Muhammad Nurudeen Nurudeen
Analysis of the Role of Immune System and Microenvironment in Leukaemia
Nurudeen, Muhammad Nurudeen
Authors
Contributors
Prof Marija Krstic-Demonacos M.Krstic-Demonacos@salford.ac.uk
Supervisor
Dr Athar Aziz A.Aziz@salford.ac.uk
Supervisor
Abstract
The immune system and microenvironment have been implicated in chemotherapy resistance
in acute lymphoblastic leukaemia (ALL). Despite the development of different therapies, the
detailed mechanism underlying chemotherapy resistance is not well understood. Hence this
study seeks to elucidate the role of the immune system and microenvironment in leukaemia
cells as well as evaluate the in vitro interactions between leukaemia cells and macrophages. To
this end, relevant microarray data from ALL cells treated with dexamethasone (Dex) – a
synthetic glucocorticoid, etoposide (Eto) – a DNA damaging drug, and conditioned media
(CM) that mimics microenvironment was retrieved from Array Express, Probeset IDs were
updated with Affymetrix Annotation, the list of new genes obtained were filtered with Fold
change ≥ 2 and q-value ≤ 0.05, then analyzed with Metascape for pathway enrichment. The
gene list was also cross matched with genes obtained from GeneCards by using seven key
terms, cBioPortal was used for the transcriptomic analysis of CD47 expression and STRING
was used to determine CD47-proteins interactions. A total of 4,188 probeset IDs previously
unannotated were matched to specific genes, the enrichment analysis report identified
pathways including polyubiquitination, receptor localization to synapse and modulation of
chemical synaptic transmission. IL-10 and CD47 genes were observed to be upregulated in
treatment groups where CM was present, and this varies depending on the presence or absence
of Dex and Eto. CD47 mRNA expression correlated positively with the age of diagnosis of
ALL, is observed in different ALL subtypes and is inversely proportional to overall survival
according to the cBioPortal analysis. CD47 interacts with several proteins such as SIRPα,
THBS, HSPA5, THSP1, BRD4, MYC, SMARCA4, MFGE8, ZEB1, GZMB and MAP2K7
which either activate or inhibit its function.
In vitro experiments were carried out to validate these findings and to elucidate details of the
role of immune system and microenvironment in leukemia. Dex and Eto cytotoxicity with and
without CM on C7-14, C1-15, and MOLT-4 ALL cell lines were evaluated using MTS and
propidium iodide staining assays. Both assays demonstrated that C7-14 cells were most
susceptible to dexamethasone, followed by C1-15 and MOLT-4. The same pattern was
observed for Eto and combined treatment, except for higher concentrations showing greater
cytotoxicity in C1-15. Propidium iodide assay particularly confirmed C7-14 to be the most
susceptible of all cell lines to Dex, whilst MOLT-4 was relatively resistant to all treatments.
Responses to other treatments were similar for C1-15 and C7-14. Across all cell lines there was
a trend for CM to reduce cytotoxicity. Given the bioinformatics-identified prognostic value of
CD47, follow-up experiments made use of anti-CD47 monoclonal antibody to determine
potential molecular mechanisms. Flow cytometry-based detection confirmed cell surface CD47
expression to be highest in C7-14 and C1-15, whilst levels were lower in MOLT-4; CM showed
increased trend in C7-14, no change in C1-15 and decrease in MOLT-4 CD47 surface
expression. Drug treatments led to small differences in CD47 surface levels. CD47 mRNA was
increased by Dex in C7-14 and decreased by Dex and Eto in C1-15, which mostly correlated
with western blot-detected protein levels. CM and Dex treatments led to an upregulation trend
of both CD47 mRNA and protein levels in C7-14 cells while Eto led to a decrease both on its
own and in combination with Dex in C1-15 which mostly correlated with western blot-detected
protein levels. CM treatment showed upregulation trends of CD47 mRNA levels in all cell
lines, whereas protein levels displayed complex patterns. Preliminary data from co-culture
experiments of leukaemia and THP-1 macrophages indicate that anti-CD47 monoclonal
antibody alone and in combination with Dex and or Eto did not seem to induce cytotoxicity or
enhance phagocytosis of C7-14, C1-15, and MOLT-4 leukaemia cell lines in vitro.
In conclusion, findings from this study suggest that CM contributes to drug resistance in all
cell lines studied. Furthermore, there is a trend of increased CD47 expression by CM in most
cell lines, potentially contributing to chemoresistance.
Thesis Type | Thesis |
---|---|
Online Publication Date | Mar 27, 2025 |
Deposit Date | Mar 20, 2025 |
Publicly Available Date | Mar 28, 2027 |
Award Date | Mar 27, 2025 |
Files
This file is under embargo until Mar 28, 2027 due to copyright reasons.
Contact M.N.Nurudeen@edu.salford.ac.uk to request a copy for personal use.
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