Association of collagen architecture with glioblastoma patient survival

Abstract

OBJECTIVE Glioblastoma (GBM) is the most malignant primary brain tumor. Collagen is present in low amounts in normal brain, but in GBMs, collagen gene expression is reportedly upregulated. However, to the authors' knowledge, direct visualization of collagen architecture has not been reported. The authors sought to perform the first direct visualization of GBM collagen architecture, identify clinically relevant collagen signatures, and link them to differential patient survival. METHODS Second-harmonic generation microscopy was used to detect collagen in a GBM patient tissue microarray. Focal and invasive GBM mouse xenografts were stained with Picrosirius red. Quantitation of collagen fibers was performed using custom software. Multivariate survival analysis was done to determine if collagen is a survival marker for patients. RESULTS In focal xenografts, collagen was observed at tumor brain boundaries. For invasive xenografts, collagen was intercalated with tumor cells. Quantitative analysis showed significant differences in collagen fibers for focal and invasive xenografts. The authors also found that GBM patients with more organized collagen had a longer median survival than those with less organized collagen. CONCLUSIONS Collagen architecture can be directly visualized and is different in focal versus invasive GBMs. The authors also demonstrate that collagen signature is associated with patient survival. These findings suggest that there are collagen differences in focal versus invasive GBMs and that collagen is a survival marker for GBM.

Keywords: ECM = extracellular matrix; GBM = glioblastoma; GSC = GBM stem-like cell; KPS = Karnofsky Performance Scale; LOCI = Laboratory for Optical and Computational Instrumentation; NOD SCID = nonobese diabetic severe combined immunodeficient; SHG = second-harmonic generation; TMA = tissue microarray; biomarker; cancer stem-like cells; collagen; glioblastoma; oncology; second-harmonic generation.

Figure 1. Visualization of collagen in GBM

Tissue specimens from 1mm core punches are used to directly visualize collagen. Masson’s trichrome, picrosirius red, and second harmonic generation microscopy detect collagen in meningioma (positive control), non-tumor brain (negative control), and GBM specimens. Corresponding H&E sections are also imaged.

Figure 2. Visualization and analysis of collagen fibers in focal vs. invasive GBM xenografts

(A) Focal tumors (12.1 GSC and 22 GSC) and invasive tumors (44 GSC and 107 GSC) are stained with picrosirius red. Collagen is visualized with polarized light. Human NUMA immunostaining of serial section delineate tumor cells (T=tumor, NT= non-tumor, red box= numa selection, black box= picrosirius selection). (B) Collagen fibers are extracted from focal tumors (12.1 GSC and 22 GSC) and invasive tumors (44 GSC and 107 GSC) using CT-FIRE software. Collagen fiber width, the angle between fibers, fiber length, and fiber straightness are calculated. Mean values are determined for each variable. There is a significant difference between fiber width, the angle between fibers, fiber length, and fiber straightness between focal vs. more invasive xenografts. (C) A Kaplan-Meier survival, curve for focal GBM xenografts (12.1 GSC and 22 GSC) and invasive GBM xenografts (44 GSC and 107 GSC) shows a statistically significant survival difference (p=0.0003). **=P<0.001, ****=P<0.0001

Figure 3. Collagen signature correlates with patient survival

A GBM tissue microarray with 1 mm specimen punches is used for second harmonic generation collagen imaging. (A) Groups A and B are observed. Group A is defined as typical fibrillar collagen fibers and group B is defined as atypical collagen fibers represented by punctate or no collagen. (B) Kaplan-Meier survival of the two collagen groups show a significant survival difference (p= 0.001). (C) Collagen fibers are extracted from the represented fibrillar and punctate signatures using CT-FIRE software. There is a significant difference between the angle between fibers and fiber length. *= P<0.05