Abstract
Introduction
Bone marrow mesenchymal stem cells (BMSCs) are effective in treating a variety of chronic pain conditions, including bone cancer pain (BCP); however, the underlying mechanism remains unclear. Microglia are the main cells involved in hyperalgesia and maintenance of BCP. Hence, their activation and polarization are key signs of neuroinflammation. The Toll-like receptor 4/ nuclear factor-kappa B (TLR4/ NF-κB) signaling pathway plays an essential role in microglial activation and neuroinflammation. Nonetheless, whether BMSCs can be used to treat BCP by regulating the TLR4/NF-κB signaling pathway and microglial activation requires further research. In this study, a series of experiments were conducted to explore the specific mechanisms underlying the beneficial effects of BMSCs on BCP.
Methods
Initially, this study demonstrated the therapeutic effect of BMSCs on BCP through behavioral and pathological evaluations. Subsequently, the effects of BMSCs on microglia and related mechanisms were detected by Western blotting, immunofluorescence, ELISA, and RNA sequencing. Finally, based on the microglia BV2-BMSCs co-culture model, the mechanism of BMSCs regulation of microglia was further verified through in vitro experiments.
Results
Intrathecal injection of BMSCs was found to alleviate BCP by inhibiting the activation of microglia in the spinal dorsal horn and the release of cytokines, which involves the regulation of the TLR4/NF-kB signaling pathway. In addition, BMSCs inhibited the nuclear transport of NF-κB in vitro.
Discussion
These results revealed that the TLR4/NF-κB signaling pathway in microglia is one of the targets of BMSCs in BCP treatment.
Keywords: BMSCs, BCP, microglia, TLR4/NF-κB signaling pathway, neuroinflammatory
Introduction
Breast cancer is the most prevalent malignant tumor in women globally. Latest US statistics indicate a rising annual incidence, with the disease accounting for the highest number of new cases and deaths among all female malignancies.1,2 Bone is the most common metastatic site of breast cancer, as 65–75% of advanced breast cancer patients develop bone metastasis.3 Cancer pain is a frequent complication in advanced cases, among which bone cancer pain (BCP) is the most severe and intractable Arising from bone metastasis and subsequent invasion of surrounding soft tissues, BCP severely impairs patients’ quality of life and imposes a heavy social burden.4 Nevertheless, the complex pathogenesis of BCP remains incompletely elucidated, and monotherapy yields suboptimal outcomes.5 Thus, effective therapeutic strategies for BCP are urgently needed.
Neuroinflammation is a hallmark and key contributor to bone cancer pain (BCP).6 In the bone metastatic microenvironment, proliferating cancer cells directly damage primary afferent nerve fibers, secrete pain mediators and inflammatory factors, and induce nociceptor activation and sensitization.7 Nociceptive signals are then transmitted to the central nervous system, triggering abnormal activation of spinal dorsal horn neurons and glial cells as well as robust neuroinflammation.8 Notably, spinal dorsal horn microglia are central to hyperalgesia development and BCP maintenance, with their activation and polarization serving as key hallmarks of neuroinflammation.9,10 Toll-like receptor 4 (TLR4), which is highly expressed in microglia, recruits the downstream adaptor MyD88 to facilitate nuclear translocation of NF-κB, thereby inducing the release of proinflammatory mediators including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and nitric oxide synthase; this signaling cascade drives microglial activation in multiple neurodegenerative diseases.11–13 Thus, inhibiting spinal dorsal horn microglial activation represents a promising therapeutic target for BCP.
Cell-based therapies have emerged as promising strategies for modulating inflammation and repairing tissue damage.14,15 Multiple studies have shown that mesenchymal stem cell (MSC) transplantation alleviates chronic pain, though the underlying mechanisms remain elusive.16,17 Bone marrow mesenchymal stem cells (BMSCs), which possess potent anti-inflammatory and immunomodulatory properties, have been widely applied in treating various diseases and demonstrated favorable analgesic efficacy and safety in preclinical and clinical settings.18,19 Previous research has confirmed that intrathecal BMSCs injection effectively relieves bone cancer pain (BCP), inflammatory pain and neuropathic pain by inhibiting glial activation and suppressing neuroinflammatory cascades.20–22 Notably, BMSCs exert anti-inflammatory effects by regulating the microglial TLR4/MyD88/NF-κB signaling pathway, thus mitigating neuroinflammation. TLR4 activation triggers MAPK and NF-κB pathway induction, driving microglial inflammatory responses; targeted inhibition of microglial activation, pro-inflammatory mediator release and TLR4-mediated signaling components holds great potential for treating neuroinflammatory disorders.23 Given the critical role of the TLR4/NF-κB pathway in neuropathic pain pathogenesis and the pain-relieving effects of its inhibition, this pathway is likely a key target of BMSCs in BCP treatment.
However, whether the therapeutic effects of BMSCs in BCP treatment are mediated by inhibition of microglial activation remains unclear. However, it remains unclear whether BMSCs can inhibit microglial activation and neuroinflammation. This study explored the therapeutic effects of BMSCs on BCP and revealed the potential molecular mechanism. These results provide insights into the selection of therapeutic targets for BCP.
Materials and Methods
Cell Culture
Identification and Cultivation of BMSCs
Rat BMSCs were purchased from the cell bank of the China Academy of Sciences (SCSP-402) and collected from Sprague–Dawley (SD) rats aged 3–6 weeks. A quality control report indicated that these cells could differentiate into fat, bone, and cartilage. The detection results for mycoplasma, bacteria, and fungi were negative and could be directly used in subsequent experiments. After thawing, the cells were cultured in α-MEM (Gibco, C12571500BT) containing 10% fetal bovine serum (FBS; Procell, 164210), penicillin, streptomycin, and amphotericin B (Solarbio, P7630). Subsequently, the cells were placed in an incubator at constant temperature and humidity (37 °C and 5% CO2). BMSCs at passages 3–5 (P3–P5) were characterized for morphology under light microscopy and immunophenotype via flow cytometry. To evaluate multilineage differentiation capacity, cells were induced toward osteogenic, adipogenic, and chondrogenic lineages using commercial induction kits from Cyagen Biosciences (Guangzhou, China; Cat. No. RAXMX-90021, RAXMX-90031, and RAXMX-90041, respectively). Differentiation was confirmed by specific staining: Alizarin Red S for calcium deposits (osteogenesis), Oil Red O solultion for lipid droplets (adipogenesis), and Alcian Blue 8GX solultion for proteoglycans (chondrogenesis). These results collectively confirm the identity and multipotency of the isolated BMSCs.
BV2 Cells and BMSCs Co-Culture Assay
The BV2 cell line was purchased from Cas9X Starfish Biological Co., Ltd. (HyCyte™ TCM-C718 Suzhou, China). The experiment was conducted using a 6-well transwell system (LABSELECT, LAB-14112), and the grouping was set as follows. (1) BV2 group, 2 mL complete medium (10% FBS+DMEM) per well with 1×106 BV2 cells/well, and the medium was changed into DMEM the next day; (2) BV2+BMSCs group, lower chamber: 2 mL complete medium (10% FBS+DMEM) per well with 1×106 BV2 cells/well, and the medium was changed into DMEM the next day; upper chamber: 1.5 mL BMSCs complete medium with 1×104 BMSCs/well; (3) BV2+LPS group, the cells were placed similarly to the BV2 group, and the medium was replaced with DMEM medium containing LPS (1 μg/mL, Sigma, L2630) the next day; (4) BV2+BMSCs+LPS group, lower chamber: 2 mL complete medium (10% FBS+DMEM) per well with 1×106 BV2 cells/well, and the medium was replaced with DMEM medium containing LPS (1 μg/mL) the next day; upper chamber: 1.5 mL BMSCs complete medium with 1×104 BMSCs/well. (5) BV2+BMSCs+LPS+PMA(phorbol-12-myristate-13-acetate) group, lower chamber: 2 mL complete medium (10% FBS+DMEM) per well with 1×106 BV2 cells/well, and the medium was replaced with DMEM medium containing LPS (1 μg/mL) and PMA (100ng/mL) the next day; upper chamber: 1.5 mL BMSCs complete medium with 1×104 BMSCs/well. After 24 h of co-culture, cells and supernatants were collected for subsequent detection.
Walker 256 Cell Line
The Walker 256 breast cancer cell line, provided by Professor Dong Changsheng (Shanghai Institute of Traditional Chinese Medicine), was utilized in accordance with the guidelines approved by the Animal Research Ethics Committee of Dalian Medical University (AEE24167). The cells were thawed and adjusted to a density of 2×107 cells/mL. Thereafter, 2×107 tumor cells were injected into the abdominal cavity of female SD mice. Approximately one week later, the mice exhibited obvious abdominal ascites, and clear and bloodless ascites were collected for analysis. The samples were processed by centrifugation at 1200 rpm for 5 min, and the supernatant was discarded. Finally, the cell density was adjusted to approximately 5×104 /μL for subsequent use.
Animal Experiments
Animals
Female SD rats weighing 180–220 g were purchased from Liaoning Changsheng Biotechnology Co. Ltd. (Dalian, China). The female SD rats were placed in sterile plastic cages and maintained in specific pathogen-free laboratories. Room temperature was maintained at 23–25 °C with a humidity of 40–60% and a 12 h/12 h alternating light and dark cycle. The animals had access to food and water ad libitum. All female SD rats underwent an adaptation training period of 3–5 days before the formal experiment to reduce stress and ensure stable behavioral performance. This study was approved by the Animal Research Ethics Committee of Dalian Medical University (AEE24167). The guidelines followed for the welfare of the laboratory animals: National Standard (Current): GB/T 42011–2022 “Laboratory animals—General code of animal welfare”.
Briefly, intrathecal catheterization was performed 3 days prior to model establishment (day −3). On day 0, Walker-256 breast cancer cells were inoculated into the left tibia to induce the BCP model. Rats were then randomly divided into different groups and subjected to corresponding interventions. Behavioral tests, including the 50% paw withdrawal threshold (PWT), ambulatory pain score, thermal withdrawal latency (TWL), and number of spontaneous foot lifts (NSF), were conducted on performed prior to BCP model establishment and on days 3, 7, 9, 11, and 13 post-modeling. Intrathecal injections of bone marrow mesenchymal stem cells (BMSCs, 2×106 cells) or normal saline (as a control) were administered on days 7, 9, and 11. X-ray imaging of the left tibia was performed on days 7 and 14 to assess bone destruction. The experimental schematic, procedures, and key time points are illustrated in Figure 1. On day 14, all rats were euthanized by cervical dislocation. Immediately after euthanasia, blood samples were collected from the abdominal aorta and centrifuged to separate serum for subsequent biochemical analysis. The left tibia was carefully harvested, fixed, decalcified, and embedded in paraffin for hematoxylin and eosin (H&E) staining to evaluate histological changes. Additionally, the L4-L6 spinal cord dorsal horn was rapidly dissected on ice, snap-frozen in liquid nitrogen, and stored at −80°C for subsequent experiments, including RNA sequencing, Western blot analysis, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence staining to detect protein expression and cellular localization.
Figure 1.
Schematic Diagram of Animal Experimental Procedures.
Establishment of BCP Model and BMSCs Administration
A total of 20 female SD rats were used in the BCP model study. Rats were intraperitoneally injected with 1% pentobarbital sodium solution (50 mg/kg) and the tibial plateau was exposed under aseptic conditions. SD rats were randomly divided into four groups (n = 5 per group): (1) Sham group, 10 µL normal saline was injected into the medullary cavity of the left tibia; (2) BCP group, Walker 256 cells (10 µL, 5×105) were injected directly into the bone marrow cavity of the left tibia; (3) BCP + PBS group, on the 7th day after the establishment of the BCP model, 20 µL PBS was injected intrathecally; and (4) BCP + BMSCs group, on the 7th day after the establishment of the BCP model, 20 µL (approximately 2×106) of BMSCs were injected intrathecally. Intrathecal injections were administered every other day until the 11th day. Throughout the experimental period, the animals were subjected to daily monitoring for general health status, and behavioral tests for the 50% paw withdrawal threshold (PWT), thermal withdrawal latency (TWL), limb use scores, and number of spontaneous flinches (NSF) were performed prior to BCP model establishment and on days 3, 7, 9, 11, and 13 post-modeling.24 50% Paw Withdrawal Threshold (PWT): Measured using calibrated von Frey filaments via the up-and-down method. This test assesses mechanical allodynia, a hallmark of cancer-induced neuropathic pain. Thermal Withdrawal Latency (TWL): Assessed using a plantar test apparatus with a focused radiant heat source. This evaluates thermal hyperalgesia. Limb Use Score: A 0–4 point scale was used to score weight-bearing and gait abnormalities during ambulation, reflecting functional impairment due to skeletal destruction or pain. Number of Spontaneous Flinches (NSF): The frequency of spontaneous withdrawal or flinching of the affected hindlimb was recorded over a 2-minute period, reflecting spontaneous pain behavior.
Exclusion criteria: We applied the following predefined exclusion criteria to ensure data validity and animal welfare: Mortality: Rats that died prior to the experimental endpoint (eg, due to surgical complications or severe distress) were excluded from analysis. The rat was replaced only if the death occurred before intervention initiation and was unrelated to the experimental treatment (eg, anesthesia overdose). Intrathecal injection failure: Rats in which the intrathecal catheter was found to be misplaced, blocked, or caused neurological damage (eg, hindlimb paralysis or unilateral paresis unrelated to the model) were excluded. Successful placement was confirmed by behavioral observation (eg, tail flick test) and failed placements rats were replaced before group allocation. Infection or severe inflammation at surgery site: Rats showing signs of severe infection, abscess, or systemic illness post-surgery were excluded and received humane euthanasia. Lack of model validation: In the BCP model, rats failing to develop significant mechanical allodynia (eg, assessed using von Frey filaments) after model establishment were excluded from further analysis.
X-Ray
Rats in the Sham, BCP, BCP+PBS, and BCP+BMSCs groups were subjected to X-ray scans on the 7th and 14th day after BCP modeling to assess bone destruction in the left hind limb. Specifically, the rats were anesthetized by intraperitoneal injection of pentobarbital sodium (40 mg/kg) and the tibia on the surgical side was scanned. The degree of bone destruction was assessed by two radiologists.
Pathology
Tissue Histological Evaluation
The left tibiae from rats in each group were collected on day 14 post-modeling and subjected to hematoxylin and eosin (H&E) staining. After euthanasia, the rats were perfused with 4% paraformaldehyde, and the L4-6 spinal cord segments and tibia of the operated side were separated. The tibia was decalcified in a 10% ethylenediaminetetraacetic acid (EDTA) solution for 2 weeks, embedded in paraffin, and sliced into 4 um sections. The slices were flattened in water at 40 °C and baked in an oven at 60 °C for 2 h. The sections were then dewaxed in xylene for 30 min and placed in ethanol for gradient dehydration. Then, HE staining was performed, and structural damage and tumor infiltration were observed under an optical microscope. For immunofluorescence staining, the sections were permeabilized with 0.5% Triton X-100 for 25 min and blocked with 5% BSA for 60 min. Subsequently, the sections were incubated overnight with primary antibodies at 4 °C. The next day, the slices were incubated with the recommended secondary antibodies for 1 h at room temperature in the dark. Following DAPI staining, the samples were photographed and analyzed using a fluorescence microscope (NIKON Eclipse Ci, Japan).
Cellular Immunofluorescence
BV2 cells in each group were collected, washed twice with PBS, and fixed with 4% paraformaldehyde for 30 min. Immunofluorescence staining was performed as described previously. The following antibodies were used for immunofluorescence staining: p-NF-κB p65 (Affinity, AF2006, 1:100), TLR4 (Abclonal, A5258, 1:100), Iba-1 (Abcam, ab178846, 0.5 μg/mL) Alexa Fluor 555-labeled Donkey Anti-Rabbit (beyotime, A0453, 1:500), Alexa Fluor 488-labeled Goat Anti-Rabbit (beyotime, A0423, 1:500).
Biochemical Analysis
To evaluate hepatic and metabolic functions, blood samples were collected from the abdominal aorta under deep anesthesia at the terminal time point (day 14 post-modeling). Following collection, whole blood was allowed to clot at room temperature for 30 minutes. Serum was then separated by centrifugation at 3000rpm for 10 minutes at 4°C and immediately aliquoted. The resulting supernatant (serum) was stored at −80°C until biochemical analysis.
The plasma concentrations of hepatic and renal function markers—including aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, and creatinine (Cre)—were determined using a Roche cobas 8000–702 automated clinical chemistry analyzer with dedicated reagents. A total of n = 5 independent biological replicates were analyzed for each experimental group. All assays were performed following the manufacturer’s standard protocols to ensure accuracy and reproducibility.
Western Blotting
Spinal cord tissues were harvested from rats in each group on day 14 post-modeling for Western blot analysis. The total protein of the L4-L6 spinal cord segments in rats was extracted using high-efficiency RIPA lysate (100 mg/mL) containing PMSF, and the protein concentration was determined using a BCA kit. Subsequently, the protein samples were separated by polyacrylamide gel electrophoresis (PAGE) gel and transferred to nitrocellulose (NC) membranes. The NC membrane was blocked with 5% skim milk powder for 2 h at room temperature, followed by overnight incubation with an appropriate concentration of primary antibodies at 4 °C. After 14–16 h, the NC membrane was incubated with the corresponding secondary antibodies for 1 h at room temperature. Finally, the bands were visualized with an ECL chromogenic solution and scanned using a ChemiScope 6000. A total of n = 5 independent biological replicates were analyzed for each experimental group. Protein bands were quantified using ImageJ (National Institutes of Health, USA) software. Briefly, the intensity of each target protein band was normalized to its corresponding GAPDH loading control. Data are presented as relative protein expression levels compared to the control group. The following antibodies were used: Iba-1 (Abcam, ab178846, 1:1000), TLR4 (Santa Cruz Biotechnology, sc-293072, 1:1000), NF-κB p65 (Bioss, bs-0465R, 1:1000), p-NF-κB p65 (Affinity, AF2006, 1:1000), and GAPDH (HUABIO, R1210-1, 1:5000).
ELISA
The supernatants of the BV2 cell culture medium and L4-L6 spinal cord segments were used for detection. The sample was ground in lysis buffer containing protease and phosphatase inhibitors. The homogenate was then incubated on ice for 5 min. The homogenate was ultrasonically lysed and centrifuged (15000 g, 4 °C, 15 min), and the supernatant was collected. All ELISA samples were run in triplicate to minimize technical variability and ensure data reliability. The mean value of the three technical replicates was used for statistical analysis. The concentrations of IL-1β, IL-4, TNF-α, and IL-10 were measured using corresponding ELISA kits (Rat IL-1β ELISA Kit, JiangLai biology, JL20884; Rat IL-10 ELISA Kit, JiangLai biology, JL13427; Rat IL-4 ELISA Kit, JiangLai biology, JL20894; Rat TNF-α ELISA Kit, JiangLai biology, JL13202; Mouse IL-1β ELISA Kit, JiangLai biology, JL18442; Mouse IL-10 ELISA Kit, JiangLai biology, JL20242; Mouse IL-4 ELISA Kit, JiangLai biology, JL20266; Mouse TNF-α ELISA Kit, JiangLai biology, JL10484).
Flow Cytometry
BMSCs were collected for fluorescence-activated cell sorting (FACS) staining. First, the cells were washed with 200 µL PBS. The cells were then centrifuged (1200 rpm, 4 °C, 3 min) and the supernatant was discarded. The cells were incubated with the recommended concentration of surface antibodies at 4 °C in the dark for 30 min, and staining was terminated using PBS. Finally, stained cells were detected using a flow cytometer. The following antibodies were used in this experiment: CD44, zenbio 783077; CD29, eBioscience, 11–0291-80; CD90.1, eBioscience, 17–0900-82; CD11b/c, eBioscience, 46–0110-80; and CD45, eBioscience, 48–0461-82.
RNA Sequencing
Rat spinal cords were isolated, and total RNA was extracted. The extracted total RNA was checked for purity, concentration, and RNA integrity number (RIN), and a cDNA library was constructed and amplified by PCR. After amplification and purification, the library was sequenced and subjected to quality control analysis. Subsequently, bead comparison was performed on the obtained data. Finally, differential expression and functional enrichment analyses were performed using R language. The RNA-seq was performed using the Illumina NovaSeq 6000 platform (Illumina, USA), which generates high-throughput paired-end reads (2 × 150 bp). An average of 40 million paired-end reads per sample were generated, ensuring sufficient coverage for transcriptome analysis. Differentially expressed genes (DEGs) were identified using the following thresholds: |log2 fold change (log2FC)| > 1 and false discovery rate (FDR) < 0.05, as calculated by DESeq2.
Statistical Analysis
All experiments were performed independently in triplicate (n=3). The experimental data were analyzed using GraphPad Prism 7.0. All data are expressed as mean ± standard deviation (SD). Normality of data distribution was verified using the Shapiro–Wilk test, and homogeneity of variances was assessed via Levene’s test. For comparisons between two independent groups with normally distributed data and homogeneous variances, an unpaired t-test was applied. For multiple group comparisons, one-way or two-way ANOVA was employed, with Tukey’s multiple comparison test used for post hoc analysis. In this study, P < 0.05 was considered statistically significant.
Results
Identification of BMSCs
The purity of the BMSCs was determined using various methods. As shown in Figure 2A, BMSCs appeared flat and spindle-shaped, similar to the fibroblasts. Moreover, a series of cell surface markers of third-generation BMSCs was analyzed and identified by flow cytometry. The MSC markers CD29, CD44, and CD90.1 were positive, whereas the hematopoietic markers CD45 and CD11b/c were negative (Figure 2B). Osteogenic, lipogenic, and chondrogenic assays were performed on BMSCs using the appropriate kits. Osteogenic differentiation experiments showed that BMSCs stained with Alizarin Red S, revealing a large number of orange plaques, indicating that calcium crystals and calcium nodules were formed in BMSCs, which could be used as osteogenic differentiation markers (Figure 2C). Adipogenic differentiation assays demonstrated that BMSCs accumulated abundant lipid droplets following induction, as evidenced by intense Oil Red O solultion staining, indicative of their adipogenic differentiation potential (Figure 2D). Chondrogenic differentiation assays demonstrated that BMSCs formed chondrospheres upon induction. Alcian Blue 8GX solultion staining revealed intense blue proteoglycan deposition within these nodules, indicating the formation of cartilaginous matrix rich in acidic glycosaminoglycans (Figure 2E).
Figure 2.
Identification of BMSCs. (A) The morphology of BMSCs was observed by microscope. (B) Flow cytometry was used to detect the surface markers of BMSCs. P1 represents the viable cell gate for BMSCs based on FSC/SSC characteristics, while P2, P3, and P4 respectively represent the percentages of CD29-positive (99.7%), CD44-positive (96.5%), and CD90.1-positive (94.4%) cells. Additionally, P5 and P6 respectively indicate the percentages of CD45-negative (0%) and CD11b/c-negative (14%) cells. (C) Alizarin Red S-positive deposits (Orange-red) in osteogenically induced BMSCs, indicating calcium nodule formation. (D) Oil Red O staining reveals abundant red lipid droplets in BMSCs following adipogenic induction, indicative of lipid accumulation and adipogenic differentiation. Unstained cells appear as gray-black background. (E) Chondrogenic nodules from BMSCs show strong Alcian Blue 8GX+ matrix (blue), confirming proteoglycan accumulation in differentiating cartilage.
Intrathecal Injection of BMSCs Can Effectively Alleviate BCP
Subsequently, this experiment verified the therapeutic effects of BMSCs using a BCP rat model of breast cancer. The experimental design is illustrated in Figure 1. Basic pain thresholds (before BCP modeling), 50% PWT, TWL, hind limb application score, and NSF were measured at 3, 7, 9, 11, and 13 days after BCP modeling. The results showed that the levels of 50% PWT and TWL in the BMSCs group were significantly higher than those in the BCP group, whereas the hind limb application score and NSF level in the BMSCs group were significantly lower than those in the BCP group (Figure 3A–D). These findings revealed that intrathecal injection of BMSCs can significantly alleviate the pain symptoms of BCP. Based on the 50%PWT, the optimal effective concentration of BMSCs was determined to be 2×106 (Figure 3E). Furthermore, the optimal onset time of BMSCs was the second hour after the intrathecal injection (Figure 3F).
Figure 3.
BMSCs can alleviate BCP in behavior. (i.t.): Denotes the time point of the first intrathecal treatment with BMSCs. (A) 50% PWT, (B) TWL, (C) Score of ambulatory pain, and (D) NSF were used to perform behavioral testing. (E) The Comparison of therapeutic effects of three different concentration of BMSCs. (F) Determine the peak time of efficacy. Mean ± SD, two-way ANOVA, *p<0.05, **p<0.01, vs BCP; #p<0.05, ##p<0.01, vs Sham.
BMSCs Failed to Improve the Bone Destruction Degree of BCP
Before further studying the effect of BMSCs on BCP, the effect of BMSCs on the serum biochemical indices of the rats was analyzed. The results indicated no significant changes in aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, or creatinine (Cre) levels after intrathecal injection of BMSCs, suggesting that intrathecal injection of 2×106 BMSCs had no significant effect on liver and kidney function in rats (Figure 4A). To determine whether BMSCs can alleviate pathological injury of the tibia, X-ray detection of the tibia was performed in the Sham, BCP, BCP+PBS, and BCP+BMSCs groups on days 7 and 14 after BCP model establishment. Compared with the Sham group, the three BCP model groups showed obvious bone destruction and tumor cell infiltration, with cancer cells completely infiltrating the bone marrow cavity, resulting in severe damage to the tibia. However, the infiltration of tumor cells and bone destruction in the BCP+BMSCs group were not significantly reduced compared to those in the BCP group (Figure 4B). HE staining of the BCP model on day 14 revealed that the tumor cells in the BCP group completely infiltrated the tibial medullary cavity, and the bone structure was seriously damaged, including the trabecular and cortical bones. Consistent with the X-ray results, the degree of tibial injury in the BCP+BMSCs group did not decrease significantly (Figure 4C).
Figure 4.
BMSCs can not significantly alleviate the pathological changes of BCP. (A) Effect of BMSCs on serum biochemical indexes in rats. (B) Days 7 and 14 post-BCP model, the tibia of different groups of rats was photographed by X-ray, and the red arrows show the main observation sites. (C) HE staining of tibia from Sham group and three BCP (14th day) groups, and the red box indicates the region magnified in the corresponding image below.
BMSCs Can Significantly Inhibit the Activation of Microglia in BCP
Microglia, as macrophages in the nervous system, play an indispensable role in the progression of BCP.25 To study the therapeutic mechanism of BMSCs on BCP, the activation of spinal microglia was detected. Compared to the Sham group, the relative expression level of Iba-1 in the spinal cord of the BCP group was significantly increased, suggesting activation of microglia in the BCP model. Following BMSCs treatment, the expression of Iba-1 in the spinal cord was significantly inhibited (Figure 5A and B). Similarly, immunofluorescence staining of the spinal cord confirmed a significant decrease in the expression level of Iba-1 in the BMSCs group (Figure 5C).
Figure 5.
BMSCs significantly inhibited the expression of microglia activation related indexes in BCP. (A and B) The relative expression level of Iba-1 was assessed by Western blotting. (C) Immunostaining of Iba-1 in different groups, and the red box indicates the region magnified on the right. (D-G) The results of ELISA showed the concentrations of IL-1β, TNF-α, IL-10, and IL-4 in spinal cord. Mean ± SD, one-way ANOVA, *p<0.05, **p<0.01.
The main pro-inflammatory cytokines derived from microglial activation in BCP animal models were IL-1β, TNF-α, and IL-6, whose inhibition effectively alleviated BCP.26 Therefore, the therapeutic effects of BMSCs on BCP were hypothesized to be mediated by the activation of microglia. ELISA was performed to detect the concentrations of various spinal inflammatory mediators, including IL-1β, TNF-α, IL-10, and IL-4, in the different groups. The results revealed that the above inflammatory cytokines were significantly increased in the BCP group and the BCP+PBS group, while BMSCs significantly reduced the concentrations of pro-inflammatory cytokines (IL-1β and TNF-α) and increased the concentrations of anti-inflammatory cytokines (IL-10 and IL-4) (Figure 5D–G).
RNA Transcriptome Sequencing Predicted the Underlying Mechanism of BMSCs in the Treatment of BCP
To further explore the mechanism by which BMSCs inhibit spinal cord microglial activation and neuroinflammation in BCP rats, the total RNA of the dorsal horn of the spinal cord on the same side of L4-6 was extracted in the Sham, BCP, and BCP+BMSCs groups for transcriptome sequencing. Sequencing results indicated that 1823 genes were upregulated and 1824 genes were downregulated in the BCP+BMSCs group compared with the BCP group. (Figure 6A). Subsequently, gene ontology (GO) annotation and Kyoto Encyclopedia of Genomes (KEGG) pathway analysis of the selected differentially expressed genes were conducted. For KEGG pathway enrichment analysis of differentially expressed genes, the top 20 most significant pathways were screened among the 300 enriched pathways and displayed from small to large according to their p-values. Among these 20 pathways, TNF and NF-κB signaling pathways were closely related to the inflammatory response, as shown in Figure 6B. In addition, the GO analysis results indicated that in terms of cell biology process (BP), the above-mentioned differentially expressed genes were mainly enriched in the regulation of inflammatory response, regulation of immune effector process, and leukocyte migration, which was consistent with the KEGG analysis results (Figure 6C). Therefore, the NF-κB signaling pathway may be a key target for BMSCs to exert inhibitory neuroinflammatory and analgesic effects in rats with BCP.
Figure 6.
Screening differentially expressed genes by RNA sequencing and related pathway enrichment analysis. (A) Volcano plot of DEGs between BCP+BMSCs and BCP groups. (B) KEGG enrichment analysis of DEGs in group BCP and BCP+BMSCs. (C) GO function annotation of DEGs in group BCP and BCP+BMSCs.
BMSCs Can Significantly Inhibit the Up-Regulated TLR4/NF-κB Signaling Pathway in BCP
Previous studies have reported that microglia can release a variety of inflammatory mediators, including cytokines and chemokines, which activate specific receptors such as TLR4. TLRs recognize pathogen or injury signals and activate downstream signaling pathways, leading to inflammation and the activation of immune cells. After activation of TLRs, a variety of signaling pathways can be activated, including NF-kB and MAPK. These pathways further promote the production of inflammatory factors such as TNF-α, ILs, etc., which play a role in regulating inflammation and immune response.27 In neuropathic pain, TLRs, particularly TLR4, are thought to be involved in pain production and maintenance by activating microglia and releasing inflammatory mediators, such as TNF-α. In addition, TLR4 and NF-κB are key components of the intracellular signaling pathway and play an important role in the occurrence and maintenance of pain.28 Activation of TLR4 and NF-κB can enhance the excitability of spinal cord neurons after nerve injury, leading to pain hypersensitivity and sensation. Therefore, the TLR4/NF-κB signaling pathway plays an essential role in the development of neuropathic pain, which has been validated in BCP.
As shown in Figure 7A and B, upregulated TLR4 expression, and NF-κB phosphorylation levels in the BCP model were significantly inhibited by BMSCs. In addition, immunofluorescence analysis showed that Iba-1 co-localized with p-NF-κB p65 and TLR4. Consistent with the Western blot results, the expression levels of p-NF-κB p65 and TLR4 were significantly upregulated in spinal cord microglia in the BCP group, whereas BMSCs was significantly inhibited in BMSCs (Figure 7C and D).
Figure 7.
BMSCs significantly inhibited the expression of TLR4/NF-κB signaling pathway related markers in BCP. (A and B) The relative expression level of TLR4, p-NF-κB p65, and NF-kB p65 proteins were determined by Western blotting. (C and D) Co-localization staining of Iba-1 and p-NF-κB p65/TLR4 in spinal dorsal horn, and the red box indicates the region magnified on the right. Mean ± SD, one-way ANOVA, *p<0.05, **p<0.01.
BMSCs Inhibit BV2 Cell Activation by Suppressing the TLR4/NF-kB Signaling Pathway in vitro
To investigate the effects of BMSCs on microglial activation in vitro, a BV2-BMSCs co-culture model was established. The experimental groups included Control, BMSCs, LPS, BMSCs+LPS, and BMSCs + LPS + PMA groups.
First, we assessed the activation state of BV2 cells. Stimulation with LPS significantly activated BV2 cells, as evidenced by significantly increased Iba-1 expression (Figure 8A and B) and morphological changes observed via immunofluorescence (Figure 8C). ELISA results showed that the secretion of pro-inflammatory cytokines ((IL-1β and TNF-α)) was significantly increased in the LPS group, whereas BMSCs effectively reversed this trend by reducing pro-inflammatory cytokines and increasing anti-inflammatory cytokines (IL-4 and IL-10) (Figure 8D–G).
Figure 8.
The effect of BMSCs on BV2 was verified in vitro. (A and B) The relative expression levels of Iba-1 in different groups by Western blotting. (C) Immunostaining of Iba-1 in different groups. (D–G) The concentration of IL-1β, TNF-α, IL-4, IL-10 was measured by ELISA. Mean ± SD, one-way ANOVA, *p<0.05, **p<0.01, ***p<0.001.
Next, to elucidate the underlying molecular mechanism, we examined the TLR4/NF-κB signaling pathway. The results revealed that the expression of p-NF-κB p65 and TLR4 in BV2 cells was significantly upregulated in the LPS group compared to the control group. Notably, BMSCs treatment significantly downregulated the expression of these proteins and inhibited the nuclear translocation of NF-κB p65 (Figure 9A–D). These findings suggest that BMSCs may exert their regulatory effects by suppressing the TLR4/NF-κB pathway.
Figure 9.
The effect of BMSCs on TLR4/NF-κB signaling pathway in BV2 cells. (A and B) The relative expression levels of TLR4, p-NF-κB p65, NF-κB p65 in different groups by Western blotting. (C and D) Immunostaining of p-NF-κB p65 and TLR4 in different groups. Mean ± SD, one-way ANOVA, *p<0.05, **p<0.01.
To further confirm this mechanism, we performed a rescue experiment using PMA, an activator of NF-κB. As shown in Figures 8 and 9, the addition of PMA significantly reversed the inhibitory effects of BMSCs. Specifically, the expression levels of Iba-1, p-NF-κB p65, and TLR4 were significantly upregulated in the BMSCs+LPS+PMA group compared to the BMSCs+LPS group, accompanied by a significant increase in pro-inflammatory cytokine secretion. These results demonstrate that BMSCs inhibit BV2 cell activation, at least in part, by suppressing the TLR4/NF-κB signaling pathway.
Discussion
Bone tissue is the metastatic site of lung cancer, breast cancer, prostate cancer, and many other cancers.29 About 75%~90% of patients with advanced cancer suffer from chronic pain symptoms, which significantly affects the daily activities of patients.30,31 Pain is mainly classified into acute pain and chronic pain, and chronic pain may be further categorized into chronic primary pain, chronic cancer-related pain, chronic postoperative or post-traumatic pain, chronic secondary musculoskeletal pain, chronic secondary visceral pain, chronic neuropathic pain, chronic secondary headache, or orofacial pain.32 Among them, cancer-related pain can generally be divided into three categories according to its cause: pain caused by cancer itself (tumor-invasive pain, metastatic pain), treatment-induced pain (chemotherapy-related pain, radiotherapy-related pain, surgery-related pain), and other causes (cancer complications, psychological factors).33 Currently, the treatment of cancer pain in clinics mainly includes drug treatment and non-drug treatment. Drug treatment remains the core of cancer pain management, especially for opioids. Opioids such as codeine and tramadol are suitable for moderate pain, while morphine, oxycodone, and fentanyl are used for moderate to severe pain.34 However, opioids can slow down or prevent bone remodeling and even increase bone fragility, which greatly hinders the treatment of bone cancer.33 In addition, these drugs have large individual differences, poor long-term analgesic effects, and a large number of complications.35 Therefore, finding an effective treatment for BCP remains an urgent issue.
According to recent research, BMSCs, a type of tissue stem cell with self-renewal ability and multi-directional differentiation potential, are considered a promising approach for treating a variety of conditions, including neuropathic pain, spinal cord injury, Alzheimer’s disease, stroke, and lung injury.22,36–39 For example, a meta-analysis by Anil et al systemically summarized 79 randomized controlled clinical trials (involving 8761 patients with knee osteoarthritis) and confirmed that over a year-long follow-up, stem cell injection exhibited the most significant improvement in pain and function in patients with knee osteoarthritis.40 Importantly, multiple studies have demonstrated that BMSCs can significantly alleviate a variety of neuropathic pain, including BCP. Yang et al found that BMSCs could sense and respond to an inflammatory microenvironment. Intrathecal BMSCs sustain the release of TSG-6 in response to nerve injury-induced neuroinflammation in the dorsal horn of the spinal cord, which downregulates the TLR2/MyD88/NF-κB signaling pathway and reduces the production of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α, resulting in sustained neuropathic pain relief.23 Although previous studies demonstrated that BMSCs can effectively alleviate BCP by inhibiting the activation of glial cells and reducing neuroinflammation, its specific molecular mechanism remains unclear.41,42 Therefore, the relieving effect of BMSCs on BCP was analyzed, exploring the mechanism of BMSCs in treating BCP.
The observed radiographic enlargement of the tibia in the BCP+BMSCs group is most likely attributable to a combination of tumor-induced bone remodeling and reactive bone formation, rather than tumor burden alone. In our BCP model, intra-tibial inoculation of tumor cells triggers osteolytic and mixed (osteolytic/osteoblastic) lesions, leading to disorganized bone architecture, cortical thickening, and periosteal reactions — all of which can contribute to increased bone diameter on X-ray. Notably, while BMSCs have been shown neuroprotective effects in our study (eg, reduced microglial activation, attenuated pain hypersensitivity), they may also modulate local bone microenvironment through paracrine signaling, potentially promoting stromal or reparative responses. This could result in increased bone matrix deposition or callus-like formation, especially in the context of ongoing tumor-bone interaction. Such changes may appear as radiographic “enlargement” even in the absence of increased tumor volume. A notable finding of this study is the dissociation between pain relief and tumor progression. Specifically, intrathecal injection of BMSCs exerted a significant analgesic effect on BCP, yet failed to significantly inhibit peripheral tumor cell infiltration or bone destruction. This observation highlights a critical concept: modulation of neuroinflammation may be sufficient to alleviate cancer pain, independent of underlying peripheral pathology. This has profound clinical implications. In advanced cancer patients, complete tumor eradication is often unattainable. Our data suggest that targeting spinal microglial activation and the associated neuroinflammatory cascade represents a viable strategy for symptomatic relief, even in the absence of disease-modifying effects. It shifts the therapeutic focus from attacking the tumor mass to modulating the host’s immune response within the central nervous system, offering a complementary approach to conventional oncological treatments. According to the literature, the mechanism of BCP primarily includes nociceptive and neurological components involving the peripheral and central nervous systems, glial cell regulation, and excessive activation of osteoclasts.43,44 Microglia play a pivotal role in the hyperactivity of the central nervous system and the changes in neurons in the dorsal horn after nerve injury.45 Specifically, when tumor-associated biomolecules cause neuronal damage or inflammation and microglia are activated, a large number of neuroactive factors and inflammatory factors are released. These include reactive oxygen species (ROS), IL-1β, IL-6, TNF-α, MMP, chemokines, ATP, and NO. On the one hand, these active substances act on the corresponding receptors on neurons, causing pain. By contrast, they activate microglia and astrocytes, produce positive feedback, and ultimately participate in the induction and maintenance of chronic pain. ATP is released from damaged cells and affects surrounding cells (especially neuronal cells) via cell-surface P2 receptors as intercellular mediators.46,47 Our preliminary studies have revealed that microglia are significantly activated and polarized towards the M1 phenotype in the spinal cords of rats with BCP. The mechanisms driving microglial activation are related to ATP released by neurons acting on the P2X7 receptor of microglia.10 Therefore, an imbalance in microglial polarization promotes the development of neuroinflammation. Activated microglia can release pro-inflammatory factors such as IL-1β and TNF-α, triggering a cascade of inflammation. Moreover, suppressing the activation of microglia in the dorsal horn of the spinal cord can effectively inhibit the neuroinflammatory response and alleviate pain.48 To explore whether BMSCs can alleviate BCP by inhibiting this process, the activation of microglia in the spinal dorsal horn was detected. The results indicated that intrathecal BMSCs significantly decreased the expression level of Iba-1 in the spinal dorsal horn and significantly inhibited the secretion of pro-inflammatory cytokines. BMSCs inhibited microglial activation in the spinal dorsal horn during BCP treatment However, mechanisms underlying the inhibitory effects of BMSCs on microglial activation and inflammation remain unclear.
Growing evidence has revealed that BMSCs can treat various neuroinflammatory diseases by regulating the TLR4/MyD88/NF-κB signaling pathway.23,49 To further confirm whether BMSCs regulate this signaling pathway during BCP treatment, total RNA in the rat spinal cord was analyzed. The differentially expressed genes in the BMSCs group were mainly involved in inflammatory response-related pathways, such as the complement and coagulation cascade, TNF, and NF-κB signaling pathways. In addition, Western blotting and immunofluorescence revealed that BMSCs significantly inhibited the upregulation of TLR4 expression and NF-κB phosphorylation in the BCP model. BMSCs may also play a role in BCP treatment by inhibiting the TLR4/NF-κB signaling pathway. TLR4, the main LPS receptor among the Toll-like receptors, can be activated by LPS.50 After recognizing LPS, TLR4 forms an activated heterodimer TLR4/MyD88 complex via the MyD88 pathway, thereby initiating intracellular signal transduction. Moreover, this process activates the inhibitor of κB (IκB) and IκB kinase (IκK) and facilitates the activated NF-κB to enter the nucleus, which promotes the release of cytokines.51 Furthermore, whether BMSCs inhibit this process was investigated using the BV2-BMSCs model. The results indicated that BV2 cells highly expressed the TLR4 protein after LPS treatment, and the phosphorylation level of NF-κB was also significantly upregulated. Notably, addition of BMSCs significantly reversed the effects of LPS. In addition, nuclear transport staining of NF-κB showed that LPS could induce the nuclear translocation of NF-κB p65 from the cytoplasm, and BMSCs could also significantly inhibit the nuclear translocation of NF-κB p65. Nuclear translocation is a key step in microglial activation and plays an essential role in the regulation of inflammatory and immune responses, cell proliferation, and apoptosis. Importantly, the NF-κB agonist (PMA) was added to the BV2 cell culture system, and the inhibitory effects of BMSCs on BV2 activation and the TLR4/NF-κB signaling pathway were significantly reversed. Collectively, BMSCs can inhibit inflammation by inhibiting the TLR4/NF-κB signaling pathway in microglia during BCP treatment, thereby relieving pain.
The therapeutic efficacy of BMSCs in alleviating BCP is well demonstrated in this study, with mechanistic evidence pointing to the suppression of spinal microglial activation and the TLR4/NF-κB signaling pathway. BMSCs exert potent anti-inflammatory and immunomodulatory effects predominantly through paracrine secretion of bioactive molecules, including growth factors, anti-inflammatory cytokines (eg, IL-10, TGF-β), and extracellular vesicles such as exosomes, which carry functional miRNAs, mRNAs, and proteins that modulate target cell activity and promote tissue repair and anti-inflammatory responses. These mediators collectively inhibit the expression and release of pro-inflammatory cytokines, reduce inflammatory cell infiltration, and thereby attenuate neuroinflammation.52–54 While the TLR4/NF-κB pathway appears central to BMSCs-mediated analgesia, it is unlikely to act in isolation. Spinal neuroinflammatory processes are orchestrated by a complex network of signaling cascades—including MAPK, JAK/STAT, and NLRP3 inflammasome pathways—that may also influence microglial polarization and pain modulation. Thus, the observed effects may involve crosstalk between multiple pathways, warranting further investigation into their potential interactions.
A key limitation of this study is the inability to identify the specific paracrine factor(s) responsible for TLR4/NF-κB inhibition in microglia. Although co-culture and cytokine array data implicate candidates such as TSG-6, PGE2, and IL-1RA, functional validation through loss-of-function approaches (eg, siRNA knockdown or neutralizing antibodies) was not performed. Therefore, future studies should aim to delineate the precise molecular mediators underlying BMSCs-induced analgesia and immunomodulation, which could reveal novel, druggable targets for BCP intervention. Notably, radiographic tibial enlargement observed in the BCP+BMSCs group did not correlate with exacerbated pain behaviors; on the contrary, pain hypersensitivity was reduced. Nevertheless, the long-term structural and functional implications of this morphological change remain unclear. To better understand the balance between tumor-driven osteolysis and potential reparative bone formation, future work should integrate longitudinal micro-computed tomography (micro-CT) with biomechanical testing and quantitative locomotor assessments. Furthermore, incorporating serial monitoring of circulating bone turnover markers (eg, CTX-I, PINP, TRACP-5b) would enable non-invasive, real-time evaluation of skeletal remodeling dynamics. Such a multimodal approach is essential to assess not only symptomatic relief but also disease-modifying potential, thereby providing a more comprehensive and clinically relevant evaluation of BMSCs-based therapies within the complex tumor-bone microenvironment.
Conclusions
In summary, our study demonstrates that intrathecal injection of BMSCs significantly alleviates BCP. Importantly, BMSCs markedly inhibit microglial activation in the spinal dorsal horn, a process closely associated with the TLR4/NF-κB signaling pathway. Although this therapy does not appear to reverse tumor progression or skeletal damage, it reveals a novel mechanism of BMSCs in treating BCP and provides deeper insights for future targeted therapies.
Funding Statement
This work was supported by the Liaoning Provincial Key Research and Development Program (2024JH2/102500038), National Natural Science Foundation of China (82474277), United Foundation for Dalian Institute of Chemical Physics, Chinese Academy of Sciences, and First Hospital of Dalian Medical University (DMU-1 & DICP UN202402), Basic Scientific Research Project of the Education Department ofLiaoning Province (LJ212510161045).
Abbreviations
ALT, alanine aminotransferase; AST, aspartate aminotransferase; BCP, bone cancer pain; BMSCs, bone marrow mesenchymal stem cells; BP, biology process; Cre, creatinine; EDTA, ethylene diamine tetraacetic acid; FACS, fluorescence-activated cell sorting; GO, gene ontology; IL, interleukin; IκB, inhibitor of κB; KEGG, Kyoto Encyclopedia of Genomes; MAPK, mitogen-activated protein kinase; MSC, Mesenchymal stem cell; NC, nitrocellulose; NSF, the number of spontaneous flinches; PAGE, polyacrylamide gel electrophoresis; PMA, phorbol-12-myristate-13-acetate; PWT, paw withdrawal threshold; RIN, RNA integrity number; ROS, reactive oxygen species; TLR4/ NF-κB, Toll-like receptor 4/ nuclear factor-kappa B; TNF-α, tumor necrosis factor-α; TWL, thermal withdrawal latency.
Data Sharing Statement
The data are made available upon request. All data generated or analyzed during this study are included in this published article files.
Author Contributions
All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.
Disclosure
The authors declare that they have no known competing financial interests or personal relationships that could influence the work reported in this study.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data are made available upon request. All data generated or analyzed during this study are included in this published article files.









