Growth and Characterization of Irradiated Organoids from Mammary Glands

Summary

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

Abstract

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Introduction

Approximately 60% of the triple negative breast cancer (TNBC) patients choose breast-conserving therapy (BCT) as a form of treatment¹. In this treatment modality, the tumor containing part of the breast tissue is removed, and the surrounding normal tissue is exposed to ionizing radiation to kill any residual tumor cells. Treatment reduces recurrence in much of the breast cancer population; however, approximately 13.5% of treated patients with TNBC experience locoregional recurrences². Therefore, studying how radiation may recruit circulating tumor cells (CTCs) will lead to important insights into local recurrence^3,4.

Previous work has shown that radiation of the normal tissue increases recruitment of various cell types⁵. In pre-clinical models of TNBC, irradiation of normal tissue increased macrophage and subsequently tumor cell recruitment to normal tissues⁵. Immune status influenced tumor cell recruitment to irradiated sites, with tumor cell migration observed in immunocompromised subjects. Recapitulating these interactions using organoids derived from mammary glands will allow the observation of cell migration and cell-stromal interactions in real time with microscopy and live cell imaging to determine the role of radiation damage in altering tumor cell behavior.

Mouse mammary organoids have helped elucidate key steps in the development of the mammary gland. A mammary organoid is a multicellular, three dimensional construct of isolated mammary epithelium that is larger than 50 μm^6,7,8,9,10. Using primary epithelial organoids, Simian et al. evaluated necessary factors for branching in the mammary gland⁷. Shamir et al. discovered that dissemination can occur without an epithelial to mesenchymal transition, providing insight into the metastatic cascade⁸. Methods for generating and characterizing organoids from mammary gland tissue are well established^6,11,12,13. However, to our knowledge, methods for growing irradiated organoids from mammary glands have not been reported. A protocol for growing and characterizing irradiated organoids would be a critical step in recapitulating radiation-induced immune and tumor cell recruitment.

In this paper, we report a method for growing and characterizing irradiated mammary epithelial organoids in low adhesion microplates coated with a hydrophilic polymer that supports the formation of spheroids. These organoids were co-cultured with macrophages to examine immune cell infiltration kinetics. This work can be extended to include co-culturing organoids with adipose cells to recapitulate mammary characteristics, breast cancer cells to visualize tumor cell recruitment, and CD8+ T cells to study tumor-immune cell interactions. Previously established protocols may be used to evaluate irradiated organoids. Earlier models co-culturing mammary organoids and immune cells have shed light on mechanisms of metastasis and dissemination. DeNardo et al. found that CD4+ T cell regulation of tumor associated macrophages enhanced a metastatic phenotype of mammary adenocarcinomas¹⁴. Co-culture models have also been used to elucidate mechanisms of biological development. Plaks et al. clarified the role of CD4+ T cells as down-regulators of mammary organogenesis¹⁵. However, our group is the first to establish a procedure of visualizing how normal tissue irradiation influences immune cell behavior. Because normal tissue irradiation has been shown to enhance tumor cell recruitment⁵, this protocol can be further developed to analyze how tumor cell behavior is altered by irradiation of normal tissue and cells, leading to a greater understanding of cancer recurrence.

Protocol

Animal studies were performed in accordance with institutional guidelines and protocols approved by the Vanderbilt University Institutional Animal Care and Use Committee.

1. Preparation of mice and cell acquisition (adapted from Nguyen-Ngoc et al.¹¹)

1. Sacrifice athymic Nu/Nu mice (8-10 weeks old) using CO­₂ asphyxiation followed by cervical dislocation. Clean the skin using 70% ethanol.
2. Resect abdominal and inguinal mammary glands from mice using pre-sterilized scissors and forceps. Remove lymph nodes before resection. Rinse in sterile 1x phosphate buffered saline (PBS) (Figure 1A).
3. Place it in 15 mL tubes with 10 mL of Dulbecco’s Modified Eagle Media/Nutrient Mixture F12 (DMEM/F12) for transport. Samples can be kept overnight at 4 °C or processed immediately. Keep on ice.
4. Irradiate samples at 20 Gy using a cesium source (Figure 1B).
5. 45 min after irradiation, place mammary glands in a 35 mm sterile cell plate and mince with scalpels (Figure 1C,D). Mince with approximately 40 strokes until the tissue relaxes and pieces are obtained that are no larger than approximately 1 mm² in area.
6. Transfer to the collagenase solution in a 50 mL centrifuge tube. Collagenase solution consists of 2 mg/mL collagenase (see Table of Materials), 2 mg/mL trypsin, 5% v/v fetal bovine serum (FBS), 5 μg/mL insulin, and 50 μg/mL gentamicin in DMEM/F12 media. Use 10 mL collagenase solution per mouse.
7. Place in a water bath at 37 °C, vortexing every 10 min for 30-60 min. Digestion is complete when the collagenase solution is cloudy (Figure 1E,F).
8. Spin down the digested solution at 450 x g for 10 min at room temperature (RT). Three layers will be observed. The supernatant is composed of fat, the middle layer is an aqueous solution, and the bottom is a pellet. The pellet will appear red as it is a mixture of epithelial cells, individual stromal cells, and red blood cells (Figure 1G).
9. Precoat all pipettes, pipette tips, and centrifuge tubes with bovine serum albumin (BSA) solution prior to contact. BSA solution consists of 2.5 w/v % BSA in Dulbecco’s Phosphate Buffered Saline (DPBS). For pre-coating, simply add then remove BSA solution to the inside of the pipette tip and tubes. BSA solution can be reused, although it should be sterile filtered before each experiment.
10. For additional recovery, transfer the supernatant to a fresh BSA coated 15 mL tube. Pipette up and down vigorously to disperse fat layer. Centrifuge at 450 x g for 10 min at RT. Aspirate the supernatant, leaving a small amount of media in the tube to avoid aspirating the cell pellet.
11. Aspirate the aqueous layer from the tube with the original pellet.
12. Add 10 mL of DMEM/F12 to the tube with the original pellet and transfer to the second tube. Pipette vigorously to combine and resuspend the two pellets.
13. Centrifuge at 450 x g for 10 min at RT. Aspirate the supernatant and add 4 mL of DMEM/F12 to the tube.
14. Add 40 μL of deoxyribonuclease (DNase) to the suspension and gently shake by hand for 2-5 min at RT. DNase solution consists of 4 U/mL DNase in DMEM/F12.
15. Add 6 mL of DMEM/F12 and pipette thoroughly. Centrifuge the tube at 450 x g for 10 min at RT.
16. Aspirate supernatant to the 0.5 mL mark. Resuspend in 10 mL of DMEM/F12 and pipette thoroughly.
17. Pulse to 450 x g and stop 4 s after reaching that speed.
18. Repeat steps 1.16-1.17 three more times to purify organoids via centrifugal differentiation. The pellet should now be an off-white color consisting of only epithelial organoids (Figure 1H).
    NOTE: Organoids can also be filtered using sterile mesh 40 μm filters. After step 1.16, pipette media containing organoids through a filter into a centrifuge tube, and then rinse with 5 to 10 mL of DMEM/F12 media. Flip the filter over a new 50 mL centrifuge tube. Pass 10 mL of DMEM/F12 media through, going the opposite way to rinse off any retentate. The retentate should consist of organoids, and the filtrate should consist mainly of stromal cells, which can be discarded or kept if desired.

2. Determining density and plating organoids

1. Resuspend pellet in 10 mL of DMEM/F12. Pipette thoroughly to create a homogenous solution.
2. Transfer 50 µL to a 30 mm Petri dish, and view under a phase contrast microscope at 20x. Count the number of organoids with a tally counter.
   NOTE: Here pipette tips have been consistently used with a minimal diameter of 457 μm, which is 5-10 times the diameter of the organoids that are seeded. For transferring volumes of 2 mL or larger (e.g., steps 1.16 and 2.1), use serological pipettes with tip diameters excess of 1,500 μm.
3. Calculate the organoid density using the following equation:
   
   The desired density is 1,000 organoids/mL to simplify further dilution. If the density is too low, centrifuge at 450 x g for 5 min and aspirate media. Add media necessary to reach 1,000 organoids/mL, and pipette thoroughly to create a homogenous mixture.
   1. To grow organoids in a protein matrix, seed organoids at a concentration of 1 organoid/μL in collagen type 1 diluted to 87% or in basement membrane extracted from Engelbreth-Holm-Swarm mouse sarcoma. While working with samples, keep on ice.
   2. To freeze organoids, transfer the desired volume to a separate centrifuge tube. Spin down at 450 x g for 5 min. Aspirate media, and then add the same volume of 90% FBS/10% DMSO. Resuspend the organoids, and then aliquot into cryotubes. Transfer to -80 ˚C, and then to liquid nitrogen within one week.
   3. To thaw, warm in a 37 ˚C water bath for one min. Centrifuge at 450 x g for 5 min, and then aspirate freezing media. Rinse with sterile DPBS, and then centrifuge again. Aspirate DPBS and add organoid media.
4. Pipette 50 µL (50 organoids) into each well of the low adhesion plate (Figure 1I).
5. Add 150 µL of organoid media to bring the total working volume to 200 µL. Organoid media consists of 1% penicillin-streptomycin and 1% insulin-transferrin-selenium (ITS) in DMEM/F12 media.
6. Every 2 days replace media carefully.
   NOTE: Low adhesion plates are not tissue culture treated; therefore, the cells can be easily detached. Aspirate media slowly by tilting the plate and inserting the pipette tip at the edge of each well. Leave a small amount of media in the bottom of the well. Add new media slowly to avoid applying unnecessary shear forces to organoids.

3. Co-culturing with macrophages

1. Maintain GFP or dTomato-labelled RAW 264.7 macrophages in DMEM media supplemented with 10% FBS and 1% penicillin-streptomycin. Seed 1 x 10⁴, 5 x 10⁴, or 1 x 10⁵ cells/mL into organoid media.
2. Use live cell phase contrast and fluorescent imaging to monitor macrophage infiltration over time.

4. Immunofluorescence staining of organoids

NOTE: Organoids can be stained in low adhesion wells or can be transferred to chamber slides. To transfer, gently pipette up and down until organoids have detached from plates. Transfer to chamber slides and incubate for 4-8 h to allow organoids to adhere to the plate surface.

1. Remove organoid medium from the wells by carefully aspirating. Fix samples with 10% neutral buffered formalin for 15 min at RT.
2. Wash 3x 5 min in 1x PBS. If desired, fixed samples can be stored at 4 °C for one week for further staining.
3. Permeabilize with 0.1% 4-(1,1,3,3-Tetramethylbutyl) phenyl-polyethylene glycol for 5 min.
   NOTE: To stain for F-actin, incubate samples with phalloidin diluted 1:1,000 and 1.67 nM bisbenzimide nuclear dye in 1% PBS/BSA for 1 h at RT. Then, proceed to step 4.8.
4. Bermeabilize with 0.5PBS. Samples can be stored at 4opy images and immunofluorescent images. mechanisms that contribute to CTC rlock with 5% normal goat serum in 0.1% PBS/Polyethylene glycol sorbitan monolaurate (PBST) for 1 h at RT. Wash 3x 5 min with PBS.
5. Incubate with Anti-Cytokeratin 14 diluted 1:1,000, E-Cadherin diluted 1:200, or Tight Junction Protein One diluted 1:100 in 1% NGS in PBST for 1 h at RT. Wash 3x 5 min in PBST.
6. Incubate with Goat Anti-Rabbit secondary diluted 1:200 with 1% NGS/PBST for 1 h at RT. Cover with foil to avoid light exposure.
7. Wash 3x 5 min in PBS. Use the nuclear dye (see Table of Materials) to stain nuclei.
8. Wash 3x 5 min in PBS. If using chamber slide, mount with a coverslip. Store wrapped in foil at 4 °C for up to 2 weeks.

Results

Irradiated epithelial mammary organoids were successfully obtained from mouse mammary glands, processed, and cultured on low-adhesion plates (Figure 1). Organoid yield was tested by seeding in different growth environments (Figure 2A-G). Seeding cells directly onto tissue culture treated 10 cm cell plates yielded an overgrowth of fibroblast cells. Fibroblasts were identified under phase contrast microscopy in or near the same plane of focus as organoids, and they quickly gre...

Discussion

In this protocol, we have developed a method for reproducible growth and characterization of irradiated mammary organoids (Figure 1). An irradiation dose of 20 Gy was applied to mirror previous in vivo models of tumor cell recruitment⁵. Irradiation of mammary glands ex vivo prior to organoid formation allowed for isolation of radiation damage effects without a corresponding infiltration of immune cells. The development of an in vitro irradiated normal tissue model ena...

Materials

Name	Company	Catalog Number	Comments
10% Neutral Buffered Formalin	VWR	16004-128
Anti-cytokeratin 14	abcam	ab181595	Lot: GR3200524-3
Bovine Serum Albumin	Sigma	A1933-25G
Collagen Type I	Corning	354236
Collagenase from Clostridium Histolyticum, Type VIII	Sigma	C2139
Collagenase I	Gibco	17018029
DMEM/F12	Thermofisher	11320-033
DNAse	Roche	10104159001
DPBS	Fisher	14190250
E-Cadherin	Cell Signaling	24E10	Lot: 13
FBS	Sigma	F0926
Gentamicin	Gibco	15750
Goat anti-rabbit secondary	abcam	ab150077	green Lot: GR3203000-1
Goat anti-rabbit secondary	abcam	ab150080	red Lot: GR3192711-1
Hoechst 33342	Fisher	62249	Lot: TG2611041
Insulin (10 mg/mL)	Sigma	I9278
Insulin-Transferrin-Selenium, 100x	Gibco	51500-056
Matrigel Basement Membrane (basement membrane extracted from Engelbreth-Holm-Swarm mouse sarcoma)	Corning	356237
Normal Goat Serum	Vector Laboratories	S-1000
Nuclon Sphera 96 well plates	Thermo	174927
PBS	VWR	10128-856
Pen/strep	Fisher	15140122
Phalloidin	abcam	ab176757	Lot: GR3214582-16
Tight Junction Protein 1	Novus	NBP1-85047	Lot: C115428
Triton X-100 (4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol)	Sigma	X100-100ML
Trypsin	Gibco	27250-018
Tween-20 (Polyethylene glycol sorbitan monolaurate)	Sigma	P1379-100ML