Global transcriptional analysis of different HT1080 cell phenotypes induced by cell proliferation and density. Homo sapiens

Following uncontrolled proliferation, a subset of primary tumor cells acquires additional traits/mutations to trigger phenotypic changes that enhance migration and are hypothesized to be the initiations of metastasis. This study reveals a novel adaptive mechanism that harnesses synergistic paracrine signaling via IL-6/8, which is amplified by cell proliferation and cell density, to directly promote cell migration. This effect occurs in metastatic human sarcoma and carcinoma cells - but not in non-metastatic cells-, through the downstream signaling of WASF3 and Arp2/3. Our RNA-seq based global transcriptional analysis showed that the transcriptional phenotype of high-density cells that emerges due to proliferation resembles that of low-density cells treated with a combination of Il-6/8. Simultaneous inhibition of IL-6/8 receptors significantly decreases the expression of WASF3 and Arp2/3 in a mouse xenograft model and consequently reduces metastasis. Overall design: Population-level RNA sequencing on five different HT1080 cell phenotypes induced by varying microenvironment. HT1080 cells were embedded in type I 3D collagen matrices in increasing cell densitieis from 10 cells/mm3 to 150 cells/mm3. RNA-seq performed on matrix embedded cells with cell densities of 10 cells/mm3 (low-density) and 50 cells/mm3 (high-density). Recombinant IL-6, IL-8 (both R&D systems), or both reconstituted in DPBS were added to matrix embedded cells with a cell density of 10 cells/mm3 (low-density with IL-6/IL-8/IL6+IL8). One collagen matrices without cells was sequenced and analyzed as a negative control. All data is obtained from single sequencing experiment. RNA-seq performed on these cell populations. All populations were incubated for 24h at 37°C, 5% CO2. The collagen matrices were exposed to cell lysis buffer and mechanically broken down using a syringe. The lysates of matrices were centrifuged and the supernatant was separately collected. Total RNA isolation was performed with RNA MiniPrep Kit (Zymo Research). Since the cells were embedded in the collagen matrices at the low cell density, the RNA-seq protocol (modified from Pan et al PNAS 2013) was implemented to capture uniformly distributed, full-length coverage sequences from low-quality and low-quantity cells. The cDNA libraries were sequenced using 50 bp single-ended reads. Data was processed using Bowtie2 for read alignment and RSEM for quantification of gene expression.