Method for detecting drug sensitivity of tumor

Abstract

A method for detecting the drug sensitivity of a tumor. The method comprises: acquiring a first active tumor section with a thickness of about 20 μm to about 300 μm on the basis of an active tumor sample; incubating the first active tumor section in the presence of a drug for a first duration to obtain a drug-treated active tumor section, wherein the first duration is about 48 hours to about 96 hours; and detecting the drug-treated active tumor section by means of nonlinear optical imaging technology, so as to detect the drug sensitivity of the tumor to the drug.

Description

Method for detecting drug sensitivity of tumor

[0001] This application claims priority to Chinese Patent Application No. 202410591743.4, filed on May 14, 2024, the disclosure of which is incorporated herein in its entirety by reference as part of the present application. TECHNICAL FIELD

[0002] The present application relates to the field of biological medicine, in particular to a method for detecting drug sensitivity of tumor. BACKGROUND

[0003] Cancer is a malignant disease that seriously threatens human health. According to the data released by the China National Cancer Center, in 2023, the number of new cancer cases in China will exceed about 4.064 million, and the number of deaths will reach about 2.413.5 million. Cancer (malignant tumor) has become one of the main causes of death affecting the health of residents.

[0004] The treatment of cancer (malignant tumor) requires the cooperation of multiple treatment strategies and factors. For different tumor treatment modes, a comprehensive judgment should be made according to the disease course, tumor size, metastatic lesions, etc. It is well known that tumors have heterogeneity and instability. This heterogeneity can occur at different locations and different times in the same patient's body, and also can occur between different patients. This also means that for a certain treatment regimen, even for patients with the same cancer or different lesions in the same patient or different disease courses of the same patient, different therapeutic effects will be produced. For example, for many different patients with the same tumor, the same drug and standard dose are traditionally used for treatment; however, due to the heterogeneity of tumors and the instability of cells, there are often great differences in treatment effects, adverse reactions, etc. between different patients, and sometimes such differences are even fatal. Or, during the treatment of tumors, new mutations often occur in tumors, and the treatment drugs that are effective in the early stage of treatment can lose efficacy due to various reasons. Therefore, in the process of tumor treatment, different treatment regimens, i.e., precision medicine or individualized medical regimens, must be selected according to different people, different tumors, and different disease stages.

[0005] Precision medicine or individualized medicine refers to individualized diagnosis and treatment strategies matched with the molecular chemistry, cell biology, and pathophysiological characteristics of patients. At present, the use of gene sequencing technology to find genetic mutation targets in patients and the selection of targeted chemotherapy or targeted drugs for precise killing of tumor cells have become a new model and research hotspot for precision treatment of malignant tumors. According to the authoritative data of the Memorial Sloan Kettering Cancer Center (MSKCC) in 2017, about 63% of patients cannot find suitable targeted treatment programs through gene detection (Zehir et al., 2017). Precision medicine for tumors not only includes gene sequencing, but also includes proteomics, individual drug response characteristics, and other multi-faceted medical technologies. In order to avoid the blindness of tumor drug use and improve the efficiency of drugs, drug sensitivity detection of tumors has become an important part of precision treatment of tumors. With the continuous improvement of the requirements of precision medicine, the demand and requirements of clinical tumor drug sensitivity detection will also be improved.

[0006] There are currently a variety of tumor drug sensitivity detection methods. Among them, the experimental models for detecting the drug sensitivity of tumors are mainly divided into in vivo detection models and in vitro detection models. The in vivo detection model is mainly a human tumor xenograft (PDX) mouse model. The traditional cell line culture method often loses the heterogeneity of tumor cells and the in vivo characteristics of tumor cells during in vitro culture, while the PDX mouse model can to some extent retain the true genomic, transcriptomic, proteomic, and abnormal signal pathway characteristics of the tumor in the patient's body, retain the in vivo activity of tumor tissue cells and interpret the mechanism, and the pharmacokinetics is closer to the human body, and has high accuracy and high sensitivity. However, the disadvantages of PDX are high modeling difficulty, inability to repeatedly obtain, long model construction time, and unstable success rate (about 20% to about 80%, with a large deviation), with limitations on the number of passages; its operation is complex, the cycle is long (up to about 2-4 months), and the cost is high (up to about 1-3 million), which is not conducive to rapid clinical diagnosis and dealing with possible mutations of tumor tissues.

[0007] To this end, various in vitro detection models have been established to provide personalized treatment of tumors, such as tumor cell-based models, such as ex vivo cultured cell lines, primary cells, circulating tumor cells, or conditionally reprogrammed cells, or in vitro organoid models, such as patient-derived organoids (PDOs), and the like. However, the tumor cell samples such as ex vivo cultured cell lines, primary cells, circulating tumor cells, or conditionally reprogrammed cells cannot simulate the in vivo tumor microenvironment, and the tumor cell heterogeneity and its microenvironment differ among different patients, which is an important reason for the great difference in drug response. Therefore, the tumor drug sensitivity detection research using the above samples as objects and the consistency of the detection results with clinical drug prognosis are poor, and the accuracy is low. In addition, in vitro tumor organoids or tissue culture mainly detects tissue activity and growth, requires a long time for culture, resulting in poor timeliness, difficulty in restoring the in vivo tumor microenvironment, low success rate of culture, and high cost; for example, the construction of PDO requires a large amount of clinical sample tissue, and the success rate of 3D culture of organoid microspheres is unstable (about 40-80%) and requires a long time for culture (about 2-4 weeks) and high cost (about 500,000-1,000,000), and the single patient tumor microenvironment (TME) properties (such as up-regulation of growth and proliferation pathways, and down-regulation of immune angiogenesis pathways) are easily lost, resulting in poor accuracy and other problems.

[0008] Therefore, the existing tumor drug sensitivity detection model cannot predict the effect of targeted and immunotherapy due to the lack or distortion of intratumoral microenvironment, and there is a need to provide a low-cost detection method for rapid (e.g., about 1 week) and accurate personalized tumor drug screening. SUMMARY

[0009] To solve the above and other technical problems, the present application provides a tumor drug sensitivity detection method.

[0010] According to an embodiment of the present application, a tumor drug sensitivity detection method is provided, which comprises: based on an active tumor sample, obtaining a first active tumor slice with a thickness of about 20 μm to about 300 μm; incubating the first active tumor slice in the presence of a drug for a first duration to obtain a drug-treated active tumor slice, the first duration being about 24 hours to about 96 hours; and detecting the drug sensitivity of the tumor to the drug by a nonlinear optical imaging technique.

[0011] For example, the tumor drug sensitivity detection method further comprises resuscitating the first active tumor slice.

[0012] For example, the nonlinear optical imaging technique comprises one or more of coherent Raman microscopy, second harmonic imaging, and two-photon fluorescence imaging.

[0013] For example, the nonlinear optical imaging technique is coherent Raman microscopic imaging, culturing the first active tumor slice in the presence of the drug comprises: incubating the first active tumor slice in the presence of a drug culture solution for a second time length, and then incubating in the presence of a Raman probe-drug culture solution for a third time length, the drug culture solution comprising a drug and a tissue culture solution, the Raman probe-drug culture solution comprising the drug, a Raman probe and the tissue culture solution, the second time length being about 0 to about 72 hours, the third time length being about 24 hours to about 96 hours, and the sum of the second time length and the third time length being equal to the first time length; detecting the drug-treated active tumor slice by a nonlinear optical imaging technique comprises detecting metabolites of the Raman probe in the drug-treated active tumor slice.

[0014] For example, the second time length is about 18 hours to about 30 hours, and the third time length is about 40 hours to about 50 hours.

[0015] For example, detecting metabolites of the Raman probe in the drug-treated active tumor slice comprises: based on the active tumor sample, obtaining a second active tumor slice with the same thickness as the first active tumor slice, wherein if the first active tumor slice is resuscitated, the second active tumor slice is resuscitated under the same resuscitation conditions; incubating the second active tumor slice in the tissue culture solution for the second time length, and then incubating the second active tumor slice in a Raman probe culture solution for the third time length to obtain a control active tumor slice, wherein the Raman probe culture solution comprises the Raman probe and the tissue culture solution; detecting metabolites of the Raman probe in the drug-treated active tumor slice and the control active tumor slice, calculating the degree of metabolic inhibition of the drug on the tumor, and detecting the drug sensitivity of the tumor to the drug.

[0016] For example, the Raman probe is a metabolic marker with Raman spectral characteristics and low biological toxicity.

[0017] For example, the Raman probe comprises one or more of heavy water, deuterated palmitic acid, deuterated oleic acid, deuterated amino acid, deuterated glucose, deuterated cholesterol and 3-O-propargyl-D-glucose.

[0018] For example, the coherent Raman microscopic imaging technique is one or more selected from stimulated Raman scattering, coherent anti-Stokes microscopic imaging technique, surface-enhanced Raman spectroscopic imaging technique and Raman spectroscopic analysis technique.

[0019] For example, the nonlinear optical imaging technique is coherent Raman microscopy, and detecting the drug-treated active tumor section by the nonlinear optical imaging technique comprises analyzing intracellular and intercellular lipids and proteins in the drug-treated active tumor section to detect the sensitivity of the tumor to the drug, wherein the intracellular lipids and the intercellular lipids are the same or different and comprise one or more of phospholipids, triglycerides, cholesterol, cholesterol esters, and glycolipids.

[0020] For example, the nonlinear optical imaging technique is second harmonic generation imaging, and detecting the drug-treated active tumor section by the nonlinear optical imaging technique comprises analyzing tumor stem cells, collagen, and / or cell microtubules in the drug-treated active tumor section to detect the sensitivity of the tumor to the drug.

[0021] For example, the nonlinear optical imaging technique is two-photon fluorescence imaging based on a fluorescent probe, and detecting the drug-treated active tumor section by the nonlinear optical imaging technique comprises labeling the drug-treated active tumor section with a fluorescent probe, detecting cell types, subtypes, specific proteins, and their spatial distribution of the drug-treated active tumor section based on two-photon fluorescence, and detecting the sensitivity of the tumor to the drug based on the detected intratumoral microenvironment image containing cell composition and distribution and the single-cell metabolic detection map.

[0022] For example, the nonlinear optical imaging technique is two-photon fluorescence imaging based on autofluorescence, and detecting the drug-treated active tumor section by the nonlinear optical imaging technique comprises imaging and detecting the subcellular distribution of autofluorescent substances in the active tumor section based on the autofluorescence signal in the drug-treated active tumor section to detect the sensitivity of the tumor to the drug.

[0023] For example, based on the active tumor sample, obtaining a first active tumor section having a thickness of between about 20 μm and about 300 μm comprises obtaining the active tumor sample from fresh ex vivo tumor tissue, preserving the active tumor sample under conditions that maintain the activity of the tumor tissue, wrapping the preserved active tumor sample with a wrapping medium to form a wrapped active tumor sample, wherein the wrapping medium is selected from agarose, silicate gum, or a combination thereof, and cutting the wrapped active tumor sample into the first active tumor section having a thickness of between about 20 μm and about 300 μm.

[0024] For example, the thickness of the first active tumor section ranges from about 50 μm to about 200 μm.

[0025] For example, the thickness of the first active tumor section ranges from about 75 μm to about 125 μm.

[0026] For example, the active tumor sample is fresh ex vivo tumor tissue having an ex vivo time of less than or equal to about 30 minutes.

[0027] For example, the fresh ex vivo tumor tissue has an ex vivo time of less than or equal to about 15 minutes.

[0028] For example, the interval between the time the fresh ex vivo tumor sample is removed and the time the first active tumor section is taken is less than or equal to about 8 hours.

[0029] For example, the active tumor sample retains at least a portion of the intratumoral microenvironment of the tumor.

[0030] For example, the three-dimensional culture is performed at a temperature of about 35 °C to about 38 °C and normoxic conditions for about 6 hours to about 18 hours.

[0031] For example, the tumor is a solid tumor.

[0032] For example, the tumor comprises one or more of bladder cancer, lung cancer, colorectal cancer, gastric cancer, breast cancer, prostate cancer, liver cancer, ovarian cancer, thyroid cancer, pancreatic cancer, esophageal cancer, and cervical cancer. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the description of the embodiments. Obviously, the drawings in the following description are only some example embodiments of the present application, and other drawings can also be obtained according to these drawings without any creative labor for those skilled in the art.

[0034] FIG. 1 shows the results of stimulated Raman scattering (SRS) microscopic imaging of active tumor sections with different thicknesses, wherein the thickness of each section in the respective panel is: (A) 100 μm, (B) 50 μm, (C) 200 μm, (D) 300 μm;

[0035] FIG. 2 shows the results of SRS microscopic imaging using different concentrations of heavy water as a Raman probe, wherein the concentration of heavy water is: 0 (control), 30 wt%, 40 wt%, and 50 wt%;

[0036] FIGS. 3A-B show the changes in the sample over time after incubation with heavy water: (A) immunofluorescence images using DAPI (4', 6-diamidino-2-phenylindole), PI (propidium iodide), Calcein, and Merged; (B) percentage of cell survival from 0 to 96 hours;

[0037] Figures 4A-B show SRS microscopic imaging results of drug-loaded probe samples and control samples (Control), respectively;

[0038] Figure 5 shows changes in heavy water (D2O) metabolism with drug activity and drug sensitivity or resistance determination therefrom;

[0039] Figure 6 shows SRS microscopic imaging results from different cases and for different drugs, wherein: (A) is an imaging image of SRS metabolic detection of a clinical sample from a PT1 bladder cancer case with gemcitabine + cisplatin; (B) is an imaging image of SRS metabolic detection of a clinical sample from another PT1 bladder cancer case with gemcitabine + cisplatin; (C) is an imaging image of SRS metabolic detection of a tumor sample with gemcitabine + cisplatin (GC); (D) is an imaging image of SRS metabolic detection of a tumor sample with gemcitabine + cisplatin (GC) and methotrexate + vinblastine + doxorubicin + cisplatin (MVAC);

[0040] Figure 7 shows multiplex detection results, wherein SRS microscopic imaging results and collagen images in tumor tissue samples obtained by second harmonic imaging are shown; and

[0041] Figure 8 shows images of a liver cell tumor active tissue section obtained by a two-photon fluorescence imaging system based on a fluorescent probe. DETAILED DESCRIPTION

[0042] For the purposes of the detailed description below, it is to be understood that various alternatives to the embodiments described can be employed in adapting the application to different uses and conditions. Also, it is to be understood that the terminology used herein is for the purpose of describing the particular embodiments only and is not intended to be limiting. In addition, it should be noted that, as used in any operation herein, the expression "coupled to" also covers the case where an element is not directly coupled to an input element through another element, but is still indirectly coupled to the input element. Also, as used herein, the terms "first," "second" and "third" can be read as taking on different meanings depending on the context. For example, the term "first" can be read to mean "second" or "third," and the term "second" can be read to mean "first" or "third." The foregoing description discloses only exemplary embodiments of the disclosure. Modifications of the above exemplary embodiments will be obvious to those with skill in the art in view of the foregoing description. Therefore, the above-described embodiments are not intended to limit the scope of the application. Rather, the scope of the application is limited only by the claims.

[0043] Notwithstanding that the numerical ranges and parameters setting forth the broadest scope of the application are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0044] Further, it should be understood that any numerical range recited herein is intended to include all sub-ranges of the same whole number recited as the minimum and the maximum. For example, a range of "1 to 10" is intended to include all sub-ranges between (and including) the recited minimum of 1 and the recited maximum of 10, that is, having a minimum of 1 and a maximum of 10.

[0045] In this application, the use of the singular includes the plural and vice- versa unless explicitly stated otherwise. In addition, in this application, the use of "or" means "and / or" unless explicitly stated otherwise. Furthermore, in this application, the use of "a" or "an" means "at least one" unless explicitly stated otherwise. For example, "a compound," "an composition," and the like mean one or more of these items.

[0046] As used herein, "comprising," "including," "containing," and the like, are to be construed as meaning "including, but not limited to," in the context of this application, and therefore should not be interpreted as being limiting.

[0047] As used herein, "consisting of" is to be construed, in the context of this application, as a closed list of elements, components, or method steps, and therefore should not be interpreted as including additional unspecified elements, components, or method steps.

[0048] In this application, the use of the singular includes the plural and vice- versa unless specifically stated otherwise. For example, although reference is made herein to "a" compound or "a" composition, combinations (i.e., multiples) of these components can be used.

[0049] In addition, in this application, the use of "or" means "and / or" unless specifically stated otherwise, although "and / or" can be explicitly used in certain instances.

[0050] In the prior art, drug sensitivity detection is usually performed using tumor cells dissociated from tumor tissues and cultured for a long time in in vivo or in vitro models. However, such tumor cell samples cultured in in vivo or in vitro models are basically detached from the original in vivo tumor microenvironment (TME), and cannot reflect the subclonal heterogeneity of the tumor and the cell interaction within the tumor microenvironment; at the same time, due to the instability of tumor cells, genotype drift often occurs after long-term culture, making it difficult to compare the detection results with clinical treatment data; in addition, specific types of tumor cells such as prostate tumor cells cannot survive in a 2D culture environment and cannot be subjected to drug sensitivity detection by the above method.

[0051] On the other hand, culturing tumors in in vivo or in vitro models is time-consuming and costly, and the success rate of culture is not stable.

[0052] Finally, the prior art does not provide a fast and accurate low-cost drug sensitivity detection method based on tumor biopsy samples.

[0053] To solve the above technical problems, the present application provides a drug sensitivity detection method based on tumor biopsy samples. The drug sensitivity detection method comprises: based on the active tumor sample, obtaining a first active tumor slice; incubating the first active tumor slice in the presence of a drug for a period of time to obtain a drug-treated active tumor slice; and detecting the drug-treated active tumor slice by nonlinear optical imaging technology to detect the drug sensitivity of the tumor to the drug. Therefore, based on the culture, slicing, drug treatment and nonlinear optical imaging of the tumor biopsy sample, the drug detection method of the present application can realize fast, accurate and low-cost drug sensitivity detection of the tumor.

[0054] As used herein, the term "drug sensitivity" also known as "drug sensitivity" refers to the sensitivity of a drug acting on a target (such as a pathogen, a cell, a tissue, an organism, etc.), i.e. the effect of the drug on the target through individual differences in the target's response to drug metabolism. Different targets have different sensitivities to different drugs. In general, if a small dose of drug can have a significant effect on the target, it means that the target is "sensitive" to the drug; on the contrary, if a large amount of drug cannot have a significant effect on the target, it means that the target is "insensitive" or even "resistant" to the drug. In clinical practice, drug sensitivity determines the therapeutic effect of the drug on the target. Drug sensitivity testing to reasonably select the drug used according to the drug sensitivity has important guiding significance for precision medicine / individualized medicine.

[0055] As used herein, the term "tumor" refers to a class of diseases formed by abnormal proliferation of cells in the body, also known as neoplasm or neoplasm. Tumors can have various classification methods, for example, according to the cell characteristics of the neoplasm and the degree of harm to the body, they can be divided into benign and malignant tumors; or according to the different primary sites, they can be divided into solid tumors and blood tumors, etc. In some examples, the method according to the present application can be used to detect the drug sensitivity of solid tumors; in other examples, the method according to the present application can be used to detect the drug sensitivity of malignant tumors; in yet other examples, the method according to the present application can be used to detect the drug sensitivity of malignant solid tumors. For example, the examples of tumors detected in the present application can include, but are not limited to, one or more of bladder cancer, lung cancer, colorectal cancer, gastric cancer, breast cancer, prostate cancer, liver cancer, ovarian cancer, thyroid cancer, pancreatic cancer, esophageal cancer and cervical cancer.

[0056] Generally, the drug sensitivity test of tumor can be performed for different targets. For example, the test can be performed for tumor cell samples such as cell lines cultured in vitro, primary cells or conditionally reprogrammed cells, however, such cell samples cultured in vitro cannot simulate the in vivo tumor microenvironment, and cannot accurately reflect the heterogeneity of tumor cells and their microenvironment, so the tumor drug sensitivity test research using the above samples as objects and the consistency of the application of the test results with clinical drug prognosis is poor, and the accuracy is low. In addition, animal in vivo animal models such as PDX models can also be used for detection, such models can retain the tumor microenvironment and subclone to some extent, but the culture and detection cycle is long (2-4 months) and the culture success rate is low (20-80% deviation is huge), which makes it difficult to meet the timeliness requirements of clinical detection and the cost (about 10-30 million) is difficult to bear; in addition, human in vivo tumor drug sensitivity test research often needs complex in vivo intervention schemes and equipment, which is difficult to implement and extremely high in cost, and at the same time, the risk to the patient is huge and it is difficult to have ethical feasibility. In vitro tumor organoids or tissue culture can also be used, however, because the detection method mainly detects tissue activity and growth, it needs a long time for culture, resulting in poor timeliness, difficulty in restoring the in vivo tumor microenvironment, and low success rate and high cost of culture. In summary, the existing tumor drug sensitivity test model is difficult to accurately and effectively predict the effect of targeted and immunotherapy due to the lack or distortion of the microenvironment.

[0057] Accordingly, the inventors have designed a method for tumor drug sensitivity detection based on an in vitro culture model of active tumor sections from a tumor biopsy. As used herein, the term "tumor biopsy" can also be referred to as an active tumor sample, and refers to a sample that retains the tissue activity of the tumor and at least a portion of the tumor microenvironment. As used herein, the term "tumor microenvironment" refers to the components and environment within the tumor tissue. Generally, in addition to cancer cells, tumor tissue also includes stromal cells, immune cells, collagenous tissue, etc., and the composition, distribution, and interaction of these components with cancer cells can contribute to tumor resistance to certain drugs. Therefore, detection of tumor tissue that retains as much of the tumor microenvironment as possible can better represent the in vivo tumor characteristics, and the detection results can more accurately reflect the characteristics of the tumor. Generally, the active tumor sample can be fresh ex vivo tumor tissue or obtained from fresh ex vivo tumor tissue. For example, the fresh ex vivo tumor tissue can be tumor tissue that is removed from a patient by surgery or biopsy sampling, and retains the tissue activity of the tumor and at least a portion (preferably most, and more preferably all) of the tumor microenvironment. As used herein, "fresh" refers to ex vivo time of the tumor tissue after surgical removal or biopsy sampling of less than or equal to about 1 hour, for example, ex vivo time of less than or equal to about 45 minutes, less than or equal to about 30 minutes, less than or equal to about 15 minutes, less than or equal to about 10 minutes, less than or equal to about 5 minutes, etc., less than or equal to about 3 minutes, less than or equal to about 1 minute; or, can refer to sample extraction immediately after surgical removal or biopsy sampling of the tumor tissue, i.e., ex vivo time of about 0. It is well known that during sample extraction, the extraction site should be avoided at the cutting (e.g., electrical cutting) edge or affected by burning, to ensure that the sample is fresh and active; as much as possible, the sample should be extracted to ensure that the tissue is representative and active; and, during extraction, the tissue should not be poked with a sharp object, and the tissue should not be squeezed too hard. Using fresh ex vivo tumor tissue as an active tumor sample can prevent significant reduction or loss of tissue activity of the tumor tissue due to prolonged ex vivo time, possible variations due to changes in the external environment, etc. The shorter the ex vivo time, the less difference between the ex vivo tumor tissue and the in vivo tumor tissue, the more active and tumor microenvironment characteristics retained by the active tumor sample, thereby making the accuracy of the drug sensitivity detection results obtained based on the sample higher.

[0058] After obtaining a fresh ex vivo tumor tissue as an active tumor sample or obtaining an active tumor sample from a fresh ex vivo tumor tissue, an active tumor section can be obtained directly from the active tumor sample, for example, when the operating room and the detection laboratory are located close enough or in the same place; or the active tumor sample can be stored for a period of time under conditions that maintain the activity of the tumor tissue before sectioning, for example, when the operating room and the detection laboratory are located in different places or far apart. The conditions for maintaining the activity of the ex vivo tumor tissue for a period of time are known or can be easily determined by those skilled in the art, for example, the active tumor sample can be placed in a suitable storage medium and stored at a temperature of about 0-4°C for a period of time of no more than about 12 hours, for example, for a period of time of no more than about 10 hours, no more than about 8 hours, no more than about 6 hours, no more than about 5 hours, no more than about 4 hours, no more than about 3 hours, no more than about 2 hours, no more than about 1 hour, etc. Suitable storage media for use in the methods of the present application can include, but are not limited to, DMEM / F12 medium containing 1 wt% penicillin-streptomycin double-antibiotic, 90 wt% Hanks balanced salt solution (containing 10 wt% fetal bovine serum), HypoThermosol FRS (HTS-FRS), and CellSafe tissue preservation solution, etc., as long as these storage media can maintain a high tissue cell survival rate of the active tumor sample for a period of time, maintain the basic physiological metabolism of the cells, and have high permeability and tissue penetration ability. Generally, in order to maintain the activity of the ex vivo tumor tissue sample and its consistency with the intratumoral microenvironment of the in vivo tumor tissue, the active tumor sample generally cannot be frozen, otherwise it will lead to loss of tissue viability or difficulty in recovery, etc., because the tumor tissue is different from general tissue samples, and freezing will seriously affect the metabolic characteristics of the tumor tissue sample. Therefore, in some aspects, the active tumor sample used in the methods of the present application does not undergo a freezing process.

[0059] As used herein, the term "slice", also referred to as "tissue slice", refers to a thin slice of biological tissue that is cut from a biological tissue sample using a specialized tool and applied to a glass slide for microscopic observation, which is widely used in the fields of biology, medicine (pathology, infectious diseases, etc.). Generally, a tissue slice suitable for observation under a microscope (e.g., optical microscope, electron microscope, etc.) is significantly different in structure from a common tissue block. For example, the thickness of a tissue slice is often much smaller than its length and width, and can even differ by several orders of magnitude. The advantage of such a tissue slice is that the significantly thinner thickness of the tissue slice allows nutrients to fully penetrate the inside of the tissue during the culture process, the culture success rate of the tissue is high, and no additional nutritional factors need to be added during the culture process, thus reducing the cost of culture; moreover, drugs can fully penetrate the tissue sample during the culture process, thus effectively acting on the entire tissue sample, and the effect of the drugs is good, so that detection can be performed after a short culture period, and the entire culture and detection period is short; the small thickness and large cross-section of the slice can better expose the internal microenvironment of the tissue, and can be prepared as a glass slide sample for detection by nonlinear optical effect microscopic imaging technology, so that the detection results based on the slice can better and more quickly reflect the characteristics of the biological tissue. In contrast, a tissue block directly obtained from a tissue (e.g., tumor tissue) often has a size that differs little (e.g., within the same order of magnitude) in the three dimensions of length, width, and thickness, and is much larger than the thickness of a tissue slice. Such a tissue block, due to its large thickness, makes it difficult for nutrients and drugs to fully penetrate during the culture process, which can result in many deficiencies, such as a low culture success rate, the need to add additional nutritional factors to increase the cost, and a poor drug effect that makes it difficult to detect after a short culture period, thus prolonging the culture and detection period and failing to timely reflect the characteristics of the tissue; in addition, the tissue block is not suitable for detection by nonlinear optical effect microscopic imaging technology.

[0060] In some examples, the thickness of the active tumor section according to the present application can range from about 20 pm to about 300 pm, for example, from about 50 pm to about 200 pm, or from about 75 pm to about 125 pm. Alternatively, the thickness of the active tumor section can be about 100 pm. The inventors have found that when the tissue section is too thin, for example, less than about 20 pm, the section can only retain a single layer of tumor tissue cells in the thickness dimension and cannot accommodate the thickness of multiple layers of cells, thus its retention of the tumor microenvironment is limited and it is difficult to fully reflect the physiological characteristics of the pathogenic tumor tissue metabolism, etc. and the effect of the pathogenic tissue section in vitro culture and imaging by nonlinear optical imaging technology is also poor (too strong background signal). In addition, the inventors have also found that if the thickness of the tissue section is too high, for example, higher than about 300 pm, it can be difficult for nutrients and drugs to be detected to effectively penetrate during the culture process, resulting in low culture success rate, increased cost due to the need to add other nutritional factors, and it can be difficult to detect after short-term culture due to poor drug effect, resulting in prolonged culture and detection period. Moreover, a tissue section with a thickness that is too high (e.g., higher than about 300 pm) can also result in the section being difficult to prepare into a slide sample for detection by a nonlinear optical device or the prepared slide sample not having a good imaging effect when used for nonlinear optical imaging, for example, the transmission signal reception of the nonlinear optical imaging technology is affected due to the sample being too thick. In some aspects, the inventors have found that a section having a thickness ranging from about 20 pm to about 300 pm as described in the present application can allow nutrients and drugs to fully penetrate, and can be detected based on nonlinear optical microscopic imaging technology in multiple dimensions, for example, but not limited to, metabolic activity, cell composition, etc. after short-term (e.g., about 3 to 5 days) culture at a low cost (without the need to add additional nutritional factors); in addition, the section within the above thickness range effectively retains at least some (e.g., most, or even all) of the intratumoral microenvironment of the tumor tissue, and when performing drug sensitivity detection, it can have high consistency with the in vivo tumor tissue, thus better reflecting the characteristics of the in vivo tumor tissue and obtaining more accurate detection results.

[0061] In the method according to the present application, any suitable method can be used to obtain the active tumor section. Examples of methods suitable for obtaining the active tumor section in the method of the present application can include: wrapping the active tumor sample or the preserved active tumor sample with a wrapping medium to form a wrapped active tumor sample; and cutting the wrapped active tumor sample into the first active tumor section having a thickness of between about 20 pm and about 300 pm to a suitable thickness. Examples of wrapping medium suitable for use in the method of the present application can be selected from, but not limited to, agarose, silicate glue, or a combination thereof. The sectioning process can be performed in any suitable medium, for example, but not limited to, in PBS.

[0062] In the methods of the present application, the active tumor section can also be optionally resuscitated according to the tissue activity or the storage time of the active tumor sample. As used herein, the term "resuscitation" refers to the process of improving the activity of an ex vivo tissue and adapting it to the in vitro culture environment by culturing after the activity and the like has been reduced. In the methods of the present application, resuscitation can be performed by culturing the active tumor section in a suitable culture environment for a period of time, so that the tissue activity of the section is improved for subsequent drug addition culture and detection. For example, the active tumor section can be resuscitated by culturing in a three-dimensional culture at a temperature of about 35°C to about 38°C and under normoxic conditions. As used herein, the term "three-dimensional culture" refers to a tissue culture technique in which the tissue is cultured in three-dimensional space to simulate the natural state in the human or animal body. For example, the active tumor section can be cultured in a three-dimensional culture medium with a semi-permeable membrane or gelatinous quality, including but not limited to culturing in a Transwell in the presence of tissue culture fluid. Resuscitation can be performed for a suitable period of time, for example, for about 6 hours to about 18 hours, for example, about 8-12 hours, and the like. The inventors have found that resuscitation of an ex vivo sample (for example, an ex vivo sample after being stored for a period of time) can be beneficial to adapt the tumor tissue to the in vitro culture environment with a good activity state after being ex vivo from surgery or biopsy and transported; otherwise, directly adding drugs to the tissue sample without adapting to the in vitro culture conditions can potentially interfere with the evaluation of the metabolic activity of the tissue due to the great change in the culture state.

[0063] After obtaining the active tumor section and optionally resuscitating it, the active tumor section can be incubated in the presence of a drug for a period of time to obtain a drug-treated active tumor section, for example, for about 48 hours to about 96 hours, and then the drug-treated active tumor section can be detected by nonlinear optical imaging technology to detect the drug sensitivity of the tumor to the drug. As used herein, the term "drug" refers to a substance that can have an effect on tumor tissue or cells, for example, a chemotherapy drug, a targeted drug, an immunological drug, and the like for tumors; and the "drug culture fluid" is a culture medium obtained by adding the drug to be detected to the tissue culture fluid. The tissue culture fluid suitable for use in the present application can be any suitable culture fluid, for example, but not limited to, 90wt% DMEM / F12 (1wt% penicillin-streptomycin double antibody) + 10wt% fetal bovine serum, and the like. The drug culture fluid suitable for use in the present application can have any suitable concentration, for example, at the maximum blood drug concentration (Cmax) of the drug to be tested, or at a concentration that is 1 / 10, 1 / 100, 1 / 1000, 1 / 10000, or the like of the Cmax of the drug to be tested. max) and a 10-fold gradient of drug concentration on both sides, etc. As used herein, the term "nonlinear optical imaging technique" refers to an imaging technique that utilizes nonlinear optical effects to cause a substance to absorb, radiate coherent light at low light intensity, and acquire information in three-dimensional space; "nonlinear optical effect" refers to the interaction of light and sample that produces nonlinear effects when the observed light wave field intensity can be comparable to the Coulomb field inside the atom or the interatomic vibration energy level in the sample, reflecting the physical quantity of the substance in the sample, and the effect is not only related to the first power of the field strength E of the light wave electromagnetic field, but also depends on the higher power term of E, resulting in many new phenomena that are not obvious in linear optics. Common nonlinear optical imaging techniques are well known in the art, such as but not limited to, coherent Raman microscopic imaging, second harmonic imaging, and two-photon fluorescence imaging, etc.

[0064] The nonlinear optical imaging technique used in the method according to the present application can be a coherent Raman microscopic imaging technique. The coherent Raman microscopic imaging technique suitable for use in the method of the present application can include but is not limited to one or more of stimulated Raman scattering (SRS), coherent anti-Stokes microscopic imaging technique (CARS), surface-enhanced Raman spectroscopic imaging technique, and Raman spectroscopic analysis technique. Among them, coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopic imaging have a signal 10 6Left and right gain, imaging speed significantly improved, while having higher spatial and temporal resolution and higher sensitivity. Unlike CARS, SRS microscopy does not have non-resonant background, has been applied to metabolic imaging of cells, tissues and model organisms. Hyperspectral stimulated Raman scattering (SRS) can record spectrum at each pixel, to distinguish SRS signal from the background. So hyperspectral stimulated Raman scattering (SRS) system can quantitatively metabolic inhibition difference observation on simple section processing complete retention of tumor microenvironment short time heavy water culture tumor tissue, realize the tumor drug sensitivity detection of accurate, fast and low cost based on the tumor drug sensitivity detection of accurate, fast and low cost. Metabolism is an important feature of life, and is crucial for elucidating and understanding the mechanistic basis of many biological processes. Organisms renew biological molecules such as proteins and lipids through metabolism, but this turnover process is difficult to measure in label-free SRS technology. To solve this problem, small molecule Raman labeling is used to distinguish newly synthesized biological molecules from original molecules, and metabolic tracking is completed. SRS microscopic imaging based on Raman probe (such as heavy water) metabolism is also applied to drug sensitivity detection. In the reduction process of NAD / NADP in cells, hydrogen in water participates in metabolism and is converted into biological matter, especially biological macromolecules such as lipids (L) and proteins (P). The C-H bond in these macromolecules has a characteristic peak (2800-3100 cm -1 ) in Raman spectrum. When some ordinary water (H2O) in the culture medium is replaced by heavy water (D2O), deuterium (D) in heavy water is used to synthesize important biological macromolecules with cell metabolism, so that C-H peak in Raman spectrum is shifted and C-D peak (2000-2300 cm -1 ) appears. After incubating cells with antitumor drugs and heavy water for a period of time, if the cells are sensitive to the drugs or the drugs are effective, the cell metabolism will be inhibited. At this time, the metabolic activity of the cells can be reflected by the presence or absence and intensity of the C-D peak, so as to know whether the drug is effective for the cells. Compared with fluorescent labeling, Raman probes have high stability (no photobleaching), near-infrared excitation to avoid phototoxicity, small label to slightly interfere with the target (chemical bond as the source), and can label small molecules such as sugars and lipids that are difficult to label with fluorescent probes, narrow spectral peak for super-multicolor multiplexing (fluorescent spectral overlap), and other advantages.

[0065] In some aspects, detecting the drug-treated active tumor section by coherent Raman microscopy can be performed as follows. The active tumor section is incubated in the presence of the drug culture for a period of time, and then incubated in the presence of the Raman probe-drug culture for another period of time. As used herein, the term "Raman probe" refers to a probe used when detected by coherent Raman microscopy, such as a metabolic marker with Raman spectral signature and low biological toxicity. Examples of Raman probes suitable for use in the methods of the present application include, but are not limited to, one or more of heavy water, deuterated palmitic acid, deuterated oleic acid, deuterated amino acid, deuterated glucose, deuterated cholesterol, and 3-0-propargyl-D-glucose. As used herein, the term "drug culture" refers to a culture medium obtained by adding a drug to be detected and a Raman probe to a tissue culture medium. A Raman probe-drug culture suitable for use in the methods of the present application can comprise, for example, about 40 wt% or less of the total weight of the culture, such as about 35 wt% or less, or about 30 wt% or less, etc. In some aspects, the active tumor section can be incubated in the presence of the drug culture for about 0 to about 72 hours, and then incubated in the presence of the Raman probe-drug culture for about 24 hours to about 96 hours, and the sum of the two incubation times is about 48 hours to about 96 hours. Among them, when the active tumor section is incubated in the presence of the drug culture for 0 hours, it means that the active tumor section does not need to be incubated in the presence of the drug culture, but is directly incubated in the Raman probe-drug culture. In other aspects, the active tumor section can be incubated in the presence of the drug culture for about 18 hours to about 30 hours, and then incubated in the presence of the Raman probe-drug culture for another about 40 hours to about 50 hours. Then, the metabolic products of the Raman probe in the drug-treated active tumor section are detected by coherent Raman microscopy. In the methods of the present application, the drug incubation is performed for a period of time, and then the corresponding sample group is subjected to drug incubation containing the Raman probe, in order to allow the drug to be detected and the sample to act first. If the drug is effective, the sample metabolism is inhibited before the heavy water is added. Compared with the culture method of adding the drug and the heavy water at the same time, the contrast of the degree of inhibition of the sample metabolism by the drug detected by SRS can be significantly improved. In some examples, the probe ratio used when the sample is incubated in the heavy water culture can be optionally within a certain range, such as not more than about 40 wt%, not more than about 35 wt%, not more than about 30 wt%, etc. Because too high a probe concentration can cause certain cell toxicity, affect the activity of the sample and the incubation time, and reduce the success rate of the culture.

[0066] In some aspects, the detection of the metabolic products of the Raman probe in the drug-treated active tumor slice can be performed as follows. In addition to the drug-incubated active tumor slice described above, another active tumor slice having the same thickness as the slice is obtained from the active tumor sample. If the drug-incubated active tumor slice described above is subjected to resuscitation, the other active tumor slice is also resuscitated under the same resuscitation conditions. The other active tumor slice is incubated under the same active conditions as the drug-incubated active tumor slice described above for the same time, except that the drug culture solution in the first incubation is replaced with tissue culture solution, and the Raman probe-drug culture solution in the second incubation is replaced with Raman probe culture solution, thereby obtaining a control active tumor slice. As used herein, the term "Raman probe culture solution" refers to a culture medium obtained by adding the Raman probe to the tissue culture solution. The detection of the metabolic products of the Raman probe in the drug-incubated active tumor slice and the control active tumor slice is performed, and the degree of metabolic inhibition of the tumor by the drug is calculated to detect the drug sensitivity of the tumor to the drug.

[0067] In other aspects, in addition to the metabolic activity detection based on the Raman metabolic markers (Raman probes), the tumor tissues and cells can also be subjected to component analysis and imaging, i.e., imaging and analysis of biological components such as proteins, nucleic acids, and lipids at different wave numbers; the analysis indicators include but are not limited to: lipid content, lipid concentration, lipid spatial distribution, protein concentration, protein content, protein spatial distribution, deoxyribonucleic acid concentration, lipid / protein content ratio, lipid / protein concentration ratio, lipid / deoxyribonucleic acid concentration ratio, lipid droplet number, lipid droplet area, lipid droplet area ratio of total cell area, lipid droplet range lipid / protein concentration ratio, lipid component / protein component area ratio, lipid component / deoxyribonucleic acid component area ratio, lipid component ratio of total cell area, protein component ratio of total cell area, and lipid component range lipid / protein concentration ratio. These data can provide more dimensional tumor tissue information for the metabolic activity detection of the tumor tissue, helping to more comprehensively and accurately judge the possible drug response of the tumor tissue. For example, in some aspects, the detection of the drug-treated active tumor slice by the coherent Raman microscopic imaging technology can also be performed by analyzing the intracellular lipids and intercellular lipids and proteins in the drug-treated active tumor slice to detect the sensitivity of the tumor to the drug. For example, the intracellular lipids and the intercellular lipids can be the same or different, and can include but are not limited to one or more of phospholipids, triglycerides, cholesterol, cholesterol esters, and glycolipids.

[0068] Alternatively, the nonlinear optical imaging technique used in the methods of the present application can also be a second harmonic imaging technique. As used herein, the term "second harmonic imaging technique" is a modern nonlinear optical microscopy that utilizes the second harmonic signal generated upon interaction of light with matter for microscopic imaging or probing. Second harmonic generation imaging is a new optical imaging technique developed in recent years, which has the high spatial resolution and high imaging depth characteristic of nonlinear optical imaging, and has received extensive attention as a new tool for biological structure detection and durable tracking markers. Second harmonic generation imaging technique avoids many inherent shortcomings encountered by classical fluorescent probes, and can avoid the fluorescence bleaching effect in two-photon fluorescence imaging, and is a non-invasive bio-imaging method. Changes in microstructure often occur in pathological changes of biological tissues, so imaging can provide a basis for the diagnosis of diseases such as tumors. Through second harmonic generation imaging, the collagen and other components and structures in the pathogenic tumor tissue of the tumor microenvironment can be imaged by simple sectioning, and the content and structure of these components can also be used as parameters for tumor drug sensitivity detection, and combined with metabolic inhibition detection to provide more accurate and reliable detection results. In some examples, detecting the drug-treated active tumor section using the second harmonic generation imaging technique can include analyzing tumor stem cells, collagen, and / or cell microtubules in the drug-treated active tumor section to detect the sensitivity of the tumor to the drug.

[0069] Alternatively, the nonlinear optical imaging technique used in the methods of the present application can also be a two-photon fluorescence imaging technique based on a fluorescent probe or a two-photon fluorescence imaging technique based on spontaneous fluorescence. As used herein, the term "two-photon fluorescence imaging technique" refers to a fluorescence imaging technique by two-photon excitation; the term "fluorescent probe" refers to a class of fluorescent molecules that have characteristic fluorescence in the ultraviolet-visible-near infrared region, and whose fluorescence properties (excitation and emission wavelength, intensity, lifetime, polarization, etc.) can be sensitively changed with the properties of the environment in which they are located, such as polarity, refractive index, viscosity, etc.; the term "spontaneous fluorescence" refers to the light naturally emitted after absorbing light of a certain range of wavelengths from certain compounds present in the organization or cells.

[0070] The two-photon fluorescence imaging technology based on fluorescent probes used in the method of the present application can also be referred to as two-photon fluorescence multiplexed immunofluorescence imaging technology. Immunofluorescence imaging technology is to label a fluorescent pigment on an antibody (or antigen) without affecting the activity of the antigen-antibody, and after binding with its corresponding antigen (or antibody), a specific fluorescence reaction is presented under a fluorescence microscope. The antigen in the biological tissue and cells is imaged through the fluorescence emission effect of the fluorescent pigment. If different types of cells are labeled by different types of fluorescent probes, different types of cells can be distinguished (as shown in the following figure). If different types of target protein molecules are labeled by different types of fluorescent probes, the content and distribution of the target protein molecules in the cells can be imaged. Compared with traditional single-photon fluorescence imaging, in two-photon fluorescence imaging, the fluorescent probe group is excited by two excitation photons before the fluorescence effect is exhibited, which has the advantages of higher imaging resolution, smaller phototoxicity, and deeper imaging depth. In some examples, detecting the active tumor section treated by the drug using the two-photon fluorescence imaging technology based on fluorescent probes can include: labeling the active tumor section treated by the drug with fluorescent probes, detecting the cell types, subtypes, specific proteins, and spatial distribution of the active tumor section treated by the drug based on two-photon fluorescence, and detecting the sensitivity of the tumor to the drug based on the detected intratumoral microenvironment image containing cell composition and distribution and the single-cell metabolic detection map.

[0071] In addition, the method of the present application can also use two-photon fluorescence imaging technology based on autofluorescence. Autofluorescence is the light naturally emitted by biological structures (such as mitochondria and lysosomes) when they absorb light, and is used to distinguish light originating from artificially added fluorescent markers (fluorophores). Autofluorescence imaging is different from fluorescence imaging based on fluorescent probes, and directly images the autofluorescence of the organism itself and the drug. Through autofluorescence imaging, the content and distribution of components and molecules with autofluorescence properties in biological tissues and cells, and even the intensity of biological reactions can be detected. Therefore, by detecting the subcellular distribution of autofluorescence substances in the tumor tissue itself or from the drug in real time, elucidating the local biological molecular changes caused by the drug, and distinguishing the reactions in different subcellular regions, more dimensional data analysis can be provided for tumor drug sensitivity detection. In some examples, detecting the active tumor section treated by the drug using the two-photon fluorescence imaging technology based on autofluorescence can include: imaging and detecting the subcellular distribution of autofluorescence substances in the active tumor section treated by the drug based on the autofluorescence signal in the active tumor section treated by the drug, to detect the sensitivity of the tumor to the drug.

[0072] The present application will be further explained in detail below with reference to the examples. However, those skilled in the art should understand that these examples are provided only for illustrative purposes, and are not intended to limit the present application.

[0073] Example

[0074] The embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are for illustrative purposes only and should not be construed as limiting the scope of the present application. Unless otherwise specified, the specific conditions in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be obtained commercially. Unless otherwise specified, all amounts listed are described in terms of weight parts or weight percentages based on the total weight. The present application should not be construed as being limited to the specific examples described. Although specific examples of the present application have been described above for the purpose of illustration, it will be apparent to those skilled in the art that various changes in the details can be made to the present application without departing from the scope defined in the appended claims.

[0075] Reagents:

[0076] Transport medium: 1 wt% penicillin-streptomycin solution (Enzyme-Linked Biological) was added to sterile DMEM / F21 medium (Merck) to prepare DMEM / F21 medium containing 1 wt% penicillin-streptomycin, which was stored as a transport medium under sterile and refrigerated conditions.

[0077] Tissue culture medium: Fetal bovine serum (GIBCO) was added to DMEM / F21 medium containing 1 wt% penicillin-streptomycin to prepare a medium containing 90 wt% DMEM / F12 (containing 1 wt% penicillin-streptomycin) + 10 wt% fetal bovine serum, which was stored as a tissue culture medium under sterile and refrigerated conditions.

[0078] Wrapping medium: Agarose (Sigma Aldrich) was dissolved in distilled water to prepare a 3 wt% agarose solution, which was sterilized and refrigerated for use as a wrapping medium.

[0079] Drug culture medium: The drug or drug combination to be tested was dissolved in the tissue culture medium to prepare a drug culture medium having a drug concentration of C max (maximum blood concentration) and a plurality of drug concentrations having a gradient of 10 times on both sides thereof.

[0080] Raman probe culture medium: The tissue culture medium was mixed with heavy water to obtain a mixture containing tissue culture medium heavy water as a Raman probe culture medium (hereinafter referred to as heavy water culture medium).

[0081] Raman probe-drug culture medium: The drug or drug combination to be tested was dissolved in the Raman probe culture medium to prepare a Raman probe-drug culture medium (hereinafter referred to as heavy water-drug culture medium) having the same drug concentration as the corresponding drug culture medium.

[0082] Fixative solution: 4wt% paraformaldehyde tissue fixative solution (Absin) was used as the fixative solution.

[0083] The above reagents were prepared or sterilized under sterile conditions, and stored and used under sterile conditions.

[0084] Instruments:

[0085] Microtome: Leica VT1000S.

[0086] Incubator: Thermo 3111.

[0087] Nonlinear optical microscopy platform: UltralView nonlinear optical microscopy platform, manufacturer: VibroniX (Suzhou) Medical Technology Co., Ltd., brand: VibroniX.

[0088] Confocal fluorescence microscope: UltralView confocal fluorescence microscope, manufacturer: VibroniX (Suzhou) Medical Technology Co., Ltd., brand: VibroniX.

[0089] Transwell plate: Transwell plate, catalog number 14212, cell culture chamber, 24-well plate (PET film, 6.5 mm, pore size 0.4 μM), film material: PET (Polyester).

[0090] Culture conditions:

[0091] Temperature: 37°C.

[0092] Pressure: atmospheric pressure.

[0093] Atmosphere: 95% air + 5% CO2.

[0094] Example 1: SRS microscopic imaging detection on tissue section samples of different thicknesses

[0095] 30 mL of transport solution was added to a 50 mL sterile centrifuge tube and cooled to 0°C. A sample with a volume of 3 cm 3 was extracted from the freshly excised bladder cancer tumor tissue to ensure tissue representativeness and activity, ensuring that the sampling site is away from the cutting edge and is not affected by burning. Avoid pricking the tissue with a sharp tool during the extraction process, and avoid excessive kneading of the tissue. After the sample is extracted, immediately place the sample in the transport solution in the centrifuge tube, then seal the centrifuge tube. Store the sealed centrifuge tube in a container with ice, then send it to the detection laboratory. The sample transportation process takes 2 hours.

[0096] Heat the 3wt% agarose solution to clear, then cool it to 40°C, then pour it into the section holder. Remove the 5 mm 3Size tissue block and immerse the tissue block completely in the agarose solution in the slice holder, and let it stand until solidification. Take out the solidified block and cut it into a cuboid tissue pellet that completely encloses the tissue block. Load the tissue pellet onto the microtome, flood it with PBS, and then carefully cut thin and even slices with thickness of 50 pm, 100 pm, 200 pm and 300 pm, respectively.

[0097] Add 500 pL of tissue culture medium into the Transwell well plate, 450 pL in the lower layer and 50 pL in the upper layer, and preheat it in the incubator. Place the slices prepared above into the preheated tissue culture medium in the Transwell well plate, and perform 3D culture in the incubator for 12 hours for recovery.

[0098] Then, replace the tissue culture medium in the Transwell well plate with heavy water culture medium containing 70 wt% tissue culture medium and 30 wt% heavy water (without adding drugs), and continue the recovery slices for 3D culture for 72 hours. Take out the slices and fix them with fixative, respectively.

[0099] Perform SRS microscopic imaging on the fixed slices using the SRS mode of the nonlinear optical microscopic imaging platform. For each slice, at least 9 typical field images are collected under the same wave number channel to ensure the representativeness of the detection results. At 2930 cm -1 and 2850 cm -1 CHP and CHL signals are collected, respectively; CDP and CDL signals are collected at 2177 cm -1 and 2135 cm -1 respectively; and Off resonance data are collected at 1902 cm -1 wave number to remove the off resonance signals in the later data processing.

[0100] The SRS microscopic imaging images were processed and displayed using ImageJ software. Fig. 1(A)-(D) shows the imaging images of the sections with thickness of: (A) 100 μm; (B) 50 μm; (C) 200 μm; and (D) 300 μm. In the figures, the CHP and CHL channel signals reflect the carbon hydrogen signal intensity of the tissues and cells, and the grayish white color other than the black background in the figures is the carbon hydrogen signal, and the brighter the signal, the stronger the signal. The CHP is the carbon hydrogen protein signal, and the CHL is the carbon hydrogen lipid signal. Similarly, the CDP and CDL channels reflect the carbon deuterium signal intensity of the tissues and cells, i.e. the carbon deuterium chemical bond contained in the cell components newly synthesized after the tissues are cultured in the culture medium containing the heavy water labeled metabolites. The grayish white color other than the black background in the figures is the carbon deuterium signal, and the brighter the signal, the stronger the signal. The signal intensity can reflect the metabolic intensity of the tissues and cells. As can be seen from Fig. 1A-1D, the images of the four different thickness sections can clearly show the signals of the proteins (CHP and CDP) and lipids (CHL and CDL) in the cells. By using the Total Intensity function in the ImageJ software, the signals can be quantitatively counted, so as to detect the metabolic intensity of the tissues and cells according to the statistical data.

[0101] As can be seen from Fig. 1A-D, when the section thickness is 100 μm (A), the SRS microscopic imaging background signal is weak, and the imaging effect is best. When the section thickness is 50 μm (B), the relative background signal of the image is slightly strong. When the section thickness is 200 μm (C) and 300 μm (D), good imaging effect can also be obtained.

[0102] Example 2: Tissue activity using culture medium containing different concentrations of heavy water

[0103] A 50 mL sterile centrifuge tube was added with 30 mL of transport solution and cooled to 0°C. A sample with a volume of 3 cm 3 was extracted from the freshly removed bladder cancer tumor tissue, ensuring that the sampling site is away from the cutting edge and is not affected by burning. During the extraction process, the tissue was avoided to be pricked by the sharp tool, and the tissue was avoided to be squeezed too hard.

[0104] A 3 wt% agarose solution was heated to be clear, and then cooled to 40°C, and then poured into a section holder. A 5 mm 3 size tissue block was taken from the extracted sample, and the tissue block was completely immersed in the agarose solution in the section holder, and was left until solidification. The solidified block was taken out and cut into a cuboid tissue clot completely wrapped in the tissue block. The tissue clot was loaded onto a microtome, which was flooded with PBS, and then carefully cut into thin and flat multiple sections with a thickness of 100 μm.

[0105] Prepare heavy water culture solution with different concentrations, in which the concentration of heavy water is 30wt%, 40wt% and 50wt% respectively.

[0106] Add 500μL of tissue culture solution and heavy water culture solution with the concentration of heavy water being 30wt%, 40wt% and 50wt% respectively into the Transwell well plate, in which 450μL is added into the lower layer and 50μL is added into the upper layer. Put the Transwell well plate into the incubator for preheating. Fluorescently dye the slices prepared above with DAPI (4', 6-diamidino-2-phenylindole), PI (propidium iodide) and Calcein respectively, and place them into different culture solutions in the preheated Transwell well plate for 3D culture in the incubator for 48 hours. Image the slices at the time points of 0, 12, 24 and 48 hours of culture respectively using confocal fluorescence microscope. Process and display the images under different fluorescent signals using ImageJ software.

[0107] Figure 2 shows the fluorescent images of the samples dyed with DAPI, PI and Calcein after 0, 12, 24 and 48 hours of culture, individually and after fusion, in which the image signal intensity of PI dyeing is proportional to the degree of apoptosis; the image signal intensity of Calcein dyeing is proportional to the degree of cell activity. As can be seen from Figure 2, as the concentration of heavy water increases, the degree of apoptosis gradually increases and the degree of cell activity gradually decreases.

[0108] Example 3: Tissue activity using heavy water culture medium with 30% concentration for different culture durations

[0109] Add 30mL of transport solution into a 50mL sterile centrifuge tube and cool it to 0℃. Extract a sample with a volume of 3cm 3 from the freshly excised bladder cancer tumor tissue, ensuring that the sampling site is away from the cutting edge and is not affected by burning. Avoid pricking the tissue with a sharp tool during the extraction process, and avoid squeezing the tissue too hard.

[0110] Heat the 3wt% agarose solution to clear, then cool it to 40℃, and then pour it into the slice holder. Take out a tissue block with a size of 5mm 3 from the extracted sample, and completely immerse the tissue block in the agarose solution in the slice holder, and let it stand until it solidifies. Take out the solidified block and cut it into a cuboid tissue clot that completely wraps the tissue block. Load the tissue clot onto the microtome, immerse it with PBS, and then carefully cut it into multiple thin and flat slices with a thickness of 100μm.

[0111] Prepare heavy water culture solution with the concentration of heavy water being 30wt%.

[0112] In the Transwell well plate, 500 μL of tissue culture solution and heavy water culture solution were added respectively, 450 μL was added to the lower layer and 50 μL was added to the upper layer. The Transwell well plate was placed in the incubator for preheating. The sections prepared as above were respectively fluorescently dyed with DAPI, PI and Calcein, and placed in different culture solutions in the preheated Transwell well plate, and stereoscopic culture was carried out in the incubator for 96 hours. Using a confocal fluorescence microscope, the sections were imaged at time points of 0, 24, 48, 72 and 96 hours of culture respectively. Using ImageJ software, the images under different fluorescent signals were processed and displayed.

[0113] Figure 3 shows the single and fused fluorescent images of the samples dyed with DAPI, PI and Calcein after 0-96 hours of culture. As can be seen from Figure 3, the degree of apoptosis gradually increases and the degree of cell activity gradually decreases over time.

[0114] Example 4: SRS microscopic imaging results of sections cultured using different concentrations of drugs

[0115] Into a 50 mL sterile centrifuge tube, 30 mL of transport solution was added and cooled to 0°C. A sample with a volume of 3 cm 3 was extracted from the freshly excised bladder cancer tumor tissue, ensuring that the sampling site is away from the cutting edge and is not affected by burning. During the extraction process, the tissue was avoided to be pricked with a sharp tool, and the tissue was avoided to be squeezed too hard.

[0116] A 3 wt% agarose solution was heated to be clear, then cooled to 40°C, and then poured into the section holder. A 5 mm 3 tissue block was taken from the extracted sample, and the tissue block was completely immersed in the agarose solution in the section holder and left until solidification. The solidified block was taken out and cut into a cuboid tissue clot that completely wrapped the tissue block. The tissue clot was loaded onto the microtome, flooded with PBS, and then carefully cut into thin and flat sections with a thickness of 100 μm.

[0117] In the Transwell well plate, 500 μL of tissue culture solution was added, 450 μL was added to the lower layer and 50 μL was added to the upper layer, and it was placed in the incubator for preheating. The sections prepared as above were respectively placed in the culture solution in the preheated Transwell well plate, and stereoscopic culture was carried out in the incubator for 12 hours for resuscitation.

[0118] Different concentrations of gemcitabine + cisplatin are used as the drug to be tested. Drug culture solutions with different drug concentrations, heavy water culture solutions, and heavy water-drug culture solutions are prepared, wherein the drug concentrations in the drug culture solutions and the heavy water-drug culture solutions correspond to each other in pairs, and are as follows: gemcitabine (0.2 μM) + cisplatin (0.1 μM), gemcitabine (2 μM) + cisplatin (1 μM), gemcitabine (20 μM) + cisplatin (10 μM), gemcitabine (50 μM) + cisplatin (25 μM), and gemcitabine (200 μM) + cisplatin (100 μM). In addition, the heavy water concentrations in the heavy water culture solutions and the heavy water-drug culture solutions are both 30 wt%.

[0119] The Transwell plates are taken out, 500 μL of fresh tissue culture solution and 500 μL of drug culture solution with different drug concentrations are respectively added to replace the tissue culture solution in the Transwell plates, and the slices are continuously cultured for 24 hours. The Transwell plates are taken out, and the culture solution in the Transwell plates is replaced with corresponding heavy water culture solution and heavy water-drug culture solution, and the culture is continued for 48 hours. The slices are taken out and fixed with fixing solution.

[0120] The fixed slices are respectively subjected to SRS microscopic imaging using the SRS mode of the nonlinear optical microscopic imaging platform. For each slice, at least 9 typical field images are collected under the same wave number channel to ensure that the detection results are representative. The wave number is 2930 cm -1 and 2850 cm -1 The CHP and CHL signals are respectively collected; the CDP and CDL signals are respectively collected at 2177 cm -1 and 2135 cm -1 ; and the Off resonance data are collected at 1902 cm -1 .

[0121] FIGS. 4A-B respectively show SRS microscopic imaging images of the drug-probe sample and the control sample, wherein (A) is a single drug concentration different region detection image, and (B) is a different drug concentration metabolic difference contrast detection image. In the figures, the CHP and CHL channel signals represent the carbon-hydrogen signal intensity of the tissue and cells, and the grayish white color other than the black background in the figures is the carbon-hydrogen signal. The brighter the signal is, the stronger the signal is. CHP is the carbon-hydrogen protein signal, and CHL is the carbon-hydrogen lipid signal. Similarly, the CDP and CDL channels represent the carbon-deuterium signal intensity of the tissue and cells, that is, the cell components containing carbon-deuterium chemical bonds newly synthesized after the tissue is cultured with the culture medium containing heavy water-labeled metabolites. The grayish white color other than the black background in the figures is the carbon-deuterium signal. The brighter the signal is, the stronger the signal is. The signal intensity can represent the metabolic intensity of the tissue and cells.

[0122] The SRS microscopic imaging images were processed and displayed using ImageJ software. The Total Intensity function of the ImageJ software was used to statistically analyze the total signal pixel density value of the CHP / CHL and CDP / CDL channels; the carbon deuterium (CD) signal pixel density value was divided by the carbon hydrogen (CH) signal pixel density value at the same drug concentration, the same imaging area and the same imaging channel to obtain the metabolic intensity ratio of the area at the drug concentration; and the Graphpad software was used to fit the drug concentration-dependent metabolic inhibition images.

[0123] Figure 5 shows the change of D2O metabolism with drug activity and the determination of drug sensitivity or resistance according to the processing and data analysis of the SRS microscopic imaging images shown in Figure 4. In the figure, the x-axis represents the drug concentration, and the drug concentrations are: C (Control) - no drug; 10 -1 -0.2 μΜ gemcitabine + 0.1 μΜ cisplatin; 10 0 -2 μΜ gemcitabine + 1 μΜ cisplatin; 10 1 -20 μΜ gemcitabine + 10 μΜ cisplatin; 10 2 -200 μΜ gemcitabine + 100 μΜ cisplatin; and the y-axis represents the metabolic intensity ratio. As can be seen from Figure 5, the metabolic intensity ratio shows different changes with the increase of drug concentration, and the error bar shows the difference in metabolic intensity ratio of different areas at the same drug concentration. According to the shape of the metabolic inhibition curve, the slope of the curve, the difference in the value of the metabolic intensity ratio between different drug concentrations, the IC 50 value, the cut-off value of the result comparison analysis, the area under the curve and other parameters, the effectiveness of the drug or drug combination for the treatment of the patient can be determined.

[0124] Example 5: SRS microscopic imaging results of slice culture from different tumor samples

[0125] A 30 mL transport solution was added to a 50 mL sterile centrifuge tube and cooled to 0°C. A sample with a volume of 3 cm 3 was extracted from the freshly resected bladder cancer tumor tissue from two different cases, ensuring that the sampling site was away from the cutting edge and was not affected by burning. During the extraction process, the tissue was not poked with a sharp tool, and the tissue was not squeezed too hard. Sample A and sample B were from different PT1 stage bladder cancer cases.

[0126] A 3 wt% agarose solution was heated to clear, then cooled to 40°C, and then poured into a slice holder. A 5 mm 3A tissue block of appropriate size was prepared and completely immersed in the agarose solution in the slice holder, and left until solidification. The solidified block was removed and cut into a cuboid tissue pellet that completely encased the tissue block. The tissue pellet was loaded onto the microtome and flooded with PBS, and then carefully cut into thin and flat sections of 100 pm thickness.

[0127] 500 pL of tissue culture medium was added to the Transwell well plate, 450 pL to the lower layer and 50 pL to the upper layer, and preheated in the incubator. The sections prepared above were placed in the preheated tissue culture medium in the Transwell well plate, and subjected to 3D culture in the incubator for 12 hours for recovery.

[0128] Gemcitabine + cisplatin (GC) was used as the drug to be tested. Drug culture media of different drug concentrations, heavy water culture media, and heavy water-drug culture media were prepared, wherein the drug concentrations in the drug culture media and the heavy water-drug culture media corresponded to each other in pairs and were 0.2 pM gemcitabine + 0.1 pM cisplatin, 2 pM gemcitabine + 1 pM cisplatin, 20 pM gemcitabine + 10 pM cisplatin, and 200 pM gemcitabine + 100 pM cisplatin, respectively; in addition, the heavy water concentrations in the heavy water culture media and the heavy water-drug culture media were both 30 wt%.

[0129] The Transwell well plate was removed, and the tissue culture medium therein was replaced with 500 pL of fresh tissue culture medium and 500 pL of drug culture medium of different drug concentrations, respectively, and the sections were continued to be cultured for 24 hours with the addition of drugs. The Transwell well plate was removed, and the culture medium therein was replaced with the corresponding heavy water culture medium and heavy water-drug culture medium, respectively, and the culture was continued for 48 hours. The sections were removed and fixed with a fixing solution, respectively.

[0130] The fixed sections were subjected to SRS microscopic imaging using the SRS mode of the nonlinear optical microscopic imaging platform. For each section, at least 9 typical field images were collected under the same wave number channel to ensure the representativeness of the detection results. At 2930 cm -1 and 2850 cm -1 CHP and CHL signals were collected, respectively; at 2177 cm -1 and 2135 cm -1 CDP and CDL signals were collected, respectively; and at 1902 cm -1 Off resonance data were collected at the wave number to remove off resonance signals in the later data processing.

[0131] FIGS. 6A-B show the SRS microscopic imaging images of sample A and sample B after drug-added culture, respectively. As shown in FIGS. 6A-B, although both sample A and sample B are at PT1 stage of bladder cancer, the responses of sample A and sample B to drugs are not the same, and the SRS images clearly show the difference.

[0132] Example 6: SRS microscopic imaging results of slice culture using different drugs

[0133] A 30 mL transport solution was added to a 50 mL sterile centrifuge tube, and cooled to 0°C. A sample with a volume of 3 cm 3 was extracted from a freshly excised bladder cancer tumor tissue, ensuring that the sampling site is away from the cutting edge and is not affected by burning. During the extraction process, the tissue was avoided to be pricked by a sharp tool, and the tissue was avoided to be squeezed too hard.

[0134] A 3 wt% agarose solution was heated to be clear, and then cooled to 40°C, and then poured into a slice holder. A 5 mm 3 size tissue block was taken from the extracted sample, and the tissue block was completely immersed in the agarose solution in the slice holder, and left until solidification. The solidified block was taken out and cut into a cuboid tissue clot that completely wrapped the tissue block. The tissue clot was loaded onto a microtome, and was flooded with PBS, and then carefully cut into multiple thin and flat slices with a thickness of 100 μm.

[0135] 500 μL of tissue culture solution was added to a Transwell well plate, 450 μL to the lower layer and 50 μL to the upper layer, and was placed in a culture incubator for preheating. The slices prepared as above were respectively placed in the tissue culture solution of the preheated Transwell well plate, and were subjected to 12 hours of three-dimensional culture in the culture incubator for recovery.

[0136] The culture solution was prepared by using (i) gemcitabine + cisplatin (GC) and (ii) methotrexate + vinblastine + doxorubicin + cisplatin (MVAC) as the drugs to be tested, and using the tissue culture solution without drugs and the heavy water culture solution as controls. The concentrations of the test drugs are as follows:

[0137] In addition, the concentration of heavy water in the heavy water culture solution and the heavy water-drug culture solution is 30 wt%.

[0138] The Transwell well plate was taken out, the tissue culture solution therein was replaced with 500 μL of fresh tissue culture solution and 500 μL of drug culture solution with different drugs and concentrations, respectively, and the slices were continued to be cultured for 24 hours with the drugs. The Transwell well plate was taken out, the culture solution therein was replaced with the corresponding heavy water culture solution and heavy water-drug culture solution, respectively, and the culture was continued for 48 hours. The slices were taken out and were fixed using a fixing solution, respectively.

[0139] SRS micro-imaging of the fixed sections was performed using SRS mode of the nonlinear optical micro-imaging platform. For each section, at least 9 typical field images were collected at the same wavenumber channel to ensure the representativeness of the detection results. At 2930 cm -1 and 2850 cm -1 CHP and CHL signals were collected respectively; at 2177 cm -1 and 2135 cm -1 CDP and CDL signals were collected respectively; and at 1902 cm -1 Off resonance data were collected at the wavenumber to remove off resonance signals in the later data processing.

[0140] FIGS. 6C-D show SRS micro-imaging images of the sections after drug incubation using GC and MVAC as test drugs respectively. As shown in FIGS. 6C-D, the drug sensitivity detection method of the present application can be applied to different drugs / drug combinations.

[0141] As can be seen from the SRS micro-imaging and data analysis results of FIGS. 6A-D, the method of the present application can quantitatively detect small metabolic inhibition differences based on the advantages of high spatiotemporal resolution and high chemical sensitivity of the SRS system, so that the clinical samples incubated in heavy water for a short time can be used for detection of the method, making the processing of the samples simple and preserving the complete tumor microenvironment of the disease source. At the same time, since the incubation time is short, small factor reagents and related tumor cell lines do not need to be added during the incubation process. Since the tumor microenvironment is completely preserved, the detection results are more accurate; since the incubation time is short, the incubation success rate is high; since small factor reagents and related tumor microenvironment cell lines do not need to be added and the incubation success rate is high, the incubation and detection cost is low. Among them, the complete preservation of the tumor microenvironment is particularly important, which can better characterize the tumor of the disease source and thus improve the accuracy of the detection results. For example, as shown in FIGS. 6A-B, the two cases are both PT1 stage bladder cancer, but the histomorphology, cell type composition and metabolic characteristics of the tumor tissues of the two cases are quite different. The above differences will lead to different characteristics of the tumor cells and their microenvironment, i.e., even the same tumor type cases at the same stage may have completely different feedbacks to the same drug or drug combination, so the preservation of the tumor microenvironment of the tumor sensitivity detection sample is very important for accurate and effective detection results. Based on this, precise and effective treatment can be achieved for different cases (including tumors of different patients or tumors of the same patient at different stages, etc.).

[0142] Examples 1-6 measure the drug sensitivity of tumors by SRS microscopic imaging technology for cell metabolic inhibition differences. However, it can be understood that other markers in the cells can also be measured using SRS microscopic imaging technology, for example, analyzing intracellular lipids and proteins and intercellular lipids and proteins in active tumor slices to detect the drug sensitivity of tumors.

[0143] Example 7: Detection using second harmonic imaging

[0144] Active tumor slices are prepared using a similar method as described in Example 3, and are resuscitated and drug-added cultured. SRS microscopic imaging is performed using the SRS mode of the nonlinear optical microscopic imaging platform, and collagen images in the tumor tissue sample are detected by the second harmonic (SHG) imaging mode. The detection parameters of the second harmonic imaging are: excitation wavelength 796 nm, and acceptance wavelength 395 nm. The imaging images are processed and displayed using ImageJ software.

[0145] Figure 7 shows the SRS microscopic imaging results and the collagen (Collagen, the rightmost column) signal images obtained by the second harmonic mode. The grayish white of the non-black background in the figure is the collagen signal. The brighter the signal in the image, the stronger the signal, and the collagen content (brightness / area) and morphology (regularity / group absorption) dimensions reflected by the image can be analyzed to provide supporting information for the drug sensitivity of tumor tissue.

[0146] Example 8: Detection using two-photon fluorescence probe mode

[0147] 30 mL of transport solution is added to a 50 mL sterile centrifuge tube, and cooled to 0°C. A sample with a volume of 3 cm 3 is extracted from the hepatoma tissue just removed, ensuring that the sampling site is away from the cutting edge and is not affected by burning. Avoid pricking the tissue with a sharp tool during the extraction process, and avoid excessive force to pinch the tissue.

[0148] A 3 wt% agarose solution is heated to clear, then cooled to 40°C, and then poured into a slice holder. A 5 mm 3 size tissue block is removed from the extracted sample, and the tissue block is completely immersed in the agarose solution in the slice holder, and left until solidification. The solidified block is removed and cut into a cuboid tissue clot that completely wraps the tissue block. The tissue clot is loaded onto a microtome, flooded with PBS, and then carefully cut into multiple thin and flat slices with a thickness of 100 μm.

[0149] The cut slices were subjected to HCC (liver cancer) cell (fluorescent probe: AFP) + CD8+T cell (fluorescent probe: CD8) double staining, and then imaged by SRS and two-photon excitation fluorescence (TPEF) modes of the nonlinear optical microscopic imaging platform, respectively, to obtain in situ neutral lipid images based on the SRS imaging mode, and in situ liver cancer cells and killer T cells (CD8+T cells) based on AFP and CD8 based on the two-photon fluorescence mode. The detection parameters are as follows: in the two-photon fluorescence mode, Dapi, GATA3, AFP and CD8 immunofluorescence imaging is performed under two-photon excitation / emission wavelengths of 750 nm / 450 nm, 800 nm / 530 nm, 1080 nm / 590 nm and 1250 nm / 690 nm; in the SRS mode, 2850 cm -1 CHL signal. The images in the SRS mode and the fluorescence mode are processed and displayed using ImageJ software.

[0150] As can be seen from FIG. 8, by stacking and fusing the in situ obtained fluorescence images of the same cell active tissue region, the spatial analysis of the images in multiple modalities is performed, and the spatial relationship between the two-photon fluorescence probe labeled tumor microenvironment (TME) image information and the metabolic content information of lipids and other metabolites is revealed; the signal threshold in the software can also be used to select the corresponding ROI to analyze the intensity, area, overlap and proportion of the fluorescence or SRS signal; thus, the data of cell types, subtypes and their spatial distribution of the active tumor slices are obtained, and the correlation between the data and the drug sensitivity of the tumor tissue is obtained based on the detected intratumoral microenvironment image containing cell composition and distribution and the single cell metabolic detection map.

[0151] In Example 8, the cell types, subtypes, specific proteins and their spatial distribution in the active tumor slices are detected by the two-photon fluorescence probe mode, so as to detect the drug sensitivity of the tumor. However, it can be understood that the two-photon autofluorescence mode can also be used to measure these and other markers of autofluorescence substances derived from the tumor tissue itself or drugs, for example, intracellular proteins, lipids, drugs and drug-induced autofluorescence in the active tumor slices are analyzed, and the spatial distribution is analyzed, so as to detect the drug sensitivity of the tumor.

[0152] As can be seen from the above experiments, the tumor drug sensitivity detection method of the present application is based on the processing and culture of the sample of the pathogenic tumor active tissue, and the imaging based on the nonlinear optical imaging technology such as coherent Raman, second harmonic and two-photon fluorescence signal, and the tumor drug sensitivity detection can be quickly, accurately and efficiently performed based on multiple microscopic imaging modes, so as to provide strong technical support and guidance for the precision medicine / individualized treatment of the tumor.

[0153] The present application has thus been fully and completely described by way of representative embodiments. Those skilled in the art will recognize that various modifications can be made to the present application without departing from the spirit and scope of the application. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0154] It is to be understood that certain features of the application that are described in the context of separate embodiments can also be provided in combination in a single embodiment. Conversely, various features of the application that are described in the context of a single embodiment can also be provided separately or in any appropriate

[0155] It is specifically contemplated that any particular feature, structure, characteristic, or combination of features described in relation to one embodiment of the application can be implemented in any other embodiment of the application. In addition, any composition of the application can be used in any method of the application, and any method of the application can be used to make or use any composition of the application. In particular, any aspect described in the claims can be used in combination with any other aspect of the claims and / or specification, alone or in combination with one or more other aspects of the claims and / or specification.

[0156] The above description is intended to be illustrative and not restrictive. Many other embodiments will be apparent to those of skill in the art upon reviewing the above description. The scope of the application should, therefore, be determined not with reference to the above description, but instead with reference to the appended claims, along with their full scope of equivalents.

Claims

1. A method for detecting drug sensitivity of a tumor, the method comprising: obtaining, based on an active tumor sample, a first active tumor slice having a thickness between about 20 pm and about 300 pm; incubating the first active tumor slice in the presence of a drug for a first duration to obtain a drug-treated active tumor slice, the first duration being about 24 hours to about 96 hours; detecting the drug-treated active tumor slice by a nonlinear optical imaging technique to detect drug sensitivity of the tumor to the drug.

2. The method of claim 1, further comprising resuscitating the first active tumor slice.

3. The method of claim 1 or 2, wherein, the nonlinear optical imaging technique comprises one or more of coherent Raman microscopic imaging, second harmonic imaging, and two-photon fluorescence imaging.

4. The method of any one of claims 1-3, wherein, the nonlinear optical imaging technique is coherent Raman microscopic imaging, incubating the first active tumor slice in the presence of a drug comprises incubating the first active tumor slice in the presence of a drug culture medium comprising a drug and a tissue culture medium, and then incubating the first active tumor slice in the presence of a Raman probe-drug culture medium comprising the drug, a Raman probe, and the tissue culture medium, the second duration being about 0 to about 72 hours, the third duration being about 24 hours to about 96 hours, and the sum of the second duration and the third duration being equal to the first duration; detecting the drug-treated active tumor slice by a nonlinear optical imaging technique comprises detecting metabolites of the Raman probe in the drug-treated active tumor slice.

5. The method of claim 3, wherein, the second duration is about 18 hours to about 30 hours, and / or the third duration is about 40 hours to about 48 hours.

6. The method of claim 4 or 5, detecting metabolites of the Raman probe in the drug-treated active tumor slice comprises: obtaining, based on the active tumor sample, a second active tumor slice having the same thickness as the first active tumor slice, wherein, if the first active tumor slice is resuscitated, the second active tumor slice is resuscitated under the same resuscitation condition; incubating the second active tumor slice in the tissue culture medium for the second duration, and then incubating the second active tumor slice in a Raman probe culture medium comprising the Raman probe and the tissue culture medium for the third duration to obtain a control active tumor slice; detecting metabolites of the Raman probe in the drug-treated active tumor slice and the control active tumor slice, and calculating a degree of metabolic inhibition of the tumor by the drug to detect drug sensitivity of the tumor to the drug.

7. The method of any one of claims 4-6, wherein, the Raman probe is a metabolic marker having Raman spectral characteristics and low biological toxicity.

8. The method of claims 4-7, wherein, the Raman probe comprises one or more of heavy water, deuterated palmitic acid, deuterated oleic acid, deuterated amino acid, deuterated glucose, deuterated cholesterol, and 3-O-propargyl-D-glucose.

9. The method of any one of claims 1-3, wherein, the nonlinear optical imaging technique is coherent Raman microscopic imaging, detecting the drug-treated active tumor section by the nonlinear optical imaging technique comprises analyzing intracellular and intercellular lipids and proteins in the drug-treated active tumor section to detect the sensitivity of the tumor to the drug, wherein the intracellular and intercellular lipids are the same or different and comprise one or more of phospholipids, triglycerides, cholesterol, cholesterol esters, and glycolipids.

10. The method of any one of claims 3-9, wherein, The coherent Raman microscopy is one or more selected from the group consisting of stimulated Raman scattering, coherent anti-Stokes Raman microscopy, surface-enhanced Raman spectroscopy, and Raman spectroscopy.

11. The method of any one of claims 1-3, wherein, The nonlinear optical imaging technique is second harmonic generation imaging, detecting the drug-treated active tumor section by the nonlinear optical imaging technique comprises analyzing intracellular and intercellular lipids and proteins in the drug-treated active tumor section to detect the sensitivity of the tumor to the drug, wherein the intracellular and intercellular lipids are the same or different and comprise one or more of phospholipids, triglycerides, cholesterol, cholesterol esters, and glycolipids.

12. The method of any one of claims 1-3, wherein, The nonlinear optical imaging technique is two-photon fluorescence imaging based on fluorescent probes, detecting the drug-treated active tumor section by the nonlinear optical imaging technique comprises labeling the drug-treated active tumor section with fluorescent probes, detecting cell types, subtypes, specific proteins, and their spatial distribution in the drug-treated active tumor section based on two-photon fluorescence, and detecting the sensitivity of the tumor to the drug based on the detected intratumoral microenvironment images comprising cell composition and distribution and single-cell metabolic detection maps.

13. The method of any one of claims 1-3, wherein, The nonlinear optical imaging technique is two-photon fluorescence imaging based on autofluorescence, detecting the drug-treated active tumor section by the nonlinear optical imaging technique comprises imaging and detecting subcellular distribution of autofluorescent substances in the drug-treated active tumor section based on autofluorescence signals in the drug-treated active tumor section to detect the sensitivity of the tumor to the drug.

14. The method of any one of claims 1-13, wherein, Based on the active tumor sample, obtaining a first active tumor section having a thickness between about 20 μm and about 300 μm comprises: obtaining the active tumor sample from fresh ex vivo tumor tissue, preserving the active tumor sample under conditions that maintain the activity of the tumor tissue, wrapping the preserved active tumor sample with a wrapping medium to form a wrapped active tumor sample, wherein the wrapping medium is selected from the group consisting of agarose, silicate gum, or a combination thereof, cutting the wrapped active tumor sample into the first active tumor section having a thickness between about 20 μm and about 300 μm.

15. The method of any one of claims 1-14, wherein, The thickness of the first active tumor section ranges from about 50 μm to about 200 μm.

16. The method of any one of claims 1-15, wherein, The thickness of the first active tumor section ranges from about 75 μm to about 125 μm.

17. The method of any one of claims 1-16, wherein, The active tumor sample is fresh ex vivo tumor tissue having an ex vivo time of less than or equal to about 30 minutes.

18. The method of claim 17, wherein, The ex vivo time of the fresh ex vivo tumor tissue is less than or equal to about 15 minutes.

19. The method of claim 18, wherein, The interval between the time when the fresh ex vivo tumor sample is removed and the time when the first active tumor section is obtained is less than or equal to about 8 hours.

20. The method of any one of claims 1-19, wherein, The active tumor sample retains at least a portion of the intratumoral microenvironment of the tumor.

21. The method of any one of claims 2-20, wherein, The recovery comprises: stereoculture for about 6 hours to about 18 hours at a temperature of about 35°C to about 38°C and normoxic conditions.

22. The method of any one of claims 1-21, wherein, The tumor is a solid tumor.

23. The method of any one of claims 1-22, wherein, The tumor comprises one or more selected from the group consisting of bladder cancer, lung cancer, colorectal cancer, gastric cancer, breast cancer, prostate cancer, liver cancer, ovarian cancer, thyroid cancer, pancreatic cancer, esophageal cancer, and cervical cancer.

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