Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

The overall goal of this analytical technique is to quantify the anti-tuberculosis drug ethambutol within tuberculosis pulmonary lesion tissue compartments to determine whether bactericidal drug concentrations are being reached. This method can answer questions in the tuberculosis drug discovery and development field by determining whether administered drugs are reaching bacterial populations at sterilizing concentrations within all granuloma tissue compartments. The main advantage of this technique is that it enables full quantification of drug concentrations at high spatial detail within specific tissue structures and cell populations.

Visual demonstration of this method is critical, as the tissue sectioning and mounting steps are difficult to learn due to the potential for tissue contamination and the fragile nature of the membrane slides. Begin this procedure with tissue collection as described in the text protocol. To perform tissue sectioning, first set the cryostat to the desired cutting temperature.

Transfer the gamma-irradiated lung biopsy from the minus 80 degree Celsius storage to the cryostat, and leave for 30 minutes to equilibrate the tissue temperature. Using tweezers, fix the biopsy to the cryostat chuck using a small amount of optimal cutting temperature adhesive to adhere the base of the tissue to the chuck. Orient the tissue so that the flat surface is the exposed surface for cutting.

Ensure the OCT does not contaminate the tissue surface, as this may interfere with the subsequent mass spectrometry analysis. Cut three tissue sections at 25 micron thickness, and mount onto PET membrane slides. Gently touch the membrane to the tissue section, and remove the section.

If too much pressure is applied, the thin membrane may tear. Avoid excessive handling of the slide prior to mounting so as not to charge the PET membrane and diminish adhesion of the tissue section. Keep the membrane slide at room temperature to enable thaw-mounting and successful adhesion of the tissue to the membrane.

Remove the slide from the cryostat, and allow the slide to air-dry for three minutes. If LCM-LC-MS/MS will not be performed immediately, seal the slide in a small airtight sealable bag and transfer to the minus 80 degree Celsius storage until required for dissection. Cut an adjacent section at 10 to 12 microns, and thaw-mount on a standard glass slide for hematoxylin and eosin staining.

Additional sections can be cut at this time for other desired histochemistry stains. Remove the sealed bag containing the slide from the minus 80 degree Celsius storage, and allow to reach room temperature for five minutes. If the cold slide is immediately exposed to the laboratory atmosphere, the tissue will become coated with condensation and the spatial integrity of the drug may be compromised.

Turn on the microscope and laser. The laser requires five to 10 minutes to warm up before cutting can commence. Load flat-cap 0.20 milliliter PCR tubes into the holder.

After removing the slide from the bag, take an optical image of the tissue section on the PET slide using a flatbed scanner. Place the slide into the slide holder, tissue side facing down. Assign separate collection tubes to specific granuloma regions of interest using the microscope software.

Typically, these will be uninvolved lung, cellular granuloma, and caseum, but may vary depending upon the specific pathology of the granuloma biopsy. Focus on the tissue using the 5X microscope objective. This magnification should provide a good overview of the tissue containing both cellular and necrotic granuloma areas.

In the software, select the tube designated caseum to move it into position under the tissue. Enter the desired dissection parameters. Typical settings for a 25-micron-thick lung section are laser power 30, speed 15, and aperture 35.

However, these will differ depending upon the microscope used and potential declining power due to the age of the laser. Select the Free Draw tool, and, using either a mouse or touchscreen pen, outline the desired region for dissection. The surface area of the region will be displayed in the software.

Keep selected regions under 500, 000 square microns to facilitate easier dissection. Repeat the dissection until three million square microns have been collected in total in the tube cap. On occasion, the dissected region may remain stuck to the surrounding membrane and not fall into the collection cap.

Remove these regions from the cumulative surface area total by manually selecting and removing within the software. Next, select the cap for cellular lesion, and collect three million square microns of tissue using the same process. Select the cap for uninvolved lung, and collect three million square microns of tissue using the same process.

Note that uninvolved lung tissue contains many bronchioles and alveolar spaces. Pay careful attention to exclude these from the defined tissue regions for dissection. Remove the cap holder, and carefully unclip, seal, and label each tube.

Protect the dissected tissues from surrounding air disturbances. Analyze the dissected tissues immediately, or store at minus 80 degrees Celsius and thaw prior to processing and LC/MS analysis. Assuming a tissue density of one gram per milliliter, prepare the homogenate stock by weighing 50 milligrams of control tissue and adding PBS buffer to dilute.

Homogenize the tissue by bead beating the lung tissue and PBS buffer for five minutes at 1750 rpm on a bead homogenizer. Next, dilute one milligram per milliliter drug stocks in one-to-one acetonitrile/water to create standard curve spiking solutions. Determine spiking standard concentrations based on the spike volume and the target tissue volume.

The illustrated example is for a 100 nanogram per milliliter standard using a 10 microliter spike volume. Remove the tubes containing the microdissected tissue from minus 80 degree Celsius storage, and allow to reach room temperature for five minutes. Add 10 microliters of one-to-one acetonitrile/water solution and two microliters of PBS buffer to the tubes containing the microdissected tissue.

For standard curve and quality control tubes, add 10 microliters of spiking solution to two microliters of control lung homogenate. Add 50 microliters of extraction solution to each tube. Vortex each tube for five minutes.

Then, sonicate them for five minutes. Next, centrifuge the tubes at 5, 000 rpm for five minutes to form a pellet of film and tissue in each tube. Following centrifugation, transfer 50 microliters of supernatant to a 96-well deep-well plate and dilute with an additional 50 microliters of deionized water in each well.

Finally, perform LC-MS/MS analysis and method validation as described in the text protocol. The LCM-LC/MS method was first validated by analyzing laser-microdissected regions from ethambutol-spiked tissue homogenate. As shown here, the method was highly reproducible and demonstrated excellent drug recovery.

Ethambutol concentrations measured by LCM-LC/MS in multiple lung tissues were further validated by direct comparison to concentrations determined by an established LC/MS approach. Equivalent concentrations were detected by both techniques. Absolute ethambutol concentrations within uninvolved lung parenchyma, cellular lesion rim, and necrotic caseum were determined by LCM-LC/MS.

Mycobacterium tuberculosis sterilizing levels were reached in all lesion compartments. The minimum concentrations required to kill 99%of extracellular replicating bacilli and 99%of intracellular bacilli in macrophages are indicated. After watching this video, you should have a good understanding of how to microdissect tissues and quantify drug levels within defined pulmonary granuloma structures.

This technique enables researchers in the field of antimicrobial drug research to accurately and quantitatively assess whether administered drugs are reaching target pathogen populations at sterilizing concentrations. This approach has broad applicability to many different tissues and disease states.