Tumor neoantigens
We have mentioned cancer immunotherapy in the section on small molecular
drug screening. This part will overview the detailed mechanism of tumor
neoantigens and cancer immunity, then discuss the application of
organoids in identifying tumor antigenic peptides.
Tumor neoantigens (also known as tumor-specific antigens) are
specifically present on tumor cells but not on normal cells and can be
recognized by T lymphocytes 99. Neoantigens are
peptides with eight to ten amino acids produced by the degradation
proteasome of intracellular proteins which are the translational
products of mutant oncogenesis100. These antigenic
peptides are then associated with major histocompatibility complex (MHC)
class Ⅰ molecules (human leukocytes antigen (HLA)) in the endoplasmic
reticulum and migrate to the cell membrane. Activated
CD8+ T lymphocytes recognize and bind to cancer cells
and kill their target cells. This process is a cycle in which the tumor
cell death results in the release of more antigens and improves the
immune response to tumor cells. Notably, tumor neoantigens are captured
by dendritic cells (DCs), then present to T lymphocytes for activating
them. Therefore, the core of cancer immunotherapy is to activate the
killing function of activated T lymphocytes or promote the presentation
of neoantigens101.
Currently, a wide range of studies focuses on identifying tumor
neoantigens, which is critical for the development of new treatment
modalities for cancers, such as peptide-based tumor vaccines and
personalized drugs to kill tumor cells102. Mass
spectrometry (MS)-based immunopeptidomics and computational predictions
are the commonly used approaches to identify possible antigenic
peptides99. The latter is based on computer-generated
algorithms to identify peptides that could be produced by mutated genes
in tumors and are likely to associate with MHC
molecules103. The selected antigenic peptides are
synthesized and used to activate T lymphocytes. The activated cytotoxic
T lymphocyte (CTL, CD8+ T lymphocytes) are then
co-cultured with tumor cells to evaluate the CTL response for neoantigen
screens104, 105. Owing to the limited source of
patient-derived tumor tissues, studies have focused on using
patient-derived organoids to find out the mutated oncogenes and
investigate whether the activated CTLs can kill cancer cells. Newey et
al. expanded patient-derived colorectal cancer (CRC) organoids and
demonstrated the feasibility of MS-based immunopeptidomics of CRC
organoids in investigating neoantigen presentation in
vitro106. Wang et al. generated patient-derived
hepatobiliary tumor organoids and found that they preserve most of the
characteristics of their parental tissues, such as genetic features and
neoantigen landscape. They used organoids as preclinical models to
identify the predicted-peptide activated CTLs that exhibited anti-tumor
activity107. This study provides evidence for the
application of tumor organoids as preclinical models for rapid antigenic
peptide validation through a prediction-based approach. Few cancer
patients share the same neoantigens, and more than 99.95% of
neoantigens are present in only one patient resulting from tumor
heterogeneity108, 109. Patient-derived tumor organoids
could avoid these issues and achieve personalized immunotherapy in the
future. Since tumor heterogeneity is also characterized by different
cell subgroups within the same tumor tissue, Demmers et al. cultured
single cell-derived CRC organoid clones from the same patient and
demonstrated that the HLA class Ⅰ peptide presentation landscape was
heterogeneous even within one individual110. They also
indicated that highly conserved antigenic peptides in HLA presentation
could be identified using the single-cell derived clonal organoids,
which may be a suitable choice for designing anti-tumor vaccines.
The current studies that used organoids to perform tumor antigenic
peptide screens are 1ow-throughput. Patient-derived organoids enable
high-throughput screening of a large number of computationally-predicted
peptides. Furthermore, organoids can undergo extensive expansion,
allowing large quantities of material for MS-based immunopeptidomics
analysis (Figure 2). Therefore, we propose that the utility of
patient-derived organoids as preclinical models to high-throughput
identify tumor neoantigens could be a powerful approach to the
development of precision therapies for cancer patients.
Cell-penetrating
peptides
Cell-penetrating peptides (CPPs) can cross tissues and cell surfaces
without causing lethal injury to the membranes. The mechanism of the
penetration process remains controversial. Most CPPs are endogenously
produced proteins and peptides, including heparin-binding proteins,
DNA-binding proteins, antimicrobial peptides and viral
peptides111. Given the characteristics of CPPs to
cross cell membranes, more and more researchers focus on whether they
can pass the blood-brain barrier (BBB)112. The BBB is
a complex microvasculature system mainly consisting of brain endothelial
cells (ECs) that are tightly lined in the cerebral vascular lumens. The
main function of BBB is to protect the brain. In addition to ECs,
astrocytic glia and neurons together are organized into well-structured
neurovascular units113. The brain ECs express high
levels of tight junction proteins, efflux pumps, and specific
transporters. The tight junctions between ECs prevent molecules in the
blood from entering the central nervous system. The efflux pumps,
including P-glycoprotein (PgP), exclude foreign substances from the
brain. Specific transporters deliver essential nutrients to the brain,
such as glucose and amino acids. Therefore, BBB is the main obstacle to
delivering drugs to neural cells and developing effective treatments for
central nervous system diseases. Although CPPs are promising vectors to
deliver drugs across the BBB, no CPP-based treatment is currently used
clinical practice, partly due to the lack of suitable preclinical models
that can accurately mimic the features of BBB to discover potential CPPs
in a high throughput way114. The widely used in vitro
models for BBB are co-culturing of brain ECs (top), astrocytes and
pericytes (bottom) in a transwell system115-117.
Microfluidics could introduce blood flow to stimulate BBB more
dynamically118. However, these model systems require
advanced equipment to establish the platform, increasing the
experimental complexity of performing high-throughput drug screens.
Researchers recently reported three-dimensional multicellular structures
through self-organization arrangement of brain ECs, pericytes and
astrocytes119. The in vitro spheroids can recapitulate
the complex interactions between each cell type, which is critical cell
behavior to maintain the essential function of BBB. Lawler’s group
modified the method and established BBB organoids that could mimic the
essential function of BBB120. They, for the first
time, investigate whether the BBB organoids are suitable for screening
BBB-penetrating drugs. BBB organoids were used to identify several CPPs
that could cross the BBB, demonstrating their feasibility and utility as
models for cost-effective and high-throughput drug screens. Many
peptides are susceptible to being degraded by proteolytic, and show a
relatively low ability to cross the BBB. The same group found that
peptide macrocyclization could increase the cell uptake of CPPs and
found that one macrocyclic analog of transpotan-10 displays improved
capacity to deliver across the BBB organoids121.
Together these studies demonstrated that three-dimensional multicellular
BBB organoids can capitulate the complex interactions and arrangements
of each cell type, and can reduce experimental complexity (Figure 2).
These advantages make BBB organoids ideal preclinical models for
high-throughput screening of CPPs that can cross
BBB114. Furthermore, CPPs are also promising carriers
to transfer therapeutic drugs into tumor cells. Therefore, it is
necessary to establish tumor organoids that are amenable to the
discovery of efficient CPPs with high stability, internalization ability
and specificity.
Host defense peptides
The primary biological functions of the naturally-produced peptides,
host defense peptides (HDPs, also known as antimicrobial peptides), are
immunomodulatory, anti-inflammatory, and
anti-bacterial122. HDP are peptides with 12–50 amino
acids composed of cationic and hydrophobic amino acids that adopt an
amphipathic conformation upon folding, usually after contact with
membranes123, 124. Increasing antimicrobial resistance
(AMR) organisms has become a severe issue for the treatment of
inflammatory diseases due to the excessive use of
antibiotics125, 126. The naturally-produced HDPs are
promising candidates for developing treatments against the global threat
caused by AMR organisms126. Similarly, there is also
an urgent need to establish novel model systems to screen HDPs as
potential drug candidates. Currently, an organoid system called
air-liquid interface (ALI) construct are widely used in investigating
HDPs126. ALI systems are comprised of a porous filter
separating the apical and basolateral compartments. Cells cultured on
the top chambers grow to multilayers and across the basal-apical
threshold, where the medium remains on the bottom of the cell layers and
the apical interface is surrounded by air. This system is ideal for
studying the biology of tissues interacting with liquid and air in
vivo127. Patients and healthy lung ALI models were
used to examine the HDP expressions. The results demonstrated that they
are suitable models to study the essential function of HDPs in
respiratory diseases and enable the identification of drug
candidates128-130. One study using primary ALI models
reported that the frog skin-derived HDPs Esc (1-21) and its synthetic
derivation both could protect the epithelial integrity when infected by
P. aeruginosa131. Ritter et al. used ALI cultures with
an aerosol delivery system to predict acute local lung toxicity through
the assessment of various combinations of HDPs and
nanocarriers132. This study also indicated the
sensitivity of ALI models compared to submerged cultures.
Current studies introducing HDPs to organoid systems are all using ALI
models, which involve complex manipulations and equipment, making them
unsuitable for high-throughput screens. We believe that real
three-dimensional organoid models (infected with microbes) are a
potential approach to discovering effective HDPs and their derivations
in a high-throughput way.