Why Tumors Must Be Dissociated
A solid tumor is a complex, heterogeneous ecosystem containing malignant cells, immune cells, and stromal cells like fibroblasts and endothelial cells. This internal diversity, known as intra-tumor heterogeneity, is a significant challenge in cancer research and treatment. To accurately study this cellular complexity, researchers must first break the tumor down into its individual components.
Traditional methods of studying tumors involve “bulk” analysis, which averages the molecular profiles of millions of cells together. This averaging effect obscures the unique characteristics of rare subpopulations, such as those that drive metastasis or drug resistance. Dissociation into a single-cell suspension bypasses this limitation, allowing scientists to examine the molecular makeup of each cell in isolation.
Analyzing the distinct genetic and phenotypic profiles of these individual cells is necessary for developing effective targeted therapies. For example, a single biopsy may contain cancer cell clones resistant to chemotherapy. Identifying these resistant populations before treatment begins leads to more precise and personalized medical strategies.
The Techniques Used for Dissociation
Breaking a solid tumor down into a single-cell suspension involves a combination of mechanical and enzymatic methods, targeting the components holding the tissue together. Mechanical dissociation physically disrupts the tissue structure, often starting by mincing the tumor sample with a scalpel. The fragments are then subjected to methods like forced filtration through mesh filters or automated systems that use shearing forces to separate the cells.
Mechanical techniques are faster, which reduces the time a cell spends outside its natural environment. However, excessive mechanical force can lead to significant cell damage, resulting in lower cell viability and inconsistent results. Therefore, mechanical disruption is often paired with enzymatic digestion, which uses specialized enzymes to break down the extracellular matrix (ECM) that acts as the tumor’s scaffolding.
Enzymatic dissociation relies on proteases, such as collagenase, trypsin, and hyaluronidase, which specifically cleave the proteins and fibers within the ECM. The choice of enzyme cocktail depends on the type of tumor, as different cancers have different matrix compositions. While enzymatic methods yield a high number of single cells, the enzymes can sometimes modify or cleave cell surface proteins, complicating downstream analysis that relies on detecting these markers.
Analyzing the Dissociated Cells
Once the tumor tissue is dissociated into a suspension of individual cells, the sample is ready for high-resolution analysis. One powerful application is single-cell RNA sequencing (scRNA-seq), which measures the entire set of RNA transcripts—the transcriptome—within thousands of individual cells simultaneously. This provides a detailed map of gene activity, revealing which genes are turned on or off, and helping to identify distinct cell types and states, such as rare immune cell populations or aggressive cancer clones.
Another widely used technique is Fluorescence-Activated Cell Sorting (FACS), a specialized form of flow cytometry. FACS uses lasers and fluorescently labeled antibodies to rapidly count, examine, and physically sort cells based on specific surface proteins. This allows researchers to isolate specific subpopulations, such as tumor-infiltrating lymphocytes (TILs) or malignant cells expressing biomarkers like PDL1, which is relevant for immunotherapy.
The data generated from these single-cell analyses are transforming personalized medicine. By identifying the specific molecular characteristics of a patient’s tumor cells and their microenvironment, doctors can move beyond broad treatment categories. These methods provide the intelligence needed to select drugs precisely targeted to the unique cellular profile of an individual patient’s cancer.
Preserving Cell Integrity During Dissociation
Maintaining the health and native state of the cells during dissociation directly impacts data quality. The goal is to maximize cell viability, which is the percentage of living cells, often targeted to be over 90% for single-cell sequencing. Prolonged dissociation times, especially those exceeding one hour, can activate cellular stress response pathways, causing cells to change their gene expression patterns. These artificial changes, or artifacts, can distort the true biological signal.
To mitigate these challenges, researchers employ strategies focused on gentle handling and environmental control. Dissociation protocols are optimized to be short, often lasting less than 30 minutes, to minimize cellular stress. Specialized buffers containing proteins like bovine serum albumin (BSA) or fetal bovine serum (FBS) protect the cells and prevent them from sticking to surfaces. Conducting the process at cold temperatures or using automated systems helps slow down cellular metabolism and enzyme activity, preserving the cells and their surface markers.