Effects of Phosphate Uptake/Metabolism on Behavior of Lung Cancer Cells Open Access

Horne, Liam (Spring 2023)

Permanent URL: https://etd.library.emory.edu/concern/etds/ks65hd57p?locale=en


Excess inorganic phosphate (Pi) availability has been suggested to have a role in cancer progression in preclinical mouse models. Cell-based studies have demonstrated that increased extracellular Pi alters proliferation, metabolism, and the migratory behavior of cells. Taken together, the results suggest that precancerous cells acquire a need for increased Pi consumption. An obstacle to assessing the Pi intake requirements of precancerous and cancerous cells is an inability to track cellular Pi consumption. In this study, a novel FRET-based fluorescent microscopy system was used with the A549 human lung adenocarcinoma cell line to track cytoplasmic Pi, as a measure of Pi consumption. The cells were sorted based on their FRET signal from the cytoplasm into subpopulations based on their Pi-consumption phenotype (high, moderate, and low PI-consuming). Assays to assess growth, migration, colony formation, and gene expression were performed to measure differences among the different Pi-consuming cell populations in normal and high Pi environments. Results showed that high Pi-consuming A549 cells demonstrated a phenotype with increased proliferation, and migration, along with upregulation of OPN, a secreted factor associated with cancer progression in various models. These findings show that high Pi-consumers have more of the characteristics associated with lung cancer progression, and thus Pi metabolism could offer a target for therapeutic intervention. The FRET sensor offers an intriguing look at how Pi consumption tracks with changes in cell behavior, particularly related to the early stages of cancer progression.

Table of Contents

Table of Contents


Goals & Hypothesis 2


Generation of FRET expression cell line 3

FACS Sorting 3

Growth Time course assay 4

Quantitative Realtime PCR (qRT-PCR) 4

Table 1 5

Migration/invasion 5

Colony formation 5

Statistics 6


Figure 1. FLIPPi FRET Biosensor 8

Figure 2. Growth and proliferation rates are different between the Pi-consuming sub-populations 9

Figure 3. High Pi consumers do not form colonies at greater rates 11

Figure 4. High Pi consumers demonstrate an increased migratory phenotype 12

Figure 5. Changes in gene expression 14


Conclusions/Future Directions 18


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