Single-cell analysis reveals context-dependent, cell-level selection of mtDNA.

Heteroplasmy occurs when wild-type and mutant mitochondrial DNA (mtDNA) molecules co-exist in single cells1. Heteroplasmy levels change dynamically in development, disease and ageing2,3, but it is unclear whether these shifts are caused by selection or drift, and whether they occur at the level of cells or intracellularly. Here we investigate heteroplasmy dynamics in dividing cells by combining precise mtDNA base editing (DdCBE)4 with a new method, SCI-LITE (single-cell combinatorial indexing leveraged to interrogate targeted expression), which tracks single-cell heteroplasmy with ultra-high throughput. We engineered cells to have synonymous or nonsynonymous complex I mtDNA mutations and found that cell populations in standard culture conditions purge nonsynonymous mtDNA variants, whereas synonymous variants are maintained. This suggests that selection dominates over simple drift in shaping population heteroplasmy. We simultaneously tracked single-cell mtDNA heteroplasmy and ancestry, and found that, although the population heteroplasmy shifts, the heteroplasmy of individual cell lineages remains stable, arguing that selection acts at the level of cell fitness in dividing cells. Using these insights, we show that we can force cells to accumulate high levels of truncating complex I mtDNA heteroplasmy by placing them in environments where loss of biochemical complex I activity has been reported to benefit cell fitness. We conclude that in dividing cells, a given nonsynonymous mtDNA heteroplasmy can be harmful, neutral or even beneficial to cell fitness, but that the 'sign' of the effect is wholly dependent on the environment.

Epigenetic profiling reveals key genes and cis-regulatory networks specific to human parathyroids.

In all terrestrial vertebrates, the parathyroid glands are critical regulators of calcium homeostasis and the sole source of parathyroid hormone (PTH). Hyperparathyroidism and hypoparathyroidism are clinically important disorders affecting multiple organs. However, our knowledge regarding regulatory mechanisms governing the parathyroids has remained limited. Here, we present the comprehensive maps of the chromatin landscape of the human parathyroid glands, identifying active regulatory elements and chromatin interactions. These data allow us to define regulatory circuits and previously unidentified genes that play crucial roles in parathyroid biology. We experimentally validate candidate parathyroid-specific enhancers and demonstrate their integration with GWAS SNPs for parathyroid-related diseases and traits. For instance, we observe reduced activity of a parathyroid-specific enhancer of the Calcium Sensing Receptor gene, which contains a risk allele associated with higher PTH levels compared to the wildtype allele. Our datasets provide a valuable resource for unraveling the mechanisms governing parathyroid gland regulation in health and disease.

Sustained and Localized Drug Depot Release Using Radiation-Activated Scintillating Nanoparticles.

Clinical treatment of cancer commonly incorporates X-ray radiation therapy (XRT), and developing spatially precise radiation-activatable drug delivery strategies may improve XRT efficacy while limiting off-target toxicities associated with systemically administered drugs. Nevertheless, achieving this has been challenging thus far because strategies typically rely on radical species with short lifespans, and the inherent nature of hypoxic and acidic tumor microenvironments may encourage spatially heterogeneous effects. It is hypothesized that the challenge could be bypassed by using scintillating nanoparticles that emit light upon X-ray absorption, locally forming therapeutic drug depots in tumor tissues. Thus a nanoparticle platform (Scintillating nanoparticle Drug Depot; SciDD) that enables the local release of cytotoxic payloads only after activation by XRT is developed, thereby limiting off-target toxicity. As a proof-of-principle, SciDD is used to deliver a microtubule-destabilizing payload MMAE (monomethyl auristatin E). With as little as a 2 Gy local irradiation to tumors, MMAE payloads are released effectively to kill tumor cells. XRT-mediated drug release is demonstrated in multiple mouse cancer models and showed efficacy over XRT alone (p < 0.0001). This work shows that SciDD can act as a local drug depot with spatiotemporally controlled release of cancer therapeutics.

Modeling the tumor microenvironment of anaplastic thyroid cancer: an orthotopic tumor model in C57BL/6 mice.

Introduction: Securing a well-established mouse model is important in identifying and validating new therapeutic targets for immuno-oncology. The C57BL/6 mouse is one of the most fully characterised immune system of any animal and provides powerful platform for immuno-oncology discovery. An orthotopic tumor model has been established using TBP3743 (murine anaplastic thyroid cancer [ATC]) cells in B6129SF1 hybrid mice, this model has limited data on tumor immunology than C57BL/6 inbred mice. This study aimed to establish a novel orthotopic ATC model in C57BL/6 mice and characterize the tumor microenvironment focusing immunity in the model. Methods: Adapted TBP3743 cells were generated via in vivo serial passaging in C57BL/6 mice. Subsequently, the following orthotopic tumor models were established via intrathyroidal injection: B6129SF1 mice injected with original TBP3743 cells (original/129), B6129SF1 mice injected with adapted cells (adapted/129), and C57BL/6 mice injected with adapted cells (adapted/B6). Results: The adapted TBP3743 cells de-differentiated but exhibited cell morphology, viability, and migration/invasion potential comparable with those of original cells in vitro. The adapted/129 contained a higher Ki-67+ cell fraction than the original/129. RNA sequencing data of orthotopic tumors revealed enhanced oncogenic properties in the adapted/129 compared with those in the original/129. In contrast, the orthotopic tumors grown in the adapted/B6 were smaller, with a lower Ki-67+ cell fraction than those in the adapted/129. However, the oncogenic properties of the tumors within the adapted/B6 and adapted/129 were similar. Immune-related pathways were enriched in the adapted/B6 compared with those in the adapted/129. Flow cytometric analysis of the orthotopic tumors revealed higher cytotoxic CD8+ T cell and monocytic-myeloid-derived suppressor cell fractions in the adapted/B6 compared with the adapted/129. The estimated CD8+ and CD4+ cell fractions in the adapted/B6 were similar to those in human ATCs but negligible in the original/B6. Conclusion: A novel orthotopic tumor model of ATC was established in C57BL/6 mice. Compared with the original B6129SF1 murine model, the novel model exhibited more aggressive tumor cell behaviours and strong immune responses. We expect that this novel model contributes to the understanding tumor microenvironment and provides the platform for drug development.

History of Thyroid Surgery in the Last Century.

Before the 20th century, thyroid surgery was regarded as "horrid butchery" such that no honest and sensible surgeon would ever engage in it. Yet, by the mid-20th century, thyroidectomy had become a respected, life-saving, safe, and increasingly practiced operation. From Kocher to Wells and onward into the 21st century, the evolution of thyroid surgery has continued, enhanced by the integration of endocrinology, genetics, immunology, physiology, technology, training, and multidisciplinary care. The ability to personalize and optimize the care of thyroid disorders has been progressively achieved through shared insights and discoveries, highlights of which are described herein.

Implementation of a New Surgeon Onboarding Program in an Academic-affiliated Community Hospital.

Best practices in onboarding are well-established, but surgeons frequently receive suboptimal introductions to new practice settings. At the same time, increasing regionalization of surgical programs and strategic alignments between academic and community hospitals have increased the demand for surgeons to practice at multiple sites with variable resources and institutional cultures. In response to this growing problem, we developed and implemented a surgeon onboarding program in an academic-affiliated community hospital. This pilot demonstrated excellent process adherence, user satisfaction, and significant improvements in preparedness to practice. We therefore conclude that robust onboarding is feasible and can be readily implemented by a local team to promote safe transitions in practice settings for surgeons.

Thyroid Cancers Exhibit Oncogene-Enhanced Macropinocytosis that Is Restrained by IGF1R and Promote Albumin-Drug Conjugate Response.

PURPOSE: Oncogene-driven macropinocytosis fuels nutrient scavenging in some cancer types, yet whether this occurs in thyroid cancers with prominent MAPK-ERK and PI3K pathway mutations remains unclear. We hypothesized that understanding links between thyroid cancer signaling and macropinocytosis might uncover new therapeutic strategies. EXPERIMENTAL DESIGN: Macropinocytosis was assessed across cells derived from papillary thyroid cancer (PTC), follicular thyroid cancer (FTC), non-malignant follicular thyroid, and aggressive anaplastic thyroid cancer (ATC), by imaging fluorescent dextran and serum albumin. The impacts of ectopic BRAFV600E and mutant RAS, genetic PTEN silencing, and inhibitors targeting RET, BRAF, and MEK kinases were quantified. BrafV600E p53-/- ATC tumors in immunocompetent mice were used to measure efficacy of an albumin-drug conjugate comprising microtubule-destabilizing monomethyl auristatin E (MMAE) linked to serum albumin via a cathepsin-cleavable peptide (Alb-vc-MMAE). RESULTS: FTC and ATC cells showed greater macropinocytosis than non-malignant and PTC cells. ATC tumors accumulated albumin at 8.8% injected dose per gram tissue. Alb-vc-MMAE, but not MMAE alone, reduced tumor size by >90% (P < 0.01). ATC macropinocytosis depended on MAPK/ERK activity and nutrient signaling, and increased by up to 230% with metformin, phenformin, or inhibition of IGF1Ri in monoculture but not in vivo. Macrophages also accumulated albumin and express the cognate IGF1R ligand, IGF1, which reduced ATC responsiveness to IGF1Ri. CONCLUSIONS: These findings identify regulated oncogene-driven macropinocytosis in thyroid cancers and demonstrate the potential of designing albumin-bound drugs to efficiently treat them.

Effectors Enabling Adaptation to Mitochondrial Complex I Loss in Hürthle Cell Carcinoma.

Oncocytic (Hürthle cell) carcinoma of the thyroid (HCC) is genetically characterized by complex I mitochondrial DNA mutations and widespread chromosomal losses. Here, we utilize RNA sequencing and metabolomics to identify candidate molecular effectors activated by these genetic drivers. We find glutathione biosynthesis, amino acid metabolism, mitochondrial unfolded protein response, and lipid peroxide scavenging to be increased in HCC. A CRISPR-Cas9 knockout screen in a new HCC model reveals which pathways are key for fitness, and highlights loss of GPX4, a defense against lipid peroxides and ferroptosis, as a strong liability. Rescuing complex I redox activity with the yeast NADH dehydrogenase (NDI1) in HCC cells diminishes ferroptosis sensitivity, while inhibiting complex I in normal thyroid cells augments ferroptosis induction. Our work demonstrates unmitigated lipid peroxide stress to be an HCC vulnerability that is mechanistically coupled to the genetic loss of mitochondrial complex I activity. SIGNIFICANCE: HCC harbors abundant mitochondria, mitochondrial DNA mutations, and chromosomal losses. Using a CRISPR-Cas9 screen inspired by transcriptomic and metabolomic profiling, we identify molecular effectors essential for cell fitness. We uncover lipid peroxide stress as a vulnerability coupled to mitochondrial complex I loss in HCC. See related article by Frank et al., p. 1884. This article is highlighted in the In This Issue feature, p. 1749.

Do Resident Coaching Programs Benefit their Coaches? Impact of a Professional Development Coaching Program on The Coaches.

BACKGROUND: Coaching has been shown to decrease physician burnout; however, coachee outcomes have been the focus. We report the impact of coaching on women-identifying surgeons who participated as coaches in a 9-month virtual program. METHODS: A coaching program was implemented in the Association of Women Surgeons (AWS) to determine the effects of coaching on well-being and burnout from 2018 to 2020. AWS members volunteered and completed training in professional development coaching. Pre- and post-study measures were assessed, and bivariate analysis performed based on burnout and professional fulfillment score. RESULTS: Seventy-five coaches participated; 57 completed both pre- and post-study surveys. There were no significant changes in burnout or professional fulfillment including the Positive Emotion, Engagement, Relationship, Meaning, and Accomplishment scale, hardiness, self-valuation, coping, gratitude, or intolerance of uncertainty scores from baseline to post-survey. On bivariate analysis, hardiness was associated with lower burnout throughout the duration of the program. Coaches with lower burnout at the end of the program met with their coachee more frequently than coaches with higher burnout [mean (SD) 3.95(2.16) versus 2.35(2.13) p = 0.0099]. DISCUSSION: Burnout and professional fulfillment demonstrated no change in women surgeons who participated as professional development coaches. Those with lower burnout and higher professional fulfillment at the end of the program were found to have higher hardiness, which may be worth future investigation. CONCLUSIONS: Acquisition of coaching skills did not directly improve well-being in faculty who participated in a resident coaching program. Future studies would benefit from control groups and exploration of qualitative benefits of coaching.

Impact of a Virtual Professional Development Coaching Program on the Professional Fulfillment and Well-Being of Women Surgery Residents: A Randomized Controlled Trial.

OBJECTIVE: Evaluate the effect of a virtual coaching program offered to women surgery residents in a surgical society. BACKGROUND: Randomized controlled experiments evaluating the effect of coaching on trainee well-being and burnout is lacking. METHODS: Women surgery residents in the Association of Women Surgeons were recruited to participate in a randomized controlled trial of the effects of a virtual coaching program on trainee well-being. Attending surgeons served as coaches after completing in-person training. Residents (n=237) were randomized to intervention (three 1:1 coaching sessions over 9 mo) or control (e-mailed wellness resources). Participants were surveyed at baseline and postintervention using validated measures of well-being, burnout, and resilience. Changes in outcome measures between presurvey and postsurvey were compared between study arms. RESULTS: Survey response rates were 56.9% (n=66) in the control group and 69.4% (n=84) in the intervention group ( P =0.05). The intervention group showed significant improvement in professional fulfillment ( P =0.021), burnout (0.026), work exhaustion (0.017), self-valuation (0.003), and well-being ( P =0.002); whereas the control group showed significant improvement in self-valuation ( P =0.015) and significant decline in resilience ( P =0.025). The intervention group had a significant improvement in well-being ( P =0.015) and intolerance of uncertainty ( P =0.015) compared to controls. CONCLUSIONS: Women surgery residents who participated in a remote coaching program offered by a surgical society demonstrated improvement in aspects of well-being relative to peers who did not receive coaching. Therefore, remote coaching offered by a professional society may be a useful component of initiatives directed at trainee well-being.

Radiation Cleaved Drug-Conjugate Linkers Enable Local Payload Release.

Conjugation of therapeutic payloads to biologics including antibodies and albumin can enhance the selectively of drug delivery to solid tumors. However, achieving activity in tumors while avoiding healthy tissues remains a challenge, and payload activity in off-target tissues can cause toxicity for many such drug-conjugates. Here, we address this issue by presenting a drug-conjugate linker strategy that releases an active therapeutic payload upon exposure to ionizing radiation. Localized X-ray irradiation at clinically relevant doses (8 Gy) yields 50% drug (doxorubicin or monomethyl auristatin E, MMAE) release under hypoxic conditions that are traditionally associated with radiotherapy resistance. As proof-of-principle, we apply the approach to antibody- and albumin-drug conjugates and achieve >2000-fold enhanced MMAE cytotoxicity upon irradiation. Overall, this work establishes ionizing radiation as a strategy for spatially localized cancer drug delivery.

Overcoming differential tumor penetration of BRAF inhibitors using computationally guided combination therapy.

BRAF-targeted kinase inhibitors (KIs) are used to treat malignancies including BRAF-mutant non-small cell lung cancer, colorectal cancer, anaplastic thyroid cancer, and, most prominently, melanoma. However, KI selection criteria in patients remain unclear, as are pharmacokinetic/pharmacodynamic (PK/PD) mechanisms that may limit context-dependent efficacy and differentiate related drugs. To address this issue, we imaged mouse models of BRAF-mutant cancers, fluorescent KI tracers, and unlabeled drug to calibrate in silico spatial PK/PD models. Results indicated that drug lipophilicity, plasma clearance, faster target dissociation, and, in particular, high albumin binding could limit dabrafenib action in visceral metastases compared to other KIs. This correlated with retrospective clinical observations. Computational modeling identified a timed strategy for combining dabrafenib and encorafenib to better sustain BRAF inhibition, which showed enhanced efficacy in mice. This study thus offers principles of spatial drug action that may help guide drug development, KI selection, and combination.

Deciphering albumin-directed drug delivery by imaging.

Albumin is the most abundant plasma protein, exhibits extended circulating half-life, and its properties have long been exploited for diagnostics and therapies. Many drugs intrinsically bind albumin or have been designed to do so, yet questions remain about true rate limiting factors that govern albumin-based transport and their pharmacological impacts, particularly in advanced solid cancers. Imaging techniques have been central to quantifying - at a molecular and single-cell level - the impact of mechanisms such as phagocytic immune cell signaling, FcRn-mediated recycling, oncogene-driven macropinocytosis, and albumin-drug interactions on spatial albumin deposition and related pharmacology. Macroscopic imaging of albumin-binding probes quantifies vessel structure, permeability, and supports efficiently targeted molecular imaging. Albumin-based imaging in patients and animal disease models thus offers a strategy to understand mechanisms, guide drug development and personalize treatments.

Survival After Adrenalectomy for Metastatic Lung Cancer.

BACKGROUND: Adrenal metastasectomy is associated with increased survival in non-small cell lung cancer (NSCLC) with isolated adrenal metastases. Although clinical use of adrenal metastasectomy has expanded, indications remain poorly defined. The aim of this study was to evaluate the clinical benefit of adrenal metastasectomy for all lung cancer subtypes. PATIENTS AND METHODS: We performed a retrospective cohort study of patients who underwent adrenal metastasectomy for metastatic lung cancer at six institutions between 2001 and 2015. The primary outcomes were disease-free survival (DFS) and overall survival (OS). Cox proportional hazards regressions and Kaplan-Meier survival analysis were performed. RESULTS: For 122 patients, the mean age was 60.5 years and 49.2% were female. Median time to detection of the metastasis was 11 months, and 41.8% were ipsilateral to the primary lung cancer. Median DFS was 40 months (1 year: 64.8%; 5 year: 42.9%). Factors associated with longer DFS included primary tumor resection [hazard ratio (HR): 0.001; p = 0.005], longer time to adrenal metastasis (HR: 0.94; p = 0.005), and ipsilateral metastases (HR: 0.13; p = 0.004). Shorter DFS corresponded with older age (HR: 1.11; p = 0.01), R1 resection (HR: 8.94; p = 0.01), adjuvant radiation (HR: 9.45; p = 0.02), and open adrenal metastasectomy (HR: 10.0; p = 0.03). Median OS was 47 months (1 year: 80.2%; 5 year: 35.2%). Longer OS was associated with ipsilateral metastasis (HR: 0.55; p = 0.02) and adjuvant chemotherapy (HR: 0.35; p = 0.02). Shorter OS was associated with extra-adrenal metastases at adrenalectomy (HR: 3.52; p = 0.007), small cell histology (HR: 15.0; p = 0.04), and lung radiation (HR: 3.37; p = 0.002). DISCUSSION: Durable survival was observed in patients undergoing adrenal metastasectomy and should be considered for isolated adrenal metastases of NSCLC. Small cell histology and extra-adrenal metastases are relative contraindications to adrenal metastasectomy.

h-Index and Academic Rank by Gender Among Breast Surgery Fellowship Faculty.

Background: Gender disparities in academic promotion and leadership are well documented. Scholarly impact is essential for promotion. The Hirsch-index (h-index) is a measure of impact using number of publications and citations. We sought to (i) evaluate breast surgery fellowship faculty in North America by academic rank and research impact using the h-index, (ii) determine whether there is a gender difference in scholarly productivity, and (iii) determine the relationship between academic rank, h-index, and gender. Materials and Methods: A retrospective cross-sectional study of h-index and academic rank disparity in breast surgery faculty by gender was performed. We reviewed the faculty of Society of Surgical Oncology-accredited breast surgery fellowships in February 2019. Rank, gender, academic appointment, years in practice, program directorship, National Cancer Institute-designated cancer center association, size of the program, and h-index (via Scopus) were recorded. Univariate and multiple linear regression analyses were performed. Results: Fifty-two programs were identified, and 209 faculty had an h-index. Of them, 69.9% were women and 30.1% were men. h-index increased with academic rank although there was considerable overlap between ranks. Women were underrepresented at the professor level (46.4%), but accounted for the majority of assistant professors (91.5%, p < 0.001), and program directors (70.7%). Men professors had a significantly higher mean h-index than women professors, p < 0.001. However, women associate professors had a higher mean h-index than men, but this did not reach significance. Conclusions: Mean h-index increased with increasing rank among breast surgery faculty for both genders. Average h-index was significantly higher for men professors compared to women professors. No significant gender difference in h-index was found for assistant professors. For associate professors, h-index for women was higher than for men. Women are underrepresented at higher academic ranks despite forming the majority of breast surgical teaching faculty.

Four-dimensional computed tomography (4D-CT) for preoperative parathyroid localization: A good study but are we using it?

BACKGROUND: Four-dimensional computed tomography (4D-CT) scan to localize abnormal parathyroid glands is diagnostically superior to ultrasound (US) and sestamibi. The implementation of 4D-CT imaging is unknown. METHODS: The Collaborative Endocrine Surgery Quality Improvement Program (CESQIP) database from 2014 to 2018 was utilized. Patients with hyperparathyroidism undergoing an initial operation were included. The rate of US, sestamibi and 4D-CT performance was calculated for the entire study population, and for each institution. RESULTS: 7,959 patients were included. In 311(3.9%) patients, no preoperative imaging was recorded. Of patients with imaging, US was performed in 6,872(86.3%), sestamibi in 5,094(64.0%), and 4D-CT in 1,630(20.4%). The combination of US and sestamibi was most frequent (3,855, 48.4%). Institutional rates of 4D-CT performance varied from 0.1% to 88.7%. CONCLUSIONS: Of the imaging modalities, 4D-CT was utilized least frequently and with greatest variability. Given the high accuracy of 4D-CT, efforts to reduce this variation may improve overall preoperative localization in patients with hyperparathyroidism.

American Association of Clinical Endocrinology And Associazione Medici Endocrinologi Thyroid Nodule Algorithmic Tool.

OBJECTIVE: The first edition of the American Association of Clinical Endocrinology/American College of Endocrinology/Associazione Medici Endocrinologi Guidelines for the Diagnosis and Management of Thyroid Nodules was published in 2006 and updated in 2010 and 2016. The American Association of Clinical Endocrinology/American College of Endocrinology/Associazione Medici Endocrinologi multidisciplinary thyroid nodules task force was charged with developing a novel interactive electronic algorithmic tool to evaluate thyroid nodules. METHODS: The Thyroid Nodule App (termed TNAPP) was based on the updated 2016 clinical practice guideline recommendations while incorporating recent scientific evidence and avoiding unnecessary diagnostic procedures and surgical overtreatment. This manuscript describes the algorithmic tool development, its data requirements, and its basis for decision making. It provides links to the web-based algorithmic tool and a tutorial. RESULTS: TNAPP and TI-RADS were cross-checked on 95 thyroid nodules with histology-proven diagnoses. CONCLUSION: TNAPP is a novel interactive web-based tool that uses clinical, imaging, cytologic, and molecular marker data to guide clinical decision making to evaluate and manage thyroid nodules. It may be used as a heuristic tool for evaluating and managing patients with thyroid nodules. It can be adapted to create registries for solo practices, large multispecialty delivery systems, regional and national databases, and research consortiums. Prospective studies are underway to validate TNAPP to determine how it compares with other ultrasound-based classification systems and whether it can improve the care of patients with clinically significant thyroid nodules while reducing the substantial burden incurred by those who do not benefit from further evaluation and treatment.

Therapeutically reprogrammed nutrient signalling enhances nanoparticulate albumin bound drug uptake and efficacy in KRAS-mutant cancer.

Nanoparticulate albumin bound paclitaxel (nab-paclitaxel, nab-PTX) is among the most widely prescribed nanomedicines in clinical use, yet it remains unclear how nanoformulation affects nab-PTX behaviour in the tumour microenvironment. Here, we quantified the biodistribution of the albumin carrier and its chemotherapeutic payload in optically cleared tumours of genetically engineered mouse models, and compared the behaviour of nab-PTX with other clinically relevant nanoparticles. We found that nab-PTX uptake is profoundly and distinctly affected by cancer-cell autonomous RAS signalling, and RAS/RAF/MEK/ERK inhibition blocked its selective delivery and efficacy. In contrast, a targeted screen revealed that IGF1R kinase inhibitors enhance uptake and efficacy of nab-PTX by mimicking glucose deprivation and promoting macropinocytosis via AMPK, a nutrient sensor in cells. This study thus shows how nanoparticulate albumin bound drug efficacy can be therapeutically improved by reprogramming nutrient signalling and enhancing macropinocytosis in cancer cells.