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Identification of biomarkers that can predict progression and severity of bladder cancers is critical to improving and refining the subsequent treatment of patients. Among the many thousands of proteins that are expressed in the bladder, how can one identify particular proteins that either correlate with bladder cancer progression or, even more importantly, cause it? You have to know where to look (and when), what you are looking for, how to look for it and how to explain why it may be there.

The paper by Lindén et al. [1] is a good example of a study aiming to identify a set of key markers that are upregulated in the urine and tumours of patients with bladder cancer compared with patients with normal bladders. The present short review will describe the steps that their study goes through to find these markers.

Where Do You Look for Markers and When?

  1. Top of page
  2. Where Do You Look for Markers and When?
  3. What Molecules Do You Look for and How Do You Look for Them?
  4. Protein Abundance Measurement by Liquid Chromatography-Mass Spectrometry (A Proteomic Technique)
  5. Western Blot Analysis
  6. Measuring Expression of the Above Markers in Tumour Tissue
  7. Immunohistochemistry
  8. Conclusions
  9. References

The key initial questions are: where should one look for the markers and when? Patients with bladder cancer will have abnormal cells that may secrete abnormal proteins into the urine. Cells shed from the cancer may also be present in the urine; therefore, urine samples are a good place to search for biomarkers. But how do you compare urine taken from different patients with cancer and what do you use as control urine? Each urine sample should be taken at the same time of day, and when patients and controls have fasted and preferably have had an equivalent intake of fluids. As biomarkers may be labile or degraded very quickly, samples should be frozen at −80oC at the time of collection and protease inhibitors should be added to prevent any protein degradation.

The tumour biopsy itself, however, will provide the best source of biomarkers. Tumour samples obtained from patients will be at different stages of the disease. If tumours from the same staging are grouped and compared with tumours obtained from later or earlier stages of the disease, this will give the best indication of markers correlated with disease progression. When tumours are biopsied, they should ideally be stored in three forms: in frozen sections, as paraffin-embedded samples and as live single-cell suspensions obtained from the tissue by disaggregation and storage at −80oC in foetal calf serum containing 10% DMSO.

What Molecules Do You Look for and How Do You Look for Them?

  1. Top of page
  2. Where Do You Look for Markers and When?
  3. What Molecules Do You Look for and How Do You Look for Them?
  4. Protein Abundance Measurement by Liquid Chromatography-Mass Spectrometry (A Proteomic Technique)
  5. Western Blot Analysis
  6. Measuring Expression of the Above Markers in Tumour Tissue
  7. Immunohistochemistry
  8. Conclusions
  9. References

Biomarkers may be proteins or genes, or they may be post-translational modifications on proteins, such as lipids or sugars. To firstly get a broad picture of which proteins or genes or post-translational modifications (lipids and sugars) are different among patients with bladder cancer, techniques that can measure changes in the genome, proteome, lipome and glycome can be performed [2-4]. These will show changes in every gene, protein, sugar and lipid in the tissue or other sample processed. It is, however, very time-consuming to perform all these array-type measurements and the information generated is vast. Usually, therefore, a single ‘-omic’ technique is applied. In the first Lindén study [5], proteomics was used.

Protein Abundance Measurement by Liquid Chromatography-Mass Spectrometry (A Proteomic Technique)

  1. Top of page
  2. Where Do You Look for Markers and When?
  3. What Molecules Do You Look for and How Do You Look for Them?
  4. Protein Abundance Measurement by Liquid Chromatography-Mass Spectrometry (A Proteomic Technique)
  5. Western Blot Analysis
  6. Measuring Expression of the Above Markers in Tumour Tissue
  7. Immunohistochemistry
  8. Conclusions
  9. References

In the accompanying paper, Lindén et al. describe their previous study [5] in which they identified changes in specific proteins in the urine that had been pooled from a collection of patients with bladder cancer and compared these with the proteins in urine pooled from normal controls. This was done using a mass spectrometric protein abundance assay, a proteomic assay where proteins from the urine have been separated out by their molecular weight using liquid chromatography (LC) and mass spectrometry (MS). These techniques give a high-resolution analysis of proteins with very small differences in their molecular weight. After the peptides were separated and their masses were determined by LC-MS, the measurements of all the masses were compared against a protein database [6], which showed which proteins are normally present in different organs and tissues. Using this technique, a number of proteins were identified that were normally present in the bladder but were expressed at much higher levels in patients with bladder cancer than in normal controls. The proteins were shown to be apolipoprotein E (APOE), fibrinogen β chain precursor (FGB), leucine-rich α-2-glycoprotein (LRG1) α-1-antitrypsin (SERPINA1) topoisomerase 2A (TOP2A) and polymerase (RNA) I polypeptide E (POLR1E).

Western Blot Analysis

  1. Top of page
  2. Where Do You Look for Markers and When?
  3. What Molecules Do You Look for and How Do You Look for Them?
  4. Protein Abundance Measurement by Liquid Chromatography-Mass Spectrometry (A Proteomic Technique)
  5. Western Blot Analysis
  6. Measuring Expression of the Above Markers in Tumour Tissue
  7. Immunohistochemistry
  8. Conclusions
  9. References

To confirm that the above-mentioned proteins were at much higher levels in the patients with cancer, the pooled patient and control samples were run on SDS-PAGE to separate the protein bands and then the proteins were blotted onto a nitrocellulose membrane and probed with antibodies to these proteins in a process known as Western blot (or WB) analysis.

Measuring Expression of the Above Markers in Tumour Tissue

  1. Top of page
  2. Where Do You Look for Markers and When?
  3. What Molecules Do You Look for and How Do You Look for Them?
  4. Protein Abundance Measurement by Liquid Chromatography-Mass Spectrometry (A Proteomic Technique)
  5. Western Blot Analysis
  6. Measuring Expression of the Above Markers in Tumour Tissue
  7. Immunohistochemistry
  8. Conclusions
  9. References
Tissue microarray

In the accompanying paper, Lindén et al. next measured the expression of the markers APOE, FGB, LRG1 and SERPINA1, TOP2A and RNA POLR1E in tumour samples from patients with different stages of bladder cancer. This was performed using a tissue microarray. A tissue microarray was prepared using very small sections (<0.5 mm2 each) of cancer tissue, embedded in paraffin after formaldehyde fixation and cut from 120 patients with three different stages of bladder cancer (360 samples in total), and placed these on slides. Several slides were prepared with the same tumour tissue panels to allow staining by antibodies to the proteins identified as being changed in the previous study [4].

Immunohistochemistry

  1. Top of page
  2. Where Do You Look for Markers and When?
  3. What Molecules Do You Look for and How Do You Look for Them?
  4. Protein Abundance Measurement by Liquid Chromatography-Mass Spectrometry (A Proteomic Technique)
  5. Western Blot Analysis
  6. Measuring Expression of the Above Markers in Tumour Tissue
  7. Immunohistochemistry
  8. Conclusions
  9. References

To detect whether the above proteins are expressed at different levels in the three different stages of bladder cancer, a technique called immunohistochemistry was performed. The slides with the tumour sections were washed with xylene to remove paraffin, and then hydrated by using wash steps in deionised water containing decreasing concentrations of alcohol (ethanol or industrial methylated spirits). After the final wash (without alcohol) the slides were boiled with an antigen-demasking solution (citrate buffer) and then blocked with a solution containing albumin to block binding of secondary antibodies to irrelevant immunoglobulins present in the tissue sections. The sections were then incubated with primary antibodies to the above proteins, washed with PBS to remove excess antibody and then stained with secondary antibodies directed toward the primary antibodies: for example, if a primary antibody was made in a mouse, the secondary antibody would be made in a second animal against the particular class of the primary antibody e.g. a goat–anti-mouse IgG1. The secondary antibodies were tagged with an enzyme called horseradish peroxidase. This enzyme can catalyse a reaction where a colourless substrate of the protein is converted to a coloured product in the presence of hydrogen peroxide, therefore, proteins that are expressed in the tissues can be detected by the development of a colour in specific areas or cells. The slides are placed under a microscope and are then photographed at a magnification sufficient to detect the architecture of the cells and differences in staining between tissue sections.

Figure 1 above is taken from the Lindén et al. paper [1]. It can be seen that the proteins APOE (panel A), FGB (panel B) and POLR1E (panel D) show greater staining at the higher tumour stages, while the expression of LRG1 (panel C) and SERPINA1 (panel E) were not different among the cancer stages.

Figure 2 below summarizes the techniques used by Lindén et al. in the accompanying paper and in their previous paper [5] to discover bladder cancer-related biomarkers.

Conclusions

  1. Top of page
  2. Where Do You Look for Markers and When?
  3. What Molecules Do You Look for and How Do You Look for Them?
  4. Protein Abundance Measurement by Liquid Chromatography-Mass Spectrometry (A Proteomic Technique)
  5. Western Blot Analysis
  6. Measuring Expression of the Above Markers in Tumour Tissue
  7. Immunohistochemistry
  8. Conclusions
  9. References

Both publications by Lindén et al. [1, 5] show that a number of proteins are upregulated in the urine of patients with bladder cancer (APOE, FGB, LRG1, SERPINA1, TOP2A and RNA POLR1E). These proteins can be divided into two classes: acute phase proteins (APOE, SERPINA1, FGB and LRG1) and proteins involved in RNA transcription (POLR1E and TOP2A). These proteins are all relevant in cancer as they can either affect cancer treatment (APOE) [7, 8], enhance anti-apoptotic mechanisms (SERPINA1) [9], growth and metastasis (FGB, POLR1E and LRG1) [10-12] or invasion (LRG1)[13] of cancer cells.

Histochemical analysis of tumour tissues showed that APOE, FGB and POLR1E are the proteins whose expression is also associated with bladder cancer progression and therefore these are the proteins that could be used as targets for bladder cancer therapy.

References

  1. Top of page
  2. Where Do You Look for Markers and When?
  3. What Molecules Do You Look for and How Do You Look for Them?
  4. Protein Abundance Measurement by Liquid Chromatography-Mass Spectrometry (A Proteomic Technique)
  5. Western Blot Analysis
  6. Measuring Expression of the Above Markers in Tumour Tissue
  7. Immunohistochemistry
  8. Conclusions
  9. References