Establishment of Patient-Derived Organoid Culture Platform of Mantle Cell Lymphoma

Background

Mantle Cell Lymphoma (MCL) is a rare incurable non-Hodgkin's lymphoma despite remarkable recent therapeutic innovations such as CAR T cell therapies. Drug sensitivity and immunotherapy efficacy assays using preclinical tumor models are important in new therapy development. As for preclinical tumor models, in addition to the commonly used mouse model such as patient-derived xenograft (PDX) and genetically engineered mouse models, a 3 ^rd tumor model, the patient-derived organoid (PDO) in 3D culture model has been established in many types of solid tumors. However, no PDO models have been generated from MCL yet. We have established MCL PDO model by optimizing cell isolation, culture add-ons, spatial and temporal conditions. Our protocol presents a versatile MCL PDO platform, suitable for quick drug screens and applicable for rapid immunotherapy evaluation. For proof of concept demonstration, we tested the therapeutic efficacy of CD19-targeted CAR T-cell in MCL-specific PDOs. The established PDO procedures can be used for diverse biopsies including whole blood, apheresis, bone marrow, lymph node and previously established PDX tumors.

Methods

For human blood, bone marrow and apheresis biospecimens, we first eliminated red blood cells using RBC lysis buffer. The cells were then spun down and cell pellets were resuspended in culture medium and aliquoted and spun-down into V-shaped 96-well plates at 1-6´10 ⁶ cells per well. After overnight incubation, the cell culture medium was replaced by 60 ml of precooled 60% Matrigel per well. The 3D formed aggregates were gently transferred into 24-well plate and feed the solidified domes with the medium containing an in-house cytokine cocktail. The developed organoids reach the drug screening optimal phase within 3-6 days or it can be further expanded for future use. For PDX tumor and human lymph node biospecimens, we first sliced tumor tissue using surgical scalpel blade, which resulted in small tissue pieces that were then resuspended in HBSS. Debris (>100 mm) were removed by brief gravity sedimentation. The resultant tissue pieces were then resuspended in 60% Matrigel in culture medium and dispensed onto pre-warmed 24-well plates. The primary tumor organoids were observed for 3-5 days before drug testing on processed for further expansion. For organoid passaging, primary organoids were extracted in pre-cooled medium by mechanically breaking the gel domes. The extracted organoids were digested by collagenase/dispase. The resultant MCL and stromal cells were enforced to aggregate in V-shape wells as described in the apheresis procedure. Organoid cell composition was examined using FACS. Drug sensitivity and T-cell activity against the MCL organoids were assessed using CellTiter-Glo 3D kit.

Results

We have successfully established and optimized the PDO procedure from diverse MCL biopsies (Fig. 1A). The success rate of MCL organoid from apheresis was > 80% with even higher success rates when using PDX tumors, lymph node and bone marrow samples (> 90%). One critical step for processing the biopsies is preserving the original stromal cells. FACS analysis showed that although <5% of total cell population is composed of macrophages, circulating fibroblast, epithelial and endothelial cells and they are indispensable for organoid formation and expansion. Addition of hematopoietic cytokines in culture medium significantly improved the organoid formation and expansion from diverse biopsies. FACS with lineage markers revealed that the cell populations of the primary and secondary organoids were not significantly different (Fig. 1B). Tumor-killing activity of the CD19-targeting CAR T cells was assessed by co-culturing the CAR T cells with MCL organoids.

Conclusion

We have established an MCL PDO platform which is time-efficient, labor-saving, cost-effective and highly reproducible. This platform provides a rapid approach for immune cell activity assays and drug screening. The organoids have been successfully used to generate PDX models. This platform can also be used for investigating the mechanism of drug resistance in the context of different TMEs.

Figure 1

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Figure 1

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