generation-an-orthotopic-xenograft-pancreatic-cancer-cells-ultrasound

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant ...

University of Florence

Barrett's esophagus (BE) is the only well-known precursor lesion of esophageal adenocarcinoma (EA). The exact estimates of the annual progression rate from BE to EA vary from 0.07% to 3.6%. The identification of BE patients at higher risk to progress to EA is hence mandatory, although difficult to accomplish. In search of novel BE biomarkers we analyzed the efficacy of hERG1 potassium channels in predicting BE progression to EA. Once tested by immunohistochemistry (IHC) on bioptic samples, hERG1 was expressed in BE, and its expression levels increased during progression from BE to esophageal dysplasia (ED) and EA. hERG1 was also over-expressed in the metaplastic cells arising in BE lesions obtained in different BE mouse models, induced either surgically or chemically. Furthermore, transgenic mice which over express hERG1 in the whole gastrointestinal tract, developed BE lesions after an esophago-jejunal anastomosis more frequently, compared to controls. A case-control study was performed on 104 bioptic samples from newly diagnosed BE patients further followed up for at least 10 years. It emerged a statistically significant association between hERG1 expression status and risk of progression to EA. Finally, a novel fluorophore- conjugated recombinant single chain variable fragment antibody (scFv-hERG1-Alexa488) was tested on freshly collected live BE biopsies: it could recognize hERG1 positive samples, perfectly matching IHC data.Overall, hERG1 can be considered a novel BE biomarker to be exploited for a novel endoscopic surveillance protocol, either in biopsies or through endoscopy, to identify those BE patients with higher risk to progress to EA.

hERG1 behaves as biomarker of progression to adenocarcinoma in Barrett's esophagus and can be exploited for a novel endoscopic surveillance.

Modern molecular imaging techniques have greatly improved tumor detection and post-treatment follow-up of cancer patients. In this context, antibody-based imaging is rapidly becoming the gold standard, since it combines the unique specificity of antibodies with the sensitivity of the different imaging technologies. The aim of this study was to generate and characterize antibodies in single chain Fragment variable (scFv) format directed to an emerging cancer biomarker, the  (hERG1) potassium channel, and to obtain a proof of concept for their potential use for  molecular imaging. The anti-hERG1scFv was generated from a full length monoclonal antibody and then mutagenized, substituting a Phenylalanine residue in the third framework of the V domain with a Cysteine residue. The resulting scFv-hERG1-Cys showed much higher stability and protein yield, increased affinity and more advantageous binding kinetics, compared to the "native" anti-hERG1scFv. The scFv-hERG1-Cys was hence chosen and characterized: it showed a good binding to the native hERG1 antigen expressed on cells, was stable in serum and displayed a fast pharmacokinetic profile once injected intravenously in nude mice. The calculated half-life was 3.1 hours and no general toxicity or cardiac toxic effects were detected. Finally, the  distribution of an Alexa Fluor 750 conjugated scFv-hERG1-Cys was evaluated both in healthy and tumor-bearing nude mice, showing a good tumor-to-organ ratio, ideal for visualizing hERG1-expressing tumor masses . In conclusion, the scFv-hERG1-Cys possesses features which make it a suitable tool for application in cancer molecular imaging.

Generation and characterization of novel recombinant anti-hERG1 scFv antibodies for cancer molecular imaging.

Cell migration exerts a pivotal role in tumor progression, underlying cell invasion and metastatic spread. The cell migratory program requires f-actin re-organization, generally coordinated with the assembly of focal adhesions. Ion channels are emerging actors in regulating cell migration, through different mechanisms. We studied the role of the voltage dependent potassium channel K 11.1 on cell migration of pancreatic ductal adenocarcinoma (PDAC) cells, focusing on its effects on f-actin organization and dynamics. Cells were cultured either on fibronectin (FN) or on a desmoplastic matrix (DM) with the addition of a conditioned medium produced by pancreatic stellate cells (PSC) maintained in hypoxia (Hypo-PSC-CM), to better mimic the PDAC microenvironment. K11.1 was essential to maintain stress fibers in a less organized arrangement in cells cultured on FN. When PDAC cells were cultured on DM plus Hypo-PSC-CM, K11.1 activity determined the organization of cortical f-actin into sparse and long filopodia, and allowed f-actin polymerization at a high speed. In both conditions, blocking K11.1 impaired PDAC cell migration, and, on cells cultured onto FN, the effect was accompanied by a decrease of basal intracellular Ca concentration. We conclude that K11.1 is implicated in sustaining pro-metastatic signals in pancreatic cancer, through a reorganization of f-actin in stress fibers and a modulation of filopodia formation and dynamics.

The Activity of Kv 11.1 Potassium Channel Modulates F-Actin Organization During Cell Migration of Pancreatic Ductal Adenocarcinoma Cells.

The antibody era has greatly impacted cancer management in recent decades. Indeed, antibodies are currently applied for both cancer diagnosis and therapy. For example, monoclonal antibodies are the main constituents of several in vitro diagnostics, which are applied at many levels of cancer diagnosis. Moreover, the great improvement provided by in vivo imaging, especially for early-stage cancer diagnosis, has traced the path for the development of a complete new class of antibodies, i.e., engineered antibody fragments. The latter embody the optimal characteristics (e.g., low renal retention, rapid clearance, and small size) which make them ideal for in vivo applications. Furthermore, the present review focuses on reviewing the main applications of antibodies and antibody fragments for solid cancer diagnosis, both in vitro and in vivo. Furthermore, we review the scientific evidence showing that ion channels represent an almost unexplored class of ideal targets for both in vitro and in vivo diagnostic purposes. In particular, we review the applications, in solid cancers, of monoclonal antibodies and engineered antibody fragments targeting the voltage-dependent ion channel Kv 11.1, also known as hERG1.

Ion Channel Targeting with Antibodies and Antibody Fragments for Cancer Diagnosis.

Pancreatic duct cells are equipped with acid/base transporters important for exocrine secretion. Pancreatic ductal adenocarcinoma (PDAC) cells may utilize such transporters to acidify extracellular tumor microenvironment, creating a niche favoring cell proliferation, fibrosis and resistance to chemotherapy-all contributing to the notoriously bad prognosis of this disease. Here, we report that gastric and non-gastric H, K-ATPases (coded by  and ) are overexpressed in human and murine pancreatic cancer and that we can target them specifically with proton pump inhibitors (PPIs) and potassium-competitive acid blockers (P-CABs) in in vitro models of PDAC. Focusing on pantoprazole, we show that it significantly reduced human cancer cell proliferation by inhibiting cellular H extrusion, increasing K conductance and promoting cyclin D1-dependent cell cycle arrest and preventing STAT3 activation. Pantoprazole also decreased collagen secretion from pancreatic stellate cells. Importantly, in vivo studies show that pantoprazole treatment of tumor-bearing mice reduced tumor size, fibrosis and expression of angiogenic markers. This work provides the first evidence that H, K-ATPases contribute to PDAC progression and that these can be targeted by inhibitors of these pumps, thus proving a promising therapeutic strategy.

Proton Pump Inhibitors Reduce Pancreatic Adenocarcinoma Progression by Selectively Targeting H, K-ATPases in Pancreatic Cancer and Stellate Cells.

Increasing evidence indicates that ion channels and transporters cooperate in regulating different aspects of tumor pathophysiology. In cancer cells, H/HCO  transporters usually invert the transmembrane pH gradient typically observed in non-neoplastic cells, which is thought to contribute to cancer malignancy. To what extent the pH-regulating transporters are functionally linked to K channels, which are central regulators of cell membrane potential (V), is unclear. We thus investigated in colorectal cancer cells the implication of the pH-regulating transporters and K11.1 (also known as hERG1) in the pH modifications stimulated by integrin-dependent cell adhesion. Colorectal cancer cell lines (HCT 116 and HT 29) were seeded onto β1 integrin-dependent substrates, collagen I and fibronectin. This led to a transient cytoplasmic alkalinization, which peaked at 90 min of incubation, lasted approximately 180 min, and was inhibited by antibodies blocking the β1 integrin. The effect was sensitive to amiloride (10 µM) and cariporide (5 µM), suggesting that it was mainly caused by the activity of the Na/H antiporter NHE1. Blocking K11.1 with E4031 shows that channel activity contributed to modulate the β1 integrin-dependent pH increase. Interestingly, both NHE1 and K11.1 modulated the colorectal cancer cell motility triggered by β1 integrin-dependent adhesion. Finally, the β1 integrin subunit, K11.1 and NHE1 co-immunoprecipitated in colorectal cancer cells seeded onto Collagen I, suggesting the formation of a macromolecular complex following integrin-mediated adhesion. We conclude that the interaction between K11.1, NHE1, and β1 integrin contributes to regulate colorectal cancer intracellular pH in relation to the tumor microenvironment, suggesting novel pharmacological targets to counteract pro-invasive and, hence, pro-metastatic behavior in colorectal cancer.

K11.1 Potassium Channel and the Na/H Antiporter NHE1 Modulate Adhesion-Dependent Intracellular pH in Colorectal Cancer Cells.

In the last decades, protein engineering has developed particularly in biotechnology and pharmaceutical field. In particular, the engineered antibody subclass has arisen. The single chain diabody format (scDb), conjugating small size with antigen specificity, offers versatility representing a gold standard for a variety of applications, spacing from research to diagnostics and therapy. Along with such advantages, comes the challenge of optimizing their production, improving expression systems, purification procedures and stability. All such parameters are detrimental for protein production in general and above all for recombinant antibody expression, which has to be fine-tuned, choosing a proper protein-expression host and adjusting expression protocols accordingly. In the present paper, we present data regarding the production and purification of a single chain diabody directed against the macromolecular complex hERG1/β1 integrin. We focus on the expression of clones deriving from the transformation of Pichia pastoris yeast cells. In particular, we compare two different clones arose from two separate transformation processes, demonstrating that both are suitable for proper protein expression. Moreover, we have set up an expression protocol and compared the yields obtained using two purification machines: Akta Pure and Akta Start, with a positive outcome.

Expression and purification of a novel single-chain diabody (scDb-hERG1/β1) from Pichia pastoris transformants.

The receptor for the luteinizing hormone (LH-R) is aberrantly over expressed in cancers of the reproductive system. To uncover whether LH-R over expression has a causative role in cancer, we generated a transgenic (TG) mouse which overexpresses the human LH-R (hLH-R) in the female reproductive tract, under the control of the oviduct-specific glycoprotein (OGP) mouse promoter (mogp-1). The transgene was highly expressed in the uterus, ovary and liver, but only in the uterus morphological and molecular alterations (increased proliferation and trans-differentiation in the endometrial layer) were detected. A transcriptomic analysis on the uteri of young TG mice showed an up regulation of genes involved in cell cycle control and a down regulation of genes related to the immune system and the metabolism of xenobiotics. Aged TG females developed tumor masses in the uteri, which resembled an Endometrial Cancer (EC). Microarray and immunohistochemistry data indicated the deregulation of signaling pathways which are known to be altered in human ECs. The analysis of a cohort of 126 human ECs showed that LH-R overexpression is associated with early-stage tumors. Overall, our data led support to conclude that LH-R overexpression may directly contribute to trigger the neoplastic transformation of the endometrium.

Transgenic mice overexpressing the LH receptor in the female reproductive system spontaneously develop endometrial tumour masses.

mAbs, either mono- or bispecific (bsAb), represent one of the most successful approaches to treat many types of malignancies. However, there are certain limitations to the use of full length mAbs for clinical applications, which can be overcome by engineered antibody fragments. The aim of this study was to develop a small bsAb, in the format of a single-chain diabody (scDb), to efficiently target two proteins, the hERG1 potassium channel and the β1 subunit of integrin receptors, which specifically form a macromolecular complex in cancer cells. We provide evidence that the scDb we produced binds to the hERG1/β1 complex in cancer cells and tissues, but does not bind to the hERG1 channel in nonpathologic tissues, in particular the heart. The scDb-hERG1-β1 (i) downregulates the formation of the hERG1/β1 complex, (ii) inhibits Akt phosphorylation and HIF-1α expression, and (iii) decreases cell survival, proliferation, and migration  These effects only occur in cancer cells (either colon, pancreatic, or breast), but not in normal cells. , the scDb-hERG1-β1 shows a good pharmacokinetic profile, with a half-life of 13.5 hours and no general, cardiac, or renal toxicity when injected intravenously up to the dose of 8 mg/kg. The scDb-hERG1-β1 accumulates into subcutaneous xenografted tumors, arising from either colon or pancreatic human cancer cells, and induces a reduction of tumor growth and vascularization. Overall, the scDb-hERG1-β1 represents an innovative single-chain bispecific antibody for therapeutic applications in solid cancers that overexpress the hERG1/β1 integrin signaling complex.

Harnessing the hERG1/β1 Integrin Complex via a Novel Bispecific Single-chain Antibody: An Effective Strategy against Solid Cancers.

Ion channels are progressively emerging as a novel class of membrane proteins expressed in several types of human cancers and regulating the different aspects of cancer cell behavior. The metabolism of cancer cells, usually composed by a variable proportion of respiration, glycolysis, and glutaminolysis, leads to the excessive production of acidic metabolic products. The presence of these acidic metabolites inside the cells results in intracellular acidosis, and hinders survival and proliferation. For this reason, tumor cells activate mechanisms of pH control that produce a constitutive increase in intracellular pH (pH) that is more acidic than the extracellular pH (pH). This condition forms a perfect microenvironment for metastatic progression and may be permissive for some of the acquired characteristics of tumors. Recent analyses have revealed complex interconnections between oncogenic activation, ion channels, hypoxia signaling and metabolic pathways that are dysregulated in cancer. Here, we summarize the molecular mechanisms of the Warburg effect and hypoxia and their association. Moreover, we discuss the recent findings concerning the involvement of ion channels in various aspects of the Warburg effect and hypoxia, focusing on the role of Na and K channels in hypoxic and metabolic reprogramming in cancer.

Article	Year
hERG1 behaves as biomarker of progression to adenocarcinoma in Barrett's esophagus and can be exploited for a novel endoscopic surveillance. Oncotarget\| PubMed ID: 27517748	2016
Generation and characterization of novel recombinant anti-hERG1 scFv antibodies for cancer molecular imaging. Oncotarget\| PubMed ID: 30405887	2018
The Activity of Kv 11.1 Potassium Channel Modulates F-Actin Organization During Cell Migration of Pancreatic Ductal Adenocarcinoma Cells. Cancers\| PubMed ID: 30678127	2019
Ion Channel Targeting with Antibodies and Antibody Fragments for Cancer Diagnosis. Antibodies (Basel, Switzerland)\| PubMed ID: 31544839	2019
Proton Pump Inhibitors Reduce Pancreatic Adenocarcinoma Progression by Selectively Targeting H, K-ATPases in Pancreatic Cancer and Stellate Cells. Cancers\| PubMed ID: 32164284	2020
K11.1 Potassium Channel and the Na/H Antiporter NHE1 Modulate Adhesion-Dependent Intracellular pH in Colorectal Cancer Cells. Frontiers in pharmacology\| PubMed ID: 32587517	2020
Expression and purification of a novel single-chain diabody (scDb-hERG1/β1) from Pichia pastoris transformants. Protein expression and purification\| PubMed ID: 33826963	2021
Transgenic mice overexpressing the LH receptor in the female reproductive system spontaneously develop endometrial tumour masses. Scientific reports\| PubMed ID: 33893331	2021
Harnessing the hERG1/β1 Integrin Complex via a Novel Bispecific Single-chain Antibody: An Effective Strategy against Solid Cancers. Molecular cancer therapeutics\| PubMed ID: 34045227	2021
Potassium and Sodium Channels and the Warburg Effect: Biophysical Regulation of Cancer Metabolism. Bioelectricity\| PubMed ID: 34471821	2019

Claudia Duranti

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