Chimeric antigen receptor–based cell therapy for treating urological tumors

Abstract

Urological tumors represent a significant global health challenge, with conventional therapies often proving insufficient to control disease progression. Recent breakthroughs in cellular immunotherapy, particularly in chimeric antigen receptor (CAR)-T cell, CAR–natural killer cell, and CAR-macrophage therapies, have demonstrated remarkable potential for treating these malignancies. Ongoing research is actively refining CAR-based strategies to enhance their precision in targeting tumor-associated antigens. This review comprehensively summarizes the applications of CAR cell therapy in the following 3 major urological tumors: renal cell carcinoma, bladder cancer, and prostate cancer. Furthermore, we analyzed the current advantages and limitations of these approaches and propose potential strategies for optimization focused on CAR-T cells. This review will provide future directions in this field and contribute to the development of more effective treatments for patients with urological cancer.

1. Introduction

Urological tumors, including renal cell carcinoma (RCC), bladder cancer (BC), and prostate cancer (PC), account for approximately 24% of all new cancer diagnoses and have a notably higher incidence in male.^[1] Prostate cancer, ranked as the second most common tumor in men, has an increasing incidence rate.^[1] Traditional therapies, such as radiotherapy, chemotherapy, and targeted therapies, primarily act directly on cancer cells but are often limited by high recurrence rates, drug resistance, and adverse effects. Circular RNAs (circRNAs) have recently emerged as promising therapeutic tools because of their stability and specificity. They play potential roles in modulating tumor invasion/metastasis, reshaping the immune tumor microenvironment (TME), and serve as the basis for circRNA-based vaccines. These applications can improve early detection, diagnosis, prognosis, and prediction of treatment response.^[2] However, the current circRNA-based strategies remain largely preclinical and are limited by off-target effects in healthy tissues.^[2] In recent years, immunotherapy has advanced rapidly as a transformative approach for the treatment of urological tumors. For instance, the bacillus Calmette-Guérin vaccine reduces the recurrence risk of high-grade nonmuscle-invasive BC by activating local mucosal immunity.^[3] Immune checkpoint inhibitors (ICIs), particularly those targeting programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) in BC, have shown encouraging results.^[4] However, Huang et al.^[5] have reported treatment-related adverse events in urological cancers, indicating that careful management and nursing strategies are required to ensure the safety of patients treated with ICIs. In addition, the identification of patients who may be sensitive to immunotherapy and enhancement of therapeutic efficacy are 2 problems of ICIs in urological cancers.^[6] Among newer modalities, chimeric antigen receptor (CAR)-based cellular therapies have gained significant attention, and multiple clinical trials are underway (Table 1).

Table 1.

Clinical trials of CAR-T, NK, and M cells therapy for RCC, BC, and PC.

#	NCT number	Tumor	Therapy	Target	Status	Research type	Research institution
1	NCT04969354	Advanced RCC	CAR-T	CAIX	Recruiting	Phase 1	Affiliated Hospital of Xuzhou Medical, China University, China
2	NCT05703854	Advanced RCC	CAR-NK	IL15	Recruiting	Phase 1/2	M D Anderson Cancer Center, US
3	NCT01218867	Renal Cancer	CAR-T	VEGFR2	Terminated	Phase 1/2	National Institutes of Health Clinical Center, US
4	NCT03638206	RCC	CAR-T	C-met	Unknown status	Phase 1/2	The First Affiliated Hospital of Zhengzhou University, China
5	NCT02830724	RCC	CAR-T	CD70	Recruiting	Phase 1/2	National Institutes of Health Clinical Center, US
6	NCT06010875	RCC	CAR-T	CD-70	Recruiting	Phase 1	The First Affiliated Hospital of Nanchang University, China
7	NCT05420519	RCC	CAR-T	CD-70	Recruiting	Phase 1	The Second People’s Hospital of Shandong Province, China
8	NCT05518253	RCC	CAR-T	CD-70	Recruiting	Phase 1	First Affiliated Hospital of Zhejiang University, China
9	NCT05420545	RCC	CAR-T	CD-70	Recruiting	Phase 1	The Second People’s Hospital of Shandong Province, China
10	NCT05468190	RCC	CAR-T	CD-70	Recruiting	Phase 1	Henan Cancer Hospital, China
11	NCT05239143	RCC	CAR-T	P-MUC1C-ALLO1	Recruiting	Phase 1	Cedars Sinai Medical Center, US. ect
12	NCT03393936	RCC	CAR-T	ROR2, AXL	Unknown status	Phase 1/2	Shanghai Public Health Clinical Center, China
13	NCT04438083	RCC	CAR-T	CD70	Active, not recruiting	Phase 1	Research site 2, 5, 4, US
14	NCT06383507	RCC	CAR-T	CD70	Not yet recruiting	Phase 1	No location data
15	NCT05103631	WT	CAR-T	Glypican 3	Recruiting	Phase 1	Houston Methodist Hospital, US
16	NCT06198296	WT	CAR-T	GPC3	Not yet recruiting	Phase 1	Houston Methodist Hospital, US
17	NCT04483778	WT	CAR-T	B7H3	Active, not recruiting	Phase 1	Seattle Children’s Hospital, US
18	NCT04897321	WT	CAR-T	B7-H3	Recruiting	Phase 1	St. Jude Children’s Research Hospital, US
19	NCT03618381	WT	CAR-T	EGFR, CD-19	Recruiting	Phase 1	Seattle Children’s Hospital, US
20	NCT06480565	ccRCC	CAR-T	CD70	Not yet recruiting	Phase 1/2	No location data
21	NCT05795595	ccRCC	CAR-T	CD70	Recruiting	Phase 1/2	Research site 3, 7, 6, US
22	NCT05672459	ccRCC	CAR-T	HLA-G	Recruiting	Phase 1/2	M D Anderson Cancer Center, US
23	NCT04696731	mccRCC	CAR-T	CD70	Recruiting	Phase 1	City of Hope.ect, US
24	NCT03740256	BC	CAR-T	HER2	Recruiting	Phase 1	Baylor St. Luke’s Medical Center, US
25	NCT03960060	BC	CAR-T	ROR2	Unknown status	Phase 1	Shanghai Zhongshan Hospital, Shanghai, China
26	NCT03185468	BC	CAR-T	FRa	Unknown status	Phase 1/2	Shenzhen Second People Hospital.ect, China
27	NCT04768608	CRPC	CAR-T	PSMA	Completed	Phase 1	The First Affiliated Hospital of Zhejiang University, China
28	NCT05354375	CRPC	CAR-T	PSMA	Recruiting	Phase 1	Affiliated Hospital of Xuzhou Medical University, China
29	NCT05805371	CRPC	CAR-T	PSCA	Recruiting	Phase 1	City of Hope Medical Center, US
30	NCT03873805	CRPC	CAR-T	PSCA	Active, not recruiting	Phase 1	City of Hope Medical Center, US
31	NCT06236139	CRPC	CAR-T	STEAP1	Not yet recruiting	Phase 1/2	Fred Hutch/University of Washington Cancer Consortium, US
32	NCT06046040	mCRPC	CAR-T	PSMA	Recruiting	Phase 1	Abramson Cancer Center of the University of Pennsylvania, US
33	NCT04249947	mCRPC	CAR-T	PSMA	Active, not recruiting	Phase 1	City of Hope Comprehensive Cancer Center.ect, US.ect
34	NCT06228404	mCRPC	CAR-T	PSMA	Recruiting	Phase 1/2	Changzheng Hospital, China
35	NCT02744287	mCRPC	CAR-T	PSCA	Suspended	Phase 1/2	Moffitt Cancer Center,ect, US
36	NCT05489991	mCRPC	CAR-T	PSMA	Terminated	Phase 1/2	Sarah Cannon Research Institute, US
37	NCT04227275	mCRPC	CAR-T	PSMA	Terminated	Phase 1	Moffitt Cancer Center.ect, US
38	NCT03692663	mCRPC	CAR-NK	PSMA	Unknown status	Phase 1	Tianjin People’s Hospital, China
39	NCT06267729	mPC	CAR-T	STEAP2	Recruiting	Phase 1/2	Research site, Duarte.ect, US
40	NCT03013712	PC	CAR-T	EpCAM	Unknown status	Phase 1/2	IEC of Chengdu Medical College, China
41	NCT05732948	PC	CAR-T	PSMA/PSCA	Recruiting	Phase 1	The First Affiliated Hospital of Soochow University, China
42	NCT04107142	PC	CAR-T	NKG2DL	Unknown status	Phase 1	Landmark Medical Centre, Malaysia
43	NCT01140373	PC	CAR-T	PSMA	Active, not recruiting	Phase 1	Memorial Sloan Kettering Cancer Center, US
44	NCT03089203	PC	CAR-T	PSMA	Active, not recruiting	Phase 1	University of Pennsylvania, US
45	NCT04633148	PC	CAR-T	PSMA	Terminated	Phase 1	Universitätsklinikum Ulm.ect, Germany
46	NCT06094842	PC	CAR-T	L1CAM	Not yet recruiting	Phase 1	Fred Hutch/University of Washington Cancer Consortium, US
47	NCT04429451	Prostate Tumor	CAR-T	PSMA	Recruiting	Phase 1/2	Shenzhen Children’s Hospital.ect, China
48	NCT05022849	Prostatic Neoplasms	CAR-T	KLK2	Active, not recruiting	Phase 1	City of Hope Cancer Center.ect, US. ect

*The clinical trials are collected from clinicaltrials.gov (accessed on September 8, 2024).

BC = bladder cancer; CAR-NK = CAR–natural killer cell; CAR-M = CAR macrophage cell; CAR-T = chimeric antigen receptor T cell; ccRCC = clear cell RCC; CRPC = castration-resistant PC; ect = additional trial sites not listed; ID = National Clinical Trial identifier; mCRPC = metastatic CRPC; mPC = metastatic PC; NCT = National Clinical Trial; PC = prostate cancer; RCC = renal cell carcinoma; WT = Wilms tumor.

Chimeric antigen receptor-T cell therapy, based on genetic engineering and immunology, can overcome histocompatibility antigen restrictions and offers a potent strategy against malignancies.^[7] Although highly effective in hematologic cancers, such as B-cell lymphomas and leukemia, its application in urological tumors targets antigens, such as prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), 6 transmembrane epithelial antigens of prostate 1 (STEAP1), epidermal growth factor receptor (EGFR), and carbonic anhydrase IX (CAIX).^[8–11] Despite ongoing clinical trials, CAR-T cells face challenges in solid tumors, including poor T-cell trafficking, an immunosuppressive TME, antigen heterogeneity, and safety concerns.^[12] By contrast, natural killer (NK) cells, important components of the innate immune system, present advantages over CAR-T cells in eliminating the risks of graft-versus-host disease, cytokine release syndrome, and neurotoxicity, while requiring no antigen priming.^[13] However, the short persistence necessitates repeated infusions.^[14] Chimeric antigen receptor-NK cell therapies have primarily been explored for PC, although they have many limitations.^[15] Alternatively, CAR-macrophage (CAR-M) therapies may offer superior tumor homing, infiltration, and TME modulation compared with CAR-T/NK cells. However, studies on their application in the treatment of urological tumors are limited.^[16] Figure 1 illustrates the generation and administration of CAR-T cells to patients. Clinical trials of these therapies are currently in the preclinical phase (Table 1).

Figure 1.

Generation and administration of CAR-T cells in patients. Autologous T cells are isolated via leukapheresis and genetically engineered to express a CAR. The CAR consists of an extracellular antigen-binding domain, transmembrane domain, and intracellular signaling. Engineered CAR-T cells are expanded, purified and quality-controlled in vivo, then reinfused into the patient. Upon antigen recognition, CAR-T cells initiate cytotoxic killing of tumor cells. CAR = chimeric antigen receptor.

We reviewed recent advances in CAR-based cell therapies (CAR-T, CAR-NK, and CAR-M) for urological tumors (RCC, BC, and PC). This review evaluates the clinical potential, limitations, and optimization strategies of CAR-T cells, with a particular focus on CAR-T applications. Our analysis offers both current perspectives and future directions for improving immunotherapy for urological tumors.

2. Structure and generations of CAR molecules

A CAR consists of the following 3 functional domains (Fig. 1): extracellular, transmembrane, and intracellular. The extracellular antigen-binding domain is composed of a single-chain variable fragment created by linking the heavy- and light-chain variable regions of an antibody via a flexible peptide linker.^[17] The transmembrane domain anchors the receptor to the cell membrane and ensures stable CAR expression. The intracellular signaling module contains both costimulatory domains (typically CD28 from the immunoglobulin superfamily or 4-1BB from the tumor necrosis factor receptor superfamily) and the primary activation domain derived from CD3ζ. This modular architecture enables CAR-expressing immune cells to recognize and respond specifically to target antigens on tumor cells.

Chimeric antigen receptor receptors have evolved over 5 generations (Fig. 2). First-generation CARs contained only a CD3ζ signaling domain without costimulatory components, resulting in limited CAR-T cell proliferation. Second-generation designs incorporated CD28 or 4-1BB costimulatory domains, significantly improving cell proliferation and reducing senescence. Third-generation CARs further enhance the functional efficacy by combining 2 distinct costimulatory signals. Clinical evidence from a phase I trial (NCT01853631) has demonstrated superior expansion and persistence of third-generation CAR-T cells compared to their second-generation counterparts in patients with relapsed/refractory non-Hodgkin lymphoma. Fourth-generation CARs feature additional intracellular domains that enable small-molecule coexpression, with interleukin (IL)-12 and IL-18 being the most investigated cytokines, and numerous other molecules are explored.^[18] The most advanced fifth-generation CARs incorporate an IL-2 receptor β-chain domain that enables antigen-dependent JAK/STAT pathway activation. Preclinical studies have shown that these next-generation constructs prevent terminal differentiation in vitro while demonstrating enhanced persistence and antitumor activity compared with second-generation CARs.^[19]

Figure 2.

Evolution of chimeric antigen receptor structures from the first to the fifth generation. Each generation includes an antigen recognition domain, transmembrane domain, and intracellular signaling domain. See Section 2 for detailed descriptions of each generation.

3. CAR cell therapy for RCC

Renal cell carcinoma treatment has advanced significantly, transitioning from high-dose cytokine therapy with tumor resection to stage-specific targeted therapies, and markedly improving patient outcomes.^[20] However, first-line immunotherapy remains insufficiently effective against advanced RCC, underscoring the need for novel therapeutic approaches. Chimeric antigen receptor cell therapy has emerged as a potential treatment option and may soon offer a viable alternative. The status of CAR therapies targeting RCC, BC, and PC is summarized in Table 2. As shown in Figure 3, current clinical trials have primarily focused on RCC and BC, with CAR-T cells dominating (95.8% of trials). Most trials were in phase 1/2, led by the US (56%) and China (40%).

Table 2.

Current status of CAR therapies targeting RCC, BC, and PC.

Tumors	Therapies	Targets	Current stage	Key findings
RCC	CAR-T	CD70	Preclinical	• Potent antitumor activity in RCC cells and NSG mice • Dual-targeting with CAIX
		c-Met	Preclinical	• Suppress tumor growth in NOD/SCID mice and enhanced efficacy with sunitinib combination
		CAIX	Preclinical	• Secretes anti-PD-L1 antibodies in NSG mice • Synergistic effect with sunitinib in RCC cells
		AXL/ROR2	Phase I/II	• Expressed in RCC and contributes to tumor growth and invasiveness • No dose-limiting toxicity in r/r metastatic RCC (NCT03393936)
		VEGFR2	Phase I/II	• No observed response (NCT01218867)
	CAR-NK	HER2	Preclinical	• Reduces pulmonary metastasis in RCC models
		CAIX	Preclinical	• Induces specific cytotoxicity and cytokine release in RCC cells and enhanced efficacy with Bortezomib combination
		EGFR	Preclinical	• Synergistic effect with cabozantinib in NOD/SCID mice
		IL-15	Phase I/II	• IL-15 enhanced persistence • Currently recruits (NCT05703854)
	CAR-M	HER2	Phase I	• 633 patients with HER2+ solid tumors (including RCC)
	CAR-Treg	EGFR	Preclinical	• Inhibits effector T cell function in NSG mice
BC	CAR-T	PD1 ligands	Preclinical	• Tumor lysis, cytokine secretion, long-term survival in mice
		MUC1	Preclinical	• Specific cytotoxicity against MUC1-positive BC organoids
		EGFR/CD44v6	Preclinical	• Enhanced cytotoxicity when combined with decitabine
		HER2	Phase I	• Overexpressed in BC, potential target for deeper investigation currently recruits, (NCT03740256)
		SIA-CIgG	Preclinical	• Effective BC cell lysis with enhanced persistence and enhanced efficacy with vorinostat combination
		uPAR	Preclinical	• Potent antitumor efficacy in immunocompetent mice
		B7H3	Preclinical	• Antitumor responses in 3D BC organoid system
		Glycosaminoglycans	Preclinical	• Increased survival in NOD/SCID mice
		PSCA (with TIGIT)	Preclinical	• Enhanced efficacy by overcoming CD155-mediated inhibition
	CAR-NK	Not Tested	Not yet applied	• Potential due to efficacy in other cancers
	CAR-M	Not Tested	Not yet applied	• Potential advantages for finding
PC	CAR-T	PSMA	Phase I/II	• Limited persistence with no observed responses (NCT01140373) • More effective, feasible, and safe with the coexpression of TGFβRDN, (NCT03089203) • Assesses the safety, tolerability, feasibility, and preliminary efficacy of TmPSMA-02 CAR-T cells (NCT06046040) • Enhanced efficacy with low-dose docetaxel and IL-23mAb combination
		PSCA	Phase I/II	• Enhanced efficacy with a 4-1BB costimulatory, (NCT05805371) • Anticancer activity with dose limiting toxicities (DLT) of cystitis • PSA declines in 4/14 patients, radiographic improvements, and limited persistence after infusion, (NCT03873805) • 56% patients with 50% PSA reduction, (NCT02744287)
		STEAP1	Preclinical	• Effective at low antigen density in NSG mice and enhanced antitumor activity with the addition of tumor-targeted IL-12
		B7-H3	Preclinical	• Effective against radiation-resistant PC stem cells
		EphA2	Preclinical	• Antigen-dependent tumor inhibition with CD28 costimulatory domain both in in vitro and vivo
		EpCAM	Preclinical	• Significant efficacy (p < 0.05) in NOD/SCID mice • Clinical relevance debated
		NKG2DL	Phase I	• Enhances expansion/reduces exhaustion in combination with IL-7
	CAR-NK	PSMA	Phase I	• Potent anti-CRPC activity and synergy with anti-PD-L1 • Assess the safety and efficacy of PSMA-CAR-NK cells (NCT03692663)
	CAR-M	Not tested	Not yet applied	• Potential TME advantages

BC = bladder cancer; CAR-M = chimeric antigen receptor macrophage; CAR-NK = chimeric antigen receptor natural killer cell; CAR-T = chimeric antigen receptor T cell; CAR-Treg = chimeric antigen receptor regulatory T cell; CRPC = castration-resistant prostate cancer; DLT = dose-limiting toxicity; IL = interleukin; NOD/SCID mice = nonobese diabetic/severe combined immunodeficiency mice; NSG mice = NOD-scid IL2Rγnull mice; PC = prostate cancer; PD-L1= programmed cell death ligand 1; PSA = prostate-specific antigen; PSMA = prostate-specific membrane antigen; RCC = renal cell carcinoma; r/r = relapsed or refractory; TME = tumor microenvironment.

Figure 3.

Distribution of tumor, therapy, stage, and country in total 48 clinical trials. (A) Tumor types (RCC, BC, or PC). (B) CAR-based therapies (CAR-T or CAR-NK). (C) Trial phases. (D) Geographic distribution. Data reflect 48 trials from Table 1. BC = bladder cancer; CAR = chimeric antigen receptor; NK = natural killer; PC = prostate cancer; RCC = renal cell carcinoma.

Current research on CAR-T therapy for RCC targets antigens, such as CD70, PD-L1, cellular mesenchymal–epithelial transition factor (c-Met), CAIX, vascular endothelial growth factor receptor 2 (VEGFR2), receptor tyrosine kinase AXL, and receptor tyrosine kinase-like orphan receptor 2 (ROR2). Panowski et al. ^[21]developed CD70-targeting CAR-T cells that demonstrated potent antitumor activity in RCC cell lines and patient-derived xenograft models (NSG mice). Suarez et al. ^[22]have reported that CAIX-CAR-T cells secrete anti-PD-L1 antibodies and suppress tumor growth in KIRC models (6- to 8-week-old male NSG mice). Furthermore, dual-targeting strategies can broaden the range of targeted cells, reduce tumor heterogeneity, and decrease adverse side effects. Mori et al. ^[23] have reported that c-Met–specific CAR-T cells infiltrated tumors in orthotopic models (6-week-old male and female NOD mice), with improved efficacy when combined with sunitinib (a multitarget inhibitor of VEGFR1, VEGFR2, VEGFR3, platelet-derived growth factor receptor β, and c-Kit). Overall, CAR-T cell therapy, particularly in combination with targeted therapies or dual-target CAR-T cells, has promising therapeutic effects in RCC. AXL, a receptor tyrosine kinase, and ROR2, a member of the tyrosine kinase orphan receptor family, are expressed in RCC and contribute to tumor growth and invasiveness.^[24] Interim results from a phase I/II trial (NCT03393936) have indicated no dose-limiting toxicity for ROR2- and AXL-CAR-T cells in r/r metastatic RCC, supporting their safety.^[25] To date, only 1 phase 1/2 clinical trial (NCT01218867) on RCC has been published. They evaluated VEGFR2-CAR-T cells in 24 patients to determine a safe number of these cells to infuse and observed the safety and effectiveness in recurrent or relapsed cancer (including RCC); however, the study was eventually terminated because of a lack of response. Nevertheless, with the continuous development of CAR T-cell therapy for RCC, more clinical trials may yield more promising outcomes.

Although CAR-NK cell therapy has emerged as a promising approach in oncology, its clinical application to RCC remains limited. A review of 6 male patients (aged 50–70 years, who had previously received treatment) revealed that RCC induces decidual-like transformation in NK cells, impairing their cytotoxic function while promoting angiogenesis.^[26] Despite these challenges, numerous studies have demonstrated the significant therapeutic potential of CAR NK cell therapy. For instance, Schönfeld et al. ^[27] engineered HER2-targeted NK-92 cells that exhibited potent cytotoxicity in vitro and reduced pulmonary metastasis in RCC models (4–6-week-old, female NSG mice), suggesting that NK-92/5.28. Z-cells may be promising candidates for RCC therapy. Additionally, NK cells can be genetically modified to reduce the expression of inhibitory receptors, increase the expression of activating receptors, and amplify the cytokine output. Kremer et al. ^[28] have reported that NK cells genetically engineered to stably express CXCR2 demonstrate enhanced migratory capacity, which improved the antitumor response following the adoptive transfer of NK cells. Cytokine engineering is another promising approach for this purpose. For instance, IL-15 modification is particularly valuable because of the strong expansion of NK and CD8+ T cell abilities and the avoidance of activation-induced cell death.^[29] This principle is being tested in an ongoing phase 1/2 trial (NCT05703854) to evaluate CD70/IL-15-modified cord blood-derived NK cells in advanced RCC.

Emerging research has indicated that CAR-M and CAR-Treg cells could also be effective in the treatment of various solid tumors, although their application in RCC remains underexplored.^[30] The natural abundance of macrophages in RCC makes CAR-M therapy particularly promising. Anti-EGFR CAR-Treg cells, when used in combination with CD28, exhibit antigen-specific infiltration and inhibited the function of effector T cells in mouse models (8-week-old female NSG mice).^[31] Moreover, CAR-T cell expansion may serve as both a biomarker for response and toxicity after CAR-T cell therapy and a regulator of CAR-T cell responses in humans, offering potential solutions to toxicity-related challenges after CAR-T cell infusion.^[32]

4. CAR cell therapy for BC

Currently, treatment paradigms for BC depend on precise staging.^[33] Intravesical bacillus Calmette-Guérin immunotherapy remains the gold standard for intermediate- and high-risk nonmuscle-invasive BC, whereas muscle-invasive BC and advanced cases benefit from expanded therapeutic options including ICIs, molecular targeted therapies, and antibody-drug conjugates. Although ICIs have become the first-line therapy for chemotherapy-ineligible patients and offer more durable efficacy and favorable safety than conventional chemotherapy, their overall efficacy remains suboptimal.^[34] This therapeutic limitation has spurred intensive investigations into novel immunotherapeutic approaches for advanced BC.

Bladder cancer expresses numerous tumor-associated antigens, making CAR-T cell therapy an important treatment option; however, current approaches primarily target pan-cancer tumor antigens. Parriott et al. ^[35] have demonstrated that chimeric PD-1 T cells exhibited effective tumor cell lysis, proinflammatory cytokine secretion, tumor burden reduction, and long-term tumor-free survival in syngeneic mouse models of BC, RCC, and PC. These findings highlight the potential of immune checkpoint-targeted CAR-T strategies for the treatment of urological malignancies. Several BC-specific targets have shown promise in recent studies. Yu et al. ^[36] have developed transmembrane glycoprotein mucin 1 (MUC1)-CAR-T cells that showed specific immune cytotoxicity against MUC1-positive BC organoids, indicating the feasibility of using MUC1-CAR-T cells to treat BC. Additionally, Grunewald et al. ^[37] have discovered that the combination of decitabine, a DNA methyltransferase inhibitor, with EGFR-CAR-T cells or CD44v6-CAR-T cells significantly enhanced cytotoxicity against urothelial carcinoma cell lines. Thus, the combined application of CAR-T cells and other drugs may result in enhanced antitumor effects. Another potential target, HER2, is overexpressed or amplified in BC.^[38] Despite its controversial prognostic value in BC, it remains a compelling therapeutic target.^[39] Moreover, Ding et al. ^[40] have revealed that sialylated cancer-derived IgG CAR-T cells can effectively lyse BC cells and demonstrate milder tumor cell lysis and enhanced persistence compared to HER2-CAR-T cells. Moreover, its efficacy is further enhanced when combined with vorinostat, a Food and Drug Administration–approved histone deacetylase inhibitor. Another target, cancer-restricted glycosaminoglycan, improves survival in BC xenograft models (8-week-old female Nu/Nu mice).^[41] Finally, Shen et al.^[42] have developed an innovative PSCA-CAR-T cell therapy by incorporating a mutated T cell immunoreceptor with immunoglobulin and tyrosine-based inhibitory motif domain coreceptor to overcome CD155-mediated inhibitory effect, significantly enhancing therapeutic efficacy in BC. In addition, the B7H3-CAR-T cell therapy was evaluated successfully in bladder cancer organoids, which was instructed to predict sensitivity and feasibility of tailored precision therapy.^[43]

Chimeric antigen receptor-NK and CAR-M cell therapies have not yet been clinically applied to urological tumors, including BC. However, the efficacy of CAR-NK cells in hematological malignancies and other solid tumors, as well as the inherent advantages of CAR-M cells in the TME, suggest their potential for BC treatment.^[44,45] Emerging immunotherapies may address the current limitations of BC treatment.

5. CAR cell therapy for PC

Current standardized treatments demonstrate limited effectiveness against metastatic prostate cancer (mPC), particularly metastatic castration-resistant prostate cancer (mCRPC).^[46] The poor response of tumors to ICIs highlights the urgent need for novel immunotherapeutic approaches to address this clinical challenge.

Prostate-specific membrane antigen is the most extensively studied target in CAR-T cell therapy for PC, along with other targets, including PSMA, PSCA, STEAP1, B7-H3, erythropoietin-producing hepatocyte receptor A2 (EphA2), epithelial cell adhesion molecule (EpCAM), and natural killer group 2D ligand (NKG2DL).^[47] Several preclinical and clinical studies have identified the inhibitory effects of PSMA-CAR-T cells on PC. Although early clinical trials of PSMA-CAR-T cells (NCT01140373) have demonstrated limited persistence and clinical responses, these findings have spurred further innovations in CAR-T cell engineering and combination strategies. Kloss et al.^[48] developed transforming growth factor-beta (TGF-β)-resistant PSMA-CAR-T cells, which is a major breakthrough. Their approach incorporating a dominant-negative TGF-β RII showed enhanced lymphocyte proliferation, cytokine secretion, and persistence in vivo, leading to complete tumor eradication in an aggressive human PC mouse model (female NSG knockout mice). This translated to promising clinical results in the first published trial (NCT03089203), where 5 of 13 patients with mCRPC experienced 2 or more cytokine release syndrome, whereas three patients achieved ≥30% PSA reduction.^[49] Another TmPSMA-02 CAR-T trial (NCT06046040) built on these findings by evaluating an optimized construct.

In addition to those targeting PSMA, CAR T cells targeting other antigens, particularly PSCA, have also been investigated. Priceman et al.^[50] have demonstrated that PSCA-CAR-T cells incorporating 4-1BB costimulatory domains exhibited superior antitumor activity compared with CD28-based constructs, indicating the importance of the intracellular costimulatory signaling domain in determining the sensitivity of CAR-T cells to tumor antigen expression. This finding is being further evaluated in an ongoing phase Ib trial (NCT05805371) of PSCA-CAR-T cell therapy for mCRPC. Clinical studies have yielded encouraging results; a phase I trial (NCT03873805) reported antitumor activity with dose-limiting toxicities of cystitis in patients with mCRPC.^[51] Extended analysis showed PSA reductions of >30% in 4 of 14 patients, radiographic improvements, and limited CAR-T persistence beyond 28 days after infusion.^[52] The BPX-601 trial (NCT02744287) has demonstrated more robust outcomes, with 56% of patients achieving a ≥50% PSA reduction and evidence of long-term CAR T-cell persistence and tumor infiltration.^[53]

Recent advances have identified several promising targets beyond PSMA and PSCA for CAR T-cell therapy in prostate cancer. Other studies have validated STEAP1 as a target for PC treatment.^[8,9] Bhatia et al.^[10] have demonstrated that STEAP1-CAR-T cells maintain reactivity even at low antigen densities and exhibit robust antitumor activity in mPC models (6- to 8-week-old male NSG). They also identified that the addition of tumor-targeted IL-12 in the form of a collagen-binding domain-IL-12 fusion protein enhanced the antitumor activity of STEAP1-CAR-T cells. Zhang et al. ^[47] have developed a second-generation CAR targeting EphA2 with CD28 as a costimulatory receptor and discovered that such cells can effectively inhibit PC growth in an antigen-dependent manner in vitro and in vivo (4-week-old male immunodeficient NCG mice). Regarding EpCAM, a preclinical study has reported that intravenous application of EpCAM-CAR-T cells showed significant efficacy compared to control cells (p < 0.05) in a PC xenograft mouse model (male NOD/SCID mice, 5–8 weeks of age).^[54] NKG2DL is also a potential target for CAR-T cell therapy for PC. He et al. ^[55] have reported that the coexpression of IL-7 Improves NKG2D-CAR-T cell therapy for PC by enhancing expansion and inhibiting apoptosis and exhaustion, providing a new adoptive cell therapy for PC, either alone or in combination with IL-7. Trials aimed at demonstrating the efficacy of EpCAM-CAR-T cells (NCT03013712) and NKG2DL-CAR-T cells (NCT04107142) in PC are currently underway.

Although CAR NK cell research on solid tumors remains exploratory, PC has emerged as a relatively promising urological application. Current efforts have primarily focused on PSMA-targeted approaches. Wang et al.^[56] successfully constructed anti-PSMA-CAR NK-92 cells that exhibited potent and specific antitumor activity in vivo, especially against CRPC. Subsequently, combination therapy of anti-PSMA-CAR-NK-92 cells with anti-PD-L1 monoclonal antibodies mitigated the immune resistance caused by the upregulation of PD-L1 when CAR-NK cells were used alone, thus enhancing their efficacy against CRPC cells.^[56] Currently, only 1 related clinical trial is underway (NCT03692663) to evaluate PSMA-CAR-NK cells (TABP EIC) for the treatment of mCRPC. This study may establish important safety and efficacy benchmarks in this field. New adoptive cell therapies, such as CAR-M cell therapy, also show potential as alternatives to CAR-T cell therapy, although their PC-specific applications are yet to be explored.

6. Challenges and potential strategies of CAR-T therapy in urological cancers

We have summarized a comprehensive comparison, mainly including the advantages, limitations, and potential improvement strategies of CAR-T, NK, and M cell therapies in solid tumors, including urological tumors (Table 3). As CAR-T cell therapy has been relatively well explored in urological cancers, this review focuses specifically on the potential challenges of CAR-T cell therapy in these cancers and discusses possible strategic solutions. The challenges of CAR-T therapy in urological tumors mainly include the immunosuppressive TME, antigen expression heterogeneity, and organ-specific barriers (Fig. 4).

Table 3.

CAR-T, CAR-NK, and CAR-M comprehensive comparison (advantages, limitations, and strategies).

Category	CAR-T cells	CAR-NK cells	CAR-M cells
Advantages	–Sufficient circulating T cells	–Natural cytotoxicity against nonself cells	–M1 proinflammatory phenotype
	-Proven efficacy in hematologic malignancies (5 FDA-approved therapies)	–Dual killing: CAR-dependent + ADCC	–Phagocytosis + antigen presentation + ECM degradation (MMP secretion)
	–Single infusion with prolonged durability	–Low CRS/GvHD risk via KIR	–Abundant in TME
	–Enhanced activation via costimulatory domains (e.g., CD28, 4-1BB)	–Multiple sources (PB, iPSC, NK92 line)	–Multiple sources (iPSC, THP-1 line)
		–Off-the-shelf potential
Limitations	–Tumor antigen heterogeneity/loss	–Limited tumor infiltration	–Low CAR transduction efficiency
	–Poor solid tumor infiltration	–Low CAR transduction efficiency	–Requires M1 polarization
	–High CRS/neurotoxicity/GvHD risk	–Poor persistence in TME	–Potential CRS/OTOT toxicity (limited clinical data)
	–Limited persistence in TME	–Challenging ex vivo expansion
Strategies	–Bispecific/multitarget CARs	–Chemokine receptor expression for homing	–Vpx-modified lentivirus/Ad5f35 vectors
	–Nanobody/chemokine receptor-enhanced infiltration	–Transduction enhancers (e.g., retronectin)	–M2-to-M1 polarization
	–IL-12/IL-15 secretion to counter TME	–Combined with TKIs/ICIs	–CAR-CD147 for TME infiltration
	–Combined with ICIs	–IL-15 expression for persistence	–Combined with anti-CD47
	–CAR design optimization (suicide gene)	–Off-the-shelf iPSC/NK92-derived products	–iPSC-derived CAR-M
Other parameters	–Signaling domains: CD3ζ + CD28/4-1BB	–Signaling domains: 2B4/DAP10/DAP12	–Signaling domains: ITAM-containing alternatives
	–Source: Autologous/MHC-matched allogeneic	–Source: Autologous/allogeneic/NK92 line	–Source: Autologous/iPSC/cell lines
	–Clinical experience: Extensive (FDA-approved)	–Clinical experience: Limited (superior safety)	–Clinical experience: Minimal (1 ongoing trial)

ADCC = antibody dependent cellular cytotoxicity; CAR-NK = chimeric antigen receptor natural killer cell; CAR-M = chimeric antigen receptor macrophage; CAR-T = chimeric antigen receptor T cell; CRS = cytokine release syndrome; ECM = extracellular matrix; FDA = Food and Drug Administration; ICI = immune checkpoint inhibitor; IL = interleukin; ITAM = immunoreceptor tyrosine-based activation motif; iPSC = induced pluripotent stem cell; KIR = killer cell immunoglobulin-like receptor; MMP = matrix metalloproteinase; TKI = tyrosine kinase inhibitor; TME = tumor microenvironment.

Figure 4.

Challenges of CAR-T therapy in urological tumors. The challenges of CAR-T therapy in urological tumors: immunosuppressive TME components, antigen heterogeneity, and organ-specific barriers (renal, bladder, prostate). See Section 6 for mechanistic details. CAR = chimeric antigen receptor; TME = tumor microenvironment.

6.1. Overcoming immunosuppressive TME

The TME is inherently immunosuppressive, primarily because of the abnormal composition of cellular components, abnormal immunosuppressive factors, and metabolic dysfunction. These stromal abnormalities collectively create formidable barriers to effective CAR T-cell therapy. Although stroma-targeting strategies have not yet been integrated into CAR T-cell therapy, their combination may represent a promising direction for future research.

6.1.1. Immunosuppressive cells

The immunosuppressive TME is characterized by an abundance of immunosuppressive cells, mainly tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), and cancer-associated fibroblasts (CAFs).

Monocyte-derived TAMs are the most abundant tumor-infiltrating immune cells in the TME. These cells facilitate tumor progression through multiple mechanisms; specifically, their immunosuppressive effects in solid tumors are primarily embodied in the secretion of chemokines, cytokines, and enzymes that inhibit T cell function.^[57] Consequently, strategies targeting TAM-mediated immunosuppression may enhance T-cell therapies. Current TAM-targeting strategies focus on macrophage depletion, inhibition of TAM activation, enhancement of macrophage phagocytic activity, inhibition of monocyte recruitment, and combination with other TAM-targeted therapies. These approaches have demonstrated varying degrees of efficacy against different tumor types.^[58,59]

Myeloid-derived suppressor cells, which are differentiated from TAMs and are potent suppressors of T lymphocytes, can induce cytokine release from macrophages.^[60] These cells promote tumor progression through the following 4 mechanisms: immune evasion, TME remodeling, establishment of a premetastatic niche, and induction of cancer stemness.^[61] Their accumulation is strongly correlated with poor prognosis and treatment resistance.^[62] Certain chemotherapeutic agents, such as cyclophosphamide, may paradoxically expand the MDSC population while impairing cytotoxic immunity.^[63,64] Given their central role in the TME, strategies to target MDSCs in combination with CAR-T cells should be considered. Key MDSC-targeting strategies include pharmacological inhibition of key immunosuppressive pathways, such as nuclear factor erythroid 2-related factor 2^[65] and phosphodiesterase-5,^[66] and differentiation modulation to inhibit pathological MDSC expansion.

The TME actively promotes Tregs differentiation through TGF-β, VEGF, and other soluble factors.^[67] These immunosuppressive CD4+ T cells mediate immune suppression primarily through adenosine and prostaglandin E2 production, thereby effectively paralyzing antitumor immunity.^[68] Clinically, Treg accumulation correlates strongly with chronic inflammatory states that frequently precede tumor development^[69] and often worsens following conventional therapies, such as radical chemoradiotherapy, which often induces persistent immune dysregulation in patients, establishing a chronic inflammatory state.^[70] This treatment-induced inflammation creates a vicious cycle by expanding Treg populations, highlighting their dual roles as immunosuppressive agents and inflammation amplifiers.^[71] Therefore, therapeutic targeting of Tregs represents a promising strategy to simultaneously boost antitumor immunity while breaking the protumorigenic cycle, potentially preventing treatment resistance and disease recurrence.

Moreover, the activation of CAFs, which can secrete extracellular matrix and form a physical barrier, impedes CAR-T cell infiltration into tumor sites.^[72] Activated by chronic stress, hypoxia, and cytokine signaling during tumor progression, these pathological fibroblasts secrete immunosuppressive factors, such as TGF-β, VEGF, and C-X-C motif ligand 12, that hinder T cell infiltration and function.^[73] Fibroblast activation protein (FAP) serves as both a key CAF marker and therapeutic target, and its overexpression in PC and urothelial carcinomas strongly correlates with poor clinical outcomes.^[74,75] The immunosuppressive mechanisms mediated by FAP include the recruitment of MDSCs through the FAP-STAT3 axis and the promotion of tumor angiogenesis.^[76,77] Current strategies to overcome CAF-mediated resistance include combining FAP-CAR-T cells with tumor-specific CAR-T constructs or cancer vaccines to enhance antitumor immunity.^[78] Moreover, although chemotherapy and radiotherapy damage tumor cells, they simultaneously induce CAF-like phenotypes in fibroblasts. Thus, combining CAF-CAR-T cell therapy with conventional chemotherapy or radiotherapy may provide synergistic antitumor efficacy by simultaneously targeting both the malignant and stromal compartments.

6.1.2. Immunosuppressive factors

Additionally, the enrichment of immunosuppressive factors such as TGF-β, IL-10, and VEGF, along with the high expression of immune checkpoint molecules such as PD-L1/PD-1 and cytotoxic T lymphocyte associated antigen 4 (CTLA-4) further impair T cell function.^[79,80]

Transforming growth factor-β serves as the main regulator of hematopoiesis and immune cell function. Tumor microenvironment impairs T cell–mediated immunity through multiple mechanisms, including direct suppression of T cells, promotion of Treg differentiation, and reinforcement of the immunosuppressive TME.^[81] Transforming growth factor-β exacerbates T-cell suppression by inhibiting dendritic cell, macrophage, and neutrophil maturation while modulating inflammatory cell polarization, thus limiting antigen presentation and suppressing T_H1-promoting cytokine secretion.^[82] Although TGF-β inhibition shows promise when combined with CAR-T therapy, its pleiotropic nature presents challenges.^[48] Systemic blockade may impair tissue-resident memory T-cell formation, paradoxically reducing tumor infiltration and worsening outcomes.^[83] This duality needs precisely engineered solutions, such as CAR-T cells incorporating TGF-β-responsive molecular switches that can selectively modulate this pathway’s effects. Such next-generation approaches aim to preserve TGF-β’s physiological functions while neutralizing its immunosuppressive actions in the tumor microenvironment, potentially overcoming current limitations of broad TGF-β inhibition strategies.

Interleukin-10 is a pleiotropic immunosuppressive cytokine that plays key roles in Treg differentiation and function of Tregs.^[84] In addition to its well-characterized anti-inflammatory properties, IL-10 contributes significantly to the immunosuppressive TME in solid tumors. Produced primarily by Tregs and MDSCs, IL-10 further suppresses CD4+ T cell proliferation and function, thereby reinforcing the immunosuppressive TME.^[84] Pegylated IL-10 (pegilodecakin) is the most commonly tested compound. Despite demonstrating antitumor activity in certain tumors, the pleiotropic nature of IL-10 poses significant therapeutic challenges.^[85] Nevertheless, IL-10-based immunotherapy maintains its clinical potential, especially in RCC, and merits further investigation through strategies, including molecular engineering, to improve its therapeutic specificity. Currently, the exploration of IL-10 in combination with CAR-T cell therapy remains limited, highlighting the critical need for systematic investigation into the IL-10–mediated enhancement of CAR-T cell efficacy.

Originally identified for its angiogenic properties, VEGF has emerged as a key immunosuppressive factor in TME. Beyond promoting vascular permeability and tumor invasion, VEGF-A directly suppresses T-cell function while recruiting MDSCs and modulating immunosuppressive myeloid population.^[86–88] These mechanisms make VEGF a multifaceted regulator of the immunosuppressive TME.^[88] Interestingly, preclinical studies and clinical observations of colorectal have demonstrated that VEGF/VEGFR inhibition promotes vascular maturation through enhanced pericyte coverage, leading to vascular normalization and potentially enhancing CAR-T cell extravasation and tumor infiltration.^[89] This dual role of immunosuppressive function and VEGF-mediated angiogenesis makes VEGF inhibition a promising strategy for simultaneously improving CAR-T cell delivery and overcoming microenvironmental immunosuppression.

Chronic antigen stimulation and cytokine upregulation induce the expression of inhibitory receptors, particularly CTLA-4 and PD-1, which actively suppress T cell function. CTLA-4 competes with CD28 for B7-1 binding, limiting T cell activation, whereas PD-1 and PD-L1 interactions inhibit proliferation and cytokine production. Unlike conventional therapies that directly kill tumor cells, ICIs function by reversing tumor-induced immune suppression and restoring T cell–mediated tumor recognition.^[90] This makes them particularly valuable when combined with CAR-T therapy, especially in advanced disease, where standard treatments often compromise immune function. Consequently, combining ICIs with CAR-T therapy may represent a promising strategy for eliminating residual disease and improving the quality of life in treatment-refractory patients.

6.1.3. Metabolic dysfunction

Metabolic abnormalities characteristic of rapidly proliferating tumors also contribute to the immunosup-pressive TME. Hypoxia-driven glycolytic metabolism generates lactate accumulation and tissue acidosis,^[72] which not only suppresses effector T and NK cell function but also promotes ferroptosis resistance through hypoxia-inducible factor-1α activation.^[91] This metabolic reprogramming has multiple immunosuppressive consequences, including lactate-induced histone lactylation that upregulates IL-10 production, whereas an acidic environment differentially impairs various immune components, including dendritic cells and antibodies.^[92,93] Thus, metabolic intervention strategies, particularly those that combine hypoxia alleviation with ferroptosis induction, could overcome resistance mechanisms in solid tumors. Importantly, neutralizing tumor acidity may restore the antitumor activity of adoptively transferred CAR-T cells, while alleviating stromalmediated immunosuppression. Such metabolic modulation approaches, when integrated with cellular immunotherapy, could significantly enhance treatment efficacy by simultaneously addressing both the biochemical and immuno-logical barriers in the TME.

6.2. Overcoming antigen expression heterogeneity

A major challenge in treating urological tumors with CAR-T cell therapy is tumor antigen heterogeneity, where the variable expression or loss of target antigens enables immune evasion.^[94] Conventional single-target CAR-T cells cannot simultaneously eliminate tumor populations that express different antigens. To overcome this limitation, researchers are developing innovative approaches, including dual CAR-T cell combination, bispecific CAR constructs, multi-CAR coexpression systems, fourth-generation CAR designs, and combination approaches. These strategies aim to broaden target recognition while preventing antigen escape, potentially improving the outcomes for heterogeneous solid tumors.

Dual CAR-T cell combination therapy involves the administration of 2 distinct CAR-T cell products targeting different tumor antigens delivered concurrently and sequentially to broaden target cell coverage. This approach has shown a promising clinical efficacy in multiple patient populations.^[95]

Another promising strategy to effectively target heterogeneous tumors is to engineer single-chain bispecific CARs containing 2 ligand-binding domains that recognize distinct tumor antigens. This strategy has shown clinical success in both leukemia and lymphoma.^[96]

An alternative approach is to coexpress multiple distinct CARs on individual T cells to enable simultaneous targeting of diverse tumor antigens. Preclinical success has been demonstrated with trivalent CAR-T cells targeting HER2, EphA2, and IL-13Rα2 in glioblastoma.^[97] However, this strategy faces technical challenges because large transgene payloads may compromise the transduction efficiency.^[98] Consequently, careful optimization of gene delivery systems is required for clinical applications.

Fourth-generation CAR-T cells, also known as T cells redirected for universal cytokine-mediated killing, represent an advanced CAR-T platform that combines direct antigen targeting of antigen-positive tumor cells with cytokine-mediated bystander effects of antigen-negative tumor cells. These engineered T cells secrete cytokines, such as IL-12 and IL-18, and have demonstrated potent antitumor activity in both xenograft and syngeneic mouse models.^[99]

Antigen loss can also be prevented by combining CAR-T cell therapy with agents that maintain target expression. For instance, the combination of γ-secretase inhibitors, which prevent B cell maturation antigen cleavage, with B cell maturation antigen-CAR-T cells has shown promising therapeutic efficacy in murine models of multiple myeloma.^[100]

Although broadening tumor recognition strategies increases the risk of on-target and off-tumor toxicity, this limitation may be overcome using hierarchical multiantigen targeting approaches. One innovative strategy involves oncolytic adenoviruses engineered to secrete EGFR-targeting BiTEs within tumors, enabling localized EGFR engagement despite widespread normal expression. An alternative strategy to address antigen heterogeneity without compromising safety utilizes engineered oncolytic viruses expressing truncated CD19 (CD19t).^[101] These oncolytic viruses infect tumor cells to express CD19t, which has no signal in infected cells but can then be targeted by CD19 CAR-T cells.

Finally, neoantigens arising from patient-specific tumor mutations are an emerging class of highly specific targets for CAR-T cell therapy. After whole-exome sequencing of tumor biopsies, computational prediction can be used to identify immunogenic neoantigens.^[102] This approach offers a promising alternative for patients who develop resistance to initial antigen-targeted CAR-T cell therapies or other ineffective treatments.

6.3. Overcoming organ-specific barriers

Organ-specific barriers, including the disordered renal blood flow, urine-bladder barrier, and blood-prostate barrier (BPB), make it difficult for CAR-T cells to effectively infiltrate cancer cells (Fig. 4).

6.3.1. Disordered renal blood flow

The kidney’s unique vascular architecture, receiving 20% of the cardiac output through serial glomerular and peritubular capillaries, exhibits hemodynamic heterogeneity, creating distinct challenges for CAR-T cell delivery.^[103] In the TME, tumor-induced vascular dysfunction manifests as abnormal arteriolar reactivity and regional hypoxia, establishing multiple barriers to CAR-T cell therapy.^[104] First, serial microcirculation in the kidney is vulnerable to flow disruption because of vasoconstriction or fibrosis, creating ischemic zones that limit CAR-T cell distribution.^[105] Secondly, peritubular capillary rarefaction, a characteristic chronic kidney disease, may also extend to RCC, impairing CAR-T cell extravasation and tumor trafficking.^[106] Third, microvascular dysfunction such as endothelial mitochondrial dysfunction or oxidative stress triggers drives profibrotic factor like TGF-β release, generating a dense extracellular matrix that that physically obstructs CAR-T cell migration.^[107] Collectively, these vascular and stromal barriers create a uniquely challenging microenvironment for CAR-T cell therapy in RCC, necessitating innovative delivery strategies to overcome these physiological obstacles.

To address the unique hemodynamic and microenvironmental challenges of RCC, the following combination strategies were considered: First, vascular normalization is performed by targeting VEGF to restore vascular integrity. For instance, low-dose anti-VEGF therapy, such as sunitinib, can reduce vascular leakage and improve perfusion, and the combined inhibition of VEGF and angiopoietin-2 may further stabilize the vascular architecture and reduce hypoxic areas.^[108,109] Second, endothelial mitochondrial dysfunction, such as aberrant H₂O₂ signaling with targeted antioxidants, is modulated.^[110] The third mechanism is fibrotic microenvironment intervention. Inhibition of TGF-β or platelet-derived growth factors receptors-β signaling pathways, such as pirfenidone may reduce fibrotic extracellular matrix deposition that impedes CAR-T cell migration, thus facilitating CAR-T cell penetration through the tumor stroma.^[111] Finally, localized delivery methods, such as renal artery infusion of CAR-T cells or oncolytic viruses, may bypass the systemic circulation barriers for direct tumor targeting.^[112] This approach may be further enhanced by coadministration of vasodilators. These strategies collectively target RCC’s unique hemodynamic and stromal barriers of RCC, with vascular normalization and fibrosis reduction potentially creating more favorable conditions for CAR-T cell infiltration and function, whereas localized delivery methods can overcome perfusion limitations inherent to the kidney’s microcirculatory architecture.

6.3.2. Mucosal barrier of the bladder

The bladder impermeable urothelium forms a key protective barrier through its specialized mucin layer composed primarily of glycosaminoglycans (GAGs) and proteoglycans.^[113] This physicochemical barrier functions via highly hydrophilic sulfated polysaccharide chains that bind water molecules to prevent solute penetration.^[114] In BC, this protective mechanism can be damaged by GAG degradation and aberrant expression.^[115] Inflammatory mediators in the TME further disrupt barrier function by neutralizing the negative charge in the mucin layer.^[116] Moreover, the anti-adhesive properties that prevent bacterial attachment may also inhibit direct contact between CAR-T cells and tumor cells, creating a unique therapeutic challenge.^[117] These findings highlight the need to develop strategies that can modulate the urothelial barrier to enhance CAR-T cell access, while preserving its essential protective functions against urinary toxins and pathogens.

Three promising approaches could address the unique barrier challenges of the bladder. First, mucin layer modulation using GAG analogs, such as pentosan polysulfate sodium, could repair damaged barriers while potentially creating temporary windows for CAR-T cell penetration.^[118] Pretreatment with topical pentosan polysulfate sodium could transiently reduce the physical barrier properties of the mucin layer, thus enhancing CAR-T cell penetration. Second, neutralizing cationic toxins such as the Tamm-Horsfall protein with Tamm-Horsfall protein antagonists may help preserve mucin integrity against urinary insults.^[116] Third, CAR-T cell penetration can be enhanced using aquaporins or hydrophilic carriers to improve migration based on the hydration-dependent properties of the barrier. These strategies, which combine barrier modification with cellular engineering, offer a multifaceted approach for enhancing CAR-T cell access while maintaining the protective functions of the urothelium. Future studies should validate these combinations in physiologically relevant bladder cancer models to assess their synergistic potential.

6.3.3. BPB

The BPB consists of prostatic ductal epithelial cells, capillary endothelial cells, and intercellular tight junctions (TJs) (Fig. 4).^[119] Tight junctions, which are localized at the apical region of cell contacts, play crucial roles in maintaining epithelial barrier function and polarity.^[120] Claudins are a family of 24 transmembrane proteins that seal intercellular gaps, form selective ion channels, and are core components of TJs.

This dynamic barrier controls substance exchange between the circulation and the prostate tissue, potentially limiting CAR-T cell penetration into tumor sites. Thus, the restrictive properties of BPB may contribute to poor tumor penetration observed in prostate CAR-T cell therapies, ultimately reducing their therapeutic efficacy against prostate malignancies.

Several strategies may improve CAR-T cell penetration of the BPB, including alternative delivery approaches, external force-assisted methods, and engineering CAR-T cells to secrete permeability-enhancing factors. First, alternative delivery methods such as intraprostatic injection can bypass the BPB entirely, enabling precise tumor targeting. This approach, which has been employed to treat prostatitis and benign prostatic hyperplasia, may significantly enhance CAR-T cell efficacy by facilitating direct tumor engagement. However, their invasiveness, potential for cancer cell dissemination, and local toxicity require further evaluation. Another approach is scaffold-based delivery. Functionalized biopolymer scaffolds can encapsulate CAR-T cells, supporting sustained proliferation and localized release when implanted at tumor resection sites.^[121] Second, external forces, such as ultrasonic sonoporation, can transiently open TJs and remodel acinar cell membranes, thus activating vesicular transport.^[122] Similarly, photodynamic therapy and hyperthermia may increase vascular permeability via cytoskeletal modulation.^[123,124] Third, engineering CAR-T cells to secrete cytokines (e.g., TGF-β1 and tumor necrosis factor α) may reduce junctional protein expression or modulate endothelial permeability.^[125,126] These complementary strategies, which combine physical, biological, and engineering approaches, may synergistically overcome the restrictive properties of BPB while maintaining treatment safety. Future studies should systematically compare these methods to optimize CAR-T cell delivery for PC therapy.

7. Conclusions

This review comprehensively summarizes the recent advancements, clinical progress, advantages, and limitations of CAR-T, NK, and M therapies for RCC, BC, and PC. More importantly, we systematically address the current challenges in CAR-T cell therapy (immunosuppressive TME, antigen expression heterogeneity, and organ-specific barriers) for urological cancers and propose corresponding optimization strategies to overcome these barriers. Innovative CAR cell therapies could serve as viable options for urological cancers in the clinical setting, ultimately expanding treatment choices and improving patient survival and quality of life.

Acknowledgments

None.

Statement of ethics

Not applicable.

Conflict of interest statement

DT is an editor-in-chief of Current Urology and confirms no involvement in any stage of this article’s review process, ensuring unbiased editorial decision making. The other authors declare no conflicts of interest.

Funding source

This work was supported by the Special Fund for the Taishan Scholars Project (tsqn202211324) and the Key Research and Development Program of Shandong Province (2021CXGC011101).

Author contributions

HT: Data acquisition, study design, and manuscript preparation;

LG: Study design, manuscript review, and editing;

WZ: Study concept and design, manuscript review, and editing;

DT: Project supervision, study concept and design, manuscript review, and editing.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.