Page 21 - Read Online

Tran et al. J Cancer Metastasis Treat 2020;6:47 I http://dx.doi.org/10.20517/2394-4722.2020.104 Page 5 of 11

(Nacalai Tesque) was prepared with dimethyl sulfoxide (DMSO), and the cells were cultured for 24 h in
poly-HEMA-treated plates containing 50 nmol/L MK2206 in 5% FBS DMEM.

Statistical analysis
Data were statistically analyzed with a two-sided Student’s t-test. P-values of less than 0.05 were considered
statistically significant.

RESULTS
ROR2 function was required for OS lung metastasis
Our recent study using the LM8 sublines (LM8-H and LM8-L) and their gene expression microarray data
revealed that noncanonical Wnt signaling is associated with LM8 extravasation into lung tissue via the
[16]
LEF1-CYGB axis . The microarray data of LM8-H and LM8-L was used to search for genes associated
[19]
with the noncanonical Wnt pathway. Gene set enrichment analysis identified several Wnt signaling-
related genes that showed higher expression in LM8-H than in LM8-L [Figure 1A]. Mapping of these genes
[20]
using Kyoto Encyclopedia of Genes and Genomes and clusterProfile package using R/Bioconductor
indicated that ROR1/2 were receptors for the noncanonical Wnt signaling that is likely involved in the lung
metastasis of LM8 [Supplementary Figure 1]. Expression levels of noncanonical Wnt signaling receptor
genes Ror1, Ror2, and Frizzled class receptor 1 (Fzd1) were examined by RT-PCR. Ror2, but not Ror1 or
Fzd1, was expressed at a significantly higher level in LM8-H than in LM8-L [Figure 1B]. Furthermore, the
mRNA level of Ror2 was well-correlated with the metastasis-free survival of OS patients [Supplementary
Figure 2]. Therefore, we focused on ROR2 for further analysis. The effect of deletion of Ror2 [Figure 1C] on
the proliferation and metastatic potential of LM8-H was first investigated. The proliferation rate of H/Ror2-
KO was similar to that of LM8-H and LM8-L [Figure 1D], indicating that ROR2 function is not involved in
proliferation. The size and number of foci in the lungs injected with H/Ror2-KO were significantly smaller
than those in the lungs injected with LM8-H [Figure 1E]. These data demonstrated the critical role of
ROR2 function in LM8 lung metastasis.

ROR2 was necessary for LM8 survival in lung capillaries
To determine how ROR2 was involved in the extravasation process in vivo, the LM8 sublines in the lungs
were examined at 30 min and 48 h post-injection because the tumor cells had reached the lungs by 30 min
and began extravasation into lung tissues by 48 h post-injection. The LM8 sublines were labeled with
a green fluorescent dye before injection, and removed lungs were observed under an inverted confocal
fluorescence microscope. Similar numbers of fluorescently labeled cells were observed in the lungs injected
with LM8-H and H/Ror2-KO 30 min after injection [Figure 2A], indicating that lack of ROR2 function
did not affect the survival of circulating LM8 cells before reaching the lungs. On the other hand, 48 h after
injection, the number of fluorescently labeled cells in lungs injected with H/Ror2-KO was significantly
reduced compared to that in lungs injected with LM8-H [Figure 2A]. The reduction reflects that more H/
Ror2-KO die in lung capillaries than LM8-H, suggesting that ROR2 function was involved in the survival of
LM8 cell in lung capillaries prior to extravasation. To confirm ROR2’s function, LM8-L, LM8-H, H/Ror2-
KO, ROR2-overexpressing LM8-L (L/ROR2), and ROR2-overexpressing H/Ror2-KO (KO/ROR2) [Figure
2B] were examined at 48 h post-injection in terms of their survival in lung capillaries. Significantly higher
fluorescence intensity was detected in lung lysates from mice injected with the LM8 sublines expressing
high ROR2 (LM8-H, L/ROR2, and KO/ROR2) compared to that in the lung lysates from mice injected with
the LM8 sublines expressing low ROR2 (LM8-L and H/Ror2-KO) [Figure 2C]. These results confirmed a
correlation between ROR2 expression and LM8 survival in lung capillaries.

The AKT signaling pathway was involved in ROR2-induced anoikis resistance in LM8
Tumor cells that have reached the lung capillaries are stressed mechanically because the blood vessels
are smaller in diameter and are less compliant . This mechanical stress causes cell death in up to more
[21]