Isolation of Pancreatic Cancer Cells from a Patient-Derived Xenograft Model Allows for Practical Expansion and Preserved Heterogeneity in Culture

Pham, Kien; Delitto, Daniel; Knowlton, Andrea E.; Hartlage, Emily R.; Madhavan, Ricky; Gonzálo, David; Thomas, Ryan M.; Behrns, Kevin E.; George, Thomas J.; Hughes, Steven J.; Wallet, Shannon M.; Liu, Chen; Treviño, José G.

doi:10.1016/j.ajpath.2016.02.009

Cited by 35 publications

(50 citation statements)

References 51 publications

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“…Thus, in order to assess TAS contributions to the unique tumor immune microenvironment, validated primary human cell culture models of TAS and PC cells were employed (Fig. 2A) (15–17). Specifically, the human TAS response to PC cell secreted factors was assessed through conditioned media transfers (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Patient-derived PC cell lines were isolated from patient-derived xenografts as previously described (16,17). Epithelial cell purity was confirmed by expression of HLA ABC, EpCam and cytokeratins 18 and 19 (Biolegend, San Diego, CA).…”

Section: Methodsmentioning

confidence: 99%

“…Primary PC cells isolated as previously described (16,17), were evaluated for HLA A2 expression using flow cytometry with a commercially validated antibody (Biolegend) and a representative HLA A2-positive line was chosen. 2.5×10 4 PC cells were seeded in 96 well plates and allowed to adhere overnight in growth medium.…”

Section: Methodsmentioning

confidence: 99%

“…Importantly, these DAMP-mediated processes affect not only immune cells within the tumor microenvironment, but stromal and epithelial cells as well (5,13,14). Using primary human cell culture systems, we discovered a MyD88-dependent TAS response to PC cell secreted factors that results in a pro-tumor polarization of the CD4 + T cell compartment and inhibition of cytotoxicity within the CD8 + T cell compartment (15–17). These in vitro findings recapitulate both the intratumoral milieu and peripheral T cell profiles observed in patients with PC.…”

Section: Introductionmentioning

confidence: 99%

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Human Pancreatic Cancer Cells Induce a MyD88-Dependent Stromal Response to Promote a Tumor-Tolerant Immune Microenvironment

Delitto

DiVita

et al. 2017

Cancer Research

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Cancer cells exert mastery over the local tumor-associated stroma (TAS) to configure protective immunity within the tumor microenvironment. The immunomodulatory character of pancreatic lysates of patients with cancer differs from those with pancreatitis. In this study, we evaluated the crosstalk between pancreatic cancer (PC) and its TAS in primary human cell culture models. Upon exposure of TAS to PC cell-conditioned media, we documented robust secretion of IL-6 and IL-8. This TAS response was MyD88-dependent and sufficient to directly suppress both CD4+ and CD8+ T cell proliferation, inducing Th17 polarization at the expense of Th1. We found that patients possessed a similar shift in circulating effector memory Th17:Th1 ratios compared to healthy controls. The TAS response also directly suppressed CD8+ T cell-mediated cytotoxicity. Overall, our results demonstrate how TAS contributes to the production of an immunosuppressive tumor microenvironment in pancreatic cancer.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Human Pancreatic Cancer Cells Induce a MyD88-Dependent Stromal Response to Promote a Tumor-Tolerant Immune Microenvironment

Delitto

DiVita

et al. 2017

Cancer Research

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“…The primary human pancreatic cancer (PPC) cell line was previously isolated, and methods described [73]. Briefly, a 2 × 2 mm viable portion of resected pancreatic tumor tissue, obtained with informed written patient consent and approved by the University of Florida Institutional Review Board, was implanted subcutaneously into immunocompromised mice and allowed to grow to 1.5 cm.…”

Section: Cellsmentioning

confidence: 99%

IL-8 Released from Human Pancreatic Cancer and Tumor-Associated Stromal Cells Signals through a CXCR2-ERK1/2 Axis to Induce Muscle Atrophy

Callaway

Delitto

D’Lugos

et al. 2019

Cancers

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Tumor-derived cytokines are known to drive the catabolism of host tissues, including skeletal muscle. However, our understanding of the specific cytokines that initiate this process remains incomplete. In the current study, we conducted multiplex analyte profiling of cytokines in conditioned medium (CM) collected from human pancreatic cancer (PC) cells, human tumor-associated stromal (TAS) cells, and their co-culture. Of the factors identified, interleukin-8 (IL-8) is released at high levels from PC cells and PC/TAS co-culture and has previously been associated with low muscle mass in cancer patients. We, therefore, treated C2C12 myotubes with IL-8 which led to the activation of ERK1/2, STAT, and Smad signaling, and induced myotube atrophy. Moreover, the treatment of mice with IL-8 also induced significant muscle wasting, confirming the in vivo relevance of IL-8 on muscle. Mechanistically, IL-8-induced myotube atrophy is inhibited by treatment with the CXCR2 antagonist, SB225002, or by treatment with the ERK1/2 inhibitor, U0126. We further demonstrate that this axis mediates muscle atrophy induced by pancreatic cancer cell CM, as neutralization of IL-8 or treatment with SB225002 or U0126 significantly inhibit CM-induced myotube atrophy. Thus, these data support a key role of IL-8 released from human PC cells in initiating atrophy of muscle cells via CXCR2-ERK1/2.

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A multi‐omics approach identifies pancreatic cancer cell extracellular vesicles as mediators of the unfolded protein response in normal pancreatic epithelial cells

Hinzman

Singh

Bansal

et al. 2022

J of Extracellular Vesicle

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Although cancer‐derived extracellular vesicles (cEVs) are thought to play a pivotal role in promoting cancer progression events, their precise effect on neighbouring normal cells is unknown. In this study, we investigated the impact of pancreatic cancer ductal adenocarcinoma (PDAC) derived EVs on recipient non‐tumourigenic pancreatic normal epithelial cells upon internalization. We demonstrate that cEVs are readily internalized and induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in treated normal pancreatic epithelial cells within 24 h. We further show that PDAC cEVs increase cell proliferation, migration, and invasion and that these changes are regulated at least in part, by the UPR mediator DDIT3. Subsequently, these cells release several inflammatory cytokines. Leveraging a layered multi‐omics approach, we analysed EV cargo from a panel of six PDAC and two normal pancreas cell lines, using multiple EV isolation methods. We found that cEVs were enriched for an array of biomolecules which can induce or regulate ER stress and the UPR, including palmitic acid, sphingomyelins, metabolic regulators of tRNA charging and proteins which regulate trafficking and degradation. We further show that palmitic acid, at doses relevant to those found in cEVs, is sufficient to induce ER stress in normal pancreas cells. These results suggest that cEV cargo packaging may be designed to disseminate proliferative and invasive characteristics upon internalization by distant recipient normal cells, hitherto unreported. This study is among the first to highlight a major role for PDAC cEVs to induce stress in treated normal pancreas cells that may modulate a systemic response leading to altered phenotypes. These findings highlight the importance of EVs in mediating disease aetiology and open potential areas of investigation toward understanding the role of cEV lipids in promoting cell transformation in the surrounding microenvironment.

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Isolation of Pancreatic Cancer Cells from a Patient-Derived Xenograft Model Allows for Practical Expansion and Preserved Heterogeneity in Culture

Cited by 35 publications

References 51 publications

Human Pancreatic Cancer Cells Induce a MyD88-Dependent Stromal Response to Promote a Tumor-Tolerant Immune Microenvironment

Human Pancreatic Cancer Cells Induce a MyD88-Dependent Stromal Response to Promote a Tumor-Tolerant Immune Microenvironment

IL-8 Released from Human Pancreatic Cancer and Tumor-Associated Stromal Cells Signals through a CXCR2-ERK1/2 Axis to Induce Muscle Atrophy

A multi‐omics approach identifies pancreatic cancer cell extracellular vesicles as mediators of the unfolded protein response in normal pancreatic epithelial cells

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