Orthotopic model of cervical cancer in NSG mice

Six-week-old female NSG mice (Cancer Research Animal Core, UTHSC, Memphis, TN) were used to generate an orthotopic model of cervical cancer. The mice were maintained in a pathogen-free environment and all procedures were carried out as approved by the UTHSC Institutional Animal Care and Use Committee (UTHSC-IACUC). All the procedures and methods were carried out in “accordance” with the approved guidelines of UTHSC-IACUC. Briefly, Caski cells (4 × 10⁶ cells/per mouse) were dispersed in 100 μL 1X PBS and 100 μL Matrigel (BD Biosciences) and injected directly into the cervix of the mice. The animals were periodically monitored for tumor development and the tumor volume was measured from day 5 after injection using a digital Vernier caliper. When the tumor volume reached ~200 mm³, CUR, Nano-CUR (100 μg/mice) and their respective vehicle controls (PBST and PLGA NPs, respectively) were injected intra-tumorally. The tumor volume was calculated using the ellipsoid volume formula: tumor volume (mm³) = π/6 × L × W × H, wherein L is length, W is width, and H is height. The tumor was regularly monitored and allowed to grow until the tumor burden reached a maximum volume of 1100 mm³. At the time of sacrifice, the mice tumors were removed, fixed in formalin, embedded in paraffin, and sliced into 5 micron sections for further processing and analysis.