Dendritic Cell Paucity Leads to Dysfunctional Immune Surveillance in Pancreatic Cancer

Abstract

Here, we utilized spontaneous models of pancreatic and lung cancer to examine how neoantigenicity shapes tumor immunity and progression. As expected, neoantigen expression during lung adenocarcinoma development leads to T cell-mediated immunity and disease restraint. By contrast, neoantigen expression in pancreatic ductal adenocarcinoma (PDAC) results in exacerbation of a fibro-inflammatory microenvironment that drives disease progression and metastasis. Pathogenic T_H17 responses are responsible for this neoantigen-induced tumor progression in PDAC. Underlying these divergent T cell responses in pancreas and lung cancer are differences in infiltrating conventional dendritic cells (cDCs). Overcoming cDC deficiency in early-stage PDAC leads to disease restraint, while restoration of cDC function in advanced PDAC restores tumor-restraining immunity and enhances responsiveness to radiation therapy.

Keywords: CD40; Flt3L; dendritic cell; immune surveillance; immunotherapy; neoantigen; pancreatic cancer; radiation therapy; vaccination.

Figure 1.. Neoantigen expression during pancreas cancer development elicits antigen-specific responses

(A) Genetic loci for KPC-OG model and immunoblot for OVA and GFP expression in KPC-OG-derived cell line 72 hours after doxycycline withdrawal. Representative of three independent cell lines. (B) Gross images (LEFT) of pancreatic tissue at 6 weeks in KPC-OG mice on or off doxycycline, and (RIGHT) immunofluorescence images of pancreatic tumors at 36 weeks in KPC-OG mice on or off doxycycline. (C) KPC-OG tumor-derived cell line depicting GFP fluorescence after 24-hour co-culture with antigen-specific (OT-I TCR) or non-specific (C57Bl/6) activated CD8⁺ T cells (CTL) consistent across three independent cell lines, n=3/group. (D) Representative images and quantification of CD8⁺ T cells, CD4⁺ T cells and B220⁺ B cells in 6-week-old KPC-OG and KPC mice. n=10 mice/group. (E) Density of CD8⁺ T cells, CD4⁺ T_H, CD4⁺ T_REG and CD19⁺CD22⁺ B cells measured by flow cytometry in early stage KPC-OG and KPC mice. n=5–8 mice/group. (F) Density of OVA-specific CD8⁺ T cells in pancreas, pancreas-dLN, and spleen of early stage KPC-OG and PC-OG mice. n=3–8 mice/group. Data were consistent across two independent experiments. Scale bar denotes 100 μm in (B) and (D). n.s., not significant; *p < 0.05. Data is presented as mean ± SEM. For comparisons between two groups, Student’s two-tailed t-test used. See also Figure S1.

Figure 2.. Neoantigen expression accelerates PDAC progression but restrains lung adenocarcinomas

(A) Representative H&E images with quantification of lesions in early stage KPC-OG and KPC mice. n=12 mice/group. (B) Lesion grades for early stage KPC-OG and KPC pancreata. n=12 mice/group. (C) Sirius Red staining with quantification in KPC-OG and KPC mice. n=12 mice/group. (D) αSMA staining with quantification in KPC-OG and KPC mice. n=12 mice/group. (E) Flow cytometric quantification of various myeloid infiltrates in KPC-OG and KPC mice. n=5–6 mice/group. (F) Kaplan-Meier survival curve for KPPC-OG mice compared to KPPC littermates. n=14–20 mice/group. (G) Representative histology of late stage KPC-OG and KPC tumors with quantification of high-grade tumors. n=14–16 mice/group. (H) Representative H&E images of late stage KPC-OG and KPC livers with quantification of metastases. n=14 mice/group. (I) Kaplan-Meier survival curve for Pdx1-Cre-ER™-driven iKPC-OG mice compared to iKPC littermates, with quantification of liver metastases. n=8–10 mice/group. (J) Density of CD8⁺ T cells in early stage KPL-OG and KPL lung lesions. n=5 mice/group. (K) Representative H&E images of early stage KPL-OG and KPL lung with quantification of lesion area and grade. Lesions demarcated by yellow line. n=5 mice/group. (L) Representative immunofluorescence images and quantification of GFP (green) expression in CK19⁺ (red) tumors of late stage KPC-OG or KPL-OG tumors. GFP-negative lesions in KPL-OG tumors are demarcated by yellow arrowhead. n=6–8 mice/group. (M) Overall survival since start of treatment for KPPC-OG mice undergoing OT-I adoptive transfer therapy, compared to untreated controls. n=10–18 mice/group. (N) Representative immunofluorescence images and quantification of GFP (green) expression in CK19⁺ (red) tumors of KPPC-OG mice subjected to OT-I adoptive transfer, compared to untreated controls. n=6 mice/group. Data were consistent across two independent experiments. Scale bar denotes 500 μm in (A), (C), (D), (G), (H) and (K); 100 μm in (L) and (N). n.s., not significant; *p < 0.05, **p < 0.01. Data is presented as mean ± SEM. For comparisons between two groups, Student’s two-tailed t-test used. For survival analyses, Log-rank (Mantel-Cox) test used. See also Figure S2, Table S1 and S2.

Figure 3.. Pro-inflammatory CD4⁺ T cell responses drive PDAC acceleration in response to neoantigen

(A) Representative H&E images with quantification of pancreatic lesion area (TOP) and grade (BOTTOM) in early stage KPC-OG mice subjected to depletion of CD4⁺ T cells or CD19⁺B220⁺ B cells. n=8–12 mice/group. (B) Sirius Red staining with quantification in early stage KPC-OG mice subjected to indicated depletions. n=8–12 mice/group. (C) αSMA staining with quantification in early stage KPC-OG mice subjected to indicated depletions. n=8–12 mice/group. (D) Representative flow cytometry plots of RORγt and GATA3 bias in T_H cells of KPC-OG and KPC tumors, with cellular density of RORγt⁺ T_H17 and GATA3⁺ T_H2 cells quantified. n=3–6 mice/group. (E) Representative flow cytometry plots of IL-17A and TNF-α expression in T_H cells of KPC-OG and KPC tumors, with cellular density quantified. n=3–6 mice/group. (F) Representative H&E image with quantification of pancreatic lesion area of early stage KPC-OG mice subjected to IL-17A and IL-17F neutralization. n=8–10 mice/group. (G) Representative Sirius Red staining of KPC-OG mice subjected to IL-17A, IL-17F neutralization, with quantification. n=8–10 mice/group. (H) Representative αSMA staining of KPC-OG mice subjected to IL-17A, IL-17F neutralization, with quantification. n=8–10 mice/group. (I) Representative p-ERK1/2, p-STAT3, p-EGFR immunohistochemistry staining in KPC-OG mice subjected to indicated depletions, with quantification over overall tissue area and lesion area. n=8–10 mice/group. Data were consistent across two independent experiments and pooled. Scale bar denotes 500 μm. n.s., not significant; *p < 0.05, **p < 0.01. Data is presented as mean ± SEM. For comparisons between two groups, Student’s two-tailed t-test used. See also Figure S3.

Figure 4.. cDCs are fewer and less functional in PDAC compared to lung cancer

(A) Heat map depicting mean density (log-scale) of major myeloid cell infiltrates in advanced KPC/OG pancreatic and KPL/OG lung tumors. n=5–10 mice/group. (B) CD103⁺ cDC1 and CD11b⁺ cDC2 density in pancreas and lung tumors, at (LEFT) early stage and (RIGHT) late stage. n=5–10 mice/group. (C) Migratory cDC1 and cDC2 density in respective draining lymph nodes of late stage KPC-OG pancreatic tumors and KPL-OG lung tumors. n=7 mice/group. (D) Immunohistochemistry for tumor cytokeratin (CK7/19) expression, and Zbtb46-GFP⁺ (pink) cDCs in late stage KPC or KPL bone marrow chimeras. RIGHT: Zbtb46-GFP⁺ cDC density in non-tumor (WT) tissue and late stage tumors. FAR RIGHT: Snx22-GFP⁺ cDC1 density in WT and late stage tumors. n=4–6 mice/group. (E) Frequency distribution of Zbtb46-GFP ⁺ cDC and Snx22-GFP⁺ cDC1 proximity to nearest CK7/19⁺ tumor cell. n=3–5 mice/group. (F) Tumoral cDC1 density (log-scale) plotted against OVA-specific CD8⁺ T cell density (log-scale) across tissue and stage. RIGHT: OVA-specific CD8⁺ T cell density in early and late stage tumors. n=5–8 mice/group. (G) Phenograph of CD45⁺ immune infiltrates from human PDAC patient CyTOF samples (pooled), with quantification of individual cellular fractions (log-scale). n=11. (H) Z-normalized cDC1 infiltration score between pancreatic (PAAD, n=177) and lung (LUAD, n=230) adenocarcinoma based on conserved cDC1 gene signature. (I) Representative histogram indicating ZsGreen in migratory cDC1s and cDC2s from respective draining lymph nodes of KPC-Z or KPL-Z tumors at denoted time points. RIGHT: percentage of migratory cDC subsets that have ZsGreen antigen in respective lymph nodes at denoted time points. n=3–4 mice/group. (J) Density of OVA-specific CD8⁺ T cells in draining lymph nodes of early stage tumors. n=5–8 mice/group. Data were consistent across two independent experiments, except in (A), (B), and (F)–(H) where they were pooled across multiple experiments. Scale bar denotes 100 μm. n.s., not significant; *p < 0.05, **p < 0.01. Data is presented as mean ± SEM, except in (H) where box plot denotes 10th to 90th percentile, middle line indicates median, range lines indicate maximal values, and data points beyond indicate outliers (>1.5X range). For comparisons between any two groups, Student’s two-tailed t-test used. Frequency distributions were compared using non-parametric Kolmogorov-Smirnov test. See also Figure S4.

Figure 5.. Mobilizing cDCs into early pancreatic lesions can reverse fibro-inflammatory responses

(A) Schematic of Flt3L administration in KPC-OG mice (starting at P30), with quantification of cDC1 and cDC2 density in pancreata of KPC-OG mice either treated or not with Flt3L and control KPC mice. n=5–6 mice/group. (B) Representative H&E images of early stage KPC-OG mice either treated or not with Flt3L, with quantification of lesion area. n=8–12 mice/group; mice from figure 2 included in this and following analyses. (C) Sirius Red staining with quantification in KPC-OG mice either treated or not with Flt3L. n=8–12 mice/group. (D) αSMA staining with quantification in KPC-OG mice either treated or not with Flt3L. n=8–12 mice/group. (E) Density of RORγt ⁺ T_H17 and GATA3⁺ T_H2 cells in early stage KPC-OG mice treated as indicated. n=3–6 mice/group. (F) Representative flow cytometry plots of IL-17A and TNF-α expression in T_H cells of KPC-OG mice either treated or not with Flt3L, with IL-17A⁺ and TNF-α⁺ IL-17A⁺ T_H cellular density quantified. n=3–6 mice/group. (G) Representative immunohistochemistry of CD8⁺ T cells (brown) and CK19⁺ tumor lesions (pink) in early stage KPC-OG mice either treated or not with Flt3L, n=6 mice/group. (H) Cumulative CD8⁺ T cell density within 30 μm of CK19⁺ lesions, and distribution of CD8⁺ T cell proximity to nearest tumor cell in KPC-OG mice treated as indicated. n=6 mice/group. (I) Density of IFN-γ⁺ TNF-α⁺ cytotoxic CD8⁺ T cells in KPC-OG mice treated as indicated. n=6 mice/group. (J) Representative H&E images of early stage KPC-OG mice treated with Flt3L and anti-CD8 or anti-IFN-γ depletion antibodies, with quantification of lesion area. n=7–12 mice/group. Data were consistent across two independent experiments, except in (B)–(D) and (H)–(J) where they were pooled across multiple experiments. Scale bar denotes 500 μm in (B), (C), (D), and (J); denotes 100 μm in (G). n.s., not significant; *p < 0.05, **p < 0.01. Data is presented as mean ± SEM. For comparisons between any two groups, Student’s two-tailed t-test used. Frequency distributions were compared using non-parametric Kolmogorov-Smirnov test. See also Figure S5.

Figure 6.. Enhancing cDC infiltration and activation in established PDAC leads to disease stabilization

(A) Schematic of Flt3L administration in ultrasound-diagnosed KPPC-OG mice. n=5–8 mice/group. (B) Density of (LEFT) CD103⁺ cDC1s, CD11b⁺ cDC2s in tumors, and (RIGHT) migratory cDC1, cDC2 populations in respective dLNs of KPPC-OG mice treated with Flt3L. n=5–8 mice/group. (C) Density of CD8⁺ T cells, CD4⁺ T_H cells and frequency of CD4⁺ T_REG in tumors of KPPC-OG mice treated with Flt3L. n=5–8 mice/group. (D) Density of cDC1s and cDC2s in tumors of KPPC-OG mice treated as described. n=7–8 mice/group. (E) Density of CD8⁺ T cells and CD4⁺ T_H cells and frequency of CD4⁺ T_REGS in tumors of KPPC-OG mice treated as described. n=7–8 mice/group. (F) Density of OVA-specific CD8⁺ T cells in tumors of treated KPPC-OG mice. n=5–8 mice/group. (G) Representative immunohistochemistry of CD8⁺ T cells (brown) and CK19⁺ tumor lesions (pink) in KPPC-OG mice treated as indicated. RIGHT: cumulative CD8⁺ T cell density within 50 μm of CK19⁺ lesions. n=5–8 mice/group. (H) Density of tumor-infiltrating NK cells, NKT cells and γδ-T cells in treated KPPC-OG mice. n=5–8 mice/group. (I) Representative flow histogram indicating ZsGreen in migratory cDC1s from draining nodes of KPPC-Z treated as indicated. RIGHT: absolute number of migratory cDC subsets that have ZsGreen. n=3–4 mice/group. (J) Density of OVA-specific CD8⁺ T cell in draining lymph nodes of treated KPPC-OG mice. n=5–8 mice/group. (K) Tumor growth quantified by ultrasound measurements over 2 weeks of treatment. RIGHT: Individual traces of untreated and anti-CD40 plus Flt3L combination cohorts. n=5–8 mice/group. (L) Representative Masson’s trichrome staining with quantification in KPPC-OG mice treated as denoted. n=5–8 mice/group. (M) Representative α-SMA staining with quantification in KPPC-OG mice treated as denoted. n=5–8 mice/group. Data were pooled across multiple independent experiments for all treatments. Scale bar denotes 100 μm in ; bar denotes 500 μm in (L) and (M). n.s., not significant; *p < 0.05, **p < 0.01. Data is presented as mean ± SEM. For comparisons between any two groups, Student’s two-tailed t-test used. See also Figure S6.

Figure 7.. cDC-directed therapy renders PDAC responsive to radiation therapy

(A) Dosage schema for administration of radiation (RT) in KPPC-OG mice treated with Flt3L and anti-CD40 upon ultrasound-based tumor diagnosis at day 0. (B) KPPC-OG tumor growth kinetics quantified by ultrasound measurements over 2 weeks of treatment. n=8 mice/group. (C) Percentage change in KPPC-OG tumor volume after RT (day 7 to day 14) with representative ultrasound images. n=8 mice/group. (D) Dosage schema for radiation (RT) in orthotopic Kras-Ink model treated with Flt3L and anti-CD40 upon tumor diagnosis. (E) Kras-Ink tumor growth kinetics quantified by ultrasound measurements over 2 weeks of treatment. n=8 mice/group. (F) Percentage change in Kras-Ink tumor volume after RT (day 6 to day 13). n=8 mice/group. (G) Kaplan-Meier survival curve for Kras-Ink orthotopic tumor-bearing mice undergoing RT-alone or RT in conjunction with Flt3L and anti-CD40. n=9–14 mice/group. (H) Dosage schema for administration of radiation (RT) in KPPC mice treated with Flt3L and anti-CD40 upon ultrasound-based tumor diagnosis at day 0. (I) KPPC tumor growth kinetics quantified by ultrasound measurements over 5 weeks since starting treatment. n=8 mice/group. (J) Kaplan-Meier survival curve for KPPC mice undergoing RT alone or RT in conjunction with Flt3L and anti-CD40. n=10–16 mice/group. Data were pooled across multiple experiments for (A)–(C), (H)–(J), and representative of two independent experiments for (D)–(G). Scale bar denotes 5 mm. n.s., not significant; *p < 0.05, **p < 0.01. Data is presented as mean ± SEM. For comparisons between any two groups, Student’s two-tailed t-test used. See also Figure S7.