Haematologica 2003 - Supplements

To confirm a direct relation between vaults and MDR and to
investigate possibel other functions of vaults, we have generated
a major vault protein knockout mouse model. The MVP-/- mice
are viable, healthy and do not show abnormalities. We
investigated the drug sensitivity of MVP-/- embryonic stem (ES)
cells and bone marrow cells derived from the MVP deficient mice
to melphalan, doxorubicin, mitoxantron, etoposide, vincristin,
dexamethasone, cisplatin and ara-C. In neither cell type an
increased sensitivity was observed as compared to wild-type
cells. The activities of MDR efflux proteins P-glycoprotein,
MRP1 and BCRP were unaffected by MVP disruption. In vivo
treatment of MVP wildtype and deficient mice with doxorubicin
resulted in similar responses and toxicity.
This study is one of the first to specifically investigate a gene that
showed a high level of expression in the myeloma gene array. No
specific role of MVP for therapy resistance could be
demonstrated.
Supported by a grant from the Multiple Myeloma Research
Foundation
219
Whole Body Green Fluorescent Protein (GFP)-Imaging
of Myeloma Tumors in Skeleton of Mice In Vivo.
Oyajobi BO 1, Munoz S1, Williams PJ1, Käkönen R1,
Williams PJ1, Gupta A1, Grubbs B1, Armstrong A2,
Dougall WC2, Garrett IR1, Mundy GR1
1Department of Cellular & Structural Biology, University of Texas
Health Science Center, San Antonio, TX 78229, 2Amgen
Washington, Seattle, WA 98101.
The prognosis of multiple myeloma (MM) patients post-diagnosis
has not improved in the last three decades and there is a
continuing and compelling need for development of novel antimyeloma
agents that significantly impact tumor burden. This has
hitherto been hindered, in part, by the lack of appropriate
preclinical models that faithfully replicate the human disease.
Unlike cells in solid tumors, myeloma cells are often spread
diffusely throughout the bone marrow cavity and in anti-tumor
efficacy studies involving currently available models of
disseminated MM, determination of overall myeloma burden
using serum titers of the monoclonal paraprotein titers is often
equivocal because of the relatively long half-life of
immunoglobulins. In an attempt to overcome this, we genetically
engineered the murine myeloma 5TGM1 cell line that we
originated to stably express enhanced green fluorescent protein
(eGFP). 5TGM1 cells, originally subcloned as a stromaindependent
variant from the Radl 5T33 myeloma, were
retrovirally transduced with the LZRS-pBMNZ vector encoding
eGFP under the control of the M-MuLV promoter. Following
single cell cloning by fluorescence activated cell sorting (FACS),
several stable subclones were isolated and one clone (H1.1+)
expressing eGFP at a very high level was further characterized.
There was no difference either in the growth rates or monoclonal
paraprotein (IgG2b) production between the eGFP-expressing
clone and parental 5TGM1 cells. eGFP expression in cultured
H1.1+ cells was analyzed by FACS repeatedly and found to be
stable in the 4-month period prior to inoculation into mice.
H1.1+ cells were inoculated intravenously into 6-9 weeks old
syngeneic C57BL/KaLwRij mice through tail veins and whole
body optical images of the live mice were obtained using an
fluorescence illuminator and a thermoelectrically-cooled color
CCD camera weekly thereafter until sacrifice. Genetically
fluorescent 5TGM1 tumors growing in situ in spine, skull and
long bones were visualized on high-resolution images.
Fluorescent tumor foci were first evident two weeks after
inoculation of myeloma cells, and always in calvariae or
scapulae. Imaging of freshly isolated whole skeleton and visceral
organs post-sacrifice revealed that the myeloma cells homed
preferentially to the skeleton in all mice with multifocal
fluorescent lesions particularly pronounced in the axial skeleton
(skull, iliac crests, scapula, lumbar and thoracic vertebrae, ribs,
sternum) but also evident in metaphyseal regions of long bones,
consistent with typical tumor distribution in MM patients. There
were also smaller fluorescent extra-medullary tumor foci
detectable infrequently in spleens, kidneys, and ovaries but not
livers of tumor-bearing mice. eGFP-positive 5TGM1 cells,
sorted by FACS from splenic cell harvests, retained the ability to
home to bone marrow when re-injected into naïve mice.
Fluorescent tumor foci were consistently associated with
increased resorptive activity assessed by staining for TRAP
activity, a recognized marker of osteoclasts. In conclusion, whole
body GFP-imaging facilitates real-time, continuous visual
monitoring of myeloma growth and spread within tumor-bearing
animals. This ability to externally and non-invasively follow
myeloma progression, combined with quantitative
histomorphometry, increases the utility of the 5TGM1 model that
has already proven to be predictive of efficacy in preclinical
studies. This should accelerate evaluation and development of
novel anti-myeloma therapies.
220
LAGλ - a SCID-hu Xenograft Model of Multiple Myeloma
Hank H. Yang, Richard A. Campbell, Haiming Chen,
Daocheng Zhu, Robert A. Vescio, and James R. Berenson
Department of Medicine, Cedars-Sinai Medical Center, UCLA
School of Medicine, Los Angeles, CA, USA
Most SCID murine myeloma models were developed from
human multiple myeloma (MM) cell lines. Success has been
limited when primary MM cells were used. We have recently
developed a new SCID-hu murine model of MM, LAGλ from the
serial passage of an intramuscularly (IM) implanted myeloma
patient’s bone marrow (BM) sample. So far, LAG has been
growing continuously for 8 passages. In this subline, we have not
only achieved nearly a 100% passing rate, we also are able to
consistently grow up visible intramuscular tumor within 3-4
weeks with a relatively similar growth rate. The mean human IgG
(hIgG) elevation and tumor growth are approximately 230 mg/dl
and 0.38 cm3 per week, respectively. A similar growth pattern
was noted when the LAGλ cells were implanted subcutaneously
with or without the use of Matrigel. LAG cells have also been
tested in the intravenous (IV) model. Approximately 70-80% of
the implanted mice showed elevated secretion of hIgG around 10
weeks after tumor cell inoculation. MM cell infiltration can be
detected in mouse BM and other organs as early as 5 weeks after
tumor cell implantation. The clonality of the LAGλ myeloma
cells was verified by both PCR and protein electrophoresis. The
level of calcium was noted to be unchanged in the IM model, but
progressively increased in the IV model. There is also a steady
increase in osteolytic lesion noted in the IV model that correlates
with the increased number of osteoclastic cells present in mouse
BM. The clinical relevance of LAGλ SCID-hu xenograft model
was tested by injection of bortezomib, a proteasome inhibitor,
intravenously into the tail vein of IM model. The mice that
received 0.5 mg/kg bortezomib showed significant inhibition of
LAGλ MM cell growth, whereas the mice that received 0.05
mg/kg bortezomib showed no inhibition, as measured by hIgG
elevation and increasing tumor size.
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