Edited by: Simona Pisanti, University of Salerno, Italy
Reviewed by: Anca Maria Cimpean, “Victor Babes” University of Medicine and Pharmacy Timisoara, Romania; Elena Ciaglia, University of Salerno, Italy
Specialty section: This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Oncology
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Metastatic brain tumors continue to be a clinical problem, despite new therapeutic advances in cancer treatment. Brain metastases (BMs) are among the most common mass lesions in the brain that are resistant to chemotherapies, have a very poor prognosis, and currently lack any efficient diagnostic tests. Predictions estimate that about 40% of lung and breast cancer patients will develop BM. Despite this, very little is known about the immunological and genetic aberrations that drive tumorigenesis in BM. In this study, we demonstrate the infiltration of mast cells (MCs) in a large cohort of human BM samples with different tissues of origin for primary cancer. We applied patient-derived BM cell models to the study of BM cell–MC interactions. BM cells when cocultured with MCs demonstrate enhanced growth and self-renewal capacity. Gene set enrichment analyses indicate increased expression of signal transduction and transmembrane proteins related genes in the cocultured BM cells. MCs exert their effect by release of mediators such as IL-8, IL-10, matrix metalloprotease 2, and vascular endothelial growth factor, thereby permitting metastasis. In conclusion, we provide evidence for a role of MCs in BM. Our findings indicate MCs’ capability of modulating gene expression in BM cells and suggest that MCs can serve as a new target for drug development against metastases in the brain.
Metastasis to the brain is a major reason of high mortality in patients with systemic cancers. Metastatic brain tumors occur in about 25% of all cancer patients and have a high mortality rate. The median survival of patients diagnosed with brain metastases (BMs) and treated with aggressive therapies is generally 4–12 months (
Brain metastasis differs in many aspects from metastases in other organs and is a treatment challenge owing to the progressive neurological disability and the lack of any effective treatment due to the unique structure of the blood–brain barrier (BBB). Even though the brain has been considered as an immune-privileged organ and not much is known about the inflammatory response by circulating tumor cells, BMs do contain infiltrating immune cells (
Mast cells (MCs) are versatile immune cells that have been implicated in various pathophysiological conditions including cancer (
In this study, we report that MCs infiltrate human BM and in line with this finding we provide evidence for a role of MCs in BM. We show that MCs
Permission for use of human tissue samples for this study was obtained from the Ethics Committee of Uppsala, Sweden (Dnr 2014/535). The study involving human tissue samples was conducted in accordance with the Declaration of Helsinki and the patients gave written informed consent for the sample collection. All human tissue samples and related patient records for research purpose (as listed in Table S1 in Supplementary Material) are part of Uppsala Biobank material and were provided to the researchers as per ethical permission and all material obtained in compliance with the Declaration of Helsinki. The researchers did not have any interaction with any patients and were not involved in the collection of human patient samples during the course of this study. Patient identity was anonymous for the researchers. All human tumor tissue sections thereafter were evaluated based on the WHO classification by experienced neuropathologists.
All cells were cultured at 37°C under 5% CO2. U3333MET, a human BM cell line was cultured in 10% FBS-containing MEM supplemented with 4 mM
The human MC line LAD2 (obtained from Prof Dean Metcalfe at NIH/NIAID, MD, USA) was cultured as described previously (
To examine the effect of MCs on BM cell growth and secretion, LAD2 cells were cocultured in 6-well format transwell (0.4 µm) with the two BM cell lines for 12, 24, and 48 h. Briefly, the BM cell lines were plated on 6-well plates in low serum (1%) conditions and allowed to attach for 2–3 hours. Overnight SCF starved LAD2 cells were suspended in medium (5 × 105 cells/ml) and added to the transwell. The cocultures are left to grow undisturbed for 12, 24, and 48 h. Stimulation experiment was done in triplicates. Appropriate negative controls were kept for each experiment.
To measure the level of MC degranulation induced by the BM cells, LAD2 cells (1 × 106 cells/ml) in triplicates were incubated at 37°C in 5% CO2 in Hanks balanced salt solution for 1 h in the presence of either 2 µM calcium ionophore A23187 (as a positive control) or for 4 h in coculture with BM cells. Samples were taken at each time point and cells were centrifuged at 300
Brain metastasis cells were seeded in triplicates and allowed to attach overnight. The following day medium was removed and replaced with either control medium (medium supplemented with 1% FBS) or 0.4 µm transwell with LAD2 cells. The cells were then grown for 12, 24, and 48 h. Cell proliferation was assayed by adding AlamarBlue (88951, ThermoFischer) according to the manufacturer’s recommendations.
Cell proliferation was assayed with the Click-iT® Plus EdU Alexa Fluor® 555 imaging kit following manufacture’s recommendation (C10638, Life Technologies). The assay is based on the principle of efficiently incorporating modified thymidine analog EdU (5-ethynyl-2′-deoxyuridine, a nucleoside analog of thymidine) into newly synthesized DNA. Visualization is achieved by fluorescent labeling with a bright, photostable Alexa Fluor® dye in a fast, highly specific, mild click reaction. Briefly, BM cells were seeded on cover slips and allowed to attach overnight. Following this, they were grown alone in normal media or in coculture with MCs for a period of 48 h. EdU was added 4–5 h prior to the end point of experiment. Cells were fixed, permeabilized, and EdU positive cells were detected with Alexa Fluor® 555-conjugated secondary antibodies and cell nuclei were detected by Hoechst 33342. This was followed by image acquisition and analysis.
Confluent monolayers of U3333MET and NCI-H1915 cells were scratched by a razor blade from more than four replicate plates for each cell type. The cells were then left in the incubator with either normal growth medium or transwell inserts with LAD2 cells on top. Images of the similar areas of scratches were taken immediately after scratching, 24 h and 48 h post-scratching by Nikon Eclipse TS 100 microscope. Quantification of wound closure was done by using the ImageJ MRI Wound Healing Tool. It measures the area of a wound in a cellular tissue on a stack of images representing a time series. Data are presented as percentage of wound closure.
To evaluate the cancer stem/progenitor cell property of the BM cells before and after MC coculture, the
The human cytokine array kit (ARY005, R&D Systems) was used according to the manufacturer’s instructions. In brief, LAD2 cells were grown alone or in coculture with BM cells. Supernatant from the cultures were collected and applied to membranes overnight, after which signals were detected after appropriate application of antibody cocktails and streptavidin–HRP solutions. Quantification of the duplicate spots on the filters was done using ImageJ software as instructed by the manufacturers.
SCF starved LAD2 cells were grown alone or in coculture with BM cells. IL-8 and IL-10 levels in culture supernatants were measured using a quantitative immunoassay ELISA kit (900K21, 900K18, Peprotech) following manufacturer’s protocol. Both ELISA were done in triplicates for each cell experiment.
RNA was extracted from control LAD2 and BM cells as well as from LAD2 and BM cells after coculture. RNA extraction was done using GENEJET RNA Purification Kit (Life Technologies) extraction method from cell pellets. cDNA was synthesized using the High-Capacity RNA-to-cDNA™ Kit (4387406, Thermo Fisher Scientific), which was then used to perform the qPCR using the PowerUp SYBR Mastermix. The target-specific primers used are listed in Table S2 in Supplementary Material. For all qPCR analysis, β-actin expression was used as endogenous control. Results are presented as fold induction. The experiments were performed three times, with triplicates in each case.
RNA quality was evaluated using the Agilent 2100 Bioanalyzer system (Agilent Technologies Inc., Palo Alto, CA, USA). In all, 100 ng of total RNA from each sample was used to generate amplified and biotinylated sense-strand cDNA from the entire expressed genome according to the GeneChip® WT PLUS Reagent Kit User Manual (P/N 703174 Rev. 1, Affymetrix Inc., Santa Clara, CA, USA). GeneChip® ST Arrays (GeneChip® Human Gene 2.0 ST Array) were hybridized for 16 h in a 45°C incubator, rotated at 60 rpm. According to the GeneChip® Expression Wash, Stain and Scan Manual (P/N 702731 Rev. 3, Affymetrix Inc., Santa Clara, CA, USA), the arrays were then washed and stained using the Fluidics Station 450 and finally scanned using the GeneChip® Scanner 3000 7G. The analysis was performed at the Array and Analysis Facility, Science for Life Laboratory at Uppsala Biomedical Centre, Uppsala, Sweden.
The raw data were normalized in Expression Console, provided by Affymetrix (
Formalin-fixed, paraffin-embedded 6 µm thick tissue sections were fixed. Thereafter, the sections were deparaffinized (in xylene on a rocking table for 1 h × 2 h followed by 2 min × 5 min incubations in 100% EtOH, 95% EtOH, 80% EtOH, distilled H2O) and subjected to pressure boiling for antigen retrieval in antigen unmasking solution (Vector Labs). Immunohistochemistry was performed using the UltraVision LP detection System (Thermo Fisher Scientific) in accordance with the manufacturer’s instructions. Briefly, after antigen retrieval, the slides were washed in PBS-T [containing 0.05% Tween (Sigma Aldrich)] and incubated with hydrogen peroxidase block. Ultra V block was subsequently applied. Primary antibody used was anti-human tryptase (sc-33676, Santa Cruz Biotechnology) diluted in 5% milk in PBS-T. Primary antibody was applied overnight at 4°C, followed by primary antibody enhancer. Slides were incubated with HRP polymer, and the signal was visualized using freshly prepared DAB plus chromogen and substrate mix. Between all the steps described above, the slides were thoroughly washed in PBS-T. After the final step, the slides were washed in distilled H2O, counterstained with hematoxylin and mounted using Immu-mount (Thermo Fisher Scientific).
For immunofluorescence staining, coverslips were rinsed in PBS, blocked in 5% milk-containing PBS-T [supplemented with 0.2% Triton X-100 (Sigma Aldrich)] for 1 h, followed by overnight incubation (4°C) with the primary antibody diluted in the blocking solution. The coverslips were subsequently incubated with appropriate secondary antibody for 45 min. Nuclei were stained with DAPI (1:5,000) for 15 min and mounted in Immu-mount. All secondary antibodies used were Alexa antibodies (Invitrogen).
IHC and IF stained slides were imaged using ZEISS AxioImager for brightfield (AxioCam color) and fluorescence (AxioCam monochrom) and Zen Blue software. Image analysis was done using ImageJ software.
In-cell ELISA (62200, Invitrogen) was used to determine the relative protein levels in whole BM cells before and after coculture with MCs according to the manufacturer’s protocol with slight modification. It is an accurate and efficient method of analysis of protein levels in cells and is ideal since it can be performed on a 96-well format with multiple repeats and less cell number. Briefly, the BM cells were seeded and allowed to attach for 3–4 h following which they were grown subsequently alone or in coculture with LAD2 cells. All experiments were performed under reduced serum condition. After the stipulated time period, the wells were washed with ice cold PBS and then proceeded according to the kit protocol. Human SOX2 (AB5603, Merck Millipore) and human CD133 (Ab19898, Abcam) antibodies were used and the detected with a horseradish peroxidase conjugate. Cell number normalization was done with the whole-cell stain, Janus Green. Absorbance was detected using an ELISA plate reader.
In order to assess the differentially expressed genes (DEGs) in the normal and LAD2 cocultured NCI-H1915 and U3333MET cells, the enrichment of functional/pathway annotations was investigated through the bioinformatic resource Database for Annotation, Visualization and Integrated Discovery (DAVID). Gene set enrichment analysis (GSEA) and pathway analysis was performed on the Broad Institute- MSigDB (The Molecular Signatures Database). To address the problem with multiple testing, the
Statistical analyses were done using the GraphPad Prism software (GraphPad Software 6.0d). For groupwise comparisons, the Student’s unpaired
To study the extent of MC infiltration in BMs, we analyzed tryptase (TPSAB1) expression profiles in BM tissues from 40 patients (Table S1 in Supplementary Material). We examined 31 metastatic adenocarcinoma nodules, 8 metastatic renal carcinoma nodules, and 1 metastatic squamous carcinoma nodule of diverse origin (lung, breast, prostate, colorectal, uterus, ovary) with hematoxylin eosin staining followed by evaluation for metastases formation and primary cancer origin (Figure
Mast cell (MC) infiltration in brain metastasis (BM).
To elucidate the possible mechanisms underlying the accumulation of MCs in BM, a number of experiments were performed. We used the two cell lines, NCI-H1915 and U3333MET, developed from BMs with primary lung carcinoma origin. To evaluate the recruitment possibility, we performed migration assay, in which SCF starved LAD2 cells were placed into hanging inserts and were allowed to actively migrate through an 8 µm porous membrane toward BM cells. MC migration toward BM cells was almost 60% as effective as migration toward SCF (Figure
Migration and activation of mast cells (MCs) in response to brain metastasis (BM) cells.
In order to evaluate the potential role of MCs in BM, we need to understand the mechanistic interplay between the cells and how this governs the metastases tissue microenvironment. The tumor cells, that cross the BBB hurdle and survive, attempt to lure the brain immune system and proliferate in the new microenvironment (
Mast cells (MCs) can significantly induce proliferation and invasion of brain metastasis (BM) cells. The BM cell lines were cocultured with MCs or cultured alone for 24 and 48 h and
Several studies have shown that the presence of tumor-initiating cell populations has a strong correlation with the development of metastases (
Enhanced self-renewal capacity in brain metastasis (BM) cells upon mast cell (MC) stimulation. BM cells were subjected to tumorsphere formation assay in order to examine the self-renewal stem-like cell population in the BM cells and the influence of MC mediators after coculture.
To identify potential MC-regulated mediators of tumor growth and metastasis, we performed a global gene expression analysis of the BM cells before and after MC coculture. U3333MET, a patient sample of BM (lung as primary cancer origin) and NCI-H1915 cells, was grown for 48 h with or without indirect MC coculture and then subjected to microarray analysis and subsequent comparative analysis. A principal component analysis of the data showed BM and BM-MC RNA expression to be differentially clustered (Figure S2 in Supplementary Material), indicating that MC mediators have an impact on the BM cells pathophysiology. The entire expression data were subsequently used for GSEA and observed enrichment related to MC activation; IL-8/CXCR1/CXCR2 pathway signaling and immune effector processes were some of the physiological pathways that were of interest (Figure S3 in Supplementary Material). The GSEA also showed that U3333MET cells showed a better enrichment status in comparison to NCI-H1915 cells. Subsequently, the expression level was set at a twofold difference cutoff and the filtered genes were then subjected to further analysis. With the set cutoff, the global gene transcription analysis identified 408 DEGs in MC cocultured NCI-H1915 cells with a significantly altered expression (
Mast cells (MCs) induce significant changes in expression levels of genes associated with brain metastasis cells.
The ESs and identification of biological modules involved in regulation of signaling pathways, inflammatory response, and cellular functionality suggest an efficient MC–BM cell cross talk.
We found that BM cells thrive in the presence of MCs and even show a better self-renewal capacity, characteristics that indicate their success in immune escape, and progression of metastasis. MCs, therefore, seem to play a role in setting up the platform for BM cell intravasation. Indeed, a cytokine array analysis of mediators secreted by MCs before and after MC–BM cell coculture shows an induction of a number of cytokines (Figure
Mast cells (MCs) secrete angiogenic and tumorigenic mediators to support immune escape and progression of metastases.
Mast cell infiltration in primary tumor has been correlated to enhance dissemination, extravasation, and metastasizing capacity of the cancer cells. Hence, the presence of MCs in primary tumors can be indicative of future metastasis. We also screened for the level of MC infiltration in the primary tumor tissue of nine patients diagnosed with BM (Table S3 in Supplementary Material). Primary tumor tissues and their matched BM tissues of the patients were evaluated by H&E staining followed by MC tryptase staining. We observed that patients with MC infiltrated BM had abundant number of MCs in their primary tumors as well (Figures
Mast cell (MC) infiltration in primary tumor microenvironment of patients with brain metastasis (BM).
Metastatic cancer to the brain shows a poor prognosis and poses a severe clinical problem due to the lack of effective therapies and knowledge of mechanisms that control metastatic growth in the brain. BM is a major cancer in the CNS with lung cancer, breast cancer, and melanoma accounting for most clinical cases of BM from non-CNS primary tumors (
Immune escape being acknowledged as a hallmark of cancer and with the CNS considered as immune privileged, the capacity of inflammatory response in the brain seem to be limited. Indeed, the knowledge about the inflammatory microenvironment of BM is still elusive. However, in spite of the BBB, immune cell infiltrations have been observed in BM with a tendency to enable the cancer cell to colonize. Infiltrating tumor-associated macrophages (TAMs) have been shown to have a metastasis promoting function by enhancing cancer cell intravasation, whereas TILs seem to have opposing effects (
Further, we observe an increased expression of VEGF by MCs in response to BM cell stimulation. VEGF can promote neoangiogenesis in the tumor tissue thereby supporting BM growth from micro- to macrometastases, which had been clinically shown to be counteracted by the use of VEGF antibody (
Mechanisms whereby MMPs influence tumor behavior have been mainly attributed to the proteolytic ability of MMPs to degrade ECM proteins. It thereby modulates the relationship between tumor cells and host tissue stroma. In our functional study, we observe an increase in proliferation and migration capacity of the BM cells, which is associated with simultaneous induction of MMP2 secretion from MCs. An enrichment of genes in BM cells involved in transmembrane protein functionality was observed upon MC coculture, along with an induction of EGFR (microarray data), involved in activation of the MAPK pathway and upregulation of CDK6, a major regulator of cell cycle. Our results suggest that MC contribution in BM development is partly mediated
The inflammatory tumor microenvironment has been under focus in the recent years and has been target for prognostic or therapeutic significance in various malignancies. Immunomodulatory drugs have shown remarkable and lasting responses in several tumor types, but their feasibility, as treatment target for BM needs to be ascertained. Brain metastatic tumors have so far hardly succumbed to conventional chemotherapy and targeted therapeutic treatments, partly due to the inability of the drugs to penetrate the BBB. Therefore, current standard treatments still include surgery and radiosurgical procedures. MCs represent an ideal candidate for targeted therapy. They can cross the BBB to infiltrate the BM environment and deliver specific mediators. This makes them perfect carriers for engineered site-specific delivery of immunostimulatory and/or tumor suppressing mediators in BM.
On the other hand, in order to suppress the negative effect of MCs in BM, MC stabilizers might be used as potential therapeutic agents to prevent MC activation. One such candidate could be the well-studied MC stabilizer, disodium cromoglycate (cromolyn), which has been previously shown to be capable of increasing BBB stability (
In summary, this is the first report demonstrating MC infiltration and their role in BM. Although single-handedly MCs are unlikely to impact durable responses in expansive refractory BMs, our results strongly support the MC mediator’s effect on sustenance and induction of the metastatic capacity of the BM cells. Finally, the present findings warrant further investigations on the role of MCs in metastatic cancer growth in the brain, with the aim to characterize the MC-dependent metastatic pathways and to identify novel drug targets.
This study involving human tissue samples was approved by the Ethics Committee of Uppsala University (Dnr 2014/535) and written informed consent was solicited prior to collection of the samples. Informed consent for the use of human brain tissue and for access to medical records for research purposes was followed as per ethical permission, and all material obtained in compliance with the Declaration of Helsinki. All human tissue samples (Table S1 in Supplementary Material) were obtained from Uppsala Biobank material. All human tumor samples have been evaluated based on the WHO classification by experienced neuropathologist.
AR and ET contributed to the conception and design of research; ET, LU, GH, FP, and IA contributed in the conceptual and practical establishment of the research; AR, SL, and IG performed experiments and analyzed data; AR, SL, HW, ET, and IA analyzed and interpreted the data; AR, ET, HW, FS, LU, and IA participated in the writing and revision of the manuscript. All authors read and approved the final version of manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer, EC, and the handling editor declared their shared affiliation, and the handling editor states that the process nevertheless met the standards of a fair and objective review.
The authors wish to thank Marianne Kastemar at the Department of Immunology, Genetics and Pathology for technical assistance with cell culturing. We are grateful to Science for Life BioVis platform in Uppsala University for assistance in image analysis. We would also like to thank the Array and Analysis Facility, Science for Life Laboratory at Uppsala Biomedical Center (BMC).
The Supplementary Material for this article can be found online at