We would like to thank Northumbria University for their support.

1. Stock solution preparation
<ol>
	<li>When preparing Calcein stocks solutions, prevent photo degradation by keeping solutions in the dark. Prepare a 1 mM solution of Calcein in Dulbecco&#39;s PBS, without magnesium or calcium14. For 5 mL of 1 mM Calcein solution, add 3.1 mg of Calcein (622.5 g/M) to 5 mL of Dulbecco&#39;s PBS. Using the 1 mM sock of Calcein, prepare 1 mL aliquots of secondary stock solutions in microfuge tube as detailed in Table 1.</li>
	<li>Prepare ferric ammonium sulphate hexahydrate stocks by dissolving 19 mg of FAS hexahydrate (392.1 g/M) in 5 mL of Dulbecco&#39;s PBS to obtain a 10 mM ferric ammonium sulphate (FAS) solution. Prepare secondary stocks of FAS by dilution. To prepare a stock of 2 mM FAS (fA), dilute 200 &#181;L of the 10 mM FAS stock with PBS with 800 &#181;L of Dulbecco&#39;s PBS. To prepare a stock of 100 &#181;M FAS (fB) dilute 50 &#181;L of stock fA with 950 &#181;L of Dulbecco&#39;s PBS.</li>
	<li>To prepare 10 mM of Deferiprone stock, dissolve 2.8 mg (139.15 g/M) in 2 mL of Dulbecco&#39;s PBS. Prepare secondary stocks of 2mM iron chelator by diluting 200 &#181;L of the 10 mM stock with 800 &#181;L of Dulbecco&#39;s PBS. 
	NOTE: In this assay the iron chelator of choice should be dissolved in Dulbecco&#39;s PBS without magnesium or calcium. As a working exemplar, we used the iron chelator Deferiprone.</li>
</ol>
2. Assay validation by determination of the linear range of Calcein fluorescence
<ol>
	<li>Prepare a range of Calcein stocks 1 mM to 1 &#181;M as described in stock solution table (see Table 1). Keep stock solutions in the dark to prevent photochemical degradation.</li>
	<li>Pipette 90 &#181;L of phosphate buffered saline (PBS) to wells A to H and 1 to 12 of a 96 well plate using a single channel manual pipette or a multichannel manual pipette.</li>
	<li>Add 10 &#181;L of 1 mM Calcein stock into column 12, rows A to H, of the 96 well plate using a single channel manual pipette.</li>
	<li>Add 10 &#181;L of the pre-prepared 800 &#181;M Calcein stock (Table 1) to a 96 well plate in column 11, rows A to H, using a single channel manual pipette.</li>
	<li>Repeat the addition procedure with the stocks from Table 1 so that column 10 contains 10 &#181;L of the 400 &#181;M stock and, column 9 contains 10 &#181;L of the 200 &#181;M stock etc. Continue this stepwise procedure across the columns until column 2 contains 10 &#181;L of the 1 &#181;M stock. 
	NOTE: The final concentration of Calcein in each well is 10-fold lower than the stock solutions due to the 1:10 dilution.</li>
	<li>Add 10 &#181;L of PBS to Column 1, row A to E.</li>
	<li>Incubate at room temperature for 30 min and set the analyzer on a microplate reader to an excitation of 490 nm and detection of 530 nm (or suitable filter set for Calcien fluorescence).</li>
</ol>
3. Assay validation by Fe2+ ion quenching to reduce Calcein fluorescence
<ol>
	<li>Prepare a Calcein (cB) stock of 10 &#181;M and a FAS stock (fA) of 2 mM (see Table of Materials). Keep the Calcein stock in the dark to prevent photodegradation.</li>
	<li>Using a single channel manual pipette or a multichannel manual pipette, add 50 &#181;L of PBS to columns 2 to 10, rows A to E. Do not add PBS to column 11.</li>
	<li>Add 100 &#181;L of a FAS (fA) stock of 2 mM into a 96 well plate in column 11 (Rows A to E) using a single channel manual pipette.</li>
	<li>Using a multichannel manual pipette remove 50 &#181;L of fA from column 11 and pipette it into the PBS already in column 10, mix thoroughly by pipetting.</li>
	<li>Continue this stepwise double dilution procedure across the columns 9-2, rows A to E of the 96 well plate, and mix by pipetting.</li>
	<li>Remove 50 &#181;L of solution by pipetting from column 2 and discard the solution to waste.</li>
	<li>Add 40 &#181;L of PBS into all wells with a solution in them, columns 2-12, rows A to E.</li>
	<li>Add 90 &#181;L of PBS to wells column 1, rows A-E. These wells act as a positive control of 1 &#181;M Calcein and 0 &#181;M FAS.</li>
	<li>Add 10 &#181;L of 10 &#181;M Calcein stock (cB) into each well with a solution in it (columns 1 to 12, rows A to E). This results in a top concentration of 1000 &#181;M FAS in column 11, with FAS concentration halving for columns 10, 9, 8 across to column 2. Each of the resultant wells from the columns contains 1 &#181;M Calcein and a dilution of FAS.</li>
	<li>Add 100 &#181;L of PBS to row F of columns 1 to 5 for the PBS alone control. Add 100 &#181;L of 2 mM of FAS to row F of columns 6 to 10 for FAS alone control. Incubate at room temperature for 10 min in the dark.</li>
	<li>Analyze on a microplate reader set to excitation of 490 nm and detection of 530 nm (or suitable filter set for Calcein fluorescence).</li>
</ol>
4. Assay for quantifying the ability of iron chelators to outcompete the weak chelator Calcein for Fe2+ ions
<ol>
	<li>Prepare a Calcein stock (cB) of 10 &#181;M, FAS stock (fB) of 100 &#181;M and the iron chelator stocks of 2 mM as outlined in Table 1. Keep Calcein stock in the dark to prevent photodegradation.</li>
	<li>Using a single channel manual pipette or a multichannel manual pipette, add 50 &#181;L of PBS to columns 2 to 10, rows A to E, of a 96 well plate. Do not add PBS to column 11.</li>
	<li>Add 100 &#181;L of the 2 mM stock (fA) of the iron chelators, into a 96 well plate in column 11, rows A to E using a single channel manual pipette.</li>
	<li>Using a multichannel manual pipette remove 50 &#181;L of fA from column 11 and pipette it into the PBS in column 10, rows A to E, mixing thoroughly by pipetting.</li>
	<li>Continue this stepwise double dilution procedure across the remaining columns 9-2, rows A to E of the plate, mix each well by pipetting. Remove 50 &#181;L from column 2 and discard. This ensures a double dilution of 2 mM of the iron chelator (Deferiprone) across the 96 well plate columns 11 to 2, rows A to E.</li>
	<li>Pipette 30 &#181;L of PBS into all wells with a solution in them (e.g., columns 2-11 rows A to E).</li>
	<li>Using a multi-channel pipette, add 10 &#181;L of 100 &#181;M FAS stock (fB) into columns 2-12, rows A to E. This results in a concentration of 1 &#181;M Calcein and 10 &#181;M FAS in each well, and a double dilution of the chelators, with the concentration halving each column across the plate, columns 11-2, with the highest concentration of iron chelator at 1000 &#181;M in column 11 wells A to E.</li>
	<li>Using a multi-channel pipette add 10 &#181;L of 10 &#181;M Calcein stock (cB) into columns 2-12, rows A to E.</li>
	<li>Pipette 80 &#181;L of PBS and 10 &#181;L of Calcein stock (cB) and 10 &#181;L of FAS stock (fB) to column 1, rows A-E using a pipette. Mix samples by pipetting. This provides the control of 1 &#181;M Calcein with 10 &#181;M FAS and no chelator.</li>
	<li>Pipette 100 &#181;L of PBS into row F columns 1 to 5. This provides the PBS negative control.</li>
	<li>Add 100 &#181;L of 2 mM of iron chelator (Deferiprone) to row F, columns 6 to 10. This provides the chelator alone control.</li>
	<li>Incubate at room temperature for 10 min in the dark. Analyze on a multimodal microplate reader set to excitation at 490 nm and detection at 530 nm (or suitable filter set for Calcien fluorescence).</li>
</ol>
5. Data analysis
<ol>
	<li>Analysis for assay validation by determination of the linear range of Calcein fluorescence.
	<ol>
		<li>Calculate the mean Relative Fluorescence Units (RFU) of rows A-H for each concentration of Calcein in the columns of the 96 well plate from step 1 and plot against the concentration. Plot the line graph of Calcein concentration on the x-axis against mean RFU on the y-axis and use a linear regression to provide line of best fit.</li>
		<li>Quantify statistical significance by comparing Calcein concentrations in each column (2-11) to the control of PBS in column 1 rows A to E. Perform an Analysis of variance (ANOVA) and apply post hoc analysis using Least Significant Difference (LSD) with a threshold of p &#60;0.001 to determine statistical significance, (denoted by ** in Figure 1).</li>
	</ol>
	</li>
	<li>Analysis for assay validation by Fe2+ ion quenching to reduce Calcein fluorescence.
	<ol>
		<li>Calculate the average Relative Fluorescence Units (RFU) of rows A to E for each concentration of FAS in columns 2-11. Plot mean RFU for each FAS concentration on the y-axis. On the x-axis plot concentrations of FAS using a Log2&#160;scale as itbetter distributes the data. Plot a line of best fit using a linear regression.</li>
		<li>Quantify statistical significance by comparing each columns mean to mean RFU of the control of PBS, 1 &#181;M Calcein, 0 &#181;M FAS (column 1 row A to E). Perform an ANOVA with LSD post hoc analysis with a threshold of p &#60;0.001 to determine statistical significance.</li>
	</ol>
	</li>
	<li>Analysis for assay: Quantifying the ability of iron chelators to outcompete the weak chelator Calcein for Fe2+ ions.
	<ol>
		<li>Calculate the average Relative Fluorescence Units (RFU) of rows A to E for each concentration of chelator in columns 2-11. Plot mean RFU for each chelator concentration on the y axis. On the x-axis plot concentrations of chelator using a Log2 scale as it better distributes the data.</li>
		<li>Quantify statistical significance by comparing each columns mean to mean RFU of the control of PBS 1 &#181;M Calcein and 10 &#181;M FAS (column 1 row A to E). Perform an ANOVA with LSD post hoc analysis with a threshold of p &#60;0.001 to determine statistical significance.</li>
	</ol>
	</li>
</ol>

Fluorescent Assay for Detection of Novel Iron Chelators

<ol>
	<li>Hsu, P. P., Sabatini, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+metabolism:+Warburg+and+beyond.">Cancer cell metabolism: Warburg and beyond.</a> Cell. 134 (5), 703-707 (2008).</li><li>Hsu, M. Y., Mina, E., Roetto, A., Porporato, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron:+An+essential+element+of+cancer+metabolism.">Iron: An essential element of cancer metabolism.</a> Cells. 9 (12), 2591 (2020).</li><li>Torti, S. V., Torti, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+and+cancer:+More+ore+to+be+mined.">Iron and cancer: More ore to be mined.</a> Nat Rev Cancer. 13 (5), 342-355 (2013).</li><li>Zhang, C., Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+homeostasis+and+tumorigenesis:+Molecular+mechanisms+and+therapeutic+opportunities.">Iron homeostasis and tumorigenesis: Molecular mechanisms and therapeutic opportunities.</a> Protein Cell. 6 (2), 88-100 (2015).</li><li>Torti, S. V., Manz, D. H., Paul, B. T., Blanchette-Farra, N., Torti, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+and+cancer.">Iron and cancer.</a> Annu Rev Nutr. 38, 97-125 (2018).</li><li>Welch, D. R., Hurst, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+hallmarks+of+metastasis.">Defining the hallmarks of metastasis.</a> Cancer Res. 79 (12), 3011-3027 (2019).</li><li>Lehmann, 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=Epigenetic+defects+of+hepatocellular+carcinoma+are+already+found+in+non-neoplastic+liver+cells+from+patients+with+hereditary+haemochromatosis.">Epigenetic defects of hepatocellular carcinoma are already found in non-neoplastic liver cells from patients with hereditary haemochromatosis.</a> Hum Mol Genet. 16 (11), 1335-1342 (2007).</li><li>Fonseca-Nunes, A., Jakszyn, P., Agudo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+and+cancer+risk--a+systematic+review+and+meta-analysis+of+the+epidemiological+evidence.">Iron and cancer risk--a systematic review and meta-analysis of the epidemiological evidence.</a> Cancer Epidemiol Biomarkers Prev. 23 (1), 12-31 (2014).</li><li>Abdelaal, G., Veuger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversing+oncogenic+transformation+with+iron+chelation.">Reversing oncogenic transformation with iron chelation.</a> Oncotarget. 12 (2), 106-124 (2021).</li><li>Brown, R. A. 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=Altered+iron+metabolism+and+impact+in+cancer+biology,+metastasis,+and+immunology.">Altered iron metabolism and impact in cancer biology, metastasis, and immunology.</a> Front Oncol. 10, 476 (2020).</li><li>Menyh&#225;rt, 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=Guidelines+for+the+selection+of+functional+assays+to+evaluate+the+hallmarks+of+cancer.">Guidelines for the selection of functional assays to evaluate the hallmarks of cancer.</a> Biochim Biophys Acta. 1866 (2), 300-319 (2016).</li><li>Basu, A., 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=Discovering+novel+and+diverse+iron-chelators+in+silico.">Discovering novel and diverse iron-chelators in silico.</a> J Chem Inf Model. 56 (12), 2476-2485 (2016).</li><li>Prus, E., Fibach, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+measurement+of+the+labile+iron+pool+in+human+hematopoietic+cells.">Flow cytometry measurement of the labile iron pool in human hematopoietic cells.</a> Cytometry A. 73 (1), 22-27 (2008).</li><li>Cold Spring Harb. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Phosphate-buffered saline (pbs). 2006 (1), (2006).</li><li>Kontoghiorghes, G. J., Pattichis, K., Neocleous, K., Kolnagou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+design+and+development+of+deferiprone+(l1)+and+other+iron+chelators+for+clinical+use:+Targeting+methods+and+application+prospects.">The design and development of deferiprone (l1) and other iron chelators for clinical use: Targeting methods and application prospects.</a> Curr Med Chem. 11 (16), 2161-2183 (2004).</li></ol>

The authors declare no conflict of interest.

The over reliance of cancers on iron to fuel their metabolism makes iron chelation a potential addition to therapeutic regimes4. However, there is a limited ability to quickly screen novel metal ion chelators for their ability to bind iron ions. The commonly used and widely available fluorescent probe Calcein is known to act as a weak iron chelator and binding by iron ions quenches Calcein fluorescence. Fluorescence can then be recovered by competing for binding to iron using a stronger iron chela...

Phenotypic changes to cells that relate to the development of cancer through a common set of altered biological capabilities are now commonly referred to as the hallmarks of cancer. Amongst them are changes resulting from the reprogramming of energy metabolism, which are widespread in cancer cell biology1. Such metabolic reprogramming includes an increased requirement for iron to support rapid proliferation and tumor growth2. This thirst for iron leads to dysregulated iron metabolism, which in and of itself is considered a hallmark of cancer3,4, with dysregulation occurring at all stages5. The hallmarks of metastasis, more recently proposed by Welch and Hurst, include a role for iron6 since iron can induce oxidative stress, and this can, in turn, mediate changes to the genome, epigenome, and proteome, enhancing the possibility of metastasis7. A link between iron levels and an increased occurrence of cancer has been demonstrated through epidemiological studies8.
Since cancer cells require large amounts of iron, they are susceptible to iron deficiency and, therefore, iron chelation. We have recently published a review article highlighting the potential for iron chelation in reversing several hallmarks of cancer through NDRG1 disrupting oncogenic signaling pathways9. However, the use of iron chelation as stand-alone cancer therapy has not yielded positive results in clinical trials due to their toxicity, short half-life, rapid metabolism, and emerging resistance mechanisms. Nevertheless, iron chelators have shown promise in in vitro and in vivo investigations, indicating that more work is needed to develop effective iron chelators for cancer therapy. Specific iron chelation is a validated strategy in anticancer drug discovery, but only a few classes have been reported to date10.
The identification and characterization of novel iron chelators requires the ability to measure their effect on several endpoints. Many of these (such as proliferation, apoptosis, reactive oxygen species formation) are routinely measured and have been outlined and reviewed in the literature as methods to evaluate the hallmarks of cancer11. When evaluating a novel iron chelator, many groups routinely examine the effect on anti-proliferative and redox activities as well as effects on iron influx or efflux. In silico prediction techniques12 further increase the growing pool of iron chelators that can be screened.
Screening of iron chelators requires the ability to measure their effect on iron levels as a means to effectively demonstrate proof of principle. Currently, the most common method to do so is flow cytometry13 which is costly, time consuming and poorly quantifiable. The choice of assay is often based on the availability of experimental equipment, speed, and cost of an assay. Therefore, the ability to chelate iron can be an overlooked end point measure due to the high costs of equipment and the challenge to quickly and reproducibly quantify the strength of chelation. Here, we describe a quantifiable and inexpensive cell free fluorescent method to confirm the ability of novel compounds to chelate iron.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ammonium iron(II) sulfate hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>215406</td>
			<td>other wise known as FAS</td>
		</tr>
		<tr>
			<td>Calcein</td>
			<td>Sigma-Aldrich</td>
			<td>C0875</td>
			<td></td>
		</tr>
		<tr>
			<td>Deferiprone</td>
			<td>Sigma-Aldrich</td>
			<td>379409</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich</td>
			<td>D5652</td>
			<td>magnesium and calcium free</td>
		</tr>
		<tr>
			<td>Greiner CELLSTAR 96 well plates</td>
			<td>Sigma-Aldrich</td>
			<td>M0812</td>
			<td>any optically transparent 96 well plate will work</td>
		</tr>
	</tbody>
</table>

quantifiable and inexpensive cell-free fluorescent method to confirm the ability of novel compounds to chelate iron

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target for anti-cancer approaches. The development of novel compounds and the identification of leads for further modification requires that proof of mechanism assays be carried out. There are many assays to evaluate the impact on proliferation; however, the ability to chelate iron is an important and sometimes overlooked end-point measure due to the high costs of equipment and the challenge to quickly and reproducibly quantify the strength of chelation. Here, we describe a quantifiable and inexpensive cell-free fluorescent method to confirm the ability of novel compounds to chelate iron. Our assay relies on the commercially available inexpensive fluorescent dye Calcein, whose fluorescence can be quantified on most fluorescent microtiter plate readers. Calcein is a weak iron chelator, and its fluorescence is quenched when it binds Fe2+/3+; fluorescence is restored when a novel chelator outcompetes Calcein for bound Fe2+/3+. The removal of fluorescent quenching and the resulting increase in fluorescence allows the chelation ability of a novel putative chelator to be determined. Therefore, we offer an inexpensive, high-throughput assay that allows the rapid screening of novel candidate chelator compounds.

For the method shown in step 2, this first experiment (Figure 1) established the linear range of the microtiter fluorescence plate reader when detecting Calcein fluorescent emissions. Our representative results show a wide linear range of Calcein fluorescence from 0-100 &#181;M. ANOVA with LSD post hoc analysis demonstrates that there is a statistically significant differences in mean RFU for all Calcein concentrations when compared to a control of 0 &#181;M of Calcein. In further experiment...

Read this Scientific Article Protocol about Quantifiable and Inexpensive Cell-Free Fluorescent Method to Confirm the Ability of Novel Compounds to Chelate Iron at JoVE.com

Author Spotlight: Assessing the Impact of Novel Iron Chelators on Cancer Cell Metabolism

We describe a fluorescent assay that can quickly and inexpensively confirm the ability of novel compounds to chelate iron. The assay measures the ability of compounds to outcompete the iron binding activity of the weak iron chelating fluorescent probe Calcein, resulting in a quantifiable increase in fluorescence when chelation occurs.

Quantifiable and Inexpensive Cell-Free Fluorescent Method to Confirm the Ability of Novel Compounds to Chelate Iron

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target ...

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Cancer Research

354 Views.  Northumbria University. We describe a fluorescent assay that can quickly and inexpensively confirm the ability of novel compounds to chelate iron. The assay measures the ability of compounds to outcompete the iron binding activity of the weak iron chelating fluorescent probe Calcein, resulting in a quantifiable increase in fluorescence when chelation occurs. 

Video: Author Spotlight: Assessing the Impact of Novel Iron Chelators on Cancer Cell Metabolism

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish (Danio Rerio)

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression. Traditional in vitro models of cancer cell invasion of endothelia typically lack the fluid dynamics that invading cells are otherwise exposed to in vivo. However, in vivo systems such as mouse models, though more physiologically relevant, require longer experimental timescales and present unique challenges associated with monitoring and data analysis. Here we describe a zebrafish assay that seeks to bridge this technical gap by allowing for the rapid assessment of cancer cell vascular invasion and extravasation. The approach involves injecting fluorescent cancer cells into the precardiac sinus of transparent 2-day old zebrafish embryos whose vasculature is marked by a contrasting fluorescent reporter. Following injection, the cancer cells must survive in circulation and subsequently extravasate from vessels into tissues in the caudal region of the embryo. Extravasated cancer cells are efficiently identified and scored in live embryos via fluorescence imaging at a fixed timepoint. This technique can be modified to study intravasation and/or competition amongst a heterogeneous mixture of cancer cells by changing the injection site to the yolk sac. Together, these methods can evaluate a hallmark behavior of cancer cells and help uncover mechanisms indicative of malignant progression to the metastatic phenotype.

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish Danio Rerio

This method utilizes zebrafish embryos to efficiently test the vascular invasive ability of cancer cells. Fluorescent cancer cells are injected into the precardiac sinus or yolk sac of developing embryos. Cancer cell vascular invasion and extravasation is assessed via fluorescence microscopy of the tail region 24 to 96 hr later.

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression.  Traditional in vitro models of cancer cell invasion of ...

Cell-Free DNA Integrity Analysis in Urine Samples

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of cancer, few studies have focused on the potential use of urine cell-free (UCF) DNA. Starting from the hypotheses that normal apoptotic cells produce highly fragmented DNA and that cancer cells release longer DNA, the potential role of UCF DNA integrity was evaluated as an early diagnostic marker capable of distinguishing between patients with prostate or bladder cancer and healthy individuals.
A UCF DNA integrity analysis is proposed on the basis of four quantitative real-time PCRs of four sequences longer than 250 bp: c-MYC, BCAS1, HER2, and AR. Sequences that frequently have an increased DNA copy number in bladder and prostate cancers were chosen for the analysis, but the method is flexible, and these genes could be substituted with other genes of interest. The potential utility of UCF DNA as a source of biomarkers has already been demonstrated for urologic malignancies, thus paving the way for further studies on UCF DNA characterization. The UCF DNA integrity test has the advantage of being non-invasive, rapid, and easy to perform, with only a few milliliters of urine needed to carry out the analysis.

A method for analyzing DNA integrity in the cell-free supernatant fraction of urine samples is proposed. The method is suitable for early detection of urological malignancies and has proven accurate for the early diagnosis of bladder cancer.

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of ...

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. Recently, impressive advances in novel therapies have dramatically prolonged survival and improved quality of life for many cancer patients. Sadly, incidence of brain metastatic recurrences is fast rising, and all current therapies are merely palliative. Hence, good experimental animal models are urgently needed to facilitate in-depth studies of the disease biology and to assess novel therapeutic regimens for preclinical evaluation. However, the standard in vivo metastasis assay via tail vein injection of cancer cells produces predominantly lung metastatic lesions; animals usually succumb to the lung tumor burden before any meaningful outgrowth of brain metastasis. Intracardiac injection of tumor cells produces metastatic lesions to multiple organ sites including the brain; however, the variability of tumor growth produced with this model is large, dampening its utility in evaluating therapeutic efficacy. To generate reliable and consistent animal models for brain metastasis study, here we describe a procedure for producing experimental brain metastasis in the house mouse (Mus musculus) via intracarotid injection of tumor cells. This approach allows one to produce large number of brain metastasis-bearing mice with similar growth and mortality characteristics, thus facilitating research efforts to study basic biological mechanisms and to assess novel therapeutic agents.

Brain metastasis has become an urgent unmet medical need as its incidence has increased while therapeutic options have remained palliative. Creating experimental animal models of brain metastasis via intracarotid arterial injection of cancer cells facilitates mechanistic studies of the disease biology and evaluation of novel intervention regimens.

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. ...

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western countries. The majority of CLL patients show an indolent course of the disease as well as an anergic phenotype of their leukemia cells, referring to a B cell receptor unresponsive to external stimulation. We have recently shown that the transcription factor NFAT2 is a crucial regulator of anergy in CLL. A major challenge in the analysis of the role of a transcription factor in different diseases is the identification of its specific target genes. This is of&#160;great significance for the elucidation of pathogenetic mechanisms and potential therapeutic interventions. Chromatin immunoprecipitation (ChIP) is a classic technique to demonstrate protein-DNA interactions and can, therefore, be used to identify direct target genes of transcription factors in mammalian cells. Here, ChIP was used to identify LCK as a direct target gene of NFAT2 in human CLL cells. DNA and associated proteins are crosslinked using formaldehyde and subsequently sheared by sonication into DNA fragments of approximately 200-500 base pairs (bp). Cross-linked DNA fragments associated with NFAT2 are then selectively immunoprecipitated from cell debris using an &#945;NFAT2 antibody. After purification, associated DNA fragments are detected via quantitative real-time PCR (qRT-PCR). DNA sequences with evident enrichment represent regions of the genome which are targeted by NFAT2 in vivo. Appropriate shearing of the DNA and the selection of the required antibody are particularly crucial for the successful application of this method. This protocol is ideal for the demonstration of direct interactions of NFAT2 with target genes. Its major limitation is the difficulty to employ ChIP in large-scale assays analyzing the target genes of multiple transcription factors in intact organisms.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. NFAT transcription factors are important regulators of development and activation in numerous cell types. Here, we present a protocol for the use of chromatin immunoprecipitation (ChIP) in human CLL cells to identify novel target genes of NFAT2.

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western ...

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few decades, the field of antibody-drug conjugates, bispecific antibodies, chimeric antigen receptors (CAR) and cancer immunotherapy has emerged as the most promising area of basic and therapeutic investigations. With numerous successful human trials targeting immune checkpoint receptors and CAR-T cells in leukemia and melanoma at a breakthrough pace, it is highly exciting times for oncologic therapeutics derived from variations of antibody engineering. Regrettably, a significantly large numbers of antibody and CAR based therapeutics have also proven disappointing in human trials of solid cancers because of the limited availability of immune effector cells in the tumor bed. Importantly, nonspecific distribution of therapeutic antibodies in tissues other than tumors also contribute to the lack of clinical efficacy, associated toxicity and clinical failure. As faithful translation of preclinical studies into human clinical trails are highly relied on mice tumor xenograft efficacy and safety studies, here we highlight a method to test the tumor and general tissue distribution of therapeutic antibodies. This is achieved by labeling the protein-A purified antibody with near Infrared fluorescent dye followed by live imaging of tumor bearing mice.

Here we present a protocol to study the in vivo localization of antibodies in mice tumor xenograft models.

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few ...

Quantifiable and Inexpensive Cell-Free Fluorescent Method to Confirm the Ability of Novel Compounds to Chelate Iron

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Chapters in this video

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish (Danio Rerio)

Cell-Free DNA Integrity Analysis in Urine Samples

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody