The cancer-prone peripheral zone of the human prostate is a fibromuscular stroma bounded by a laminin-rich smooth muscle capsule. Aggressive tumors traverse the muscle to escape organ confinement, but the determinants of cell migration through this complex and highly elastic environment are not well understood. We modeled smooth muscle invasion using a unique in vivo model to test the role of mechanosensing, laminin-binding integrins (α6β1 and α3β1), collect cells successful in traversing smooth muscle, and identify transcriptomes involved in this complex invasive process.
From the DU145 prostate cancer cell line (expressing integrins α3 and α6), we generated homozygous knockouts for genes ITGA6 (α6KO) and ITGA3 (α3KO). We tested for the ability to create 3D invasive cellular networks and measured biophysical features by electric cell-substrate impedance sensing. Muscle invasion was tested in vivo by injecting cells into the intraperitoneal cavity of male NSG mice. The cells colonized the inferior side of the respiratory diaphragm and invaded through to the superior surface after 6 weeks. We analyzed diaphragms by number of invasion sites and average invasion depth. A series of clones were also grown from wild-type, DU145 cells that had completely traversed the diaphragm muscle.
Cells expressing integrins α6 and α3 produced 3D invasive networks on laminin-containing Matrigel and invaded into the diaphragm muscle in vivo. α6KO cells did not produce networks or invade muscle. Surprisingly, knockout of α3 increased 3D invasive networks, decreased cell-cell impedance, and altered in vivo muscle invasion. The results suggest that the integrins α6 and α3 are involved in the invasion of prostate cancer cells through a dynamic smooth muscle layer.
The mechanosensing integrins are central to a variety of cell signaling programs. To further explore the transcriptional regulators of the muscle invasion process, we isolated RNA from cancer cells colonizing the inferior, superior surface and the intact diaphragm. Whole genome RNAseq revealed 1330 differentially expressed genes (DEGs) between inferior cells and cancer cells within the diaphragm, 788 DEGs between cells on the inferior and superior diaphragm surface, and 241 DEGs between cancer cells within the diaphragm and cells that reached the superior surface (padj <0.05, |fold change| >=1.3). Ingenuity Pathway Analysis revealed alterations in pathways including prostate cancer signaling, integrin signaling, other known cancer-related processes, and others.
Through this in vivo assay we have the unique ability to parse out unexplored patterns of changes that occur in early muscle invasive disease to improve prostate cancer patient selection for curative treatment versus active surveillance.
These studies were supported by NCI F30CA247106, P30CA23074, and R01CA24226. K.D.M is additionally supported by the ARCS Foundation Phoenix Chapter.
Citation Format: Kendra D. Marr, Hina Arif-Tiwari, Jaime M. Gard, Noel E. Warfel, Raymond B. Nagle, Anne E. Cress. Cancer cell invasion through a complex and dynamic smooth muscle microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB233.