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. 2025 Jul 1;15:20507. doi: 10.1038/s41598-025-02588-6

Sphingosine 1-phosphate derived from tumor-educated hepatic stellate cells combining with S1PR4 promotes tumor associated macrophages differentiation through FAO modulation

Rui Feng 1, Zilin Cui 1,✉, Long Yang 1, Zirong Liu 1
PMCID: PMC12218931  PMID: 40592997

Abstract

The tumor immune microenvironment (TIME) is significance to the occurrence and development of tumors. Macrophages, making great contributes to TIME, develop into tumor-associated macrophages (TAM) under the influence of the tumor microenvironment (TME), resulting in altered metabolic pathways. Sphingosine 1-phosphate (S1P) is involved in immune regulation as a lipid metabolite. The role of S1P in the differentiation and metabolic regulation of tumor-associated macrophages is unknown. Meanwhile, the source of S1P in TME is not very clear. Our research found that hepatic stellate cells co-cultured with tumor cells could prompt macrophages to the M2 phenotype of TAM differentiation. It was further discovered that S1P activated peroxisome proliferator-activated receptor α (PPARα) by binding to S1P receptor 4 (S1PR4) of macrophages, upregulating lipid metabolism and inducing the TAM differentiation. Ultimately, tumor cells activated nuclear factor erythroid 2-related factor 2 (Nrf2) in hepatic stellate cells (HSCs), enhancing sphingosine kinase 1 (SphK1) expression and elevating S1P production and secretion. This study has demonstrated a possible interaction pathway among tumor cells, HSCs and macrophages. It has revealed that tumor cells activate the Nrf2/SphK1 pathway in HSCs to secrete S1P, which subsequently bound S1PR4, triggered PPARα activation, and drove macrophage polarization toward pro-tumor M2-type TAMs.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-02588-6.

Subject terms: Cancer metabolism, Cancer microenvironment

Introduction

The tumor microenvironment (TME) is the structure of the cells in which the tumor is situated. The composition of the TME is influenced by immune and nonimmune cells, the extracellular matrix (ECM) as well as soluble and physical characteristics such as pH acidity and hypoxia1–3. As a result of the heterogeneity of immune cells within the TME, the biological properties of tumor are various, and treatment resistance and failure are common outcomes4. Within the tumor microenvironment, macrophages participate in the immune response of tumors exhibiting a variety of phenotypes5. The classical or M1 phenotype of tumor-associated macrophages (TAMs) is characterized by the secretion of IL-1 and IL-6, which has a pro-inflammatory characteristic. The alternative or M2 macrophage phenotype, which primarily expresses Arg1 and CD163, is known for its anti-inflammatory feature. It is believed that TME potentiates immune suppressive M2 TAMs through the production of cytokines, thereby facilitating the growth of tumors. According to current research, many soluble mediators present in the tumor microenvironment contribute to the differentiation of macrophages to the M2 phenotype6.

It is essential that lipid metabolism plays a role in the production of cellular biomass and energy. The availability of lipids and their metabolites in the TME alters a number of immune related pathways in the TME that allow tumors growth and progression7. In particular, lipids influence the production, differentiation, and function of TAMs in the TME. According to pervious reports, macrophages with an immunosuppressive phenotype prefer fatty acid oxidation (FAO) over glycolysis as an energy source8. There has been an established correlation between the metabolic activities of lipids and the differentiation of the M2 phenotype in previous studies9. It has been showed that FAO was necessary to promote the polarization of macrophages in M210. Additionally, TAM subpopulations can be distinguished on the basis of the transcription factor pivotal for FAO11. As a result of these findings, it is apparent that the interplay between lipid metabolism and immune modulation in the TME is complex, and indicates potential therapeutic targets for the treatment of cancer. Nonetheless, it is unclear exactly how lipid metabolism plays a role during macrophage phenotype differentiation in the TME.

Sphingosine 1-phosphate (S1P), which results from the metabolism of sphingolipids by sphingosine kinase 1/2 (SphK1/2), participates in TME remodeling both intracellularly and extracellularly. In addition to interacting with specific intracellular target proteins, S1P can be released from cells to bind to S1P-specific G protein-coupled receptors (S1PR1-5), causing cell proliferation, migration, differentiation, and immune responses (including monocytes, dendritic cells, and macrophages). As a result of inflammatory responses, high S1P levels and sphingolipid signaling activation can be observed in tissues and organs that are closely associated with inflammation modulation, such as certain cancers that are associated with chronic inflammation. There is evidence that S1P may reduce inflammatory factors such as IL-1β and IL-6 or increase IL-10 expression12. Based on the previous studies, there appears to be a relationship between S1P-related signal and macrophage phenotype13. However, there is rare research on whether S1P is related to macrophage differentiation.

This work involved phenotypes validation after stimulating macrophages with conditioned media from hepatocellular carcinoma (HCC) cells and hepatic stellate cells (HSCs) microspheres. Then the role of S1P of macrophage differentiation and metabolism modulation were detected. Furthermore, the mechanism of S1P secretion were detected during cancer cells and HSCs communication.

Methods

Tissue samples

In the period from January 1, 2021 to December 31, 2021, tissue samples were collected from patients who underwent hepatectomy for HCC. The tissues were all stored at 80 °C. Tianjin First Central Hospital’s Ethics Committee approved the study involving human samples (Registration number: 2022N077KY). Research involving human research participants must have been performed in accordance with the Declaration of Helsinki.

Cell isolation

Tumor tissues (1–2 cm3 in size) from the surgical patients were placed into a sterile culture dish containing 10 mL of RPMI-1640 medium (serum-free), and remove non-tumor and necrotic tumor tissue. The tumor tissues were cut the tumor into small pieces (2–3 mm3 in size) in a new culture dish and re-suspend in 20 mL of serum-free RPMI-1640 medium. After centrifugation at 1200 rpm for 6 min at room temperature, the collected tumor fragments were digested with 10 mL of tumor tissue digestion solution at 37 °C for 30 min. The digested suspension was filtrated, and the precipitate is then collected by centrifugation. The cell suspensions were spread over the surface of the tumor cell purification solution. After centrifugation, the purified solution containing tumor cells at the bottom was collected. The collected cells were cultured in DMEM containing 10% fetal bovine serum for 3–4 days for use.

Normal liver tissues around the patient’s tumor were obtained and placed in DMEM/F-12 culture medium, and cut into small pieces of approximately 2 mm3 in size. The divided pieces were placed in collagenase solution (3 ml of 0.075% collagenase in 24 ml of Grignard’s balanced salt solution, 0.002% DNase I) and digested at 37 °C for 20–30 min. The cells were suspended in 1 ml of Gey’s balanced salt solution and 3 ml of Optiprep was added as a bottom layer, which was covered with 5 ml of 11.5% OptiPrep solution and the top layer was covered with 2 ml of Gey’s balanced salt solution. The cells were centrifuged at 3000 rpm for 17 min, and the cells between the Gey’s balanced salt solution and the 11.5% OptiPrep solution were collected. The separated cells were identified by detecting the expression of FAP and a-SMA.

The microspheres of HCC and HSC is formed by co-culture with HCC cells (104 cells per-well) and HSCs ((2*104 cells per-well)) in low-attachment 96-well dish for 72 h.

Human monocyte cell line, THP-1, gotten from Culture Collections of Public Health England, grew in RPMI-1640 containing 10% FBS and 10 mM HEPES. THP-1 cells were treated with 150 nM phorbol-12-myristate-13-acetate (PMA) (Sigma, St. Louis, MO, USA) for 48 h to induce to macrophages.

Conditioned medium (CM) of cells collection

Following two times washes with pre-warmed PBS, the cells were incubated with the FBS-free medium. Incubation medium was collected after 24 h, centrifuged (3000 rpm) for 5 min, and filtered through 0.2 microns filters. In this study, it was not necessary to supplement the conditioned medium with additional FBS.

Cell viability

A Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Tokyo, Japan) was used to detect the viability or proliferation of the cells. The cells were cultured with 10% CCK-8 culture media for a duration of 1–4 h in an incubator. The plate reader (SpectraMax i3; Molecular Devices, Tokyo, Japan) was employed to measure the absorbance at 450 nm.

Flow cytometry

The samples of the cells were collected and washed with PBS (phosphate-buffered sodium). Staining of the cells was carried out using the FITC Annexin-V Apoptosis Detection Kit (R&D Systems, Minneapolis, MN) in accordance with the manufacturer’s instructions. The collected cells stained with Annexin-V-FITC (0.25 µg/ml) and PI for 15 min at room temperature in the absence of light. Within 30 min of staining, the labeled cells were analyzed by flow cytometry using a large gate established based on forward and side scatters.

The Fc receptors of the cells were inhibited with 100 μg/mL rat IgG (R5381, Sigma) for 10 min at 4 °C. After dilution to a concentration of 1*106 cells in a milliliter, the suspension was stained with APC-CD163 (ab134416, Abcam) and FITC-CD206 (ab270647, Abcam) at a concentration of 5 μL per million cells in accordance with the manufacturer’s instructions. Labelled cells were examined using flow cytometry.

Polymerase chain reaction

In accordance with the manufacturer’s instructions, total RNA was extracted from each sample using the RNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA was generated with the reverse transcription kit from Applied Biosystems, Thermo Fisher Scientific Inc., Waltham, MA, USA. The following primers from TaqMan gene expression assays (assay identification number) were used: PPARα (Hs00947536_m1), S1PR4 (Hs02330084_s1), CD206 (Hs07288635_g1), IL-10 (Hs00961622_m1) and Arg-1 (Hs00163660_m1). The internal control was GAPDH (Hs02786624_g1). The qRT-PCR was conducted using the StepOnePlus Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific Inc., Waltham, MA, USA).

Western blotting

Cell samples were extracted and analyzed for protein concentration using a BCA kit (ab102536, Abcam). Protein concentrations were standardized prior to separation on 12% SDS-PAGE gels and subsequently transferred to Polyvinylidene difluoride (PVDF) membranes (#1,620,264, Bio-Rad). The membranes were incubated with primary antibodies, specifically ACAA1 (44,276), ALOX5 (3289), CPT2 (52,552), IDO1 (8137), BAX (2772), Bcl2 (15,071), Caspase3 (6992), SphK1 (12,071), α-SMA (19,245), Vimentin (5471), Nrf2 (20733S), HO-1 (70,081), NQO-1 (62,262) and GAPDH (2118), from Cell Signaling, and PPARα (ab24509), ACAA2 (ab128929) from Abcam, S1PR4 (BGT-ANT-36320,Biogradetech, USA) at a dilution of 1:100. The proteins were identified using chemiluminescence following a one-hour incubation with the secondary antibody.

Oxygen consumption

The oxygen consumption rate was determined using the Seahorse XFe bioanalyzer. The cells were seeded into 96-well Seahorse plates with RPMI medium (containing 10 mM glutamines, 10 mM sodium pyruvate, and 25 mM glucose) and cultured at 37 °C. The following compounds were administered: 1 μM oligomycin, 1.5 μM fluorocarbonyl cyanide phenylhydrazone, and 100 nM rotenone combined with 1 μM antimycin A. The Extracellular Flus Analyzer (Seahorse Bioscience) was utilized for measurements.

Extracellular acidification rate measurement

The sample cells were inoculated in a Seahouse XF24 cell culture plate at density 30,000 cells per-well overnight at 37℃ with 5% CO2. After the first day of culture, the culture medium was changed to Seahorse XF RPMI Medium (Agilent, Cat. 103,576–100) supplemented with 11 mM glucose and 2 mM L-glutamine. After one hour of culture in a CO2-free incubator, the assay was conducted. In the following three measurements (3–0-3, mix-wait-measure cycle), the extracellular acidification rates (ECAR) were obtained after the instrument calibration.

Statistical analysis

Data were presented as mean ± SD, and statistical analyses were conducted using SPSS 21.0 (IBM). For the comparison the differences between the two groups, the Student’s t-test was used. The differences among more than two groups were analyzed using the one-way analysis of variance followed by Bonferroni’s test. The significance of the result was determined by a P-value below 0.05.

Results

To ascertain if HCC-HSC microspheres or the conditioned medium of HCC-HSC microspheres can facilitate the differentiation of macrophages into the M2 phenotype, we initially fabricated cell microspheres utilizing isolated liver cancer cells from clinical samples and HSCs from liver tissues. M0 macrophages were subsequently grown with the HCC-HSC microspheres or conditioned medium from HCC-HSC microspheres for 48 h. Macrophages did not exhibit death remarkably during the culture period, and the cell cycle ratio shown no notable change in comparison to the control group (Fig. 1A–B). Examination of apoptotic indicators demonstrated that each culture condition did not show significant apoptosis (Fig. 1C–D). Upon eliminating the influences of cell cycle modulation and proliferation, the cell counts that exhibited M2 phenotype indicators were identified. A significant elevation of CD163 and CD206 positive cells was noted in the co-cultured and CM stimulation groups, in contrast to the control group and tumor cells and HSC single cell culture media (Fig. 1E–F). Likewise, macrophages in the conditioned medium of HCC-HSC microspheres and co-culture with HCC-HSC microspheres exhibited a markedly elevated expression of M2 subtype marker markers (Fig. 1G). The data suggested that extracellular components significantly influenced macrophage differentiation.

Fig. 1.

Fig. 1

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HCC-HSC microspheres influenced macrophage differentiation. (A) The cell viability of macrophages cultured with normal medium (Ctrl), stimulated with mixed conditioned medium of HCC and HSC (HCC-CM + HSC-CM), stimulated with conditioned medium of HCC-HSC microspheres (HCC-HSC-CM) or co-cultured with HCC-HSC microspheres (HCC-HSC) (n = 5). (B) The cell cycle analysis of macrophages cultured with normal medium (Ctrl), stimulated with mixed conditioned medium of HCC and HSC (HCC-CM + HSC-CM), stimulated with conditioned medium of HCC-HSC microspheres (HCC-HSC-CM) or co-cultured with HCC-HSC microspheres (HCC-HSC) (n = 3). (C) and (D) The protein levels of Bax, Bcl-2 and Caspase3 of macrophages cultured with normal medium (Ctrl), stimulated with mixed conditioned medium of HCC and HSC (HCC-CM + HSC-CM), stimulated with conditioned medium of HCC-HSC microspheres (HCC-HSC-CM) or co-cultured with HCC-HSC microspheres (HCC-HSC) (n = 3). (E) and (F) The CD163 positive (CD163-APC) and CD206 positive (CD206-FITC) cell counts by flow cytometry (n = 3). (G) The mRNA levels of Arg-1, CD206 and IL-10 of macrophages cultured with normal medium (Ctrl), stimulated with mixed conditioned medium of HCC and HSC (HCC-CM + HSC-CM), stimulated with conditioned medium of HCC-HSC microspheres (HCC-HSC-CM) or co-cultured with HCC-HSC microspheres (HCC-HSC) (n = 3). The differences were analyzed using the one-way analysis of variance followed by Bonferroni’s test. * Represents the significant difference between two groups (p < 0.05).

Prior research has demonstrated a strong correlation in macrophages between lipid metabolism and cellular differentiation14. It revealed that both the conditioned medium from HCC-HSC microspheres and the co-culture of HCC-HSC microspheres remarkedly enhanced the expression of lipid metabolism-related genes in macrophages compared to the control group (Fig. 2A–B). Lipid metabolism in cellular energy metabolism utilizes more oxygen than glycolysis15. It was confirmed that the conditioned medium from HCC-HSC microspheres and co-culture of HCC-HSC microspheres greatly enhanced oxygen consumption (Fig. 2C). In a standard cell culture condition, the ECAR is primarily influenced by lactic acid produced during glycolytic energy consumption. The increase in ECAR indicates a pronounced glycolytic metabolic profile16. Figure 2D demonstrates that, in comparison to the control group, the extracellular acidification rate of macrophages stimulated with HCC-HSC conditioned medium and co-culture of HCC-HSC microspheres were markedly diminished, suggesting that HCC-HSC conditioned medium enhanced the lipid metabolism of macrophages. The data suggest that extracellular components modulated the lipid metabolism of macrophages.

Fig. 2.

Fig. 2

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HCC-HSC microspheres modulated the lipid metabolism of macrophages. (A) and (B) The protein levels of ACAA1, ACAA2, ALOX5, CPT2 and IDO1 of macrophages stimulated with normal culture medium (Ctrl), co-cultured with HCC-HSC microspheres (HCC-HSC) or stimulated with conditioned medium of HCC-HSC microspheres (HCC-HSC-CM) (n = 3). (C) The oxygen consumption rates of macrophages stimulated with normal culture medium (Ctrl), co-cultured with HCC-HSC microspheres (HCC-HSC) or stimulated with conditioned medium of HCC-HSC microspheres (HCC-HSC-CM) (n = 3). (D) The extracellular acidification rate of macrophages stimulated with normal culture medium (Ctrl), co-cultured with HCC-HSC microspheres (HCC-HSC) or stimulated with conditioned medium of HCC-HSC microspheres (HCC-HSC-CM) (n = 3). The differences were analyzed using the one-way analysis of variance followed by Bonferroni’s test. * Represents the significant difference between two groups (p < 0.05).

To identify the components derived from HCC-HSC microspheres involved in macrophage differentiation, the HCC-HSC microsphere conditioned medium underwent three thermal cycling treatment (− 80 °C, 60 °C). This method was employed to denature the tertiary and quaternary structures of protein components in the culture medium and to inactivate proteins in various research17. The conditioned medium subjected to thermal cycling was found to still facilitate macrophage differentiation, suggesting that the non-protein constituents in the medium were responsible for this process (Fig. 3A). The conditioned media were also filtered to remove protein components using molecular sieves. The differentiation of macrophages into the M2 phenotype was also not influenced by the protein removed (Fig. 3 A). S1P is abundantly expressed in the tumor microenvironment, and possesses immunomodulatory effects18. The result of ELISA revealed that HCC-HSC microspheres released a greater quantity of S1P within 24 h compared to culture alone (Fig. 3B).

Fig. 3.

Fig. 3

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S1P derived from HCC-HSC microspheres might influence macrophage differentiation. (A) The mRNA levels of Arg-1, CD206 and IL-10 of macrophages stimulated with HCC-HSC microsphere conditioned medium (HCC-HSC-CM), HCC-HSC microsphere conditioned medium underwent three freeze–thaw cycles (− 80 °C, 60 °C) (Thermo-cycle-CM) or filtrated HCC-HSC microsphere conditioned medium (HCC-HSC-CM) (Protein-RM-CM) (n = 3). (B) The S1P levels of 12 and 24 h-cultured medium of HCC (HCC-CM), HSC (HSC-CM) or HCC-HSC microsphere (HCC-HSC-CM) (n = 3). The differences among more than two groups were analyzed using the one-way analysis of variance followed by Bonferroni’s test. The Student’s t-test was used to compare the difference between the two groups. * Represents the significant difference between two groups (p < 0.05).

S1P modulates intracellular downstream pathways primarily through its interaction with S1P receptors (S1PR)1–5. S1PR4 is present on immune cells, such as T cells and macrophages, suggesting that S1P in conditioned media may facilitate macrophage development via S1PR419. To evaluate the role of S1P-S1PR4 in macrophage differentiation, shRNAs were employed to downregulate S1PR4 expression in macrophages (Fig. 4 A). Following the confirmation that S1PR4 knockdown did not influence macrophage proliferation and apoptosis, the conditioned media from HCC-HSC microspheres were employed to activate S1PR4−/− macrophages (Fig. 4B–E). In comparison to wild-type macrophages, M2 phenotype markers showed significant down-regulation (Fig. 4F). Also, the number of CD163 and CD206 positive cells markedly decrease (Fig. 4 G–H). These findings demonstrated that HCC-HSC microspheres could facilitate the differentiation of macrophages into the M2 phenotype via S1P-S1PR4.

Fig. 4.

Fig. 4

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HCC-HSC microspheres facilitated the differentiation of macrophages via S1P-S1PR4. (A) The protein levels of S1PR4 macrophages after different short hairpin RNA transfection. (B) The cell viability of macrophages with or without S1PR4 knockdown stimulated with S1P (S1P), conditioned medium of HCC-HSC microspheres (HCC-HSC-CM) and conditioned medium of HCC-HSC microspheres with S1P supply (HCC-HSC-CM + S1P) (n = 5). (C) The cell cycle analysis of macrophages with or without S1PR4 knockdown stimulated with S1P (S1P), conditioned medium of HCC-HSC microspheres (HCC-HSC-CM) and conditioned medium of HCC-HSC microspheres with S1P supply (HCC-HSC-CM + S1P) (n = 3). (D) and (E) The protein of BAX, Bcl-2 and Caspase3 of macrophages with or without S1PR4 knockdown stimulated with S1P (S1P), conditioned medium of HCC-HSC microspheres (HCC-HSC-CM) and conditioned medium of HCC-HSC microspheres with S1P supply (HCC-HSC-CM + S1P) (n = 3). (F) The mRNA level of Arg-1, CD206 and IL-10 of macrophages with or without S1PR4 knockdown stimulated with S1P (S1P), conditioned medium of HCC-HSC microspheres (HCC-HSC-CM) and conditioned medium of HCC-HSC microspheres with S1P supply (HCC-HSC-CM + S1P) (n = 3). (G) The CD163 positive cell counts by flow cytometry (n = 3). (H) The CD206 positive cell counts by flow cytometry (n = 3). The Student’s t-test was used to compare the difference between the two groups. * Represents the significant difference between two groups (p < 0.05).

It has been demonstrated that HCC-HSC microspheres facilitated the differentiation of macrophages into the M2 phenotype in the experiment above, characterized by elevated expression of proteins associated with macrophage lipid metabolism. To investigate the relationship between lipid metabolism and the role of S1P in supporting the differentiation of macrophages into the M2 phenotype, wild-type and S1PR4-/- macrophages were treated with maintenance medium supplemented with S1P or conditioned medium from tumor cell-HSC microspheres. The results indicated that, in contrast to wild-type, S1PR4−/− macrophages did not elevate the expression of lipid-related proteins when stimulated by S1P in maintenance medium or tumor cell HSC conditioned medium (Fig. 5A–B). Simultaneously, the findings regarding oxygen consumption and extracellular acidification rate indicated that S1P facilitated lipid metabolism and stimulated M2 subtype differentiation of macrophages via S1PR4 (Fig. 5C–D).

Fig. 5.

Fig. 5

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HCC-HSC microspheres modulated the FAO of macrophages via S1P-S1PR4. (A) and (B) The protein levels of ACAA1, ACAA2, ALOX5, CPT2 and IDO1 of macrophages with or without PPARα knockdown (shCtrl or shPPARα) stimulated with S1P or HCC-HSC microsphere conditioned medium (HCC-HSC-CM) (n = 3). (C) The oxygen consumption rates of macrophages with or without S1PR4 knockdown (shCtrl or shS1PR4) stimulated with S1P or HCC-HSC microsphere conditioned medium (HCC-HSC-CM) (n = 3). (D) The extracellular acidification rate of macrophages with or without S1PR4 knockdown (shCtrl or shS1PR4) stimulated with S1P or HCC-HSC microsphere conditioned medium (HCC-HSC-CM) (n = 3). The Student’s t-test was used to compare the difference between the two groups. *Represents the significant difference between two groups (p < 0.05).

Peroxisome proliferator-activated receptor α (PPARα) is a fundamental lipid metabolism route that regulates the expression of various lipid metabolism enzymes, including CPT2, IDO1/2, and ACAA120. S1P has been observed to enhance PPARα expression to modulate lipid metabolism21. Following the knockdown of PPARα (Fig. 6A), S1P was administered in maintenance medium or HCC-HSC microsphere conditioned medium to stimulate wild-type or PPARα-/- macrophages. The results indicated that the number of CD163+ and CD206+ cells remained unchanged after S1P or conditioned medium stimulation, demonstrating that the differentiation effect of S1P on M2 macrophages was abolished (Fig. 6 B–C). Concurrently, markers associated with lipid metabolism decreased comparing with wild-type group (Fig. 6D). The oxygen consumption and extracellular acidification rate showed that S1P facilitated lipid metabolism and stimulated via PPARα (Fig. 6E–F) It was found that the expression of PPARα decreased in S1PR4−/− macrophages and not conversely (Fig. 6G). The findings indicated that S1P/S1PR4 facilitated lipid metabolism and stimulated M2 macrophage differentiation via PPARα.

Fig. 6.

Fig. 6

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S1P/S1PR4 facilitated lipid metabolism and stimulated M2 macrophage differentiation via PPARα. (A) The protein levels of PPARα macrophages after different short hairpin RNA transfection. (B) and (C) The CD163 positive (CD163-APC) and CD206 positive (CD206-FITC) cell counts by flow cytometry (n = 3). (D) The protein levels of ACAA1, ACAA2, ALOX5, CPT2 and IDO1 of macrophages with or without PPARα knockdown (shCtrl or shPPARα) stimulated with S1P or HCC-HSC microsphere conditioned medium (HCC-HSC-CM) (n = 3). (E) The oxygen consumption rates of macrophages with or without PPARα knockdown (shCtrl or shPPARα) stimulated with S1P or HCC-HSC microsphere conditioned medium (HCC-HSC-CM) (n = 3). (F) The extracellular acidification rate of macrophages with or without PPARα knockdown (shCtrl or shPPARα) stimulated with S1P or HCC-HSC microsphere conditioned medium (HCC-HSC-CM) (n = 3). (G) The mRNA levels of PPARα and S1PR4 of macrophages with S1PR4 knockdown (shS1PR4) or PPARα knockdown (shPPARα) after stimulated with S1P or HCC-HSC microsphere conditioned medium (HCC-HSC-CM) (n = 3). The Student’s t-test was used to compare the difference between the two groups. * Represents the significant difference between two groups (p < 0.05).

The HCC-HSC microspheres exhibit significant secretion of S1P. SphK1 serves as the primary enzyme responsible for S1P production. Comparing with HCC cells, HSC highly expressed S1P and SphK1 (Fig. 7A–B). While SphK2 another enzyme responsible for S1P was not changed significantly (Supplement Fig. 1). Nrf2 is an important factor in the transformation of cancer associated fibroblast (CAF)22. Likewise, it was observed that HSCs within microspheres exhibited elevated expression of Nrf2 compared to those cultivated alone (Fig. 7C). The protentional binding sites located within the promoter of SphK1 were analysis by using JASPAR database (Supplement Table 1). According to the bioinformatics analysis of “Transcription Factor ChIP-seq” experiment in Encode23, Nrf2 might be involved in SphK1 transcription and to enhance S1P synthesis (Supplement Fig. 2). To elucidate the relationship between Nrf2 and SphK1 overexpression in HSCs derived from HCC-HSC microspheres, Nrf2 in HSCs was knocked-down prior to the formation of HCC-HSC microspheres (Fig. 7D). Following Nrf2 knockdown, the expression of tumor-associated fibroblast marker molecules of HSCs educated by tumor cells decreased (Fig. 7E). Concurrent with down-regulation of Nrf2 downstream molecules, SphK1 expression was also diminished (Fig. 7F). The secretion of S1P were decrease following the knockdown of Nrf2, confirming that Nrf2-induced SphK1 can enhance S1P secretion. It demonstrated that tumor cells educated HSCs to increase S1P secretion through Nrf2/SphK1. Furthermore, after SnpK1 knocked down, the HSCs, even educated with HCCs, could not increase S1P secretion and induce the CD163 and CD206 expression of macrophages (Supplement Fig. 3A–C).

Fig. 7.

Fig. 7

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HCC cells educated HSCs to increase S1P secretion through Nrf2/SphK1. (A) The S1P levels of 24 h-cultured medium of HCC cells and HSC cells in microspheres (n = 3). (B) The protein levels of SphK1 of HCC cells and HSC cells in microspheres. (C) The protein levels of Nrf2 and SphK1 of HSC after mono-culture or in HCC-HSC microspheres. (D) The protein levels of Nrf2 of HSC cells after different short hairpin RNA transfection. (E): The protein levels of α-SMA, FAP and Vimentin of HSC in HCC-HSC microspheres (HSC), HSC which transfected with control short hairpin RNA (shCtrl HSC) and HSC which transfected with Nrf2 short hairpin RNA (shNrf2 HSC) (n = 3). (F) The protein levels of HO-1, NQO-1 and SphK1 of HSC in HCC-HSC microspheres (HSC), HSC which transfected with control short hairpin RNA (shCtrl HSC) and HSC which transfected with Nrf2 short hairpin RNA (shNrf2 HSC) (n = 3). (G) The S1P levels of 24 h-cultured medium of HSC in HCC-HSC microspheres (HSC), HSC which transfected with control short hairpin RNA (shCtrl HSC) and HSC which transfected with Nrf2 short hairpin RNA (shNrf2 HSC) (n = 3). The Student’s t-test was used to compare the difference between the two groups. The differences among more than two groups were analyzed using the one-way analysis of variance followed by Bonferroni’s test. * Represents the significant difference between two groups (p < 0.05).

Discussion

In this study, we uncovered a novel axis linking tumor-educated HSCs to macrophage polarization via S1P-driven metabolic reprogramming The result of this study demonstrated that S1P generated from HCC-HSC microspheres enhanced macrophage lipid metabolism and facilitated M2 phenotype differentiation via S1PR4/PPAR pathway. Tumor cells induced Nrf2 expression in HSCs to promote SphK1 expression and S1P secretion (Fig. 8).

Fig. 8.

Fig. 8

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The graph illustrates the main finding of the study.

The role of lipid metabolism on phenotype changes of macrophages

The tumor microenvironment is abundant in lipids, such as fatty acids, lipoproteins, and cholesterol, resulting from the vigorous lipogenesis activity and the buildup of adipocytes in TMEs24. Lipid metabolism is essential for numerous functions of macrophages, including signal transduction and gene expression related to different phenotypes25. Previous studies have demonstrated that within the TME, gene expression profiles linked to fatty acid (FA) β-oxidation and FAO-related genes are significantly upregulated in TAMs, particularly in the M2 phenotype, in comparison to control macrophages (MΦs)26. This indicates a metabolic reprogramming characterized by enhanced FA uptake and FAO in M2 TAMs, as opposed to glycolysis. Several studies demonstrated that M1 macrophages depend on glycolysis and the pentose phosphate pathway (PPP) for the synthesis of inflammatory cytokines, whereas M2 macrophages predominantly utilize the tricarboxylic acid (TCA) cycle and FAO to suppress the inflammatory response14. In our study, during co-culture with HCC-HSC microspheres or stimulation with conditioned medium of HCC-HSC, the FAO related genes expressions of macrophages were increase along with M2 phenotype markers overexpression. The evidence of which lipid metabolism modulates M2 phenotype polarization has been revealed in kinds of studies. Malandrino et al. demonstrated that the inhibition of FAO using etomoxir, a mitochondrial carnitine palmitoyl-transferase 1 inhibitor, obstructed the differentiation of M2 macrophages triggered by IL-427. While, enhancing mitochondrial fatty acid oxidation of macrophages which coculture with adipose-derived stem cells facilitated the early transition to M2 phenotype28.

Our study suggested the role of PPARα-dominated changes in lipid metabolism in the phenotype differentiation of TAMs. Our results showed that the PPARα knockdown inhibited the FAO and M2 phenotype markers expression. PPARα is a transcriptional regulator of genes involved in peroxisomal and mitochondrial β-oxidation and FA transport20. As previous study showed, a novel PPARα activator, BP-2, exhibited the ability of promotion the expression of the M2-like macrophage marker29. The PPAR-FAO axis has been proved that it could enhance M2 polarization of macrophages at the transcriptional level due to the substantial downregulation of Silent Information Regulator 4 (SIRT4) in hepatocellular carcinoma30. PPARα knockdown prevents M2 macrophage polarization facilitated by Oleoylethanolamide31. CPT1, as a downstream molecular of PPARα is essential for mitochondrial-dependent oxidation of long-chain FAs. CPT1a overexpression increased the anti-inflammatory M2 phenotype macrophage which reduced kidney inflammatory profile32.

S1P/S1PR4 as a metabolic checkpoint and phenotype changer for TAMs

S1P is a lipidic mediator that regulates a number of physiological processes. The S1P/S1PRs signaling are linked to the pathophysiology of immune-related disorders, including psoriasis, multiple sclerosis (MS), inflammatory bowel disease (IBD), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and malignancies33–35. In macrophages, all five S1PRs are expressed in normal and inflammatory conditions36. The transcriptome profile of circulating cells indicated that monocytes, which overexpressed S1P related pathway, had immunosuppressive characteristics37. The presence of S1P inhibited M1 macrophage-mediated phagocytosis of tumor cells in diffuse large B cell lymphoma by modulating the M1/2 phenotype ratio18. Moreover, S1P induced a dose-dependent increase in M2 markers in LPS-stimulated primary macrophages, promoting an IL-4-dependent M2-like phenotype in the cells38. Our study found that S1P in the conditioned medium played role in the differentiation of macrophage phenotype, as inhibiting S1P/S1PR4 diminished the differentiation of M2 phenotype.

The results of our study revealed that S1P/S1PR4 signal modulate the expression of genes associated with lipid metabolism and FAO. The effect of S1P on lipid metabolism has been revealed in some previous studies. In Chao’s study, it was concluded that SPNS2/S1P signaling prevents the immunosuppressive properties of macrophages by modulating the metabolism of phospholipase A239. FTY720, an S1P receptor modulator, could enhance fatty acid oxidation in neutrophils through the PPARα–CPT1a pathway40. It has been demonstrated that PPAR related fat acid metabolism pathway, which correlated with S1P signaling, increased the polarization of M2 macrophages in a FGF21KO metabolic dysfunction-associated steatohepatitis model41.

Nrf2/SphK1-S1P axis on HSCs

Our study has identified Nrf2 activation in HSCs after tumor cell stimulation. Nrf2 is an important factor in the transformation of fibroblasts into myofibroblasts42. As the study of the DEN-induced HCC model of mice showed, SphK1 was overexpressed in HCC tissues43. In the tumor microenvironment, HSCs will highly express Nrf2 under a variety of stimulation. In lung tumorigenesis, Nrf2 upregulation activated activating transcription factor 6 which induced CAF activation22. In HCC, a majority of CAFs originate from hepatic stellate cells that have shed their lipid droplets and are expressing specific markers, such as smooth muscle actin (SMA), fibroblast activation protein (FAP), and fibroblast specific protein 1 (FSP1)44. Exo-miR-1290-induced by COX-2 activated CAFs through Nrf2 pathway45.

The SphK1 and S1P overexpression were observed in our study. SphK overexpression have been observed in kinds of tumor stroma. Earlier studies have shown SPHK1 is highly expressed in the tumor stroma of high-grade serous ovarian cancers (HGSC). Previous studies showed that HSC activation consistent with SphK expression46. As a result of an activation of hepatic stellate cells in a liver fibrosis model, SphK1 was overexpressed in these cells47. Corneal fibroblasts are similar to HSCs in that they express a-SMA and assemble an extracellular matrix with TGF-B stimulation. Following TGF-b activation, the levels of the signaling sphingolipids ceramide (Cer) and S1P increased48.

Our study identified Nrf2 as a driver of SphK1 overexpression in HSCs. Tumor cells induce Nrf2 activation in HSCs (Fig. 7C), which directly enhances SphK1 expression and S1P secretion (Fig. 7B, G). There are relatively few studies on the relationship between Nrf2 and SphK. Previous study showed Nrf2 and SphK1 were up-regulated concurrently following FGF receptor activation at neuromuscular junctions during oxidative stress49. On account of the role of transcription factor of Nrf2, we checked the protentional binding sites of Nrf2 located within the promoter of SphK1. As the bioinformatics analysis of “Transcription Factor ChIP-seq” experiment showed, Nrf2 could bind to promoter of SnpK1. This result could explain Nrf2 regulated the SphK1 expression by binding to the promoter of SnpK1 as a transcription factor.

Clinical implications and limitations

The Nrf2/SphK1-S1P axis offers dual therapeutic targets: stromal S1P production (via Nrf2/SphK1 inhibition) and macrophage S1PR4/PPARα/FAO signaling. The S1PR modulators such as Ozanimod and Siponimod have been applied on treatment of MS. There are also kinds of lipid metabolism modulators for hyperlipidemia. Whether these drugs have synergistic effect with immune checkpoint inhibitor through reversing immunosuppressive microenvironment.

There are some limitations in our study. First, in our experiment, the reason why S1P was identified as a key factor in M2 macrophage differentiation was relied on preliminary experimental outcomes. The other potential non-protein components which have significant different concentration between HSC conditioned medium and HCC-HSC conditioned medium, were not included. Second, as for lipid metabolism, the lipid substrate and metabolites play an essential role in the physiology of the cell within the context of lipid metabolism. The investigation that we conducted does not provide any clarity regarding the precise lipid substrates and metabolites that are involved in the PPARα pathway. It is planned to incorporate the particular lipid acid into subsequent research. Last, the HCC-HSC microspheres were utilized in the research is in order to simulate that tumor tissues in vivo. In a number of studies, it was demonstrated that the contact stereoscopic culture could accurately represent the microenvironment of the actual tumor50. However, it does not provide any evidence for explanation the difference between the HCC-HSC microspheres and the conventional two dimensional co-culture procedure.

In conclusion, tumor-induced HSCs, characterized by elevated SphK1 and S1P expression, enhance macrophage lipid metabolism and promote M2 phenotypic differentiation through S1PR4.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (4.9MB, docx)
Supplementary Material 2 (16.2KB, docx)
Supplementary Material 3 (6.5MB, docx)

Author contributions

R.F. gave the idea of the study and wrote the main manuscript text. Long Yang prepared Figs. 1–4. Zirong Liu prepared Figs. 5–7. Z.C. supervised the study. Each author has approved the submitted version and agreed both to be personally accountable for the author’s own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.

Funding

The study was supported by The Fund of the Natural Science Foundation of Tianjin (Grand No. 24JCYBJC01820) and Health Science and Technology Youth Project (Grand No. TJWJ2023QN028).

Data availability

The datasets generated and/or analysed during the current study are not publicly available due to that the research program the data of this article belonging to was not finished but are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

This study involving human samples was approved by Tianjin First Central Hospital’s Ethics Committee (Registration number: 2022N077KY). It was confirmed that informed consent was obtained from all participants and/or their legal guardians. Research involving human research participants have been performed in accordance with the Declaration of Helsinki.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  • 1.Anderson, N. M. & Simon, M. C. The tumor microenvironment. Curr. Biol.30, R921–R925. 10.1016/j.cub.2020.06.081 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Gajewski, T. F., Schreiber, H. & Fu, Y. X. Innate and adaptive immune cells in the tumor microenvironment. Nat. Immunol.14, 1014–1022. 10.1038/ni.2703 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Sadeghi Rad, H. et al. Understanding the tumor microenvironment for effective immunotherapy. Med. Res. Rev.41, 1474–1498. 10.1002/med.21765 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Roma-Rodrigues, C., Mendes, R., Baptista, P. V. & Fernandes, A. R. Targeting tumor microenvironment for cancer therapy. Int. J. Mol. Sci.10.3390/ijms20040840 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Pan, Y., Yu, Y., Wang, X. & Zhang, T. Tumor-associated macrophages in tumor immunity. Front. Immunol.11, 583084. 10.3389/fimmu.2020.583084 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Gao, J., Liang, Y. & Wang, L. Shaping polarization of tumor-associated macrophages in cancer immunotherapy. Front. Immunol.13, 888713. 10.3389/fimmu.2022.888713 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Yang, K. et al. The role of lipid metabolic reprogramming in tumor microenvironment. Theranostics13, 1774–1808. 10.7150/thno.82920 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Nomura, M. et al. Macrophage fatty acid oxidation inhibits atherosclerosis progression. J. Mol. Cell. Cardiol.127, 270–276. 10.1016/j.yjmcc.2019.01.003 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Viola, A., Munari, F., Sanchez-Rodriguez, R., Scolaro, T. & Castegna, A. The metabolic signature of macrophage responses. Front. Immunol.10, 1462. 10.3389/fimmu.2019.01462 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Huang, C. et al. Ketone body beta-hydroxybutyrate ameliorates colitis by promoting M2 macrophage polarization through the STAT6-dependent signaling pathway. BMC Med.20, 148. 10.1186/s12916-022-02352-x (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Liu, S. et al. S100A4 enhances protumor macrophage polarization by control of PPAR-gamma-dependent induction of fatty acid oxidation. J. Immunother. Cancer9, e002548. 10.1136/jitc-2021-002548 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Kuai, F. et al. Exploring the protective role and the mechanism of sphingosine 1 phosphate in endotoxic cardiomyocytes. Shock52, 468–476. 10.1097/SHK.0000000000001270 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Okamoto, Y. et al. Sphingosine 1-phosphate receptor type 2 positively regulates interleukin (IL)-4/IL-13-induced STAT6 phosphorylation. Cell. Signal.88, 110156. 10.1016/j.cellsig.2021.110156 (2021). [DOI] [PubMed] [Google Scholar]
  • 14.O’Neill, L. A., Kishton, R. J. & Rathmell, J. A guide to immunometabolism for immunologists. Nat. Rev. Immunol.16, 553–565. 10.1038/nri.2016.70 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Plitzko, B. & Loesgen, S. Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in culture cells for assessment of the energy metabolism. Bio Protoc.8, e2850. 10.21769/BioProtoc.2850 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Wu, M. et al. Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells. Am. J. Physiol. Cell Physiol.292, C125-136. 10.1152/ajpcell.00247.2006 (2007). [DOI] [PubMed] [Google Scholar]
  • 17.Sousa, C. M. et al. Pancreatic stellate cells support tumour metabolism through autophagic alanine secretion. Nature536, 479–483. 10.1038/nature19084 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Perry, T. A. et al. The oncogenic lipid sphingosine-1-phosphate impedes the phagocytosis of tumor cells by m1 macrophages in diffuse large b cell lymphoma. Cancers (Basel)16, 574. 10.3390/cancers16030574 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Schuster, C. et al. S1PR4-dependent CCL2 production promotes macrophage recruitment in a murine psoriasis model. Eur. J. Immunol.50, 839–845. 10.1002/eji.201948349 (2020). [DOI] [PubMed] [Google Scholar]
  • 20.Pawlak, M., Lefebvre, P. & Staels, B. Molecular mechanism of PPARalpha action and its impact on lipid metabolism, inflammation and fibrosis in non-alcoholic fatty liver disease. J. Hepatol.62, 720–733. 10.1016/j.jhep.2014.10.039 (2015). [DOI] [PubMed] [Google Scholar]
  • 21.Zhang, J. et al. SPHK1/S1PR1/PPAR-alpha axis restores TJs between uroepithelium providing new ideas for IC/BPS treatment. Life Sci Alliance10.26508/lsa.202402957 (2025). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Kang, J. I. et al. p62-induced cancer-associated fibroblast activation via the Nrf2-ATF6 pathway promotes lung tumorigenesis. Cancers (Basel)13, 864. 10.3390/cancers13040864 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Consortium, E. P. An integrated encyclopedia of DNA elements in the human genome. Nature489, 57-74, 10.1038/nature11247 (2012). [DOI] [PMC free article] [PubMed]
  • 24.Koundouros, N. & Poulogiannis, G. Reprogramming of fatty acid metabolism in cancer. Br. J. Cancer122, 4–22. 10.1038/s41416-019-0650-z (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Xu, D. et al. The gluconeogenic enzyme PCK1 phosphorylates INSIG1/2 for lipogenesis. Nature580, 530–535. 10.1038/s41586-020-2183-2 (2020). [DOI] [PubMed] [Google Scholar]
  • 26.Su, P. et al. Enhanced lipid accumulation and metabolism are required for the differentiation and activation of tumor-associated macrophages. Can. Res.80, 1438–1450. 10.1158/0008-5472.CAN-19-2994 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Malandrino, M. I. et al. Enhanced fatty acid oxidation in adipocytes and macrophages reduces lipid-induced triglyceride accumulation and inflammation. Am. J. Physiol. Endocrinol. Metab.308, E756-769. 10.1152/ajpendo.00362.2014 (2015). [DOI] [PubMed] [Google Scholar]
  • 28.Li, J. et al. Adipose stem cells regulate lipid metabolism by upregulating mitochondrial fatty acid beta-oxidation in macrophages to improve the retention rate of transplanted fat. Stem Cell Res. Ther.15, 328. 10.1186/s13287-024-03953-4 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Marques, P. et al. Anti-inflammatory effects and improved metabolic derangements in ob/ob mice by a newly synthesized prenylated benzopyran with pan-PPAR activity. Pharmacol. Res.: Off. J. Italian Pharmacol. Soc.187, 106638. 10.1016/j.phrs.2022.106638 (2023). [DOI] [PubMed] [Google Scholar]
  • 30.Li, Z. et al. SIRT4 silencing in tumor-associated macrophages promotes HCC development via PPARdelta signalling-mediated alternative activation of macrophages. J. Exp. Clin. Cancer Res.38, 469. 10.1186/s13046-019-1456-9 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Chen, Z. et al. Oleoylethanolamide stabilizes atherosclerotic plaque through regulating macrophage polarization via AMPK-PPARalpha pathway. Biochem. Biophys. Res. Commun.524, 308–316. 10.1016/j.bbrc.2020.01.103 (2020). [DOI] [PubMed] [Google Scholar]
  • 32.Jativa, S. et al. The phagocytosis dysfunction in lupus nephritis is related to monocyte/macrophage CPT1a. Immunol. Lett.266, 106841. 10.1016/j.imlet.2024.106841 (2024). [DOI] [PubMed] [Google Scholar]
  • 33.Zhao, Y. et al. Pathogenic sphingosine 1-phosphate pathway in psoriasis: a critical review of its pathogenic significance and potential as a therapeutic target. Lipids Health Dis.22, 52. 10.1186/s12944-023-01813-3 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Dal Buono, A. et al. Sphingosine 1-phosphate modulation in inflammatory bowel diseases: keeping lymphocytes out of the intestine. Biomedicines10.3390/biomedicines10071735 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Pyne, N. J. & Pyne, S. Recent advances in the role of sphingosine 1-phosphate in cancer. FEBS Lett.594, 3583–3601. 10.1002/1873-3468.13933 (2020). [DOI] [PubMed] [Google Scholar]
  • 36.Heo, J. Y. & Im, D. S. Pro-inflammatory role of S1P(3) in macrophages. Biomol. Ther. (Seoul)27, 373–380. 10.4062/biomolther.2018.215 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Terlizzi, M., Falanga, A., Colarusso, C., Pinto, A. & Sorrentino, R. Sphingosine-1-phosphate shapes healthy monocytes into an immunosuppressive phenotype. Cellular Physiol. Biochem.: Int. J. Exp. Cell. Physiol., Biochem., Pharmacol.58, 156–171. 10.33594/000000691 (2024). [DOI] [PubMed] [Google Scholar]
  • 38.Park, S. J. et al. Sphingosine 1-phosphate induced anti-atherogenic and atheroprotective M2 macrophage polarization through IL-4. Cell. Signal.26, 2249–2258. 10.1016/j.cellsig.2014.07.009 (2014). [DOI] [PubMed] [Google Scholar]
  • 39.Fang, C. et al. Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE(2) production. Cell Commun. Signal22, 463. 10.1186/s12964-024-01851-z (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Wang, R. X. et al. FTY720 ameliorates experimental MPO-ANCA-associated vasculitis by regulating fatty acid oxidation via the neutrophil PPARalpha-CPT1a pathway. Rheumatology (Oxford)63, 2578–2589. 10.1093/rheumatology/keae320 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Shi, X. et al. Compromised macrophages contribute to progression of MASH to hepatocellular carcinoma in FGF21KO mice. Sci. Adv.10, eao9311. 10.1126/sciadv.ado9311 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Hiebert, P. et al. Nrf2-mediated fibroblast reprogramming drives cellular senescence by targeting the matrisome. Devel. Cell46, 145–161110. 10.1016/j.devcel.2018.06.012 (2018). [DOI] [PubMed] [Google Scholar]
  • 43.Liu, X. T. et al. Targeting the SphK1/S1P/PFKFB3 axis suppresses hepatocellular carcinoma progression by disrupting glycolytic energy supply that drives tumor angiogenesis. J. Transl. Med.22, 43. 10.1186/s12967-023-04830-z (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Chen, X. et al. Interplay of Helicobacter pylori, fibroblasts, and cancer cells induces fibroblast activation and serpin E1 expression by cancer cells to promote gastric tumorigenesis. J. Transl. Med.20, 322. 10.1186/s12967-022-03537-x (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Bai, X. et al. Exo-miR-1290-induced by COX-2 overexpression promotes cancer-associated fibroblasts activation and tumor progression by CUL3-Nrf2 pathway in lung adenocarcinoma. Cell Commun. Signal21, 242. 10.1186/s12964-023-01268-0 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Beach, J. A. et al. Sphingosine kinase 1 is required for TGF-beta mediated fibroblastto- myofibroblast differentiation in ovarian cancer. Oncotarget7, 4167–4182. 10.18632/oncotarget.6703 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Zhan, W. et al. Leucine-rich repeats and immunoglobulin 1 (LRIG1) ameliorates liver fibrosis and hepatic stellate cell activation via inhibiting sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P) pathway. Iran J. Allergy Asthma Immunol.19, 397–408. 10.18502/ijaai.v19i4.4114 (2020). [DOI] [PubMed] [Google Scholar]
  • 48.Qi, H. et al. Analysis of sphingolipids in human corneal fibroblasts from normal and keratoconus patients. J. Lipid Res.58, 636–648. 10.1194/jlr.M067264 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Kim, S. & Sieburth, D. A receptor tyrosine kinase network regulates neuromuscular function in response to oxidative stress in caenorhabditis elegans. Genetics211, 1283–1295. 10.1534/genetics.119.302026 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Manduca, N., Maccafeo, E., De Maria, R., Sistigu, A. & Musella, M. 3D cancer models: One step closer to in vitro human studies. Front. Immunol.14, 1175503. 10.3389/fimmu.2023.1175503 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

Supplementary Material 1 (4.9MB, docx)
Supplementary Material 2 (16.2KB, docx)
Supplementary Material 3 (6.5MB, docx)

Data Availability Statement

The datasets generated and/or analysed during the current study are not publicly available due to that the research program the data of this article belonging to was not finished but are available from the corresponding author on reasonable request.

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