Immunogenic cell death mediated TLR3/4-activated MSCs in U87 GBM cell line

Abstract

Background and aims: Glioblastoma (GBM) is an aggressive primary brain cancer with no promising curative therapies. It has been indicated that MSCs can interact with the tumour microenvironment (TME) through the secretion of soluble mediators regulating intercellular signalling within the TME. TLRs are a multigene family of pattern recognition receptors with evolutionarily conserved regions and are widely expressed in immune and other body cells. MSCs by TLRs can recognize conserved molecular components (DAPMPs and PAPMPs) and activate signalling pathways, which regulate immune and inflammatory responses. MSCs may exert immunomodulatory functions through interaction with their expressed toll-like receptors (TLRs) and exert a protective effect against tumour antigens. As an emerging approach, we aimed to monitor the U87 cell line growth, migration and death markers following specific TLR3/4-primed-MSCs-CMs treatment.

Methods and results: We investigated the phenotypic and functional outcomes of primed-CMs and glioma cell line co-culture following short-term, low-dose TLR3/4 priming. The gene expression profile of target genes, including apoptotic markers and related genes, was analyzed by qRT-PCR. MicroRNA-Seq examined the miRNA expression patterns, and flow cytometry evaluated the cell viability and cycle stages. The results showed significant changes in apoptosis and likely necroptosis-related markers following TLR3/4-primed-MSCs-CMs exposure in the glioma cell line. Notably, we observed a considerable induction of selective pro-apoptotic markers and both the early and late stages of apoptosis in treated U87 cell lines. Additionally, the migration rate of glioma cells significantly decreased following MSCs-CM treatment.

Conclusion: Our findings confirmed that the exposure of TLR3/4-activated-MSCs-CMs with glioma tumour cells possibly changes the immunogenicity of the tumour microenvironment and induces immunogenic programmed cell death. Our results can support the idea that TLR3/4-primed-MSCs can lead to innate immune-mediated cell death and modify tumour cell biology in invasive and metastatic cancers.

Keywords: Cell death; Mesenchymal stem cells; Priming; TLR3; TLR4; miRSeq.

Fig. 1

A) Schematic representation of four classical cell surface markers expressed in MSCs by enzymatic digestion method was validated by flowcytometry among freshly isolated and expanded MSCs. Illustrative histograms of surface marker expression compared to unstained MSCs (negative control). MSCs expressed CD44, CD73, CD90 and CD105, but did not express CD34 and CD45. B) MSCs have a spindle and fibroblast-like shape, illustrating the typical shape of MSCs and the U87 cell line has an epithelial morphology. Cells differentiated for 12 days have altered morphology and contains lipid drops. Differentiated cells stained with Oil Red O had well-defined lipid inclusions. Phase contrast microscopy allowed us to observe large lipid drops. Oil red O staining shows cells grown in the defined inductive medium differentiate into adipogenic lineages, for osteogenic differentiation induction was validated by alizarin red staining. Cells grown in a defined inductive medium differentiate into osteogenic lineages and finally various calcium depositions were seen. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

The expression level of TLR3/4 genes in MSCs elevated following exposure to LPS and Poly (I:C) at various dosages/times exposure. A) Expression level of TLR3 following Poly (I:C) exposure in a dose-dependent manner. B) Expression level of TLR3 at different exposure time points. C) Expression level of TLR4 following LPS exposure in a dose-dependent manner. D) Expression level of TLR4 at various time points following exposure to LPS. Values depicted are mean ± SD from 3 independent cell cultures and duplicate repeated RT-PCR. The GAPDH and β-actin were used to normalize data in qRT-PCR. ANOVA with Fisher's least significant difference (LSD) was applied to compare gene expression levels (*P < .05; **P < .01; ***P < .001; ****P < .0001).

Fig. 3

A) Generally, the results showed the increased caspase-3 mRNA levels in GBM exposed to TLRs-primed groups. U87 cells pretreated with 1, 2.5, and 5 μg/ml poly (I:C) primed MSCs-CM significantly increased caspase-3 expression level (2.5 and 5 μg/ml ****P < .0001). Also, we observe significantly enhanced expression in the TLR4 primed group at LPs 10 ng/ml (***P < 0. 001). Meaningfully, a light correlation between TLR3, TLR4, and caspase-3 mRNA expression was observed in the U87 cell line. These observations demonstrate that TLR3 and TLR4 activation possibly drive cell death in tumour cells. Our results confirmed in vitro overexpression of caspase-3, a key mediator of apoptosis. Similarly, the expression of caspase-8 and caspase-9 at the mRNA level, strongly related to the apoptosis signalling pathway, was elevated at specific concentrations of TLR agonists (*: p < 0.05, #: p < 0.01, &: p < 0.001, $: p < 0.0001). B) The Bcl-2 expression significantly decreased in selective groups. Apoptosis is regulated by key regulators of anti-apoptotic and pro-apoptotic family members of the B cell lymphoma 2 (Bcl-2). Bcl-2 can inhibit apoptosis by preserving the outer membrane integrity. All the primed groups considerably decreased the expression level of Bcl-2 in U87 cell line (****P value < .0001).

Fig. 4

Volcano plot presenting varied miRNA expressions detected using miRNA-seq in the U87 cell line. The y-axis shows the log 10 of the P-values, and the x-axis is the FC (calculated as the log-ratio transformed ratio of the expression between TLR3/4-primed and unprimed groups). miRNAs with logFC < − 1 and logFC >1 (with a p-value <.05) are displayed in green. The green and red labels show the top miRNAs with high levels of dysregulation. Unique DE miRNAs (hsa-miR-16-5p, hsa-miR-15a-5p, and hsa-miR-7641), whose targets were found to be associated with cell survival and apoptosis have been identified in treated U87 cell line. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Combination of imaging flow cytometry and Annexin V/PI staining protocol used to detect cell death induced by TLRs-primed-MSCs-CMs for 48 h in the U87 cell line. The level of significance was denoted with an asterisk corresponding to the p-value (*: p < 0.05, #: p < 0.01, &: p < 0.001, $: p < 0.0001).

Fig. 6

Cell cycle analysis and quantitative measurement of cell cycle phase through PI staining and following flow cytometry for the U87 cells following different CMs treatment (*: p < 0.05, #: p < 0.01, &: p < 0.001, $: p < 0.0001).

Fig. 7

Wound healing assay to evaluate cell migration. The assay was performed at 0, 24, 48, and 72 h in TLRs-primed-MSCs-CMs treated and unprimed U87 cell lines. (A) Illustrative phase-contrast microscope images presenting the wound closure by the cells at 0, 24, 48, and 72 h and graph of scratch wound healing assay in untreated, LPS (10 ng/ml), TAK-242(1 μM) and Poly (I:C) (1 μg/ml) groups. (B) Representative images of scratch wound healing assay of Untreated, LPS (10μg/ml), and Poly (I:C) (5 μg/ml) groups. (C) Scratch-wound closure was monitored over time in Untreated, LPS (1 μg/ml), and Poly (I:C) (2.5 μg/ml) groups. Cell migration was measured by the rate of cells moving towards the scratched area upon time using ImageJ ™ software. Data were analyzed over time (vs. time = 0 h) using a one-way ANOVA followed by Uncorrected Fisher's LSD test (*P < .05 and * *P < .01, ** *P < .001).

Fig. 8

A) Interaction of mir15a/16-5p cluster and mir-7641 with targeted genes involved in intrinsic and extrinsic signalling pathways of apoptosis. As depicted above, the mir-15a/16 cluster contributes to apoptosis induction by suppressing anti-apoptotic genes such as Bcl-2 and Mc-1. Moreover, miR-16-5p probably inhibits glioma cell viability and invasion through miR-16-5p/Talin-1 (TLN1) Axis. It is worth mentioning that miR-15a and miR-16 can induce apoptosis and cell cycle arrest by suppressing cyclin D1 (CCND1). On the other hand, miR-7641, as an oncogenic miR, promotes tumour cell proliferation by improving intercellular communication. B) Network construction of top differentially expressed miRNAs (DE-miRNAs) and their target mRNAs.

Fig. 9

Caspase expression profiles in U87 cell lines in response to TLRs-primed-MSCs-CMs and their inhibitors treatment (*: p < 0.05, #: p < 0.01, &: p < 0.001, $: p < 0.0001).

Fig. 10

TLR signalling leads to apoptosis in tumour cells. Following TLR3 and TLR4 priming in MSCs, cascades of molecular interactions result in the secretion of various molecules in CM, known as secretome. It comprises different biomolecules, such as regulatory RNAs, miRNAs, and proteins. After removing cell derisions, CM was transferred to wells, including U87 cells. Evaluation of apoptotic markers showed us that secretome ingredients following TLRs preconditioning significantly induce apoptosis in treated u87 cell lines. The extrinsic apoptotic pathway is triggered by the ligation of TNF-α to its DRs.

Fig. 11

Comparison of U87 cell migration following co-culture with CMs of various TLRs and TAK-242-primed-MSCs groups. Generally, migration is decreased in TLRs-primed groups at different time points and dosages. Interestingly, the TAK-242 group did not show significant changes compared to the control group. Differences were determined over time (vs. time = 0 h) using a two-way ANOVA followed by an Uncorrected Fisher's LSD test (*: p < 0.05, #: p < 0.01, &: p < 0.001, $: p < 0.0001).