Cells and Tissues

Cell and tissue-based applications generally require an affinity ligand which is able to recognise the target when it is ‘surrounded’ by other materials (e.g. other proteins, cell walls etc). This can be problematic for reagents such as antibodies, as they are usually isolated against a recombinant form of the target protein. If the antibody binding epitope is occluded when the protein is presented in a cell membrane, then the antibody will not bind to the cell / tissue.

Aptamers can be selected directly against cells or tissues expressing the target of interest. This direct selection approach means that only cell surface exposed epitopes can be used for binding; completely bypassing the antibody problem.

This benefit gives aptamers a significant advantage in cell and tissue based applications such as fluorescent imaging and microscopy, flow cytometry and Fluorescence Activated Cell Sorting (FACS), as well as therapeutic applications such as targeted delivery in Aptamer Drug Conjugate (ApDC) development.

Isolation of aptamers against cells or tissues, follows many of the same principals of protein based aptamer selection.

  1. An aptamer library is incubated with the target (cells, tissues etc in this case).
  2. Counter selections can be included to ensure that the resulting aptamers are specific to a cell / tissue etc.
  3. Non-binding sequences are partitioned and discarded; the binders are recovered and amplified.
  4. The process is repeated using different selection pressures to ensure that the resulting aptamer population has the required properties (specificity etc).
  5. Individual aptamers are isolated from the final population and characterised

Aptamer-Based Cell And Tissue Targeting Reagents Have A Number Of Advantages Over Antibody-Based Reagents

  • The aptamers can be isolated directly using intact cells / tissues – there is no need to produce a recombinant protein (although this can be used to help specific targeting)
  • The nature of the target protein does not need to be known – aptamer selection can be ‘hypothesis-free’ and target all cell surface proteins simultaneously.
  • Several different cell lines / tissues can be used (from different models, patients etc) for both positive and negative selection targets – this can help to target features which are common to all patients, or to isolate aptamers specific to a disease state or stage (e.g. patient stratification) etc
  • Additional steps can be included during the aptamer selection process, to ensure that aptamers are internalised – this is may be essential for use as a targeted therapy (e.g. Aptamer Drug Conjugates (ApDCs))

Aptamers Can Be Used For Hypothesis-Free Selection And Subsequent Stratification Of Patient Samples

Careful application of cell-based aptamer selection processes, in conjunction with the correct choice of representative cell-lines (or tissues), can lead to the isolation of aptamer populations which discriminate between closely related materials. This tuned specificity has many potential applications, including patient stratification. Here, four cell-line models were chosen which represent different stages of a cancer development; Healthy (far left), Early Stage (middle left), Late Stage (middle right) and a related (but histopathologically indistinguishable) cancer (far right). Using each of these cell-lines in different combinations as either target or counter target, lead to the development of three aptamer populations with very distinct target binding. The nature of this discrimination is as yet unknown but is clearly present, demonstrating the power of ‘hypothesis-free’ aptamer selection.

Aptamers Can Be Selected To Remain On A Cell Surface Or Be Internalised

Additional steps can be included in cell-based aptamer selection, to ensure that the resulting aptamers have the necessary properties to serve as a targeted drug delivery agent. Here we demonstrate that these processes can be applied to isolate aptamers which remain on the cell surface (left) or get internalised by the cells (right).

Aptamers Can Target Specific Cell Types Within A Tissue

The principals of cell-based aptamer selection can also be applied to tissues. Careful choice of appropriate positive and negative target material can result in selection of aptamers with useful properties; again in a ‘hypothesis free’ approach. Here aptamers were selected against ‘injured’ tissue samples. Tissue sections were stained with the resulting aptamers (red) and show higher density staining around the site of injury.

Cell and Tissue Imaging

Antibodies generated against highly purified, recombinant proteins often underperform when used in the context of a whole cell or tissue imaging application. This is largely due to the significant differences in epitope availability between a recombinant protein and the same protein in a cell surface and in tissue.

These issues are compounded when used in applications such as Immunohistochemistry (IHC), where the epitopes are further modified through various fixation, embedding and antigen retrieval methods.

It is perhaps unsurprising that antibodies (or other affinity ligands) have difficulty with IHC applications as they are isolated and characterised using purified recombinant protein, but are then required to recognise the same protein when it is;

  1. Presented in a cell surface – masking some epitopes
  2. Contained with a tissue – limiting access to the protein
  3. Formalin fixed – changing the protein epitope and cross-linking it to surrounding material
  4. Embedded in wax – limiting access to the protein
  5. Put through antigen retrieval – often involving boiling in buffer, which will likely denature the target

One of the biggest advantages of the aptamer isolation process, is that it is in vitro in nature. This means that we can readily work with more complex targets including whole cells, tissues, FFPE sections etc.

At Aptamer Group, we start with the end in mind. In applications such as this, we isolate the aptamers directly against the cells / tissues of interest; after they have undergone all of 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.

Aptamers Out Perform Antibodies in Complex Systems

Comparative FFPE tissue staining with a commercial antibody (upper panels) and an aptamer (lower panels) against the protein target. The images clearly show a stronger staining from the aptamer.

References:

  1. PLoS One. 2013;8(2):e57613. doi: 10.1371/journal.pone.0057613. Epub 2013 Feb 27. The use of sensitive chemical antibodies for diagnosis: detection of low levels of EpCAM in breast cancer. Shigdar S1, Qian C, Lv L, Pu C, Li Y, Li L, Marappan M, Lin J, Wang L, Duan W.