The 3D spatiotemporal dynamics of tubular membrane protrusions are crucial for understanding phagocytosis, cellular communication and mechanobiology. Confocal microscopy, despite its prevalent use in membrane protrusion studies, presents limitations due to its inherently low axial resolution and high phototoxicity, which significantly hinder live imaging of tubular protrusion along the axial plane. We discovered that rotational oblique interference scattering (RO-iSCAT) leverages off-axis illumination to induce a larger lateral shift in out-of-focus iSCAT signals compared to in-focus signals. This phenomenon generates speckle-free widefield interferometric scattering signals with a 10-fold signal-to-noise ratio improvement, eliminating the need for any background subtraction. RO-iSCAT enables real-time, label-free imaging of diverse nanoparticles and tubular membrane protrusions, thus providing biophysical profiling of tubular membrane protrusions across multiple cell types and in complex co-cultures. RO-iSCAT empowers rapid quantitative dissection of the axial spatiotemporal complexities of membrane protrusions at tens to hundreds of nanometer displacements without requiring 3D volumetric imaging.

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.

ABSTRACT Background Pancreatic ductal adenocarcinoma (PDAC) is a particularly lethal malignancy with few treatment options available. Extensive remodelling of extracellular matrix (ECM) generates a highly fibrotic tumour landscape, which impairs therapeutic response. Objective We investigated whether stromal priming via the highly specific Focal Adhesion Kinase (FAK) inhibitor narmafotinib (AMP945) in combination with the two major standard-of-care chemotherapies in PDAC, gemcitabine/Abraxane and FOLFIRINOX, reduces fibrosis and enhances treatment efficacy. Design 3D organotypic matrices, intravital imaging, and in vivo subcutaneous and orthotopic PDAC models were used to provide a rationale for a first-line priming regimen of narmafotinib prior to chemotherapy. Results Neoadjuvant chemotherapy induces fibrosis in PDAC indicating a need for upfront first-line priming of the ECM to normalise the stroma for optimal treatment response. Narmafotinib is a new potent small molecule FAK inhibitor. Phase I safety data shows excellent safety, tolerability, and pharmacokinetics following oral administration in humans. We reveal that narmafotinib treatment during early ECM remodelling (‘priming’) reduces fibrosis, while limiting subsequent PDAC invasion. Moreover, intravital imaging demonstrates real-time FAK inactivation and cell cycle stalling, leading to improved chemotherapeutic efficacy upon narmafotinib priming in vivo . Long-term assessment in patient-derived models shows that narmafotinib priming prior to gemcitabine/Abraxane or FOLFIRINOX reduces PDAC progression and extends survival in both chemotherapy settings. Conclusions Our results using these Phase II-ready drug combinations strongly support the clinical assessment of narmafotinib in PDAC. Narmafotinib is currently in Phase Ib/IIa trials, assessing a pulsed dosing regimen prior to gemcitabine/Abraxane, and warrants further clinical assessment in combination with FOLFIRINOX. SIGNIFICANCE OF THIS STUDY What is already known on this topic Pancreatic cancer (PC) is one of the most lethal malignancies and is characterised by a dense, fibrotic stroma, which impairs chemotherapy efficacy. The non-receptor tyrosine kinase FAK is known to promote cancer fibrosis and therefore represents a therapeutic target to normalise the PC stroma and to improve chemotherapy performance. What this study adds Neoadjuvant chemotherapy induces early fibrosis indicating a need for upfront first-line priming of the ECM to blunt or normalise stromal fibrosis for optimal response to therapy. The small molecule inhibitor narmafotinib (which is currently under Phase Ib/IIa clinical trial assessment) shows high specificity towards FAK as well as desirable pharmacokinetics and pharmacodynamics in healthy human volunteers. Early short-term narmafotinib priming reduces fibrosis and improves the efficacy of subsequent standard-of-care gemcitabine/Abraxane chemotherapy. FOLFIRINOX (oxaliplatin, irinotecan, leucovorin and 5-fluorouracil) is a multi-agent chemotherapy preferentially used in PDAC patients with good performance status. Our results demonstrate that narmafotinib priming also improves FOLFIRINOX efficacy, leading to extended survival in patient-derived PDAC models. How this study might affect research, practice, or policy This study supports the clinical development of narmafotinib in combination with both gemcitabine/Abraxane (ACCENT trial) and further FOLFIRINOX standard-of-care chemotherapies for PDAC patient treatment. The first-line priming strategy and early ECM normalisation used in this study may also be applicable to other combination therapy settings and warrants further investigation in ongoing clinical studies.

Supplementary Figure 2 shows figures related to the Consensus clustering analyses. (A) A delta area plot displays the relative change in the cumulative distribution function (CDF) curve comparing k and k-1 clusters from our cohort. (B) The silhouette plot displays the silhouette width for the four clusters determined by consensus clustering within this cohort. (C) Kaplan-Meier plot of the recurrence-free survival rates of the combined clusters (Clusters 1 with 3 and Clusters 2 with 4).

Supplementary Figure 6 shows a forest plot detailing the hazard ratio of the proteomic risk score and clinically relevant variables for PDA within our study cohort using multivariable Cox regression modeling.

Pancreatic cancer (PC) is a highly metastatic malignancy. More than 80% of patients with PC present with advanced-stage disease, preventing potentially curative surgery. The neuropeptide Y (NPY) system, best known for its role in controlling energy homeostasis, has also been shown to promote tumorigenesis in a range of cancer types, but its role in PC has yet to be explored. We show that expression of NPY and NPY1R are up-regulated in mouse PC models and human patients with PC. Moreover, using the genetically engineered, autochthonous KPR172HC mouse model of PC, we demonstrate that pancreas-specific and whole-body knockout of Npy1r significantly decreases metastasis to the liver. We identify that treatment with the NPY1R antagonist BIBO3304 significantly reduces KPR172HC migratory capacity on cell-derived matrices. Pharmacological NPY1R inhibition in an intrasplenic model of PC metastasis recapitulated the results of our genetic studies, with BIBO3304 significantly decreasing liver metastasis. Together, our results reveal that NPY/NPY1R signaling is a previously unidentified antimetastatic target in PC.

Despite the crucial importance of dynamic membrane protrusions for understanding phagocytosis, cellular communication and mechanobiology, current imaging modalities struggle to quantitatively track their real-time, 3D spatiotemporal dynamics with sufficient molecular specificity and minimal perturbation. Many membrane protrusions studies still utilize confocal microscopy where its axial resolution and high phototoxicity remains a key limiting factor for live axial imaging. We discovered that multiple rotational oblique interference scattering (RO-iSCAT) leverages off-axis illumination to induce a larger lateral shift in out-of-focus iSCAT signals compared to in-focus signals. This phenomenon provides a foundation to generate speckle-free widefield interferometric signals with a 10-fold signal to noise ratio improvement, eliminating the need for any background subtraction. RO-iSCAT enables real-time, label-free, and minimally invasive imaging of diverse membrane protrusions within complex co-cultures. RO-iSCAT enables nanoscale-sensitive tracking of membrane protrusion dynamics along the axial direction. This allows for the construction of dynamic axial variance maps, facilitating quantitative measurements of membrane protrusion formation at tens to hundreds of nanometer displacements, without requiring 3D volumetric imaging. RO-iSCAT empowers real time quantitatively dissection of the axial spatiotemporal complexities of membrane protrusions and unlock future insights into fundamental processes like cell migration, durotaxis, and intercellular communication. ### Competing Interest Statement The authors have declared no competing interest.

Supplementary Figure 9: Kaplan-Meier plot for patients dichotomized by a proteomic risk score that uses only the ten proteins detected in blood by mass spectrometry (PURB, GALM, SERPINA3, OAS3, KRT2, NUDT2, SERPINA4, CUTA, POSTN, CLEC11A).

Supplementary Figure 7 shows the proteomic signature performance based on the Receiver operator characteristic curve analysis. (A) Receiver operator characteristic curve (ROC) at 1 year of follow-up for the proteomic signature (purple) and other clinically relevant variables for PDA within our cohort. (B) An Area Under the Curve (AUC) plot displays the AUC as a function of time for the proteomic risk score (purple) and other clinically relevant variables for PDA within our cohort.

Atypical chemokine receptors (ACKRs) are a subclass of chemokine receptors that internalise and degrade chemokines instead of eliciting chemotaxis. Scavenging by ACKRs reduces the local bioavailability of chemokines and can thus reshape chemokine gradients that direct leukocyte trafficking during inflammation and anticancer responses. In pancreatic ductal adenocarcinoma (PDAC), chemokine axes, such as CXCL12-CXCR4, are co-opted by cancer-associated fibroblasts (CAFs) for tumour growth and escape, and immunosuppression. Here, we explore the use of ACKRs to reshape chemokine gradients within the PDAC tumour microenvironment. ACKR2, previously only known to scavenge inflammatory CC chemokines, was recently shown to be able to interact with CXCL10 and CXCL14. Here, using a chemokine binding assay and cytometric bead arrays, we reveal that ACKR2 scavenges additional CXC chemokines CXCL12 and CXCL1. ACKR2 scavenges CXCL12 with reduced efficiency compared to ACKR3, previously reported to bind CXCL12. Finally, we demonstrate that the overexpression of ACKR2 on bystander cells protects primary murine cytotoxic T lymphocytes from PDAC CAF-mediated chemoattraction. These findings reveal new CXC chemokine ligands of ACKR2 and indicate that ACKR overexpression may protect T cells from misdirection by CAFs.

Supplementary Figure 16 shows a Forest plot presentation of the hazard ratios for each KRT protein detected in the tumor samples from the study cohort, derived from a univariate Cox regression analysis with overall survival as the outcome of interest. Note that this figure excludes KRT75, which was detected in only two samples.

A schematic representing (A) data collection from 30-μm sections of 115 PDA and 61 adjacent normal fresh-frozen tissues that were prepared (19) for (B) DIA-MS. C, Protein quantification from DIA-MS data files using DIA-NN and MaxLFQ software for data normalization, QC, and peptide-to-protein inference and (D) the downstream analyses of the protein data. FC, fold change.

The distribution of clinical variables across the four clusters.

Supplementary Figure 12 shows a volcano plot displaying the differentially abundant proteins between tumors harboring a KRAS-G12D mutation versus those with any other KRAS-G12 mutation

Proteomic risk score for mortality. A, Forest plot detailing the multivariable HRs for the overall survival of each of the proteins used to build the proteomic risk score. B, Kaplan–Meier curve displaying the overall survival probability of the low- and high-risk groups within our cohort. C, Kaplan–Meier curve displaying the survival probability of the low- and high-risk groups within the CPTAC validation dataset.

Supplementary Table 9 shows the differential abundance of the KRT protein family across the different clinical variables of interest, where “Up” means upregulated and “Down” means downregulated in the group of interest versus others. *Note that KRT9, KRT10, KRT20, KRT23, KRT77, KRT74, KRT79 and KRT80 were not differentially abundant among any subgroup

Supplementary Table 4 shows previous evidence regarding each protein included in the risk score in terms of their association with different types of cancer diagnosis and prognosis. *Based on data extracted from the Human Protein Atlas (https://www.proteinatlas.org/)

Proteomic subtypes of PDA. A, A clustered heatmap displaying the highly variable protein intensities across the four clusters detected within this cohort. B, Kaplan–Meier plots of the survival rates of the four clusters. C, Kaplan–Meier plot of the survival rates of the clusters combined according to prognosis (cluster 1 with 3 and cluster 2 with 4).

Supplementary Figure 10 shows the differential abundance and pathway enrichment analyses based on KRAS mutations. (A) Volcano plot displaying the differentially abundant proteins between KRAS mutant PDA and KRAS wild-type PDA. (B) Pathways enriched in Gene Ontology molecular function database from upregulated proteins in tumors that harbored KRAS mutations.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer, with a five-year survival rate of just 13%1. Over 80% of patients present with advanced, metastatic disease and are, therefore, ineligible for surgical resection. Systemic chemotherapy, particularly FOLFIRINOX, offers modest survival benefits (median 11.1 months vs. 6.8 months with gemcitabine)2, however, we and others have shown that co-targeting pancreatic cancer tumours in combination with chemotherapy can improve outcomes in pre-clinical models3-7. Thus, this project aims to uncover the mechanisms by which PDAC acquires resistance to FOLFIRINOX, and to identify strategies to restore or enhance treatment sensitivity. To model clinical treatment dynamics, we have subjected orthotopically implanted patient-derived xenografts (PDXs) from the Australian Pancreatic Cancer Matrix Atlas (APMA)8 to 11–15 rounds of FOLFIRINOX or vehicle in vivo. Similarly, orthotopic tumours derived from the KPC mouse model (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre) have undergone 5–6 cycles of treatment. Tumours were then profiled using data-independent acquisition (DIA) mass spectrometry and RNA sequencing to identify chemotherapy-induced vulnerabilities in these patient-derived tumours. This dual analysis revealed a strong DNA repair signature in the chemotherapy-treated PDXs, and I am now investigating the top candidates as co-targets alongside FOLFIRINOX. In parallel, we have established matched treatment-naïve and FOLFIRINOX-treated patient-derived cell lines (PDCLs) and KPC tumour cell lines. These models will be used to further validate candidate resistance pathways through genetic and pharmacological approaches. Functional assays including 3D organotypic matrix cultures will assess tumour cell invasion and drug response3-7. Furthermore, in vivo subcutaneous and orthotopic models, coupled with intravital imaging and biosensors, will allow dynamic monitoring of our co-targeting strategy in live primary pancreatic tumours and metastatic sites (eg. liver)3-7. Overall, by identifying resistance-associated proteins and pathways, this work aims to inform the development of co-targeting approaches that can restore and/or enhance the therapeutic efficacy of FOLFIRINOX in PDAC. References: 1. Siegel et al. CA: A Cancer Journal for Clinicians 75, 10-45 (2025). 2. Conroy et al. New England Journal of Medicine 364, 1817-1825 (2011). 3. Vennin et al. Science Translational Medicine 9 9(384):eaai8504 (2017). 4. Vennin et al. Nature Communications 10, 3637 (2019). 5. Murphy et al. Science Advances 7, eabh0363 (2021). 6. Chitty et al. Nature Cancer 4, 1326–1344 (2023) 7. Pereira et al. Science Advances 10, eadl1197 (2024). 8. Australian Pancreatic Cancer Matrix Atlas (APMA), https://www.pancreaticcancer.net.au/apma/ Brooke A. Pereira, Katie Gordon, Victoria M. Tyma, Ying Fei Liew, Alice M H. Tran, Anna E. Howell, Shona Ritchie, Kendelle J. Murphy, Marina Pajic, Thomas R. Cox, David Herrmann, Paul Timpson. Exploiting chemotherapy-induced vulnerabilities in pancreatic ductal adenocarcinoma (PDAC) to improve treatment outcomes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr B005.