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Tumour immune evasion presents a significant challenge to the effectiveness of cancer immunotherapies. Recent advances in high-throughput screening techniques have uncovered that loss of antigen presentation and cytokine signalling pathways are central mechanisms by which tumours evade T cell immunity. To uncover additional vulnerabilities in tumour cells beyond the well-recognized antigen presentation pathway, we conducted a genome-wide CRISPR/Cas9 screen to identify genes that mediate resistance to chimeric-antigen receptor (CAR)-T cells, which function independently of classical antigen presentation. Our study revealed that loss of core-binding factor subunit beta (CBFβ) enhances tumour cell resistance to T cell killing, mediated through T cell-derived TNF. Mechanistically, RNA-sequencing and elemental analyses revealed that deletion of CBFβ disrupts numerous pathways including those involved in zinc homoeostasis. Moreover, we demonstrated that modulation of cellular zinc, achieved by supplementation or chelation, significantly altered tumour cell susceptibility to TNF by regulating the levels of inhibitor of apoptosis proteins. Consistent with this, treatment of tumour cells with a membrane-permeable zinc chelator had no impact on tumour cell viability alone, but significantly increased tumour cell lysis by CD8+ T cells in a TNF-dependent but perforin-independent manner. These results underscore the crucial role of intracellular zinc in regulating tumour cell susceptibility to T cell-mediated killing, revealing a novel vulnerability in tumour cells that might be exploited for the development of future cancer immunotherapeutics., Competing Interests: Competing interests: The authors declare no competing interests. Ethics approval and consent to participate: All animal studies were performed in accordance with the NHMRC Australian Code for the Care and Use of Animals for Scientific Purposes 8th edition (2013) and with approval from the Peter MacCallum Cancer Centre Animal Experimentation Ethics Committee (Ethics approvals E548, E638, 2023-17)., (© 2024. The Author(s).)

Despite the clinical success of cancer immunotherapies including immune checkpoint blockade and adoptive cellular therapies across a variety of cancer types, many patients do not respond or ultimately relapse; however, the molecular underpinnings of this are not fully understood. Thus, a system-level understating of the routes to tumor immune evasion is required to inform the design of the next generation of immunotherapy approaches. CRISPR screening approaches have proved extremely powerful in identifying genes that promote tumor immune evasion or sensitize tumor cells to destruction by the immune system. These large-scale efforts have brought to light decades worth of fundamental immunology and have uncovered the key immune-evasion pathways subverted in cancers in an acquired manner in patients receiving immune-modulatory therapies. The comprehensive discovery of the main pathways involved in immune evasion has spurred the development and application of novel immune therapies to target this process. Although successful, conventional CRISPR screening approaches are hampered by a number of limitations, which obfuscate a complete understanding of the precise molecular regulation of immune evasion in cancer. Here, we provide a perspective on screening approaches to interrogate tumor-lymphocyte interactions and their limitations, and discuss further development of technologies to improve such approaches and discovery capability., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Cancer immunotherapies have demonstrated remarkable success; however, the majority of patients do not respond or develop resistance. Here, we conduct epigenetic gene-targeted CRISPR-Cas9 screens to identify epigenomic factors that limit CD8 + T cell-mediated anti-tumor immunity. We identify that PRMT1 suppresses interferon gamma (Ifnγ)-induced MHC-I expression, thus dampening CD8 + T cell-mediated killing. Indeed, PRMT1 knockout or pharmacological targeting of type I PRMT with the clinical inhibitor GSK3368715 enhances Ifnγ-induced MHC-I expression through elevated STAT1 expression and activation, while re-introduction of PRMT1 in PRMT1-deficient cells reverses this effect. Importantly, loss of PRMT1 enhances the efficacy of anti-PD-1 immunotherapy, and The Cancer Genome Atlas analysis reveals that PRMT1 expression in human melanoma is inversely correlated with expression of human leukocyte antigen molecules, infiltration of CD8 + T cells, and overall survival. Taken together, we identify PRMT1 as a negative regulator of anti-tumor immunity, unveiling clinical type I PRMT inhibitors as immunotherapeutic agents or as adjuncts to existing immunotherapies., Competing Interests: Declaration of interests S.J.H. is an employee of AbbVie and holds stock in the company. R.W.J. receives research support from Roche, BMS, Astra-Zeneca, Pfizer, and MecRx and is a scientific consultant and shareholder in MecRx., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

CXCL9 expression is a strong predictor of response to immune checkpoint blockade therapy. Accordingly, we sought to develop therapeutic strategies to enhance the expression of CXCL9 and augment antitumor immunity. To perform whole-genome CRISPR-Cas9 screening for regulators of CXCL9 expression, a CXCL9-GFP reporter line is generated using a CRISPR knockin strategy. This approach finds that IRF1 limits CXCL9 expression in both tumor cells and primary myeloid cells through induction of SOCS1, which subsequently limits STAT1 signaling. Thus, we identify a subset of STAT1-dependent genes that do not require IRF1 for their transcription, including CXCL9. Targeting of either IRF1 or SOCS1 potently enhances CXCL9 expression by intratumoral macrophages, which is further enhanced in the context of immune checkpoint blockade therapy. We hence show a non-canonical role for IRF1 in limiting the expression of a subset of STAT1-dependent genes through induction of SOCS1., Competing Interests: Declaration of interests P.A.B. declares the following conflicts: research funding from AstraZeneca, Bristol-Myers-Squibb, and Gilead Sciences. P.K.D. declares the following conflicts: research funding from Myeloid Therapeutics, Prescient Therapeutics, Bristol-Myers-Squibb, and Juno Therapeutics., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Applications involving ultrasound treatment as a therapeutic strategy have gained interest due to its enhanced tissue penetration, broad availability, and minimal invasiveness. Recently, ultrasound treatment has been utilized for applications such as controlled drug delivery, enhanced drug penetration, sonodynamic therapy for generating ROS species, and targeted tissue ablation. However, our ability to study and explore applications is limited by the lack of in vitro models that enable efficient and representative screening of ultrasound-based therapeutic strategies. There is a need for cell culture approaches that mimic the mechanical environment of native tissues, which can prevent uncontrolled cell lysis due to ultrasonic energy. We developed two-dimensional and three-dimensional collagen-based materials for culturing cells in vitro that withstand ultrasound treatment. We hypothesized that the collagen matrix mimics the extracellular matrix and absorb most of the energy from ultrasound treatment - similar to in vivo effects - thereby preventing uncontrolled cell lysis. In this study, we developed a strategy for fabricating both the 2D coatings and 3D hydrogels coatings and tested the viability of the cultured cells post different durations of ultrasound treatment., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 Published by Elsevier B.V.)

Bacterial biofilms are a major healthcare concern resulting in refractory conditions such as chronic wounds, implant infections and failure, and multidrug-resistant infections. Aggressive and invasive strategies are employed to cure biofilm infections but are prone to long and expensive treatments, adverse side-effects, and low patient compliance. Recent strategies such as ultrasound-based therapies and antimicrobial nanomaterials have shown some promise in the effective eradication of biofilms. However, maximizing therapeutic effect while minimizing healthy tissue damage is a key challenge that needs to be addressed. Here a combination treatment involving ultrasound and antimicrobial polymeric nanoparticles (PNPs) that synergistically eradicate bacterial biofilms is reported. Ultrasound treatment rapidly disrupts biofilms and increases penetration of antimicrobial PNPs thereby enhancing their antimicrobial activity. This results in superior biofilm toxicity, while allowing for a two- to sixfold reduction in both the concentration of PNPs as well as the duration of ultrasound. Furthermore, that this reduction minimizes cytotoxicity toward fibroblast cells, while resulting in a 100- to 1000-fold reduction in bacterial concentration, is demonstrated., (© 2022 Wiley-VCH GmbH.)

Diabetic foot ulcers (DFU) are chronic wounds sustained by pathological fibroblasts and aberrant extracellular matrix (ECM). Porous collagen-based scaffolds (CS) have shown clinical promise for treating DFUs but may benefit from functional enhancements. Our previous work showed fibroblasts differentiated from induced pluripotent stem cells are an effective source of new ECM mimicking fetal matrix, which notably promotes scar-free healing. Likewise, functionalizing CS with this rejuvenated ECM showed potential for DFU healing. Here, we demonstrate for the first time an approach to DFU healing using biopsied cells from DFU patients, reprogramming those cells, and functionalizing CS with patient-specific ECM as a personalized acellular tissue engineered scaffold. We took a two-pronged approach: 1) direct ECM blending into scaffold fabrication; and 2) seeding scaffolds with reprogrammed fibroblasts for ECM deposition followed by decellularization. The decellularization approach reduced cell number requirements and maintained naturally deposited ECM proteins. Both approaches showed enhanced ECM deposition from DFU fibroblasts. Decellularized scaffolds additionally enhanced glycosaminoglycan deposition and subsequent vascularization. Finally, reprogrammed ECM scaffolds from patient-matched DFU fibroblasts outperformed those from healthy fibroblasts in several metrics, suggesting ECM is in fact able to redirect resident pathological fibroblasts in DFUs towards healing, and a patient-specific ECM signature may be beneficial.

The mechanism of action of eprenetapopt (APR-246, PRIMA-1 MET ) as an anticancer agent remains unresolved, although the clinical development of eprenetapopt focuses on its reported mechanism of action as a mutant-p53 reactivator. Using unbiased approaches, this study demonstrates that eprenetapopt depletes cellular antioxidant glutathione levels by increasing its turnover, triggering a nonapoptotic, iron-dependent form of cell death known as ferroptosis. Deficiency in genes responsible for supplying cancer cells with the substrates for de novo glutathione synthesis ( SLC7A11 , SHMT2 , and MTHFD1L ), as well as the enzymes required to synthesize glutathione ( GCLC and GCLM ), augments the activity of eprenetapopt. Eprenetapopt also inhibits iron-sulfur cluster biogenesis by limiting the cysteine desulfurase activity of NFS1, which potentiates ferroptosis and may restrict cellular proliferation. The combination of eprenetapopt with dietary serine and glycine restriction synergizes to inhibit esophageal xenograft tumor growth. These findings reframe the canonical view of eprenetapopt from a mutant-p53 reactivator to a ferroptosis inducer.

Background: Interferon gamma (IFNγ) is a pro-inflammatory cytokine that directly activates the JAK/STAT pathway. However, the temporal dynamics of chromatin remodeling and transcriptional activation initiated by IFNγ have not been systematically profiled in an unbiased manner. Herein, we integrated transcriptomic and epigenomic profiling to characterize the acute epigenetic changes induced by IFNγ stimulation in a murine breast cancer model., Results: We identified de novo activation of cis-regulatory elements bound by Irf1 that were characterized by increased chromatin accessibility, differential usage of pro-inflammatory enhancers, and downstream recruitment of BET proteins and RNA polymerase II. To functionally validate this hierarchical model of IFNγ-driven transcription, we applied selective antagonists of histone acetyltransferases P300/CBP or acetyl-lysine readers of the BET family. This highlighted that histone acetylation is an antecedent event in IFNγ-driven transcription, whereby targeting of P300/CBP acetyltransferase activity but not BET inhibition could curtail the epigenetic remodeling induced by IFNγ through suppression of Irf1 transactivation., Conclusions: These data highlight the ability for epigenetic therapies to reprogram pro-inflammatory gene expression, which may have therapeutic implications for anti-tumor immunity and inflammatory diseases., (© 2022. The Author(s).)

Cytotoxic lymphocytes are essential for anti-tumor immunity, and for effective responses to cancer immunotherapy. Natural killer cell granule protein 7 (NKG7) is expressed at high levels in cytotoxic lymphocytes infiltrating tumors from patients treated with immunotherapy, but until recently, the role of this protein in cytotoxic lymphocyte function was largely unknown. Unexpectedly, we found that highly CD8+ T cell-immunogenic murine colon carcinoma (MC38-OVA) tumors grew at an equal rate in Nkg7 +/+ and Nkg7 -/- littermate mice, suggesting NKG7 may not be necessary for effective CD8+ T cell anti-tumor activity. Mechanistically, we found that deletion of NKG7 reduces the ability of CD8+ T cells to degranulate and kill target cells in vitro . However, as a result of inefficient cytotoxic activity, NKG7 deficient T cells form a prolonged immune synapse with tumor cells, resulting in increased secretion of inflammatory cytokines, including tumor necrosis factor alpha (TNF). By deleting the TNF receptor, TNFR1, from MC38-OVA tumors, we demonstrate that this hyper-secretion of TNF compensates for reduced synapse-mediated cytotoxic activity against MC38-OVA tumors in vivo , via increased TNF-mediated tumor cell death. Taken together, our results demonstrate that NKG7 enhances CD8+ T cell immune synapse efficiency, which may serve as a mechanism to accelerate direct cytotoxicity and limit potentially harmful inflammatory responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lelliott, Ramsbottom, Dowling, Shembrey, Noori, Kearney, Michie, Parish, Jordan, Baxter, Young, Brennan and Oliaro.)

Pharmacologic inhibition of epigenetic enzymes can have therapeutic benefit against hematologic malignancies. In addition to affecting tumor cell growth and proliferation, these epigenetic agents may induce antitumor immunity. Here, we discovered a novel immunoregulatory mechanism through inhibition of histone deacetylases (HDAC). In models of acute myeloid leukemia (AML), leukemia cell differentiation and therapeutic benefit mediated by the HDAC inhibitor (HDACi) panobinostat required activation of the type I interferon (IFN) pathway. Plasmacytoid dendritic cells (pDC) produced type I IFN after panobinostat treatment, through transcriptional activation of IFN genes concomitant with increased H3K27 acetylation at these loci. Depletion of pDCs abrogated panobinostat-mediated induction of type I IFN signaling in leukemia cells and impaired therapeutic efficacy, whereas combined treatment with panobinostat and IFNα improved outcomes in preclinical models. These discoveries offer a new therapeutic approach for AML and demonstrate that epigenetic rewiring of pDCs enhances antitumor immunity, opening the possibility of exploiting this approach for immunotherapies., Significance: We demonstrate that HDACis induce terminal differentiation of AML through epigenetic remodeling of pDCs, resulting in production of type I IFN that is important for the therapeutic effects of HDACis. The study demonstrates the important functional interplay between the immune system and leukemias in response to HDAC inhibition. This article is highlighted in the In This Issue feature, p. 1397., (©2022 The Authors; Published by the American Association for Cancer Research.)

Poly(globalide) (PGl), an aliphatic polyester derived from unsaturated macrocylic lactone, can be cross-linked during electrospinning and drug-loaded for regenerative medicine applications. However, it lacks intrinsic recognition sites for cell adhesion and proliferation. In order to improve their cell adhesiveness, and therefore their therapeutic potential, we aimed to functionalize electrospun PGl fibers with RGD sequence generating a biomimetic scaffold. First, an amine compound was attached to the surface double bonds of the PGl fibers. Subsequently, the amino groups were coupled with RGD sequences. X-ray photoelectron spectroscopy (XPS) analysis confirmed the functionalization. The obtained fibers were more hydrophilic, as observed by contact angle analysis, and presented smaller Young's modulus, although similar tensile strength compared with non-functionalized cross-linked fibers. In addition, the functionalization process did not significantly alter fibers morphology, as observed by scanning electron microscopy (SEM). Finally, in vitro analysis evidenced the increase in human mesenchymal stromal cells (hMSC) adhesion (9.88 times higher DNA content after 1 day of culture) and proliferation (3.57 times higher DNA content after 8 days of culture) compared with non-functionalized non-cross-linked fibers. This is the first report demonstrating the functionalization of PGl fibers with RGD sequence, improving PGl therapeutic potential and further corroborating the use of this highly versatile material toward regenerative medicine applications., (© 2021 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

Targeting chromatin binding proteins and modifying enzymes can concomitantly affect tumor cell proliferation and survival, as well as enhance antitumor immunity and augment cancer immunotherapies. By screening a small-molecule library of epigenetics-based therapeutics, BET (bromo- and extra-terminal domain) inhibitors (BETi) were identified as agents that sensitize tumor cells to the antitumor activity of CD8 + T cells. BETi modulated tumor cells to be sensitized to the cytotoxic effects of the proinflammatory cytokine TNF. By preventing the recruitment of BRD4 to p65-bound cis -regulatory elements, BETi suppressed the induction of inflammatory gene expression, including the key NF-κB target genes BIRC2 (cIAP1) and BIRC3 (cIAP2). Disruption of prosurvival NF-κB signaling by BETi led to unrestrained TNF-mediated activation of the extrinsic apoptotic cascade and tumor cell death. Administration of BETi in combination with T-cell bispecific antibodies (TCB) or immune-checkpoint blockade increased bystander killing of tumor cells and enhanced tumor growth inhibition in vivo in a TNF-dependent manner. This novel epigenetic mechanism of immunomodulation may guide future use of BETi as adjuvants for immune-oncology agents., (©2021 American Association for Cancer Research.)

Tissue engineering solutions have been widely explored for enhanced healing of skin wounds. Diabetic foot ulcers (DFU) are particularly challenging wounds to heal for a variety of reasons, including aberrant ECM, dysregulation of vascularization, and persistent inflammation. Tissue engineering approaches, such as porous collagen-based scaffolds, have shown promise in replacing the current treatments of surgical debridement and topical treatments. Collagen-glycosaminoglycan scaffolds, which are FDA approved for diabetic foot ulcers, can benefit from further functionalization by incorporation of additional signaling factors or extracellular matrix molecules. One option for this is to incorporate matrix from a rejuvenated cell source, as wounds in younger patients heal more quickly. Induced pluripotent stem cells (iPS) are generated from somatic cells and share many functional similarities with embryonic stem cells (ES), while avoiding the ethical concerns. Fibroblasts differentiated from iPS cells have been shown to enrich their ECM with glycosaminoglycan (GAGs), collagen Type III and fibronectin, to have an increased ECM production, and to be pro-angiogenic. Here we describe a technique to grow matrix from post-iPS fibroblasts, and to develop a scaffold from this matrix, in combination with collagen, with the goal of enhancing wound healing. By activating scaffolds with extracellular matrix (ECM) from fibroblasts derived from an iPS source (post-iPSF), the scaffolds are enriched with beneficial elements like GAGs, collagen type III, fibronectin, and VEGF. We believe these scaffolds can enhance skin regeneration and that the techniques can be modified for other tissue engineering applications., (© 2021. Springer Science+Business Media, LLC.)

Tumor necrosis factor (TNF) is a proinflammatory cytokine that is produced and secreted by cytotoxic lymphocytes upon tumor target recognition. Depending on the context, TNF can mediate either pro-survival or pro-death signals. The potential cytotoxicity of T cell-produced TNF, particularly in the context of T cell-directed immunotherapies, has been largely overlooked. However, a spate of recent studies investigating tumor immune evasion through the application of CRISPR-based gene-editing screens have highlighted TNF-mediated killing as an important component of the mammalian T cell antitumor repertoire. In the context of the current understanding of the role of TNF in antitumor immunity, we discuss these studies and touch on their therapeutic implications. Collectively, we provide an enticing prospect to augment immunotherapy responses through TNF cytotoxicity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The success of cancer immunotherapy is limited to a subset of patients, highlighting the need to identify the processes by which tumors evade immunity. Using CRISPR/Cas9 screening, we reveal that melanoma cells lacking HOIP, the catalytic subunit of LUBAC, are highly susceptible to both NK and CD8 + T-cell-mediated killing. We demonstrate that HOIP-deficient tumor cells exhibit increased sensitivity to the combined effect of the inflammatory cytokines, TNF and IFN-γ, released by NK and CD8 + T cells upon target recognition. Both genetic deletion and pharmacological inhibition of HOIP augment tumor cell sensitivity to combined TNF and IFN-γ. Together, we unveil a protective regulatory axis, involving HOIP, which limits a transcription-dependent form of cell death that engages both intrinsic and extrinsic apoptotic machinery upon exposure to TNF and IFN-γ. Our findings highlight HOIP inhibition as a potential strategy to harness and enhance the killing capacity of TNF and IFN-γ during immunotherapy., (© 2021 The Authors.)

Pharmacologic inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) are an approved treatment for hormone receptor-positive breast cancer and are currently under evaluation across hundreds of clinical trials for other cancer types. The clinical success of these inhibitors is largely attributed to well-defined tumor-intrinsic cytostatic mechanisms, whereas their emerging role as immunomodulatory agents is less understood. Using integrated epigenomic, transcriptomic, and proteomic analyses, we demonstrated a novel action of CDK4/6 inhibitors in promoting the phenotypic and functional acquisition of immunologic T-cell memory. Short-term priming with a CDK4/6 inhibitor promoted long-term endogenous antitumor T-cell immunity in mice, enhanced the persistence and therapeutic efficacy of chimeric antigen receptor T cells, and induced a retinoblastoma-dependent T-cell phenotype supportive of favorable responses to immune checkpoint blockade in patients with melanoma. Together, these mechanistic insights significantly broaden the prospective utility of CDK4/6 inhibitors as clinical tools to boost antitumor T-cell immunity. SIGNIFICANCE: Immunologic memory is critical for sustained antitumor immunity. Our discovery that CDK4/6 inhibitors drive T-cell memory fate commitment sheds new light on their clinical activity, which is essential for the design of clinical trial protocols incorporating these agents, particularly in combination with immunotherapy, for the treatment of cancer. This article is highlighted in the In This Issue feature, p. 2355 ., (©2021 American Association for Cancer Research.)

Extracellular matrix is a key component of all tissues, including skin and it plays a crucial role in the complex events of wound healing. These events are impaired in chronic wounds, with chronic inflammation and infection often present in these non-healing wounds. Many tissue engineering approaches for wound healing provide a scaffold to mimic the native matrix. Fibroblasts derived from iPS cells (iPSF) represent a novel source of matrix rich in pro-regenerative components, which can be used for scaffold fabrication to improve wound healing. However, in vitro production of matrix by cells for scaffold fabrication requires long cell culturing times which increases cost. The aim of this work is to optimize the iPSF matrix production by boosting matrix deposition, without affecting its composition. A good candidate technique to achieve this goal is macromolecular crowding, which is known to promote conversion of procollagen into mature collagen and its accumulation. We tested two molecular crowders, Ficoll and Carrageenan-in combination with ascorbic acid-over a prolonged period of time. Ficoll in combination with ascorbic acid notably increased collagen deposition and matrix dry weight compared to ascorbic acid alone, and did not affect matrix composition as measured by RT-PCR. Interestingly, Carrageenan did not affect collagen quantity, but it significantly increased glycosaminoglycan deposition. Finally, we successfully fabricated scaffolds from harvested matrix and confirmed their ability for cell growth and viability. This work lays the foundation for development of a time and cost effective protocol for novel iPSF ECM production for tissue engineering scaffolds., (© 2021 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

The function of MR1-restricted mucosal-associated invariant T (MAIT) cells in tumor immunity is unclear. Here we show that MAIT cell-deficient mice have enhanced NK cell-dependent control of metastatic B16F10 tumor growth relative to control mice. Analyses of this interplay in human tumor samples reveal that high expression of a MAIT cell gene signature negatively impacts the prognostic significance of NK cells. Paradoxically, pre-pulsing tumors with MAIT cell antigens, or activating MAIT cells in vivo, enhances anti-tumor immunity in B16F10 and E0771 mouse tumor models, including in the context of established metastasis. These effects are associated with enhanced NK cell responses and increased expression of both IFN-γ-dependent and inflammatory genes in NK cells. Importantly, activated human MAIT cells also promote the function of NK cells isolated from patient tumor samples. Our results thus describe an activation-dependent, MAIT cell-mediated regulation of NK cells, and suggest a potential therapeutic avenue for cancer treatment., (© 2021. Crown.)

Hydrogels are perfectly suited to support cell and tissue growth in advanced tissue engineering applications as well as classical wound treatment scenarios. Ideal hydrogel materials for these applications should be easy to produce, biocompatible, resorbable and antimicrobial. Here we report the fabrication of degradable covalent antimicrobial lysine and tryptophan containing copolypeptide hydrogels, whereby the hydrogel properties can be independently modulated by the copolypeptide monomer ratio and chiral composition. Well-defined statistical copolypeptides comprising different overall molecular weights as well as ratios of l- and d-lysine and tryptophan at ratios of 35 : 15, 70 : 30 and 80 : 20 were obtained by N-carboxyanhydride (NCA) polymerisation and subsequently crosslinked by the selective reaction of bifunctional triazolinedione (TAD) with tryptophan. Real-time rheology was used to monitor the crosslinking reaction recording the fastest increase and overall modulus for copolypeptides with the higher tryptophan ratio. Water uptake of cylindrical hydrogel samples was dependent on crosslinking ratio but found independent of chiral composition, while enzymatic degradation proceeded significantly faster for samples containing more l-amino acids. Antimicrobial activity on a range of hydrogels containing different polypeptide chain lengths, lysine/tryptophan composition and l/d enantiomers was tested against reference laboratory strains of Gram-negative Escherichia coli (E. coli; ATCC25922) and Gram-positive, Staphylococcus aureus (S. aureus; ATCC25923). log reductions of 2.8-3.4 were recorded for the most potent hydrogels. In vitro leachable cytotoxicity tests confirmed non-cytotoxicity as per ISO guidelines.

Natural bioactive cue profiles are generally transient with cues switching on/off to coordinate successful outcomes. Dysregulation of these sequences typically leads to disease. Successful wound healing, for example, should progress sequentially through hemostasis, inflammation, granulation tissue formation, and maturation. Chronic wounds, such as diabetic foot ulcers, suffer from uncoordinated signaling, and arrest and cycle between the inflammation and granulation stages. Traditionally, therapeutic delivery in tissue engineering has focused on sustaining delivery of key signaling factors; however, temporal and sequential delivery have increasingly come into focus. To fully take advantage of these signaling systems, a scaffold or matrix material that can house the delivery system is desirable. In this work, we functionalized a collagen-based scaffold - which has proven regenerative potential in wounds - with on-demand delivery of nanoparticles. Building on our previous work with ultrasound-responsive alginate that shows near-zero baseline release and a rapid release in response to an ultrasound trigger, we developed two novel scaffolds. In the first version, homogeneously-distributed microparticles of alginate were incorporated within the collagen-glycosaminoglycan (GAG) scaffold; ultrasound-triggered release of platelet derived growth factor (PDGF) loaded gold nanoparticles was demonstrated; and their maintained bioactivity confirmed. In the second version, pockets of alginate that can be individually loaded and triggered with ultrasound, were incorporated. The ability to sequentially release multiple therapeutics within these scaffolds using ultrasound was successfully confirmed. These platforms offer a precise and versatile way to deliver therapeutic nanoparticles within a proven regenerative template, and can be used to deliver and probe timed therapeutic delivery in wound healing and other tissue engineering applications.

Gene expression by RNA polymerase II (RNAPII) is tightly controlled by cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The pausing checkpoint following transcription initiation is primarily controlled by CDK9. We discovered that CDK9-mediated, RNAPII-driven transcription is functionally opposed by a protein phosphatase 2A (PP2A) complex that is recruited to transcription sites by the Integrator complex subunit INTS6. PP2A dynamically antagonizes phosphorylation of key CDK9 substrates including DSIF and RNAPII-CTD. Loss of INTS6 results in resistance to tumor cell death mediated by CDK9 inhibition, decreased turnover of CDK9 phospho-substrates, and amplification of acute oncogenic transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill both leukemic and solid tumor cells, providing therapeutic benefit in vivo. These data demonstrate that fine control of gene expression relies on the balance between kinase and phosphatase activity throughout the transcription cycle, a process dysregulated in cancer that can be exploited therapeutically., Competing Interests: Declaration of interests The Johnstone laboratory receives funding support from Roche, BMS, Astra Zeneca, and MecRx. R.W.J. is a shareholder in MecRx and receives consultancy payments., (Copyright © 2021 Elsevier Inc. All rights reserved.)

To separate causal effects of histone acetylation on chromatin accessibility and transcriptional output, we used integrated epigenomic and transcriptomic analyses following acute inhibition of major cellular lysine acetyltransferases P300 and CBP in hematological malignancies. We found that catalytic P300/CBP inhibition dynamically perturbs steady-state acetylation kinetics and suppresses oncogenic transcriptional networks in the absence of changes to chromatin accessibility. CRISPR-Cas9 screening identified NCOR1 and HDAC3 transcriptional co-repressors as the principal antagonists of P300/CBP by counteracting acetylation turnover kinetics. Finally, deacetylation of H3K27 provides nucleation sites for reciprocal methylation switching, a feature that can be exploited therapeutically by concomitant KDM6A and P300/CBP inhibition. Overall, this study indicates that the steady-state histone acetylation-methylation equilibrium functions as a molecular rheostat governing cellular transcription that is amenable to therapeutic exploitation as an anti-cancer regimen., Competing Interests: Declaration of interests The Johnstone laboratory receives funding support from Roche, Bristol Myers Squibb (BMS), AstraZeneca, and MecRx. R.W.J. is a shareholder in and consultant for MecRx. A.L. and K.D.B. are employees of and shareholders in AbbVie. A.S. has participated on advisory boards for and received research funding from Celgene, Juno, BMS, Janssen-Cilag, Novartis, Amgen, Haemalogix, Abbvie, and Takeda. J.S. has participated on advisory boards for and received honoraria from Celgene. O.A.-W. has served as a consultant for H3B Biomedicine, Foundation Medicine Inc., Merck, Prelude Therapeutics, and Janssen; is on the scientific advisory boards of Envisagenics Inc., Pfizer Boulder, and AIChemy Inc.; and has received prior research funding from Loxo Oncology and H3 Biomedicine. J.D.L. is a scientific adviser to the Samuel Waxman Cancer Research Foundation. All other authors declare no competing interests., (Crown Copyright © 2021. Published by Elsevier Inc. All rights reserved.)

Background: The incidence and prevalence of pressure ulcers in critically ill patients in intensive care units (ICUs) remain high, despite the wealth of knowledge on appropriate prevention strategies currently available., Methods: The primary objective of this systematic review was to examine the economic impact of pressure ulcers (PU) among adult intensive care patients. A systematic review was undertaken, and the following databases were searched; Medline, Embase, CINAHL, and The Cochrane Library. Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines was used to formulate the review. Quality appraisal was undertaken using the Consensus on Health Economic Criteria (CHEC)-list. Data were extracted using a pre-designed extraction tool, and a narrative analysis was undertaken., Results: Seven studies met the inclusion criteria. Five reported costs associated with the prevention of pressure ulcers and three explored costs of treatment strategies. Four main PU prevention cost items were identified: support surfaces, dressing materials, staff costs, and costs associated with mobilisation. Seven main PU treatment cost items were reported: dressing materials, support surfaces, drugs, surgery, lab tests, imaging, additional stays and nursing care. The overall validities of the studies varied between 37 and 79%, meaning that there is potential for bias within all the included studies., Conclusion: There was a significant difference in the cost of PU prevention and treatment strategies between studies. This is problematic as it becomes difficult to accurately evaluate costs from the existing literature, thereby inhibiting the usefulness of the data to inform practice. Given the methodological heterogeneity among studies, future studies in this area are needed and these should use specific methodological guidelines to generate high-quality health economic studies., (Copyright © 2020 Tissue Viability Society. Published by Elsevier Ltd. All rights reserved.)

Multimodal single-cell RNA sequencing enables the precise mapping of transcriptional and phenotypic features of cellular differentiation states but does not allow for simultaneous integration of critical posttranslational modification data. Here, we describe SUrface-protein Glycan And RNA-seq (SUGAR-seq), a method that enables detection and analysis of N-linked glycosylation, extracellular epitopes, and the transcriptome at the single-cell level. Integrated SUGAR-seq and glycoproteome analysis identified tumor-infiltrating T cells with unique surface glycan properties that report their epigenetic and functional state., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Tissue engineering products, like collagen-glycosaminoglycan scaffolds, have been successfully applied to chondrogenic defects. Inducible Pluripotent Stem cell (iPS) technology allows reprograming of somatic cells into an embryonic-like state, allowing for redifferentiation. We postulated that a fibroblast cell line (BJ cells - 'pre-iPSF') cycled through iPS reprogramming and redifferentiated into fibroblasts (post-iPSF) could lubricate collagen-glycosaminoglycan scaffolds; fibroblasts are known to produce lubricating molecules (e.g., lubricin) in the synovium. Herein, we quantified the coefficient of friction (CoF) of collagen-glycosaminoglycan scaffolds seeded with post-iPSF; tested whether cell-free scaffolds made of post-iPSF derived extracellular matrix had reduced friction vs. pre-iPSF; and assessed lubricin quantity as a possible protein responsible for lubrication. Post-iPSF seeded CG had 6- to 10-fold lower CoF versus pre-iPSF. Scaffolds consisting of a collagen and pre-/post-iPSF extracellular matrix blend outperformed these cell-seeded scaffolds (~5-fold lower CoF), yielding excellent CoF values close to synovial fluid. Staining revealed an increased presence of lubricin within post-iPSF scaffolds (confirmed by western blotting) and on the surface of iPSF-seeded collagen-glycosaminoglycan scaffolds. Interestingly, when primary cells from patient biopsy-derived fibroblasts were used, iPS reprogramming did not further reduce the already low CoF of these cells and no lubricin expression was found. We conclude that iPS reprogramming activates lubricating properties in iPS-derived cells in a source cell-specific manner. Additionally, lubricin appears to play a lubricating role, yet other proteins also contribute to lubrication. This work constitutes an important step for understanding post-iPSF lubrication of scaffolds and its potential for cartilage tissue engineering., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Commercial cell-based skin regenerative products are highly expensive, carry the risk of rejection and require a long cell culture period to manufacture. This work describes the synthesis of bilayer films from poly(globalide) (PGl) and regenerated cellulose nanofibers (rCNFs) and their use as a cell-free scaffold to support keratinocyte attachment and proliferation. The method is simple, eco-friendly (as the cellulose precursor is obtained from agricultural waste) and of low cost. The rCNFs were produced by acid hydrolysis and PGl was obtained via enzymatic ring-opening polymerization. The bilayer films were synthesized by layer-by-layer casting at ambient temperature. All the films showed a well-defined interface between PGl and cellulose. The produced rCNF/PGl bilayer films showed cell metabolic activity far superior in comparison with pristine PGl regarding the keratinocyte growth, which illustrates the potential use of these materials in skin tissue engineering., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The circadian clock is a collection of endogenous oscillators with a periodicity of ~ 24 h. Recently, our understanding of circadian rhythms and their regulation at genomic and physiologic scales has grown significantly. Knowledge of the circadian influence on biological processes has provided new possibilities for novel pharmacological strategies. Directly targeting the biological clock or its downstream targets, and/or using timing as a variable in drug therapy are now important pharmacological considerations. The circadian machinery mediates many aspects of the inflammatory response and, reciprocally, an inflammatory environment can disrupt circadian rhythms. Therefore, intense interest exists in leveraging circadian biology as a means to treat chronic inflammatory diseases such as sepsis, asthma, rheumatoid arthritis, osteoarthritis, and cardiovascular disease, which all display some type of circadian signature. The purpose of this review is to evaluate the crosstalk between circadian rhythms, inflammatory diseases, and their pharmacological treatment. Evidence suggests that carefully rationalized application of chronotherapy strategies - alone or in combination with small molecule modulators of circadian clock components - can improve efficacy and reduce toxicity, thus warranting further investigation and use., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Diabetic foot ulcers (DFUs) are chronic wounds, with 20% of cases resulting in amputation, despite intervention. A recently approved tissue engineering product-a cell-free collagen-glycosaminoglycan (GAG) scaffold-demonstrates 50% success, motivating its functionalization with extracellular matrix (ECM). Induced pluripotent stem cell (iPSC) technology reprograms somatic cells into an embryonic-like state. Recent findings describe how iPSCs-derived fibroblasts ("post-iPSF") are proangiogenic, produce more ECM than their somatic precursors ("pre-iPSF"), and their ECM has characteristics of foetal ECM (a wound regeneration advantage, as fetuses heal scar-free). ECM production is 45% higher from post-iPSF and has favorable components (e.g., Collagen I and III, and fibronectin). Herein, a freeze-dried scaffold using ECM grown by post-iPSF cells (Post-iPSF Coll) is developed and tested vs precursors ECM-activated scaffolds (Pre-iPSF Coll). When seeded with healthy or DFU fibroblasts, both ECM-derived scaffolds have more diverse ECM and more robust immune responses to cues. Post-iPSF-Coll had higher GAG, higher cell content, higher Vascular Endothelial Growth Factor (VEGF) in DFUs, and higher Interleukin-1-receptor antagonist (IL-1ra) vs. pre-iPSF Coll. This work constitutes the first step in exploiting ECM from iPSF for tissue engineering scaffolds., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

CRISPR/Cas9 technologies have revolutionized our understanding of gene function in complex biological settings, including T cell immunology. Current CRISPR-mediated gene editing strategies in T cells require in vitro stimulation or culture that can both preclude the study of unmanipulated naive T cells and alter subsequent differentiation. In this study, we demonstrate highly efficient gene editing within uncultured primary naive murine CD8 + T cells by electroporation of recombinant Cas9/sgRNA ribonucleoprotein immediately prior to in vivo adoptive transfer. Using this approach, we generated single and double gene knockout cells within multiple mouse infection models. Strikingly, gene deletion occurred even when the transferred cells were left in a naive state, suggesting that gene deletion occurs independent of T cell activation. Finally, we demonstrate that targeted mutations can be introduced into naive CD8 + T cells using CRISPR-based homology-directed repair. This protocol thus expands CRISPR-based gene editing approaches beyond models of robust T cell activation to encompass both naive T cell homeostasis and models of weak activation, such as tolerance and tumor models., (Copyright © 2020 by The American Association of Immunologists, Inc.)

A number of natural polymer biomaterial-based nerve guidance conduits (NGCs) are developed to facilitate repair of peripheral nerve injuries. Cross-linking ensures mechanical integrity and desired degradation properties of the NGCs; however, common methods such as formaldehyde are associated with cellular toxicity. Hence, there is an unmet clinical need for alternative nontoxic cross-linking agents. In this study, collagen-based NGCs with a collagen/chondroitin sulfate luminal filler are used to study the effect of cross-linking on mechanical and structural properties, degradation, biocompatibility, and immunological response. A simplified manufacturing method of genipin cross-linking is developed, by incorporating genipin into solution prior to freeze-drying the NGCs. This leads to successful cross-linking as demonstrated by higher cross-linking degree and similar tensile strength of genipin cross-linked conduits compared to formaldehyde cross-linked conduits. Genipin cross-linking also preserves NGC macro and microstructure as observed through scanning electron microscopy and spectral analysis. Most importantly, in vitro cell studies show that genipin, unlike the formaldehyde cross-linked conduits, supports the viability of Schwann cells. Moreover, genipin cross-linked conduits direct macrophages away from a pro-inflammatory and toward a pro-repair state. Overall, genipin is demonstrated to be an effective, safe, biocompatible, and anti-inflammatory alternative to formaldehyde for cross-linking clinical grade NGCs., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

One of the hallmarks of cancer cells is their ability to evade cell death via apoptosis. The inhibitor of apoptosis proteins (IAPs) are a family of proteins that act to promote cell survival. For this reason, upregulation of IAPs is associated with a number of cancer types as a mechanism of resistance to cell death and chemotherapy. As such, IAPs are considered a promising therapeutic target for cancer treatment, based on the role of IAPs in resistance to apoptosis, tumour progression and poor patient prognosis. The mitochondrial protein smac (second mitochondrial activator of caspases), is an endogenous inhibitor of IAPs, and several small molecule mimetics of smac (smac-mimetics) have been developed in order to antagonise IAPs in cancer cells and restore sensitivity to apoptotic stimuli. However, recent studies have revealed that smac-mimetics have broader effects than was first attributed. It is now understood that they are key regulators of innate immune signalling and have wide reaching immuno-modulatory properties. As such, they are ideal candidates for immunotherapy combinations. Pre-clinically, successful combination therapies incorporating smac-mimetics and oncolytic viruses, as with chimeric antigen receptor (CAR) T cell therapy, have been reported, and clinical trials incorporating smac-mimetics and immune checkpoint blockade are ongoing. Here, the potential of IAP antagonism to enhance immunotherapy strategies for the treatment of cancer will be discussed., Competing Interests: The authors declare no conflicts of interest.

Porous collagen-glycosaminoglycan (collagen-GAG) scaffolds have shown promising clinical results for wound healing; however, these scaffolds do not replace the dermal and epidermal layer simultaneously and rely on local endogenous signaling to direct healing. Functionalizing collagen-GAG scaffolds with signaling factors, and/or additional matrix molecules, could help overcome these challenges. An ideal candidate for this is platelet-rich plasma (PRP) as it is a natural reservoir of growth factors, can be activated to form a fibrin gel, and is available intraoperatively. We tested the factors released from PRP (PRPr) and found that at specific concentrations, PRPr enhanced cell proliferation and migration and induced angiogenesis to a greater extent than fetal bovine serum (FBS) controls. This motivated us to develop a strategy to successfully incorporate PRP homogeneously within the pores of the collagen-GAG scaffolds. The composite scaffold released key growth factors for wound healing (FGF, TGFβ) and vascularization (VEGF, PDGF) for up to 14 days. In addition, the composite scaffold had enhanced mechanical properties (when compared to PRP gel alone), while providing a continuous upper surface of extracellular matrix (ECM) for keratinocyte seeding. The levels of the factors released from the composite scaffold were sufficient to sustain proliferation of key cells involved in wound healing, including human endothelial cells, mesenchymal stromal cells, fibroblasts, and keratinocytes; even in the absence of FBS supplementation. In functional in vitro and in vivo vascularization assays, our composite scaffold demonstrated increased angiogenic and vascularization potential, which is known to lead to enhanced wound healing. Upon pro-inflammatory induction, macrophages released lower levels of the pro-inflammatory marker MIP-1α when treated with PRPr; and released higher levels of the anti-inflammatory marker IL1-ra upon both pro- and anti-inflammatory induction when treated with the composite scaffold. Finally, our composite scaffold supported a co-culture system of human fibroblasts and keratinocytes that resulted in an epidermal-like layer, with keratinocytes constrained to the surface of the scaffold; by contrast, keratinocytes were observed infiltrating the PRP-free scaffold. This novel composite scaffold has the potential for rapid translation to the clinic by isolating PRP from a patient intraoperatively and combining it with regulatory approved scaffolds to enhance wound repair., (Copyright © 2019 do Amaral, Zayed, Pascu, Cavanagh, Hobbs, Santarella, Simpson, Murphy, Sridharan, González-Vázquez, O'Sullivan, O'Brien and Kearney.)

Despite the clinical success of cancer immunotherapies, the majority of patients fail to respond or develop resistance through disruption of pathways that promote neo-antigen presentation on MHC I molecules. Here, we conducted a series of unbiased, genome-wide CRISPR/Cas9 screens to identify genes that limit natural killer (NK) cell anti-tumor activity. We identified that genes associated with antigen presentation and/or interferon-γ (IFN-γ) signaling protect tumor cells from NK cell killing. Indeed, Jak1-deficient melanoma cells were sensitized to NK cell killing through attenuated NK cell-derived IFN-γ-driven transcriptional events that regulate MHC I expression. Importantly, tumor cells that became resistant to T cell killing through enrichment of MHC I-deficient clones were highly sensitive to NK cell killing. Taken together, we reveal the genes targeted by tumor cells to drive checkpoint blockade resistance but simultaneously increase their vulnerability to NK cells, unveiling NK cell-based immunotherapies as a strategy to antagonize tumor immune escape., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Extracellular vesicles (EVs) are emerging as a promising alternative approach to cell-therapies. However, to realize the potential of these nanoparticles as new regenerative tools, healthcare materials that address the current limitations of systemic administration need to be developed. Here, two technologies for controlling the structure of alginate based microgel suspensions are used to develop sustained local release of EVs, in vitro. Microparticles formed using a shearing technique are compared to those manufactured using vibrational technology, resulting in either anisotropic sheet-like or spheroid particles, respectively. EVs harvested from preosteoblasts are isolated using differential ultracentrifugation and successfully loaded into the two systems, while maintaining their structures. Promisingly, in addition to exhibiting even EV distribution and high stability, controlled release of vesicles from both structures is exhibited, in vitro, over the 12 days studied. Interestingly, a significantly greater number of EVs are released from the suspensions formed by shearing (69.9 ± 10.5%), compared to the spheroids (35.1 ± 7.6%). Ultimately, alterations to the hydrogel physical structures have shown to tailor nanoparticle release while simultaneously providing ideal material characteristics for clinical injection. Thus, the sustained release mechanisms achieved through manipulating the formation of such biomaterials provide a key to unlocking the therapeutic potential held within EVs., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mutation of Dedicator of cytokinesis 8 (DOCK8) has previously been reported to provide resistance to the Th17 cell dependent EAE in mice. Contrary to expectation, we observed an elevation of Th17 cells in two different DOCK8 mutant mouse strains in the steady state. This was specific for Th17 cells with no change in Th1 or Th2 cell populations. In vitro Th cell differentiation assays revealed that the elevated Th17 cell population was not due to a T cell intrinsic differentiation bias. Challenging these mutant mice in the EAE model, we confirmed a resistance to this autoimmune disease with Th17 cells remaining elevated systemically while cellular infiltration in the CNS was reduced. Infiltrating T cells lost the bias toward Th17 cells indicating a relative reduction of Th17 cells in the CNS and a Th17 cell specific migration disadvantage. Adoptive transfers of Th1 and Th17 cells in EAE-affected mice further supported the Th17 cell-specific migration defect, however, DOCK8-deficient Th17 cells expressed normal Th17 cell-specific CCR6 levels and migrated toward chemokine gradients in transwell assays. This study shows that resistance to EAE in DOCK8 mutant mice is achieved despite a systemic Th17 bias., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The bone infection osteomyelitis (typically by Staphylococcus aureus) usually requires a multistep procedure of surgical debridement, long-term systemic high-dose antibiotics, and - for larger defects - bone grafting. This, combined with the alarming rise in antibiotic resistance, necessitates development of alternative approaches. Herein, we describe a one-step treatment for osteomyelitis that combines local, controlled release of non-antibiotic antibacterials with a regenerative collagen-based scaffold. To maximise efficacy, we utilised bioactive glass, an established osteoconductive material with immense capacity for bone repair, as a delivery platform for copper ions (proven antibacterial, angiogenic, and osteogenic properties). Multifunctional collagen-copper-doped bioactive glass scaffolds (CuBG-CS) were fabricated with favourable microarchitectural and mechanical properties (up to 1.9-fold increase in compressive modulus over CS) within the ideal range for bone tissue engineering. Scaffolds demonstrated antibacterial activity against Staphylococcus aureus (up to 66% inhibition) whilst also enhancing osteogenesis (up to 3.6-fold increase in calcium deposition) and angiogenesis in vitro. Most significantly, when assessed in a chick embryo in vivo model, CuBG-CS not only demonstrated biocompatibility, but also a significant angiogenic and osteogenic response, consistent with in vitro studies. Collectively, these results indicate that the CuBG-CS developed here show potential as a one-step osteomyelitis treatment: reducing infection, whilst enhancing bone healing., (Copyright © 2019. Published by Elsevier Ltd.)

Macrophages are the first responders to biomaterial implantation and determine the success or failure of an implant through their polarization into proinflammatory (M1) and anti-inflammatory (M2) states. It is known that material properties such as stiffness can influence this response, with these properties typically modulated using a cross-linking agent. However, the cellular response comparing different cross-linking agents is often not analyzed. In this study, collagen scaffolds were cross-linked with one physical (DHT) and two chemical cross-linking methods (EDAC and genipin) in order to independently modulate the stiffness of scaffolds. The physical and structural properties of the scaffolds were thoroughly characterized to ensure that macrophage behaviors toward scaffold stiffness and cross-linking agent employed could be evaluated independent of each other. Through gene expression and protein secretion analysis of THP1 cultures, we demonstrate that the macrophage response to collagen scaffold stiffness is dependent on the cross-linking agent used. Macrophages respond similarly to scaffolds of increasing stiffness generated using the same cross-linking agent. However, when exposed to scaffolds of similar bulk modulus and degradation characteristics cross-linked using different cross-linkers, the cells responded to the cross-linking agent used rather than to the bulk modulus of the scaffolds. Moreover, while genipin cross-linking suppressed both proinflammatory and anti-inflammatory responses from macrophages, EDAC cross-linking promoted a robust proinflammatory and anti-inflammatory response to M1 and M2 factors, respectively. The results demonstrate the potential of using tailored individual cross-linking treatments depending on the clinical indication. Taken together, the results from this study highlight the importance of understanding the macrophage response to both chemical and physical properties of scaffolds in order to promote positive remodeling outcomes after biomaterial implantation.

Chimeric antigen receptor (CAR) T-cell therapy has proven successful in the treatment of hematological malignancies, notably acute lymphoblastic leukemia and B-cell lymphoma. However, the efficacy of CAR T cells against solid tumors is poor, likely due to tumor-associated immunosuppression. Here, we demonstrated that antagonizing the "inhibitor of apoptosis proteins" with the clinical smac-mimetic, birinapant, significantly enhanced the antitumor activity of CAR T cells in a tumor necrosis factor (TNF)-dependent manner. Enhanced tumor cell death occurred independently of the perforin-mediated granule exocytosis pathway, underscoring the cytotoxic potential of CAR T-cell-derived TNF. Combining CAR T-cell therapy with birinapant significantly reduced established tumor growth in vivo, where either therapy alone was relatively ineffective. Using patient biopsy-derived tumoroids, we demonstrated the synergistic potential of combining CAR T-cell therapy with smac-mimetics. Taken together, we identified CAR T-cell-derived TNF as a potent antitumor effector, which can be further harnessed by smac-mimetics., (©2019 American Association for Cancer Research.)

Collagen-glycosaminoglycan (CG) scaffolds have been widely developed for a range of regenerative medicine applications. To enhance their efficacy, CG scaffolds have previously been prevascularised in vitro using human umbilical vein endothelial cells and human mesenchymal stromal cells (hMSCs); however, at later timepoints, a regression of vascularisation is observed. This is undesirable for longer preculture periods (e.g., for partial/full organ regeneration) and for in vitro vascularised tissue model systems (e.g., for drug testing/modelling). We hypothesised that delayed platelet-derived growth factor (PDGF)-BB addition could stabilise vessels, preventing their regression. In 2D, we identified 25 ng/ml as a suitable dose that enhanced hMSC metabolic activity and proliferation, without affecting endothelial cells, or migration in either cell type. In our 3D model of CG scaffold vascularisation, early addition of PDGF (Day 3) behaved similarly to no PDGF controls. However, PDGF addition at later timepoints (i.e., Days 4 and 5), with a second addition on Day 10, prevented vascular regression. In quantifying our observations, we identified a need for a tool to measure in vitro vascularisation in porous scaffolds. This was a second key objective of this work. A novel ImageJ macro was developed, which allowed us to analyse vessel-like structures, evaluating their number and morphology, and confirmed our qualitative observations. Finally, upregulation of angiogenic genes (ANG1, KDR, and TEK2) involved in vessel maturation illustrated how PDGF addition contributed to vascular stability. Taken together, the results suggest that addition of PDGF at specific timepoints can be used to stabilise vasculature in CG scaffolds., (© 2018 John Wiley & Sons, Ltd.)

Immunotherapy has revolutionized outcomes for cancer patients, but the mechanisms of resistance remain poorly defined. We used a series of whole-genome clustered regularly interspaced short palindromic repeat (CRISPR)-based screens performed in vitro and in vivo to identify mechanisms of tumor immune evasion from cytotoxic lymphocytes [CD8 + T cells and natural killer (NK) cells]. Deletion of key genes within the tumor necrosis factor (TNF) signaling, interferon-γ (IFN-γ) signaling, and antigen presentation pathways provided protection of tumor cells from CD8 + T cell-mediated killing and blunted antitumor immune responses in vivo. Deletion of a number of genes in the TNF pathway also emerged as the key mechanism of immune evasion from primary NK cells. Our screens also identified that the metabolic protein 2-aminoethanethiol dioxygenase (Ado) modulates sensitivity to TNF-mediated killing by cytotoxic lymphocytes and is required for optimal control of tumors in vivo. Remarkably, we found that tumors delete the same genes when exposed to perforin-deficient CD8 + T cells, demonstrating that the dominant immune evasion strategy used by tumor cells is acquired resistance to T cell-derived cytokine-mediated antitumor effects. We demonstrate that TNF-mediated bystander killing is a potent T cell effector mechanism capable of killing antigen-negative tumor cells. In addition to highlighting the importance of TNF in CD8 + T cell- and NK cell-mediated killing of tumor cells, our study also provides a comprehensive picture of the roles of the TNF, IFN, and antigen presentation pathways in immune-mediated tumor surveillance., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)