Visualizing Engrafted Human Cancer and Therapy Responses in Immunodeficient Zebrafish.

Xenograft cell transplantation into immunodeficient mice has become the gold standard for assessing pre-clinical efficacy of cancer drugs, yet direct visualization of single-cell phenotypes is difficult. Here, we report an optically-clear prkdc-/-, il2rga-/- zebrafish that lacks adaptive and natural killer immune cells, can engraft a wide array of human cancers at 37°C, and permits the dynamic visualization of single engrafted cells. For example, photoconversion cell-lineage tracing identified migratory and proliferative cell states in human rhabdomyosarcoma, a pediatric cancer of muscle. Additional experiments identified the preclinical efficacy of combination olaparib PARP inhibitor and temozolomide DNA-damaging agent as an effective therapy for rhabdomyosarcoma and visualized therapeutic responses using a four-color FUCCI cell-cycle fluorescent reporter. These experiments identified that combination treatment arrested rhabdomyosarcoma cells in the G2 cell cycle prior to induction of apoptosis. Finally, patient-derived xenografts could be engrafted into our model, opening new avenues for developing personalized therapeutic approaches in the future.

Factors associated with first-time and repeat blood donation: Adverse reactions and effects on donor behavior.

BACKGROUND: Blood centers have a dual mission to protect donors and patients; donor safety is paramount to maintaining an adequate blood supply. Elucidating donor factors associated with adverse reactions (AR) is critical to this mission. STUDY DESIGN/METHODS: A retrospective cohort analysis of whole blood donors from 2003 to 2020 was conducted at a single blood center in northern California. Adjusted odds ratios (AORs) with 95% CIs for ARs were estimated via multivariable logistic regression on demographics, donation history, and physical examination data. Where appropriate, Wilcoxon-Rank Sum and chi-squared tests were used to determine significance. RESULTS: First-time blood donors (FTD) exhibited a higher AR rate than repeat donors (4.4% vs. 1.9% p < .0001). When compared with FTDs without AR, FTDs with ARs (FT-AR) were less likely to return (30.0% vs. 47.3%, p < .0001), and, of those who returned, had a higher rate of reaction 20.2% versus 2.8% (p < .001). Factors found to be associated with FT-AR (younger age, increased heart rate, and higher diastolic blood pressure) still correlated positively with AR on return donation, but to a lower degree. FTD who potentially witnessed an AR had a lower return rate (44.6% vs. 47.3%, p = <.001) and donated fewer units (2.38 vs. 3.37, p < .001) when compared to FTD who did not witness an AR. CONCLUSION: The AR on FTD increases the AR likelihood of return donation. Longitudinal analysis shows that a time-based deferral policy targeted at FT-AR young donors can reduce the number of ARs while not dramatically impacting the blood supply.

Race and Ethnicity and Prehospital Use of Opioid or Ketamine Analgesia in Acute Traumatic Injury.

Importance: Racial and ethnic disparities in pain management have been characterized in many hospital-based settings. Painful traumatic injuries are a common reason for 911 activations of the EMS (emergency medical services) system. Objective: To evaluate whether, among patients treated by EMS with traumatic injuries, race and ethnicity are associated with either disparate recording of pain scores or disparate administration of analgesia when a high pain score is recorded. Design, Settings, and Participants: This cohort study included interactions from 2019 to 2021 for US patients ages 14 to 99 years who had experienced painful acute traumatic injuries and were treated and transported by an advanced life support unit following the activation of the 911 EMS system. The data were analyzed in January 2023. Exposures: Acute painful traumatic injuries including burns. Main Outcomes and Measures: Outcomes were the recording of a pain score and the administration of a nonoral opioid or ketamine. Results: The study cohort included 4 781 396 EMS activations for acute traumatic injury, with a median (IQR) patient age of 59 (35-78) years (2 497 053 female [52.2%]; 31 266 American Indian or Alaskan Native [0.7%]; 59 713 Asian [1.2%]; 742 931 Black [15.5%], 411 934 Hispanic or Latino [8.6%], 10 747 Native Hawaiian or other Pacific Islander [0.2%]; 2 764 499 White [57.8%]; 16 161 multiple races [0.3%]). The analysis showed that race and ethnicity was associated with the likelihood of having a pain score recorded. Compared with White patients, American Indian and Alaskan Native patients had the lowest adjusted odds ratio (AOR) of having a pain score recorded (AOR, 0.74; 95% CI, 0.71-0.76). Among patients for whom a high pain score was recorded (between 7 and 10 out of 10), Black patients were about half as likely to receive opioid or ketamine analgesia as White patients (AOR, 0.53; 95% CI, 0.52-0.54) despite having a pain score recorded almost as frequently as White patients. Conclusions and Relevance: In this nationwide study of patients treated by EMS for acute traumatic injuries, patients from racial or ethnic minority groups were less likely to have a pain score recorded, with Native American and Alaskan Natives the least likely to have a pain score recorded. Among patients with a high pain score, patients from racial and ethnic minority groups were also significantly less likely to receive opioid or ketamine analgesia treatment, with Black patients having the lowest adjusted odds of receiving these treatments.

Single-cell imaging of T cell immunotherapy responses in vivo.

T cell immunotherapies have revolutionized treatment for a subset of cancers. Yet, a major hurdle has been the lack of facile and predicative preclinical animal models that permit dynamic visualization of T cell immune responses at single-cell resolution in vivo. Here, optically clear immunocompromised zebrafish were engrafted with fluorescent-labeled human cancers along with chimeric antigen receptor T (CAR T) cells, bispecific T cell engagers (BiTEs), and antibody peptide epitope conjugates (APECs), allowing real-time single-cell visualization of T cell-based immunotherapies in vivo. This work uncovered important differences in the kinetics of T cell infiltration, tumor cell engagement, and killing between these immunotherapies and established early endpoint analysis to predict therapy responses. We also established EGFR-targeted immunotherapies as a powerful approach to kill rhabdomyosarcoma muscle cancers, providing strong preclinical rationale for assessing a wider array of T cell immunotherapies in this disease.

Single-cell imaging of human cancer xenografts using adult immunodeficient zebrafish.

Zebrafish are an ideal cell transplantation model. They are highly fecund, optically clear and an excellent platform for preclinical drug discovery studies. Traditionally, xenotransplantation has been carried out using larval zebrafish that have not yet developed adaptive immunity. Larval engraftment is a powerful short-term transplant platform amenable to high-throughput drug screening studies, yet animals eventually reject tumors and cannot be raised at 37 °C. To address these limitations, we have recently developed adult casper-strain prkdc-/-, il2rgα-/- immunocompromised zebrafish that robustly engraft human cancer cells for in excess of 28 d. Because the adult zebrafish can be administered drugs by oral gavage or i.p. injection, our model is suitable for achieving accurate, preclinical drug dosing. Our platform also allows facile visualization of drug effects in vivo at single-cell resolution over days. Here, we describe the procedures for xenograft cell transplantation into the prkdc-/-, il2rgα-/- model, including refined husbandry protocols for optimal growth and rearing of immunosuppressed zebrafish at 37 °C; optimized intraperitoneal and periocular muscle cell transplantation; and epifluorescence and confocal imaging approaches to visualize the effects of administering clinically relevant drug dosing at single-cell resolution in vivo. After identification of adult homozygous animals, this procedure takes 35 d to complete. 7 days are required to acclimate adult fish to 37 °C, and 28 d are required for engraftment studies. Our protocol provides a comprehensive guide for using immunocompromised zebrafish for xenograft cell transplantation and credentials the model as a new preclinical drug discovery platform.