Name: in vitro imaging and quantification of the drug targeting efficiency of fluorescently labeled gnrh analogues
Uploaded: 2017-03-21T15:00:00
Description: Watch this Scientific Journal Video about In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues at JoVE.com

The project was supported by National Research Fund OTKA (K 104045).

1. Preparation of Cell Cultures and Reagents
<ol>
	<li>Maintain the cell cultures in the manufacturer&#39;s recommended medium, supplemented with 10% (v/v) fetal bovine serum and antibiotics (called complete medium). Keep the cell culturing flask in a humidified, 5% CO2 atmosphere incubator at 37 &#176;C. Follow the proliferation and confluency of cells by inverted microscope (using 10X phase contrast objective).</li>
	<li>When cells reach adequate confluency, remove the medium, and wash the culture with 2-3...

<ol>
	<li>Gilad, Y., Firer, M., Gellerman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Innovations+in+Peptide+Based+Targeted+Drug+Delivery+to+Cancer+Cells.">Recent Innovations in Peptide Based Targeted Drug Delivery to Cancer Cells.</a> Biomedicines. 4 (2), 11 (2016).</li><li>Majumdar, S., Siahaan, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide-mediated+targeted+drug+delivery.">Peptide-mediated targeted drug delivery.</a> Med Res Rev. 32 (3), 637-658 (2012).</li><li>Ramakrishnappa, N., Rajamahendran, R., Lin, Y. M., Leung, P. C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GnRH+in+non-hypothalamic+reproductive+tissues.">GnRH in non-hypothalamic reproductive tissues.</a> Anim Reprod Sci. 88 (1-2), 95-113 (2005).</li><li>Schally, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothalamic+hormones+and+cancer.">Hypothalamic hormones and cancer.</a> Front Neuroendocrinol. 22 (4), 248-291 (2001).</li><li>Limonta, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GnRH+receptors+in+cancer:+From+cell+biology+to+novel+targeted+therapeutic+strategies.">GnRH receptors in cancer: From cell biology to novel targeted therapeutic strategies.</a> Endocr Rev. 33 (5), 784-811 (2012).</li><li>Ma, Y. T., Zhou, N., Liu, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+drug+delivery+system+based+on+gonadotropin-releasing+hormone+analogs:+Research+advances.">Targeting drug delivery system based on gonadotropin-releasing hormone analogs: Research advances.</a> J of Int Pharm Res. 41 (2), 140-148 (2014).</li><li>Manea, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-vivo+antitumour+effect+of+daunorubicin-GnRH-III+derivative+conjugates+on+colon+carcinoma-bearing+mice.">In-vivo antitumour effect of daunorubicin-GnRH-III derivative conjugates on colon carcinoma-bearing mice.</a> Anti-Cancer Drugs. 23 (1), 90-97 (2012).</li><li>Argyros, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide-Drug+conjugate+gnrh-sunitinib+targets+angiogenesis+selectively+at+the+site+of+action+to+inhibit+tumor+growth.">Peptide-Drug conjugate gnrh-sunitinib targets angiogenesis selectively at the site of action to inhibit tumor growth.</a> Cancer Res. 76 (5), 1181-1192 (2016).</li><li>Karampelas, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GnRH-Gemcitabine+conjugates+for+the+treatment+of+androgen-independent+prostate+cancer:+pharmacokinetic+enhancements+combined+with+targeted+drug+delivery.">GnRH-Gemcitabine conjugates for the treatment of androgen-independent prostate cancer: pharmacokinetic enhancements combined with targeted drug delivery.</a> Bioconjug Chem. 25 (4), 813-823 (2014).</li><li>Millar, R. P., Pawson, A. J., Morgan, K., Rissman, E. F., Lu, Z. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+actions+of+GnRHs+mediated+by+ligand-induced+selective+signaling.">Diversity of actions of GnRHs mediated by ligand-induced selective signaling.</a> Front Neuroendocrinol. 29 (1), 17-35 (2008).</li><li>Millar, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gonadotropin-releasing+hormone+receptors.">Gonadotropin-releasing hormone receptors.</a> Endocr Rev. 25 (2), 235-275 (2004).</li><li>Olberg, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+in+vitro+evaluation+of+small-molecule+[18F]+labeled+gonadotropin-releasing+hormone+(GnRH)+receptor+antagonists+as+potential+PET+imaging+agents+for+GnRH+receptor+expression.">Synthesis and in vitro evaluation of small-molecule [18F] labeled gonadotropin-releasing hormone (GnRH) receptor antagonists as potential PET imaging agents for GnRH receptor expression.</a> Bioorg Med Chem Lett. 24 (7), 1846-1850 (2014).</li><li>Zoghi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Ga-68+labeled+triptorelin+analog+for+GnRH+receptor+imaging.">Development of a Ga-68 labeled triptorelin analog for GnRH receptor imaging.</a> Radiochimica Acta. 104 (4), 247-255 (2016).</li><li>Guo, H., Gallazzi, F., Sklar, L. A., Miao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+indium-111-labeled+gonadotropin-releasing+hormone+peptide+for+human+prostate+cancer+imaging.">A novel indium-111-labeled gonadotropin-releasing hormone peptide for human prostate cancer imaging.</a> Bioorg Med Chem Lett. 21 (18), 5184-5187 (2011).</li><li>Nederpelt, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+12+GnRH+peptide+agonists+-+A+kinetic+perspective.">Characterization of 12 GnRH peptide agonists - A kinetic perspective.</a> Br J Pharmacol. 173 (1), 128-141 (2016).</li><li>Szabo, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+in+vitro+biological+evaluation+of+daunorubicin+containing+GnRH-I+and+GnRH-II+conjugates+developed+for+tumor+targeting.">Comparative in vitro biological evaluation of daunorubicin containing GnRH-I and GnRH-II conjugates developed for tumor targeting.</a> J Pept Sci. 21 (5), 426-435 (2015).</li><li>Leurs, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GnRH-III+based+multifunctional+drug+delivery+systems+containing+daunorubicin+and+methotrexate.">GnRH-III based multifunctional drug delivery systems containing daunorubicin and methotrexate.</a> Eur J Med Chem. 52, 173-183 (2012).</li><li>Mur&#225;nyi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+characterization+and+systematic+comparison+of+FITC-labelled+GnRH-I,+-II+and+-III+analogues+on+various+tumour+cells.">Synthesis, characterization and systematic comparison of FITC-labelled GnRH-I, -II and -III analogues on various tumour cells.</a> J Pept Sci. 22 (8), 552-560 (2016).</li></ol>

Experiments described herein use selectively labeled GnRH analogues for screening adherent cell cultures in vitro. Confocal microscopy and flow cytometry methods are suitable for tracking and quantifying the cellular uptake of these GnRH-FITC conjugates in a time and concentration dependent manner. These experiments have the following critical steps: 1) maintain a sterile, healthy cell culture; 2) GnRH peptides must be of high quality; 3) reasonable concentrations and incubation times must be followed; ...

Peptide based targeted drug delivery systems have become a fast developing and promising area in cancer therapy over the past few years1,2. Human gonadotropin releasing hormone receptor type I (GnRH-I-R) is primarily located in the pituitary gland but is also present in several other tissues which are responsible for self-reproduction3. GnRH-I-R is also expressed in a number of cancer tissues, related or unrelated to the reproductive system4,5. The high expression of GnRH-I-R on several malignant tumor cells compared with healthy tissues provides an opportunity for targeted therapy5,6.
Many gonadotropin-releasing hormone (GnRH) analogues have been developed in the last few decades, which could be used as targeting moieties7,8,9. These peptides are able to deliver anticancer agents with high selectivity into malignant tumor cells which over-express GnRH-I-R6. Several GnRH conjugated anti-tumor drugs with higher selectivity and better efficiency than the corresponding unconjugated free drug have been reported7,8,9.
Previous publications about GnRH peptides and their receptors reported that the GnRH-I-R can assume various conformations which have different selectivity for GnRH analogues10. The highly variable GnRH-I-R has complex and various signaling pathways are endowed with different activity against their natural and artificial ligands11. These facts make investigation of GnRH-based systems challenging. On the other hand, they possess promising therapeutic potential. Several experiments with radiolabeled GnRH peptides were previously reported12,13,14,15, but experiments in which fluorescently labeled GnRH analogues were used are still limited. While radioactive labeling offers high sensitivity, fluorescent labeling has several other advantages, for example the easier handling, and the ability to counterstain with different fluorophores. Three common GnRH analogues which have successfully been used for drug delivery are the [D-Lys6]-GnRH-I, [D-Lys6]-GnRH-II and GnRH-III, but the effectiveness of these peptides as targeting moieties is rarely compared16,17. On the other hand, results from separate experiments in which different cancer cells and GnRH analogues were used is diverse.
Based on these considerations, we focused on the tumor targeting and drug delivery potential of these GnRH peptides, and thereby synthesized and characterized the [D-Lys6(FITC)]-GnRH-I, [D-Lys6(FITC)]-GnRH-II and [Lys8(FITC)]-GnRH-III peptide conjugates18. These analogues are selectively labeled with FITC on the side chain of their Lys or D-Lys (peptide-FITC ratio 1:1 at each conjugate). The idea was that the selective fluorescent labeling can offer novel information about these peptides, and allows their good tracking and reliable quantification. These conjugates have safe handling and reliable detectability, which make it easier to compare their tumor targeting efficiency, and the screening of numerous types of malignant tumor cells. We hope that up-to date experiments with these peptide conjugates could contribute to the development of novel cancer targeting GnRH-drug conjugates, and help to identify new therapeutic targets as well.
The present manuscript demonstrates some well reproducible and fast experiments with GnRH-FITC conjugates. The cell surface expression of GnRH-R is a determinative condition regarding GnRH uptake, therefore we simultaneously investigated the cell surface level of GnRH-I-R on the tested cell lines. We visualized the GnRH-I-R and GnRH-FITC conjugates by confocal laser scanning microscopy (CLSM) and quantified the cellular uptake of GnRH-FITC conjugates using fluorescence-activated cell sorting (FACS).

<table border="0" cellpadding="0" cellspacing="0" width="496">
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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">EBC-1</td>
			<td style="width:79px;">JCRB Cell Bank</td>
			<td style="width:79px;">JCRB0820</td>
			<td style="width:191px;">Human lung squamous cell carcinoma</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">BxPC-3</td>
			<td style="width:79px;">ATCC</td>
			<td style="width:79px;">CRL-1687</td>
			<td style="width:191px;">Human pancreatic adenocarcinoma</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:149px;">Detroit-562</td>
			<td style="width:79px;">ATCC</td>
			<td style="width:79px;">CCL-138</td>
			<td style="width:191px;">Human pharyngeal carcinoma</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">RPMI-1640 Medium with L-glutamine</td>
			<td style="width:79px;">Lonza</td>
			<td style="width:79px;">BE12-702F</td>
			<td style="width:191px;">Culture medium for BxPC-3 cells</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:149px;">Eagle&#39;s Minimum Essential Medium</td>
			<td style="width:79px;">Lonza</td>
			<td style="width:79px;">&#160;BE12-611F</td>
			<td style="width:191px;">Culture medium for EBC-1 cells, and complemented with 0,1% sodium pyruvate for Detroit-562 cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">Fetal Bovine Serum</td>
			<td style="width:79px;">Euro Clone</td>
			<td style="width:79px;">ECS0180L</td>
			<td style="width:191px;">Complements the culture medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">MycoZap Plus-CL&#160;</td>
			<td style="width:79px;">Lonza</td>
			<td style="width:79px;">VZA-2012</td>
			<td style="width:191px;">Complements the culture medium (antibiotics)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">Standard Line Cell Culture Flasks</td>
			<td style="width:79px;">VWR</td>
			<td style="width:79px;">10062-872</td>
			<td style="width:191px;">Provide consistent, sterile growth environment for cells</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:149px;">Trypsin-EDTA Mixture&#160;</td>
			<td style="width:79px;">Lonza</td>
			<td style="width:79px;">BE17-161E</td>
			<td style="width:191px;">Remove attached cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:79px;">Lonza</td>
			<td style="width:79px;">BE17-516F</td>
			<td style="width:191px;">Solvent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">Dimethyl sulfoxide</td>
			<td style="width:79px;">Sigma-Aldrich</td>
			<td style="width:79px;">D8418-100ML</td>
			<td style="width:191px;">Solvent</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:149px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:79px;">Thermo Fisher Scientific</td>
			<td style="width:79px;">15250061</td>
			<td style="width:191px;">Used to counting cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">[D-Lys6(FITC)]-GnRH-I</td>
			<td style="width:79px;">made by us</td>
			<td style="width:79px;">-</td>
			<td style="width:191px;">Purity &#8805;98% (HPLC)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">[D-Lys6(FITC)]-GnRH-II&#160;</td>
			<td style="width:79px;">made by us</td>
			<td style="width:79px;">-</td>
			<td style="width:191px;">Purity &#8805;98% (HPLC)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">[Lys8(FITC)]-GnRH-III</td>
			<td style="width:79px;">made by us</td>
			<td style="width:79px;">-</td>
			<td style="width:191px;">Purity &#8805;98% (HPLC)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">glass bottom 8-well microscopic slide</td>
			<td style="width:79px;">Ibidi</td>
			<td style="width:79px;">80826</td>
			<td style="width:191px;">Use in CLSM experiment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">Corning Costar cell culture plate, 12 well</td>
			<td style="width:79px;">Sigma-Aldrich</td>
			<td style="width:79px;">CLS3513-50EA</td>
			<td style="width:191px;">Use in FACS experiment</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:149px;">Fixing solution (10% neutral buffered formalin)</td>
			<td style="width:79px;">Bio-Optica</td>
			<td style="width:79px;">01V60P</td>
			<td style="width:191px;">Fix adherent cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">Bovine Serum Albumin</td>
			<td style="width:79px;">Sigma-Aldrich</td>
			<td style="width:79px;">A7906-10G</td>
			<td style="width:191px;">Prevent the nonspecific binding of the antibodies</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:149px;">GnRHR Antibody</td>
			<td style="width:79px;">Proteintech</td>
			<td style="width:79px;">19950-1-AP</td>
			<td style="width:191px;">Immunocytochemistry reagent, bind to the human type-I GnRH receptors</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:149px;">Secondary Antibody, Alexa Fluor 546 conjugate</td>
			<td style="width:79px;">Thermo Fisher Scientific</td>
			<td style="width:79px;">A11035</td>
			<td style="width:191px;">Immunocytochemistry reagent, bind to the primary antibody</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:149px;">Fluorescent probe solution (Draq5)</td>
			<td style="width:79px;">Thermo Fisher Scientific</td>
			<td style="width:79px;">62254</td>
			<td style="width:191px;">Counterstain nuclei</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">Fluoromount Aqueous Mounting Medium</td>
			<td style="width:79px;">Sigma-Aldrich</td>
			<td style="width:79px;">F4680-25ML</td>
			<td style="width:191px;">Use in CLSM experiment, preserving fluorescence</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">Inverted microscope</td>
			<td style="width:79px;">Elektro-Optika Ltd.&#160;</td>
			<td style="width:79px;">Alpha XDS-2T</td>
			<td style="width:191px;">Check the phenotype and confluency of cell cultures</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">Inverted confocal laser scanning microscope</td>
			<td style="width:79px;">Zeiss</td>
			<td style="width:79px;">LSM-710</td>
			<td style="width:191px;">Use in CLSM experiment</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:149px;">Flow cytometer</td>
			<td style="width:79px;">BD Biosciences</td>
			<td style="width:79px;">BD FACSCalibur</td>
			<td style="width:191px;">Use in FACS experiment</td>
		</tr>
	</tbody>
</table>

in vitro imaging and quantification of the drug targeting efficiency of fluorescently labeled gnrh analogues

GnRH analogues are effective targeting moieties and able to deliver anticancer agents selectively into malignant tumor cells which highly express GnRH receptors. However, the quantitative analysis of GnRH analogues&#39; cellular uptake and the investigated cell types in GnRH-based drug delivery systems are currently limited. Previously introduced, selectively labeled fluorescent GnRH I, -II and -III derivatives provide great detectability, and they have suitable chemical properties for reproducible and robust experiments. We also found that the appropriate up-to-date methods with these labeled GnRH analogues could offer novel information about the GnRH-based drug delivery systems. This manuscript introduces some simple and fast experiments regarding the cellular uptake of [D-Lys6(FITC)]-GnRH-I, [D-Lys6(FITC)]-GnRH-II and [Lys8(FITC)]-GnRH-III on the EBC-1 (lung), the BxPC-3 (pancreas) and on the Detroit-562- (pharynx) malignant tumor cells. In parallel with these GnRH-FITC conjugates, the cell surface level of GnRH-I receptors was also examined on these cell lines before and after the GnRH treatment by confocal laser scanning microscopy. The cellular uptake of GnRH-FITC conjugates was quantified by fluorescence-activated cell sorting. In these experiments minor differences among GnRH analogues and major differences among cell types was observed. The significant differences among cell lines are correlated with their distinct level of cell surface GnRH-I receptors. The introduced experiments contain practical methods to visualize, quantify and compare the uptake efficiency of GnRH-FITC conjugates in a time- and concentration-dependent manner on various adherent cell cultures. These results could predict the drug targeting efficiency of GnRH conjugates on the given cell culture, and offer a good basis for further experiments in the examination of GnRH-based drug delivery systems.

Images obtained by confocal laser scanning microscopy (CLSM) offer spectacular information about the uptake of GnRH-FITC conjugates on the given cell culture in a time and concentration dependent manner. In parallel with these GnRH-FITC conjugates, the presence of the GnRH-I-R on the cell surface is also verifiable by the CLSM experiment. Furthermore, by using a far-red DNA staining fluorescent probe, it is possible to counterstain the nuclei of cells besides GnRH and GnRH-I-R. However, w...

Watch this Scientific Journal Video about In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues at JoVE.com

In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

Selectively labeled fluorescent GnRH-I, -II and -III derivatives are reliable tools for tracking and quantifying their cellular uptake. This manuscript introduces experiments to visualize, quantify and compare the uptake efficiency of these GnRH conjugates in various cell lines.

In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

GnRH analogues are effective targeting moieties and able to deliver anticancer agents selectively into malignant tumor cells which highly express GnRH ...

The overall goal of this procedure is to investigate and predict the efficacy of GnRH-based drug delivery systems in human cancer cell cultures. This method can help answer key questions in the targeted anti-cancer therapy field, such as how efficiently cells of different human cancer types take of GnRH targeted anti-cancer drugs. The main advantage of this method is that it uses new fluorescently labeled GnRH analogues that allow simultaneous use of confocal microscopy and flow cytometry analysis.The implications of this technique extends toward anti-cancer therapy because GnRH receptors are highly expressed on several cancer cell types, enabling the selective targeting of them. Demonstrating the procedure will be Bianka Gurbi, a Ph.D.student from my laboratory. To begin, remove the cultured medium from each of the seven wells of a seeded microscopic slide.Then, add 250 microliters of the preheated complete medium to the negative control well and 250 microliters of the preheated GnRH fit seed treating media to each of the remaining six wells. Incubate the slide in an incubator at 37 degrees Celsius, five percent carbon dioxide for five hours. After five hours of incubation, aspirate the medium from each well and wash the cells with 250 microliters of PBS.Subsequently, add 250 microliters of fixing solution into each well and incubate the slide at room temperature for 10 minutes. Once the cells are fixed, discard the fixing solution and wash the cells with 250 microliters of PBS. Add 250 microliters of five micromolar fluorescent probe solution prepared in PBS into each well and incubate the slide at room temperature for 10 minutes.After removing the fluorescent probe solution, carefully wash the cells twice with 250 microliters of PBS. Finally, add three to four drops of mounting medium into each well. Keep the slide in dark at room temperature until imaged under a microscope.Remove the cultured medium from each of the five wells of a seeded microscopic slide. Next, add 250 microliters of the preheated complete medium to the negative control well and to the second well with the untreated cells. Pretreat the cells in the three remaining wells with the preheated treating media containing 10 micromolar GnRH FITC.Incubate the slide in an incubator at 37 degrees Celsius, five percent carbon dioxide for one hour. Afterward remove the media only from the treated cells and wash the cells with 250 microliters of the preheated complete medium. Next, remove the wash medium.Then, add 250 microliters of the preheated complete medium into each of the three wells. Incubate the slide in an incubator at 37 degrees Celsius, five percent carbon dioxide for one hour. After incubation, discard the media from all five wells and wash the cells with 250 microliters of PBS.Then, remove the PBS and fix the cells with 250 microliters of fixing solution at room temperature for 10 minutes. Next, discard the fixing solution and wash the cells with 250 microliters of PBS. Add 250 microliters of blocking solution into each well and incubate the slide at room temperature for one hour.Aspirate blocking solution from each well except the negative control. After washing the cells with 250 microliters of PBS, add 250 microliters of primary antibody solution to each well. Incubate the slide at room temperature for one hour in a dark place.Remove solutions from each of the five wells and wash the cells with 250 microliters of PBS. Then, remove the PBS and add 250 microliters of secondary antibody solution. Incubate the slide at room temperature for one hour in the dark.Aspirate the solution from each well and wash the cells with 250 microliters of PBS. Remove the wash PBS and then add 250 microliters of PBS containing five micromolar fluorescent probe into each well. Incubate the slide for 10 minutes at room temperature.Remove the fluorescent probe solution from each well and carefully wash the cells twice with 250 microliters of PBS. Finally, add three to four drops of mounting medium into each well. Keep the slide in the dark at room temperature until imaged under a microscope.Image the cells under an inverted confocal laser scanning microscope. Turn on the 488 nanometer and the 633 nanometer lasers. Check the laser amplification and the pinhole size.Image the negative control sample. Optimize the signal observed for the nucleus and adjust the signal of FITC and Alexa 546 so that it is close to zero. Then, using the same parameters, image the treated samples.Discard the complete medium from all seven wells of a seeded culture plate. Then, add one milliliter of preheated complete medium into the negative control well and one milliliter of the preheated treating media to the remaining six wells. Incubate the plate in an incubator at 37 degrees Celsius, five percent carbon dioxide for five hours.After discarding the medium from all wells, carefully wash the cells twice with two milliliters of PBS. Remove the PBS and then add 500 microliters of Trypsin EDTA solution into each well. Incubate the plate at 37 degrees Celsius until cells detach, which will take approximately 10 minutes.Using one milliliter of complete medium, neutralize the Trypsin in each well and shake the plate. Afterward suspend the cells by gentle pipetting. Transfer the suspensions into separate FACS tubes and centrifuge the samples at 150 times G at four degrees Celsius for four minutes.Decant the supernatants carefully. Then, add 500 microliters of ice-cold PBS into each FACS tube and gently resuspend the cells. Keep the tubes on ice until analyzed by FACS.Presented here is the analysis of GnRH uptake in pharynx carcinoma cells. Five hour treatment with one micromolar of fluorescent complex of GnRH resulted in slight, but distinguishable intake manifested as green fluorescent signal localized in pharynx cancer cells. Additional increase of complex dosage led to enhanced uptake, demonstrating dose dependency of GnRH uptake in pharynx carcinoma cells.Receptor dependent uptake of GnRH and the cellular distribution of the ligand in its receptor in lung cancer cells were analyzed by simultaneous treatment with fluorescent GnRH complex and staining of the type one GnRH receptor and nuclei. The results show that the type one GnRH receptor is associated with the cell membrane while the GnRH protein is internalized in cells and remains in their cytoplasm. The GnRH uptake in pharynx carcinoma cells was also studied using FACS analysis.The results show that the uptake of GnRH observed when cells were treated with one micromolar of GnRH conjugate was strongly enhanced when the dose applied to the cells was increased to 10 micromolar. Once mastered, this technique can be finished in eight hours if it is performed properly. While attempting this procedure, it's important to remember to protect the treated samples and fluorescent dyes from direct light.Following this procedure, other methods like cell viability or apoptosis assays can be performed in order to answer additional questions like how efficiently the GnRH targeted drug can inhibit the growth of a given type of cancer. After this development, this technique paved the way for researchers in the field of cancer therapy to explore the efficacy of GnRH-based drug delivery systems in adhering cancer cells. After watching this video, you should have a good understanding of how to use these fluorescently labeled GnRH conjugates to explore the susceptibility of cancer cells for GnRH targeted therapies.

Research

JoVE Journal

Cancer Research

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