heteromulticellular-stromal-cells-scaffold-free-3d-cultures

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

Baylor University

Extracellular matrix (ECM) protein expression/deposition within and stiffening of the breast cancer microenvironment facilitates disease progression and correlates with poor patient survival. However, the mechanisms by which ECM components control tumorigenic behaviors and responses to therapeutic intervention remain poorly understood. Fibronectin (FN) is a major ECM protein controlling multiple processes. In this regard, we previously reported that DHPS-dependent hypusination of eIF5A1/2 is necessary for fibronectin-mediated breast cancer metastasis and epithelial to mesenchymal transition (EMT). Here, we explored the clinical significance of an interactome generated using hypusination pathway components and markers of intratumoral heterogeneity. Solute carrier 3A2 (SLC3A2 or CD98hc) stood out as an indicator of poor overall survival among patients with basal-like breast cancers that express elevated levels of DHPS. We subsequently discovered that blockade of DHPS or SLC3A2 reduced triple negative breast cancer (TNBC) spheroid growth. Interestingly, spheroids stimulated with exogenous fibronectin were less sensitive to inhibition of either DHPS or SLC3A2 - an effect that could be abrogated by dual DHPS/SLC3A2 blockade. We further discovered that a subset of TNBC cells responded to fibronectin by increasing cytoplasmic localization of eIF5A1/2. Notably, these fibronectin-induced subcellular localization phenotypes correlated with a G0/G1 cell cycle arrest. Fibronectin-treated TNBC cells responded to dual DHPS/SLC3A2 blockade by shifting eIF5A1/2 localization back to a nucleus-dominant state, suppressing proliferation and further arresting cells in the G2/M phase of the cell cycle. Finally, we observed that dual DHPS/SLC3A2 inhibition increased the sensitivity of both Rb-negative and -positive TNBC cells to the CDK4/6 inhibitor palbociclib. Taken together, these data identify a previously unrecognized mechanism through which extracellular fibronectin controls cancer cell tumorigenicity by modulating subcellular eIF5A1/2 localization and provides prognostic/therapeutic utility for targeting the cooperative DHPS/SLC3A2 signaling axis to improve breast cancer treatment responses.

Fibronectin, DHPS and SLC3A2 Signaling Cooperate to Control Tumor Spheroid Growth, Subcellular eIF5A1/2 Distribution and CDK4/6 Inhibitor Resistance.

Aqueous humor (AH) and blood levels of transforming growth factor β (TGFβ) are elevated in idiopathic primary open angle glaucoma (POAG) representing a disease biomarker of unclear status and function. Tsk mice display a POAG phenotype and harbor a mutation of fibrillin-1, an important regulator of TGFβ bioavailability. AH TGFβ2 was higher in Tsk than wild-type (WT) mice (by 34%; p = 0.002; ELISA); similarly, AH TGFβ2 was higher in human POAG than controls (2.7-fold; p = 0.00005). As in POAG, TGFβ1 was elevated in Tsk serum (p = 0.01). Fibrillin-1 was detected in AH from POAG subjects and Tsk mice where both had similar levels relative to controls (p = 0.45). 350 kDa immunoblot bands representing WT full-length fibrillin-1 were present in human and mouse AH. A 418 kDa band representing mutant full-length fibrillin-1 was present only in Tsk mice. Lower molecular weight fibrillin-1 antibody-reactive bands were present in similar patterns in humans and mice. Certain bands (130 and 32 kDa) were elevated only in human POAG and Tsk mice (p ≤ 0.04 relative to controls) indicating discrete isoforms relevant to disease. In addition to sharing a phenotype, Tsk mice and human POAG subjects had common TGFβ and fibrillin-1 features in AH and also blood that are pertinent to understanding glaucoma pathogenesis.

Aqueous humor TGFβ and fibrillin-1 in Tsk mice reveal clues to POAG pathogenesis.

Patient-derived organoids (PDOs) developed ex vivo and in vitro are increasingly used for therapeutic screening. They provide a more physiologically relevant model for drug discovery and development compared to traditional cell lines. However, several challenges remain to be addressed to fully realize the potential of PDOs in therapeutic screening. This paper summarizes recent advancements in PDO development and the enhancement of PDO culture models. This is achieved by leveraging materials engineering and microfabrication technologies, including organs-on-a-chip and droplet microfluidics. Additionally, this work discusses the application of PDOs in therapy screening to meet diverse requirements and overcome bottlenecks in cancer treatment. Furthermore, this work introduces tools for data processing and analysis of organoids, along with their microenvironment. These tools aim to achieve enhanced readouts. Finally, this work explores the challenges and future perspectives of using PDOs in drug development and personalized screening for cancer patients.

Patient-Derived Organoids as Therapy Screening Platforms in Cancer Patients.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy, with a median survival of less than 12 months and a 5-year survival of less than 10 %. Here, we have established an image-based screening pipeline for quantifying single PDAC spheroid dynamics in genetically and phenotypically diverse PDAC cell models. Wild-type KRas PDAC cells formed tight/compact spheroids - compaction of these structures was completely blocked by cytoplasmic dynein and focal adhesion kinase (FAK) inhibitors. In contrast, PDAC cells containing mutant KRas formed loosely aggregated spheroids that grew significantly slower following inhibition of polo-like kinase 1 (PLK1) or focal adhesion kinase (FAK). Independent of genetic background, multicellular PDAC-mesenchymal stromal cell (MSC) spheroids self-organized into structures with an MSC-dominant core. The inclusion of MSCs into wild-type KRas PDAC spheroids modestly affected their compaction; however, MSCs significantly increased the compaction and growth of mutant KRas PDAC spheroids. Notably, exogenous collagen 1 potentiated PANC1 spheroid compaction while ITGA1 knockdown in PANC1 cells blocked MSC-induced PANC1 spheroid compaction. In agreement with a role for collagen-based integrin adhesion complexes in stromal cell-induced PDAC phenotypes, we also discovered that MSC-induced PANC1 spheroid growth was completely blocked by the ITGB1 immunoneutralizing antibody mAb13. Finally, multiplexed single-cell immunohistochemical analysis of a 25 patient PDAC tissue microarray revealed a relationship between decreased variance in Spearman r correlation for ITGA1 and PLK1 expression within the tumor cell compartment of PDAC in patients with advanced disease stage, and elevated expression of both ITGA1 and PLK1 in PDAC was found to be associated with decreased patient survival. Taken together, this work uncovers new therapeutic vulnerabilities in PDAC that are relevant to the progression of this stromal cell-rich malignancy and which may reveal strategies for improving patient outcomes.

Multidimensional screening of pancreatic cancer spheroids reveals vulnerabilities in mitotic and cell-matrix adhesion signaling that associate with metastatic progression and decreased patient survival.

SMADs are the canonical intracellular effector proteins of the TGF-β (transforming growth factor-β). SMADs translocate from plasma membrane receptors to the nucleus regulated by many SMAD-interacting proteins through phosphorylation and other post-translational modifications that govern their nucleocytoplasmic shuttling and subsequent transcriptional activity. The signaling pathway of TGF-β/SMAD exhibits both tumor-suppressing and tumor-promoting phenotypes in epithelial-derived solid tumors. Collectively, the pleiotropic nature of TGF-β/SMAD signaling presents significant challenges for the development of effective cancer therapies. Here, we review preclinical studies that evaluate the efficacy of inhibitors targeting major SMAD-regulating and/or -interacting proteins, particularly enzymes that may play important roles in epithelial or mesenchymal compartments within solid tumors.

Targeting SMAD-Dependent Signaling: Considerations in Epithelial and Mesenchymal Solid Tumors.

Editorial: Mechanisms of microenvironment governed plasticity and progression in solid tumors.

Neuroendocrine tumors (NETs) of the pancreas are rare neoplasms that present complex challenges to diagnosis and treatment due to their indolent course. The incidence of pancreatic neuroendocrine tumors has increased significantly over the past two decades. A limited number of pancreatic neuroendocrine cell lines are currently available for the research. Here, we present 3D-iNET ORION, a novel 3-dimensional (spheroid) cell line, isolated from human pancreatic neuroendocrine tumor liver metastasis. Three-dimensionally grown (3D) cancer cell lines have gained interest over the past years as 3D cancer cell lines better recapitulate the in vivo structure of tumors, and are more suitable for in vitro and in vivo experiments. 3D-iNET ORION cancer cell line showed high potential to form tumorspheres when embedded in Matrigel matrix and expresses synaptophysin and EpCAM. Electron microscopy analysis of cancer cell line proved the presence of dense neurosecretory granules. When xenografted into athymic mice, 3D-iNET ORION cells produce slow-growing tumors, positive for chromogranin and synaptophysin. Human Core Exome Panel Analysis has shown that 3DiNET ORION cell line retains the genetic aberration profile detected in the original tumor. In conclusion, our newly developed neuroendocrine cancer cell line can be considered as a new research tool for in vitro and in vivo experiments.

Development of 3D-iNET ORION: a novel, pre-clinical, three-dimensional in vitro cell model for modeling human metastatic neuroendocrine tumor of the pancreas.

Article	Year
Fibronectin, DHPS and SLC3A2 Signaling Cooperate to Control Tumor Spheroid Growth, Subcellular eIF5A1/2 Distribution and CDK4/6 Inhibitor Resistance. bioRxiv : the preprint server for biology\| PubMed ID: 37090582	2023
Aqueous humor TGFβ and fibrillin-1 in Tsk mice reveal clues to POAG pathogenesis. Scientific reports\| PubMed ID: 38347040	2024
Patient-Derived Organoids as Therapy Screening Platforms in Cancer Patients. Advanced healthcare materials\| PubMed ID: 38359321	2024
Multidimensional screening of pancreatic cancer spheroids reveals vulnerabilities in mitotic and cell-matrix adhesion signaling that associate with metastatic progression and decreased patie... Biochemical and biophysical research communications\| PubMed ID: 38382357	2024
Targeting SMAD-Dependent Signaling: Considerations in Epithelial and Mesenchymal Solid Tumors. Pharmaceuticals (Basel, Switzerland)\| PubMed ID: 38543112	2024
Editorial: Mechanisms of microenvironment governed plasticity and progression in solid tumors. Frontiers in cell and developmental biology\| PubMed ID: 38590776	2024
Development of 3D-iNET ORION: a novel, pre-clinical, three-dimensional in vitro cell model for modeling human metastatic neuroendocrine tumor of the pancreas. Human cell\| PubMed ID: 39103560	2024

Jonathan A. Kelber

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