TLR3 stimulation improves the migratory potency of adipose-derived mesenchymal stem cells through the stress response pathway in the melanoma mouse model

Mesenchymal stem cells (MSCs) are utilized as a carrier of anti-tumor agents in targeted anti-cancer therapy. Despite the improvements in this area, there are still some unsolved issues in determining the appropriate dose, method of administration and biodistribution of MSCs. The current study aimed to determine the influence of toll-like receptor 3 (TLR3) stimulation on the potential of MSCs migration to the neoplasm environment in the mouse melanoma model. Adipose-derived MSCs (ADMSCs) were isolated from the GFP+ transgenic C57BL/6 mouse and treated with different doses (1 µg/ml and 10 µg/ml) of polyinosinic-polycytidylic acid, the related TLR3 agonist, at various time points (1 and 4 h). Following the treatment, the expression of targeted genes such as α4, α5, and β1 integrins and TGF-β and IL-10 anti-inflammatory cytokines was determined using real-time PCR. In vivo live imaging evaluated the migration index of the intraperitoneally (IP) injected treated ADMSCs in a lung tumor-bearing mouse (C57BL/6) melanoma model (n = 5). The presented findings demonstrated that TLR3 stimulation enhanced both migration of ADMSCs to the tumor area compared with control group (n = 5) and expression of α4, α5, and β1 integrins. It was also detected that the engagement of TLR3 resulted in the anti-inflammatory behavior of the cells, which might influence the directed movement of ADMSCs. This research identified that TLR3 activation might improve the migration via the stimulation of stress response in the cells and depending on the agonist concentration and time exposure, this activated pathway drives the migratory behavior of MSCs.


Introduction
Cell-based therapy with mesenchymal stem cells (MSCs) has been considered a key approach in regenerative medicine and many clinical situations [1][2][3]. Because of the various promising features of MSCs like multilineage differentiation capability [4], easy isolation from the multitude of sources [5,6], migration to the damaged area [7,8] and, also immunomodulatory responses [9], there has been increasing consideration in taking advantages of these cells in therapeutic applications.
MSCs also have been proven to be employed in targeted anti-cancer therapy as a carrier of anti-tumor drugs due to their innate tropism to the neoplasm areas [10][11][12]. The tumor microenvironment, an "unhealed wound," can induce MSCs migration because of its typical inflammatory state comparable to the injured site [13]. This MSCs feature is undertaken in stress conditions such as mechanical injury (wounds), inflammation, infection, or cancer. Even though the mechanism of MSCs tumor tropism is unknown, the trans-endothelial migration of MSCs toward tumors is comparable to that of leukocytes, including processes such as rolling, adhesion, and extravasation. Consequently, it makes the MSCs egress from their niche and allows them to migrate into the circulation, invade vessels, and engraft the injured site to perform their repair function [14]. Some evidence supports the involvement of inflammatory signals in cancer as environmental cues in MSCs migration and biodistribution paradigm [15]. Previous research has shown that reducing tumor load and extending the lifespan of animal studies of melanoma, lung, breast cancer, and glioma using MSC expressing IL-12, interferon-β (IFN-β), and prodrugs [16][17][18].
While advancement in MSC-based therapy in treating numerous pathologic conditions and improving the quality of life, there remain some unclear challenges in establishing optimal dosage, route of administration, determination of their biodistribution, long-term viability, and biological fate [19]. Developing strategies to achieve the desired therapeutic efficacy for future practical MSCs application is required. Many findings have proposed that the preconditioning of MSCs using various factors in different conditions, including inflammatory cytokines [20,21], hypoxic preconditioning [22,23], Toll-like receptors (TLRs)-ligand priming [24], can enhance the migratory and homing behavior of these cells.
TLRs are a family of conserved receptors that identify pathogen-associated molecular patterns (PAMPs) and damaged-associated molecular patterns (DAMPs) and induce immune cell activation. TLR agonist activation induces the production of inflammatory cytokines or co-stimulatory molecules through the myeloid differentiation primary response 88 (MyD88)-dependent or MyD88-independent signaling pathway, which may increase the stimulated cell's chemotaxis [25][26][27].
Numerous reports have investigated MSCs as cells that express TLRs. According to previous findings on mesenchymal and hematopoietic stem cells, TLRs engagement may function in stem cell biology, particularly enhancing their migratory ability. As a critical factor in innate immune response, TLRs have been introduced to drive MSC homing by regulating a range of chemokine receptors such as CXC and CC receptors and activating integrins and other adhesion molecules involved in cell migration. As a result, TLR activation might be one strategy that promotes MSC recruitment and migration in wounded or stressed regions [28][29][30][31]. The discovery of TLRs as essential mediators of stress responses inside MSCs establishes a new component of their biology. It gives a unique target for improving stem cell-based treatment technics.
Previous studies did not investigate the involvement of TLR3 in significant migratory responses of stem cells in a tumor model, which we are focusing on here. In the current study, relying on the TLR-ligand-activated pathway, we aimed to determine whether TLR3 stimulation influences the potential of MSCs migration and recruitment to the neoplasm environment in a melanoma mouse model.

Adipose-derived MSCs (ADMSCs) preparation and culture
Using collagenase type I digestion protocol [32], ADM-SCs were prepared from 6 to 8 weeks male C57BL/6 GFPtransgenic mice (purchased from Stem Cell Technology Research Center, Tehran, Iran) abdominal fat. Briefly, after cervical dislocation of the animal, isolated abdominal fat was collected in sterile condition and washed with phosphate buffer saline (PBS) (Sigma-Aldrich, St. Louis, Missouri, USA) containing 1% penicillin/streptomycin antibiotic (Sigma-Aldrich; St. Louis, Missouri, USA). After that, samples were cut into small pieces and then digested with 0.075% collagenase type I (Gibco, Waltham, Massachusetts, USA) diluted in Dulbecco's Modified Eagle's Medium-high glucose (DMEM), (Sigma-Aldrich, St. Louis, Missouri, USA) without fetal bovine serum (FBS) (Gibco, Waltham, Massachusetts, USA) for 30 min at 37 °C cell culture incubator [33]. The digestive process was neutralized by adding an appropriate volume (5 ml) of DMEM high glucose supplemented with 10% heat-inactivated FBS and centrifuged for 15 min at 300×g. Cellular pellet was resuspended and washed two times under the same previous condition to remove undigested tissues. Finally, the cellular pellet was resuspended and cultured in tissue culture flasks in a complete cell culture medium consisting of high glucose concentration containing 10% FBS, 2 mmol/l l-glutamine (Sigma-Aldrich, St. Louis, Missouri, USA), and 1% penicillin/streptomycin at 37 °C with 5% CO 2 humidified incubator [34]. The medium was replaced with a fresh medium to remove non-adherent cells after 72 h of starting culture. The fibroblast-like appearance of the cells isolated from fat was confirmed by an inverted microscope (Zeiss, Jena, Germany). After reaching the 80% confluency, the cells were passaged using 0.25% Trypsin/0.02% EDTA (Gibco, Waltham, Massachusetts, USA) for further expansion. ADMSCs were used for 2 weeks expansions at passages 2-3 for all experiments to maintain consistency.

Immunophenotyping of ADMSCs
Flow cytometry was used to validate the MSCs phenotype, which was determined by the presence of CD105, CD90, and CD73 antigens in the lack of CD45, CD34 antigens using purchased antibodies [BD Biosciences (San Diego, CA, USA)]. According to the standard protocol, the surface staining of the cells was performed, and samples were characterized using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, New Jersey, USA). The flow Jo software (Ashland, Oregon, USA) was used to analyze the data.

Preconditioning of TLR3
TLR3 preconditioning of ADMSCs was performed using poly(I:C) (1 µg/ml, 10 µg/ml) (Sigma-Aldrich, St. Louis, Missouri, USA) as the agonist [35,36]. 5 × 10 4 cells were seeded in 24 well plates per well to reach 70-80% confluency. After that, the culture medium was removed, and the cells were washed with PBS and treated using poly(I:C) prepared in a complete culture medium for 1 and 4 h for all mentioned concentrations. The control cells were incubated with only a complete medium. After incubation time points, cells were washed with PBS three times and incubated in a complete medium for further experiments.

Cell proliferation assay
MTT (3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyl tetrazolium bromide) (Sigma-Aldrich, St. Louis, Missouri, USA) was utilized to monitor the cell growth. MSCs are pre-conditioned under a variety of settings, as previously stated. After that, each control and poly(I:C)-pre-treated cell received 500 µl of 5 mg/ml MTT solution 24 h, 48 h, and 72 h after treatment. After a 4 h incubation period, the medium was sucked off, and the crystals of formazan were dissolved in 150 µl dimethyl sulfoxide (DMSO) (Merck, Readington, New Jersey, USA). The absorption spectrum at 570 nm was measured using an ELISA plate analyzer. Samples were examined in triplicate in this investigation.

RNA purification and quantitative real-time PCR
Following the manufacturer's instruction, the TriPure isolation reagent (Roche, Basel, Switzerland) was used to isolate the total RNA after priming the cells with poly(I:C).
A NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, Massachusetts, USA) was used to quantify RNA concentration, and the DNA decontamination process with a DNase kit (Thermo Scientific, Waltham, Massachusetts, USA) eradicated any potential DNA contamination in the RNA sample. After then, cDNA was made from 1 µg of RNA using a RevertAid First Strand cDNA Synthesis kit (Thermo Scientific, Waltham, Massachusetts, USA). The Rotor-Gene 6000 Real-Time PCR System (Corbett Research, Hilden, Germany) was used to perform the test using the Maxima SYBR green master mix (Fermentas, Waltham, Massachusetts, USA). Denaturation at 95 °C for 15 s and annealing and elongation at 60 °C for 60 s was used to amplify DNA in a 40-cycle PCR reaction. The fold change was computed compared to the untreated control cells after correcting for the TATA-Box binding protein (TBP) reference gene. Relative gene expression levels were calculated utilizing the 2 −ΔΔCt method [33]. Representative data sets are provided for each sample examined in triplicate. Primer sequences are available in supplementary Table 1.

Transwell migration/invasion assay
For invasion assay, pre-treated ADMSCs were plated in Matrigel (Corning, New York, USA) pre-coated 24-well transwell inserts with 8.0 μm pore membrane filters (Corning, New York, USA). Briefly, the insert's filters were coated with diluted Matrigel to imitate the basement membrane and then incubated for 24 h at 37 °C. Pre-conditioned and untreated MSCs were seeded on the upper chamber of inserts (10 4 cells in 250 µl serum-free culture medium). 5 × 10 4 B16-F10 melanoma cells were plated in the lower chamber of the transwell containing the 10% FBS culture medium. After 24 h of incubation at 37 °C and 5% CO 2 , the upper chamber culture medium was removed and washed with PBS three times. The cells that reached the lower surface of the filter were fixed with methanol and stained with 2% crystal violet (Sigma-Aldrich, St. Louis, Missouri, USA) [37]. The migrated MSCs were detected using a microscope in × 10 magnification (Zeiss, Jena, Germany), and the number of cells was determined using the ImageJ software (National Institutes of Health, USA) [38].

Western blot analysis
After preconditioning, the MSCs were rinsed with a cold PBS/phosphatase inhibitor/protease inhibitor cocktail (Sigma-Aldrich, St. Louis, Missouri, USA). Then the nuclear extract was made by adding 250 µl of hypotonic buffer to the 4 × 10 6 treated MSCs. The lysed cells were centrifuged at 14,000×g for 1 min at 4 °C. The supernatant was removed, then the cellular pellet was resuspended in 50 µl complete lysis buffer, protease/phosphatase inhibitor cocktail, and centrifuged at 14,000×g for 15 min at 4 °C. The supernatant was collected, and protein concentration was measured by Bradford protein assay (Bio-Rad, Hercules, CA, USA).
Equal quantities of extracted nuclear fraction were separated on 10% SDS-PAGE. Then electro-transferred onto a nitrocellulose membrane (Hybond-ECL, Amersham Corp, UK) using a semidry transfer cell (Bio-Rad, Hercules, CA, USA) for subsequent immunoblotting. Membranes were then blocked for 1 h at room temperature with 5% nonfat dry milk in TBS containing 0.1% (v/v) Tween-20 (TBST) prior to being analyzed with diluted specified primary antibodies (Amersham Pharmacia Biotech, United Kingdom) overnight at 4 °C. The membranes were rinsed three times in TBST and then treated with horse radish peroxidase-conjugated (HRP) secondary antibodies. Then we used the chemiluminescence detection method to view the proteins (Amersham ECL Advance Kit, GE Healthcare, UK) [39]. ImageJ software (National Institutes of Health, USA) was used for densitometric quantification.

In vivo experiment
Six to eight weeks male C57BL/6 mice were supplied from the Iran University of medical sciences, center for experimental and comparative studies. The animals were cared by the ethical principles of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the ethics committee of the Iran University of Medical Sciences (Code of Ethics: IR.IUMS. REC.1397.1172). The experiment was repeated twice with 10 mice (5 mice for laboratory experiments and 5 for survival) in each group. Each mouse received 3 × 10 5 B16-F10 cells via tail vein injection to establish a melanoma tumor in the lung. Tumors formed in the lungs after 10 days. To verify the neoplasm formation in the lung, the small animal positron emission tomography scan (PETs) technic was employed with a micro-PET scanner (Xtrim PET) at the Preclinical Core Facility (TPCF) based at the Tehran University of Medical Sciences. The mice were injected through the tail vein with about 300 µCi of the 18FDG under general anesthesia. For each small animal PET scan, qualitative analysis was performed to examine the model or metastasis. Subsequently, after confirming the tumor establishment in the lungs, mice were intraperitoneally (IP) injected with 10 6 cells from two different GFP + ADMSC groups separately: untreated ADMSCs (n = 10) and poly(I:C)-treated ADMSCs (n = 10) (10 µg/ml for 1 h treatment).

GFP tracking using live imaging
The in vivo imaging was performed using the KODAK imaging system (FX Pro, Rochester, New York, USA) with florescent mode and 5 min exposure time to track the localization of GFP + ADMSCs 3, 10, and 14 days after IP injection (n = 5 in each group). The excitation and emission filters were set to 470 and 535 nm, respectively. The light emitted from the mice was captured, integrated, processed, and displayed by the KODAK camera system. The black-white and color photos were merged to capture fluorescence signaling. Then, the pseudo color format overlapped on the standard light image. Also, due to the high autofluorescence generated from the animal's hair, only fluorescent signals from the region of interest are shown.

GFP tracking using real-time PCR
Tumor-bearing animals (n = 5 in each group) injected with two different treated and untreated GFP + MSCs were sacrificed at 3, 10, and 14 days after the administration of the GFP + cells, and lung tissue was harvested from all groups. The homogenizer was used to homogenize the taken tissue, and using the QIAamp DNA Mini Kit (Hilden, Germany), DNA was extracted according to the manufacturer's procedure. The GFP amount was quantified using the Maxima SYBR green master mix (Fermentas, Waltham, Massachusetts, USA) and Rotor-Gene 6000 Real-Time PCR System (Corbett Research, Hilden, Germany). In a 40-cycle PCR reaction, DNA was amplified by denaturation at 95 °C for 15 s, annealing, and elongation at 60 °C for 60 s. Internal control was TATA-Box binding protein (TBP). Primer sequences are available in supplementary Table 2.

Statistical analysis
GraphPad Prism software was employed to do statistical analysis (GraphPad, San Diego, CA, USA). Student's t-tests (between two groups) and one-way and two-way ANOVAs (for more than two groups) were used to determine statistical significance. Statistical significance was defined as a P value of less than 0.05. The mean and ± standard deviation are used to show the data.

Immunophenotyping confirmed the characteristic of ADMSCs
Following 4-5 days after initial culture, single adherent cells with spindle fibroblast-like appearance derived from adipose mass expanded. After two passages, the isolated cells had shown identical morphology and proliferated ( Fig. 1a  and b). Flow cytometry was utilized to assess the immunophenotypic pattern of cells. As a result, the separated cells expressed CD105, CD90, and CD73, while CD45, CD34 were not. The data have determined the specifications required to identify isolated cells as MSCs (Fig. 1c).

Poly(I:C) had minimal effect on the cell growth potential of ADMSCs
The MTT test was used 24 h following the treatment with poly(I:C) to evaluate the proliferation of ADMSCs. According to our findings, after 1, 2, and 3 days, treated cells exhibited a minimal decreasing effect on cell growth compared to untreated control ADMSCs. Notably, the treated cells with 10 µg/ml poly(I:C) for 4 h demonstrated a higher decrease in the proliferation rate, as shown in the data (Fig. 2). Fig. 1 a and b Mesenchymal stem cells isolated from adipose mass demonstrated fibroblast-like shapes under a fluorescent and inverted microscope. c According to flow cytometry analyses, the cells were positive for CD105, CD90, and CD73 while negative for CD34, and CD45 Stimulation of TLR3 using poly(I:C) prompted the NF-κB activation It was expected that following the TLRs stimulation, NF-κB, a downstream factor, would be activated in the related pathway. The western blot examination clarified the stimulation of TLR3 using related agonists, resulting in the increased amount of p65 subtype of NF-κB protein in the nuclear fraction, which confirmed the appropriate stimulation of TLR3 related receptor (Fig. 3a). The ratios of NF-κB p65 protein to β-Actin were graphed (Fig. 3b).

Poly(I:C) treatment enhanced the integrin cell-adhesion molecules and, anti-inflammatory cytokine expression in ADMSCs
The qPCR was performed to demonstrate the expression level of IL-10, TGF-β anti-inflammatory cytokines, and α4, α5, β1 integrins in ADMSCs previously pre-conditioned with poly(I:C) as a TLR3 agonist. TATA Box Binding Protein (TBP) primer was utilized as a housekeeping gene to normalize data. The present research used three different doses and time points of poly(I:C) treatment conditions. Firstly, the α4, α5, and β1 integrins expression was measured since the essential function of integrins in MSCs migration was previously shown [40]. It was discovered that MSCs exposed to 10 µg/ml poly(I:C) for 1 h expressed more α4, α5, and β1 integrins compared to the untreated cells (Fig. 4a). It was also found that poly(I:C) treated ADMSCs with the mentioned concentration above express an amplified amount of IL-10, known as an antiinflammatory cytokine but not significantly TGF-β mRNA level (Fig. 4b).

Poly(I:C) treatment enhanced the migration index of ADMSCs in vitro
The transwell invasion experiment evaluated the influence of treatment on the invasion ability of the pre-conditioned ADMSCs in reply to B16-F10 cell line stimuli. According to the test, we identify that the poly(I:C) treatment (10 µg/ml for 1 h) had a statistically significant impact on MSCs invasion compared with the untreated cells, as reported in Fig. 5. The migrated cells under a microscope (× 10 magnification) (Zeiss, Jena, Germany) were evaluated ( Fig. 5a and b). The ImageJ software (National Institutes of Health, USA) was applied to count the migrated cell numbers. Finally, the ratio of migrated poly(I:C) treated to migrated untreated control cells is regarded as the migration index (Fig. 5c).

Poly(I:C) treatment increased the ADMSCs homing in lung melanoma tumor
We employed the micro-PET technic to ensure the formation of melanoma tumor in the lung subsequently to tail intravenous (IV) injection of the B16-F10 melanoma cell line in C57BL/6 mice. It was found that tumors had developed in the lung 10 days after the cell line inoculation (Supplementary Fig. 1a). To realize the movement of ADMSCs, we infused GFP + cells into melanoma-affected mice intraperitoneally. The injected cells were tracked on days 3, 10, and 14 after administration using the KODAK imaging system with fluorescent mode. As shown in this study, poly(I:C) exposure (10 µg/ml for 1 h) promoted the migration of stimulated cells when compared to the untreated cells. GFP + MSCs produced a distinctive biodistribution pattern in the peritoneum within Fig. 2 The poly(I:C) treatment had a statistically minimal impact on MSCs' growth rate. Proliferation assay indicated that MSC subjected to 10 µg/ml poly(I:C) for 4 h (poly10-4) showed significantly decreased proliferation compared to untreated cells and other treatment conditions. (1 µg/ml poly(I:C) for 1 h (poly1-1), 1 µg/ml poly(I:C) for 4 h (poly1-4), 10 µg/ml poly(I:C) for 1 hour (poly10-1), control (ctl)). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. untreated control Fig. 3 a The activation of the TLR3 signaling pathway after poly(I:C) treatment was investigated using the western blot assay. b Western blotting showed that the nuclear fraction of p65 NF-κB enhanced following TLR3 stimulation. MSCs treated with 10 µg/ml poly(I:C) for 4 h exhibited a slightly lower amount of p65 NF-κB than other conditions. The ratios of p65 to β-Actin are presented. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. untreated control three days. The overall signal was considerably diminished 10 days after cell injection. On day 10, the fluorescent signal measured at the tumor location with poly(I:C) treated cell infusion was higher than that of the untreated control cells. Noticeably, there was no discernible signal in mice 14 days following the treated and controlled ADMSCs administration ( Supplementary Fig. 1b).

Detected genomic GFP DNA isolated from the tumor-bearing lungs confirmed the homing of ADMSCs using GFP tracking Real-time PCR
In the current study, real-time PCR was used to examine for any trace of infused cells in lung tumor melanoma to confirm and improve the sensitivity of in vivo imaging findings. DNA was isolated from the lung tissues from different groups of animals, and quantity of GFP DNA was reported utilizing real-time PCR. The DNA sample is taken from tissues, displaying GFP concordance with in vivo imaging on days 3 and 10 after cell injection. Notably, it was discovered that the tissues harvested from the groups treated with poly(I:C) had mild positive GFP signals on day 14 after infusion compared with untreated cells (Supplementary Fig. 2).

Discussion
Experimental research has examined the use of MSCs in tissue regeneration in various conditions and cancers as anticancer treatment carriers [41,42]. The therapeutic effectiveness of MSCs as anti-tumor vehicles relies on their capacity to migrate and home to the desired neoplasm region, and comprehending the mechanisms that control MSCs recruitment to tumors is critical for achieving this purpose [19].
The current research examined TLR3 stimulation and consequent effects on MSCs' migratory potential to learn about the influence of stress signals via TLR's ligands on Fig. 4 TLR ligand engagement resulted in specific cytokines conditions and cell-adhesion molecules as detected by real-time PCR expression analysis. a TLR3 stimulation enhanced the expression level of integrins and b anti-inflammatory cytokines in various conditions. Notably, the poly(I:C) treatment with 10 µg/ml for 1 h sub-stantially affects the expression pattern of intended genes. TATA Box Binding Protein (TBP) was utilized as a housekeeping gene to normalize data. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. untreated control the mobilization of cells and engraftment at inflamed tumor locations in a mouse-bearing lung melanoma tumor. The presented findings demonstrated that TLR3 priming with poly(I:C) (10 µg/ml for 1 h) as the related agonist led to enhanced migration of ADMSCs to tumor areas compared with untreated control cells. Here, the approach to using the TLRs ligands to precondition the MSCs was based on prior research that found that activation of MSCs may enhance the movement of these cells. For example, according to existing evidence, MSC stimulation with proinflammatory cytokines (e.g., IL-β1 and TNF-α) before administration improves theirs in vivo migratory and adhesion potential via activation of the vascular cell adhesion molecule-1/very late antigen-4 (VCAM-1/VLA-4) adhesion pathway (27). Moreover, several growth stimuli induce MSC migration. As previously reported, the Insulin-like growth factor 1 (IGF-1) promotes migration by increasing the expression of chemokine receptors, including CCR5-the RANTES (CCL5) on MSCs [43]. It was also demonstrated that growth factors, including basic fibroblast growth factor (b-FGF) and vascular endothelial growth factor (VEGF), which were released under hypoxemic stress, enhanced migratory propensity through a phosphoinositide-3 kinase/AKT (PI3-kinase/AKT) pathway downstream of the b-FGF receptor on the MSCs [44]. Additionally, it has been shown that the combined potential of VEGF and platelet-derived growth factor ab (PDGFab) PDGF acting as chemo-attractants induce MSCs migration; this composition of growth factors is more effective than the each of individual growth factors [45].
On the other hand, earlier studies found that humans and mice express TLRs in immune and other non-immune hematopoietic and mesenchymal stem cells [24]. TLRs signaling activates numerous pathways, including mitogenactivated protein kinase (MAPK), myeloid differentiation primary response 88 (MyD88), c-Jun N-terminal kinase (JNKs), an inhibitor of kB kinase (IkB kinase), which leads to the stimulation of transcription factors nuclear factor-kB (NF-kB) and activator protein-1 (AP-1), which then leads to the production of pro-inflammatory or anti-inflammatory chemokines and cytokines [27]. According to these researches, TLRs are involved in MSCs' stress response and migration. It has also been found that TLR3 activation causes human BM-MSCs to migrate in vitro, according to Tomchuck et al. [24].
In this experiment, we examined four different poly(I:C), the TLR3 agonist, statuses based on concentration and exposure time. Because of the previous finding on the essential function of integrins in MSCs migration, we employed realtime PCR to identify the expression of β1, α4, and α5 integrins. In contrast to the critical involvement of C-X-C motif chemokine receptor 4/stromal derived factor 1 (CXCR4/ SDF1) in hematopoietic stem cells (HSCs) migration and the role in directing the migration of various tumor cell lines to metastatic locations, Ip and colleagues found that inhibiting the CXCR4 receptor did not affect MSC migration. They discovered integrin β1 but not integrin α4 or CXCR4 as an element for BM-MSC intramyocardial migration and engraftment [40]. Integrins have been essential in cell adhesion, migration, and chemotaxis. They also implied that similar MSCs and leukocyte extravasation to reach the site of inflammation required repeated bonding and deadhesive processes using integrins [40]. After being driven Fig. 5 The transwell invasion experiment showed that the a poly(I:C) treatment with 10 µg/ml concentration for 1 h (poly10-1) significantly influenced ADMSC migration behavior compared with b the control cells. Different groups of invading cells under a phasecontrast microscope (× 10 magnification) were illustrated. c The ratio of migrated poly(I:C) treated to migrated untreated control cells is regarded as the migration index. ***P < 0.001 vs. untreated control by a chemotactic shift, the leukocytes must pass the extracellular matrix (ECM) via impermanent contacts between integrin receptors and ECM components that serve as adhesive ligands. Neutrophil motility has been linked to CD29 (β1-integrin) and CD18 (β2-integrin) family groups. CD29 is also implicated in cell-to-cell adhesion, which might be essential for the attachment of engrafted cells [40]. To determine the contribution of preferred chosen molecules, they have assessed the inhibiting impact of monoclonal antibodies against CXCR-4, CD29, and CD49. They discovered that pretreatment with an antibody against CD29 dramatically reduced the amount of BM-MSCs that engrafted and moved into the ischemic myocardium, indicating that CD29 is essential for stem cell cardiac engraftment [40]. Similar to this, prior research found that CD29 inhibition reduced neutrophil migration to the inflammatory lung tissue [46]. As detected here, pre-conditioning with poly(I:C) (10 µg/ml for 1 h) resulted in upregulation of β1, α4, and α5 integrin in mRNA. The presented real-time PCR findings also imply that poly(I:C) pre-treatment of MSCs is likely to produce an anti-inflammatory milieu due to the significant enhancement in the anti-inflammatory IL-10 expression. Initially, it was postulated that the anti-inflammatory property of MSCs consequent with TLR3 ligand exposure might influence the mobility tendency to the tumor region as the unhealed inflamed environment. This conclusion is consistent with an earlier study that suggested that MSCs migration can occur considering the cytokine milieu [47]. Remarkably, we demonstrated that at the same time, the integrins might coordinate with the anti-inflammatory feature of MSCs, which proposed that the cytokine context of cells could determine the potential cell migration. These findings finally led us to select 10 µg/ml poly(I:C) for the 1 h treatment condition for our following experiments.
In addition, it was discovered that MSCs express distinct amounts of anti-inflammatory cytokines related to the concentration and exposure time of poly(I:C). Interestingly, in high concentrations with an extended time of treatment (10 µg/ml for 4 h), expression of α4, α5, β1 integrins and, also IL-10 and TGF-β were decreased as compared with other conditions. It was previously discovered that the stimulant concentration and the length of time exposed impact human MSCs (hMSCs) migration [35]. As evidenced by the prior report, poly(I:C) (1 µg/ml) treatment for 48 h has no significant enhancement in the migration of cells in a rat ligament model. It was also reported that TLR3 stimulation over 24 h in hMSCs resulted in reduced migration, while activation for a short time increased migration [48]. Similar research used poly(I:C) to stimulate the hMSCs in vitro for even less time (4 h) and found enhanced cell migration [24]. In other studies, on porcine MSCs, researchers employed a high concentration of poly(I:C) (4 µg/ml), and they found no difference in migration in vitro after 24 h of exposure to poly(I:C). Moreover, after exposing hMSCs to poly(I:C) (10 µg/ml) for 6 h, researchers observed the expression of two critical molecules involved in cell migration (CXCR4, CXCR7) was drastically down-regulated [49].
Additionally, an investigation indicated that administration of poly(I:C) (4 µg/ml for 24 h) had a statistically insignificant effect on MSCs migration in vitro. Western blot analysis displayed that poly(I:C) dramatically reduced integrin β1 expression, but CXCR4, integrin α5, and integrin αV were unaffected [49]. According to the abovementioned investigations, higher doses or longer exposures to poly(I:C) may reduce migratory capacities in vitro. They believe that the short-time, low-concentration of related agonist poly(I:C), simulates the gradient of danger signals that endogenous MSCs confront, react and attract to the proper distant location.
Notably, in the present investigation, the short exposure time (1 h) along with a partially low concentration (10 µg/ ml) of poly(I:C) along with the elevated expression level of β1, α4, and α5 integrins enhanced the migratory quality of ADMSCs in transwell migration/invasion experiment and the melanoma mouse model. It is also comparable with prior findings that demonstrated the critical role of β1-integrin in MSCs movement.
As displayed in the current investigation, following intraperitoneal (IP) administration, TLR3 priming with poly(I:C) leads to enhanced migration of ADMSCs to tumor areas compared with the untreated control cells. In vivo, live imaging surveys were conducted 3, 10, and 14 days after cell infusion to investigate the homing and migration rate of treated and untreated ADMSCs in the mouse model. We found that poly(I:C) pretreated cells with overexpression of β1, α4, and α5 integrins exhibited more frequency in the tumor region than untreated cells.
We reported the fluorescent signals related to the migrated cells to the tumor site. However, due to the different characteristics of photon absorption and scattering with tissue depth in fluorescent optical imaging systems, the reliable existence of GFP + ADMSCs is not attainable in the tumor region. As a result, the lack of fluorescent signals from GFP + ADMSCs does not certainly translate to the absence of the cells and is thus not comparable [50].
We quantified the migrated cells towards a developed lung melanoma tumor by employing a real-time PCR procedure for tracking the administrated ADMSCs GFP DNA. We discovered that the results coincided with in vivo animal imaging. However, there was evidence of migrated GFP + ADMSCs after 2 weeks of administration that we could not detect by live fluorescent imaging.

Conclusion
The current research has identified that TLR activation causes the stress response in MSCs, leading to increased migration ability in the tumor model. Depending on the TLR-ligand used, these activated pathways drive the distinct cytokine expression patterns and migratory behavior of MSCs.
Different factors, including the receptors and their ligands (chemokines, cytokines, growth factors, and small molecules) and cell adhesion molecules, have been identified for MSCs migration. However, they have not been recognized as a single primary process; instead, they may work together in a complementary way. Additionally, a better knowledge of the signaling axes that govern MSCs trafficking may enhance the overall effectiveness of cell-based treatment approaches.