Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Summary

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Abstract

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).

In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Introduction

Multiple myeloma (MM) is characterized by proliferation of malignant plasma cells in the bone marrow, bone lesions and immunodeficiency ¹. Although new treatment options such as proteasome inhibitors (bortezomib) and immune modulatory drugs (pomalidomide and lenalidomide) are available, MM still remains an incurable malignancy with a grim prognosis ². The bad prognosis might be explained by the extraordinary heterogeneity of MM cell clones that contributes to variable responses to therapy, in particular under long time treatment and selection pressure of MM clones ³.

Preclinical testing of new drugs and their combinations in vitro and in vivo is a critical and time-consuming step for future drug development. Thus, useful in-vivo models of MM are required to gain a better understanding of the biology of the disease and to enable the discovery of new drugs. Actually, the best xenotransplantation models for hematological malignancies and therapeutics are immune-deficient mice, such as the severe-combined immunodeficient (SCID) mice ^4-7, the non-obese diabetic/SCID (NOD/SCID) mice ^8,9 or the β-microglobulin-knockout NOD/SCID mice ^10,11.

Although murine models of human MM in some aspects can resemble the phenotype of human disease, immune-deficient mice are inbred, therefore simulate only one individual response to a drug and costs are very high. Due to immunosuppression animals require special maintenance conditions and the engraftment of human MM in mice requires 6 weeks to 2 months ^9,12, unless cells are grafted directly to the bone marrow using a technically demanding procedure with lower rates of animal survival ^7,13. Therefore, new methods using stem-cell based organoid models ¹⁴, tissue engineering ¹⁵ or sophisticated 3D cell culture models ¹⁶ have been established. They will compete in the near future with classical animal experiments for preclinical drug testing, but cannot replace systemic toxicity tests in living organisms.

The chicken embryo has been demonstrated before to be a suitable organism for xenotransplantation of human cells and tissues due to lack of adaptive immune response until hatching ^17-19. Moreover, each chicken embryo reflects an individual reaction to applied drugs or tumor cells due to genetic diversity within the chicken population. The chorioallantoic membrane (CAM) is a well-established system to study tumor-dependent angiogenesis ^20-22. When solid tumors are grafted to the CAM, they display many characteristics of cancers in vivo, including proliferation, invasion, angiogenesis and metastasis ^23-27.

Based on the previous experience of our group with CAM xenograft models^20,26,27, a human MM model was established that combines the advantage of a human 3D culture system with the model of ex ovo developing chicken embryos. This MM model system allows real time monitoring of MM growth progression, quantification of cell mass and preclinical drug testing.

Protocol

According to the Austrian law, and the Office of Laboratory Animal Welfare of the US public health service avian embryos are not considered as live vertebrate animals until hatching.The NIH Office of Laboratory Animal Welfare has provided written guidance in this area (http://www.grants.nih.gov/grants/olaw/references/ilar91.htm and NIH Publication No.: 06-4515).

1. Cell Culture and Lentiviral Transfection

1. Culture MM cell lines OPM-2, RPMI-8226 and human mesenchymal stem cells from bone marrow in RPMI1640 medium, supplemented with 10% bovine fetal calf serum and 100 IU/ml penicillin, 100 μg/ml streptomycin and 2 mM glutamine in the presence of 5% CO₂ at 37°C.
2. Transfect 5 x 10⁶ HEK 293FT cells with viral packaging mix (9 µg) DNA and 3 µg pLenti6/V5 dest eGFP vector by the use of 30 µl liposomal transfection reagent and transfection medium (10 ml). Remove transfection medium after 12 h and add 10 ml DMEM medium with 10% bovine calf serum and 1% non-essential amino acids (NEAA).
3. After 5 days, collect supernatants of HEK293FT cells after centrifugation of swimming cells (1,000 x g, 5min). Determine viral titer by real time PCR as described elsewhere ²⁸.
4. Transfect 1 x 10⁶ MM cells with eGFP lentiviral particles (1 x 10⁵ particles) in a 24 well plate in complete growth medium. After 3 days, start selection process by adding 2 µg/ml blasticidin to the culture medium. For the commercially available eGFP lentivirus, use 500 µg/ml neomycin.
5. After 2 weeks of selection, clusters of eGFP expressing MM cells will appear; collect cells by centrifugation (1,000 x g, 5 min) and expand them as OPM-2^eGFP and RPMI-8226^eGFP sublines for experiments (section 2 and 3).

2. 3D-Multiple Myeloma Spheroid Model

1. Chill collagen type-I solution and 10 x DMEM on ice.
2. Mix 1/10 Volume of 10 x DMEM medium into collagen matrix; add NaOH (0.2 N) to neutralize acidic collagen solution to a pH value of 7.4; store collagen/medium solution on ice.
3. Mix transgenic MM cell lines (OPM-2^eGFP or RPMI-8226 ^eGFP; 250,000 per spheroid,) with human mesenchymal cells (50,000 cells/ spheroid, i.e., 30 µl drop).
4. Centrifuge cell mixture in 15 ml tubes (1000 x g, 5min), add cold prepared collagen mixture (1mL) to the cell pellet and mix well (with 1,000 µl tip).
5. Immediately pipette 30 µl of the collagen/cell mixture (with 100 µl tip) in a 24 well plate on sterile paraffin film and allow cell/collagen mixture to polymerize for 30 min at 37°C (see Figure 1A).
6. Overlay MM spheroids with 1mL culture medium containing 1, 10 and 100 nM bortezomib (see Figure 1B).
7. After 72 hr of incubation at 37°C, document spheroids by fluorescence stereomicroscopy (see Figure 1C).
8. Transfer each spheroid by the use of forceps with wide flat jaws in a reaction tube for measurement of GFP (see Figure 1D).

3. 3D Multiple Myeloma Xenograft Model in the CAM

1. Incubate chicken eggs in a special incubator for avian eggs at 37°C and 70% humidity for three days.
2. Thereafter, open eggs and transfer embryos to sterilized with ethanol, square, 10-cm plastic weighing boats with cell culture plate lid and incubate “ex ovo” for further six days, so that the CAM is able to develop (see Figure 2A).
3. Chill collagen type-I solution and 10 x DMEM on ice, mix 1/10 Volume of 10 x DMEM medium into collagen matrix, Add NaOH (0.2 N) to neutralize acidic collagen solution to a pH value of 7.4; store collagen/medium solution on ice.
4. Mix transgenic MM cell lines (OPM-2^eGFP or RPMI-8226^eGFP; 250,000 per spheroid,) with human mesenchymal cells (50,000 cells/ spheroid).
5. Centrifuge cells in 15 ml tubes (1,000 x g, 5min; for each test compound 1 vial), add 1mL cold prepared collagen mixture with drug (at desired working concentration) to the cell pellet and mix well (with 1,000 µl tip).
6. Place collagen drops (30 µl each) on parafilm in a 6 well pate for 30 min to allow polymerization of the extracellular matrix at 37°C.
7. Transfer “onplants” from step 3.6 with the use of forceps to the untreated surface of the CAM (2 cm away from embryo) of 9-day-old chicken embryos (4 onplants for each chicken embryo, see Figure 2B)
8. After 5 days of in vivo growth in an egg incubator at 37°C and 70% humidity, document xenografts by fluorescence stereomicroscopy (see Figure 2C). Euthanize chicken embryos by hypothermia at 4°C in the fridge for 5h.
9. Remove xenografts with subjacent CAM tissue by an ophthalmic scissor and forceps with wide flat jaws. Use them for measurement of GFP (section 4, see Figure 2D) or for immunohistochemical analysis of blood vessels and/or invading tumor cells (section 5).

4. Quantification of eGFP Protein by ELISA

1. Transfer each MM spheroid or excised xenograft into 0.5 ml RIPA Buffer containing 200 µg/ml protease inhibitors.
2. Homogenize spheroid/xenograft with a tissue homogenizer on ice.
3. Perform three freezing/thawing-cycles in liquid nitrogen and 37°C water bath.
4. Centrifuge homogenate at 4°C for 20 min (12,000 g) and store supernatants.
5. Dilute samples 1:20 in assay buffer (200 µl) of the ELISA kit. Measure GFP-levels by a commercial GFP ELISA Kit using biotinylated anti-GFP antibodies, according to the manufacturer’s protocol.

5. Immunohistochemical Analysis of Blood Vessels and Invading Tumor Cells

1. Fix excised xenografts with CAM area in 4% paraformaldehyde O/N at 4°C.
2. Place fixed xenografts into embedding cassettes and transfer them into a tissue embedding station with an increasing graded alcohol series (50%, 70%, 80%, 95% ethanol, xylol and paraffin; each step 60 min).
3. Section xenografts (5 µm) by the use of a benchtop rotary microtome. Bake paraffin sections on glass slides O/N at 56°C.
4. Deparaffinize sections by a decreasing graded alcohol series to double-distilled water (xylol, 95%, 80%, 70%, 50% ethanol, double-distilled water; each step 10 min).
5. Perform antigen retrieval in a water bath (95°C, 20 min) with an antigen retrieval solution (citrate- buffer; pH 7.0; volume 100 µl).
6. Block endogenous peroxidase activity with 100 µl 3% H₂O₂/methanol for 30 min.
7. Block sections in PBS containing 10% fetal calf serum for 45 min (volume 100 µl).
8. Stain for 1 hr with 100 µl of primary antibody (1µg/ml) diluted in PBS containing 1% fetal calf serum at RT.
9. After washing 3-times in PBS, incubate for 1h with biotinylated secondary antibody (0.1 µg/ml) in PBS containing 1% fetal calf serum at RT.
10. After washing 3-times in PBS perform color reaction by the avidin/biotin-complex (ABC) and the diaminobenzidine (DAB) substrate solution according to manufacturer’s instructions.
11. 5.11. Stop reaction by transferring sections to double-distilled water, counterstain with hematoxylin and mount sections with a synthetic mounting medium.

Results

In vitro analysis of target compounds in 3D multiple myeloma spheroid assays

Due to the limitation of culturing primary human MM cells in vitro we established new 3D in vitro culture models for human MM cell lines making use of an extracellular growth matrix and supportive primary human mesenchymal cells from bone marrow (Figure 1A, B). EGFP transgenic MM cell lines allow visualization and quantification of MM tumor ...

Discussion

The development of new therapeutic agents for refractory MM requires less time consuming and expensive in-vivo systems to evaluate sensitivity of human MM cells to drugs. Hitherto, only few in-vivo systems are available for the preclinical evaluation of new anti-myeloma therapies. All of them have their limitations for large scale screening of compound libraries ^29. 

The best current models for human MM cells are highly immune-deficient mice ^7,13,30 and turkey embryos ²⁹...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI-8226 cells	DSMZ	ACC 9	STR profiled
OPM-2 cells	DSMZ	ACC 50	STR profiled
Human mesenchymal stem cells	PromoCell	PC-C-12974
HEK293FT cells	Invitrogen	R700-07
RPMI1640 Medium	Sigma Aldrich	R0883
Fetal Bovine Serum  HyClone	ThermoScientific	SH30070.03
L-Glut- Pen- Strep solution	Sigma	G6784
DMEM Medium	Gibco	31966
NEAA	Sigma Life Sciences	M7145
Transfection Medium/Opti-MEM	Gibco	51985
eGFP lentiviral particles	GeneCopoeia	LPP-EGFP-LV105	Ready to use viral particles
pLenti6/V5Dest6 eGFP vector	Invitrogen	PN 35-1271	from authors
ViralpowerTM packaging mix	Invitrogen	P/N 35-1275
Transfection reagent/ Lipofectamin 2000	Invitrogen	11668-027
Blasticidin	Invitrogen	R210-01
Neomycin	Biochrom	A2912
Collagen-Type1  Rat Tail	BD Biosciences	354236
DMEM powder	Life Technologies	Art.Nr. 10338582
plitidepsin	Pharmamar
bortezomib	LKT Lab., Inc.	B5871
SPF-white hen eggs	Charles River		Fertilized  white Leghorn  chicken eggs
Plastic weighing boats	neoLab	Art.Nr. 1-1125	for ex-ovo culture
Petridish square (Lids)	Simport	D210-16	for ex-ovo culture
RIPA Buffer (10x)	Cell Signaling	#9806
Protease Inhibitor Tablets	Roche	11 836 170 001
Complete Mini EDTA-free
GFP ELISA	Cell Biolabs, Inc.	AKR-121
Histocette II	Simport	M493-6
PFA  37%	Roth	7398.1
DPBS	Lonza	BE17-512F
Ethanol absolut	Normapur	20,821,321
Roti-Histol	Roth	Art.Nr.6640.4
Paraplast	Sigma	A6330
SuperFrost Microscope Slides	R. Langenbrinck	Art.-Nr.
Labor- u. Medizintechnik	03-0060
DakoCytomation Wash Buffer 10x	DakoCytomation	Code-Nr.
S 3006
Target Retrieval Solution (10x)  pH 6,1	DAKO	Code-Nr.
S 1699
H2O2	Merck
m-a-hu ASMA clone 1A4	DAKO	M0851
m-a-hu CD138 clone MI15	DAKO	M7228
m-a-hu Vimentin clone V9	DAKO	M0725
m-a-hu Desmin clone D33	DAKO	M0760
m-a-hu Ki67  clone MIB-1	DAKO	M7240
biotinylated goat- anti-mouse IgG	Vector Laboratories Inc.	BA-9200
Vectastain Elite ABC Kit	Vector Laboratories Inc.	# PK-6100
FAST DAB Tablet Set.	Sigma Biochemicals	# D4293
Mayer’s haemalaun solution	Merck	1,092,490,500
Roti Histokitt	Roth	Art.Nr.6638.2
Bench top rotary microtome	Thermo Electron, Shandon Finesse ME+
Tissue embedding station	Leica, TP1020
Egg-Incubator	Grumbach	BSS160
Stereo fluorescence microscope equipped with an connected with a digital camera (Olympus E410) and flexible cold light	Olympus, SZX10
Ultra Turrax	IKA T10		Homogenizer