Highlights d Hypoxia/HIF signaling converts collective invasion to blebbing-amoeboid migration d Calpain-2 cleaves talin-1, inactivates b1 integrins, and induces amoeboid conversion d Amoeboid movement occurs with reduced oxidative metabolism and glycolysis d Calpain-2 inhibition prevents amoeboid transition and HIFinduced metastasis

Hypoxia, through hypoxia inducible factor (HIF), drives cancer cell invasion and metastatic progression in various cancer types, leading to poor prognosis. In epithelial cancer, hypoxia further induces the transition to amoeboid cancer cell dissemination, yet the molecular mechanisms, relevance for metastasis, and effective interventions to combat hypoxia-induced amoeboid reprogramming remain unclear. Here, we identify calpain-2 as key regulator and anti-metastasis target of hypoxia-induced transition from collective to amoeboid dissemination of breast and head and neck (HN) carcinoma cells. Hypoxia-induced amoeboid dissemination occurred through low ECM-adhesive, bleb-based amoeboid movement, which effectively invaded into 3D collagen with low-oxidative and -glycolytic energy metabolism, revealing an microenvironmentally-induced, energy-conserving dissemination route in epithelial cancers. Hypoxia-induced calpain-2 mediated amoeboid conversion by deactivating beta1 integrins, through enzymatic cleavage of the focal adhesion adaptor protein talin-1.Consequently, targeted downregulation of calpain-2 or pharmacological intervention restored talin-1 integrity, beta1 integrin engagement and reverted blebbing-amoeboid to elongated phenotypes under hypoxia. Calpain-2 activity was required for hypoxia-induced blebbing-amoeboid conversion in the orthotopic mouse dermis, and upregulated in invasive HN tumor xenografts in vivo, and attenuation of calpain activity prevented hypoxia-induced metastasis to the lungs. This identifies the calpain-2/talin-1/beta1 integrin axis as mechanosignaling program and promising intervention target of plasticity of cancer cell invasion and metastasis formation in epithelial cancers under hypoxia. IntroductionCancer cell invasion initiates a multistep cascade to metastasis, which converts local neoplasia into a life-threatening systemic disease. 1-3 Invading cancer cells migrate away from the primary tumor and penetrate blood and lymph vessels, followed by systemic spreading and metastatic colonization of distant organs. 1,3 For tissue invasion, cancer cells deploy a range of collective and individual cell 3 migration strategies. Collective invasion of multicellular groups occurs when cells are held together by cell-cell adhesion, whereas single-cell migration lacks cell-cell cohesion and connectivity. 4 Mesenchymal single-cell migration depends on effective integrin-mediated adhesion to the extracellular matrix (ECM), which supports spindle-like cell elongation and directs matrix metalloproteases (MMPs) for ECM remodeling and path generation. 5 Amoeboid movement of roundish or ellipsoid cells engages only weak adhesion to the ECM, lacks ECM remodeling and, instead deploys kinetic deformation of the cell body for passage through 3D tissue. 6 Besides the cell shape and the strength of ECM interactions, protrusion types differ between amoeboid migration modes. Amoeboid-moving leukocytes and cancer cells develop actin-rich pseudopodia and filopodia at the leading edge, which generate protrusive...

Gene expression analysis of rare or heterogeneous cell populations such as disseminated cancer cells (DCCs) requires a sensitive method allowing reliable analysis of single cells. Therefore, we developed and explored the feasibility of a quantitative PCR (qPCR) assay to analyze single-cell cDNA pre-amplified using a previously established whole transcriptome amplification (WTA) protocol. We carefully selected and optimized multiple steps of the protocol, e.g. re-amplification of WTA products, quantification of amplified cDNA yields and final qPCR quantification, to identify the most reliable and accurate workflow for quantitation of gene expression of the ERBB2 gene in DCCs. We found that absolute quantification outperforms relative quantification. We then validated the performance of our method on single cells of established breast cancer cell lines displaying distinct levels of HER2 protein. The different protein levels were faithfully reflected by transcript expression across the tested cell lines thereby proving the accuracy of our approach. Finally, we applied our method to breast cancer DCCs of a patient undergoing anti-HER2-directed therapy. Here, we were able to measure ERBB2 expression levels in all HER2-protein-positive DCCs. In summary, we developed a reliable single-cell qPCR assay applicable to measure distinct levels of ERBB2 in DCCs.

2Gene expression analysis of rare or heterogeneous cell populations such as disseminated cancer 3 cells (DCCs) requires a sensitive method allowing reliable analysis of single cells. Therefore, we 4 developed and explored the feasibility of a quantitative PCR (qPCR) assay to analyze single-cell 5 cDNA pre-amplified using a previously established whole transcriptome amplification (WTA) 6 protocol. We carefully selected and optimized multiple steps of the protocol, e.g. re-7 amplification of WTA products, quantification of amplified cDNA yields and final qPCR 8 quantification, to identify the most reliable and accurate workflow for quantitation of gene 9 expression of the ERBB2 gene in DCCs. We found that absolute quantification outperforms 10 relative quantification. We then validated the performance of our method on single cells of 11 established breast cancer cell lines displaying distinct levels of HER2 protein. The different 12 protein levels were faithfully reflected by transcript expression across the tested cell lines 13 thereby proving the accuracy of our approach. Finally, we applied our method on patient-derived 14 breast cancer DCCs. Here, we were able to measure ERBB2 expression levels in all HER2-15 positive DCCs. In addition, we could detect ERBB2 transcript expression even in HER2-negative 16 DCCs, suggesting post-transcriptional mechanisms of HER2 loss in anti-HER2-treated DCCs. In 17 summary, we developed a reliable single-cell qPCR assay applicable to measure distinct levels of 18 ERBB2 in DCCs. 19 66(27). Moreover, protocols for preparing qPCR samples are simpler and result in higher 67 sensitivity and reproducibility as compared to NGS-based approaches (7, 27). Importantly, 68 single-cell qPCR workflows exhibit high levels of reliability and wide and dynamic detection 69 ranges, making them exceptionally well-suited for targeted gene expression analyses in singles 70 cells, where sensitivity is essential and the amount of target genes is low (7, 27). Therefore, the 71 present study aimed to develop a single-cell qPCR assay to quantify gene expression changes in 72 single cells, specifically in patient-derived DCCs. We established a workflow comprised of 73 single-cell WTA, re-amplification of single-cell cDNA, post-WTA normalization of cDNA 74 quantities and qPCR-based data analysis. The new assay provides means for measuring 75 expression levels of individual pre-selected genes in WTA products generated from single cells 76 in an accurate and reliable fashion.77 6 78 Materials and methods 79 Cell lines 80 BT-474 (ACC 64) and MCF-7 (ACC 115) breast cancer cell lines were obtained from German 81 Collection of Microorganisms and Cell Cultures (DSMZ). MCF-10A (CRL-10317), a non-82 tumorigenic mammary epithelial cell line was obtained from American Type Culture Collection 83 (ATCC). ZR-75-1 (CRL1500, ATCC) and MDA-MB-453 (ACC 65, DSMZ) cells were 84 purchased from DSMZ. The identity of all cell lines was confirmed by DNA finger printing 85 analysis utilizing the GenePrint® 10 System (Promega). B...