Introduction

Pancreatic ductal adenocarcinoma (PDAC) is a systemic disease that resists almost any available therapies and is strikingly associated with thrombosis. We hypothesize that cell membrane of PDAC exhibits a high level of plasticity through exocytosis/endocytosis coupling-associated exosomes (or “extracellular vesicles”), leading to the cancer-related systemic effects, and reducing the biogenesis, release, and/or function of exosomes could offer a new therapeutic approach for PDAC.

Aims & Methods

We aimed to test whether pharmacological inhibition of exosomes might suppress the progression of PDAC. We first predicted the protein-protein interactions in our proteomics datasets with the focus on botulinum toxin A (BTX-A) and/or ivermectin (IVM) targets. We obtained PDAC cells from a genetically engineered C57BL/6 mouse model with KrasG12D/Pdx1-Cre for in vitro and in vivo tests. We implanted the cells in 521 mice (C57BL/6) orthotopically (in the pancreas), subcutaneously, through hemi-spleen injection or tail vein injection, and treated the mice with BTX-A, IVM, gemcitabine plus paclitaxel (Gem/Pac) or their combinations. Of note, BTX-A was given before the cell transplantation. In addition, we used PDAC mouse and human organoids. Measurements included survival rate, tumor size, growth of organoids, proteomics, exosomes, immunohistochemistry, electron microscopy, and bioinformatics.

Results

In response to orthotopical injection of PDAC cells, all 18 mice died within 12 days after receiving 1.0 x 10⁶ cells and all 13 mice died within 18 days after receiving 2.25 x 10⁵ cells due to thrombosis. Treatments with BTX-A and/or IVM in vitro suppressed the plasma membrane plasticity of PDAC cells (e.g., neurite-like outgrowth and expression of plasticity protein GAP43) in response to nerve growth factor or conditioned media of PDAC cells. BTX-A in vitro exerted no effect on PDAC cell proliferation, but inhibited the SNARE protein complex signaling pathway, whereas IVM eliminated the formation of membrane microvilli (volume density 0.5% vs 10% saline) and inhibited cell proliferation, clathrin-mediated endocytic signaling and particularly Rab27a. BTX-A+IVM treated PDAC cells showed impaired formation and release of exosomes and down-regulation of proteins involved in exocytosis/endocytosis coupling and biogenesis of exosomes. The exosomes of PDAC cells contained 301 proteins involved in axonal guidance signaling, clathrin-medicated endocytosis signaling, neuroinflammation signaling pathway, epithelial adherents junction signaling, EIF2 signaling and integrin signaling, and toxicities in neuro-immune, kidney, liver, and heart. In vivo, treatment with IVM showed down-regulation of serum exosome proteins. Treatments with BTX-A, IVM or combination had no effects on the tumor size and the cell proliferation rate (Ki67 and DCLK1), but enhanced the inhibitory effects of Gem/Pac on tumors implanted in the pancreas, liver, lung or under the skin. Importantly, BTX-A and/or IVM increased median survivals of PDAC mice, i.e., 26 days without treatment vs. 21 days with Gem/Pac, 40 days with BTX-A, 89.5 days with IVM, and 90.5 days with BTX-A+IVM+Gem/Pac, and of mice with PDAC liver or lung metastasis (>135 days). IVM inhibited the growths of PDAC mouse organoids and human organoids which were derived from a PDAC patient at T2N2MO stage III without neoadjuvant therapy.

Conclusion

Inhibition of exosomes with IVM and/or BTX-A increased the survival rate in PDAC mice (probably by retardation of thrombosis) and the growth of human PDAC organoids, suggesting a translational potential.

Disclosure

Yes

Introduction

While advanced pancreatic cancer is a malignant tumor with a high mortality rate and poor prognosis, early-stage pancreatic cancer, including carcinoma in situ (CIS), has a favorable prognosis and may be treatable with curative resection. However, several recent reports have shown that even in some cases of early-stage pancreatic cancer, recurrence in the remnant pancreas can occur. Pathologically, the tumor occasionally spreads laterally along the main pancreatic duct, presenting challenges in determining the extent of surgical resection. Recently, two types of changes in pancreatic parenchymal volume, called focal pancreatic parenchymal atrophy (FPPA) and upstream pancreatic atrophy (UPA), have been reported to indicate the presence of early-stage pancreatic cancer. It has been reported that in cases of advanced pancreatic cancer with FPPA in previous imaging studies, greater extension along the pancreatic duct has been found. However, in early-stage pancreatic cancer, a comprehensive pathological evaluation of the correlation between cancer location and pancreatic atrophy has not been adequately explored.

Aims & Methods

In early-stage pancreatic cancer without mass, preoperative assessment of intraductal cancer extension along the main pancreatic duct is necessary to determine the extent of surgical resection. This study aimed to investigate the association between the pattern of pancreatic parenchymal atrophy and intraductal cancer extension using a substantial number of resected specimens of early-stage pancreatic cancer. Thirty-two patients with early-stage pancreatic cancer who underwent surgery at five participating centers were enrolled. Of the patients, twenty-seven had CIS, and five had microinvasive carcinoma. Pancreatic atrophy was defined as narrowing of the parenchyma compared to the surrounding parenchyma and was classified as either FPPA (partial atrophy surrounding the pancreatic duct stenosis) or UPA (global atrophy caudal to the site of duct stenosis). We evaluated the correspondence between atrophy on preoperative computed tomography (CT) images and pathologic cancer extension in detail. The length of cancer extension along the main pancreatic duct was calculated as 5 mm per section of a surgically resected pathology specimen (5 mm wide) in which cancer was present. Intraductal cancer extension was defined as an extension exceeding 10 mm.

Results

Preoperative CT revealed FPPA, UPA, and no parenchymal atrophy in 13, 13, and 6 patients. The lateral distance of cancer extension was significantly longer in cases with FPPA than in those without atrophy (median: 20.0 mm vs. 5.0 mm, P = 0.005). Furthermore, a significant difference was observed in the distance of cancer extension between the UPA group and the group without atrophy (median: 20.0 mm vs. 5.0 mm, P = 0.03). Intraductal cancer extension was present in all but one case of FPPA. 69% (9/13) of the cases with UPA showed intraductal cancer extension, whereas cases without atrophy showed no intraductal cancer extension. Importantly, two patients with FPPA or UPA showed positive resection margins during surgery, and three patients with FPPA or UPA showed recurrence in the remnant pancreas.

Conclusion

The presence of FPPA and UPA indicates lateral cancer extension in early-stage pancreatic cancer. In cases presenting with FPPA and UPA, caution should be exercised while determining the extent of surgical resection. Preoperative assessment of the pancreatic parenchyma may provide valuable insights for determining the extent of surgical resection.

Introduction

​Pancreas inflammation is frequently brought on by sustained intraductal pressure, which is mostly caused by gallstones or ERCP, a minimally invasive diagnostic technique. It has been demonstrated that in mice, this brief pressure reduces tight junction integrity through calcineurin. Another effector protein of intraluminal pressure is the mechanosensitive Ca²⁺ channel PIEZO1, which senses cell elongation, increases Ca²⁺ influx, and hence controls both epithelial cell division and apoptosis. However, the development of treatment approaches that specifically target post-ERCP and gallstone-induced pancreatitis has been hampered in recent years due to challenges in in vitro modeling of pressure-related changes.

Aims & Methods

Therefore, our aim was to use apical-out organoids (hPOCs) generated from human pancreas to demonstrate the pressure-related disease processes ex vivo.
Pancreatic tissue samples were collected from 3 cadaver donors and used for establishment of hPOCs. Polarity switching of organoids was induced by Matrigel removal. Conventional and apical-out hPOCs were compared by RNA-sequencing and fluorescent live cell imaging. Gene expression changes in response of 1 µM PIEZO1 activator (YODA1) treatment were measured by qRT-PCR in apical-out hPOCs.

Results

Polarity switching reduced PIEZO1 expression, although decreasing intraluminal pressure and therefore cell elongation reduced expression of cytoskeletal and cell cycle related genes such as ACTB, VCL, LPXN, LMNA, and cyclin gene family members. In the same experiment, pro-inflammatory cytokines like CXCL1, CXCL2, CXCL3, and CXCL8 were found to be downregulated. We demonstrated that polarity switching resulted in enhanced Cl^- efflux via CFTR and ANO1 due to the absence of sustained intraluminal pressure maintained by basal secretion in the closed lumen of conventional organoids. The production of pro-inflammatory cytokines in apical-out hPOCs was reversed upon pharmacological activation of PIEZO1 by YODA1, indicating that PIEZO1 is responsible for the development of the inflammation-like pre-stimulated phenotype of apical-in hPOCs.

Conclusion

Altogether, our results suggest that conventional organoid cultures, previously used as physiologically relevant models, may represent a pressure-induced inflammatory state by overexpressing elements of the epithelial innate immune response, while polarity switching of organoids may overcome the previous limitations and open new perspectives in modelling pressure-induced pancreatitis in vitro.

Introduction

Pancreatic cancer (PC) is characterized by an aggressive biology and high tumor heterogeneity causing considerable variations in therapy response. Patient-derived organoids (PDOs) reflect parental tumor features and may represent a powerful preclinical tool to predict drug response and advance personalized treatment.

Aims & Methods

We have generated a living organoid biobank with >100 PDO lines derived from treatment-naïve and pretreated PC patient primary tumor and metastases with a reliable efficacy, and previously developed a pharmacotyping-guided prediction model to prognosticate patient therapy response. Following up our initial feasibility trial (Beutel, 2021), we now validated the model accuracy in real-life in a higher number of PC patients (41 cases). Additionally, a limited set of 6 PDO pairs were derived from the same patients at two distinct timepoints, followed by pharmacotyping and whole exome sequencing (WES).

Results

Real-life chemotherapy responses in 41 patients matched to pharmacotyping-guided predictions with overall 80.0% accuracy, 89.3% sensitivity, and 64.7% specificity. Particularly, successful response prediction for first and second-line regimens culminated with an accuracy of 85.7% in naïve patients. Prediction power was nevertheless lower in pretreated patients with a precision of 58.3% for subsequent chemotherapy lines. Retrospective analysis of patient clinical data finally showed that the administration of a therapy prognosticated to be efficient ultimately translated into a longer progression-free survival. Moreover, the implementation of an automated pipetting system and the subsequent miniaturization of screenings enhanced our process capacity, conferring the possibility to extend our drug panel to a second list of approved targeted agents and significantly reducing the time before pharmacotyping.
The access to longitudinal biopsies allowed to conduct WES on 12 PDOs to capture a comprehensive genetic profiling over the time of treatment, notably revealing a CHEK2-mutated patient responding over time upon PARP inhibitor maintenance therapy, in line with our PDO-based prediction, highlighting the robustness of our method and algorithm. Tracking clonal evolution in longitudinal biopsies by WES revealed lower mutational burden and therapy-driven genetic alterations upon progression. Finally, to decipher molecular mechanisms driving PC chemoresistance with an unprecedented accuracy level, we took advantage of our phenotypically and therapeutically-profiled PDO living biobank and employed single-nucleus multiomic approaches, capturing simultaneously the transcriptome as well as the chromatin landscape, of chemoresponsive and non-responsive PC PDOs and of a unique longitudinally-collected case study. This allowed us to identify transcriptomic and epigenetic variabilities in a longitudinal setting across several sequential therapeutic lines.

Conclusion

We report a robust and clinically-relevant companion tool for therapy-response prediction, one more step towards a PDO therapeutic profiling-guided personalized medicine in clinical routine. Additionally, uncovering expression signatures driving chemoresistance may strikingly enhance patient stratification and therefore advance precision oncology in PC.

Introduction

Recent studies using single-cell transcriptomic analysis have reported several distinct clusters of neoplastic epithelial cells and cancer-associated fibroblasts in the pancreatic cancer tumor microenvironment. However, their molecular characteristics and biological significance have not been clearly elucidated due to intra- and inter-tumoral heterogeneity.

Aims & Methods

We performed single cell RNA sequencing using enriched epithelial cells and fibroblasts from 17 pancreatic cancer tissues. Then, to resolve the spatial information of heterogeneous cancer subclusters and cancer-associated niches in human pancreatic cancer tissues, we performed RNA in situ hybridization and spatial transcriptome analysis using 10X Visium technique in paired tumor tissues.

Results

We identified five distinct functional subclusters of pancreatic cancer cells and six distinct cancer-associated fibroblast subclusters. We deeply profiled their characteristics, and we found that these subclusters successfully deconvoluted most of the features suggested in bulk transcriptome analysis of pancreatic cancer. Among those subclusters, we identified a novel cancer cell subcluster, Ep_VGLL1, showing intermediate characteristics between the extremities of basal-like and classical dichotomy, despite its prognostic value. Molecular features of Ep_VGLL1 suggest its transitional properties between basal-like and classical subtypes. In the spatial transcriptome analysis, we constructed an adjacency network graph reflecting the neighborhood enrichment relationships across diverse cell types and draw a putative cancer progression axis from Ep_FXYD2, ADM population, to Ep_TRIM54, Ep_VGLL1, and Ep_KRT6A, supporting our model of pancreatic cancer cell dynamics. In addition, we also discovered that the spatial distributions of Fb_LRRC15, representing the myCAF population, were highly correlated with pancreatic cancer cell distributions, whereas Fb_SFRP1, representing the iCAF population, were located distal to the cancer-associated niche.

Conclusion

This integrative analysis not only provides a comprehensive landscape of pancreatic cancer and fibroblast population, but also suggests a novel insight to the dynamic states of pancreatic cancer cells and unveils potential therapeutic targets.

References

Collisson, E. A. et al. Subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy. Nat. Med. 17, 500-503, (2011).
Moffitt, R. A. et al. Virtual microdissection identifies distinct tumor- and stroma-specific subtypes of pancreatic ductal adenocarcinoma. Nat Genet 47, 1168-1178, (2015).
Collisson, E. A. et al. Molecular subtypes of pancreatic cancer. Nat. Rev. Gastroenterol. Hepatol. 16, 207-220, doi:10.1038/s41575-019-0109-y (2019).
Chan-Seng-Yue, M. et al. Transcription phenotypes of pancreatic cancer are driven by genomic events during tumor evolution. Nat Genet 52, 231-240, (2020).
Porter, R. L. et al. Epithelial to mesenchymal plasticity and differential response to therapies in pancreatic ductal adenocarcinoma. Proc. Natl. Acad. Sci. U. S. A., doi:10.1073/pnas.1914915116 (2019).
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Liu, X. et al. Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens. Nat Protoc 12, 439-451, doi:10.1038/nprot.2016.174 (2017).

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal human cancers. In recent years, bacteria resembling gut microbiota have been found within pancreatic cancer tissues, suggesting their potential influence on tumor growth and prognosis. However, the details remain insufficiently elucidated. Therefore, we clarified the characteristics of the tumor microbiota using resected pancreatic cancer specimens from our institution and to analyze their relationship with the tumor microenvironment and prognosis.

Aims & Methods

We analyzed formalin-fixed paraffin-embedded (FFPE) sections from 162 PDAC patients who underwent surgery between April 2008 and March 2017 in our institution.
DNA were extracted from FFPE using Quantitative polymerase chain reaction (qPCR) and in situ hybridization (ISH) targeting 16S rRNA. Microbial community was explored by using 16S metagenomics from FFPE. The profile of intratumoral bacteria was compared with clinical information, pathological findings including tumor-infiltrating T cells, tumor-associated macrophage, fibrosis, and alterations in four main driver genes (KRAS, TP53, CDKN2A/p16, SMAD4) in tumor genomes.

Results

Among 162 cases, 52 showed amplification in qPCR targeting 16S rRNA and positive ISH reactions targeting 16S rRNA within tumor tissues. The presence of bacteria commonly found in gut microbiota such as Proteobacteria and Firmicutes was observed within tumor tissues of these 52 cases. Comparison with paired normal tissues (N=26) revealed differences in αdiversity (P<0.01) and βdiversity (P<0.01), suggesting the presence of a unique tumor microbiota. Subsequently, we divided the 52 cases into two groups based on the median survival period. We identified one bacterial species was dominant in the long-term survival group and ten were in the long-term survival group using Linear Discriminant Analysis Effect Size. Comparison of bacterial profiles between cases with good and poor prognosis revealed a significant positive correlation between a shorter survival time and the presence of anaerobic bacteria such as Bacteroides, Lactobacillus, and Peptoniphilus. 24 cases which were positive for at least one of the three prognostic bacteria were compared with 28 cases negative for all of them, there were not signicfficant correlation with tumor size, pathological stage, fibrosis,and alterations in four main driver genes,but signicfficant correlation with the lower degree of tumor-infiltrating T cells positive for CD4, CD8, and CD45RO (P<0.01, P<0.01, P<0.01, respectively)

Conclusion

We identified multiple bacterial species within pancreatic cancer tumors associated with prognosis. Increased abundance of Bacteroides, Lactobacillus and Peptoniphilus along with the progression of pancreatic cancer suggests their involvement in worsening prognosis through tumor immunity.