The entity of isolated pancreatic metastases is very rare in metastatic renal cell carcinoma and is characterized by unusual features: 1. The isolated occurrence of pancreatic metastases itself; 2. the long interval between treatment of renal cell carcinoma and the occurrence of pancreatic metastases (9.6 years); 3. the frequent multiple occurrence of isolated pancreatic metastases (36%); and 4. the favourable prognosis after surgical therapy . Some of the causes of this protracted course can already be traced back to specific genetic/epigenetic changes.Isolated pancreatic metastases of renal cell carcinoma (isPMRCC) are a rare manifestation of metastatic renal cell carcinoma (mRCC) characterized by two peculiarities: (1). The definite or at least long-term exclusive occurrence of metastases in the pancreas and (2). an unusual low tumour aggressiveness with slow tumour progression and consecutive, good treatment results. According to current knowledge, the exclusive occurrence of pancreatic metastases is due to a highly specific and highly selective seed and soil mechanism, which does not allow metastases settlement outside the pancreas, and whose detailed genetic/epigenetic causes are not yet elucidated. Recent studies have shed light on some of the pathways involved for the protracted course of the disease and highlighted a special genetic profile , which deviates from the conventional mRCC profile. Finally, the question of the reasons for the long-term relative genetic stability of the involved cell clones, which is an essential prerequisite for a favourable prognosis, remains unanswered. In rare cases, the pancreas may be the site of distant metastases of extrapancreatic primary tumours. These tumours include, in particular, clear cell renal cell carcinomas, lung carcinomas, colorectal carcinomas, melanomas, and sarcomas ,4,5,6,7.The aim of the review is therefore to present the particularities of isPMRCC by means of a systematic literature review with meta-analysis and, as far as is currently known, to show the genetic and epigenetic mechanisms that might explain this unexpected course.The repeatedly presented MEDLINE (PubMed)-based database on isPMRCC  ,21,22 waInclusion criteria for the database were reports of isPMRCC, i.e., synchronous or metachronous occurring pancreatic metastases (PM) of renal cell carcinoma without metastases in other organs at the time of PM diagnosis, or within 6 months before or after isPM diagnosis. Observations were excluded: (1). If the results did not clearly differentiate PM from RCC and PM from other  primary tumours; (2). if cases with additional (oligometastatic) extrapancreatic distant metastases were included in the collective of isPMRCC; and 3. if histology or detailed clinical data on the isPMRCC observations were missing. These observations were separated into those that presented observations in casuistic form and those that reported the summarized results of single institution or multicentre analyses .For calculations of the influence of PM number, only those reports that cited an exact number or reported unquestionably on single or multiple PM were analysed. An analogous procedure was applied to synchronous or metachronous metastases. To determine the localization of the metastases within the pancreas, only those isPMRCC that explicitly indicated the localization in the head, body, or tail region were used. In the few cases of one institution repeatedly reporting their isPMRCC observations ), the most detailed report was selected for analysis.Continuous data is presented as mean (standard deviation). Differences were calculated with Fisher’s exact test  and non-parametric binominal distribution test (dichotomous variables). Survival was calculated according to the Kaplan–Meier method and differences among subgroups were compared by log-rank test. The level of statistical significance was set to <0.05.In addition to the exclusive occurrence of metastases in the pancreas, isPMRCC are characterized by: (1). a long interval between RCC treatment and manifestation of isPMRCC, (2). the frequent occurrence of multiple metastases in the pancreas, and (3). the favourable treatment results .In only 7.4% of cases does the isPMRCC occur synchronously with the RCC. In the majority 92.6%), a metachronous occurrence was observed. In those metachronous observations, a long interval from RCC therapy to the manifestation of PM is a characteristic of isPMRCC. A mean interval of 9.6 years (SD 6.5) is calculated from 855 observations. In single and multi-centre reports 2.6%, a m,248,250 A frequency of solitary PM of 63.6% can be calculated from 733 observations, whereas multiple PM were observed in 36.4%—a surprisingly high incidence in the pancreas measuring only 80 mL . The lar(a)The independence of the distribution of metastases within the pancreas from the site of the RCC in the right or left kidney . This be(b)p = 0.206).The distribution of metastases within the pancreas  corresponds to the volume proportions of caput (46%) versus corpus and cauda (54%)  and does(c)In the few observations in which RCC metastases had to be removed before or that occurred after isPMRCC treatment, there was a clear dominance of systemic hematogenic metastases. Of the metastases that had to be removed between RCC and isPMRCC therapy, 78.3% (N = 36) were undoubted systemic hematogenic metastases ,206,236.(d)The rate of regional peripancreatic lymph node metastases is low in this entity. According to literature data, it is only 6.2% ,241,247.(e)The rate of subsequent liver metastases is also not increased in isPMRCC. Literature data shows a value of only 8.0% ,194,226.In early publications, it was suggested that the topographical proximity of the ren and pancreas is responsible for isPMRCC via direct lymphatic anastomoses after blockade of regional lymph nodes (LN) ,178,191;However, if one is willing to accept the high impact of a systemic MP in this entity as derived from epidemiological studies, the question inevitably arises: Why do metastases occur only and exclusively in the pancreas in spite of systemic tumour cell spread? This is even more true if one takes into account the multiple pancreatic metastases observed in around 36%, which are undoubtedly caused by multiple cell embolisms. That all these cell embolisms by chance only reach the pancreas with a size of only 80 mL  is absolAs early as 1889, Paget  showed fIn summary, these absolutely necessary interactions between the embolized tumour cells and the host organ result in a selection process in metastasis settlement, whereby the different organ tumours have different predilection organs for metastases . Such a selective metastasis mechanism must also be suspected for isPMRCC , howeverFor many years, surgical removal was the only effective therapy for isPMRCC. Depending on the location and number of metastases within the pancreas, this can be performed as partial duodenopancreatectomy, distal pancreatectomy, total DP, central pancreatectomy, and local tumour resection. In general, the surgical procedures provide a surprisingly good result for metastasis surgery. From 415 casuistically reported observations, a cumulative 5-year and 10-year survival rate of 75.7% and 47.3% respectively was calculated .These values are consistent with the results of single and multicentre reports. For the median OS ,241,246,Following isPMRCC therapy, tumour recurrence with metastases occurred in 125 of 301 sufficiently documented casuistic observations (41.5%) after an interval of 30 months (SD 25 months). The longest reported interval to tumour recurrence—it was a resectable recurrence in the lung—was 10 years . These mInstitutional studies  report sThe observation that 10.3% of the single organ relapses after isPMRCC therapy occurred in endocrine organs is noteworthy, since endocrine organs only have about 2% of the volume of the large metastatic organs  . This maIn recent decades, a highly effective drug therapy for metastatic RCC (mRCC) was established in the form of targeted therapy with tyrosine kinase inhibitors (TKI); mTor inhibitors; and immune checkpoint inhibitors such as anti-PD1, anti-PD L1, and anti-CTLA4 ,272,273,Research over the past decades has shown that risk factors associated with tumour volume and tumour growth are also of prognostic importance with advanced mRCC ,280,281,It is therefore all the more remarkable that these risk factors do not have a prognostic significance in the isPMRCC. The increasing number of case studies (N = 573) but also newer voluminous single institution and multicentre reports revealed the ineffectiveness of established clinical risk factors that reflect tumour volume and tumour growth rate . These rIn summary, the clinical course of isPMRCC, both by the long interval to the occurrence of PM and even more by the favourable treatment results, suggests that isPMRCC is composed of cell clones characterized by a low aggressiveness, which is maintained for many years.The occurrence of PM allows the identification and comparison of three courses in mRCC: mRCC without PM, mRCC with PM, and observations with isolated PM . An analThe clinical course of isPMRCC is characterized by two peculiarities: a low aggressiveness and a pronounced organotropism (the exclusive occurrence of pancreatic metastases).The long interval to the onset of isPMRCC (~10 years); the favourable treatment results ; as well as the slow spontaneous course  as well as the absence of alterations of the PBRM1 gen p = 0.0035.The genome of the clear cell RCC was decoded in 2013 . It is cIn 2018, the first studies were presented looking for genetic changes that control the metastasis behaviour of the RCC ,306. TurIn summary, these studies prove that the occurrence of PM in mRCC is associated with cell clones that have a lower aggressiveness and that can be distinguished from observations with extrapancreatic metastases by lack of loss of 9p, lower weight genome instability index, low frequency of BAP1 alterations, and a high frequency of PBRM 1 loss. Genetic studies thus provide a correlate for the clinical experience of the more favourable course of mRCC with pancreatic metastases than observations without PM ,286,287.p = 0.007; and normal vs. activated stroma subtype p = 0.037); and Waddell [p = 0,041) and disease-free survival (p = 0.008). Finally, loss of function mutation of suppressor gene SMAD4 is highly associated with increased propensity to metastasize and poor prognosis [The observation that a subtype of a metastatic solid tumour—the isPMRCC—manifests itself by an extravagant organotropism as well as by a favourable clinical course, and that this independent behaviour has now been supported by genetic studies is extremely unusual. In this pronounced form, a similar peculiarity has not been demonstrated in other solid tumours. Even for adenocarcinoma of the pancreas (DAPA)—just to give a comparative example—decisive genetic/epigenetic characteristics have already been deciphered. ACPA is a cancer with a low overall tumour mutation burden, the majority of which is less than 50 mutations . The gen Waddell —stable,  Waddell  observed Waddell  reportedrognosis ,322.A subtype of ACPA, which can be separated analogously to isPMRCC by highly specific organotropism, very favourable prognosis, and specific genetic profile, is not reported so far in ACPA. On the contrary, exome sequencing of a cohort of eight very long-term survivors of ACPA found no differences in somatic mutations to explain the improved biology of tumours from these rare patients compared to the majority of patients with ACPA .The SSM postulated and suspected for the development of the isPMRCC is also suitable to contribute to the favourable course of the isPMRCC observations. As shown, there is evidence for addressing the exclusive growth of RCC metastases in the pancreas as a result of a highly selective and effective SSM, which allows circulating tumour cells to grow and mature to metastases only in the pancreas. However, if tumour cells outside the pancreas cannot survive—or, as is equally conceivable, are forced into a dormant, non-growth state for years ,325—an aThe extrapancreatic absence of viable metastatic tumour cells may also explain the lack of relevance of the risk factors mentioned at the beginning. These risk factors only describe the probability that after removal of the macroscopically detected PM, the presence of occult micrometastases in distant organs can be expected, from which tumour progression will occur later. However, if outside the pancreas no further occult micro-settlement can take place in isPMRCC, these risk factors must necessarily be negative.In addition to the low aggressiveness of the tumour cells, which manifest themselves in a limited tendency to metastasize and slow tumour growth, another characteristic of the cell clones is important, which is decisive for the favourable course: According to the data so far, the progressive dedifferentiation of the tumour cells with increasing tumour age is unusually low.As the study of Turajlic shows , there aThe epidemiological data presented under recurrence  further The genetic changes discussed above are quite likely to provide a plausible explanation for the better prognosis of isPMRCC due to low aggressiveness and metastasis potential. However, the lower aggressiveness does not explain the exclusive growth of metastases in the pancreas. This unusual metastasis behaviour requires a highly selective selection mechanism, which allows the occurrence of metastases only in the pancreas and completely prevents extrapancreatic metastasis. The cell clones leading to the isPMRCC thus have two unusual characteristics: On the one hand, a low level of aggressiveness, the specific genetic causes of which have already been elucidated at least in part in recent years. On the other hand, these cell clones of the non-inflammatory mRCC subtype have also acquired properties that—by epigenetic mechanisms—trigger a highly specific SSM in possible host organs, which ultimately allows the settlement of metastases only in the pancreas. Biochemical studies on isPMRCC observations—whether genetic or epigenetic—that explain the exact mechanism that leads to the exclusive settlement of metastases in the pancreas have not been presented to our knowledge due to the rarity of this entity. Therefore, only analogical conclusions of more frequent and better studied tumour entities can be used as possible clues. Currently, at least five mechanisms can be defined that can trigger an organotropism  in metasBased on the efficacy of an SSM, primary tumours that produce a large number of cells with different properties will gain an advantage in metastases formation, as this increases the chance that a suitable circulating tumour cell will reach a suitable host organ to mature for metastasis. The RCC is characterized by a great heterogeneity ,327,328,MiRNA are a group of small RNA (16–22 nucleotides) that control the expression of target genes. These miRNAs can also influence the metastasis process, e.g., by influencing epithelial-mesenchymal transformation, migration, and settlement ,340,341.The pmN is the result of an epigenetic phenomen: the ability of tumours to influence a target organ long before metastases develop in such a way that they form a special microenvironment, a “fertile soil”, which enables subsequent metastasis formation by supporting the seeding, colonisation, survival, and outgrowth of the metastasizing tumour cells ,351,352.The development of pmN thus takes place in an ordered sequence of interactions of tumour cell and host organ properties. Since the profile of these special properties varies in both individual primary tumours and host organs, this inevitably leads to organotropism in metastasis development, which is a characteristic of the pmN ,359. ForThe cells of the primary tumour as well as circulating and metastasizing tumour cells are recognised and combated to varying degrees as foreign cells by the immune system. Thus, immunity is an effective defence against metastasis . A fatalIt is, therefore, all the more surprising that in the previously cited study by Singla , IT in PThe insensitivity to IT with simultaneous sensitivity to antiangiogenetic therapy with TKI could be explained in the above-mentioned study  by the bA successful interaction of a chemokine receptor located at the tumour cell surface and a suitable ligand of the host organ is a necessary prerequisite for the activation of numerous signal transforming pathways, which are critical in the early metastatic process ,365. SinIn the stage of early avascular tumour growth, micrometastasis goes through a critical phase, as the supply of energy carriers by diffusion alone is critical. Each host organ presents the embolized tumour cells with the challenge of coping with the different energy carriers and oxygen or oxygen stress. Only those tumour cell clones that are able to use all energy carriers provided by the respective host organ through metabolic adaptation will remain successful ,370,371.2, which forms with H2O the volatile acid H2CO3. As a result of their increased growth, the tumour cells modify the microenvironment to acidic pH [Tumour-specific rapid cell proliferation is associated with an increase in cell metabolism, which in turn affects the microenvironment. On the one hand, poor perfusion/hypoxia-induced glycolysis leads to the release of lactate; and on the other hand, the oxidative energy metabolism leads to the release of COcidic pH , which icidic pH ,375. HowThe limitations of the presented review with meta-analysis are the retrospective nature of the analysed casuistic reports, the long period of investigation, and the possibility that a bias in the compiled papers cannot be excluded. However, this methodological limitation is partially compensated by the confirmatory results of current large single and multicentre analyses.isPMRCC is a tumour entity with a unique disease biology  that tri


During the crystallisation period, the nucleation possibility in droplets, crystal numbers, and cell growth and cell density were observed and analysed. The results indicated the strong interaction between the lysozyme molecules and the cell wall of the S. cerevisiae, proved by the crystallization of lysozyme with fluorescent labels. The biotemplates demonstrated positive influence or negative influence on the nucleation, i.e. shorter or longer induction time, dependent on the concentrations of the lysozyme and the S. cerevisiae cells, and ratios between them. In the biomanufacturing process, target proteins were various cells were commonly mixed with various cells, and this work provides novel insights of new design and application of live cells as biotemplates for purification of macromolecules.Macromolecular protein crystallisation was one of the potential tools to accelerate the biomanufacturing of biopharmaceuticals. In this work, it was the first time to investigate the roles of biotemplates, Saccharomyces cerevisiae live cells, in the crystallisation processes of lysozyme, with different concentrations from 20 to 2.5 mg/mL lysozyme and different concentrations from 0 to 5.0 × 10 The purification of therapeutic proteins is very expensive, due to the high cost of the chromatography method2 for recovering the resin and scaling up4, as well as a large footprint for the product and environment6. Crystallisation has been a potential purification and isolation method11, which widely used in small organic molecular pharmaceutical purification. Macromolecular protein crystallisation12 has the potential to significantly simplify the downstream biomanufacturing process, achieving a significant waste reduction. Therapeutic proteins in the crystalline form have multiple advantages over their solution counterpart including higher stability15. However, due to the difficulty of large and flexible proteins forming highly ordered structure17, nucleation and crystal growth are still challenging in industrial applications. More research on protein crystallisation was reported and with attempts to accelerate the nucleation and as the purification process is limited by the crystallisation time18. Different solid templates (i.e. heterogeneous particles and solid surfaces) have been investigated to improve the protein crystallisation efficiency26 and shorten the crystallisation process. Protein crystallisation becomes more difficult due to the presence of biological impurities such as cell debris, host cell protein, DNA and virus, among which host cell protein normally has the highest concentration and hence poses the greatest hindrance to the success of crystallisation27. In order to better connect the upstream bioprocess and downstream bioprocess, it is critical to understand the influence of the impurities such as cells on the crystallisation process.Therapeutic proteins provide effective treatments to a wide range of medical conditions and their demands have grown rapidly over the past two decades. The global sale of mAb products almost doubled from 2008 to 2013, reaching nearly $75 billion, and is expected to increase faster in this decade30. Its diameter is 5–10 μm, and the cells are spherical or oval. The cell wall of yeast, for maintaining cell morphology and intercellular recognition, including 30% dry weight of mannan, 30% of β-Dextran, 20% of glycoprotein and chitin, and others31. Mannan has a three-dimensional spiral structure in space, and it also has a variety of physiological functions such as cell recognition and control of cell wall pore size. Mannan is chemically stable, with a relatively stable pH between 2.5 and 8.033. Lysozyme can act on the β-1,4 bond between N-acetylmuramic acid and N-acetylglucosamine in the peptide polysaccharide molecule, which destroys the cell wall of bacteria, losing its protective effect on cells, and finally makes bacteria dissolve and die34. However, lysozyme has limited impact on the cell wall lysis of the S. cerevisiae, due to the lack of lysozyme sites in its cell wall36.Saccharomyces cerevisiae (S. cerevisiae) with a short growth cycle and strong fermentation capacity, is widely used in the large-scale production of wine, foods, chemical products and pharmaceuticalsTo the best knowledge of the authors, the current study is the first to apply the biotemplates, live-cell templates, on protein crystallisation, this research will fill the technology gap by demonstrating the feasibility of yeast cells, S. cerevisiae, as a heterogeneous nucleant to promote the crystallisation of a model protein, lysozyme. The work also demonstrated the crystallisation technology has the ability to isolate protein in the complex solution environment with various concentrations of the live cells. The knowledge provided in this study can be transformed into future applications of biotemplate design for bioseparation and purification of therapeutic proteins or other macromolecules.SC[Low] of the biotemplates had much more crystals, and the crystals were obviously smaller, as well as more uniformed, than the crystals obtained in the droplets with biotemplates at CSC[High]. It is obvious that plenty of S. cerevisiae cells were observed in the droplets with biotemplates at CSC[High], much fewer S. cerevisiae was observed in the droplets with biotemplates at CSC[Low]. The crystals had light agglomerations with a lower concentration of biotemplates, but there was no agglomeration observed in the droplets without and with a high concentration of S. cerevisiae. There was no obvious trend in the change of the crystal shape in the droplets with different concentrations of the biotemplates.Figure Lys was 2.5 mg/mL, the nucleation time increased dramatically, only 20% of droplets nucleated in 60 h crystallisation time with or without biotemplates. When the CLys increased to 7.5 mg/mL and above, nucleation occurred in more than 50% of droplets in 20 h, and there was nucleation in less than 4 h in all the droplets with the CLys of 20 mg/mL. However, it is noted that with different concentrations of biotemplates, the crystallisation percentages were different in the droplet with the same CLys. Figure Lys was 2.5 mg/mL, no nucleation was observed in 24 h, and with an increase in the CLys, more droplets with crystals were observed, and there were crystals in almost all of the droplets with CLys above 7.5 mg/mL at 24 h.Figure Lys of 10 or 20 mg/mL: with CSC[Low] > with CSC[High] > with CSC[Ref], and at an equal CLys of 5 or 7.5 mg/mL, the order changed to: with CSC[Low] > with CSC[Ref] > with CSC[High]. At 2.5 mg/mL, the order of the nucleation possibility in the droplets became: with CSC[Ref] > with CSC[Low] > with CSC[High], shown in Fig. Lys, S. cerevisiae as biotemplates increased the nucleation rate, at the middle range of the CLys, only a low concentration of S. cerevisiae prompted the nucleation, and the high concentration of S. cerevisiae hindered the nucleation compared with the droplets without S. cerevisiae. The trend was consistent that with the CLys decreased to 2.5 mg/mL, both high and low concentrations of S. cerevisiae slowed the nucleation rates. The trend indicates that the biotemplates would accelerate the nucleation possibility, but too many templates would lead to an opposite effect. The results were also in the agreement with the median induction time shown in Fig. Lys at 2.5 mg/mL. The high concentration of the biotemplates tended to be of less effect or negative effect in the droplets with lower CLys. The overall median induction time increased with a decrease in the CLys as expected.It is obvious that the S. cerevisiae as biotemplate had strong influences on the nucleation and crystallisation process, and positive or negative influences were dependent on the concentrations of the lysozyme and the biotemplates. The possibility of the nucleation at 24 h follow the order in the droplets at an equal CLys were compared. At CLys of 20 mg/mL, the average number of crystals in each droplet were all over one hundred in less than 8 h, and the differences between the droplets with different concentrations of biotemplates were not obviously observed. Figure Lys, there was few droplets with crystals at 24 h, and at 96 h, there were only a few crystals in each droplet without big differences for droplets with different concentrations of biotemplates. In all the conditions, the average numbers of crystals in the droplets with biotemplates at CSC[Low] were much higher than the droplets without biotemplates. The average numbers of crystals in the droplets with biteplates at CSC[Low] were higher than those without biotemplates, but the order tended to be reversed in the droplets with low CLys , the influences of changes in S. cerevisiae cell number were very limited. Therefore, the differences in induction time and crystals formation in the droplets, shown in Figs. Most of the crystallisation experiments were completed at 96 h, during which period the density of S. cerevisiae increased less than 5 folds. The increase in the number of the biotemplates would influence heterogeneous nucleation, due to an increase in heterogeneous surface areas in the crystallisation solution. This can be one of the explanations of the trend shown in Fig. SC[Low] and CSC[High]. However, a further large increase in the concentration of the biotemplates (at concentrations from CSC[Low] to CSC[High]) led less positive influences, i.e. the slower nucleation rates with the biotemplates at CSC[High] than with the biotemplates at CSC[Low]. Moreover, the ratio between the concentrations of the cells and the lysozyme would also determine the influence on the nucleation process, as negative influences were observed in the crystallisation solution of very low lysozyme concentration at 2.5 mg/mL with biotemplates at both low and high concentrations. The higher ratio between the concentration of the biotemplates to the lysozyme, the weaker the positive influence of biotemplates tended to be, turning into negative influences at very high ratios. It is noticed that the influences on the crystallisation process were not linear correlated to the ratio between concentrations of biotemplates and lysozymes. The possible reason can be that a large number of the S. cerevisiae would attract huge amount of the lysozyme molecules, diluting or hindering the formation of aggregations which could result in relatively low concentrations of lysozyme near or attach on the cell surface of each cell. Therefore, the high ratio between the number of biotemplates to the lysozyme molecules lead to the decrease in the driving force of the nucleation, hindering the crystallisation processes.There were positive influences on the nucleation and crystal growth in the crystallisation solution of all the lysozyme concentrations, except 2.5 mg/mL, with biotemplates at both CVarious protein crystallisation processes with biotemplates of live cells were first time observed, proved to be contributed by the interactions between cell walls and lysozyme. The interactions were observed by the fluorescence lysozyme attached on the cell walls and aggregated around cells. The biotemplates of S. cerevisiae at low concentrations could facilitate the nucleation, with more droplets having crystals inside during the same period, and crystal growth of lysozyme, with the formation of more crystals. With the lysozyme in the solution, the cells would still grow without lysis but the density of the cells only slowly increased, indicating strong interactions between the lysozyme and the cell wall of S. cerevisiae. Protein molecules adhered on the cell wall of S. cerevisiae and aggregated around, and then the cell wall as heterogeneous surface decreased the nucleation free energy, leading to positive influences on the nucleation. These positive influences became weaker with a decrease in the concentration of lysozyme or a large increase in the concentration of biotemplates. With much lower ratios between concentration of the lysozyme to the biotemplates, the nucleation was hindered with a longer induction time. The crystal shape was not obviously influenced by the biotemplates at both high and low concentrations, but with low concentration of the biotemplates, there were always times more crystals formed in the droplets with more uniform size distributions.Lysozyme (≥ 90%) was purchased from Sigma Aldrich ; FITC-labelled fluorescent lysozyme (> 90%) was purchased in Ruixi Biology); Chinasodium chloride, acetic acid glacial (> 99.7%) was purchased from Beijing Chemical Works ; sodium acetate (> 99.7%) was purchased from Xilong Scientific . Surfactant-free cellulose acetate membrane filter (0.22 µm) was purchased from Green Mall . FDA/PI staining kits were purchased from BestBio . De-ionised water was used, and all chemicals were used as received without further purification.S. cerevisiae was activated with LB medium, placed in a constant temperature incubator at 20 °C 6 cfu/mL) of the S. cerevisiae, shown in Table The sodium acetate buffer (0.14 M) was prepared by dissolving anhydrous sodium acetate in de-ionised water and followed by the addition of acetic acid to adjust the pH to 4.2. The sodium acetate buffer was filtered through a 0.22 µm filter. The preparation of protein solution was done by dissolving the protein  in the sodium acetate buffer. For example, to prepare a lysozyme solution with a target concentration of 20 mg/mL, lysozyme (20 mg) was dissolved in sodium acetate buffer (1 mL). Other solutions with concentrations of lysozyme from 10.0 to 2.5 mg/mL were prepared by dissolving corresponding amounts of lysozyme. Once lysozyme was fully dissolved, the solution was filtered through a 0.22 µm filter. Protein crystallization experiments were conducted using a conventional hanging-drop vapor diffusion technique. Crystallization drops were made by mixing 1.0 μL of protein solution and an equal volume of precipitation solution (80 mg/mL sodium chloride), with three different concentrations  .Supplementary Information.


Patients with gastric and oesophageal adenocarcinomas (GOCs) have short life expectancies as their tumours spread to other sites early. This is facilitated by the increased expression of the urokinase plasminogen activation system (uPAS); a feature of the majority of GOCs. There is increasing appreciation of the importance of uPAS expression in a range of cell types within the tumour microenvironment. Abundant clinical evidence indicates that altered expression of uPAS proteins is associated with worse outcomes, including time to tumour recurrence and patient survival. Emerging technologies, including liquid biopsy, suggest a role of uPAS for the detection of circulating tumour cells, which are responsible for the dissemination of cancers. We review and summarise pre-clinical and clinical data that supports the use of uPAS as a biomarker in GOC.Gastric and oesophageal cancers (GOCs) are lethal cancers which metastasise early and recur frequently, even after definitive surgery. The urokinase plasminogen activator system (uPAS) is strongly implicated in the invasion and metastasis of many aggressive tumours including GOCs. Urokinase plasminogen activator (uPA) interaction with its receptor, urokinase plasminogen activator receptor (uPAR), leads to proteolytic activation of plasminogen to plasmin, a broad-spectrum protease which enables tumour cell invasion and dissemination to distant sites. uPA, uPAR and the plasminogen activator inhibitor type 1 (PAI-1) are overexpressed in some GOCs. Accumulating evidence points to a causal role of activated receptor tyrosine kinase pathways enhancing uPAS expression in GOCs. Expression of these components are associated with poorer clinicopathological features and patient survival. Stromal cells, including tumour-associated macrophages and myofibroblasts, also express the key uPAS proteins, supporting the argument of stromal involvement in GOC progression and adverse effect on patient survival. uPAS proteins can be detected on circulating leucocytes, circulating tumour cells and within the serum; all have the potential to be developed into circulating biomarkers of GOC. Herein, we review the experimental and clinical evidence supporting uPAS expression as clinical biomarker in GOC, with the goal of developing targeted therapeutics against the uPAS. GOC will refer to adenocarcinomas arising from any location within the oesophagus or stomach, otherwise individual locations will be identified.Gastroesophageal cancers (GOC) are amongst the leading causes of cancer related morbidity and mortality worldwide . GastricThe plasminogen activation system is a multi-component regulatory system that, under normal conditions, functions in the clearance of blood clots and degradation of the extracellular matrix (ECM) and basement membranes (BM) during tissue remodelling processes such as wound healing ,7,8,9,10Overexpression of components of the uPA system (uPAS) in GOCs, on tumour cells and/or associated stromal cells in the tumour microenvironment (TME), is strongly associated with worse tumour staging ,18,19,20The key components of the uPAS include uPA, uPAR, plasminogen and specific uPA and plasmin inhibitors. uPA is a single stranded extracellular protein, secreted as an inactive double stranded zymogen (pro-urokinase), which is produced by leucocytes, fibroblasts and the urogenital system in normal physiological conditions . Upon biuPAR is a heavily glycosylated protein and is either membrane-bound via a glycosyl-phosphatidylinositol anchor or found in its soluble forms . uPAR coSoluble uPAR (suPAR) is produced through cleavage of the membrane bound uPAR; this cleavage occurs between the glycosylphosphatidylinositol (GPI)-anchor molecule and domain 3 of uPAR facilitated by plasmin, cathepsin G and GPI-specific phospholipase-D and can be identified in plasma, ascites and urine . VasculaA key level of control in the regulation of plasmin activity arises through inhibition of uPA (and tPA) via the serine proteinase inhibitors (serpins) plasminogen activator inhibitor (PAI)-1 and PAI-2 . While tThe expression and activity of the uPAS is tightly regulated during physiological processes to prevent unnecessary ECM remodelling through the production of excessive plasmin at the cell surface and dysregulated downstream signalling ,50,51. CKey cancer signalling pathways also alter uPAS mRNA and protein expression in GOC cell lines and xenografts. uPAS expression can be modulated by targeting key pathways with drug blockade ,67, tranExposure to exogenous growth factors such as epidermal growth factor increases uPAR mRNA expression, and this appears to occur through the mitogen-activated protein kinase (MAPK)/extracellular signal-related kinases (ERK) signalling pathway . uPA andThe expression of uPAS in tumour tissue, stroma and liquid biopsies correlates to both clinicopathological features of tumours ,19,20 anA meta-analysis by Brungs et al. evaluated uPAS expression in GOCs, which demonstrated the following expression levels: uPA 52.8%, uPAR 56.8%, PAI-1 53.3% and PAI-2 57.5% of all patients with GOC . ReportiActivation of the uPAS system is a requirement for tumour cells to invade deeper into the ECM or seed at distant metastatic sites . TherefoSERPINE1 gene expression (encoding for PAI-1) was higher in those patients with increased nodal disease  [Relative uPA expression levels are biologically important: where >20% of primary tumour cells stained positive for uPA, higher tumour staging and histological grading was seen . As will tissue) . It can  tissue) .To date, PAI-2 alone has not been associated with any clinicopathological features as described in The peritoneum of patients with GOC peritoneal metastases shows generalised uPAS upregulation compared to uninvolved peritoneum of patients with GOC metastases at other sites . uPAS exRetrospective analysis of GOCs with lymph node metastases showed uPAS protein expression in the primary tumour was correlated with lymph nodal metastases . 82% of uPAS overexpression is associated with poorer disease-free and overall survival (OS) of patients with GOCs  [uPAS expression shows significant intra-tumoural heterogeneity in GOC and can vary widely within patients, within the same tumour, between the primary tumour and its metastatic tumour or between different tumour histology types. For example, Alpízar-Alpízar et al. demonstrated uPAR overexpression at the invading front of gastric adenocarcinomas but not the tumour core . This ex= 0.011) .We have investigated uPAR expression at the tumour core and invasion front in an Australian cohort of GOC patients . uPAR IHuPAR overexpression at the invading front of tumours has been supported in a number of other studies ,21. IncrThe invasion of cancer cells into normal tissues relies on interactions between the tumour and the surrounding stroma. There is increasing evidence of the importance of stroma in initiating and regulating the speed of invasion ,94,95. Tp = 0.0002) [As expected, the most critical tumour region for uPAS expression in the stroma is at the advancing tumour front. Macrophages and myofibroblasts at the invading front of GOCs express increased uPAR compared to the tumour core ,18. In a 0.0002) . Conflic 0.0002) . In addiuPAR-expressing myofibroblasts (defined by expression of α-smooth actin) are not significantly associated with patient outcomes in GOC ,18. Howep = 0.002), and a tendency to worse OS (p = 0.07) [Similarly, in other solid tumours, stromal uPAS expression is significantly linked with tumour-associated stromal cells, and in the case of colon cancer poorer clinical outcomes . There i = 0.07) .There are many MMPs with different functional roles, with variable association with cancer occurrence and progression . In one Cathepsin B is a cysteine protease which has indirect proteolytic activity through interactions with pro-uPA, pro-MMPs, TGF-β and toll-like receptor 3, therefore it has an important role in cell proliferation, differentiation and angiogenesis ,111. CatPeripheral blood sampling allows for minimally invasive assessment of patient’s tumour and immune response. The uPAS has been assessed in serum ,116,117,To date, only two studies have investigated the role of serum uPA levels in GOC with inconsistent findings. Herszényi, et al. showed serum uPA levels were associated with a diagnosis of GOC and the severity of disease . Howeverp < 0.0001), and significantly higher in those with metastatic disease compared to non-metastatic disease  [ELISA  and time > 0.05) ,36. In v > 0.05) .suPAR has been better characterised in other gastrointestinal cancers. In colon cancer, increased pre-operative suPAR levels are significantly associated with poorer prognosis . InteresPeripheral blood mononuclear cells (PBMCs), identified by gradient centrifugation of blood, includes the majority of leukocytes. In malignancy, monocytes may display both pro-tumoural and anti-tumoural effects on cancers . As suchp = 0.014) [p = 0.014) [uPA mRNA assessed in peripheral blood monocytes in treatment naïve patients, following gastrectomy, demonstrated that patients with more advanced disease showed higher relative levels prior to adjuvant chemotherapy  . OS was = 0.014) .In addition to leukocytes, the PBMC layer also contains CTCs, which are a critical link in the development of distant metastases. High CTCs numbers in GOCs show worse prognosis and poor response to therapy ,127.Current food and drug administration agency approved CTC isolation utilises epithelial cell adhesion molecule (EpCAM) expression as a positive marker for CTCs . EpCAM iBrungs et al. assessed 43 patients from whom CTCs were isolated using the standard EpCAM isolation methods at any clinical stage of GOC. In 93% of patients, where EpCAM selected CTCs were identified, a proportion also co-expressed surface uPAR (CK+/EpCAM+/DAPI+/CD45-/uPAR+ CTCs) . In furtThis data shows the feasibility of uPAR detection on CTCs captured targeting cell surface EpCAM. Given the proposed role of uPAR in promoting aggressive tumour phenotypes, further investigation of uPAR in CTC development and their ability to form metastasis is certainly warranted.It is clear that in many carcinoma types, including GOC, the uPAS is a driver of tumour aggressiveness. Not surprisingly, several experimental anti-cancer and imaging approaches targeting various components of the uPAS have been pursued  stromal macrophage uPAR expression is associated with a poorer prognosis. The role of the uPAS in the stroma is under-investigated in GOCs; larger cohorts and prognostic assessment are required to understand the role of the uPAS protein expression in the stroma.An optimal biomarker for GOCs would offer real time prognostic and/or predictive qualities, as such liquid biopsy is of keen interest. suPAR shows promise as a diagnostic biomarker with increased expression reported in patients with GOCs. Unfortunately, to date, suPAR has failed to yield a prognostic association in GOCs in the same way as it has for colon cancer. For this reason, it is not currently considered a useful predictive biomarker. uPAR mRNA isolated from circulating immune cells from the peripheral blood monocyte layer has been shown to have prognostic potential, when assessing reduction in OS. These findings offer potential of a uPAS-related prognostic biomarker being identified in the circulating blood.In summary, uPAS has a highly active role in the progression of GOC, and compelling evidence of its relationship with prognosis and clinicopathological features regardless of its assessment in the primary tissue or as a circulating biomarker. GOC uPAS expression in tumour-associated stroma requires further investigation to further specify the stromal role in tumour progression. We have demonstrated primary tissue assessment of the uPAS as a useful prognostic biomarker in GOCs and highlighted the exciting potential of liquid biopsies to be added to the list of prognostic biomarkers. Through ongoing investigation and drug development to target this pathway, there is significant potential for the uPAS as a predictive biomarker of uPAS directed therapies.


Oncolytic adenovirus therapy is gaining importance as a novel treatment option for the management of various cancers. Different concepts of modification within the adenovirus vector have been identified that define the mode of action against and the interaction with the tumour. Adenoviral vectors allow for genetic manipulations that restrict tumour specificity and also the expression of specific transgenes in order to support the anti-tumour effect. Additionally, replication of the virus and reinfection of neighbouring tumour cells amplify the therapeutic effect. Another important aspect in oncolytic adenovirus therapy is the virus induced cell death which is a process that activates the immune system against the tumour. This review describes which elements in adenovirus vectors have been identified for modification not only to utilize oncolytic adenovirus vectors into conditionally replicating adenoviruses (CRAds) that allow replication specifically in tumour cells but also to confer specific characteristics to these viruses. These advances in development resulted in clinical trials that are summarized based on the conceptual design. In recent years, the development of oncolytic viruses and their implementation in clinical trials have gained increased attention. Adenoviruses are among the best-studied viruses in the field of oncolytic virotherapy and several molecular biological discoveries can be traced back to adenovirus research ,2. They http://hadvwg.gmu.edu/ (accessed on 23 September 2021)). Serotypes 2 and 5 are most commonly used in research.The life cycle of adenoviruses is extrachromosomal and they are therefore considered as non-integrating vectors that can infect both replicating and non-replicating cells . AccordiGene therapy with replication-deficient adenoviruses is limited to the expression of a transgene and, in the case of suicide gene therapy, to the killing of the infected cell. In the oncolytic approach, on the other hand, the viruses replicate, followed by lysis of the infected cell and release of new infectious units into the immediate tumour environment. An important aspect of adenoviral oncolysis is that it is also effective in tumour stem cells, which often cause tumour recurrences due to intrinsic resistance to therapy .Since the early 1990s, sophisticated methods and advances in molecular biology and virology led to a resumption of virus vector development ,11. In pThis review article focuses primarily on strategies of genetic engineering of oncolytic adenovirus vectors for tumour-specific replication, the main prerequisite for oncolysis. It also describes aspects of transgene expression and stimulation of the immune system. These concepts are currently being tested in clinical trials that are summarized based on adenovirus vector design which will determine as a decisive aspect for further improvements in vector design and combination with other treatment options in the future.The replication cycle of adenoviruses can be divided into two phases. Accordingly, the adenoviral genome is organised into early (E1–E4) and late (L1–L5) transcription units, the latter encoding structural proteins . The earE1 deleted adenoviruses are considered as replication-deficient and are used as shuttle vectors in gene therapy or for vaccination. Replication-competent adenoviruses that are restricted in their replication to cancer cells are therefore referred to as CRAd   and are There are four general strategies in current clinical adenoviral vectors to achieve conditional replication: one concept is the replacement or overlay of the native E1 promoter with cancer cell-specific promoters, the other three concepts involve modification of the early transcription units E1A and E1B. In a number of constructs, an E1A modification is combined with a tumour specific promoter. However, there are several other promising approaches that have not yet been tested in clinical trials.Tumour-specific promoters are characterized by their ability to cause high expression of specific genes that are crucial for a malignant phenotype. Control of E1A-mediated viral replication by a tumour- or tissue-specific promoter restricts replication to the appropriate cells. Deletions in the E1A regulatory region should not interfere with either the inverted terminal repeat  or the packaging signal (194–358 bp) that overlaps with the E1A enhancer region.Therefore, exogenous promoters are mostly inserted without any or with small deletions in the E1A regulatory region, leaving the E1A enhancer/encapsidation intact as in CRAd-Survivin-pk7 . For furRestriction of adenoviral replication to specific tumour entities and tissues was also successfully achieved. For prostate tissue, the probasin promoter (to drive E1A) and the enhancer element of prostate-specific antigen (PSA) (to drive E1B) were used  and for A synthetic fusion construct of core promoter and enhancers of the human tyrosinase was engineered for Ad2xTyr, an oncolytic adenovirus in which parts of the E1A regulatory region as well as the E4 promoter were replaced. Ad2xTyr showed tumour selectivity against melanoma cells versus normal fibroblasts and keratinocytes .Another approach to achieve tumour-specific adenoviral replication is to alter the protein-coding genes in the E1 region by introducing loss-of-function mutations. A cancer cell with specific molecular features can compensate for this defect, thus enabling replication.In a non-replicating cell, the retinoblastoma protein (pRB) interacts as a negative regulator with proteins of the E2F transcription factor family and thus control cell cycle progression . This paIn various further developments of Ad5-Delta-24, the modified E1A region is regulated via a tumour-specific promoter. In AdCN103, the 24 bp deletion of E1A is combined with a modified hTERT promoter  and in SThe adenoviral E2 promoter can be divided into an early and a late promoter for the regulation of the E2 transcription unit, which includes the DNA polymerase. The late E2 promoter region contains Y-boxes, binding sites for the transcription factor YB-1 (Y-box binding protein-1) ,35. A seThe E1B region of the adenovirus genome encodes a 55-kD protein (E1B55K) that binds and inactivates p53. This binding is thought to be essential for virus replication, supposedly because E1A triggers p53-dependent apoptosis . Dl1520 The induction of immunogenic cell death has proved to be a key aspect in virotherapy  Figure . Based oBesides tissue specific replication, vector systems have been developed that improve oncolytic and cytotoxic properties of adenoviruses. Thus, deletion of the E1B19K protein—a Bcl-2 homologue that blocks apoptosis induction—has been shown to increase viral spread and enhance TNF-alpha-mediated cell death ,56.Apart from strategies that warrant cancer-specific replication, genes that promote oncolysis or stimulate of the immune system have also been introduced.Derived from early studies for the development of non-replicating vector systems, most oncolytic adenoviruses use deletions in the E3 region, as this region is non-essential for viral replication that allows inserts of up to 8.3 kb . ExpressTo enhance the therapeutic effect of oncolytic adenoviruses, various approaches have been employed such as gene-directed enzyme prodrug therapy (GDEPT), enhancement of virus-induced apoptosis and viral spread, expression of specific genes and attraction of the immune system.The advantage of GDEPT is the local conversion of non-toxic compounds into cytotoxic drugs, thus minimizing systemic toxicity . Some exBioselection of Ad5 mutants with enhanced anti-tumour activity in an in vivo tumour environment as selection pressure resulted in the variant AdT1 with a single adenine insertion within the retention domain of the endoplasmic reticulum (ER) of E3 gp19K that causes an enhanced release of the virus from the infected cell . As descSystemic application of adenoviral vectors leads to viral uptake in all organs and especially in the liver . After iTo ensure a potent anti-tumour response of oncolytic adenoviruses, modifications and delivery methods must be considered to achieve sufficient viral uptake into tumour cells.Cellular entry of adenoviruses is a two-step process consisting of binding of the viral fibre knob to the cell via the coxsackievirus and adenovirus receptor (CAR)  and inteSince adenoviral cell entry receptors are ubiquitously expressed in various tissues of mammals, one strategy to ensure tumour-exclusive therapy effects aims to ablate the natural viral tropism by altering the RGD motif in the penton base , incorpoTo introduce a new tropism, Douglas et al. chemically conjugated an antibody fragment against the fibre protein with a folate molecule and obtained a targeted adenovirus . The folBesides local administration of oncolytic virus into the lesion, cell-mediated delivery is a novel and promising approach to improve the clinical usage of oncolytic adenoviruses. Here, neural  or mesenAdenoviruses are the most commonly used oncolytic viruses in clinical trials . At the Telomelysin (OBP-301) is an oncolytic adenovirus in which the hTERT promoter regulates both E1A and E1B, which are linked by an IRES element . In an iThe first AdDelta-24 derivative being tested in clinical trials was DNX-2401 (Delta-24-RGD)  mainly fICOVIR-5 (Ad5-E2F-Delta-24-RGD) was administered systemically intravenously in a phase I trial in melanoma patients (NCT01864759) and was well tolerated . AlthougVCN-01 (Ad5-E2F-Delta-24-RGD-PH20) expresses hyaluronidase (PH20), which enhances intratumoural spread of the virus . It is cONYX-015 (dl1520) was the first oncolytic adenovirus in clinical trials for the treatment of head and neck cancer . AlthougA novel adenoviral vector, enadenotucirev (Colo-Ad1), is derived from a pool of different serotypes of species B to F by directed evolution and was selected to replicate only in colon cancer cells . Three pThe clinical application of therapies based on oncolytic adenoviruses has proven their enormous potential, but is still in its infancy. In particular, the wide-ranging compatibility with already established therapies (without increased toxicity), the multiple cell-killing effects, and the induction of an immunogenic cell death makes it highly attractive in the clinical arena.Three aspects are of crucial relevance in the generation of novel viral vectors: (a) tissue specificity, (b) replication and lysis capability; and (c) triggering of the immune system in order to achieve a systemic therapy response. There are several sophisticated methods to increase the selectivity of infection of tumour cells to obtain a higher level of safety and efficacy—e.g., by adding certain transgenes. However, selectively replicating oncolytic adenoviruses are often attenuated in their replicative capacity compared to wild type . This noThe most promising aspect of the development of new adenoviral vectors is the activation of the immune system by the virus, which would represent a vaccination strategy against the tumour. A better understanding of mutually supporting effects of introducing certain transgenes or combining them with immune checkpoint inhibitors will allow novel directions in treatment strategies to increase oncolytic or immune stimulatory efficacy. Several clinical trials are currently underway, and it will gradually become clear which strategies and modifications will provide the best results. An important aspect in these trials will be a thorough patient stratification that addresses the characteristics of the used oncolytic adenovirus. As we have only recently begun to translate the knowledge gained mainly in the laboratory into the clinic, new findings will automatically generate a great deal of interest in the enormous possibilities offered by the great versatility in developing new treatment strategies for cancer with adenoviruses.


V subunit. The activation of latent TGF-β by cancer-cell-expressed αV re-shapes the tumour microenvironment, and this could affect patient responses to PD-1-targeting therapy. Here we show, using multiplex immunofluorescence staining in cohorts of anti-PD-1 and anti-PD-L1-treated lung cancer patients, that decreased expression of cancer cell αV is associated with improved immunotherapy-related, progression-free survival, as well as with an increased density of CD8+CD103+ tumour-infiltrating lymphocytes. Mechanistically, tumour αV regulates CD8 T cell recruitment, induces CD103 expression on activated CD8+ T cells and promotes their differentiation to granzyme B-producing CD103+CD69+ resident memory T cells via autocrine TGF-β signalling. Thus, our work provides the underlying principle of targeting cancer cell αV for more efficient PD-1 checkpoint blockade therapy.TGF-β is secreted in the tumour microenvironment in a latent, inactive form bound to latency associated protein and activated by the integrin α V integrin expression leads to better tumour growth control by anti-PD-1 via reduced TGF-β activation and hence increased infiltration of anti-tumour CD8+ T cells.Response to PD-1 checkpoint blockade is unpredictable in lung cancer patients. Here authors show in human lung and mouse tumour models that low or absent α However, given the limited therapeutic benefit of anti-PD-(L)1 antibodies as single agents, it is crucial to identify the immune mechanisms involved in tumour resistance to immune checkpoint blockade (ICB) and develop more effective combinatorial approaches. Resistance to ICB has been associated with defects in genes relating to antigen presentation by major histocompatibility complex class I (MHC-I) molecules to CD8+ T lymphocytes4, mutations in the Janus kinase (JAK)1/JAK2 and interferon (IFN) signalling pathways6, and clonal deletion of tumour-specific T cells7. Accumulating evidence indicates that tumour regression following PD-1 blockade requires pre-existing CD8+ T lymphocytes that are negatively regulated by PD-1-mediated resistance8. Consequently, tumours that are weakly infiltrated by CD8+ T cells are unlikely to respond to such therapeutic interventions. The quality of CD8+ tumour-infiltrating lymphocytes (TIL), especially their reactivity toward the cognate target, is also directly associated with the efficacy of anti-PD-1. In this regard, expression of PD-1 on CD8+ TIL appeared to define clonally expanded tumour neoantigen-specific T cells detected in cancer patients10. More recently, expression of CD103 (αE(CD103)β7) integrin, together with CD39 and CD137 (4-1BB), has been reported to identify truly tumour-reactive CD8+ T cells in human solid tumours12.Immunotherapy targeting the T cell inhibitory receptor programmed death-1 (PD-1) and its ligand PD-L1 holds promise in lung cancer treatment+ resident memory T (TRM) cells, which stably reside in many human solid tumours where they likely orchestrate a local immune response to cancer cells13. Human non-small-cell lung cancer (NSCLC) CD103+CD8+ TRM cells frequently express the activation marker CD69 and a panel of T cell inhibitory receptors, including PD-114. They are enriched with tumour-reactive T lymphocytes able to kill autologous tumour cells upon blockade of PD-1 with neutralizing antibodies15. This CD103+CD8+ TRM subset emerges as a predictive marker of survival in several cancers, including NSCLC18. CD103+CD8+ TRM expands during anti-PD-1 immunotherapy, and their accumulation in tumours is associated with the improved outcome of anti-PD-(L)1-treated patients19.CD103 delineates a subtype of CD8+ T lymphocytes and persistence of CD103+CD8+ TRM in epithelial tissues requires transforming growth factor-beta (TGF-β). TGF-β is important for tissue remodelling and repair at sites of inflammation20, but is also an immunosuppressive mediator used by malignant cells to escape from the immune system22. This cytokine is secreted in the tumour microenvironment (TME) in its inactive (latent) form bound to latency-associated protein (LAP), and is activated by metalloproteinases (MMP)24 and RGD-binding integrins, such as αVβ6 and αVβ826. It has been reported that αVβ8-expressing tumours evade host immunity by activating latent TGF-β on adjacent immune cells27. In contrast, activation of TGF-β by αVβ8 integrin on tumour-infiltrating dendritic cells (DC) induced CD103 expression on CD8+ T cells resulting in inhibition of cancer progression28. Thus, the role of αV integrins in shaping the tumour ecosystem and regulating the anti-tumour immune response is controversial and needs to be better understood.It is now widely recognized that induction of CD103 on activated CD8V expression is associating with worse immunotherapy-related progression-free survival (PFS) in anti-PD-(L)1-treated NSCLC patients, which correlates with the decreased density of CD103+CD8+ TIL. In vivo therapeutic blockade of PD-1 in a mouse model greatly improves growth control of αV-knockout tumours via a mechanism involving increased tumour infiltration by activated tumour-specific CD103+CD8+ T cells. Thus, targeting tumour αV integrin to prevent endogenous TGF-β maturation is a promising approach for more effective ICB.Here, we show that increased tumour αV expression on survival in patients with lung cancer, we used a retrospective cohort of 113 patients with treatment-naïve early-stage NSCLC15. Tumour sections from formalin-fixed, paraffin-embedded samples were stained with anti-αV monoclonal antibodies (mAb) and evaluated by immunohistochemistry (IHC) for the expression of the integrin in epithelial tumour regions. Variability in tumour αV expression was seen, with 11% of tumours displaying a αVhigh profile and 89% displaying a αVlow profile, among which 36% were negative for αV expression (αVneg)  Fig. . We did urs Fig. , with a urs Fig. . These dV levels on survival in patients treated with ICB. We established a retrospective cohort (cohort 1) of 106 patients with advanced NSCLC treated with a single-agent anti-PD-(L)1 as a second-line treatment  after the first immunotherapy administration was assessed. Notably, chemotherapy and radiation therapy prior to immunotherapy had no significant impact on tumour αV levels (Supplementary Table Vlow tumours (including 18% αVneg) had increased PFS with a HR = 0.47  and a median PFS of 8.2 months , who displayed strongly decreased PFS, with a median PFS of 1.5 months. Association between αVlow tumours and better PFS of anti-PD-(L)1-treated patients was then investigated in a second cohort (cohort 2) of 51 NSCLC (Supplementary Table n = 9) of αVhigh tumours in cohort 2. Results showed a trend toward statistical significance for the association between αVlow tumours and better PFS in anti-PD-(L)1-treated patients , there was a trend toward a worse PFS for patients with αVhigh tumours  RT-PCR to evaluate the expression of ITGAV, ITGB6, and ITGB8 mRNA in 15 freshly resected primary NSCLC. Log scale analyses showed that ITGAV and ITGB8 transcripts were more strongly expressed in four out of 15 tumour samples compared to autologous proximal healthy lung tissues. In contrast, the ITGB6 transcript was much less frequently expressed  and that ITGB3 was more strongly expressed in two tumours than in healthy lungs  peripheral blood mononuclear cells (PBMC) only rarely expressed the integrin  to suppress all αV integrins  from IGR-B2T or IGR-B2T-KO cells, and evaluated CD103 expression on CD8+ T cells. Activation of T lymphocytes with anti-CD3 plus CM from IGR-B2T resulted in induction of CD103 on up to 70% of CD8+ T cells TGF-β, used as a positive control, induced expression of the integrin in a dose-dependent manner  to mimic epithelial tumours. We selected B16F10 because it expressed αV and β8 subunits, produced constitutively LAP-TGF-β  which displayed similar expression levels of E-cadherin as the parental clone  and confocal microscopy  administration of neutralizing anti-PD-1 mAb greatly improved control of B16F10E-KO tumour growth, but not B16F10E growth , have been developed for treating neoplasms, including lung cancer38. Targeting the ITGAV gene has also been used as a therapeutic approach for metastatic cancer39. Notably, antagonists of αV observed promising preclinical results with anti-angiogenic and anti-metastatic effects40. However, most of the clinical trials with inhibitors targeting αV integrin failed to demonstrate therapeutic benefits32. Along the same lines, our data indicate that decreased expression of αV in lung tumours had no effect on survival in treatment-naïve NSCLC patients. In addition, although tumour αV-knockout greatly increased tumour infiltration by CD8 T lymphocytes in our mouse model, it failed to delay tumour progression. Indeed, our in vivo studies showed similar growth kinetics of αV-proficient and -deficient tumours even though the latter were much more strongly infiltrated by proliferative PD-1+CD8+ effector T cells. Thus, it is clear that targeting tumour αV as single therapeutic agent is not sufficient in the clinical setting to reliably eradicate cancer. TGF-β inhibitors as single therapeutic agents also observed limited clinical success, likely due to restricted targeting of TGF-β-responsive cells and inadequate effects on cancer cell proliferation41. Consistently, targeting TGF-β with neutralizing mAb had no effect on B16F10E tumour growth. Moreover, the development of cancer therapies targeting TGF-β signalling has been hindered by dose-limiting toxicities42. Thus, targeting tumour αV in combination with ICB appears a promising strategy to selectively target non-immune cells and ensure a superior safety profile compared to TGF-β inhibitors.Targeting tumour αV integrin is considered as a serious cause of resistance to ICB by activating TGF-β and thereby excluding CD8+ T cells from the TME. Therefore, manipulating αV seems a promising approach to improve the clinical success of ICB. In this context, we show that decreased expression of tumour αV correlates with improved outcome in NSCLC patients treated with anti-PD-(L)1. Our data indicate that αVlow tumour-bearing patients displayed improved PFS compared to αVhigh tumour-bearing patients associated with an increase in tumour infiltration by CD103+CD8+ lymphocytes. Moreover, our in vivo mouse model revealed that tumour αV knockout combined with anti-PD-1 resulted in delayed tumour progression likely associated with decreased TGF-β activation and concomitant T cell recruitment. Consistently, blockade of αVβ8 and PD-1 synergistically promoted better anti-tumour response and more beneficial effects27. The present study further shows that beneficial effects are likely associated with improved CD8 T cell immunity and increased tumour infiltration by tumour-specific CD103+CD69+ TRM cells. Indeed, tumour growth control was dependent on anti-tumour CTL response and correlated with an increase in tumour infiltration by proliferative tumour-reactive CD8+ T lymphocytes producing granzyme B. CD8+ T cell-mediated cytotoxicity against the cognate target correlated with upregulation of MHC-I and PD-L1 on B16F10E-KO cells, excluding an association of the beneficial effect with a reduced PD-L1 expression on αV-knockout tumours as previously reported43. Of note, αV-deficient cancer cells displayed dramatic morphological modifications probably due to alteration in cell-adhesive capacities. However, these changes did not affect tumour cell susceptibility to CTL-mediated killing in vitro, suggesting that in vivo αV modulation would not have an impact on target cell sensitivity to CD8+ T cells. Preclinical mouse models revealed that TGF-β blockade also unleashed an anti-tumour CTL response, rendering tumours more susceptible to anti-PD-(L)122. Thus, co-administration of blocking anti-TGF-β and anti-PD-L1 antibodies facilitated T cell recruitment and promoted anti-tumour immunity leading to tumour regression44. Selective targeting of LAP-TGF-β overcomes primary resistance to ICB by increasing intra-tumoural CD8+ T cells and decreasing immunosuppressive myeloid cells42. Moreover, simultaneous targeting of TGF-β and PD-L1 has been described to enhance anti-tumour activity by increasing the influx and functionality of CD8+ T lymphocytes in the TME46. However, targeting TGF-β and PD-1 with specific blocking antibodies had no effect in our in vivo mouse model, suggesting that a minimal level of the cytokine is needed for TRM cell formation and control of tumour progression.Tumour α+CD8+ TRM cells in peripheral tissues and solid tumours, at least in part by triggering CD103 expression. Along the same lines, we show that by activating TGF-β, tumour αV promotes CD103 expression on activated CD8+ T lymphocytes in vitro, and that integrin knockout inhibits this process at least in part, by compromising activation of Smad2/3, critical regulators of the ITGAE gene, which encodes CD10347. Although αV knockout induced a slight decrease in the frequency of CD103+CD8+ TIL in anti-PD-1-treated mice, an increase in the percentage of CD103+CD69+CD8+ TRM cells was observed. These data suggest that by inhibiting TGF-β activation, αV knockout in anti-PD-1-treated tumours results in recruitment and expansion of CD8+ T cells, and subsequent induction of CD103 on TCR-engaged CTL as we previously reported48. These CD103+CD69+CD8+ TRM cells may correspond to truly tumour-reactive T cells the differentiation of which is associated with TGF-β that might be activated in the TME by a mechanism dependent of MMP on tumour cells or αV integrins on immune cells. Although tumour αV-dependent active TGF-β may participate in CD103+CD8+ TRM differentiation, higher levels of TGF-β may compromise T cell recruitment in tumours and their expansion at the memory phase. We consistently observed in anti-PD-1-treated NSCLC patients that the density of CD8+CD103+ TRM cells in tumours with αVlow profile was higher than in tumours with αVhigh.TGF-β is an important regulator of local immune responses via its participation in the differentiation of CD103+CD8+ T cells, our in vivo studies showed a strong decrease in the frequency of CD103+FoxP3+CD4+ Treg in αV-knockout tumours, suggesting that CD103 expression in FoxP3+CD4+ and CD8+ T cells is regulated by distinct mechanisms. CD103+CD4+ Treg were described as suppressing T-effector cell activation more strongly than their CD103neg counterparts, and that silencing TGF-β reduced their frequency and suppressive activity49. Targeting LAP on Treg cells also promoted anti-tumour immunity by decreasing TGF-β production and increasing tumour infiltration with activated CTL50. Consistent with this, we observed that an optimized local anti-tumour CD8 T cell immunity correlates with a decreased frequency of CD103+CD4+ Treg cells in anti-PD-1-treated-αV-knockout tumours. Blockade of αV on human Treg cells has also been shown to inhibit Treg-mediated immune suppression by inhibiting TGF-β maturation52. This integrin is also expressed by CD103+ DC and confers the capacity to generate Treg by activating TGF-β53. Thus, targeting αV integrin in tumour and immune cells would permit inhibition of Treg generation and promote CD8+ T cell recruitment and effector functions within the TME.As opposed to CD103RM recruitment within epithelial tumour regions, and that TGF-β enhanced CD103-dependent T cell adhesion and migration and promoted anti-tumour CTL functions54. CD103 also participates in TRM residency in tumours, and targeting this integrin or TGF-β has been shown to decrease the number of intra-tumoural TRM cells55. CD103 favours T cell retention in epithelial tumour islets through binding to its ligand, the epithelial cell marker E-cadherin56. Thus, TGF-β is involved in the retention of TRM at least in part through the induction of CD103 and CD6957. Our in vivo studies showed that αV knockout combined with PD-1 blockade induced an increase in intra-tumoural granzyme-B-producing CD103+CD69+CD8+ T cells with a parallel optimization of CTL-mediated target cell killing. Moreover, blockade of CD103 inhibited growth control of anti-PD-1-treated-αV-knockout tumours associated with a decrease in T cell-mediated cytotoxicity toward target cells. Thus, by activating TGF-β, αV integrin plays a dual role in regulating anti-tumour T cell response; on the one hand by controlling CD8+ TRM differentiation and retention in epithelial tumour regions, and on the other, by participating in T cell exclusion and dysfunction. The capacity of αV on stromal cells in controlling the formation and epithelial residence of CD8+ TRM cells by activating TGF-β has been demonstrated in healthy tissues58. Moreover, the expression of αV by DC has been reported to prepare naïve CD8+ T cells for the formation of epithelial TRM cells36. These findings support the hypothesis that αV on tumour-infiltrating immune cells participate in the formation of CD103+CD8+ TRM in the TME and in regulating T cell recruitment and functions.We previously demonstrated that CD103 is directly involved in TV on tumour cells shapes the tumour immune infiltrate and dictates the cellular and cytokine components of the TME by activating TGF-β and thereby by regulating CD8 T cell immunity and response to anti-PD-1. This integrin could be considered as a potential biomarker of response to T cell-based cancer immunotherapies and targeting it may improve the clinical benefit from ICB.Overall, we conclude that α15. The monocentric retrospective study at Gustave Roussy describes a first cohort (cohort 1) of 106 and a second cohort (cohort 2) of 51 advanced NSCLC patients treated between 2012 and 2020, with anti-PD-(L)1 administered in a variety of settings. Demographic, clinical, pathological, and molecular data were collected  during 1 h at room temperature. Amplification and detection were performed with an Ultraview kit using amplification and 3,3′-diaminobenzidine as a chromogen. Integrin αV staining was evaluated by a pathologist as the prominent intensity for each tumour. Tumours were considered αVlow when cancer cells were negative for αV expression (αVneg) or when their αV expression level was weaker than on stromal cells and infiltrating immune cells. They were considered αVhigh, when αV expression on more than 75% of cancer cells was stronger than on stromal and immune cells. Multiplexed fluorescent IHC for CD8+CD103+ and CD8+CD103neg lymphocytes was performed by sequential staining of a single tissue section with anti-CD8 , anti-CD103 , and anti-cytokeratin 14. For each staining, the HRP-conjugated amplification system was associated with a tyramide-coupled fluorophore: Opal 690, Opal 250, and Opal 570, respectively. Multispectral fluorescent images were captured using the Vectra 3 microscope (PerkinElmer) and regions of interest were selected. Image analysis using InForm software (PerkinElmer) included spectral unmixing, nuclei detection based on DAPI staining, and cell segmentation followed by cell phenotyping for identification of cell populations defined by the combination of individual markers. The density (number of cells per square mm) of CD8+, CD8+CD103+, and CD8+CD103neg was determined for each tumour sample in the total tumour area based on tissue segmentation. Results from image analysis were validated for all cases.IHC was performed on archived FFPE tumour tissue specimens using Ventana Benchmark and Discovery automated platforms. After deparaffinization and epitope retrieval in CC1 buffer , tissue sections were incubated with primary mAb for α+, CD8+CD103+, and CD8+CD103neg TIL was assessed using the log-rank maximization method59. Survival analyses were performed using the Kaplan−Meier method and the log-rank test. All p-values inferior to 0.05 were considered statistically significant.OS for treatment-naïve patients was calculated from the date of primary tumour resection until death due to any cause. PFS for anti-PD-(L)1-treated patients was calculated from the date of the first immunotherapy administration until disease progression or death due to any cause, whichever occurred first. The best cut-point for total CD8p-value cut-off = 0.10). The proportional hazard hypothesis was verified with the Schoenfeld residual method. Predictive factors of disease control were tested with logistic regression in univariate and multivariate analyses. The alpha level was 5%. Statistical analyses were performed with RStudio v1.1.463 .A Cox proportional hazards regression model was used to evaluate independent prognostic factors for OS and PFS. Variables included in the final multivariate model were selected according to their clinical relevance and statistical significance in univariate analysis (n = 70). Gene-expression data were automatically computed to generate Kaplan–Meier plots 60.Using http://kmplot.com, analysis of gene-expression datasets derived from NSCLC patients was performed on a group of stage I surgically managed patients who did not receive radiotherapy or chemotherapy  and mouse  genes . cDNA was synthesized with the Maxima First Strand cDNA Synthesis Kit (ThermoFischer Scientific). qRT-PCR was performed on a Step-One Plus (Applied Biosystems) using Maxima SYBR Green Master Mix (ThermoFischer Scientific). Expression levels of transcripts were normalized to V , -β6 , -β8 , anti-E-cadherin  and -EpCAM  mAb were used. Cell surface and intracellular staining of mouse cells was performed on single-cell suspensions using antibodies specific to the following molecules: CD51 , E-cadherin , H2-Kb/H2-Db , PD-L1 , integrin β5 , integrin β3 , CD3 , CD4 , FoxP3 , CD8α , CD62L , CD44 , PD-1 , CD69 , CD103 , KLRG1 , Ki-67 , IFN-γ , granzyme B , pSmad2/3 , MHC-II  and CD11c . Dead cells were excluded using the Live/Dead Fixable Blue Dead Cell Stain Kit (Invitrogen 186684). Staining of MMP14 was performed with specific mAb  followed by secondary antibody staining .For human tumour cell surface and intracellular staining, anti-αFor intracellular staining, cells were fixed, permeabilized with the FoxP3 staining buffer set according to the manufacturer’s instructions (eBioscience 00-5523-00). Flow cytometry was conducted on an LSR Fortessa (BD) and analyzed using FlowJo software V10 (Tree Star).HD blood samples were collected from the French blood bank (Etablissement Français du Sang (EFS); agreement number N°12/EFS/079. PBMC were isolated by a Ficoll-Hypaque gradient.NSCLC tumours were obtained from the Centre chirurgical Marie Lannelongue. For freshly isolated TIL and cancer cells, human lung tumours were dissociated mechanically and enzymatically using a Tumour Dissociation Kit . Mononuclear cells were then isolated by a Ficoll-Hypaque gradient. Tumour cells were isolated by magnetic separation using Tumour Cell Isolation Kit . Healthy donors and patients provided their written informed consent prior to inclusion in this study. All human experiments were approved by the Institutional Review Board of Gustave Roussy.30. The allogeneic NSCLC cell lines IGR-Pub, IGR-Heu, ADC-Coco, ADC-Tor, and ADC-Let were derived from tumour specimens in one of our laboratories. H1355 (adenocarcinoma), H460, and H1155 (large cell carcinoma) were a generous gift from Dr. S. Rogers , and A549 (adenocarcinoma), SK-Mes, Ludlu  and DMS53  were purchased from the European Collection of Cell Cultures30. Lung tumour cells lines were grown in DMEM/F-12 medium (ThermoFischer Scientific) supplemented with 10% fetal calf serum (FCS), 1% Ultroser G , 1 mM sodium pyruvate, and antibiotics (50 U/ml penicillin and 50 μg/ml streptomycin). All the cell lines are mycoplasma-free and were regularly tested for mycoplasma contamination. We regularly authenticate IGR-B2 cell line by testing recognition by autologous B90 CTL clone, and HLA-A2 expression.The IGR-B2 cell line and the autologous B90 CTL clone were derived from patient Bla large cell carcinomaThe IGR-B2 NSCLC cell line was stably transfected with pcDNA3.1-hTGFβ-LAP plasmid (from one of our laboratories) using Lipofectamine 2000 (ThermoFischer Scientific) according to the manufacturer’s instructions. The cell line obtained was cloned, and one clone, IGR-B2T, was selected for its capacity to produce TGF-β detected in cell supernatant by ELISA .V expression in the IGR-B2T cell clone was performed using CRISPR-Cas9 technology. The cells were double transfected with integrin αV CRISPR-Cas9 KO plasmid  and integrin αV HDR plasmid . The transfection efficiency was confirmed by flow cytometry for double GFP and RFP expression. The GFP+RFP+ cells were cell sorted and treated with puromycin . The generated cell line was tested for αV knockout by FACS, and then cloned by cell sorting. A clone, thereafter named IGR-B2T-KO, was retained for further studies.Homozygous knockout of αb) was purchased from the American Type Culture Collection (ATCC CRL-6475) and grown in DMEM-F12 medium supplemented with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, and antibiotics. It was stably transfected with pcDNA-E-cadherin plasmid  and a clone, named B16F10E, was isolated and used in all experiments. Homozygous knockout of αV expression was performed as above using the CRISPR-Cas9 technology (sc-421169 and sc-421169-HDR), and a clone, named B16F10E-KO, was selected for further experiments.The B16F10 melanoma cell line . CM was used to measure the concentration of total TGF-β by ELISA using human and mouse TGF-β DuoSet ELISA kits . For MMP activity inhibition, tumour cells were cultured in the presence of the MMP inhibitor GM6001  or DMSO control, and four days later, CM was harvested.V-knockout tumour cells was evaluated using the thymic mink lung epithelial cells Mu.1LV , cultured with 24 h CM. First, Mu.1LV cells were resuspended in DMEM/F12 medium supplemented with 10% FCS, plated for 24 h, and then transfected with the pGL3-(CAGA)9-Lux reporter construct, which contains concatemerized CAGA elements that bind Smad3 and Smad4 complexes and are transactivated by both TGF-β and Smad3/4 expression61. Twenty-four hours after Mu.1LV transfection, CM was added to serum-free Mu.1LV cells, and cells were lysed and assayed for luciferase activity using Bright Glo Luciferase Assay System (Promega). For quantification of active and total TGF-β in mouse B16F10E and B16F10E-KO, tumours were crushed and rinsed with phosphate-buffered saline (PBS), and the solution was used for active and total TGF-β quantification .Activation of LAP-TGF-β by parental and α5 PBMC/well were added and cultured for 3 days at 37 °C and 5% CO2 with CM from B16F10E or B16F10E-KO and IGR-B2T or IGR-B2T-KO cells in the absence or presence of a low (1 ng/ml) or high (5 ng/ml) dose of rTGF-β  or blocking anti-TGF-β mAb . Expression of CD103  and KLRG1  on CD3+CD8+ cells  was then determined by flow cytometry.Plates were coated with 1 µg/ml of anti-CD3 blocking mAb  for 24 h at 4 °C. 5 × 10V and LAP-TGF-β interaction. PLA was performed on fixed and permeabilized tumour cells according to the manufacturers’ instructions . After blocking, anti-αV  and anti-LAP-TGF-β  antibodies were used. PLA-minus and PLA-plus probes, containing the secondary antibodies conjugated with oligonucleotides (dilution in Antibody diluent 1/5), were added and incubated for 1 h at 37 °C. After hybridization, oligonucleotide ligation and a rolling circular amplification were performed. Cell membranes were stained with WGA red conjugate , nuclei with Fluoromount-G mounting media containing DAPI (eBioscience), and then analyzed with a confocal microscope SP8 (Leica). The number of PLA signals, marked as green dots on the cell surface, was counted in 25−30 fields containing 500−600 cells, by image analysis .The green PLA signal was observed for αTumour cells were labelled with 5 µM Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) dye (Stemcell Technologies) for 5 min at 37 °C. Cells were then washed twice with FCS-free medium and cultured for 6 days. The proliferation index was calculated every other day by flow cytometric analysis using ModFit LT™ programme v5.0.nu mice (6 weeks old) were inbred in-house. For each experiment, groups of four to eight mice, 7−9 weeks of age received 2 × 105 tumour cells subcutaneously in the right flank. Tumour volume was measured using a calliper every second day and estimated using the following formula: π/6 × length × width × thickness (mm3). The mice were sacrificed when the tumour size exceeded the acceptable institutional limit of 2000 mM3 by CO2 inhalation.Animal experiments were performed in accordance with Gustave Roussy’s relevant ethical regulations for animal testing and research. This study received ethical approval from the institutional animal committee (CEEA no. 026: 2018-056-16280) after receiving the legal approval from French Minister of Higher Education, Research and Innovation under the procedure number APAFIS#16281-2018072515064652v2. Female C57BL/6 mice were purchased from Envigo. The female athymic nude Crl:NU(NCr)-Foxn1+ or CD8+ cells using anti-CD45 or anti-CD8 microbeads , and then purified using the POSSEL2 programme on MultiMACS. The positive fraction was recovered for TIL analysis and the negative fraction for tumour cell analysis by flow cytometry and ex vivo cytotoxicity assay.For TIL isolation, tumours were harvested at day 14 and digested for 30 min at 37 °C according to the Tumour Dissociation kit protocol . Tumours were crushed on 100 μm cell strainers and washed twice with PBS 2% FCS. Single-cell suspensions were enriched for CD45For in vivo blockade with neutralizing mAb, mice received i.p. 100 μg/mouse of anti-CD8 , intra-tumoural (i.t.) 50 μg/mouse of anti-CD103 , i.t. 25 μg/mouse of anti-TGF-β  and/or 200 μg/mouse of anti-PD-1  mAb or isotype control . For tumour outgrowth experiments with anti-PD-1, mice were treated on days 4, 7, and 11 after tumour inoculation, whereas with anti-CD8 and/or anti-PD-1, mice were treated on days 4, 7, 10, and 13. For experiments with anti-CD103 and/or anti-PD-1, mice were treated on days 4, 7, 11, and 15, whereas for experiments with anti-TGF-β and/or anti-PD-1, mice were treated on days 4, 8, 11, and 14. TIL were sorted and analyzed on day 14 or 15. For experiments with anti-CD103, anti-TGF-β, and/or anti-PD-1, mice were treated on days 4, 7, 11, and 14. TIL were sorted and analyzed on day 15 or 17.51Cr) release assay30. For cytotoxicity with mouse TIL, CD45+ cells were isolated and cultivated overnight in a medium containing 50 U IL-2. CD8+ T cells were purified using microbeads and co-cultured for 4 h with syngeneic 51Cr-labelled B16F10E or B16F10E-KO tumour cells.The cytotoxic activity of B90 CTL clone against autologous IGR-B2, IGR-B2T, and IGR-B2T-KO tumour cells was measured using a conventional 4-h chromium  test with Tukey’s correction, and two-way ANOVA. Statistical analyses were performed with GraphPad Prism software V8 .Statistical significance was determined with the unpaired or paired Student Further information on research design is available in the Supplementary InformationReporting Summary


Yeung and Wong's study is an example of how modern information retrieval technology can produce good results. It seems to clearly indicate that Hayes and Patterson  is the oPaterson instead of Patterson). Therefore, it is difficult to obtain an overview of the GRS from this paper alone. If we seek further information, we notice that the authors belong to Scott Co. Moreover, it is easy to see that Scott is the famous Walter Dill Scott by referring to Freyd  should be Scott and Hayes  in termsIf one still wants to name the sole inventor, Walter Dill Scott will be it.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hypoglycemia is a common, symptom seen in individuals. Hypoglycemia in the elderly is both under-recognized and misdiagnosed due to nonspesific hypoglycemic symptoms and accompanying comorbidities in this population. In diabetic individuals, hypoglycemia is most commonly caused by administering insulin or sulphonylureas and insulin secretagogues. Other drugs, such as antibiotics or beta-blockers, have been reported to reduce blood glucose to abnormally low levels. Hypoglycemia in non-diabetic patients is considered a rare event, and the possible reasons may be reactive hypoglycemia, insulin-secreting tumours and other malignancies, hypopituitarism, hypocortisolism, alcohol abuse, inappropriate insulin self-administration, malnutrition, renal failure and sepsis.An 86- year- old male was admitted to the emergency department with hypoglycemia diagnosed with non-pancreatic neuroendocrine tumour (NET) on lung secreting insulin. No surgical intervention or chemotherapy was planned due to patients age and comorbidities so best supportive care was planned. We used prednisone for symptomatic treatment of hypoglycemia and the patient has been followed up periodically. In this period he had no hypoglycemic attack.For patients with hypoglycemia who are unable/decline to receive any further treatment, low dose glucocorticoid is a good choice to achieve normoglycemia. It seems to be more cost effective compared to other treatment options. Furthermore hospitalisation rates may decrease due to decreased hypogylcemic attacks. Hypoglycemia is a common, under-recognized symptom seen in individuals. Elderly individuals are more vulnerable to complications of hypoglycemia than others because of co-morbidities and senility. Furthermore, autoregulatory responses are impaired through aging, which means the symptoms of hypoglycemia are often less specific and are therefore either missed or incorrectly diagnosed as transient ischemic attacks or other cerebrovascular events. The elderly are also more prone to the effects of hypoglycemia such as the increased risk of accidents, falls, fractures, hospitalizations, in-hospital mortality and long-term impairment ocognition. Thus it is very important to evaluate the patient for hypoglycemia when there is a clinical suspicion.The most common endogenous cause of hypoglycemia is hyperinsulinemia secondary to islet cell tumours of the pancreas. An 86- year- old male was admitted to the emergency department with altered mental status. He had a similar episode nine months ago and was told that this situation was due to hypoglycemia but no further tests were done. He had a history of coronary bypass surgery, myocardial infarction, heart failure, systemic hypertension, chronic obstructive lung disease but no diabetes. He had not been prescribed insulin or oral hypoglycemic agents before. No one in the household was diabetic or using any medication for diabetes. He was immobile for two years due to vertebral fracture. His vital signs were stable . He was agitated, disoriented and uncooperative on physical examination. His neurological examination was normal. His venous glucose level was 34 mg/dl (N: 74 – 106 mg/dl), his laboratory values were otherwise unremarkable on admission. He was admitted to the internal medicine department for further evaluation of hypoglycemia. His capillary glucose level was monitored and intravenous dextrose infusion planned when hypoglycemia was detected. His plasma glucose level was 53 mg/dl (N: 74 – 106 mg/dl), insulin was 5.21 uIU/ml (N: 1.9 – 23 uIU/mL), c- peptide was 3.5 ng/ml (N: 0.8 – 4.2 ng/ml). Even though these values were in normal ranges for our hospital laboratory, they were consistent with endogenous hyperinsulinemia according to Endocrine Society Guideline. 2A CT – guided biopsy from this lesion was performed by invasive radiologists and histopathology revealed high grade neuroendocrine carcinoma. The patient was referred to the medical oncology department; they did not suggest any surgical intervention or chemotherapy due to his advanced age and comorbidities and recommended supportive care. The patient has been followed up by our internal medicine outpatient clinic periodically. He died of decompensated heart failure after 3 months. He had used prednisone 10 mg/day for one month and 5mg/day thereafter. In this period he had no hypoglycemic attack.In diabetic individuals, hypoglycemia is most commonly caused by administering insulin or sulphonylureas and insulin secretagogues. Other drugs, such as antibiotics or beta-blockers, have occasionally been reported to reduce blood glucose to abnormally low levels. 11Initial evaluation of hypoglycemia should proceed according to usual practice. The Endocrine Society Guidelines recommend investigation in patients in whom Whipple's triad is fulfilled.Malignancies causing hypogylcemia can be divided into several categories according to underlying mechanisms: tumour insulin secretion pancreatic islet b-cell tumours, insulinomas or non-islet-cell tumours such as bronchial carcinoid or gastrointestinal stromal tumours; tumour IGF2 precursors secretion (big IGF2) ‘IGF2-oma’ ; tumour somatostatin secretion ‘Somatostatinoma’ (pancreatic neuroendocrine tumour); tumour IGF1 secretion ‘IGF1-oma’ (large-cell carcinoma of the lung); tumour glucagon-like peptide 1 (GLP1) secretion ‘GLP1-oma’ (ovarian neuroendocrine tumour); autoantibodies to insulin or its receptor ‘Tumour autoimmune hypoglycemia’; or other tumour-related factors including massive tumour burden, massive liver tumour infiltration or pituitary and/or adrenal glands tumour destruction.If there are clues to NICTH , this can be pursued early. Otherwise, we would consider this rare diagnosis if the workup of the preceding causes was unrevealing.Initial treatment of hypoglycemia is accomplished by oral glucose and/or iv glucose or dextrose-containing fluids as necessary. In many cases, this suffices to avoid further hypoglycemia. Once NICTH is identified and a primary tumour is found, the mainstay of treatment is surgical resection, which is curative for hypoglycemia if resection is complete. 23In our case an elderly patient experiencing altered consciousnes due to hypoglycemia was diagnosed with high grade neuroendocrine carcinoma. He was suitable neither for any chemotherapy nor surgical intervention because of his accompanying comorbidities and patients' preference. For such patients with hypoglycemia who are unable/decline to receive any further treatment, low dose glucocorticoid is a good choice to achieve normoglycemia. It seems to be more cost effective method compared to other treatment options. Furthermore hospitalisation rates may decrease due to decreased hypogylcemic attacks.