Dissecting the intra- and intertumoral heterogeneity of adrenocortical carcinoma by single-cell multi-omics analyses

Adrenocortical carcinoma (ACC) is a rare but aggressive malignancy originating in the adrenal cortex, characterized by significant intra- and intertumoral heterogeneity. In this study, we employed a combination of whole-genome sequencing, single-cell RNA sequencing, T cell receptor sequencing, spatial transcriptome sequencing, and multiple fluorescent staining to construct a comprehensive multi-omics landscape of ACC. Our findings demonstrated that, although all tumor cells exhibited a "confused cell identity" feature, distinct subpopulations were present in each ACC patient. Among these, the *LDLR*+ and *MKI67*+ subpopulations expressed elevated levels of C1A signature genes and established robust communication with endothelial cells through *NECTIN*-*PVR* and *NOTCH2*-*JAG* pairs, both significantly associated with poor prognosis. Interestingly, the *PCDH15*+ subpopulations displayed moderate levels of both C1A signature genes and extracellular matrix related genes. On the other hand, the *HLA-B*+ and subpopulation exhibited the highest levels of antigen-processing related genes, a characteristic not previously reported. The amalgamation of these distinct subpopulations facilitated the stratification of ACC patients into subtypes with varying prognoses. Patients (Group II) dominated by *LDLR*+ and *MKI67*+ subpopulations exhibited the worst prognosis, while those (Group III) with a higher proportion of *PCDH15*+ subpopulation demonstrated the most favorable outcomes. Significantly, patients (Group I) with a higher prevalence of the *HLA-B*+ subpopulation also showed notably increased *LAG3*+ memory CD8 T cells, indicating their potential suitability for immunotherapy. Despite responding averagely to conventional treatment, this subgroup was predicted to be responsive to immunotherapy. Notably, one patient from Group I exhibited a positive response to camrelizumab treatment after relapse with mitotane, leading to a successful outcome, while two patients from Group II did not respond favorably. Our study offers insights into the single-cell level heterogeneity of ACC and provides valuable implications for precision treatments for this disease.