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

Abstract

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel therapeutics are urgently required. The ability of dosed TB drugs to reach and sterilize bacteria within poorly-vascularized necrotic regions (caseum) of pulmonary granulomas is crucial for successful therapeutic intervention. Effective therapeutic regimens must therefore contain drugs with favorable caseum penetration properties. Current LC/MS methods for quantifying drug levels in biological tissues have limited spatial resolution capabilities, making it difficult to accurately determine absolute drug concentrations within small tissue compartments such as those found within necrotic granulomas. Here we present a protocol combining laser capture microdissection (LCM) of pathologically-distinct tissue regions with LC/MS quantification. This technique provides absolute quantification of drugs within granuloma caseum, surrounding cellular lesion and uninvolved lung tissue and, therefore, accurately determines whether bactericidal concentrations are being achieved. In addition to tuberculosis research, the technique has many potential applications for spatially-resolved quantification of drugs in diseased tissues.

Storage lipid studies in tuberculosis reveal that foam cell biogenesis is disease-specific

“…Equal areas (totaling 5 million μm 2 ) of cellular (300 μM-wide regions surrounding the caseum) and caseous regions of necrotizing lung granulomas were dissected from three serial sections of lung tissue using a Leica LMD6500 system (Buffalo Grove, IL, USA) ( S1 Fig ). The mass of each pooled sample was calculated based on the surface area and tissue thickness of the pooled sections and an assumed tissue density of 1 g/ml, as previously described [ 98 , 99 ]. Pooled dissected material was collected into 0.25 ml standard PCR tubes and immediately transferred to -80°C for storage.…”

High-resolution mapping of fluoroquinolones in TB rabbit lesions reveals specific distribution in immune cell types

“…Tissue sections of appropriate thickness were cut from γ-irradiated rabbit lung biopsies using a Microm HN505 N (Walldorf, Germany) and thaw-mounted onto either stainless steel slides for MALDI-MSI (12 μm thick), PET-Membrane FrameSlides (Leica) for LCM analyses shown in Figure 1—figure supplement 2C (25 μm thick as described in Zimmerman et al, 2018 ), or standard glass microscope slides for H and E staining of adjacent slides (6 μm thick) shown in Figure 1 . For same-section MALDI-MSI and H and E staining, 12 μm thick sections were cut and processed as described in Blanc et al, 2018 and Figure 3—figure supplement 1 .…”

Penetration of Ibrexafungerp (Formerly SCY-078) at the Site of Infection in an Intra-abdominal Candidiasis Mouse Model

“…Laser-capture microdissection. Distinct subcompartments of infected liver tissue (e.g., abscesses and surrounding uninvolved tissue) totaling 1 million to 3 million m 2 were carefully dissected from 4 to 6 serial liver tissue sections using a Leica LMD 6 (CC7000) system (Buffalo Grove, IL) (40). Lesion areas were identified optically from the bright-field image scan and by comparison to the adjacent sectioned GMS-and H&E-stained tissue.…”