IGFBP3-mediated M2 Macrophage Polarization Enhances Resistance to Rosiglitazone and Cisplatin in Breast Cancer

doi:10.21203/rs.3.rs-5174260/v1

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IGFBP3-mediated M2 Macrophage Polarization Enhances Resistance to Rosiglitazone and Cisplatin in Breast Cancer

https://doi.org/10.21203/rs.3.rs-5174260/v1

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Background

Rosiglitazone (PPARγ agonist, RGZ) combined with chemotherapy drugs has proven effective in treating clinical breast cancer patients. However, the underlying targets and resistance mechanisms remain unclear, posing challenges in maintaining long-term efficacy. This study aimed to investigate the mechanisms of RGZ in macrophage polarization and evaluate its effects within the TME.

Methods

In vivo experiments were conducted using a patient-derived xenograft (PDX) nude mouse model resistant to chemotherapy, which simulates the clinical immune microenvironment. In vitro, the Thp-1 human monocytic cell line was induced to differentiate into macrophages and cocultured with MDA-MB-468 breast cancer cells. The effects of PPARγ agonists on the drug resistance of breast cancer cells in a coculture model were explored.

Results

An increase in M2 macrophages was observed in combination-resistant mice, with PPARγ activation by RGZ inducing macrophage differentiation toward the M2 phenotype. Knockdown of IGFBP3 in macrophages alleviated breast cancer cell resistance in the microenvironment by reversing polarization.

Conclusion

Our study revealed that targeting macrophage polarization through IGFBP3 can reverse the effects of PPARγ on macrophage polarization, promote cancer cell apoptosis and collectively reverse the resistance of tumor cells to combination therapy. These findings provide a new theoretical basis for anti-breast cancer treatment.

macrophage polarization

PPARγ

IGFBP3

combination therapy resistance

breast cancer

Breast cancer is the most commonly diagnosed malignancy in women, and its burden has been increasing globally [1]. Surgery is the mainstay of breast cancer therapy; however, it also causes some side effects, including lymphoedema and psychosocial fallout [2]. Therefore, exploring new and effective approaches is crucial for breast cancer treatment. Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated nuclear receptor [3]. PPARγ agonists, such as rosiglitazone (RGZ) and pioglitazone, are used to treat type 2 diabetes (T2D) with insulin resistance (IR) [4]. These drugs have clinical importance because of their widespread and safe use in treating hypercholesterolemia (hyperlipidemia) and diabetes. The safety and cost-effectiveness of these drugs make them promising candidates for repurposing to inhibit tumor growth. In previous studies, RGZ displayed an antitumor effect in preclinical studies but a beneficial limit effect in breast cancer clinical trials [5, 6]. This could be due to the antagonism of cell-specific effects between tumor cells and immune cells [7].

Tumor-associated macrophages (TAMs) constitute the major component of all immune cells present in the tumor microenvironment (TME) [8], constituting up to 50% of the tumor mass in most human solid tumors, including breast cancer [9]. TAMs can change their phenotype in response to stimuli from the surrounding TME [10, 11]. In general, macrophages are immunoregulatory and suppress immune responses to tumor-derived antigens [12]. Even worse, they can induce resistance to multiple treatments in preclinical breast cancer models [13–15]. In some specific situations, macrophages can be antitumoral, offering support such as the suppression of angiogenesis or the stimulation of the T-cell response [16, 17]. However, the role of transferring macrophages remains unclear, and a better understanding of the polarization of macrophages in breast tumors will contribute to the further development of cancer immunotherapies.

In the present study, a chemoresistant PDX nude model with an anti-inflammatory and tumor-promoting phenotype of TAMs and M2 macrophages that advanced gradually was established. In addition, the effects of chemotherapy combined with RGZ on breast tumor cells were analyzed. RGZ also promotes the transformation of macrophages to the beneficial type of tumor. The potential mechanism of RGZ in the regulation of macrophage polarization in breast cancer was subsequently explored through the activation of IGFBP3, which is driven by PPARγ. These findings suggest that IGFBP3 is a potential target for TAM immunotherapy in breast cancer.

Mice

In this study, female BALB/c background nude mice (Shanghai Experimental Animal Center, Chinese Academy of Sciences, China) from 4--6 weeks were selected to establish a PDX model. The experimental mice were housed in individual ventilated cages under specific-pathogen-free conditions. All animal care and experimental protocols were carried out according to the Chinese Animal Management Rules of the Ministry of Health. All of the procedures were authorized by the Animal Ethics Committees of Nantong University.

PDX mice were inoculated subcutaneously with patient-derived tumor tissue fragments and were used for the experiment when they passed to the F3 generation. All the mice used were age matched and were assigned randomly to groups. After the tumor volume reached 100–200 mm³, PDX mice were injected intraperitoneally (ip) with chemotherapeutic agents as indicated by the administration of cisplatin (5 mg/kg/3d), paclitaxel (5 mg/kg/3d), docetaxel (2 mg/kg/3d) or saline.

Cell culture

THP-1 and MDA-MB-468 cells were purchased from the Cell Bank of the Institute of Biochemistry and Cell Biology (Shanghai, China). The THP-1 cells were incubated in RPMI 1640 medium (Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Gibco, Waltham, USA). The human breast cancer cell line MDA-MB-468 was maintained in Dulbecco’s modified Eagle’s medium (Gibco, Thermo Fisher Scientific) supplemented with 10% FBS. Both cell lines were maintained at 37 °C in an atmosphere containing 5% CO₂. Both cell lines were used within 20 passages and confirmed to be mycoplasma free before use in studies.

Real-time quantitative PCR

TRIzol reagent (Invitrogen, Carlsbad, USA) was used to extract total RNA from the cell lines or tissue samples. Real-time quantitative PCR (qPCR) was performed in a total volume of 20 μL of SYBR Green PCR Master Mix (Roche, Germany). All reactions were performed in duplicate. The mRNA expression levels of the target genes were normalized to the GAPDH mRNA levels via the 2^-ΔΔCT method. The primer sequences are listed in Table 1:

Gene	Primer sequences
GAPDH-F	5’-GCACCGTCAAGGCTGAGAAC-3’
GAPDH-R	5’-TGGTGAAGACGCCAGTGGA-3’
IL1-β-F	5’- TCCAGGATGAGGACATGAGCAC -3’
IL1-β-R	5’- GAACGTCACACACCAGCAGGTTA -3’
TNF-α-F	5’- GTTCTATGGCCCAGACCCTCAC -3’
TNF-α-R	5’- GGCACCACTAGTTGGTTGTCTTTG -3’
iNOS -F	5’- CAAGCACCTTGGAAGAGGAG -3’
iNOS -R	5’- AAGGCCAAACACAGCATACC -3’
CD16-F	5’-TTTGGACACCCAGATGTTTCAG-3’
CD16-R	5’-GTCTTCCTTGAGCACCTGGATC-3’
CD206-F	5’-CAAGGAAGGTTGGCATTTGT-3’
CD206-R	5’-CCTTTCAGTCCTTTGCAAGC-3’
TGF-β-F	5’-TGCGCTTGCAGAGATTAAAA-3’
TGF-β-R	5’-CGTCAAAAGACAGCCACTCA-3’
IL-10-F	5’-CCAAGCCTTATCGGAAATGA-3’
IL-10-R	5’-TTTTCACAGGGGAGAAATCG-3’

Western blotting

The cells or tissues were harvested in lysis buffer containing 1% phosphatase inhibitor. Total proteins separated by SDS‒PAGE were transferred to nitrocellulose membranes (Millipore, USA), which were blocked in 5% w/v nonfat milk powder in TBST (0.1 M Tris, 1.2 M NaCl, pH 7.4 and 0.1% v/v Tween 20) for 2 h at room temperature and then incubated with primary antibody overnight at 4°C. The membrane was washed with TBST adequately and incubated with the secondary antibody (Jackson, West Grove, USA). Proteins were visualized with enhanced chemiluminescence (ECL) detection reagents. The primary and secondary antibodies used were as follows: B7-2/CD86 [Unconjugated] (NOVUS, USA), CD11b (NOVUS, USA), PPARγ (Abcam, USA), IGFBP3 (Abcam, USA), monoclonal anti-β-actin (Sigma, USA), goat anti-rabbit secondary (Bioworld, USA), rabbit anti-goat secondary (Bioworld, USA), and goat anti-mouse secondary (Jackson, USA) antibodies.

Macrophage polarization

THP-1 monocytes were differentiated into macrophages by 24 h of incubation with 150 nM phorbol 12-myristate 13-acetate (PMA) (Sigma, USA) in RPMI medium. Next, the macrophages were stimulated with 20 ng/ml IFNγ (PeproTech, USA) and 100 ng/ml LPS (Sigma, USA) for 48 h to obtain the M1 phenotype. The M2 phenotype was obtained by incubation with 20 ng/ml interleukin 4 (IL-4) (Sigma, USA) and 20 ng/ml interleukin 13 (IL-13) (PeproTech, USA) for 48 h. Afterwards, the cells were collected and analyzed.

Apoptosis analysis by flow cytometry

The cells in the exponential growth phase were trypsinized and resuspended in fresh culture medium at a density of 1×10⁵ cells/well in a 6-well plate for 24 h at 37°C. After treatment, the cells were collected with 5% trypsin, washed twice with phosphate-buffered saline (PBS), and resuspended in binding buffer at a concentration of 5×10⁵ cells/ml. The cells (100 μl) were stained with 5 μl of fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody specific for Annexin V and 5 μl of propidium iodide (PI) (BD, USA) and then incubated in the dark at 25°C for 15 min. Subsequently, cell apoptosis was measured via flow cytometry (Beckman Coulter, USA). The data were analyzed with FlowJo 10.

Cytotoxicity assays

Cellular cytotoxicity was assessed by the level of lactate dehydrogenase (LDH) activity. THP-1 cells were seeded on inverted transwell plates, differentiated into macrophages for 24 h, washed and treated with IFNγ combined with LPS or IL-4 combined with IL-13 for 48 h. MDA-MB-468 cells were seeded on 24-well plates and incubated with polarized macrophages. After 48 h of exposure, the cocultured cells were washed for detection of LDH. LDH release was measured via a Cytotoxicity Detection Kit (Roche Diagnostics, Germany) according to the manufacturer's instructions.

Cell Counting Kit-8 (CCK-8) assay

Cell viability was measured via the Cell Counting Kit-8 (CCK-8) (D Dojindo Molecular Technologies, Japan) assay following the manufacturer’s protocol. The cells were seeded into 96-well plates at a density of 3000 cells/well. After treatment with different doses of cisplatin for 24 h, 48 h or 72 h, 10 µL of CCK-8 solution was added to the cells. One hour later, the absorbance was calculated with a microplate spectrophotometer (Multiskan™ GO, Thermo Fisher Scientific) at 450 nm.

Infection with lentiviral shRNA

For the lentiviral infection experiments, recombinant lentivirus (GenePharma, China) was used according to the manufacturer's protocol. THP-1 cells were seeded at 1 × 10⁵cells/well in a 12-well plate and incubated overnight. The next day, control shRNA lentivirus and IGFBP3 knockdown shRNA lentivirus were combined with 10 μg/ml polybrene (Sigma, USA) and then added to the THP-1 cells. At 48 h after transduction, THP-1 cells generated with the IGFBP3 shRNA lentivirus were cultured with puromycin-containing (4 to 8 μg/ml) medium for 2 weeks to select stable clones.

Immunohistochemistry

The tumor tissue was fixed in formalin, embedded in paraffin, sectioned and heated. Subsequently, the sections were deparaffinized, rehydrated, and subjected to antigen retrieval. After being blocked with 2% goat serum, the sections were incubated with the indicated antibodies against CD11b (NOVUS, USA) and CD86 (NOVUS, USA) overnight at 4°C. The next day, the sections were washed with PBS and then incubated with the corresponding secondary antibodies for 1 hour at room temperature. The sections were treated with DAB working solution and then visualized by microscopy.

Immunofluorescence

ICC staining was performed as described previously(11). The cells were processed routinely. For ICC staining, the following antibodies were used: B7-2/CD86 [Unconjugated] (NOVUS, USA), CD11b (NOVUS, USA), PPARγ (Abcam, USA), IGFBP3 (Abcam, USA), β-Tubulin (Cell Signaling Technology, USA), Caspase 3 (Invitrogen, UK) and goat anti-rabbit secondary antibodies (West Grove, USA). Nuclei were counterstained with DAPI (Beyotime, China). Immunostaining was visualized with a confocal laser scanning microscope (SP8, Leica, Germany).

Dual-luciferase reporter assays

TheIGFBP3 promoter fragments were amplified and cloned and inserted into the pGL3 basic vector. Luciferase activity was examined via a dual luciferase assay (Promega, Madison, WI, USA) following the manufacturer's instructions.

Statistical analysis

Data analysis was conducted via GraphPad Prism 9 (GraphPad Software, San Diego, CA). The results are presented as the means ± SDs. One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test was used to determine significant differences between groups. For data involving only two groups, an unpaired Student’s t test was employed. P values < 0.05 were considered statistically significant (*p < 0.05, **p < 0.01).

The synergistic effects of cisplatin and RGZ yield significant antitumor effects

To investigate the impact of immune cells on the breast cancer microenvironment, we utilized patient-derived xenograft (PDX) models for in vivo experiments. The subcutaneous tumor model was derived from a patient with triple-negative breast cancer (TNBC). PDX mice were treated with RGZ (5 mg/kg) or cisplatin (5 mg/kg) either separately or in combination. As shown in Fig. 1A and B, there was no significant difference in tumor volume between the RGZ-treated group and the control group. However, the cisplatin-treated group presented a marked reduction in tumor burden. Notably, the combination treatment of RGZ and cisplatin resulted in more pronounced inhibition of the tumor burden than did cisplatin alone (Fig. 1C). The safety of RGZ and cisplatin was assessed by monitoring the weights of the mice (Fig. 1D). Our findings demonstrate that the synergistic effect of cisplatin and RGZ leads to enhanced antitumor efficacy.

RGZ enhances the proapoptotic effects of chemotherapy

To further validate the antitumor effects of RGZ in vitro, we treated MDA-MB-468 breast cancer cells with RGZ in the presence or absence of cisplatin. CCK-8 assays revealed a time- and dose-dependent increase in the cytotoxic effects of cisplatin (Supplemental Fig. 1A-C). Cotreatment with 25 µM RGZ and various doses of cisplatin for 48 hours significantly reduced cell viability (Fig. 2A). Serum-free growth experiments indicated that 25 µM RGZ alone significantly inhibited cell proliferation (Fig. 2B). Additionally, cotreatment with cisplatin and RGZ for 48 hours markedly increased the expression of the apoptosis marker caspase-3 in MDA-MB-468 cells (Fig. 2C and D). Flow cytometry analysis revealed a significant increase in the number of apoptotic cells in the cisplatin/RGZ cotreatment group compared with the cisplatin-only group (Fig. 2E-G). Lactate dehydrogenase (LDH) release, a marker of damage to the integrity of the cell membrane, was significantly greater in the RGZ-treated group than in the adriamycin (ADR) single-treatment group (Fig. 2H). These results indicate that RGZ enhances the sensitivity of breast cancer cells to cisplatin, leading to increased cytotoxicity.

Increased TAMs (M2) in cisplatin- and RGZ-resistant PDX model mice

Given that cisplatin and RGZ have the most promising antitumor effects, we implanted tumor tissues from the cisplatin + RGZ group into nude mice and treated them with RGZ (5 mg/kg) and cisplatin (5 mg/kg) to establish resistant patient-derived xenograft (R-PDX) models. For the first two weeks, the R-PDX tumors presented a steady volume under RGZ treatment, indicating initial sensitivity to the drug. However, by the third week, a rapid increase in tumor volume was observed, suggesting the development of drug tolerance (Fig. 3A). Previous studies have identified TAMs as critical drivers of chemoresistance. In our study, we examined both the infiltration and polarization of TAMs within the R-PDX tumor environment. We noted a decrease in the M1 macrophage marker IL-1β during the drug tolerance stage. Conversely, M2 macrophage markers, including TGF-β and CD206, were significantly elevated (Fig. 3B-D). The frequency of CD206-positive M2 macrophages was greater in resistant tumors than in resistant tumors, whereas the percentage of CD86-positive M1 macrophages was lower (Fig. 3E-G). These findings suggest that macrophage polarization plays a role in chemotherapy resistance.

RGZ promotes M2 macrophage polarization in vitro

To elucidate the mechanism underlying macrophage polarization, we established fully polarized macrophages derived from human monocytic THP-1 cells. CD11b is a primary marker for macrophages (M0), while CD86 and CD16 are markers for M1 macrophages, and CD206 is a marker for M2 macrophages. After a 24-hour incubation with 150 nM PMA, monocytes adhered to the dish bottom and displayed M0 characteristics. Stimulating M0 cells with 10 pg/mL LPS and 20 µg/mL IFN-γ for 24 hours resulted in detectable CD16 expression via immunocytochemistry. Similarly, incubating M0 cells with 20 µg/mL IL-4 and 20 µg/mL IL-13 for 24 hours increased CD206 levels (Fig. 4A). RGZ treatment significantly increased the protein expression of CD206 and PPARγ in macrophages but reversed the increase in the expression of CD86 (Fig. 4B and C). The mRNA levels of the M1-related genes INOS and IL-1β were significantly decreased in the RGZ-treated M1 group. In contrast, the expression of the M2-related genes CD206 and TGF-β was upregulated in both the M0 + RGZ and M1 + RGZ groups (Fig. 4D). Compared with that in primary macrophages, CD206 expression was greater in the M0 + RGZ and M1 + RGZ groups (Fig. 4E and G). Nile red staining revealed increased lipid content in macrophages exposed to RGZ (Fig. 4F and H). These results indicate that RGZ promotes macrophage polarization toward the M2 phenotype and reverses the characteristics of M1 macrophages.

M1 macrophages promote the apoptosis of breast cancer cells

Our comprehensive analysis demonstrated that the activation of PPARγ by RGZ inhibits tumor cell proliferation, increases sensitivity to chemotherapy drugs, and promotes apoptosis in breast cancer cells. Additionally, RGZ treatment encouraged macrophages to polarize toward the M2 type, shifting their function from promoting inflammation to inhibiting inflammation, thus promoting the development of tumor cells. To better understand this interaction, we established a coculture system of macrophages and breast cancer cells to simulate the human TME (Fig. 5A). After inducing macrophage polarization and coculturing them with breast cancer cells for 48 hours, RGZ was added to the coculture model for an additional 24 hours. LDH release assays indicated that in the absence of RGZ, M1-type macrophages induced increased LDH release from MDA-MB-468 cells because of the release of inflammatory factors. Conversely, M2-type macrophages, which release anti-inflammatory factors, downregulate LDH release from breast cancer cells. Upon RGZ treatment, LDH release increased in all groups, suggesting that the cytotoxic effect of RGZ on breast cancer cells outweighs the anti-inflammatory and drug resistance effects induced by M2-type polarization (Fig. 5B). We further analyzed the combined effects of cisplatin and RGZ on tumor cell apoptosis via flow cytometry in a coculture system. The results revealed a significant increase in apoptosis in MDA-MB-468 cells treated with RGZ compared with those not in coculture (Fig. 5C and E). Compared with M2-type macrophages, M1-type macrophages significantly increased early tumor cell apoptosis in the untreated group without RGZ (Fig. 5D). There was no significant change in tumor cell apoptosis in the RGZ-treated group compared with the untreated group.

IGFBP3 regulates M2 macrophage polarization induced by RGZ

Previous studies have indicated that macrophages can be regulated by specific genetic targets. To identify the genes involved in RGZ-induced M2 macrophage polarization, we analyzed publicly available ChIP-seq data from RGZ-treated PMA-differentiated THP-1 cells and conducted additional ChIP-seq experiments on classic M1- and M2-polarized macrophages. We integrated data showing genes upregulated in the RGZ-treated group and those active in M2 but not M1 macrophages. Using the Venn tool (https://bioinfogp.cnb.csic.es/tools/venny/index.html), we identified six candidate genes: IFI27, IGFBP3, ISG15, MX1, MX2, and XAF1 (Fig. 6A). Among these genes, IGFBP3 was the only gene with a significant positive correlation (r > 0.3) with the M2 macrophage marker CD163 and no positive correlation with the M1 marker CD86, as shown in the TCGA breast cancer database (Supplemental Figure S2, http://timer.cistrome.org/). A volcano plot further revealed high IGFBP3 expression in classic M2 macrophages (Fig. 6B). In the GSE25608 dataset, THP-1 cells cultured with RGZ also presented elevated IGFBP3 expression (Fig. 6C). Verification of IGFBP3 protein levels in THP-1 cells revealed that it was overexpressed in IL4-induced macrophages and primary macrophages (Fig. 6D). To determine the role of IGFBP3 in macrophage polarization and tumor cell promotion, we constructed IGFBP3-knockdown THP-1 cell lines (Fig. 6E). Cotreatment with IGFBP3 knockdown and cisplatin significantly increased late apoptotic tumor cell proportions, whereas early apoptosis remained unaffected (Supplemental Figure S3). In subsequent apoptosis assays, we evaluated the proportion of apoptotic tumor cells within a coculture system of IGFBP3-knockdown macrophages and breast cancer cells. Compared with the control group, the IGFBP3 knockdown group (shIGFBP3 + CIS) presented an increased proportion of early apoptotic breast cancer cells. Further treatment with RGZ (shIGFBP3 + ROSI + CIS) further increased early apoptosis (Fig. 6F and G). Notably, compared with the RGZ-only group, the shIGFBP3 + ROSI + CIS group presented a significantly greater proportion of apoptotic cells (Fig. 6H). These findings demonstrate that IGFBP3 knockdown combined with RGZ treatment significantly promotes apoptosis, thereby increasing tumor cell sensitivity to chemotherapeutic agents.

IGFBP3 expression and clinical outcomes

We further investigated the protein expression of IGFBP3 following RGZ treatment and found that low-dose RGZ (5 µM) significantly increased IGFBP3 protein levels (Fig. 7A‒C). Furthermore, macrophages treated with a relatively high dose of RGZ (10 µM) presented a 2-fold increase in IGFBP3 reporter gene activity (Fig. 7D). These findings indicate that PPARγ activation by RGZ enhances IGFBP3 expression.

To validate the clinical relevance of IGFBP3 expression in breast cancer, we analyzed IGFBP3 levels in a cohort of 64 breast cancer patients. Immunohistochemical analysis revealed that high IGFBP3 expression was present in 46% of the tumor samples. High IGFBP3 expression was significantly correlated with poorer overall survival (OS) and number at risk (p = 0.033, Fig. 7E and F).

Clinical data indicate that combining tumor activity regulators with low-dose chemotherapy agents can produce impressive tumor responses, leading to sustained complete remission. The combination of RGZ and chemotherapeutic agents has demonstrated therapeutic potential. However, the utilization of PPARγ agonists in tumor therapy has experienced several failures. The target of RGZ, PPARγ, is multifaceted [18]. It is a major regulator of adipocyte differentiation. It enhances macrophage lipid uptake and lipid efflux and possesses anti-inflammatory properties. PPARγ is expressed in various cancers, including colon, prostate, and breast cancers [19]. PPARγ ligands generally exhibit antiproliferative effects in these contexts, but some studies have suggested that they may exacerbate the growth of certain tumors. Specifically, in breast cancer, RGZ promotes G0‒G1 cell cycle arrest, increases caspase-9 activity, and induces growth arrest and apoptosis in breast cancer cells [20–22]. This finding is consistent with our observations that RGZ induces apoptosis in the breast cancer cell line MDA-MB-468 in vitro. Recent studies have reported that RGZ exhibits antitumor activity in the early stages of tumor progression in vivo but fails in later stages of treatment [23]. Here, we demonstrate that the combination of RGZ and cisplatin has significant therapeutic effects in the early stages of breast cancer, whereas resistance develops in later stages in mice. This resistance may be related to changes in macrophage polarization within the TME.

Macrophages exhibit plasticity [24]. They can be polarized into the classically activated M1 type through proinflammatory stimuli (e.g., IFN-γ and LPS) or the alternatively activated M2 type through anti-inflammatory stimuli (e.g., IL-4 and IL-13), depending on the growth factors in the immune environment. The M1-like phenotype is immunostimulatory and can inhibit tumor development and progression, whereas the M2-like phenotype promotes malignant tumor proliferation and metastasis, facilitates tumor immune evasion, and induces drug resistance. Most tumor-associated macrophages (TAMs) share similar markers, including CD206, CD163, CCL8, and Arg1 [25]. The M2 subtype is widely recognized as the predominant phenotype of protumor TAMs (pTAMs). The activation of various cellular signaling pathways, such as the NF-κB, P38 MAPK, Notch, and JAK/STAT signaling pathways, can direct macrophage polarization [26–29]. PPARγ is also considered to play crucial roles in promoting M2 macrophage polarization. We investigated the impact of RGZ on the expression of macrophage polarization-related markers. RGZ inhibited the expression of the M1 phenotype marker CD86. RGZ treatment increased the levels of the M2 markers CD206 and TGFβ. These findings further indicate that RGZ can reverse the M0 and M1 phenotypes of macrophages to the protumor M2 phenotype. Consistent with these results, RGZ induced the release of inflammatory factors (e.g., INOS and IL-1β), enhanced the phagocytic capacity of macrophages (an M2 marker), and increased the CD206/CD11b ratio. Above all, we demonstrated the paradoxical effects of RGZ in breast cancer. An in vitro coculture system of breast cancer cells and macrophages was established to simulate the human microenvironment. In isolated breast cancer cell cultures, treatment with RGZ increased LDH release and upregulated the expression of the apoptotic factor Caspase3. However, in coculture with macrophages, the apoptotic effect on breast cancer cells was diminished. This attenuation is presumed to be due to the polarization shift from M1 to M2 macrophages, which promotes tumor cell proliferation and resistance, counteracting RGZ-induced cell damage.

To mitigate the reduction in RGZ efficacy due to macrophage polarization to the M2 phenotype observed during clinical administration, we conducted an extensive database analysis. This analysis revealed that insulin-like growth factor binding protein-3 (IGFBP3) was differentially expressed during both the macrophage polarization state and RGZ treatment. These findings suggest that IGFBP3 could be a potential target to counteract the regulation of macrophage polarization by PPARγ. IGFBP3 is a member of the IGFBP family that is capable of binding to IGF-I and IGF-II with high affinity, thus inhibiting cell actions mediated by IGF-I receptor activation [30, 31]. The overexpression of IGFBP3 has been observed in gliomas [32], nasopharyngeal carcinoma [33], esophageal squamous cell carcinoma [34], pancreatic ductal adenocarcinoma [35] and breast cancer [36, 37]. IGFBP3 expression is associated with poor patient prognosis [32, 36]. Previous reports have shown that high IGFBP3 expression in liver cancer patients is associated with an immunosuppressive phenotype [38]. T-cell infiltration is reduced in breast tumor environments deficient in IGFBP3 [39]. Our findings are consistent with this observation. Low doses of RGZ increase IGFBP3 expression and promote macrophage polarization toward the M2 phenotype. IGFBP3 knockdown reversed macrophage polarization to the M1 phenotype and indirectly enhanced the apoptosis of MDA-MB-468 cells in a coculture system. Dual-luciferase assays revealed that low doses of DAC significantly increased the binding affinity of IGFBP3 for the PPARγ promoter, supporting the role of IGFBP3 in macrophage polarization.

To date, this is the first study to reveal the indispensable role of IGFBP3 in M2 macrophage polarization. This research reveals the potential mechanism of resistance development with long-term administration of the chemical drug cisplatin in combination with RGZ. Therefore, incorporating IGFBP3-targeted therapies could effectively reverse PPARγ-induced macrophage polarization and thereby alleviate the occurrence of resistance. Despite these significant findings, our study has several limitations. First, in the PDX mouse resistance model, we observed changes in macrophage infiltration with a high enrichment of M2 macrophages, suggesting that macrophage polarization mediates the development of resistance. However, other cells in the TME, such as T cells and endothelial cells, may also contribute. Additionally, the precise role of tumor IGFBP3 in different cancers remains contradictory [39], which limits the application of IGFBP3 inhibitors in combination therapies. The potential mechanisms involving PPARγ and IGFBP3 also require further investigation. Nonetheless, we believe that IGFBP3-targeted therapies will offer new insights for patients with breast cancer who frequently experience disease recurrence or refractory conditions in combination treatments.

Our study provides compelling evidence that IGFBP3-mediated M2 macrophage polarization plays a critical role in modulating the TME and influencing the sensitivity of breast cancer cells to chemotherapy. By promoting an immunosuppressive environment, IGFBP3 enhances tumor cell survival and resistance to treatment (Fig. 8). Targeting IGFBP3 in combination with PPARγ agonists such as RGZ could offer a novel therapeutic strategy to improve outcomes for patients with breast cancer, particularly those with high IGFBP3 expression. Further clinical validation and mechanistic studies are warranted to fully realize the potential of this approach.

Ethics approval and consent to participate: All of the animal procedures were authorized by the Animal Ethics Committees of Nantong University. BC tissues were from patients in Affiliated Hospital of Nantong University. Ethical clearance for the research was acquired from the institution's ethics committee.

Consent for publication: Not applicable.

Availability of data and materials: The data that involved in this study are available in the supplementary material.

Competing interests: The authors declare that they have no competing interests.

Funding: This work was supported by the following funding:

1. Open research Project of the Key Laboratory of Prevention and Treatment of Cardiovascular and Cerebrovascular Diseases (Ministry of Education), Gannan Medical University (XN202005);

2. The Scientific Research Foundation of State Key Laboratory of Vaccines for Infectious Diseases，Xiang An Biomedicine Laboratory (2023XAKJ0101005);

3. Basic Research Program of Jiangsu Province (BE2018778).

Authors' contributions: Concept and design: GW, TH, CG; Performing experiments and analysis of data: CG, GL; Contributing reagents, materials and other analytical tools: GW, TH; Writing the manuscript: CG, GW. All authors read and approved the final manuscript.

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No competing interests reported.

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IGFBP3-mediated M2 Macrophage Polarization Enhances Resistance to Rosiglitazone and Cisplatin in Breast Cancer

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Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Methods

Results

The synergistic effects of cisplatin and RGZ yield significant antitumor effects

RGZ enhances the proapoptotic effects of chemotherapy

Increased TAMs (M2) in cisplatin- and RGZ-resistant PDX model mice

RGZ promotes M2 macrophage polarization in vitro

M1 macrophages promote the apoptosis of breast cancer cells

IGFBP3 regulates M2 macrophage polarization induced by RGZ

IGFBP3 expression and clinical outcomes

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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