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

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. Overall design: With approval from the Ethics Committee of West China Hospital, Sichuan University, we collected 3 adjacent normal adrenal tissues from 3 aldosterone producing adenoma patients who are diagnosed with adrenal venous sampling and CT, and ACC tumor samples from 8 pathologically diagnosed ACC patients during surgical resections. Their ages varied from 18 to 64, with a median of 50. Clinical characteristics including sex, age, plasma levels of aldosterone, ACTH, cortisol, metanephrine, normetanephrine, testosterone, estradiol, progesterone, hormone activity, 24h urine levels of cortisol, duration from diagnosis to surgery, specimen type, tumor size, ENST stage and TNM stage are listed in Supplementary Table 1. Three patients were included in our clinical trial (Registration number: ChiCTR1900028111) and received ICI plus apatinib treatment.