Single-cell sequencing technologies have revealed an unexpectedly broad repertoire of cells required to mediate complex functions in multicellular organisms. Despite the multiple roles of adipose tissue in maintaining systemic metabolic homeostasis, adipocytes are thought to be largely homogenous with only 2 major subtypes recognized in humans so far. Here we report the existence and characteristics of 4 distinct human adipocyte subtypes, and of their respective mesenchymal progenitors. The phenotypes of these distinct adipocyte subtypes are differentially associated with key adipose tissue functions, including thermogenesis, lipid storage, and adipokine secretion. The transcriptomic signature of “brite/beige” thermogenic adipocytes reveals mechanisms for iron accumulation and protection from oxidative stress, necessary for mitochondrial biogenesis and respiration upon activation. Importantly, this signature is enriched in human supraclavicular adipose tissue, confirming that these cells comprise thermogenic depots in vivo, and explain previous findings of a rate-limiting role of iron in adipose tissue browning. The mesenchymal progenitors that give rise to beige/brite adipocytes express a unique set of cytokines and transcriptional regulators involved in immune cell modulation of adipose tissue browning. Unexpectedly, we also find adipocyte subtypes specialized for high-level expression of the adipokines adiponectin or leptin, associated with distinct transcription factors previously implicated in adipocyte differentiation. The finding of a broad adipocyte repertoire derived from a distinct set of mesenchymal progenitors, and of the transcriptional regulators that can control their development, provides a framework for understanding human adipose tissue function and role in metabolic disease.

Materials/Methods: Women with HREC (FIGO-stage I grade 3 with deep myometrial invasion and/or LVSI; stage II or III; or serous/ clear cell histology) were randomized (1:1) to CTRT (two cycles of cisplatin 50 mg/ m 2 in week 1&4 of RT, followed by four cycles of carboplatin AUC5 and paclitaxel 175 mg/m 2 at 3-week intervals) or RT alone (48.6 Gy in 1.8 Gy fractions). The co-primary endpoints were overall survival (OS) and failure-free survival (FFS). Secondary endpoints vaginal, pelvic, and distant recurrence were analyzed according to first site of recurrence. The Kaplan-Meier method, log-rank test, and Cox regression analysis were used according to intention-to-treat, and competing risk methods for FFS and recurrence. Analysis of the primary endpoints was adjusted for the stratification factors (participating group, lymphadenectomy, stage of cancer and histological type). PORTEC-3 is registered with ISRCTN (ISRCTN14387080) and ClinicalTrials.gov (NCT00411138). Results: Six hundred eighty-six women were enrolled between 2006 and 2013; 26 were excluded for immediate informed consent withdrawal or ineligibility, leaving 660 patients in the final analysis, 330 CTRT and 330 RT. Median follow-up was 72.6 months (IQR 59.9-85.6). 5-year OS was 81.4% vs 76.1% for CTRT vs RT [HR 0.70, 95% CI 0.51-0.97, pZ0.034], and 5-year FFS was 76.5% vs 69.1% [HR 0.70, 95% CI 0.52-0.94, pZ0.016]. Women with serous cancers had significantly lower OS compared with other histologies (62.0% vs 81.9% at 5 years), while 5-year OS for serous cancer was 71.4% with CTRT vs 52.8% with RT [HR 0.48, 95% CI 0.24-0.96, pZ0.037], and 5-year FFS was 59.7% vs 47.9% [HR 0.42, 95% CI 0.22-0.80, pZ0.008]. For women with stage III disease an absolute 5-year OS improvement of 10% (HR 0.63, 95% CI 0.41-0.99, pZ0.043) and FFS improvement of 12.5% at (HR 0.61 (95% CI 0.42-0.89, pZ0.011) was found with CTRT. Distant metastases were the first site of recurrence in the majority of patients, 21.4% (CTRT) vs 29.1% (RT) (pZ0.047), and most patients received chemotherapy for recurrence. Survival after recurrence was 1.2 vs 1.4 years (pZ0.7). Pelvic control was high in both arms with isolated vaginal or pelvic recurrence in only 1.2%. Conclusion: This updated analysis with median FU of 6 years showed a significantly improved OS and FFS with combined adjuvant chemotherapy and radiation therapy for HREC. The largest improvement was found for women with stage III and/or serous cancers. Shared decision making remains essential to weigh the costs and benefits for individual patients.

Human thermogenic adipose tissue mitigates metabolic disease, raising much interest in understanding its development and function. Here, we show that human thermogenic adipocytes specifically express a primate-specific long non-coding RNA, LINC00473 which is highly correlated with UCP1 expression and decreased in obesity and type-2 diabetes. LINC00473 is detected in progenitor cells, and increases upon differentiation and in response to cAMP. In contrast to other known adipocyte LincRNAs, LINC00473 shuttles out of the nucleus, colocalizes and can be crosslinked to mitochondrial and lipid droplet proteins. Up- or down- regulation of LINC00473 results in reciprocal alterations in lipolysis, respiration and transcription of genes associated with mitochondrial oxidative metabolism. Depletion of PLIN1 results in impaired cAMP-responsive LINC00473 expression and lipolysis, indicating bidirectional interactions between PLIN1, LINC00473 and mitochondrial oxidative functions. Thus, we suggest that LINC00473 is a key regulator of human thermogenic adipocyte function, and reveals a role for a LincRNA in inter-organelle communication and human energy metabolism.

Obesity and type 2 diabetes are associated with disturbances in insulin-regulated glucose and lipid fluxes and severe comorbidities including cardiovascular disease and steatohepatitis. Whole body metabolism is regulated by lipid-storing white adipocytes as well as “brown” and “brite/beige” adipocytes that express thermogenic uncoupling protein 1 (UCP1) and secrete factors favorable to metabolic health. Implantation of brown fat into obese mice improves glucose tolerance, but translation to humans has been stymied by low abundance of primary human beige adipocytes. Here we apply methods to greatly expand human adipocyte progenitors from small samples of human subcutaneous adipose tissue and then disrupt the thermogenic suppressor gene NRIP1 by CRISPR. Ribonucleoprotein consisting of Cas9 and sgRNA delivered ex vivo are fully degraded by the human cells following high efficiency NRIP1 depletion without detectable off-target editing. Implantation of such CRISPR-enhanced human or mouse brown-like adipocytes into high fat diet fed mice decreases adiposity and liver triglycerides while enhancing glucose tolerance compared to implantation with unmodified adipocytes. These findings advance a therapeutic strategy to improve metabolic homeostasis through CRISPR-based genetic enhancement of human adipocytes without exposing the recipient to immunogenic Cas9 or delivery vectors.

ImportanceSpine metastasis can be treated with high-dose radiation therapy with advanced delivery technology for long-term tumor and pain control.ObjectiveTo assess whether patient-reported pain relief was improved with stereotactic radiosurgery (SRS) as compared with conventional external beam radiotherapy (cEBRT) for patients with 1 to 3 sites of vertebral metastases.Design, Setting, and ParticipantsIn this randomized clinical trial, patients with 1 to 3 vertebral metastases were randomized 2:1 to the SRS or cEBRT groups. This NRG 0631 phase 3 study was performed as multi-institutional enrollment within NRG Oncology. Eligibility criteria included the following: (1) solitary vertebral metastasis, (2) 2 contiguous vertebral levels involved, or (3) maximum of 3 separate sites. Each site may involve up to 2 contiguous vertebral bodies. A total of 353 patients enrolled in the trial, and 339 patients were analyzed. This analysis includes data extracted on March 9, 2020.InterventionsPatients randomized to the SRS group were treated with a single dose of 16 or 18 Gy (to convert to rad, multiply by 100) given to the involved vertebral level(s) only, not including any additional spine levels. Patients assigned to cEBRT were treated with 8 Gy given to the involved vertebra plus 1 additional vertebra above and below.Main Outcomes and MeasuresThe primary end point was patient-reported pain response defined as at least a 3-point improvement on the Numerical Rating Pain Scale (NRPS) without worsening in pain at the secondary site(s) or the use of pain medication. Secondary end points included treatment-related toxic effects, quality of life, and long-term effects on vertebral bone and spinal cord.ResultsA total of 339 patients (mean [SD] age of SRS group vs cEBRT group, respectively, 61.9 [13.1] years vs 63.7 [11.9] years; 114 [54.5%] male in SRS group vs 70 [53.8%] male in cEBRT group) were analyzed. The baseline mean (SD) pain score at the index vertebra was 6.06 (2.61) in the SRS group and 5.88 (2.41) in the cEBRT group. The primary end point of pain response at 3 months favored cEBRT (41.3% for SRS vs 60.5% for cEBRT; difference, −19 percentage points; 95% CI, −32.9 to −5.5; 1-sided P = .99; 2-sided P = .01). Zubrod score (a measure of performance status ranging from 0 to 4, with 0 being fully functional and asymptomatic, and 4 being bedridden) was the significant factor influencing pain response. There were no differences in the proportion of acute or late adverse effects. Vertebral compression fracture at 24 months was 19.5% with SRS and 21.6% with cEBRT (P = .59). There were no spinal cord complications reported at 24 months.Conclusions and RelevanceIn this randomized clinical trial, superiority of SRS for the primary end point of patient-reported pain response at 3 months was not found, and there were no spinal cord complications at 2 years after SRS. This finding may inform further investigation of using spine radiosurgery in the setting of oligometastases, where durability of cancer control is essential.Trial RegistrationClinicalTrials.gov Identifier: NCT00922974