Johannes David Schultz

Evaluation of the effects of different culture media on the myogenic differentiation potential of adipose tissue- or bone marrow-derived human mesenchymal stem cells

The creation of functional muscles/muscle tissue from human stem cells is a major goal of skeletal muscle tissue engineering. Mesenchymal stem cells (MSCs) from fat/adipose tissue (AT-MSCs), as well as bone marrow (BM-MSCs) have been shown to bear myogenic potential, which makes them candidate stem cells for skeletal muscle tissue engineering applications. The aim of this study was to analyse the myogenic differentiation potential of human AT-MSCs and BM-MSCs cultured in six different cell culture media containing different mixtures of growth factors. The following cell culture media were used in our experiments: mesenchymal stem cell growth medium (MSCGM)™ as growth medium, MSCGM + 5-azacytidine (5-Aza), skeletal muscle myoblast cell growth medium (SkGM)-2 BulletKit™, and 5, 30 and 50% conditioned cell culture media, i.e., supernatant of human satellite cell cultures after three days in cell culture mixed with MSCGM. Following the incubation of human AT-MSCs or BM-MSCs for 0, 4, 8, 11, 16 or 21 days with each of the cell culture media, cell proliferation was measured using the alamarBlue® assay. Myogenic differentiation was evaluated by quantitative gene expression analyses, using quantitative RT-PCR (qRT-PCR) and immunocytochemical staining (ICC), using well-defined skeletal markers, such as desmin (DES), myogenic factor 5 (MYF5), myosin, heavy chain 8, skeletal muscle, perinatal (MYH8), myosin, heavy chain 1, skeletal muscle, adult (MYH1) and skeletal muscle actin-α1 (ACTA1). The highest proliferation rates were observed in the AT-MSCs and BM-MSCs cultured with SkGM-2 BulletKit medium. The average proliferation rate was higher in the AT-MSCs than in the BM-MSCs, taking all six culture media into account. qRT-PCR revealed the expression levels of the myogenic markers, ACTA1, MYH1 and MYH8, in the AT-MSC cell cultures, but not in the BM-MSC cultures. The muscle-specific intermediate filament, DES, was only detected (by ICC) in the AT-MSCs, but not in the BM-MSCs. The strongest DES expression was observed using the 30% conditioned cell culture medium. The detection of myogenic markers using different cell culture media as stimuli was only achieved in the AT-MSCs, but not in the BM-MSCs. The strongest myogenic differentiation, in terms of the markers examined, was induced by the 30% conditioned cell culture medium.

CD44 as a stem cell marker in head and neck squamous cell carcinoma

In the recent past, evidence is increasing indicating the existence of a subpopulation of resistant tumor cells in head and neck squamous cell carcinoma (HNSCC) that cannot be eradicated by established antineoplastic treatments. These cancer stem cells (CSCs) have features of somatic stem cells such as selfrenewal, proliferation and differentiation. CD44+ cells in tumors of the head and neck are referred to as CSCs of HNSCC. Expression profiling of CD44 in 29 HNSCC tumors was performed by fluorescence microscopy. ELISA analysis was performed to detect concentration of soluble CD44 in the peripheral blood of 29 HNSCC patients and 11 healthy controls. Expression of CD44 was determined in all HNSCC tissue samples (n=29). In all samples a surface staining pattern was found. The concentration of CD44 in the peripheral blood of HNSCC patients was significantly higher compared to a healthy control group (mHNSCC =13.5 ± 0.5 ng/ ml; mCont = 9.3 ± 0.6 ng/ml; P=0.6 x 10(-12)). The role of CD44 as a marker for CSCs in HNSCC remains to be ascertained. Further experiments might reveal its role as a diagnostic and prognostic factor, and possibly as a therapeutic target.

SDF-1-CXCR4 axis: Cell trafficking in the cancer stem cell niche of head and neck squamous cell carcinoma

Stromal cell-derived factor-1α (SDF-1α), also known as CXCL12, has variable effects on a plurality of cells. CXCR4 has been identified as its corresponding receptor. The SDF-1-CXCR4 axis is postulated to be a crucial key pathway in the interaction between (cancer) stem cells and their surrounding supportive cells in the cancer stem cell niche. We evaluated the expression of CD44 as a cancer stem cell marker and of CXCR4 in human HNSCC tissue samples. Afterwards, we monitored the concentration of SDF-1 in peripheral blood samples of HNSCC patients and healthy donors. We showed that CD44 and CXCR4 are expressed in human HNSCC tissues. Markedly, CD44 showed a high expression in HNSCC cells bordering cancer stromal cells. CXCR4 was mainly expressed in HNSCC tumor nests, but not in the surrounding stromal cells. No significant difference was noted between the SDF-1 concentration in the peripheral blood of HNSCC patients compared to healthy donors. We showed that CD44, as a stem cell marker in HNSCC, is located mainly at the borderline of HNSCC tumor nests with the surrounding cells. In addition, we demonstrated that CXCR4 as the corresponding receptor to SDF-1 is highly expressed in HNSCC tumor nests, but not in the tumor stroma. We collected evidence that SDF-1-CXCR4 interaction may be a crucial pathway in cell trafficking in the cancer stem cell niche of HNSCC, while SDF-1 was not detected in the peripheral blood of HNSCC patients. The SDF-1-CXCR4 axis may play an important role in the cancer stem cell theory of HNSCC. As SDF-1α also exhibits a multitude of functional effects on HNSCC cells, such as migration and polarization, it may be possible that the SDF-1-CXCR4 axis is also involved in the pathophysiology of the progression, recurrence and metastasis of malignant disease. Understanding these interactions may help to gain further insight into these mechanisms and as such help to discover new strategies of therapy.

Down-regulation of MMP-2 expression due to inhibition of receptor tyrosine kinases by imatinib and carboplatin in HNSCC.

Squamous cell carcinoma of the head and neck (HNSCC) is the most common neoplasm arising in the upper aerodigestive tract. Unfortunately, the survival for this type of cancer has not improved significantly in the past 25 years. To enhance the survival rate multimodal therapy regimens have been set up. In these regimens chemotherapy plays a pivotal role in the majority of advanced cases. Transmembrane protein- tyrosine kinases (PTK) are fundamental elements of the signal transduction. In consequence, they might be promising targets for cancer therapy. Imatinib (STI 571) was originally designed to inhibit the BCR-ABL tyrosine kinase in chronic myeloid leukemia. But imatinib also has an inhibitory impact on the PTK receptor c-kit and on its PTK activity. Furthermore, growth and invasion of HNSCC are strongly influenced by the extracellular matrix (ECM). The ECM is altered by matrix metalloproteinases (MMP). In this study, we incubated different HNSCC cell lines with rising concentrations of imatinib and/or carboplatin. After an incubation time of up to 10 days, we evaluated c-kit, MMP-2 and MMP-14 by ELISA techniques and immunohistochemical methods. Especially the combination of 7.5 μmol carboplatin with 30 μmol imatinib resulted in a significant decrease in MMP-2 expression in all observed cell lines (p<0.05). We did not demonstrate a significant alteration in c-kit expression by imatinib and carboplatin. We observed an increase in apoptosis in HNSCC cells by the combination of the two observed chemotherapeutic drugs. In all cell lines tested, expression of c-kit and MMP could be demonstrated. Our results indicate that MMP-2 expression was suppressed in the presence of imatinib. Thus, imatinib may exert in part its inhibitory effect on malignant cell growth via the blockage of the signal transduction of PTK receptors. Further studies are warranted, especially one keeping in mind the moderate toxicity of imatinib.

Chemotherapeutic alteration of VEGF-/PDGF- and PDGF-Rα/β expression by imatinib in HPV-transformed squamous cell carcinoma compared to HPV-negative HNSCC in vitro

Head and neck squamous cell carcinoma (HNSCC) is an aggressive epithelial malignancy known to be the most common neoplasm appearing in the upper aerodigestive tract. The poor five-year survival rate has remained unchanged in the last decades despite the emergence of improved techniques in surgery, radiation and chemotherapy. In the last 20 years awareness of a subset of squamous cell carcinomas induced by oncogenic forms of the human papilloma virus (HPV) (high-risk types 16 and 18) has increased. The incidence of HPV-associated oropharyngeal cancer is rising, indicating the increased importance of the viral etiology. Cell proliferation, migration, induction of tumor vascularization and carcinogenesis, as well as invasion facilitation is regulated by a variety of angiogenic peptides like PDGF, PDGF-R and VEGF. They might be an encouraging target for biological anticancer therapy by inhibiting disrupted cellular signaling pathways. Imatinib has been shown to target specific tyrosine kinases, inhibiting proliferation in various cancer entities. The purpose of this study was to evaluate the expression pattern of angiogenic factors (VEGF, PDGF and PDGF-R) in HPV-positive (p16-CERV196 SCC) and (-negative squamous cell carcinoma (HNSCC). The study also evaluated the vulnerability of anti-angiogenesis therapy depending on the HPV status as potential treatment modality compared to established platinum-based chemotherapeutic drugs. The different squamous tumor cell lines were incubated with increasing concentrations of carboplatin (3 and 7.5 µmol) and imatinib (18 and 30 µmol). ELISA immunohistochemical methods were carried out after 48, 72, 120, 192 and 240 h. We demonstrated a significant reduction of VEGF and PDGF-Rα/β expression patterns after incubation of imatinib in ELISA and immunohistochemical methods, irrespective of the HPV status of the tumor cells, whereas the application of carboplatin had no impact on the expression of angiogenic peptides. Viral oncogen-transformed squamous cell carcinoma (CERV196) cells were characterized by a reduced susceptibility for an imatinib-altered VEGF expression. Further studies are planned to investigate this observance in HPV-positive HNSCC in vitro. The implementation of a selective molecular anti-angiogenic therapy in established chemotherapeutic regimens may enhance the efficacy of platinum-based chemotherapy without an increased toxicity profile and could thus improve the clinical outcome in HNSCC, irrespective of the HPV status.

Clinical Outcome and Quality of Life After a Multimodal Therapy Approach to Ear Keloids.

IMPORTANCE Keloids are fibroproliferative scars that can cause a huge psychological burden and severe problems for patients, such as depression. Many treatment options exist; however, recurrence rates, especially with monotherapy, remain high. OBJECTIVE To investigate the recurrence rate and changes in quality of life after multimodal therapy. DESIGN, SETTING, AND PARTICIPANTS A total of 33 patients with 42 auricle keloids (24 female and 9 male patients; mean [SD] age, 27 [17] years) were enrolled in a prospective cohort study and underwent intramarginal keloid excision and multimodal therapy. Patients were observed postoperatively in the outpatient Department of Otorhinolaryngology-Head and Neck Surgery, University Hospital of Mannheim, from August 1, 2007, through September 30, 2014, with a mean (SD) follow-up of 30 (19) months (through August 31, 2014). A retrospective analysis of clinical outcomes was performed from September 1 through November 15, 2014. INTERVENTIONS Excision followed by 6 intralesional corticosteroid injections at 4- to 6-week intervals and individually customized pressure splints applied at least 5 nights a week for 6 months. MAIN OUTCOMES AND MEASURES Keloid recurrence rate and subjective handling of the pressure splint were evaluated during clinical visits. Quality of life was measured after the end of therapy with a 3-part questionnaire, including the Glasgow Benefit Inventory (GBI). RESULTS After excluding 4 patients (with 5 keloids) for nonadherence to treatment, 3 of 37 keloids recurred, for a recurrence rate of 8% among 29 patients. Insecure handling of the pressure splint significantly correlated with a higher relapse rate (mean subjective handling score in patients with a relapse, 3.60; P = .02). Four of 8 patients with recurrent keloids had poor adherence to adjuvant pressure therapy, which suggests an association between keloid recurrence and adherence to adjuvant pressure therapy. Patients received the 3-part questionnaire by mail to collect data on quality of life. Of 43 patients approached, 33 treated with multimodal therapy completed the questionnaire for a return rate of 77%. Improvement in quality of life after keloid treatment was significant in recurrence-free patients, with a mean GBI score of 22.53 (P < .001). CONCLUSIONS AND RELEVANCE The present study showed an improvement in quality-of-life scores after multimodal therapy for keloids. Because poor adherence to the use of ear splints correlated with a higher recurrence rate of keloids, efforts are needed to improve adherence and minimize recurrence. LEVEL OF EVIDENCE 3.

Imatinib-associated matrix metalloproteinase suppression in p16-positive squamous cell carcinoma compared to HPV-negative HNSCC cells in vitro

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide. The growth and invasion of HNSCC are strongly influenced by the extracellular matrix (ECM), which is modified by matrix metalloproteinases (MMPs). The MMP family is still relevant to cancer research, as it promotes malignant transformation, cell proliferation and modulation of angiogenesis even in the early stages of cancer. The proteolytic processing of bioactive molecules by MMP-14 (MT1-MMP) causes severe abnormalities in connective tissue, defective angiogenesis and premature death. MMP-2 (gelatinase A) and MMP-14 also play a role in degradation of basement membrane and cell carcinoma invasion. Imatinib blocks the PTK receptor c-kit and forestalls its PTK activity. The aim of the present study was to investigate the expression pattern of MMP-14 and MMP-2 in human papilloma virus (HPV)-negative and p16-positive SCC and to evaluate the chemosensitivity of the tumour cells to the chemotherapeutic agents, imatinib and 5-fluorouracil (5-FU). We incubated the SCC cell lines with imatinib (18 and 30 µmol/ml) and 5-FU (1 and 5 µmol/ml) and detected MMP-14 and MMP-2 by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) after 48, 72, 120, 192 and 240 h. We detected expression of MMP-2 and MMP-14 in all incubated tumour cell lines. With imatinib in particular, we found a reliable trend towards decreased MMP-2 and MMP-14 expression levels in p16-positive and p16-negative SCC tumour cell lines in addition to an induced apoptotic effect. We found statistically significant imatinib-induced suppression of MMP-2- and MMP-14, dependent on the incubation time and the cell line. We detected a significant suppression of MMP-2 and MMP-14 especially in p16-negative HNSCC14C cells after prolonged treatment time with imatinib. Dose escalation of imatinib and 5-FU had no statistically significant effect on the expression of MMP-2 or MMP-14. The p16-positive SCC cells exhibited higher expression of total protein. We detected a significant suppression of MMP-2 and MMP-14 in all the incubated SCC cell lines, partially after treatment with imatinib. We found higher suppression of MMP-2 in the CERV196 cells after incubation with imatinib. We detected a reliable trend towards increased chemosensitivity of p16-positive tumour cells in vitro after treatment with imatinib. Extended studies and clinical trials are needed to further investigate these findings in HPV-associated HNSCC.

Interleukin 4, interleukin 6 and osteopontin‑serological markers of head and neck malignancy in primary diagnostics: A pilot study

The progression of head and neck squamous cell carcinoma (HNSCC) is stimulated by various angiogenic peptides and growth factors. A correlation between tumor progression and the secretion of various serological mediators in patients with malignant tumors of the head and neck is of major interest for tumor diagnostics, evaluation of the therapy response and it may predict prognosis by specifying the individual tumor biology. Established chemotherapeutic regimes for head and neck tumors usually consist of platinum-based chemotherapeutic drugs and 5-fluorouracil (5-FU). The present pilot study sought to assess the eligibility of seven serological factors as biomarkers for malignant tumors of the head and neck: Platelet-derived growth factor, vascular endothelial growth factor, epidermal growth factor receptor, osteopontin, granulocyte-colony stimulating factor, interleukin-4 (IL-4) and IL-6. The serum levels of each factor in 20 patients receiving concomitant radiochemotherapy with cisplatin or carboplatin and 5-FU with curative intent were determined prior and subsequent to chemotherapy and were compared with 40 healthy controls. Another aim of the pilot study was to investigate whether the serum of patients showed significant differences in the concentrations of the analyzed factors at the start of concomitant radiochemotherapy compared with the controls, whether those markers indicated a neoplastic process and whether concomitant radiochemotherapy with cisplatin or carboplatin and 5-FU induced significant alterations of concentration compared with pre-therapeutic levels. The included patients were histopathologically diagnosed with HNSCC and the average age was 62.3 years. The serum samples of the patients were obtained during the course of regular pre- and post-chemotherapeutic blood draws one week prior to the start of radiochemotherapy and one week following the completion of chemotherapy. The healthy controls were collected from patients of the Sleep Laboratory of the Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital (Mannheim, Germany) without clinical evidence or laboratory signs of inflammation or history of a malignant disease. The average age was 50.3 years. The serological level of each factor was ascertained by enzyme-linked immunosorbent assay in duplicate. Serum levels of IL-4, IL-6 and osteopontin were significantly increased in patients with HNSCC compared with those in chemotherapy-naive healthy controls. IL-4 and osteopontin showed no significant therapy-associated alterations. Notably, IL-6 levels significantly increased post-therapeutically. Using logistic regression with osteopontin and IL-4, an individual risk-profile for random samples was calculated. IL-4, IL-6 and osteopontin appear to be suitable indicators of the neoplastic process as they are significantly increased in HNSCC patients compared with the control group. With the exception of IL-6, whose levels were in fact increased following therapy, a significant therapy-associated alteration of these factors was missing. Therefore, these serological markers failed to predict the therapy response, but they may be valuable as a screening instrument in primary diagnostics.

Lapatinib‑induced mesenchymal‑epithelial transition in squamous cell carcinoma cells correlates with unexpected alteration of β‑catenin expression

The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer progression, and may promote resistance of cancer cells to therapy. Inhibiting EMT appears to be crucial to inhibit drug resistance. The mesenchymal-epithelial transition (MET), which is the reverse program of EMT in metastases, is characterized by the upregulation of epithelial adhesive proteins such as E-cadherin, and downregulation of mesenchymal proteins such as vimentin. The sensitivity of cancer cells to epithelial growth factor receptor (EGFR) inhibitor may be increased by inducing MET in these cells. Therefore, it is of clinical importance to specify the phenotype of cancer cells in order to overcome the phenomenon of drug resistance. The aim of the present study was to investigate the expression pattern of specific markers in squamous cell carcinoma (SCC) cells following stimulation with lapatinib and gefitinib. For this purpose, the head and neck (HN) SCC cell lines HNSCC22B and HNSCC11A were incubated with 0.5 and 2 µg/ml lapatinib and gefitinib, and the levels of E-cadherin, vimentin, matrix metalloproteinase-14, c-kit and β-catenin were detected by immunocytochemistry and enzyme-linked immunosorbent assay at 5, 24 and 96 h post-incubation. The results indicated that, compared with HNSCC22B cells, the protein expression levels of vimentin increased, whereas those of E-cadherin reduced, in non-stimulated HNSCC11A cells. In addition, the protein expression levels of β-catenin were altered in the epithelial- and mesenchymal-associated SCC cell lines following treatment with lapatinib and gefitinib. Furthermore, lapatinib induced the downregulation of vimentin and upregulation of E-cadherin in HNSCC11A cells in a time-dependent manner. This suggests that the sensitivity of cancer cells to lapatinib may be improved by inducing MET in these cells. In summary, the results of the present study demonstrated that lapatinib-induced MET led to an unexpected alteration of the protein expression levels of β-catenin in SCC cells. Further studies on the mechanistic role of MET are required in order to increase the sensitivity of cancer cells to EGFR inhibitor and block the EMT process in these cells.

Perioperative Antibiotikaprophylaxe in der HNO

ZusammenfassungDie perioperative Antibiotikaprophylaxe (POAP) verfolgt im Gegensatz zur therapeutischen Gabe von Antibiotika nicht das Ziel, eine definierte Infektion zu bekämpfen. Es soll vielmehr verhindert werden, dass Keime, die in den meisten Fällen zur physiologischen Normalflora gehören, im Gebiet des Hautschnitts bzw. des operativen Zugangswegs ins Gewebe eindringen und sich dort ansiedeln können. Die Frage, bei welchem spezifischen Eingriff und bei welchem Patienten eine POAP sinnvoll ist, kann im Fachbereich der HNO-Heilkunde allerdings nur zum Teil mithilfe experimenteller Daten beantwortet werden. Ziel dieser Arbeit war es, die aktuelle Literatur nach Empfehlungen zur antibiotischen Prophylaxe im HNO-Bereich zu analysieren und auf der Basis der gefundenen Arbeiten einen rationalen Standard zu entwickeln.AbstractSurgical antibiotic prophylaxis (SAP) is defined as the administration of an antimicrobial agent prior to contamination in previously sterile spaces and fluid. SAP should not be confused with the therapeutic use of antibiotics. There are a growing number of studies with the goal of answering the question which patients benefit most from SAP during which specific surgical procedure. However, in the specific surgical field of head and neck surgery and otolaryngological surgery, there are only a few guidelines answering that question for specific procedures. The aim of this study was to analyze the evidence found in the literature and to develop a standard operating procedure, which specifically addresses head and neck and otolaryngeal surgical procedures.Surgical antibiotic prophylaxis (SAP) is defined as the administration of an antimicrobial agent prior to contamination in previously sterile spaces and fluid. SAP should not be confused with the therapeutic use of antibiotics. There are a growing number of studies with the goal of answering the question which patients benefit most from SAP during which specific surgical procedure. However, in the specific surgical field of head and neck surgery and otolaryngological surgery, there are only a few guidelines answering that question for specific procedures. The aim of this study was to analyze the evidence found in the literature and to develop a standard operating procedure, which specifically addresses head and neck and otolaryngeal surgical procedures.