Automation’s Role in a CRISPR Screening Pipeline using Underrepresented Patient Derived Tumor Organoids (Cancer Research article)
Explore how CRISPR-based genetic screens, supported by automation, offer a broad view into cancer vulnerabilities that can inform precision medicine approaches in drug screening and discovery.
Personalized medicine remains a dynamic work in progress. Whereas some successes have reshaped cancer approaches, they also revealed that not all therapies offer the same advantages to different people. This is especially true in underrepresented populations where the lack or low rate of inclusion in clinical trials and studies means a poor therapy response characterization. At the same time, cell models long-accustomed to non-physiologic environments may react quite differently than fresh patient-derived models when challenged with compounds, thus skewing outcomes.
Recently, researchers in the United States and Ghana collaborated on a study that took a functional precision medicine approach to unlock potential anti-breast cancer targets using patient-derived tumor organoids (PDTO) from women with African ancestry. PDTO retain many in vivo functions, including cell-cell and cell-matrix interactions, to more closely reflect the original tumor characteristics. Their work is summarized in the original research article, “Kinome-Focused CRISPR-Cas9 Screens in African Ancestry Patient-Derived Breast Cancer Organoids Identify Essential Kinases and Synergy of EGFR and FGFR1 Inhibition”, published by Cancer Research.
KINOME-FOCUSED CRISPR-Cas9 SCREENING
Functional precision medicine is one of many research fields positively impacted by the emergence of CRISPR-based technologies. The multi-national team used CRISPR-Cas9 technology, aided by a guide RNA (gRNA) library, to disrupt kinase genes. Kinases in these 3D cell models were chosen because they are actionable targets and because previous independent studies demonstrated many kinases to be upregulated in triple-negative breast cancer tumors in women of African ancestry compared with their European American counterparts.
In preparation for screening, the PDTO were cultured and fully characterized. Clones were then created, transduced, and enriched to express Cas9 activity, and the gRNA library and drug compounds were prepared.
THE HIGH-THROUGHPUT DRUG SCREENING SYSTEM
This important study relied on a fully automated system from HighRes Biosolutions to perform high-throughput screens. The system integrated the Prime® liquid handler to perform cell seeding in 384-well plates and MicroSpin™ automated centrifuge from HighRes. It also included a second automated liquid handler, plate incubators, a plate reader, and a high content imager.
The automated system was powered and controlled through Cellario® whole lab workflow automation software. Beyond connecting and controlling the integrated devices, this powerful software platform allowed users to design automated experimental protocols and simulate tests before introducing the PDTO clones. Cellario also empowered user-friendly order creation with real-time run control.
One function of this automated system was screening the PDTO clones against a library of 156 FDA-approved drugs and other targeted compounds. The drug compounds were tested alone and in combination with the EGFR inhibitor, gefitinib. EGFR, or epidermal growth factor receptor, is a protein often overexpressed in triple negative breast cancer and associated with resistance to hormonal therapy and a poor prognosis.
Subsequent screens included testing candidate kinases against select inhibitors in several other breast cancer cell models and identifying synergistic interactions between drug compounds.
CONCLUSION
In addition to expected results, the team identified multiple previously unidentified putative therapeutic targets. This information, along with the compound synergies characterized, may be used to inform drug screening and discovery insights moving forward. Ultimately, the team foresees a broadened scope of diverse clinical information, especially as PDTO CRISPR methods progress, as genes may be differentially expressed across different patient populations.
The automated high-throughput drug screening system played a pivotal role in this CRISPR-based screen study, supplying highly robust performance in nuanced workflows along with walkaway convenience so that the researchers could focus on high-value tasks.
For details regarding use of CRISPR-Cas9 methods to screen 3D tumor organoids, read the full paper at https://doi.org/10.1158/0008-5472.CAN-24-0775. To develop your own automated screening solution, contact your local HighRes expert.