Cellular Oncology

https://doi.org/10.1007/s13402-024-00960-8

REVIEW

A systematic review on the culture methods and applications of 3D

tumoroids for cancer research and personalized medicine
Jessica Kalla1 · Janette Pfneissl1 · Theresia Mair1 · Loan Tran1,2 · Gerda Egger1,2,3

Accepted: 11 May 2024

© The Author(s) 2024

Abstract
Cancer is a highly heterogeneous disease, and thus treatment responses vary greatly between patients. To improve therapy
efficacy and outcome for cancer patients, more representative and patient-specific preclinical models are needed. Organ-
oids and tumoroids are 3D cell culture models that typically retain the genetic and epigenetic characteristics, as well as
the morphology, of their tissue of origin. Thus, they can be used to understand the underlying mechanisms of cancer
initiation, progression, and metastasis in a more physiological setting. Additionally, co-culture methods of tumoroids and
cancer-associated cells can help to understand the interplay between a tumor and its tumor microenvironment. In recent
years, tumoroids have already helped to refine treatments and to identify new targets for cancer therapy. Advanced cultur-
ing systems such as chip-based fluidic devices and bioprinting methods in combination with tumoroids have been used
for high-throughput applications for personalized medicine. Even though organoid and tumoroid models are complex in
vitro systems, validation of results in vivo is still the common practice. Here, we describe how both animal- and human-
derived tumoroids have helped to identify novel vulnerabilities for cancer treatment in recent years, and how they are
currently used for precision medicine.

Keywords Cancer · 3D models · Preclinical models · Organoids · Tumoroids · Precision medicine · Co-culture ·
Bioprinting · Fluidic devices

Abbreviations EMT Epithelial to mesenchymal transition

ALI Air-liquid interface GEMM Genetically engineered mouse model
BlCa Bladder cancer HNSCC Head and neck squamous cell carcinoma
BrCa Breast cancer iPSC Induced pluripotent stem cell
CAF Cancer-associated fibroblast NF Normal fibroblast
CRC Colorectal cancer NK Natural killer
CTC Circulating tumor cell PaCa Pancreatic cancer
CTG Cell Titer Glo PCa Prostate cancer
ECM Extracellular matrix PDT Patient-derived tumoroid
SCLC Small cell lung cancer
TAM Tumor-associated macrophage
Jessica Kalla and Janette Pfneissl have contributed equally to this TME Tumor microenvironment
work. TOBisMC Thiol-reactive Organoid Barcoding in situ
Mass Cytometry
Gerda Egger
[email protected]
1
Department of Pathology, Medical University of Vienna,
Vienna, Austria
2
Ludwig Boltzmann Institute Applied Diagnostics, Vienna,
Austria
3
Comprehensive Cancer Center, Medical University of
Vienna, Vienna, Austria

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 J. Kalla et al.

1 Background 1.2.1 Establishment and culture

1.1 Model systems for research Although a lot of research has been done on 3D models
derived from stem cells in the past [15], the group of Hans
One of the biggest challenges in science is the representative Clevers pioneered the successful establishment of organoids
modeling of multicellular tissues, dynamic interactions, and in the modern era [14]. These organoids were generated in
the complex mechanisms underlying disease. Therefore, 2009 from murine intestinal stem cells, and since then many
scientists mostly rely on different model systems ranging different research groups have adapted the establishment
from cell lines to animal models, to representative models protocol to generate an extensive collection of organoid
derived from primary tissue [1]. models from both animals and humans [16].
2D cell lines have been established from a diverse range Typically, primary surgical resection material is cut into
of malignant diseases but also from a selection of cell types small fragments using scalpels, followed by an enzymatic
in the healthy human body and are a widely used model digestion to dissociate the tissue into single cells or small
system. Because of easy handling for in vitro cell culture cell clumps [17, 18]. However, as access to tissue samples
and simple introduction of genetic modifications, they can can be anatomically and logistically limited, stem cells
deliver meaningful insights on specific research questions derived from fine needle aspirates of tissues [19–21], fluid
[2]. However, these cell lines consist of only one cell type samples such as urine [22], ascites [23], broncho-alveolar
optimized for long-term culture. Additionally, as the culti- lavage fluid [24], and bile [25], or enriched circulating
vation in a monolayer can alter the shape, polarization, and tumor cells (CTCs) [26] might provide a minimally invasive
global signalling of cells, 2D cell lines do not adequately alternative for organoid generation.
reflect the physiological situation in organs [3, 4]. There- Organoids are mostly cultivated in 3D ECM hydrogel
fore, research relying solely on cell lines does not success- domes such as basement membrane extract, Matrigel® [27],
fully translate from bench to bedside [5, 6]. or Geltrex®, in addition to a variety of xeno-free synthetic
Animal models, which better reflect the cellular complex- hydrogels [28]. Organoid culture medium provides a vari-
ity and molecular crosstalk of organs in the human body, are ety of niche-specific growth factors matched to the tissue
frequently used to study cells in their physiological context. of origin to ensure optimal growth conditions [17, 29, 30].
However, animals and humans are still inherently different Common key components include activators of the canoni-
so that not all biological processes can be modeled accu- cal WNT pathway (R-Spondin), MAPK pathway (EGF),
rately. Moreover, animal models are more complicated and and inhibitors of TGF-β (A83-01) as well as BMP signaling
time consuming to work with than in vitro cell lines, in addi- (Noggin) to allow stem cell renewal and proliferation [11,
tion to the ethical issues connected to the use of animals for 14, 17].
research [7]. Stable organoid lines can be expanded long-term (> 10
passages) in vitro, and cryopreserved [31]. Of note, even
1.2 Organoids though conventional submerged organoid cultures exclu-
sively enrich for epithelial cells and fail to retain stromal
Organoids are one model system that combines several components [32], organoids do recapitulate the tissue archi-
advantages of in vitro 2D cell culture and in vivo animal tecture on a microscale, and can mimic the function of the
models [8]. Even though commonly used 3D spheroids are a organ they resemble, while stably maintaining mutational
promising tool for cancer research, they are cell line-derived and epigenetic states in line with cellular signaling [33–35].
aggregates that do not self-organize or differentiate into
multiple cell types [9]. In contrast, organoids are 3D self- 1.2.2 Organoids for cancer research: tumoroids
organizing cell clusters that can be derived from embryonic
stem cells, tissue-specific adult stem cells, or from induced Organoids are used in many different research areas, includ-
pluripotent stem cells (iPSCs) from both animals and ing reconstructive medicine, evolutionary biology, and
humans. These stem cells, which are commonly embedded pathogen-host interactions, summarized in previously pub-
in an extracellular matrix (ECM), can then further differen- lished reviews [36–41]. In recent years, tumoroids derived
tiate into different types of epithelial cells specific for the from both animals and humans have been intensively used
tissue of interest [10–14]. In addition to stem cell-derived for preclinical cancer modeling, with a focus on cancers
organoids, tumor-derived organoids, so called tumoroids, originating from epithelial cells (Fig. 1). However, efforts
have been successfully generated for various cancer entities have been made to also establish tumoroids from non-epi-
and provide valuable tools for cancer research. thelial cancers and rare cancer types, for example melanoma

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A systematic review on the culture methods and applications of 3D tumoroids for cancer research and…

Fig. 1 3D model systems in cancer research. Applications of both Comparison of different 3D model systems, including organoids, co-
human- and animal-derived tumoroids in cancer research (top). culture models, microfluidic devices, and in vivo models, highlighting
Tumoroids can be used for either cancer modeling with a focus on the the advantages and disadvantages of each model (bottom). Created
tumor microenvironment (TME), or as a tool for personalized medi- with BioRender.com
cine and drug discovery based on the mutational landscape of patients.

[42], glioblastoma [43, 44], rhabdomyosarcoma [45], epi- organoids and tumoroids could be used for both answering
thelioid sarcoma [46], and rhabdoid cancer [47]. basic questions of tumorigenesis and developing a personal-
Disease modeling using tumoroids allows for the inves- ized medicine approach to help advance the field of cancer
tigation of different hurdles of oncology such as tumor ini- research.
tiation and driver events of cancer, tumor heterogeneity or Given recent advancements in organoid technologies, we
development of efficient therapies [10, 48–50]. By estab- here summarize the latest developments in the field, out-
lishing healthy organoids and altering cancer type specific lining various methods and practical applications of these
genes in vitro, the transformation from healthy to cancerous innovative model systems. We will focus on current uses for
cells can be studied in a 3D model focusing on different cancer research including both human- and animal-based
tissues and diseases. Additionally, by generating organoids methods. Furthermore, we will highlight recent work that
and tumoroids directly from both healthy and tumor tissue, has improved preclinical cancer models by using tumoroids
cancer driver mutations can be identified and used to dis- in combination with other cell types to model the tumor
cover patient-specific therapeutic targets [33, 51]. This way, microenvironment (TME). Moreover, studies focusing on

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 J. Kalla et al.

advanced 3D culture systems based on fluidic devices and models have been established and used across all fields of
bioprinting, and grafting tumoroid models into animals for cancer research (Table 1). Importantly, the availability of
in vivo based studies, will be summarized. Finally, existing normal tissue samples and thus healthy organoid lines from
limitations are discussed, and future developments of the animal models allows for the evaluation of adverse side
field are highlighted. effects of cancer therapies on healthy tissue [52].
In the past, cancer research mainly relied on genetically
modified mouse models (GEMMs) targeting genes com-
2 In vitro culture methods and applications monly mutated in human cancers [93]. Thus, a variety of
for cancer research organoids and tumoroids from these mouse models exist,
which can further be easily genetically modified in vitro.
2.1 Conventional ECM-drop culture Two recent studies focused on the development of preclini-
cal 3D models for ovarian cancer based on murine cancer
2.1.1 Animal-derived tumoroids in cancer research and models [94, 95]. The knock-out of ovarian cancer-specific
their application genes was induced using either a Cre recombinase-based
system or CRISPR/Cas9 in organoids derived from healthy
As tissue samples from animals often are easier accessible murine tissue in vitro, leading to their malignant transfor-
than most human biopsies, and as a big variety of murine mation. The group of Zhang et al. hereby focused on the
cancer models exist, animal-derived tumoroids can be espe- influence of the mutational heterogeneity on treatment
cially useful when human-derived tumoroids are not avail- response, and showed that both tumoroids and mice with
able. Therefore, many murine and other animal-derived 3D the same mutational background can be treated success-
fully with identical combination therapies [94]. The group
Table 1 Summary of animal-derived tumoroid models, including of Löhmussaar et al. established a tumor progression model
murine, canine, and porcine 3D models for the most commonly diag- for high-grade serous ovarian carcinoma from two different
nosed cancer types
epithelial lineages of mouse ovaries. Their findings support
Species Cancer Type Year References
Mouse Breast Cancer 2023 [53, 54]
the dual-origin hypothesis of these tumors and confirmed
2022 [55] lineage-dependent differences in drug sensitivities [95].
2020 [56] Thus, these studies highlight how murine tumoroids can be
2016 [57] used as a model for identifying novel genotype-informed
Lung Cancer 2021 [58] treatments, and for investigating the differences and vulner-
2020 [59, 60]
2018 [61] abilities of tumors arising from distinct cell types, leading to
2017 [62] more patient-specific and efficient therapy options.
Colorectal Cancer 2022 [63–65] Another approach using tumoroids derived from a pros-
2020 [66, 67] tate cancer (PCa)-specific mouse model was reported by
Prostate Cancer 2022 [68] Chan et al., who investigated genetic driver mutations of
2019 [69]
2018 [70]
treatment-induced transformation and plasticity [68]. The
2016 [71] group induced malignant transformation of healthy organ-
2015 [72] oids by knocking out the PCa-specific genes Pten, Rb1, and
2014 [73, 74] Tp53 in vitro and showed that over time the resulting tumor-
Gastric Cancer 2022 [75] oids acquired an intermediate luminal-basal phenotype. An
2021 [76]
2019 [34, 77] epithelial to mesenchymal (EMT) signature was further sup-
2014 [78] porting tumoroid plasticity, a phenomenon that has recently
Liver Cancer 2021 [79] also been investigated in pancreatic cancer (PaCa) organ-
2020 [80] oids [96]. Of note, the authors reported that the PCa tumor-
2019 [81, 82]
oid plasticity was further induced by androgen ablation (the
Dog Breast Cancer 2022 [83]
2017 [84] suppression or blockage of the androgen pathway using
Lung Cancer 2023 [85] chemical compounds), the most common treatment for PCa,
Colorectal Cancer 2019 [86] and identified JAK/STAT and FGFR signaling as the main
Prostate Cancer 2017 [87] drivers [68]. Since the inhibition of these two pathways con-
Bladder Cancer 2020 [88] verted the tumoroids back to an androgen ablation-sensitive
2019 [89] more luminal phenotype, PCa patients may benefit from the
Thyroid Cancer 2023 [90] same treatment. The researchers also tried to confirm their
2021 [91]
results in human PCa tumoroids. However, it is difficult to
Pig Intestinal Cancer 2017 [92]

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A systematic review on the culture methods and applications of 3D tumoroids for cancer research and…

maintain human prostate organoids and tumoroids in long- 3D models were then used to show that trametinib, a drug
term cultures, and the results did not overlap [68, 97, 98]. mainly used for melanoma treatment [103], and extracts
More studies are needed to verify these findings, but PCa is from the Chaga mushroom [104], could be a potential
a good example for how animal-derived 3D models could therapy option for patients with BlCa. Recently, the same
be used as an alternative model for human cancer. researchers also established canine healthy lung and lung
To investigate metastasis formation in addition to tumor cancer 3D models that resemble human disease as a new
heterogeneity, two of the main factors for poor overall sur- model for molecular analysis and drug testing of lung can-
vival of breast cancer (BrCa) patients, tumoroids from the cer [85]. It can be expected that additional organoid systems
C3(1)-TAg BrCa mouse model were used [54]. Individual will be established from non-conventional animals in the
tumoroid lines established from the same primary tumor near future, some of which might have high translational
showed heterogeneous invasive mechanisms that were clas- impact for human cancer [105, 106].
sified into collective invasion or dissemination of single
cells. Interestingly, KRAS expression was required for 2.1.2 Human-derived tumoroids in cancer research and
both mechanisms, and ERK inhibition blocked both inva- their application
sive processes [54]. Additionally, it was shown that col-
lective migration of BrCa cells is mediated by a keratin 14 Patient-derived tumoroids (PDTs) maintain the characteris-
and cadherin 3-positive subpopulation of leader cells [53]. tics of the primary tumor and can thus be used as a model
These cells have an enhanced protrusive activity and inter- to identify the most efficient cancer treatment for individual
act with the TME to initiate invasion, and could become patients, laying the foundation for personalized medicine
a novel target for preventing or treating BrCa progression. [107]. Therefore, tremendous efforts have been made to
However, murine 3D cell models can not only be used to generate organoids and tumoroids from diverse tissues and
test the effectiveness of drugs, but also of other anti-cancer tumor types. Until now, PDTs from more than 20 different
treatments like radiation. After irradiating murine intestinal carcinoma types, including BrCa, lung cancer, and colorec-
organoid models, Du et al. demonstrated that the induction tal cancer (CRC) have been established (Table 2).
of inflammation with Zymosan-A promoted the regenera- Due to their advantages over 2D cell culture, PDTs are
tion of intestinal stem cells by up-regulation of ASCL2 [52]. widely used for drug screening and therapy response pre-
Thus, Zymosan-A may be an effective radio-protective drug diction of patients [17]. Additionally, as it was shown that
for the prevention of harmful side effects on surrounding both animal- and human-derived tumoroids do not acquire
healthy tissue and treatment of ionizing-radiation-induced additional mutations during long-term culture in vitro [148],
intestinal injury. numerous studies have already proven the usefulness of
In general, the results obtained using murine organoid such 3D systems for precision medicine including CRC
systems that mimic human disease adequately reflect human [113, 149–151], PaCa [152–154], as well as rare cancer
physiology and mechanisms [52–54, 68, 94, 95]. However, types such as Merkel cell carcinoma [147]. In CRC PDTs,
rodents and mammals are inherently different, which can the response of patients to oxaliplatin was predicted with
lead to unexpected discrepancies during translation to the a sensitivity of 70% and a specificity of 71% [155]. Addi-
clinic [99, 100]. Recently, the use of organoids and tumor- tionally, transcriptome analysis revealed that differences
oids derived from farm or companion animals has become in oxaliplatin response were mediated by distinct genetic
more popular in the field of cancer research, as animals like features, and 18 specific genetic alterations were identified
cattle, horses, pigs, monkeys, dogs, and cats have a more as a potential biomarker panel for oxaliplatin resistance
similar anatomy to humans [101]. One big difference to in CRC patients to aid clinical decision making. Another
rodents is that these animals can develop spontaneous can- study focusing on the resistance of PaCa patients to neo-
cer lesions, which makes them more suitable to study the adjuvant chemotherapy confirmed a high heterogeneity in
mechanisms of cancer initiation and metastasis [83]. For patient responses, supporting the notion that personalized
example, in dogs bladder cancer (BlCa) arises spontane- medicine approaches might be more efficient [156]. Hennig
ously with similar pathology and genetics to human disease et al. hypothesized that the observed resistance is mediated
[89], while murine tumoroids do not fully reflect the char- by resistant clones residing in the tumor that get enriched
acteristics of human BlCa [102]. Therefore, Elbadawy et under systemic treatment. In addition to intrinsic resistance
al. focused on canine tumoroids derived from CTCs from of tumors, acquired resistance can also hinder efficient treat-
urine samples to generate a more representative model to ment. Most research on the underlying mechanisms of this
study BlCa [89]. Later the same group also established phenomenon have been generated using 2D cell lines, but
canine healthy bladder organoids to study the transforma- recently resistant tumoroid models have been established as
tion of healthy cells to cancer cells [102]. The canine BlCa a more representative system [157–159].

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Table 2 Summary of human tumoroid models for the most commonly of ovarian cancer PDTs derived from different sites of the
diagnosed cancer types
same primary tumor for a small patient cohort [162]. The
Cancer type Year Reference
group reported that individual tumoroids showed a differen-
Breast cancer 2022 [108]
2020 [109] tial response of 31%, defined as a more than 10-fold change
2018 [110] in IC50 value, to the same treatment, proving inter-patient
Lung cancer 2019 [24, 111] heterogeneity. Additionally, individual tumoroids from
2017 [112] the same primary tumor showed high differences in drug
Colorectal cancer 2022 [113] response in six out of seven cases, suggesting that PDTs
2016 [114]
2011 [11] genetically maintain the heterogeneity and drug sensitiv-
Prostate cancer 2022 [97] ity of the original tumor [162]. Interestingly, it was recently
2021 [115] shown that a more physiological culture medium changes
2014 [26] the drug response of ovarian tumoroids compared to com-
Gastric cancer 2018 [116–118] monly used organoid medium, highlighting the necessity for
Liver cancer 2018 [19]
careful selection of experimental conditions [166]. Using a
2017 [119]
Cervical cancer 2022 [120]
similar approach, the intra-tumoral drug response differ-
2021 [121] ences of liver cancer were investigated [163]. In this study,
Esophageal cancer 2021 [122] 27 PDTs were generated from different areas of five primary
2019 [123] lesions and a total of 129 cancer drugs were tested. Even
2018 [124] though a high inter-patient and intra-tumoral heterogeneity
Thyroid cancer 2023 [125]
was confirmed, the researchers identified several pan-effec-
2022 [126]
2021 [127] tive drugs that showed an inhibitory effect for most of the
Bladder cancer 2023 [128] tested tumoroids [163].
2018 [129] The high heterogeneity of cancer is a significant prob-
Pancreatic cancer 2018 [130] lem for efficient treatment; however, metastasis formation
2017 [112] is still the main reason for cancer related death. To eluci-
2015 [131, 132]
date the differential drug responses between primary tumors
Endometrial cancer 2023 [133]
2019 [134] and matched liver metastases of CRC, Mo et al. generated
2017 [112] tumoroids from both samples of 25 patients, and showed
Ovarian cancer 2020 [135, 136] that the intra- and inter-patient heterogeneity is reflected by
2019 [23] the in vitro 3D models [167]. Interestingly, although tran-
Glioblastoma 2020 [137]
scriptomic analysis revealed differences between primary
2019 [138]
2016 [139] and metastatic tumoroids, drug sensitivities were highly
Head and neck squamous cell carcinoma 2023 [140] consistent. Thus, the authors suggested that the response of
2019 [141] metastatic lesions to specific drugs could be predicted using
2018 [142] tumoroids derived from primary tissue for a personalized
Mesothelioma 2018 [143] medicine approach [167].
Renal cancer 2022 [144]
The predictive potential of PDTs and their translation to
2021 [145]
2019 [146] the clinics has also been investigated for various other can-
Merkel cell carcinoma 2022 [147] cer types, such as PaCa [168], and brain cancer [169], two
highly aggressive cancer types with limited or inefficient
As confirmed by the recent improvements of transcrip- treatment options. In a translational approach, tumoroids
tome and proteome analysis on single cell level, one of the of different stages of PaCa and brain tumors were used to
remaining problems for efficient cancer treatment is the predict the optimal treatment for each patient in a clinically
intra-tumoral and inter-patient heterogeneity [160, 161]. relevant time frame [168, 169]. Even though both studies
Studies showed that between different regions of the same confirmed that tumoroids reflect inter-patient heterogeneity
tumor, drugs can have varying inhibitory effects [162, 163]. and patient-specific drug responses, limitations of varying
This highlights the fact that biopsies might not be repre- efficiencies of PDT generation, time management, but also
sentative for the drug response of the whole tumor, and cost, have to be overcome for direct translation of tumoroid
that specific combinatorial therapies might be necessary to research to the clinics. Especially for rare cancers, where
target all tumor subclones [164, 165]. To analyze whether a major problem for efficient treatment is the lack of prog-
PDTs can reflect the intra-tumoral heterogeneity of a pri- nostic and diagnostic biomarkers due to small patient num-
mary tumor, De Witte et al. compared the drug responses bers [170, 171], tumoroids might be suitable to improve

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A systematic review on the culture methods and applications of 3D tumoroids for cancer research and…

treatment response and survival rates of patients [172]. For 2.2 Co-cultures and advanced culturing systems
example, PDTs of PD-L1 negative mucinous adenocarci-
noma of the appendix were used to predict response to che- Recent advances in the field of cancer research highlight the
motherapeutic drugs and targeted therapy for one individual critical importance of not only the tumor itself but also its
patient [173]. PDT drug response correlated well with the surrounding TME. The interplay between the tumor and the
patient response, and the tyrosine kinase inhibitor dasatinib TME is a key determinant in tumor growth and progres-
was identified as a possible treatment option for this patient. sion, making it an essential focus for comprehensive stud-
In addition to evaluating the efficiency of commonly used ies [180–182]. Traditional 2D or 3D cell culture models are
treatments for patient-specific precision medicine, PDTs can limited in their ability to fully capture these complex inter-
also be used to test the efficacy of novel treatment regimes. actions, prompting the development of more sophisticated
As an example, lung cancer PDTs are highly sensitive to the methods. In recent years, three innovative approaches have
tyrosine kinase inhibitors dabrafenib and trametinib, which emerged to model the TME in a more physiologically rel-
are commonly used for melanoma treatment, suggesting evant manner. This chapter will discuss: (1) the integration
these drugs as a novel therapy for lung cancer [174]. of co-culture techniques with an air-liquid interface (ALI),
In summary, several studies have provided clinically rel- which allows for relevant interactions between tumor cells
evant evidence that PDTs can be a suitable model for a pre- and their environment; (2) the application of bioprinting to
cision medicine approach to overcome the problem of high create tumoroids including cells of the TME, which offer
heterogeneity in drug responses between patients. In line a more realistic, 3D structure for studying tumor behavior;
with this, PDTs have also been used to identify pre-existing 3) the incorporation of microfluidic channels, which can be
and acquired resistance to commonly used drugs, to avoid used independently or combined with bioprinting, to simu-
treatment of patients with inefficient therapies. late dynamic factors like blood flow or targeted drug admin-
PDTs are not only suitable to predict the response of istration (Fig. 2A).
tumor cells to chemotherapy or targeted drugs, but also to
other treatments like radiotherapy [141, 175–178]. Radia- 2.2.1 Co-cultures
tion directly leads to the induction of single-strand and
double-strand breaks in DNA, as well as the generation of To investigate the interplay between cancer cells and their
reactive oxygen species and the upregulation of oxidative surrounding environment, advanced co-culture models,
stress signaling pathways, and is thus commonly used as including the culturing of tumoroids with cancer-associated
a combinatorial treatment with chemotherapy [179]. For fibroblasts (CAFs) [187, 188], lymphocytes [189], and
patients with locally advanced rectal cancer, the standard myeloid cells such as macrophages [190–192], or even bac-
treatment is neoadjuvant chemoradiation followed by total teria [193] have been used. Indirect cell interactions can be
mesorectal excision of the tumor [175]. Drug response of mimicked by the addition of cell-conditioned media, which
rectal PDTs matched chemoradiation responses in patients contains secreted molecules, to another cell type. However,
with an accuracy of up to 84% [175, 176], and a sensitiv- this co-culture system only allows for single-directional
ity of 78.01% with 91.97% specificity [176]. The predictive response, which can be overcome by the use of transwell
potential of PDTs for radiotherapy has also been confirmed cultures. Individual cell types are separated via a mesh
for head and neck squamous cell carcinoma (HNSCC), leading to diffusion of secreted molecules and therefore
where the combination of the EGFR inhibitor cetuximab to a multi-directional interaction [13, 188]. However, as
and radiotherapy resulted in increased cell death in vitro, direct cell-cell contact is essential for various physiological
and showed higher efficacy compared to radiotherapy alone processes, indirect co-culture systems can only be used to
[141]. A recent study from the same researchers confirmed investigate the effect of metabolites, cytokines, and other
the predictive potential of HNSCC tumoroids by correlat- secreted molecules. In direct co-culture systems different
ing tumoroid therapy response with patient clinical response cell types are embedded in the same ECM and then either
to model the efficacy of chemoradiation for these patients, submerged in culture medium or maintained as an ALI.
and the further use for biomarker selection and validation Because of the direct cell-cell contact, different aspects
[177]. In addition, PDTs offer an advanced model to study of the interaction between the tumor and its TME can be
the dynamics and mechanisms of radioresistance. Glio- studied. However, as it is more complex to isolate specific
blastoma PDTs reflected in vivo resistance mechanisms cell types for downstream analysis, single cell read outs or
mediated by treatment-induced senescence to combination optical techniques are required to efficiently analyze cell-
treatment with temozolomide and radiation, suggesting the cell dependencies or their crosstalk [187, 190, 194, 195].
use of PDTs for studying the underlying mechanisms of As important factors normally found in the TME are lost
drug resistance [178]. upon dissociation of the tumor tissue and the subsequent

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 J. Kalla et al.

Fig. 2 Co-culture techniques for tumoroid models. (A) Overview of sections of bioprinted tumoroids stained for CD31 (yellow), VIM
of state-of-the-art co-culture techniques in cancer research. (B) ALI (red) and KRT8/18 (green) (bottom) [185]. (D) Schematic (middle)
tumoroid models of different cancer types for TME studies on day 30. and immunofluorescence stainings (left, right) of a vascularized BrCa
Phase contrast (top), H&E (middle), and immunofluorescent stain- tumoroid-on-a-chip model for studying the TME and drug sensibility
ing (bottom) of ALI PDTs for DAPI (purple) and VIM, CA9, S100 of cancer cells [186]. B. Copyright Elsevier, C. Copyright Wiley-VCH
or CK7, respectively (green) [183]. (C) Schematic depicting the gen- GmbH, D. Copyright Royal Society of Chemistry. Reproduced with
eration of a bioprinted tumoroid co-culture model including fibro- permission. Created with BioRender.com
blasts and endothelial cells (top) [184]. Immunofluorescent staining

artificial assembly of the isolated cells for direct co-culture exposed to the atmosphere [183, 188] (Fig. 2A). ALI mod-
systems, Ootani et al. established a method to culture cells els have already been established for several cancer types,
in their original environment by preserving the TME using such as salivary gland cancer and kidney cancer (Fig. 2B).
ALI models [196]. Dissociated tissue is hereby directly This method leads to the differentiation and maturation of
embedded into an ECM such as collagen type I and/or a the cell types present in the tissue, resulting in organoids or
hydrogel, seeded onto a transwell mesh, and then cultured tumoroids containing epithelial cells, as well as stromal and
in a way that only the basal part of the ECM or tissue is in immune cells [13, 183, 197]. This way, PDTs can be cul-
contact with the culturing medium while the other part is tured for months while mostly preserving the original TME,

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A systematic review on the culture methods and applications of 3D tumoroids for cancer research and…

thus reflecting the physiological tumor site. However, the how stroma cells could be targeted for anti-cancer therapies
limited availability of primary tumor tissue, the slow growth [204].
of a tissue ALI culture, and the partial loss of stromal cells
during long-term culture, makes this method unsuitable for The importance of specific fibroblast subtypes and the
high-throughput analyses such as drug screens [198]. Tissue synergistic cross-talk between the tumor and its TME for
ALI culture can nevertheless be a powerful direct co-culture cancer progression is well understood. However, how tumor
model for studying processes like tumor progression, ther- cells reprogram NFs into CAFs is still largely unknown. To
apy response to selected drugs, or the presence and infiltra- investigate whether the mutational status of a tumor could
tion of immune cells in the original tumor tissue. reprogram and determine the fibroblast phenotype, Shaas-
hua et al. analyzed the differences between NFs indirectly
2.2.1.1 Stromal cells As mentioned above, stromal cells in co-cultured with either BRCA wild-type or germline BRCA
the TME are involved in many tumor promoting processes. mutated PaCa tumoroids [205]. Different fibroblast sub-
CAFs can accelerate tumor growth by synthesizing and types were indeed derived from the NFs depending on the
remodeling the ECM, and by producing growth factors that BRCA mutational status, thus suggesting that each tumor
induce angiogenesis. Additionally, by negatively affecting might shape its individual TME.
drug access to the tumor they can influence therapy response In summary, germline mutations can not only change
[187, 199–201]. We recently developed a PDT fibroblast the behavior of the tumor itself but can also influence the
co-culture model using matched tumoroids and CAFs or surrounding stromal cells [205]. Thus, to overcome stroma-
normal fibroblasts (NFs) isolated from CRC patients [188]. mediated resistance, therapies have to target multiple cell
Both NFs and CAFs were able to support the growth and types in the TME simultaneously, and personalized medi-
differentiation of tumoroids without the addition of specific cine approaches are necessary for efficient treatment.
niche factors, suggesting a better reflection of the in vivo
tumor heterogeneity than conventional PDT cultures. In 2.2.1.2 Leukocytes Immune cells are another important
addition, we observed an enhanced cancer-promoting phe- cell type in the TME inducing both anti- and pro-tumori-
notype of NFs upon co-culture with the tumoroids, high- genic effects, which represent promising targets for efficient
lighting the dynamic and interdependent cross-talk between cancer treatment. Even though co-cultures of immortalized
the tumor and its microenvironment [188]. These co-culture 2D cell lines with immune cells, or animal models, already
models not only reflect the microenvironment but can also led to the development of promising cancer treatments
be used to model the influence of the TME on cancer cell targeting the immune system, tumoroids co-cultured with
drug response. By testing the response of both human and immune cells might be another suitable model for testing
murine liver tumoroids to conventional anti-cancer drugs patient-specific responses to various immunotherapies [32,
such as regorafenib, sorafenib, and 5-FU, it was shown that 40, 206, 207].
the direct co-culture with CAFs or the addition of CAF-
conditioned medium to the liver tumoroids led to a higher Many groups have already established co-cultures of
resistance of the cancer cells [79]. Also, PaCa PDTs, which tumoroids with lymphocytes focusing on T cells and natural
were directly co-cultured with CAFs, showed higher resis- killer (NK) cells to improve the modeling of immunother-
tance against commonly used PaCa drugs when compared apies like CAR T cells and immune checkpoint blockade
to tumoroid-only cultures [187]. Single cell RNA-Sequenc- [208]. CAR T cell therapy has proven successful for leu-
ing revealed differences in gene expression in the PDTs kemia, but models for investigating the cytotoxic effect
induced by the CAFs, which led to the induction of EMT of these cells on solid tumors like CRC are still needed.
in the cancer cells. The authors suggested that this EMT Therefore, Schnalzger et al. developed a CRC PDT co-cul-
induction contributed to the chemo-protectant effect seen in ture model as a sensitive in vitro platform to study patient-
their co-culture system [187]. Similar EMT-inducing effects specific treatment responses [207]. Other groups focused
have been seen in other studies where EMT was partially on investigating the mechanism of PD-1/PD-L1 immune
induced in cancer cells by direct co-culturing of CAFs and checkpoint blockade using advanced co-culture models, and
human CRC tumoroids mimicking either early or late stage showed that human and murine lymphoma tumors and their
CRC [202, 203]. Further on, inhibition of WNT signaling in TME could be cultivated for several weeks using an adapted
CAFs was shown to induce different CAF subtypes result- ALI model [183, 209]. The system reflected the physiologi-
ing in repression of EMT in CRC tumoroids, highlighting cal properties of the tissue, and cell composition including
lymphoid cells and supporting T helper cells was preserved.
Importantly, the ALI cultures functionally recapitulated
PD-1/PD-L1 dependent immune checkpoint blockade,

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 J. Kalla et al.

highlighting the potential of this model to develop personal- time consuming, and high-throughput methods still have
ized immunotherapies for lymphomas [183, 209]. to be improved. Automated methods like bioprinting could
Similarly, a recent human tumoroid and immune cell co- help to overcome some of these limitations.
culture model for gastric cancer was developed [189]. In
this model, dendritic cells were first activated by a patient- 2.2.2 Bioprinting
derived tumor antigen, and then used to prime CD8 + T
cells. The authors suggest that the direct T cell activation Bioprinting allows for the exact positioning of different
in their model is superior to activation by anti-CD28-coated cell types and biomaterials in a 3D space with the help
plates, which was used in a similar co-culture model [210]. of a mechanical and computer-assisted system to mimic
Moreover, focusing on immune checkpoint blockade the in vivo spatial architecture of a tissue or tumor and its
therapies, novel bispecific antibodies for PD-1 and PD-L1 microenvironment [216]. The biggest advantage of using a
were used to restore NK and CD8 + T cell activity in ovar- bioprinting-based approach to generate preclinical cancer
ian PDT co-cultures with intra-tumoral immune cells [211]. models is the standardization of cell dispensing [217], and
Immune cell reactivation was shown to be mediated by the the possibility to construct an artificial 3D tumor including
partial downregulation of BRD1, and inhibition of BRD1 different cell types, structures, and ECMs for more precise
had a similar anti-tumoral effect in vitro and in vivo. Thus, personalized medicine approaches [218].
BRD1 inhibition could provide a new therapy option for Early studies focused on generating such models by
ovarian cancer patients to circumvent immune cell evasion using bioprinted cancer cell lines or single cell suspensions
[211]. [184]. Recently, the dispersion of organoids and tumoroids
Myeloid immune cells such as tumor-associated macro- together with stromal cells allowed for the development of
phages (TAMs) play an important role in the TME to sup- co-culture models including the TME (Fig. 2C) [219].
port tumor growth, and thus represent important therapeutic CRC microtissues were produced according to patient-
targets. Therefore, both conventional organoid [212] and specific colonoscopy images by printing PDTs surrounded
ALI co-culture models combining tumoroids and macro- by healthy organoids to model the interaction of a tumor
phages have been developed [213]. with normal adjacent tissue [220]. As the in vitro treatment
By including multiple immune cell types in one co- response of these microtissues to the standard 5-FU therapy
culture model, it is possible to further analyze the complex reflected the patient response, the model could be used as a
interplay between the tumor, and between different kinds of more physiological drug screening platform. Additionally,
immune cells. This way, it was shown that reprogramming the patient-specific risk of tumor invasion into the surround-
of TAMs following monoamine oxidase A inhibitor (MAO- ing tissues was calculated in this model by correlating the
Ai) treatment, a widely used class of drugs for the treatment number and distance of invading tumor cells. This could
of depression and Parkinson’s disease, enabled the induc- provide a real-time quantitative readout for analyzing can-
tion of anti-tumor T cell reactivity in a melanoma co-culture cer progression and metastasis [220].
model [214]. MAO-Ai could thus be used in combination In another recent study, microtissues consisting of
with PD-1/PD-L1 inhibitors to reactivate the anti-tumor patient-derived lung tumoroids in co-culture with matched
immune response as a new potential treatment for several CAFs and endothelial cells were generated [221]. After
cancer types. printing the vessel structures and seeding the CAFs, the
However, co-cultures can not only be used to study the tumoroids suspended in a hydrogel derived from por-
interactions between tumoroids and multiple immune cells cine lung tissue were printed into the same compartment.
but also to analyze more complex interactions between cells An active fusion of the stromal cells and tumoroids was
in the TME, including tumor cells, immune cells, and stro- observed, and microvessels formed to directly interact with
mal cells. For this, Sufi et al. developed the Thiol-reactive the other cell types. After administering the drug poziotinib
Organoid Barcoding in situ Mass Cytometry (TOBis MC) through the vessel structures it was seen that both the endo-
protocol, which allows for the analysis of organoid and thelial cells and CAFs, but also the CAF-secreted matrix,
tumoroid lines in co-culture with leukocytes and fibroblasts protected the lung tumoroids from the treatment. Thus, this
on single cell level to study the interactions between TAMs model could be used to further test the influence of cell-cell
and CAFs in high-resolution [215]. or cell-matrix interactions on the efficacy of drug delivery to
In summary, organoid and tumoroid models in co-cul- the tumor tissue [221].
ture with other cell types can be used to model the complex Apart from printing models mimicking the TME, bio-
interactions of the TME. Even though the benefit of these printing can be used for standardized cell dispensing for
co-culture models for improving or finding novel treatment high-throughput applications. However, one drawback
options has been proven, the assembly of these models is is the difficulty of printing bioink into small wells as the

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A systematic review on the culture methods and applications of 3D tumoroids for cancer research and…

utilized ECMs can spread in the well and thus do not main- treatment. Aside from its suitability for physiological drug
tain the shape necessary for 3D cell growth. To circumvent testing, this chip design could be a useful tool to study and
the spreading, patient-derived glioblastoma or sarcoma cells easily visualize differences in angiogenesis, proliferation,
were mixed with a bioink consisting of hyaluronic acid as and migration [186]. Importantly, it has already been shown
well as collagen, and then printed into wells coated with before that fluidic devices provide a more physiological
gelatin, which was later removed and substituted with model to study cancer cell migration [229].
medium [222]. In addition, acoustic bioprinting of small Using microfluidic devices with tumoroids alone, recent
droplets onto a hydrophobic substrate was used to generate papers already described the advantages of chip-based mod-
BlCa-derived tumoroids consisting of both cancer cells and els for a personalized medicine approach and more rep-
CAFs [223]. This method allows for the generation of large resentative drug testing compared to static models. Both
numbers of uniform tumoroids that mimic the TME, which Mazzocchi et al. [143] and Jung et al. [230] used PDTs to
can easily be dispensed into small wells and be used in high- perform medium-throughput drug screens for mesothelioma
throughput drug screens for personalized therapy. or small cell lung cancer (SCLC) patients, respectively. Drug
Thus, the combination of 3D cell models and bioprinting response to specific chemotherapeutic therapies was differ-
could help to facilitate reproducible drug testing, and the ent between the tumoroid lines with different mutational
investigation of cellular processes in a 3D space with mul- backgrounds but correlated well with the patient response.
tiple cell types and structures. However, these printed mod- Importantly, SCLC tumoroids on the chip maintained a che-
els are still static and do not reflect the effect of mechanical motherapy resistant core, which might be due to insufficient
forces like dynamic flow, or chemical gradients, which drug delivery also observed in in vivo tumors [230].
influence the tumor cell phenotype in vivo [224]. To facilitate the translation of this drug screening method
to the clinics, Schuster et al. developed an automated high-
2.2.3 Fluidic devices throughput screening platform on a chip [231]. The authors
constructed a microfluidic system with which 20 indepen-
The important physical and chemical characteristics of the dent experimental conditions could be tested on up to 10
TME can be modeled with microfluidic systems, where different patient-derived 3D cell lines. A customizable soft-
cells are cultured in chambers connected to microchannels ware allows for the stimulation, assaying, and imaging of
supplying oxygen, growth factors, or drugs via dynamic the tumoroids without human intervention. Interestingly,
perfusion [225–227] (Fig. 2D). In a proof of concept study, combinatorial treatments and time-dependent adminis-
CRC PDTs cultured on fluidic devices showed a higher via- tration with multiple rounds of treatment showed greater
bility, proliferation rate, and tumoroid formation efficiency, therapy efficacy on PaCa tumoroids compared to single
compared to tumoroids cultured in static ECM drops on a treatments [231].
plate [228]. Additionally, chip-grown tumoroids were big- To investigate the influence of the tumor stroma and the
ger in size and showed a multilayered morphology with method of drug delivery on treatment efficiency, a fluidic
crypt-like projections similar to the human colon, while system mimicking the TME for drug testing on PaCa PDTs
the conventional tumoroids formed more cystic monocel- has recently been established [232]. The vasculature was
lular structures. Interestingly, when testing the response of modeled by growing endothelial cells in a perfusable micro-
PDTs to 5-FU no significant differences were seen between fluidic scaffold. Tumoroids co-cultured with fibroblasts,
the culturing methods, suggesting that chip-grown 3D cells which resulted in increased proliferation of cancer cells and
maintain their phenotype, while having a growth advantage, enhanced tissue stiffness, were then added into adjacent
and thus validating the use of fluidic devices for tumoroid compartments. When administering the chemotherapeutic
culture and disease modeling [228]. drug gemcitabine to the co-culture via diffusion through
However, the tumor niche consists of multiple cell types, the vessel-like structures, the drug had a reduced efficiency
and an extensive vasculature, through which nutrients, oxy- compared to the administration to tumoroid single cultures
gen or drugs get delivered to the cancer cells. In order to or to static co-cultures. Thus, the authors were able to reca-
mimic this vascular transport of factors and metabolites to pitulate the microenvironment of a vascularized pancreatic
the tumor, endothelial cells together with fibroblasts were tumor and to demonstrate the inhibitory effect of the vascu-
grown in microfluidic chambers to produce a 3D micro- lature cell barrier and the stroma on drug efficiency [232].
vascular network, before adding primary BrCa tumoroid- Interestingly, another PaCa-on-a-Chip model was used
like structures into a separate adjacent compartment [186]. to show that stroma-targeting agents do not influence the
Using this setup, vessel outgrowth towards the tumor cell cell viability of tumoroids in monoculture, but lead to a sig-
chamber was observed in addition to tumor cells invading nificant increase of chemotherapeutic anti-cancer effects in
the vasculature chamber after inducing EMT via TGF-β a fluidic device co-culture [233]. Using a similar model, it

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 J. Kalla et al.

was reported that different drugs show different efficacies in 3 In vivo methods and applications
targeting PaCa cells in normoxic versus hypoxic conditions
[234]. Thus, both publications highlight the importance of 3.1 Modeling of tumor biology
including cells of the TME and relevant oxygen levels for
microfluidic drug response studies. Even though tumoroid models can deliver meaningful trans-
In conclusion, tumoroid-on-a-chip models provide a latable results for basic cancer research and patient therapy
powerful tool to investigate biological processes and to options, the assessment of systemic effects and the influence
assess drug responses in an efficient and more physiological of a more complex in vivo TME can only be validated in ani-
manner compared to static tumoroid cultures. mal models [51] (Fig. 3A). In the past, whole tissue pieces,
digested primary tissue, or 2D cell lines were grafted into

Fig. 3 In vivo methods for tumoroid models. (A) Overview of in vivo treatment of patient-derived xenograft mice representing a pretreat-
applications of both human and murine tumoroids. (B) Light micros- ment tumor with either patient-matched neoadjuvant therapy (includes
copy pictures of tumoroids with different phenotypes (Scale bar the drugs doxorubicin hydrochloride (Adriamycin) and cyclophospha-
200 μm) and H&E staining of corresponding tumors after re-inoculation mide, followed by treatment with paclitaxel (Taxol) = AC-T) (left) or
of subcutaneous tumor-derived tumoroids (Scale bar 50 μm) [237]. (C) with drugs selected from a xenograft-derived tumoroid screen (right)
Pearson correlation coefficient plots of gene expression between pros- [108]. B, C, E adapted from corresponding citations; Springer Nature
tate tumors and matched tumoroid lines [238]. (D) Dynamic changes (Creative Commons Attribution 4.0 International). D adapted from
of immune cell populations during early, middle, and late stages in Wiley (Creative Commons Attribution 4.0 International). Created with
a novel tumoroid-based liver metastasis model [239]. (E) Real-time BioRender.com

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A systematic review on the culture methods and applications of 3D tumoroids for cancer research and…

animals as allografts (between same species) or xenografts heterogeneity and plasticity hindering efficient treatment,
(between different species) either subcutaneously or ortho- metastasis formation is one of the leading causes of cancer-
topically [235]. These models have lately been expanded related deaths [242]. CTCs can spread to distant secondary
to transplant organoid and tumoroid lines into animals as sites to induce metastasis [243]. As CTCs are a rare and het-
allografts and xenografts. Importantly, the pathological and erogeneous cell population, De Angelis et al. established a
invasive features of the original tumor and drug response of model to study the biological features and possible drug sen-
the patient are retained in these models, providing a suitable sitivities of these cells [244]. The group derived tumoroids
alternative to investigate mechanisms of malignant transfor- from both orthotopic CRC xenografts and CTCs isolated
mation, tumor plasticity, and metastasis in vivo [236]. from the blood of the same mice. As expected, CTC-derived
tumoroids showed a more aggressive and migratory pheno-
3.1.1 Cancer initiation and progression type in addition to higher stem cell and EMT marker expres-
sion compared to the xenograft-derived tumoroids. The
To study the tumorigenic potential of cancer cells based authors also showed that CTC-derived tumoroids are highly
on common mutational patterns specific for endometrial sensitive to drugs targeting the Survivin pathway including
cancer, murine tumoroids that overexpress KrasG12D and YM155 and quercetin, providing a new possible treatment
carry genetic deletions of various tumor suppressor genes to inhibit metastasis formation. As this model reflected the
(Pten, Tp53) were allografted subcutaneously into nude properties of CTCs isolated from CRC patients, De Angelis
mice [237]. Different mutational backgrounds led to the et al. hypothesized that in the future patient-specific drug
development of specific tumor subtypes with correspond- sensitivities could be studied in a metastasis xenograft
ing tumoroids having an either more cystic or spindle-like model established from the patient’s primary tumor even
morphology because of an irreversible EMT during trans- before the onset of metastatic disease, and thus prevent
formation. This also correlated with a less or more aggres- tumor progression [244].
sive phenotype, respectively [237] (Fig. 3B). The same group also developed another preclinical
As the specific cancer subtype also plays an important metastasis model for CRC [245]. After orthotopically graft-
role in the aggressiveness and treatment of PaCa, Miya- ing patient-derived cells into immunodeficient mice, the
bayashi et al. established an in vivo model to investigate the authors generated tumoroids from the primary tumor site
role of patient-specific genetic and epigenetic aberrations and from spontaneous liver metastases and analyzed the
for PaCa heterogeneity [240]. The authors derived tumor- EMT state together with the drug response. As expected,
oids from patients and xenografted them orthotopically into metastatic tumoroids displayed a more mesenchymal phe-
the pancreatic ducts of immunodeficient mice. Tumors were notype and increased chemoresistance. Interestingly, high
either indolent with a luminal morphology or progressed numbers of CTCs in patients correlated with an increased
to a more aggressive basal-like subtype based on epigen- engraftment efficiency of the corresponding tumor-derived
etic aberrations and deregulation of KRAS. In contrast to cells, again highlighting the important role of CTCs for can-
Maru et al. [237], this group argued that cellular plasticity cer progression [245].
is mainly influenced by the stroma and microenvironment EMT is not only an important characteristic of CTCs, but
rather than cellular clonality, thus suggesting that allo- and also of the cancer cells in tumor lesions. Recently it was
xenografts should be performed orthotopically to recapitu- shown that hybrid cells, presenting both epithelial and mes-
late the corresponding tumor niche for translatable results enchymal characteristics, can be found in human tumor tis-
[240]. sues, GEMMs, and tumoroids of BrCa [246]. The number of
these cells correlated with worse overall survival in patients,
3.1.2 Cellular plasticity and metastasis while driving invasion in vitro. Using tail vein injection or
orthotopic engraftment of tumor cells or tumoroids, respec-
Cellular plasticity and clonality can not only be influenced tively, heterogeneous EMT states were identified within
by the microenvironment, but also by specific therapies the same tumor. Sequential molecular EMT programs were
[241]. To investigate resistance mechanisms of androgen essential for tumor cell invasion and colonization during the
pathway directed therapy, Lee et al. generated tumoroids metastatic process. Thus, both models could be used to fur-
from patient-derived xenograft models of bone metastatic ther investigate the influence of the EMT status on patient
PCa. Androgen ablation led to the development of revers- survival [246].
ibly dormant basal-luminal-like hybrid cells, highlighting To further understand the mechanism of metastasis for-
how some cancer therapies might even support the pro- mation, the role of the immune system in the metastatic
gression of the disease, thus suggesting that treatment regi- niche was also recently investigated in a PCa model of liver
mens might have to be reviewed [241]. Apart from tumor metastasis [239]. Murine tumoroids generated from GEMMs

13
 J. Kalla et al.

with PCa specific mutations were orthotopically allografted case study showed that drug testing using xenografted PDTs
into immunocompetent mice, which resulted in metastatic is feasible in real time and can lead to beneficial results
spread to the liver after only 30 days. This model is not for patients [108]. Drug screens with extensive compound
only less time consuming compared to original GEMMs libraries can also help to further elucidate the mechanism
and xenografts, and more reliable than patient-derived cell of cancer progression. Even though many epigenetic aber-
lines, but importantly also allows for the investigation of rations play a role in the progression of BlCa, the use of
the immune cells in the metastatic niche. The authors found epigenetic drugs as a potential treatment has so far only
that an immune suppressive microenvironment mediated been investigated in five clinical trials [249]. The histone
mainly by neutrophils, anti-inflammatory macrophages, and deacetylase activator SRT1720, which specifically targets
CD4 + T cells was essential for the survival of tumor cells SIRT1, was recently identified as a potent drug to inhibit
and progression of metastases (Fig. 3D). This model might the growth of both BlCa tumoroids and xenografts [249].
be relevant for testing drugs targeting immune cells, and for Mechanistically, SIRT1 activation resulted in deacetylation
elucidating cell-cell crosstalk in the metastatic niche [239]. of HIF1α and subsequent repression of the hypoxia path-
In summary, organoids and tumoroids can be used in way. This pathway has been correlated with poor prognosis
an in vivo setting to investigate many aspects of cancer in patients, highlighting the potential of epigenetic therapies
development and progression, highlighting the advantages for BlCa [249]. In contrast, SIRT1 was also reported to pro-
of orthotopic grafts and allografts. Thus, these models are mote the progression of BlCa [250, 251]. However, these
highly suitable for drug testing and the development of studies were performed on 2D cell lines and might thus be
novel therapies. less representative compared to 3D models and xenografts
[249].
3.2 Therapy development and drug testing in vivo Apart from mouse models, rats are a widely used animal
model for cancer research and treatment development. By
Patient-derived in vitro models in combination with matched orthotopically transplanting kidney tumoroids derived from
in vivo models represent a suitable model system for per- patient-specific iPSCs into rats, a fully vascularized xeno-
sonalized medicine applications. Multiple groups have used graft model for angiomyolipoma, a rare kidney tumor, was
this approach to study cancer subtypes, patient-specific generated [252]. Using this model, the therapeutic efficacy
drug responses, and to identify potential alternative thera- of mTOR inhibition by rapamycin-loaded nanoparticles, the
pies. Importantly, while both 3D in vitro and in vivo models main treatment for angiomyolipoma, was confirmed, sug-
retain the genetic and epigenetic background of the primary gesting that this model would also be suitable to identify
tumor (Fig. 3C), xenograft-derived 2D cell lines can acquire novel alternative treatments for this rare cancer type [252].
additional mutations in culture, suggesting that 3D culture Additionally, zebrafish models are well established to
systems are more stable and representative models for drug study tumor metastasis and are also commonly used for in
screening [247]. vivo drug testing [253]. A comparative analysis of relapsed
Several recent studies provided evidence for the usabil- pediatric malignancies used patient-derived spheroid cul-
ity of PDTs for different tumor types, including endometrial tures, tumor cells isolated from corresponding mouse
cancer [248], glioma [247], BrCa [108], and metastatic PCa xenografts, long-term organoid-like cultures, and zebrafish
[238]. These “living” biobanks reflected patient-specific xenografts to compare drug responses in the different model
drug response, mimicked tumor metastasis [248], and were systems. The zebrafish model proved to be a promising addi-
used to identify alternative treatment options such as the tion to current drug testing tools to rapidly assess potential
chemotherapeutic dianhydrogalactitol (VAL-083) for gli- treatments in vivo, and could be included for personalized
oma [247], or multi-kinase inhibitors for therapy resistant medicine approaches in the future [253].
metastatic PCa [238]. In summary, a variety of different animal- and patient-
Additionally, Guillen et al. reported a first proof that derived in vitro and in vivo models are available, which can
results generated by patient-specific tumoroids in vitro could be tailored to answer specific questions for basic and trans-
be translated to the clinic [108] (Fig. 3E). The group had lational research.
already generated a xenograft model with a matched tumor-
oid line of a BrCa patient and performed a drug screen iden-
tifying eribulin as a novel drug that showed effective growth
inhibition in vitro and complete regression with no recur-
rence in the mice. The patient went into complete remission
after being treated with the same drug, however due to other
complications later unfortunately died. Nonetheless, this

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A systematic review on the culture methods and applications of 3D tumoroids for cancer research and…

4 Limitations a time-consuming process that generates large amounts of

data difficult to store and analyze [270, 271]. Additionally,
Even though various studies have shown that PDTs are a computational analysis methods that are optimized for imag-
reliable model system for the evaluation and discovery of ing of 3D structures with defined parameters for organoid
anti-cancer drugs, several limitations have to be considered and tumoroid phenotypes are needed [270, 272]. Compared
for tumoroid-based research. to imaging analysis, metabolic assays like CTG 3D are less
Depending on the tumor type, only limited patient tis- time consuming and the readout is easier to process and
sue material might be available. In addition, some ethical interpret [270]. However, less information can be obtained
concerns regarding consent, ownership, and data integrity with this method as it is an endpoint-measurement repre-
should be considered for the use and application of PDTs senting the result of all cells in one well. Thus, a potential
[254]. Also, the establishment success rates vary immensely heterogeneity in drug response between the cells of a tumor-
between different tumor types, and whether PDTs are oid cannot be analyzed [270]. Even though both metabolic
established from fresh or frozen tissue. This additionally and imaging methods are suitable for high-throughput 3D
negatively influences the translation to the clinics [255]. In applications, further standardization for both assay develop-
general, establishment of 3D cell lines is more time- and ment and data analysis have to be implemented.
labor-consuming compared to 2D cell lines. The different The total number of cells needed, and the culturing
ECMs, and growth media containing specific niche factors, method of organoids additionally hinder the performance
used for organoid and tumoroid culture do not adequately of high-throughput screens, as it is difficult to work with
reflect the molecular features and stiffness of the primary ECMs in commonly used 384-well plates [222]. Further-
TME, and one major drawback is the high variability in more, it has been shown that the artificial ECMs can nega-
their composition. Additionally, ECMs are mostly derived tively influence drug penetration by forming a protective
from mice [256], which raises the issue of possible influ- barrier around the tumor cells, and thus lead to unreliable
ence of cross species interactions, when culturing human- results during drug screening [230]. To circumvent these
derived 3D cell lines in murine-derived ECMs [257, 258]. hurdles, drug screening protocols with tumoroid suspension
Another problem with 3D cell culture is the overgrowth of cultures have been developed [219]. However, most studies
tumor cells by normal cells that were present in the tissue of present a far too small sample size for reliable translation of
origin. This is common in lung [259] and PCa [73] tumoroid results into clinical practice [107].
cultures, presenting a limitation for cultivating pure tumor- The cells of the TME can also greatly influence the drug
oid cultures over extended periods. response of tumor cells [186]. However, as tumoroid cul-
It has been shown that organoid and tumoroid models tures only maintain tumor cells of epithelial origin, the role
stably reflect inter-patient and intra-tumor heterogeneity of the TME cannot be modeled with conventional 3D organ-
[148, 260, 261]. However, tumoroid lines established from oid systems, highlighting the importance of using advanced
different sites of the same primary tumor show highly vari- co-culture and organ-on-a-chip models. However, establish-
able drug responses [162], raising the question whether ing these advanced chip-based systems requires technical
sampling issues might limit the representative use of knowledge and specific instrumentation. Also, there are
tumoroids for personalized drug screens. High-throughput no standardized organoid-on-a-chip systems available yet,
screening using tumoroids as model systems is still limited which makes validation of published data difficult.
by the fact that analysis methods are not standardized, and Since the establishment and long-term cultivation of
automation is needed for reliable high-throughput applica- patient-derived organoids and PDTs has not been successful
tions. Some commonly used readouts for these models are for some human tissues and cancer types, many researchers
metabolic cell viability assays such as Cell Titer Blue, a still rely on animal models. Besides using animal-derived
resazurin-based fluorescent measurement [262], Cell Titer tumoroids for cancer research, one of the most important
Glo (CTG) 3D, a luminescent-based agent that measures model systems is the establishment of tumoroid xenografts
ATP content in spheroid [263, 264] and tumoroid screenings to investigate the initiation and progression of human
[219, 265, 266]. Phenotypic readouts can be performed on tumoroid-derived lesions in vivo. Although one of the limi-
fluorescently-labelled or stained organoids and tumoroids tations is the high number of tumoroids required for suc-
either over a period of time [231, 267], or at a pre-defined cessful xenografting, established tumors stably recapitulate
endpoint [268, 269]. Using a multiparametric microscopy- the morphology of the primary tumor. Additionally, this
based readout, more information up to single cell resolu- method leads to the development of cancer-specific symp-
tion can be acquired [270]. However, because of the size toms in mice that can be seen less frequently in cell line-
and 3D structure of organoids and tumoroids, Z-stacks have derived xenografts [248]. Animal models are a valuable tool
to be used to generate high-resolution imaging data; this is

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 J. Kalla et al.

for assessing the complex biological effects of drugs, which have already been established by the National Cancer Insti-
cannot yet be fully replaced by PDT models. tute in the US [289], and by a consortium of several Euro-
pean institutions (Human cancer models initiative) [290].
Recently, the Organoid Cell Atlas, which in addition to
5 Conclusion and outlook tumor samples and tumoroids also includes healthy organ-
oids, was launched as a comprehensive database [291, 292].
As cancer is a highly heterogeneous disease, versatile Similarly, OrganoidDB has already collected bulk and sin-
models that can easily be manipulated in vitro, and stably gle cell transcriptomic data of more than 16.000 organoid
reflect the characteristics of the primary tumor, are needed. and tumoroid models for both human and mouse samples, in
Thus, tumoroids have a great potential to replace standard addition to data generated from primary tissue and cell lines
2D cell culture or animal models. However, to adequately for comparison [293].
model the inter- and intra-patient heterogeneity, several Lastly, 3D cell models are a promising tool to reduce the
tumoroid lines would have to be generated from the same number of animals used for preclinical cancer research in
tissue biopsy, which is not the standard practice [162, 163]. concordance with the 3R principles [294]. Due to a recent
Additionally, limitations regarding tumoroid cultivation ground-breaking new law, the US Food and Drug Adminis-
methods still need to be overcome. For example, artificial tration can now approve drugs for clinical trials on humans
ECM hydrogels [257, 273, 274], or human-derived ECMs that were tested only in vitro on organ-on-a-chip models or
[275], have been developed to limit negative cross-species organoid and tumoroid models [295]. Although alternative
interactions for PDTs. Additionally, cultures with no matrix methods for drug testing, such as 3D cell models, are not
at all [276], or bioreactors [277] could be used for tumor- yet standard practice, they hold great promise for redefin-
oid expansion and drug screens in the future. Importantly, ing preclinical cancer research and precision medicine in the
many groups have focused on the automation of 3D cell future, due to their molecular and phenotypic similarities to
culturing and analysis methods for high-throughput appli- patient tumors.
cations to achieve a better translation of results to the clinic
[278–280]. For high-throughput drug screens, protocols Acknowledgements The authors want to thank Milena Mijović, Kata-
rina Mišura, and Luisa Grader for compiling references. The authors
that either use tumoroid-derived single cells, or small- to also want to thank Maša Zrimšek, Kristina Draganić, Katarina Mišura,
medium-sized tumoroids in suspension, have already been and Gabriel Wasinger for providing high-level feedback on the draft.
developed [219]. Additionally, bioinformatic approaches
and mathematical modeling have been combined with 3D Author contributions Writing - original draft: JK, JP, TM, LT; Writ-
cancer models to test for drug sensitivities faster [281–283]. ing - reviewing and editing: JK, JP, GE; Conceptualization: JK, JP,
GE; Visualization: JP, JK; Supervision: GE. All authors have read and
To further improve the model systems available for devel- approved the final manuscript.
oping and testing novel treatment options, multi-organ chip-
based systems were developed, which are used to study the Funding This work was supported by the Austrian Science Founda-
systemic effects of anti-cancer drugs on healthy tissue or tion (FWF) project P 32771 (Gerda Egger, Jessica Kalla); Austrian
other organs [284, 285], and thus could reduce the use of Science Foundation (FWF) SFB F83 (Gerda Egger, Janette Pfneissl);
Austrian Science Foundation (FWF), doc.funds grant DOC59 (Gerda
animal models for drug testing. Egger, Theresia Mair); City of Vienna Fund for Innovative Interdisci-
In the future, living biobanks of organoid and tumoroid plinary Cancer Research, 21118 (Gerda Egger); City of Vienna Fund
models will be essential for real-time personalized treat- for Innovative Interdisciplinary Cancer Research, 21209 (Loan Tran);
ment of cancer patients, as summarized in a recent review Austrian Academy of Sciences, Doc Fellowship 25276 (Loan Tran).
Open access funding provided by Medical University of Vienna.
[286]. A number of clinical trials are already investigating
the outcome of personalized therapy selection based on Data availability No datasets were generated or analysed during the
tumoroid drug sensitivity testing for several cancer types, current study.
including PaCa (NCT04931394, NCT04931381), BrCa
(NCT04450706, NCT03544047, NCT05177432), lung Declarations
cancer (NCT05136014), BlCa (NCT05024734), and sev-
eral different cancers (NCT04279509) [287]. However, the Ethics approval and consent to participate Not applicable.
standardization of methods has to be addressed by regula-
Copyright Where specified, figures were reproduced from cited pub-
tory authorities and put into practice in the industry to suc- lications with permission of the respective journals, or were adapted
cessfully implement 3D models in the clinics. Open data from cited publications under the Creative Commons Attribution 4.0
platforms that include repositories of protocols, donors, International (https://creativecommons.org/licenses/by/4.0/.
organoids and tumoroids, and ethical information, are
needed [288]. Such biobanks for human-derived 3D models Consent for publication Not applicable.

13
A systematic review on the culture methods and applications of 3D tumoroids for cancer research and…

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