This document summarizes an investigation into the effects of tissue stiffness on miRNA content within extracellular vesicles secreted by colorectal cancer cells. The introduction provides background on the tumor microenvironment and extracellular vesicles. The study aims to determine how tissue stiffness impacts miRNA content in EVs and the stem cell characteristics of colorectal cancer cells. Preliminary results show higher transglutaminase 2 expression, a protein that maintains stem cell phenotype, in EVs from softer microenvironments. Further analysis of EV cargo may help understand cancer stem cell and extracellular matrix crosstalk.

1. Investigating the Effects of
Tissue Stiffness on miRNA
Content Within Extracellular
Vesicles Secreted by Colorectal
Cancer Cells
By Jamie Cooper
Supervisors : Dr Nick Peake, Dr Lucy Crooks

2. Introduction – Tumor Microenvironment
 The tumor microenvironment (TME) comprises of the
extracellular matrix (ECM), myofibroblasts, fibroblasts,
neuroendocrine, adipose cells, immune-inflammatory cells,
blood and lymphatic networks (Wang et al., 2017).
 Mediators within the TME include growth factors, cytokines
and proteolytic enzymes which are initially thought to be
beneficial to tumorigenesis (Xie et al., 2019).
 Within the TME, ECM accumulation is a frequent
phenomenon that leads to an intense fibrotic response
termed tumour stiffening (Gkretsi & Stylianopoulos, 2018).
 This phenomenon has also been established to contribute
to cell-ECM interaction and thereby promote cancer
invasion to surrounding tissues (Grasset et al., 2018).
(Wang et al., 2017).

3. A biological mediator within the TME which has had had increasing attention over
the last decade are extracellular vesicles (EVs).
Extracellular vesicles (EVs) are small membrane-surrounded structures released
by all type of cells including normal and disease cells (Wu et al., 2018).
They contain significant bioactive cargo in the form of proteins, lipids, metabolites,
DNA, RNA and non-coding RNA’s (miRNA, tRNA, rRNA) for local and distant
intercellular communications (Chuo et al., 2018).
EVs are classified into three distinct categories including exosomes, microvesicles
and apoptotic bodies (Raposo & Stoorvogel, 2013).
Introduction – Extracellular Vesicles
(Hartjes et al., 2019)
(Jabalee et al., 2018)

4. Within a pathological microenvironment there is a difference between the
population and the contents of EVs than that of a normal microenvironment
(Tkach & Théry, 2016).
When cells are exposed to various responses of stress (acidity, hypoxia and
structural deficiencies), the overall biogenesis, content sorting and release of EVs
can change and thereby induce phenotypic differences (Xie et al., 2019).
Differences of stress responses such as tissue stiffness within the pathological
microenvironment may affect the overall biogenesis and content sorting within
EVs secreted by cells and thereby inducing phenotypic changes that may support
tumorigenesis.
Introduction – Tissue Stiffness and EVs
(Yamada, 2016)

5. Establish the Effects of Tissue Stiffness On
miRNA content Within Extracellular Vesicles
Secreted by Colorectal Cancer Cells.
Determining the Differences of Stem Cell
Characteristics Between A Soft and Stiff Tumour
Microenvironment in Colorectal Cancer Cells.
Establishing The Stem Cell Phenotypic Effects
of EVs Derived from Different Stiffness
Microenvironments.
Aim
Future!

6. Method
SDS Page Western Blot Add Primary Antibody Add Secondary Antibody Develop Colour

7. Soft - 1.2% Stiff - 2.2%
Day3Day1Day5
Results – Manipulating Cell Density In 3D Models
Soft - 1.2% Stiff - 2.2%
Day3Day1Day5
1.2% 2.2%
Day3Day1Day5
5x105 Cells/mL 2.5x106 Cells/mL 1x107 Cells/mL

8. Results – Manipulating Cell Density In 3D Models

9. Results –
 Within a tumour there are a small subpopulation of cells termed cancer stem cells which have
the ability of self-renewal and differentiation.
 An important protein in maintaining stem cell phenotype within colorectal cancer cells is
transglutaminase 2 (TG2) by regulation of the EMT(Ayinde et al., 2019).
 Cancer stem cells can perceive the differences of stiffness and reorganise their ECM creating a
local niche (d’Angelo et al., 2019).

10.  A higher yield and purity of miRNA will allow global assessment to be performed by next
generation sequencing to determine the overall impact of a stiffer microenvironment on
EVs cargo. Establishing EVs cargo in different microenvironments may enable potential
biomarkers to support earlier detection and identification of cancers with poorer outcomes
such as colorectal caner
 Although studies have started to establish TG2 in stem cell phenotype, the understanding
between the crosstalk of between cancer stem cells and ECM is lacking. On this basis, TG2
expression was qualitatively confirmed in both microenvironments, with higher expression
reported in the softer microenvironment when compared to stiffer microenvironment.
 Establishing this crosstalk may help future stem-cell based regenerative approaches in the
therapeutics of colorectal cancer.
 Future Publications?
Conclusive Remarks

11. References
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