Introduction
Tumors are highly complex tissues consisting of malignant cells besides a variety of stromal cell populations, all of which act in concert to propel tumor growth and metastasis. Tumor-infiltrating macrophages (TIMs) are recruited from a circulating pool of monocytes and once present in the vicinity of the tumor cells are re-educated for a phenotype promoting tumor growth and metastasis and are accordingly, referred to as Tumor-associated macrophages (TAMs). TAMs constitute the predominant stromal cells populating solid tumors and support tumor growth and facilitate metastatic dissemination [
1,
2]. There are considerable clinical studies that demonstrate a strong correlation between a high population of TAMs and poor prognosis or survival in lung, breast, ovarian, and cervical cancers [
3‐
6]. However, the molecular mechanisms by which tumor cells interact with macrophages and re-educate them for a phenotype beneficial for tumor progression and metastasis remains poorly defined. A symbiotic relationship between tumor cells and TAMs has been proposed wherein tumor derived molecules attract TAMs, promote their survival and in reciprocation receive signals and factors that promote tumor progression and metastasis [
7].
Secretory proteins have long been studied to act as a source of communication among various cell types in tumor microenvironment [
8]. Considering the key role of such proteins in mediating intercellular interactions, it can therefore be envisaged that the secretory proteins establish a diabolic liaison between tumor cells and TAMs, leading to monocyte recruitment, their subsequent polarization and consequent tumor progression. Analysis of the secretome, therefore offers an attractive approach to elucidate the mechanism driving interactions between cancer cells and the TAMs. Although a few studies have been conducted to screen the cancer cell secreted factors that activate macrophages [
9], none of the studies have comprehensively carried out the secretome profiling of human cancer cell-monocyte microenvironmnent. In this context, the present study was designed and a SILAC based quantitative proteomic approach was adopted to identify these secretory proteins in tumor microenvironment simulating co-culture models involving model human lung adenocarcinoma cell line A549 and monocytic cell line, THP-1. We demonstrated that the interaction between lung carcinoma cells and monocytes is a two-way process and involves cancer cell-derived proteins that are capable of modulating the monocytes to generate a milieu congenial for metastatic growth in vitro.
Materials and methods
Cell culture
Human lung adenocarcinoma cell line, A549 and human monocytic leukemia cell line, THP-1 used in the study were purchased from National Centre for Cell Science, Pune, India. Cell line authentication at the repository was carried out by short tandem repeat (STR) profiling. A549 and THP-1 cells were maintained in DMEM and RPMI‐1640 respectively supplemented with 10% FBS and 100 U/ml penicillin. Cells were cultured under 5% CO2 and 37 °C temperature [
10].
Cytokine quantification
TNF-α, IL-6 and IL-10 levels were quantified from culture supernatants using commercially available kits following manufacturer’s instructions. Briefly, a 96-well microplate (Maxisorp, Genetix) was coated with 50 µl capture antibody (diluted 1: 250 in 100mM carbonate buffer, pH 9.5) and kept overnight at 4 °C. Then plate was washed 3 times with PBS-Tween (PBST) and blocked with PBS-BSA-1% (100 µl/well) for 1 h at 37 °C. After washing, samples were added to each well and the plate was incubated for 1 h at 37 °C. Subsequently, the plate was washed and incubated with detection reagent mix (detection antibody + avidin-HRP) diluted 1: 250 in PBS-BSA 1%. After 1 h of incubation, the plate was washed and the enzyme activity was determined by adding freshly prepared substrate solution containing TMB/TBABH/H2O2 (50 µl/well). The reaction was stopped with 50 µl of 2 N H2SO4 and the absorbance was read at 450 nm (or as advised in the manufacturer’s instructions). All the assays were performed in triplicate.
Migration assay
A549 cells were plated in 30 mm dishes and grown upto 90% confluency in DMEM supplemented with 10% FBS. The media was then removed and the monolayer was scratched with a 200 ml pipette tip, washed twice with PBS to remove detached cells and photographed (t = 0). Cells were then incubated in co-culture conditioned medium or homotypic conditioned medium for 24 h. Then the wounds were observed and photographed (t = 24). The assay was repeated three independent times. The percentage of wound closure was estimated as reported in [
11].
Invasion assay
The polycarbonate filter inserts (8 mm pore size, Corning) precoated with Matrigel were pre-incubated in DMEM supplemented with 1% FBS for 2 h before the cells were plated. A549 cells (5 × 104) in 200 µl of co-culture conditioned medium or homotypic conditioned medium were seeded in the upper chamber. Then 500 µl DMEM supplemented with 10% FBS was added to the lower chamber as a chemotactic agent. After 24 h incubation, non-migrating cells in the upper chamber of the filters were removed using cotton swabs. The cells that migrated and adhered to the other side of the filter were fixed in 3.7% formaldehyde for 20 min, stained with crystal violet and counted per five fields.
Anchorage independent growth in soft agar was used to assess the tumorigenic potential imparted by secretory Fibronectin and its associated EDA in vitro. The soft agar assay was performed in 6-well plates containing two layers of Agar. The bottom layer consisted of 0.8% agar in 1 ml of DMEM supplemented with 10% FBS. A549 cells (1 × 104/well) were placed in the top layer containing 0.4% agar in the same medium as the bottom. Cells were cultured for 14 days under different conditions and colonies were photographed and counted per four fields under a microscope.
Western blot
The samples to be analyzed were separated on 10% or 12% SDS-PAGE and transferred to a PVDF membrane by semi-dry blotting for 1.5 h at a constant current of 250 mA, using a semi-dry transfer apparatus (Siplast, UK). The transfer of proteins was ascertained by staining the PVDF membrane with Ponceau-S (1X). The membrane was blocked overnight at 4ºC with 1% BSA prepared in PBS and subsequently probed with the appropriate primary antibody at recommended dilutions and time points, followed by three washings with PBST. Finally the membrane was incubated in HRP or IR-labeled labeled secondary antibody for 1 h. After three washings with PBST, the membrane was developed using Enhanced Chemiluminescence reagents or scanned on an Infrared imager, Odyssey Licor (Licor Biosciences, USA).
Immunofluorescence
A549 cells were seeded in 24 well tissue culture plates and grown overnight in DMEM supplemented with 10% FBS. After washing with serum free medium, cells were incubated in conditioned medium containing Fibronectin or medium supplemented with recombinant EDA for 24 h. Then the cells were fixed with 4% formaldehyde and permeabilized with PBS containing 0.1% Triton X-100 for 30 min. This was followed by blocking with 1% BSA in PBS for 1 h and incubation in primary antibody overnight (or as recommended) at 4 °C. After washings with PBST three times, cells were incubated in secondary anti-mouse IgG antibody conjugated with Alexa Fluor for 1 h, observed under Evos cell imaging system (Life Technologies, USA) and photographed.
Quantitative real-time PCR
Total RNA was isolated from A549 and THP-1 cells as reported in [
12]. Real-time PCR was performed using gene specific primers and SYBR Green (Kapa Biosystems, SA).The reaction mixture was run in lightCycler 480-II (Roche, Germany) and the Gene expression was quantified using 2
−ΔΔCT method [
13].
Labeling with stable isotopes
For stable isotopic labeling, DMEM was supplemented with stable heavier isotopes (13C6 L-Arginine[R]-HCl and 13C6 L-Lysine[K]-2HCl) and RPMI with corresponding lighter isotopes (regular 12C6 L-arginine[R] and 12C6 L-lysine[K]) (Thermo Scientific Rockford, USA) at 0.1 g/L concentration in accordance with the SILAC kit instructions. A549 cells were cultured in the DMEM and THP-1 in RPMI containing heavier and lighter isotopes of L-Arginine and L-Lysine respectively. In order to achieve approximately 99% incorporation of the heavy and light isotopes into the proteins, A549 and THP-1 were repeatedly sub-cultured for ~ 7 passages (~ 5 weeks). Repeated seeding of cells marked as AhdD10 and TldR10 grown in T-75 flasks was performed. Every other day, the cells were supplemented with labeled media. A549 cells were dislodged by Enzyme-free Cell Dissociation Buffer (Invitrogen Grand Island, USA) after achieving confluence, whereas THP-1 cells were centrifuged for 5 min at 800 g. After the adaptation phase, when cells reached around 80% confluence, they were handled individually by centrifuging and washing them thrice using RPMI devoid of Arginine (R) and Lysine (K). Finally, the cells were sorted into three different categories: A549 monoculture (1 × 106), THP-1 monoculture (10 × 106), and a co-culture mix of 1:10 ratios (1 × 106 [A549]: 10 × 106 [THP-1]) and were cultured for 48 h in RPMI (lacking arginine and lysine).
Collection and processing of the Secretome
Conditioned media from each of the three categories were harvested after 48 h and centrifuged for 30 min at 800 g to remove any floating and unattached cells. In order to remove the cellular debris, the supernatants were again filtered by running through a Millex-GP 0.22 μm filter (Millipore, Ireland). Following this, filtered secretomes were concentrated using a 3 kDa cut-off Millipore Amicon Ultra filters (Millipore, Billerica, MA, USA) by centrifugation at 4000 g till the secretome was condensed to 1mL, and subsequently centrifuging at 14,000 g using the 3 kDa cut-off filters till a condensed volume of 500µL was obtained. Pierce microBCA kit (Thermo Scientific) was used to determine the protein concentration.
Preparation of samples and trypsin (in-solution) digestion
Before MS analysis, each sample was subjected to in-solution digestion. 40 µg protein was lyophilized per sample and resuspended in 100mM TEABC (pH = 8.0). After being reduced using 5mM dithiothreitol at 60℃ for 30 min, the samples were alkylated at room temperature with 20mM iodoacetamide. Proteins were digested at 37 °C for 16 h with an enzyme – protein ratio of 1:20 (w/w) utilizing sequencing grade trypsin (modified sequencing grade; Promega, Madison, WI). Peptides were dried in a vacufuge concentrator after trypsin digestion, desalted with C18 Stage Tips, and kept at -80 °C till LC-MS/MS analysis could be performed.
LC-MS/MS
Digested samples were inspected by LC-MS/MS analysis using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific, Bremen, Germany) coupled to an Easy nLC-1000 system (Thermo Scientific, Odense, Denmark). The already digested peptides were first reconstituted in 0.1% formic acid before being mounted into a trap column (75 μm × 2 cm; 3 μm C18 100 A˚, Thermo Scientific, Acclaim Pepmap 100) utilizing solvent A (i.e., 0.1% formic acid) and then resolved on an analytical column (75 μm × 50 cm; 2 μm C18 100 A˚, Thermo Scientific, Acclaim PepMap RSLC). For peptide resolution, a linear gradient spanning 10–32% of solvent B (i.e., 0.1% formic acid in 95% acetonitrile) was run at a flow rate of 300 nl/min for 105 min (total run time: 120 min). The data was retrieved using the data-dependent acquisition approach, with a scan spectrum ranging between 400 and 1600, a mass resolution of 120,000, a maximum injection duration of 50 ms, and an AGC target of 2 × e6 ions. Utilizing an isolation window adjusted at 1.6 m/z in a quadrupole mass filter, the top ten precursor ions were separated. To facilitate the dissociation of the ions in the precursor pool, they were exposed to greater energy collision-induced dissociation with 34% normalised collision energy. The MS/MS scans were generated at a maximal ion injection time of 200 ms, with a mass resolution of 30,000, as well as an AGC target of 1 × e6 ions. In order to have mass measurement precision, the polydimethylcyclosiloxane (m/z, 445.1200025) ion was utilized, and the lock mass was enabled. Independent triplicate runs of each sample were performed on an Orbitrap Fusion mass spectrometer.
Analysis of LC-MS/MS data
Raw data generated from LC-MS/MS was investigated using proteome discoverer [PD] (Version 2.1.1) software (Thermo Fisher Scientific, Bremen, Germany) employing the Mascot and Sequest search engine algorithms when compared to the Human refseq-81 database. Carbamidomethylation at the cysteine residue was used as a static modification, whereas oxidation of methionine and SILAC (C6-arginine/lysine) were used as variable modifications in the database search variables. For both the precursor and the fragment ions, a mass error of 10 ppm and 0.05 Da, respectively, was permitted. Trypsin, a designated protease, and a single missed cleavage was considered acceptable. The false discovery rate (FDR) was calculated by carrying out decoy database searches and FDR cut-off for peptide and protein identification was set to 0.01. For quantification, exclusively unique peptides were taken into account. Peptide Spectrum Match (PSM) entries with associated peak regions were exported from PD. For the co-culture setting, the PSM entry was divided up into two independent files: heavy and light, which corresponded to heavy-labelled cancer cells and light-labelled monocytes, respectively. Peptide and protein lists were created using an in-house tool. For further analysis those proteins were further taken into consideration that were present in at least two of the three replicates. Protein normalization for individual cell lines between mono-culture and their corresponding co-culture (e.g. A549 mono & A549 co-culture) was done based on summed peak area of all the proteins. The normalization factor was calculated by taking the ratio of total peak area in mono and co-culture. Proteins ascertained from each cell line in a monoculture scenario were compared with those ascertained in a co-culture scenario (i.e., A549 monoculture versus A549 co-culture; THP-1 monoculture versus THP-1 co-culture). Using these two comparative sets of data, three protein lists were generated: (i) Proteins that were exclusively found in A549 and THP-1 mono-cultures, respectively, (ii) Proteins that were exclusively found in A549 and THP-1 co-cultures, respectively and (iii) Proteins that were found in both mono- and co-culture scenarios. Fold change was computed for the third group of proteins by evaluating the area of each proteins in mono- as well as co-culture scenarios. Identified proteins that have at least two unique peptides and four PSMs with a %CV of ≤ 30 are documented.
Several bioinformatics algorithmic methods were used for shortlisting and ascertaining the candidature of the identified proteins [
14]. JVenn was employed across all six scenarios to determine which proteins were unique and which were shared among the groups. Morpheus, a versatile matrix visualization and analysis software [
15] was employed to generate the heatmaps. With the help of Morpheus, heat map of all the proteins in six different scenarios were generated firstly based on %CV of < 10 and/or fold-change of > 3, and then secondly based on SecretomePv2.0 [
16] and SignalPv4.1 [
17] respectively… To further illustrate the function of all secretory proteins in each scenario, Cytoscapev3.8.2 platform was utilized and the software packages STRINGv8.3 to display the interaction of narrowed down secretory proteins across and within the different scenarios. BiNGOv3.0.5 (Biological Networks Gene Ontology) was used to visualize the involvement of the narrowed down secretory proteins. STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) is a repository of protein interactions, both established and presumed whereas BiNGO integrates the prominent functional features of a particular gene/protein set onto the GO hierarchical order and produces same data as a Cytoscape diagram. To depict the connection between proteins and their respective pathways, tool bundle in the Reactomev79 was used. Reactome offers easy bioinformatics tools for pathway viewing, inference and analysis, e.g., molecular interactions between nucleic acids, proteins, complexes and small compounds [
18]. The cancer hallmarks analytics tool [CHAT] [
19](
http://chat.lionproject.net/) is a text-mining assessment of several millions of PubMed articles that enables users to rapidly and simply examine the degree of correlation between gene/protein of interest and cancer hallmarks across literatures. CHAT was implemented to assess the shortlisted secretory proteins in light of previously recognized cancer hallmarks based on scientific literature.
Discussion
To evaluate the mechanisms by which lung cancer cells and monocytes modulate biological properties of each other in a reciprocal manner, we established an in vitro tumor microenvironment simulating model by co-culturing human model lung carcinoma cells, A549 and human monocytes, THP-1 in varying ratios. Expression of various cytokines commonly known to support tumor growth and metastatic dissemination was quantified to get an account of the interaction(s) occurring within the co-cultures [
21‐
23]. All the tested co-cultures showed significant production of TNF-α, IL-6 and IL-10. However, the most effective response was observed at a co-culture ratio of 1:10, thus pointing at the abundance of monocyte activator(s) in the co-culture milieu in this ratio. The levels of the prototypical pro-inflammatory cytokines, TNF-α and IL-6 were observed to increase with time, with response peaking at 48 h time point, however, IL-10, an anti-inflammatory cytokine, showed an optimal response at 48 h. Early release of TNF-α and IL-6 as well as the expression of IL-10 at the highest time point hints at the differentiation of monocytes in co-culture towards a phenotype that resembles M2 macrophages as compared to the M1 phenotype observed at earlier time points. Both TNF-α and IL-6 have been shown to be classical indicators of macrophage activation [
9] and in vivo demonstrated to mediate tumor promotion in various human cancers [
24,
25]. In our settings, accentuated migration, invasion, colony formation as well as transition of A549 cells to mesenchymal state when cultured in 1:10 co-culture conditioned medium can be attributed to such characteristics of these cytokines.
Taking the above findings into consideration, we further sought to identify secretory proteins that mediate the interactions between lung carcinoma cells and monocytes in co-culture by using SILAC based proteomic approach. We applied the ‘double labeled’ SILAC approach to investigate protein secretion and regulation during co-culture of lung carcinoma cell and monocytes. The advantage of the SILAC-based experimental strategy is that the proteins secreted by each cell type in co-culture can be distinguished and identified by the stable isotope amino acids that had been incorporated [
26,
27]. We identified a total of 414 potentially secreted proteins which were subjected to various filters of %CV, fold-change and neural-network probability algorithmic tools, SecretomeP and SignalP to select authentic secretory proteins across all the scenarios. Overall, 39 proteins were shortlisted among which 20 were classical secretory proteins (CSPs) while 19 were non-classical secretory proteins (NCSPs). Which were finally were analyzed through STRING protein database and Reactome knowledgebase system respectively to investigate the intrinsic interactions exhibited by them and to analyze the pathways in which these proteins might be involved [
28‐
30]. Through the analysis of biological processes, cellular component and molecular function using BINGO, it was revealed that the 39 selected proteins are mainly localized in extracellular region and are involved in protein binding, catalytic activity and ion binding, identical protein and receptor binding. The molecular functions attributed to these proteins primarily included metabolic process, response to stimulus and stress.
Among the 39 selected secretory proteins we focused only on the 7 unique proteins in the co-culture scenarios for downstream analysis because of their peculiar secretion in these scenarios. These included RNH1 and IFI30 in A549 Co-CM and CLEC3B, VCAN, IGFBP2, C2, TUBB4B in THP-1-Co CM scenarios. Validation of the gene expression of these protein candidates by qRT-PCR analysis depicted a general consonance between the proteins identified and their expression at transcriptional level. In addition, qRT-PCR revealed that induction in the gene expression of all the candidate proteins occurs after 24 h of co-culture except IGFBP2 whose expression was seen to rise only after 48 h of co-culture. The finding provides an understanding that the release of these proteins occurs due to their transcriptional upregulation. In addition, we demonstrated the proteins uniquely secreted in A549 and THP-1 co-cultures except TUBB4 are associated with one or the other hallmark of cancer which supports their candidature as the proteins involved in lung cancer pathogenesis. This candidature was further evidenced by evaluating their prognostic potential using the related RNA-seq data available in the TCGA, EGA, and GEO databases. The high expression of the VCAN, IGFBP2, C2, and TUBB4 (proteins uniquely secreted in THP-1 co-culture) and IFI30 (proteins uniquely secreted in A549 co-culture) were associated with poor prognosis and correlated negatively with disease outcomes. Intriguingly such features of these proteins were specific to lung adenocarcinoma compared to lung squamous cell carcinoma, gastric, ovarian and breast carcinomas.
The secretion of versican (VCAN) by monocytes due to their interaction with lung carcinoma cells is being reported in this study for the first time. Versican is an extracellular aggregating proteoglycan involved in the assembly of the extracellular matrix [
31]. Versican inhibits cancer cell attachment to stromal matrix, thereby facilitating cancer cell migration and invasion [
32,
33]. Ligation of TLR2 by versican has been reported to activate stromal cell populations in tumor microenvironment and subsequent inflammatory cytokine secretion [
34]. In our study, the augmented migration, invasion and EMT of A549 cells, when grown in the co-culture conditioned medium, can therefore be related to the action of THP-1 secreted versican in the co-culture conditioned medium. Conversely, versican secreted by THP-1 cells can ligate TLR-2 on these cells to induce the production of inflammatory cytokines in an autocrine fashion which generates an inflammatory microenvironment congenial for tumor progression [
9]. Versican expression has been correlated with poor prognosis, increased TAM infiltration, poor tumor differentiation, and a higher tumor-grade metastasis (TNM stage) in a variety of cancers [
35]. Therefore our findings are in line with these studies and lay further impetus on the importance of Versican in lung cancer progression.
Tetranectin (CLEC3B) is a lectin with specific binding affinity for plasminogen. This binding of Tetranectin to plasminogen leads to the activation of plasminogen-cascade that triggers the proteolytic processing and degradation of the extracellular matrix and thereby cancer cell migration and invasion [
36]. In our experimental setup, prometastatic effects of the co-culture medium can be ascribed to the presence of Tetranectin in the co-culture medium secreted by monocytes due to their interaction with lung carcinoma cells. The results further provide clue that in lung cancers, presence of monocytes in tumor microenvironment triggers the release of Tetranectin from cancer cells, thereby promoting cancer progression.
Insulin like growth factor binding protein 2 (IGFBP2) is the protein secreted by THP-1 cells in co-culture with A549 cells. IGFBP2 promotes tumor cellular proliferation, migration, invasion, angiogenesis, epithelialtomesenchymal transition and is highly elevated in serum or tissue in patients with malignant tumors. It has been found to be overexpressed in a broad spectrum of tumors including lung cancer wherein higher plasma levels of IGFBP2 have been positively associated with tumor size, lymph node metastasis, advanced tumor stage, and shorter overall survival [
37]. In our co-culture medium set-up, observed metastatic effects could be related to the secretion of IGFBP2 by monocytes. Overall the finding provide an insight into the role played by IGFBP2 in lung cancer metastasis.
TUBB4B is an isotype of β-tubulin, a member of the tubulin family that forms the building blocks of the microtubule networks. This protein is reported to be overexpressed in glioblastoma, ovarian and prostate cancers [
38‐
40], although its downregulation has been shown to initiate EMT in colon cancer [
41]. A recent study has implicated TUBB4B as an essential factor required for the maintenance of cancer stem cell niche via its interaction with Ephrin-B1. In the context of our study, we assume that the TUBB4B secreted by THP-1 cells during co-culture might interact with Ephrin-B1 on A549 cells which in turn by ligating ephrin receptor might promote cell migration and invasion [
42,
43]. Thus the finding provides evidence that lung cancer cells modulate monocytes to release TUBB4B which in turn promotes cancer metastasis.
C2, a complement protein was secreted by THP-1 cells in co-culture with A549 cells. In solid tumors both cancer and stromal cells have the ability to produce complement proteins. Amplification of the complement system has been shown to promote proliferation, migration, and epithelial-mesenchymal transition [
44,
45]. Therefore it can be argued that the presence of C2 in the co-culture conditioned medium supports the metastasis properties of A549 cells.
Interferon-gamma-inducible protein 30 (IFI30) was secreted by A549 cells in co-culture with THP-1. Dysregulated expression of IFI30 has been associated with human cancers. IFI30 is upregulated in breast cancer and melanoma and has been linked to disease progression in prostate cancer [
46]. Gene Ontology (GO) analysis shows that IFI30 is associated with enhanced leukocyte-mediated immune and inflammatory responses. In addition, studies on the tumor microenvironment show that increased infiltration of M2-type macrophages was associated with high IFI30 expression [
47]. Our results in line with these findings suggest that lung cancer cell secreted IFI30 promotes inflammatory response observed in co-culture scenario.
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