(W1430-02-08) In Vitro Method Using Circulating Tumor Cells-Derived Organoids from Patients with Solid Tumors to Transform Oncology Drug Development

Purpose: In the landscape of oncology drug development, new drug screening methods are needed to help improve the success rate. Patient derived organoids have started gaining attention as a new promising tool. Among them, Rapid, Reproducible, Rare Cell Expansion (R3CE) system, a novel platform that could culture and expand circulating tumor cells into organoids, emerges as a revolutionary solution, bringing unparalleled benefits to address existing challenges. Traditional methods of in vitro organoid growth, while crucial for translational science and drug evaluation, have been hampered by their invasive nature, necessitating tumor needle biopsies. By utilizing peripheral blood, R3CE sidesteps the need for invasive procedures, offering a patient-friendly alternative. The system excels in generating viable representative cells of a patient's tumor with metastasis potential, which can be expanded and stored. This also eliminates limitations imposed by material availability, enabling an unrestricted number of characterization assays. Moreover, R3CE's capability to support 3D organoid growth, in contrast to traditional 2D cell growth, underscores its advanced potential. This shift in dimensionality not only mimics the physiological context more accurately but also contributes to more reliable drug response predictions. The benefits extend further with the abundance of organoids generated, facilitating extensive screening of new drug candidates within an exceptionally rapid timeframe. This accelerated pace of drug screening holds the promise of expediting the drug development pipeline, potentially bringing life-saving treatments to patients sooner. The work presented will show that the modified surface combined with proprietary medium of R3CE supports 3D organoid growth compared to traditional 2D cell growth, the results of ADC candidate screening using R3CE grown organoids, and results from breast cancer patient drug screening.

Methods: Patient peripheral blood is drawn and processed to remove red blood cells. The remaining peripheral blood mononuclear cells are counted and diluted in proprietary medium and seeded onto proprietary coated plates. Growth of organoids are monitored and passaged for two weeks. Subsequently, organoid banks are frozen and stored in vapor phase of liquid nitrogen. For screening drug candidates, organoids are thawed and seeded onto proprietary plates, grown for several days and then screened for drug resistance or susceptibility by assessing the relative viability by luminescent measurements by normalizing to a mock control.

Results: The R³CE grown organoids from colorectal HCT116 cell line maintain their 3D configuration when maintained in the proprietary coated plates and medium. After transferred to standard tissue culture plates and regular FBS-supplemented DMEM medium, the cells of the organoids transition to single layers gradually starting from 3rd day, but the R³CE culture system can maintain the organoid structures over time (up to three weeks when the organoids need to be passaged). Antibody-drug conjugates (ADC) candidates that target Galectin 3, a β-galactoside-binding protein (Ahmed and AlSadek, 2015), were tested for their effectiveness of killing cancer organoids. The ADC was tested at a concentration predetermined from screening work and incubated with the organoids for 4 days and generated a killing curve showing addition of Galenctin 3 enhanced the killing for all three ADCs while ADC 2 has the best killing (Image 2.) The ability to use the R³CE culture system as a Precision Medicine platform for cancer treatment was tested using peripheral blood from stage IV breast cancer patients. The peripheral blood from five cancer patients were used in the R³CE culture system to grow patient specific organoids. The following drugs were tested for each patient: Cisplatin, Paclitaxel, 5-FU (fluorouridine), Vinorebline, Gemcitibine. Cell viability was measured by luminescent readout for each drug tested and normalized to a mock infected control to generate a relative cell viability. The results are shown in Image3. A cut-off was set to distinguish patient organoids resistance or susceptibility to the drug tested.  The R³CE culture system showed 100% concordance with the clinical result.

Conclusion: By harnessing the power of the R³CE platform, we not only cultivate and expand 3-D organoids derived from circulating tumor cells in the blood of cancer patients but also establish a comprehensive bank crucial for large-scale drug screening initiatives. The non-invasive nature of this approach allows amassing of a diverse collection of organoids per solid tumor, like 50 patients, providing a rich representation of the true disease spectrum within the target population. This patient-derived organoid system emerges as a groundbreaking tool, extending beyond current methodologies and exhibiting the potential to yield results closely aligned with clinical outcomes. Furthermore, the integration of real-world data from R³CE's concurrent use in screening individual cancer patients' organoids for drug sensitivity, through collaborative clinical efforts, adds another layer of validation. The seamless collaboration between cutting-edge technology and clinical practice will build the credibility of this platform and will likely lead to advancements in drug development. As we persistently refine and validate our approach through clinical collaborations, the promise of revolutionizing how we predict and understand clinical outcomes in cancer treatment becomes increasingly tangible.

References: Ahmed, H and DM AlSadek. 2015. Clinical Med Insights Oncol 9: 113-121.

Organoids cultured in R3CE (A) and transitioned to regular tissue culture plate with standard media (10% FBS in DMEM) (B)

Drug sensitivity time course of Liver cancer organoids to three antibody-drug conjugates with or without added Galectin 3

Cultured CTC-derived organoids in drug surveillance for breast cancer patients