Deubiquitinating enzyme USP48 mediates pyroptosis and meliorates immune evasion in tumors by stabilizing GSDME expression

wang yunshan (  wangyunshansd@sdu.edu.cn ) The Second Hospital of Shandong University Yidan Ren The Second Hospital of Shandong University Maoxiao Feng The Second Hospital of Shandong University Xiaoyan Liu Department of Human Anatomy and Key Laboratory of Experimental Teratology Xiaodong Hao The Second Hospital of Shandong University Juan Li Department of Clinical Laboratory, The Second Hospital of Shandong University Peilong Li The Second Hospital, Cheeloo College of Medicine, Shandong University https://orcid.org/0000-00027441-6514 Jie Gao Shandong University Qiuchen Qi Shandong University Lutao Du Shandong University Chuanxin Wang The Second Hospital, Cheeloo College of Medicine, Shandong University https://orcid.org/0000-00023796-6293


Introduction 24
Pyroptosis is a type of programmed cell death mediated by the gasdermin family of proteins, which 25 has been discovered in recent years 1 . Different proteases or granzymes mediate the cleavage of the 26 gasdermin family proteins to release the N-terminal domain, which is then transported to the cell 27 membrane to form multimeric pores, causing changes in membrane permeability, and leads to cell 28 swelling and membrane rupture 2-4 . Pyroptosis is an important immune response of the body, which 29 plays an important role in antagonizing infection and endogenous danger signals 5 . It is widely 30 involved in the occurrence and development of tumors, infectious diseases, metabolic diseases, 31 nervous system-related diseases and atherosclerotic diseases 6 . 32 33 Recent studies have found that gasdermin E (GSDME, also known as DFNA5) is mutated in familial 34 aging-related hearing loss. GSDME can be cleaved by caspase 3, thereby converting non-35 inflammatory apoptosis into pyroptosis in GSDME-expressing cells 7 . In many cancers, the 36 expression of GSDME is suppressed 8 . Studies have shown that reduced GSDME levels are 37 associated with reduced survival rates caused by breast cancer, which suggests that GSDME may 38 be a tumor suppressor 9 . The evidence that GSDME may be used as a tumor suppressor also includes promoter DNA methylation in many primary cancers that can inactivate GSDME; GSDME inhibits 1 colony formation, proliferation and invasiveness of gastric cancer, melanoma, and colorectal cancer 2 cells; and the decreased expression of GSDME is closely related to worse 5-year survival rates and 3 poor metastasis of many patients with cancer [10][11][12][13] . Recent studies have revealed that the expression 4 of GSDME enhances the phagocytosis of tumor-associated macrophages on tumor cells, as well as 5 the number and function of tumor-infiltrating natural killer cells and CD8 + T lymphocytes. 6 Granzyme B from natural killer cells can also activate caspase-independent pyroptosis in target cells 7 by directly cleaving GSDME at the same site as caspase 3 14,15 . Therefore, GSDME in tumor tissues 8 acts as a tumor suppressor by activating pyroptosis and enhancing anti-tumor immunity. 9 10 Ubiquitin is an evolutionarily conserved 8.5 kDa protein, which is covalently linked to the N-11 terminal or lysine residue of the substrate protein through the sequence reaction of ubiquitin 12 activating enzyme (E1), ubiquitin coupling enzyme (E2) and ubiquitin ligase (E3) 16  repositioning, promoting protein-protein interactions and regulating signal events 17 . Ubiquitination 17 is regulated by more than 1000 human proteins, accounting for about 4% of the proteome, including 18 two E1s, about 40 E2s, more than 600 E3s and about 100 deubiquitinases (DUBs) 18 . It has been 19 reported that there are more than 8000 ubiquitination sites on thousands of proteins 19 . Studies have 20 shown that ubiquitin-related proteins play a key regulatory role in many biological processes, such 21 as the cell cycle, DNA damage, apoptosis and autophagy 20-23 . However, the roles and molecular 22 mechanisms of ubiquitin-related proteins in pyroptosis have not been well identified. 23 The latest progress of CRISPR-Cas9 gene screening technology makes it possible to measure gene 24 importance, cancer cell dependence, and genetic interactions in human cells in a high-throughput 25 manner 24 . Here, we screened and identified USP48, a ubiquitinase that plays an important role in 26 regulating cell pyroptosis by applying the CRISPR-Cas9 gene screening technology, combined with 27 the detection of related secretory factors. In terms of molecular mechanism, USP48 stabilizes its 28 expression by causing deubiquitination of GSDME, achieving its regulatory effect on pyroptosis. 29 Clinical tumor tissue testing confirmed that the expression of USP48 has a significant positive 30 correlation with GSDME and pyroptosis-related factors. The single-cell sequencing results showed 31 that the immune microenvironment in the tumor tissues of the mice after USP48 gene knockout was 32 significantly changed. Consistent with recent reports, the functions of T cells and tumor-associated 33 macrophages in the tumor microenvironment are inhibited to varying degrees after pyroptosis. 34 Finally, the tumor formation experiment in mice confirmed that overexpression of USP48 could 35 effectively improve the therapeutic effect of PD-1 inhibitors. 36 37

USP48 is involved in the regulation of pyroptosis
Pyroptosis is a newly identified pattern of programmed cell death, which is mainly mediated by 1 inflammasomes through the activation of a variety of caspases (including caspase-1), resulting in 2 the shear and multimerization of a variety of gasdermin family members( including GSDMD), 3 which caused cell perforation and cell death 25 . In order to find related genes involved in the 4 regulation of pyroptosis, we conducted CRISPR-Cas9 gene screening in 293T cells, using the 5 secretion of LDH after Raptinal (a fast caspase-3 activator)treatment as a readout，and the 6 obtained genes were further screened and verified by single siRNA screening technology (Figure  7 1A). The screening library consists of 384 unique sgRNAs，knocking down the expression of 96 8 related genes. According to the fold change of LDH secretion after sgRNA transfection, 39 genes 9 were selected as the main genes. Among them, the silencing of 18 candidate genes increased the 10 secretion of LDH by more than 2-fold, while the silencing of the other 21 genes inhibited the 11 secretion of LDH ( Figure 1B). In order to further screen the obtained genes, we designed four 12 independent small interfering RNAs (siRNAs) for each gene. The results showed that the 13 interference of USP48 had the most significant inhibitory effect on the release of LDH in cells 14 ( Figure 1C). 15

16
Ubiquitination is an important item in post-translation. Its most well-known function is to guide the 17 degradation of proteins. Ubiquitination is reversible and can be reversed by a large group of 18 proteases called deubiquitinases (DUBs) 26 . USP48 is an important member of the USP family of 19 DUBs. In order to further clarify the role of USP48 in pyroptosis, we silenced the expression of 20 USP48 by transfecting siRNA into the 293T cell line ( Figure 1D) and treated the cells with Raptinal 21 to detect the effect of USP48 silencing on LDH, IL-1β, and IL-18 in 293T cells ( Figure 1G). The 22 results showed that the silence of USP48 could significantly inhibit the release of LDH, IL-1β and 23 IL-18 in 293T cells. In addition, we also observed the morphological changes and the absorption of 24 SYTOX green in 293T cells transfected with siRNA (si-NC) or USP48-specific siRNA (si-USP48) 25 after Raptinal treatment by time-lapse microscopy, and the results showed that USP48 silencing 26 significantly inhibited Raptinal-induced cell membrane rupture and SYTOX green absorption 27 ( Figure 1F-I). The above results were further confirmed in L929 cells, which is a mouse fibroblast 28 cell line (Supplementary Figure 1A-F). In addition, we also found that USP48 silencing inhibited 29 the Raptinal-induced release of HMGB1 (Figure 1J，Supplementary Figure 1G). In addition, 30 upregulating the expression of USP48 in 293T cells also obtained consistent results with the above 31 (Supplementary Figure 1H-K). In summary, we have preliminarily confirmed that USP48 may be 32 involved in the regulation of pyroptosis. 33 34 USP48 regulates the expression of GSDME 35 In order to further clarify the specific mechanism of USP48 regulating cell pyroptosis, we performed 36 proteomic sequencing in 293T cells overexpressing USP48 or transfected with an empty plasmid 37  Table 2). In addition, the results also showed that 1 USP48 was involved in the regulation of various cell biological processes, including pyroptosis and 2 cell metabolism (Supplementary Figure 2C and D). To further identify the proteins physically 3 associated with USP48, we used anti-Flag affinity purification mass spectrometry to identify 4 potential USP48-interacting proteins in 293T cells expressing Flag-tagged USP48 and found 59 5 proteins that could directly interact with USP48 (Supplementary Table 3). After combined analyses 6 of both mass spectrometry and proteomics results, we obtained eight related proteins ( Figure 2B  7 and C). In order to further verify the above results, we tested the changes in the expression levels of 8 these eight proteins in 293T cells overexpressing USP48 and knocking down USP48 and found that 9 the changes in GSDME were the most significant ( Figure 2D and E). 10 11 GSDME is a newly discovered important protein involved in the regulation of pyroptosis and an 12 important tumor suppressor. In cells expressing GSDME, GSDME can be cleaved and activated by 13 caspase-3, resulting in cell pyroptosis. Subsequently, we further confirmed the physical interaction 14 between USP48 and GSDME by CO-IP ( Figure 2F and G). Subsequently, we confirmed the 15 regulatory effect of USP48 on GSDME through immunohistochemistry and Western blotting in 16 human pancreatic cancer and paraneoplastic tissues, as well as human liver cancer and 17 paraneoplastic tissues, and confirmed the positive correlation between USP48 and GSDME 18 (Supplementary Figure 3A-F). In addition, we also found that USP48 did not affect the cleavage of 19 GSDME ( Figure 2H). In summary, we found that USP48 could regulate the expression of GSDME 20 (but does not affect its cleavage) and can be directly combined with GSDME. 21

USP48 affects pyroptosis by regulating the expression of GSDME 23
Due to the key role of GSDME in activating cell pyroptosis, we speculate that GSDME also plays 24 an important role in the regulation of cell pyroptosis by USP48. To confirm this hypothesis, we 25 established a 293T cell line that simultaneously expressed USP48 and shRNA targeting GSDME 26 ( Figure 3A). As expected, the downregulation of GSDME substantially rescued the promotion of 27 overexpression of USP48 on the production of LDH ( Figure 3B), IL-18 ( Figure 3C), and IL-1β 28 ( Figure 3D) in 293T cells after treatment with Raptinal. Conversely, in 293T cell lines expressing 29 both shUSP48 and GSDME ( Figure 3E), it was also confirmed that overexpression of GSDME 30 could rescue the inhibitory effect of USP48 knockdown on the production of LDH ( Figure 3F), IL-31 1β ( Figure 3G), and IL-18 ( Figure 3H) in 293T cells after treatment with Raptinal. Subsequently, 32 we also used time-lapse microscopy to observe the morphology and absorption of SYTOX green in 33 the 293T cell line expressing USP48 and shRNA targeting GSDME after treatment with Raptinal 34 and consistent results were obtained ( Figure 4I and J). In addition, we also obtained results 35 consistent with the above in L929 cells (Supplementary Figure 4). Interestingly, although the study 36 confirmed that GSDME can be cleaved by caspase-3 and induce pyroptosis, our results suggest that 37 USP48 did not achieve regulatory effects on GSDME, as well as pyroptosis, through caspase-3. 38 Activating or overexpressing caspase-3 in 293T cells with USP48 knockdown, respectively, did not affect the ability of USP48 to regulate GSDME and pyroptosis levels (Supplementary Figure 5). In 1 conclusion, we confirmed that USP48 may promote the expression of GSDME in a way that was 2 independent of caspase-3, thereby promoting the occurrence of pyroptosis. 3 4 USP48 prevents the degradation of GSDME by deubiquitinating it 5 The above results have preliminarily confirmed that USP48 realizes its regulation through physical 6 interaction with GSDME, but the specific mechanism is still unclear. USP48 is an important member 7 of the deubiquitinating enzyme family. Studies have confirmed that it is involved in mediating Gli1, 8 TRAF2, Mdm2 and many other proteins 27-30 . Therefore, we speculate that USP48 deubiquitinates 9 GSDME, thereby inhibiting the proteasome degradation of GSDME. The ubiquitin-proteasome 10 pathway is an important protein degradation regulatory system in cells. 11

12
In order to further confirm whether USP48 affects the stability of GSDME, we found that the 13 proteasome-specific inhibitor MG132 could effectively reverse the impact of USP48 knockdown 14 on GSDME ( Figure 4A) and through cycloheximide (CHX) chase analysis to evaluate the potential 15 of USP48 in regulating GSDME protein turnover rate, which shows that the reduction of the USP48 16 level is obviously related to the reduction of the half-life of GSDME ( Figure 4B). Interestingly, we 17 found that the change of USP48 expression had no effect on GSDME at the RNA level 18 (Supplementary Figure 6A and B), which was also consistent with USP48 affecting the protein 19 stability of GSDME. In view of the above observations, we established 293T cells with Dox-induced 20 wild-type USP48 (USP48/WT) and catalytically inactive mutant USP48 (USP48/C98A). It was 21 found that only wild-type USP48 gradually increased GSDME levels in a Dox dose-dependent 22 manner ( Figure 4C), while no significant changes in GSDME protein levels were detected in cells 23 expressing USP48/C98A ( Figure 4D). Through further experiments, we found that the 24 overexpression of wild-type USP48 significantly reduced the ubiquitination level of GSDME, while 25 293T cells expressing USP48/C98A had no effect on the ubiquitination of GSDME ( Figure 4E). In 26 contrast, knockdown of USP48 in 293T cells led to the accumulation of ubiquitinated GSDME 27 Besides, we also found that overexpression of USP48/C98A in 293T cells had no effect on (K63) 31 . We also found that USP48 affects K48-linked ubiquitin in GSDME, but it has no effect on 37 K63-linked ubiquitination ( Figure 4G). Then, we predicted the ubiquitination sites of GSDME 38 through the website(http://plmd.biocuckoo.org/index.php) and verified the seven predicted sites (K30, K39, K120, K161, K189, K240, and K248) through in vitro ubiquitination experiments. To 1 examine the K48-linked ubiquitination sites, HEK293T cells were co-transfected with the seven 2 Myc tagged mutants of GSDME and HA-ubiquitin K48. An in vitro ubiquitination assay showed 3 that ubiquitin K48 ubiquitinated GSDME at the K30, K120 and K189 sites ( Figure 4H and I). Next, 4 in order to identify the target residues of GSDME modified by USP48, we constructed 5 corresponding point mutants at positions K30, K120 and K189 of GSDME ( Figure 4J), HEK293T 6 cells were co-transfected with the GSDME mutant labeled with Myc, HA-ubiquitin K48, and Flag-7 USP48. The results showed that USP48 inhibited the K48-linked ubiquitination of GSDME at K120 8 ( Figure 4K). Taken together, these results indicate that USP48 prevents the degradation of GSDME 9 by inhibiting K48-linked ubiquitination at position K120 of GSDME. 10 11 USP48 affects anti-tumor immunity by regulating the expression of GSDME 12 Our above results have preliminarily confirmed that USP48 can promote the expression of GSDME, 13 thereby promoting the occurrence of pyroptosis in cells. In order to further clarify this result, we 14 hybridized USP48 deficient mice (USP48 flox / flox ) with KRAS G12D ;PDX1-Cre mice to obtain mice 15 models with specific deletion of USP48 in the pancreas. The expression of USP48 and GSDME in 16 the tissues was then detected by immunohistochemistry and immunofluorescence using pancreatic 17 tissue from the above mice and laboratory-preserved liver tissue from mice lacking USP48 18 specificity 32 , and the results showed that the expression levels of USP48 were positively correlated 19 with the expression of GSDME ( Figure 5A used to detect the expression of USP48 and GSDME in the liver and pancreas of the above mice for 25 further detection and consistent results were obtained ( Figure 5G and H). The above results further 26 confirmed the regulatory effect of USP48 on GSDME in vivo. 27

28
Pyroptosis is a pro-inflammatory form of cell death. Unlike apoptosis, pyroptosis is a pathological 29 death in which cells swell until their membranes rupture, resulting in leakage of cytoplasmic content 30 and a strong inflammatory immune response 33 . Previous studies have shown that GSDME inhibits 31 tumor growth in mice by promoting cell anti-tumor immunity. Compared with control mice, mice 32 expressing GSDME had more natural killer cells and CD8 + cytotoxic T killer cells in tumors and 33 expressed more toxic proteins and cytokines 14 . Therefore, we speculate that USP48 may exert its 34 anti-tumor effect by promoting the expression of GSDME and then promoting the anti-tumor 35 immune response of cells. 36

37
In order to confirm this hypothesis, we explored the effect of the loss of USP48 on the immune cell 38 population in PDAC tissues. The immunofluorescence results showed that the tumor-infiltrating CD8+ and CD4+ T cells were significantly reduced in KUC mice ( Figure 5I-J). The proportion of 1 natural killer cells and CD8+ cytotoxic T killer cells was significantly reduced, but the proportion 2 of tumor-associated macrophages (TAMs) and Treg cells was significantly increased ( Figure 5K). 3 Together, these data imply that USP48 can affect the anti-tumor immunity of cells by regulating the 4 expression of GSDME. In order to further clarify the anti-tumor immune function of USP48, we performed single-cell 10 sequencing in pancreatic tissues of KC and KUC mice. Single-cell RNA sequencing was performed 11 using the 10x chromium method. Consistent with the enrichment approach, all cells were positive 12 for the CD45 gene (PTPRC) irrespective of treatment ( Figure 6A). We applied the size clustering 13 algorithm as a quality control indicator for cells. Figure 6B shows the cell distribution in pancreatic 14 tumor tissues of KC and KUC mice. Then, we applied the size clustering algorithm as the quality 15 control index of the cells, divided the cells into groups, and defined five cell populations that capture 16 the TAMs, dendritic cells (DCs), T cells, monocyte-1 cells and monocyte-2 cells ( Figure 6B and C). 17 By comparing the differences of cell subgroups in KC and KUC pancreatic cancer tissues, we found 18 that there were significant differences in the number of cells in TAMs and monocyte-2 cells. 19 Specifically, the downregulation of USP48 expression significantly increased the number of TAMs 20 and monocyte-2 cells ( Figure 6D). We showed the difference of marker genes in each cell subgroup 21 through heat maps and dot maps (Cd3d = T cells; Cd14 = monocytes; C1qb = TAMs; S100a3 and 22 Thbs1 = monocytes; and Clec9a = DCs) ( Figure 6E and F) and obtained the number and proportion 23 of each cell subgroup in KC and KUC pancreatic cancer tissues ( Figure 6G). 24

25
In order to further determine the immune cell subpopulations regulated by USP48, we first 26 performed a subpopulation analysis of T cells in KC and KUC pancreatic cancer tissues ( Figure 7A). 27 It was found that the proportion of exhaustible T (Tex) cells and Treg cells in KUC pancreatic cancer 28 tissues increased significantly, indicating that the reduction of USP48 expression inhibited the 29 occurrence of tumor immunity ( Figure 7B). We also used UMAP to show the expression of standard 30 marker genes for T cell subgroup classification and the ratio of T cell subgroups in KC and KUC 31 pancreatic cancer tissues, further confirming the above results. (Figure 7C and D). Subsequently, 32 we used multicolor immunofluorescence experiments to detect the distribution of Tex cells and Treg 33 cells in pancreatic cancer tissues of KC and KUC mice, and the results obtained were consistent 34 with the sequencing results ( Figure 7E and F). 35

36
In the above results, we have initially found that knockdown of USP48 significantly increased the 37 proportion of TAM subgroups ( Figure 7G and H). Therefore, further analysis of TAMs was 38 performed, and the same results were obtained. Multicolor immunofluorescence experiments also confirmed that the lack of USP48 increased the distribution of TAMs in pancreatic cancer tissues 1 ( Figure 7I). In summary, we applied 10x single-cell sequencing technology to confirm that 2 USP48 deletion inhibited the anti-tumor immunity of pancreatic cancer cells. 3 4 USP48-GSDME modulates the sensitivity of mice to anti-PD-1 immunotherapy 5 In order to further clarify the role of USP48 in anti-tumor immunity, Pan02 cells overexpressing 6 USP48 or empty vectors were subcutaneously implanted into C57 mice. The mice were treated with 7 aPD-1 (an anti-PD-1 antibody) on days 7, 11 and 15, and the growth of the mice was continuously 8 observed and killed on day 30 ( Figure 8A). PD-1 antibody is an important immunotherapy. It has a 9 good therapeutic effect in the treatment of more than 10 malignant tumors, such as lung cancer, 10 lymphoma, liver cancer, gastric cancer and pancreatic cancer 34 . The results showed that, compared 11 with the empty vector, overexpression of USP48 significantly inhibited the growth of tumors in 12 mice. In addition, overexpression of USP48 also significantly improved the sensitivity of mice to 13 anti-PD-1 treatment ( Figure 8B-E). To further clarify its mechanism, we expressed shGSDME or 14 scramble in Pan02 cells overexpressing USP48 and injected them subcutaneously into C57 mice. inflammatory factors 25 . Up to this point, the mechanism of pyroptosis had been studied clearly. In 11 this study, we applied CRISPR-Cas9 high-throughput screening technology and found that USP48 12 had a significant regulatory effect on pyroptosis. 13 14 GSDME is also an important member of the gasdermin family. Previously, it was shown that the 15 promoter of GSDME was methylated in a variety of cancer cells, and this epistatic modification 16 inhibited its expression in cancer cells. In addition, it was also found that GSDME could inhibit the 17 development of a variety of cancers, including breast cancer and melanoma 38,39 . In 2017, Shao Feng 18 et al. revealed its key role in pyroptosis and found that GSDME could be cleaved and activated by 19 caspase-3, which in turn converts apoptosis into pyroptosis 7 . Interestingly, our results found that 20 USP48 can affect the expression of GSDME (and thus pyroptosis) through physical interaction with 21 GSDME and does not affect the activation and cleavage of GSDME by caspase-3. 22

23
Our study revealed the regulatory role of USP48 on pyroptosis and its molecular mechanism, and 24 we found that USP48 promotes pyroptosis by deubiquitinating GSDME, inhibiting its ubiquitinated 25 degradation, promoting its expression, and ultimately promoting the development of pyroptosis. 26 Unlike immunosilencing apoptosis, pyroptosis is characterized by cell membrane rupture in which 27 numerous cytokines and danger signaling molecules are released, activating the immune system and 28 leading to an inflammatory response 40 . In 2020, Judy Lieberman's group at Harvard Medical School 29 and Boston Children's Hospital found that granzyme B from natural killer cells can directly cleave 30 GSDME and activate pyroptosis, which occurs to further activate the anti-tumor immune response 31 and inhibit tumor growth 14 . Given the important role of pyroptosis in anti-tumor immunity, we 32 speculate that USP48 also has a regulatory role on cellular anti-tumor immunity, which was 33 confirmed by both single-cell sequencing technology and flow cytometry. We found that USP48 34 could regulate the ratio and number of TAMs, NK cells, Tregs and other immune cells. 35

36
The tumor immune microenvironment is a highly complex system. It has also become an important 37 research hotspot in recent years. With more and more in-depth research on immunotherapy and the 38 continuous improvement of immunotherapy efficacy, immunotherapy has begun to be well-known to the public. PD-1 inhibitors are a new class of drugs that block PD-1 and are used to treat certain 1 types of cancer by activating the immune system to attack tumors 41,42 . We found that changes in 2 USP48 expression significantly modulated the efficacy of PD-1 inhibitors by constructing a 3 xenograft tumor model in C57 mice, and increased USP48 expression significantly improved the 4 therapeutic effect of PD-1 inhibitors in mouse tumors. 5 6 In conclusion, our study identified the key regulatory role of USP48 on pyroptosis and elucidated 7 its molecular mechanism, in addition to the role of USP48 in anti-tumor immunity and 8 immunotherapy. Therefore, specifically targeting the USP48 or USP48-GSDME axis may be a 9 potential future therapeutic strategy. Nevertheless, our study leaves much to be desired, as we have 10 only described the regulatory role of USP48 in anti-tumor immunity and immunotherapy. But, the 11 specific molecular mechanisms need to be further elucidated.

Retroviral infection and overexpression of USP48 and GSDME 21
The siRNA used for the knockdown of USP48 and GSDME in the cells was purchased from 22 GenePharma (Shanghai, China). The siRNAs were transfected into cells using Lipofectamine 2000 23 (Invitrogen) according to the manufacturer's instructions. Lentivirus-mediated overexpression and 24 knockdown of USP48 and GSDME against cells was purchased from GeneChem (Shanghai, China). 25 Cells (30% confluence) were incubated in medium containing optimal dilutions of lentivirus mixed 26 with polybrene. After 48 hours of transfection, cells were subjected to puromycin selection (5 27 mg/mL) to obtain stably transfected cells. 28 29

Tissue specimens 30
The collection of adjacent normal tissue specimens from a standard distance (3 cm) from the edge 31 of the tumor tissue removed from patients with PDAC or HCC undergoing surgical resection was 32 performed. All clinical specimens used in this study were histopathologically and clinically 33 diagnosed. In order to use these clinical data for research purposes, the consent and approval of the A combined library of thousands of defined single-guide RNA (sgRNA or gRNA) sequences can 7 disrupt (or "knock out") hundreds of genes in an entire population of cells in a single experiment. 8 The cell population is then screened for the release of lactate dehydrogenase, so the specific gene 9 driving that phenotype can be identified. The CRISPR Knockout Pooled Lentiviral sgRNA Library 10 was purchased from Dharmacon. 11 12

Immunohistochemistry 13
The specimens were fixed with formalin, paraffin-embedded sections, and the protein to be detected

Western Blotting and co-immunoprecipitation (co-IP) 29
The cells were treated with RIPA buffer containing 1x protease inhibitor and phosphatase inhibitor, Total RNA was isolated from cells cultured with TRIzol reagent (Invitrogen) as directed. According 7 to the manufacturer's instructions for the reagent, the PrimeScript TM RT Reverse Transcription Kit 8 (TaKaRa) was used to reverse the RNA (1 μg) to cDNA (20 μL). The experiment was performed at 9 least three times and repeated twice. Endogenous GAPDH was used as a standardized control. The cells transfected with the corresponding lentivirus or plasmid were made into a cell suspension 9 with a density of 1 x 10 7 /mL. C57 mice aged 8-12 weeks were selected and 100 μL of the cell 10 suspension was subcutaneously injected into the left armpit. Tumor growth was observed according 11 to the experimental needs condition. 12 13