Nuclear transport proteins are secreted by cancer cells and identified as potential novel cancer biomarkers.

Previous studies have identified increased expression of members of the nuclear transport protein family in cancer cells. Recently, certain nuclear transport proteins have been reported to be secreted by cells and found in the serum. The aims of our study were to investigate the levels of multiple nuclear transport proteins secreted from cancer cells, and to determine their potential as diagnostic markers for cervical and oesophageal cancer. Mass spectrometry identified 10 nuclear transport proteins in the secretome and exosomes of cultured cancer cells, and Western blot analysis confirmed increased secreted levels in cancer cells compared to normal. To investigate their presence in patient serum, enzyme-linked immunosorbent assays were performed and revealed significantly increased levels of KPNß1, CRM1, CAS, IPO5 and TNPO1 in cervical and oesophageal cancer patient serum compared to non-cancer controls. Significantly elevated KPNα2 and RAN levels were also identified in oesophageal cancer serum samples. Logistics regression analyses revealed IPO5 and TNPO1 to be the best performing individual candidate biomarkers in discriminating between cancer cases and controls. The combination of KPNß1, CRM1, KPNα2, CAS, RAN, IPO5 and TNPO1 as a panel of biomarkers had the highest diagnostic capacity with an area under the curve of 0.944 and 0.963, for cervical cancer and oesophageal cancer, and sensitivity of 92.5% at 86.8% specificity and 95.3% sensitivity at 87.5% specificity, respectively. These results suggest that nuclear transport proteins have potential as diagnostic biomarkers for cervical and oesophageal cancers, with a combination of protein family members being the best predictor.

Fibroblast-Derived Extracellular Matrix Induces Chondrogenic Differentiation in Human Adipose-Derived Mesenchymal Stromal/Stem Cells in Vitro.

Mesenchymal stromal/stem cells (MSCs) represent an area being intensively researched for tissue engineering and regenerative medicine applications. MSCs may provide the opportunity to treat diseases and injuries that currently have limited therapeutic options, as well as enhance present strategies for tissue repair. The cellular environment has a significant role in cellular development and differentiation through cell-matrix interactions. The aim of this study was to investigate the behavior of adipose-derived MSCs (ad-MSCs) in the context of a cell-derived matrix so as to model the in vivo physiological microenvironment. The fibroblast-derived extracellular matrix (fd-ECM) did not affect ad-MSC morphology, but reduced ad-MSC proliferation. Ad-MSCs cultured on fd-ECM displayed decreased expression of integrins α2 and ß1 and subsequently lost their multipotency over time, as shown by the decrease in CD44, Octamer-binding transcription factor 4 (OCT4), SOX2, and NANOG gene expression. The fd-ECM induced chondrogenic differentiation in ad-MSCs compared to control ad-MSCs. Loss of function studies, through the use of siRNA and a mutant Notch1 construct, revealed that ECM-mediated ad-MSCs chondrogenesis requires Notch1 and ß-catenin signaling. The fd-ECM also showed anti-senescence effects on ad-MSCs. The fd-ECM is a promising approach for inducing chondrogenesis in ad-MSCs and chondrogenic differentiated ad-MSCs could be used in stem cell therapy procedures.

BTK drives neutrophil activation for sterilizing antifungal immunity.

We describe a previously-unappreciated role for Bruton's tyrosine kinase (BTK) in fungal immune surveillance against aspergillosis, an unforeseen complication of BTK inhibitors (BTKi) used for treating B-cell lymphoid malignancies. We studied BTK-dependent fungal responses in neutrophils from diverse populations, including healthy donors, BTKi-treated patients, and X-linked agammaglobulinemia patients. Upon fungal exposure, BTK was activated in human neutrophils in a TLR2-, Dectin-1-, and FcγR-dependent manner, triggering the oxidative burst. BTK inhibition selectively impeded neutrophil-mediated damage to Aspergillus hyphae, primary granule release, and the fungus-induced oxidative burst by abrogating NADPH oxidase subunit p40phox and GTPase RAC2 activation. Moreover, neutrophil-specific Btk deletion in mice enhanced aspergillosis susceptibility by impairing neutrophil function, not recruitment or lifespan. Conversely, GM-CSF partially mitigated these deficits by enhancing p47phox activation. Our findings underline the crucial role of BTK signaling in neutrophils for antifungal immunity and provide a rationale for GM-CSF use to offset these deficits in susceptible patients.

Isolation of Mouse Neutrophils.

Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited or acquired qualitative and quantitative neutrophil defects are at high risk for developing bacterial and fungal infections and suffering adverse outcomes from these infections. Therefore, research aiming at defining the molecular factors that modulate neutrophil effector function under homeostatic conditions and during infection is essential for devising strategies to augment neutrophil function and improve the outcomes of infected individuals. This article describes reproducible density-gradient-centrifugation-based as well as positive and negative immunomagnetic selection protocols that can be applied in any laboratory to harvest large numbers of highly enriched and highly viable neutrophils from the bone marrow of mice. In another protocol, we also present a method that combines gentle enzymatic tissue digestion with a positive immunomagnetic selection technique or fluorescence-activated cell sorting (FACS) to harvest highly pure and highly viable preparations of neutrophils directly from mouse tissues such as the kidney, the liver, or the spleen. Mouse neutrophils isolated by these protocols can be used to examine several aspects of cellular function ex vivo, including pathogen binding, phagocytosis, and killing, neutrophil chemotaxis, oxidative burst, degranulation, and cytokine production, and for performing neutrophil adoptive transfer experiments. © 2023 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol 1: Isolation of Neutrophils from Mouse Bone Marrow Using Positive Immunomagnetic Separation Alternate Protocol 1: Purification of Neutrophils from Bone Marrow Using Negative Immunomagnetic Separation Alternate Protocol 2: Purification of Neutrophils from Bone Marrow Using Histopaque-Based Density Gradient Centrifugation Basic Protocol 2: Isolation of Neutrophils from Mouse Tissues Using Positive Immunomagnetic Separation Alternate Protocol 3: Isolation of Neutrophils from Mouse Tissues Using FACS.

Decellularized extracellular matrix scaffolds identify full-length collagen VI as a driver of breast cancer cell invasion in obesity and metastasis.

The extracellular matrix (ECM), a major component of the tumor microenvironment, promotes local invasion to drive metastasis. Here, we describe a method to study whole-tissue ECM effects from disease states associated with metastasis on tumor cell phenotypes and identify the individual ECM proteins and signaling pathways that are driving these effects. We show that decellularized ECM from tumor-bearing and obese mammary glands drives TNBC cell invasion. Proteomics of the ECM from the obese mammary gland led us to identify full-length collagen VI as a novel driver of TNBC cell invasion whose abundance in tumor stroma increases with body mass index in human TNBC patients. Last, we describe the mechanism by which collagen VI contributes to TNBC cell invasion via NG2-EGFR cross-talk and MAPK signaling. Overall, these studies demonstrate the value of decellularized ECM scaffolds obtained from tissues to identify novel functions of the ECM.

Cell shape, and not 2D migration, predicts extracellular matrix-driven 3D cell invasion in breast cancer.

Metastasis, the leading cause of death in cancer patients, requires the invasion of tumor cells through the stroma in response to migratory cues, in part provided by the extracellular matrix (ECM). Recent advances in proteomics have led to the identification of hundreds of ECM proteins, which are more abundant in tumors relative to healthy tissue. Our goal was to develop a pipeline to easily predict which ECM proteins are more likely to have an effect on cancer invasion and metastasis. We evaluated the effect of four ECM proteins upregulated in breast tumor tissue in multiple human breast cancer cell lines in three assays. There was no linear relationship between cell adhesion to ECM proteins and ECM-driven 2D cell migration speed, persistence, or 3D invasion. We then used classifiers and partial-least squares regression analysis to identify which metrics best predicted ECM-driven 2D migration and 3D invasion responses. We find that ECM-driven 2D cell migration speed or persistence did not predict 3D invasion in response to the same cue. However, cell adhesion, and in particular cell elongation and shape irregularity, accurately predicted the magnitude of ECM-driven 2D migration and 3D invasion. Our models successfully predicted the effect of novel ECM proteins in a cell-line specific manner. Overall, our studies identify the cell morphological features that determine 3D invasion responses to individual ECM proteins. This platform will help provide insight into the functional role of ECM proteins abundant in tumor tissue and help prioritize strategies for targeting tumor-ECM interactions to treat metastasis.