Search

MET amplification (METamp), a mechanism of acquired resistance to EGFR tyrosine kinase inhibitors, occurs in up to 30% of patients with non-small-cell lung cancer (NSCLC) progressing on first-line osimertinib. Combining osimertinib with a MET inhibitor, such as tepotinib, an oral, highly selective, potent MET tyrosine kinase inhibitor, may overcome METamp-driven resistance. INSIGHT 2 (NCT03940703), an international, open-label, multicenter phase II trial, assesses tepotinib plus osimertinib in patients with advanced/metastatic EGFR-mutant NSCLC and acquired resistance to first-line osimertinib and METamp, determined centrally by fluorescence in situ hybridization (gene copy number ≥5 and/or MET/CEP7 ≥2) at time of progression. Patients will receive tepotinib 500 mg (450 mg active moiety) plus osimertinib 80 mg once-a-day. The primary end point is objective response, and secondary end points include duration of response, progression-free survival, overall survival and safety. Trial registration number: NCT03940703 (clinicaltrials.gov). [ABSTRACT FROM AUTHOR]

We investigated the effect of cyclooxygenase (COX) activity on the regulation of the renin-angiotensin-aldosterone system by salt intake. Therefore, Sprague-Dawley rats were subjected to different salt diets [0.02, 0.6, and 8% NaCl (wt/wt)] and treated with the selective COX-2 inhibitor rofecoxib (10 mg * kg body [wt.sup.-1] * [day.sup.-1]) or with ketorolac at a dose selective for COX-1 inhibition (2 mg * kg body [wt.sup.-1] * [day.sup.-1) for 3, 7, 14, and 21 days. Rofecoxib and ketorolac caused a similar reduction of renocortical PG[E.sub.2] formation with a low-salt diet. Rofecoxib did not change plasma renin activity or renocortical renin mRNA abundance with any of the diets but clearly lowered plasma aldosterone concentration. In contrast, ketorolac delayed the increase in plasma renin activity and of renin mRNA in response to low salt intake but did not change plasma aldosterone concentration. Prolonged treatment with rofecoxib but not with ketorolac caused an upregulation of COX-2 expression while COX-1 mRNA abundance remained unchanged. These findings suggest that COX-l-derived, but not COX-2-derived, prostanoids are of relevance for the regulation of the renin system by salt intake. cyclooxygenase; kidney

Renal cells are used in basic research, disease models, tissue engineering, drug screening, and in vitro toxicology. In order to provide a reliable source of human renal cells, we developed a protocol for the differentiation of human embryonic stem cells into renal epithelial cells. The differentiated stem cells expressed markers characteristic of renal proximal tubular cells and their precursors, whereas markers of other renal cell types were not expressed or expressed at low levels. Marker expression patterns of these differentiated stem cells and in vitro cultivated primary human renal proximal tubular cells were comparable. The differentiated stem cells showed morphological and functional characteristics of renal proximal tubular cells, and generated tubular structures in vitro and in vivo. In addition, the differentiated stem cells contributed in organ cultures for the formation of simple epithelia in the kidney cortex. Bioreactor experiments showed that these cells retained their functional characteristics under conditions as applied in bioartificial kidneys. Thus, our results show that human embryonic stem cells can differentiate into renal proximal tubular-like cells. Our approach would provide a source for human renal proximal tubular cells that are not affected by problems associated with immortalized cell lines or primary cells. [ABSTRACT FROM AUTHOR]

In the organism epithelia perform perfect barrier functions. Strong rheological and mechanical influences constitute the normal environment of this tissue throughout life. Most epithelia are exposed to different fluids at the luminal and basal sides. To obtain realistic information about tissue development in modern biomaterial testing and tissue engineering it is necessary to mimik the natural environment of epithelia. Cultured cells are brought in contact with an artificial extracellular matrix to determine whether proper development into a functional epithelium occurs. As under natural conditions the cultures have to withstand mechanical and fluid stress over a prolonged period of time in close contact to a selected biomaterial. However, development of tissue‐specific features such as polarization, tightness and transport under in vitro conditions will only occur, if the biomaterial and the culture conditions support tissue development. Leakage, edge damage and pressure differences during culture have to be avoided so that the natural functions of the growing epithelium can develop. Our aim is to generate functional epithelia derived from renal explants containing stem cells, which are microsurgically isolated and placed into specific O‐ring carriers for optimal handling. The cells develop in combination with a collagenous matrix from an embryonic into a functional collecting duct (rCD) epithelium. To achieve optimal culture conditions the tissue is placed in a gradient culture container. A typical environment can be simulated by superfusing different culture media at the luminal and basal sides. Within days epithelia growing inside the gradient container build up a physiological barrier, which is maintained during the whole culture period. The described method allows to investigate the influence of new biomaterials over prolonged periods of time. [ABSTRACT FROM AUTHOR]

During kidney development a multitude of tubular portions is formed. Little knowledge is available by which cellbiological mechanism a cluster of embryonic cells is able to generate the threedimensional structure of a tubule. However, this know-how is most important in tissue engineering approaches such as the generation of an artificial kidney module or for the therapy of renal diseases using stem cells. To obtain cellbiological insights in parenchyme development we elaborate a new technique to generate under in vitro conditions renal tubules derived from the embryonic cortex of neonatal rabbits. The aim of the experiments is to establish a specific extracellular environment allowing optimal threedimensional development of renal tubules under serum-free culture conditions. In the present paper we demonstrate features of the renal stem cell niche and show their isolation as intact microcompartiments for advanced tissue culture. Perfusion culture in containers exhibiting a big dead fluid volume results in the development of a flat collecting duct (CD) epithelium at the surface of the tissue explant. In contrast, by fine-tuning the dead fluid volume within a perfusion culture container by an artificial interstitium made of a polyester fleece shows the generation of tubules. It is an up to date unknown morphogenetic information which tells the cells to form tubular structures. [ABSTRACT FROM AUTHOR]

Epithelia perform barrier functions being exposed to different fluids on the luminal and basal side. For long-term testing of new biomaterials as artificial basement membrane substitutes, it is important to simulate this fluid gradient. Individually-selected biomaterials can be placed in tissue carriers and in gradient containers, where different media are superfused. Epithelia growing on the tissue carriers form a physiological barrier during the whole culture period. Frequently however, pressure differences between the luminal and basal compartments occur. This is caused by a unilateral accumulation of gas bubbles in the container compartments resulting in tissue damage. Consequently, the occurence of gas bubbles has to be minimized. Air bubbles in the perfusion culture medium preferentially acumulate at sites where different materials come into contact. The first development is new screw caps for media bottles, specifically designed to allow fluid contact with only the tube and not the cap material. The second development is the separation of remaining gas bubbles from the liquid phase in the medium using newly-developed gas expander modules. By the application of these new tools, the yield of embryonic renal collecting duct epithelia with intact barrier function on a fragile natural support material can be significantly increased compared to earlier experiments. [ABSTRACT FROM AUTHOR]

Optimal results in biomaterial testing and tissue engineering under in vitro conditions can only be expected when the tissue generated resembles the original tissue as closely as possible. However, most of the presently used stagnant cell culture models do not produce the necessary degree of cellular differentiation, since important morphological, physiological, and biochemical characteristics disappear, while atypical features arise. To reach a high degree of cellular differentiation and to optimize the cellular environment, an advanced culture technology allowing the regulation of differentiation on different cellular levels was developed. By the use of tissue carriers, a variety of biomaterials or individually selected scaffolds could be tested for optimal tissue development. The tissue carriers are to be placed in perfusion culture containers, which are constantly supplied with fresh medium to avoid an accumulation of harmful metabolic products. The perfusion of medium creates a constant microenvironment with serum-containing or serum-free media. By this technique, tissues could be used for biomaterial or scaffold testing either in a proliferative or in a postmitotic phase, as is observed during natural development. The present paper summarizes technical developments, physiological parameters, cell biological reactions, and theoretical considerations for an optimal tissue development in the field of perfusion culture. [ABSTRACT FROM AUTHOR]

The prognosis of patients with glioblastoma (GBM) remains poor despite current treatments. Targeted therapy in GBM has been the subject of intense investigation but has not been successful in clinical trials. The reasons for the failure of targeted therapy in GBM are multifold and include a lack of patient selection in trials, the failure to identify driver mutations, and poor blood-brain barrier penetration of investigational drugs. Here, we describe a case of a durable complete response in a newly diagnosed patient with GBM with leptomeningeal dissemination and PTPRZ1-MET fusion who was treated with tepotinib, a brain-penetrant MET inhibitor. This case of successful targeted therapy in a patient with GBM demonstrates that early molecular testing, identification of driver molecular alterations, and treatment with brain-penetrant small molecule inhibitors have the potential to change the outcome in select patients with GBM., (© The Author(s) 2024. Published by Oxford University Press.)

Purpose: Primary analysis of VISION showed tepotinib had durable clinical activity in patients with MET exon 14 (METex14) skipping non-small cell lung cancer (NSCLC). We present updated outcomes for clinically relevant subgroups., Patients and Methods: This phase II, open-label, multi-cohort study of 500 mg (450 mg active moiety) tepotinib in patients with METex14 skipping NSCLC assessed efficacy and safety in predefined subgroups according to age, prior therapies (chemotherapy and immune checkpoint inhibitors), and brain metastases. An ad hoc retrospective analysis using Response Assessment in Neuro-Oncology Brain Metastases (RANO-BM) criteria assessed intracranial activity., Results: 152 patients were evaluable for efficacy (median age: 73.1). Overall, objective response rate (ORR) was 44.7% [95% confidence interval (CI): 36.7-53.0]. Patients aged <75 (n = 84) and ≥75 (n = 68) had ORRs of 48.8% (95% CI: 37.7-60.0) and 39.7% (95% CI: 28.0-52.3), respectively. Treatment-naïve (n = 69) versus previously treated (n = 83) patients showed consistent efficacy [ORR (95% CI): 44.9% (32.9-57.4) vs. 44.6% (33.7-55.9); median duration of response (95% CI): 10.8 (6.9-not estimable) vs. 11.1 (9.5-18.5) months]. Of 15 patients analyzed by RANO-BM (12 received prior radiotherapy), 13 achieved intracranial disease control; 5 of 7 patients with measurable brain metastases had partial intracranial responses. Of 255 patients evaluable for safety, 64 (25.1%) experienced grade ≥3 treatment-related adverse events (TRAE), leading to discontinuation in 27 patients (10.6%). Rates of adverse events (AE) were broadly consistent irrespective of prior therapies., Conclusions: Tepotinib showed meaningful activity across subgroups by age, prior therapies, and brain metastases, with a manageable safety profile and few treatment discontinuations. See related commentary by Rosner and Spira, p. 1055., (©2021 The Authors; Published by the American Association for Cancer Research.)

MET amplification (MET amp), a mechanism of acquired resistance to EGFR tyrosine kinase inhibitors, occurs in up to 30% of patients with non-small-cell lung cancer (NSCLC) progressing on first-line osimertinib. Combining osimertinib with a MET inhibitor, such as tepotinib, an oral, highly selective, potent MET tyrosine kinase inhibitor, may overcome MET amp-driven resistance. INSIGHT 2 (NCT03940703), an international, open-label, multicenter phase II trial, assesses tepotinib plus osimertinib in patients with advanced/metastatic EGFR -mutant NSCLC and acquired resistance to first-line osimertinib and MET amp, determined centrally by fluorescence in situ hybridization (gene copy number ≥5 and/or MET/CEP7 ≥2) at time of progression. Patients will receive tepotinib 500 mg (450 mg active moiety) plus osimertinib 80 mg once-a-day. The primary end point is objective response, and secondary end points include duration of response, progression-free survival, overall survival and safety. Trial registration number: NCT03940703 (clinicaltrials.gov).

The presentation of bioactive ligands on biomaterial surfaces is often confounded by the adsorption of proteins present in the biological milieu, rendering any type of cellular response nonspecific. We have engineered a polyelectrolyte complex membrane that demonstrates specific adhesion of various cell types for both two-dimensional (2D) and three-dimensional (3D) cell culture systems. Specific cell adhesion is achieved by a three-tiered structure: a silica cross-linked polycation as the bottom (first) tier, a nonfouling polyanion-poly(ethylene glycol) (PEG) conjugate as the intermediate (second) tier, and the cell-adhesion ligand as the top (third) tier. Each tier of the membrane was characterized in terms of chemical composition and dimensions. Epithelial cells (primary human cortical renal cells and a hepatocellular carcinoma cell line) cultured on the membranes exhibited little cell attachment on the polyanion-PEG second tier and good cell adhesion on the RGD-modified third tier. Thus, the second tier allowed the effect of cell adhesion due to the ligand (third tier) to be isolated and distinguished from nonspecific cell attachment to the first tier. For the culturing of cells in three dimensions, the three-tiered membrane system was applied using a highly swellable chitosan membrane as the first tier. The resulting cell-membrane construct was uniformly dispersed and centrifuged to form a matrix that interacted intimately with cells in the form of a pellet. Presentation of RGD in the latter format enhanced the viability of human mesenchymal stem cells (hMSCs) over controls without RGD.

Cultured precision-cut liver tissue slices are useful for studying the metabolism and toxicity of xenobiotics in liver. They may also be used to investigate the behavior of and interaction between different cell types in an intact histo-architecture. Because cultured liver tissues undergo a loss of function and morphology because of their separation from the blood supply, we investigated changes in key protein marker expressions in parenchymal and non-parenchymal cells, as well as in the extracellular matrix (ECM) at different time points. We also compared conventional culture methods such as static and dynamic cultures with perfusion culture, which allows a continuous exchange of the culture medium. In conventional culture methods, the expression of vimentin and collagen type IV decreased after 5 h in the non-parenchymal cells and the ECM, respectively, whereas the hepatocyte nuclear factor 4 alpha (HNF4alpha) staining in the hepatocytes remained constant. In perfusion culture, on the other hand, vimentin, collagen type IV, and HNF4alpha staining were clearly detectable after 5 h. The histo-architecture obtained from perfusion culture was also more compact than those obtained from conventional culture methods. After 24 h, only the perfusion cultured sample retained protein marker expression in all components of the liver tissue. Our results suggest that, to develop improved culture techniques for liver slices, changes at the early time-points should be taken into consideration. Our results also show that culture techniques that enable a continuous exchange of the culture medium seem to be superior to static or dynamic cultures in terms of maintaining the protein expression and the histo-architecture.

In the present paper, a modular system to generate artificial tissues under optimal perfusion culture conditions is described. The microenvironment within the culture containers can be fine-tuned to meet the physiologic needs of individual tissues. An optimal physiologic environment is created by modulating a liquid phase as well as an artificial interstitium surrounding the growing construct. For example, the generation of tubular structures derived from renal stems cells under in vitro conditions is shown for the first time.

The efficiency of cell or tissue cultures is usually judged by how quickly confluence is reached within a Petri dish or on a scaffold. Growth factors and fetal bovine serum are employed to drive cultured cells from one mitosis to the next as quickly as possible. The tissue specific interphase is extremely short under these conditions, so that the degree of differentiation desired in tissue engineering cannot be achieved. To reach the goal of functional differentiation in vitro mitosis and interphase must be separated experimentally and tailored to the specific requirements of the cell-type used. This could be achieved by a three step concept for tissue-engineering in vitro as we present here. The expansion phase is followed by a phase in which tissue differentiation is initiated. The final phase serves to express and maintain histotypical differentiation of the generated tissue.