Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Summary

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

Abstract

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient’s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Introduction

Three-dimensional cultured organoids have drawn attention for their potential to recapitulate the in vivo architecture, cellular functionality and genetic signature of their original tissues^1,2,3,4,5. Most importantly, 3D organoids established from patient tumor tissues or patient derived xenograft (PDX) models provide invaluable opportunities to understand mechanisms of cellular signaling upon tumorigenesis and to determine the effects of drug treatment on each cell population^{6,7,8,9,10,11,12,13}. Drost et al.⁵ developed a standard protocol for establishment of human and mouse prostate organoids, which has been widely adopted in the field of urology. In addition, significant effort has been dedicated for further characterization of 3D organoids and to understand the detailed mechanisms of tumorigenesis and metastasis^4,12,14,15. In addition to the previously established and widely accepted protocol for 3D organoids cultures, we describe here a step-by-step protocol for the 3D culture of PDO using three different doming methods in optimized culture conditions.

In this manuscript, 3D organoids were established as an ex vivo model of bone metastatic prostate cancer (BMPC). The cells used for these cultures came from the Prostate Cancer San Diego (PCSD) series and were derived directly from patient prostate cancer bone metastatic tumor tissues (PCSD18 and PCSD22) or patient derived xenograft (PDX) tumor models (samples named PCSD1, PCSD13, and PCSD17). Because spontaneous bone metastasis of prostate cancer cells is rare in genetically engineered mouse models¹⁶, we used direct intra-femoral (IF) injection of human tumor cells into male Rag2^-/-γc^-/- mice to establish the PDX models of bone metastatic prostate cancer¹⁷.

Once 3D organoids are established from heterogeneous patient tumor cells or patient derived xenografts, it is essential to confirm their identity as prostate tumor cells and to determine their phenotypes in the 3D organoid cultures. Immunofluorescence chemistry (IFC) allows the visualization of protein expression in situ in each cell, often indicating the potential functions for specific cell populations^2,4. In general, IFC protocols for a vast majority of samples including tissues and cells are straightforward and fully optimized. However, the cell density and number of organoids can be significantly lower than that of conventional culture. Therefore, the IFC protocol for organoids requires additional steps to ensure proper processing and embedding in paraffin for all organoids in the samples. We describe additional steps for an agarose pre-embedding process and tips to label the location of sectioned organoids on the slide that increases the success rate of IFC on organoids especially when the samples of organoids have lower cell density than desired.

Protocol

This study was carried out in strict accordance with the recommendations in the Guide for the University of California San Diego (UCSD) Institutional Review Board (IRB). IRB #090401 Approval was received from the UCSD Institutional Review Board (IRB) to collect surgical specimen from patients for research purposes. An informed consent was obtained from each patient and a surgical bone prostate cancer metastasis specimen was obtained from orthopedic repair of a pathologic fracture in the femur. Animal protocols were performed under the University of California San Diego (UCSD) animal welfare and Institutional Animal Care and Use Committee (IACUC) approved protocol #S10298. Cells from mechanically and enzymatically dissociated patient tumor tissue were intra-femorally injected into 6 to 8 week old male Rag2^-/-;γ_c^-/- mice as previously described¹⁷. Xenograft tumor volume was determined using an in vivo bioluminescence imaging system and caliper measurements. Upon tumor growth up to 2.0 cm (the maximal allowable size approved by IACUC), the tumor was harvested for 3D organoids establishment.

NOTE: Figure 1 shows the workflow for establishing 3D Organoids and a protocol number for each step of the experimental procedures.

1. Processing of patient derived xenograft (PDX) tumor tissues

NOTE: This is an initial step for organoid establishment for a tumor derived from a xenograft mouse model. This protocol is adapted from a previous publication by Drost et al.⁵ and we have modified the media conditions to include serum supplementation to organoid media.

1. Process the tumor specimen as described below.
   1. Mince tumor samples to 1-3 mm³ sized pieces and digest the samples with 10 mL of cell dissociation solution for 45 min at room temperature.
   2. To terminate digestion, add 20 mL of DMEM complete media to the samples.
   3. Filter the suspension through a 70 μm cell strainer. Use a sterile plunger flange to push any leftover tissue on the top of 70 μm cell strainer.
   4. Centrifuge at 300 x g for 5 min at 4 °C.
   5. Wash the cell pellet three times with fresh adDMEM complete media. Match the volume of media for wash and resuspension with the volume of media suggested in Table 3. For example, for a 24 well plate culture condition, the volume for the wash should be 500 µL.
      NOTE: As shown in Table 3, the protocol is applicable to different culture conditions.
   6. Determine final cell counts using Trypan blue dye and a hemocytometer.
   7. After obtaining cell counts, re-suspend cell pellet in 80 µL of 2% FBS in PBS per 2 x 10⁶ tumor cells.
   8. Add 20 µL of Mouse Cell Depletion Cocktail per 2 x 10⁶ tumor cells. Mix well and incubate for 15 min at 2-8 °C.
   9. Adjust the volume to 500 µL with 2% FBS in PBS buffer per 2 x 10⁶ tumor cells.
      NOTE: Up to 1 x 10⁷ tumor cells in 2.5 mL of cell suspension can be processed on one LS column.
   10. Load the LS columns on the magnetic column separator and place a 15 mL conical tube on a rack underneath to collect the flow-through.
   11. Rinse each column with 3 mL of 2% FBS in PBS buffer. Discard the conical tube with the wash flow-through and replace with a new, sterile 15 mL conical tube.
   12. Add the cell suspension (up to 2.5 mL of 1 x 10⁷ tumor cells) onto the column. Collect flow-through that will be the enriched with human tumor cells.
   13. Wash the column twice with 1 mL of 2% FBS in PBS buffer.
       NOTE: It is important to perform wash steps as soon as the column is empty. Also, try to avoid forming air bubbles.
2. Aliquot the appropriate volume of cell suspension to a 1.5 mL tube for the desired culture set up (Table 3).
3. Centrifuge the 1.5 mL tube at 300 x g and 4 °C for 5 min.
4. Carefully remove and discard the supernatant.

2. Processing of patient primary tumor tissues

NOTE: This is an initial step for organoid establishment.

1. Follow all of step 1 except the mouse cell depletion process, which is not necessary for processing of patient primary tumor tissues.

3. Forming an attached round dome on the plate

NOTE: This manuscript describes three ways to make a dome from a mixture of the cell pellet and the basement membrane (e.g., Matrigel) as shown in Figure 1 and Figure 2. In Steps 2-4, the cells and the basement membrane should be kept on ice to prevent solidification of the basement membrane.

1. Resuspend the cell pellet in the appropriate volume of basement membrane (e.g., 40 µL) for a 24 well plate set up (Table 3).
2. Pipette up and down gently to ensure that the cells are re-suspended well in the basement membrane.
3. Pipette the appropriate volume (Table 3) of the cell-basement membrane mixture (and optional 10 µL of adDMEM complete media) into the center of the pre-warmed tissue culture plate.
4. Invert the plate and immediately place the plate upside down in the CO₂ cell culture incubator set at 5% CO₂, 37 °C for 15 min. This prevents cells from settling and adhering to the plate bottom while allowing the basement membrane to solidify.
5. Pipette the appropriate volume (Table 3) of pre-warmed medium containing 10 μM Y-27632 dihydrochloride into each well.
6. Place the plate right side up inside the CO₂ cell culture incubator (5% CO₂, 37 °C).
7. Change the media every 3-4 days. After 5-7 days, use culture medium without 10 μM Y-27632 dihydrochloride to maintain the cultures.

4. Forming a floating dome from an attached round dome on the plate

1. After step 3.7, detach the dome using a cell scraper.

5. Forming floating beads

NOTE: This protocol is named as floating beads since the mixture of basement membrane, media, and organoids look like beads.

1. Cut a 2 inch x 4 inch piece of paraffin film.
2. Place the paraffin film on the top of the divots of an empty tip-holding rack from a 1000 µL plastic pipette tip box.
3. Gently press down on the paraffin film to trace the divots using a gloved index finger but without breaking through the paraffin film.
4. Spray the paraffin film with 70% ethanol and turn on the UV lamp in the cell culture hood to sterilize the prepared paraffin film for at least 30 min.
5. Prepare a mixture of cells and 20 µL of basement membrane. Seeding density can be 50,000 - 250,000 cells per dome.
6. Pipette the mixture of cells processed from step 1 or 2, and 20 µL of basement membrane into the mold of the divot formed in the prepared paraffin film.
7. Resuspend the cell pellet in basement membrane and pipette the cell suspension in the prepared paraffin filmed divots.
8. Place the solidified beads and paraffin film into a 6-well plate. One well in a 6-well plate can fit up to 5 beads.
9. Pipette 3-5 mL of pre-warmed medium containing 10 μM Y-27632 dihydrochloride into each well while gently brushing beads off of the paraffin film.
   NOTE: As a minimum volume, 3 mL is recommended. For a maximum number of beads (N=5) per well, 5 mL of medium is recommended.
10. Place the plate inside a CO₂ incubator (5% CO₂, 37 °C).
11. Change the organoid media every 3-4 days. After 5-7 days, use culture medium without 10 μM Y-27632 dihydrochloride to maintain the cultures.

6. In vivo organoids image stitching using microscope⁸

NOTE: Certain microscopes are unable to reach the outer perimeter of the cell plate (edge wall); therefore, we suggest using the wells close to the perimeter of the cell plate when image stitching.

1. Place the cell culture plate in an upward position into the plate holder in the Keyence microscope.
2. Place the lens on the center of the target dome.
3. Set up the automatic stitching process by selecting number of the frames. For examples, 3 x 3 or 5 x 5 can be chosen to generate 9 images or 25 images total.
4. Press the capture button to initiate imaging process.
5. Open the image viewer software and load a group of images taken by step 4.
6. Click Image Stitching to create a high-resolution stitched image.
   NOTE: Capturing of serial 9 or 25 images can be performed either by manual or automatic set up to focus the cells.

7. Organoid processing for histology: the agarose spin down method

NOTE: This protocol is adapted from a previous publication by Vlachogiannis et al.⁷. We have added a step involving agarose embedding to successfully embed all populations of organoids.

1. Remove existing media from the well. Be careful not to aspirate the basement membrane domes.
2. Add an equal (equal to the volume of media removed from step 1) volume of cell recovery solution and incubate for 60 min at 4 °C.
3. Dislodge the basement membrane dome using a pipette and crush the basement membrane dome using a pipet tip. Collect the dissociated dome and cell recovery solution in a 1.5 mL tube.
4. Centrifuge at 300 x g and 4 °C for 5 min.
5. Remove the supernatant (cell recovery solution). Save all supernatants in separate tubes until the end when the presence of organoids is confirmed in the final pelleting step.
6. Add desired volume (Table 3) of cold PBS and gently pipette up and down to mechanically disturb pellet.
7. Centrifuge at 300 x g and 4 °C for 5 min.
8. Remove the supernatant (PBS).
9. Fix the pellet in a matched volume (e.g., 500 µL for one pellet from the 24 well plate culture condition, Table 3) of 4% PFA for 60 min at room temperature.
10. Following fixation, centrifuge at 300 x g and 4 °C for 5 min.
11. Remove the supernatant (PFA).
12. Wash with matched volume (e.g., 500 µL for one pellet from the 24 well plate culture condition, Table 3) of PBS and centrifuge at 300 x g and 4 °C for 5 min.
13. Prepare warm agarose (2% agarose in PBS).
    NOTE: Here, cell pellets for frozen sections can be directly re-suspended in 200 µL of OCT compound without further steps in Protocol 7.
14. Re-suspend the cell pellet in 200 µL of agarose (2% in PBS).
    1. Immediately after adding agarose, gently detach the cell pellet from the wall of the 1.5 mL tube using the 25 G needle attached to 1 mL syringe. As shown in Figure 3, if the cell pellet is not physically detached from the wall of the 1.5 mL tube, then there is a risk of losing all or part of the cell pellet during the agarose embedding process.
15. Wait until the 2% agarose in PBS is completely solidified.
16. Detach the solidified agarose block from the 1.5 mL tube using a 25 G needle attached to the 1 mL syringe.
17. Transfer the detached agarose block containing the cell pellet to a new 1.5 mL tube.
18. Fill the tube with 70% EtOH and proceed further using the conventional protocol for tissue dehydration and paraffin embedding.

8. Histology and Immunofluorescent cytochemistry (IFC) of organoids

1. Select the slide(s) for histology or IFC.
2. Before initiating the staining process, find out where the cells are located on the slide and draw a circle around the cells on the slide using a marker.
3. Draw the perimeter around the edge or boarder of the slide and where circles are located on the slide in a laboratory notebook to record their locations.
4. Perform desired staining.
   NOTE: During this process, marked circles disappear since regular maker is not resistant to the chemicals. Even some histology permanent markers may be erased during staining process.
5. After the staining process, place the slide over the drawing in the laboratory notebook to find the locations of cells on the slide.

Results

3D organoids were successfully established from a patient derived xenograft (PDX) model of bone metastatic prostate cancer (BMPC) as well as directly from patient bone metastatic prostate cancer tissue (Figure 4). Briefly, our PDX models of BMPC were established by intra-femoral (IF) injection of tumor cells into male Rag2^-/- c^-/- mice and then PDX tumors were harvested and processed as described in this manuscript. As shown in Figure 4, PD...

Discussion

3D organoids derived from patient bone metastasis prostate cancer cells are still relatively rare. Here, we describe strategies and further optimized protocol to successfully established serial 3D patient derived organoids (PDOs) of BMPC. In addition, protocols are described to secure the organoids in samples with lower cell density for IFC and IHC analysis. Differential phenotypes in the form of cyst, spheroids and more complex organoids indicate that this protocol provides culture conditions that allows for heterogonou...

Materials

Name	Company	Catalog Number	Comments
1 mL Pipettman	Gilson	F123602
1 mL Syringe	BD Syringe	329654
1.5 mL tube	Spectrum Lab Products	941-11326-ATP083
25G Needle	BD PrecisionGlide Needle	305122
4% Paraformaldehyde (PFA)	Alfa Aesar	J61899
70% Ethanol (EtOH)	VWR	BDH1164-4LP
A83-01	Tocris Bioscience	2939
Accumax	Innovative Cell Technologies, Inc.	AM105
adDMEM	Life Technologies	12634010
Agarose	Lonza	50000
Antibody -for Cytokeratin 5	Biolegend	905901
Antibody for Cytokeratin 8	Biolegend	904801
B27	Life Technologies	17504044
Bioluminescence imaging system, IVIS 200	Perkin Elmer Inc	IVIS 200
Cell Culture Plate - 24 well	Costar	3524
Cell Culture Plate - 48 well	Costar	3548
Cell Culture Plate - 6 well	Costar	3516
Cell Dissociation Solution, Accumax	Innovative Cell Technologies, Inc.	AM105
Cell Recovery Solution	Corning	354253
Cell Scraper	Sarstedt	83.180
Cell Strainer	Falcon (Corning)	352350
CO2 incubator	Fisher Scientific	3546
DAPI	Vector Vectashield	H-1200
DHT	Sigma-Aldrich	D-073-1ML
dPBS	Corning/Cellgro	21-031-CV
EGF	PeproTech	AF-100-15
FBS	Gemini Bio-Products	100-106
FGF10	PeproTech	100-26
FGF2	PeproTech	100-18B
Forceps	Denville Scientific	S728696
Glutamax	Gibco	35050-061
HEPES	Gibco	15630-080
LS Columns	Miltenyi	130-0420401
Magnetic Column Seperator: QuadroMACS Separator	Miltenyi	130-090-976
Marker	VWR	52877-355
Matrigel (Growth Factor Reduced)	Mediatech Inc. (Corning)	356231
Matrigel (High Concentration)	BD (Fisher Scientific)	CB354248
Microscope Imaging Software, Keyence	BZ-X800 (newest software) BZ-X700 (old software)
Microscope, Keyence	BZ-X700 (model 2016-2017)/BZ-X710 (model 2018-2019)
Mouse Cell Depletion Kit	Miltenyi	130-104-694
N-Acetylcysteine	Sigma-Aldrich	A9165-5G
Nicotinamide	Sigma-Aldrich	N0636-100G
Noggin	PeproTech	120-10C
OCT Compound	Tissue-Tek	4583
Parafilm	American National Can	N/A
Pen-Strep	Mediatech Inc. (Corning)	30-002-CI-1
Pipette tipes for 1 mL (Blue Tips)	Fisherbrand Redi-Tip	21-197-85
Plunger (from 3 mL syringe)	BD Syringe	309657
Prostaglandin E2	Tocris Bioscience	2296
R-Spondin 1	Trevigen	3710-001-01
SB2021190	Sigma-Aldrich	S7076-25MG
Small Table Top Centrifuge	ThermoFisher Scientific	75002426
Water Bath	Fisher Sci	2320
Y-27632 Dihydrochloride	Abmole Bioscience	M1817