Cetuximab induces mitochondrial translocalization of EGFRvIII, but not EGFR: involvement of mitochondria in tumor drug resistance?

Dysregulation of growth factor receptors such as the epidermal growth factor receptor (EGFR) and of its truncated form EGFRvIII is frequently found in human tumors. EGFRvIII is a promising target for selective molecular tumor therapy because it is exclusively expressed by tumor cells. Cetuximab/Erbitux is a monoclonal antibody which targets EGFR and EGFRvIII. The effects of cetuximab on EGFRvIII but still the exact function and mechanism of cetuximab in relation to EGFR and EGFRvIII are incompletely understood. Therefore, we investigated the influence of cetuximab on EGFRvIII signaling and cellular survival. We found that cetuximab leads to increased internalization of EGFRvIII in NR6M cells but is unable to inhibit neither the activation of EGFRvIII nor its downstream signaling pathways. Incubation with cetuximab also did not alter the survival and proliferation of EGFRvIII-expressing cells. However, it caused increased mitochondrial activity and an increase in co-localization of EGFRvIII with mitochondria. These results demonstrate that interaction of EGFRvIII with mitochondria could play a role in survival of cetuximab-treated NR6M cells. Thus, a role of mitochondria in resistance to cetuximab has to be considered.

Ovine carotid artery-derived cells as an optimized supportive cell layer in 2-D capillary network assays.

BACKGROUND: Endothelial cell co-culture assays are differentiation assays which simulate the formation of capillary-like tubules with the aid of a supportive cell layer. Different cell types have been employed as a supportive cell layer, including human pulmonary artery smooth muscle cells (PASMCs) and human mammary fibroblasts. However, these sources of human tissue-derived cells are limited, and more readily accessible human or animal tissue-derived cell sources would simplify the endothelial cell co-culture assay. In the present study, we investigated the potential use of alternative, accessible supportive cells for endothelial cell co-culture assay, including human umbilical cord and ovine carotid artery. METHODS AND RESULTS: Human umbilical artery SMCs (HUASMCs) and ovine carotid artery-derived cells were seeded into 96-well plates, followed by addition of human umbilical vein endothelial cells (HUVECs). Nine days after co-culture, cells were fixed, immunostained and analysed using an in vitro angiogenesis quantification tool. Capillary-like structures were detected on ovine carotid artery-derived supportive cell layers. The initial cell number, as well as pro- and anti-angiogenic factors (VEGF, PDGF-BB and Bevacizumab), had a positive or negative influence on the number of capillary-like structures. Furthermore, HUVECs from different donors showed distinct levels of VEGF receptor-2, which correlated with the amount of capillary-like structures. In the case of HUASMC supportive cell layers, HUVECs detached almost completely from the surface. CONCLUSIONS: Cells of different origin have a varying applicability regarding the endothelial cell co-culture assay: under the conditions described here, ovine carotid artery-derived cells seem to be more suitable than HUASMCs for an endothelial co-culture assay. Furthermore, the ovine carotid artery-derived cells are easier to obtain and are in more abundant supply than the currently used dermal or breast tissue cells. The use of ovine carotid artery-derived cells simplifies the endothelial co-culture assay with respect to testing large amounts of pro- and anti-angiogenic factors.

Biofunctionalized microfiber-assisted formation of intrinsic three-dimensional capillary-like structures.

OBJECTIVES: A vascular supply network is essential in engineered tissues >100-200-µm thickness. To control vascular network formation in vitro, we hypothesize that capillarization can be achieved locally by using fibers to position and guide vessel-forming endothelial cells within a three-dimensional (3D) matrix. MATERIALS AND METHODS: Biofunctionalization of poly-(L-lactic acid) (PLLA) fibers was performed by amino-functionalization and covalent binding of RGD peptides. Human foreskin fibroblasts (HFFs) and human umbilical vein endothelial cells (HUVECs) were seeded on the fibers in a mould and subsequently embedded in fibrin gel. After 9-21 days of coculture, constructs were fixed and immunostained (PECAM-1). Capillary-like structures with lumen in the 3D fibrin matrix were verified and quantified using two-photon microscopy and image analysis software. RESULTS: Capillary-like networks with lumen formed adjacent to the PLLA fibers. Increased cell numbers were observed to attach to RGD-functionalized fibers, resulting in enhanced formation of capillary-like structures. Cocultivation of HFFs sufficiently supported HUVECs in the formation of capillary-like structures, which persisted for at least 21 days of coculture. CONCLUSIONS: The guidance of vessel growth within tissue-engineered constructs can be achieved using biofunctionalized PLLA microfibers. Further methods are warranted to perform specified spatial positioning of fibers within 3D formative scaffolds to enhance the applicability of the concept.

Axon growth-promoting properties of human bone marrow mesenchymal stromal cells.

Mesenchymal stromal cells are promising candidate donor cells for promoting functional tissue repair following traumatic spinal cord injury (SCI), however, the mechanism(s) of action remain poorly defined. Here, we describe an in vitro study of the axon growth-promoting properties of highly enriched populations of adult human mesenchymal stromal cells (hMSC). A random, non-oriented pattern of neuritic outgrowth was observed from dissociated adult rat DRG neurons seeded onto confluent A431 cells and PLL/laminin positive control substrata. Confluent hMSC formed arrays of similarly orientated cell bodies and processes which supported the regeneration of significantly more primary neurites but a slightly lower overall neuritic length than was observed over the PLL/laminin control substrate. The hMSC exerted a strong influence on the direction of neuritic outgrowth, with many regenerating processes following the orientation of underlying hMSC. The production of extracellular matrix appeared to be responsible for neuritic directionality, but the release of growth factors was a significant promoter for DRG neuritic outgrowth. This suggests that further investigations into the properties of hMSC may be of particular interest in the development of transplant-mediated strategies intending to promote functional axonal regeneration after SCI.