The current study reports the first noninvasive real-time PA-molecular detection of single CD44+ CTCs (i.e.
, with cancer stem phenotype) among other (bulk) CTCs in bloodstream of breast cancer bearing mice in vivo
. We also demonstrated the poof-of-concept that this diagnosis can be integrated with targeted eradication of very aggressive metastasis initiating tumor cells using clinically-relevant multifunctional PAFC-PT-nanotechnology-based platform. No other currently available in vivo
diagnostic techniques, such as magnetic resonance imaging (MRI), positron emission tomography (PET) or optical assays, have similar dynamic capability. In vivo
fluorescent flow cytometry [5
] only showed detection of bulk CTCs targeted by fluorescent conjugates (e.g.
, folate) [7
] with no apparent application for identification and counting of stem CTCs. Moreover, compared to the robust PA and PT technique that can easily integrate diagnosis and therapy with the same laser, the fluorescent flow cytometry is primarily used for diagnostics. In the fluorescent technique, it is really hard to use the same laser simultaneously for label excitation and therapy of the fast moving CTCs because of different requirements to laser power and spectral range. Recently we also introduced in vivo
Raman flow cytometry [15
], which can be integrated with PA and PT technique, allowing detection of CTCs targeted by NPs with strong Raman contrast properties (e.g.
, CNTs or SERS). However, all these techniques require further sensitivity improvement so lesser laser energy can be applied to avoid potential photodamage of normal tissue.
Various strongly absorbing NPs have been used for both PA diagnostics and PT cancer therapy, including gold nanospheres, nanoshells, nanorods, nanocages, CNTs and others [39
]. To the best of our knowledge, we demonstrate here for the first time that NPs, especially advanced low toxic GNTs with one of the highest PA/PT contrast properties, can be used for ultra-sensitive molecular detection and ablation of single stem CTCs in vivo
. The population of CSCs are likely heterogenous [17
] and identification of stem CTCs requires the use of several biomarkers (e.g.
, CD44, CD24, CD133, ALDH) among bulk CTCs. In this preliminary study, as a first-step of mimicking the real situations, folic acid and Abs to CD44 and combination of GNTs with conventional MNPs as multimodal agents were selected. Although we focused on intrinsic PA/PT contrast properties of MNPs, they can be also used for magnetic capturing of stem CTCs directly in bloodstream [33
] (with their possible ablation or isolation) or MRI detection [42
] in static condition (due to relatively slow MRI signal acquisition algorithm) after CTC extravasations and localization in specific organs. Because the clinical potential of PA devices and the required low laser energy within safety standard (45–100 mJ/cm2
at 700–1,100 nm, respectively [43
]) has been demonstrated in several pilot trials in humans [44
], and some gold and magnetic NPs are also approved for pilot clinical trials (see discussion in [33
]), the PA diagnosis and PT ablation of stem CTCs has high potential for use on humans.
The important questions are the threshold sensitivity and the targeting efficiency of stem CTC in real biological environment in vivo
. We demonstrated the threshold sensitivity of PAFC around 1 CTC/mL in blood in vivo
, which was primarily limited by the relatively small blood pool volume (~2 mL) present in mice and realistic monitoring times (a few hrs), rather than by PAFC parameters [4
]. We predict that the use of large animals and especially translation of this technology to humans with much larger blood volume can significantly increase the detection sensitivity of stem CTCs up to 103
fold as the ratio of assessed blood volumes in vivo
to that ex vivo
. This can also reduce the examination time because the blood volume of ~5 L circulates through a ~3 mm in diameter human vein within 1 hr [33
]. Such threshold of sensitivity is unachievable with other existing techniques and represents sufficient level to rapidly detect extremely rare stem CTCs among bulk CTCs (see above) disseminated with a large background of blood cells. For MNPs with less intrinsic NIR absorption, higher concentrations should be required compared to GNTs. However, as we shown in this study, PA/PT response from cells with MNPs can be significantly amplified by magnet-induced MNP clustering, or/and by developing of hybrid MNPs with gold or other strongly NIR absorbing layers as discussed in [33
The requirements for the labeling of CTCs, and especially stem CTCs in the bloodstream are much less strict than those for targeting primary tumors because of 1) lower NP concentration required due to lower CTC concentration, 2) faster labeling in flow (up to 5-10 min [33
]), and 3) easy optical access to a peripheral blood vessels. The high efficiency of CTC labeling is related with frequent NP-CTC collisions in blood flow with the local turbulence [33
] even at similar NP and CTC velocity; on the contrary, for the cancer cell targeting in solid tumor, fast moving NPs even in capillary have to adhere to endothelial cells, penetrate through vessel wall, and migrate to nearest malignant tissue. The low background signals from unbound NPs can be achieved by selecting optimal NP concentration for injection to minimize signals in normal blood but generate detectable signals from each CTC targeted by NPs. Indeed, according to recent data [33
], injection of 109
NPs/mL in 50-100 μL of PBS into the mouse blood circulation provided low background signal from unbound NPs (just few NPs in the irradiated blood volume) and nonspecifically bound NPs by blood macrophages compared to the signals from single CTCs with high local concentration of NPs. In addition, the identification of signals from targeted stem CTCs in the presence of background signals from unbound NPs (e.g.
, at high NP concentration injected) in blood is possible due to the fast clearance of unbound NPs from circulation (15–30 min, which can be adjusted by proper selection of NP properties) compared to the longer circulation of labeled CTCs [33
]. In this study, the optimal time for detecting stem CTCs is 30-40 min after NP injection to allow unbound NP to wash-out.
In our previous [33
] and current study, PA technique demonstrated both high sensitivity and high dynamic linear range, allowing to detect single CTCs targeted by various number of NPs from minimal level of 10-50 to maximum up to 104
depending on marker expression, amount of injected NPs, targeting efficiency, and sizes of NPs and biomarkers. As a result, PAFC has potential to determine the marker expression in individual cells in vivo
because PA signal amplitude is proportional to total number of NPs targeting specific biomarkers in one CTC assuming that NP amount per one biomarker (likely 1-3) can be determined. Because PA signal amplitude also depends on laser energy, this parameter should be carefully optimized for identification of cancer stem cells. For example, at increased laser energy (300-500 mJ/cm2
), we could detect PA signals from 30-50% of cancer cells labeled by NP-CD44 complexes even at low CD44 expression. However, only small portion of cells provided strong PA signals indicating significantly higher biomarker expression (overexpression).
The interpretation of the observed rate of CD44+cells in vivo
is difficult at current stage of research because the literature data are not quite sufficient. Thus, we demonstrated a novel biophotonic noninvasive platform and its potential for detection of rare stem CTCs rather than the exact quantitative data of percentage of stem CTTs in the circulation. Indeed, the values of CSCs expressed CD44 (e.g., with the phenotype of CD44+/CD24-) varies from a few persantage to 30-80% in bulk breast cancer cells [18
]. Moreover, it was recently suggested that the proportion of CSCs varies widely with various factors, including 2-fold change during tumor development [48
]. In addition, to estimate the rate of false-positive signals the expression of CD44 in WBCs and/or GNT-CD44 nonspecific binding to other cells should be estimated. The proportion and role of CD44+/Folate+ cells are also unclear yet and it has not yet been determined the level of possible folate receptor expression in breast stem CTCs. Further study, to address significance of various marker(s) for stem CTCs, is currently in progress in our laboratories using an advanced time-resolved multi-spectral PAFC with multicolor NPs for multiplex biomarker targeting [33
]. In the current study, we used only one biomarker, i.e.
, CD44, to identify CSCs because this marker is most relevant to breast CSCs (as reported by other groups [17
]). However, as discussed above, biomarker expressions may be heterogeneous among stem and another CTCs [17
]. Therefore, as the next step, we plan to test the labeling of breast CSCs with Abs specific to other known markers (e.g., CD133 and ALDH) [33
]. To discriminate PA signals from CD44+ stem CTCs in the background of other potentially CD44+ cancer cells (i.e.
, bulk CTCs and leukocytes), multi-molecular targeting can be applied using CD44, CD45 and CD24 markers. We assume that CD44+/CD24-/CD45- cells could be related to cancer stem cells, and CD44+/ CD45+ could be leukocytes.
In this pilot study, to make sure that CTCs and, especially, stem CTCs (tumor-initiating cells) were present in the blood and disseminated to distant organs at the point of PA detection, we chose later (not early) stage of cancer with visible hematogenous metastases in liver. However, the high sensitivity of PAFC technique may allow assessing the value of stem CTCs for metastasis prevention that should hold a tremendous importance in cancer research [4
]. Our planned future investigations of earliest stages of breast cancer (i.e.
, the stages of sentinel lymph node metastasis [12
] and even before overt metastasis) would elucidate this issue.
In general, the developed biophotonic technology may fill some gaps in in vivo cancer stem cell research related to (1) stem CTC molecular targeting without the potential influence of the novel labels on the host microenvironment, (2) counting of stem CTCs in low concentrations against a background of blood, (3) estimation of the proportion of stem CTCs within the entire CTC population, (4) estimation of CTC/CSC lifetime in circulation, (5) correlation of stem CTC number with metastasis stages, and (6) significance of stem CTC for early metastasis diagnosis and prevention.
Besides breast tumors, the presented strategy may be broadly applicable to all metastatic cancers and would contribute to a better understanding of cancer stem cell biology at the molecular and cellular levels in vivo
, and it would accelerate progress towards attacking the earliest cancer metastasis. We predict that such comprehensive understanding along with this relatively simple technology platform using safe laser parameters and low toxic molecular contrast agents can be quickly translated to humans, possibly as a portable wrist device (e.g.
, bracelet: see the Supplementary notes in [4
]) for non-invasive diagnosis, specific targeted therapies, individualized selection of therapeutic modalities for a specific patient and monitoring of cancer recurrence and disease-free status.