Introduction

Pancreatic cancer (PC) is emerging as one the most lethal types of cancer with only a 10% five-year survival rate [1]. Pancreatic ductal adenocarcinoma (PDAC) being the most common among PC. Low survival rate is attributed to the fact that most incidences of PDACs are diagnosed in advanced or metastatic stages [2]. In such cases, gemcitabine remains one the first-line drugs used for treatment [2] but the efficacy of the treatment can fall short due to PDAC tumour heterogeneity and its acquired resistance to said chemotherapy [3]. The aryl hydrocarbon receptor (AHR) is a transcription factor that is commonly upregulated in pancreatic ductal adenocarcinoma [4]. AHR can be linked to Human antigen R (ELAVL1) whose expression in cell cytoplasm is positively associated with PDAC response to gemcitabine [5]. AHR can block ELAVL1 from shuttling from nucleus to cytoplasm [6] where it stabilizes its target messenger RNAs (mRNAs) and increases protein expression of said mRNAs. Among those target mRNAs are those induced by gemcitabine treatment. Frequently with upregulated AHR expression, ELAVL1 would be sequestered in the nucleus which would lead to increased chemoresistance, however the relationship between AHR and ELAVL1 in PDAC is still poorly understood.

Aims & Methods

The aim of the study was to investigate how AHR and ELAVL1 genes and proteins interacts with each other and change their cellular localization in PDAC cells.
Two PDAC cell lines were used (BxPC-3, Su.86.86). AHR and ELAVL1 genes were silenced for 24-hours by lipofectamine mediated siRNA transfection. Cellular localization change of AHR and ELAVL1 proteins was illucidated by fluorescent microscopy. After silencing of AHR or ELAVL1 genes, RNA and proteins were extracted and real-time polymerase chain reaction (RT-PCR) and Western Blot (WB) analysis were performed. Direct binding between ELAVL1 protein and AHR mRNA was investigated by immunoprecipitation assay followed by RT-PCR and WB assays.

Results

The results of gene silencing showed that silencing of AHR can increase ELAVL1 mRNA and protein and silencing of ELAVL1 can decrease AHR mRNA and protein expression. The results of immunoprecipitation assay showed that ELAVL1 protein binds AHR mRNA resulting in mRNA stabilization. This shows a direct link between these two molecules. Moreover, silencing of AHR resulted in increased ELAVL1 protein concentration in cytoplasm which indicates that AHR blocks ELAVL1 shuttling from nucleus to cytoplasm. Silencing of ELAVL1 did not alter the cellular localization of AHR protein.

Conclusion

A direct relationship between AHR and ELAVL1 can be seen in PDAC. Moreover, by decreasing AHR expression it is possible to increase ELAVL1 shuttling from nucleus to cytoplasm which might contribute to PDAC cells responding better to chemotherapy.

References

[1] Hu JX, Zhao CF, Chen WB, Liu QC, Li QW, Lin YY, Gao F. Pancreatic cancer: A review of epidemiology, trend, and risk factors. World journal of gastroenterology. 2021 Jul 7;27(27):4298.
[2] Kamisawa T, Wood LD, Itoi T, Takaori K. Pancreatic cancer. The Lancet. 2016 Jul 2;388(10039):73-85.
[3] Juiz NA, Iovanna J, Dusetti N. Pancreatic cancer heterogeneity can be explained beyond the genome. Frontiers in oncology. 2019 Apr 5;9:246.
[4] Koliopanos A, Kleeff J, Xiao Y, Safe S, Zimmermann A, Büchler MW, Friess H. Increased arylhydrocarbon receptor expression offers a potential therapeutic target for pancreatic cancer. Oncogene. 2002 Sep;21(39):6059-70
[5] Richards, N. G., Rittenhouse, D. W., Freydin, B., Cozzitorto, J. A., Grenda, D., Rui, H., ... & Witkiewicz, A. K. (2010). HuR status is a powerful marker for prognosis and response to gemcitabine-based chemotherapy for resected pancreatic ductal adenocarcinoma patients. Annals of surgery, 252(3), 499-506.
[6] Zago, M., Sheridan, J. A., Nair, P., Rico de Souza, A., Gallouzi, I. E., Rousseau, S., ... & Baglole, C. J. (2013). Aryl hydrocarbon receptor-dependent retention of nuclear HuR suppresses cigarette smoke-induced cyclooxygenase-2 expression independent of DNA-binding. PLoS One, 8(9), e74953.

Introduction

Despite advances in cancer treatment, pancreatic adenocarcinoma continues to have a poor prognosis and remains a leading cause of death among all tumors [1]. Most pancreatic adenocarcinoma patients are diagnosed in stages with metastasis. Moreover, pancreatic adenocarcinoma has a restricted ability to respond to immunotherapy due to low immune infiltration. Prior studies have demonstrated a correlation between tumor endothelial cells (TECs) and angiogenesis, which has been indicated to play a critical role in T-cell-mediated immunosurveillance [2]. Identifying the specific subpopulation of TEC that correlates with immune evasion in pancreatic adenocarcinoma remains a challenge. However, to clarify this subpopulation may bring novel hope to patients with pancreatic adenocarcinoma.

Aims & Methods

Our study aimed to identify the specific subpopulation of TECs associated with increased immunoevasion. Analysis based on the TCGA database, scRNA-seq, the online tool Kaplan Meier plotter, and spatial transcriptomics data were utilized in our study. Moreover, we cultured human umbilical vein endothelial cells (HUVECs) and performed siRNA transfection and Western blotting to confirm the function of RASIP1 in endothelial cells.

Results

Genes related to patient survival in pancreatic adenocarcinoma were identified by the analysis from the Cancer Genome Atlas (TCGA). On the other hand, based on scRNA-seq data, the genes exclusively expressed in endothelial cells were identified. Our analysis revealed that RASIP1 (Ras Interacting Protein 1) was specifically expressed in endothelial cells and linked to improved pancreatic cancer patient survival. Subsequently, the gene ontology analysis of RASIP1-positive TECs was performed based on the scRNA-seq data, and it turned out that RASIP1-positive TECs were related to leukocyte transendothelial migration. Intriguingly, based on the analysis of the TCGA database and spatial transcriptomics data, it was revealed that RASIP1-positive TECs are correlated with increased T cell infiltration and elevated T-effector and IFN-gamma gene signatures in pancreatic adenocarcinoma. These findings suggest that RASIP1-positive TECs may contribute to the "hot" tumor phenotype by promoting T cell infiltration. To further investigate the relationship between RASIP1 and leukocyte transendothelial migration, we knocked down RASIP1 in human umbilical vein endothelial cells (HUVECs) and found a decrease in the expression of the key adhesion molecule intercellular cell adhesion molecule (ICAM). Moreover, the potential related signaling pathway was also investigated, including Erk and p38. Intriguingly, the survival analysis based on patients with pan-cancer immunotherapy also indicated the expression of RASIP1 was proportional to the survival time of cancer patients receiving immunotherapy. In the patients with high expression of RASIP1, longer survival was shown, with a p-value of 0.014 and HR of 0.74. And particularly, in patients treated with PD-L1, significantly longer survival was shown in the group with high expression of RASIP1, with a p-value of 0.01 and HR of 0.74

Conclusion

All in all, our study revealed a potential novel type of endothelial cell in pancreatic adenocarcinoma, which correlated with increased leukocyte transendothelial migration and may provide a potentially effective therapeutic mechanism.

References

1. Ullman NA, Burchard PR, Dunne RF, Linehan DC. Immunologic Strategies in Pancreatic Cancer: Making Cold Tumors Hot. J Clin Oncol. 2022 Aug 20;40(24):2789-2805.
2. Nagl L, Horvath L, Pircher A, Wolf D. Tumor Endothelial Cells (TECs) as Potential Immune Directors of the Tumor Microenvironment - New Findings and Future Perspectives. Front Cell Dev Biol. 2020 Aug 19;8:766.

Introduction

Pancreatic cancer (PC) is one of the fatal malignant tumors, with a 5-year survival rate is approximately 6%, and is expected to become the second leading cause of cancer deaths by 2030. The poor prognosis of PC is associated with the tumour microenvironment, characterized by excessive fibrosis and extracellular matrix deposition, known as desmoplasia. PC’s highly desmoplastic property not only increases the malignancy of the tumor but also inhibits the penetration and diffusion of anti-cancer drugs, related to drug resistance. Chauhan et al. demonstrated that the angiotensin inhibitor (ASI), losartan reduced stromal collagen and hyaluronan production, thus improving the efficacy of chemotherapy in PC models. Several retrospective studies have reported that the ASI usage group showed higher overall survival (OS) and progression-free survival compared to the non-ASI usage group in PC patients. However, most of the previous studies on ASI in PC patients were based on retrospective data from single institutions. Recently, real-world big data study could explain this subject.

Aims & Methods

In this study, we aimed to conduct whether ASI use in PC patients improve OS using National Health Information Database of the National Health Insurance Service, which represents the entire Korean population. We assessed 209,723 participants for eligibility, diagnosed with pancreatic cancer between 2002 and 2020. A total of 64,219 PC patients were enrolled and divided into 3 groups [chronic ASI users with hypertension (HTN), n=13,239; non-ASI users with HTN, n=5,963; non-HTN group, n=45,017]. We performed propensity score matching to balance the baseline characteristics of the 3 groups and to reduce potential confounders using a logistic regression model with adjustment for the following: age; sex; region of residence (urban or rural); household income; underlying disease of HTN, diabetes mellitus, cardiovascular disease, cerebrovascular disease, chronic kidney disease or chronic pancreatitis; Charlson Comorbidity Index (0, 1 or ≥2); concurrent use of aspirin, metformin, statin; PC surgery type; radiotherapy; and chemotherapy.

Results

Multivariate analysis using Cox regression analysis for matched cohort presented ASI usage, female, younger, history of chronic pancreatitis, CCI ≥2, concurrent use of statin, and receiving distal pancreatectomy were significantly associated with longer OS. In addition, Kaplan-Meier curve for OS of 3 groups according to ASI usage showed that chronic ASI users presented significantly longer OS than non-ASI users and non-HTN group [Hazard ratio, chronic ASI users (control) versus non-ASI users 1.447; chronic ASI users (control) versus non-HTN group 1.518; P value < 0.001].

Conclusion

We found that the use of ASI after PC diagnosis is associated with longer survival in PC patients. Additional well-designed randomized controlled studies are needed to confirm these findings in the future.

References

1. Rahib, L., B. D. Smith, R. Aizenberg, A. B. Rosenzweig, J. M. Fleshman and L. M. Matrisian (2014). "Projecting cancer incidence and deaths to 2030: the unexpected burden of thyroid, liver, and pancreas cancers in the United States" Cancer Res 74(11): 2913-2921.
2. Encarnación-Rosado, J. and A. C. Kimmelman (2021). "Harnessing metabolic dependencies in pancreatic cancers." Nature Reviews Gastroenterology & Hepatology 18(7): 482-492.
3. Thomas, D. and P. Radhakrishnan (2019). "Tumor-stromal crosstalk in pancreatic cancer and tissue fibrosis." Molecular cancer 18: 1-15.
4. Chauhan, V. P., J. D. Martin, H. Liu, D. A. Lacorre, S. R. Jain, S. V. Kozin, T. Stylianopoulos, A. S. Mousa, X. Han, P. Adstamongkonkul, Z. Popović, P. Huang, M. G. Bawendi, Y. Boucher and R. K. Jain (2013). "Angiotensin inhibition enhances drug delivery and potentiates chemotherapy by decompressing tumour blood vessels." Nat Commun 4: 2516.
5. Pinter, M. and R. K. Jain (2017). "Targeting the renin-angiotensin system to improve cancer treatment: Implications for immunotherapy." Sci Transl Med 9(410).

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease refractory to all targeted and immune therapies. However, our understanding of PDAC microenvironment especially the metastatic microenvironment is very limited partly due to the inaccessibility to metastatic tumor tissues. Here, we aim to carry out the first single-cell transcriptome analysis of PDAC metastatic microenvironment in matched primary tumor and liver metastasis tissues.

Aims & Methods

We collected synchronously resected PDAC primary tumors and matched liver metastases from four PDAC patients and performed single-cell RNA sequencing. We performed comparative analysis on both cellular composition and functional phenotyping between primary and metastatic tumors. We also performed trajectory analysis and cellular interactome analysis in the metastasis microenvironment.

Results

Epithelial cancer cells exhibited distinct transcriptomic profile in liver metastasis with clearly defined evolutionary routes from primary tumor. We identified specific subtypes of stromal and immune cells critical to the formation of the pro-tumor microenvironment in metastatic lesions, including RGS5⁺ cancer-associated fibroblasts, CCL18⁺ lipid-associated macrophages, S100A8⁺ neutrophils and FOXP3⁺ regulatory T cells. We also identified several new biomarkers associated with PDAC metastasis and poor prognosis, such as LITAF, SPRR1B, KLK7 and LGALS9. Cellular interactome analysis further revealed that the lack of tumor-immune cell interaction in metastatic tissues contributes to the formation of the immunosuppressive microenvironment.

Conclusion

Our study provides a comprehensive characterization of the transcriptional landscape of PDAC liver metastasis, which deepens our understanding of the mechanisms underlying the immunosuppressive microenvironment in metastatic PDAC and serves as a valuable resource for the design of novel targeted therapies.

Disclosure

No

Introduction

Pancreatic cancer (PC) is characterized by an exceptionally aggressive biology and high tumor heterogeneity causing considerable variations in therapy response. Patient-derived organoids (PDOs) reflect parental tumor features and represent a powerful preclinical tool to predict drug response and harness personalized treatment. In clinical practice, PDO-guided selection of effective drugs may provide substantial benefit and improve outcomes.

Aims & Methods

We have derived >40 PDOs from treatment-naïve and pretreated PC patient primary tumor and metastases (after ultrasound-guided biopsy, endoscopic ultrasound-guided biopsy or surgical resection) with a reliable efficacy. PDO drug response was evaluated using a cytotoxicity assay. Using Jenks natural breaks classification method, we clustered response (AUC) for each drug and correlated it to patient response.

Results

The implementation of an automated pipetting system and the subsequent miniaturization of drug screenings significantly enhanced our process capacity, conferring the possibility to extend our panel to a second list of approved targeted substances and further reducing the time before pharmacotyping. The validity of our approach was assessed via re-pharmacotyping a set of PDOs. Following up our feasibility trial (Beutel, 2021), we validated the accuracy of our model on a greater number of PC patients. Our model allowed overall a successful drug-response prediction in naïve patients with an accuracy of 85.7% for both first and second-line regimens. Prediction power remained lower for pretreated patients, with a precision of 57.1% for subsequent lines. A trend was also observed towards a better performance of the system in prognosticating chemoresponsiveness vs unresponsiveness (89.5% vs 68.8%). Finally, the administration of a regimen predicted to be efficient translated into a significantly longer progression-free survival. The access to longitudinal biopsies allowed us to conduct whole exome sequencing on 14 PDOs to capture a comprehensive genetic profiling over the time of treatment, notably revealing a CHEK2-mutated patient who responded over time upon PARP inhibitor maintenance therapy, in line with our PDO-based response prediction, further highlighting the robustness of our method and algorithm.

Conclusion

Overall, we report a robust and clinically-relevant preclinical tool for drug response prediction, paving the way towards an PDO-based true precision medicine in clinical routine.

Introduction

Pancreatic cancer (PC) accounts for 0.5 million new cases and 4.7% of the world's cancer-related deaths in 2020^1,2. Endoscopic ultrasound (EUS) guided Fine Needle Biopsies (FNB) are the current standard of care for confirming suspicious lesions, however the diagnostic accuracy is reported below 70%^3-5. Rapid on-site evaluation (ROSE) improves the diagnostic performances of biopsies via an on-site pathological assessment to reduce the number of needle passes, complication rates, and the need for additional procedures⁶. However, availability of an on-site pathologist is often not feasible. NovaScan has pioneered the use of an impedance spectroscopy biomarker for cancer detection in different tissues based on the Cole Relaxation Frequency (CRF). NovaScan has developed a ”smart ROSE” device that assesses PC presence in FNBs obtained during endoscopic procedures ex vivo, in real-time and on-site, without altering the tissue sample or affecting the workflow.

Aims & Methods

Data were collected in a double-blind, IRB approved study from a cohort of 21 patients (9 males, 12 females) undergoing EUS procedures for suspected pancreatic lesions at the Texas International Endoscopy Center. FNBs were performed with a 22 gauge biopsy from a transduodenal approach. Each biopsy sample was transferred onto a disposable strainer allowing for any excess fluid to drain. An electrode for scanning was placed onto the core samples for data collection with the "smart ROSE," a low cost, portable device that can be used in the operating room. The device measures impedance spectroscopy in the 1 kHz - 10 MHz frequency range and derives a bio impedance biomarker via custom-designed hardware. The assessment of the sample is completed in a few seconds and subsequently, the sample is flushed into a pathology jar to undergo the standard workflow for cytology. The algorithm for extracting the bioimpedance biomarker from the measurement was developed based on our previous preclinical study with transgenic (KPC) mice⁷; pancreas samples with a CRF biomarker below 1 MHz were considered noncancerous, while biomarker values above 1MHz or the absence of a biomarker reading were considered cancerous.

Results

PC assessment provided by nsCanary was validated against pathology outcomes for each biopsy sample. Findings showing a sensitivity of 80% and a specificity of 87.5% are reported in Table 1.

Conclusion

We believe the "smart ROSE" device will provide immediate feedback to the endoscopist on cancer presence and on sample adequacy. This device is intended to support the decision-making process by enabling clinicians to overcome the challenge of proving cancer presence during the first biopsy procedure, ultimately avoiding revisits and unnecessary trauma.

References

1. S. Kato and K. Honda. Use of biomarkers and imaging for early detection of pancreatic cancer. Cancers, 12(7):1965, 2020.
2. M. R. Young, N. Abrams, S. Ghosh, J. A. S. Rinaudo, G. Marquez, and S. Srivastava. Prediagnostic image data, artificial intelligence, and pancreatic cancer: a tell-tale sign to early detection. Pancreas, 49(7):882–886,2020.
3. S. Varadarajulu and M. B. Wallace. Applications of endoscopic ultrasonography in pancreatic cancer. Cancer Control, 11(1):15–22, 2004.
4. P. Michl et al. UEG position paper on pancreatic cancer. Bringing pancreatic cancer to the 21st century: Prevent, detect,and treat the disease earlier and better. UEG journal, 9(7):860–871, 2021
5. D. T. de Moura et al. Evaluation of endoscopic ultrasound fine-needle aspiration versus fine-needle biopsy and impact of rapid on-site evaluation for pancreatic masses. Endoscopy International Open, 8(06):E738–E747, 2020.
6. J. B. Klapman, R. Logrono, C. E. Dye, and I. Waxman. Clinical impact of on-site cytopathology interpretation on endoscopic ultrasound-guided fine needle aspiration. The American journal of gastroenterology, 98(6):1289–1294, 2003.
7. Bogdanowicz, Les, et al. Cole Relaxation Frequency: A Parameter for Pancreatic Cancer Detection. The American Journal of Gastroenterology 117(10S):p e24-e25, October 2022