Physical activity modulates macrophage phenotype in mammary tissue and inhibits tumor progression

We generated RNAseq results from the mammary tissues of mice from a 2 x 2 factorial design experiment (running wheels vs. control condition X clodronate liposomes vs. PBS liposomes) where EO771 mammary cancer cells had been engrafted in the mammary tissue for nascent tumor formation. RNAseq results were used to complete digital cytometry with the CIBERSORTx platform to analyze relative proportions of M1-like and M2-like macrophages in the mammary tissue. Overall design: Total RNA was purified from mouse mammary tissues with engrafted EO771 cells from a 2 x 2 factorial design experiment: running wheels vs. control condition X clodronate liposomes vs. PBS liposomes. Control+PBS (n=6), Wheel+PBS (n=6), Control+Clodronate (n=5), Wheel+Clodronate (n=5). Mice used were 7-8 week-old female C57BL/6J mice (Jackson Laboratory) and were housed on an individually ventilated cage (IVC) rack in dual filter disposable cages (Innovive, Inc.), with corn cob bedding, nesting tissue, and ad libitum access to food and water on a 12:12 light:dark cycle at 22oC. Mice were given access to an angled running wheel-hub apparatus (Fast-Trac Mouse Igloo®, Bio-Serve, Flemington, NJ) in each home cage for two weeks or only to a hub if control mice. At the conclusion of the two-week period, the left 4th mammary fat pad was engrafted with 1 × 10^5 cells of the syngeneic mammary cancer cell line, EO771 (CH3 Biosystems, LLC), which had been transduced to express firefly luciferase. Mice were also injected intravenously with liposomes (Liposoma BV) at both 24 hours before and again at the time of tumor engraftment. Mice continued to have wheel access for another 24 hours after engraftment before euthanasia for mammary tissue collection. Each left 4th mammary tissue was dissected and lysed so that total RNA could be extracted (Qiagen RNeasy Mini Kit, #74104), cleared of contaminating DNA with on-column DNase digestion (Qiagen RNase-Free DNase Set, #79254), and quantified by spectrophotometry (NanoDrop ND-1000, Thermo Scientific). Total RNA was converted to cDNA (Lexogen QuantSeq 3'FWD), sequenced using an Illumina HiSeq 4000 instrument in the UCLA Neuroscience Genomics Core Laboratory, and low-level sequencing data was mapped to the RefSeq mouse genome sequence and normalized to counts per million mapped reads (CPM). All procedures were carried out under protocols approved by the Institutional Animal Use and Care Committee of the University of California, Los Angeles.