Real-time particle-by-particle detection of erythrocyte-camouflaged microsensor with extended circulation time in the bloodstream.

Personalized medicine offers great potential benefits for disease management but requires continuous monitoring of drugs and drug targets. For instance, the therapeutic window for lithium therapy of bipolar disorder is very narrow, and more frequent monitoring of sodium levels could avoid toxicity. In this work, we developed and validated a platform for long-term, continuous monitoring of systemic analyte concentrations in vivo. First, we developed sodium microsensors that circulate directly in the bloodstream. We used "red blood cell mimicry" to achieve long sensor circulation times of up to 2 wk, while being stable, reversible, and sensitive to sodium over physiologically relevant concentration ranges. Second, we developed an external optical reader to detect and quantify the fluorescence activity of the sensors directly in circulation without having to draw blood samples and correlate the measurement with a phantom calibration curve to measure in vivo sodium. The reader design is inherently scalable to larger limbs, species, and potentially even humans. In combination, this platform represents a paradigm for in vivo drug monitoring that we anticipate will have many applications in the future.

Flow-regulated endothelial glycocalyx determines metastatic cancer cell activity.

Cancer metastasis and secondary tumor initiation largely depend on circulating tumor cell (CTC) and vascular endothelial cell (EC) interactions by incompletely understood mechanisms. Endothelial glycocalyx (GCX) dysfunction may play a significant role in this process. GCX structure depends on vascular flow patterns, which are irregular in tumor environments. This work presents evidence that disturbed flow (DF) induces GCX degradation, leading to CTC homing to the endothelium, a first step in secondary tumor formation. A 2-fold greater attachment of CTCs to human ECs was found to occur under DF conditions, compared to uniform flow (UF) conditions. These results corresponded to an approximately 50% decrease in wheat germ agglutinin (WGA)-labeled components of the GCX under DF conditions, vs UF conditions, with undifferentiated levels of CTC-recruiting E-selectin under DF vs UF conditions. Confirming the role of the GCX, neuraminidase induced the degradation of WGA-labeled GCX under UF cell culture conditions or in Balb/C mice and led to an over 2-fold increase in CTC attachment to ECs or Balb/C mouse lungs, respectively, compared to untreated conditions. These experiments confirm that flow-induced GCX degradation can enable metastatic CTC arrest. This work, therefore, provides new insight into pathways of secondary tumor formation.

Introduction to the Optical Molecular Probes, Imaging and Drug Delivery 2023 feature issue.

A feature issue is being presented by a team of guest editors containing papers based on studies presented at the Optical Molecular Probes, Imaging and Drug Delivery conference as part of the Optica Biophotonics Congress in Vancouver, Canada from April 24-27, 2023.

Lipid nanoparticle encapsulated large peritoneal macrophages migrate to the lungs via the systemic circulation in a model of clodronate-mediated lung-resident macrophage depletion.

Rationale: A mature tissue resident macrophage (TRM) population residing in the peritoneal cavity has been known for its unique ability to migrate to peritoneally located injured tissues and impart wound healing properties. Here, we sought to expand on this unique ability of large peritoneal macrophages (LPMs) by investigating whether these GATA6+ LPMs could also intravasate into systemic circulation and migrate to extra-peritoneally located lungs upon ablating lung-resident alveolar macrophages (AMs) by intranasally administered clodronate liposomes in mice. Methods: C12-200 cationic lipidoid-based nanoparticles were employed to selectively deliver a small interfering RNA (siRNA)-targeting CD-45 labeled with a cyanine 5.5 (Cy5.5) dye to LPMs in vivo via intraperitoneal injection. We utilized a non-invasive optical technique called Diffuse In Vivo Flow Cytometry (DiFC) to then systemically track these LPMs in real time and paired it with more conventional techniques like flow cytometry and immunocytochemistry to initially confirm uptake of C12-200 encapsulated siRNA-Cy5.5 (siRNA-Cy5.5 (C12-200)) into LPMs, and further track them from the peritoneal cavity to the lungs in a mouse model of AM depletion incited by intranasally administered clodronate liposomes. Also, we stained for LPM-specific marker zinc-finger transcription factor GATA6 in harvested cells from biofluids like broncho-alveolar lavage as well as whole blood to probe for Cy5.5-labeled LPMs in the lungs as well as in systemic circulation. Results: siRNA-Cy5.5 (C12-200) was robustly taken up by LPMs. Upon depletion of lung-resident AMs, these siRNA-Cy5.5 (C12-200) labeled LPMs rapidly migrated to the lungs via systemic circulation within 12-24 h. DiFC results showed that these LPMs intravasated from the peritoneal cavity and utilized a systemic route of migration. Moreover, immunocytochemical staining of zinc-finger transcription factor GATA6 further confirmed results from DiFC and flow cytometry, confirming the presence of siRNA-Cy5.5 (C12-200)-labeled LPMs in the peritoneum, whole blood and BALF only upon clodronate-administration. Conclusion: Our results indicate for the very first time that selective tropism, migration, and infiltration of LPMs into extra-peritoneally located lungs was dependent on clodronate-mediated AM depletion. These results further open the possibility of therapeutically utilizing LPMs as delivery vehicles to carry nanoparticle-encapsulated oligonucleotide modalities to potentially address inflammatory diseases, infectious diseases and even cancer.

Two-color diffuse in vivo flow cytometer.

Significance: Hematogenous metastasis is mediated by circulating tumor cells (CTCs) and CTC clusters (CTCCs). We recently developed "diffuse in vivo flow cytometry" (DiFC) to detect fluorescent protein (FP) expressing CTCs in small animals. Extending DiFC to allow detection of two FPs simultaneously would allow concurrent study of different CTC sub-populations or heterogeneous CTCCs in the same animal. Aim: The goal of this work was to develop and validate a two-color DiFC system capable of non-invasively detecting circulating cells expressing two distinct FPs. Approach: A DiFC instrument was designed and built to detect cells expressing either green FP (GFP) or tdTomato. We tested the instrument in tissue-mimicking flow phantoms in vitro and in multiple myeloma bearing mice in vivo. Results: In phantoms, we could accurately differentiate GFP+ and tdTomato+ CTCs and CTCCs. In tumor-bearing mice, CTC numbers expressing both FPs increased during disease. Most CTCCs (86.5%) expressed single FPs with the remainder both FPs. These data were supported by whole-body hyperspectral fluorescence cryo-imaging of the mice. Conclusions: We showed that two-color DiFC can detect two populations of CTCs and CTCCs concurrently. This instrument could allow study of tumor development and response to therapies for different sub-populations in the same animal.

In Vivo Labeling and Detection of Circulating Tumor Cells in Mice Using OTL38.

PURPOSE: We recently developed an optical instrument to non-invasively detect fluorescently labeled circulating tumor cells (CTCs) in mice called 'Diffuse in vivo Flow Cytometry' (DiFC). OTL38 is a folate receptor (FR) targeted near-infrared (NIR) contrast agent that is FDA approved for use in fluorescence guided surgery of ovarian and lung cancer. In this work, we investigated the use OTL38 for in vivo labeling and detection of FR + CTCs with DiFC. PROCEDURES: We tested OTL38 labeling of FR + cancer cell lines (IGROV-1 and L1210A) as well as FR- MM.1S cells in suspensions of Human Peripheral Blood Mononuclear cells (PBMCs) in vitro. We also tested OTL38 labeling and NIR-DIFC detection of FR + L1210A cells in blood circulation in nude mice in vivo. RESULTS: 62% of IGROV-1 and 83% of L1210A were labeled above non-specific background levels in suspensions of PBMCs in vitro compared to only 2% of FR- MM.1S cells. L1210A cells could be labeled with OTL38 directly in circulation in vivo and externally detected using NIR-DiFC in mice with low false positive detection rates. CONCLUSIONS: This work shows the feasibility of labeling CTCs in vivo with OTL38 and detection with DiFC. Although further refinement of the DiFC instrument and signal processing algorithms and testing with other animal models is needed, this work may eventually pave the way for human use of DiFC.

A computer vision approach to rare cell in vivo fluorescence flow cytometry.

Noninvasive enumeration of rare circulating cell populations in small animals is of great importance in many areas of biomedical research. In this work, we describe a macroscopic fluorescence imaging system and automated computer vision algorithm that allows in vivo detection, enumeration and tracking of circulating fluorescently-labeled cells from multiple large blood vessels in the ear of a mouse. This imaging system uses a 660 nm laser and a high sensitivity electron-multiplied charge coupled device camera (EMCCD) to acquire fluorescence image sequences from relatively large (∼5 × 5 mm(2) ) imaging areas. The primary technical challenge was developing an automated method for identifying and tracking rare cell events in image sequences with substantial autofluorescence and noise content. To achieve this, we developed a two-step image analysis algorithm that first identified cell candidates in individual frames, and then merged cell candidates into tracks by dynamic analysis of image sequences. The second step was critical since it allowed rejection of >97% of false positive cell counts. Overall, our computer vision IVFC (CV-IVFC) approach allows single-cell detection sensitivity at estimated concentrations of 20 cells/mL of peripheral blood. In addition to simple enumeration, the technique recovers the cell's trajectory, which in the future could be used to automatically identify, for example, in vivo homing and docking events.

Evaluation of a fast single-photon avalanche photodiode for measurement of early transmitted photons through diffusive media.

We tested the performance of a fast single-photon avalanche photodiode (SPAD) in measurement of early transmitted photons through diffusive media. In combination with a femtosecond titanium:sapphire laser, the overall instrument temporal response time was 59 ps. Using two experimental models, we showed that the SPAD allowed measurement of photon-density sensitivity functions that were approximately 65% narrower than the ungated continuous wave case at very early times. This exceeds the performance that we have previously achieved with photomultiplier-tube-based systems and approaches the theoretical maximum predicted by time-resolved Monte Carlo simulations.

Maximum likelihood tomographic reconstruction of extremely sparse solutions in diffuse fluorescence flow cytometry.

We apply reparameterization and the maximum likelihood method to a specific fluorescence-mediated tomography problem where the solution is known a priori to be extremely sparse (i.e., all image values are zero except for one). Our algorithm performs significantly better than a standard image reconstruction method, particularly for deep-seated targets, and achieves close to 150 µm accuracy in a 3 mm diameter cross-sectional area with only 12 measurements. Moreover, results do not depend on the selection of a regularization parameter or other ad hoc values, and since reconstructions can be computed very quickly, the algorithm is also suitable for real-time implementation.

Special Section Guest Editorial: Seeing Inside Tissue with Optical Molecular Probes.

The editorial introduces the Special Section on Seeing Inside Tissue with Optical Molecular Probes.

Ratiometric fluorescence sensing and quantification of circulating blood sodium sensors in mice in vivo.

In this work, we introduce ratiometric diffuse in vivo flow cytometry (R-DiFC) for quantitative measurement of circulating fluorescent red blood cell (fRBC) sensors for systemic blood sodium levels. Unlike in our previous work in measuring circulating fRBC sensors, R-DiFC allows simultaneous measurement of two fluorophores encapsulated in the sensor, the ratio of which enables self-calibration of the fluorescence signal with different fRBC depths in biological tissue. We show that the R-DiFC signal varies significantly less than either fluorescence signal alone. This work holds promise for personalized monitoring of systemic sodium for bipolar patients in the future.

Dual-ratio approach for detection of point fluorophores in biological tissue.

SIGNIFICANCE: Diffuse in-vivo Flow Cytometry (DiFC) is an emerging fluorescence sensing method to non-invasively detect labeled circulating cells in-vivo. However, due to Signal-to-Noise Ratio (SNR) constraints largely attributed to background tissue autofluorescence, DiFC's measurement depth is limited. multiplies Aim: The Dual-Ratio (DR) / dual-slope is a new optical measurement method that aims to suppress noise and enhance SNR to deep tissue regions. We aim to investigate the combination of DR and Near-InfraRed (NIR) DiFC to improve circulating cells' maximum detectable depth and SNR. APPROACH: Phantom experiments were used to estimate the key parameters in a diffuse fluorescence excitation and emission model. This model and parameters were implemented in Monte-Carlo to simulate DR DiFC while varying noise and autofluorescence parameters to identify the advantages and limitations of the proposed technique. RESULTS: Two key factors must be true to give DR DiFC an advantage over traditional DiFC; first, the fraction of noise that DR methods cannot cancel cannot be above the order of 10% for acceptable SNR. Second, DR DiFC has an advantage, in terms of SNR, if the distribution of tissue autofluorescence contributors is surface-weighted. CONCLUSIONS: DR cancelable noise may be designed for (e.g. through the use of source multiplexing), and indications point to the autofluorescence contributors' distribution being truly surface-weighted in-vivo. Successful and worthwhile implementation of DR DiFC depends on these considerations, but results point to DR DiFC having possible advantages over traditional DiFC.

Dual-ratio approach for detection of point fluorophores in biological tissue.

Significance: Diffuse in vivo flow cytometry (DiFC) is an emerging fluorescence sensing method to non-invasively detect labeled circulating cells in vivo. However, due to signal-to-noise ratio (SNR) constraints largely attributed to background tissue autofluorescence (AF), DiFC's measurement depth is limited. Aim: The dual ratio (DR)/dual slope is an optical measurement method that aims to suppress noise and enhance SNR to deep tissue regions. We aim to investigate the combination of DR and near-infrared (NIR) DiFC to improve circulating cells' maximum detectable depth and SNR. Approach: Phantom experiments were used to estimate the key parameters in a diffuse fluorescence excitation and emission model. This model and parameters were implemented in Monte Carlo to simulate DR DiFC while varying noise and AF parameters to identify the advantages and limitations of the proposed technique. Results: Two key factors must be true to give DR DiFC an advantage over traditional DiFC: first, the fraction of noise that DR methods cannot cancel cannot be above the order of 10% for acceptable SNR. Second, DR DiFC has an advantage, in terms of SNR, if the distribution of tissue AF contributors is surface-weighted. Conclusions: DR cancelable noise may be designed (e.g., through the use of source multiplexing), and indications point to the AF contributors' distribution being truly surface-weighted in vivo. Successful and worthwhile implementation of DR DiFC depends on these considerations, but results point to DR DiFC having possible advantages over traditional DiFC.

Near-infrared diffuse in vivo flow cytometry.

Significance: Diffuse in vivo flow cytometry (DiFC) is an emerging technique for enumerating rare fluorescently labeled circulating cells noninvasively in the bloodstream. Thus far, we have reported red and blue-green versions of DiFC. Use of near-infrared (NIR) fluorescent light would in principle allow use of DiFC in deeper tissues and would be compatible with emerging NIR fluorescence molecular contrast agents. Aim: We describe the design of a NIR-DiFC instrument and demonstrate its use in optical flow phantoms in vitro and in mice in vivo. Approach: We developed an improved optical fiber probe design for efficient collection of fluorescence from individual circulating cells and efficient rejection of instrument autofluorescence. We built a NIR-DiFC instrument. We tested this with NIR fluorescent microspheres and cell lines labeled with OTL38 fluorescence contrast agent in a flow phantom model. We also tested NIR-DiFC in nude mice injected intravenously with OTL38-labeled L1210A cells. Results: NIR-DiFC allowed detection of circulating tumor cells (CTCs) in flow phantoms with mean signal-to-noise ratios (SNRs) of 19 to 32 dB. In mice, fluorescently labeled CTCs were detectable with mean SNR of 26 dB. NIR-DiFC also exhibited orders significantly lower autofluorescence and false-alarm rates than blue-green DiFC. Conclusions: NIR-DiFC allows use of emerging NIR contrast agents. Our work could pave the way for future use of NIR-DiFC in humans.

Signal and measurement considerations for human translation of diffuse in vivo flow cytometry.

SIGNIFICANCE: "Diffuse in vivo flow cytometry" (DiFC) is an emerging technology for fluorescence detection of rare circulating cells directly in large deep-seated blood vessels in mice. Because DiFC uses highly scattered light, in principle, it could be translated to human use. However, an open question is whether fluorescent signals from single cells would be detectable in human-scale anatomies. AIM: Suitable blood vessels in a human wrist or forearm are at a depth of ∼2 to 4 mm. The aim of this work was to study the impact of DiFC instrument geometry and wavelength on the detected DiFC signal and on the maximum depth of detection of a moving cell. APPROACH: We used Monte Carlo simulations to compute fluorescence Jacobian (sensitivity) matrices for a range of source and detector separations (SDS) and tissue optical properties over the visible and near infrared spectrum. We performed experimental measurements with three available versions of DiFC (488, 640, and 780 nm), fluorescent microspheres, and tissue mimicking optical flow phantoms. We used both computational and experimental data to estimate the maximum depth of detection at each combination of settings. RESULTS: For the DiFC detection problem, our analysis showed that for deep-seated blood vessels, the maximum sensitivity was obtained with NIR light (780 nm) and 3-mm SDS. CONCLUSIONS: These results suggest that-in combination with a suitable molecularly targeted fluorescent probes-circulating cells and nanosensors could, in principle, be detectable in circulation in humans.

Early photon tomography allows fluorescence detection of lung carcinomas and disease progression in mice in vivo.

Imaging of targeted fluorescent probes offers significant advantages for investigating disease and tissue function in animal models in vivo. Conversely, macroscopic tomographic imaging is challenging because of the high scatter of light in biological tissue and the ill-posed nature of the reconstruction mathematics. In this work, we use the earliest-transmitted photons through Lewis Lung Carcinoma bearing mice, thereby dramatically reducing the effect of tissue scattering. By using a fluorescent probe sensitive to cysteine proteases, the method yielded outstanding imaging performance compared with conventional approaches. Accurate visualization of biochemical abnormalities was achieved, not only in the primary tumor, but also in the surrounding tissue related to cancer progression and inflammatory response at the organ level. These findings were confirmed histologically and with ex vivo fluorescence microscopy. The imaging fidelity demonstrated underscores a method that can use a wide range of fluorescent probes to accurately visualize cellular- and molecular-level events in whole animals in vivo.

Comparison of fluorescence tomographic imaging in mice with early-arriving and quasi-continuous-wave photons.

The highly diffuse nature of light propagation in biological tissue is a major challenge for obtaining high-fidelity fluorescence tomographic images. In this work we investigated the use of time-gated detection of early-arriving photons for reducing the effects of light scatter in mice relative to quasi-cw photons. When analyzing sinographic representations of the measured data, it was determined that early photons allowed a reduction in the measured FWHM of fluorescent targets by a factor of approximately 2-3, yielding a significant improvement in the tomographic image reconstruction quality.

Short-Term Circulating Tumor Cell Dynamics in Mouse Xenograft Models and Implications for Liquid Biopsy.

MOTIVATION: Circulating tumor cells (CTCs) are widely studied using liquid biopsy methods that analyze fractionally-small peripheral blood (PB) samples. However, little is known about natural fluctuations in CTC numbers that may occur over short timescales in vivo, and how these may affect detection and enumeration of rare CTCs from small blood samples. METHODS: We recently developed an optical instrument called "diffuse in vivo flow cytometry" (DiFC) that uniquely allows continuous, non-invasive counting of rare, green fluorescent protein expressing CTCs in large blood vessels in mice. Here, we used DiFC to study short-term changes in CTC numbers in multiple myeloma and Lewis lung carcinoma xenograft models. We analyzed CTC detections in over 100 h of DiFC data, and considered intervals corresponding to approximately 1%, 5%, 10%, and 20% of the PB volume. In addition, we analyzed changes in CTC numbers over 24 h (diurnal) periods. RESULTS: For rare CTCs (fewer than 1 CTC per ml of blood), the use of short DiFC intervals (corresponding to small PB samples) frequently resulted in no detections. For more abundant CTCs, CTC numbers frequently varied by an order of magnitude or more over the time-scales considered. This variance in CTC detections far exceeded that expected by Poisson statistics or by instrument variability. Rather, the data were consistent with significant changes in mean numbers of CTCs on the timescales of minutes and hours. CONCLUSIONS: The observed temporal changes can be explained by known properties of CTCs, namely, the continuous shedding of CTCs from tumors and the short half-life of CTCs in blood. It follows that the number of cells in a blood sample are strongly impacted by the timing of the draw. The issue is likely to be compounded for multicellular CTC clusters or specific CTC subtypes, which are even more rare than single CTCs. However, we show that enumeration can in principle be improved by averaging multiple samples, analysis of larger volumes, or development of methods for enumeration of CTCs directly in vivo.

Fluorescence Labeling of Circulating Tumor Cells with a Folate Receptor-Targeted Molecular Probe for Diffuse In Vivo Flow Cytometry.

PURPOSE: We recently developed a new instrument called "diffuse in vivo flow cytometry" (DiFC) for enumeration of rare fluorescently labeled circulating tumor cells (CTCs) in small animals without drawing blood samples. Until now, we have used cell lines that express fluorescent proteins or were pre-labeled with a fluorescent dye ex vivo. In this work, we investigated the use of a folate receptor (FR)-targeted fluorescence molecular probe for in vivo labeling of FR+ CTCs for DiFC. PROCEDURES: We used EC-17, a FITC-folic acid conjugate that has been used in clinical trials for fluorescence-guided surgery. We studied the affinity of EC-17 for FR+ L1210A and KB cancer cells. We also tested FR- MM.1S cells. We tested the labeling specificity in cells in culture in vitro and in whole blood. We also studied the detectability of labeled cells in mice in vivo with DiFC. RESULTS: EC-17 showed a high affinity for FR+ L1210A and KB cells in vitro. In whole blood, 85.4 % of L1210A and 80.9 % of KB cells were labeled above non-specific background with EC-17, and negligible binding to FR- MM.1S cells was observed. In addition, EC-17-labeled CTCs were readily detectable in circulation in mice with DiFC. CONCLUSIONS: This work demonstrates the feasibility of labeling CTCs with a cell-surface receptor-targeted probe for DiFC, greatly expanding the potential utility of the method for pre-clinical animal models. Because DiFC uses diffuse light, this method could be also used to enumerate CTCs in larger animal models and potentially even in humans.

Heterogeneity of circulating tumor cell dissemination and lung metastases in a subcutaneous Lewis lung carcinoma model.

Subcutaneous (s.c.) tumor models are widely used in pre-clinical cancer metastasis research. Despite this, the dynamics and natural progression of circulating tumor cells (CTCs) and CTC clusters (CTCCs) in peripheral blood are poorly understood in these models. In this work, we used a new technique called 'diffuse in vivo flow cytometry' (DiFC) to study CTC and CTCC dissemination in an s.c. Lewis lung carcinoma (LLC) model in mice. Tumors were grown in the rear flank and we performed DiFC up to 31 days after inoculation. At the study endpoint, lungs were excised and bioluminescence imaging (BLI) was performed to determine the extent of lung metastases. We also used fluorescence macro-cryotome imaging to visualize infiltration and growth of the primary tumor. DiFC revealed significant heterogeneity in CTC and CTCC numbers amongst all mice studied, despite using clonally identical LLC cells and tumor placement. Maximum DiFC count rates corresponded to 0.1 to 14 CTCs per mL of peripheral blood. In general, CTC numbers did not necessarily increase monotonically over time and were poorly correlated with tumor volume. However, there was a good correlation between CTC and CTCC numbers in peripheral blood and lung metastases. We attribute the differences in CTC numbers primarily due to growth patterns of the primary tumor. This study is one of the few reports of CTC shedding dynamics in sub-cutaneous metastasis models and underscores the value of in vivo methods for continuous, non-invasive CTC monitoring.