A protocol for selection in mice of highly metastatic ovarian cancer cell with omental tropism

Summary

Preclinical models mimicking spontaneous omental metastasis from ovarian cancer (OC) can benefit the study of anti-metastatic therapies for OC patients. Here, we present a protocol to establish a highly metastatic (HM) mouse model with omental tropism by in vivo selection. We describe the processes of implanting OC cells in the ovaries of mice and obtaining HM sublines from their omental metastases. HM cells can metastasize from the ovary to the omentum within 2 weeks.

For complete details on the use and execution of this protocol, please refer to Ying et al.¹

Graphical abstract

Highlights

• •Steps to establish an ovarian cancer mouse model of omental metastasis
• •Protocol is adaptable to both immunodeficient and immunocompetent mice
• •In vivo selection enables enrichment of highly metastatic cells with omental tropism
• •Efficient modeling of spontaneous omental metastasis by using highly metastatic cells

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Preclinical models mimicking spontaneous omental metastasis from ovarian cancer (OC) can benefit the study of anti-metastatic therapies for OC patients. Here, we present a protocol to establish a highly metastatic (HM) mouse model with omental tropism by in vivo selection. We describe the processes of implanting OC cells in the ovaries of mice and obtaining HM sublines from their omental metastases. HM cells can metastasize from the ovary to the omentum within 2 weeks.

Before you begin

Patients with ovarian cancer (OC) frequently present with omental metastasis, which represents a major challenge in treatment. Mouse models harboring orthotopic OC xenografts can mimic the natural course of tumor progression, but with uncertainty in omental metastasizing. Here, we describe protocols for in vivo selection of highly metastatic (HM) tumor cells with omental tropism and establishment of mouse models of spontaneous omental metastases. This protocol is demonstrated using OC cell lines, and presumably, it can be employed for patient-derived models as well.

The protocol contains three main sections: obtainment of tumor tissue blocks, in vivo selection of HM OC cells, and isolation and primary culture of HM cells from omental metastasis.

Institutional permissions

All animal experiments were conducted in accordance with the guidelines for animal experimentation at Huazhong University of Science and Technology and approved by the Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology. The experiments were approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. Readers who use this protocol will need to acquire permission from their relevant institutions.

Labeling of the OC cells

Timing: 1–2 weeks

For in vivo imaging system (IVIS) imaging, OC cell lines (ES2 and ID8 are used in this protocol) should be labeled with luciferase. All cell lines should be authenticated using short tandem repeat profiling before experiment starts.

• 1.Seed OC cells into a 96-well plate at a quantity of 5000/well.
• 2.Infect the cells with LV5-LUCIFERASE lentivirus for 16 h.

Note: The multiplicity of infection and the time of incubation should be optimized for each cell line

• 3.Screen the cells with 5 μg/mL puromycin for 7 days.

Note: The screening antibiotic depends on the lentivirus used. The concentration of antibiotic and the screening time should be optimized for each cell line.

• 4.Establish the single cell-derived clones through serial dilution of the cells into 96-well plates.
• 5.Apply the bioluminescent imaging to verify the expression of luciferase in OC cells.

Note: For a more detailed process, refer to Guo J. et al.²

Preparation of the surgical instruments and objects

Timing: 24 h

• 6.Prepare the surgical kit.

Note: The kit contains 2 ophthalmic forceps, 2 ophthalmic scissors, microsurgical instruments (2 forceps, 1 scissor, 1 needle holder, 7-0 sutures with round needle), needles (1 round and 1 angled), 5-0 sutures, small cotton balls, 2 small stainless-steel cups and several operating towels. All instruments should be kept in autoclavable containers.

• 7.Sterilize the surgical kit using a gravity autoclave set at 121°C for a minimum of 30 min.
• 8.Exsiccate the kit and let cool to room temperature (20°C–24°C).

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Bacterial and virus strains
LV5-Luciferase lentivirus	Suzhou GenePharma	N/A
Chemicals, peptides, and recombinant proteins
D-luciferin potassium salt	PerkinElmer	#122799
Collagenase type 1	Biosharp	BS163
Isoflurane	RWD	R510-22-10
Puromycin	Shanghai BasalMedia Technologies	S250J0
Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F12)	Shanghai BasalMedia Technologies	L310KJ
Phosphate-buffered saline (PBS)	Shanghai BasalMedia Technologies	B320KJ
Trypsin-ethylenediaminetetraacetic acid (EDTA)	Shanghai BasalMedia Technologies	S310KJ
Fetal bovine serum (FBS)	Nanjing BioChannel Biotechnology	BC-SE-FBS07
Experimental models: Cell lines
ES-2	China Center for Type Culture Collection	GDC0322
ID8-luc	Xiao Haihua’s laboratory, Chinese Academy of Sciences, Beijing	N/A
Experimental models: Organisms/strains
Mouse: 4-week-old female BALB/c nude mice: CAnN.Cg-Foxn1nu/Crl	Charles River	401
Mouse: 4-week-old female C57BL/6N: C57BL/6NCrl	Charles River	213
Software and algorithms
Living Image Software	Caliper Life Science	4.3.1 https://www.perkinelmer.com.cn/lab-products-and-services/resources/in-vivo-imaging-software-downloads.html
Other
Anesthesia apparatus	Shenzhen RWD	R500
Syringe 29G	Becton Dickinson	Cat# 324702
Sterile sutures with round needle (7-0 3/8)	Ningbo Medical Needle	N/A
Sterile sutures (5-0)	Yangzhou Jinhuan Medical Appliance Factory	N/A
Round needle (1/2 6 × 14)	Shanghai Pudong Jinhuan Medical Products	N/A
Angled needle (3/8 6 × 17)	Shanghai Pudong Jinhuan Medical Products	N/A
Microsurgical forceps	Suzhou Stronger Medical Instruments	XTN1405
Microsurgical scissor	Suzhou Stronger Medical Instruments	XTJ1410
Microsurgical needle holder	Suzhou Stronger Medical Instruments	XGQ1400
Ophthalmic forceps	Suzhou Stronger Medical Instruments	JYN1020
Ophthalmic scissor	Suzhou Stronger Medical Instruments	JYJ1040
Cell filter (40 μm)	Biosharp	BS-40-CS
Culture dish (65 mm)	NEST Biotechnology	#705001
Culture flask (T25)	NEST Biotechnology	#707003

Materials and equipment

• •10% Complete DMEM/F12 medium: add 50 mL FBS in 450 mL DMEM/F12 medium.

[Store at 4°C for up to 2 weeks].

• •15 mg/mL D-luciferin solution: add 1 g D-luciferin potassium salt in 66.66 mL PBS.

[Store at –20°C for up to 1 year].

• •150 μg/mL D-luciferin solution: add 10 μL 15 mg/mL D-luciferin solution in 1 mL PBS.

[Dilute when it will be used].

• •1% Collagenase type 1 Solution: add 1 g Collagenase type 1 in 100 mL DMEM/F12 medium.

[Store at –20°C for up to 1 year].

• •IVIS software setting: Luminescent (Exposure time: Auto, Binning: 8, F/stop: 1), Photograph (Binning: 2, F/Stop: 8), Field of View: D, Subject height: 1.5 cm (Figure 1).

Figure 1.

The software setting of IVIS

The screenshot of Living Image Software for in vivo bioluminescent imaging.

Step-by-step method details

Obtainment of tumor tissue blocks

Timing: 2–3 weeks

Timing: 1 h (for steps 16–22)

This section describes how to obtain the tumor tissue-blocks from the subcutaneous tumor or patient-derived tissues for orthotopic transplantation.

Harvest luciferase labeled OC cells

Note: For patient-derived xenograft (PDX) models, steps 1 to 19 can be skipped.

• 1.Expand the OC cells labeled with luciferase in complete DMEM/F12 medium.

Note: ES2 and ID8 cells labeled with luciferase are cultured in complete DMEM/F12 medium. Different cells prefer different media.

• 2.For inoculation, collect the OC cells at 80%–90% confluence.
• 3.Prewarm the culture medium, PBS and trypsin-EDTA solution at room temperature for 30 min before operation.
• 4.Remove the medium in the culture dishes and gently rinse the cells with PBS.
• 5.Add 2 mL trypsin-EDTA solution and gently shake the flasks. Allow enzymatic reaction for 30 s–60 s.

CRITICAL: Optimize the enzymatic reaction time for each cell lines to reduce the influence of digestion on the cell viability.

• 6.Add 2 mL complete medium, gently suspend the cells. Mix all cell suspensions up in a new centrifuge tube.
• 7.Calculate the total number of cells: Total = cell concentration (determined by a cell counter after 0.4% trypan blue staining) × volume. After a 300 × g centrifugation at room temperature for 5 min, discard the supernatant and obtain the cell pellet.
• 8.Wash the cells with PBS, resuspend the cells with 1 mL serum-free medium and calculate the total number of viable cells.
• 9.Dilute the cell suspension with serum-free medium to the concentration of 1–3 × 10⁷/mL in a new 1.5 mL Eppendorf tube.

CRITICAL: Keep the tube on ice during transportation to maintain the viability of cells. Inoculate the mice within 1 hour.

Subcutaneous inoculation and tumor growth

Note: The subcutaneous tumor serves as the donor of the tumor tissue blocks for subsequent orthotopic transplantation. For the tumor derived from OC cell lines and the primary cells of OC patients, steps 10 to 19 should be conducted. For the PDX derived from the solid tumor tissues of OC patients, these steps can be skipped.

• 10.Sterilize all materials and equipment using 75% ethanol and a 30 min ultraviolet irradiation in a superclean bench.

Note: This step can be carried out at the same time as harvesting the OC cells

• 11.Inhale the suspension into the syringe, 150 μL (1.5–4.5 × 10⁶ cells) each time. Mix the cell suspension before each extraction.
• 12.Disinfect the scapular area of the mouse skin with 75% ethanol and wait for the ethanol to dry out.
• 13.Inject the cell suspension subcutaneously.

Note: Successful injection will generate a skin bulge in the inoculation area.

Note: For most human OC cell lines, BALB/c Nude mice are used for inoculation. For ID8 cells, C57BL/6N mice are used for inoculation. For PDX derived from the primary cells and cell lines with poor tumorigenicity, mice with higher levels of immunodeficiency should be used, such as NOD-SCIDs, NSG, etc.

CRITICAL: Ensure that there is no fluid leak out.

• 14.Return the mice to their original cages if they do not exhibit any significant discomfort.
• 15.Use a slide caliper to measure the tumor diameters when subcutaneous tumors can be observed. Calculate the tumor volume as Volume = length × width × width / 2. Troubleshooting 1.

Note: For subcutaneous tumors, a suitable volume of the tumor is 0.5 cm³.

CRITICAL: We recommend to use two mice for individual cell line in this step to ensure that enough tissue blocks can be obtained for the following steps.

Dissociation of subcutaneous tumors and preparation of tumor tissue blocks for orthotopic transplantation model

Note: Tissue blocks can be acquired from subcutaneous tumors derived from OC cell lines or the primary cells of OC patients, or directly from the surgical specimens of tumor tissues from OC patients.

• 16.When the volume of subcutaneous tumor reaches approximately 0.5 cm³, euthanize the mouse by cervical dislocation under isoflurane anesthesia.
• 17.Place the mouse in a prone position and disinfect the mouse skin with 75% ethanol.
• 18.Lift and cut the skin in the middle line of the tumor surface.
• 19.Dissociate the tumor from the mouse and place it in sterile PBS solution at 4°C.

CRITICAL: If transplantations cannot be started immediately, the tumor from subcutaneous model or the tissue from OC patient should be kept in pre-cooled serum-free culture medium at 4°C, and the tissue blocks should be transplanted to mice within 24 hours.

• 20.Remove the tumor capsule with forceps and scissors, and wash the tumor with cold PBS three times.
• 21.
  Check the tumor carefully to identify the tumor tissue sections with better vitality (tissues without bleeding or necrosis).

  • a.
    Obtain 3-4 pieces of tumor tissue from these sections.
  • b.
    Place them into a new dish containing fresh cold PBS.

Note: As the central area of tumor is usually more likely to be necrotic due to an insufficient supply of blood and oxygen, the solid tissues located in the peripheral regions generally tend to be more active.

• 22.
  Cut the selected tumor tissues into small tissue-blocks with a maximum diameter of no more than 1 mm.

  • a.
    Wash the tissue blocks with fresh PBS three times.
  • b.
    Keep them in pre-cooled serum-free culture medium at 4°C.
  • c.
    Transplant the tissue blocks to mice within 4 h.

In vivo selection of HM OC cells

Timing: 3 months

Timing: 10–15 min for each mouse (for steps 23–40)

Timing: 20 min for each mouse (for steps 41–48)

Timing: 3–4 weeks for each cycle (for steps 58–60)

This section aims to enrich the OC cells with highly metastatic ability through the in vivo selection, which involves establishing the orthotopic model, in vivo bioluminescent imaging, and dissociating the omental metastases.

Establishing the orthotopic transplantation model

• 23.Place the surgical instruments and objects (the stereomicroscope, sterilized surgical kit, mouse mask, and operating platform) on a clean bench.
• 24.Disinfect the surfaces of all objects with 75% ethanol spray followed by 30 min UV irradiation (Figure 2).
• 25.Open the surgical kit on the clean bench, and cover the operating platform by the sterile operational towel.
• 26.Place the sterile surgical instruments and objects on the platform (Figure 3).
• 27.Set the gas flow of induction cage at 1.5 L/min, and the concentration of isoflurane at 2.5%.
• 28.Weigh the mice and put them in the induction cage, and then wait for them to be fully anesthetized.

Note: For most human OC cell lines, BALB/c nude mice are used for transplantation. For ID8 cells, C57BL/6 mice are used for transplantation. For PDXs and cells with poor tumorigenicity, mice with higher levels of immuno-deficiency should be used, such as NOD-SCIDs, NSG etc. The mice aged 5–6 weeks are used for orthotopic transplantation. The body weight should be more than 16 g in order to guarantee that they can tolerate the surgery.

CRITICAL: We recommend utilizing three mice for this step, considering the possibility of unexpected unsuccessful modelling. If all succeeded, in addition to the orthotopic xenograft for the next passage mice, the extra omental tumor lesions obtained are recommended for primary cell isolation, paraffin embedding and cryopreservation.

• 29.Placed the mouse on the right side after it is fully anesthetized.
• 30.Modify the gas flow of mask at 0.75 L/min and keep the mouse anesthetized by isoflurane through the respiratory mask.

Note: In the step 28, all three mice are anesthetized in the induction cage simultaneously. While in the step 29, the mice receive surgery one by one. The mice waiting for transplantation are left in the induction cage to keep anesthetized.

• 31.
  Open the abdominal cavity.

  • a.
    Disinfect the skin of its left abdomen with 75% ethanol.
  • b.
    Use curved ophthalmic forceps to lift the skin at the lowest point and make a 0.4–0.8 cm incision with ophthalmic scissors.

CRITICAL: In this position, a depression can be observed on the left lateral abdomen, between the rib bow and the thigh, near the spine (Figure 4).

• 32.Search a fat pad sized approximately 0.3 cm × 0.4 cm under the abdominal incision near the spine side.
• 33.Gently grip the edge of the fat pad with micro forceps and slowly pull it outside.

Note: A red spherical organization wrapped in the center can be observed, which is the ovary of the mouse (Figure 5A). Troubleshooting 2.

• 34.Keep the fat pad moist with normal saline throughout the surgery. Place the mouse under the stereomicroscope.
• 35.
  Open the ovarian bursa.

  • a.
    Gently grab the capsular membrane surrounding the ovary along the borderline of the ovarian bursa and the adipose tissue with micro forceps.
  • b.
    Make an incision with a micro scissor whose size can just match or is slightly smaller than the transplanting tissue-block with a micro scissor (Figures 5B and 5C).
• 36.Insert a tissue-block into the ovarian bursa with micro forceps, and push it deeply into the bursa as much as possible. Troubleshooting 3.
• 37.Close the incision with a 7-0 suture (Figure 5D).
• 38.Gently place the fat pad back to the abdominal cavity.
• 39.Close the abdominal cavity with a 5-0 suture. Clean and disinfect the wound with 75% ethanol.
• 40.Turn off the gas flow of mask and wait for the mouse to awaken from the anesthesia, and then return it back to the cages.

Note: It usually takes the mouse around 3 min to awaken. And consider the small incision and their quick recovery, the pain relief isn’t required after surgery. No significant discomfort is observed after the surgery. No infection is observed in our experiments due to the SPF circumstance and aseptic technique.

Figure 2.

The surgical instruments and objects

(A) The stereomicroscope.

(B) The operating platform.

(C) The mouse mask.

(D) The sterilized surgical kit.

(E) The sterilized stainless-steel cups.

Figure 3.

Surgical setup

(A) The sterile operational towel.

(B) The round and angled needles.

(C) The small stainless-steel cup with 75% ethanol and sterile cotton swabs (D) The small stainless-steel cup with normal saline and sterile cotton balls (E) The 5-0 sutures.

(F) The 7-0 sutures with round needle.

(G) The microsurgical scissor.

(H) The microsurgical needle holder.

(I) The ophthalmic scissors.

(J) The ophthalmic forceps.

(K) The microsurgical forceps.

(L) The stereomicroscope.

(M) The respiratory mask.

Figure 4.

The position of the incision

Left: the lowest point in the side view (the yellow arrow). Right: the incision should be made at the lowest point in the depression area between the rib bow and the thigh near the spine on a left lateral position. Circle: the spleen (white); the kidney (blue); the suitable area for incision (red). Figures originally published in Ying F. et al., iScience.¹

Figure 5.

The process of implanting the tissue blocks

(A) The ovary of the mouse, circle: the fat pad (yellow), the ovary (red).

(B) The incision of the bursa of ovary, green arrow.

(C) Insert the tissue block into the bursa of ovary, circle: the tissue block (blue).

(D) Stitch the incision of bursa with a 7-0 suture.

In vivo bioluminescent imaging

Note: The first imaging should be performed 7 days after the implantation.

Optional: For tumors that cannot be labeled, such as PDX, disease progression can be detected by other equipment, such as X-ray, magnetic resonance imaging, computed tomography, and ultrasound. Ovarian tumor size can be estimated by abdominal palpation. In advanced diseases, ascites may also be observed.

• 41.
  Prepare the IVIS and the luciferase substrate working solution.

  • a.
    Initiate the IVIS and set the imaging mode to bioluminescence.
  • b.
    Disinfect the imaging room with 75% ethanol and UV irradiation.
  • c.
    Thaw the luciferase substrate working solution at room temperature or in a 37°C incubator while protecting it from light.

Note: The solution should be mixed up before injection.

• 42.Transport the mice to the imaging room in sterile cages.
• 43.Initiate the gas anesthesia system. Set the gas flow of induction cage at 1.5 L/min, concentration of isoflurane at 2.5% and oxygen pressure at 0.1 MPa.
• 44.Put the mice into the anesthesia induction cage 4 min prior to the injection time. Then wait for the mice to be fully anesthetized.
• 45.Weigh the mice and calculate the substrate dosage for each mouse.
• 46.Intraperitoneally inject the 15 mg/mL D-luciferin solution into the mice at a dose of 150 mg/kg (10 μL/g) and allow the mice to metabolize for 15 min before imaging.

Note: The peak imaging time is 10–20 min after injection. The bioluminescence signals will reach a plateau during this period. Usually there are several mice waiting for imaging. We recommend to intraperitoneally administer the substrate to different mice at 3-min intervals, and record the time point for each individual, so as to have enough time to complete imaging for each mouse.

• 47.When the imaging time is reached, set the gas flow of the imaging chamber to 0.5 L/min, place the mouse in the chamber, and acquire the image.
• 48.After successfully imaging, close the isoflurane flow and allow the mouse to wake up on the heat pad and then back to the cages. Troubleshooting 4.

Note: The imaging interval is inversely related to the malignancy of the grafts. The more rapidly the tumors progress, the more frequently the imaging should be done. In our experiments, the imaging is performed every 7 days for ID8, while every 3 days on the mice bearing ES2 due to their rapid growth.

Dissociating the omental metastases

• 49.Euthanize the mouse by cervical dislocation under isoflurane anesthesia when peritoneal metastasis is observed.

Note: The typical bioluminescent image of metastasis can refer to Figure 6A. Due to the difference between OC cells, it is difficult to assign an exact imaging signal count.

• 50.Lay the mouse on a piece of sterile gauze, and use 75% ethanol to disinfect the whole abdominal skin of the mouse.
• 51.Make a horizontal “H” shaped incision to expose the abdominal organs.

Note: The ophthalmic forceps and scissors should be sterilized in advance.

• 52.Check the left ovary to confirm tumor formation.
• 53.Search the omentum between the stomach and the spleen. Troubleshooting 5.
• 54.Dissociate the omentum and wash with PBS three times.
• 55.Soak the omentum in 150 μg/mL D-luciferin solution in a 60 mm dish and incubate at 37°C for 2–3 min.
• 56.Put the dish into the imaging chamber to conduct ex vivo imaging. Based on the image result, determine the tissue areas with strong bioluminescent signals.

Note: The strong signals indicated that these parts of omental tissue contain many metastatic tumor cells.

• 57.Harvest these parts of tissue and remove the bleeding or necrosis sections to obtain tumor tissue blocks for orthotopic transplantation to the mice of next passage.

Figure 6.

The progress of the orthotopic mouse model

(A) The representative bioluminescent images of the tumor progress of the orthotopic model. Figures originally published in Ying F. et al., iScience.¹

(B) The representative photograph of metastatic omentum and other abdominal organs. Arrows: red (omentum), orange (stomach), yellow (spleen), blue (pancreas).

In vivo selection cycle

• 58.Transplant the tissue-blocks obtained from step 57 into the ovary bursas of passage 2 mice.
• 59.The subsequent processing and maintenance of the omentum tissues are the same as in steps 23 to 58.
• 60.Wait for the formation of peritoneal metastasis. Two more cycles of in vivo selection are conducted until passage 3 mice. Troubleshooting 6.

Note: It usually takes 3–4 weeks for each cycle. The tumors in passage 2 and 3 mice will progress more rapidly than those in passage 1 mice.

CRITICAL: The in vivo selection should be conducted until at least passage 3 at least. More passages can be accepted to enrich HM cells with enhanced metastatic ability.

Isolation and primary culture of HM cells from omental metastasis

Timing: 1–2 weeks

This section describes the procedure of isolating the HM cells from the omental metastasis and cultivating the cells ex vivo, all of which must be done in a sterile setting.

Isolating the HM cells from omental metastasis from passage 3 mouse

• 61.Obtain the omentum from the passage 3 mouse as previously described (dissociating the omental metastases). Wash it with PBS three times.
• 62.Put the tissue in a new 5 mL Eppendorf tube, and cut the tissue into pieces of 0.1 mm³ in size.
• 63.Prepare collagenase type 1 working solution by adding 200 μL of 1% collagenase type 1 solution in 1800 μL of serum-free medium at a final concentration of 1 mg/mL.
• 64.Add 2 mL collagenase type 1 working solution and incubate the tube on a thermostat shaker at 37°C for 1 h.
• 65.Add 2 mL fresh medium to dilute the enzyme.
• 66.Mix the suspension and infiltrate with a 40 μm cell filter.
• 67.Obtain the precipitation through a 200 × g centrifugation at room temperature for 5 min.
• 68.Resuspend the precipitation in 5 mL complete DMEM/F12 medium and seed into a 25 cm² culture flask.

Note: No antibiotics are used in the culture media.

Primary culture of HM cells

• 69.Wash the cells with PBS and add fresh culture medium after 24 h of incubation at 37°C.
• 70.Observe the adherent cells under an inverted microscope.
• 71.Change the culture medium every three days.
• 72.Passage cells at 85%–90% confluence.

Expected outcomes

Spontaneous omental metastasis and ascites formation can be observed in orthotopic models. The omentum will be thicker after metastasis formation (Figure 6).

Tumor progression was gradually accelerated, and the survival of tumor-bearing mice was shortened during in vivo selection. The ES2-HM and ID8-HM cells were derived from the omental metastases of passage 3 mice. Morphologically, HM cells were significantly slenderer and more refractive (Figure 7).

Figure 7.

The morphology of highly metastasis (HM) cells

(A) The morphology of ES2 and ES2-HM cells under light and fluorescence microscope Right: light microscope, Left: fluorescence microscope, magnification 200×, scale bar: 50 μm.

(B and C) The morphology of ID8 and ID8-HM cells under light microscope, magnification 100×, scale bar: 100 μm (B). magnification 200×, scale bar: 50 μm (C). Figures originally published in Ying F. et al., iScience.¹

The mice bearing ES2-HM xenografts exhibited widespread intraperitoneal metastasis, while the disease progression in the ES2 group was much milder. Similarly, ID8-HM cell-derived orthotopic OC xenografts developed intraperitoneal metastases more rapidly and more widely than ID8 tumors (Figure 8).

Figure 8.

The metastatic abilities of HM cells in vivo

(A) The representative bioluminescent images of orthotopic models of ES2-HM and ES2 cells. Right: the curves of tumor burden by bioluminescent imaging, data are represented as mean ± SD.

(B) The representative bioluminescent images of orthotopic models of ID8-HM and ID8 cells. Right: the curves of tumor burden by bioluminescent imaging, data are represented as mean ± SD. Figures originally published in Ying F. et al., iScience.¹

Limitations

This protocol requires a long experimental period and high equipment requirements. The in vivo model does not reproduce the initial stage of tumorigenesis. This model requires the engrafted cells or grafts to have a certain degree of metastatic capacity. If the cell line’s ability to metastasize is too weak, omental metastasis may not occur in passage 1 mice. As the basis of in vivo selection is enrichment, studies have shown that the proportion of cancer stem cell-like side populations after enrichment declines rapidly upon passaging.³ Similarly, HM cells may lose their highly metastatic properties under long-term in vitro culturing. Therefore, it is necessary to regularly repeat in vivo selection to maintain the enrichment state. The cells used in this protocol are transfected with luciferase, which may influence the cell phenotype by introducing changes in the genome.

Troubleshooting

Problem 1

The subcutaneous tumor grows slowly or there is no tumor growing subcutaneously (related to Step 15).

Potential solution

• •Check the passage of the cells used, and avoid using cells passaged too many times in vitro.
• •Check the tumorigenesis ability of the cells, and ensure that the cells used are able to form tumors subcutaneously.
• •Avoid using mice that are too old.

Problem 2

It is difficult to find the ovary of the mouse (related to Step 35).

Potential solution

• •Enlarge the incision appropriately.

Problem 3

It is difficult to insert the tissue block into the ovarian bursa of mouse (related to Step 36).]

Potential solution

• •Enlarge the incision appropriately.
• •Reduce the volume of the tissue block if needed.

Problem 4

There is no signal when performing in vivo bioluminescent imaging (related to Step 48).]

Potential solution

• •Ensure that the luciferase substrate is injected into the abdominal cavity of the mouse.
• •Extend the imaging time appropriately.
• •Sometimes, the tumor is too small to be detected, especially a few days after xenograft. Wait for 3–7 days and perform bioluminescent imaging again.

Problem 5

It is difficult to recognize the omentum when metastasis occurs (related to Step 53).

Potential solution

• •Find the tissue between the stomach and the spleen. Look for the omental tissue by the blood vessel across the omentum.
• •Choose the central part of the omentum to ensure that the tissue used contains only omentum.

Problem 6

On occasion, you may need to suspend the in vivo selection cycle due to certain emergency matters. For instance, the demise of the mice during the experiment or the machinery malfunctioning unexpectedly (related to Step 60).

Potential solution

• •The in vivo selection could be paused at any passage by culturing and storing the primary cells from the omentum. Once the in vivo selection is paused, the process should be restarted from step 10 and the number of passages should be reduced by 1 accordingly. For example, the in vivo selection is paused at passage 2, then the cell collected should be recognized as passage 1. The process should be conducted until at least passage 3.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Zehua Wang (zehuawang@hust.edu.cn).

Materials availability

This study did not generate new unique reagents.

Data and code availability

The protocol doesn’t include any datasets generated or analyzed during this study. All the other data reported in this paper will be shared by the lead contact upon request.

There is no original code in this study.

Any additional information required to reanalyze the data reported in this protocol is available from the lead contact upon request.