Method and device for unmasking biological cells via plasmin activation

Abstract

The invention relates to a method (500) and a device (100) for unmasking biological cells, in particular circulating tumor cells, with plasmin, wherein a reagent (10) for converting plasminogen to plasmin is added to a sample containing plasminogen and biological cells, in particular a blood sample.

Description

State of the art

[0001] The most common cause of death after a cancer diagnosis is not the primary tumor itself, but in around 90% of cases, metastases that appear later (Jemal et al. “Global cancer statistics”. CA Cancer J Clin. 61, 2011, pp. 69-90). These can occur even years after treatment or removal of the primary tumor. Therefore, extensive research is being conducted into methods that make it possible to detect the disease before metastases develop and to adapt treatment accordingly. Metastases are widely believed to arise from cells called circulating tumor cells (CTCs), which detach from the primary tumor, invade the peripheral bloodstream, and then reach surrounding tissue. Approximately 0.1% of all CTCs can leak into microvessels in the surrounding tissue or persist in the bloodstream for a long time.These few cells still present in the blood despite surgery and therapy are considered the starting point for metastases and recurrences. Therefore, their detection is relevant for both prognostic statements and clinical management, as described, for example, in Pantel et al., "Detection and clinical importance of micrometastatic disease." J Natl Cancer Inst. 91, 1999, 13, pp. 1113-24.

[0002] The analysis of CTCs from blood is based on different physical and biological properties. Biological characteristics ensure greater selectivity and specificity of the method. Epithelial CTCs express various cytokeratins CK8, CK18, and CK19 (intracellular structural proteins) and the surface protein epithelial cell adhesion molecule (EpCAM). These surface proteins can be detected on cells in blood samples using specific, fluorescently labeled antibodies, allowing the CTCs to be positively selected and counted. For example, the published patent application DE 10 2021 203 897 A1 describes a microfluidic cartridge with a microfluidic chip with a multitude of etched wells into which CTCs can be sedimented and subsequently counted based on their fluorescent labels.Various studies have shown that by determining the number of epithelial CTCs in the blood, conclusions can be drawn about treatment response or the development of resistance, as shown, for example, in Camara et al., “The relevance of circulating epithelial tumor cells (CETC) for therapy monitoring during neoadjuvant (primary systemic) chemotherapy in breast cancer.” Ann Oncol. 18, 2007, Vol. 9, pp. 1484-1492.

[0003] A major challenge in the detection of CTCs is their low number in the blood compared to healthy / normal blood cells (10 1 -10 3 CTCs on 10 6 -10 7Leukocytes / mL blood). Furthermore, the surface proteins through which CTCs can be detected are often hidden by the attachment of platelets, leukocytes, fibrin, proteins, or carbohydrates from the blood, which is also referred to as CTC masking. Tumor cells can also activate platelets and the blood coagulation system through their ability to produce procoagulant substances or inhibitors. This leads to detection antibodies being sterically prevented from binding to the cells. This requires a so-called unmasking step. There is evidence in the literature that unmasking can occur after a certain standing time of the blood sample by proteases from dying blood samples, so that more EpCAM-positive cells are detected after 24 hours of standing time than in the first hours after collection, as described in Hekimian et al."Demasking of epithelial cell adhesion molecule (EpCAM) on circulating epithelial tumor cells by Tween®20 treatment in breast cancer patients" Clinical Chemistry and Laboratory Medicine, vol. 50, no. 4, 2012, pp. 701-708. https: / / doi.org / 10.1515 / cclm.2011.812. Another possibility is the addition of detergents. However, the effect these have on the cells beyond unmasking is not fully understood. Furthermore, detergents in excessively high concentrations can lead to undesirable complete cell lysis. Disclosure of the inventionAdvantages of the invention

[0004] Against this background, the invention relates to a method for unmasking biological cells, in particular circulating tumor cells, with plasmin. A reagent for converting plasminogen to plasmin is added to a sample containing plasminogen and biological cells.

[0005] The sample can be a blood sample, in particular a whole blood sample, i.e. a sample that contains blood, in the case of whole blood, blood with all its components.

[0006] Biological cells can be understood as animal or human cells that are masked, as described above, i.e., in particular, cells whose surface proteins are at least partially covered by other entities, and in which this covering can be reduced or completely removed by plasmin. In particular, these biological cells are masked cells found in the blood, especially masked immune cells, such as masked plasma cells or B cells, or circulating tumor cells (CTCs).

[0007] Plasmin, a serine protease, is the proteolytically active form of plasminogen and plays an active role in fibrinolysis and the prevention of blood clots. Inactive plasminogen circulates in the body at a concentration of approximately 180 µg / mL (2 µM). The invention exploits the fact that the proteolytically inactive form is also present in a blood sample, with a half-life of approximately 2 days in plasma. Furthermore, it utilizes the finding that the activation of plasmin by adding a plasminogen-activating reagent, in particular a streptokinase, is an effective method for making surface proteins on cells, particularly on CTCs, accessible to detection antibodies (e.g., EpCAM antibodies in the case of CTCs) through the degradation or detachment of platelets, fibrin, proteins, or carbohydrates deposited on the cell surface.

[0008] The invention thus advantageously enables a defined and controlled unmasking of cells, particularly CTCs, in blood samples taken by adding a reagent that specifically converts the plasminogen into active plasmin. This reagent thus indirectly exposes the surface antigens on these cells according to the invention.

[0009] The conversion of plasminogen to plasmin can be understood as the activation of plasminogen. The reagent is therefore a plasminogen activator, particularly a kinase such as streptokinase or urokinase (especially uPA - urokinase plasminogen activator), tissue plasminogen activator (tPA), fibrin, or coagulation factor XII (Hageman factor).

[0010] The invention thus makes it possible to avoid the alternative direct addition of active plasmin for unmasking. This avoids, in particular, the direct binding of directly added plasmin by plasmin inhibitors present in the blood, particularly serpins or serine protease inhibitors.

[0011] In a particularly advantageous development of the invention, a second reagent is added to the sample to inhibit the conversion of plasminogen to plasmin. The second reagent can comprise one or more plasminogen inactivators and / or a plasminogen activator inhibitor. This has the advantage that the activation of the plasminogen and thus the unmasking can be stopped in a controlled manner. The plasminogen inactivators can be, for example, aprotinin, ε-aminocaproic acid, para-aminomethylbenzoic acid (PAMBA), tranexamic acid, alpha-2-antiplasmin, and / or alpha-2-macroglobulin. The plasminogen activator inhibitors can be, in particular, plasminogen activator inhibitor type 1 (PAI-1) and / or one of the other types 2 to 4. In particular, depending on the plasminogen activator initially added, an effective antagonist can be added as a plasminogen activator inhibitor.

[0012] According to a further advantageous development of the invention, the reagent is at least partially separated from the sample by centrifugation. The cells can then preferably be resuspended in a physiological buffer. This can also be done alternatively or in addition to the above-described preferred addition of plasminogen inactivators and / or plasminogen activator inhibitors and has the advantage that the resulting dilution inhibits the conversion of plasminogen to plasmin.

[0013] The invention also relates to a device, in particular a microfluidic cartridge, for unmasking biological cells, in particular circulating tumor cells. The device comprises an input opening for receiving the sample containing plasminogen and biological cells, as well as the reagent for converting plasminogen to plasmin. The reagent can preferably be stored upstream of the device, in particular in a chamber in the case of the cartridge. After addition of the sample, the sample and the reagent can be mixed, in particular by conveying the sample to the reagent and / or vice versa. According to a preferred development, the device can also comprise the second reagent described above, for example likewise stored upstream in a, preferably different, chamber in the case of the cartridge. This enables a high degree of automation for carrying out the method according to the invention.The device, in particular the microfluidic cartridge, can comprise further functionalities for counting and / or analyzing CTCs, as described in particular in the above-mentioned document DE 10 2021 203 897 A1. In particular, the device can comprise a carrier substrate, preferably with microcavities, for sedimentation and subsequent counting and / or analysis of the unmasked cells. Short description of the drawings

[0014] Embodiments of the invention are illustrated schematically in the drawings and explained in more detail in the following description.

[0015] It shows Fig. 1 a flowchart of an embodiment of the method according to the invention, for example using the device according to the invention and Fig. 2 an embodiment of the device according to the invention. Embodiments of the invention

[0016] In the following, a preferred embodiment of the method according to the invention is described, for which purpose the flow chart 500 in Fig. 1 shows the corresponding process steps schematically.

[0017] According to a first step 501 of the method 500, a sample containing biological cells and plasminogen is provided. The sample is, in particular, whole blood from a human or animal, for which, in this example, a count of the CTCs contained in this sample is to be performed. For example, a device and method as described in published patent application DE 10 2021 203 897 A1 can be used for this purpose. The sample, which comprises, for example, a few milliliters (mL) of whole blood, can be obtained, in particular, by a conventional blood draw from a patient.

[0018] In a second step of method 502, the reagent for converting plasminogen to plasmin is added to the sample, thus starting the unmasking reaction. The reagent can be added, in particular, into a vessel containing the sample with the biological cells and the plasminogen. The vessel can be a conventional vessel for (possibly only temporarily) storing a blood sample. Alternatively, the addition can also take place within a microfluidic device. For this purpose, the reagent can also be stored upstream of the device. Fig. 2 shows an embodiment of the device 100 according to the invention, which comprises an input opening 101 for receiving the sample and the reagent 10. For example, the device 100 is a microfluidic cartridge as described in DE 10 2021 203 897 A1, into which the reagent 10 is introduced or in which the reagent 10 is already stored, for example in a first chamber 110 of the device.

[0019] For example, the reagent is streptokinase. It is advantageous to add 10,000–30,000 IU (International Units) of streptokinase per mL of blood, followed by incubation for 10 minutes to 12 hours at a sample temperature between 20–37°C. When using a different reagent than plasmin activator, for example, as described above, a similar amount can be used.

[0020] According to a third method step 503, the unmasking reaction is deliberately stopped again at a suitable time, preferably by adding a second reagent 20. For this purpose, a plasminogen activator inhibitor, in particular PAl1, can be added as the second reagent 20, which inhibits the conversion of plasminogen to plasmin. An advantageous concentration for this would be, for example, the concentration of 15-35 ng / mL usually present in blood plasma. The second reagent 20 can, as in Fig. 2, can also be stored upstream of the device 100, for example, in a second chamber 120, and, if necessary, can be conveyed to the sample for mixing with the sample by means of fluidic processes. Alternatively or additionally, the second reagent can comprise other plasminogen activator inhibitors and / or plasminogen inactivators, as described above, for example, in a similar concentration.

[0021] Alternatively or additionally, the sample can be centrifuged after incubation with the plasminogen activator, and preferably the portion of the centrifuged sample containing the CTCs can then be resuspended with a physiological buffer to remove the activator from the sample. This is particularly advantageous when the first three method steps are performed outside of the device 100.

[0022] Subsequently, in a preferred fourth method step 504, the blood sample can be treated with an erythrocyte lysis buffer, wherein the erythrocyte lysis buffer can also be stored upstream in the device 100. Optionally, the erythrocytes can be removed from the solution after lysis, in particular by centrifugation. The lysed erythrocytes remain in the supernatant, and the leukocytes and CTCs contained therein are pelleted at the bottom of the vessel. CTCs contained in the sample can then be stained and detected using specific fluorescence-coupled antibodies and other dyes (e.g., for live / dead detection), wherein the antibodies and / or other dyes can also be stored upstream in the device, in particular in the cartridge. Due to the unmasking, it is advantageously possible to specifically stain a larger number of CTCs than without this treatment.This is particularly advantageous in a point-of-care assay, as it shortens the time-to-result factor and eliminates the need to wait for the natural unmasking of the cells after 12-24 hours, but rather this can be triggered in a targeted and controlled manner.

[0023] The device 130 can further comprise a detection chamber 130 with a carrier substrate 131 for sedimentation of the unmasked cells and subsequent counting and / or analysis of the sedimented cells in a fifth method step 505. For this purpose, the carrier substrate 131 can preferably have microcavities for accommodating individual cells, for example as a silicon array structured with microcavities, as described, for example, in DE 10 2021 203 897 A1. Microcavities are understood to be recesses on a surface of the carrier substrate, in particular of the silicon substrate, wherein the recesses have widths and depths in the single- to double-digit micrometer range for accommodating at least one cell. These microcavities can be created in the silicon substrate using semiconductor technology or microsystem technology methods, for example, by deep plasma etching processes ("Bosch-DRIE").

[0024] The invention can also be advantageously used for other samples containing cells and plasminogen that can be demasked via plasmin. QUOTES CONTAINED IN THE DESCRIPTION

[0000] This list of documents submitted by the applicant was generated automatically and is included solely for the convenience of the reader. This list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions. Cited patent literature

[0000] DE 10 2021 203 897 A1 [0002, 0013, 0017, 0018, 0023] Cited non-patent literature

[0000] Jemal et al. “Global cancer statistics”. CA Cancer J Clin. 61, 2011, pp. 69-90

[0001] Pantel et al. “Detection and clinical importance of micrometastatic disease”. J Natl Cancer Inst. 91, 1999, 13, pp. 1113-24

[0001] Camara et al. „The relevance of circulating epithelial tumor cells (CETC) for therapy monitoring during neoadjuvant (primary systemic) chemotherapy in breast cancer.“ Ann Oncol. 18, 2007, Bd. 9, S. 1484-1492

[0002] Hekimian et al. „Demasking of epithelial cell adhesion molecule (EpCAM) on circulating epithelial tumor cells by Tween®20 treatment in breast cancer patients“ Clinical Chemistry and Laboratory Medicine, vol. 50, no. 4, 2012, pp. 701-708. https: / / doi.org / 1 0.1 515 / cclm.2011.812

[0003]

Claims

[1] Method (500) for unmasking biological cells, in particular circulating tumor cells, with plasmin, wherein a reagent (10) for converting plasminogen to plasmin is added to a sample containing plasminogen and biological cells, in particular a blood sample. [2] Method (500) according to claim 1, wherein the reagent (10) comprises kinase, in particular urokinase and / or streptokinase. [3] Method (500) according to one of the preceding claims, wherein the sample comprises blood, in particular whole blood, and wherein between 10,000 and 30,000 IU of reagent (10), in particular kinase, preferably urokinase and / or streptokinase, are added per milliliter of blood. [4] The method (500) of any preceding claim, wherein a second reagent (20) is added to the sample to inhibit the conversion of plasminogen to plasmin. [5] The method (500) of claim 4, wherein the second reagent (20) comprises a plasminogen inactivator. [6] The method (500) of claim 4 or 5, wherein the second reagent (20) comprises a plasminogen activator inhibitor. [7] Method (500) according to one of the preceding claims, wherein the reagent (10) is at least partially separated from the sample by centrifugation and preferably the cells are resuspended in a physiological buffer. [8] Device (100), in particular a microfluidic cartridge (100), for unmasking biological cells, in particular circulating tumor cells, with an input opening (101) for receiving a sample containing plasminogen and biological cells, in particular a blood sample, wherein the device (100) comprises a reagent (10) for converting plasminogen to plasmin. [9] Device (100) according to claim 8, wherein the device (100) further comprises a second reagent (20) for inhibiting the conversion of plasminogen to plasmin, in particular a plasminogen inactivator and / or a plasminogen activator inhibitor. [10] Device (100) according to claim 8 or 9, further comprising a carrier substrate (131), in particular with microcavities, for sedimentation of the unmasked cells.

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