Adipocyte Interactions and Immunomodulation in Triple-Negative Breast Cancer
Adipocyte Interactions and Immunomodulation in Triple-Negative Breast Cancer
Adipocyte Transformation in the Tumour Microenvironment
Triple-negative breast cancer (TNBC) is defined by a lack of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2) expression [1]. This subtype is known for high metastatic potential and poor prognosis compared to other forms of breast cancer [1]. Within the tumour microenvironment (TME), which is the ecosystem comprising cancer cells, immune cells, and stromal components, adipocytes are a major constituent [2]. Adipocytes serve as energy storage units and endocrine cells [2]. In the presence of cancer cells, these adipocytes are transformed into cancer-associated adipocytes (CAA) [1].
CAA are observed at the invasive front of human breast tumours [2]. These cells exhibit distinct morphological changes, such as a smaller size and irregular shapes [2]. Lipid accumulation is reduced in CAA compared to normal mature adipocytes [1]. In laboratory models, CAA are established by co-culturing 3T3-L1-induced adipocytes with 4T1 breast cancer cells [1]. These cells lose markers of mature adipocyte differentiation, such as peroxisome proliferator-activated receptor gamma (PPAR-γ), CCAAT enhancer-binding protein alpha (C/EBPα), and fatty acid binding protein 4 (FABP4) [2, 7]. Simultaneously, markers of undifferentiated cells, such as alpha-smooth muscle actin (α-SMA) and vimentin, are increased [2, 7].
Adipose-derived mesenchymal stem cells (ADMSC) also respond to the TNBC secretome [6]. These cells can differentiate into several lineages, but when exposed to media from MDA-MB-231 cells, they acquire a CAA-like phenotype [6]. This transition involves the acquisition of a pro-inflammatory state characterised by the secretion of many cytokines [6]. The presence of crown-like structures (CLSs), which consist of macrophages surrounding dying adipocytes, is a histologic marker of this pro-inflammatory transition [2].
Signalling Pathways and Epithelial-Mesenchymal Transition
The progression of TNBC is often driven by the epithelial-mesenchymal transition (EMT), a sequence where epithelial cells lose polarity and acquire mesenchymal traits [1]. CAA promote this transition through the activation of several intracellular pathways [1]. One such pathway is the PI3K/AKT signalling cascade [1]. In tumour tissues where CAA are present, E-cadherin expression is downregulated, while Ki67 expression is increased, indicating higher proliferation [1].
Another major pathway involved in CAA-mediated progression is the Stat3 (signal transducer and activator of transcription 3) pathway [7]. Granulocyte colony-stimulating factor (G-CSF), secreted by CAA, activates Stat3 in TNBC cells [7]. This activation leads to the expression of pro-invasive genes, such as matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) [7]. Laboratory data display that targeting the G-CSF/Stat3 axis with neutralizing antibodies or siRNAs can abrogate the migration and invasion induced by CAA [7].
Snail is a biomarker that controls the acquisition of the CAA phenotype in ADMSC [6]. When ADMSC are exposed to the TNBC secretome, Snail expression is induced [6]. This induction is associated with the activation of Smad2 and NF-κB pathways [6]. These pathways coordinate the expression of inflammatory and immunomodulatory genes that support a tumour-promoting environment [6]. The use of epigallocatechin-3-gallate (EGCG), a polyphenol from green tea, has been shown to inhibit the activation of these pathways and prevent the onset of the CAA phenotype [6].
Cytokine-Mediated Communication and Metabolic Changes
CAA act as endocrine and paracrine cells by releasing a variety of cytokines and chemokines, collectively called adipokines [7]. These substances comprise interleukin-6 (IL-6), interleukin-8 (IL-8, also known as CXCL8), interleukin-1 beta (IL-1β), and C-C motif chemokine ligand 5 (CCL5) [2]. IL-6 is a pleiotropic cytokine that stimulates the invasion and migration of breast tumour cells through the Cofilin-1 pathway [2]. Other members of the IL-6 family, such as leukaemia inhibitory factor (LIF) and oncostatin M (OSM), also participate in metastatic transition and the degradation of the extracellular matrix (ECM) [2].
CXCL8 is a chemokine secreted by CAA that has been linked to tumour growth and metastasis [3]. High levels of CXCL8 are found in the secretome of CAA isolated from TNBC patients [3]. This chemokine promotes the proliferation and migration of cancer cells [3]. Separately, CCL2 and CCL5 are induced in ADMSC by the TNBC secretome [6]. These chemokines are associated with the recruitment of immune cells and the promotion of an invasive phenotype in cancer cells [2, 6].
Metabolic changes are also a feature of the interaction between cancer cells and adipocytes [2]. CAA provide metabolic substrates, such as free fatty acids (FFAs), to fuel the growth of cancer cells [1, 2]. This metabolic rewiring allows cancer cells to survive in the hypoxic conditions often found within solid tumours [2]. The secretion of metabolites like lactate, pyruvate, and ketone bodies by CAA further supports this adaptive state [6]. These changes contribute to an environment that facilitates both tumour expansion and the evasion of therapeutic agents [2].
Modification of the Tumour Immune Microenvironment
The tumour immune microenvironment (TIME) is a complex system of immune cells that can either inhibit or promote tumour growth [3]. CAA contribute to an immunosuppressive TIME by modulating the function of T cells and other immune populations [1]. One mechanism is the upregulation of programmed death-ligand 1 (PD-L1, also known as CD274) on the surface of TNBC cells [1, 3]. Increased PD-L1 expression allows cancer cells to evade detection by the immune system [1].
In tumours co-existing with CAA, a reduction in the infiltration of CD8+ T cells is observed [1]. These cells are the primary effectors of the anti-tumour immune response. The secretome of CAA, particularly CXCL8, has been shown to suppress both CD4+ T and CD8+ T cell infiltration [3]. For this reason, targeting the CXCL8 pathway may be a strategy to enhance the efficacy of immune checkpoint inhibitors, such as anti-PD-1 therapies [3]. In mice, the combination of a CXCL8 inhibitor and anti-PD-1 therapy resulted in a synergistic inhibition of tumour progression [3].
Immune evasion is also supported by the recruitment of immunosuppressive cells [2]. CAA-derived cytokines stimulate the recruitment of macrophages and promote their polarisation into a pro-tumour M2 phenotype [2]. This recruitment is mediated by factors such as CCL2 and VEGF [2]. The resulting environment is one where anti-tumour immunity is suppressed, and pro-inflammatory signals continue to drive malignancy [1, 2].
Extracellular Vesicles and Paracrine Regulation
Extracellular vesicles (EVs) are small, lipid-delimited particles that facilitate cell-to-cell communication by transferring proteins, lipids, and nucleic acids [4]. Exosomes are a subtype of EVs with a diameter between 30 and 150 nm [4]. TNBC cells, such as the MDA-MB-231 line, secrete EVs that can pre-condition the surrounding adipose tissue [4]. These vesicles trigger a pro-inflammatory phenotype in ADMSC, characterised by increased expression of CXCL8, CCL2, and IL-1β [4].
Recent data indicate that EVs from TNBC cells also carry mitochondrial components and mitochondrial DNA (mtDNA) [4]. The horizontal transfer of these components can modulate the metabolic function and viability of recipient cells [4]. In ADMSC, exposure to TNBC-derived EVs induces markers of senescence, such as p21 and beta-galactosidase [4]. This senescence is associated with the activation of signaling pathways like AKT and GSK-3β [4].
Dietary polyphenols like EGCG can alter the content and effect of these vesicles [4]. Treatment of MDA-MB-231 cells with EGCG modifies the genetic material found within their secreted EVs [4]. EGCG-treated EVs have a lower mitochondrial content and a reduced capacity to induce inflammation and senescence in ADMSC [4]. This suggests that diet-derived substances can interfere with the paracrine regulation exerted by TNBC cells on their local environment [4].
Single-Cell Analysis of Adipocyte Heterogeneity
White adipose tissue (WAT) is a metabolic organ found at several anatomic sites, including subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) [5]. It comprises mature adipocytes and adipose stem and progenitor cells (ASPCs) [5]. Single-nucleus RNA sequencing (snRNA-seq) and single-cell RNA sequencing (scRNA-seq) have allowed for a detailed mapping of these cell populations in both mice and humans [5]. These methods reveal that adipocytes in the tumour-adipose microenvironment (TAME) are highly heterogeneous [5].
Several subtypes of cancer-associated adipocytes have been identified [5]. DPP4+ adipocytes are found in VAT, while ADIPOQ+ adipocytes are present in SAT [5]. High levels of these subtypes are associated with poor outcomes in adipose-associated cancers, including breast cancer [5]. These subtypes interact with immune cells and other stromal components to create an ecosystem that supports tumour development [5].
Bioinformatics screening of single-cell data has identified possible drugs to target these adipocyte subtypes [5]. These comprise Trametinib, Selumetinib, and Ulixertinib [5]. Separately, the knockdown of adiponectin receptor 1 (AdipoR1) and AdipoR2 has been shown to impair the proliferation and invasion of breast cancer cells in vitro [5]. Patients with high expression of AdipoR2 in breast cancer tissues have been observed to have shorter relapse-free survival compared to those with low expression [5]. This knowledge provides a basis for the development of targeted therapies that focus on exact adipocyte subpopulations within the TME [5].
Experimental Models and Future Directions
The study of CAA often relies on orthotopic mammary tumour models [1, 3]. In these models, TNBC cells and CAA are co-injected into the mammary fat pad of mice [1]. Observations from such experiments show that the presence of CAA accelerates primary tumour growth and promotes lung metastasis [1]. Histological analysis of the collected tissues confirms the activation of EMT and the remodelling of the immune environment [1, 3]. These organismal-level findings validate the observations made in cell culture systems [1].
The use of 6-well Transwell plates with polycarbonate membranes allows for the isolation of the effects of secreted factors from direct cell contact [1, 3]. This method has been essential for identifying the secretome of CAA and understanding its effect on cancer cell behavior [1, 3]. Furthermore, transcriptome sequencing of CAA versus normal adipocytes has revealed many differentially expressed genes that could serve as therapeutic targets [3, 7].
In summary, the interaction between TNBC cells and adipocytes is a major driver of tumour progression and immune escape [1, 2]. Adipocytes undergo a transformation into CAA, which then secrete cytokines like IL-6, IL-8, and G-CSF [2, 7]. These factors activate pathways like Stat3 and PI3K/AKT, leading to EMT and increased metastasis [1, 7]. CAA also reshape the TIME by upregulating PD-L1 and reducing T cell infiltration [1, 3]. Interventions such as EGCG or the targeting of exact chemokines like CXCL8 represent possible strategies to block these pro-tumour interactions [3, 4, 6]. The use of single-cell technologies continues to refine the knowledge of these cell populations and their parts in breast cancer biology [5].
References
Zhang, X., Zhong, S., Liu, S., Mu, X., Chen, G., & Chen, W. (2025). Cancer-associated adipocyte promote progression and immunosuppression in triple-negative breast cancer. PubMed Central. https://pmc.ncbi.nlm.nih.gov/articles/PMC13128427/
Yang, Z., Zeng, H., Li, J., et al. (2024). Dissecting the emerging role of cancer-associated adipocyte-derived cytokines in remodeling breast cancer progression. Heliyon, 10, e35200. https://doi.org/10.1016/j.heliyon.2024.e35200
Huang, R., Wang, Z., Hong, J., et al. (2023). Targeting cancer-associated adipocyte-derived CXCL8 inhibits triple-negative breast cancer progression and enhances the efficacy of anti-PD-1 immunotherapy. https://doi.org/10.21203/rs.3.rs-2419604/v1
Gonzalez Suarez, N., Fernandez-Marrero, Y., Hébert, M. P. A., et al. (2023). EGCG inhibits the inflammation and senescence inducing properties of MDA-MB-231 triple-negative breast cancer (TNBC) cells-derived extracellular vesicles in human adipose-derived mesenchymal stem cells. Cancer Cell International, 23. https://doi.org/10.1186/s12935-023-03087-2
Liu, S.-Q., Chen, D.-Y., Li, B., et al. (2023). Single-cell analysis of white adipose tissue reveals the tumor-promoting adipocyte subtypes. Journal of Translational Medicine, 21. https://doi.org/10.1186/s12967-023-04256-7
Gonzalez Suarez, N., Fernandez-Marrero, Y., Torabidastgerdooei, S., & Annabi, B. (2022). EGCG Prevents the Onset of an Inflammatory and Cancer-Associated Adipocyte-like Phenotype in Adipose-Derived Mesenchymal Stem/Stromal Cells in Response to the Triple-Negative Breast Cancer Secretome. Nutrients, 14, 1099. https://doi.org/10.3390/nu14051099
Liu, L., Wu, Y., Zhang, C., et al. (2020). Cancer-associated adipocyte-derived G-CSF promotes breast cancer malignancy via Stat3 signaling. Journal of Molecular Cell Biology, 12, 723-737. https://doi.org/10.1093/jmcb/mjaa016
FAQ
This specific type of breast cancer is identified by the absence of estrogen receptor, progesterone receptor, and HER2 expression. It is frequently associated with high metastatic capacity and lower survival rates. The ecosystem surrounding these tumours consists of malignant cells, immune cells, and stromal parts. Within this space, adipocytes are found. They usually function as storage for energy or as endocrine cells. Cells are transformed. When cancer cells are present, these fat cells are converted into a different state. This occurs locally. The lack of standard receptors makes treatment choices more difficult. Aggression is expected. Many patients face a more aggressive disease course. The ways in which surrounding cells support the growth of the cancer are focused on by research. This knowledge is needed if new ways to treat the condition are to be found by us.
The conversion into cancer-associated adipocytes is observed at the edges where tumours invade healthy tissue. These cells are small. Irregular shapes are seen in these cells when compared to normal fat cells. Lipid storage is reduced. In laboratory settings, this state is created by placing specific fat cells together with breast cancer cells. The cells lose maturity. New markers associated with undifferentiated cells like α-SMA are then expressed by the cells. Stem cells also change. A pro-inflammatory state is acquired by these cells when they are exposed to the substances released by the tumour. This change is permanent. Histological markers like crown-like structures are often seen in these areas. Macrophages surround dying cells. This observation confirms the presence of inflammation. The state is driven by the close proximity of the malignant cells.
Transformed adipocytes lose several protein labels that are typically found in mature cells. Labels are missing. These labels include PPAR-γ, C/EBPα, and FABP4. The reduction of these proteins indicates a loss of differentiation. Simultaneously, the cells start to produce other proteins such as α-SMA and vimentin. These are undifferentiated. Mesenchymal stem cells from fat tissue also show changes in protein expression. Snail is induced. When these stem cells are exposed to the cancer secretome, the biomarker Snail is produced. This protein is linked to the activation of the Smad2 and NF-κB pathways. Internal routes coordinate genes. Researchers are helped by the presence of these markers to find transformed cells within the tissue. The survival of the malignancy is supported by every change. Detailed lab analysis is used.
The epithelial-mesenchymal transition is a sequence where epithelial cells lose their polarity and gain traits of mesenchymal cells. This sequence is often observed during the advancement of triple-negative breast cancer. Certain internal routes are triggered by transformed adipocytes to assist this change. The PI3K/AKT signalling cascade is one such route. In areas where these fat cells exist, the expression of E-cadherin is lowered. Higher levels of Ki67 expression are seen in these same regions. This indicates that the cells are dividing more rapidly. Cancer cells are allowed to become more mobile by this transition. They can then be spread to other parts of the body more easily. This movement is a main feature of aggressive cancers. Interactions help. The transition is made possible by the interaction between different cell groups. It is considered a major part of how tumours grow.
The Stat3 pathway is a main driver of tumour advancement when triggered by transformed fat cells. This activation is caused by G-CSF, which is a substance released by these adipocytes. Once the pathway is active, it leads to the production of pro-invasive genes. MMP2 and MMP9 are two examples of these genes. The breakdown of the surrounding tissue is assisted by these proteins. This allows the cancer cells to migrate and invade other areas. Laboratory tests show that this movement can be stopped by blocking this specific axis. Neutralising antibodies or siRNAs are used to achieve this effect. This suggests that the G-CSF/Stat3 axis is a possible target for therapy. Without this signalling, the aggressive nature of the cancer is reduced. A bridge between the fat cells and the tumour is formed by the pathway.
Transformed adipocytes act as endocrine cells by releasing several substances known as adipokines. These include IL-6, CXCL8, IL-1β, and CCL5. IL-6 is a substance that encourages the invasion of tumour cells through the Cofilin-1 pathway. Other family members assist. LIF and OSM also participate in the spread and degradation of the material that holds cells together. Growth is linked to CXCL8. High levels of this substance are found in the secretions of fat cells from patients. Malignant cells are supported. The movement and division of cancer cells is helped by this chemokine. CCL2 and CCL5 are also induced. Immune cells are helped into the area by these chemokines. This chemical communication is a constant feature of the environment.
The immune environment is modified by transformed adipocytes to help the tumour avoid detection. One way this happens is through the increased expression of PD-L1 on cancer cells. This protein allows the cells to hide. A reduction in the number of CD8+ T cells is also observed in these tumours. These cells are defenders. The secretions from the fat cells, especially CXCL8, prevent these immune cells from entering the area. Targeting this pathway might improve the success of other treatments. In mouse models, the disease was slowed by combining a CXCL8 inhibitor with anti-PD-1 therapy. Transformed fat cells recruit macrophages. These macrophages are changed into a form that supports the tumour. Inflammation continues. A space where the cancer can grow without being attacked is created.
Extracellular vesicles are small particles that allow cells to talk to one another by sharing proteins and genetic material. Exosomes are a type of these vesicles with a very small diameter. These particles are released by cancer cells to prepare the surrounding fat tissue for the tumour. An inflammatory state is triggered in the stem cells found in the fat. Mitochondrial parts and DNA can be carried by these vesicles. When these components are transferred, the function of the receiving cells is changed. The stem cells can be caused to stop dividing by exposure to these particles. This state of senescence is linked to the activation of the AKT pathway. The contents of these vesicles can be changed by dietary substances. This may reduce their ability to cause inflammation. Paracrine regulation is a main part of how the cancer interacts with its surroundings.
A substance from green tea known as EGCG has been studied for its effects on this sequence. Paths are blocked. It can inhibit the activation of the Smad2 and NF-κB pathways. This prevents the stem cells in fat from becoming transformed. The contents of the vesicles released by cancer cells are also modified by EGCG. Vesicles are changed. The vesicles treated with this substance have fewer mitochondrial components. They are also less likely to cause inflammation or senescence in other cells. This suggests that certain items in the diet can interfere with the signals sent by the tumour. Research is ongoing. The use of such polyphenols is being investigated in laboratory research. It offers a way to block the support system of the cancer. By stopping the transformation of fat cells, the growth of the tumour might be slowed. This remains an active area of study.
Single-cell analysis methods allow for a detailed mapping of the different cells found in fat tissue. Cells are diverse. These techniques show that the fat cells in the tumour environment are not all the same. Several versions of transformed adipocytes have been found in different locations. Outcomes are affected. High levels of these specific subtypes are linked to worse outcomes for patients. Drugs are found. Bioinformatics tools have been used to find drugs that might target these cells. Trametinib and Ulixertinib are examples. It has also been shown by research that lowering certain receptors on cancer cells can stop them from growing. Survival is shortened. Patients with high levels of AdipoR2 often have shorter survival times. The creation of therapies that focus on exact cell groups is helped by this knowledge. A clearer picture of the environment around the tumour is offered.
