Commentary: Characteristics of tertiary lymphoid structures in prostate cancer and the impact of neoadjuvant therapy on their formation and maturation

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We read with great interest the article by Liu and colleagues (1), which systematically characterizes tertiary lymphoid structures (TLS) in prostate cancer (PCa) and demonstrates that neoadjuvant hormone therapy (NHT) promotes TLS formation, maturation, and immune cell infiltration. This is among the first studies to establish TLS as a favorable prognostic biomarker in PCa and, to our knowledge, the first to provide direct clinical and experimental evidence that androgen deprivation therapy (ADT) can convert prostate cancer from an immunologically "cold" tumor into a more immuneresponsive "hot" tumor. We hold great admiration for the academic contributions of the research team. At the same time, we would like to offer several constructive suggestions regarding potentially extensible directions in this study, hoping to provide reference for future investigations.A distinctive strength of this study lies in its establishment of a complete chain of evidence spanning from clinical pathological specimens to transcriptomics and ultimately to animal models. The authors first systematically characterized the spatial distribution, density, and maturation of TLS in three independent clinical cohorts using H&E staining and multiplex immunohistochemistry (mIHC). Subsequently, they leveraged public transcriptomic databases (2,3) to compare TLS gene signature expression between NHTtreated and treatment-naïve patients via single-sample gene set enrichment analysis (ssGSEA) (4). Critically, paired pre-and post-NHT biopsy-prostatectomy specimens provided direct evidence of NHT-induced TLS enhancement within the same patients.Finally, the authors established an orthotopic immunocompetent mouse model using CRISPR-Cas9 gene editing of prostate organoids (TP53, PTEN, and RB1 triple knockout with MYC amplification) (5), and validated in vivo that ADT remodels the tumor immune microenvironment and synergizes with anti-PD-1 therapy. This research paradigm-"clinical observation → pathological quantification → transcriptomic validation → animal model verification"-not only substantially enhances the rigor and reliability of the study's conclusions but also provides a methodological template for similar investigations of therapy-induced immune modulation.The study goes beyond a simple "TLS presence or absence" dichotomy by performing a refined maturation staging of TLS-distinguishing early TLS (E-TLS), primary follicle-like TLS (PFL-TLS), and secondary follicle-like TLS (SFL-TLS), the latter characterized by CD21⁺ follicular dendritic cell networks and active germinal centers. A key finding is that only mature TLS, particularly SFL-TLS, significantly correlate with prolonged progression-free survival (PFS: P = 0.0364, HR = 0.1745), whereas immature TLS lack significant prognostic value. Similarly, higher intra-tumoral TLS density (P = 0.0016, HR = 0.079) was associated with better prognosis compared to peri-tumoral TLS. This nuanced stratification aligns with the emerging consensus in genitourinary cancers that mature TLS with functional germinal centers are critical drivers of effective anti-tumor immunity (6,7), whereas immature TLS may fail to elicit adequate immune responses and may even be associated with an immunosuppressive phenotype (8).This study is the first to demonstrate, in both clinical specimens and an orthotopic mouse model, that NHT promotes TLS formation and maturation. Patients in the NHT group exhibited significantly increased TLS density and maturity, along with enhanced infiltration of CD4⁺, CD8⁺, CD20⁺, and CD21⁺ immune cells. Paired biopsy analysis revealed that the detection rate of SFL-TLS increased from 0% to 65% following NHT.Transcriptomic analysis confirmed the upregulation of multiple TLS gene signatures after NHT. In animal experiments, ADT increased CD45⁺ immune cell infiltration and the proportion of CD3⁺ T cells, with a shift toward CD8⁺ predominance. The combination of anti-PD-1 and ADT exerted the most potent anti-tumor effect, with one mouse achieving complete tumor regression-an outcome not observed in any monotherapy group. This finding carries significant translational implications, suggesting that TLS induction may represent a core mechanism linking ADT to enhanced immunotherapy responsiveness in prostate cancer, corroborating the observations by Hawley et al. (9), who reported enhanced immune infiltration with anti-PD-1 plus ADT in metastatic hormone-sensitive prostate cancer.Notwithstanding the notable strengths outlined above, upon careful examination of the study, we believe that several aspects warrant further exploration and refinement to enhance mechanistic depth and the robustness of the conclusions.While the study convincingly demonstrates that NHT promotes TLS formation and maturation at the phenotypic level, the underlying molecular mechanisms remain largely unknown. Multiple potential pathways merit in-depth investigation. First, chemokine networks are critical for TLS formation-CXCL13 recruits B cells and T follicular helper cells, while CCL19 and CCL21 organize T-cell zones and promote high endothelial venule (HEV) formation (10). Does NHT upregulate these chemokines? To address this question, we propose that multiplex cytokine/chemokine profiling of pre-and post-NHT specimens be performed to identify TLS-inducing factors. Second, androgen receptor (AR) signaling in immune cells directly influences anti-tumor immunity. Guan et al. (11) demonstrated that AR activity in T cells limits checkpoint blockade efficacy, and that AR inhibition enhances CD8⁺ T-cell function and prevents exhaustion. Yang et al. (11) showed that AR regulates CD8⁺ T-cell stemness through epigenetic and transcriptional differentiation programs. Chesner et al. (12) further found that AR inhibition upregulates MHC-I expression and improves T-cell responses. However, whether these AR-mediated T-cell effects represent upstream events of TLS formation or operate through independent pathways remains unclear. Therefore, conditional knockout of AR in T cells or stromal cells could be employed to dissect cell-type-specific contributions. Third, stromal components-including fibroblasts, HEVs, and lymphatic vessels-serve as essential scaffolds for TLS formation. Does NHT modulate the fibroblast-derived lymphotoxin-β receptor (LTβR) pathway or promote vascular remodeling to facilitate TLS neogenesis (13)? Spatial transcriptomics could be utilized in future studies to map TLSassociated molecular gradients within the tumor microenvironment, thereby addressing these knowledge gaps.The study primarily quantifies TLS based on morphological and immunophenotypic criteria (CD4⁺, CD8⁺, CD20⁺, and CD21⁺ staining). However, the functional quality of these TLS -that is, whether they are capable of supporting effective anti-tumor immune responses-remains incompletely characterized. Key unresolved questions include: Do NHT-induced TLS contain tumor-specific T cells? Are B cells within TLS capable of producing anti-tumor antibodies (14)? Is the functional status of CD8⁺ T cells within TLS fully ascertained-specifically, whether they are functionally active (expressing Granzyme B, Perforin, IFN-γ) or exhausted (PD-1⁺, TIM-3⁺)? While the transcriptomic analysis in the present study revealed upregulation of GZMB and PRF1 following NHT (Figure 6C The authors acknowledge that, in Cohort 2, patients in the NHT group had statistically higher Gleason scores than those in the non-NHT group-a baseline imbalance that may introduce selection bias. Although the paired pre-and post-NHT biopsy-to-prostatectomy design in Cohort 3 partially addresses this issue by using each patient as their own control, the sample size for the paired analysis (n = 20) remains relatively small, which may limit the generalizability of the findings. Furthermore, the mouse model utilized bicalutamide as the ADT agent, whereas clinical NHT regimens often encompass a variety of agents, including GnRH agonists, anta