Type 2 diabetes (T2D) is a chronic disease currently affecting around 500 million people worldwide with often severe health consequences. Yet, histopathological analyses are still inadequate to infer the glycaemic state of a person based on morphological alterations linked to impaired insulin secretion and β-cell failure in T2D. Giga-pixel microscopy can capture subtle morphological changes, but data complexity exceeds human analysis capabilities. In response, we generate a dataset of pancreas whole-slide images from living donors with multiple chromogenic and multiplex immunofluorescence stainings and train deep learning models to predict the T2D status. Using explainable AI, we make the learned relationships interpretable, quantify them as biomarkers, and assess their association with T2D. Remarkably, the highest prediction performance is achieved by simultaneously focusing on islet α- and δ-cells and neuronal axons, alongside subtle pancreatic alterations in T2D donors such as larger adipocyte clusters, altered islet-adipocyte proximity and smaller islets. This data-driven approach provides a foundation for future research into relevant diagnostic and therapeutic targets, refining several hypotheses regarding tissue alterations associated with T2D.

(1) Background: Laparoscopic staging is essential in gastric cancer (GC) to rule out peritoneal metastasis (PM). Hypericin, a plant-derived fluorescent compound, has been suggested to improve laparoscopic visualization of PM from GC. This prospective, single-arm, open-label clinical trial aimed to assess the feasibility and safety of oral hypericin administration as well as the suitability of fluorescence-guided laparoscopy (FGL) for improving the sensitivity and specificity of staging in GC patients (EudraCT-Number: 2015-005277-21; clinicaltrials.gov identifier: NCT-02840331). (2) Methods: GC patients received Laif® 900, an approved hypericin-containing phytopharmaceutical, once orally two to four hours before white light and ultraviolet light laparoscopy. The peritoneal cancer index was evaluated, biopsies taken and hypericin concentrations in serum and peritoneal tissue were determined by mass spectrometry. (3) Results: Between 2017 and 2021, out of 63 patients screened for eligibility, 50 patients were enrolled and treated per protocol. The study intervention was shown to be feasible and safe in all patients. Standard laparoscopy revealed suspicious lesions in 27 patients (54%), among whom 16 (59%) were diagnosed with PM. FGL identified suspicious areas in 25 patients (50%), among whom PM was confirmed in 13 cases (52%). Although hypericin concentrations in serum reached up to 5.64 ng/mL, no hypericin was detectable in peritoneal tissue biopsies. (4) Conclusions: FGL in patients with GC was shown to be feasible but futile in this study. Sufficient levels of hypericin should be ensured in target tissue prior to reassessing FGL with hypericin.

Patient-derived xenograft (PDX) tumor models are essential for identifying new biomarkers, signaling pathways and novel targets, to better define key factors of therapy response and resistance mechanisms. Therefore, this study aimed at establishing pancreas carcinoma (PC) PDX models with thorough molecular characterization, and the identification of signatures defining responsiveness toward drug treatment. In total, 45 PC-PDXs were generated from 120 patient tumor specimens and the identity of PDX and corresponding patient tumors was validated. The majority of engrafted PDX models represent ductal adenocarcinomas (PDAC). The PDX growth characteristics were assessed, with great variations in doubling times (4 to 32 days). The mutational analyses revealed an individual mutational profile of the PDXs, predominantly showing alterations in the genes encoding KRAS, TP53, FAT1, KMT2D, MUC4, RNF213, ATR, MUC16, GNAS, RANBP2 and CDKN2A. Sensitivity of PDX toward standard of care (SoC) drugs gemcitabine, 5-fluorouracil, oxaliplatin and abraxane, and combinations thereof, revealed PDX models with sensitivity and resistance toward these treatments. We performed correlation analyses of drug sensitivity of these PDX models and their molecular profile to identify signatures for response and resistance. This study strongly supports the importance and value of PDX models for improvement in therapies of PC.

<p>Complete supplementary figures containing: Suppl. Fig. 1: HLA-coverage (Caucasian); Suppl. Fig. 2: Maximum attainable quantities of source proteins; Suppl. Fig. 3: Comparsion with complete benign dataset; Suppl. Fig. 4: Legend PANTHER analysis; Suppl. Fig. 5: Legend KEGG analysis; Suppl. Fig. 6: Representation analysis HLA class II</p>

<p>Supplementary Tables 1-5, 7, 9-12 containing: Suppl. Table 1: Sample characteristics; Suppl. Table 2: Patient characteristics; Suppl. Table 3: Control peptides Suppl. Table 4: Peptide-specific properties Suppl. Table 5: Over-represented Peptides HLA class I; Suppl. Table 7: Over-represented Peptides HLA class II; Suppl. Table 9: STRING results Suppl. Table 9: Protein-protein interactions (STRING) Suppl. Table 10: Over-represented pathways (STRING) Suppl. Table 11: Vaccine peptide selection Suppl. Table 12: Candidate vaccine peptides</p>

<p>Statistics over-represented peptides HLA class I</p>

Statistics over-represented peptides HLA class II

Hepatotoxicity is among the most frequent reasons for drug withdrawal from the market. Therefore, there is an urgent need for reliable predictive in vitro tests, which unfailingly identify hepatotoxic drug candidates, reduce drug development time, expenses and the number of test animals. Currently, human hepatocytes represent the gold standard. However, the use of hepatocytes is challenging since the cells are not constantly available and lose their metabolic activity in culture. To solve these problems many different approaches have been developed in the past decades. The aim of this review is to present these approaches and to discuss the possibilities and limitations as well as future opportunities and directions.

Background: Radiofrequency ablation (RFA) is an established treatment option for malignancies located in the liver. RFA-induced irreversible coagulation necrosis leads to the release of danger signals and cellular content. Hence, RFA may constitute an endogenous in situ tumor vaccination, stimulating innate and adaptive immune responses, including tumor-antigen specific T cells. This may explain a phenomenon termed abscopal effect, namely tumor regression in untreated lesions evidenced after distant thermal ablation or irradiation. In this study, we therefore assessed systemic and local immune responses in individual patients treated with RFA. Methods: For this prospective clinical trial, patients with liver metastasis from colorectal carcinoma (mCRC) receiving RFA and undergoing metachronous liver surgery for another lesion were recruited (n = 9) during a 5-year period. Tumor and non-malignant liver tissue samples from six patients were investigated by whole transcriptome sequencing and tandem-mass spectrometry, characterizing naturally presented HLA ligands. Tumor antigen-derived HLA-restricted peptides were selected by different predefined approaches. Further, candidate HLA ligands were manually curated. Peripheral blood mononuclear cells were stimulated in vitro with epitope candidate peptides, and functional T cell responses were assessed by intracellular cytokine staining. Immunohistochemical markers were additionally investigated in surgically resected mCRC from patients treated with (n = 9) or without RFA (n = 7). Results: In all six investigated patients, either induced immune responses and/or pre-existing T cell immunity against the selected targets were observed. Multi-cytokine responses were inter alia directed against known tumor antigens such as cyclin D1 but also against a (predicted) mutation contained in ERBB3. Immunohistochemistry did not show a relevant influx of immune cells into distant malignant lesions after RFA treatment (n = 9) as compared to the surgery only mCRC group (n = 7). Conclusions: Using an individualized approach for target selection, RFA induced and/or boosted T cell responses specific for individual tumor antigens were more frequently detectable as compared to previously published observations with well-characterized tumor antigens. However, the witnessed modest RFA-induced immunological effects alone may not be sufficient for the rejection of established tumors. Therefore, these findings warrant further clinical investigation including the assessment of RFA combination therapies e.g., with immune stimulatory agents, cancer vaccination, and/or immune checkpoint inhibitors.

OBJECTIVE:to report the first case of resection and partial liver segment 2-3 transplantation with delayed total hepatectomy (RAPID) from living donor in a patient affected of irresectable colorectal liver metastases (i-CRLM) BACKGROUND:: A renaissance of liver transplantation (LT) for i-CRLM has been recently observed. The Norwegian SECA trial demonstrated a 5-year overall survival rate of approximately 60%, notwithstanding early tumor recurrence. The RAPID technique was recently introduced as alternative to whole deceased donor LT, but it is limited by poor availability of splittable organs and many organisational aspects. In this context left lateral living donor LT may be the ideal solution.METHODS:Report about the technique and results of living donor RAPID procedure.TECHNIQUE:A 49 years old woman affected with i-CRLM from adenocarcinoma of right colon, underwent a left hepatectomy with ligation of right portal vein maintaining the right hepatic artery patent. Subsequently, the left lateral lobe from her son was implanted as auxiliary partial orthotopic LT. Two weeks later completion of hepatectomy was performed.RESULTS:The donor postoperative course was uneventful. The recipient developed postoperatively a slight small for size syndrome which spontaneously resolved. No graft dysfunction and no rejection were observed. At POM 5 micrometastases occurred in bones and lungs, which were treated with radiotherapy and chemotherapy, respectively. Almost 2 years later the patient is alive, in good general condition, although slight progression of bone and lung metastases.CONCLUSIONS:LT poses a valid treatment option for i-CRLM. In times of organ paucity, "living donor-RAPID" procedure may represent a paradigm shift in the management of i-CRLM.

Abstract Immune cell infiltrates have proven highly relevant for colorectal carcinoma prognosis, making colorectal cancer a promising candidate for immunotherapy. Because tumors interact with the immune system via HLA-presented peptide ligands, exact knowledge of the peptidome constitution is fundamental for understanding this relationship. Here, we comprehensively describe the naturally presented HLA ligandome of colorectal carcinoma and corresponding nonmalignant colon (NMC) tissue. Mass spectrometry identified 35,367 and 28,132 HLA class I ligands on colorectal carcinoma and NMC, attributable to 7,684 and 6,312 distinct source proteins, respectively. Cancer-exclusive peptides were assessed on source protein level using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein analysis through evolutionary relationships (PANTHER), revealing pathognomonic colorectal carcinoma–associated pathways, including Wnt, TGFβ, PI3K, p53, and RTK-RAS. Relative quantitation of peptide presentation on paired colorectal carcinoma and NMC tissue further identified source proteins from cancer- and infection-associated pathways to be overrepresented merely within the colorectal carcinoma ligandome. From the pool of tumor-exclusive peptides, a selected HLA-ligand subset was assessed for immunogenicity, with the majority exhibiting an existing T-cell repertoire. Overall, these data show that the HLA ligandome reflects cancer-associated pathways implicated in colorectal carcinoma oncogenesis, suggesting that alterations in tumor cell metabolism could result in cancer-specific, albeit not mutation-derived, tumor antigens. Hence, a defined pool of unique tumor peptides, attributable to complex cellular alterations that are exclusive to malignant cells, might comprise promising candidates for immunotherapeutic applications. Significance: Cancer-associated pathways are reflected in the antigenic landscape of colorectal cancer, suggesting that tumor-specific antigens do not necessarily have to be mutation-derived but may also originate from other alterations in cancer cells. Cancer Res; 78(16); 4627–41. ©2018 AACR.

Introduction: For patients with otherwise non-resectable primary and secondary cancer manifestations within the liver or contraindications against surgery, radiofrequency ablation (RFA) is a potentially curative treatment option. Heat causes coagulative necrosis and breaks local immune tolerance with subsequent release of cellular content like soluble danger signals which may reshape adaptive anti-tumor immune responses. The induction of effective functional tumor-specific T cells and in situ tumor-recognition remain a major challenge for immunotherapy, frequently limiting therapeutic effects. In this study, the induction of antigen-specific T cells against patient-individual, naturally presented tumor antigens and the infiltration of immune cells into distant liver metastasis after RFA were evaluated.

Donor organ quality affects long term outcome after renal transplantation. A variety of prognostic molecular markers is available, yet their validity often remains undetermined. A network-based molecular model reflecting donor kidney status based on transcriptomics data and molecular features reported in scientific literature to be associated with chronic allograft nephropathy was created. Significantly enriched biological processes were identified and representative markers were selected. An independent kidney pre-implantation transcriptomics dataset of 76 organs was used to predict estimated glomerular filtration rate (eGFR) values twelve months after transplantation using available clinical data and marker expression values. The best-performing regression model solely based on the clinical parameters donor age, donor gender, and recipient gender explained 17% of variance in post-transplant eGFR values. The five molecular markers EGF, CD2BP2, RALBP1, SF3B1, and DDX19B representing key molecular processes of the constructed renal donor organ status molecular model in addition to the clinical parameters significantly improved model performance (p-value = 0.0007) explaining around 33% of the variability of eGFR values twelve months after transplantation. Collectively, molecular markers reflecting donor organ status significantly add to prediction of post-transplant renal function when added to the clinical parameters donor age and gender.

Introduction: The underlying cellular mechanisms of successful immunotherapy are still insufficiently characterized. Especially, priming of tumor-specific T cells against relevant antigens and in situ tumor-recognition is crucial for therapy success in the development of active immunotherapeutic approaches, but remain a serious challenge. Thermal ablation of cancer, e.g. percutaneous radiofrequency ablation (RFA) is a well-established treatment option, particularly for patients with contraindications for surgical resection or in non-resectable primary or secondary liver cancers. RFA induces local tumor destruction by coagulation necrosis, leading to the release of danger signals (e.g. heat shock proteins) and cellular content that can induce and stimulate adaptive and innate immune responses, including tumor-antigen specific T cells. By breaking local immune tolerance, RFA may support the development of antigen-specific anti-tumor responses. Here, we analyzed patient-individual immune responses against autologous HLA-presented tumor antigens after RFA treatment.

Introduction: Peptide-basedcancer vaccines targeting tumor-associated antigens comprised of synthetic peptides have shown promising clinical results and have progressed to Phase III clinical testing in several tumor entities, including colorectal carcinoma (CRC). As of yet, these vaccines have typically been limited to HLA-A*02 positive patients, which means that more than half of the patient pool will not be eligible for those treatment strategies. The development of novel strategies for vaccine design, which are applicable to a larger fraction of potential patients, is therefore required. CRC seems to be particularly suitable for antigen-specific immunotherapy as it is among those cancers with highest rates of genetic mutations leading to alterations in protein metabolism. In this study, we aimed to identify HLA class I and II tumor-associated antigens from CRC covering the most frequent HLA-allotypes, in order to develop a peptide warehouse containing HLA-specific peptide panels that can be compiled in a patient-individualized manner, according to the respective patient's HLA-typing.

Availability of an individualized preselection of oncolytic viruses to be used for virotherapy of tumor patients would be of great help. Using primary liver tumor resection specimens we evaluated the precision-cut liver slice (PCLS) technology as a novel in vitro test system for characterization of paramount tumor infection parameters of individual patients. PCLS slices from resection specimens of 20 liver tumor patients were cultivated in vitro for up to 5 days and infected with 5 different oncolytic measles vaccine virus (MeV) strains. Effectiveness of tumor infection was monitored by viral nucleocapsid (N) protein detection in immunofluorescence staining or Western blot analysis or by detection of GFP marker gene expression. MeV spreading in PCLS cultures was visualized by confocal microscopy. Oncolytic MeV vaccine particles were demonstrated to efficiently infect PCLS slices originating from different primary and secondary tumors of the liver with MeV strains Moraten/Edmonston Zagreb and AIK-C showing highest infection rates (75% of all tested tumor specimens). Employing mixed liver tissue slices (exhibiting both tumorous and non-tumorous tissue areas on one and the same sample) a distinct tumor area favouring pattern of MeV infections was observed being in accordance with our finding that primary human hepatocytes are also permissive to MeV particles, albeit at a much lower rate and with a much less pronounced cytopathic effect. Furthermore, confocal microscopy demonstrated virus penetration throughout tumor tissues into deep cell layers. In conclusion, the PCLS technology is suitable to perform a tumor-patient individualized preselection of oncolytic agents prior to clinical virotherapeutic applications.

Background Polyadenosine diphosphate-ribose (poly(ADP-ribose)) is a nuclear polymer which is derived from nicotinamide adenine dinucleotide (NAD(+)) catalysed by poly(ADP-ribose) polymerase 1 (PARP-1). Aside from the well known role of poly(ADP-ribosyl)ation (pADPR) in DNA repair, pADPR is also involved in other cellular processes such as apoptosis and gene expression. However, the factors that regulate the level of pADPR are not fully elucidated. In view of the fact that healing wounds contain high concentrations of lactate (10-15 mM) and exogenous lactate reduce the NAD(+) pool in cultured fibroblasts, we propose that high lactate lowers the level of nuclear pADPR.Materials and methods Neonatal human dermal fibroblasts (NHDF) were plated to subconfluence and allowed to adhere. Cells were treated with 15 mM L-lactate and pADPR production was assessed by immunofluorescence analysis using 10H antibody. Difference in pADPR production was determined by calculation of positively stained cells compared to total cell numbers. Inhibition of PARP activity was tested by treatment with 100 mu M 3-aminobenzamide (3-AB). Specificity of the lactate effect on pADPR synthesis was verified by using the analogue D-lactate. The contents of nicotinamide adenine dinucleotide (NAD(+)) and its reduced form (NADH) in lactated and non-lactated cell cultures were quantified by the enzymatic cyclic assay.Results We found that exogenous L-lactate (15 mM) can significantly depress pADPR content in cultured fibroblasts. PARP-1 activity was inhibited by 3-AB and analogue D-lactate showed no effect on pADPR synthesis. NAD(+)/NADH ratio was significantly lowered in lactated compared to non-lactated cell culture.Conclusions Exogenous L-lactate (15 mM) can depress pADPR content in cultured fibroblasts. In view of the fact that healing wounds contain such high concentrations of lactate, we propose that down regulation of pADPR is associated with elevated tissue repair via pADPR dependent gene expression. This observation is important in understanding the stimulation of lactate-mediated protein expression during wound healing.

Background. Suppressive dendritic cells (DCs) are a promising tool for tolerance induction in transplantation. A human DCs subpopulation, which constitutively expresses indoleamine-2,3-dioxygenase (IDO), a molecule shown to prevent the rejection of fetus during pregnancy, has recently been described. This subset, characterized by nonadherence and CD123/CCR6 expression, exhibited sustained IDO production if exposed to interleukin (IL)-10. In the present work, we generated human nonadherent, CD123(+)/CCR6(+) DCs secreting IL-10.Methods. Monocytes were separated by plastic adherence and differentiated to DCs in the presecence of IL-3 and IL-4. Expression of IDO was determined by reverse-transcriptase polymerase chain reaction and enzyme activity by reverse-phased high-performance liquid chromatography. Mixed lymphocyte cultures were performed with allogeneic nylon wool-purified T-cells.Results. Contradicting previous findings, CD123+/CCR6+ DCs did not express IDO. Maturation of these cells with inducer-cytokines up-regulated IDO, but the allogeneic T-cell stimulatory capacity of these DCs was even stronger than that of immature IDO- DCs, and chemical abrogation of IDO activity did not increase T-cell proliferation. In parallel, we generated mature IDO- DCs, but these cells also did not induce stronger T-cell stimulation than their IDO+ counterpart.Conclusions. In conclusion, CD123(+)/CCR6(+) DCs do not constitutively express IDO and "induced" IDO+ DCs, even coexpressing anti-inflammatory IL-10, do not suppress allogeneic T-cell responses.

Hypoxia serves as a physiologic cue to drive an angiogenic response via HIF- dependent mechanisms. Interestingly, minor elevation of lactate levels in the tissue produces the same effect under aerobic conditions. Aerobic glycolysis contributes to lactate accumulation in the presence of oxygen, especially under inflammatory conditions. We previously postulated that aerobic lactate accumulation, already known to stimulate collagen deposition, will also stimulate angiogenesis. If substantiated, this concept would advance understanding of wound healing and aerobic angiogenesis because lactate accumulation has many aerobic sources. In this study, Matrigel plugs containing a powdered, hydrolyzable lactate polymer were implanted into the subcutaneous space of mice. Lactate monomer concentrations in the implant were consistent with wound levels for more than 11 days. They induced little inflammation but considerable VEGF production and were highly angiogenic, as opposed to controls. Arterial hypoxia abrogated angiogenesis. Furthermore, inhibition of lactate dehydrogenase by using oxamate also prevented the angiogenic effects of lactate. Lactate monomer, at concentrations found in cutaneous wounds, stabilized HIF-1 alpha and increased VEGF levels in aerobically cultured human endothelial cells. Accumulated lactate, therefore, appears to convey the impression of " metabolic need" for vascularization, even in well- oxygenated and pH-neutral conditions. Lactate and oxygen together stimulate angiogenesis and matrix deposition.