Metabolite profiling of human renal cell carcinoma reveals tissue-origin dominance in nutrient availability.

The tumor microenvironment is a determinant of cancer progression and therapeutic efficacy, with nutrient availability playing an important role. Although it is established that the local abundance of specific nutrients defines the metabolic parameters for tumor growth, the factors guiding nutrient availability in tumor compared to normal tissue and blood remain poorly understood. To define these factors in renal cell carcinoma (RCC), we performed quantitative metabolomic and comprehensive lipidomic analyses of tumor interstitial fluid (TIF), adjacent normal kidney interstitial fluid (KIF), and plasma samples collected from patients. TIF nutrient composition closely resembles KIF, suggesting that tissue-specific factors unrelated to the presence of cancer exert a stronger influence on nutrient levels than tumor-driven alterations. Notably, select metabolite changes consistent with known features of RCC metabolism are found in RCC TIF, while glucose levels in TIF are not depleted to levels that are lower than those found in KIF. These findings inform tissue nutrient dynamics in RCC, highlighting a dominant role of non-cancer-driven tissue factors in shaping nutrient availability in these tumors.

Cancer cells convert nutrients into energy differently compared to healthy cells. This difference in metabolism allows them to grow and divide more quickly and sometimes to migrate to different areas of the body. The environment around cancer cells ­ known as the tumor microenvironment ­ contains a variety of different cells and blood vessels, which are bathed in interstitial fluid. This microenvironment provides nutrients for the cancer cells to metabolize, and therefore influences how well a tumor grows and how it might respond to treatment. Recent advances with techniques such as mass spectrometry, which can measure the chemical composition of a substance, have allowed scientists to measure nutrient levels in the tumor microenvironments of mice. However, it has been more difficult to conduct such studies in humans, as well as to compare the tumor microenvironment to the healthy tissue the tumors arose from. Abbott, Ali, Reinfeld et al. aimed to fill this gap in knowledge by using mass spectrometry to measure the nutrient levels in the tumor microenvironment of 55 patients undergoing surgery to remove kidney tumors. Comparing the type and levels of nutrients in the tumor interstitial fluid, the neighboring healthy kidney and the blood showed that nutrients in the tumor and healthy kidney were more similar to each other than those in the blood. For example, both the tumor and healthy kidney interstitial fluids contained less glucose than the blood. However, the difference between nutrient composition in the tumor and healthy kidney interstitial fluids was insignificant, suggesting that the healthy kidney and its tumor share a similar environment. Taken together, the findings indicate that kidney cancer cells must adapt to the nutrients available in the kidney, rather than changing what nutrients are available in the tissue. Future studies will be required to investigate whether this finding also applies to other types of cancer. A better understanding of how cancer cells adapt to their environments may aid the development of drugs that aim to disrupt the metabolism of tumors.

VHL loss reprograms the immune landscape to promote an inflammatory myeloid microenvironment in renal tumorigenesis.

Clear cell renal cell carcinoma (ccRCC) is characterized by dysregulated hypoxia signaling and a tumor microenvironment (TME) highly enriched in myeloid and lymphoid cells. Loss of the von Hippel Lindau (VHL) gene is a critical early event in ccRCC pathogenesis and promotes stabilization of HIF. Whether VHL loss in cancer cells affects immune cells in the TME remains unclear. Using Vhl WT and Vhl-KO in vivo murine kidney cancer Renca models, we found that Vhl-KO tumors were more infiltrated by immune cells. Tumor-associated macrophages (TAMs) from Vhl-deficient tumors demonstrated enhanced in vivo glucose consumption, phagocytosis, and inflammatory transcriptional signatures, whereas lymphocytes from Vhl-KO tumors showed reduced activation and a lower response to anti-programmed cell death 1 (anti-PD-1) therapy in vivo. The chemokine CX3CL1 was highly expressed in human ccRCC tumors and was associated with Vhl deficiency. Deletion of Cx3cl1 in cancer cells decreased myeloid cell infiltration associated with Vhl loss to provide a mechanism by which Vhl loss may have contributed to the altered immune landscape. Here, we identify cancer cell-specific genetic features that drove environmental reprogramming and shaped the tumor immune landscape, with therapeutic implications for the treatment of ccRCC.

Metabolite profiling of human renal cell carcinoma reveals tissue-origin dominance in nutrient availability.

The tumor microenvironment is a determinant of cancer progression and therapeutic efficacy, with nutrient availability playing an important role. Although it is established that the local abundance of specific nutrients defines the metabolic parameters for tumor growth, the factors guiding nutrient availability in tumor compared to normal tissue and blood remain poorly understood. To define these factors in renal cell carcinoma (RCC), we performed quantitative metabolomic and comprehensive lipidomic analyses of tumor interstitial fluid (TIF), adjacent normal kidney interstitial fluid (KIF), and plasma samples collected from patients. TIF nutrient composition closely resembles KIF, suggesting that tissue-specific factors unrelated to the presence of cancer exert a stronger influence on nutrient levels than tumor-driven alterations. Notably, select metabolite changes consistent with known features of RCC metabolism are found in RCC TIF, while glucose levels in TIF are not depleted to levels that are lower than those found in KIF. These findings inform tissue nutrient dynamics in RCC, highlighting a dominant role of non-cancer driven tissue factors in shaping nutrient availability in these tumors.

Differential Effects of Glutamine Inhibition Strategies on Antitumor CD8 T Cells.

Activated T cells undergo metabolic reprogramming to meet anabolic, differentiation, and functional demands. Glutamine supports many processes in activated T cells, and inhibition of glutamine metabolism alters T cell function in autoimmune disease and cancer. Multiple glutamine-targeting molecules are under investigation, yet the precise mechanisms of glutamine-dependent CD8 T cell differentiation remain unclear. We show that distinct strategies of glutamine inhibition by glutaminase-specific inhibition with small molecule CB-839, pan-glutamine inhibition with 6-diazo-5-oxo-l-norleucine (DON), or by glutamine-depleted conditions (No Q) produce distinct metabolic differentiation trajectories in murine CD8 T cells. T cell activation with CB-839 treatment had a milder effect than did DON or No Q treatment. A key difference was that CB-839-treated cells compensated with increased glycolytic metabolism, whereas DON and No Q-treated cells increased oxidative metabolism. However, all glutamine treatment strategies elevated CD8 T cell dependence on glucose metabolism, and No Q treatment caused adaptation toward reduced glutamine dependence. DON treatment reduced histone modifications and numbers of persisting cells in adoptive transfer studies, but those T cells that remained could expand normally upon secondary Ag encounter. In contrast, No Q-treated cells persisted well yet demonstrated decreased secondary expansion. Consistent with reduced persistence, CD8 T cells activated in the presence of DON had reduced ability to control tumor growth and reduced tumor infiltration in adoptive cell therapy. Overall, each approach to inhibit glutamine metabolism confers distinct effects on CD8 T cells and highlights that targeting the same pathway in different ways can elicit opposing metabolic and functional outcomes.

Perspectives in Melanoma: meeting report from the Melanoma Bridge (December 2nd - 4th, 2021, Italy).

Advances in immune checkpoint and combination therapy have led to improvement in overall survival for patients with advanced melanoma. Improved understanding of the tumor, tumor microenvironment and tumor immune-evasion mechanisms has resulted in new approaches to targeting and harnessing the host immune response. Combination modalities with other immunotherapy agents, chemotherapy, radiotherapy, electrochemotherapy are also being explored to overcome resistance and to potentiate the immune response. In addition, novel approaches such as adoptive cell therapy, oncogenic viruses, vaccines and different strategies of drug administration including sequential, or combination treatment are being tested. Despite the progress in diagnosis of melanocytic lesions, correct classification of patients, selection of appropriate adjuvant and systemic theràapies, and prediction of response to therapy remain real challenges in melanoma. Improved understanding of the tumor microenvironment, tumor immunity and response to therapy has prompted extensive translational and clinical research in melanoma. There is a growing evidence that genomic and immune features of pre-treatment tumor biopsies may correlate with response in patients with melanoma and other cancers, but they have yet to be fully characterized and implemented clinically. Development of novel biomarker platforms may help to improve diagnostics and predictive accuracy for selection of patients for specific treatment. Overall, the future research efforts in melanoma therapeutics and translational research should focus on several aspects including: (a) developing robust biomarkers to predict efficacy of therapeutic modalities to guide clinical decision-making and optimize treatment regimens, (b) identifying mechanisms of therapeutic resistance to immune checkpoint inhibitors that are potentially actionable, (c) identifying biomarkers to predict therapy-induced adverse events, and (d) studying mechanism of actions of therapeutic agents and developing algorithms to optimize combination treatments. During the Melanoma Bridge meeting (December 2nd-4th, 2021, Naples, Italy) discussions focused on the currently approved systemic and local therapies for advanced melanoma and discussed novel biomarker strategies and advances in precision medicine as well as the impact of COVID-19 pandemic on management of melanoma patients.

Uropathogenic Escherichia coli subverts mitochondrial metabolism to enable intracellular bacterial pathogenesis in urinary tract infection.

Urinary tract infections are among the most common human bacterial infections and place a significant burden on healthcare systems due to associated morbidity, cost and antibiotic use. Despite being a facultative anaerobe, uropathogenic Escherichia coli, the primary cause of urinary tract infections, requires aerobic respiration to establish infection in the bladder. Here, by combining bacterial genetics with cell culture and murine models of infection, we demonstrate that the widely conserved respiratory quinol oxidase cytochrome bd is required for intracellular infection of urothelial cells. Through a series of genetic, biochemical and functional assays, we show that intracellular oxygen scavenging by cytochrome bd alters mitochondrial physiology by reducing the efficiency of mitochondrial respiration, stabilizing the hypoxia-inducible transcription factor HIF-1 and promoting a shift towards aerobic glycolysis. This bacterially induced rewiring of host metabolism antagonizes apoptosis, thereby protecting intracellular bacteria from urothelial cell exfoliation and preserving their replicative niche. These results reveal the metabolic basis for intracellular bacterial pathogenesis during urinary tract infection and identify subversion of mitochondrial metabolism as a bacterial strategy to facilitate persistence within the urinary tract.

Stimulating TAM-mediated anti-tumor immunity with mannose-decorated nanoparticles in ovarian cancer.

BACKGROUND: Current cancer immunotherapies have made tremendous impacts but generally lack high response rates, especially in ovarian cancer. New therapies are needed to provide increased benefits. One understudied approach is to target the large population of immunosuppressive tumor-associated macrophages (TAMs). Using inducible transgenic mice, we recently reported that upregulating nuclear factor-kappaB (NF-κB) signaling in TAMs promotes the M1, anti-tumor phenotype and limits ovarian cancer progression. We also developed a mannose-decorated polymeric nanoparticle system (MnNPs) to preferentially deliver siRNA payloads to M2, pro-tumor macrophages in vitro. In this study, we tested a translational strategy to repolarize ovarian TAMs via MnNPs loaded with siRNA targeting the inhibitor of NF-κB alpha (IκBα) using mouse models of ovarian cancer. METHODS: We evaluated treatment with MnNPs loaded with IκBα siRNA (IκBα-MnNPs) or scrambled siRNA in syngeneic ovarian cancer models. ID8 tumors in C57Bl/6 mice were used to evaluate consecutive-day treatment of late-stage disease while TBR5 tumors in FVB mice were used to evaluate repetitive treatments in a faster-developing disease model. MnNPs were evaluated for biodistribution and therapeutic efficacy in both models. RESULTS: Stimulation of NF-κB activity and repolarization to an M1 phenotype via IκBα-MnNP treatment was confirmed using cultured luciferase-reporter macrophages. Delivery of MnNPs with fluorescent payloads (Cy5-MnNPs) to macrophages in the solid tumors and ascites was confirmed in both tumor models. A three consecutive-day treatment of IκBα-MnNPs in the ID8 model validated a shift towards M1 macrophage polarization in vivo. A clear therapeutic effect was observed with biweekly treatments over 2-3 weeks in the TBR5 model where significantly reduced tumor burden was accompanied by changes in immune cell composition, indicative of reduced immunosuppressive tumor microenvironment. No evidence of toxicity associated with MnNP treatment was observed in either model. CONCLUSIONS: In mouse models of ovarian cancer, MnNPs were preferentially associated with macrophages in ascites fluid and solid tumors. Evidence of macrophage repolarization, increased inflammatory cues, and reduced tumor burden in IκBα-MnNP-treated mice indicate beneficial outcomes in models of established disease. We have provided evidence of a targeted, TAM-directed approach to increase anti-tumor immunity in ovarian cancer with strong translational potential for future clinical studies.

The therapeutic implications of immunosuppressive tumor aerobic glycolysis.

In 2011, Hanahan and Weinberg added "Deregulating Cellular Energetics" and "Avoiding Immune Destruction" to the six previous hallmarks of cancer. Since this seminal paper, there has been a growing consensus that these new hallmarks are not mutually exclusive but rather interdependent. The following review summarizes how founding genetic events for tumorigenesis ultimately increase tumor cell glycolysis, which not only supports the metabolic demands of malignancy but also provides an immunoprotective niche, promoting malignant cell proliferation, maintenance and progression. The mechanisms by which altered metabolism contributes to immune impairment are multifactorial: (1) the metabolic demands of proliferating tumor cells and activated immune cells are similar, thus creating a situation where immune cells may be in competition for key nutrients; (2) the metabolic byproducts of aerobic glycolysis directly inhibit antitumor immunity while promoting a regulatory immune phenotype; and (3) the gene programs associated with the upregulation of glycolysis also result in the generation of immunosuppressive cytokines and metabolites. From this perspective, we shed light on important considerations for the development of new classes of agents targeting cancer metabolism. These types of therapies can impair tumor growth but also pose a significant risk of stifling antitumor immunity.

Apoptolidin family glycomacrolides target leukemia through inhibition of ATP synthase.

Cancer cells have long been recognized to exhibit unique bioenergetic requirements. The apoptolidin family of glycomacrolides are distinguished by their selective cytotoxicity towards oncogene-transformed cells, yet their molecular mechanism remains uncertain. We used photoaffinity analogs of the apoptolidins to identify the F1 subcomplex of mitochondrial ATP synthase as the target of apoptolidin A. Cryogenic electron microscopy (cryo-EM) of apoptolidin and ammocidin-ATP synthase complexes revealed a novel shared mode of inhibition that was confirmed by deep mutational scanning of the binding interface to reveal resistance mutations which were confirmed using CRISPR-Cas9. Ammocidin A was found to suppress leukemia progression in vivo at doses that were tolerated with minimal toxicity. The combination of cellular, structural, mutagenesis, and in vivo evidence defines the mechanism of action of apoptolidin family glycomacrolides and establishes a path to address oxidative phosphorylation-dependent cancers.

Leptin Augments Antitumor Immunity in Obesity by Repolarizing Tumor-Associated Macrophages.

Although obesity can promote cancer, it may also increase immunotherapy efficacy in what has been termed the obesity-immunotherapy paradox. Mechanisms of this effect are unclear, although obesity alters key inflammatory cytokines and can promote an inflammatory state that may modify tumor-infiltrating lymphocytes and tumor-associated macrophage populations. To identify mechanisms by which obesity affects antitumor immunity, we examined changes in cell populations and the role of the proinflammatory adipokine leptin in immunotherapy. Single-cell RNAseq demonstrated that obesity decreased tumor-infiltrating lymphocyte frequencies, and flow cytometry confirmed altered macrophage phenotypes with lower expression of inducible NO synthase and MHC class II in tumors of obese animals. When treated with anti-programmed cell death protein 1 (PD-1) Abs, however, obese mice had a greater absolute decrease in tumor burden than lean mice and a repolarization of the macrophages to inflammatory M1-like phenotypes. Mechanistically, leptin is a proinflammatory adipokine that is induced in obesity and may mediate enhanced antitumor immunity in obesity. To directly test the effect of leptin on tumor growth and antitumor immunity, we treated lean mice with leptin and observed tumors over time. Treatment with leptin, acute or chronic, was sufficient to enhance antitumor efficacy similar to anti-PD-1 checkpoint therapy. Further, leptin and anti-PD-1 cotreatment may enhance antitumor effects consistent with an increase in M1-like tumor-associated macrophage frequency compared with non-leptin-treated mice. These data demonstrate that obesity has dual effects in cancer through promotion of tumor growth while simultaneously enhancing antitumor immunity through leptin-mediated macrophage reprogramming.

Cell-programmed nutrient partitioning in the tumour microenvironment.

Cancer cells characteristically consume glucose through Warburg metabolism1, a process that forms the basis of tumour imaging by positron emission tomography (PET). Tumour-infiltrating immune cells also rely on glucose, and impaired immune cell metabolism in the tumour microenvironment (TME) contributes to immune evasion by tumour cells2-4. However, whether the metabolism of immune cells is dysregulated in the TME by cell-intrinsic programs or by competition with cancer cells for limited nutrients remains unclear. Here we used PET tracers to measure the access to and uptake of glucose and glutamine by specific cell subsets in the TME. Notably, myeloid cells had the greatest capacity to take up intratumoral glucose, followed by T cells and cancer cells, across a range of cancer models. By contrast, cancer cells showed the highest uptake of glutamine. This distinct nutrient partitioning was programmed in a cell-intrinsic manner through mTORC1 signalling and the expression of genes related to the metabolism of glucose and glutamine. Inhibiting glutamine uptake enhanced glucose uptake across tumour-resident cell types, showing that glutamine metabolism suppresses glucose uptake without glucose being a limiting factor in the TME. Thus, cell-intrinsic programs drive the preferential acquisition of glucose and glutamine by immune and cancer cells, respectively. Cell-selective partitioning of these nutrients could be exploited to develop therapies and imaging strategies to enhance or monitor the metabolic programs and activities of specific cell populations in the TME.

Perspectives in immunotherapy: meeting report from the "Immunotherapy Bridge" (December 4th-5th, 2019, Naples, Italy).

Over the last few years, numerous clinical trials and real-world experience have provided a large amount of evidence demonstrating the potential for long-term survival with immunotherapy agents across various malignancies, beginning with melanoma and extending to other tumours. The clinical success of immune checkpoint blockade has encouraged increasing development of other immunotherapies. It has been estimated that there are over 3000 immuno-oncology trials ongoing, targeting hundreds of disease and immune pathways. Evolving topics on cancer immunotherapy, including the state of the art of immunotherapy across various malignancies, were the focus of discussions at the Immunotherapy Bridge meeting (4-5 December, 2019, Naples, Italy), and are summarised in this report.

Fine-Needle Aspiration-Based Patient-Derived Cancer Organoids.

Patient-derived cancer organoids hold great potential to accurately model and predict therapeutic responses. Efficient organoid isolation methods that minimize post-collection manipulation of tissues would improve adaptability, accuracy, and applicability to both experimental and real-time clinical settings. Here we present a simple and minimally invasive fine-needle aspiration (FNA)-based organoid culture technique using a variety of tumor types including gastrointestinal, thyroid, melanoma, and kidney. This method isolates organoids directly from patients at the bedside or from resected tissues, requiring minimal tissue processing while preserving the histologic growth patterns and infiltrating immune cells. Finally, we illustrate diverse downstream applications of this technique including in vitro high-throughput chemotherapeutic screens, in situ immune cell characterization, and in vivo patient-derived xenografts. Thus, routine clinical FNA-based collection techniques represent an unappreciated substantial source of material that can be exploited to generate tumor organoids from a variety of tumor types for both discovery and clinical applications.

Macrophages Promote Aortic Valve Cell Calcification and Alter STAT3 Splicing.

OBJECTIVE: Macrophages have been described in calcific aortic valve disease, but it is unclear if they promote or counteract calcification. We aimed to determine how macrophages are involved in calcification using the Notch1+/- model of calcific aortic valve disease. Approach and Results: Macrophages in wild-type and Notch1+/- murine aortic valves were characterized by flow cytometry. Macrophages in Notch1+/- aortic valves had increased expression of MHCII (major histocompatibility complex II). We then used bone marrow transplants to test if differences in Notch1+/- macrophages drive disease. Notch1+/- mice had increased valve thickness, macrophage infiltration, and proinflammatory macrophage maturation regardless of transplanted bone marrow genotype. In vitro approaches confirm that Notch1+/- aortic valve cells promote macrophage invasion as quantified by migration index and proinflammatory phenotypes as quantified by Ly6C and CCR2 positivity independent of macrophage genotype. Finally, we found that macrophage interaction with aortic valve cells promotes osteogenic, but not dystrophic, calcification and decreases abundance of the STAT3ß isoform. CONCLUSIONS: This study reveals that Notch1+/- aortic valve disease involves increased macrophage recruitment and maturation driven by altered aortic valve cell secretion, and that increased macrophage recruitment promotes osteogenic calcification and alters STAT3 splicing. Further investigation of STAT3 and macrophage-driven inflammation as therapeutic targets in calcific aortic valve disease is warranted.

Student Perspectives on the "Step 1 Climate" in Preclinical Medical Education.

The United States Medical Licensing Examination Step 1 was implemented in the 1990s as the most recent version of the National Board of Medical Examiners' preclinical licensing examination originally created in the late 1960s. For the purposes of state licensure, the exam is pass/fail, but the Step 1 numeric score has in recent years become central to the residency application and selection process. Consequently, a medical student's Step 1 score is increasingly viewed as a key outcome of preclinical medical education.In this Invited Commentary, students from various institutions across the country draw on their shared experiences to argue that the emphasis on Step 1 for residency selection has fundamentally altered the preclinical learning environment, creating a "Step 1 climate." The authors aim to increase awareness of the harms and unintended consequences of this phenomenon in medical education. They outline how the Step 1 climate negatively impacts education, diversity, and student well-being, and they urge a national conversation on the elimination of reporting Step 1 numeric scores.

DNA Instability Maintains the Repeat Length of the Yeast RNA Polymerase II C-terminal Domain.

The C-terminal domain (CTD) of RNA polymerase II in eukaryotes is comprised of tandemly repeating units of a conserved seven-amino acid sequence. The number of repeats is, however, quite variable across different organisms. Furthermore, previous studies have identified evidence of rearrangements within the CTD coding region, suggesting that DNA instability may play a role in regulating or maintaining CTD repeat number. The work described here establishes a clear connection between DNA instability and CTD repeat number in Saccharomyces cerevisiae First, analysis of 36 diverse S. cerevisiae isolates revealed evidence of numerous past rearrangements within the DNA sequence that encodes the CTD. Interestingly, the total number of CTD repeats was relatively static (24-26 repeats in all strains), suggesting a balancing act between repeat expansion and contraction. In an effort to explore the genetic plasticity within this region, we measured the rates of repeat expansion and contraction using novel reporters and a doxycycline-regulated expression system for RPB1 In efforts to determine the mechanisms leading to CTD repeat variability, we identified the presence of DNA secondary structures, specifically G-quadruplex-like DNA, within the CTD coding region. Furthermore, we demonstrated that mutating PIF1, a G-quadruplex-specific helicase, results in increased CTD repeat length polymorphisms. We also determined that RAD52 is necessary for CTD repeat expansion but not contraction, identifying a role for recombination in repeat expansion. Results from these DNA rearrangements may help explain the CTD copy number variation seen across eukaryotes, as well as support a model of CTD expansion and contraction to maintain CTD integrity and overall length.