Atomic Force Microscope Nanoindentation Analysis of Diffuse Astrocytic Tumor Elasticity: Relation with Tumor Histopathology

Abstract

This study aims to investigate the influence of isocitrate dehydrogenase gene family (IDH) mutations, World Health Organization (WHO) grade, and mechanical preconditioning on glioma and adjacent brain elasticity through standard monotonic and repetitive atomic force microscope (AFM) nanoindentation. The elastic modulus was measured ex vivo on fresh tissue specimens acquired during craniotomy from the tumor and the peritumoral white matter of 16 diffuse glioma patients. Linear mixed-effects models examined the impact of tumor traits and preconditioning on tissue elasticity. Tissues from IDH-mutant cases were stiffer than those from IDH-wildtype ones among anaplastic astrocytoma patients (p = 0.0496) but of similar elasticity to IDH-wildtype cases for diffuse astrocytoma patients (p = 0.480). The tumor was found to be non-significantly softer than white matter in anaplastic astrocytomas (p = 0.070), but of similar elasticity to adjacent brain in diffuse astrocytomas (p = 0.492) and glioblastomas (p = 0.593). During repetitive indentation, both tumor (p = 0.002) and white matter (p = 0.003) showed initial stiffening followed by softening. Stiffening was fully reversed in white matter (p = 0.942) and partially reversed in tumor (p = 0.015). Tissue elasticity comprises a phenotypic characteristic closely related to glioma histopathology. Heterogeneity between patients should be further explored.

Nanomechanical and Morphological AFM Mapping of Normal Tissues and Tumors on Live Brain Slices Using Specially Designed Embedding Matrix and Laser-Shaped Cantilevers

“…The first task is to fix the tissue sample for subsequent scanning. Most major professionals use a thin layer of biocompatible glue [ 29 , 36 , 37 ]. The second well-known problem is the high heterogeneity of the tissue and its uneven surface [ 29 , 37 ].…”

“…This problem was solved in one of the works by using AFM with a large z-range [ 29 ], although this method has several disadvantages, including the rendering high throughput mapping impossible. Another described method is the use of a probe with a large radius: up to 25 microns [ 36 ]. At the same time, increasing the probe radius helps to solve the problem of measuring the mechanical properties by averaging the result over a large area of the surface [ 36 ].…”

“…Another described method is the use of a probe with a large radius: up to 25 microns [ 36 ]. At the same time, increasing the probe radius helps to solve the problem of measuring the mechanical properties by averaging the result over a large area of the surface [ 36 ]. Previously, highly adhesive uneven surfaces with low elasticity and stiffness were analyzed only at microscale level.…”

“…Recently, the utility of the embedding medium has been proposed to improve the quality of slices [ 39 , 40 ]; because pre-fixed tissue [ 41 ] and infiltrating medium [ 40 ] were used, the advantages of a vibratomy were negated due to these procedures causing cell death. However, agarose as an embedding matrix can preserve the tissue alive, and is suitable for AFM investigation [ 36 ]. To provide AFM investigation in the mode of combined morphological and nanomechanical mapping, slices should be obtained in an embedding matrix with certain composition and viscoelastic properties, because they influence slice quality [ 39 ].…”

“…Thirdly, the matrix rheological properties are optimized in order to obtain high-quality slices by protecting the tissue from tearing and crushing under the vibratome blade. Agar and collagen were chosen as the base components of the matrix because they are highly compatible with live objects and have been used for tissue embedding many times [ 36 , 42 , 43 , 44 , 45 ]. Agarose-only hydrogels have already been used as an embedding matrix for the vibratomy of live brain tissue [ 36 ], but they can be employed to obtain slices of live tissue suitable only for the Ramp Mode of AFM.…”