Related Experiment Video
Updated: Sep 1, 2025
A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies
Published on: April 12, 2017
Methods for Establishing a Renal Cell Carcinoma Tumor Spheroid Model With Immune Infiltration for Immunotherapeutic
Leonard Lugand1, Guillaume Mestrallet1, Rebecca Laboureur1
1Hemato-Immunology Research Department, CEA, DRF-Francois Jacob Institute, Saint-Louis Hospital, Paris, France - U976 HIPI Unit, IRSL, Paris University, Paris, France.
Abstract:
Tumor spheroids play an increasingly important role in cancer research. Their ability to recapitulate crucial features of tumor biology that are lost in the classically used 2D models along with their relative simplicity and handiness have made them the most studied 3D tumor model. Their application as a theranostic tool or as a means to study tumor-host interaction is now well-established in various cancers. However, their use in the field of Renal Cell Carcinoma (RCC) remains very limited. The aim of this work is to present methods to implement a basic RCC spheroid model. These methods cover the steps from RCC tumor dissociation to spheroid infiltration by immune cells. We present a protocol for RCC dissociation using Liberase TM and introduce a culture medium containing Epithelial Growth Factor and Hydrocortisone allowing for faster growth of RCC primary cells. We show that the liquid overlay technique allows for the formation of spheroids from cell lines and from primary cultures. We present a method using morphological criteria to select a homogeneous spheroid population based on a Fiji macro. We then show that spheroids can be infiltrated by PBMCs after activation with OKT3 or IL-15. Finally, we provide an example of application by implementing an immune spheroid killing assay allowing observing increased spheroid destruction after treatment with PD-1 inhibitors. Thus the straightforward methods presented here allow for efficient spheroid formation for a simple RCC 3D model that can be standardized and infused with immune cells to study immunotherapies.
Insights
This study introduces a simple 3D model for Renal Cell Carcinoma (RCC) research using tumor spheroids. The protocol facilitates spheroid formation and immune cell infiltration for studying immunotherapies.
Area of Science:
- Oncology
- Biotechnology
- 3D Cell Culture
Background:
- Three-dimensional (3D) tumor spheroids are valuable models in cancer research, recapitulating in vivo tumor characteristics often lost in 2D cultures.
- While widely used, 3D spheroid models for Renal Cell Carcinoma (RCC) are underutilized.
- This study addresses the limited application of RCC spheroids by presenting a standardized methodology.
Purpose of the Study:
- To establish a basic 3D Renal Cell Carcinoma (RCC) spheroid model.
- To provide a detailed protocol for RCC spheroid formation and immune cell infiltration.
- To enable the study of RCC tumor biology and immunotherapy responses in a 3D context.
Main Methods:
- Developed an RCC tumor dissociation protocol using Liberase TM.
- Introduced a specialized culture medium with Epithelial Growth Factor and Hydrocortisone for enhanced RCC primary cell growth.
- Utilized the liquid overlay technique for spheroid formation from cell lines and primary cultures.
- Implemented a Fiji macro for morphological selection of homogeneous spheroid populations.
- Demonstrated immune cell (PBMC) infiltration into spheroids following activation with OKT3 or IL-15.
Main Results:
- Successfully established a reproducible method for generating RCC spheroids.
- Showcased faster growth of RCC primary cells using the specialized culture medium.
- Validated the infiltration of activated PBMCs into RCC spheroids.
- Successfully implemented an immune spheroid killing assay to evaluate immunotherapy efficacy.
- Observed enhanced spheroid destruction with PD-1 inhibitor treatment in the assay.
Conclusions:
- The presented methods enable efficient formation of a simple, standardized RCC 3D spheroid model.
- This model facilitates immune cell infusion for studying cancer immunotherapies.
- The protocol supports the advancement of 3D modeling in Renal Cell Carcinoma research and therapeutic development.
