Metabolic Reprogramming Research Articles

The role of tumor-associated macrophages in tumor immune evasion.

Tumor growth is closely linked to the activities of various cells in the tumor microenvironment (TME), particularly immune cells. During tumor progression, circulating monocytes and macrophages are recruited, altering the TME and accelerating growth. These macrophages adjust their functions in response to signals from tumor and stromal cells. Tumor-associated macrophages (TAMs), similar to M2 macrophages, are key regulators in the TME. We review the origins, characteristics, and functions of TAMs within the TME. This analysis includes the mechanisms through which TAMs facilitate immune evasion and promote tumor metastasis. Additionally, we explore potential therapeutic strategies that target TAMs. TAMs are instrumental in mediating tumor immune evasion and malignant behaviors. They release cytokines that inhibit effector immune cells and attract additional immunosuppressive cells to the TME. TAMs primarily target effector T cells, inducing exhaustion directly, influencing activity indirectly through cellular interactions, or suppressing through immune checkpoints. Additionally, TAMs are directly involved in tumor proliferation, angiogenesis, invasion, and metastasis. Developing innovative tumor-targeted therapies and immunotherapeutic strategies is currently a promising focus in oncology. Given the pivotal role of TAMs in immune evasion, several therapeutic approaches have been devised to target them. These include leveraging epigenetics, metabolic reprogramming, and cellular engineering to repolarize TAMs, inhibiting their recruitment and activity, and using TAMs as drug delivery vehicles. Although some of these strategies remain distant from clinical application, we believe that future therapies targeting TAMs will offer significant benefits to cancer patients.

The cAMP-dependent phosphorylation footprint in response to heat stress.

cAMP modulates the phosphorylation status of highly conserved phosphosites in RNA-binding proteins crucial for mRNA metabolism and reprogramming in response to heat stress. In plants, 3',5'-cyclic adenosine monophosphate (3',5'-cAMP) is a second messenger that modulates multiple cellular targets, thereby participating in plant developmental and adaptive processes. Although its role in ameliorating heat-related damage has been demonstrated, mechanisms that govern cAMP-dependent responses to heat have remained elusive. Here we analyze the role cAMP-dependent phosphorylation during prolonged heat stress (HS) with a view to gain insight into processes that govern plant responses to HS. To do so, we performed quantitative phosphoproteomic analyses in Nicotiana tabacum Bright Yellow-2 cells grown at 27°C or 35°C for 3days overexpressing a molecular "sponge" that reduces free intracellular cAMP levels. Our phosphorylation data and analyses reveal that the presence of cAMP is an essential factor that governs specific protein phosphorylation events that occur during prolonged HS in BY-2 cells. Notably, cAMP modulates HS-dependent phosphorylation of proteins that functions in mRNA processing, transcriptional control, vesicular trafficking, and cell cycle regulation and this is indicative for a systemic role of the messenger. In particular, changes of cAMP levels affect the phosphorylation status of highly conserved phosphosites in 19 RNA-binding proteins that are crucial during the reprogramming of the mRNA metabolism in response to HS. Furthermore, phosphorylation site motifs and molecular docking suggest that some proteins, including kinases and phosphatases, are conceivably able to directly interact with cAMP thus further supporting a regulatory role of cAMP in plant HS responses.

Positive feedback regulation between glycolysis and histone lactylation drives oncogenesis in pancreatic ductal adenocarcinoma

BackgroundMetabolic reprogramming and epigenetic alterations contribute to the aggressiveness of pancreatic ductal adenocarcinoma (PDAC). Lactate-dependent histone modification is a new type of histone mark, which links glycolysis metabolite to the epigenetic process of lactylation. However, the role of histone lactylation in PDAC remains unclear.MethodsThe level of histone lactylation in PDAC was identified by western blot and immunohistochemistry, and its relationship with the overall survival was evaluated using a Kaplan-Meier survival plot. The participation of histone lactylation in the growth and progression of PDAC was confirmed through inhibition of histone lactylation by glycolysis inhibitors or lactate dehydrogenase A (LDHA) knockdown both in vitro and in vivo. The potential writers and erasers of histone lactylation in PDAC were identified by western blot and functional experiments. The potential target genes of H3K18 lactylation (H3K18la) were screened by CUT&Tag and RNA-seq analyses. The candidate target genes TTK protein kinase (TTK) and BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B) were validated through ChIP-qPCR, RT-qPCR and western blot analyses. Next, the effects of these two genes in PDAC were confirmed by knockdown or overexpression. The interaction between TTK and LDHA was identified by Co-IP assay.ResultsHistone lactylation, especially H3K18la level was elevated in PDAC, and the high level of H3K18la was associated with poor prognosis. The suppression of glycolytic activity by different kinds of inhibitors or LDHA knockdown contributed to the anti-tumor effects of PDAC in vitro and in vivo. E1A binding protein p300 (P300) and histone deacetylase 2 were the potential writer and eraser of histone lactylation in PDAC cells, respectively. H3K18la was enriched at the promoters and activated the transcription of mitotic checkpoint regulators TTK and BUB1B. Interestingly, TTK and BUB1B could elevate the expression of P300 which in turn increased glycolysis. Moreover, TTK phosphorylated LDHA at tyrosine 239 (Y239) and activated LDHA, and subsequently upregulated lactate and H3K18la levels.ConclusionsThe glycolysis-H3K18la-TTK/BUB1B positive feedback loop exacerbates dysfunction in PDAC. These findings delivered a new exploration and significant inter-relationship between lactate metabolic reprogramming and epigenetic regulation, which might pave the way toward novel lactylation treatment strategies in PDAC therapy.

OTUB2 silencing promotes ovarian cancer via mitochondrial metabolic reprogramming and can be synthetically targeted by CA9 inhibition

Ovarian cancer is an aggressive gynecological tumor characterized by a high relapse rate and chemoresistance. Ovarian cancer exhibits the cancer hallmark of elevated glycolysis, yet effective strategies targeting cancer cell metabolic reprogramming to overcome therapeutic resistance in ovarian cancer remain elusive. Here, we revealed that epigenetic silencing of Otubain 2 (OTUB2) is a driving force for mitochondrial metabolic reprogramming in ovarian cancer, which promotes tumorigenesis and chemoresistance. Mechanistically, OTUB2 silencing destabilizes sorting nexin 29 pseudogene 2 (SNX29P2), which subsequently prevents hypoxia-inducible factor-1 alpha (HIF-1α) from von Hippel-Lindau tumor suppressor-mediated degradation. Elevated HIF-1α activates the transcription of carbonic anhydrase 9 (CA9) and drives ovarian cancer progression and chemoresistance by promoting glycolysis. Importantly, pharmacological inhibition of CA9 substantially suppressed tumor growth and synergized with carboplatin in the treatment of OTUB2-silenced ovarian cancer. Thus, our study highlights the pivotal role of OTUB2/SNX29P2 in suppressing ovarian cancer development and proposes that targeting CA9-mediated glycolysis is an encouraging strategy for the treatment of ovarian cancer.

Persistent gene expression and DNA methylation alterations linked to carcinogenic effects of dichloroacetic acid

BackgroundMechanistic understanding of transient exposures that lead to adverse health outcomes will enhance our ability to recognize biological signatures of disease. Here, we measured the transcriptomic and epigenomic alterations due to exposure to the metabolic reprogramming agent, dichloroacetic acid (DCA). Previously, we showed that exposure to DCA increased liver tumor incidence in B6C3F1 mice after continuous or early life exposures significantly over background level.MethodsUsing archived formalin-fixed liver samples, we utilized modern methodologies to measure gene expression and DNA methylation levels to link to previously generated phenotypic measures. Gene expression was measured by targeted RNA sequencing (TempO-seq 1500+ toxicity panel: 2754 total genes) in liver samples collected from 10-, 32-, 57-, and 78-week old mice exposed to deionized water (controls), 3.5 g/L DCA continuously in drinking water (“Direct” group), or DCA for 10-, 32-, or 57-weeks followed by deionized water until sample collection (“Stop” groups). Genome-scaled alterations in DNA methylation were measured by Reduced Representation Bisulfite Sequencing (RRBS) in 78-week liver samples for control, Direct, 10-week Stop DCA exposed mice.ResultsTranscriptomic changes were most robust with concurrent or adjacent timepoints after exposure was withdrawn. We observed a similar pattern with DNA methylation alterations where we noted attenuated differentially methylated regions (DMRs) in the 10-week Stop DCA exposure groups compared to the Direct group at 78-weeks. Gene pathway analysis indicated cellular effects linked to increased oxidative metabolism, a primary mechanism of action for DCA, closer to exposure windows especially early in life. Conversely, many gene signatures and pathways reversed patterns later in life and reflected more pro-tumorigenic patterns for both current and prior DCA exposures. DNA methylation patterns correlated to early gene pathway perturbations, such as cellular signaling, regulation and metabolism, suggesting persistence in the epigenome and possible regulatory effects.ConclusionLiver metabolic reprogramming effects of DCA interacted with normal age mechanisms, increasing tumor burden with both continuous and prior DCA exposure in the male B6C3F1 rodent model.

Metabolic reprogramming of tumor-associated macrophages using glutamine antagonist JHU083 drives tumor immunity in myeloid-rich prostate and bladder cancer tumors.

Glutamine metabolism in tumor microenvironments critically regulates anti-tumor immunity. Using glutamine-antagonist prodrug JHU083, we report potent tumor growth inhibition in urologic tumors by JHU083-reprogrammed tumor-associated macrophages (TAMs) and tumor-infiltrating monocytes (TIMs). We show JHU083-mediated glutamine antagonism in tumor microenvironments induces TNF, pro-inflammatory, and mTORC1 signaling in intratumoral TAM clusters. JHU083-reprogrammed TAMs also exhibit increased tumor cell phagocytosis and diminished pro-angiogenic capacities. In vivo inhibition of TAM glutamine consumption resulted in increased glycolysis, a broken TCA cycle, and purine metabolism disruption. Although the anti-tumor effect of glutamine antagonism on tumor-infiltrating T cells was moderate, JHU083 promoted a stem cell-like phenotype in CD8+ T cells and decreased Treg abundance. Finally, JHU083 caused a ubiquitous shutdown in glutamine utilizing metabolic pathways in tumor cells, leading to reduced HIF-1alpha, c-MYC phosphorylation, and induction of tumor cell apoptosis, all key anti-tumor features.

Abstract PO3-08-07: Licochalcone A as a risk reducing agent against luminal and non-luminal breast cancers

Abstract Background: Breast cancer risk reducing drugs with proven efficacy have adverse side effects, significantly minimizing their uptake and impact. Further, they do not prevent ER- breast cancer. Effective alternative strategies with lower toxicity are needed. Previously, we have shown that licochalcone A (LicA) suppresses aromatase expression and activity, enhances the activity of detoxifying enzymes, and reduces estrogen genotoxic metabolism in cell lines and animal models. These data led us to hypothesize that LicA creates a tumor preventive environment in the breast by reprogramming metabolism and antioxidant/anti-inflammatory responses in the breast leading to decreased proliferation and tumor suppression. We now report on the breast tumor preventive effects of LicA in xenograft models, its oral bioavailability, and its biologic effects on human breast microstructures from women at increased risk of breast cancer. Methods: We prepared microstructures from the fresh tissue of contralateral unaffected mastectomy specimens of 6 postmenopausal women with incident unilateral breast cancer. After exposing them to DMSO (control) or LicA (5 µM), we performed total RNA sequencing. Differentially expressed genes were identified and analyzed by gene ontology and pathway membership. The RNA-seq data was also utilized to conduct metabolism flux analysis. Combined enrichment scores > 4 and FDR < 0.05 was considered significant. The NanoString metabolism panel was employed in 6 additional subjects. We performed live cell imaging to monitor proliferation of pre-malignant DCIS.COM, DCIS.COM/ER+ PR+; and malignant MDA-MB-231 (ER- PR-), MCF-7 (ER+ PR+), MCF-7aro, and BRCA1 defective HCC-1937, and HCC3153 cells. Xenograft models of MCF-7aro and MDA-MB-231 tumors were established in female nude mice and the animals treated for 28 days with vehicle or LicA (80 mg/kg.day, s.c.). We measured the rate of tumor growth. We also conducted a PK/PD study with oral LicA (100 mg/kg) in intact BALB/c female mice. Results: We observed significant (FDR < 0.05) upregulation of antioxidant genes (up to 8-fold), consistent with upregulation of NRF2 and the thioredoxin system, the major regulators of antioxidant pathways. This was accompanied by significant downregulation of RELA- and NF-kB1-dependent inflammatory pathways. In addition, we observed decreased expression of PI3K-AKT genes and the pro-adipogenic transcription factors SREBF1 and SREBF2, which may explain the downregulation (4 to 32-fold) of cholesterol biosynthesis and transport, and lipid metabolism genes. Metabolism studies confirmed these data and demonstrated a robust increase in the pentose phosphate shunt and NAD(P)H generation without enhancing ribose 5 phosphate formation, suggesting an antioxidant and anti-proliferative environment. LicA also suppressed proliferation of pre-malignant and malignant cells, with sustained effects on aggressive cells at doses < 10 µM. LicA significantly reduced tumor growth in luminal (P = 0.008) and triple negative (P = 0.001) in vivo models (unpaired t-test with Welch’s correction for unequal variances). Promising serum and breast bioavailability, equivalent to low micromolar concentrations sufficient to show efficacy was demonstrated as well. Conclusion: Our data suggest that LicA is a good candidate for breast cancer prevention in both ER+ and ER- breast cancers through reprogramming metabolism and antioxidant pathways leading to decreased proliferation. We will study LicA in intraductal models of ER+ and ER- precancer lesions in immunocompetent mice and will monitor their progression to invasive breast cancer to further establish its preventive efficacy. Citation Format: Atieh Hajirahimkhan, Elizabeth Bartom, Sriram Chandrasekaran, Susan Clare, Seema Khan. Licochalcone A as a risk reducing agent against luminal and non-luminal breast cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-08-07.

Abstract PO2-01-01: Biophysical simulation using DCE-MRI to forecast response to NAT in HER2+ patients, with glucose characterization and orthogonal validation using FDG-PET

Abstract Background: Metabolic reprogramming and tumor angiogenesis are two tightly linked hallmarks of cancer. Regions of high metabolic activity within a tumor can become hypoxic or nutrient starved, eliciting a cascade of pro-angiogenic signals that can lead to increased tumor perfusion and in turn greater metabolic activity. Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) and 18Fluorodeoxyglucose (FDG) positron emission tomography (PET) are two imaging methods commonly used in the diagnosis of cancer; the former can be also used to identify the perfusion of the tumor microenvironment (TME) and the latter provides a coarse readout of glucose metabolic activity. Here, we are presented with a unique cohort of patients who underwent DCE-MRI, 18FDG-PET and 64Cu-DOTA-Traztuzumab offering potential to perform a pilot analysis. First, we characterize the performance of our perfusion model SimBioSys- Microvasculature (SBS-MV) through a stability analysis, as well as comparison with two PET modalities. Finally, we use this model in 3D simulations of individual HER2+ breast cancer patients to forecast responses to neoadjuvant chemotherapy (NAT). Methods: SBS-MV is a modified Tofts model for pharmacokinetic modeling of DCE-MRI data, utilizing a tissue segmentation model developed in-house to inform parameter fits and ensure that the derived parameters fall within acceptable values on a tissue-by-tissue level, in addition its function for standard fitting of DCE time-course data. We first evaluated the stability of the SBS-MV model by analyzing a separate cohort of 10 patients who underwent ultrafast DCE-MRI. Temporal undersampling was performed on the original high temporal resolution data to mimic low temporal resolution from standard-of-care (SoC) DCE studies. Both datasets were processed with SBS-MV model. Next, we analyzed data from a cohort of 18 patients, each of whom underwent a pre-NAT SoC DCE-MRI study, a mid-treatment FDG PET/CT study, and a mid-treatment 64Cu-DOTA-Trastuzumab/CT study during treatment. Tissue segmentation and SBS-MV fits were performed on the pre-NAT DCE MRI. Tightly cropped boxes encompassing the tumor in the 3D SBS-MV volumes as well as the PET volumes were created. Using these identified regions, all summary statistics were calculated and cross-correlated between imaging modalities. Results: SBS-MV was stable to SoC-mimicking temporally undersampled ultrafast DCE MRI series, demonstrating relatively little variability in the output parameters (a voxel wise median deviation of approximately 0.005 min-1 ). Further, we demonstrate that SBS-MV parameters (Ktrans, ve) are correlated with FDG SUVSA and 64Cu SUVSA. In addition, the glucose concentrations used in the biophysical simulations were correlated with FDG SUVSA, indicating that downstream applications of the SBS-MV models still carry this information about perfusion. Finally, using SBS-MV, our biophysical simulations of HER2-targeted therapy accurately predict patient response. In this small cohort, our biophysical simulation platform performed well, yielding predictive accuracy of 0.83, with sensitivity and specificity of 0.83 and 0.83, respectively. These performance metrics are in line with previously published reports. Conclusion: Our MV model is capable of extracting biologically meaningful perfusion parameters from standard clinical DCE MRI time series, providing the same benefits of a comprehensive kinetic analysis, without impacting current clinical workflow. This approach could be used in both the research and clinical settings, offering actionable information on what drives individual patient therapeutic response for a more personalized care. Citation Format: John Whitman, Vikram Adhikarla, Russell Rockne, Lusine Tumyan, Joanne Mortimer, Wei Huang, Joseph Peterson, Dorys Lopez-Ramos, John Cole. Biophysical simulation using DCE-MRI to forecast response to NAT in HER2+ patients, with glucose characterization and orthogonal validation using FDG-PET [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-01-01.

Abstract PO4-08-01: Methylsterol monooxygenase 1 (MSMO1) modulates endoplasmic reticulum stress through cholesterol metabolic reprogramming to alter chemotherapy sensitivity in breast cancer

Abstract Background Endoplasmic reticulum stress (ER stress) is a process in which cells activate unfolded protein response and other signaling pathways in response to misfolded and unfolded protein aggregation and calcium ion balance disturbance in the endoplasmic reticulum cavity. Depending on the intensity of endoplasmic reticulum stress, it can not only induce chaperone expression to play a protective effect, but also induce apoptosis independently. Metabolic reprogramming is one of the characteristics of tumor, which refers to the metabolic changes that cells make in response to various stimuli. Here, we demonstrate that methylsterol monooxygenase 1 (MSMO1) regulates cellular cholesterol metabolism and influences breast cancer susceptibility to chemotherapy by regulating the relative intracellular T-MAS content. Methods RNA-seq was performed on core needle biopsy samples from 26 patients receiving neoadjuvant chemotherapy for breast cancer to screen for genes that may influence neoadjuvant chemotherapy sensitivity. Cell proliferation, CCK8 assay and apoptosis assay were performed for MSMO1 phenotype study. Transmission electron microscopy (TEM) was conducted to visualize the structure of endoplasmic reticulum. Western Blot was used to detect the activation level of the unfolded protein response pathway. The relative content of sterols in MSMO1 knockdown cells was detected by targeted lipid mass spectrometry. AO/PI staining of breast cancer organoids was performed to verify the effect of MSMO1 metabolic substrate T-MAS on chemotherapy drug sensitivity. Results RNA-seq of core needle biopsy samples from breast cancer patients receiving neoadjuvant chemotherapy suggested that MSMO1 was highly expressed in non-pCR patients, and clinical survival data suggested that MSMO1 was associated with poor prognosis in patients with all types of breast cancer, suggesting that MSMO1 might be related to chemotherapy sensitivity of breast cancer. Phenotypic experiments revealed that MSMO1 regulates chemotherapy sensitivity of breast cancer both in vivo and in vitro. Ectopic overexpression of MSMO1 promoted cancer cell resistance to chemotherapy. To uncover the underlying mechanisms, RNA-seq of MSMO1 knock-down cells were carried out and identified that MSMO1 was associated with ER stress. TEM and western blot assay indicated that downregulation of MSMO1 expression leads to endoplasmic reticulum swelling and activation of the unfolded protein response. Considering that MSMO1 is a cholesterol metabolizing enzyme, targeted lipids mass spectrometry was performed to detect the changes in the contents of various intermediate metabolites in the cholesterol pathway, which indicated that the relative content of T-MAS, a metabolic substrate of MSMO1, was significantly up-regulated in MSMO1 knockdown cells. Then, in order to verify whether this metabolite can independently regulate ER stress, breast cancer cells were given T-MAS externally, and TEM and Western Blot analysis indicated that T-MAS could induce ER stress and activate the unfolded protein response independently. Apoptosis assay and AO/PI staining of breast cancer organoids revealed that T-MAS can improve the sensitivity of breast cancer to chemotherapy drugs. Conclusion In summary, these results shed light on the role of MSMO1 in cholesterol metabolism reprogramming, which adjust the endoplasmic reticulum stress state of cells by changing the relative content of intracellular T-MAS, and thereby influencing the sensitivity of breast cancer chemotherapy. As an intermediate metabolite of cholesterol metabolism, T-MAS has potential clinical application value of chemotherapy sensitization. Citation Format: Hengyu Ren, Liren Wangxu, Yayun Chi, Jiong Wu. Methylsterol monooxygenase 1 (MSMO1) modulates endoplasmic reticulum stress through cholesterol metabolic reprogramming to alter chemotherapy sensitivity in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-08-01.

Abstract PO1-26-04: Patient-derived organotypic tissue cultures for organoid isolation and probing unique metabolic features of individual breast cancer patients

Abstract Patient-derived organotypic tissue cultures (PD-OTC) are unique models for probing how individual patient’s tumor microenvironment (TME) influences cancer development and treatment responses. They retain the patient tumor architecture and TME, that are difficult to recapitulate in other models, while affording flexible treatment options and comparison of cancer (CA) versus matched non-cancer (NC) tissue responses to treatments. We have developed a culturing method for establishing PD-OTC of breast cancer patients that exhibit CA tissue growth and extensive metabolic reprogramming between CA versus matched NC tissues while enabling patient-derived organoids (PDO) and fibroblasts to be isolated. Pairs of CA and NC tissues freshly resected from six patients bearing early-stage estrogen receptor/progesterone receptor positive ductal carcinoma were sliced at 750 µm, embedded in Matrigel, and cultured in a 6-well plate with a Biopore membrane insert and custom medium at 37°C/5% CO2 with gentle rocking (16-40 mg wet weight; n=3). Excess tissue slices were cryopreserved and kept at -196 °C. Culture media were sampled periodically for metabolite analysis by NMR. Microscopic examination revealed significant tissue, organoid-like, and fibroblast outgrowth after 1-1.5 month of culturing. Two to three days prior to the harvest of two patients’ (CZ16 and 17) OTC, the culture medium was replaced with [U-2H]-glucose + [U-13C,15N]-glutamine-containing medium to allow the two stable isotope tracers to be metabolized, followed by Stable Isotope-Resolved Metabolomic (SIRM) analysis by NMR and ion chromatography coupled with ultra high-resolution Fourier transform mass spectrometry (IC-UHRFTMS). Two separate pieces of the OTC were cut and each was assayed for glucose uptake and mitochondrial membrane potential by live fluorescence spectroscopy via a portable custom-built fluorescence microscope, and by histochemical analysis. Histological analysis showed that both CA and NC OTC of all patients maintained their structural integrity. SIRM analysis of two patients’ OTC revealed active catabolic and anabolic metabolism including glucose uptake/glycolysis, the Krebs cycle/mitochondrial membrane potential, the pentose phosphate pathway (PPP), gluconeogenesis, glycogen synthesis, and purine/pyrimidine/sugar nucleotides synthesis. The CA OTC of both patients showed enhanced glucose uptake, glycolysis, mitochondrial membrane potential/Krebs cycle, PPP, glycogen synthesis, and purine nucleotide synthesis, when compared with the matched NC OTC. Treatment-naïve CA OTC of CZ16 showed much more significant metabolic reprogramming (NAD+ metabolism, in particular) than CA OTC of CZ17 who has undergone neoadjuvant treatment, chemotherapy, and hormone therapy prior to surgery. Distinct metabolic features of breast CA tissues can be exploited as therapeutic targets. Organoids and fibroblasts were isolated from CA and/or NC OTC cultures and their characterization is in progress. When revived, cryopreserved OTC exhibited active metabolism and tissue/organoid/fibroblast outgrowth similarly to fresh OTC. In conclusion, our culturing method enables PD-OTC models to be established efficiently for early-stage breast cancer patients for downstream functional studies including live cell imaging and SIRM analysis. Organoids and fibroblasts can also be derived from < 20 mg of tissues for further studies. Future direction includes therapeutic studies on these patient-derived models to better understand and predict individual patient’s responses to therapy. Citation Format: Teresa Fan, Carlos Goncalves, Jing Yan, Jahid Islam, Penghui Lin, Mohamed Kaddah, Richard Higashi, Andrew Lane, Caigang Zhu. Patient-derived organotypic tissue cultures for organoid isolation and probing unique metabolic features of individual breast cancer patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-26-04.

Abstract ED03-01: Breaking the Obesity-Breast Cancer Link: Comparing Diet, Drug and Surgical Approaches

Abstract The prevalence of obesity, an established risk and progression factor for at least 15 types of cancer (including breast cancer), continues to rise in the US and many other countries. Lifestyle approaches to weight loss have proven challenging for most people to maintain, and bariatric surgery, while effective, is expensive and has numerous adverse effects. The recent availability of highly effective incretin therapies for weight loss raises several key questions that we will address, including: i) do these agents drive weight loss in obese female mice?; ii) do they reverse obesity-induced metabolic dysregulation?; iii) can they reverse obesity-driven cancer?; iv) how heterogeneous is the response to these agents? We will report our study of the effects of extended (13 weeks) tirzepatide (TZP, a dual GLP1R/GIPR agonist) treatment in obese female mice on weight loss, metabolic hormones, and mammary tumor growth. We found that—similar to reports in the literature using male mice—obese female mice lost >20% body weight by 4 weeks of TZP treatment. Moreover, TZP-induced weight loss was maintained through 13 weeks of treatment by progressively escalating the dose up to 40 nmol/kg. Congruent with reports in male rodents and humans, we found the majority of weight loss in female mice was attributed to loss of adipose tissue, rather than lean mass. The effects of TZP on body weight and composition were associated with reversal of obesity-induced changes in glycemic control and circulating levels of metabolic hormones, particularly leptin, insulin, and IGF-1. TZP also significantly reversed the protumor effects of obesity in our murine E0771 orthotopic transplant model of TNBC.Furthermore, daily 30% calorie restriction, which we previously reported has strong weight loss-inducing, metabolic reprogramming and anticancer effects in multiple mouse models of breast and other cancers, reversed body weight, adiposity, and TNBC tumor growth and altered metabolic hormones to a greater extent than all groups studied, including the never-obese control mice and TZP-treated mice. Citation Format: S. Hursting. Breaking the Obesity-Breast Cancer Link: Comparing Diet, Drug and Surgical Approaches [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr ED03-01.

Abstract PO1-16-03: GDP-mannose enhances the efficacy of PARP inhibitors and immunotherapy in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) patients with high HRD scores benefit from poly (ADP-ribose) polymerase (PARP) inhibitors, while those with low HRD scores still lack therapeutic options. Homologous recombination deficiency (HRD) induction is an effective strategy to broaden the indications of PARP inhibitors. Metabolic reprogramming is a critical feature of TNBC. In this study, we aimed to explore novel metabolic biomarkers for HRD and new strategy for sensitizing PARP inhibitors. Methods: We utilized TNBC metabolomics to systematically evaluate metabolites that were correlated with HRD. A crucial metabolite, GDP-mannose (GDP-M), that impedes homologous recombination repair (HR) and sensitizes PARP inhibitors was identified and functionally validated. We further explored the detailed mechanism and proposed a potential treatment strategy utilizing GDP-M for TNBC in preclinical models. Results: Systematic metabolomic analysis revealed that GDP-mannose (GDP-M) was significantly enriched in basal-like tumors with HRD. GDP-M promoted cisplatin-induced DNA double-strand breaks by inducing HRD. Mechanistically, the low expression of the upstream enzyme GMPPA led to the endogenous upregulation of GDP-M, which further promoted the ubiquitin-mediated degradation of BRCA2 to inhibit HR. GMPPA expression and GDP-M could serve as predictive biomarkers for HRD and the response to PARP inhibitors. Therapeutically, we validated that the combination of GDP-M and PARP inhibitors synergistically inhibit tumor growth in multiple preclinical models. Moreover, GDP-M supplementation plus PARP inhibition activated STING-dependent antitumor immunity and further augmented the efficacy of anti-PD-1 antibodies. Conclusions: GDP-M promotes the degradation of BRCA2 and thus enhances the sensitivity of PARP inhibitors in TNBC. The combination of GDP-M, PARP inhibitors and anti-PD-1 immunotherapy may be a potential treatment strategy for HRD-low TNBC. Citation Format: Yi Xiao, Jiahan Ding, Yi-Zhou Jiang, Zhi-Ming Shao, Genhong Di, Fan Yang, Xiaoqing Song. GDP-mannose enhances the efficacy of PARP inhibitors and immunotherapy in triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-16-03.

Von Hippel-Lindau protein signalling in clear cell renal cell carcinoma.

The distinct pathological and molecular features of kidney cancer in adaptation to oxygen homeostasis render this malignancy an attractive model for investigating hypoxia signalling and potentially developing potent targeted therapies. Hypoxia signalling has a pivotal role in kidney cancer, particularly within the most prevalent subtype, known as renal cell carcinoma (RCC). Hypoxia promotes various crucial pathological processes, such as hypoxia-inducible factor (HIF) activation, angiogenesis, proliferation, metabolic reprogramming and drug resistance, all of which contribute to kidney cancer development, growth or metastasis formation. A substantial portion of kidney cancers, in particular clear cell RCC(ccRCC), are characterized by a loss of function of Von Hippel-Lindautumour suppressor (VHL), leading to the accumulation of HIF proteins, especially HIF2α, a crucial driver of ccRCC. Thus, therapeutic strategies targeting pVHL-HIF signalling have been explored in ccRCC, culminating in the successful development of HIF2α-specific antagonists such as belzutifan (PT2977), an FDA-approved drug to treat VHL-associated diseases including advanced-stage ccRCC. An increased understanding of hypoxia signalling in kidney cancer came from the discovery of novel VHL protein (pVHL) targets, and mechanisms of synthetic lethality with VHL mutations. These breakthroughs can pave the way for the development of innovative and potent combination therapies in kidney cancer.

Abstract PO3-24-05: Cysteine metabolism related ferroptosis sensitivity in trastuzumab resistant HER2 positive breast cancer

Abstract Background Trastuzumab has shown great effectiveness in HER2 positive breast cancer treatment, but about 50% patients would undergo resistance during or after treatment. Although previous research has suggested several potential reasons for trastuzumab resistance, the metabolic reprogramming during resistance formation remains largely unclear. Here we identified aberrant ferroptosis associated cysteine metabolism in trastuzumab resistant HER2 positive breast cancer, which might become a novel target for overcoming resistance. Methods Trastuzumab sensitive HER2 positive breast cancer cell SKBR3 and resistant JITM1 were obtained for transcriptomics, proteomics, metabolomics and epigenomics analysis. Gene silencing was mediated by siRNAs. CUT&Tag was applied to compare H3K4me3 and H3K27me3 binding regions. DNA methylation levels and different methylated regions were evaluated by WGBS-seq. CRISPRi with dCas9-DNMT3A was applied to regulate specific DNA methylation in CpG islands. Lipid ROS was measured by flow cytometry with BODIPY-C11. Results Joint analyses of transcriptomics and proteomics according to ferroptosis pathways revealed downregulated glutathione metabolism, glutamate transmembrane and homocysteine metabolism processes, as well as upregulated fatty acid metabolism and iron metabolism pathways in JIMT1. Metabolomics verified that JIMT1 increased cysteine metabolism and decreased glutathione metabolism. SLC7A11 expression and GSH/GSSG ratio were increased in JIMT1, while no difference was observed in free cysteine. JIMT1 featured significant higher UGC codon usage bias and increased cysteinyl-tRNA synthetase. The abundance of H3K4me3 other than H3K27me3 in SLC7A11 promoter region was found increased in JIMT1, and the 5-mC level of CpG islands in SLC7A11 promoter region was shown decreased. Using dCas9-DNMT3A, the methylation of SLC7A11 promoter was enhanced and SLC7A11 expression was reduced in JIMT1. Inhibition of SLC7A11 by siRNAs, CRISPRi or Erastin all indicated a higher ferroptosis sensitivity in JIMT1. Conclusion Trastuzumab resistant HER2 positive breast cancer features aberrant cysteine metabolism resulting from altered H3K4me3 modification and DNA methylation in SLC7A11 promoter region. This might provide novel targets for further anti-HER2 treatment. Citation Format: Yongmei Yin, Yijia Hua, Ningjun Duan, Chunxiao Sun, Fan Yang. Cysteine metabolism related ferroptosis sensitivity in trastuzumab resistant HER2 positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-05.

Abstract PS03-02: Single-cell metabolic profiling uncovers new precision immunotherapy strategy in triple-negative breast cancer

Abstract Background: Immunotherapy has emerged as a novel cornerstone in the treatment of triple-negative breast cancer (TNBC), while its benefits have been observed in only a limited subset of patients. The intricate metabolic interplay between cancer cells and microenvironmental cells remodels the tumor microenvironment and exerts a significant influence on the responses to immunotherapy. However, there is still a lack of clarity regarding the strategies to target this metabolic crosstalk in order to predict efficacy and enhance sensitivity for immunotherapy. Methods: We conducted single-cell RNA sequencing (scRNA-seq) analysis on a cohort of 27 patients with TNBC receiving either immunotherapy combination regimen or chemotherapy alone. Building upon this foundation, we embarked on a comprehensive investigation of the single-cell metabolic landscape from three distinct perspectives: metabolic genes, metabolic pathways, and metabolic flux, represented by the flux balance analysis (FBA) algorithm Compass. Furthermore, we identified crucial cell subgroups that exhibited a strong correlation with immunotherapy efficacy and elucidated their metabolic characteristics to postulate potential metabolic crosstalk. Then, we employed the in vitro, in vivo, and patient-derived models to unravel the underlying mechanisms of metabolic crosstalk and proposed the translational strategies. Results: Utilizing newly-developed single-cell FBA method, we illustrated that different cell types have distinct metabolic features. Specifically, tumor cells and macrophages had more active metabolic reprogramming and higher metabolic heterogeneity. Furthermore, we illustrated that a subset of tumor cells having active antioxidant metabolism (featured by the upregulation of GSTP1) and macrophage subset depending on glutamine metabolism (characterized by CCL3+) were negatively and positively predictive of immunotherapy responses, respectively. Mechanistically, GSTP1-mediated excessive consumption of glutamine by tumor cells competitively restrained the intake of glutamine into CCL3+ macrophages to promote their ferroptosis and reduce their infiltration. Clinically, we demonstrated that targeting the GSTP1, which is specifically expressed in tumor cells, remodeled immune-metabolic microenvironment and enhanced immunotherapy efficacy. Furthermore, we established the utility of plasma CCL3 abundance, GSTP1 protein abundance, and CCL3 protein abundance, as robust predictors of response to immunotherapy, in several independent immunotherapy cohorts. Conclusion: Our study presented a comprehensive and multidimensional analysis of the single-cell metabolic landscape. Identifying the crucial metabolic crosstalk remodeling the immune microenvironment, our promising finding highlights the potential significance of modulating cell-cell metabolic interactions in sensitizing TNBC to immunotherapy, providing valuable insights for future therapeutic interventions. Citation Format: Ying Xu, Yi Xiao, Han Wang, Zhimin Shao, Yi-Zhou Jiang. Single-cell metabolic profiling uncovers new precision immunotherapy strategy in triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS03-02.

Abstract PO3-24-04: Altered epigenetic modifications and genome architecture reshape lipid metabolism during secondary trastuzumab resistance formation of HER2-positive breast cancer

Abstract Title Altered epigenetic modifications and genome architecture reshape lipid metabolism during secondary trastuzumab resistance formation of HER2-positive breast cancer Background Secondary trastuzumab resistance seriously affects the treatment of HER2-positive breast cancer. Although studies have demonstrated several key cellular properties that are closely accompanied with trastuzumab resistance formation, we still have poor knowledge on the genetic and epigenetic variations that promote such transformation. Here we suggested that the epigenetic modification changes on histone H3 together with altered chromatin architecture promote lipid metabolism reprogramming during secondary trastuzumab formation. Materials and methods Induced secondary trastuzumab-resistant SKBR3_HR cell line together with the original trastuzumab-sensitive SKBR3 cell line were applied in this study. Total RNA was collected for transcriptome analysis. Anti-H3K4me3 and K27me3 antibodies were chosen for CUT&Tag sequencing library preparation. Total genome DNA was prepared for Micro-C sequencing library preparation. Activity score of metabolism pathway was calculated as relative gene expression value averaged over all genes in this pathway in certain cell types. Results Upregulation of arachidonic acid metabolism and downregulation of unsaturated fatty acid synthesis, which are mainly characterized by the activation of PTGS1 and PTGES genes and the repression of FASN and SCD genes, respectively, were observed during the formation of secondary trastuzumab resistance from SKBR3 cell to SKBR3_HR cells. Variations of H3k4me3 instead of H3k27me3 regulate the expression of these 4 genes. The accumulation of H3k4me3 was detected at the promoters of PTGS1 and PTGES genes while they were removed from the promoters of FASN and SCD genes in SKBR3_HR cells. More intra-chromosomal interactions were constituted during resistance formation. In detail, 4626 and 4394 topological associated domains, 3125 and 5824 DNA loops were founded in SKBR3 and SKBR3_HR cells, respectively. Furthermore, the lost and gained DNA loops between SCD and PTGS1 genes and distant genome regions may indicate the weaken and strengthen interactions with transcriptional regulators located there. Conclusions During trastuzumab resistance formation, altered histone modifications as well as higher genome structure could regulate the expression of key genes in lipid metabolism pathways, which may further affect cell properties and interactions with cells in tumor microenvironment. Citation Format: Ningjun Duan, Yijia Hua, Yongmei Yin. Altered epigenetic modifications and genome architecture reshape lipid metabolism during secondary trastuzumab resistance formation of HER2-positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-04.

Metabolic Rewiring in Cancer: Small Molecule Inhibitors in Colorectal Cancer Therapy

Alterations in cellular metabolism, such as dysregulation in glycolysis, lipid metabolism, and glutaminolysis in response to hypoxic and low-nutrient conditions within the tumor microenvironment, are well-recognized hallmarks of cancer. Therefore, understanding the interplay between aerobic glycolysis, lipid metabolism, and glutaminolysis is crucial for developing effective metabolism-based therapies for cancer, particularly in the context of colorectal cancer (CRC). In this regard, the present review explores the complex field of metabolic reprogramming in tumorigenesis and progression, providing insights into the current landscape of small molecule inhibitors targeting tumorigenic metabolic pathways and their implications for CRC treatment.

Abstract PO3-25-12: Tigecycline-Induced Metabolic Reprogramming and Cytokine Modulation Suppress the Characteristics of Breast Cancer Cells

Abstract Objective: Patients with advanced breast cancer are prone to recurrence and metastasis. Hence, there is still a need to find new potential drugs or adjuvants to improve prognosis. The Warburg effect, which is highly dependent on glycolysis, has been found to be present in the energy metabolism of tumors, including breast cancer. The effect also promotes the production of cytokines, to adapt the inflammatory microenvironment of tumor. However, drugs targeting Warburg effect and inflammatory environment are rarely reported in the treatment of breast cancer. Tigecycline (Tige) is a commonly used glycylcycline antibiotic. Previous research has indicated it impede mitochondrial oxidative phosphorylation (OXPHOS) in various solid tumors. Based on the team's previous research, our study aimed to investigate whether Tige could inhibit breast cancer development by interfering with the Warburg effect and the inflammatory environment. Methods: MB231, MB468 and MCF7, T47D, which represent triple-negative and estrogen receptor positive breast cancers, were treated with Tige at different concentrations for different periods of time. CCK8, colony formation and sphere formation assays were used to evaluate the effects on proliferation and tumor stemness in vitro, respectively. The effects on migration and invasion were evaluated by wound healing assay and Transwell. RNA sequencing was used to explore the genes altered and analyze the pathways involved. After labeling with probes, the changes of reactive oxygen species (ROS) were detected by fluorescence microscope, microplate reader, and flow cytometry. Cell cycle and apoptosis were detected by flow cytometry. Liquid cytokine chip was used to analyze the secretion of cytokines. Electron microscopy was performed to observe the changes in mitochondrial morphology. RT-PCR was used to detect the expression of rate-limiting enzymes in glycolysis pathway. Glucose consumption, lactate and pyruvate production were measured using glucose, lactate and pyruvate detection kits, respectively. Western blot was used to detect the changes of respiratory chain protein complex and other related pathway proteins in OXPHOS. Results: Four breast cancer cells showed a significant decrease in cell proliferation, invasion and migratory ability after Tige treatment. Sphere-forming assays also showed that Tige had an effect on the stemness of breast cancer. RNA-sequencing showed that glycolysis, cell cycle, and immune-related pathways were inhibited after Tige treatment, and ROS production analysis revealed a decrease in ROS production. Flow cytometry also revealed that the cell cycle was arrested in S phase without significant apoptosis. Cytokine liquid chip analysis showed that the secretion of many cytokines, mainly interleukin (such as IL-1B), was inhibited after Tige intervention. RT-PCR results showed that the expression of several rate-limiting enzymes of the glycolytic pathway decreased. Glucose, lactate and pyruvate assay results showed that Tige reduced glucose uptake and lactate, pyruvate production. Electron microscopy results revealed that mitochondria in Tige treated cells showed significant contractures. WB results also showed a decrease in the expression of OXPHOS-related proteins. However, due to the heterogeneity of the four breast cancer cells, the degree of change in these capacities was not entirely consistent. Conclusions: Tige not only inhibits mitochondrial OXPHOS, but also inhibits the glycolysis pathway, which results in the inhibition of glucose uptake and the production of pyruvate and lactate. The secretion of early pro-inflammatory cytokines also affected. Therefore, this study suggests that Tige may change the glucose metabolism and energy metabolism, and also change the inflammatory environment of breast tumors, which provides new evidence for the anti-tumor effect of Tige in breast cancer. Citation Format: Haochen Yu, Lingfeng Tang, Shengchun Liu. Tigecycline-Induced Metabolic Reprogramming and Cytokine Modulation Suppress the Characteristics of Breast Cancer Cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-12.

Abstract PO4-13-09: Paclitaxel affects the efficacy of PD-1 blockade by interfering with lipid metabolism reprogramming in patients with breast cancer

Abstract Background: The question of how chemotherapeutic drugs affect the efficacy of PD-1/PD-L1 antibodies in patients with breast cancer is still pending. In a previously single-cell sequencing study, we have demonstrated that paclitaxel reduces the level of CXCL13+ T cells and other immune cells critical for atezolizumab to work, leading to inferior efficacy of atezolizumab in triple negative breast cancer (TNBC). How paclitaxel affects these important immune cells remains obscure. Recently, plasma metabolomic and proteomics analysis have shown that patients’ systemic metabolic signatures is correlated with the response of PD-1/PD-L1 antibody. As such, understanding the differential metabolic influences of the diverse chemotherapeutic drugs that comprise an immune response to TNBC offers an opportunity to appropriately select chemotherapeutic backbones for ICIs. Methods: We collected pre- and post-treatment plasma samples from breast cancer patients for metabolomic and proteomic analysis, including four patient cohorts: two independent cohorts (discovery cohort and validation cohort) in which patients were treated with ICIs, one cohort in which patients were treated with paclitaxel, and one cohort of patients treated with nab-paclitaxel. Patients were divided into responder group (R, tumor shrinkage) or non-responder group (NR, tumor increase) according to the change in tumor size after treatment relative to baseline. This was followed by integrating metabolomics and proteomics with single-cell transcriptome, along with in vitro experiments, to shed light on the mechanisms of action leading to ICIs resistance in breast cancer patients. Results: In the discovery cohort, the metabolites that mostly differed between the R and NR groups at baseline were lysoglycerophospholipids. These results were further validated in the validation cohort of patients. The dynamic changes of lysoglycerophospholipids were still significantly different between the R and the NR groups after ICIs treatment in the discovery cohort. It’s worth noting that the level of lysophosphatidylcholines (LPCs), the most prominent lysoglycerophospholipids, increased significantly in the R group, while no significant changes among LPCs were found in the NR group after ICIs treatment. In the validation cohort, the median PFS of LPClow patients was 1.8 months versus 6.8 months in the LPChigh group (P = 0.0068). After one cycle of paclitaxel treatment, the metabolic status of patients changed dramatically, especially the levels of lipids. It is notable that most LPCs were significantly downregulated after paclitaxel treatment. However, in patients who received nab-paclitaxel, few significantly changed lipids were detected after one cycle nab-paclitaxel treatment compared with their baseline conditions. Interestingly, the levels of LPCs were upregulated after one cycle nab-paclitaxel treatment, but decreased after paclitaxel treatment. Single-cell omics results showed that in patients' PBMCs, CXCR4+ NK cells were the most closely related to plasma LPC levels, while FGFBP2+ NK cells were in the tumor microenvironment. We isolated and expanded NK cells from PBMCs for in vitro experiments, and found that LPC stimulation can significantly upregulate the expression of CXCR4+ in NK cells, and further increase the chemotaxis and cytotoxicity of NK cells. Conclusions: In breast cancer patients receiving ICIs, the systemic lipid metabolism status, especially lysoglycerophospholipids, is closely related to the efficacy of immunotherapy. The key molecule LPC, the most prominent lysoglycerophospholipids, may promote the efficacy of immunotherapy by activating NK cells in breast cancer patients. Paclitaxel may impair the efficacy of immunotherapy by affecting the systemic lipid metabolism status, especially by reducing plasma LPC levels, so it is not a suitable partner for ICIs. Citation Format: Hongnan Mo, Jiashu Han, Jingtong Zhai, Xiaoying Sun, Xiuwen Guan, Haili Qian, Fei Ma. Paclitaxel affects the efficacy of PD-1 blockade by interfering with lipid metabolism reprogramming in patients with breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-13-09.

An Update on MR Spectroscopy in Cancer Management: Advances in Instrumentation, Acquisition, and Analysis.

MR spectroscopy (MRS) is a noninvasive imaging method enabling chemical and molecular profiling of tissues in a localized, multiplexed, and nonionizing manner. As metabolic reprogramming is a hallmark of cancer, MRS provides valuable metabolic and molecular information for cancer diagnosis, prognosis, treatment monitoring, and patient management. This review provides an update on the use of MRS for clinical cancer management. The first section includes an overview of the principles of MRS, current methods, and conventional metabolites of interest. The remainder of the review is focused on three key areas: advances in instrumentation, specifically ultrahigh-field-strength MRI scanners and hybrid systems; emerging methods for acquisition, including deuterium imaging, hyperpolarized carbon 13 MRI and MRS, chemical exchange saturation transfer, diffusion-weighted MRS, MR fingerprinting, and fast acquisition; and analysis aided by artificial intelligence. The review concludes with future recommendations to facilitate routine use of MRS in cancer management. Keywords: MR Spectroscopy, Spectroscopic Imaging, Molecular Imaging in Oncology, Metabolic Reprogramming, Clinical Cancer Management © RSNA, 2024.