Main findings and interpretation
Previous studies have demonstrated that SPOP can suppress or promote tumorigenesis in a variety of malignancies, including lung, colon, gastric, prostate, and liver cancers [
10]. However, few studies have focused on SPOP in the development of CC, and only two reports have also shown dual effects [
17,
18]. Traditional procedures have been used to elucidate the mechanism by regularly exploring such molecular pathways that would destroy the spatial structure [
62,
63]. By the m-IF and HALO systems, the spatial orientation interrelation of immune cells and immune markers would be preserved [
22,
64]. Through a series of experiments, we demonstrated for the first time that SPOP promotes CC pLN metastasis by promoting PD-1 movement away from PD-L1.
Beginning with this study, we found that the expression of SPOP was increased in CC in the pLN-positive group compared with the pLN-negative group. This suggests that SPOP may be associated with pLN metastasis in CC. Second, further analysis found that the High-SPOP group had poorer OS and RFS. In conclusion, these results demonstrated that SPOP is upregulated in CC with pLN metastasis and negatively associated with patient outcomes.
Next and most importantly, we needed to conduct in vitro or in vivo experiments to prove a causal relationship between SPOP and CC cell migration and invasion. We showed in vitro that knockdown or overexpression of SPOP can significantly inhibit or promote, respectively, CC cell proliferation, cloning, cell cycle, wound healing, and Transwell assays. These data suggest that SPOP can promote the migration and invasion of CC cells. Moreover, our in vivo experiments demonstrated that SPOP knockdown can significantly inhibit the metastasis of CC cells.
From bench to bedside, we analysed the immune network environment of the CC TMA again to determine the potential mechanism of pLN metastasis. PD-L1 expression has been detected in the majority of CC using IHC analysis of tumour cells [
65], suggesting that anti-PD-1 therapies may be effective in CC. As a result, a series of clinical trials for the anti-PD-1-based treatment of advanced or recurrent CC have emerged, such as Keynote 028, Keynote 158, and Checkmate 358 [
66‐
68]. We can conclude that pembrolizumab (an anti-PD-1 drug) provides durable antitumor activity in partial patients, regardless of PD-L1 expression, and has manageable toxicity. Afterwards, pembrolizumab received approval from the Food and Drug Administration (FDA) for the treatment of patients with recurrent or metastatic CC with disease progression during or after chemotherapy (whose tumours express PD-L1) based on the objective response rate and durability of responses [
67]. However, the response rate of PD-1 in tumors is still low in general, which requires further study. Here, we have discovered a new mechanism of immune checkpoint inhibitors (ICIs) in the treatment of CC. Through quantitative pathology and tissue space analysis of the HALO system, the tumour area, PD-1 numbers, and PD-L1 numbers of each point on TMA representing tissue samples from different patients were presented. According to statistical principles, if continuous variables with more than 30 cases conform to the normal distribution,
t test can be used to analyse the differences between the two groups; our analysis showed that the differences were not statistically significant [
69]. However, the number of PD-1 molecules in the 100 µm PD-L1 range decreased significantly with increasing SPOP expression. Therefore, the core idea of the whole paper also emerged. With the increase in SPOP expression, PD-1 was significantly farther away from PD-L1 in terms of spatial distance.
Lastly, to further verify the causal relationship between the PD-1/PD-L1 axis and SPOP, a Co-IP-MS experiment was conducted. We dissected how the cellular components of the tumour, immune compartments interact through chemical-receptor signaling and spatial barrier levels. It has been previously reported that tumour cells can potentially mediate T cell recruitment via the CXCL16-CXCR6 axis [
57,
58,
70]. In addition, CAFs can create a physical barrier that blocks the access of T cells to the tumour epithelium [
60,
61,
71]. Our study revealed that SPOP can bind CXCL16 and promote its degradation, resulting in CAFs increasing the spatial distance between immune cells and tumour cells. Moreover, we compared the control group and SPOP knockdown group with different concentrations of PD-1 in HeLa and SiHa cell lines in vitro and found that knocking down SPOP could significantly reduce the IC50 value of PD-1. Further knockdown of CXCL16 in SPOP-shRNA1 cell lines could reverse these effects.
These results suggest that SPOP can achieve immune tolerance by promoting PD-1 sequestration from PD-L1.