This study was supported by funding provided by METAvivor, the Peter Carlson Trust, Theresa&#39;s Research Foundation, and the NCI/UTSW Simmons Cancer Center P30 CA142543. We acknowledge the assistance of the University of Texas Southwestern Tissue Management Shared Resource, a shared resource at the Simmons Comprehensive Cancer Center, which is supported in part by the National Cancer Institute under award number P30 CA142543. Special thanks to all members of the Chan Lab.

All mouse tissue utilized in this manuscript has been ethically collected in accordance with the Institutional Animal Care and Use Committee (IACUC) regulations and guidelines of the University of Texas Southwestern Medical Center. Likewise, all the patients consented prior to tissue donation under the oversight of an Institutional Review Board (IRB), and the samples were deidentified.
NOTE: This protocol describes the generation of organoids from primary tissue.
1. Overnight preparation of materials
<ol>
	<li>For embedding in basement extracellular matrix (BECM), thaw the BECM aliquot by leaving it at 4 &#176;C.</li>
</ol>
2. Preparing collagenase and bovine serum albumin (BSA) coating solution
<ol>
	<li>Prepare 2.64% BSA/PBS coating solution (BSA Solution): Sterile filter 50 mL of phosphate-buffered saline (PBS) and 4.10 mL of 30% BSA solution using a 50 mL/0.2 &#181;m filter flask. This BSA solution can be filtered and used again.</li>
	<li>Prepare collagenase solution for mouse mammary tissue: Sterile filter 27 mL of basal cell medium, 1.5 mL of fetal bovine serum (FBS), 30 &#181;L of 50 mg/mL gentamicin, 15 &#181;L of 10 mg/mL insulin, 600 &#181;L of 0.1 g/mL collagenase A, and 600 &#181;L of 0.1 g/mL trypsin using a 50 mL/0.2 &#181;m filter flask. Allocate between 10 mL and 30 mL of collagenase solution per mouse, depending on tissue mass.</li>
	<li>Prepare collagenase solution for human breast tissue: Sterile filter 18 mL of RPMI-1640, 200 &#181;L of 100x penicillin-streptomycin solution (pen/strep), 200 &#181;L of 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) Buffer, 1 mL of FBS, and 400 &#181;L of 0.1 g/mL Collagenase A using a 50 mL/0.2 &#181;m filter flask. Allocate between 10 mL and 20 mL per tissue sample, depending on the tissue mass.</li>
</ol>
3. Preparing media
<ol>
	<li>Prepare organoid media: Sterile filter basal cell medium with 1% pen/strep, and 1% Insulin-Transferrin-Selenium (ITS) using a 50 mL/0.2 &#181;m filter flask. To make organoid growth factor media, add basic fibroblast growth factor (bFGF) to a final concentration of 2.5 nM.</li>
	<li>Prepare mammary epithelial media: To make 100 mL of mammary epithelial media, sterile filter high-glucose common basal medium with 1 mL of 100x glutamine supplement, 1 mL of pen/strep, 1 mL of 10 mM HEPES buffer (7.3 pH), 250 &#181;L of 30% BSA stock, 1 &#181;L of 1 mg/mL cholera toxin stock, 1 mL of 50 &#181;g/mL hydrocortisone stock in PBS, 50 &#181;L of 10 mg/mL human insulin solution, 10 &#181;L of 50 &#181;g/mL epidermal growth factor (EGF) stock, and 1% FBS using a 150 mL/0.2 &#181;m filter flask. Store the media at 4 &#176;C and use for up to 2 weeks.</li>
	<li>Prepare amphotericin wash: Sterile filter PBS with 2% pen/strep and 2% amphotericin B. Store at 4 &#176;C.</li>
</ol>
4. Collecting and digesting tissue
<ol>
	<li>Mouse mammary tissue
	<ol>
		<li>Euthanize the mouse by placing in a CO2 chamber for 2-5 min, and then follow with cervical dislocation. With the ventral side facing upward, splay mouse limbs and use four 19 G needles to pin the mouse by the paws to a board covered with an absorbent pad. Spray with 70% ethanol to smooth the fur down and sanitize the skin. Wipe away feces with a gauze pad or tissue.</li>
		<li>Starting just above the anogenital region, cut upward from the midline using surgical scissors, taking care not to pierce through the peritoneum. Upon reaching the chin, make lateral cuts down both clavicles and down hind legs, and pin the skin of the mouse taut to the board to expose mammary fat pads.</li>
		<li>Locate the inguinal mammary fat pads on wild-type mice by identifying the inguinal lymph node located near the hindlimbs. Locate the thoracic mammary fat pads on wild-type mice as the thicker, vascularized tissue beneath the front limbs.</li>
		<li>Use forceps to elevate the mammary fat pad. Avoid collecting underlying muscle. Using the blunt end of sharp-blunt scissors, create a pocket underneath the mammary fat pad, away from the skin. Cut away the mammary fat pad in one complete piece.</li>
		<li>Once mammary fat pads have been removed, rinse in PBS before placing them in a sterile tissue culture dish. Quickly transfer to a tissue culture hood. 
		NOTE: For mammary tumors, use a cotton swab wetted with PBS to gently roll the tumor away from the skin. Be certain to avoid darkened or soft areas, as dark discoloration indicates necrosis, which will not yield healthy organoids.</li>
		<li>Mince the mammary tumors with a #10 or #11 scalpel to loosen tissue until it reaches a paste-like consistency. Transfer the minced tissue into a conical tube containing 10-30 mL of collagenase solution using a scalpel. To ensure all tissue is collected, pipette 1 mL of collagenase solution onto the tissue culture plate and back into the conical tube. 
		NOTE: To produce larger organoids, mechanical digestion should be minimized, leaving some small visible pieces of tissue intact. Alternatively, tissue can be stored frozen for later organoid preparation with minimal loss in viability. To do so, wash tissue in a solution of PBS or basal cell media + 1% FBS, and then coarsely mince to increase surface area (keeping the tissue in one or two solid pieces). Move the tissue to a cryo-vial, top with freezing media (90% FBS + 10% dimethyl sulfoxide (DMSO)). Freeze the vials in a controlled-rate freezing container overnight before moving to storage in liquid nitrogen.</li>
		<li>Place the conical tube into a 37 &#176;C benchtop shaking incubator at 180 RPM until the tissue becomes stringy and the collagenase solution becomes cloudy. For example, a large tissue mass (around 500-800 mg) from a tumor takes 30-60 min. A smaller tissue mass (around 100-300 mg) from wild-type mammary fat pads takes about 20-30 min. If uncertain, check at 5 min intervals.</li>
		<li>After incubation, centrifuge the conical tube for 10 min at 550 x g at room temperature (RT).</li>
		<li>Carefully aspirate the supernatant from the conical tube. 
		NOTE: Moving forward, pre-coat all pipette tips, serological pipets, and conical tubes with BSA solution to avoid loss of organoids due to adhesion to plastic.</li>
		<li>Add 8 mL of basal cell media and 80 &#181;L of DNase solution [2 U/&#181;L] to the tube. Invert gently for 1-3 min.</li>
		<li>Add 12 mL of basal cell media to the tube and carefully mix by pipetting or gently rotate the tube 15 times to mix.</li>
		<li>Centrifuge for 10 min at 550 x g at RT.</li>
		<li>Aspirate the supernatant, and then resuspend the pellet in 12 mL of basal cell media.</li>
		<li>Let the heaviest tissue fragments settle to the bottom. Collect the supernatant with a serological pipet and transfer to a 15 mL conical tube. This suspension contains epithelial organoids and stromal/immune cells.</li>
	</ol>
	</li>
	<li>Human breast tissue
	<ol>
		<li>Process the human breast tissue samples for organoids or store them within 24 h of collection. Store the samples at 4 &#176;C in CO2-Independent media with 1% 100x antibiotic-antimycotic.</li>
		<li>Transfer the tissue into a tissue culture plate. Tilt the plate and aspirate the excess storage media, and then rinse the tissue with 5 mL of the amphotericin B wash. Remove the amphotericin B wash immediately.</li>
		<li>Mince the tissue sample with a #10 or #11 scalpel to loosen the tissue until it reaches a paste-like consistency. Transfer the minced tissue into a conical tube containing 10-30 mL of collagenase solution using a scalpel. To ensure all tissue is collected, pipette 1 mL of collagenase solution onto the tissue culture plate and return to the conical tube. 
		NOTE: To produce larger organoids, mechanical digestion should be minimized, leaving some small visible pieces of tissue intact. Starting tissue mass for this protocol ranged between 100-250 mg. Alternatively, tissue can be stored frozen for later organoid preparation with minimal loss in viability after step 4.2.2 in 90% FBS + 10% DMSO as above.</li>
		<li>Place the conical tube into a 37 &#176;C benchtop shaking incubator at 180 RPM until the tissue becomes smaller and collagenase becomes cloudy. Check the tissue at 5 min intervals. Collagenase dissociation of human samples takes roughly 5-20 min.</li>
		<li>After incubation, spin down the conical tube for 10 min at 550 x g at RT.</li>
		<li>Carefully aspirate the supernatant from the conical tube. 
		NOTE: Moving forward, pre-coat all the pipette tips, serological pipets, and conical tubes with BSA solution to avoid loss of organoids due to adhesion to plastic.</li>
		<li>Add 4 mL of basal cell media and 40 &#181;L of DNase solution [2 U/&#181;L] to the conical tube. Invert gently for 3 min.</li>
		<li>Add 6 mL of basal cell media to the tube and carefully mix by pipetting or gently rotate the conical tube 15 times to mix.</li>
		<li>Centrifuge for 10 min at 550 x g at RT.</li>
		<li>Aspirate the supernatant, and then resuspend the pellet in 10 mL of basal cell media.</li>
		<li>Let the heaviest tissue fragments settle to the bottom. Collect the supernatant and transfer it to a 15 mL conical tube. This suspension contains epithelial organoids and stromal/immune cells.</li>
	</ol>
	</li>
</ol>
5. Differential centrifugation
<ol>
	<li>Pulse to 550 x g for 3-4 s at RT, aspirate the supernatant, and resuspend the pellet in 10 mL of basal cell media. Repeat this step three more times (a total of four spins).</li>
	<li>After each centrifugation, ensure that the pellet becomes increasingly opaque. This remaining pellet will be epithelial organoids.</li>
</ol>
6. Collecting small organoids
<ol>
	<li>Pulse to 80-100 x g for 3 s at RT. Collect the supernatant into a fresh BSA-coated tube, and then pulse to 550 x g for 3 s at RT to pellet the small organoids.</li>
</ol>
7. Embedding organoids in BECM
<ol>
	<li>Aliquot appropriate suspension of organoids into microcentrifuge tubes according to the organoid density relative to the amount of BECM per well (Table 1). For example, use 50-100 organoids for 20 &#181;L of BECM in one well of a 96-well plate. 
	NOTE: Organoid density can be counted using the following formula: ((Average number of organoids)/50 &#181;L) x dilution factor.</li>
	<li>Perform all the steps on ice. Place the thawed BECM on ice in a tissue culture hood. While preparing the hood, place all the pipette tips on ice to cool.</li>
	<li>Centrifuge the microcentrifuge tubes for 10 min at 300 x g at RT. Discard the supernatant from the tubes. Move the tubes with organoid pellets to ice and add the appropriate volume of BECM to each microcentrifuge tube (Table 1). 
	NOTE: Since BECM is viscous, add in additional volumes (approximately 10%-20% extra of dome volume) of BECM to account for the loss in the pipet tip.</li>
	<li>Gently pipette up and down to resuspend the organoids in BECM, taking care not to produce bubbles.</li>
	<li>Slowly and carefully pipette the BECM-suspended organoids onto the plating surface. While pipetting, slowly bring up the pipette to create a dome. Fill all the empty wells with PBS to maintain humidity.</li>
	<li>Place the plate into a 37 &#176;C incubator for 1 h to allow BECM to solidify, and then cover with the appropriate volume of media (Table 1).</li>
</ol>
8. Embedding organoids in collagen
<ol>
	<li>Perform all the steps on ice. Prepare the collagen solution by combining, in sequential order, 375 &#181;L of 10x DMEM, 100 &#181;L of 1 N sodium hydroxide (NaOH), and 3 mL of rat tail collagen I solution in a 15 mL conical tube. Pipette mix, taking care not to make bubbles. Titrate the solution to a pH of 7.2-7.4 with small amounts (increments of 1-3 &#181;L) of NaOH or 10x DMEM as needed.</li>
	<li>Coat the bottom of the wells with the minimum amount of collagen required to fully cover the bottom of the well. One approach is to place a small amount of collagen and rock the plate from side to side to coat. Allow the collagen underlays to set at 37 &#176;C for 30 min-2 h.</li>
	<li>Aliquot the appropriate suspension of organoids into microcentrifuge tubes according to the organoid density relative to the amount of collagen per well (Table 1). Place in a 37 &#176;C incubator.</li>
	<li>Store the collagen solution at 4 &#176;C and monitor polymerization by checking for fiber formation under a microscope every 10 min. Collagen will reach appropriate polymerization between 30 min-2 h. 
	NOTE: Proper polymerization can be determined by the presence of fibers branching throughout the matrix. Proceed to step 8.5 immediately once a few fibers are observed in the field of view.</li>
	<li>Centrifuge the microcentrifuge tubes at 300 x g for 10 min&#160;at RT. Discard the supernatant from the tubes. Move the organoid pellets to ice and add the appropriate volume of collagen to each microcentrifuge tube (Table 1). 
	NOTE: Since collagen is viscous, add in additional volumes (approximately 10%-20% extra of dome volume) of collagen to account for the loss in the pipet tip.</li>
	<li>Gently pipette up and down to resuspend organoids in collagen, taking care not to produce bubbles.</li>
	<li>Slowly and carefully pipette the collagen-suspended organoids onto the plating surface. While pipetting, slowly bring up the pipette to create a dome. Fill all the empty wells with PBS to maintain humidity.</li>
	<li>Place the plate into a 37 &#176;C incubator for 1 h to allow collagen to solidify, and then cover with the appropriate volume of media (Table 1).</li>
	<li>NOTE : Organoids maintain viability in 3D matrix for up to 7 days. If imaging is being used for analysis, proceed to steps 9 and 10.</li>
</ol>
9. Fixing embedded organoids
<ol>
	<li>Use a pipette to remove all media from the wells without making contact with ECM domes.</li>
	<li>Fix with 4% paraformaldehyde (PFA)/PBS solution for 5 min.</li>
	<li>Wash two to three times by applying PBS to wells after placing them on a slowly moving shaker. After washes, apply fresh PBS to the domes and store the plate at 4 &#176;C.</li>
</ol>
10. Immunofluorescent staining of embedded organoids
<ol>
	<li>Preparing solutions for immunofluorescent staining
	<ol>
		<li>Permeabilization buffer: Prepare a PBS solution with 10% Triton X-100.</li>
		<li>Blocking buffer: Prepare a PBS solution with 10% FBS and 0.2% Triton X-100.</li>
		<li>Antibody dilution buffer: Prepare a PBS solution with 2% FBS and 0.2% Triton X-100.</li>
	</ol>
	</li>
	<li>Permeabilization and blocking
	<ol>
		<li>Pipette enough permeabilization buffer to cover the ECM domes. Incubate for 1 h at RT on a slowly moving shaker.</li>
		<li>Remove the permeabilization buffer with a pipette and apply the same volume of blocking buffer for 3 h.</li>
	</ol>
	</li>
	<li>Primary antibody incubation
	<ol>
		<li>Remove the blocking buffer with a pipette and apply the antibody dilution buffer containing the primary antibody at manufacturer-specified concentrations. Incubate the sample at 4 &#176;C for 12-16 h on a slowly-moving shaker.</li>
		<li>Remove the primary antibody solution and wash the samples three times for 10 min with PBS at RT on a slowly moving shaker.</li>
	</ol>
	</li>
	<li>Secondary antibody incubation and nuclear stain
	<ol>
		<li>Aspirate the remaining PBS wash and apply the antibody dilution buffer containing secondary antibody at manufacturer-specified concentrations. Additionally, add DAPI or Hoechst (to label nuclei) or phalloidin (to label actin) to the buffer at a 1:250 ratio. Incubate for 2-4 h at RT on a slowly-moving shaker.</li>
		<li>Remove the secondary antibody solution and wash samples three times for 10 min in PBS at RT on a shaker. Store the samples in PBS at 4 &#176;C until imaging.</li>
	</ol>
	</li>
</ol>

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T., Wolfe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+processing+and+hematoxylin+and+eosin+staining.">Tissue processing and hematoxylin and eosin staining.</a> Methods in Molecular Biology. 1180, 31-43 (2014).</li><li>LeSavage, B. L., Suhar, R. A., Broguiere, N., Lutolf, M. P., Heilshorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+cancer+organoids.">Next-generation cancer organoids.</a> Nature Materials. 21 (2), 143-159 (2022).</li><li>Nguyen-Ngoc, K. 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=3D+culture+assays+of+murine+mammary+branching+morphogenesis+and+epithelial+invasion.">3D culture assays of murine mammary branching morphogenesis and epithelial invasion.</a> Methods in Molecular Biology. 1189, 135-162 (2015).</li><li>Padmanaban, 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=Organotypic+culture+assays+for+murine+and+human+primary+and+metastatic-site+tumors.">Organotypic culture assays for murine and human primary and metastatic-site tumors.</a> Nature Protocols. 15 (8), 2413-2442 (2020).</li></ol>

The authors declare no conflicts of interest.

Different methods have been described in the literature to generate tumor organoids. This protocol highlights a method for generating tumor organoids directly from the tumor without passaging. Using this method, tumor organoids are producible within hours of initiating the procedure and generate close to 100% viable organoids compared to 70% reported in the literature31. In comparison, other methods require serial passaging of cells into organoids over several weeks. Thus, the downstream applicati...

The ability to model epithelial cells in vitro has been the foundation of modern biomedical research because it captures cellular features that are not accessible in vivo. For instance, growing epithelial cell lines in a two-dimensional plane can provide an assessment of the molecular changes that occur in an epithelial cell during proliferation1. Furthermore, measuring the dynamic regulation between signaling and gene expression is limited in in vivo systems2. In cancer research, cancer epithelial cell line modeling has enabled the identification of molecular drivers of disease progression and potential drug targets3. However, growing cancer epithelial cell lines on a two-dimensional plane has limitations, as most are genetically immortalized and modified, often clonal in nature, selected for their ability to grow in non-physiologic conditions, limited in their assessment of three-dimensional (3D) tumor tissue architecture, and do not adequately model microenvironment interactions within a realistic tissue environment4. These constraints are particularly evident in modeling metastasis, which in vivo includes several distinct biological stages, including invasion, dissemination, circulation, and colonization at the distant organ site5.
Cancer epithelial organoids have been developed to better recapitulate the 3D environment and behavior of tumors6,7,8. Organoids were first developed from single LRG5+ intestinal crypt cells and differentiated to represent the 3D structure of crypt-villus units that maintained the hierarchical structure of the small intestine in vitro9. This approach permitted real-time visualization and characterization of self-organizing tissue architecture under homeostatic and stress conditions. As a natural extension, cancer epithelial organoids were developed to model many different cancer types, including colorectal10, pancreatic11, breast12, liver13, lung14, brain15, and gastric cancers16. Cancer epithelial organoids have been exploited to characterize cancer evolution17,18 and metastatic spatiotemporal behaviors19,20 and interrogate tumor heterogeneity21, and test chemotherapies22. Cancer epithelial organoids have also been isolated and collected during ongoing clinical trials to predict patient response to anticancer agents and radiation therapy ex vivo8,23,24,25. Furthermore, systems incorporating cancer epithelial organoids can be combined with other non-cancer cells, such as immune cells, to form a more comprehensive model of the tumor microenvironment to visualize interactions in real-time, uncover how cancer epithelial cells change the fundamental nature of cytotoxic effector immune cells such as natural killer cells, and test potential immunotherapies and antibody-drug dependent cytotoxic activity26,27,28. This article demonstrates a method of generating epithelial organoids without passaging and embedding in collagen and basement extracellular matrix (ECM). Additionally, techniques for downstream imaging of isolated organoids are also shared.

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			<td>10 mM HEPES Buffer</td>
			<td>Gibco&#160;</td>
			<td>15630080</td>
			<td></td>
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			<td>100x Antibiotic-Antimycotic&#160;</td>
			<td>Gibco&#160;</td>
			<td>15240-096</td>
			<td></td>
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			<td>100x Glutamax</td>
			<td>Life Technologies&#160;</td>
			<td>35050-061</td>
			<td>Glutamine supplement</td>
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			<td>100x Insulin-Transferrin-Selenium (ITS)&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>51500-056</td>
			<td></td>
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			<td>100x Penicillin/Streptomycin (Pen/Strep)</td>
			<td>Sigma&#160;</td>
			<td>P4333</td>
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			<td>10x DMEM</td>
			<td>Sigma&#160;</td>
			<td>D2429</td>
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			<td>50 mL/0.2 &#181;m filter flask</td>
			<td>Fisher&#160;</td>
			<td>#564-0020</td>
			<td></td>
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			<td>Amphotericin B</td>
			<td>Life Technologies&#160;</td>
			<td>15290-018</td>
			<td></td>
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			<td>bFGF</td>
			<td>Sigma</td>
			<td>F0291</td>
			<td></td>
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			<td>BSA Solution (32%)</td>
			<td>Sigma&#160;</td>
			<td>#A9576</td>
			<td></td>
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			<td>Cholera Toxin&#160;</td>
			<td>Sigma&#160;</td>
			<td>C8052</td>
			<td></td>
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			<td>CO2-Independent Medium&#160;</td>
			<td>Gibco</td>
			<td>18045-088</td>
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			<td>Collagenase A&#160;</td>
			<td>Sigma&#160;</td>
			<td>C2139</td>
			<td></td>
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			<td>Deoxyribonuclease I from bovine pancreas (DNase)</td>
			<td>Sigma</td>
			<td>D4263</td>
			<td></td>
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			<td>DMEM with 4500 mg/L glucose, sodium pyruvate, and sodium bicarbonate, without L-glutamine, liquid, sterile-filtered, suitable for cell culture</td>
			<td>Sigma</td>
			<td>D6546</td>
			<td>Common basal medium</td>
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			<td>D-MEM/F12&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>#10565-018</td>
			<td>Basal cell medium</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (D-PBS)&#160;</td>
			<td>Sigma</td>
			<td>#D8662</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma&#160;</td>
			<td>#F0926</td>
			<td></td>
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			<td>Gentamicin&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>#15750-060</td>
			<td></td>
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			<td>Human epidermal growth factor (EGF)</td>
			<td>Sigma&#160;</td>
			<td>E9644</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone&#160;</td>
			<td>Sigma&#160;</td>
			<td>H0396</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin&#160;</td>
			<td>Sigma&#160;</td>
			<td>#I9278</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel&#160;</td>
			<td>Corning&#160;</td>
			<td>#354230</td>
			<td>Basement Extracellular Matrix (BECM)</td>
		</tr>
		<tr>
			<td>NaOH (1 N)</td>
			<td>Sigma&#160;</td>
			<td>S2770</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat Tail Collagen I</td>
			<td>Corning&#160;</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 media</td>
			<td>Fisher&#160;</td>
			<td>SH3002701</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>27250-018</td>
			<td></td>
		</tr>
	</tbody>
</table>

generating and imaging mouse and human epithelial organoids from normal and tumor mammary tissue without passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

The images featured in Figure 1 provide an example of wild-type and tumorous mammary epithelial organoids from human and mouse tissues. An at-a-glance illustration of the method for isolating epithelial organoids through differential centrifugation is provided in the cartoon workflow in Figure 1A, showing that primary tissues from different species can be processed in near-identical ways while yielding epithelial tissue as shown in the brightfield images (<stron...

Watch this Scientific Journal Video about Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging at JoVE.com

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

generating-imaging-mouse-human-epithelial-organoids-from-normal-tumor

Research

JoVE Journal

Cancer Research

2.7K Views.  University of Texas Southwestern Medical Center. This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.

Video: Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary epithelium using transplantation techniques takes advantage of the hormonal environment provided by the adolescent rodent host. These studies are ideally suited to explore the impact of various biological manipulations on mammary gland development and dissect many aspects of mammary gland biology. A common, but limiting, feature is that transplanted epithelial cells are strongly influenced by the surrounding stroma and outcompeted by endogenous epithelium; to utilize native mammary tissue, the abdominal-inguinal white fat pad must be cleared to remove host mammary epithelium prior to the transplantation. A major obstacle when using the rat model organism is that clearing the developing mammary tree in post-weaned rats is not efficient. When transplanted into gland-free fat pads, donor epithelial cells can repopulate the cleared host fat pad and form a functional mammary gland. The interscapular fat pad is an alternative location for these grafts. A major advantage is that it lacks ductal structures yet provides the normal stroma that is necessary to promote epithelial outgrowth and is easily accessible in the rat. Another major advantage of this technique is that it is minimally invasive, because it eliminates the need to cauterize and remove the growing endogenous mammary tree. Additionally, the interscapular fat pad contains a medial blood vessel that can be used to separate sites for grafting. Because the endogenous glands remain intact, this technique can also be used for studies comparing the endogenous mammary gland to the transplanted gland. This paper describes the method of mammary epithelial cell transplantation into the interscapular white fat pad of rats.

This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique.

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary ...

Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional (3D) tumor architecture and low relevance to human cancer. Researchers have recently developed a 3D in vitro cancer model referred to as tumor organoids that can mimic the characteristics of a native tumor in a culture dish. Here, experimental procedures are described in detail for the establishment of bladder tumor organoids from a carcinogen-induced murine bladder tumor, including culture, passage, and maintenance of the resulting 3D tumor organoids in vitro. In addition, protocols to manipulate the established bladder tumor organoid lines for genetic engineering using lentivirus-mediated transduction are described, including optimized conditions for the efficient introduction of new genetic elements into tumor organoids. Finally, the procedure for orthotopic transplantation of bladder tumor organoids into the wall of the murine bladder for further analysis is laid out. The methods described in this article can facilitate the establishment of an in vitro model for bladder cancer for the development of better therapeutic options.

This protocol provides detailed experimental steps to establish a three-dimensional in vitro culture of bladder tumor organoids derived from carcinogen-induced murine bladder cancer. Culture methods including passaging, genetic engineering, and orthotopic transplantation of tumor organoids are described.

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional ...

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 ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

Pseudomyxoma peritonei (PMP) is a rare condition that results from the dissemination of a mucinous primary tumor and the resultant accumulation of mucin-secreting tumor cells in the peritoneal cavity. PMP can arise from various types of cancers, including appendiceal, ovarian, and colorectal, though appendiceal neoplasms are by far the most common etiology. PMP is challenging to study due to its (1) rarity, (2) limited murine models, and (3) mucinous, acellular histology. The method presented here allows real-time visualization and interrogation of these tumor types using patient-derived ex vivo organotypic slices in a preparation where the tumor microenvironment (TME) remains intact. In this protocol, we first describe the preparation of tumor slices using a vibratome and subsequent long-term culture. Second, we describe confocal imaging of tumor slices and how to monitor functional readouts of viability, calcium imaging, and local proliferation. In short, slices are loaded with imaging dyes and are placed in an imaging chamber that can be mounted onto a confocal microscope. Time-lapse videos and confocal images are used to assess the initial viability and cellular functionality. This procedure also explores translational cellular movement, and paracrine signaling interactions in the TME. Lastly, we describe a dissociation protocol for tumor slices to be used for flow cytometry analysis. Quantitative flow cytometry analysis can be used for bench-to-bedside therapeutic testing to determine changes occurring within the immune landscape and epithelial cell content.

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

We describe a protocol for the production, culture, and visualization of human cancers, which have metastasized to the peritoneal surfaces. Resected tumor specimens are cut using a vibratome and cultured on permeable inserts for increased oxygenation and viability, followed by imaging and downstream analyses using confocal microscopy and flow cytometry.

Pseudomyxoma peritonei (PMP) is a rare condition that results from the dissemination of a mucinous primary tumor and the resultant accumulation of ...

In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection

Mouse mammary glands comprise ductal trees, which are lined by epithelial cells and have one opening at the tip of each nipple. The epithelial cells play a major role in mammary gland function and are the origin of most mammary tumors. Introducing genes of interest into mouse mammary epithelial cells is a critical step in evaluating gene function in epithelial cells and generating mouse mammary tumor models. This goal can be accomplished through the intraductal injection of a viral vector carrying the genes of interest into the mouse mammary ductal tree. The injected virus subsequently infects mammary epithelial cells, bringing in the genes of interest. The viral vector can be lentiviral, retroviral, adenoviral, or adenovirus-associated viral (AAV). This study demonstrates how a gene of interest is delivered into mammary epithelial cells through mouse mammary intraductal injection of a viral vector. A lentivirus carrying GFP is used to show stable expression of a delivered gene, and a retrovirus carrying Erbb2 (HER2/Neu) is used to demonstrate oncogene-induced atypical hyperplastic lesions and mammary tumors.

In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection

The present protocol describes intraductal injection of viral vectors via the teat to deliver genes of interest into the mammary epithelial cells.

Mouse mammary glands comprise ductal trees, which are lined by epithelial cells and have one opening at the tip of each nipple. The epithelial cells play ...

Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts from Fresh Tissue

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

Author Spotlight: Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...

Radiation Response in Patient-Derived Tumor Organoids Using a Clonogenic Assay

Live Imaging to Quantify Cellular Radiosensitivity in Patient-Derived Tumor Organoids

Radiation therapy (RT) is one of the mainstays of modern clinical cancer management. However, not all cancer types are equally sensitive to irradiation, often (but not always) because of differences in the ability of malignant cells to repair oxidative DNA damage as elicited by ionizing rays. Clonogenic assays have been employed for decades to assess the sensitivity of cultured cancer cells to ionizing irradiation, largely because irradiated cancer cells often die in a delayed manner that is difficult to quantify with short-term flow cytometry- or microscopy-assisted techniques. Unfortunately, clonogenic assays cannot be employed as such for more complex tumor models, such as patient-derived tumor organoids (PDTOs). Indeed, irradiating established PDTOs may not necessarily abrogate their growth as multicellular units, unless their stem-like compartment is completely eradicated. Moreover, irradiating PDTO-derived single-cell suspensions may not properly recapitulate the sensitivity of malignant cells to RT in the context of established PDTOs. Here, we detail an adaptation of conventional clonogenic assays that involves exposure of established PDTOs to ionizing radiation, followed by single-cell dissociation, replating in suitable culture conditions and live imaging. Non-irradiated (control) PDTO-derived stem-like cells reform growing PDTOs with a PDTO-specific efficiency, which is negatively influenced by irradiation in a dose-dependent manner. In these conditions, PDTO-forming efficiency and growth rate can be quantified as a measure of radiosensitivity on time-lapse images collected until control PDTOs achieve a predefined space occupancy.

Author Spotlight: Radiotherapy and Clonogenic Assays for Advancing Cancer Research and Personalized Medicine

Here, we detail a straightforward live imaging approach for quantifying the sensitivity of patient-derived tumor organoids to ionizing radiation.

Radiation therapy (RT) is one of the mainstays of modern clinical cancer management. However, not all cancer types are equally sensitive to irradiation, ...