse. Data are mean ± SEM; n = 3–4 mice per group;****p < 0.0001; Student’s t test.

(S) Proportion of each monocyte wave that gives rise to FRβ⁺ macrophages. Each dot is an independent mouse. Data are mean ± SEM; n = 3–4 mice per group.