The human BC cell line MDA-MB-231 (cat. no. iCell-h133), the mouse BC 4T1 cells (cat. no. iCell-m067), mouse RAW264.7 macrophages (cat. no. iCell-m047) and human monocytic leukemia THP-1 cells (cat. no. iCell-h213) were obtained from iCell Bioscience Inc. All cells were cultured in RPMI-1640 medium supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.; cat. no. 10099-14) and 1% penicillin-streptomycin at 37°C with 5% CO₂. THP-1 cells were differentiated into macrophages through the treatment with 100 ng/ml PMA (16). To induce M2 macrophage polarization, THP-1-derived macrophages were stimulated with 20 ng/ml IL-4 for 36 h (17).

For co-culture experiments, RAW264.7 macrophages and 4T1 cells were seeded into a Transwell chamber with 0.4 μm pore. For next-generation RNA sequencing analysis, RAW264.7 macrophages were subjected to high-energy X-ray irradiation.

For hypoxic treatment and exosome isolation, MDA-MB-231 cells were incubated in a tri-gas incubator of 1% O₂, 5% CO₂ and 94% N₂ with contained exosome-depleted FBS medium for 12 or 24 h, followed by co-culture with THP-1-derived macrophages for 24 h (18). To inhibit exosome biogenesis, MDA-MB-231 cells were pretreated with 10 μM GW4869 prior to further experimentation (19). To further confirm the role of NRG1 in modulating M2 polarization, an anti-NRG1 blocking antibody (10 μg/ml; Ab-2, LabVision) was used for cell treatment (20).

The pc-DNA3.1 vectors containing the complete sequence of NRG1 (Ov-NRG1), an empty vector (Ov-NC), the small interfering RNA targeting NRG1 (si-NRG1) and the corresponding negative control (si-NC) were purchased from Shanghai GenePharma Co., Ltd. and transfected into THP-1-derived macrophages. The sequences were: si-NRG1#1: 5'-CCCGATTGAAAGAGATGAAAAGC-3'; si-NRG1#2: 5'-TGGGAATGAATTGAATCGAAAAA-3'; si-NRG1#3: 5'-TGGCTGATTCTGGAGAGTATATG-3'; si-NC: 5'-AAGACAUUGUGUGUCCGCCTT-3'.

The pc-DNA3.1 vectors containing the complete sequence of CAMTA1 (Ov-CAMTA1), the empty vector (Ov-NC), the short hairpin RNA targeting CAMTA1 (sh-CAMTA1) and the corresponding negative control (sh-NC) were obtained from Shanghai GenePharma Co., Ltd. and transfected into MDA-MB-231 cells. The target sequences were: sh-CAMTA1#1: 5'-TGAGGAAATTGCGGCTTATTT-3'; sh-CAMTA1#2: 5'-CCCGACTGTTTCCTCAATAAT-3'; sh-CAMTA1#3: 5'-TCGGTCTGAACCCTCTAATTA-3'; sh-NC: 5'-GGAATCTCATTCGATGCATAC-3'.

Cell transfection was performed using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.

Exosomes were isolated using a standard differential centrifugation protocol. Initially, the conditioned medium was centrifuged at 300 × g for 10 min at 4°C to remove cell debris, 2,000 × g for 20 min at 4°C to remove the dead cells and 30 min at 10,000 × g at 4°C to further remove cell debris (21). The pellet of exosomes was collected through the ultracentrifugation at 100,000 × g for 70 min at 4°C (22). The exosome particle size was determined by nanoparticle tracking analysis using ZetaView PMX 110 (Particle Metrix).

For fluorescent labeling, purified exosomes were stained with PKH26 red fluorescent labeling kits (MilliporeSigma) according to the manufacturer's instructions. Briefly, ultracentrifuged exosomes were re-suspended in diluent C (100 μl) after ultracentrifugation at 100,000 × g for 70 min at 4°C and then incubated with PKH26 dye solution (100 μl) at room temperature for 5 min, followed by the addition of 200 μl serum. Subsequently, the labeled exosomes were washed by PBS at 4°C and then incubated with MDA-MB-231 cells. Fluorescence microscopy was used to visualize exosome uptake.

Exosomes were fixed with 2.5% glutaraldehyde at 4°C, placed onto the copper grids and negatively stained with 2% phosphotungstic acid for 2 min at room temperature. After air-drying, the samples were observed using a transmission electron microscope. Images were analyzed using ImageJ software (version 1.53, National Institutes of Health).

Total RNA was extracted from sample cells seeded at a density of 1×10⁶ cells per well using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) strictly according to the manufacturer's protocol. 1 μg of RNA was reverse-transcribed into cDNA using a commercial RevertAid cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The reverse transcription was performed at 42°C for 60 min, followed by 70°C for 5 min to inactivate the reverse transcriptase. qPCR was conducted using SYBR Green PCR Master Mix on the 7500 Fast Real-time PCR system according to the manufacturer's instructions. Thermocycling conditions were as follows: Initial denaturation at 95°C for 2 min. Denaturation at 94°C for 15 sec, annealing and extension at 60°C for 30 sec, 40 cycles. The relative gene expression was determined using 2^−ΔΔCq method (23). GAPDH was used as the internal reference gene. All RT-qPCR experiments were performed with three independent biological replicates. The following were the primer sequences: NRG1 (human) forward, 5'-GATTCCTACCGAGACTCTCCTC-3' and reverse, 5'-TGGAAGGCATGGACACCGTCAT-3'; NRG1 (mouse) forward, 5'-GCTCATCACTCCACGACTGTCA-3' and reverse, 5'-TGCCTGCTGTTCTCTACCGATG-3'; CAMTA1 (human) forward, 5'-AGTGCAGAAAATGAAGAATGCG-3' and reverse 5'-CAAAATTCTCCTGCTTGATTCG-3'; CAMTA1 (mouse) forward, 5'-CGGTGGTGTTTGAGTACAAGGC-3' and reverse 5'-CCTCCTTTCCATCTGCTCCAGA-3'; GAPDH (human) forward, 5'-GAAGGTGAAGGTCGGAGTC-3' and reverse, 5'-GAAGATGGTGATGGGATTTC-3', or GAPDH (mouse) forward, 5'-AGGTCGGTGTGAACGGATTTG-3' and reverse, 5'-TGTAGACCATGTAGTTGAGGTCA-3'.

The total proteins were extracted from sample cells using RIPA lysis buffer (Beyotime Biotechnology, China) and the protein concentration was quantified using BCA assay kits (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Separated by 10% SDS-PAGE, equal amounts of protein (30 μg per lane) were transferred to PVDF membranes (MilliporeSigma). Blocked by 5% BSA (MilliporeSigma) for 1 h at room temperature, the membranes were incubated with primary antibodies against NRG1 (cat. no. 10527-1-AP; 1:1,000; Proteintech Group, Inc.), hypoxia-inducible factor-1α (HIF-1α; cat. no. 20960-1-AP; 1:2,000; Proteintech Group, Inc.), CD63 (cat. no. 25682-1-AP; 1:2,000; Proteintech Group, Inc.), CD81 (cat. no. 27855-1-AP; 1:1,000; Proteintech Group, Inc.), CD9 (cat. no. 20597-1-AP; 1:2,000; Proteintech Group, Inc.), ALIX (cat. no. 12422-1-AP; 1:5,000; Proteintech Group, Inc.) and GAPDH (cat. no. 10494-1-AP; 1:5,000; Proteintech Group, Inc.) overnight at 4°C. On the next day, the membranes were incubated with goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (cat. no. SA00001-2; Proteintech Group, Inc.) at room temperature for 3 h. The protein bands were visualized using ECL regent (Thermo Fisher Scientific, Inc.) and protein density was semi-quantified via ImageJ software (version 1.53; National Institutes of Health, USA).

For the Transwell migration assay, MDA-MB-231 cells were suspended in serum-free medium (200 μl) and seeded into the upper chamber, while THP-1-derived macrophages were cultured in medium (800 μl) containing 10% FBS in the lower chamber. After 24 h of co-culture, the non-migrated cells were gently removed while the migrated cells were stained by 0.1% crystal violet for 30 min. The migrated cells in five randomly selected non-overlapping fields were observed under a light microscope.

The THP-1 macrophages were suspended in pre-cooled PBS and then centrifuged at 200 × g for 5 min at 4°C (24). After discarding the supernatant, cells were fixed with 0.2 ml of fixation buffer for 5 min at room temperature and then 0.5 ml of the permeabilization wash buffer was added for 5 min at room temperature. Subsequently, the cells were incubated with CD163 antibody (cat. no. 16627; 1:50; Cell Signaling Technology, Inc.) at room temperature for 30 min. A BD FACSCanto II flow cytometer (BD Biosciences) was used to assess CD163 expression and the data analysis was processed using FlowJo software (version 10.8.1; FlowJo LLC; BD Biosciences).

The MDA-MB-231 cells were washed by PBS and centrifuged to remove the supernatant (25). For cell cycle analysis, MDA-MB-231 cells were treated with 50 μl RNase A and 200 μl PI and incubated in the dark at room temperature for 20 min, followed by flow cytometry analysis. For apoptosis analysis, MDA-MB-231 cells were suspended in 500 μl PBS and then incubated with 5 μl Annexin-V-FITC and 5 μl PI in the dark at room temperature for 15 min, followed by flow cytometry analysis. All samples were analyzed using a BD FACSCanto II flow cytometer (BD Biosciences). Data acquisition and analysis were performed using FlowJo software (version 10.8.1; FlowJo LLC; BD Biosciences). The apoptotic rate was calculated as follows: Apoptotic rate (%)=early apoptotic cells (Annexin V⁺/PI⁻) + late apoptotic cells (Annexin V⁺/PI⁺).

Cell viability was detected using MTT assay and colony formation assay. For MTT detection, cells were incubated with MTT solution, followed by the aspiration of supernatant and the addition of DMSO (100 μl). The optical density was measured at 490 nm using a microplate reader.

For colony formation assays, the colonies were fixed with methanol at room temperature for 15 min and stained by 0.1% crystal violet at room temperature for 20 min. The number of colonies was counted using under a light microscope (magnification, ×100). Five randomly selected, non-overlapping fields per well were examined.

The supernatant was collected by centrifugation at 2,000 × g for 5 min at 4°C. The concentrations of IL-10 and IL-12 were detected using ELISA-related IL-10 assay kits and IL-12 assay kits according to the manufacturer's instructions. The optical density was measured at 450 nm using a microplate reader.

All animal experiments approved by the Animal Ethics Committee of Jiangsu Cancer Hospital and Jiangsu Institute of Cancer Research and The Affiliated Cancer Hospital of Nanjing Medical University and conducted in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (approval no. IACUC-20210216-01). A total of 50 female BALB/c nude mice (4-5 weeks old, weighing 18-22 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd.. Mice were housed under SPF conditions at a controlled temperature of 22±2°C, relative humidity of 50-60%, and a 12-h light/dark cycle, with free access to food and water. The mice were subcutaneously injected with 1×10⁶ MDA-MB-231 cells transfected with sh-CAMTA1 or Ov-CAMTA1 and then randomly divided into five groups (n=10): PBS, Normoxic exo, Hypoxic exo, Hypoxic exo + Ov-CAMTA1 and Hypoxic exo + sh-CAMTA1 groups. Mice in PBS group received 30 μl PBS, while mice in remaining groups were treated with 30 μg exosomes. Local radiotherapy in the tumors was performed for consecutive 5 days (2 Gy/d). The tumor growth was monitored every week. After five weeks, the mice were sacrificed by intraperitoneal injection of pentobarbital sodium (150 mg/kg). Death was confirmed based on the disappearance of pain response, no response when pressing the toes with hands or forceps and observation of cardiac and respiratory arrest. Tumor tissues were removed, images captured and collected for histological and immunohistochemistry analysis.

The collected tumor issues were fixed with 10% formalin at room temperature for 24 h. After fixation, tissues were dehydrated through a graded ethanol series, cleared in xylene, and subsequently embedded in paraffin according to standard protocol. Paraffin-embedded tissues were then sectioned into 7 μm-thick slices. Subsequently, the slices were stained with H&E at room temperature, including hematoxylin staining for 5 min followed by eosin staining for 2 min. The stained sections were observed under a light microscope.

Immunohistochemistry was performed to assess the expression of NRG1, CD163, and cleaved caspase-3 in tumor tissues. Briefly, antigen retrieval was performed by heating the tissue samples in 1X sodium citrate buffer using a microwave, followed by the incubation with 3% hydrogen peroxide for 10 min and subsequent inhibition with 3% normal goat serum (Solarbio, China) for 1 h at room temperature (26). The slices were then incubated with primary antibody against NRG1 (cat. no. 83323-6-RR; 1:500; Proteintech Group, Inc.), CD163 (cat. no. 83285-4-RR; 1:2,000; Proteintech Group, Inc.) and Caspase-3 (cat. no. 19677-1-AP; 1:500; Proteintech Group, Inc.) overnight at 4°C. On the next day, the slices were incubated with HRP-labeled secondary antibody (cat. no. ab6721; 1:1,000; Abcam) for 1 h at room temperature. Following the treatment with DAB solution, the counterstaining to hematoxylin was performed for 1 min at room temperature and a light microscope was used for observation.

To explore the expressions of CAMTA1 and NRG1 in BC and validate their association with macrophage polarization, several public databases were used. Gene expression levels of CAMTA1 and NRG1 in The Cancer Genome Atlas-Breast Invasive Carcinoma (TCGA-BRCA) samples were obtained from the UCSC Xena platform (https://xenabrowser.net/). Spearman's correlation between CAMTA1 and NRG1 was analyzed using R software (v4.2.2; https://www.r-project.org/). The correlation between NRG1 expression and M2 macrophage marker CD163 was assessed via the Gene Expression Profiling Interactive Analysis (GEPIA)2 platform (http://gepia2.cancer-pku.cn/) based on TCGA-BRCA tumor data. Kaplan-Meier survival analysis was performed using the KMplot database (https://kmplot.com) with the overall survival in Affymetrix breast cancer cohorts stratified by NRG1 expression.

The collected data was processed using GraphPad Prism 8 software (Dotmatics) and presented as mean ± SD. Statistical comparisons between two groups were performed using unpaired Student's t-test (for two groups) while comparisons among three or more groups were conducted using one-way ANOVA with Tukey's test. P<0.05 was considered to indicate a statistically significant difference.

Through next-generation RNA sequencing analysis, NRG1 expression was found to be increased in RAW264.7 macrophages co-cultured with 4T1 cells. By contrast, radiotherapy treatment markedly suppressed NRG1 expression (Fig. 1A). These findings were further confirmed by RT-qPCR and immunoblotting analysis. Compared with the RAW264.7 macrophages group, both the mRNA and protein expressions of NRG1 in RAW264.7 macrophages were markedly increased following the co-culture with 4T1 cells, whereas radiotherapy treatment effectively reversed these changes (Fig. 1B and C).

Under hypoxic conditions, the protein expression of HIF-1α in MDA-MB-231 cells was assessed using immunoblotting analysis. As shown in Fig. 2A, HIF-1α protein level progressively increased with prolonged hypoxia exposure (0, 12 and 24 h). Based on this observation, MDA-MB-231 cells pre-treated with hypoxia for 24 h were subsequently co-cultured with THP-1-derived macrophages for another 24 h. Following co-culture, the increased HIF-1α expression induced by hypoxia was markedly decreased, yet remained above normoxic control (Fig. 2B). Transwell assays demonstrated that the migration of THP-1 macrophages was markedly increased after co-culture with hypoxia-pretreated MDA-MB-231 cells (Fig. 2C). Flow cytometry analysis further showed that hypoxia stimulation increased the expression of CD163, a well-established M2 macrophage marker, in a time-dependent manner (Fig. 2D and E). IL-12 is a cytokine associated with the M1 phenotype, while IL-10 is indicative of M2 polarization. In addition, ELISA results revealed that hypoxia promoted IL-10 secretion while concomitantly reducing IL-12 production in THP-1 macrophages (Fig. 2F and G). Taken together, these findings suggested that hypoxic MDA-MB-231 cells could facilitate the M2 polarization of THP-1-derived macrophages.

To investigate the radio-resistance of MDA-MB-231 cells, all cells were exposed to X-ray irradiation at doses of 1, 2 and 3 Gy. Given that hypoxic conditions can induce the differentiation of M0 macrophages into M2 macrophages, MDA-MB-231 cells were co-cultured with THP-1 macrophages under hypoxia for varying durations. The cells were divided into MDA-MB-231, MDA-MB-231 + M2, MDA-MB-231 + THP-1 + hypoxia 0 h, MDA-MB-231 + THP-1 + hypoxia 12 h and MDA-MB-231 + THP-1 + hypoxia 24 h groups. MDA-MB-231 cell viability was first assessed using the MTT assay. Compared with the 0 Gy group, MDA-MB-231 cell viability was reduced with increasing radiation dose, whereas co-culture with M2 macrophages conferred the strongest radiation resistance (Fig. 3A). Based on these findings, subsequent experiments were conducted using 2 Gy radiation. Flow cytometry revealed that M2 polarization markedly inhibited the apoptosis of MDA-MB-231 cells. Moreover, hypoxic co-culture with THP-1 macrophages suppressed apoptosis in a time-dependent manner compared with the MDA-MB-231 + THP-1 + hypoxia 0 h group (Fig. 3B and C). To assess whether the reduced apoptosis was associated with altered cell proliferation, cell-cycle distribution was analyzed by flow cytometry. The results showed that M2 polarization markedly increased the proportion of cells in S phase while decreasing the proportion in G₀/G₁ phase, suggesting accelerated DNA synthesis and proliferation potential. Similarly, hypoxic co-culture with THP-1 macrophages increased the S-phase fraction in a time-dependent manner (Fig. 3D and E). In addition, colony formation assays demonstrated that M2 polarization greatly increased the number of colonies compared with the MDA-MB-231 group and hypoxic co-culture with THP-1 macrophages further promoted clonogenic survival (Fig. 3F-G).

Analysis of the TCGA database revealed that NRG1 expression was markedly upregulated in BC samples (Fig. 4A). GEPIA2 database analysis further demonstrated a positive correlation between NRG1 expression and the M2 macrophage marker CD163 (Fig. 4B). Moreover, Kaplan-Meier survival analysis showed that high NRG1 expression was associated with low overall survival in BC patients (Fig. 4C).

To assess the effects of hypoxia on NRG1 expression, MDA-MB-231 cells were exposed to hypoxia for 24 h and co-cultured with THP-1 macrophages. RT-qPCR analysis revealed that hypoxia markedly upregulated NRG1expression compared with the Normoxia group (Fig. 5A). To upregulate or downregulate NRG1 expression, Ov-NRG1 or si-NRG1 was transfected into cells and the transfection efficacy was examined using RT-qPCR and immunoblotting analysis (Fig. 5B and C). si-NRG1#2 (hereinafter referred as si-NRG1) was selected for subsequent experiments due to its more prominent transfection efficacy. Then, the cells were assigned into M2, M2 + Ov-NRG1, M2 + si-NRG1, Hypoxic MDA-MB-231 + THP-1, Hypoxic MDA-MB-231 + THP-1 + Ov-NRG1 and Hypoxic MDA-MB-231 + THP-1 + si-NRG1 groups. ELISA results showed that Ov-NRG1 elevated IL-10 secretion, whereas si-NRG1 reduced IL-10 level compared with the M2 or Hypoxic MDA-MB-231 + THP-1 groups (Fig. 5D). Consistently, flow cytometry analysis demonstrated that CD163 expression was increased by Ov-NRG1 and decreased by si-NRG1 (Fig. 5E and F). To further verify whether the reduction in IL-10 was specifically attributable to impaired NRG1 signaling, hypoxic MDA-MB-231/THP-1 co-cultures were treated with an anti-NRG1 blocking antibody. Cells were divided into Hypoxic MDA-MB-231 + THP-1, Hypoxic MDA-MB-231 + THP-1 + IgG and Hypoxic MDA-MB-231 + THP-1 + anti-NRG1 antibody groups. Blocking NRG1 markedly decreased IL-10 secretion (Fig. 5G) and consistently reduced CD163 expression (Fig. 5H and I). These findings indicated that hypoxia-induced NRG1 upregulation is a key driver of M2 macrophage polarization, at least in part by enhancing IL-10 secretion and CD163 expression.

In the tumor microenvironment, macrophages play crucial roles in tumor initiation and progression. To investigate whether exosomes contribute to M2 polarization, MDA-MB-231 cells were treated with the exosome biogenesis inhibitor GW4869 and then co-cultured with THP-1 macrophages. Transwell assays showed that hypoxic MDA-MB-231 cells markedly enhanced the migration of THP-1 macrophages, whereas this effect was notably attenuated by GW4869 treatment (Fig. 6A). Moreover, GW4869 treatment markedly reduced CD163 expression and IL-10 secretion, suggesting that exosomes derived from hypoxic MDA-MB-231 cells are pivotal in promoting M2 polarization (Fig. 6B and C). Transmission electron microscopy further revealed abundant exosomes with typical bilayer membrane structures in the supernatant of hypoxic MDA-MB-231 cells. Nanoparticle size analysis showed that the average exosome diameter was ~100 nm (Fig. 6D and E). Immunoblotting analysis confirmed the presence of exosomal marker proteins CD9, ALIX, CD63 and CD81 in exosome extracts (Fig. 6F).

Exosomes extracted from hypoxic or normoxic MDA-MB-231 cells were subsequently labeled with PKH67 and incubated with THP-1 macrophages. Fluorescence imaging confirmed the successful intake of MDA-MB-231-derived exosomes by THP-1 macrophages (Fig. 6G). Consistently, flow cytometry analysis demonstrated that hypoxia-derived exosomes markedly increased CD163 expression in THP-1 macrophages (Fig. 6H). The above results indicated that hypoxic MDA-MB-231 cells promoted the M2 polarization of THP-1 macrophages via exosomes.

Analysis of the TCGA database revealed that CAMTA1 expression was markedly elevated in BC samples (Fig. 7A). Consistently, RT-qPCR results showed that hypoxic stimulation markedly increased CAMTA1 expression in both MDA-MB-231 cells and their secreted exosomes (Fig. 7B and C). Furthermore, co-culture experiments demonstrated a substantial increase in CAMTA1 expression with recipient THP-1 macrophages after exposure to hypoxia-derived exosomes (Fig. 7D). To reduce CAMTA1 expression, sh-CAMTA1 was transfected into MDA-MB-231 cells and the transfection efficacy was examined using RT-qPCR and immunoblotting analysis (Fig. 7E). sh-CAMTA1#1 (hereinafter referred as sh-CAMTA1) was selected for subsequent experiments due to its improved transfection efficacy. As Fig. 7F and G shows, CAMTA1 expression was markedly reduced in both MDA-MB-231 cells and exosomes following the transfection of sh-CAMTA1. Exosomes from CAMTA1-silenced MDA-MB-231 cells were then incubated with THP-1 macrophages and then divided into Normoxic MDA-MB-231 + exo + sh-NC, Normoxic MDA-MB-231 + exo + sh-CAMTA1, Hypoxic MDA-MB-231 + exo + sh-NC and Hypoxic MDA-MB-231 + exo + sh-CAMTA1 groups. Functional assays revealed that CAMTA1 knockdown markedly impaired the migration of THP-1 macrophages (Fig. 7H). Moreover, CAMTA1 depletion also led to a notable reduction in the expressions of CD163 and IL-10 (Fig. 7I-K).

To upregulate CAMTA1 expression, Ov-CAMTA1 was transfected into MDA-MB-231 cells and the transfection efficacy was examined (Fig. 8A). To investigate the role of CAMTA1 in vivo, MDA-MB-231 cells transfected with sh-CAMTA1 or Ov-CAMTA1 were subcutaneously injected into mice for the establishment of MDA-MB-231 tumor-bearing nude mice model. The appearance of tumor was shown in Fig. 8B. As illustrated in Fig. 8C and D, CAMTA1 overexpression increased the tumor volume and weight, whereas CAMTA1 knockdown produced the opposite effects. Throughout the present study, the maximum tumor volume measured was ~850 mm³, with a corresponding maximum diameter of ~10 mm. RT-qPCR analysis confirmed that CAMTA1 expression in tumor tissues was elevated by CAMTA1 overexpression and reduced by CAMTA1 silencing compared with the Hypoxia exo group (Fig. 8E). Furthermore, the expression levels of IL-10 and CD163, which were elevated in the Hypoxia exo group, were further increased upon CAMTA1 overexpression, suggesting that CAMTA1 promoted M2 polarization in vivo (Fig. 8F and G). Additionally, CAMTA1 overexpression increased tumor cell viability and suppressed Caspase 3 expression, while CAMTA1 silencing exerted the opposite effects (Fig. 8H and I).

Correlation analysis revealed a positive association between CAMTA1 and NRG1 expression (Fig. 8J). Immunohistochemistry further confirmed that CAMTA1 overexpression markedly upregulated NRG1 expression in tumor tissues, suggesting that CAMTA1 may positively regulate NRG1 in BC (Fig. 8K).