Reliable detection of disseminated tumor cells and of the biodistribution of tumor-targeting therapeutic antibodies within the entire body has long been needed to better understand and treat cancer ...metastasis. Here, we developed an integrated pipeline for automated quantification of cancer metastases and therapeutic antibody targeting, named DeepMACT. First, we enhanced the fluorescent signal of cancer cells more than 100-fold by applying the vDISCO method to image metastasis in transparent mice. Second, we developed deep learning algorithms for automated quantification of metastases with an accuracy matching human expert manual annotation. Deep learning-based quantification in 5 different metastatic cancer models including breast, lung, and pancreatic cancer with distinct organotropisms allowed us to systematically analyze features such as size, shape, spatial distribution, and the degree to which metastases are targeted by a therapeutic monoclonal antibody in entire mice. DeepMACT can thus considerably improve the discovery of effective antibody-based therapeutics at the pre-clinical stage.
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•DeepMACT is a deep learning-based pipeline for comprehensive analysis of metastases•DeepMACT identifies micrometastases and single cancer cells in full-body 3D scans•DeepMACT reveals the efficacy of antibody-drug targeting in the entire body•DeepMACT indicates that the tumor microenvironment affects drug targeting efficacy
Deep learning-based automated detection and quantification of micrometastases and therapeutic antibody targeting down to the level of single disseminated cancer cells provides unbiased analysis of multiple metastatic cancer models at the full-body scale.
Carbonic anhydrase XII (CAXII) is a membrane‐tethered ectoenzyme involved in intracellular pH regulation and overexpressed across various types of human cancer. Because CAXII inhibition shows ...antitumor activity in vitro, it is thought that the enzyme is mandatory for maximum tumor growth, above all under hypoxic conditions. Recently, it has been shown that CAXII is co‐expressed along with the P‐glycoprotein (P‐GP) on many tumor cells and that both proteins physically interact. Of interest, blocking CAXII activity also decreases P‐GP activity in cancer cells both in vitro and in vivo. Previously, we have reported on the development of a monoclonal antibody, termed 6A10, which specifically and efficiently blocks human CAXII activity. Here, we demonstrate that 6A10 also indirectly reduces P‐GP activity in CAXII/P‐GP double‐positive chemoresistant cancer cells, resulting in enhanced chemosensitivity as revealed by enhanced accumulation of anthracyclines and increased cell death in vitro. Even more important, we show that mice carrying human triple‐negative breast cancer xenografts co‐treated with doxorubicin (DOX) and 6A10 show a significantly reduced number of metastases. Collectively, our data provide evidence that the inhibition of CAXII with 6A10 is an attractive way to reduce chemoresistance of cancer cells and to interfere with the metastatic process in a clinical setting.
What's new?
Through the reversible hydratation of carbon dioxide, carbonic anhydrases (CAs) produce bicarbonate, a critical mediator of pH homeostasis. In human cancers, however, certain forms of CA are overexpressed and may provide a survival advantage for hypoxic tumor cells in acidic tumor environments. Here, using a monoclonal antibody, 6A10, the authors show that inhibition of cancer‐associated CAXII enhances chemosensitivity in resistant cancer cells. In an orthotopic breast cancer mouse model, co‐treatment with 6A10 and doxorubicin had no inhibitory effect on primary tumor growth. In tumor‐bearing animals, however, co‐treatment with the drugs significantly reduced the number of lung metastases.
Proliferative vitreoretinopathy (PVR) is a blinding disease frequently occurring after retinal detachment surgery. Adhesion, migration and matrix remodeling of dedifferentiated retinal pigment ...epithelial (RPE) cells characterize the onset of the disease. Treatment options are still restrained and identification of factors responsible for the abnormal behavior of the RPE cells will facilitate the development of novel therapeutics. Galectin-3, a carbohydrate-binding protein, was previously found to inhibit attachment and spreading of retinal pigment epithelial cells, and thus bares the potential to counteract PVR-associated cellular events. However, the identities of the corresponding cell surface glycoprotein receptor proteins on RPE cells are not known. Here we characterize RPE-specific Gal-3 containing glycoprotein complexes using a proteomic approach. Integrin-β1, integrin-α3 and CD147/EMMPRIN, a transmembrane glycoprotein implicated in regulating matrix metalloproteinase induction, were identified as potential Gal-3 interactors on RPE cell surfaces. In reciprocal immunoprecipitation experiments we confirmed that Gal-3 associated with CD147 and integrin-β1, but not with integrin-α3. Additionally, association of Gal-3 with CD147 and integrin-β1 was observed in co-localization analyses, while integrin-α3 only partially co-localized with Gal-3. Blocking of CD147 and integrin-β1 on RPE cell surfaces inhibited binding of Gal-3, whereas blocking of integrin-α3 failed to do so, suggesting that integrin-α3 is rather an indirect interactor. Importantly, Gal-3 binding promoted pronounced clustering and co-localization of CD147 and integrin-β1, with only partial association of integrin-α3. Finally, we show that RPE derived CD147 and integrin-β1, but not integrin-α3, carry predominantly β-1,6-N-actyl-D-glucosamine-branched glycans, which are high-affinity ligands for Gal-3. We conclude from these data that extracellular Gal-3 triggers clustering of CD147 and integrin-β1 via interaction with β1,6-branched N-glycans on RPE cells and hypothesize that Gal-3 acts as a positive regulator for CD147/integrin-β1 clustering and therefore modifies RPE cell behavior contributing to the pathogenesis of PVR. Further investigations at this pathway may aid in the development of specific therapies for PVR.
Carbonic anhydrase 9 (CA9) and carbonic anhydrase 12 (CA12) were proposed as potential targets for cancer therapy more than 20 years ago. However, to date, there are only very few antibodies that ...have been described to specifically target CA9 and CA12 and also block the enzymatic activity of their targets. One of the early stage bottlenecks in identifying CA9- and CA12-inhibiting antibodies has been the lack of a high-throughput screening system that would allow for rapid assessment of inhibition of the targeted carbon dioxide hydratase activity of carbonic anhydrases. In this study, we show that measuring the esterase activity of carbonic anhydrase offers a robust and inexpensive screening method for identifying antibody candidates that block both hydratase and esterase activities of carbonic anhydrase's. To our knowledge, this is the first implementation of a facile surrogate-screening assay to identify potential therapeutic antibodies that block the clinically relevant hydratase activity of carbonic anhydrases.
Carbonic anhydrase XII (CA XII) is a membrane-tethered cell surface enzyme that is highly expressed on many human tumor cells. Carbonic anhydrase members in this class of exofacial molecules ...facilitate tumor metabolism by facilitating CO2 venting and intracellular pH regulation. Accordingly, inhibition of exofacial CAs has been proposed as a general therapeutic strategy to target cancer. The recent characterization of 6A10, the first CA XII-specific inhibitory monoclonal antibody, offered an opportunity to evaluate this strategy with regard to CA XII-mediated catalysis. Using functional assays, we showed that 6A10 inhibited exofacial CA activity in CA XII-expressing cancer cells. 6A10 reduced spheroid growth in vitro under culture conditions where CA XII was active (i.e., alkaline pH) and where its catalytic activity was likely rate-limiting (i.e., restricted extracellular HCO3-supply). These in vitro results argued that the antibody exerted its growth-retarding effect by acting on the catalytic process, rather than on antigen binding per se. Notably, when administered in a mouse xenograft model of human cancer, 6A10 exerted a significant delay on tumor outgrowth. These results corroborate the notion that exofacial CA is critical for cancer cell physiology and they establish the immunotherapeutic efficacy of targeting CA XII using an inhibitory antibody.
Human carbonic anhydrase (CA) IX has emerged as a promising anticancer target and a diagnostic biomarker for solid hypoxic tumors. Novel fluorinated CA IX inhibitors exhibited up to 50 pM affinity ...towards the recombinant human CA IX, selectivity over other CAs, and direct binding to Zn(II) in the active site of CA IX inducing novel conformational changes as determined by X-ray crystallography. Mass spectrometric gas-analysis confirmed the CA IX-based mechanism of the inhibitors in a CRISPR/Cas9-mediated CA IX knockout in HeLa cells. Hypoxia-induced extracellular acidification was significantly reduced in HeLa, H460, MDA-MB-231, and A549 cells exposed to the compounds, with the
values up to 1.29 nM. A decreased clonogenic survival was observed when hypoxic H460 3D spheroids were incubated with our lead compound. These novel compounds are therefore promising agents for CA IX-specific therapy.
We investigated whether gene transfer of insulin‐like growth factor I (IGF‐I) to the hepatic tissue was able to improve liver histology and function in established liver cirrhosis. Rats with liver ...cirrhosis induced by carbon tetrachloride (CCl4) given orally for 8 weeks were injected through the hepatic artery with saline or with Simian virus 40 vectors encoding IGF‐I (SVIGF‐I), or luciferase (SVLuc). Animals were sacrificed 8 weeks after vector injection. In cirrhotic rats we observed that, whereas IGF‐I was synthesized by hepatocytes, IGF‐I receptor was predominantly expressed by nonparenchymal cells, mainly in fibrous septa surrounding hepatic nodules. Rats treated with SVIGF‐I showed increased hepatic levels of IGF‐I, improved liver function tests, and reduced fibrosis in association with diminished α‐smooth muscle actin expression, up‐regulation of matrix metalloproteases (MMPs) and decreased expression of the tissue inhibitors of MMPs TIM‐1 and TIM‐2. SVIGF‐I therapy induced down‐regulation of the profibrogenic molecules transforming growth factor beta (TGFβ), amphiregulin, platelet‐derived growth factor (PDGF), connective tissue growth factor (CTGF), and vascular endothelium growth factor (VEGF) and induction of the antifibrogenic and cytoprotective hepatocyte growth factor (HGF). Furthermore, SVIGF‐I‐treated animals showed decreased expression of Wilms tumor‐1 (WT‐1; a nuclear factor involved in hepatocyte dedifferentiation) and up‐regulation of hepatocyte nuclear factor 4 alpha (HNF4α) (which stimulates hepatocellular differentiation). The therapeutic potential of SVIGF‐I was also tested in rats with thioacetamide‐induced liver cirrhosis. Also in this model, SVIGF‐I improved liver function and reduced liver fibrosis in association with up‐regulation of HGF and MMPs and down‐regulation of tissue inhibitor of metalloproteinase 1 (TIMP‐1). Conclusion: IGF‐I gene transfer to cirrhotic livers induces MMPs and hepatoprotective factors leading to reversion of fibrosis and improvement of liver function. IGF‐I gene therapy may be a useful alternative therapy for patients with advanced cirrhosis without timely access to liver transplantation. (HEPATOLOGY 2010;51:912–921.)
Proliferative vitreoretinopathy (PVR) is a blinding disease frequently occurring after retinal detachment surgery. Adhesion, migration and matrix remodeling of dedifferentiated retinal pigment ...epithelial (RPE) cells characterize the onset of the disease. Treatment options are still restrained and identification of factors responsible for the abnormal behavior of the RPE cells will facilitate the development of novel therapeutics. Galectin-3, a carbohydrate-binding protein, was previously found to inhibit attachment and spreading of retinal pigment epithelial cells, and thus bares the potential to counteract PVR-associated cellular events. However, the identities of the corresponding cell surface glycoprotein receptor proteins on RPE cells are not known. Here we characterize RPE-specific Gal-3 containing glycoprotein complexes using a proteomic approach. Integrin-beta1, integrin-alpha3 and CD147/EMMPRIN, a transmembrane glycoprotein implicated in regulating matrix metalloproteinase induction, were identified as potential Gal-3 interactors on RPE cell surfaces. In reciprocal immunoprecipitation experiments we confirmed that Gal-3 associated with CD147 and integrin-beta1, but not with integrin-alpha3. Additionally, association of Gal-3 with CD147 and integrin-beta1 was observed in co-localization analyses, while integrin-alpha3 only partially co-localized with Gal-3. Blocking of CD147 and integrin-beta1 on RPE cell surfaces inhibited binding of Gal-3, whereas blocking of integrin-alpha3 failed to do so, suggesting that integrin-alpha3 is rather an indirect interactor. Importantly, Gal-3 binding promoted pronounced clustering and co-localization of CD147 and integrin-beta1, with only partial association of integrin-alpha3. Finally, we show that RPE derived CD147 and integrin-beta1, but not integrin-alpha3, carry predominantly beta-1,6-N-actyl-D-glucosamine-branched glycans, which are high-affinity ligands for Gal-3. We conclude from these data that extracellular Gal-3 triggers clustering of CD147 and integrin-beta1 via interaction with beta1,6-branched N-glycans on RPE cells and hypothesize that Gal-3 acts as a positive regulator for CD147/integrin-beta1 clustering and therefore modifies RPE cell behavior contributing to the pathogenesis of PVR. Further investigations at this pathway may aid in the development of specific therapies for PVR.
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