Meline Stölting

Myoblasts Inhibit Prostate Cancer Growth by Paracrine Secretion of Tumor Necrosis Factor-α

PURPOSE Myoblasts can form muscle fibers after transplantation. Therefore, they are envisioned as a treatment for urinary incontinence after radical prostatectomy. However, to our knowledge the safety of this treatment and the interaction of myoblasts with any remaining neighboring cancer are unknown. We investigated the interactions between myoblasts and prostate carcinoma cells in vitro and in vivo. MATERIALS AND METHODS Myoblasts isolated from the rectus abdominis were used in a series of co-culture experiments with prostate cancer cells and subcutaneously co-injected in vivo. Cell proliferation, cell cycle arrest and apoptosis of cancer in co-culture with myoblasts were assessed. Tumor volume and metastasis formation were evaluated in a mouse model. Tissue specific markers were assessed by immunohistochemistry, fluorescence activated cell sorting analysis, Western blot and real-time quantitative polymerase chain reaction. RESULTS Myoblasts in proximity to tumor provided paracrine tumor necrosis factor-α to their microenvironment, decreasing the tumor growth of all prostate cancer cell lines examined. Co-culture experiments revealed induction of cell cycle arrest, tumor death by apoptosis and increased myoblast differentiation. This effect was largely blocked by tumor necrosis factor-α inhibition. The same outcome was noted in a mouse model, in which co-injected human myoblasts also inhibited the tumor growth and metastasis formation of all prostate cancer cell lines evaluated. CONCLUSIONS Myoblasts restrict prostate cancer growth and limit metastasis formation by paracrine tumor necrosis factor-α secretion in vitro and in vivo.

Skeletal Muscle Regeneration for Clinical Application

Comprising nearly 50% of the human body [1] skeletal muscles compose the machinery that sets the body in movement. When well-trained, muscles have the capability to protect joints and bones from daily waste and trauma [2]. They hold an intrinsic protective mechanism against cancer formation and metastasis settling [3] and are at the same time the main energy reservoir of the body storing more than 80% of our glycogen reserve [4]. Hence, muscle tissue is associated to several functions and networks with different parts of the body. It is composed of muscle fibers, the contractile units, which are bound together by connective tissue. Most importantly, skeletal muscles display an astonishing regenerative capacity [5]. Due to resident stem cells, one week after severe trauma new myotubes are already being formed, and within 28 days after trauma muscle regeneration is almost complete [6]. These intrinsic features turn the skeletal muscle into a very interesting topic of study in regenerative medicine. Taking advantage of the regenerative potential of stem and precursor cells, skeletal muscle is con‐ stantly renewed in response to injury, damage or aging. It is this natural process that research‐ ers are about to harness in order to help patients with many muscle diseases and diseases that causes weakness or destruction of the muscle for instance stress urinary incontinence (SUI), muscular dystrophy. In this chapter, the focus will be on the regeneration of the skeletal muscle and especially in the case of incontinence. Urinary incontinence is the involuntary loss of urine and is a major medical problem affecting millions of people worldwide. It impairs the quality of life of patients and involves high healthcare costs. The main reason provoking SUI is the damage of the sphincter muscle due to childbirth, surgical treatments (as prostatectomy) or as an effect of aging. Current treatment encompasses behavioral training, pelvic floor exercising, drugs, medical devices and surgery. Unfortunately, all these options permit only limited recovery: short-term relief and are often accompanied with complications. The ultimate goal will be to prevent disease progression and to restore the tissue and its functions.

The role of donor age and gender in the success of human muscle precursor cell transplantation

Autologous cell transplantation for the treatment of muscle damage is envisioned to involve the application of muscle precursor cells (MPCs) isolated from adult skeletal muscle. At the onset of trauma, these cells are recruited to proliferate and rebuild injured muscle fibres. However, a variety of donor‐specific cues may directly influence the yield and quality of cells isolated from a muscle biopsy. In this study, we isolated human MPCs and assessed the role of donor gender and age on the ability of these MPCs to form functional bioengineered muscle. We analysed the cell yield, growth and molecular expression in vitro, and the muscle tissue formation and contractility of the bioengineered muscle, from cells isolated from men and women in three different age groups: young (20–39 years), adult (40–59 years) and elderly (60–80 years). Our results suggest that human MPCs can be successfully isolated and grown from patients of all ages and both genders. However, young female donors provide fast‐growing cells in vitro with an optimum contractile output in vivo and are therefore an ideal cell source for muscle reconstruction. Taken together, these findings describe the donor‐related limitations of MPC transplantation and provide insights for a straightforward and unbiased clinical application of these cells for muscle reconstruction. Copyright

Magnetic stimulation supports muscle and nerve regeneration after trauma in mice

Introduction: Magnetic stimulation (MS) has the ability to induce muscle twitch and has long been proposed as a therapeutic modality for skeletal muscle diseases. However, the molecular mechanisms underlying its means of action have not been defined. Methods: Muscle regeneration after trauma was studied in a standard muscle injury mouse model. The influence of MS on the formation of motor units, posttrauma muscle/nerve regeneration, and vascularization was investigated. Results: We found that MS does not cause systemic or muscle damage but improves muscle regeneration by significantly minimizing the presence of inflammatory infiltrate and formation of scars after trauma. It avoids posttrauma muscle atrophy, induces muscle hypertrophy, and increases the metabolism and turnover of muscle. It triples the expression of muscle markers and significantly improves muscle functional recovery after trauma. Conclusions: Our results indicate that MS supports muscle and nerve regeneration by activating muscle–nerve cross‐talk and inducing the maturation of neuromuscular junctions. Muscle Nerve 53: 598–607, 2016

1063 In vivo electromagnetic stimulation supports muscle regeneration after stem cell injection by boosting muscular metabolism and stimulating nerve ingrowth

INTRODUCTION & OBJECTIVES: Muscle Precursor Cells (MPCs) are adult stem cells present on skeletal muscle fibers. Responsible for normal muscle regeneration, they are envisioned as excellent cell sources for muscle tissue engineering with promising results in preclinical studies. Optimal muscular reconstruction involves formation of new muscle fibers capable of performing tonic contractions which is only possible if the same oxidative muscle fiber type, nerve ingrowth and formation of new neuromuscular junctions (NMJ) can be induced. In this study, we evaluate whether Noninvasive electromagnetic stimulation (NMS) improves tissue engineered muscle regeneration after MPC implantation, by investigating the presence of synapses, clustering of acetylcholine receptors (AChRs) and muscular metabolic adaptations caused by muscle contraction.

411 MUSCLE PRECURSOR CELLS ARE SAFE FOR THE TREATMENT OF URINARY INCONTINENCE AFTER SURGERY FOR PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: Skeletal muscle encloses sources of Muscle Precursor Cells (MPCs), which are capable of reconstructing muscle fiber upon injury. MPCs are studied for the treatment of urinary incontinence, also for patients after prostatectomy. However, the safety of injecting these cells in proximity of a potential location of tumor recurrence has not yet been investigated. METHODS: We have injected human MPCs, isolated from muscle biopsies harvested from the rectus abdominis, together with different types of prostate cancer, DU145, PC3 and LnCAP (ATCCLGC Standard) in vivo. Tumor sizes were measured with caliper twice a week for 6 weeks. Lymph node metastasis and tumor phenotypes were analyzed by histology. Tissue physiological activity was assessed by PET scan with F-choline. After harvest tissues were examined by IHC evaluating myogenic differentiation apoptosis and cell cycle arrest of the retrieved tumor. Data was analyzed with SPSS v11 (SPSS Inc, Chicago, IL) by independent samples t-tests or one way ANOVA (p 0.05 is considered significant). RESULTS: When co-injected with MPCs, Prostate carcinoma growth was reduced up to 5 fold (fig.1), while lymph node metastases were reduced from 100% after 6 weeks to only 10%. Lymph node and bone metastasis were identified by PET scan with F-choline (Fig.2) on the animals injected with tumor alone. In contrast, no animal bearing tumor co-injected with MPCs presented positive lymph node or metastasis. Histology demonstrates that MPCs differentiated in vivo developing into organized and functional muscle, while the tumor in its proximity were undergoing apoptosis (Caspase3 positive), cell cycle arrest (p21 positive) and expressing the tumor suppressor (BIN1). CONCLUSIONS: In this study, we report that the use of MPCs in proximity of prostate cancer is safe in vivo. Further, it decreased tumor growth and metastasis formation could be demonstrated. These results suggest that MPCs can be safely injected on the pelvic floor after prostatectomy.

1000 MUSCLE PRECURSOR CELLS ARE SAFE FOR THE TREATMENT OF URINARY INCONTINENCE AFTER SURGERY FOR PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: Skeletal muscle encloses sources of Muscle Precursor Cells (MPCs), which are capable of reconstructing muscle fiber upon injury. MPCs are studied for the treatment of urinary incontinence, also for patients after prostatectomy. However, the safety of injecting these cells in proximity of a potential location of tumor recurrence has not yet been investigated. METHODS: We have injected human MPCs, isolated from muscle biopsies harvested from the rectus abdominis, together with different types of prostate cancer, DU145, PC3 and LnCAP (ATCCLGC Standard) in vivo. Tumor sizes were measured with caliper twice a week for 6 weeks. Lymph node metastasis and tumor phenotypes were analyzed by histology. Tissue physiological activity was assessed by PET scan with F-choline. After harvest tissues were examined by IHC evaluating myogenic differentiation apoptosis and cell cycle arrest of the retrieved tumor. Data was analyzed with SPSS v11 (SPSS Inc, Chicago, IL) by independent samples t-tests or one way ANOVA (p 0.05 is considered significant). RESULTS: When co-injected with MPCs, Prostate carcinoma growth was reduced up to 5 fold (fig.1), while lymph node metastases were reduced from 100% after 6 weeks to only 10%. Lymph node and bone metastasis were identified by PET scan with F-choline (Fig.2) on the animals injected with tumor alone. In contrast, no animal bearing tumor co-injected with MPCs presented positive lymph node or metastasis. Histology demonstrates that MPCs differentiated in vivo developing into organized and functional muscle, while the tumor in its proximity were undergoing apoptosis (Caspase3 positive), cell cycle arrest (p21 positive) and expressing the tumor suppressor (BIN1). CONCLUSIONS: In this study, we report that the use of MPCs in proximity of prostate cancer is safe in vivo. Further, it decreased tumor growth and metastasis formation could be demonstrated. These results suggest that MPCs can be safely injected on the pelvic floor after prostatectomy.