A CRISPRi/a screening platform to study cellular nutrient transport in diverse microenvironments

Abstract

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 CRISPRi/a screening platform to systematically interrogate the contribution of specific nutrient transporters to support cancer cell proliferation in environments ranging from standard culture media to tumor models. We applied this platform to identify the transporters of amino acids in leukemia cells and found that amino acid transport is characterized by high bidirectional flux that is dependent on the composition of the microenvironment. While investigating the role of transporters in cystine starved cells, we uncovered a novel role for serotonin uptake in preventing ferroptosis. Finally, we identified transporters essential for cell proliferation in subcutaneous tumors and found that levels of glucose and amino acids can restrain proliferation in that environment. This study provides a framework for the systematic identification of critical cellular nutrient transporters, characterizing the function of such transporters, and studying how the tumor microenvironment impacts cancer metabolism.

ASCT2 is the primary serine transporter in cancer cells

“…The design and construction of transporter CRISPRi and CRISPRa sgRNA libraries has been reported elsewhere 40 . The multiplicity of infection (MOI) of lentiviral supernatant produced from each library was determined by titration onto MCF7 CRISPRi and CRISPRa cell lines and quantification of the percentage of puromycin resistant cells.…”

“…To prepare complete RPMI medium with amino acids present at physiological levels screens (RPMI-PAA), base medium lacking amino acids was prepared as above, except that 965 mL deionized H 2 O was used. To that, amino acids were added to their concentration in human plasma using the same stock solutions as for RPMI above and as previously described 40 . All media were preheated and equilibrated to 37 °C/5% CO 2 before use.…”

“…At screen end, 6–12 million cells were collected by centrifugation, washed 1 × with PBS and stored at −80 °C. The abundance of each sgRNA in individual samples was determined as previously reported 40 . Briefly, genomic DNA (gDNA) was extracted from cell pellets using the QIAamp DNA Blood Mini Kit (Qiagen) and typical yields ranged from 40 to 80 μg gDNA.…”

“…We have recently discovered that luminal/ER+ breast cancer cells are auxotrophic for serine 20 and reasoned that their dependence on exogenous serine would make them an ideal model for identifying serine transporters. Because there are numerous transporters that have the potential to transport serine, we made use of a two-part CRISPRi and CRISPRa screen with a library targeting 489 human transporter genes belonging to the SLC and ABC families 40 . This library has recently been used to identify transporters for a variety of amino acids, but these efforts were less successful for identifying serine transporters given that they were screened in cells that are not auxotrophic for serine 40 .…”

“…Cells in RPMI and RPMI low Ser underwent 6.8 and 1.3 population doublings, respectively.At screen end, 6-12 million cells were collected by centrifugation, washed 1 × with PBS and stored at −80 °C. The abundance of each sgRNA in individual samples was determined as previously reported40 .…”