Abstract

Background

   Emerging evidence underscores the pivotal role of M2-polarized
   tumor-associated macrophages (M2-TAMs) in orchestrating
   immunosuppressive tumor microenvironments that fuel metastatic
   dissemination in prostate cancer (PCa), yet the fundamental mechanisms
   governing M2-TAM trafficking in lethal PCa progression remain poorly
   understood.

Methods

   Multi-cohort transcriptomic analyses were performed to identify
   metastasis-associated genes, with CTSZ prioritized as a key cathepsin
   linked to prostate cancer progression. Circulating tumor and bone
   metastatic mouse models were employed to investigate CTSZ-driven M2-TAM
   infiltration and metastatic behavior. Mechanistic studies included
   proteasomal degradation assays, IL32 pre-mRNA splicing analysis, and
   IL-32 binding experiments using RGD motif-dependent interactions.
   Therapeutic efficacy was tested with the ITGA5 inhibitor GLPG0187 in
   preclinical models.

Results

   Elevated CTSZ expression shows strong clinical association with
   advanced pathological progression. CTSZ overexpression in PCa cells
   drives lung metastasis dissemination and bone metastatic in vivo model
   but fails to enhance cell-intrinsic oncogenic behaviors in vitro
   systems. Overexpression of CTSZ promotes M2-TAM infiltration and
   metastasis by inducing TRA2A degradation via the proteasome pathway,
   which alleviates TRA2A-mediated suppression of IL32 alternative
   splicing. Enhanced IL-32 secretion facilitates M2-TAM recruitment
   through binding to macrophage integrin ITGA5. Pharmacological
   inhibition of ITGA5 with GLPG0187 significantly reduced metastatic
   burden and M2-TAM infiltration in vivo.

Conclusions

   The CTSZ/TRA2A/IL-32/ITGA5 axis orchestrates protumoral immunity in PCa
   metastasis by driving M2-TAM recruitment. Targeting this pathway,
   particularly through ITGA5 blockade, represents a promising therapeutic
   strategy to inhibit metastatic progression and remodel the
   immunosuppressive tumor microenvironment.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12967-025-06865-w.

   Keywords: CTSZ, IL-32, TRA2A, ITGA5, Macrophage, Tumor microenvironment

Introduction

   Prostate cancer (PCa) represents a formidable clinical challenge as the
   second most lethal malignancy in men globally, with over 1.4 million
   new cases diagnosed annually. The 5 year survival rate exhibits a
   dramatic decline—from nearly 90% in localized stages to below 30% in
   metastatic disease [[48]1, [49]2]. The immunosuppressive tumor
   microenvironment (TME), characterized by dysfunctional cytotoxic T
   cells and paradoxical accumulation of protumoral M2-polarized
   macrophages, has emerged as a key driver of therapeutic resistance and
   metastatic progression [[50]3, [51]4]. Recent single-cell analyses
   reveal that M2 tumor-associated macrophages (TAMs) constitute the
   predominant immune infiltrate in advanced PCa. These cells form
   feedforward regulatory loops with cancer cells through androgen
   receptor co-activation and suppress natural killer (NK) cell function
   via the IL-10/PD-L1 signaling axis [[52]5–[53]7]. However, the
   molecular underpinnings governing their preferential recruitment to the
   TME remain poorly characterized, creating a critical knowledge gap in
   developing macrophage-targeted therapies.

   Cathepsin Z (CTSZ), a lysosomal cysteine protease with unique RGD-motif
   dependent extracellular signaling capacity, has emerged as a key
   mediator of tumor-stroma crosstalk [[54]8, [55]9]. The catalytically
   mature CTSZ isoform is restricted to carboxypeptidase activity via its
   C-terminal monopeptidase domain, which mediates proteolytic processing
   of substrate C-terminal residues [[56]10–[57]12]. In addition, relevant
   evidence shows that in the studies of triple negative breast cancer and
   hepatocellular carcinoma, the overexpression of CTSZ is correlates with
   advanced disease and low patient survival rate [[58]13, [59]14].
   However, the functional landscape of CTSZ in metastatic PCa remains
   unknown: its mechanistic contribution to the metastatic cascade,
   crosstalk with TAMs, and immunomodulatory role within the TME require
   experimental dissection. This metastasis-associated expression pattern,
   coupled with its undefined role in PCa immunology, prompted
   investigation into CTSZ-mediated TME reprogramming.

   Our findings establish CTSZ as a non-cell-autonomous driver of prostate
   cancer metastasis, mediating M2-polarized TAM recruitment through
   proteolytic regulation of the TRA2A/IL-32/ITGA5 axis. Mechanistically,
   CTSZ-mediated degradation of TRA2A liberates IL32 mRNA from splicing
   repression, leading to increased secretion of IL-32 that engages ITGA5
   receptors on macrophages. This ligand-receptor interaction activates
   PI3K/AKT signaling in macrophages, driving chemotaxis and M2
   polarization. Pharmacological inhibition of ITGA5 using the antagonist
   GLPG0187 significantly attenuated tumor metastasis in syngeneic mouse
   models. These results decode a tumor cell-macrophage crosstalk circuit
   and propose a dual therapeutic strategy targeting both CTSZ protease
   activity and IL-32/ITGA5 signaling to dismantle the immunosuppressive
   TME in advanced PCa.

Materials and methods

Online bioinformatics analysis and transcriptome sequencing

   Transcriptome data from [60]GSE32269, [61]GSE101607, and [62]GSE210729
   datasets were analyzed through GEO database. Clinical correlations of
   CTSZ with nodal metastasis and Gleason score in PCa patients were
   evaluated using UALCAN platform. Genomic profiling and survival
   analysis across nine prostate cancer cohorts were performed via
   cBioPortal, where SU2C mPCa cohort data further revealed CTSZ
   correlations with macrophage markers and IL-32/TRA2A interactions.
   Immune infiltration patterns were assessed using Time2.0 database,
   while CTSZ expression profiles across disease stages were retrieved
   from Cambridge CRUK dataset.For experimental validation,
   TRIzol-extracted RNA from PC3 cells (sh-CTSZ vs sh-NC groups) underwent
   RNA-seq analysis (Majorbio Co.) using SMARTer mRNA-seq library prep kit
   per manufacturer’s protocol.

Single-cell sequencing and immune infiltration analysis

   Single-cell RNA sequencing data from [63]GSE143791 (10 bone metastatic
   cases) were processed through the Seurat pipeline (v3.1.2) [[64]15].
   This was followed by doublet removal via the DoubletFinder R package,
   with a 10% doublet formation rate [[65]16]. After doublet removal using
   DoubletFinder, datasets underwent anchor-based integration with
   subsequent scaling and PCA dimensionality reduction. Graph-based
   clustering analysis was performed before UMAP visualization of cell
   populations. The cell type enrichment analysis tool CIBERSORT was used
   to calculate the gene set enrichment scores of the 22 immune cell
   subtypes via the GSVA package.

Cell acquisition and culture

   All the cell lines used in this study were sourced from the American
   Type Culture Collection (ATCC) and were accompanied by species
   verification and mycoplasma contamination detection certificates. The
   cell cultures were meticulously maintained in a CO2 incubator at 37 °C
   with either RPMI 1640 medium or DMEM supplemented with 10% FBS and 1%
   penicillin‒streptomycin solution.

Cell transfection

   CTSZ-, IL-32-, and TRA2A-targeting siRNAs (sequences in Table S1) and
   lentiviral constructs were obtained from Tsingke Biotech. siRNA
   transfection in PC3/22RV1 cells followed Lipo3000 protocol, with
   RNA/protein harvested at 24/48 h post-transfection. For stable CTSZ
   overexpression, 22RV1/RM1 cells were transduced with
   pLenti-CMV-Puro-Luciferase-CTSZ (vector-modified by CTSZ insertion)
   followed by titer-optimized puromycin selection. Empty vector served as
   negative control.

Lung metastasis model and bioluminescence imaging

   All procedures complied with AAALAC (Association for Assessment and
   Accreditation of Laboratory Animal Care, International) guidelines
   under Army Medical University IACUC (Institutional Animal Care and Use
   Committee) approval (No. AMUWEC20232943). Male BALB/c nude mice
   (4–5 weeks, 18–22 g; n = 6/group) were injected via tail vein with
   1 × 10^6 PC3 cells suspended in PBS. Metastatic progression was
   monitored by bioluminescence imaging (IVIS Spectrum CT) at days 3 and
   21 post-injection following intraperitoneal D-luciferin administration
   (150 mg/kg; 10 μL/g, Beyotime ST198). Lungs harvested at endpoint were
   fixed in bitter acid for 10 min, rinsed in PBS, and metastatic nodules
   (> 1 mm) quantified macroscopically before 4% PFA fixation and H&E
   validation.

Bone metastasis model

   Ten nude mice, aged 5–6 weeks, were prepared for the experiment using
   CTSZ-OE and Vector cells. The mice were sedated with isoflurane gas
   anesthesia throughout the procedure. Tumor cells, 10^5 in number and
   suspended in 20 μl of PBS, were injected into the tibia of each mouse
   after disinfecting the injection site. An insulin needle was inserted
   gently along the tibial plateau to reach the bone marrow cavity. After
   the injection, the area was disinfected. On day 21, the mice were
   euthanized, and their tibiae were collected. The samples were fixed for
   at least 24 h and subjected to a bone micro-CT scan in Kontich,
   Belgium. Relevant indicators were assessed via H&E staining,
   immunohistochemical staining, and trap staining after decalcification.

Isolation of macrophages from tumor tissues

   Fresh tumor tissue was dissected into small 1 mm pieces in a 10 cm dish
   and then digested with a solution containing 1 g/L type IV collagenase
   (#[66]LS004188, Worthington), 0.1 g/L hyaluronidase (Sigma), and
   0.01 g/L DNase I (#D8071, Solarbio, China) in a cell incubator for 2 h.
   After dispersion, the cell suspensions were collected and centrifuged
   at 600 × g for 5 min, the supernatant was discarded, ACK lysis buffer
   was added for cell lysis, and the mixture was washed through
   centrifugation at 600 × g for 5 min, which was repeated three times.
   The cell pellet was then resuspended in complete culture medium and
   filtered through a 100-μm filter to collect the cells for further
   isolation of the macrophages.

Transwell assay

   Invasion/migration assays were performed in triplicate using
   Matrigel-coated Transwell chambers (8 μm pores). PCa cells
   (3 × 10^3/well, counted via automated cell counter) in serum-free
   medium were seeded in upper chambers, with complete medium as
   chemoattractant in lower chambers. After 18 h incubation, traversed
   cells were fixed in 4% PFA, stained with crystal violet, and quantified
   by microscopic examination of five random fields (20 × objective).
   Parallel migration assays omitted Matrigel coating. Three biological
   replicates with duplicate technical repeats were conducted.

Macrophage migration assays

   Vector- or CTSZ-OE-transfected 22RV1 cells were cultured to 90%
   confluence in T25 flasks. Conditioned medium (CM) was collected after
   48 h, centrifuged (300 × g, 5 min), and used for Transwell assays.
   THP-1-derived macrophages were polarized with PMA (100 nM) followed by
   LPS (100 ng/ml, M1) or IL-4/IL-13 (20 ng/ml each, M2). Polarized
   macrophages (5 × 10^4 cells/well) were seeded in 8-μm Transwells with
   CM in lower chambers. Migrated cells were fixed (4% paraformaldehyde)
   and stained (0.1% crystal violet) after 24 h.

Real-time PCR

   Total RNA was extracted via TRIzol reagent (Ambion 317908) and then
   converted into cDNA using PrimeScript (DRR047A; TaKaRa, China). Gene
   expression analysis was conducted via real-time qPCR in a Bio-Rad CFX
   Connect real-time system utilizing SYBR Green Master Mix from Thermo
   Fisher Scientific (A25742, China) and the primers listed in
   Supplementary Table S2 from Sangon Biotech. The gene expression levels
   were determined via the 2^−ΔΔCt method.

Western blot

   Cell proteins were extracted via radioimmunoprecipitation assay (RIPA)
   lysis buffer (#P0013, Beyotime, China) and quantified using a BCA kit
   (#P0009, Beyotime, China). For SDS‒PAGE analysis, 2 µg of protein was
   added to each well of a 6/10/15% SDS‒PAGE gel after BCA quantification.
   The proteins were then transferred to Immune-Blot PVDF membranes
   (#1620177, Bio-Rad, USA). The membranes were blocked with protein-free
   rapid sealing solution (#G2052, Servicebio, China) for 10 min, followed
   by overnight incubation with the primary antibody at 4 °C. After the
   membranes were washed three times for 15 min each with PBST, they were
   incubated at room temperature for 1 h with the corresponding secondary
   antibodies. The signals were detected via the Ultrasensitive ECL
   Western HRP Substrate (#17047, Zenbio, China) and a Bio-Rad ChemiDoc MP
   System (170–8280). The primary antibodies used are listed in Table S3.

Immunohistochemistry and immunofluorescence

   The tumor tissue microarray of patients with prostate cancer used in
   this study was provided by Changsha Yaxiang Biotechnology Co., Ltd.
   Mouse orthotopic, lung, and tibial tumor tissues were sectioned into
   5-mm-thick slices. The tissue microarrays and mouse tumor tissue slices
   were deparaffinized at 70 °C for 2 h. EDTA repair was then performed at
   a high temperature for 3 min, followed by washing with PBS and blocking
   with serum for 1 h. The primary antibody was added to the samples in
   the dark and stored overnight at 4 °C. After the samples were washed
   with PBS, secondary antibodies were added, and the samples were
   incubated at room temperature for 30 min. The immunofluorescence
   procedure was similar to that used for immunohistochemistry, with the
   addition of DAPI for nuclear staining. Finally, the slides were sealed
   with mounting medium containing an anti-fluorescence quencher, and all
   the slides were scanned via a whole-slide scanner. All tumor samples
   were processed in the same manner.

EMSA

   Biotin labels were introduced into the 5′-TCTGCTGGAAACGACTCGGAG-3′
   sequence of IL-32 immature mRNA, which includes the GAAARGARR sequence
   and has been confirmed as the binding site for TRA2A. In the sequence
   from positions 602 to 767, the GAAACGA sequence was subsequently
   mutated to AGGCCTA, whereas in the sequence from positions 2297 to
   2489, the GAAACAA sequence was subsequently mutated to TAGCGAT. To
   assess whether TRA2A binds to this probe and whether the corresponding
   sequence mutation affects binding, a chemiluminescent EMSA kit (#GS009,
   Beyotime, China) was used. The sample was loaded onto a BeyoGel™ EMSA
   Precast PAGE gel (#GS301S, Beyotime, China) for electrophoresis. The
   supershift group consisted of 1 μg of anti-TRA2A antibody and
   20 pmol/μL wild-type probe. The experimental procedures were conducted
   according to the Chemiluminescent EMSA Kit protocol. Signal detection
   was performed via a Bio-Rad ChemiDoc MP System after exposure to the
   nylon membrane.

RNA immunoprecipitation

   An RNA immunoprecipitation (RIP) kit (#p0102, Geneseed, China) was used
   to obtain RNA bound to TRA2A according to the manufacturer’s protocol.
   The RNA concentrations were determined via Nanodrop Lite (Thermo
   Science, Serial Number: LT1613). Because TRA2A may bind to the intron
   region of IL-32 pre-mRNA, it is necessary to use random primers to
   complete reverse transcription (#151600, Toyobo, China). qPCR was
   performed to determine the expression of IL-32 in the
   reverse-transcribed IgG and IP group cDNAs. The relative expression of
   the target genes was calculated according to the 2^−ΔΔCt formula, with
   ΔΔCt = (Ct[IP]–Ct[Input])–(Ct[IgG]–Ct[Input]). Using the Ct value of
   the input group as the reference CT value, the gene expression level of
   the IP group of the target gene was 2^−X greater than that of the IgG
   group.

RNA pulldown

   Biotin-labeled RNA probes (602-767, 602-767-M, 2297-2489, 2297-2489-M)
   were synthesized by Shanghai Shenggong Biotechnology. PC3 lysates
   (> 2 mg/mL) were prepared for RNA pull-down using a commercial kit
   (#DW1127, Dowobio, China). Magnetic beads were washed twice with 20 mM
   Tris (50 μL), resuspended in 1 × RNA capture buffer (50 μL), and
   incubated with RNA pull-down mix (100 μL, 4 °C rotation, 3 h). Beads
   were further incubated in capture buffer (50 μL, RT, 2 h), washed
   thrice with wash buffer, eluted (50 μL elution buffer, 1 h rotation),
   and supernatants collected for Western blot.

ELISA

   The supernatants were collected from PCa cells with either knockdown or
   overexpression of CTSZ. After centrifugation at 2000 × g, the
   supernatant was collected for further use. Next, we prepared the
   standard gradient working solution, 1 × Biotin-antibody,
   streptavidin-HRP, and washing buffer. The prepared samples (100 μL)
   were added to each well of a 96-well plate and incubated for 1 h in a
   cell incubator. After the liquid was removed, 100 μL of
   1 × biotin-antibody was added to each well and incubated at 37 °C for
   1 h. The wells were washed three times with washing buffer, and 100 μL
   of streptavidin-HRP solution was added to each well and incubated at
   37 °C for 1 h. After washing three times, 90 μL of substrate (TMB) was
   added to each well and incubated at 37 °C for 15 min in the dark. After
   incubation, the plate was removed, and 50 μL of stop solution was added
   to each well. The optical density (OD) of each well was measured
   immediately at 450 nm. The purchased reagent kits included the
   following products (#JONLOBIO, China): Cat No. JL19279 (IL32), JL14142
   (CTSZ), JL19334 (CCL2), and JL1102 (CXCL10).

Protease degradation reaction

   CTSZ protease activity was measured via Octet 2.0. The sensor
   (Biosensors, NI–NTA, Tray) was prewetted with PBST for 10 min and then
   successfully immobilized with recombinant TRA2A protein (#TP762583,
   ORIGENE, USA) containing a histidine tag. The reaction was performed at
   37 °C with varying concentrations of recombinant CTSZ (#934-CY, R&D
   Systems, Minneapolis, MN, USA) to detect its ability to hydrolyze the
   immobilized TRA2A protein in 0.20 ml per working volume. Hydrolytic
   events were quantified by measuring the shift in the optical
   interference pattern.

Mass spectrometry analysis

   Matrix-assisted laser desorption and ionization time-of-flight
   (MALDI-TOF) was used to analyze pure TRA2A and enzymatically cleaved
   TRA2A. Recombinant CTSZ (#934-CY; R&D Systems, Minneapolis, MN, USA)
   was generated according to the manufacturer’s instructions. A mixture
   of CTSZ and TRA2A (#TP762583, ORIGENE, USA) at a final concentration of
   1 μg/μL was incubated at 37 °C for 2 h. Subsequently, the reaction was
   terminated at − 20 °C and analyzed via a Bruker Daltonics Ultraflex
   MALDI‒TOF mass spectrometer (Bremen, Germany). Calibration was
   performed using Bruker protein standards.

Fluorescence in situ hybridization (FISH)

   PC3 cells were fixed on coverslips with 4% paraformaldehyde for 20 min
   and then washed three times with PBS for 5 min each. Proteinase K
   (20 μg/mL) was added, and the mixture was digested for 8 min. The cells
   were subsequently washed three times with PBS for 5 min each.
   Prehybridization solution was added and incubated at 37 °C for 1 h. The
   prehybridization solution was removed, and the hybridization solution
   containing the IL-32 probe was added and incubated overnight at 37 °C.
   The hybridization mixture was washed with 2 × SSC at 37 °C for 10 min,
   followed by 1 × SSC at 37 °C for 10 min and then 0.5 × SSC at 37 °C for
   10 min. The hybridization mixture was removed, and the TRA2A antibody
   was added and incubated overnight at 37 °C. The DAPI staining solution
   was added to the slides, which were subsequently incubated for 8 min in
   the dark. After washing, the slides were sealed with anti-fade mounting
   medium. The slides were examined under a fluorescence microscope, and
   images were captured.

Statistical analysis

   All data presented in this study are representative of similar results
   obtained from a minimum of three replicates unless stated otherwise.
   The Shapiro‒Wilk normality test was used to assess the data
   distribution. The quantitative data are expressed as the
   means ± standard deviations (SDs) and were analyzed via GraphPad Prism
   8 software. Unpaired t tests were used to compare differences between
   two groups, whereas one-way ANOVA and log-rank trend tests were
   employed to determine statistically significant differences among three
   or more groups. A p value < 0.05 was considered statistically
   significant.

Results

Aberrant CTSZ upregulation correlates with adverse prognosis in metastatic
PCa

   To identify molecular drivers of PCa metastasis, we analyzed three
   independent GEO datasets ([67]GSE32269, [68]GSE101607, [69]GSE210729)
   using stringent criteria (adjusted p < 0.05, |log2FC|> 1).
   Intersectional analysis revealed two candidate genes: CTSZ and COCH
   (Fig. [70]1A). Notably, CTSZ was the only gene showing consistent
   upregulation across all metastatic cohorts (Fig. [71]1B, S1A).
   Moreover, the expression of CTSZ was increased in metastatic PCa (mPCa)
   cell lines such as PC3, LNCAP, C4-2B, and DU145 than in non-metastatic
   PCa cell lines 22RV1 (Fig. [72]1C). Immunohistochemical validation
   using tissue microarrays (42 metastatic vs. 40 localized PCa cases)
   showed markedly elevated CTSZ protein abundance in metastatic specimens
   (positive cell proportion: 5.911 ± 4 vs 10.44 ± 8; p < 0.001, unpaired
   t-test), demonstrating progressive CTSZ upregulation through disease
   advancement (Fig. [73]1D). Clinical relevance analyses using
   GEPIA-based TCGA datasets revealed that CTSZ overexpression strongly
   correlated with advanced lymph node metastasis and higher Gleason
   scores (Fig. [74]1E, F). Critically, quantitative analysis of CTSZ
   expression in 42 mPCa specimens versus 40 localized PCa controls
   revealed that elevated CTSZ levels were significantly associated with
   diminished clinical outcomes, demonstrating a median reduction of
   survival times in both overall survival (OS) and disease-free survival
   (DFS) (log-rank p < 0.05) (Fig. [75]1G, H). This association was
   validated in the TCGA-PRAD cohort (n = 498), where CTSZ overexpression
   correlated with reduced survival duration (HR = 2.7) (Fig. [76]1I).
   Genomic profiling in the SU2C/PCF metastatic cohort identified
   increased CTSZ amplification frequency in metastatic lesions (Figure
   S1B), with amplified cases showing poorer survival outcomes (p < 0.05,
   Figure S1C). Collectively, multi-cohort analyses establish CTSZ
   overexpression as a robust biomarker of metastatic progression in PCa,
   underscoring the need for functional studies to dissect its mechanistic
   role.

Fig. 1.

   [77]Fig. 1
   [78]Open in a new tab

   CTSZ overexpression correlates with metastatic progression and poor
   prognosis in prostate cancer. A Venn diagram of differentially
   expressed genes (DEGs) across three PCa progression datasets
   ([79]GSE32269, [80]GSE101607, [81]GSE210729). B CTSZ mRNA expression in
   PCa progression datasets. C CTSZ protein (Western blot) and mRNA
   (qRT-PCR) levels in PCa cell lines (DU145, PC3, LNCaP, 22Rv1, C4-2B). D
   Representative H&E and CTSZ IHC staining in localized (n = 42) versus
   metastatic (n = 40) PCa specimens (scale bar: 50 μm). E Expression of
   CTSZ in PRAD based on nodal metastasis status in the UALCAN database. F
   CTSZ expression in PRAD patients on the basis of the patient Gleason
   score in the UALCAN database. G, H Kaplan–Meier analysis of overall
   survival (OS) and disease-free survival (DFS) stratified by CTSZ
   expression (high vs low, log-rank test). I TCGA-PRAD cohort analysis of
   CTSZ-associated DFS (Cox proportional hazards model). Data represent
   mean ± SD. Statistics: unpaired t-test (B, D); one-way ANOVA (C, E, F).
   Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001

CTSZ overexpression drives prostate cancer metastasis independent of
cell-autonomous invasion and migration

   To determine the role of cancer cell-derived CTSZ in PCa metastasis, we
   modulated CTSZ expression in human PCa cell lines (PC3, 22RV1) and a
   mouse PCa line (RM1). Transcriptional and translational validation
   confirmed successful CTSZ knockdown/overexpression (Fig. [82]2A, B and
   S1A). Notably, CTSZ manipulation in PC3 (knockdown) and 22RV1
   (overexpression) did not significantly affect cellular
   invasion/migration capacities in Transwell assays (Fig. [83]2C) or
   alter epithelial-mesenchymal transition (EMT) markers (CDH1, CDH2,
   VIMENTIN) as assessed by western blot analysis. (Fig. [84]2D, E). These
   in vitro findings collectively demonstrate that CTSZ manipulation does
   not directly regulate malignant cellular phenotypes.

Fig. 2.

   [85]Fig. 2
   [86]Open in a new tab

   Overexpression of CTSZ in PCa cells promotes PCa metastasis in vivo. A,
   B Validation of CTSZ knockdown (PC3) and overexpression (22Rv1)
   efficiency by qRT-PCR and immunoblotting (mean ± SD; one-way ANOVA). C
   Transwell migration/invasion assays (representative images and
   quantification; three biological replicates). D, E EMT marker
   expression (CDH1, CDH2, VIMENTIN) in CTSZ-modulated cells
   (immunoblots). F Longitudinal bioluminescence imaging (BLI) of systemic
   metastasis (n = 6/group; days 3–21). G, H Ex vivo and H&E stain of lung
   metastasis (H&E; scale bars: 2.5 mm). I Quantification of metastatic
   nodules (mean ± SD; unpaired t-test). J Micro-CT reconstruction of
   proximal tibiae (red box: ROI; 3D trabecular analysis). K
   Histomorphometry (H&E, TRAP, CD206 staining; scale bars: 50 μm). L Bone
   structural parameters: BMD, BV/TV, Tb.Th, Tb.N, Tb.Sp, SMI
   (n = 3/group; one-way ANOVA). *p < 0.05; **p < 0.01. ns, not
   significant. BLI: Bioluminescence imaging

   Given the lack of in vitro phenotypic effects, we subsequently employed
   xenograft models to investigate CTSZ's potential role in metastatic
   dissemination. Bioluminescence imaging of the tail vein-injected mice
   revealed that substantially higher metastatic burden was observed in
   the mice inoculated with CTSZ-OE cells than in the mice inoculated with
   Vector cells at day 21 (Fig. [87]2F). Picric acid staining confirmed
   increased tumor burden in CTSZ-OE tissues (red arrows, Fig. [88]2G).
   Histopathological analysis of H&E-stained lung sections revealed an
   8.5-fold increase in metastatic foci density in CTSZ-OE mice (red
   arrows, p < 0.001, Fig. [89]2H, I). Since bone metastasis is an
   important cause of mortality in patients with advanced PCa, we
   inoculated RM1 (vector control and CTSZ-OE) cells into the tibia of
   male nude mice. Micro-CT analysis (2D/3D reconstruction) showed that
   CTSZ-OE inoculation significantly reduced trabecular bone number
   (dashed red boxes, Fig. [90]2J). H&E and TRAP staining confirmed
   decreased trabecular bone mass and increased osteoclast formation in
   CTSZ-OE tibiae (red arrows, Fig. [91]2K). Parameters representing the
   structural integrity of trabecular bone, such as percent trabecular
   area (BV/TV), trabecular thickness (Tb.Th), and trabecular number
   (Tb.N) were significantly decreased in the CTSZ-OE group than that in
   the Vector group, whereas parameters representing trabecular separation
   (Tb.Sp) and the structural model index and (SMI) were notably elevated
   in the CTSZ-OE group (Fig. [92]2L). Collectively, these findings
   suggest that overexpression of CTSZ in tumor cells facilitates PCa
   metastasis in vivo.

CTSZ correlates with M2 macrophage enrichment in PCa microenvironment

   Prostate cancer is increasingly recognized as a process driven by
   immunosuppressive microenvironment reprogramming, characterized by
   tumor-associated macrophage (TAM) enrichment and cytotoxic T cell
   depletion [[93]3]. To investigate which cell subtype drives the
   immune-evasive phenotype, we performed single-cell transcriptomic
   profiling on metastatic PCa lesions ([94]GSE143791). This analysis
   revealed significantly higher macrophage infiltration in metastatic
   versus primary tumors (Fig. [95]3A and B). Strikingly, M2-polarized
   TAMs dominated these infiltrates (78% vs. 22% M1; Fig. [96]3C).

Fig. 3.

   [97]Fig. 3
   [98]Open in a new tab

   CTSZ correlates with M2 macrophage enrichment in PCa microenvironment.
   A Single-cell transcriptomic analysis ([99]GSE143971) of immune cell
   heterogeneity in localized vs metastatic PCa (Seurat v4.0). B, C
   Proportional distribution of 16 immune subsets (left) and M1/M2
   macrophage ratios (right; ***p < 0.001, χ^2 test). D ssGSEA enrichment
   scores for M1/M2 signatures in [100]GSE32269 cohort (n = 21 localized
   vs 29 metastatic; two-way ANOVA). E, F Top CTSZ-correlated genes (E)
   and M1/M2 marker correlations (F) in SU2C-mPCa cohort (cBioPortal). G
   CIBERSORT-ABS quantification of CTSZ-M2 macrophage association in
   TCGA-PRAD (TIMER2.0). H Multiplex IF staining of CTSZ^+ (magenta),
   CD206^+ (M2, red), and CD86^+ (M1, green) cells in clinical specimens
   (H&E: 100 μm; IF: 25 μm). Data presented as mean ± SD. Statistics:
   unpaired t-test (D, H), Pearson correlation (E, F). *p < 0.05,
   **p < 0.01; ns = not significant

   Using RNA-seq data from localized/metastatic PCa tissues
   ([101]GSE32269), we quantified 22 immune cell subsets and found M2
   macrophages to be the most differentially enriched cell type in
   metastatic specimens (Fig. [102]3D). The CTSZ-M2 axis was further
   validated in the SU2C metastatic PCa cohort, where CTSZ expression
   exhibited strong correlation with pan-macrophage marker CD68
   (P < 0.001) and preferential associations with canonical M2 markers
   (CD163, CD115, CD301, CSF1R, TGFβ1) over M1 signatures (TLR2, TNF,
   NOS2, CD80, CD86; Fig. [103]3E, F). CIBERSORT-ABS analysis of TCGA-PRAD
   data further confirmed that M2 macrophages showed the strongest
   correlation with CTSZ among all immune subsets (Fig. [104]3G, S2B).
   Spatial histopathological analysis (n = 38 localized vs 40 metastatic)
   demonstrated that metastatic PCa lesions exhibited elevated CTSZ
   expression concomitant with CD206^+ M2 macrophage enrichment,
   contrasting with concurrent reduction of CD86^+ M1 populations
   (Fig. [105]3H). These findings collectively position CTSZ as a
   molecular orchestrator of M2-dominated immunosuppressive niches in
   advanced PCa.

CTSZ-mediated M2 macrophage infiltration facilitates tumor metastasis

   To delineate the tumor cell-macrophage crosstalk mediated by CTSZ, we
   evaluated the impact of CTSZ-OE in tumor cells on the behavior of
   macrophages. In vitro co-culture models revealed that conditioned media
   from 22RV1-CTSZ-OE cells preferentially chemoattracted M2-polarized
   macrophages, with no significant effect on M1 subsets (Fig. [106]4A).
   To validate these findings in vivo, we investigated whether prostate
   cancer cells overexpressing CTSZ could recruit M2 macrophages. Western
   blot analysis of metastatic lesions confirmed elevated expression of
   the M2 macrophage marker arginase-1 (ARG1) and suppressed levels of the
   M1 marker TNF-α. These results suggest that CTSZ induces metabolic
   reprogramming favoring an M2-like polarization in the tumor
   microenvironment (Fig. [107]4B). Similarly. Immunofluorescence and
   immunohistochemical analyses of tumor tissues confirmed that CTSZ-OE
   tumors exhibited increased CTSZ expression, enhanced CD206 + M2
   macrophage infiltration, and reduced CD86 + M1 macrophage density
   compared to vector controls (Fig. [108]4C, E). To further validate the
   pivotal role of CTSZ-mediated M2 macrophages in promoting tumor
   metastasis, we established a lung metastasis model using CTSZ-OE PCa
   cells. Bioluminescence imaging (BLI) quantification revealed a
   significant reduction in pulmonary tumor fluorescence intensity in the
   clodronate-treated group compared to both untreated controls and
   liposome-only controls (Fig. [109]4F). Picric acid staining of isolated
   lungs confirmed fewer metastatic foci in the clodronate group (red
   arrows, Fig. [110]4G). The number of metastatic lesions is indicated by
   the red arrow and quantified in Fig. [111]4H for statistical analysis.
   Immunohistochemical analysis revealed that clodronate treatment
   attenuated epithelial-mesenchymal transition (EMT) markers (reduced
   CDH2, VIMENTIN) and Ki67 proliferation index, accompanied by restored
   E-cadherin (CDH1) expression (Fig. [112]4I). Collectively, these
   results demonstrate that CTSZ-driven PCa metastasis is critically
   dependent on M2 macrophage infiltration.

Fig. 4.

   [113]Fig. 4
   [114]Open in a new tab

   CTSZ-mediated M2 macrophage infiltration facilitates tumor metastasis.
   A Transwell migration of THP-1 cells toward PCa-conditioned media (CM:
   Vector vs CTSZ-OE; n = 3; mean ± SD; scale bar = 50 μm). B Immunoblot
   analysis of M2 (ARG1) and M1 (TNFα) markers in xenograft tumors. C -E
   Representative images of H&E staining, immunohistochemistry, and
   immunofluorescence staining for CD86 and CD206 in the CTSZ-OE and
   Vector groups. CD206 (Red), and CD86 (green) staining in the CTSZ-OE
   and Vector groups. Scale bar = 50 μm. F Longitudinal bioluminescence
   imaging (BLI) of systemic metastasis (n = 3/group). G Ex vivo lung
   metastasis. H Quantification of metastatic nodules (one-way ANOVA;
   unpaired t-test). I Representative image of immunohistochemistry for
   CDH1, CDH2, VIMENTIN and Ki67 in the Li-Clodronate, Liposome and
   CTSZ-OE groups. Scale bar = 50 μm. *p < 0.05; **p < 0.01. CM,
   conditioned medium

CTSZ^+PCa cells promote the recruitment of M2 macrophages via IL-32 secretion

   To investigated the molecular mechanisms by which CTSZ orchestrates M2
   macrophage recruitment, we performed transcriptomic profiling of
   CTSZ-knockdown (CTSZ-KD) versus control PC3 cells. RNA-seq analysis
   revealed distinct transcriptional alterations in cytokine-mediated
   signaling pathways (Fig. [115]5A, B). In the cytokine signaling
   pathway, ten secreted proteins were identified. Heatmap analysis of
   transcriptome sequencing data revealed that IL-32 exhibited the great
   fold change in expression and the lowest p-value among these proteins.
   (Fig. [116]5C, S3A). Simultaneously, qPCR and Western Blot experiments
   confirmed that knocking down CTSZ resulted in downregulation of IL-32
   expression in PC3 cells, while overexpression of CTSZ resulted in
   upregulation of IL-32 expression in 22RV1 cells (Fig. [117]5D–G). ELISA
   assays confirmed that IL-32 secretion was significantly altered in the
   supernatants of CTSZ-knockdown or -overexpressing cells compared to
   controls. There was no difference in secretion between CTSZ and known
   molecules that regulate macrophage infiltration, such as CXCL10 and
   CCL2 (Fig. [118]5H). Functional rescue experiments validated the
   necessity and sufficiency of IL-32 in CTSZ-driven macrophage
   chemotaxis. Conditioned media from CTSZ-OE cells enhanced macrophage
   migration, an effect abolished by IL-32 neutralization. Conversely,
   impaired macrophage recruitment in CTSZ-KD models was rescued by
   exogenous IL-32 supplementation (Fig. [119]5I, J). Collectively, these
   findings define a CTSZ-IL-32 paracrine axis that governs protumoral M2
   macrophage infiltration. Although IL-32 is established as the key
   mediator, the precise mechanisms underlying CTSZ-regulated IL-32
   secretion, including post-transcriptional modification or secretory
   pathway modulation, require further investigation.

Fig. 5.

   [120]Fig. 5
   [121]Open in a new tab

   CTSZ + PCa cells promote the recruitment of M2 macrophages via IL-32
   secretion. A Volcano plot of differentially expressed genes in PC3
   cells following CTSZ knockdown. B KEGG pathway enrichment analysis
   comparing shCTSZ and shNC groups in PC3 cells. C Hierarchical
   clustering heatmap of cytokine signaling-related genes identified in
   KEGG analysis. D–G Validation of CTSZ-mediated IL-32 regulation:
   qRT-PCR (D, E) and immunoblotting. H ELISA quantification of secreted
   IL-32, CTSZ, CCL2, and CXCL10 in CM from PC3 cells with CTSZ knockdown
   (shCTSZ) or 22RV1 overexpression (CTSZ-OE). I THP-1 macrophage
   migration assay: Comparative effects of CM from shNC, shCTSZ, and
   shCTSZ + IL-32 rescue conditions. J Transwell migration assay
   demonstrating IL-32-dependent chemotaxis: CM from Vector, CTSZ-OE, and
   CTSZ-OE + siIL32 groups. Data expressed as mean ± SD (n = 3 biological
   replicates). Significance determined by one-way ANOVA with post hoc
   Tukey test. *p < 0.05; **p < 0.01. CM: conditioned media

CTSZ degrades TRA2A protein expression through peptidase activity

   While intracellular CTSZ is established as a peptidase through
   C-terminal protein cleavage, our study uncovers a novel role for CTSZ
   in upregulating IL-32 expression and secretion in tumor cells. The
   positive correlation between CTSZ and IL-32 production implies an
   indirect regulatory mechanism. To investigate CTSZ-mediated proteolytic
   targets, comparative proteomics of 22RV1 Vector vs. CTSZ-OE groups
   identified 31 downregulated and 22 upregulated differential expressed
   proteins (Fig. [122]6A). Notably, TRA2A, a top-ranked
   CTSZ-downregulated RNA-binding protein, is a known suppressor of
   pre-mRNA splicing and mature mRNA biogenesis [[123]17]. CatRAPID
   analysis predicted TRA2A-IL-32 regulatory interactions (Figure S3C).
   Additionally, SU2C analysis revealed that negative correlations between
   TRA2A and both IL-32/CTSZ (Figure S3D, E, ). Functional validation in
   PCa cells showed that CTSZ knockdown increased TRA2A protein levels
   without altering mRNA expression (Fig. [124]6B, C), indicating
   post-transcriptional regulation. MALDI-TOF analysis of TRA2A-CTSZ
   co-incubation revealed enhanced proteolytic cleavage, with recombinant
   CTSZ (500 μg/mL) generating 31 specific peptide peaks versus 20 in
   controls (Fig. [125]6D). Octet-based dose–response assays showed
   concentration-dependent TRA2A degradation, with 10–50 nM activated CTSZ
   efficiently cleaving sensor-immobilized TRA2A (Fig. [126]6E). These
   findings conclusively establish CTSZ as a direct protease catalyzing
   TRA2A degradation.

Fig. 6.

   [127]Fig. 6
   [128]Open in a new tab

   CTSZ promotes the splicing and expression of IL-32 by degrading TRA2A.
   A Volcano plot of proteomic sequencing comparing 22RV1-Vector vs.
   22RV1-CTSZ-OE cells. B, C CTSZ knockdown validation: TRA2A mRNA (B) and
   protein (C) levels in PC3 cells. D MALDI-TOF MS analysis of
   CTSZ-mediated TRA2A proteolytic cleavage products. E Reflectometric
   interference spectroscopy quantification of CTSZ
   concentration-dependent TRA2A hydrolysis efficiency (Δλ shift). F In
   silico prediction of IL-32-TRA2A binding motifs via CatRAPID algorithm.
   G RIP-qPCR validation of TRA2A binding to IL-32 pre-mRNA regulatory
   sequences. H RNA pulldown assay demonstrating TRA2A interaction with
   IL-32 pre-mRNA probes. I Dual-color FISH co-localization of TRA2A
   (green) and IL-32 pre-mRNA (red). Scale bar = 10 μm. J EMSA showing
   nuclear protein binding affinity to IL-32 RNA probes: wild-type (WT),
   cold competitor (CC), and mutant (Mut) probes. K, L TRA2A knockdown
   effects on IL-32 expression: qRT-PCR K and immunoblot (L). The data are
   presented as the means ± SDs and were analyzed via two-tailed unpaired
   Student’s t test. Figure K was analyzed via one-way ANOVA; * p < 0.05;
   ** p < 0.01. ns: not significant

TRA2A binds to IL-32 precursor mRNA and inhibits its splicing

   To define the specific TRA2A binding sites on IL-32 pre-mRNA,
   computational prediction using CatRAPID identified three potential
   interaction regions (nt 602–767, 1668–1833, 2297–2489) in IL-32
   pre-mRNA (Fig. [129]6F). RNA immunoprecipitation (RIP) assays in PC3
   cells confirmed preferential enrichment of nt 602–767 and 2298–2489
   regions by TRA2A-specific antibodies compared to isotype controls
   (Fig. [130]6G). These regions contain a conserved GAAARGARR motif, a
   known recognition element for TRA2A’s RNA recognition motif (RRM) and
   arginine-rich domains [[131]17, [132]18]. Subsequently, RNA pull-down
   assays using biotinylated probes further validated direct interaction:
   TRA2A bound to wild-type nt 602–767 and 2297–2489 sequences but showed
   negligible binding to motif-mutated probes (Fig. [133]6H).
   Consistently, RNA fluorescence in situ hybridization (RNA FISH)
   revealed nuclear co-localization of TRA2A with IL-32 pre-mRNA at these
   motifs, with binding significantly reduced upon motif mutation
   (Fig. [134]6I). Moreover, Electrophoretic mobility shift assays (EMSA)
   showed stronger TRA2A binding to wild-type versus mutated or cold
   probes for both regions (Fig. [135]6J). Functional validation in PC3
   cells showed that TRA2A silencing significantly upregulated IL-32 mRNA
   and protein levels (Fig. [136]6K, L). Collectively, these results
   demonstrate that TRA2A binds to the GAAARGARR motif in IL-32 pre-mRNA
   to suppress splicing. Elevated CTSZ promotes IL-32 maturation and
   secretion by proteolytically degrading TRA2A, thereby relieving
   translational repression.

Targeting IL-32/ITGA5-PI3K/AKT axis disrupts protumoral M2 macrophage
recruitment

   While the cellular receptor for IL-32 remains undefined, previous
   literature indicates that all IL-32 isoforms contain an RGD motif.
   Given the canonical role of RGD motifs in integrin binding, we
   performed integrin interaction screening in SU2C-metastatic PCa
   cohorts. This analysis identified ITGA5 as the strongest correlate of
   IL-32 expression, establishing a potential ligand-receptor relationship
   (Spearman = 0.43; Fig. [137]7A). Biochemical validation via co-IP
   confirmed direct IL-32/ITGA5 binding, which was RGD-dependent as GRGDNP
   treatment reduced affinity (Fig. [138]7B, C). Molecular docking
   (Gram-X) identified high-affinity interaction (ΔG = − 18.5kcal/mol)
   through LEU706/THR38 hydrogen bonds (Fig. [139]7D).

Fig. 7.

   [140]Fig. 7
   [141]Open in a new tab

   Targeting IL-32/ITGA5-PI3K/AKT axis disrupts protumoral M2 macrophage
   recruitment. A Correlation analysis of IL-32 and RGD-binding integrin
   receptors in the SU2C metastatic prostate cancer cohort (cBioPortal).
   B, C IL-32/ITGA5 interaction validation: (B) Co-immunoprecipitation
   (Co-IP) of IL-32 and ITGA5 in THP-1 macrophage lysates. C RGD motif
   dependency confirmed via GRGDNP inhibitor treatment. D Computational
   docking model of IL-32(Blue)/ITGA5(Yellow) complex. E KEGG pathway
   enrichment of ITGA5-associated genes in TCGA-PRAD cohort. F GLPG0187
   dose-dependent effects on macrophage polarization: Immunoblot analysis
   of M1 (iNOS) and M2 (CD206) markers, PI3K/AKT pathway inhibition
   assessed by p-AKT (Ser473) levels. G Bioluminescence imaging (BLI) of
   lung metastasis in mice injected with 22RV1-CTSZ-OE cells ± GLPG0187
   (n = 4/group). H Metastatic burden quantification: (H) Sirius
   Red-stained lung sections showing metastatic nodules (yellow arrows). I
   H&E quantification of lung metastases (blue arrows). J Statistical of
   tumor metastasis numbers. K Multiplex immunofluorescence of CD206 (M2,
   red), CD86 (M1, green), and DAPI (blue) in lung metastases. L IHC
   validation of CD206/CD86 expression across treatment groups. The data
   are presented as the means ± SDs and were analyzed via one-way ANOVA;
   *p < 0.05; **p < 0.01. ***p < 0.01; ns: not significant. 50 μm ( K, L);
   2.5 mm (I). BE: binding energy

   Activation of the ITGA5-PI3K/AKT signaling axis has been established to
   promote tumor progression [[142]19–[143]22]. KEGG pathway analysis of
   ITGA5-associated differentially expressed genes in TCGA-PRAD confirmed
   PI3K/AKT as the top enriched signaling pathway (Fig. [144]7E).
   Pharmacological inhibition with GLPG0187 demonstrated dose-dependent
   suppression of PI3K/AKT phosphorylation and M2 macrophage polarization,
   with maximal inhibitory efficacy observed at a concentration of 40
   ng/mL (Fig. [145]7F). To explore the effect of GLPG0187 on ITGA5 and
   downstream signaling in PCa cells. Western blot analysis revealed no
   significant changes in ITGA5 protein expression across all treatment
   groups compared to the wildtype cells (Figure S4A). Concomitantly,
   phosphorylation levels of PI3K/AKT (pPI3K/AKT) remained unchanged in
   GLPG0187-treated cells, as confirmed by densitometric quantification
   (Figure S4B). In vivo validation using a tail vein injection model with
   CTSZ-OE PCa cells showed that GLPG0187(100 mg/kg) treatment
   significantly reduced lung metastasis burden, as confirmed by
   bioluminescence imaging (Fig. [146]7G). Compared with the untreated and
   PBS-treated groups, the GLPG0187-treated group exhibited significantly
   reduced PCa lung metastasis with bitter acid and HE staining
   (Fig. [147]7H–J). Additionally, immunofluorescence and
   immunohistochemical analyses of lung tumor sections revealed that
   GLPG0187 markedly decreased CD206^+ M2 macrophage infiltration while
   increasing CD86^+ M1 macrophage populations (Fig. [148]7K, L).
   Collectively, our data suggest that the ITGA5/PI3K axis in macrophages
   mediates CTSZ-induced M2 macrophage infiltration and PCa metastasis.

Discussion

   Our findings elucidate a non-canonical role for CTSZ in driving
   prostate cancer metastasis through orchestration of M2-polarized
   macrophage recruitment. Multi-omics analyses established CTSZ as a
   metastasis-associated protease that remodels the immunosuppressive
   niche without intrinsic tumor cell effects. Mechanistically,
   CTSZ-mediated degradation of the RNA splicing repressor TRA2A licenses
   IL-32 isoform maturation, which engages macrophage ITGA5 via its RGD
   motif. This interaction triggers PI3K/AKT-dependent chemotaxis and M2
   macrophage infiltration. Therapeutic disruption of this axis using
   ITGA5 inhibitor GLPG0187 significantly attenuated lung metastasis in
   vivo, validating the CTSZ/TRA2A/IL-32/ITGA5 pathway as a druggable
   effector of metastatic progression.

   While the cathepsin family has been extensively studied for its matrix
   metalloproteinase (MMP)-dependent roles in extracellular matrix (ECM)
   remodeling and tumor invasion [[149]8], CTSZ represents a functional
   dichotomy. As a carboxypeptidase lacking direct ECM degradation
   capacity [[150]23], CTSZ exerts dual functionality through its unique
   structural determinants: (1) The extracellular RGD motif facilitates
   integrin receptor binding to modulate cell adhesion/migration; (2)
   Intracellularly, it serves as a key lysosomal enzyme mediating
   proteolytic processing of PolyQ peptides, chemokines (CXCL12), and
   pathogenic proteins (huntingtin) in disease contexts [[151]12,
   [152]24–[153]27]. This dual nature positions CTSZ as a molecular
   rheostat coordinating extracellular signaling and intracellular
   proteostasis. Despite documented CTSZ upregulation in multiple
   malignancies, its tumor-intrinsic regulatory mechanisms remain
   incompletely understood. Current paradigms emphasize CTSZ-integrin
   interactions in regulating angiogenesis and metastatic dissemination,
   yet critical knowledge gaps persist regarding its tumor
   microenvironment (TME) remodeling capacity. Seminal work by Akkari et
   al. revealed macrophage-derived CTSZ promotes pancreatic cancer
   invasion via tumor cell integrin engagement [[154]9]. Contrastingly,
   our findings establish an inverse regulatory axis: Tumor cell-intrinsic
   CTSZ orchestrates TME immunosuppression through
   TRA2A/IL-32/ITGA5-mediated M2 macrophage recruitment—a mechanism
   substantiated by three lines of evidence. First, CTSZ-mediated TRA2A
   degradation relieves suppression on IL-32 mRNA splicing. Second,
   secreted IL-32 activates macrophage ITGA5/PI3K signaling to drive M2
   macrophage infiltration. Third, pharmacological ITGA5 inhibition
   reverses CTSZ-driven metastasis in vivo.

   The TRA2A/IL-32/ITGA5 signaling axis uncovered in this study introduces
   new dimensions to cancer immunology, yet warrants deeper mechanistic
   interrogation. While IL-32 exists as nine isoforms with divergent
   tumor-modulatory roles [[155]28], our focus on its
   macrophage-polarizing function aligns with consensus findings in
   esophageal cancer and myeloma, where IL-32β/γ isoforms drive M2
   polarization [[156]29]. Importantly, we identified TRA2A as a
   gatekeeper of IL-32 mRNA maturation—a regulatory layer distinct from
   canonical transcriptional control. Three lines of evidence substantiate
   this paradigm: (1) Transcriptome profiling of CTSZ-knockdown PCa cells
   revealed IL-32 downregulation, implicating post-transcriptional
   regulation; (2) Proteomic analysis pinpointed TRA2A among top
   CTSZ-downregulated RBPs (RNA-binding proteins); (3) Biochemical
   validation confirmed CTSZ-mediated TRA2A proteolysis and its steric
   hindrance on IL-32 pre-mRNA splicing via conserved GAAARGARR motif
   binding. We detected IL-32mRNA levels in non-tumoral prostate
   epithelial cells (RWPE-1) and prostate cancer cells (PC3, DU145)
   (Figure S3B). The results revealed that IL-32 expression was
   significantly lower in RWPE-1 cells compared to PC3 and DU145 cells.
   While this confirms minimal IL-32 contribution from non-tumoral
   epithelium, we did not assess IL-32 expression in other TME components,
   such as immune or stromal cells. This finding helps to preliminarily
   exclude the potential influence of non-cancerous IL-32 on our
   experimental results.

   Current research on ITGA5 predominantly explores its translational
   potential in clinical therapeutics, with GLPG0187 and volociximab
   emerging as promising targeted agents. Preclinical evaluation through a
   phase I clinical trial demonstrated GLPG0187’s inhibitory effects on
   tumor progression and metastasis across multiple solid tumor models
   [[157]30]. Notably, while volociximab has exhibited therapeutic
   efficacy in metastatic lung and breast cancer management [[158]31,
   [159]32], emerging evidence highlights GLPG0187’s specific potency in
   suppressing osseous metastasis of prostate carcinoma [[160]33]. Our
   novel finding through orthotopic prostate tumor analysis reveals M2
   macrophages as the primary cellular target of GLPG0187, providing a
   distinct mechanistic perspective. It is noteworthy that neither
   compound has entered clinical application for prostate cancer
   treatment, thereby limiting direct assessment of their therapeutic
   efficacy in this malignancy. This evidence gap underscores the
   significance of our study in establishing theoretical foundations for
   future clinical translation of these agents in prostate cancer
   management. Our findings position the CTSZ/IL-32/ITGA5 axis as a novel
   immunomodulatory target distinct from current macrophage-focused
   therapies. While CSF1R inhibitors aim to deplete TAMs broadly, their
   clinical efficacy in solid tumors has been limited by compensatory
   immune evasion and stromal resistance [[161]34]. In contrast, targeting
   ITGA5 specifically disrupts CTSZ-driven M2-TAM recruitment without
   globally suppressing myeloid populations, potentially preserving
   antitumor immunity mediated by dendritic cells and M1 macrophages.
   Furthermore, the observed immunotherapy resistance in prostate cancer,
   characterized by diminished immune cell infiltration (excluding
   macrophages) as evidenced by our database analysis, suggests
   macrophage-mediated immune evasion mechanisms. This finding reinforces
   the therapeutic rationale for targeting tumor-associated macrophages
   within the tumor microenvironment to overcome treatment resistance.
   From a diagnostic perspective, the potential utility of CTSZ as a
   circulating biomarker for metastatic prostate cancer warrants further
   investigation. We propose that integrating macrophage-targeted
   therapeutic strategies with existing treatment modalities may yield
   synergistic antitumor effects, offering new avenues for combinatorial
   therapy development.

Conclusion

   This study elucidates a CTSZ-driven metastatic axis in cancer. Tumor
   cell–expressed CTSZ promotes TRA2A degradation, thereby unleashing
   IL-32-mediated activation of macrophage ITGA5/PI3K signaling to
   orchestrate protumoral M2 macrophage infiltration. Therapeutically,
   targeting this axis with GLPG0187 specifically disrupts tumor
   microenvironment (TME) immunosuppression by blocking ITGA5 in M2
   macrophages. This mechanism differs from conventional tumor
   cell–directed therapies. These findings not only reveal a novel
   proteolytic–immune crosstalk pathway governing metastasis but also
   establish ITGA5 inhibition as a clinically actionable strategy for
   reprogramming the metastatic niche.

Supplementary Information

   [162]12967_2025_6865_MOESM1_ESM.jpg^ (2MB, jpg)

   Supplementary Material 1: Figure S1..Expression of COCH in GSE32269 and
   GSE101607 datasets.Frequency of genomic alterations in the CTSZ in nine
   prostate cancer genomic sequencing datasets from the cBioPortal
   database. Relationships between CTSZ genomic alterations and overall
   patient survival.Survival analysis of patients in gene amplification
   group and non- amplification group. Data presented as mean ± SD.
   Statistics: unpaired t-test; *p<0.05
   [163]12967_2025_6865_MOESM2_ESM.jpg^ (2.8MB, jpg)

   Supplementary Material 2: Figure S2..Western Blot assay to verify the
   efficiency of CTSZ overexpression in RM1 cells.Validation of CTSZ
   expression and its correlation with T cell, B cell, and Treg cell
   infiltration in the TIMER2.0 database
   [164]12967_2025_6865_MOESM3_ESM.jpg^ (1.9MB, jpg)

   Supplementary Material 3: Figure S3. TRA2A may be a key protein that
   negatively regulates IL-32 expression.Original expression matrix of 10
   differentially secreted proteins in the cytokine signaling pathway of
   transcriptome sequencing.Top ten ranked RNA-binding protein prediction
   scores for IL-32.Correlation between IL-32 and TRA2A levels in the
   SU2C-mPCa database.Correlation between TRA2A and CTSZ expression in the
   SU2C-mPCa database.Transwell experiment detects the effect of
   conditioned medium overexpressing TRA2A on M2 macrophage recruitment in
   cells overexpressing CTSZ
   [165]12967_2025_6865_MOESM4_ESM.jpg^ (850.6KB, jpg)

   Supplementary Material 4: Figure S4. GLPG0187 did not affect the
   expression of ITGA5 and activation of the PI3K/AKT pathway in PC3
   cells.Western Blot experiment verifies the expression of ITGA5 in PC3
   treated with different concentrations of GLPG0187.Western Blot
   experiment verifies the changes in PI3K/AKT phosphorylation levels of
   PC3 treated with different concentrations of GLPG0187

Acknowledgements