Society for Endocrinology
Bioengineered in vitro three-dimensional tumor models in endocrine cancers
Aleksander Skardal®1,2,3, Hemamylammal Sivakumar1, Marco A Rodriguez1, Liudmila V Popova4 and Priya H Dedhia2,3,4
1Department of Biomedical Engineering, The Ohio State University, Columbus, Ohio, USA
2The Ohio State University and Arthur G. James Comprehensive Cancer Center, Columbus, Ohio, USA
3Center for Cancer Engineering, The Ohio State University, Columbus, Ohio, USA
4Division of Surgical Oncology, The Ohio State University and Arthur G. James Comprehensive Cancer Center, Columbus, Ohio, USA
Correspondence should be addressed to A Skardal or P Dedhia: skardal.1@osu.edu or priya.dedhia@osumc.edu
Graphical abstract
Adrenocortical Carcinoma
Thyroid Cancer
Pheochromocytoma
Neuroendocrine Tumors
Tumor Spheroids
ECM Hydrogel-based Organoids
Tumor-on-a-Chip Invasion and Metastasis Models
Microfluidic Multi-Organoid Metastasis Models
Labeled Tumor Cell
Matrix-Filled Channels
Engrafted Metastatic Tumor cells
2
Tumor Biospecimen
Flow
Cell Line
Mets
Tumor Organoid
Complexity
Abstract
Endocrine tumors are a heterogeneous cluster of malignancies that originate from cells that can secrete hormones. Examples include, but are not limited to, thyroid cancer, adrenocortical carcinoma, and neuroendocrine tumors. Many endocrine tumors are relatively slow to proliferate, and as such, they often do not respond well to common antiproliferative chemotherapies. Therefore, increasing attention has been given to targeted therapies and immunotherapies in these diseases. However, in contrast to other cancers, many endocrine tumors are relatively rare, and as a result, less is understood about their biology, including specific targets for intervention. Our limited understanding of such tumors is in part due to a limitation in model systems that accurately recapitulate and
enable mechanistic exploration of these tumors. While mouse models and 2D cell cultures exist for some endocrine tumors, these models often may not accurately model nuances of human endocrine tumors. Mice differ from human endocrine physiology and 2D cell cultures fail to recapitulate the heterogeneity and 3D architectures of in vivo tumors. To complement these traditional cancer models, bioengineered 3D tumor models, such as organoids and tumor-on-a- chip systems, have advanced rapidly in the past decade. However, these technologies have only recently been applied to most endocrine tumors. In this review we provide descriptions of these platforms, focusing on thyroid, adrenal, and neuroendocrine tumors and how they have been and are being applied in the context of endocrine tumors.
Keywords: bioengineered; in vitro; three-dimensional; models; organoids
Introduction
Endocrine tumors are a heterogeneous collection of malignancies that originate from cells that can secrete hormones (Latteyer et al. 2016). These are generally found in the tissues of the endocrine system, such as the thyroid, adrenal, pancreas, parathyroid, and pituitary glands. Specific examples include thyroid cancers, adrenocortical carcinoma (ACC), neuroendocrine tumors (NETs), paraganglioma, and pheochromocytoma (Belfiore & Perks 2013).
With incidences of several endocrine tumors increasing at rates more rapidly than most other cancer types in the USA and many other countries (Dasari et al. 2017, Darba & Marsa 2019), there is a crucial need to identify new therapeutic targets and treatments for these diseases. In the case of some endocrine tumors, few if any reliable preclinical models exist to identify and study potential therapeutic interventions.
In this mini-review, we focus our attention on describing several different approaches to creating 3D tumor models of endocrine cancers. Specifically, we focus primarily on thyroid cancer and the rare endocrine neoplasms - neuroendocrine tumors, ACC, and pheochromocytoma.
Overview of tumor organoids and other 3D tumor platforms
Bioengineered 3D platforms using human cells for mechanistic research can complement traditional animal models and 2D cell cultures. The creation of accurate 3D models that mimic human cell-cell, cell-extracellular matrix (ECM), and mechanical interactions of in vivo tissue, fills a critical experimental gap (Mazzocchi et al. 2017, Devarasetty et al. 2018). Animal models and 2D cell cultures have been used in cancer research for decades and have been crucial tools for countless scientific discoveries and medical advances. However, for the study of many biological phenomena, animals and 2D cultures have notable limitations (Schmeichel & Bissell 2003, Prestwich 2008). Animals are inherently different than humans, often having distinct physiologies, metabolic functions, and immune systems. Furthermore, xenograft
mouse models have compromised immune systems, limiting investigations of the tumor microenvironment and immunotherapies. On the other hand, 2D cultures fail to recapitulate the heterogeneity of human tumors or the complex 3D architecture of in vivo tissues. In addition, the synthetic mechanical properties and surface topographies of in vitro culture vessels place stresses on cells that drive phenotypic and even genetic changes (Ben-David et al. 2019). These differences in accurately recapitulating human tissue physiology can result in experimental findings that do not represent biological outcomes in humans. For example, drug responses and metabolism can vary drastically between some 2D and 3D biological systems (Skardal et al. 2015, 2016b, 2017a, 2020). In many applications, bioengineered 3D human models, such as organoids and organ-on-a-chip systems offer complementary representations of human physiology compared to traditional models (Bhise et al. 2014, Polini et al. 2014).
Spheroids without ECM
Three-dimensional tumor models occupy a range of complex platforms that recapitulate various aspects of 3D architecture and cell-cell or cell-ECM interactions. The simplest models are spheroids comprised of single cell line populations with no ECM component. These are generally formed using hanging drop cultures or low adhesion round-bottom well plates, and occasionally using rotating wall vessel (RWV) bioreactors or spinner flasks (Fig. 1A). In these scenarios, the cells are unable to adhere to a scaffold and instead aggregate together (Mehta et al. 2012). In endocrine cancers, spheroids - or tumorspheres - have been utilized in several studies. In the case of thyroid cancer, cell line-based spheroids were used as part of much larger study to demonstrate that the knockdown of the metastasis suppressor gene RCAN1-4 resulted in increased proliferation (Wang et al. 2017). In ACC, spheroids formed from the NCI-H295R cell line were deployed to test a drug delivery strategy in which the drug mitotane was delivered in micelles (Haider et al. 2020). To increase complexity, spheroids incorporating multiple cell lines can also be studied. Although such investigations have yet to be pursued for endocrine tumors, such experiments can begin to recapitulate some aspects of tumor cell heterogeneity
A
B
Cell Encapsulation in Basement Membrane Extract Hydrogel
Organoids
Homogeneous Spheroid
Heterogeneous Spheroid
Basement Membrane Extract Organoids
C
D
Labeled Tumor Cell
Tumor Biospecimen
Matrix-Filled Channels
Cytokines/Chemokines to Drive Gradient Formation and Cell Migration
Cell Line
Engineered Hydrogel Tumor Construct/Organoid
Directed 3D Tumor Invasion/ Migration Model
Recirculating Media Flow
E
F
Engrafted Metastatic Tumor cells
Labeled Tumor Cells in Circulation
2
Downstream Tissue Organoids
Mets
Labeled 3D Tumor Organoid
Flow
Microfluidic Two-Organoid Metastasis Model
Microfluidic Multi-Organoid Metastasis Model
observed in patient-derived models (Sivakumar et al. 2020). A limitation of experiments implementing spheroids is that not all primary cells are proficient at self-assembling through cell-cell interactions to form spheroids. Furthermore, these cultures are often limited to 1-2 weeks in timeframe due to the lack of ECM components.
Basement membrane extract biomaterials
As an alternative, hydrogel biomaterials can be used to encapsulate cells, removing the requirement of self-assembly. In cancer biology, the most common approach is to encapsulate tumor cells - either cell
lines or patient-derived tumor cells - in basement membrane extract (BME) hydrogels such as Matrigel, the industry leader. Matrigel, and most other BMEs, is ECM isolated from murine sarcoma tumors that has been decellularized and processed. Due to their origin, these BMEs contain large amounts of cytokines, chemokines, and growth factors and as a result are incredibly potent. BMEs effectively drive self-assembly of tumor cell cultures into organoids by providing signals from the BME (Fig. 1B). In addition, because of BME potency, such organoids can be made from most tumor types - even otherwise difficult to maintain tumor cells - and passaged for extended periods of time. While its use is nearly ubiquitous across laboratories working with
3D tumor models, its composition - undefined growth factors, cytokines, and likely miRNAs and exosomes (Panek et al. 2018) - pose a barrier for translational efforts that require eventual FDA approval to move toward clinical use, either for direct use in patients or as patient- specific diagnostic tools. Furthermore, Matrigel-based components have been shown to contaminate recovered organoid material, confounding proteomic analyses (Wang et al. 2022c). Consequently, these limitations must be considered when implementing Matrigel-cultured cells for any subsequent clinical applications.
Engineered hydrogel biomaterials
As alternatives to Matrigel, some laboratories synthesize ECM-based materials from their individual components. These materials include single component materials such as raw type I collagen (Baltazar et al. 2022), photo- crosslinkable collagen (Berger et al. 2020), gelatin (Pedron et al. 2015, Pedron et al. 2017), or more complex biomaterials such as hyaluronic acid (HA) (Wang et al. 2022a), synthetic polyethylene glycol (Jabbari et al. 2015), or self-assembling peptide hydrogels (Hainline et al. 2019). These biomaterials are considerably simplified compared to BMEs like Matrigel and lack growth factors and cytokines found in BMEs. As a result, these simple hydrogels are well suited for short-term investigations of tumor cell lines in 3D (Fig. 1C). Long-term passage of cell lines or maintenance of patient-derived tumor cells is often more challenging. In order to overcome these difficulties; however, many labs add well-defined components, such as growth factors or cytokines, to simple hydrogels in order to support the growth of patient-derived tumor cells. Determining growth factor or cytokine supplements that support maintenance of patient-derived tumor cells in simple hydrogels has been an important direction of current research.
Defined hydrogel biomaterial systems can also be successful in creating patient-derived tumor organoids (PTOs) and other 3D tumor platforms. With combined expertise in biomaterials, biofabrication, and ECM hydrogel technologies based on chemically modified polymers and proteins such as HA, collagen, gelatin, and adhesion proteins and peptides with complementary chemical groups for cross-linking (Skardal et al. 2012, Skardal 2016, Clark et al. 2019, Kyriakopoulou et al. 2023), a broad range of 3D tissue constructs have been generated (Skardal et al. 2017b, Zhang et al. 2017). These include PTOs, which can retain the important heterogeneity of in vivo tumors (Dedhia et al. 2016, Rodrigues et al. 2021). Our team generated PTOs from a multitude of tumor types, including intestinal cancers, mesothelioma, melanoma, sarcoma, Merkel cell carcinoma, lung adenocarcinoma, and gliomas (Mazzocchi et al. 2018, Forsythe et al. 2019, Mazzocchi et al. 2019, Maloney et al. 2020, Votanopoulos et al. 2019a, 2020). Furthermore, by integrating microfluidic device technologies with organoids and 3D models, organ-on-a-chip - or in the
case of cancer research, tumor-on-a-chip - systems can be generated (Fig. 1D) (Bhise et al. 2014, Skardal et al. 2016a, Liu et al. 2021, Zheng et al. 2016). These enable the introduction of fluid flow, an important environmental variable in many biological phenomena. Introduction of microfluidic devices further advances the complexity of such tumor models, thus generating a variety of tumor- on-a-chip platforms (Mazzocchi et al. 2018, Shirure et al. 2018, Rajan et al. 2020a,b). These have been deployed across a number of applications, including drug and toxin screening in organoids (Skardal et al. 2017a, Zhang et al. 2017, Rajan et al. 2020b, Skardal et al. 2020), modeling of tumor metastasis (Fig. 1E and F) (Skardal et al. 2016b, Aleman & Skardal 2018, Aleman et al. 2019), and correlation of drug responses between patients and PTOs derived from those patients’ tumors (Mazzocchi et al. 2018, Votanopoulos et al. 2019a, 2020).
Implementation of organoids and other 3D platforms in endocrine tumors
Tumor organoid and other 3D tumor platforms have been deployed widely in common cancers such as breast cancer, lung cancer, melanoma, and colorectal cancer. Until recently, there have been a limited number of studies in which organoids and other 3D in vitro model systems have been generated from endocrine tumors. Here we highlight recent notable advancements in generating organoids from endocrine tumors including rare endocrine cancers.
Thyroid cancer
There have been a number of studies using organoid or organoid-like technologies in thyroid cancer, especially differentiated thyroid cancers, given their higher incidence compared to other endocrine tumors such as neuroendocrine tumors (Chew et al. 2020). An early example of 3D culture of thyroid cancer employed papillary thyroid cancer (PTC) and ATC cell lines as well as primary patient-derived papillary and follicular thyroid cancer (FTC) tissues to generate spheroids by growing aggregates on agarose. These spheroids were then cocultured with endothelial cells to demonstrate that FTC and ATC cells migrated out of spheroids toward endothelial cells more quickly than PTC cells (Grimm et al. 1995). In later studies, the FTC cell line, FTC-133, was found to generate larger spheroids under microgravity conditions compared to the normal thyroid cell line Nthy-ori 3-1 due to increased expression of pro- growth and angiogenesis factors (Kopp et al. 2015). Cell line spheroids from multiple thyroid cancer cell lines were used to parallel murine models and demonstrate that knocking out RCAN1-4 in thyroid cancer cells results in highly metastatic cells with increased proliferative capabilities (Wang et al. 2017). Furthermore, culture
of ATC cell lines in 3D collagen type I demonstrated that suppression of membrane type 1 matrix metalloproteinase (MT1-MMP) reduced proliferation and invasion, while suppressing ERK signaling (Yoshida et al. 2020). These studies demonstrate the utility of even cell line-based 3D platforms when used to complement 2D cell cultures and murine studies.
Matrigel has prominently been used in thyroid cancer organoid based studies. For example, in one Matrigel-based study, PTC organoids from patient specimens were generated to serve as a potential diagnostic tool for radioactive iodine (I131)-resistant patients. Substantial differences in gene and protein expression were seen between the PTC organoids and radioactive iodine-refractory thyroid cancer organoids, illuminating potential biomarkers that could allow early identification of I131-resistent patients prior to treatment (Sondorp et al. 2020). In another Matrigel-based study, PTC organoids, which could be passaged in vitro long term, demonstrated patient- specific genetic landscape and drug responses (Chen et al. 2021). In a study using PTC or ATC cell lines, spheroids were generated in Matrigel in order to test responses to the drug dabrafenib. This study demonstrated that morphology, cell signaling, and drug sensitivities to dabrafenib could vary from 2D to 3D culture (Lee et al. 2020). Another team used Matrigel to generate patient-derived PTC organoids and normal thyroid organoids to compare to novel Hashimoto’s thyroiditis organoids. In this study, the use of Matrigel- cultured organoids in conjunction with primary patient tissues demonstrated a stepwise progression of expression of certain biomarkers from normal thyroid to Hashimoto’s thyroiditis to papillary thyroid cancer (Xiao et al. 2021). Matrigel-based PTC organoids have also been cocultured with tumor-infiltrating T lymphocytes resulting in increased interferon-y and TNF-a secretion from the T lymphocytes (Baregamian et al. 2023).
With regards to medullary thyroid cancer (MTC), a neuroendocrine tumor of the thyroid, the MTC cell lines, TT and MZ-CRC-1, have been propagated in bioreactors. However, additional studies are needed to determine how these cultures compare to 2D culture. Matrigel- based MTC patient-derived organoids have been generated, but these were only able to secrete calcitonin after 2-3 passages, indicating a potential limitation to short term experiments using these MTC organoids (Baregamian et al. 2023, Sondorp et al. 2023).
By combining the aforementioned thyroid organoid and 3D cultures with microfluidic organ-on-a-chip or metastasis-on-a-chip platforms (Skardal et al. 2016b, Aleman & Skardal 2018), one can capture stages of metastasis in vitro. Because of the transparent walls of a microfluidic device, compared to an animal model, there is unparalleled ability for direct observation of metastasizing cells from tumor to tissue organoids. These advantages enable evaluation of the effect of drugs or genetic manipulations on metastasis kinetics of
tumor cells into target site organoids. Such technology platforms have immense utility in future research, and we are actively integrating thyroid cancer organoids into these platforms (Nairon et al. 2023).
Neuroendocrine tumors
NETs most commonly originate in the gastrointestinal system, lung, and pancreas but also include thyroid and adrenal NETs such as MTC and pheochromocytoma (described in the indicated section), respectively. As with other tumor types, 3D culture of NETs include development of spheroids using cell lines or ultralow- attachment cultures or bioreactors to culture primary patient tissue (Wong et al. 2012, Bresciani et al. 2019, Ear et al. 2019, April-Monn et al. 2021, Herring et al. 2021, Gulde et al. 2022). Together, these studies demonstrated differential drug sensitivities between 2D and 3D cultures and variable drug sensitivities in patient- derived tumor organoids. Matrigel-based investigations using colon NET organoids demonstrated similar synaptophysin and chromogranin A expression to the primary tumor and a distinct gene signature from colon adenocarcinoma organoids (Fujii et al. 2016). Similarly, patient-derived pancreatic NET organoids cultured in Matrigel demonstrated variable sensitivities to sunitinib, everolimus, and temozolomide (April- Monn et al. 2021). Another study of Matrigel-based NET organoids, which developed 22 neuroendocrine carcinoma (NEC) and 3 NET organoid lines that resembled corresponding primary patient tumors. This study demonstrated that combined deletion of TP53 and RB1 conferred features of neuroendocrine neoplasm to normal colon organoids (Kawasaki et al. 2020). Other Matrigel studies focused on a rare NET, duodenal gastrinoma by demonstrating that normal duodenal organoids expressed NET genes, SYO, CHGA, and NKX6.3 after treatment with TNF-a (Rico et al. 2021). Together, these 3D models have provided unique insights into NET biology and are likely to become a routine part of preclinical evaluation of therapeutic targets in NET.
Pheochromocytoma/paraganglioma
As described earlier, studies in pheochromocytoma and paraganglioma (PPGL) are rare. Spheroids generated from murine PPGL cell lines (monocyte chemoattractant protein (MPC)/monocyte chemoattractant protein/3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were implemented to demonstrate a combinatorial sensitivity to BYL719 and everolimus (Fankhauser et al. 2019). Similarly, patient-derived spheroids from four PPGL patients demonstrated differential drug sensitivity (Wang et al. 2022b). Patient-derived PPGL organoids demonstrated hormonal profiles similar to the corresponding primary tumor (Calucho et al. 2023). Generation of additional cell line or patient- derived organoids and pairing such models with
metastasis-on-a-chip platforms, has the potential to improve our understanding of these diseases including drivers, biomarkers of malignancy, and targeted therapies.
Adrenocortical carcinoma
With respect to ACC, few ACC organoid or 3D tumor platform studies have been published from either cell lines or patient-derived tissues. One study utilized 3D ACC cell line spheroids to test drug delivery efficiency of mitotane versus mitotane delivered in the form of micelles (Haider et al. 2020). Another study demonstrated that Matrigel-based patient-derived tumor organoids initially secreted cortisol, but the secretion was nearly or completely abrogated by passage 5 (Baregamian et al. 2023). The parental H295 ACC cell line, which was established in 1980, is a stalwart preclinical model to study ACC. H295R cell line spheroids secreted higher levels of aldosterone (Lichtenauer et al. 2013). In addition, H295R spheroid generation was shown to be affected by estrogen receptor a expression or treatment with sorafenib (Avena et al. 2022). To date, no 3D studies implementing extracellular matrix have been performed using the H295 cell line or its more commonly used derivative, H295R (Wang & Rainey 2012). Our team created viable 3D H295R tumor constructs using chemically modified hyaluronic acid and gelatin hydrogels and found that the H295R tumor constructs
express a variety of biomarkers expected in and associated with ACC, including ß-catenin, steroidal factor 1 (SF-1), inhibin alpha, and IGF2 (Dedhia et al. 2023). These cell linetumor constructs proliferated over time and secreted cortisol in response to stimulation similar to 2D cell culture. These 3D ACC tumor constructs were significantly less responsive to common ACC therapies such as mitotane and EDP (etoposide, doxorubicin, and cisplatin) compared to 2D cell culture suggesting that 3D architecture may influence H295R drug sensitivity and therefore may represent a more accurate model for future drug screening efforts. Finally, we developed a metastasis-on-a-chip model implementing the H295R cell line and target lung organoids and found that inhibition of matrix metalloproteinases, which are highly active in H295R cells, decreased H295R invasion into lung organoids (Dedhia et al. 2023).
In addition to working with the H295R ACC cell line, our team has generated viable ACC PTOs from clinical biospecimens (Sivakumar et al. 2021, 2023). We have performed a series of characterization studies, including immunohistochemistry stains for the biomarkers described earlier, viability assessment over time (Fig. 2G), followed by deployment in drugs screening assays and steroid quantification assays (Fig. 2H). Future studies will assess the efficacy of targeting specific pathways, including Wnt, IGF2, and TP53, in a concerted manner to develop new therapeutic strategies. In addition, we are utilizing
A
H295R Cell Line ACC Organoids
B
C
D
E
+
H295R ACC Cells
400 um
ECM Hydrogel
100 um
100 um
100 μm
Highly viable H295R ACC tumor constructs on day 5 - LIVE/DEAD
Beta-Catenin/DAPI
Steroidal Factor 1/ Alpha Inhibin
IGF2/DAPI
ACC Patient-Derived Tumor Organoids
F
G
H
ACC PTO Drug Response
DA- Mitotane, 8 ug/mL
2.0×107
DB- Mitotane, 20 ug/mL
Relative Viability ATP Activity (RLUs)
1.5×107
T
DC- EDP, (0.25 ug/ml E, 0.1 ug/mL D, 0.1 ug/mL P)
DD- EDP, (25 ug/ml. E, 10 ug/mL D, 10 ug/mL P)
+
1.0×107
5.0×106
DE- Mitotane, 8 ug/mL+EDP, (0.25 ug/mL E, 0.1 ug/mL. D, 0.1 ug/mL. P)
ACC Tumor
**
Patient-Derived Tumor Cells
DF-Mitotane, 20 ug/mL+ EDP, (25 ug/mL E, 10 ug/mL D. 10 ug/mL. P)
400 um
0.0
ECM Hydrogel
Control
DA
08
DC
00
DE
of
Drug Treatments
Figure 2 Generation of cell line and patient-derived adrenocortical carcinoma organoids. (A) H295R ACC cell line organoids were created by encapsulating H295R cells within covalently cross-linked hyaluronic acid and collagen hydrogels. (B) LIVE/DEAD staining shows high viability on day 5 of culture. Green - Calcein AM-stained viable cells; red - ethidium homodimer 1-stained dead cell nuclei. (C-E) Immunofluorescent staining of common ACC biomarkers, including (C) beta-catenin, (D) steroidal factor 1 and alpha inhibin, and (E) IGF2. (F) ACC patient-derived tumor organoids were created by encapsulating cells from ACC tumor biospecimens in the same hydrogel system. (G) LIVE/DEAD staining shows high viability of ACC PTOs. (H) An example of deployment of ACC PTOs in a drug response experiment using clinically relevant drugs and drug cocktails. Statistical significance: * P < 0.05; ** P < 0.01.
our metastasis-on-a-chip platform to recapitulate ACC metastasis (Dedhia et al. 2023) and identify key differences using RNA sequencing to between tumor cells that can and cannot metastasize in order to identify novel targets of intervention. We hope that with these ongoing efforts we will gain an increased understanding of a type of cancer that compared to most others is poorly understood.
A look to the future
The bulk of cancer research has been based on 2D cancer cell line cultures and animal tumor models. These platforms have yielded incredible and crucial discoveries that have led to improved clinical care for cancer patients. Three-dimensional in vitro human tumor models differ from the status quo in several particular distinctions: (i) They do not use animal derived components which can alter human physiology; (ii) They are inherently 3D, unlike 2D cell cultures that immediately drive cells to change; and (iii) They more closely mimic the tissue biospecimens from which they were derived because of advances in biofabrication technologies. However, like traditional models, 3D human cell-based tumor models still have limitations. Most organoids and other 3D tumor models are overly simplistic and fail to recapitulate the complexity and heterogeneity of in vivo human tumors. Ongoing work aims to address these limitations. For example, numerous laboratories are integrating various immune cell populations to enable study of tumor-immune interactions and screening of immunotherapies (Dijkstra et al. 2018, Votanopoulos et al. 2019b).
These model systems - some of which have been applied to other more common cancer types - are now being applied to the heterogeneous family that comprises endocrine tumors. Between existing and developing biomaterials to support 3D tumor cultures and advancement in microfluidic device-based tumor- on-a-chip and organ-on-a-chip platform technologies, researchers in the endocrine tumor space have a toolbox of new tools to develop preclinical models and to make an impact mechanistically and clinically.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector.
Acknowledgements
AS and PHD acknowledge funds from NIH grant R21CA277083 and the Ohio State University Comprehensive Cancer Center. PHD acknowledges funds from the Society of University Surgeons and the American Association
of Endocrine Surgeons. We acknowledge the use of BioRender Scientific Image and Illustration Software for generating cartoon components of the figures in this document.
References
Aleman J & Skardal A 2018 A multi-site metastasis-on-a-chip microphysiological system for assessing metastatic preference of cancer cells. Biotechnology and Bioengineering 116 936-944. (https://doi. org/10.1002/bit.26871)
Aleman J, George SK, Herberg S, Devarasetty M, Porada CD, Skardal A & Almeida-Porada G 2019 Deconstructed microfluidic bone marrow On-A- chip to study normal and malignant hemopoietic cell-niche interactions. Small 15 e1902971. (https://doi.org/10.1002/smll.201902971)
April-Monn SL, Wiedmer T, Skowronska M, Maire R, Schiavo Lena M, Trippel M, Di Domenico A, Muffatti F, Andreasi V, Capurso G, et al. 2021 Three-dimensional primary cell culture: a novel preclinical model for pancreatic neuroendocrine tumors. Neuroendocrinology 111 273-287. (https://doi.org/10.1159/000507669)
Avena P, De Luca A, Chimento A, Nocito MC, Sculco S, La Padula D, Zavaglia L, Giulietti M, Hantel C, Sirianni R, et al. 2022 Estrogen related receptor alpha (ERRalpha) a bridge between metabolism and adrenocortical cancer progression. Cancers 14. (https://doi.org/10.3390/ cancers14163885)
Baltazar T, Kajave NS, Rodriguez M, Chakraborty S, Jiang B, Skardal A, Kishore V, Pober JS & Albanna MZ 2022 Native human collagen type I provides a viable physiologically relevant alternative to xenogeneic sources for tissue engineering applications: a comparative in vitro and in vivo study. Journal of Biomedical Materials Research 110 2323-2337. (https://doi.org/10.1002/jbm.b.35080)
Baregamian N, Sekhar KR, Krystofiak ES, Vinogradova M, Thomas G, Mannoh E, Solorzano CC, Kiernan CM, Mahadevan-Jansen A, Abumrad N, et al. 2023 Engineering functional 3-dimensional patient-derived endocrine organoids for broad multiplatform applications. Surgery 173 67-75. (https://doi.org/10.1016/j.surg.2022.09.027)
Belfiore A & Perks CM 2013 Grand challenges in cancer endocrinology: endocrine related cancers, an expanding concept. Frontiers in Endocrinology 4 141. (https://doi.org/10.3389/fendo.2013.00141)
Ben-David U, Beroukhim R & Golub TR 2019 Genomic evolution of cancer models: perils and opportunities. Nature Reviews. Cancer 19 97-109. (https://doi.org/10.1038/s41568-018-0095-3)
Berger AJ, Renner CM, Hale I, Yang X, Ponik SM, Weisman PS, Masters KS & Kreeger PK 2020 Scaffold stiffness influences breast cancer cell invasion via EGFR-linked Mena upregulation and matrix remodeling. Matrix Biology 85-86 80-93. (https://doi.org/10.1016/j. matbio.2019.07.006)
Bhise NS, Ribas J, Manoharan V, Zhang YS, Polini A, Massa S, Dokmeci MR & Khademhosseini A 2014 Organ-on-a-chip platforms for studying drug delivery systems. Journal of Controlled Release 190 82-93. (https://doi.org/10.1016/j.jconrel.2014.05.004)
Bresciani G, Hofland LJ, Dogan F, Giamas G, Gagliano T & Zatelli MC 2019 Evaluation of spheroid 3D culture methods to study a pancreatic neuroendocrine neoplasm cell line. Frontiers in Endocrinology 10 682. (https://doi.org/10.3389/fendo.2019.00682)
Calucho M, Cheng Z, Nguyen HT, Shihabi AA, Gonzalez-Cantu H, Guo Q, Thaker M, Bechmann N, Eisenhofer G, Ding Y, et al. 2023 Abstract 195: establishment and validation of pheochromocytoma organoids for high- throughput drug screening. Cancer Research 83 195. (https://doi. org/10.1158/1538-7445.AM2023-195)
Chen D, Tan Y, Li Z, Li W, Yu L, Chen W, Liu Y, Liu L, Guo L, Huang W, et al. 2021 Organoid cultures derived from patients with papillary
thyroid cancer. Journal of Clinical Endocrinology and Metabolism 106 1410-1426. (https://doi.org/10.1210/clinem/dgab020)
Chew D, Green V, Riley A, England RJ & Greenman J 2020 The changing face of in vitro culture models for thyroid cancer research: a systematic literature review. Frontiers in Surgery 7 43. (https://doi.org/10.3389/ fsurg.2020.00043)
Clark CC, Aleman J, Mutkus L & Skardal A 2019 A mechanically robust thixotropic collagen and hyaluronic acid bioink supplemented with gelatin nanoparticles. Bioprinting 16. (https://doi.org/10.1016/j.bprint.2019.e00058)
Darba J & Marsa A 2019 Exploring the current status of neuroendocrine tumours: a population-based analysis of epidemiology, management and use of resources. BMC Cancer 19 1226. (https://doi.org/10.1186/ s12885-019-6412-8)
Dasari A, Shen C, Halperin D, Zhao B, Zhou S, Xu Y, Shih T & Yao JC 2017 Trends in the incidence, prevalence, and survival outcomes in patients with neuroendocrine tumors in the United States. JAMA Oncology 3 1335-1342. (https://doi.org/10.1001/jamaoncol.2017.0589)
Dedhia PH, Bertaux-Skeirik N, Zavros Y & Spence JR 2016 Organoid models of human gastrointestinal development and disease. Gastroenterology 150 1098-1112. (https://doi.org/10.1053/j. gastro.2015.12.042)
Dedhia PH, Sivakumar H, Rodriguez MA, Nairon KG, Zent JM, Zheng X, Jones K, Popova LV, Leight JL & Skardal A 2023 A 3D adrenocortical carcinoma tumor platform for preclinical modeling of drug response and matrix metalloproteinase activity. Scientific Reports 13 15508. (https://doi.org/10.1038/s41598-023-42659-0)
Devarasetty M, Mazzocchi AR & Skardal A 2018 Application of bioengineered 3D tissue and tumor organoids in drug development and precision medicine: current and future. BioDrugs 32 53-68. (https://doi. org/10.1007/s40259-017-0258-x)
Dijkstra KK, Cattaneo CM, Weeber F, Chalabi M, van de Haar J, Fanchi LF, Slagter M, van der Velden DL, Kaing S, Kelderman S, et al. 2018 Generation of tumor-reactive T cells by co-culture of peripheral blood lymphocytes and tumor organoids. Cell 174 1586-1598.e12. (https://doi. org/10.1016/j.cell.2018.07.009)
Ear PH, Li G, Wu M, Abusada E, Bellizzi AM & Howe JR 2019 Establishment and characterization of small bowel neuroendocrine tumor spheroids. Journal of Visualized Experiments 152. (https://doi. org/10.3791/60303)
Fankhauser M, Bechmann N, Lauseker M, Goncalves J, Favier J, Klink B, William D, Gieldon L, Maurer J, Spottl G, et al. 2019 Synergistic highly potent targeted drug combinations in different pheochromocytoma models including human tumor cultures. Endocrinology 160 2600-2617. (https://doi.org/10.1210/en.2019-00410)
Forsythe S, Mehta N, Devarasetty M, Sivakumar H, Gmeiner W, Soker S, Votanopoulos K & Skardal A 2019 Development of a colorectal cancer 3D micro-tumor construct platform from cell lines and patient tumor biospecimens for standard-of-care and experimental drug screening. Annals of Biomedical Engineering 48 940-952. (https://doi.org/10.1007/ s10439-019-02269-2)
Fujii M, Shimokawa M, Date S, Takano A, Matano M, Nanki K, Ohta Y, Toshimitsu K, Nakazato Y, Kawasaki K, et al. 2016 A colorectal tumor organoid library demonstrates progressive loss of niche factor requirements during tumorigenesis. Cell Stem Cell 18 827-838. (https:// doi.org/10.1016/j.stem.2016.04.003)
Grimm D, Bauer J, Kromer E, Steinbach P, Riegger G & Hofstadter F 1995 Human follicular and papillary thyroid carcinoma cells interact differently with human venous endothelial cells. Thyroid 5 155-164. (https://doi.org/10.1089/thy.1995.5.155)
Gulde S, Foscarini A, April-Monn SL, Genio E, Marangelo A, Satam S, Helbling D, Falconi M, Toledo RA, Schrader J, et al. 2022 Combined
targeting of pathogenetic mechanisms in pancreatic neuroendocrine tumors elicits synergistic antitumor effects. Cancers 14. (https://doi. org/10.3390/cancers14225481)
Haider MS, Schreiner J, Kendl S, Kroiss M & Luxenhofer R 2020 A micellar mitotane formulation with high drug-loading and solubility: physico-chemical characterization and cytotoxicity studies in 2D and 3D in vitro tumor models. Macromolecular Bioscience 20 e1900178. (https:// doi.org/10.1002/mabi.201900178)
Hainline KM, Gu F, Handley JF, Tian YF, Wu Y, de Wet L, Vander Griend DJ & Collier JH 2019 Self-assembling peptide gels for 3D prostate cancer spheroid culture. Macromolecular Bioscience 19 e1800249. (https://doi. org/10.1002/mabi.201800249)
Herring B, Jang S, Whitt J, Goliwas K, Aburjania Z, Dudeja V, Ren B, Berry J, Bibb J, Frost A, et al. 2021 Ex Vivo modeling of human neuroendocrine tumors in tissue surrogates. Frontiers in Endocrinology 12 710009. (https://doi.org/10.3389/fendo.2021.710009)
Jabbari E, Sarvestani SK, Daneshian L & Moeinzadeh S 2015 Optimum 3D matrix stiffness for maintenance of cancer stem cells is dependent on tissue origin of cancer cells. PLoS One 10 e0132377. (https://doi. org/10.1371/journal.pone.0132377)
Kawasaki K, Toshimitsu K, Matano M, Fujita M, Fujii M, Togasaki K, Ebisudani T, Shimokawa M, Takano A, Takahashi S, et al. 2020 An organoid biobank of neuroendocrine neoplasms enables genotype- phenotype mapping. Cell 183 1420-1435.e21. (https://doi.org/10.1016/j. cell.2020.10.023)
Kopp S, Warnke E, Wehland M, Aleshcheva G, Magnusson NE, Hemmersbach R, Corydon TJ, Bauer J, Infanger M & Grimm D 2015 Mechanisms of three-dimensional growth of thyroid cells during long- term simulated microgravity. Scientific Reports 5 16691. (https://doi. org/10.1038/srep16691)
Kyriakopoulou K, Koutsakis C, Piperigkou Z & Karamanos NK 2023 Recreating the extracellular matrix: novel 3D cell culture platforms in cancer research. FEBS Journal 290 5238-5247. (https://doi.org/10.1111/ febs.16778)
Latteyer S, Tiedje V, Schilling B & Fuhrer D 2016 Perspectives for immunotherapy in endocrine cancer. Endocrine-Related Cancer 23 R469-R484. (https://doi.org/10.1530/ERC-16-0169)
Lee MA, Bergdorf KN, Phifer CJ, Jones CY, Byon SY, Sawyer LM, Bauer JA & Weiss VL 2020 Novel three-dimensional cultures provide insights into thyroid cancer behavior. Endocrine-Related Cancer 27 111-121. (https:// doi.org/10.1530/ERC-19-0374)
Lichtenauer UD, Shapiro I, Osswald A, Meurer S, Kulle A, Reincke M, Riepe F & Beuschlein F 2013 Characterization of NCI-H295R cells as an in vitro model of hyperaldosteronism. Hormone and Metabolic Research 45 124-129. (https://doi.org/10.1055/s-0032-1323810)
Liu X, Fang J, Huang S, Wu X, Xie X, Wang ], Liu F, Zhang M, Peng Z & Hu N 2021 Tumor-on-a-chip: from bioinspired design to biomedical application. Microsystems and Nanoengineering 7 50. (https://doi. org/10.1038/s41378-021-00277-8)
Maloney E, Clark C, Sivakumar H, Yoo K, Aleman J, Rajan SAP, Forsythe S, Mazzocchi A, Laxton AW, Tatter SB, et al. 2020 Immersion bioprinting of tumor organoids in multi-well plates for increasing chemotherapy screening throughput. Micromachines (Basel) 11. (https://doi. org/10.3390/mi11020208)
Mazzocchi AR, Soker S & Skardal A 2017 Biofabrication technologies for developing in vitro tumor models. In Tumor Organoids, pp 51-70. Eds. S Soker, A Skardal. Berlin, Germany: Springer Nature. (https://doi. org/10.1007/978-3-319-60511-1_4)
Mazzocchi AR, Rajan SAP, Votanopoulos KI, Hall AR & Skardal A 2018 In vitro patient-derived 3D mesothelioma tumor organoids facilitate patient-centric therapeutic screening. Scientific Reports 8 2886. (https:// doi.org/10.1038/s41598-018-21200-8)
Mazzocchi A, Devarasetty M, Herberg S, Petty WJ, Marini F, Miller LD, Kucera GL, Dukes DK, Ruiz J, Skardal A, et al. 2019 Pleural Effusion Aspirate for use in 3D Lung Cancer Modeling and Chemotherapy Screening. ACS Biomaterials Science and Engineering 5 1937-1943. (https://doi.org/10.1021/acsbiomaterials.8b01356)
Mehta G, Hsiao AY, Ingram M, Luker GD & Takayama S 2012 Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy. Journal of Controlled Release 164 192-204. (https://doi.org/10.1016/j.jconrel.2012.04.045)
Nairon KG, Rajan N, Ringel MD & Skardal A 2023 RCAN1-4 suppresses metastatic invasion and tumor cell proliferation in a 3D thyroid metastasis-on-a-chip model. In Tissue Engineering & Regenerative Medicine International Society - Americas 2023 Annual Conference and Exhibition. Boston, MA, USA: TERMIS. (https://doi.org/10.1089/ten. tea.2023.29041.abstracts)
Panek M, Grabacka M & Pierzchalska M 2018 The formation of intestinal organoids in a hanging drop culture. Cytotechnology 70 1085-1095. (https://doi.org/10.1007/s10616-018-0194-8)
Pedron S, Becka E & Harley BA 2015 Spatially gradated hydrogel platform as a 3D engineered tumor microenvironment. Advanced Materials 27 1567-1572. (https://doi.org/10.1002/adma.201404896)
Pedron S, Polishetty H, Pritchard AM, Mahadik BP, Sarkaria JN & Harley BAC 2017 Spatially graded hydrogels for preclinical testing of glioblastoma anticancer therapeutics. MRS Communications 7 442-449. (https://doi.org/10.1557/mrc.2017.85)
Polini A, Prodanov L, Bhise NS, Manoharan V, Dokmeci MR & Khademhosseini A 2014 Organs-on-a-chip: a new tool for drug discovery. Expert Opinion on Drug Discovery 9 335-352. (https://doi.org/1 0.1517/17460441.2014.886562)
Prestwich GD 2008 Evaluating drug toxicity and efficacy in three dimensions: using synthetic extracellular matrices in drug discovery. Accounts of Chemical Research 41 139-148. (https://doi.org/10.1021/ ar7000827)
Rajan SAP, Skardal A & Hall AR 2020a Multi-domain photopatterned 3D tumor constructs in a micro-physiological system for analysis, quantification, and isolation of infiltrating cells. Advanced Biosystems 4 e1900273. (https://doi.org/10.1002/adbi.201900273)
Rajan SAP, Aleman J, Wan M, Pourhabibi Zarandi N, Nzou G, Murphy S, Bishop CE, Sadri-Ardekani H, Shupe T, Atala A, et al. 2020b Probing prodrug metabolism and reciprocal toxicity with an integrated and humanized multi-tissue organ-on-a-chip platform. Acta Biomaterialia 106 124-135. (https://doi.org/10.1016/j.actbio.2020.02.015)
Rico K, Duan S, Pandey RL, Chen Y, Chakrabarti JT, Starr J, Zavros Y, Else T, Katona BW, Metz DC, et al. 2021 Genome analysis identifies differences in the transcriptional targets of duodenal versus pancreatic neuroendocrine tumours. BMJ Open Gastroenterology 8. (https://doi. org/10.1136/bmjgast-2021-000765)
Rodrigues J, Heinrich MA, Teixeira LM & Prakash J 2021 3D in vitro model (R)evolution: unveiling tumor-stroma interactions. Trends in Cancer 7 249-264. (https://doi.org/10.1016/j.trecan.2020.10.009)
Schmeichel KL & Bissell MJ 2003 Modeling tissue-specific signaling and organ function in three dimensions. Journal of Cell Science 116 2377-2388. (https://doi.org/10.1242/jcs.00503)
Shirure VS, Bi Y, Curtis MB, Lezia A, Goedegebuure MM, Goedegebuure SP, Aft R, Fields RC & George SC 2018 Tumor-on-a-chip platform to investigate progression and drug sensitivity in cell lines and patient-derived organoids. Lab on a Chip 18 3687-3702. (https://doi.org/10.1039/c8lc00596f)
Sivakumar H, Devarasetty M, Kram DE, Strowd RE & Skardal A 2020 Multi-cell type glioblastoma tumor spheroids for evaluating sub- population-specific drug response. Frontiers in Bioengineering and Biotechnology 8 538663. (https://doi.org/10.3389/fbioe.2020.538663)
Sivakumar H, Miller BS, Nairon KG, Zheng XG, Phay JE, Kirschner LS, Skardal A & Dedhia PH 2021 Generation of an organoid model of adrenocortical carcinoma. American Association of Endocrine Surgeons 41st Annual Meeting. Virtual. AAES.
Sivakumar H, Dedhia PH & Skardal A 2023 Establishment of an organoid model for adrenal cortical carcinoma. In Tissue Engineering & Regenerative Medicine International Society - Americas 2023 Annual Conference and Exhibition. Boston, MA, USA: TERMIS. (https://doi. org/10.1089/ten.tea.2023.29041.abstracts)
Skardal A 2016 Biopolymers for in vitro tissue model biofabrication. In Biopolymers for Medical Applications. Eds. JM Ruso & PV Messina. Boca Raton, FL, USA: CRC Press (https://doi.org/10.1201/9781315368863).
Skardal A, Smith L, Bharadwaj S, Atala A, Soker S & Zhang Y 2012 Tissue specific synthetic ECM hydrogels for 3-D in vitro maintenance of hepatocyte function. Biomaterials 33 4565-4575. (https://doi. org/10.1016/j.biomaterials.2012.03.034)
Skardal A, Devarasetty M, Rodman C, Atala A & Soker S 2015 Liver- tumor hybrid organoids for modeling tumor growth and drug response in vitro. Annals of Biomedical Engineering 43 2361-2373. (https://doi. org/10.1007/s10439-015-1298-3)
Skardal A, Shupe T & Atala A 2016a Organoid-on-a-chip and body-on-a- chip systems for drug screening and disease modeling. Drug Discovery Today 21 1399-1411. (https://doi.org/10.1016/j.drudis.2016.07.003)
Skardal A, Devarasetty M, Forsythe S, Atala A & Soker S 2016b A reductionist metastasis-on-a-chip platform for in vitro tumor progression modeling and drug screening. Biotechnology and Bioengineering 113 2020-2032. (https://doi.org/10.1002/bit.25950)
Skardal A, Murphy SV, Devarasetty M, Mead I, Kang HW, Seol Y], Shrike ZY, Shin SR, Zhao L, Aleman J, et al. 2017a Multi-tissue interactions in an integrated three-tissue organ-on-a-chip platform. Scientific Reports 7 8837. (https://doi.org/10.1038/s41598-017-08879-x)
Skardal A, Murphy SV, Devarasetty M, Mead I, Kang HW, Seol YJ, Shrike Zhang Y, Shin SR, Zhao L, Aleman J, et al. 2017b Multi-tissue interactions in an integrated three-tissue organ-on-a-chip platform. Scientific Reports 7 8837. (https://doi.org/10.1038/s41598-017-08879-x)
Skardal A, Aleman J, Forsythe S, Rajan S, Murphy S, Devarasetty M, Pourhabibi Zarandi N, Nzou G, Wicks R, Sadri-Ardekani H, et al. 2020 Drug compound screening in single and integrated multi-organoid body-on-a-chip systems. Biofabrication 12 025017. (https://doi. org/10.1088/1758-5090/ab6d36)
Sondorp LHJ, Ogundipe VML, Groen AH, Kelder W, Kemper A, Links TP, Coppes RP & Kruijff S 2020 Patient-derived papillary thyroid cancer organoids for radioactive iodine refractory screening. Cancers 12. (https://doi.org/10.3390/cancers12113212)
Sondorp LHJ, Jager EC, Antunes IF, Maturi R, Jansen L, Zandee WT, Brouwers AH, Links TP, Coppes RP & Kruijff S 2023 Patient-derived medullary thyroid cancer organoids; a model for patient-tailored drug and PET-tracer screening. bioRxiv. (https://doi. org/10.1101/2023.09.18.558266)
Votanopoulos KI, Mazzocchi A, Sivakumar H, Forsythe S, Aleman J, Levine EA & Skardal A 2019a Appendiceal cancer patient-specific tumor organoid model for predicting chemotherapy efficacy prior to initiation of treatment: a feasibility study. Annals of Surgical Oncology 26 139-147. (https://doi.org/10.1245/s10434-018-7008-2)
Votanopoulos KI, Forsythe S, Sivakumar H, Mazzocchi A, Aleman J, Miller L, Levine E, Triozzi P & Skardal A 2019b Model of patient-specific immune-enhanced organoids for immunotherapy screening: feasibility study. Annals of Surgical Oncology 27 1956-1967. (https://doi. org/10.1245/s10434-019-08143-8)
Votanopoulos KI, Forsythe S, Sivakumar H, Mazzocchi A, Aleman J, Miller L, Levine E, Triozzi P & Skardal A 2020 Model of patient-specific
immune-enhanced organoids for immunotherapy screening: feasibility study. Annals of Surgical Oncology 27 1956-1967. (https://doi. org/10.1245/s10434-019-08143-8)
Wang T & Rainey WE 2012 Human adrenocortical carcinoma cell lines. Molecular and Cellular Endocrinology 351 58-65. (https://doi. org/10.1016/j.mce.2011.08.041)
Wang C, Saji M, Justiniano SE, Yusof AM, Zhang X, Yu L, Fernandez S, Wakely P, Jr, La Perle K, Nakanishi H, et al. 2017 RCAN1-4 is a thyroid cancer growth and metastasis suppressor. JCI Insight 2 e90651. (https:// doi.org/10.1172/jci.insight.90651)
Wang J, Xu W, Qian J, Wang Y, Hou G & Suo A 2022a Photo-crosslinked hyaluronic acid hydrogel as a biomimic extracellular matrix to recapitulate in vivo features of breast cancer cells. Colloids and Surfaces. B, Biointerfaces 209 112159. (https://doi.org/10.1016/j.colsurfb.2021.112159)
Wang K, Schutze I, Gulde S, Bechmann N, Richter S, Helm J, Lauseker M, Maurer J, Reul A, Spoettl G, et al. 2022b Personalized drug testing in human pheochromocytoma/paraganglioma primary cultures. Endocrine- Related Cancer 29 285-306. (https://doi.org/10.1530/ERC-21-0355)
Wang M, Yu H, Zhang T, Cao L, Du Y, Xie Y, Ji J & Wu J 2022c In-depth comparison of Matrigel dissolving methods on proteomic profiling of organoids. Molecular and Cellular Proteomics 21 100181. (https://doi. org/10.1016/j.mcpro.2021.100181)
Wong C, Vosburgh E, Levine AJ, Cong L & Xu EY 2012 Human neuroendocrine tumor cell lines as a three-dimensional model for the study of human neuroendocrine tumor therapy. Journal of Visualized Experiments 66 e4218. (https://doi.org/10.3791/4218)
Xiao H, Liang J, Liu S, Zhang Q, Xie F, Kong X, Guo S, Wang R, Fu R, Ye Z, et al. 2021 Proteomics and organoid culture reveal the underlying pathogenesis of Hashimoto’s thyroiditis. Frontiers in Immunology 12 784975. (https://doi.org/10.3389/fimmu.2021.784975)
Yoshida T, Suganuma N, Sato S, Toda S, Nakayama H, Masudo K, Okubo Y, Hayashi H, Yokose T, Koshikawa N, et al. 2020 Membrane type 1 matrix metalloproteinase regulates anaplastic thyroid carcinoma cell growth and invasion into the collagen matrix. Biochemical and Biophysical Research Communications 529 1195-1200. (https://doi. org/10.1016/j.bbrc.2020.06.043)
Zhang YS, Aleman J, Shin SR, Kilic T, Kim D, Mousavi Shaegh SA, Massa S, Riahi R, Chae S, Hu N, et al. 2017 Multisensor-integrated organs-on- chips platform for automated and continual in situ monitoring of organoid behaviors. PNAS 114 E2293-E2302. (https://doi.org/10.1073/ pnas.1612906114)
Zheng F, Fu F, Cheng Y, Wang C, Zhao Y & Gu Z 2016 Organ-on-a-chip systems: microengineering to biomimic living systems. Small 12 2253-2282. (https://doi.org/10.1002/smll.201503208)