![]() ![]() Specifically, we evaluated effects of CAFs on tumor cells in response to erlotinib, a small molecule targeting the Epidermal Growth Factor Receptor (EGFR) that is often used in clinical treatment of NSCLC. CAFs have been implicated in tumor initiation, progression, and therapeutic response therefore, performing phenotypic assays on tumor cells in the absence of CAFs can be misleading 7, 8, 9. We focus on the interaction between Non-Small Cell Lung Cancer (NSCLC) cells and Cancer-Associated Fibroblasts (CAFs), the most prevalent stromal cell type found in tumors. In this manuscript, we apply our quantitative imaging pipeline to highlight the evolutionary dynamics of heterogeneous cell populations within a tumor. Microscopy-based imaging is an alternative method for profiling cellular phenotypes in a multiplexed, quantitative, and robust manner. altering inherent cell properties, time-consuming) and therefore additional methods to classify cell types are advantageous. Fluorescently labeling cells has downfalls ( i.e. ![]() Additionally, one can use differences in morphological features to discriminate between subpopulations in co-culture systems. Hence, it is of great interest to study these cellular features and how they relate to specific environmental perturbations. Furthermore, tumor cell morphology has been shown to correlate with tumor subtypes 5 and aggressiveness 6. Profiling cell states through morphological characterization and mapping similarities across samples is a powerful, unbiased tool with the ability to provide novel insights into many aspects of basic and translational research, including basic cell biology and drug discovery 4. Other important cellular phenotypes, including morphological changes, occur in response to environmental stimuli but are not captured by traditional cell-based assays. While flow cytometry methods have the ability to distinguish between multiple cell populations, cell labels are required, dynamic sampling is challenging, and when using adherent cells, this application becomes time-consuming and error prone. Another major disadvantage of these techniques is their inability to distinguish between multiple cell types – most biological systems are heterocellular. However, the metabolic activity of cells can be affected by different culture conditions (such as media or oxygen concentration), which leads to inaccurate results and prevents standardization across cell types and conditions 2, 3. MTT and MTS assays indirectly measure cell viability through measurements of mitochondrial metabolic rate. Trypan blue exclusion assays, while simple and inexpensive, require a large number of cells, are time consuming, and are often influenced by user bias 1. Traditional cell-based assays that are used to measure cell viability include: trypan blue exclusion assays, MTT/MTS, and Annexin V-FITC flow cytometry staining. Here, we present a novel method for utilizing quantitative imaging to simultaneously analyze heterocellular phenotypes in response to relevant, co-occurring environmental stimuli. However, the limitations of these standard assays have become increasingly apparent with the discordance between in vitro and clinical data and the failure of most drugs to receive FDA approval. This protocol allows one to identify subpopulations within a co-culture system and characterize the particular response of each to external stimuli.Ĭell-based assays have been a workhorse in basic research and drug development settings. We demonstrate this application using response to drug in a cancer model however, it can easily be applied more broadly to other physiological processes. However, in some cases, cell populations may be difficult to identify and quantitate based on complex cellular features and will require additional troubleshooting we highlight some of these circumstances in the protocol. This platform allows for a more comprehensive characterization of subpopulation response to perturbation while utilizing shorter time, smaller amounts of reagents, and lower likelihood of error than traditional cell biology assays. We highlight our ability to distinguish between cell types based upon either fluorescence intensity or inherent morphology features depending on the application. morphology changes, proliferation, apoptosis) of heterogeneous cell populations to changes in environmental stimuli. Using a quantitative imaging-based approach, we present a high-content protocol for characterizing the dynamic phenotypic responses ( i.e. Existing cell biology techniques often do not allow for accurate interpretation of this interplay. ![]() Cellular processes are complex and result from the interplay between multiple cell types and their environment. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |