Fig 2: Rab6 expression in mammary luminal and basal cells isolated from adult virgin and pregnant females. (A) Flow cytometry analysis of mammary cells isolated from 12-week-old virgin mice (left; V-12w) and 15-day pregnant mice (right, P15). The gated subsets within the CD24+ epithelial cell pool are: CD24low ICAM1+ basal cells (Ba), CD24high ICAM1− and CD24high ICAM1+ luminal cells (Lu; HR+ and HR−, respectively) (left); CD24low CD49fhigh basal cells (Ba) and CD24high CD49flow (Lu) (right). (B) Heat map based on microarray data (Chiche et al., 2019) showing the relative gene expression of 58 RAB GTPases in the HR+ and HR− luminal cell populations isolated from adult virgin mice as shown in A (left). Genes expressed at a significant level appear in pink to red. The heat map includes the reference genes Icam1, Elf5, Pgr (encoding PR) and Esr1 (encoding ER) used to characterize the luminal cell populations. Pgr and Esr1 are highly expressed in the hormone-responsive HR+ cells, whereas Elf5 indicates the HR− luminal secretory lineage. Microarray data are from seven separate sorting experiments. Each cell preparation was obtained from a pool of mammary glands taken from 3-4 females. (C) qPCR analysis of Rab6a and Rab6a′ expression in mammary basal and luminal cells isolated from adult virgin and pregnant mice as shown in A. Data are mean±s.e.m. of three separate cell preparations. For the virgin state, each preparation was obtained from a pool of mammary glands taken from 3-4 adult virgin females (total number=10). At pregnancy, each preparation was obtained from a pool of mammary glands taken from one female (total number=3). *P=0.039. ns, not significant. (D) RAB6 immunolocalization in a lactating mammary gland. Actin marks basal myoepithelial cells and the apical pole of luminal cells facing the alveolar lumen (L). Nuclei are stained with DAPI. Some of them are out of the focal plane. The lowest panel shows an enlarged view of the boxed area in the middle panel. Arrows point to the apical presence of RAB6. Scale bars: 10 µm in D (top and middle); 5.5 µm in D (bottom).

Fig 3: Loss of p53 induces expansion of luminal progenitors overexpressing Met. a Flow cytometry analysis of CD24 and ICAM-1 expression in mammary cells isolated from Trp53F/F (Ctrl, control) and K5Cre;Trp53F/F (Mut, mutant) adult virgin mice. b qPCR analysis of Krt18 and Trp53 expression in Lu-neg and Lu-pos cells isolated from control and mutant adult virgin mice. Data are the mean ± SEM of 4 separate cell preparations. **p ≤ 0.01. c Microphotographs of the colonies formed by 500 control and mutant Lu-neg and Lu-pos cells. d Left: Percentages of clonogenic cells in control and mutant Lu-pos cell populations. Data are the mean ± SEM of 3 distinct assays. Right: Percentages of Lu-pos cells in control and mutant luminal cell populations calculated from flow cytometry data. Values shown represent the mean ± SEM of 7 separate analyses of distinct cell preparations. e Expression of Cdkn1a, Ccng1, and Gadd45a (encoding p21, cyclin G1, and Gadd45a, respectively) in Lu-pos cells isolated from control and mutant adult virgin mice, evaluated by qPCR. Data are shown as mean ratios (± SEM) between gene expression levels in mutant and control Lu-pos cells from 4 separate preparations. **p ≤ 0.01. f Left: K8 and BrdU immunodetection in colonies derived from control and mutant Lu-pos cells. DAPI-stained nuclei appear in blue. Bar, 20 μm. Right: Percentages of BrdU+ cells. Each point represents counting of one microscope field comprising 100–300 DAPI-stained nuclei. Data are shown as mean ± SEM of countings from 2 distinct experiments. **p ≤ 0.01. g Expression levels of the luminal-specific regulators, Elf5, Hey1, Gata3, and Met in control and mutant Lu-pos cells, evaluated by qPCR. Data are the mean ± SEM of 3–4 separate preparations. **p ≤ 0.01

Fig 4: Therapeutic efficacy of the hydrogel microsphere vaccine in combination with IRE ablation for pancreatic cancer.a Schematic diagram illustrates different therapeutic combinations for in situ immune modulation. b, c Representative images of the bioluminescence experiment demonstrating local tumour recurrence and progression in tumour-bearing mice treated with the hydrogel microsphere vaccine or different control hydrogel microspheres (b). Analysis of the radiance intensity indicating significantly attenuated tumour recurrence in hydrogel microsphere vaccine-treated mice (c). The sample size in (c) corresponds to that in (b). Data are presented as mean values +/- SEM. Two-tailed unpaired t test. d Survival analysis of mice bearing orthoptic tumours after different combination therapies. The therapeutic strategies and sample size of each group are labelled in the figure. Survival data are analysed using Kaplan–Meier Method. Two-tailed log-rank test. e, f Representative images of flow cytometry analysis showing the proportion of circulating CD11c+ MHC II+ cells (e) and CD8+ T (f) cells in control, IRE monotherapy-treated and IRE+ hydrogel microsphere vaccine-treated mice. g The hydrogel microsphere vaccine significantly amplified the cellular density of circulating CD11c+MHC II+ cells (left), CD8+ T-cell-to-CD4+ T-cell ratio (middle left), cellular density of CD24+CD64- cells (middle right) and cellular density of CD8+ T cells (right) in KPC tumour-bearing mice. h Schematic diagram showing the in vivo experiments for determination of the abscopal effect. The upper panel indicates experiments performed using mice bearing KPC orthotopic pancreatic tumours. The lower panel indicates experiments performed using mice bearing Panc02 orthotopic pancreatic tumours. i, j Comparison of the weight (i) and volume (j, k) of distant metastatic tumours across different groups. l Line chart showing the growth of individual tumours in each treatment group. g, i, j, k n = 5 biologically independent samples for each group. Data are presented as mean values +/- SEM. Two-tailed unpaired t test. Source data are provided as a Source data file.

Fig 5: The hydrogel microsphere vaccine amplified the recruitment, activation and migration of CD103+CD11b- cDC1s.a Schematic diagram of the in vivo experiments. b Representative images of H&E staining and CD45-labelling IHC staining of pancreatic cancer tissues after different combination therapy. Five biologically independent samples were performed to confirm data stability. c Analysis of the intratumour release of FLT3L and CD40L by the hydrogel microsphere vaccine or direct intratumour injection of dual cytokines. n = 3 biologically independent samples. d Representative images of flow cytometry analysis showing subpopulations of tumour-resident DCs across different groups. e Quantification of the proportion of tumour-resident CD24+CD64- total DCs and CD103+CD11b- cDC1s across different treatment groups (n = 5 for each group). f Quantification of the expression of CD86 and CD40 on the surface tumour-resident cDC1s across different groups (n = 5 for each group). g Quantification of the cellular density of tumour-resident CD24+CD64- DCs and CD103+CD11b- cDC1s across different treatment groups (n = 5 for each group). The hydrogel microsphere vaccine significantly amplified the cellular density of tumour-resident cDC1s and increased the cDC1-to-cDC2 ratio in pancreatic cancer. h Identification of the correlation between the cellular density of tumour-resident total DCs or cDC1s and the tumour weight across all tumour-bearing mice. i, j Representative images of flow cytometry analysis showing total CD11c+MHC II+ myeloid cells and subpopulations of DCs in the TdLNs and spleens across different groups. k, l Quantification of the density of CD11c+MHC II+ myeloid cells and CD103+CD11b- cDC1s in TdLNs (k) and spleens (l) in mice bearing pancreatic cancer. The hydrogel microsphere vaccine significantly amplified the density of cDC1s and increased the cDC1-to-cDC2 ratio in the TdLNs and spleens in mice bearing pancreatic cancer. m Representative images showing IF-stained CD103, CD31, CD4 or CD8-positive cells in pancreatic tumour tissue (left panel), TdLNs and spleens in tumour-bearing mice treated with the hydrogel microsphere vaccine or Group1 control microspheres. n Quantification of the density of tumour-resident OVA257-246-H-2Kb-specific cDC1s in hydrogel microsphere vaccine-treated mice and Group1-treated mice. e–g, k, l, n n = 5 biological independent samples for each group. Data are presented as mean values +/- SEM. Two-tailed unpaired t test. h n = 25 biological independent samples, data are tested using Pearson’s test with two-tailed P values. Source data are provided as a Source data file.