Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

Bregenzer, Michael E.; Davis, Ciara; Horst, Eric N.; Mehta, Pooja; Novak, Caymen; Raghavan, Shreya; Snyder, Catherine S.; Mehta, Geeta

doi:10.3791/59696

Cited by 8 publications

(12 citation statements)

References 21 publications

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“…Given the heterogeneity in the patient samples, the IC 50 values differed significantly between the heterospheroids generated with the OVCAR3 CSC and patient-derived CSC. These results are expected based on the previous characterization of patient-derived heterogeneity in the hanging drop spheroids [18][19][20][21]55].…”

Section: Csc/msc Heterospheroids Demonstrate a Platinum-resistant Phesupporting

confidence: 77%

“…To assess the impact of MSC on cancer cell stemness, we evaluated aldehyde dehydrogenase (ALDH) activity within heterospheroids as a marker of CSC, consistent with our prior work [28]. Previous work has established ovarian cancer cells that express CD133 highly and have elevated ALDH activity, as the ALDH+ CD133+ CSC, which are at the apex of the CSC hierarchy [8,9,11,[18][19][20][21]55,59]. These CSC and can self-renew and asymmetrically divide to create daughter progenies that are either ALDH+ or CD133+.…”

Section: Csc/msc Heterospheroids Demonstrate a Platinum-resistant Phementioning

confidence: 85%

“…CSC were isolated from the OVCAR3 cell line, or two high-grade serous ovarian carcinomas (Patient 1 and Patient 2), using protocols outlined previously [19,[55][56][57][58]. Briefly, single cell suspensions were incubated with ALDEFLUOR reagent (Stem Cell Technologies, Vancouver, BC, Canada), and CD133 antibody.…”

Section: Isolation Of Ovarian Cscs and Ca-mscs From Ovarian Cancer Pamentioning

confidence: 99%

“…Mono-or heterospheroids were generated on an in vitro 384 well hanging drop array platform as described previously [19,[55][56][57][58]. Briefly, to generate CSC monospheroids, single cell suspensions of CSC were diluted, in such a manner that a 20 µL drop contained 12 CSCs.…”

Section: Formation Of Mono-and Heterospheroids From Csc and Mscmentioning

confidence: 99%

“…In this study, we utilized a three-dimensional (3D) hanging drop array spheroid platform, to bring CSC and human adipose MSC (MSC) or carcinoma-associated primary MSC (CA-MSC) in close association in a non-adherent 3D in vitro structure, similar to their presence and interactions within the malignant ascites [19,50,[55][56][57][58]. Using this model, we aimed to investigate the hypothesized PDGF-BB/PDGFR-β signaling and its downstream effects on functional CSC behaviors, including migratory potential, tumorigenicity and platinum resistance.…”

Section: Introductionmentioning

confidence: 99%

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Carcinoma-Associated Mesenchymal Stem Cells Promote Chemoresistance in Ovarian Cancer Stem Cells via PDGF Signaling

Raghavan

Snyder

Wang

et al. 2020

Cancers

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Within the ovarian cancer tumor microenvironment, cancer stem-like cells (CSC) interact with carcinoma associated mesenchymal stem/stromal cells (CA-MSC) through multiple secreted cytokines and growth factors. These paracrine interactions have been revealed to cause enrichment of CSC and their chemoprotection; however, it is still not known if platelet-derived growth factor (PDGF) signaling is involved in facilitating these responses. In order to probe this undiscovered bidirectional communication, we created a model of ovarian malignant ascites in the three-dimensional (3D) hanging drop heterospheroid array, with CSC and CA-MSC. We hypothesized that PDGF secretion by CA-MSC increases self-renewal, migration, epithelial to mesenchymal transition (EMT) and chemoresistance in ovarian CSC. Our results indicate that PDGF signaling in the CSC-MSC heterospheroids significantly increased stemness, metastatic potential and chemoresistance of CSC. Knockdown of PDGFB in MSC resulted in abrogation of these phenotypes in the heterospheroids. Our studies also reveal a cross-talk between PDGF and Hedgehog signaling in ovarian cancer. Overall, our data suggest that when the stromal signaling via PDGF to ovarian CSC is blocked in addition to chemotherapy pressure, the tumor cells are significantly more sensitive to chemotherapy. Our results emphasize the importance of disrupting the signals from the microenvironment to the tumor cells, in order to improve response rates. These findings may lead to the development of combination therapies targeting stromal signaling (such as PDGF and Hedgehog) that can abrogate the tumorigenic, metastatic and platinum resistant phenotypes of ovarian CSC through additional investigations.

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Section: Csc/msc Heterospheroids Demonstrate a Platinum-resistant Phesupporting

confidence: 77%

Section: Csc/msc Heterospheroids Demonstrate a Platinum-resistant Phementioning

confidence: 85%

Section: Isolation Of Ovarian Cscs and Ca-mscs From Ovarian Cancer Pamentioning

confidence: 99%

Section: Formation Of Mono-and Heterospheroids From Csc and Mscmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Carcinoma-Associated Mesenchymal Stem Cells Promote Chemoresistance in Ovarian Cancer Stem Cells via PDGF Signaling

Raghavan

Snyder

Wang

et al. 2020

Cancers

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The Role of the Tumor Microenvironment in CSC Enrichment and Chemoresistance: 3D Co-culture Methods

Bregenzer

Horst

Mehta

et al. 2021

Methods in Molecular Biology

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Cancer stem-like cells (CSC) are responsible for tumor progression, chemoresistance, recurrence, and poor outcomes in many cancers, making them critical research and therapeutic targets. One of the critical components potentiating CSC chemoresistance is the interactions between CSC and the surrounding cells in the tumor microenvironment. Our lab has developed several 3D co-culture models to study ovarian CSC interactions with stromal or immune cells found in ovarian tumor microenvironments. In this chapter, we use ovarian cancer as a model to describe the methodologies developed in our lab; however, these techniques are applicable to a wide range of cancers. First, we discuss our method for isolating CSC from heterogeneous tumors and for creating 3D self-assembled tumoroids in hanging drop plates, in either monoculture or co-culture with mesenchymal stem cells or monocytes/macrophages. We then discuss methods for analyzing these models with a focus on isolating cell-type-specific changes and mechanism investigation. Specifically, we describe lentiviral transduction and flow cytometry as established and robust methods to identify and separate each cell type for downstream analysis. We then describe methods to examine CSC functionality with transwell migration assays and colorimetric MTS-based proliferation assays. Finally, we demonstrate enzyme-linked immunosorbent assays (ELISA) and quantitative polymerase chain reaction (qPCR) methods as mechanistic investigation tools to decouple paracrine and juxtacrine interactions. These methods have wide-reaching applications in cancer research from basic biological investigations, to drug discovery, and personalized drug screening for precision medicine.

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Co-Delivery Polymeric Poly(Lactic-Co-Glycolic Acid) (PLGA) Nanoparticles to Target Cancer Stem-Like Cells

Snyder,

Repetto,

Burkhard

et al. 2024

Methods in Molecular Biology

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Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

Cited by 8 publications

References 21 publications

Carcinoma-Associated Mesenchymal Stem Cells Promote Chemoresistance in Ovarian Cancer Stem Cells via PDGF Signaling

Carcinoma-Associated Mesenchymal Stem Cells Promote Chemoresistance in Ovarian Cancer Stem Cells via PDGF Signaling

The Role of the Tumor Microenvironment in CSC Enrichment and Chemoresistance: 3D Co-culture Methods

Co-Delivery Polymeric Poly(Lactic-Co-Glycolic Acid) (PLGA) Nanoparticles to Target Cancer Stem-Like Cells

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