INTRODUCTION: In breast cancer patients with the most common metabolic disorder, Type 2 Diabetes (T2D), it has been established that the mortality rate is increased when juxtaposed to breast cancer patients without T2D. The mortality rate further increases when the T2D diagnosis occurs on or post the breast cancer diagnosis. Amongst the breast cancer types, Triple Negative Breast Cancer (TNBC) is a negative diagnosis, resulting from the lack of expression of receptors, and is by far the most heterogenous type of breast cancer and exhibits the greatest difficulties for treatment and worst outcomes.
Although solid tumors have been studied by pathologists, surgeons, molecular biologists and other experts for many decades, important questions remain about the role of metabolism and tumor microenvironment in the emergence of cancer metastasis, which is the focus of this thesis.
METHODS: The 4T1 cells were cultured in RPMI media at 11 mM glucose concentration and at RPMI at 4 mM glucose, to mimic the diabetic and non-diabetic condition respectively. The cells in both glucose concentrations are cultured parallelly in 10 cm dishes until they reach 60-70% confluence, Nile reed was used to stain the lipid in the cells and was observed under fluorescent microscope. The cells were then treated once with exosomes collected from mature, differentiated, insulin sensitive (IS) and insulin resistant (IR) 3T3-L1 cells, in 6cm dishes and grown for 3 days. They were then plated in a Seahorse plate and analyzed using Seahorse Cell Mito-stress test assay to measure the real-time change in their Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECR), to study the effect the exosome treatment and their response to acute injections of Oligomycin, FCCP and Antimycin A during the assay.
Cells were also parallelly extracted with SDS-PAGE sample buffer, separated by electrophoresis and transferred to PVDF membrane for western blotting after SDS PAGE electrophoresis. Phosphorylated and unphosphorylated acetyl-CoA carboxylase (ACC), AMP-activated protein kinase (AMPK) and Ki67 (nuclear protein- biomarker of proliferation) were detected with specific rabbit antibodies (Cell Signaling). Protein bands were detected on a GE LAS-4000 gel imager using enhanced chemiluminescence. Bands were analyzed using ImageJ software (Schneider, Rasband and Eliceiri 2012) and the intensity of the bands was measured and analyzed with Adobe photoshop.
RESULTS: There was a noticeable loss of lipid accumulation when the 4T1 cells were shifted from 11 mM glucose to 4 mM glucose. The cell count in both 11 mM glucose and 4 mM glucose was significantly higher in the IR treatment group when compared to the other groups.
In 11 mM glucose cells, the treatment with IR exosomes drove up the baseline OCR and overall response to the injections when compared to the controls. In 4 mM glucose cells, the IR exosome treatment seems to have inhibited the baseline OCR and the overall response to the injections.
Western blot analysis, it revealed that the IR exosome treatment increased the phosphorylation of AMPK in 11 mM glucose cells whereas it drastically reduced it in 4 mM glucose cells. The levels of phosphorylation of ACC and AKT remained mostly unchanged amongst all treatment groups of 11 mM and 4 mM glucose cells whereas the IR treatment ramped up the expression of Ki67 only in 11 mM glucose cells and not in 4 mM glucose cells.
CONCLUSION: The results obtained reveal that there may be a correlation with the lipid stores that are found in the 11 mM glucose cells that may have played a role in the effectiveness of the exosome treatment, in terms of the intensity and direction of the effect. Similarly, the 4 mM glucose cells respond in a different way possibly due to a difference in the metabolism of the exosomes themselves. 4 mM glucose cells are depleted of the lipid stores that the 11 mM glucose cells have and the exosomes that are used for the treatment originate from the adipocytes, indicating that the cross-talk that is implied between the adipose tissues surrounding the breast cancer cells does more than just deliver possible nutrients but is greatly involved in the metabolic reprogramming that is long standing and possibly irreversible making the breast cancer cells of the triple negative type in T2D patients proliferate rapidly, indicating more metastasis and rendering it even more dangerous.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/48453 |
| Date | 19 March 2024 |
| Creators | Darga, Anoushka |
| Contributors | Denis, Gerald V., Deeney, Jude T. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
| Rights | Attribution 4.0 International, http://creativecommons.org/licenses/by/4.0/ |
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