RESUMO
Personalized cancer therapy is based on a patient's tumor lineage, histopathology, expression analyses, and/or tumor DNA or RNA analysis. Here, we aim to develop an in vitro functional assay of a patient's living cancer cells that could complement these approaches. We present methods for developing cell cultures from tumor biopsies and identify the types of samples and culture conditions associated with higher efficiency of model establishment. Toward the application of patient-derived cell cultures for personalized care, we established an immunofluorescence-based functional assay that quantifies cancer cell responses to targeted therapy in mixed cell cultures. Assaying patient-derived lung cancer cultures with this method showed promise in modeling patient response for diagnostic use. This platform should allow for the development of co-clinical trial studies to prospectively test the value of drug profiling on tumor-biopsy-derived cultures to direct patient care.
Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias/tratamento farmacológico , Medicina de Precisão/métodos , Cultura Primária de Células/métodos , Acrilamidas , Aminopiridinas , Quinase do Linfoma Anaplásico , Compostos de Anilina , Biomarcadores Tumorais/metabolismo , Biópsia , Crizotinibe , Receptores ErbB/genética , Receptores ErbB/metabolismo , Cloridrato de Erlotinib/uso terapêutico , Células Alimentadoras/citologia , Imunofluorescência/métodos , Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Queratina-18/genética , Queratina-18/metabolismo , Queratina-8/genética , Queratina-8/metabolismo , Lactamas , Lactamas Macrocíclicas/uso terapêutico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Mutação , Neoplasias/classificação , Neoplasias/genética , Neoplasias/patologia , Piperazinas/uso terapêutico , Pirazóis/uso terapêutico , Piridinas/uso terapêutico , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Células Tumorais CultivadasRESUMO
Tendon injuries are common and heal poorly. Strategies to regenerate or replace injured tendons are challenged by an incomplete understanding of normal tendon development. Our previous study showed that embryonic tendon elastic modulus increases as a function of developmental stage. Inhibition of enzymatic collagen crosslink formation abrogated increases in tendon elastic modulus at late developmental stages, but did not affect increases in elastic modulus of early stage embryonic tendons. Here, we aimed to identify potential contributors to the mechanical properties of these early stage embryonic tendons. We characterized tendon progenitor cells in early stage embryonic tendons, and the influence of actin cytoskeleton disruption on tissue elastic modulus. Cells were closely packed in embryonic tendons, and did not change in density during early development. We observed an organized network of actin filaments that seemed contiguous between adjacent cells. The actin filaments exhibited a crimp pattern with a period and amplitude that matched the crimp of collagen fibers at each developmental stage. Chemical disruption of the actin cytoskeleton decreased tendon tissue elastic modulus, measured by atomic force microscopy. Our results demonstrate that early developmental stage embryonic tendons possess a well organized actin cytoskeleton network that contributes significantly to tendon tissue mechanical properties.
