Name: creating matched in vivo/in vitro patient-derived model pairs of pdx and pdx-derived organoids for cancer pharmacology research
Uploaded: 2021-05-05T13:00:00
Description: Watch this Scientific Journal Video about Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research at JoVE.com

The authors would like to thank Dr. Jody Barbeau, Federica Parisi and Rajendra Kumari for critical reading and editing of the manuscript. The authors would also like to thank the Crown Bioscience Oncology in vitro and in vivo team for their great technical efforts.

All the protocols and amendment(s) or procedures involving the care and use of animals were reviewed and approved by the Crown Bioscience Institutional Animal Care and Use Committee (IACUC) prior to conducting the studies. The care and use of animals was conducted in accordance with AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) International guidelines as reported in the Guide for the Care and Use of Laboratory Animals, National Research Council (2011). All animal experimental procedures...

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The preliminary data for PDX-/PDXO-CR2110 in this report supports the biological equivalence between PDX and its derivative, PDXO, with regards to genomics, histopathology and pharmacology, since both models represent the disease forms from the original CSC of patient. Both models are patient-derived disease models, potentially predictive of the clinical response of patients10,11,12,21. The mat...

Cancers are a collection of diverse genetic and immunological disorders. Successful development of effective treatments is highly dependent on experimental models that effectively predict clinical outcomes. Large libraries of well-characterized patient-derived xenografts (PDXs) have long been viewed as the translational in vivo system of choice to test chemo- and/or targeted therapies due to their ability to recapitulate patient tumor characteristics, heterogeneity and patient drug response1, thus enabling Phase II-like mouse clinical trials to improve clinical success2,3. PDXs are generally considered as cancer stem cell diseases, featuring genetic stability, in contrast to cell line derived xenografts2. Over the last few decades, large collections of PDXs have been created throughout the world, becoming the workhorse of cancer drug development today. Although widely used and with great translational value, these animal models are intrinsically costly, time consuming and low throughput, therefore inadequate for large scale screening. PDX are also undesirable for immuno-oncology (IO) testing due to an immune-compromised nature4. It is thus impractical to take full advantage of the available large library of PDXs.
Recent discoveries, pioneered by the Hans Clevers&#8217; laboratory5, have led to the establishment of in vitro cultures of organoids generated from adult stem cells in most human organs of epithelial origin5. These protocols have been further refined to allow the growth of organoids from assumed CSCs in human carcinomas of various indications6,7. These patient-derived organoids (PDOs) are genomically-stable8,9 and have been shown to be highly predictive of clinical treatment outcomes10,11,12. In addition, the in vitro nature of PDOs enables high-throughput screening (HTS)13, thus potentially offering an advantage over in vivo models and leveraging large organoid libraries as a surrogate of the patient population. PDOs are poised to become an important discovery and translational platform, overcoming the many limitations of PDXs described above.
Both PDO and PDX are patient-derived and CSC-driven models, with the ability to evaluate therapeutics in the context of either personalized treatment or clinical trial format. Existing large libraries of PDXs, like the proprietary collection of &#62;3000 PDXs14,15,16,17, are therefore suitable for the rapid generation of libraries of tumor organoids (PDX-derived organoids, or PDXO), resulting in a matched library of paired PDX and PDXO models. This report describes the procedure to create and characterize colorectal cancer PDXO-CR2110 in relation to its parental PDX-CR2110 model16.

<table>
	<tbody>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Life Technologies</td>
			<td>12634028</td>
			<td>Base medium</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Hyclone</td>
			<td>SH30243.01</td>
			<td>Washing medium</td>
		</tr>
		<tr>
			<td>Collagenese type II</td>
			<td>Invitrogen</td>
			<td>17101015</td>
			<td>Digest tumor</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Organoid culture matrix (Basement Membrane Extract, growth factor reduced)</td>
		</tr>
		<tr>
			<td>N-Ac</td>
			<td>Sigma</td>
			<td>A9165</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>A83-01</td>
			<td>Tocris</td>
			<td>2939</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>Peprotech</td>
			<td>120-10C</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma</td>
			<td>N0636</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>SB202190</td>
			<td>Sigma</td>
			<td>S7076</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>Gastrin</td>
			<td>Sigma</td>
			<td>G9145</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>Rspondin</td>
			<td>Peprotech</td>
			<td>120-38-1000</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Life Technologies</td>
			<td>35050038</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Life Technologies</td>
			<td>15630056</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>penicillin-streptomycin</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Abmole</td>
			<td>M1817</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Life Technologies</td>
			<td>17105041</td>
			<td>Screening assay</td>
		</tr>
		<tr>
			<td>CellTiter-Glo 3D</td>
			<td>Promega</td>
			<td>G9683</td>
			<td>Screening assay (luminescent ATP indicator)</td>
		</tr>
		<tr>
			<td>Multidrop dispenser</td>
			<td>Thermo Fisher</td>
			<td>Multidrop combi</td>
			<td>Plating organoids/CellTiter-Glo 3D addition</td>
		</tr>
		<tr>
			<td>Digital dispener</td>
			<td>Tecan</td>
			<td>D300e</td>
			<td>Compound addition</td>
		</tr>
		<tr>
			<td>Envision Plate reader</td>
			<td>Perkin Elmer</td>
			<td>2104</td>
			<td>Luminescence reading</td>
		</tr>
		<tr>
			<td>Balb/c nude mice</td>
			<td>Beijing HFK Bio-Technology Co</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RNAeasy Mini kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td>tRNA purification kit</td>
		</tr>
		<tr>
			<td>DNAeasy Blood &#38; Tissue Kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td>DNA purification kit</td>
		</tr>
		<tr>
			<td>Histogel</td>
			<td>Thermo Fisher</td>
			<td>HG-4000-012</td>
			<td>Organoid embedding</td>
		</tr>
	</tbody>
</table>

creating matched in vivo/in vitro patient-derived model pairs of pdx and pdx-derived organoids for cancer pharmacology research

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) for conventional cancer drug evaluation. A large library of PDXs is reflective of the diversity of patient populations and thus enables population based preclinical trials (&#8220;Phase II-like mouse clinical trials&#8221;); however, PDX have practical limitations of low throughput, high costs and long duration. Tumor organoids, also being patient-derived CSC-driven models, can be considered as the in vitro equivalent of PDX, overcoming certain PDX limitations for dealing with large libraries of organoids or compounds. This study describes a method to create PDX-derived organoids (PDXO), thus resulting in paired models for in vitro and in vivo pharmacology research. Subcutaneously-transplanted PDX-CR2110 tumors were collected from tumor-bearing mice when the tumors reached 200-800 mm3, per an approved autopsy procedure, followed by removal of the adjacent non-tumor tissues and dissociation into small tumor fragments. The small tumor fragments were washed and passed through a 100 &#181;m cell strainer to remove the debris. Cell clusters were collected and suspended in basement membrane extract (BME) solution and plated in a 6-well plate as a solid droplet with surrounding liquid media for growth in a CO2 incubator. Organoid growth was monitored twice weekly under light microscopy and recorded by photography, followed by liquid medium change 2 or 3 times a week. The grown organoids were further passaged (7 days later) at a 1:2 ratio by disrupting the BME embedded organoids using mechanical shearing, aided by addition of trypsin and the addition of 10 &#181;M Y-27632. Organoids were cryopreserved in cryo-tubes for long-term storage, after release from BME by centrifugation, and also sampled (e.g., DNA, RNA and FFPE block) for further characterization.

Morphology of PDXOs, typical of organoids under light microcopy, and consistent with parental PDX per H&#38;E staining 
Under light microscopy, PDXO-CR2110 demonstrates typical cystic morphology (Figure 1A), as described previously for patient-derived organoids (PDO), evidence supporting the similarity between PDXO and PDO under the same culture conditions.
Histopathological examination by H&#38;E staining reveals that the tissue structures a...

Watch this Scientific Journal Video about Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research at JoVE.com

Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research

A method is described to create organoids using patient-derived xenografts (PDX) for in vitro screening, resulting in matched pairs of in vivo/in vitro models. PDX tumors were harvested/processed into small pieces mechanically or enzymatically, followed by the Clevers&#8217; method to grow tumor organoids that were passaged, cryopreserved and characterized against the original PDX.

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) ...

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