Scaffolds for in vivo capture of metastasizing cells

An orthotopic model of human breast cancer metastasis was employed to investigate the capture of metastatic cells at a biomaterial implant. tdTomato- and luciferase-expressing MDA-MB-231BR (231BR) cells, a highly metastatic variant of the MDA-MB-231 cell line21, were transplanted into the right mammary fat pads of female NOD/SCID-IL2Rγ−/− (NSG) mice (Fig. 1c). One week after tumour inoculation, microporous PLG scaffolds (5-mm diameter, 2-mm height, Supplementary Fig. 1a–c) were implanted into the peritoneal fat pads, a site to which 231BR cells are not reported to colonize, yet supports the vascularization of PLG scaffolds. Bioluminescence imaging and histological analysis of peritoneal fat pads removed 28 days after tumour inoculation demonstrated the presence of tumour cells within the implanted PLG scaffold (Fig. 1a,d) and the absence of tumour cells in fat pads without scaffolds (Fig. 1b,e), indicating that the local environment generated by implantation of the scaffold enabled recruitment of metastatic cells. Primary tumour growth was not affected by either implantation of scaffolds or a mock surgery (Supplementary Fig. 2). Staining for fibronectin, a matrix protein reported to be involved in establishment of the pre-metastatic niche8, indicated that fibronectin was present in scaffolds implanted in both healthy and tumour-bearing mice as early as 7 days post implantation (Supplementary Fig. 1d). Interestingly, recruitment of cells to the scaffold was not site-specific, as tumour cells were detected in scaffolds implanted in the subcutaneous tissue (Supplementary Fig. 3).

Figure 1: PLG scaffolds recruit metastatic tumour cells. Tissues were isolated at day 28 post-tumour inoculation (21 days after scaffold implantation or mock surgery). (a,b) Bioluminescence imaging (BLI) of peritoneal fat pads receiving scaffold implants (a) or mock surgeries (b). (c-e) Hematoxylin and eosin (H&E) staining of the primary tumour (c) a fat pad containing a scaffold (white circle indicates metastatic cluster) (d) and a fat pad without a scaffold (e). Scale bars, 100 μm. Full size image

Scaffolds reduce tumour burden in solid organs

We subsequently investigated whether capturing tumour cells in scaffolds would reduce colonization of standard metastatic sites, such as the lung and liver. At 28 days post-tumour inoculation, the relative abundance of tumour cells, reported as the ratio of tdTomato-positive tumour cells to total cells, was determined. For mice that received scaffolds, the relative abundance of tumour cells in the lung was 1:5,400, compared with 1:645 for mice receiving a mock surgery (Fig. 2a, Supplementary Fig. 4). Thus, the presence of a scaffold reduced the tumour burden for the lung by 88±7% (average±s.e.m.). Histological analysis of lung sections confirmed a reduction in the tumour cell burden with scaffold implantation (Fig. 2b,c), with an average of 1.7±0.5 metastatic lesions per section observed in the lungs of scaffold-bearing mice, compared with 5.5±1.7 lesions per section in mice receiving mock surgeries. Furthermore, flow cytometric analysis of cells isolated from the liver showed detectable tumour cells in eight out of eight mice receiving mock surgeries, while mice receiving scaffold implants only exhibited detectable tumour cells in two of eight livers (P<0.01, Fisher’s exact test).

Figure 2: Recruitment of tumour cells to scaffolds reduces tumour burden in lung. (a) Flow cytometric analysis of the percentage of tdTomato-positive tumour cells in cells isolated from lungs at day 28 post-tumour inoculation. Data shown as mean±s.e.m. (n=8, 2 independent biological replicates). *P<0.01 compared with mock surgery (Mann–Whitney test). (b) H&E staining of lung section (black circles indicate metastatic clusters). Scale bar, 200 μm. (c) Histological analysis of H&E-stained lung sections to determine the number of tumour clusters per section. Data shown as mean±s.e.m. (n=12, 2 independent biological replicates). *P<0.05 compared with mock surgery (Mann–Whitney test). Full size image

Early detection of tumour cells in scaffold

The potential to use scaffolds for early detection of metastasis was determined by quantifying the percentage of tumour cells in intraperitoneal and subcutaneous scaffolds compared with the lung and liver at day 14 post-tumour inoculation. In a group of eight mice, most intraperitoneal scaffolds (15/16) contained tumour cells at this time point, while none of the mice had detectable tumour cells in the lung and liver (Fig. 3a). In a separate group of mice, all subcutaneous scaffolds (10/10) contained tumour cells. The incidence of detectable metastatic disease at this early time point was lower than at day 28 post-tumour inoculation. At day 28 post-tumour inoculation in scaffold-bearing mice, the lung and liver exhibited tumour cells in 8 and 2 of the eight mice, respectively. Furthermore, for mice receiving mock surgeries instead of scaffold implants, the incidence of metastatic cells in both the lung and liver increased to eight out of eight mice. Importantly, at day 14 post inoculation, while none of the lungs and livers exhibited detectable tumour cells, both intraperitoneal and subcutaneous scaffolds had a detectable percentage of tumour cells (0.019±0.005% for intraperitoneal scaffolds and 0.044±0.017% for subcutaneous scaffolds) (Fig. 3b, Supplementary Fig. 5). This ability to detect tumour cells in the scaffold before detection in the lungs and liver may enable the early detection of metastatic disease through imaging the scaffold.

Figure 3: Early detection of tumour cells in scaffolds. Flow cytometric analysis of tdTomato-positive tumour cells in tissues isolated from mice at day 14 and 28 post-tumour inoculation. (a) Number of mice with tumour cells detectable in each tissue in a group of 8 mice at day 14 or 28 post-tumour inoculation. Each mouse received two intraperitoneal (IP) scaffolds. P values from Fisher’s exact test. (b) Percentage of tdTomato-positive cells in the total cell population isolated from IP scaffolds and subcutaneous (SQ) scaffolds at day 14 post-tumour inoculation. Data shown as mean±s.e.m. (n=16 for IP scaffold, n=10 for SQ scaffold, two independent biological replicates). *P<0.05 compared with IP scaffold (Mann–Whitney test). Full size image

Label-free detection of metastasis at the scaffold

ISOCT22 was applied to directly visualize the scaffold architecture and provide quantitative measurement of the ultrastructural changes induced by the cancer cells. ISOCT is a light scattering-based technique capable of non-invasive three-dimensional (3D) imaging of tissue morphology with micron-level resolution and millimeter-level penetration depth23,24. In addition, for each 3D resolution voxel (15 × 15 × 2 μm) ISOCT also performs a spectroscopic analysis and quantifies the power of the spectra by a scattering model I(λ)∝λD-4 22. D is the shape factor that physically defines the macromolecular density correlation function for a range of length scales from ∼40 to 350 nm (ref. 25), with higher D values indicating a more clumped structure. It has been demonstrated that D is a ubiquitous marker of the ultrastructural alterations in the early stages of various cancer types despite their different etiologies26,27,28,29,30, with both neoplastic cells and the surrounding stroma exhibiting an increase in D in part due to chromatin condensation and collagen remodelling, respectively27,30,31. Given the nanoscale sensitivity of measuring D and the tissue-level imaging capability, we hypothesized that ISOCT could be an effective approach for detection of cancer cells within the scaffold.

In vitro studies were performed to demonstrate that ISOCT could capture changes in D for cells and matrices, and the technique was subsequently applied to in situ imaging of scaffolds. ISOCT analysis of 231BR cell pellets confirmed that they had a higher D than normal mammary epithelial cells (MCF10A) and cells isolated from lungs of tumour-free NSG mice (3.49±0.12, 2.74±0.15 and 3.00±0.13, respectively, Supplementary Fig. 6a). Changes to collagen remodelling by 231BR cells were evaluated by culturing 231BR cells in collagen gels for 3 days, after which cells were extracted and the gels were analysed using ISOCT. 231BR-conditioned matrices exhibited a D value of 1.69±0.08 compared with a D value of 1.29±0.04 for gels cultured with media (Supplementary Fig. 6b). With confirmation of the technique in vitro, in situ ISOCT analysis was applied to scaffolds, which demonstrated that scaffolds implanted in the subcutaneous tissue of tumour-bearing mice also had an increase in D at day 14 post-tumour inoculation compared with control scaffolds in tumour-free mice (Fig. 4), with an average D value of 5.77±0.38 in tumour-bearing mice compared with 4.71±0.17 in tumour-free mice. This increase in D is consistent with the changes associated with the presence of cancerous cells and the ensuing reorganization of the extracellular matrix. These results indicate that this method can be used for label-free detection of micrometastases within the scaffold at the early stages of metastatic disease.

Figure 4: Detection of tumour cells in scaffold using ISOCT. (a,b) Representative 3D maps of D generated from in situ ISOCT analysis of subcutaneous scaffolds implanted in tumour-free (a) and tumour-bearing (b) mice at day 14 post-tumour inoculation. Scale bars, 200 μm. (c) Average D value for subcutaneous scaffolds in tumour-free (‘No Tumor’) and tumour-bearing (‘Tumor’) mice. Data shown as mean±s.e.m. (n=6, 2 independent biological replicates). *P<0.05 compared with tumour-free mice (Mann–Whitney test). Full size image

Immune cells contribute to tumour cell recruitment

Given the critical role of various immune cells types in establishing the pre-metastatic niche8,10,12,16,19,20, we hypothesized that the immune response to the scaffold was mediating recruitment of tumour cells. For analysis of the immune environment within the scaffolds, we used an immune-competent mouse model in addition to the NSG model to account for effects of both the innate and adaptive immune response. Scaffolds implanted into BALB/c mice inoculated with 4T1 mouse breast cancer cells also demonstrated metastatic cells within the scaffold, indicating that the scaffold could still achieve homing within the context of an intact immune system (Supplementary Fig. 7).

Inflammatory cells proposed to be involved in recruiting tumour cells were characterized within the peritoneal fat pads of mice in the presence and absence of a scaffold. A high density of CD45-positive leukocytes was present in histological sections of the scaffold, with no observed CD45-positive leukocytes present in fat pads of mice receiving mock surgeries (Fig. 5a,b). The ability of CD45-positive leukocytes to influence homing of tumour cells was investigated through migration assays using media conditioned by splenocytes isolated from spleens of tumour-bearing mice, as the spleen contains a large number of immune cells and has a distribution of immune cells similar to the scaffolds, with the predominant cell type being Gr1hiCD11b+ cells (Supplementary Fig. 8). Migration of both cell types was significantly increased in the presence of splenocyte-conditioned media relative to unconditioned media (Fig. 5c,d), with 261±35 migrating 231BR cells per well in conditioned media compared with 137±20 cells in unconditioned media and 521±22 migrating 4T1 cells per well in conditioned media compared with 292±23 cells in unconditioned media. These results indicate that paracrine signalling from immune cell populations similar to those present in the scaffold can induce migration of tumour cells.

Figure 5: Evaluation of the immune environment within scaffolds. (a,b) CD45 immunolabeling (green) at day 28 post-tumour inoculation of fat pads receiving mock surgery (a) or scaffold implant (b). Nuclei are blue. Scale bar, 100 μm. (c,d) Number of migrating 231BR (c) or 4T1 (d) cells in the presence of splenocyte-conditioned media (SCM) or non-conditioned media (non-SCM). Data shown as mean±s.e.m. (n=6, 2 independent biological replicates). *P<0.05 and **P<0.005 compared with non-conditioned media (Mann–Whitney test). (e–h) Flow cytometric analysis of cells removed from lungs (e,f) and scaffolds (g,h) of tumour-free and tumour-bearing (day 14 and 28) NSG (e,g) or BALB/c (f,h) mice. The model used for mice with tumours involved the inoculation of tumour cells at day 0, with scaffolds implanted at day 7. The evaluation of scaffolds in tumour-free mice was performed on day 7 post implantation. Cell populations are reported as percentage of total CD45-positive leukocytes. Data shown as mean±s.e.m. (n=5 for lungs, n=10 for scaffolds, 2 independent biological replicates). *P<0.05 and **P<0.005 compared with no tumour (Mann–Whitney test). Full size image

Before and after the introduction of cancer cells, we analysed the local immune environment of the implanted scaffold, which was compared with that of the lung, a common site of breast cancer metastasis. Flow cytometric analysis demonstrated that during disease progression, the most notable change in the relative distribution of immune cells was an increase in Gr1hiCD11b+ cells (Fig. 5e–h, Supplementary Figs 9–11). This increase was consistent in both the NSG and BALB/c mouse models, and was found in both the lungs and scaffolds. In the lungs of the NSG mice, the percentage of Gr1hiCD11b+ cells increased from 45±2% at day 0 (no tumour) to 52±3% at day 14 post-tumour inoculation, to 84±1% at day 28 post-tumour inoculation (Fig. 5e). Following this trend, the percentage of Gr1hiCD11b+ cells found in the scaffolds from the NSG mice increased from 21±1% (tumour-free mice), to 35±5% at day 14 post-tumour inoculation, to 62±2% at day 28 post-tumour inoculation (Fig. 5g). In the lungs of BALB/c mice, Gr1hiCD11b+ cells increased from 1±0.1% at day 0 (tumour-free mice) to 57±1% at day 14 post-tumour inoculation, to 89±2% at day 28 post-tumour inoculation (Fig. 5f). Likewise, within the scaffolds from the BALB/c mice, Gr1hiCD11b+ cells increased from 0.1±0.01% (no tumour) to 32±3% at day 14 post-tumour inoculation, to 66±5% at day 28 post-tumour inoculation (Fig. 5h). These results highlight the correlation between disease exposure time and the relative abundance of Gr1hiCD11b+ cells found in both the lungs and scaffolds of affected animals, and show that the immune environment within a metastatic organ site is similar to that of an implanted scaffold.

We subsequently investigated the hypothesis that modulation of the inflammatory microenvironment within the scaffold site would influence recruitment of metastatic cells. Lentiviral vectors were delivered from the scaffold to promote localized transgene expression for the duration of the study (Supplementary Fig. 12). The chemokine CCL22 was selected for expression as it induces migration of splenocytes harvested from NSG mice (Supplementary Fig. 13a) yet does not influence the migration of tumour cells (Supplementary Fig. 13b). Flow cytometric analysis of CCL22 scaffolds at day 7 post implantation indicated an increase in Gr1hiCD11b+ cells, which have been implicated in the pre-metastatic niche10,14,15, from 20±2% to 29±3% in NSG mice and from 14±1% to 19±1% in BALB/c mice (Fig. 6a,c). In addition, F4/80+CD11b+ inflammatory macrophages, which are recruited to circulating metastatic cells as they undergo extravasation and begin colonization12, increased from 28±2% to 34±2% in the NSG model and from 10±1% to 14±1% in the BALB/c model (Fig. 6a,c). Furthermore, CCL22 expression increased the number of tdTomato-positive tumour cells present in the scaffold in both mouse models at day 7 post implantation (day 14 post-tumour inoculation), with an average of 92±16 cells compared with an average of 23±4 cells for β-galactosidase expression in NSG mice and an average of 62±5 cells compared with an average of 20±2 cells for β-galactosidase expression in BALB/c mice (Fig. 6b,d).

Figure 6: Immunomodulation of the scaffold microenvironment influences recruitment of tumour cells. Flow cytometric analysis at day 14 post-tumour inoculation of cells removed from scaffolds containing a CCL22 or β-galactosidase (control) vector implanted in NSG (a,b) or BALB/c (c,d) mice. Cell populations are reported as percentage of total CD45-positive leukocytes (a,c) or total number of tumour cells in the scaffold (b,d). Data shown as mean±s.e.m. (n=10, 2 independent biological replicates). *P<0.05, **P<0.005 and ***P<0.001 compared with β-galactosidase scaffolds (Mann–Whitney test). (e) Number of migrating 231BR cells in the presence of Gr1hiCD11b+ cell-conditioned media (Gr1hiCD11b+ CM) or non-conditioned media (non-SCM). Data shown as mean±s.e.m. (n=6, 2 independent biological replicates). ***P<0.001 compared with non-conditioned media (Mann–Whitney test). (f,g) Flow cytometric analysis at day 14 post-tumour inoculation of cells removed from blank scaffolds or scaffolds seeded with Gr1hiCD11b+ cells. Cell populations are reported as the percentage of Gr1hiCD11b+ cells in the total leukocyte population (f) or total number of tumour cells in the scaffold (g). Data shown as mean±s.e.m. (n=10, 2 independent biological replicates). *P<0.05 compared with blank scaffold (Mann–Whitney test). Full size image

A cell population upregulated by CCL22 delivery, Gr1hiCD11b+ cells, was next transplanted on the scaffold to directly modulate the local immune environment and further demonstrate that the immune environment mediates metastatic cell recruitment. Gr1hiCD11b+ cells were selected for these studies given their established role in the pre-metastatic niche, along with migration assays showing soluble factors secreted by Gr1hiCD11b+ cells isolated from spleens of tumour-bearing NSG mice induced migration of 231BR cells (Fig. 6e). Interestingly, the Gr1hiCD11b+ cells induced migration to a greater extent than splenocyte-conditioned media (Supplementary Fig. 14). Implantation of scaffolds seeded with Gr1hiCD11b+ cells significantly increased the percentage of Gr1hiCD11b+ cells in the total leukocyte population (from 20±2 to 26±2%, Fig. 6f) as well as the number of tumour cells (from 29±10 to 74±13 cells, Fig. 6g) present per scaffold, indicating that Gr1hiCD11b+ cells contributed to recruitment of tumour cells to the scaffold.