Effects of tumor treating fields (TTFields) on human mesenchymal stromal cells

Purpose Mesenchymal stromal cells (MSCs) within the glioblastoma microenvironment have been shown to promote tumor progression. Tumor Treating Fields (TTFields) are alternating electric fields with low intensity and intermediate frequency that exhibit anti-tumorigenic effects. While the effects of TTFields on glioblastoma cells have been studied previously, nothing is known about the influence of TTFields on MSCs. Methods Single-cell RNA sequencing and immunofluorescence staining were employed to identify glioblastoma-associated MSCs in patient samples. Proliferation and clonogenic survival of human bone marrow-derived MSCs were assessed after TTFields in vitro. MSC’ characteristic surface marker expression was determined using flow cytometry, while multi-lineage differentiation potential was examined with immunohistochemistry. Apoptosis was quantified based on caspase-3 and annexin-V/7-AAD levels in flow cytometry, and senescence was assessed with ß-galactosidase staining. MSCs’ migratory potential was evaluated with Boyden chamber assays. Results Single-cell RNA sequencing and immunofluorescence showed the presence of glioblastoma-associated MSCs in patient samples. TTFields significantly reduced proliferation and clonogenic survival of human bone marrow-derived MSCs by up to 60% and 90%, respectively. While the characteristic surface marker expression and differentiation capacity were intact after TTFields, treatment resulted in increased apoptosis and senescence. Furthermore, TTFields significantly reduced MSCs’ migratory capacity. Conclusion We could demonstrate the presence of tumor-associated MSCs in glioblastoma patients, providing a rationale to study the impact of TTFields on MSCs. TTFields considerably increase apoptosis and senescence in MSCs, resulting in impaired survival and migration. The results provide a basis for further analyses on the role of MSCs in glioblastoma patients receiving TTFields. Supplementary Information The online version contains supplementary material available at 10.1007/s11060-024-04740-0.


Introduction
Mesenchymal stromal cells (MSCs) can be found in different tissues including the microenvironment of various cancers [1][2][3].In glioblastoma, the number of glioma-infiltrating MSCs have been shown to inversely correlate with patient survival [3].Glioblastoma-associated MSCs promote proliferation, invasiveness and angiogenesis of glioblastoma cells [4,5], and mitochondrial transfer from MSCs to glioblastoma stem cells contributes to resistance against temozolomide [6].The origin of glioblastoma-associated MSCs remains a matter of debate: differentiation from glioblastoma stem cells, epithelial-to-mesenchymal transitionlike processes from astrocytes, transdifferentiation from pericytes and vascular smooth muscle cells, or migration of bone marrow-MSCs towards glioblastomas have been discussed [7].
Given the pro-tumorigenic effects of glioblastoma-associated MSCs, we aimed to examine the role of MSCs in glioblastoma patients and to explore the influence of TTFields on MSCs in vitro.

Single-cell RNA sequencing
For single-cell analysis, we used the GBMap reference dataset [25,26].Single-cell data were processed using the Seurat (v5.0) package in R software.To identify the MSC population within the single cell data, we first filtered the cells by the expression of classical positive marker genes for MSCs (NT5E, THY1, ENG, and ITGB1) [27].We built a meta score using the Seurat function Seurat::AddModuleScore().Next, we removed all cells containing high expression (> 50% quantile) of MSC-negative marker genes (CD14, CD34, PTPRC) and subsequently, all cells bearing aneuploid chromosomal alterations (based on the inferCNV [copy number variations] estimations) to avoid contamination with tumor cells.

Immunofluorescence staining
In a cohort of 11 glioblastoma patients, all treated with surgical resection, (chemo)radiation and TTFields treatment, the expression of the MSC markers CD73, CD90 and CD105 was analyzed at initial diagnosis.Formalin-fixed paraffinembedded tissue sections of 4 μm thickness were mounted on glass slides for deparaffinization and heat-induced antigen retrieval at pH 9, subsequent blocking, indirect immunofluorescence staining (Supplementary Table 1) and DAPI staining (Carl Roth, Karlsruhe, Germany).Randomly selected representative images from the lesion area were acquired on a Keyence BZ-X810 fluorescence microscope (Keyence Corporation, Osaka, Japan) in quadruplets.Total cell numbers were determined using the particle analyzation plug-in for ImageJ (National Institutes of Health, Bethesda, USA).Cells larger than 10 μm and equally expressing CD73, CD90 and CD105 were counted.

Cell culture
Human MSCs were isolated from the bone marrow of three healthy donors and cultured in StemMACS™ human MSC Expansion Medium (Miltenyi Biotec, Bergisch-Gladbach, Germany), supplemented with 1% (v/v) penicillin/streptomycin [28,29].Written consent was obtained before bone marrow sampling, and the investigation was approved in advance by the Heidelberg University Ethics Committee (S-384/2004) and the Freiburg University Ethics Committee (436/20).
In order to compare the results obtained with MSCs, HS68 (RRID: CVCL_0839) fibroblasts were used as control cell population.HS68 fibroblasts were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco's Modified Eagle Medium (DMEM), containing 1 g/L glucose and GlutaMAX™ (Gibco, Carlsbad, USA), supplemented with 10% fetal bovine serum (FCS) and 1% penicillin/streptomycin.
All cells were maintained at 37 °C in a humidified incubator with 5% CO 2 , and the medium was changed twice a week.

TTFields
TTFields were applied using the inovitro™ system (Novocure, Haifa, Israel) [30].Cells were exposed to the electric fields for 72 h prior to analyses.2 × 10 4 cells were plated on cover slips and left to adhere overnight prior to TTFields application.For proliferation and viability analyses, TTFields were applied at an intermediate (~ 1,33 V/cm root-mean-square (RMS)) and a high (~ 1,62 V/cm RMS) field intensity, and several frequencies within the range of 100-500 kHz were used.For all other endpoints, cells were treated at an intensity of 1.33 V/cm and the clinically applied frequency of 200 kHz.

Proliferation, clonogenic survival, and viability
After TTFields treatment, cells were detached using trypsin and counted using a Neubauer chamber.Trypan blue staining was performed to select viable cells.
For clonogenic survival analyses, cells were treated with TTFields and then replated in T25 cell culture flasks.Cells were allowed to divide for 10-14 days before fixation and staining using a methanol/crystal violet solution, as described before [31].Colonies with ≥ 50 cells were counted with a light microscope.Cellular survival fractions were calculated using the formula (no. of colonies/ no. of cells plated) treated /(no. of colonies/ no. of cells plated) untreated .
Cellular viability was determined using resazurin assays.After TTFields treatment, 2 × 10 3 cells, suspended in 100 µL of the respective cell culture medium, were replated in a 96-well plate.After 96 h, 10 µL of 0.3 mg/mL resazurin (PromoCell, Heidelberg, Germany) was added to each well and incubated for 4 h.Colorimetric analyses were performed by measuring light absorbance at 570 nm and 600 nm using a VersaMax microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA).To quantify cellular capacity to metabolize resazurin, background absorbance at 600 nm was subtracted from absorbance at 570 nm.
To further differentiate between early and late apoptosis, annexin V/7-aminoactinomycin D (7-ADD) measurements were performed.Viable MSCs and fibroblasts were harvested after being exposed to TTFields for 72 h and stained with the PE-Annexin-V Apoptosis Detection Kit with 7-AAD (BioLegend, London, UK) following the manufacturer's protocol.
Flow cytometry measurements were performed using a FACSVerse™ (BD Biosciences, San Jose, CA, USA) and quantified using FlowJo™ v10 (FlowJo LLC).Senescence 2 × 10 3 were plated on glass cover slips and exposed to TTFields for 72 h, before cover slips were transferred to a 24-well plate to be fixed and stained using the Senescence β-Galactosidase Staining Kit (Cell Signaling Technology, Danvers, MA, USA).Nuclei were counterstained with 1 µg/ mL DAPI/PBS.Five randomly chosen regions of interest (ROI) per technical replicate were photographed using an Olympus IX51 microscope (Olympus Corporation, Tokyo, Japan).Senescent cells were manually counted in each ROI, while the total number of cells were automatically determined using ImageJ.

Cell cytoskeleton
As TTFields are known to alter the cytoskeletal structure of glioblastoma cells, thereby impairing the migratory potential, we performed F-actin immunofluorescence staining to examine the influence of TTFields on the cytoskeleton of MSCs.After TTFields treatment, 1 × 10 3 cells were plated on glass cover slips in 24-well plates.Cells were fixed using a 3% PFA/PBS solution, permeabilized with 70% ethanol at -20 °C for 30 min, and stained with AF488-conjugated phalloidin (Invitrogen, Waltham, MA, USA), diluted in 1% BSA/PBS, for 60 min at room temperature.Cell nuclei were counterstained with 1 µg/mL DAPI/PBS.

Migration
Boyden chamber migration assays based on an FCS-gradient were performed to examine the effect of TTFields on MSCs' migratory potential.To sensitize the cells to the gradient, MSCs and fibroblasts were cultured in FCS-free DMEM for 48 h. 2 × 10 3 FCS-starved cells were then pipetted into a transwell insert (24 well ThinCert-TC Inserts, pore size 8 μm, Greiner Bio-One, Frickenhausen, Germany) and transferred to inovitro™ high wall dishes containing DMEM supplemented with 10% FCS (for MSCs) and 20% FCS (for HS68).Afterwards, high wall dishes were connected to TTFields base plates to initiate the treatment.After 24-hours TTFields treatment, cells that had not migrated were cleared off the upper membrane surface.Transwells were washed in PBS and placed in a 4% PFA/PBS solution for 15 min.Nuclei were stained with 1 µg/mL DAPI/PBS, included with StemMACS ™ ChondroDiff Media (Miltenyi Biotech).After 21 days, spheroids were fixed with 4% PFA/ PBS solution for 30 min, frozen at -20 °C and sectioned using a cryomicrotome.Sections were incubated in 1% alcian blue dissolved in 3% acetic acid solution for 30 min, and washed with 0.1 M hydrochloric acid, PBS and deionized water.

Statistics
At least three replicates were used for all experiments.Values are presented as mean with standard deviations.Potential differences between the TTFields and control group were examined using unpaired t-tests.Statistical analyses and visualization of the results were performed using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA).A p-value < 0.05 was considered as statistically significant.

Abundance of MSCs in glioblastoma
We accomplished MSC identification through single-cell RNA-sequencing and a dual marker strategy, utilizing positive (NT5E, THY1, ENG, ITGB1) and negative markers (CD14, CD34, PTPRC), followed by the exclusion of cells with aneuploid chromosomal alterations (Fig. 1a-c).After demonstrating the presence of these multipotent stromal cells within the glioblastoma microenvironment, we conducted immunofluorescence analyses of CD73, CD90 and CD105 in glioblastoma patient samples at initial surgery in order to validate these findings (Fig. 1d-e, Supplementary Table 2).Here, CD73/CD90/CD105-positive cells were found in 10 out of the 11 analyzed patients, with a mean abundance value of 0.5% (range, 0.0-2.2%).

TTFields reduce MSCs' proliferation and clonogenic survival
In vitro, TTFields reduced the proliferation rates of human bone marrow-derived MSCs by about 50% (Fig. 2a).While there was a frequency optimum at 100-200 kHz in HS68 fibroblasts, no frequency optimum could be found for MSCs.Proliferation curves showed no significant differences between the two tested field intensities in MSCs (p = 0.27 for MSC1, p = 0.06 for MSC2, p = 0.60 for MSC3, paired t-tests) and HS68 fibroblasts (p = 0.73).
MSCs exhibited reduced clonogenic survival rates after TTFields by up to 88.3% in MSC1, 75.0% in MSC2 and 73.1% in MSC3 (Fig. 2b).At 200 kHz, which is clinically used in glioblastoma treatment [33], HS68 fibroblasts and images of 4 ROIs per transwell were taken.Total cell number in these sections was determined using the particle analyzation plug-in for ImageJ.

Adhesion
TTFields-associated increase of cellular adhesion was investigated as previously described [13].The average time needed for detachment of untreated cells was determined for each cell line individually.Subsequently, TTFieldstreated cells were incubated in trypsin for exactly this period of time, and cells detached after this time span were counted using a Neubauer chamber.To compensate for generally lower cell numbers in TTFields-treated specimens, a second trypsinization step was performed until all treated cells were detached, and cells were counted again.The detachment rate was calculated by the formula (No. of cells detached) 1st trypsinization /((No. of cells detached) 1st trypsinization +(No. of cells detached) 2nd trypsinization ).

Surface marker expression
To examine whether TTFields affect the expression of the defining MSC surface marker pattern [27], flow cytometric measurements were performed as reported before [32].After harvesting, specimens were washed and resuspended in FACS buffer containing 0.5% BSA/2 mM ethylenediaminetetraacetic acid in PBS. 10 µL of MSC Phenotyping solution (MSC Phenotyping Kit, human, Miltenyi Biotec) was added, followed by incubation for 10 min.MSC markers were targeted using anti-CD73-APC, anti-CD90-FITC and anti-CD105-PE antibodies, whereas PerCP-conjugated anti-CD14/CD20/CD34/CD45-antibodies were used regarding MSC negative markers.Fluorescence signatures were measured on a FACSVerse™ and analyzed with FlowJo™ v10.
To analyze chondrogenic differentiation, 1 × 10 5 MSCs were transferred to each well of 96-well plates and allowed to form spheroids.Chondrogenic differentiation was Furthermore, the number of ß-galactosidase-positive cells was higher in all MSCs (p < 0.05 for MSC1 and MSC3, p < 0.01 for MSC2) following 72 h of TTFields treatment (Fig. 3c).The strongest increase was observed in MSC2 in which senescence levels doubled from 4.8 to 11.9%.
After TTFields treatment for 72 h, treatment effects on the cell cycle distribution of MSCs were small.While TTFields resulted in a minor G1 phase arrest in MSC1 cells (p < 0.01), there was no G1 phase arrest in MSC2 and MSC3 cells (Fig. 2C).

Discussion
In this comprehensive preclinical and translational study, we could demonstrate the presence of MSCs within the glioblastoma microenvironment.Preclinically, human MSCs were found relatively sensitive towards TTFields.Their migratory potential and adhesive abilities were hampered, potentially related to alterations in the MSC actin cytoskeleton caused by TTFields.Both apoptosis and senescence levels were found to be increased after TTFields in MSCs, whereas the differentiation ability and surface marker expression of MSCs were unaltered.
To the best of our knowledge, this is the first study in which the effects of TTFields on human MSCs were studied.Both single-cell RNA sequencing and immunofluorescence analyses identified CD73/CD90/CD105-positive cells in the glioblastoma microenvironment, providing a rationale

MSCs' stem cell characteristics remain unaltered after TTFields
All three investigated MSC samples were positive for CD73, CD90 and CD105.TTFields exposure for 72 h led to higher expression of these surface markers in MSC1-3 (Fig. 5a).As observed in flow cytometry analyses, cells were significantly larger after TTFields exposure, potentially contributing to the increased surface marker expression (Supplementary Material).As examined with immunohistochemical staining, exposure to TTFields did not mitigate the potential of MSCs to differentiate into adipocytes, osteocytes, and chondrocytes (Fig. 5b).RMS, root-mean-square also been described for other cell types, including glioblastoma, osteosarcoma, and liposarcoma cells [13,40,41].It has been demonstrated in a previous study that TTFields alter the organization and dynamics of microtubules and actin, thereby hampering both cellular adhesion and migration [13].These findings provide a preclinical rationale that TTFields also impair tumor cell metastatic activity, as observed in an in vivo study by Kirson and colleagues [18].Considering the fact that MSCs exhibit a strong tropism towards glioblastoma cells, one may hypothesize that the reduced migration ability of MSCs after TTFields could negatively impact MSCs' glioblastoma tropism [38].
In contrast to several cancer cells, MSCs did not exhibit a clear TTFields frequency optimum [12,42,43].It has been shown that the optimal frequency is dependent on cellular morphology and size; Kirson et al. observed an inverse relationship between cellular size and the optimal TTFields to study the influence of TTFields on MSCs [3,4,34,35].The majority of previous studies about TTFields focused on different cancer cells, while few studies examined TTFields' effects on normal tissue cells such as astrocytes and neurons [36].Previous studies could show that TTFields result in immunogenic cell death and furthermore induce STING and AIM2 inflammasome activation, thereby activating the adaptive immunity [15,16].As MSCs have been reported to exhibit immunosuppressive functions in the glioblastoma microenvironment, the anti-proliferative effects of TTFields on MSCs may further enhance the immunogenic potential of TTFields [37].
The migration of MSCs towards glioblastoma potentially contributes to the pro-tumorigenic ability of MSCs in the glioblastoma microenvironment [38,39].In our analysis, TTFields considerably impaired the migration capability of MSCs.A reduced migratory capacity after TTFields has In conclusion, we could show for the first time that TTFields considerably impair survival and migratory capacity of human MSCs.Our findings provide a basis for further analyses on the role of MSCs in glioblastoma patients undergoing TTFields treatment.Our results may also have implications for other cancer types in which TTFields are currently investigated, as tumor-associated MSCs are also found in the stromal compartment of many cancer entities [48].
frequency [42].A previous preclinical investigation in which the effects of TTFields on head-and-neck squamous cell carcinoma cells were examined, did not show a distinct frequency optimum in terms of maximal reduction of cell proliferation either [44].Although there was no clear frequency optimum, TTFields treatment with 200 kHz, as used for glioblastoma treatment in clinical settings, resulted in considerably reduced proliferation rates (about 50%) and clonogenic survival (80-90%) in MSCs.
A limitation of our study is the fact that we used bone marrow-derived MSCs instead of glioblastoma-derived MSCs.Bone marrow-derived MSCs have been discussed as potential source of glioblastoma-associated MSCs due to MSCs' strong glioma tropism, and there are some similarities described between the bone marrow and the glioblastoma stem cell niche [45][46][47].However, further studies  if changes were made.The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/.

Fig. 1
Fig. 1 Single-cell RNA sequencing and immunofluorescence reveal MSCs within the glioblastoma microenvironment.(a) Illustration of the workflow regarding identification of glioblastoma-associated MSCs.(b) Scatter plot of the dimensional reduction (UMAP) of the GBMap single cell dataset.Colors indicate the MSCs-score after filtering for positive and negative marker.(c) UMAP representation of the MSC cells after removal of cells containing chromosomal alterations.(d) Representative immunofluorescence staining images of CD73/

Fig. 2
Fig. 2 TTFields result in reduced proliferation and clonogenic survival in human MSCs.(a) Relative number of viable cells after 72 h of TTFields treatment depending on the electric field frequency in different MSCs and HS68 fibroblasts.Two different intensities (1.33 V/ cm RMS and 1.62 V/cm RMS) were used.(b) Clonogenic survival of MSCs and HS68 fibroblasts after TTFields with an intensity of 1.33 V/

Fig. 3
Fig. 3 TTFields lead to increased apoptosis and senescence in human MSCs.(a) Percentage of early and late apoptotic cells after TTFields as assessed by annexin-V/7-AAD flow cytometry analyses.Representative scatter plots show the shift towards more annexin-V-and 7-AADpositive cells after TTFields treatment compared with untreated controls.(b) Relative expression of cleaved caspase-3 in TTFields-treated

Fig. 5
Fig. 5 MSCs maintain their stem cell characteristics after TTFields.(a) Representative histograms of flow cytometry staining for the characteristic surface marker pattern of MSCs are shown, and mean fluorescence intensity (MFI) values of both MSC-negative (CD14, CD20, CD34, CD45) and MSC-positive marker (CD73, CD90, CD105) are presented.(b) Representative immunohistochemical images of adipogenic, osteogenic and chondrogenic differentiation of MSCs after