UEG Journal Abstracts 2022 - 168

Tuesday, October 11, 2022
Monday, October 10, 2022
Sunday, October 9, 2022
The respective effects of TRAIL and the PARP inhibitor olaparib or of their
combination were assessed in pancreatic cancer cell lines and patientderived
organoids. In addition, BRCA2-knockout and BRCA2-complementation
models were used. The effects of these agents on apoptosis, DNA
damage, and cell cycle were assessed by immunofluorescence, western
blot, and flow cytometry. The effect of PARP inhibition on the surface expression
of receptors for TRAIL was assessed by flow cytometry.
Results: PARP inhibition and TRAIL synergized to cause cell death in four
pancreatic cancer cell lines. This effect proved independent of BRCA2
gene status in three independent models. These results were confirmed
by using patient-derived PDAC organoids. Olaparib and TRAIL in combination
caused a detectable increase of DNA damage and a concentration- dependent
cell cycle arrest in the G2/M and S cell cycle phases. Olaparib also
significantly increased the proportion of membrane-bound TRAIL receptors
2. In BRCA2-mutated cells, PARP-inhibition increased the sensitivity
to TRAIL by enhancing the activation of caspase 8 without affecting the
intrinsic proapoptotic signaling pathway.
Conclusion: Our results provide a preclinical rationale for combining PARP
inhibitors and TRAIL receptor agonists for treatment of pancreatic cancer,
and suggest that the use of PARP inhibitors could be extended to patients
without BRCA2 mutations if used in combination with these agents.
References:
1. Mattiuzzi C, Lippi G. Current Cancer Epidemiology. J Epidemiol Glob
Health. 2019;9(4):217-222.
2. Sohal DPS, Kennedy EB, Cinar P, Conroy T, Copur MS, et al. Metastatic
Pancreatic Cancer: ASCO Guideline Update. Journal of Clinical Oncology.
2020;38(27):3217-30.
3. Waddell N, Pajic M, Patch A-M, Chang DK, Kassahn KS, et al. Whole genomes
redefine the mutational landscape of pancreatic cancer. Nature.
2015;518(7540):495-501.
4. Bailey P, Chang DK, Nones K, Johns AL, Patch A-M, et al. Genomic
analyses identify molecular subtypes of pancreatic cancer. Nature.
2016;531(7592):47-52.
5. Holter S, Borgida A, Dodd A, Grant R, Semotiuk K, et al. Germline BRCA
Mutations in a Large Clinic-Based Cohort of Patients With Pancreatic Adenocarcinoma.
J Clin Oncol. 2015;33(28):3124-9.
6. Golan T, Hammel P, Reni M, Van Cutsem E, et al. Maintenance Olaparib
for Germline BRCA-Mutated Metastatic Pancreatic Cancer. New England
Journal of Medicine. 2019;381(4):317-27.
7. Golan T, Hammel P, Reni M, Van Cutsem E, et al. Overall survival from
the phase 3 POLO trial: Maintenance olaparib for germline BRCA-mutated
metastatic pancreatic cancer. Journal of Clinical Oncology. 2021;39(3_
suppl):378-.
8. De Toni EN, Ziesch A, Rizzani A, Török HP, et al. Inactivation of BRCA2
in human cancer cells identifies a subset of tumors with enhanced
sensitivity
towards death
receptor-mediated apoptosis. Oncotarget.
2016;23;7(8):9477-90.
Disclosure: Nothing to disclose.
OP222
HARNESSING PLURIPOTENT STEM CELL DERIVED
TRILINEAGE-COMPETENT PANCREATIC PROGENITORS TO
GENERATE DUCT-LIKE ORGANOIDS
S. Merz1, M.K. Melzer1
A. Kleger1
1
, M. Breunig1, J. Krüger1, M. Hohwieler1
University Hospital Ulm, Internal Medicine I, Ulm, Germany
Contact E-Mail Address: sarah.merz@uni-ulm.de
Introduction: Current protocols for directed pancreatic differentiation of
human pluripotent stem cells (hPSCs) are mainly built upon efforts to generate
insulin-producing beta cells via PDX1 and NKX6-1 expressing pancreatic
progenitors (PPs) and are thus biased towards the endocrine lineage.
However, proper formation of multipotent precursors enabling the generation
of endocrine and exocrine cells from the same pool of progenitors is
of utmost interest for studying developmental routes and disease such as
early cancer events and related genes affecting different cells of origin.
Therefore, a broader marker spectrum applied for the prediction of lineage
competence as well as lineage tracking and purification tools are
warranted for improved pancreas in vitromodels.
Aims & Methods: Here we performed a comprehensive compound testing
to tailor the four-step differentiation of human embryonic stem cells
(hESCs) to Glycoprotein-2 (GP2) positive PPs, thereby expanding the commonly
applied markers PDX1 and NKX6-1 to label multipotent progenitor
cells. The fine-tuned PPs were further differentiated into the endocrine
and exocrine lineage in vitro and in vivo. A duct-specific KRT7-Venus reporter
hESC line was generated by a CRISPR/Cas9 approach and phenotypically
characterized in an in vitro pancreatic duct-like organoid (PDLO)
system and on porcine urinary bladder, a recently published ex vivo matrix
(Melzer et al., 2022).
Results: Based on the stage-specific compound testing we compiled an
improved protocol to generate PPs. These progenitor cells reveal constantly
high levels of NKX6-1 and PDX1 but most importantly of the trilineage
label GP2, hence outperforming currently used protocols in the
field. Multipotency of the progenitor cells was proven in vitro by further
differentiation towards the endocrine lineage as well as towards PDLOs. In
addition, freeze-thaw cycles at the pancreatic endoderm stage preserved
differentiation capacity. Multipotency was further substantiated by orthotopic
transplantation into mice showing that PPs specialized into acinar,
ductal and endocrine cells in vivo, resembling 18 to 21 weeks post conception
of human fetal pancreatogenesis.
Finally, we generated a unique KRT7 reporter stem cell line to specifically
track ductal differentiation. An extensive in vitro and ex vivo characterization
of this ductal reporter line underpins advantages of our refined PP
differentiation pipeline, here exemplified for the subsequent formation of
ductal organoids.
Conclusion: In this study, we systematically fine-tuned the differentiation
towards pancreatic progenitors expressing GP2 and could successfully
demonstrate multipotency in vitro as well as in vivo.
Further, we engineered a duct-specific KRT7 reporter hESC line to track
and purify pancreatic ductal cells which displays a unique tool for studying
pancreatic ductal diseases and early oncogenic events in our PDLO
system.
References: Melzer, Michael, et al. " Organoids at the PUB: the porcine urinary
bladder serves as a pancreatic niche for advanced cancer modeling. "
Advanced Healthcare Materials (2022): 2102345.
Disclosure: Nothing to disclose.
,
168
UEG Journal | Abstract Book
UEG Journal Abstracts 2022

Table of Contents for the Digital Edition of UEG Journal Abstracts 2022
