automated-imaging-analysis-for-quantification-fluorescently-labeled

Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and ...

NCI-designated Cancer Center

Uptake of nutrients, such as glucose and amino acids, is critical to support cell growth and is typically mediated by cell surface transporters. An alternative mechanism for the bulk uptake of nutrients from the extracellular space is macropinocytosis, a nonclathrin, and nonreceptor-mediated endocytic process, in which extracellular fluid is taken up into large intracellular vesicles called macropinosomes. Oncogenic transformation leads to the increased metabolic activity of tumor cells, and in the Ras-driven tumor part of this enhanced activity is the stimulation of macropinocytosis. To measure oncogene-dependent macropinocytosis, we used HeLa cells expressing oncogenic HRAS(G12D) driven from a Tet-regulated promoter. Upon oncogenic HRAS expression, the cells undergo metabolic changes that include the elevation of macropinocytosis. We detected macropinocytosis through the uptake of lysine-fixable tetramethyl rhodamine (TMR)-Dextran (70 kDa) from the cell media into nascent intracellular macropinosomes. These macropinosomes were quantified by image-based high-content analysis, with the size, intensity, and position of macropinosomes measured. Using this model system, we ran a full genome-wide siRNA screen (siGenome™; GE) to identify genes involved in controlling oncogenic HRAS-dependent macropinocytosis. Hits from the primary screen were confirmed with siRNA reagents from a different library (GE, OTP), which allowed us to mitigate potential off-target effects. Candidate genes from this screen include known regulators of macropinocytosis as well as novel targets. 

High-content, full genome siRNA screen for regulators of oncogenic HRAS-driven macropinocytosis.

Oncogenic mutations, such as Ras mutations, drive not only enhanced proliferation but also the metabolic adaptations that confer to cancer cells the ability to sustain cell growth in a harsh tumor microenvironment. These adaptations might represent metabolic vulnerabilities that can be exploited to develop novel and more efficient cancer therapies. Macropinocytosis is an evolutionarily conserved endocytic pathway that permits the internalization of extracellular fluid  large endocytic vesicles known as macropinosomes. Recently, macropinocytosis has been determined to function as a nutrient-scavenging pathway in Ras-driven cancer cells. Macropinocytic uptake of extracellular proteins, and their further degradation within endolysosomes, provides the much-needed amino acids that fuel cancer cell metabolism and tumor growth. Here, we review the molecular mechanisms that govern the process of macropinocytosis, as well as discuss recent work that provides evidence of the important role of macropinocytosis as a nutrient supply pathway in cancer cells.

Macropinocytosis: A Metabolic Adaptation to Nutrient Stress in Cancer.

Macropinocytosis Fuels Prostate Cancer.

Macropinocytosis is a mechanism of fluid-phase endocytosis that functions in the nonspecific internalization of extracellular fluid. This uptake pathway has specialized roles in different cell types and organisms, and its importance has recently been established in several diseases, including cancer. In cancer, macropinocytosis is stimulated by oncogenes, such as Ras, and macropinocytic cargo is targeted to lysosomes for degradation, providing a catabolic route for tumor cells to obtain amino acids from the tumor microenvironment. Here, we describe a protocol to employ fluorescently labeled dextran molecules in order to visualize and quantify the extent of macropinocytosis in pancreatic tumors. Multiple samples can be processed in parallel by this method, and the protocol can be easily customized for pancreatic tumor tissue isolated from subcutaneous, orthotopic and genetically engineered mouse models (GEMM), or human patients.

Detection and Quantification of Macropinosomes in Pancreatic Tumors.

Macropinocytosis has emerged as an important nutrient supply pathway that sustains cell growth of cancer cells within the nutrient-poor tumor microenvironment. By internalizing extracellular fluid through this bulk endocytic pathway, albumin is supplied to the cancer cells, which, after degradation, serves as an amino acid source to meet the high nutrient demands of these highly proliferating cells. Here, we describe a streamlined protocol for visualization and quantitation of macropinosomes in adherent cancer cells grown in vitro. The determination of the "macropinocytic index" provides a tool for measuring the extent to which this internalization pathway is utilized within the cancer cells and allows for comparison between different cell lines and treatments. The protocol provided herein has been optimized for reproducibility and is readily adaptable to multiple conditions and settings.

Quantitation of Macropinocytosis in Cancer Cells.

In tumour cells, macropinocytosis functions as an amino acid supply route and supports cancer cell survival and proliferation. Initially demonstrated in oncogenic KRAS-driven models of pancreatic cancer, macropinocytosis triggers the internalization of extracellular proteins via discrete endocytic vesicles called macropinosomes. The incoming protein cargo is targeted for lysosome-dependent degradation, causing the intracellular release of amino acids. These protein-derived amino acids support metabolic fitness by contributing to the intracellular amino acid pools, as well as to the biosynthesis of central carbon metabolites. In this way, macropinocytosis represents a novel amino acid supply route that tumour cells use to survive the nutrient-poor conditions of the tumour microenvironment. Macropinocytosis has also emerged as an entry mechanism for a variety of nanomedicines, suggesting that macropinocytosis regulation in the tumour setting can be harnessed for the delivery of anti-cancer therapeutics. A slew of recent studies point to the possibility that macropinocytosis is a pervasive feature of many different tumour types. In this review, we focus on the role of this important uptake mechanism in a variety of cancers and highlight the main molecular drivers of macropinocytosis in these malignancies. This article is part of the Theo Murphy meeting issue 'Macropinocytosis'.

The pervasiveness of macropinocytosis in oncological malignancies.

Macropinocytosis is an important nutrient-scavenging pathway in numerous cancer types, including pancreatic, lung, prostate, and bladder. This Forum highlights recent work identifying the key regulators of macropinocytosis that support tumor cell fitness in different contexts, providing a unique framework for strategies to target macropinocytosis in the treatment of cancer.

Macropinocytosis in Cancer: A Complex Signaling Network.

Macropinocytosis has emerged as an important nutrient-scavenging pathway that supports tumor cell fitness. By internalizing extracellular protein and targeting it for lysosomal degradation, this endocytic pathway functions as an amino acid supply route, permitting tumor cell growth and survival despite the nutrient-poor conditions of the tumor microenvironment. Here, we provide evidence that a subset of pancreatic ductal adenocarcinoma (PDAC) tumors are wired to integrate contextual metabolic inputs to regulate macropinocytosis, dialing up or down this uptake pathway depending on nutrient availability. We find that regional depletion of amino acids coincides with increased levels of macropinocytosis and that the scarcity of glutamine uniquely drives this process. Mechanistically, this stimulation of macropinocytosis depends on the nutrient stress-induced potentiation of epidermal growth factor receptor signaling that, through the activation of Pak, controls the extent of macropinocytosis in these cells. These results provide a mechanistic understanding of how nutritional cues can control protein scavenging in PDAC tumors.

EGFR-Pak Signaling Selectively Regulates Glutamine Deprivation-Induced Macropinocytosis.

Cancer cells reprogram their metabolism to meet elevated energy demands and favor glycolysis for energy production. This boost in glycolytic flux supports proliferation, but also generates acid in the form of hydrogen ions that must be eliminated from the cytoplasm to maintain the alkaline intracellular pH (pHi) associated with transformation. To cope with acid production, tumor cells employ ion transport systems, including the family of sodium-hydrogen exchangers (NHE). Here, we identify NHE7 as a novel regulator of pHi in pancreatic ductal adenocarcinoma (PDAC). We determine that NHE7 suppression causes alkalinization of the Golgi, leading to a buildup of cytosolic acid that diminishes tumor cell fitness mainly through the dysregulation of actin. Importantly, NHE7 knockdown  leads to the abrogation of tumor growth. These results identify Golgi acidification as a mechanism to control pHi and point to the regulation of pHi as a possible therapeutic vulnerability in PDAC. SIGNIFICANCE: NHE7 regulates cytosolic pH through Golgi acidification, which points to the Golgi as a "proton sink" for metabolic acid. Disruption of cytosolic pH homeostasis via NHE7 suppression compromises PDAC cell viability and tumor growth...

Golgi Acidification by NHE7 Regulates Cytosolic pH Homeostasis in Pancreatic Cancer Cells.

Nutrient stress driven by glutamine deficiency activates EGFR signaling in a subset of KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) cells. EGFR signaling in the context of glutamine starvation is thought to be instigated by the transcriptional upregulation of EGFR ligands and functions as an adaptation mechanism to allow PDAC cells to maintain metabolic fitness. Having a clear view of the intricate signaling cascades potentiated by the metabolic induction of EGFR is important in understanding how these effector pathways influence cancer progression. In this study, we examined the complex signaling that occurs in PDAC cells when EGFR is activated by glutamine deprivation. We elucidate that the metabolic activation of EGFR is principally mediated by HB-EGF, and that other members of the ErbB receptor tyrosine kinase family are not activated by glutamine starvation. Additionally, we determine that glutamine depletion-driven EGFR signaling is associated with a specific receptor phosphorylation known to participate in a feedback loop, a process that is dependent on Erk. Lastly, we determine that K-Ras is required for glutamine depletion-induced Erk activation, as well as EGFR feedback phosphorylation, but is dispensable for Akt activation. These data provide important insights into the regulation of EGFR signaling in the context of metabolic stresses.

Metabolic regulation of EGFR effector and feedback signaling in pancreatic cancer cells requires K-Ras.

The interplay between nutrient scarcity and signal transduction circuits is an important aspect of tumorigenesis that regulates many aspects of cancer progression. Glutamine is a critical nutrient for cancer cells, as it contributes to biosynthetic reactions that sustain cancer proliferation and growth. In tumors, because nutrient utilization can often outpace supply, glutamine levels can become limiting and oncogene-mediated metabolic rewiring triggers signaling cascades that support nutrient stress survival. Recently, we identified that in pancreatic ductal adenocarcinoma (PDAC) cells, glutamine depletion can trigger p21-activated kinase (Pak) activation through EGFR signaling as a means to circumvent metabolic stress. Here, we elucidate that glutamine starvation, as well EGF stimulation, can enhance the presence of many different Pak phosphoforms, and that this activation only occurs in a subset of PDAC cells. Pak is a well-established effector of Rac1, and while Rac1 mutant variants can modulate the metabolic induction of Pak phosphorylation, Rac1 inhibition only partially attenuates Pak activation upon glutamine depletion. We decipher that in order to efficiently suppress metabolic activation of Pak, both EGFR and Rac1 signaling must be inhibited. These results provide a mechanistic understanding of how glutamine-regulated signal transduction can control Pak activation in PDAC cells.

Rac1 and EGFR cooperate to activate Pak in response to nutrient stress.

Although pancreatic ductal adenocarcinoma (PDAC) cells are exposed to a nutrient-depleted tumor microenvironment, they can acquire nutrients via macropinocytosis, an endocytic form of protein scavenging that functions to support cancer metabolism. Here, we provide evidence that macropinocytosis is also operational in the pancreatic tumor stroma. We find that glutamine deficiency triggers macropinocytic uptake in pancreatic cancer-associated fibroblasts (CAF). Mechanistically, we decipher that stromal macropinocytosis is potentiated via the enhancement of cytosolic Ca and dependent on ARHGEF2 and CaMKK2-AMPK signaling. We elucidate that macropinocytosis has a dual function in CAFs-it serves as a source of intracellular amino acids that sustain CAF cell fitness and function, and it provides secreted amino acids that promote tumor cell survival. Importantly, we demonstrate that stromal macropinocytosis supports PDAC tumor growth. These results highlight the functional role of macropinocytosis in the tumor stroma and provide a mechanistic understanding of how nutrient deficiency can control stromal protein scavenging. SIGNIFICANCE: Glutamine deprivation drives stromal macropinocytosis to support CAF cell fitness and provide amino acids that sustain PDAC cell survival. Selective disruption of macropinocytosis in CAFs suppresses PDAC tumor growth..

Macropinocytosis in Cancer-Associated Fibroblasts Is Dependent on CaMKK2/ARHGEF2 Signaling and Functions to Support Tumor and Stromal Cell Fitness.

Cancer cells exhibit increased glycolytic flux and adenosine triphosphate (ATP) hydrolysis. These processes increase the acidic burden on the cells through the production of lactate and protons. Nonetheless, cancer cells can maintain an alkaline intracellular pH (pHi) relative to untransformed cells, which sets the stage for optimal functioning of glycolytic enzymes, evasion of cell death, and increased proliferation and motility. Upregulation of plasma membrane transporters allows for H and lactate efflux; however, recent evidence suggests that the acidification of organelles can contribute to maintenance of an alkaline cytosol in cancer cells by siphoning off protons, thereby supporting tumor growth. The Golgi is such an acidic organelle, with resting pH ranging from 6.0 to 6.7. Here, we posit that the Golgi represents a "proton sink" in cancer and delineate the proton channels involved in Golgi acidification and the ion channels that influence this process. Furthermore, we discuss ion channel regulators that can affect Golgi pH and Golgi-dependent processes that may contribute to pHi homeostasis in cancer.

Article	Year
High-content, full genome siRNA screen for regulators of oncogenic HRAS-driven macropinocytosis. Assay and drug development technologies\| PubMed ID: 26267765	2015
Macropinocytosis: A Metabolic Adaptation to Nutrient Stress in Cancer. Frontiers in endocrinology\| PubMed ID: 29085336	2017
Macropinocytosis Fuels Prostate Cancer. Cancer discovery\| PubMed ID: 29967075	2018
Detection and Quantification of Macropinosomes in Pancreatic Tumors. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 30378054	2019
Quantitation of Macropinocytosis in Cancer Cells. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 30725454	2019
The pervasiveness of macropinocytosis in oncological malignancies. Philosophical transactions of the Royal Society of London. Series B, Biological sciences\| PubMed ID: 30967003	2019
Macropinocytosis in Cancer: A Complex Signaling Network. Trends in cancer\| PubMed ID: 31208695	2019
EGFR-Pak Signaling Selectively Regulates Glutamine Deprivation-Induced Macropinocytosis. Developmental cell\| PubMed ID: 31257175	2019
Golgi Acidification by NHE7 Regulates Cytosolic pH Homeostasis in Pancreatic Cancer Cells. Cancer discovery\| PubMed ID: 32200349	2020
Metabolic regulation of EGFR effector and feedback signaling in pancreatic cancer cells requires K-Ras. Biochemical and biophysical research communications\| PubMed ID: 32972751	2020
Rac1 and EGFR cooperate to activate Pak in response to nutrient stress. Biochemical and biophysical research communications\| PubMed ID: 32972756	2020
Macropinocytosis in Cancer-Associated Fibroblasts Is Dependent on CaMKK2/ARHGEF2 Signaling and Functions to Support Tumor and Stromal Cell Fitness. Cancer discovery\| PubMed ID: 33653692	2021
The Golgi as a "Proton Sink" in Cancer. Frontiers in cell and developmental biology\| PubMed ID: 34055797	2021

Cosimo Commisso

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