Native isolation of proteins in high yield and purity is a major bottleneck for analysis of their three-dimensional structure, function, and interactome. Here, we present a streamlined workflow for the rapid production of proteins or protein complexes using lentiviral transduction of human suspension cells, combined with highly-specific nanobody-mediated purification and proteolytic elution. (1) First, generation of a plasmid coding for a protein of interest fused to an N- or C-terminal GFP or ALFA peptide tag is rapidly achieved using the lentiviral plasmid toolkit we have designed. (2) Human suspension cell lines stably expressing the tagged fusion protein can be generated in <5 days using lentiviral transduction. (3) Leveraging the picomolar affinity of the GFP and ALFA nanobodies for their respective tags, proteins expressed even at low levels can be specifically captured from the resulting cell lysate in a variety of conditions, including detergents and mild denaturants. (4) Finally, rapid and specific elution of tagged or untagged proteins under native conditions is achieved within minutes at 4˚C using an engineered SUMO protease. We demonstrate the wide applicability of the method by purifying multiple challenging soluble and membrane protein complexes to high purity from human cells. Our strategy is also directly compatible with many widely used GFP expression plasmids, cell lines and transgenic model organisms; is faster than alternative approaches, requiring ~8 days from cloning to purification; and results in substantially improved yields and purity.
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer with few effective treatments. Despite recent advances in in other cancers, the five-year survival rate of PDAC is still only 10%, with most patients succumbing to their disease within the first year. One of the main factors responsible for this poor outcome is the development of chemoresistance in nearly all clinical cases. While the intrinsic factors that facilitate chemoresistance in the tumor cells have been studied extensively, fewer studies have elucidated how the complex and unique microenvironment surrounding the tumor affects therapeutic responses. The abundant stromal cells and the stiff desmoplastic microenvironment constitute more than 90% of the primary tumor bulk. However, there is a lack of high-throughput, easily tunable models to recapitulate this complex microenvironment, in order to identify a critical cell-extrinsic factors that could drive acquired chemoresistance in PDAC cells. To address this issue, we have developed a Matrigel-based, orthogonally tunable 3-dimensional (3D) culture system to co-culture mouse derived PDAC organoids and host-matching cancer-associated fibroblasts (CAFs). Using this system, we found that matrix-activated CAFs readily remodel the desmoplastic matrix through lysol-oxidase dependent crosslinking. Moreover, our novel system demonstrates how collagen-I and matrix stiffness affect chemoresistance via the hypersecretion CAF-derived exosomes. Interestingly, our data show that it is CAF-derived exosomes, not the organoid-derived exosomes, that are most important in regulation of proliferation and chemoresistance. Lastly, we identified key cell surface integrins for collagen-I as therapeutic targets to prevent desmoplastic matrix-mediated chemo-resistance in PDAC. Our study provides insights into how matrix composition and stiffness affect therapeutic outcome in PDAC. Moving forward, therapies aimed at interrupting how PDAC cells and stomal cells sense the matrix microenvironment could be utilized to eventually overcome matrix-mediated chemoresistance in PDAC. Citation Format: Weikun Xiao, Chae-Young Eun, Xinyu Zhang, Charlene DeKalb, Mahsa Pahlavan, Bayan Mahgoub, Hanaa Knaneh, Alireza Sohrabi, Stephanie K. Seidlits, Reginald Hill. Increased extracellular matrix stiffness induces hypersecretion of chemoresistance-promoting cancer associated fibroblast-derived exosomes in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1567.
Exosomes play a critical role in promoting proliferation and survival of cancer cells. These extracellular vesicles that range in size from 30-100nm in diameter are released by many different cell types and contain proteins, nucleic acids, and micro RNAs that can modify the content and state of recipient cells. Our previous studies have shown that cancer-associated fibroblasts (CAFs), which make up the bulk of pancreatic ductal adenocarcinoma (PDAC) tumors, hypersecrete exosomes upon chemotherapy treatment. When these exosomes were taken up by surrounding cancer cells, we observed more cell proliferation and drug resistance. Considering these findings, there is clearly a need of therapeutic strategies that target exosome biogenesis and secretion. Here, we investigate the effectiveness of several compounds, including farnesyl transferase inhibitors, imidazoles, and nonsteroidal anti-inflammatory drugs (NSAIDs), in blocking exosome secretion and suppressing chemoresistance in PDAC CAF and cancer cell lines. We observed decrease in exosome secretion upon ketoconazole, tipifarnib, and neticonazole treatment, using GW4869 (a neutral sphingomyelinase inhibitor) as our control. Furthermore, we established a 3D organoid model from our PDAC mouse models to better understand the impact of these drugs on exosome secretion in tumors with different genetic alterations. Together, our 2D and 3D models illuminate the potential for pharmacological targeting of exosome secretion as a treatment option that can improve the current standard-of-care treatment in PDAC. Citation Format: Chae Young Eun, Charlene DeKalb, Weikun Xiao, Xinyu Zhang, Reginald Hill. Using 3D models to test the efficacy of targeting cancer-associated fibroblast exosomes to overcome chemoresistance in pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6414.
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal and malignant type of cancer with few effective treatments. Despite recent advancement in knowledge of cancer treatment, median survival of PDAC patients remains within 2 years, and less than 8% patients live beyond 5 years after diagnosis. The main factor that drives PDAC lethality is the development of chemoresistance in nearly all clinical PDACs. While intrinsic factors that facilitates chemoresistance have been studied extensively, few studies have illustrated how complex and unique microenvironment surrounding the PDAC tumor affects therapeutic responses. Moreover, a physiologically relevant co-culture model of PDAC and stromal cells remained challenging. To address this issue, we have developed a Matrigel-based, orthogonally tunable 3-dimensional (3D) culture system to culture established human PDAC cell lines, cancer-associated fibroblast (CAF) cells and mouse model-derived organoids. Individual matrix parameters can be customized to mimic actual tumor matrix composition and mechanical properties. Moreover, we have optimized a co-culture medium to maintain viability of both organoid tumor and cancer associated fibroblasts. Through our 3D culture system, our data suggests that matrix-induced exosome secretion directly facilitate gemcitabine resistance in PDAC cells. By directly increasing stiffness of our 3D culture platform, we have observed that PDAC cells potentially display more invasive phenotype. Moreover, in the co-culture systems, we have seen that CAF cells cultured in different stiffness substrates affect PDAC drug response in organoid cultures. Our investigation provides insight into how matrix composition and stiffness together affect therapeutic outcome in PDAC. We believe that specific therapeutic strategies aimed at targeting the matrix microenvironment will be instrumental in order to overcome matrix-mediated chemoresistance in PDAC. Citation Format: Weikun Xiao, Chae-Young Eun, Charlene Dekalb, Kexin Zhang, Reginald Hill. Investigation of how extracellular matrix composition affects pancreatic cancer chemoresistance through the use of biomimetic models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5108.
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