Location: Children's Nutrition Research Center
2024 Annual Report
Objectives
We will: 1)study the effect of enteral nutrition on the downstream signaling pathways and metabolism;2)study if increased FGF19 availability controls rate of growth, tissue protein synthesis and intestinal development;3)study if being born prematurely blunts protein and glucose metabolic responses to the feeding-induced rise in amino acids and insulin; 4)identify by which amino acids, regulate protein synthesis, degradation, and accretion and how responses change with age;5)removed due to investigator departure;6)study molecular mechanisms and functional significance of differences in gene expression identified in satellite cell-derived myoblasts;7)study the impact of maternal dietary protein level during lactation;8)study if vitamin D receptors in the brain are critical for glucose regulation;9)study if leptin is involved in the regulation of gluconeogenesis via the leptin receptor and if leptin agonist and small doses of hypoglycin-A/B reduces gluconeogenesis;10)study the role of the SIRT3 in regulation of pyruvate carboxylase and the gluconeogenesis pathway;11)alter DNA methylation in specific subpopulations of hypothalamic neurons and evaluate lifelong effects on energy metabolism, food intake, and PA;12)find the causes of interindividual epigenetic variation and consequences for human energy balance;13)study the functional impact of folic acid supplementation and in intestinal carcinogenesis;14)study the effect of adiposity, adipokine dysregulation, insulin resistance and vitamin D concentrations on bone and endothelial function; 15)study the effect of vitamin D therapy on change in bone and endothelial function;16)removed due to investigator departure;17)study the CNS circuit architecture and explore circuit complexities that regulate non-homeostatic feeding behaviors via environmental signals transduced by epigenetic mechanisms;18)study the tumorigenic effects of HFCS on a humanized colon tumor mouse model;19)study the effects of HFCS on the gut microbiota of a humanized colon tumor mouse model;20)study the role of HFCS-induced gut microbiota in CRC development; create multi-omic nutritional data share portal to resolve the unmet demand for an efficient access to the large volumes of heterogeneous multi-omic data across various research labs;21)integrate heterogeneous multi-omic datasets such as genetic (SNPs), transcriptomic, epigenetic, proteomic, metabolomic and microbiome to infer molecular network structures illustrating eating disorder dynamics;&22)decode genetic and epigenetic patterns of disordered eating using machine learning methods.
Approach
This research will be accomplished using a variety of models and scientific tools to simulate the human newborn and/or child. Researchers will use neonatal piglet and rodent models to fill these knowledge gaps. We will determine whether being born prematurely blunts the anabolic response to feeding and identify mechanisms by which amino acids, particularly leucine, regulate lean growth. Additionally we will use various rodent models to test leptin's effect on gluconeogenesis that is independent of body weight, and will utilize in vitro experiments employing primary hepatocytes. Scientists will also integrate both detailed studies of animal models and characterization of epigenetic mechanisms in humans. We will use mouse models of developmental epigenetics in the hypothalamus to understand cell type-specific epigenetic mechanisms mediating developmental programming of body weight regulation. Mouse models will also be used to investigate how folic acid intake affects epigenetic mechanisms regulating intestinal epithelial stem cell (IESC) development and characterize the involvement of these mechanisms in metabolic programming related to obesity, inflammation, and gastrointestinal cancer. In human studies, we will identify human genomic loci at which interindividual variation in DNA methylation is both sensitive to maternal nutrition in early pregnancy and associated with risk of later weight gain. We will also examine whether restoration of vitamin D sufficiency, in a randomized placebo controlled study design, has a positive effect on bone microarchitecture, bone biomarkers and endothelial function. Studies will be conducted in mice that will uncover the molecular basis of interrelationships among dietary sugar, gut microbiota, and CRC development and identify sugar-induced metabolites and/or microbes that can serve as new biomarkers and targets. Researchers will also conduct a multi-omic integrative study to systematically decipher the molecular interplay of disordered eating and neuron specific brain circuits that control feeding behavior.
Progress Report
To review the progress made during the year, please refer to the following projects: 3092-10700-070-010S (Project #1), 3092-10700-070-020S (Project #2), 3092-10700-070-030S (Project #3) and 3092-10700-070-040S (Project #4).
Accomplishments
1. How premature birth impacts infant nutrition and growth. In the United States, a substantial number of infants are born prematurely, leading to reduced capacity to digest, absorb, and metabolize nutrients essential for healthy growth and development. Researchers at the Children's Nutrition Research Center in Houston, Texas, used infant pigs as a model for human infants to investigate how premature birth adversely impacts metabolism and growth. We revealed that premature birth results in a reduced secretion of cortisol, a key stress hormone normally released near full-term birth. This diminished cortisol level is linked to a decreased production of hepatic bile and reduced intestinal secretion of the hormone fibroblast growth factor 19 (FGF19). This immaturity in bile metabolism may explain why premature infants have difficulty digesting fat, a critical source of nutrition for their growth and development. This finding may elucidate why the therapeutic use of stress hormones, often administered to hospitalized premature infants to induce lung development, may also improve their overall nutrient metabolism and growth.
2. Targeted nutritional therapy can overcome the anabolic resistance of preterm birth. Preterm infants gain less body weight than full-term infants due to reduced muscle growth, but not fat accumulation. This contributes to long-term adverse health outcomes, such as obesity and type 2 diabetes. Research at the Children's Nutrition Research Center in Houston, Texas, showed the synthesis of protein in skeletal muscle after a meal is lower in preterm pigs than in those that are born at full term. They also showed that supplementing formula with the amino acid leucine, which targets key cell signaling pathways, can overcome this anabolic resistance in preterm muscle. These studies provide vital information on the mechanisms underlying reduced muscle growth in preterm infants and may lead to targeted nutritional therapies to improve lean growth in premature infants.
3. Youth-onset type 2 diabetes is not suppressed by a combination therapy of metformin and liraglutide. Youth-onset type 2 diabetes is a rapidly progressive disease characterized by increased glucose production from the liver, rapid failure of pancreatic cells (B-cell) that produce insulin, and severe insulin resistance. This chronic disease is magnified among African American youth who have the highest disease prevalence and burden of metabolic complications. Since increased rates of glucose production from the liver is an important primary factor in youth-onset type 2 diabetes, researchers at the Children's Nutrition Research Center in Houston, Texas, investigated the effectiveness of a combination therapy of metformin with a glucagon like peptide-1 receptor agonist (liraglutide), in reducing glucose production from the liver and improving B-cell function. We showed that youth-onset type 2 diabetes is associated with dysregulated and high rates of glucose production from the liver which were not suppressed by metformin alone or in combination with liraglutide. Increased glucose clearance was related to improvements in B-cell function. These findings show that early combination therapies that will abrogate multiple pathways that contribute to high rates of glucose production and improve B-cell function in youth-onset type 2 diabetes are needed.
4. Obesity-associated glycine deficiency impairs the human detoxification system which can be improved when glycine supply is restored. Glycine is an amino acid that is central to human metabolism, is integral of the human detoxification system, and is required in large amounts by the human body. Plasma glycine concentration is low in individuals with severe obesity and is associated with impaired glycine synthesis. Interestingly, both glycine synthesis and glycine levels improve after bariatric surgery. Researchers at the Children's Nutrition Research Center in Houston, Texas, examined the impact of obesity-associated glycine deficiency and bariatric surgery on the detoxification system in humans. We showed that obesity-associated glycine deficiency impairs the human detoxification system, and this impairment is improved when glycine supply is restored after bariatric surgery. This finding proposes that dietary glycine supplementation could treat obesity-associated metabolic complications due to the accumulation of toxic metabolites by helping the body get rid of harmful substances more efficiently.
5. High dietary vitamin D protects aging mice against blood sugar abnormalities only in males. Vitamin D is important for health but there are conflicting results on its impact in humans. Most research does not analyze sex-specific effects of vitamin D, although some data exists that vitamin D may only be protective against diabetes in males. Researchers at the Children's Nutrition Research Center in Houston, Texas, found that middle-aged mice on high vitamin D levels had improved blood sugar levels compared to those on normal or low vitamin D diets. This may be secondary to differences in gene expression of important glucose-regulating proteins in the liver; males on a high vitamin D diet had higher expression levels of the insulin receptor but no effect of diet was seen in the females. These findings give evidence that males and females may respond to vitamin D differently and support human studies showing that vitamin D, as currently administered, may only be beneficial for glucose control in males. Future research is needed to determine how to improve female response to dietary vitamin D.
6. Dietary folate enhances colon cancer risk in animal models. There is an unresolved debate about the extent to which the environment contributes to cancer risk. Although epidemiological studies suggest that environmental factors such as diet can certainly contribute to this risk, especially for colon cancer, how dietary factors could tip the scale in favor of cancer is not known. Researchers at the Children's Nutrition Research Center in Houston, Texas, developed the first mouse model of engineered p16 promoter hypermethylation, leading to accelerated p16 epimutation in somatic tissues during aging. This work investigates the link between age-related p16 epimutation which is regulated by folate and intestinal tumorigenesis, identifying potential targets for colorectal cancer treatment. It also sheds light on the connection between diet and epigenetic regulation in cancer development. Importantly, these findings highlight the need to monitor the long-term safety of folate fortification in high-risk individuals.
7. Discovering novel splicing changes with new software. Novel splicing events in genes, called cryptic splicing events, are often missed by traditional tools but are crucial for understanding the biological phenomenon. To address this gap, specialized computational tools are necessary to accurately identify cryptic splicing. Researchers at the Children's Nutrition Research Center in Houston, Texas, developed a new software that finds these hidden changes and shows how diet affects them, offering new ways to manage metabolic diseases like diabetes. Researchers have used this tool to uncover genetic changes in heart failure and mental health disorders, leading to new diagnostic markers and treatments influenced by diet. This work broadens our understanding of gene regulation and its nutritional impacts, benefiting public health. By identifying diet-related splicing changes, the software opens avenues for personalized nutrition plans to prevent and treat metabolic conditions including obesity and diabetes.
8. The effect of cold on body fat and health. Brown fat helps keep us warm by burning energy, and cold temperatures make it more active. Researchers at the Children's Nutrition Research Center in Houston, Texas, studied the effect of temperature on body fat and health by housing mice at different temperatures, from comfortable to very cold and profiling their molecular changes. We saw a decrease in DNA methylation and increases in certain protein modifications, which together aid in regulating how brown fat genes turn on or off to produce heat in response to cold. This discovery suggests new ways to treat obesity and diabetes by understanding body heat and energy use and could lead to treatments that mimic cold effects on brown fat. This research impacts how we manage body fat and energy and highlights the potential for environmental and lifestyle changes to play a significant role in combating metabolic diseases.
9. Sexual dimorphism in the relationship of obesity and dysglycemia with bone microarchitecture in youth. In adults, individuals with type 2 diabetes are known to have worse bone quality and a higher risk of fractures. The relationship between obesity, type 2 diabetes and bone health in youth however is unclear and this is important since the majority of bone formation occurs during the adolescent years. Researchers at the Children's Nutrition Research Center in Houston, Texas, examined the relationship of adiposity and glucose metabolism to bone quality and strength measures in adolescents with obesity, with and without type 2 diabetes, and compared with their normal weight counterparts. We found that bone mineral content, quality, and strength measures were worse in boys with abnormalities in their glucose compared with obesity but normal glucose than those who are of normal weight. The negative effects of high blood sugar on bone health may be more evident in adolescent boys than in girls and additional studies are needed to better understand what factors may explain the effect.
Review Publications
Dietsche, K.B., Magge, S.N., Dixon, S.A., Davis, F.S., Krenek, A., Chowdhury, A., Mabundo, L., Stagliano, M., Courville, A.B., Yang, S., Turner, S., Cai, H., Kasturi, K., Sherman, A.S., Ha, J., Shouppe, E., Walter, M., Walter, P.J., Chen, K.Y., Brychta, R.J., Peer, C., Zeng, Y., Figg, W., Cogen, F., Estrada, D., Chacko, S., Chung, S.T. 2023. Glycemia and gluconeogenesis with metformin and liraglutide: A randomized trial in youth-onset type 2 diabetes. Journal of Clinical Endocrinology and Metabolism. https://doi.org/10.1210/clinem/dgad669.
Yang, L., Peery, R.C., Farmer, L.M., Gao, X., Zhang, Y., Creighton, C.J., Zhang, L., Shen, L. 2024. Dietary folate and cofactors accelerate age-dependent p16 epimutation to promote intestinal tumorigenesis. Cancer Research Communications. 4(1):164-169. https://doi.org/10.1158/2767-9764.CRC-23-0356.
Rudar, M., Naberhuis, J.K., Suryawan, A., Nguyen, H.V., Fiorotto, M.L., Davis, T.A. 2023. Prematurity blunts protein synthesis in skeletal muscle independently of body weight in neonatal pigs. Pediatric Research. 94(1):143-152. https://www.nature.com/articles/s41390-022-02456-3.
Quaye, E., Chacko, S., Startzell, M., Brown, R.J. 2023. Leptin decreases gluconeogenesis and gluconeogenic substrate availability in patients with lipodystrophy. Journal of Clinical Endocrinology and Metabolism. 109(1):e209-e215. https://doi.org/10.1210/clinem/dgad445.
Tang, H., Hsu, J.W., Tai, E., Chacko, S., Kovalik, J., Jahoor, F. 2024. The impact of obesity-associated glycine deficiency on the elimination of endogenous and exogenous metabolites via the glycine conjugation pathway. Frontiers in Endocrinology. 15. Article 1343738. https://doi.org/10.3389/fendo.2024.1343738.
Sanyal, S., Calarge, C., Rowan, P.J., Aparasu, R.R., Abughosh, S., Sisley, S., Chen, H. 2023. Adherence to recommended metabolic monitoring of children and adolescents taking second-generation antipsychotics. Psychiatric Services. 75(4):342-348. https://doi.org/10.1176/appi.ps.20220584.
The RADIANT Study Group. 2023. The Rare and Atypical Diabetes Network (RADIANT) study: Design and early results. Diabetes Care. 46(6):1265-1270. https://doi.org/10.2337/dc22-2440.
Wasserman, H., Jenkins, T., Inge, T., Ryder, J., Michalsky, M., Sisley, S., Xie, C., Kalkwarf, H.J. 2024. Bone mineral density in young adults 5 to 11 years after adolescent metabolic and bariatric surgery for severe obesity compared to peers. International Journal of Obesity. 48:575-583. https://doi.org/10.1038/s41366-023-01453-8.
Baumert, B.O., Fischer, F.C., Nielsen, F., Grandjean, P., Bartell, S., Stratakis, N., Walker, D., Valvi, D., Kohli, R., Inge, T., Ryder, J., Jenkins, T., Sisley, S., Xanthakos, S., Rock, S., La Merrill, M.A., Conti, D., McConnell, R., Chatzi, L. 2023. Paired liver: Plasma PFAS concentration ratios from adolescents in the teen-LABS study and derivation of empirical and mass balance models to predict and explain liver PFAS accumulation. Environmental Science and Technology. 57:14817-14826. https://doi.org/10.1021/acs.est.3c02765.
Kubota-Mishra, E., Huang, X., Minard, C.G., Astudillo, M., Refaey, A., Montes, G., Sisley, S., Ram, N., Winter, W.E., Naylor, R.N., Balasubramanyam, A., Redondo, M.J., Tosur, M., and RADIANT Study Group. 2024. High prevalence of A-B+ ketosis-prone diabetes in children with type 2 diabetes and diabetic ketoacidosis at diagnosis: Evidence from the Rare and Atypical Diabetes Network (RADIANT). Pediatric Diabetes. Article 5907924. https://doi.org/10.1155/2024/5907924.
Baumert, B.O., Eckel, S.P., Goodrich, J.A., Li, Z., Stratakis, N., Walker, D.I., Zhao, Y., Fischer, F.C., Bartell, S., Valvi, D., Lin, X., Fuentes, Z.C., Inge, T., Ryder, J., Jenkins, T., Kohli, R., Sisley, S., Xanthakos, S., Rock, S., La Merrill, M.A., McConnell, R., Conti, D.V., Chatzi, L. 2024. Changes in plasma concentrations of per- and polyfluoroalkyl substances after bariatric surgery in adolescents from the Teen-Longitudinal Assessment of Bariatric Surgery (Teen-LABS) study. Science of the Total Environment. 930. Article 172840. https://doi.org/10.1016/j.scitotenv.2024.172840.
Rubbo, B., Li, Z., Tachachartvanich, P., Baumert, B.O., Wang, H., Pan, S., Rock, S., Ryder, J.R., Jenkins, T., Sisley, S., Lin, X., Bartell, S., Inge, T.H., Xanthakos, S., McNeil, B., Robuck, A.R., La Merrill, M.A., Walker, D.I., Conti, D.V., McConnell, R., Eckel, S.P., Chatzi, L. 2024. Exposure to 4,4'-DDE in visceral adipose tissue and weight loss in adolescents from the Teen-LABS cohort. Obesity. 32(5):1023-1032. https://doi.org/10.1002/oby.24009.
Li, Y., Baumert, B.O., Stratakis, N., Goodrich, J.A., Wu, H., He, J., Zhao, Y., Aung, M.T., Wang, H., Eckel, S.P., Walker, D.I., Valvi, D., La Merrill, M.A., Ryder, J.R., Inge, T.H., Jenkins, T., Sisley, S., Kohli, R., Xanthakos, S., Baccarelli, A.A., McConnell, R., Conti, D.V., Chatzi, L. 2024. Circulating microRNA expression and nonalcoholic fatty liver disease in adolescents with severe obesity. World Journal of Gastroenterology. 30(4):332-345. https://doi.org/10.3748/wjg.v30.i4.332.
Garcia Castro, D.R., Mazuk, J.R., Heine, E.M., Simpson, D., Pinches, R., Lozzi, C., Hoffman, K., Morrin, P., Mathis, D., Lebedev, M.V., Nissley, E., Hoo Han, K., Farmer, T., Merry, D.E., Tong, Q., Pennuto, M., Montie, H.L. 2023. Increased SIRT3 combined with PARP inhibition rescues motor function of SBMA mice. iScience. 26(3). Article 107375. https://doi.org/10.1016/j.isci.2023.107375.
Bacha, F., Gupta, R., Jenkins, T.M., Brandt, M.L., Inge, T.H., Kleiner, D.E., Xanthakos, S.A., for the Teen-Longitudinal Assessment of Bariatric Surgery (Teen-LABS) Consortium. 2024. Prognostic factors in resolution of nonalcoholic fatty liver disease post bariatric surgery in adolescents. Elsevier. 20(4):367-375. https://doi.org/10.1016/j.soard.2023.11.004.
Pham, D.T., Westerman, K.E., Pan, C., Chen, L., Srinivasan, S., Isganaitis, E., Vajravelu, M., Bacha, F., Chernausek, S., Gubitosi-Klug, R., Divers, J., Pihoker, C., Marcovina, S.M., Manning, A.K., Chen, H. 2023. Re-analysis and meta-analysis of summary statistics from gene-environment interaction studies. Bioinformatics. 39(12). Article btad730. https://doi.org/10.1093/bioinformatics/btad730.
Bacha, F., El-Ayash, H., Mohamad, M., Sharma, S., Puyau, M., Kanchi, R., Coarfa, C. 2024. Distinct amino acid profile characterizes youth with or at risk for type 2 diabetes. Diabetes. 73(4):628–636. https://doi.org/10.2337/db23-0375.
Koren, D., Knutson, K.L., Burke, B.K., Drews, K.L., Bacha, F., Katz, L., Marcus, M.D., McKay, S., Nadeau, K., Mokhlesi, B., and on behalf of the TODAY Study Group. 2024. The association of self-reported sleep and circadian measures with glycemic control and diabetes complications among young adults with type 2 diabetes: Results from the TODAY2 study. American Journal of Physiology - Heart and Circulatory Physiology. 326(6):H1386–H1395. https://doi.org/10.1152/ajpheart.00550.2023.
Jonnakuti, V.S., Wagner, E.J., Maletic-Savatic, M., Liu, Z., Yalamanchili, H.K. 2024. PolyAMiner-Bulk is a deep learning-based algorithm that decodes alternative polyadenylation dynamics from bulk RNA-seq data. Cell Reports Methods. 4. Article 100707. https://doi.org/10.1016/j.crmeth.2024.100707.
Khan, M., Chen, X.X., Dias, M., Santos, J.R., Kour, S., You, J., Van Bruggen, R., Youssef, M.M., Wan, Y., Liu, Z., Rosenfeld, J.A., Tan, Q., Pandey, U.B., Yalamanchili, H.K., Park, J. 2024. MATR3 pathogenic variants differentially impair its cryptic splicing repression function. FEBS Letters. 598:415-436. https://doi.org/10.1002/1873-3468.14806.
Chadchan, S.B., Popli, P., Liao, Z., Andreas, E., Dias, M., Wang, T., Gunderson, S.J., Jimenez, P.T., Lanza, D.G., Lanz, R.B., Foulds, C.E., Monsivais, D., Demayo, F.J., Yalamanchili, H.K., Jungheim, E.S., Heaney, J.D., Lydon, J.P., Moley, K.H., O'Malley, B.W., Kommagani, R. 2024. A GREB1-steroid receptor feedforward mechanism governs differential GREB1 action in endometrial function and endometriosis. Nature Communications. 15. Article 1947. https://doi.org/10.1038/s41467-024-46180-4.
Taylor, B.C., Steinthal, L.H., Dias, M., Yalamanchili, H.K., Ochsner, S.A., Zapata, G.E., Mehta, N.R., McKenna, N.J., Young, N.L., Nuotio-Antar, A.M. 2024. Histone proteoform analysis reveals epigenetic changes in adult mouse brown adipose tissue in response to cold stress. Epigenetics and Chromatin. 17. Article 12. https://doi.org/10.1186/s13072-024-00536-8.
Elefson, S.K., Stoll, B., Davis, T., Fiorotto, M.L., El Kadi, S.W., Genovese, K.J., Thymann, T., Sangild, P.T., Burrin, D.G. 2024. Adverse metabolic phenotypes in parenterally fed neonatal pigs do not persist into adolescence. Journal of Nutrition. 154(2):638-647. https://doi.org/10.1016/j.tjnut.2023.12.048.
Maj, M.A., Burrin, D.G., Manjarin, R. 2023. Decreased FXR agonism in the bile acid pool is associated with impaired FXR signaling in a pig model of pediatric NAFLD. Biomedicines. 11(12). Article 3303. https://doi.org/10.3390/biomedicines11123303.
Foster, K.L., Lee, D.J., Witchel, S.F., Gordon, C.M. 2024. Ovarian insufficiency and fertility preservation during and after childhood cancer treatment. Journal of Adolescent and Young Adult Oncology. https://doi.org/10.1089/jayao.2023.0111.
Posey, E.A., Davis, T.A. 2023. Review: Nutritional regulation of muscle growth in the neonatal swine. Animal-The International Journal of Animal Biosciences. 17(3). Article 100831. https://doi.org/10.1016/j.animal.2023.100831.
Rudar, M., Suryawan, A., Nguyen, H.V., Chacko, S.K., Vonderohe, C., Stoll, B., Burrin, D.G., Fiorotto, M.L., Davis, T.A. 2023. Pulsatile leucine administration during continuous enteral feeding enhances skeletal muscle mechanistic target of rapamycin complex 1 signaling and protein synthesis in a preterm piglet model. Journal of Nutrition. 154(2):505–515. https://doi.org/10.1016/j.tjnut.2023.12.034.
Suryawan, A., Rudar, M., Naberhuis, J.K., Fiorotto, M.L., Davis, T.A. 2022. Preterm birth alters the feeding-induced activation of Akt signaling in the muscle of neonatal piglets. Pediatric Research. 93(7):1891-1898. https://doi.org/10.1038/s41390-022-02382-4.
Chang, W., Baker, M.S., Laritsky, E., Gunasekara, C.J., Maduranga, U., Galliou, J.C., McFadden, J.W., Waltemyer, J.R., Berggren-Thomas, B., Tate, B.N., Zhang, H., Rosen, B.D., Van-Tassell, C.P., Liu, G.E., Coarfa, C., Ren, Y., Waterland, R.A. 2024. Systemic interindividual DNA methylation variants in cattle share major hallmarks with those in humans. Genome Biology. 25. Article 185. https://doi.org/10.1186/s13059-024-03307-6.
Rasmussen, M., Holgersen, K., Pankratova, S., Baek, O., Burrin, D.G., Thymann, T., Sangild, P. 2023. Gut development following insulin-like growth factor-1 supplementation to preterm pigs. Pediatric Research. 95:1528-1535. https://doi.org/10.1038/s41390-023-02949-9.
Yang, L., Peery, R.C., Farmer, L.M., Gao, X., Zhang, Y., Creighton, C.J., Zhang, L., Shen, L. 2024. Dietary folate and cofactors accelerate age-dependent p16 epimutation to promote intestinal tumorigenesis. Cancer Research Communications. 4(1):164–169. https://doi.org/10.1158/2767-9764.CRC-23-0356.