KR100892980B1 - Method of screening for anticancer agent or method of anticancer treatment using calcein-AM in multicellular layer - Google Patents

Abstract

The present invention relates to a method for screening an anticancer substance or anticancer treatment method using calcein-AM in a multicellular cancer cell (multicellular layer, MCL), specifically, one of the screening systems for the discovery of anticancer candidates effective for solid cancer The anticancer agent was treated by the anticancer candidate substance or candidate anticancer treatment method in the MCL model, followed by successive treatments with calcein-AM (calcein-acetoxymethyl ester), and by measuring the fluorescence intensity distribution of calcein-AM that has passed through the MCL. A method of screening a substance or anticancer treatment method. Using the method of the present invention can know the tissue permeability and anticancer effect of the anticancer candidate, it can be usefully used to search for or evaluate the efficacy of a new anticancer or anticancer therapy for solid cancer.

Solid cancer, multicellular layer of cancer cell, colorectal cancer cell line (DLD-1), calcein-AM (calcein-acetoxymethyl ester)

Description

Method of screening for anticancer agent or method of anticancer treatment using calcein-AM in multicellular layer}

1 is a diagram illustrating a chamber for culturing a multicellular layer (MCL).

Figure 2 is a graph showing the growth of colorectal cancer cell line (DLD-1) cultured with MCL.

3 is a diagram showing the growth of colorectal cancer cell line (HT29) cultured with MCL.

4 is a graph showing fluorescence density according to calcein-AM concentration and cell number.

5 is a photograph showing the distribution of calcein-AM cultured at room temperature in DLD-1 cultured with MCL:

    A: phase difference image of DLD-1; And

    B: Distribution of calcein-AM of DLD-1.

Figure 6 is a photograph showing the distribution of calcein-AM cultured in a pre-culture method of colorectal cancer cell lines (HT29 and DLD-1) cultured with MCL:

    A: phase difference image of HT-29;

    B: distribution of calcein-AM of HT-29;

    C: phase difference image of DLD-1; And

    D: distribution of calcein-AM of DLD-1;

7 is a graph measuring the fluorescence density of fragments of various depths from the surface of calcein-AM cultured at room temperature in DLD-1 cultured with MCL.

FIG. 8 is a graph measuring the fluorescence density of fragments of various depths from the surface of calcein-AM cultured by cooling pre-culture method in colorectal cancer cell lines (HT29 and DLD-1) cultured with MCL:

Figure 9 is a photograph showing the distribution of DOX and calcein-AM after exposure to DOX (Doxorubicin) and calcein-AM in DLD-1 incubated with MCL:

    A: phase difference image;

    B: distribution of DOX; And

    C: distribution of calcein-AM.

10 is a photograph showing the calcein-AM distribution after PTX-Rd (Paclitaxel-rhodamine) and calcein-AM were exposed to HT-29 incubated with MCL:

    A: distribution of calcein-AM prior to PTX-Rd treatment in HT-29;

    B: distribution of calcein-AM after PTX-Rd treatment in HT-29; And

    C: Distribution of calcein-AM before and after PTX-Rd treatment in HT-29.

11 is a photograph showing the distribution of calcein-AM after exposure to PTX-Rd (Paclitaxel-rhodamine) and calcein-AM in DLD-1 cultured with MCL:

    A: distribution of calcein-AM prior to PTX-Rd treatment in DLD-1;

    B: distribution of calcein-AM after PTX-Rd treatment in DLD-1; And

    C: Distribution of calcein-AM before and after PTX-Rd treatment in DLD-1.

12 is a photograph showing the distribution of PTX-Rd over time after PTX-Rd (Paclitaxel-rhodamine) and calcein-AM were exposed to HT-29 and DLD-1 incubated with MCL.

The present invention relates to a screening method of an anticancer substance or an anticancer treatment method using calcein-AM in a multicellular cancer cell (multicellular layer, MCL), and more particularly in a screening system for the discovery of an anticancer candidate substance effective for solid cancer. After treatment with a candidate anticancer agent or a candidate anticancer treatment method in one MCL model, and subsequently treated with calcein-AM (calcein-acetoxymethyl ester), measuring the fluorescence intensity distribution of calcein-AM that has passed through the MCL The present invention relates to a method for screening an anticancer substance or an anticancer treatment method.

The key to preclinical studies to develop anticancer drugs or new chemotherapy is to accurately and quickly assess anticancer activity. Although the in vitro laboratory system is economical and superior to the in vivo laboratory system in terms of cost and speed for developing new anticancer drugs and chemotherapy, the efficacy of the efficacy evaluation in the in vitro laboratory system is improved. It depends on whether I can fully represent my situation and demonstrate clinically meaningful efficacy. To date, about 90% of anti-cancer candidates that have entered clinical trials through the preclinical development phase have failed in final development because of the lack of adequate experimental systems that have failed to identify compounds with potential for clinical efficacy (Staquet MJ et. al., Cancer Treat Rep : 67, 753-765, 1983; Twentyman PR, Ann Oncol : 5, 394-396, 1994; von Hoff DD, Clin Cancer Res : 4, 1079-1086, 1998).

In vitro chemosensitivity assays which are widely used to date include colony assay, dye exclusion assay, and tetrazolium salt 3- (4,5-dimethylthiazol- 2-yl) -2,5-diphenyl tetrazolium bromide assay or sulforhodamine B assay. The above method is meaningful as a primary screening system that requires high-throughput in anticancer drug development, but conditions are very different from in vivo conditions of human solid cancer, and it is difficult to predict the reaction rate for solid cancer. . In the case of solid cancer, the low response rate to chemotherapy is due to the three-dimensional organization of solid cancer cells and the interaction between cells and extracellular matrix (ECM) resulting from the multicellular system. It is known to induce drug resistance due to various mechanisms such as lowered penetration of cancer drug and lower concentration in tissue (Wolfgang MK, Crit Rev Oncol Hematol : 36, 123-139, 2000; Bernard; D. et al., Crit Rev Oncol Hematol: 36, 193-207, 2000).

In order to solve the problems of the anticancer drugs due to the drug penetration disorder of the solid cancer cells and the structural characteristics of the cancer cells, many scholars have developed an in vitro experimental system model that reflects the characteristics of solid cancers that cause the resistance of solid cancers in vivo. We tried to predict the clinical results more accurately as a preclinical efficacy screening system for anticancer candidates.

As an early model, a spherical multicellular spheroid (MCS) model was first reported by Inch et al. (Inch WR et al., Growth : 34, 271-282, 1970). This experimental system focuses on the differences in the concentration of essential ingredients, oxygen partial pressure, drug concentration, cell growth rate, and growth fraction according to cell sites. It is an experimental system mainly used for the expression of molecular genetic factors related to drug efficacy in solid cancer. Later, a multicellular layer (MCL) was developed for the first time by Cowan et al. (Cowan DS et al., Br J Cancer Suppl : 27, S28-31, 1996), and has been modified or simplified by many scholars. It has been usefully used in studies such as a comparison between drug penetration, drug metabolism and drug inactivation.

Since Cowan et al. Reported for the first time that cancer cell permeability of DNA intercalators was low using MCL in the late 1990s, Tannock et al. These expressions were similar and the tissue permeability of anticancer drugs in MCL was specific for cell lines and drugs (Tannock IF et al., Clin Cancer Res : 8, 878-884, 2002). Phillips et al. Also found that MCL permeability varies depending on the drug, and one of the factors that influence this is metabolism of intracellular drugs (Phillips RM et al., Br J Cancer : 77, 2112-2119, 1998). Hicks et al. Used MCL to demonstrate that the reason for the impairment of penetration of DAPA, one of the DNA intercalators, was drug capture in lysosomes (Hicks KO et al., Br J Cancer : 76, 894-903, 1997). . In addition, Hicks et al. Mathematically modeled MCL permeation and combined it with in vivo pharmacokinetics (PK) models to predict drug concentrations in cancer tissues in vivo, leading to excellent DNA intercalators. It has been reported that it can be applied to the design (Hicks KO et al., J Pharmacol Exp Ther: 297, 1088-1098, 2001), and it is hypothaxin, a tyra pazamine, which is active at the hypoxic part of the solid cancer tissue. Based on the results of studies that the activity of (tirapazamine) decreases as the penetration distance into the cancer tissue increases, in vitro MCL permeation model-in vivo PK-vascular structure in actual cancer tissue A model was developed to predict drug concentrations in cancer tissues (Hicks KO et al., Int J Radiat Oncol Biol Phys : 42, 641-649, 1998). In other words, by suggesting the characteristics of excellent hypotoxin candidates, we demonstrated the possibility of MCL as an information feedback method in drug development (Hicks KO et al., Cancer Res : 15, 5970-5977, 2003; Hicks). KO et al., American Association for Cancer Research, 2003 Jul 11-14; Wilson WR, American Association for Cancer Research , 2003 Jul 11-14).

Since the evaluation of the permeation and post-permeation activity of solid cancer tissues of anticancer drugs using the above methods is a method of directly quantifying the concentration of the drug, there was a great difficulty in terms of cost and time for the compounds to be screened at the stage of drug development. Therefore, if the present inventors apply a new bioassay that directly evaluates anticancer activity after tissue permeation of the anticancer agent, some disadvantages of the existing MCL method will be compensated for, and the economics and applicability of the anticancer drug development will be greatly increased. As a screening system for discovering effective anticancer candidates for solid cancer, MCL model using human colon cancer cell line DLD-1 can be directly assessed for anticancer activity after tissue penetration of anticancer agent by cytotoxicity assay. A method for measuring the activity of anticancer substances designed to be developed was developed (Korean Patent No. 0689107).

On the other hand, calcein-AM (calcein-AM) is a hydrophobic dye that identifies viable cells and shows fluorescence from calcein (calcein) that passes through the cell membrane and is released by the esterase present in the cell (B Jonsson, et al ., European Journal of Cancer , 32A (5), 883-997, 1996).

Accordingly, the present inventors selected paclitaxel and doxorubicin, which are frequently used in clinical practice, as models of anticancer drugs, and their changes in cancer tissue permeation distribution and calcein-AM (calcein-acetoxymethyl ester) tissue distribution coincide. Since the distribution of calcein-AM may represent cell viability in cancer tissues induced under anticancer treatment (an anticancer treatment or other anticancer treatment such as radiation), it may be effectively used for screening anticancer substances or anticancer treatment methods. Confirmed and completed the present invention.

An object of the present invention is to provide a screening method of an anticancer substance or an anticancer treatment method using calcein-AM in a multicellular cancer cell (multicellular layer, MCL).

In order to achieve the above object, the present invention provides a screening method of an anticancer substance or an anticancer treatment method using calcein-AM (calcein-acetoxymethyl ester) in a multilayer cancer cell culture system.

Hereinafter, the present invention will be described in detail.

The present invention

1) inserting a chamber consisting of a pore membrane at the bottom and an impermeable material at the side into a well of a cell culture dish, and then culturing cancer cells in the chamber to establish a multicellular layer (MCL);

2) treating an anticancer candidate substance or performing a candidate anticancer treatment method in the chamber of step 1);

3) after performing step 2), continuously treating calcein-AM (calcein-acetoxymethyl ester) in the chamber;

4) preparing the MCL of step 3) into fragments;

5) measuring the fluorescence intensity distribution of the segment; And

6) provides a screening method of an anticancer substance or an anticancer treatment method comprising the step of selecting an anticancer candidate substance or a candidate anticancer treatment method in which the fluorescence intensity distribution is significantly reduced compared to the control group.

The chamber of step 1) is preferably a transwell insert (Transwell insert ^® , Corning Costar, Acton, MA), but is not limited thereto.

The multi-layered cancer cell culture system of step 1)

Iii) inoculating a chamber consisting of a pore membrane and an impermeable material at the bottom into a well of a cell culture dish, and then inoculating passaged cancer cell lines in the chamber;

Ii) attaching the inoculated cancer cell line; And

Iii) adding the medium to the cell culture dish and incubating in a CO ₂ incubator at 37 ° C. (see FIG. 1).

The cancer cell line of step iii) may use solid cancer cell lines such as gastric cancer cell line, lung cancer cell line, liver cancer cell line, uterine cancer cell line or breast cancer cell line, and in the following preferred embodiment of the present invention, human colon cancer cell lines DLD-1 and HT-29 Use

The medium of step iii) was RPMI-1640 (Gibco BRL) containing 10% FBS (Sigma, USA), penicillin 100 unit / ml (Sigma, USA), streptomycin 100 μg / ml (Sigma, USA) , Grand Island, NY).

Multi-layered cancer cell culture system (multicellular layer) established in such a step is referred to as "MCL". The thickness of the MCL continuously cultured for 8 days in the same step as described above is about 80 ~ 190 ㎛ (paraffin fragment), the growth rate is slowed down after 5 days of culture (see Figs. 2 and 3), In the following preferred embodiment, MCL cultured from 5 days to 7 days is used.

When treated with calcein-AM in monolayer cell culture, the fluorescence density increased in calcein-AM treated with high concentration and increased with increasing cell number (see FIG. 4), which was calcein as an active probe. -AM can be used.

Anticancer candidates of step 2) include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts or animal tissue extracts, but are not particularly limited now, preferred embodiments of the present invention In the treatment of paclitaxel (hereinafter abbreviated as "PTX") and doxorubicin (abbreviated as "DOX") which is a kind of anticancer substance. The temperature to be treated may vary depending on the candidate material but is preferably 37 ° C., similar to in vivo conditions.

The candidate anticancer treatment method of step 2) means a treatment method by radiotherapy or a physical method in addition to the anticancer candidate substance.

As soon as step 2) is performed, calcein-AM (calcein-acetoxymethyl ester) is successively treated in the upper chamber of the transwell insert (step 3 above). The concentration of calcein-AM is preferably 20 to 60 µM, more preferably 30 to 50 µM, and more preferably 40 µM. The treatment time of calcein-AM is preferably 30 minutes to 3 hours, more preferably 1 to 2 hours, and most preferably 1 hour.

Calcein-AM was added to MCL, incubated for 60 minutes at room temperature (room temperature) with MCL for 45 minutes at 4 ° C, and 15 minutes at room temperature (cold pre-incubation) in MCL. The calcein-AM distribution ranges for are similar, but wider in pre-culture (see FIGS. 5 and 6), and graphs of fluorescence density versus relative depth from the section surface show similar shapes, but pre-culture The degree of fluorescence signal confusion is somewhat reduced (see FIGS. 7 and 8). Therefore, the treatment temperature of calcein-AM is preferably at room temperature, and more preferably cold pre-incubation as described above.

Most preferably, the section of step 4) has a thickness of 20 µm and may be 10-70 µm, and most preferably a cryo-section.

The fluorescence intensity distribution of step 5) is preferably measured by fluorescence microscopy in the section, most preferably in the frozen section, and in the preferred embodiment of the present invention, AX70TR-62A02 of Olympus (Japan) was used.

In the method of the present invention, the distribution of DOX and calcein-AM can be measured simultaneously in the MCL exposed to DOX (see FIG. 9), and can be measured simultaneously in the MCL exposed to PTX-Rd (FIGS. 10 to 12). Reference). Even after treatment with the two anticancer agents, the cells remaining viable were stained with calcein-AM, so that the change of MCL permeability distribution of the drug and the change of calcein-AM distribution coincide. After exposure of PTX-Rd to HT-29 and DLD-1, the distribution of PTX-Rd over time was observed, indicating that the distribution of PTX-Rd in DLD-1 was smaller than that of HT-29 ( This may be due to the resistance of DLD-1 to PTX.

Therefore, after treatment with the anticancer candidate or after the candidate anticancer treatment method, the value of the measured fluorescence intensity distribution treated with calcein-AM and transmitted through the MCL and the anticancer candidate substance are not treated or the candidate anticancer treatment method is not performed. By comparing the fluorescence intensity distribution of calcein-AM of the non-control group, it can be usefully used to select the efficacy of the candidate substance or the anticancer treatment method on the anti-cancer, in particular on the solid cancer.

Hereinafter, the present invention will be described in detail by way of examples.

The following examples are only examples for describing the present invention in detail, and the present invention is not limited by the following examples.

Example 1 Establishment of a Multicellular Layer (MCL)

<1-1> Cell Culture

In order to establish a multi-layered cancer cell culture system for evaluating the permeation and post-permeation activity of cancer drug substance on cancer tissue, cancer cells were first cultured.

The cancer cells used in the present invention were distributed by the Korean Cell Line Bank (Seoul, Korea) as human colorectal cancer cell lines (DLD-1 and HT29). The human colorectal cancer cell line was RPMI-1640 (Gibco BRL, 10% FBS (Sigma, USA), penicillin 100 unit / ㎖ (Sigma, USA), streptomycin 100 ㎍ / ㎖ (Sigma, USA) Grand Island, NY). The incubator was supplied with 5% CO ₂ and 95% air, and maintained at a temperature of 37 ° C.

<1-2> Culture of Multicellular Layer (MCL)

Culture in Example <1-1> to six pore size 0.4 ㎛ the transwell inserts ^{(Transwell insert ⓡ, Corning Costar,} Acton, MA) collagen (collagen) of the upper chamber (top chamber, TC) coated with a a 3 × 10 ⁵ pieces / inoculated of 100 ㎕ cancer cells, 150 ㎖ added to the RPMI-1640 medium in the lower chamber (bottom chamber, BC) was adhered for 3 to 4 hours at 37 ℃ the transwell insert CO Culture by stirring in _two incubators (RM Phillips, et, al., Br J. Cancer , 77, 2112-9, 1998; DS Cowan, et, al., Br J. Cancer Suppl, 27, S28-31, 1996) (FIG. 1). 4% of MCL formed from the above procedure for each culture day from 1 to 8 days Formaldehyde was fixed and embedded in paraffin, cut into 5 μm thick sections, and the sections were stained with hematoxylin-eosin (H & E) to observe MCL formation and culture thickness.

As a result, it was observed that DLD-1 and HT29 incubated with MCL grow flat and evenly in a multi-layered form. In addition, there is a difference in the shape of the cells depending on the distance from the pore membrane (Microporous membrane, pore size = 0.4 ㎛), the top layer was grown while forming a flat spread shape. When the continuous culture for 8 days, the thickness was about 80 ~ 190 ㎛. In addition, the growth doubling time (TD) measured by increasing the thickness of the multi-layered cancer cell culture system was about 2 days at the beginning of culture (up to 4 days), and the growth rate was about 4 days as time passed (after 5 days). Slowed down (FIGS. 2 and 3).

As described above, since the growth rate is slowed down after 5 days of culturing, MCL was used for 5 days.

Example 2 Investigation of Calcein-AM Signal in Monolayer Cell Culture

Cells cultured in the same manner as in Example <1-1> were cultured overnight at 0.1 × 10 ⁵ , 0.5 × 10 ⁵ , 1 × 10 ⁵ , 1.5 × 10 ⁵ per well. Next, after exposure to calcein-AM (calcin-AM) -containing medium (2 μM and 4 μM) for 45 minutes, the cells were washed twice with ice-cooled PBS (phosphate buffered saline) and the fluorescence density Was measured using a fluorescence measuring device (VICTOR ³ , PerKin Elmer LAS, MA) at λ _{Ex / Em} = 494/517 nm (K. Yazawa, et al., Cancer Sci., 96, 93-9, 2005). . The results are expressed as mean ± standard error (SE).

As a result, the fluorescence density increased from 4 μM calcein-AM to 2 μM calcein-AM but was less than twice. In addition, the fluorescence density increased as the number of cells increased, but increased in a straight line form up to 1.5 × 10 ⁵ cells only (FIG. 4).

<Example 3> Calcein-AM using cryo-section in MCL Activity

Calcein-AM was added to the upper chamber of the MCL prepared in Example <1-2> to a final concentration of 40 μM, incubated for 60 minutes at room temperature and incubated for 45 minutes at 4 ° C., followed by 15 minutes at room temperature. The culture was divided into MCL to be cultured. In the following, MCL incubated for 60 minutes at room temperature was referred to as "room temperature incubation", and after 45 minutes incubation, MCL incubated for 15 minutes at room temperature was referred to as "cold pre-incubation". Next, the MCL was embedded with a freezing embedding agent (OCT compound, Leica, Germany) and rapidly frozen in liquid nitrogen. The sections were then obtained at relative depths of 0%, 20%, 40%, 60%, 80% and 100% from the frozen section surface using an ultrathin sectioner (Cryostat microtome, CM 1800, Leica, Germany), Fluorescence density was measured at λ _{Ex / Em} = 488/517 nm using a fluorescence microscope (Olympus, AX70TR-62A02, Japan). Images were analyzed using an image analysis program (Optimas ver. 6.5, Media Cybernetics, Inc., USA).

As a result, the calcein-AM distribution was wider in pre-culture than in room temperature culture (FIGS. 5 and 6), and the fluorescence density was increased from the section surface to a section of relatively deeper depth in the cooling pre-culture than in room temperature culture. It was possible to measure, and the degree of fluorescence signal confusion was reduced (FIGS. 7 and 8).

Example 4 Drug Distribution in MCL distribution ) And Calcein - AM  Survey of distribution

To investigate the relationship between drug distribution and calcein-AM distribution in MCL, MCL was added to the upper chamber of the transwell insert containing MCL cultured for 5 days in the same manner as in Example 3 above. rhodamine, 100 μM) or DOX (Doxorubicin, 100 μM) was exposed for 72 hours in a CO ₂ incubator at 37 ° C., followed by subsequent exposure to calcein-AM (40 μM) using the above pre-cooling culture method. . Next, 20 µm of frozen sections were obtained by the same method as in Example 4, and calcein-AM was OX _{Ex / Em} = 488/517 nm using a fluorescence microscope (Olympus, AX70TR-62A02, Japan). And PTX-Rd (Paclitaxel-rhodamine) measured fluorescence density at λ _{Ex / Em} = 482/505 nm, respectively.

As a result, DOX-exposed MCL was able to observe the distribution of DOX and Calcein-AM at the same time (FIG. 9) and PTX-Rd distribution and Calcein-AM distribution were also observed in PTX-Rd-exposed MCL. Could be (FIGS. 10-12). In addition, compared to HT-29, DLD-1 showed a smaller change in the fluorescence distribution of PTX-Rd over time (FIG. 12), since DLD-1 is resistant to PTX.

In this case, the cells stained with calcein-AM are living cells, and a large number of cells stained with calcein-AM are resistant to the anticancer agent or the anticancer treatment method treated by the cancer cell, or the treated anticancer agent or It shows that the anticancer treatment did not penetrate the lower layers of the multi-layered cancer cells. On the contrary, the small number of cells stained with calcein-AM is not highly resistant to the anticancer agent or anticancer treatment treated by the cancer cells, or the treated anticancer agent or anticancer treatment penetrates to the lower layers of the multi-layered cancer cells. To show that it can be exercised. Therefore, the method of the present invention can confirm the effect of the treated anticancer drug candidate or anticancer treatment method by confirming the staining of calcein-AM in various fragments of the MCL, it can be usefully used for effective anticancer screening .

As described above, the method of the present invention for screening an anticancer substance or an anticancer treatment method using calcein-AM (calcein-acetoxymethyl ester) in a multicellular cancer cell culture (multicellular layer, MCL) is an MCL-permeated calcein- By measuring AM fluorescence intensity distribution, it is possible to determine the degree of MCL penetration and anticancer effect of a candidate anticancer substance or a candidate anticancer treatment method, which is useful for quickly and conveniently searching for or evaluating the efficacy of a new anticancer agent or anticancer therapy on solid cancer. Can be used.

Claims (7)

1) inserting a chamber consisting of a pore membrane at the bottom and an impermeable material at the side into a well of a cell culture dish, and then culturing cancer cells in the chamber to establish a multicellular layer (MCL); 2) treating an anticancer candidate substance or performing a candidate anticancer treatment method in the chamber of step 1); 3) after performing step 2), continuously treating calcein-AM (calcein-acetoxymethyl ester) in the chamber; 4) preparing the MCL of step 3) into fragments; 5) measuring the fluorescence intensity distribution of the segment; And 6) Screening method of anti-cancer substance or anti-cancer treatment method effective in vivo, comprising the step of screening the anti-cancer candidate substance or candidate anti-cancer treatment method with significantly reduced fluorescence intensity distribution compared to the control group . delete According to claim 1, wherein the multicellular cancer cell culture (multicellular layer) of step 1) Iii) inoculating a chamber consisting of a pore membrane and an impermeable material at the bottom into a well of a cell culture dish, and then inoculating passaged cancer cell lines in the chamber; Ii) attaching the inoculated cancer cell line; And Iii) adding the medium to the cell culture dish and culturing in a CO ₂ incubator at 37 ° C. The method of claim 3, wherein the cancer cell line of step iii) is a human colorectal cancer cell line (DLD-1 or HT-29). The method of claim 1, wherein the anticancer candidate of step 2) is a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, a cell extract, a plant extract or an animal tissue extract. The method of claim 1, wherein the candidate anticancer treatment method of step 2) is radiotherapy. The method of claim 1, wherein the fluorescence intensity distribution of step 5) is measured using a fluorescence microscope.

Images