Growing Desmoplastic Three-Dimensional Pancreatic Cancer Spheroids from Co-Culture
September 27th, 2024
Pancreatic cancer remains one of the toughest cancers to treat. Therefore, it is critical that pre-clinical models evaluating treatment efficacy are reproducible and clinically relevant. This protocol describes a simple co-culture procedure to generate reproducible, clinically relevant desmoplastic spheroids.
3D spheroid cultures are increasingly used to answer mechanistic questions about tumor growth and serve to screen new therapeutics. Therefore, it is critical that these models are clinically representative. We focus on replicating the desmoplasia seen in many cancers, including pancreatic cancer, using this technique.
Spheroid cultures have evolved to incorporate patient-derived cells, complex co-cultures with immune cells, and physiological structures like vasculature. Additionally, microfluidic technology is employed for high-throughput screening and creating organ on-a-chip systems. Multi-omic profiling in spheroids is now utilized to gain mechanistic insights.
The described method can be used to create reproducible 3D co-cultured spheroids with a robust ECM, free of synthetic matrix materials. The advantage is that the ECM closely replicates clinical tumors without the relevant synthetic matrix interfering with therapeutic screening applications. This model will enable the valuation of ECM response to therapeutics and answer important mechanistic questions on the dynamic barrier that the ECM presents in numerous cancers.
We plan to expand this model to include immune cell and macrophage components, in addition to expanding the model used to understand ECM as a barrier to therapeutics and diseases outside of cancer, such as tuberculosis granulomas. To begin, obtain PANC-1 cell culture, and wash the cells with nine milliliters of Hanks'Balanced Salt Solution in three milliliter increments. Incubate the cells with two milliliters of trypsin for about 10 minutes.
To neutralize the suspension, add three milliliters of DMEM supplemented with 10%FBS. After washing the cells seven times with one-milliliter media increments, collect each neutralized aliquot in a 15-milliliter tube. Similarly, obtain trypsinized human pancreatic stellate cells, or HPaSteC, using trypsin neutralization solution and stellate cell media.
Mix the required amount of both the cell suspensions in 11 milliliters of DMEM with 10%FBS, avoiding froth formation. Gently dispense 100 microliters of cell suspension mix into the culture plate, and incubate it at 37 degrees Celsius with 5%carbon dioxide. On day three, add 50 microliters of DMEM with 10%FBS to each well along the side of the well.
On day 10 or 11, aspirate the spent media without disturbing the solution near the halo, and add 100 microliters of fresh media along the side of each well. On day 14 or 17, using a one-milliliter pipette, remove media from each well until the halo is reached. Align the plate against the background of the hood to locate the spheroids at the bottom.
Gently pipette around 50 microliters of media near the spheroid to disturb it for pooling. The spheroid obtained on day 14 were found to grow up to an average volume of 0.048 cubic millimeters.
Overview
This protocol outlines a co-culture procedure to create desmoplastic spheroids that are clinically relevant and reproducible for pancreatic cancer research. The use of 3D spheroid cultures allows for mechanistic studies of tumor growth and therapeutic screening.
Key Study Components
Area of Science
- Neuroscience
- Oncology
- Cell Biology
Background
- Pancreatic cancer is challenging to treat due to its aggressive nature.
- Desmoplasia is a common feature in many cancers, including pancreatic cancer.
- 3D spheroid cultures can incorporate patient-derived cells and immune cells.
- Microfluidic technology enhances high-throughput screening capabilities.
Purpose of Study
- To develop a reproducible model for studying pancreatic cancer.
- To evaluate treatment efficacy in a clinically relevant context.
- To replicate the desmoplastic environment of tumors.
Methods Used
- Co-culture procedures to generate spheroids.
- Integration of patient-derived cells and immune cells.
- Utilization of microfluidic technology for screening.
- Multi-omic profiling for mechanistic insights.
Main Results
- Successful generation of desmoplastic spheroids.
- Demonstrated relevance to clinical pancreatic cancer environments.
- Facilitated high-throughput screening of therapeutics.
- Provided insights into tumor growth mechanisms.
Conclusions
- The co-culture procedure is a valuable tool for pancreatic cancer research.
- 3D spheroid models can enhance the understanding of tumor biology.
- This approach may lead to improved therapeutic strategies.
Frequently Asked Questions
Executive Industry Relevance
Desmoplastic 3D pancreatic cancer spheroids generated from co-culture provide a clinically relevant, reproducible model that closely mimics the tumor microenvironment, including the extracellular matrix (ECM) barrier. This system enables mechanistic de-risking and predictive confidence for therapeutic screening, addressing a critical inflection point where preclinical models often fail to translate to clinical outcomes. The model's alignment with clinical tumor characteristics supports risk-adjusted portfolio decisions in early discovery and preclinical research.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of therapeutic hypotheses in a disease-relevant ECM context.
- Supports functional target validation by replicating clinical desmoplasia.
- Facilitates mechanistic de-risking of candidate therapies targeting the tumor microenvironment.
- Improves predictive confidence for advancing compounds through the pipeline.
Screening & Assay Development
- Provides a standardized, scaffold-free 3D system for quantitative drug response assessment.
- Ensures reproducibility and scalability for high-throughput screening applications.
- Generates robust ECM components, enabling reliable evaluation of ECM-targeting agents.
- Prepares validated biological systems for downstream mechanistic and efficacy studies.
Translational & Preclinical Research
- Aligns preclinical models with clinical tumor ECM features for translational continuity.
- Supports biomarker discovery and validation in a physiologically relevant setting.
- Enables risk-adjusted advancement decisions based on clinically predictive data.
- Facilitates mechanistic studies of ECM as a barrier to therapy.
Pipeline & Workflow Integration
This co-culture spheroid model bridges early discovery, lead identification, and preclinical validation by providing a clinically relevant platform for hypothesis testing and drug screening.
- Discovery Biology: Supports hypothesis testing and pathway clarification in a desmoplastic microenvironment.
- Screening: Delivers reproducible, quantitative outputs for compound evaluation against ECM-rich spheroids.
- Analytics: Enables measurement of spheroid volume and ECM composition to compare therapeutic responses.
- Translational Research: Maintains continuity with clinical tumor characteristics for biomarker and efficacy studies.
- Enterprise Reuse: Offers a reusable, standardized platform adaptable to additional cell types and disease models.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in ECM-targeted therapy development.
- Operational Value: Enhances standardization, reproducibility, and scalability for cross-functional R&D teams.
- Strategic Value: Improves go/no-go decisions and capital efficiency by aligning preclinical models with clinical outcomes.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of candidates with higher translational potential.
Implementation Considerations
- Requires expertise in 3D cell culture and co-culture techniques.
- Needs access to imaging and quantitative analysis infrastructure for spheroid assessment.
- Demands cross-team standardization for reproducibility across screening campaigns.
- Adaptable to additional cell types or disease models with protocol optimization.
- Limited to ECM-rich tumor contexts as supported by current validation data.
