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Spencer et al. J Cancer Metastasis Treat 2022;8:2 https://dx.doi.org/10.20517/2394-4722.2021.174 Page 5 of 15

Transfection of 143B cells, with a luciferase-Tomato reporter gene (pFULT) expressing firefly luciferase 2,
allowed for the development of murine orthotopic models of OS that are easily traceable in vivo. Also, the
cells were labelled with tdTomato making it possible to distinguish between tumour cells and host cells
during immunohistochemical analysis. Histological characterisation of tissue samples generated from this
orthotopic model revealed that they mimicked human disease, with a strong expression of MT1-MMP in
tumour cells excised from the tibia and from metastatic lesions that had developed in the lungs. In addition
to this, utilising Crispr/Cas9 gene editing techniques, the researchers generated MT1-MMP knockout and
wildtype cells lines (also expressing luciferase and tdTomato), both of which were screened for MT1-MMP
protein expression by western blot and genotyped. Comparison of these cell lines revealed that loss of MT1-
MMP expression resulted in a reduction in the ability of 143B cells to degrade cellular collagen. Further
investigation showed that there was no difference in the extent of bone degradation or the formation of lung
metastases in orthotopic models of the newly derived cell lines, however. This suggests that MT1-MMP
does not play a direct role in bone degradation or in the lung colonisation processes in 143B orthotopic
models of OS, but rather acts via alternative methods .
[30]
The differential expression of MT1-MMP in OS cells and clinical samples, when compared to controls,
provides compelling evidence for its involvement in OS development. Many of the clinical studies are
limited by small sample sizes, a common problem encountered when evaluating protein expression in OS
tumours (given the rarity of the disease), and a more comprehensive study with a larger sample size could
provide a better understanding of the role of MT1-MMP in OS. Within the tumour microenvironment, the
complex network of pathways, many of which could be involved in the regulation of MT1-MMP,
determines the rate of progression and metastasis in OS. These pathways, summarised in Figure 2, will be
considered in the following section.

ROLE OF MT1-MMP IN OS PROGRESSION AND METASTASIS
The regulation of MT1-MMP expression by the key SRC/ERK1/2 signalling pathway (a major player in OS
progression, Figure 2) in response to WNT5A has been investigated by Wang et al. . They found that
[31]
WNT5A-induced MG-63 cell migration and invasion was in part due to the activation of SRC/ERK1/2
signalling pathways that were shown to upregulate downstream MT1-MMP. Previous studies have reported
that WNT5A-induced invasion in osteosarcoma cell lines (U2OS and Saos-2) is mediated by SRC through
upregulation of MMP-13 [32,33] . The authors speculated that activation of the SRC/ERK1/2 signalling pathway
is responsible for the increased expression of both MT1-MMP and MMP-13 in response to WNT5A.

The activity of a Wnt co-receptor, low-density lipoprotein receptor-related protein 5 (LRP5), has been
linked with OS metastasis and poorer prognosis. Canonical Wnt signalling is responsible for the
downstream regulation of a broad spectrum of cell cycle regulators, oncogenes and matrix-degrading
enzymes. The transfection of a soluble form of LRP5 (sLRP5) into SaOS-2 cells significantly reduced the
protein expression of MT1-MMP and MMP-2, with the effect on MMP-2 being more pronounced. MT1-
MMP is a known Wnt/β-catenin target gene and membrane-bound activator of MMP-2 . The authors did
[34]
note that due to the differences in genetic backgrounds and mutational profiles of OS cell lines, the results
here in Saos-2 cells cannot be generalised. Therefore further investigation of sLRP5 activity in additional cell
[35]
lines that are stable following sLRP5 transfection is required .
α1-Antitrypsin Portland (α1-PDX), a furin inhibitor, has been shown to affect the migration and invasion of
MG-63 and Saos-2 OS cell lines . This study suggested that α1-PDX may play a key role in the inhibition of
[36]
migration and invasion through the non-canonical Wnt pathway-mediated downregulation of MT1-MMP
expression. It would be interesting to see what further exploration into the effect α1-PDX has on the activity