Tubulocystic renal cell carcinoma with aggressive behavior-insights from molecular profiling and single-cell analysis: A case report

Abstract

BACKGROUND

Tubulocystic renal cell carcinoma (TRCC) is a rare subtype of renal cell carcinoma that is generally regarded as an indolent malignancy with a favorable prognosis.

CASE SUMMARY

We report an exceptional case of aggressive TRCC characterized by rapid postoperative recurrence, distant metastasis, and resistance to multiple therapeutic modalities. Next-generation sequencing (NGS) was performed to identify genomic alterations in the patient, while single-cell RNA sequencing (scRNA-seq) was used to delineate the phenotypic heterogeneity of tumor cells and characterize the tumor microenvironment. Targeted NGS identified a germline missense fumarate hydratase mutation and somatic mutations in AFF3, MDM2, and LRP1B. scRNA-seq revealed marked activation of angiogenesis-related pathways in recurrent TRCC cells, along with a tumor microenvironment characterized by a high proportion of naïve T cells and SPP1⁺ tumor-associated macrophages. These findings suggest that enhanced angiogenic signaling in tumor cells, together with an immunosuppressive macrophage-rich microenvironment, may contribute to the aggressive behavior of the tumor and its resistance to therapy.

CONCLUSION

This study provides a comprehensive characterization of the molecular landscape and tumor microenvironment of aggressive TRCC, offering insights into its biological behavior and identifying potential therapeutic targets for future clinical intervention.

Key Words: Metastatic tubulocystic renal cell carcinoma; Tumor microenvironment; Genetic profiling; Single-cell RNA sequencing; Case report

Core Tip: Tubulocystic renal cell carcinoma (TRCC) is generally considered an indolent malignancy. However, we report an exceptional aggressive case characterized by rapid recurrence and metastasis. Using next-generation sequencing and single-cell RNA sequencing, we identified a germline missense mutation in fumarate hydratase and somatic alterations in AFF3, MDM2, and LRP1B. The recurrent tumor exhibited enhanced angiogenic activity and an immunosuppressive microenvironment enriched in naïve T cells and SPP1⁺ tumor-associated macrophages. These findings provide insights into the molecular mechanisms underlying aggressive TRCC and highlight potential therapeutic targets for future clinical management.

INTRODUCTION

Tubulocystic renal cell carcinoma (TRCC) is a rare and enigmatic subtype of renal cell carcinoma (RCC), accounting for less than 1% of all RCC cases[1]. First described in the medical literature in 1956[2], TRCC has attracted increasing attention due to its distinctive histopathological features and clinical behavior. Histologically, TRCC is characterized by a unique architecture composed of tubules and cysts lined by bland, hobnail-like epithelial cells, which often results in underdiagnosis or misdiagnosis as a benign cystic lesion[3]. Generally, TRCC is considered an indolent neoplasm with low metastatic potential, and surgical resection remains the primary treatment strategy. For the majority of patients, this approach results in favorable outcomes, with a minimal risk of recurrence or metastasis[4-6]. However, recent case reports have challenged this traditionally indolent perception. Increasing evidence indicates that TRCC can exhibit aggressive behavior in rare cases, including rapid local recurrence and widespread multi-organ metastasis[7-10]. These rare but clinically significant events underscore the need for a more comprehensive understanding of the biological spectrum of TRCC.

Despite these emerging insights, our understanding of TRCC remains limited, particularly regarding its therapeutic responsiveness and tumor microenvironment. The rarity of reported cases and the absence of comprehensive datasets have hindered the development of robust clinical trials and molecular investigations. Consequently, limited information is available regarding how TRCC responds to various treatment modalities, including targeted therapies and immunotherapies[7]. Moreover, the tumor microenvironment of TRCC, which plays a critical role in tumor progression and therapeutic efficacy in other malignancies, remains largely unexplored.

This knowledge gap is particularly concerning given the potential for TRCC to exhibit aggressive behavior, as demonstrated in our exceptional case. In this study, we aim to address these critical gaps by presenting a comprehensive analysis of an aggressive TRCC case. Comprehensive genomic profiling using targeted next-generation sequencing (NGS) and single-cell RNA sequencing (scRNA-seq) was performed to elucidate the genetic alterations and unique microenvironmental characteristics of the tumor. Our findings not only highlight the underrecognized malignant potential of TRCC but also provide a foundation for future investigations into its therapeutic vulnerabilities and microenvironmental interactions.

CASE PRESENTATION

Chief complaints

A 27-year-old female patient was referred to our hospital following the incidental detection of a left renal mass on abdominal ultrasonography performed during a routine health examination.

History of present illness

She was asymptomatic, with no family history of RCC or clinical features suggestive of hereditary leiomyomatosis and RCC (HLRCC) syndrome. A comprehensive clinical evaluation, including a detailed dermatological and gynecological history and physical examination, revealed no evidence of cutaneous or uterine leiomyomas. The diagnostic and therapeutic course of this patient is illustrated in Figure 1A. Contrast-enhanced computed tomography (CT) of the abdomen and pelvis confirmed the presence of a left renal mass measuring 5.4 cm × 5.0 cm (Figure 1B). The patient underwent laparoscopic radical left nephrectomy. Histopathological examination revealed a diagnosis of TRCC (Figure 1C), staged as pT1bNx according to the American Joint Committee on Cancer criteria. The diagnosis was confirmed through multidisciplinary review by multiple genitourinary pathologists based on the World Health Organization Classification of Renal Tumors. The tumor exhibited the classic histological features of TRCC, including an exclusively tubulocystic architecture composed of small cystic spaces lined by bland, hobnail-like epithelial cells with minimal nuclear atypia. Notably, the characteristic high-grade cytomorphological features of FH-deficient RCC, specifically prominent eosinophilic nucleoli with perinucleolar haloes, were absent, further supporting the diagnosis of TRCC rather than an FH-deficient RCC phenotype.

Figure 1 The diagnostic and therapeutic course of this patient. A: Clinical timeline depicting the diagnostic and therapeutic course. Arrows indicate treatment duration; vertical bars denote key clinical events; B: Enhanced computed tomography examination revealing disease progression in the patient; C: Hematoxylin and eosin staining of surgical and puncture specimens from the patient.

Nineteen months after surgery, surveillance CT revealed local recurrence in the renal fossa and retroperitoneal lymph node metastases. First-line therapy with pazopanib was initiated at a dose of 600 mg daily. One month after treatment initiation, the dose was reduced to 400 mg daily due to the development of grade 2 palmar-plantar erythrodysesthesia (PPE) and drug-induced hypertension. No treatment interruption occurred; the toxicities were managed with symptomatic supportive care, and the patient demonstrated good compliance, continuing therapy until radiographic disease progression was confirmed in February 2023. At that time, disease progression was observed, characterized by enlargement of tumors in the left renal surgical bed, retroperitoneal lymph nodes, and left posterior abdominal wall (Figure 1B). These lesions were confirmed as fluorodeoxyglucose-avid by positron emission tomography-CT (PET-CT). Percutaneous biopsy of the left abdominal wall metastasis further confirmed recurrent TRCC (Figure 1C). The patient subsequently underwent CyberKnife radiosurgery and radioactive seed implantation therapy; however, no significant therapeutic response was observed on follow-up CT scans (Figure 1B). Given the resistance to pazopanib, treatment was switched to axitinib (5 mg twice daily). Despite adverse effects including PPE (erythema and desquamation), CT imaging demonstrated stable disease (SD) without evident tumor progression during the initial treatment period (Figure 1B). In January 2024, new liver lesions were detected, with mildly enhanced lesions identified in hepatic segments V and VIII on contrast-enhanced CT (Figure 1B). To determine the pathological nature of these hepatic lesions, laparoscopic partial hepatectomy (segments II, III, V, and VIII) was performed, and histopathological examination confirmed metastatic TRCC (Figure 1C). Postoperatively, the patient received combination targeted immunotherapy consisting of axitinib (5 mg once daily) and camrelizumab (a PD-1 inhibitor; 200 mg intravenous infusion every 3 weeks). CT imaging demonstrated SD according to Response Evaluation Criteria in Solid Tumors (RECIST) criteria, with slight tumor shrinkage in the retroperitoneal lymph nodes, hepatic segment VIII, and omentum (Figure 1B). However, in October 2024, CT imaging revealed enlargement of tumor lesions at the aforementioned sites (Figure 1B), indicating resistance to the combination therapy. Concurrently, the patient presented with melena and hematemesis. Gastroscopy revealed diffuse gastric congestion accompanied by petechial hemorrhages. The patient declined further treatment and died one month later due to progressive disease after discontinuation of therapy.

History of past illness

The patient was previously healthy, with no significant medical history.

Personal and family history

The patient had no prior history of malignancy and no family history of cancer.

Physical examination

Comprehensive clinical evaluation, including a detailed dermatological and gynecological history and physical examination, revealed no evidence of cutaneous or uterine leiomyomas. Physical examination showed no palpable abdominal masses or other abnormalities. No clinical features suggestive of hereditary HLRCC syndrome were identified.

Laboratory examinations

TRCC is generally considered an indolent malignancy with low metastatic potential[11,12]. However, in our patient, the tumor exhibited a highly aggressive phenotype characterized by rapid recurrence, widespread metastasis, and substantial resistance to multiple therapies. To elucidate the molecular basis underlying this unusual behavior, we performed NGS and scRNA-seq analyses on recurrent TRCC samples obtained from an abdominal wall metastasis. Targeted NGS identified three somatic mutations in AFF3, MDM2, and LRP1B, as well as a germline missense mutation in FH (Table 1). Concurrently, the analysis revealed microsatellite stability and a low tumor mutational burden in the patient. Immunohistochemical staining was performed to evaluate FH and 2-succinocysteine expression levels, confirming preserved FH function (Supplementary Figure 1A). Immunohistochemical analysis of PD-L1 and CD8 expression demonstrated a tumor proportion score of 5% for PD-L1 expression in tumor cells and a 30% positivity rate for CD8 expression in tumor-infiltrating lymphocytes (Supplementary Figure 1B). Although the precise functional significance of these mutations remains to be fully elucidated, their identification suggests that they may contribute to the aggressive behavior and therapeutic resistance of this tumor.

Table 1 Genetic testing results.

	Base alteration	Amino acid alteration	Mutation frequency (%)
Germline mutation
FH	c.1106C>G	p.P369R	69.14
Somatic mutations
AFF3	c.2800G>A	p.A934T	24.27
MDM2	c.1165A>G	p.T389A	14.29
LRP1B	c.11856T>A	p.H3952Q	13.70

To comprehensively characterize the tumor microenvironment, we integrated our scRNA-seq data with publicly available scRNA-seq datasets derived from two clear cell RCC samples, one chromophobe RCC sample, one papillary RCC (PRCC) sample, and one normal renal tissue sample. Following stringent quality control, a total of 23903 cells were obtained and annotated into eight major cell types: Epithelial cells, endothelial cells, fibroblasts, macrophages, monocytes, T/NK cells, B cells, and mast cells (Figure 2A and B). Due to insufficient epithelial cell numbers, the PRCC sample was excluded from further analysis. The cellular composition of the TRCC sample differed from that of other renal carcinoma subtypes, indicating potential heterogeneity in the tumor microenvironment among different tumor types (Figure 2C). Using epithelial cells from normal renal tissue as a reference, we identified tumor cells in each RCC sample based on inferCNV analysis (Figure 2D and Supplementary Figure 2). GSVA enrichment analysis of tumor cells revealed significant activation of the HALLMARK_ANGIOGENESIS pathway in recurrent TRCC compared with other RCC subtypes (Figure 2E). This finding may explain the initial response to axitinib, an antiangiogenic targeted therapy, as the tumor exhibited strong angiogenic activity (Supplementary Figure 1C).

Figure 2 Single-cell analysis reveals the characteristics of tubulocystic renal cell carcinoma. A: UMAP plots showing 23903 cells grouped into 8 groups; B: Heatmap illustrating signature genes across distinct subpopulations; C: The bar chart comparing cell type proportions across various subtypes of renal cell carcinoma and normal renal tissue; D: Copy number variation analysis distinguishing normal cells from tumor cells within epithelial populations; E: Gene set variation analysis enrichment analysis of tumor cells showing highly elevated angiogenesis scores in tubulocystic renal cell carcinoma compared to other renal cell carcinoma subtypes. CRCC: Chromophobe renal cell carcinoma; TRCC: Tubulocystic renal cell carcinoma; CCRCC: Clear cell renal cell carcinoma; UMAP: Uniform manifold approximation and projection.

Comparative analysis of the immune microenvironment demonstrated that TRCC exhibited relatively lower immune cell infiltration compared with other RCC subtypes (Figure 2C). T/NK cells were stratified into four distinct subpopulations, including naïve T cells, effector T cells, exhausted T cells, and NK cells (Figure 3A and Supplementary Figure 3A). Naïve T cells constituted a high proportion of T/NK cells, whereas effector T cells represented a relatively small fraction (Figure 3B and C). The high proportion of naïve T cells may reflect a relatively non-exhausted immune compartment; however, whether this feature directly contributed to the observed clinical response remains speculative due to the lack of longitudinal functional data and the potential confounding effects of prior therapies[13]. We next focused on tumor-associated macrophages (TAMs), which were categorized into four subclusters and annotated as SPP1⁺ TAMs, ISG15⁺ TAMs, IL1B⁺ TAMs, and FOLR2⁺ TAMs based on canonical marker gene expression (Figure 3D and E). Notably, TRCC specimens showed enrichment of SPP1⁺ TAMs. GSVA of macrophage subclusters revealed significantly higher HALLMARK_ANGIOGENESIS scores in SPP1⁺ TAMs than in other macrophage subclusters (Figure 3F). Moreover, SPP1⁺ TAMs exhibited high expression levels of VCAN and FCN1 (Supplementary Figure 3B). Previous studies have demonstrated that angiogenesis-associated macrophages express high levels of these markers[14]. The enrichment of SPP1⁺ TAMs may therefore contribute to the enhanced angiogenic activity of the tumor and its resistance to targeted therapies and immunotherapies[15,16].

Figure 3 Single-cell analysis delineates the immune microenvironment characteristics of tubulocystic renal cell carcinoma. A: UMAP plots illustrating signature genes across distinct T/NK cell subpopulations: Naïve T cells, Effector T cells, Exhausted T cells, and NK cells; B: Bar chart comparing proportions of T/NK cell subpopulations across different histological subtypes of renal cell carcinoma; C: UMAP plots demonstrating the distribution of T/NK cell subpopulations across various types of renal cell carcinoma; D: UMAP plots illustrating signature genes across distinct macrophage subpopulations; E: Density plots depicting the distribution of signature genes across macrophage populations; F: GSVA enrichment analysis of macrophage subclusters showing elevated HALLMARK_ANGIOGENESIS scores in SPP1+ TAMs compared to other macrophage subclusters. CRCC: Chromophobe renal cell carcinoma; TRCC: Tubulocystic renal cell carcinoma; CCRCC: Clear cell renal cell carcinoma; TAM: Tumor-associated macrophage; UMAP: Uniform manifold approximation and projection; GSVA: Gene set variation analysis.

To determine whether the angiogenic and immune characteristics observed in the recurrent metastatic lesion were already present at initial diagnosis, we performed immunohistochemical staining of the primary tumor specimen. The primary tumor exhibited strong CD31 positivity, indicating active angiogenesis, along with enrichment of CD8⁺ tumor-infiltrating lymphocytes, consistent with the immune-active microenvironment observed in the metastatic lesion (Supplementary Figure 4A). Notably, SPP1⁺ TAMs were nearly absent in the primary tumor (Supplementary Figure 4B), suggesting that SPP1⁺ TAM enrichment may represent a therapy-induced microenvironmental adaptation rather than an intrinsic characteristic of primary TRCC.

Collectively, our findings suggest that the initial response to immunotherapy may have been associated with a higher proportion of naïve T cells, whereas the subsequent enrichment of immunosuppressive SPP1⁺ TAMs may represent a therapy-induced adaptation contributing to acquired treatment resistance.

Imaging examinations

A left renal mass measuring 5.4 cm × 5.0 cm was incidentally detected by abdominal ultrasonography and subsequently confirmed by contrast-enhanced CT. Following radical nephrectomy, the patient remained disease-free for 19 months, after which surveillance CT revealed local recurrence and retroperitoneal lymph node metastases. Despite sequential therapies, including pazopanib, CyberKnife radiosurgery, radioactive seed implantation, and axitinib, the disease continued to progress, with the emergence of new metastatic lesions in the left posterior abdominal wall and liver (segments V and VIII), as demonstrated by serial CT and PET-CT imaging. Although combination therapy with axitinib and camrelizumab initially achieved SD with slight tumor regression, subsequent CT imaging revealed enlargement of all target lesions by October 2024, indicating the development of acquired resistance to combined targeted immunotherapy.

FINAL DIAGNOSIS

TRCC of the left kidney.

TREATMENT

Initial treatment

The patient underwent laparoscopic radical left nephrectomy for the left renal mass. Histopathological examination confirmed the diagnosis of TRCC.

First-line targeted therapy

Nineteen months postoperatively, after the detection of local recurrence in the renal fossa and retroperitoneal lymph node metastases, first-line therapy with pazopanib was initiated at a dose of 600 mg daily. The dose was reduced to 400 mg daily one month after treatment initiation due to grade 2 PPE and drug-induced hypertension. No treatment interruption occurred; toxicities were managed with symptomatic supportive care.

Radiotherapy

Following disease progression characterized by enlargement of tumors in the left renal surgical bed, retroperitoneal lymph nodes, and left posterior abdominal wall, the patient underwent CyberKnife radiosurgery and radioactive seed implantation therapy. However, no significant therapeutic response was observed on follow-up CT scans.

Second-line targeted therapy

Given the resistance to pazopanib, treatment was switched to axitinib (5 mg twice daily). Adverse effects included PPE (erythema and desquamation). CT imaging demonstrated SD without evident tumor progression during the initial treatment period.

Surgical intervention for metastasis

In January 2024, new liver lesions were detected. Laparoscopic partial hepatectomy involving segments II, III, V, and VIII was performed, and histopathological examination confirmed metastatic TRCC.

Combined targeted therapy and immunotherapy

Postoperatively, the patient received combination targeted therapy and immunotherapy consisting of axitinib (5 mg once daily) and camrelizumab (a PD-1 inhibitor; 200 mg administered by intravenous infusion every 3 weeks). CT imaging demonstrated SD according to RECIST criteria, with slight tumor shrinkage observed in the retroperitoneal lymph nodes, hepatic segment VIII, and omentum.

Treatment discontinuation

In October 2024, CT imaging revealed enlargement of tumor lesions, indicating resistance to the combination therapy. Concurrently, the patient presented with melena and hematemesis. Gastroscopy demonstrated diffuse gastric congestion accompanied by petechial hemorrhages. The patient declined further treatment and died one month later due to progressive disease after discontinuation of therapy.

OUTCOME AND FOLLOW-UP

The overall survival from the initial diagnosis to death was approximately 6.5 years. The recurrence-free interval following radical nephrectomy was 19 months.

DISCUSSION

Given the limited understanding of TRCC’s biological heterogeneity and the scarcity of comprehensive molecular studies, we aimed to provide a detailed characterization of an exceptionally aggressive TRCC case to elucidate the mechanisms underlying its recurrence, metastasis, and therapeutic resistance. In this study, we report a patient with TRCC who experienced rapid local recurrence and progressive multi-organ metastasis despite multimodal treatment, including surgical resection, targeted therapy, and immunotherapy. Comprehensive molecular profiling identified three somatic mutations (AFF3, MDM2, and LRP1B) and a germline missense mutation in FH in this patient. Furthermore, scRNA-seq revealed key biological features distinguishing this aggressive TRCC from other RCC subtypes, including marked activation of angiogenesis pathways in recurrent tumor cells, a high proportion of naïve T cells, and enrichment of pro-angiogenic SPP1⁺ TAMs. The significance of this case lies in demonstrating the underrecognized malignant potential of TRCC and highlighting the need for a more comprehensive understanding of its molecular and microenvironmental characteristics. Notably, although the patient harbored a germline FH missense variant (p.P369R), the combination of classic TRCC morphology, absence of HLRCC-associated clinical features, retained FH protein expression, and negative 2-succinocysteine staining strongly supports the diagnosis of true TRCC rather than FH-deficient RCC. However, the functional significance of this germline variant warrants further investigation.

The molecular profile identified in this case provides plausible mechanistic insights into the aggressive behavior of this tumor. Despite retained FH protein expression, the germline FH variant (p.P369R) may confer subtle metabolic alterations; hypomorphic FH variants can induce pseudohypoxia and angiogenesis through partial enzymatic dysfunction, even in the absence of canonical HLRCC morphology[17,18]. Among the identified somatic alterations, MDM2 mutations are well-established drivers of p53 suppression, promoting genomic instability and therapeutic resistance across various malignancies[19]. AFF3 is involved in chromatin remodeling and transcriptional regulation, and its dysregulation may contribute to aberrant gene expression programs[20,21]. Loss-of-function alterations in LRP1B may enhance growth factor signaling and extracellular matrix remodeling, thereby facilitating metastatic dissemination[22]. Collectively, these molecular alterations may converge to promote the aggressive phenotype observed in this case. However, given the rarity of TRCC, these interpretations remain hypothetical and require validation in future studies.

While TRCC has traditionally been considered an indolent malignancy with low metastatic potential and favorable outcomes following surgical resection[12], recent reports have increasingly highlighted its potential for aggressive behavior[23]. Several studies have documented cases of TRCC exhibiting rapid recurrence and widespread metastasis, challenging the perception that TRCC is uniformly benign. For example, Choi et al[7] reported a case of TRCC with early recurrence and multi-organ metastasis, which showed similarities to our patient’s clinical course. Additionally, Zhao et al[24] described a TRCC case with rapid progression despite initial surgical management. These reports, together with our findings, underscore the need to reassess the malignant potential and biological heterogeneity of TRCC. In our case, the patient developed local recurrence in the renal fossa and retroperitoneal lymph node metastases only 19 months after surgical resection. This short recurrence-free interval suggests that long-term postoperative surveillance may be warranted for TRCC, as even tumors diagnosed at an early stage can exhibit aggressive behavior. Furthermore, the initial response to pazopanib (lasting 36 months) and subsequent axitinib-based therapy (lasting 12 months) indicates that, although tyrosine kinase inhibitors (TKIs) may provide temporary disease control, acquired resistance ultimately develops. Notably, the occurrence of PPE and hypertension during TKI therapy reflected typical on-target toxicities, suggesting adequate drug exposure and supporting the hypothesis that treatment resistance was driven by tumor biology rather than insufficient drug delivery. Moreover, the limited durability of response to combined axitinib and camrelizumab therapy (lasting only 9 months) suggests that immune checkpoint inhibitors may have limited efficacy in TRCC. The clinical course of our patient, characterized by rapid recurrence, transient responses to targeted therapies, and short-lived benefit from immunotherapy, further highlights the aggressive nature of this TRCC case.

At the molecular and microenvironmental levels, our scRNA-seq analysis provides critical insights into the aggressive behavior of this TRCC case and its response to therapy. Three key findings merit emphasis. First, the marked activation of angiogenesis pathways in tumor cells provides a plausible explanation for the transient responsiveness to TKIs[25]. The efficacy of TKIs, such as axitinib, is closely associated with tumor angiogenic activity. Given that our analysis revealed significant upregulation of angiogenesis-related pathways in tumor cells, the initial response to axitinib was consistent with its mechanism of action in suppressing angiogenesis. This finding underscores the importance of angiogenesis as a potential therapeutic target in TRCC, particularly in cases exhibiting high angiogenic activity. Second, the high proportion of naïve T cells in the tumor microenvironment represents both a potential opportunity and a challenge for immunotherapy. However, this observation should be interpreted as hypothesis-generating rather than as definitive evidence of immune competence. Although naïve T cells possess high activation potential and a lower risk of exhaustion[13], their presence in this case cannot be directly attributed to the clinical benefit observed with camrelizumab, particularly given that the patient received this agent after multiple prior therapies and achieved only a limited response duration of 9 months. Moreover, comparisons with other RCC subtypes using the GSE152938 dataset were not matched for treatment history or disease stage, precluding definitive conclusions regarding the unique immune landscape of TRCC. Instead, more robustly quantified parameters, including a PD-L1 tumor proportion score of 5% and a CD8⁺ tumor-infiltrating lymphocyte density of 30%, provide more reliable, albeit modest, evidence of an immune-active microenvironment. Whether the naïve T-cell population represents a reservoir for therapeutic activation or merely reflects ineffective antigen priming in the context of SPP1⁺ TAM-mediated immunosuppression requires further investigation in prospectively designed studies. The initial disease stabilization followed by rapid progression suggests that, although naïve T cells may be activated, their antitumor efficacy is ultimately constrained by the immunosuppressive microenvironment dominated by SPP1⁺ TAMs[26]. Third, the presence of a distinct SPP1⁺ TAM population characterized by strong activation of angiogenesis pathways may represent a critical factor contributing to resistance to both targeted therapy and immunotherapy[27,28]. Preclinical studies have implicated SPP1 in promoting angiogenesis and mediating immunotherapy resistance through modulation of the tumor microenvironment[29,30]. The enrichment of SPP1⁺ TAMs in our patient’s tumor may have contributed to the rapid progression and treatment resistance observed during the disease course. These findings suggest that SPP1⁺ TAMs may represent a potential therapeutic target in recurrent TRCC, particularly in cases resistant to TKIs. Targeting these pro-angiogenic macrophages may provide a strategy to overcome resistance and improve therapeutic outcomes. The minimal SPP1 expression in macrophages from the primary tumor, in contrast to the prominent SPP1⁺ TAM population observed in the recurrent metastatic lesion, further supports the hypothesis that these macrophages may represent a therapy-induced adaptive response. This distinction has important therapeutic implications: While anti-angiogenic therapy may be effective from the outset due to the intrinsic angiogenic potential of TRCC, targeting SPP1⁺ TAMs may be particularly relevant in the setting of acquired resistance following TKI and immunotherapy exposure.

These findings suggest a sequential therapeutic strategy for aggressive TRCC. Anti-angiogenic TKIs may provide initial disease control due to the intrinsic angiogenic activity of the tumor; however, close monitoring for early resistance is warranted. Upon disease progression, combining immune checkpoint inhibitors with TKI therapy may leverage the preserved immune potential suggested by the high proportion of naïve T cells. Nevertheless, the limited 9-month response observed in this case indicates that immunosuppressive SPP1⁺ TAMs may constrain the durability of therapeutic efficacy. In this context, SPP1⁺ TAM-targeted strategies, such as CSF1R inhibition or blockade of the SPP1-CD44 axis, may represent rational approaches to overcoming macrophage-mediated resistance. Prospective validation of this sequential treatment paradigm in larger TRCC cohorts is urgently needed.

Several limitations of this study should be acknowledged. First, the scRNA-seq analysis was performed on a single biopsy specimen obtained from an abdominal wall metastasis after multiple lines of systemic therapy, including pazopanib, axitinib, radiotherapy, and combination axitinib plus camrelizumab therapy. Therefore, the observed activation of angiogenesis pathways and enrichment of SPP1⁺ TAMs may reflect therapy-induced microenvironmental remodeling rather than intrinsic biological characteristics of primary TRCC. Notably, immunohistochemical analysis of the primary tumor revealed robust CD31 positivity and enrichment of CD8⁺ T cells, indicating that angiogenesis and immune cell infiltration were already present at diagnosis. However, SPP1 expression in macrophages was minimal in the primary tumor, supporting the hypothesis that SPP1⁺ TAM enrichment likely emerged under therapeutic selective pressure. Without single-cell profiling of the primary tumor, we cannot definitively determine whether these characteristics were present at diagnosis or developed as a consequence of treatment-induced selection. Second, the absence of longitudinal sampling precludes evaluation of dynamic changes in the tumor microenvironment during treatment. Future studies incorporating paired primary and metastatic specimens, as well as serial biopsies collected throughout treatment, will be essential to distinguish intrinsic tumor biology from therapy-related microenvironmental adaptations in aggressive TRCC. Furthermore, it is important to emphasize that the TRCC sample analyzed in this study was derived from a recurrent metastatic lesion following multiple lines of therapy, whereas samples from other RCC subtypes in the public dataset GSE152938 were not matched for treatment history or disease stage. Consequently, differences in naïve T-cell proportions may be influenced by therapy-induced immune microenvironmental remodeling, disease progression, and prior treatment exposure, rather than representing intrinsic biological features of TRCC. These comparative findings should therefore be interpreted cautiously and not overinterpreted.

CONCLUSION

In conclusion, our findings emphasize the importance of understanding both tumor cell characteristics and the microenvironmental landscape of TRCC. The activation of angiogenesis pathways and enrichment of SPP1⁺ TAMs may contribute to resistance to TKIs and combination therapies. Although this study is limited by the single-case design, these observations highlight the need for further prospective validation in larger cohorts. By identifying specific molecular and microenvironmental features associated with aggressive TRCC, we may facilitate early identification of high-risk cases and enable the development of more tailored and intensive treatment strategies. This work provides a foundation for future investigations aimed at improving therapeutic approaches for recurrent TRCC.