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5-Fluorouracil resistance due to sphingosine kinase 2 overexpression in colorectal cancer is associated with myeloid-derived suppressor cell-mediated immunosuppressive effects

BMC Cancer volume 24, Article number: 983 (2024) Cite this article

Abstract

Purpose

Colorectal cancer (CRC) is one of the top five cancer-related causes of mortality globally. Acquired resistance has hindered the effectiveness of 5-fluorouracil (5-FU), the main chemotherapeutic drug used to treat CRC. Sphingosine kinase 2 (SphK2) may be a cancer treatment target and involved in 5-FU resistance.

Methods

Cell growth was examined using MTT and clone formation assays for SphK2 expression. To identify immune cells in mice, flow cytometry was performed. West blotting demonstrated alterations in cell division and inflammation-related proteins. SphK2 levels and inflammation-related variables were studied using Elisa.

Results

Due to SphK2 overexpression, immunosuppression, and 5-FU resistance are caused by the development of myeloid-derived suppressor cells (MDSCs) subsequent to IL-6/STAT3 activation and alterations in the arginase (ARG-1) protein. After therapy, the combination of SphK2 inhibitors and 5-FU can effectively suppress MDSCs while increasing CD4+ and CD8+ T cell infiltration into the tumor microenvironment, lowering tumor burden, and exhibiting a therapeutic impact on CRC.

Conclusions

Our findings suggest that 5-FU treatment combined with simultaneous Spkh2 inhibition by ABC294640 has anti-tumor synergistic effects by influencing multiple effects on tumor cells, T cells, and MDSCs, potentially improving the poor prognosis of colorectal cancer patients.

Peer Review reports

Introduction

Globally, Colorectal cancer (CRC) has been the second leading cause of cancer death in recent years [1, 2]. The main chemotherapy drug used to treat CRC (5-fluorouracil, 5-FU) has shown less success due to acquired resistance [3]. The progression of the tumor after treatment resistance is the main cause of CRC death. Therefore, to increase the survival rate of patients treated with 5-FU, it is imperative to look into the processes by which CRC develops resistance to 5-FU therapy.

Cancer is characterized by immune system evasion. The complex tumor microenvironment (TME) aids immune evasion, tolerance, and tumor growth. TME cancer cells use myeloid-derived suppressor cells (MDSC) to evade immune attacks [4]. Colon adenomas are associated with higher MDSC levels than healthy controls [5]. MDSC activity and recruitment variables develop [6]. Giving 5-FU slows nucleic acid synthesis, raises T cells in malignant areas, and lowers MDSCs [7]. MDSCs prevent CD8+ and CD4+ T lymphocytes from activating and proliferating when cocultured with their target antigen [8]. Cancer produces low levels of myeloid growth factors and inflammatory mediators, changing the microenvironment and inhibiting myeloid differentiation [9,10,11], the mechanism is unclear.

The function of Sphingosine kinases 2 (SphK2) among isoforms (SphKs) is highly controversial, but a growing number of studies have demonstrated that it plays a role in malignant diseases and is associated with a poor prognosis for patients [12]. SphK2 knockdown makes CRC cancer cells more sensitive to chemotherapy [13], while chemoinhibition reduces cancer cell proliferation in vitro [14, 15] and animal models [16]. The results of our last experiment [17] and other relevant research [18] shows that overexpression of SphK2 in colon cancer cells is linked to resistance to 5-FU.

As research progresses, it has become increasingly evident that SphK2 regulates inflammation in addition to its impact on tumor cell proliferation. Its pro-inflammatory actions are intimately linked to the immune response [19, 20]. Using in vitro cellular experiments as a starting point, the current study used a transgenic mouse model with SphK2 overexpression to look at the immune-related effects of SphK2 gene overexpression on the development of colorectal cancer (CRC) in mice and how it responds to 5-FU treatment. Wild-type (WT) mice were used as a control group. By increasing the precision of clinical treatment scenarios, this method investigates the connection between drug resistance and immunity.

Materials and methods

Web-based data queries

Published datasets were bioinformatically analyzed using these databases: The Cancer Genome Atlas (TCGA) (https://portal.gdc.cancer.gov/) showed colon cancer level 3’RNAseq data and clinical information. The Genomics of Drug Sensitivity in Cancer (GDSC, https://www.cancerrxgene.org) database was used to predict the therapeutic response. Single Cell RNA Sequencing dataset was created using a meta-analysis of sequencing studies of single-cell types (https://www.proteinatlas.org) and healthy human tissue single-cell files. The fraction of genes in clusters was used to compute confidence levels in future clustering. Immune cell type specificity was analyzed using the protein atlas’ immune-cell section. This investigation examined T-cells, granulocytes, monocytes, NK-cells, dendritic cells, B-cells, progenitors, and total peripheral blood mononuclear cells (PBMC). The HA and Monaco datasets were used to examine SphK2 expression in distinct blood cell lineages. TIMER (http://cistrome.org/TIMER) was used to investigate the correlation between the SphK2 gene expression and immune infiltrates in 457 RNA-seq human colon adenocarcinoma (COAD) samples from The Cancer Genome Atlas (TCGA) [21]. The scatterplot shows the purity-corrected partial Spearman’s correlation and statistical significance that are provided within the data sets obtained through the TIMER module called “Exploration-Immune-Gene.” We use database data to assess cancer tissue immunological infiltrates (quantities of CD4+ T cells, CD8+ T cells, neutrophils, macrophages, B cells, and dendritic cells).

Cell lines, lentivirus transduction, and cell culture

American Type Culture Collection-approved HCT116 human colon cancer cells were obtained from a Chinese cell bank (ATCC® CCL-247TM). Cell culture was conducted in RPMI-1640 media (Sigma-Aldrich, USA) with 10% FBS (Invitrogen, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin. The cells were grown in a 37 °C, 5% CO2 humidified incubator. To create stable SphK2 transfectants, HCT 116 was transfected using lentiviral vectors. A lentivirus technique created stable overexpressing and knockdown cell lines. GeneChem (Shanghai, China) provided the 108 IU/mL negative control lentivirus vector SphK2. HitransG and puromycin were used to transfect the stable cell lines HCT116SphK2 and HCT116shSphK2 with the negative control virus vector and target gene vector, respectively, according to the manufacturer’s instructions. Overnight culture of 1 × 105 cells in 6-well plates (plated volume). Treatment involved dilution of 20µL of lentivirus in 2 mL of Opti-MEM medium with 5µg/mL of polybrene (Sigma-Aldrich, USA). After 24 h, the transduced cells were cultured in a fresh medium and exposed to 2 µg/mL puromycin (Invitrogen, USA) to select positively. At 14 days following selection, almost 90% of cells fluoresced green at 587 and 610 nanometers.

Cell viability assay

The first cell viability test used tetrazolium bromide (3,5-diphenyl-2 H-trimethyl-2-thiazolyl, MTT). 5000 cells/well of HCT116SphK2 and HCT116shSphK2 cells were plated in a 96-well plate with RPMI-1640 media to test 5-FU’s cytotoxic effects. After treating cells with 5-FU (1.25–20 µg/mL) for 24 h, the medium was carefully removed. Then, the treated cells were incubated in 0.5 mg/mL MTT solution (Sigma, USA) at 37 °C for 4 h without light. After careful disposal of the supernatant, formazan crystals were dissolved in DMSO (Sigma, USA). At 570 nanometers, a microplate reader recorded absorbance. Cell survival was calculated using the control solvent proportion. Plate clone formation is measured second. HCT116SphK2 and HCT116shSphK2 cells were put into 6-well plates at 300 cells per well after 5-FU treatment. The plates were incubated at 37 °C for 7 days. Colonies were fixed for 10 min with 95% ethanol and stained for 20 with 0.1% crystal violet. After that, the colonies were thrice rinsed with PBS. The plate was gently washed three times with PBS. After adding sample colonies for photography, ImageJ counted the visible colonies on the plate. Colony inhibition rate= [1−(number of colonies in experimental groups/control group)] ×100%.

Mice used as CRC models

The C57BL/6J mice (wild-type, WT) were purchased from Beijing Huafukang Biotechnology Co. The transgenic mice overexpressing Sphingosine Kinase 2 (SphK2 Tg) used in this study were generously provided by Professor Xianjun Qu from the School of Basic Medical Sciences at Capital Medical University in Beijing, China. The ethical approval for animal research was obtained from Shandong First Medical University and Shandong Academy of Medical Sciences Ethics Committee (approval number: SDTHEC2023003122). Groups of 5 mice sorted by sex were kept on a 12-hour day/night cycle for 8–10 weeks. All animals were maintained in aseptic laminar air flow units. There was abundant food and drink. Mice were anesthetized with 5% isoflurane, then 1% when the right reflex disappeared. Mice were immobilized on a head holder and heating plate (37 °C) for optimal body temperature. Azoxymethane (AOM) and frequent dextran sulfate sodium (DSS) administration caused colorectal cancer. Age-matched mice (8–10 weeks old) received a single intraperitoneal injection of AOM (10 mg/kg; Sigma, Germany). Drinking water with 1% DSS (MP Biomedicals, USA) was given for 7 days, then regular water for 14 days. Two further DSS cycles were administered. FTY720 was given intraperitoneally at 1 mg/kg or ABC294640 orally at 5 mg/kg. Additionally, 10 mg/kg 5-FU was intraperitoneally administered twice a week. After 28 days of treatment, all animals reached the experimental endpoint and were euthanized by placing them in a carbon dioxide anesthesia chamber, opening the gas valve, increasing the carbon dioxide concentration to 100%, rendering them unconscious, and deciding they were dead after 2 min of ventilation. After opening the mice’s colon longitudinally and rinsing it with cold phosphate-buffered saline (PBS), the tumors were examined.

Stains with hematoxylin and eosin

After 4 h in 4% PFA, mice’s colon samples were placed in 70% ethanol. The samples were dried with alcohol gradients and placed on paraffin wax blocks. Tissue slices were cleansed of wax using xylene, rehydrated with ethanol in progressive quantities, and rinsed in PBS before immunostaining. Hematoxylin and eosin (Beyotime Biotechnology) colored the samples. The tinted pieces were dried with additional ethanol and xylene. Photos were taken with a Leica DM2500 with a 100 × lens. To evaluate immunological infiltration, eight high-magnification fields (×400) were examined. Of these fields, the five with the most immune cells were chosen to assess their immune cell counts. Tissues with cell counts above the norm were considered robust immune infiltration, whereas those below the average were weak.

Isolation of MDSCs from spleens for 5-FU in vitro killing assays

After dissociating spleens, individual splenocytes were collected using 30 μm nylon, centrifuged, counted, and cleaned with PBS. G-MDSCs (Gr-1 high Ly-6G +) and M-MDSCs (Gr-1 dim Ly-6G −) cells were isolated from the spleens of SphK2 Tg mice or WT mice using the Myeloid-Derived Suppressor Cell Isolation Kit (Miltenyi Biotech, Germany) with anti-Ly-6G-biotin and anti-biotin beads and anti-Gr-1-biotin and streptavidin beads, respectively, (indirectly magnetic labeling of the cells) using MACS® Columns (LS Columns and MS Columns) and MACS Separators according to the manufacturer. Collecting, centrifuging, counting, and transferring floating cells to RPMI-1640 complete media. Following a 24-hour incubation period with varying amounts of 5-FU (0.25–2 µg/mL), 10 µL of Cell Counting Kit-8 (CCK-8, Beyotime, China) was added to each well. The cells were then incubated for an additional two hours in a cell culture incubator, and the absorbance was recorded using a microplate reader at 450 nm.

Flow cytometry evaluation

For flow cytometry immunotypic analysis, blood samples were collected in heparin and peripheral blood mononuclear cells (PBMCs) were diluted 1:2 with PBS 1X and stratified by density gradient. We overlaid diluted whole blood samples on Histopaque and centrifuged them at 450×g for 30 min at room temperature without braking. After transferring the lymphocyte-enriched ring to a fresh tube, wash it with PBS 1X by centrifuging at 450×g for 10 min. Fc receptor block (PN 422301, TruStain FcX, BioLegend) stopped nonspecific antibody binding in cells for 10 min. The cells were stained with monoclonal antibodies, Brilliant Violet 421™ anti-mouse Ly-6G (BioLegend, #127627), PE anti-mouse Ly-6 C (BioLegend, #128007), FlTC anti-mouse/human CD11b (BioLegend, #101205), APC anti-mouse CD31 (BioLegend, #102510), Alexa Fluor® 647 anti-mouse (BioLegend, #100426), and Alexa Fluor® 700 anti-mouse CD8a (BioLegend, #100730), all at a dilution of 1:200. A live-dead marker recognized living cells. A live-dead marker recognized living cells. Gating CD3+ cells allowed quantification of CD4+ and CD8+ cells in this population. Both G-MDSC and M-MDSC populations were also subjected to phenotyping. G-MDSC was defined as CD11b+Ly6G+Ly6C−, and M-MDSC was defined as CD11b+Ly6C+Ly6G−.

Western blot analysis

About 30 µg of proteins were separated using a 10% SDS-PAGE gel. Bedford, Massachusetts-based Millipore deposited these proteins on PVDF membranes. After blocking for an hour with 10% non-fat dry milk in TBST, PVDF membranes were incubated overnight at 4 °C with specified antibodies. The following antibodies were used: anti-SphK2 (Proteintech, 17096-1-AP), anti-iNOS (Abcam, ab178945), anti-ARG (Abcam, ab124917), anti-STAT3 (Abcam, ab119352), anti-p-STAT3 (Abcam, ab76315) (The primary antibody mentioned above was diluted 1:2000) and anti-actin (Sigma, A2066) (1:5000 dilutions). Membranes were incubated with secondary antibodies (1:5,000 dilutions) in TBST for another hour at room temperature. An enhanced chemiluminescence (ECL) kit from Pierce, Shanghai, China, was utilized to see the blots. The FluorChem FC3 (ProteinSimple, USA) enhanced chemiluminescent detection system was used to view the bound antibodies, and AlphaView (3.4.0.0, ProteinSimple, USA) software was utilized to assess the band density. Protein intensity ratios were calculated in relation to β-actin.

Real-time RT-PCR

TRIzol Reagent (Invitrogen, USA) was utilized for the extraction of RNA. Synergy Brands Green RT-PCR was used to detect the mRNA levels, and the 2 − ΔΔCt technique was used to determine them. The following were the primers: GAPDH, 5′-TGCACCACCAACTGCTTAGC-3′ (forward) and 5′-GGCATGGACTGTGGTCATGAG-3′ (reverse); SphK2, 5′-GGTTGCTTCTATTGGTCAATCC-3′ (forward) and 5′-GTTCTGTCGTTCTGTCTGGATG-3′ (reverse).

Enzyme-linked immunosorbent assay (ELISA)

TNF-α (Boster Biological Technology, Wuhan, China, EK0527), IFN-γ (Boster, EK0375), IL-6 (Boster, EK0411), and IL-10 (Boster, EK0417) were measured in the hippocampus using a mouse ELISA kit in accordance with the manufacturer’s instructions. The results were expressed as pg/mL.

Statistics analysis

In this research, experiments were repeated at least three times. ANOVA and Tukey’s multiple comparisons were used to evaluate the statistics (SPSS 22.0, Chicago, CA); continuous data with normal distributions were compared with the mean ± standard deviation (SD). The threshold of significance was set at P<0.05. The IC50 score dispersion. The abscissa shows sample groups and the ordinate shows IC50 score dispersion. Top-left displays the significance p-value test technique, colors represent groups. *p < 0.05, **p < 0.01, ***p < 0.001, asterisks (*) indicate importance. Wilcox test assessed two groups’ statistical differences, Kruskal-Wallis test examined three groups’ significant differences. GraphPad Prism 9 was used for graphing.

Results

The expression of SphK2 is inversely correlated with 5-FU sensitivity

To clarify the role of SphK2 in 5-FU therapy for colorectal cancer, we used the GDSC database to predict SphK2 expression and 5-FU chemotherapeutic response for 620 colorectal cancers in the TCGA dataset using level 3 RNAseq data and clinical information. A rise in SphK2 gene expression was followed by an increase in IC50 values, and upland expressions differed strongly statistically (p<0.001, Fig. 1A). A model was then created using GDSC cell expression patterns from cancer databases to predict 5-FU’s IC50 on 43 colorectal cancer cell lines. Thus, 5-FU administration may affect colorectal cancer cell lines. The medication became more effective as the IC50 fell. Since HCT116 cells (IC50 = 2.69 μm) were the most vulnerable to 5-FU treatment, they were used to study how SphK2 expression affects 5-FU resistance (Fig. 1B).

Fig. 1
figure 1

SphK2 levels are negatively correlated with 5-FU sensitivity. (A) The expression of the SphK2 gene and the IC50 score for 5-FU were correlated using the Spearman correlation function. The range of IC50 scores is represented by the vertical coordinates, while the axes represent the sample groups. The IC50 score range is represented by the right density curve and gene expression is depicted in the upper density curve. These are the p-values, coefficients, and calculations for the optimal correlation. (B) The treatment sensitivity of the HCT116 colon cancer cell line was determined by examining the IC50 values for 5-FU. (C) RT-PCR was used to measure the SphK2 mRNA levels in stable transfected HCT116 SphK2, HCT116 shSphK2 cell line, and HCT116 cells. (D) Western blotting was used to measure the SphK2 protein levels in stable transfected HCT116, HCT116 shSphK2, and HCT116 SphK2 cells. Protein levels of SphK2. On the right is a densitometric study of SphK2 normalized to β-actin. (E) The sensitivity of HCT116 SphK2, HCT116 shSphK2, and HCT116 cells to a 5-FU dose gradient ranging from 0 µL to 20 µg/mL was determined using the MTT assay. (F) A single 6-well plate in a plate cloning experiment is shown with the 5-FU dosage indicated in the top left corner of the figure. The HCT116 SphK2, HCT116 shSphK2, and HCT116 cell plate clone creation test results are displayed in the photos. (G) Cell clone formation rate = (number of cell clones formed / number of inoculated cells) × 100%

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Intracellular green fluorescence from puromycin screening and GFP tagging on lentiviral vectors showed transfection efficiency above 90% in both cell lines. We found no statistical difference between empty vector-carrying cells and untransfected cells. To maintain a constant control group, we used untransfected HCT116. The WB results were validated by the matching gene expression results of PCR, as shown in Fig. 3C and D. Using the MTT test, SphK2’s influence on CRC proliferation was examined in the presence of 5-FU. Overexpression cell lines proliferated faster than suppression cells in all cases, reaching statistical significance. Unlike the HCT116 growth control group, 5-FU doses below 5 µg/mL did not reduce HCT116 SphK2 cell proliferation. However, treatment with 1.25 µg/mL 5-FU significantly reduced the value-added rate of HCT116 shSphK2 cells (Fig. 1E). Then, these cells were treated with 5-FU for 7 days to determine clone creation (Fig. 1F). In each concentration group, significant differences were found, except for 20 µg/mL, where HCT116 SphK2 cell colonies were higher than the control group at the same 5-FU concentration (Fig. 1F and G). Compared to the control group, HCT116 shSphK2 cell colonies were lower. After SphK2 overexpression, colon cancer cells were resistant to 5-FU. In addition, 5-FU concentration inversely correlated with cell growth and clone formation after SphK2 knockdown.

A chemically induced colon cancer model showed more tumors in SphK2 Tg mice than WT mice

We initially investigated how SphK2 gene overexpression may affect CRC proliferation and progression. To determine SphK2 expression in colon cells, single-cell sequencing data from colon tissues was analyzed. Figure 2A shows that distal intestinal cells expressed more SphK2. Due to this difference in expression, we compared SphK2 Tg animals to WT mice in a chemically-induced colon cancer model. After one dosage of AOM and three cycles of 1% DSS, WT, and SphK2 Tg mice developed lower to intermediate colon cancers. Mice treated with carcinogens got 100% tumors. AOM/DSS-treated mice with SphK2 overexpression are more likely to develop colon cancer than wild mice (Fig. 2). Less than 1 mm, 1–2 mm, and 3 mm colon cancers were categorized by size. Figure 2B shows the macroscopic colon of SphK2 Tg mice induced with AOM/DSS compared to WT mice. Figure 2C shows a substantial difference (p < 0.001) in tumor count between the SphK2 Tg group (35.67 ± 2.08) and the WT control group (22.67 ± 0.58). The SphK2 Tg group had considerably more tumors (12.67 ± 1.53) than the wild control group (7.60 ± 0.58) in the 1–2 mm and 3 mm tumor volume ranges, with significant differences (p = 0.001 and p = 0.009, respectively). We found no significant increase in tumor volume across groups with tumor sizes of 2–3 mm (p = 0.238) (Fig. 2D). WB showed that SphK2 Tg mice expressed more SphK2 than WT mice (Fig. 2E). SphK2-overexpressing transgenic animals have enlarged spleens (Fig. 2F). According to the data, AOM/DSS-induced SphK2 Tg mice had bigger and more colon tumors than normal mice, suggesting that overexpression of this gene may cause distal colorectal cancer.

Fig. 2
figure 2

Colon cancers were more likely to develop in SphK2 Tg animals exposed to AOM/DSS than in wild mice. (A) Single-cell analysis of SphK2 expression in the colon. The cells’ color scale points showed groups of different cells. The order of the clusters was shown by a number code from 0 to 10. The scatter plot showed how the cells in different groups of cells behaved. (B) SphK2 Tg mice generated by AOM/DSS develop larger colonic tumors than WT mice, n = 5 for each group. (C) Statistical analysis of SphK2 Tg and WT mice tumor numbers was very significant. (D) The tumor diameter size distribution in each group of mice (1–2 mm, 2–3 mm, > 3 mm) was statistically evaluated, and there were statistically significant differences between the two groups, except for 2–3 mm. Data were expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. (E) SphK2 expression of extracted proteins from colonic tumors in WT and SphK2 Tg mice, 3 mice from each group. (F) AOM/DSS tumor model mice had bigger spleens than blank animals without tumor induction, and SphK2 Tg mice had a greater enlargement. (G) Images of HE-stained slices of colon cancers from WT and SphK2 Tg group were taken with a light microscope (magnification: 100; scale bar: 200 m)

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The SphK2 Tg mice exhibit resistance to 5-fluorouracil (5-FU) treatment in the context of colon cancer

We gave 5-FU to groups of mice with colon cancer models so we could study SphK2 gene overexpression and how it is linked to resistance to treatment for CRC. AOM/DSS colon cancer model (Figs. 2B and 3A) reveals a significant difference in tumor number (p = 0.002) but not tumor size (WT control group: 22.67 ± 0.58) vs. 5-FU treatment group (12.33 ± 0.58). 5-FU responded less in SphK2 Tg mice than in WT mice. WT mice demonstrated higher 5-FU effectiveness than SphK2 Tg mice. There was a modest decrease in tumors in the SphK2 Tg 5-FU group (28.67 ± 2.81) compared to the control group (35.67 ± 2.08), but no significant change in total tumor number or tumor size distribution (p = 0.059). These data imply that SphK2 overexpression causes 5-FU resistance.

Fig. 3
figure 3

Comparison of colorectal tumor response to 5-FU after 4 weeks of treatment in SphK2 Tg mice and WT mice. (A) Mouse CRC tumors with high expression of SphK2 tolerate 5-FU treatment, and tumors became sensitive to the effects of 5-FU treatment after inhibitor SphK2. n = 5 for each group. Each group of mice had its tumor count (B) and size distribution (1–2 mm, 2–3 mm, > 3 mm) statistically examined (C). (D) is 5-FU at 20 mg/kg and FTY720 at 1 mg/kg were administered intraperitoneally, and ABC294640 was given orally at a dose of 5 mg/kg. Images of HE-stained slices of colon cancers from each group were taken with a light microscope (magnification: 100; scale bar: 200 m). Areas of immunological infiltration are shown by yellow arrows

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Inhibition of SphK2 sensitization to 5-FU colon cancer treatment in SphK2 Tg mice

Given that 5-FU therapy didn’t work as well as it could in the group of mice with too much SphK2, it was thought that the increased growth of tumors caused by SphK2 overexpression played a role in this. In order to investigate this matter, two inhibitors of SphK2 were chosen for the experimental study. These inhibitors include FTY720, which functions as a competitive antagonist of S1P, and ABC294640, a non-lipid-competitive inhibitor of SphK2. Significant tumor decrease was found in SphK2 Tg mice treated with 5-FU and FTY720 (15.67 ± 4.73) or ABC294640 (10.00 ± 1.73), compared to the control group (35.67 ± 2.08) (p < 0.001) (Fig. 3A). Statistical analysis showed significant differences in tumor size (1–2 mm, 2–3 mm, and > 3 mm) in the 5-FU and FTY720 group (p < 0.001). The treatment group receiving 5-FU combined ABC294640 showed significant differences in tumor size levels: 1–2 mm (p < 0.001), 2–3 mm (p = 0.001), and tumors over 3 mm (p < 0.001). SphK2 Tg mice treated with 5-FU and FTY720 (15.67 ± 4.73) or ABC294640 (10.00 ± 1.73) showed no significant difference from the WT group treated with 5-FU alone (12.33 ± 0.58). The SphK2 Tg 5-FU group showed a slight reduction in tumor count (28.67 ± 2.81) compared to the control group (35.67 ± 2.08). There was no significant difference between the two groups in tumor count or size distribution (p = 0.059) (Fig. 3B and C). The findings suggest that SphK2 overexpression causes 5-FU resistance. These differences were extremely statistically significant, demonstrating that 5-FU + ABC294640 suppressed tumors more than 5-FU + FTY720. As anticipated by the GDSC database, SphK2 expression and 5-FU treatment response in each sample imply that reducing SphK2 expression can make CRC more responsive to 5-FU.

The histopathological alterations observed in colon tumors in mice

Intestinal tissues were then histopathologically examined. The WT control model group mice in Fig. 2G had modest to moderate cellular heterogeneity and heterogeneous glands, typical of intramucosal hyperdifferentiated carcinomas. Figure 3D shows how wild model 5-FU-treated mice acquired intramucosal cancer. A histological analysis found moderately developed adenocarcinoma in the SphK2 Tg control group’s mucosal layer. Some lymphocytic infiltration was also found in surrounding mesenchymal tissue. Figures 2G and 3D reveal that the SphK2 Tg 5-FU single-agent group had similar pathology to the control group. SphK2 Tg 5-FU + FTY720 had moderately differentiated adenocarcinomas and little lymphocytic infiltration. SphK2 Tg 5-FU + ABC294640 tumor histology was similar to WT 5-FU, with well-differentiated adenocarcinoma. Pathologic studies showed enhanced SphK2, decreased differentiation, and increased malignancy. These findings match clinical 5-FU treatment’s poor results. In SphK2 Tg mice with colon cancers, 5-FU and ABC294640 were more effective than FTY720 at suppressing the tumors. In particular, SphK2 Tg mice colon tumors demonstrated immune infiltration.

SphK2 gene expression is likely related to the immune response to CRC

The findings of the aforementioned experiments suggest a potential association between the expression of SphK2 and immunization. Consequently, we proceeded to further examine this impact. HPA and Monaco datasets confirmed that hematopoietic organs have specific immune cell types. The HPA information showed that SphK2 expression was high in granulocytes, monocytes, and T-cells (Fig. 4A). The results of the study of the Monaco database were the same as those of the HPA dataset. Monocytes and T-cells had the most SphK2 expression among blood immune cells (Fig. 4B). According to the findings from TIMER analysis, we found a statistically significant negative correlation between the expression level of SphK2 and the presence of CD8+ T cells, neutrophils, and B cells in colorectal adenocarcinoma (COAD, n = 457), as illustrated in Fig. 4C-E (p < 0.001), and 4 F (p = 0.044). while the number of CD4+ T cells was significantly positively correlated with SphK2 expression (p < 0.001). There was no statistically significant difference observed in the correlation between SphK2 and macrophages and dendritic cells in the analyzed samples (Fig. 4G–H). Neutrophils and monocytes have a tight association with myeloid-derived suppressor cells (MDSCs). Hence, it can be inferred that the influence of SphK2 on the incidence and progression of CRC is mediated through the regulation of immune cell infiltration, thereby suggesting a promising avenue for further investigation.

Fig. 4
figure 4

The number of cytotoxic T cells is reduced as a result of SphK2 overexpression’s impact on immunological infiltration. (A)-(F) Correlation analysis between SphK2 and quantities of CD8+ T cells, CD4+ T cells, neutrophils, macrophages, B cells, and dendritic cells

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SphK2 overexpression-mediated resistance is associated with expanded MDSCs

More immunoassays were done on MDSCs in order to get a more complete picture of the relationship between the regulation of SphK2 gene expression and the anti-tumor immune response. CD11b, Ly6G, and Ly6C staining determined MDSC subpopulations [22], as shown in Fig. 5A-B. SphK2 Tg mice had higher G- and M-MDSC levels than WT hormonal animals (p < 0.01). After 5-FU therapy, SphK2 Tg mice showed greater G-MDSC and lower M-MDSC levels than controls (p = 0.032 and 0.011, respectively). Inhibiting SphK2 with ABC294640 significantly reduced G- and M-MDSC levels (p < 0.001) compared to 5-FU-treated WT mice (Fig. 5C-D). According to research, 5-FU kills MDSC which impairs anti-tumor immunity [7]. Our experiments showed that the 5-FU treatment did not affect the WT group. After 5-FU therapy, SphK2 Tg had a larger percentage of G-MDSC than the no-dose control group, showing that MDSCs may alter 5-FU effectiveness. To test the effect of SphK2 on 5-FU treatment in MDSCs, we extracted G-MDSC and M-MDSC from the spleens of the above animals and added different doses of 5-FU to the in vitro MDSC culture medium based on the tumor cell-to-MDSC ratio as described [7]. Both Gr-1highLy-6G+ and Gr-1dimLy-6G− cells in the SphK2 Tg group showed no changes when treated with less than 2 µg/mL of 5-FU compared to WT mice, and were insensitive to 5-FU treatment (Fig. 5E-F). After treatment with ABC294640 and 5-FU, the SphK2 Tg group had considerably less G-MDSC and M-MDSC than the no-dose control group, showing that MDSC inhibition may reverse resistance.

Fig. 5
figure 5

SphK2’s effects on MDSCs and the cytokines and proteins linked to them in CRC mice’s tumor microenvironment (TME). (A) Flow cytometry study of G-MDSC (CD11b+Ly6C+/−Ly6G+) and (B) M-MDSC (CD11b+Ly6G−Ly6C+) subpopulations in SphK2 Tg and WT mice in each drug group. (C) Compared to control mice, the percentage of G-MDSC subsets significantly increased in the tumor-bearing mice. (D) Compared to control mice, the percentage of M-MDSC subsets significantly increased in the tumor-bearing mice. (E) G-MDSC (Gr-1highLy-6G+) activity assay for 5-FU treatment in vitro. (F) M-MDSC (Gr-1dimLy-6G−) in vitro 5-FU treatment activity assay. Following treatment, serum samples were taken from mice in each group, and the enzyme-linked immunosorbent assay (ELISA) method was used to measure the amounts of limiting and active cytokines in each group. Levels of IL-6 (G), TNF-α (H), IL-10 (I), and IFN-γ (J). (K) Western blotting was used to find the levels of the immunosuppressant proteins Arg-1, iNOS, p-STAT3, and STAT3 that are linked to MDSCs. (L)Mice treated with 5-FU and ABC294640 had much lower Arg-1 protein expression compared to SphK2 Tg mice treated with only monotherapy. SphK2 Tg mice had higher levels of iNOS protein production when given 5-FU and ABC294640. (N) The amount of p-STAT3 protein changed a lot in the SphK2 Tg treatment group, but the amount of STAT3 protein didn’t change much in the WT group (P). Data were expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control mice, ns, no significance

Full size image

Effect of MDSC activation and inhibitory cytokine levels on SphK2 expression in mice

Multiple signaling pathways play a crucial role in the development, amplification, and control of MDSC activity. Subsequently, an evaluation was conducted to determine the impact of SphK2 on the release of pro-inflammatory cytokines, such as IL6 and TNF-α. Serum samples were collected from mice that had been treated and grouped according to the presence of SphK2 Tg or WT. The concentrations of several cytokines (IL-6, TNF-α, IL-10, and IFNγ) were measured using the Elisa test method. Significantly greater expression levels of IL-6, TNF-α, and IL-10 were seen in SphK2 Tg mice compared to control mice (Fig. 5G, H, and I), with the exception of the IFNγ (Fig. 5J). The administration of ABC294640 in conjunction with 5-FU treatment resulted in a significant reduction in the levels of IL-6 (4.66-fold), TNF-α (1.33-fold), and IL-10 (2.34-fold), while simultaneously increasing the levels of IFNγ (1.92-fold). Previous research has demonstrated that tumor-derived growth factors, such as IL-6, are the primary cause of MDSC proliferation. Our findings suggest that SphK2 can modulate the increase or decrease of MDSCs in the circulation by influencing cytokines such as IL-6.

Proteins change in MDSC are related to immunosuppression

We examined the protein expression levels of arginase (ARG-1), inducible nitric oxide synthase (iNOS), phosphorylated signal transducer and activator of transcription 3 (p-STAT3), and STAT3 in murine colon cancers during treatment in SphK2 Tg mice to determine key signaling pathway protein expression. 5-FU and ABC294640-treated animals showed much less Arg-1 protein than SphK2 Tg-expressing mice (Fig. 6A, B). The mice that received 5-FU and ABC294640 had higher iNOS protein expression than the SphK2-transgenic control mice (Fig. 5K-M). The SphK2 Tg control and 5-FU-treated groups had no statistically significant changes in either protein. Arg-1 was significantly different between the WT control group and the 5-FU-treated group, whereas iNOS was not. Overactivation of the STAT3 pathway promotes p-STAT3 and MDSC accumulation. p-STAT3 was substantially different between SphK2 Tg mice treated with 5-FU and ABC294640 (Fig. 5K, N), although STAT3 protein was not. Statistical significance was evaluated by comparing findings to the control group at P <0.05. Arg-1, iNOS, and p-STAT3 expression were most affected by SphK2-mediated 5-FU therapy on MDSC proliferation and activity.

Fig. 6
figure 6

SphK2 decreases cytotoxic T lymphocyte tumor invasion. After CRC therapy in SphK2 Tg and WT mice, single-cell suspensions were prepared for flow cytometry. The SphK2 affects the number of CD4+T cells (A) and CD8+ T cells (B) infiltrating. Percentage of tumor infiltrating CD4+T cells (A, C) and CD8+ T cells (B, D). 5 mice per group. Results are representative of two independent experiments. Data were expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control mice, ns, no significance

Full size image

5-FU activates systemic anti-tumor immunity following SphK2 inhibition

With the elimination of the aforementioned immunosuppressive factors on MDSCs, we were finally able to investigate how SphK2 inhibitors reverse 5-FU treatment resistance and stimulate systemic antitumor immunity. Experiments were conducted on the PBMCs of rodents with in situ colon cancer. Specifically, we focused on the analysis of CD4+ T cells (CD3+CD4+ T cells) (Fig. 6A) and CD8+ T cells (cytotoxic T lymphocytes, CTLs, CD3+CD8+ T cells) across different treatment groups (Fig. 6B). No substantial alterations in the percentages of CD4+ and CD8+ T cells were observed following 5-FU treatment in the tumor group of WT mice. This indicates that the dosage of 5-FU commonly employed is inadequate to modify the immune tumor microenvironment. Nevertheless, the percentages of CD4+ and CD8+ T cells were observed to be lower in the tumor group of SphK2 Tg mice compared to the control group following treatment with 5-FU. Notably, the decrease in CD8+ T cells was found to be statistically significant (p = 0.0126). It is relevant that the percentage of CD4+ and CD8+ T cells in the SphK2 Tg 5-FU + ABC294640 group (1.6% / 3.6%) was significantly higher (p = 0.0002 / p = 0.0091) compared to the SphK2 Tg control group (0.5% / 1.6%), as depicted in Fig. 6C and D. These findings imply that by inhibiting the immune-tumor microenvironment, SphK2 expression can deregulate immunosuppression in terms of limiting MDSCs and eliminating resistance to 5-FU so that the sensitizing effect of ABC294640 on 5-FU results in an immune microenvironment that promotes immune response by SphK2 Tg antitumor therapy.

Discussion

The main treatment for CRC has been chemotherapy, especially 5-FU, for years. However, at least half of CRCs tolerate 5-FU-based chemotherapies [23]. Thus, research has focused on identifying variables that contribute to 5-FU-based treatment tolerance and biomarkers that can help select patients most likely to benefit [17, 23, 24]. In CRC cells, overexpression of SphK2 has been documented and is linked to treatment resistance [25]. Consistent with our finding that overexpression of the SphK2 gene increases resistance to CRC and 5-FU. Additionally, previous studies have demonstrated the significance of the SphK2 inhibitor ABC294640 in the etiology of CRC [26]. Resistance mechanisms have been investigated with an emphasis on combined DNA repair damage, pro-apoptotic, and anti-proliferative effects [25, 27, 28]. Though little is known about the immunological function of SphK2 in CRC, it is necessary for the release of proinflammatory cytokines [29]. According to this study, immune cells and cytokines are involved in the synergistic impact of treating colon cancer with 5-FU and the SphK2 inhibitor ABC294640. This finding calls for more research in the next studies. Furthermore, we agree that the differential expression of SphK2 in primary colon cancer cells explains the poorer sensitivity of the SphK2 inhibitor ABC294640 utilized in this work against colon cancer than reported in the literature [18]. This has to be confirmed under precise experimental settings.

The tumor microenvironment is the main battleground between tumor cells and the host immune system. Tumor-infiltrating immune cells are major immunological markers that strongly predict clinical outcomes [30]. The TIMER database found a substantial connection between SphK2 expression and immune cell infiltration in colorectal adenocarcinoma (COAD), including CD4 + T cells, CD8 + T cells, and neutrophils. Human samples in the database showed a positive link between SphK2 expression and increased CD4 + T cell levels, and a negative correlation with CD8 + T cell levels. This suggests that SphK2 may regulate the immunological response involved in CRC formation. According to the previous study, animals lacking Sphk2 (Sphk2−/−) have enhanced CD4+ T cell activity and proliferation, which boosts CD8+ T cell immunology [31]. There is evidence that T-cell depletion reduces 5-FU’s in vivo efficacy [32]. In SphK2 Tg mice, CD4+ T and CD8+ T cells decreased, especially in 5-FU-resistant animals. The co-administration of 5-FU and the suppression of SphK2 resulted in a substantial elevation in the quantities of CD4+ and CD8+ T cells. However, the heterogeneity in CD4+ T cell expression among species may be due to their differentiation into different cell types in differing SphK2 tumor microenvironments, which regulate the immune response. The experimental results match database studies on CD8+ T cell expression, demonstrating that CD8+ T cells have the greatest influence on the immune system. Thus, TME factors that inhibit CD8+ T cells may explain why 5-FU doesn’t operate on CRC with high SphK2 expression.

MDSCs comprise the preponderance of immune cells that infiltrate the TME [33]. Chemotherapy removes TME cell immunosuppression [34]. High-dose 5-FU selectively kills MDSCs and activates CD8+ T cells in the spleen and tumor [7]. SphK2 Tg mice could not take large 5-FU dosages, thus we used a small-dose multiple-dose protocol. SphK2 Tg mice were poorly anticancer after 5-FU treatment, and we identified a large density of immunosuppressive MDSCs in the TME, which sustained 5-FU-induced T-cell immunosuppression and exacerbated therapeutic resistance. MDSCs exhibit variability in the composition of immunosuppressive molecules, as different subsets of MDSCs possess distinct levels of arginase [35] and inducible nitric oxide synthase [36]. G-MDSCs preferentially use arginase I (ARG1) to mediate immunosuppression and are independent of inducible nitric oxide synthase (iNOS) [37], while M-MDSC-mediated inhibition mostly relies on nitric oxide (NO) and inhibitory cytokines for their suppressive activities [36, 38]. We found that targeting SphK2, which causes 5-FU resistance, by ABC294640 reduced MDSC accumulation in vivo, especially G-MDSC, in the SphK2 Tg mice CRC model. When all of these medicines are taken together, they can greatly reduce the expression of ARG-1, which makes it harder for G-MDSC to suppress the immune system. Due to the presence of iNOS and ARG-1, it is unclear whether additional factors can inhibit M-MDSC.

MDSC proliferation has been extensively studied, although its mechanism remains unknown. A growing number of studies support the dual-signaling concept, which posits that MDSC creation is a sequential process initiated by proliferation and activation [36, 39]. IL-6, TNF-α, IFN-γ, and vascular endothelial growth factor can regulate the production and activation of MDSCs [39]. These chemicals normally activate the immunological response to acute inflammation, but their persistence can cause MDSC buildup and activation. Just as elevated systemic IL-6 promotes extramucosal malignancy development, MDSCs are also mobilized [40]. IL-6, p-STAT3, and ARG-1 were positively correlated with SphK2, showing that SphK2 is enhanced due to MDSC proliferation. MDSC development may be modulated by IL-6 via the downstream JAKs/STAT3 pathway. The first transcription factor linked to malignant MDSC development is STAT3 [41]. IL-6 stimulated Arg1 to promote MDSCs-mediated immunosuppression, while SphK2 overexpression elevated IL-6 and IL-10 to increase drug-resistant tumor microenvironment. 5-FU induces targeted cell death in MDSCs and activates CD8+ T lymphocytes to generate IFN-γ, especially in response to antigens [7]. Effects were consistent in our experiments. IL-6/STAT3/ARG-1 controls MDCS proliferation and activation, hence inhibiting SphK2 restores 5-FU resistance. In response, CD8+ T cells in the tumor microenvironment release IFN-γ, leading to reduced tumor burden.

This study has some shortcomings. The research notes that Gr-1, used to screen medicines that specifically reduce MDSC, is also expressed in granulocytes, therefore opportunistic infections in hormone hosts may arise utilizing this antibody [7]. Our objective of researching SphK2 resistance to TME was incompatible with our extended animal experiment cycle, hence we did not inject a monoclonal antibody against Gr-1 in vivo. In addition, not just MDSCs, but also macrophages and other cell lines express ARG-1, iNOS, and STAT3. Recent investigations have revealed that MDSCs may develop into tumor-associated macrophages in the tumor environment and divide into the tumor-inhibiting M1 and tumor-promoting M2 subpopulations [42]. Future research will include macrophage-associated tests, which this study did not.

Conclusions

The interaction between tumor-infiltrating cells and the disease’s microenvironment is the new focus of therapy beginning in 5-FU-resistant colon cancer caused by SphK2 overexpression. Different delivery methods have varying effects on prognosis. Consequently, concomitant 5-FU anticancer treatment targeting host SphK2 and accurate screening of patients with high SphK2 expression can improve therapeutic effectiveness and reduce therapeutic resistance.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

The transgenic mice overexpressing Sphingosine Kinase 2 (SphK2 Tg) used in this study were generously provided by Professor Xianjun Qu from the School of Basic Medical Sciences at Capital Medical University in Beijing, China.

Funding

This project was supported by a grant from the Shandong Medical and Health Technology Development Project (Grant No. 202113010434), Traditional Chinese Medicine Science and Technology Project of Shandong Province (Grant No. M-2023184).

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Author notes

  1. Xiuyun Li and Yungao Chen contributed equally to this work.

Authors and Affiliations

  1. Maternal and Child Health Development Research Center, Shandong Provincial Maternal and Child Health Care Hospital Affiliated to Qingdao University, Jinan, China

    Xiuyun Li

  2. Human Resources Department, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China

    Yungao Chen

  3. School of Nursing, Peking Union Medical College, Beijing, China

    Yulin Liang

  4. Department of Pharmacy and Shandong Provincial key Traditional Chinese Medical Discipline of Clinical Chinese pharmacy, Shandong Cancer Hospital and Institute, Shandong First Medical University, Shandong Academy of Medical Sciences, Jinan, China

    Wenna Shi

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Contributions

Wenna Shi conceived and devised the investigation.Yungao Chen wrote the paper.Xiuyun Li edited the manuscript.Wenna Shi and Xiuyun Li were responsible for the propagation and identification of transgenic mice, as well as the completion of the animal model.Yungao Chen and Yulin Liang participated in the Western blot and flow cytometry investigations.All authors contributed to the paper’s creation and authorized its ultimate form.

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Li, X., Chen, Y., Liang, Y. et al. 5-Fluorouracil resistance due to sphingosine kinase 2 overexpression in colorectal cancer is associated with myeloid-derived suppressor cell-mediated immunosuppressive effects. BMC Cancer 24, 983 (2024). https://doi.org/10.1186/s12885-024-12742-4

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BMC Cancer

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