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">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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.

in-vitro-imaging-quantification-drug-targeting-efficiency

Research

JoVE Journal

Cancer Research

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce decellularized tongue extracellular matrix (TEM) which provides functional scaffolds for TSCC construction. TEM provides an in vitro niche for cell growth, differentiation, and cell migration. The microstructures of native extracellular matrix (ECM) and biochemical compositions retained in the decellularized matrix provide tissue-specific niches for anchoring cells. The fabrication of TEM can be realized by deoxyribonuclease (DNase) digestion accompanied with a serious of organic or inorganic pretreatment. This protocol is easy to operate and ensures high efficiency for the decellularization. The TEM showed favorable cytocompatibility for TSCC cells under static or stirred culture conditions, which enables the construction of the TSCC model. A self-made bioreactor was also used for the persistent stirred condition for cell culture. Reconstructed TSCC using TEM showed the characteristics and properties resembling clinical TSCC histopathology, suggesting the potential in TSCC research.

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

A method is shown here for the preparation of the tongue extracellular matrix (TEM) with efficient decellularization. The TEM can be used as functional scaffolds for the reconstruction of a tongue squamous cell carcinoma (TSCC) model under static or stirred culture conditions.

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

Live Cell Imaging of the TGF-	&beta;/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System

Transforming Growth Factor &#946; (TGF-&#946;) signaling regulates many important functions required for cellular homeostasis and is commonly found overexpressed in many diseases, including cancer. TGF-&#946; is strongly implicated in metastasis during late stage cancer progression, activating a subset of migratory and invasive tumor cells. Current methods for signaling pathway analysis focus on endpoint models, which often attempt to measure signaling post-hoc of the biological event and do not reflect the progressive nature of the disease. Here, we demonstrate a novel adenovirus reporter system specific for the TGF-&#946;/Smad3 signaling pathway that can detect transcriptional activation in live cells. Utilizing an Ad-CAGA12-Td-Tom reporter, we can achieve a 100% infection rate of MDA-MB-231 cells within 24 h in vitro. The use of a fluorescent reporter allows for imaging of live single cells in real-time with direct identification of transcriptionally active cells. Stimulation of infected cells with TGF-&#946; displays only a subset of cells that are transcriptionally active and involved in specific biological functions. This approach allows for high specificity and sensitivity at a single cell level to enhance understanding of biological functions related to TGF-&#946; signaling in vitro. Smad3 transcriptional activity can also be reported in vivo in real-time through the application of an Ad-CAGA12-Luc reporter. Ad-CAGA12-Luc can be measured in the same manner as traditional stably transfected luciferase cell lines. Smad3 transcriptional activity of cells implanted in vivo can be analyzed through conventional IVIS imaging and monitored live during tumor progression, providing unique insight into the dynamics of the TGF-&#946; signaling pathway. Our protocol describes an advantageous reporter delivery system allowing for quick high-throughput imaging of live cell signaling pathways both in vitro and in vivo. This method can be expanded to a range of image based assays and presents as a sensitive and reproducible approach for both basic biology and therapeutic development.

Live Cell Imaging of the TGF-	&amp;beta;/Smad3 Signaling Pathway &lt;em&gt;In Vitro&lt;/em&gt; and &lt;em&gt;In Vivo&lt;/em&gt; Using an Adenovirus Reporter System

Here, we present a protocol for live cell imaging of TGF-&#946;/Smad3 signaling activity using an adenovirus reporter system. This system tracks transcriptional activity in real-time and can be applied to both single cells in vitro and in live animalmodels.

Transforming Growth Factor &#946; (TGF-&#946;) signaling regulates many important functions required for cellular homeostasis and is commonly found ...

Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic spread has already occurred. However, ovarian cancer has a unique pattern of metastatic spread, in that tumor implants are initially contained within the peritoneal cavity. This feature could enable, in principle, the complete resection of tumor implants with curative intent. Many of these metastatic lesions are microscopic, making them hard to identify and treat. Neutralizing such micrometastases is believed to be a major goal towards eliminating tumor recurrence and achieving long-term survival. Raman imaging with surface enhanced resonance Raman scattering nanoprobes can be used to delineate microscopic tumors with high sensitivity, due to their bright and bioorthogonal spectral signatures. Here, we describe the synthesis of two &#39;flavors&#39; of such nanoprobes: an antibody-functionalized one that targets the folate receptor &#8212; overexpressed in many ovarian cancers &#8212; and a non-targeted control nanoprobe, with&#160;distinct spectra. The nanoprobes are co-administered intraperitoneally to mouse models of metastatic human ovarian adenocarcinoma. All animal studies were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. The peritoneal cavity of the animals is surgically exposed, washed, and scanned with a Raman microphotospectrometer. Subsequently, the Raman signatures of the two nanoprobes are decoupled using a Classical Least Squares fitting algorithm, and their respective scores divided to provide a ratiometric signal of folate-targeted over untargeted probes. In this way, microscopic metastases are visualized with high specificity. The main benefit of this approach is that the local application into the peritoneal cavity &#8212; which can be done conveniently during the surgical procedure &#8212; can tag tumors without subjecting the patient to systemic nanoparticle exposure. False positive signals stemming from non-specific binding of the nanoprobes onto visceral surfaces can be eliminated by following a ratiometric approach where targeted and non-targeted nanoprobes with distinct Raman signatures are applied as a mixture. The procedure is currently still limited by the lack of a commercial wide-field Raman imaging camera system, which once available will allow for the application of this technique in the operating theater.

Ovarian cancer forms metastases throughout the peritoneal cavity. Here, we present a protocol to make and use folate-receptor targeted surface-enhanced resonance Raman scattering nanoprobes that reveal these lesions with high specificity via ratiometric imaging. The nanoprobes are administered intraperitoneally to living mice, and the derived images correlate well with histology.

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic ...

A Data Integration Workflow to Identify Drug Combinations Targeting Synthetic Lethal Interactions

A synthetic lethal interaction between two genes is given when knock-out of either one of the two genes does not affect cell viability but knock-out of both synthetic lethal interactors leads to loss of cell viability or cell death. The best studied synthetic lethal interaction is between BRCA1/2 and PARP1, with PARP1 inhibitors being used in clinical practice to treat patients with BRCA1/2 mutated tumors. Large genetic screens in model organisms but also in haploid human cell lines have led to the identification of numerous additional synthetic lethal interaction pairs, all being potential targets of interest in the development of novel tumor therapies. One approach is to therapeutically target genes with a synthetic lethal interactor that is mutated or significantly downregulated in the tumor of interest. A second approach is to formulate drug combinations addressing synthetic lethal interactions. In this article, we outline a data integration workflow to evaluate and identify drug combinations targeting synthetic lethal interactions. We make use of available datasets on synthetic lethal interaction pairs, homology mapping resources, drug-target links from dedicated databases, as well as information on drugs being investigated in clinical trials in the disease area of interest. We further highlight key findings of two recent studies of our group on drug combination assessment in the context of ovarian and breast cancer.

Large genetic screens in model organisms have led to the identification of negative genetic interactions. Here, we describe a data integration workflow using data from genetic screens in model organisms to delineate drug combinations targeting synthetic lethal interactions in cancer.

A synthetic lethal interaction between two genes is given when knock-out of either one of the two genes does not affect cell viability but knock-out of ...

In Vivo Targeting of Xenografted Human Cancer Cells with Functionalized Fluorescent Silica Nanoparticles in Zebrafish

Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal models are required for bridging the nanotechnology to its biomedical application. The mouse represents the traditional animal model for preclinical testing; however, mice are relatively expensive to keep and have long experimental cycles due to the limited progeny from each mother. The zebrafish has emerged as a powerful model system for developmental and biomedical research, including cancer research. In particular, due to its optical transparency and rapid development, zebrafish embryos are well suited for real-time in vivo monitoring of the behavior of cancer cells and their interactions with their microenvironment. This method was developed to sequentially introduce human cancer cells and functionalized nanoparticles in transparent Casper zebrafish embryos and monitor in vivo recognition and targeting of the cancer cells by nanoparticles in real time. This optimized protocol shows that fluorescently labeled nanoparticles, which are functionalized with folate groups, can specifically recognize and target metastatic human cervical epithelial cancer cells labeled with a different fluorochrome. The recognition and targeting process can occur as early as 30 min postinjection of the nanoparticles tested. The whole experiment only requires the breeding of a few pairs of adult fish and takes less than 4 days to complete. Moreover, zebrafish embryos lack a functional adaptive immune system, allowing the engraftment of a wide range of human cancer cells. Hence, the utility of the protocol described here enables the testing of nanoparticles on various types of human cancer cells, facilitating the selection of optimal nanoparticles in each specific cancer context for future testing in mammals and the clinic.

Described here is a method for utilizing zebrafish embryos to study the ability of functionalized nanoparticles to target human cancer cells in vivo. This method allows for the evaluation and selection of optimal nanoparticles for future testing in large animals and in clinical trials.

Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal ...

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach in&#160;vaccine design. Antigen is delivered to DC via antibodies specific for endocytosis receptors such as DEC-205 that induce uptake, processing, and MHC class I- and II-presentation.
Efficient and reliable conjugation of the desired antigen to a suitable antibody is a critical step in DC targeting and among other factors depends on the format of the antigen. Chemical conjugation of&#160;full-length protein to purified antibodies is one possible strategy. In the past, we have successfully established cross-linking of the model antigen ovalbumin (OVA) and a DEC-205-specific IgG2a antibody (&#945;DEC-205)&#160;for in vivo DC targeting studies in mice. The first step of the protocol is the purification of the antibody from the supernatant of the NLDC (non-lymphoid dendritic cells)-145 hybridoma by affinity chromatography. The purified antibody is activated for chemical conjugation by sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) while at the same time the sulfhydryl-groups of the OVA protein are exposed through incubation with TCEP-HCl (tris (2-carboxyethyl) phosphine hydrochloride). Excess TCEP-HCl and sulfo-SMCC are removed and the antigen is mixed with the activated antibody for overnight coupling. The resulting &#945;DEC-205/OVA conjugate is concentrated and freed from unbound OVA. Successful conjugation of OVA to &#945;DEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA).
We have successfully used chemically crosslinked &#945;DEC-205/OVA to induce cytotoxic T cell responses in the liver and to compare different adjuvants for their potential in inducing humoral and cellular immunity following in vivo targeting of DEC-205+ DC. Beyond that, such chemically coupled antibody/antigen conjugates offer valuable tools for the efficient induction of vaccine responses to tumor antigens and have been proven to be superior to classical immunization approaches regarding the prevention and therapy of various types of tumors.

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

We describe a protocol for the chemical conjugation of the model antigen ovalbumin to an endocytosis receptor-specific antibody for in vivo dendritic cell targeting. The protocol includes purification of the antibody, chemical conjugation of the antigen, as well as purification of the conjugate and the verification of efficient conjugation.

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...

Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural microenvironment and stromal cell interaction, cannot be entirely replicated with in vitro assays; thus, animal models are critical for investigating and understanding the effects of therapeutic intervention. However, most brain tumor xenografting methods do not produce brain metastases consistently in terms of the time frame and tumor burden. Brain metastasis models generated by intracardiac injection of cancer cells can result in unintended extracranial tumor burden and lead to non-brain metastatic morbidity and mortality. Although intracranial injection of cancer cells can limit extracranial tumor formation, it has several caveats, such as the injected cells frequently form a singular tumor mass at the injection site, high leptomeningeal involvement, and damage to brain vasculature during needle penetration. This protocol describes a mouse model of brain metastasis generated by internal carotid artery injection. This method produces intracranial tumors consistently without the involvement of other organs, enabling the evaluation of therapeutic agents for brain metastasis.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Most brain metastasis mouse models are complicated by systemic metastases confounding analysis of mortality and therapeutic intervention outcomes. Presented here is a protocol for internal carotid injection of cancer cells that produces consistent intracranial tumors with minimal systemic tumors.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

This protocol describes a semi-automated method for medium- to high-throughput organoid drug screenings and microscope-agnostic, automated image analysis software to quantify and visualize multiparametric, single-organoid drug responses to capture intratumor heterogeneity.

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...