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Microenvironmental changes in the early metastatic niche may be exploited to identify therapeutic targets to inhibit secondary tumor formation and improve disease outcomes. We dissected the developing lung metastatic niche in a model of metastatic, triple-negative breast cancer using single-cell RNA-sequencing. Lungs were extracted from mice at 7-, 14-, or 21 days after tumor inoculation corresponding to the pre-metastatic, micro-metastatic, and metastatic niche, respectively. The progression of the metastatic niche was marked by an increase in neutrophil infiltration (5% of cells at day 0 to 81% of cells at day 21) and signaling pathways corresponding to the hallmarks of cancer. Importantly, the pre-metastatic and early metastatic niche were composed of immune cells with an anti-cancer phenotype not traditionally associated with metastatic disease. As expected, the metastatic niche exhibited pro-cancer phenotypes. The transition from anti-cancer to pro-cancer phenotypes was directly associated with neutrophil and monocyte behaviors at these time points. Predicted metabolic, transcription factor, and receptor-ligand signaling suggested that changes in the neutrophils likely induced the transitions in the other immune cells. Conditioned medium generated by cells extracted from the pre-metastatic niche successfully inhibited tumor cell proliferation and migration in vitro and the in vivo depletion of pre-metastatic neutrophils and monocytes worsened survival outcomes, thus validating the anti-cancer phenotype of the developing niche. Genes associated with the early anti-cancer response could act as biomarkers that could serve as targets for the treatment of early metastatic disease. Such therapies have the potential to revolutionize clinical outcomes in metastatic breast cancer.

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.

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

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.

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

Description of additional methods and procedures used in the study. Also includes Supplementary References.

Sacrifice end points observed in the post-surgical model in the mock and scaffold groups with resection performed at day 10 without Gr-1 depletion.

Micro-porous PCL scaffolds persist and maintain a space for extended times in vivo (S1); Host response following implantation of micro-porous PCL scaffolds in the dorsal subcutaneous space of an NSG mouse in vivo (S2); Dynamics of immune cell populations in the spleen of BALB/c mice with a PCL scaffold implant at day 0, 5, 10, and 15 post tumor inoculation (S3); Micro-porous PCL scaffolds enable recruitment of human MDA-MD-231BR cells in a chronic model of scaffold implantation (S4); Micro-porous PCL scaffolds do not significantly improve survival in a post-surgical model of breast cancer metastasis with day 6 post tumor inoculation resection (S5); Anti-Gr-1 depletion significantly reduces the presence of CD11b+Gr-1hiLy6C- MDSCs in the spleen primary tumor and scaffold (S6).

The onset of distant organ metastasis from primary breast cancer marks the transition to a stage IV diagnosis. Standard imaging modalities often detect distant metastasis when the burden of disease is high, underscoring the need for improved methods of detection to allow for interventions that would impede disease progression. Here, microporous poly(ϵ-caprolactone) scaffolds were developed that capture early metastatic cells and thus serve as a sentinel for early detection. These scaffolds were used to characterize the dynamic immune response to the implant spanning the acute and chronic foreign body response. The immune cell composition had stabilized at the scaffold after approximately 1 month and changed dramatically within days to weeks after tumor inoculation, with CD11b+Gr1hiLy6C− cells having the greatest increase in abundance. Implanted scaffolds recruited metastatic cancer cells that were inoculated into the mammary fat pad in vivo, which also significantly reduced tumor burden in the liver and brain. Additionally, cancer cells could be detected using a label-free imaging modality termed inverse spectroscopic optical coherence tomography, and we tested the hypothesis that subsequent removal of the primary tumor after early detection would enhance survival. Surgical removal of the primary tumor following cancer cell detection in the scaffold significantly improved disease-specific survival. The enhanced disease-specific survival was associated with a systemic reduction in the CD11b+Gr1hiLy6C− cells as a consequence of the implant, which was further supported by Gr-1 depletion studies. Implementation of the scaffold may provide diagnostic and therapeutic options for cancer patients in both the high-risk and adjuvant treatment settings. Cancer Res; 76(18); 5209–18. ©2016 AACR.

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.

Breast cancer is the most commonly diagnosed cancer in the USA. Although advances in treatment over the past several decades have significantly improved the outlook for this disease, most women who are diagnosed with estrogen receptor positive disease remain at risk of metastatic relapse for the remainder of their life. The cellular source of late relapse in these patients is thought to be disseminated tumor cells that reactivate after a long period of dormancy. The biology of these dormant cells and their natural history over a patient’s lifetime is largely unclear. We posit that research on tumor dormancy has been significantly limited by the lack of clinically relevant models. This review will discuss existing dormancy models, gaps in biological understanding, and propose criteria for future models to enhance their clinical relevance.

Pancreatic cancer has the worst prognosis of all cancers due to disease aggressiveness and paucity of early detection platforms. We developed biomaterial scaffolds that recruit metastatic tumor cells and reflect the immune dysregulation of native metastatic sites. While this platform has shown promise in orthotopic breast cancer models, its potential in other models is untested. Herein, we demonstrate that scaffolds recruit disseminated pancreatic cells in the KPCY model of spontaneous pancreatic cancer prior to adenocarcinoma formation (3-fold increase in scaffold YFP + cells). Furthermore, immune cells at the scaffolds differentiate early- and late-stage disease with greater accuracy (0.83) than the natural metastatic site (liver, 0.50). Early disease was identified by an approximately 2-fold increase in monocytes. Late-stage disease was marked by a 1.5-2-fold increase in T cells and natural killer cells. The differential immune response indicated that the scaffolds could distinguish spontaneous pancreatic cancer from spontaneous breast cancer. Collectively, our findings demonstrate the utility of scaffolds to reflect immunomodulation in two spontaneous models of tumorigenesis, and their particular utility for identifying early disease stages in the aggressive KPCY pancreatic cancer model. Such scaffolds may serve as a platform for early detection of pancreatic cancer to improve treatment and prognosis.

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.

Metastasis represents the greatest challenge to treatment of cancer patients. Biomaterial scaffolds that recruit tumor cells to a defined site in vivo are an emerging platform for the diagnosis, treatment, and study of metastasis. Recruitment of immune cells and metastatic tumor cells to a defined location provides a precision health platform to assess current clinical cancer biomarkers in a metastatic setting, and to define the next generation of biomarkers. These platforms represent an opportunity to create a molecular staging of metastasis that could aid in both the early diagnosis and treatment of metastasis.

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.

Pre-metastatic niches in distant tissue facilitate metastasis from the primary tumor. Cargo-free porous polymer scaffolds implanted in tumor-bearing mice act as synthetic metastatic niches recruiting metastasizing cancer cells. Herein, we investigated the mechanisms by which these implants attract cancer cells from circulation. Scaffolds attract cancer cells in part via S100A8/A9 secreted by Gr1+ myeloid cells in a mechanism that mimics lung metastasis. Further, cancer cells attracted to the scaffold have a lung-tropic gene expression signature regardless of their tissue of origin. The scaffold implant reduces metastasis to the lung suggesting otherwise lung-tropic cancer cells are diverted to the scaffold. The suppression of metastatic spread by the scaffold suggests this mechanism may be exploited for novel therapies, and may broadly influence the design of scaffold-based drug delivery system for anti-cancer therapy.

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.

For female cancer survivors, premature ovarian insufficiency (POI) is a common complication of anticancer treatments. Ovarian tissue cryopreservation before treatment, followed by auto-transplantation after remission is a promising option to restore fertility and ovarian endocrine function. However, auto-transplantation is associated with the risk of re-introducing malignant cells harbored in the stroma of the ovarian autograft. To mitigate this risk, we investigated in this pilot study whether an immuno-isolating dual-layered poly(ethylene glycol)(PEG) capsule can retain cancer cells, while supporting folliculogenesis. The dual PEG capsule loaded with 1000 4T1 cancer cells retained 100% of the encapsulated cells in vitro for 21 days of culture. However, a greater cell load of 10,000 cells/capsule led to capsule failure and cells’ release. To assess the ability of the capsule to retain cancer cells, prevent metastasis, and support folliculogenesis in vivo we co-encapsulated cancer cells with ovarian tissue in the dual PEG capsule and implanted subcutaneously in mice. Control mice implanted with 2000 non-encapsulated cancer cells had tumors formed within 14 days and metastasis to the lungs. In contrast, no tumor mass formation or metastasis to the lungs was observed in mice with the same number of cancer cells encapsulated in the capsule. Our findings suggest that the immuno-isolating capsule may prevent the escape of the malignant cells potentially harbored in ovarian allografts and, in the future, improve the safety of ovarian tissue auto-transplantation in female cancer survivors.

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.

Pancreatic cancer is a lethal malignancy with a 5-year survival rate of less than 10%. This poor prognosis is, in part, due to patients most often presenting with already metastatic disease. Pancreatic cancer is characterized by an abundant, reactive fibroinflammatory stroma, consisting of cancer-associated fibroblasts (CAFs) and infiltrating immune cells that send and receive signals to and from other nearby cells, including the tumor cells. The crosstalk between tumor cells and elements of the stroma results in an immunosuppressive tumor microenvironment both at the primary tumor and the sites of metastases. A key limitation in studying metastatic disease in mouse models is the inability to predict when the cancer cells will ultimately metastasize. In this study biomaterial scaffolds that mimic the premetastatic niche in vivo, by attracting immune cells followed by tumor cells, were used to longitudinally follow the metastasis process in individual animals. Biomaterial scaffolds were implanted subcutaneously to mimic the premetastatic niche in both syngeneic and spontaneous mouse (iKras* p53*) models of pancreatic cancer. Scaffolds were harvested 21 days post implantation and taken for histologic analysis to characterize the cellular infiltrate in response to the primary tumor. Histologic analysis (H&E, Gomori’s trichrome, and multiplex IHC) shows tumor cell colonization in the scaffold along with a robust immune and fibrotic response. To further characterize the scaffold infiltrate in control versus tumor-bearing mice, mass cytometry (CyTOF) and single-cell RNA sequencing (scRNA seq) analysis was performed. CyTOF analysis revealed the most abundant cell population is heterogenous myeloid cells, which are immunosuppressive in nature. Scaffolds from tumor-bearing animals have an increase in fibroblasts, and subsequently have fewer cytotoxic, CD8+ T cells. scRNA seq analysis of the scaffold infiltrate reveals CD8+ T cells from tumor-bearing animals express higher levels of the immune checkpoint, programmed cell death protein 1 (PD-1) and lower levels of the functional markers, perforin 1 and granzyme B, suggesting the CD8+ T cells are unable to perform their cytotoxic functions. Further analysis revealed a chemokine signaling axis, where fibroblasts recruit distinct subsets of myeloid cells. Taken together, the premetastatic niche (recapitulated by the biomaterial scaffolds) in pancreatic cancer has a high abundance of immunosuppressive myeloid cells, an increase in fibroblasts, and fewer CD8+ T cells, creating an immunosuppressive niche that allows for tumor cell colonization and growth. Future directions include blocking myeloid cell chemotaxis to the scaffold to prevent tumor cell dissemination and reduce metastatic burden. Citation Format: Samantha Kemp, Nina Steele, Eileen Carpenter, Veerin Sirihorachai, Grace Bushnell, Aaron Morris, Carlos Espinoza, Fatima Lima, Zeribe Nwosu, Sophia Orbach, Lonnie Shea, Filip Bednar, Howard Crawford, Marina Pasca di Magliano. Using biomaterial scaffolds to study the genesis of the immunosuppressive premetastatic niche in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A25.

The onset of distant organ metastasis from primary breast cancer marks the transition to a stage IV diagnosis. Standard imaging modalities often detect distant metastasis when the burden of disease is high, underscoring the need for improved methods of detection to allow for interventions that would impede disease progression. Here, microporous poly(ϵ-caprolactone) scaffolds were developed that capture early metastatic cells and thus serve as a sentinel for early detection. These scaffolds were used to characterize the dynamic immune response to the implant spanning the acute and chronic foreign body response. The immune cell composition had stabilized at the scaffold after approximately 1 month and changed dramatically within days to weeks after tumor inoculation, with CD11b+Gr1hiLy6C− cells having the greatest increase in abundance. Implanted scaffolds recruited metastatic cancer cells that were inoculated into the mammary fat pad in vivo, which also significantly reduced tumor burden in the liver and brain. Additionally, cancer cells could be detected using a label-free imaging modality termed inverse spectroscopic optical coherence tomography, and we tested the hypothesis that subsequent removal of the primary tumor after early detection would enhance survival. Surgical removal of the primary tumor following cancer cell detection in the scaffold significantly improved disease-specific survival. The enhanced disease-specific survival was associated with a systemic reduction in the CD11b+Gr1hiLy6C− cells as a consequence of the implant, which was further supported by Gr-1 depletion studies. Implementation of the scaffold may provide diagnostic and therapeutic options for cancer patients in both the high-risk and adjuvant treatment settings. Cancer Res; 76(18); 5209–18. ©2016 AACR.

Most synthetic polymer hydrogel tissue adhesives and sealants swell considerably in physiologic conditions, which can result in mechanical weakening and adverse medical complications. This paper describes the synthesis and characterization of mechanically tough zero- or negative-swelling mussel-inspired surgical adhesives based on catechol-modified amphiphilic poly(propylene oxide)-poly(ethylene oxide) block copolymers. The formation, swelling, bulk mechanical, and tissue adhesive properties of the resulting thermosensitive gels were characterized. Catechol oxidation at or below room temperature rapidly resulted in a chemically cross-linked network, with subsequent warming to physiological temperature inducing a thermal hydrophobic transition in the PPO domains and providing a mechanism for volumetric reduction and mechanical toughening. The described approach can be easily adapted for other thermally sensitive block copolymers and cross-linking strategies, representing a general approach that can be employed to control swelling and enhance mechanical properties of polymer hydrogels used in a medical context.

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.

Background Intraoperative rupture (IOR) is a rare, but potentially morbid complication of endovascular aneurysm coil embolization. Yet, IOR predictors have remained relatively uninvestigated in relation to coil design. Objective To develop a novel in vitro aneurysm model to characterize forces exerted by coils of different design on the aneurysm during endovascular embolization that are hypothesized to contribute to IOR. Methods A 3-mm saccular aneurysm model was developed with flat latex membrane at the dome apex. Membrane deflection was observed throughout simulated embolization and converted to force measurement. Simultaneous coil insertion and force measurement were accomplished with a compression strength-testing machine. Membrane and insertion forces across coil type, microcatheter tip placement, and insertion rate were evaluated. Results Insertion force and force directly on the aneurysm wall exhibited a difference, with framing coils exerting greatest force, followed by filling and finishing coils. Regarding microcatheter placement, a similar graded response in membrane and insertion forces was observed with positioning in the top-third of the aneurysm generating the greatest force compared with central and bottom-third placement. Insertion rate was also a factor with the slowest rate (10 mm/min) exhibiting the greatest membrane force, followed by lower forces at 30 and 50 mm/min. A multiple linear regression model was created to assess the contributions of each factor toward aneurysm forces. Conclusion Increased force on the aneurysm is associated with framing coil use, microcatheter placement proximal to aneurysm dome, and slow insertion rate. Further characterization remains necessary to reduce IOR risk, especially concerning the contributions of insertion rate.