## 2 Materials and Methods
### Human Imaging Dataset Acquisition and Post-Processing  
All patient participants in the primary enrollment cohort provided informed consent prospectively (IRB #1097531). Patients presenting with volume-occupying brain lesions on previous computed tomography (CT) to a single tertiary care center were approached for consent to undergo a research-specific dwMRI imaging protocol incorporating standard single-shot EPI spatial encoding on a siemens 3T scanner (b-value = 1000 s/mm\({}^{2}\), 64 gradient directions, echo time = 95 ms, voxel size 1.8 \(\times\) 1.8 \(\times\) 4.0 mm), in combination with anatomical MRI. These scanning parameters were selected to maximize the likelihood of observing any structures with coherent diffusional property oriented nearby the tumor, while endeavoring to minimize the scan time given that all diffusion data were acquired as add-on research sequences during standard-of-care preoperative scans after the diagnosis of new brain tumors. Of successfully enrolled subjects (\(n\) = 13), WHO Grade 4 glioblastoma diagnosis was confirmed on postoperative pathology in \(n\) = 7 cases, confirming inclusion for the present study. Tumor region(s) of interest (ROI) were manually segmented within diffusion spectrum imaging studio (DSI Studio, Version Date: 3 October 2019) after registration of QSI and post-contrast T1- or T2-weighted images. Tumor ROI were generated by manually tracing a single boundary encompassing the enhancing tumor and necrotic core, but excluding any evident peritumoral edema, on each axial slice. In cases of multiple tumor foci, each was segmented and investigated individually. T1-weighted contrast-enhanced images or T2-weighted images were favored for aiding tumor segmentation when available. While explicit distortion correction of raw dwMRI data was not employed as part of pre-processing, the bad slice inspector tool was used when loading data into DSI Studio, prior to masking to filter out background, in an effort to identify and eliminate possible head motion or eddy current-related artifacts. \"Bad slices\" were identified and excluded from further processing in only a minority of cases. The post-processing of QSI data was then completed in DSI studio for each scan to generate tumor-associated tractograms. After setting tracking parameters including a tract length threshold of 0-300 mm, angular threshold of \(\geq\)45\({}^{\circ}\), and step size of 0.3 mm, diffusional tracts which explicitly intersected the manually segmented tumor ROI were displayed. Tumor-intersecting tractography or \"tumor tracts\" were observed qualitatively across the study cohort, and metrics quantified within DSI Studio included mean tract length and mean projecting tract length. The mean tract length was defined as the mean length of all post-processed tumor-intersecting tracts. The mean projecting tract length was defined as the mean length of those tracts which extended outward from the tumor ROI rather than terminating within the segmented mass or adhering closely to the surface of the mass, as described by [PERSON] et al. as \"core\" and \"surface\" tracts, respectively [40]. Such tracts were manually selected from the generated tractograms for further quantification. Thus, the mean projecting tract length specifically excluded any tracts which were confined exclusively within or circumferentially around the tumor ROI. Both tract length metrics were computed directly by DSI Studio. Tumor volumes were additionally quantified using 3D Slicer (v4.10.2). Patient demographics, receipt of surgery and/or chemoradiation, MGMT promotor methylation status, IDH mutation status, tumor location, number of tumor foci at presentation, and patient outcomes were obtained from the electronic medical record. For the confirmation that observed tractography patterns were also seen more generally across a larger cohort, and for a more rigorous analysis of association with clinical outcomes, a de-identified historical comparison dataset from University of Texas, MD Anderson Cancer Center (MDACC) was utilized.