The term autophagy refers to the engulfment and degradation of cytoplasmic components within the lysosome. This process can benefit cells and organisms by removing damaged, superfluous, or harmful cellular components, and by generating a supply of recycled macromolecules that can support biosynthesis or energy production. Recent interest in autophagy has been driven by its potential role in several disease-related phenomena including neurodegeneration, cancer, immunity and aging. Drosophila provides a valuable animal model context for these studies, and work in this system has also begun to identify novel developmental and physiological roles of autophagy. Here, we provide an overview of methods for monitoring autophagy in Drosophila, with a special emphasis on the larval fat body. These methods can be used to investigate whether observed vesicles are of autophagic origin, to determine a relative rate of autophagic degradation, and to identify specific step(s) in the autophagic process in which a given gene functions.

Degradation of cellular material by autophagy is essential for cell survival and homeostasis, and requires intracellular transport of autophagosomes to encounter acidic lysosomes through unknown mechanisms. Here, we identify the PX-domain-containing kinesin Klp98A as a new regulator of autophagosome formation, transport and maturation in Drosophila. Depletion of Klp98A caused abnormal clustering of autophagosomes and lysosomes at the cell center and reduced the formation of starvation-induced autophagic vesicles. Reciprocally, overexpression of Klp98A redistributed autophagic vesicles towards the cell periphery. These effects were accompanied by reduced autophagosome-lysosome fusion and autophagic degradation. In contrast, depletion of the conventional kinesin heavy chain caused a similar mislocalization of autophagosomes without perturbing their fusion with lysosomes, indicating that vesicle fusion and localization are separable and independent events. Klp98A-mediated fusion required the endolysosomal GTPase Rab14, which interacted and colocalized with Klp98A, and required Klp98A for normal localization. Thus, Klp98A coordinates the movement and fusion of autophagic vesicles by regulating their positioning and interaction with the endolysosomal compartment.

The self-degradative process of autophagy is important for energy homeostasis and cytoplasmic renewal. This lysosome-mediated pathway is negatively regulated by the target of rapamycin kinase (TOR) under basal conditions, and requires the vesicle trafficking machinery regulated by Rab GTPases. However, the interactions between autophagy, TOR and Rab proteins remain incompletely understood Here, we identify Rab6 as a critical regulator of the balance between TOR signaling and autolysosome function. Loss of Rab6 causes an accumulation of enlarged autophagic vesicles resulting in part from a failure to deliver lysosomal hydrolases, rendering autolysosomes with a reduced degradative capacity and impaired turnover. Additionally, Rab6-deficient cells are reduced in size and display defective insulin-TOR signaling as a result of mis-sorting and internalization of the insulin receptor. Our findings suggest that Rab6 acts to maintain the reciprocal regulation between autophagy and TOR activity during distinct nutrient states, thereby balancing autophagosome production and turnover to avoid autophagic stress.

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Autophagy is a housekeeping mechanism tasked with eliminating misfolded proteins and damaged organelles to maintain cellular homeostasis. Autophagy deficiency results in increased oxidative stress, DNA damage and chronic cellular injury. Among the core genes in the autophagy machinery, ATG7 is required for autophagy initiation and autophagosome formation. Based on the analysis of an extended pedigree of familial cholangiocarcinoma, we determined that all affected family members had a novel germline mutation (c.2000C>T p.Arg659* (p.R659*)) in ATG7. Somatic deletions of ATG7 were identified in the tumors of affected individuals. We applied linked-read sequencing to one tumor sample and demonstrated that the ATG7 somatic deletion and germline mutation were located on distinct alleles, resulting in two hits to ATG7. From a parallel population genetic study, we identified a germline polymorphism of ATG7 (c.1591C>G p.Asp522Glu (p.D522E)) associated with increased risk of cholangiocarcinoma. To characterize the impact of these germline ATG7 variants on autophagy activity, we developed an ATG7-null cell line derived from the human bile duct. The mutant p.R659* ATG7 protein lacked the ability to lipidate its LC3 substrate, leading to complete loss of autophagy and increased p62 levels. Our findings indicate that germline ATG7 variants have the potential to impact autophagy function with implications for cholangiocarcinoma development.

Objective: Appendiceal mucinous neoplasms (AMN) start in the appendix. Following appendiceal perforation, these tumor metastasize, leading to pseudomyxoma peritonei (PMP). We characterized the distinct cell types and states of AMN and PMP. Design: Single-cell RNA-seq was conducted on 31 samples including AMNs, PMP metastases and a subset of matched normal appendix or omental tissues. We validated the findings with immunohistochemistry, mass spectrometry on malignant ascites from PMP patients and gene expression data from an independent set of 63 PMPs. Results: AMNs and PMPs had epithelial tumor cells with goblet features including the elevated expression of mucin genes. Among these tumors, we identified developmental cell states of the epithelium that ranged from progenitor phase to goblet cell differentiation. Metastatic PMP cells had a distinct cell state with gene expression signatures indicative of mTOR and RAS signaling pathways. A series of cancer genes defined this metastatic cell state. Matched PMP metastases from the same patient differed in their expression signatures. The cell state signatures were validated in external datasets containing 63 PMP patients. AMN and PMP tumor microenvironment (TME) contained CD4 T follicular helper-like cells and cytotoxic CD8 T cells. Conclusion: AMN and PMP tumors consisted of progenitor epithelial cells that differentiate into goblet cells. PMP cells had a distinct cell state indicative of metastasis. The TME was infiltrated with T cells, suggesting that immunotherapy is a potential treatment.

Understanding the cellular mechanisms of novel immunotherapy agents in the human tumor microenvironment (TME) is critical to their clinical success. We examined GITR and TIGIT immunotherapy in gastric and colon cancer patients using ex vivo slice tumor slice cultures derived from cancer surgical resections. This primary culture system maintains the original TME in a near-native state. We applied paired single-cell RNA and TCR sequencing to identify cell type specific transcriptional reprogramming. The GITR agonist was limited to increasing effector gene expression only in cytotoxic CD8 T cells. The TIGIT antagonist increased TCR signaling and activated both cytotoxic and dysfunctional CD8 T cells, including clonotypes indicative of potential tumor antigen reactivity. The TIGIT antagonist also activated T follicular helper-like cells and dendritic cells, and reduced markers of immunosuppression in regulatory T cells. Overall, we identified cellular mechanisms of action of these two immunotherapy targets in the patients' TME.

The tumor microenvironment (TME) contains diverse cell phenotypes. This heterogeneity influences the cancer immunity cycle and response to immunotherapy. We examined the effects of immunotherapy in a unique patient-derived ex vivo system that maintains the TME in its near-native state. We leveraged single-cell RNA sequencing (scRNA-seq) to conduct an unbiased analysis of transcriptional responses in heterogenous TME cell types. We established slice cultures using a vibratome from five surgical resections of colorectal or gastric cancer. Ex vivo slice cultures (‘TME-models') were treated with isotype control antibody, GITR agonist, TIGIT inhibitor or PMA/Ionomycin for 24 hours. TME-models and original surgical resection (‘T0') were dissociated into single-cell suspensions and subjected to scRNA-seq. Following quality control, we performed dimensionality reduction and differential expression analysis. We sequenced 219000 single cells detecting an average of 841 median genes per cell. All cell lineages identified in the T0 resection including tumor epithelium, CD4, CD8, Treg, NK, B, plasma, mast, dendritic cells, macrophages, fibroblasts and endothelial cells were maintained in the corresponding ex vivo TME-model. No consistent differences in proportions or transcriptional profiles were observed, indicating that original TME is preserved in the near-native state. PMA/ionomycin led to an activation phenotype in CD8, CD4 and regulatory T cells including a significant increase in expression of effector cytokines (GZMB, IFNG, PRF1, CCL3, etc.) , activation markers (IL2RA, CD69), immune checkpoints (PDCD1, TIGIT, LAG3, etc.) and co-stimulatory molecules (TNFRSF18, TNFRSF9, etc.). PMA/Ionomycin also led to upregulation of chemokines (CXCL9, CXCL5, etc.) and interferon response genes (ISG20, IRF1, etc.) in fibroblasts and tumor epithelial cells potentially indicative of indirect T-cell mediated effects. GITR agonist and TIGIT inhibitor led to an increase in cytotoxic genes including IFNG in effector CD8 T cells but not in naïve cells. Moreover, the extent of transcriptional response varied across tumors. Experiments on additional tumors are ongoing. Ex vivo TME-models derived from surgical resections maintain all TME components in their near native state. In combination with scRNA-seq, this system can be utilized to test targets in the TME and provide insights into their mechanisms of action and resistance. Using this approach, we successfully identified heterogenous patient responses to GITR stimulation and TIGIT inhibition in gastrointestinal cancers. Citation Format: Anuja Sathe, Jiamin Chen, Susan M. Grimes, Carlos I. Ayala, George Poultsides, Hanlee P. Ji. Patient-derived ex vivo TME-models and single-cell sequencing reveal transcriptional responses to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1680.