PDF(Pubmed)
Abstract：
UNASSIGNED: Cancer cells remodel their local physical environment through processes of matrix reorganisation, deposition, stiffening and degradation. Urokinase-type plasminogen activator (uPA), which is encoded by the PLAU gene, is an extracellular proteolytic enzyme known to be involved in cancer progression and tumour microenvironment (TME) remodelling. Perturbing uPA therefore has a strong potential as a mechano-based cancer therapy. This work is a bioengineering investigation to validate whether 1) uPA is involved in matrix degradation and 2) preventing matrix degradation by targeting uPA can reduce cancer cell invasion and metastasis.
UNASSIGNED: To this aim, we used an engineered 3D in vitro model, termed the tumouroid, that appropriately mimics the tumour\'s native biophysical environment (3 kPa). A CRISPR-Cas9 mediated uPA knockout was performed to introduce a loss of function mutation in the gene coding sequence. Subsequently, to validate the translational potential of blocking uPA action, we tested a pharmacological inhibitor, UK-371,801. The changes in matrix stiffness were measured by atomic force microscopy (AFM). Invasion was quantified using images of the tumouroid, obtained after 21 days of culture.
UNASSIGNED: We showed that uPA is highly expressed in invasive breast and colorectal cancers, and these invasive cancer cells locally degrade their TME. PLAU (uPA) gene knock-out (KO) completely stopped matrix remodelling and significantly reduced cancer invasion. Many invasive cancer gene markers were also downregulated in the PLAU KO tumouroids. Pharmacological inhibition of uPA showed similarly promising results, where matrix degradation was reduced and so was the cancer invasion.
UNASSIGNED: This work supports the role of uPA in matrix degradation. It demonstrates that the invasion of cancer cells was significantly reduced when enzymatic breakdown of the TME matrix was prevented. Collectively, this provides strong evidence of the effectiveness of targeting uPA as a mechano-based cancer therapy.
UNASSIGNED: To this aim, we used an engineered 3D in vitro model, termed the tumouroid, that appropriately mimics the tumour\'s native biophysical environment (3 kPa). A CRISPR-Cas9 mediated uPA knockout was performed to introduce a loss of function mutation in the gene coding sequence. Subsequently, to validate the translational potential of blocking uPA action, we tested a pharmacological inhibitor, UK-371,801. The changes in matrix stiffness were measured by atomic force microscopy (AFM). Invasion was quantified using images of the tumouroid, obtained after 21 days of culture.
UNASSIGNED: We showed that uPA is highly expressed in invasive breast and colorectal cancers, and these invasive cancer cells locally degrade their TME. PLAU (uPA) gene knock-out (KO) completely stopped matrix remodelling and significantly reduced cancer invasion. Many invasive cancer gene markers were also downregulated in the PLAU KO tumouroids. Pharmacological inhibition of uPA showed similarly promising results, where matrix degradation was reduced and so was the cancer invasion.
UNASSIGNED: This work supports the role of uPA in matrix degradation. It demonstrates that the invasion of cancer cells was significantly reduced when enzymatic breakdown of the TME matrix was prevented. Collectively, this provides strong evidence of the effectiveness of targeting uPA as a mechano-based cancer therapy.
摘要：
■癌细胞通过矩阵重组过程重塑其局部物理环境，沉积,硬化和降解。尿激酶型纤溶酶原激活物(uPA),由PLAU基因编码，是已知参与癌症进展和肿瘤微环境（TME）重塑的细胞外蛋白水解酶。因此,干扰uPA作为基于机械的癌症疗法具有强大的潜力。这项工作是一项生物工程研究，以验证1）uPA是否参与基质降解和2）通过靶向uPA防止基质降解可以减少癌细胞的侵袭和转移。
■为此，我们使用了一个工程3D体外模型，被称为肿瘤，适当地模拟肿瘤的天然生物物理环境（3kPa）。进行CRISPR-Cas9介导的uPA敲除以在基因编码序列中引入功能缺失突变。随后，为了验证阻断uPA作用的翻译潜力，我们测试了一种药物抑制剂，英国-371,801。通过原子力显微镜（AFM）测量基体刚度的变化。使用肿瘤的图像量化侵袭，培养21天后获得。
■我们发现uPA在浸润性乳腺癌和结直肠癌中高表达，这些侵袭性癌细胞局部降解它们的TME。PLAU（uPA）基因敲除（KO）完全停止了基质重塑，并显着减少了癌症侵袭。许多浸润性癌基因标记物也在PLAUKO肿瘤中下调。uPA的药理学抑制显示出类似的有希望的结果，基质降解减少，癌症侵袭也减少了。
■这项工作支持uPA在基质降解中的作用。这表明,当阻止TME基质的酶促分解时,癌细胞的侵袭显著降低。总的来说，这为靶向uPA作为基于机械的癌症治疗的有效性提供了强有力的证据.
■为此，我们使用了一个工程3D体外模型，被称为肿瘤，适当地模拟肿瘤的天然生物物理环境（3kPa）。进行CRISPR-Cas9介导的uPA敲除以在基因编码序列中引入功能缺失突变。随后，为了验证阻断uPA作用的翻译潜力，我们测试了一种药物抑制剂，英国-371,801。通过原子力显微镜（AFM）测量基体刚度的变化。使用肿瘤的图像量化侵袭，培养21天后获得。
■我们发现uPA在浸润性乳腺癌和结直肠癌中高表达，这些侵袭性癌细胞局部降解它们的TME。PLAU（uPA）基因敲除（KO）完全停止了基质重塑，并显着减少了癌症侵袭。许多浸润性癌基因标记物也在PLAUKO肿瘤中下调。uPA的药理学抑制显示出类似的有希望的结果，基质降解减少，癌症侵袭也减少了。
■这项工作支持uPA在基质降解中的作用。这表明,当阻止TME基质的酶促分解时,癌细胞的侵袭显著降低。总的来说，这为靶向uPA作为基于机械的癌症治疗的有效性提供了强有力的证据.
