Detection of human brain tumor infiltration with quantitative stimulated Raman scattering microscopy

Abstract

Differentiating tumor from normal brain is a major barrier to achieving optimal outcome in brain tumor surgery. New imaging techniques for visualizing tumor margins during surgery are needed to improve surgical results. We recently demonstrated the ability of stimulated Raman scattering (SRS) microscopy, a nondestructive, label-free optical method, to reveal glioma infiltration in animal models. We show that SRS reveals human brain tumor infiltration in fresh, unprocessed surgical specimens from 22 neurosurgical patients. SRS detects tumor infiltration in near-perfect agreement with standard hematoxylin and eosin light microscopy (κ = 0.86). The unique chemical contrast specific to SRS microscopy enables tumor detection by revealing quantifiable alterations in tissue cellularity, axonal density, and protein/lipid ratio in tumor-infiltrated tissues. To ensure that SRS microscopic data can be easily used in brain tumor surgery, without the need for expert interpretation, we created a classifier based on cellularity, axonal density, and protein/lipid ratio in SRS images capable of detecting tumor infiltration with 97.5% sensitivity and 98.5% specificity. Quantitative SRS microscopy detects the spread of tumor cells, even in brain tissue surrounding a tumor that appears grossly normal. By accurately revealing tumor infiltration, quantitative SRS microscopy holds potential for improving the accuracy of brain tumor surgery.

Fig. 1. SRS microscopy workflow and imaging of normal gray and white matter

All imaged specimens were collected from patients undergoing anterior temporal lobectomy for intractable epilepsy. (A) Experimental setup of SRS microscopy. The Stokes beam was modulated at high frequency (10 MHz), and the weak stimulated Raman loss signal was demodulated by a lock-in amplifier. A transmission mode detection scheme was used for ex vivo imaging on fresh tissues. DC, dichroic mirror; EOM, electro-optical modulator; FL, optical filter; GM, galvanometer mirror. (B) Raman spectra from fresh sections of human glioblastoma biopsy show white matter, cortex, and tumor. The marked frequencies (dashed lines) at 2845 and 2930 cm⁻¹ were chosen for two-color SRS imaging. (C) SRS imaging of normal gray matter at high magnification showing neuronal soma with pyramidal architecture filled with lipofuscin-rich granules (left), that stain positively for the neuronal nuclei antigen (NeuN) within the neuronal cell body (right). (D) SRS imaging of white matter (left) demonstrates individual axons appearing as linear, lipid-rich structures that correspond well with neurofilament immunohistochemical staining (right). (E) An SRS image of the gray-white junction (left) demonstrates parallel bundles of lipid-rich white matter tracts that are not visible with H&E staining (right). (F) Capillaries filled with protein-rich erythrocytes appear blue on SRS imaging (left) and eosinophilic on H&E-stained section (right). (G) At low magnification, the biochemical differences between protein-rich gray matter (blue) and myelinated white matter (green) are apparent.

Fig. 2. SRS and traditional microscopy of intrinsic brain tumors

(A) SRS imaging of a GBM (arrowhead) demonstrating ring enhancement on MRI. (B) Hypercellularity and nuclear atypia of viable tumor is apparent on both SRS (left) and H&E (right) microscopy. (C) Microvascular proliferation creates tortuous vascular complexes evident on SRS microscopy (left, arrowheads) and highlighted with periodic acid Schiff staining (right, arrowhead). (D) Mitotic figures are also visible (arrowheads) with SRS microscopy (left) and H&E staining (right). (E and F) A non-enhancing, low-grade oligodendroglioma (arrowhead, E) consists of hypercellular tissue with nests of “fried-egg” morphology (arrowheads, F) causing minimal axonal disruption on SRS imaging (left), as confirmed through neurofilament immunostaining (right). (G and H) “Chicken wire” blood vessels (arrowheads, G) imaged with SRS (left) and H&E (right) microscopy, and perineuronal satellitosis is visible in both SRS (left) and H&E (right) microscopy (H).

Fig. 3. SRS microscopy of tissue at the periphery of high- and low-grade gliomas

(A) SRS images of the margin of an infiltrating glioblastoma within cortex depicting a transition from densely tumor-infiltrated brain to minimally infiltrated brain (left to right). (B to D) Cellularity and protein:lipid ratio vary in high-magnification images acquired in densely infiltrated tissue (B), moderately infiltrated tissue (C), and minimally infiltrated tissue (D). (E) SRS imaging of an oligodendroglioma infiltrating within white matter, depicting a transition from densely tumor-infiltrated brain to minimally infiltrated brain (left to right). (F to H) Variation in axonal density, cellularity, and protein:lipid ratio is apparent when comparing high-magnification images from densely infiltrated tissue (F), moderately infiltrated tissue (G), and minimally infiltrated tissue (H).

Fig. 4. Quantitative analysis of an infiltrative tumor margin imaged with SRS microscopy

(A) Cellularity was quantified manually and with automated methods in 20 representative fields of view, drawn from 6 patients with varying degrees of tumor infiltration (2 controls without tumor infiltration, 2 with infiltrating tumor and 2 with dense tumor infiltration). Data are averages ± SEM (B) The variability in cellularity, axonal density, protein:lipid raio, and classifier values at a brain tumor margin. SRS microscopy lipid and protein channels were overlaid. Heat maps show calculated axon densities (arbitrary units) for all FOVs, nuclei per FOV, calculated protein:lipid ratio for all FOVs, and classifier values for all FOVs. Insets are FOVs with high (red), average (yellow), and low (blue) classifier values.

Fig. 5. Nuclear density, axonal density and protein:lipid ratio are quantified from SRS images

(A) Measurements were taken from 1477 300×300 μm² FOVs from 51 fresh tissue biopsies from 18 patients (3 epilepsy patients, 15 brain and spine tumors encompassing 8 distinct histologic subtypes). Each point on the scatterplot represents the average value of each biopsy. Biopsies were classified as predominantly normal to minimally hypercellular (n = 21), infiltrating tumor (n=14), or high-density tumor (n = 16) by a board-certified neuropathologist based on H&E staining. Marker color indicates the mean classifier value for each biopsy, with 0 (most likely normal) depicted in cyan and 1 (most likely tumor) depicted in red. Representative FOVs from normal cortex, normal white matter, low-grade glioma, and high-grade glioma are shown. (B and C) Relationship of classifier values with tumor density (B) and histologic subtype (C). All parameters are normalized to the maximum measurement obtained of that variable and displayed in arbitrary units. Data are means ± SEM.

Fig. 6. SRS microscopy within and surrounding a glioblastoma

(A) A coronal slice of cadaveric brain from a patient who expired with glioblastoma was sampled at the points indicated in green, shown along 5-mm iso-distance lines (as measured from the tumor margin). (B) FOVs captured from the gross tumor margin (0 mm), 5 mm outside the tumor margin (center), and 15 mm outside the tumor margin reveal dense tumor, infiltrating tumor, and normal tissue by SRS, H&E staining, EGFR immunohistochemistry, and neurofilament immunostaining. Scale bars, 50 μm. (C) Tukey boxplots showing quantified axonal density, nuclear density, protein:lipid ratio, and classifier values for all FOVs taken from the necrotic tumor core, viable tumor edge, and at 5-mm increments from 5–30 mm from the gross tumor margin (n = 8). Outlier cutoff defined as median ±1.5 interquartile range.