Most Crohn’s disease-associated small bowel carcinomas (CrD-SBCs) are diagnosed in advanced stage and have poor prognosis. To improve diagnosis and therapy, a better knowledge of tumour precancerous lesions, histotypes and prognostic factors is needed. We investigated histologically and immunohistochemically 52 CrD-SBCs and 51 small bowel carcinomas unrelated to inflammatory disease, together with their tumour-associated mucosa, looking for Crohn-selective changes. Histologic patterns and phenotypic markers potentially predictive of CrD-SBC histogenesis and prognosis were analysed. Cytokeratin 7 or MUC5AC-positive metaplastic changes were found in about half of investigated CrD-SBCs, significantly more frequently than in CrD-unrelated SBCs. They correlated with metaplastic changes of their associated mucosa, while being absent in normal ileal mucosa. Histologic patterns suggestive for progression of some cytokeratin 7 and/or MUC5AC-positive metaplastic lesions into cancer of the same phenotype were also observed. Patient survival analyses showed that tumour cytokeratin 7 or MUC5AC expression and non-cohesive histotype were adverse prognostic factors at univariable analysis, while cytokeratin 7 and non-cohesive histotype were also found to predict worse survival in stage- and age-inclusive multivariable analyses. Besides conventional dysplasia, hyperplasia-like non-conventional lesions were observed in CrD-SBC-associated mucosa, with patterns suggestive for a histogenetic link with adjacent cancer. In conclusion the cytokeratin 7 and/or MUC5AC-positive metaplastic foci and the non-conventional growths may have a role in cancer histogenesis, while tumour cytokeratin 7 and non-cohesive histotype may also predict poor patient survival. Present findings are worth being considered in future prospective histogenetic and clinical studies.The online version contains supplementary material available at 10.1007/s00428-021-03109-2. Most Crohn’s disease-associated small bowel carcinomas (CrD-SBCs) show unfavourable outcome due to a highly invasive pattern coupled with advanced stage at diagnosis –3. AttemIn a recent study on small bowel cancers, we were impressed by the large prevalence of non-intestinal tumour cell phenotypes among CrD-SBCs, partly mimicking gastric and/or pancreatobiliary duct cells, as well as by the occurrence, within their associated non-tumour mucosa, of focal changes showing the same “metaplastic” cell phenotypes as the cancer tissue itself . We wondIn this study, we analysed the tumour cell phenotype, as well as putative precursor lesions in the background mucosa, of a large cohort of CrD-SBCs, collected and followed through the Small Bowel Cancer Italian Consortium and correlated these data with patients’ clinical outcome. Histologic and immunohistochemical findings in these cases were compared with those obtained in sporadically arising SBCs, in the absence of predisposing chronic immune-inflammatory diseases.Fifty-two cases of CrD-SBCs , were collected and followed by 18 tertiary referral centres participating in the Small Bowel Cancer Italian Consortium. The patients had surgical resection with accurate tumour staging according to AJCC  and exteAll cancers were classified histologically according to criteria previously found to be pathologically and clinically informative for SBCs , 21, 22.In accordance with previous studies –23, diffFor immunohistochemistry, 4-μm-thick tissue sections were stained using the following antibodies: CK7 , MUC5AC , MUC6 , MUC2 , CDX2  and CK20 ; in order to assess tumour cell phenotype, for each one of the previously listed markers, only tumours with at least 10% of immunoreactive cells were regarded as positive, as previously reported , 26. CumFor the assessment of mismatch repair (MMR)/MSI status, MLH1 , MSH2 , MSH6  and PMS2 , immunostainings were performed and tumours were evaluated as MMR-proficient or MMR-deficient/MSI. Tumours were classified MMR-proficient when they retained the expression of MMR proteins and MMR-deficient/MSI (absent expression) when they lost MMR nuclear staining in the totality of neoplastic cells but not in the internal positive control (intra-tumour or peri-tumour inflammatory cells or non-neoplastic mucosa). Tumour-infiltrating lymphocytes (TILs) were stained using CD3 antibody  and quantified as previously described [Cancer-associated mucosae were evaluated for CK7 and MUC5AC expression, and they were scored as positive when at least one focus of more than five CK7-reactive or MUC5AC-reactive cells was found.A central pathology review was performed by two gastrointestinal pathologists (AV and ES).P-value <0.05 was considered statistically significant.The distribution of biomarkers was reported as counts and percentages, and Fisher’s exact test was used to compare them across clinical groups. Cumulative cancer-specific survival was plotted according to the Kaplan–Meier method. The cause of death was identified by the treating physician and/or from the corresponding code in the death certificate. The follow-up extended from the date of surgery to the date of death or last follow-up. The association of candidate prognostic factors and tumour-related death was estimated by means of univariable and multivariable Cox regression. Hazard ratios (HR) and 95% confidence intervals (CI) were computed. The following known non-collinear predictors of tumour death were included in the multivariable model in addition to CK7 or MUC5AC: stage, histotype and patient age at SBC diagnosis. Given the low number of events, no further adjustment for confounding factors could be performed. Hazard ratios and their 95% confidence intervals were computed. The proportional hazard assumption was tested, based on Schoenfeld residuals. A two-sided morphologic histotypes  showed at least one of the two markers and 29% (15 cases) both markers. Of interest was the significantly lower expression by tumour cells  of MUC6, a specific marker of pyloric and Brunner glands. The three “canonical” intestinal markers CDX2, CK20 and MUC2 were individually expressed by 56%, 54% and 54% of tumours, respectively, with 79% of all cases showing at least one of such markers. Although the expression of metaplastic or canonical markers inside the same tumour cell or tumour area was often mutually exclusive, some hybrid cells expressing both types of markers were also found. Only a limited, non-significant association was observed between the CK7 and MUC5AC expression by tumour cells , while a significant association was found between MUC5AC and MUC6 expression (p=0.003), with all but one MUC6-positive cancers also showing MUC5AC reactivity. In addition, an inverse association was noted between the expression of CDX2 and that of CK7 (p=0.029) or MUC5AC (p<0.001).Data on the expression of intestinal  and non-intestinal markers  in 52 CrD-SBCs are shown in Table p<0.001) and among cohesive cancers . A peculiarity of CK7 expression was its concentration in budding cells and in poorly differentiated clusters at the tumour invasive front of glandular cancers, a pattern suggesting worse behaviour [p=0.005), which was however not seen in the three signet-ring cell SBCs. Eight of 29 cohesive cancers showed relevant desmoplasia, usually at the tumour-invasive front. As a whole, considering the 23 tumours with desmoplasia, 19 of them (83%) also showed CK7 expression of their epithelial component.CK7 turned out to be more expressed among mixed-type cancers (92%), while being significantly less expressed among purely diffuse cancers . Tumour MSI/MMR-deficiency was detected in 10 (19%) CrD-SBCs, only 5 of which with high TIL density.p53 protein immunoreactivity of more than 50% of cancer cells was found in 27 of the 52 (52%) cases (Suppl Table p<0.001), in the absence, however, of significant tumour stage differences (p=0.573). Only two diffuse-type cancers occurred among no-PID-SBCs, versus 11 among CrD-SBCs (p=0.008), while the non-cohesive histology accounted for only 25% of no-PID-SBC cases (Table p=0.046). By comparing each other Table p=0.008) and MUC5AC (p=0.044) than no-PID-SBCs. Notably, such differences remain highly significant (p=0.004 for CK7 and p=0.009 for MUC5AC) when the analyses were restricted to jejunal-ileal tumours only (i.e. 50 CrD-SBCs vs 45 no-PID-SBCs). No relevant difference was noted among the two etiologically different tumour groups concerning cumulative intestinal marker expression (p=0.556); however, the percentage of cases with CDX2 expression turned out to be significantly lower (p=0.007) in CrD-SBCs. MUC6 was rarely expressed in both groups.Mean patient age at tumour diagnosis of the 52 CrD-SBCs was 57.6 ±12.9 years, significantly lower than that of no-PID-SBC cases . Tumour MSI/MMR deficiency was detected in 20 (39%) no-PID-SBC cases, a significantly higher frequency (p=0.031) than that of CrD-SBCs. In keeping with this finding, a high TILs density was more frequently (p=0.023) observed in no-PID-SBCs , rather than in CrD-SBCs. Furthermore, in no-PID-SBCs only, an inverse correlation (p=0.01) was found between widespread p53 expression and MSI status.p53 immunoreactivity of more than 50% of cancer cells was observed in 14 (42%) of no-PID-SBC cases investigated, without significant difference between the two groups . CK7 and MUC5AC were frequently expressed by the same mucosa  and often associated with focal loss of intestinal markers. Such metaplastic foci were more frequently observed in juxta-tumoural areas, where patterns suggestive for direct transition to neoplasia of the same phenotype, as well as more atypical CK7-positive foci, with buttons or cords of non-polarized and crowded cells with large nuclei and prominent nucleoli, were occasionally identified. In addition, CK7-positive ductular-type structures unrelated to adjacent intestinal crypts or villi and lacking signs of cellular differentiation were identified. On the other hand, MUC6-positive pyloric-type glands, which usually showed a well-differentiated mucin-rich pattern lacking atypical changes, were as a rule CK7-negative, and most of them were MUC5AC-negative.In addition to conventional dysplastic lesions with well-known histology –7, CrD-Sp<0.001) and (ii) a highly significant (p<0.001) association existed between CK7 expression by CrD-SBCs and their respective mucosae, while such an association was lacking among no-PID-SBC cases. No CK7-reactive foci were observed in histologically normal jejunal-ileal mucosa from tumour-free sections of no-PID patients.A detailed analysis of CK7 distribution among the non-neoplastic mucosa associated with CrD-SBCs or non-PID-SBCs is outlined in Table While searching for metaplastic changes in CrD-SBC juxta-tumour mucosa, we observed several non-conventional lesions, often forming papillary-to-polypoid protrusions, with either mucinous or columnar eosinophilic cell differentiation. Direct continuity of such non-conventional, hyperplasia-like and mildly atypical lesions with cancer, without interposition of conventional adenomatous dysplasia, was ascertained in seven of the 46 cases investigated for this purpose. All, but one, cancers showed glandular histology and all, but two, strong and widespread (>50% of cells) p53 reactivity, while the precursor component lacked frank, conventional (adenomatous) dysplasia and showed only scattered p53 reactivity  was covered by a MUC2-reactive “hypermucinous” non-conventional lesion . At univariable analysis, MUC5AC-reactive cases also showed an association with a worse prognosis  . Patients with CrD-SBC positive for CK7 had significantly worse cancer-specific survival compared to those with CrD-SBC negative for CK7  Fig. . In addi38) Fig. .Fig. 3Kp=0.35). However, CDX2 expression alone was found to correlate with better overall survival . When CK7 and CDX2 expressions were combined, patients with CK7-positive and CDX2-negative tumours showed the worst outcome, while patients with CK7-negative and CDX2-positive tumours showed the best survival, with intermediate figures for the double-positive or double-negative tumours  showed no significant correlation with patient survival , whereas MMR-d was not associated with patient cancer-specific survival .An association of tumour non-cohesive histotype with worse patient survival was also observed .Importantly, a multivariable analysis including tumour CK7 (positive versus negative), histotype (cohesive versus non-cohesive), stage (I+II versus III+IV) and patient age at diagnosis showed an independent prognostic power for each of the four parameters Table . MUC5AC,In this study, we found that two non-intestinal markers, i.e. CK7 and MUC5AC, were expressed at significantly higher percentages in both tumour tissue and associated mucosa of CrD-SBC cases compared to ordinary no-PID-SBC cases. This finding, in addition to a significant increase among CrD-SBCs of the non-cohesive histotype and a lack of CK7 and MUC5AC expression in histologically normal ileal mucosa, suggests that a distinctive carcinogenic process plays a role in a relevant fraction of CrD-SBC cases. This hypothesis is reinforced by the highly significant association of CK7 expression between tumour tissue and respective mucosa, an association lacking in no-PID-SBC cases, as well as by the occasional finding of direct topographic continuity between CK7 and/or MUC5AC-positive mucosal lesions and the invasive cancers of the same phenotype, which we identified in some CrD-SBC cases. The additional observation that tumour CK7 positivity was an independent adverse prognostic factor points to some mechanism underlying the metaplastic growth which may favour both tumour development and progression.Metaplastic changes of gastric type, with special reference to MUC6-positive “pyloric” glands, have long been reported in Crohn’s disease intestinal mucosa , 11. WhiCrD-SBC patient survival analysis highlighted an important prognostic influence of CK7-positive metaplastic phenotype. In particular, we found a significant association between CK7 expression by SBC and worse patient survival, which was confirmed at multivariable analysis inclusive of patient age, stage and histotype. Present findings suggesting a relationship between CK7 expression and CrD-SBC progression fit with previous observations on SBC as a whole  and on cIn both CrD-SBCs and associated mucosa, metaplastic marker expression was commonly associated with loss of canonical intestinal markers, with special reference to the CDX2 transcription factor. This seems interesting as, in keeping with previous findings on gastrointestinal cancers, including SBCs as a whole , 41, 42,In addition to classic/conventional dysplastic lesions –7, severIn conclusion, our fairly large retrospective CrD-SBC series showed important heterogeneity of histologic structure and tumour cell phenotype, also paralleled in part by a polymorphism of mucosal precancerous changes. Among the latter, in addition to classic  dysplasia, CK7-positive and MUC5AC-positive metaplastic lesions, on one hand, and non-conventional, atypical growths, on the other hand, may have a role in Crohn’s disease-promoted cancer histogenesis. In addition, tumour CK7-positive metaplastic phenotype and non-cohesive histotype were both found to predict CrD-SBC patient adverse prognosis. These findings, besides suggesting a distinctive CrD-SBC natural history, are worth considering in future prospective and endoscopic/bioptic studies aiming to gain diagnosis of early tumourigenic lesions, as well as in attempts to provide more appropriate and patient-personalized treatments , 42, 51.ESM 1(PNG 10035 kb)Supplementary Figure 1. A minute intramucosal CrD-SBC , well demarcated by massive nuclear p53 immunoreactivity , arises in the background of an extensive non-conventional growth of columnar, eosinophilic cells, which entirely surrounds it, a pattern highly suggestive for a histogenetic link between the two growths. For comparison, note in C), bottom left, a fragment of normal ileal mucosa with goblet cells and in D) the sparse and scattered p53 reactivity of the columnar cells in the non-conventional growth. High resolution image (TIFF 135498 kb)ESM 2(PNG 12387 kb)Supplementary Figure 2. A hypermucinous, non-conventional papillary growth , MUC2-reactive  generates (see a cancer focus in B on the right) and covers a signet-ring type diffuse cancer  composed of MUC2-positive cells . High resolution image (TIFF 170428 kb)ESM 3(DOCX 9 kb)


Immune evasion is key to cancer initiation and later at metastasis, but its dynamics at intermediate stages, where potential therapeutic interventions could be applied, is undefined. Here we show, using multi-dimensional analyses of resected tumours, their adjacent non-tumour tissues and peripheral blood, that extensive immune remodelling takes place in patients with stage I to III hepatocellular carcinoma (HCC). We demonstrate the depletion of anti-tumoural immune subsets and accumulation of immunosuppressive or exhausted subsets along with reduced tumour infiltration of CD8 T cells peaking at stage II tumours. Corresponding transcriptomic modification occur in the genes related to antigen presentation, immune responses, and chemotaxis. The progressive immune evasion is validated in a murine model of HCC. Our results show evidence of ongoing tumour-immune co-evolution during HCC progression and offer insights into potential interventions to reverse, prevent or limit the progression of the disease. In order to design cancer immune therapies, it is important to understand how tumours evade the immune response that is mounted against them. Authors here analyse the distribution and properties of immune cells in hepatocellular carcinoma and describe a progressive tumour-immune co-evolution programme from early to late stage cancer. The immune microenvironment plays a critical role in tumour development, particularly in HCC, where long-standing liver inflammation or chronic hepatitis infection often precedes carcinogenesis4. Moreover, the immune composition of the tumour microenvironment (TME) contributes to disease prognosis9, evidencing its importance in determining clinical outcomes of HCC patients. Indeed, tumour initiation and progression require escape from the immune system, representing an ongoing arms race between cancer and host. Studies have defined immune evasion as an early event in lung cancer11 or late event during metastasis in advanced colorectal cancer12. Thus both early and late phases of immune evasion could exist, with different characteristics and mechanisms to pave the way towards tumour progression. However, what happens in between these points throughout the intermediate stages of cancer is unknown.Hepatocellular carcinoma (HCC) remains a critical cancer burden ranking as the fourth most common cancer-related mortality worldwideBy exploring the changing immune landscapes in patients with TNM  stage I–III HCC spanning across tumours, adjacent non-tumour tissues and blood, we show early and sustained immune escape. A second wave of immune evasion and concurrent tumour evolution peaks at stage II HCC. Our findings demonstrate continual immune-tumour co-evolution throughout tumour progression and identify intermediate tumour stage as a potential key interventional point for immunotherapy to prevent widespread HCC.n = 16 S1, n = 12 S2 and n = 10 S3), who underwent resection as first-line therapy (Supplementary Table T = 136) to account for intratumoural heterogeneity reported previously14, along with 38 matched adjacent non-tumour liver tissues (N) and peripheral blood (P) samples. All patients were clinically comparable, except for those parameters related to disease stage (i.e. tumour size and microvascular invasion)  I–III S1-S3) HCC   to measure protein expression of 33 immune markers on or within individual single cells (Supplementary Table 35) Fig.  that wer35) Fig. . UMAP di35) Fig. . Indeed,35) Fig. .Fig. 1Di+GBloCD45RO+CD8+ memory T (C1), CD69+CD8+ memory T cells (C9) and PD-L1+CD45RO+CD4+ memory T cells (C13) were enriched in both N and T compared to P  and HLA-DR+CD14+ myeloid cells (C10), as well as CD27+CD45RO−CD4+ naïve T cells (C17) were significantly reduced in both N and T compared to P  (C4), PD1+CD103+CD45RO+CD8+ resident memory T cells (TRM) (C7) and PD1+ CD45RO+CD4+ memory T cells (C19)  and GB+CD56+CD8+ NKT cells (C18) were significantly lower in T than N or P  Fig. . ConversTaken together, these data indicate that immune evasion is established early and maintained throughout the following stages of tumour development.+CD27+CD45RO− naïve T cells (C17) and CD3+Ki67+ proliferating T cells (C20) were less abundant in S2 compared to S1 or S3  coupled with reduced CD3+CD56+ natural killer (NK) T cells (C15) were observed in S2 , PD-1−GB+CD45RO+ , and PD-1−CD69+CD45RO+  active memory CD8+ T cells , Treg , and PD-1+GBlowCD45RO+CD4+ exhausted memory T cells  were significantly higher in S2 tumours than in either S1 or S3  and co-expressed high level of CTLA-4 (Median 66.2% ± 21.71%), Tim3 (Median = 72.4% ± 17.61%) and to a lesser extent Lag3 (Median 36.3% ± 17.07%)  and enriched PDCD1 (PD-1), FOXP3 and CTLA4 compared to N or S1 and S3 tumours  that promotes M2 polarization facilitating immune escape23 and TREM2, which marks the highly immunosuppressive TAMs24. In contrast, CD38, whose expression on myeloid cells predicts favourable prognosis hence its anti-tumoural activity in HCC25 and proinflammatory cytokine TNF, were both reduced in S2 and S3 tumours  revealed that the greatest differences in gene expression were observed between N and T from any stage, while S2 tumours showed the greatest differences from N, S1 or S3 tumours Fig. , consist26   in samples of the three tumour stages  Fig. , which i11. We quantified neoantigens in tumour samples from each stage using whole-genome sequencing (Methods) and found significantly more heterogenous , but not ubiquitous  neoantigens in S3 than S1 tumours , CXCR2 (receptor for CXCL1 and CXCL6) and CXCR3  showed a consistent trend in correlation with the key immune subsets  to detect tumour infiltration of CD8+ or exhausted PD-1+CD8+ T cells. Indeed, we observed a progressive reduction in the density of total CD8+ T cells from S1 to S3 HCC tumours  on FFPE tissues from an independent HCC cohort  post DEN-treatment are generally small and monomorphic, while larger tumours emerge from eight to 12 mos post-injection37 -induced HCC model, which as similar to the human condition, develops HCC from a background of chronic liver inflammationn37 Fig. , b, reprion Fig. . This suhi, PD-1lo and PD-1− populations among CD8+ T cells with a significant accumulation of the PD-1hi and depletion of PD-1lo population within CD8+ T cells from intermediate 8 mos post-DEN HCC tumours compared to 6 or 12 mos post-DEN tumours 14. In addition, single-cell transcriptomic analysis of TILs in HCC also revealed immune evasion with T cell exhaustion45, myeloid/lymphoid cross-interaction46 and onco-foetal reprogramming of endothelial cells and macrophages19. In this study, we revealed an unprecedented progressive immune evolution alongside tumour progression peaking at the intermediate stage of HCC.In colorectal cancer, a similar inverse relationship between tumour-infiltrating CD812, at least at the very early or very late disease stages captured in these studies. Similar to other human solid cancer studies, our current study has been limited to inferring the parallel cancer and immune evolution using specimens taken from different patients at various stages of tumour48. To demonstrate the trajectory of tumour landscapes evolution, both scRNA pseudotime trajectory prediction on tumour-infiltrating immune cells as well as early to late stages DEN-induced HCC model validated our discovery on a peak of phenotypic immune evasion at the intermediate tumour stage and a progressively immune excluded TME during tumour progression.Longitudinal studies of tumour progression and its relationship to the immune landscape in human colorectal and lung cancer do suggest that evolution of these tumours is immune-related or immune-drivenTo conclude, our in-depth analysis of intratumoural immune dynamics during HCC progression has profound implications for our understanding of HCC evolution and will help guide the development of potential immunotherapeutic treatments.n = 38) were isolated by Ficoll-Paque Plus  density gradient centrifugation. Tumour-infiltrating leucocytes (TIL) (n = 135) and non-tumour tissue-infiltrating leucocytes (NIL) (n = 38) were isolated from each tissue separately using enzymatic digestion with 500 μg/mL collagenase IV  and 50 μg/mL DNase I  for 30 min in 37 °C. Cells were stored in liquid nitrogen with 10% DMSO in foetal bovine serum (FBS) until further analysis by CyTOF.The study was approved by the Central Institution Review Board (CIRB) of SingHealth, of which all National Cancer Centre Singapore, Singapore General Hospital and National University Hospital were constituent members (CIRB Ref: 2016/2626 and 2018/2112). Tumour (T), adjacent non-tumour liver tissue (N) and peripheral blood (P) were collected from 38 HCC patients, each provided written informed consent, with demographics and clinical characteristics as described in (Supplementary Table 2 and 3 μg/mL Brefeldin A (eBiosience) and 2 μM monesin (BioLegend) was added at the last 3.5 h of the incubation. The cells were then washed and stained with cisplatin-Live/Dead stain (Fludigm) before labelling with a combination of barcoding anti-CD45 antibody conjugated to three different metals, for simultaneous analysis of multiple samples49. Cells were then combined and labelled with metal-conjugated antibodies targeting surface markers before fixing with 1.6% paraformaldehyde, permeabilizing with 100% methanol and staining with metal-conjugated antibodies targeting intracellular molecules. All 33 antibodies and barcoding anti-CD45 antibodies were either conjugated in-house according to the manufacturer’s instructions (Fluidigm) or purchased pre-conjugated directly from Fluidigm .TILs, NILs and PBMCs were thawed and rested for at least 30 min in RPMI medium with 10% FBS and 1% penicillin/streptomycin. Only samples with >100,000 live cells after thawing will be subjected to downstream CyTOF analysis (note: only two tumour sectors with insufficient cell numbers were excluded from the entire cohort). Data was collected from at least two sectors of each tumour in this study. The cells were unstimulated or stimulated with 150 ng/mL PMA  and 100 ng/mL ionomycin (Sigma) and incubated for a total of 5 h in 37 °C and 5% CO16. We also performed batch normalization, data visualization and statistical tests16. Clustering was performed using the Phenograph algorithm (v1.5.2)15 or FlowSOM algorithm17 and dimension reduction using tSNE. To compare the frequencies of cell populations between tissue types and clinical HCC stages, we applied one way ANOVA test with post-pairwise Mann–Whitney U-test. Of note, we averaged cluster frequencies from multiple tumour sectors to represent each patient for all statistical tests. Validation using manual gating was performed with FlowJo (v.10.2). Immune diversity was quantified by calculating the multivariate beta-dispersion of the Bray–Curtis distances between immune clusters for each tissue type using the ‘vegan’ R package v2.5. The beta-dispersion were compared among tissues and p-values were determined using Tukey’s HSD (honestly significant difference) test.The generated files were analysed by FlowJo : live single-cells  were debarcoded to each sample file based on their unique CD45 barcodes. Each file was then down-sampled to 10,000 cells and further analysed using our in-house Extended Polydimensional Immunome Characterization (EPIC) analysis pipeline with data visualization using the browser-based R shiny app ‘SciAtlasMiner’n = 7), S2 (n = 4) and S3 (n = 3) HCC, followed the same enzymatic digestion as described previously19. Briefly, dead cells were removed using dead cell removal kit . CD45+ cells were enriched using CD45 MicroBeads  before CD45+ and CD45− cells were processed using the Chromium Single Cell 30 (v2 Chemistry) platform  as previously described19. All data were then aggregated using cellranger aggr by normalizing all runs to the same sequencing depth. Downstream analysis was performed using Scanpy . For clustering, best matched k-Nearest Neighbour is automatically weighted by the algorithm to compute the best UMAP topology . The Louvain method (scanpy.api.tl.louvain) is then used to detect a community of similar cells resolution parameter 0.6 to 1 as illustrated previously19. Pseudotime trajectory analysis of tumour-infiltrating immune cells were performed using the Monocle R package (version 3.0)20, on T cells (further sub-divided to CD3+CD8- and CD3+CD8+ T cells), NK cells and MNPs clusters. A Cell Data Set object was created according to Monocle’s pipeline. Log-normalisation and scaling was done using Monocle’s pre-processing function. Principle Component Analysis (PCA), dimension reduction and clustering were performed using the default parameters. Pseudotime trajectories were plotted for each partition using Monocle’s reversed graph embedding algorithm. For pseudotime ordering, cells from the Stage 1 (S1) tumour samples were taken as the starting point.Single cells were isolated from tumour and adjacent non-tumour tissues from 14 patients with S1 (TM Multiplex immunohistochemistry Detection Kit and images were acquired using Vectra 3.0 pathology imaging system microscope (Perkin Elmer) according to manufacturer’s instructions. We used antibodies: anti-CD8  and anti-PD-1  and detection dye: Opal690 dye (CD8) and Opal650 dye (PD-1) with DAPI as nuclear counterstain. Quantification of stained cells was performed on the whole 1 mm core (Area = 0.785 mm2) and the average values of two cores from each patient sample was calculated and shown as the number of cells/mm2. Separately, IHC staining was done on FFPE tissues from DEN-induced mice (See description below). Adjacent non-tumour (N) and tumour samples from 4 mos (n = 7), 8 mos (n = 9) and 10 mos (n = 9) post-DEN mice were stained with Anti-CD8 , anti-PD-1  and detection dye: Opal650 dye (CD8) and Opal620 dye (PD-1) with DAPI as nuclear counterstain. Quantification was performed and analyzed from at least 2–6 (limited by tumour size) random 0.6 × 0.5 mm2 regions.Formalin-fixed paraffin-embedded (FFPE) sections were from an independent cohort of 102 HCC patients who underwent curative resection from 1991 to 2009, and were obtained from the Department of Anatomical Pathology, Division of Pathology, Singapore General Hospital. Two cores of tumour and non-tumour FFPE tissues from each patient were stained with OpalTotal RNAs from multiple tumour sectors and each non-tumour sector were isolated using Picopure RNA-Isolation kit , and cDNA was generated using the SMART-Seq® v4 UltraTM Low Input RNA Kit for Sequencing . Illumina indexed libraries were created using the Nextera XT DNA Library Prep Kit  and multiplexed for 2×101 bp-sequencing. RNA sequencing was performed on a HiSeq High output platform at the Genome Institute of Singapore (GIS).51 to the Human Reference Genome hg19, and the gene-level expected counts were calculated using RSEM52. Only protein-coding genes with at least one count per million reads in 5% or more of the samples were retained, and the data were normalized using DEseq253. Differential expression analysis was performed using R package Limma54 with the false discovery rate (FDR) adjusted for multiple testing using Benjamini–Hochberg procedure55. Differentially expressed genes (DEGs) were identified at an FDR < 0.05 with pairwise comparison. Pathway enrichment analyses were performed using integrated Differential Expression and Pathway analysis (iDEP)56 v0.91 and DAVID pathway analysis v.6.8, both with adjusted p-value < 0.01. For validation of selected genes of interest, the raw counts for the Liver HCC, The Cancer Genome Atlas (TCGA) dataset were downloaded from FireBrowse57. Only protein-coding genes with raw counts > 1 (TCGA dataset) in ≥5 samples were retained and data were normalized using DEseq2 .The raw reads were aligned via STAR33, and the relative expression levels of these genes which were calculated for each tumour according to the median expression of each gene. The expression levels of genes of interest were compared among three tumour stages using one-way ANOVA test with post-hoc Tukey’s pairwise test. Correlation between genes and immune subsets were computed with Spearman’s rho and p-values and visualized using R package “corrplot”: Visualization of a Correlation Matrix (Version 0.84) and “network”: Butts C (2020).Genes over- or under-expressed in CTNNB1-mutation signature were previously reported in HCC58. After removal of duplicated reads by PICARD (http://broadinstitute.github.io/picard/), base quality recalibration and realignment were performed using the Genome Analysis Tool Kit59. Comparison of tumour against non-tumour tissue was performed using Mutect (version 1.1.7)60 to call for somatic variants.DNA from tumour and adjacent non-tumour tissues was extracted using Qiagen AllPrep kit and sonicated into shorter fragments using the Covaris system. Quality check was performed using Agilent 2100 Bioanalyser. DNA fragments were end-repaired, ligated, amplified, and sequenced using the Illumina sequencing platform at GIS. Raw reads were mapped to the Human Reference Genome hg19 using the Burrows–Wheeler Aligner61, with variant calling information obtained from MuTect60. The variant calls were annotated using VEP: 8- to 11-mer epitopes with <500 nM predicted binding affinity to MHC-class 1. The ubiquitous  and heterogenous  neoantigens as defined previously62 were computed.Neoantigen prediction was achieved using personalized Variant Antigens by Cancer Sequencing(pVacSeq)37. Mice were weaned at 21 days of age and maintained on the Chow diet and 12 h light/12 h dark cycles at 23 °C co-housed in pathogen-free animal facilities. Mice were euthanized by carbon dioxide-induced asphyxia. Tumours of various sizes corresponding to different stages of development were harvested at 4 or 6 mos (early stage), 8 mos (intermediate stage) and 12 mos (late stage) post DEN induction. Immune cells isolated from tumours, adjacent non-tumour liver tissues and spleens were analysed with flow cytometry (as detailed below). Additionally, FFPE tissues from adjacent non-tumour and tumour specimens were separately analysed with IHC (as detailed above). Tumours showed varying sizes with the median diameters as 3 mm.The animal procedure was approved by and conducted in accordance with the regulations and guidelines of the Institutional Animal Care and Use Committee (IACUC) of the National University of Singapore and SingHealth Institute (IACUC reference number: R019-0666 and 2016/SHS/1228). HCC was induced by intraperitoneal injection of a single dose of diethylnitrosamine (DEN) (Sigma-Aldrich) at 25 mg per Kg body weight to 15 days old wild-type male C57BL/6 J mice, consistent with previously published methodImmune cells were isolated from mouse liver tumours or healthy livers by digestion with 500μg/mL collagenase IV (Thermo Fisher Scientific) and 50 μg/mL DNase I  and spleen from mechanical isolation. The isolated cells were analysed using antibodies listed in Supplementary Table p-values, in GraphPad Prism (V7.0d). The edgeR package63 was used for differential expression analysis of the RNA-seq data. Spearman’s correlation analysis was performed with the rho and p-values reported.Statistical comparisons of cell frequencies between groups were performed using the parametric one-way ANOVA test (OWA) with Tukey post-hoc multiple comparison test (human data) or unpaired two-sided Mann–Whitney U (MWU) tests for pairwise comparisons and non-parametric one-way ANOVA Kruskal–Wallis test with Dunn’s post-hoc multiple comparison test (murine data), both with two-sided Further information on research design is available in the Supplementary InformationPeer Review FileReporting summary


The tsl2 mutant showed a weak yellowish phenotype at normal growth temperature (22°C), and the phenotype was more pronounced at low growth temperature (16°C) and largely rescued at high growth temperature (29°C). Bulk Segregant Analysis (BSA) revealed that TSL2 encodes FtsH-Inactive Protein 5 (FtsHi5). Genetic complementation analysis confirmed that complemented expression of FtsHi5 rescued the chlorophyll content and thylakoid development defects observed in tsl2 mutants at 16°C. Quantitative mass spectrometry analysis with Tandem Mass Tag (TMT) isobaric labeling revealed broad changes in the chloroplast proteome of tsl2 mutant plants at low temperature, which is agreed with the impaired chloroplast biogenesis and function in tsl2 plants. Together, our data demonstrates that FtsHi5/TSL2 plays an important role in chloroplast development and protein accumulation in chloroplasts, especially at low environmental temperatures in Arabidopsis.Chloroplasts are indispensable for higher plants. The growth and development of plants are very sensitive to environmental temperature changes, and chloroplast development is also regulated by adverse environmental temperatures. However, the molecular mechanism of how plants coordinate chloroplast development and environmental temperature changes remains largely unknown. Here, a temperature-conditioned chloroplast development defective mutant  Chloroplast biogenesis affects plant growth and development due to its functions in photosynthesis and biosynthesis of amino acids, fatty acids, nucleotides, and phytohormones . ChloropArabidopsis thaliana, mutation of DELAYED GREENING1 (DG1) impairs chloroplast RNA editing and chloroplast development at elevated temperature (29°C) (Oryza sativa) (Light-regulating chloroplast development requires transcription factors such as ELONGATED HYPOCOTYL 5 (HY5), GOLDEN2-LIKEs (GLKs), GATA NITRATE-INDUCIBLE CARBON-METABOLISM-INVOLVED (GNC) which appear to regulate the expression of genes essential for chloroplast development, chlorophyll biosynthesis, and photosynthesis . At the e (29°C) . DG1 is e (29°C) . Low tem sativa) . CP31A,  sativa) . DG1 andthermo-sensitive mutant in leaf color 2 (tsl2), which was obtained from an ethyl methane sulfonate (EMS) mutant screening. The tsl2 mutant has chloroplast biogenesis defects at low ambient temperatures and TSL2 is allelic to FtsHi5, encoding a FtsH-like protein lacking the zinc-binding motif. Further proteomic analyses revealed that chloroplast ultrastructure and protein homeostasis are impaired in tsl2 mutant especially at low ambient temperatures. Thus, our results demonstrate the important role of TSL2/FtsHi5 in protein accumulation in chloroplast, which is associated with chloroplast development especially at low temperatures.Filamentation temperature sensitive protein H (FtsH) protease belongs to a member of the ATPases associated with diverse cellular activities (AAA) protease family and is conserved among bacteria, mitochondria, and chloroplasts . FtsH prArabidopsis thaliana plants used in this study were in the Columbia (Col-0) background. The seeds were surface sterilized for 5 min with 75% ethanol and then were washed three times with sterilized water. After sterilization, seeds were sown on 1/2 Murashige and Skoog (MS) medium  and stratified at 4°C for 2 days. Then they were transferred to 22°C under a 16-h light/8-h dark cycle. For phenotypic assays, seedlings were firstly grown at 22°C, and then kept at 22°C or transferred to different temperature conditions as indicated.All the 665−6.88 × OD649) plus chlorophyll b (24.96 × OD649−7.32 × OD665). Content of chlorophyll = (CT × V)/W, where V is the volume of 95% ethanol and W is the weight of fresh tissues. Each sample performed with three independent replicates.Fresh leaves of plants were weighted and immediately incubated in 1 mL of 95% ethanol in 1.5 mL microfuge tubes at 4°C in the dark. After 24 h, the absorbance of the supernatant was measured at 649 and 665 nm. The chlorophyll concentration (CT) was the sum of chlorophyll a (13.95 × ODtsl2 mutant in Col-0 background was crossed to the WT Landsberg (Ler) ecotype. About three hundred seedlings whose leaves were yellow at 16°C from the F2 segregation population were sampled, and their genomic DNA was pooled for deep sequencing using the Illumina HiSeq 4000 platform  according to the standard Illumina protocol.For genome resequencing, the tsl2 mutants, the 8.5-kb wild-type genomic DNA fragment of AT3G04340 including the upstream promoter and coding sequences was amplified by PCR and inserted into the pCAMBIA1300 plasmid. The construct was subsequently transformed into Agrobacterium tumefaciens strain GV3101 via the freeze-thaw method and introduced into tsl2 mutant plants via the floral dip method. All the primers used in the present study are listed in TSL2-FLAG, the promoter and the coding sequence of TSL2 were amplified and inserted into pCAMBIA1306, respectively, and the FLAG tag was fused to TSL2 at the C-terminus. The mutated form of TSL2m was amplified from cDNA derived from tsl2 mutant plants. Both plasmids were transformed into GV3101 and introduced into WT plants via the floral-dip method. Primers were listed in For the complementation of tsl2 mutant seedlings were grown at 22°C for 4 days and then transferred to 16 and 29°C or maintained at 22°C for 10 days. Subsequently, true leaves were detached and fixed overnight at 4°C in 2.5% glutaraldehyde and 0.1 M phosphate buffer (pH 7.0) and processed for electron microscopy as described . For reverse transcription, 2 μg of total RNA with oligo(dT) and random 6-mer primers were used to synthesize cDNA in a 20 μL reaction using M-MLV reverse transcriptase . The cDNA was used to perform RT-PCR and qRT-PCR. qRT-PCR was performed using the SuperReal PreMix Color kit  in a CFX96 real-time system . Each comparison consisted of three biological replicates, and the expression of Total proteins were extracted from plants with extraction buffer , separated by 10% SDS-PAGE gels, and analyzed using anti-FLAG  or anti-Tubulin .3). The suspension was filtered through three layers of Miracloth  and centrifuged for 5 min at 1,500 g to pellet the crude chloroplast fraction. The pellet was resuspended in 300 mM sorbitol, 20 mM HEPES-KOH (pH 7.6), 5 mM MgCl2, and 2.5 mM EDTA. The suspension was layered on top of a step gradient consisting of (bottom to top): 2 mL of 80% (v/v) Percoll/grinding buffer, 4 mL of 35% (v/v) Percoll/grinding buffer in a 15-mL polycarbonate tube. The gradient was centrifuged for 25 min at 3,000 g and then the lower band was collected and diluted with 3 volumes of dilution buffer . The chloroplasts were centrifuged for 2 min at 1,000 g. The pellet was resuspended in 25 mL dilution buffer and centrifuged for 1 min at 1,000 g. The chloroplast pellet was resuspended in 300 μL Lysis buffer .Chloroplasts were isolated according to previous methods  with modg at 4°C for 10 min. The supernatant was precipitated with cold 20% trichloroacetic acid for 2 h at −20°C. After centrifugation at 12,000 g at 4°C for 10 min, the supernatant was discarded and the remaining precipitate was washed with cold acetone for three times. Finally, the protein was dissolved in 8 M urea and the protein concentration was determined with a BCA kit, according to the manufacturer’s instructions. Before digestion, the protein sample was diluted by adding 100 mM triethylammonium bicarbonate (TEAB). For digestion, trypsin was added at 1:50 trypsin-to-protein mass ratio for digestion overnight and the protein solution was reduced with 5 mM DTT for 30 min at 56°C and then alkylated with 11 mM iodoacetamide for 15 min at room temperature in the dark. For TMT labeling, peptide was desalted with Strata X C18 SPE column  and vacuum dried. After that, peptide was reconstituted in 0.5 M TEAB and processed according to the manufacturer’s protocol for the TMT kit. Briefly, one unit of TMT reagent was thawed and reconstituted in acetonitrile and added to the peptide sample. Then, the peptide mixtures were incubated for 2 h at room temperature and pooled, desalted, and dried by vacuum centrifugation.Proteomics analyses were carried out in the company PTM BIO . Briefly, the chloroplast pellet was resuspended in 300 μL Lysis buffer , followed by sonication three times on ice using a high intensity ultrasonic processor  and centrifugation at 20,000 1. The mass error for precursor ions was less than 20 ppm in the first search and 5 ppm in the main search, and the mass error for fragment ions was less than 0.02 Da. Differentially regulated peptides were calculated and their corresponding proteins were subjected to GO analysis .High performance liquid chromatography (HPLC) fractionation was performed with high pH reverse-phase HPLC. Briefly, peptides were dissolved in 0.1% formic acid, and then directly loaded onto reversed-phase analytical columns (Agilent 300Extend C18). The peptides have a stepwise gradient of 8∼32% acetonitrile, pH 9.0, 60 min to separate 60 components, then the peptides were combined into six components, and vacuum freeze-dried for subsequent analyses. The peptides are dissolved in the mobile phase A (0.1% formic acid and 2% acetonitrile) of liquid chromatography and then separated using the EASY-nLC 1200 ultra-high-performance liquid system. The gradient was increased from 7 to 11% (0.1% formic acid in 90% acetonitrile) over 4 min, 11 to 32% in 49 min, 32 to 80% in 4 min, and then held at 80% for the last 3 min, all at a constant flow rate (500 nL/min) on an EASY-nLC 1000 UPLC system. The peptides were sent to NSI source followed by tandem mass spectrometry (MS/MS) in Orbitrap Exploris 480  coupled online to the UPLC in the Orbitrap with routine setting parameters. The resulting MS/MS data were processed using the Proteome Discoverer (v2.4.1.15) against the Arabidopsis TAIR databasethermo-sensitive mutant in leaf color 2 (tsl2) through screening an EMS-mutated population of Arabidopsis plants. The tsl2 mutant showed a weak yellowish phenotype at 22°C (tsl2 was similar to that of the wild type (WT) when the temperature was increased to 29°C  showed that there was a guanine (G) to an adenine (A) transition at the 9th exon of FtsHi5 (AT3G04340) in the tsl2 mutant, which led to an amino acid change from Ala to Thr (A939T) in AAA domain of the protein . Under normal growth temperature condition (22°C), chloroplasts in the tsl2 mutant showed less organized thylakoid membrane systems compared to that of the WT plants  analysis to detect the expression of TSL2 in WT and tsl2 plants grown under different temperature conditions. The results showed that there was no obvious reduction in terms of TSL2 transcript level in tsl2 to that of WT at 16 or 22°C, at which temperature the tsl2 showed defects in chloroplast development  and 281 proteins that were downregulated in tsl2 at 18°C . While at 26°C, there were 180 proteins upregulated and 99 proteins downregulated in tsl2 mutant, respectively  involved in chlorophyll b biosynthesis , NAD(P)H-quinone oxidoreductase subunits (ndhS and ndhU), Photosynthetic NAD(P)H dehydrogenase subunits (NDH18 and NDH48), Photosystem I reaction center subunits   . TherefoFtsHi5 and carried out quantitative proteomic analysis of tsl2 mutant, further demonstrated the important function of FtsHi5/TSL2 in chloroplast protein homeostasis. Our current results further revealed substantial changes at chloroplast proteome level in tsl2 mutant plants at 18°C, which is associated with chloroplast development and function. Similar pale-green phenotypes with defects in chloroplast have also been found in knock-down lines of FtsHi4 . The previously reported ftshi5-1 mutant containing a mutation (D400N) located near the DUF5401 domain also showed chloroplast development defects  and FtsH5 function in the degradation of photodamaged photosystem II (PSII) reaction center proteins D1 and D2 . The vart manner . The chlt manner . The prot manner . These rOur results demonstrated the important function of FtsHi5/TSL2 in chloroplast protein accumulation, which is required for chloroplast development, especially at low environmental temperatures.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD030185.J-YL and J-XL designed the experiments, analyzed the data, and wrote the manuscript. J-YL, J-LS, and Y-YT performed the experiments. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Pleural effusion, defined as an abnormal accumulation of fluid in pleural space, can be of two types: transudative and exudative. The primary aim of the study was to assess the predictive accuracy of procalcitonin (PCT) and pentraxin-3 (PTX-3) in comparison to other biochemical markers such as C-reactive protein (CRP), and adenosine deaminase (ADA) in the differential diagnosis of pleural effusions.A cross-sectional analytical study was conducted on patients with pleural effusion. Multiple comparisons and receiver-operating characteristics (ROC) analyses were made to evaluate the diagnostic significance of biochemical markers.Sixty-six patients with exudative pleural effusion classified as malignant, tuberculous, and parapneumonic effusions  were included. Significant differences in pleural fluid levels in both PCT  and PTX-3, as well as serum levels of PCT , were observed between the three groups. ROC analysis showed both PTX-3 and PCT having favorable discrimination ability with high sensitivity (≥90%) and specificity to predict PPE from TPE and MPE.Evaluation of serum and pleural fluid PCT and levels of PTX-3 in the pleural fluid may be used as an early biomarker to differentiate the etiology of pleural effusion. Pleural effusion is defined as an abnormal accumulation of fluid in pleural space . It can Recently, two further biomarkers have been evaluated in the early diagnosis of pleural effusions: pentraxin-3 (PTX-3) and procalcitonin (PCT). PTX-3 pleural fluid levels distinguish PPE and other exudative effusions , 10], , , 12]. P. P12]. PThus, much research has been carried out in the last few years to validate biomarkers able to differentiate pleural effusions’ etiology. However, an adequate diagnostic accuracy of these biomarkers has not yet been demonstrated . We planPatients who underwent diagnostic or therapeutic thoracocentesis during the past year in the Department of Pulmonary Medicine, Himalaya Institute of Medical Science, Swami Rama Himalaya University, Dehradun, Uttrakhand, India, were enrolled in the study. Only patients with exudative pleural effusion were included in the study. Patients with pyothorax, hemothorax, or transudative effusion were excluded. Pleural fluid and serum samples from such patients were collected after obtaining informed written consent. Approval of the Institutional Review Board (IRB) was received before the study, and the IRB approval number was HIMS/RC/2017/40 dated 31/01/2017.–○Tuberculous effusions were confirmed by the presence of acid-fast bacilli (AFB) and TB-PCR testing.Group 1: Patients with TPE: 33 patients–○Malignant effusions were confirmed by cytology/histology examination. All 23 MPE cases were cytologically confirmed and were further divided into primary and secondary.Group-2: Patients with MPE: 23 patients–○PPE was confirmed by purulent sputum and response to antibiotics.Group-3: Patients with PPE: 10 patientsThis is an analytical cross-sectional study. All patients with exudative pleural effusion were divided into three groups after routine pleural fluid analysis based on widely accepted Light’s criteria .–Group 1Samples were collected with stringent aseptic precaution from the Department of Pulmonary Medicine and send to the central diagnostic laboratory for evaluation.Biochemical parameters, including protein, lactate dehydrogenase (LDH), CRP, and pentraxin, were performed immediately on automated analyzers. Serum/pleural fluid PCT sample was preserved at −20 °C before analysis as recommended by the kit insert. After routine cytological and microbiological examination, samples of serum and pleural fluid were analyzed for PTX3 , CRP , and PCT  using commercial kits on MR-96A ELISA reader and COBAS-6000 modular analyzer. ADA was estimated by the enzymatic method on Unicel DXC800 (Beckman Coulter India [Pvt.] Ltd). Assay kits were calibrated, and quality controls were analyzed daily at the National Accreditation Board for testing and calibration laboratories (NABL) accredited lab in Himalaya Institute of medical science. The diagnostic labs also participate in EQAS  assurance programs to maintain the accuracy and precision of laboratory results.Data were analyzed by using statistical software SPSS 20. Categorical data were expressed as frequency and percentage. Quantitative data were expressed as mean ± SD and median (min–max) for normal and skewed distribution. Spearman correlation coefficient was used to find the correlation between biomarkers in serum and pleural fluid. Kruskal Wallis H test followed by Dunn’s posthoc multiple comparison tests to compare median values among the three groups. Receiver operating characteristics (ROC) curve was used to find cut-off values for pneumonia considering TB and lung cancer reference one by one. A ROC analysis was performed, and the area under the curve (AUC) was calculated.Out of the total 80 patients enrolled, 14 were excluded after diagnosis of pyothorax, hemothorax, or transudative effusion. The remaining study group included 66 patients  with a mean age of 53.9 years. Out of these patients, 33 (50%) had TPE, 23 (34%) had MPE, and 10 (15%) had PPE. Out of 23 MPE, 19 were due to primary lung cancer . The remaining four effusions were secondary to other malignancies. The pleural fluid characteristics of the 66 patients are shown in Analysis of serum biomarkers revealed variations across the three groups . However, only PCT showed a substantial difference between all three groups  by multiple comparisons. ADA showed a significant difference only between TPE and PPE. Serum CRP and PTX-3 levels showed no statistical differences between the groups .ADA, PCT, and PTX-3 levels in pleural fluid revealed notable differences across the groups. Only PCT showed a significant difference between all three groups  by multiple comparisons. ADA and PTX-3 levels were significantly different between two groups (TPE vs. MPE and MPE vs. PPE) and , respectively. CRP levels showed no statistically significant difference between the groups .To differentiate PPE from TPE, pleural fluid PCT levels at the cut-off value of 6,173 had a sensitivity and specificity of 90%, and PTX-3 levels at the cut-off 9.2 had a sensitivity and specificity of 90 and 88%, respectively. To differentiate the PPE from MPE, pleural fluid PCT levels at the cut-off value of 2,189.7 had a sensitivity and specificity of 90 and 87%, respectively; PTX-3 levels at the cut off 7.19 had a sensitivity and specificity of 100 and 95.6%, respectively . CorrelaThe purpose of our study was to find unsurpassed abilities of biochemical markers in diagnosing and differentiating pleural effusion. We evaluated two established  and two more recent biomarkers (PTX-3 and PCT) in serum and pleural fluid separately to find their accuracy and efficiency to discriminate pleural effusion. Our study’s novelty was the analysis of different biomarkers simultaneously in the serum and pleural fluid, revealing that PTX-3 measurement in the pleural fluid is more accurate than in the serum. In contrast, both serum and pleural fluid levels of PCT provided a higher accuracy rate in discrimination of pleural effusions of different etiologies.The prior literature shows that PCT in serum and pleural fluid can differentiate between PPE and MPE . ElevateIn the present study, biomarker analyses in the pleural fluid revealed that ADA, PCT, and PTX-3 in pleural fluid significantly differentiate PPE from TPE and MPE. ADA levels were found to be significantly higher in tuberculous exudative vs. nontuberculous exudative pleural effusions , 22], , . SimilarAfter determining the area under the curve of PCT and PTX-3 levels in the pleural fluid at a particular cut-off value, we found both biomarkers suitable to differentiate PPE from TPE. We set the cut-off value based on literature data. Wang et al.  reported1c, albumin and serum/fluid cytology were not considered. A possible influence of the treatment applied  on the laboratory results cannot be excluded.Our study also has certain limitations. Firstly, the sample size was relatively small, and there was a variation in cases accretion. Thus, our data has exploratory value, and the obtained cut-offs from ROC must be re-assessed in a large population to validate the findings. Secondly, sampling bias may have been present during the pleural fluid tapping procedure, which may have influenced the validity of laboratory measurements. We did not analyze the demographic and baseline patient characteristic . Further laboratory data such as serum/pleural levels of glucose/HbAThere is no gold standard diagnostic technique for differential diagnosis of pleural effusion. The available traditional methods like cytology and microbiology are time-consuming. Depending on the laboratory consultant’s expertise, there is a chance of subjective bias affecting the patients’ management. Radiological investigations (USG/CT/MRI) are rapid but cannot distinguish the cause of the pleural effusion. Radiological studies are costlier, require specific technical infrastructure as well as diagnostic confirmation. The present study shows that newer biomarkers might be sensitive and specific enough compared to traditional methods. Although we did not evaluate these aspects, biomarker testing might be more cost-effective, rapid, reliable, and easy to perform than available comparators, improving the capacity to differentiate pleural effusion. Serum and pleural fluid PCT and PTX-3 levels might be useful early biomarkers to diagnose pleural effusions’ etiology. However, more extensive, adequately powered prospective studies are required to validate our findings.


Intraoperative graft assessment with tools like Transit Time Flow Measurement (TTFM) is imperative for quality control in coronary surgery. We investigated the variation of TTFM parameters before and after protamine administration to identify new benchmark parameters for graft quality assessment.The database of the REQUEST  study was retrospectively reviewed. A per graft analysis was performed. Only single grafts  where both pre- and post-protamine TTFM values were recorded with an acoustical coupling index > 30% were included. Grafts with incomplete data and mixed grafts (arterio-venous) were excluded. A second analysis was performed including single grafts only in the same MAP range pre- and post- protamine administration.p = 0.009) and decrease in PI  were observed after the administration of protamine. These changes were especially notable for venous conduits and for CABG procedures performed on-pump.After adjusting for MAP, we found a small increase in MGF (29 mL/min to 30 mL/min, The small changes in TTFM parameters observed before and after protamine administration seem to be clinically irrelevant, despite being statistically significant in aggregate. Our data do not support a need to perform TTFM measurements both before and after protamine administration. A single TTFM measurement taken either before or after protamine may suffice to achieve reliable data on each graft’s performance. Depending on the specific clinical situation and intraoperative changes, more measurements may be informative.NCT02385344, registered February 17th, 2015.Clinical Trials Number: The online version contains supplementary material available at 10.1186/s13019-021-01575-y. Intra-operative assessment of graft patency is crucial to perform high-quality coronary artery bypass graft (CABG) procedures. Occlusion rates as high as 20.0 and 8.0% have been reported at 1-year follow-up for venous and arterial conduits, respectively , 4, whicTransit-Time Flow Measurement (TTFM) is an intra-operative tool to assess the patency and the quality of a graft in order to prevent errors from going unnoticed. TTFM relies on specific flow parameters, namely mean graft flow (MGF), pulsatility index (PI), diastolic filling (DF) and backflow (BF). Importantly, no single-parameter measurements have been demonstrated to predict the quality and long-term patency of a graft.TTFM has been reported to have a 2–4% detection rate of intra-operative errors, and despite the recommendation for its use (class IIa) in the 2018 European guidelines for myocardial revascularization, its adoption has not yet become widespread , 7 with Performing a retrospective review of the large, multicenter REQUEST  registry , we inveThe Registry for Quality Assessment with Ultrasound Imaging and TTFM in Cardiac Bypass Surgery (REQUEST) is an international, multicenter, prospective registry that included 1016 patients in seven cardiac surgery centers (four in Europe and three in North America) between April 2015 and December 2017. These patients underwent isolated coronary artery bypass grafting (CABG) with intraoperative assessment of multiple potential surgical sites, including the ascending aorta , coronary targets, conduits and finally proximal and distal anastomoses using high frequency ultrasound (HFUS) and graft assessment using transit time flowmetry (TTFM) with the MiraQ™ or VeriQ C™ devices .The registry was designed to capture information on any changes in the proposed surgical procedure based on HFUS and/or TTFM findings. These results, together with the study protocol, were reported in a previous publication .The original REQUEST study was funded by Medistim, yet this current study received no funding from any source. The principal investigators and authors had complete scientific freedom. The original REQUEST study is registered at ClinicalTrials.gov (NCT02385344).Patients diagnosed with multivessel coronary artery disease and scheduled for isolated CABG were eligible to be included in the REQUEST registry. Patients were excluded from enrolment if undergoing emergency surgery, when concomitant surgical procedures were planned , if the medical history included the presence of a myopathy, or when the patient was known to be suffering from any psychological, developmental, or emotional disorder. From the original REQUEST trial 1016 patients were included out of the total 1046. Out of the 30 patients excluded, 8 were due to screening failure, 11 because surgical team members were not trained according to the REQUEST study protocol, and 11 because TTFM or HFUS images were unavailable for analysis.The decision between performing the CABG operation with vs. without cardiopulmonary bypass  was left to the discretion of the operating surgeon.It was highly recommended, but not mandatory, to intra-operatively assess with TTFM each conduit used for CABG. Only TTFM measurements with an acoustic coupling index (ACI)  above 30%  were included in the analysis.The following four TTFM parameters were measured and recorded: mean graft flow (MGF), pulsatility index (PI), diastolic filling (DF) and backflow (BF).The systemic mean arterial pressure (MAP) at the time of TTFM was recorded and classified in six ranges: < 53 mmHg, 53–63 mmHg, 64–74 mmHg, 75–85 mmHg, 86–96 mmHg, or > 96 mmHg.In the first stage of the analysis, we considered only single grafts  with pre- and post-protamine TTFM performed and pre- and post-protamine ACI both above 30%. There were 702 patients with a total of 1335 grafts that met these requirements. Sequential grafts were excluded because their presence could add confounders to the analysis. Our analysis remained tailored to single grafts only as TTFM parameters before and after protamine were still undefined and unpublished.We then repeated the analysis only for those grafts with the same MAP  pre- and post-protamine. There were 368 patients with 567 grafts that met these criteria for analysis.p-values < 0.05 deemed statistically significant. Analyses were performed using the open-access software R (https://www.r-project.org/).Continuous data were reported as median , and categorical data as number (percentage). Comparisons were performed with the chi square, the exact Fisher and the Wilcoxon rank-sum tests, with individual Graft and patient selection as previously described are illustrated in Fig. The pre- and post-protamine TTFM variables for the eligible grafts are reported in Table There were statistically significant differences in pre- vs. post-protamine MAPs in the 768 grafts , but remained unchanged for arterial grafts . Concerning the single grafts with the same MAP range pre and post protamine, the 95th percentile for MGF exceeded 25 mL/min. No correlation was found between the outliers  and graft or anastomosis revision , and was numerically unchanged, but statistically significant for arterial grafts . DF decreased post-protamine for arterial grafts  but was unchanged for venous grafts . Finally, BF was unchanged for both arterial  and venous grafts   after protamine administration, once MAP had been corrected for. The 95th percentile for MGF was 25 mL/min . PI slightly decreased   with cutThe diagnostic accuracy of TTFM is still a matter of debate with different values of sensitivity and specificity reported , 11. FurUsing a patency prediction model based on virtual machine learning, Mao et al. have recently demonstrated that the implementation of TTFM parameters with clinical and hemodynamic characteristics significantly increased its sensitivity and specificity values . Though The recently published REQUEST trial  was the Mean arterial pressure (MAP) has been known to effect MGF. Except at the extremes of MAP, MGF is directly proportional to MAP when coronary vascular resistance is unaltered. For instance, Balacumaraswami et al. , when reAs previously mentioned, despite TTFM’s potential to provide valuable data in coronary surgery , it is underutilized. Using a retrospective analysis of the REQUEST trial, we aimed to add another parameter to TTFM and to improve TTFM’s sensitivity and specificity. Based on clinical observations of a change in TTFM parameters after the administration of protamine, we retrospectively analyzed the REQUEST trial to quantify this change in parameters. To reduce the effects of MAP on graft flow we compared the various TTFM parameters using only single grafts with similar MAP range before and after the administration of protamine. Sequential grafts were excluded to minimize confounding, since the effect of protamine on single grafts had not yet been quantified. We found no reduction in flow in the post-protamine measurements . There was also some improvement in the PI post–protamine . DF decreased post-protamine for arterial grafts but was unchanged for venous grafts and BF was unchanged for both graft types. Of note, reports on flows in arterial vs. venous conduits usually report higher flows in venous conduits , but thiProtamine causes a well-known series of transient effects during reversal of heparin anticoagulation, including systemic hypotension, pulmonary hypertension, bradycardia and bronchoconstriction . BelboulThis study was a retrospective review of the data from the original REQUEST trial that was not designed to answer the question of how TTFM parameters change after protamine administration. Use of TTFM requires committed training both for the application of the device and the interpretation of the data. Only in-hospital outcomes of patients were tracked so no long-term clinical outcomes or angiographic outcomes are available to correlate with our findings. We did not examine the different effects of protamine administration on graft flow by coronary territory, in preticular compering the left anterior descending to non-left anterior descending targets. Finally, only single grafts were analyzed because we wanted to reduce any additional confounding with the inclusion of sequential grafts, yet sequential grafts are routinely performed in clinical practice.We found no clinically significant difference in TTFM parameters before and after the administration of protamine. Furthermore, even when examining outliers , we did not find a higher rate of graft revision. This finding might obviate the need for performing TTFM before and after protamine administration since a single TTFM measurement may suffice to achieve reliable data on each graft’s performance. Depending on the specific clinical situation and intraoperative changes, more measurements may be informative .Additional file 1.


The physical appearance of material parameters on the flow field, temperature, concentration, drag force, and Nusselt number was discussed through plots. The numerical results were obtained for limiting circumstances. The unsteadiness factor thinned the velocity boundary layer but decreased the thermal and concentration boundary layers. By increasing the Eckert number, the nondimensional temperature profile was enhanced. The novelty of the present study is that no one has numerically investigated the magnetized Casson fluid over a moving wedge in the presence of a chemical reaction and thermal radiation.The influence of the chemical interaction and dynamic micropolar convective heat transfer flow of Casson fluid caused by a moving wedge immersed in a porous material was explored. The Joule heating owing to magnetized porous matrix heating was also deliberated. The mathematical formulation for mass conservation, momentum, energy, and concentration profiles was expressed in the form of partial differential equations. The dimensionless set of ordinary equations was reduced from modeled equations via a transformation framework and then solved by the RK4 built-in function in MATLAB SOFTWARE by taking a step size of  The analysis and various features of non-Newtonian fluid have attracted an excessive amount of consideration due to their rigorous forms of solicitation in numerous manufacturing products and industrial goods, mostly in the abstraction of fossil fuels from crude goods, the manufacture of treacle drags, and plastic products. In the viscous and incompressible fluids class, Casson fluid has numerous choices. In this class of viscous fluid, the Casson fluid is one of which performs folding compacted at the same time, and it has the shear stress inside the critical equation for such a fluid. Non-Newtonian transmission phenomena are occurring in numerous branches of mechanics and the production of chemicals, and jointly in food processors. The different properties of the non-Newtonian liquid are exposed by some common ingredients, including ink, milk, tomato ketchup, and sugar solution. The analysis of Casson  defined In nuclear plants, steam turbines, solar energy generation, molten fluids, elevated plasmas, groundwater engineering, and other disciplines, the subject of hydromagnetic boundary layer movement of the position of thermal radiation arises. Applications of these kinds of fields include the melting of metal during an electric arc furnace, as well as the chilling of the first wall within a nuclear plant reactor core, where the initial plasma is segregated from the wall via a generating magnetism. Furthermore, when a maximum temperature is necessary, the effect of thermal irradiation cannot be overlooked, particularly if the existing network is housed in a thermally confined space. Furthermore, energy storage is an important worry in a number of energy-scarce places. To solve the scarcity problem, a more efficient model that can replace the current one must be devised. The liquid becomes conductive when it undergoes thermal performance, most likely as a result of the ionization induced by high temperature. The influence of heat irradiation on the convection moment of a radiated magnetohydrodynamic fluid passing a body is poorly understood. Chamkha et al.  considerIn the ferromagnetic materials, Mohyud et al.  inspecteInvasive mass and operational conditions that migrate owing to movement bodies frequently cause a chemical process. A chemical process might be instantaneous or take place over a protracted era of time. Chemical reactions could be of any degree, but the smallest would be a first chemical reaction, wherein the reaction rate and the proportion of the entities are precisely proportionate. In the context of first-order reactions, Magyari and Chamkha  investigScientists, statisticians, and mathematicians, on the other hand, have recently been concerned with the analysis of boundary layer movement of non-Newtonian fluids. It is largely attributable to its extensive uses in a variety of technical and manufacturing fields. In liquid thermal performance convection and cooling operations, the understanding of these types of fluxes is critical. The normal rhythm generates a variety of properties as a result of its assortment, for example, the viscoelastic fluids model, power-law fluid model, Jeffrey fluid model, Williamson fluid model, and Eyring–Powell fluid model. The Casson model of fluid is indeed a confined rheological fluid used to explain non-Newtonian liquid movement features with a strain rate. Casson fluid has benefited a variety of industries and technical processes, including pharmaceuticals, astronautics, food production, and paper making. The Casson flow rate in crescent bodies is also useful for oil extraction, radioactive wastes’ storage, and blood vessels. Mukhopadhyay and Mandal  completeThe goal of this study was to learn more about the Falkner–Skan mixed convection flow through a wedge, which was inspired by the above-mentioned papers and its implementations in a variety of sciences and industries. In addition, it is clear from the literature review that the unsteady movement of Casson fluid via a rotating wedge mostly in the vicinity of a porous medium and heat generation has received very little attention. Several scholars ,47,48,49In an applied magnetic field, an unsteady 2D mixed convection flow of Casson fluid over a moving wedge in the presence of porous media was examined. The wedge is moving with velocity The governing equations for continuity, momentum, energy, and mass equations are ,36:Continuity equation:Momentum equation:Energy equation:Mass equation:The extreme conditions are:Here, ented as ,40:(8)qrUsing Equations (8) and (9) in Equation (3), we obtain: We introduce the following similarity transformations:Using Equation (11), Equation (1) is satisfied identically, while Equations (2), (3), and (10) become dimensionless forms as:Equation (1) is true automatically, while Equations (2)–(5) have the forms:The physical quantitates reported by Mukhopadhyay and Mandal  are:(16)The system of Equations (12)–(15) was solved numerically using the Runge–Kutta fourth-order technique. The accuracy of this approach is fourth order, and it is operationally feasible. Equations (13)–(16) were first transformed to a first-order system. The generated mathematical equations were therefore liberalized and represented in matrix form utilizing Newton’s method. Finally, using the block-tridiagonal evacuation technique, the linear set of equations was computed. In order to obtain numerical results, MATLAB software (7.10.0.499 (R2010a), The MathWorks, Inc., Natick, MA, USA) was used to create an algorithm. Furthermore, the scale size a et al.  and Kumaa et al.  with theo et al.  and Mukho et al. , which co et al.  and Chamo et al.  for diffA, m, M, K, N, and m = 0 (flat plate), m = 1/5 (m = 1 (stagnation point flow), m = 5 , and A, Casson parameter m, magnetic parameter M, porosity parameter K, thermal buoyancy parameter λ, buoyancy ratio factor N, Prandtl number Pr, radiation parameter Rd, Eckert number Ec, Schmidt number Sc, reaction factor R, moving wedge factor γ, slip parameter δ, and Biot numbers Bi1, Bi2 were deliberated graphically. The motivating conclusions of this work are:A, b, M, and δ decreased the moment boundary layer thickness.Increases in The influence of k on the flow field was more prominent in γ = 0 and γ < 0 cases.The impact of δ on the flow field was less noticeable in the presence of a magnetic field.The nonlinear thermal radiation along with concentration thickness boosted with A.Rises in λ and N intensely influenced the fluid flow; however, the influence of these factors on heat and concentration field was found slighter.The barrier yield stress was detected to decrease with The temperature and rate of heat transfer were perceived higher with the increase in Rd.The mass transfer rate was observed to be greater with the variation in chemical reaction.The heat and mass transfer exploration for an unsteady Casson magnetic field flow through a moving wedge in a permeable sheet with nonlinear thermal radiation, chemical reaction, velocity field, as well as nonlinear convective boundary constraints was examined numerically. The basic nonlinear PDEs were transformed to nonlinear ODEs using the similarity variable and then elucidated numerically by the RK4 method. The current method was authenticated through assessment with the consequences of previous authors as a preventive case. The effects of physical factors, such as unsteadiness factor


The magnitude of the host immune response can be regulated by either stimulatory or inhibitory immune checkpoint molecules. Receptor-ligand binding between inhibitory molecules is often exploited by tumours to suppress anti-tumour immune responses. Immune checkpoint inhibitors that block these inhibitory interactions can relieve T-cells from negative regulation, and have yielded remarkable activity in the clinic. Despite this success, clinical data reveal that durable responses are limited to a minority of patients and malignancies, indicating the presence of underlying resistance mechanisms. Accumulating evidence suggests that tumour hypoxia, a pervasive feature of many solid cancers, is a critical phenomenon involved in suppressing the anti-tumour immune response generated by checkpoint inhibitors. In this review, we discuss the mechanisms associated with hypoxia-mediate immunosuppression and focus on modulating tumour hypoxia as an approach to improve immunotherapy responsiveness. Immune checkpoint inhibitors (ICIs) sit at the forefront of cancer immunotherapy. They are emerging as the primary treatment option for many advanced stage cancer patients as a result of their clinical success. Within the last decade, seven ICIs targeting the immune checkpoint receptors cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed death protein 1 (PD-1), and the ligand to the checkpoint receptor PD-1 programme death protein ligand 1 (PD-L1) have been approved by the FDA for the treatment of a variety of malignancies ,3,4,5,6.In general, immunotherapies are designed to increase the number and functionality of anti-tumour effector cells. The majority of such treatments target activating antigen-specific T-cells to selectively eliminate tumours through the recognition of unique or aberrantly expressed antigens presented as peptides by major histocompatibility complex (MHC) molecules on the tumour cell surface. ICIs such as anti-CTLA-4, anti-PD-1 and anti-PD-L1 monoclonal antibodies relieve T-cells from negative regulation governed by immune checkpoints by blocking the interactions between immune checkpoint receptors and their respective ligands on tumour cells, infiltrating myeloid cells or T-cells themselves . The immTumours with high expression of PD-L1 respond better to ICIs targeting the PD-1/PD-L1 pathway and are also found to carry more tumour somatic mutations ,13. ThisTumour resistance to ICIs can be classified into three broad categories: (i) primary resistance, in which an anti-tumour immune response cannot be elicited in the patient; (ii) adaptive immune resistance, in which an active anti-tumour immune response is present and can recognise cancerous cells but is unable to eliminate them due to immune evasion mechanisms established within the tumour milieu; and (iii) acquired resistance, in which the tumour initially responds to immunotherapy but subsequently becomes resistant and progresses on therapy ,11,15. TImmunotherapy functions by stimulating the host immune system; an effective immunotherapy must be capable of generating a robust T-cell response that can overcome tumour and TME induced immunosuppression. Apart from co-stimulatory molecules, immune cells also express surface inhibitory receptors that are a part of their intrinsic regulatory mechanism, sanctioned to act as checkpoints that prevent over-activation of the immune response . Such ovIpilimumab (Yervoy™) is a monoclonal antibody that targets the CTLA-4 inhibitory receptor. It was the first checkpoint inhibitor to be approved by the FDA in 2011 for the treatment of patients with unresectable or advanced stage metastatic melanoma . CTLA-4 Since the FDA approval of ipilimumab, six more ICIs targeting the PD-1/PD-L1 inhibitory pathway have been approved for clinical use . Nivolum+ T-cells. The TME can also lead to the upregulation of PD-1 on T-cells themselves, which further impairs the anti-tumour immune response . T. T144]. To date, a limited series of bespoke HAPs have been evaluated in clinical trials, including porfiromycin, banoxantrone, tirapazamine, evofosfamide, PR-104 and tarloxotinib ,147,148.Evofosfamide (TH-302) is a 2-nitroimidazole triggered HAP of a DNA-crosslinking phosphoramidate mustard, which demonstrated considerable hypoxia-selective cytotoxicity in vitro across a range of human cancer cell lines and in vivo anti-tumour efficacy in multiple tumour xenograft models ,150,151.+ T-cells [+ T-cells in the tumour, and allowed evofosfamide, in combination with CTLA-4/PD-1 blockade, to cure most animals bearing TRAMP-C2 tumours  [+ T-cell proliferation and a significant drop in MDSC proliferation [Using a syngeneic murine TRAMP-C2 prostate model, it was demonstrated that the improved blood vessel density observed following evofosfamide administration correlated with a reduction in hypoxic fraction and a simultaneous increase in tumour infiltration of CD3 T-cells . When suurvival) . Evofosfferation . Synergyferation . Overallferation . In immuferation . Promisiferation .2 in gas phase). Under hypoxic conditions, tarloxotinib undergoes controlled fragmentation, leading to the release of a potent, irreversible, pan-human epidermal growth factor receptor (pan-HER) inhibitor (tarloxotinib-TKI) [Tarloxotinib is a molecularly targeted HAP developed from the prototype tyrosine kinase inhibitor prodrug SN29966 ,159. Tarnib-TKI) . The relPreclinical studies have shown the superior anti-tumour activity of tarloxotinib compared to various clinical-stage EGFR-TKIs. Notably, tumour regressions were more profound and durable compared to afatinib (irreversible EGFR inhibitor) and cetuximab  in murine xenograft models of epidermoid carcinoma (A431) and HNSCC (FaDu) . Through+ T-cell/MDSC and CD8+ T-cell/Treg ratios after the administration of tarloxotinib. Further studies showed increased antigen-release to the lymph nodes, increased T-cell function in the tumour and changes in cytokine production profiles after tarloxotinib administration [Tarloxotinib has been demonstrated to enhance the efficacy of ICI. In vivo studies showed that treatment with tarloxotinib reduced the hypoxic fractions within syngeneic murine tumours and delayed tumour growth when combined with ICIs . Investistration ,165. OveCP-506 is a second generation analogue of PR-104 that is resistant to aerobic activation by aldo-keto reductase 1C3 ,167. CP-2 in gas phase). Hypoxia-selective cytotoxicity ratios up to 200-fold were observed in 2D and 3D cell culture models, and CP-506 inhibits the growth of a variety of tumour xenograft models. The observed therapeutic effect was selective for hypoxic cells and causally related to tumour oxygenation. For example, the administration of CP-506 in mice bearing the triple negative breast cancer tumour model MDA-MB-468 is observed to eliminate tumour hypoxia detected by pimonidazole and EF5 binding 24 h later  led to reduced HIF-1-mediated upregulation of CCL28 expression, which can potentially lead to reduced Treg recruitment and Treg-induced tumour angiogenesis . Furthert cancer . The PhaThe novel HIF-2 allosteric inhibitor belzutifan   is curre+ T-cells (increased IFN-γ) [Additional evidence for this hypothesis is illustrated by the targeting of the HIF-1-induced cell surface pH regulatory enzyme, carbonic anhydrase IX (CAIX). CAIX contributes to the acidification of the TME by hydrating carbon dioxide to produce protons, and its expression is associated with poor prognosis . A studyd IFN-γ) .Replicating biological vectors such as viruses or bacteria have been shown to selectively infect, replicate in, and lyse hypoxic tumour tissue, providing an alternative means to eliminate tumour hypoxia. Infection of the tumour microenvironment can often provide an immunostimulatory effect, with the potential for anti-tumour immune responses and synergy with current cancer immunotherapy strategies.Clinicaltrials.gov (accessed 31 March 2021).An oncolytic virus preferentially infects and lyses cancer cells, resulting in the spread of progeny virus particles to adjacent tumour cells where the oncolytic process is repeated . TropismWhilst hypoxia typically inhibits adenoviral replication , other tSalmonella typhimurium (VNP20009) is thought to target tumours due to its preference for hypoxia in tumour cores, in contrast to the more oxygenated outer regions [Many genera of bacteria have been shown to specifically and preferentially target solid tumours and cause tumour cell lysis. Tumour selectivity is usually achieved by utilising the existing characteristics of the tumour microenvironment, such as necrosis and hypoxia, with these areas providing a haven for anaerobic and facultative anaerobic bacteria to germinate ,192. A g regions . FacultaClostridium novyi-NT spores germinate in necrotic regions of tumours and have resulted in objective responses in induced tumours in mouse models and naturally developing neoplasia in companion dogs. When injected intratumourally, C. novyi-NT resulted in increased phagocytosis and NK cell-like function after treatment, while intravenous injection increased LPS-induced TNF-α production, lipoteichoic acid (LTA)-induced IL-10 production and NK cell-like function post-treatment [C. novyi-NT is currently undergoing Phase I evaluation with pembrolizumab (NCT03435952).reatment . C. novyImmune checkpoint inhibitors are undoubtedly a promising treatment for cancer, but their efficacy is often impeded by complex sets of tumour-intrinsic and -extrinsic resistance factors which constrain their broader therapeutic potential. The concept that tumour hypoxia within the TME is one of the major drivers for immunosuppression is becoming well established. Several studies have modulated tumour hypoxia and hypoxia-driven pathways to improve immunotherapy responsiveness with promising results. The hypoxia-targeted therapeutic strategies discussed in this review provide additional therapeutic opportunities to combine with clinical stage ICIs to increase the number of responders amongst cancer patients and improve patient outcome. More preclinical studies are needed to ensure that novel therapies that overcome hypoxia-mediated immunosuppression do so without detrimental effects on the immune system. For example, the strict oxygen-dependence of HAP activation (termed the k-curve) is an important feature given the presence of mild physiological hypoxia in various lymphoid organs ,195,196.


This study evaluated the effect of bovine pulmonary surfactant (BPS) on the C. freundii, E. cloacae, E. coli, H. influenzae, H. parainfluenzae, K. pneumoniae, methicillin-susceptible S. aureus, M. catarrhalis, S. pneumoniae, and S. pyogenes. Isolates were tested with the appropriate broth microdilution method for each organism as specified by CLSI. For most organisms, MICs were determined in cation-adjusted Mueller-Hinton broth (CAMHB). CAMHB was supplemented with 2.5-5% lysed horse blood for streptococci and Haemophilus Test Medium broth for Haemophilus spp. Daptomycin (DAP) was tested against S. aureus ATCC 29213 as a positive control.A total of 10 isolates recovered from patients with infections in 2018 were tested for antimicrobial susceptibility to TBP and ETP in the absence or presence of 1%, 5%, or 10% BPS . These isolates included the following species: Haemophilus strains growing in the presence of 5% or 10% BPS. For these conditions, resazurin was added to establish an MIC value. The MIC values found with this method did not differ from the MIC values found in either HTM media or HTM media with 1% BPS. As expected, the addition of BPS shifted DAP S. aureus MIC values to >8 mg/L for all 3 BPS concentrations.All isolates displayed TBP MIC values ranging from ≤0.004 to 0.06 mg/L in media without BPS. There were no observed MIC increases >2-fold in the presence of BPS. 4 of the 10 isolates displayed slightly higher (≥4-fold) ETP than TBP MIC values. The ETP MIC values ranged from 0.015-0.25 mg/L in media without BPS. Similarly, there were no observed instances of a >2-fold shift toward lower potency in the presence of BPS. For both TBP and ETP, MIC endpoint values were easily determined, except for in the case of the 2 in vitro MIC values of TBP or ETP against these species.TBP displayed potent activity against all isolates tested, as all observed MIC values were ≤0.06 mg/L. The addition of BPS to the testing medium did not affect the S J Ryan Arends, PhD, AbbVie (formerly Allergan) (Research Grant or Support)GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Abby L. Klauer, n/a, Cidara Therapeutics, Inc. (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Nicole Cotroneo, Spero Therapeutics  Ian A. Critchley, Ph.D., Spero Therapeutics  Rodrigo E. Mendes, PhD, AbbVie (Research Grant or Support)AbbVie (formerly Allergan) (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)ContraFect Corporation (Research Grant or Support)GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support)


ABSTRACT IMPACT: NJ ACTS provides mentored coaching in NIH grant writing for early stage investigators. OBJECTIVES/GOALS: Launching an independent academic careers requires the ability to effectively communicate the purpose and impact of biomedical research in order to obtain extramural funding. We sought to develop and evaluate an interactive grant writing group of junior faculty and senior postdoctoral fellows mentored by trained coaches. METHODS/STUDY POPULATION: Participants meet monthly for 1 hour to peer review Specific Aims pages for grant applications to NIH and private foundations. Sessions are moderated by two senior faculty trained as coaches by the National Research Mentoring Network. Participant grant submission and review of the program are collected annually. RESULTS/ANTICIPATED RESULTS: From 2019-2020, 15 faculty and 2 postdoctoral fellows participated in the grant writing group with an average of 5.7 participants each month. Over the year, participants submitted 53 grant applications (68% submitted to NIH with the majority being R21 or R01 grant mechanisms). Half of grants submitted were discussed during peer review sessions. Of the grants reviewed, 42% were funded or near/below the funding payline. Using a 5-point Likert scale, participants highly rated the quality of coaching (mean/SD: 4.9 ±0.2), time discussing their research (mean/SD: 4.5 ±0.8), and the input from other participants (mean/SD: 4.5 ±0.5). DISCUSSION/SIGNIFICANCE OF FINDINGS: In conclusion, a monthly meeting of junior investigators hosted by two grant writing coaches is an effective means to receive peer review of grant application aims and support submissions for extramural funding.