The authors would like to thank the MSKCC Tissue Procurement Service Team (TPS), specifically, Maria Corazon Mariano, Priscilla McNeil, Anas Idelbi, Daniel Navarrete and Katrina Allen, in all of their efforts in the successful pursuit of this project and funding from the following sources: 5 R21 CA158609-02 and the Conquer Cancer Foundation and the Breast Cancer Research Foundation. In addition, the authors would like to thank Eric Cottington PhD, Vice President of the Office of Research and Project Administration, the Office of Technology Development, Research Outreach and Compliance and RTM Information Systems Support, in the support of the submission of this manuscript.

NOTE: Patient tissue procurement was authorized through institutionally review board (IRB)-approved Biospecimen and Clinical Protocols (Protocol numbers 09-121 and 11-041, respectively) at Memorial Sloan Kettering Cancer Center.
1. Tissue Procurement
<ol>
	<li>Procurement of patient primary tumor/metastasis 
	NOTE: To date, this protocol has been performed on surgically removed pancreatic, gastric and breast tumor types, as well as, lymphoma metastases.
	<ol>
		<li>Direct the Surgical team to deliver the specimen by courier or pneumatic tube system to&#160;the Pathology department in tightly sealed and sterile leak-proof plastic specimen bag within&#160;sterile&#160;spill-proof container. Direct the Surgical team to transport the&#160;specimen&#160;in the fresh state with no formalin or fixative.</li>
		<li>Direct the pathologist or pathologist assistant to harvest the fresh specimen using&#160;sterile&#160;technique, which includes use of sterile gloves and instruments under a laminar flow hood.</li>
		<li>Record the harvesting time of the procured specimen, which is kept rigorously under 30 min from the completion of the surgical procedure. Taking into consideration effects of cold ischemia, do not take samples from resected specimens with a cold ischemic time of &#62; 30 min18.</li>
		<li>Direct the Pathology Department personnel to remove a primary tumor specimen of approximately 0.5 cm3 to 1.0 cm3 in a laminar flow hood to maintain a sterile environment. When possible, choose tumor tissue from the periphery of the index lesion to avoid potential frank central necrosis (cell death). 
		NOTE: The necrotic tissue may be grossly recognizable by any of the following criteria: loss of color or paleness of the tissue; loss of strength in which necrotic tissue is soft and friable; a distinct demarcation between the necrotic and viable tissue.</li>
		<li>Direct the pathologist or pathologist assistant to provide peripheral tissue that is in excess (surgical discard) after immediate sampling of the tumor for diagnostic evaluation. Place the specimen in a 15 ml sterile conical centrifuge tube containing approximately 5 ml of minimum essential media (MEM) containing 1% antibiotics (penicillin and streptomycin).</li>
		<li>If available (e.g., lymph node of axillary tail of mastectomy specimen), procure a grossly positive associated lymph node specimen of the same size (0.5 cm3 to 1.0 cm3) and compare to drug response of primary tumor. Like the primary tumor specimen, place the associated lymph node specimen in a 15 ml sterile conical centrifuge tube containing approximately 5 ml of minimum essential media (MEM) containing 1% antibiotics (penicillin and streptomycin).</li>
		<li>If size of surgical specimen permits (e.g., mastectomy specimen), remove (distant from primary tumor) a sample of normal tissue (i.e., normal dense/fibrous breast parenchyma) and place in a 15 ml sterile conical centrifuge tube containing approximately 5 ml of minimum essential media (MEM) containing 1% antibiotics for transfer purposes only. Following transfer to the laboratory facilities, transfer the &#8216;normal&#8217; specimen to a cryovial, &#8216;snap&#8217; freeze and store in a -80 &#176;C freezer for future molecular analyses.</li>
		<li>Place all specimens on wet ice and immediately transport to the laboratory space for ex vivo fresh tissue sectioning. Do not section the normal specimen but rather store in a -80 &#176;C freezer for future molecular analyses. Transfer a portion of the primary tumor and associated metastasis to a cryovial, &#8216;snap&#8217; freeze and store in a -80 &#176;C freezer for future molecular analyses.</li>
	</ol>
	</li>
	<li>Procurement of tissue from clinical studies (e.g., optional core needle biopsies (CNB)) 
	NOTE: We obtain tumor tissues from patients enrolled on clinical protocols who give their consent to undergo a CNB of an accessible tumor metastasis prior to the start of therapy. CNBs, to date, have been performed on patients with breast, marginal zone lymphoma, mantle cell lymphoma, metastatic paraganglioma, cervix (squamous cell) and ovarian cancers.
	<ol>
		<li>Have clinical personnel from Interventional Radiology or relevant department perform a pre-treatment core needle biopsy and place the specimen in a 15 ml sterile conical centrifuge tube containing approximately 5 ml of minimum essential media (MEM) containing 1% antibiotics (penicillin and streptomycin).</li>
		<li>Place the biopsy specimen on wet ice and immediately transport to the laboratory space for ex vivo fresh tissue sectioning.</li>
	</ol>
	</li>
</ol>
2. Preparation of Materials for Sectioning and Vibratome Settings
<ol>
	<li>Preparation of Materials for Sectioning
	<ol>
		<li>Make a 4% agarose solution by adding 4 g of agarose to 100 ml of PBS. Autoclave (cycle settings: liquid, 121 &#176;C, 30 min, 15 pounds per square inch) the solution before first use. For subsequent uses, place the 4% agarose in a microwave to liquefy (High heat for 30 sec intervals) prior to each use and cool appropriately to ~ 25 &#176;C (i.e., warm to touch) mindful that agarose solidifies at RT.</li>
		<li>Use the 4% agarose as a non-permanent tissue embedding media. When the 4% agarose has cooled sufficiently and is still in a liquid state, with sterile forceps place the tissue specimen in an embedding mold and pour in the 4% agarose.</li>
		<li>Position the tissue specimen close (~ 2 mm distance) but not directly on the cutting surface wall of the embedding mold. The 4% agarose will begin to solidify quickly, therefore appropriately position the tissue specimen quickly. During sectioning, keep the 4% agarose in a 55 &#176;C water bath to keep in solution.</li>
		<li>To a 24-well plate for section analysis, add 1 ml of complete media (i.e., MEM + 10% fetal bovine serum + 1% antibiotics) to each well. Keep the 24-well plate at RT (~25 &#176;C) throughout the duration of sectioning.</li>
	</ol>
	</li>
	<li>Preparation of Vibratome Settings
	<ol>
		<li>Using the vibrating microtome, set the speed at which the blade approaches the specimen [0 (0.00 mm/sec) to 10 (2.5 mm/sec)] and the frequency of the blade [0 (0 Hz) to 10 (100 Hz)]. Adjust the settings according to the texture of the tissue (i.e., density/firmness).</li>
		<li>For a primary breast tumor of invasive ductal carcinoma, adjust settings to 6 and 3, speed and frequency respectively. For less dense/firm tumor tissue, decrease speed and increase frequency accordingly.</li>
		<li>Set the section thickness within the instrument limits (1 &#8211; 999 &#181;m). For these analyses, set the section thickness at 200 &#181;m.</li>
		<li>Mount the agarose-embedded specimen using a thin layer of liquid adhesive on the specimen disc that is submersed in a reservoir of media that is encased in wet ice.</li>
	</ol>
	</li>
</ol>
3. Tissue Sectioning, Treatment &#38; Analyses
<ol>
	<li>Remove the precision-sliced sections immediately upon sectioning using sterile forceps and place in a multi-well plate with 1 ml of complete media. When sufficient sections are acquired for response and correlative analyses, place the multi-well plate for treatment response in a tissue culture incubator set at standard tissue culturing conditions.</li>
	<li>Take multiple control sections that bookend the treated sections in an effort to confirm dose-dependent response as opposed to cultured artifacts. Perform tissue sectioning rapidly (~30 &#8211; 40 min), well within any detrimental effects of cold ischemic time (~2 &#8211; 4 hr)18. Perform treatment of sections only following a 1 hr acclimation period.</li>
	<li>Following the 1 hr acclimation period, add increasing doses (therapeutically relevant; e.g., 0 &#8211; 10 &#956;M concentrations) of drug to the sections and house under standard tissue culture conditions for the treatment period. For this study, use 24 and/or 48 hr assessment periods. However, control (vehicle only) tissue sections will remain viable, as tested in this study, for periods up to and including 96 hr assessment periods.</li>
	<li>Following treatment, remove all sections under sterile conditions and formalin-fix by placing the tissue section in a tube containing either 10% buffered formalin and place at RT O/N or in a tube containing 4% paraformaldehyde (dilute 16% paraformaldehyde from a vial with PBS for a final concentration of 4%) and place at RT for 2 hr.
	<ol>
		<li>Fix larger tissue sections (e.g., ~6 mm x 4 mm) 200 &#956;m thick in 10% buffered formalin O/N at RT. Fix smaller sections (e.g., 1 mm x 1 mm) 200 &#956;m thick in 4% paraformaldehyde for 2 hr at RT. 
		NOTE: For efficient penetration of fixative the volume of fixative should be 20x the volume of tissue. Given that this protocol results in extremely small tissue sizes (e.g., 6 mm3 tissue = 6 &#956;l volume), for assurance of proper fixation use 1 ml of fixative for all tissue sizes.</li>
		<li>Following fixation have the tissue specimens paraffin embedded by the Pathology Department personnel. Section specimen paraffin blocks onto charged slides to be hematoxylin and eosin stained and assessed immunohistochemically (e.g., apoptosis).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Abercrombie, M., Ambrose, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interference+Microscope+Studies+of+Cell+Contacts+in+Tissue+Culture.">Interference Microscope Studies of Cell Contacts in Tissue Culture.</a> Experimental Cell Research. 15 (58), 332-345 (1958).</li><li>Coriell, L. L., McAllister, R. M., Wagner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Criteria+for+Determining+Malignancy+in+Tissue-Culture+Cell+Lines+in+the+Albino+Rat.">Criteria for Determining Malignancy in Tissue-Culture Cell Lines in the Albino Rat.</a> New York Academy of Science. 5, 351-355 (1957).</li><li>Paul, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cancer+Cell+in+Vitro:+A+Review.">The Cancer Cell in Vitro: A Review.</a> Cancer Research. 22, 431-440 (1962).</li><li>Dean, J. L., 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=Therapeutic+response+to+CDK4/6+inhibition+in+breast+cancer+defined+by+ex+vivo+analysis+of+human+tumors.">Therapeutic response to CDK4/6 inhibition in breast cancer defined by ex vivo analysis of human tumors.</a> Cell Cycle. 11 (14), 2756-2761 (2012).</li><li>Bray, K., 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=Bcl-2+Modulation+to+Activate+Apoptosis+in+Prostate+Cancer.">Bcl-2 Modulation to Activate Apoptosis in Prostate Cancer.</a> Molecular Cancer Research. 7 (9), 1487-1496 (2009).</li><li>Quail, D. F., Joyce, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+regulation+of+tumor+progression+and+metastasis.">Microenvironmental regulation of tumor progression and metastasis.</a> Nature Medicine. 19 (11), 1423-1437 (2013).</li><li>Nakasone, E. 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=Imaging+Tumor-Stroma+Interactions+during+Chemotherapy+Reveals+Contributions+of+the+Microenvironment+to+Resistance.">Imaging Tumor-Stroma Interactions during Chemotherapy Reveals Contributions of the Microenvironment to Resistance.</a> Cancer Cell. 21, 488-503 (1016).</li><li>Shi, Y., Hogue, J., Dixit, D., Koh, J., Olson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+genetic+studies+of+isolated+cells+from+parathyroid+tumors+reveal+the+complex+pathogenesis+of+parathyroid+neoplasia.">Functional and genetic studies of isolated cells from parathyroid tumors reveal the complex pathogenesis of parathyroid neoplasia.</a> PNAS. 111 (8), 3092-3097 (2014).</li><li>Vidal, S. 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=Isolation+of+cancer+stem+cells+from+human+prostate+cancer+samples.">Isolation of cancer stem cells from human prostate cancer samples.</a> J. Vis. Exp. (85), e51332 (2014).</li><li>Wanping, X., Neckers, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+Molecular+Chaperone+Heat+Shock+Protein+90+Provides+a+Multifaceted+Effect+on+Diverse+Cell+Signaling+of+Cancer+Cells.">Targeting the Molecular Chaperone Heat Shock Protein 90 Provides a Multifaceted Effect on Diverse Cell Signaling of Cancer Cells.</a> Clinical Cancer Research. 13, 1625-1629 (2007).</li><li>Moulick, K., 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=Affinity-based+proteomics+reveal+cancer-specific+networks+coordinated+by+Hsp90.">Affinity-based proteomics reveal cancer-specific networks coordinated by Hsp90.</a> Nature Chemical Biology. 7 (11), 818-826 (1038).</li><li>Alpaugh, M. L., Tomlinson, J. S., Ye, Y., Barsky, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+sialyl-Lewis(x/a)+underexpression+and+E-cadherin+overexpression+in+the+lymphovascular+embolus+of+inflammatory+breast+cancer.">Relationship of sialyl-Lewis(x/a) underexpression and E-cadherin overexpression in the lymphovascular embolus of inflammatory breast cancer.</a> American Journal of Pathology. 161 (2), 619-628 (2002).</li><li>Chu, K., Boley, K. M., Moraes, R., Barsky, S. H., Robertson, 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=The+paradox+of+E-cadherin:+role+in+response+to+hypoxia+in+the+tumor+microenvironment+and+regulation+of+energy+metabolism.">The paradox of E-cadherin: role in response to hypoxia in the tumor microenvironment and regulation of energy metabolism.</a> Oncotarget. 4 (3), 446-462 (2013).</li><li>Vaira, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+model+of+organotypic+culture+for+pharmacodynamic+profiling+of+human+tumors.">Preclinical model of organotypic culture for pharmacodynamic profiling of human tumors.</a> PNAS. 107 (18), 8352-8356 (2010).</li><li>Centenera, M. M., Raj, G. V., Knudsen, K. E., Tilley, W. D., Butler, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+culture+of+human+prostate+tissue+and+drug+development.">Ex vivo culture of human prostate tissue and drug development.</a> Nature Reviews Urology. 10, 483-487 (2013).</li><li>Jhaveri, K., 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=Heat+shock+protein+90+inhibitors+in+the+treatment+of+cancer:+current+status+and+future+directions.">Heat shock protein 90 inhibitors in the treatment of cancer: current status and future directions.</a> Expert Opinion on Investigational Drugs. 5, 611-628 (2014).</li><li>Gerecitano, J. F., 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=Using+124-PU-H71PET+imaging+to+predict+intratumoral+concentration+in+patients+on+a+phase+I+trial+of+PU-H71.">Using 124-PU-H71PET imaging to predict intratumoral concentration in patients on a phase I trial of PU-H71.</a> Journal of Clinical Oncology. 31, 11076-11 (2013).</li><li>Yildiz-Aktas, I., Dabbs, D. J., Bhargava, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cold+ischemic+time+on+the+immunohistochemical+evaluation+of+estrogen+receptor,+progesterone+receptor,+and+HER2+expression+in+invasive+breast+carcinoma.">The effect of cold ischemic time on the immunohistochemical evaluation of estrogen receptor, progesterone receptor, and HER2 expression in invasive breast carcinoma.</a> Modern Pathology. 25, 1098-1105 (2012).</li></ol>

No conflicts of interest disclosed.

Cancer biologists face significant challenges when attempting to develop efficacious therapeutic strategies. Testing drugs in development on established cancer cell lines cannot accurately reflect in vivo response and in vivo experiments on PDX models are labor intensive and very expensive. Given the above, the application of ex vivo techniques of primary patient tumors14, 15 is now positioned beside the molecular analysis of established cancer cell lines and patient-derived xenograf...

Developing efficacious cancer therapeutics has proven to be extremely challenging. Cancer cell lines and tumor explants - as well as xenografts have been used in cancer research for over half a century1,2,3. To date, the molecular analysis of drug sensitivity and resistance in both established cancer cell lines and patient-derived xenografts (PDX) is indispensable. However, testing of compounds in established cancer cell lines is not often predictive of in vivo efficacy, and corresponding in vivo studies in animals, especially in PDX models, is very expensive and time consuming. The limitations of these model systems, namely the inability to inform on the influence of the native microenvironment in tumor progression and response to therapeutic strategies, has led the research field to develop additional methods to compliment these analyses. Of recent, heightened attention is being paid towards ex vivo analysis of patient tumor explants 4, 5 due to the greater understanding that cancer therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment 6, 7 a feature that cannot be recapitulated by traditional culturing methods and/or PDX. Ex vivo analysis in the above context (i.e., influence of adjacent surrounding tumor cell microenvironment) implies assessment of viable primary tumor/metastasis sections, rather than ex vivo analysis of cellular isolates 8, 9.
We report here on an ex vivo technique (i.e., precision-sliced fresh tissue sections) of both patient primary tumors and associated metastases (i.e., lymph nodes) that faithfully informs on response (IC50), off-target effects and allows for molecular analysis of resistance and feedback mechanisms. Additionally, a correlative analysis of therapeutic sensitivity/resistance versus biomarker and gene expression profile can be performed in an effort to identify patients more likely to respond to the experimental drug of interest (i.e., high drug response matches patient with particular biological profile). Application of the ex vivo technique and assessment in a multi-parameter fashion is movement towards patient selection and overall improvement of clinical outcomes.
Ex vivo treatment response analysis could become a standard tool in the preclinical and clinical development of cancer therapeutics and is envisioned as a step towards a personalized medicine approach in therapeutic development strategies.

<table><tbody><tr><td>Vibratome Leica VT1000s</td><td>Leica</td><td>14047235613</td><td></td></tr><tr><td>UltraPure agarose</td><td>Invitrogen</td><td>16500500</td><td>Prepare 4% and 6% before use</td></tr><tr><td>Injector blade</td><td>Ted Pella</td><td>121-4</td><td></td></tr><tr><td>MEM with Penicillin&amp;nbsp; + Streptomycin</td><td>Media Core Facilities (MSKCC)</td><td></td><td>The media is prepared at Memorial Sloan Kettering Cancer Center&amp;nbsp;</td></tr><tr><td>Scalpel no. 10</td><td>Thermo Scientific</td><td>31-200-32</td><td></td></tr><tr><td>Disposable forceps</td><td>Cole-Parmer</td><td>84011182</td><td></td></tr><tr><td>Embedding mold</td><td>Electron Microscopy Science</td><td>70181</td><td></td></tr><tr><td>FBS (heat inactivated)</td><td>Gemini</td><td>100106</td><td></td></tr><tr><td>24 well plates</td><td>Corning</td><td>3524</td><td></td></tr><tr><td>Formalin (10%)</td><td>Sigma Diagnostics</td><td>SDHT501128&amp;nbsp;</td><td></td></tr><tr><td>16% Formaldehyde solution</td><td>Thermo Scientific</td><td>28908</td><td></td></tr><tr><td>Embedding microsettes</td><td>Simport</td><td>M503-2</td><td></td></tr><tr><td>Ethanol (70%)</td><td>Fisher Scientific</td><td>A405P-4</td><td></td></tr><tr><td>Waterbath</td><td>Fisher Scientific</td><td>15-462-2SQ</td><td></td></tr><tr><td>Microwave</td><td>General Electric</td><td>ModelJES2051DNBB</td><td></td></tr><tr><td>Adhesive (Ethyl Cyanoacrylate)</td><td>Sigma-Aldrich</td><td>E1505-5G</td><td></td></tr><tr><td>10 mm dishes</td><td>BD Falcon</td><td>353003</td><td></td></tr><tr><td>15 ml tubes</td><td>BD Falcon</td><td>352096</td><td></td></tr></tbody></table>

ex vivo treatment response of primary tumors and/or associated metastases for preclinical and clinical development of therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

For this study, the ex vivo technique was used in a correlative analysis of therapeutic sensitivity/resistance of a heat shock protein 90 inhibitor (Hsp90i). In a preclinical assessment of this Hsp90i, the breast cancer primary tumor, an ER+ invasive ductal carcinoma (IDC), and associated lymph node metastasis were analyzed ex vivo for treatment response&#160;(Figure 1). Multiple 200 &#956;m serial sections were treated with vehicle only and increasing doses (0.25, 0.50...

Watch this Scientific Journal Video about Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics at JoVE.com

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes.

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

ex-vivo-treatment-response-primary-tumors-andor-associated-metastases

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Medicine

14.7K Views.  Memorial Sloan Kettering Cancer Center. Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes.

Video: Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Cancer Research

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu constitutes the tumor microenvironment (TME) and can modulate response to therapy in vivo or drug response ex vivo. Conventional cancer drug discovery screens are carried out on cells cultured in a monolayer, a system critically lacking the influence of TME. Thus, experimental systems that integrate sensitive and high-throughput assays with physiological TME will strengthen the preclinical drug discovery process. Here, we introduce ex vivo tumor tissue slice culture as a platform for medium-high-throughput drug screening. Organotypic tissue slice culture constitutes precisely-cut, thin tumor sections that are maintained with the support of a porous membrane in a liquid-air interface. In this protocol, we describe the preparation and maintenance of tissue slices prepared from mouse tumors and tumors from patient-derived xenograft (PDX) models. To assess changes in tissue viability in response to drug treatment, we leveraged a biocompatible luminescence-based viability assay that enables real-time, rapid, and sensitive measurement of viable cells in the tissue. Using this platform, we evaluated dose-dependent responses of tissue slices to the multi-kinase inhibitor, staurosporine, and cytotoxic agent, doxorubicin. Further, we demonstrate the application of tissue slices for ex vivo pharmacology by screening 17 clinical and preclinical drugs on tissue slices prepared from a single PDX tumor. Our physiologically-relevant, highly-sensitive, and robust ex vivo screening platform will greatly strengthen preclinical oncology drug discovery and treatment decision making.&#160;

We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions.

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

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 ...

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and treatment response to targeted inhibitors in cancer. Pioneering work in gastrointestinal and pancreatic cancers has highlighted the promise of patient-derived organoids (PDOs) as a patient-proximate culture system, with an increasing number of models emerging. Similarly, work in other cancer types has focused on establishing organoid models and optimizing culture protocols. Notably, 3D cancer organoid models maintain the genetic complexity of original tumor specimens and thus translate tumor-derived sequencing data into treatment with genetically informed targeted therapies in an experimental setting. Further, PDOs might foster the evaluation of rational combination treatments to overcome resistance-associated adaptation of tumors in the future. The latter focuses on intense research efforts in non-small-cell lung cancer (NSCLC), as resistance development ultimately limits the treatment success of targeted inhibitors. An early assessment of therapeutically targetable mechanisms using NSCLC PDOs could help inform rational combination treatments. This manuscript describes a standardized protocol for the cell culture plate-based assessment of drug sensitivities to targeted inhibitors in NSCLC-derived 3D PDOs, with potential adaptability to combinational treatments and other treatment modalities.

This protocol describes a standardized evaluation of drug sensitivities to targeted signaling inhibitors in NSCLC patient-derived organoid models.

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

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

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

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

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

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

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