Laser Capture Microdissection (LCM) - A Strategy for Success

Micro-sampling by laser capture microdissection is a powerful tool allowing specific analysis from whole tissues, in heterogeneous microenvironments such as tumours, where less well represented target cells are dwarfed by tissue stroma and other cell types. Whilst the application is potentially powerful for target discovery and mechanistic understanding, the process is notoriously difficult, particularly in clinical specimens.

Stage 1 – Development of Staining Protocols

Epistem has developed a modified H&E staining protocol that preserves RNA integrity. In many cases this enables us to extract target cells using morphology alone.

If available - by providing Epistem with H&E stained tissues prior to commencing the study we will evaluate the probability of obtaining RNA from specific cells based on morphological staining.

h&e stained tissue

Immunohistochemical staining is a powerful method to discriminate cells based on the presence of specific target proteins.

Epistem has developed RNA-sympathetic immunostaining in a number of targets, including CDH1 in colon (Fig), fibrosis markers in lung, liver and kidney, inflammation markers (CD4) in skin and CD8a/CD3e for tumour infiltrating lymphocytes

RNA-sympathetic staining of CDH1 in colon

Case Study – Immunostaining of tumour infiltrating lymphocytes (TILs) in CRC tissue

Tumour infiltrating leukocytes are implicated in survivability for cancer patients, but are also under investigation as a therapy for solid tumours such as adoptive cell transfer. Samples from three different colorectal cancer (CRC) donors were histologically examined and samples acquired by laser capture immunostaining and highly sensitive RNA amplification techniques to transcriptionally profile TILs compared to T-cells in normal adjacent tissue (NAT).

Matched tumour and normal adjacent tissue obtained from 3 individuals were embedded in OCT and sectioned by cryostat. Two different T-cell markers, CD3e and CD8a, were used individually to select for T-cells from both healthy and diseased tissue for each donor.

Differential gene expression analysis resulted in confirmation that targeted selection results in discrete data sets, and that relevant biological signal can be missed in gross tissue preparation.

fluorescent anti-CD8a staining on human colon NAT
bright field image with fluorescent CD8a+ cells overlaid

Representative fluorescent anti-CD8a staining on human colon NAT and matched bright field image with fluorescent CD8a+ cells overlaid.

Epistem also routinely evaluates proposed targets for specific studies. We will evaluate your targets for morphological staining or develop RNA-friendly IHC staining protocols.

Positive confirmation results in Stage Progression

Stage 2 - Capture and analyse relevant material

Samples representative to the study you wish to conduct should be supplied to Epistem. This is especially relevant to longitudinal studies where target tissue/cells may become less abundant over time.

We will confirm the developed staining protocols are reproducible under study conditions.

We will also estimate time required to capture all samples in the main study so that we can supply the most accurate costings and timelines.

We will confirm the reproducibility and quality of RNA.

We can also confirm that the relevant target tissue/cells are being captured by analysing appropriate markers by qPCR to triage samples.

RNA recovered from CHD1 immuno-stained tissue sections (blue) with comparable quality to unstained sections (red).

Case Study – Expression of CD3a and CD8 markers in captured TILs

CD3a and CD8 immune-positive cells were specifically isolated from each tissue for analysis. Gross whole sections were immuno-stained and processed in parallel as comparator samples. RNA was extracted from all samples using Epistem's proprietary methodology with surrogate RNA quality evaluated by Bioanalyser. RNA yields for whole sections were evaluated by Nanodrop. RNA normalisation downstream was achieved by controlling laser capture area size.

RT-qPCR of immune-stain markers CD3e against background KRT6
RT-qPCR of immune-stain markers CD8 against background KRT6

RT-qPCR of immune-stain markers CD3e and CD8 against background KRT6 indicates higher levels of marker expression in the LCM samples compared to the whole slide samples and lower expression of the background marker KRT6.

Positive confirmation results in Stage Progression

Stage 3 - Main study and downstream processing

We work with Sponsors to establish protocols best suited to their project requirements. We use a Palm MicroBeam 4 LCM platform to efficiently identify, cut and specifically capture target tissue/cells. Our technique allows multiple capture types per slide, sample pooling when required, high capture throughput and capture image documentation. We routinely assess capture areas of approximately 50 cells and have experience of capturing cells from a large variety of tissue types, both healthy and diseased. RNA from LCM is suitable for RT-qPCR, NGS and microarray analysis and Epistem's Single Cell RNA-Amp™ provides robust and linear amplification of RNA enabling comparative analysis in applications such as target discovery and pharmacodynamics.

Case Study – Microarray analysis of captured TIL RNAs

Direct comparisons in the profiles obtained following differential immune-stain selection were conducted to evaluate transcriptional differences between activation status in CD3e+ and CD8a+ T-cells. cDNA amplification of RNA samples was carried out using Epistem's proprietary amplification kits (Single Cell RNA-Amp™).

The Affymetrix HG-U133 Plus 2.0 microarray platform was used to determine differentially expressed genes in gross tissue and laser capture samples between NAT and tumour. The array contained transcripts to the entire human genome: 47,000 transcripts and variants. Duplicate individual captures and post-stain gross tissue samples in each group were assessed by microarray analysis.

Bioinformatic analysis of the CD3a laser capture dataset

Bioinformatic analysis of the CD3a laser capture dataset above shows enrichment of genes associated with lymphocyte activity. The whole slide dataset (not shown) has enriched genes associated with oncogenic pathways with many of the changes in immune-associated genes undetected due to the abundance of gross tissue expression.

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Epistem Biomarker Discovery and Validation

Epistem is your ideal partner for biomarker discovery and validation. We have leveraged over 15 years of histology and IHC expertise to develop RNA-friendly stains for specific cell types making them amenable for gene expression studies using LCM. All of our histology and gene expression, LCM and hair IHC applications are GCLP-compliant and we have participated in many clinical studies. We have in-house bioinformatics support for all of our gene expression studies and have extensive experience identifying biomarkers or biomarker signatures in a variety of tissues.

About Epistem

Epistem's contract research service is committed to providing reliable, innovative and transferable pre-clinical models and services to support decision making throughout the drug discovery and development pipeline.

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