Abstract
Neoplastic pancreatic epithelial cells are widely believed to die via Caspase 8-dependant apoptotic cell death and chemotherapy is thought to further promote tumor apoptosis1. Conversely, disruption of apoptosis is a basic modality cancer cells exploit for survival2,3. However, the role of necroptosis, or programmed necrosis, in pancreatic ductal adenocarcinoma (PDA) is uncertain. There are a multitude of potential inducers of necroptosis in PDA including ligation of TNFR1, CD95, TRAIL receptors, Toll-like receptors, ROS, and Chemotherapeutics4,5. Here we report that the principal components of the necrosome, RIP1 and RIP3, are highly expressed in PDA and are further upregulated by chemotherapy. Blockade of the necrosome in vitro promoted cancer cell proliferation and induced an aggressive oncogenic phenotype. By contrast, in vivo RIP3 deletion or RIP1 inhibition was protective against oncogenic progression and was associated with the development of a highly immunogenic myeloid and T cell infiltrate. The immune-suppressive tumor microenvironment (TME) associated with intact RIP1/RIP3 signaling was in-part contingent on necroptosis-induced CXCL1 expression whereas CXCL1 blockade was protective against PDA. Moreover, we found that cytoplasmic SAP130 was expressed in PDA in a RIP1/RIP3-dependent manner, and Mincle – its cognate receptor – was upregulated in tumor-infiltrating myeloid cells. Mincle ligation by SAP130 promoted oncogenesis whereas Mincle deletion was protective and phenocopied the immunogenic reprogramming of the TME characteristic of RIP3 deletion. Cellular depletion experiments suggested that whereas inhibitory macrophages promote tumorigenesis in PDA, they lose their immune-suppressive effects in the context of RIP3 or Mincle deletion. As such, T cells which are dispensable to PDA progression in hosts with intact RIP3 or Mincle signaling become reprogrammed into indispensable mediators of anti-tumor immunity in absence of RIP3 or Mincle. Our work describes parallel networks of necroptosis-induced CXCL1 and Mincle signaling which critically promote macrophage-induced adaptive immune suppression enabling PDA progression.
Keywords: Pancreatic cancer, inflammation, C-type lectin receptors
We found that RIP1 and RIP3 are highly expressed in human PDA (Fig. 1a, b). Western blotting confirmed higher RIP1 and RIP3 expression in human PDA compared with surrounding normal pancreas (Fig. 1c). Similarly, FADD, which complexes with RIP1/RIP3 to form the necrosome, MLKL, a downstream mediator of necroptosis, and Caspase 8, a principal driver of apoptosis, were upregulated in PDA (Fig. 1c)5. Immune fluorescence microscopy showed evidence of RIP1–RIP3 co-localization in human (Fig. 1d) and murine (Fig. 1e) PDA suggesting necrosome complex formation. To test whether the necrosome was inducible with chemotherapy, we treated PDA-bearing mice with Gemcitabine. Chemotherapeutics increased PDA expression of RIP1 and RIP3 in vivo (Fig. 1f, g). Similarly, Gemcitabine increased RIP1 and RIP3 expression and RIP1–RIP3 co-association in vitro in PDA cells (Fig. 1h, i). Expression of components of the necrosome were also inducible by chemotherapeutics in human PDA cells whereas MLKL inhibition prevented chemotherapy-induced cell death (Fig. 1j, k).
Since necroptosis is a pro-inflammatory process, we postulated that it would support peri-tumoral inflammation6. We found that CXCL1 is one of the most highly expressed chemokines in murine PDA (Fig. 2a). Similarly, CXCL1 was robustly expressed in human PDA (Fig. 2b–d). Gemcitabine upregulated PDA expression of CXCL1 in mice (Fig. 2e), whereas RIP3 deletion mitigated CXCL1 expression in vivo (Fig. 2f, g) and in vitro (Fig. 2h). High RIP3 also correlated with higher CXCL1 in a human PDA RNAseq database (Fig. 2i). Further, upregulation of CXCL1 by Gemcitabine was mitigated by RIP3 deletion in vivo (Fig. 2j) and by RIP1 or RIP3 inhibition in vitro (Fig. 2k). Collectively, these data suggest necrosome-dependent upregulation of CXCL1 in PDA.
We studied the effects of RIP3 deletion on the properties of in vitro cultured KrasG12D-transformed pancreatic ductal epithelial cells (KrasG12D PDEC). Predictably, RIP3 deletion increased the proliferative rate of KrasG12D PDEC in vitro (Extended-Data-Fig. 1a). Moreover, KrasG12D;RIP3−/− PDEC exhibited a distinct phenotype including loss of CDK4 and elevated expression of Bcl-xL and c-Myc (Extended-Data-Fig 1b) which have selectively been associated with aggressive tumor-biology in PDA7–11.
Since RIP3 deletion increased proliferation of PDA cells, we postulated that blockade of necroptosis in vivo would accelerate tumorigenesis. To test this, we compared the rate of oncogenic progression in p48Cre;KrasG12D(KC);RIP3+/+ versus KC;RIP3−/− pancreata. Contrary to our hypothesis and belying our in vitro findings, RIP3 deletion was protective. KC;RIP3−/− pancreata exhibited a diminished rate of acinar replacement by dysplastic ducts, slower PanIN progression, and reduced fibro-inflammatory changes compared with KC;RIP3+/+ (Fig. 3a and Extended-Data-Fig. 1c). Accordingly, aged-matched KC;RIP3−/− pancreata weighed less than controls and RIP3 deletion extended survival (Fig. 3b, c). The proliferative rate was similar in KC;RIP3+/+ and KC;RIP3−/− pancreatic epithelial cells in vivo (Fig. 3d). To test whether abrogation of RIP1 signaling similarly protected against PDA, we treated 6 week-old KC mice for 8 weeks with Nec-1s. RIP1 blockade protected against oncogenic progression based on pancreas weight and histology (Fig. 3e, f).
Since necroptosis can modulates inflammation12, we postulated that RIP3 deletion offers tumor-protection by enhancing peri-tumoral immunogenicity. RIP3 deletion diminished infiltration of tumor-associated macrophages (TAMs; Extended-Data-Fig. 2a). Conversely, the fraction of T cells and B cells were increased in KC;RIP3−/− pancreata (Extended-Data-Fig. 2b, c). Analysis of the myeloid compartment showed a decreased fraction of MDSC and DC in KC;RIP3−/− pancreata (Extended-Data-Fig. 2d, e). Further, consistent with our immunohistochemical data, bulk tumor-infiltrating TAMs and their M2-like Arg1+CD206+ subset were diminished in the context of RIP3 deletion (Extended-Data-Fig. 2f–h). Macrophage expression of PD-L1 was also reduced by RIP3 deletion (not shown). Collectively, these data suggest that RIP3 deletion increases lymphocyte infiltration in PDA and reduces infiltration of immune-suppressive myeloid cellular subsets. Similarly, in human PDA high RIP1/RIP3 co-expression correlated with elevated expression of the myeloid cell marker CD11b (Extended-Data-Fig. 2i).
To determine whether deletion of RIP3 in the epithelial compartment alone is sufficient to protect against oncogenesis, we challenged cohorts of WT mice with an orthotopic injection of either KrasG12D;RIP3+/+ PDEC or KrasG12D;RIP3−/− PDEC. Similar to our findings using pan-RIP3 deletion, KrasG12D;RIP3−/− tumors grew at slower rates than KrasG12D;RIP3+/+ (Fig. 4a) suggesting that RIP3 blockade in the epithelial compartment alone protects against PDA progression.
Since inflammatory cells within the PDA TME express the components of the necrosome (Figs. 1a and 4b, c), we investigated whether RIP3 deletion in the extra-epithelial compartment would similarly mitigate PDA progression. WT and RIP3−/− mice were challenged with an orthotopic intra-pancreatic injection of KrasG12D PDEC or Pdx1Cre;KrasG12D;Tp53R172H (KPC)-derived PDA cells, which express both mutant Kras and p53, and tumor size was measured at 3 weeks. RIP3−/− mice developed smaller KrasG12D (not shown) and KPC-derived tumors (Fig. 4d) implying that blockade of necroptosis in the extra-epithelial compartment alone is protective against PDA.
To determine whether deletion of RIP3 in the extra-epithelial compartment similarly bolsters peri-tumoral immunogenicity, we analyzed the inflammatory infiltrate in orthotopic KPC tumors in WT and RIP3−/− hosts. RIP3 deletion resulted in elevated T cell and B cell infiltrates (Fig. 4e, f) and peri-tumoral T cells expressed lower IL-10 and PD-1, higher CD44, and exhibited a lower fraction of Tregs compared with control (Fig. 4g, h; Extended-Data-Fig. 3a–d). Analysis of the myeloid compartment again revealed a reduction in the fraction of peri-tumoral MDSC (Fig. 4i) and TAMs (Fig. 4j) with a shift toward an M1-like phenotype (Fig. 4k, l) and reduced PD-L1 expression (Fig. 4m). These data ostensibly contrast with a recent report which found that RIP1 signaling can enhance CD8+ T cell cross-priming. However, effects in PDA may be unique to the immunologic milieu of the pancreatic TME13. Accordingly, RIP3 deletion was not protective against B16 melanoma or subcutaneously-implanted KPC cells (Extended-Data-Fig. 3e, f).
Since CXCL1 expression in PDA is contingent on the necrosome (Fig. 2) and we found that CXCR2 is widely expressed on peri-tumoral leukocytes (Extended-Data-Fig. 4a, b), we postulated that CXCL1 may be responsible for the pro-tumorigenic immune-suppression associated with RIP3 signaling by mobilizing myeloid cells14,15. To test this hypothesis, we challenged WT mice with orthotopic PDA whilst blocking CXCL1 in select cohorts. CXCL1 blockade protected against tumorigenesis in both the orthotopic KrasG12D PDEC (not shown) and KPC (Extended-Data-Fig. 4c) models. However, αCXCL1 treatment did not further enhance tumor protection in RIP3−/− animals (Extended-Data-Fig. 4d). Moreover, similar to RIP3 deletion, CXCL1 blockade reduced MDSC and TAM accumulation (Extended-Data-Fig. 4e, f). Tumor-infiltrating T cells were also more activated in the context of CXCL1 blockade as evidenced by higher CD44 and TNFα expression (Extended-Data Fig. 4g, h). However, CXCL1 inhibition was not significantly associated with higher infiltration of peri-tumoral T cells (Extended-Data-Fig. 4i), nor did it diminish Treg accumulation or lower IL-10 expression (not shown), each characteristic of RIP3 deletion. Taken together, these data suggest that CXCL1 overexpression alone may not account for the entire immune-suppressive phenotype associated with intact necroptosis signaling in PDA.
We postulated that necroptotic tumor cells release soluble factors which induce peri-tumoral immune-suppression. Mincle, a C-type lectin receptor (CLR) critical in mycobacterial immunity, can promote sterile inflammation in vitro by ligating SAP130, a nuclear protein released from dying cells16,17. We discovered high cytoplasmic SAP130 expression in human PDA (Extended-Data-Fig. 5a). SAP130 expression was also upregulated by chemotherapeutics in human PDA cell lines (Extended-Data-Fig. 5b). Further, SAP130 was highly expressed in KC;RIP3+/+ pancreata whereas its expression was reduced in KC;RIP3−/− (Extended-Data-Fig. 5c). Similarly, Gemcitabine-induced upregulation of Sap130 was partially mitigated by Nec-1s (Extended-Data-Fig. 5d). SAP130 expression in PDA was evident in both epithelial and inflammatory cells (Extended-Data-Fig. 5e, f). Moreover, confocal microscopy suggested SAP130 co-localization with RIP1/RIP3 in human (Extended-Data-Fig. 5g) and murine (not shown) PDA. SAP130 similarly correlated with high RIP1/RIP3 coexpression in a human RNAseq database (Extended-Data-Fig. 5h). Notably, there was a trend toward an association between high SAP130 expression and diminished survival in human PDA (Extended-Data-Fig. 5i).
We postulated that Mincle ligation by SAP130 drives necrosome-induced accelerated oncogenesis. Accordingly, immunoprecipitation experiments suggested that Mincle co-associates with SAP130 in PDA (Extended-Data-Fig. 5j). Mincle was expressed in inflammatory cells in the human PDA TME, but absent in transformed ductal cells and in normal pancreas (Extended-Data-Fig. 6a). Overall, ~10% of tumor-infiltrating leukocytes expressed Mincle in human PDA compared with minimal expression in PBMC (Extended-Data-Fig. 6b). Subset analysis revealed high Mincle expression in human CD14+CD15+ tumor-infiltrating monocytes compared with lower expression in their counterparts in PBMC (Extended-Data-Fig. 6c). Similarly, in KC mice 10–15% of pancreatic leukocytes expressed Mincle compared with low expression in the spleen or in parenchymal cells (Extended-Data-Fig. 6d). Immunofluorescence microscopy confirmed Mincle expression in enriched PDA-infiltrating leukocytes (Extended-Data-Fig. 6e). Subset analysis suggested that Mincle was highly expressed in PDA-infiltrating MDSC, DC, and macrophages compared with lower expression in spleen (Extended-Data-Fig. 6f). Western blotting showed elevated expression of Mincle-related signaling intermediates in PDA in a RIP3-dependant manner (Extended-Data-Fig. 6g). Accordingly, p-Syk+ leukocytes were comparatively scarce in KC;RIP3−/− and KC;Mincle−/− pancreata (Extended-Data-Fig. 6h). However, Mincle deletion in PDA did not mitigate CXCL1 expression (Extended-Data-Fig. 6i) and CXCL1 blockade did not alter expression of Mincle-associated signaling intermediates (not shown).
To determine whether Mincle signaling accelerates oncogenesis, we serially treated 6 week-old KC mice with the Mincle ligand TDB, which we confirmed induced Syk phosphorylation in vivo (Extended-Data-Fig. 7a). Mincle ligation accelerated tumorigenesis resulting in higher grade PanIN lesions, extensive fibrosis, and scattered foci of invasion (Extended-Data-Fig. 7b). TDB also accelerated the growth-rate of orthotopically implanted KPC-derived tumors in WT (not shown) and RIP3−/− animals (Extended-Data-Fig. 7c), suggesting that the pro-tumorigenic effects of Mincle activation in PDA are either independent or downstream of RIP3 signaling. Moreover, the inflammatory TME in TDB-treated RIP3−/− pancreata recapitulated the immune-suppressive milieu associated with an intact necroptosis signaling mechanism. Specifically, TDB-treated pancreata trended toward a lower fraction of tumor-infiltrating T cells (Extended-Data-Fig. 7d) and exhibited increased recruitment of both MDSC (Extended-Data-Fig. 7e) and M2-like TAMs which expressed high PD-L1 (Extended-Data-Fig. 7f–i). Similarly, direct inoculation of orthotopic PDA tumors with recombinant SAP130 accelerated PDA growth in WT and RIP3−/− hosts but not in Mincle−/− animals (Extended-Data-Fig. 7j) and recruited an immune-suppressive infiltrate (Extended-Data-Fig. 7k, l).
To determine whether Mincle signaling is required for PDA progression, we crossed Mincle−/− with KC mice and interrogated pancreata at serial intervals. Mincle deletion slowed the rate of oncogenesis based on histological analysis, pancreas weight, and animal survival (Extended-Data-Fig. 8a–c). Similarly, orthotopic KPC-derived tumor implantation in Mincle−/− pancreata resulted in smaller tumors and prolonged survival compared with implantation in WT hosts (Extended-Data-Fig. 8d, e). However, the survival benefit afforded to Mincle−/− mice was not as pronounced as RIP3−/−. Moreover, KrasG12D PDEC orthotropic implantation experiments suggested that CXCL1+Mincle blockade had additive protective effects whereas combined blockade of RIP3+Mincle or RIP3+CXCL1 did not confer additional protection over RIP3 blockade alone (Extended-Data-Fig. 8f).
To determine whether Mincle deletion mimics the immunogenic reprogramming of the TME associated with RIP3 deletion, we assayed the pancreatic infiltrate in KC;Mincle−/− mice. KC;Mincle−/− pancreata exhibited diminished TAM infiltration but increased T cell recruitment on IHC (Extended-Data-Fig. 9a). Flow cytometry confirmed that Mincle deletion was associated with a higher immunogenic T cell infiltrate (Extended-Data-Fig. 9b–d), diminished MDSC infiltration (Extended-Data-Fig. 9e), a trend toward reduced DC (Extended-Data-Fig. 9f), a decreased fraction of TAMs (Extended-Data-Fig. 9g) with M1-like polarization (Extended-Data-Fig. 9h, i) and reduced PD-L1 expression (not shown). These changes recapitulate the immunogenic reprogramming of the TME characteristic of RIP3 deletion.
To investigate whether tumor-protection in absence of RIP3 or Mincle signaling is T cell dependent, we depleted T cells coincident with orthotopic KPC tumor administration in cohorts of WT, RIP3−/−, and Mincle−/− animals. T cell depletion did not influence PDA growth in WT mice. However, tumor protection was abrogated in RIP3−/− and Mincle−/− cohorts (Extended-Data-Fig. 10a). Conversely, depletion of macrophages in WT mice led to T cell activation and tumor protection; however, macrophage-depletion had no effect on further enhancing T cell activation or tumor protection in RIP3−/− and Mincle−/− animals (Extended-Data-Fig. 10b, c). These data suggest that in WT hosts TAMs promote PDA progression whereas T cells are dispensable to outcome; conversely, in absence of RIP3 or Mincle signaling, macrophages surrender their tumor-promoting effects and T cells are reprogrammed into indispensable mediators of anti-tumor immunity. Collectively, our work suggests that necroptosis-induced CXCL1 and Mincle signaling promote myeloid cell induced adaptive immune suppression in PDA. Each of these networks represent novel targets for experimental therapeutics (Extended-Data-Fig. 10c).
Methods
Animals and In Vivo Models
C57BL/6 (H-2Kb) mice were purchased from Jackson Labs (Bar Harbor, ME). Mincle−/− mice were obtained from the MMRRC (San Diego, CA)17. RIP3−/− mice were obtained from Genentech (San Francisco, CA)18. KC (gift of D. Bar-Sagi) and KPC (gift of Mark Philips, both New York University) mice develop pancreatic neoplasia endogenously by expressing mutant Kras alone or mutant Kras and p53, respectively, in the progenitor cells of the pancreas19,20. Both male and female mice were used but animals were gender and age matched in each experiment. Randomization was not performed. There were no specific inclusion or exclusion criteria. Sample sizes for experiments were determined without formal power calculations. Survival data for control KC mice were previously reported21. For orthotopic pancreatic tumor challenge, mice were administered intra-pancreatic injections of either KrasG12D PDEC or FC1242 tumor cells derived from KPC mice. KrasG12D PDEC and FC1242 cells were generated as previously described21,22. In preparation for intra-pancreatic injection, cells were suspended in PBS with 50% Matrigel (BD Biosciences, Franklin Lakes, NJ) at 1×106 cells/mL and 1×105 cells were injected into the body of the pancreas via laparotomy. Age-matched mice were used between 8–10 weeks of age for orthotopic tumor experiments. Mice were sacrificed 3–6 weeks later and tumor volume recorded. To study the effects of Mincle ligation, mice were administered TDB (4mg/kg; Invivogen, San Diego, CA) by i.p. injection thrice weekly for 8 weeks in endogenous tumor models and for 3 weeks in the orthotopic tumor models. In other experiments, orthotopic tumors were serially treated with direct inoculation of recombinant SAP130 (22µg; MyBioSource, San Diego, CA) at one week intervals via mini-laparotomy. In select experiments, cohorts of mice were treated daily with the RIP1 inhibitor Nec-1s (2mg/kg, i.p.; BioVision, Milpitas, CA) or a neutralizing α-CXCL1 mAb (4mg/kg, i.v.; R&D Systems). Gemcitabine (100mg/kg, i.p.; Hospira, Lake Forest, IL) was administered in vivo to KPC mice for 3 doses at 72h intervals unless otherwise specified. T cells (T24/31) and macrophages (F4/80, both BioXcell) were depleted with neutralizing mAbs using regimens we have previously described23. In some experiments, mice were subcutaneously administered FC1242 cells (1×106) or B16 melanoma (1×106; gift of Ronald DeMatteo, Memorial Sloan-Kettering Cancer Center) and sacrificed at 18 days. Investigators were not blinded to group allocation but were blinded when assessing outcome. All animal procedures were approved by the New York University School of Medicine IACUC. The maximum tumor size permitted is 3cm3 and this was not exceeded.
Cell lines and In Vitro Experiments
The human PDA cell lines AsPC1, PANC1, and MIA PaCa-2 cells (gifts of Dafna Bar-Sagi, originally obtained from ATCC) were maintained in complete RPMI (RPMI 1640 with 10% heat-inactivated FBS, 2 mM L-glutamine, 1% Penicillin/Streptomycin). Cell lines were not authenticated. Cells were free of mycoplasma. In selected experiments, cells were treated with Gemcitabine (10–50µM), Nec-1s (50µM), a RIP3 inhibitor (GSK872; 6 µM), or a MLKL inhibitor (Necrosulphonamide, 1µM, both EMD Millipore, Billerica, MA). Cellular viability was determined by PI staining. Cellular proliferation was assessed using the XTT II assay according to the manufacturer’s protocol (Roche, Pleasanton, CA) and expressed as % proliferation compared to control. Inflammatory mediators in cell culture supernatant were measured using the Milliplex Immunoassay (Millipore, Billerica, MA). CXCL1 was additionally measured using Flexbeads (BD Biosciences) and ELISA (R&D Systems).
Cellular Harvest and Flow Cytometry
Human or murine single cell suspensions for flow cytometry were prepared as described previously with slight modifications24. Briefly, pancreata were placed in cold RPMI 1640 with 1 mg/mL Collagenase IV (Worthington Biochemical, Lakewood, NJ) and 2 U/mL DNAse I (Promega, Madison, WI) and minced with scissors to sub-millimeter pieces. Tissues were then incubated at 37°C for 30 minutes with gentle shaking every 5 minutes. Specimens were passed through a 100µm mesh, and centrifuged at 350g for 5 minutes. The cell pellet was resuspended in cold PBS with 1% FBS. After blocking FcγRIII/II with an anti-CD16/CD32 mAb (eBioscience, San Diego, CA), cell labeling was performed by incubating 106 cells with 1 µg of fluorescently conjugated mAbs directed against murine CD44 (IM7), CD206 (C068C2), PD-L1 (10F.9G2), PD-1 (29F.1A12), CD3 (17A2), CD4 (RM4-5), CD8 (53-6.7), CD45 (30-F11), CD11b (M1/70), Gr1 (RB6-8C5), CD11c (N418), MHC II (M5/114.15.2), IL-10 (JES5-16E3), IFN-γ (XMG1.2), TNFα (MP6-XT22), F4/80 (BM8), CD19 (6D5; all Biolegend, San Diego, CA), p-Syk (moch1ct, eBioscience), and CD204 (2F8; Acris Antibodies, San Diego, CA). mAbs directed against Mincle (4A9, MBL International Corporation, Woburn, MA) were conjugated to FITC using the FITC Conjugation Kit (Abcam, Cambridge, MA). Human pancreas and PBMC were stained with mAbs directed against CD45 (HI30), CD14 (HCD14), CD15 (W6D3), CD19 (H1B19), CD11b (M1/70), CD11c (3.9), MHC II (L243; all Biolegend) and Mincle (AT16E3; Acris Antibodies). Intracellular cytokine staining was performed using the Fixation/Permeabilization Solution Kit (BD Biosciences). Flow cytometry was carried out on the LSR-II flow cytometer (BD Biosciences). Data were analyzed using FlowJo v.7.6.5 (Treestar, Ashland, OR).
Western Blotting and Immunoprecipitation
For protein extraction from tissues, 15–30 mg of tissue was homogenized in 150–300µL (i.e. 10 times the weight) ice-cold RIPA buffer. Total protein was quantified using the BioRad DC Protein Assay according to the manufacturer’s instructions (BioRad, Hercules, CA). Western blotting was performed as previously described with minor modifications24. Briefly, 10 % Bis-Tris polyacrylamide gels (NuPage, Invitrogen) were equiloaded with 10–30µg of protein, electrophoresed at 200 V, and electrotransferred to PVDF membranes. After blocking with 5% BSA, membranes were probed with primary antibodies to β-actin (8H10D10), FADD (Polyclonal), RIP1 (D94C12), Caspase-8 (D35G2), PLC-γ (polyclonal), p-PLC-γ (polyclonal), Bcl-XL (54H6; all Cell Signaling, Beverly, MA), RIP3 (Polyclonal; Abgent, San Diego, CA), c-Myc (9E10), CDK4 (C-22), CARD9 (polyclonal), Syk (polyclonal), p-Syk (polyclonal), Rb (C-15), SAP130 (H-300), Mincle (H-46; all Santa Cruz Biotechnologies, Dallas, TX), and MLKL (polyclonal; Abcam). Blots were developed by ECL (Thermo Scientific, Asheville, NC). For immunoprecipitation experiments, RIP1 or SAP130 was precipitated with protein G-agarose from cells. Immunoprecipitates were re-suspended and heated in loading buffer under reduced conditions, and resolved by 10% SDS-PAGE before transfer to PVDF membranes. The presence of co-immunoprecipitated RIP3 or Mincle, respectively, were determined by western blotting.
Histology, Immunohistochemistry, and Microscopy
For histological analysis, pancreatic specimens were fixed with 10% buffered formalin, dehydrated in ethanol, embedded with paraffin, and stained with H&E or Gomori’s Trichrome. The fraction of preserved acinar area was calculated as previously described24. Pancreatic ductal dysplasia was graded according to established criteria25. Immunohistochemistry in mouse tissues was performed using antibodies directed against F4/80 (CI:A3-1; Abcam), CD3 (Polyclonal; Abcam), Arg1 (EPR6671(B); Abcam), SAP130 (Polyclonal; Abcam), Mincle (AT16E3; Abcam), p-Syk (Polyclonal; Abcam), Ki67 (Polyclonal; Abcam), and CXCL1 (Polyclonal, Abcam). For analysis of human tissues, de-identified paraffin-embedded human PDA specimens and surrounding non-tumorous tissues from 10 consecutive surgically resected PDA patients at NYU Medical Center were probed with antibodies directed against RIP1 (D94C12; Cell Signaling), RIP3 (Q9Y572; Abgent), Mincle (AT16E3; Abcam), CXCL1 (Polyclonal; Abcam), and SAP130 (Polyclonal; Abcam). All human tissues were collected using an IRB approved protocol and donors of de-identified specimens gave informed consent. Sample sizes for human experiments were not determined based on formal power calculations. Quantifications were performed by assessing 10 high-power fields (HPF; 40×) per slide in a blinded manner. Immunofluorescent staining in frozen mouse tissues or cells was performed using antibodies against Mincle (AT16E3, Acris Antibodies), RIP1 (Polyclonal, Bioss), RIP3 (Polyclonal, Bioss), CD45 (30-F11; BD Biosciences), CK19 (Clone 13, Abnova), CXCL1 (Polyclonal; Abcam), CXCR2 (SA045E1; BioLegend), SAP130 (Polyclonal; Abcam), and DAPI (Vector Labs, Burlingame, CA). Immunofluorescent images were acquired using the Zeiss LSM700 confocal microscope with ZEN 2010 software (Carl Zeiss, Thornwood, New York).
PCR
RNA extraction was performed using the RNeasy Mini kit (Qiagen, Germantown, MD) as per manufacturer’s instructions. RNA was converted to cDNA using the RT2 First Strand Kit (Qiagen). qPCR was performed using the RT2 SYBR Green qPCR mastermix (Qiagen) on the Stratagene MX3005P (Stratagene, La Jolla, CA) according to the respective manufacturers’ protocols. Primers employed for human and mouse samples (RIP1, RIP3, CASP8, FADD, CXCL1, and SAP130) were purchased from Qiagen. Expression levels were normalized to β-actin and expressed as fold change compared to control.
Human Database and Statistical Analysis
Human RNAseq data and clinical correlations were performed using the UCSC Cancer Genomics Browser (https://genome-cancer.ucsc.edu/)26. Data is presented as mean +/− standard error. Survival was measured according to the Kaplan-Meier method. Statistical significance was determined by the Student’s t test and the log-rank test using GraphPad Prism 6 (GraphPad Software, La Jolla, CA). P-values <0.05 were considered significant.
Extended Data
Supplementary Material
Acknowledgments
This work was supported by grants for the German Research Foundation (LT), the National Pancreas Foundation (CPZ), the Pancreatic Cancer Action Network (GM), the Lustgarten Foundation (GM), and National Institute of Health Awards CA155649 (GM), CA168611 (GM), and CA193111 (GM, ATH). We thank the New York University Langone Medical Center (NYU LMC) Histopathology Core Facility, the NYU LMC Flow Cytometry Core Facility, the NYU LMC Microscopy Core Facility, and the NYU LMC BioRepository Center, each supported in part by the Cancer Center Support Grant P30CA016087 and by grant UL1 TR000038 from the National Center for the Advancement of Translational Science (NCATS).
Footnotes
Author Contributions
LS (In vivo experiments, flow cytometry; analysis and interpretation; manuscript preparation; statistical analysis; co-first author), GW (In vivo experiments, flow cytometry; analysis and interpretation; manuscript preparation; statistical analysis; co-first author), ST (in vivo experiments, IHC), NNGL (western blotting), SA (IHC), DA (flow cytometry), AA (tissue culture, cell line generation), RB (technical assistance, critical review), DD (flow cytometry, critical review), SHG (mouse breeding, critical review), ATH (technical assistance, critical review), MP (western blotting, flow cytometry, critical review), AO (Immunoprecipitation), CPZ (technical advice, PCR, flow cytometry), MP (western blotting), MR (genotyping), DT (animal breeding, in vivo tumor experiments), CH (histological analysis), MH (FACS, data analysis), VRM (FACS, data analysis), DE (cell line creation, in vivo experiments), GM (analysis and interpretation; study concept and design; study supervision; critical review).
None of the authors has any potential conflict of interest related to this manuscript.
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