Perspective Chapter: Investigating Cancer Tumor Microenvironment In Vitro – Co-Culture Studies on Adipocytes and Cancer Cells

Abstract

The tumor microenvironment increases the growth and invasion of cancer cells, makes classical chemotherapy applications inadequate, and is associated with a poor cancer prognosis. Recent studies reveal that cancer stroma supports tumor growth and metastasis and develops resistance to chemotherapy. In vitro co-culture techniques are widely used to study cross-talk between tumor microenvironment cells such as adipocytes, endothelial cells, fibroblasts, macrophages, and cancer cells. Co-culture techniques are classified into two main categories: indirect and direct methods. Transwell (indirect) co-culture of mature adipocytes with cancer cells has shown cancer cell viability, growth, proliferation, invasion, and metastases. This chapter covers the general methods of co-culture studies and will emphasize the results obtained on the co-culture of adipocytes and cancer cells.

1. Introduction

Cancer is a multidisciplinary disease that occurs when the balance between cell proliferation and death is disrupted in the direction of proliferation. After excessive proliferation, tumor mass invades vessels and metastasizes to adjacent tissues [1]. In other words, a tumor colony is a group of cells that have gained a higher proliferation rate and can invade other tissues and metastasis [2]. Due to its high metastasis capacity, although it changes according to the type, the mortality rate is high in many cases [3]. Cancer is named according to the tissue or organ it originates from. Indeed, this terminology is insufficient most of the time, and each cancer type is also subdivided in compliance with molecular marker, histological staining status, and cell type. About 200 cancer subtypes have been identified by the National Cancer Institute (NCI), but the exact number of subtypes is still a mystery. Subtyping in cancer cases is essential because the subtype affects the therapy model [4]. The accumulation of genetic and environmental risk factors triggers the initiation of carcinogenesis. Although there are cancer-type-specific markers, KRAS, BRAF, PIK3CA, epithelial growth factor receptor (EGFR), p53, and c-myc are the most common mutated genes in various cancers [5].

Surgery supported by chemotherapy is still the primary treatment method for solid tumors. Still, the adverse effects of chemotherapy reduce the life quality of patients because of the toxicity of drugs to healthy cells and the tumor cells. Radiation therapy and immunotherapy can also be used for treatment. The main drawback of developing new therapies with minimal adverse effects is the intra- and peri-tumor heterogeneity [3]. The tumor contains highly proliferating cancerous cells and is surrounded by different types of cells and tissues, defined as a tumor microenvironment (TME). Recent studies concerning tumor biology showed that TME has a significant role in drug resistance, tumor development, and malignancy [6]. Intra-tumor heterogeneity also affects the classification and subtyping of tumors. In some cases, intra-tumor solid heterogeneity complicates the type and prevents identifying the case, whether a known subtype or a new one [4]. Therefore, it is essential to define the tumor microenvironment to assess the subtype, malignancy, and metastasis profile of a tumor and study in cultures for new therapeutic approaches.

This chapter summarizes the important concepts of TME and potential applications in cell culture. As TME is quite complex, this chapter focuses on the studies of TME in adipocytes and cancer cells. This chapter summarizes the components of TME and the developing cell culture applications that show great potential to model various cancers. We also summarize the co-culture applications in cancer research. This chapter also provides further insights into novel cell-culture-based TME studies.

2. Cellular and non-cellular components of tumor microenvironment (TME)

TME, defined as the environment of a tumor that tumor cells interact with, is composed of cellular and noncellular compartments [7]. The cellular compartment contains mainly fibroblasts, endothelial cells, adipocytes, stem cells, stellate cells, and the innate and adaptive immune cells such as B lymphocytes, T lymphocytes, macrophages, dendritic cells, natural killer (NK) cells, neutrophils, and myeloid-derived suppressor cells (MDSC) (Figure 1) [8]. These cells are laid on a scaffold formed by an extracellular matrix (ECM), which includes proteins, proteoglycans, glycoproteins, and chemicals secreted by cellular compartments such as growth factors, cytokines, and chemokines [9]. Tumor cells interact with other cellular components, and the extracellular matrix provides a structure to hold all these elements together. Interactions between cellular immune cells and non-cellular components account for the tumor heterogeneity and complexity of TME and limitations for targeted therapies in patients [10]. As a result of this interaction, the cellular environment also shows some genetic and morphological changes. It causes conditions supporting the tumor’s development, contributing to metastasis and drug resistance [11]. Due to its contribution to tumor development, metastasis, and drug resistance, the tumor microenvironment is an important topic that has been popular recently by researchers and offers a new perspective on cancer treatment [8].

Figure 1.

Illustration of cellular and non-cellular of the tumor microenvironment.

It was first discovered in the 1890s when tumor and tumor microenvironment interacted, emerging with the theory “the seeds and the soil” of English surgeon Stephen Paget [12]. This theory that metastasis depends on communication between certain cancer cells as the “seeds” and specific tissue microenvironments as the “soil” still retains its thought [13]. In further studies, it has become important to investigate the role of TME-associated cellular responses in carcinogenesis. Dysregulated immune responses are the main characteristics of inflammatory tissue microenvironment. TME exhibits an increase in inflammatory regulators such as tumor-infiltrating cells, T cells, macrophages, dendritic cells, and natural killer cells that are the cause of chronic inflammation to promote tumor progression, carcinogenesis, and metastasis [14]. A suppressed immune system, defined as immunosuppression, is one of the potential adverse effects of several cancers.

2.1 Cellular components of tumor microenvironment

2.1.1 Fibroblasts

Cells of connective tissue that are neither epithelial nor endothelial nor immune cells are referred to as fibroblasts [15]. Fibroblasts are substantial cells involved in extracellular matrix formation by synthesizing collagen, laminin, and fibronectin and thus contribute significantly to wound healing [16]. Resident fibroblasts, resident endothelial cells, and epithelial cells can be activated in tumorigenesis and differentiate into cancer-associated fibroblasts (CAFs) in response to growth factors and cytokines. Cancer-associated fibroblasts are the primary cell type in the stromal part of the tumor microenvironment [17]. These differentiated fibroblasts contribute to inflammation, angiogenesis, TME remodeling, epithelial-mesenchymal transition (EMT), metastasis and anti-drug resistance via producing matrix metalloproteinases (MMPs), growth factors (TGF-β, IGF, HGF, VEGF), cytokines and chemokines (IL-6, CCL7, CXCL2) and inhibiting natural killer cells, and cytotoxic T lymphocytes [9, 16, 18, 19]. Therefore, they promote tumor development and progression and are shown to be related to poor prognosis in pancreatic and breast cancers [17].

2.1.2 Endothelial cells

Endothelial cells are the chief cells that line the blood vessels, forming a cellular layer between the blood and the tissue, which organizes the interchange of materials from each side. Endothelial cells differentiate into tumor endothelial cells (TECs) in tumors that organize and play critical roles in immune system regulation, tumor cell proliferation, invasion, and metastasis by expression of inhibitory receptors for immune cells, vascular cell adhesion molecule 1 (VCAM1), and Notch1 signaling [10, 17].

2.1.3 Adipocytes

Adipocytes convert glucose and fatty acid to triglycerides in a mechanism called lipogenesis and, in this way, provide the energy demand of other cells [20]. In the tumor microenvironment, cancer-associated adipocytes (CAAs) are likewise energy storage cells [21] by supplying lipids to tumorigenic cells [10]. Apart from lipid and energy supply, they release various metabolites (fatty acids), hormones (leptin, adiponectin), proteins (collagen, VEGF, and matrix metalloproteinases), inflammatory factors (CCL2), and cytokines (IL-8), which are referred to as adipokines [9, 22, 23]. Through these adipokines, they regulate macrophage differentiation [9], immune cell homeostasis [23], immune evasion and tumor progression [9, 22], ECM remodeling [22], metastasis [24], and angiogenesis via increasing vascularization [25]. Many cancer types, such as breast, ovarian, colon, and prostate cancer, are primarily known for high adipocyte concentration; therefore, it is important to understand the interactions of the cells in TME with adipocytes to improve the therapeutics [26].

2.1.4 B lymphocytes

Although B lymphocytes’ primary function is antibody secretion upon antigen recognition, in the tumor microenvironment, they activate mast cells and promote the production of cytokines when they interact with the antigens of tumor cells [27]. B lymphocytes have mainly antitumor functions, whereas when specific chemokines (CXCL13) and cytokines (IL-10) are present, they promote tumorigenesis and induce angiogenesis [9]. Thus, the role and function of B lymphocytes in the tumor microenvironment differ in accordance with the chemical secretions in the environment and tumor type, which affects the tumor’s prognosis status [28].

2.1.5 T lymphocytes

T lymphocytes constitute one of the immune system’s main groups of adaptive immune response. T lymphocytes have many subgroups, of which CD8⁺ cytotoxic T lymphocytes (T_c) are responsible for the detection of tumor antigens and for destroying the tumor cells [18]. In the tumor microenvironment, as well as T_c lymphocytes, CD4⁺ T helper lymphocytes (T_h) also play essential promoting roles as both stimulation of T_c lymphocytes and B lymphocytes via secreting various cytokines [9]. On the contrary, another subgroup of T helper lymphocytes, T_h17 lymphocytes, exerts double-sided effects. T_h17 lymphocytes trigger inflammation and angiogenesis on the one hand and have anti-tumorigenic effects on the other [29].

The common immunosuppressor T cells, regulatory T cells (T_regs), are typically identified near tumors and suppress anticancer immune responses [30]. In normal cells, T_regs sustain self-tolerance mechanisms by preventing the proliferation of T lymphocytes and induction of immunity against self-antigens [31]. Studies indicated that the FOXP3⁺CD4⁺CD25⁺ subtype of T_reg cells is instrumental in controlling immune responses in the tumor microenvironment. Tumor cells and macrophages in the tumor microenvironment secrete CCL22 chemokine, whose receptor, CCR4, was expressed by T_reg cells and causes the accumulation of T_reg cells in TME [30]. T_regs contribute to immunosuppression by secreting immune inhibitory cytokines such as transforming growth factor (TGF-β) and interleukin 10 (IL-10), which inhibit the anti-tumor activity of killer cells [27].

2.1.6 Macrophages

Macrophages are the phagocytic cells in the innate immune system [32]. However, when recruited to the tumor microenvironment with certain cytokines or differentiated from circulating monocytes, they can acquire different phenotypes and are referred to as tumor-associated macrophages (TAMs) after that [17]. These different phenotypes are known as M1 anti-tumor macrophages and M2 pro-tumor macrophages, which are determined by the polarization status of the TAMs [9]. TAMs, particularly the M2 phenotype, are the cells that are the most infiltrated immune system cells in TME [33]. Besides, since they secrete extracellular matrix components, they participate in ECM remodeling [19].

2.1.7 Dendritic cells

Dendritic cells are the antigen-presenting cells in the immune system that detect foreign antigens, present them to T lymphocytes and induce adaptive immune response [34]. In the tumor microenvironment, different subtypes of dendritic cells can be infiltrated. Classical dendritic cells and plasmacytoid dendritic cells are responsible for anti-tumorigenic response. On the other hand, tumor cells secrete CCL-2, CXCL1, and CXCL5, thus preventing dendritic cell maturation [9]. Further, dendritic cells promote T_reg cell differentiation by secreting cytokines [10]. In this manner, dendritic cells’ function depends on the chemicals secreted in TME [22].

2.1.8 Natural killer (NK) cells

Natural killer (NK) cells are the primary defense cells of the immune system against tumor cells [35]. When they encounter tumor cells in the bloodstream, natural killer cells essentially destroy them, whereas they cannot function effectively in the tumor microenvironment [22]. Although the dendritic cells recruit natural killer cells to the tumor microenvironment through various chemokines such as IFN-γ, they cannot correctly mature [9].

2.1.9 Neutrophils

Neutrophils, widespread in circulation [35], are the cells of the innate immune system responsible for the phagocytosis of dead cells or pathogens and for presenting antigens to adaptive immune system cells [9, 36]. In the case of tumorigenesis, neutrophils are recruited to the tumor site. In the early stages of tumorigenesis, these neutrophils are differentiated into the anti-tumor N1 subtype under the influence of IFN-γ [37, 38]. N1 subtype neutrophils release cytokines and reactive oxygen species, thus inducing apoptotic death of tumor cells [22]. In later stages of tumorigenesis, cancer-associated fibroblasts (CAFs) secrete TGF-β that promotes the differentiation of N2 subtypes and suppression of N1 subtype [28]. These N2 neutrophils secrete cytokines as IL-1β, growth factors as vascular endothelial growth factor (VEGF), and matrix metalloproteinases as MMP-9, in this way, stimulate inflammation, angiogenesis, matrix remodeling, and invasion [22, 28, 37].

2.1.10 Myeloid-derived suppressor cells (MDSCs)

MDSCs secrete cytokines and growth factors; in this way, they suppress immune cells and induce tumor cell dissemination and renewal of tumor cells [27]. They originate from myeloid progenitor cells in the bone marrow and then migrate to the tumor microenvironment to support tumorigenesis, angiogenesis, and metastasis [9]. In TME, they also differentiate into tumor-associated macrophages (TAMs), which act together responsible for resistance to anti-angiogenic treatments [17].

2.2 Non-cellular components of tumor microenvironment

Non-cellular components of the tumor microenvironment involve mainly the extracellular matrix (ECM), which is different from the matrix of normal tissue in that it enables the neoplastic cells to metastasize. Collagen and fibronectin are the main components of ECM in solid tumors and ensure the endurance of the matrix [17]. Factors secreted from cellular components of the tumor microenvironment, such as cytokines, chemokines, proteases, growth factors, polysaccharides, and integrins, are also significant members, and these components provide the communication and interaction of cells with each other in TME [39, 40]. The importance of non-cellular components of the tumor microenvironment is that they organize not only the scaffold where the tumor mass resides but also regulate the polarization and the fate of the cellular components [41]. Non-cellular components of ECM also participate in drug resistance by providing a dense environment and disallowing the drugs to penetrate [17].

To summarize, the cancer tumor microenvironment is characterized by abundant stroma, extracellular matrix, stellate cells, cancer-associated fibroblasts, various immune cells, and cytokines are associated with a hypoxic environment, high vascularity and intense immunosuppression, proliferation, metastasis, and drug resistance of several cancers [36]. Various studies explained the direct interaction between tumor cells and immune cells, but the relationship between immune cells and TME needs to be clarified in the cell fate decision.

3. Cell culture applications studying TME

To improve personalized therapy and the prognosis of cancer, extensive and varied investigations are performed from experimental to clinical applications. However, the complexity and heterogeneity of TME contribute to cancer development, progression, and drug resistance. Thus, developing cell culture applications is required to enlighten the interaction network of TME and cancer cells.

Since it was first introduced in 1907 for nerve fibers [42], cell culture techniques have been eased in cancer research to mimic the in vivo evolvement and reaction of tumors in vitro [43]. Classical two-dimensional (2D) cell cultures allow the cell to grow only in two dimensions; in other words, cells can only grow along the surface of the culture flask (Figure 2) [44]. These cultures may be conducted in adherent cultures and suspension cultures, of which the latter imitates the non-adhesive cells, such as hematologic malignancies [45], to resemble the natural environment in blood or lymph [43]. In adherent cultures, cells are attached to the plastic surface of flasks or dishes as a single layer and grow, interacting with the surface’s neighboring cells and protein coating. These cultures provide a cost-effective, high-performance, and reproducible way to study in vitro [46]. For many years, these cultures have been used to investigate the cytotoxicity of drugs in pharmacology studies, the migration and invasion characteristics of the cells in understanding the underlying mechanisms, the genome modifications in enlightening the genetic basis of cancer cells, and so on [47]. However, studying just one cell type in a 2D cell culture is nowhere near the interactions between cellular and non-cellular components of the tumor microenvironment [48]. Therefore, new techniques and applications must be developed to mimic the in vivo tumor microenvironment better.

Figure 2.

Comparison of 2D vs. 3D cell culture.

3.1 In vitro co-culture applications for TME and cancer cell interactions

The co-culture method is the direct or indirect interaction of multiple cell types in an identical culture environment [49]. Through the interaction of various cell types, co-culture systems regulate the properties of individual cells. Cellular mechanisms, including paracrine signaling, cell-to-cell communication, and modeling epithelial-mesenchymal transition (EMT), are better estimated by co-culturing numerous cell lines into a single three-dimensional (3D) model [50].

Two types of cells are defined as “target cells” and “assisting cells” responsible for the co-culture environment. Target cells act to build up the engineered tissue and are responsible for the mechanical and metabolical functions of the tissue. Target cells are guided by assisting cells to exhibit various desired characteristics such as proliferation, differentiation, matrix production, or secretion [49].

Co-culture aims to form tissue-like characters of cells in vitro through cell–cell communication of multiple cell types in direct or indirect interaction. Co-cultures are used to explore three cell-to-cell communication patterns: cell–cell adhesion, including adherence, gap and tight junctions, cell-ECM adhesion, and paracrine signaling via soluble factors.

Co-culture techniques have been essential for researching the interaction between different cell types under diverse conditions, such as cancer cell growth, differentiation, proliferation, and metastasis. The complexity of the in vivo environment, where several cells interact and signal concurrently, is not fully mimicked by one cell type-based in vitro cell cultures due to inadequate prediction of signaling cascades. Therefore, several co-culture models have been improved to mimic TME, which are less expensive than in vivo animal models.

3.1.1 Direct co-cultures

Direct co-cultures allow two or more different cells to make physical connections with each other while mixing in the 2D or 3D cell culture environment. Mixed populations of monolayer cells on slides or in dishes are standard 2D cell culture methods [51].

Due to their simplicity and ease of control, 2D culture systems are acceptable as the easiest way to investigate cellular interactions, such as examining adhesion molecules, cytokine production, and juxtracrine signals. However, the composition of native tissues can be modeled using 3D culture systems, which is accomplished by culturing multiple cells in materials such as fibrin, agarose, collagen, or alginate [49].

3.1.2 Indirect co-cultures

Additionally, conditioned medium is widely employed in indirect co-cultures. Multiple cell types are separated through good inserts like Transwell or Boyden chamber permeable membranes or pre-conditioned media. Utilizing permeable membrane inserts allows secreted soluble factors to pass through the membrane, only enabling signaling via the cell secretome. Alternatively, utilizing previously created conditioned media from one of the co-culture cell types are used [52].

Indirect 2D co-culture systems allow a better explanation of specific cellular functions and features that occur specifically to cell types. However, in indirect 3D co-culture systems, multiple cell types are separated using hydrogel or collagen matrix, which allows better mimicking of native tissue structures and eliminates the requirement for direct cell-to-cell contact between cell types [49].

Transwell plates [53, 54], microfluidic platforms [55, 56, 57, 58], or solid supports like Petri dishes [59, 60], 3D scaffolds [61], hydrogels [62], or microarrays [63] are often utilized choices for co-culturing populations that are somewhat separated from one another (Figure 3). For co-cultures, many custom microfluidic devices have been developed [65, 66, 67, 68, 69]. The possibility for high throughput in microfluidics is one of its key benefits. Microfluidic setups might not represent larger volumes as complexity rises [56], and this benefit may be undermined. Techniques used in monoculture modified for co-culture include cell movement experiments. Most traditional laboratory cultivation focuses on maintaining pure cultures of certain species [65, 70]. Although there have been recent developments that could be further utilized, such as microcarrier beads, the micro-Petri dish [71], diffusion chambers, dialysis reactors, and other techniques [70, 72], the cultivation of monocultures on agar plates and in liquid medium has always followed the same paradigm [71, 72].

Figure 3.

Available technologies for co-culture [64].

Cell-to-cell direct co-cultures have some limitations and need more optimization steps. Therefore, indirect co-cultures are preferred as they provide reproducible results. However, indirect co-cultures cannot capture cell–cell adhesion, integrin, and notch-type signaling.

3.2 Adipocytes-related cancer studies

Since TME has become widely recognized as a significant contributor to tumor progression and cancer, it has gained importance to determine the roles of individual TME components in cancer. Adipocytes, endothelial cells, fibroblasts, macrophages, and muscle cells are cell types commonly studied in co-culture systems [73]. Adipocytes were subsequently recognized as a significant TME component in tumor development, angiogenesis, and metastasis through the secretion of soluble factors [74]. Adipocytes were once considered the body’s energy reserves, but today, it is recognized that they have a role in various pathological processes, such as inflammation. In particular, they impact gene expression and cell motility in TME [75].

Mammalian epithelial cells are surrounded by an extracellular environment comprising adipocytes and other stromal cells, including fibroblasts, and endothelial and inflammatory cells. Adipocytes and adipocyte precursor cells are a substantial percentage of breast TME, which may play a significant role in the interaction between stromal and ductal epithelial cells and function in the secretion of growth factors, hormones, and adipokines [76].

According to the studies in cancer metastasis, it was determined that free fatty acids (FFAs) released from adipocytes through lipolysis may have been an energy source for cancer cells through mitochondrial fatty acid oxidation (FAO) [77]. In culture conditions, FFAs from adipocytes to cancer cells enhance FAO and support tumor cell proliferation, migration, and invasion. Elevating lipid load in adipocytes increases FFA transfer and is stored in tumor cells with lipid droplets, which is also associated with enhancing CAA-mediated proliferative effects in the TME [78].

In the first studies investigating the role of TME in breast cancer, the role of fibroblasts, macrophages, and other inflammatory cells was investigated. Still, the role of adipocytes was not recognized. Further studies in breast cancer have shown that the adipocyte-rich environment can promote cancer proliferation. It was shown that murine mammary carcinoma SP-1 cells injected subcutaneously into adipose-rich tissues in mice promoted tumor growth and metastasis. Still, neither growth nor metastasis is observed in tissues with low levels of adipose. These findings were reinforced by the discovery that estrogen regulates the adipocyte-associated growth and proliferation of SP-1 cells [79, 80].

It has been determined by DNA microarray studies that adipokines secreted from adipocytes regulate cell survival, proliferation, invasion, and angiogenesis by inducing IGF2, FOS, JUN, cyclin D1, MMP1, ATF3, and NFkβ transcriptional target molecules. In addition, the induction of these genes was specific to factors released by adipocytes and cannot be observed in other breast stromal cells [74].

Previous studies showed that co-culture of differentiated murine preadipocytes with mammary tumor cells caused the delipidation of adipocytes with the increase of proinflammatory cytokines and matrix remodeling proteins while decreasing adipocyte differentiation markers, including resistin, adiponectin, and hormone-sensitive lipase (HSL). These adipocyte cells, triggered by tumor cells and changed phenotypically and functionally, are considered cancer-associated adipocytes (CAA) [81]. In addition, bidirectional contact between adipocytes and tumor cells changes the shape of adipocytes into adipocyte-derived fibroblasts, which are the parts of the CAAs and induce invasion, migration, and metastasis [82]. Studies have shown that interleukins produced by CAAs such as IL6, IL1β, TNFα, and MMP-11 are functionally responsible for supporting the invasion capacity of tumor cells and augmenting the proinflammatory TME properties. It was shown that pancreatic cancer cells linked to obesity release cytokines such as IL1β, which promotes infiltration of immune cells and leads to cancer progression and drug resistance [83]. Numerous pro-inflammatory cytokines, such as tumor necrosis factor alfa (TNF-α) and plasminogen activator inhibitor-1 (PAI-1), are produced by adipocytes and overexpressed in obese patients [84].

The methods by which stromal cells in TME alter tumor properties in different microenvironments are initiated to be clarified by several direct or indirect studies. Stromal cell-secreted factors such as matrix metalloproteases (MMPs) and growth factors regulate the cancer cell decision. As mentioned in previous studies, the interaction between adipocytes and cancer cells causes the change in the phenotype of adipocytes into CAA and delipidation with the overexpression of inflammatory cytokines but increases in the adipocyte differentiation markers [81]. Secretion of inflammatory cytokines such as IL-6, IL1β, or leptin promotes cancer development and metastasis in several cancer cells.

Even if the interaction between adipocytes and cancer cells is known, these cell–cell interactions are complicated, and it is also known that they cannot occur by the effect of a single secreted molecule or a signal. Therefore, molecular mechanisms of heterotypic signaling in bidirectional crosstalk between adipocytes and cancer cells are frequently investigated with in vitro and in vivo studies to enlighten carcinogenesis and metastasis [76]. Adipocytes and breast cancer cells are immediately near one another due to invasive breast tumors that penetrate the basement membrane and infiltrate fibrous tissue barriers, enabling paracrine interaction between two cell types [85].

To understand how adipocytes affected the motility of epithelial and cancer cells and the role of adipocytes on the cancer-related gene expressions on co-culture systems in TME, adipocyte-conditioned media treated MCF-10A mammary epithelial cells and invasive MCF-10CA1 carcinoma cells were used. The first step of the study was the differentiation of primary human breast preadipocytes into lipid-accumulating mature adipocytes. Preadipocytes were cultured in a differentiation medium for seven days to do this experiment. After differentiation, adipocytes were maintained in DMEM-F12 containing 1% (penicillin–streptomycin fungizone) PSF, 10% (fetal bovine serum) FBS, and five ng/ml insulin for a week. To compare the role of TME in normal and cancer cell motility, cells were treated with adipocyte-conditioned media. For the formation of conditioned medium (CM), preadipocytes were serum-starved in DMEM: F12 containing 1%PSF for 24 h. Meanwhile, MCF-10A and MCF-10CA1 cells were serum-starved overnight and treated with CM from preadipocytes and mature adipocytes for 24 h. It was found that mature adipocyte-conditioned media increases cancer cell motility and migration. In addition, mature adipocyte-CM increases phosphorylated-Akt levels in MCF-10CA cells. However, angiogenesis (fibroblast growth factor receptor-2, interferon-a, and platelet-derived growth factor β-peptide) and matrix metalloproteinases (MMP-1, −2, and − 9) genes were downregulated in adipocyte CM-treated MCF-10A cells, in adipocyte-CM treated MCF-10ACA1 cancer cells, PAI-1, TGF-B-1 expression upregulated but MMP1 and angiopoietin-1 were downregulated. These data confirmed the positive effect of adipocytes on cell survival, proliferation, migration, and tumorigenesis in breast cancer (Figure 4) [86].

Figure 4.

A workflow example in adipocytes-related cancer studies.

The dysregulation of adipokine synthesis and secretion is a significant risk factor for obesity, and Type 2 diabetes mellitus (T2DM) results from obesity. Given that T2DM is caused by insulin resistance, insulin promotes adipokine production, leading to cancer development [87]. Considering T2DM is related to cancer, metformin is used as an oral antidiabetic drug explaining the role of adipocytes in inflammation-associated cancer development. To find out how adipokine production is associated with obesity and T2DM, the molecular mechanism in insulin resistance and breast cancer development needs to be clarified. Similar to other studies, preadipocytes were differentiated for 14 days, and after differentiation, adipocyte cells were propagated in low glucose (5 mM) conditions for 48 h. Then, MCF-7 cells were treated with preadipocyte and adipocyte-CM for 72 h. It was found that adipocyte-derived CM significantly induced MCF-7 cell proliferation that preadipocyte-derived CM did not. In addition, IL-6 expression was increased in differentiated mature adipocytes, which are downregulated by metformin. Findings suggested that conditioned media from adipocytes increase cell proliferation and pro-inflammatory adipokine expression, which are decreased by metformin treatment (Figure 4) [88].

Utilizing direct or indirect 2D co-culture, in which tumor and stromal cells are cultured together on a flat, has begun to be inadequate for explaining the complex intercellular interactions between heterogeneous cancer, immune and stromal cells, and between cells and ECM [89]. However, compared to 2D culture, 3D culture is the most commonly effective method for constructing tissue architecture lacking monolayer cultures and better recapitulating the effect of TME. The 3D cell culture enables the investigation of cell function, gene expression, and paracrine signaling effects.

Organoids are one of the improved in vitro tissue engineering and 3D-cell culture applications that represent the structure and function of the organ in vivo, such as a population of self-organizing stem cells developing into specific complex organs [90]. Organoids can be produced through cell signaling pathways regulating self-organization and differentiation of embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and adult stem cells (ASCs) for the establishment of complex organs. For successful organoid formation and growth, improved cell culture conditions activate cellular signaling pathways triggered by intrinsic factors and extrinsic mediators, such as extracellular matrix (ECM), to maintain stem cell function [91]. Since the cancer organoids have the same characteristics and genetic background as the original tumors, co-culture cancer organoid models have been sufficiently developed to mimic the heterogeneous environment in vitro to investigate the complex interaction network of cellular and non-cellular components in TME. However, maintaining different cancer organoids requires cancer-specific procedures and methods.

In recent years, many studies have been conducted on organoids for cancer research. In such a study, biopsies of metastatic lesions of colorectal cancer patients were collected, and patient-derived organoids (PDOs) were generated in cultures. These PDOs were tested to consider the sensitivity of patients to chemotherapy and observed that PDOs can be used to foresee which patients bearing metastatic colorectal cancer are insensitive to irinotecan-based chemotherapy [92]. In another study, organoid models were formed from human breast epithelial samples and used to understand the molecular mechanisms underlying breast cancer oncogenesis. Four breast cancer-associated tumor suppressor genes were knocked out by CRISPR-Cas9 in mammary progenitor cells, and observed that at least three tumor suppressor genes must be blocked for breast tumorigenesis [93]. Wang et al. carried out a study with PDOs from hepatocellular carcinoma (HCC) patients to demonstrate the relationship between the presence of CD44 surface marker and resistance to sorafenib treatment in HCC patients. They also indicated that blocking Hedgehog signaling can reverse this sorafenib resistance [94]. Another study used PDOs generated from colorectal cancer patients for genomic and transcriptomic analysis to assess multi-drug response. It is indicated that these PDOs can be used to predict the response of the patient to drug treatment and develop personalized therapies [95].

The method is frequently initiated by mechanical or enzymatic digestion of tumor tissues obtained from the minimum necrotic margins into pieces about 1 mm in diameter. Following the breaking down of tumor samples, it is required to seed the cells with supplemented media onto a 3D Matrigel scaffold matrix, which consists of ECM proteins such as laminin, entactin, proteoglycan, and collagen IV and can be improved by tumor-specific growth factors such as Wnt3A, transforming growth factor beta (TGF-B) receptor inhibitor, and Noggin of epidermal growth factor (EGF) [96]. Organoid cells are co-cultured with TME components through this process.

4. Conclusion

Identifying the complex network between stromal cells and cancer cells in the TME is a potential hallmark to enlighten cancer progression and metastasis. This chapter summarizes the role of TME in various cancers by describing current co-culture cell culture applications.

The critical ideas of TME and its possible uses in cell culture are outlined in this chapter. This chapter concentrates on the research on TME in adipocytes and cancer cells since TME is highly complicated. The components of TME and emerging cell culture applications that have a great deal of promise to represent different malignancies are outlined in this chapter. We also provide a summary of co-culture’s uses in cancer research. Additionally, this chapter offers more information on cutting-edge TME investigations based on cell culture.

Acknowledgments

We acknowledge İrem Nur Ateş and Deniz Turan for their valuable contribution to constructing this chapter.

Conflict of interest

The authors declare no conflict of interest.