Fibrinogen-like protein 1 induces the formation of an immunosuppressive microenvironment by upregulating fibronectin 1 to promote immune evasion in bladder cancer

Cells, transfection, and samples

Bladder cancer cell lines 5637 (HTB-4), RT4 (HTB-2), T24 (HTB-9), and UMUC3 (CRL-1749) and immortalized human ureteral epithelial cell line SV-HUC-1 (CRL-9621) were acquired from the American Type Culture Collection (Manassas, VA). The cells were cultured with Roswell Park Memorial Institute medium (RPMI)-1640 medium (90022, Solarbio, Beijing) containing 10% fasting blood sugar (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified incubator (Eppendorf) supplied with 5% CO₂ air at 37°C. Peripheral blood mononuclear cells (PBMCs) were isolated from the venous blood of three healthy volunteer donors using density gradient centrifugation. Written informed consent was obtained from all the participants. The identity of the cell lines was confirmed, and cross-contamination was ruled out through short tandem repeat (STR) analysis. Routine testing was performed using the MycoSEQ^™ Plus Kit (A55124) following the manufacturer’s protocol to ensure the absence of mycoplasma contamination. Overexpression plasmids for FN1 (pcDNA-FN1) and FGL1 (pcDNA-FGL1) and their negative control (pcDNA3.1) were purchased from RiboBio Ltd. Small interfering RNA (siRNA) for FGL1 (si-FGL1) and FN1 (si-FN1) and their negative control (si-NC) were obtained from GenePharma (Shanghai, China). The target sequences were as follows: si-FGL1 F5'-GGG AAG UUC UAC AAU UCU AAU-3' and R5'-UAG AAU GUA GAA CUC CCA G-3'; FN1-siRNA F5'-CCA UUU CAC CUU CAG ACA ATT-3' and F5'-UUG UCU GAA GGU GAA AUG GTT-3'; and NC-siRNA F5'-UUC UCC GAA CGU GUC ACG UTT-3' and R5'-ACG UGA CAC GUU CGG AGA ATT-3'. After reaching 60% confluence, the cells were transfected with pcDNAs or siRNAs in accordance with the instructions of the Lipofectamine^™ 2000 kit (11668-027; Invitrogen, USA). The instructions of the purchased reagent kit were strictly followed while implementing cell transfection. Tumor tissue and adjacent tissue were collected from 30 patients with bladder cancer who received treatment in Yantaishan Hospital (ethical approval number: LL-2025-080-L). This study was conducted in accordance with the ethical principles outlined in the 2024 version of the Declaration of Helsinki.^[16] All the subjects signed the informed consent form.

Animals

Human PBMC (huPBMC) non-obese diabetic (NOG) mice (weight 20 ± 5 g, 6 weeks) were obtained from Beijing Charles River Laboratory Animal Technology Co., Ltd. This mouse model of human immune system reconstitution was constructed by transplanting huPBMCs into immunodeficient NOG mice. The mice were housed in an SPF environment with a 12/12 h light/dark cycle and unrestricted water and food and were randomly assigned to two experimental groups (n = 8 per group): the si-NC group, in which RT4 cells transfected with si-NC were subcutaneously implanted and the si-FGL1 group, in which RT4 cells transfected with siFGL1 were administered through the same route. The cells were digested with trypsin and added to phosphate-buffered saline (PBS) to prepare a cell suspension. A total of 10⁵ cells were injected subcutaneously into the right armpit of the mice. Tumor length and width were assessed using a vernier caliper every 7 days a week after cell injection to estimate tumor volume. The mice were euthanized after the 35^th day, and tumor tissues were stripped for subsequent studies. The euthanasia protocol was as follows: The mice were placed in a euthanasia chamber filled with 70% CO₂ and maintained under these conditions for 5 min. Following the cessation of breathing and heartbeat, cervical dislocation was carried out to confirm and ensure euthanasia. This study was approved by the Animal Ethics Committee of Affiliated Hospital of Qingdao University (FFSY_2024_05_78).

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from the tumor tissues and their adjacent non-tumor tissues using TRIzol^® Reagent (Invitrogen, Carlsbad, CA, USA). Approximately 50 mg of tissue was homogenized in 1 mL of TRIzol reagent using a tissue homogenizer. The homogenate was then subjected to phase separation by adding chloroform, followed by centrifugation at 12,000 × g for 15 min at 4°C. Complementary DNA was synthesized from 1 µg of total RNA using Takara’s PrimeScript^™ RT Master Mix (Takara Bio Inc., Kusatsu, Shiga, Japan). RT-qPCR was performed using SYBR^® Green Premix Ex Taq^™ (Takara) on a StepOnePlus real-time polymerase chain reaction system (Applied Biosystems, Foster City, CA, USA). The primers were as follows: FGL1 forward, 5'-GCA AGG AGT CTG CTT CTG CT-3', reverse, 5'-TGC CAT GTT CCC CCT TGA AA-3' and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward, 5'-GGA GCG AGA TCC CTC CAA AAT-3', reverse, 5'-GGC TGT TGT CAT ACT TCT CAT GG-3' (Sangon Biotech, Shanghai, China). The RT-PCR cycling conditions were 95°C for 3 min, followed by 35 cycles of amplification for 20 s at 95°C, 30 s at 55°C, and 15 s at 72°C, and a final extension for 1 min at 72°C. The messenger RNA (mRNA) expression levels of FGL1 were normalized to the housekeeping gene GAPDH, which served as an endogenous control. Relative gene expression was calculated using the 2^−ΔΔCt method, where the ΔCt values were obtained by subtracting the Ct value of GAPDH from that of FGL1 for each sample and the ΔΔCt values were calculated by comparing the ΔCt of each tumor sample to that of a reference calibrator sample. All reactions were run in triplicate, and data were presented as fold change relative to the control group.

Western blot

RIPA lysis buffer (P0013B, Beyotime, China) was used to obtain the total protein from tissues and cells. Protein concentration was quantified using the bicinchoninic acid protein assay kit (23227, Thermo Fisher Scientific, USA). According to the molecular weight of the target protein, 12% polyacrylamide gel was used to separate the protein through SDS-PAGE. Following electrophoretic separation, the proteins were transferred onto a polyvinylidene fluoride membrane (IPVH00010, Merck Millipore, Germany) using a wet transfer apparatus at a constant voltage (typically 100 V for 90 min) in transfer buffer containing 20% methanol. The membrane was placed in a blocking solution consisting of 5% nonfat dry milk or 3% bovine serum albumin dissolved in Tris-buffered saline containing 0.1% Tween-20 and then incubated for 2 h with gentle shaking to prevent nonspecific binding. The primary antibodies used were as follows: Rabbit polyclonal anti-β-actin antibody (1:3000, ab8227, Abcam, Cambridge, UK), rabbit polyclonal anti-FGL1 antibody (1:2000, ab236552, Abcam, Cambridge, UK), and rabbit monoclonal anti-FN1 antibody (1:1000, ab2413, Abcam, Cambridge, UK). The membranes were treated with horseradish peroxidase-labeled secondary antibody goat anti-rabbit immunoglobulin G (IgG) (1:4000, ab205718, Abcam) and incubated for 2 h at ambient temperature. Electrochemiluminescence detection reagents (K-12049-D50, Advansta, USA) were applied to the membrane, which was then placed into a chemiluminescent imaging system (Bio-Rad, model 1708280). Protein bands were quantified using ImageJ software 2.x version (National Institutes of Health, USA). β-actin was used as an internal control to account for variations in protein loading.

Isolation of huPBMCs

In brief, 15 mL of fresh heparin anticoagulated healthy human peripheral blood was extracted under sterile conditions, added with the same amount of PBS, and mixed well. The volume ratio of the lymphocyte separation solution to the diluted blood was 1:1. The sample was centrifuged at 400 × g in a –20°C centrifuge tube for 20 min. After centrifugation, the top layer of the centrifuge tube was plasma, the middle was the separation solution, and the bottom layer was red blood cells and granulocytes. A white film, that is, the enriched white blood cells, formed between the separation solution and the plasma. The white membrane layer was aspirated, transferred to another 15 mL centrifuge tube, added with more than 5 times the volume of PBS buffer, mixed well, and centrifuged at 400 × g for 10 min. The cells were washed twice. Resuspend the cells in culture medium and adjust the cell concentration to 1 × 10⁶/mL.

Construction of a coculture system comprising RT4 cells and PBMCs

According to the cultivation procedure, RT4 cells were incubated with PBMCs when they reached 60% fusion. The RPMI 1640 medium used for incubating PBMCs was supplemented with 10% FBS and 300U/mL interleukin (IL)-2 (PEProTech, USA). In addition, PBMCs were stimulated with anti-human CD3 monoclonal antibody (555340, BD Biosciences, 200 µL/well) and anti-human CD28 monoclonal antibody (555739, BD Biosciences, 200 µL/well) for 72 h. After activation, Transwell cells inoculated with 1 × 10⁶ PBMCs were placed in an RT4 cell culture plate and cocultured for 72 h. Then, flow cytometry (BD FACSVerse, BD Biosciences) was used to analyze the proportion of CD4+T cells, CD8+T cells, and Treg cells, and the apoptosis rate of CD8+T cells.

Detection of T cell proportion

The PBMCs were collected and washed using PBS, followed by cell resuspension using PBS and transfer to labeled tubes. The corresponding antibodies of CD4+T cells (555346, BD Biosciences), CD8+T cells (557085, BD Biosciences), and Treg cells (Foxp3-Alexa Fluor 648: 561184; CD25-PerCPCy5:555433, BD Biosciences) were added to the flow tube and fully mixed, and the cells were placed in a dark environment at room temperature for 30 min. The cells were washed and resuspended in PBS. Flow cytometry tubes were prepared, added with cell fluid, and detected using equipment.

Transwell invasion assay

An RPMI-1640 medium, which did not contain serum components, was used to starve RT4 cells for 12 h. The cells were then washed, resuspended, and counted.

The upper chamber of the Transwell system was coated with Matrigel before cell seeding. A suitable volume of cell suspension was added to the upper chamber, and RPMI-1640 medium supplemented with 10% FBS was placed in the lower chamber as a chemoattractant. The assembled Transwell plates were incubated for 48 h under standard culture conditions. Following incubation, the Transwell chambers were retrieved, and the remaining medium was aspirated. Non-migrated cells on the upper surface of the membrane were gently removed with a cotton swab. The cells that had migrated to the lower surface were fixed by submerging the chamber in 4% formaldehyde solution for 10 min. Stain with 0.1% crystal violet, after 20 min, wash off the excess dye from the membrane. Visualize and quantitatively stain cells under a microscope (Olympus IX71; Tokyo, Japan).

Plate clonogenic assay

RT4 cells were cultured in the culture plate for 14 days, and each well contained 1 × 10³ cells. On day 15, the culture medium was carefully removed, and the cell monolayers were gently rinsed with PBS. The cells were fixed by adding 4% paraformaldehyde solution to each well and incubated for 15 min at room temperature. Afterward, soak in crystal violet solution for 2 min. The solution was removed from the hole, the cells were washed repeatedly with PBS, and the number of colonies with a cell number >50 in the plate was counted under a microscope (Olympus IX71; Tokyo, Japan).

Immunohistochemical assay

Paraffin-embedded sections of the tumor and adjacent non-tumor tissues were prepared following previously established protocols. The tissue sections were subjected to antigen retrieval by incubation in antigen recovery solution. Afterward, the slides were rinsed with PBS and treated with 3% H₂O₂ for 2 min to block endogenous peroxidase activity, followed by three PBS washes. Each section was applied with 50 µL of normal goat serum to reduce non-specific binding, and the slides were incubated at room temperature for 30 min. The primary antibody working solution (FGL1: PA5-89420, 1:100, Thermo Fisher Scientific, USA; Ki67: ab8191, 1:200, Abcam, UK) was then added, and the sections were incubated overnight at 4°C in a humidified chamber. Following incubation, the slides were returned to room temperature and allowed to equilibrate for 30 min. Immunostaining was developed by applying DAB (D4293, Sigma-Aldrich, USA) chromogen for 5 min. The sections were then counterstained with hematoxylin, dehydrated through a graded ethanol series, and mounted for microscopic evaluation. Finally, the sections were sealed with neutral resin. High-resolution photographs were taken under the Olympus BX53 microscope (Olympus Corporation, Japan), and the number of brown positive cells was automatically identified and calculated using ImageJ/Fiji (National Institutes of Health, USA).

Wound healing assay

First, the cells were seeded into a six-well plate. After the cells spread all over the hole, two vertical lines with uniform width were drawn in the hole with a sterile pipette gun head. The old medium was aspirated, and the scratched cells were gently rinsed 3 times with PBS, added with serum-free medium, and continued to be cultured. Cell migration status was observed and recorded under a microscope (Olympus BX53) at 0 and 24 h.

Enzyme-linked immunosorbent assay (ELISA)

The supernatant of PBMCs was collected and centrifuged at 1500 rpm/min for 3 min to remove the sediment. The standard was diluted by concentration gradient in accordance with the kit instructions. Volume 100 µL of the standard and the sample to be tested were dropwise added to the microplate plate and reacted at 37°C for 2 h. Biotin-labeled interferon gamma (IFN-γ) (ab46070, Abcam, UK) and IL-10 (ab46077, Abcam, UK) antibody working solutions were then added to the well, which was then placed in a 37°C for 1 h. The sample was added with the colorimetric solution and placed in a dark environment for 0.5 h. After the stop solution was added, the absorbance value at 450 nm wavelength was detected using a microplate reader (Multiskan GO; Thermo Fisher Scientific). The calibration curve was employed to calculate the levels of IFN-γ and IL-10.

CO-IP assay

The cells were washed twice with PBS, added to precooled lysate, and lysed. The lysate was transferred to a centrifuge tube, which was then placed on ice for 10 min, and ultrasonic fragmentation was performed on the cells. After centrifuging the sample, take the supernatant and add an appropriate amount of PBS, then add the labeled binding beads. The mixture was then centrifuged at 5000 rpm for 5 min, and the resulting pellet was retained. Protein lysate was subsequently added to the precipitate, and the suspension was incubated at 4°C for 12 h. Centrifugation was performed at 5000 rpm for 5 min, and the precipitate was retained. Finally, 10 µL of 6× loading and 35 µL of lysate were mixed for 10 min. Western blot was used to detect protein expression.

Immunofluorescence

Antigen retrieval was performed using an antigen recovery solution. Add Triton X-100 (T8787, Sigma Aldrich, USA) dropwise onto the slices and incubate for 10 min. Add goat serum to the slices and incubate at room temperature for 1 h to block non-specific binding sites. Afterward, the sections were washed 3 times with PBS. A diluted primary antibody solution (rabbit monoclonal CD4: 14-0041-82, 1:100, Cell Signaling Technology, USA; mouse monoclonal CD8: 300902, 1:200, BioLegend, USA) was then applied, and the sections were incubated overnight at 4°C in a humidified chamber. Upon removal from the cold, the sections were equilibrated at room temperature for 60 min, followed by five PBS washes. The appropriate secondary antibody (CD4: goat anti-rabbit IgG, A-11008; CD8: goat anti-mouse IgG, A-11005; 1:500, Thermo Fisher Scientific, USA) diluted in blocking buffer was applied to the sections, which were then incubated for an additional 60 min at room temperature and washed 5 times with PBS. Excess liquid was carefully removed, and 10 µL of an antifade mounting medium containing DAPI (62248, Thermo Fisher Scientific, USA) was added to each slide. A coverslip was applied, and the sections were allowed to stand for 15 min before being examined under a fluorescence microscope (Olympus IX71; Tokyo, Japan). Images were captured and stored for subsequent analysis.

Statistical analysis

All data were analyzed with the Statistical Package for the Social Sciences (SPSS) software (version 22.0, SPSS, Chicago). The values derived from three independent experiments were expressed as mean ± standard deviation. Comparisons between the two groups were analyzed by t-tests. For comparison between multiple groups, oneway analysis of variance was applied to assess variance homogeneity, and non-parametric tests were used to assess uneven variance. For non-normally distributed data, the Mann–Whitney U-test was used for comparison between two groups, and the Kruskal–Wallis test was used for comparison between multiple groups. n = 3 in cell experiment; n = 8 in animal experiments. Values with P < 0.05 were statistically significant.

FGL1 expression in bladder cancer tissues and cells

We collected 30 pairs of tumor tissues and paracancerous tissues from patients with bladder cancer and detected the mRNA expression level of FGL1 by RT-qRCR. The results showed that FGL1 was upregulated in the tumor tissues [Figure 1a]. In addition, the protein expression of FGL1 was increased in the bladder cancer cell lines including 5637, RT4, T24, and UMUC3 compared with that in the human ureteral epithelial immortalized cell line SV-HUC-1 [Figures 1b and c]. Immunohistochemical analysis of the paracancerous tissues and tumor tissues of three patients showed that the number of FGL1-positive cells was significantly increased in the bladder cancer tissues [Figure 1d and e].

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Figure 1:

Fibrinogen-like protein 1 (FGL1) expression in bladder cancer tissues and cell lines is upregulated. (a) The messenger RNA expression of FGL1 in adjacent and tumor tissues from bladder cancer patients was detected by reverse transcription quantitative polymerase chain reaction paired sample t-test. n = 30. (b and c) Western blotting was used to detect the protein expression of FGL1 in bladder cancer cell lines including 5637, RT4, T24, and UMUC3, and human ureteral epithelial immortalized cell line SV-HUC-1 was used as a control. n = 3. The difference between groups was analyzed by one-way analysis of variance. (d and e) Immunohistochemical images of FGL1 from adjacent and tumor tissues of three bladder cancer patients. The difference between samples was analyzed by paired sample t-test. ^✶✶P < 0.01, ^✶✶✶P < 0.001. Scale bar = 50 µm.

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FGL1 promotes RT4 cell growth

We transfected pcDNA3.1-FGL1 and si-FGL1 into RT4 cells and observed that the FGL1 overexpression vector promoted FGL1 protein expression, whereas FGL1 siRNA inhibited FGL1 protein expression [Figure 2a and b]. Next, we studied the association of FGL1 expression with the invasion and migration of RT4 cells. When FGL1 was overexpressed, it promoted the physiological activities of the cells. Meanwhile, FGL1 silencing inhibited the migration [Figures 2c and d], invasion [Figure 2e and f], and clonogenic ability [Figure 2g and h] of RT4 cells.

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Figure 2:

Fibrinogen-like protein 1 (FGL1) promotes the growth of bladder cancer cells. The RT4 cells were transfected with pcDNA3.1-FGL1 or si-FGL1 for 48 h, respectively. (a and b) The transfection efficiency of pcDNA3.1-FGL1 and si-FGL1 was detected by reverse transcription quantitative polymerase chain reaction. (c and d) Cell migration was determined by wound-healing assay. (e and f) Transwell assay was used to detect the changes of invasion ability of RT4 cells after overexpression or knockdown of FGL1. (g and h) Cell clonogenic ability assay. ^✶✶P < 0.01, ^✶✶✶P < 0.001. n = 3. Scale bar = 50 µm. The difference between samples was analyzed by one-way analysis of variance.

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FGL1 promotes the formation of immune microenvironment in bladder cancer

We co-incubated PBMCs and RT4 cells transfected with pcDNA3.1-FGL1 to simulate the immune microenvironment in this malignant tumor. After completing the coculture operation, we used relevant techniques to detect the T-cell proportion in different groups. The proportion of CD4+T cells [Figure 3a and b] and CD8+T cells [Figure 3c and d] in the PBMCs cocultured with the FGL1-silenced RT4 cells was increased, whereas that of Tregs cells were decreased [Figure 3e and f]. ELISA showed that FGL1 overexpression significantly reduced the secretion level of IFN-γ and increased that of IL-10. By contrast, the secretion level of IFN-γ increased [Figure 3g] and the secretion of IL-10 decreased [Figure 3h] in the PBMCs incubated with FGL1-silenced RT4 cells.

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Figure 3:

Fibrinogen-like protein 1 (FGL1) promotes immune evasion of bladder cancer cells. The RT4 cells overexpressing or knocking down FGL1 were co-cultured with peripheral blood mononuclear cells (PBMCs). The proportions of (a and b) CD4+T cells, (c and d) CD8+ T cells, and (e and f) Treg cells in PBMCs after co-culture were detected by (g and h) flow cytometry. The secretion levels of factors interferon gamma (immune effector cytokines) and interleukin-10 (immunosuppressive cytokines) in the supernatant of PBMCs after co-culture were detected by enzyme-linked immunosorbent assay. ^✶P < 0.05, ^✶✶P < 0.01, ^✶✶✶P < 0.001. n = 3. The difference between samples was analyzed by one-way analysis of variance.

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FGL1 upregulates FN1

Through the retrieval and analysis of online bioinformatics databases, we found that FN1 may be a potential binding protein of FGL1 ([Figure 4a], https://cn.string-db.org/). Coimmunoprecipitation test confirmed the binding relationship between FGL1 and FN1 [Figure 4b]. We then transfected pcDNA3.1-FGL1 and si-FGL1 into RT4 cells and observed that FN1 expression was promoted by FGL1 overexpression vector but inhibited by FGL1 siRNA [Figure 4c and d]. We collected three pairs of tumor tissues and adjacent tissues from patients with malignant tumors. Compared with adjacent tissues, the malignant tumor tissues had significantly higher expression levels of FN1 protein [Figure 4e and f].

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Figure 4:

Fibrinogen-like protein 1 (FGL1) upregulates fibronectin 1 (FN1) protein expression. (a) Through online biological database search, we screened FN1 as a potential protein that may interact with FGL1 (https://cn.string-db.org/). (b) The binding relationship of FGL1 and FN1 was confirmed by CO-IP assay. n = 3. (c and d) Western blotting was used to detect the effect of overexpression or knockdown of FGL1 on FN1 protein expression. n = 3. (e and f) The protein expression of FN1 protein in adjacent and tumor tissues from bladder cancer patients (3 pairs) was detected by Western blotting. ^✶✶P < 0.01, ^✶✶✶P < 0.001. The difference between samples was analyzed by a paired sample t-test (f) or one-way analysis of variance.

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FN1 promotes the migration, invasion, and clonogenesis of RT4 cells

We transfected pcDNA3.1-FN1 and si-FN1 into RT4 cells and observed that the FN1 protein expression in the cells was promoted by the FN1 overexpression vector but inhibited by FN1 siRNA [Figure 5a and b]. Analysis on the relationship between FN1 expression and the invasion and migration of RT4 cells revealed that when FN1 overexpression promotes the physiological activities of the cells. By contrast, FN1 silencing inhibited the migration [Figure 5c and d], invasion [Figure 5e and f], and clonogenic ability [Figure 5g and h] of RT4 cells.

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Figure 5:

Fibronectin 1 (FN1) promotes the growth of bladder cancer cells. The RT4 cells were transfected with pcDNA3.1-FN1 or small interfering FN1 (si-FN1) for 48 h, respectively. (a and b) The transfection efficiency of pcDNA3.1-FN1 and si-FN1 was detected by reverse transcription quantitative polymerase chain reaction. (c and d) Cell migration was determined by wound-healing assay. (e and f) Transwell assay was used to detect the changes in invasion ability of RT4 cells after overexpression or knockdown of FGL1. (g and h) Cell clonogenic ability assay. ^✶P < 0.05, ^✶✶P < 0.01, ^✶✶✶P < 0.001. n = 3. Scale bar = 50 µm. The difference between samples was analyzed by one-way analysis of variance.

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FGL1 induces the immunosuppressive microenvironment through FN1

To study the relationship between the immunosuppressive microenvironment in bladder cancer and FGL1 and determine whether FN1 plays a mediating role in this process, we hydrolyzed the blocking experiment in vitro. RT4 cells overexpressing FGL1 were added to the lumen of Transwell, followed by FN1-neutralizing antibodies to block FN1. Flow cytometry showed that FGL1 overexpression decreased the proportion of CD4+T cells [Figure 6a and b] and CD8+T cells [Figure 6c and d] but increased the proportion of Treg cells [Figure 6e and f] in the PBMCs. Meanwhile, the effect of FGL1 overexpression was reversed by the FN1-neutralizing antibodies. ELISA results indicated that FN1-neutralizing antibodies can reverse the decrease in IFN-γ secretion [Figure 6g] and the increase in IL-10 secretion [Figure 6h] caused by FGL1 overexpression.

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Figure 6:

Fibrinogen-like protein 1 (FGL1) promotes immune evasion of bladder cancer cells through fibronectin 1 (FN1). The RT4 cells overexpressing FGL1 were co-cultured with peripheral blood mononuclear cells (PBMCs), and FN1 neutralizing antibody (anti-FN1) was added to the co-culture system. The proportions of (a and b) CD4+T cells, (c and d) CD8+ T cells, and (e and f) Treg cells in PBMCs after co-culture were detected by (g and h) flow cytometry. The secretion levels of factors interferon gamma (immune effector cytokines) and interleukin-10 (immunosuppressive cytokines) in the supernatant of PBMCs after co-culture were detected by enzyme-linked immunosorbent assay. n = 3. ^✶P < 0.05, ^✶✶P < 0.01, ^✶✶✶P < 0.001. The difference between samples was analyzed by one-way analysis of variance.

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FGL1 silencing inhibits the formation of an immune microenvironment in mice with bladder cancer

A mouse model of subcutaneously transplanted bladder cancer tumor was established to explore the relationship between the immune microenvironment in bladder cancer and FGL1-FN1 molecular regulation axis. Compared with those of the control group, the subcutaneously transplanted tumors of the FGL1-silenced mice grew slower, the volume of the formed tumor was smaller [Figure 7a], and the tumor weight was lower [Figure 7b]. The representative image is shown in Figure 7c. A reduction was observed in FGL1 and FN1 protein expression [Figure 7d and e] and the number of Ki67 dye positive [Figure 7f and g]. Immunofluorescence results of the transplanted tumor tissues in mice showed that the localization of CD4 [Figure 7h and i] and CD8 [Figure 7j and k] was more localized in the cytoplasm.

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Figure 7:

Fibrinogen-like protein 1 (FGL1) silencing inhibits tumor formation in nude mice. The logarithmic phase cells of each group were collected, adjusted to 100 µL single-cell suspension with phosphate-buffered saline, and injected subcutaneously into the right armpit of nude mice. The tumorigenesis of mice was observed every 7 days, and the mice were euthanized on the 35^th day, and the tumor tissues were collected for subsequent research. (a) Growth curve of transplanted tumor on days 7, 14, 21, 28, and 35. (b) The weight of the transplanted tumor at day 35. (c) Representative images of tumor tissues at day 35. (d and e) The protein expressions of FGL1 and fibronectin 1 were detected by Western blotting. (f and g) Immunohistochemical images of Ki67 in tumor tissues after knockdown of FGL1 expression. Scale bar = 50 µm. Immunofluorescence images of CD4 (h and i) and CD8 (j and k) in tumor tissues. Scale bar = (h) 100 µm and (j) 25 µm. ^✶P < 0.05, ^✶✶P < 0.01, ^✶✶✶P < 0.001. n = 8. The difference between samples was analyzed by one-way analysis of variance.

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