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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.

This study uses biomaterial scaffolds to identify a macrophage gene signature defined by high expression of C1qa, C1qb, and Trem2 that is elevated systemically in mouse and human pancreatic cancer., Pancreatic ductal adenocarcinoma (PDA) is accompanied by reprogramming of the local microenvironment, but changes at distal sites are poorly understood. We implanted biomaterial scaffolds, which act as an artificial premetastatic niche, into immunocompetent tumor-bearing and control mice, and identified a unique tumor-specific gene expression signature that includes high expression of C1qa, C1qb, Trem2, and Chil3. Single-cell RNA sequencing mapped these genes to two distinct macrophage populations in the scaffolds, one marked by elevated C1qa, C1qb, and Trem2, the other with high Chil3, Ly6c2 and Plac8. In mice, expression of these genes in the corresponding populations was elevated in tumor-associated macrophages compared with macrophages in the normal pancreas. We then analyzed single-cell RNA sequencing from patient samples, and determined expression of C1QA, C1QB, and TREM2 is elevated in human macrophages in primary tumors and liver metastases. Single-cell sequencing analysis of patient blood revealed a substantial enrichment of the same gene signature in monocytes. Taken together, our study identifies two distinct tumor-associated macrophage and monocyte populations that reflects systemic immune changes in pancreatic ductal adenocarcinoma patients.

OBJECTIVES/GOALS: Breast cancer metastases are stochastic and difficult to detect. Therapy is often ineffective due to phenotypic changes of tumor cells at these sites. We engineered a synthetic metastatic niche to study the role of phenotypic transitions in the microenvironment on tumor cell phenotype. METHODS/STUDY POPULATION: The engineered metastatic niche is composed of a porous polycaprolactone scaffold implanted subcutaneously in Balb/c mice. The mice received an orthotopic inoculation of 4T1 cells (murine triple negative breast cancer) in the fourth right mammary fat pad and the disease was allowed to progress for 7-21 days (pre-metastatic to overt metastatic disease). The scaffolds and lungs (native metastatic site) were explanted and analyzed by single cell RNA-seq via Drop-seq. Cell phenotypes were identified and tracked over time with the Seurat and Monocle3 pipelines. Assessment of the impact of these cell populations on tumor cell phenotype was conducted through Transwell co-cultures. RESULTS/ANTICIPATED RESULTS: Healthy scaffolds are primarily composed of macrophages, dendritic cells, and fibroblasts – consistent with a foreign body response. Despite differences in the lung and scaffold prior to tumor inoculation, both tissues were marked by >5-fold increase in neutrophils/MDSCs. Additionally, 79% of genes at the scaffold that significantly changed over time were also identified in the lung, indicating key similarities in niche maturation. However, many immune cells at the scaffold had distinct phenotypes, with pro-inflammatory/cytotoxic characteristics. These changes clearly impacted tumor cell phenotype, as cells from the scaffold increased tumor cell migration and apoptosis in vitro. DISCUSSION/SIGNIFICANCE OF IMPACT: Early phenotypic changes at the engineered metastatic niche can identify signs of metastasis prior to colonization of tumor cells. Furthermore, dynamics of immune and stromal cells change throughout niche maturation, influencing tumor cell phenotype and may suggest targeted therapies. CONFLICT OF INTEREST DESCRIPTION: Lonnie Shea, Jacqueline Jeruss, and Grace Bushnell are named inventors on patents or patent applications.

Metastases are preceded by stochastic formation of a hospitable microenvironment known as the premetastatic niche, which has been difficult to study. Herein, we employ implantable polycaprolactone scaffolds as an engineered premetastatic niche to independently investigate the role of interleukin-10 (IL10), CXCL12, and CCL2 in recruiting immune and tumor cells and impacting breast cancer cell phenotype via lentiviral overexpression. Lentivirus delivered from scaffolds in vivo achieved sustained transgene expression for 56 days. IL10 lentiviral expression, but not CXCL12 or CCL2, significantly decreased tumor cell recruitment to scaffolds in vivo. Delivery of CXCL12 enhanced CD45+ immune cell recruitment to scaffolds while delivery of IL10 reduced immune cell recruitment. CCL2 did not alter immune cell recruitment. Tumor cell phenotype was investigated using conditioned media from immunomodulated scaffolds, with CXCL12 microenvironments reducing proliferation, and IL10 microenvironments enhancing proliferation. Migration was enhanced with CCL2 and reduced with IL10-driven microenvironments. Multiple linear regression identified populations of immune cells associated with tumor cell abundance. CD45+ immune and CD8+ T cells were associated with reduced tumor cell abundance, while CD11b+Gr1+ neutrophils and CD4+ T cells were associated with enhanced tumor cell abundance. Collectively, biomaterial scaffolds provide a tool to probe the formation and function of the premetastatic niche.

Metastatic tumor cells colonize the pre-metastatic niche, which is a complex microenvironment consisting partially of extracellular matrix (ECM) proteins. We sought to identify and validate novel contributors to tumor cell colonization using ECM-coated poly(e-caprolactone) (PCL) scaffolds as mimics of the pre-metastatic niche. Utilizing orthotopic breast cancer mouse models, fibronectin and collagen IV-coated scaffolds implanted in the subcutaneous space captured colonizing tumor cells, showing a greater than 2-fold increase in tumor cell accumulation at the implant site compared to uncoated scaffolds. As a strategy to identify additional ECM colonization contributors, decellularized matrix (DCM) from lungs and livers containing metastatic tumors were characterized. In vitro, metastatic cell adhesion was increased on DCM coatings from diseased organs relative to healthy DCM. Furthermore, in vivo implantations of diseased DCM-coated scaffolds had increased tumor cell colonization relative to healthy DCM coatings. Mass-spectrometry proteomics was performed on healthy and diseased DCM to identify candidates associated with colonization. Myeloperoxidase was identified as abundantly present in diseased organs and validated as a contributor to colonization using myeloperoxidase-coated scaffold implants. This work identified novel ECM proteins associated with colonization using decellularization and proteomics techniques and validated candidates using a scaffold to mimic the pre-metastatic niche. Statement of Significance The pre-metastatic niche consists partially of ECM proteins that promote metastatic cell colonization to a target organ. We present a biomaterials-based approach to mimic this niche and identify ECM mediators of colonization. Using murine breast cancer models, we implanted microporous PCL scaffolds to recruit colonizing tumor cells in vivo . As a strategy to modulate colonization, we coated scaffolds with various ECM proteins, including decellularized lung and liver matrix from tumor-bearing mice. After characterizing the organ matrices using proteomics, myeloperoxidase was identified as an ECM protein contributing to colonization and validated using our scaffold. Our scaffold provides a platform to identify novel contributors to colonization and allows for the capture of colonizing tumor cells for a variety of downstream clinical applications.

Immune signaling within a metastatic niche drives recruitment and colonization, and the subsequent tumor cell fate within the niche. We employed a biomaterial scaffold as a synthetic metastatic niche to dissect the signals driving the responses within the niche relative to the native metastatic niche, which was the lung in these models. For both the natural and the synthetic niche, Gr-1+ cells secreting S100A8/A9 were the major factors contributing to tumor cell recruitment. Interestingly, upon recruitment, tumor cells in the synthetic niche were observed to undergo senescence and were cleared whereas cells within the native niche accumulated and ultimately proliferated to form macrometastases. The synthetic niche has underlying inflammation due to the foreign body responses (FBR) consisting of N1-polarized Gr1+ myeloid cells, M1-polarizedmacrophage, mature dendritic cells and highly cytotoxic CTLs and NK cells, which were not observed in the lung. Furthermore, we found two proteins (osteopontin and decorin) that are associated with developing FBR greatly improve the cytotoxicity of CTLs and NK cells in the lung from stage 3 breast cancers by increasing their production of Perforin/Granzyme, Fas/TRAIL and IFNr/TNFa. Our results highlight the potential of FBR-associated biomolecules to be used as immune-editing anticancer drugs and also inspire the development of therapeutic implants to actively catch and eradicate metastatic cells. Citation Format: Jing Wang, Matthew Hall, Grace Bushnell, Joseph Decker, Sophia Orbach, Ravi Raghani, Yining Zhang, Aaron Morris, Jeffery Ma, Pridvi Kandagatla, Jacqueline Jeruss, Lonnie Shea. Foreign body responses create a highly active anti-tumor environment to recruit and eradicate metastatic cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-360.

Background: Current technology limits the detection of metastatic lesions until the patient becomes symptomatic or radiological evidence is identified, and thus invasive cancer cells are not detectable prior to their colonization in distant organs. We have developed a biomaterial implant that serves as a synthetic metastatic niche that recruits tumor cells prior to their accumulation in the lung, a natural metastatic niche. We aimed to dissect the heterogeneity of stromal and immune cells at the synthetic and lung metastatic niches over time, thereby identifying changes in cell populations, phenotypes, and gene expression that could be used for the molecular staging of metastatic disease. Methods: Porous polycaprolactone scaffolds (5 mm diameter x 2 mm height) were implanted subcutaneously into Balb/c mice two weeks before triple negative murine breast cancer cells (4T1) were inoculated into the mammary fat pad. We tracked disease progression in the lung from a pre-metastatic niche (7 days post-inoculation) to micrometastases (14 days) to overt metastatic disease (21 days). Ten genes were selected from the TaqManTM Open Array Mouse Inflammation Panel to develop a PCR-based signature and single value decomposition (SVD) score in the scaffold that successfully delineated metastatic staging. Furthermore, the scaffold and lung were analyzed through single cell RNA-sequencing (Drop-seq) to identify heterogeneity and contributions of each cell type. Results: Ten genes were identified from Open Array of the scaffolds that directly correlated to disease stage in the lungs – S100a8, S100a9, Pglyrp1, Ltf, Camp, Ela2, and Chi3l3 increased with metastatic progression, while Bmp15, Ccl22 and Ccr7 decreased. SVD was applied to establish a single score from the scaffold that could be used for accurate molecular staging. Scaffolds were scored on a scale from 0 to 1, where a value of 0.2 – 0.3 generally correlated to pre-metastatic disease, 0.4 - 0.8 indicated micrometastases and 0.8 – 1.0 demarked metastasis. This model successfully predicted disease stage with an area under the curve of 0.878. When the scaffold-derived gene signature was applied to the lung metastases (21 days), the output was comparable to the scaffold, further demonstrating the physiological relevance of the implants. Single cell analysis indicated that the genes in the signature were primarily expressed by macrophages and neutrophils. After 7 days, 22% of macrophages and 83% of neutrophils were classified as tumor associated cells in the scaffold. In contrast, only 16% of macrophages and 44% of neutrophils in the lung were tumor associated at this time point. These findings support the hypothesis that the scaffolds could be used as early surrogates for the pre-metastatic niche, becoming responsive prior to their biological metastatic counterpart. Moreover, this finding was demonstrated through changes in T cell phenotype, specifically loss of cytotoxic Cd8+ T cells. After 7 days, Cd8+ T cells in the scaffolds decreased by 35%. Whereas Cd8+ T cells in the lungs decreased by 35% after 14 days. Conclusions: A biomaterial scaffold was employed as a synthetic niche that recruits metastatic tumor cells. These scaffolds established a molecular signature of metastatic breast cancer using a 10-gene panel. Additionally, single cell analysis identified the cell types that induce this signature and the disease-associated changes that occur in the scaffold earlier than the lung. In summary, we have shown the potential of a biomaterial scaffold to act as a surrogate metastatic niche that can be used to delineate disease progression and understand the role of specific cell types in the formation of this niche. Mechanistic investigation into the synthetic niche may identify novel therapeutic targets that could be activated to inhibit the advent of metastasis. Citation Format: Sophia M Orbach, Robert S Oakes, Michael D Brooks, Grace G Bushnell, Pridvi Kandagatla, Max S Wicha, Jacqueline S Jeruss, Lonnie D Shea. Early detection of metastatic breast cancer through gene expression and cell heterogeneity at an engineered metastatic niche [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-07.

The pre-metastatic niche - the accumulation of aberrant immune cells and extracellular matrix proteins in target organs - primes the initially healthy organ microenvironment and renders it amenable for subsequent metastatic cell colonization. By attracting metastatic cancer cells, mimics of the pre-metastatic niche offer both diagnostic and therapeutic potential. However, deconstructing the complexity of the niche by identifying the interactions between cell populations and the mediatory roles of the immune system, soluble factors, extracellular matrix proteins, and stromal cells has proved challenging. Experimental models need to recapitulate niche-population biology in situ and mediate in vivo tumour-cell homing, colonization and proliferation. In this Review, we outline the biology of the pre-metastatic niche and discuss advances in engineered niche-mimicking biomaterials that regulate the behaviour of tumour cells at an implant site. Such oncomaterials offer strategies for early detection of metastatic events, inhibiting the formation of the pre-metastatic niche, and attenuating metastatic progression.

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.