This study was funded in part by grants from the R01(CA258113), SPORE (CA261608), and P01 (CA114046). Figure 1 was prepared in BioRender.com.

Human melanoma tissues were obtained from patients receiving treatment at the University of Pennsylvania using the tissue collection protocol (UPCC08607) that is approved by the Institutional Review Board of the University of Pennsylvania. All patients have signed informed consent. Following resection of the human melanoma tissues, the tumor tissue is placed in Dulbecco&#39;s Modified Eagle Medium (DMEM) and kept at 4 &#176;C until processing (within 6 h). Melanoma patient-derived organoids (MPDOs) were derived from a fresh tumor as illustrated in Figure 1.
1. Isolation of cells from human melanoma tissue
<ol>
	<li>Preparations before the start of the procedure 
	NOTE: Two culture methods (Matrigel and collagen gel) can be used to culture dissociated melanoma tissue:
	<ol>
		<li>For the Matrigel method: Prepare a coating solution of Growth Factor Reduced (GFR) Basement Membrane Matrix by diluting GFR Basement Membrane Matrix with organoid culture medium (see Table 1) at a 1:2 ratio. Add approximately 115 &#181;L of the thawed solution to each of several wells of a 48-well plate and leave it to solidify for 30 min at 37 &#176;C in a humidified incubator.</li>
		<li>For the collagen gel method: Add 1 mL of Collagen gel (see Table 1) to a 30 mm inner membrane insert. Allow the gel to solidify for 30 min at 37 &#176;C in a humidified incubator.</li>
	</ol>
	</li>
	<li>Prepare the L-WRN conditioned medium15. L-WRN conditioned medium is produced from an L-WRN cell line engineered to secrete Wnt3a, R-spondin 3, and Noggin into a single conditioned medium that is widely used for cell culture.
	<ol>
		<li>Thaw a cryotube of L-WRN cells in the 37 &#176;C water bath. Add 5 mL of L-cell medium (see Table 1) and the L-WRN cell suspension to a 15 mL conical tube, and centrifuge at 120 &#215; g for 5 min at 4 &#176;C. Aspirate the supernatant, ensuring that ~500 &#181;L is left at the bottom to resuspend the pellet of cells.</li>
		<li>Transfer the cell pellet in the supernatant into 30 mL of L-cell medium on ice and add 150 &#181;L of hygromycin (500 &#181;g/mL) and 300 &#181;L of G418 (500 &#181;g/mL). Seed the cells at a density of 3 &#215; 106 on a T-150 flask and incubate at 37 &#176;C in a humidified incubator with 5% CO2 until ~90% confluent.</li>
		<li>Once the cells are ~90% confluent, split cells into five T-150 flasks by washing with 20 mL of phosphate-buffered saline (PBS), aspirating, and digesting with 3 mL of trypsin substitute. After 5 min, neutralize with 9 mL of L-cell medium followed by centrifugation for 5 min at 120 &#215; g at 4 &#176;C . When ~100% confluent, split the five T-150 flasks into 10 T-175 3-layer flasks.</li>
		<li>Once confluent, wash cells with 2 x 50 mL of PBS and then with 25 mL of washing medium (see Table 1).</li>
		<li>Add 75 mL of primary culture medium to each T-175 3-layer flask and incubate for 3 days at 37 &#176;C in a humidified incubator with 5% CO2 (see Table 1).</li>
		<li>After 3 days, harvest the conditioned medium into a 50 mL conical and spin at 1,100 &#215; g for 5 min at 4 &#176;C. Collect the supernatant (conditioned medium), ensuring that 5 mL is left behind.</li>
		<li>Add fresh 75 mL of primary cell culture medium to each T-175 3-layer flask and incubate for an additional 3 days to collect additional conditioned medium.</li>
		<li>To the collected conditioned medium, add an equal volume of primary cell culture medium (50% volume) and vacuum filter the contents using a 0.22 &#181;m filter. Store aliquots into 50 mL tubes and store at -20 &#176;C for long-term usage or 4 &#176;C for up to 2-3 weeks. Use this medium to supplement the organoid culture media (see Table 1). 
		NOTE: Thaw aliquots at 4 &#176;C overnight prior to use.</li>
	</ol>
	</li>
	<li>Harvesting the cellular mixture from fresh melanoma tissue
	<ol>
		<li>Wash the freshly resected melanoma tissue in a 10 mL Petri dish containing washing medium. After the first rinse, use sterile tweezers to transfer the tumor into a second Petri dish for further washing and repeat this process 3x on ice to preserve tumor integrity and viability. Throughout these rinsing cycles, use sterile scissors and a blade to remove excess connective tissue, fat, and residual blood. After the three washes, place the tumor in an empty Petri dish and use sterile blades to finely mince it. 
		NOTE: After mincing, the tumor should exhibit a gel-like consistency. The finer the mincing of the tumor, the higher the cell yield will be. This is because larger clumps of the tumor will be more challenging to digest and may not pass through the 70 &#181;m Nylon cell strainer in the subsequent step.</li>
		<li>Once the tumor tissue has been thoroughly washed and minced, transfer it into a 50 mL conical tube containing 10 mL of the prepared digestion medium (see Table 1). Place the conical in a water bath at 37 &#176;C, ensuring the contents are vortexed every 5 min.</li>
		<li>After 25 min, add 30 mL of the ADMEM/F12 medium containing 10% FBS to stop digestion. 
		NOTE: These specified quantities are intended for tumors weighing no more than 0.2 g.</li>
		<li>Filter the digested cell suspension through a 70 &#181;m Nylon cell strainer, then rinse the strainer with ADMEM/F12 (1x) Reduced Serum Medium (1:1) containing 10% FBS. Following the washing step, use a pipette to retrieve any remaining media on the bottom of the filter to ensure that the highest possible number of cells is retrieved. Then, centrifuge at 300 &#215; g for 7 min at 4 &#176;C. Discard the supernatant and resuspend the pellet in organoid culture medium (see Table 1).</li>
		<li>Culture the melanoma single-cell suspension in either Matrigel or collagen culture systems:
		<ol>
			<li>Matrigel: Count the single-cell suspension to ensure that approximately 2 &#215; 105 cells are seeded in the prewarmed Matrigel-coated wells supplemented with 200 &#181;L of organoid culture medium. 
			NOTE: Matrigel-based organoids are used to study pathophysiology, screen drug efficacy, and test drug toxicity16.</li>
			<li>Collagen gel: Count the single-cell suspension to ensure that approximately 106 cells are mixed with 1 mL of collagen gel. Add the mixture to the pre-solidified collagen gel membrane inserts and place these in a 6-well plate. Add enough organoid culture medium to submerge the inserts containing the cells and collagen gel. 
			NOTE: Collagen gel organoids are used to measure T cell migration and study tissue development16.</li>
		</ol>
		</li>
		<li>Allow the organoids to grow while ensuring that fresh medium is added every 3 days.</li>
		<li>Passage the organoids once they reach 150-200 &#181;m in size or cryopreserve them until further usage.
		<ol>
			<li>For passage of organoids, transfer the organoids into a 15 mL conical and wash with 1x DPSB. Centrifuge the mixture at 300 &#215; g for 7 min at 4 &#176;C. Remove the supernatant and add trypsin substitute (50-100 &#181;L per organoid) for 10 min in a 37 &#176;C water bath, ensuring that contents are vortexed every 3 min.</li>
			<li>Stop the digestion using ADMEM/F12 (1x) Reduced Serum Medium (1:1) medium containing 10% FBS. Centrifuge the mixture at 300 &#215; g for 7 min and resuspend in organoid culture medium.</li>
			<li>Seed the single-cell suspension into a new Matrigel/Collagen-coated well at the same initial seeding density (2 &#215; 105 cells in 200 &#181;L of organoid culture media for Matrigel-coated wells in a 48 well plate or 106 cells in 1 mL of organoid culture media for collagen coated 30 mm inner membrane insert).</li>
			<li>For cryopreservation, digest MPDOs using the trypsin substitute into single-cell suspensions followed by resuspension of 106 cells into 1 mL of freezing medium (see Table 1).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

Patient-Derived Melanoma Organoids: A Frontier in Cancer Immunotherapy

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The authors have no conflicts of interest to declare.

The emergence of PDOs has addressed multiple limitations posed by other previously established cancer research methods while introducing transformative potential applications in the field. This organoid technology was initially proposed in 2009 by Hans Clevers and colleagues, who were able to successfully establish an intestinal organoid culture system by culturing Lgr5+ stem cells derived from the intestine of mice in a 3D matrix gel containing R-sponsin, EGF, and Noggin factors20. A f...

Conventional 2-D cell culture models are essential tools in cancer research for studying cancer progression and therapy1. These models not only allow for controlled experimental conditions to investigate the molecular and cellular mechanisms underlying cancer development and progression but also provide cost-effective and relatively rapid experimental results. Their usage, however, is restricted due to the limited cellular diversity in the tumor microenvironment (TME), which cannot be recapitulated in a coplanar nature of mono-culture models2. Additionally, cell culture models offer an oversimplified environment compared to the human body3. Thus, to better understand cancer progression and develop effective treatments, researchers have turned to innovative models that aim to recapitulate the TME in nature closely. Consequently, maintaining fidelity to native tumors is paramount in these models. Given the intricate nature of cancer, any significant deviation from the original tumor could introduce unforeseen variables that might confound meaningful conclusions about tumor behavior. 
While patient-derived xenografts (PDX) have emerged as a model system that allows for monitoring of tumor progression in vivo, some limitations question how well the developed tumor recapitulates human cancer biology4. For instance, melanoma TME contains tumor-infiltrating immune cells and stromal cells. When passaged as a xenograft, the non-cancer cells from the patient will be gradually lost, which can select adaptations that differ from those in patient tumors. While these limitations can be mitigated by performing limited passages of the PDX in vivo, the differences between the species can still pose a risk of genetic changes to the PDX that deviate from that of the native tumor5. Moreover, PDX models are typically established in immunocompromised mice to prevent rejection of human tissues. This limits the ability to study the role of the host immune system in cancer, especially in immunotherapeutic studies6. We and others have engrafted PDXs in "humanized" mice7,8. However, establishing PDX models in humanized mice is time-consuming and expensive. It can take several months to years to develop a single PDX model from a patient's tumor specimen, thus limiting the speed and scale of research. Despite these limitations, PDX models have been shown to accurately represent the biology of the TME, thus having been extensively used for the development of cancer therapeutics, personalized medicine, and immunotherapy, among others9. 
An alternative 3D culture technique that has emerged is the patient-derived organoid (PDO), where a freshly resected patient tumor is cultured to create models that maintain phenotypic heterogeneity, histoarchitecture, and intercellular communications10. Its ability to closely resemble native tumors has been previously demonstrated where cultured organoids from non-muscle invasive (NMIBC) and muscle-invasive bladder cancer (MIBC) successfully recapitulated key aspects of the parental tumors11. Due to their potential and advantages over other models, PDOs offer a wide variety of tailored applications that other models lack, including but not limited to the study and development of immune checkpoint inhibitors along with cellular and targeted therapies. For example, we have previously used melanoma patient-derived organoids (MPDOs) to study the ability of tumor-infiltrating lymphocytes (TILs) to be expanded by IL-2 and anti-PD1 antibodies (&#945;PD-1). Results showed that the TILs expanded by IL-2 and &#945;PD-1 not only had a higher number but could also successfully infiltrate MPDOs and kill melanoma cells with higher efficiency than TILs expanded using other methods12. Other groups have shown similar results where the usage of anti-PD-1 and/or anti-PD-L1 leads to TILs remaining functional in PDOs, which subsequently leads to tumor killing13. Additionally, our group has also used MPDOs to assess &#947;&#948; T-cell infiltration and killing ability14. PDOs' applicability spans a wide range of areas, including TIL expansion, cytotoxicity studies, and small molecule screening. All these applications highlight the potential and usability that this model confers. As such, we describe the detailed protocol for the culture of MPDOs.

<table><tbody><tr><td>2 mL Red Cap Internal Threaded Polypropylene Cryogenic Vial</td><td>Corning</td><td>431420</td><td></td></tr><tr><td>50 mL Polypropylene Conical Tube</td><td>Corning</td><td>352070</td><td></td></tr><tr><td>100 mm TC-treated Cell Culture Dish</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>105 mm Polystyrene Forceps Sterile</td><td>Caplugs Evergreen&amp;nbsp;</td><td>222-1121-B1I</td><td></td></tr><tr><td>150 cm&lt;sup&gt;2 &lt;/sup&gt;Cell Culture Flask</td><td>Corning</td><td>CLS430825</td><td></td></tr><tr><td>A83-01</td><td>Tocris</td><td>2939</td><td></td></tr><tr><td>Advanced DMEM/F12 (1x) Reduced Serum Medium (1:1)</td><td>Gibco</td><td>12634-010</td><td></td></tr><tr><td>B-27 Supplement (50x)</td><td>Gibco</td><td>17504-044</td><td></td></tr><tr><td>Brilliant Violet 510 anti-human Perforin Antibody</td><td>BioLegend</td><td>308120</td><td></td></tr><tr><td>Brilliant Violet 605 anti-human Ki-67 Antibody</td><td>BioLegend</td><td>350522</td><td></td></tr><tr><td>Brilliant Violet 650 anti-human CD366 (Tim-3) Antibody</td><td>BioLegend</td><td>345028</td><td></td></tr><tr><td>Brilliant Violet 711 anti-human CD45 Antibody</td><td>BioLegend</td><td>304050</td><td></td></tr><tr><td>Cell Culture Inserts 0.4 &amp;micro;m, 30 mm Diameter</td><td>Millipore Sigma</td><td>PIHP03050</td><td></td></tr><tr><td>Cell Staining Buffer</td><td>BioLegend</td><td>420201</td><td></td></tr><tr><td>Cell Strainer 40 &amp;micro;m Nylon</td><td>Corning&amp;nbsp;</td><td>352340</td><td></td></tr><tr><td>Cell Strainer 70 &amp;micro;m Nylon</td><td>Corning&amp;nbsp;</td><td>352350</td><td></td></tr><tr><td>Collagenase, Type IV, powder</td><td>Gibco</td><td>17104019</td><td></td></tr><tr><td>Cultrex Rat Collagen I</td><td>Trevigen</td><td>3440-100-01</td><td></td></tr><tr><td>Culture Plate, PS, 48 wells, TC treated with lid, sterile</td><td>Max Scientific&amp;nbsp;</td><td>07-6048</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO) Hybri-Max</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>DMEM - high glucose 4.5 mg/mL</td><td>Corning</td><td>MT10-0130CV</td><td></td></tr><tr><td>Dnase I - Grade II</td><td>Millipore Sigma</td><td>10104159001</td><td></td></tr><tr><td>DPBS, 1%&amp;nbsp;</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>Fetal Bovine Serum, FBS</td><td>Corning</td><td>MT35-010-CV</td><td></td></tr><tr><td>FITC Annexin V Apoptosis Detection Kit I</td><td>BD Biosciences&amp;nbsp;</td><td>556547</td><td></td></tr><tr><td>Forskolin</td><td>Tocris</td><td>1099</td><td></td></tr><tr><td>Geneticin Selective Antibiotic (G418 Sulfate) (50 mg/mL)</td><td>ThermoFisher</td><td>10131035</td><td></td></tr><tr><td>GlutaMAX Supplement</td><td>ThermoFisher</td><td>35050061</td><td></td></tr><tr><td>Ham&#039;s F12 Nutrient Mix</td><td>ThermoFisher</td><td>11765054</td><td></td></tr><tr><td>HEPES (1 M)</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>Hygromycin B (50 mg/mL)</td><td>ThermoFisher</td><td>10687010</td><td></td></tr><tr><td>L-Glutamine 200 mM (100x)</td><td>Gibco</td><td>25030-081</td><td></td></tr><tr><td>L-WRN cell</td><td>ATCC</td><td>CRL-3276</td><td></td></tr><tr><td>Matrigel Phenol Free &amp;amp; Growth Fact. Reduced</td><td>Corning</td><td>356231</td><td></td></tr><tr><td>Millex PVDF syringe filter, 0.22 &amp;mu;M</td><td>Millipore Sigma</td><td>SLGVR33RB</td><td></td></tr><tr><td>N-Acetylcysteine</td><td>Sigma-Aldrich</td><td>A9165-5G</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma-Aldrich</td><td>N0636-100G</td><td></td></tr><tr><td>Nunc TripleFlask Treated Cell Culture Flask</td><td>ThermoFisher</td><td>132867</td><td></td></tr><tr><td>PE/Cyanine5 anti-human CD8a Antibody</td><td>BioLegend</td><td>301010</td><td></td></tr><tr><td>PE-Cy 7 Mouse anti-Human CD279 (PD-1)</td><td>BD Biosciences&amp;nbsp;</td><td>561272</td><td></td></tr><tr><td>Pen Strep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Recombinant Human EGF</td><td>Peprotech</td><td>AF-100-15-1MG</td><td></td></tr><tr><td>Recombinant Human FGF-acidic</td><td>Peprotech</td><td>100-17A</td><td></td></tr><tr><td>Sodium Bicarbonate Solution (NaHCO&lt;sub&gt;3&lt;/sub&gt;) (7.5%)</td><td>Quality Biological&amp;nbsp;</td><td>118-085-721</td><td></td></tr><tr><td>Stainless Steel Surgical Blades no. 22</td><td>Integra</td><td>4-322</td><td></td></tr><tr><td>TrypLE Express&amp;nbsp;</td><td>Gibco</td><td>12604-021</td><td></td></tr></tbody></table>

unveiling therapeutic opportunities with melanoma patient-derived organoid models

With the development of immunotherapy, there is an ongoing need to develop models that can recapitulate the tumor microenvironment of native tumors. While traditional two- and three-dimensional models can offer insights into cancer development and progression, these lack crucial aspects that hinder a faithful mimic of native tumors. An alternative model that has gained a lot of attention is the patient-derived organoid. The development of these organoids recapitulates the complex intercellular communication, tumor microenvironment, and histoarchitecture of tumors. This paper describes the protocol for establishing melanoma patient-derived organoid (MPDO) models. To validate these models, we assessed the immune cell composition, including the expression levels of T-cell activation markers, to confirm the cellular heterogeneity of the organoids. Additionally, to describe the potential utility of MPDOs in cellular therapies, we evaluated the cytotoxic capabilities of treating the organoids with &#947;&#948; T-cells. In conclusion, the MPDO models offer promising avenues for understanding tumor complexity, validating therapeutic strategies, and potentially advancing personalized treatment.

The shape and size of the MPDOs were monitored over time to study their growth dynamics. As seen in Figure 2, during the initial growth period, the organoid size increased substantially throughout the 7 days depicted. Following the observation of MPDO growth dynamics, attention turned to evaluating the immune cell composition to verify the faithful recapitulation of organoids to the original tumors. This assessment, primarily focusing on &#945;&#946; T cell abundance, provided insights into ...

Author Spotlight: Recreating Melanoma Complexity with Patient-Derived Organoids for Immunotherapy Evaluation

Here, we present a protocol to generate melanoma patient-derived organoids by culturing disassociated cell suspensions from fresh melanoma tissues. These organoids faithfully recapitulate patient-specific tumors in vitro, offering an innovative approach to exploring tumor immunosuppressive mechanisms, drug screening, drug resistance mechanisms, and cancer surveillance approaches.

Unveiling Therapeutic Opportunities with Melanoma Patient-derived Organoid Models

With the development of immunotherapy, there is an ongoing need to develop models that can recapitulate the tumor microenvironment of native tumors. While ...

unveiling-therapeutic-opportunities-with-melanoma-patient-derived

Research

JoVE Journal

Cancer Research

1.3K Views.  University of Pennsylvania. We ontwikkelen en valideren organoïden afkomstig van melanoompatiënten. Het heeft tot doel de micro-omgeving van de tumor nauwkeurig na te bootsen, interacties tussen immuuncellen te bestuderen en therapeutische reacties te beoordelen, met name op immunotherapieën. We proberen het begrip van tumorbiologie te vergroten, strategieën voor de ontwikkeling van geneesmiddelen te verbeteren en mogelijk gepersonaliseerde kankerbehandelingen mogelijk te maken....

Video: Auteur in de schijnwerpers: Melanoomcomplexiteit nabootsen met van de patiënt afgeleide organoïden voor evaluatie van immunotherapie

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

A Melanoma Patient-Derived Xenograft Model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

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.

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) ...

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

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 ...

Establishment and Culture of Patient-Derived Breast Organoids

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor organoids developed in our laboratory consist of a mix of multiple tumor-derived cell populations, and thus represent a better approximation of tumor cell diversity and milieu than the established 2D cancer cell lines. Organoids serve as an ideal in vitro model, allowing for cell-extracellular matrix interactions, known to play an important role in cell-cell interactions and cancer progression. Patient-derived organoids also have advantages over mouse models as they are of human origin. Furthermore, they have been shown to recapitulate the genomic, transcriptomic as well as metabolic heterogeneity of patient tumors; thus, they are capable of representing tumor complexity as well as patient diversity. As a result, they are poised to provide more accurate insights into target discovery and validation and drug sensitivity assays. In this protocol, we provide a detailed demonstration of how patient-derived breast organoids are established from resected breast tumors (cancer organoids) or reductive mammoplasty-derived breast tissue (normal organoids). This is followed by a comprehensive account of 3D organoid culture, expansion, passaging, freezing, as well as thawing of patient-derived breast organoid cultures.

A detailed protocol is provided here for establishing human breast organoids from patient-derived breast tumor resections or normal breast tissue. The protocol provides comprehensive step-by-step instructions for culturing, freezing, and thawing human patient-derived breast organoids.

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor ...

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. ...

Ovarian Cancer Patient-Derived Organoid Models for Pre-Clinical Drug Testing

Ovarian cancer is a fatal gynecologic cancer and the fifth leading cause of cancer death among women in the United States. Developing new drug treatments is crucial to advancing healthcare and improving patient outcomes. Organoids are in-vitro three-dimensional multicellular miniature organs. Patient-derived organoid (PDO) models of ovarian cancer may be optimal for drug screening because they more accurately recapitulate tissues of interest than two-dimensional cell culture models and are inexpensive compared to patient-derived xenografts. In addition, ovarian cancer PDOs mimic the variable tumor microenvironment and genetic background typically observed in ovarian cancer. Here, a method is described that can be used to test conventional and novel drugs on PDOs derived from ovarian cancer tissue and ascites. A luminescence-based adenosine triphosphate (ATP) assay is used to measure viability, growth rate, and drug sensitivity. Drug screens in PDOs can be completed in 7-10 days, depending on the rate of organoid formation and drug treatments.

We present a protocol that can be used to conduct therapeutic drug testing with patient-derived ovarian cancer organoids.

Ovarian cancer is a fatal gynecologic cancer and the fifth leading cause of cancer death among women in the United States. Developing new drug treatments ...

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...

Immunology and Infection

A Preclinical Human-Derived Autologous Gastric Cancer Organoid/Immune Cell Co-Culture Model to Predict the Efficacy of Targeted Therapies

Tumors expressing programmed cell death-ligand 1 (PD-L1) interact with programmed cell death protein 1 (PD-1) on CD8+ cytotoxic T lymphocytes (CTLs) to evade immune surveillance leading to the inhibition of CTL proliferation, survival, and effector function, and subsequently cancer persistence. Approximately 40% of gastric cancers express PD-L1, yet the response rate to immunotherapy is only 30%. We present the use of human-derived autologous gastric cancer organoid/immune cell co-culture as a preclinical model that may predict the efficacy of targeted therapies to improve the outcome of cancer patients. Although cancer organoid co-cultures with immune cells have been reported, this co-culture approach uses tumor antigen to pulse the antigen-presenting dendritic cells. Dendritic cells (DCs) are then cultured with the patient's CD8+ T cells to expand the cytolytic activity and proliferation of these T lymphocytes before co-culture. In addition, the differentiation and immunosuppressive function of myeloid-derived suppressor cells (MDSCs) in culture are investigated within this co-culture system. This organoid approach may be of broad interest and appropriate to predict the efficacy of therapy and patient outcome in other cancers, including pancreatic cancer.

The goals of the protocol are to use this approach to 1) understand the role of the immunosuppressive gastric tumor microenvironment and 2) predict the efficacy of patient response, thus increasing the survival rate of patients.

Tumors expressing programmed cell death-ligand 1 (PD-L1) interact with programmed cell death protein 1 (PD-1) on CD8+ cytotoxic T lymphocytes (CTLs) to ...

Unveiling Therapeutic Opportunities with Melanoma Patient-derived Organoid Models

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