In Vivo Fluorescent Imaging Using Fluorescent Proteins
By R.M Hoffman
Springer Protocols, Methods in Molecular Biology,
872, XIV, 258p, 2011
Transdifferentiation of glioblastoma cells into vascular
Soda, Y., Marumoto, T., Friedmann-Morvinski, D., Soda,
M., Liu, F., Michiue, H., Pastorino, S., Yang, M., Hoffman, R.M., Kesari, S.,
Proc. Natl. Acad. Sci. USA 108, 4274-4280, 2011.
UV light killing efficacy of fluorescent protein-expressing cancer cells
in vitro and in vivo.
Kimura, H., Lee, C., Hayashi, K., Yamauchi, K., Yamamoto, N., Tsuchiya, H.,
Tomita, K., Bouvet, M., and Hoffman, R.M.
J. Cell. Biochem 110, 1439-1446, 2010.
Metronomic gemcitabine in combination with Sunitinib inhibits multisite
metastasis and increases survival in an orthotopic model of pancreatic cancer.
Tran Cao, H.S., Bouvet, M., Kaushal, S., Keleman, A., Romney, E., Kim, G.,
Fruehauf, J., Imagawa, D.K., Hoffman, R.M., and Katz, M.
Molecular Cancer Therapeutics 9, 2068-2078, 2010.
(Highlights Section. Molecular Cancer Therapeutics 9, 1929, 2010).
Complementary use of fluorescence and magnetic resonance imaging of
metastatic esophageal cancer in a novel orthotopic mouse model.
Gros, S.J., Dohrmann, T., Peldschus, K., Schurr, P.G., Kaifi, J.T., Kalinina,
T., Reichelt, U., Mann, O., Strate, T.G., Adam, G., Hoffman, R.M., and Izbicki,
International Journal of Cancer 126, 2671-2681, 2010.
Systemic targeting of primary bone tumor and lung metastasis of high-grade
osteosarcoma in nude mice with a tumor-selective strain of Salmonella
Hayashi, K., Zhao, M., Yamauchi, K., Yamamoto, N., Tsuchiya, H., Tomita, K.,
Kishimoto, H., Bouvet, M., and Hoffman, R.M.
Cell Cycle 8, 870-875, 2009.
In vitro/in vivo biorecognition of
lectin-immobilized fluorescent nanospheres for human colorectal cancer cells.
Sakuma, S., Yano, T., Masaoka, Y., Kataoka,
M., Hiwatari, K., Tachikawa, H., Shoji, Y., Kimura, R., Ma, H., Yang, Z., Tang,
L., Hoffman, R.M., Yamashita, S.
Journal of Controlled Release 134, 2-10, 2009.
Glowing proteins with promising
biological and medical applications.
by Linda Zajac
Chem Matters 12-14, December
Dual-color imaging of angiogenesis and its
inhibition in bone and soft tissue sarcoma.
K., Yamauchi, K., Yamamoto, N., Tsuchiya, H., Tomita, K., Amoh, Y., Hoffman,
R.M., and Bouvet, M.
J. Surg. Res. 140, 165-170,
cells genetically labeled with GFP in the nucleus and RFP in the cytoplasm for
imaging cellular dynamics. T
P., Yamauchi, K., Yang., Tsuji, K., Xu, M., Maitra, A., Bouvet, M., and
5, 1198-1201, 2006.
Dual-color fluorescent cells with one color fluorescent protein in the
nucleus and another color fluorescent protein in the cytoplasm were genetically
engineered. The dual-color cancer cells enable real-time nuclear-cytoplasmic
dynamics to be visualized in living cells in vivo as well as in vitro. To
obtain the dual-color cells, red fluorescent protein (RFP) was expressed in the
cytoplasm of a series of human and rodent cancer cells, and green fluorescent
protein (GFP) linked to histone H2B was expressed in the nucleus. Nuclear GFP
expression enabled visualization of nuclear dynamics, whereas simultaneous
cytoplasmic RFP expression enabled visualization of nuclear-cytoplasmic ratios
as well as simultaneous cell and nuclear shape changes. Using the Olympus OV100
Whole-Mouse Imaging System, total sub-cellular dynamics can be visualized in
the living dual-color cells in real time in the live mouse after cell
injection. Highly elongated cancer cells and nuclei in narrow capillaries were
visualized where both the nuclei and cytoplasm deform. Both cytoplasm and
nuclei were visualized to undergo extreme deformation during extravasation with
cytoplasmic processing exiting vessels first and nuclei following along these
processes. The dual-color cells described here thus enable the sub-cellular
dynamics of cancer celltrafficking to be imaged in the living animal.
Highlights of the Recent Literature
[BIOTECHNIQUES 36, No. 1 January, 19,
development of tumor-induced vasculature is essential for both tumor growth
and the formation of metastases. Newly formed blood vessels thus provide
promising potential therapeutic targets. However, the role of host tissues
and their interactions with the tumor in this angiogenesis has remained
unclear. Yang et al. have developed a technique for delineating tumor-induced
host processes by exploiting a dual-color fluorescent system. They
transplanted tumors expressing red fluorescent protein (RFP) into green
fluorescent protein (GFP)-expressing nude mice. All of the tissues from these
transgenic mice fluoresce green, except for their hair and erythrocytes. This
dual-color fluorescence imaging is unique in that it can depict morphological
events during tumor-host interactions at the cellular level in fresh tissue
in addition to whole-body imaging. They visualized macrophages engulfing tumor
cells, dendritic cells contacting tumor cells, and lymphocytes attacking the
tumor. The insights gleaned from this study and others like it should be
important in elucidating the role of host cells in tumor growth, progression,
and invasion. Proceedings of the National Academy of Sciences of the USA
100 (24): 14259-14262 (2003)
Host-derived GFP-expressing blood
vessels visualized in live RFP-expressing mouse melanoma.
Galectin-3 Inhibits Tumor Growth and Metastasis in Orthotopic Nude Mouse Model
of Human Breast Cancer.
C.M., Leffler, H., Kahl-Knutsson, B., Svensson, I., Jarvis, G.A.
Cancer Research 9, 2374-2383, 2003]
cancer xenograft imaged with green fluorescent protein in nude mouse treated
with truncated galectin-3 (galectin-3C). The mammalian lectin galectin-3 has
been suggested to promote cancer by stimulation of cell adhesion and inhibition
of apoptosis reulting in increased tumor growth and metastasis. Galectin-3C, a
fragment of galectin-3 retaining its carbohydrate binding activity but lacking
the cross linking ability and thereby possibly acting as a dominant negative
inhibitor, reduced tumor growth and metastasis and showed no toxic side
effects. For details, see C.M. John et
al., in this issue.
M. John, Hakon Leffler, Barbro Kahl-Knutsson, Inga Svensson, and Gary A.
Jarvis. Truncated Galectin-3 Inhibits Tumor Growth and Metastasis
in Orthotopic Nude Mouse Model of Human Breast Cancer. Clin
Cancer Res 9: 2374-2382, 2003.
Green fluorescent protein imaging of tumour growth, metastasis and
angiogenesis in mouse models.
Robert M. Hoffman
[Lancet Oncology 3, 546-556, 2002]
Green fluorescence protein imaging of tumour growth, metastasis, and
angiogenesis in mouse models by Robert M Hoffman. The figure below shows a
whole-body fluorescent image of a mouse with bone metastases in the (a)
skull, (b) scapula, and (c) spine.
Dissecting p53 tumor suppressor functions in vivo
Clemens A. Schmitt, Jordan S. Fridman, Meng Yang, Eugene Baranov, Robert
M. Hoffman, and Scott W. Lowe
[CANCER CELL 1, 289-298 April 2002]
Whole body fluorescence imaging of lymphoma
progression in live mice. The cover shows the temporal and spatial
progression of Em-myc lymphoma cells tagged with green
fluorescent protein in a live mouse (with time progression from top to
bottom). Note that the lymphomas first expand within the lympoid compartments
and bone. In the absence of p53 or following Bcl-2 overexpression, these
lympomas readily disseminate into nonlympoid compartments. For details see
Schmitt et al. (pp. 289-298) in this issue.
Although the p53 tumor supressor acts in plethora of processes that influence
cellular proliferation and survival, it remains unclear which p53 functions
are essential for tumor suppression and, as a consequence, are selected
against during tumor development. Using a mouse model harbouring primary,
genetically modified myc-driven lympomas, we show that disruption of apoptosis
downstream of p53 by Bcl2 or a dominant-negative caspase 9 confers-like p53
loss-a selective advantage, and completely alleviates pressure to inactivate
p53 during lymphomagenesis. Despite their p53-null-like aggressive phenotype,
apoptosis-defective lymphomas that retain intact p53 genes do not display the
checkpoint defects and gross aneuploidy that are charcteristic of p53 mutant
tumors. Therefore, apoptosis is the only p53 function selected against during
lymphoma development, whereas defective cell-cycle checkpoints and aneuploidy
are mere byproducts of p53 loss.
Figure 4. Whole body fluorescence imaging
allows visualisation of lymphoma dissemination.
Lymphomas with indicated
genotypes and transduced
with a GFP-coexpressing retrovirus were
into recipients to monitor lymphoma dissemination in whole
viable animals by GFP fluorescence.
A Senescence Program Contolled by p53 and p16INK4a
Contribures to the Outcome of Cancer Therapy
Clemens A. Schmitt, Jordan S. Fridman, Meng Yang, Soyoung Lee, Eugene
Baranov, Robert M. Hoffman, and Scott W. Lowe
[CELL 109, 335-346, May 3, 2002]
p53 andINK4a/ARF mutations promote tumorogenesis and drug resistance,
in part, by disabling apoptosis. We show that primary murine lympomas also
respond to chemotherapy by engaging a senescence program controlled by p53
and p16INK4a. Hence, tumors with p53 or INK4a/ARF mutations-but
not those lacking ARF alone - respond poorly to cyclophosphamide therapy in
vivo. Morover, tumors harboring a Bcl2-mediated apoptotic block undergo a
drug-induced cytostasis involving the accumulation of p53, p16INK4a,
and senescence markers, and typically acquire p53 or INK4a mutations upon
progression to a terminal stage. Finally, mice bearing tumors capable of
drug-induced senescence have a much better prognosis following chemotherapy
than those harboring tumors with senescence defects. Therefore, cellular senescence
contributes to treatment outcome in vivo.
Figure 1. Contribution of
p53 and Bcl2 to Treatment Responses
Mice harboring ctrl.-MSCV, p53 null-MSCV,
and ctrl.-bcl2 lymphomas were treated at comparable tumor burdens (day 0)
with a single dose of cyclophosphamide (CTX) and monitored by whole-body
fluorescence imaging. Representative examples are shown.
Metastatic-Cancer Mouse Models.
By Robert M. Hoffman
[In: Teicher, B.,
ed. Tumor Models in Cancer Research.
Totowa, NJ: Humana Press Inc., 99-112, 2002].
Green Fluorescent Protein Imaging of Tumor Cells in Mice
By Robert M. Hoffman
[LAB ANIMAL 31, No. 4, 34-41 April 2002]
Real-Time Optical Imaging of Primary Tumor Growth and Multiple Metastatic
Events in a Pancreatic Cancer Orthotopic Model
Michael Bouvet, Jinwei Wang, Stephanie R. Nardin, Rounak Nassirpour, Meng
Yang, Eugene Baranov, Ping Jang, A. R. Moosa and Robert M. Hoffman
[CANCER RESEARCH 62, 1534-1540, March 1,
ABSTRACT: We report here whole-body
optical imaging, in real time, of genetically fluorescent pancreatic tumors
growing and metastasizing to multiple sites in live mice. The whole-body
optical imaging system is external and noninvasive. Human pancreatic tumor
cell lines, BxPC-3 and MiaPaCa-2, were engineered to stably express
high-levels of the Acqurea victoria green fluorescent protein (GFP).
The GFP-expressing pancreatic tumor cell lines were surgically orthotopically
implanted as tissue fragments in the body of the pancreas of nude mice.
Whole-body optical images visualized real-time primary tumor growth and
formation of metastatic lesions that developed in the spleen, bowel, portal
lymph nodes, omentum, and liver. Intravital imaging was used for
quatification of growth of micrometastasis on the liver and stomach.
Whole-body imaging was carried out with either a trans-illuminated
epi-fluorescence microscope or a fluorescent light box, both with a
thermoelectrically cooled color CCD camera. The simple, noninvasive, and
highly selective imaging made possible by the strong GFP fluorescence allowed
detailed simultaneous quantitative imaging of tumor growth and multiple
metastasis formation of pancreatic cancer. The GFP imaging affords
unprecendented contionuous visual monitoring of malignant growth and spread
within intact animals without the need for anesthesia, substrate injection,
control agents, or restraint of animals required by the other imaging
methods. The GFP imaging technology presented in this report will facilitate
studies of modulators of pancreatic cancer growth, including inhibition by
potential chemotherapeutic agents.
The human pancreatic tumor cell line,
BxPC-3, was engineered to stably express high-levels of the Aquorea victoria
green fluorescent protein (GFP). The GFP-expressing pancreatic tumor cell
line was surgically orthotopically implanted (SOI) as tissue fragments in the
body of the pancreas of nude mice. Intravital imaging was used for
quantification of growth metastasis on the liver. The diameters of the three
micrometastases were 288
mm and 208
mm the right lobe and 344
mm on the left lobe as quantified by image analysis
at day-70 post SOI.
Visualization of GFP-Expressing Tumors and Metastasis In Vivo
Robert M. Hoffman
[BIOTECHNIQUES 30, No. 5 May,
ABSTRACT: We have developed mouse
models of metastatic cancer with genetically fluorescent tumors that can be
imaged in fresh tissue, in situ, as
well as externally. To achieve this
capability, we have transduced the green fluorescent protein (GFP) gene,
cloned from the bioluminescent jellyfish Aequorea
victoria, into a series of human and rodent cancer cell lines that were
selected in vitro to stably express
GFP in vivo after transplantation
to metastatic rodent models. Techniques were also developed for transduction
of tumors by GFP in vivo. With this fluorescent tool, we detected and
visualized for the first time tumors and metastasis in fresh viable tissue or
in situ in host organs down to the single cell level. GFP tumors on the colon, prostate, breast,
brain, liver, lymph nodes, lung, pancreas, bone and other organs can also be
visualized externally, transcutaneously by quantitative whole-body
fluorescence optical imaging.
Real-time tumor and metastatic growth and angiogenesis and inhibition
by representative drugs can be imaged and quantified for rapid anti-tumor,
anti-metastatic and anti-angiogenesis drug screening. The GFP-transfected tumor cells enabled a
fundamental advance in the visualization of tumor growth and metastasis in
real time in vivo.
Three-dimensional histoculture: Origins and applications in cancer
research. Cancer Cells 3, 86-92, 1991.
Robert M. Hoffman
Cancer Cells 3, 86-92, 1991. (675
Cover: Confocal photomicrograph of
human colon cancer in three-dimensional histoculture on collagen containing
sponges. The culture on the right has been treated with 5-fluorouracil.
Viable cells are stained with BCEF-AM (green) and nonviable cells are
stained with propidium iodide (red). For a review of methods and
applications of three-dimensional histoculture, see the article.