OE33 R cells are more metabolically robust.

<p>(<b>A</b>) OE33 P and OE33 R cells display similar sensitivities to the glycolytic inhibitor 2-deoxyglucose (55 µg/mL). (<b>B</b>) ECAR is significantly increased in OE33 R cells following oligomycin treatment (2 µg/mL), when compared to OE33 P. (<b>C</b>) OE33 R cells have significantly enhanced clonogenic survival following treatment with oligomycin (2 µg/mL) for 1.5 h, when compared to OE33 P cells. Data are presented as mean ± SEM from at least 3 independent experiments. Statistical analysis was performed by 2-tailed Student’s <i>t</i>-test, *<i>P</i><0.05.</p

Patient cohort characteristics

<p>Abbreviations:^aValues given are mean (range); TNM, Tumour-node-metastasis clinical staging classification; NS, not specified; TRG, Tumour regression grade; N0, indicates lymph node metastasis negative; N1, lymph node metastasis positive.</p

ATP5B expression is increased in the tumour epithelium of poor responders.

<p>ATP5B expression was assessed by immunohistochemistry in pre-treatment tumour biopsies from OAC patients who subsequently received neoadjuvant CRT (<i>n</i> = 23). Representative images of ATP5B staining in tumours from good (<b>A</b>) and poor (<b>B</b>) responders, magnification 10×. (<b>C</b>) ATP5B positivity in the epithelium is significantly increased in tumours of patients with a poor response to neoadjuvant CRT (TRG 3–5), when compared to good responders (TRG 1 and 2). (<b>D</b>) ATP5B positivity in the epithelium is significantly increased in tumours of patients with no evidence of regression (TRG 5), when compared to patients achieving a full (TRG 1) or major response (TRG 2). Statistical analysis was performed by unpaired 2-tailed Student’s <i>t</i>-test, *<i>P</i><0.05.</p

Radioresistant OE33 R cells have increased mitochondrial mutagenesis and altered mitochondrial function.

<p>(<b>A</b>) OE33 R cells demonstrate significantly elevated basal levels of random mitochondrial mutations, when compared to OE33 P. (<b>B</b>) OE33 R cells have significantly increased basal ROS levels, when compared to OE33 P. ROS levels are significantly increased in OE33 P cells at 24 h post irradiation with 2 Gy. (<b>C</b>) Mitochondrial mass is significantly increased in OE33 P cells at 24 h post irradiation with 2 Gy, this effect is not seen in OE33 R. Data are presented as mean±SEM from 3 independent experiments. Statistical analysis was performed by 2-tailed Student’s <i>t</i>-test, *<i>P</i><0.05.</p

OE33 R cells have higher ATP turnover and reduced proton leak.

<p>(<b>A</b>) OE33 P and OE33 R cells demonstrate similar sensitivity to antimycin (2.6 µM). (<b>B</b>) OE33 R cells were significantly more sensitive to the effects of oligomycin (2 µg/mL) on OCR, when compared to OE33 P cells. At 24 h post 2 Gy, OE33 P cells were significantly more sensitive to the effects of oligomycin on OCR, when compared to unirradiated cells. (C) OE33 R cells demonstrate significantly higher percentage of mitochondrial respiration coupled to ATP synthesis, when compared to OE33 P. (<b>D</b>) OE33 R cells demonstrate significantly lower proton leak, when compared to OE33 P. Data are presented as mean± SEM from at least 3 independent experiments. Statistical analysis was performed by 2-tailed Student’s <i>t</i>-test, *<i>P</i><0.05. The contribution of mitochondrial respiration to ATP synthesis in OE33 P (<b>E</b>), OE33 R (<b>F</b>) at basal level and at 24 h post irradiation with 2 Gy (<b>G</b> and <b>H</b>).</p

OE33 R cells display alterations in mitochondrial-associated gene expression, total cellular energy and energy metabolism pathways.

<p>(A) Basal expression of 15 mitochondrial function and energy metabolism-associated genes were altered 1.5-fold in OE33 R cells, when compared to OE33 P. Data are presented as mean ± SEM from 2 independent experiments. (B) OE33 R cells have significantly increased basal intracellular ATP levels, when compared to OE33 P. ATP levels significantly decrease in OE33 P cells at 24 h post irradiation with 2 Gy, when compared to basal levels. Data are presented as mean ± SEM from 1 independent experiment. (C) Basal oxygen consumption rates (OCR) are significantly increased in OE33 R cells, when compared to OE33 P. Data are presented as mean ± SEM from 4 independent experiments. (D) OE33 R and OE33 P cells demonstrate similar extracellular acidification rates (ECAR). Data are presented as mean ± SEM from 4 independent experiments. Statistical analysis was performed by 2-tailed Student’s <i>t</i>-test, *<i>P</i><0.05, **<i>P</i><0.01.</p

Additional file 1: Table S1. of LC3B globular structures correlate with survival in esophageal adenocarcinoma

Clinical and histopathological data from both Group 1 (neoadjuvant-naïve) and Group 2 (neoadjuvant therapy) esophageal adenocarcinoma patients. (DOCX 15 kb

Additional file 2: Figure S1. of LC3B globular structures correlate with survival in esophageal adenocarcinoma

Evaluation of the autophagy marker LC3B in esophageal cancer cell lines following 5-fluorouracil (5-FU) treatment. Untreated and treated (40 μM 5-FU for 48 h) (A) OE21 and (B) KYSE450 cells were prepared as agrose cell pellets which were fixed, processed and stained by standard immunohistochemistry. Mild staining of LC3B is detected before and after treatment in OE21 cells, while in KYSE450 cells, staining is mild in pre-treatment sections, with strong staining observed following treatment (magnification 100×). Untreated and treated (40 μM 5-FU for 48 h) (C) OE21 and (D) KYSE450 cells were fixed and stained for LC3B. Immunofluorescence analysis of OE21 cells shows little if any staining with anti-LC3B, either pre- or post-treatment. In contrast, a small number of KYSE450 cells display LC3B staining, prior to treatment, while the extent and intensity of LC3B staining is significantly increased post treatment (magnification 400×). (Cytospins were fixed in 4 % PFA for 20 min and washed with PBS. Permeabilization was carried out with 0.2 % Triton X prior to staining with anti-LC3B). (TIFF 2351 kb

Characterising the prognostic potential of HLA‑DR during colorectal cancer development

HLA-DR, an MHC class II molecule that mediates antigen presentation, is a favourable prognostic indicator in colorectal cancer (CRC). However, the dynamics and location of HLA-DR expression during CRC development are unclear. We aimed to define HLA-DR expression by immunohistochemistry in colorectal epithelium and stromal tissue at different stages of cancer development, assessing non-neoplastic colorectal adenocarcinoma–adjacent tissue, adenomas and carcinoma tissues, and to associate HLA-DR levels with clinical outcomes. Patients with higher than median HLA-DR expression survived at least twice as long as patients with lower expression. This association was significant for HLA-DR staining in the colorectal carcinoma epithelium (n = 152, p = 0.011, HR 1.9, 95% CI 1.15–3.15) and adjacent non-neoplastic epithelium (n = 152, p < 0.001, HR 2.7, 95% CI 1.59–4.66), but not stroma. In stage II cases, however, the prognostic value of HLA-DR expression was significant only in adjacent non-neoplastic tissues, for both epithelium (n = 63, p = 0.015, HR 3.6, 95% CI 1.279–10.25) and stroma (n = 63, p = 0.018, HR 5.07, 95% CI 1.32–19.49). HLA-DR was lower in carcinoma tissue compared to matched adenomas (n = 35), in epithelium (p < 0.01) and stroma (p < 0.001). HLA-DR was further reduced in late-stage carcinoma (n = 101) compared to early stage (n = 105), in epithelium (p < 0.001) and stroma (p < 0.01). HLA-DR expression was lower (p < 0.05) in the adjacent non-neoplastic epithelium of patients with cancer recurrence. We demonstrate a progressive loss of HLA-DR in epithelial and stromal tissue compartments during CRC development and show prognostic ability in carcinoma–adjacent non-neoplastic tissues, highlighting the importance of this molecule in the anti-cancer immune response. These findings may have wider implications for immunotherapeutic interventions

Effect of the rehabilitation program, ReStOre, on serum biomarkers in a randomized control trial of esophagogastric cancer survivors

Background: The Rehabilitation Strategies Following Esophagogastric cancer (ReStOre) randomized control trial demonstrated a significant improvement in cardiorespiratory fitness of esophagogastric cancer survivors. This follow-up, exploratory study analyzed the biological effect of exercise intervention on levels of 55 serum proteins, encompassing mediators of angiogenesis, inflammation, and vascular injury, from participants on the ReStOre trial. Methods: Patients >6 months disease free from esophagogastric cancer were randomized to usual care or the 12-week ReStOre program (exercise training, dietary counselling, and multidisciplinary education). Serum was collected at baseline (T0), post-intervention (T1), and at 3-month follow up (T2). Serum biomarkers were quantified by enzyme-linked immunosorbent assay (ELISA). Results: Thirty-seven patients participated in this study; 17 in the control arm and 20 in the intervention arm. Exercise intervention resulted in significant alterations in the level of expression of serum IP-10 (mean difference (MD): 38.02 (95% CI: 0.69 to 75.35)), IL-27 (MD: 249.48 (95% CI: 22.43 to 476.53)), and the vascular injury biomarkers, ICAM-1 (MD: 1.05 (95% CI: 1.07 to 1.66)), and VCAM-1 (MD: 1.51 (95% CI: 1.04 to 2.14)) at T1. A significant increase in eotaxin-3 (MD: 2.59 (95% CI: 0.23 to 4.96)), IL-15 (MD: 0.27 (95% CI: 0 to 0.54)) and decrease in bFGF (MD: 1.62 (95% CI: -2.99 to 0.26)) expression was observed between control and intervention cohorts at T2 (p Conclusions: Exercise intervention significantly altered the expression of a number of serum biomarkers in disease-free patients who had prior treatment for esophagogastric cancer. Impact: Exercise rehabilitation causes a significant biological effect on serum biomarkers in esophagogastric cancer survivors. Clinical trial registration: ClinicalTrials.gov (NCT03314311).</p