Cancer cell-intrinsic inflammasome protein ASC links innate immunity with mitochondrial metabolism in driving pancreatic cancer

Domain: chemistry
Published: 2026-02-07
Editorial outcome: minor_revision

Abstract
Pancreatic ductal adenocarcinoma (PDAC) is driven by genetic alterations in the pancreatic epithelium (e.g., KRAS) coupled with dysregulated innate immunity that triggers tumor-promoting chronic inflammation. However, the identity of innate immune molecular regulators as therapeutic targets in PDAC is ill-defined. Here, we show in PDAC patients that elevated tumoral expression of the inflammasome adaptor protein ASC and its downstream effector Caspase-1 is primarily colocalized to the pancreatic ductal epithelium and prognostic for poor survival. In the mutant Kras-driven KPC PDAC mouse model, global and conditional (pancreatic epithelial) ablation of ASC, or nanobody-mediated targeting of extracellular ASC, suppresses pancreatic tumorigenesis. Whole transcriptome profiling and multiplex immunofluorescence reveal that the tumor-promoting activities of epithelial-derived ASC align with molecular pathways for mitochondrial respiration, metabolism (glycolysis), and immune responses. Our discovery that ASC-containing inflammasomes promote PDAC by acting as a molecular bridge between innate immunity, mitochondrial dysfunction and metabolic reprogramming provides the rationale to therapeutically target ASC in cancers.

Introduction
Pancreatic cancer is predicted to be the second most lethal cancer in Western countries by 2030 and is associated with a low overall 5-year survival rate of 10–12%1–3. Poor survival outcomes for pancreatic cancer are linked to late detection in 80% of patients diagnosed with inoperable locally advanced or metastatic late-stage disease, and the limited effectiveness of current standard-of-care chemotherapy regimens, and more recently immunotherapy3,4. The overwhelming majority (~90%) of cases present as pancreatic ductal adenocarcinoma (PDAC), driven by genetic alterations in the pancreas epithelium (e.g., KRAS, 90%; TP53, <70%) coupled with a highly heterogenic tumor microenvironment (TME) that invariably exists in an immunosuppressive state3–5.

Chronic inflammation driven by uncontrolled activation of the innate arm of the immune system is associated with at least 30% of all cancers, including PDAC, and is one of the hallmarks of cancer6–9. In PDAC, the chronically inflamed TME is enriched in innate immune cells (e.g., tumor-associated macrophages, myeloid-derived suppressor cells (MDSCs), neutrophils, dendritic cells), which synergize with the transformed pancreatic epithelium and other stromal cellular components (i.e. cancer-associated fibroblasts, endothelial cells) to release a myriad of inflammatory cytokines (e.g. IL-1β, IL-6, IL-10, TNFα), chemokines (e.g., CCL2, CXCL1, CXCL12), growth (e.g., TGFβ), and angiogenic (e.g., VEGF) factors that propagate a pro-tumorigenic TME4,5,10,11. Despite the burgeoning evidence for innate immunity promoting the pathogenesis of PDAC, immune-based therapies for PDAC patients have been restricted to inhibitors targeting immune checkpoints of adaptive immunity, namely PD-1, PD-L1 and CTLA-4, with minimal clinical benefit4,5.

Innate immune-driven inflammatory responses to microbial infection and sterile injury depend on a series of cell surface, endosomal and cytosolic pattern recognition receptors (PRRs) that are expressed in immune and non-immune (e.g., epithelial) cells12,13. PRRs are classified into several structurally and functionally conserved subfamilies, including Toll-like receptors (TLRs), Absent in melanoma 2 (AIM2)-like receptors (ALRs), and nucleotide-binding domain and leucine-rich repeat containing protein receptors (NLRs)14–16. PRRs have also gained recent attention as key oncogenic drivers in many cancer types, including upper gastrointestinal12. A critical role for microbial-sensing innate immune PRRs in PDAC is also suggested by the discovery that pancreatic tumors house a distinct gut-derived microbiome that promotes tumor growth and influences patient survival17,18.

A subset of NLR family members, namely NLR pyrin domain-containing 1 (NLRP1), NLRP3, NLRP6, NLRP12, NLR CARD domain-containing 4 (NLRC4), and NLR apoptosis inhibitory protein (NAIP), as well as AIM2 and pyrin, are best characterised for driving chronic inflammatory responses via multiprotein complexes called ‘inflammasomes’19. Upon ligand sensing, these PRRs associate with the adaptor protein Apoptosis-associated Speck-like protein containing a CARD (ASC; encoded by the PYCARD gene) and oligomerize to form inflammasome complexes (ASC “specks”) that recruit Caspase-1 to catalyze maturation of inactive pro-IL-18 and pro-IL-1β into their bioactive cytokines19. The extracellular secretion of IL-1β and IL-18 across the plasma membrane is facilitated by the pore-forming protein gasdermin D (GSDMD), which is itself activated by Caspase-1-mediated cleavage20. ASC can possess both intracellular and extracellular functions, the latter aligned to a sustained pathologic response in inflamed tissues that can be selectively targeted with camelid-derived anti-ASC nanobodies (VHHmASC), thus distinguishing between intracellular and extracellular ASC function21,22.

Definitive studies on ASC-containing inflammasomes in cancer are still in their relative infancy and have been largely restricted to the colon, stomach, and skin23–26. Intriguingly, ASC has been assigned opposing tumor promoter or suppressor roles, which likely depend on the cell (e.g., myeloid versus epithelial) and tissue type and disease stage in which ASC is expressed, as well as its intra- versus extra-cellular location, downstream inflammasome effectors (IL-1β versus IL-18), and engagement with specific inflammasome-associated PRR(s)12,26,27. Here, we reveal that tumor (i.e., epithelial)-cell intrinsic ASC and its inflammasome effectors Caspase-1 and IL-18 (but not IL-1β) are upregulated in PDAC, and demonstrate in the KPC spontaneous mouse model for PDAC that global and conditional (pancreatic epithelial) genetic targeting of ASC, as well as nanobody-mediated neutralization of extracellular ASC, suppressed pancreatic tumorigenesis. Furthermore, in KPC mice, ASC-driven PDAC aligned with enrichment of molecular pathways involved in mitochondrial dysfunction and a metabolic shift towards glycolysis. Collectively, we discover a targetable pro-tumorigenic function of ASC-containing inflammasomes with clinical significance in PDAC.

Results
Elevated expression and activation of inflammasome components ASC, Caspase-1, and IL-18 in human PDAC are prognostic for poor survival
Global transcriptomics on PDAC patient primary tumor versus non-tumor pancreas tissues in independent datasets (Victorian Pancreatic Cancer Biobank, VPCB; The Cancer Genome Atlas, TCGA; GSE15471) identified that PYCARD mRNA levels were significantly upregulated in PDAC tumor biopsies at all stages of disease (Fig. 1a, Supplementary Fig. S1a, b). In addition, PYCARD gene expression was elevated in PDAC patient tumors irrespective of distinct molecular subtypes previously assigned to PDAC28 (Supplementary Fig. S1c, d). Furthermore, elevated PYCARD gene expression significantly correlated with impaired survival in both VPCB and TCGA patient cohorts, indicating that PYCARD is a poor prognostic indicator in PDAC (Fig. 1b, Supplementary Fig. S1e).

Increased ASC protein expression levels were also detected in PDAC patient tumors by immunoblotting and immunohistochemistry, the latter revealing pronounced staining of ASC-positive cells predominantly in the pancreatic ductal epithelium (Fig. 1c, d). Opal multiplex immunofluorescence staining of PDAC patient tumors confirmed that ASC strongly colocalized predominantly with CK19-positive epithelial cells, while ASC staining in CD45-positive pan-immune cells was not as pronounced (Fig. 1e, f, Supplementary Fig. S1f, g). Notably, throughout PDAC patient tumors, ASC staining in epithelial (and immune) cells localized to both the nucleus and cytoplasm, and frequently appeared punctate, suggestive of ASC “specks’, with evidence also of ASC extracellular staining (Fig. 1e, f, Supplementary Fig. S1f, g).

The above observations suggest that heightened activation of ASC-containing inflammasomes may be a common feature of human PDAC. Indeed, mRNA levels for CASP1 were significantly elevated in PDAC patient tumors, which positively correlated with PYCARD and were indicative of poor patient survival (Supplementary Fig. S2a–c). Gene expression levels for inflammasome-associated PRRs, except for NLRP3, were also significantly upregulated in PDAC patient tumors (Supplementary Fig. S2d). Immunoblotting indicated that protein expression levels of pro (p45) and cleaved (p20) forms of Caspase-1, the latter indicative of ASC inflammasome activation, were significantly elevated in PDAC patient tumor versus non-tumor tissues (Fig. 1c). Immunohistochemistry also demonstrated significantly increased numbers of cleaved Caspase-1-positive cells in the ductal epithelium of human PDAC tumor versus non-tumor tissue sections, with immunofluorescence depicting strong nuclear colocalization of cleaved Caspase-1 largely in the cytokeratin-positive epithelium of PDAC tumors (Fig. 1e–g, Supplementary Fig. S1f, g). Moreover, strong nuclear, and to a lesser extent cytoplasmic, colocalization of cleaved Caspase-1 with ASC was observed in PDAC patient tumors (Fig. 1e, f, Supplementary Fig. S1f, g).

We next investigated whether the expression levels of inflammasome effector cytokines IL-1β and IL-18 were upregulated in independent PDAC patient cohorts. ELISA revealed that protein levels of pro and mature forms of IL-18, but not IL-1β, were significantly increased in serum and/or tumor tissue lysates from PDAC patients, with an incremental increase in IL-18 protein levels observed from early (I/II) through to advanced (III, IV) stages (Fig. 1h, i, Supplementary Fig. S3a). Furthermore, IL18, but not IL1B, tumoral mRNA levels were consistently and significantly upregulated in PDAC patient cohorts, with only elevated IL18 mRNA levels prognostic for impaired patient survival (Fig. 1j, k, Supplementary Fig. S3b, f). In addition, serum IL-18 protein levels positively correlated with tumoral PYCARD and CASP1 mRNA levels in PDAC patients (Fig. 1l). Taken together, these data support the presence of activated inflammasome complexes in human PDAC.

Elevated expression of ASC, activated Caspase-1 and mature IL-18 are features of PDAC in KPC mice
We next investigated the inflammasome activation status in the KPC mouse strain which expresses conditionally-induced activating KrasG12D and dominant-negative Trp53R172H mutations controlled by the pancreas-specific Pdx1 promoter, and mimics the molecular and cellular immunologic and oncogenic processes, as well as tumor heterogeneity, of human PDAC29. At 6-8 weeks of age, KPC mice develop precursory pancreatic intraepithelial neoplasia (PanIN) accompanied by an influx of inflammatory cells that progresses to localized (10-12 weeks) and metastatic (16-18 weeks) ductal (adeno) carcinoma29. In 10wo and 20wo lesion-bearing KPC pancreatic tissues, qPCR revealed that Pycard and Casp1 mRNA levels, as well as inflammasome-associated PRRs, were significantly elevated compared to lesion-free pancreas tissues from wild-type (WT) littermate controls (Fig. 2a, b, Supplementary Fig. S4a, b). Consistent with our clinical findings, immunoblotting revealed significantly higher protein expression levels of ASC, pro and cleaved Caspase-1 in pancreatic tissues from 10wo and 20wo KPC mice compared to age-matched WT controls, with immunohistochemistry suggesting nuclear and cytoplasmic staining of cleaved Caspase-1-positive cells throughout the dysplastic and hyperproliferative pancreatic ductal epithelium of KPC mice (Fig. 2c, d, Supplementary Fig. S4c, d). Furthermore, multiplex immunofluorescence confirmed the predominant nuclear and cytoplasmic colocalization of ASC and cleaved Caspase-1 in ductal epithelial cells, which became more apparent as pancreatic lesions progressed from early to intermediate to late stages in 6wo, 10wo, and 20wo KPC mice, respectively (Fig. 2e, Supplementary Fig. S4e). Notably, ASC and cleaved Caspase-1 also colocalized in extracellular spaces as punctate ASC-containing specks indicative of inflammasome activation (Supplementary Fig. S4e). In further support of excessive inflammasome activation during PDAC, ELISA indicated that secreted IL-18, but not IL-1β, serum protein levels were significantly elevated (~2.5-fold) in 20wo KPC versus WT mice, despite levels of mRNA and pro (inactive) protein forms for both cytokines being similarly upregulated in the pancreas of KPC mice (Fig. 2f–h). Moreover, ELISA indicated a significant 2-fold increase in mature IL-18 levels in pancreatic tissues from 10wo and 20wo KPC mice versus age-matched WT controls (Fig. 2i). Collectively, these data indicate that elevated activation of ASC-containing inflammasomes is also a feature of the KPC PDAC model.

ASC-containing inflammasomes promote PDAC in KPC mice via selective upregulation of mature IL-18
To investigate whether ASC-containing inflammasomes promoted PDAC, we generated KPC mice globally deficient for ASC (KPC:ASCKO) (Supplementary Fig. S5a). At 10 and 20 weeks of age, the pancreas size and mass of KPC mice were markedly elevated compared to WT mice (Fig. 3a, b, Supplementary Fig. S5b, c). By contrast, compared to age- and sex-matched KPC littermates, the pancreas of 10wo and 20wo KPC:ASCKO mice were visibly smaller and significantly reduced in mass by ~65% and ~75%, respectively, with no observable sex bias (Fig. 3a, b, Supplementary Fig. S5b, c). Histopathological assessment of 10wo H&E-stained pancreas cross-sections indicated a marked suppression in the early development of PDAC in KPC:ASCKO mice, with a 7-fold reduction in the area of pancreas affected by lesions (49% KPC versus 7% KPC:ASCKO) comprising only PanIN versus ductal carcinomas of various differentiation grades in KPC mice (Supplementary Fig. S5d–g). Similarly, 20wo KPC:ASCKO mice displayed a 3.5-fold reduction in pancreas area (65% KPC versus 18% KPC:ASCKO) comprising lower grade well and moderate differentiated lesions compared to the more severe poor differentiated lesions in 50% of age-matched KPC mice (Fig. 3c–e, Supplementary Fig. S5g). Furthermore, metastatic lesions were observed in the livers of ~19% (7/37) of KPC mice aged 20 or more weeks (Fig. 3a), compared to 0% (0/33) in age-matched KPC:ASCKO mice. Therefore, these data indicate that elevated ASC-containing inflammasome activity promotes the onset and progression of PDAC in KPC mice.

The suppressed PDAC phenotype in KPC:ASCKO mice associated with reduced inflammasome activation, with immunohistochemistry and immunofluorescence showing that elevated numbers of cleaved Caspase-1-positive cells within the transformed pancreatic epithelium of 10wo and 20wo KPC mice were significantly reduced in the pancreas of age-matched KPC:ASCKO mice (Fig. 3f, g, Supplementary Fig. S5h). Immunoblotting also confirmed that cleaved Caspase-1 protein levels were markedly lower in the pancreas of 20wo KPC:ASCKO compared to KPC mice (Fig. 3h). Similarly, total GSDMD cellular staining was significantly upregulated in the pancreatic ductal epithelium of KPC compared to WT and KPC:ASCKO mice (Fig. 3i). Intriguingly, immunoblotting showed that the increased GSDMD levels in the pancreas of KPC mice (versus WT and KPC:ASCKO) corresponded to full-length GSDMD protein, rather than the N-terminal cleaved GSDMD p30 fragment (lowly expressed among all genotypes) which facilitates plasma membrane pore formation and the extracellular release of cytosolic contents (Fig. 3j). In further support of reduced inflammasome activity in KPC:ASCKO mice, ELISA demonstrated that protein levels of both secreted IL-18 in serum and mature IL-18 in pancreatic tissues from KPC:ASCKO mice were significantly reduced compared to elevated levels in KPC mice (Fig. 3k, l, Supplementary Fig. S5i). Taken together, these data strongly suggest the requirement of ASC for the inflammasome-mediated processing of mature IL-18 during pancreatic tumorigenesis.

ASC-driven PDAC in KPC mice is associated with elevated pancreatic epithelial cell proliferation, apoptosis and inflammation
We next assessed the cellular processes by which ASC inflammasome activation promoted pancreatic tumorigenesis in KPC mice. Consistent with amelioration of the hyperplastic PDAC phenotype of KPC mice upon genetic ablation of ASC, elevated numbers of PCNA- and Ki67-positive proliferating cells in the pancreatic epithelium of 10wo and 20wo KPC mice were significantly reduced in age-matched KPC:ASCKO mice (Fig. 4a, Supplementary Fig. S6a, b). ASC also promoted dysregulated apoptosis of the transformed pancreatic epithelium that is a feature of PDAC in KPC mice30, as evidenced by the paucity of apoptotic cleaved Caspase-3-positive cells in the pancreas of 10wo and 20wo KPC:ASCKO mice (similar to that seen in WT mice) compared to the significantly higher numbers of cleaved Caspase-3-positive cells in the pancreas of age-matched KPC mice (Fig. 4b, Supplementary Fig. S6c).

ASC deficiency also markedly suppressed chronic pancreatic inflammation in KPC mice, with immunohistochemical analyses indicating significantly reduced numbers of CD45+ leukocyte infiltrates, comprising F4/80+ macrophages, B220+ B cell, CD3+ T cell and NK1.1+ NK cell immune subsets, in the pancreas of KPC:ASCKO versus KPC mice (Fig. 4c–e, Supplementary Fig. S6d, S7a, b). The suppressed infiltration of immune cells also coincided with reduced mRNA levels of numerous inflammatory genes in the pancreas of KPC:ASCKO versus KPC mice (Fig. 4f). By contrast, the expression of representative genes encoding tumor-promoting angiogenic factors, namely Cxcl1, Cxcl2, Mmp2, Mmp9 and Vegf, was significantly elevated in the pancreas of KPC and KPC:ASCKO mice versus WT mice (Supplementary Fig. S7c). Collectively, these data suggest that ASC primarily promotes pancreatic tumor cell proliferation, apoptosis and inflammation in KPC mice.

ASC ablation in the pancreatic ductal epithelium of KPC mice suppresses PDAC
Considering that elevated expression of ASC and activated Caspase-1 in pancreatic tumors predominantly colocalize to the transformed ductal epithelium, we investigated whether epithelial-derived ASC-containing inflammasomes contributed to PDAC by generating KPC mice carrying conditionally targeted ASC-floxed alleles (KPC:ASCfl/fl) under control of the Pdx1 promoter (also driving mutant Kras and Trp53 expression). Immunohistochemistry and immunofluorescence confirmed the marked loss of ASC expression in the pancreatic ductal epithelium of KPC:ASCfl/fl mice, which coincided with reduced expression of activated Caspase-1 in the pancreatic epithelium (Fig. 5a–c). Notably, the pancreas of KPC:ASCfl/fl mice displayed significantly reduced overall mass (2.5-fold) and lesion burden (2-fold), along with a markedly higher proportion of low-grade well differentiated ductal lesions in the absence of any high-grade poor differentiated lesions, compared to age-matched KPC counterparts (Fig. 5d–h). Furthermore, proliferating Ki67-positive epithelial cell numbers were significantly reduced (2-fold) in ductal lesions of KPC:ASCfl/fl mice compared to KPC mice, while remaining significantly elevated compared to WT mice (Fig. 5i). Surprisingly the numbers of CD45-positive immune cell infiltrates were comparable (Fig. 5j). These data therefore implicate the pancreatic ductal epithelium as a major cellular source of tumor-promoting ASC-containing inflammasomes in PDAC, while suggesting that activation of ASC (and inflammasomes) in the non-epithelial compartment of the TME (e.g., immune cells) is also likely to contribute to tumorigenesis.

Nanobody-mediated therapeutic targeting of extracellular ASC specks (eASC) suppresses pancreatic tumorigenesis in KPC mice
We have previously discovered that ASC-containing inflammasomes display extracellular pro-inflammatory activities22 and the specific targeting of extracellular ASC specks (eASC) with camelid-derived nanobodies against murine ASC (VHHmASC) suppresses joint inflammation in in vivo models of gout and rheumatoid arthritis21,31. Importantly, VHHmASC binds to the pyrin domain of mouse ASC with high specificity, but not to human ASC or the pyrin domain of other inflammasome components (e.g., NLRP3), and no off-target effects have been observed21. Our observations that eASC appear in pancreatic lesions of KPC mice prompted us to target these complexes using VHHmASC. Acute treatment of 10wo KPC mice over 24 hours with VHHmASC markedly reduced Caspase-1 activation in the pancreas, supporting the notion that eASC contributes to Caspase-1 activation in PDAC (Fig. 6a). To evaluate whether eASC promotes PDAC, we administered VHHmASC or vehicle control to 6wo KPC mice (early onset of lesions) over an 8-week period. VHHmASC treatment significantly reduced the pancreas mass and tumor burden of KPC mice, with a 2-fold decrease in ductal pancreatic lesions of a lower grade (PanIN and moderately differentiated) compared to vehicle-treated (moderately and poorly differentiated) KPC mice (Fig. 6b-e).

Consistent with reduced tumor burden, the numbers of proliferating Ki67-positive ductal epithelial cells in the pancreas of VHHmASC-treated KPC mice were significantly reduced, concomitant with markedly reduced Caspase-1-positive cells and ASC speck formation in the ductal epithelium (Fig. 6f-j, Supplementary Fig. S8a, b). In the pancreas of VHHmASC-treated KPC mice, there was also a reduction in the number of infiltrating CD45-positive immune (i.e., inflammatory) cells which were predominantly CD3-positive T cells (Fig. 6k–n). Together, these findings reveal that nanobody-mediated therapeutic targeting of eASC in KPC mice can suppress PDAC.

ASC promotes PDAC via molecular pathways associated with immune responses, mitochondrial function, and energy metabolism
To investigate the molecular mechanisms downstream of ASC-containing inflammasomes that promote PDAC, we profiled the whole transcriptome of the pancreas from 20wo WT, KPC and KPC:ASCKO mice by RNA-seq. The pancreatic gene expression profile of tumor-bearing KPC mice was clearly distinguished from WT controls, with 1093 and 1094 genes significantly downregulated and upregulated, respectively, in KPC pancreatic tissues (Fig. 7a, Supplementary Data 1). A distinct differential gene expression profile was also observed between pancreatic tissues from KPC versus KPC:ASCKO mice (480 downregulated and 219 upregulated genes), the latter strikingly comparable to WT mice (Fig. 7a, Supplementary Data 1). Competitive gene set testing revealed that downregulated genes in the pancreas of KPC mice were primarily enriched in molecular pathways associated with energy metabolism and mitochondrial function (e.g., oxidative phosphorylation (OXPHOS), electron transport chain (ETC), cellular respiration), while upregulated genes were largely associated with activation of the immune (innate and adaptive) system (Fig. 7b, c, Supplementary Fig. S9, S10, Supplementary Data 2 and 3). Consistent with a downregulation of mitochondrial respiration pathways in the pancreas of KPC versus KPC:ASCKO and WT mice, expression of both mitochondrial- and nuclear-encoded genes for mitochondrial respiratory chain complexes I-V, in particular those of complex I (e.g., mt-Nd1, mt-Nd2, mt-Nd3, Ndufb8, Ndufs6), which is the ETC entry point, was significantly reduced in KPC mice (Fig. 7c, d). Furthermore, immunoblotting confirmed reduced expression levels of proteins representative of four mitochondrial OXPHOS complexes, namely NDUFB8 (complex I), UQCRC2 (complex III), MTCO1 (complex IV) and ATP5A (complex V), in the pancreas of 10wo and 20wo KPC mice compared to WT and KPC:KOmice (Fig. 8a, b, Supplementary Fig. S11a, b). Taken together, these molecular observations indicate a potential role for ASC in modulating mitochondrial activities needed for energy metabolism in PDAC.

ASC-driven PDAC is associated with impaired mitochondrial biogenesis and metabolic reprogramming towards aerobic glycolysis
Considering mitochondrial biogenesis encapsulates the transcription and translation of mitochondrial- and nuclear-encoded genes32, the impaired expression of mitochondrial ETC components in KPC mice (Fig. 8a, b) suggests that over-activation of ASC-containing inflammasomes in the pancreas may disrupt mitochondrial homeostasis. Indeed, mitochondrial DNA (mtDNA) copy number, which is a biomarker of mitochondrial biogenesis33, was significantly reduced in the pancreas of KPC mice compared to the higher pancreatic mtDNA copy number of KPC:ASCKO (and WT) mice (Fig. 8c). We next explored the ASC-mediated functional consequences of impaired mitochondrial biogenesis by assessing levels of reactive oxygen species (ROS), which in many cancer types, including oncogenic Kras-induced PDAC, accumulate in response to dysfunctional mitochondrial respiration and OXPHOS34–36. Immunodetection of carbonyl groups on proteins, an indicator of protein oxidation by ROS, demonstrated that significantly elevated levels of protein oxidation in pancreas lysates from tumor-bearing KPC mice (versus WT controls) were reduced towards wild-type levels in KPC:ASCKO mice (Fig. 8d, e). Furthermore, immunohistochemical staining for the cellular marker of oxidative stress, 4-hydroxy-2-nonenal (4-HNE), which is upregulated in PDAC35,37, was more pronounced in pancreatic epithelial ductal regions of KPC mice versus WT and KPC:ASCKO mice, as well as KPC:ASCfl/fl mice (Fig. 8f).

The downregulation of mitochondrial-associated OXPHOS in the transformed pancreas of KPC mice is consistent with the hypoxic state of the TME in Kras-addicted PDAC, which fosters a switch in mitochondrial-driven energy metabolism from OXPHOS to aerobic glycolysis (‘Warburg effect’)38–40. Indeed, elevated lactate levels in pancreatic tissues of KPC (versus WT) mice were substantially lower in KPC:ASCKO mice (Fig. 8g). Collectively, these data suggest that ASC-containing inflammasomes can contribute to the metabolic reprogramming of cancer cells in PDAC by influencing mitochondrial biogenesis, resulting in skewed energy metabolism towards aerobic glycolysis.

To determine the spatial cellular context of changes to mitochondrial biogenesis within the pancreatic TME and their localization with activated ASC-containing inflammasomes, we next performed multiplexed immunofluorescence on KPC versus WT pancreas sections with an antibody panel against ASC, cleaved Caspase-1, ductal epithelial (CK19) versus immune (CD45) cells, NDUFB8 (complex I), and UQCRC2 (complex III). Consistent with our earlier findings, the comparable staining pattern of ASC and cleaved Caspase-1 predominantly colocalized with CK19-positive ductal epithelial (~30%) versus CD45-positive immune (~8%) cells in the pancreas of KPC mice, which was not observed in the pancreas of WT mice comprising >80% CK19-negative acinar cells (Fig. 8h, i, Supplementary Fig. S11c–e). Moreover, colocalization of dual-labeled ASC and cleaved Caspase-1 cells with mitochondrial markers NDUFB8 and UQCRC2 (~20%) was observed in CK19-positive ductal epithelial lesions in KPC, but not WT, mice (Fig. 8h, i, Supplementary Fig. S11c–e). Furthermore, both the conditional and nanobody-mediated therapeutic targeting of ASC in KPC mice resulted in a marked reduction in the mitochondrial colocalization of ASC and cleaved Caspase-1 with NDUFB8 and UQCRC2 in the CK19-positive pancreatic ductal epithelium, with the suppressive effect on inflammasome activity more pronounced upon nanobody (VHHmASC) treatment (Supplementary Fig. S12, S13). Therefore, these in vivo data support the notion that the expression (and activation) of ASC-containing inflammasomes is upregulated in the pancreatic ductal epithelium and localizes with mitochondrial markers during PDAC.

To translate key in vivo mechanistic findings to human PDAC cells, we engineered two human Kras-mutant PDAC cell lines, PANC-1 and MIA PaCa-2, by siRNA-mediated knockdown of PYCARD to express reduced levels of ASC (ASCKD), concomitant with lower levels of active Caspase-1 (Supplementary Fig. S14a, b). Indeed, the proliferation, viability, and colony formation of PANC-1/ASCKD and MIA PaCa-2/ASCKD cells were significantly reduced compared to their respective control cells transfected with non-targeted scrambled siRNA (Supplementary Fig. S14c–e). Importantly, the lower proliferative potential of PANC-1/ASCKD and MIA PaCa-2/ASCKD cells coincided with their significantly reduced lactate and mitochondrial superoxide levels, along with increased mitochondrial-related gene expression and DNA copy number (Supplementary Fig. S14f–i). These observations are consistent with our in vivo data (e.g. Figs. 4a, 5i, 7, 8a–g) that genetic ablation of ASC in KPC mice reduced cellular proliferation within the pancreatic ductal epithelium which associated with elevated mitochondrial gene networks and DNA copy number, and a reversion of energy metabolism back towards mitochondrial respiration (OXPHOS), as seen in non-cancerous (wild-type) mice. Considering the reliance of cancer cells on aerobic glycolysis for proliferation and viability (survival), we further investigated the interplay between metabolic reprogramming and ASC-mediated cell proliferation and/or viability by treating human PDAC cells with two glycolysis inhibitors, STF-31 and 2-deoxyglucose (2-DG). Notably, consistent with ASC deficiency metabolically reprogramming cells towards mitochondrial OXPHOS, PANC-1/ASCKD and MIA PaCa-2/ASCKD cells were markedly less sensitive to the impaired viability (proliferation) induced by glycolysis inhibitors compared to their respective control cells (Supplementary Fig. S14j). Finally, mitochondrial-associated metabolic changes mediated by ASC in human PDAC cells were assessed by measuring key parameters of mitochondrial respiration (i.e., oxygen consumption rate; OCR) using the Seahorse XF Mito Stress Test. As shown in Supplementary Fig. S14k, l, both basal OCR and maximal OCR capacity, as well as the spare respiratory capacity (a measure of mitochondrial fitness in response to energy demands), were increased in ASCKD versus control scrambled siRNA-transfected human PDAC cells. Collectively, these data further indicate that ASC deficiency during PDAC rewires cellular energy needs for viability and proliferation from a glycolytic state back towards mitochondrial OXPHOS (i.e. respiration).

Methods
Ethical considerations
Full and informed patient consent was obtained for procedures under approval from the Monash Health Human Research Ethics Committee (HREC/15/MonH/117 and LNR/17/MonH/335). Patient studies were conducted in accordance with the World Medical Association Declaration of Helsinki statement on the ethical principles for medical research involving human subjects.

All animal studies were approved by the Monash University Monash Medical Centre “B” Animal Ethics Committee (approval numbers MMCB-2018-26 and MMCB-2022-23), and the University of Adelaide Animal Ethics Committee (AEC Medical; approval number M-2024-044). Disease progression in mice aged <16 weeks and >16 weeks was assessed once and twice weekly, respectively, for symptoms associated with advanced-stage cancer (e.g., weight loss, abdominal bloating, discolored stools). Experimental endpoints were set at pre-determined age groups based on disease stages (6 weeks, early; 10 weeks, intermediate; 20 weeks, late).

Humansamples
Primary PDAC tumor (n=83) tissue biopsies stored in the Victorian Pancreatic Cancer Biobank (VPCB) were collected by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) from patients at Monash Medical Centre (Melbourne, Australia), along with non-tumor pancreatic tissues (n=14) collected by Whipple surgical resection in individuals with non-cancerous conditions (Supplementary Table S1)41,58. Biopsies were stored in 10% formalin for blinded histopathological assessment and snap-frozen in liquid nitrogen for molecular analyses. Whole blood from the same PDAC patients and cancer-free individuals (i.e., non-tumor controls) (Supplementary Table S1) was collected and serum processed by coagulation at room temperature and centrifugation prior to storage at -80°C.

Mice
KPC (LSL-KrasG12D/+:LSL-Trp53R172H/+:Pdx1-Cre) mice29, along with ASC (encoded by Pycard) homozygous knockout (KO) mice59 and floxed ASCfl/fl mice (D. Golenbock, University of Massachusetts Chan Medical School, Worcester, MA, USA)24, were used to generate KPC:ASCKO and KPC:ASCfl/fl mice, respectively, on a mixed 129Sv × C57BL/6 background. Mice were housed under specific pathogen-free (SPF) conditions on a 12-hour light/dark cycle. As published previously59, ASC-deficient mouse strains do not exhibit microbial-driven adverse events (e.g., increased susceptibility to bacterial infections) when housed under a steady-state SPF environment. For therapeutic studies with camelid-derived mouse-specific ASC nanobody (VHHmASC), 6-week-old (6wo) male and female KPC mice were randomly assigned to treatment over 8 weeks by intraperitoneal injection, 3× weekly, with either VHHmASC (5mg/kg) or vehicle (saline) control. Experiments throughout our study comparing different mouse strains on a mixed 129Sv × C57BL/6 background, including where appropriate wild-type (WT) control mice, involved randomly assigned genetic- and age-matched littermates with no sex bias, and were conducted in accordance with ARRIVE guidelines60.

Cell lines and treatments
Human PDAC cell lines PANC-1 (CRL-1469) and MIA PaCa-2 (CRL-1420) were obtained from the American Type Culture Collection and grown in a humidified incubator at 37°C with 5% CO2. PANC-1 cells were maintained in Dulbecco’s Modified Eagle Media (DMEM; Gibco) supplemented with 10% fetal calf serum (FCS; Bovogen), L-glutamine (4mM) and glucose (4500mg/L), and MIA PaCa-2 cells were maintained in the above media also containing 2.5% horse serum (Thermo Fisher Scientific). Cell lines were routinely tested (negative) for mycoplasma contamination (MycoAlertTM PLUS Mycoplasma Detection Kit, Lonza).

Cell lines were transfected with the following siRNAs (Thermo Fisher Scientific) using Lipofectamine™ RNAiMAX Transfection Reagent (Thermo Fisher Scientific) in 6-well plates as per the manufacturer’s protocol: siRNAs for GAPDH positive control (AM4605) and non-targeting Negative Control No. 1 (4390843), along with two pre-designed siRNAs against PYCARD (PYCARD/ASC1, IDs 26508; PYCARD/ASC2 IDs 195168).

For treatments with metabolic inhibitors (in full-serum media), following 24-hour serum starvation and siRNA transfection, cells were treated with 2-Deoxy-D-glucose (2-DG; 10mM) or STF-31 (1µM), along with their respective vehicle controls (PBS, 1% final concentration or DMSO, 0.01% final concentration).

Cell proliferation, viability, and colony formation assays
For cell proliferation and viability assays, PANC-1 and MIA PaCa-2 cells were seeded in experimental triplicate into 96-well plates (2×103 cells/well). At 72 hours following either siRNA transfection or treatments, CellTitre Glo 2.0 (Promega) or alamarBlue™ HS (Invitrogen) reagent was added to each well, and luminescence or fluorescence values, respectively, were determined using a CLARIOstar Plus (BMG LABTECH) microplate reader. For colony formation assays, PANC-1 and MIA PaCa-2 cells were seeded in experimental triplicate into 24-well plates (100 cells/well) at 24 hours after siRNA transfection. After 96 hours, all wells were transfected again with siRNA to maintain the knockdown. At 7 days post seeding, the media was removed, and the cells were washed in PBS prior to fixing and staining in 0.1% crystal violet in 10% formalin for 20 minutes. Plates were washed and left to air-dry prior to manual counting of colonies (containing ≥50 cells).

RNA isolation and real-time PCR
Total RNA was isolated from snap-frozen mouse and human pancreas tissues using TRI Reagent® Solution (Sigma) followed by on-column RNeasy® Mini Kit RNA clean-up and DNase treatment (Qiagen), and from cells using the AllPrep DNA/RNA Mini Kit (Qiagen). Quantitative (q) real-time PCR was performed in technical triplicates on cDNA (synthesized by the Transcriptor High Fidelity cDNA Synthesis Kit; Roche) with SYBR Green chemistry (Life Technologies) using the 7900HT Fast Real-Time System (Applied Biosystems) and the ViiA7 Real-Time PCR System (Thermo Fisher Scientific). Data acquisition and analyses were undertaken using the respective Sequence Detection System Version 2.4 (Applied Biosystems) and ViiATM 7 Software (Thermo Fisher Scientific) packages. Forward and reverse primer sequences for mouse and human genes analyzed in this study have been previously published23,61,62. Additional primer pairs used were for human mt-ND1 (F, 5-CTACTCCTCATTGTACCCAT-3; R, 5-GTGAA GAGTTTTATGGCGTC-3), and UQCRC2 (F, 5-CCGTGGAATTGAAG CAGTTGGTG-3; R, 5-CTGTGGTGACATTGAGCAGGAAC-3).

Human PDAC patient datasets
Gene expression and patient survival data from the VPCB patient dataset, comprising 83 PDAC patient tumor samples and 14 non-tumor samples from cancer-free individuals, were generated and interrogated41. RNA sequencing (RNA-seq) data was extracted from The Cancer Genome Atlas (TCGA) for 148 primary tumor (PDAC) and 14 non-tumor pancreatic tissue samples in the TCGA-PAAD dataset, and reads per kilobase of exon model per million mapped reads (RPKM) was used to quantify mRNA levels for PYCARD, CASP1, IL18 and IL1B from each PDAC patient. For the GSE15471 PDAC patient dataset (36 paired tumor and non-tumor samples), gene expression data was downloaded and interrogated41. To assess PYCARD gene expression across distinct molecular subtypes of PDAC, normalized RNA-seq data was retrieved from PDAC patient tumors (n=96) and samples were grouped based on transcriptional signatures, segregating into four molecular subtypes (squamous, ADEX (abnormally differentiated endocrine exocrine), pancreatic progenitor, immunogenic)28. PYCARD mRNA levels were overlayed across the four subtypes, and pairwise independent t-tests were performed on PYCARD expression level comparing each subtype to another. Statistical significance was evaluated based on two-tailed P values.

ELISA and immunoblotting
Total protein lysates from snap-frozen tissues were prepared23,61,62. ELISA for human total IL-1β (R&D Systems), and mouse and human total IL-18 (MBL International Corporation) were performed on serum according to the manufacturer’s instructions. Mature and free mouse and human IL-18 were detected with sandwich ELISAs on lysates23. Immunoblotting was performed with antibodies against ASC (AdipoGen, AL177, 1:200), human Caspase-1 (Cell Signaling Technologies, D7F10 or 2225, 1:1000), mouse Caspase-1 (AdipoGen, AG-20B-0042-C100, 1:500), GSDMD (Abcam, ab219800, 1:2000) and α-Tubulin (Abcam, ab6160, 1:6667), as well as the OxPhos Rodent WB Antibody Cocktail (Invitrogen, 45-8099, 1:1000). Total protein staining was performed using the Revert 700 Total Protein Stain (LI-COR Biotechnology). Caspase-1 protein bands were visualized with horseradish peroxidase-conjugated secondary antibodies using the ChemiDoc Imaging System (Bio-Rad Laboratories), and for all other proteins with secondary antibodies conjugated to Alexa Fluor 680 or DyLight 800 using the Odyssey XF Imaging System (LI-COR Biotechnology). Immunoblots with the OxPhos Rodent WB Antibody Cocktail were also performed using the TrueBlot DyLight 680 conjugated anti-mouse IgG secondary antibody (Rockland Immunochemicals) to avoid interference from the mouse Ig heavy chain. For immunodetection of oxidized proteins, 10μg of protein was derivatized with the OxyBlot Protein Oxidation Detection Kit (Sigma-Aldrich) as per manufacturer’s protocols, with exception of using a goat anti-rabbit secondary conjugated to Alexa Fluor 680 prior to imaging using the Odyssey XF Imaging System. Protein bands were quantified using ImageJ software (National Institutes of Health, USA) or Empiria Studio (LI-COR Biotechnology).

Histology, immunohistochemistry, and immunofluorescence
FFPE mouse and human pancreas tissue sections (5μm) were stained with hematoxylin and eosin (H&E) for blinded histological evaluation. Immunohistochemistry was performed with antibodies against ASC (AdipoGen, AG-25B-0006-C100, 1:200), cleaved (p10) Caspase-1 (Invitrogen, PA5-105049, 1:200), cleaved Caspase-3 (Cell Signaling Technologies, ASP175, 1:200), CD3 (Abcam, ab11089, 1:100), CD45 (Abcam, ab10558, 1:100), CD45R/B220 (BD Pharmingen, 550286, 1:100), F4/80 (Abcam, ab111101, 1:100), GSDMD (Abcam, ab219800, 1:1000), Ki67 (Abcam, AB16667, 1:200), NK1.1/CD161 (Cell Signaling Technologies, 24395, 1:200) and proliferating cell nuclear antigen (PCNA; Cell Signaling Technologies, 2586, 1:200) using the VECTASTAIN Elite ABC HRP Kit (Vector Laboratories) and the Liquid DAB+ Substrate Chromogen System (Agilent)23,61,63. Immunohistochemistry with an antibody against 4-Hydroxynonenal (4-HNE; Abcam, ab48506, 1:100) was performed using the Mouse on Mouse (M.O.M.) Detection Kit (Vector Laboratories). Slide scanning was performed using the NanoZoomer 2.0-HT (Hamamatsu Photonics), and positive-stained cells were counted61.

Multiplex immunofluorescence and multispectral image analysis
Human and mouse FFPE pancreas tissues were subjected to multi-label immunofluorescence using the Opal 7-Plex Manual Detection Kit and Anti-Rb HRP Kit (Akoya Biosciences). In mouse studies, the primary antibodies used, and the corresponding fluorescent detection signal for each antibody, were as follows: ASC (AdipoGen, AG-25B-0006-C100, 1:500) with Opal 690, cleaved (p10) Caspase-1 (Invitrogen, PA5-105049, 1:500) with Opal 780, CD45 (Cell Signaling Technology, D3F8Q, 1:500) or Cytokeratin-19 (CK19; Invitrogen, MA5-31977, 1:500) with Opal 520, NDUFB8 (Abcam, EPR15961, 1:500) with Opal 620, and UQCRC2 (Abcam, EPR13051, 1:200) with Opal 570. In some experiments, ASC and Caspase-1 (p10) were detected with Opal 570 and Opal 690, respectively. In human studies, the primary antibodies used, and the corresponding fluorescent detection signal for each antibody, were ASC (AdipoGen, AG-25B-0006-C100, 1:500) with Opal 620, cleaved (p10) Caspase-1 (Invitrogen, PA5-105049, 1:500) with Opal 690, and CD45 (Abcam, AB10558, 1:500) or CK19 (Invitrogen, SA30-06, 1:500) with Opal 520. Images were acquired on the Confocal Laser Scanning Microscope 980 instrument (Zeiss) at 20× and 63× magnification.

The BgNorm method was used to normalise image intensities. Raw TIFF images were first denoised by applying a 3×3 median filter to each channel, divided by a cofactor of 150 to deemphasize low intensities, and then log transformed64. For each marker channel, the distribution of log-transformed intensities across all pixels was fit to a three-component Gaussian mixture model (GMM; G=3) using the mclust package (v6.1) in R (v4.4). Components were interpreted as: background (U1), non-specific binding (U2), and true signal (U3). The intensities were adjusted using a deconvolution formula where the true signal component (U3), for each observed X3 is:
E(U3|X3=x3) = π3μ3 + σ32/σ32+σ22min(σ32,σ22) * (x3 - μ3)
where π3, μ3, σ32 are the weight, mean, and variance, respectively, of the third Gaussian, and μ2, σ2 are those of the second.

Cell segmentation was carried out with InstanSeg65 on the raw TIFF, and the resulting masks were overlaid on each adjusted intensity channel to extract cell-level summaries. Per-cell marker intensity was defined as the median of all adjusted pixel intensities within the mask. Finally, cells were classified as positive for a given marker if their median adjusted intensity was more than 0.65. CK19⁺ (epithelial) and CD45⁺ (immune) cells were first identified on their binary positivity (median adjusted intensity >0.65). Within each immune or epithelial cell, the presence or absence of inflammasome (ASC, cleaved (p10) Caspase-1) and mitochondrial (NDUFB8, UQCRC2) markers was determined by applying the same threshold. Each cell was then assigned to one of four colocalization categories: Inflammasome + Mitochondrial (all four markers positive), Mitochondrial (only both mitochondrial markers positive), Inflammasome (only both inflammasome markers positive), Negative (all four markers negative). The proportion of epithelial or immune cell populations falling into each colocalization category was computed, and plots were produced in R (v4.4) with the ggplot2 (v3.2) package.

RNA sequencing and analysis
Libraries were generated from 20ng of total RNA input using multiplex RNA-seq methodology, and sequencing was performed on a NextSeq 2000 System using a P2 100 Cycle Reagent Kit (Illumina)66. RNA-seq read alignment and UMI counting were performed in R (v4.1.2)67, and the scPipe package (v1.14.0) was employed to process data68. Multiplex RNA-seq FASTQ files had already been demultiplexed and contained the I1 index and UMI sequences in the read headers, but headers were reordered using awk for compatibility with scPipe. Read alignment was performed using the RSubread package (v2.6.4)69, and an index was built using the Ensembl Mus musculus GRCm38 primary assembly genome file. Aligned reads were mapped to exons using the sc_exon_mapping function with the Ensembl Mus musculus GRCm38 v100 GFF3 genome annotation file. BAM files were processed, and reads mapping to exons were associated with each individual sample using the sc_demultiplex function, taking the UMI into account, and an overall count for each gene per sample was generated using the sc_gene_counting function (with UMI_cor=1).

To identify differentially expressed genes, quantified gene counts were processed using the edgeR quasi-likelihood pipeline70, and genes with low counts per group were filtered out using the filterByexpr function in the edgeR R/Bioconductor package. Normalization factors for each sample were computed using the TMM method to account for library size biases71. A linear model was fit using edgeR, with trended dispersion estimated for each gene. Prior degrees of freedom for dispersion and quasi-dispersion were estimated using in-built robust estimators. Differentially expressed genes were called at the false discovery rate (FDR) of 0.05.

Pathway analysis was performed using the fry method implemented in the limma R/Bioconductor package72. Mouse-specific gene-sets were derived from the molecular signatures database (MSigDB v7.5) through the msigdbR/Bioconductor package73,74. The following collections and sub-collections were used for the analysis: Hallmark gene-sets (h), Gene Ontology molecular function (GO:MF), Gene Ontology biological processes (GO:BP), KEGG pathways (CP:KEGG), Reactome pathways (CP:REACTOME), Biocarta pathways (CP:BIOCARTA), and Wikipathways (CP:WIKIPATHWAYS). Gene-sets with fewer than 5 genes overlapping with the data were triaged.

Pathways with an FDR < 0.05 were aggregated into higher-order biological themes using the vissE tool74. The network of significant gene-sets was identified by applying an ARI threshold of 0.35. Clusters with > 15 gene-sets were identified for further study using the walktrap community detection algorithm, and clusters associated with metabolic/mitochondrial respiration and immune responses, as assessed through the word clouds, were further aggregated to form a single cluster representing the respective process.

Protein-protein interaction (PPI) networks were accessed through the vissE package which provided pre-processed networks from the International Molecular Exchange (IMEx) database (version 2022-02-03)75. APPI was induced for genes belonging to each cluster of processes identified using vissE (or aggregated manually as described above). Gene importance was determined by measuring the degree of centrality of each gene.

Mitochondrial DNA copy number
Genomic (g)DNA was isolated from either snap-frozen pancreas tissues or human cell lines using the PureLink Genomic DNA Mini Kit (Invitrogen). qPCR was performed on the QuantStudio 7 Pro system in technical quadruplicates using Power SYBR Green PCR Master Mix (Applied Biosystems), 50nM of each primer, and 10ng gDNA template. Primer pairs used were for mouse mtDNA (F, 5-CTCCGTGCTACC TAAACACCT-3; R, 5-GATGGCAAGGGTGATAGGATT-3) and B2m (F, 5- CTCCGTGCTACCTAAACACCT-3; R, 5-GATGGCAAGGGTGATAGGATT-3), and human mtDNA (F, 5-CACCCAAGAACAGGGTTTGT-3; R, 5- TGGCCATGGGTATGTTGTTA-3) and B2M (F, 5- TGCTGTCTCCA TGTTTGATGTATCT-3; R, 5-TCTCTGCTCCCCACCTCTAAGT-3). The relative quantification (RQ) of mitochondrial DNA (mtDNA) copy number was defined as the ratio of mtDNA gene copy number to the amount of nuclear DNA (B2m reference gene) internal control and is proportional to mtDNA copy number per sample.

Lactate assay
Lactate was assessed in protein lysates from mouse pancreas tissues and human cell line supernatants (1:5 dilution) using the L-lactate Colorimetric Assay kit (Abcam, ab65331), according to manufacturer’s instructions. Tissue samples were lysed in 6× volume (6µL buffer/1mg tissue) using a Dounce homogenizer and deproteinized using the Deproteinizing Sample Preparation Kit – TCA (Abcam, ab204708) prior to the assay. Readings were normalized to cell protein concentrations measured by Pierce BCA Protein Assay (Thermo Fisher Scientific).

Mitochondrial superoxide assay
The Mitochondrial Superoxide Assay Kit (Fluorometric) (Abcam) was used as per the manufacturer’s protocols on siRNA-transfected cells that were seeded at 48 hours post transfection in 96-well plates (2×104 cells/well) and assayed at 72 hours post transfection. Assay media contained FluoroBrite DMEM (Thermo Fisher Scientific) and NucBlue Live ReadyProbes Reagent (Hoechst 33342) (Invitrogen). Measurements were performed on a CLARIOstar Plus microplate reader using the 15 × 15 well scan mode at 3mm diameter, with multichromatic detection set to Ex540-15/Di auto 563.8/Em590-20 for mitochondrial superoxide detection and Ex355-20/Di auto 402.5/Em455-30 for Hoechst 33342. Fluorescence intensity for mitochondrial superoxide was normalized to Hoechst intensity.

Bioenergetic assay
Mitochondrial respiration parameters in human cell lines were assessed with the Seahorse XF Mito Stress Test Kit on a Seahorse XFe96 Analyser (Agilent) according to the manufacturer’s protocol. Briefly, siRNA-transfected cells were seeded in XFe96 cell culture microplates at 48 hours post transfection (2 × 104 cells/well) and assayed at 72 hours post transfection. Drugs injected during the assay were oligomycin (1.5μM), FCCP (1μM), rotenone (1μM), and antimycin A (0.5μM). Normalization of Seahorse metabolic assay data to account for differences in cell number was performed as above using Hoechst staining of nuclei and well-scan mode on a CLARIOstar Plus microplate reader.

Statistics & reproducibility
Statistical analyses were performed using GraphPad Prism V10.1.0 software. Data normality was assessed using the D’Agostino-Pearson omnibus K2 normality test. Statistical significance (P<0.05) between the means of two groups was determined using Student’s t-tests or Mann-Whitney U-tests (two-tailed), and significance between the means of multiple groups was determined using ordinary one-way analysis of variance (ANOVA) or Kruskal-Wallis tests, as appropriate. The log-rank (Mantel-Cox) test was used to calculate the statistical significance of the difference in survival between two groups. The tests used to identify significance between the means of two or multiple groups are presented in the relevant figure legends. The statistical tool G*Power was used to predetermine minimal sample sizes. No data were excluded from analyses. Data are expressed as the mean ± standard error of the mean (SEM) from at least three biological replicates. P values <0.0001 are presented as “P<0.0001”.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Chey et al is an original, elegant and complete study reporting a role for ASC in promoting PDAC initiation and progression. The findings are shown to be relevant in human cancer and have therapeutic implications since blockade of extracelllar ASC partially recapitulates the phenotypes observed in total and conditional KO animals. The study also brings mechanistic data, showing that ASC impacts on metabolic programming of pancreatic malignant cells. This said, the study would benefit from establishing causal links between and parameters such as proliferation and/or control of apoptosis within malignant cells.\nMajor comments.\n1) Is metabolic reprogramming needed to support ASC-dependent malignant cell proliferation?\n2) Are oxygen consumption and extracellular acidification rates modulated by ASC in malignant cells?\n3) Does extracellular ASC control OXPHOS and glycolysis?\nMinor issues.\n1) Assessing mitochoindrial superoxide anion production is needed to link mitochondrial dysfunction to ROS production.\n2) The qulality of Figure 1E-F should be improved. The fluorescence is saturated and it is difficult to assess colocalization in those conditions.\n3) The discussion is quite long. It should be shortened.", "reviewer_2": "In this study by Chey et al., entitled “Cancer cell-intrinsic ASC links innate immunity with mitochondrial metabolism in driving inflammasome-associated pancreatic tumorigenesis”, the authors convincingly demonstrate that ASC (apoptosis-associated speck-like protein containing a CARD) plays a critical role in pancreatic ductal adenocarcinoma (PDAC) by modulating the tumorigenic process through three independent approaches: global genetic ablation, epithelial-specific conditional knockout, and nanobody-mediated targeting of extracellular ASC in vivo. This multifaceted strategy unambiguously establishes the relevance of ASC in PDAC development. However, despite this strength, several important aspects of the analyses and their interpretations are either insufficiently developed, potentially misleading, or require further clarification or experimental validation to reinforce the conclusions drawn.\nMajor comments.\n1. The authors analyze PYCARD/ACS expression in human datasets. Is ACS expression associated more to a classical/ductal tumor subtype or also to a basal subtype? The authors should probe other datasets (i.e., Bailey et al.), classifying patients into subtypes to determine if ACS expression is more enriched in the classical versus the basal subtype, for example.\n2. Fig 2. The authors state that “that elevated activation of ASC-containing inflammasomes is also a feature of the KPC PDAC model.” However, ASC staining in KPC mouse at early, intermediate and late stages is needed to really understand the dynamics of ACS expression and its kinetics across KPC tumor evolution.\n3. Do the KPC ACKO and ACSfl/fl mouse models present any adverse effects? Modulation of the inflammasome pathway would likely make these mice more susceptible to bacterial infections. Information alone these lines should be mentioned in the M&Ms or cited if this information exists.\n4. Figs. 3D & 5H: In both panels, a similar analysis to compare both models should be performed. Based on the data presented, it appears as though the KPC and KPC APCKO models used in Fig 3D are less aggressive than those shown in Fig 5H, yet PanINs were not assessed in Fig. 3D.\n5. Why is the tumor evolution very different between the ACS total ablation (KO) KPC model and the ACS conditional (fl/fl) KPC model. Particularly for the wt KPC mice, where in the ACS total ablation KPC model shown in Fig 2-3, the mice were sacrificed at 20 weeks, but in the ACS conditional KPC model, the mice were sacrificed at 12 weeks. In Figure 2-3, the KPC wt mice used and scarified at 20weeks have a mean tumor weight of 0.45g and 60% area of lesions, but the KPC wt mice used in the comparison with the KPC APC fl/fl mice in Figure 5 have a mean tumor weight of 1g and 70% area of lesions at 12 weeks. That is a big discrepancy between the control KPC wt mice for both comparisons. All models (KPC wt, KPC APCKO KPC APCfl/fl) need to be analyzed at the same time point- either 12 or 20 weeks. Are the difference due to mixed 129Sv × C57BL/6 background?\n6. Page 13: The statement “These data therefore implicate the pancreatic ductal epithelium as the predominant cellular source of tumor-promoting ASC inflammasome activation in PDAC.” Is overstated. It appears as through the cellular source of tumor-promoting ASC inflammasome activation is also TME-based.\n7. The image shown in Fig 6C is misleading and not representative of the tumor burden calculated in Fig 6D, where in the pancreata analyzed more than 50% was moderate PDAC.\n8. The authors performed RNA-seq comparing KPC and KPC:ASCKO mice, but the complete ablation of APC in the KPC:ASCKO mice can confound the analysis. Specifically, in KPC:ASCKO mice, as shown in Figure 3, the pancreas is practically normal, with very little tumor burden. Thus, it is almost like comparing KPC to a normal pancreas. Likewise, the total ablation of APC in the KPC:ASCKO mice could affect the results. The authors should confirm that the effects observed on mt- and nuclear-encoded genes are also observed in the APCfl/fl mice at 20 weeks (not 12 weeks), where one would assume more tumor burden in the KPC APCfl/fl mice. RTqPCR for the target genes can be performed in lieu of another RNAseq. Likewise, the authors need to sort tumor cells from both KPC and KPC:ASCfl/fl mice to even more specifically show that the effects are tumor cell specific and not influenced by the TME.\n9. Fig 8. In line with the above comments, the data shown in Fig 8 should also be performed with 20 week old KPC wt and KPC fl/fl mice or KPC tumor cells isolated from the pancreas of these two models.\n10. For many of the statistical comparisons, the KPC APCKO (and future KPC APCfl/fl) groups should be compared to the wt group. The fact that for many panels, there is likely no significant difference between KPC APCKO and wt suggests even more that the lack of tumor development in the KPC APCKO mice results in a pancreas that is almost normal, which undermines the analyses to determine how ASC promotes PDAC via molecular pathways.\n11. Fig 8: The authors performed multiplexed immunofluorescence on KPC versus WT pancreas sections to determine the spatial cellular context of the changes in mitochondrial biogenesis (and protein expression of UQCRC2, NDUFB8, etc) within the pancreatic TME and their localization with activated ASC-containing inflammasomes. It is necessary to perform these analyses as well in the areas of poor, moderate and/or well PDAC in the KPC APC fl/fl pancreas and in the pancreas from the VHHACS-treated mice to show that the proteins and parameters are not observed in tumor cells in the absence of APV. The analyses in the pancreas of VHHACS-treated mice are particularly relevant to show that the MOA of the treatment is similar. The authors do not take full advantage of the pancreas samples from the VHHACS-treated mice to support their claims and mechanistic analyses.\n12. Discussion: The authors state in the Discussion “novel cancer (epithelial) cell-intrinsic role for ASC-containing inflammasomes in promoting the initiation and progression of PDAC”; however, the data, as is, does not really support this claim. The completion of the above experiments in the KPC APC fl/fl mice and in the pancreas from the VHHACS-treated mice will better support this claim.\n13. Discussion: Limitations of the study need to be discussed.\nMinor points\n1. The images in Fig 1 E-F and accompanying supplementary images are over exposed. Lower exposure images should be shown.\n2. Panel 5H and 6D: “PanIN” instead of “PanIn” in the legend.\n3. Mouse Background Clarification: The use of a mixed 129Sv × C57BL/6 background is briefly mentioned. Given that this can influence tumor progression and immune responses, the authors should clarify whether littermate controls were used throughout and whether background effects may explain phenotype differences.\n4. Nanobody Specificity: More data or citation supporting the specificity of VHHASC for extracellular ASC specks in vivo would strengthen the therapeutic relevance and rule out off-target effects.\n5. PRR Partner for ASC: Although the Discussion mentions the uncertainty regarding the PRR responsible for ASC inflammasome activation in PDAC, no experiments or strong hypotheses are proposed. Even correlative analysis of PRR expression in epithelial vs. immune compartments would be informative."}, "author_response": "We thank the Reviewer for their positive comments on our study and manuscript, and trust that our new data sufficiently reinforces our mechanistic findings and conclusions regarding the role of ASC in contributing to PDAC.\nMajor comments\n1) Is metabolic reprogramming needed to support ASC-dependent malignant cell proliferation?\nAuthor response:\nWe appreciate the Reviewer’s insightful comment which is based on our in vivo data (in the KPC PDAC mouse model) demonstrating that ASC-mediated pancreatic tumourigenesis (incorporating augmented cellular proliferation in the pancreatic epithelium) is associated with metabolic reprogramming. Given the brevity of this comment, we have assumed that the Reviewer is now requesting in vitro data (consistent with their other comments below). Accordingly, we have performed experimentation with two widely-used transformed human PDAC cell lines, PANC-1 and MIA PaCa-2, whereby we have knocked-down ASC (encoded by PYCARD) with siRNA (ASCKD). As shown in the new Supplementary Figures S14a-h, successful siRNA-mediated knockdown of ASC expression (along with the expected reduced cleaved Caspase-1 levels) significantly reduced cell proliferation, colony formation and viability coincident with reduced lactate levels and increased mitochondrial DNA copy number and gene expression in human PDAC cells. This is consistent with our in vivo data (e.g. Figures 4a, 5i, 7, 8c-g) that genetic ablation of ASC in KPC mice reduced cellular proliferation within the pancreatic epithelium which associated with elevated mitochondrial gene networks and DNA copy number, and a reversion of energy metabolism from aerobic glycolysis (as seen in cancer cells; Warburg effect) towards mitochondrial respiration (as seen in non-cancerous (wild-type) mice with normal, lowly-proliferating cells).\nTo further assess whether metabolic reprogramming in PDAC cells aligns with ASC-mediated cell proliferation and/or viability, we next treated ASCKD and control (scramble siRNA) transfected PANC-1 and MIA PaCa-2 cells with two glycolysis inhibitors, STF-31 and 2-deoxyglucose (2-DG), noting that cancer cells heavily rely on aerobic glycolysis for proliferation and viability (survival). In contrast to negative control (scramble siRNA) transfected PDAC cells, ASCKD PDAC cells were markedly less sensitive to impaired viability (proliferation) induced by glycolysis inhibitors (Supplementary Figure S14j), which supports the notion that ASC deficiency switches the cellular energy needs for viability and proliferation towards OXPHOS (i.e. mitochondrial respiration).\nWhile being mindful of the Reviewer’s comment below to shorten the Discussion, we have briefly referred to these new human cell line data in the Discussion on pages 19 (lines 443-447) and 21 of (lines 483-485), as well as in the Results (pages 17-18, lines 393-414, 419-422) and Methods (pages 26-27, lines 602-631, 788, 794-796, 802-803) sections.\n2) Are oxygen consumption and extracellular acidification rates modulated by ASC in malignant cells?\nAuthor response:\nA key finding of our study was that ASC-containing inflammasomes promoted mitochondrial dysfunction resulting in a bias towards aerobic glycolysis (Warburg effect) in PDAC, as evidenced by the blockade of ASC (in the KPC PDAC model) which switched energy metabolism back towards mitochondrial-driven OXPHOS (i.e. cellular respiration). To address this comment by the Reviewer, we have therefore performed the Seahorse XF Mito Stress Test to measure key parameters of mitochondrial respiration (oxygen consumption rate; OCR) in the above human PDAC cells subjected to siRNA-mediated knockdown of ASC (ASCKD).\nAs shown in the new Supplementary Figures 14k and l, both basal OCR and maximal OCR capacity (induced by the proton ionophore FCCP, a potent uncoupler of mitochondrial OXPHOS) were increased in ASCKD versus control (scramble siRNA) transfected human PDAC cells, which further supports our notion that ASC deficiency during PDAC (e.g. in KPC:ASCKO mice) rewires energy metabolism back towards mitochondrial respiration. Furthermore, the spare respiratory capacity (SRC) of ASCKD PDAC cells was also elevated versus control PDAC transfected cells (Supplementary Figures 14k and l), indicating that ASC deficiency enhanced mitochondrial fitness in response to energy demands. We also refer to our new above-mentioned data (in response to comment #1) showing that extracellular lactate production – which is a primary source of extracellular acidification during glycolysis – was significantly reduced in PDAC cells upon siRNA-mediated knockdown of ASC (Supplementary Figure 14h). Collectively, these data indicate that oxygen consumption and extracellular acidification rates are indeed modulated by ASC in transformed (pancreatic) cancer cells.\nThese new data are now briefly referred to in the Discussion (lines 483-485) on page 21, as well as in the Results (page 18, lines 414-422) and Methods (page 35, lines 821-829) sections.\n3) Does extracellular ASC control OXPHOS and glycolysis?\nAuthor response:\nIn line with the above comments by the Reviewer referring to “malignant cells”, we assume here the Reviewer is also referring to the context of human PDAC cells. Therefore, to address this comment, we have treated human PDAC cells with an ASC-specific nanobody (VHH ) and performed assays for lactate production which (as mentioned above) is an indicator of glycolysis (versus OXPHOS). We note that these experiments were performed with a limiting amount of available human-specific ASC nanobody (VHH ) which is different to the murine ASC (VHH ) used in our study on KPC mice because i) VHH is specific for mouse only ASC, and ii) VHH is specific for human only ASC (also see new text in the Results on page 13, lines 298-301 referring to this - in response to Minor comment #4 by Reviewer #2).\nAs shown in the figure below for the Reviewer, these experiments (n = 3; each experiment comprising technical triplicates is coded with a specific colour) revealed that VHH reduced lactate production (albeit not significantly; P = 0.0665, paired t-test) which is consistent with our new in vitro data (Supplementary Figure 14) showing that ASC modulates mitochondrial OXPHOS and glycolysis. The observation that the reduction in lactate production in human PDAC cells upon VHH treatment was not as pronounced as that seen in siRNA-mediated knockdown of ASC is analogous to the in vivo situation where the suppressed PDAC phenotype in VHH -treated KPC mice was not as pronounced as that observed upon whole-body ASC deletion in KPC:ASCKO mice (e.g. compare Figure 6d and Supplementary Figure 5e). This could be explained, at least in part, by the global blockade of intracellular and extracellular ASC activity (KPC:ASCKO mice, ASCKD PDAC cells) versus the nanobody-mediated blockade of (predominantly) extracellular ASC activity: note, we have now included new text in the Discussion on page 24, lines 561-565 referring to this). One must also consider that differences in the bioactivity of VHH versus VHH may account for any differential effects observed on mouse versus human cells with these nanobodies. Since these data are preliminary and subject to our ongoing detailed characterization of the mechanisms of action of ASC-targeted nanobodies in cancer, we believe it best to include these data for the Reviewers only.\nMinor issues.\n1) Assessing mitochoindrial superoxide anion production is needed to link mitochondrial dysfunction to ROS production.\nAuthor response:\nTo address this comment from the Reviewer, we have assessed mitochondrial superoxide production in the above-mentioned PANC-1/ASCKD and MIA PaCa-2/ASCKD human PDAC cell lines. As shown in the new Supplementary Figure 14i, siRNA-mediated knockdown of ASC expression significantly reduced mitochondrial superoxide levels compared to elevated levels observed in negative control transfected cells. Importantly, these data correlate with the increased mitochondrial DNA (mtDNA) copy number and reduced lactate production that was also observed in ASDKD cells compared to control cells. Moreover, our new in vitro data further validate our in vivo findings in KPC mice that ASC deficiency in the pancreas i) suppresses oxidative stress, ii) normalizes mitochondrial biogenesis/function by increasing mtDNA copy number and gene expression, and iii) reduces lactate production as an indicator of a switch from aerobic glycolysis back towards OXPHOS (see Figure 8). We have included new text in the Results (page 17, lines 399-402) and Methods (pages 34-35, lines 810-819) sections referring to these new analyses.\n2) The qulality of Figure 1E-F should be improved. The fluorescence is saturated and it is difficult to assess colocalization in those conditions.\nAuthor response:\nAs requested by the Reviewer, we have replaced the images in Figure 1e and 1f with new images obtained via Opal multilabel immunofluorescence for ASC, cleaved Caspase-1, cytokeratin-19 (epithelial cells) and CD45 (pan-immune cells), along with DAPI (nuclear staining). The figure legend for these new images, along with the Methods section (under subsection “Multiplex immunofluorescence and multispectral image analysis”; page 31, lines 715-719), has also been updated accordingly.\n3) The discussion is quite long. It should be shortened.\nAuthor response:\nAs requested by the Reviewer, we have omitted text and condensed sentences and paragraphs throughout the Discussion section, where possible, without comprising the integrity and readability of the Discussion. We also note the request by Reviewer #2 for additional text be added to the Discussion (such as “Limitations of the study”, see Major point #13 by Reviewer #2) where we have been mindful to keep such additional text as succinct as possible.\nReviewer #2 (Remarks to the Author):\nIn this study by Chey et al., entitled “Cancer cell-intrinsic ASC links innate immunity with mitochondrial metabolism in driving inflammasome-associated pancreatic tumorigenesis”, the authors convincingly demonstrate that ASC (apoptosis-associated speck-like protein containing a CARD) plays a critical role in pancreatic ductal adenocarcinoma (PDAC) by modulating the tumorigenic process through three independent approaches: global genetic ablation, epithelial-specific conditional knockout, and nanobody-mediated targeting of extracellular ASC in vivo. This multifaceted strategy unambiguously establishes the relevance of ASC in PDAC development.\nAuthor response:\nWe thank the Reviewer for acknowledging the strength of our study in uncovering the important role of ASC (via inflammasomes) in promoting PDAC, and trust that our new data sufficiently reinforces our mechanistic findings and conclusions.\nMajor comments\n1. The authors analyze PYCARD/ACS expression in human datasets. Is ACS expression associated more to a classical/ductal tumor subtype or also to a basal subtype? The authors should probe other datasets (i.e., Bailey et al.), classifying patients into subtypes to determine if ACS expression is more enriched in the classical versus the basal subtype, for example.\nAuthor response:\nAs suggested by the Reviewer, we have now interrogated the gene expression profile of PYCARD (encoding ASC) against distinct molecular subtypes – based on transcriptome classification – previously assigned to PDAC in the study by Bailey et al (now included as new reference 28). We note that the four molecular subtypes of PDAC are classified as ‘squamous’, ‘pancreatic progenitor’ (previously termed ‘classical’), ‘ADEX’ (abnormally differentiated endocrine exocrine) and ‘immunogenic’. As shown in the new Supplementary Figures 1c and 1d, PYCARD gene expression was comparable among all molecular subtypes. We have included new text in the Results (page 7, lines 135-137) and Methods (pages 28-29, lines 658-665) sections referring to these new analyses.\n2. Fig 2. The authors state that “that elevated activation of ASC-containing inflammasomes is also a feature of the KPC PDAC model.” However, ASC staining in KPC mouse at early, intermediate and late stages is needed to really understand the dynamics of ACS expression and its kinetics across KPC tumor evolution.\nAuthor response:\nIn the original manuscript we included immunofluorescence staining showing elevated expression of ASC in the pancreas of KPC mice aged 20 weeks (late stage) compared to age-matched wild-type (WT) mice (Figure 2e). This supported our qPCR and immunoblotting data showing elevated ASC expression at the mRNA and protein levels in KPC mice at 10 weeks (intermediate stage) and 20 weeks compared to age-matched WT mice (Figures 2a and 2c, Supplementary Figures 4a and 4c). These qPCR and immunoblotting data indicated that the increased levels of ASC expression in KPC mice were comparable at 10 and 20 weeks of age (e.g. compare immunoblots of 10-week-old (wo) (Supplementary Figure 4c) and 20wo (Figure 2c) KPC mice.\nAs requested by the Reviewer, we have now included new data in Supplementary Figure 4e showing representative Opal immunofluorescence staining of ASC and cleaved Caspase-1 in the pancreas of KPC mice aged 6 weeks (early stage), 10 weeks (intermediate stage) and 20 weeks (late stage). These new data demonstrate elevated ASC expression across all age groups (i.e. during tumour evolution) and notably confirm the predominant nuclear and cytoplasmic colocalization of ASC and cleaved Caspase-1 in ductal epithelial cells, which became more apparent as pancreatic lesions progress from early to intermediate to late stages in KPC mice. These new data are now referred to in the text in the Results section on page 9 (lines 194-197) of the revised manuscript.\n3. Do the KPC ACKO and ACSfl/fl mouse models present any adverse effects? Modulation of the inflammasome pathway would likely make these mice more susceptible to bacterial infections. Information alone these lines should be mentioned in the M&Ms or cited if this information exists.\nAuthor response:\nThe ASCKO mice used in our current study (mixed 129Sv × C57BL/6 background) harbouring global ablation of ASC were generated nearly 20 years ago (see reference 59), and as stated in this referred paper by Ö zö ren et al “ASC-KO mice were born at the expected Mendelian ratio, were fertile, showed no gross abnormalities, and appeared healthy when housed in a standard specific pathogen-free environment.”. Our laboratory has housed ASCKO mice under specific pathogen-free (SPF) conditions for nearly 10 years, and we have not encountered any adverse effects (such as an increase in bacterial infections), which is consistent with many other laboratories worldwide who house these mice in SPF facilities. Similarly, we have not encountered any unexpected susceptibility to infections in KPC:ASCKO mice, or for that matter KPC:ASCfl/fl mice, housed in our SPF facility for 4 years and 2 years, respectively (we note that the original ASCfl/fl mice were generated over 10 years ago – see reference 24 – and again have been used worldwide without any health issues). It is widely known and acknowledged that modulation of inflammasome activity does not impact on susceptibility to bacterial infection because of the functional redundancy that exists among the many PRRs (including those associated with inflammasomes) that constitute the innate immune system’s role in host defence against infection. For instance, in ASCKO mice engineered for the functional absence of inflammasome complexes comprising a PRR that recognizes bacterial components, other PRRs (such as those belonging to the Toll-like receptor family) can still recognize and respond to bacteria, thus minimising risk of infection. As requested by the Reviewer, in the Methods section under the subsection “Mice”, we have now added a brief sentence (page 25, lines 589-591 referring to the absence of microbial-driven adverse events (increased susceptibility to bacterial infections) in mice lacking ASC.\n4. Figs. 3D & 5H: In both panels, a similar analysis to compare both models should be performed. Based on the data presented, it appears as though the KPC and KPC APCKO models used in Fig 3D are less aggressive than those shown in Fig 5H, yet PanINs were not assessed in Fig. 3D.\nAuthor response:\nIt appears that the Reviewer has both misinterpreted the data shown in Figures 3d and 5h, and not seen the Supplementary Figures. Firstly, pancreas lesions are less aggressive in 10-week-old (wo) KPC mice (Figure 5h and Supplementary Figure 5e) compared to 20wo KPC mice (Figure 3d). For example, early PanIN lesions are only observed in 10wo KPC mice (Figure 5h and Supplementary Figure 5e), and the more severe poor differentiated ductal carcinomas are observed in only 20-30% of 10wo KPC mice (Figure 5h and Supplementary Figure 5e) compared to 50% of 20wo KPC mice (Figure 3d).\nSecondly, data shown in Figure 3d is for the KPC:ASCKO mouse model (global knockout of ASC), whereas data shown in Figure 5h is for the KPC:ASCfl/fl mouse model (conditional epithelial-specific knockout of ASC), so the Reviewer is referring to and comparing different mouse strains (models) in which ASC has been genetically targeted.\nThirdly, we have already performed these comparable analyses between 10wo and 20wo KPC and KPC:ASCKO mice: we refer the Reviewer to Supplementary Figure 5e (10wo data) and Figure 3d (20wo data), as well as existing text (page 10, lines 217-225) referring to these analyses in the Results section.\nLastly, PanINs were not reported in 20wo KPC mice in Figure 3d because at this late stage KPC mice predominantly display PDAC tumours (i.e. little to no PanIN) which is consistent with other literature on this model (e.g. see reference 29, as well as Lee JW et al, Curr Protoc Pharmacol. 2016;73:14.39.1-14.39.20. doi: 10.1002/cpph.2. PMID 27248578).\n5. Why is the tumor evolution very different between the ACS total ablation (KO) KPC model and the ACS conditional (fl/fl) KPC model. Particularly for the wt KPC mice, where in the ACS total ablation KPC model shown in Fig 2-3, the mice were sacrificed at 20 weeks, but in the ACS conditional KPC model, the mice were sacrificed at 12 weeks. In Figure 2-3, the KPC wt mice used and scarified at 20weeks have a mean tumor weight of 0.45g and 60% area of lesions, but the KPC wt mice used in the comparison with the KPC APC fl/fl mice in Figure 5 have a mean tumor weight of 1g and 70% area of lesions at 12 weeks. That is a big discrepancy between the control KPC wt mice for both comparisons. All models (KPC wt, KPC APCKO KPC APCfl/fl) need to be analyzed at the same time point- either 12 or 20 weeks. Are the difference due to mixed 129Sv × C57BL/6 background?\nAuthor response:\nFirstly, we refer the Reviewer to the age-matched pancreatic mass (weight) and lesion data that is already present in Supplementary Figures 5c-e (now updated) for 10-week-old (wo) WT, KPC and KPC:ASCKO (i.e. harbouring global ablation of ASC) mice, and in Figures 5e, g and h (now updated) for 10wo WT, KPC and KPC:ASCfl/fl mice (i.e. harbouring conditional ablation of ASC). Therefore, all these models have already been analysed at the same 10wo time point.\nSecondly, it is well established that the pancreas tumour phenotype of the KPC mouse strain on a mixed 129Sv x C57BL/6 background displays a high degree of heterogeneity (variability) which mirrors that of human PDAC (reference 29), and we now refer to this in new text on page 9 (line 182) of the revised manuscript.\nThirdly, the 10wo and 20wo WT, KPC and KPC:ASCKO pancreas weight data points presented in the original Supplementary Figure 5c and Figure 3b (n = 12/genotype), respectively, were representative of a randomised subset of overall mouse data in order to be consistent with the smaller sample sizes (i.e. data points) of other genotypes (e.g. KPC:ASCfl/fl; n = 8) and treatment groups (e.g. VHH ; n = 7) used in our study. However, in acknowledging the tumour heterogeneity of PDAC, and to address the Reviewers’ comment on “tumor evolution”, we have now included additional pancreas weight data points in graphs to increase the sample sizes to n = 20/genotype for WT, KPC and KPC:ASCKO mice aged 10 weeks (revised Supplementary Figure 5c) and 20 weeks (revised Figure 3b) – this accounts for virtually all of the weight data collected for the duration of the study. We have also increased the number of 10wo WT, KPC and KPC:ASCfl/fl pancreas weight data points to n = 10/genotype in revised Figure 5e. Importantly, these datasets reflect the expected high degree of heterogeneity in the KPC pancreas tumour phenotype. KPC pancreas weights are as follows: 0.886g and 0.525g for 10wo comparative KPC:ASCfl/fl (n = 10) and KPC:ASCKO (n = 20) cohorts, respectively; 0.809g for 20wo comparative KPC:ASCKO (n = 20) cohort.\nFourthly, with respect to lesion percentages of “control” KPC mice mentioned by the Reviewer, we emphasise that not all lesions are the same grade within a genotype and age group (due to tumour heterogeneity), as well as between age groups for a given genotype. Therefore, one must compare lesion percentages while also considering the type of lesions present. In this regard, the severity of lesions (65% of pancreas area) in 20wo KPC mice (revised Figure 3c and d) is substantially greater than lesions in 10wo KPC control mice (63% and 49% of pancreas area) from the comparative KPC:ASCfl/fl (revised Figures 5g and h) and KPC:ASCKO (revised Supplementary Figures 5d and e) cohorts. For example, 50% of 20wo KPC mice (revised Figure 3d) versus only 20-30% of 10wo KPC mice (revised Figure 5h and Supplementary Figure 5e) display the more severe poor differentiated PDAC phenotype, with only 10wo KPC mice displaying PanIN lesions.\nLastly, despite the heterogeneity of the PDAC phenotype in KPC mice, our revised data (with increased sample sizes) further confirms that the genetic ablation of ASC (either total or conditional knockout) significantly supresses pancreatic tumourigenesis, thus supporting the critical role of ASC in promoting the development of PDAC.\n6. Page 13: The statement “These data therefore implicate the pancreatic ductal epithelium as the predominant cellular source of tumor-promoting ASC inflammasome activation in PDAC.” Is overstated. It appears as through the cellular source of tumor-promoting ASC inflammasome activation is also TME-based.\nAuthor response:\nAs requested by the Reviewer, we have now amended the sentence in the revised manuscript (page 13, lines 287-291) to read as follows: “These data therefore implicate the pancreatic ductal epithelium as a major cellular source of tumor-promoting ASC-containing inflammasomes in PDAC, while suggesting that activation of ASC (and inflammasomes) in the non-epithelial compartment of the TME (e.g. immune cells) is also likely to contribute to tumorigenesis.”. We also note that, despite the need to reduce the Discussion section (Reviewer #1, Minor point #3), we have included new text in the Discussion referring to the notion that additional cellular sources (e.g. immune cells, fibroblasts) of tumor-promoting ASC (inflammasome) activation likely exist in the TME (pages 19-20, lines 447-452 of revised manuscript).\n7. The image shown in Fig 6C is misleading and not representative of the tumor burden calculated in Fig 6D, where in the pancreata analyzed more than 50% was moderate PDAC.\nAuthor response:\nAs requested by the Reviewer, in the revised Figure 6c we have now replaced the VHH -treated KPC mouse pancreas image with a new image representative of the pancreas comprising moderate differentiated lesions (along with a greater composition of acinar cells consistent with the lower tumour burden in the VHH -treated KPC group). We have also amended the legend for Figure 6c to reflect the new image.\n8. The authors performed RNA-seq comparing KPC and KPC:ASCKO mice, but the complete ablation of APC in the KPC:ASCKO mice can confound the analysis. Specifically, in KPC:ASCKO mice, as shown in Figure 3, the pancreas is practically normal, with very little tumor burden. Thus, it is almost like comparing KPC to a normal pancreas. Likewise, the total ablation of APC in the KPC:ASCKO mice could affect the results. The authors should confirm that the effects observed on mt- and nuclear-encoded genes are also observed in the APCfl/fl mice at 20 weeks (not 12 weeks), where one would assume more tumor burden in the KPC APCfl/fl mice. RTqPCR for the target genes can be performed in lieu of another RNAseq. Likewise, the authors need to sort tumor cells from both KPC and KPC:ASCfl/fl mice to even more specifically show that the effects are tumor cell specific and not influenced by the TME.\nAuthor response:\nImportantly, we point out our new in vitro data generated from human PDAC (i.e. tumour epithelial) cells in response to comments by Reviewer #1. Here, the siRNA-driven targeting of ASC (PYCARD) in these cancer cells directly modulated numerous intrinsic cellular processes comparable to our observations in KPC mice upon genetic ablation of ASC, including: i) reduced cell proliferation and viability, ii) reduced lactate production and increased mitochondrial respiration/oxygen consumption rate (indicative of a switch from glycolysis towards OXPHOS for energy metabolism), iii) increased mtDNA copy number and iv) reduced oxidate stress (i.e. mitochondrial superoxide production) (new Supplementary Figure S14). Moreover, in relation to the Reviewer’s comment, we show in the new Supplementary Figure S14g that siRNA-mediated knockdown of ASC in human PDAC cells directly increased mRNA expression levels of mitochondrial- and nuclear-encoded genes. Taken together with our existing Opal multiplex immunofluorescence analyses (Figures 8h,i, Supplementary Figures S11-S13) which provides spatial context for the predominant localization of ASC-containing inflammasomes with mitochondrial markers in the pancreatic ductal epithelium, we believe these data provide compelling evidence for ASC-mediated tumour (cancer) cell specific effects during PDAC.\nIn further response to this Reviewer’s comment, firstly we note that a health issue arising with aged (~20-week-old (wo)) KPC mice is the appearance of anogenital and facial warts because of the “leakiness” of the Pdx1-Cre allele which can lead to activation of mutant-Kras in other tissues. As a consequence of this, over the last 6 months we have been directed by our University Veterinarian and Animal Ethics Committee to perform studies on KPC mouse strains only aged up to 12 weeks, so we are currently unable to generate 20wo mice. Secondly, cell sorting for tumour cells is suboptimal because i) spatial context within the TME (of the pancreas) is lost, and ii) in the absence of a highly specific and robust tumour/ductal epithelial cell marker (i.e. antibody) it cannot be assumed that a generic epithelial-specific antibody alone for sorting will capture only tumour cells. For these reasons, we are currently undertaking a combinatorial approach of spatial transcriptomics (archived 12wo and 20wo mouse FFPE pancreas samples) coupled with single-cell RNASeq (fresh 12wo) on WT, KPC, KPC:ASCKO and KPC:ASCfl/fl mice to align the expression of mitochondrial- and nuclear-encoded ASC-dependent genes and molecular pathways to distinct cellular compartments (i.e. tumour/ductal epithelial cells, acinar cells, immune cell subsets, fibroblasts etc) within pancreatic lesions. As the Reviewer can appreciate, this is quite an undertaking and is well beyond the scope of the current study: we envisage our ongoing combinatorial transcriptomics studies will form the basis of a future independent manuscript.\n9. Fig 8. In line with the above comments, the data shown in Fig 8 should also be performed with 20 week old KPC wt and KPC fl/fl mice or KPC tumor cells isolated from the pancreas of these two models.\nAuthor response:\nFirstly, please also see our response to the Reviewer’s comment #11 below, whereby as requested with respect to Figure 8, we have now included new data (new Supplementary Figure 12) from Opal multiplex immunofluorescence performed on the pancreas of KPC:ASCfl/fl mice to match our existing data on KPC mice (Figure 8h and 8i).\nSecondly, we have now added new data in Figure 8f showing reduced 4-HNE staining (i.e. oxidative stress) in pancreas lesions from KPC:ASCfl/fl mice (similar to that observed in KPC:ASCKO mice) compared to KPC mice.\n10. For many of the statistical comparisons, the KPC APCKO (and future KPC APCfl/fl) groups should be compared to the wt group. The fact that for many panels, there is likely no significant difference between KPC APCKO and wt suggests even more that the lack of tumor development in the KPC APCKO mice results in a pancreas that is almost normal, which undermines the analyses to determine how ASC promotes PDAC via molecular pathways.\nAuthor response:\nAs requested by the Reviewer, we have now included data from WT mice, where relevant, for statistical comparisons against other mouse genotypes in revised datasets and graphs, as follows: Figure 3f (20wo mouse p10 Caspase-1 immunohistochemistry), Figure 5e (10wo mouse pancreas weights), Figure 5i (10wo mouse Ki67 immunohistochemistry), Figure 5j (10wo mouse CD45 immunohistochemistry) and Supplementary Figure 5h (10wo mouse p10 Caspase-1 immunohistochemistry). These data further confirm that total ablation of ASC in KPC mice leads to a significant reduction in activation of Caspase-1 (i.e. inflammasomes) in the pancreatic epithelium back to WT levels (Figure 3f, Supplementary Figure 5h). For Figure 5i, we note that the reduced numbers of proliferating Ki67-positive epithelial cells in the pancreas of KPC:ASCfl/fl mice (compared to KPC mice) remained significantly elevated compared to WT mice (see new text in Results section on page 13, lines 285-286), and this reduction was not as pronounced as that observed for proliferating cells in pancreas of KPC:ASCKO mice. As per our above response to point #6 by the Reviewer, these data suggest that activation of ASC-containing inflammasomes in the non-epithelial compartment of the TME (e.g. immune cells) may also contribute to tumorigenesis, which is now referred to in new text in the Discussion section of the revised manuscript (pages 19-20, lines 447-450).\n11. Fig 8: The authors performed multiplexed immunofluorescence on KPC versus WT pancreas sections to determine the spatial cellular context of the changes in mitochondrial biogenesis (and protein expression of UQCRC2, NDUFB8, etc) within the pancreatic TME and their localization with activated ASC-containing inflammasomes. It is necessary to perform these analyses as well in the areas of poor, moderate and/or well PDAC in the KPC APC fl/fl pancreas and in the pancreas from the VHHACS-treated mice to show that the proteins and parameters are not observed in tumor cells in the absence of APV. The analyses in the pancreas of VHHACS-treated mice are particularly relevant to show that the MOA of the treatment is similar. The authors do not take full advantage of the pancreas samples from the VHHACS-treated mice to support their claims and mechanistic analyses.\nAuthor response:\nAs requested by the Reviewer, we have now included new data (new Supplementary Figures 12 and 13) from Opal multiplex immunofluorescence performed on the pancreas of KPC:ASCfl/fl mice, as well as KPC mice treated with VHH (nanobody) versus saline control. As expected, both the conditional and nanobody-mediated therapeutic targeting of ASC in KPC mice resulted in a marked reduction in the mitochondrial colocalization of ASC and cleaved Caspase-1 (i.e. inflammasome activation) with NDUFB8 and UQCRC2 in the CK19-positive pancreatic ductal epithelium, with the suppressive effect on inflammasome activity more pronounced upon nanobody (VHH ) treatment. These data, together with our existing findings, therefore, strongly support the notion that the expression and activation of ASC-containing inflammasomes is upregulated in the pancreatic ductal epithelium and localizes with mitochondrial markers during PDAC. We now mention these new findings in the text on page 17 (lines 385-390) of the Results section of the revised manuscript.\n12. Discussion: The authors state in the Discussion “novel cancer (epithelial) cell-intrinsic role for ASC-containing inflammasomes in promoting the initiation and progression of PDAC”; however, the data, as is, does not really support this claim. The completion of the above experiments in the KPC APC fl/fl mice and in the pancreas from the VHHACS-treated mice will better support this claim.\nAuthor response:\nAs detailed in the above responses to the Reviewer’s comments, we have provided new experimental data (as requested), as well as alerted the Reviewer to existing data in the Supplementary Figures, which collectively reinforce our identification of a hitherto unknown role for cancer cell-intrinsic ASC-containing inflammasomes in promoting the development of PDAC. This is also supported by our new in vitro human PDAC cell line data in Supplementary Figure 14 (in response to comments by Reviewer #1) indicating human PDAC cancer-cell intrinsic effects. We also refer again to our above responses to points #6 and #10 by the Reviewer, whereby we acknowledge our data implying that cancer cell extrinsic activation of ASC-containing inflammasomes, such as in immune cells within the TME, may also contribute to PDAC. This notion is referred to in new text in the Discussion section of the revised manuscript (pages 19-20, lines 447-450).\n13. Discussion: Limitations of the study need to be discussed.\nAuthor response:\nWhile being mindful of the comment by Reviewer #1 (Minor point #3) to reduce the Discussion section, as requested by the Reviewer we have included new text in the last paragraph of the Discussion (page 24, lines 550-557) referring to some limitations (which despite being out of scope of our current study, are the focus of ongoing work in our laboratory).\nMinor points\n1. The images in Fig 1 E-F and accompanying supplementary images are over exposed. Lower exposure images should be shown.\nAuthor response:\nAs requested by the Reviewer, we have replaced the PDAC patient tumour section images in Figures 1e and 1f with new images obtained via Opal multiplex immunofluorescence for ASC, cleaved Caspase-1, cytokeratin-19 (epithelial cells) and CD45 (pan-immune cells), along with DAPI (nuclear staining). In addition, we have also added new images of the corresponding Opal multiplex immunofluorescence performed on an independent PDAC patient tumor in the new Supplementary Figures 1f and 1g (noting that these replace the original Supplementary Figure 1d, 1e and 2e-g images). The figure legends for these new images, along with the Methods section (under subsection “Multiplex immunofluorescence and multispectral image analysis”), has also been updated accordingly.\n2. Panel 5H and 6D: “PanIN” instead of “PanIn” in the legend.\nAuthor response:\nAs requested, we have now amended the legends in these panels to read “PanIN”.\n3. Mouse Background Clarification: The use of a mixed 129Sv × C57BL/6 background is briefly mentioned. Given that this can influence tumor progression and immune responses, the authors should clarify whether littermate controls were used throughout and whether background effects may explain phenotype differences.\nAuthor response:\nThe KPC mouse strain on a mixed 129Sv x C57BL/6 background has been extensively characterised and reported in the literature over the last 20 years (see reference 29 by Hingorani et al, Cancer Cell, 2005) as the gold standard preclinical mouse model to study the molecular and cellular events that promote the initiation and progression of PDAC. As stated on pages 24-25 in the Methods section under the subsection “Mice”, the mice used in our study are “on a mixed 129Sv × C57BL/6 background” and “Experiments comparing different mouse strains, including where appropriate wild-type (WT) control mice, involved randomly assigned genetic- and age-matched littermates with no gender bias…….”. These elements are incorporated into the experimental design of our study to negate any confounding phenotypic effects that can occur from variations in genetic background. While we believe the existing text articulates that littermate controls (on a mixed 129Sv × C57BL/6 background) are used throughout the study, we have nonetheless modified the text on page 24 (lines 594-597) to further reiterate and emphasize this point.\n4. Nanobody Specificity: More data or citation supporting the specificity of VHHASC for extracellular ASC specks in vivo would strengthen the therapeutic relevance and rule out off-target effects.\nAuthor response:\nWe refer the Reviewer to two cited publications (manuscript references 21 and 31) by co-authors Bernardo Franklin and Florian Schmidt who have previously demonstrated the specificity of the ASC nanobody (VHH ) in blocking the activities of extracellular ASC specks in vitro and in vivo. For example, in reference 31 (e.g. Figure 1, Schmidt FI et al. J Exp Med), experimental data are presented whereby recombinant VHH (targeting human ASC) is used to immunoprecipitate a single specific band of ASC from cell lysates, and fluorescently labelled VHH specifically stains ASC specks. These and other data in this study by Schmidt et al verify the high degree of specificity of VHH against (human) ASC. More recently, experimental data presented in reference 21 (e.g. Figure EV3, Bertheloot D et al. EMBO Mol Med) show that VHH (targeting murine ASC – used in our current study) specifically binds to mouse ASC (via its pyrin domain), but not to human ASC or the pyrin domain of other inflammasome components (e.g. NLRP3). Moreover, Bertheloot et al (e.g. Figures 4 and EV4) demonstrate that VHH blocks the pro‐inflammatory functions of extracellular ASC specks and predominantly targets ASC specks in the extracellular space. Importantly, we also note that no off-targets effects have been observed in vivo with VHH in inflammatory disease models (i.e. gout and arthritis, see Figures 6 and 7 in Bertheloot et al), similar to our own current observations in the KPC PDAC model.\nIn line with the comments by the Reviewer, we have now modified the text and added a statement in the Results section on page 13 (lines 298-301) of the revised manuscript emphasizing the specificity of VHH against extracellular ASC specks and the absence of any known off-target effects.\n5. PRR Partner for ASC: Although the Discussion mentions the uncertainty regarding the PRR responsible for ASC inflammasome activation in PDAC, no experiments or strong hypotheses are proposed. Even correlative analysis of PRR expression in epithelial vs. immune compartments would be informative.\nAuthor response:\nAs the Reviewer can appreciate, the discovery of the ASC inflammasome-associated PRR(s) that promotes PDAC is a significant undertaking that is beyond the scope of this current manuscript, and in fact constitutes a major study being performed in our laboratory at present. In this regard, to screen for ASC-interacting PRR(s) that promote PDAC, we have performed a proteomics-based approach, termed Rapid Immunoprecipitation Mass Spectrometry of Endogenous Protein (RIME), on pancreatic tissues from the KPC mouse model (and appropriate controls including WT and ASC-deficient KPC mice) at defined time points. RIME is a robust proteomics strategy that is used to identify protein-protein interactions, including previously by our laboratory (Balic JJ et al, Oncogene; PMID 31481496). Having identified several candidate ASC-interacting PRRs, we are currently performing validation studies that include genetic targeting of each candidate in KPC mice, along with correlative expression (mRNA, protein) analyses in different cellular compartments within the TME of KPC mice and clinical biopsies and datasets. Therefore, we strongly believe it would be prematurely speculative and potentially compromise novelty of our ongoing studies (and subsequent manuscript(s)) to include such correlative analyses in our current study.\nImportantly, we refer the Reviewer to existing data presented in main Figure 2a,b and Supplementary Figure 2d showing the expression profile of numerous inflammasome-associated PRRs in pancreatic tumours from KPC mice and PDAC patients, respectively. These data demonstrate that upregulation of several inflammasome-associated PRRs in pancreatic tumours aligns with that observed for ASC (and Caspase-1). To address the Reviewer’s comment, while being mindful to shorten the Discussion (see comment by Reviewer #1), we have briefly added new text in the Discussion section on page 22 (lines 545-547) which refers to our current experimental strategy (RIME) to identify the PRR(s) responsible for ASC inflammasome activation in PDAC."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors answered satisfactorily all the issues raised by the reviewer", "reviewer_2": "The authors have adequately addressed all of the concerns raised by this reviewer."}, "author_response": ""}]

Decision letter