Bone marrow stroma influences metastatic prostate cancer (PCa) progression, latency, and recurrence. At sites of PCa bone metastasis, cancer-associated fibroblasts and tumor-associated macrophages ...interact to establish a perlecan-rich desmoplastic stroma. As a heparan sulfate proteoglycan, perlecan (HSPG2) stores and stabilizes growth factors, including heparin-binding Wnt3A, a positive regulator of PCa cell growth. Because PCa cells alone do not induce CAF production of perlecan in the desmoplastic stroma, we sought to discover the sources of perlecan and its growth factor-releasing modifiers SULF1, SULF2, and heparanase in PCa cells and xenografts, bone marrow fibroblasts, and macrophages. SULF1, produced primarily by bone marrow fibroblasts, was the main glycosaminoglycanase present, a finding validated with primary tissue specimens of PCa metastases with desmoplastic bone stroma. Expression of both HSPG2 and SULF1 was concentrated in αSMA-rich stroma near PCa tumor nests, where infiltrating pro-tumor TAMs also were present. To decipher SULF1's role in the reactive bone stroma, we created a bone marrow biomimetic hydrogel incorporating perlecan, PCa cells, macrophages, and fibroblastic bone marrow stromal cells. Finding that M2-like macrophages increased levels of SULF1 and HSPG2 produced by fibroblasts, we examined SULF1 function in Wnt3A-mediated PCa tumoroid growth in tricultures. Comparing control or SULF1 knockout fibroblastic cells, we showed that SULF1 reduces Wnt3A-driven growth, cellularity, and cluster number of PCa cells in our 3D model. We conclude that SULF1 can suppress Wnt3A-driven growth signals in the desmoplastic stroma of PCa bone metastases, and SULF1 loss favors PCa progression, even in the presence of pro-tumorigenic TAMs.
Perlecan/heparan sulfate proteoglycan 2 (HSPG2), a large HSPG, is indispensable for the development of musculoskeletal tissues, where it is deposited within the pericellular matrix (PCM) surrounding ...chondrocytes and disappears nearly completely at the chondro‐osseous junction (COJ) of developing long bones. Destruction of perlecan at the COJ converts an avascular cartilage compartment into one that permits blood vessel infiltration and osteogenesis. Mutations in perlecan are associated with chondrodysplasia with widespread musculoskeletal and joint defects. This study elucidated novel signaling roles of perlecan core protein in endochondral bone formation and chondrocyte behavior. Perlecan subdomains were tested for chondrogenic properties in ATDC5 cells, a model for early chondrogenesis. A region within domain IV of perlecan (HSPG2 IV‐3) was found to promote rapid prechondrocyte clustering. Introduction of the mutation (R3452Q) associated with the human skeletal disorder Schwartz‐Jampel syndrome limited HSPG2 IV‐3‐induced clustering. HSPG2 IV‐3 activity was enhanced when thermally unfolded, likely because of increased exposure of the active motif(s). HSPG2 IV‐3‐induced clustering was accompanied by the deactivation of key components of the focal adhesion complex, FAK and Src, with increased messenger RNA (mRNA) levels of precartilage condensation markers Sox9 and N‐cadherin (
Cdh2), and cartilage PCM components collagen II (
Col2a1) and aggrecan (
Acan). HSPG2 IV‐3 reduced signaling through the ERK pathway, where loss of ERK1/2 phosphorylation coincided with reduced FoxM1 protein levels and increased mRNA levels cyclin‐dependent kinase inhibitor 1C (Cdkn1c) and activating transcription factor 3 (
Atf3), reducing cell proliferation. These findings point to a critical role for perlecan domain IV in cartilage development through triggering chondrocyte condensation.
This study identified a novel active chondrogenic region within domain IV of the perlecan core protein with direct relevance to cartilage development and dysplasia. We showed that HSPG2 IV‐3 induced cell‐cell interactions reminiscent of mesenchyme stem cell condensation and elevated major markers associated with chondrocyte differentiation. Introduction of a mutation associated with Schwartz‐Jampel syndrome abrogated the clustering activity of HSPG2 IV‐3, which may partially explain the cartilage dysmorphisms seen in patients with this mutation. Together, these results point to a key role for the perlecan core protein in chondrocyte condensation in the developing growth plate cartilage, which is disrupted in certain chondrodysplasias.
Cancer occurs when cells acquire genomic instability and inflammation, produce abnormal levels of epigenetic factors/proteins and tumor suppressors, reprogram the energy metabolism and evade immune ...destruction, leading to the disruption of cell cycle/normal growth. An early event in carcinogenesis is loss of polarity and detachment from the natural basement membrane, allowing cells to form distinct three-dimensional (3D) structures that interact with each other and with the surrounding microenvironment. Although valuable information has been accumulated from traditional in vitro studies in which cells are grown on flat and hard plastic surfaces (2D culture), this culture condition does not reflect the essential features of tumor tissues. Further, fundamental understanding of cancer metastasis cannot be obtained readily from 2D studies because they lack the complex and dynamic cell–cell communications and cell–matrix interactions that occur during cancer metastasis. These shortcomings, along with lack of spatial depth and cell connectivity, limit the applicability of 2D cultures to accurate testing of pharmacologically active compounds, free or sequestered in nanoparticles. To recapitulate features of native tumor microenvironments, various biomimetic 3D tumor models have been developed to incorporate cancer and stromal cells, relevant matrix components, and biochemical and biophysical cues, into one spatially and temporally integrated system. In this article, we review recent advances in creating 3D tumor models employing tissue engineering principles. We then evaluate the utilities of these novel models for the testing of anticancer drugs and their delivery systems. We highlight the profound differences in responses from 3D in vitro tumors and conventional monolayer cultures. Overall, strategic integration of biological principles and engineering approaches will both improve understanding of tumor progression and invasion and support discovery of more personalized first line treatments for cancer patients.
Many advanced cancer models, such as patient‐derived xenografts (PDXs), offer significant benefits in their preservation of the native tumor's heterogeneity and susceptibility to treatments, but face ...significant barriers to use in their reliance on a rodent host for propagation and screening. PDXs remain difficult to implement in vitro, particularly in configurations that enable both detailed cellular analysis and high‐throughput screening (HTS). Complex multilineage co‐cultures with stromal fibroblasts, endothelium, and other cellular and structural components of the tumor microenvironment (TME) further complicate ex vivo implementation. Herein, the culture of multiple prostate cancer (PCa)‐derived PDX models as 3D clusters within engineered biomimetic hydrogel matrices, in a HTS‐compatible multiwell microfluidic format, alongside bone marrow‐derived stromal cells and a perfused endothelial channel. Polymeric hydrogel matrices are customized for each cell type, enabling cell survival in vitro and facile imaging across all conditions. PCa PDXs demonstrate unique morphologies and reliance on TME partners, retention of known phenotype, and expected sensitivity or resistance to standard PCa therapeutics. This novel integration of technologies provides a fully human model, and expands the information to be gathered from each specimen, while avoiding the time and labor involved with animal‐based testing.
Patient‐derived xenografts (PDXs) are an exciting testbed for drug screening, as they often present a more complete picture than immortalized cell lines. This work describes hydrogels for in vitro coculture of prostate cancer PDXs, bone‐derived stroma, and endothelial cells, in a perfusable microfluidic 3D culture system, suitable for high‐throughput drug screening.
Silicon‐based micro and nanoparticles are ideally suited for use as biomedical imaging agents because of their biocompatibility, biodegradability, and simple surface chemistry that facilitates drug ...loading and targeting. A method to hyperpolarize silicon particles using dynamic nuclear polarization (DNP), which increases magnetic resonance (MR) imaging signals by several orders‐of‐magnitude through enhanced nuclear spin alignment, was developed to allow silicon particles to function as contrast agents for in vivo magnetic resonance imaging. In this review, we describe the application of the DNP technique to silicon particles and nanoparticles for background‐free real‐time molecular MR imaging. This review provides a summary of the state‐of‐the‐science in silicon particle hyperpolarization with a detailed protocol for hyperpolarizing silicon particles. This information will foster awareness and spur interest in this emerging area of nanoimaging and provide a path to new developments and discoveries to further advance the field.
This article is categorized under:
Diagnostic Tools > In Vivo Nanodiagnostics and Imaging
Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease
Therapeutic Approaches and Drug Discovery > Emerging Technologies
Hyperpolarized silicon MRI as a platform imaging technology for nanomedicine.
Validation of a high‐throughput compatible 3D hyaluronic acid hydrogel coculture of cancer cells with stromal cells. The multilayered hyaluronic acid hydrogels improve drug screening predictability ...as evaluated with a panel of clinically relevant chemotherapeutics in both prostate and endometrial cancer cell lines compared to 2D culture.
Development of an artificial salivary gland will benefit patients with xerostomia after radiation therapy for upper respiratory cancer. The goal is to devise a three-dimensional (3D) culture system ...in which salivary cells differentiate into polarized acini that express essential biomarkers and directionally secrete alpha-amylase. Differentiated acini-like structures in a 3D biomaterial-based scaffold will mimic salivary gland functions.
Cells were seeded onto HA-based hydrogels containing PlnDIV peptide and allowed to differentiate into acini-like structures. Cell viability and phenotype were examined.
Laboratory-based tissue procurement study.
Salivary gland tissue was obtained from patients undergoing surgery. Marker expression established the phenotype of salivary gland cells. Perlecan/HSPG2, an important component of the basement membrane, was highly expressed in salivary gland tissue. A culture system consisting of hyaluronic acid (HA) hydrogel and a coupled bioactive peptide derived from domain IV of perlecan (PlnDIV) was used. Prior studies demonstrated differentiation of acinar cells into lobular structures that mimicked intact glands when cultured on PlnDIV peptide-coated surfaces.
Lobular acini-like structures formed on hydrogels and expressed tight junction components such as zona occludens 1. Acini-like structures were stained for the presence of alpha-amylase. Live/dead staining revealed the presence of apoptotic cells in the center of the acini-like structures, indicative of lumen formation.
A novel system supporting acini-like assembly in a 3D culture system was established. Presence of biomarkers and secretion of salivary enzymes confirms functionality in vitro. Future experiments will test the 3D system in an animal model.
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