- Research Letter
- Open Access
Macrophage polarization by MSC-derived CXCL12 determines tumor growth
Cellular & Molecular Biology Letters volume 26, Article number: 30 (2021)
Phenotypic and functional heterogeneity of macrophages is known to be the main reason for their ability to regulate inflammation and promote tumorigenesis. Mesenchymal stem cells (MSCs) are one of the principal cells commonly found in the tumor stromal niche, with capability of macrophage phenotypic switching. The objective of this study was to evaluate the role of C-X-C motif chemokine ligand 12 (CXCL12) produced by marrow-derived MSCs in the phenotypic and functional pattern of bone marrow-derived macrophages (BMDMs).
First, the CRISPR/Cas9 system was used for the CXCL12 gene knock-out in MSCs. Then, coculture systems were used to investigate the role of MSCsCXCL12−/− and MSCsCXCL12+/+ in determination of macrophage phenotype. To further analyze the role of the MSC-derived CXCL12 niche, cocultures of 4T1 mammary tumor cells and macrophages primed with MSCsCXCL12−/− or MSCsCXCL12+/+ as well as in-vivo limiting dilution assays were performed.
Our results revealed that the expression of IL-4, IL-10, TGF-β and CD206 as M2 markers was significantly increased in macrophages co-cultured with MSCsCXCL12+/+ , whereas the expression of IL-6, TNF-α and iNOS was conversely decreased. The number and size of multicellular tumor spheroids were remarkably higher when 4T1 cells were cocultured with MSCCXCL12+/+-induced M2 macrophages. We also found that the occurrence of tumors was significantly higher in coinjection of 4T1 cells with MSCCXCL12+/+-primed macrophages. Tumor initiating cells were significantly decreased after coinjection of 4T1 cells with macrophages pretreated with MSCsCXCL12−/−.
In conclusion, our findings shed new light on the role of MSC-derived CXCL12 in macrophage phenotypic switching to M2, affecting their function in tumorigenesis.
Tissue macrophages are members of the mononuclear phagocyte system, signifying their ontogeny and phagocytic activity. Great functional and phenotypic diversity was reported within these cells. Stage-specific functional dynamism of macrophages ranging from injury initiation to inflammation and finally instructing repair is crucial for tissue homeostasis [1,2,3]. It is now clear that this developmental dynamism results from a complex interplay between intrinsic differentiation pathways and environmental signals received from neighboring cells . It has been demonstrated that the functional dynamism of macrophages depends on their phenotypic polarization . Macrophages are able to oscillate between pro-inflammatory M1 and debris-scavenging/tissue-remodeling alternatively activated M2 states . This broad range of functional dynamism results in macrophages being involved in several chronic pathologic conditions within the tumor environment 
It has been reported that macrophages represent a prominent component (up to 50% of cells) of infiltrated leukocytes in malignancies . The presence of M2 macrophages has been correlated with worse clinical consequences in a series of neoplastic conditions [8, 9].
Therefore, recognition of macrophage polarization and biological factors involved in this process has now become the area of many investigations. Hypoxia and the various signals derived from stromal niche cells profoundly regulate macrophage polarization . Generally interferon gamma, lipopolysaccharide, tumor necrosis factor (TNF) and granulocyte–macrophage colony-stimulating factor are known as M1 inducers while colony stimulating factor-1, IL-4, IL-13 and IL-10 and hypoxia are involved in M2 polarization .
Mesenchymal stromal cells (MSCs) are immature, adherent stromal cells residing in various tissues and commonly found at injury sites and in tumors . Emerging data suggest that MSCs can promote tumorigenic processes, including neoplastic tissue formation, maintenance, and chemoresistance, as well as tumor growth [11, 12]. Some recent evidence demonstrated that significant functional interactions can occur between MSCs and macrophages, such that MSCs regulate the function of macrophages and induce macrophage differentiation/polarization via secreting various types of chemokines [13,14,15].
Among MSC-released chemoattractants, C–X–C motif chemokine ligand 12 (CXCL12), also known as stromal cell derived factor 1 (SDF-1), is known to be highly inducible in some pathologic states involving ischemia or hypoxia and in proangiogenic environments, such as tumors and wounds [16,17,18]. CXCL12 is a small molecule with immunomodulatory properties as it is a highly effective lympho-monocyte chemoattractant [6, 17, 19,20,21]. A series of G protein-coupled chemokine receptors, which are characterized by seven transmembrane domains, can be activated by the N-terminus part of CXCL12. CXCR4, a 352-amino acid molecule, is the main receptor for CXCL12, which transduces multiple signals after induction, resulting in a number of context-dependent biological functions such as proliferation, cell chemotaxis, migration, apoptosis, survival and differentiation [17, 22,23,24].
Sánchez-Martín et al. reported that monocyte-derived CXCL12 led monocytes to differentiate into a distinct subtype of macrophages, characterized by enhanced expression of CD14 and CD163 and the secretion of proangiogenic factors, such as vascular endothelial growth factor and CCL1 . Moreover, CXCL12 in the tumor microenvironment was reported to induce macrophage mobilization and vasculogenesis .
Considering the importance of macrophage polarization with subsequent functional changes in different physiopathological conditions, and with respect to the role of MSC as an effector cell in macrophage polarization dynamism, this study was designed to evaluate the role of MSC-derived CXCL12 in macrophage polarization and phagocytosis. In this regard, we applied the CRISPR/Cas9 system to knock out the CXCL12 gene in bone marrow (BM)-derived MSCs in order to discover the biological effect of the MSC-derived CXCL12 niche on BM macrophages.
Eight-week-old female BALB/c mice were used in this study. The mice were treated according to the Guide for the Care and Use of Laboratory Animals (NIH Publication, 8th edition, revised 2011). All steps of this study received consent from the Animal Care Committee of the University of Tehran.
Cells and culture conditions
Isolation and culture of BM macrophages and MSCs were performed using standardized protocols with some modifications [25,26,27,28]. Briefly, we flushed the femurs of mice with PBS and harvested the bone marrow mononuclear cells (BM-MNCs) by centrifugation on a Ficoll (Cat No: 10771; Sigma Aldrich, USA) gradient. Afterwards, the total cell count was carried out using a cell counter machine (Model: MEK-6450k, Nihon Kohden; Japan). For expansion of bone marrow derived macrophages (BMDMs), MNCs were cultured in RPMI-1640 containing 10% FBS, 3% penicillin–streptomycin and 7.5 mL of L929 cell-conditioned medium at 37 °C, 5% CO2, and 95% relative humidity for one week. Cells were washed and passaged after 70% confluency .
For the purpose of MSC expansion, BM-MNCs were plated into 24-well dishes and cultured in Dulbecco's modified Eagle's medium (DMEM) (Cat. No: 11-995-040; Gibco) high glucose, supplemented with 10% FBS and 3% penicillin–streptomycin at 37 °C, 5% CO2, and 95% relative humidity. Cells of the third passage were used for our experiments.
For experimental tumor induction, the 4T1 breast cancer cell line (which is a triple-negative breast cancer tumor-initiating cell (TNBC-TIC) with similar aggressive phenotype to the human disease) were purchased from ATCC. Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) at 37 °C humidified atmosphere with 5% CO2. Cells were then washed and passaged at almost 80% confluency .
Flow cytometry and characterization
Flow cytometric analysis was performed to characterize cultured MSCs and macrophages isolated from bone marrow. For this work, cells were incubated with 5% BSA solution at 4 °C for 20 min. MSCs were then incubated with a panel of antibodies, including PE-conjugated anti-mouse CD34 (Cat. No: ab187284; Abcam, Inc.), PE-conjugated anti-mouse CD45 (Cat. No.: 12-0451-81; eBioscience, Inc.), PE-conjugated anti-mouse CD105 (Cat. No.: 120407; BioLegend, Inc.), PE-conjugated anti-mouse CD90 (Cat. No.: 12-0900-81; eBioscience, Inc.), and macrophages were incubated with PE-conjugated anti-mouse F4/80 (Cat. No.: ab105156; Abcam) at 4 °C for 30 min . Ultimately, the cells were washed twice with PBS and analyzed by BD FACSAria II (BD Biosciences). For CD206 staining, Alexa Fluor 488-conjugated anti-mouse CD206 (Cat. No.: 141709; BioLegend Inc.) was used. In flow cytometry analyses, cell viability was first performed by staining cells with 7-AAD or DAPI. Data were analyzed using FlowJo v7.6.5 software.
CXCL12 gene knock-out in MSCs
The CRISPR/Cas9 system was utilized for knocking out the CXCL12 gene in BM-derived MSCs. Accordingly, we seeded 2 × 105 cells in a 6-well culture plate and incubated in DMEM containing 10% FBS. After 60–80% confluency, we transfected the cells with CXCL12 CRISPR/Cas9 KO Plasmid (Cat No: sc-422854, Santa Cruz, Inc. USA) using a commercial transfection reagent (Cat No: sc-395739, Santa Cruz, Inc. USA) in accordance with the manufacturer's instructions. Transfected cells expressing plasmids were recognized by emergence of green fluorescent protein (GFP) signals resulting from a GFP reporter gene in the plasmid. Western blot analyses were performed to confirm the CXCL12 gene knock-out in MSCs. A mock plasmid was used in our transfection experiments as a negative control.
Co-cultures of BMDMs and MSCs
For transwell co-cultures, BMDMs were seeded into a 24-well plate. The next day, 0.4-mm-pore size Corning Transwell inserts (Cat. No: 35324; SPLInsert) containing 2 × 105 MSCsCXCL12−/− or 2 × 105 MSCsCXCL12+/+ were placed into the macrophage containing plates. After 48 h incubation, macrophages were harvested for a series of experiments to investigate the phenotype switching by ELISA tests, western blotting and flow cytometry.
Enzyme-linked immunosorbent assay (ELISA)
The conditioned medium from each culture was centrifuged at 6000g for 5 min at 4 °C to exclude cell and cell debris contamination. The level of IL-10/IL4/IL6/ transforming growth factor beta (TGF-β) / tumor necrosis factor alpha (TNF-α) in the conditioned medium was quantified using Quantikine ELISA kits for mouse IL-10 (Cat No: DY417; R&D), IL-4 (Cat No: M4000B; R&D), IL-6 (Cat No: M6000B; R&D), TGF-β (Cat No: DY1679; R&D), and TNF-α (Cat No: MTA00B; R&D) according to the manufacturer’s instructions.
Cells to be subjected to western blot analysis were lysed in a lysis buffer (Cat No: R0278, Sigma Aldrich, USA). Proteins were quantified using the Bradford assay. We relocated lysates (20 μg) to SDS-PAGE after 5 min boiling and after that transferred them to 0.2 μm Immun-Blot polyvinylidene difluoride membranes (Cat No: 162-017777; Bio-Rad Laboratories, CA, USA). Next, the membranes were blocked with 3% non-fat dry milk (Cat No: 1.15363.0500; Merck KGaA, Darmstadt, Germany) in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h. Then, we incubated the membranes with primary antibodies, including anti-CXCL12 (Cat No: sc-28876, Santa Cruz, Inc. USA), anti-inducible nitric oxide synthase (iNOS) (Cat No: sc-28876, Santa Cruz, Inc. USA) and anti-β actin-loading control (Cat No: ab8224; Abcam) at room temperature for 1 h, washed them three times with TBST, and afterward incubated them with HRP-conjugated secondary antibody, which is a rabbit anti-mouse IgG-HRP (1:4000; Cat No: ab6728; Abcam), for 1 h at room temperature. The membranes were incubated with enhanced chemiluminescence (ECL) for 1–2 min. Protein expression was normalized to β-actin. Densitometry of protein bands was performed using the gel analyzer Version 2010a software (NIH, USA), so that the percentage area under the curve of each band was divided by the percentage area under the curve of its corresponding actin band, and then calculated values were compared between groups.
Fluorescein isothiocyanate- (FITC-) conjugated dextran (Cat. No: 68059; Sigma Aldrich, USA) was used to determine the phagocytic function of macrophages. Bone marrow macrophages co-cultured with MSCsCXCL12+/+ or MSCsCXCL12−/− were adjusted to a concentration of 3 × 105 cells in 100 μL of complete RPMI 1640 medium and pre-incubated on ice for 30 min. Then, the above cells were incubated with 20 mg/L dextran-FITC for 30 min at 37 °C or at 4 °C to detect nonspecific binding. Cells were washed three times with 500 μL of complete RPMI 1640 medium and fixed in 10% (vol/vol) formaldehyde-PBS. Median fluorescence intensities (MFIs) and the percentage of dextran-positive cells were determined by flow cytometry using the BD FACSAria II system (BD Biosciences).
For 3D co-cultures, a commercially available polysaccharide hydrogel system (Cat. No: TWG001; Vitrogel—TheWell Bioscience) which mimics the natural ECM (extracellular matrix) was used. MSCCXCL12+/+ and MSCCXCL12−/−-primed macrophages harvested from the bone marrow were stained with CFSE (Cat. No: 65-0850; Thermo Fisher, Scientific). For this purpose, 1 × 105 cells were incubated in 1 µM dye solution for 10 min at room temperature in the dark. 4T1 cells were also labeled with CM-DiI (Cat. No: V-22888; Molecular probes) at 5 µM concentration according to the manufacturer’s instructions. After labeling, 4 × 104 macrophages (MSCCXCL12+/+ or MSCCXCL12−/−-primed macrophages) were mixed with 4 × 104 4T1 cells in a final volume of 40 µl of culture medium (DMEM/F12). The Vitrogel solution was first diluted with distilled water (DW) at a 1:4 (V/V) ratio. Then, the cell mixture (40 µl) was mixed with 160 µl of diluted Vitrogel and the final mixture was transferred to a 96-well culture plate with 50 µl in each well. The plate was then incubated for 15 min at room temperature for soft gel formation. Finally, the culture medium was added to cover the cell-containing hydrogel and incubated at 37 °C/95% O2/5% CO2 with more than 90% humidity. Spheroid formation was assessed under an inverted microscope after 72 h.
In order to perform the quantitative evaluation of multicellular tumor spheroids (MCTS), we used the Image Processing toolbox in the MATLAB software. For this purpose, image segmentation was implemented, where MCT images were divided into two segments, including red pixels and background pixels. As the images were in true color RGB with three color bands, namely red, green and blue, the first step was to convert the images to grayscale. Each pixel then had three grayscales corresponding to its color band. By considering three thresholds for each color band, a binary mask image was created, as shown in Fig. 5C. The pixels with a value of 1 in the mask images represent red pixels and those with a value of 0 correspond to background pixels. Considering the corresponding array of the mask images, the sum total of the array values equals the number of all red pixels in each image. To implement the process, a script was written and run in the software.
Twelve 8-week-old female BALB/c mice were divided into three groups in which a mixture of 4T1 cells (1 × 105, 2 × 105 or 4 × 105 cells) and MSCCXCL12-/–primed macrophages (1 × 105 cells) was injected into the right inguinal mammary fat pads in 10 µL of Matrigel (Cat No: 354248; Corning). For each mouse a corresponding mixture of 4T1 cells (1 × 105, 2 × 105 or 4 × 105 cells) and MSCCXCL12+/+-primed macrophages (1 × 105 cells) was also injected into the left inguinal mammary fat pads in Matrigel. Tumor development was checked every day. After two weeks, the mice were sacrificed by carbon dioxide asphyxiation and the mammary tumors excised from the mice. We then measured tumor dimensions with a caliper to calculate the tumor volume using the following formula: 0.52 × long diameter × short diameter2 .
Data are represented as mean ± SD. Student's t-test was used for statistical analyses after confirming the homogeneity of variances and normal distribution of data. For tumor initiation assay Fisher's exact test and the Cochran–Mantel–Haenszel (CMH) test were used. Statistical analyses were conducted using GraphPad InStat software version 2.02. Values of P < 0.05 were considered statistically significant.
Phenotypic polarization of BMDMs by MSC-derived CXCL12
Macrophages and MSCs were expanded from the BM mononuclears. BMDMs were morphologically round with eccentric nuclei and foamy cytoplasm (Fig. 1A). Macrophages were characterized as cells with F4/80 expression [32, 33] (Fig. 1B). MSCs were found to be positive for the expression of CD90 and CD105 and negative for CD34 and CD45 (Fig. 1C). CXCL12 chemokine was found to be expressed by MSCs. We first knocked out the CXCL12 gene in MSCs using a mouse CRISPR-Cas9 CXCL12 knockout system. As shown in Fig. 1D, the cells were efficiently transfected with the GFP reporter containing the CRISPR/Cas9 vector, with transfection efficiency of more than 90%. Furthermore, CXCL12 knockout was confirmed in MSCs by western blotting, as there was no CXCL12 protein expression in these cells (Fig. 1E; Additional file 1: Fig. S1). CXCL12 secretion was measured in the serum-free conditioned media of MSCsCXCL12+/+ whereas CXCL12 was undetectable in the conditioned media of MSCsCXCL12−/− (Fig. 1F). There was no morphological difference in MSCsCXCL12+/+ and MSCsCXCL12−/− as microscopic analyses consistently revealed the outgrowth of mesenchymal cells with a spindle-like morphology in both MSCCXCL12+/+ and MSCCXCL12−/− populations (data is not shown).
We then assessed the role of the MSC-derived CXCL12 niche in BMDM phenotypic switching in non-contact co-culture systems (Fig. 2A). We measured the protein levels of IL-4, IL-10 and TGF-β as M2 macrophage markers as well as IL-6 and TNF-α as M1 macrophage markers in the macrophage cell culture media by ELISA. Intriguingly, our findings showed that the deletion of CXCL12 from the MSC niche significantly reduced the expression of M2 macrophage markers in the BMDM cells, whereas the M1 macrophage markers were markedly increased (Fig. 2B).
We also evaluated the expression of the surface receptor CD206, a specific M2 macrophage marker, in BMDMs co-cultured with MSCsCXCL12+/+ or MSCsCXCL12−/− using flow cytometry. Our results showed that the percentage of CD206 positive cells was decreased in BMDMs co-cultured with MSCsCXCL12−/− (Fig. 2E). Moreover, the expression level of iNOS was remarkably increased in BMDMs co-cultured with MSCsCXCL12−/− (Fig. 2C, D; Additional file 2: Fig. S2), further confirming that M1 macrophage phenotypic switching is induced by coculturing with MSCsCXCL12−/−.
Thus, these results demonstrated that the BM MSC-derived CXCL12 niche was able to induce an M2 phenotype in BMDMs.
MSC-derived CXCL12 effect on BMDM function
We investigated the phagocytic properties of macrophages primed with MSCsCXCL12+/+ compared with macrophages primed with MSCsCXCL12−/−. As shown in Fig. 3, macrophages co-cultured with MSCsCXCL12−/− exhibited enhanced phagocytic potency compared with MSCCXCL12+/+-primed macrophages. Our results showed that CXCL12 is a significant inhibitor of macrophage phagocytic activity.
Augmentary effect on tumor induction by BMDMs primed with MSC-derived CXCL12
To assess whether BMDM phenotypic switching by MSC-derived CXCL12 might have an impact on tumor induction by 4T1 cells, a mixture of 4T1 cells plus BMDM co-cultured with MSCsCXCL12+/+ or MSCsCXCL12−/− was respectively injected into the left and right inguinal mammary fat pads (four fat pads in each group) (Fig. 4A). For this purpose, different numbers of 4T1 cells (1 × 105, 2 × 105, 4 × 105) were co-injected with 1 × 105 macrophages into each fat pad.
Our results showed remarkably augmented tumor induction potency by 4T1 cells after co-injection with MSCCXCL12+/+-primed macrophages. While coinjection of 1 × 105 4T1 cells with MSCCXCL12−/−-primed macrophages precluded orthotropic tumor induction, coinjection of similar 4T1 cell numbers with MSCCXCL12+/+-primed macrophages resulted in tumor induction in two out of four mammary fat pads (mean tumor size, 217 mm3) (Fig. 4B). While the tumor induction rate was three out of four fat pads with co-injection of higher concentrations of 4T1 cells (2 × 105 or 4 × 105) and MSCCXCL12+/+-primed M2 macrophages (mean tumor size in the 2 × 105 group, 103 mm3; mean tumor size in the 4 × 105 group, 347 mm3), these concentrations of 4T1 cells had a tumor induction rate of just one out of four fat pads after co-injection with MSCCXCL12−/−-primed M1 macrophages with tumor sizes of 27 mm3 and 125 mm3 in the 2 × 105 and 4 × 105 groups, respectively (Fig. 4B).
The results of in vivo limiting dilution assay using ELDA software are depicted in Fig. 4C. Furthermore, the tumor-initiating cell frequencies were calculated as 23 450 (CI = 10 613–51 814) and 124 331 (CI = 31,020–498,340) in MSCCXCL12+/+-primed M2 and MSCCXCL12−/−-primed M1 macrophage groups, respectively.
Our analysis using Fisher's exact test and the CMH test revealed that occurrence of tumors was significantly higher in the MSCCXCL12+/+-primed group than the MSCCXCL12−/−-primed group (chi-squared = 6.54, P = 0.011). Considering the occurrence of tumor in a single mouse/100 000 injected cells as one fold, average occurrences of tumor across three independent experiments in MSCCXCL12+/+-primed and MSCCXCL12−/−-primed macrophage groups were 1.3 and 0.25 fold, respectively (Fig. 4D).
Next, we confirmed BMDM phenotypic switching by three-dimensional coculture of 4T1 and MSCCXCL12−/− or MSCCXCL12+/+-primed macrophages. Spheroid formation was assessed under an inverted microscope after 72 h (Fig. 5A, B). Multicellular tumor spheroids (MCTS) were formed one day after culture and rapidly increased in size and number. Three days after co-culturing 4T1 cells with MSCCXCL12+/+-primed macrophages, large MCTS of 4T1 cells were noted with a scattered population of CFSE-labeled macrophages within the gel (Fig. 5B). A number of macrophages were seen in close association with MCTS. In order to quantitatively evaluate the differences between groups, image processing was performed in the MATLAB software (Fig. 5C). According to the obtained results, the number and size of 4T1 MCTS were remarkably lower in 4T1-MSCCXCL12−/−-primed macrophage co-cultures than in 4T1-MSCsCXCL12+/+-primed macrophage co-cultures (Fig. 5D).
In this study, we focused on the effect of CXCL12 produced by BM-MSCs on BMDMs. As polarized macrophages differ in terms of receptor expression, chemokine production and effector functions , to observe the effect of MSC-derived CXCL12 on BMDM phenotype, we analyzed secretion of phenotype-specific cytokines, expression of a phenotype-specific CD marker, spheroid formation rate in three dimensional coculture assays and tumor initiation rate after in vivo tumor induction. Our findings revealed that BMDM underwent phenotypic switching into M2 cells following co-culture with MSCCXCL12+/+, which was documented by augmented expression of IL-4, IL-10, TGF-β, and CD206 as M2 markers, and decreased expression of M1 specific markers, such as IL-6, TNF-α and iNOS. Macrophage M2 phenotype is associated with tumor initiation, development and progression, so that tumor-associated macrophages (TAMs) are recognized as M2 macrophages .
Hence, we confirmed our in vitro findings by a three dimensional coculture assay and an in vivo experiment assessing tumor induction rate. According to these tests, the number and size of MCTS were much higher in 4T1 cells co-cultured with MSCCXCL12+/+-primed (M2) macrophages. Likewise, tumor induction rate was enhanced when 4T1 cells were co-injected into the mice fat pads with these MSCCXCL12+/+-primed M2 macrophages.
Our findings showed down-regulation of macrophage phagocytic activity after exposure to the MSC-derived CXCL12 niche. Although the potential effect of the MSC CXCL12 niche on macrophage functions has not been investigated yet, M1 macrophages were reported to have greater phagocytic activity [34, 35].
Clinical studies have shown that macrophages can release cytotoxic molecules, such as reactive nitrogen intermediates, reactive oxygen intermediates, and a migration inhibitory factor during chronic inflammation, leading to DNA mutation and defective p53 activity in the surrounding epithelial cells, thereby predisposing these cells to premalignant transformation and tumor initiation. Moreover, TAMs were able to transmit pro-survival signals to these premalignant cells through production of several cytokines, including TNF-α, IL-1, and IL-6 .
Tumor-associated macrophages were also found to regulate tumor cell migration/invasion through the extracellular matrix (ECM). Passing through the ECM is a necessary aspect of cell migration. Macrophages can regulate composition of the ECM by depositing ECM components, including various types of collagens. These cells are also a major source of various proteolytic enzymes such as cathepsins, matrix metalloproteinases, serine proteases and urokinase plasminogen activator. Macrophage-derived proteases degrade the surrounding ECM, providing a suitable matrix for cancer cell invasion. Furthermore, TAMs were found to significantly increase the migration and invasion of cancer cells via overexpression of epidermal growth factor [6, 37].
As a matter of fact, it has recently been demonstrated that there is a correlation between M1/M2 ratio and overall survival of ovarian cancer patients, such that the M1/M2 ratio was positively correlated with overall survival and inversely correlated with cancer stage. Iron removal from tumor cells by M1 macrophages and promotion of the antitumor T1 response and cytotoxic T lymphocyte recruitment to tumors by M1 macrophages were suggested to be the main reasons for improvement of the prognosis of patients with ovarian cancer containing an M1-rich microenvironment [38, 39].
With regard to previous studies, MSCs are considered to be responsible for macrophage phenotypic switching [13, 40], but the underlying mechanisms are poorly clarified. Cardiac adipose tissue-derived MSCs were reported to release IL-6, which stimulates macrophages to release M2 inducer cytokines such as IL-10 and IL-13 . Moreover, Vasandan et al. proposed a mechanistic role for bioactive factors such as MSC-derived prostaglandin E2 in instructing M1-M2 polarization shifts . In another study, umbilical cord-derived MSC was found to alter the phenotype and function of monocyte-derived dendritic cells through lactate-mediated metabolic reprogramming. UC-MSC derived lactate can induce M2-macrophage phenotypic switching in terms of morphology, surface markers, migratory properties and antigen presentation capacity .
In spite of the fact that CXCL12 is considered as a critical component of the MSC niche [43,44,45,46], as yet, it has not been investigated whether MSC-derived CXCL12 might induce phenotypic alteration of macrophages. Stromal cell-derived factor-1 is a small chemotactic, angiogenic factor  that was first identified as a molecule involved in B-cell lymphopoiesis using an expression cloning program . Human and mouse CXCR4  and CXCR7  are known as CXCL12 receptors. CXCR4 expression is found in blood monocytes, neutrophils, lymphocytes, hematopoietic progenitors  and tissue macrophages , and CXCR7 is induced during monocyte-to-macrophage differentiation . Expression of CXCL12 receptors on immune-system cells may be indicative of CXCL12’s role as an important soluble factor operating on effector cells in inflammatory situations and the tumor microenvironment. Interestingly, previous evidence indicated CXCL12 as a critical mediator for monocyte extravasation [16, 20], regulation of monocyte function, survival and differentiation into macrophages  and macrophage phagocytosis enhancement .
In conclusion, our results suggest that the MSC-derived CXCL12 niche is involved in macrophage polarization dynamics as it could prompt BMDM phenotypic switching into M2 macrophages, which could have a decisive role in the tumor microenvironment. It is noteworthy that all the immunosuppressive circumstances occurring in the cancer stromal niche can result in suppression of cancer immunity, thereby enabling malignant cells to escape immune surveillance and promote tumor growth and metastasis . Thus, investigation of mechanisms interfering with immune suppression and tumor growth may be of great interest in finding new remedies to control and treat cancers.
Availability of data and materials
All data are contained in the article.
mesenchymal stem cell
Bone marrow-derived macrophage
C–X–C motif chemokine ligand 12
Stromal cell-derived factor-1
Inducible nitric oxide synthase
Tumor necrosis factor alpha
Transforming growth factor beta
Cluster of differentiation
Carboxyfluorescein succinimidyl ester
Multicellular tumor spheroids
- 3D coculture:
Three dimensional coculture
Vasandan AB, Jahnavi S, Shashank C, Prasad P, Kumar A, Prasanna SJ. Human Mesenchymal stem cells program macrophage plasticity by altering their metabolic status via a PGE 2-dependent mechanism. Sci Rep. 2016;6:38308.
Elchaninov AV, Fatkhudinov TK, Vishnyakova PA, Lokhonina AV, Sukhikh GT. Phenotypical and functional polymorphism of liver resident macrophages. Cells. 2019;8:1032.
Elliott LA, Doherty GA, Sheahan K, Ryan EJ. Human tumor-infiltrating myeloid cells: phenotypic and functional diversity. Front Immunol. 2017;8:86.
Mantovani A, Sozzani S, Locati M, Allavena P, Sica A. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol. 2002;23:549–55.
Kim YH, Tabata Y. Recruitment of mesenchymal stem cells and macrophages by dual release of stromal cell-derived factor-1 and a macrophage recruitment agent enhances wound closure. J Biomed Mater Res Part A. 2016;104:942–56.
Ruffell B, Affara NI, Coussens LM. Differential macrophage programming in the tumor microenvironment. Trends Immunol. 2012;33:119–26.
Goswami KK, Ghosh T, Ghosh S, Sarkar M, Bose A, Baral R. Tumor promoting role of anti-tumor macrophages in tumor microenvironment. Cell Immunol. 2017;316:1–10.
Erlandsson A, Carlsson J, Lundholm M, Fält A, Andersson SO, Andrén O, Davidsson S. M2 macrophages and regulatory T cells in lethal prostate cancer. Prostate. 2019;79:363–9.
Helm O, Held-Feindt J, Schäfer H, Sebens S. M1 and M2: there is no “good” and “bad”—How macrophages promote malignancy-associated features in tumorigenesis. Oncoimmunology. 2014;3:e946818.
Koh Y-C, Yang G, Lai C-S, Weerawatanakorn M, Pan M-H. Chemopreventive effects of phytochemicals and medicines on M1/M2 polarized macrophage role in inflammation-related diseases. Int J Mol Sci. 2018;19:2208.
Kamdje AHN, Kamga PT, Simo RT, Vecchio L, Etet PFS, Muller JM, Bassi G, Lukong E, Goel RK, Amvene JM. Mesenchymal stromal cells’ role in tumor microenvironment: involvement of signaling pathways. Cancer Biol Med. 2017;14:129.
Liang W, Chen X, Zhang S, Fang J, Chen M, Xu Y, Chen X. Mesenchymal stem cells as a double-edged sword in tumor growth: focusing on MSC-derived cytokines. Cell Mol Biol Lett. 2021;26:1–25.
Cho D-I, Kim MR, Jeong H-Y, Jeong HC, Jeong MH, Yoon SH, Kim YS, Ahn Y. Mesenchymal stem cells reciprocally regulate the M1/M2 balance in mouse bone marrow-derived macrophages. Exp Mol Med. 2014;46:e70–e70.
Lin T, Pajarinen J, Kohno Y, Huang J-F, Maruyama M, Romero-Lopez M, Nathan K, Yao Z, Goodman SB. Trained murine mesenchymal stem cells have anti-inflammatory effect on macrophages, but defective regulation on T-cell proliferation. FASEB J. 2019;33:4203–11.
Liu W, Yu M, Xie D, Wang L, Ye C, Zhu Q, Liu F, Yang L. Melatonin-stimulated MSC-derived exosomes improve diabetic wound healing through regulating macrophage M1 and M2 polarization by targeting the PTEN/AKT pathway. Stem Cell Res Ther. 2020;11:1–15.
Sánchez-Martín L, Estecha A, Samaniego R, Sánchez-Ramón S, Vega MÁ, Sánchez-Mateos P. The chemokine CXCL12 regulates monocyte-macrophage differentiation and RUNX3 expression Blood. 2011;117:88–97.
Kryczek I, Wei S, Keller E, Liu R, Zou W. Stroma-derived factor (SDF-1/CXCL12) and human tumor pathogenesis. Am J Physiol Cell Physiol. 2007;292:C987–95.
Guo R, Chai L, Chen L, Chen W, Ge L, Li X, Li H, Li S, Cao C. Stromal cell-derived factor 1 (SDF-1) accelerated skin wound healing by promoting the migration and proliferation of epidermal stem cells. Vitro Cell Dev Biol Anim. 2015;51:578–85.
Lataillade J-J, Clay D, Dupuy C, Rigal S, Jasmin C, Bourin P, Bousse-Kerdilès M-CL. Chemokine SDF-1 enhances circulating CD34+ cell proliferation in synergy with cytokines: possible role in progenitor survival Blood. 2000;95:756–68.
Tseng D, Vasquez-Medrano D, Brown J. Targeting SDF-1/CXCR4 to inhibit tumour vasculature for treatment of glioblastomas. Br J Cancer. 2011;104:1805–9.
Wang S-C, Yu C-F, Hong J-H, Tsai C-S, Chiang C-S. Radiation therapy-induced tumor invasiveness is associated with SDF-1-regulated macrophage mobilization and vasculogenesis. PloS ONE. 2013;8:e69182.
Jin W, Zhao Y, Hu Y, Yu D, Li X, Qin Y, Kong D, Wang H. Stromal cell-derived factor-1 enhances the therapeutic effects of human endometrial regenerative cells in a mouse sepsis model. Stem Cells Int. 2020;2020:78.
Sabbagh P, Karkhah A, Nouri HR, Javanian M, Ebrahimpour S. The significance role of regulatory T cells in the persistence of infections by intracellular bacteria. Infect Genet Evol. 2018;62:270–4.
Gheisari Y, Azadmanesh K, Ahmadbeigi N, Nassiri SM, Golestaneh AF, Naderi M, Vasei M, Arefian E, Mirab-Samiee S, Shafiee A. Genetic modification of mesenchymal stem cells to overexpress CXCR4 and CXCR7 does not improve the homing and therapeutic potentials of these cells in experimental acute kidney injury. Stem Cells Dev. 2012;21:2969–80.
Boltz-Nitulescu G, Wiltschke C, Holzinger C, Fellinger A, Scheiner O, Gessl A, Förster O. Differentiation of rat bone marrow cells into macrophages under the influence of mouse L929 cell supernatant. J Leukoc Biol. 1987;41:83–91.
Zhang X, Goncalves R, Mosser DM. The isolation and characterization of murine macrophages. Curr Protoc Immunol. 2008;83:14.
Bailey JD, Shaw A, McNeill E, Nicol T, Diotallevi M, Chuaiphichai S, Patel J, Hale A, Channon KM, Crabtree MJ. Isolation and culture of murine bone marrow-derived macrophages for nitric oxide and redox biology. Nitric Oxide. 2020;8:44.
Matluobi D, Araghi A, Maragheh BFA, Rezabakhsh A, Soltani S, Khaksar M, Siavashi V, Feyzi A, Bagheri HS, Rahbarghazi R. Carvacrol promotes angiogenic paracrine potential and endothelial differentiation of human mesenchymal stem cells at low concentrations. Microvasc Res. 2018;115:20–7.
Medeiros PJ, Al-Khazraji BK, Novielli NM, Postovit LM, Chambers AF, Jackson DN. Neuropeptide Y stimulates proliferation and migration in the 4T1 breast cancer cell line. Int J Cancer. 2012;131:276–86.
Ben-Mordechai T, Holbova R, Landa-Rouben N, Harel-Adar T, Feinberg MS, Abd Elrahman I, Blum G, Epstein FH, Silman Z, Cohen S. Macrophage subpopulations are essential for infarct repair with and without stem cell therapy. J Am Coll Cardiol. 2013;62:1890–901.
Kim EJ, Choi M-R, Park H, Kim M, Hong JE, Lee J-Y, Chun HS, Lee KW, Park JHY. Dietary fat increases solid tumor growth and metastasis of 4T1 murine mammary carcinoma cells and mortality in obesity-resistant BALB/c mice. Breast Cancer Res. 2011;13:1–13.
Lin H-H, Faunce DE, Stacey M, Terajewicz A, Nakamura T, Zhang-Hoover J, Kerley M, Mucenski ML, Gordon S, Stein-Streilein J. The macrophage F4/80 receptor is required for the induction of antigen-specific efferent regulatory T cells in peripheral tolerance. J Exp Med. 2005;201:1615–25.
McCauley J, Bitsaktsis C, Cottrell J. Macrophage subtype and cytokine expression characterization during the acute inflammatory phase of mouse bone fracture repair. J Orthopaed Res. 2020;38:1693–702.
Varin A, Mukhopadhyay S, Herbein G, Gordon S. Alternative activation of macrophages by IL-4 impairs phagocytosis of pathogens but potentiates microbial-induced signalling and cytokine secretion. Blood. 2010;115:353–62.
Martinez FO, Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000 Prime Reports. 2014;6:2.
Biswas SK, Sica A, Lewis CE. Plasticity of macrophage function during tumor progression: regulation by distinct molecular mechanisms. J Immunol. 2008;180:2011–7.
Chanmee T, Ontong P, Konno K, Itano N. Tumor-associated macrophages as major players in the tumor microenvironment. Cancers. 2014;6:1670–90.
Macciò A, Gramignano G, Cherchi MC, Tanca L, Melis L, Madeddu C. Role of M1-polarized tumor-associated macrophages in the prognosis of advanced ovarian cancer patients. Sci Rep. 2020;10:1–8.
Madeddu C, Gramignano G, Kotsonis P, Coghe F, Atzeni V, Scartozzi M, Macciò A. Microenvironmental M1 tumor-associated macrophage polarization influences cancer-related anemia in advanced ovarian cancer: key role of interleukin-6. Haematologica. 2018;103:e388.
Takizawa N, Okubo N, Kamo M, Chosa N, Mikami T, Suzuki K, Yokota S, Ibi M, Ohtsuka M, Taira M. Bone marrow-derived mesenchymal stem cells propagate immunosuppressive/anti-inflammatory macrophages in cell-to-cell contact-independent and-dependent manners under hypoxic culture. Exp Cell Res. 2017;358:411–20.
Adutler-Lieber S, Ben-Mordechai T, Naftali-Shani N, Asher E, Loberman D, Raanani E, Leor J. Human macrophage regulation via interaction with cardiac adipose tissue-derived mesenchymal stromal cells. J Cardiovasc Pharmacol Ther. 2013;18:78–86.
Selleri S, Bifsha P, Civini S, Pacelli C, Dieng MM, Lemieux W, Jin P, Bazin R, Patey N, Marincola FM. Human mesenchymal stromal cell-secreted lactate induces M2-macrophage differentiation by metabolic reprogramming. Oncotarget. 2016;7:30193.
Keshavarz S, Nassiri SM, Siavashi V, Alimi NS. Regulation of plasticity and biological features of endothelial progenitor cells by MSC-derived SDF-1. BBA Mol Cell Res. 2019;1866:296–304.
Rahbarghazi R, Nassiri SM, Khazraiinia P, Kajbafzadeh A-M, Ahmadi SH, Mohammadi E, Molazem M, Zamani-Ahmadmahmudi M. Juxtacrine and paracrine interactions of rat marrow-derived mesenchymal stem cells, muscle-derived satellite cells, and neonatal cardiomyocytes with endothelial cells in angiogenesis dynamics. Stem Cells Dev. 2013;22:855–65.
Vafaei R, Nassiri SM, Siavashi V. β3-Adrenergic regulation of EPC features through manipulation of the bone marrow MSC niche. J Cell Biochem. 2017;118:4753–61.
Nassiri SM, Rahbarghazi R. Interactions of mesenchymal stem cells with endothelial cells. Stem Cells Dev. 2014;23:319–32.
Wan X, Xia W, Gendoo Y, Chen W, Sun W, Sun D, Cao C. Upregulation of stromal cell-derived factor 1 (SDF-1) is associated with macrophage infiltration in renal ischemia-reperfusion injury. PLoS ONE. 2014;9:e114564.
Ma Q, Jones D, Borghesani PR, Segal RA, Nagasawa T, Kishimoto T, Bronson RT, Springer TA. Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4-and SDF-1-deficient mice. Proc Natl Acad Sci. 1998;95:9448–53.
Burns JM, Summers BC, Wang Y, Melikian A, Berahovich R, Miao Z, Penfold ME, Sunshine MJ, Littman DR, Kuo CJ. A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development. J Exp Med. 2006;203:2201–13.
Verani A, Pesenti E, Polo S, Tresoldi E, Scarlatti G, Lusso P, Siccardi AG, Vercelli D. Cutting edge: CXCR4 is a functional coreceptor for infection of human macrophages by CXCR4-dependent primary HIV-1 isolates. J Immunol. 1998;161:2084–8.
Ma W, Liu Y, Ellison N, Shen J. Induction of CXC chemokine receptor type 7 (CXCR7) switches stromal cell-derived factor-1 (SDF-1) signaling and phagocytic activity in macrophages linked to atherosclerosis. J Biol Chem. 2013;288:15481–94.
Chatterjee M, von Ungern-Sternberg SN, Seizer P, Schlegel F, Büttcher M, Sindhu N, Müller S, Mack A, Gawaz M. Platelet-derived CXCL12 regulates monocyte function, survival, differentiation into macrophages and foam cells through differential involvement of CXCR4–CXCR7. Cell Death Dis. 2015;6:e1989–e1989.
We are grateful to Zahra Mohseni for technical assistance and the members of the Royan animal laboratory for help.
This work was supported by a research grant from the University of Tehran.
Ethics approval and consent to participate
Experimental animal research in this study was performed in accordance with the Basel Declaration. Mice were treated according to the Guide for the Care and Use of Laboratory Animals (NIH Publication, 8th edition, revised 2011). All steps of this study were approved by the Animal Care Committee of the University of Tehran (No. 341-2018).
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Babazadeh, S., Nassiri, S.M., Siavashi, V. et al. Macrophage polarization by MSC-derived CXCL12 determines tumor growth. Cell Mol Biol Lett 26, 30 (2021). https://doi.org/10.1186/s11658-021-00273-w
- Mesenchymal stem cells (MSCs)
- C-X-C motif chemokine ligand 12 (CXCL12)
- Tumor microenvironment