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Interested in our full process? Read our thesis article! This article provides all the information in the poster and padlet explained in greater depth.

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Western blotting is an analytical technique to separate and identify a specific target protein in a complicated mixture of proteins extracted from cells based on molecular weight.

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1. Make a membrane and load the sample.

2. Running gel will make the samples sink to the right molecular weight.

3. Transfer the samples on the membrane to another sheet.

4. Incubate with the antibodies and wash remains.

5. Image with the use of machine.

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With the use of detecting light, the protein bindings are shown if they are present.

GAPDH = is our housekeeping gen, meaning it is the control which shows the right molecular weight and if each sample is correct. We had good results, which means that our research is valid.

Protein A = located on DAOY and HDMB03 (+ it's diluted sample). This means that Protein A is more noticeably present on HDMB03.

Protein B = is not located on anything.

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FACS stands for Fluorescence-Activated Cell Sorting, which is a type of flow cytometry used to identify cells in a diverse cell population. Antibodies that bind with the proteins are labelled with different types of fluorescent light, so the flow cytometry picks up the unique binding signal.

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1. Cell counting and preparing samples with labelled antibodies (unstained, isotype, stained)

2. A laser is emitted through the single cells going through the machine. Signal is detected if the right wavelength (depending on the type of fluorescent light) strikes the labelled antibody.

3. This signal is translated into a voltage pulse in the graph.

4. The protein is present on the tumour cell if the signal matches with the marker on the antibody, meaning the antibody and protein have bonded.

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For each protein and cell line three graphs were generated to see their presence on medulloblastoma. An example is provided below, further results are seen in the thesis article.

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Unstained = the control sample of a protein. This means the labelled antibody is not yet added.

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Isotype = shows the amount of non-specific bindings, which occurs when proteins stick to one another without pattern.

If the peak of the isotype control shifts to the right compared to the unstained control, then non-specific bindings are present.
In this example, only a little bit of non-specific bindings are present.

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In order to read the thesis article more clearly, here are some abbreviations used:

MB stands for medulloblastoma

NKC stands for Natural Killer Cell

IC stands for immune checkpoint

ICI stands for immune checkpoint inhibitor

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The location of the different subgroups of medulloblastoma; 

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Medulloblastoma is the most common and aggressive brain tumour in infants. The tumour is mostly located in the posterior fossa region, which is located between the brainstem and cerebellum.

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Medulloblastoma has several subgroups with different molecular characteristics. We examined SHH (Sonic Hedgehog) and Group 3 for our research.

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Current treatments (like surgery, chemotherapy and radiation), contain low survival rates with high relapse rates and are too invasive for children. Additionally, medulloblastoma has a diverse nature, which means that each distinct tumour reacts differently to the same treatment method. This possibly leads to increasing tumour invasion and progression. A possible solution for this problem is immunotherapy, which can adjust to each patient and tumour type.

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Stained = shows the amount of specific bindings, meaning the binding between the labelled antibody and IC ligand on the tumour cell.

If the peak of the stained control shifts to the right compared to the isotype control (=non-specific bindings), that means specific bindings are present. This confirms the present of the protein on the cell line, meaning the immune checkpoint is present.

In this example, a shift is shown, meaning that the protein is present on this cell line because specific bindings between the two are shown.

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To what extent do the immune checkpoints Protein A, Protein B, Protein C and Protein D come to expression in medulloblastoma and natural killer cells?

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We expected all proteins to be present on all the medulloblastoma cell lines, and not on the immune cells. Except Protein C, which was only expected to be present on subgroup SHH.

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Western blotting

FACS

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Cell lines = a population of cells that have been produced in the lab, originating from one single cell.

The cell lines we used are:

DAOY = a medulloblastoma cell line originating from SHH

D341 = a medulloblastoma cell originating from group 3

HD-MB03 = a medulloblastoma cell line originating from group 3

NK92 = a Natural Killer Cell cell line

YT Indy = a Natural Killer Cell cell line

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Our research was performed at the Bachelor Research Hub at UMC Utrecht. This is a research facility used by Utrecht University to educate Medicine and Biomedical Science students to become more familiar in a laboratorium.

We spent three days with amazing researchers Niels Bovenschen, Emma van Pijnappel, Toine ten Broeke and Sandra Crnko. We are very grateful for this experience.

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Final version thesisposter C09 - Immunotherapy.V2

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Our research was performed at the Bachelor Research Hub at UMC Utrecht. This is a research facility used by Utrecht University to educate Medicine and Biomedical Science students to become more familiar in a laboratorium.

We spent three days with amazing researchers Niels Bovenschen, Emma van Pijnappel, Toine ten Broeke and Sandra Crnko. We are very grateful for this experience.

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The immune system is a network in the body that defends the body against diseases. The body can remember which diseases the body is familiar with by producing antibodies. These antibodies detect and defend against diseases. Immune cells like Natural Killer Cells and T-cells are used to destroy an unfamiliar disease.

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In order to protect the surrounding healthy cells, the immune system needs to be kept in balance with the activation and deactivation of T-cells. Immune checkpoints make sure that the immune response is too weak to harm this cell. The checkpoint binds to a specific protein on the activated T-cell and the attack is prevented. However, tumour cells use this to their advantage to escape the immune system.

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The binding between the T-Cell receptor’s immune checkpoint and the tumour's immune checkpoint can be blocked with the use of so-called blocking inhibitors. Immune checkpoint inhibitor immunotherapy uses antibodies to prevent the immune response from being stopped.

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Plus = present on that cell line
Minus = absence on that cell line

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Table 1 Presents the combined results of Western blotting and FACS. Cell lines of MB and NKC are portrayed on the side, and the ICs are portrayed on the top. The plus (+) suggests expression of the IC on the cell lin

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A protein has potential if the protein is located in the medulloblastoma and not on the immune cell, because otherwise the blocking inhibitors will block the binding with immune cells and healthy cells.

With our gathered results, we can conclude that:

Protein D has the most potential, with it being present on all medulloblastoma cell lines.

Protein A has the second most potential, with it being present on two of the medulloblastoma cell lines.

Protein C has some potential, with it being present on one of the medulloblastoma cell lines.

Protein B has no potential, because it is not present on any cell line.

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For more reliable results, all proteins need to be researched again, preferably with an additional research method.

Western blotting:

Protein A & B another time

Additionally research Protein C and Protein D

FACS:

Protein B another time to confirm absence

Additionally research Protein A and the Natural Killer Cells

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If the present immune checkpoints have been confirmed by multiple researchers, then the following steps can proceed.

1. Examine if the immune checkpoints are not present on healthy cells and immune cells.

2. Examine the range of overexpression on tumour cells, making sure the inhibitor will block the ligand located on the tumour.

3. Making sure that the specific blocking inhibitors can pass the brain-blood barrier and other hinders.

When the blocking inhibitors satisfy each requirement, they can be tested in the clinics and will soon improve the immune system of infected patients!

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These are the complete results of FACS in a simplified graph. The bars show how many immune checkpoints of each protein are present on the three medulloblastoma cell lines.

The number of events of the isotype controls are extracted from the events of the stained to acquire the relative amount of specific bindings for the results. If there is a significant amount of events in the diagram, then the IC is present.