Abstract
Prostate cancer is the most common cancer in over 50% of the countries
and the third most common malignancy worldwide. The tumor
microenvironment denotes the non-cancerous cells and components present
in the tumor, including the molecules they produce and release. Prostate
cancer proliferation, angiogenesis, metastasis and drug resistance are
closely associated with the tumor microenvironment. With the continuous
development of in vitro tumor models, they have gradually become an
important tool for recapitulating parental tumors in vivo and studying
the reciprocal interactions between tumors and their microenvironment.
In this review, we describe significant in vitro models of prostate
cancer, analyze the research results on incorporating the tumor
microenvironment into these models, and compare their advantages and
disadvantages. Furthermore, we highlight the future developmental
direction of prostate cancer in vitro model research according to the
hotspots of in vitro model research in other cancer types to facilitate
precision medicine in prostate cancer.
Keywords: Prostate cancer; Tumor microenvironment; In-vitro
model; organoid
1 Introduction
Prostate cancer (PCa) is the most frequently diagnosed cancer among men
in more than half of the countries worldwide. It is the leading cause of
cancer-related deaths among men in a quarter of the world’s countries
[1]. Tumor microenvironment (TME) denotes the non-cancerous cells
and components present in the tumor, including the molecules they
produce and release [2]. Interactions between the TME and tumor
cells play a central role in the process of tumor initiation,
progression, metastasis, and response to therapies. In vivo models for
PCa research, including knockout and transgenic mouse models,
patient-derived xenografts, and circulating tumor cell explants, have
several limitations, such as long tumor latencies and high expense
[3]. Furthermore, a small number of patient PCa specimens obtained
from clinics cannot support the establishment of large numbers of in
vivo models. By contrast, in vitro model has the advantages of infinite
growth, cost-effectiveness, and low demand for clinical specimens,
making it promising for PCa research. However, in vitro tumor models
primarily consist of a single cell type, the tumor cell, lacking
non-cancerous cell types and the extracellular matrix. These models fail
to recapitulate 3D organization and reciprocal interactions between the
tumor and its microenvironment, contributing to the gap between the
research results and real in vivo conditions. Therefore, modeling the
TME in vitro to accurately recapitulate patient tumors is crucial for
understanding PCa mechanisms and testing new therapeutic agents. In this
review, we provide an overview of tumor-mimicking in vitro models,
analyze their implications for PCa, and highlight directions for future
development.
2 Simple PCa models consisting of cancer cells
2.1 Two-dimensional (2D) cell lines
PCa research initially relied on 2D monoculture models, almost
exclusively dependent on immortalized cell lines [3]. These cell
lines constitute a spectrum of diseases with various types and stages,
partially reflecting the heterogeneity of malignancy [4]. At first,
LNCaP, DU-145, and PC-3 cell lines established from metastatic brain,
vertebral and lymph node foci were used for PCa investigation [5].
After that, nearly 200 cell lines and sublines derived from primary
tumors, metastases and xenografts have been used in PCa research
[6].
Moreover, immortalized cell lines can help predict drug responses
[7]. Tran et al tested several AR blockers and anti-androgens in
LNCAP-AR cells, where enzalutamide was found to be the most effective
drug [8].
The steady accessibility, simplicity, reproducibility has led to the
widespread use of PCa cell lines [3]. However, the immortalized cell
line is absent of ECM deposition and TME with high serum concentrations,
which make them distinctly different from in vivo tumor biology [3].
Furthermore, these PCa cell lines gradually lose the characteristics of
patient tumors because of their extended culture and repeated passages.
Cells at increasing passages exhibit progressive changes in genotypes
and phenotypes including alterations in growth rates, morphology,
protein expression, response to drugs and migration [9-11]. The
great limitations of the widely used 2D model gave rise to the
establishment of 3D models, making models closer to in vivo conditions.
2.2 3D tumor spheroids
Tumor spheroids are defined as spherical clusters of tumor cells, with
or without other cell types, in a 3D system [12]. Cell lines and
patient-specific cells are used to establish PCa spheroids in
scaffold-free or scaffold-based methods[13].
In comparison with homogeneity of the cell line, PCa spheroids show
intratumoral heterogeneity and are used for cell-origin detection. A
CD49b high, CD29 high and CD44 high cell population was found to be
self-renewing tumor-initiating cells with a high propensity for
invasion, migration and tumorigenicity by measuring the
prostasphere-forming capacity [14, 15].
Moreover, spheroids allow cell cultivation and cell-cell and cell-matrix
interactions in a spatially 3D manner [16]. These interactions lead
to changes in morphological and cellular characteristics compared to 2D
cell lines, reducing the difference between in vitro and in vivo
conditions [12]
PCa spheroids also establish oxygen and nutrient gradient inside the
model [17] and shows necrosis if the spheroid grows to a size
> 100 μm on average, which mimics in vivo conditions
[18].
As a 3D patient derived model, PCa spheroid is more reliable than cell
line in drug sensitivity test. Johannes et al. showed that PCa spheroids
established from radical prostatectomy specimens responded sensitively
to bicalutamide and enzalutamide but are resistant to abiraterone and
docetaxel [18].
However, because PCa spheroids are derived from clusters of tumor cells
and not stem cells, they lack the ability to self-organize and cannot
fully recapitulate intratumoral and intertumoral heterogeneity. As a
result, drug sensitivity results based on them may lack uniformity and
cannot precisely predict the therapeutic response in clinical settings.
Furthermore, simple PCa spheroids consisting of tumor cells fail to
mimic heterotypic solid tumors, excluding reciprocal interactions
between cancerous and non-cancerous cells. The limitation of the lack of
self-organization gives rise to the patient-derived organoid model, and
the limitation of the absence of non-cancerous cells gives rise to a
complicated co-culture model, which will be discussed in detail later.
2.3 Patient-derived organoids
Patient-derived organoids (PDOs) are self-organizing 3D models
established from diseased cells of organs with stem cell properties
[19]. The difference between tumor spheroids and organoids is that
spheroids are derived from a cluster of tumor cells that may include
cells with stem properties [20, 21]. In contrast, organoids process
multiple genetic sub-clones and tissue structures derived from single
epithelial or mesenchymal stem cell [22]. Tumor PDOs can be
established by single malignant cells, minced micro tissues, or inducing
normal tissue organoids.
Dong Gao et al. were the first to establish advanced PCa organoids in
long-term cultures derived from tissue biopsies or circulating tumor
cells. These PCa organoids harbor the genetic and epigenetic
characteristics of primary PCa and recapitulate the phenotypic diversity
of CRPC, making them amenable to drug testing [22].
Since then, PCa organoids have been widely used to study the cells of
origin, screen for genomic mutations, and conduct drug sensitivity
assays in PCa (Table 1). Long-term expansion of primary mouse and human
organoids revealed a luminal multi-lineage progenitor cell that gave
rise to the formation of prostate glands [23]. Furthermore, Park et
al. found that both luminal and basal progenitor cells initiate
tumorigenesis but contribute to different phenotypes in PCa organoids
[24]. Based on the inheritance of various genetic mutations
previously reported in distinct subtypes of prostate cancer, such as
PTEN, SPOP, TMPRSS2-ERG, LRP5, and CTNNB1 [22, 25], PCa organoid
lines further revealed the functions of genes, including ERG, c-Myc,
SPOP, and CHD-1, in PCa development[26]. Simultaneously, PCa
organoid cell lines are used to evaluate the efficacy of novel drugs
found in basic research or clinical trials. Dong Gao et al. found that
only one in seven CRPC organoid lines responded sensitively to
enzalutamide [22], consistent with cancer heterogeneity. Beshiri et
al. found that the response of mCRPC PCa organoids derived from
patient-derived xenografts (PDXs) to olaparib correlated with clinical
outcomes in patients [25], demonstrating the potential of PCa
organoids in precision medicine.
PDOs can accurately recapitulate the intra- and intertumoral biological
heterogeneity of tumors. Specifically, PDOs can preserve various cell
sub-clones with distinct genetic and phenotypic characteristics
intratumorally and recapitulate patient-specific characteristics
intertumorally [27]. Therefore, in basic and translational research,
PCa organoids have a unique advantage over cell lines and spheroids.
However, several limitations preclude their clinical applications. The
average success rate of PCa organoid establishment (< 20%)
still requires improvement [26]. Moreover, simple PCa organoids do
not include the TME, which differs from in vivo tumors. Co-culture
models and air-liquid-interface PDO have emerged to address the absence
of a TME in PCa organoids, which will be discussed in detail later.
3 Complex PCa models integrating tumor microenvironment components into
cancer cells
Cancers are intricate ecosystems composed of tumor and non-cancerous
cells embedded in a modified extracellular matrix [28]. TME cell
types include immune cells, endothelial cells, pericytes,
cancer-associated fibroblasts, and other tissue-resident cell types.
These host cells play central roles in cancer pathogenesis and are
considered novel targets for cancer therapy [28]. The in vitro
prostate cancer model mainly consists of cancer cells without TME
components differing from in vivo tumors. Consequently, several studies
have attempted to co-culture cancer cells, either cell lines or
patient-derived cells, with non-cancerous cells in the TME to discover
phenotypic and genomic alterations in cancer cells and examine their
potential for drug sensitivity assays. Based on different culturing
methods, we classified them into 2D co-culture, spheroid-based,
tissue-slice-culture-based, organoid-based, microfluidic
organ-on-a-chip-based models.
3.1 2D co-culture system
The 2D co-culture model refers to cancer cells without certain spatial
structures interacting with their surrounding microenvironments on the
same surface [29].
2D co-culture model is ideal for researchers to study direct and
indirect interactions between various types of cells. CAFs gave BPH a
more elongated and invasive phenotype than non-malignant prostate tissue
fibroblasts (NPFs) through direct contact [30, 31]. PCa cells
co-cultured with bone marrow stromal cells attenuated endoglin
expression and TGF-B signaling in the stromal and promoted proliferation
of cancer cells [32]. Teng et al. showed that HMC-1-SAMD14+
secretions inhibited pro-tumorigenic prostate epithelial morphology and
decreased the deposition and arrangement of the matrix generated by CAFs
in a co-culture model of primary prostatic CAFs and prostate epithelium
[33]. Additionally, the extracellular matrices surrounding tumors
have been modelled in a 2D plane. A monolayer ECM deposited from
patient-derived CAFs was established, to which BPH-1 cells were added.
This showed that the ECM architecture derived from CAFs was stiffer
where BPH-1 cells exhibited a more elongated and invasive phenotype
compared with NPF [30].
Due to the convenience and ease of 2D cell culture [34], many
studies have attempted to recapitulate TME interactions under 2D
conditions. However, a significant limitation of the 2D co-culture model
is the absence of a spatial structure that plays a central role in cell
behavior in vivo [35]. Consequently, the results obtained from the
2D co-culture models may differ from those obtained in vivo. The
phenotypic changes in cancer cells co-cultured with non-cancerous cells
in the 2D model are sometimes similar to those in the 3D model [30,
36]. Still, gene expression alterations in cancer cells in 2D models
are frequently not verified in clinical specimens. Drug screening for
cancer treatment utilizing cell lines has seldom led to the discovery of
a therapeutically effective agent [34].
3.2 3D co-culture system
3.2.1 Spheroids-based
Spheroids-based co-culture model is a cluster of different types of
cells with a spatial structure, either spherical or tissue-shaped.
The model has been further used to study the interaction between cancer
cells and non-cancerous in addition to the past use of 2D co-culture
models. Compared with 2D models, it is a valuable tool for drug
sensitivity study. Eder et al. co-cultured PCa cell lines (LNCAP, DuCaP,
and LAPC4) with CAFs in 3D scaffold-free hanging drops [37]. They
found that the addition of CAFs affected cancer cells’ sensitivity to
anti-androgens. and spheroids’ anti-androgen resistance could be
reversed by PI3K inhibitors [37].
Moreover, resistance mechanism can be explored. Neuwirt et al. found
that CAFs contributed to upregulating the HMGCS2 gene, AKR1C3 gene, and
the biosynthesis pathway of steroids and cholesterol, which enabled
cancer cells to escape androgen deprivation. Simvastatin, which targets
cholesterol and steroid biosynthesis with an AKR1C3 inhibitor, could
potentially reverse it by conducting gene expression analysis of
spheroids[38]. Kato, M et al. found that ADT led to an increase in
the CD105+ fibroblastic subpopulation and downstream SFRP1 in a
spheroid-based co-culture model, which induced neuroendocrine
differentiation of prostate cancer cells in a paracrine manner [39].
Spheroid models have also been used to mimic the metastatic environment
in PCa combined with bioengineered materials. Paindelli et al. further
modified a tissue-engineered bone model by seeding human mesenchymal
stem cells (hMECs) on a calcium phosphate polycaprolactone scaffold
(mPCL-CaP). They found that tumors became resistant to the
chemotherapeutic drug docetaxel in a bone stroma-dependent way, and
Radium-223 targeting the bone stroma could induce cytotoxicity [40].
Similarly, PCa cells in bone-mimetic environment models derived from
culturing osteoprogenitor cells on polymer scaffolds displayed molecular
and functional features consistent with in vivo bone metastatic
condition [41, 42].Anti-androgen drug sensitivity assays were
further conducted in this type of model, where enzalutamide contributed
to stronger adaptive responses of cancer cells, osteomimicry, and a
better treatment response than bicalutamide, correlating with
enzalutamide delaying the onset of bone-related events and prolonging
survival in mCRPC[42]. Bioengineering models integrating prostate
cancer spheroids into bone-mimetic environments have demonstrated their
potential in precision medicine for late-stage prostate cancer where
bone metastasis occurs.
Owing to their accessibility and cost-effectiveness, spheroid-based
co-culture models have been widely used to study the interactions
between tumor and non-cancerous cells and model the pre-metastatic niche
in bone. Although it has a 3D structure, the spheroid lacks
self-organization and intertumoral heterogeneity. Thus, it is not the
most ideal model for mimicking patient tumors in vitro.
3.2.2 Organoids-based
PDOs are a revolutionary model for heterogeneous recapitulation and
personalized medicine which preserve the features of the parent tumor at
the morphological, genetic, proteomic, and pharmaceutical levels
[27]. Recent studies have emphasized the development of organoid
culture methods that can accurately recapitulate TME cell heterogeneity
and model heterotypic cell interaction. Modeling ECM in organoids widely
used naturally derived hydrogels, such as the EHS matrix, which are
poorly tunable and ill-defined [43]. A recent study integrated
microscopy, spatial omics, proteomics, and transcriptomics on patient
biopsies to define the ECM tumor environment in castration-resistant
prostate cancer adenocarcinoma (CRPC-Adeno) as neuroendocrine prostate
cancer (CRPC-NEPC) [44]. Based on these findings, synthetic
hydrogels were developed to grow CRPC-NEPC organoids, where the
tumor-specific ECM differentially regulated the mobilized genes,
epigenetic methylation, and therapeutic response to drugs of the
organoids. Using synthetic hydrogel-grown organoids, they discovered a
putative therapeutic drug, the DRD2 inhibitor ONC-201, to treat
CRPC-NEPC. They clarified how ECM-integrin interactions could make these
tumors vulnerable to DRD2 and EZH2 antagonist activity [44].
Integrating other cell-based factors in the TME, such as CAFs and immune
cells, into organoids has also been performed. The direct co-culture of
organoids with other cell populations enables studying cell interactions
and drug targeting within the TME. Human prostate cancer organoids
co-cultured with cancer-associated fibroblasts purified from the
patient-derived xenograft model, CWR22Pc, showed increased resistance to
androgen deprivation therapy [45]. Furthermore, NRG1 secreted by
CAFs improves ADT resistance [45]. On the other hand, androgen
induced the migration of CAFs to cancer cells and, finally, an enlarged
cancer organoid size in the co-culture mode. While co-culturing TME
components with patient-derived organoids has been widely used in
various cancer types, such as esophageal, colorectal, and pancreatic
cancer [46-48], attempts to develop a co-culture model for prostate
cancer are limited. This limitation is partially due to the relatively
low success rate of prostate cancer organoid establishment itself, which
remains at 16% [49]. Moreover, simultaneously supporting the growth
viability of cancer organoids and specific TME components is in high
demand for ECMs and culture media. For example, to maintain the
viability of CD8+ T-cells in clear renal cell carcinoma organoids, a
series of chemokines, including IL-2, must be added to the ECM and
culture medium at specific concentrations [50].
Despite organoids from purely patient-derived epithelial cell
populations, the air-liquid-interface patient-derived organoid (ALI-PDO)
cultures both epithelial and non-epithelial cell populations by
embedding millimeter-scale tumor fragments in a 3D matrix [51]. The
ALI-PDO model was established for various types of cancers to study the
preservation of the TME and its potential for personalized medicine. Li
et al. cultured both epithelial and mesenchymal cell populations in the
organoids from mouse colonic, gastric, and pancreatic malignancies
[52]. The organoids exhibited extremely detailed histologic
endpoints for transformation and dysplasia after oncogenic
reprogramming, which were not previously observed in epithelial-only
organotypic modeling in the past [52]. Neal et al. successfully
co-cultured tumor epithelia and stromal components with endogenous
syngeneic immune cells in an organoid model from more than 100 human or
mouse tumor biopsies, including human pancreatic, colon, lung, and
ampullary adenocarcinomas and so on [53]. Furthermore, they
demonstrated that organoids accurately recapitulated the T cell
repertoire and modeled responses to immune checkpoint inhibitors in
patient tumors [53]. The ALI-PDO platform has also been developed to
predict the response of tumors to immunotherapy in other studies [47,
50, 54], promoting precision medicine in cancers. However, no
organoids utilize air-liquid interface methodology in PCa to allow for
the co-culture of epithelial and non-epithelial cells. We look forward
to breakthroughs in establishing air-liquid-interface PCa organoids,
given the unique advantages of the methodology in TME heterogeneity
recapitulation.
3.2.3 Microfluidic organ-on-a-chip system
Microfluidic-based systems enable the introduction of physiological
factors, such as mechanical stress, flow, pressure, and tissue-to-tissue
interfaces, into cancer models in vitro [55]. These systems employ
microchips, often engineered with various lateral channels and chambers
with fluidic flow [56]. Microfluidic systems have demonstrated
unique advantages for PCa modeling in vitro. Hsiao et al. utilized a
microfluidic system to culture tumor spheroids composed of PC-3 cells,
endothelial cells, and osteoblasts to mimic the bone metastatic
environment, which led to uniform cell distribution and easy cell
tracking during the culture course [57]. Padmyastuti et al. found
that microRNAs promoting PCa progression were mostly upregulated,
whereas PSA secretion remained constant in spheroids derived from LNCAP
cells when cultured in a microfluidic system, which showed that the
micro-physiological system could induce significant phenotypic changes
in PCa models [58]. Furthermore, the microfluidic device aided
precision medicine in PCa by simultaneously delivering drugs with
reproducible concentration gradients to patient derived PCa spheroids of
multiple sizes [59]. PCa spheroids cultivated in a Microwell Flow
Device (MFD) demonstrated decreased necrotic cores, downregulated cell
stress genes, enhanced proliferation, improved cellular structural
integrity, and better recapitulation of chemotherapy responses, which
prompted feasible research into hypoxia modulation and cancer metabolism
under pathophysiological conditions [60]. Hence, more research into
PCa TME remodeling using microfluidic devices considering physiological
factors is warranted.
Microfluidic systems give rise to an organ-on-a-chip (OoC) model,
designed to model the functional units of single or multiple human
organs [61]. The OoC comprises simplified essential elements
necessary for the physiological functions of one or multiple organs in a
microfabricated device [61]. Owing to the multiple biomechanical
stimuli and complex connectivity between the elements inside the device,
OoCs are often regarded as ideal in vitro models for recapitulating
self-regulating biochemical and biophysical networks in vivo[62].
Several organs, including the heart, lungs, kidneys, and liver, have
been established in OoCs [55]. The notable features, such as gut
microbiome enzymatic activity in the intestine, secretion of anionic
drugs in the kidney, and biliary duct formation in the liver, have been
recapitulated [58]. Although an OoC model composed of human
osteogenically differentiated MSCs, PCa spheroids, and collagen matrices
for further research and drug testing has been established [63],
more attempts to establish OoC models to study invasion, progression,
metastasis, and interactions with the TME and other organs in PCa are
needed.
Recently, a strategy termed organoid-on-a-chip combines organ-on-a-chip
with organoids. These two complementary approaches mimic the complexity
of human tissues in vitro [61]. Organoid-on-a-chip allows the
presence of other biochemical and biophysical elements in the TME, such
as cell-secreted soluble factors, mechanical flow, and blood vessels,
with precise control, to study organoids changes in genes, phenotypes,
and drug sensitivity [61]. In an intestinal organoid-on-a-chip
system, opposing gradients of BMP and WNT signaling were used to culture
self-renewing epithelial cells, finally leading to the
compartmentalization of non-proliferative and proliferative cells
[64]. The vascularization and perfusion of tumor organoids were
observed under physiological flow conditions in a breast cancer
organoid-on-a-chip [65]. Fluid shear stress promotes the maturation
and vascularization of kidney organoids, as validated by the high
expression of vascular markers and increased vascular density in kidney
organoids-on-a-chip [66]. Drug discovery is among the most promising
applications of the organoids-on-a-chip technology. The correlation
between organoids and actual organs makes them more effective for
therapeutic target identification, while organs-on-a-chip are more
controllable and reproducible engineered devices, better suitable for
efficient screening [61]. Currently, there are no PCa
organoid-on-a-chip of great interest for future research.
3.2.4 Tissue slice culture
To maintain the architecture and heterogeneity of the TME, a tissue
slice culture (TSC) was developed by precisely cutting the slices to a
certain thickness in vitro [67]. Precision-cut slices were first
developed for pharmacological metabolism studies in the liver and kidney
and were extended to other organs, including the prostate [68].
Initially, several issues, including secretory cell degeneration, basal
cell hyperplasia, and poor in vitro survival, were observed in prostate
TSC culture [69]. Maund et al. demonstrated a PCa TSC model
recapitulating patient tumors’ genetic, cellular, and structural
characteristics during a 5-day culture period by optimizing the culture
condition [70]. The survival of monocytes, macrophages, and
endothelial cells was shown in the model for the first time, and ADT
drug sensitivity assays were conducted [70]. Centenera et al.
evaluated a novel HSP90 inhibitor in organotypic PCa TSC as well as 2D
cell lines and found that tissue cultures offered insights into the drug
response not previously seen in animal models or cell lines before
[71]. Zhang et al. tested enzalutamide and olaparib, a PRAP-1
inhibitor, in a PCa TSC model whose viability was maintained for 6 days
[72]. Besides drug testing, TSC culture has a unique advantage in
spatial transcriptomic methods because co-detection by indexing (CODEX)
imaging enables the examination of more than 60 markers in one TSC model
[67].
TSC is patient-specific, maintains the local microenvironment, and holds
promise for basic translational research. A major limitation of TSC
culture is that its structure and cell viability cannot be maintained
for more than 1 week [70, 72], which hampers reproducible and
high-throughput drug screening. When treated with samples with mixed
pathology, commonly observed in clinics, one specific subtype of cancer
cells often outgrows and finally homogenizes the entire tissue in the
TSC culture [67]. Additionally, unlike spheroids and organoids,
studying the interactions between cancerous and non-cancerous cells in
the TME using a TSC culture model is difficult.
The advantages, limitations, and applications of the different in vitro
PCa models are summarized in Table 2.
4 Discussion
With the rapid development of in vitro cancer models from cell lines and
spheroids to organoids, models that integrate the TME into in vitro
tumor cells to recapitulate parental tumors have advanced significantly
from 2D, spheroid-based, organoid-based co-culture to
air-liquid-interface organoids, and finally to microfluidic
organoid-on-a-chip systems (Figure 1). These models are becoming
increasingly intricate, incorporating an increasing number of TME
components, including the ECM, non-cancerous cellular components, and
physiological factors, into cancer cells.
However, challenges associated with PCa organoid cultures have impeded
the development of in vitro models. No ALI-PDO and organoids-on-a-chip
model has been reported in PCa research, underscoring the need for more
research in this area. Although the use of drug screening in advanced in
vitro models has been a heat map in recent years, the correlation
between the results of drug sensitivity assays and clinical outcomes is
seldom verified in clinical settings. Possible reasons for this include
unapproved medication combinations based on drug screening results used
in real patients and long follow-up times required to observe clinical
outcomes. Consequently, there remains a gap between the design of
complicated in vitro models and their implementation in precision
medicine.
In conclusion, research on establishing more efficient and complex
organoids should be conducted in PCa to produce in vitro models that
recapitulate patient tumors regarding TME components for basic and
translational research.