Label-free surface-enhanced Raman spectroscopy (SERS) can interrogate systems by directly fingerprinting their components' unique physicochemical properties. In complex biological systems however, ...this can yield highly overlapping spectra that hinder sample identification. Here, we present an artificial-nose inspired SERS fingerprinting approach where spectral data is obtained as a function of sensor surface chemical functionality. Supported by molecular dynamics modeling, we show that mildly selective self-assembled monolayers can influence the strength and configuration in which analytes interact with plasmonic surfaces, diversifying the resulting SERS fingerprints. Since each sensor generates a modulated signature, the implicit value of increasing the dimensionality of datasets is shown using cell lysates for all possible combinations of up to 9 fingerprints. Reliable improvements in mean discriminatory accuracy towards 100% are achieved with each additional surface functionality. This arrayed label-free platform illustrates the wide-ranging potential of high-dimensionality artificial-nose based sensing systems for more reliable assessment of complex biological matrices.
Colorectal carcinoma is the third most common cancer in developed countries and the second leading cause of cancer-related mortality. Interest in the influence of the intestinal microbiota on CRC ...emerged rapidly in the past few years, and the close presence of microbiota to the tumour mass creates a unique microenvironment in CRC. The gastrointestinal microbiota secrete factors that can contribute to CRC metastasis by influencing, for example, epithelial-to-mesenchymal transition. Although the role of EMT in metastasis is well-studied, mechanisms by which gastrointestinal microbiota contribute to the progression of CRC remain poorly understood. In this review, we will explore bacterial factors that contribute to the migration and invasion of colorectal carcinoma and the mechanisms involved. Bacteria involved in the induction of metastasis in primary CRC include
Fusobacterium nucleatum
,
Enterococcus faecalis
, enterotoxigenic
Bacteroides fragilis
,
Escherichia coli
and
Salmonella enterica
. Examples of prominent bacterial factors secreted by these bacteria include Fusobacterium adhesin A and Bacteroides fragilis Toxin. Most of these factors induce EMT-like properties in carcinoma cells and, as such, contribute to disease progression by affecting cell-cell adhesion, breakdown of the extracellular matrix and reorganisation of the cytoskeleton. It is of utmost importance to elucidate how bacterial factors promote CRC recurrence and metastasis to increase patient survival. So far, mainly animal models have been used to demonstrate this interplay between the host and microbiota. More human-based models are needed to study the mechanisms that promote migration and invasion and mimic the progression and recurrence of CRC.
Extracellular vesicles (EVs) secreted by cancer cells provide an important insight into cancer biology and could be leveraged to enhance diagnostics and disease monitoring. This paper details a ...high-throughput label-free extracellular vesicle analysis approach to study fundamental EV biology, toward diagnosis and monitoring of cancer in a minimally invasive manner and with the elimination of interpreter bias. We present the next generation of our single particle automated Raman trapping analysisSPARTAsystem through the development of a dedicated standalone device optimized for single particle analysis of EVs. Our visualization approach, dubbed dimensional reduction analysis (DRA), presents a convenient and comprehensive method of comparing multiple EV spectra. We demonstrate that the dedicated SPARTA system can differentiate between cancer and noncancer EVs with a high degree of sensitivity and specificity (>95% for both). We further show that the predictive ability of our approach is consistent across multiple EV isolations from the same cell types. Detailed modeling reveals accurate classification between EVs derived from various closely related breast cancer subtypes, further supporting the utility of our SPARTA-based approach for detailed EV profiling.
Treatment resistance is the main cause of adverse disease outcome in breast cancer patients. Here, we aimed to investigate common features in tamoxifen-resistant and radioresistant breast cancer, as ...tamoxifen-resistant breast cancer cells are cross-resistant to irradiation
RNA sequencing of tamoxifen-resistant and radioresistant breast cancer cells was performed and validated by quantitative PCR. Pathways were further investigated
and in breast cancer patient cohorts to establish their relation with treatment resistance.
Both tamoxifen-resistant and radioresistant breast cancer cells had increased expression levels of genes involved in type I IFN signaling compared with nonresistant cells. IFN-stimulated genes (ISG) were induced in a dose-dependent and time-dependent manner after tamoxifen treatment and irradiation. Tamoxifen treatment also led to ssDNA presence in the cytoplasm, which is known to induce expression of ISGs, a phenomenon that has already been described for irradiation. Moreover, in a breast cancer patient cohort, high expression levels of ISGs were found in the primary tumor in around half of the patients. This was associated with a tumor-infiltrating lymphocyte (TIL) expression signature, although the ISGs were also expressed by the tumor cells themselves. Importantly, the expression of ISGs correlated with outcome in breast cancer patients treated with adjuvant tamoxifen or radiotherapy, but not in systemically untreated patients or chemotherapy-treated patients.
Our data indicate that expression of ISGs by tumor cells is involved in acquired, treatment-induced resistance to tamoxifen and radiotherapy, and might play a role in intrinsic resistance via interaction with TILs.
.
The hypoxia-inducible factor (HIF)-1 pathway can stimulate tumor cell migration and metastasis. Furthermore, hypoxic tumors are associated with a poor prognosis. Besides the HIF-1 pathway, the ...unfolded protein response (UPR) is also induced by hypoxic conditions. The PKR-like ER kinase (PERK)/activating transcription factor 4 (ATF4)-arm of the UPR induces expression of lysosomal-associated membrane protein 3 (LAMP3), a factor that has been linked to metastasis and poor prognosis in solid tumors. In this study the role of UPR-induced LAMP3 in hypoxia-mediated migration of breast cancer cells was examined.
A number of in vitro metastasis models were used to study the migration and invasion of MDA-MB-231 breast cancer cells under hypoxic conditions. PERK, ATF4 and their downstream factor LAMP3 were knocked down to examine their role in cell migration. In addition, multicellular tumor spheroids were used to study the involvement of the tumor microenvironment in invasion.
Using transwell assays, migration of different breast cancer cell lines was assessed. A direct correlation was found between cell migration and baseline LAMP3 expression. Furthermore, moderate hypoxia (1% O2) was found to be optimal in stimulating migration of MDA-MB-231 cells. siRNA mediated knockdown of PERK, ATF4 and LAMP3 reduced migration of cells under these conditions. Using gap closure assays, similar results were found. In a three-dimensional invasion assay into collagen, LAMP3 knockdown cells showed a diminished capacity to invade compared to control cells when collectively grown in multicellular spheroids.
Thus, the PERK/ATF4/LAMP3-arm of the UPR is an additional pathway mediating hypoxia-induced breast cancer cell migration.
Clinical use of human mesenchymal stem cells (hMSCs) is limited due to their rapid clearance, reducing their therapeutic efficacy. The inflammatory cytokine IL-1β activates hMSCs and is known to ...enhance their engraftment. Consequently, understanding the molecular mechanism of this inflammation-triggered adhesion is of great clinical interest to improving hMSC retention at sites of tissue damage. Integrins are cell-matrix adhesion receptors, and clustering of integrins at the nanoscale underlies cell adhesion. Here, we found that IL-1β enhances adhesion of hMSCs via increased focal adhesion contacts in an α5β1 integrin-specific manner. Further, through quantitative super-resolution imaging we elucidated that IL-1β specifically increases nanoscale integrin α5β1 availability and clustering at the plasma membrane, whilst conserving cluster area. Taken together, these results demonstrate that hMSC adhesion via IL-1β stimulation is partly regulated through integrin α5β1 spatial organization at the cell surface. These results provide new insight into integrin clustering in inflammation and provide a rational basis for design of therapies directed at improving hMSC engraftment.
Enabling concurrent, high throughput analysis of single nanoparticles would greatly increase the capacity to study size, composition and inter and intra particle population variance with applications ...in a wide range of fields from polymer science to drug delivery. Here, we present a comprehensive platform for Single Particle Automated Raman Trapping Analysis (SPARTA) able to integrally analyse nanoparticles ranging from synthetic polymer particles to liposomes without any modification. With the developed highly controlled automated trapping process, single nanoparticles are analysed with high throughput and sensitivity to resolve particle mixtures, obtain detailed compositional spectra of complex particles, track sequential functionalisations, derive particle sizes and monitor the dynamics of click reactions occurring on the nanoparticle surface. The SPARTA platform opens up a wide range of new avenues for nanoparticle research through label-free integral high-throughput single particle analysis, overcoming key limitations in sensitivity and specificity of existing bulk analysis methods.
It has been demonstrated that some commonly used Extracellular Vesicle (EV) isolation techniques can lead to substantial contamination with non-EV factors. Whilst it has been established that this ...impacts the identification of biomarkers, the impact on apparent EV bioactivity has not been explored. Extracellular vesicles have been implicated as critical mediators of therapeutic human mesenchymal stem cell (hMSC) paracrine signalling. Isolated hMSC-EVs have been used to treat multiple in vitro and in vivo models of tissue damage. However, the relative contributions of EVs and non-EV factors have not been directly compared. The dependence of hMSC paracrine signalling on EVs was first established by ultrafiltration of hMSC-conditioned medium to deplete EVs, which led to a loss of signalling activity. Here, we show that this method also causes depletion of non-EV factors, and that when this is prevented proangiogenic signalling activity is fully restored in vitro. Subsequently, we used size-exclusion chromatography (SEC) to separate EVs and soluble proteins to directly and quantitatively compare their relative contributions to signalling. Non-EV factors were found to be necessary and sufficient for the stimulation of angiogenesis and wound healing in vitro. EVs in isolation were found to be capable of potentiating signalling only when isolated by a low-purity method, or when used at comparatively high concentrations. These results indicate a potential for contaminating soluble factors to artefactually increase the apparent bioactivity of EV isolates and could have implications for future studies on the biological roles of EVs.
The application of microfluidic devices as next-generation cell and tissue culture systems has increased impressively in the last decades. With that, a plethora of materials as well as fabrication ...methods for these devices have emerged. Here, we describe the rapid prototyping of microfluidic devices, using micromilling and vapour-assisted thermal bonding of polymethyl methacrylate (PMMA), to create a spheroid-on-a-chip culture system. Surface roughness of the micromilled structures was assessed using scanning electron microscopy (SEM) and atomic force microscopy (AFM), showing that the fabrication procedure can impact the surface quality of micromilled substrates with milling tracks that can be readily observed in micromilled channels. A roughness of approximately 153 nm was created. Chloroform vapour-assisted bonding was used for simultaneous surface smoothing and bonding. A 30-s treatment with chloroform-vapour was able to reduce the surface roughness and smooth it to approximately 39 nm roughness. Subsequent bonding of multilayer PMMA-based microfluidic chips created a durable assembly, as shown by tensile testing. MDA-MB-231 breast cancer cells were cultured as multicellular tumour spheroids in the device and their characteristics evaluated using immunofluorescence staining. Spheroids could be successfully maintained for at least three weeks. They consisted of a characteristic hypoxic core, along with expression of the quiescence marker, p27
. This core was surrounded by a ring of Ki67-positive, proliferative cells. Overall, the method described represents a versatile approach to generate microfluidic devices compatible with biological applications.
Lysosome-associated membrane protein 3 (LAMP3) is a member of the LAMP-family of proteins, which are involved in the process of autophagy. Autophagy is induced by tamoxifen in breast cancer cells and ...may contribute to tamoxifen resistance. In this study, the significance of LAMP3 for tamoxifen resistance in breast cancer was examined. The methods employed included use of clonogenic assays to assess the survival of MCF7 breast cancer cells with LAMP3 knockdown after tamoxifen treatment and of quantitative real-time PCR of LAMP3 to evaluate its predictive value for first-line tamoxifen treatment in patients with advanced breast cancer. Results show that tamoxifen treatment of MCF7 cells induced LAMP3 mRNA expression. LAMP3 knockdown in these cells increased tamoxifen sensitivity. Evaluation of expression of the autophagy markers, LC3B and p62, after LAMP3 knockdown showed increased expression levels, indicating that cells with LAMP3 knockdown have a suppressed ability to complete the autophagic process. In addition, knockdown of autophagy-associated genes resulted in sensitization to tamoxifen. Next, tamoxifen-resistant MCF7 cells were cultured. These cells had a sevenfold higher LAMP3 mRNA expression, showed elevated basal autophagy levels, and could be significantly resensitized to tamoxifen by LAMP3 knockdown. In patients treated with first-line tamoxifen for advanced disease (n=304), high LAMP3 mRNA expression was associated with shorter progression-free survival (P=0.003) and shorter post-relapse overall survival (P=0.040), also in multivariate analysis. Together, these results indicate that LAMP3 contributes to tamoxifen resistance in breast cancer. Tamoxifen-resistant cells are resensitized to tamoxifen by the knockdown of LAMP3. Therefore, LAMP3 may be clinically relevant to countering tamoxifen resistance in breast cancer patients.
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