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

Because of their high incidence and mortality solid cancers are a major health problem worldwide. Although several new biomarkers and potential targets for therapy have been identified through biomolecular research in the last years, the effects on patients' outcome are still unsatisfactory. Increasing evidence indicates that hERG1 potassium channels are overexpressed in human primary cancers of different origin and several associations between hERG1 expression and clinicopathological features and/or outcome are emerging. Aberrant hERG1 expression may be exploited either for early diagnosis (especially in those cancers where it is expressed in the initial steps of tumor progression) or for therapy purposes. Indeed, hERG1 blockage impairs tumor cell growth both in vitro and in vivo in preclinical mouse model. hERG1-based tumor therapy in humans, however, encounters the major hindrance of the potential cardiotoxicity that many hERG1 blockers exert. In this review we focus on recent advances in translational research in some of the most frequent human solid cancers (breast, endometrium, ovary, pancreas, esophagus, stomach, and colorectum) that have been shown to express hERG1 and that are a major health problem. 

hERG1 Potassium Channels: Novel Biomarkers in Human Solid Cancers.

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.

A 51 year-old woman was diagnosed with endometrial cancer (EC) and underwent surgical staging. Pathological evaluation showed a 2 cm × 1 cm G2 endometrioid EC with a 30% myometrial deep invasion (FIGO Stage 1A). The patient was classified as low risk of recurrence, and no adjuvant treatment was offered. Six months after surgery, the patient developed an early vescico-vaginal recurrence, and chemotherapy treatment was started. Few months later, a subsequent involvement of vaginal wall, ileum, and omentum was detected, and the patient underwent second surgery.

LH/hCG-Receptor Expression May Have a Negative Prognostic Value in Low-Risk Endometrial Cancer.

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.

Gastric cancer (GC) is still a relevant health issue worldwide. The identification of prognostic factors for progression of gastric dysplasia (GD), the main pre-cancerous lesion of the intestinal-type GC, is hence mandatory.

The hERG1 Potassium Channel Behaves As Prognostic Factor In Gastric Dysplasia Endoscopic Samples.

Acute tissue injury causes DNA damage and repair processes involving increased cell mitosis and polyploidization, leading to cell function alterations that may potentially drive cancer development. Here, we show that acute kidney injury (AKI) increased the risk for papillary renal cell carcinoma (pRCC) development and tumor relapse in humans as confirmed by data collected from several single-center and multicentric studies. Lineage tracing of tubular epithelial cells (TECs) after AKI induction and long-term follow-up in mice showed time-dependent onset of clonal papillary tumors in an adenoma-carcinoma sequence. Among AKI-related pathways, NOTCH1 overexpression in human pRCC associated with worse outcome and was specific for type 2 pRCC. Mice overexpressing NOTCH1 in TECs developed papillary adenomas and type 2 pRCCs, and AKI accelerated this process. Lineage tracing in mice identified single renal progenitors as the cell of origin of papillary tumors. Single-cell RNA sequencing showed that human renal progenitor transcriptome showed similarities to PT1, the putative cell of origin of human pRCC. Furthermore, NOTCH1 overexpression in cultured human renal progenitor cells induced tumor-like 3D growth. Thus, AKI can drive tumorigenesis from local tissue progenitor cells. In particular, we find that AKI promotes the development of pRCC from single progenitors through a classical adenoma-carcinoma sequence.

Acute kidney injury promotes development of papillary renal cell adenoma and carcinoma from renal progenitor 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.

Little is known about the metabolic regulation of cancer stem cells (CSCs) in cholangiocarcinoma (CCA). We analyzed whether mitochondrial-dependent metabolism and related signaling pathways contribute to stemness in CCA.

Mitochondrial oxidative metabolism contributes to a cancer stem cell phenotype in cholangiocarcinoma.

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.

Cholangiocarcinoma (CCA) is characterized by high resistance to chemotherapy and poor prognosis. Several oncogenic pathways converge on activation of extracellular signal-regulated kinase 5 (ERK5), whose role in CCA has not been explored. The aim of this study was to investigate the role of ERK5 in the biology of CCA.

Extracellular Signal-Regulated Kinase 5 Regulates the Malignant Phenotype of Cholangiocarcinoma Cells.

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.

Article	Year
hERG1 Potassium Channels: Novel Biomarkers in Human Solid Cancers. BioMed research international\| PubMed ID: 26339650	2015
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
LH/hCG-Receptor Expression May Have a Negative Prognostic Value in Low-Risk Endometrial Cancer. Frontiers in oncology\| PubMed ID: 27610354	2016
Generation and characterization of novel recombinant anti-hERG1 scFv antibodies for cancer molecular imaging. Oncotarget\| PubMed ID: 30405887	2018
The hERG1 Potassium Channel Behaves As Prognostic Factor In Gastric Dysplasia Endoscopic Samples. OncoTargets and therapy\| PubMed ID: 31807018	2019
Acute kidney injury promotes development of papillary renal cell adenoma and carcinoma from renal progenitor cells. Science translational medicine\| PubMed ID: 32213630	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
Mitochondrial oxidative metabolism contributes to a cancer stem cell phenotype in cholangiocarcinoma. Journal of hepatology\| PubMed ID: 33484774	2021
Transgenic mice overexpressing the LH receptor in the female reproductive system spontaneously develop endometrial tumour masses. Scientific reports\| PubMed ID: 33893331	2021
Extracellular Signal-Regulated Kinase 5 Regulates the Malignant Phenotype of Cholangiocarcinoma Cells. Hepatology (Baltimore, Md.)\| PubMed ID: 33959996	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

Tiziano Lottini

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