Bulk RNAseq of K7M2 osteosarcoma cells in engineered bone marrow treated with Doxorubicin

Osteosarcoma (OS) is the most common primary malignant bone cancer in children and adolescents. While numerous other cancers now have promising therapeutic advances with immunotherapies, targeted therapies, and stem cells, treatment options for patients with OS have remained unchanged for more than four decades since the advent of standard chemotherapeutics and offer less than a 25% 5-year survival rate for those with metastatic disease. This dearth of clinical progress underscores a lack of understanding of OS progression and metastagenesis and necessitates the study of this disease in a novel system. Here, we adapt a previously described engineered bone marrow (eBM) construct for use as a 3D platform to study how microenvironmental and immune factors affect OS tumor progression. We form eBM by implanting acellular bone-forming materials in mice and explanting the cellularized constructs for study after eight weeks. We interrogate the influence of the anatomical implantation site on the quality of generated tissue, test ex vivo stability under normoxic (5% O2) and standard (21% O2) culture conditions, and culture OS cells within these eBM constructs. We show that eBM accurately and stably recapitulates the composition of native bone marrow. OS cells exhibit differential behavior dependent on metastatic potential when cultured in eBM, thus closely mimicking in vivo conditions. Furthermore, we highlight the clinical applicability of eBM as a drug-screening platform through doxorubicin treatment of OS cultured in monolayer, Matrigel, or eBM, and we show that eBM confers a protective effect on OS cells that parallel clinical responses. Combined, this work presents eBM as a cellular construct that mimics the complex bone marrow environment that may be useful for mechanistic bone cancer research and drug screening.

Methods
eBM was minced into <= 0.5mm cubes and collected in 500 micro liter of TRIzol Reagent (Invitrogen). Samples were then homogenized with a Tissue-tearor (BioSpec, Bartlesville, OK) at highest speed for 10 s intervals totaling 30 s, followed by sonication for another 10 s. Following RNA isolation, sequencing libraries were prepared using Illumina Stranded mRNA Prep kit using manufacturer's recommendations and then sequenced on an Illumina NextSeq 500 sequencer. Conversion of Illumina base calls to sequences and sample demultiplexing was performed using bcl2fastq.