key: cord-0705922-ur5dhsio
authors: Schikora, Detlef
title: In vivo detection of circulating tumour cell clusters by photodiagnostic spectroscopy
date: 2020-04-13
journal: Photodiagnosis Photodyn Ther
DOI: 10.1016/j.pdpdt.2020.101755
sha: 69aedd78e1e142120aa6b2f424b95ff5337895b7
doc_id: 705922
cord_uid: ur5dhsio

Abstract Background We demonstrate a new diagnostic method, the Photodiagnostic Infrared Spectroscoppy (PDIS), which is able to detect circulating tumor clusters and circulating tumor cells in the circulatory system. Methods The PDIS method is based on photodiagnostic physics and high resolution spectroscopy and is using calibrated spectroscopic data for the diagnostic analysis of the blood screening spectra. Using Confocal Laser Scanning Mikcroscopy the Indocyangreen-uptake of different cell lines of breast cancer cells is studied. Results The PDIS supplies calibrated diagnostic data about the presence or absence of CTCs and CTC clusters in the bloodstream with a sensitivity of 98 %. Therefore, the PDIS is suited to control the blood of cancer patients with respect of CTC and CTC clusters with an resolution of one CTC and one CTC cluster per blood volume. PDIS distinguishes the different phenotypes of CTC clusters. Conclusion Circulating tumor cell slusters play a key role in the metastatic process and are formed only in solid tumors, they are appropriate objects to validate cancer treatments and to recognice cancer formation. The PDIS is a calibrated diagnostic method which allows to evaluate the results of solid tumor treatments and of chemotherapy treatments and to optimize the individual cancer treatment strategies.

The PDIS method is based on photodiagnostic physics and high resolution spectroscopy and is using calibrated spectroscopic data for the diagnostic analysis of the blood screening spectra.

Using Confocal Laser Scanning Mikcroscopy the Indocyangreen-uptake of different cell lines of breast cancer cells is studied.

Results: The PDIS supplies calibrated diagnostic data about the presence or absence of CTCs and CTC clusters in the bloodstream with a sensitivity of 98 % . Therefore, the PDIS is suited to control the blood of cancer patients with respect of CTC and CTC clusters with an resolution of one CTC and one CTC cluster per blood volume. PDIS distinguishes the different phenotypes of CTC clusters.

Conclusion: Circulating tumor cell slusters play a key role in the metastatic process and are formed only in solid tumors, they are appropriate objects to validate cancer treatments and to recognice cancer formation. The PDIS is a calibrated diagnostic method which allows to evaluate the results of solid tumor treatments and of chemotherapy treatments and to optimize the individual cancer treatment strategies. key words: photodiagnostic diagnosis; circulating tumor cell clusters; fluorescence spectroscopy; high resolution blood screening; photodiagnostic infrared spectroscopy BACKGROUND Metastases are the main cause of cancer-related deaths; however, the mechanisms underlying metastatic spread are not completely understood. It is a complicated, multistep process that requires detachment of cancer cells from the primary tumour, intravasation into the bloodstream, the survival of tumour cells and tumour clusters in the bloodstream, and finally extravasation to distant organs [1] . Besides circulating tumour cells, tumour clusters seem to play a key role in the metastatic process [2] . In 1954, Watanabe showed that these clustered cells have high metastatic potential [3] . More recent studies indicated that clusters have distinct features compared to single tumour cells, including phenotype, gene expression signature, and dissemination mode. Thus, establishing the role and significance of circulating tumour cell clusters in the spread of cancer is of extreme importance. Circulating tumour cell clusters are defined as a group of more than two or three tumour cells with strong cell-cell contacts moving in the blood circulation and in the lymphatic system. They are rare, but highly metastatic. Despite representing only 2-5% of all circulating tumour cells, clusters were shown to form about 50% of breast cancer metastases, and their metastatic potential was estimated to be 23-50 times higher than that of single cells [4] . Circulating tumour cell clusters represent a conglomerate of tumour cells and other types of cells including platelets, immune cells and cancer-associated fibroblasts. It seems that tumour clusters are epithelial-mesenchymal hybrids and therefore possess enormous plasticity [5] . This composi-J o u r n a l P r e -p r o o f tion provides a local microenvironment that is thought to protect tumour clusters from death in the circulation by minimising shear stress and immune attack, and facilitating colonisation [6] . Au et al. have shown that clusters can actually traverse capillary-sized vessels [7] . It appears that upon constriction, tumour cell clusters undergo rapid reorganisation, forming a chain-like structure that reduces hydrodynamic resistance and allows them to pass through a small vessel. As concequence, the tumor cluster chains move preferentially parallel oriented to the vessel axis to maintain the lowest hydrodynamic resistance. This reorganisation was shown to be reversible, since after exiting constriction, clusters rearrange into their typical organisations. Aceto et al. [4] have demonstrated that the invasive potential of tumour clusters is higher than that of single tumour cells. Cheung et al. obtained similar results and estimated that approximately 97% of metastases arise from clusters [8] . Other data suggested that intravascular aggregation and proliferation could be excluded as potential sources of tumour clusters due to the unfavourable conditions present in the bloodstream [9] . Very recently, Gkountela et al. [10] demonstrated that -in strong contrast to circulating tumour cells -the pattern of transcription factors in tumour clusters reveals similarities to embryonic stem cells along with increased proliferation. However, some cluster processes are still unknown regarding their genesis,transit and settlement and about the precise cellular and molecular mechanism. Nevertheless, the clinical data so far indicate the prognostic value of CTC cluster analysis in predicting treatment resistance and survival outcomes in cancer patients. (16) Tumour clusters are of particular diagnostic importance because they only occur when a primary tumour exists. Tumour clusters thus may serve as indicators for the formation and existence of primary tumours, and conversely, they may also serve as indicators for complete or incomplete removal of primary tumours [4] , as long metastases have not been formed. Cancer metastases arise mainly from circulating tumour cell clusters. Therefore, they should be the most important targets for metastasis prevention and reduction [8] .The existing liquid biopsy methods of circulating tumour cell detection, like Cell Search [12] , cannot be applied as reference for tumour cluster enumeration because they do not preserve their morphological and molecular status and are sensitive only for epithelial cells. Clearance rates of the labeled clusters and single CTCs from the bloodstream were established in mouse experiments by in vivo flow cytometry [7] . A photoacoustic in vivo method for circulating melanoma tumor cell cluster detection is described in (15) .

There is no gold-standard method of detecting tumour clusters that enables a comparison of results obtained by different methods. Therefore, there is an urgent need for a standardised method for isolating and detecting circulating tumour clusters [2] . Photodiagnostic Infrared Spectroscopy (PDIS) is based on photodynamic physics, which was discovered and described for the first time by Jablonski in 1933 [11] . Photodynamic excitation is a physical principle that is explained in the Jablonski Diagram. The most important characteristic feature of a photodynamic process in general, and with liposomal indocyanine green (ICG) as a photosensitiser in particular, is the simultaneous emission of fluorescence and phosphorescence radiation. This is a kind of "fingerprint" and proves without a doubt the photodynamic origin of the signals. Therefore, in spectroscopic peak analyses, photon peaks generated by the environment can be clearly distinguished from photon peaks generated by a photodynamic process. The internal conversion of photon energy to heat can be neglected, it does not influence the detected signature of the signals. The amount of heat is very low due to the low excitation energies of about 10 J o u r n a l P r e -p r o o f mW/cm2 and the low concentration of ICG. We have used ICG as a specific liposomal formulation, which has an absorption wavelength of 785 nm, a fluorescence wavelength of 830 nm, and a phosphorescence wavelength of 940 nm, as reported by Bäumler et al [13] . The advantage of liposomal ICG compared to normal ICG, which is used in angiography, is the much higher lifetime of liposomal ICG in blood and tissue ( ~ 36 h) compared to normal ICG (~ 10 min) . After intravenous injection, normal ICG binds within 1-2 sec almost completely (98%) to serum proteins, therefore an accumulation in peripheral tissue or organs or even tumors is practically impossible.

Methods for in vivo detection of circulating tumour clusters do not exist. Due to the lack of evidencebased reference procedures and standards regarding in vivo tumour cluster detection, we have calibrated the PDIS. in all experiments, the flow speed was kept constant at 10 cm/s. At the end of the optical fibre detector, J o u r n a l P r e -p r o o f an external laser source, emitting at 785 nm, is placed to generate an infrared light field within the silicon tube. When an ICG-incubated tumour cell or tumour cluster flows through the light field, fluorescence radiation at 830 nm and phosphorescence radiation are emitted and guided via optical fibre to the spectrometer. After 4 h incubation in 15 µM ICG solution, the cells, which were plated in a 6-well tissue culture plate at a concentration of 10 cells/well, were injected into the upper nutrition reservoir. The ICG uptake of MDA-231 cancer cell clusters was studied by haematological investigation using Confocal Scanning Laser Microscopy (CSLM). CLSM images were acquired by a VisiScope (Visitron Systems, Germany). Liposomal ICG was purchased from Burg-Apotheke, Koenigstein, Germany; the spectroscopic device was a C9505CB with a detection range from 400 nm to 1100 nm, produced by Hamamatsu Ltd, Hamamatsu, Japan. The laser catheter was purchased from Webermedical GmbH (Lauenfoerde,Germany); the numerical aperture was 0.3. The recorded spectra were statistically analysed with respect to peak emission wavelength (nm), peak "Full Width at Half Maximum" (FWHM)-broadness (s), peak intensity (counts), and peak origin (simultaneous fluorescence and phosphorescence emission). The infusion concentration of liposomal ICG was always 15 µM. In contrast, human leukocytes exposed to 15 µM ICG solution did not show any photosensitiser accumulation [14] . Figure 2 shows clearly that liposomal ICG was accumulated in breast tumour cells and clusters at diagnostic concentrations of 15 J o u r n a l P r e -p r o o f µM. Hence, the prerequisite for photodiagnostic excitation of the breast cancer cells, as well for diagnostics for therapy, is fulfilled in light of these results. Our haematologic investigations with normal human leukocyte cells have shown that these cells do not accumulate liposomal ICG at concentrations of 15 µM. [14] Therefore, there are two components in the bloodstream which emit 830 nm fluorescence: on the one hand, there is the infused liposomal ICG, which is to 98% bound to plasma proteins such as albumin and which forms a constant, continuous fluorescence radiation background. On the other hand, there are the low numbers of circulating breast tumour cells and tumour clusters, which have accumulated the liposomal ICG and which supply discrete fluorescence signals upon excitation. This is how it is possible to unambiguously distinguish the continuous background from the discrete tumour cluster peaks in PDIS blood screening. Before starting the PDIS blood screening, a "reference" light intensity is measured within 10 -3 s. This reference spectrum is permanently subtracted from each "verum" spectrum (every 10 -3 s) measured during the screening cycle of 10 3 s duration. As a result, only the additional fluorescence and phosphorescence peaks from tumour cells and clusters are recorded above the noise. The calibration procedures were performed in a dark environment. The CTC clusters were externally prepared by adding plakoglobin to the tumor cell suspension. After passing the laser spot, the single clusters werer filtered out by a microcavity array filter and investigated by CSLM, to correlate the cluster morphology and the corresponding cluster fluorescence. When passing the 785 nm laser, the ICGincubated cancer cells emitted 830 nm fluorescence peaks, which were guided to the spectrometer via optical laser catheter. The DMEM nutrition solutions used here were optically inert. The spectroscopic background radiation was compensated for with a special subtracting software, as described above, so that the starting intensity was always near the zero-fluorescence intensity value. The advantage is that only the photons emitted from the moving tumour clusters are displayed above the spectral noise. The flow speed was held constant at 10 cm/s, which is close to the blood flow velocity in the peripheral vena basilica, which has always been used in clinical PDIS screenings. It must be pointed out that in all 120 calibration experiments, almost constant peak intemsities for all MDA 231 cells were measured Obviously, the liposomal ICG uptake of breast cancer cells is relatively stable and not significantly different in blood and in calibration experiments. We never found peak intensities of single breast cancer cells above the limits, given in the peak parameter set.

The PDIS calibration peak of a moving tumour cluster cloud of seven MDA-231 cells in Figure 3 [7] . Even so, the clinical relevance is not dependent upon cluster morphology. When a tumour cluster, chain, or cloud is detected in the bloodstream, there must be a solid tumour in the body, and there is a significant risk of formation of metastases. To destroy these clusters, Photodynamic Therapy was performed using liposomal ICG. The photo-oxidative killing of tumour cells by ICG was reported by Bäumler et.al. [13] . After LED irradiation of a vein with an external 785 nm infrared LED radiation head of 40 mW power and 30 J/cm 2 , the circulating tumour cells and tumour clusters were photo-oxidatively killed, and the final PDIS diagnosis of Figure 6 showed only noise, with no peak-signals from circulating tumour cells or tumour clusters. The decrease of the fluorescence intensity during the measuring cycle is mainly due to the photodynamic destruction of tumour cells and clusters, which decreases the ICG concentration in the blood. The influence of bleaching effects was determined in separate experiments that showed intensity reductions of liposomal ICG during the screening cycle due to bleaching effects on the order of 3%.

The discontinuous emission of CTC clusters reported by Aceto et al [4] is in agreement with the PDIS screening shown in Figure 4 . It is obvious that the primary tumour emits the breast cancer clusters discontinuously. (17) that the dye Riboflavin binds to corona viruses, which enables the detection and destruction by PDIS using 450 nm blue light emitters.

In summary, we have developed and calibrated the diagnostic PDIS blood screening method. PDIS supplies calibrated diagnostic data about the presence or absence of circulating tumour clusters, which,

in combination with other diagnostic information, allows for fast and easy estimation of the success of applied cancer therapies. The PDIS procedure represents a new diagnostic method to monitor and control the most important transport channels for distant metastases formation with regard to circulating J o u r n a l P r e -p r o o f tumour cell clusters: the circulatory system and the lymphatic system. A PDIS blood screening can be performed and analyzed within 45 min. PDIS diagnostics allows for the verification of the results of surgery and chemotherapy, and for the targeting of treatment strategies for circulating tumour cluster dissolution to keep the blood of cancer patients free from tumour clusters and cells, and consequently, to prevent metastasis formation and to improve the overall survival. The PDIS method can be applied also for corona virus detection and destruction using Riboflavin as photosensitizer.

Ethical approval and consent to participate All PDIS diagnostics performed on patients were initiated after each patient gave written consent. Patients with brain tumours and leukaemia were excluded, and patients with breast cancer of a different stage were included in prospective investigations, which were performed in two German clinics. The Authors' contributions D.S. designed the calibration apparatus, performed the screenings, and wrote the manuscript.

The author declares no conflicts of interest.

Metastatic colonization by circulating tumour cells

CTC clusters in cancer prgression and metastasts

The metastasizability of tumour cells

Circulating tumour cell clusters are oligoclonal precursors of breast cancer metastasis

En route to metastasis: circulating tumour cell clusters and epithelial-to-mes-enchymal transition

Circulating tumour cell clusters: what we know and what we expect (review)

Clusters of circulating tumour cells traverse capillary-sized vessels

Polyclonal breast cancer metastases arise from collective dissemination of keratin 14-expressing tumour cell clusters

Neutrophils escorting circulating tumour clusters to enable cell cycle progression

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Szeimies RM Photo-oxidative killing of human colonic cancer cells using indocyanine green and infrared light

Labelfree high-throughput detection and quantification of circulating melanoma tumor cell clus ters by linear-array-based photoacoustic tomography

Trivedi Perspective on Circulating Tumor Cell Clusters: Why it takes a village to metastasize Canc

Inactivation of Middle East respiratory syndrome coronavirus (MERS-CoV) in plasma products using a riboflavin-based and ultraviolet light-based photochemical treatment

The author would like to thank the University of Paderborn for technical support. The collaboration with Dr M. Weber (Laserclinic Lauenförde) with regard to clinical support is gratefully acknowledged.J o u r n a l P r e -p r o o f