Biomaterial Scaffolds as Pre-metastatic Niche Mimics Systemically Alter the Primary Tumor and Tumor Microenvironment

Abstract

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.

Keywords: biomaterial; immunomodulation; metastasis; pre-metastatic niche; tumor microenvironment.

Figure 1

Transcriptomics analysis of primary tumor cells reveal differentially regulated genes and signaling pathways in response to PLG scaffold implants. A) Schematic of experimental design. B) Volcano plot showing 892 genes (in red) with most significantly altered gene expression of scaffold PT cells relative to mock PT cells (n = 3, p < 0.01, FDR < 0.1). A complete list of significantly altered genes is provided in Table S1 (Supporting Information). C,D) Metascape analysis of C) upregulated and D) downregulated genes (p < 0.01, FDR < 0.1).

Figure 2

CD45⁺ immune cell conditioned media from mock and scaffold mice alter MDA-MB-231 and 4T1 cell invasion. A) Schematic of experimental design. B) Representative bright-field images of invading tumor cells in various media. Scale bar on all images indicate 500 μm. C,D) Tumor cell invasion counts in Control, mock CD45⁺ conditioned media, and scaffold CD45⁺ conditioned media (n = 12) for C) MDA-MB-231 and D) 4T1 tumor cells. Letters “a,” “b,” and “c” denote groups that are statistically distinct (p < 0.05) according to the one-way ANOVA with Bonferroni testing for multiple comparisons. Data are shown as box-and-whisker plots with minimum values, maximum values, and interquartile range.

Figure 3

Scaffold implants alter the CD45⁺ immune cell secretome, which changes transcription factor activity in MDA-MB-231 cells. A) Scatter plot showing the average peptide spectral match (PSM) values of 161 secreted factors present in both CD45⁺ mock and scaffold media (n = 3). The red points represent PSM values with a log2 fold change greater than 1.5 in the scaffold CD45⁺ media, and the blue points represent PSM values with a log2 fold change greater than 1.5 in the mock CD45⁺ media. A complete list of identified secreted factors is provided in Table S2 (Supporting Information). B) Concentration of CCL2 measured using ELISA in mock and scaffold CD45⁺ media (n = 12), *p < 0.001. C) Concentration of decorin measured using ELISA in mock and scaffold CD45⁺ media (n = 12), *p < 0.01. Data are shown as box-and-whisker plots with minimum values, maximum values, and interquartile range. D) Heat map of normalized TF activity values for MDA-MB-231 cells cultured in scaffold CD45⁺ conditioned media relative to mock CD45⁺ conditioned media over 8 h. Significant changes in TF activity for at least one time point indicated in bold (n = 3 arrays, n = 12 total measurements per time point, p < 0.05).

Figure 4

Characterization of immune cell populations and phenotypes at the primary tumor in response to scaffold implants. A) Percent of CD45⁺ leukocytes (Gr1^hiCD11b⁺Ly6C⁻ MDSCs, Ly6C⁺F4/80⁻ inflammatory monocytes, CD11c⁺ dendritic cells, and F4/80⁺CD11b⁺ macrophages) present in different tissues from PLG implanted mice (n = 5). B) Percent of CD45⁺ leukocytes present at the PT from mock surgery and PLG scaffold-implanted mice (n = 5, *p < 0.05). C) Gating strategy for stratifying macrophage populations (F4/80⁺Vcam1⁺ (denoted as F⁺/V⁺, recruited macrophages), F4/80⁺Vcam1⁻ (F⁺/V⁻, resident macrophages), or F4/80⁻Vcam1⁻ (F⁻V⁻, other leukocytes)). D) Percent of leukocytes for each macrophage population (n = 5). Data are shown as mean ± SEM. E) Fold change in invasion and metastasis associated genes in recruited versus resident TAM populations in mock and scaffold mice (n = 6). All genes shown at a confidence level of 90% or higher, with *p < 0.05, **p < 0.01 according to the Student’s t-test.