scholarly journals Murine- and Human-Derived Autologous Organoid/Immune Cell Co-Cultures as Pre-Clinical Models of Pancreatic Ductal Adenocarcinoma

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3816
Author(s):  
Loryn Holokai ◽  
Jayati Chakrabarti ◽  
Joanne Lundy ◽  
Daniel Croagh ◽  
Pritha Adhikary ◽  
...  
Keyword(s):  
T Cell ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Immune Cell ◽  
The United States ◽  
Ductal Adenocarcinoma ◽  
Checkpoint Inhibition ◽  
Arginase 1 ◽  
Programmed Death

Purpose: Pancreatic ductal adenocarcinoma (PDAC) has the lowest five-year survival rate of all cancers in the United States. Programmed death 1 receptor (PD-1)-programmed death ligand 1 (PD-L1) immune checkpoint inhibition has been unsuccessful in clinical trials. Myeloid-derived suppressor cells (MDSCs) are known to block anti-tumor CD8+ T cell immune responses in various cancers including pancreas. This has led us to our objective that was to develop a clinically relevant in vitro organoid model to specifically target mechanisms that deplete MDSCs as a therapeutic strategy for PDAC. Method: Murine and human pancreatic ductal adenocarcinoma (PDAC) autologous organoid/immune cell co-cultures were used to test whether PDAC can be effectively treated with combinatorial therapy involving PD-1 inhibition and MDSC depletion. Results: Murine in vivo orthotopic and in vitro organoid/immune cell co-culture models demonstrated that polymorphonuclear (PMN)-MDSCs promoted tumor growth and suppressed cytotoxic T lymphocyte (CTL) proliferation, leading to diminished efficacy of checkpoint inhibition. Mouse- and human-derived organoid/immune cell co-cultures revealed that PD-L1-expressing organoids were unresponsive to nivolumab in vitro in the presence of PMN-MDSCs. Depletion of arginase 1-expressing PMN-MDSCs within these co-cultures rendered the organoids susceptible to anti-PD-1/PD-L1-induced cancer cell death. Conclusions: Here we use mouse- and human-derived autologous pancreatic cancer organoid/immune cell co-cultures to demonstrate that elevated infiltration of polymorphonuclear (PMN)-MDSCs within the PDAC tumor microenvironment inhibit T cell effector function, regardless of PD-1/PD-L1 inhibition. We present a pre-clinical model that may predict the efficacy of targeted therapies to improve the outcome of patients with this aggressive and otherwise unpredictable malignancy.

scholarly journals Gut-derived lipopolysaccharide remodels tumoral microenvironment and synergizes with PD-L1 checkpoint blockade via TLR4/MyD88/AKT/NF-κB pathway in pancreatic cancer

2021 ◽  
Vol 12 (11) ◽  
Author(s):  
Hanlin Yin ◽  
Ning Pu ◽  
Qiangda Chen ◽  
Jicheng Zhang ◽  
Guochao Zhao ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Escape ◽  
Adaptive Immune System ◽  
Bioinformatic Analysis ◽  
Ductal Adenocarcinoma ◽  
Promoter Regions ◽  
T Cell Infiltration ◽  
Programmed Death

AbstractLipopolysaccharide (LPS) as an important inflammatory mediator activates the innate/adaptive immune system. The existence of LPS in pancreatic ductal adenocarcinoma (PDAC) has been reported, however, its biological function in PDAC remains unclear. Here, we demonstrated that circulating and tumoral LPS was significantly increased by intestinal leakage in the orthotopic murine PDAC model, and LPS administration promoted T cell infiltration but exhaustion paradoxically in the subcutaneous murine PDAC model. By bioinformatic analysis, Toll-like receptor 4 (TLR4), LPS receptor, was further found to enrich in immune tolerance signaling in PDAC tissues. Then, a significant positive correlation was found between TLR4 and programmed death ligand-1 (PD-L1) in clinical PDAC tissues, as well as serum LPS and tumoral PD-L1. Meanwhile, LPS stimulation in vitro and in vivo obviously upregulated tumor PD-L1 expression, and effectively promoted cancer cells resistance to T cell cytotoxicity. Mechanistically, the activation of TLR4/MyD88/AKT/NF-κB cascade was found to participate in LPS mediated PD-L1 transcription via binding to its promoter regions, which was enhanced by crosstalk between NF-κB and AKT pathways. Finally, PD-L1 blockade could significantly reverse LPS-induced immune escape, and synergized with LPS treatment. Taken together, LPS can remodel tumor microenvironment, and synergize with PD-L1 blockade to suppress tumor growth, which may be a promising comprehensive strategy for PDAC.

scholarly journals Loss of Rnf31 and Vps4b sensitizes pancreatic cancer to T cell-mediated killing

2021 ◽  
Author(s):  
Nina Frey ◽  
Luigi Tortola ◽  
David Egli ◽  
Sharan Janjuha ◽  
Kim Fabiano Marquart ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Cell ◽  
Cd8 T Cell ◽  
Ductal Adenocarcinoma ◽  
Pancreatic Tumors ◽  
T Cell Infiltration ◽  
Desmoplastic Stroma ◽  
Tumor Cell Lysis

Pancreatic ductal adenocarcinoma (PDA) is an inherently immune cell deprived tumor, characterized by desmoplastic stroma and suppressive immune cells. Here we systematically dissected PDA intrinsic mechanisms of immune evasion by in vitro and in vivo CRISPR screening, and identified Rnf31 and Vps4b as essential factors required for escaping CD8+ T cell-killing. Using murine PDA cells and human PDA organoids, we demonstrate that Rnf31 protects from TNF-mediated caspase 8 cleavage and subsequent apoptosis induction. For Vps4b we found that inactivation impairs autophagy, resulting in increased accumulation of CD8+ T cell-derived granzyme B and subsequent tumor cell lysis. Orthotopic transplantation of Rnf31- or Vps4b deficient pancreatic tumors, moreover, revealed increased CD8+ T cell infiltration and effector function, and markedly reduced tumor growth in mice. Our work uncovers vulnerabilities in PDA that might be exploited to render these tumors more susceptible to the immune system.

scholarly journals Combining in Vitro Diagnostics with in Vivo Imaging for Earlier Detection of Pancreatic Ductal Adenocarcinoma: Challenges and Solutions

Radiology ◽  
2015 ◽  
Vol 277 (3) ◽  
pp. 644-661 ◽  
Author(s):  
Paul F. Laeseke ◽  
Ru Chen ◽  
R. Brooke Jeffrey ◽  
Teresa A. Brentnall ◽  
Jürgen K. Willmann
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
In Vivo Imaging ◽  
Ductal Adenocarcinoma ◽  
In Vitro Diagnostics

scholarly journals GLRX3, a novel cancer stem cell-related secretory biomarker of pancreatic ductal adenocarcinoma

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jung Hyun Jo ◽  
Sun A Kim ◽  
Jeong Hoon Lee ◽  
Yu Rang Park ◽  
Chanyang Kim ◽  
...  
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
Cancer Progression ◽  
Biomarker Discovery ◽  
Disease Free Survival ◽  
Tumor Formation ◽  
Ductal Adenocarcinoma ◽  
Curative Surgery ◽  
In Vitro Proliferation

Abstract Background Cancer stem cells (CSCs) are implicated in carcinogenesis, cancer progression, and recurrence. Several biomarkers have been described for pancreatic ductal adenocarcinoma (PDAC) CSCs; however, their function and mechanism remain unclear. Method In this study, secretome analysis was performed in pancreatic CSC-enriched spheres and control adherent cells for biomarker discovery. Glutaredoxin3 (GLRX3), a novel candidate upregulated in spheres, was evaluated for its function and clinical implication. Results PDAC CSC populations, cell lines, patient tissues, and blood samples demonstrated GLRX3 overexpression. In contrast, GLRX3 silencing decreased the in vitro proliferation, migration, clonogenicity, and sphere formation of cells. GLRX3 knockdown also reduced tumor formation and growth in vivo. GLRX3 was found to regulate Met/PI3K/AKT signaling and stemness-related molecules. ELISA results indicated GLRX3 overexpression in the serum of patients with PDAC compared to that in healthy controls. The sensitivity and specificity of GLRX3 for PDAC diagnosis were 80.0 and 100%, respectively. When GLRX3 and CA19–9 were combined, sensitivity was significantly increased to 98.3% compared to that with GLRX3 or CA19–9 alone. High GLRX3 expression was also associated with poor disease-free survival in patients receiving curative surgery. Conclusion Overall, these results indicate GLRX3 as a novel diagnostic marker and therapeutic target for PDAC targeting CSCs.

scholarly journals Pancreatic Ductal Adenocarcinoma: Preclinical in vitro and ex vivo Models

2021 ◽  
Vol 9 ◽  
Author(s):  
Beate Gündel ◽  
Xinyuan Liu ◽  
Matthias Löhr ◽  
Rainer Heuchel
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
Ex Vivo ◽  
Treatment Options ◽  
3D Cell Culture ◽  
Therapy Resistance ◽  
Ductal Adenocarcinoma ◽  
The Past ◽  
Slow Progress

Pancreatic ductal adenocarcinoma (PDAC) is one of the most overlooked cancers despite its dismal median survival time of 6 months. The biggest challenges in improving patient survival are late diagnosis due to lack of diagnostic markers, and limited treatment options due to almost complete therapy resistance. The past decades of research identified the dense stroma and the complex interplay/crosstalk between the cancer- and the different stromal cells as the main culprits for the slow progress in improving patient outcome. For better ex vivo simulation of this complex tumor microenvironment the models used in PDAC research likewise need to become more diverse. Depending on the focus of the investigation, several in vitro and in vivo models for PDAC have been established in the past years. Particularly, 3D cell culture such as spheroids and organoids have become more frequently used. This review aims to examine current PDAC in vitro models, their inherent limitations, and their successful implementations in research.

scholarly journals Positive Crosstalk Between Hedgehog and NF-κB Pathways Is Dependent on KRAS Mutation in Pancreatic Ductal Adenocarcinoma

2021 ◽  
Vol 11 ◽  
Author(s):  
Yuqiong Wang ◽  
Dan Wang ◽  
Yanmiao Dai ◽  
Xiangyu Kong ◽  
Xian Zhu ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Signaling Pathways ◽  
Kras Mutation ◽  
Biological Effects ◽  
Ductal Adenocarcinoma ◽  
Luciferase Reporter ◽  
Hh Signaling

It has been shown that aberrant activation of the Hedgehog (Hh) and nuclear factor-kappa B (NF-κB) signaling pathways plays an important role in the pancreatic carcinogenesis, and KRAS mutation is a hallmark of pancreatic ductal adenocarcinoma (PDAC). Until now, the role of KRAS mutation in the context of crosstalk between Hh and NF-κB signaling pathways in PDAC has not been investigated. This study was to determine whether the crosstalk between the Hh and NF-κB pathways is dependent on KRAS mutation in PDAC. The correlation between Gli1, Shh, NF-κB p65 expression and KRAS mutation in PDAC tissues was firstly examined by immunohistochemistry. Next, Western blotting, qPCR, and immunofluorescence were conducted to examine the biological effects of interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α) as NF-κB signaling agonists, Shh as an Hh ligand alone or in combination with KRAS small interfering RNA (si-KRAS) in KRAS-mutant PDAC cells (MT-KRAS; SW1990 and Panc-1), wild-type KRAS PDAC cells (WT-KRAS; BxPC-3) and mutant KRAS knock-in BxPC-3 cells in vitro as well as tumor growth in vivo. KRAS mutation-dependent crosstalk between Hh and NF-κB in PDAC cells was further assessed by Ras activity and luciferase reporter assays. The aberrant Hh and NF-κB pathway activation was found in PDAC tissues with KRAS mutation. The same findings were confirmed in MT-KRAS PDAC cells and MT-KRAS knock-in BxPC-3 cells, whereas this activation was not observed in WT-KRAS PDAC cells. However, the activation was significantly down-regulated by KRAS silencing in MT-KRAS PDAC cells. Furthermore, MT-KRAS cancer cell proliferation and survival in vitro and tumor growth after inoculation with MT-KRAS cells in vivo were promoted by NF-κB and Hh signaling activation. The pivotal factor for co-activation of NF-κB and Hh signaling is MT-KRAS protein upregulation, showing that positive crosstalk between Hh and NF-κB pathways is dependent upon KRAS mutation in PDAC.

scholarly journals Checkpoint Blockade in Combination with CD33 Chimeric Antigen Receptor T Cell Therapy and Hypomethylating Agent Against Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1383-1383 ◽  
Author(s):  
Tongyuan Xue ◽  
Marissa Del Real ◽  
Emanuela Marcucci ◽  
Candida Toribio ◽  
Sonia Maryam Setayesh ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Myeloid Leukemia ◽  
T Cell Therapy ◽  
Programmed Death ◽  
Car T ◽  
Acute Myeloid

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. The cure rate for primary AML patients is only 35% and decreases with age. Novel and effective immunotherapies for patients with relapsed and/or refractory (r/r) AML remain an urgent unmet need. CD33 is an attractive immunotherapeutic target for myeloid malignancies given its expression on more than 85% of AML patient samples. We therefore set out to design and test CD33 chimeric antigen receptor (CD33CAR) T cells preclinically as a single agent and in combinational therapy. To assess antileukemic responses of CD33CAR T cells in vitro and in vivo, we enriched CD4/CD8 T cells from peripheral blood mononuclear cells (PBMCs) and genetically modified them to express a second-generation CD33CAR. CD33CAR T cells exhibited potent antigen dependent CD107a degranulation, IFN-γ production and killing activities against AML cells in vitro. Using a NOD-SCID-IL2Rgnull (NSG) xenograft model engrafted with MOLM-14-ffluc, a CD33 expressing AML cell line transduced with lentivirus carrying firefly luciferase (ffluc) and enhanced green fluorescent protein (eGFP), 3 million CD33CAR or mock T cells were introduced intravenously. CD33 CAR T cell-treated group displayed 98.2% leukemic regression 4 days post CAR T infusion, and 99.6% reduction on day 31. Bioluminescent imaging (BLI) and Kaplan-Meier analysis demonstrated that CD33CAR T cells significantly decreased leukemic burden and prolonged overall survival compared to mock T cells in vivo. Decitabine, a DNA hypomethylating agent (HMA), is a main therapeutic agent for treating AML. We observed HMA treatment led to increased CD33 expression on MOLM-14 cells in vitro. We hypothesized that decitabine can potentiate CD33CAR T cell-mediated AML killing by increasing CD33 expression. MOLM-14 cells were treated with either decitabine alone, CD33CAR T cells alone, or sequential treatment using various concentrations of decitabine or DMSO followed by CD33CAR or mock T cells in an E:T ratio of 1:100. We determined the target specific killing activities in each group using flow cytometric based analysis 48 and 96 hours later. The decitabine followed by CD33CAR T cells treatment reproducibly resulted in the most robust antileukemic activity with 80.6% MOLM-14 cells killed. In comparison, CD33CAR T cells or decitabine monotherapy resulted in 11.5% and 50.9% killing, respectively. In vivo testing of the combinational effects of decitabine and CD33CAR T cells are underway and will be updated at the meeting. Finally, checkpoint blockade targeting programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) has shown survival benefits, particularly in combination with HMA, for patients with r/r AML (Daver et al. 2019). We observed elevated PD-L1 expression on residual AML blasts that survived the treatment with decitabine in combination with CD33CAR T cells. Therefore, we hypothesized that blockade of PD-1/PD-L1 interaction might further improve the antileukemic effect of CD33CAR T cells against AML cells post antigen induction by decitabine. MOLM-14 cells were treated with decitabine for 2 days and CD33CAR T cells were added in an E:T ratio of 1:75. Anti-PD-1 or IgG4 antibody was added to the culture at various concentrations. The most robust CD33 specific killing was seen in the culture with anti-PD-1 antibody added. Further characterization are underway and will be presented. Taken together, our preclinical findings have demonstrated the potency of the CD33CAR T cell therapy and ways to optimize its efficacy. Our results support clinical translation of CD33CAR T cells for patients with AML. Disclosures Budde: F. Hoffmann-La Roche Ltd: Consultancy.

scholarly journals The ATR Inhibitor AZD6738 Synergizes with Gemcitabine In Vitro and In Vivo to Induce Pancreatic Ductal Adenocarcinoma Regression

2018 ◽  
Vol 17 (8) ◽  
pp. 1670-1682 ◽  
Author(s):  
Yann Wallez ◽  
Charles R. Dunlop ◽  
Timothy Isaac Johnson ◽  
Siang-Boon Koh ◽  
Chiara Fornari ◽  
...  
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
Ductal Adenocarcinoma

scholarly journals KIF4A Regulates the Progression of Pancreatic Ductal Adenocarcinoma through Proliferation and Invasion

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Jing Chen ◽  
Cui-Cui Zhao ◽  
Fei-Ran Chen ◽  
Guo-Wei Feng ◽  
Fei Luo ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cell Proliferation ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Disease Free Survival ◽  
High Expression ◽  
Ductal Adenocarcinoma ◽  
Migration And Invasion

Background. Pancreatic cancer is a malignant tumor of the digestive tract, which is difficult to diagnose and treat due to bad early diagnosis. We aimed to explore the role of kinesin superfamily 4A (KIF4A) in pancreatic ductal adenocarcinoma (PDAC). Methods. We first used the bioinformatic website to screen the data of pancreatic cancer in TCGA, and KIF4A protein was detected among the 86 specimens of patients in our hospital combined with clinic-pathological characteristics and survival analysis. KIF4A loss-expression cell lines were established by RNA interference (RNAi). In addition, we performed in vitro cell assays to detect the changes in cell proliferation, migration, and invasion. The proteins involved in the proliferation and metastasis of cancer cells were also detected by western blot. The above results could be proved in vivo. Further, the correlation between KIF4A and CDC5L was analyzed by TCGA and IHC data. Results. We first found a high expression of KIF4A in pancreatic cancer, suggesting a role of KIF4A in the development of pancreatic cancer. KIF4A was found to be differentially expressed ( P < 0.05 ) among the 86 specimens of patients in our hospital and was significantly associated with PDAC TNM stages and tumor size. High KIF4A expression also significantly worsened overall survival (OS) and disease-free survival rate (DFS) ( P < 0.05 , respectively). In addition, cell proliferation, migration, and invasion were inhibited by the KIF4A-shRNA group compared with the control ( P < 0.05 , respectively). In the end, knockdown of KIF4A could inhibit tumor development and metastasis in vivo. Further, the positive correlation between KIF4A and CDC5L existed, and KIF4A might promote pancreatic cancer proliferation by affecting CDC5L expression. Conclusion. In conclusion, the high expression level of KIF4A in PDAC was closely related to poor clinical and pathological status, lymphatic metastasis, and vascular invasion. KIF4A might be involved in promoting the development of PDAC in vitro and in vivo, which might be a new therapeutic target of PDAC.

Combined SHP2 and ERK inhibition for the treatment of KRAS-driven Pancreatic Ductal Adenocarcinoma

2021 ◽  
Author(s):  
Katrin J Ciecielski ◽  
Antonio Mulero-Sanchez ◽  
Alexandra Berninger ◽  
Laura Ruiz Canas ◽  
Astrid Bosma ◽  
...  
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
Tumor Regression ◽  
Mapk Pathway ◽  
Single Agent ◽  
Ductal Adenocarcinoma ◽  
Good Tolerability ◽  
Recent Emergence ◽  
Erk Inhibition

Mutant KRAS is present in over 90% of pancreatic as well as 30-40% of lung and colorectal cancers and is one of the most common oncogenic drivers. Despite decades of research and the recent emergence of isoform-specific KRASG12C-inhibitors, most mutant KRAS isoforms, including the ones frequently associated with pancreatic ductal adenocarcinoma (PDAC), cannot be targeted directly. Moreover, targeting single RAS downstream effectors induces adaptive mechanisms leading to tumor recurrence or resistance. We report here on the combined inhibition of SHP2, a non-receptor tyrosine phosphatase upstream of KRAS, and ERK, a serine/threonine kinase and a key molecule downstream of KRAS in PDAC. This combination shows synergistic anticancer activity in vitro, superior disruption of the MAPK pathway, and significantly increased apoptosis induction compared to single-agent treatments. In vivo, we demonstrate good tolerability and efficacy of the combination. Concurrent inhibition of SHP2 and ERK induces significant tumor regression in multiple PDAC mouse models. Finally, we show evidence that 18F-FDG PET scans can be used to detect and predict early drug responses in animal models. Based on these compelling results, we will investigate this drug combination in a clinical trial (SHERPA, SHP2 and ERK inhibition in pancreatic cancer, NCT04916236), enrolling patients with KRAS-mutant PDAC.

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