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The pancreatic tumor microenvironment drives deregulated nutrient availability. Accordingly, pancreatic cancer cells require metabolic adaptations to survive and proliferate. Pancreatic cancer subtypes have been characterized by transcriptional and functional differences, with subtypes reported to exist within the same tumor. However, it remains unclear if this diversity extends to metabolic programming. Here, using metabolomic profiling and functional interrogation of metabolic dependencies, we identify two distinct metabolic subclasses among neoplastic populations within individual human and mouse tumors. Furthermore, these populations are poised for metabolic cross-talk, and in examining this, we find an unexpected role for asparagine supporting proliferation during limited respiration. Constitutive GCN2 activation permits ATF4 signaling in one subtype, driving excess asparagine production. Asparagine release provides resistance during impaired respiration, enabling symbiosis. Functionally, availability of exogenous asparagine during limited respiration indirectly supports maintenance of aspartate pools, a rate-limiting biosynthetic precursor. Conversely, depletion of extracellular asparagine with PEG–asparaginase sensitizes tumors to mitochondrial targeting with phenformin.

SUMMARYPD-1 blockade unleashes the potent antitumor activity of CD8 cells but can also promote immunosuppressive T regulatory (Treg) cells, which may worsen response to immunotherapy. Tumor Treg inhibition is a promising strategy to overcome therapeutic resistance; however, the mechanisms supporting tumor Tregs during PD-1 immunotherapy are largely unexplored. Here, we report that PD-1 blockade increases tumor Tregs in mouse models of immunogenic tumors, including melanoma, and metastatic melanoma patients. Unexpectedly, Treg accumulation was not caused by Treg-intrinsic inhibition of PD-1 signaling but instead depended on an indirect effect of activated CD8 cells. CD8 cells colocalized with Tregs within tumors and produced IL-2, especially after PD-1 immunotherapy. IL-2 upregulated the anti-apoptotic protein ICOS on tumor Tregs, causing their accumulation. ICOS signaling inhibition before PD-1 immunotherapy resulted in increased control of immunogenic melanoma. Thus, interrupting the intratumor CD8:Treg crosstalk is a novel strategy that may enhance the efficacy of immunotherapy in patients.

The pancreatic ductal adenocarcinoma microenvironment is composed of a variety of cell types and marked by extensive fibrosis and inflammation. Tumor-associated macrophages (TAMs) are abundant, and they are important mediators of disease progression and invasion. TAMs are polarized in situ to a tumor promoting and immunosuppressive phenotype via cytokine signaling and metabolic crosstalk from malignant epithelial cells and other components of the tumor microenvironment. However, the specific distinguishing features and functions of TAMs remain poorly defined. Here, we generated tumor-educated macrophages (TEMs) in vitro and performed detailed, multiomic characterization (i.e., transcriptomics, proteomics, metabolomics). Our results reveal unique genetic and metabolic signatures of TEMs, the veracity of which were queried against our in-house single-cell RNA sequencing dataset of human pancreatic tumors. This analysis identified expression of novel, metabolic TEM markers in human pancreatic TAMs, including ARG1, ACLY, and TXNIP. We then utilized our TEM model system to study the role of mutant Kras signaling in cancer cells on TEM polarization. This revealed an important role for granulocyte–macrophage colony-stimulating factor (GM-CSF) and lactate on TEM polarization, molecules released from cancer cells in a mutant Kras-dependent manner. Lastly, we demonstrate that GM-CSF dysregulates TEM gene expression and metabolism through PI3K–AKT pathway signaling. Collectively, our results define new markers and programs to classify pancreatic TAMs, how these are engaged by cancer cells, and the precise signaling pathways mediating polarization.

Pancreatic Ductal Adenocarcinoma (PDA) is the 3rd leading cause of cancer related deaths in the US with a 5-year survival of 11%. Most patients die from disseminated disease, with liver being the most frequently colonized metastatic site. In primary PDA tumors, the tumor microenvironment has been extensively linked to therapy resistance and immune suppression. However, the extent that the microenvironment in the liver metastatic niche promotes immune evasion compared to primary PDA lesions remains unclear. Importantly, strategies to effectively target metastatic disease will provide the most immediate benefit for clinical treatment of PDA. The heterogeneous tumor microenvironment has been recognized as a primary barrier to PDA treatment. Here, the immune and stromal cells are not functioning in a manner beneficial for the host. Numerous cell types have been shown to have a pro-tumor function including regulatory T cells, tumor associated macrophages (TAMs), myeloid derived suppressor cells, and cancer associated fibroblasts (CAFs). Recently, we and others have shown that there are metabolic crosstalk interactions between cancer cells, TAMs, and CAFs. These interactions function to support primary tumor growth, immune suppression, and resistance to chemotherapy. However, the degree that the cancer cell heterogeneity, immune infiltration, and stromal populations present in metastatic lesions mirror primary tumors remains unclear. To directly compare primary vs. metastatic tumors in PDA tumors, we have optimized a syngeneic experimental model to contrast primary vs. metastatic lesions. Using this system, we have identified differential programming among the different cell populations between pancreas and liver PDA lesions using single cell RNA sequencing. Further, we directly compared immune infiltration between primary and metastatic tumors using mass cytometry. Our data indicate alternative mechanisms of immune suppression in liver PDA lesions, which can be directly targeted in combination with checkpoint inhibition strategies. Overall, these data have the potential to lead to new avenues to activate or target the immune system to treat metastatic PDA, an urgent clinical need for pancreatic cancer patients. Citation Format: Rima Singh, Christopher Halbrook, Nina Steele. Deconvoluting the metastatic PDA tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C083.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with a devastating 5-year survival rate of 11%. A lack of durable responses to standard-of-care chemotherapy combinations renders its treatment particularly challenging and largely contributes to the devastating prognosis. Gemcitabine, a pyrimidine anti-metabolite, is a cornerstone in PDAC therapy, but resistance remains a major hurdle. Multiple mechanisms of chemoresistance have been suggested to be mediated by rewired metabolism in PDAC cells. Accordingly, we hypothesize that metabolic reprogramming can be targeted to re-sensitize PDAC tumors to anti-metabolite chemotherapy. To define the spectrum of targetable metabolic and transcriptomic programs that drive cell-intrinsic resistance to gemcitabine, we established organoids from treatment-naïve PDAC patients. Gemcitabine high versus low responders, as assessed by dose response viability assays, were selected for metabolomic profiling and RNA sequencing. To define real-time reprogramming during the acquisition of resistance, we generated gemcitabine resistant murine pancreatic cancer cell lines and collected a time course series of metabolomic and transcriptomic datasets of sensitive, intermediate resistant and resistant cells. Integration of these dataset through a systems biology approach to define primary and de novo resistance is being used to characterize metabolic gemcitabine resistance mechanisms. These are functionally validated to provide novel approaches to re-sensitize resistant PDAC cells to gemcitabine in vitro and in vivo. The improvement of current chemotherapy combinations represents a promising approach, with potential to immediately translate into a clinical benefit and improve survival in this deadly disease. Citation Format: Alica Katrin Beutel, Rima Singh, Cecily Anaraki, Gregory Tong, Thomas Martinez, Alexander Kleger, Zeljka Jutric, Christopher James Halbrook. Targeting gemcitabine resistance in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR023.

Over the previous years, the use of an animal model has become very common for the screening of novel drugs. Animal model represents the complex problems of humans into the simplest forms, so these can be extended further to be included in the experimental procedure. The most successful models in neuroscience, rats and mice, are undoubtedly considered as one of the best models to understand the psychology of the mammalian brain and its associated functions involved in behavioral repertoire. Moreover, recently researchers in behavioral neuroscience are focusing more on the use of aquatic animals, especially fish, as model species due to their simplicity and cost-effectiveness. Zebrafish (Danio rerio) is a tropical fish from the minnow family, a genetic structure surprisingly 84% similar to humans. It is gaining popularity as a model to study the mechanism in behavioral neuropharmacology. Moreover, zebrafish has numerous advantages over other rodent models like the ease in maintenance due to their small size, more breeding power, transparency of embryos, overall reduced cost of experimentation, and many more. Nowadays, it is considered an ideal model to study the neurobehavioral aspects with relevance to humans. It is also used in a variety of scientific studies like genetics, neuroscience, pharmacology, and toxicology. In this manuscript, we have described the feasibility and importance of zebrafish as a model for the screening of novel drugs for different neurological disorders.

In the original article, the co-author name "Jennifer Kim" has been inadvertently missed during the publication process. The complete author group is given in this correction.

Mutations can occur throughout the virus genome and may be beneficial, neutral or deleterious. We are interested in mutations that yield a C next to a G, producing CpG sites. CpG sites are rare in eukaryotic and viral genomes. For the eukaryotes, it is thought that CpG sites are rare because they are prone to mutation when methylated. In viruses, we know less about why CpG sites are rare. A previous study in HIV suggested that CpG-creating transition mutations are more costly than similar non-CpG-creating mutations. To determine if this is the case in other viruses, we analyzed the allele frequencies of CpG-creating and non-CpG-creating mutations across various strains, subtypes, and genes of viruses using existing data obtained from Genbank, HIV Databases, and Virus Pathogen Resource. Our results suggest that CpG sites are indeed costly for most viruses. By understanding the cost of CpG sites, we can obtain further insights into the evolution and adaptation of viruses., Evolutionary Ecology, 34, ISSN:0269-7653, ISSN:1573-8477

Adenoid cystic carcinoma (ACC) is an uncommon malignant neoplasm composed of basaloid epithelial and myoepithelial cells. The palate is the most commonly involved intraoral site for ACC. Here, we document the case of an advanced ACC arising from the hard palate that presented with right-sided sixth nerve palsy in a 75-year-old male with no other systemic illnesses. ACC of the head and neck involving the cavernous sinus and presenting as isolated sixth nerve palsy is exceedingly rare. In the absence of vasculopathic or ischemic risk factors, regardless of the age of the patient; neuroimaging should be performed in cases of isolated nontraumatic sixth nerve palsy.