Orthotopic and subcutaneous xenograft studies

HT1080 cells (a human fibrosarcoma cell line), U-87 MG cells (a human glioblastoma cell line), and SK-UT-1 and SK-LMS-1 cells (human LMS cell lines) were obtained from the ATCC and cultured using methods provided by the ATCC. Prior to implantation, the SK-LMS-1 cells were serial passaged seven cycles in male athymic nude mice to generate a more aggressive tumor model. The D-09–0500 MG xenograft line has been previously described (23) and was generated from a treatment-naïve 58-year-old male glioblastoma (GBM) patient undergoing surgery at Duke University Medical Center (Durham, NC) and was maintained by serial subcutaneous passage in athymic nude mice.

All in vivo studies were performed at an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-certified facility under Institutional Animal Care and Use Committee (IACUC)-approved protocols. The day treatment initiated was denoted as day 0. An animal was taken off study when it attained endpoint (e.g., death, moribundity, or tumor volume endpoint) or if it exhibited a body weight loss ≥20% of its initial day 0 body weight. For dose optimization studies, HT1080 fibrosarcoma or U-87 MG GBM tumor cells (5 × 10⁶ cells in 0.2 mL 50% Matrigel) were inoculated subcutaneously into the right flank of athymic nude mice. For the orthotopic GBM study, athymic nude mice were injected intracranially with U-87 MG tumor cells (2 × 10⁵ in 2 μL PBS) at the right frontal lobe, 2 mm lateral and 0.5 mm anterior from bregma at a depth of 3.5 mm. For the patient-derived GBM xenograft study, D-09–0500 MG tumors were removed from host animals under sterile conditions, homogenized into a cell suspension, and injected subcutaneously (50 μL) into the right flank of athymic nude mice.

For subcutaneous xenograft studies, tumors were measured using digital calipers. Tumor volumes were calculated as (l x w²)/2, where l was the longest tumor measurement and w was the shortest tumor measurement. When tumors reached the appropriate size, mice were randomized into groups so that the average initial tumor size was the same across groups within a study.

For subcutaneous xenograft studies, mice were taken off study when individual tumor volumes were ≥1,000 mm³, mice were moribund or found dead, or on the final day of the study, whatever came first. The time for the tumor in an individual mouse to reach 1,000 mm³ was calculated using the FORECAST function in Excel and then the median time across the group determined. Differences in the distribution of time-to-endpoint (time to 1000 mm³ or death in days) were analyzed by the Kaplan–Meier method and the log-rank test. Mean tumor growth curves were plotted and truncated when 50% of test animals were taken off study. For the orthotopic GBM survival study, the MST was determined for each treatment group and the increase in lifespan, expressed as a percentage of the control, was calculated. Graphical presentations and statistical analyses were performed using GraphPad Prism version 8.4.3 for Windows, (GraphPad Software).

The interaction between PTC596 and the various compounds was evaluated using the fractional product method (24). The expected effect of PTC596 and a compound acting independently was calculated as the product of the unaffected fractions after treatment with either drug alone. Then, the potential interaction of the combined drugs was determined by calculating a Synergy Score as the ratio of observed to expected, where Synergy Score <0.7 indicated synergy.