Circulating tumor cells (CTCs) are shed into the bloodstream from primary and metastatic tumor deposits. Their isolation and analysis hold great promise for the early detection of invasive cancer and ...the management of advanced disease, but technological hurdles have limited their broad clinical utility. We describe an inertial focusing-enhanced microfluidic CTC capture platform, termed "CTC-iChip," that is capable of sorting rare CTCs from whole blood at 10(7) cells/s. Most importantly, the iChip is capable of isolating CTCs using strategies that are either dependent or independent of tumor membrane epitopes, and thus applicable to virtually all cancers. We specifically demonstrate the use of the iChip in an expanded set of both epithelial and nonepithelial cancers including lung, prostate, pancreas, breast, and melanoma. The sorting of CTCs as unfixed cells in solution allows for the application of high-quality clinically standardized morphological and immunohistochemical analyses, as well as RNA-based single-cell molecular characterization. The combination of an unbiased, broadly applicable, high-throughput, and automatable rare cell sorting technology with generally accepted molecular assays and cytology standards will enable the integration of CTC-based diagnostics into the clinical management of cancer.
Chemotherapy treatment of metastatic colon cancer ultimately fails due to development of drug resistance. Identification of chemotherapy-induced changes in tumor biology may provide insight into drug ...resistance mechanisms.
We studied gene expression differences between groups of liver metastases that were exposed to preoperative chemotherapy or not. Multiple patient-derived colonosphere cultures were used to assess how chemotherapy alters energy metabolism by measuring mitochondrial biomass, oxygen consumption, and lactate production. Genetically manipulated colonosphere-initiated tumors were used to assess how altered energy metabolism affects chemotherapy efficacy.
Gene ontology and pathway enrichment analysis revealed significant upregulation of genes involved in oxidative phosphorylation (OXPHOS) and mitochondrial biogenesis in metastases that were exposed to chemotherapy. This suggested chemotherapy induces a shift in tumor metabolism from glycolysis towards OXPHOS. Indeed, chemotreatment of patient-derived colonosphere cultures resulted in an increase of mitochondrial biomass, increased expression of respiratory chain enzymes, and higher rates of oxygen consumption. This was mediated by the histone deacetylase sirtuin-1 (SIRT1) and its substrate, the transcriptional coactivator PGC1α. Knockdown of SIRT1 or PGC1α prevented chemotherapy-induced OXPHOS and significantly sensitized patient-derived colonospheres as well as tumor xenografts to chemotherapy.
Chemotherapy of colorectal tumors induces a SIRT1/PGC1α-dependent increase in OXPHOS that promotes tumor survival during treatment. This phenomenon is also observed in chemotherapy-exposed resected liver metastases, strongly suggesting that chemotherapy induces long-lasting changes in tumor metabolism that potentially interfere with drug efficacy. In conclusion, we propose a novel mechanism of chemotherapy resistance that may be clinically relevant and therapeutically exploitable.
Abstract In epithelial tumors, the platelet-derived growth factor receptor B (PDGFRB) is mainly expressed by stromal cells of mesenchymal origin. Tumor cells may also acquire PDGFRB expression ...following epithelial-to-mesenchymal transition (EMT), which occurs during metastasis formation. Little is known about PDGFRB signaling in colorectal tumor cells. We studied the relationship between PDGFRB expression, EMT, and metastasis in human colorectal cancer (CRC) cohorts by analysis of gene expression profiles. PDGFRB expression in primary CRC was correlated with short disease-free and overall survival. PDGFRB was co-expressed with genes involved in platelet activation, transforming growth factor beta (TGFB) signaling, and EMT in three CRC cohorts. PDGFRB was expressed in mesenchymal-like tumor cell lines in vitro and stimulated invasion and liver metastasis formation in mice. Platelets, a major source of PDGF, preferentially bound to tumor cells in a non-activated state. Platelet activation caused robust PDGFRB tyrosine phosphorylation on tumor cells in vitro and in liver sinusoids in vivo . Platelets also release TGFB, which is a potent inducer of EMT. Inhibition of TGFB signaling in tumor cells caused partial reversion of the mesenchymal phenotype and strongly reduced PDGFRB expression and PDGF-stimulated tumor cell invasion. These results suggest that PDGFRB may contribute to the aggressive phenotype of colorectal tumors with mesenchymal properties, most likely downstream of platelet activation and TGFB signaling.
Fragility ankle fractures in elderly have a rising incidence and hospitalization may be prolonged due to pre-existing comorbidities, compromised soft tissue and postoperative difficulties in the ...rehabilitation process. The aim of this retrospective cohort study was to investigate risk factors for longer total hospitalization duration in elderly patients with surgically treated fragility (Lauge Hansen supination external rotation type 4) fractures. We included all patients ≥ 70 years with a fragility fracture, who were treated surgically between 2011 and 2019 (n = 97) in a level 1 and 2 trauma center. Data on patient demographics, fracture characteristics, surgical treatment strategies and postoperative complications were retrieved from medical records. Multivariate regression analysis was performed to identify independent risk factors for longer hospitalization duration. The mean age of the included patients was 78.27 (± 6.56) years; 71 patients (73.20%) were female. Ten fractures (10.30%) were classified as open and 49 (50.50%) as a luxation type fracture. Fifty-nine patients (60.80%) were hospitalized after admission to the emergency department. External fixation was performed in 34 patients (35.10%) and served as bridge to definitive fixation in 29 patients (85.30%). The mean total hospital length of stay of all patients was 7.04 (± 6.58) days. Multivariate regression analysis demonstrated that the use of external fixation (p < .001) and the postoperative discharge destination (p < .001) were independently associated with a prolonged hospital stay. External fixation and discharge destination were independent risk factors for a prolonged hospital stay in elderly patients with a fragility fracture.
Deployment of geriatric care would be more sustainable if we could limit geriatric co-management to older hip fracture patients who benefit most from it. We assumed that riding a bicycle is a proxy ...of good health and hypothesized that older patients with a hip fracture due to a bicycle accident have a more favorable prognosis than patients whose hip fracture was caused by another type of accident.
Retrospective cohort study of hip fracture patients ≥ 70 years admitted to hospital. Nursing home residents were excluded. Primary outcome was length of hospital stay (LOS). Secondary outcomes were delirium, infection, blood transfusion, intensive care unit stay and death during hospitalization. The group with a bicycle accident (BA) was compared to the non-bicycle accident (NBA) group using linear and logistic regression models, with correction for age and sex.
Of the 875 patients included, 102 (11.7%) had a bicycle accident. BA patients were younger (79.8 versus 83.9 years, p < 0.001), less often female (54.9 versus 71.2%, p = 0.001) and lived independently more often (100 versus 85.1%, p < 0.001). Median LOS in the BA group was 0.91 times the median LOS in the NBA group (p = 0.125). For none of the secondary outcomes the odds ratio favored the BA group, except for infection during hospital stay (OR = 0.53, 95%CI 0.28-0.99; p = 0.048).
Although older hip fracture patients who had a bicycle accident appeared more healthy than other older hip fracture patients, their clinical course was not more favorable. Based on this study, a bicycle accident is not an indicator that geriatric co-management can be omitted.
Drug-resistant cancer stem cells (CSCs) have been implicated in tumor recurrence following chemotherapy. However, the contribution of CSCs to drug-resistance in colorectal cancer is unclear and ...CSC-intrinsic drug-resistance mechanisms are ill-defined. Here, we address these issues by proteomic analysis of the secretomes of CSCs and isogenic differentiated tumor cells (DTCs) isolated from three distinct metastasized colon tumors.
Mass spectrometry-based proteomics identified 1254 unique proteins in the conditioned media of the paired CSC and DTC cultures. Ingenuity Pathway Analysis revealed that proteins governing ‘Cell Death’ were most significantly enriched in the CSC secretome. The vast majority of these (37/43) promote cell survival. The CSC secretome is also characterized by a pro-survival Nrf2 antioxidant signature. Interestingly, proteome-maintenance networks are highly enriched in the CSC secretome. CSCs also secrete high levels of drug-metabolizing enzymes, including aldehyde dehydrogenase 1 (ALDH1A1) and bleomycin hydrolase (BLMH). We show that these enzymes cause extracellular detoxification of maphosphamide and bleomycin respectively.
We conclude that colorectal CSCs are characterized by extensive survival and anti-oxidant networks, which are likely to contribute to CSC-intrinsic drug-resistance. In addition, CSCs may modulate drug responses in nearby tumor cells by detoxifying chemotherapeutic drugs in the extracellular space.
Cancer stem cells are thought to play an important role in mediating drug resistance and tumor recurrence following chemotherapy. Therefore, it is important to identify the factors that are secreted by them. Our results provide novel insights into the pathways that govern the intrinsic resistance of CSCs to chemotherapy and, furthermore, demonstrate that they can also inactivate chemotherapeutic drugs in the extracellular space. A better understanding of the pathways that govern drug resistance in CSCs may help in developing effective CSC-targeting drugs.
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•Secretome analysis of cancer stem cells and differentiated tumor cells•Survival proteins are enriched in the cancer stem cell secretome.•Cancer stem cells secrete high levels of drug-metabolizing enzymes.•Secreted drug-metabolizing enzymes detoxify drugs in the extracellular space.•Cancer stem cells may protect nearby non-CSCs from cytotoxic chemotherapy.
Background & Aims Stem cells of normal tissues have resistance mechanisms that allow them to survive genotoxic insults. The stem cell–like cells of tumors are defined by their tumor-initiating ...capacity and may have retained these resistance mechanisms, making them resistant to chemotherapy. We studied the relationship between resistance to the topoisomerase I inhibitor irinotecan and tumor-initiating potential in human colonosphere cultures and in mice with colorectal xenograft tumors. Methods Colonosphere cultures were established from human colorectal tumor specimens obtained from patients who underwent colon or liver resection for primary or metastatic adenocarcinoma. Stem cell and differentiation markers were analyzed by immunoblotting and fluorescence-activated cell sorting. Clone- and tumor-initiating capacities were assessed by single-cell cloning and in immune-deficient mice. Sensitivity to irinotecan was assessed in vitro and in tumor-bearing mice. The relationship between drug resistance and tumor-initiating capacity was tested by fluorescence-activated cell sorting of colonosphere cells, based on expression of ABCB1 and aldehyde dehydrogenase (ALDH) activity. Results Colonosphere cultures had a high capacity to initiate tumors in mice and were resistant to irinotecan. Inhibition of the drug-efflux pump ABCB1 by PSC-833 allowed irinotecan to eradicate tumor-initiating cells. However, ABCB1 was expressed only by a subpopulation of differentiated tumor cells that did not form clones or tumors. Conversely, tumor-initiating cells were ABCB1-negative and were identified by high ALDH activity. Tumorigenic ALDHhigh /ABCB1negative cells generated nontumorigenic ALDHlow /ABCB1positive daughter cells in vitro and in tumor xenografts. PSC-833 increased the antitumor efficacy of irinotecan in mice. Conclusions The resistance of colorectal tumors to irinotecan requires the cooperative action of tumor-initiating ALDHhigh /ABCB1negative cells and their differentiated, drug-expelling, ALDHlow /ABCB1positive daughter cells.
Intramedullary fixation using a fibular nail is a minimally invasive alternative to conventional plate fixation that provides superior biomechanical strength and allows immediate full weightbearing ...postoperatively. The study aim was to compare the postoperative complications of minimally invasive intramedullary fibular nail fixation to plate fixation for Lauge-Hansen supination external rotation type 4 (Weber B) fractures in patients aged 65 years or older treated in a single geriatric trauma unit in the Netherlands. A retrospective cohort study was performed including patients aged 65 years or older with a Lauge-Hansen supination external rotation type 4 (Weber B) fracture treated with either intramedullary fibular fixation or plate fixation between January 2017 and January 2019. A total number of 58 patients were included with a mean age of 73.9 years (range 65-95). The intramedullary fixation-cohort (n = 13) had a significantly higher mean age (82.5 vs 71.4 years, p = .002) and Charlson Co-morbidity Index (4.7 vs 3.6, p = .005) compared to the plate fixation-cohort (n = 45). The total number of postoperative complications was lower after intramedullary fixation (n = 2, 15%) compared to plate fixation (n = 15, 33%), although this relative difference was not significant (p = .307). All 2 complications observed after intramedullary fixation were wound infections demanding no debridement or implant removal. No implant related complications, hospital-acquired complications or mortality were observed after intramedullary fixation. Despite the higher mean age and co-morbidity status of patients treated with minimally invasive intramedullary fibular nailing, the total number of postoperative complications was lower after intramedullary fixation compared to plate fixation. This technique might be a promising alternative in selected patients.
Wnt/β-catenin signalling controls development and adult tissue homeostasis and causes cancer when inappropriately activated. In unstimulated cells, an Axin1-centred multi-protein complex ...phosphorylates the transcriptional co-activator β-catenin, marking it for degradation. Wnt signalling antagonizes β-catenin proteolysis, leading to its accumulation and target gene expression. How Wnt stimulation alters the size distribution, composition and activity of endogenous Axin1 complexes remains poorly understood. Here, we employed two-dimensional blue native/SDS-PAGE to analyse endogenous Axin1 and β-catenin complexes during Wnt signalling. We show that the size range of Axin1 complexes is conserved between species and remains largely unaffected by Wnt stimulation. We detect a striking Wnt-dependent, cytosolic accumulation of both non-phosphorylated and phosphorylated β-catenin within a 450 kDa Axin1-based complex and in a distinct, Axin1-free complex of 200 kDa. These results argue that during Wnt stimulation, phosphorylated β-catenin is released from the Axin1 complex but fails to undergo immediate degradation. Importantly, in APC-mutant cancer cells, the distribution of Axin1 and β-catenin complexes strongly resembles that of Wnt-stimulated cells. Our findings argue that Wnt signals and APC mutations interfere with the turnover of phosphorylated β-catenin. Furthermore, our results suggest that the accumulation of small-sized β-catenin complexes may serve as an indicator of Wnt pathway activity in primary cancer cells.
To assess the contribution of hypoxia and bone marrow-derived cells to aggressive outgrowth of micrometastases after liver surgery.
Liver surgery generates a microenvironment that fosters aggressive ...tumor recurrence. These areas are characterized by chronic hypoxia and influx of bone marrow-derived cells.
The contribution of hematopoietic cell types was studied in mice lacking specific components of the immune system and in irradiated mice lacking all bone marrow-derived cells. Tumor cells were derived from colorectal cancer patients and from a metastatic tumor cell line. Hypoxia-induced changes in stem cell and differentiation marker expression, clone-forming potential, and metastatic capacity were assessed. The effect of vascular clamping on cancer stem cell (CSC) characteristics was performed in mice bearing patient-derived liver metastases.
Immune cells and bone marrow-derived cells were not required for aggressive outgrowth of micrometastases in livers treated with surgery. Rather, hypoxia was sufficient to promote invasion and accelerate metastatic outgrowth. This was associated with a rapid loss of differentiation markers and increased expression of CSC markers and clone-forming capacity. Likewise, metastases residing in ischemia-reperfusion-injured liver lobes acquired CSC characteristics. Despite their renowned general resistance to chemotherapy, clone-forming CSCs were readily killed by the hypoxia-activated prodrug tirapazamine.
Surgery-generated hypoxia in the liver causes rapid dedifferentiation of tumor cells into immature CSCs with high clone- and metastasis-forming capacity. The results help explain the phenomenon of aggressive local tumor recurrence after liver surgery and offer a potential strategy to kill aggressive CSCs by hypoxia-activated prodrugs.
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