Volume of xenograft tumors is an important metric of disease progression and response to therapy in preclinical cancer studies. Noninvasive imaging technologies suitable for measuring xenograft volume are increasingly available, yet bi-dimensional volumetric assessment with manual calipers, which are susceptible to inaccuracy and bias, remain the standard of practice. We sought to quantify and compare the accuracy, precision, and inter-observer variability of xenograft tumor volume assessment by caliper measurements, surface bioluminescence (BLI) and ultrasound (US) imaging. Romidepsin (R) and Pralatrexate (P) are two drugs recently approved for treatment of relapsed/refractory T-cell lymphomas but despite these advances the prognosis for most patients still remains poor. Measurements obtained from these modalities were utilized to determine synergistic interactions of R and P in our novel mouse models of T-cell lymphomas.
Stably transfected human tumor derived H9 sezary cells constitutively expressing mcherry and firefly luciferase were implanted into the flank of 5–7 week old female NOD/Shi-scid/IL-2Rγnull (NOG) mice via subcutaneous (s.c) injection. After confirmation of a bioluminescent signal, the mice were assigned to the vehicle control group or one of the treatment groups: R (2 mg/kg on day1, 8 and 15, ip); P (15mg/kg on day1, 4, 8 and 11, ip); combination [R (2 mg/kg on day1, 8 and 15, ip) plus P (15mg/kg on day1, 4, 8 and 11, ip)]. In vivo bioluminescence imaging was performed twice weekly for 4 weeks from the start of treatment under standardized conditions. In parallel a second xenograft tumor experiment was conducted where untransfected H9 cells were implanted into the flank of 5–7 week old female NOG mice. Tumor-bearing mice were imaged on US and after they achieved a mean diameter ranging between 3–4 mm, mice were assigned to similar control and treatment groups as described. US imaging was performed twice weekly for 4 weeks after the start of treatment to measure tumor burden using 3D volumetric analysis. After 3 weeks of treatment tumors were harvested for immunostaining and western blot analysis from all mice. Conventional tumor volume measurements with manual calipers were also collected for comparison in this experiment. Imaging- and caliper-derived volumes were compared with the direct tumor mass, determined after resection of final tumors.
Sequential surface BLI outputs (photons/s) from s.c xenografts over the 4-week period were significantly lower in the mice treated with R and P compared with the control group and single agent groups suggesting potential synergistic effect in low tumor disease state. Moreover, all the mice that received the combination treatment remained in complete remission even after completion of treatment highlighting durability and depth of response. Mice treated with combination drugs exhibited minimal toxicity as demonstrated by <10% weight loss. 3D tumor volumetric measurements with US confirmed the BLI findings highlighting the synergy even in states of higher tumor burden. The average deviation from the true volume and inter-observer differences were significantly lower for ultrasound volumes compared with caliper volumes. Immunohistochemical studies revealed higher BrdU incorporation in control, R and P treated mice compared with mice that received both R and P. TUNEL assay demonstrated greater percentage of apoptotic cells in mice that received combination drugs compared with control, R treated and P treated mice.
Our complimentary imaging modalities (US and BLI) confirm synergy of R with P in xenograft mouse models of T-cell lymphoma across a spectrum of tumor volumes thereby providing a platform for translation to early phase clinical trial in this challenging disease. Ultrasound imaging more accurately, precisely, and reproducibly reflects xenograft tumor volume compared with caliper measurements. Surface BLI demonstrated high correlations with accurate 3D US volumetric measurements albeit at small tumor volumes, suggesting its usefulness in tracking early tumor response quantitatively in drug intervention studies. A complementary imaging modality, like US, is recommended to monitor tumor burden and response to novel compounds in advanced stages. Ongoing co-relative pharmacokinetic and molecular studies will elucidate the mechanistic basis for synergy between R and P.
Amengual:Acetylon Pharmaceuticals, Inc: Research Funding. O'Connor:Allos Pharmaceuticals, Inc: Consultancy; TG Therapeutics, Inc: Consultancy; Millenium: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees.
Asterisk with author names denotes non-ASH members.