Page 23 - Read Online

Tran et al. J Cancer Metastasis Treat 2020;6:47 I http://dx.doi.org/10.20517/2394-4722.2020.104 Page 7 of 11

Figure 2. ROR2 regulated the survival of LM8 in lung capillaries. A: fluorescently labeled LM8 cells in the lungs were quantified using
an inverted confocal fluorescence microscope (top). Data are shown as the mean ± SD of three fields of fluorescently labeled cells in
each lung on the indicated days. n = 3, **P < 0.01. Representative image of LM8 cells in lungs on the indicated days (bottom). Red: blood
vessel, Green: tumor cell, Black: lung tissue. Scale: 50 μm; B: ROR2 protein level in the indicated LM8 sublines. The numbers below the
bands indicate the corresponding ROR2 expression levels relative to GAPDH; C: fluorescence intensity in lung lysates prepared from
mice injected with the indicated LM8 sublines was quantified on day 0 (30 min) and day 2 (48 h). Data are shown as the ratio of day 2
fluorescence intensity normalized to day 0. n = 3, *P < 0.05. ROR2: receptor tyrosine kinase-like orphan receptor 2; LM8-H: LM8 cell line
with high metastatic ability; LM8-L: LM8 cell line with low metastatic ability; H/Ror2-KO: LM8-H knocked out of Ror2; L/ROR2: LM8-L
expressing ROR2; KO/ROR2: H/Ror2-KO expressing ROR2; n.s: not significant

[22]
than 90% of tumor cells entering the capillaries . The LM8 sublines were examined for sensitivity to
mechanical stress in vitro using a hypotonic buffer that causes hypotonic cell swelling, an established
perturbation method that examines the strength against mechanical stress by inducing elongation of the
[23]
plasma membrane in a well-controlled manner . Incubation of LM8-H and H/Ror2-KO in hypotonic
buffer for 30 min reduced viability by less than half in both sublines compared to incubation in isotonic
buffer [Supplementary Figure 3], indicating that ROR2 was not involved in the resistance to mechanical
stress in the lung capillaries. The differences in anoikis resistance among the LM8 sublines were examined
in terms of viability after culturing for 24 h on poly-HEMA-coated dishes (under low adhesion conditions)
using fluorescent dyes that differentially label live and dead cells. The LM8 sublines with low ROR2
expression (LM8-L and H/Ror2-KO) significantly increased anoikis, and those with high ROR2 expression
(L/ROR2 and KO/ROR2) significantly decreased anoikis [Figure 3A]. It has been reported that the activity
[24]
of Akt was upregulated in anoikis resistant human OS cells . Therefore, we investigated the involvement
of AKT signaling in LM8 ROR2-induced anoikis resistance. First, AKT activation (Ser473 phospho-