The authors wish to thank Dr. Veronica Zanoni for editing the manuscript.

NOTE: This protocol has been specifically adjusted for the analysis of the mechanisms underlying acquired resistance to trastuzumab of HER-positive gastric cancer cell lines. All laboratory instruments needed are reported in the Table of Materials.
1. Generation of Targeted Therapy Resistant Subclones
NOTE: This is the most critical and difficult to standardize step in the protocol due to the fact that different cell lines may exhibit different sensitivities to the targeted therapeutic inhibitor independently from their tumor histotype or drug concentration used.
<ol>
	<li>Culture NCI-N87 cell line in RPMI medium supplemented with fetal calf serum (10%) and glutamine (2 mM, 1%), as a monolayer at 37 &#176;C, and seed it onto 25 cm2 culture flasks.</li>
	<li>Add to the culture medium in each flask 10 &#181;g/mL of trastuzumab as the starting dose. Expose the cells to the starting dose until they resume growing. 
	NOTE: The starting dose of the targeted therapy inhibitor should be 10-fold lower than its plasma peak concentration. Plasma peak concentration is the highest level of drug concentration detected in patient blood usually following multiple doses of standard therapy. This value can be retrieved from studies on pharmacokinetics.</li>
	<li>Monitor cell growth with the trypan blue exclusion method; when cell growth is resumed (typically after a period of two to three weeks), expose cells to a 10-fold higher dose concentration of trastuzumab.</li>
	<li>Expose cells to a gradually increasing dose of trastuzumab (from 100 &#181;g/mL to 400 &#181;g/mL) until cells keep growing (after about 2 weeks) despite the presence of a high drug concentration (at least 2.5 -&#160;4-fold higher than the plasma peak).</li>
	<li>Perform a clonogenic assay to evaluate the level of resistance to targeted therapy at every change in the concentration of the target therapy inhibitor in the culture medium, and define relative resistance IC50 index (RR IC50) as follows.
	<ol>
		<li>Seed 500 cells in 24 multi-well culture plates. 
		NOTE: 5 wells must be prepared for each condition set.</li>
		<li>Expose cells to increasing target therapy inhibitor (trastuzumab) concentrations from 100 &#181;g/mL up to 400 &#181;g/mL.</li>
		<li>Incubate cells in a CO2 incubator at 37 &#176;C for 15 days.</li>
		<li>After incubation, fix and stain samples as follows.
		<ol>
			<li>Remove medium and rinse cells with 10 mL 1x PBS. Remove&#160;1x PBS&#160;and add 2 - 3 mL of fixation solution (acetic acid:methanol, 1:7, (vol/vol)). Remove the fixation solution and add 0.5% crystal violet solution. Incubate at room temperature for 2 h</li>
			<li>Remove the crystal violet solution carefully by aspirating with a pipette and immerse plates in tap water to rinse off any crystal violet solution residues. Air-dry at room temperature for up to 2 days.</li>
		</ol>
		</li>
		<li>Count colonies of more than 50 cells using an inverted microscope (4X magnification). 
		NOTE: Two independent observers are needed for this.</li>
		<li>Calculate RR IC50 as the ratio between the IC50 value of target-therapy-resistant subclone cells and the value of the original/na&#239;ve cells.</li>
	</ol>
	</li>
</ol>
2. Validation Protocol for the Candidate Targeted Therapy Resistance Gene (R-gene)
NOTE: After the characterization of gene expression, signatures associated with the acquired resistance to target therapy and any candidate gene as driver of resistance will be investigated through: a) RT-PCR, and b) Western blot analysis.
<ol>
	<li>Real-time RT-PCR
	<ol>
		<li>Extract total RNA from cell lines using acid guanidinium thiocyanate-phenol-chloroform mixture (see Table of Materials).</li>
		<li>Perform reverse transcription (RT) reactions using random hexamer primers and set the thermal cycler as follows: priming at 25 &#176;C for 5 min, RT at 46 &#176;C for 20 min, and RT inactivation at 95 &#176;C for 1 min.</li>
		<li>Use 400 ng of total RNA for a 20 &#181;L reaction. Prepare the reaction mixture for sample as follows: 7 &#181;L RNase free H2O, 2 &#181;L cDNA (10 ng/&#181;L), 1 &#181;L 20x fluorescent-labeled target-specific probe, and 10 &#181;L 2x Mix containing buffer, dNTPs, and DNA polymerase (see Table of Materials).</li>
		<li>Add 20 &#181;L of the reaction mixture to each well plate and run the following program: holding stage at 50 &#176;C for 2 min, at 95 &#176;C for 10 min (1 cycle); then 40 cycles at 95 &#176;C for 15 s and at 60 &#176;C for 1 min.</li>
	</ol>
	</li>
	<li>Western blot
	<ol>
		<li>Recover cell suspensions by adding 2 mL of 0.05% trypsin/EDTA solution and allowing 2 - 3 min for trypsin to work.</li>
		<li>Add 2 volumes of medium containing 10% fetal bovine serum to neutralize trypsin (e.g., 2 mL of medium for each 1 mL of trypsin) and centrifuge at 1,200 x g for 5 min. Discard supernatant and wash cells with cold 1x PBS.</li>
		<li>Centrifuge at 1,200 x g for 5 min and discard supernatant. Resuspend the cell pellet with lysis buffer and incubate on a rocker at 4 &#176;C for 30 min.
		<ol>
			<li>The amount of lysis buffer depends on the number of cells (e.g., 100 &#181;L for 1 x 106 cells); make sure to prepare fresh lysis buffer each time. For 1 mL of lysis buffer preparation (leave on ice): use 975 &#181;L M-PER Mammalian lysate buffer, 15 &#181;L protease and phosphatase inhibitors, and 10 &#181;L 100 &#181;M PMSF inhibitors.</li>
		</ol>
		</li>
		<li>Recover the supernatant protein by cell lysate centrifugation at 13,000 x g at 4 &#176;C for 15 min.</li>
		<li>Determine the protein concentrations by using a BCA protein assay.</li>
		<li>Add 4x LaemmLi Sample Buffer to protein samples (at least 20 - 30 &#181;g of protein) and heat at 100 &#176;C for 3 min.</li>
		<li>Use precast 4 - 20% gels and load 20 - 30 &#181;g of LaemmLi protein solution and 5 - 10 &#181;L of protein standard per sample.</li>
		<li>Fill the Western blot ring with TRIS/GLICYNE /SDS 1x buffer and run gel at 110 V for 30 - 60 min (when time stops check that the dye has reached the end of the gel, otherwise run for a few additional minutes)</li>
		<li>Electroblot to transfer protein into a PVDF membrane (see Table of Materials) by using a semi-dry system.</li>
		<li>Block the membrane by soaking in the appropriate protein solution (milk/BSA 5%) on the shaker at room temperature for 1 h.</li>
		<li>Make a 1:400 solution with anti-IQGAP1 primary antibody in a 5% milk solution (see Table of Materials).</li>
		<li>Place the membrane in primary antibody solution on a shaker in a cold room overnight.</li>
		<li>The following day, discard the antibody solution and soak in T-TBS 1x&#160;solution (1x&#160;T-TBS: 1x&#160;Tris Saline Buffer added by Tween20 0.1%) on a shaker at room temperature for 7 min.</li>
		<li>Discard T-TBS 1x&#160;solution and replace. Repeat 3 times.</li>
		<li>Make a 1:10,000 secondary mouse-antibody solution by adding antibody for standard Western blot detection (see Table of Materials) and place the membrane on a shaker; leave at room temperature for 2 h.</li>
		<li>Discard the antibody solution and soak in T-TBS 1x&#160;solution on a shaker at room temperature for 7 min.</li>
		<li>Discard T-TBS 1x&#160;solution and replace. Repeat 3 times.</li>
		<li>Soak membrane for 5 min in a chemiluminescent solution, and image the membrane on a chemiluminescence imager.</li>
	</ol>
	</li>
</ol>
3. Restoring Target-therapy Inhibitor Sensitivity by Candidate R-gene Knock-down
<ol>
	<li>Cell preparation
	<ol>
		<li>Prepare single cell suspension by trypsinization. Perform transfection on the day that the cells are plated.
		<ol>
			<li>Wash NCI-N87 cells with PBS and incubate at 37 &#176;C with 0.05% trypsin/EDTA solution for 5 - 10 min. Add 2 volume of RPMI medium containing 10% fetal bovine serum to neutralize trypsin (e.g., 2 mL of medium for each 1 mL of trypsin).</li>
			<li>Count cells with a hemocytometer using trypan blue staining. Seed 2.5 x 105 cells (60% confluence) onto a T25 cm2 flask for each condition. 
			NOTE: The appropriate number of cells for seeding is determined by the doubling time of the cell line and the duration of the experiment. The aim is to achieve the target gene knock-down for the entire duration of the experiment. Nine T25 cm2 flasks are ideal for transfection conditions and the clonogenic assay (3 flasks for controls, 3 flasks for negative transfection controls, 3 flasks for gene-target transfection).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Reverse transfection
	<ol>
		<li>Prepare gene-target and negative control transfection mix for each T25 cm2 flask as follows.
		<ol>
			<li>Dilute 12.5 &#181;L of each gene-targeting siRNA oligonucleotides or Negative Control siRNA oligonucleotides in minimal essential transfection medium (ratio 1:55; see Table of Materials).</li>
			<li>Add a cationic liposome transfection reagent (1:1 ratio with gene-targeting siRNA oligonucleotides; see Table of Materials). Incubate at room temperature for 20 min. 
			NOTE: The total volume of the transfection mix is 700 &#181;L.</li>
			<li>Add 700 &#181;L of the transfection mix to each T25 cm2 flask. Incubate cells at 37 &#176;C for 1 - 3 days.</li>
		</ol>
		</li>
		<li>Verify gene knock-down by RT-PCR and Western blot analysis. Double-check the impact of the putative R-gene knock-down on the sensitivity restoration to the targeted therapeutic agent through colony formation experiments (repeat Step 1.5 ).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Gerber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+therapies:+a+new+generation+of+cancer+treatments.">Targeted therapies: a new generation of cancer treatments.</a> Am Fam Physician. 77 (3), 311-319 (2008).</li><li>Zhang, J., Yang, P. L., Gray, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+cancer+with+small+molecule+kinase+inhibitors.">Targeting cancer with small molecule kinase inhibitors.</a> Nat Rev Cancer. 9 (1), 28-39 (2009).</li><li>Nelson, A. L., Dhimolea, E., Reichert, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+trends+for+human+monoclonal+antibody+therapeutics.">Development trends for human monoclonal antibody therapeutics.</a> Nat Rev Drug Discov. 9 (10), 767-774 (2010).</li><li>Chabner, B. A., Roberts, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timeline:+Chemotherapy+and+the+war+on+cancer.">Timeline: Chemotherapy and the war on cancer.</a> Nat Rev Cancer. 5 (1), 65-72 (2005).</li><li>Gilbert, L. A., Hemann, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapeutic+resistance:+surviving+stressful+situations.">Chemotherapeutic resistance: surviving stressful situations.</a> Cancer Res. 71 (15), 5062-5066 (2011).</li><li>Izar, B., Rotow, J., Gainor, J., Clark, J., Chabner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetics,+clinical+indications,+and+resistance+mechanisms+in+molecular+targeted+therapies+in+cancer.">Pharmacokinetics, clinical indications, and resistance mechanisms in molecular targeted therapies in cancer.</a> Pharmacol Rev. 65, 1351-1395 (2013).</li><li>Ellis, L. M., Hicklin, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+Targeted+Therapies:+Refining+Anticancer+Therapy+in+the+Era+of+Molecular+Oncology.">Resistance to Targeted Therapies: Refining Anticancer Therapy in the Era of Molecular Oncology.</a> Clin Cancer Res. 15 (24), 7471-7478 (2009).</li><li>Asi&#263;, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dominant+mechanisms+of+primary+resistance+differ+from+dominant+mechanisms+of+secondary+resistance+to+targeted+therapies.">Dominant mechanisms of primary resistance differ from dominant mechanisms of secondary resistance to targeted therapies.</a> Crit Rev Oncol Hematol. 97, 178-196 (2016).</li><li>Arienti, C., 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=Preclinical+evidence+of+multiple+mechanisms+underlying+trastuzumab+resistance+in+gastric+cancer.">Preclinical evidence of multiple mechanisms underlying trastuzumab resistance in gastric cancer.</a> Oncotarget. 7 (14), 18424-18439 (2016).</li><li>Tanner, 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=Amplification+of+HER-2+in+gastric+carcinoma:+association+with+Topoisomerase+IIalpha+gene+amplification,+intestinal+type,+poor+prognosis+and+sensitivity+to+trastuzumab.">Amplification of HER-2 in gastric carcinoma: association with Topoisomerase IIalpha gene amplification, intestinal type, poor prognosis and sensitivity to trastuzumab.</a> Ann Oncol. 16 (2), 273-278 (2005).</li><li>Bang, Y. 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=Trastuzumab+in+combination+with+chemotherapy+versus+chemotherapy+alone+for+treatment+of+HER2-positive+advanced+gastric+or+gastro-oesophageal+junction+cancer+(ToGA):+a+phase+3,+open-label,+randomised+controlled+trial.">Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial.</a> Lancet. 376 (9742), 687-697 (2010).</li><li>Shimoyama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+trastuzumab+and+lapatinib+inefficiency+in+gastric+cancer:+Future+steps+(Review).">Unraveling trastuzumab and lapatinib inefficiency in gastric cancer: Future steps (Review).</a> Mol Clin Oncol. 2 (2), 175-181 (2014).</li><li>Kelly, C. M., Janjigian, Y. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genomics+and+therapeutics+of+HER2-positive+gastric+cancer-from+trastuzumab+and+beyond.">The genomics and therapeutics of HER2-positive gastric cancer-from trastuzumab and beyond.</a> J Gastrointest Oncol. 7 (5), 750-762 (2016).</li><li>Wong, S. S., 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=Genomic+landscape+and+genetic+heterogeneity+in+gastric+adenocarcinoma+revealed+by+whole-genome+sequencing.">Genomic landscape and genetic heterogeneity in gastric adenocarcinoma revealed by whole-genome sequencing.</a> Nat Commun. 5, 5477 (2014).</li><li>Oshima, Y., 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=Lapatinib+sensitivities+of+two+novel+trastuzumab-resistant+HER2+gene-amplified+gastric+cancer+cell+lines.">Lapatinib sensitivities of two novel trastuzumab-resistant HER2 gene-amplified gastric cancer cell lines.</a> Gastric Cancer. 17 (3), 450-462 (2014).</li><li>Pignatta, S., 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=Prolonged+exposure+to+(R)-bicalutamide+generates+a+LNCaP+subclone+with+alteration+of+mitochondrial+genome.">Prolonged exposure to (R)-bicalutamide generates a LNCaP subclone with alteration of mitochondrial genome.</a> Mol Cell Endocrinol. 382 (1), 314-324 (2014).</li></ol>

The authors have nothing to disclose.

While personalized and targeted therapies have elicited growing enthusiasm, these so-called &#39;smart&#39; therapies face the same major hurdle as traditional chemotherapy drugs, i.e., the rapid and unpredictable acquisition of drug resistance. To improve the results of targeted therapy, drug-resistance problems must be solved through a better understanding of the mechanisms that cause them. Here, we presented a widely accessible in vitro methodology to investigate the mechanisms of acquired resistance...

Following up on the crucial discoveries in molecular and cellular biology, a number of novel synthesized anticancer molecules have been developed to selectively target oncogenic signaling pathways in a broad array of tumor types. In particular, two categories of targeted therapeutic agents with unique properties in oncology, namely synthetic chemical compounds and recombinant monoclonal antibodies, are known to have achieved clinical successes and dramatically altered cancer care in recent decades1,2,3.
Nevertheless, despite their impressive initial response to treatment, the majority of cancer patients have developed drug resistance to all targeted therapeutic agents, both monoclonal antibodies and kinase inhibitors. As a consequence, drug resistance, the major hurdle for classic anti-cancer medicines4, is still a great challenge for emerging targeted therapies5,6.
Resistance to targeted therapy may be intrinsic (i.e., primary) or acquired (i.e., secondary). Intrinsic resistance describes a de novo lack of response to therapy, whereas secondary resistance occurs after a period of response to drug treatment7. The latter is caused by outbreaks of small cohorts of tumor cells within the bulk of the primitive tumor or nestled in distal cryptic anatomical niches, exhibiting up to 90% resistance to one or more targeted therapeutic agents. Molecular mechanisms underlying a resistance development to targeted therapy mainly result from target gene mutations and redundant activation of other pro-survival signaling pathways, whose understanding is still far from complete8.
In this work, we proposed a time- and cost-effective approach to investigate in vitro mechanisms of acquired resistance to targeted therapeutic agents, starting from the generation of drug-resistant cellular subclones to the description of silencing procedures used for restoring the sensitivity to the inhibitor, a crucial tool used for testing and validating the working hypotheses. In particular, our approach was used to investigate, in gastric cancer, the resistance mechanisms to trastuzumab (e.g., Herceptin), a humanized monoclonal antibody targeting the extracellular domain of HER2 protein9. Trastuzumab + chemotherapy is widely accepted as the standard first-line treatment for HER2-positive metastatic breast cancer. Thanks to recent preclinical studies showing that anti-HER2 therapies have significant activity in both in vitro and in vivo HER2-positive gastric cancer models10,11, molecular drugs targeting HER2 have been extensively examined in clinical trials, some of which are still ongoing, on patients with gastroesophageal cancer12,13,14,15.
These studies have emphasized the rising number of patients experiencing resistance to trastuzumab16, similarly to what is observed for other targeted therapeutic inhibitors. In particular, the response rate of trastuzumab was 47%, and the median overall survival for patients on trastuzumab + chemotherapy was only 2.7 months longer than for patients on chemotherapy alone17. This suggested that while primary trastuzumab resistance is prevalent, secondary trastuzumab resistance is inevitable. For this reason, there is urgent need to clarify the mechanisms causing HER2-targeted therapy resistance in gastric cancer, which is even more problematic given the intra-tumoral genetic heterogeneity of this neoplasia18.
In addition, mechanisms of resistance to trastuzumab in gastric cancer have proven challenging to explain, partially due to the difficulty in obtaining reliable preclinical models representative of this condition. Oshima et al. reported an in vivo selection method of trastuzumab-resistant gastric cancer cell lines, consisting of a repeated culture of a small residual peritoneal metastasis after treatment with trastuzumab19. However, the need of an enclosure, of specific animal handling expertise, and of the approval of the Animal Ethics Committee contributed to making it a time- and cost-consuming method. The approach we describe in this work is simple and widely applicable, and could be easily extended to investigate the resistance mechanisms to other therapeutic drugs in different tumor histotypes20.

<table>
	<tbody>
		<tr>
			<td>Trizol Reagent</td>
			<td>Invitrogen</td>
			<td>15596026</td>
			<td>TRIzol Reagent is a reagent for isolating high-quality total RNA or simultaneously isolating RNA, DNA, and protein from a variety of biological samples</td>
		</tr>
		<tr>
			<td>ISCRIPT CDNA SYNTHESIS KIT</td>
			<td>Bio-Rad</td>
			<td>1708891</td>
			<td>The iScript cDNA synthesis kit is a sensitive and easy-to-use first-strand cDNA synthesis kit for gene expression analysis using real-time qPCR.</td>
		</tr>
		<tr>
			<td>TaqMan gene expression assay</td>
			<td>Life Technologies</td>
			<td>4331182</td>
			<td>Applied Biosystems TaqMan Gene Expression Assays consist of a pair of unlabeled PCR primers and a TaqMan probe with an Applied Biosystems FAM or VIC dye label on the 5&#8217; end and minor groove binder (MGB) and nonfluorescent quencher (NFQ) on the 3&#8217; end.</td>
		</tr>
		<tr>
			<td>M-PER Mammalian Protein Extraction Reagent</td>
			<td>Thermo Scientific</td>
			<td>78501</td>
			<td>Thermo Scientific M-PER Mammalian Protein Extraction Reagent is designed to provide highly efficient total soluble protein extraction from cultured mammalian cells.</td>
		</tr>
		<tr>
			<td>Halt Protease and Phosphatase Inhibitor Cocktail (100X)</td>
			<td>Thermo Scientific</td>
			<td>78440</td>
			<td>Thermo Scientific Halt Protease and Phosphatase Inhibitor Cocktail (100X) provides the convenience of a single solution with full protein sample protection for cell and tissue lysates.</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td>The Thermo Scientific Pierce BCA Protein Assay Kit is a two-component, high-precision, detergent-compatible assay reagent set to measure (A562nm) total protein concentration compared to a protein standard.</td>
		</tr>
		<tr>
			<td>4x Laemmli Sample Buffer</td>
			<td>Bio-Rad</td>
			<td>1610747</td>
			<td>Use 4x Laemmli Sample Buffer for preparation of samples for SDS PAGE. For reduction of samples, add a reducing agent such as 2-mercaptoethanol to the buffer prior to mixing with the sample.</td>
		</tr>
		<tr>
			<td>4&#8211;20% Mini-PROTEAN TGX Precast Protein Gels</td>
			<td>Bio-Rad</td>
			<td>456-1094</td>
			<td>Long-life TGX (Tris-Glycine eXtended) Gels have a novel formulation and can be used for both standard denaturing protein separations as well as native electrophoresis.</td>
		</tr>
		<tr>
			<td>Precision Plus Protein WesternC Blotting Standards</td>
			<td>Bio-Rad</td>
			<td>1610376</td>
			<td>Precision Plus Protein Prestained Standards are available in Dual Color, All Blue, Kaleidoscope, and Dual Xtra formats. All four have the same gel migration patterns, with 3 high-intensity reference bands (25, 50, and 75 kD).</td>
		</tr>
		<tr>
			<td>10x Tris/Glycine/SDS</td>
			<td>Bio-Rad</td>
			<td>1610732</td>
			<td>10x Tris/glycine/SDS is a premixed running buffer for separating protein samples by SDS-PAGE.</td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo Mini PVDF Transfer Packs</td>
			<td>Bio-Rad</td>
			<td>1704156</td>
			<td>Trans-Blot Turbo Mini PVDF Transfer Packs for fast, efficient transfer of proteins from mini gels using the Trans-Blot Turbo Transfer System. Each vacuum sealed, ready-to-use transfer pack contains two buffer-saturated ion reservoir stacks and a prewetted PVDF membrane.</td>
		</tr>
		<tr>
			<td>Precision Protein StrepTactin-HRP Conjugate</td>
			<td>Bio-Rad</td>
			<td>1610381</td>
			<td>StrepTactin-Horseradish Peroxidase (HRP) Conjugate for chemiluminescent or colorimetric detection of Precision Plus Protein Unstained Protein Standards or Precision Plus Protein WesternC Protein Standards on western blots.</td>
		</tr>
		<tr>
			<td>Immun-Star WesternC solution</td>
			<td>Bio-Rad</td>
			<td>5572</td>
			<td>Immun-Star WesternC solution, a method of protein detection , detects mid-femtogram amounts of protein.</td>
		</tr>
		<tr>
			<td>Universal Negative Control</td>
			<td>Invitrogen</td>
			<td>12935300</td>
			<td>Negative Control siRNA has no significant sequence similarity to mouse, rat, or human gene sequences. The control has also been tested in cell-based screens and proven to have no significant effect on cell proliferation, viability, or morphology.</td>
		</tr>
		<tr>
			<td>opti-MEM Glutamax Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>51985026</td>
			<td>Opti-MEM Medium is an improved Minimal Essential Medium (MEM) that allows for a reduction of Fetal Bovine Serum supplementation by at least 50% with no change in growth rate or morphology.</td>
		</tr>
		<tr>
			<td>Silencer Select Pre-Designed &#38; Validated siRNA</td>
			<td>Ambion</td>
			<td>4390824</td>
			<td>RNA interference (RNAi) is the best way to effectively knock down gene expression to study protein function in a wide range of cell types.</td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMAX Transfection Reagent</td>
			<td>Invitrogen</td>
			<td>13778075</td>
			<td>Lipofectamine RNAiMAX Transfection Reagent offers an advanced, efficient solution for siRNA delivery. No other siRNA specific transfection reagent provides such easy and efficient siRNA delivery in a wide variety of cell lines including common cell types, stem cells and primary cells, as well as traditionally hard-to-transfect cell types.</td>
		</tr>
		<tr>
			<td>Mouse anti-IQGAP1</td>
			<td>Invitrigen</td>
			<td>33-8900</td>
			<td>working concentration: 1:400 in 5% milk solution</td>
		</tr>
		<tr>
			<td>goat anti-mouse IgG-HRP: sc-2005</td>
			<td>Santa Cruz</td>
			<td>sc-2005</td>
			<td>working concentration: 1:10000 in 5% milk solution</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma Aldrich</td>
			<td>P 7626</td>
			<td>this is an inhibitor of serine proteases and acetylcholinesterase</td>
		</tr>
		<tr>
			<td>Thermocycler for RT-PCR</td>
			<td>MJ Research</td>
			<td>MJ Research PTC-200 Thermal Cycler</td>
			<td>it allows temperature homogeneity and a ramping speed of up to 3&#176;C/sec. The protocol can be performed also with other thermalcyclers</td>
		</tr>
		<tr>
			<td>Real Time RT-PCR instrument</td>
			<td>Applied Biosystems</td>
			<td>7500 RT-PCR system</td>
			<td>This is a five-color platform that uses fluorescence-based polymerase chain reaction (PCR) reagents to provide a relative quantification using comparative CT assay type. The protocol can be performed also with other thermalcyclers</td>
		</tr>
		<tr>
			<td>Microvolume Spectrophotometers</td>
			<td>Thermo Scientific</td>
			<td>Nanodrop</td>
			<td>It provides an accurate and fast acid nucleid measurement using little volume of sample</td>
		</tr>
		<tr>
			<td>Blotting system</td>
			<td>Bio-Rad</td>
			<td>Trans-Blot Turbo system</td>
			<td>It perfoms a semy-dry transfer in a few minutes</td>
		</tr>
		<tr>
			<td>Image acquisition system and gel documentation</td>
			<td>Bio-Rad</td>
			<td>Chemidoc image system</td>
			<td>It is easy to use for chemiluminescent detection</td>
		</tr>
	</tbody>
</table>

looking for driver pathways of acquired resistance to targeted therapy: drug resistant subclone generation and sensitivity restoring by gene knock-down

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more impressive clinical efficacy and minimized adverse effects than traditional treatments, drug resistance has become the main limitation to their benefits. Several preclinical in vitro/in vivo models of acquired resistance to targeted agents in clinical practice have been developed mainly by using two strategies: i) genetic manipulation for modeling genotypes of acquired resistance, and ii) in vitro/in vivo selection of resistant models. In the present work, we propose a unifying framework, for investigating the underlying mechanisms responsible for acquired resistance to targeted therapeutic agents, starting from the generation of drug-resistant cellular subclones to the description of silencing procedures used for restoring the sensitivity to the inhibitor. This simple time- and cost-effective approach is widely applicable, and could be easily extended to investigate resistance mechanisms to other targeted therapeutic drugs in different tumor histotypes.

Figure 1 outlines the framework of the experimental procedure. Three gastric cancer cell-lines expressing a high level of HER2 and sensitive to trastuzumab (IC50 &#60; peak plasma level of drug, Figure 2A, left graph) were grown in culture medium containing the targeted therapeutic agent. A dose 10-fold lower than the peak plasma concentration of trastuzumab (10 &#181;g/mL) was set as the starting dose, and gradually i...

Watch this Scientific Journal Video about Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down at JoV

Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

This is a time- and cost-effective in vitro protocol investigating the mechanisms of acquired resistance to targeted therapeutic agents, which is a highly unmet medical need in cancer management.

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more ...

looking-for-driver-pathways-acquired-resistance-to-targeted-therapy

Research

JoVE Journal

Cancer Research

7.1K Views.  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS. This is a time- and cost-effective in vitro protocol investigating the mechanisms of acquired resistance to targeted therapeutic agents, which is a highly unmet medical need in cancer management.

Video: Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

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 ...

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light.
To increase the therapeutic effect of PDT, we developed both photosensitizer- and anticancer agent-loaded lung cancer-targeted nanoparticles. Both enhanced permeability and retention (EPR) effect-based passive targeting and hyaluronic-acid-CD44 interaction-based active targeting were applied. CD44 is a well-known hyaluronic acid receptor that is often introduced as a biomarker of non-small cell lung cancer. 
In addition, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may increase anticancer therapeutic effects and reduce adverse reactions. 
We chose hypocrellin B (HB) as a novel photosensitizer in this study. It has been reported that HB causes higher anticancer efficacy of PDT compared to hematoporphyrin derivatives1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for lung cancer2. 
The antitumor efficacies of photosensitizer (HB) solution, photosensitizer encapsulated hyaluronic acid-ceramide nanoparticles (HB-NPs), and both photosensitizer- and anticancer agent (paclitaxel)-encapsulated hyaluronic acid-ceramide nanoparticles (HB-P-NPs) after PDT were compared both in vitro and in vivo. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.
The HB-P-NP treatment group showed the most effective anticancer effect after PDT. In conclusion, the HB-P-NPs prepared in the present study represent a potential and novel photosensitizer delivery system in treating lung cancer with PDT.

Photodynamic therapy (PDT) is an alternative choice for lung cancer treatment. To increase the therapeutic effect of PDT, lung cancer-targeted nanoparticles combined with chemotherapy were developed. Both in vitro and in vivo anticancer efficacies of PDT with prepared nanoparticles were evaluated.

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. ...

Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a common mechanism of disease progression and a prognostic-determinant feature of malignant tumors. In prostate cancer (PC), resistance to MTAs such as the taxane Docetaxel dictates treatment failure as well as progression towards lethal stages of disease that are defined by a poor prognosis and high mortality rates. Though studied for decades, the array of mechanisms contributing to acquired resistance are not completely understood, and thus pose a significant limitation to the development of new therapeutic strategies that could benefit patients in these advanced stages of disease. In this protocol, we describe the generation of Docetaxel-resistant prostate cancer cell lines that mimic lethal features of late-stage prostate cancer, and therefore can be used to study the mechanisms by which acquired chemoresistance arises. Despite potential limitations intrinsic to a cell based model, such as the loss of resistance properties over time, the Docetaxel-resistant cell lines produced by this method have been successfully used in recent studies and offer&#160;the opportunity to advance our molecular understanding of acquired chemoresistance in lethal prostate cancer.

Resistance to cancer therapies contributes to disease progression and death. Determining the mechanistic underpinnings of resistance is crucial for improving therapeutic response. This manuscript details the protocol to generate taxane-resistant cell models of prostate cancer (PC) to help dissecting the pathways involved in progression to Docetaxel resistance in PC patients.

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a ...

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates approaching 85% are common. Unfortunately, patients often become refractory to the treatment by altering their signal transduction pathways. An implementation of the expression profiling with microarrays can identify the overall mRNA-level changes, and proteomics can identify the overall changes in protein levels or can identify the proteins involved, but the activity of the signal transduction pathways can only be established by interrogating post-translational modifications of the proteins. As a result, the ability to identify whether a drug treatment is successful or whether resistance arose, or the ability to characterize any alterations in the signaling pathways, is an important clinical challenge. Here, we provide a detailed explanation of antibody arrays as a tool which can identify system-wide alterations in various post-translational modifications (e.g., phosphorylation). One of the advantages of using antibody arrays includes their accessibility (an array does not require either an expert in proteomics or costly equipment) and speed. The availability of arrays targeting a combination of post-translational modifications is the primary limitation. In addition, unbiased approaches (phosphoproteomics) may be more suitable for the novel discovery, whereas antibody arrays are ideal for the most widely characterized targets.

Here, we present a protocol for antibody arrays to identify alterations in signaling pathways in various cellular models. These changes, caused by drugs/hypoxia/ultra-violet light/radiation, or by overexpression/downregulation/knockouts, are important for various disease models and can indicate whether a therapy will be effective or can identify mechanisms of drugs resistance.

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates ...

Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples from cancer patients often directly influences diagnosis, prognosis, and/or therapy selection. As a result, the accurate detection of gene fusions has become a critical component of clinical management for many disease types. Until recently, clinical gene fusion detection was predominantly accomplished through the use of single-gene assays. However, the ever-growing list of gene fusions with clinical significance has created a need for assessing fusion status of multiple genes simultaneously. Next generation sequencing (NGS)-based testing has met this demand through the ability to sequence nucleic acid in massively parallel fashion. Multiple NGS-based approaches that employ different strategies for gene target enrichment are now available for use in clinical molecular diagnostics, each with its own strengths and weaknesses. This article describes the use of anchored multiplex PCR (AMP)-based target enrichment and library preparation followed by NGS to assess for gene fusions in clinical solid tumor specimens. AMP is unique among amplicon-based enrichment approaches in that it identifies gene fusions regardless of the identity of the fusion partner. Detailed here are both the wet-bench and data analysis steps that ensure accurate gene fusion detection from clinical samples.

This article details the use of an anchored multiplex polymerase chain reaction-based library preparation kit followed by next-generation sequencing to assess for oncogenic gene fusions in clinical solid tumor samples. Both wet-bench and data analysis steps are described.

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Discovery of Driver Genes in Colorectal HT29-derived Cancer Stem-Like Tumorspheres

Cancer stem cells play a vital role against clinical therapies, contributing to tumor relapse. There are many oncogenes involved in tumorigenesis and the initiation of cancer stemness properties. Since gene expression in the formation of colorectal cancer-derived tumorspheres is unclear, it takes time to discover the mechanisms working on one gene at a time. This study demonstrates a method to quickly discover the driver genes involved in the survival of the colorectal cancer stem-like cells in vitro. Colorectal HT29 cancer cells that express the LGR5 when cultured as spheroids and accompany an increase CD133 stemness markers were selected and used in this study. The protocol presented is used to perform RNAseq with available bioinformatics to quickly uncover the overexpressed driver genes in the formation of colorectal HT29-derived stem-like tumorspheres. The methodology can quickly screen and discover potential driver genes in other disease models.

Presented here is a protocol to discover the overexpressed driver genes maintaining the established cancer stem-like cells derived from colorectal HT29 cells. RNAseq with available bioinformatics was performed to investigate and screen gene expression networks for elucidating a potential mechanism involved in the survival of targeted tumor cells.

Cancer stem cells play a vital role against clinical therapies, contributing to tumor relapse. There are many oncogenes involved in tumorigenesis and the ...

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the &#34;Murine Ommaya.&#34; As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.

Here, we describe a murine xenograft model that functionally resembles an Ommaya reservoir in patients. We developed the Murine Ommaya to study novel therapeutics for the universally fatal leptomeningeal disease.

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers ...

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable preclinical models that can accurately predict patient responses. One of the disadvantages of existing preclinical models is the inability to contextually preserve the human tumor microenvironment (TME) and accurately represent intratumoral heterogeneity, thus limiting the clinical translation of data. By contrast, by representing the culture of live fragments of human tumors, the patient-derived explant (PDE) platform allows drug responses to be examined in a three-dimensional (3D) context that mirrors the pathological and architectural features of the original tumors as closely as possible. Previous reports with PDEs have documented the ability of the platform to distinguish chemosensitive from chemoresistant tumors, and it has been shown that this segregation is predictive of patient responses to the same chemotherapies. Simultaneously, PDEs allow the opportunity to interrogate molecular, genetic, and histological features of tumors that predict drug responses, thereby identifying biomarkers for patient stratification as well as novel interventional approaches to sensitize resistant tumors. This paper reports PDE methodology in detail, from collection of patient samples through to endpoint analysis. It provides a detailed description of explant derivation and culture methods, highlighting bespoke conditions for particular tumors, where appropriate. For endpoint analysis, there is a focus on multiplexed immunofluorescence and multispectral imaging for the spatial profiling of key biomarkers within both tumoral and stromal regions. By combining these methods, it is possible to generate quantitative and qualitative drug response data that can be related to various clinicopathological parameters and thus potentially be used for biomarker identification.

This paper describes methods for the generation, drug treatment, and analysis of patient-derived explants for assessing tumor drug responses in a live, patient-relevant, preclinical model system.

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable ...