Routine diagnostic practice for cancer and metastasis relies on a time-consuming staining process and the use of antibodies to detect selected molecular markers and is hence limited by a lack of real-time data and the availability of molecular information. Against this background, techniques based on rapid chemical analysis to identify migratory properties are highly desirable. Fourier-Transform Infrared (FTIR) microspectroscopy has a long history in the label-free identification of infrared marker bands for cancer detection. However, it requires extensive postprocessing of the acquired spectra, is of limited suitability for analysis in aqueous environments, and has poor spatial resolution. To overcome these challenges, we are using a new method termed Optical Photothermal Infrared (O-PTIR) spectroscopy to detect local absorption to establish potential IR tumor markers and classification models. We report on experimental outcomes using machine learning and FTIR microspectroscopy for the classification of cells and the analysis of spectral features reflecting cancer and migratory properties, comparing a commercial FTIR microspectrometer to a custom-built O-PTIR instrument dedicated to spectroscopic measurement and imaging in microfluidic channels.

Jagged1-Notch2 and –Notch3 signaling are important for smooth muscle cell (SMC) development. However, a comprehensive understanding of the Jagged1-Notch2/3 signaling in SMCs has been missing. By combining in vitro , in vivo , and in silico analyses, we here show that Notch2 and Notch3 have overlapping roles in differentiating SMCs upon Jagged1 interaction. However, Notch3 is a less potent inducer of Notch signaling activity compared to Notch2, due to weaker interactions between the Notch3 intracellular domain and RBPJκ. Accordingly, unlike Notch3, Notch2 depletion causes an almost total loss of Notch activity and differentiation in large arteries. In smaller arteries, the loss of Notch2 is partially compensated for by a higher SMC expression of Notch3. Our results, therefore, indicate that SMCs have artery-typical vulnerability to altered Notch2 and Notch3 signaling, contributing to our mechanistic understanding of the Notch-related vascular disease onset. HIGHLIGHTS ### Competing Interest Statement The authors have declared no competing interest.

The extracellular matrix (ECM) in the tumor microenvironment plays an essential role in cancer progression. High expression of Jagged1 correlates with poor patient survival and promotes in vivo tumor growth and invasion in triple-negative breast cancer (TNBC). Using transcriptomics, proteomics, and imaging of cancer cell/fibroblast co-cultures, both in vitro and in vivo , we demonstrate that Jagged1-mediated crosstalk between TNBC cells and fibroblasts enhances myofibroblast activation, collagen deposition, and alignment of ECM fibers. Jagged1 increases transforming growth factor beta (TGFβ) activity in fibroblast co-cultures and the Jagged1-induced ECM alignment can be prevented by a TGFβ inhibitor. Thus, Jagged1 regulates ECM remodeling upstream of TGFβ. Furthermore, higher substrate stiffness increases Jagged1 expression, pointing towards a feed-forward loop between Jagged1, ECM stiffness, and TGFβ. With the emergence of safe therapeutics targeting specific Notch ligands and receptors, Jagged1 modulation may offer a path to therapeutically target invasive breast cancer. ### Competing Interest Statement The authors have declared no competing interest. European Union, https://ror.org/019w4f821, 953234 Academy of Finland, https://ror.org/05k73zm37, 307133, 309373 Research Council of Finland, 337531, 357911 Stiftelsen för Åbo Akademi Cancer Foundation Finland Jane and Aatos Erkko Foundation, https://ror.org/03vxy9y38 Sigrid Jusélius Foundation, https://ror.org/00ckakm23 Finnish Cultural Foundation, https://ror.org/027xav248 Swedish Cultural Foundation K. Albin Johanssons stiftelse, https://ror.org/03arxxe13 Ida Montinin Säätiö, https://ror.org/0269hs475

Cancer cells undergo major epigenetic alterations and transcriptomic changes, including ectopic expression of tissue- and cell-type-specific genes. Here, we show that the germline-specific RNA helicase DDX4 forms germ-granule-like cytoplasmic ribonucleoprotein granules in various human tumors, but not in cultured cancer cells. These cancerous DDX4 complexes contain RNA-binding proteins and splicing regulators, including many known germ granule components. The deletion of DDX4 in cancer cells induces transcriptomic changes and affects the alternative splicing landscape of a number of genes involved in cancer growth and invasiveness, leading to compromised capability of DDX4-null cancer cells to form xenograft tumors in immunocompromised mice. Importantly, the occurrence of DDX4 granules is associated with poor survival in patients with head and neck squamous cell carcinoma and higher histological grade of prostate cancer. Taken together, these results show that the germ-granule-resembling cancerous DDX4 granules control gene expression and promote malignant and invasive properties of cancer cells.

Mesoporous silica nanoparticles (MSNs) have emerged as promising drug carriers that can facilitate targeted anticancer drug delivery, but efficiency studies relying on active targeting mechanisms remain elusive. This study implements in vitro 3D cocultures, so‐called microtissues, to model a physiologically relevant tumor microenvironment (TME) to examine the impact of surface‐modified MSNs without targeting ligands on the internalization, cargo delivery, and cargo release in tumor cells and cancer‐associated fibroblasts. Among these, acetylated MSNs most effectively localized in tumor cells in a 3D setting containing collagen, while other MSNs did so to a lesser degree, most likely due to remaining trapped in the extracellular matrix of the TME. Confocal imaging of hydrophobic model drug‐loaded MSNs demonstrated effective cargo release predominantly in tumor cells, both in 2D and 3D cocultures. MSN‐mediated delivery of an anticancer drug in the microtissues exhibited a significant reduction in tumor organoid size and enhanced the tumor‐specific cytotoxic effects of a γ‐secretase inhibitor, compared to the highly hydrophobic drug in free form. This inherent targeting potential suggests reduced off‐target effects and increased drug efficacy, showcasing the promise of surface modification of MSNs as a means of direct cell‐specific targeting and delivery for precise and successful targeted drug delivery.

Metastasis is a multi-step process that is critically affected by cues from the tumor micro-environment (TME), such as from the extracellular matrix (ECM). The role of the ECM in the onset of metastasis, invasion, is not yet fully understood. A further complicating factor is that the ECM in the TME is mostly heterogeneous, in particular presenting a basement membrane (BM) directly enveloping the tumor, which acts as a barrier to invasion into the surrounding stromal ECM. To systematically investigate the role of ECM in invasion, appropriate in vitro models with control over such ECM heterogeneity are essential. We present a novel high-throughput microfluidic approach to build such a model, which enables to capture the invasion of cancer cells from the tumor, through the BM and into the stromal tissue. We used a droplet-maker device to encapsulate cells in beads of a primary hydrogel mimicking BM, Matrigel, which were then embedded in a secondary hydrogel mimicking stromal ECM, collagen I. Our technology ultimately provides control over parameters such as tissue size, cell count and type, and ECM composition and stiffness. As a proof-of-principle, we carried out a comparative study with two breast cancer cell types, and we observed typical behavior consistent with previous studies. Highly invasive MDA-MB-231 cells showed single cell invasion behavior, whereas poorly invasive MCF-7 cells physically penetrated the surrounding matrix collectively. A comparative analysis conducted between our heterogeneous model and previous models employing a single type of hydrogel, either collagen I or Matrigel, has unveiled a substantial difference in terms of cancer cell invasion distance. Our in vitro model resembles an in vivo heterogeneous cancer microenvironment and can potentially be used for high throughput studies of cancer invasion.

Cancer is a global public health concern. Alterations in epigenetic processes are among the earliest genomic aberrations occurring during cancer development and are closely related to progression. Unlike genetic mutations, aberrations in epigenetic processes are reversible, which opens the possibility for novel pharmacological treatments. Non-coding RNAs (ncRNAs) represent an essential epigenetic mechanism, and emerging evidence links ncRNAs to carcinogenesis. Epigenetic drugs (epidrugs) are a group of promising target therapies for cancer treatment acting as coadjuvants to reverse drug resistance in cancer. The present review describes central epigenetic aberrations during malignant transformation and explains how epidrugs target DNA methylation, histone modifications and ncRNAs. Furthermore, clinical trials focused on evaluating the effect of these epidrugs alone or in combination with other anticancer therapies and other ncRNA-based therapies are discussed. The use of epidrugs promises to be an effective tool for reversing drug resistance in some patients with cancer.

Current biomedical models fail to replicate the complexity of human biology. Consequently, almost 90% of drug candidates fail during clinical trials after decades of research and billions of investments in drug development. Despite their physiological similarities, animal models often misrepresent human responses, and instead, trigger ethical and societal debates regarding their use. The overall aim across regulatory entities worldwide is to replace, reduce, and refine the use of animal experimentation, a concept known as the Three Rs principle. In response, researchers develop experimental alternatives to improve the biological relevance of in vitro models through interdisciplinary approaches. This article highlights the emerging organ‐on‐a‐chip technologies, also known as microphysiological systems, with a focus on models of the vasculature. The cardiovascular system transports all necessary substances, including drugs, throughout the body while in charge of thermal regulation and communication between other organ systems. In addition, we discuss the benefits, limitations, and challenges in the widespread use of new biomedical models. Coupled with patient‐derived induced pluripotent stem cells, organ‐on‐a‐chip technologies are the future of drug discovery, development, and personalized medicine.

The mechanical regulation of Notch signaling is an emerging area of interest in cell biology. Notch is essential in many physiological processes in which mechanical stress plays an important role. This review provides an overview of the mechanoregulation of Notch signaling in multiple steps of the pathway. First, we discuss the current knowledge on the direct mechanoregulation of Notch receptor maturation and localization to the membrane and the effect of mechanical stress on the Notch components. Next, we explore how ligand-receptor interactions and membrane dynamics are possible subjects to mechano-regulation, emphasizing the role of cytoskeletal interactions, membrane stiffness, and endocytic complex formation. We further delve into the necessity of tension generation for negative regulatory region (NRR) domain unfolding, facilitated by ligand endocytosis and other microforces. Additionally, we examine the indirect mechano-regulation of S2 and S3 cleavages. Finally, we discuss the mechanoregulation of the Notch intracellular domain (NICD) trafficking and nuclear entry and the impact of mechanical stress on heterochromatin dynamics and nuclear NICD interactions. This review aims to draw attention to the intricate interplay between mechanical cues and Notch signaling regulation, offering novel insights into the multifaceted nature of cellular mechanobiology.

Hydrogels play an important role in tissue engineering due to their native extracellular matrix-like characteristics, but they are insufficient in providing the necessary stimuli to support tissue formation. Efforts to integrate bioactive cues directly into hydrogels are hindered by incompatibility with hydrophobic drugs, issues of burst/uncontrolled release, and rapid degradation of the bioactive molecules. Skeletal muscle tissue repair requires internal stimuli and communication between cells for regeneration, and nanocomposite systems offer to improve the therapeutic effects in tissue regeneration. Here, the versatility of mesoporous silica nanoparticles (MSN) was leveraged to formulate a nanoparticle-hydrogel composite and to combine the benefits of controlled delivery of bioactive cues and cellular support. The tunable surface characteristics of MSNs were exploited to optimize homogeneity and intracellular drug delivery in a 3D matrix. Nanocomposite hydrogels formulated with acetylated or succinylated MSNs achieved high homogeneity in 3D distribution, with succinylated MSNs being rapidly internalized and acetylated MSNs exhibiting slower cellular uptake. MSN-hydrogel nanocomposites simultaneously allowed efficient local intracellular delivery of a hydrophobic model drug. To further study the efficiency of directing cell response, a Notch signaling inhibitor (DAPT) was incorporated into succinylated MSNs and incorporated into the hydrogel. MSN-hydrogel nanocomposites effectively downregulated the Notch signaling target genes, and accelerated and maintained the expression of myogenic markers. The current findings demonstrate a proof-of-concept in effective surface engineering strategies for MSN-based nanocomposites, suited for hydrophobic drug delivery in tissue regeneration with guided cues.

Recent experimental evidence indicates a role for the intermediate filament vimentin in regulating cellular mechanical homeostasis, but its precise contribution remains to be discovered. Mechanical homeostasis requires a balanced bi-directional interplay between the cell's microenvironment and the cellular morphological and mechanical state-this balance being regulated via processes of mechanotransduction and mechanoresponse, commonly referred to as mechanoreciprocity. Here, we systematically analyze vimentin-expressing and vimentin-depleted cells in a swatch of in vitro cellular microenvironments varying in stiffness and/or ECM density. We find that vimentin-expressing cells maintain mechanical homeostasis by adapting cellular morphology and mechanics to micromechanical changes in the microenvironment. However, vimentin-depleted cells lose this mechanoresponse ability on short timescales, only to reacquire it on longer time scales. Indeed, we find that the morphology and mechanics of vimentin-depleted cell in stiffened microenvironmental conditions can get restored to the homeostatic levels of vimentin-expressing cells. Additionally, we observed vimentin-depleted cells increasing collagen matrix synthesis and its crosslinking, a phenomenon which is known to increase matrix stiffness, and which we now hypothesize to be a cellular compensation mechanism for the loss of vimentin. Taken together, our findings provide further insight in the regulating role of intermediate filament vimentin in mediating mechanoreciprocity and mechanical homeostasis.

The Notch signaling pathway is a crucial regulator of cell differentiation as well as tissue organization, whose deregulation is linked to the pathogenesis of different diseases. NOTCH1 plays a key role in breast cancer progression by increasing proliferation, maintenance of cancer stem cells, and impairment of cell death. NOTCH1 is a mechanosensitive receptor, where mechanical force is required to activate the proteolytic cleavage and release of the Notch intracellular domain (NICD). We circumvent this limitation by regulating Notch activity by light. To achieve this, we have engineered an optogenetic NOTCH1 receptor (optoNotch) to control the activation of NOTCH1 intracellular domain (N1ICD) and its downstream transcriptional activities. Using optoNotch we confirm that NOTCH1 activation increases cell proliferation in MCF7 and MDA-MB-468 breast cancer cells in 2D and spheroid 3D cultures, although causing distinct cell-type specific migratory phenotypes. Additionally, optoNotch activation induced chemoresistance on the same cell lines. OptoNotch allows the fine-tuning, ligand-independent, regulation of N1ICD activity and thus a better understanding of the spatiotemporal complexity of Notch signaling.

The canonical members of the Jagged/Serrate and Delta families of transmembrane ligands have an extracellular, amino-terminal C2 domain that binds to phospholipids and is required for optimal activation of the Notch receptor. Somatic mutations that cause amino substitutions in the C2 domain in human JAGGED1 (JAG1) have been identified in tumors. We found in reporter cell assays that mutations affecting an N-glycosylation site reduced the ligand’s ability to activate Notch. This N-glycosylation site located in the C2 domain is conserved in the Jagged/Serrate family but is lacking in the Delta family. Site-specific glycan analysis of the JAG1 amino terminus demonstrated that occupancy of this site by either a complex-type or high-mannose N-glycan was required for full Notch activation in reporter cell assays. Similarly to JAG1 variants with defects in Notch binding, N-glycan removal, either by mutagenesis of the glycosylation site or by endoglycosidase treatment, reduced receptor activation. The N-glycan variants also reduced receptor activation in a Notch signaling–dependent vascular smooth muscle cell differentiation assay. Loss of the C2 N-glycan reduced JAG1 binding to liposomes to a similar extent as the loss of the entire C2 domain. Molecular dynamics simulations suggested that the presence of the N-glycan limits the orientation of JAG1 relative to the membrane, thus facilitating Notch binding. These data are consistent with a critical role for the N-glycan in promoting a lipid-binding conformation that is required to orient Jagged at the cell membrane for full Notch activation.

Hyperactive Notch signalling is frequently observed in breast cancer and correlates with poor prognosis. However, relatively few mutations in the core Notch signalling pathway have been identified in breast cancer, suggesting that as yet unknown mechanisms increase Notch activity. Here we show that increased expression levels of GIT1 correlate with high relapse-free survival in oestrogen receptor-negative (ER(-)) breast cancer patients and that GIT1 mediates negative regulation of Notch. GIT1 knockdown in ER(-) breast tumour cells increased signalling downstream of Notch and activity of aldehyde dehydrogenase, a predictor of poor clinical outcome. GIT1 interacts with the Notch intracellular domain (ICD) and influences signalling by inhibiting the cytoplasm-to-nucleus transport of the Notch ICD. In xenograft experiments, overexpression of GIT1 in ER(-) cells prevented or reduced Notch-driven tumour formation. These results identify GIT1 as a modulator of Notch signalling and a guardian against breast cancer growth.

The opportunity to manipulate cell functions by regulating bioactive surfaces is a potentially promising approach for organic bioelectronics. Here, the tuning of the orientation of charged peptides by means of an electrical input observed via optical tensiometry is reported. A stimuli‐responsive self‐assembled monolayer (SAM) with specially designed charged peptides is used as a model system to switch between two separate hydrophilic states. The underwater contact angle (UCA) technique is used to measure changes in the wetting property of a dichloromethane droplet under electrical stimuli. The observed changes in the UCA of the bio‐interface can be understood in terms of a change in the surface energy between the ON and OFF states. Molecular dynamics simulations in an electric field have been performed to verify the hypothesis of the orientational change of the charged peptides upon electrical stimulation. In addition, X‐ray photoelectron spectroscopy (XPS) is performed to clarify the stability of the functionalized electrodes. Finally, the possibility of using such a novel switching system as a tool to characterize bioactive surfaces is discussed.

The Notch signaling pathway is a direct cell-cell communication system involved in a wide variety of biological processes, and its disruption is observed in several pathologies. The pathway is comprised of a ligand-expressing (sender) cell and a receptor-expressing (receiver) cell. The canonical ligands are members of the Delta/Serrate/Lag-1 (DSL) family of proteins. Their binding to a Notch receptor in a neighboring cell induces a conformational change in the receptor, which will undergo regulated intramembrane proteolysis (RIP), liberating the Notch intracellular domain (NICD). The NICD is translocated to the nucleus and promotes gene transcription. It has been demonstrated that the ligands can also undergo RIP and nuclear translocation, suggesting a function for the ligands in the sender cell and possible bidirectionality of the Notch pathway. Although the complete mechanism of ligand processing is not entirely understood, and its dependence on Notch receptors has not been ruled out. Also, ligands have autonomous functions beyond Notch activation. Here we review the concepts of reverse and bidirectional signalization of DSL proteins and discuss the characteristics that make them more than just ligands of the Notch pathway.

Abstract The oncogenic PIM kinases and the tumor-suppressive LKB1 kinase have been linked to regulation of cell growth and proliferation. Here we have investigated their potential interactions as well as their relative impacts on tumorigenic growth of prostate cancer cells. The cellular functions of PIM and LKB1 kinases were evaluated using either pan-PIM inhibitors or CRISPR/Cas9-based genomic editing, with which all three PIM family members and/or LKB1 were knocked out from PC3 prostate cancer cells. In addition to cell-based 2D proliferation assays, we examined tumor growth using the chick embryo chorioallantoic membrane (CAM) xenograft model. In this report, we show that inhibition of PIM expression or activity results in increased phosphorylation of AMPK at Thr172 in an LKB1-dependent fashion. With in vitro kinase assays, we demonstrate that LKB1 is a novel direct substrate for PIM kinases and that Ser334 is one of the PIM target sites in LKB1. Accordingly, wild-type LKB1, but not the phosphodeficient S334A mutant can restore PIM inhibitor-induced AMPK phosphorylation in LKB1 knock-out cells. Whereas loss of LKB1 exaggerates formation of PC3-based tumors in the CAM xenograft model, co-deletion of PIM kinases attenuates it. The impairment of cell proliferation and tumor growth in cells lacking both PIM and LKB1 kinases not only underscores the potential crosstalk in signaling between these kinases, but also suggest that PIM inhibitors could be used to restrain growth of LKB1-deficient tumors. Citation Format: Kwan Long Mung, William Eccleshall, Niina M. Santio, Adolfo Rivero-Müller, Cecilia Sahlgren, Päivi J. Koskinen. Crosstalk of PIM and LKB1 kinases in driving growth of prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 79.