Biomedicines 11 00890

biomedicines
Review
Pancreatic Cancer Organoids: An Emerging Platform for
Precision Medicine?
Evangelia Sereti 1, * , Irida Papapostolou 2 and Konstantinos Dimas 3, *
1 Department of Translational Medicine, Lund University, 22363 Lund, Sweden

2 Department of Biochemistry and Molecular Medicine, 3012 Bern, Switzerland
3 Department of Pharmacology, University of Thessaly, Biopolis, 41500 Larissa, Greece
* Correspondence: evangelia.sereti@med.lu.se (E.S.); kdimas@uth.gr (K.D.)
Abstract: Despite recent therapeutic advances, pancreatic ductal adenocarcinoma (PDAC) remains
one of the most aggressive malignancies, with remarkable resistance to treatment, poor prognosis,
and poor clinical outcome. More efficient therapeutic approaches are urgently needed to improve
patients’ survival. Recently, the development of organoid culture systems has gained substantial
attention as an emerging preclinical research model. PDAC organoids have been developed to study
pancreatic cancer biology, progression, and treatment response, filling the translational gap between
in vitro and in vivo models. Here, we review the rapidly evolving field of PDAC organoids and
their potential as powerful preclinical tools that could pave the way towards precision medicine for
pancreatic cancer.
Keywords: pancreatic organoids; pancreatic cancer; precision medicine
1. Introduction
Despite significant progress regarding our knowledge of cancer biology, cancer re-
mains a leading cause of death worldwide. In the European Union (EU-27), according to
recent data (22 July 2020) released from the European Cancer Information System (ECIS),
cancer burden has risen to 2.7 million new cases (all types, excluding non-melanoma skin
Citation: Sereti, E.; Papapostolou, I.; cancer) with 1.3 million deaths in 2020 [1]. The above also records that pancreatic cancer
Dimas, K. Pancreatic Cancer (PC) is the seventh most common cancer for both sexes, with an incidence of approximately
Organoids: An Emerging Platform 3.5%, and the fourth most common cause of cancer related death for both sexes and all ages
for Precision Medicine? Biomedicines (with an estimated percentage of 7.1%) while it rises to the third place among individuals
2023, 11, 890. https://doi.org/ over the age of 65. Pancreatic ductal adenocarcinoma (PDAC) represents the 85% of all
10.3390/biomedicines11030890 cases of pancreatic cancer [2]. Smoking, even after cessation, diabetes mellitus, obesity, age,
Academic Editor: David R. Wallace
and genetic predisposition are the main risk factors identified so far to be implicated in the
development of PDAC [3–6]. Despite constant efforts to develop novel diagnostic tools and
Received: 3 January 2023 treatment approaches, it is estimated that PDAC will rise to the second most common cause
Revised: 14 February 2023 of cancer death by 2030 [6,7]. The prognosis for PDAC patients remains unfavorable, with
Accepted: 19 February 2023 a 5-year overall survival rate of less than 5% [8]. Surgical resection followed by adjuvant
Published: 14 March 2023
therapy is the only therapeutic option; however, less than 15% of patients present local
and potentially operable disease, while it is well known that operability and survival are
better in patients with smaller lesions [9]. Patients with advanced disease at presentation
Copyright: © 2023 by the authors.
or recurrence may receive palliative chemotherapy, although the rates of response and
Licensee MDPI, Basel, Switzerland. overall benefit are modest. The aggressive nature of PDAC and primary or secondary drug
This article is an open access article resistance [10] contribute significantly to this dismal prognosis. It has become evident that
distributed under the terms and PDAC has a genetic component, with several relevant mutations, while its progression is
conditions of the Creative Commons characterized by high heterogeneity [11–13]. KRAS, TP53, CDKN2A, and SMAD4 have
Attribution (CC BY) license (https:// been identified as recurrently mutated genes in pancreatic cancer [14]. Pancreatic cancer
creativecommons.org/licenses/by/ progression and resistance are complex processes involving multiple mechanisms, includ-
4.0/). ing genetic and epigenetic alterations, tumor microenvironment, metabolic reprogramming,
Biomedicines 2023, 11, 890. https://doi.org/10.3390/biomedicines11030890 https://www.mdpi.com/journal/biomedicines

Biomedicines 2023, 11, 890 2 of 19
immune evasion, and tumor heterogeneity. Recent studies have shown that several protein
kinases, like cyclin-dependent kinase 1 (CDK1) [15–18], play a significant role in pancreatic
cancer resistance by modifying proteins involved in key signaling pathways. These modi-
fications can lead to the activation of cellular processes that promote cancer cell growth,
survival, and resistance to chemotherapy.
Due to the reasons stated above, the scientific community is focusing on prevention,
by identifying and minimizing environmental risk factors, and of course on early diagnosis,
which holds the promise of improved outcomes. Chemotherapy remains the cornerstone
for PC management along with tumor resection, radiotherapy, and more recently im-
munotherapy. However, chemotherapy is severely restricted by multiple constraints, such
as severe side effects and toxicity leading to non-compliance with prolonged treatment,
drug resistance, incomplete cure, and low patient quality of life. Thus, there is an urgent
need to develop novel and more effective therapies capable of improving PC patients’
survival rates.
To overcome these constraints and improve the overall outcome of chemotherapy, one
of the major advances in cancer treatment focuses on the application of more personalized
approaches. Personalized or precision medicine is a rapidly evolving approach that allows
clinicians to select treatments that carry the potential to greatly improve both treatment
effectiveness as well as patients’ quality of life. Precision medicine (which is the preferred
term nowadays, for more details over the debate see [19]) is the result of many years of
research on tumor biology that has led scientists to understand that individual differences
in genetics, lifestyle, and environment will influence the way cancer patients respond
to treatment.
2. Current Status
Beyond any doubt, the most successful example of precision medicine is pharmacoge-
nomics. Pharmacogenomics is “the study of how genomic variation within the individual
or their disease (including gene expression, epigenetics, germline, and somatic mutations)
influences his/her response to drugs”, as described in the Precision Medicine Glossary
of ESMO [19]. In pharmacogenomics, as explained in the Glossary, genomic variation is
correlated with pharmacodynamics and pharmacokinetics. Pharmacogenomics aims to
optimize drug therapy by maximizing therapeutic effects and minimizing adverse effects.
However, these therapies are very often limited to narrow subsets of patients with a specific,
molecularly defined tumor profile [20].
The efforts to develop chemosensitivity tests that could predict the response of tumors
to anticancer drugs are not new. They date back to the mid-50s when various techniques for
testing chemosensitivity were developed using tumor cells from cancer patients in in vitro
experiments [21]. Human-derived cell lines are the most widely used models for these
experiments. Such experiments are described in the paper by Keepers et al. [16], where the
authors compare two assays commonly used for drug screening, the tetrazolium (MTT)
and the sulforhodamine B (SRB) assays, in two established cancer cell lines: the HT29, a
colorectal cancer cell line and the 11B, a squamous cell carcinoma cell line of the head and
neck. The first human pancreatic cancer cell line was generated in 1963 [22], and since then
many human PDAC cell lines have been established. Cell lines possess critical advantages,
as they are easy to manipulate, they can grow indefinitely at low cost, and are suitable
for high throughput pharmacological screening and genetic testing. However, cell line
models also have significant drawbacks, the most important of which is their inability to
recapitulate the histological and biological complexity of a tumor. Especially the absence of
the tumor microenvironment, mainly consisting of extracellular matrix (ECM) components
and various other cell types, such as fibroblasts, nerves, immune cells, adipocytes, and
endothelial cells, is a critical limitation of the use of cell lines [23].
Nonetheless, efforts have been made towards the development of chemotherapy
sensitivity and resistance assays with the use of patient-derived tumor cells that could be
part of routine oncology practice [24]. These assays differ from the older approaches that
quantify drug sensitivity by growth inhibition of tumor cells (such as tumor clonogenic and
[3 H]thymidine incorporation assays) to the newer approaches, i.e., the differential staining
assay and the ATP bioluminescence assay (ATP-TCA), that measure the rate of tumor cell
death as a readout [25]. These latter assays use the quantification of cell viability as a
measure of anticancer drug effectiveness, whereas growth inhibition assays often promote
the development of single clones, thus failing to reflect the in vivo situation [26]. ATP-TCA
has been shown to comprise high sensitivity and reproducibility [27]. Prospective studies
using this approach revealed a correlation between in vitro sensitivity and in vivo tumor
response in metastasized breast cancer [28] and recurrent ovarian cancer [29]. However,
the American Society of Clinical Oncology Working Group on Chemotherapy Sensitivity
and Resistance Assays stated that chemosensitivity assays should not be recommended for
clinical use since the current evidence is not enough to justify the use of any of these assays
as a guide to personalized treatment of cancer patients [24].
Animal cancer models derived from human cancer cell-developed models (xenografts)
are also widely used in translational research. However, these animal models have several
serious pitfalls (limited potential to mirror the actual tumor microenvironment, intratu-
moral clonal heterogeneity and human stromal cells properties), parameters that greatly
affect tumor invasion, migration, recurrence, and drug resistance. As an alternative, Patient-
Derived Xenografts (PDX) developed by the engraftment of patients’ excised tumors di-
rectly into immune-deficient animal models, are novel preclinical tools that could achieve a
greater similarity to human cancer genetics, tumor heterogeneity, and microenvironment.
However, so far, only a limited number of studies have assessed PDX models as preclinical
models for targeted chemotherapy and precision medicine. These studies have documented
that even these sophisticated animal models entail serious disadvantages, such as the cost,
tumor take-rate limitations and slow growth, that are important drawbacks for real-time
personalized medicine approach [30].
A methodology that aims to bridge the utilization of human cancer cells cultures (or
2D cultures) and xenografts, which gained increasing popularity in basic and translational
cancer research lately, is the development of three-dimensional cultures (3D cultures). These
cultures aim to rebuild the 3D microenvironment of a tumor to cultivate an “identical twin”
of a tumor in a culture dish that could be used for basic and translational research purposes,
such as the anticancer drug testing. The first approach of this method is the cultivation
of formations called “spheroids”. Historically, the first spheroid model was reported by
Sutherland in 1986 [31]. Sutherland developed this model to study several microenviron-
mental factors associated with tumor growth. Since then, spheroids have been grown from
a variety of normal cells, established tumor cell lines, or patient-derived tumor cells using
different culture techniques and various artificial and natural extracellular matrices (ECM)
and mechanical methods to generate spherical cell clusters [32–35]. Several studies have
tested the usefulness of multicellular spheroids in cancer biology and the development of
anticancer drugs. Importantly, these studies documented significant differences between
2D and 3D multicellular spheroid cultures in cancer drug sensitivity [36]. Spheroids have
also been used for screening putative new anticancer compounds and for testing individual
chemosensitivity in the context of cancer precision chemotherapy [37]. It remains to be
comprehensively demonstrated that chemosensitivity data derived from 3D cell cultures
capture clinically relevant responses. Moreover, the major limitation of these systems is
that they cannot completely mimic the complex tissue architecture and the high degree of
variability of individual tumors. Having noted that, 3D systems undoubtedly increase the
number of cell-to-cell interactions and more closely resemble the architectural organization
of tissues in vivo. Hence, the data gathered so far support the use of spheroids as reliable
models of in vivo solid tumors and as drug screening platforms.
As spheroids have indeed proven to be useful in the development of anticancer drugs,
researchers put effort towards improving this model. One of the most recent advances in
this field is the development and introduction of more advanced forms of spheroids, which
are called organoids.
3. Organoids
The main strategy to develop 3D cultures is to avoid cell attachment to the bottom of
the culture dishes. This can be achieved simply by either keeping cells in suspension or
by culturing them in special matrices. 3D cultures derived this way from monolayer cell
cultures, are referred to as spheroids and share some characteristics with tumors in vivo
(e.g., the production of ECM, increased chemo-resistance, the appearance of polarized cell
junctions, etc.). However, the use of monolayers in this 3D cell culture system is reported
to have limited in vivo relevance [38]. In a breakthrough study in 2009, Sato et al. [39]
described the establishment of long-term culture conditions under which organoids could
be developed and cultured by stem cells. They utilized a culture system in which specific
niche factors were artificially supplied, including the Wnt agonist R-spondin 1, epidermal
growth factor (EGF), and Noggin (a protein that is involved in the development of many
body tissues, such as nerve tissue, muscles, and bones). To prevent cells undergoing
anoikis, they used laminin rich Matrigel to support intestinal epithelial growth. This
was the first successful attempt to grow organ-like structures for an extended period, up
to 14 months. A month later, Ootani et al. [40] described the development of a robust
long-term methodology for culturing small and large intestines of mouse origin (C57BL/
6 mice), incorporating an air-liquid interface (ALI) and underlying stromal elements. In
their method, Ootani and colleagues used a type I collagen gel as the scaffold to support
the growth of intestinal cells from minced tissues, along with residual stromal components
from tissue fragments that provided the essential niche factors. In 2014, two groups, Li
et al. and Gao et al., described breakthrough advances in the use of organoid models in
cancer research. Li et al. [41] used a single ALI culture method without modification to
engineer oncogenic mutations into primary epithelial and mesenchymal organoids from
the mouse colon, stomach, and pancreas. Their studies demonstrated the general utility
of a primary organoid system (of mice origin though) for cancer modelling and driver
oncogene validation in diverse gastrointestinal tissues. Gao et al. [42] used a 3D organoid
system that allowed them the successful long-term culture of prostate cancer organoids
from biopsy specimens and circulating tumor cells. Interestingly, when these organoids
were grafted into SCID mice, the tumors recapitulated both the histological and immuno-
histological patterns of the patient samples. The authors also performed growth assays
to determine the sensitivity of organoids to enzalutamide and two PI3-kinase pathway
inhibitors, everolimus, and BKM-120. This was practically the first attempt to incorporate
organoids into the anticancer drug development research and precision medicine frame.
The most important pitfall of this study was the low initial success rate, with only 6 out of
32 attempts from seven patients resulting in successful organoid development, yielding a
success rate of only 15–20%.
Based on the findings of these pioneer studies, an “organoid” refers to a group of
cells growing in a 3D structure that is generated directly from primary tissues (healthy
or diseased, like in tumors), embryonic stem cells, circulating tumor cells, or pluripotent
stem cells. Organoids are capable of self-renewal and self-organization, enabling them to
recapitulate the histology and function of the tissue of origin. Nowadays, organoids can
be maintained through an indefinite passage, stored in liquid nitrogen for future use, and,
most importantly, preserve genetic stability [43]. 3D organoids share many characteristics
with spheroids (3D spheres); however, the two systems are quite different. Spheroids
originate from monolayers of epithelial cells (usually from established cell lines and rarely
from patients’ tumor cells) with no specific supplements and with the major requirement
to be cultured under low-adhesion culture conditions that promote cell self-aggregation.
On the contrary, 3D organoids come directly from tissues (normal or diseased), adult stem
cells, or embryonic stem cells, following specific culture protocols (Table 1). They retain
self-renewal/self-organization capacities and can very closely recapitulate the architecture
and characteristics of the tissue of origin. Organoids derived from clinical tumor specimens
can also be passaged over time and maintain some of the molecular characteristics of the
original tumor and are named tumoroids or tumor organoids [19].
Table 1. Human PDAC organoid culture methodology.
Targeted
Transplantation of Success of
Reference [No] Sample Matrix Culture Medium Passage Treatment on Storage
Organoids Organoid Culture
Organoids
AdDMEM/F12 supplemented with: (1) Glutamax 1X,
PDOX creation by
(2) Hepes 1X, (3) Noggin 10% v/v or recombinant protein
orthotopical
Fresh tumors from 0.1 µg/mL, (4) Gastrin 10 nM, (5) Nicotinamide 10 mM, Indefinite
GFR Matrigel injection of human Organoids can be
Boj et al. 2015 [44] surgical resection (6) R-spondin-1 10% v/v, (7) EGF 50 ng/mL, (8) FGF-10 75% passaging Not mentioned
(embedded) PDAC organoids cryo-preserved
and FNA biopsies 100 ng/mL, (9) N-acetyl-L-cysteine 1 mM, (10) B27 capability
to Nu/Nu mice,
supplement, (11) Wnt3a 50% v/v, (12) primocin 1 mg/mL,
PanIN like lesions
(13) Penicillin-streptomycin 1X, (14) A83-01 0.5 µM
Pancreatic Progenitor and Tumor Organoid Media (PTOM)
consisted of DMEM with (1) 1% B27, (2) 50 ug/mL ascorbic
acid, (3) 20 µg/mL insulin, (4) 0.25 µg/mL hydrocortisone, Tumor organoids
At day 16,
(5) 100 ng/mL FGF2, (6) 100 nM all-trans retinoic acid, could be
organoids are
(7) 10 µM Υ267632. At day 8 in 3D culture, culture medium Drugs targeting freezed-thawed
treated with
was replaced with fresh pancreatic organoid maintenance epigenetic and still
0.3–0.5 million collagenase for
medium (POMM, which contains 1% B27 and 50 µg/mL regulators were re-establish
cells were injected 2 hours and then
Huang et al. Fresh tumors from ascorbic acid) with 5% Matrigel. Culture media was replaced 85% tested, i.e A366 cultures that
Bed of Matrigel into the mammary additionaly with
2015 [45] surgical resection every 4 days. Pancreatic Organoid Differentation Media 1 (17/20 samples) and UNC1999, preserve phase
glands of female trypsin for another
(PODM 1) consisted of DMEM supplemented with (1) 1% alone or in and hematoxylin
NOD/SCID mice 10–30 min. The
B27, (2) 300 µM 2-phospho ascorbic acid, (3) 100 ng/ml combination with and eosin stain
cells are collected
FGF7, (4) 10 ng/mL EGF, (5) 1 µM A8301 and (6) 1 µM DBZ. Gemcitabine (H&E)
and re-seeded in
Pancreatic Organoid Differentation Medium 2 (PODM 2) morphology across
3D culture.
consisted of DMEM supplemented with (1) 1% B27, passages
(2) 300 µM 2-phospho ascorbic acid, (3) 100 ng/mL FGF7 and
(4) 10 ng/mL EGF.
Organoids were
cultured for 3 days
and treated with
(1) DMSO
(control), (2)
Gemcitabine
(25 µg/mL),
Biopsy from a (3) AZD1480
patient with poorly Matrigel RPMI supllemented with (1) 10% FBS, (2) 1% (100 nM), (4)
Walsh et al.
differentiated (embedded at Penicillin/Streptomycin, (3) 10 ng EGFR. Culture medium Not reported Not reported Not mentioned AZD6244 (4 µM), Not reported
2016 [46]
pancreatic ductal 1:2 ratio) was replaced every 3 days. (5) XL147
adenocarcinoma (25 nM) and (6)
combination of
gemcitabine plus
(a) AZD1480,
(b) AZD1480 +
AZD6244,
(c) AZD1480 +
AZD6244 + XL147
Table 1. Cont.
Targeted
Organoids
Chemotherapeutic
Organoids were drugs tested:
Complete media [Intesticult (Stemcell Technologies, 6005), passaged via (1) gemcitabine
Biopsies (n = 10)
(1) A83-01 (0.5 mmol/L), (2) fibroblast growth factor 10 mechanical (3 nmol/L,
from PDAC
Romero-Calvo GFR Matrigel (FGF10, 100 ng/mL), dissociation with 10 nmol/L,
patients and PDX Not reported 100% Not reported
et al. 2019 [22] (embedded) (3) gastrin I (10 nmol/L), (3) N-acetyl-L-cysteine TrypLE Express 30 nmol/L)
derived organoid
(10 mmol/L), (4) nicotinamide (10 mmol/L), (5) B27 1X, and passage was (2) FOLFIRINOX
cultures
(6) primocin (1 mg/mL), and (7) Y-27632 (10.5 mmol/L) performed weekly (10 nmol/L)
with a 1:2 ratio. (3) Abraxane
(10 nm/L)
After continued
growth
(>4 passages,
>100 days) some
PDOs did not
ADMEM/F12 supplemented with 50% Wnt3a, RSPO1,
maintain the
Noggin-conditioned media (L-WRN) with HEPES (1 mM),
100 patients complex tissue 80%
Glutamax (1X), Nicotinamide (10 mM), N-Acetylcysteine (1
representing 19 73% (at 1-month architecture (cryo-recovered
Neal et al. mM), B-27 without vitamin A (1X), A83-01 (0.5 mM),
distinct tissue sites ALI method Not reported culture across exhibiting a Not reported and serially
2018 [47] Pen-Strep Glutamine (1X), Gastrin (10 nM), SB-202190
and 28 unique tumor histologies) simple, cystic re-propagated
(10 mM), and EGF (50 ng/mL). The transwell containing
disease subtypes morphology. every few weeks)
tumor tissue and collagen was placed into an outer 60 mm
PDOs could be
cell culture dish containing 1.0 mL of medium.
xenografted into
immunocompro-
mised mice and
re-derived as
organoids.
Biopsies of liver
metastasis from
patients with
Organoids were
PDAC were used
treated with
to develop PDOX
(1) DMSO
in female Advanced DMEM/F12 with The researchers
(control),
Choi et al. Hsd:athymic (1) B27, (2) N-acetylcysteine, (3) EGF, (4) FGF-10, (5) used first passage
GFR Matrigel Not reported Not mentioned (2) Gemcitab- Not mentioned
2019 [48] nude-Foxn1 mice R-spondin 1 and (6) Noggin. Medium was replaced every cells isolated from
ine-HCL and
aged 5–8 weeks. 3 days. F1 PDOX tissues
(3) Albumin-b-
Then, first passage
ound paclitaxel for
cells isolated from
7 days
F1 PDX tissues
were used to
develop organoids.
Table 1. Cont.
Targeted
Organoids
Chemotherapeutic
drugs tested:
(1) Gemcitabine at
1 µM, 200 nM,
100 nM, 50 nM,
Human primary 25 nM, 10 nM and
tumor samples 1 nM,
from (2) 5-Fuorouracil at
31 treatment-naïve Advanced DMEM/F12 suplemented with (1) WNT3a 50% 50 µM, 25 µM,
PDAC patients: v/v, (2) noggin 10% v/v, (3) Rspondin1 10% v/v, (4) B27 1X, 71% (68% from 10 µM, 5 µM,
Hennig et al. 25 specimens from (5) nicotinamide 10 mM, (6) Gastrin 1 nM, surgical resections 1 µM, 100 nM and Repeated
GFR Matrigel Not reported Not mentioned
2019 [11] surgical tumor (7) n-acetyl-L-cysteine 1 mM, (8) primocin 1 mg/mL, and 83% from fine 10 nM, freeze-thaw cycles
resections and (9) mEGF 50 ng/mL, (10) hFGF10 100 ng/mL, needle aspirations) (3) Oxaliplatin at
6 from endoscopic (11) A83-01 0.5 µM, (12) N2 1X 250 µM, 25 µM,
ultrasound- (EUS-) 10 µM, 1 µM,
guided fine needle 100 nM, 10 nM and
aspiration (FNA) 1 nM and
(4) Irinotecan at
250 µM, 25 µM,
10 µM, 1 µM,
100 nM, 10 nM and
1 nM
Table 2. Supplements used for human pancreatic organoid culture and their function.
Supplements Function
2-phospho
Biomedicines 2023, 11, 890 ascorbic acid Maintains cellular differentiation 8 of 19
A 83-01 TGF-b inhibitor

B27 supplement
Methods of PDAC Organoids Development Vitamins and growth factors mix
EGF A summary of the procedures followed by researchers Mitogen. to create pancreatic organoids
is presented in Table 1. For an organoid to be created, samples from patients with PDAC
EGFRare required. The samples are biopsies collected after Mitogen
the surgical removal of the tumor.
FGF-10 There is a specific procedure for manipulating
Stimulatesthe cell proliferation
specimen collected from the patient
to successfully create PDAC organoids. Currently, there are two main approaches for
FGF-2 Stimulates cell proliferation
developing organoids (Figure 1). In one approach, developed and established by Boj S
FGF-7
et al., researchers cultivated organoidsStimulates cell patients
not directly from proliferation
but by using PDX as an
intermediate step [44,48]. According to this approach,
Gastrin after the tumor was surgically re-
TGF-b inhibitor
moved, it was transferred into a 50 mL tube containing RPMI 1640 (Roswell Park Memorial
Glutamax Substitute for L-glutamine
Institute) supplemented with 1% penicillin/streptomycin and 1% fungizone DCU. The
HEPESsample was subsequently cut into Provides a buffered
small pieces pH environment
to create fragments with a size < 2 mm. It
should be noted that all procedures must take place within 24 h to maintain maximum
IGF-1 Mitogen
viability of tumor cells. The next step was the in vivo development of the PDX. Specific
N-acetyl-L-cysteine
strains of mice were chosen, such Precursor in glutathione
as the NSG mice, for the tumor tosynthesis
grow. The injection of
the tumor fragments was performed
N2 supplement either orthotopically
Vitamins and growth (meaning that the
factors mixfragments are
directly injected into the organ of interest) or fragments were subcutaneously implanted.
Nicotinamide Maintain
Hereafter, the mice were observed cell
for the stemnessofand
development cystic
tumors. Thephenotype
tumors were mea-
Noggin
sured regularly until they reached the desired, Inhibits BMP-4
non-harming for mice, size. At the end
of the procedure, mice are humanely euthanized, and the tumors are removed. After the
Penicillin/streptomycin Avoid bacterial contamination
extraction, the tumors were washed with special buffers for contaminating particles to be
PGE2removed. Next, the tumorsMaintain
were slicedcell
intostemness
small piecesandwith cystic phenotype
sterile forceps and blades.
The pieces were digested with a special buffer for
Primocin around 15 min at room temperature
Antibiotic
(RT). After digestion, the tumor pieces were passed through a strainer, and the process was
RSPO-1
repeated 2–3 times with washing buffers. Wnt These signaling activator
steps are needed to isolate as many cells as
SB202190
possible. The tube containing the desired cellsp38was MAPK inhibitor
centrifuged at 1000 RPM for 5 min at
4 ◦ C. After the centrifugation, the medium was carefully removed to avoid pellet disruption.
Wnt3A conditioned Medium Wnt signaling activator
The procedure followed from now on is similar for both techniques used. One difference
Y-27632
is the consistency of the medium ROCKused for inhibitor
the organoidto prevent
culture. The anoikis
difference lies in the
presence or absence of specific factors in the medium (Table 2). Some of these factors are the
following: (1) EGFR, (2) L-glutamine, (3) amphotericin b, (4) Insulin transferrin selenium,
(5) PDGF, and (6) IGF-1. Organoid cultures generally require a change of medium once per
week and can be expanded by passaging at a 1:3 to 1:6 ratio once per week.
Figure 1. Schematic representation of patient-derived organoids (PDO) development for precision

cancer medicine.
Figure 1. Schematic representation of patient-derived organoids (PDO) development for precision

cancer medicine.
Table 2. Supplements used for human pancreatic organoid culture and their function.
Supplements Function
2-phospho ascorbic acid Maintains cellular differentiation
A 83-01 TGF-b inhibitor
B27 supplement Vitamins and growth factors mix
EGF Mitogen.
EGFR Mitogen
Gastrin TGF-b inhibitor
Glutamax Substitute for L-glutamine
HEPES Provides a buffered pH environment
IGF-1 Mitogen
N-acetyl-L-cysteine Precursor in glutathione synthesis
N2 supplement Vitamins and growth factors mix
Nicotinamide Maintain cell stemness and cystic phenotype
Noggin Inhibits BMP-4
Penicillin/streptomycin Avoid bacterial contamination
PGE2 Maintain cell stemness and cystic phenotype
Primocin Antibiotic
RSPO-1 Wnt signaling activator
SB202190 p38 MAPK inhibitor
Wnt3A conditioned Medium Wnt signaling activator
Y-27632 ROCK inhibitor to prevent anoikis
4. Organoids and Tumoroids in the Battle against Pancreatic Cancer

The first to describe the system of organoids for pancreatic tissue was once again
the Clever’s lab. In 2013, Huch et al. published their pioneer work, where they devel-
oped pancreatic organoids by isolating pancreatic ducts from the bulk of the pancreas of
mice [49]. Initially they mixed, seeded, and cultured these pieces with Matrigel, using a
culture medium based on AdDMEM/F12 and supplemented with B27, N-Acetylcysteine,
gastrin, and the growth factors EGF, RSPO1-conditioned media, Noggin, FGF10, and Nicoti-
namide. They were able to expand and passage these organoids for at least 9 months and
store them in cell culture freezing medium. Interestingly, the organoids were ready to be
passaged within one week after seeding and culturing primary isolated ducts in Matrigel.
Two years later, Boj et al. reported the first tumor organoids from human PDAC. To achieve
this, the team substantially modified the procedure and culture conditions compared to
mouse-derived organoids (Table 1) [44]. The material they used to develop human PDAC
organoids was collected either from surgically excised tumors or by FNA (fine needle
aspiration) biopsies, which is important considering that the vast majority of PDAC pa-
tients are inoperable [50]. The success rate of organoid formation was up to 85%, and
they could be passaged indefinitely and cryopreserved for future use. Even more, these
organoids showed complex morphology with differing degrees of dysplastic tall columnar
cells resembling low-grade pancreatic intraepithelial neoplasias (PanINs). Further thor-
ough characterization of these organoids suggested that they were indeed very closely
mirroring neoplastic human pancreatic ductal cells, hence offering a model system to
explore human pancreatic cancer biology. The orthotopic transplantation into Nu/Nu mice
resulted in infiltrative carcinoma comprised of poorly defined and invasive glands, while a
prominent desmoplastic reaction was observed in PanIN-like structures and PDAC. Boj
et al. used these organoids to also identify novel genes that were upregulated in hu-
man PDAC, showing for the first time that they can serve as a tool for the discovery and
dissection of pathways driving human pancreatic carcinogenesis. This could be rather
helpful in the development of novel therapeutic and diagnostic approaches. A few months
later, Muthuswamy’s lab reported the second successful attempt to develop pancreatic
cancer tumoroids [45]. Fresh tissues of primary tumors were digested from patients and
re-suspended initially in a special culture medium, developed by the authors, and called
PTOM (progenitor and tumor organoid medium), and plated on a bed of Matrigel. As
shown in Table 1, the group followed a rather complicated system to maintain, expand,
and passage tumoroids, and their culture medium was mainly supplemented with B27,
ascorbic acid, insulin, hydrocortisone, fibroblast growth factor 2, all-trans retinoic acid,
and Y267632 [a cell-permeable, highly potent and selective inhibitor of Rho-associated,
coiled-coil containing protein kinase (ROCK) inhibitor] without any stimulants of Wnt
signaling (unlike in the Boj et al. method). The success rate was high (17 out of the
20 samples resulted in organoids, reaching 85% of success rate, as in the Boj et al. method),
and tumor organoids showed similar morphological and cytological features, as well as
patterns of differentiation markers, as the primary tumors they were derived from. All
samples could be kept in long-term cultures, frozen, and thawed to re-establish cultures
while maintaining phase and morphology across passages. Tumoroids could also gener-
ate tumors in xenograft models when injected at low inoculation numbers (50,000 cells)
into NSG mice within a period of 4 to 7 weeks. Importantly, the developed tumors were
reported to maintain the histoarchitectures present in the primary tumors from which the
organoids originated and could also be used to re-establish organoid cultures. Following
these remarkable studies, Walsh et al. reported in 2016 a simpler approach to develop
human pancreatic cancer organoids (Table 1) [46]. They used a simple medium consisting
of RPMI supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and
epidermal growth factor receptor at a concentration of 10 ng/mL. Authors reported the
development of xenografts from just one patient with poorly differentiated pancreatic
ductal adenocarcinoma. The sample was taken from a primary tumor biopsy during a
Whipple procedure. Organoids were generated by mechanical dissociation with surgical
scissors and a scalpel. The authors do not mention digestion with enzymes, passage of
organoids, storage, subculture, or long-term self-renewal potential, nor animal testing
to assess their ability to develop tumors. Although they do report that organoids were
heterogeneous in their morphology, however; they did not compare them to the primary
tumor to assess whether organoids recapitulate the morphological and cytological features
of the tumor of origin. The authors cultured these organoids for three days and then used
them for drug testing and optical imaging. Based on the information provided in this
manuscript, it is difficult to understand whether these 3D cultures are real tumoroids or
merely spheroids. In an extraordinary publication in 2018, the group of Kuo reported
the development of organoids from 100 patients representing 19 distinct tissue sites and
28 unique disease subtypes using an improved approach of the ALI method of Ootani
et al. [40,47]. In this work, authors reported that organoids could be formed even when
using media supplemented with fetal calf serum alone; however, the best outcome was
reached when the culture medium was supplemented with WNT3A, EGF, NOGGIN, and
RSPO1 (WENR), which was used to expand and serially passage mechanically processed
tumor fragments as ALI organoids (Table 1). These organoids could be further passaged
with a high success rate (73%) and cryopreserved at a very high success rate of 80%. No-
tably, following this approach, authors were able to preserve both the non-immune and the
immune stromal elements, which suggests that these organoids very closely mimic the real
tumor. A drawback of this approach was that upon long term culture, i.e., over 4 passages
of over 100 days in culture, some of the organoids did not retain the initial complex tissue
architecture, which suggests that the PDOs developed using this method should carefully
be monitored to ensure their reliability compared to the parental tumor.
A different approach for the development of organoids was reported by Romero-
Calvo et al. [22]. In this study, researchers dissociated cells from biopsies and PDX in
a two-step procedure. First, they used collagenase XI and dispase, and subsequently
TrypLE® Express (Thermo Fisher Scientific, Waltham, MA, USA, #12605-010) and DNAse I.
To develop organoids, dissociated cells were embedded in growth factor–reduced (GFR)
Matrigel and cultured in a basal medium called Intesticult (Stemcell Technologies, #6005),
supplemented with: A83-01, fibroblast growth factor 10 (FGF10), gastrin I, N-acetyl-L-
cysteine, Nicotinamide, B27 supplement, primocin and Y-27632 (Table 1). These organoids
were passaged weekly in a 1:2 subculturing ratio via mechanical dissociation with TrypLE®
Express. The success rate was 100%, as all 10 specimens (five directly from patients’
biopsies and another five from PDX) developed organoids. Subsequent H&E histological
analysis showed that a high degree of correlation was maintained between the morphologic
structure of the primary tumors and the corresponding organoids. For the PDX-derived
organoids, although these organoids recapitulated the microscopic features of the primary
tumors, their architecture was more complex and atypical compared with the organoid
models, in part due to the presence of mouse stroma within the tumor, as authors reported.
Analysis of the differential gene expression in organoids from one patient also revealed a
high overlap, close to 90%, with the primary tumor. Thus, the overall results of this study
indicated strong concordance at the morphological and molecular levels.
5. Tumor Organoids in Precision Medicine for PDAC

The first work of anticancer drug testing using tumoroids took place at Muthuswamy’s
lab [45]. The first test they did was to analyze the response of tumoroids from five indepen-
dent patient tumors to the standard drug for PDAC, gemcitabine. According to their report,
all organoid tests showed a poor response to the drug, with an average growth inhibition of
30%. Unfortunately, they did not correlate the responses of these “personalized” organoids
with that of the patients, as all patients had undergone surgery and were disease-free at
the time of the study. The group proceeded to use the same five personalized organoids in
a drug-screening approach to test five different epigenetic inhibitors. In a well-designed
experiment, they first tested these inhibitors against non-tumor pancreatic organoids for
toxicity, and the two least toxic were further tested against the tumoroids as a monotherapy
or in combination with gemcitabine. Through this approach, they identified at least one
inhibitor that resulted in a dose-dependent decrease in the proliferation rate of four out
of the five tumoroids when combined with gemcitabine. The most active inhibitor was
found to be the UNC1999, an inhibitor of EZH2 [a ‘writer’ of trimethylation of histone
H3 at Lys27 (H3K27me3)]. Interestingly, all sensitive tumoroids, as well as the matching
tumors and non-tumor organoids, were positive for the H3K27me3 mark, suggesting a
strong correlation between the organoids and the primary tumor. It would have been
of great interest to see how these studies would correlate with in vivo studies in NSG
xenografts developed with the use of these organoids, but the group did not perform
these experiments. Authors also report that tumor organoids retained patient-specific
traits, such as repressive epigenetic marks, oxygen consumption, and EZH2 dependence,
observations that underline the usefulness of their system for further drug screening. Walsh
et al. [46] also used the organoids they developed to test their response in gemcitabine and
AZD1480 (a novel ATP-competitive JAK2 inhibitor) alone or in combination. Organoids
were treated for 24 h with gemcitabine, AZD1480, and their combination. As they report,
while a significant reduction in the OMI index (optical metabolic imaging, used to evaluate
the effect on organoids proliferation) was detected with gemcitabine and their combination,
AZD1480 alone failed to induce a significant reduction. Additionally, the authors did not
report any correlation between patients’ treatment and response. In this direction, Tiriac
et al. attempted to correlate tumor organoid drug response to patients’ clinical outcomes.
They developed a cohort of 66 PDAC patient-derived organoids and established a drug
testing platform, suggesting that drug testing in PDO cultures could be used for treatment
selection in patients within a clinically relevant timeframe. The organoid response to
drug testing, termed pharmacotyping, was performed on PDOs treated with 5 common
clinically used chemotherapeutic agents: gemcitabine, nab-paclitaxel, irinotecan (SN-38),
5-fluorouracil (5-FU) and oxaliplatin. The authors collected retrospective clinical follow-up
from 9 patients and reported that eight out of nine (89%) organoids responded similarly
to the corresponding patients. More specifically, 6 patients treated with at least one drug
to which PDOs were found to be sensitive showed an improved progression-free survival
(PFS), whereas two out of three patients treated with a drug to which the corresponding
PDOs were resistant rapidly progressed. Authors suggested that the PDOs’ response to
drug screening parallels patients’ sensitivity profiles to chemotherapy [51]. The authors fur-
ther evaluated the efficacy of other targeted agents as potential anticancer drugs for PDAC
using these PDOs. Targeted agents like selumetinib (MEK inhibitor), afatinib, everolimus,
and LY2874455 (fibroblast growth factor receptor inhibitor) were tested. Authors found
that PDOs harboring ERBB2 amplifications and EGFR mutations were sensitive to the
tyrosine kinase inhibitor afatinib, whereas a PDO carrying an oncogenic PIK3CA allele was
sensitive to the mTOR agent everolimus. A similar correlation between clinical response
and gemcitabine sensitivity was reported by Driehuis et al. [52]. The authors exposed
24 established pancreatic PDOs to an extensive drug screening with 76 chemotherapeutic
agents aiming to assess PDOs’ drug sensitivity and correlate it to the patients’ clinical
outcomes. However, clinical data were available only for 4 patients and all of them were
treated with gemcitabine. Authors reported that one patient, with the corresponding PDO
being resistant to gemcitabine, developed distant metastasis during treatment, while an-
other patient with a PDO sensitive to gemcitabine had stable disease and a decrease in
liver metastasis after gemcitabine treatment. The corresponding PDOs from the two other
patients showed an intermediate response in the in vitro assays, and in accordance with this
finding, the two patients showed stable disease during gemcitabine treatment. Based on
these findings, authors suggest that the in vitro sensitivity of the PDOs to gemcitabine cor-
relates to patients’ clinical response. However, it should be noted that the patients’ cohort
and the number of corresponding PDOs in this study were relatively small [52]. In the same
study, authors used PDOs to evaluate the potential of a new targeted agent, EZP01556,
as an anticancer agent. This inhibitor specifically targets the arginine methyltransferase
5 (PRMT5) protein. PRMT5 is reported as a synthetic lethal gene in methylthioadenosine
phosphorylase deficient (MTAP− ) cells [53].
In their study, Romero-Calvo et al. [22] reported the testing of organoids for dosage-
dependent and drug-specific responses. They studied the response of organoids from
two patients to gemcitabine and a combination of gemcitabine and abraxane. The authors
reported a dose-response relationship to gemcitabine in both cases; however, this did
not reflect the patients’ clinical outcome, as both patients were unresponsive to gemc-
itabine. Organoids from one patient were also tested with a combination of gemcitabine
plus abraxane and showed a very good response consistent with the corresponding PDX.
Nevertheless, this could not be correlated with clinical data since this patient did not
receive this regimen. However, the key finding of the study is the isolation of two clones
from the organoids, which underline the potential of these models to provide valuable
information about clonal populations. Another study from Seppälä et al. [54] aimed to
determine whether the PDOs’ pharmacotyping could guide the patients’ postoperative
chemotherapeutic selection within a clinically relevant time frame. Authors first studied
PDOs’ drug sensitivity in early-, as compared to late-passage organoids derived from
the same primary tumor. Interestingly, they found that the established clones showed
a similar pharmacotyping profile, which was consistent across over a dozen of samples
(7–89 days between passages). Since they confirmed the PDOs’ pharmacotyping stability,
authors reported the days in culture before pharmacotyping for 13 PDOs and found that
they managed to complete the drug screening within a range of 18–102 days (median 48
days). Considering the median time between surgery and the initiation of chemotherapy,
which was 62 days, authors suggest that rapid PDOs’ pharmacotyping could be a feasible
strategy with clinical relevance in PDAC treatment. However, once again in this study,
authors did not report any correlation between patients’ response and outcome [54]. In
another study, Farshadi et al. [55] developed 5 PDOs from pancreatic cancer patients who
has undergone eight cycles of the FOLFIRINOX regimen and 5 PDOs from treatment-naive
patients. The authors studied the PDOs’ drug response to oxaliplatin, 5-FU, and SN38 as
monotherapies. The results suggest that the clinical drug response for three out of five
treatment-naive patients, who received FOLFIRINOX treatment, mirrored the in vitro drug
sensitivity of their corresponding PDOs. Interestingly, one out of five FOLFIRINOX-treated
PDOs showed higher sensitivity to oxaliplatin in vitro compared to the others, and comput-
erized tomography of this patient’s tumor showed that this was the only patient with a
partial response (PR) to FOLFIRINOX. Congruently, one out of five treatment-naive PDOs
showed less sensitivity to different concentrations of FOLFIRINOX in vitro, mirroring the
observed clinical resistance of this patient to adjuvant FOLFIRINOX. However, these results
have been observed in a very small number of patients to draw definite conclusions and
must be validated in a larger cohort. In a more recent study, Grossman et al. [56] established
12 PDOs suitable for drug testing from 12 patients with pancreatic cancer enrolled in the
study. PDOs sensitivities to various drugs such as gemcitabine, 5-FU, oxaliplatin, SN-38,
and paclitaxel were tested. The drugs tested in each PDO were selected according to the
treatments each patient received or was likely to receive. Researchers calculated the area
under the curve (AUC) for each drug on each PDO as an index for the clinical response pre-
diction, and they observed a high degree of correlation between clinical and PDO responses.
For example, in one patient, the developed PDO showed the highest degree of sensitivity
to irinotecan, followed by trametinib, and in accordance with this finding, the patient
from whom this PDO was derived showed stable disease (SD) on the agent FOLFIRINOX
(contains irinotecan) and partial response (PR) on trametinib/lapatinib, suggesting that
the AUC values calculated from the PDOs could provide useful insights into the clinical
responses to treatment [56].
6. Discussion
In cancer research, human cell lines, mouse models, and patient-derived xenografts
(PDXs) have been traditionally used as the main experimental research platforms. However,
extrapolating results derived from these cancer models to patients has become a major
bottleneck, especially in the drug discovery process. Patient-derived organoids (PDOs)
have recently emerged as a powerful model for precision medicine applications. Currently,
there is an increasing number of organoids used in xenograft formation and molecular
profiling, and patient-derived organoid models are extensively used as in vitro screening
platforms with the potential to predict the best therapeutic options for individual patients.
Numerous studies have been performed in the field of PDO drug screening; however,
it remains unclear whether and how this approach could match the patients’ clinical
responses and, more importantly, whether these results can be translated into a clinical
decision strategy.
PDOs are establishing their place in the effort to bridge the gap between animal
models and human patients, as multiple studies are currently aiming to optimize their use,
focusing on their benefits and their potential applications. Several benefits of the PDOs over
previous models lie in their relative rapidness, robustness, high success rate, reproducibility,
and affordability. Animal models, which are still the most used preclinical models, require
much longer times to be developed and established compared to organoids, which usually
can be established within a few weeks. However, the time required for the organoids’
establishment might vary greatly. There are studies where PDOs were established from
4 up to 33 days [57] or within a range of 18–102 days [54] showing that the time-consuming
aspect of organoids’ development is a parameter that cannot be overlooked.
The success rate of organoids is an issue that needs to be further addressed. Al-
though the reported rate of organoid development is high (70–73%) [45], it might vary
depending on the tumor type or the amount of available material, which is usually limited.
Boj et al., [44] report that they failed to establish organoids from a patient who underwent
neo-adjuvant chemotherapy due to extensive necrosis in the sample, as revealed by histo-
logical examination. They also failed to establish organoids from a patient sample highly
enriched in stromal cells, and thus tumor cells were not enough for the formation of the
tumor organoid. Similarly, Huang et al. managed to isolate 17 organoids from 20 PDAC
patient samples. The three samples that didn’t lead to organoid formation were poorly to
moderately differentiated PDAC and intraductal papillary mucinous cystic neoplasms [45].
The methods used for tumor sample acquisition (i.e., biopsies vs surgical resections) can
also impact the efficacy of organoid generation. In the work of Driehuis et al., it is re-
ported that the generation of organoids from biopsies was limited to 31%. The authors
also concluded, through whole-genome sequencing (WGS) of the generated organoids,
that specific genes, such as SMAD4, and their mutation status can influence organoids’
growth in specific media. Therefore, they suggest testing samples to identify appropriate
organoid culture media, as specific growth factors may be required for the establishment
of patient derived organoids [52]. Indeed, although the culture of organoids is generally
straightforward, the issue of culture conditions is critical and requires proper consideration.
Various protocols from different groups exist, resulting in a variety of outcomes due to
the lack of a widely accepted standardized methodology (see Tables 1 and 2). This raises
concerns regarding the validity of the models and increases the difficulty of comparing the
results across different studies and research groups.
The maintenance of organoids remains an unresolved challenge within the scientific
community. In a recently published study by Mäkinen et al., authors report a 100% success
rate in organoid formation, yet only approximately 50% of the organoids could be cultured
beyond passage three. Storage of organoids towards developing a biobank for future use
doesn’t seem to be quite successful, as the success rate for re-establishing stored organoids
is relatively poor, not exceeding 50% [58]. These limitations pose significant obstacles to
the widespread use of organoids in precision medicine.
Another consideration regarding organoid models is the lack of inter-organ commu-
nication. Organoids are limited to replicating tissue-specific physiology and to overcome
this limitation researchers started to focus on the development of models able to connect
multiple organoids and study the communication between them. For example, in a recently
published study, researchers studied the communication between pancreas, liver, and
gastrointestinal organoid systems derived from human pluripotent stem cells (PSCs), and
they managed to develop a functionally and structurally integrated organoid system [59].
In that direction, it would be of great interest and usefulness the development of models
that will offer the ability to study multi-organ communication in vitro, aiming ultimately
towards personalized therapeutic approaches.
In pancreatic cancer research, organoids are currently studied as model systems for
the development of novel precision medicine strategies. Several studies aim to evaluate
patient-derived organoids as predictive platforms for the choice of chemotherapy regimens
and entirely novel drugs and combinations for the treatment of PDAC. Researchers aim to
develop pancreatic human organoids that could become a tool for rapid drug assessment
of the chemosensitivity of individual tumors (i.e., patients) before or in parallel with
implementing treatment in patients with PDAC. Currently, there are very few studies
that correlate pancreatic PDOs’ drug screening results to patients’ clinical responses, as
we summarized above. In these studies, researchers have shown that PDOs’ response to
drug testing was able to parallel patients’ clinical response [52,55,56,60]. However, these
studies have been performed on relatively small cohorts, highlighting the importance of
well-planned, more extensive co-clinical trials in larger patient cohorts.
Another important aspect to consider is that organoids are mainly constituted of
epithelial cells and lack numerous tumor microenvironment (TME) elements, including
different types of cells such as fibroblasts, endothelial, immune, and other stromal cells.
This is an important limitation of the organoid model system, and efforts have been made
to establish co-cultures of organoids with other cell types in order to generate an advanced
model able to recapitulate tumor heterogeneity, structure, and putative cell-to-cell interac-
tions. In cancer, it is well known that the tumor microenvironment including extracellular
matrix (ECM), stromal elements, vascular structures as well as infiltrating immune cells is
of critical importance for tumor progression and metastasis. Also, many drug responses on
human tumors are related and eventually strongly affected by the tumor microenvironment
elements. In that context, several variations to PDO cultures are under development, and
the tumor stroma and immune system are being incorporated in an increasing number of
studies. For example, in a recent study, Tsai et al. reported the co-culture of pancreatic
cancer organoids with patient derived CAFs and peripheral blood lymphocytes to assess
lymphocyte migration and the activation status of CAFs [61]. Organoid vascularization is
another aspect that should be considered. Organoids lack vascular circulation, and to over-
come this limitation, researchers have been co-culturing organoids with endothelial cells
in microfluidic devices [62]. Nevertheless, the maintenance of different cell populations
over time and the long-term preservation of stromal components and immune cells in the
organoid culture are still under investigation. The culture medium composition needs to be
optimized to support the diverse cell types while avoiding clone selection. The significance
of these specific issues is underscored by the work of Raghavan et al. in which authors
clearly show that the ex vivo TME and the media formulation may not only drive the state
of the pancreatic cancer cells grown as PDOs in a non-genetic manner, but also distinctly
affect the drug responses. In this work, researchers, by simply omitting or including TGF-β
in the culture medium, managed to shift the sensitivity of the tested organoids from agents
targeting DNA-damage repair pathways to inhibitors of the mitogen-activated protein
kinase (MAPK) pathway [63].
Given the detailed discussion above, it is evident that organoids are currently not
suitable to support clinical decision-making. The extended time required to establish
organoids, the moderate success rate of their development, the dependence on the method-
ology used to obtain the material, the challenges in culturing, maintaining, and storing
them for future use, the lack of inter-organ communication, and the lack of numerous
tumor microenvironment elements remain significant barriers that the scientific community
must overcome for organoids to find their place in the clinical setting.
Efforts are currently underway to overcome these limitations. In 2021, Osuna de la
Pena et al. reported the use of a non-Matrigel based ECM, much easier to be standardized.
This ECM was based on hydrogels developed by amphiphilic polypeptides that could be
self-assembled and supplemented with multiple PDAC ECM components like collagen
type I, fibronectin, laminin, hyaluronan, and components of the TME like patient-derived
pancreatic stellate cells (PSCs) and primary macrophages. The subsequent testing of PDOs
developed in this matrix revealed that the responses of the organoids against nab-paclitaxel
and gemcitabine were correlated with these of the corresponding patients. Although this
testing was limited to only three organoids, it is an essential step forward in addressing
issues related to organoid culture [64]. Recent advances in technology aiming to merge
the fields of organoid research and organ-on-a-chip research are also particularly excit-
ing. The aim is the development of an ‘organoid-on-a-chip’ technology by integrating
human organoids with organ-on-a-chip engineering. This could provide a superior in vitro
microfluidic platform, that will enable the separate culture of distinct organoid types, per-
mitting their communication, for preclinical drug screening [65]. However, these models
are expensive, still largely under development, constantly being optimized and improved,
and difficult to use for high-throughput drug screening experiments and obviously for the
purposes of precision medicine.
Despite the remaining challenges, human organoids represent a dynamically devel-
oping technology with enormous potential in the field of basic and clinical translational
research. Numerous studies underscore this potential. The investigation of mechanisms
that result in drug resistance and strategies for overcoming them is a field of immense use
of PDOs. Ponz-Sarvise et al., in an effort to study and overcome the resistance observed in
PDAC upon combined MAPK and PI3K inhibition, identified the overactivation of ERBB
as a critical parameter in this pathway. They further demonstrated that the combined MEK
and ERBB inhibition using the MEK inhibitor selumetinib and the pan-ERBB inhibitor nera-
tinib of human organoid orthotopic xenografts resulted in tumor regression in short-term
intervention studies [66]. Another example is the investigation and comprehension of the
underlying mechanism of resistance development of PDAC against FOLFIRINOX regimen,
as demonstrated in a recent study by Bachir et al., with the aid of PDOs [67]. Besides the
use of PDOs for drug studies, these models are already in use to uncover and validate novel
targets and signaling pathways involved in pancreatic cancer progression [51,52,56,68].
Organoids are extensively used to shed light on the mechanisms driving the metastatic
potential of PDAC. With the aid of PDOs, Huang et al., identified SMAD4 as the tran-
scription factor responsible for promoting EMT and orchestrating the invasion program in
PDAC [69].
Their use in the development of more clinically relevant animal models of cancer is
another field where organoids are gaining ground. PDO xenografts are reported to better
represent the cancer cell state of the parental tumor, and thus serve as more effective models
for drug testing. They can be easily genetically modified, allowing for broad applications
in basic cancer research and show a higher success rate as compared to PDX models at a
more reasonable time and cost (for a more comprehensive review on this see [70]).
Precision medicine is an approach to medical treatment that considers individual
variability in genes, environment, and lifestyle for each person to tailor the most effective
treatment and prevention strategies for that individual. PDOs are 3D cell culture systems
that can replicate the structure and function of specific organs and tumors to a significant
extent while retaining individual gene signatures, allowing researchers to study disease
development and drug responses in a more physiologically relevant environment.
Considering the rapid technological and methodological advances in the field, we
believe that human organoid systems have the potential to guide personalized therapeutic
approaches. Future advancements are anticipated to achieve a robust cancer model system
and a powerful tool not only for understanding cancer biology, biomarker research and
drug screening but also able to guide clinical decisions, improve cancer chemotherapies,
and become a cornerstone for a more precise oncology approach.
Author Contributions: Conceptualization, K.D. and E.S.; writing—original draft preparation, K.D.,
E.S. and I.P.; writing—review and editing, K.D., E.S. and I.P. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: Authors would like to thank V. Trachana (Department of Biology, Faculty of
Medicine, University of Thessaly) for critically editing the revised version of this manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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biomedicines

1 Department of Translational Medicine, Lund University, 22363 Lund, Sweden

Keywords: pancreatic organoids; pancreatic cancer; precision medicine

Biomedicines 2023, 11, 890. https://doi.org/10.3390/biomedicines11030890 https://www.mdpi.com/journal/biomedicines

Table 1. Human PDAC organoid culture methodology.

A 83-01 TGF-b inhibitor

Figure 1. Schematic representation of patient-derived organoids (PDO) development for precision

Figure 1. Schematic representation of patient-derived organoids (PDO) development for precision

4. Organoids and Tumoroids in the Battle against Pancreatic Cancer

5. Tumor Organoids in Precision Medicine for PDAC

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