We have reported a new molecular-targeted cancer phototherapy, photoimmunotherapy (PIT), which
killed implanted tumors in mice without side-effects. To understand the mechanism of cell killing
with PIT, three-dimentional dynamic low-coherence quantitative phase microscopy (3D LC-QPM),
a device developed by Hamamatsu Photonics K.K, was used to detect morphologic changes in
cancer cells during PIT. 3T3/HER2 cells were incubated with anti-HER2 trastuzumab-IR700 (10
μg/mL, 0.1 μM as IR700) for 24 hours, then, three-dimensionally imaged with the LC-QPM during
the exposure of two different optically filtered lights for excitation of IR700 (500-780 nm) and
imaging (780-950 nm). For comparison with traditional PDT, the same experiments were performed
with Photofrin (10 and 1 μM).
Serial changes in the cell membrane were readily visualized on 3D LC-QPM. 3T3/HER2 cells began
to swell rapidly after exposure to 500-780 nm light excitation. The cell volume reached a maximum
within 1 min after continuous exposure, and then the cells appeared to burst. This finding suggests
that PIT damages the cell membrane by photo-reaction inducing an influx of water into the cell
causing swelling and bursting of the cells. Interestingly, even after only 5 seconds of light exposure,
the cells demonstrated swelling and bursting albeit more slowly, implying that sufficient cumulative
damage occurs on the cell membrane to induce lethal damage to cells even at minimal light exposure.
Similar but non-selective membrane damage was shown in PDT-treated cells Photofrin.
Thus, PIT induces sufficient damage to the cell membrane within 5 seconds to induce rapid necrotic
cell death which can be observed directly with 3D LC-QPM. Further investigation is needed to
evaluate the biochemical mechanisms underlying PIT-induced cellular membrane damage.
The microdistribution of therapeutic monoclonal antibodies within a tumor is important for determining clinical response.
Nonuniform microdistribution predicts therapy failure. Herein, we developed a semiquantitative method for measuring
microdistribution of an antibody within a tumor using in situ fluorescence microscopy and sought to modulate the
microdistribution by altering the route and timing of antibody dosing. The microdistribution of a fluorescently-labeled
antibody, trastuzumab (50-μg and 150-μg intraperitoneal injection (i.p.), and 100-μg intravenous injection (i.v.)) was
evaluated in a peritoneal dissemination mouse model of ovarian cancer. In addition, we evaluated the microdistribution
of concurrently-injected (30-μg i.p. and 100-μg i.v.) or serial (two doses of 30-μg i.p.) trastuzumab using in situ
multicolor fluorescence microscopy. After the administration of 50-μg i.p. and 100-μg i.v. trastuzumab fluorescence
imaging showed no significant difference in the central to peripheral signal ratio (C/P ratio) and demonstrated a
peripheral-dominant accumulation, whereas administration of 150-μg i.p. trastuzumab showed relatively uniform, central
dominant accumulation. With concurrent-i.p.-i.v. injections trastuzumab showed slightly higher C/P ratio than
concurrently-injected i.p. trastuzumab. Moreover, in the serial injection study, the second injection of trastuzumab
distributed more centrally than the first injection, while no difference was observed in the control group. Our results
suggest that injection routes do not affect the microdistribution pattern of antibody in small peritoneal disseminations.
However, increasing the dose results in a more uniform antibody distribution within peritoneal nodules. Furthermore, the
serial i.p. injection of antibody can modify the microdistribution within tumor nodules. This work has implications for
the optimal delivery of antibody based cancer therapies.
KEYWORDS: Luminescence, Tumors, In vivo imaging, Molecules, Molecular imaging, Cancer, Lung, Imaging systems, Signal detection, Fluorescence resonance energy transfer
Molecular imaging probes rely on high target-to-background ratios (TBR) to achieve maximum sensitivity
and specificity. We utilized "quenchers" to turn off the background signal from the unbound probe and
investigated the ability of specific fluorophore-quencher pairs to activate at target tissues. Both fluorophore
and quencher were conjugated to a single cancer targeting molecule, either avidin or antibody. Fluorescence
signal from these targeting molecules was "turned off" by the quencher in the unbound state, but was "turned
on" only when the molecules bound to the cell surface target and was internalized.
We tested the following fluorophore-quencher combinations based on fluorescence resonance energy transfer
(FRET) pairs; OregonG-BHQ1, RhodG-BHQ1/ATTO540Q, TAMRA-QSY7/QSY21, TexRed-QSY21,
Alexa647-QSY21, Cy5.5-QSY21/BHQ3 and Alexa680-QSY21/BHQ3. Among these, only RhodGATTO540Q
and TAMRA-QSY7/21 pair showed activation upon cell binding/internalization. Among these
combinations, TAMRA-QSY7 pair showed the highest activation (40-fold and 13-fold for avidin and
antibody conjugate, respectively) as measured with an in vitro dissociation assay. The activation was
dependent on the method used to conjugate fluorophores and quenchers to the targeting molecule. In vitro
microscopic studies with TAMRA-QSY7 pair conjugated to avidin or antibody showed high fluorescent
signal inside the target cancer cells, indicating activation after internalization. In vivo imaging studies in
tumor bearing mice demonstrated that tumors could be clearly detected with low background.
Although the precise quenching mechanism remains to be determined, this activation system can achieve high
TBR in vivo molecular imaging.
Commonly used in flow cytometry, multiplexed optical probes can diagnose multiple types of cell surface marker, potentially leading to improved diagnosis accuracy in vivo. Herein, we demonstrate the targeting of two different tumor markers in models of disseminated ovarian cancer. Two ovarian cancer cell lines (SKOV3 and SHIN3) were employed; both overexpress D-galactose receptor (D-galR), but only SKOV3 overexpresses HER2/neu. Additionally, fusion tumors composed of SKOV3 and SHIN3/RFP were evaluated. Both galactosyl serum albumin-rhodamine green (GSA-RhodG), which binds D-galR, and trastuzumab-Alexa680, which binds HER2/neu, were administered to tumor-bearing mice for in vivo fluorescence imaging and in situ fluorescence microscopy. In vivo fluorescence imaging depicted 64 of 69 SKOV3 tumors (94.2%) based on their dual spectra corresponding to both RhodG and Alexa680, while all 71 SHIN3 tumors (100%) were detected based on their single spectrum corresponding only to RhodG. All 59 SHIN3 and 36 SKOV3 tumors were correctly diagnosed with in situ microscopy. Additionally, in the mixed tumor model, all tumors could be depicted using the RhodG spectrum, but only SKOV3 components also showed the Alexa680 spectrum. In conclusion, multitargeted multicolor optical imaging enabled specific in vivo diagnosis of tumors expressing distinct patterns of receptors, leading to improved diagnostic accuracy.
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