RESUMEN
Blocking the import of nutrients essential for cancer cell proliferation represents a therapeutic opportunity, but it is unclear which transporters to target. Here we report a CRISPR interference/activation screening platform to systematically interrogate the contribution of nutrient transporters to support cancer cell proliferation in environments ranging from standard culture media to tumours. We applied this platform to identify the transporters of amino acids in leukaemia cells and found that amino acid transport involves high bidirectional flux dependent on the microenvironment composition. While investigating the role of transporters in cystine starved cells, we uncovered a role for serotonin uptake in preventing ferroptosis. Finally, we identified transporters essential for cell proliferation in subcutaneous tumours and found that levels of glucose and amino acids can restrain proliferation in that environment. This study establishes a framework for systematically identifying critical cellular nutrient transporters, characterizing their function and exploring how the tumour microenvironment impacts cancer metabolism.
Asunto(s)
Proliferación Celular , Microambiente Tumoral , Humanos , Animales , Sistemas CRISPR-Cas , Nutrientes/metabolismo , Línea Celular Tumoral , Transporte Biológico , Glucosa/metabolismo , Aminoácidos/metabolismo , Serotonina/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Ratones , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente EspaciadasRESUMEN
Rapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases. Current state of the art diagnostics are highly sensitive and specific, but slow, and require expensive equipment. Here we report the development of a molecular diagnostic test for SARS-CoV-2 based on an enhanced recombinase polymerase amplification (eRPA) reaction. eRPA has a detection limit on patient samples down to 5 viral copies, requires minimal instrumentation, and is highly scalable and inexpensive. eRPA does not cross-react with other common coronaviruses, does not require RNA purification, and takes ~45 min from sample collection to results. eRPA represents a first step toward at-home SARS-CoV-2 detection and can be adapted to future viruses within days of genomic sequence availability.
Asunto(s)
Betacoronavirus/genética , Betacoronavirus/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/métodos , Prueba de COVID-19 , Técnicas de Laboratorio Clínico , Infecciones por Coronavirus/diagnóstico , Humanos , ARN/metabolismo , ARN Viral/genética , ARN Viral/aislamiento & purificación , ADN Polimerasa Dirigida por ARN/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Recombinasas/metabolismo , SARS-CoV-2 , Saliva/virología , Virión/genéticaRESUMEN
Rapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases. Current state of the art diagnostics are highly sensitive and specific, but slow, and require expensive equipment. We developed a molecular diagnostic test for SARS-CoV-2, FIND (Fast Isothermal Nucleic acid Detection), based on an enhanced isothermal recombinase polymerase amplification reaction. FIND has a detection limit on patient samples close to that of RT-qPCR, requires minimal instrumentation, and is highly scalable and cheap. It can be performed in high throughput, does not cross-react with other common coronaviruses, avoids bottlenecks caused by the current worldwide shortage of RNA isolation kits, and takes ~45 minutes from sample collection to results. FIND can be adapted to future novel viruses in days once sequence is available. ONE SENTENCE SUMMARY: Sensitive, specific, rapid, scalable, enhanced isothermal amplification method for detecting SARS-CoV-2 from patient samples.
