Unveiling Therapeutic Opportunities with Melanoma Patient-derived Organoid Models

Summary

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.

Abstract

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 γδ T-cells. In conclusion, the MPDO models offer promising avenues for understanding tumor complexity, validating therapeutic strategies, and potentially advancing personalized treatment.

Introduction

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" mice^7,8. 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 (αPD-1). Results showed that the TILs expanded by IL-2 and αPD-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 γδ 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.

Protocol

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's Modified Eagle Medium (DMEM) and kept at 4 °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

1. Preparations before the start of the procedure
   NOTE: Two culture methods (Matrigel and collagen gel) can be used to culture dissociated melanoma tissue:
   1. 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 µL of the thawed solution to each of several wells of a 48-well plate and leave it to solidify for 30 min at 37 °C in a humidified incubator.
   2. 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 °C in a humidified incubator.
2. 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.
   1. Thaw a cryotube of L-WRN cells in the 37 °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 × g for 5 min at 4 °C. Aspirate the supernatant, ensuring that ~500 µL is left at the bottom to resuspend the pellet of cells.
   2. Transfer the cell pellet in the supernatant into 30 mL of L-cell medium on ice and add 150 µL of hygromycin (500 µg/mL) and 300 µL of G418 (500 µg/mL). Seed the cells at a density of 3 × 10⁶ on a T-150 flask and incubate at 37 °C in a humidified incubator with 5% CO₂ until ~90% confluent.
   3. 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 × g at 4 °C . When ~100% confluent, split the five T-150 flasks into 10 T-175 3-layer flasks.
   4. Once confluent, wash cells with 2 x 50 mL of PBS and then with 25 mL of washing medium (see Table 1).
   5. Add 75 mL of primary culture medium to each T-175 3-layer flask and incubate for 3 days at 37 °C in a humidified incubator with 5% CO₂ (see Table 1).
   6. After 3 days, harvest the conditioned medium into a 50 mL conical and spin at 1,100 × g for 5 min at 4 °C. Collect the supernatant (conditioned medium), ensuring that 5 mL is left behind.
   7. 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.
   8. 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 µm filter. Store aliquots into 50 mL tubes and store at -20 °C for long-term usage or 4 °C for up to 2-3 weeks. Use this medium to supplement the organoid culture media (see Table 1).
      NOTE: Thaw aliquots at 4 °C overnight prior to use.
3. Harvesting the cellular mixture from fresh melanoma tissue
   1. 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 µm Nylon cell strainer in the subsequent step.
   2. 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 °C, ensuring the contents are vortexed every 5 min.
   3. 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.
   4. Filter the digested cell suspension through a 70 µm 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 × g for 7 min at 4 °C. Discard the supernatant and resuspend the pellet in organoid culture medium (see Table 1).
   5. Culture the melanoma single-cell suspension in either Matrigel or collagen culture systems:
      1. Matrigel: Count the single-cell suspension to ensure that approximately 2 × 10⁵ cells are seeded in the prewarmed Matrigel-coated wells supplemented with 200 µL of organoid culture medium.
         NOTE: Matrigel-based organoids are used to study pathophysiology, screen drug efficacy, and test drug toxicity¹⁶.
      2. 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.
   6. Allow the organoids to grow while ensuring that fresh medium is added every 3 days.
   7. Passage the organoids once they reach 150-200 µm in size or cryopreserve them until further usage.
      1. For passage of organoids, transfer the organoids into a 15 mL conical and wash with 1x DPSB. Centrifuge the mixture at 300 × g for 7 min at 4 °C. Remove the supernatant and add trypsin substitute (50-100 µL per organoid) for 10 min in a 37 °C water bath, ensuring that contents are vortexed every 3 min.
      2. Stop the digestion using ADMEM/F12 (1x) Reduced Serum Medium (1:1) medium containing 10% FBS. Centrifuge the mixture at 300 × g for 7 min and resuspend in organoid culture medium.
      3. Seed the single-cell suspension into a new Matrigel/Collagen-coated well at the same initial seeding density (2 × 10⁵ cells in 200 µL 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).
      4. 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).

Results

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 αβ T cell abundance, provided insights into ...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
2 mL Red Cap Internal Threaded Polypropylene Cryogenic Vial	Corning	431420
50 mL Polypropylene Conical Tube	Corning	352070
100 mm TC-treated Cell Culture Dish	Corning	353003
105 mm Polystyrene Forceps Sterile	Caplugs Evergreen	222-1121-B1I
150 cm2 Cell Culture Flask	Corning	CLS430825
A83-01	Tocris	2939
Advanced DMEM/F12 (1x) Reduced Serum Medium (1:1)	Gibco	12634-010
B-27 Supplement (50x)	Gibco	17504-044
Brilliant Violet 510 anti-human Perforin Antibody	BioLegend	308120
Brilliant Violet 605 anti-human Ki-67 Antibody	BioLegend	350522
Brilliant Violet 650 anti-human CD366 (Tim-3) Antibody	BioLegend	345028
Brilliant Violet 711 anti-human CD45 Antibody	BioLegend	304050
Cell Culture Inserts 0.4 µm, 30 mm Diameter	Millipore Sigma	PIHP03050
Cell Staining Buffer	BioLegend	420201
Cell Strainer 40 µm Nylon	Corning	352340
Cell Strainer 70 µm Nylon	Corning	352350
Collagenase, Type IV, powder	Gibco	17104019
Cultrex Rat Collagen I	Trevigen	3440-100-01
Culture Plate, PS, 48 wells, TC treated with lid, sterile	Max Scientific	07-6048
Dimethyl Sulfoxide (DMSO) Hybri-Max	Sigma-Aldrich	D2650
DMEM - high glucose 4.5 mg/mL	Corning	MT10-0130CV
Dnase I - Grade II	Millipore Sigma	10104159001
DPBS, 1%	Corning	21-031-CV
Fetal Bovine Serum, FBS	Corning	MT35-010-CV
FITC Annexin V Apoptosis Detection Kit I	BD Biosciences	556547
Forskolin	Tocris	1099
Geneticin Selective Antibiotic (G418 Sulfate) (50 mg/mL)	ThermoFisher	10131035
GlutaMAX Supplement	ThermoFisher	35050061
Ham's F12 Nutrient Mix	ThermoFisher	11765054
HEPES (1 M)	Gibco	15630-080
Hygromycin B (50 mg/mL)	ThermoFisher	10687010
L-Glutamine 200 mM (100x)	Gibco	25030-081
L-WRN cell	ATCC	CRL-3276
Matrigel Phenol Free & Growth Fact. Reduced	Corning	356231
Millex PVDF syringe filter, 0.22 μM	Millipore Sigma	SLGVR33RB
N-Acetylcysteine	Sigma-Aldrich	A9165-5G
Nicotinamide	Sigma-Aldrich	N0636-100G
Nunc TripleFlask Treated Cell Culture Flask	ThermoFisher	132867
PE/Cyanine5 anti-human CD8a Antibody	BioLegend	301010
PE-Cy 7 Mouse anti-Human CD279 (PD-1)	BD Biosciences	561272
Pen Strep	Gibco	15140-122
Recombinant Human EGF	Peprotech	AF-100-15-1MG
Recombinant Human FGF-acidic	Peprotech	100-17A
Sodium Bicarbonate Solution (NaHCO3) (7.5%)	Quality Biological	118-085-721
Stainless Steel Surgical Blades no. 22	Integra	4-322
TrypLE Express	Gibco	12604-021