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RT-qPCR data including: Raw Non-Normalized Cq, Reference Gene Normalized Î"Cq. Format similar to NCBI GEO template with ID_REF, Gene Symbol, Reference/Target use, Catalog Number, Assay Type, Gene Name, Alias, RefSeq Accession Number, GenBank mRNA Numbers, Target Species, Amplicon Length, and Platform.

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.Significance:These findings suggest that metastatic tumor cells captured by a biomaterial scaffold may serve as a diagnostic for molecular staging of metastasis.

NCBI GEO formatted submission for OpenArray{trade mark, serif} RT-qPCR data including: ID_REF, Gene Symbol, Reference/Target use, Catalog Number, Assay Type, Gene Name, Alias, RefSeq Accession Number, GenBank mRNA Numbers, Target Species, Amplicon Length, Platform, Raw Non-Normalized Cq, Reference Gene Normalized Î"Cq and Î"Î"Cq Fold Changes.

Kaplan-Meier survival curves correlated with high and low gene expression from breast cancer patient samples. (a) Microarray (GEO via KMPlot) and prognosis data were queried for genes selected for the 10-gene panel derived from tissue within implants to determine the expression level relevance in primary tumors on the clinical outcome of systemically untreated breast cancer patients (n=818). Kaplan-Meier plots indicate as separation of two profiles separating high and low gene expression that are automatically divided by the median expression of the genes for all samples. Plots indicate the Hazard Ratio (HR) which is highest for S100a9 and logrank significance comparing high and low expression as a function of recurrence-free survival. FDR 10% corrected significance (p

Additional scRNA-seq plots of the gene panel analyzed in Fig. 3. (a) Heatmap of fold-change between scaffolds in tumor-bearing mice and tumor-free controls. Data is analogous to that presented in Fig. 3c and Fig. S7b violin plots. (b) Violin plots of single-cell gene expression from TFC and TB mice including B cells, dendritic cells (DCs), macrophages, monocytes, neutrophils, natural killer (NK) cells, and T cells. Each dot represents the gene expression (x-axis) of a single cell within a cell type and health state (TB or TFC, y-axis). Bmp15 is absent as it was undetectable by scRNA-seq.

Gating scheme for flow cytometry analysis of adoptive transferred cells. Supporting Fig. 2 these plots indicate the (a) cells and (b) singlet gating prior to (c) gating for DiO and DiD fluorescent lipophilic dyes. Axes in panel C are identical to those in panels D-F. Numbers on plots indicate percentage of gated cells in comparison to a) total events, b) gated cells, and c) single cells. Representative flow cytometry plots and gating for (d) PBS injected control mice and adoptively transferred Ly6G+ and Gr1+Ly6G- populations into (e) TFC and (f) TB mice.

Scaffolds from (a) left and (b) right side of a mouse inoculated with parental E0771 which developed a high metastatic burden which metastases present in the (c) brain and (d) lungs. Metastatic lines Br.1 and Lu.1 were derived from the tumor cells from these brain and lung metastases, respectively. Serial inoculation via intracardiac injection and isolation of developed metastases (e) indicated a propensity for the lung derived metastases have an organotropic bias toward lung tissue. (f) Confirmation of fluorescent tumor cells in the scaffolds from a-b was verified by fluorescent microscopy of scaffold tissue sections. (g) In vivo imaging of Br.1 and Lu.1 line 11 days following intracardiac inoculation.

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

Monitoring metastatic events in distal tissues is challenged by their sporadic occurrence in obscure and inaccessible locations within these vital organs. A synthetic biomaterial scaffold can function as a synthetic metastatic niche to reveal the nature of these distal sites. These implanted scaffolds promote tissue ingrowth, which upon cancer initiation is transformed into a metastatic niche that captures aggressive circulating tumor cells. We hypothesized that immune cell phenotypes at synthetic niches reflect the immunosuppressive conditioning within a host that contributes to metastatic cell recruitment and can identify disease progression and response to therapy. We analyzed the expression of 632 immune-centric genes in tissue biopsied from implants at weekly intervals following inoculation. Specific immune populations within implants were then analyzed by single-cell RNA-seq. Dynamic gene expression profiles in innate cells, such as myeloid-derived suppressor cells, macrophages, and dendritic cells, suggest the development of an immunosuppressive microenvironment. These dynamics in immune phenotypes at implants was analogous to that in the diseased lung and had distinct dynamics compared with blood leukocytes. Following a therapeutic excision of the primary tumor, longitudinal tracking of immune phenotypes at the implant in individual mice showed an initial response to therapy, which over time differentiated recurrence versus survival. Collectively, the microenvironment at the synthetic niche acts as a sentinel by reflecting both progression and regression of disease.Significance:Immune dynamics at biomaterial implants, functioning as a synthetic metastatic niche, provides unique information that correlates with disease progression.

Experimental timeline, gene expression analysis, singular value decomposition, and bagged decision tree analysis and computational pipelines. Map of computational processes and the data outputs that are represented in the manuscript. For example, the calculation of the Euclidean distance from the principle components of the singular value decomposition (SVD) represented in Fig. 4b can be traced back through its processing steps to the total RNA isolation, quality control, and cDNA synthesis.

Implant microenvironment exhibits good tissue ingrowth and facilitates surgical and core-needle biopsies to acquire RNA for transcriptomic or gene expression analysis. (a) A surgically-biopsied implant illustrating the intact, frozen condition prior to RNA isolation and RT-qPCR assessment. (b) A sample derived from a scaffold using a core needle biopsy (CNB, Bard® Mission® Disposable Core Biopsy Instrument) which enables minimally invasive retrieval of samples, similar to clinical approaches for sampling suspicious tissue. In both samples tissue ingrowth into the microporous structure is evident but especially in the CNB as the cutting plane transected the inner core of the implant. Scaffolds as shown are in -80{degree sign}C frozen state. Diameter of the black background circle is 7.5 mm. (c) Comparing the RNA isolated from the full surgically biopsied scaffold compared to the CNB sample, there is a significant -1.812 log-transformed fold change, but sufficient material for numerous gene expression assessments (RT-qPCR, RNAseq, etc.) with the CNB samples containing an average of 4376 ng of total RNA. * indicates differences between surgically biopsied and CNB samples from an unpaired t-test (p

Development of the pre-metastatic niche (preMN), metastatic niche (MN), and synthetic diagnostic site. Circulating tumor cells exhibit a tropism for specific distal microenvironments, thus indicating that metastasis is pre-determined. Development of these predetermined sites is driven by (1) systemic conditioning from the primary tumor through secretion of factors and exosomes, which is amplified by simultaneous conditioning of the bone-marrow and alterations in bone-marrow derived cells. (2) This process conditions distal tissues and contributes to the (3) aggregation of pre-metastatic immune cells and extracellular matrix proteins. (4) These primed distal microenvironments are denoted as the preMN and facilitate homing then colonization of (5) circulating tumor cells which (6) extravasate and (7) migrate into distal organs where they may become a (8) disseminated tumor cells if the conditions are hospitable. This disseminated tumor cell may then undergo a phenotypic shift to initiate a (9) metastatic secondary growth, thus leading to development of a metastatic niche. Given that the preMN contains a unique mixture of soluble factors, extracellular matrix, stromal and immune cells (e.g., cancer-associated fibroblasts and myeloid-derived suppressor cells, MDSCs), it is reasonable that preMN function could be synthesized through the recapitulation these unique factors at a synthetic site in vivo. This rationale underpins the development of material based synthetic diagnostic site that (2.1) is conditioned, (3.1) populated by disease associated immune cells, (4.1) develops characteristics of a preMN, and (7.1) captures metastatic tumor cells.

Statistical plots to support heatmaps in main text and supplementary materials. (a) Box plots for gene panel heatmap in Fig. 1f show the median, 25th-75th percentiles and most extreme data points that were not considered outliers (outliers are visually indicated by red +). TB cohort expression is centered relative to the time-matched TFC cohort median. Scale depicts the log2-transformed fold change. A two-way MANOVA showed a significant interaction effect between condition and time (Pillai's Trace=1.276, F(20,28)=2.465, p=0.014). Post hoc univariate ANOVA showed significant differences within the diseased cohort over time (indicated by #, df=(2,22), p

Annotated Matlab output for OpenArray{trade mark, serif} and CFX gene expression analysis and signature development via singular value decomposition and bagged decision tree ensemble including raw figures from manuscript to correlate with associated code.

Additional data from post-excision model demonstrating the cohort survival curve and gene expression trajectories. (a) Kaplan-Meier survival analysis comparing TFC and TB mice that received mammary gland excisions. (b-k) Time course analysis for 10 gene panel. Error bars in line plots for gene expression and signature trajectory indicate SEM. The cohort size for each group is decreased by one at Day 21 due recurrence and animal censorship. For longitudinal data, statistics were performed via a linear mixed model. Post hoc simple effects analysis indicates significant differences (p

Supplementary Materials and Methods, Figures, and Descriptions of Supplementary Data

Monitoring metastatic events in distal tissues is challenged by their sporadic occurrence in obscure and inaccessible locations within these vital organs. A synthetic biomaterial scaffold can function as a synthetic metastatic niche to reveal the nature of these distal sites. These implanted scaffolds promote tissue ingrowth, which upon cancer initiation is transformed into a metastatic niche that captures aggressive circulating tumor cells. We hypothesized that immune cell phenotypes at synthetic niches reflect the immunosuppressive conditioning within a host that contributes to metastatic cell recruitment and can identify disease progression and response to therapy. We analyzed the expression of 632 immune-centric genes in tissue biopsied from implants at weekly intervals following inoculation. Specific immune populations within implants were then analyzed by single-cell RNA-seq. Dynamic gene expression profiles in innate cells, such as myeloid-derived suppressor cells, macrophages, and dendritic cells, suggest the development of an immunosuppressive microenvironment. These dynamics in immune phenotypes at implants was analogous to that in the diseased lung and had distinct dynamics compared with blood leukocytes. Following a therapeutic excision of the primary tumor, longitudinal tracking of immune phenotypes at the implant in individual mice showed an initial response to therapy, which over time differentiated recurrence versus survival. Collectively, the microenvironment at the synthetic niche acts as a sentinel by reflecting both progression and regression of disease. Significance: Immune dynamics at biomaterial implants, functioning as a synthetic metastatic niche, provides unique information that correlates with disease progression.

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. Significance: These findings suggest that metastatic tumor cells captured by a biomaterial scaffold may serve as a diagnostic for molecular staging of metastasis.

Timely assessment of patient-specific prognosis is critical to oncology care involving a shared decision-making approach, but clinical prognostic factors traditionally used in NSCLC have limitations. We examine a proteomic test to address these limitations.This study examines the prognostic performance of the VeriStrat blood-based proteomic test that measures the inflammatory disease state of patients with advanced NSCLC. A systematic literature review (SLR) was performed, yielding cohorts in which the hazard ratio (HR) was reported for overall survival (OS) of patients with VeriStrat Poor (VSPoor) test results versus VeriStrat Good (VSGood). A study-level meta-analysis of OS HRs was performed in subgroups defined by lines of therapy and treatment regimens.Twenty-four cohorts met SLR criteria. Meta-analyses in five subgroups (first-line platinum-based chemotherapy, second-line single-agent chemotherapy, first-line EGFR-tyrosine kinase inhibitor (TKI) therapy, and second- and higher-line TKI therapy, and best supportive care) resulted in statistically significant (Advanced NSCLC patients classified VSGood have significantly longer OS than those classified VSPoor. The summary effect size for OS HRs around 0.4-0.5 indicates that the expected median survival of those with a VSGood classification is approximately 2-2.5 times as long as those with VSPoor. The robust prognostic performance of the VeriStrat test across various lines of therapy and treatment regimens has clinical implications for treatment shared decision-making and potential for novel treatment strategies.

Engineered cell-based therapies comprise a promising, emerging biomedical technology. Broad utilization of this strategy will require new approaches for implementing sophisticated functional programs, such as sensing and responding to the environment in a defined fashion. Towards this goal, we investigated whether our self-contained receptor and signal transduction system (MESA) could be multiplexed to evaluate extracellular cues, with a focus on elucidating principles governing the integration of such engineered components. We first developed a set of hybrid promoters that exhibited AND gate activation by two transcription factors. We then evaluated these promoters when paired with two MESA receptors and various ligand combinations. Unexpectedly, although the multiplexed system exhibited distinct responses to ligands applied individually and in combination, the same synergy was not observed as when promoters were characterized with soluble transcription factors. Therefore, we developed a mechanistic computational model leveraging these observations, to both improve our understanding of how the receptors and promoters interface and to guide the design and implementation of future systems. Notably, the model explicitly accounts for the impact of intercellular variation on system characterization and performance. Model analysis identified key factors that affect the current receptors and promoters, and enabled an in silico exploration of potential modifications that inform the design of improved logic gates and their robustness to intercellular variation. Ultimately, this quantitative design-driven approach may guide the use and multiplexing of synthetic receptors for diverse custom biological functions beyond the case study considered here.

For most cancers, metastasis is the point at which disease is no longer curable. Earlier detection of metastasis, when it is undetectable by current clinical methods, may enable better outcomes. We have developed a biomaterial implant that recruits metastatic cancer cells in mouse models of breast cancer. Here, we investigate spectral ultrasound imaging (SUSI) as a non-invasive strategy for detecting metastasis to the implanted biomaterial scaffolds. Our results show that SUSI, which detects parameters related to tissue composition and structure, identified changes at an early time point when tumor cells were recruited to scaffolds in orthotopic breast cancer mouse models. These changes were not associated with acellular components in the scaffolds but were reflected in the cellular composition in the scaffold microenvironment, including an increase in CD31 + CD45-endothelial cell number in tumor bearing mice. In addition, we built a classification model based on changes in SUSI parameters from scaffold measurements to stratify tumor free and tumor bearing status. Combination of a linear discriminant analysis and bagged decision trees model resulted in an area under the curve of 0.92 for receiver operating characteristics analysis. With the potential for early non-invasive detection, SUSI could facilitate clinical translation of the scaffolds for monitoring metastatic disease.

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 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.

Background: Immune cells at the primary tumor (PT) and pre-metastatic niche (PMN) sites are critical to metastasis progression. Recently, synthetic biomaterial scaffolds used as PMN mimics were shown to capture both immune and metastatic tumor cells in vivo (1-3). The redirection of tumor cells toward the implant also reduces tumor burden and provides a survival benefit in orthotopic breast cancer mouse models (4). Given the scaffold reduces tumor burden, we hypothesized the scaffold modulates PT and immune cell phenotypes to generate an invasion-suppressive tumor microenvironment (TME). Methods: Female NOD scid gamma (NSG) mice inoculated with tdTomato+ MDA-MB-231BR cells in the mammary fat were used as orthotopic human breast cancer models. Poly(lactide-co-glycolide) scaffolds were implanted in the intraperitoneal fat pad 7 days post-tumor inoculation (3). Mock surgery mice did not receive an implant. After 28 days, mock vs. scaffold PTs were harvested, digested, and live-cell sorted to obtain tdTomato+ tumor cells and CD45+ immune cells. The PT cell transcriptome was analyzed using RNAseq and GO analysis was performed in Metascape. CD45+ immune cells were cultured to obtain conditioned media (CM) for secretomics analysis, in vitro tumor cell invasion assays, and transcription factor activity arrays (2). Immune cells, specifically tumor associated macrophages (TAMs) (5), were characterized using flow cytometry and RT-PCR. Results: RNAseq analysis identified 892 differentially expressed genes in tumor cells in response to the scaffold implant, and Metascape GO analysis revealed signaling pathways relevant to invasion. The scaffold immune-CM decreased tumor cell invasion more than two-fold relative to mock CM. Secretomics analysis showed an increase in the pan-metastasis inhibitor decorin and a decrease in invasion-promoting CCL2 in the scaffold immune cell CM relative to mock CM. Reduced NFkB, SRF, and RAR transcription factor activity in tumor cells treated with scaffold CM relative to mock CM indicate immune secreted factors contribute to the invasion-suppressive scaffold-influenced TME. Finally, analysis of PT immune cells identified a recruited TAM population whose transcriptomic profile may contribute to the invasion-suppressive TME in scaffold bearing mice. Discussion: We demonstrate that implanted scaffolds can distally modulate secretomic and transcriptional profiles of immune cells in the TME and may reduce PT cell invasion. Our work suggests scaffolds have an active role in modulating tumor burden and may provide a foundation for developing an effective implantable therapeutic tool. References: (1) Aguado et al, Acta Biomater 33, 13-24, 2016. (2) Aguado et al, Sci Rep 5, 17566, 2015. (3) Azarin et al, Nat Commun 6, 8094, 2015. (4) Rao et al, Cancer Res 76, 5209-5218, 2016. (5) Franklin et al, Science 344, 921-925, 2014. Citation Format: Brian A. Aguado, Rachel M. Hartfield, Grace G. Bushnell, Joseph T. Decker, Samira M. Azarin, Dhaval Nanavati, Matthew J. Schipma, Shreyas S. Rao, Robert S. Oakes, Yining Zhang, Jacqueline S. Jeruss, Lonnie D. Shea. A synthetic pre-metastatic niche mimic alters the primary tumor and tumor microenvironment [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 1106.

Primary tumor (PT) immune cells and pre-metastatic niche (PMN) sites are critical to metastasis. Recently, synthetic biomaterial scaffolds used as PMN mimics are shown to capture both immune and metastatic tumor cells. Herein, studies are performed to investigate whether the scaffold-mediated redirection of immune and tumor cells would alter the primary tumor microenvironment (TME). Transcriptomic analysis of PT cells from scaffold-implanted and mock-surgery mice identifies differentially regulated pathways relevant to invasion and metastasis progression. Transcriptomic differences are hypothesized to result from scaffold-mediated modulations of immune cell trafficking and phenotype in the TME. Culturing tumor cells with conditioned media generated from PT immune cells of scaffold-implanted mice decrease invasion in vitro more than two-fold relative to mock surgery controls and reduce activity of invasion-promoting transcription factors. Secretomic characterization of the conditioned media delineates interactions between immune cells in the TME and tumor cells, showing an increase in the pan-metastasis inhibitor decorin and a concomitant decrease in invasion-promoting chemokine (C-C motif) ligand 2 (CCL2) in scaffold-implanted mice. Flow cytometric and transcriptomic profiling of PT immune cells identify phenotypically distinct tumor-associated macrophages (TAMs) in scaffold-implanted mice, which may contribute to an invasion-suppressive TME. Taken together, this study demonstrates biomaterial scaffolds systemically influence metastatic progression through manipulation of the TME.