|Year : 2021 | Volume
| Issue : 3 | Page : 174-182
Progress in the application of ovarian and fallopian tube organoids
Yi-Lin Dai1, Jin-Song Wei2, Jing Xu3, Ke Li1, Jing Wang3, Ran-Ran Cha1, Yu Kang3
1 Department of Gynecology, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011, China
2 State Key Laboratory of Genetic Engineering, School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, China
3 Department of Gynecology, Obstetrics and Gynecology Hospital, Fudan University; Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Shanghai 200011, China
|Date of Submission||12-Feb-2021|
|Date of Decision||21-Mar-2021|
|Date of Acceptance||13-Apr-2021|
|Date of Web Publication||30-Jul-2021|
Obstetrics and Gynecology Hospital, Fudan University, No. 419, Fangxie Road, Shanghai 200011
Source of Support: None, Conflict of Interest: None
As an innovative in vitro culture model, organoids have been established by cell sorting and subsequent culture in three-dimensional culture systems. Organoids can be derived from induced pluripotent stem cells or organ-restricted adult stem cells. Compared with traditional two-dimensional cell culture models and patient-derived xenograft models, organoids possess long-term genetic stability and can better retain the characteristics of source tissues or organs. These advantages have led to the increased use of ovarian and fallopian tube organoids in various fields of research, including cell differentiation and development, establishment of disease occurrence and progression models, tissue regeneration and reconstruction, individual drug screening, immune cell co-culture, and maternal–fetal medicine. This review briefly summarizes the recent progress in the application of ovarian and fallopian tube organoids in the field of obstetrics and gynecology.
Keywords: Organoid; Personalized Therapy; Stem Cell; Three-Dimensional Culture
|How to cite this article:|
Dai YL, Wei JS, Xu J, Li K, Wang J, Cha RR, Kang Y. Progress in the application of ovarian and fallopian tube organoids. Reprod Dev Med 2021;5:174-82
|How to cite this URL:|
Dai YL, Wei JS, Xu J, Li K, Wang J, Cha RR, Kang Y. Progress in the application of ovarian and fallopian tube organoids. Reprod Dev Med [serial online] 2021 [cited 2021 Dec 8];5:174-82. Available from: https://www.repdevmed.org/text.asp?2021/5/3/174/322840
| Introduction|| |
In 2009, Sato et al. reported a breakthrough in cell culture: leucine-rich repeat-containing G-protein-coupled receptor 5-positive intestinal stem cells from the base of the intestinal crypts could be induced to produce a “mini-gut” within an in vitro three-dimensional (3D) culture system. These “mini-guts”, also known as intestinal organoids, were shown to contain Paneth, goblet, and enteroendocrine cells, with a structure similar to that of the native lumen of the gut.,
The concept of organoids was systematically proposed for the first time in 2014. Organoids are derived from induced pluripotent stem cells (iPSCs) or organ-restricted adult stem cells (ASCs) through cell sorting and spatially restricted lineage commitment. Organoids self-assemble to generate cell aggregates consisting of multiple cell types that are similar in spatial form to their respective source organs.
The characteristics and advantages of organoids have led to their extensive application in numerous research fields. The ovaries and fallopian tubes serve as female reproductive organs and are directly regulated by multiple hormones. Organoids are uniquely applied in research fields involving the ovaries and fallopian tubes.
To date, a total of 24 articles on ovarian and fallopian tube organoids have been published [Supplementary Table 1], including 12 articles on organoids representing the fallopian tube epithelium (FTE),,,,,,,,,,,, four articles on organoids of the ovarian surface epithelium (OSE),,,, and 13 articles on ovarian cancer (OC) organoids,,,,,,,,,,,, involving multiple cell types [Figure 1]. The present review summarizes the recent progress in the application of ovarian and fallopian tube organoids.
|Figure 1: Cells in the ovary and fallopian tube. (a) Fallopian tube epithelium contains two principal types: secretory cells and ciliated cells. Epithelial stem/progenitor cells are basally located. Immune cells are present in both the epithelium and stroma. (b) The ovary contains several cell types, including ovarian surface epithelium, ovarian stromal cells, and ovarian follicles at different stages (composed of oocytes, granulosa cells, and theca cells). (c) The endometrium comprises the endometrial epithelium and endometrial stroma.|
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| Organoid Formation and Categorization|| |
Organoids can be generated from both normal and tumor tissues, and organoids derived from normal tissues involve embryonic stem cells (ESCs), iPSCs, and ASCs.
ESCs are derived from the inner cell mass of the blastocyst; these are useful in vitro models for exploring interactions between cells from different germ layers during organogenesis.,
iPSCs are reprogrammed from somatic cells via the overexpression of pluripotent transcription factors, which avoids embryo-related ethical issues., Organoids derived from iPSCs provide an ideal platform to study organ development and evolution.
Maintaining ASC-derived organoids depends upon the activation of the required growth factors, and different activation sequences of signaling pathways in ESCs and iPSCs lead to different organoid types.
| Applications of Ovarian and Fallopian Tube Organoids|| |
Cell differentiation and development
The mechanisms by which cells differentiate and are maintained within the FTE remain to be fully elucidated. FTE organoid models represent an invaluable tool for investigating cell differentiation and development in vitro.
Human FTE can be digested into single cells and cultured into monoclonal organoids comprising both secretory and ciliated cells. Following 2 weeks of growth, cilia can be observed on previously nonciliated progenitor cells, which demonstrate the bipotency of FTE progenitors.
FTE organoids from Pax8rtTA; TetOCre: Rosa26-tdTomato mice have been used to demonstrate that Tomato + cells can co-localize with PAX8-positive FTE secretory cells after 2 days of exposure to doxycycline (Dox) and that after cessation of Dox treatment for 2 months, both the secretory and ciliated cells remain as Tomato+, indicating that PAX8-positive secretory cells differentiate into ciliated cells in vivo.
iPSC-derived organoids provide a systematic platform to study organ development and evolution. Previous studies have reported that long-term FTE organoid models can be established using three female human iPSCs. Investigators have demonstrated that Wnt and BMP signals are regulated to induce the differentiation of iPSCs into Müllerian cells; follistatin can then be used to promote the differentiation of FTE precursor cells. The lumen, cilia structure, and cell markers of the resultant cultures indicate that these organoids consist of both ciliated and secretory epithelium.
Establishment of disease models
Infectious disease models
The long-term co-culture of normal tissue-derived organoids with infection source pathogens affords the opportunity to directly investigate the infection process and chronic tissue alterations associated with infection in vitro.
For example, fallopian tubes are prone to scarring and infertility due to Chlamydia trachomatis (CTR) infection; however, the mechanism underlying CTR-induced fallopian tube inflammation remains unclear. A 9-month in vitro infection analysis of human fallopian tubes and serotypes D, K, and E of genital CTR indicated that the epithelial monolayer actively discharges bacteria into the lumen and promotes cell proliferation. This analysis also demonstrated that CTR infection activates Leukemia inhibitory factor signaling, which induces organoids to exhibit poorly differentiated phenotypes and a higher cell stemness score, and that CTR facilitates DNA hypermethylation, an indicator of accelerated molecular aging. These heritable changes may contribute to the pathological progression of fallopian tubes, including the occurrence of high-grade serous ovarian carcinoma (HGSOC).
Normal organoids can be used to explore tissue origins and their corresponding tumor characteristics. It is now widely recognized that distal FTE is the origin of most HGSOCs,,,,,, while the OSE is considered a possible source of nonfallopian tube-derived HGSOC.,, Investigators have induced Tp53R172H/fl along with Rb inactivation in mouse FTE and OSE organoids and have suggested that OSE-derived tumors have longer latency and lower formation rates than those derived from FTE. The use of CRISPR-Cas9 technology to target TP53 + BRCA1 + PTEN/TP53 + BRCA1 + NF1 within mouse FTE and OSE organoids demonstrated that double-mutant and triple-mutant FTE organoids have tumorigenic potential during orthotopic transplantation, while no tumors were formed by the OSE organoids.
Organoids derived from normal tissues can be utilized to establish tumor models with different genotypes to more clearly observe the distinct roles of different oncogenic mutations during tumor initiation. For example, in the case where Tp53R172H/fl is induced along with Rb inactivation in mouse organoids, a single Rb-family inactivation can induce serial tubal intraepithelial carcinoma, the precursor of HGSOC, within FTE organoids, and restrict FTE differentiation to the secretory lineage. Human FTE organoids retain the ability to differentiate into ciliated cells in the case of combined TP53, PTEN, and Rb knockdown, suggesting that these molecular defects are insufficient to induce complete malignant transformation.
Tissue regeneration and reconstruction
Organoids serve as a promising source of transplant material in regenerative medicine. Organoids have the potential to confer cells with reconstructive functions and improve the adaptability of grafts to the pathological microenvironment.,
Fallopian tubes play an important role in sperm transport, oocyte capture and transport, fertilization, and early embryo development. The prevalence of fallopian tube disease in infertile patients is reported to be approximately 30–35%., It is possible for the physiological function of the fallopian tube to be repaired by reconstructing the ciliated epithelium of the FTE by organoids, thereby avoiding the occurrence of infertility and other adverse events.
iPSCs have the advantage of being derived from human origin and the ability to form any cell type in vitro. They are relatively easy to obtain and are not subject to ethical issues surrounding embryo-derived stem cells. For this reason, iPSCs are also considered a favorable source of cells for in vitro functional reconstruction. Previous studies have reported the establishment of long-term FTE organoid models using three female human iPSCs.
Estrogen receptor-α (ESR-α) and progesterone receptor (PGR) expressed by FTE cells are crucial for the development and differentiation of FTE. FTE organoids represent a useful resource for investigating the effects of estrogen and progesterone on FTE. At present, it has been confirmed that ESR-α and PGR are expressed in both human and mouse organoids., They are responsive to estradiol or progesterone, since part of gene expression pattern exhibits an opposite trend after treatment with estradiol or progesterone. Several regulated genes are reported to be inversely regulated during different menstrual phases, such as PGR and tumor necrosis factor (TNF)., Moreover, oviductal glycoprotein 1 (OVGP1) is highly upregulated by estradiol in human organoid models.
Drug reaction and their mechanisms
Predicting drug sensitivity
The composition of the in vitro cell culture model has a profound impact on drug reaction studies. For example, drugs that can strongly retard growth with only limited induction of cell death are more effective in 3D organoid cultures than in two-dimensional (2D) cell monolayers.
Organoids can reflect both the heterogeneity between patients (interpatient) and between spatially and temporally separated tissues from the same patient (intrapatient).,, Compared with patient-derived xenograft (PDX) models, organoids have a moderate culture time and cost, and therefore, they represent a favorable platform for accurately screening individualized drugs and studying mechanisms of drug action.
The clinical reactions of patients to drugs can be predicted by observing the drug reactions of organoids. In a previous study, 88% of organoids derived from HGSOC patients demonstrated a strong response to at least one drug, and a significant relationship was reported between the carboplatin–paclitaxel sensitivity of these organoids and the clinical sensitivity of their respective patients to these agents.
Moreover, HGSOC organoid sensitivity to carboplatin, paclitaxel, poly (ADP-ribose) polymerase inhibitors), and other drugs has been related to their homologous recombination deficiency (HRD) scores, CHORD classification (a genome-wide mutational scar-based pan-cancer classifier of homologous recombination deficiency), and degree of HR function as determined by RAD51 foci assays., Replication fork protection disorders of HGSOC organoids, tested using DNA fiber assays, are also related to their sensitivity to carboplatin, CHK1, and ATR inhibitors. However, not all of these correlations can be detected in monolayer 2D cultures.
Mechanisms of drug action
Normal organoids can be used to determine drug sensitivity, depending on specific tumorigenic mutations. For example, the genotype dependence of immunotherapy has been demonstrated using tumorigenic FTE organoids. Tumorigenic FTE organoids overexpressing CCNE1, AKT2, and KRAS in mice demonstrated a complete response to the combination therapy of gemcitabine, anti-cytotoxic lymphocyte-associated antigen 4 (CTLA4) antibody, and anti-PD-L1 antibody. This was accompanied by increased CD8+ cell infiltration and T lymphocyte activation. Conversely, the same treatment strategy failed to demonstrate efficacy in organoids with decreased PTEN and NF1 expression.
It is impossible to completely restore the microenvironment of the original tissues using organoids alone. In traditional organoids, epithelial cells from tissues become enriched, whereas matrix components gradually decrease. However, technical improvements have been made to these models.
The functions of cytokines/chemokines can be evaluated in vitro using organoids. Previous studies have demonstrated that changes in cytokine/chemokine secretion spectra of FTE organoids in response to specific genetic changes can be observed by detecting the levels of cytokines, chemokines, and growth factors in the conditional medium of mouse FTE organoids. The influence of cytokines on organoids can be evaluated by the addition of recombinant cytokines into the organoid extracellular matrix (ECM)., The migration of specific immune cells induced by different cytokines/chemokines can be assessed in vitro via Transwell assays, where corresponding neutralizing antibodies are added into the conditioned medium of mouse FTE organoids [Figure 2]a.
|Figure 2: Summary of current microenvironment remodeling methods of organoids. (a) Evaluating the functions of cytokines/chemokines in conditioned medium of organoids using ELISA or Transwell assay. (b) A holistic method of microenvironment remodeling by air–liquid interface culture. (c) A holistic method of microenvironment remodeling by packing primary tissues and niche cells as a whole in collagen gels in three-dimensional microfluidic culture devices. (d) The organoids of primary epithelial cells were independently cultured and then co-cultured with immune cells.|
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In vivo studies have reported the chemotactic effect of different mutation types and cytokines/chemokines on specific immune cell populations using flow cytometry after allogenic mice were injected in situ with tumorigenic FTE organoids together with the corresponding neutralizing antibody.
The methodology described above was not designed to remodel the microenvironment of organoids in vitro. Currently, two main methods are employed for remodeling the microenvironment of organoids: holistic and reduction [Figure 2]b, [Figure 2]c, [Figure 2]d.
The holistic method refers to culturing all cells (including immune cells and other nonepithelial cells) with the primary tissues. The advantage of this approach is that the native state of the microenvironment in vivo can be better restored [Figure 2]b and [Figure 2]c.,
The holistic method can be further divided into two main types. Collagen gels were first used to pack primary tissues and niche cells as a whole and were then treated with culture medium in 3D microfluidic culture devices [Figure 2]c. The second involves the use of air–liquid interface (ALI) cultures. The primary tissues and niche cells are placed on collagen inside a tissue culture dish, with the culture medium outside the dish permeating through holes on the bottom into the interior. As the top of the collagen layer is exposed to air through ALI, cells can obtain sufficient air [Figure 2]b.
In the reduction method, the primary epithelial cells are independently cultured into organoids, which are subsequently co-cultured with immune cells from the peripheral blood or spleen of the same patient or animal. This provides the advantage of a longer co-culture time [Figure 2]d.,
The holistic method has been used to remodel the microenvironment of organoids in obstetrics and gynecological studies. When normal human FTE organoids, mesenchymal stem cells, and human umbilical vein endothelial cells are co-cultured at a ratio of 10:7:2, the FTE may induce the formation of “contracted” organoids, which can be prevented by inhibiting the Wnt signaling pathway by DKK1.
Moreover, epithelial tumors and complete intratumoral immune cells can be isolated from the affected tissues of patients with HGSOC and can be short-term co-cultured (for 96 h). The growth factors in the culture medium are low in quantity, which helps prevent possible hybrid changes in the immune microenvironment during long-term culture, including clonal selection of T cells. Components of the co-cultured cells can be verified against the parental tumor using flow cytometry and sequencing.
Organoids and immune checkpoint inhibitors
Immune checkpoint proteins include CTLA-4 and programmed death receptor (PD-1). When PD-1 is combined with its ligands, PD-L1 and PD-L2, it is regarded as a common modulator of T cell survival and proliferation as well as a modulator of the phosphorylation of downstream T cells and levels of regulatory T cell populations.
Organoids can be used to assess the efficacy and mechanisms of immune checkpoint inhibitors in vitro. Investigators have discussed the use of bispecific anti-PD-1/PD-L1 antibodies in HGSOC organoids co-cultured with immune cells. These induce a relatively active and cytotoxic state in the NK cells and CD8+ T lymphocytes, experiencing a state of inertia and exhaustion. Its effects are partly attributed to the decline in the levels of bromodomain-containing protein. The transcription factors that are able to regulate NK cells are altered to allow them to interact with chromatin in key immunoregulatory genes.
Application of organoids in maternal–fetal medicine
Maternal–fetal medicine has received considerable research attention in recent years, and it includes aspects of reproductive physiology, pregnancy pathology, and placental defects. Organoids serve as a promising platform for investigating maternal–fetal interactions.
Based on organ-on-a-chip technology, researchers have cultured mouse ovarian tissue together with human fallopian tube, uterus, cervix, and liver in an integrated 3D microfluidic device, which can be employed to analyze tissue–tissue interactions in the field of reproductive physiology and pregnancy pathology. This microfluidic platform supports follicle development (follicle growth, maturation, ovulation, and granulosa cell luteinization) and accurately mimics the hormone secretion pattern (estrogen, progesterone, and inhibin) of the follicles and the corpus luteum during pregnancy by providing follicle-stimulating hormone, luteinizing hormone, and human chorionic gonadotropin at appropriate times. The co-cultured explants of the fallopian tube, uterus, cervix, and liver respond to the hormonal signals imitating the human menstrual cycle and pregnancy.
It is difficult to investigate maternal–fetal interactions between decidua and trophoblasts (including cytotrophoblasts [CTBs], syncytiotrophoblasts [STBs], and extravillous CTB [EVT]) in vitro, as some trophoblast cell lines differ from the original tissue in expression pattern or HLA status., Co-culture of decidua and trophoblast organoids therefore affords the opportunity to analyze the secretome of these cells and represents a promising approach to examine disorders related to placental insufficiencies, such as preeclampsia, recurrent abortion, and preterm labor., Researchers have already generated long-term trophoblast organoids., These organoids mimic the complex structure, transcriptome pattern, and methylation profile of villous placenta. They can differentiate into STBs that secrete placental-specific peptides and hormones. At the same time, they generate migratory and invasive HLA-G+ EVT cells. In the future, trophoblast organoids may also be combined with organoids of the ovary, fallopian tube, uterus, cervix, and liver in a microfluidic platform based on organoids-on-a-chip technology.
| Advantages and Disadvantages of Organoids|| |
As a sophisticated 3D in vitro culture system, organoids represent an opportunity to remedy the deficiencies of the traditional cell culture models, such as traditional 2D culture systems, PDXs, and other in vitro 3D culture models, including organotypic multicellular spheroids, multicellular spheroids, and tumor-derived spheroids.
Traditional two-dimensional cell culture models
Traditional 2D culture systems have been used to assess cell interactions by growing cells on flat surfaces, typically plastics. Within these 2D environments, the natural structure of source tissues cannot be imitated, especially cell–cell and cell–matrix interactions.
Cell lines – the immortalized cell products of powerful in vitro selection – have been established and maintained over several generations and can be perpetuated in culture over decades, inevitably resulting in the deviation of their characteristics from the original source tissue, including differences in mutational and copy number variation (CNV) profiles.,
Compared with cell lines, PDX models have the advantage of generating structured tissues and maintaining tissue microenvironments, including the proportions of stromal cells and vascular invasion.,
However, PDX models do not accurately represent the heterogeneity of the primary tissues. The human stromal cell components within PDX models, such as the vascular system, immune cells, and fibroblasts, are substituted by mouse stromal cells over multiple passages in culture.
In addition, the success rate of PDX model establishment is limited. Combined with the high latency and resource costs of PDX model establishment (PDX models usually take over 4 months to establish), they also have limited application with regards to high-throughput drug screening studies.
Other three-dimensional culture models
Other in vitro 3D culture models include organotypic multicellular spheroids, multicellular spheroids, and tumor-derived spheroids [Figure 3].
|Figure 3: Three-dimensional culture models in vitro. Three-dimensional culture models in vitro contain organotypic multicellular spheroids, multicellular spheroids, tumor-derived spheroids, and organoids. Organotypic multicellular spheroids are gently dissociated and can precisely replicate the initial microenvironment. Multicellular spheroids are a three-dimensional extension of traditional two-dimensional cell lines. Tumor-derived spheroids enrich cancer stem cells, while organoids comprise stem cells and their downstream progeny.|
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After gentle mechanical dissociation of tissues, organotypic multicellular spheroids can be established to faithfully replicate the interstitial components in the tissue microenvironment. In contrast, multicellular spheroids represent a 3D extension of traditional 2D cell culture systems.
Tumor-derived spheroids, through the enrichment of cancer stem cells (CSCs), are suitable for drug screening to identify new agents targeting the CSC population. For example, HGSOC-derived spheres can express CSC markers (ALDH1A1, CD133, and SOX2) and show the capacity for differentiation and oncogenicity in a xenograft. Compared to spheroids, organoids, including stem cells and downstream progeny with higher differentiation, demonstrate more favorable long-term viability in culture.
Advantages of organoids
In contrast to other in vitro culture models, an organoid is distinguished from simpler models by possessing the following characteristics:
- Organoids are capable of self-assembly, and cell organization within the organoid is similar to that of the source organ. For example, FTE organoids have polar columnar epithelium, cell junctions, and folded mucosal structures
- Organoids comprise multiple cell types from the source organs. For example, FTE organoids contain both secretory and ciliated cells
- Organoids exhibit unique physiological functions in the source organ. For example, the apical surface of FTE organoids is covered by abundant microvilli and fully developed cilia; ruffling of the apical membrane on the nonciliated cells is highly suggestive of active substance secretion into the lumen.
In addition, stem cells inside the organoid maintain cell renewal, enabling long-term in vitro culture, and maintenance of genetic stability. This advantage of organoids promotes their use in investigations of interpatient heterogeneity at both the histological and genomic levels, even after prolonged culture and passage. Organoids derived from HGSOC tumor material demonstrate CNV, somatic single nucleotide variants, structural variation, and DNA methylation patterns consistent with source tissues.
Similarly, organoids can reflect the spatial and temporal heterogeneity within a single patient. With respect to time, it has been reported that organoids derived from HGSOC in relapsed patients with platinum/paclitaxel resistance are themselves resistant to platinum/paclitaxel, while organoids derived before therapy resistance demonstrate therapy sensitivity consistent with the primary lesion. Spatially, the expression of factors related to epithelial–mesenchymal transition and other tumor metastasis-related pathways is upregulated in organoids derived from malignant pleural fluid and ascites. Different responses to a single treatment were observed in 31% of OC organoids obtained from the same patient at the same time point.
Disadvantages of organoids
Despite their advantages over traditional culture methods, organoids have a number of limitations.
Currently, ovarian organoids are derived from OSE. However, there are still a large number of ovarian stromal cells and follicle cells at different stages inside the ovary, including oocytes, granulosa cells, and theca cells. These cells respond to the hypothalamic–pituitary–ovarian axis to perform reproductive and endocrine functions. There is still a large research space surrounding ovarian organoids, which have the potential to improve models of ovary maturation and endocrine remodeling in vitro.
The culture of traditional organoids has been hindered by the lack of an intrinsic vascular system in vitro; however, such limitations have been overcome in recent years. For example, reports of co-culture of breast cancer organoids and human blood vessels have demonstrated the dynamic relationship between cancer cells and blood vessels.
Although there is an increase in the number of approaches to remodel the immune system within organoids, the complexity of the original microenvironment is usually underestimated in vitro. For instance, immune selection may lead to the overgrowth of certain cell clones, leading to a shift in the underlying immune composition of the organoid.,
| Conclusions and Prospects|| |
Although organoids themselves have some limitations, they represent more comprehensive in vitro research models than traditional models. In addition, organoid culture technology continues to develop rapidly.
Organoids-on-a-chip is a promising development direction for organoids and has a close connection with bioengineering. These allow for strict control of the physical and biochemical environment in the culture process of organoids, also facilitating the monitoring of signal transduction and cell migration among different components of the organoids. This represents an ideal platform for studying the ovary, harboring multiple cell types, and displaying responses to a variety of hormones. Combined with organ-on-a-chip or other microfluidic devices, micro-physiological vascular perfusion or lymphatic systems may be restored.,
The combination of organoids and 3D printing technology facilitates the establishment of more precise relationships between cells and the surrounding matrix. Studies on centimeter-level human heart organoids using 3D printing technology have demonstrated a normal pumping function within these models.
In consideration of these promising development directions, we have reason to be optimistic about the use of organoids to study tissue development, investigate disease pathogenesis, individualize treatment for precision medicine, and impact clinical decision-making.
Supplementary information is linked to the online version of the paper on the Reproductive and Developmental Medicine website.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]