Hypoxia stimulates CTC-platelet cluster formation to promote breast cancer metastasis

Abstract

Circulating tumor cell clusters/micro-emboli (CTM) possess greater metastatic capacity and survival advantage compared to individual circulating tumor cell (CTC). However, the formation of CTM subtypes and their role in tumor metastasis remain unclear. In this study, we used a microfluidic Cluster-Chip with easy operation and high efficiency to isolate CTM from peripheral blood, which confirmed their correlation with clinicopathological features and identified the critical role of CTC-platelet clusters in breast cancer metastasis. The correlation between platelets and CTM function was further confirmed in a mouse model and RNA sequencing of CTM identified high-expressed genes related to hypoxia stimulation and platelet activation which possibly suggested the correlation of hypoxia and CTC-platelet cluster formation. In conclusion, we successfully developed the Cluster-Chip platform to realize the clinical capture of CTMs and analyze the biological properties of CTC-platelet clusters, which could benefit the design of potential treatment regimens to prevent CTM-mediated metastasis and tumor malignant progression.

Keywords: Biotechnology; Cancer; Cell biology.

Graphical abstract

Scheme 1

Schematic diagram of the Cluster-Chip developed for CTM isolation Schematic diagram of the Cluster-Chip developed for CTM isolation. Sequential validation of the clinical applicability of the Cluster-Chip and mechanism of circulating tumor cell micro-embolus (CTM) in breast cancer metastasis.

Figure 1

Performance and optimization experiments for determining capture and release efficiency (A) Diagram of the overall and internal structure of the Cluster-Chip. Scale bar: 100 μm. (B) Capture efficiency at different flow rates measured at 4°C. The Cluster-Chip was filled with Pluronic F127 (1% w/v in PBS) to minimize cell adhesion using artificial clusters of A549 cells spiked in whole blood. (C and D) 4T1 cells that were stained by CFSE ranged from 20, 45, 90, and 180 in total number and were spiked into 1 mL of healthy blood. Fluorescent images of the captured cell clusters on the chip are shown. Scale bar: 100 μm (C). Captured vs. input cell number (D). (E) Release efficiency and integrity of cell clusters from the chip as a function of the reverse flow rate at 4°C. (F) Images of the intact cell clusters captured following release into the solution from the Cluster-Chip operated at 250 mL/h. Scale bar: 100 μm and 20 μm. (G) Percentage of viable cell clusters released at different flow rates using Cluster-Chip against the control group. (H) Fluorescent images of cell clusters treated with two-color viability (green)/cytotoxicity (red) assay. The cells are divided in two batches including control and cell clusters captured and released from Cluster-Chip at 250 mL/h. Scale bar: 100 μm and 50 μm. Data were presented as mean ± SD. ∗∗p < 0.01.

Figure 2

Characterization and enumeration of CTM isolated from blood samples from breast cancer patients (A and B) Representative fluorescent micrographs of homotypic and heterotypic CTM in different sizes including neutrophils or platelets, respectively. Captured cells were stained with Cytokeratin (green), CD45 or P-selectin (red), DAPI (nuclei, blue). Scale bar: 50 μm. (C and D) CTM counts per 4 mL of peripheral blood samples from BC patients with non-metastasis (n = 6), lymph node metastasis (n = 17), and distant metastasis (n = 20). (E) CTM counts of BC patients with CEA <4.7 ng/mL and CEA ≥4.7 ng/mL. (F) Percentages of BC patients with different detectable types of CTM in distant metastasis (n = 20) and locoregional BC patients (n = 12). (G) Various CTM percentages from distant metastasis (n = 8) and locoregional BC patients (n = 5). (H) CTM counts for patients with different disease states across different types of CTM, corresponding to (G). Data are represented as mean ± SEM. ∗0.01 < p ≤ 0.05; ∗∗∗p ≤ 0.001.

Figure 3

Disturbing the interaction between platelets and CTM led to fewer metastases (A) IVIS images demonstrating metastasis of 4T1-Luc cells in lungs at the 16th day after injection. BALB/c female mice divided into 4 groups were injected with 1 × 106 cells from the tail vein. Two days prior to 4T1-Luc cell injection, mice were treated with vehicle, aspirin (ASA), U46619, and ASA plus U46619 for 3 weeks (n = 7, 9, 8, and 8, respectively). (B) The total flux of the cells in the mice in different groups. (C) Representative images of CTC clusters isolated from mice with metastatic breast cancer to the lungs. CTC clusters were stained with Pan-cytokeratin (green), CD45 (red), and DAPI (nuclei, blue). Scale bar: 50 μm. (D) Quantitation of CTC clusters per 1 mL of blood in mice of the 4 groups. (E and F) H&E staining of lung sections (left) and physical images of the lung (right) (E) The number of mouse lung metastases in different groups (F). Scale bar: 100 μm. (G) Change in body weight of mice. (H) Overall survival curves of the 4 groups of mice. Data are represented as mean ± SEM. ∗0.01 < p ≤ 0.05; ∗∗0.001 < p ≤ 0.01; ∗∗∗p ≤ 0.001.

Figure 4

Specific genes related to hypoxia stimulation and platelet activation were enriched in the CTM group versus the control group (A) Images of CTM released from the Cluster-Chip and live stained with FITC anti-mouse CD326 (EPCAM) (green) and Alex594-conjugated antibodies against CD45 (red). (B) Heatmap of gene expression related to epithelial status, mesenchymal status, cell stemness, leukocytes, cell junctions, and platelets in samples from each group. (C) Gene Ontology enrichment analysis in treatment vs. control groups. (D) Heatmap of gene expression associated with CTM-mediating metastasis in the two groups of samples, with some genes marked with red and some marked with green relating to hypoxia stimulation and platelet activation, respectively. (E) Verification of key genes that are differently expressed in treatment vs. control group by qPCR. (F) Representative fluorescent micrographs of 4T1 cell clusters captured on the chip following culture in ultra-low adhesion well plates with or without platelets under the hypoxic or normoxic conditions. CTC-clusters stained for Pan-cytokeratin and EPCAM (green), P-selectin (red), and DAPI (nuclei, blue). Scale bar: 50 μm and 20 μm. (G) The statistic quantification of the different types of captured CTM cultured in 4 conditions as mentioned in (F). Data were presented as mean ± SD. ∗0.01 < p ≤ 0.05; ∗∗0.001 < p ≤ 0.01; ∗∗∗p ≤ 0.001.