Imaging mass spectrometry enables molecular profiling of mouse and human pancreatic tissue.

AIMS/HYPOTHESIS: The molecular response and function of pancreatic islet cells during metabolic stress is a complex process. The anatomical location and small size of pancreatic islets coupled with current methodological limitations have prevented the achievement of a complete, coherent picture of the role that lipids and proteins play in cellular processes under normal conditions and in diseased states. Herein, we describe the development of untargeted tissue imaging mass spectrometry (IMS) technologies for the study of in situ protein and, more specifically, lipid distributions in murine and human pancreases. METHODS: We developed matrix-assisted laser desorption/ionisation (MALDI) IMS protocols to study metabolite, lipid and protein distributions in mouse (wild-type and ob/ob mouse models) and human pancreases. IMS allows for the facile discrimination of chemically similar lipid and metabolite isoforms that cannot be distinguished using standard immunohistochemical techniques. Co-registration of MS images with immunofluorescence images acquired from serial tissue sections allowed accurate cross-registration of cell types. By acquiring immunofluorescence images first, this serial section approach guides targeted high spatial resolution IMS analyses (down to 15 µm) of regions of interest and leads to reduced time requirements for data acquisition. RESULTS: MALDI IMS enabled the molecular identification of specific phospholipid and glycolipid isoforms in pancreatic islets with intra-islet spatial resolution. This technology shows that subtle differences in the chemical structure of phospholipids can dramatically affect their distribution patterns and, presumably, cellular function within the islet and exocrine compartments of the pancreas (e.g. 18:1 vs 18:2 fatty acyl groups in phosphatidylcholine lipids). We also observed the localisation of specific GM3 ganglioside lipids [GM3(d34:1), GM3(d36:1), GM3(d38:1) and GM3(d40:1)] within murine islet cells that were correlated with a higher level of GM3 synthase as verified by immunostaining. However, in human pancreas, GM3 gangliosides were equally distributed in both the endocrine and exocrine tissue, with only one GM3 isoform showing islet-specific localisation. CONCLUSIONS/INTERPRETATION: The development of more complete molecular profiles of pancreatic tissue will provide important insight into the molecular state of the pancreas during islet development, normal function, and diseased states. For example, this study demonstrates that these results can provide novel insight into the potential signalling mechanisms involving phospholipids and glycolipids that would be difficult to detect by targeted methods, and can help raise new hypotheses about the types of physiological control exerted on endocrine hormone-producing cells in islets. Importantly, the in situ measurements afforded by IMS do not require a priori knowledge of molecules of interest and are not susceptible to the limitations of immunohistochemistry, providing the opportunity for novel biomarker discovery. Notably, the presence of multiple GM3 isoforms in mouse islets and the differential localisation of lipids in human tissue underscore the important role these molecules play in regulating insulin modulation and suggest species, organ, and cell specificity. This approach demonstrates the importance of both high spatial resolution and high molecular specificity to accurately survey the molecular composition of complex, multi-functional tissues such as the pancreas.

Immune characterization of a xenogeneic human lung cross-circulation support system.

Improved approaches to expanding the pool of donor lungs suitable for transplantation are critically needed for the growing population with end-stage lung disease. Cross-circulation (XC) of whole blood between swine and explanted human lungs has previously been reported to enable the extracorporeal recovery of donor lungs that declined for transplantation due to acute, reversible injuries. However, immunologic interactions of this xenogeneic platform have not been characterized, thus limiting potential translational applications. Using flow cytometry and immunohistochemistry, we demonstrate that porcine immune cell and immunoglobulin infiltration occurs in this xenogeneic XC system, in the context of calcineurin-based immunosuppression and complement depletion. Despite this, xenogeneic XC supported the viability, tissue integrity, and physiologic improvement of human donor lungs over 24 hours of xeno-support. These findings provide targets for future immunomodulatory strategies to minimize immunologic interactions on this organ support biotechnology.

Beyond Programmed Death-Ligand 1: B7-H6 Emerges as a Potential Immunotherapy Target in SCLC.

INTRODUCTION: The programmed death-ligand 1 (PD-L1) immune checkpoint inhibitors, atezolizumab and durvalumab, have received regulatory approval for the first-line treatment of patients with extensive-stage SCLC. Nevertheless, when used in combination with platinum-based chemotherapy, these PD-L1 inhibitors only improve overall survival by 2 to 3 months. This may be due to the observation that less than 20% of SCLC tumors express PD-L1 at greater than 1%. Evaluating the composition and abundance of checkpoint molecules in SCLC may identify molecules beyond PD-L1 that are amenable to therapeutic targeting. METHODS: We analyzed RNA-sequencing data from SCLC cell lines (n = 108) and primary tumor specimens (n = 81) for expression of 39 functionally validated inhibitory checkpoint ligands. Furthermore, we generated tissue microarrays containing SCLC cell lines and patient with SCLC specimens to confirm expression of these molecules by immunohistochemistry. We annotated patient outcomes data, including treatment response and overall survival. RESULTS: The checkpoint protein B7-H6 (NCR3LG1) exhibited increased protein expression relative to PD-L1 in cell lines and tumors (p < 0.05). Higher B7-H6 protein expression correlated with longer progression-free survival (p = 0.0368) and increased total immune infiltrates (CD45+) in patients. Furthermore, increased B7-H6 gene expression in SCLC tumors correlated with a decreased activated natural killer cell gene signature, suggesting a complex interplay between B7-H6 expression and immune signature in SCLC. CONCLUSIONS: We investigated 39 inhibitory checkpoint molecules in SCLC and found that B7-H6 is highly expressed and associated with progression-free survival. In addition, 26 of 39 immune checkpoint proteins in SCLC tumors were more abundantly expressed than PD-L1, indicating an urgent need to investigate additional checkpoint targets for therapy in addition to PD-L1.

Fine-Needle Aspiration-Based Patient-Derived Cancer Organoids.

Patient-derived cancer organoids hold great potential to accurately model and predict therapeutic responses. Efficient organoid isolation methods that minimize post-collection manipulation of tissues would improve adaptability, accuracy, and applicability to both experimental and real-time clinical settings. Here we present a simple and minimally invasive fine-needle aspiration (FNA)-based organoid culture technique using a variety of tumor types including gastrointestinal, thyroid, melanoma, and kidney. This method isolates organoids directly from patients at the bedside or from resected tissues, requiring minimal tissue processing while preserving the histologic growth patterns and infiltrating immune cells. Finally, we illustrate diverse downstream applications of this technique including in vitro high-throughput chemotherapeutic screens, in situ immune cell characterization, and in vivo patient-derived xenografts. Thus, routine clinical FNA-based collection techniques represent an unappreciated substantial source of material that can be exploited to generate tumor organoids from a variety of tumor types for both discovery and clinical applications.

Rapidly fatal pneumonitis from immunotherapy and concurrent SARS-CoV-2 infection in a patient with newly diagnosed lung cancer.

Immune checkpoint inhibitors (ICIs) are used for the treatment of numerous cancers, but risks associated with ICI-therapy during the COVID-19 pandemic are poorly understood. We report a case of acute lung injury in a lung cancer patient initially treated for ICI-pneumonitis and later found to have concurrent SARS-CoV-2 infection. Post-mortem analyses revealed diffuse alveolar damage in both the acute and organizing phases, with a predominantly CD68+ inflammatory infiltrate. Serum was positive for anti-SARS-CoV-2 IgG, suggesting that viral infection predated administration of ICI-therapy and may have contributed to a more fulminant clinical presentation. These data suggest the need for routine SARS-CoV-2 testing in cancer patients, where clinical and radiographic evaluations may be non-specific.

Alteration in prehospital drug concentration after thermal exposure.

OBJECTIVE: The aim of the study was to determine the remaining concentration of 23 commonly carried emergency medical services medications used in the United States after they have experienced thermal extremes that have been documented in the prehospital environment for a period of 1 month. METHODS: Pharmaceuticals were thermally cycled (-6 degrees C and 54 degrees C) every 12 hours and then assayed by high-performance liquid chromatography. RESULTS: Eight (35%) of 23 prehospital pharmaceuticals revealed ending concentrations of less than 90% with strong correlation to thermal exposure time. These included lidocaine, diltiazem, dopamine, nitroglycerin, ipratropium, succinylcholine, haloperidol, and naloxone. CONCLUSION: A decrease in concentration was found to be statistically significant in 8 (35%) of 23 commonly carried emergency medical services pharmaceuticals. These results provide new information and perspective regarding stability of emergency drugs in the prehospital environment by evaluating a broad range of pharmaceuticals as well as by using thermal exposure points that have been documented in the United States.

A clinical trial to investigate the effect of silver nylon dressings on mediastinitis rates in postoperative cardiac sternotomy incisions.

Mediastinitis is a rare but serious postoperative complication of cardiac surgery that increases mortality rates, hospital length of stay, and medical costs. A clinical trial was conducted to investigate whether the type of postoperative surgical dressing (silver nylon or standard gauze) affects the rate of mediastinal infections. The sample consisted of 1,600 surgical cardiac patients. Infection rates in the standard gauze group (control, n = 1,235) were collected retrospectively from 24 months of infection control records. In the prospective treatment arm of the study, the wounds of all consecutive surgical patients (n = 365) were covered with a silver nylon dressing and patients were assessed during the 3-week postoperative visit. Thirteen (13) patients in the control group (1%) and none of the patients in the treatment group developed mediastinitis (chi2 [1, N = 1,600] = 3.88, P <0.05). Study findings support the need for a large, prospective, controlled clinical study to confirm the effects of these dressings on mediastinitis, resultant morbidity, and costs of care.

Wound closure after split-thickness skin grafting is accelerated with the use of continuous direct anodal microcurrent applied to silver nylon wound contact dressings.

Wound healing after graft closure of excised burn wounds is a critical factor in the recovery process after thermal injury. Processes that speed time to stable wound closure should lead to improved outcomes, shorter lengths of hospital stays, and decreased complications. A randomized clinical trial to test the ability of continuous direct anodal microcurrent application to silver nylon wound contact dressings was designed. Time for wound closure after split-thickness skin grafting was observed. Thirty patients with full-thickness thermal burns were randomized into two groups. The control group received postoperative dressing care using moistened silver nylon fabric covered with gauze after tangential burn wound excision and split-thickness skin grafting. The study group received an identical protocol with the addition of continuous direct anodal microcurrent application. Time to 95% wound closure was measured using digital photography. The digital photographs were evaluated by a burn surgeon blinded to the patient's randomization. An independent t-test was used to analyze the data. The study group experienced a 36% reduction in time to wound closure (mean of 4.6 days) as compared to the control group (mean of 7.2 days). This was statistically significant at a P value of <.05. The use of continuous direct anodal microcurrent decreased time to wound closure after split-thickness skin grafting.