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Purpose: Tumor hypoxia imposes a main obstacle to the efficacy of anti-cancer therapy. Understanding the cellular dynamics of individual hypoxic cells before, during and post-treatment has been hampered by the technical inability to identify and trace these cells over time., Methods and Materials: Here, we present a novel lineage-tracing reporter for hypoxic cells based on the conditional expression of a HIF1a-CreER T2 -UnaG biosensor that can visualize hypoxic cells in a time-dependent manner and trace the fate of hypoxic cells over time. We combine this system with multiphoton microscopy, flow cytometry, and immunofluorescence to characterize the role of hypoxic cells in tumor relapse after irradiation in H1299 tumor spheroids and in vivo xenografts., Results: We validate the reporter in monolayer cultures and we show that tagged cells colocalize in spheroids and human tumor xenografts with the hypoxic marker pimonidazole. We found that irradiation of H1299-HIFcreUnaG spheroids leads to preferential outgrowth of cells from the hypoxic core. Similarly, in xenografts tumors, although initially UnaG-positive-cells coincide with pimonidazole-positive tumor areas and they are merely quiescent, upon irradiation UnaG-positive cells enrich in regrowing tumors and are mainly proliferative., Conclusions: Collectively, our data provide clear evidence that the hypoxic cells drive tumor relapse after irradiation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Pancreatic ductal adenocarcinoma (PDAC) is characterized by increasing fibrosis, which can enhance tumor progression and spread. Here, we undertook an unbiased temporal assessment of the matrisome of the highly metastatic KPC ( Pdx1-Cre , LSL-Kras G12D/+ , LSL-Trp53 R172H/+ ) and poorly metastatic KP fl C ( Pdx1-Cre , LSL-Kras G12D/+ , Trp53 fl/+ ) genetically engineered mouse models of pancreatic cancer using mass spectrometry proteomics. Our assessment at early-, mid-, and late-stage disease reveals an increased abundance of nidogen-2 (NID2) in the KPC model compared to KP fl C, with further validation showing that NID2 is primarily expressed by cancer-associated fibroblasts (CAFs). Using biomechanical assessments, second harmonic generation imaging, and birefringence analysis, we show that NID2 reduction by CRISPR interference (CRISPRi) in CAFs reduces stiffness and matrix remodeling in three-dimensional models, leading to impaired cancer cell invasion. Intravital imaging revealed improved vascular patency in live NID2-depleted tumors, with enhanced response to gemcitabine/Abraxane. In orthotopic models, NID2 CRISPRi tumors had less liver metastasis and increased survival, highlighting NID2 as a potential PDAC cotarget.

Human and non-human primates have strikingly similar genomes, but they strongly differ in many brain-based processes (e.g., behaviour and cognition). While the functions of protein-coding genes have been extensively studied, rather little is known about the role of non-coding RNAs such as long non-coding RNAs (lncRNAs). Here, we predicted lncRNAs and analysed their expression pattern across different brain regions of human and non-human primates (chimpanzee, gorilla, and gibbon). Our analysis identified shared orthologous and non-orthologous lncRNAs, showing striking differences in the genomic features. Differential expression analysis of the shared orthologous lncRNAs from humans and chimpanzees revealed distinct expression patterns in subcortical regions (striatum, hippocampus) and neocortical areas while retaining a homogeneous expression in the cerebellum. Co-expression analysis of lncRNAs and protein-coding genes revealed massive proportions of co-expressed pairs in neocortical regions of humans compared to chimpanzees. Network analysis of co-expressed pairs revealed the distinctive role of the hub-acting orthologous lncRNAs in a region- and species-specific manner. Overall, our study provides novel insight into lncRNA driven gene regulatory landscape, neural regulation, brain evolution, and constitutes a resource for primate's brain lncRNAs., (© 2024. The Author(s).)

Although initially successful, treatments with chemotherapy often fail because of the recurrence of chemoresistant metastases. Since these tumors develop after treatment, resistance is generally thought to occur in response to chemotherapy. However, alternative mechanisms of intrinsic chemoresistance in the chemotherapy-naïve setting may exist but remain poorly understood. Here, we study drug-naïve murine breast cancer brain metastases (BCBMs) to identify how cancer cells growing in a secondary site can acquire intrinsic chemoresistance without cytotoxic agent exposure. We demonstrate that drug-naïve murine breast cancer cells that form cancer lesions in the brain undergo vascular mimicry and concomitantly express the adenosine 5'-triphosphate-binding cassette transporter breast cancer resistance protein (BCRP), a common marker of brain endothelial cells. We reveal that expression of BCRP by the BCBM tumor cells protects them against doxorubicin and topotecan. We conclude that BCRP overexpression can cause intrinsic chemoresistance in cancer cells growing in metastatic sites without prior chemotherapy exposure.

The lysyl oxidase family represents a promising target in stromal targeting of solid tumors due to the importance of this family in crosslinking and stabilizing fibrillar collagens and its known role in tumor desmoplasia. Using small-molecule drug-design approaches, we generated and validated PXS-5505, a first-in-class highly selective and potent pan-lysyl oxidase inhibitor. We demonstrate in vitro and in vivo that pan-lysyl oxidase inhibition decreases chemotherapy-induced pancreatic tumor desmoplasia and stiffness, reduces cancer cell invasion and metastasis, improves tumor perfusion and enhances the efficacy of chemotherapy in the autochthonous genetically engineered KPC model, while also demonstrating antifibrotic effects in human patient-derived xenograft models of pancreatic cancer. PXS-5505 is orally bioavailable, safe and effective at inhibiting lysyl oxidase activity in tissues. Our findings present the rationale for progression of a pan-lysyl oxidase inhibitor aimed at eliciting a reduction in stromal matrix to potentiate chemotherapy in pancreatic ductal adenocarcinoma., (© 2023. The Author(s).)

Although treatment with taxanes does not always lead to clinical benefit, all patients are at risk of their detrimental side effects such as peripheral neuropathy. Understanding the in vivo mode of action of taxanes can help design improved treatment regimens. Here, we demonstrate that in vivo, taxanes directly trigger T cells to selectively kill cancer cells in a non-canonical, T cell receptor-independent manner. Mechanistically, taxanes induce T cells to release cytotoxic extracellular vesicles, which lead to apoptosis specifically in tumor cells while leaving healthy epithelial cells intact. We exploit these findings to develop an effective therapeutic approach, based on transfer of T cells pre-treated with taxanes ex vivo, thereby avoiding toxicity of systemic treatment. Our study reveals a different in vivo mode of action of one of the most commonly used chemotherapies, and opens avenues to harness T cell-dependent anti-tumor effects of taxanes while avoiding systemic toxicity., Competing Interests: Declaration of interests J.F.D. is named as inventor on a patent related to organoid technology. C.V., J.vR. and S.C.L. are named as inventors on a patent related to taxane treatment of T cells. The authors have no other competing interests to declare., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The basement membrane (BM) around tumor lobes forms a barrier to prevent cancer cells from invading the surrounding tissue. Although myoepithelial cells are key producers of the healthy mammary epithelium BM, they are nearly absent in mammary tumors. To study the origin and dynamics of the BM, we developed and imaged a laminin beta1-Dendra2 mouse model. We show that the turnover of laminin beta1 is faster in the BMs that surround the tumor lobes than in the BMs that surround the healthy epithelium. Moreover, we find that epithelial cancer cells and tumor-infiltrating endothelial cells synthesize laminin beta1 and that this production is temporarily and locally heterogeneous, leading to local discontinuity of the BM laminin beta1. Collectively, our data draw a new paradigm for tumor BM turnover in which the disassembly happens at a constant rate, and a local misbalance of compensating production leads to reduction or even complete disappearance of the BM., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

An increasing number of breast cancer patients develop brain metastases (BM). Standard-of-care treatments are largely inefficient, and breast cancer brain metastasis (BCBM) patients are considered untreatable. Immunotherapies are not successfully employed in BCBM, in part because breast cancer is a "cold" tumor and also because the brain tissue has a unique immune landscape. Here, we generate and characterize immunocompetent models of BCBM derived from PyMT and Neu mammary tumors to test how harnessing the pro-senescence properties of doxorubicin can be used to prime the specific immune BCBM microenvironment. We reveal that BCBM senescent cells, induced by doxorubicin, trigger the recruitment of PD1-expressing T cells to the brain. Importantly, we demonstrate that induction of senescence with doxorubicin improves the efficacy of immunotherapy with anti-PD1 in BCBM in a CD8 T cell-dependent manner, thereby providing an optimized strategy to introduce immune-based treatments in this lethal disease. In addition, our BCBM models can be used for pre-clinical testing of other therapeutic strategies in the future., Competing Interests: Declaration of interests P.L.J.d.K. is a co-founder and shareholder of Cleara Biotech BV, the Netherlands. D.A.P. works as a scientist at Cleara Biotech BV, the Netherlands., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Upon chronic stress, a fraction of individuals shows stress resilience, which can prevent long-term mental dysfunction. The underlying molecular mechanisms are complex and have not yet been fully understood. In this study, we performed a data-driven behavioural stratification together with single-cell transcriptomics of the hippocampus in a mouse model of chronic social defeat stress. Our work revealed that in a sub-group exhibiting molecular responses upon chronic stress, the dorsal hippocampus is particularly involved in neuroimmune responses, angiogenesis, myelination, and neurogenesis, thereby enabling brain restoration and homeostasis after chronic stress. Based on these molecular insights, we applied rapamycin after the stress as a proof-of-concept pharmacological intervention and were able to substantially increase stress resilience. Our findings serve as a data resource and can open new avenues for further understanding of molecular processes underlying stress response and for targeted interventions supporting resilience.

(1) Background: an increasing number of breast cancer patients develop lethal brain metastases (BM). The complete removal of these tumors by surgery becomes complicated when cells infiltrate into the brain parenchyma. However, little is known about the nature of these invading cells in breast cancer brain metastasis (BCBM). (2) Methods: we use intravital microscopy through a cranial window to study the behavior of invading cells in a mouse model of BCBM. (3) Results: we demonstrate that BCBM cells that escape from the metastatic mass and infiltrate into brain parenchyma undergo epithelial-to-mesenchymal transition (EMT). Moreover, cells undergoing EMT revert to an epithelial state when growing tumor masses in the brain. Lastly, through multiplex immunohistochemistry, we confirm the presence of these infiltrative cells in EMT in patient samples. (4) Conclusions: together, our data identify the critical role of EMT in the invasive behavior of BCBM, which warrants further consideration to target those cells when treating BCBM.

Purpose: Extensive work in preclinical models has shown that microenvironmental cells influence many aspects of cancer cell behavior, including metastatic potential and their sensitivity to therapeutics. In the human setting, this behavior is mainly correlated with the presence of immune cells. Here, in addition to T cells, B cells, macrophages, and mast cells, we identified the relevance of nonimmune cell types for breast cancer survival and therapy benefit, including fibroblasts, myoepithelial cells, muscle cells, endothelial cells, and seven distinct epithelial cell types., Experimental Design: Using single-cell sequencing data, we generated reference profiles for all these cell types. We used these reference profiles in deconvolution algorithms to optimally detangle the cellular composition of more than 3,500 primary breast tumors of patients that were enrolled in the SCAN-B and MATADOR clinical trials, and for which bulk mRNA sequencing data were available., Results: This large data set enables us to identify and subsequently validate the cellular composition of microenvironments that distinguish differential survival and treatment benefit for different treatment regimens in patients with primary breast cancer. In addition to immune cells, we have identified that survival and therapy benefit are characterized by various contributions of distinct epithelial cell types., Conclusions: From our study, we conclude that differential survival and therapy benefit of patients with breast cancer are characterized by distinct microenvironments that include specific populations of immune and epithelial cells., (©2021 The Authors; Published by the American Association for Cancer Research.)

Glioma is the most common form of malignant primary brain tumours in adults. Their highly invasive nature makes the disease incurable to date, emphasizing the importance of better understanding the mechanisms driving glioma invasion. Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is characteristic for astrocyte- and neural stem cell-derived gliomas. Glioma malignancy is associated with changes in GFAP alternative splicing, as the canonical isoform GFAPα is downregulated in higher-grade tumours, leading to increased dominance of the GFAPδ isoform in the network. In this study, we used intravital imaging and an ex vivo brain slice invasion model. We show that the GFAPδ and GFAPα isoforms differentially regulate the tumour dynamics of glioma cells. Depletion of either isoform increases the migratory capacity of glioma cells. Remarkably, GFAPδ-depleted cells migrate randomly through the brain tissue, whereas GFAPα-depleted cells show a directionally persistent invasion into the brain parenchyma. This study shows that distinct compositions of the GFAPnetwork lead to specific migratory dynamics and behaviours of gliomas., (© 2022. The Author(s).)

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic, chemoresistant malignancy and is characterized by a dense, desmoplastic stroma that modulates PDAC progression. Here, we visualized transient manipulation of focal adhesion kinase (FAK), which integrates bidirectional cell-environment signaling, using intravital fluorescence lifetime imaging microscopy of the FAK-based Förster resonance energy transfer biosensor in mouse and patient-derived PDAC models. Parallel real-time quantification of the FUCCI cell cycle reporter guided us to improve PDAC response to standard-of-care chemotherapy at primary and secondary sites. Critically, micropatterned pillar plates and stiffness-tunable matrices were used to pinpoint the contribution of environmental cues to chemosensitization, while fluid flow–induced shear stress assessment, patient-derived matrices, and personalized in vivo models allowed us to deconstruct how FAK inhibition can reduce PDAC spread. Last, stratification of PDAC patient samples via Merlin status revealed a patient subset with poor prognosis that are likely to respond to FAK priming before chemotherapy.

The concept that extracellular vesicles (EVs) from the diet can be absorbed by the intestinal tract of the consuming organism, be bioavailable in various organs, and in-turn exert phenotypic changes is highly debatable. Here, we isolate EVs from both raw and commercial bovine milk and characterize them by electron microscopy, nanoparticle tracking analysis, western blotting, quantitative proteomics and small RNA sequencing analysis. Orally administered bovine milk-derived EVs survive the harsh degrading conditions of the gut, in mice, and is subsequently detected in multiple organs. Milk-derived EVs orally administered to mice implanted with colorectal and breast cancer cells reduce the primary tumor burden. Intriguingly, despite the reduction in primary tumor growth, milk-derived EVs accelerate metastasis in breast and pancreatic cancer mouse models. Proteomic and biochemical analysis reveal the induction of senescence and epithelial-to-mesenchymal transition in cancer cells upon treatment with milk-derived EVs. Timing of EV administration is critical as oral administration after resection of the primary tumor reverses the pro-metastatic effects of milk-derived EVs in breast cancer models. Taken together, our study provides context-based and opposing roles of milk-derived EVs as metastasis inducers and suppressors.

Double strand breaks (DSBs) are highly toxic to a cell, a property that is exploited in radiation therapy. A critical component for the damage induction is cellular oxygen, making hypoxic tumor areas refractory to the efficacy of radiation treatment. During a fractionated radiation regimen, these hypoxic areas can be re-oxygenated. Nonetheless, hypoxia still constitutes a negative prognostic factor for the patient's outcome. We hypothesized that this might be attributed to specific hypoxia-induced cellular traits that are maintained upon reoxygenation. Here, we show that reoxygenation of hypoxic non-transformed RPE-1 cells fully restored induction of DSBs but the cells remain radioresistant as a consequence of hypoxia-induced quiescence. With the use of the cell cycle indicators (FUCCI), cell cycle-specific radiation sensitivity, the cell cycle phase duration with live cell imaging, and single cell tracing were assessed. We observed that RPE-1 cells experience a longer G1 phase under hypoxia and retain a large fraction of cells that are non-cycling. Expression of HPV oncoprotein E7 prevents hypoxia-induced quiescence and abolishes the radioprotective effect. In line with this, HPV-negative cancer cell lines retain radioresistance, while HPV-positive cancer cell lines are radiosensitized upon reoxygenation. Quiescence induction in hypoxia and its HPV-driven prevention was observed in 3D multicellular spheroids. Collectively, we identify a new hypoxia-dependent radioprotective phenotype due to hypoxia-induced quiescence that accounts for a global decrease in radiosensitivity that can be retained upon reoxygenation and is absent in cells expressing oncoprotein E7.

Metastasis is a highly dynamic process during which cancer and microenvironmental cells undergo a cascade of events required for efficient dissemination throughout the body. During the metastatic cascade, tumor cells can change their state and behavior, a phenomenon commonly defined as cellular plasticity. To monitor cellular plasticity during metastasis, high-resolution intravital microscopy (IVM) techniques have been developed and allow us to visualize individual cells by repeated imaging in animal models. In this review, we summarize the latest technological advancements in the field of IVM and how they have been applied to monitor metastatic events. In particular, we highlight how longitudinal imaging in native tissues can provide new insights into the plastic physiological and developmental processes that are hijacked by cancer cells during metastasis., (Copyright © 2020 Cold Spring Harbor Laboratory Press; all rights reserved.)

Breast cancer is a major public health problem and the leading world cause of women death by cancer. Both the recurrence and mortality of breast cancer are mainly caused by the formation of metastasis. The long non-coding RNA H19 , the precursor of miR-675, is involved in breast cancer development. The aim of this work was to determine the implication but, also, the relative contribution of H19 and miR-675 to the enhancement of breast cancer metastatic potential. We showed that both H19 and miR-675 increase the invasive capacities of breast cancer cells in xenografted transgenic zebrafish models. In vitro, H19 and miR-675 enhance the cell migration and invasion, as well as colony formation. H19 seems to induce the epithelial-to-mesenchymal transition (EMT), with a decreased expression of epithelial markers and an increased expression of mesenchymal markers. Interestingly, miR-675 simultaneously increases the expression of both epithelial and mesenchymal markers, suggesting the induction of a hybrid phenotype or mesenchymal-to-epithelial transition (MET). Finally, we demonstrated for the first time that miR-675, like its precursor H19, increases the stemness properties of breast cancer cells. Altogether, our data suggest that H19 and miR-675 could enhance the aggressiveness of breast cancer cells through both common and different mechanisms.

Introduction : Pancreatic cancer (PC) is responsible for significant worldwide cancer-associated mortality and has one of the lowest five-year survival rate post-diagnosis of all epithelial cancers. A major contributor to this dismal outcome is the extensive stromal reaction that occurs during PC progression. As such, targeting key components of the pancreatic tumor stroma in combination with standard-of-care chemotherapy has been a recent focus in both the pre-clinical and clinical settings. Areas Covered : In this commentary, we highlight how perlecan was identified as a new potential target for this disease. Expert Opinion : Perlecan is deposited by cancer-associated fibroblasts (CAFs) in the pancreatic tumor stroma, and work from our laboratory group recently demonstrated that depleting perlecan reduces metastatic spread, while also improving chemotherapy efficacy in pancreatic tumors harboring a gain-of-function p53 mutation. We also discuss potential strategies to therapeutically target perlecan which could be tested in pre-clinical models prior to translation into the clinic.

Background: The use of in vitro cell cultures is a powerful tool for obtaining key insights into the behaviour and response of cells to interventions in normal and disease situations. Unlike in vivo settings, in vitro experiments allow a fine-tuned control of a range of microenvironmental elements independently within an isolated setting. The recent expansion in the use of three-dimensional (3D) in vitro assays has created a number of representative tools to study cell behaviour in a more physiologically 3D relevant microenvironment. Complex 3D in vitro models that can recapitulate human tissue biology are essential for understanding the pathophysiology of disease., Aim: The development of the 3D coculture collagen contraction and invasion assay, the "organotypic assay," has been widely adopted as a powerful approach to bridge the gap between standard two-dimensional tissue culture and in vivo mouse models. In the cancer setting, these assays can then be used to dissect how stromal cells, such as cancer-associated fibroblasts (CAFs), drive extracellular matrix (ECM) remodelling to alter cancer cell behaviour and response to intervention. However, to date, many of the published organotypic protocols are low-throughput, time-consuming (up to several weeks), and work-intensive with often limited scalability. Our aim was to develop a fast, high-throughput, scalable 3D organotypic assay for use in oncology screening and drug development., Methods and Results: Here, we describe a modified 96-well organotypic assay, the "Mini-Organo," which can be easily completed within 5 days. We demonstrate its application in a wide range of mouse and human cancer biology approaches including evaluation of stromal cell 3D ECM remodelling, 3D cancer cell invasion, and the assessment of efficacy of potential anticancer therapeutic targets. Furthermore, the organotypic assay described is highly amenable to customisation using different cell types under diverse experimental conditions., Conclusions: The Mini-Organo high-throughput 3D organotypic assay allows the rapid screening of potential cancer therapeutics in human and mouse models in a time-efficient manner., (© 2019 The Authors Cancer Reports Published by Wiley Periodicals, Inc.)

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease; the identification of novel targets and development of effective treatment strategies are urgently needed to improve patient outcomes. Remodeling of the pancreatic stroma occurs during PDAC development, which drives disease progression and impairs responses to therapy. The actomyosin regulatory ROCK1 and ROCK2 kinases govern cell motility and contractility, and have been suggested to be potential targets for cancer therapy, particularly to reduce the metastatic spread of tumor cells. However, ROCK inhibitors are not currently used for cancer patient treatment, largely due to the overwhelming challenge faced in the development of anti-metastatic drugs, and a lack of clarity as to the cancer types most likely to benefit from ROCK inhibitor therapy. In 2 recent publications, we discovered that ROCK1 and ROCK2 expression were increased in PDAC, and that increased ROCK activity was associated with reduced survival and PDAC progression by enabling extracellular matrix (ECM) remodeling and invasive growth of pancreatic cancer cells. We also used intravital imaging to optimize ROCK inhibition using the pharmacological ROCK inhibitor fasudil (HA-1077), and demonstrated that short-term ROCK targeting, or 'priming', improved chemotherapy efficacy, disrupted cancer cell collective movement, and impaired metastasis. This body of work strongly indicates that the use of ROCK inhibitors in pancreatic cancer therapy as 'priming' agents warrants further consideration, and provides insights as to how transient mechanical manipulation, or fine-tuning the ECM, rather than chronic stromal ablation might be beneficial for improving chemotherapeutic efficacy in the treatment of this deadly disease.

Cancer-associated fibroblasts (CAFs) are one of the most significant components in the tumour microenvironment (TME), where they can perform several protumourigenic functions. Several studies have recently reported that CAFs are more heterogenous and plastic than was previously thought. As such, there has been a shift in the field to study CAF subpopulations and the emergent functions of these subsets in tumourigenesis. In this review, we explore how different aspects of CAF heterogeneity are defined and how these manifest in multiple cancers, with a focus on pancreatic ductal adenocarcinoma (PDAC). We also discuss therapeutic approaches to selectively target protumourigenic CAF functions, while avoiding normal fibroblasts, providing insight into the future of stromal targeting for the treatment of PDAC and other solid tumours., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Neural stem cells (NSCs) in the adult mouse hippocampus occur in a specific neurogenic niche, where a multitude of extracellular signaling molecules converges to regulate NSC proliferation as well as fate and functional integration. However, the underlying mechanisms how NSCs react to extrinsic signals and convert them to intracellular responses still remains elusive. NSCs contain a functional endocannabinoid system, including the cannabinoid type-1 receptor (CB1). To decipher whether CB1 regulates adult neurogenesis directly or indirectly in vivo, we performed NSC-specific conditional inactivation of CB1 by using triple-transgenic mice. Here, we show that lack of CB1 in NSCs is sufficient to decrease proliferation of the stem cell pool, which consequently leads to a reduction in the number of newborn neurons. Furthermore, neuronal differentiation was compromised at the level of dendritic maturation pointing towards a postsynaptic role of CB1 in vivo. Deteriorated neurogenesis in NSC-specific CB1 knock-outs additionally resulted in reduced long-term potentiation in the hippocampal formation. The observed cellular and physiological alterations led to decreased short-term spatial memory and increased depression-like behavior. These results demonstrate that CB1 expressed in NSCs and their progeny controls neurogenesis in adult mice to regulate the NSC stem cell pool, dendritic morphology, activity-dependent plasticity, and behavior.