Visualization of Macropinocytosis in Prostate Fibroblasts
Related Experiment Video
Updated: Jan 20, 2026
Isolation, Culture, and Characterization of Prostate Cancer-Associated Fibroblasts
Published on: August 1, 2025
Rajeev Mishra1, Neil A Bhowmick2,3
1Department of Biosciences, Manipal University Jaipur, Rajasthan, India.
Abstract:
Macropinocytosis has emerged as an important mechanism for non-selective route to internalize extracellular fluids and dissolved molecules in eukaryotic cell. As fundamental cellular behavior, macropinocytosis plays specific and distinct roles in many physiological and pathological processes, such as nutrients uptake, antigen presentation, pathogen capture, and tumorigenesis. It supports tumorigenesis by providing metabolic needs to dividing cells in Ras driven cancer. In recent years, macropinocytosis has gained considerable interest in physiology and various diseases, including cancer, neurodegenerative diseases and atherosclerosis, which in turn has led to the discovery of new endocytic recycling systems. Approaches to assess macropinocytosis will provide insight into its underlying regulatory molecular mechanisms and enable the physiological control of macropinocytosis for controlled drug delivery and targeted cancer therapy. Macropinocytosis is an important phenomenon in Ras-expressing cancer cells and, recently, we have revealed a functional role for macropinocytosis in cancer associated fibroblasts (CAFs) fueling cancer cell growth. Here, we describe a protocol for detection of macropinocytosis in prostatic fibroblasts in vitro by utilizing fluorescently-labeled, lysine-fixable, 70 kDa high molecular weight dextran. Macropinosomes are visualized as fluorescent intracellular puncta either by confocal or fluorescent microscopy. To follow, subsequent intracellular events and their underlying mechanisms after macropinosomes formation, we perform co-localization of quenched BSA (DQ™-BSA) along with dextran labeling in cancer associated fibroblasts. Our protocol provides a consistent way to understand macropinocytosis in wild type or genetic manipulated prostatic fibroblast.
Insights
This study presents a new protocol to detect macropinocytosis in prostatic fibroblasts using fluorescent dextran. This method helps understand how cancer-associated fibroblasts fuel tumor growth and aids in developing targeted cancer therapies.
Area of Science:
- Cell Biology
- Cancer Research
- Molecular Mechanisms
Background:
- Macropinocytosis is a cellular process for internalizing extracellular fluids and molecules, crucial in physiological and pathological contexts like cancer.
- It plays a role in nutrient uptake, immune responses, and tumorigenesis, particularly in Ras-driven cancers.
- Recent interest focuses on macropinocytosis in diseases such as cancer, neurodegenerative disorders, and atherosclerosis, leading to discoveries in endocytic recycling.
Purpose of the Study:
- To establish a reliable in vitro protocol for detecting macropinocytosis in prostatic fibroblasts.
- To investigate the role of macropinocytosis in cancer-associated fibroblasts (CAFs) that contribute to tumor growth.
- To provide a method for understanding macropinocytosis in both wild-type and genetically modified fibroblasts for cancer therapy research.
Main Methods:
- Utilized fluorescently labeled, lysine-fixable, 70 kDa high molecular weight dextran to detect macropinocytosis in prostatic fibroblasts.
- Visualized macropinosomes as fluorescent intracellular puncta using confocal or fluorescent microscopy.
- Employed co-localization of quenched Bovine Serum Albumin (DQ™-BSA) with dextran labeling to track subsequent intracellular events in CAFs.
Main Results:
- Successfully developed and demonstrated a protocol for visualizing macropinocytosis in prostatic fibroblasts.
- The method allows for the consistent detection and analysis of macropinosome formation and trafficking.
- Co-localization experiments provide insights into the intracellular fate of internalized material within CAFs.
Conclusions:
- The described protocol offers a consistent method for studying macropinocytosis in prostatic fibroblasts.
- This technique can be applied to wild-type or genetically manipulated cells, advancing research into fibroblast-mediated tumor growth.
- Understanding macropinocytosis in CAFs is vital for developing targeted drug delivery and cancer therapies.
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