For most cancers, metastasis is the point at which clinical treatment shifts from curative intent to extending survival. Biomaterial implants acting as a synthetic premetastatic niche recruit metastatic cancer cells and provide a survival advantage, and their use as a diagnostic platform requires assessing their relevance to disease progression. Here, we showed that scaffold-captured tumor cells (SCAF) were 30 times more metastatic to the lung than primary tumor (PT) cells, similar to cells derived from lung micrometastases (LUNG). SCAF cells were more aggressive in vitro, demonstrated higher levels of migration, invasion, and mammosphere formation, and had a greater proportion of cancer stem cells than PT. SCAF cells were highly enriched for gene expression signatures associated with metastasis and had associated genomic structural changes, including globally enhanced entropy. Collectively, our findings demonstrate that SCAF cells are distinct from PT and more closely resemble LUNG, indicating that tumor cells retrieved from scaffolds are reflective of cells at metastatic sites.

<div>Abstract<p>For most cancers, metastasis is the point at which clinical treatment shifts from curative intent to extending survival. Biomaterial implants acting as a synthetic premetastatic niche recruit metastatic cancer cells and provide a survival advantage, and their use as a diagnostic platform requires assessing their relevance to disease progression. Here, we showed that scaffold-captured tumor cells (SCAF) were 30 times more metastatic to the lung than primary tumor (PT) cells, similar to cells derived from lung micrometastases (LUNG). SCAF cells were more aggressive <i>in vitro</i>, demonstrated higher levels of migration, invasion, and mammosphere formation, and had a greater proportion of cancer stem cells than PT. SCAF cells were highly enriched for gene expression signatures associated with metastasis and had associated genomic structural changes, including globally enhanced entropy. Collectively, our findings demonstrate that SCAF cells are distinct from PT and more closely resemble LUNG, indicating that tumor cells retrieved from scaffolds are reflective of cells at metastatic sites.</p>Significance:<p>These findings suggest that metastatic tumor cells captured by a biomaterial scaffold may serve as a diagnostic for molecular staging of metastasis.</p></div>

<p>1. Fig S1: Gating strategy for flow cytometric assays 2. Fig S2: Metastatic lung tumor burden for LUNG and LUNG2 lines developed from mice with and without scaffolds 3. Fig S3: Mutational analysis of cell lines by whole exome sequencing 4. Table S1: Table of unique mutations identified for each cell line by whole exome sequencing 5. Table S2: Table of Gene ontology terms significantly altered in genes with mutations in SCAF, PT and LUNG cell lines 6. Fig S4: RNAseq supplemental information 7. Fig S5: Schematic of multiscale Hi-C analysis 8. Fig S6: Hi-C does not reveal additional copy number variations, translocations, or breakpoint mutations between SCAF and PT cell line. 9. Table S3: Table of KEGG pathways investigated for structure/function changes</p>

<p>1. Fig S1: Gating strategy for flow cytometric assays 2. Fig S2: Metastatic lung tumor burden for LUNG and LUNG2 lines developed from mice with and without scaffolds 3. Fig S3: Mutational analysis of cell lines by whole exome sequencing 4. Table S1: Table of unique mutations identified for each cell line by whole exome sequencing 5. Table S2: Table of Gene ontology terms significantly altered in genes with mutations in SCAF, PT and LUNG cell lines 6. Fig S4: RNAseq supplemental information 7. Fig S5: Schematic of multiscale Hi-C analysis 8. Fig S6: Hi-C does not reveal additional copy number variations, translocations, or breakpoint mutations between SCAF and PT cell line. 9. Table S3: Table of KEGG pathways investigated for structure/function changes</p>

Objective: For most cancers, the formation of distant metastasis is the point at which clinical treatment shifts from curative intent to palliative care. At present, there is no clinical method to detect metastatic dissemination and colonization until radiologically evident, at which point organ function has already been compromised. The Shea laboratory has developed a biomaterial implant that acts as a synthetic pre-metastatic niche and recruits metastatic cancer cells in xenogeneic human and syngeneic mouse models of breast cancer. Scaffold implantation has facilitated detection of metastasis prior to colonization of organs and has been shown to reduce metastatic burden, resulting in enhanced survival with surgical intervention. Methods: Triple-negative breast cancer models were used in which tdTomato+ 4T1 or MDA-MB-231-BR cells were orthotopically inoculated into balb/c or NSG mice. Microporous polycaprolactone (PCL) scaffolds were fabricated using gas-foaming and poragen leaching and were subcutaneously implanted one month prior to tumor inoculation. High frequency ultrasound (US) was used to probe the scaffold for early detection of tumor cells prior to metastasis to organs. Human tumor cells were isolated from the scaffold (SCAF), primary tumor (PT), and a bone metastasis via MACS mouse cell depletion (Miltenyi) and grown into stable cell lines in vitro. Scratch, migration, invasion, mammosphere, cancer stem cell markers, RNA-seq, qRT-PCR, and HiC assays were performed to investigate behavioral differences between cell lines in vitro. Additionally, cell lines were inoculated into mice and investigated for metastatic ability. Results: Initial studies applied US for detection of metastatic cell arrival at the scaffold. The analysis of spectral imaging distinguished scaffolds from tumor bearing relative to tumor free mice. A cell line created from the scaffold (SCAF) was more aggressive in vitro, demonstrating higher levels of migration, invasion, mammosphere formation, and proportion of cancer stem cells compared to PT. SCAF cells were also found to be ~30x more metastatic to the lung in vivo compared to PT cells (SCAF 10,150±7792 PT 354±296 cells per lung). RNA-seq identified 14232 genes with measured expression, 2901 of which were differentially expressed (p&lt;0.05 and log fold change &gt; 0.6) between scaffold and primary tumor cells. Conclusions: Biomaterial scaffolds capable of recruiting metastatic tumor cells in vivo can serve as a platform for early detection and intervention, and also provide a tool to study metastasis in vivo and the properties of the early metastatic cells. Citation Format: Grace G. Bushnell, Tejaswini P. Hardas, Rachel M. Hartfield, Yining Zhang, Robert S. Oakes, Adeline Hong, Jacqueline S. Jeruss, Lonnie D. Shea. Biomaterial scaffolds that capture metastatic tumor cells in vivo to detect, treat, and study mechanisms of the premetastatic niche and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5000.