Doctor of Philosophy, Stanford University, BIOE-PHD (2022)
Master of Science, Stanford University, BIOE-MS (2018)
Sarah Heilshorn, Postdoctoral Faculty Sponsor
Cancer-associated mesothelial cells promote ovarian cancer chemoresistance through paracrine osteopontin signaling.
The Journal of clinical investigation
2021; 131 (16)
Ovarian cancer is the leading cause of gynecological malignancy-related deaths, due to its widespread intraperitoneal metastases and acquired chemoresistance. Mesothelial cells are an important cellular component of the ovarian cancer microenvironment that promote metastasis. However, their role in chemoresistance is unclear. Here, we investigated whether cancer-associated mesothelial cells promote ovarian cancer chemoresistance and stemness in vitro and in vivo. We found that osteopontin is a key secreted factor that drives mesothelial-mediated ovarian cancer chemoresistance and stemness. Osteopontin is a secreted glycoprotein that is clinically associated with poor prognosis and chemoresistance in ovarian cancer. Mechanistically, ovarian cancer cells induced osteopontin expression and secretion by mesothelial cells through TGF-beta signaling. Osteopontin facilitated ovarian cancer cell chemoresistance via the activation of the CD44 receptor, PI3K/AKT signaling, and ABC drug efflux transporter activity. Importantly, therapeutic inhibition of osteopontin markedly improved the efficacy of cisplatin in both human and mouse ovarian tumor xenografts. Collectively, our results highlight mesothelial cells as a key driver of ovarian cancer chemoresistance and suggest that therapeutic targeting of osteopontin may be an effective strategy for enhancing platinum sensitivity in ovarian cancer.
View details for DOI 10.1172/JCI146186
View details for PubMedID 34396988
Next-generation cancer organoids.
Organotypic models of patient-specific tumours are revolutionizing our understanding of cancer heterogeneity and its implications for personalized medicine. These advancements are, in part, attributed to the ability of organoid models to stably preserve genetic, proteomic, morphological and pharmacotypic features of the parent tumour in vitro, while also offering unprecedented genomic and environmental manipulation. Despite recent innovations in organoid protocols, current techniques for cancer organoid culture are inherently uncontrolled and irreproducible, owing to several non-standardized facets including cancer tissue sources and subsequent processing, medium formulations, and animal-derived three-dimensional matrices. Given the potential for cancer organoids to accurately recapitulate the intra- and intertumoral biological heterogeneity associated with patient-specific cancers, eliminating the undesirable technical variability accompanying cancer organoid culture is necessary to establish reproducible platforms that accelerate translatable insights into patient care. Here we describe the current challenges and recent multidisciplinary advancements and opportunities for standardizing next-generation cancer organoid systems.
View details for DOI 10.1038/s41563-021-01057-5
View details for PubMedID 34385685
Engineered Matrices Enable the Culture of Human Patient-Derived Intestinal Organoids.
Advanced science (Weinheim, Baden-Wurttemberg, Germany)
2021; 8 (10): 2004705
Human intestinal organoids from primary human tissues have the potential to revolutionize personalized medicine and preclinical gastrointestinal disease models. A tunable, fully defined, designer matrix, termed hyaluronan elastin-like protein (HELP) is reported, which enables the formation, differentiation, and passaging of adult primary tissue-derived, epithelial-only intestinal organoids. HELP enables the encapsulation of dissociated patient-derived cells, which then undergo proliferation and formation of enteroids, spherical structures with polarized internal lumens. After 12 rounds of passaging, enteroid growth in HELP materials is found to be statistically similar to that in animal-derived matrices. HELP materials also support the differentiation of human enteroids into mature intestinal cell subtypes. HELP matrices allow stiffness, stress relaxation rate, and integrin-ligand concentration to be independently and quantitatively specified, enabling fundamental studies of organoid-matrix interactions and potential patient-specific optimization. Organoid formation in HELP materials is most robust in gels with stiffer moduli (G' ≈ 1 kPa), slower stress relaxation rate (t1/2 ≈ 18 h), and higher integrin ligand concentration (0.5 × 10-3-1 × 10-3 m RGD peptide). This material provides a promising in vitro model for further understanding intestinal development and disease in humans and a reproducible, biodegradable, minimal matrix with no animal-derived products or synthetic polyethylene glycol for potential clinical translation.
View details for DOI 10.1002/advs.202004705
View details for PubMedID 34026461
View details for PubMedCentralID PMC8132048
- Engineered Matrices Enable the Culture of Human Patient-Derived Intestinal Organoids ADVANCED SCIENCE 2021
Microrheology reveals simultaneous cell-mediated matrix stiffening and fluidization that underlie breast cancer invasion.
2021; 7 (8)
Living tissues embody a unique class of hybrid materials in which active and thermal forces are inextricably linked. Mechanical characterization of tissues demands descriptors that respect this hybrid nature. In this work, we develop a microrheology-based force spectrum analysis (FSA) technique to dissect the active and passive fluctuations of the extracellular matrix (ECM) in three-dimensional (3D) cell culture models. In two different stromal models and a 3D breast cancer spheroid model, our FSA reveals emergent hybrid dynamics that involve both high-frequency stress stiffening and low-frequency fluidization of the ECM. We show that this is a general consequence of nonlinear coupling between active forces and the frequency-dependent viscoelasticity of stress-stiffening networks. In 3D breast cancer spheroids, this dual active stiffening and fluidization is tightly connected with invasion. Our results suggest a mechanism whereby breast cancer cells reconcile the seemingly contradictory requirements for both tension and malleability in the ECM during invasion.
View details for DOI 10.1126/sciadv.abe1969
View details for PubMedID 33597244
- Defined matrices bring IBD to 3D. Nature materials 2021; 20 (2): 124–25
Neural Progenitor Cells Alter Chromatin Organization and Neurotrophin Expression in Response to 3D Matrix Degradability.
Advanced healthcare materials
Neural progenitor cells (NPCs) are promising therapeutic candidates for nervous system regeneration. Significant efforts focus on developing hydrogel-based approaches to facilitate the clinical translation of NPCs, from scalable platforms for stem cell production to injectable carriers for cell transplantation. However, fundamental questions surrounding NPC-hydrogel interactions remain unanswered. While matrix degradability is known to regulate the stemness and differentiation capacity of NPCs, how degradability impacts NPC epigenetic regulation and secretory phenotype remains unknown. To address this question, NPCs encapsulated in recombinant protein hydrogels with tunable degradability are assayed for changes in chromatin organization and neurotrophin expression. In high degradability gels, NPCs maintain expression of stem cell factors, proliferate, and have large nuclei with elevated levels of the stemness-associated activating histone mark H3K4me3. In contrast, NPCs in low degradability gels exhibit more compact, rounded nuclei with peripherally localized heterochromatin, are non-proliferative yet non-senescent, and maintain expression of neurotrophic factors with potential therapeutic relevance. This work suggests that tuning matrix degradability may be useful to direct NPCs toward either a more-proliferative, stem-like phenotype for cell replacement therapies, or a more quiescent-like, pro-secretory phenotype for soluble factor-mediated therapies.
View details for DOI 10.1002/adhm.202000754
View details for PubMedID 32743903
Bioprinting Cell- and Spheroid-Laden Protein-Engineered Hydrogels as Tissue-on-Chip Platforms.
Frontiers in bioengineering and biotechnology
2020; 8: 374
Human tissues, both in health and disease, are exquisitely organized into complex three-dimensional architectures that inform tissue function. In biomedical research, specifically in drug discovery and personalized medicine, novel human-based three-dimensional (3D) models are needed to provide information with higher predictive value compared to state-of-the-art two-dimensional (2D) preclinical models. However, current in vitro models remain inadequate to recapitulate the complex and heterogenous architectures that underlie biology. Therefore, it would be beneficial to develop novel models that could capture both the 3D heterogeneity of tissue (e.g., through 3D bioprinting) and integrate vascularization that is necessary for tissue viability (e.g., through culture in tissue-on-chips). In this proof-of-concept study, we use elastin-like protein (ELP) engineered hydrogels as bioinks for constructing such tissue models, which can be directly dispensed onto endothelialized on-chip platforms. We show that this bioprinting process is compatible with both single cell suspensions of neural progenitor cells (NPCs) and spheroid aggregates of breast cancer cells. After bioprinting, both cell types remain viable in incubation for up to 14 days. These results demonstrate a first step toward combining ELP engineered hydrogels with 3D bioprinting technologies and on-chip platforms comprising vascular-like channels for establishing functional tissue models.
View details for DOI 10.3389/fbioe.2020.00374
View details for PubMedID 32411691
View details for PubMedCentralID PMC7198818
- Bioprinting of stem cell expansion lattices ACTA BIOMATERIALIA 2019; 95: 225–35
Matrix Remodeling Enhances the Differentiation Capacity of Neural Progenitor Cells in 3D Hydrogels
2019; 6 (4): 1801716
Neural progenitor cells (NPCs) are a promising cell source to repair damaged nervous tissue. However, expansion of therapeutically relevant numbers of NPCs and their efficient differentiation into desired mature cell types remains a challenge. Material-based strategies, including culture within 3D hydrogels, have the potential to overcome these current limitations. An ideal material would enable both NPC expansion and subsequent differentiation within a single platform. It has recently been demonstrated that cell-mediated remodeling of 3D hydrogels is necessary to maintain the stem cell phenotype of NPCs during expansion, but the role of matrix remodeling on NPC differentiation and maturation remains unknown. By culturing NPCs within engineered protein hydrogels susceptible to degradation by NPC-secreted proteases, it is identified that a critical amount of remodeling is necessary to enable NPC differentiation, even in highly degradable gels. Chemical induction of differentiation after sufficient remodeling time results in differentiation into astrocytes and neurotransmitter-responsive neurons. Matrix remodeling modulates expression of the transcriptional co-activator Yes-associated protein, which drives expression of NPC stemness factors and maintains NPC differentiation capacity, in a cadherin-dependent manner. Thus, cell-remodelable hydrogels are an attractive platform to enable expansion of NPCs followed by differentiation of the cells into mature phenotypes for therapeutic use.
View details for PubMedID 30828535
Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D.
Journal of visualized experiments : JoVE
Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.
View details for PubMedID 29863669
Maintenance of neural progenitor cell stemness in 3D hydrogels requires matrix remodelling.
2017; 16 (12): 1233–42
Neural progenitor cell (NPC) culture within three-dimensional (3D) hydrogels is an attractive strategy for expanding a therapeutically relevant number of stem cells. However, relatively little is known about how 3D material properties such as stiffness and degradability affect the maintenance of NPC stemness in the absence of differentiation factors. Over a physiologically relevant range of stiffness from ∼0.5 to 50 kPa, stemness maintenance did not correlate with initial hydrogel stiffness. In contrast, hydrogel degradation was both correlated with, and necessary for, maintenance of NPC stemness. This requirement for degradation was independent of cytoskeletal tension generation and presentation of engineered adhesive ligands, instead relying on matrix remodelling to facilitate cadherin-mediated cell-cell contact and promote β-catenin signalling. In two additional hydrogel systems, permitting NPC-mediated matrix remodelling proved to be a generalizable strategy for stemness maintenance in 3D. Our findings have identified matrix remodelling, in the absence of cytoskeletal tension generation, as a previously unknown strategy to maintain stemness in 3D.
View details for PubMedID 29115291