Understanding cellular processes underlying early lung adenocarcinoma (LUAD) development is needed to devise intervention strategies. While most if not all single-cell RNA sequencing (scRNA-seq) studies of lung cancer provided details on immune and stromal states, little insight is drawn to epithelial cells given their paucity (~4%) when performing unbiased scRNA-seq analysis without prior enrichment. Here, we performed in-depth scRNA-seq of enriched (by sorting for EPCAM+) epithelial cell subsets from 16 early-stage LUADs and 47 matching normal lung (NL) tissues. We also studied tissues from the same LUADs and adjacent NL by whole exome sequencing and a subset by high-resolution spatial protein and transcriptome analysis. We also performed scRNA-seq analysis of murine lungs from a human-relevant model of LUAD development following exposure to tobacco carcinogen, including strains with an alveolar type II (AT2) cell-specific lineage reporter. After extensive quality control, we retained 246,102 high quality human epithelial cells which comprised diverse normal alveolar and airway lineages as well as cancer cell populations. Diversity among cancer cells was strongly linked to LUAD oncogenic drivers. KRAS-mutant cancer cells were unique in their transcriptional features, strikingly reduced differentiation, low levels of copy number changes, and increased variability amongst the cells themselves. The local epithelial niche of LUADs, relative to that of NL, was enriched with intermediary cells in lung alveolar differentiation. A subset of these cells displayed elevated KRT8 expression (KRT8+ alveolar cells; KACs), increased plasticity and frequency of KRASG12D mutations, and its gene expression profiles were enriched in lung precancer and LUAD and signified poor survival. Notably, KACs harboring KRAS mutations were only found in the ecosystem of KRAS-mutant LUADs. Murine KACs were evident in lungs of tobacco carcinogen-exposed mice that develop KRAS-mutant LUADs but not in the saline-treated control group. While murine KACs emerged prior to tumor onset, they persisted for months after carcinogen cessation, and like their human counterparts, acquired driver Kras mutations, were poorly differentiated, and harbored KRAS-specific transcriptional programs. Spatial transcriptomics analysis showed that KAC and KRAS signatures were elevated in both murine and human tumors as well as in KACs that were in the local spatial vicinity of the LUADs. Organoids derived from lungs of tumor-bearing reporter mice were markedly enriched with KACs and were conspicuously sensitive to targeted inhibition of KRAS-G12D. This study provides new insights into the landscape of normal epithelial and malignant cells in LUAD, the role of alveolar intermediate subsets in development of the malignancy, particularly that driven by mutant KRAS, and, thus, potential targets for early interception. Citation Format: Guangchun Han, Ansam Sinjab, Warapen Treekitkarnmongkol, Zahraa Rahal, Yuejiang Liu, Alejandra G. Serrano, Jiping Feng, Ke Liang, Khaja Khan, Wei Lu, Sharia Hernandez, Xuanye Cao, Enyu Dai, Yunhe Liu, Guangsheng Pei, Jian Hu, Lorena I. Gomez Bolanos, Edwin R. Parra, Tina Cascone, Boris Sepesi, Seyed Javad Moghaddam, Paul Scheet, Marcelo V. Negrao, John V. Heymach, Mingyao Li, Jichao Chen, Steven M. Dubinett, Junya Fujimoto, Luisa M. Solis, Ignacio I. Wistuba, Christopher S. Stevenson, Avrum Spira, Linghua Wang, Humam Kadara. An atlas of epithelial cell states and plasticity in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 113.

Introduction: Millions of Americans infected with the severe acute respiratory syndrome-associated coronavirus-19 (COVID-19) need oncologic surgery. Patients with acute or resolved COVID-19 illness complain of neuropsychiatric symptoms. How surgery affects postoperative neuropsychiatric outcomes such as delirium is unknown. We hypothesize that patients with a history of COVID-19 could have an exaggerated risk of developing postoperative delirium after undergoing major elective oncologic surgery. Methods: We conducted a retrospective study to determine the association between COVID-19 status and antipsychotic drugs during postsurgical hospitalization as a surrogate of delirium. Secondary outcomes included 30 days of postoperative complications, length of stay, and mortality. Patients were grouped into pre-pandemic non-COVID-19 and COVID-19-positive groups. A 1:2 propensity score matching was used to minimize bias. A multivariable logistic regression model estimated the effects of important covariates on the use of postoperative psychotic medication. Results: A total of 6003 patients were included in the study. Pre- and post-propensity score matching demonstrated that a history of preoperative COVID-19 did not increase the risk of antipsychotic medications postoperatively. However, respiratory and overall 30-day complications were higher in COVID-19 individuals than in pre-pandemic non-COVID-19 patients. The multivariate analysis showed that the odds of using postoperative antipsychotic medication use for the patients who had COVID-19 compared to those who did not have the infection were not significantly different. Conclusion: A preoperative diagnosis of COVID-19 did not increase the risk of postoperative antipsychotic medication use or neurological complications. More studies are needed to reproduce our results due to the increased concern of neurological events post-COVID-19 infection.

OBJECTIVES/GOALS: Leigh Syndrome, French Canadian-Type (LSFC) is a neurometabolic disorder caused by mutation of mitochondria-related gene, LRPPRC. White matter lesions and demyelination in central nervous system are common in LSFC. LRPPRC is enriched in myelinating glial cells, yet its role is not known. Our goal is to elucidate its mechanistic role in myelination. METHODS/STUDY POPULATION: We crossed C57BL/6N mice bearing a LRPPRC-loxP allele with mice bearing a Plp-CreERT2 allele. Mice with the Plp-CreERT2 allele expresses a tamoxifen-inducible Cre under the control of the Plp promoter, which drives expression in oligodendrocytes. Using these strains, we can target the deletion of LRPPRC, via tamoxifen injection, in both newly formed myelin and mature myelin. Plp-CreERT2; LRPPRCL/L (LRPPRC-KO) or control littermate mice will be injected for LRPPRC deletion at developmental and maturation stages of myelin. Immunofluorescence and electron microscopy of isolated brain tissues will be used for myelin integrity analysis. Cognitive functions of the mice will be measured via behavioral tests. Lastly, we will submit tissues for lipidomic analyses to observe any lipid metabolite variation. RESULTS/ANTICIPATED RESULTS: Behavioral and motor defects would be expected in LRPPRC-KO mice performing in cognitive function tasks across myelin maturation stages. Electron microscopy-based structure analysis of optic nerve, corpus callosum, and spinal cord should reveal thin or loss of myelin on the axons of LRPPRC-KO compared to control. Immunofluorescence staining of major myelin structural proteins, including myelin proteolipid protein (PLP), myelin basic protein (MBP), and myelin-associated glycoprotein (MAG) would be expected have lower levels in LRPPRC deficient tissues. Since myelin is a lipid-rich species, we would also expect lipid concentrations to be affected. LRPPRC-KO lipidomic analyses of myelin-related lipids should depict lower levels in comparison to control, which would imply dysfunctional lipid metabolism. DISCUSSION/SIGNIFICANCE: There are limited studies in ameliorating neural deficits caused by LS and LSFC. Successful completion of this project would help elucidate the functions of LRPPRC in myelination and lipid metabolism and potentially provide insights for developing novel therapeutic strategies for alleviating the demyelination and neural deficits in LSFC.

Glioblastoma (GBM) is the most frequent and deadliest primary brain tumor, with a <10% 5-year overall survival rate. Despite promising results in other disease contexts, deployment of immune system-targeted therapies against GBM has not been successful thus far. In this regard, few preclinical models effectively recapitulate the human GBM immune microenvironment, hindering our ability to identify potentially targetable vulnerabilities. In this study, we characterized tumor immune microenvironment of the Qk/trp53/Pten (QPP) triple-knockout mouse model, harboring alterations common in human GBM and found that tumor infiltrates contain a predominantly myeloid cell population of monocytes, macrophages, and resting dendritic cells, with minor populations of T, B, and NK cells. Notable differences between spontaneous QPP tumors and tumors derived by implanting established QPP cell lines included T-cell enrichment and a larger proportion of myeloid-derived suppressive cells (MDSCs) in implanted tumors. Profiles of myeloid cells subtypes in QPP tumors paralleled findings in human GBMs, suggesting that this model effectively recapitulates the complexity of the myeloid-cell compartment and other human GBM immune cell populations.

Background The lack of murine glioblastoma models that mimic the immunobiology of human disease has impeded basic and translational immunology research. We therefore developed murine glioblastoma stem cell lines derived from Nestin-CreERT2QkL/L; Trp53L/L; PtenL/L (QPP) mice driven by clinically relevant genetic mutations common in human glioblastoma. This study aims to determine the immune sensitivities of these QPP lines in immunocompetent hosts and underlying mechanisms. Methods The differential responsiveness of QPP lines was assessed in the brain and flank in untreated, anti-PD-1, or anti-CTLA-4 treated mice. The impact of genomic landscape on responsiveness of each tumor was measured through whole exome sequencing. The immune microenvironments of sensitive (QPP7) versus resistant (QPP8) lines were compared in the brain using flow cytometry. Drivers of flank sensitivity versus brain resistance were also measured for QPP8. Results QPP lines are syngeneic to C57BL/6J mice and demonstrate varied sensitivities to T cell immune checkpoint blockade ranging from curative responses to complete resistance. Infiltrating tumor immune analysis of QPP8 reveals improved T cell fitness and augmented effector to suppressor ratios when implanted subcutaneously (sensitive), which are absent upon implantation in the brain (resistant). Upregulation of PD-L1 across the myeloid stroma acts to establish this state of immune privilege in the brain. In contrast, QPP7 responds to checkpoint immunotherapy even in the brain likely resulting from its elevated neoantigen burden. Conclusions These syngeneic QPP models of glioblastoma demonstrate clinically-relevant profiles of immunotherapeutic sensitivity and potential utility for both mechanistic discovery and evaluation of immune therapies.