Engineered matrices reveal stiffness-mediated chemoresistance in patient-derived pancreatic cancer organoids.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by its fibrotic and stiff extracellular matrix. However, how the altered cell/extracellular-matrix signalling contributes to the PDAC tumour phenotype has been difficult to dissect. Here we design and engineer matrices that recapitulate the key hallmarks of the PDAC tumour extracellular matrix to address this knowledge gap. We show that patient-derived PDAC organoids from three patients develop resistance to several clinically relevant chemotherapies when cultured within high-stiffness matrices mechanically matched to in vivo tumours. Using genetic barcoding, we find that while matrix-specific clonal selection occurs, cellular heterogeneity is not the main driver of chemoresistance. Instead, matrix-induced chemoresistance occurs within a stiff environment due to the increased expression of drug efflux transporters mediated by CD44 receptor interactions with hyaluronan. Moreover, PDAC chemoresistance is reversible following transfer from high- to low-stiffness matrices, suggesting that targeting the fibrotic extracellular matrix may sensitize chemoresistant tumours. Overall, our findings support the potential of engineered matrices and patient-derived organoids for elucidating extracellular matrix contributions to human disease pathophysiology.

Human enteroids as a tool to study conventional and ultra-high dose rate radiation.

Radiation therapy, one of the most effective therapies to treat cancer, is highly toxic to healthy tissue. The delivery of radiation at ultra-high dose rates, FLASH radiation therapy (FLASH), has been shown to maintain therapeutic anti-tumor efficacy while sparing normal tissues compared to conventional dose rate irradiation (CONV). Though promising, these studies have been limited mainly to murine models. Here, we leveraged enteroids, three-dimensional cell clusters that mimic the intestine, to study human-specific tissue response to radiation. We observed enteroids have a greater colony growth potential following FLASH compared with CONV. In addition, the enteroids that reformed following FLASH more frequently exhibited proper intestinal polarity. While we did not observe differences in enteroid damage across groups, we did see distinct transcriptomic changes. Specifically, the FLASH enteroids upregulated the expression of genes associated with the WNT-family, cell-cell adhesion, and hypoxia response. These studies validate human enteroids as a model to investigate FLASH and provide further evidence supporting clinical study of this therapy. Insight Box Promising work has been done to demonstrate the potential of ultra-high dose rate radiation (FLASH) to ablate cancerous tissue, while preserving healthy tissue. While encouraging, these findings have been primarily observed using pre-clinical murine and traditional two-dimensional cell culture. This study validates the use of human enteroids as a tool to investigate human-specific tissue response to FLASH. Specifically, the work described demonstrates the ability of enteroids to recapitulate previous in vivo findings, while also providing a lens through which to probe cellular and molecular-level responses to FLASH. The human enteroids described herein offer a powerful model that can be used to probe the underlying mechanisms of FLASH in future studies.

VEGF-attenuated platelet-rich plasma improves therapeutic effect on cartilage repair.

Autologous platelet-rich plasma (PRP) has gained popularity as a less invasive treatment for various musculoskeletal tissue injuries and conditions due to its favorable safety profile, minimal manipulation and cost-effectiveness. Although PRP treatment has been clinically used for the treatment of osteoarthritis (OA) and damaged cartilage, evidence on therapeutic efficacy has been inconsistent, which calls for a methodology to achieve consistent and improved treatment outcomes. Given that PRP contains numerous proteins, we hypothesized that attenuation of a growth factor known to be detrimental to the healing tissue would enhance efficacy of PRP treatment. Considering that VEGF-mediated angiogenesis inhibits the repair of articular cartilage, we developed VEGF-attenuated PRP by sequestering VEGF in PRP using VEGF-binding microspheres. We demonstrated that VEGF attenuation in PRP did not inhibit the effect of PRP on chondrogenic differentiation of stem cells in vitro. In addition, healing of rat OA cartilage was significantly improved after treatment with VEGF-attenuated PRP when compared to the PRP treatment group or PBS control group. We expect that attenuation of unwanted biological activity using growth factor-binding microspheres could provide a new PRP customization method broadly applicable to various tissue repair processes.

Elastin-like Proteins to Support Peripheral Nerve Regeneration in Guidance Conduits.

Synthetic nerve guidance conduits (NGCs) offer an alternative to harvested nerve grafts for treating peripheral nerve injury (PNI). NGCs have been made from both naturally derived and synthesized materials. While naturally derived materials typically have an increased capacity for bioactivity, synthesized materials have better material control, including tunability and reproducibility. Protein engineering is an alternative strategy that can bridge the benefits of these two classes of materials by designing cell-responsive materials that are also systematically tunable and consistent. Here, we tested a recombinantly derived elastin-like protein (ELP) hydrogel as an intraluminal filler in a rat sciatic nerve injury model. We demonstrated that ELPs enhance the probability of forming a tissue bridge between the proximal and distal nerve stumps compared to an empty silicone conduit across the length of a 10 mm nerve gap. These tissue bridges have evidence of myelinated axons, and electrophysiology demonstrated that regenerated axons innervated distal muscle groups. Animals implanted with an ELP-filled conduit had statistically higher functional control at 6 weeks than those that had received an empty silicone conduit, as evaluated by the sciatic functional index. Taken together, our data support the conclusion that ELPs support peripheral nerve regeneration in acute complete transection injuries when used as an intraluminal filler. These results support the further study of protein engineered recombinant ELP hydrogels as a reproducible, off-the-shelf alternative for regeneration of peripheral nerves.

Engineered Matrices Enable the Culture of Human Patient-Derived Intestinal Organoids.

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.

Adipose-derived stem cells delay muscle atrophy after peripheral nerve injury in the rodent model.

INTRODUCTION: Injuries to peripheral nerves cause distal muscle atrophy. The effects of adipose-derived stem cell (ASC) injections into a muscle after injury were examined. METHODS: A 1.5 cm defect in the rat sciatic nerve was created, resulting in gastrocnemius muscle atrophy. The nerve defect was repaired with autograft; DiR-labeled ASCs were injected into the gastrocnemius immediately postoperatively. Quantitation of gross musculature and muscle fiber area, cell survival, fibrosis, lipid deposition, inflammation, and reconstructive responses were investigated. RESULTS: ASCs were identified in the muscle at 6 weeks, where injections showed increased muscle mass percentage retained, larger average fiber area, and less overall lipid content accumulated throughout the musculature. Muscles having received ASCs showed increased presence of interlukin-10 and Ki67, and decreased inducible nitric oxide synthase (iNOS). DISCUSSION: This investigation is suggestive that an ASC injection into denervated muscle post-operatively is able to delay the onset of atrophy. Muscle Nerve 59:603-603, 2019.

The Influence of Naproxen on Biological Factors in Leukocyte-Rich Platelet-Rich Plasma: A Prospective Comparative Study.

PURPOSE: To quantify and compare normative catabolic and anabolic factor concentrations in leukocyte-rich platelet-rich plasma (LR-PRP) at various time points, including baseline, 1 week after initiating naproxen use, and after a 1-week washout period. METHODS: Asymptomatic healthy donors aged between 18 and 70 years were recruited (average age, 36.6 years; range, 25-64 years). Subjects were excluded from the study if they were actively taking any prescribed medications or nonsteroidal anti-inflammatory drugs (NSAIDs) or if they had any of the following at present or previously: blood or immunosuppression disorders, cancer, osteonecrosis, rheumatoid arthritis, avascular necrosis, NSAID intolerance, gastrointestinal or peptic ulcer disease, or kidney dysfunction. The anabolic factors vascular endothelial growth factor, fibroblast growth factor 2, platelet-derived growth factor AB (PDGF-AB), and platelet-derived growth factor AA (PDGF-AA) and the catabolic factors interleukin (IL) 1ß, IL-6, IL-8, and tumor necrosis factor α in LR-PRP were measured. Peripheral blood was drawn at 3 time points: baseline, after 1 week of naproxen use, and after a 1-week washout period. RESULTS: The angiogenic factors PDGF-AA (44% decrease in median) and PDGF-AB (47% decrease) significantly declined from baseline (P < .05) after 1 week of naproxen use. There was a significant recovery (P < .05) of PDGF-AA (94% increase) and PDGF-AB (153% increase) levels after the 1-week washout period, with a return to baseline levels. The catabolic factor IL-6 also had a significant decline from baseline (77% decrease in median, P < .05) after 1 week of naproxen use. After a 1-week washout period, the IL-6 level was similar to the baseline level (130% increase, P < .05). CONCLUSIONS: Naproxen use diminished several biological factors in LR-PRP; however, a 1-week washout period was sufficient for the recovery of PDGF-AA, PDGF-AB, and IL-6 to return to baseline levels. Tumor necrosis factor α, IL-1ß, IL-8, vascular endothelial growth factor, and fibroblast growth factor 2 did not show differences between the 3 time points of data collection. Discontinuing NSAIDs for a minimum of 1 week before LR-PRP treatment may improve certain biological factor levels. LEVEL OF EVIDENCE: Level II, prospective comparative study.

Controlled dexamethasone delivery via double-walled microspheres to enhance long-term adipose tissue retention.

Current materials used for adipose tissue reconstruction have critical shortcomings such as suboptimal volume retention, donor-site morbidity, and poor biocompatibility. The aim of this study was to examine a controlled delivery system of dexamethasone to generate stable adipose tissue when mixed with disaggregated human fat in an athymic mouse model for 6 months. The hypothesis that the continued release of dexamethasone from polymeric microspheres would enhance both adipogenesis and angiogenesis more significantly when compared to the single-walled microsphere model, resulting in long-term adipose volume retention, was tested. Dexamethasone was encapsulated within single-walled poly(lactic-co-glycolic acid) microspheres (Dex SW MS) and compared to dexamethasone encapsulated in a poly(lactic-co-glycolic acid) core surrounded by a shell of poly-l-lactide. The double-walled polymer microsphere system in the second model was developed to create a more sustainable drug delivery process. Dexamethasone-loaded poly(lactic-co-glycolic acid) microspheres (Dex SW MS) and dexamethasone-loaded poly(lactic-co-glycolic acid)/poly-l-lactide double-walled microspheres (Dex DW MS) were prepared using single and double emulsion/solvent techniques. In vitro release kinetics were determined. Two doses of each type of microsphere were examined; 50 and 27 mg of Dex MS and Dex DW MS were mixed with 0.3 mL of human lipoaspirate. Additionally, 50 mg of empty MS and lipoaspirate-only controls were examined. Samples were analyzed grossly and histologically after 6 months in vivo. Mass and volume were measured; dexamethasone microsphere-containing samples demonstrated greater adipose tissue retention compared to the control group. Histological analysis, including hematoxylin and eosin and CD31 staining, indicated increased vascularization (p < 0.05) within the Dex MS-containing samples. Controlled delivery of adipogenic factors, such as dexamethasone via polymer microspheres, significantly affects adipose tissue retention by maintaining healthy tissue formation and vascularization. Dex DW MS provide an improved model to former Dex SW MS, resulting in notably longer release time and, consequently, larger volumes of adipose retained in vivo. The use of microspheres, specifically double-walled, as vehicles for controlled drug delivery of adipogenic factors therefore present a clinically relevant model of adipose retention that has the potential to greatly improve soft tissue repair.