The tumor micro-environment often contains stiff and irregular-bundled collagen fibers that are used by tumor cells to disseminate. It is still unclear how and to what extent, extracellular matrix (ECM) stiffness versus ECM bundle size and alignment dictate cancer cell invasion. Here, we have uncoupled Collagen-I bundling from stiffness by introducing inter-collagen crosslinks, combined with temperature induced aggregation of collagen bundling. Using organotypic models from mouse invasive ductal and invasive lobular breast cancers, we show that increased collagen bundling in 3D induces a generic increase in breast cancer invasion that is independent of migration mode. However, systemic collagen stiffening using advanced glycation end product (AGE) crosslinking prevents collective invasion, while leaving single cell invasion unaffected. Collective invasion into collagen matrices by ductal breast cancer cells depends on Lysyl oxidase-like 3 (Loxl3), a factor produced by tumor cells that reinforces local collagen stiffness. Finally, we present clinical evidence that collectively invading cancer cells at the invasive front of ductal breast carcinoma upregulate LOXL3. By uncoupling the mechanical, chemical, and structural cues that control invasion of breast cancer in three dimensions, our data reveal that spatial control over stiffness and bundling underlie collective dissemination of ductal-type breast cancers.

Cidofovir (CDV) is an antiviral agent with antiproliferative properties. The aim of our study was to investigate the efficacy of CDV in HPV-positive and -negative head and neck squamous cell carcinoma (HNSCC) cell lines and whether it is caused by a difference in response to DNA damage. Upon CDV treatment of HNSCC and normal oral keratinocyte cell lines, we carried out MTT analysis (cell viability), flow cytometry (cell cycle analysis), (immuno) fluorescence and western blotting (DNA double strand breaks, DNA damage response, apoptosis and mitotic catastrophe). The growth of the cell lines was inhibited by CDV treatment and resulted in γ-H2AX accumulation and upregulation of DNA repair proteins. CDV did not activate apoptosis but induced S- and G2/M phase arrest. Phospho-Aurora Kinase immunostaining showed a decrease in the amount of mitoses but an increase in aberrant mitoses suggesting mitotic catastrophe. In conclusion, CDV inhibits cell growth in HPV-positive and -negative HNSCC cell lines and was more profound in the HPV-positive cell lines. CDV treated cells show accumulation of DNA DSBs and DNA damage response activation, but apoptosis does not seem to occur. Rather our data indicate the occurrence of mitotic catastrophe.

Background Oral squamous cell carcinoma (OSCC) frequently invades the jaw. The exact mechanism of bone invasion remains unclear. This study investigates the role of osteoclasts and RANKL/OPG/RANK in the development of bone invasion in OSCC. Methods OSCC-patients treated with resection were included and divided in three groups; Non-invasion (NI-group), erosion (E-group) and bone invasion (I-group). Tissue-sections were stained with Cathepsin K (for counting osteoclasts), RANKL, OPG and RANK. Staining intensity was scored in tumor-front, tumor-center, tumor-backside and normal mucosa. Immunohistochemistry and qPCR for RANKL/OPG/RANK was performed in five head-and-neck SCC organoids to correlate protein and mRNA-expression levels. Results The mean number of osteoclasts in Cathepsin K stained sections in the NI-group was 3.09 (1.12-5.05; 95%CI), in the E-group 6.15 (4.04–8.25; 95%CI) and in the I-group 10.58 (5.81–15.34; 95%CI). Compared to normal mucosa, the expression in all tumor regions was higher for RANKL, in most tumor regions for OPG and not higher for RANK. RANKL-expression in tumor-front was higher than expression in tumor-backside (I-group). RANK-expression in the tumor-front and the tumor-center was higher than expression in tumor-backside in all groups. qPCR showed a 20-43x higher RANKL-mRNA expression in 3/5 tumor organoid samples compared to a normal squamous cell organoid line and no higher mRNA-expression of OPG and RANK. There was no correlation between protein and mRNA expression in the HNSCC organoids. Conclusion The number of osteoclasts and their regulating proteins RANKL/OPG/RANK differ between OSCC patients with and without bone invasion. Bone invasive OSCCs have more osteoclasts and express more RANKL in tumor-front, which suggest that OSCC’s induce bone invasion by stimulating osteoclast activation by regulating the production of RANKL/OPG/RANK proteins.

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