Pancreatic ductal adenocarcinoma (PDAC) remains a lethal malignancy with an immunosuppressive microenvironment that is resistant to most therapies. IL17 is involved in pancreatic tumorigenesis, but ...its role in invasive PDAC is undetermined. We hypothesized that IL17 triggers and sustains PDAC immunosuppression. We inhibited IL17/IL17RA signaling using pharmacological and genetic strategies alongside mass cytometry and multiplex immunofluorescence techniques. We uncovered that IL17 recruits neutrophils, triggers neutrophil extracellular traps (NETs), and excludes cytotoxic CD8 T cells from tumors. Additionally, IL17 blockade increases immune checkpoint blockade (PD-1, CTLA4) sensitivity. Inhibition of neutrophils or Padi4-dependent NETosis phenocopies IL17 neutralization. NMR spectroscopy revealed changes in tumor lactate as a potential early biomarker for IL17/PD-1 combination efficacy. Higher expression of IL17 and PADI4 in human PDAC corresponds with poorer prognosis, and the serum of patients with PDAC has higher potential for NETosis. Clinical studies with IL17 and checkpoint blockade represent a novel combinatorial therapy with potential efficacy for this lethal disease.
Biomedical devices such as islet‐encapsulating systems are used for treatment of type 1 diabetes (T1D). Despite recent strides in preventing biomaterial fibrosis, challenges remain for biomaterial ...scaffolds due to limitations on cells contained within. The study demonstrates that proliferation and function of insulinoma (INS‐1) cells as well as pancreatic rat islets may be improved in alginate hydrogels with optimized gel%, crosslinking, and stiffness. Quantitative polymerase chain reaction (qPCR)‐based graft phenotyping of encapsulated INS‐1 cells and pancreatic islets identified a hydrogel stiffness range between 600 and 1000 Pa that improved insulin Ins and Pdx1 gene expression as well as glucose‐sensitive insulin‐secretion. Barium chloride (BaCl2) crosslinking time is also optimized due to toxicity of extended exposure. Despite possible benefits to cell viability, calcium chloride (CaCl2)‐crosslinked hydrogels exhibited a sharp storage modulus loss in vitro. Despite improved stability, BaCl2‐crosslinked hydrogels also exhibited stiffness losses over the same timeframe. It is believed that this is due to ion exchange with other species in culture media, as hydrogels incubated in dIH2O exhibited significantly improved stability. To maintain cell viability and function while increasing 3D matrix stability, a range of useful media:dIH2O dilution ratios for use are identified. Such findings have importance to carry out characterization and optimization of cell microphysiological systems with high fidelity in vitro.
The study reports on the phenotypic and secretion response of insulinoma cell‐ and primary rat islet‐laden alginate hydrogels as a function of storage moduli to elucidate an optimal micro‐environment for insulin secretion. Effects of barium chloride exposure on cell viability as well as storage modulus loss of alginate in media over time are considered with respect to optimization for in vitro studies.
Introduction
A major challenge in cancer medicine is the safe and effective delivery of drugs to the right tissue at the right time. Despite being designed for greater target specificity, many drugs ...still result in side effects and lack of safety in patients following global dissemination. Therefore, to develop new, more effective formulations capable of improving specificity and reducing off-target effects, here we describe formulation of drug crystals, from even a very hydrophobic and otherwise difficult to solubilize small molecule chemical compound, capable of providing constant drug release for weeks following a single injection.
Methods
We chose to utilize the multi-tyrosine kinase inhibitor and multi-modal (anti-angiogenic and tumor cell cytotoxic) agent sorafenib, to combat aberrant angiogenesis and tumor growth which contribute to metastasis, ultimately responsible for poor patient outcomes. We tuned crystal size (surface area:volume ratios), imaged by SEM, to display controllability of drug delivery kinetics in
in vitro
drug release assays.
Results
Single and powder crystal X-ray diffraction (XRD) established that all crystals were the same polymorph and drug form. When utilized against an orthotopic triple negative breast cancer (TNBC) mouse model (4T1 in syngeneic BALB/c mice), we established anti-tumor activity from a single local, subcutaneous injection of crystalline sorafenib.
Conclusion
From our findings, we support that engineering crystalline drug delivery systems has implications in the treatment of cancer or other diseases where high enough constitutive drug levels are needed to maintain target saturation and inhibition while also preventing emergence of drug resistance, which is a consequence often seen with suboptimal dosing.
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•Metronomic chemotherapy is optimized with tuned drug doses and drug-free breaks.•Innate immunity can be just as essential in antitumor efficacy as adaptive.•Conventional maximum ...tolerated dose administration can often be immunosuppressive.•Optimal drug scheduling may vary based on drug mechanism/target and cancer type.•Metronomic chemotherapy may be delivered with current biomaterials for clinical ease.
Metronomic chemotherapy has been shown to elicit anti-tumor immune response and block tumor angiogenesis distinct from that observed with maximal tolerated dose (MTD) therapy. This review delves into the mechanisms behind anti-tumor immunity and seeks to identify the differential effect of dosing regimens, including daily low-dose and medium-dose intermittent chemotherapy (MEDIC), on both innate and adaptive immune populations involved in observed anti-tumor immune response. Given reports of VEGF/VEGFR blockade antagonizing anti-tumor immunity, drug choice, dose, and selective delivery determined by advanced formulations/vehicles are highlighted as potential sources of innovation for identifying anti-angiogenic modalities that may be combined with metronomic regimens without interrupting key immune players in the anti-tumor response. Engineered drug delivery mechanisms that exhibit extended and local release of anti-angiogenic agents both alone and in combination with chemotherapeutic treatments have also been demonstrated to elicit a potent and potentially systemic anti-tumor immune response, favoring tumor regression and stasis over progression. This review examines this interplay between various cancer models, the host immune response, and select anti-cancer agents depending on drug dosing, scheduling/regimen, and delivery modality.
We describe the staining methods used for simultaneous detection of tumor microenvironment components as well as the automated quantification methodologies. This method uses mouse formalin-fixed ...paraffin-embedded tissues and multiplex immunofluorescence (Multiplex IF) followed by multispectral imaging. Currently, this methodology has shown to have a valuable role in murine immunoprofiling, and can be useful when evaluating the changes incurred on the tumor microenvironment upon various immunopreventive strategies.
Progressive stiffening of the extracellular matrix (ECM) is observed in tissue development as well as in pathologies such as cancer, cardiovascular disease, and fibrotic disease. However, methods to ...recapitulate this phenomenon in vitro face critical limitations. Here, we present a poly(ethylene glycol)-based peptide-functionalized ECM-mimetic hydrogel platform capable of facile, user-controlled dynamic stiffening. This platform leverages supramolecular interactions between inverse-electron demand Diels–Alder tetrazine–norbornene click products (TNCP) to create pendant moieties that undergo non-covalent crosslinking, stiffening a pre-existing network formed via thiol–ene click chemistry over the course of 6 h. Pendant TNCP moieties have a concentration-dependent effect on gel stiffness while still being cytocompatible and permissive of cell-mediated gel degradation. The robustness of this approach as well as its simplicity and ease of translation give it broad potential utility.
The discovery of tetrazine click‐induced secondary interactions is reported as a promising new tool for polymeric biomaterial synthesis. This phenomenon is first demonstrated as a tool for ...poly(ethylene glycol) (PEG) hydrogel assembly via purely non‐covalent interactions and is shown to yield robust gels with storage moduli one to two orders of magnitude higher than other non‐covalent crosslinking methods. In addition, tetrazine click‐induced secondary interactions also enhance the properties of covalently crosslinked hydrogels. A head‐to‐head comparison of PEG hydrogels crosslinked with tetrazine‐norbornene and thiol‐norbornene click chemistry reveals an approximately sixfold increase in storage modulus and unprecedented resistance to hydrolytic degradation in tetrazine click‐crosslinked gels without substantial differences in gel fraction. Molecular dynamic simulations attribute these differences to the presence of secondary interactions between the tetrazine‐norbornene cycloaddition products, which are absent in the thiol‐norbornene crosslinked gels.
Herein, the discovery of secondary interactions between the products of the inverse electron demand Diels–Alder tetrazine‐norbornene click reaction is leveraged to produce purely non‐covalently crosslinked hydrogels. In covalently crosslinked poly(ethylene glycol) hydrogels, the presence of these secondary interactions results in increased storage modulus and resistance to hydrolytic degradation compared to gels crosslinked with radical‐mediated thiol‐norbornene chemistry
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Abstract
Pancreatic ductal adenocarcinoma (PDAC) continues to be an aggressive disease with an immunosuppressive tumor microenvironment, which is recalcitrant to most therapies. Interleukin-17 ...(IL-17), a potent pro-inflammatory cytokine, has been implicated in initiation of pancreatic premalignant lesions but its role in invasive PDAC has not been widely explored. We hypothesized that IL-17 triggers and sustains immunosuppression during PDAC. We employed pharmacological and genetic strategies to inhibit IL-17/IL-17RA signaling axis and characterized the tumor microenvironment with RNA sequencing, mass & flow cytometry and quantitative multiplex immunofluorescence. Our analysis revealed that IL-17 signaled through tumor cells to recruit neutrophils into the tumor microenvironment and spatially exclude cytotoxic CD8 T cells. Furthermore, IL-17 signaling in tumor cells triggered neutrophils to form extracellular traps (NETs), a primary mechanism mediating PDAC immunosuppression. Genetic or pharmacological inhibition of IL-17 overcame resistance to immune checkpoint blockade (PD-1, CTLA-4), in a CD8 T cell dependent manner. Inhibition of neutrophils or Padi4-dependent NETosis phenocopied IL-17 neutralization. Higher expression of IL-17 and PADI4 in human PDAC corresponded with poorer prognosis, according to TCGA data. Serum from PDAC patients had a higher capacity for NET formation and diminished ability for NET degradation in control neutrophils, in comparison to serum from healthy donors. In conclusion, the cross-talk of IL-17 with pancreatic cancer cells orchestrates neutrophil recruitment and activation of Padi4 dependent NETosis, ultimately resulting in spatial remodeling of the stroma and immunosuppression. IL-17 inhibition overcomes resistance to checkpoint blockade in a CD8 T cell dependent fashion. Clinical studies with IL-17 inhibitors and checkpoint blockade represent a promising novel combinatorial therapy for this lethal disease.
Citation Format: Vidhi Chandra, Yu Zhang, Erick Riquelme, Prasanta Dutta, Pompeyo R. Quesada, Amanda Rakoski, Michelle Zoltan, Nivedita Arora, Seyda Baydogan, William Horne, Jared Burks, Hanwen Xu, Perwez Hussain, Huamin Wang, Sonal Gupta, Anirban Maitra, Jennifer M. Bailey, Seyed J. Moghaddam, Sulagna Banerjee, Ismet Sahin, Pratip Bhattacharya, Florencia McAllister. Interleukin 17 induced- neutrophil extracellular traps (NETs) mediate resistance to checkpoint blockade in pancreatic cancer abstract. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-014.
Biomedical devices comprise a major component of modern medicine, however immune-mediated fibrosis and rejection can limit their function over time. Here, we describe a humanized mouse model that ...recapitulates fibrosis following biomaterial implantation. Cellular and cytokine responses to multiple biomaterials were evaluated across different implant sites. Human innate immune macrophages were verified as essential to biomaterial rejection in this model and were capable of cross-talk with mouse fibroblasts for collagen matrix deposition. Cytokine and cytokine receptor array analysis confirmed core signaling in the fibrotic cascade. Foreign body giant cell formation, often unobserved in mice, was also prominent. Last, high-resolution microscopy coupled with multiplexed antibody capture digital profiling analysis supplied spatial resolution of rejection responses. This model enables the study of human immune cell-mediated fibrosis and interactions with implanted biomaterials and devices.
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