Biomolecular imaging of colorectal tumor lesions using a FITC-labeled scFv-Cκ fragment antibody

AbstractFor the sensitive diagnosis of colorectal cancer lesions, advanced molecular imaging techniques using cancer-specific targets have emerged. However, issues regarding the clearance of unbound probes and immunogenicity remain unresolved. To overcome these limitations, we developed a small-sized scFv antibody fragment conjugated with FITC for the real-time detection of colorectal cancer by in vivo molecular endoscopy imaging. A small-sized scFv fragment can target colon cancer secreted protein-2 (CCSP-2), highly expressed in colorectal adenocarcinoma tissues; moreover, its full-length IgG probe has been used for molecular imaging previously. To assess the efficacy of anti-CCSP-2 scFv-FITC, surgical specimens were obtained from 21 patients with colorectal cancer for ex vivo molecular fluorescence analysis, histology, and immunohistochemistry. Orthotopic mice were administered with anti-CCSP-2 scFv-FITC topically and intravenously, and distinct tumor lesions were observed by real-time fluorescence colonoscopy. The fluorescence imaging of human colon cancer specimens allowed the differentiation of malignant tissues from non-malignant tissues (p < 0.05), and the CCSP-2 expression level was found to be correlated with the fluorescence intensity. Here, we demonstrated the feasibility and safety of anti-CCSP-2 scFv-FITC for molecular imaging as well as its potential in real-time fluorescence colonoscopy for the differential diagnosis of tumor lesions.

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Biomolecular Imaging of Colorectal Tumor Lesion Using FITC Labeled scFv-Cκ Fragment Antibody

10.21203/rs.3.rs-128088/v1 ◽

2020 ◽

Author(s):

Hyung il Kim ◽

Jinhyeon Kim ◽

Hyori Kim ◽

Yong Sik Yoon ◽

Sung Wook Hwang ◽

...

Keyword(s):

Colorectal Cancer ◽

Colon Cancer ◽

Molecular Imaging ◽

Real Time ◽

Ex Vivo ◽

Imaging Techniques ◽

Antibody Fragment ◽

Fluorescence Analysis ◽

Human Colon Cancer

Abstract For the sensitive diagnosis of colorectal cancer lesions, advanced molecular imaging techniques using cancer-specific targets were emerged. However, issues for the clearance of unbound probes and immunogenicity remain unresolved. To overcome these limitations, we developed a small sized antibody fragment conjugated with FITC for the real-time detection of colorectal cancer by in vivo molecular endoscopy imaging. A small-sized scFv fragment targets colon cancer secreted protein-2 (CCSP-2), highly expressed in colorectal adenocarcinoma tissue, and its full-length IgG probe has subjected to study for molecular imaging previously. To assess the advanced efficacy of anti-CCSP-2 scFv-FITC, surgical specimens were obtained from 21 patients with colorectal cancer for ex vivo molecular fluorescence analysis, histology, and immunohistochemistry. Orthotopic mice were administered with anti-CCSP-2 scFv-FITC topically and intravenously, and distinct tumor lesions were observed by real-time fluorescence colonoscopy. The fluorescence imaging of human colon cancer specimens allowed the distinction of malignant tissues from non-malignant tissues (p < 0.05), and the CCSP-2 expression level was found to be correlated with the fluorescence intensity. Here, we demonstrated the feasibility and safety of anti-CCSP-2 scFv-FITC for molecular imaging as well as its potential in real-time fluorescence colonoscopy for the differential diagnosis of tumor lesions.

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EANM recommendations based on systematic analysis of small animal radionuclide imaging in inflammatory musculoskeletal diseases

EJNMMI Research ◽

10.1186/s13550-021-00820-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Erik H. J. G. Aarntzen ◽

Edel Noriega-Álvarez ◽

Vera Artiko ◽

André H. Dias ◽

Olivier Gheysens ◽

...

Keyword(s):

Molecular Imaging ◽

Radionuclide Imaging ◽

Musculoskeletal Diseases ◽

Ex Vivo ◽

Imaging Techniques ◽

Large Body ◽

Therapeutic Interventions ◽

Histopathological Analysis ◽

Complementary Value

AbstractInflammatory musculoskeletal diseases represent a group of chronic and disabling conditions that evolve from a complex interplay between genetic and environmental factors that cause perturbations in innate and adaptive immune responses. Understanding the pathogenesis of inflammatory musculoskeletal diseases is, to a large extent, derived from preclinical and basic research experiments. In vivo molecular imaging enables us to study molecular targets and to measure biochemical processes non-invasively and longitudinally, providing information on disease processes and potential therapeutic strategies, e.g. efficacy of novel therapeutic interventions, which is of complementary value next to ex vivo (post mortem) histopathological analysis and molecular assays. Remarkably, the large body of preclinical imaging studies in inflammatory musculoskeletal disease is in contrast with the limited reports on molecular imaging in clinical practice and clinical guidelines. Therefore, in this EANM-endorsed position paper, we performed a systematic review of the preclinical studies in inflammatory musculoskeletal diseases that involve radionuclide imaging, with a detailed description of the animal models used. From these reflections, we provide recommendations on what future studies in this field should encompass to facilitate a greater impact of radionuclide imaging techniques on the translation to clinical settings.

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Gold Nanoparticle-Based Fluorescent Theranostics for Real-Time Image-Guided Assessment of DNA Damage and Repair

International Journal of Molecular Sciences ◽

10.3390/ijms20030471 ◽

2019 ◽

Vol 20 (3) ◽

pp. 471 ◽

Cited By ~ 2

Author(s):

Shriya S. Srinivasan ◽

Rajesh Seenivasan ◽

Allison Condie ◽

Stanton L. Gerson ◽

Yanming Wang ◽

...

Keyword(s):

Molecular Imaging ◽

Real Time ◽

Targeted Delivery ◽

Specific Binding ◽

Imaging Techniques ◽

Cancer Cell Line ◽

Molecular Probe ◽

Biodistribution Studies

Chemotherapeutic dosing, is largely based on the tolerance levels of toxicity today. Molecular imaging strategies can be leveraged to quantify DNA cytotoxicity and thereby serve as a theranostic tool to improve the efficacy of treatments. Methoxyamine-modified cyanine-7 (Cy7MX) is a molecular probe which binds to apurinic/apyrimidinic (AP)-sites, inhibiting DNA-repair mechanisms implicated by cytotoxic chemotherapies. Herein, we loaded (Cy7MX) onto polyethylene glycol-coated gold nanoparticles (AuNP) to selectively and stably deliver the molecular probe intravenously to tumors. We optimized the properties of Cy7MX-loaded AuNPs using optical spectroscopy and tested the delivery mechanism and binding affinity using the DLD1 colon cancer cell line in vitro. A 10:1 ratio of Cy7MX-AuNPs demonstrated a strong AP site-specific binding and the cumulative release profile demonstrated 97% release within 12 min from a polar to a nonpolar environment. We further demonstrated targeted delivery using imaging and biodistribution studies in vivo in an xenografted mouse model. This work lays a foundation for the development of real-time molecular imaging techniques that are poised to yield quantitative measures of the efficacy and temporal profile of cytotoxic chemotherapies.

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Multi-Modal Multi-Spectral Intravital Macroscopic Imaging of Signaling Dynamics in Real Time during Tumor–Immune Interactions

Cells ◽

10.3390/cells10030489 ◽

2021 ◽

Vol 10 (3) ◽

pp. 489 ◽

Cited By ~ 1

Author(s):

Tracy W. Liu ◽

Seth T. Gammon ◽

David Fuentes ◽

David Piwnica-Worms

Keyword(s):

Molecular Imaging ◽

Real Time ◽

Bioluminescence Imaging ◽

Immune Cell ◽

Imaging Techniques ◽

Spectral Unmixing ◽

Fluorescent Reporters ◽

Signaling Dynamics ◽

Cell Enzyme

A major obstacle in studying the interplay between cancer cells and the immune system has been the examination of proposed biological pathways and cell interactions in a dynamic, physiologically relevant system in vivo. Intravital imaging strategies are one of the few molecular imaging techniques that can follow biological processes at cellular resolution over long periods of time in the same individual. Bioluminescence imaging has become a standard preclinical in vivo optical imaging technique with ever-expanding versatility as a result of the development of new emission bioluminescent reporters, advances in genomic techniques, and technical improvements in bioluminescence imaging and processing methods. Herein, we describe an advance of technology with a molecular imaging window chamber platform that combines bioluminescent and fluorescent reporters with intravital macro-imaging techniques and bioluminescence spectral unmixing in real time applied to heterogeneous living systems in vivo for evaluating tumor signaling dynamics and immune cell enzyme activities concurrently.

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5-Fluorouracil Loaded Orally Administered WGA-decorated Poly(lactic-co-glycolic acid) Nanoparticles for Treatment of Colorectal Cancer: In Vivo Evaluation

Current Nanomedicine ◽

10.2174/2468187310999201123195233 ◽

2020 ◽

Vol 10 ◽

Author(s):

Aditya Nath Pandey ◽

Kuldeep Rajpoot ◽

Sunil K. Jain

Keyword(s):

Colorectal Cancer ◽

Colon Cancer ◽

Oral Delivery ◽

Wheat Germ ◽

Glycolic Acid ◽

Organ Distribution ◽

In Vivo Evaluation ◽

Distribution Study ◽

Prolonged Retention

Background:: Several studies have suggested potential aptitude of polylactic-co-glycolic acid (PLGA)-derived nanoparticles (NPs) to improve the antitumor efficacy of anticancer drugs against colon cancer. Further, conjugation of lectins over the surface of the NPs may ameliorate interaction and thus enhance attachment of NPs with receptors. Objective:: The main goal of the study was to prepare and evaluate targeting potential (in vivo) of the optimized NPs against colorectal cancer. Methods:: The 5-fluorouracil (5-FU) loaded and wheat germ agglutinin (WGA)-conjugated PLGA-NPs (WFUNPs) were prepared and then they were evaluated in vivo for targeting aptitude of formulation using gamma scintigraphy after oral delivery. The WGA-conjugated and non-conjugated optimized NPs were compared for any significant results. Further, optimized formulations were also assessed for different parameters such as radiolabeling efficiency, sodium pertechnetate uptake, stability of NPs, and organ distribution study. Results:: Findings suggested prolonged retention of 99mTc-tagged WFUNPs in the colonic region after 24 h study. Eventually, the outcome from conjugated formulation revealed enhanced bioavailability of the drug in blood plasma for up to 24 h. Conclusion:: In conclusion, WGA-conjugation to NPs could improve the performance of the PLGA-NPs in the treatment of colorectal cancer.

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Cytotoxicity and Apoptosis Effects of Curcumin Analogue (2E,6E)-2,6-Bis(2,3-Dimethoxybenzylidine) Cyclohexanone (DMCH) on Human Colon Cancer Cells HT29 and SW620 In Vitro

Molecules ◽

10.3390/molecules26051261 ◽

2021 ◽

Vol 26 (5) ◽

pp. 1261

Author(s):

Nurul Fattin Che Rahim ◽

Yazmin Hussin ◽

Muhammad Nazirul Mubin Aziz ◽

Nurul Elyani Mohamad ◽

Swee Keong Yeap ◽

...

Keyword(s):

Colorectal Cancer ◽

Colon Cancer ◽

Cell Death ◽

Cancer Cells ◽

Cell Lines ◽

Curcuma Longa ◽

Metastatic Colon Cancer ◽

Colon Cancer Cells ◽

Human Colon Cancer ◽

Sw620 Cell

Colorectal cancer (CRC) is the third most common type of cancer worldwide and a leading cause of cancer death. According to the Malaysian National Cancer Registry Report 2012–2016, colorectal cancer was the second most common cancer in Malaysia after breast cancer. Recent treatments for colon cancer cases have caused side effects and recurrence in patients. One of the alternative ways to fight cancer is by using natural products. Curcumin is a compound of the rhizomes of Curcuma longa that possesses a broad range of pharmacological activities. Curcumin has been studied for decades but due to its low bioavailability, its usage as a therapeutic agent has been compromised. This has led to the development of a chemically synthesized curcuminoid analogue, (2E,6E)-2,6-bis(2,3-dimethoxybenzylidine) cyclohexanone (DMCH), to overcome the drawbacks. This study aims to examine the potential of DMCH for cytotoxicity, apoptosis induction, and activation of apoptosis-related proteins on the colon cancer cell lines HT29 and SW620. The cytotoxic activity of DMCH was evaluated using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) cell viability assay on both of the cell lines, HT29 and SW620. To determine the mode of cell death, an acridine orange/propidium iodide (AO/PI) assay was conducted, followed by Annexin V/FITC, cell cycle analysis, and JC-1 assay using a flow cytometer. A proteome profiler angiogenesis assay was conducted to determine the protein expression. The inhibitory concentration (IC50) of DMCH in SW620 and HT29 was 7.50 ± 1.19 and 9.80 ± 0.55 µg/mL, respectively. The treated cells displayed morphological features characteristic of apoptosis. The flow cytometry analysis confirmed that DMCH induced apoptosis as shown by an increase in the sub-G0/G1 population and an increase in the early apoptosis and late apoptosis populations compared with untreated cells. A higher number of apoptotic cells were observed on treated SW620 cells as compared to HT29 cells. Human apoptosis proteome profiler analysis revealed upregulation of Bax and Bad proteins and downregulation of Livin proteins in both the HT29 and SW620 cell lines. Collectively, DMCH induced cell death via apoptosis, and the effect was more pronounced on SW620 metastatic colon cancer cells, suggesting its potential effects as an antimetastatic agent targeting colon cancer cells.

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Olaparib-mediated enhancement of 5-fluorouracil cytotoxicity in mismatch repair deficient colorectal cancer cells

BMC Cancer ◽

10.1186/s12885-021-08188-7 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Helena de Castro e Gloria ◽

Laura Jesuíno Nogueira ◽

Patrícia Bencke Grudzinski ◽

Paola Victória da Costa Ghignatti ◽

Temenouga Nikolova Guecheva ◽

...

Keyword(s):

Colorectal Cancer ◽

Colon Cancer ◽

Dna Damage ◽

Cancer Cells ◽

Mismatch Repair ◽

Cell Cycle Progression ◽

Combined Therapy ◽

Combined Treatment ◽

Human Colon ◽

Human Colon Cancer

Abstract Background The advances in colorectal cancer (CRC) treatment include the identification of deficiencies in Mismatch Repair (MMR) pathway to predict the benefit of adjuvant 5-fluorouracil (5-FU) and oxaliplatin for stage II CRC and immunotherapy. Defective MMR contributes to chemoresistance in CRC. A growing body of evidence supports the role of Poly-(ADP-ribose) polymerase (PARP) inhibitors, such as Olaparib, in the treatment of different subsets of cancer beyond the tumors with homologous recombination deficiencies. In this work we evaluated the effect of Olaparib on 5-FU cytotoxicity in MMR-deficient and proficient CRC cells and the mechanisms involved. Methods Human colon cancer cell lines, proficient (HT29) and deficient (HCT116) in MMR, were treated with 5-FU and Olaparib. Cytotoxicity was assessed by MTT and clonogenic assays, apoptosis induction and cell cycle progression by flow cytometry, DNA damage by comet assay. Adhesion and transwell migration assays were also performed. Results Our results showed enhancement of the 5-FU citotoxicity by Olaparib in MMR-deficient HCT116 colon cancer cells. Moreover, the combined treatment with Olaparib and 5-FU induced G2/M arrest, apoptosis and polyploidy in these cells. In MMR proficient HT29 cells, the Olaparib alone reduced clonogenic survival, induced DNA damage accumulation and decreased the adhesion and migration capacities. Conclusion Our results suggest benefits of Olaparib inclusion in CRC treatment, as combination with 5-FU for MMR deficient CRC and as monotherapy for MMR proficient CRC. Thus, combined therapy with Olaparib could be a strategy to overcome 5-FU chemotherapeutic resistance in MMR-deficient CRC.

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Farnesyl dimethyl chromanol targets colon cancer stem cells and prevents colorectal cancer metastasis

Scientific Reports ◽

10.1038/s41598-020-80911-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kazim Husain ◽

Domenico Coppola ◽

Chung S. Yang ◽

Mokenge P. Malafa

Keyword(s):

Colorectal Cancer ◽

Colon Cancer ◽

Cancer Metastasis ◽

Annexin V ◽

Cancer Cell Growth ◽

Ki 67 ◽

Sox2 Expression ◽

Tumour Metastasis

AbstractThe activation and growth of tumour-initiating cells with stem-like properties in distant organs characterize colorectal cancer (CRC) growth and metastasis. Thus, inhibition of colon cancer stem cell (CCSC) growth holds promise for CRC growth and metastasis prevention. We and others have shown that farnesyl dimethyl chromanol (FDMC) inhibits cancer cell growth and induces apoptosis in vitro and in vivo. We provide the first demonstration that FDMC inhibits CCSC viability, survival, self-renewal (spheroid formation), pluripotent transcription factors (Nanog, Oct4, and Sox2) expression, organoids formation, and Wnt/β-catenin signalling, as evidenced by comparisons with vehicle-treated controls. In addition, FDMC inhibits CCSC migration, invasion, inflammation (NF-kB), angiogenesis (vascular endothelial growth factor, VEGF), and metastasis (MMP9), which are critical tumour metastasis processes. Moreover, FDMC induced apoptosis (TUNEL, Annexin V, cleaved caspase 3, and cleaved PARP) in CCSCs and CCSC-derived spheroids and organoids. Finally, in an orthotopic (cecum-injected CCSCs) xenograft metastasis model, we show that FDMC significantly retards CCSC-derived tumour growth (Ki-67); inhibits inflammation (NF-kB), angiogenesis (VEGF and CD31), and β-catenin signalling; and induces apoptosis (cleaved PARP) in tumour tissues and inhibits liver metastasis. In summary, our results demonstrate that FDMC inhibits the CCSC metastatic phenotype and thereby supports investigating its ability to prevent CRC metastases.

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Multi-Modal Multi-Spectral Intravital Microscopic Imaging of Signaling Dynamics in Real-Time during Tumor–Immune Interactions

Cells ◽

10.3390/cells10030499 ◽

2021 ◽

Vol 10 (3) ◽

pp. 499

Author(s):

Tracy W. Liu ◽

Seth T. Gammon ◽

David Piwnica-Worms

Keyword(s):

Molecular Imaging ◽

Single Cell ◽

Real Time ◽

Bioluminescence Imaging ◽

Emission Spectra ◽

Ccd Camera ◽

Microscopic Imaging ◽

Signaling Dynamics ◽

Window Chamber

Intravital microscopic imaging (IVM) allows for the study of interactions between immune cells and tumor cells in a dynamic, physiologically relevant system in vivo. Current IVM strategies primarily use fluorescence imaging; however, with the advances in bioluminescence imaging and the development of new bioluminescent reporters with expanded emission spectra, the applications for bioluminescence are extending to single cell imaging. Herein, we describe a molecular imaging window chamber platform that uniquely combines both bioluminescent and fluorescent genetically encoded reporters, as well as exogenous reporters, providing a powerful multi-plex strategy to study molecular and cellular processes in real-time in intact living systems at single cell resolution all in one system. We demonstrate that our molecular imaging window chamber platform is capable of imaging signaling dynamics in real-time at cellular resolution during tumor progression. Importantly, we expand the utility of IVM by modifying an off-the-shelf commercial system with the addition of bioluminescence imaging achieved by the addition of a CCD camera and demonstrate high quality imaging within the reaches of any biology laboratory.

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A novel method for visualizing and tracking endogenous mRNA in a specific cell population in pathological neovascularization

Scientific Reports ◽

10.1038/s41598-021-81367-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Md Imam Uddin ◽

Tyler C. Kilburn ◽

Sara Z. Jamal ◽

Craig L. Duvall ◽

John S. Penn

Keyword(s):

Real Time ◽

Disease Burden ◽

Ex Vivo ◽

Expression Patterns ◽

Fluorescence Emission ◽

High Sensitivity ◽

Living Systems ◽

Specific Cell ◽

Potential Marker

AbstractDiabetic retinopathy, retinopathy of prematurity and retinal vein occlusion are potentially blinding conditions largely due to their respective neovascular components. The development of real-time in vivo molecular imaging methods, to assess levels of retinal neovascularization (NV), would greatly benefit patients afflicted with these conditions. mRNA hybridization techniques offer a potential method to image retinal NV. The success of these techniques hinges on the selection of a target mRNA whose tissue levels and spatial expression patterns correlate closely with disease burden. Using a model of oxygen-induced retinopathy (OIR), we previously observed dramatic increases in retinal endoglin that localized to neovascular structures (NV), directly correlating with levels of neovascular pathology. Based on these findings, we have investigated Endoglin mRNA as a potential marker for imaging retinal NV in OIR mice. Also of critical importance, is the application of innovative technologies capable of detecting mRNAs in living systems with high sensitivity and specificity. To detect and visualize endoglin mRNA in OIR mice, we have designed and synthesized a novel imaging probe composed of short-hairpin anti-sense (AS) endoglin RNA coupled to a fluorophore and black hole quencher (AS-Eng shRNA). This assembly allows highly sensitive fluorescence emission upon hybridization of the AS-Eng shRNA to cellular endoglin mRNA. The AS-Eng shRNA is further conjugated to a diacyl-lipid (AS-Eng shRNA–lipid referred to as probe). The lipid moiety binds to serum albumin facilitating enhanced systemic circulation of the probe. OIR mice received intraperitoneal injections of AS-Eng shRNA–lipid. Ex vivo imaging of their retinas revealed specific endoglin mRNA dependent fluorescence superimposed on neovascular structures. Room air mice receiving AS-Eng shRNA–lipid and OIR mice receiving a non-sense control probe showed little fluorescence activity. In addition, we found that cells in neovascular lesions labelled with endoglin mRNA dependent fluorescence, co-labelled with the macrophage/microglia-associated marker IBA1. Others have shown that cells expressing macrophage/microglia markers associate with retinal neovascular structures in proportion to disease burden. Hence we propose that our probe may be used to image and to estimate the levels of retinal neovascular disease in real-time in living systems.

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