Modelling Pathology in Laboratory

For exams - what would be the most appropriate assay to use if we're researching this...

What 2 laws must be abided when using patient data in research

General Data Protection Regulations (EU law)

Health Research Regulations (Irish law)

What happened Henrietta Lacks

When was each paper retracted

Why is the level of stromal tumour infiltrating lymphocytes (sTILs) used in treatment

It may be a predictor of response to chemotherapy

Does high/low sTILs → good/bad response to chemotherapy

Patients with low levels of sTILs do significantly worse.

Explain how patients with low levels of sTILs do significantly worse.

Hypothesis is that when patients received chemotherapy, their tumour cells undergo apoptosis which releases tumour neoantigens

The tumour neoantigens are seen as “foreign” and so immune cells help to clear the tumour

If there is a lack of sTILs in the tumour microenvironment – process can’t occur

Of relevance to the action of immune check point inhibitors (e.g. anti-PD-L1)

How does neoantigen load have an effect on possible therapies

How can AI improve Our Ability to Reliably assess tumours

May Improve Our Ability to Reliably Quantify sTILs

Give 2 examples of Target Manipulation tests

shRNA

CRISPR

Give 2 examples of Functional Assays tests

Cell Invasion

Toxicity Assays

Give 2 examples of OMIC tests

RNA sequencing

Proteomics

What is Triple Negative Breast Cancer & what does it mean for treatment

Characterised by absence of ER, PR, HER2 → no response to targeted chemotherapeutic treatment

High HERV-K & iNOS are predictive of good/bad prognosis

Bad

What kind of treatment could HERV-K & iNOS be used for

Potential targets for the treatment of high grade hormone independent breast cancer

HERV-K is an important predictor for what in breast cancer

Metastasis in TNBC (Triple-negative breast cancer)

At what stage of progression are HERV-K & iNOS seen

In what ways does NO influence cancer formation & progression

NO influences all processes relevant to cancer formation and progression

What are Mobile DNA elements (name 2 types)

Pieces of DNA that can move around within the genome

• DNA transposons

• Retrotransposons

Differentiate between DNA transposons & Retrotransposons

2 types of Retrotransposons

• Non-LTR retrotransposons -These don't have LTRs (Long Terminal Repeats).

• LTR retrotransposons

3 types of Non-LTR retrotransposons

LINEs

SINEs (Alus)

SVAs

What are ERVs & why do we care about them

Endogenous Retroviruses - A type of LTR (Long Terminal Repeat) retrotransposons

Some ERV sequences become more active/expressed in certain diseases

(HERV-K is a type of ERV)

What % of the human genome is made up of ERVs (endogenous retroviruses)

8%

What class & subclass of retrovirus does HERV-K belong to

Class II retroviruses

• beta-retrovirus-like

How many HERV-K HML-2 loci are there

How many of these are:

• solo-LTR insertions

• provirus insertions

• unfixed polymorphic loci

1063 total

• 973 solo-LTR insertions

• 90 provirus insertions

• 69 of these loci are unfixed polymorphic loci

HERV-K HML-2 can produce several proteins through alternative splicing - name 3

1× spliced → Env

2× spliced → Rec

2× spliced → Np9

How might HERV-K contribute to cancer?

HERV-K products can alter immune responses, promote inflammation, evade immune surveillance and activate pathways involved in cell proliferation and migration, potentially contributing to tumour progression.

How would you assess iNOS and HERV-K Protein Expression in TNBC Patient Samples

Immunohistochemistry

Explain how IHC can be used to assess for iNOS and HERV-K

In IHC, you use an antibody against the protein you want to detect:

Anti-iNOS antibody → binds iNOS

Anti-HERV-K antibody → binds HERV-K protein

The antibody is then detected using an enzyme system, commonly horseradish peroxidase (HRP). When the DAB chromogen is added, HRP converts DAB into an insoluble brown precipitate at the location where the target protein is present

What is HERV-K Env Expression used to predict

Survival

High HERV-K Env expression is associated with metastasis to what 2 tissues

Liver

Soft tissue

Dual HERV-K Env and iNOS Expression leads to what fold increase in

• most distant metastases

• liver metastases

Doubles most distant metastases

5x Liver metastases

What is shRNA & how can it be used to investigate the role of HERV-K in breast cancer?

Short hairpin RNA (shRNA) is an artificial RNA molecule with a tight hairpin turn used to silence target gene expression through RNA interference

shRNA can be used to destroy HERV-K mRNA, reducing production of HERV-K protein

How does shRNA reduce HERV-K expression (MOA)

A complementary shRNA sequence binds to the HERV-K mRNA, causing the mRNA to be degraded and therefore reducing the corresponding protein.

How was successful reduction of HERV-K expression confirmed?

Using Western blotting in MDA-MB-231 TNBC cells following stable lentiviral shRNA treatment.

What does a stronger band on a Western blot indicate

A greater amount of protein.

What are the main stages of a Western blot

SDS-PAGE – separates proteins according to size.

Protein transfer to a membrane.

Immunodetection/staining using antibodies against the protein of interest.

Visualisation of the bands to determine protein expression

What does Western blotting allow researchers to determine

Whether the amount of a specific protein has changed, such as whether HERV-K protein expression has been reduced after shRNA treatment.

Name 3 methods for Analysing Cell Proliferation

You could physically count the cells

XTT assay

Luminescent Assay

How can cell proliferation be measured by physically counting cells?

Trypan blue is added to the sample (which will be taken up by dead cells, but not live ones - allows live to be counted) & cells can be counted directly

What is a disadvantage of physically counting cells with trypan blue?

It is not efficient.

What does an XTT assay measure?

It measures metabolically active cells through MTS reduction.

What is the sensitivity and time frame of the XTT assay ?

Approximately 800 cells, measured over 1–4 hours, using a colourimetric readout

What does a luminescent assay measure

ATP, which reflects metabolically active cells

What is the sensitivity and time frame of the luminescent assay

Approximately 50 cells in 10 minutes, using luminescence.

What effect did HERV-K knockdown have on TNBC cell proliferation?

TNBC proliferation was reduced

Cell migration of HERV-K Knockdown MDA-MB-231 cells were also measured. How?

Using a Boyden chamber, where cells migrate through 8 μm (micron) pores

The Boyden Chamber's has been coated with a thin layer of matrigel. The cells must degrade it and then access the 8 micron pores to migrate through.

They are visualised with Crystal Violet stain

What effect did HERV-K knockdown have on cell migration in MDA-MB-231 cells?

HERV-K knockdown reduced cell migration potential

Name a more realistic way of assessing tumour cell invasion

Tumour invasion is multidimensional in reality, so 3D tumour spheroids may provide a more realistic model of tumour cell invasion

Using 3D tumour spheroids we can assess the ability of tumour cells to invade out of the spheroid

How can researchers investigate signalling pathways regulated by iNOS and HERV-K?

RNA sequencing (RNA-seq)

Why is interpreting RNA-seq data difficult

RNA-seq provides expression information for approximately 60,000 genes, including mRNA, lncRNA, snoRNA and miRNA.

How can researchers make sense of thousands of RNA-seq results?

They can group genes into biological pathways/hallmarks and examine which pathways are upregulated or downregulated

How were these hallmarks affected by high DETA/NO (high iNOS)

• Inflammatory response

• TNFA signalling via NFκB

• KRAS signalling

• p53

• G2M checkpoint

Upregulated:

• Inflammatory response

• TNFA signalling via NFκB

• KRAS signalling

• p53

Downregulated

• G2M checkpoint

How were these hallmarks affected by high HERV-K (EnvHIGH)?

• Inflammatory response

• TNFA signalling via NFκB

• KRAS signalling

All downregulated

What does “more than additive” mean in the analysis of iNOS and HERV-K

The combined effect of high iNOS and high HERV-K is greater than would be expected from simply adding their individual effects.

If a patient is diagnosed with breast cancer, they are assessed for estrogen receptor, progesterone receptor and HER2 receptor status using what test

IHC

1 Limitation of Immunohistochemistry

We are limited to identifying one protein at a time

Name an alternative approach to allow for staining with multiple antibodies on the one tissue

Immunofluorescence

Once tumour punches are obtained from therapeutic excision specimens, what is constructed for their analysis

A tissue microarray (TMA).

What is the MACSima Spatial Multi-Omics Platform used for?

It allows detailed spatial analysis of tissue, including cell phenotyping, cellular neighbourhoods, cell-cell interactions and functional states of cells.

How does tissue imaging & marking work

Tissue is stained, imaged, the markers are erased, and the tissue can then be stained and imaged again with additional markers

This allows many different markers to be assessed sequentially on the same tissue section, enabling extensive multiplex spatial profiling

What test is used for each of these research questions:

Research question: Does a target affect tumour cell proliferation?

XTT/MTS assay or ATP luminescence assay

Research question: Does a target affect tumour cell migration?

Use: Boyden chamber migration assay

Research question: Does a target affect tumour cell invasion?

Use: Matrigel-coated Boyden chamber

Research question: Want a more realistic 3D model of tumour invasion?

Use: 3D tumour spheroid model

Research question: Does knocking down a target reduce its protein expression?

Use: Western blot

Research question: What genes/pathways are affected by manipulating a target?

Use: RNA sequencing

Research question: Where is a protein expressed in patient tissue?

Use: Immunohistochemistry (IHC)

Research question: Want to detect multiple proteins in the same tissue section?

Use: Immunofluorescence / multiplexing

Research question: Want to study spatial interactions between many cell types/proteins?

Use: Spatial multi-omics / multiplex imaging

Research question: Is a biomarker associated with patient survival

Use: Patient cohort + biomarker measurement + survival/outcome analysis

Research question: Does a biomarker actually cause a disease phenotype

Use: Manipulate the biomarker in a laboratory model and assess the phenotype

Research question: Does a diagnostic marker give consistent results between observers

Assess: Reproducibility

Research question: Does an antibody detect only the intended target?

Assess: Antibody specificity