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Mesenchymal stem cells (MSCs) are multipotent cells known for their immunomodulatory properties [1,2]. They play a pivotal role in tissue remodelling, wound healing and homeostasis [3,4]. Since these cells form an integral part of the stroma and are intimately in contact with the tissue undergoing tumorigenesis, their role in cancer development is taking centre stage. MSCs exert their effects either by direct contact or by paracrine effects with immune cells, induce migration of suppressor cells in the tumour microenvironment and create an area of immune suppression. Bone marrow derived mesenchymal stem cells (BM-MSC) can be mobilized into circulation, migrate towards the tumour and aid in tumour progression [5,6]. Clinical studies indicate migration of BM-MSC towards tumours to be a marker of poor prognosis [7].

Wound healing properties of MSCs are well documented and solid tumours are now regarded as chronic wounds, i.e. wounds which never heal [8]. It is now becoming clear that tumour progression does not involve only the tumour cells, but more likely similar to a "soil and seed" hypothesis, wherein the "seed/tumour cell" strongly interacts with the tumour microenvironment, comprising of stromal cells, BM-MSC, adipocytes and endothelial cells, which secrete growth factors and a wide array of mediators to aid in tumour progression [9].

Transforming Growth Factor (TGFβ) is a pleiotropic cytokine and is over expressed in the tumour microenvironment and suppresses the immune system and also mediates tumour progression [10-12]. One of the main escape mechanisms of neoplasm is the recruitment of T_reg in the tumour microenvironment and subsequent increase in TGFβ levels which results in immune suppression [13]. Modulation of this immune suppression could lead to favourable response to cancer immunotherapy.

Autologous activated T cell based immunotherapy (ACT) involves infusion of (non-specifically activated) T cells into patients and demonstrated promise in several cancer conditions [14]. Effects of ACT on TGF β levels in the tumour microenvironment would help understand the outcomes of immune-therapy and treatment endpoints.

The study explores the interplay of HeLa cells and BM-MSC and Transforming Growth Factors Beta (TGF β) levels. Activated T cell supernatant derived from PHA activated T cells were also co-cultured to detect any change in TGF β levels.

Cell lines

HeLa cells (Human cervical cancer cells) were obtained from NCCS, Pune. The cells were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco). Human BM-MSCs were obtained from Lonza. BM-MSCs were maintained in DMEM supplemented with 20% FBS. BM-MSCs between passages 2-3 were used for the experiments. All cultures were incubated at 37˚C in 5% humidified CO₂ incubator.

Preparation of T cells conditioned medium

15 ml human blood was collected after venous puncture (standard blood bank procedures) in healthy subject, in EDTA vacutainer. Institutional ethical clearance was obtained under reference number EC/363/17/09 for the study and informed consents were given prior blood collection. Mononuclear cells were isolated by density gradient using Ficoll-Hypaque and were cultured in DMEM supplemented with 10% FBS with mitogen, Phytohemagglutin (PHA, Gibco). After 72 h, the supernatant was collected and centrifuged at 1500rpm for 10 mins. The supernatant (T sup) was collected and preserved at -80˚C until used.

Cell culture in different microenvironment

HeLa cells and BM-MSCs were cultured and co-cultured on a 3D scaffold (patent application number: 201841037897). After 24 h, the complete medium was changed according to respective groups, as shown in Table 1. Further the cells were incubated at 37˚C in 5% humidified CO2 incubator for 5 days and the supernatant was collected for cytokine analysis.

Table 1: Groups showing different culture conditions used for the experiment. Group (1) HeLa cells, (2) HeLa cells supplemented with activated T cell Supernatant, (3) Bone Marrow Mesenchymal Stem cells, (4) Bone Marrow Mesenchymal Stem cells supplemented with activated T cell supernatant, (5) HeLa cells co-cultured with Bone Marrow Mesenchymal Stem cells, (6) HeLa cells co-cultured with Bone Marrow Mesenchymal Stem cells supplemented with activated T cell Supernatant.

Cytokines detection

TGF-beta levels in all the groups were determined by ELISA (RayBio ELISA Kit).

Statistical analysis

All values were expressed as mean ± standard error mean (SEM). Differences of the parameters between patients and controls were analyzed by using unpaired Student’s -test. P value less than 0.05 were considered to be statistically significant.

Three independent experiments were performed and TGFβ cytokine levels were measured in cell supernatant cultured in different 3D microenvironment by ELISA. Concentration of TGFβ measured is summarized in Table 2. Unpaired student test was done on group 1 and 2, 3 and 4, 5 and 6. Significant decrease in TGFβ levels were observed in HeLa cells supplemented with T sup when compared to HeLa cells with p<0.05. Significant increase in TGFβ levels were seen in BM-MSCs cells supplemented with T sup when compared to BM-MSCs with p<0.05. No significant changes were seen in co-cultured groups i.e., HeLa + BM-MSC and HeLa + BM-MSC + T sup as shown in Figure 1.

Table 2: Summary of TGFβ levels (ng/mL) in different groups. Group (1) HeLa cells, (2) HeLa cells supplemented with activated T cell Supernatant, (3) Bone Marrow Mesenchymal Stem cells, (4) Bone Marrow Mesenchymal Stem cells supplemented with activated T cell supernatant, (5) HeLa cells co-cultured with Bone Marrow Mesenchymal Stem cells, (6) HeLa cells co-cultured with Bone Marrow Mesenchymal Stem cells supplemented with activated T cell Supernatant.

Figure 1: Graphical representation of TGFβ levels (ng/mL) in different groups. Group (1) HeLa cells, (2) HeLa cells supplemented with activated T cell Supernatant, (3) Bone Marrow Mesenchymal Stem cells (BM-MSC), (4) Bone Marrow Mesenchymal Stem cells (BM-MSC) supplemented with activated T cell supernatant (T sup), (5) HeLa cells co-cultured with Bone Marrow Mesenchymal Stem cells (BM-MSC), (6) HeLa cells co-cultured with Bone Marrow Mesenchymal Stem cells (BM-MSC) supplemented with activated T cell Supernatant (T sup).

The tumour microenvironment appears to be an important component and one of the key factors which determines the final outcome of disease [15,16]. Tumour stroma comprise of MSC, endothelial cells, adipocytes, myo-fibroblasts, Cancer Associated Fibroblasts (CAF), as well as immune cells like T cells, NK cells macrophages and neutrophils [17]. CAF has been pronounced as an important if not singular component to promote epithelialmesenchymal transition (EMT) of tumour cells, aids in tumour progression, induces aggressive phenotype of tumour cells promote metastasis and angiogenesis [18-21]. The EMT transition could be due to the paracrine effects of TGF β signalling [21]. In vitro studies with MSC and tumour conditioned medium induced differentiation of MSC into CAF via TGF β signalling [22,23]. Due to their inherent plastic nature, fusion with tumour cells or their trans-differentiation due to soluble factors associated with the tumour, results in remodelling of tumour microenvironment leading to a pro tumourigenic stroma, as seen in lung cancer [24], human melanoma [25], breast cancer and ovarian adenocarcinoma cells [26]. These studies suggest a major role for MSC towards promoting a pro tumoural stroma. TGF β mediated immune suppression mediates a chronic inflammatory microenvironment encouraging other suppressor cells like MDSC to migrate in the region [27].

These results are contrary to previous studies by Ping-Jin [28], where they showed a polarization of the stroma towards a Th1 type phenotype on co culturing with TNFα and IFNγ, which are produced by activated TILs. Th1 polarization of tumour microenvironment is conducive to adoptive T cell therapy [29], vaccination [30] and immune checkpoint inhibitors [31,32]. A strong Th1 response is critical to induce tumour rejection, following immunotherapy [31,33-37]. Our study demonstrated no reduction of TGFβ levels in HeLa- BM MSC co culture group, implying involvement of additional factors not solely dependent on cytokine circuitry, within the tumour environment. Most importantly, it suggests that the optimal period for adoptive T cell immune-therapy could be preferably after surgical resection, as surgery results in massive reduction tumour mass and significant reduction in TGFβ levels in the tumour microenvironment.

Further studies to explore modalities to mitigate the TGFβ mediated immunosuppressive tumour microenvironment will be conducive to cellular immune-therapies. Also, elucidation of MSC in the tumour microenvironment and analysis of CAF associated molecular markers will help understand the implications of TGFβ induced tumour remodelling, cancer progression and metastasis. Analysis of Th1 versus Th2 cytokine profiles will help elucidate the larger picture at play in terms of our existing TGFβ data.