Biology subjects

Harley, B. A. C.

Publications and source records attributed to Harley, B. A. C..

13 recordsLinked to original sources

bioRxiv2026

TUMOR HETEROGENEITY AND STEMNESS CAN BE SHAPED BY MECHANO-STRUCTURAL PARAMETERS IN OSTEOSARCOMA

Chemoresistance and recurrence of osteosarcoma (OS) can be attributed to a subpopulation of OS cells known as OS Cancer Stem-like Cells (OS-CSCs). We hypothesized that certain mechano-structurally distinct niches within the bone and tumor might be more conducive to OS-CSC formation. Using biomaterial-based 3D scaffolds of distinct structural and mechanical properties (anisotropic soft [~]10 kPa vs isotropic stiff [~]35 kPa), we discovered that OS cells growing in softer, anisotropic scaffolds were rounder, softer, had higher stemness gene expression, showed increased chemoresistance to doxorubicin and cisplatin, and were more tumorigenic in vivo. Mechanistically, biological reprogramming of OS cells on these scaffolds occurred through niche-driven transcriptional changes and differences in chemoresistance-associated epigenetic pathways. Our study showed that a softer, anisotropic niche is more conducive for maintaining OS-CSCs and these findings could be translated to designing therapeutic strategies targeting heterogeneous tumor populations or modifying the microarchitecture to reduce CSC-favoring niches.

cancer biology↗

bioRxiv2025

Size-dependent invasion and therapeutic phenotype of 42MGBA glioblastoma spheroids

Glioblastoma (GBM) is one of the most common malignant brain tumors, with patient mortality driven by invasion into the surround brain microenvironment and drug resistance. Multicellular spheroids are increasingly a common model to study GBM invasion and drug response in engineered biomaterials. However, a key design feature of tumor spheroid studies is the size of each spheroid (number of cells, diameter). Given the heterogenous growth of GBM cells at the surgical margin, spheroids of different sizes may also have clinical relevance. Here, we define shifts in behavior and drug response of wild type and temozolomide (TMZ) resistant GBM spheroids as a function of initial spheroid size. GBM spheroids ranging from 100 to 12,000 cells in size were embedded into a methacrylamide-functionalized gelatin (GelMA) hydrogel. GBM spheroid size had an inverse relationship with the number of apoptotic cells. We observed significant spheroid size dependent effects on TMZ efficacy for both TMZ resistant and wild type cells. Interestingly, high single doses of TMZ were more effective in reducing three-dimensional migration from smaller spheroids than metronomic dosing while high single dose and metronomic dosing were equally effective in reducing invasion for large TMZ-resistant spheroids. Our study highlights the importance of considering and reporting spheroid size for cancer tissue engineering studies considering invasion and drug resistance. It also informs future studies of residual GBM at the tumor margins most responsible for patient relapse and mortality.

bioengineering↗

bioRxiv2025

Hydrogel microdroplet based glioblastoma drug screening platform

Glioblastoma is the most common primary malignant brain tumor with a five-year survival rate less than 5%. The standard of care involves surgical resection followed by treatment with the alkylating agent temozolomide (TMZ). GBM cells that evade surgery eventually become resistant to TMZ and lead to recurrence of tumors in patients. With only four drugs currently FDA-approved for GBM treatment, there is a need for a clinically relevant model capable of accelerating the identification of new therapies. Microgels are microscale ([~]10-1,000 m) hydrogel particles that can be used to encapsulate cells in a tailorable 3D matrix. Microdroplets offer short diffusion lengths relative to conventional hydrogel constructs (>1 mm) to limit spatial distributions of hypoxia and potentially screen therapeutics in a controlled and physiologically relevant environment. Here, we establish a method to encapsulate GBM cells in gelatin and polyethylene glycol (PEG) microgels. We show that microgel composition can affect cell morphology and further, that collections of GBM-laden hydrogels can be used to quantify the effect of single vs. metronomic doses of TMZ. GBM metabolic activity is maintained in microgel culture and GBM cells display drug response kinetics similar to previously established literature using macro-scale hydrogel constructs. Finally, we show microgels can be integrated with a liquid handler to enable high-throughput screening using cell-laden microgels.

bioengineering↗

bioRxiv2025

Multicellular model of temozolomide resistance in glioblastoma reveals phenotypic shifts in drug response and migratory potential

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults, with limited survival outcomes due to tumor recurrence, mainly driven by GBM cell invasion and therapy resistance. Although temozolomide (TMZ) remains the standard-of-care chemotherapeutic, its long-term efficacy is often compromised by rapid emergence of acquired resistance, largely mediated by the DNA repair enzyme, methylguanine methyltransferase (MGMT). To investigate the interplay between tumor heterogeneity, drug resistance, and the extracellular matrix (ECM) microenvironment, we adapted a 3D methacrylamide-functionalized gelatin (GelMA) hydrogel model to study the behavior of mixed populations of TMZ-sensitive and TMZ-resistant GBM cells. Using both single-cell distributions and multicellular spheroids, we report the impact of heterogeneous cell populations and TMZ dosing regimens, including physiological, supraphysiological, and metronomic TMZ schedules, on drug response and migration. We show that the combination therapy of TMZ with an MGMT inhibitor, lomeguatrib, can modulate TMZ resistance in vitro. This hydrogel model enables systematic investigation of GBM heterogeneity, "go-or-grow" phenotypic plasticity, and therapeutic resistance in an ECM-rich microenvironment, offering a valuable platform for future translational research.

bioengineering↗

bioRxiv2025

Resorbable Poly-(D,L)-Lactide Anchorage of Nanoparticulate Mineralized Collagen Materials Maximizes In Vivo Skull Regeneration

BackgroundA clinical demand exists for bone biomaterials that mirror tissue-specific extracellular matrix (ECM) properties and regulate progenitor cell fate. Nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) scaffolds have demonstrated to promote skull regeneration in vivo without addition of growth factors or exogenous progenitor cells, offering a materials-only solution for cranial defect reconstructions. Further enhancement of the safety and regenerative potential of MC-GAG is however necessary for clinical translation. Here, we investigated the regenerative effect of MC-GAG scaffold fixation with a resorbable poly(D,L-lactide) (PDLLA) implant for cerebral protection. MethodsFourteen-millimeter cranial defects were created in New Zealand white rabbits, divided into four groups: 1) defect only, 2) defect with PDLLA, 3) MC-GAG, and 4) MC-GAG with PDLLA. Initial bone healing assessment was conducted at 3 and 9 months using microcomputed tomography (microCT), histology, reference point indentation, and scanning electron microscopy. Long-term effects were evaluated through in vivo microCT imaging at 3, 6, and 9 months, with biomechanical testing of explanted skulls at 9 months. ResultsAt 3 months, MC-GAG significantly enhanced mineralization compared to empty and PDLLA-treated defects, with even greater mineralization observed for MC-GAG-PDLLA. Histologically, MC-GAG demonstrated trabecular mineralization, while MC-GAG-PDLLA had a structural organization resembling native bone. Biomechanical assessment showed enhanced toughness and stiffness for both MC-GAG and MC-GAG-PDLLA. At 6 months, MC-GAG-PDLLA displayed the highest bone mass, and at 9 months, both MC-GAG-containing scaffolds demonstrated superior bone regeneration compared to controls. By 9 months, PDLLA implants were completely resorbed and MC-GAG-PDLLA exhibited mechanical properties surpassing those of all other groups. ConclusionsFixation of MC-GAG scaffolds with PDLLA implants improved mineralization and bone regeneration compared to either material alone for cranial defect reconstruction. Clinical Relevance StatementCranioplasties are essential for cerebral protection and neurological restoration, but current materials have limitations, necessitating alternative bone biomaterials for improved outcomes.

bioengineering↗

bioRxiv2025

Matrix tropism influences endometriotic cell attachment patterns

Due to the extended period for clinical diagnosis, the etiology of endometriotic lesion initiation is not well understood or characterized. Endometriotic lesions are most often found on pelvic tissues and organs, especially the ovaries. To investigate the role of tissue tropism on ovarian endometrioma initiation, we adapted a well-characterized polyacrylamide microarray system to investigate the role of tissue-specific extracellular matrix and adhesion motifs on endometriotic cell attachment, morphology, and size. We report the influence of cell origin (endometriotic vs. non-endometriotic), substrate stiffness mimicking aging and fibrosis, and the role of multicellular (epithelial-stromal) cohorts on cell attachment patterns. We identify multiple ovarian-specific attachment motifs that significantly increase endometriotic (vs. non-endometriotic) cell cohort attachment that could be implicated in early disease etiology.

bioengineering↗

bioRxiv2025

Radiation damage to a three-dimensional hydrogel model of the brain perivascular niche

Glioblastoma (GBM) is a highly aggressive and recurrent brain cancer characterized by diffuse metastasis at the tumor margins. Radiation therapy is a standard component of current treatment and offers potential for improved patient outcomes. While radiation therapy targets GBM cells in the tumor margins, it may also significantly damage adjacent non-cancerous tissues, leading to reduced quality of life and potentially creating a tumor-supportive microenvironment. The perivascular niche (PVN) in the tumor margins is believed to play a significant role in regulating the glioblastoma stem cell subpopulation as well as serving as a site for cancer recurrence and migration. Understanding the impact of radiation on the PVN can better inform radiation schemes and improve our understanding of GBM recurrence, but is difficult in vivo. Here we adapt a previously developed three-dimensional hydrogel model of the brain perivascular niche to investigate the impact of radiation dosage and delivery rate on perivascular niche properties in vitro. Effects of radiation on vessel architecture can be measured in this hydrogel-based model, suggesting an approach that can provide insight into the effects of radiation on a shorter time scale relative to in vivo experiments. IMPACT STATEMENTGlioblastoma (GBM) is a highly aggressive and recurrent brain cancer characterized by diffuse metastasis at the tumor margins. The perivascular niche (PVN) in the tumor margins plays a significant role in GBM progression and is a target for radiation therapy. We report a method to use three-dimensional hydrogel models of the brain perivascular niche to benchmark the impact of radiation dosage and delivery rate on perivascular niche properties in vitro. This approach provides new insight into the effects of radiation on a shorter time scale relative to in vivo experiments.

bioengineering↗

bioRxiv2021

Beta-Catenin Limits Osteogenesis on Regenerative Materials in a Stiffness-Dependent Manner

Targeted refinement of regenerative materials requires mechanistic understanding of cell-material interactions. The nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) scaffold is a porous biomaterial that promotes regenerative healing of calvaria defects in vivo without addition of exogenous growth factors or progenitor cells, suggesting its potential as an off-the-shelf implant for reconstructing skull defects. In this work, we evaluate the relationship between material stiffness, a tunable MC-GAG property, and activation of the canonical Wnt (cWnt) signaling pathway. Primary human bone marrow-derived mesenchymal stem cells (hMSCs) were differentiated on two MC-GAG scaffolds varying by stiffness (non- crosslinked, NX-MC, 0.3 kPa vs. conventionally crosslinked, MC, 3.9 kPa). hMSCs exhibited increased expression of activated {beta}-catenin, the major cWnt intracellular mediator, and the mechanosensitive YAP protein with near complete subcellular colocalization in stiffer MC scaffolds. Small molecule Wnt pathway inhibitors reduced activated {beta}-catenin and YAP protein quantities and colocalization, osteogenic differentiation, and mineralization on MC, with no effects on NX-MC. Concomitantly, Wnt inhibitors increased BMP4 and phosphorylated Smad1/5 (p-Smad1/5) expression on MC, but not NX-MC. Unlike non-specific Wnt pathway downregulation, isolated canonical Wnt inhibition with {beta}-catenin knockdown increased osteogenic gene expression and mineralization specifically on the stiffer MC. {beta}-catenin knockdown also increased p-Smad1/5, Runx2, and BMP4 expression only on the stiffer MC material. Our data indicates stiffness-induced activation of the Wnt and mechanotransduction pathways promotes osteogenesis in MC-GAG scaffolds. However, activated {beta}-catenin is a limiting agent and may serve as a useful target or readout for optimal modulation of stiffness in skeletal regenerative materials. One Sentence Summary{beta}-Catenin limits stiffness-induced osteogenenic differentiation on nanoparticulate mineralized collagen glycosaminoglycan materials

bioengineering↗

bioRxiv2021

Hydrogels Containing Gradients in Vascular Density Reveal Dose-Dependent Role of Angiocrine Cues on Stem Cell Behavior

Biomaterials that replicate patterns of microenvironmental signals from the stem cell niche offer the potential to refine platforms to regulate stem cell behavior. While significant emphasis has been placed on understanding the effects of biophysical and biochemical cues on stem cell fate, vascular-derived or angiocrine cues offer an important alternative signaling axis for biomaterial-based stem cell platforms. Elucidating dose-dependent relationships between angiocrine cues and stem cell fate are largely intractable in animal models and two-dimensional cell culture. In this study, we leverage microfluidic mixing devices to generate three-dimensional hydrogels containing lateral gradients in vascular density alongside murine hematopoietic stem cells (HSCs). Regional differences in vascular density can be generated via embossed gradients in cell, matrix, or growth factor density. HSCs co-cultured alongside vascular gradients reveal spatial patterns of HSC phenotype in response to angiocrine signals. Notably, decreased Akt signaling in high vessel density regions led to increased expansion of lineage-positive hematopoietic cells. This approach offers a combinatorial tool to rapidly screen a continuum of microenvironments with varying vascular, biophysical, and biochemical cues to reveal the influence of local angiocrine signals on HSC fate.

bioengineering↗

bioRxiv2020

The role of pregnancy-specific glycoproteins on trophoblast motility in three-dimensional gelatin hydrogels

Trophoblast invasion is a complex biological process necessary for establishment of pregnancy; however, much remains unknown regarding what signaling factors coordinate the extent of invasion. Pregnancy-specific glycoproteins (PSGs) are some of the most abundant circulating trophoblastic proteins in maternal blood during human pregnancy, with maternal serum concentrations rising to as high as 200-400 g/mL at term. Here, we employ three-dimensional (3D) trophoblast motility assays consisting of trophoblast spheroids encapsulated in 3D gelatin hydrogels to quantify trophoblast outgrowth area, viability, and cytotoxicity in the presence of PSG1 and PSG9 as well as epidermal growth factor and Nodal. We show PSG9 reduces trophoblast motility whereas PSG1 increases motility. Further, we assess bulk nascent protein production by encapsulated spheroids to highlight the potential of this approach to assess trophoblast response (motility, remodeling) to soluble factors and extracellular matrix cues. Such models provide an important platform to develop a deeper understanding of early pregnancy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/314195v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@9062aaorg.highwire.dtl.DTLVardef@13dbc87org.highwire.dtl.DTLVardef@2473eaorg.highwire.dtl.DTLVardef@1362fba_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Freeform printing of heterotypic tumor models within cell-laden microgel matrices

The tissue microenvironment is comprised of a complex assortment of multiple cell types, matrices, membranes and vessel structures. Emulating this complex and often hierarchical organization in vitro has proved a considerable challenge, typically involving segregation of different cell types using layer-by-layer printing or lithographically patterned microfluidic devices. Bioprinting in granular materials is a new methodology with tremendous potential for tissue fabrication. Here, we demonstrate the first example of a complex tumor microenvironment that combines direct writing of tumor aggregates, freeform vasculature channels, and a tunable macroporous matrix as a model to studying metastatic signaling. Our photocrosslinkable microgel suspensions yield local stiffness gradients between particles and the intervening space, while enabling the integration of virtually any cell type. Using computational fluid dynamics, we show that removal of a sacrificial Pluronic ink defines vessel-mimetic channel architectures for endothelial cell linings. Pairing this vasculature with 3D printing of melanoma aggregates, we find that tumor cells within proximity migrated into the prototype vasculature. Together, the integration of perfusable channels with multiple spatially defined cell types provides new avenues for modelling development and disease, with scope for fundamental research and drug development.

bioengineering↗

bioRxiv2020

Tuning trophoblast invasion in a gelatin hydrogel via soluble cues from the maternal-fetal interface

Trophoblast cells play multiple critical roles in pregnancy, notably modulating blastocyst attachment to the endometrium as well as invading into and actively remodeling the endometrium to facilitate biotransport needs of the growing embryo. Despite the importance of trophoblast invasion for processes essential at early stages of pregnancy, much remains unknown regarding the balance of signaling molecules that may influence trophoblast invasion into the endometrium. The goal of this study was to use a three-dimensional trophoblast spheroid invasion assays to examine the effect of cues from the maternal-fetal interface on trophoblast invasion. We report use of a methacrylamide-functionalized gelatin (GelMA) hydrogel to support quantitative analysis of trophoblast outgrowth area and cell viability. We show this multidimensional model of trophoblast invasion can resolve quantifiable differences in outgrowth area and viability in the presence of a known invasion promoter, epidermal growth factor, and a known invasion inhibitor, transforming growth factor {beta}1. We then investigate the sensitivity of trophoblast invasion to cortisol, a hormone associated with exogenous stressors. Together, this approach provides a toolset to investigate the coordinated action of physiological and pathophysiological processes on early stages of trophoblast invasion. IMPACT STATEMENTTrophoblast cells from the invading blastocyst play crucial roles in pregnancy, including remodeling endometrial structure to support embryonic biotransport needs; however, much remains unknown regarding the balance of signaling molecules that may influence trophoblast invasion. We show this multidimensional model of invasion can resolve quantifiable differences in outgrowth area and viability in the presence of known invasion promoters and inhibitors and then investigate invasion sensitivity to cortisol, a hormone associated with exogenous stressors. This approach provides a toolset to investigate the coordinated action of molecules from the maternal-fetal interface on trophoblast invasion that may be challenging to study in humans.

bioengineering↗

bioRxiv2018

The influence of cyclic tensile strain on multi-compartment collagen-GAG scaffolds for tendon-bone junction regeneration

Orthopedic injuries often occur at the interface between soft tissues and bone. The tendon-bone junction (TBJ) is a classic example of such an interface. Current clinical strategies for TBJ injuries prioritize mechanical reattachment over regeneration of the native interface, resulting in poor outcomes. The need to promote regenerative healing of spatially-graded tissues inspires our effort to develop new tissue engineering technologies that replicate features of the spatially-graded extracellular matrix and strain profiles across the native TBJ. We recently described a biphasic collagen-glycosaminoglycan (CG) scaffold containing distinct compartment with divergent mineral content and structural alignment (isotropic vs. anisotropic) linked by a continuous interface zone to mimic structural and compositional features of the native TBJ. Here, we report application of physiologically relevant levels of cyclic tensile strain (CTS) to the scaffold via a bioreactor leads to non-uniform strain profiles across the spatially-graded scaffold. Further, combinations of CTS and matrix structural features promote rapid, spatially-distinct differentiation profiles of human bone marrow-derived mesenchymal stem cells (MSCs) down multiple osteotendinous lineages. CTS preferentially upregulates MSC activity and tenogenic differentiation in the anisotropic region of the scaffold. Further, there are no negative effects of CTS on MSC osteogenic potential in the mineralized region previously shown to promote robust bone regeneration. Together, this work demonstrates a tissue engineering approach that couples instructive biomaterials with physiological stimuli as a mean to promote regenerative healing of orthopedic interfaces.

bioengineering↗