Cancer-associated fibroblasts (CAF) in the tumor microenvironment have a decisive influence on tumor growth and metastatic behavior. The cellular origins as well as the stimuli leading to CAF formation are heterogenous, impeding a precise characterization. Aim of this study was to analyze the influence of cytokines secreted in the process of wound healing, tumor cell-associated paracrine-secreted factors, and direct cell-cell contact on the expression of the CAF-associated markers fibroblast activation protein (FAP), α-smooth muscle actin (α-SMA), thrombospondin-1 (THBS1), and tenascin-c (TNC) by RT-PCR in mesenchymal stem cells (MSC). Cells developed different morphological characteristics after incubation with wound fluid (WF). Moreover, expression of FAP and α-SMA in MSC was significantly reduced after WF compared to tumor-conditioned medium and in co-culture with tumor cells; THBS1 and TNC were not significantly altered after any of the different incubation methods. There were no alterations of expression patterns of FAP and α-SMA in the immunohistochemical analysis. Differ­ences in the cytokine composition of the media were found in the dot blot. The heterogeneity of the results emphasizes the complexity of the interactions of tumor cells and cells of the microenvironment, particularly through the addition of human-derived WF.

Tumor tissue is composed of malignant and nonmalignant cells, of which the nonmalignant cells in the tumor stroma play a crucial role in disease progression in terms of tumor growth and metastatic spread. In addition to components of the extracellular matrix (ECM), the tumor microenvironment (TME) consists of a diversity of different cell types that can be divided into stromal cells and immune cells. Stromal cells include for example pericytes, adipocytes, endothelial cells, or mesenchymal stem cells (MSC) [Belli et al., 2018]. Another important stromal element of the TME is a certain subpopulation of fibroblasts called cancer-associated fibroblasts (CAF), which are considered active players in tumor progression. Tumor growth is a consequence of cell hyperplasia and uncontrolled proliferation. The resulting hypoxia, oxidative stress, and acidosis induce remodeling processes of the ECM in terms of an increased stiffness, and the formation of blood and lymphatic vessels. These alterations are orchestrated by autocrine and paracrine signal transduction between the components of the TME [Roma-Rodrigues et al., 2019]. Tumor cells can stimulate CAF to trigger these specific modifications in the TME like the homeostasis of the ECM. By the activation of pro-inflammatory signaling pathways, including the upregulation of nuclear factor kappa B (NF-κB), signal transducer and activator of transcription 1 and 3 (STAT-1 and -3), and transforming growth factor (TGF)-β/SMAD, changes of the composition of fibronectin, collagens, and ECM-degrading proteases, namely MMPs, are induced [Erez et al., 2010]. This leads to hyperplastic tissue growth and ameliorates cell migration and metastatic spread [Kessenbrock et al., 2013; Affo et al., 2021]. Furthermore, CAF promote an immunosuppressive environment [Costa et al., 2018]. These mechanisms are especially important for the development of drug resistance, making the specific targeting of CAF an interesting approach for the improvement of therapeutic regimes [Sahai et al., 2020].

However, the interaction with cancer cells is reciprocal. CAF have the potential to influence cancer cells by secreting factors like interleukin (IL)-6, IL-8, or TGF-β, which reduce their drug-sensitivity for chemotherapeutic agents and enhance their metastatic potential [Ostman and Augsten, 2009; New et al., 2017; Wei et al., 2021]. Labernadie et al. [2017] described that CAF empower the invasiveness of cancer cells by the upregulation of N-cadherin.

Regular fibroblasts are particularly involved in wound healing as they invade into lesions and produce ECM that serves as a scaffold for other cells. Fibroblasts from wound healing sites show higher proliferation rates and secrete more of normal ECM constituents than their resting counterparts and are therefore referred to as “activated.” In accordance, fibroblasts at the site of a tumor are permanently activated as a tumor can be considered a wound that does not heal [Dvorak, 1986; Kalluri and Zeisberg, 2006]. The majority of CAF in the tumor stroma are derived from resident fibroblasts, yet there is evidence that also other cells can undergo a transdifferentiation into CAF, including endothelial cells, adipocytes, smooth muscle cells, or MSC [Karnoub et al., 2007]. Another source for CAF are epithelial cells, which acquire mesenchymal features via epithelial-mesenchymal transition and become fibroblasts [Cirri and Chiarugi, 2011]. In addition to the diversity of their origin, both interindividual differences between various tumor entities but also within one tumor make CAF an extraordinarily heterogeneous cell population aggravating a precise definition by means of expression patterns, molecular features, or phenotypic characteristics [Ohlund et al., 2014]. A variety of different markers are regularly used to define CAF, yet it always must be considered that each of them represents different pathophysiological characteristics. For example, tumor-supportive CAF overexpress markers such as α-smooth muscle actin (α-SMA) [Liao et al., 2019], whereas low intratumoral fibroblast activation protein (FAP)+ CAF counts are significantly correlated with a worse prognosis in pancreatic ductal adenocarcinoma as compared with high counts, indicating a tumor-inhibitory effect [Park et al., 2017]. Tenascin-c (TNC) is an extracellular matrix glycoprotein, which has been shown to contribute to cancer cell migration, invasion, and metastasis and is considered a potential biomarker for CAF [Qian et al., 2022]. Another marker often used for the identification of CAF is thrombospondin-1 (THBS1), which is associated with cancer aggressiveness [Spaeth et al., 2009].

MSC are multipotent cells and possess the potential to differentiate into various types of mesenchymal tissue, including cartilage, tendon, muscle, bone, or fat [Pittenger et al., 1999]. After tissue damage, MSC are recruited to the site to assist in the wound healing process [Fox et al., 2007]. At the site of damage, they initiate their anti-inflammatory activity to remove injured tissue, facilitate the migration of reparative cell types, and promote vascularization and nutrient delivery [Toh et al., 2018]. Furthermore, MSC show a distinct tumor tropism, they migrate toward tumor tissue and integrate into the TME. In the TME, MSC can differentiate into CAF [Mishra et al., 2008; Quante et al., 2011]. The mechanisms involved in the process of cell transdifferentiation have been extensively studied. A variety of stimuli has been identified to induce CAF activation like direct contact to cancer cells, DNA-damage, or physical changes in the composition of ECM [Fordyce et al., 2012; Calvo et al., 2013; Strell et al., 2019]. Moreover, cancer cell-derived growth factors activate local fibroblasts into resident CAF. MSC could be activated to CAF by conditioned medium taken from a neuroblastoma cell line (NBCM) in vitro [Hashimoto et al., 2016] and in co-culture with esophageal cancer cell lines [Higashino et al., 2019]. Rubinstein-Achiasaf et al. [2021] were able to induce conversion of MSC into CAF by persistent inflammatory stimulation with tumor necrosis factor (TNF)-α and IL-1β [Rubinstein-Achiasaf et al., 2021].

Exposure to paracrine secreted tumor-derived factors, such as osteopontin, could lead to a conversion of MSC to CAF [Weber et al., 2015]. Furthermore, inflammatory modulators like TGF-β, TNF-α, IL-1 and -6 are able to initiate cell transdifferentiation into CAF [Sanz-Moreno et al., 2011]. These cytokines also play an essential role mediating the process of wound healing and can be found in large amounts in wound fluid (WF), which accumulates after surgical procedures.

It is well known, that a chronic inflammatory milieu facilitates tumorigenesis as for example in hepatocellular carcinoma or gastric cancer [Grivennikov et al., 2010]. Previous studies have shown that WF enhances proliferation in head and neck squamous cell carcinoma cells in vitro [Scherzad et al., 2019]. The local wound environment in the immediate postoperative period is characterized by the presence of pro-inflammatory cells, cytokines, and growth factors orchestrating the wound healing process. This pro-inflammatory milieu can serve as a driving factor for local tumor progression. Residual tumor cells receive direct proliferative stimuli, but it also seems conceivable that the environment leads to a pro-oncogenic transformation of originally benign cells of the TME. There is a lack of knowledge about the influence of WF on MSC and its possible ability to induce a transdifferentiation into CAF. The aim of this study was to investigate the influence of wound healing processes and tumor cell-associated paracrine-secreted factors on the potential of MSC to transdifferentiate into CAF. A combination of the CAF-typical biomarkers α-SMA, FAP, TNC, and THBS1, as well as morphological features, and genetic mutations were analyzed to investigate, whether the transformation into CAF can be detected using these methods.

MSC Isolation and Culture

MSC were harvested from spongiosa of femoral heads of patients after total hip replacement procedure at the Department of Orthopedic Surgery at the University Hospital Wuerzburg with written informed consent. The study was approved by the Ethics Committee of the Medical Faculty of the University of Wuerzburg (91/19-me). Cell isolation was performed as previously described by our group [Scherzad et al., 2015]. Cells were then cultivated in 175-cm2 flasks (Greiner Bio-One GmbH, Frickenhausen, Germany) in expansion medium consisting of Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies Corp. [Gibco], Carlsbad, CA, USA), 10% fetal calf serum (Linaris, Wertheim, Germany), and 1% penicillin/streptomycin (Biochrom AG, Berlin, Germany). Cells were incubated at 37°C and 5% CO2, medium was changed twice a week. Prior to each experiment cells were characterized according to the criteria of the International Society for Cellular Therapy [Dominici et al., 2006] and analyzed for their potential of differentiation.

Cell Line Culture and Preparation of Tumor-Conditioned Medium

The human squamous cell carcinoma cell line Cal27 was cultivated in 75- and 175-cm2 flasks in DMEM expansion medium under the same culture conditions as described for the bone marrow MSC.

For the acquisition of tumor-conditioned medium (TCM), cells were cultivated to a confluence of 80%. Medium was then extracted and replaced with fresh DMEM for 16 h. The medium was centrifuged for 5 min at 3,000 rpm and frozen at −80°C after sterile filtration.

Preparation of Wound Fluid

WF was collected from 5 male patients (aged 46–75 years) who underwent a planned neck dissection at the Department of Otorhinolaryngology, Plastic, Aesthetic and Reconstructive Head and Neck Surgery at Julius Maximilian University of Wuerzburg (Wuerzburg, Germany) after written informed consent. Fluid from vacuum drainages extracted 48–72 h after surgery was centrifuged with 1,000 g for 10 min at 4°C to eliminate cell detritus. Then, a second centrifugation step was performed in leucosep medium (Ficoll Paque Plus GE Healthcare, Freiburg, Germany) to eliminate immune cells. Subsequently WF was filtered using a 0.45-μm syringe filter (Sarstedt, Inc., Newton, NC, USA), and 100 U/mL penicillin and 100 mg/mL streptomycin (1% penicillin/streptomycin) were added to avoid contamination. The WF from the 5 donors was pooled and frozen at −80°C. For the further experiments it was diluted to a concentration of 40% as this had previously been shown to be the optimal concentration for promoting cell proliferation [Scherzad et al., 2019].

Exposure of MSC with WF, TCM, and Co-culture Conditions with Cal27

MSC were seeded in a 6-well round bottom plate (Greiner Bio-One GmbH) with a density of 2.25 × 105 cells per well and allowed to attach overnight. Culture medium was then removed and replaced with DMEM without supplementation with fetal calf serum for another 24 h. Finally, the TCM or 40% diluted WF was applied to the cells for a duration of either 24 or 48 h.

For the co-culture, 2.25 × 105 Cal27 cells were placed in a 6-well insert of a transwell plate system (ThinCerts, pore diameter 0.4 μm, pore density: 4 × 106/cm2; Greiner Bio-One GmbH).

Quantitative Real-Time PCR Analysis

MSC of 10 donors were each incubated with WF, TCM or in co-culture with Cal27 cells for 24 and 48 h. After the exposure period, RNA was extracted from cells using a RNeasy Mini Kit (Quiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. SuperScript VILO Mastermix (Applied Biosystems, Life Technologies GmbH, Darmstadt, Germany) was used to synthesize cDNA. The Taq-Man Gene Expression Assays (Thermo Fisher Scientific, Waltham, MA, USA) used were fibroblast activation protein alpha (FAP, Assay-ID: Hs00990791_m1), alpha-smooth muscle actin (HS00426835_g1), thrombospondin-1 (Hs00962908_m1), and tenascin-c (Hs01115665_m1). The expression of the selected gene was normalized to the GAPDH gene (glyceraldehyde-3-phosphate dehydrogenase, Hs02758991_g1), which was used as an internal control. The 2−ΔΔCt method was used to quantify mRNA expression.

Immunofluorescence Analysis

To investigate the protein expression profile of CAF-associated markers, indirect immunofluorescence was performed. MSC were incubated with TCM, WF or in co-culture with tumor cells as previously described. Cells were cultivated on cover slips with a diameter of 15 mm (A. Hartenstein GmbH, Würzburg, Germany) after previous autoclaving of the cover glasses (Laboklav, SHP Steriltechnik AG, Magdeburg, Germany). Therefore, 1 cover slip was transferred in each well of a 12-well round-bottom plate (Greiner Bio-One GmbH). Then, 250 μL of cell suspension containing 2 × 104 cells was carefully placed on the cover slip. Each sample was set up as a triplicate. For the co-culture experiment, 12-well plates with suitable inserts (12-well inserts, Transwell Permeable Supports, pore size 0.4 μm, Corning Inc., Corning, NY, USA) were used and each filled with 1 mL cell suspension containing 2 × 104 Cal27 tumor cells.

After 48 h of incubation, cells were gradually treated with paraformaldehyde (PFA, 4% solution). For this, 30 μL of PFA were added for 2 min in each well. Cells were then treated with 100 μL pure PFA. After 30 min of incubation, fixation was complete and PFA could be removed. Each well was then filled with 2 mL TBST buffer solution (200 mM Tris-base pH 8, 8% NaCl, 1% Tween-20; Sigma-Aldrich, Steinheim, Germany) and stored at 4°C until staining.

For the staining, cover slips were washed 3 times with 1× TBST for 5 min, blocked with 10% BSA in 1× TBST for 30 min, and then incubated with primary antibodies overnight at 4°C. Cells were immunostained for FAP alpha (1:250; rabbit monoclonal, ab207178 Abcam RabMab, Berlin, Germany) and α-SMA (1:200; mouse monoclonal, Sigma A5228, Sigma-Aldrich). After washing the slips, staining with the secondary antibodies, diluted in 1% BSA in 1× TBST, was carried out. Antibodies used were 4′,6-diamidino-2-phenylindole (DAPI) (1:3,000; Sigma-Aldrich), Alexa Fluor Plus 555 (1:1,000; donkey anti-rabbit IgG, Thermo Fisher Scientific), and Alexa Fluor Plus 488 (1:1,000; donkey anti-mouse IgG, Thermo Fisher Scientific). After staining was completed, the cover slips were again rinsed with TBST for 3 times. Then they were covered with Mowiol (Sigma-Aldrich) on microscope slides (Langenbrinck, Emmendingen, Germany). For microscopy and photography, a microscope (Leica DMI 4000 B, Leica Microsystems GmbH, Wetzlar, Germany) with a fluorescent lamp (Leica EL6000) and a microscope camera (LEICA DFC 320) was used. The evaluation was carried out using the software LAS Leica Application Suite (Leica Microsystems GmbH). Overlay images were created using ImageJ 1.53a (Rasband WS, US National Institutes of Health, Bethesda, MD, USA).

Cytokine Analysis of Different Incubation Media

To analyze the cytokine patterns of WF, TCM, and the supernatants after co-culture experiment, a dot blot assay was applied. Materials and reagents used, including the C-Series Human Cytokine Antibody Array 3 kit (cat. No. AAH-CyT-3-4) were obtained from RayBiotech Inc. (Norcross, GA, USA).

First, the membranes were incubated with blocking buffer for 30 min at room temperature. Then 1 mL of each medium was applied and incubated at 4°C overnight on a shaking incubator. After several washing steps, incubation with 1 mL biotin-conjugated antibodies (prefabricated solution) and horseradish peroxidase-conjugated streptavidin (1:1,000) was conducted for 2 h at room temperature. After treatment with detection buffer and exposure to x-ray film, the labeled proteins were detected via chemiluminescence. The samples were evaluated semi-quantitatively with the program ImageJ 1.53a by analyzing the densitometric intensity of each dot normalizing it to the positive control dots.

Statistical Analysis

Statistical analysis was performed using Graphpad Prism 9 (GraphPad Software, San Diego, CA, USA). Friedman test was applied to analyze differences between the different incubation media. A post-hoc Dunn’s test and Wilcoxon test were used to determine the significance between groups. p < 0.05 was considered statistically significant.

Analysis of Morphological Changes and Cell Growth Patterns

Before the staining procedure for the immunohistochemical analysis, cells were examined with the light microscope after cultivation with WF, TCM, and in co-culture with Cal27 tumor cells. Slight differences in cell morphology and growth patterns were apparent between the samples (Fig. 1). Untreated MSC grew with a spindle-shaped morphology forming extensions in all directions (Fig. 1a). Figures 1b–d show treated MSC. The cells in Figure 1b after incubation with WF show a higher confluence with elongated cell bodies and extensions. Cells in Figure 1c (after TCM) and 1d (after co-culture) show a higher confluence as well, but overall a less altered growth pattern.

Fig. 1.

Light microscopy images of mesenchymal stem cells (MSC) after different culture conditions for 48 h. a Native MSC (treated with expansion medium) grew with a spindle-shaped morphology forming extensions in all directions. b–d Treated MSC. b After incubation with wound fluid, the cells showed a higher confluence with elongated cell bodies and extensions. c, d Treatment with tumor-conditioned medium (c) and co-culture with Cal27 tumor cells (d) did not alter morphology distinctively. Scale bars, 200 μm.

Fig. 1.

Light microscopy images of mesenchymal stem cells (MSC) after different culture conditions for 48 h. a Native MSC (treated with expansion medium) grew with a spindle-shaped morphology forming extensions in all directions. b–d Treated MSC. b After incubation with wound fluid, the cells showed a higher confluence with elongated cell bodies and extensions. c, d Treatment with tumor-conditioned medium (c) and co-culture with Cal27 tumor cells (d) did not alter morphology distinctively. Scale bars, 200 μm.

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Analysis of Expression of CAF-Associated Markers

To evaluate, whether different culture conditions lead to a transformation of MSC to CAF, mRNA expression of typical CAF markers was measured by RT-PCR. As there are no markers that exclusively specify CAF, a variety of markers was chosen that are associated with CAF. Therefore, the fibroblast marker FAP, the activation marker α-SMA, and the invasion markers THBS1 and TNC were analyzed.

Gene Expression of FAP

All examined samples revealed only slight changes in the expression of FAP (Fig. 2). Relative gene expression of FAP was reduced compared to the control group after treatment with WF and in co-culture after 24 h. After incubation with TCM and in co-culture, FAP mRNA was significantly higher compared to WF after 24 and 48 h. After an incubation period of 48 h, the expression was reduced in all groups compared to the negative control.

Fig. 2.

Gene expression of cancer-associated fibroblast-linked markers in human mesenchymal stem cells (MSC) of 10 donors. Comparison of the relative gene expression of fibroblast activation protein (FAP), alpha-smooth muscle actin (α-SMA), thrombospondin-1 (THBS1), and tenascin-c (TNC) in MSC after cultivation with wound fluid (WF), tumor-conditioned medium (TCM), and in co-culture with Cal27 tumor cells after 24 and 48 h. Evaluation was performed using the 2−ΔΔCT method. The results are presented as the fold change of target gene expression in the target sample relative to a reference sample, normalized to a reference gene. DMEM expansion medium was used for the reference sample and would usually be set to 1. For the purpose of a better overview it has been left out of the figure. Data are presented with box plots, margins of them illustrate the 25th and 75th percentiles. Statistical significance of the different samples based on a pvalue <0.05 is indicated by asterisks. Square, circle, and triangle mark results outside 1.5 SD from median.

Fig. 2.

Gene expression of cancer-associated fibroblast-linked markers in human mesenchymal stem cells (MSC) of 10 donors. Comparison of the relative gene expression of fibroblast activation protein (FAP), alpha-smooth muscle actin (α-SMA), thrombospondin-1 (THBS1), and tenascin-c (TNC) in MSC after cultivation with wound fluid (WF), tumor-conditioned medium (TCM), and in co-culture with Cal27 tumor cells after 24 and 48 h. Evaluation was performed using the 2−ΔΔCT method. The results are presented as the fold change of target gene expression in the target sample relative to a reference sample, normalized to a reference gene. DMEM expansion medium was used for the reference sample and would usually be set to 1. For the purpose of a better overview it has been left out of the figure. Data are presented with box plots, margins of them illustrate the 25th and 75th percentiles. Statistical significance of the different samples based on a pvalue <0.05 is indicated by asterisks. Square, circle, and triangle mark results outside 1.5 SD from median.

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Gene Expression of α-SMA

Treatment of MSC with WF, TCM, and in co-culture led to only minor changes regarding the gene expression of the activation marker α-SMA (Fig. 2). In both settings, after 24 h and after 48 h, a decrease in the expression of α-SMA based on the median values was noticeable, especially after exposure to WF. After 24 and 48 h, gene expression was significantly less after WF compared to co-culture conditions and after 48 h also compared to TCM.

Gene Expression of THBS1

Different treatment conditions of MSC led to only minor alterations in the expression of THBS1 (Fig. 2). Friedman test showed no significant differences between samples, both after 24 h and after 48 h.

Gene Expression of TNC

Expression of the marker tenascin-c was as well only slightly influenced in relation to untreated control cells (Fig. 2). Friedman test revealed no statistically significant alterations after 24 and 48 h. The median expression was increased after 48 h in co-culture with Cal27 cells.

In summary, gene expression of CAF-associated markers was only slightly altered after different cultivation conditions compared to the control. Influence of the different media was greater on the expression of FAP and α-SMA than on THBS1 and TNC. Expression of FAP and α-SMA was significantly less after WF compared to TCM and co-culture. Expression patterns showed a broad variability overall.

Immunhistochemical Evaluation of CAF-Associated Markers

The formation of CAF can be assessed by the examination of specific markers. Typical CAF-associated markers are FAP and α-SMA. The gene expression of these characteristics was measured by RT-PCR as described previously. Immunohistochemical staining was carried out to evaluate whether these markers were expressed at the protein level.

Fluorescence microscopy showed heterogeneous results as there was a broad fluctuation in the signal intensity between donors. After 48 h of incubation only minor changes were apparent between groups (Fig. 3). There were only traces of FAP (red color) detectable whereas the green dye of α-SMA was clearly found in all cells, including untreated MSC.

Fig. 3.

Fluorescence microscope images after indirect immunofluorescence staining. Cancer-associated fibroblast markers alpha-smooth muscle actin (α-SMA) (green, intracellular), fibroblast activation protein (FAP) (red, membrane-bound), and DAPI nuclear stain (blue) were investigated. Mesenchymal stem cells (MSC) were treated for 48 h with different media. a Untreated MSC (incubation with expansion medium DMEM). b MSC treated with 40% wound fluid. c MSC treated with tumor-conditioned medium. d MSC incubated in co-culture with Cal27 tumor cells. α-SMA was detectable in all cells, including DMEM-treated cells, FAP was only found in traces. Scale bars, 200 μm.

Fig. 3.

Fluorescence microscope images after indirect immunofluorescence staining. Cancer-associated fibroblast markers alpha-smooth muscle actin (α-SMA) (green, intracellular), fibroblast activation protein (FAP) (red, membrane-bound), and DAPI nuclear stain (blue) were investigated. Mesenchymal stem cells (MSC) were treated for 48 h with different media. a Untreated MSC (incubation with expansion medium DMEM). b MSC treated with 40% wound fluid. c MSC treated with tumor-conditioned medium. d MSC incubated in co-culture with Cal27 tumor cells. α-SMA was detectable in all cells, including DMEM-treated cells, FAP was only found in traces. Scale bars, 200 μm.

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Cytokine Analysis of Different Incubation Media

In total, 80 cytokines involved in angiogenesis, proliferation, inflammation, and immunomodulation were investigated. In this way, specific patterns of the different incubation media could be analyzed. DMEM without additives was used as a negative control.

First, the dots on x-ray film were examined using the instructions of the manufacturer’s protocol (Fig. 4).

Fig. 4.

Dot blot assay with correlation of the dots to the respective cytokines based on the instructions of the manufacturer’s protocol. The images on the left show the x-ray films of the dot blot assay. The figures on the right display the instructions. Fields with darker background mark cytokines with stronger expression. a Negative control (DMEM without additives). b Wound fluid. c Tumor-conditioned medium. d Supernatant of the co-culture after 24 h.

Fig. 4.

Dot blot assay with correlation of the dots to the respective cytokines based on the instructions of the manufacturer’s protocol. The images on the left show the x-ray films of the dot blot assay. The figures on the right display the instructions. Fields with darker background mark cytokines with stronger expression. a Negative control (DMEM without additives). b Wound fluid. c Tumor-conditioned medium. d Supernatant of the co-culture after 24 h.

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As expected, in the negative control with DMEM without additives only the control dots were detected as positive (Fig. 4a). The assays of the different media, however, showed relevant differences compared to the negative control. In all 3 media, the growth-regulated oncogene (GRO), the inflammatory cytokines IL-6, IL-8, as well as the protein TIMP metallopeptidase inhibitor 2 (TIMP-2) were detected.

The analysis of the WF showed that monocyte chemotactic protein 1 (MCP-1), RANTES, angiogenin, insulin-like growth factor binding protein 2 (IGFBP-2), neutrophil activating peptide 2 (NAP-2), and osteopontin were found (Fig. 4b).

In the TCM assay, in addition to the cytokines present in all media, granulocyte-macrophage colony-stimulating factor (GM-CSF), growth-regulated oncogene alpha (GRO-α), and angiogenin were detected (Fig. 4c).

Between the results of the co-culture supernatant after 24 h and 48 h there were no optical differences notable. The supernatants included higher levels of epithelial-derived neutrophil-activating protein 78 (ENA-78), GRO-α, MCP-1, IGFBP-1, -2, -4, as well as osteoprotegerin and TIMP-1 (Fig. 4d).

The densitometric intensity of the individual spots was then determined semi-quantitatively. The results in Figure 5 are presented as relative intensity in percent based on the intensity of the positive control. The relative intensity of the cytokines, which were detectable as positive dots on the x-ray films is shown. Levels of GRO, IL-8, and TIMP-2 barely differed between the media. The concentration of IL-6 was lowest in TCM and highest in the supernatants of the co-culture after 48 h. Furthermore, it could be seen that concentrations of ENA-78 and MCP-1 in supernatants of co-culture were considerably higher after 24 h compared to 48 h. In contrast, IL-6 was more intense after 48 h than after 24 h. TIMP-1 was hardly detectable in the supernatants after 48 h.

Fig. 5.

Semiquantitative analysis of the dot blot assays. The relative intensity of the dots based on the intensity of the positive control is shown in percent. Differences between the cytokine concentrations of different cultivation media could be observed. Wound fluid (WF), tumor-conditioned medium (TCM), and supernatants after co-culture of mesenchymal stem cells with Cal27 cells for 24 and 48 h were analyzed.

Fig. 5.

Semiquantitative analysis of the dot blot assays. The relative intensity of the dots based on the intensity of the positive control is shown in percent. Differences between the cytokine concentrations of different cultivation media could be observed. Wound fluid (WF), tumor-conditioned medium (TCM), and supernatants after co-culture of mesenchymal stem cells with Cal27 cells for 24 and 48 h were analyzed.

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ENA-78, IGFBP-1 and -4, and osteoprotegerin could only be found in the supernatants of the co-cultures, GM-CSF only in TCM, and RANTES, NAP-2, and osteopontin only in WF.

Cancer development and progression is a consequence of interaction between malignant and TME-associated nonmalignant cells. CAF are among the most dominant cellular components of the TME in both primary and metastatic tumors. Studies have shown that the detection of a great quantity of CAF in solid tumors correlates with a poor prognosis, as their interaction with cancer cells may create a tumor-supportive environment [Liu et al., 2016]. Despite the advances made in the treatment of many tumor entities, the prognosis of patients with HNSCC is still poor in many cases [Svider et al., 2017]. HNSCC characteristically possess a broad inter- and intratumoral heterogeneity, although the stromal composition seems to be relatively consistent between patients [Puram et al., 2017]. Therefore, targeting CAF seems to be an interesting approach for cancer research and therapy. Nevertheless, there are still many unknown factors in the process of CAF formation that need to be elucidated.

The immediate postoperative phase is exceedingly vulnerable with regard to the local processes taking place in the tissue. WF contains large amounts of growth factors and proliferation-promoting cytokines, which can directly stimulate tumor cells. However, the influence on the other, originally benign cells in the TME is unclear, although their strong influence in tumorigenesis has been previously illustrated. The aim of this study was to investigate whether WF can induce a transformation of MSC to CAF, which could result in an enhancement of the local protumorigenic environment. The cultivation of MSC with WF represents an approximation to the pathophysiological processes taking place in vivo and is an extension to the specific stimulation with selected cytokines. Profound knowledge of the crosstalk between tumor cells and MSC is essential for multiple reasons. Stem cell-based therapies are promising in various disciplines ranging from the treatment of hematooncological to metabolic diseases. However, an application in reconstructive medicine for patients with cancer history might be impeded. Hypotheses state that paracrine factors secreted by MSC may activate a rare subpopulation of persisting cancer stem cells that could facilitate cancer relapse and progression [Maj et al., 2022]. On the other hand, cytokines released by cancer cells lead to an activation of MSC. The complex interactions between these cells are not yet fully understood and require further research. In this study, TCM was chosen to investigate the influence of tumor cell-secreted paracrine factors on MSC. To distinguish the effects elicited by direct cell-cell contact or by tumor cell-secreted factors, the different media were analyzed.

After treatment with WF, MSC developed different phenotypical characteristics. They showed higher confluence in their collective and elongated, widely branched cell bodies, which might indicate an activation into myofibroblasts. The conversion of MSC into myofibroblasts has been shown previously [Barbosa et al., 2010]. In general, myofibroblasts exhibit contractile features by the expression of β- and γ-cytoplasmic actins as well as α-SMA and are therefore important for the wound contraction in the process of wound healing [Tomasek et al., 2002]. CAF are considered myofibroblast-like cells, and the expression of α-SMA is commonly used for their characterization. In contrast to the morphological changes, RT-PCR did not reveal higher gene expression of α-SMA in MSC treated with WF. On the other hand, there was a significantly higher α-SMA expression in the cells treated with TCM and in co-culture with Cal27 tumor cells compared to WF after 24 and 48 h. mRNA expression of FAP after cultivation in WF was significantly less than in co-culture conditions or after TCM. Overall, the incubation with WF led to a suppression of the CAF-typical markers FAP and α-SMA. The expression of THBS1 and TNC was not significantly altered after any of the different incubation methods. In general, the interindividual variations were quite large, what can be attributed to the fact that primary MSC were used instead of cell lines. Immunofluorescence microscopy revealed that MSC did not express FAP on the protein level, which is consistent with RT-PCR results. α-SMA occurred at similar levels in all cells. This could be due to the fact that not enough time had elapsed for a change in gene expression to be shown at the protein level as well.

Although morphological changes could be visualized microscopically after WF incubation, no clear identification of CAF based on molecular genetic alterations or the expression of surface markers was possible. One explanation for the distinct changes could be the relatively short incubation period. Rubinstein-Achiasaf et al. [2021] stimulated MSC with the cytokines TNF-α and IL-1β for up to 19 days. They showed that gene expression was considerably less after short-term stimulation for only 48 h. In contrast, Werner et al. [2019] measured elevated α-SMA amounts on mRNA and protein level in MSC after 24- and 48-h incubation with TCM from HCT8 cells. In particular, TNF-α and IL-1β seem to play an important role in the process of cell transdifferentiation into CAF. The dot blot assay revealed that none of the media showed high concentrations of these specific cytokines. WF contained the highest concentration of osteopontin of the different media. Osteopontin is a multifunctional cytokine that is strongly expressed in healing wounds and is necessary for the formation of myofibroblasts [Lenga et al., 2008]. Moreover, cancer-derived osteopontin is considered a potent driver for the generation of CAF [Butti et al., 2021]. Although myofibroblast-like morphological changes could be observed microscopically, there was a suppression of the CAF-associated markers FAP and α-SMA measurable on the molecular level. Gene expression of these two markers was significantly higher after incubation with TCM and in co-culture with tumor cells, which may indicate that tumor cell-associated stimuli in the culture medium are required for CAF formation.

CAF play a central role in tumor formation, growth, and metastatic spread. A better understanding of the pathomechanisms of CAF formation is therefore of crucial importance in order to enable advances in tumor therapy. In this study, morphological changes and alterations in CAF-typical markers on a molecular level could be observed by incubating MSC with different culture media. However, an exact phenotypic and molecular characterization of CAF was not possible, and the natural individual heterogeneity of primary cells makes a precise definition even more difficult. The only minor changes observed could have various causes. In the incubation media used, the crucial induction factors for the transformation of MSC into CAF could have been missing. In addition, the incubation period may not have been long enough. As a precise characterization of CAF was not possible with the methods used, the statement that WF induces CAF cannot be made unreservedly. However, it became evident that different culture conditions influence MSC in diverse ways. Even the previously reported CAF formation through TCM and co-culture could not be reproduced explicitly. The Cal27 cell line was chosen, which is derived from a tongue SCC, as this tumor entity is more immunologically active as for example larynx carcinomas. Escobar et al. [2015] demonstrated that MSC secreted higher levels of chemokines after treatment with TCM from aggressive breast cancer cells compared to less aggressive breast cancer cells. This shows that the response can vary even within the same tumor entity and suggests that comparisons between different tumors are difficult to make. In this study, a simulation of the complex pathophysiological processes in the local postoperative environment was aimed for. It has been demonstrated that the modification by adding human-derived WF leads to more heterogeneous results than co-culture experiments or specific cytokine stimulation. Moreover, the motive of the study, that residual tumor cells are stimulated by the pro-inflammatory postoperative milieu, can, however, also be discussed to the contrary. Immunogenic cell death as a type of regulated cell death has increasingly become a focus of research. Damage-associated molecular patterns (DAMPs) are stress signals released by dying cells. In the oncologic setting tumor cell-associated DAMPs bind to innate immune receptors on antigen-presenting cells, which results in a tumor-specific activation of T cells in lymph nodes. These tumor-reactive T cells traffic to the tumor and eliminate cancer cells [Birmpilis et al., 2022]. It can therefore be argued that tissue damage through surgical intervention could lead to the release of tumor-specific DAMPs, which in turn could result in immunogenic cell death of tumor cells. A methodical limitation of this study was the usage of a 2D cell culture. 2D models are a simple and reproducible method to approximate in vivo conditions. However, cell-cell and cell-matrix interactions are often impaired due to a distortion of spatial arrangement of cells. 3D cultured cells like spheroids better imitate the in vivo architecture of tumors [Antoni et al., 2015]. Especially for continuative research on the crosstalk of tumor cells and cells of the TME, this form of culture should be taken into consideration. The dot blot assay was used as it offers a broad test spectrum for cytokines and a semi-quantitative evaluation, which was suitable for the purpose of this study. It allowed the comparison of cytokine levels between the used media. For more distinct questions multiplex panels could be used if measurements of exact concentrations of certain cytokines are of interest. Incubation times were chosen based on data from previous studies. However, it must be mentioned again that the cellular processes and cell-cell interactions seem to depend significantly on the tumor entity. In further studies an approach could be pursued in which different incubation times and tumor cell lines are examined.

The current standard of care for patients with head and neck cancer is associated with significant limitations in quality of life and comorbidities. However, resistance to radio-/chemotherapy and a lack of targeted therapy options limit the prognosis, especially in recurrent or metastatic situations. This highlights the need for innovative treatment modalities to ameliorate the prognosis while reducing undesirable treatment effects. Despite intensive ongoing research, there are many unanswered questions about the relationship between tumor and microenvironment [Alsahafi et al., 2019]. The results shown in this study confirm the complexity of these interactions and should encourage to continue investigating the interplay of tumor cells and cells of the TME.

We thank Michael Kessler and Silke Hummel for their technical support.

The study was approved by the Ethics Committee of the Medical Faculty of the University of Wuerzburg (91/19-me) and conforms to the standards of the Declaration of Helsinki. Written informed patient consent has been obtained before.

The authors have no conflicts of interest to declare.

There was no funding.

Helena Moratin performed conceptualization and methodology together with Agmal Scherzad and drafted the work. Sonja Böhm acquired and contributed to the interpretation of data. Stephan Hackenberg and Rudolf Hagen reviewed the process of editing the manuscript and approved the final version to be published.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

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