Cholesteryl ester (CE) accumulation in intracellular lipid droplets (LDs) is an essential signature of clear cell renal cell carcinoma (ccRCC), but its molecular mechanism and pathological significance remain elusive. Enabled by the label-free Raman spectromicroscopy, which integrated stimulated Raman scattering microscopy with confocal Raman spectroscopy on the same platform, we quantitatively analyzed LD distribution and composition at the single cell level in intact ccRCC cell and tissue specimens in situ without any processing or exogenous labeling. Since we found that commonly used ccRCC cell lines actually did not show the CE-rich signature, primary cancer cells were isolated from human tissues to retain the lipid signature of ccRCC with CE level as high as the original tissue, which offers a preferable cell model for the study of cholesterol metabolism in ccRCC. Moreover, we established a patient-derived xenograft (PDX) mouse model that retained the CE-rich phenotype of human ccRCC. Surprisingly, our results revealed that CE accumulation was induced by tumor suppressor VHL mutation, the most common mutation of ccRCC. Moreover, VHL mutation was found to promote CE accumulation by upregulating HIFα and subsequent PI3K/AKT/mTOR/SREBPs pathway. Inspiringly, inhibition of cholesterol esterification remarkably suppressed ccRCC aggressiveness in vitro and in vivo with negligible toxicity, through the reduced membrane cholesterol-mediated downregulations of integrin and MAPK signaling pathways. Collectively, our study improves current understanding of the role of CE accumulation in ccRCC and opens up new opportunities for treatment. This work was supported by National Natural Science Foundation of China (No. U23B2046 and No. 62027824), National Key R&D Program of China (No. 2023YFC2415500), Fundamental Research Funds for the Central Universities (No. YWF-22-L-547), PKU-Baidu Fund (No. 2020BD033), Peking University First Hospital Scientific and Technological Achievement Transformation Incubation Guidance Fund (No. 2022CX02), and Beijing Municipal Health Commission (No. 2020-2Z-40713).

Background: Studies on biological functions of N6‐methyladenosine (m⁶A) modification in mRNA have sprung up in recent years. Previous studies have reported m⁶A can determine mRNA fate and play a pivotal role in tumour development and progression. The zinc finger protein 677 (ZNF677) belongs to the zinc finger protein family and possesses transcription factor activity by binding sequence‐specific DNA.Methods: The expression of ZNF677 and its clinicopathological impact were evaluated in renal cell carcinoma (RCC) patients. The m⁶A level of ZNF677 was determined by m⁶A methylated RNA immunoprecipitation‐sequencing (MeRIP‐seq) and MeRIP‐qPCR in RCC tissues and adjacent normal tissues. RNA immunoprecipitation‐qPCR (RIP‐qPCR) and luciferase assays were performed to identify the targeted effect of IGF2BP2 and YTHDF1 on ZNF677. RCC cells and subcutaneous models uncovered the role of ZNF677 methylated by CRISPR/dCas13b‐METTL3 in tumour growth. ZNF677‐binding sites in the CDKN3 promoter were investigated by chromatin immunoprecipitation (ChIP) and luciferase assays.Results: ZNF677 is frequently downregulated in RCC tissues and its low expression is associated with unfavourable prognosis and decreased m⁶A modification level. Further, we find the m⁶A‐modified coding sequence (CDS) of ZNF677 positively regulates its translation and mRNA stability via binding with YTHDF1 and IGF2BP2, respectively. Targeted specific methylation of ZNF677 m⁶A by CRISPR/dCas13b‐METLL3 system can significantly increase the m⁶A and expression level of ZNF677, and dramatically inhibit cell proliferation and induce cell apoptosis of RCC cells. In addition, ZNF677 exerted its tumour suppressor functions in RCC cells through transcriptional repression of CDKN3 via binding to its promoter. In vitro and clinical data confirm the negative roles of ZNF677/CDKN3 in tumour growth and progression of RCC.Conclusion: ZNF677 functions as a tumour suppressor and is frequently silenced via m⁶A modification in RCC, which may highlight m⁶A methylation‐based approach for RCC diagnosis and therapy.

Previous reports about laparoscopic total pelvic exenteration (LTPE) are still limited. In the present study, we described our single-center experience of the initial 11 cases. Between April 2011 and September 2015, eight males and three females diagnosed as pelvic malignancies underwent LTPE by the same operation team. We retrospectively collected all cases’ parameters about surgical technique. Thirty-seven patients who received open surgery were also retrospectively collected. A comparison between LTPE and open surgery was performed to evaluate the feasibility and safety of LTPE. Eleven cases successfully underwent the LTPE without any intraoperative complication. No open conversion was required. Eight patients underwent Bricker’s procedure. Three patients were performed with the cutaneous ureterostomy. Anus preservation operation was performed in three patients. Compared with open surgery, LTPE had longer mean operative time (565.2 vs 468.2 min, p = 0.004) but less mean blood loss (547.3 vs 1033.0 ml, p < 0.001) and shorter postoperative hospitalization time (15.3 vs 22.4 days, p = 0.004). One patient died of pulmonary embolism in the 7th month of follow-up time. One patient died of recurrence in the 12th month of follow-up time. Nine patients are still alive without recurrence and metastasis. The mean follow-up time was 11.1 months. The technique of LTPE seems to be feasible and safe in the treatment of carefully selected patients of pelvic malignancies. LTPE can also decrease the blood loss, the recovery time, and the hospital stay. But the oncological safety and long-term outcome of LTPE still need to be explored.