(A) mCRC patients on regorafenib treatment underwent biopsies at baseline (BL), partial response/stable disease stage (PR/SD), or post-treatment (PD). An early reduction (15 days) in functional imaging (DCE-MRI) parameters correlated with changes in micro-vasculature assessed by CD31 staining and clinical benefit from regorafenib (right panel). (B) Changes in micro-vasculature in response to regorafenib were assessed in PDO mouse xenografts by quantification of tumor-associated CD31-positive vessels. Data show PDO xenografts from a primary resistant (R-009) and a long-term responder (R-005) to regorafenib. Mean ± SD from indicated number of mice (n) in a representative experiment is shown; significance was determined using Student’s unpaired t-test. (C) Reduction in fractional blood volume (fBV) in regorafenib-treated mice carrying long-term regorafenib responder (R-005) PDO-xenografts. A total of ten animals were analyzed (five in each arm); data represent the mean ± SD of an individual experiment. Day 0 fBV values could not be obtained for two animals due to respiratory movement. Significance was determined using Student’s paired t-test for fBV and unpaired t-test for CD31 and necrosis. (D) Schematic representation of animal experiment using PDOs from patient R-011, established pre- and post-treatment with regorafenib. Mice carrying liver orthotopic R-011 pre-treatment (BL) and post-treatment (PD) PDO-xenografts were randomized to control and treatment arms, and treated with vehicle or regorafenib for 10 days. Following-treatment, each arm was further randomized to a cohort culled for histopathological analysis and a survival cohort which was monitored over time. (E) CD31 immunostaining in the parental patient baseline (BL), stable disease (SD), and post-treatment (PD) biopsies, demonstrating an initial reduction in tumor microvasculature in response to regorafenib. Data represent mean ± SD calculated by scoring ten high-power field tumor areas. (F) Representative images of CD31 immunostaining in the baseline (BL) and post-treatmen (PD) R-011 PDO-xenografts. Data represent mean ± SD calculated by scoring at least ten high-power field tumor areas per animal in an individual experiment; n= number of animals analyzed in each group. Significance was determined using Student’s unpaired t-test. (G) Kaplan-Mayer curves of regorafenib- or vehicle-treated mice bearing baseline (BL) and post-treatment (PD) PDO-xenografts from patient R-011 from an individual experiment. (n= number of mice analyzed). Significance was determined using the Mantel-Cox log-rank test.