Immortalized Human Bone Marrow Mesenchymal Cells-hTERT

The p53 Isoform Delta133p53 Drives High Level of HGF Secretion in Ewing Sarcoma

Ewing sarcoma (EWS) represents a challenging cancer with limited success in current treatments due to its aggressive nature and poor prognosis in advanced stages. Aberrant expression of HGF, driven by delta133p53, facilitates tumor growth and metastasis. To address this, Charan et al.explored the therapeutic potential of combining an HGF receptor neutralizing antibody (AMG102) with GD2-directed CAR-T therapy.

They examined the underlying molecular mechanism of high levels of HGF expression in EWS. Ewing sarcoma (EWS) is characterized by the expression of the EWS/FLI fusion protein, a key driver mutation disrupting gene regulation related to cancer. They hypothesized that EWS/FLI leads to high HGF expression. To test this, they used siRNA to knock down EWS/FLI1 in cell lines and analyzed HGF expression. Surprisingly, as shown in Figure 1a, HGF levels remained unchanged, suggesting its regulation is independent of EWS/FLI. In Ewing sarcomas, only 10% show genetic lesions in TP53, with most expressing functional wild-type p53. Surprisingly, the oncogenic p53 isoform Δ133p53 is abnormally expressed in EWS. They investigated whether Δ133p53 influences HGF expression using Ewing cell lines ES2 and ES4 with inducible vectors to inhibit Δ133p53. Figures 1b and 1c show that reducing Δ133p53 significantly decreased HGF levels. To confirm their findings further, they genetically engineered immortalized human bone marrow mesenchymal cells (hMSC-hTERT), thought to be the cellular origin of EWS, by overexpressing Δ133p53 or EWS/FLI. Overexpressing Δ133p53 in hMSC-hTERT increased HGF expression, unlike EWS/FLI (Fig.1f-g). Taken together, their data demonstrate that high level of HGF expression and secretion are driven by Δ133p53 and independent of EWS/FLI1 expression.

Fig. 1. Δ133p53 expression drives HGF expression and secretion (Charan M, Dravid P, et al., 2019).

MMC Stabilized MVNs in Microfluidic Devices

Microvessels are key in molecular exchange and disease processes. Current two-dimensional and in vivo models lack detailed spatiotemporal insights. While engineered MVNs in microfluidics show promise, their instability limits long-term studies. Wan et al. introduces macromolecular crowding (MMC) in microfluidic devices as a method to stabilize MVNs by mimicking naturally crowded in vivo conditions, thereby obviating the need for auxiliary cells or protease inhibitors.

Utilizing a design with three channels separated by permeable partitions (Fig. 2A), HUVECs and immortalized human bone marrow mesenchymal cells – hTERT (hTERT-MSCs), acting as pericytes, were seeded into the central channel in a fibrinogen solution (Fig. 2B). Thrombin facilitated rapid fibrin hydrogel formation, serving as a 3D matrix for the cells. Protease inhibitors were excluded to avoid affecting biological processes. Cells formed tubule-like structures and interconnected MVNs within two days (Fig. 2C). One day post-seeding, macromolecular crowding (MMC) was optionally added to the culture medium. The MMC cocktail, using two sizes of Ficoll macromolecules, has shown to promote ECM deposition in both 2D and 3D cultures.

Fig. 2. HUVECs and MSCs form interconnected MVNs within the microfluidic device (Wan H Y, Chen J C H, et al., 2023).