Cancer histopathology is currently the preferred method for detecting microscopic anatomical changes in tissue sections, making the discovery of cancer biomarkers critical for early diagnosis and treatment. Antibodies have been used extensively as the molecular probes; however, the production of antibodies requires a series of complicated and time-consuming processes.

There is no question that aptamers can offer benefits over antibodies, as they have been demonstrated as excellent probes for immunostaining of frozen and formalin fixed and paraffin embedded (FFPE) tissues. Aptamers have unique properties that make them advantageous in future clinical settings by overcoming the limitations of antibodies. These include:

  • Isolated in vitro, without the need of animals
  • Smaller size which allows increased penetration into cells and tissues
  • Non-immunogenicity
  • Significantly lower batch to batch variation
  • Reduced cost and faster production
  • Potentially increased stability and convenient storage

Moreover, in direct comparison with antibodies in IHC, aptamer probes provide:

  • Optimal staining with shorter reaction times
  • Less restricted antigen retrieval conditions
  • Reduced time to perform the aptamer histochemistry technique
  • Lower background staining (negligible non-specific binding)

The flexibility of aptamers means they can be easily conjugated with a wide range of tags for imaging without affecting selectivity or affinity. In this view, the following researchers have reported the use of fluorescent/biotin tagged aptamers in aptahistochemistry for the characterization of the histological structure of cancer tissues.

Selective staining of Lung Adenocarcinoma Tissue

Zamay et al. selected three aptamers LC-17, LC-18 and LC-224 to postoperative lung adenocarcinoma tissue for histochemical analysis of biomarkers, tumour cells, blood vessels and connective tissues. The aptamers showed binding to various tumour specific biomarkers and tumour structures such as elastic fibers, tumour cells, blood vessels, and elastin all of which play important roles in tumour progression. Also, these aptamers did not stain normal lung tissue confirming that they only have affinity to elastic fibers that have been modified due to a malignant process (Figure 1).

Figure 1. Laser scanning imaging showing comparison of binding specificity of antibodies and aptamers (LC-18, LC-224 and LC-17) to protein biomarkers associated with Lung adenocarcinoma. The aptamers showed binding selectivity of aptamers to transformed elastic fibers of blood vessels along with tumour cells, glands of tumour tissues.

Selective staining of Breast Cancer Tissue

Ahirwar et al. attempted to ascertain the clinical applicability of breast cancer specific aptamer in evaluating the ERα expression, by selective staining of ERα-positive cells, to assist in breast cancer diagnosis and grading. An aptahistochemistry assay was performed using biotinylated-ERaptD4 on deparaffinised and hydrated tissue sections of breast cancer patients. Aptamer ERaptD4 showed specific staining in the nuclei of ERα positive breast cancer cells where it specifically stained the malignant duct cancer cells without any cross reactivity to fibroblasts, adipocytes, inflammatory cells or extracellular components (Figure 2).

Figure 2. Comparative staining analysis of ERα antibody and biotinylated ERaptD4 aptamer on ERα positive cells in breast cancer tissue samples.

Selective staining of glioma tissues

Similarly, Aptekar et al. determined the effectiveness of aptamers in recognizing and differentiating between different grades of glioma and non-cancerous brain tissues. Staining of tissue sections was graded using a UK NEQAS scoring system. The aptamer SA43 showed moderate to strong nuclear staining in tumour tissues with an average score ranging from 4–6.6, whereas, in non-cancerous tissues, the average score was only 2.9. The random sequence aptamer showed comparatively negligible binding to the non-cancerous and tumour tissues. Moreover, the aptamer showed selective binding to endothelial cells in tumour tissues (Figure 3).

Figure 3. Aptahistochemistry analysis of non-cancerous and different pathological grade glioma patients stained with biotinylated DNA aptamers.

Overall, these findings have opened the door to aptamer histochemistry applications in both research and clinical settings, including intraoperative diagnostics in which speed and accuracy are paramount.

At Aptamer Group Ltd, we start with the end in mind by designing the aptamers according to the customer specific requirements, so that they are optimized for end applications. For such IHC applications, we isolate the aptamers directly against the cells / tissues of interest; after they have undergone all the necessary treatments (fixation, embedding, antigen retrieval etc). Taking this approach means that we isolate the aptamers against the antigen, as it will be presented in the final application. This helps to ensure that the aptamers are fit for purpose faster than traditional technologies. If you want more information on exploring applications of aptamers in IHC, please contact us using the form below.

References:

Ahirwar R, Vellarikkal SK, Sett A, Sivasubbu S, Scaria V, Bora U, et al. (2016) Aptamer-Assisted Detection of the Altered Expression of Estrogen Receptor Alpha in Human Breast Cancer. PLoS ONE 11(4): e0153001. https://doi.org/10.1371/journal.pone.0153001

Aptekar S, Arora M, Lawrence CL, Lea RW, Ashton K, Dawson T, et al. (2015) Selective Targeting to Glioma with Nucleic Acid Aptamers. PLoS ONE 10(8): e0134957. https://doi.org/10.1371/journal.pone.0134957

Zamay GS, Ivanchenko TI, Zamay TN, et al. (2017). DNA Aptamers for the Characterization of Histological Structure of Lung Adenocarcinoma. Mol Ther Nucleic Acids.6:150–162. doi:10.1016/j.omtn.2016.12.004

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