Cell jamming in a collagen-based interface assay is tuned by collagen density and proteolysis.

Tumor cell invasion into heterogenous interstitial tissues consisting of network-, channel- or rift-like architectures involves both matrix metalloproteinase (MMP)-mediated tissue remodeling and cell shape adaptation to tissue geometry. Three-dimensional (3D) models composed of either porous or linearly aligned architectures have added to the understanding of how physical spacing principles affect migration efficacy; however, the relative contribution of each architecture to decision making in the presence of varying MMP availability is not known. Here, we developed an interface assay containing a cleft between two high-density collagen lattices, and we used this assay to probe tumor cell invasion efficacy, invasion mode and MMP dependence in concert. In silico modeling predicted facilitated cell migration into confining clefts independently of MMP activity, whereas migration into dense porous matrix was predicted to require matrix degradation. This prediction was verified experimentally, where inhibition of collagen degradation was found to strongly compromise migration into 3D collagen in a density-dependent manner, but interface-guided migration remained effective, occurring by cell jamming. The 3D interface assay reported here may serve as a suitable model to better understand the impact of in vivo-relevant interstitial tissue topologies on tumor invasion patterning and responses to molecular interventions.

Formalin-Fixed, Paraffin-Embedded-Targeted Locus Capture: A Next-Generation Sequencing Technology for Accurate DNA-Based Gene Fusion Detection in Bone and Soft Tissue Tumors.

Chromosomal rearrangements are important drivers in cancer, and their robust detection is essential for diagnosis, prognosis, and treatment selection, particularly for bone and soft tissue tumors. Current diagnostic methods are hindered by limitations, including difficulties with multiplexing targets and poor quality of RNA. A novel targeted DNA-based next-generation sequencing method, formalin-fixed, paraffin-embedded-targeted locus capture (FFPE-TLC), has shown advantages over current diagnostic methods when applied on FFPE lymphomas, including the ability to detect novel rearrangements. We evaluated the utility of FFPE-TLC in bone and soft tissue tumor diagnostics. FFPE-TLC sequencing was successfully applied on noncalcified and decalcified FFPE samples (n = 44) and control samples (n = 19). In total, 58 rearrangements were identified in 40 FFPE tumor samples, including three previously negative samples, and none was identified in the FFPE control samples. In all five discordant cases, FFPE-TLC could identify gene fusions where other methods had failed due to either detection limits or poor sample quality. FFPE-TLC achieved a high specificity and sensitivity (no false positives and negatives). These results indicate that FFPE-TLC is applicable in cancer diagnostics to simultaneously analyze many genes for their involvement in gene fusions. Similar to the observation in lymphomas, FFPE-TLC is a good DNA-based alternative to the conventional methods for detection of rearrangements in bone and soft tissue tumors.

Identification of Fusion Genes and Targets for Genetically Matched Therapies in a Large Cohort of Salivary Gland Cancer Patients.

Introduction: Salivary gland cancer (SGC) is a rare cancer for which systemic treatment options are limited. Therefore, it is important to characterize its genetic landscape in search for actionable aberrations, such as NTRK gene fusions. This research aimed to identify these actionable aberrations by combining NGS-based analysis of RNA (gene fusions) and DNA (single and multiple nucleotide variants, copy number variants, microsatellite instability and tumor mutational burden) in a large cohort of SGC patients. Methods: RNA and DNA were extracted from archival tissue of 121 patients with various SGC subtypes. Gene fusion analysis was performed using a customized RNA-based targeted NGS panel. DNA was sequenced using a targeted NGS panel encompassing 523 cancer-related genes. Cross-validation of NGS-based NTRK fusion detection and pan-TRK immunohistochemistry (IHC) was performed. Results: Fusion transcripts were detected in 50% of the cases and included both known (MYB-NFIB, MYBL1-NFIB, CRTC1-MAML2) and previously unknown fusions (including transcripts involving RET, BRAF or RAD51B). Only one NTRK fusion transcript was detected, in a secretory carcinoma case. Pan-TRK IHC (clone EPR17341) was false positive in 74% of cases. The proportion of patients with targets for genetically matched therapies differed among subtypes (salivary duct carcinoma: 82%, adenoid cystic carcinoma 28%, mucoepidermoid carcinoma 50%, acinic cell carcinoma 33%). Actionable aberrations were most often located in PIK3CA (n = 18, 15%), ERBB2 (n = 15, 12%), HRAS and NOTCH1 (both n = 9, 7%). Conclusions: Actionable genetic aberrations were seen in 53.7% of all SGC cases on the RNA and DNA level, with varying percentages between subtypes.

Impact of DNA damage repair defects on response to radium-223 and overall survival in metastatic castration-resistant prostate cancer.

PURPOSE: Radium-223 is a targeted alpha radiation therapy for metastatic castration-resistant prostate cancer. DNA damage repair (DDR) defective prostate cancers, specifically genetic aberrations leading to homologous recombination deficiency (HRD), accumulate irreparable DNA damage following genotoxic treatment. This retrospective study assessed DDR mutation status in patients treated with radium-223, investigating their association with efficacy and overall survival (OS). PATIENTS AND METHODS: Included patients were treated with radium-223 and had results from primary or metastatic tumour tissue of a comprehensive next-generation sequencing panel of DDR genes, including canonical HRD genes. Patients were grouped by presence (DDR+) or absence (DDR-) of pathogenic somatic or germline aberrations in DDR genes. We evaluated OS, time to ALP progression (TAP), time to initiation of subsequent systemic therapy (TST) and biochemical responses between DDR groups. RESULTS: Ninety-three patients were included. Twenty-eight (30%) patients had DDR mutations, most frequently in ATM (8.6%), BRCA2 (7.5%) and CDK12 (4.3%) genes. DDR+ patients showed prolonged OS (median 36.3 versus 17.0 months; HR 2.29; P = 0.01). Median TAP and TST in the DDR+ and DDR- patients was 6.9 versus5.8 months (HR = 1.48; P = 0.15), and 8.9 versus7.3 months (HR = 1.58; P = 0.08), respectively. DDR+ patients more frequently completed radium-223 therapy (79% versus 47%; P = 0.05). No difference in biochemical responses were seen. CONCLUSION: Patients harbouring DDR aberrations showed significant OS benefit, and more commonly completed radium-223 therapy. These findings need prospective confirmation and support strategies of genotoxic agents such as radium-223 in patients harbouring DDR defects.

Effect of mechanical loading and substrate elasticity on the osteogenic and adipogenic differentiation of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are highly sensitive to biomechanics of their extracellular environment. Generally, a higher elasticity of culture substrates can drive cells into the osteogenic lineage, whereas low substrate elasticity results in adipogenesis. Applied mechanical loading by cyclic strain is another major variable influencing cell fate. Yet, little is known about the simultaneous effect of both cues. Therefore, the present study investigated the relative importance of both cues on differentiation. MSCs were cultured in an osteogenic and also an adipogenic environment on soft polyacrylamide (PAAm; E = 23 ± 0.3 kPa), stiff PAAm (111 ± 2 kPa), and polydimethylsiloxane (PDMS; E = 1,5 ± 0.07 MPa) either unstrained or with 8% cyclic strain at 1 Hz. Without strain, the relative expression of the early osteogenic marker alkaline phosphatase (ALP) was significantly higher (78%) on PDMS than on both PAAm. With 8% cyclic strain, ALP expression increased for all groups in comparison with unstrained controls. The highest increase was observed for the soft PAAm by 36%. Moreover, relative oil red O (ORO) expression-indicating adipogenesis-was the highest for unstrained soft PAAm. On the other hand, the percentage of ORO positive cells significantly decreased by 57% and 69% for soft and stiff PAAm when strained. In conclusion, biomaterial elasticity and mechanical loading can act simultaneously on cell differentiation. Substrate elasticity is an important factor, regulating the differentiation, but cyclic strain can drive MSCs towards the osteogenesis even on the softest substrate. As such, the osteogenic effect of mechanical loading can overrule the adipogenic effect of soft substrates, thereby acting as an inhibitor.

Mechanoreciprocity in cell migration.

Cell migration is an adaptive process that depends on and responds to physical and molecular triggers. Moving cells sense and respond to tissue mechanics and induce transient or permanent tissue modifications, including extracellular matrix stiffening, compression and deformation, protein unfolding, proteolytic remodelling and jamming transitions. Here we discuss how the bi-directional relationship of cell-tissue interactions (mechanoreciprocity) allows cells to change position and contributes to single-cell and collective movement, structural and molecular tissue organization, and cell fate decisions.

Hypoxia Induces a HIF-1-Dependent Transition from Collective-to-Amoeboid Dissemination in Epithelial Cancer Cells.

Cancer metastases arise from a multi-step process that requires metastasizing tumor cells to adapt to signaling input from varying tissue environments [1]. As an early metastatic event, cancer cell dissemination occurs through different migration programs, including multicellular, collective, and single-cell mesenchymal or amoeboid migration [2-4]. Migration modes can interconvert based on changes in cell adhesion, cytoskeletal mechanotransduction [5], and/or proteolysis [6], most likely under the control of transcriptional programs such as the epithelial-to-mesenchymal transition (EMT) [7, 8]. However, how plasticity of tumor cell migration and EMT is spatiotemporally controlled and connected upon challenge by the tumor microenvironment remains unclear. Using 3D cultures of collectively invading breast and head and neck cancer spheroids, here we identify hypoxia, a hallmark of solid tumors [9], as an inducer of the collective-to-amoeboid transition (CAT), promoting the dissemination of amoeboid-moving single cells from collective invasion strands. Hypoxia-induced amoeboid detachment was driven by hypoxia-inducible factor 1 (HIF-1), followed the downregulation of E-cadherin, and produced heterogeneous cell subsets whose phenotype and migration were dependent (∼30%) or independent (∼70%) of Twist-mediated EMT. EMT-like and EMT-independent amoeboid cell subsets showed stable amoeboid movement over hours as well as leukocyte-like traits, including rounded morphology, matrix metalloproteinase (MMP)-independent migration, and nuclear deformation. Cancer cells undergoing pharmacological stabilization of HIFs retained their constitutive ability for early metastatic seeding in an experimental model of lung metastasis, indicating that hypoxia-induced CAT enhances cell release rather than early organ colonization. Induced by metabolic challenge, amoeboid movement may thus constitute a common endpoint of both EMT-dependent and EMT-independent cancer dissemination programs.

Strain Stiffening of Fibrillar Collagen during Individual and Collective Cell Migration Identified by AFM Nanoindentation.

The multistep process of cell migration requires cells to dynamically couple to extracellular interfaces and generate traction force or friction for displacement of the cell body. When deformed, biopolymer networks, including fibrillar collagen and fibrin, undergo a nonlinear elasticity change that is termed strain stiffening and is commonly measured by bulk rheology. It remains poorly characterized, however, whether forces generated by moving cells suffice to induce strain stiffening. To detect strain stiffening at the leading edge of normal and tumor cells moving across fibrillar type I collagen, we combined AFM nanoindentation and differential field probing with confocal reflection microscopy. In different cell models, gradient-like fiber realignment, densification, and elevation of Young's modulus ahead of the leading edge were observed, with peak increases of up to 1.15 kPa near the leading edge. Moving fibroblasts generated a larger anterograde strain field with a higher amplitude and up to 6-fold increased cumulative strain stiffening (52 kPa) compared with mesenchymal HT1080 fibrosarcoma cells (8.8 kPa) and epithelial SCC38 cancer cells (9.8 kPa). Collectively moving SCC38 cells produced 4-fold increased cumulative strain stiffening (38 kPa) compared with individually moving SCC38 cells in a ß1 integrin- and actomyosin-dependent manner. This indicates that the extent of strain stiffening by the leading edge of moving cells scales with cell type, multicellular cooperativity, integrin availability, and contractility. By straining, migrating cells realign and densify fibrillar extracellular matrix and thus adopt an autonomous strategy to move on a "traveling wave" of stiffened substrate, which reaches levels sufficient for mechanosensory activation and self-steering of migration.