Platelets play a crucial role in tumor metastasis by being recruited and activated by tumor cells, and subsequently binding to circulating tumor cells (CTCs) to facilitate the spread of tumor cells to distant organs. Therefore, nanoparticles that hitchhike to activated platelets are believed to have excellent targeting ability for primary tumors, CTCs, and metastasis to distant organs. However, activated tumor-homing platelets release transforming growth factor-β (TGF-β), which promotes tumor metastasis and creates an immunosuppressive microenvironment. Therefore, a strategy is needed to balance accurate tumor tracking with the alleviation of immunosuppressive signals.

Hence, the featured blog discusses the research titled “Efficient Treatment of Metastatic Cancer through Fucoidan-Functionalized Activated Platelet-Hitchhiking Micelles that Track Tumor Cells and Modify the Immunosuppressive Microenvironment”. It was conducted by Rong Guo et al., where they developed micelles (FD/DOX – Doxorubicin) functionalized with fucoidan. These micelles effectively bind to activated platelets through P-selectin.

Platelets play an important role in cancer development. As shown in Figure 1A and B, we observed that in mice bearing 4T1 (Mouse breast cancer cells) tumors, a large number of platelets were recruited to the primary tumor and metastatic sites in the lungs and liver, and the platelet counts were higher than those in tumor-free mice. These results confirmed that tumors promote platelet production, and that activated platelets have tumor and metastasis-homing ability.

P-selectin is a glycoprotein that is overexpressed in activated platelets. According to the findings in Figure 1C, the in vitro detection indicated that thrombin-activated platelets exhibited a significant increase in P-selectin expression. Thus, P-selectin was deemed a potential target by activated platelets. In addition, various inflammatory factors, such as TNF-α, secreted by tumor cells could stimulate vascular endothelium to increase the expression of P-selectin. As shown in Figure 1D and Supporting Information Figure S1, after being cultured in the culture medium of 4T1 cells, HUVECs showed higher expression of P-selectin. Also, in Figure 1E, the expression of P-selectin on vascular endothelium around primary tumors and metastases was significantly increased, promoting the accumulation of P-selectin-targeted nanoparticles in tumor and metastatic sites.

The researchers successfully developed micelles that are functionalized with fucoidan and can effectively bind to activated platelets through the use of P-selectin, also known as FD/DOX. So, then to confirm that FD/DOX binds to activated platelets via P-selectin, half of the activated platelets were preincubated with anti-CD62P antibody and then incubated with FD/DOX. As shown in Figure 2G, pretreatment with anti-CD62P (p-selection) antibody weakened the fluorescence quantified by the flow cytometer, indicating that FD/DOX specifically binds to P-selectin on activated platelets.

Additionally, the researchers conducted cellular uptake assays in 4T1 cells and observed that the uptake of FD/DOX was hindered by low temperature and several inhibitors. These findings suggest the involvement of multiple uptake mechanisms that could potentially enhance the cellular uptake of FD/DOX.

Activated platelets have been reported to adhere to CTCs (circulating tumor cells) as a “protective coat” to protect cells from loss and promote cancer development. Hence, it was important to investigate the cellular uptake of micelles in the presence of activated platelets. 4T1 cells were preincubated with activated platelets for 30 minutes before incubation with DOX-loaded micelles (DD/DOX) or FD/DOX. The uptake of DD/DOX by cells was significantly decreased when they were preincubated with activated platelets. This is because the adhesion of platelets to the surface of 4T1 cells prevents tumor cells from contacting DD/DOX. However, preincubation with activated platelets increased the cellular uptake of FD/DOX. This is likely because activated platelets act as a “bridge” between FD/DOX and tumor cells, increasing the contact of FD/DOX with 4T1 cells.

They also established a 4T1 spontaneous metastasis model with 4T1-Luc cells and investigated the antitumor and antimetastatic effects in vivo. Compared with other groups, the luminescence signals in the tumor area of ​​mice treated with FD/DOX were significantly reduced, indicating a strong inhibitory effect on 4T1 tumors. Moreover, the luminescent signals in the lungs of mice that received DOX and DD/DOX treatments were significantly brighter compared to those in mice treated with Fu. On the other hand, mice treated with FD/DOX exhibited almost no luminescent signal in their lung area. The results suggest that FD/DOX showed exhibited the best inhibitory effect on metastasis.

The anti-metastatic activity of the formulations was evaluated in the lungs and liver, respectively. Many metastatic nodules were observed on the surface of the lungs treated with Glu, DOX, and DD/DOX, while few nodules were visible in the lungs treated with FD/DOX. This can be attributed to its capability to attach to activated platelets. By hitchhiking to activated platelets, FD/DOX can not only enhance the targeting effect on tumors and metastasis but also track and kill CTCs in the blood circulation. Fu also exhibited an anti-metastatic effect, which may be because the binding of P-selectin on the vascular endothelium with fucoidan can competitively inhibit CTCs from attaching to the vascular endothelium and transplanting to distant organs.

In order to study the prolonged anti-metastatic impact of FD/DOX, we utilized the lungs and livers of mice that had been injected with 4T1-Luc cells for H&E staining. There was still no obvious luminescent signal in the lung area of ​​the mice. The results of H&E staining also showed that there was no serious metastasis in the lungs and liver. Therefore, it was considered that FD/DOX had a long-lasting anti-metastatic effect.

Overall, the utilization of fucoidan-functionalized activated platelet-targeting micelles presents a promising approach for treating metastatic cancer. This approach involves accurately monitoring tumor cells and modifying the microenvironment of primary tumors and pre-metastatic organs.

Source: Acta Pharm Sin B. 2022 Jan; 12(1): 467–482. doi: 10.1016/j.apsb.2021.05.012