CN118169379B

CN118169379B - Drug sensitivity detection method for tumor

Info

Publication number: CN118169379B
Application number: CN202410591743.4A
Authority: CN
Inventors: 岳蜀华; 杨彬; 姚林; 王璞
Original assignee: Beijing Weiao Medical Laboratory Co ltd; Beihang University; Peking University First Hospital
Current assignee: Beijing Weiao Medical Laboratory Co ltd; Beihang University; Peking University First Hospital
Priority date: 2024-05-14
Filing date: 2024-05-14
Publication date: 2024-09-06
Anticipated expiration: 2044-05-14
Also published as: WO2025236610A1; CN118169379A

Abstract

The present application discloses a method for detecting drug sensitivity of a tumor, comprising: obtaining a first active tumor slice with a thickness between 20 μm and 300 μm based on an active tumor sample, and optionally resuscitating the first active tumor slice; incubating the first active tumor slice in the presence of a drug for a first period of time to obtain an active tumor slice treated with the drug, the first period of time being 48 to 96 hours; detecting the active tumor slice treated with the drug by nonlinear optical imaging technology to detect the drug sensitivity of the tumor to the drug.

Description

Drug sensitivity detection method for tumor

Technical Field

The application relates to the field of biological medicine, in particular to a drug sensitivity detection method for tumors.

Background

Cancer is a malignant disease that severely threatens human health. According to the data issued by the cancer center of China, the number of new cancer cases in China exceeds 406.40 ten thousand cases in 2023 years, and the number of death cases reaches 241.35 ten thousand cases. Cancer (malignant tumor) has become one of the leading causes of death affecting resident health.

Treatment of cancer (malignant tumor) requires a variety of therapeutic strategies and factors to be coordinated. For the treatment modes of different tumors, the treatment modes should be comprehensively judged according to the disease course, the tumor size, the metastasis and other conditions. It is well known that tumors present heterogeneity and instability, which can occur at different locations and at different times within the same patient, as well as between different patients. This also means that for a certain treatment regimen, different differences in efficacy occur even for patients with the same cancer or different lesions in the same patient or during different courses of the same patient. For example, for many different patients with the same tumor, the same drug and standard dose would be traditionally used for treatment; however, due to tumor heterogeneity and cell instability, there are often substantial differences in therapeutic effects, adverse reactions, etc. among patients in practice, and sometimes such differences are even fatal. Or because new mutation appears in the tumor treatment process, the effective therapeutic drug in the early treatment stage can possibly reduce the drug effect and even lose the curative effect due to various reasons. Thus, during tumor treatment, different treatment protocols must be selected according to different people, different tumors, and different stages of the course, i.e., precision or personalized medical protocols.

Precision or personalized medicine refers to personalized diagnostic and therapeutic strategies that match the molecular chemistry, cell biology, and pathophysiological characteristics of a patient. At present, gene mutation targets of patients are searched by utilizing a gene sequencing technology, and targeted chemotherapy or targeted drugs are selected to accurately kill tumor cells to become a new mode and a research hotspot of accurate treatment of malignant tumors. The authoritative data from the 2017 mechanical slot KETTERING CANCER CENTER (MSKCC, commemorative ston-ketchun cancer center) showed that: 63% of patients fail to find an appropriate targeted therapeutic regimen by gene detection (Zehir et al, 2017). Accurate medical treatment of tumors not only comprises gene sequencing, but also comprises multi-layer medical technologies such as proteomics, response characteristics of individuals to drugs and the like. In order to avoid blindness of tumor drug use and improve the effective rate of the drug, drug sensitivity detection on tumors becomes an important component of accurate tumor treatment. Along with the continuous improvement of the accurate medical requirements, the clinical requirements and requirements for tumor drug sensitivity detection can be improved.

There are currently a variety of drug sensitive detection methods for tumors. The experimental model for detecting the drug sensitivity of the tumor is mainly divided into an in-vivo detection model and an in-vitro detection model. The in vivo detection model is mainly a mouse model of human tumor xenograft (PDX), the heterogeneity of tumor cells and the in-vivo characteristics of the tumor cells are often lost in the in-vitro culture process by the traditional cell line culture method, and the PDX mouse model can reserve the true genome, transcriptome, proteomics and abnormal signal path characteristics of tumors in patients to a certain extent, reserve the in-vivo activity and interpretation mechanism of tumor tissue cells, and has the pharmacokinetics more similar to that of human bodies, high accuracy, high sensitivity and the like. However, PDX has the disadvantages of high modeling difficulty, incapability of repeatedly acquiring, long model construction time, unstable success rate (20% to 80%, large deviation) and limitation on passage times; the method has the defects of complex operation, long period (up to 2-4 months) and high cost (up to 10-30 ten thousand), and is unfavorable for clinical rapid diagnosis and possible mutation of tumor tissues.

For this purpose, various in vitro test models have been successively established to provide personalized treatment of tumors, for example, tumor cell-based models such as ex vivo cultured cell lines, primary cells, circulating tumor cells or conditionally reprogrammed cells, etc., or in vitro organoid models such as pathogenic organoids (PDO), etc. However, the tumor cell samples such as cell lines, primary cells, circulating tumor cells or conditional reprogramming cells which are cultured in vitro at present cannot simulate in-vivo tumor microenvironment, and the heterogeneity of tumor cells and the difference of the microenvironment among different patients are important reasons for causing great difference of drug response, so that the consistency of the detection results and clinical medication prognosis of tumor drug sensitivity detection research and application taking the samples as objects is poor, and the accuracy is low. In addition, in vitro tumor organoids or tissue culture mainly detects tissue activity and growth in a detection mode, and needs long-time culture, so that the culture has poor timeliness, a sample is difficult to be reduced to an in-vivo tumor microenvironment, and the culture success rate is low and high; for example, the construction of PDO requires a large amount of clinical sample tissue, the success rate of 3D culture of organoid microspheres is unstable (40-80%) due to various factors, and the organoid microspheres need to be cultured for a long time (2-4 weeks), have high cost (5-10 ten thousand), and are prone to losing the properties of Tumor Microenvironment (TME) of a single patient (such as up-regulation of pathway expression such as growth and proliferation, down-regulation of pathways such as immune angiogenesis), thus causing problems such as poor accuracy.

Therefore, the existing tumor drug sensitivity detection model is difficult to predict the drug effect of targeting and immunotherapy due to the lack or distortion of the intratumoral microenvironment, and a low-cost detection method capable of rapidly (for example, within 1 week) and accurately performing personalized tumor drug accurate screening is needed.

Disclosure of Invention

In order to solve the above and other technical problems, the application provides a drug-sensitive detection method for tumors, which uses an in-vitro culture model of a tumor living tissue sample to directly slice and culture isolated tumor tissues and then detect the isolated tumor tissues through a nonlinear optical imaging technology, and relatively perfectly reserves tissue structures and other types of cells in microenvironments, including immune cells and interstitial cells, in the culture process, thereby reserving at least a part of intratumoral microenvironments in the detected tumor sample, further realizing accurate, rapid and low-cost detection of tumor drug sensitivity and realizing multiple benefits of high timeliness, economy and medical quality.

According to an embodiment of the present application, there is provided a drug sensitive detection method of a tumor, the method comprising: obtaining a first active tumor section having a thickness of between 20 and 300 μm based on an active tumor sample, and optionally resuscitating the first active tumor section; incubating the first active tumor section in the presence of a drug for a first period of time to obtain a drug-treated active tumor section, the first period of time being 24-96 hours; detecting the drug-treated active tumor section by nonlinear optical imaging technology to detect drug sensitivity of the tumor to the drug.

Optionally, the nonlinear optical imaging technique includes one or more of coherent raman microscopy imaging, second harmonic imaging, and two-photon fluorescence imaging.

Optionally, the nonlinear optical imaging technique is coherent raman microscopy imaging, and culturing the first active tumor section in the presence of a drug comprises: incubating the first active tumor section in the presence of a drug culture solution for a second period of time, then incubating in the presence of a raman probe-drug culture solution comprising a drug and a tissue culture solution, the raman probe-drug culture solution comprising the drug, the raman probe, and the tissue culture solution for a third period of time, the second period of time being 0-72 hours, the third period of time being 24-96 hours, and the sum of the second period of time and the third period of time being equal to the first period of time; detecting the drug-treated active tumor section by nonlinear optical imaging techniques includes detecting metabolites of the raman probe in the drug-treated active tumor section.

Optionally, the second time period is 18-30 hours, and the third time period is 40-50 hours.

Optionally, detecting the metabolites of the raman probe in the drug-treated active tumor section comprises: acquiring a second active tumor section with the same thickness as the first active tumor section based on the active tumor sample, wherein if the first active tumor section is resuscitated, the second active tumor section is resuscitated under the same resuscitating condition; incubating the second active tumor section in the tissue culture solution for the second period of time, and then incubating the second active tumor section in a raman probe culture solution for the third period of time to obtain a control active tumor section, 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 section and the control active tumor section, and calculating the degree of metabolic inhibition of the drug on the tumor to detect the drug sensitivity of the tumor to the drug.

Optionally, the raman probe is a metabolic characterizer having raman spectral characteristics and low biotoxicity.

Optionally, the raman probe comprises one or more of heavy water, deuterated palmitic acid, deuterated oleic acid, deuterated amino acids, deuterated glucose, deuterated cholesterol, and 3-O-propynyl-D-glucose.

Optionally, the coherent raman microscopy imaging technique is one or more selected from the group consisting of stimulated raman scattering, coherent anti-stokes microscopy imaging technique, surface enhanced raman spectroscopy imaging technique and raman spectroscopy analysis technique.

Optionally, the nonlinear optical imaging technique is coherent raman microscopy, and detecting the drug-treated active tumor section by nonlinear optical imaging technique comprises analyzing intracellular lipids and proteins and intercellular lipids and proteins in the drug-treated active tumor section to detect 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 esters, and glycolipids.

Optionally, the nonlinear optical imaging technique is second harmonic imaging, and detecting the drug-treated active tumor slice by the nonlinear optical imaging technique comprises: tumor stem cells, collagen and/or cellular microtubules in the drug-treated active tumor sections are analyzed to detect the sensitivity of the tumor to the drug.

Optionally, the nonlinear optical imaging technique is fluorescence probe-based two-photon fluorescence imaging, and detecting the drug-treated active tumor slice by the nonlinear optical imaging technique comprises: labeling the active tumor section treated by the drug with a fluorescent probe, detecting the cell type, subclass, specific protein 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 constitution and distribution and single cell metabolism detection map.

Optionally, the nonlinear optical imaging technique is autofluorescence-based two-photon fluorescence imaging, and detecting the drug-treated active tumor slice by the nonlinear optical imaging technique comprises: imaging and detecting subcellular distribution of autofluorescent substances in the active tumor section based on autofluorescent signals in the drug-treated active tumor section to detect sensitivity of the tumor to the drug.

Optionally, based on the active tumor sample, obtaining a first active tumor section having a thickness between 20 μm and 300 μm comprises: obtaining the active tumor sample from fresh, ex vivo tumor tissue, preserving the active tumor sample under conditions that preserve 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, diatomaceous earth, or a combination thereof, and cutting the wrapped active tumor sample into the first active tumor section having a thickness of between 20 μm and 300 μm.

Optionally, the thickness of the first active tumor section ranges from 50 μm to 200 μm.

Optionally, the thickness of the first active tumor section ranges from 75 μm to 125 μm.

Optionally, the active tumor sample is fresh ex vivo tumor tissue, and the ex vivo time of the fresh ex vivo tumor tissue is less than or equal to 30 minutes.

Optionally, the ex vivo time of the fresh ex vivo tumor tissue is less than or equal to 15 minutes.

Optionally, the interval between the time the fresh ex vivo tumor sample is removed and the time the first active tumor section is obtained is less than or equal to 8 hours.

Optionally, the active tumor sample retains at least a portion of the intratumoral microenvironment of the tumor.

Alternatively, the three-dimensional culture is performed at a temperature of 35 to 38℃and under normoxic conditions for 6 to 18 hours.

Alternatively, the tumor is a solid tumor.

Optionally, the tumor comprises one or more of the following: 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.

Drawings

In order to more clearly illustrate the technical solution of the embodiments of the present invention, the drawings that are used in the description of the embodiments will be briefly described. It should be apparent that the drawings in the following description are merely exemplary embodiments of the present invention, and that other drawings may be obtained from these drawings without inventive effort to those of ordinary skill in the art.

Fig. 1 shows the results of stimulated raman scattering (Stimulated RAMAN SCATTERING, SRS) microscopic imaging of active tumor sections of different thickness, where the section thickness in each panel is: (A) 100 μm, (B) 50 μm, (C) 200 μm, (D) 300 μm;

Fig. 2 shows SRS microscopy imaging results using different concentrations of heavy water as raman probe, wherein the heavy water concentrations are respectively: 0 (control), 30wt%, 40wt% and 50wt%;

Figures 3A-B show the change in samples over time after addition of heavy water culture: (A) Immunofluorescence images using DAPI (4', 6-diamidino-2-phenylindole), PI (propidium iodide), calcein (Calcein), and fusion (Merged); (B) percentage of cell survival between 0 and 96 hours;

FIGS. 4A-B show SRS microscopy imaging results of a drug-loaded probe sample and a Control sample (Control), respectively;

FIG. 5 shows the change in heavy water (D ₂ O) metabolism with drug activity and the judgment of drug sensitivity or resistance made therefrom;

Fig. 6 shows SRS microscopy imaging results from different cases and for different drugs, wherein: (A) Is an imaging image of SRS metabolic detection with gemcitabine + cisplatin on a clinical specimen from a PT1 bladder cancer case; (B) Is an imaging image of SRS metabolic detection with gemcitabine + cisplatin on a clinical specimen from another PT1 bladder cancer case; (C) Is an imaging image of SRS metabolic detection of tumor samples with gemcitabine + cisplatin (GC); (D) Is an imaging image of SRS metabolic detection of a tumor sample with Gemcitabine+cisplatin (GC) versus Methotrexate+vinblastine+doxorubicin+cisplatin (MVAC);

FIG. 7 shows multiplexed detection results, showing SRS microscopic imaging results and collagen images in tumor tissue samples obtained by second harmonic imaging; and

Fig. 8 shows images of hepatoma-active tissue sections obtained by a fluorescent probe-based two-photon fluorescence imaging system.

Detailed Description

For the purposes of the following detailed description, it is to be understood that the application may assume various alternative variations and step sequences, except where expressly specified to the contrary. Furthermore, except in any operating examples, or where otherwise indicated, all numbers expressing, for example, quantities of ingredients used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties to be obtained by the present application. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forth the broad 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.

Furthermore, it should be understood that any numerical range recited herein is intended to include all sub-ranges subsumed therein. For example, a range of "1 to 10" is intended to include all subranges between (and including) the minimum value of 1 and the maximum value of 10, i.e., having a minimum value equal to or greater than 1 and a maximum value of equal to or less than 10.

In the present application, the use of the singular includes the plural and plural encompasses singular, unless explicitly stated otherwise. Furthermore, in the present application, "or" is used to mean "and/or" unless explicitly stated otherwise, even though "and/or" may be explicitly used in certain instances. In addition, in the present application, "a" or "an" is used to mean "at least one" unless explicitly stated otherwise. For example, "a" compound, "a" composition, etc., refers to any one or more of these items.

As used herein, "comprising," "including," and similar terms are to be understood in the context of the present application to be synonymous with "including," and thus are open-ended and do not exclude additional unredescribed or unrecited elements, materials, components, or method steps.

As used herein, "consisting of … …" is understood in the context of the present application to exclude the presence of any unspecified elements, components or method steps.

In the present application, the use of the singular includes the plural and plural encompasses singular, unless specifically stated otherwise. For example, while reference is made herein to "a" compound or "a" composition, a combination (i.e., multiple) of these components may be used.

In addition, in the present application, unless specifically stated otherwise, the use of "or" means "and/or", although "and/or" may be explicitly used in some cases.

In the prior art, drug sensitivity detection is generally performed by using tumor cells that are dissociated from tumor tissue and cultured for a long period of time in an in vivo or in vitro model. However, such in vivo or in vitro model cultured tumor cell samples are substantially free of the original in vivo tumor microenvironment (tumor microenvironment, TME), failing to demonstrate subcloning heterogeneity of the tumor and cellular interactions within the tumor microenvironment; meanwhile, due to the instability of tumor cells, genotype drift often occurs after long-term culture, so that the detection result is difficult to compare with clinical treatment data; furthermore, tumor cells of a specific type, such as prostate tumor cells, cannot survive in a 2D culture environment and cannot be drug-sensitive detected by the above method.

On the other hand, in vivo or in vitro models, the time and cost for culturing the tumor are long, and the success rate of culturing is not stable.

Finally, there is no provision in the prior art for a rapid and accurate low cost drug sensitive detection method based on tumor biopsy samples.

In order to solve the technical problems, the application provides a drug sensitivity detection method based on a tumor biopsy sample. The drug sensitivity detection method comprises the following steps: obtaining a first active tumor section based on the active tumor sample; incubating the first active tumor section in the presence of a drug for a period of time to obtain a drug-treated active tumor section; the drug-treated active tumor sections were examined by nonlinear optical imaging techniques to detect the drug sensitivity of the tumor to the drug. Therefore, the drug detection method can realize rapid, accurate and low-cost drug sensitivity detection of tumors based on culture, slicing, drug adding treatment and nonlinear optical imaging of tumor living tissue samples.

As used herein, the term "drug sensitivity" also known as "drug sensitivity" refers to the degree of sensitivity of a drug to a target (e.g., pathogen, cell, tissue, body, etc.), i.e., the effect of a drug on a target through individual differential responses of the target to drug metabolism. Different targets have different sensitivities to different drugs. In general, a target is "sensitive" to a drug if a small dose of the drug can exert a significant effect on the target; conversely, if a larger amount of drug does not exert a significant effect on the target, it indicates that the target is "insensitive" to the drug or even "drug resistant". In clinical practice, drug sensitivity determines the therapeutic effect of a drug on a target. The drug sensitivity detection is carried out to reasonably select the used drugs according to the drug sensitivity, so that the method has important guiding significance for accurate medical treatment/individuation medical treatment.

As used herein, the term "tumor" refers to a group of diseases formed by the abnormal proliferation of body cells, also known as a neoplasm or neoplasm. Tumors can be classified in various ways, for example, benign and malignant tumors, depending on the cellular nature of the neoplasm and the degree of harm to the body; or may be classified into solid tumors, hematological tumors, etc. according to the primary site. In some examples, methods according to the application may be used to detect drug sensitivity of solid tumors; in other examples, the method according to the application may be used to detect drug sensitivity of a malignancy; in still other examples, the method according to the application may be used to detect drug sensitivity of malignant solid tumors. For example, examples of tumors detected in the present application may 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.

In general, drug sensitive detection of tumors can be performed against different targets. For example, it is possible to detect tumor cell samples such as cell lines, primary cells or conditional reprogramming cells which are cultured ex vivo, however, such ex vivo cell samples cannot simulate in vivo tumor microenvironments and cannot accurately reflect heterogeneity of tumor cells and microenvironments thereof, so that tumor drug sensitivity detection studies and application of the samples as objects have poor consistency with clinical drug prognosis and low accuracy. In addition, animal in-vivo animal models such as a PDX model and the like can be used for detection, and the model can keep tumor microenvironment and subcloning to a certain extent, but has long culture and detection period (2-4 months) and low culture success rate (large deviation of 20-80 percent), so that the model is difficult to meet the timeliness requirement of clinical detection and is difficult to bear in cost (10-30 ten thousand); in addition, human body in-vivo tumor drug sensitivity detection research often needs to pass through complicated in-vivo intervention schemes and equipment, so that the implementation difficulty is high, the cost is extremely high, and the risk of patients is huge, so that the method is difficult to have ethical feasibility. In-vitro tumor organoids or tissue culture can be used, however, the detection mode is mainly used for detecting tissue activity and growth, and long-time culture is needed, so that the timeliness is poor, the sample construction is difficult to reduce in-vivo tumor microenvironment, and the culture success rate is low and high. In a word, the existing tumor drug sensitivity detection model is difficult to accurately and effectively predict the drug effect of targeting and immunotherapy due to the lack or distortion of the microenvironment.

In view of this, the inventors devised a method for tumor drug sensitivity detection based on an in vitro culture model of active tumor sections of tumor biopsy samples. As used herein, the term "tumor biopsy sample" may also be referred to as an active tumor sample, meaning a sample that retains the tissue activity and intratumoral microenvironment of the tumor. As used herein, the term "intratumoral microenvironment" refers to the constituent parts and environment within the tumor tissue. In general, in addition to cancer cells, tumor tissues include interstitial cells, immune cells, collagen tissues and the like, and the composition, distribution, interaction with cancer cells and the like of these components may cause the tumor to be resistant to specific drugs, so that detection of tumor tissues which retain as many intratumoral microenvironments as possible can more characterize in-vivo tumor characteristics, and detection results can also more accurately reflect the characteristics of tumors. In general, the active tumor sample may be or be obtained from fresh ex vivo tumor tissue. For example, the fresh, ex vivo tumor tissue may be tumor tissue obtained from a patient by surgical excision or biopsy sampling that retains tumor tissue activity and at least a portion (preferably a majority, more preferably all) of the intratumoral microenvironment. As used herein, "fresh" refers to tumor tissue having an ex vivo time of less than or equal to 1 hour after surgical excision or biopsy sampling, e.g., an ex vivo time of less than or equal to 45 minutes, less than or equal to 30 minutes, less than or equal to 15 minutes, less than or equal to 10 minutes, less than or equal to 5 minutes, etc., less than or equal to 3 minutes, less than or equal to 1 minute; or may refer to sample extraction immediately after surgical excision or biopsy sampling of tumor tissue, i.e., an ex vivo time of about 0. It is well known that during sample extraction, it is desirable to avoid extraction of tissue at the edges of the cut (e.g., cut) or affected by the burn, to ensure that the sample is fresh and active; samples are extracted as much as possible, so that tissue representativeness and activity are guaranteed; in addition, the tissue is prevented from being poked by a sharp tool in the extraction process, and the tissue is prevented from being excessively pinched with force. The use of fresh in vitro tumor tissue as an active tumor sample can prevent the tumor tissue from significantly decreasing or losing tissue activity due to too long in vitro time, and from possible variation due to external environmental changes, etc. The shorter the in vitro time is, the smaller the difference between the in vitro tumor tissue and the in vivo tumor tissue is, the more the activity and intratumoral microenvironment characteristics of the obtained active tumor sample are reserved, and therefore the higher the accuracy of the drug sensitivity detection result obtained based on the sample is.

After taking fresh ex vivo tumor tissue as the active tumor sample or taking an active tumor sample from fresh ex vivo tumor tissue, an active tumor slice may be obtained directly from the active tumor sample, for example, when the operating room is located sufficiently close to or at the same site as the detection laboratory; alternatively, the active tumor sample may be sectioned after being stored under conditions that preserve tumor tissue activity for a period of time, for example, when the operating room is located at a different location or remote from the detection laboratory. The conditions under which the ex vivo tumor tissue remains active for a period of time are known to or can be readily determined by those skilled in the art, e.g., the active tumor sample may be placed in a suitable preservation medium and preserved at a temperature of 0-4 ℃ for a period of no more than 12 hours, e.g., no more than 10 hours, no more than 8 hours, no more than 6 hours, no more than 5 hours, no more than 4 hours, no more than 3 hours, no more than 2 hours, no more than 1 hour, etc. Preservation media suitable for use in the methods of the application may include, but are not limited to, DMEM/F12 medium containing 1wt% penicillin-streptomycin diabody, 90wt% hanks balanced salt solution (containing 10wt% fetal calf serum), hypoThermosol FRS (HTS-FRS), and maytansinoid tissue preservation fluid, among others, provided that such preservation media maintain a high tissue cell viability of the active tumor sample over a period of time, maintain basic physiological metabolism of the cells, and have high permeability and tissue penetration. In general, in order to maintain the activity of an isolated tumor tissue sample and its consistency with the intratumoral microenvironment of the tumor tissue in vivo, the active tumor tissue sample is generally unable to be frozen, otherwise, the tissue activity is lost or difficult to recover, etc., because the freezing seriously affects the metabolic characteristics of the tumor tissue sample unlike the general tissue sample. Thus, in some aspects, the active tumor samples used in the methods of the application do not undergo a freezing process.

As used herein, the term "slice" also referred to as "tissue slice" refers to a slice of biological tissue that is cut from biological tissue by a special tool and applied to a slide for microscopic observation, which is widely used in the fields of biology, medicine (pathology, infectious disease, etc.), and the like. In general, tissue sections suitable for observation by a microscope (e.g., optical microscope, electron microscope, etc.) are significantly different in structure from common tissue pieces. For example, tissue slices tend to be much smaller in thickness than their length and width, possibly even orders of magnitude different. The advantage of such tissue slices is that the significantly thinner thickness of the tissue slices enables sufficient access of nutrients into the tissue during the culture process, the success rate of tissue culture is high, and no other nutrient factors need to be added during the culture process, thus reducing the culture cost; in addition, the medicine can fully penetrate into the tissue sample in the culture process, so that the medicine can effectively act on the whole tissue sample, and has good effect, so that the detection can be performed after short-time culture, and the whole culture and detection period is short; the smaller thickness and larger cross section of the sections better exposes the internal microenvironment of the tissue and can be prepared as slide samples for detection by nonlinear optical effect microscopy imaging techniques, so that the detection results based on the sections can better and more rapidly reflect the characteristics of biological tissue. In contrast, tissue masses obtained directly from tissue (e.g., tumor tissue) tend to be of comparable size (e.g., on the same order of magnitude) in three dimensions of length, width, and thickness, and are much greater than tissue slices. Such tissue blocks are difficult to sufficiently permeate nutrient substances and medicines in the culture process due to the large thickness, and may cause various defects, such as low culture success rate, increased cost due to the need of adding other nutrient factors, and difficult detection after short-time culture due to poor medicine effect, prolonged culture and detection period, incapability of timely reflecting the characteristics of tissues, and the like; in addition, tissue masses are not suitable for detection by nonlinear optical effect microscopy imaging techniques.

In some examples, the thickness of an active tumor slice according to the application may be in the range of 20 μm to 300 μm, for example in the range of 50 μm to 200 μm, or in the range of 75 μm to 125 μm. Or the thickness of the active tumor section may be about 100 μm. The inventors found that when the tissue slice is too thin, for example, less than 20 μm, the slice can only retain a single layer of tumor tissue cells in the thickness dimension, but cannot accommodate the thickness of multiple layers of cells, so that the retention of tumor microenvironment is limited, physiological characteristics such as disease-derived tumor tissue metabolism and the like are difficult to comprehensively reflect, and the effects of in-vitro culture and imaging by a nonlinear optical imaging technology of the disease-derived tissue slice are poor (the background signal is too strong). In addition, the inventors have found that if the thickness of a tissue slice is too high, for example, higher than 300 μm, it may be difficult for nutrients and drugs to be detected to penetrate efficiently during the culture, resulting in low success rate of culture, increased cost due to the need to add other nutritional factors, and difficulty in detection after short-time culture due to poor effects of the drugs, resulting in prolonged culture and detection period. Moreover, tissue slices with too high a thickness (e.g., above 300 μm) can also result in the slice being difficult to prepare as a slide sample for detection by a nonlinear optical device or the prepared slide sample having poor imaging performance when used for nonlinear optical imaging, e.g., because the sample is too thick, which affects the transmission signal reception of nonlinear optical imaging techniques. In some aspects, the inventors have found that sections with a thickness in the range of 20 μm to 300 μm, as described herein, allow adequate penetration of nutrients and drugs, allowing multi-dimensional assays based on nonlinear optical microscopy, such as but not limited to assays in metabolic activity, cellular components, etc., through short-time (e.g., 3 to 5 days) culture at low cost (without the need to add additional nutritional factors); in addition, sections within the above thickness range effectively preserve at least some (e.g., most, or even all) of the intratumoral microenvironment of the tumor tissue, enabling higher consistency with in vivo tumor tissue when performing drug sensitive assays, better reflecting the characteristics of in vivo tumor tissue and yielding more accurate assay results.

In the method according to the application, any suitable method may be used for obtaining active tumor sections. Examples of methods for obtaining active tumor sections suitable for use in the methods of the application may 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 slices having a thickness between 20 μm and 300 μm to a suitable thickness. Examples of encapsulation media suitable for use in the methods of the present application may be selected from, but are not limited to, agarose, diatomaceous earth, or combinations thereof. The slicing process may be performed in any suitable medium, such as, but not limited to, in PBS.

In the method according to the application, the active tumor sections may optionally also be resuscitated depending on the tissue activity or the preservation time of the active tumor sample. As used herein, the term "resuscitation" refers to the process by which ex vivo tissue is cultured to increase its activity and adapt to an in vitro culture environment after physiological functions such as activity and the like are reduced. In the method of the application, the active tumor slices can be revived by culturing the slices in a suitable culture environment for a period of time, thereby enhancing the tissue activity of the slices for subsequent medicated culture and detection. For example, active tumor sections can be subjected to stereoscopic culture at a temperature of 35-38℃under normoxic conditions for resuscitation. As used herein, the term "stereoscopic culture" refers to tissue culture techniques that culture tissue in three-dimensional space to mimic the natural state in a human or animal body. For example, active tumor sections may be cultured in a stereospecific culture medium with a semipermeable membrane or gel mass, including but not limited to culturing in a Transwell in the presence of tissue culture fluid. The resuscitation may be performed for a suitable period of time, for example, 6 to 18 hours, for example, 8 to 12 hours, or the like. The inventors have found that resuscitating an ex vivo sample (e.g., an ex vivo sample after a period of storage) may facilitate adapting tumor tissue to an in vitro culture environment in a state of good activity after surgery or biopsy ex vivo and transportation; otherwise, the direct addition of drugs under conditions where the tissue sample is not adapted to in vitro culture may lead to an evaluation of the metabolic activity of the tissue that is disturbed by a large change in the state of culture.

After the active tumor slices are obtained and optionally resuscitated, the active tumor slices can be incubated in the presence of a drug for a period of time to obtain drug-treated active tumor slices, e.g., 48-90 hours, which can then be detected by nonlinear optical imaging techniques to detect the drug sensitivity of the tumor to the drug. As used herein, the term "drug" refers to a substance that can exert a certain effect on tumor tissue or cells, e.g., chemotherapeutic drugs, targeted drugs, immune drugs, etc. against tumors; the "drug culture solution" is a culture medium obtained by adding a drug to be detected to a tissue culture solution. Tissue culture fluids suitable for use in the present application may be any suitable culture fluid such as, but not limited to, 90wt% DMEM/F12 (1 wt% penicillin-streptomycin diabody) +10wt% fetal bovine serum, and the like. The drug culture solution suitable for use in the present application may have any suitable concentration, for example, a drug concentration at the highest blood concentration (C _max) of the drug to be tested and a gradient of 10-fold on both sides thereof, and the like. As used herein, the term "nonlinear optical imaging technique" refers to an imaging technique that uses nonlinear optical effects to absorb and radiate coherent light from a substance at low light intensities to obtain information in three dimensions; "nonlinear optical effect" means that when the intensity of the light wave field of the observed excitation light is comparable to the coulomb field or the interatomic vibration energy level inside the atoms in the sample, the interaction of the light with the sample will produce a nonlinear effect reflecting the physical quantity of the substance in the sample, and this 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 apparent in linear optics. Common nonlinear optical imaging techniques are well known in the art, such as, but not limited to, coherent raman microscopy imaging, second harmonic imaging, two-photon fluorescence imaging, and the like.

The nonlinear optical imaging technique used in the method according to the application may be a coherent raman microscopy imaging technique. Coherent raman microscopy imaging techniques suitable for use in the methods of the present application may include, but are not limited to, one or more of Stimulated Raman Scattering (SRS), coherent anti-stokes microscopy imaging techniques (CARS), surface-enhanced raman spectroscopy imaging techniques, and raman spectroscopy analysis techniques. The coherent anti-stokes raman scattering (CARS) and Stimulated Raman Scattering (SRS) are used for microscopic imaging, compared with spontaneous raman scattering, the signal is obtained with about 10 ⁶ gain, the imaging speed is remarkably improved, and the space-time resolution and the sensitivity are higher. Unlike CARS, SRS microscopy does not have a non-resonant background, and has been applied to metabolic imaging of cells, tissues and model organisms. Hyperspectral Stimulated Raman Scattering (SRS) can record spectra at each pixel to distinguish SRS signals from the background. Therefore, the hyperspectral Stimulated Raman Scattering (SRS) system can conduct quantitative metabolism inhibition difference observation on short-time rehydration cultured disease source tumor tissues with the tumor microenvironment completely reserved by simple slice treatment, so that the disease source tumor samples are more than 95% of culture success rate, 100% of culture and detection cycle of reserved tumor tissue microenvironment, less than 5 days of culture and detection period and more than 90% of detection result accuracy rate are realized, and accurate, rapid and low-cost tumor drug sensitivity detection is realized based on the detection result. Metabolism is an important feature of life and is critical to the fundamental elucidation and understanding of the mechanisms of many biological processes. Organisms update biomolecules such as proteins and lipids by metabolism, but the measurement of such a new replacement process is difficult to achieve in the label-free SRS technique. In order to solve the problem, a small molecule Raman labeling mode is adopted to distinguish newly synthesized biomolecules from original molecules, so that metabolic tracking is completed. SRS microscopy imaging based on raman probe (e.g. heavy water) metabolism is also used in drug sensitive assays. During the intracellular NAD/NADP reduction process, hydrogen in water is metabolized and converted into biomass, especially biomacromolecules such as lipid (L) and protein (P) therein. The C-H bonds in these macromolecules have characteristic peaks in the Raman spectrum (between 2800 and 3100cm ^-1). When heavy water (D ₂ O) is used instead of some of the normal water (H ₂ O) in the medium, deuterium (D) in the heavy water is used to synthesize important biomacromolecules along with the metabolism of the cells, so that the important biomacromolecules enter the cells, the C-H peak in the Raman spectrum is shifted, and the C-D peak (2000-2300 cm ^-1) appears. After incubating the cells with an anti-tumor agent and heavy water for a period of time, if the cells are sensitive to the agent or the agent is effective, the metabolism of the cells is inhibited. In this case, the presence or absence and intensity of the C-D peak can reflect the metabolic activity of the cell, and thus it is known whether or not the drug is effective on the cell. Compared with fluorescent labeling, the Raman probe has the advantages of high stability (no light bleaching), near infrared excitation to avoid phototoxicity, small interference of a marker on a target (a source is a chemical bond), capability of labeling small molecules which are difficult to label by the fluorescent probe such as glycolipid, capability of performing super-polychromatic multiplexing (overlapping of fluorescence spectra) due to narrow spectrum peak, and the like.

In some aspects, detection of the drug-treated active tumor sections by coherent raman microscopy techniques can be performed as follows. The active tumor sections are incubated in the presence of the drug broth for a period of time and then in the presence of the raman probe-drug broth for an additional period of time. As used herein, the term "raman probe" refers to a probe used in detection by coherent raman microscopy imaging techniques, e.g., a metabolic characterization having raman spectral characteristics and low biotoxicity. Examples of raman probes suitable for use in the methods of the application include, but are not limited to, one or more of heavy water, deuterated palmitic acid, deuterated oleic acid, deuterated amino acids, deuterated glucose, deuterated cholesterol, and 3-O-propynyl-D-glucose. As used herein, the term "drug broth" refers to a culture medium obtained by adding a drug to be detected and a raman probe to a tissue culture medium. The raman probe-drug culture solution suitable for use in the method of the present application may contain, for example, heavy water in an amount of 40wt% or less, for example, 35wt% or less, 30wt% or less, etc., based on the total weight of the culture solution. In some aspects, the active tumor sections may be incubated in the presence of the drug broth for 0-72 hours followed by incubation in the presence of the raman probe-drug broth for 24-96 hours, and the sum of the two incubation times is 48-96 hours. Wherein, when an active tumor section is incubated for 0 hours in the presence of a drug culture solution, it means that the active tumor section does not need to be incubated in the presence of a drug culture solution, but is directly incubated in a raman probe-drug culture solution. In other aspects, the active tumor sections may be incubated in the presence of the drug broth for 18-30 hours followed by an additional incubation in the presence of the raman probe-drug broth for 40-50 hours. The metabolites of the raman probe in the drug-treated active tumor sections were then detected by coherent raman microscopy. In the method, after the medicine adding incubation is carried out for a period of time, the medicine adding incubation containing the Raman probe is carried out on the corresponding sample group, so that the medicine to be detected and the sample act in advance, if the medicine is effective, the metabolism of the sample is inhibited before the heavy water is added, and compared with the culture mode of adding the medicine and the heavy water at the same time, the contrast of the SRS detection medicine on the inhibition degree of the metabolism of the sample can be obviously improved. In some examples, the proportion of probe used when the sample is added to the heavy water culture is optionally within a certain range, for example, not more than 40wt%, not more than 35wt%, not more than 30wt%, etc., because too high a concentration of probe may cause some cytotoxicity, affect the activity of the sample and the length of time of culture, and reduce the success rate of culture.

Among them, the detection of metabolites of raman probes in active tumor sections treated with drugs can be performed as follows. In addition to the active tumor section of the drug-loaded culture described above, another active tumor section having the same thickness as the section is obtained from the active tumor sample. If the active tumor section of the drug-added culture is subjected to resuscitation, the other active tumor section is also resuscitated under the same resuscitation conditions. The other active tumor section was incubated under the same active conditions as the above-described dosing culture for the same time, except that the drug culture solution in the first section of incubation was replaced with tissue culture solution and the raman probe-drug culture solution in the second section of incubation was replaced with raman probe culture solution, thereby obtaining a control active tumor section. As used herein, the term "raman probe broth" refers to a culture medium obtained by adding raman probes to a tissue culture broth. Detecting metabolites of the Raman probe in the active tumor slices and the control active tumor slices which are subjected to dosing culture, and calculating the metabolic inhibition degree of the drug on the tumor so as to detect the drug sensitivity of the tumor on the drug.

In other aspects, in addition to the detection of metabolic activity based on raman metabolic markers (raman probes), tumor tissues and cells can be subjected to component analysis and imaging, i.e., imaging and analysis at different wavenumbers for biological components such as proteins, nucleic acids, and lipids; analytical criteria 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 to cell total area ratio, lipid droplet range lipid/protein concentration ratio, lipid component/protein component area ratio, lipid component/deoxyribonucleic acid component area ratio, lipid component to cell total area ratio, protein component to cell total area ratio, and lipid component range lipid/protein concentration ratio. The data can provide more dimensional tumor tissue information for detecting the metabolic activity of the tumor tissue, and help to judge the possible drug response of the tumor tissue more comprehensively and accurately. For example, in some aspects, detection of a drug-treated active tumor section by coherent raman microscopy can also be performed by analyzing intracellular and intercellular lipids and proteins in the drug-treated active tumor section to detect sensitivity of the tumor to the drug. For example, the intracellular and intercellular lipids may be the same or different and may include, but are not limited to, one or more of phospholipids, triglycerides, cholesterol esters, and glycolipids.

Alternatively, the nonlinear optical imaging technique used in the method of the present application may also be a second harmonic imaging technique. As used herein, the term "second harmonic imaging technique" is a modern nonlinear optical microscopy that uses the second harmonic signals generated when light interacts with a substance for microscopic imaging or detection. Second harmonic imaging is a new optical imaging technique developed in recent years, with high spatial resolution and high imaging depth that are characteristic of nonlinear optical imaging, and has received widespread attention as a new tool for biological structure detection and durable tracking marks. The second harmonic imaging technology avoids a plurality of inherent defects encountered by classical fluorescent probes, can avoid fluorescence bleaching effect in two-photon fluorescence imaging, and is a non-invasive living organism imaging method. Lesions in biological tissues often cause microstructure changes, so imaging can provide basis for diagnosis of diseases such as tumors. Through the second harmonic imaging, components such as collagen in the pathogenic tumor tissue of the tumor microenvironment and the structure can be completely reserved for imaging observation through simple slicing treatment, the content of the components, the structure and other factors can also be used as parameters for tumor drug sensitivity detection, and the combined metabolic inhibition detection can provide more accurate and reliable detection results. In some examples, detecting the drug-treated active tumor slice using second harmonic imaging techniques may include: tumor stem cells, collagen and/or cellular microtubules in active tumor sections treated with the drug are analyzed to detect the sensitivity of the tumor to the drug.

Alternatively, the nonlinear optical imaging technique used in the method of the present application may be a two-photon fluorescence imaging technique based on a fluorescent probe or a two-photon fluorescence imaging technique based on autofluorescence. As used herein, the term "two-photon fluorescence imaging technique" refers to a fluorescence imaging technique by excitation of two excitation photons; the term "fluorescent probe" refers to a class of fluorescent molecules that have characteristic fluorescence in the ultraviolet-visible-near infrared region and whose fluorescent properties (excitation and emission wavelengths, intensities, lifetimes, polarizations, etc.) can change sensitively with changes in the properties of the environment in which they are located, such as polarity, refractive index, viscosity, etc.; the term "autofluorescence" refers to the natural emission of light from a tissue or cell upon absorption of light of a range of wavelengths due to certain compounds present therein.

The two-photon fluorescence imaging technique based on fluorescent probes used in the method of the present application may also be referred to as a multiple immunofluorescence imaging technique of two-photon fluorescence. The immunofluorescence imaging technology is to label fluorescent pigment which does not affect the activity of antigen and antibody on antibody (or antigen), combine with the corresponding antigen (or antibody), and then present a specific fluorescence reaction under a fluorescence microscope, and image the biological tissue and the antigen in the cells through the fluorescence effect of the fluorescent pigment, if multiple fluorescent probes respectively label the specific biological antigens of different types of cells, different types of cells can be distinguished (the following diagram), if multiple fluorescent probes label different types of target protein molecules, the content and distribution of the target protein molecules in the cells can be imaged. Meanwhile, compared with the traditional single photon fluorescence imaging, the fluorescent probe group in the two-photon fluorescence imaging is excited by two excitation photons to show a fluorescence effect, and has the advantages of higher imaging resolution, smaller phototoxicity, deeper imaging depth and the like. In some examples, detecting the drug-treated active tumor section using a fluorescent probe-based two-photon fluorescence imaging technique may include: and marking the active tumor section treated by the drug by using a fluorescent probe, detecting the cell type, subclass, specific protein 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 constitution and distribution and a single cell metabolism detection map.

In addition, the method of the present application may also use two-photon fluorescence imaging techniques based on autofluorescence. Autofluorescence is the light that biological structures (e.g., mitochondria and lysosomes) emit naturally as they absorb light, and is used to distinguish light from artificially added fluorescent labels (fluorophores). Autofluorescence imaging is distinguished from fluorescence imaging based on fluorescent probes, which directly image fluorescence from both the organism itself and the drug source. By autofluorescence imaging, the content and distribution of components and molecules having autofluorescence properties in biological tissues and cells, as well as the intensity of biological reactions, can be detected. Therefore, by detecting the subcellular distribution of the tumor tissue itself or the autofluorescent substance derived from the drug in real time, the local biomolecule changes caused by the drug are clarified, and the reactions of different subcellular regions are distinguished, so that more dimensional data analysis can be provided for tumor drug sensitivity detection. In some examples, detecting the drug-treated active tumor section using an autofluorescence-based two-photon fluorescence imaging technique can include: subcellular distribution with autofluorescent substances in active tumor sections is imaged and detected based on autofluorescent signals in active tumor sections treated with drugs to detect sensitivity of tumors to drugs.

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

Examples

Embodiments of the present application will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present application and should not be construed as limiting the scope of the present application. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. All amounts listed are described in parts by weight or percent by weight based on total weight unless otherwise indicated. The application should not be construed as being limited to the particular embodiments described. Although specific examples of the application have been described above for illustrative purposes, it will be apparent to those skilled in the art that various changes in detail may be effected therein without departing from the spirit and scope of the application as defined in the appended claims.

Reagent:

Carrying and delivering liquid: a1 wt% penicillin-streptomycin double antibody solution (ELISA) was added to a sterile DMEM/F21 medium (Merck), and a DMEM/F21 medium containing 1wt% penicillin-streptomycin double antibody was prepared and stored as a transport fluid under sterile and refrigerated conditions.

Tissue culture solution: a medium containing 90wt% DMEM/F12 (containing 1wt% penicillin-streptomycin diabody) +10wt% fetal bovine serum was prepared by adding fetal bovine serum (GIBCO) to a DMEM/F21 medium containing 1wt% penicillin-streptomycin diabody, and stored as a tissue culture broth under aseptic and refrigerated conditions.

Wrapping medium: agarose (SIGMA ALDRICH) was dissolved in distilled water to prepare a 3wt% agarose solution, sterilized and refrigerated for use as a coating medium.

Drug culture solution: the drug or the drug combination to be tested is dissolved in the tissue culture solution, so that the drug concentration is C _max (highest concentration of blood drug) of single drug in the drug or the drug combination and a plurality of drug concentrations with 10 times gradient on two sides of the drug or the drug combination respectively, and the drug culture solution is prepared.

Raman probe culture solution: the tissue culture solution is mixed with heavy water to obtain a mixture containing the heavy water of the tissue culture solution as a Raman probe culture solution (hereinafter referred to as a heavy water culture solution).

Raman probe-drug culture solution: the drug or combination of drugs to be tested is dissolved in the raman probe culture liquid so that the concentration of the drug therein is the same as that of the corresponding drug culture liquid to prepare a raman probe-drug culture liquid (hereinafter referred to as a weight water-drug culture liquid).

Fixing solution: as the fixing solution, 4wt% paraformaldehyde tissue fixing solution (Absin) was used.

All of the above reagents are prepared or sterilized under aseptic conditions and stored and used under aseptic conditions.

Instrument:

Slicing machine: leika VT1000S

Incubator: thermo 3111

Nonlinear optical microscopy imaging platform: ultralView nonlinear optical microscopy imaging platform, manufacturer: vibrating electric (su state) medical science and technology limited, brand: vibroniX A

Confocal fluorescence microscope: ultralView confocal fluorescence microscope, manufacturer: vibrating electric (su state) medical science and technology limited, brand: vibroniX A

Transwell plates: screening, numbering, 14212, cell culture chamber, 24 well plate (PET membrane, 6.5mm, pore size 0.4 μm), membrane material: PET (Polyester) A

Culture conditions:

Temperature: 37 DEG C

Pressure: atmospheric pressure

Atmosphere: 95% air+5% CO ₂

Example 1: SRS microscopic imaging detection for tissue slice samples of different thicknesses

To a50 mL sterile centrifuge tube was added 30mL of a shipping solution and cooled to 0 ℃. Samples of 3cm ³ volume were taken on freshly resected bladder cancer tumor tissue to ensure tissue representation and viability, ensuring that the sampling site was far from the cutting edge and unaffected by the burn. The tissue is prevented from being poked by a sharp tool in the extraction process, and the tissue is prevented from being excessively pinched. Immediately after the sample is extracted, the sample is placed in the transport fluid within the centrifuge tube, and the tube is then sealed. The sealed centrifuge tube is stored in a container containing ice cubes and then sent to a testing laboratory. The sample transfer process took 2 hours.

The 3wt% agarose solution was heated to clear and then cooled to 40℃and then poured into a slice tray. Tissue pieces of size 5mm ³ were removed from the preserved samples and the pieces were completely immersed in agarose solution in a slice tray and allowed to stand until coagulated. The coagulated mass is removed and cut into rectangular solid tissue clots that completely encapsulate the tissue mass. The tissue clot was loaded onto a microtome, submerged with PBS, and then carefully cut into thin and flat sections with thicknesses of 50 μm, 100 μm, 200 μm and 300 μm, respectively.

500. Mu.L of tissue culture medium was added to a Transwell plate, wherein 450. Mu.L was added to the lower layer and 50. Mu.L was added to the upper layer, and the mixture was placed in an incubator for preheating. The sections prepared as above were placed in the culture solution of the preheated Transwell plate, respectively, and three-dimensional culture was performed in an incubator for 12 hours for resuscitation.

Then, the tissue culture solution in the Transwell plate was replaced with a heavy water culture solution containing 70wt% of the tissue culture solution and 30wt% of heavy water (no drug was added thereto), and resuscitated sections were continued for 72 hours of stereoscopic culture. The sections were removed and fixed with a fixing solution, respectively.

And respectively performing SRS microscopic imaging on the fixed sections by using an SRS mode of the nonlinear optical microscopic imaging platform. For each slice, at least 9 representative field-of-view images were acquired at the same wavenumber channel to ensure that the detection results were representative. CHP and CHL signals were acquired at 2930cm ^-1 and 2850cm ^-1, respectively; CDP and CDL signals were acquired at 2177cm ^-1 and 2135cm ^-1, respectively; and non-resonance (Off resonance) data was acquired at 1902cm ^-1 wavenumbers to remove non-resonance signals during later data processing.

SRS microscopy imaging images were processed and presented using ImageJ software. Fig. 1 (a) to (D) show thicknesses of: (A) 100 μm; (B) 50 μm; (C) 200 μm; and (D) imaging images of 300 μm slices. The CHP and CHL channel signals in the figure reflect the hydrocarbon signal intensity of tissues and cells, the grey-white color of the non-black background in the figure is the hydrocarbon signal, the brighter the signal is, the stronger the signal is, the CHP is the hydrocarbon protein signal, and the CHL is the hydrocarbon lipid signal; the same CDP and CDL channels react with the carbon deuterium signal intensity of tissues and cells, namely, the cell components which are newly synthesized and contain carbon deuterium chemical bonds after the tissues are cultured by a culture medium containing heavy water calibration metabolites, the off-white color of a non-black background in the figure is the carbon deuterium signal, the brighter the signal is, the stronger the signal intensity can reflect the metabolic intensity of the tissue cells. As can be seen from FIGS. 1A-1D, the signals of proteins (CHP and CDP) and lipids (CHL and CDL) in cells are clearly shown in the images of 4 different thickness sections. By using the global signal pixel density value (Total density) function in ImageJ software, quantitative statistics can be performed on the signals, thereby detecting the metabolic Intensity of tissue cells based on the statistical data.

As can be seen from fig. 1A to D, when the slice thickness is 100 μm (a), the SRS microscopic imaging background signal is weak, and the imaging effect is optimal; when the slice thickness is 50 μm (B), the relative background signal of the image is slightly stronger; and when the slice thickness is 200 μm (C) and 300 μm (D), a good imaging effect can be obtained.

Example 2: tissue activity by culture with medium containing different concentrations of heavy water

To a 50mL sterile centrifuge tube was added 30mL of a shipping solution and cooled to 0 ℃. Samples of 3cm ³ volume were taken on freshly resected bladder cancer tumor tissue, ensuring that the sampling site was away from the cutting edge and unaffected by the burn. The tissue is prevented from being poked by a sharp tool in the extraction process, and the tissue is prevented from being excessively pinched.

The 3wt% agarose solution was heated to clear and then cooled to 40℃and then poured into a slice tray. Tissue pieces of size 5mm ³ were removed from the extracted samples and the pieces were completely immersed in agarose solution in a slice tray and allowed to stand until coagulated. The coagulated mass is removed and cut into rectangular solid tissue clots that completely encapsulate the tissue mass. The tissue clot was loaded onto a microtome, submerged with PBS, and then carefully cut into thin and flat sections 100 μm thick.

Heavy water culture solutions with different concentrations are prepared, wherein the concentration of the heavy water is 30wt%, 40wt% and 50wt% respectively.

Mu.L of tissue culture solution and heavy water culture solution with the concentration of 30wt%, 40wt% and 50wt% of heavy water are respectively added into a Transwell plate, wherein 450 mu.L of each culture solution is added into the lower layer, and 50 mu.L of each culture solution is added into the upper layer. The Transwell plates were placed in an incubator for preheating. Sections prepared as above were subjected to fluorescent staining using DAPI (4', 6-diamidino-2-phenylindole), PI (propidium iodide) and Calcein (Calcein), respectively, and placed in different culture solutions of a preheated Transwell plate, and subjected to three-dimensional culture in an incubator for 48 hours. The sections were imaged separately at time points of incubation for 0, 12, 24 and 48 hours using confocal fluorescence microscopy. Images under different fluorescent signals were processed and displayed using ImageJ software.

FIG. 2 shows fluorescence images of individual and fused samples after incubation for 0, 12, 24 and 48 hours using DAPI, PI and Calcein staining, where the image signal intensity of PI staining is proportional to the degree of apoptosis; the intensity of the image signal of Calcein staining is proportional to the degree of cell activity. As can be seen from fig. 2, the degree of apoptosis gradually increases and the degree of cell activity gradually decreases with increasing heavy water concentration.

Example 3: tissue activity for different culture duration using heavy water medium containing 30% concentration

A heavy water culture broth was prepared, in which the heavy water concentration was 30wt%.

Mu.L of tissue culture solution and heavy water culture solution were added to each of the Transwell plates, respectively, wherein 450. Mu.L of each culture solution was added to the lower layer and 50. Mu.L of each culture solution was added to the upper layer. The Transwell plates were placed in an incubator for preheating. Sections prepared as above were stained with DAPI, PI and Calcein, respectively, and placed in different culture solutions of a preheated Transwell plate for 96 hours of stereoscopic culture in an incubator. The sections were imaged separately at time points of incubation for 0, 24, 48, 72 and 96 hours using confocal fluorescence microscopy. Images under different fluorescent signals were processed and displayed using ImageJ software.

FIG. 3 shows separate and fused fluorescence images of samples stained with DAPI, PI and Calcein after incubation for 0-96 hours. As can be seen from fig. 3, the degree of apoptosis gradually increases and the degree of cell activity gradually decreases over time.

Example 4: SRS microscopy imaging results using sections cultured with different concentrations of drug

Different concentrations of gemcitabine + cisplatin were used as the drug to be tested. Preparing a medicine culture solution, a heavy water culture solution and a heavy water-medicine culture solution with different medicine concentrations, wherein the medicine concentrations in the medicine culture solution and the heavy water-medicine culture solution correspond to each other in pairs, and are respectively 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), gemcitabine (200 μm) +cisplatin (100 μm); in addition, the concentration of heavy water in both the heavy water culture solution and the heavy water-drug culture solution was 30wt%.

The Transwell plate was removed, the tissue culture solution therein was replaced with 500. Mu.L of fresh tissue culture solution and 500. Mu.L of drug culture solution of different drug concentrations, respectively, and each slice was continued for 24 hours of drug culture. And taking out the Transwell plate, and respectively replacing the culture solution in the Transwell plate with the corresponding heavy water culture solution and the corresponding heavy water-medicine culture solution, and continuing to culture for 48 hours. The sections were removed and fixed with a fixing solution, respectively.

Fig. 4A-B show SRS microimaging images of a drug added probe sample and a Control sample (Control), respectively, wherein (a): single drug concentration different region assay (B): comparison of metabolic differences between different drug concentrations. The CHP and CHL channel signals in the figure reflect the hydrocarbon signal intensity of tissues and cells, the grey-white color of the non-black background in the figure is the hydrocarbon signal, the brighter the signal is, the stronger the signal is, the CHP is the hydrocarbon protein signal, and the CHL is the hydrocarbon lipid signal; the same CDP and CDL channels react with the carbon deuterium signal intensity of tissues and cells, namely, the cell components which are newly synthesized and contain carbon deuterium chemical bonds after the tissues are cultured by a culture medium containing heavy water calibration metabolites, the off-white color of a non-black background in the figure is the carbon deuterium signal, the brighter the signal is, the stronger the signal intensity can reflect the metabolic intensity of the tissue cells.

SRS microscopy imaging images were processed and presented using ImageJ software. Counting the quantitative data of the integral signal pixel density values (Total density) of the CHP/CHL and CDP/CDL channels through the integral signal pixel density value (Total density) function of imageJ software; dividing the Carbon Deuterium (CD) signal pixel density value of the same drug concentration, the same imaging region and the same imaging channel by the hydrocarbon (CH) signal pixel density value to obtain the metabolic intensity proportion of the region under the drug concentration; drug concentration-dependent metabolic inhibition image fitting was performed using Graphpda software.

Fig. 5 shows the change in D ₂ O metabolism with drug activity and the determination of drug sensitivity or drug resistance made therefrom, as a result of processing and data analysis from the SRS microscopy image shown in fig. 4. Wherein the x-axis represents drug concentration, respectively: c (Control ) -non-dosed; 10 ^-1 -0.2 μm gemcitabine+0.1 μm cisplatin; 10 ⁰ -2 μm gemcitabine+1 μm cisplatin; 10 ¹ -20 μM gemcitabine+10 μM cisplatin; 10 ² -200 μM gemcitabine+100 μM cisplatin; the y-axis represents the ratio of metabolic intensities. As can be seen from fig. 5, the ratio of metabolic intensities shows different changes as the concentration of the drug increases, wherein the error bars show the difference in ratio of metabolic intensities in different regions at the same concentration of the drug. The effectiveness of a drug or combination of drugs for the treatment of the patient can be determined based on the shape of the metabolic inhibition curve, the slope of the curve, the degree of difference in the metabolic intensity ratio values between different drug concentrations, the IC ₅₀ value, the result comparison analysis cut-off value (cut-off), the area under the curve, and other parameters.

Example 5: SRS microscopy imaging results from section cultures of different tumor samples 30mL of transport fluid was added to a 50mL sterile centrifuge tube and cooled to 0 ℃. Samples of 3cm ³ volume were taken on freshly resected bladder cancer tumor tissue from two different cases, ensuring that the sampling site was far from the cutting edge and unaffected by the burn. The tissue is prevented from being poked by a sharp tool in the extraction process, and the tissue is prevented from being excessively pinched. Wherein sample a and sample B are from different cases of PT1 bladder cancer.

Gemcitabine + cisplatin (GC) was used as the drug to be tested. Preparing a medicine culture solution, a heavy water culture solution and a heavy water-medicine culture solution with different medicine concentrations, wherein the medicine concentrations in the medicine culture solution and the heavy water-medicine culture solution correspond to each other in pairs, and are respectively as follows: 0.2 mu M gemcitabine+0.1 mu M cisplatin, 2 mu M gemcitabine+1 mu M cisplatin 20 mu M gemcitabine+10 mu M cisplatin 200 mu M gemcitabine +100 μM cisplatin; in addition, the concentration of heavy water in both the heavy water culture solution and the heavy water-drug culture solution was 30wt%.

Fig. 6A-B show SRS microscopy imaging images of sample a and sample B, respectively, after drug addition culture. As shown in fig. 6A-B, although the responses of sample a and sample B to the drug were not the same as in stage PT1 of bladder cancer, SRS images clearly showed this difference. Example 6: SRS microscopy imaging results for section culture using different drugs

The method adopts the following steps: (i) gemcitabine + cisplatin (GC); and (ii) methotrexate + vinblastine + doxorubicin + cisplatin (MVAC) as the drug to be tested, and using non-medicated tissue culture broth and heavy water culture broth as controls. Wherein, the concentration of the test drug is as follows:

In addition, the concentration of heavy water in both the heavy water culture solution and the heavy water-drug culture solution was 30wt%.

The Transwell plate was removed, the tissue culture solution therein was replaced with 500. Mu.L of fresh tissue culture solution and 500. Mu.L of drug culture solution of different drugs and concentrations, respectively, and each section was continued for 24 hours of drug culture. And taking out the Transwell plate, and respectively replacing the culture solution in the Transwell plate with the corresponding heavy water culture solution and the corresponding heavy water-medicine culture solution, and continuing to culture for 48 hours. The sections were removed and fixed with a fixing solution, respectively.

Fig. 6C-D show SRS microscopy imaging images after drug addition culture of sections using GC and MVAC as test drugs, respectively. As shown in fig. 6C-D, the drug sensitive detection method of the present application can be applied to different drugs/drug combinations.

From the results of the SRS microscopic imaging and data analysis shown in fig. 6A to D, the method of the present application can quantitatively detect the small metabolic inhibition difference based on the advantages of high spatial-temporal resolution and high chemical sensitivity of the SRS system, so that the clinical sample cultured in short time and heavy water can be used for the detection of the method, the sample can be simply processed, the complete pathogenic tumor microenvironment can be maintained, and meanwhile, the small factor reagent and the relevant tumor cell line are not required to be added in the culture process due to the short culture time. The tumor microenvironment is completely reserved, so that the detection result is more accurate; because the culture time is short the success rate of the culture is high due to detection; the culture and detection cost is low because small factor reagents and related tumor microenvironment cell lines are not needed to be added and the culture success rate is high. The method has the advantages that the tumor microenvironment is completely reserved, and the disease-source tumor can be better characterized, so that the accuracy of the detection result is improved. For example, two cases shown in fig. 6A-B are the same as the PT1 bladder cancer, but the histomorphology, cell type composition and metabolic characteristics of the tumor tissue of the two cases are greatly different, and the differences can lead to different characteristics of the biochemistry and physiology of the tumor cells and the microenvironment thereof, even though the treatment feedback of the same drug or drug combination may be completely different for the same tumor cases in the same period, so that the tumor microenvironment retention of the tumor drug-sensitive detection sample is very important for accurate and effective detection results. Based on the above, accurate and effective treatment can be realized for different cases (including tumors of different patients or tumors of the same patient in different periods, etc.).

Examples 1 to 6 were examined for drug sensitivity of tumors by measuring differences in cell metabolism inhibition by SRS microscopy. However, it will be appreciated that SRS microscopy can also be used to measure other markers in cells, for example, to analyze intracellular lipids and proteins and intercellular lipids and proteins in active tumor sections to detect drug sensitivity of tumors.

Example 7: detection using second harmonic imaging

Active tumor sections were prepared using methods similar to those described in example 3 and resuscitated and medicated cultures were performed. SRS microscopy imaging was performed using SRS mode of a nonlinear optical microscopy imaging platform, and collagen images in tumor tissue samples were detected by Second Harmonic (SHG) imaging mode. The detection parameters of the second harmonic imaging are as follows: excitation wavelength 796nm, acceptance wavelength 395nm. The imaged image was processed and presented using ImageJ software.

Fig. 7 shows SRS microscopic imaging results and a Collagen (right-most column) signal image obtained in a second harmonic mode, wherein the grey-white color of a non-black background in the image is a Collagen signal, and the brighter the signal in the image is, the stronger the brighter the signal in the image is, the dimensions such as the Collagen component content (brightness/area) and the morphology (uniformity/group absorption) reflected by the image can be analyzed, so that support information of tumor tissue drug sensitivity is provided.

Example 8: detection using two-photon fluorescent probe mode

To a 50mL sterile centrifuge tube was added 30mL of a shipping solution and cooled to 0 ℃. Samples of 3cm ³ volume were taken on freshly resected hepatoma tissue, ensuring that the sampling site was far from the cutting edge and unaffected by the burn. The tissue is prevented from being poked by a sharp tool in the extraction process, and the tissue is prevented from being excessively pinched.

And (3) performing double staining on the cut sections by using HCC (liver cancer) cells (fluorescent probes: AFP) +CD8+ T cells (fluorescent probes: CD 8), then respectively imaging by using SRS and two-photon excited fluorescence (TPEF) modes of a nonlinear optical microscopic imaging platform, obtaining neutral lipid images in situ based on the SRS imaging mode, and obtaining liver cancer cells and killer T cells (CD8+ T cells) based on APF and CD8 in situ in the two-photon fluorescence mode. The detection parameters are as follows: in a two-photon fluorescence mode, performing Dapi, GATA3, AFP and CD8 immunofluorescence imaging under the conditions that the two-photon excitation/emission wavelengths are 750nm/450nm, 800nm/530nm, 1080nm/590nm and 1250nm/690nm respectively; 2850cm ^-1 CHL signal in SRS mode. Images in SRS mode and fluorescence mode were processed and presented using ImageJ software.

As can be seen from fig. 8, by performing stacking fusion on fluorescence images obtained in situ from the same cell living tissue region, in-situ spatial analysis of images under multiple modes is performed, so as to reveal the correlation between two-photon fluorescence probe-labeled Tumor Microenvironment (TME) image information and lipid and other metabolite content information in spatial positions; the correlation of the intensity, the area, the overlapping, the proportion and the like of fluorescence or SRS signals can be analyzed by respectively selecting corresponding ROIs by utilizing signal thresholds in software; so as to obtain data about cell types, subclasses and spatial distribution of the active tumor sections, and obtain correlation between the data and tumor tissue drug sensitivity based on the detected intratumoral microenvironment images containing cell constitution and distribution and single cell metabolism detection patterns.

Example 8 drug sensitivity of tumors was tested by detecting cell types, subclasses, specific proteins and their spatial distribution in active tumor sections by two-photon fluorescence probe mode. However, it will be appreciated that two-photon autofluorescence modes can also be used to measure these and other markers of self-or drug-derived autofluorescence in tumor tissue, for example, to analyze intracellular proteins, lipids, drugs and drug-induced autofluorescence and spatial distribution in active tumor sections to detect drug sensitivity of tumors.

According to the experiment, the tumor drug sensitivity detection method is used for processing and culturing samples based on the pathogenic tumor living tissue, imaging based on nonlinear optical imaging technologies such as coherent Raman, second harmonic and two-photon fluorescence signals, and the like, and can be used for rapidly, accurately and efficiently detecting tumor drug sensitivity based on various microscopic imaging modes, so that powerful technical support and guidance are provided for accurate medical treatment/individuation treatment of tumors.

The present application has been fully disclosed and described in accordance with the representative embodiments described above. Those skilled in the art will recognize that various modifications may be made to the present application without departing from the spirit and scope of the application. The terms used in the present application should be understood to have their ordinary meanings in the art; in case of conflict, the present application is defined or interpreted in control.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

It is specifically contemplated that any of the limitations discussed in relation to one embodiment of the present invention may be applied to any other embodiment of the present invention. Furthermore, any of the compositions of the present invention may be used in any of the methods of the present invention, and any of the methods of the present invention may be used in making or using the compositions of the present invention. In particular, any aspect described in the claims, alone or in combination with one or more aspects of the claims and/or the specification, is to be understood as being combinable with other aspects of the invention set forth elsewhere in the claims and/or the specification.

Claims

1. A method of drug sensitive detection of a tumor for non-therapeutic, non-diagnostic purposes, the method comprising:

Obtaining a first active tumor section having a thickness between 75 μm and 125 μm based on the active tumor sample;

Incubating the first active tumor section for a first time period in the presence of a drug to obtain a drug-treated active tumor section, wherein the first time period is 24-96 hours;

Detecting the active tumor slices treated by the drug by nonlinear optical imaging technology to detect the drug sensitivity of the tumor to the drug,

Wherein the nonlinear optical imaging technique is a coherent Raman microscopic imaging technique,

Incubating the first active tumor section in the presence of a drug comprises: incubating the first active tumor section in the presence of a drug culture solution for a second time period, and then incubating in the presence of a raman probe-drug culture solution for a third time period, wherein the drug culture solution comprises a drug and a tissue culture solution, the raman probe-drug culture solution comprises the drug, a raman probe and the tissue culture solution, the second time period is 0-72 hours, the third time period is 24-96 hours, and the sum of the second time period and the third time period is equal to the first time period;

Detecting the drug-treated active tumor section by nonlinear optical imaging technique comprises:

Based on the active tumor sample, obtaining a second active tumor section having the same thickness as the first active tumor section;

Incubating the second active tumor section in the tissue culture solution for the second period of time, and then incubating the second active tumor section in a raman probe culture solution for the third period of time to obtain a control active tumor section, 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 section and the control active tumor section, calculating a degree of metabolic inhibition of the drug on the tumor to detect drug sensitivity of the tumor to the drug;

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-propynyl-D-glucose.

2. The method of claim 1, wherein after obtaining the first active tumor section and prior to incubating the first active tumor section in the presence of a drug, the method further comprises:

resuscitating the first active tumor section; and

Resuscitating the second active tumor section,

Wherein the resuscitation of the first active tumor section and the manner of the second active tumor section are performed under the same resuscitation conditions.

3. The method of claim 1, wherein the second duration is 18-30 hours and/or the third duration is 40-48 hours.

4. The method of claim 1 or 2, wherein the coherent raman microscopy imaging technique is one or more selected from the group consisting of stimulated raman scattering, coherent anti-stokes microscopy imaging techniques, and surface enhanced raman spectroscopy imaging techniques.

5. The method of claim 1, wherein obtaining a first active tumor slice having a thickness between 75 μιη and 125 μιη based on the active tumor sample comprises:

The active tumor samples were obtained from fresh ex vivo tumor tissue,

Preserving said active tumor sample under conditions that preserve tumor tissue activity,

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, diatomaceous earth, or a combination thereof,

The wrapped active tumor sample is cut into the first active tumor section having a thickness between 75 μm and 125 μm.

6. The method of claim 1 or 5, wherein the active tumor sample is fresh ex vivo tumor tissue and the ex vivo time of the fresh ex vivo tumor tissue is less than or equal to 30 minutes.

7. The method of claim 6, wherein the ex vivo time of the fresh ex vivo tumor tissue is less than or equal to 15 minutes.

8. The method of claim 7, wherein the interval between the time the fresh ex vivo tumor sample is removed and the time the first active tumor section is obtained is less than or equal to 8 hours.

9. The method of claim 1 or 5, wherein the active tumor sample retains at least a portion of the intratumoral microenvironment of the tumor.

10. The method of claim 2, wherein the resuscitating comprises: and carrying out three-dimensional culture for 6-18 hours at the temperature of 35-38 ℃ under the condition of normal oxygen.

11. The method of claim 1 or 2, wherein the tumor is a solid tumor.

12. The method of claim 11, wherein 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.