Abstract

Background

   The aberrant upregulation of PVT1 in gastric cancer (GC) has emerged as
   a critical molecular marker, while its mechanism of remodeling the
   tumor microenvironment (TME) through metabolic regulation remains
   unclear. This study aims to unveil the novel mechanism by which PVT1
   regulates the fate determination of tumor-associated neutrophils (TANs)
   via the calcium signaling pathway.

Methods

   Clinical analysis of 90 GC cases revealed a correlation between PVT1
   expression and neutrophil infiltration in tumor tissues.
   Mechanistically, multi-omics integration of transcriptomics and
   4D-label-free quantitative proteomics identified PVT1-associated
   pathways driving GC progression. Functional validation through in vitro
   and in vivo models demonstrated PVT1’s oncogenic role, while
   transcriptomic profiling further decoded neutrophil activation
   mechanisms regulated by PVT1.

Results

   The expression of PVT1 was significantly elevated in GC tissues
   compared to adjacent non-tumor tissues (p < 0.001), with 73.3% of cases
   showing an increased proportion of PVT1-positive cells. High PVT1
   expression negatively correlated with the infiltration of neutrophils
   (r = − 0.3554, p = 0.0012), and patients in the PVT1 + CD66b- subgroup
   exhibited the poorest prognosis, characterized by a median survival of
   20.5 months and a mortality rate of 72.5%. Silencing PVT1 upregulated
   calcium signaling pathway-related genes (RYR2 and RYR3) in GC cells,
   accompanied by a marked increase in intracellular Ca^2⁺ concentration
   (p < 0.01). Proteomic analysis revealed that PVT1 knockdown
   significantly enhanced the secretion of synaptopodin (SYNPO), which
   demonstrated co-expression and physical interactions with calcium
   channel proteins RYR2/RYR3. PVT1 knockdown markedly suppressed GC cell
   proliferation, migration, and invasion (p < 0.05) while elevating the
   expression of γ-H2AX (p < 0.05). In xenograft models, tumors with PVT1
   knockdown displayed reduced volume and decreased Ki67 expression
   (p < 0.01). Conditioned medium from PVT1-knockdown GC cells promoted
   neutrophil activation, delayed apoptosis, and amplified intracellular
   Ca^2⁺ signaling. Exogenous SYNPO supplementation mimicked these effects
   and attenuated neutrophil replicative senescence by modulating p53
   pathway-associated genes (TP53, CHEK1, and SERPINE1).

Conclusions

   Overall, this study provides the first evidence that PVT1 establishes a
   metabolic checkpoint by inducing calcium overload, thereby regulating
   both the senescence process and functional phenotype of neutrophils,
   which provides a novel metabolic intervention target for GC
   immunotherapy.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12967-025-06912-6.

   Keywords: Gastric cancer, PVT1, Tumor-associated neutrophils, Calcium
   signaling pathway, Replicative senescence

Introduction

   Gastric cancer (GC) remains one of the most prevalent malignancies,
   ranking fifth in incidence and fourth as cause of cancer death globally
   [[40]1]. Although chemotherapy has been the primary treatment for
   advanced GC for many years, we are now entering an era of more targeted
   drugs targeting abnormally expressed genes and signaling pathways
   [[41]2–[42]5]. Efficient cancer management heavily relies on the
   identification and investigation of key mediators involved in
   tumorigenesis. With the advent of whole-transcriptome sequencing (WTS)
   and computational sciences, profound insights into non-coding RNAs have
   been gained [[43]6]. Abnormally expressed lncRNAs might be potential
   molecular biomarkers for GC diagnosis. Plasmacytoma variant
   translocation 1 (PVT1), which is located on chromosome 8q24, was found
   to be amplified in double minutes of GC cells and overexpressed in GC
   tissues [[44]7, [45]8]. The level of PVT1 in tissues or in serum can
   help to assess the risk of lymphatic metastasis in GC patients [[46]9,
   [47]10]. PVT1 could also regulate the viability, invasion and cell
   cycle progression of GC cells as a ceRNA [[48]11]. Besides,
   overexpressed PVT1 could enhance the 5-fluorouracil resistance and
   anti-apoptosis of GC cells [[49]12]. These findings that elucidate the
   regulatory mechanisms of PVT1 may provide novel insights for the
   therapeutics of tumors. However, the exact mechanism of PVT1 in GC
   still needs to be elucidated.

   Cancer-related inflammation serves as the primary driver of tumor
   initiation and progression, facilitating cancer cells to evade immune
   surveillance and infiltration of diverse immune cells in tumor
   microenvironment (TME) has been demonstrated to play a pivotal role in
   tumorigenesis [[50]13, [51]14]. Tumor-associated neutrophils (TANs) are
   defined as neutrophils that have infiltrated the TME and constitute a
   significant component of the inflammatory milieu within tumors. The
   presence of neutrophil infiltration in tumorigenesis is widely
   recognized as a crucial factor governing tumor progression,
   angiogenesis, and modulation of immune responses [[52]15, [53]16]. TANs
   exhibit diverse associations, either promoting or inhibiting the
   progression of various cancers [[54]17]. The study suggests that
   neutrophils may undergo phenotypic transformation in response to
   specific cytokines or metabolites within the TME, thereby directly
   influencing on tumor cell proliferation and metastasis [[55]18].

   The occurrence and prognosis of GC are intricately associated with the
   infiltration of immune cells within the TME. An increase in infiltrated
   immune cells is indicative lower incidence of lymph node metastasis and
   overall survival for GC patients [[56]19]. Notably, elevated levels of
   neutrophil infiltration have been observed in the cancerous tissues of
   individuals diagnosed with GC, accompanied by an extended lifespan of
   neutrophils activated by GC cells [[57]20]. Senescence-targeting may
   ultimately affect neutrophil states. In turn, the identification of
   specific senescence-associated neutrophil subsets may open a new avenue
   of treatments that limit senescence and cancer by targeting
   neutrophils, without affecting neutrophilic response against pathogens
   [[58]21]. An increasing number of studies have unveiled the involvement
   of abnormal gene expression in GC in regulating neutrophil infiltration
   [[59]22–[60]24]. Studies indicate that, when constructing a risk
   prediction model for GC based on TCGA dataset, PVT1 was identified as a
   crucial gene for predicting immune cell infiltration (T cells,
   monocytes, and neutrophils) and patient prognosis in GC [[61]25].

   Therefore, gaining a comprehensive understanding of the regulatory role
   of neutrophils within the TME in GC and exploring the association
   between PVT1 and neutrophils could offer novel insights for therapeutic
   interventions and prognostic assessments in GC.

Materials and methods

Gastric cancer tissues chip of patients, blood in healthy population

   The tissue chip consisted of 90 cases of human gastric cancer tissues
   and their corresponding adjacent tissues, totaling 180 sites (number:
   HStmA180Su11, lot number: XT17-035), obtained from Shanghai Outdo
   Biotech Co., Ltd. (Shanghai, China). The chip was accompanied by
   matching HE staining information. Among the samples, there were 32
   patient fatalities and 58 survivors. The peripheral blood samples were
   obtained from 10 healthy subjects at the Second Affiliated Hospital of
   Harbin Medical University following routine examination, collected in
   EDTA-K3 anticoagulant tubes. This study received ethical approval from
   the Medical Ethics Committee of the Second Affiliated Hospital of
   Harbin Medical University prior to commencement (Approval number:
   YJSKY-2022-517).

Cell culture, transfection and treatment

   The human gastric mucosal epithelial cell line GES-1 and the human
   gastric cancer cell lines HGC-27, AGS, NCI-N87 and MKN-45 were
   maintained in accordance with a previous report [[62]26]. GC cell line
   SNU-16 was purchased from American Type Culture Collection (ATCC,
   Manassas, VA, USA) and cultured in RPMI-1640 (Gibco BRL, Waltham, MA,
   USA) with 10% fetal bovine serum at 5% CO[2] in a 37 ℃ incubator. The
   genotypically validated of cell lines were authenticated by Beijing
   Microread Genetics Co., Ltd. (Beijing, China) using short tandem repeat
   (16-STR) analysis. The antisense oligonucleotide (ASO) for PVT1 and
   negative control (NC) were purchased from Guangzhou RiboBio Co., Ltd.
   (lnc6180719010843-1-5, Guangzhou, China). Cells were seeded in six-well
   plates, and transfected with PVT1-ASO or NC using riboFECTTMCP
   Transfection kit (C10511-1, RiboBio, Guangzhou, China) according to the
   instructions. The transfection efficiency was measured by real-time PCR
   at 48 h and 72 h after transfection. For tumorigenicity assay in vivo,
   lentiviral particles for PVT1-shRNA(Catalog #:
   LPP-CS-SH3617-LVRU6GP-a-500) and for negative control (Catalog #:
   LPP-CSHCTR001-LVRU6GP-500) were purchased and infected to HGC-27 cells
   using the Lentiviral granulosa Cell Infection Experiment User's Manual
   from GeneCopoeia, Inc. Recombinant Human TNF-α was purchased from
   Beyotime Biotechnology (p5318, Shanghai, China) and recombinant Human
   SYNPO protein was purchased from Zeye Biotechnology (ZY685Hu01P,
   Shanghai, China).

Isolation, extraction, and identification of neutrophils from healthy
individuals

   The isolation of peripheral blood neutrophils was conducted following
   the protocol from the human peripheral blood neutrophil isolation kit
   (Solarbio, P9040, Beijing, China). Briefly, 4 mL of reagent A was
   layered with 2 mL of reagent C in a 15 mL tube, followed by overlaying
   5 mL of anticoagulated blood. The mixture was centrifuged at 2500 rpm
   for 30 min at room temperature to separate the mononuclear cell layer
   (upper) and neutrophil layer (lower). Neutrophils were aspirated,
   washed with 10 mL washing solution (1200 rpm for 10 min), lysed with
   2 mL lysis buffer for 10 min, neutralized with 5 mL washing solution,
   centrifuged, and resuspended in culture medium.

   After isolation, cell viability was assessed using the Trypan Blue
   staining kit (Beyotime, C0011, Shanghai, China) and an automated cell
   counter (LUNA-II™, Logos Biosystems, Shanghai, China). Neutrophil
   purity was determined by Wright’s stain solution (Solarbio, G1040,
   Beijing, China), followed by observation and photography under an
   optical microscope (ZEISS Axio Vert.A1, Germany).

Dual hybridization of fluorescence in situ hybridization (FISH) and
Immunofluorescence (IF)

   To account for inter-individual variability in GC patients, the
   percentage of positive cells was used to evaluate the expression levels
   of PVT1 and neutrophil infiltration in both tumor tissues and adjacent
   tissues obtained from the same GC patient. The tissues were
   deparaffinized and subjected to antigen retrieval according to standard
   protocols. Subsequently, the samples were digested with proteinase K,
   pre-hybridized, and hybridized with a PVT1 fluorescent probe (1:67
   dilution, Genepharma) combined with the RNA FISH SA-Biotin
   amplification system kit (Genepharma). The tissues area was sealed at
   room temperature, followed by the addition of the anti-CD66b primary
   antibody (1:500, Abcam, ab300122). After overnight hybridization at
   4 ℃, incubation with FITC-labeled goat anti-rabbit secondary antibody
   (1:500, Immunoway, RS0004) was performed. Finally, the DAPI dye
   solution was added. The tissues were examined using a fluorescence
   microscope (NIKON, YS100, Japan) and images were acquired.
   CD66b-positive cells emitted green fluorescence, PVT1 produced red
   fluorescence, and DAPI-stained nuclei showed blue fluorescence.

Whole transcriptome sequencing (WTS)

   WTS was conducted by Shanghai Outdo Biotech Co., Ltd. (Shanghai,
   China). Briefly, the total RNA was extracted using the RNeasy Mini Kit
   (Qiagen, 74106, Germany). Sequencing libraries were generated using
   VAHTS^™ Stranded mRNA-seq Library Prep Kit for Illumina (Vazyme, NR612,
   Nanjing, China) according to the manufacturer’s instructions. The
   sequencing was performed on Illumina NovaSeq 6000 platform (Illumina,
   San Diego, CA, USA). Raw data were processed through Seqtk. In this
   step, the raw data were cleaned by removing adapter sequences,
   contaminant reads, and low-quality bases. Additionally, the Q30, and GC
   content were calculated to estimate the quality of clean reads. Next,
   the clean reads were aligned to the reference genome (GRCh38) using
   HISAT2. The Read Counts of transcripts and ncRNAs were calculated by
   String Tie. And the expression of mRNA and lncRNA was normalized to
   FPKM. FPKM refers to the number of fragments per kilobase length from a
   gene per million fragments mapped considering the effect of both
   sequencing depth and gene length on fragments count. The sequencing
   depth (in total reads) of WTS is summarized in Table S1.

4D-Label-free quantitative proteome analysis by LC–MS/MS

   LC–MS/MS was conducted by Shanghai Outdo Biotech Co., Ltd. (Shanghai,
   China). It comprised protein extraction and peptide hydrolysis, as well
   as LC–MS/MS data collection. The samples were lysed by SDT (4% (w/v)
   SDS, 100 mM Tris–HCl pH 7.6, 0.1 M DTT). Each sample was separated
   using Brucker nanoElute, a ultra performance liquid chromatography
   (UPLC) liquid phase system with a nanoliter flow rate. Samples
   separated by nanoflow ultra-high performance liquid chromatography
   (nanoUHPLC) were analyzed by data-dependent acquisition (DDA) mass
   spectrometry using timsTOF Pro mass spectrometer (Bruker). The
   detection mode was positive ion mode, and the ion source voltage was
   set to 1.5 kv. Time of flight (TOF) was used for detection and analysis
   of MS and MS/MS. The mass spectrum scanning range was set to
   100–1700 m/z. Parallel accumulation aerial fragmentation (PASEF) mode
   was used for data acquisition.

Real time quantitative polymerase chain reaction (Real-time PCR)

   Total RNA were extracted from cells using the SevenFast^™ Total RNA
   Extraction Kit (SEVEN, SM130, Beijing, China), and first-strand cDNA
   was synthesized using the All-in-one First Strand cDNA Synthesis Kit
   (SEVEN, SM131, Beijing, China) according to the manufacturer’s
   protocol. The cDNA was subjected to real-time PCR in SLAN-96P real-time
   PCR system (Hongshitech, Shanghai, China) using 2 × SYBR Green qPCR
   Master Mix (SEVEN, SM133, Beijing, China) with fluorescence detection.
   Genes expression levels were normalized to β-actin by the 2^−△△Ct
   method. The sequence of primers are listed in Table S2.

Western blot analysis

   Total protein was extracted from the GC cells using RIPA buffer
   (Beyotime, P0013B). The protein samples were separated by sodium
   dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and then
   transferred onto polyvinylidene difluoride (PVDF) membranes. The
   membranes were blocked with 5% nonfat milk for 2 h at room temperature
   and then hybridized with primary antibodies: anti-Synaptopodin
   (1:1,000; Affinity, DF12173), anti-MMP9 (1:1,000; Beyotime, AF5234) and
   anti-β-actin (1:10,000; Affinity, AF7018) overnight at 4 °C, followed
   by incubation with a horseradish peroxidase HRP-conjugated goat
   anti-rabbit secondary antibody (1:10,000; ZSGB-BIO, ZB-5301). Blots
   were imaged using chemiluminescence imaging system (ChemiScope 6300,
   CLiNX).

CCK-8 assay and cell viability analysis

   Cells were seeded into 96-well plates at a density of 2000 cells per
   well. Cell proliferation was monitored daily over 5 consecutive days,
   while viability was assessed at 16 h or 24 h using the Cell Counting
   Kit-8 (SEVEN, SC119, Beijing, China) according to the manufacturer’s
   protocol. Optical density (OD) was measured at 450 nm using a
   microplate reader (BIO-RAD, iMark, Japan). Each experiment included six
   biological replicates, and data are expressed as mean ± standard
   deviation (SD).
   graphic file with name d33e590.gif

Colony formation assay

   Cells were seeded in 6-well plates at a density of 200 cells per well.
   Following 10 days of culture, the medium was removed, and cells were
   washed twice with phosphate-buffered saline (PBS). Subsequently, cells
   were fixed with methanol for 20 min at room temperature, followed by
   PBS washing and staining with Giemsa solution (Beyotime, C0133,
   Shanghai, China) for 30 min. The assay was performed in three
   independent replicates. Colonies were imaged and quantified with ImageJ
   software.

Wound healing assay and transwell migration assay

   Cell migration ability was evaluated through wound healing and
   transwell assays.

Wound healing assay

   Cells were seeded in 6-well plates and cultured until reaching 90%
   confluency. A uniform scratch was created using a sterile 200 μL
   pipette tip. After washing with PBS to remove detached cells, cells
   were maintained in medium containing 2% FBS (to suppress
   proliferation). Wound closure was monitored at 0 h and 24 h using an
   inverted microscope (ZEISS, Axio Vert.A1, Germany).

Transwell migration assay

   Transwell chambers (8 μm pore size, Corning, NY, USA) were placed in a
   24-well plate. The upper chamber was loaded with 200 μL serum-free
   medium containing 3 × 10^4 cells, while the lower chamber contained
   800 μL medium with 10% FBS as a chemoattractant. After 24 h incubation,
   non-migrated cells on the upper membrane surface were removed with a
   cotton swab. Migrated cells on the lower surface were fixed with 4%
   methanol for 20 min, stained with Giemsa solution (Beyotime, C0133,
   China) for 30 min, and imaged under an optical microscope (ZEISS, Axio
   Vert.A1, Germany).

IF, apoptosis/necrosis, and Fluo-4 calcium assays

IF

   Cells were cultured in 6-well plates. DNA damage was assessed using the
   DNA Damage Assay Kit (Beyotime, C2035S) according to the manufacturer’s
   protocol. For protein detection, cells were fixed with 4%
   paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked
   with 5% BSA. Subsequently, cells were incubated with anti-γ-H2AX
   antibody (Beyotime, C2035S-4), anti-SYNPO (1:500, Affinity, DF12173),
   anti-SERPINE1 (1:200, Immunoway, YT3569), anti-TP53 (1:200, Immunoway,
   YT3528) and anti-CHEK1 (1:200, Immunoway, YT0902) overnight at 4 °C.
   After washing, cells were incubated with either Alexa Fluor
   488-conjugated anti-rabbit IgG (Beyotime, C2035S-5) or Goat Anti Rabbit
   IgG (H + L) (AbFluor 594) (1:200, Immunoway, RS3611) for 1 h at room
   temperature. Cells were counterstained with DAPI (Beyotime, C1002) or
   Phalloidin (YEASEN, 40774ES03) and imaged using a fluorescence
   microscope (NIKON, YS100, Japan).

Apoptosis and necrosis assay

   Cell apoptosis and death was analyzed using the Apoptosis and Necrosis
   Assay Kit (Beyotime, C1056). Briefly, cells were stained with Hoechst
   33,342 (10 μg/mL) and propidium iodide (PI) (5 μg/mL) in staining
   buffer for 20 min at 37 °C. After PBS washing, apoptotic
   (Hoechst-positive) and necrotic (PI-positive) cells were quantified
   under a fluorescence microscope (NIKON, YS100, Japan).

Fluo-4 calcium assay

   Intracellular calcium levels were measured with the Fluo-4 Calcium
   Assay Kit (Beyotime, S1061S). Cells were loaded with Fluo-4 AM in
   staining solution at 37 °C for 30 min. Green fluorescence signals
   (Ex/Em = 490/525 nm) were captured using a fluorescence microscope
   (NIKON, YS100, Japan). Data were normalized to baseline fluorescence
   intensity.

Tumorigenicity assay in vivo

   Female BALB/c Nude mice aged 4–6 weeks were purchased from Beijing
   Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and
   were housed under pathogen-free conditions. Mice were randomly divided
   into NC groups (n = 5) and experimental groups (n = 5). Subcutaneous
   injections of HGC-27 cells (1 × 10^7) with or without stable PVT1
   silencing were administered to establish xenografts. Tumor growth was
   monitored for 4 weeks before mice were sacrificed. Mice were sacrificed
   by cervical dislocation and tumor tissues were obtained for
   pathological examination. Paraffin sections of tumors were prepared by
   conventional methods, and the tumor tissues were observed and examined
   via hematoxylin and eosin (HE) staining. Ki67 expression was assessed
   by immunohistochemistry using an anti-Ki67 antibody (1:100, Affinity,
   AF0198) in tumor tissues among different groups. All animal studies
   were performed in accordance with the ethical principles of animal
   protection and welfare, and approved by the Ethics Committee of the
   Second Affiliated Hospital of Harbin Medical University (Ethical review
   approval number: YJSDW2022-252).

Statistical analysis

   Data are expressed as mean ± SD and analyzed using GraphPad Prism
   software (v10.1.2). Comparisons between two groups were performed using
   the Wilcoxon rank-sum test or Student’s t-test. A p < 0.05 was
   considered statistically significant unless otherwise stated. Sequence
   data visualization was generated by
   [63]https://www.bioinformatics.com.cn (last accessed on 20 Feb 2023),
   an online platform for data analysis and visualization.

Results

PVT1 expression negatively correlates with neutrophil infiltration and
predicts poor prognosis in gastric cancer

   The correlation between PVT1 and neutrophil infiltration was observed
   in gastric cancer (GC). The somatic copy number alterations (SCNA)
   analysis module of the TIMER database was utilized to compare
   neutrophil infiltration levels in GC tissues with different PVT1 SCNA
   statuses. Paradoxically, high amplification, arm-level gain and
   deletion of PVT1 all seem to be associated with the decreased
   neutrophil infiltration compared to the diploid/normal status
   (Fig. [64]1A). Therefore, RNA FISH for PVT1 and IF for CD66b
   (Neutrophils marker) were performed in 90 GC cases and paired adjacent
   tissues to investigate the differential expression of PVT1 and
   neutrophil infiltration (Fig. S1A). Notably, CD66b exhibited robust
   specificity for neutrophils in GC tissues (Fig. S1B). The findings
   demonstrated a significant increase in the proportion of PVT1-positive
   cells in GC tissues compared to non-adjacent cancerous tissues
   (Fig. [65]1B, p < 0.001), and 66 (73.3%) of the patients exhibited a
   higher abundance of PVT1-positive cells in GC tissues compared to the
   adjacent tissues (Fig. [66]1D). In contrast, 53 (61.1%) of the patients
   showed fewer CD66b-positive cells in GC tissues compared to adjacent
   tissues, while 3 (3.3%) exhibited deficient expression of CD66b in
   tissues (Fig. [67]1E). However, no significant difference in CD66b
   expression was observed between GC and non-adjacent cancerous tissues
   (Fig. [68]1C, p > 0.05). These findings suggest a decrease in the
   number of neutrophil infiltrates in GC tissues exhibiting high
   expression levels of PVT1 (Fig. S1C).

Fig. 1.

   [69]Fig. 1
   [70]Open in a new tab

   The expression of PVT1 demonstrated an inverse correlation with
   neutrophil infiltration in GC. A Box plots are presented to show the
   distributions of neutrophil infiltration levels for each copy number
   status of PVT1 in GC. The infiltration level for each SCNA category is
   compared with the normal using a two-sided wilcoxon rank-sum test. B
   Positive cells of PVT1 in tissues. C Positive cells of CD66b in
   tissues. D The positive cell rate of PVT1 in 90 GC tissues. E The
   positive cell rate of CD66b in 90 GC tissues. F The proportion in
   combinations of PVT1 expression and neutrophil infiltration. The cases
   of GC with a higher number of PVT1 positive cells in cancer tissues
   compared to adjacent tissues were designated as PVT1 +, while those
   with a lower number were designated as PVT1-. Similarly, GC cases with
   a higher number of CD66b-positive cells in cancer tissues compared to
   adjacent tissues were labeled as CD66b +, while those with lower or no
   expression were labeled as CD66b-. G The representative cases in
   combinations of PVT1 expression and neutrophil infiltration. H The
   proportion of survival and death in groups. I The mean survival time in
   groups. J The fraction survival proportion in groups. K Correlation
   analysis was performed on the PVT1 + CD66b- group. (ns no significance,
   **p < 0.01, and ***p < 0.001)

   Four PVT1/CD66b expression patterns were identified: PVT1 + CD66b + (26
   cases, 28.9%), PVT1 + CD66b- (40 cases, 44.4%), PVT1-CD66b + (8 cases,
   8.9%), and PVT1-CD66b- (16 cases, 17.8%). Notably, the combination of
   PVT1 + CD66b- exhibited the highest proportion among all observed
   patterns in GC tissues (Fig. [71]1F and G). Besides, of the 40 patients
   in PVT1 + CD66b- group, 11 survived and 29 died, accounting for 72.5%
   of deaths (Fig. [72]1H), the median survival time was 20.5 months
   (Fig. [73]1I). This suggests that GC patients with high PVT1 expression
   and low neutrophil infiltration have a poor prognosis (Fig. [74]1J).
   The Pearson correlation analysis revealed a negative correlation
   between PVT1 expression and neutrophil infiltration in PVT1 + CD66b-
   group (r = − 0.3554, p = 0.0012) (Fig. [75]1K). The present findings
   further validate the inverse correlation between PVT1 expression and
   neutrophil infiltration in GC.

Whole transcriptome sequencing revealed activation of the calcium signaling
pathway following PVT1 silencing in gastric cancer cells

   To investigate PVT1’s regulatory mechanism in GC, we first evaluated
   PVT1 expression levels across GC cell lines and selected HGC-27 for
   further experiments (Fig. S2A, p < 0.001). PVT1 knockdown in HGC-27
   cells (Fig. S2B, p < 0.001) enabled identification of differential
   expressed genes (DEGs) through whole transcriptome sequencing (WTS)
   between negative control (NC) and PVT1-knockdown groups. The quality
   control of sequencing data is shown in Fig. S3A-3B. Differential
   expression analysis revealed 15 PVT1 transcript variants, with 11
   downregulated (PVT1-229, PVT1-233, PVT1-234, PVT1-239, PVT1-258,
   PVT1-273, PVT1-287, PVT1-304, PVT1-329, PVT1-333, PVT1-367) were shown
   in Fig. [76]2A, Fig. S3C. The overall distribution of DEGs were
   visually shown in the volcano map (Fig. [77]2B). After knockdown of
   PVT1 in GC cells, the expression of histone 3′ UTR stem-loop region was
   increased (Fig. S3D). The peak chromosome distribution map has
   exhibited the distribution of DEGs on each chromosome and changes in
   abundance of tRNA expression (Fig. S3E-3F). A total of 1,126 DEGs (968
   upregulated, 158 downregulated;|log2FC|> 2.0, p < 0.05) were visualized
   via clustering heatmap (Fig. [78]2C). Gene Ontology (GO) and Kyoto
   Encyclopedia of Genes and Genomes (KEGG) analyses highlighted
   enrichment in calcium channel complexes (Fig. [79]2D, Fig. S3G-3H), and
   calcium signaling pathway (Fig. [80]2E). Fluo-4 AM assays confirmed
   increased intracellular Ca^2⁺ concentration in PVT1-ASO-treated cells
   (Fig. [81]2F, p < 0.01). Transcriptome sequencing further demonstrated
   calcium pathway activation through upregulated RYR2 and RYR3 expression
   (Fig. [82]2G), validated by PCR in PVT1-knockdown cells (Fig. [83]2H,
   p < 0.05).

Fig. 2.

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

   Activation of calcium signaling pathway in GC cells. A Differential
   expression of PVT1 transcripts variants. B The overall distribution of
   DEGs in the volcano map. C The clustering heatmap visualization of
   DEGs. D GO and E KEGG pathway enrichment analyses of DEGs. F The
   intracellular Ca^2+ concentration in HGC-27 cells (NC vs PVT1-ASO)
   measured by fluorescence intensity of Fluo-4 AM calcium indicator. G
   Upregulation of RYR2 and RYR3 in calcium signaling pathway revealed by
   transcriptome sequencing. H Expression levels of RYR2 and RYR3
   validated by real-time PCR. (ns no significance, *p < 0.05, **p < 0.01,
   and ***p < 0.001)

   To further explore the contribution of PVT1 knockdown to the phenotypic
   plasticity of GC cells, we performed in vitro and in vivo experiments.
   Cell proliferation (Fig. [86]3A) and colony formation (Fig. [87]3B,
   p < 0.05) were both significantly decreased after PVT1 knockdown in
   HGC-27 cells. Transwell and wound healing assays revealed that the
   migration (Fig. [88]3C, p < 0.05) and invasion (Fig. [89]3D, p < 0.01)
   capabilities of HGC-27 cells were markedly impaired following PVT1
   knockdown. MMP9 expression was also downregulated upon PVT1 silencing
   (Fig. [90]3E, p < 0.01). Genomic stability assessment via γ-H2AX assay
   showed substantially increased nuclear green fluorescence intensity in
   PVT1-knockdown cells (Fig. [91]3F, p < 0.05). In subcutaneous xenograft
   models established using PVT1-shRNA-transfected HGC-27 cells (Fig.
   S2C), tumor volume in the PVT1-shRNA group was significantly smaller
   than the NC group (Fig. [92]3G), with immunohistochemical analysis
   showing reduced Ki67 expression in tumor tissues (Fig. [93]3H,
   p < 0.01). These findings demonstrate that PVT1 knockdown critically
   regulates multiple malignant phenotypes of GC cells.

Fig. 3.

   [94]Fig. 3
   [95]Open in a new tab

   PVT1 silencing modulates phenotypic plasticity in GC cells. A
   Proliferation capacity of HGC-27 cells post-PVT1-ASO transfection
   assessed by CCK-8 assay. B Clonogenic ability of PVT1-knockdown HGC-27
   cells. C Transwell and D wound healing assays evaluating migratory
   capacity. E Western blot analysis of MMP9 expression pre- and post-PVT1
   knockdown. F Genomic stability assessment via γ-H2AX foci formation
   assay, the average number of γ-H2AX foci per cell was obtained by
   counting in 100 cells. G Subcutaneous xenograft model (n = 5/group)
   showing tumor growth inhibition in PVT1-ASO group, with HE staining
   confirming malignancy (100 ×), and tumor volume were calculated as
   volume = 0.5 × a × b^2 mm^3, a: length, b: width. H Ki67 expression
   levels across groups quantified by immunohistochemistry (100 ×). (ns no
   significance, *p < 0.05, **p < 0.01, and ***p < 0.001)

Proteomic profiling revealed that SYNPO secretion may be a key activator of
calcium signaling in gastric cancer cells

   4D-label-free quantitative proteomic analyses were performed via
   LC–MS/MS on cell culture supernatants from NC and PVT1-knockdown GC
   cells. We detected 18,470 proteins and obtained 188,030 peptide
   spectra, with 9,122 proteins (92,250 unique peptides) identified in the
   NC group and 9,348 proteins (95,780 unique peptides) in the PVT1-ASO
   group (Fig. S4A-4D). The overall distribution of differentially
   expressed proteins (DEPs) were shown in the volcano plot (Fig. [96]4A),
   of which 460 DEPs were identified with p < 0.05. Further, 46 DEPs
   with|log2FC|> 1.0 and p < 0.05 (37 DEPs up-regulated and 9 DEPs
   down-regulated) were identified and visualized in the hierarchical
   clustering heat map (Fig. [97]4B). InterProScan domain prediction
   showed significant enrichment of C2H2-type zinc fingers and EGF-like
   domains (Fig. [98]4C). GO analysis demonstrated DEP enrichment in
   clathrin-associated vesicular structures (Fig. [99]4D), while KEGG
   pathway analysis highlighted involvement in endoplasmic reticulum
   protein processing and endocrine-regulated calcium reabsorption
   (Fig. [100]4E).

Fig. 4.

   [101]Fig. 4
   [102]Open in a new tab

   4D-Label-free quantitative proteome analysis in GC cells. A Volcano
   plot showing the overall distribution of DEPs. B Hierarchical
   clustering of identified DEPs. C Top 20 statistically enriched protein
   domain classifications. D GO and E KEGG enrichment analyses of DEPs. F
   Venn diagram of transcriptomic-proteomic DEP intersections. G
   Expression-level comparison of overlapping DEPs. H PVT1-SYNPO
   correlation analysis. I Western blot analysis of SYNPO expression in GC
   cells. J GENEMANIA-based interaction network (SYNPO, RYR2, RYR3)
   showing co-expression, physical interactions, and correlations
   ([103]http://genemania.org/search/homo-sapiens/). (*p < 0.05)

   Correlation analysis between DEGs and DEPs, based on mRNA-protein
   translational relationships, identified six genes (SYNPO, MBTPS1,
   APOOL, ENO2, THBS3, CAPN7) (Fig. [104]4F). SYNPO exhibited significant
   upregulation at both transcriptional and protein levels (Fig. [105]4G).
   Furthermore, correlation analysis showed that SYNPO seems to be
   negatively correlated with PVT1 expression in GC cells (r = − 0.216,
   p < 0.001) (Fig. [106]4H), which was consisted with the elevated SYNPO
   expression in PVT1 knockdown GC cells (F[107]ig. [108]4I, Fig. S4E).
   Notably, SYNPO showed positive correlations with calcium channel
   proteins RYR2 (r = 0.412, p < 0.001) and RYR3 (r = 0.481, p < 0.001).
   GeneMANIA database analysis ([109]http://genemania.org/) further
   indicated direct/indirect interactions between SYNPO and RYR2/RYR3
   through co-expression networks and physical binding (Fig. [110]4J).
   These findings collectively suggest that SYNPO upregulation may trigger
   calcium signaling activation in GC cells.

Whole transcriptome sequencing revealed gene expression profiles in
neutrophils activated by PVT1-knockdown gastric cancer cells

   To investigate the negative correlation between PVT1 expression and
   neutrophil infiltration in GC tissues, we developed an in vitro tumor
   microenvironment (TME) model. Peripheral blood neutrophils were
   isolated from healthy donors (Fig. S1D-1F) and co-cultured with 48 h
   conditioned medium (CM) from HGC-27 GC cells (with/without PVT1
   knockdown). The results demonstrated that neutrophils co-cultured with
   GC CM for 16 h exhibited aggregation. A subset of cells transitioned
   from round/oval morphology to a polarized phenotype with surface
   irregularities and pseudopodia formation (Fig. [111]5A). Cell viability
   assays showed prolonged survival of neutrophils exposed to CM from
   PVT1-knockdown GC cells compared to control CM (Fig. [112]5B,
   p < 0.05). Combined apoptosis/necrosis assays further confirmed reduced
   cell death in the PVT1-knockdown group (Fig. [113]5C). Hoechst staining
   detected neutrophil extracellular traps (NETs) in CM-treated
   neutrophils, with TNF-α as a positive control (Fig. [114]5D). These
   findings suggest PVT1 knockdown in GC cells enhances neutrophil
   survival and activation through secreted factors.

Fig. 5.

   [115]Fig. 5
   [116]Open in a new tab

   PVT1 knockdown in GC cells enhances neutrophil activation via secreted
   factors. A Neutrophil morphology before and after CM induction. B
   Viability comparison of neutrophils exposed to CM from NC vs
   PVT1-knockdown HGC-27 cells. C Apoptosis/necrosis staining of
   CM-induced neutrophils. D NETs formation assay with TNF-α positive
   control. (*p < 0.05)

   To investigate the mechanism of PVT1-mediated neutrophil activation, we
   performed WTS on healthy donor neutrophils (n = 3) exposed to CM (Fig.
   S5A-5C). Sequencing identified 16,024 DEGs, with volcano plot
   visualization showing distinct expression patterns (Fig. [117]6A).Using
   stringent thresholds (p < 0.05,|log2FC|> 2), we identified 714 DEGs
   (384 upregulated, 330 downregulated) associated with neutrophil
   activation, as demonstrated by hierarchical clustering (Fig. [118]6B).
   GO enrichment analysis revealed three key functional categories: (1)
   Biological processes: cell adhesion mediated by integrin, regulation of
   chromatin silencing, chromatin assembly, replicative senescence and
   negative regulation of chromatin silencing. (2) Cellular components:
   microtubule end, sarcoplasmic reticulum, microtubule plus − end and
   sarcoplasmic reticulum membrane. (3) Molecular functions: sodium
   antiporter activity and calcium: cation antiporter activity
   (Fig. [119]6C). KEGG pathway analysis identified two mechanistically
   relevant pathways for further investigation: (1) Calcium signaling
   pathway (aligned with cation transport functions in GO). (2) p53
   signaling pathway (consistent with senescence regulation in GO)
   (Fig. [120]6D). These pathways were prioritized for further
   investigation based on mechanistic relevance.

Fig. 6.

   [121]Fig. 6
   [122]Open in a new tab

   Transcriptomic profiling of neutrophils induced by GC CM. A Volcano
   plot displaying the overall distribution of DEGs. B Hierarchical
   clustering of DEGs. C GO enrichment analysis (biological processes,
   cellular components, molecular functions). D KEGG pathway enrichment
   analysis

Suppression of PVT1 in gastric cancer cells enhances neutrophil calcium
signaling and delays replicative senescence

   The analysis showed that the DEGs in the expression profile of
   neutrophils involved in the calcium signaling pathway, covering the
   regulation of promoting or inhibiting the increase the concentration of
   cytoplasmic Ca^2+ (Fig. [123]7A). The cells of each group were stained
   with Fluo-4 AM, revealing an increased cytoplasmic calcium signal in
   neutrophils subsequent to co-culturing with PVT1-knockdown GC cells.
   (Fig. [124]7B).

Fig. 7.

   [125]Fig. 7
   [126]Open in a new tab

   Mechanistic validation of calcium and p53 signaling in neutrophils. A
   Regulatory node mapping in calcium signaling pathway. B Intracellular
   Ca^2⁺ levels measured by Fluo-4 AM fluorescence intensity. C
   Dose-dependent Ca^2⁺ response to SYNPO. D SYNPO internalization (red)
   and F-actin (green) via immunofluorescence. E Venn diagram of p53
   pathway-senescence gene intersection. F Transcriptome sequencing
   analysis of TP53, CHEK1, and SERPINE1 RNA levels. G Immunofluorescence
   of TP53, CHEK1, and SERPINE1 protein expression

   Given the observed increase in SYNPO secretion from PVT1-knockdown GC
   cells, we validated its role using recombinant human SYNPO protein.
   Neutrophils exposed to 1.0 ng/mL SYNPO showed maximal calcium signal
   enhancement (Fig. [127]7C). Immunofluorescence confirmed SYNPO
   internalization by neutrophils, with signal intensity correlating with
   exogenous SYNPO concentration (Fig. [128]7D). These findings suggest
   that SYNPO upregulation in PVT1-knockdown GC CM may regulate neutrophil
   calcium signaling. Intersection analysis identified TP53, CHEK1, and
   SERPINE1 through overlapping replicative senescence genes and p53
   pathway DEGs (Fig. [129]7E). Notably, the expression levels of p53 and
   CHEK1 were found to be decreased, while the expression level of
   SERPINE1 was observed to increase (Fig. [130]7F). Consistent with the
   transcriptomic sequencing results, immunofluorescence analysis also
   revealed a significant decrease in TP53 and CHEK1 expression levels in
   neutrophils from both the co-cultured group consisting of
   PVT1-knockdown GC cells and the SYNPO-supplemented group, while there
   was an observed increase in SERPINE1 expression (Fig. [131]7G).
   Meanwhile, clinical correlation analysis revealed a significant
   negative association (r = − 0.307, p < 0.001) between SYNPO expression
   and CHEK1 expression, as well as a positive correlation (r = 0.296,
   p < 0.001) with SERPINE1 expression in GC tissues (Fig. S5D-5F). The
   findings of this study suggest that co-culture with PVT1-knockdown GC
   cells delays neutrophil replicative senescence, and the increased
   presence of SYNPO in the supernatant of PVT1-knockdown GC cells may be
   serves as one of the regulatory factors implicated in this process.

Discussion

   While anti-angiogenesis and immunotherapy have advanced GC treatment
   [[132]27], tumor heterogeneity and dynamic TME remodeling continue to
   impede therapeutic efficacy [[133]13, [134]28]. Recent investigations
   reveal various TME remodeling modulators. For instance, CD4⁺ T cells
   can reshape the TME following oncogene inactivation [[135]29], while
   carcinogenic EGFR signals remodel the TME and trigger immune escape
   mechanisms [[136]30]. Similar to other tumors, GC occurrence and
   prognosis correlate strongly with immune cell infiltration. Elevated
   immune cell infiltration levels correlate with increased lymph node
   metastasis and reduced overall survival rates in GC [[137]19]. TANs,
   referring to neutrophil infiltration into the TME, represent a pivotal
   component of the tumor inflammatory milieu [[138]31]. Studies
   demonstrate that RAI14 gene and Notch receptor expression in GC
   correlate with neutrophil recruitment in tissues, indicating
   unfavorable prognosis in GC patients [[139]23, [140]24].

   Neutrophils constitute a prominent population of infiltrating immune
   cells within the TME of GC [[141]20]. To elucidate the correlation
   between PVT1 expression and neutrophil infiltration in GC tissues, we
   selected CD66b (a specific neutrophil marker [[142]32]) and employed IF
   combined with FISH. In 90 GC tissue samples exhibiting PVT1 and CD66b
   co-detection, we observed diverse PVT1 expression patterns with
   neutrophil infiltration. Intriguingly, the PVT1⁺CD66b⁻ subgroup
   accounted for the highest proportion (44.4%). Moreover, GC patients
   with high PVT1 expression and low neutrophil infiltration displayed
   poor prognosis. Our findings revealed a negative correlation between
   PVT1 expression levels and neutrophil infiltration density.

   Mechanistically, PVT1 silencing induced cytoplasmic calcium overload.
   Our study suggests that this phenomenon may result from SYNPO
   upregulation, which further activates endoplasmic reticulum (ER) Ca^2⁺
   release channels (RYR2 and RYR3), thereby promoting Ca^2⁺ efflux. These
   findings indicate that PVT1 knockdown in GC cells disrupts calcium
   homeostasis. As a ubiquitous signaling molecule, intracellular Ca^2⁺
   controls fundamental cellular processes including proliferation,
   differentiation, and cell death [[143]33, [144]34]. Consequently, ER
   Ca^2⁺ homeostasis maintenance is critical for cellular survival
   [[145]35]. Notably, calcium overload can induce immunogenic cell death
   through tumor-associated antigen release while simultaneously promoting
   calreticulin exposure an "eat me" signal that enhances immunotherapy
   via Ca^2⁺-mediated immune cell recruitment [[146]36, [147]37].
   Intriguingly, recent preclinical models utilizing CaCl₂/CaCO₃ mixtures
   demonstrated remarkable anti-tumor efficacy through calcium overload
   induction [[148]38]. Importantly, this study also noted that modulating
   cellular calcium homeostasis could synergize with calcium
   signaling-targeting drugs to enhance therapeutic outcomes [[149]38].
   Therefore, we speculate that PVT1 may play an important role in the
   alleviation of calcium overload alleviation during GC progression.

   As expected, PVT1 knockdown in HGC-27 cells significantly inhibited
   malignant phenotypes in vitro and suppressed tumor growth in vivo.
   Surprisingly, reduced PVT1 expression markedly elevated γH2AX (a DNA
   damage biomarker) nuclear levels in GC cells, indicating enhanced
   genomic instability (GI) [[150]39]. Increasing evidence showed that
   point mutations or genome rearrangements caused by defects in genome
   maintenance, can cause neo-antigens presentation and thus be recognized
   by immune cells, making tumor cells susceptible to recognition by the
   immune system [[151]40]. This suggests that PVT1 knockdown in GC cells
   may promote immune cell infiltration.

   Subsequently, we conducted in vitro observations to assess neutrophil
   activation under PVT1-regulated TME conditions through co-culture
   systems. After 16 h co-culture with GC cells culture supernatants, both
   experimental groups exhibited significant neutrophil morphological
   changes. Notably, neutrophils exposed to PVT1-knockdown GC cell
   supernatants showed reduced apoptosis/necrosis rates and extended
   survival duration. Studies have demonstrated that the half-life of
   neutrophils in the systemic circulation of healthy individuals is
   approximately 7 h [[152]41], while cancer patients may exhibit a
   prolonged half-life of up to 17 h [[153]42]. These results indicate
   successful in vitro replication of PVT1-regulated TME, with the
   PVT1-deficient GC microenvironment showing enhanced neutrophil survival
   capacity.

   GO and KEGG pathway enrichment analyses revealed that DEGs in activated
   neutrophils were predominantly enriched in calcium signaling pathways
   and cellular senescence-related pathways. Intracellular calcium
   homeostasis is mechanistically linked to cellular senescence. For
   instance, EDTA-mediated calcium chelation inhibits DNA synthesis in
   phytohemagglutinin (PHA)-stimulated lymphocytes, suggesting cytoplasmic
   calcium influx regulates proliferation. Senescent WI-38 fibroblasts
   exhibit either intracellular Ca^2⁺ store release or reduced
   extracellular inflow, implying diminished cytoplasmic Ca^2⁺ levels may
   suppress proliferation and accelerate aging [[154]43, [155]44]. In this
   study, we observed co-culture with PVT1-knockdown GC cells supernatants
   augmented cytoplasmic Ca^2⁺ signaling in neutrophils, indicating a
   potential enhancement in intracellular Ca^2+ flux, augmented release of
   calcium stores, or delayed expulsion of Ca^2+ to compensate for
   cytoplasmic calcium deficiency during cellular senescence, which is
   consistent with the phenotype associated with aging of neutrophils. By
   integrating the findings of quantitative proteomic analysis, our
   results suggest that increased expression of SYNPO protein in
   PVT1-knocked down GC cells and subsequent phagocytosis by neutrophils
   may potentially act as regulatory factors mediating enhanced
   cytoplasmic Ca^2+ signaling in neutrophils.

   Cell senescence represents a prolonged and irreversible state of the
   cell cycle arrest [[156]45]. Under normal physiological conditions,
   proliferating cells undergo follow regular cell cycle progression
   [[157]46]. It has been reported that the aging process in neutrophils
   may impact their immunosuppressive effect within tumors [[158]47], and
   potentially compromise their ability to release NETs [[159]48]. Further
   analysis of the expression of TP53, CHEK1, and SERPINE1 in the
   senescence pathway revealed a gradual decrease in p53 and CHEK1
   expression in neutrophils co-cultured with supernatant and SYNPO
   protein derived from knockdown PVT1 GC cells. Conversely, there was an
   observed increase in SERPINE1 expression. The P53, which encodes a
   transcriptional regulatory protein, which encodes as a DNA
   transcription regulator to induce G1 phase cell cycle arrest for DNA
   damage repair [[160]49]. Specifically, D40p53, D133p53α, and p53β
   isoforms are primarily involved in cellular senescence during its early
   stages [[161]50]. CHEK1 can induce a delay in the cell cycle process,
   allowing sufficient time for DNA damage repair [[162]51]. SERPINE1
   belongs to the serine protease inhibitor family [[163]52] and has
   recently been discovered to exhibit pro-angiogenic, proliferative,
   migratory, and anti-apoptotic activities in tumors [[164]53]. To
   summarize, neutrophils co-cultured with culture supernatant and SYNPO
   protein derived from PVT1-knocked down GC cells showed postponed
   senescence.

   In conclusion, this study elucidates the dual oncogenic mechanisms of
   PVT1 in GC progression (Fig. [165]8): (1) Remodel the TME by
   suppressing neutrophil infiltration/activation, and (2) disrupt genomic
   stability and induce calcium overload via PVT1 knockdown to establish a
   metabolic checkpoint. We provide the first evidence that PVT1
   orchestrates calcium homeostasis to regulate both neutrophil senescence
   and functional polarization, with SYNPO emerging as a critical
   downstream mediator of PVT1-driven calcium signaling in immune-stromal
   crosstalk. The discovery of this PVT1-centered metabolic-immune axis
   not only reveals a novel therapeutic target for GC immunotherapy but
   also offers innovative strategies to overcome TME-mediated therapeutic
   resistance by simultaneously addressing calcium dysregulation and
   neutrophil dysfunction.

Fig. 8.

   [166]Fig. 8
   [167]Open in a new tab

   Possible underlying mechanism of PVT1 knockdown in GC cells. This
   figure was created with Figdraw (ID:ASOTT28c1d)

Supplementary Information

   [168]Supplementary material 1. ^(9.2MB, zip)
   [169]Supplementary material 2. ^(23.7KB, zip)

Acknowledgements