Organoids as Sophisticated Tools for Renal Cancer Research: Extensive Applications and Promising Prospects

Abstract

Background

Kidney cancer is a significant global health problem that affects nearly 1 in 25 of cancer patients. Prevalence, morbidity and mortality data associated with kidney cancer continue to increase every year, revealing a heavy economic and social burden. Organoid culture is a new research tool with great potential for many applications, particularly in cancer research. The integration of organoids with other emerging technologies has simultaneously expanded their potential applications. However, there is no thorough assessment of organoids in the field of renal cancer research.

Objectives

This paper presents a comprehensive review of the current development of renal cancer organoids and discusses the corresponding solutions and future directions of renal cancer organoids.

Methods

In this study, we have compared the operating procedures of different organoid culture protocols and proposed a summary of constituents in culture media. Extensive discussions of renal cancer organoids, including generation and maintenance approaches, application scenarios, current challenges and prospects, have also been made. The information required for this study is extracted from literature databases such as PubMed, SCOPUS and Web of Science.

Results

In this article, we systematically review thirteen successful methods for generating organoids to kidney cancer and provide practical guidelines for their construction as a reference. In addition, we also elucidate the clinical application of organoids, address the existing challenges and limitations, and highlight promising prospects.

Conclusion

Ultimately, we firmly believe that as kidney tumour organoids continue to develop and improve, they will become a crucial tool for treating kidney cancer.

Introduction

The kidney is a vital retroperitoneal organ in the human body that plays crucial roles in filtration, reabsorption, and endocrine functions [1]. Unfortunately, several million individuals succumb to various kidney diseases annually [2–6]. The global incidence and mortality rates of kidney cancer have significantly increased in recent years, particularly in the aftermath of the COVID-19 pandemic [7]. According to statistics from the International Agency for Research on Cancer, kidney cancer is the 14th most common malignancy globally in 2022, accounting for 434,419 new cancer diagnoses and 155,702 deaths [8]. The main pathological subtypes of RCC include clear cell RCC (ccRCC), papillary RCC (pRCC) and chromophobe RCC (chRCC) [9]. The progression of kidney cancer may occur several decades prior to the initial diagnosis [10]. The efficacy of chemotherapy and radiotherapy in treating RCC is limited. For localized RCC, partial or radical nephrectomy can eradicate the disease; however, a significant proportion of patients present with metastases at an early stage after diagnosis [11]. Indeed, approximately one-third of patients exhibit metastatic disease upon presentation, and approximately one-quarter will eventually experience relapse [12]. Treatment of metastatic RCC (mRCC) has made significant advancements, transitioning from therapies involving interleukin-2 and interferon-α to targeted therapy [13]. By inhibiting specific signal transduction pathways and associated proteins involved in tumour growth and progression, molecular targeted drugs such as immune checkpoint inhibitors (ICIs), VEGF receptor tyrosine kinase inhibitors (VEGFR TKIs) and mTOR inhibitors have demonstrated remarkable efficacy in prolonging the survival rate of patients with mRCC [14].

Therefore, investigating the mechanisms underlying the occurrence and progression of RCC and establishing a precise model for this malignancy hold significant implications for guiding recurrence prediction and subsequent treatment strategies. RCC exhibits limited sensitivity to chemotherapy and radiotherapy, while extensive heterogeneity resulting from multiple genetic and epigenetic alterations contributes to increased resistance and aggressiveness of this disease [15, 16]. Although various cell lines and laboratory animals are currently used to study the pathogenic features of human kidney disease, develop therapeutics, and evaluate drug effectiveness, these models are often unable to accurately recreate the disease and do meet current medical research needs. Traditional 2D culture models and animal experimental models have proven insufficient for facilitating the development of novel drug treatment strategies for RCC patients and clinical translational research due to the limited understanding of the biological mechanisms underlying RCC.

Organoids have emerged as a valuable tool to overcome these limitations [17]. Within the past decade, renal cancer organoids have garnered significant interest in both preclinical and translational settings of renal cancer research. Compared to traditional two-dimensional (2D) cell line cultures and animal models, organoids cultivated under three-dimensional (3D) systems in vitro conditions more accurately reflect in vivo scenarios, effectively simulating the cellular heterogeneity, tumor microenvironment, and disease progression associated with renal cancer. Pre-clinically, they serve as robust research models that hold promise for providing novel insights into renal cancer pathogenesis and tumor biology. And in the clinic, they play a pivotal role in guiding drug discovery efforts and the development of innovative therapeutic strategies for renal cancer, representing a crucial avenue for precision medicine. Organoids are 3D structure models derived from pluripotent stem cells, progenitors, and/or differentiated cells that self-organize through cell‒cell and cell-matrix interactions to model aspects of the native tissue architecture and function in vitro [18]. In general, spheroids cultured from a single cell type, such as tumour cells, are free-floating 3D cultures formed in the absence of a predefined culture substrate for adherence [19]. The term “organoids” refers to 3D cultures constructed from multiple cell types, including epithelial organoids, multitissue organoids, and multiorgan organoids. Hence, organoids are capable of replicating the authentic spatial organization and diverse physiological processes of bodily tissues in vitro, thus improving disease research methods previously limited to the cell and animal models.

Compared with 2D cultures, 3D organoid cultures derived from patients exhibit a greater degree of resemblance to renal carcinoma in terms of morphology and specific biomarker expression [20, 21]. The renal organoid model has made remarkable advancements toward understanding cancer progression in recent years. In vitro 3D culture conditions for RCC which reproduce the molecular and histological phenotype of the parental tumour more precisely than standard 2D culture conditions may represent a solution for basic tumour biology studies and for efficiently assessing targeted and patient-specific personalized therapies [22]. Generally speaking, the commonly utilized models for renal cancer research encompass 2D cell lines, animal models, and 3D organoids. 2D Cell lines are employed to investigate the biological characteristics of renal cancer through culturing specific kidney cancer cell lines (such as 786-O, Caki-1, ACHN and so on), which are suitable for preliminary fundamental research. Animal models simulate the occurrence and development of kidney cancer by transplanting kidney cancer cells or using genetically modified mice (such as VHL-deficient mice), thereby playing a pivotal role in elucidating the mechanisms underlying renal cancer. Organoid models, on the other hand, involves cultivating three-dimensional renal cancer organoids from patient-derived tissues or cells in vitro and is considered closer to clinical practice (Fig 1). Moreover, the application prospects of renal cancer organoids are extensive in the fields of drug screening, drug toxicology research, precision medicine, and gene editing. The combination of organoids and diseases modeling, gene editing, drug testing, organ-on-a-chip, and 3D bioprinting has enabled numerous progressive breakthroughs to advance studies on tumor occurrence, invasion, metastasis and treatment, as well as the underlying mechanisms of multidrug resistance (Table 1). In this article, we systematically review successful methods for generating organoids to kidney cancer and provide practical guidelines for their construction as a reference. In addition, we also elucidate the clinical application of organoids, address the existing challenges and limitations, and highlight promising prospects. Ultimately, we firmly believe that as kidney tumour organoids continue to develop and improve, they will become a crucial tool for treating kidney cancer (Table 2).

Fig. 1.

The primary models utilized in renal cancer research are 2D cell lines, animal models and renal cell carcinoma organoids. 2D cell cultures are easily obtainable and simple to culture, but they fail to replicate the intricate structure and functionality of tumor tissue accurately. While mouse models can effectively mimic the tumor microenvironment, they lack species specificity. Organoids represent an advanced tool in renal cancer research that overcomes these limitations and offers a wide range of applications with promising prospects

Table 1.

Progressive breakthroughs in the development of renal cancer organoids

Histological Type	Cell/Tissue Collection	Establishment	Success rate	Maximum passage	Applications	Published Year	References
RCC	Patient-derived kidney tumor tissue and normal kidney tissue distal to the tumor	Seeded on scaffolds and cultured in organoid medium	80% (20/25)	N/A	Assessment of the ability of 3D scaffolds to support RCC growth and to maintain the phenotype over time	2015	Batchelder et al. [22]
Normal tissue			100% (22/22)
ccRCC	Patient-derived kidney tumor tissue and normal kidney tissue	Embedded in growth factor reduced matrigel and cultured in basic organoid medium	67% (10/15)	15	Cytotoxicity assay and drug testing	2019	Grassi et al. [30]
Normal tissue			100% (13/13)	15
Wilms tumours	Patient-derived kidney tumor tissue and matching normal kidney tissue	Cultured in kidney organoid medium then transfected by using lipofection	75% (40/53)	> 20(Wilms tumours)	Gene editing and high-throughput drug screens	2020	Calandrini et al. [36]
MRTK			100% (7/7)	> 20(MRTKs)
RCC			75% (3/4)	~ 10(chemo-treated RCCs)
CMNs			100% (2/2)	> 20(chemo-naive RCC)
Metanephric adenoma			100% (1/1)	N/A
Nephrogenic rest			100% (1/1)	N/A
Normal tissue			100% (47/47)	N/A
ccRCC	Patient-derived kidney tumor tissue	Embedded in Matrigel and cultured in organoid medium	N/A	N/A	Investigation of the pharmacological inhibition of WNT and NOTCH signaling of CSC in ccRCC organoids	2020	Fendler et al. [60]
ccRCC	Patient-derived kidney tumor tissue	Resuspended inmixed Type I collagen solution and cultured in a collagen-based 3D, ALI culture system	77% (20/26)	N/A	Drug response Rates of cabozantinib and nivolumab	2020	Esser et al. [82]
pRCC			80%(4/5)
Upper urinary tract urothelial carcinomas			88%(7/8)
Oncocytoma			33%(1/3)
chRCC			0%(0/1)
ccRCC	Patient-derived kidney tumor tissue	Plated in Matrigel and cultured in organoid medium	100% (3/3)	N/A	Gene editing and Drug validation	2021	Zhang et al. [98]
mRCC	Patient-derived kidney tumor lung metastases tissue	Embedded in Matrigel and cultured in organoid medium	N/A	N/A	Investigation of The biological roles and mechanisms of METTL14/BPTF in promoting lung metastasis	2021	Zhang et al. [99]
AML and cystic disease	TSC2 − / − hiPSCs patient-derived-hiPSCs	Differentiated and Inducted with IL-1β	N/A	N/A	Modeling TSC-associated human kidney diseases	2021	Hernandez et al. [43]
ccRCC	Patient-derived kidney tumor tissue	Mixed with Matrigel and cultured in hepatocyte culture medium	74% (14/19)	15	Drug testing	2021	Kazama et al. [47]
chRCC			100% (1/1)
Wilms tumor	WT1 knockout iPSCs	Cultured in ultra-low-attachment 96-well plates	N/A	N/A	Modeling pediatric kidney cancer Wilms tumor	2021	Waehle et al. [46]
RCC	Patient-derived kidney tumor tissue	Mixed with Matrigel and cultured in organoid medium	N/A	N/A	Drug testing	2021	Hamdan et al. [94]
ccRCC	Patient-derived kidney tumor tissue and adjacent non-tumor kidney tissue	Mixed with matrigel and cultured in organoid mediums with seven niche factors	77%(30/39)	N/A	Drug response and Assessment of CAR‐mediated cytotoxicity	2022	Li et al. [35]
pRCC			67%(2/3)
chRCC			100%(1/1)
Normal tissue			100%(10/10)
ccRCC	Patient-derived kidney tumor tissue	Placed in low attachment cell culture plates and cultured in spheroid medium	43% (3/7)	N/A	Modeling ccRCC of various grades	2022	Nyga et al. [79]
ccRCC	Patient-derived kidney tumor tissue	Resuspended in cold collagen matrix and cultured with PDO culture medium	88% (7/8)	N/A(not consider passaging or prolonged preservation)	Recapitulation of the response to immune checkpoint blockades	2022	Xue et al. [95]
Metastatic PRCC-TFE3 fusion tRCC	Patient-derived kidney tumor tissue	Seeded into growth factor-reduced Matrigel and cultured in organoid medium	N/A	N/A	High-throughput compound library screening	2022	Cao et al. [50]
ccRCC	Patient-derived kidney tumor tissue	Resuspended in cold organoid culture medium	50% (10/20)	N/A	Investigation of the potential role of P2XR4 in regulating mitochondrial activity in patient derived organoids	2023	Rupert et al. [92]
RCC	Patient-derived kidney tumor tissue	Embedded in BME and cultured with human renal cancer organoid medium	N/A	N/A	Investigation of the Carcinogenic effects of piR-1742 on renal cancer cells	2023	Zhang et al. [96]
RCC	Patient-derived kidney tumor tissue	suspended in 80% matrigel and 20% histology-specific media	100% (1/1)	N/A	The efficacy of T cell selection tools in adoptive cell therapy	2023	Parikh et al. [93]
RCC	Patient-derived kidney tumor tissue	Embedded in Matrigel and cultured in organoid medium	N/A	N/A	Investigation of the function of SATB2-mediated antioxidative pathway in tumorigenesis and chemotherapy resistance	2023	Jin et al. [97]
Normal tissue	Normal kidney tissue	Embedded in Matrigel and cultured in organoid medium	100% (2/2)	N/A	Toxicity testing of environmental toxicants	2024	Caipa et al. [52]
Tubuloid established from adult mouse kidney single epithelial cells	Whole adult mouse kidney tissues	Seeded in Matrigel and cultured in tubuloid medium	N/A	12	Investigation of the involvement of stem cell-associated Wnt and Notch signaling in development of human ccRCC	2024	Myszczyszyn et al. [62]
ccRCC	Patient-derived kidney tumor tissue	Embedded in Matrigel and cultured in organoid medium	100% (2/2)	N/A	High throughput screening	2024	Ortiz et al. [49]

Table 2.

Summary of constituents in culture media based on methodology described on thirteen papers documenting successful RCCO culture

Reagent Name	Function	Concentrations
		Human Normal kidney organoid		Human RCC organoid
References		Li et al. (2022) [35]	Caipa et al. (2024) [52]	Li et al. (2022) [35]	Parikh et al. (2023) [93]	Xue et al. (2022) [95]	Jin et al. (2023) [97]
Basal medium
Advanced DMEM/F12	Providing basal nutrition	1x	1x	1x	35%	1x	1x
Supplementary ingredients
Antibiotic-Antimycotic	Preventing microbial contamination	1x		1x	100 U/mL(Penicillin - streptomycin) + 100 μg/mL(Primocin)	1x(Normocin) + 1x(Primocin)
Heparin	Regulating cellular adhesion to the extracellular matrix as well as cell-cell interactions
B-27/N2	Suppressing cell differentiation, supporting the growth	1x	1.50%(with vitamin A)	1x	1.5% B27	1x	2% B27
HEPES	Maintaining the osmotic pressure	10 mM	10 mM	10 mM	1% HEPES
Amino acids
N-Acetyl-L-cysteine	Regulate cell proliferation, differentiation and apoptosis	1.25 mM	1 mM	1.25 mM	1 mM	1 mM	1.25 mM
GlutaMAX	Common substitution of L-glutamine, reducing ammonia accumulation during cellular metabolism	1x	1x	1x	1% GlutaMAX	1x
Nicotinamide	The components of coenzyme NAD, regulating cellular energy metabolism	10 mM		10 mM		10 mM
Cytokines
Wnt3A	Ligand of canonical Wnt/β-catenin pathway, regulate cell development, lipogenesis, differentiation, adhesion, and polarity				50% Wnt3A conditioned medium
EGF	Growth factor, promoting tumor growth	50 ng/mL	50 ng/mL	50 ng/mL	50 ng/mL	50 ng/mL
FGF	Growth factor, promoting the occurrence, growth, invasion, and migration of tumor cells	20 μg/L(FGF-10)	100 μg/L(FGF-10)		100 ng/mL(FGF-10)
HGF	Growth factor, promoting the occurrence, invasion, and angiogenesis of tumor cells
IL-2	T cell growth factor, enhancing the cytolytic activity of NK cells and promoting immunoglobulin production by B cells					600 IU/ml
IGF-1	Affecting glycogen metabolism, DNA synthesis and glucose uptake by binding to the IGF-I receptor, and acting as a mitogen instead of insulin for anti-apoptosis
Inhibitors
A83-01	Activin/NODAL/TCF-β bate pathway inhibitor; Inhibiting differentiation of iPSCs and maintaining the self-renewal Inhibiting differentiation and maintaining the self-renewal of stem cells in vitro	500 nM	5 µM		5 μM	0.5 μM	200 nM
Noggin	Inhibitor of BMP-4 and BMP-7; to inhibit the differentiation of stem cells	100 μg/L				1X	100 ng/mL
SB202190	P38 MAPK inhibitor; Inhibiting the proliferation and migration of cancer cells	10 mM		10 mM		10 μM
Y-27632	Rock inhibitor, facilitate the attachment of primary cells in vitro, inhibit the apoptosis of embryonic stem cells and promote the self-renewal and proliferation of stem cells	10 mM		10 mM	10 μM	10 μM	10 μM
Activators
R-spondin-1	Wnt pathway activator, promoting self-renewal of stem cells	500 μg/L	10%		10% R-spondin-conditioned medium	1X
Hormone
Dihydrotestosterone	Inhibiting mature dendritic cells differentiation, and promoting the differentiation of human mesenchymal stem cells						1 nM
Prostaglandin E2 (PGE2)	Activating differen PGE−2 receptor subtypes with distinct functions
Gastrin	Growth factor, prolonging the survival time of the intestine and liver, and pancreatic cancer organoids

Reagent Name	Human RCC organoid						Human Normal kidney organoid and RCC organoid
References	Fendler et al. (2020) [60]	Rupert et al. (2023) [92]	Zhang et al. (2021) [98]	Zhang et al. (2021) [99]	Esser et al. (2020) [82]	Cao et al. (2022) [50]	Calandrini et al. (2020) [36]	Grassi et al. (2019) [30]
Basal medium
Advanced DMEM/F12	1x	1x	1x	1x	1x	1x	1x	1x
Supplementary ingredients
Antibiotic-Antimycotic	1x(Penicillin-Streptomycin) + 1.25 µg/ml Amphotericin B	1x			1x (Penicillin/Streptomycin) + 100 μg/ml(Normocin)	200 U/ml(penicillin/streptomycin)	1x(Penicillin - streptomycin)	1x(Penicillin - streptomycin)
Heparin	4 µg/µl
B-27/N2	1x	1x	2% B27		1x(without vitamin A)	1x	1.5% B27	1x
HEPES		10 mM			1 mM		10 mM	10 mM
Amino acids
N-Acetyl-L-cysteine		1.25 mM	1.25 mM		1 mM	1 mM	1.25 mM	1 mM
GlutaMAX		1x			1x		1x	1x
Nicotinamide		10 mM	4 mM	4 mM	10 mM
Cytokines
Wnt3A			100 ng/ml	100 ng/ml	50% Wnt-3A	20% afamin/Wnt3a conditional medium
EGF	20 ng/ml	50 ng/mL	50 ng/ml		50 ng/ml		50 ng/mL	20 μg/ml
FGF	20 ng/µl		10 ng/ml(FGF-2) + 10 ng/ml(FGF-10)	10 ng/ml(FGF-2) + 10 ng/ml(FGF-10)		50 ng/ml(FGF-2)	100 ng/mL(FGF-10)	10 μg/ml(FGF-2)
HGF			20 ng/ml	20 ng/ml
IL-2					600 IU/ml
IGF-1						100 ng/ml
Inhibitors
A83-01			0.5 µM	0.5 µM	0.5 µM	500 nM	5 μM	500 mM
Noggin			100 ng/ml	100 ng/ml	25 ng/ml	100 ng/ml
SB202190		10 mM	5 µM	5 µM	10 µM
Y-27632		10 mM	10 µM	10 µM		10 µM	10 μM	10 nM
Activators
R-spondin-1			500 ng/ml	500 ng/ml	50% R-spondin 1 conditioned medium	1 µg/ml	10% R-spondin-conditioned medium
Hormone
Dihydrotestosterone			1 nM
Prostaglandin E2 (PGE2)			1 µM	1 µM
Gastrin			10 nM	10 nM	10 nM	10 nM

For detailed technical information and culture methods not suitable for tabular format, such as timing of media use, constituents of the mediums for passage and methods of validation, we recommend referring to the original articles.

Generation and Maintenance of Renal Tumour Organoids

The development of organoids derived from various tissues has progressed since the pioneering work of Hans Clevers, who first established epithelial organoids using mouse intestinal tissue [23]. The cell sources of organoids can be primarily categorized into adult stem cells (ASCs, also known as somatic stem cells, SSCs), induced pluripotent stem cells (iPSCs), and embryonic stem cells (ESCs) [24]. The type of source cell dictates the level of organoid maturation in vitro. PSCs, including iPSCs and ESCs, possess unlimited replication and differentiation potential. Compared with embryonic stem cells, which are associated with significant ethical concerns, the utilization of iPSCs to develop organoids are optimal for constructing kidney organoids. PSC-derived organoids are mainly used to study organogenesis and developmental events that lead to tissue formation, but PSC-derived organoids rarely reach the adult tissue stage in vitro and often resemble foetal tissue [25].

Differentiation via mesenchymal to epithelial transition (MET) determines the generation of nephrons during development, which can potentially give rise to oncogenic stem cells and drive the progression of paediatric kidney cancer [26]. Moreover, kidney organoids derived from iPSCs have been found to possess heightened potential for carcinogenesis due to the molecular-level similarities between iPSCs and RCCs, as well as the unique metabolic environment provided by the specific anatomic niche of the kidney which facilitates the oncogenic transformation of partially differentiated cells and iPSCs [27]. Organoids derived from ASCs or adult tissue fragments are thought to mimic the homeostatic or regenerative conditions of the tissue from which they originate and can, therefore, be used to investigate issues affecting adult tissue biology [28].

Li et al. employed mouse and human nephron progenitor cells (NPCs) to successfully generate nephron organoids that are consistently replicated in vitro over an extended period, providing a readily accessible system for modelling kidney development, conducting nephrotoxicity testing, performing gene editing, and facilitating disease modeling [29].

The utilization of adult stem cell-derived cancer organoids significantly expands the applicability of organoid models, and can serve as effective in vitro tools for mimicking primary cancers, with extensive applications in kidney cancer research. Grassi et al. initially established and characterized organoid cultures derived from undifferentiated enriched heterogeneous tissues from patients with normal kidneys and from patients with ccRCC, demonstrating their significant potential for both basic research and clinical translation [30].

According to the scaffold materials and culture equipment used, there are four main methods typically used to construct organoids: suspension culture, gel scaffold culture, rotational bioreactor, and gas‒liquid interface culture (Table 3). In addition, novel technologies such as bioprinting technology and organ-on-a-chip technology are emerging methods for constructing organoid models [31] (Table 4).

Table 3.

List of methodologies for generating renal cancer organoids

Methodologies	Scaffold-free culture	Scaffold-based culture
Culture environment	Growing freely in the suspension and formed aggregates or spheres in the culture medium	Growing in a matrix that provides physical support and naturally form a three-dimensional structure
Categorization	Air-Liquid Interface, Microfluidic Culture, Spinner Bioreactor, Free-floating Culture	Matrigel, Collagen Gel, Alginate Gel, Fibrin Gel, PEG, ECM
Advantages	Providing extracellular matrix and mimicing the natural growth environment of tumor cells in vivo	Straightforward procedures and high experimental efficacy
Disadvantages	Intricate operations and substantial maintenance cost	Lack of simulation of the tumor microenvironment, and the type and concentration of growth factors in the culture medium need to be precisely controlled
Applications	Personalized drug screening and tumor biology research	High-throughput screening and large-scale basic experiments

Table 4.

Comparison of advantages and disadvantages between emerging organoid technologies

Comparison	Organoid coculture system	Bioprinting	Organ-on-a-chip	CRISPR gene editing	High-throughput screening platform
Competitive edge	Replicating the microenvironment within the organism	Fabricating three-dimensional (3D) tissues that closely mimics human organs	Integrating multiple organoids to facilitate multi-system research	Investigating the association between gene mutations and diseases by precisely editing specific genes	Accelerating the drug screening process
Technical complexity	Medium	Difficult	Medium	Medium	Easy
Application scenarios	Tumor immunotherapy research	Customized organizational design	Drug testing and toxicity assessment	Tailored treatment guided by genetic mutations	Personalized drug screening
Resource costs	Medium	High	High	High	Medium
Development Thresholds	Great heterogeneity in data interpretation	High technical difficulty and low generalizability	Complex system design and high maintenance costs	Off-target effects	Huge substantial computational capabilities

Generally, organoid cultivation requires the following conditions: first, stem cells are derived from tissues; second, these stem cells are seeded into an appropriate culture medium; third, specific culture conditions are implemented to maintain the tissue-specific characteristics of the cultured stem cells [32](Fig 2).

Fig. 2.

Overview of the main protocols in RCC: 2D hydrogel coating culture, 2D ECM coating culture, 3d hydrogel culture culture, 3d ECM culture culture and droplet-based 3D culture. The establishment and generation of organoids from primary tissues can be divided into the following steps: firstly, the tissue is dissected into small fragments; secondly, enzymatic digestion using various enzymes such as collagenase, trypsin, hyaluronidase, etc., is employed to obtain single cells. Subsequently, in the third step, these cells are mixed with Matrigel and inoculated. In the fourth step, a growth medium is added for cultivation. Following different processing methods, organoid samples need to be cultured in vitro to construct 3D culture models. The available extracellular matrix methods for cultivation may vary but can provide an appropriate microenvironment for organoid growth. A Hydrogel and medium mixture are first plated for adhesion after solidification, followed by the addition of cells on the surface. B Hydrogel is firstly mixed with the cell suspension and subsequently transferred to the culture dish. Once the hydrogel underwent gelation or solidification, a covering medium is added for organoid formation during cultivation. C After combining hydrogel with cell suspension and allowing soft gel formation, the culture medium is introduced in the form of liquid droplets to generate organoids. D The orifice plate is equipped with a dome-shaped structure, where natural extracellular matrix (ECM) is incorporated. Additionally, cells are seeded onto the surface of the dome and covered with a medium. E The natural extracellular matrix (ECM) and cell suspension are first mixed, then added in the form of a dome-shaped structure to an orifice plate, followed by the addition of a cover medium after curing

Different tissues require distinct media components, while diverse culture protocols are implemented across laboratories. Nonetheless, Advanced DMEM/F12 is typically used as the basal medium and the supplemented with essential constituents such as antibiotics, B27 supplements, N2 supplements, HEPES buffer, and amino acids. However, the specific cytokines, small molecule inhibitors, small molecule agonists, and hormones required to replicate stem cell niche signaling differ. The ROCK inhibitor Y-27632 can suppress stem cell apoptosis, enhance cloning efficiency, and extend cell passage, making it a crucial component of kidney organoid culture reagents [33]. However, previous reports have indicated that Y-27632 can impede the growth of subcutaneous ccRCC tumours in mice, suggesting its potential as a therapeutic agent for VHL-deficient ccRCC [34]. Some studies have reported that R‐spondin1 and A83‐01 may lead to the overgrowth of normal kidney organoids. Therefore, removing these nonessential components from media can greatly reduce production cost without diminishing the effect of the organoid medium [35](Table 2).

Applications of Renal Tumour Organoids

As an emerging and sophisticated research tool, renal cancer organoids offer crucial support for drug development, fundamental research, and clinical applications. Specific prospects for application are outlined in Figure 3.

Fig. 3.

Contemporary applications of organoids in renal cancer research: renal cell carcinoma biobank, disease modeling, drug screening, personalized medicine, tumour biology and xenograft models

Renal Cancer Organoid Biobank

Calandrini et al. established the first organoid biobank for paediatric renal cancers, which provided the impetus for subsequent research on kidney cancer in children [36]. By incorporating matching organoids from healthy tissue into controls, multiomics analysis of kidney organoid biobanks can further clarify the phenotypic characteristics of different molecular subclasses of renal carcinoma. Similar studies have yielded significant findings in the field of digestive system tumors [37–39]. Ji et al. established a patient-derived liver cancer organoid biobank (LICOB) that comprehensively represents the histological and molecular characteristics of various types of liver cancer, as identified through multi-omics analyses including genomics, epigenomics, transcriptomics, and proteomics [40]. Grunwald et al. discovered “subTME” by integrating large-scale histology-guided regional multiomic data with clinical information, histologically definable tissue states anchored in fibroblast plasticity, with regional relationships to tumor immunity, subtypes, differentiation, and treatment response, which also unveiled the relationship between the malignant biology of pancreatic cancer and the specific tumor microenvironment phenotype exhibited by individual patients [41]. However, it should be noted that the genetic characteristics of organoid biobanks change during cryopreservation due to long-term culture. Specific culture mechanisms may favour the growth of certain subpopulations, thereby destroying the heterogeneity of the entire tumour. Moreover, the application of radiotherapy and chemotherapy to tumour tissue also greatly affects the applicability of organoid biobanks.

Disease Modelling

Kidney organoids offer a distinct advantage in addressing rare kidney diseases, including uncommon renal tumours and congenital kidney disorders [42, 43]. Hwang et al. established a patient-specific model of pRCC with a c-MET mutation using embryoid bodies from MET-induced pluripotent stem cells, thereby providing a genetically accurate representation of the disease. This model successfully expressed renal markers, generated tumours in NSG mice, and exhibited pRCC marker expression in vivo and in vitro, accurately capturing the molecular characteristics of human RCC [44].

Hernandez et al. utilized TSC2-type − / − hiPSCs to generate in vitro 3D kidney organoids exhibiting features of renal AML and demonstrated that rapamycin effectively eradicates orthotopic TSC2 − / − AML xenografts in immunodeficient nude rats [43].

Gerli et al. identified the presence of gastrointestinal, renal, and pulmonary epithelial progenitor cells in amniotic fluid (AF), from which kidney tubule amniotic fluid organoids (KAFOs) expressing nephron progenitor cells and tubular markers were generated. These rudimentary organoids are expandable and capable of functional maturation, providing the possibility to simulate various developmental tissues during pregnancy. This approach eliminates the need to obtain samples from terminated pregnancies, allowing donors to generate organoids while continuing their pregnancy, thereby offering prenatal disease modelling for patients with congenital hereditary renal cancer [45].

Homozygous deletion of the tumour suppressor gene WT1 is a prevalent cause of Wilms tumour, the most common type of renal cancer in children. However, paediatric oncogenesis remains largely unexplored in humans. Waehle et al. developed a human kidney organoid phenotype model to investigate Wilms tumour formation and discovered that loss of WT1 during organoid development leads to excessive growth of renal progenitor cells at the expense of differentiated glomeruli and tubules, thereby elucidating the role of WT1 in promoting renal progenitor cell progression and suppressing tumorigenesis [46].

Drug Screening, Personalized Medicine, and Drug Toxicology Tests

Drug resistance in advanced renal cancer poses a challenging clinical dilemma that significantly impacts patient survival rates.

Kazama et al. established 15 patient-derived RCC tumour organoid cultures and assessed the drug response to five standard-of-care tyrosine kinase inhibitors, including sunitinib, axitinib, pazopanib, sorafenib, and cabozantinib, in four patient-derived RCCO cultures [47].

Additionally, exploring optimized combinations of multiple drugs represents a promising avenue for research [48]. Employing high-throughput screening techniques on kidney organoids enables the investigation of both the efficacy and selectivity of multidrug combinations in drug-resistant RCC.

Precision medicine approaches for various cancer types, such as colorectal cancer, rely on next-generation sequencing (NGS) since extensive research suggests a relationship between genetic alterations and drug response. However, identifying biomarkers that predict drug response is hindered by the significant tumour heterogeneity observed in RCC patients, thereby limiting the effectiveness of NGS in RCC precision medicine.

As a tool to guide precision medicine and personalized medicine, the use of patient-derived tumour organoids enables the selection of chemotherapy drugs based on tumour characteristics, and the combination with high-throughput screening (HTS) methods can mitigate the limitations of NGS [35, 49]. Consequently, employing the patient-derived cancer organoid (PDCO) model can serve as a precision medicine method for RCC by elucidating tumour tissue responses to chemotherapy drugs and targeted therapies. HTS was used by Cao et al. to validate the promising antitumour activity of crizotinib on metastatic tRCC organoids harbouring PRCC-TFE3 fusions [50].

The kidney organoids also serve as highly effective in vitro platforms for conducting drug toxicology studies on multiple renal toxicities, thereby circumventing any potential ethical concerns [51]. Garcia et al. investigated the oncogenic effects of aristolochic acid I (AAI) using kidney organoids [52].

Tumour Biology

The relationship between the occurrence and development of each RCC subtype and related genes has been rigorously elucidated in various studies [53, 54]. Aberrant epigenetic remodelling can serve as a molecular hallmark for the investigation of tumorigenesis and progression. The utilization of organoids as an advanced preclinical model presents a formidable tool for investigating the nephrogenesis and molecular biology of RCC. In the context of technological breakthroughs facilitated by the advancement of diverse emerging technologies, such as multiple omics technology, HTS, and organ chips, the integration of organoids with other cutting-edge technologies will further expand the application prospects of organoids [55].

Single cell RNA sequencing (ScRNA‐seq) can reveal cell heterogeneity within RCC organoids, which is helpful for investigating the tumour microenvironment (TME) of renal carcinoma organoids [35]. The occurrence of unrepaired DNA damage can result in genetic mutations that facilitate the progression of kidney cancer [56, 57]. The utilization of kidney organoids can offer valuable insights into the response of specific renal cell types, such as RCC and Wilms tumour cells, to DNA damage [58].

Bulk RNA‐seq of RCC organoids and matched normal kidney organoids allows comparisons of the average expression of genes between different samples. Li et al. found several upregulated genes and downregulated genes among the genes differentially expressed between RCC organoids and normal kidney organoids, some of which have been confirmed in previous studies [35]. These findings suggest that RCC organoids have the ability to identify potential tumour biomarkers, which may play an important role in the early diagnosis of kidney cancer and the discovery of advanced metastases.

Wang et al. used high spatial resolution MALDI-MSI to establish a cell type-specific spatial dynamic metabolomics platform combined with single-cell transcriptomics to describe the metabolic trajectory during human kidney development at high spatial resolution, which may be helpful for further research on kidney regenerative medicine [17].

Xu et al. established chimeric organoids by combining embryonic kidney cells with renal cancer cells, which can serve as a novel model to investigate the behavioural role of renal cancer cells within the context of emergent complex tissue structures, allowing for the examination of their impact on normal cell proliferation, differentiation, and morphogenesis in an environment closely resembling in vivo conditions. Additionally, 3D RCC-MM chimaera organoids can also be used as an innovative tool for elucidating the involvement of coregulatory genes in kidney development and renal carcinogenesis, identifying potential oncogenes, and monitoring how silencing oncogenic genes influences cancer growth mediated by normal renal cells [59].

Previous studies have shown that the involvement of Wnt and Notch signalling in the development of human ccRCC can be attributed to their regulation of ccRCC patient-derived stem cells [60]. Myszczyszyn et al. successfully generated tubular organoids (tubuloids) resembling the adult tubular epithelial renewal process from total single adult mouse kidney epithelial cells; these tubuloids exhibited characteristics of adult renal cells with stem/progenitor cell-like functionality, such as activation of Wnt and Notch signalling pathways, sustained proliferation, and expression of markers specific to proximal and distal nephritic lineages, resulting in high self-renewal but without concomitant elevation of proliferation and neoplasia [61]. Moreover, tightly regulated endogenous Wnt and Notch signalling has been observed to drive kidney regeneration but can contribute to chronic kidney disease (CKD) when genetically dysregulated [62].

Xenograft Models

The renal cancer organoid xenograft model serves as a valuable in vivo preclinical tool for investigating the proliferation, growth, invasion, and metastasis characteristics of renal cancer tissues. The efficacy of patient-derived renal cancer organoids has been demonstrated through their successful engraftment into the subcapsular kidney of immunocompromised mice, where they maintain a proliferative tumour-propagating population, exhibit robust implantation rates, display disorganized growth structures, possess invasive capabilities into normal tissues, and can be effectively maintained in vivo [30, 60].

The functionality of glomerular and tubular structures in renal organoids determines their suitability for application in regenerative medicine. Recent studies have demonstrated that transplantation under the renal capsule in immunodeficient mice can lead to vascularization and subsequent maturation of kidney organoids [63–65]. By employing a titanium imaging window, Berg et al. assessed posttransplantation renal organoids in immunized mice and confirmed functional aspects of the kidney, such as glomerular filtration and proximal tubular reabsorption, were intact [66].

Limitations and Current Challenges

Tumour organoid models have some limitations. The field of basic research continues to face numerous challenges, and the process of clinical translation remains distant.

Highly Heterogeneous Culture Protocols

A widely recognized protocol for establishing organoid systems to study renal cancer does not exist. Numerous articles have detailed the distinct methodologies employed by various research laboratories to generate kidney cancer organoids, which leads to variability.

Culturing tumour organoids is a costly and labour-intensive process that requires substantial quantities of materials, workforce, and time. The cytokines essential for formulating organoid media are expensive and the absence of standardized culture protocols frequently necessitates repetitive experiments, resulting in a significant financial burden on researchers and severely restricting the utilization of organoids in large-scale studies.

The culture success rate of tumour tissue-derived organoids is comparatively lower, and their maintenance poses greater challenges than that of normal tissues. Simultaneously, the high heterogeneity of the culture protocol will increasingly impact the reproducibility of experiments and the interpretation of results, particularly as more novel organoid systems are developed.

Limitations Regarding the Reproducibility of the Authentic Tumour State

Organoid techniques often fail to preserve all different tumour compartments, including cancer-associated fibroblasts (CAFs), immune cells, and tumour blood vessels. Since the majority of renal cancer organoids are derived from epithelial cells, current studies predominantly focus on the cellular origin and carcinogenic factors of renal cancer, with limited systematic research on the tissue microenvironment. This lack of preservation of tumour blood vessels and the TME, including stroma and immune cells, hinders their application in evaluating the efficacy of antiangiogenic drugs and immune checkpoint inhibitors.

Ock et al. demonstrated that the cRCC TME exhibited the highest degree of immune infiltration among solid tumours in a pancancer analysis [67]. The tumour immune niche, which is composed of various immune cells, comprises cytotoxic T cells, tumour-infiltrating dendritic cells, regulatory T cells, tumour-associated macrophages, and myeloid-derived suppressor cells.

The significance of immunotherapy must be underscored as immunotherapy is recommended as the primary treatment option for patients with metastatic RCC. However, due to the dynamic nature of the tumour immune microenvironment, which may vary across different tumour types and individual patients, modelling the immune microenvironment surrounding tumours in certain organs is challenging.

The basement membrane (BM) and adhesion molecules also play a crucial role in facilitating tumour metastasis [68]. The BM is a complex network of macromolecules that form a supportive structure for all successive layers of cells, and is mainly comprised of collagen type IV and laminin [69]. The assembly and regulation of the BM have vital impacts on the development, differentiation, and maturation of renal organoids and have significant implications for cancer invasion and metastasis [68]. However, the dynamics of the BM in renal cancer remain poorly understood, thereby limiting our understanding of the mechanism of metastasis and posing a significant challenge to the advancement of renal cancer organoids [70].

Limitations of Culture Techniques

Culturing renal cancer organoids often leads to an imbalanced proliferation of nonmalignant cells due to the use of media additives, thereby limiting the comprehensive representation of the entire disease spectrum in the study. More importantly, it is not feasible to simultaneously sustain the long-term viability of multiple lineage cells and maintain synchronized growth rates utilizing identical culture media during renal cancer organoid cultivation.

Genetic stability may also be influenced by variations in kidney type during batch augmentation. The efficacy of medical therapy in patients with RCC is indisputable, particularly for those with compromised performance status or unresectable RCC, and systemic therapy should be considered the appropriate treatment option. However, due to the absence of interorgan interactions, renal cancer organoids fail to accurately replicate the overall systemic response during systemic therapy.

Prospects and Future Directions

Establishing a Standard Culture Protocol

The standardization and validation of renal cancer organoids are crucial for advancing the development of these models. First, researchers should provide a comprehensive description of the organoids, including detailed information on the donor's physiology, tissue source, and specific characteristics of the original cells. Moreover, it is imperative to employ HE staining, immunofluorescence staining, whole‐exome sequencing, RNA sequencing, scRNA-seq and other techniques to thoroughly characterize cultured organoids at various levels, including morphology, gene expression, and functionality. Additionally, the inclusion of both transverse and longitudinal contrasts is crucial, encompassing matching organoids derived from adjacent normal tissues and previously established systems.

Although a generic protocol for establishing PDCOs has been described and utilized for semiautomated chemotherapeutic drug screening, the implementation of a universal cultivation protocol can only offer limited reference value due to the inherent variability in cell types, structures, and functions across different organizations [32].

Therefore, we urge experts in relevant fields to promptly reach a consensus on these aforementioned aspects to establish a standardized culture protocol and nomenclature system for renal cancer organoids that can effectively guide future research.

Coculture

There are three modes of organoid coculture. First, the effects of immune cell-derived cytokines on epithelial cells can be evaluated by using recombinant cytokines present in the extracellular matrix-processing organs. Second, organs can be digested into individual cells and then regrown in the presence of immune cells to assess the impact of immune cells and their cytokines (soluble or membrane-bound) on organ growth and differentiation, as well as the influence of epithelial cells on immune cell phenotypes. Third, activated immune cells such as T cells or innate lymphoid cells (ILCs) can be added to the ECM or growth medium (suspension culture) containing intact organs to evaluate the interaction between immune cells and epithelial cells. The immune cells utilized in these cocultures were either directly isolated from tissues, directly extracted from peripheral blood, or first differentiated in vitro.

A study conducted by Wang et al. employed a coculture system of hiPSCs and fibroblastic reticular cells (FRCs) to generate high endothelial venule organoids (HEVOs). Subsequent implantation of these HEVOs into immunodeficient mice resulted in the formation of lymph node (LN)-like structures and third lymph node structures (TLSs), which effectively recruited antigen-presenting cells and transplanted lymphocytes, thereby enhancing the adaptive immune response and antitumour activity. Consequently, the coculture of HEVOs with renal cancer organoids holds promise as a potential application in cancer immunotherapy [71].

Organ-on-a-Chip

Although there are some challenges in technical implementation and standardization, the combination of renal cancer organoids and Organ-on-a-chip technology offers a robust tool for renal cancer research, enabling better simulation of the tumor microenvironment and driving advancements in drug development and personalized therapy [72, 73]. A microfluidic-based mini-tumour chip method combined with RCC organoids proposed by Ao et al. enables real-time observation of interactions among immune cells, between immune cells and tumour cells, and the preservation of original tumour cell components in tumour organoids, facilitating the measurement of tumour response to cancer immunotherapy [74].

Organoid Scaffolds

A considerable body of prior research has demonstrated the indispensability of cellular biochemical signals and microenvironmental mechanical signals in facilitating the maturation of nephrons within organoids [75, 76]. Previous studies have elucidated the role of biochemical signals, while matrix mechanical signals, such as those pertaining to the mechanical properties of the extracellular matrix (ECM) can effectively regulate cell behaviours such as proliferation, differentiation, and migration to drive nephrogenesis.

Hence, the selection of a matrix adhesive is crucial for the cultivation of renal cancer organoids. Matrigel is a naturally derived soluble basement membrane matrix obtained from Engelbreth-Holm-Swarm mouse sarcoma cells and is enriched for ECM proteins [77]. Owing to the uncertainty surrounding its immunogenicity and composition, which leads to considerable variability in cultured organoids from one batch to another, it becomes challenging to elucidate the matrix-specific factors that control organoid development [78].

Normal tissue-derived renal organoids can thrive in both rigid and flexible matrices, maintaining favourable passage times, while RCC organoids cultured in Matrigel often exhibit reduced proliferation capacity and appear to be more prone to losing their ability to form cohesive structures during long-term culture. Interestingly, RCC organoids exhibit a preference for stiff gels that mimic the stiffness of the TME in vivo [79].

Synthetic scaffold stiffness can be effectively enhanced by incorporating relevant extracellular matrix components and other biomaterials into the scaffold, offering a viable approach to sustain long-term culture of RCC organoids while increasing passage numbers.

A study conducted by Nerger et al. revealed that the mechanical and soluble signals derived from 3D encapsulation in alginate saline gels, including stiffness and viscoelasticity, can significantly influence nephron patterning and morphology in renal organoids. Furthermore, the extent of hydrogel deformation induced by cells broadly governs epithelial morphogenesis during 3D culture of organoids [80].

Batchelder et al. assessed the capacity of 3D scaffolds to support RCC growth and to maintain phenotype over time by employing renal ECM from decellularized kidneys and a patented polysaccharide scaffold (PSS) and demonstrated that both types of 3D scaffolds effectively preserved the gene expression phenotype and significantly influenced cell morphology. Natural materials such as PSS may be particularly advantageous for long-term investigations involving stromal or nonneoplastic cell interactions with malignant cells. Conversely, the distinct interactions facilitated by the renal medullary components of the ECM in relation to RCC could prove valuable for future studies on the metastatic origin of RCC, as well as specific renal tubular characteristics that might impact tumour proliferation [22].

Microfluidic Device

A microfluidic chip operates by controlling fluid movement in a tiny channel. Changing the size and shape of the channel affects the motion behaviour of the fluid, which can achieve fast, efficient and automated complex analysis and detection. Ozcelik et al. designed a simple microfluidic device and successfully established size-controlled ccRCC organoids within alginate hydrogels, which can be used to mimic the drug effects of cisplatin and TME responses [81]. With the continuous combination and development of microfluidic technology and organoid culture methods, mass manufacturing of customized standard kidney cancer organoids will likely become a reality soon.

Air-Liquid Interface Medium

The combination of renal cancer organoids and air-liquid interface technology offers a novel experimental platform for renal cancer research, enabling better replication of tumor biological characteristics and enhancing the reliability of drug screening and mechanism investigation. Nevertheless, there still exist obstacles in optimizing culture conditions and ensuring model stability. A patient-derived kidney tumour ALI PDO biobank, established by Esser et al., was characterized through multiple methods to assess the treatment response rate of individual ALI PDOs to cabozantinib and nivolumab [82].

CRISPR-Cas9 Genome Editing

It is worth mentioning that genetic engineering technology, especially CRISPR-Cas9 genome editing technology, when combined with renal cancer organoids, shows broad application prospects in the field of renal cancer [83, 84]. CRISPR-Cas9 genome editing is a technology that uses artificially designed sgRNA to recognize the genomic sequence of the target gene and guide the Cas9 protein enzyme to effectively cut the double strand of DNA, thereby achieving gene deletion or insertion [84]. Through gene deletion or insertion, CRISPR-Cas9 genome editing can gain a deeper understanding of the role of specific genes in the development of renal cancer and provide new targets for targeted therapy [85, 86]. By analyzing the genomic information of renal cancer patients, CRISPR-Cas9 genome editing can be used to target and repair renal cancer-related gene mutations and develop personalized gene therapy to inhibit tumor growth [87]. By combining CRISPR-Cas9 genome editing technology, specific gene mutations in renal cancer organoids can be constructed, providing more precise tools for renal cancer research [88]. CRISPR-Cas9 genome editing also faces limitations like ethical issues, off-target effects, and safety issues [89]. But it can be predicted that, as CRISPR-Cas9 genome editing and organoid technology continue to converge, a comprehensive analysis of gene expression changes in renal cancer organoids and a deep understanding of the molecular mechanisms of renal cancer will become a reality in the near future, greatly advancing the progress of organoid research in renal cancer [89, 90].

Cancer Treatment

The metabolic reprogramming of RCC significantly influences tumour progression and distant metastasis, which has profound implications for guiding RCC treatment strategies. The identification of pivotal epigenetic drivers with high mutation frequencies in RCC tumours through HTS, such as VHL, SETD2, BAP1, PBRM1 and KAT2A, among others, has sparked significant interest due to their potential as therapeutic targets for epigenetic interventions [91].

Wang et al. established patient-derived organoids to demonstrate the pivotal role of P2XR4 as a mediator of the interplay between lysosomes and mitochondria in tumour metabolism through Ca²⁺ homeostasis, offering a promising therapeutic approach for a specific subgroup of ccRCC patients exhibiting elevated mitochondrial activity [92].

Parikh et al. established RCCO to screen for tumour-infiltrating lymphocytes (TILs) with neoantigen reactivity and isolated individualized T-cell receptors (TCRs), thereby providing novel insight into the immune evasion mechanisms associated with renal cancer and offering potential applications in adoptive cell therapy [93].

Hamdan et al. cultivated patient-derived tumor organoids to develop an oncolytic adenovirus that produces a cross-hybrid Fc fusion peptide targeting PD-L1, thereby activating both IgG1 and IgA1 effector mechanisms to engage multiple immune pathways. This approach may lead to a substantial enhancement in tumour eradication while minimizing unnecessary cellular toxicity and ensuring drug stability and safety, thus offering a promising avenue for immunotherapy against kidney cancer [94].

Fendler et al. reported a well-characterized organoid model from human primary ccRCCs and demonstrated a positive correlation between the frequency of cancer stem cells (CSCs) expressing CXCR4⁺MET⁺CD44⁺ and tumour aggressiveness in ccRCC, along with activation of the WNT and NOTCH signalling pathways. Inhibition of WNT and NOTCH signalling effectively impedes CSC proliferation and self-renewal in RCC organoids. These findings suggest that CSCs play a pivotal role in the therapeutic resistance and metastatic potential of tumours, thereby offering a novel approach for treating ccRCC by targeting key mechanisms involved in CSCs that are crucial for this disease [60].

Xue et al. successfully established a tumour-infiltrating lymphocyte-preserved PDO model based on the ALI system, which holds great potential for predicting immunotherapy response and guiding personalized medication in patients [95].

Zhang et al. reported high expression of piRNA-1742 in RCC tumours, and the inhibition of piR-1742 significantly reduced tumour growth in RCC organoid models by destabilizing USP8 mRNA and facilitating its degradation [96].

Jin et al. utilized a substantial number of renal cancer organoids to demonstrate that the YAP-SATB2-NRF2 regulatory axis can facilitate chromatin remodelling and enhance antioxidative stress signalling, thereby contributing to tumorigenesis. The design of targeted inhibitors against SATB2 holds promise for potential therapeutic strategies in YAP-high RCC patients [97].

Zhang et al. discovered that aberrant expression of p300 and subsequent p300-mediated H3K27ac modification may partially contribute to and explain elevated JMJD6 levels in patient‐derived organoids, thereby modulating the oncogenic transcriptome in RCC cells to facilitate tumorigenesis. The administration of SKLB325, a specific inhibitor targeting JMJD6, exhibits significant potential in impeding the progression of RCC and augmenting the responsiveness of RCC towards sunitinib [98].

Zhang et al. demonstrated that downregulation of METTL14 resulted in increased stability of BPTF in patient‐derived organoids, leading to a dysregulated oncogenic transcriptome and enhanced superenhancers. This aberrant regulation further activated the aerobic glycolysis pathway and facilitated lung metastasis in renal cancer. Importantly, inhibition of BPTF using a specific inhibitor (AU1) significantly suppressed distant metastasis in RCC [99].

Conclusion

Although significant progress has been made in the research on renal cancer mechanisms and drug therapies, it is imperative to acknowledge that early diagnosis and treatment following tumour recurrence and metastasis remain pressing issues to be addressed.

The value of traditional models, such as 2D cell culture and experimental animal models, is undeniable. However, due to the inherent heterogeneity of tumour and cancer biology within kidney organoids, this technique provides a more accurate representation of the spatial structure and pathophysiological characteristics associated with tumour occurrence and metastasis, making it an invaluable platform for studying the initiation, progression, recurrence, metastasis, and treatment of RCC.

In addition, the application of novel technologies such as HTS, air-liquid interface culture, tissue engineering, organ-on-a-chip, and microfluidic devices can compensate for their limitations and ultimately render them more suitable for clinical settings. Therefore, despite numerous challenges, we believe that renal cancer organoids represent a highly promising research tool that may play a pivotal role in renal cancer investigation and treatment. Moreover, groundbreaking achievements in combating kidney tumours are highly likely to be achieved in the foreseeable future.

Abbreviations

RCC

Renal cell carcinoma

RCCO

Renal cell carcinoma organoid

ccRCC

Clear cell renal cell carcinoma

pRCC

Papillary renal cell carcinoma

chRCC

Chromophobe renal cell carcinoma

ICIs

Immune-checkpoint inhibitors

VEGFR TKIs

VEGF receptor tyrosine kinase inhibitors

mRCC

Metastatic renal cell carcinoma

ASCs

Adult stem cells

SSCs

Somatic stem cells

iPSCs

Induced Pluripotent stem cells

ESCs

Embryonic stem cells

MET

Mesenchymal to epithelial transition

NPCs

Nephron progenitor cells

RCCO

Renal cell carcinoma organoid

CRC

Consecutive colorectal carcinoma

TSC

Tuberous sclerosis complex

AML

Angiomyolipoma

hiPSCs

Human induced pluripotent stem cells

AF

Amniotic fluid

KAFOs

Kidney tubule amniotic fluid organoids

NGS

Next-generation sequencing

PDCO

Patient-derived cancer organoid

AAI

Aristolochic acid I

HTS

High-throughput screening

scRNA-seq

Single cell RNA sequencing

MALDI-MSI

Matrix-assisted Laser Desorp-tion Ionization Mass Spectro-metric Imaging

RCC-MM

Renal cell carcinoma-metanephric mesenchyme

CKD

Chronic kidney disease

CAFs

Cancer-associated fibroblasts

TME

Tumor microenvironment

BM

Basement membrane

FRCs

Fibroblastic reticular cells

HEVOs

High endothelial venule organoids

TLS

Lymph node structures

ILCs

Innate lymphoid cells

ECM

Extracellular matrix

PSS

Polysaccharide scaffold

ALI

Air-liquid interface

TILs

Tumor-infiltrating lymphocytes

TCRs

T-cell receptors

CSCs

Cancer stem cells

MRTK

Malignant rhabdoid tumours of the kidney

CMN

Congenital mesoblastic nephromas

Author Contributions

XL and CS conceived and designed this article. JH, XW and SG contributed to the conceptualization, manuscript writing, revisions, and literature review. All authors have read and agreed to the published version of the manuscript.

Data availability

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

Declarations

Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.