Alexander Kirchmair

The Fontan circulation is associated with progressive multisystem complications, yet its underlying molecular mechanisms remain incompletely understood. We aimed to characterize systemic proteomic alterations and identify biological pathways associated with Fontan physiology, long-term adaptation, and clinically unfavorable phenotypes. Peripheral vein serum proteomics was performed using mass spectrometry in 48 Fontan patients and matched controls. Differential protein expression and pathway enrichment analyses were applied across the overall cohort and clinically defined subgroups according to Fontan duration and composite clinical risk phenotyping. More than one third of quantified proteins were differentially expressed, demonstrating extensive systemic proteomic remodeling. Extracellular matrix remodeling emerged as the dominant signature, characterized by fibrosis-associated proteins and enrichment of TGF-β-related profibrotic pathways. Prolonged Fontan duration and clinically unfavorable status were not associated with substantially amplified extracellular matrix signatures. Instead, long-term Fontan patients demonstrated increasing immune, complement, and endothelial activation, whereas high-risk patients showed metabolic and redox-related alterations. These findings indicate that extracellular matrix remodeling and profibrotic signaling are dominant features of the Fontan circulation, while increasing Fontan duration and clinical deterioration are associated with immune activation, endothelial stress, and metabolic dysregulation with oxidative stress.

The total cavopulmonary anastomosis (Fontan procedure), a palliative procedure for single-ventricle congenital heart disease, improves survival but is associated with progressive multiorgan complications and high long-term morbidity. Prior blood-based proteomic studies in adults have been limited to targeted antibody-based panels or focused on methodological comparisons. Systemic molecular alterations in younger, clinically heterogeneous patients, particularly in untargeted pathways, remain incompletely characterized. Serum samples from 48 Fontan patients and 48 age- and sex-matched healthy controls were analyzed using mass spectrometry with TMT labeling. 2228 proteins were quantified, of which 124 were significantly differentially abundant (fold change > 1.5 or p < 0.05). Network analysis identified three major functional clusters: extracellular matrix (ECM) organization (predominantly increased), actin cytoskeleton organization, and platelet-related pathways (both predominantly decreased). Stratified analyses showed reduced ECM protein abundance in high-risk patients, suggesting a shift from active remodeling toward a more established fibrotic state, and uniquely elevated cytochrome b5 reductase 3 (CYB5R3), implicating altered redox homeostasis, nitric oxide metabolism, and cellular aging. Overall, our findings extend prior targeted analyses, reveal potential biomarkers such as CYB5R3 and underscore the complexity of the Fontan circulation, with implications for risk stratification and therapeutic targeting.

Macrophages are recruited to sites of infection contributing to the killing of bacteria, but also to malignant tumors, where they promote angiogenesis and suppress antitumor immune responses. The metabolic microenvironment in tumors is frequently depleted of important nutrients such as glucose. Here, we investigated metabolic adaptation strategies of macrophages to glucose deprivation using stable isotopic tracing. Lactate production was decreased, potentially indicating a reduction of glycolysis. In contrast, the contribution of glutamine to the tricarboxylic acid cycle via α-ketoglutarate and reductive carboxylation were increased. Moreover, gluconeogenesis, the reverse pathway of glycolysis, was activated in glucose-deprived macrophages, proceeding partially to the generation of glycolytic intermediates and glycerol-3-phosphate. The partial gluconeogenesis pathway was abrogated in human and murine macrophages lacking the initial gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PCK2, mitochondrial isoform). Partial gluconeogenesis was higher in anti-inflammatory, interleukin-4-stimulated compared to proinflammatory, interferon-γ/lipopolysaccharide-stimulated macrophages. Single-cell analysis and immunostaining revealed expression of PCK2 in macrophages from both lung cancer and normal lung. Low glucose conditions only partially modulated macrophage phenotypes, leading to reduced CD80 surface marker levels in proinflammatory, and enhanced vascular endothelial growth factor expression in anti-inflammatory macrophages. Our study reveals partial gluconeogenesis in glucose-deprived macrophages and shows that this versatile type of immune cells exhibits remarkable metabolic flexibility.

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Resistance to cyclin-dependent kinase 4/6 (CDK4/CDK6) inhibitors leads to treatment failure and disease progression in women with hormone receptor⁺HER2⁻ (HR⁺HER2⁻) breast cancer (BC). We delineated a hypoxia-sensitive, CCL2-dependent pathway recruiting interleukin-17A (IL-17A)-secreting γδ T cells to mouse HR⁺HER2⁻ BCs following CDK4/CDK6 inhibition, resulting in repolarization of tumor-associated macrophages (TAMs) toward an immunosuppressive CX3CR1⁺ phenotype associated with resistance. Increased IL-17A signaling and intratumoral γδ T cell abundance positively correlated with advanced grade and/or reduced survival in two cohorts of individuals with HR⁺HER2⁻ BC. Circulating γδ T cells and plasma CCL2 levels negatively correlated with progression in an independent series of individuals with HR⁺HER2⁻ BC receiving CDK4/CDK6 inhibitors. Intratumoral γδ T cells were increased in post- versus pretreatment biopsies from individuals with HR⁺HER2⁻ BC relapsing on CDK4/CDK6 inhibitors. CX3CR1⁺ TAMs had negative prognostic impact in women with HR⁺HER2⁻ BC receiving neoadjuvant PD-1 blockage and radiotherapy. Thus, γδ T cells and CX3XR1⁺ TAMs may favor resistance to CDK4/CDK6 inhibitors in individuals with HR⁺HER2⁻ BC.

SUMMARY: The immune composition of the tumor microenvironment (TME) has a major impact on the therapeutic response and clinical outcome in patients with colorectal cancer (CRC). Here, we comprehensively characterize the TME at the single-cell level by first building a large-scale atlas that integrates 4.27 million single cells from 1,670 patient samples. We then complemented the atlas with single-cell profiles from four CRC cohorts with 266 patients, including cells with low mRNA content, spatial transcriptional profiles from 3.7 million cells, and protein profiles from 0.7 million cells. The analysis of the atlas allows refined tumor classification into four immune phenotypes: immune desert, B cell enriched, T cell enriched, and myeloid cell enriched subtypes. Within the myeloid compartment we uncover distinct subpopulations of neutrophils that acquire new functional properties in blood and in the TME, including anti-tumorigenic capabilities. Further, spatial multimodal single-cell profiling reveals that neutrophils are organized in clusters within distinct functional niches. Finally, using an orthotopic mouse model we show that cancer-derived systemic signals modify neutrophil production in the bone marrow, providing evidence for tumor-induced granulopoiesis. Our study provides a big data resource for the CRC and suggests novel therapeutic strategies targeting neutrophils.

Naïve T cells remain in an actively maintained state of quiescence until activation by antigenic signals, upon which they start proliferation and generation of effector cells to initiate a functional immune response. Metabolic reprogramming is essential to meet the biosynthetic demands of the differentiation process, and failure to do so can promote the development of hypofunctional exhausted T cells. Here we used ¹³ C metabolomics and transcriptomics to study the metabolic dynamics of CD8 ⁺ T cells in their complete course of differentiation from naïve over stem-like memory to effector cells. The quiescence of naïve T cells was evident in a profound suppression of glucose oxidation and a decreased expression of ENO1 , downstream of which no glycolytic flux was detectable. Moreover, TCA cycle activity was low in naïve T cells and associated with a downregulation of SDH subunits. Upon stimulation and exit from quiescence, the initiation of cell growth and proliferation was accompanied by differential expression of T cell regulatory genes and metabolic reprogramming towards aerobic glycolysis with high rates of nutrient uptake, respiration and lactate production. High flux in anabolic pathways imposed a strain on NADH homeostasis, which coincided with engagement of the proline cycle for mitochondrial redox shuttling. With acquisition of effector functions, cells increasingly relied on glycolysis as opposed to oxidative phosphorylation, which paradoxically was not linked to changes in mitochondrial abundance. We further investigated the metabolic phenotype of exhausted T cells, finding that decreased effector function concurred with a reduction in mitochondrial metabolism, glycolysis and amino acid import, and an upregulation of suppressive and quiescence-associated genes, including TXNIP and KLF2 . Thus, these results identify multiple features critical for the metabolic reprogramming that supports quiescence, proliferation and effector function of CD8 ⁺ T cells during differentiation. Further, an impairment of the same processes in exhaustion suggests that targeting these control points may be useful for both modulation of differentiation and prevention of exhaustion.

The standard of care therapies for metastatic colorectal cancer (mCRC) patients with wild-type (WT) RAS or BRAF consists of chemotherapy combined with targeted anti-EGFR antibodies. Still, only half of the RAS WT mCRC patients are sensitive to anti-EGFR therapy¹, and eventually some of them stop responding due to intrinsic and acquired resistance to anti-EGFR treatment². Moreover, recent clinical studies have also shown that left-sided mCRC respond better to anti-EGFR treatment for reasons that are still unknown. Studies from our lab and others using genetically engineered mouse models (GEMMs) showed that EGFR expressing tumor-associated myeloid cells release inflammatory cytokines that have a pro-tumorigenic role in CRC^3,4. Deletion of EGFR in tumor cells of these mice had no effect in tumor size, whereas deletion of EGFR in myeloid cells impaired CRC development³. Moreover, human CRC biopsies confirmed that presence of EGFR⁺ myeloid cells is associated with a worse overall survival of mCRC patients. From our results in GEMMs, it seems that the therapeutic action of EGFR inhibitors is mediated via EGFR inhibition in myeloid cells. Assuming that tumor cells are not the primary target of anti-EGFR therapies, it is puzzling that KRAS mutations in tumor cells confer resistance to anti-EGFR treatment in mCRC patients. We hypothesize that epithelial KRAS mutations alter the tumor microenvironment (TME) towards an immunosuppressive milieu. We are testing this hypothesis by comparing expression signatures derived from tumor and stroma of human mCRC biopsies that are WT or mutant for KRAS to elucidate the molecular signature driven by KRAS that explains anti-EGFR treatment resistance. In parallel, we employ CRC mouse models generated by orthotopic implantation of WT and RAS mutant mouse CRC organoids into GEMMs lacking the EGFR in different cell types to mimic right and left-sided metastatic disease. Targets identified in human mCRC patients are being mechanistically validated in organoids and derived tumors by CRISPR/Cas9-mediated genetic engineering. The results of these experiments will be presented. The expected results will provide a better understanding of how anti-EGFR antibodies work, how resistance mechanisms are developed, and possible new treatment combinations for KRAS mutated CRC. References: 1. Hsu, H. C. et al. Mutations of KRAS/NRAS/BRAF predict cetuximab resistance in metastatic colorectal cancer patients. Oncotarget 7, 22257–22270 (2016). 2. Martini, G. et al. Present and future of metastatic colorectal cancer treatment: A review of new candidate targets. World J. Gastroenterol. 23, 4675–4688 (2017). 3. Srivatsa, S. et al. EGFR in Tumor-Associated Myeloid Cells Promotes Development of Colorectal Cancer in Mice and Associates With Outcomes of Patients. Gastroenterology 153, 178-190.e10 (2017). 4. Hardbower, D. M. et al. EGFR-mediated macrophage activation promotes colitis-associated tumorigenesis. Oncogene 36, 3807–3819 (2017). Citation Format: Emi Adachi-Fernandez, Veronica Moreno-Viedma, Dana Krauss, Alexander Kirchmair, Zlatko Trajanoski, Maria Sibilia. Role of EGFR in KRAS-mediated resistances in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A013.