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Patients with pancreatic cancer often suffer from cachexia and experience gastrointestinal symptoms that may be related to intestinal smooth muscle cell (SMC) dysfunction. We hypothesized that pancreatic tumor organoids from cachectic patients release factors that perturb the SMC's contractile characteristics. Human visceral SMCs were exposed to conditioned medium (CM) from the pancreatic tumor organoid cultures of cachectic ( n = 2) and non-cachectic ( n = 2) patients. Contractile proteins and markers of inflammation, muscle atrophy, and proliferation were evaluated by qPCR and Western blot. SMC proliferation and migration were monitored by live cell imaging. The Ki-67-positive cell fraction was determined in the intestinal smooth musculature of pancreatic cancer patients. CM from the pancreatic tumor organoids of cachectic patients did not affect IL-1β , IL-6 , IL-8 , MCP-1 , or Atrogin-1 expression. However, CM reduced the α-SMA, γ-SMA, and SM22-α levels, which was accompanied by a reduced SMC doubling time and increased expression of S100A4 , a Ca 2 +-binding protein associated with the synthetic SMC phenotype. In line with this, Ki-67-positive nuclei were increased in the intestinal smooth musculature of patients with a low versus high L3-SMI. In conclusion, patient-derived pancreatic tumor organoids release factors that compromise the contractile SMC phenotype and increase SMC proliferation. This may contribute to the frequently observed gastrointestinal motility problems in these patients.

Organoids are increasingly used to investigate patient-specific drug responsiveness, but organoid culture is complex and expensive, and carried out in rich, non-physiological media. We investigated reproducibility of drug-responsiveness of primary cell cultures in 2D versus 3D and in conventional versus physiological cell culture medium. 3D pancreatic ductal adenocarcinoma organoid cultures PANCO09b and PANCO11b were converted to primary cell cultures growing in 2D. Transformed 2D cultures were grown in physiological Plasmax medium or Advanced-DMEM/F12. Sensitivity towards gemcitabine, paclitaxel, SN-38, 5-fluorouacil, and oxaliplatin was investigated by cell viability assays. Growth rates of corresponding 2D and 3D cultures were comparable. PANCO09b had a shorter doubling time in physiological media. Chemosensitivity of PANCO09b and PANCO11b grown in 2D or 3D was similar, except for SN-38, to which PANCO11b cultured in 3D was more sensitive (2D: 8.2 ×10 -3 ± 2.3 ×10 -3 vs. 3D: 1.1 ×10 -3 ± 0.6 ×10 -3 , p = 0.027). PANCO09b and PANCO11b showed no major differences in chemosensitivity when cultured in physiological compared to conventional media, although PANCO11b was more sensitive to SN-38 in physiological media (9.8 × 10 -3 ± 0.7 × 10 -3 vs. 5.2 × 10 -3 ± 1.8 × 10 -3 , p = 0.015). Collectively, these data indicate that the chemosensitivity of organoids is not affected by culture medium composition or culture dimensions. This implies that organoid-based drug screens can be simplified to become more cost-effective.

Background: Previous preclinical and clinical research has investigated the role of intestinal microbiota in carcinogenesis. Growing evidence exists that intestinal microbiota can influence breast cancer carcinogenesis. However, the role of intestinal microbiota in breast cancer needs to be further investigated. This study aimed to identify the microbiota differences between postmenopausal breast cancer patients and controls., Patients and Methods: This prospective cohort study compared the intestinal microbiota richness, diversity, and composition in postmenopausal histologically proven ER+/HER2- breast cancer patients and postmenopausal controls. Patients scheduled for (neo)adjuvant adriamycin, cyclophosphamide (AC), and docetaxel (D), or endocrine therapy (tamoxifen) were prospectively enrolled in a multicentre cohort study in the Netherlands. Patients collected a faecal sample and completed a questionnaire before starting systemic cancer treatment. Controls, enrolled from the National Dutch Breast Cancer Screening Programme, also collected a faecal sample and completed a questionnaire. Intestinal microbiota was analysed by amplicon sequencing of the 16S rRNA V4 gene region., Results: In total, 81 postmenopausal ER+/HER2- breast cancer patients and 67 postmenopausal controls were included, resulting in 148 faecal samples. Observed species richness, Shannon index, and overall microbial community structure were not significantly different between breast cancer patients and controls. There was a significant difference in overall microbial community structure between breast cancer patients scheduled for adjuvant treatment, neoadjuvant treatment, and controls at the phylum ( p = 0.042) and genus levels ( p = 0.015). Dialister ( p = 0.001) and its corresponding family Veillonellaceae ( p = 0.001) were higher in patients scheduled for adjuvant treatment, compared to patients scheduled for neoadjuvant treatment. Additional sensitivity analysis to correct for the potential confounding effect of prophylactic antibiotic use, indicated no differences in microbial community structure between patients scheduled for neoadjuvant systemic treatment, adjuvant systemic treatment, and controls at the phylum ( p = 0.471) and genus levels ( p = 0.124)., Conclusions: Intestinal microbiota richness, diversity, and composition are not different between postmenopausal breast cancer patients and controls. The increased relative abundance of Dialister and Veillonellaceae was observed in breast cancer patients scheduled for adjuvant treatment, which might be caused by a relative decrease in other bacteria due to prophylactic antibiotic administration rather than an absolute increase.

Systemic inflammation is thought to underlie many of the metabolic manifestations of cachexia in cancer patients. The complement system is an important component of innate immunity that has been shown to contribute to metabolic inflammation. We hypothesized that systemic inflammation in patients with cancer cachexia was associated with complement activation. Systemic C3a levels were higher in cachectic patients with inflammation ( n = 23, C-reactive protein (CRP) ≥ 10 mg/L) as compared to patients without inflammation ( n = 26, CRP < 10 mg/L) or without cachexia ( n = 13) (medians 102.4 (IQR 89.4-158.0) vs. 81.4 (IQR 47.9-124.0) vs. 61.6 (IQR 46.8-86.8) ng/mL, respectively, p = 0.0186). Accordingly, terminal complement complex (TCC) concentrations gradually increased in these patient groups (medians 2298 (IQR 2022-3058) vs. 1939 (IQR 1725-2311) vs. 1805 (IQR 1552-2569) mAU/mL, respectively, p = 0.0511). C3a and TCC concentrations were strongly correlated (r s = 0.468, p = 0.0005). Although concentrations of C1q and mannose-binding lectin did not differ between groups, C1q levels were correlated with both C3a and TCC concentrations (r s = 0.394, p = 0.0042 and r s = 0.300, p = 0.0188, respectively). In conclusion, systemic inflammation in patients with cancer cachexia is associated with the activation of key effector complement factors. The correlations between C1q and C3a/TCC suggest that the classical complement pathway could play a role in complement activation in patients with pancreatic cancer.