Figure 5.

Targeting CX3CR1 in a model of established metastases and transcriptome analyses of metastatic tumors. A, CX3CR1 was conditionally silenced by an inducible-CRISPRi system. Complete protein repression was achieved 72 hours after exposing cells to 2μg/ml of doxycycline. B, Mice were inoculated via the left cardiac ventricle with MDA-231-TRE-CRISPRi_CX3CR1 cells, stably expressing both GFP and luciferase. Animals were left untreated for one week after the inoculation of cancer cells; then, a doxycycline-containing diet was administered to the treatment group for the next following 3 weeks before euthanasia. Tumor progression was monitored weekly by in vivo bioluminescence imaging. Mice in both control and treatment groups imaged at 4 weeks are shown. C, Quantification of the number of metastatic lesions and overall tumor burden based on the detection of bioluminescent signal in both control and CRISPRi groups. D, Mice bearing one-week tumors in bones and soft-tissues were treated daily with JMS-17-2 (10mg/Kg - i.p. twice a day) for the next following 3 weeks before euthanasia. Control animals received only vehicle. E, Quantification of number of metastatic lesions and overall tumor burden based on bioluminescent signal in both control and JMS-17-2 treated groups. F, Two representative images of a mouse tissue section containing a bone tumor, identified by the emitted green fluorescence, before (left) and after (right) harvesting by LCM. G, Tumor tissues from four animals for each experimental groups (Control, JMS-17-2 and CRISPRi) were harvested and pooled for RNA isolation; of the 730 human genes with demonstrated implication in tumorigenesis and progression included in the nCounter PanCancer panel, approximately 330 genes were significantly altered in tumors collected from JMS-17-2 treated and CX3CR1-silenced tumors as compared to tumors in control animals. Of these genes, 46 for the JMS-17-2 group and 35 from the CX3CR1-silenced group showed at least 3-fold change. Nine of these genes were found to be affected in a similar fashion between the two experimental groups. Statistical significance was established by using the t-test module included in the nSolver software.