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Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39223212
71014
10.1038/s41598-024-71014-0
Article
Impact of anti leukemia inhibitory factor antibody on immune related gene expression in breast cancer Balb/c mouse model
Seifati Seyed Mohammad 12
Zare Fateme 1
Bafghi Seyed Ali Mirghanizadeh 2
Hadinedoushan Hossein hhadin@ssu.ac.ir
hhadin2000@gmail.com

12
1 https://ror.org/03w04rv71 grid.411746.1 0000 0004 4911 7066 Reproductive Immunology Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Postal Code: 8916181635 Iran
2 https://ror.org/03w04rv71 grid.411746.1 0000 0004 4911 7066 Department of Immunology, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
2 9 2024
2 9 2024
2024
14 2040321 5 2024
23 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Leukemia inhibitory factor (LIF) is involved in the progression of different cancers. In this study, we investigated the effect of anti-LIF antibodies on immune-related gene expression in the Balb/c mouse model of breast cancer. To immunize mice against LIF, recombinant LIF with Freund adjuvant was injected into the test group, whereas the control group received phosphate-buffered saline with adjuvant. Tumor induction (4T1 cell line) was performed by increasing the antibody titer. The expression of immune-related genes was evaluated by real-time PCR. The anti-LIF titer was significantly increased in the immunized group. The expression of genes related to the differentiation of T helper (Th)-1, Th-2, and Th-17 cells was significantly higher in the immunized group than in the control group. In addition, anti-LIF did not have a significant effect on the expression of genes related to the differentiation of regulatory T cells, and immune checkpoint-associated genes. Additionally, the test group had higher survival and lower tumor development rates. The results demonstrated that the anti-LIF antibody may potentially play a role in the differentiation of immune cells or immune responses. However, further studies utilizing advanced techniques are necessary to validate its function.

Keywords

Breast cancer
Immunity
Immunization
Leukemia inhibitory factor
Neutralizing antibodies
Subject terms

Cancer
Immunology
issue-copyright-statement© Springer Nature Limited 2024
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pmcLeukemia inhibitory factor (LIF) is a pleiotropic cytokine with diverse activities, that belongs to the interleukin (IL)-6 family and has multiple functions in different tissues1. For the first time, LIF was extracted from serum-free conditioned medium produced by culturing normal L929 fibroblast cell lines2. This protein was named after its ability to inhibit the growth and differentiation of M1 myeloid leukemia cells into macrophages and granulocytes3. The functions of LIF include blastocyst implantation, development and differentiation of nerve cells, regulation of mesenchymal-to-epithelial transition during kidney development, potential immune tolerance in the maternal–fetal relationship bone metabolism, and the occurrence of various types of cancer4–6. LIF acts on target cells by binding to the LIF receptor (LIFR)/glycoprotein 130 (gp130) complex. Altogether, Several signaling pathways are activated when LIF binds to the LIFR complex, including MAPK, JAK/STAT, AKT, and mTOR7. LIF is implicated in a variety of physiological and pathological processes8, and its expression is associated with poor prognosis in a number of tumor types, including nasopharyngeal carcinoma9, lung cancer10, colorectal cancer11, glioblastoma multiforme12, and breast cancer13.

Breast cancer has a high frequency in women and is one of the main causes of cancer-related deaths worldwide14. The American Cancer Society projects that by 2024, breast cancer is expected to be the most prevalent cancer among women, with an estimated 310,720 new cases and 42,250 fatalities15. The symptoms of breast cancer include the presence of a lump in the breast, loss of breast structure, or asymmetry. Further investigation is performed using ultrasound, magnetic resonance imaging, or core needle biopsy16. Despite significant advances in the treatment of breast cancer, a notable rise in mortality rates globally over the past 25 years may be linked to increased incidence and prevalence of the disease17. Therefore, understanding the molecular pathways underlying the development and metastasis of breast cancer is crucial.

According to recent studies, LIF and LIFR expression is strongly increased in breast cancer and is related to the biological characteristics of breast cancer18. LIF and Oncostatin M (OSM) can function as growth factors in breast cancer and promote the development of breast tumor epithelial cells through a paracrine or autocrine pathway19. Signal transducer and activator of transcription-3 (STAT-3) can be activated by LIFR and OSM receptors, and this activation is associated with the malignant phenotype. These effects cause cell growth and apoptosis to be suppressed through an increase in Bcl-xL expression20. Additionally, LIF can activate the AKT-mTOR signaling pathway and increase the invasion and metastasis of breast cancer cell13. Fibroblasts adjacent to the tumor are stimulated by growth factors and cytokines released into the tumor microenvironment. High levels of LIF secreted by tumor cells stimulate fibroblast activation and promote cancer cell invasion10. Recent research has demonstrated that tumor growth in mouse model of cancer is inhibited by LIF-neutralizing antibodies21,22. It has been observed that the blockage of LIF is associated with an increase in C-X-C motif chemokine ligand 9 production by macrophages and a concurrent decrease in CD163, CD206, and CCL22622. Additionally, LIF inhibition enhanced CD8+ T-cell infiltration in a tumor model22. Exogenous LIF enhances cell invasion, migration, and proliferation by triggering the AKT/mTOR pathway. This effect is inhibited by the addition of an LIF-neutralizing antibody or shRNA knock-down of LIF13.

There have been several studies on the inhibition of LIF, LIFR, and its signaling pathways using different molecules and techniques, and they have produced encouraging results. In this study, the effect of anti-LIF antibodies produced in immunized mice on immune-related gene expression in a breast cancer Balb/c mouse model was investigated.

Materials and methods

Reagents and materials

This study used the recombinant LIF that was synthesized in our laboratory23. Terrific Broth (TB) medium was obtained from Sigma-Aldrich USA; isopropyl-ß-D-1-thiogalactopyranoside (IPTG) was sourced from CinnaGen, Iran; Ni-NTA columns were acquired from Qiagen, USA; RPMI-1640 was purchased from Sigma-Aldrich, USA; Fetal Bovine Serum (FBS) was supplied by Gibco, USA; and both penicillin (100 U/mL) and streptomycin (100 μg/mL) were provided by Gibco, USA. GM-CSF (20 ng/mL) was obtained from Peprotech, UK; commercial rLIF was sourced from Royan Institute, Tehran, Iran; Freund’s adjuvant was provided by Sigma, USA; and 96-well Enzyme-Linked Immunosorbent Assay (ELISA) plates were purchased from SPL Life Sciences, Korea. The sheep anti-mouse immunoglobulin antibody conjugated with HRP (Horseradish Peroxidase) was acquired from Sina Biotech Co., Iran; TMB substrate (3,3',5,5'-Tetramethylbenzidine) was sourced from Pishtaz-Teb Co., Iran. TF-1 and 4T1 cell lines were obtained from the National Cell Bank of Iran. Additionally, the RNeasy Mini Kit was sourced from Qiagen, USA, cDNA synthesis was conducted using products from Parstous, Iran, and SYBR Green was obtained from Ampliqon, Denmark.

Production and functional evaluation of recombinant LIF

The recombinant LIF (rLIF) protein was constructed within a pColdI vector (Takara, Japan) and subsequently expressed in Escherichia coli (E. coli) Origami-(DE3). Details regarding the construction, expression, and purification of the rLIF protein have been previously described23. Briefly, the transformation process utilized TB media with specific antibiotic concentrations: 50 μg/mL of ampicillin, 15 μg/mL of kanamycin, and 12.5 μg/mL of tetracycline. To start the culture, the TB medium with 1% glucose and the antibiotics mentioned above, along with 100 μL of the construct, was incubated overnight at 37◦C in a shaker incubator. The next day, the culture was diluted at a ratio of 1:50 in TB medium with the same composition as previously. The lac operon was induced using IPTG once the culture reached the appropriate optical density (OD = 0.9 at 600 nm). Upon completion of the induction process, the culture was incubated overnight at 15◦C in a shaker incubator. Subsequently, on the final day of the procedure, cell pellets were harvested via centrifugation and subsequently stored at a temperature of -20 °C until further utilization. The cells were lysis through a sonication system, and the recombinant protein was subsequently purified using Ni–NTA column (Qiagen, USA) chromatography. Bradford's method determined the concentration of each fraction. Purified fractions underwent overnight dialysis. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was then used to confirm them.

The functional analysis of rLIF produced using the proliferation method of the TF-1 (erythroleukemia cell line, National Cell Bank of Iran, Code: C602) was evaluated. Briefly, in a 96-well plate (SPL Life Sciences, Korea), 2 × 103 cells were cultured with RPMI-1640 (Sigma-Aldrich, USA) containing 10% FBS, penicillin (100 U/mL, Gibco, USA) and streptomycin (100 μg/mL, Gibco, USA) and 20 ng/mL GM-CSF (Peprotech, UK). The cell pellet was then incubated at 37 °C and 5% CO2 for 24 h. Subsequently, serial dilution of purified rLIF (from 0.625 to 25 ng/mL) was added to the wells and the incubation was repeated for over 96 h. Positive control cells were treated with commercial rLIF (Royan Institute, Tehran, Iran), while negative control cells were not given GM-CSF. Following this, 0.5 mg/mL MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) solution was added to all wells and incubated for 4 h. Finally, 100 μL of dimethyl sulfoxide (DMSO) was added and incubated for 10 min. The absorbance of the wells was measured at a wavelength of 570 nm. The proliferation effect of rLIF and positive control on TF-1 cells was determined by dividing the O.D. 570 from the treated well by the O.D. 570 in the untreated well.

Animal study

The interventional study was performed on 25 female Balb/c mice (6–8 weeks old). The mice were obtained from the Animal Laboratory of the Pasteur Institute of Iran. All mice were housed under standard laboratory conditions (12/12 h light/dark cycle, temperature 22 °C, humidity 55%, and free access to water and food). All experiments were conducted in accordance with the relevant guidelines (including the ARRIVE guidelines). The use of animals’ guidelines was approved by the Shahid Sadoughi University of Medical Sciences Ethics Committee in Yazd, Iran (Ethics Code No: IR.SSU.MEDICINE.REC.1400.096).

Mice immunization

In this study the mice were allocated to three groups: the experimental groups (EI: non-tumor vaccinated mice, N = 5; EII: tumor vaccinated mice, N = 10) and a control group (N = 15) (Supplementary Fig. S1). The test group (EI and EII) received Freund’s adjuvant (Sigma, USA) and rLIF protein, whereas the control group received phosphate-buffered saline (PBS) with adjuvant. In this study, the mice were immunized with rLIF protein injected subcutaneously four times every two weeks and intraperitoneal for the final injection. Complete Freund's adjuvant was used for first injection of 40 μg of antigen (rLIF), and incomplete Freund's adjuvant was used for the subsequent injections of half the original protein concentration. Before each immunization, blood was collected from the tail vein of the mice.

Evaluate the production of anti-LIF antibody in immunized mice

To evaluate the production of antibodies against rLIF in immunized mice serum, the following method was used. In a 96-well ELISA plate (SPL Life Sciences, Korea), 2 μg of pure rLIF was coated and incubated for 90 min at 37 °C. The wells were then blocked with 100 µL of a 3% Skim milk solution dissolved in PBS for 60 min. In the following, 50 µL of the control sample and serum sample dilutions (prepared by diluting the samples in PBS at a 1:200 ratio) were dispensed into the wells. This was followed by a two-fold serial dilution process, progressing up to 1:1600 for each sample. The wells were incubated for 90 min at 37 °C. Next, sheep anti-mouse immunoglobulin antibody conjugated with HRP (Horseradish Peroxidase, Sina Biotech Co., Iran) at 1:500 dilutions (diluted with PBS) was added to all wells and incubated for 60 min at 37 °C. Subsequently, 50 µL of TMB substrate (Pishtaz-Teb Co., Iran) was added to each well and incubated for 15 min at room temperature and in the dark. The reaction in all wells was stopped by adding 50 µL of 1 Molar H2SO4 stop solution, and the light absorption at a wavelength of 450 nm was measured using an ELISA Reader (StateFax 3200 Awareness, USA). All wells were washed three times with PBS plus 0.05% Tween 20 solution after coating, blocking, adding sample, and detection steps. The results of the antibody titer test were determined using the formula E/C (experiment/control). Results above two were considered positive23.

E/C = absorbance of wells coated with antigen at 450 nm/absorbance of a well not coated with antigen (negative control) at 450 nm.

Western blot analysis

The specificity and purity of rLIF were evaluated using Western blot and SDS-PAGE analysis. Western blot analysis was performed to confirm the binding of antibodies produced in mice to rLIF. For this purpose, serum obtained from immunized mice with higher titer was used for purified polyclonal antibody (pAb) with protein A column. For western blot analysis, 10 μg of rLIF was run on SDS-PAGE and after separation on the gel, proteins were transferred onto a nitrocellulose membrane. The membrane after being exposed to blocking buffer and wash, was placed overnight at 4 °C in anti-rLIF pAb (purified from immunized mice) with 1:1000 dilution. After that, the membrane was washed and incubated with secondary antibodies, HRP-conjugated sheep anti-mouse (Sina Biotech, Iran). Finally, specific protein bands were visualized by adding 100 μL of the TMB (Sigma-Aldrich, USA).

Tumor induction in Balb/c mice

Tumor induction was performed on mice with high antibody titers (as per the above formula), while the remaining mice were excluded from the study. The induction involved injecting 1 × 106 cells from a 4T1 cell line subcutaneously into the back region of each mouse ten days after the last injection25. Half of the mice were sacrificed after tumor pulp formation at 10 ± 2 days after the induction of tumor cells. Spleen and lymph nodes adjacent to the tumor were isolated to evaluate the expression of the desired genes. The remaining mice were monitored to measure tumor size and survival rate. Tumor size and survival rate were calculated using the following formula, respectively: (length × width × height) × 0.5 mm3, and (mean survival time of the treated group/mean survival time of the control group × 100)26. It is worth mentioning, five immunized mice (EI) were dissected before the induction of a tumor cell line to compare the expression levels of the target genes after tumor induction, and their spleen and lymph node tissues were separated (Supplementary Fig. S1).

Gene expression

The total RNA of the spleen and the lymph nodes near the tumor was extracted using RNeasy Mini Kit (Qiagen, USA) according to the kit instructions. cDNA synthesis was performed according to the kit protocol (Parstous, Iran). The primers required for the investigated genes were designed using Primer3 input (version 0.4.0) (genes associated with cytotoxicity T lymphocytes (Granzyme B), T helper (Th)-1 (IFN-γ, STAT-1, STAT-4, and T-bet), Th-2 (IL-4, IL-10, GATA-3 and STAT-6), Th-17 (IL-17, IL-6, ROR-γt, TGF-β and STAT-3), regulatory T cells (Treg) (FOXP-3 and STAT-5b), and immune checkpoints (CTLA-4, PD-1, TIM-3, and LAG-3)) (Supplementary Table S1). Gene expression was carried out using the SYBR Green method (Ampliqon, Denmark) and the Real-Time PCR system (ABI, USA), after the primer was designed and the annealing temperature of the primers was set. The relative expression of mRNAs in all samples compared to that of the control gene was calculated using the ΔΔCt method.

Statistical analysis

The relative expression of mRNAs in all samples compared to the control gene was calculated by‏ ‏the following formulas according to Livak method:ΔCT=CT\,of\,target\,gene-CT\,of\,housekeeping\,gene.

ΔΔCT=ΔCT\,of\,target\,gene-average\,of\,control\,groupΔCT.

Relative\,Fold\,ChangeRFC=2-ΔΔCT

Initially, the RFC for each sample was computed using Microsoft Excel. Data were analyzed with GraphPad Prism version 8.00 (GraphPad Software, San Diego, CA, USA) using either parametric, un-paired t-test, or nonparametric, Wilcoxon and Mann–Whitney tests. The normality of data distribution was assessed using the Shapiro–Wilk and Kolmogorov–Smirnov tests. P < 0.05 was regarded as a significant value. Mean and standard error of the mean (SEM) were used to express the results. All graphs are drawn with GraphPad Prism version 8.00 and heat map plots analyzed with “https://www.bioinformatics.com.cn/en”, a free online platform for data analysis and visualization.

Ethical approval

The use of animals’ guidelines was approved by the Shahid Sadoughi University of Medical Sciences Ethics Committee in Yazd, Iran (Ethics Code no: IR.SSU.MEDICINE.REC.1400.096).

Results

Production and characterization of rLIF

rLIF, with a molecular weight of 22 kDa, was produced in the pColdI/LIF construct. After purification, its expression was confirmed by SDS-PAGE (Supplementary Fig. S2A). The findings from the investigation into the function of rLIF on TF-1 indicated that both produced and commercial rLIF can stimulate cell proliferation through increasing concentration (Supplementary Fig. S2B).

Evaluate the production of anti-LIF antibody in immunized mice

The rLIF coating results in the 96-well ELISA plate indicated a significant increase in anti-rLIF antibody titer in the experimental group compared to the control group (Supplementary Fig. S3A). Western blot analysis showed that the antibodies produced in mice serum specifically bind to the rLIF (Supplementary Fig. S3B).

The effect of anti-LIF antibody on tumor volume and survival rate

The tumor volume in the tumor-immunized mice (EII) was significantly lower than that in the control group (P = 0.0005) (Fig. 1A,B). Additionally, the survival rate of the immunized group was higher than that of the control group, however, this difference was not statistically significant (P = 0.06) (Fig. 1C).Fig. 1 The results of tumor induction in the studied mice‏. EII (Experimental group II): Tumor-immunized mice. (A) The tumor volume in the tumor immunized mice group was significantly reduced compared to the control group (P = 0.0005). (B) The results regarding the tumor's growth rate during a 60-day period. (C) The survival rate in the group immunized with LIF protein was higher than the control group, but this difference was not statistically significant (P = 0.06).

Differentiation of immune cells to provide an immune response against the tumor cells

In this study, the expression levels of genes involved in the differentiation of tumor-related immune cells were investigated to evaluate the immune response against tumors. The expression of Granzyme B, which is involved in the process of cell death, was increased in the lymph nodes in the test groups compared to that in the control group. This increase was significant in the EI group in lymph nodes (P = 0.02) (Fig. 2).Fig. 2 The relative expression level of Granzyme B gene in the spleen and lymph nodes of the studied mice. EI (Experimental group I): Non-tumor immunized mice, EII (Experimental group II): Tumor-immunized mice. In the spleen, the expression of this gene was significantly different between EI and EII groups (P = 0.01). Also, its expression was decreased in the EII group compared to the EI and control groups. In the lymph node, although this gene's expression increased in the test groups, this difference was statistically significant between EI and control groups (P = 0.02). (Results are reported as the mean ± SEM).

Regarding genes associated with Th-1 (IFN-γ, T-bet, and STAT-1) in the spleen, there was increased in experimental groups compared to control group. Meanwhile, only group EI showed a significant increase in comparison to the control group (P = 0.006, P = 0.02 and P = 0.04 respectively). Furthermore, among the genes related to Th-1, only STAT-4 gene expression was decreased in test groups. The expression patterns of genes related to Th-1 in the lymph nodes were slightly different. Regarding the IFN-γ gene, we observed an increase in expression similar to that in the spleen in the two immunized groups and the expression in the immunized tumor group was significant compared to that in the control (P = 0.04 and P = 0.002 respectively). In addition, the pattern of STAT-1 expression was completely different from that in the spleen, and in the test groups, the expression of this gene was significantly reduced (P = 0.002 and P = 0.0007 respectively). STAT-4 gene expression was also lower in the experimental group than in the control group. The decrease was observed between group EII and the significant control (P = 0.02) (Fig. 3).Fig. 3 The relative expression level of genes related to Th-1 in the spleen and lymph node of the studied mice. EI (Experimental group I): Non-tumor immunized mice, EII (Experimental group II): Tumor-immunized mice. The expression of IFN-γ gene in the EI and EII groups was increased compared to the control group (in spleen, EI vs control, P = 0.06 and in lymph node test groups vs control, P = 0.04 and P = 0.002 respectively). The level of T-bet gene expression increased in the tested group compared to the control. In addition, The STAT-1 gene showed higher expression in the test group compared to controls in the spleen but lower in the lymph node. (Results are reported as the mean ± SEM).

In the spleen, the relative expression of genes related to Th-2 (IL-4, Il-10, GATA-3 and STAT-6) was higher in the experimental groups than in the control group. Meanwhile, only the expression of GATA-3 and STAT-6 genes in the EII group was significant compared to the control group (P = 0.0009 and P = 0.01 respectively). In contrast to the spleen, in the lymph node IL-4 gene expression was significantly decreased in the study groups compared to the control group (P = 0.002 and P = 0.002 respectively). IL-10 gene expression in the lymph nodes was increased in the two experimental groups compared to the control group, and this increase was statistically significant between the non-tumor immunized and control groups (P = 0.01). Additionally, the expression level of GATA-3 in the experimental groups was lower than that in the control group. However, this difference in GATA-3 was significantly observed in the EII group only (P = 0.02). The expression of STAT-6 in the lymph nodes increased significantly in the studied groups compared to control group (P = 0.03 and P = 0.001 respectively) (Fig. 4).Fig. 4 Relative expression levels of genes related to Th-2 in the spleen and lymph nodes of the mice. EI (Experimental group I): Non-tumor immunized mice, EII (Experimental group II): Tumor-immunized mice. IL-4, IL-10, GATA-3 and STAT-6 genes were associated with an increase in expression in the test groups compared to the control group in the spleen. In lymph nodes, the levels of IL-4 gene expression showed significant reduce in test groups (P = 0.002 and  P = 0.002 respectively). The GATA-3 gene was significantly different in the spleen and Lymph node between the test and control groups (P = 0.01 and  P = 0.02 respectively). (Results are reported as the mean ± SEM).

In the investigations that were conducted on the genes related to Th-17 (IL-17, IL-6, ROR-γt, TGF-β and STAT-3), the results showed that IL-17, IL-6, ROR-γt, and TGF-β gene expression in the spleen in test groups were significantly higher than the control group, Except IL-6, which was not statistically significant (P = 0.00006 for all of them). In lymph nodes, the IL-17, IL-6, and ROR-γt gene expression level significantly increased in the immunized group compared to that in the control group (P = 0.03 and P = 0.005, P = 0.0005 and P = 0.004, P = 0.002 and P = 0.0002 respectively). The TGF-β gene expression in lymph nodes decreased in the test groups, unlike in spleen cells, but this difference was not significant (P = 0.06). In addition, in this investigation, The STAT-3 gene expression level was reduced in the two target tissues, However, this decrease was only notable in the lymph nodes (P = 0.02 and P = 0.0005 respectively) (Fig. 5).Fig. 5 The relative expression level of genes related to Th-17 in the spleen and lymph node of the studied mice. EI (Experimental group I): Non-tumor immunized mice, EII (Experimental group II): Tumor-immunized mice. The difference in the expression of IL-17 genes in EI vs control group in the spleen were significant (P = 0.0007), but in the lymph node, significantly increased ( P = 0.03 and P = 0.005 respectively). The expression of IL-6 gene in the two experimental groups compared to the control group was associated with an increase in both tissues, but this increase was statistically significant in lymph node only (P = 0.004 and P = 0.0005 respectively). In spleen and lymph node tissues, the expression of ROR-γt also increased significantly in the experimental groups compared to that in the control group (P < 0.0001). (Results are reported as the mean ± SEM).

In the investigations on the expression of genes related to Tregs (FOXP-3 and STAT-5b), there was significant increase in the expression of FOXP-3 in the spleen (P = 0.003 for both of them), although there was a decrease in the lymph node (Fig. 6).Fig. 6 The relative expression level of genes related to T regulatory cell in the spleen and lymph node of the studied mice. EI (Experimental group I): Non-tumor immunized mice, EII (Experimental group II): Tumor-immunized mice. The expression of FOXP-3 and STAT-5b genes in the spleen significantly increased in the test groups compared to the control group (P = 0.001 and P = 0.005 respectively). However, in the lymph node the expression of FOXP-3 was reduced. (Results are reported as the mean ± SEM).

Finally, the genes related to immune checkpoints that were investigated in this study (CTLA-4, PD-1, TIM-3, and LAG-3) in the spleen, showed an increase in the test groups. Unlike the spleen, in the lymph node, all gene expression was significantly decreased in the test groups compared to the control group (P = 0.01, P = 0.00004, P = 0.003, and P = 0.0003 respectively) (Fig. 7).Fig. 7 The relative expression level of genes related to immune checkpoints in the spleen and lymph node of the studied mice. EI (Experimental group I): Non-tumor immunized mice, EII (Experimental group II): Tumor-immunized mice. CTLA-4 expression in the spleen was significantly different among the three groups (P = 0.02). The PD-1 gene was increased in the spleen, but in the lymph node significantly reduced ( P= 0.0005 and P = 0.0001 respectively). TIM-3 and LAG-3 gene expression were higher than control group, However, it was the reverse in the lymph node. (Results are reported as the mean ± SEM).

Overall, if we consider the expression levels of the studied genes in the control group and compare the test groups with those for each gene, Supplementary Figs. S4 and S5 are obtained. In the spleen, the relative expression of all genes in the test group increased compared to that in control group, except for STAT-4, STAT-3, and Granzyme B, which decreased (Supplementary Fig. S4).

In the lymph nodes, some genes associated with Th-1 and Th-17 showed increased expression in the test group compared to the control group. The remaining genes were similar to those in the control group (Supplementary Fig. S5). Also, the gene-expression profiles in each group represented as heat-map plot in Supplementary Fig. S6.

Discussion

According to the American Cancer Society, breast cancer is the most prevalent cancer in women, with an estimated 310,720 new cases and 42,250 deaths by 202415. Although breast cancer mortality rates have decreased with advances in prevention, surgery, and adjuvant therapies, global breast cancer mortality is significantly increasing17. Therefore, there is an urgent need to understand the molecular mechanisms underlying breast cancer tumorigenesis and metastasis. Overexpression of LIF is observed in a variety of cancers including: breast cancer, but its role in cancer is not well understood. Previous studies have suggested a potential role of LIF in some cancers. This study aimed to produce active antibodies against LIF in mice. Following induction of the 4T1 cell line in the mice, the antibody's impact on immune cell differentiation genes associated with the tumor was assessed. Finally, the study showed that anti-LIF antibodies may potentially increase immune responses against tumor cells and delay breast tumor progression, improving survival in mice.

Research has been conducted on the production and identification of LIF protein and the evaluation of the contraceptive effect of antibodies against it in Balb/c mice in 202023,27. Similar to the present research, it also discussed the active production of antibodies against LIF in Balb/c mice. The study's researchers observed that the antibody titer reached an acceptable level after six weeks of active immunization.

In a study by Ghanei et al., immunization of Balb/c mice against LIF and its receptor was found to suppress mammary tumor formation in immunized mice21. The results showed the production of neutralizing antibodies and the secretion of IFN-γ and IL-2 in response to immunization. In the present study, anti-LIF production was detected in the sera of Balb/c mice. The researchers of this study reported that LIFR immunotherapy was a more effective approach than LIF for inhibiting tumor growth. According to the findings of this study, LIF and LIFR can be used to target cancer, especially tumors expressing these proteins.

Recent research actively produced anti-LIF antibodies in Balb/c mice by injecting them with LIF protein. The rate of embryo implantation in mice was assessed to examine the neutralizing function of the produced antibody. The results showed that the produced antibody prevented 80.49% of embryo implantation21,23. The present study found that the rLIF protein may be able to induce TF-1 cells proliferation. As well as, the increase of the anti-rLIF antibody titer in mice serum was examined with coated rLIF in an ELISA plate, and its specific binding to rLIF was analyzed by western blotting.

The tumor pulp became palpable 10 ± 2 days after following the induction of the tumor cell line, however, it took a little longer in the immunized group. In addition, the tumor growth process was slower in the test group during the two months’ follow-up. Based on these findings, the survival rate of tumor immunized mice was markedly greater. In the study of Ghanei et al.21, in the control group, after 28 days, tumor pulp was formed. In addition, in the group that had a high anti-LIF titer in their serum, out of 8 induced mice, only 6 developed tumors. The time for formation of the tumor pulp in this study was longer than that in the current study. This difference may be due to the use of a tumor cell line, which is different from that used in our study. Another reason could be the higher antibody titers against LIF in those mice. In this research, tumor growth and survival rates were evaluated, and the results were similar to those of our study.

According to the analysis of the expression of genes associated with Th-1 and Th-2 in this study, the test groups appeared to have more differentiated T cells than the control group. Davis et al.28 observed that LIF directly reduced IL-12 p40 release from pro-inflammatory macrophages in rats and suppressed IL-12/ IFN-γ/IP-10 expression. As a result, the differentiation towards Th-1 was inhibited. In this study, LIF was found to inhibit the maturation of CD8+ T cells and the migration of immune cells. Conversely, an increase in the differentiation of T cells towards cytotoxic T cells (as indicated by increased Granzyme B gene expression) and Th-1 was observed in the current study, possibly due to the production of anti-LIF antibody in tumor immunized mice. This antibody may have neutralized LIF and enhanced the immune response to tumor cells. Therefore, our study results are in line with the aforementioned study.

Examining the expression of genes related to Th-17 and Treg cells in the present study, it seems that the differentiation towards Th-17 increased significantly, but this was not the case with Tregs. According to previous studies on human lymphocytes, LIF release appears to be specific to T cells, especially CD4+ T cells29. Tregs were observed as the main source of LIF secretion30. On the other hand, STAT-5b is one of the Treg-related genes that can directly regulate LIF gene expression in myeloid cell lines. Thus, STAT-5b binds to the LIF promoter and induces LIF expression following the activation of the JAK2/STAT5 pathway31. In a recent study, the Tregs population in the spleen was analyzed following the injection of anti-LIF into mice with breast cancer. The study revealed that the cell population in the experimental group showed no significant difference compared to the control group32. Similar to our study, the existence of anti-LIF may be able to result in progress towards inflammatory cell differentiation, while the differentiation into anti-inflammatory cells, like Tregs, may be reduced.

Quaglino et al. studied various breast tumor cell lines by inducing Balb/c mice to investigate the activation of STAT-3 by LIF. A specific anti-LIF antibody was used in this study. The results showed that the activation of STAT-3 by LIF was inhibited in the presence of anti-LIF33. Additionally, research on the pre-invasive activation of stromal fibroblasts by LIF in cancer revealed that LIF-neutralizing antibodies blocked STAT-3 activation and remodeling of pro-invasive extracellular matrix in stromal fibroblasts and inhibited the invasion of cancer cells10. These findings also aligned with our study. The STAT-3 gene expression decreased in the spleen and lymph node, with a significant decrease observed in the lymph node. This decrease in expression might be attributed to the presence of anti-LIF antibodies.

Immune checkpoints are molecules that contribute to inhibitory pathways in the immune system and play a fundamental role in the escape of tumor cells from the immune response34. Recent reports have shown that immune checkpoints are increased in the breast tumor microenvironment35, and their expression is related to tumor progression36. According to studies that have focused on the suppression of immune checkpoints for cancer treatment, LIF functions as a treatment resistance factor. By using an LIF neutralizing antibody along with inhibiting immune checkpoints such as anti-PD-1, they observed that the response to treatment improves and their simultaneous use has a synergistic effect on tumor suppression. These investigations have been carried out at the preclinical and clinical trial levels. In a separate study, the anti-LIF antibody was injected to treat breast cancer in mice. They observed that the immune checkpoints in the group receiving the anti-LIF antibody increased significantly compared to the control group32. In our research, following the production of the anti-LIF antibody in mice, a significant reduction was observed in the lymph node. Conversely, in the spleen, there was an elevation in gene expression levels of the immune checkpoint. This implies that anti-LIF antibodies might not effectively regulate immune checkpoints in the immune cell population in the spleen. Nonetheless, they may potentially influence the expression level of these genes in the lymph node. Therefore, it is recommended to use anti-LIF in conjunction with immune checkpoint inhibitors for more effective treatment of breast cancer.

Considering the important role of LIF in cancer progression in various solid tumors, such as breast tumor, LIF holds promise as a therapeutic target in these types of cancers. As a cytokine that interacts with gp130/LIFR on the cell membrane, LIF signaling can be obstructed by anti-LIF antibodies, effectively counteracting multiple functions of LIF in various research studies37–39. Since LIF play a crucial role in immune cell differentiation. They impact signaling pathways, including JAK/STAT3, MAPK, PI3K/AKT, ERK1/2, and mTOR. In breast cancer, the AKT-mTOR pathway is significant, especially in triple-negative breast cancer (TNBC). TNBC lacks estrogen receptor, progesterone receptor, and HER2 expression, making it aggressive. Researchers are exploring targeted therapies to improve TNBC treatment outcomes 40.

LIF promotes tumorigenesis, invasion, and migration of breast cancer cells in vitro. In vivo studies show that LIF contributes to breast cancer metastasis, particularly to vital organs like the lung, bone, and brain1. LIF mediates Epithelial-Mesenchymal Transition, a process crucial for cancer cell invasion and metastasis. It enhances the pro-invasive activation of stromal fibroblasts. Also, LIF serves as a significant biomarker for breast tumors. Detecting LIF levels could aid in early diagnosis and monitoring disease progression. Given its role in breast cancer progression, LIF represents a potential therapeutic target. Developing LIF-specific antibodies or inhibitors could help manage the disease. However, in case there as some limitations including: (1) LIF modulates multiple signaling pathways, making it challenging to precisely target without affecting other essential processes41. (2) Despite recent advancements in immunotherapy (e.g., anti-PD-L1 antibodies), immunologic treatment of breast tumors remains a challenge. LIF-based therapies may face similar problems42. (3) While LIF’s involvement in breast cancer is evident, more research is needed to understand its precise mechanisms and validate its clinical utility41.

Conclusion

In summary, according to our data in the spleen, the relative expression of all genes in the test group increased compared to the control group, except for IL-4, IL-10, STAT -3, and Granzyme B, which decreased. In the lymph node, the result was that some genes such as IFN-γ, ROR-γt, IL-6, and LAG-3 had increased expression in the test group compared to the control group, and the rest of the genes were similar to the control group. In the present study, the findings demonstrated that the anti-LIF antibodies may be able to differentiate immune cells to the immune response against breast cancer such as Th-1, Th-2, and Th-17. However, it had no significant effect on genes related to Tregs and immune checkpoints. It may also control cells and response-inhibiting factors, ultimately reducing tumor progression. However, according to the results of the present and recent studies, LIF plays an important but complex role in many physiological and pathological processes, although the exact role and mechanism of LIF in these processes are not completely understood. LIF orchestrates immune cell fate by modulating these pathways, but further research is needed to uncover precise mechanisms. A better understanding of the role and mechanism of LIF in these processes will be very important for future applications in targeting LIF in the treatment of cancers and other diseases.

Supplementary Information

Supplementary Information.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-71014-0.

Acknowledgements

The authors appreciate technical help by staff in the Department of Immunology and the Reproductive Immunology Research Center at the Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Author contributions

H.H. and F.Z. contributed to the conception and design of this study. This manuscript is a part of the M.Sc. thesis of S.M.S. in Immunology course. F.Z. and S.A.M.B. supervised this study. S.M.S. and F.Z. performed material preparation, data collection and analysis. The first draft of the manuscript was written by S.M.S. and all authors commented on the previous versions of the manuscript. All authors have read and approved the final manuscript.

Funding

Financial support was provided by a grant from the Shahid Sadoughi University of Medical Sciences, Yazd, Iran (Grant no.: 10646).

Data availability

The data generated in this study is made available both within the article itself and in its Supplementary Data.

Competing interests

The authors declare no competing interests.

Publisher's note

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