CN113892463A

CN113892463A - Peritoneal irrigation fluid tumor model for screening peritoneal and pelvic malignant tumor drugs, construction method and application

Info

Publication number: CN113892463A
Application number: CN202111123981.5A
Authority: CN
Inventors: 彭思颖; 孔毅荣; 李红伍
Original assignee: Beijing Idmo Co ltd
Current assignee: Beijing Idmo Co ltd
Priority date: 2021-09-24
Filing date: 2021-09-24
Publication date: 2022-01-07
Anticipated expiration: 2041-09-24
Also published as: CN113892463B

Abstract

A method for constructing a tumor model of an abdominal cavity flushing fluid for screening medicines of an abdominal cavity malignant tumor comprises the following steps: collecting abdominal cavity washing liquid of an abdominal cavity malignant tumor patient, purifying the abdominal cavity washing liquid by a magnetic bead method to obtain non-immune cells, then inoculating the obtained non-immune cells into a high immune deficiency animal body, feeding the animal inoculated with the non-immune cells to form a tumor body in the animal body, and screening the animal with the tumor body to obtain the animal model with the abdominal cavity malignant tumor characteristic. The obtained human source allograft animal model with the property of the abdominal and pelvic malignant tumor can be used for testing various medicines and combined medicines and effectively screening the abdominal and pelvic malignant tumor medicines.

Description

Peritoneal irrigation fluid tumor model for screening peritoneal and pelvic malignant tumor drugs, construction method and application

Technical Field

The application belongs to the technical field of biological medicines, and particularly relates to an abdominal cavity flushing fluid tumor model for screening of medicines for abdominal and pelvic malignant tumors, a construction method and application.

Background

At present, the incidence of malignant tumors in China is increased year by year, and the abdominal and pelvic malignant tumors become main diseases threatening the life and health of people. The clinically common treatment mode is surgery plus postoperative intravenous chemotherapy. Although patients may experience different degrees of remission from initial treatment, most tumors will recur and have poor results with re-surgery and chemotherapy. The postoperative implantation, recurrence and drug resistance of anticancer drugs of the abdominal and pelvic malignant tumors are always one of the main factors influencing the prognosis of the tumors, and how to prevent and treat the tumors becomes a research hotspot of scholars at home and abroad and is also a concern of tumor patients.

In recent years, a new tumor treatment method, namely intraperitoneal thermal perfusion chemotherapy (IPHC), has been developed at home and abroad, namely, after an operation is performed on a patient with middle and late malignant tumors, doctors perform intraperitoneal thermal perfusion chemotherapy on free cancer cells remaining in abdominal cavities and widely scattered tiny focuses which cannot be excised after the operation, and the purpose is to fully utilize the synergistic effect of the thermotherapy and the chemotherapy, integrate the characteristics of selective regional chemotherapy and obviously enhance the effect of killing the tumor cells. Has positive effects of preventing and treating abdominal and pelvic malignant tumors, greatly reduces toxic and side effects of chemotherapy of patients, and improves the quality of life of patients. Reduce recurrence rate and improve survival rate. The selection of the chemotherapy scheme aiming at the abdominal cavity thermal perfusion also becomes a problem which is concerned by doctors and patients.

Disclosure of Invention

In view of the above, in one aspect, some embodiments disclose a method for constructing a tumor model of an abdominal cavity washing fluid for screening an abdominal cavity and pelvic cavity malignant tumor drug, the method comprising:

collecting abdominal cavity washing liquid of a patient with abdominal cavity and pelvic cavity malignant tumor;

purifying the peritoneal washing liquid by a magnetic bead method to obtain non-immune cells;

inoculating the obtained non-immune cells into a high immunodeficiency animal;

raising the animal inoculated with the non-immune cells, forming a tumor body in the animal body, and screening the animal with the tumor body to obtain the animal model with the property of the abdominal and pelvic malignant tumor.

Further, in the methods for constructing a tumor model of the peritoneal irrigation solution for screening the drugs for the pelvic and abdominal malignant tumors disclosed in some embodiments, the non-immune cells are human CD45 cells obtained by magnetic bead negative screening.

Some embodiments disclose methods for constructing a tumor model of an abdominal cavity washing liquid for screening medicines of an abdominal cavity and pelvic cavity malignant tumor, wherein an animal is an NPI mouse.

Some embodiments disclose abdominal cavity washing liquid tumor model construction methods for screening of medicines for pelvic cavity malignant tumor, wherein the tumor volume is controlled to be 600-800 mm³In the meantime.

Some embodiments disclose methods for constructing a tumor model of an abdominal cavity washing liquid for screening drugs for the pelvic cavity malignant tumor, and animals with tumor bodies are screened through two-color flow analysis.

Some embodiments disclose methods for constructing a tumor model of an abdominal cavity flushing fluid for screening medicines of an abdominal cavity and a pelvic cavity malignant tumor, and the screening of animals further comprises STR detection.

In the methods for constructing the tumor model of the peritoneal irrigation solution for screening the medicines for the pelvic cavity malignant tumor, the feeding period of animals is controlled to be 60-90 days.

Some embodiments disclose methods for constructing a tumor model of an abdominal washing liquid for screening an abdominal and pelvic malignant tumor drug, wherein the obtained non-immune cells are inoculated to the subcutaneous part of a high immunodeficiency animal.

On the other hand, some embodiments disclose an abdominal cavity washing liquid tumor model for screening of an abdominal cavity and pelvic cavity malignant tumor medicament, wherein the tumor model is an animal model with the characteristics of the abdominal cavity and pelvic cavity malignant tumor, which is obtained by the abdominal cavity washing liquid tumor model construction method for screening of the abdominal cavity and pelvic cavity malignant tumor medicament.

In another aspect, some embodiments disclose the use of a tumor model of an abdominal cavity washing fluid in screening drugs for an abdominal cavity and pelvic cavity malignancy, specifically comprising:

obtaining an animal model of the characteristics of the abdominal and pelvic cavity malignant tumors;

stripping a tumor body, and cutting the tumor body into tumor body samples meeting the inoculation requirement;

and (3) inoculating the tumor body sample into an animal body, performing pharmacodynamic test on the abdominal and pelvic cavity malignant tumor medicament, and screening the abdominal and pelvic cavity malignant tumor medicament with the medicament effect meeting the requirement.

The method for constructing the tumor model of the abdominal cavity flushing fluid for screening the medicines for the abdominal cavity and pelvic cavity malignant tumors, disclosed by the embodiment of the application, can be used for testing various medicines and combined medicines and effectively screening the medicines for the abdominal cavity and pelvic cavity malignant tumors to obtain the human source allograft animal model with the characteristics of the abdominal cavity and pelvic cavity malignant tumors.

Drawings

FIG. 1 example 1 flow analysis of peritoneal irrigation fluid

FIG. 2 flow analysis of example 1 after purification

FIG. 3 tumor growth curves of example 1;

FIG. 4 example 1 two-color flow analysis chart for determining tumor CD19/CD 45;

FIG. 5 example 1 determination of pathological diagnosis HE staining pattern of tumor body;

FIG. 6 is a graph showing the results of drug screening using an animal model of pelvic cavity malignancy in example 2.

Detailed Description

The word "embodiment" as used herein, is not necessarily to be construed as preferred or advantageous over other embodiments, including any embodiment illustrated as "exemplary". Performance index tests in the examples of this application, unless otherwise indicated, were performed using routine experimentation in the art. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; other test methods and techniques not specifically mentioned in the present application are those commonly employed by those of ordinary skill in the art.

The terms "substantially" and "about" are used herein to describe small fluctuations. For example, they may mean less than or equal to ± 5%, such as less than or equal to ± 2%, such as less than or equal to ± 1%, such as less than or equal to ± 0.5%, such as less than or equal to ± 0.2%, such as less than or equal to ± 0.1%, such as less than or equal to ± 0.05%. Numerical data represented or presented herein in a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a numerical range of "1 to 5%" should be interpreted to include not only the explicitly recited values of 1% to 5%, but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values, such as 2%, 3.5%, and 4%, and sub-ranges, such as 1% to 3%, 2% to 4%, and 3% to 5%, etc. This principle applies equally to ranges reciting only one numerical value. Moreover, such an interpretation applies regardless of the breadth of the range or the characteristics being described.

In this document, including the claims, conjunctions such as "comprising," including, "" carrying, "" having, "" containing, "" involving, "" containing, "and the like are understood to be open-ended, i.e., to mean" including but not limited to. The conjunctions "consisting of … …" and "consisting of … …" are closed conjunctions.

In the following detailed description, numerous specific details are set forth in order to provide a better understanding of the present disclosure. It will be understood by those skilled in the art that the present application may be practiced without some of these specific details. In the examples, some methods, means, instruments, apparatuses, etc. known to those skilled in the art are not described in detail in order to highlight the subject matter of the present application.

On the premise of no conflict, the technical features disclosed in the embodiments of the present application may be combined arbitrarily, and the obtained technical solution belongs to the content disclosed in the embodiments of the present application.

In some embodiments, the method for constructing a tumor model of an abdominal cavity washing liquid for screening medicines for an abdominal cavity and pelvic cavity malignant tumor comprises the following steps:

inoculating the obtained non-immune cells into a high immunodeficiency animal;

The inventor researches and discovers that non-immune cells purified from an abdominal cavity flushing fluid are inoculated into an immunodeficient mouse body, a human source allograft tumor model with the property of the abdominal cavity and pelvic cavity malignant tumor can grow in the mouse body, a mouse individual with the abdominal cavity and pelvic cavity malignant tumor can be screened on the basis of keeping the biological characteristics and the reaction characteristics to the drugs of the tumor model, the mouse model with the abdominal cavity and pelvic cavity malignant tumor property can be obtained, the mouse model can be used for testing various drugs and a combined drug administration method, an accurate individual drug administration scheme can be found, the best clinical curative effect can be obtained, the individual cancer treatment of the abdominal cavity and pelvic cavity malignant tumor patient can be effectively guided, and the life cycle of the patient can be prolonged.

The mouse is used as an inoculated animal, the growth period is short, the tumor growth speed is high, the model construction time can be effectively shortened, and the success rate of model construction is improved. Further as an optional embodiment, the inoculated animal is an NPI mouse which has high immunodeficiency, single background, low rejection rate to xenotransplantation, high survival rate of the xenotransplantation and long service life, is favorable for the success rate of model construction, and is more favorable for the later-stage drug evaluation test.

As an optional embodiment, in the process of constructing the tumor model of the peritoneal irrigation solution, the volume of the tumor growing in the mouse is controlled to be 600-800 mm³The tumor body is easy to be accurately judged, if the tumor body volume is too small, the case identification degree of the tumor body is not high, the classification of the tumor body is difficult to be accurately judged, and if the tumor body volume is too large, the tumor is subjected to ischemic necrosis.

The inventor finds that in the process of constructing the peritoneal irrigation fluid tumor model, the feeding period of the mouse is controlled to be 60-90 days, and the tumor volume can be effectively controlled to be 600-800 mm³In the meantime.

As an alternative example, the non-immune cells are human CD45 cells obtained by magnetic bead negative selection.

The inventor finds that the non-immune cells are obtained by purifying the abdominal cavity washing liquid by using the magnetic beads, so that the non-immune cells can grow rapidly in the body of the inoculated animal, and the success rate and the construction rate of model construction are improved.

The inventor further finds that a mouse with a tumor body is screened by two-color flow analysis, for example, by two-color flow dyeing analysis of CD19/CD45, the two-color flow analysis result of CD19/CD45 can provide immune cell infiltration information, whether the tumor body is derived from non-immune cells or not can be judged from the immune cell infiltration angle, the accuracy is high, the success rate of obtaining a mouse model with abdominal and pelvic cavity malignant tumors is high, the subsequent mouse model is used for screening drugs, and the reliability of the drug screening result is further improved.

The inventor further finds that the screening of the mice is further carried out through STR detection, the STR detection can judge the source of the tumor sample and the gene expression information of the sample from the gene perspective, and further can judge the type of the tumor sample more accurately from the cell morphology, the biochemical information and the gene expression information simultaneously, so that the failure of a more serious drug screening experiment caused by the error construction of an animal model is avoided. The STR detection can be used for judging whether non-immune cells obtained by purifying tumor bodies and the abdominal cavity flushing fluid are from the same individual from the gene perspective, so that the success rate of model construction is further improved. The constructed mouse model is subsequently used for screening medicaments for the pelvic cavity and abdominal malignant tumor, and the reliability of the screening result can be further improved.

As an alternative example, the obtained non-immune cells are inoculated subcutaneously into highly immunodeficient mice. The subcutaneous inoculation is beneficial to the survival and growth of the inoculated cells in the inoculated animal body, and is beneficial to further improving the success rate of model construction.

The tumor model is a human source allograft mouse model with the property of the abdominopelvic malignant tumor.

Some embodiments disclose applications of a peritoneal irrigation fluid tumor model in screening of medicines for pelvic and abdominal malignant tumors, which specifically include:

obtaining a mouse model of the characteristics of the abdominal and pelvic malignant tumors;

and (3) inoculating the tumor body sample into a mouse body, performing pharmacodynamic test on the abdominal and pelvic cavity malignant tumor medicament, and screening the abdominal and pelvic cavity malignant tumor medicament with the medicament effect meeting the requirement.

The inventor finds that the mouse model serving as an abdominal cavity flushing fluid tumor model can be used for effectively screening western medicines, traditional Chinese medicines, combined medicines and combined medicines.

The inventor finds that, as an optional embodiment, a mouse model obtained by using the abdominal cavity flushing fluid tumor model construction method can effectively test multiple drugs and combined drug administration schemes, finds out an accurate individual drug administration scheme to obtain the best treatment effect on the abdominal cavity and pelvic cavity malignant tumor, can effectively guide individualized cancer treatment of the abdominal cavity and pelvic cavity malignant tumor patients, and reduces the blindness of clinical drug administration and the error rate of clinical drug administration.

In the embodiment disclosed in the invention, the inventor inoculates non-immune cells obtained by purifying fresh abdominal cavity washing liquid obtained in a patient operation into an immunodeficient mouse body, and after tumor formation, the tumor formation mouse is screened, and the obtained abdominal cavity and pelvic cavity malignant tumor mouse model can be used for carrying out an abdominal cavity and pelvic cavity malignant tumor drug screening experiment, and an accurate individual medication scheme is found out to obtain the best clinical curative effect. Non-immune cells obtained by purifying fresh peritoneal washing liquid obtained in the operation of a patient are inoculated to an immunodeficient mouse, and a tumor model of the patient is reconstructed in the mouse, so that the biological characteristics of tumor tissues and the reaction characteristics to drugs can be kept best, the curative effect of the drugs can be predicted more accurately, and ineffective chemotherapy and possible toxic and side effects can be avoided.

It should be noted that the "inoculation" operation described herein is not particularly limited and can be easily performed by a person skilled in the art without special professional training.

The peritoneal irrigation solution tumor model disclosed herein is used for screening drugs or drug combinations for treating the peritoneal and pelvic malignant tumors on one hand, and on the other hand, the construction method for the peritoneal irrigation solution tumor model for the peritoneal and pelvic malignant tumors is also indirectly used for prognosis judgment of patients with the peritoneal and pelvic malignant tumors.

In addition, when screening an anti-tumor treatment plan using the mouse model of an abdominopelvic malignancy obtained by the method disclosed herein, the screening of the anti-tumor treatment plan used includes screening of an abdominopelvic malignancy drug, screening of an administration dose, screening of a chemotherapy plan, screening of a radiotherapy, management of an administration period, and the like. Wherein, the technicians in this field can adopt any conventional in this field or in the development of drugs as candidate therapy, in addition, technicians in this field can also use different doses of existing drugs for different dosing cycles and dose studies. The antitumor drugs include western drugs and traditional Chinese drugs, the types of which are not particularly limited, and drugs suitable for chemotherapy, molecular targeting therapy, immunotherapy, and the like are included in the antitumor drugs disclosed herein. According to the clinically established treatment scheme, the administration modes of experimental animals, including oral administration, subcutaneous injection, intravenous injection and the like, can be determined according to the administration routes of different medicines, and the administration frequency is carried out according to the clinical administration modes of patients.

The technical details are further illustrated in the following examples.

Example 1

Construction of abdominal cavity flushing fluid tumor model for screening abdominal cavity and pelvic cavity malignant tumor medicine

In this embodiment 1, the method for constructing a tumor model of an abdominal cavity washing fluid for screening a medicine for a pelvic cavity malignant tumor comprises:

experimental Material

Clinically provides an abdominal cavity flushing fluid for a patient with a fresh abdominal cavity and pelvic cavity malignant tumor, and transports the flushing fluid at 4 ℃.

Laboratory animal

Variety: NPI mice

Age: 6-8 weeks old

The supplier: beijing Edmo Biotechnology Ltd

Breeding environment: SPF experimental animal house of Beijing Edmo biotechnology Limited company

Laboratory temperature 22-25 deg.C

Relative humidity: 40 to 70 percent

Relative air pressure: 10 to 20Pa

Air exchange frequency: 10 to 15 times/hour

Illumination cycle: 12 hours of light (8:00-20:00), 12 hours of darkness (20: 00-8: 00)

Animal feeding: 5 mice/cage, central air-conditioning and air-filtering mechanical equipment, animal cages, drinking water, padding and the like are sterilized by high-pressure steam, and are fed with SPF-level special granulated feed to drink water freely.

Reagent

Anticoagulant, tissue culture fluid, antibiotic, etc.

Content of the experiment

Material taking:

abdominal cavity washing fluid is obtained from patients with abdominal and pelvic malignant tumors by means of surgery;

immediately after the material was taken, the tube was placed in a liquid collection tube, transported at 4 ℃ and delivered within 24 hours.

And (3) purification:

1250ml of ascites are dispensed into 50ml × 25 centrifuge tubes and centrifuged at 1620rpm (500g) for 10 min;

sucking the supernatant, resuspending the lower layer cells with 2ml PBS, adding 8ml ammonium chloride solution, mixing the cell fluid uniformly, and placing the cell fluid on ice to crack red for 5 min;

1620rpm (500g) centrifugation, washing the cells 1 times with PBS;

discard supernatant, cell count after resuspension of cells: the number of viable cells was 1.13X 10⁸；

Take 5X 10⁴Carrying out flow detection on the cells;

CD45-beads kit (STEMCELL) sorting CD 45-cells;

CD45- (non-immune) cells completed the inoculation work;

mice were inoculated subcutaneously with 5E6 per standard. Generally, 3-6 inoculated mice are used, and the specific number is determined according to the cell amount; in example 1, 3 NPI mice were transplanted with CD 45-cells (double-sided single-point, 8.1E6 cells/transplant point);

observation and measurement:

observing the state of the animals daily after inoculation and before the end of the experiment, and if the animals are found to be sick or abnormally dead, handling or dissecting the animals by a veterinarian;

measurement of tumor growth: measuring the volume of the tumor by using a vernier caliper once a week and weighing the weight of the tumor-bearing mouse;

tumor volume calculation formula: v (mm)³)＝(a×b²) A is the tumor major diameter, b is the tumor minor diameter;

ending the experiment and taking materials:

if the inoculated first-generation animal P0 has tumor growth, selecting the tumor body with the fastest growth volume to 600-800 mm³Stripping tumor body tissues, fixing and storing one piece of the tumor body tissues, and storing the rest of the slow freezing liquid by nitrogen;

if there is no sign of growth of the P0 tumor at 90 days post inoculation, the batch of mice is sacrificed and the model is terminated.

Sample detection:

and carrying out CD19/CD45 bicolor flow analysis and staining, HE staining analysis and STR detection on the fixed tumor body tissues P0 and P1.

And (4) analyzing results:

comparing the HE staining of P0 and P1 with STR detection reports, and determining the type of tumor mass, with reference to the HE staining results of the original tumor tissue. Fig. 1 is a flow analysis diagram of a peritoneal irrigation solution, fig. 2 is a flow analysis diagram after purification, fig. 3 is a tumor body growth curve, as shown in fig. 4, a tumor body CD19/CD45 double-color flow analysis diagram is determined, and fig. 5, pathological diagnosis HE staining diagram data of the tumor body is determined, wherein tumor body tissues are shown in tumor cell diffuse distribution, the cell size is consistent, the tumor cells are circular, oval, nucleolar and oval, nucleolus is not obvious, more nuclear division is seen, and abnormal B lymphocyte proliferation is avoided.

Example 2

Drug screening

In this example 2, a successfully constructed mouse model of peritoneal irrigation fluid tumor was used for screening therapeutic drugs, and the experimental process included:

p1 generation tumor forming animal, when tumor body reaches 600-800 mm³Dissecting tumor body, cutting into 3 × 3mm³Size;

inoculating according to the standard of 1 block/mouse, and inoculating 40 mice;

when the tumor body reaches 150mm³And then, carrying out pharmacodynamic test in random groups, and screening out an optimal scheme for treating the abdominal and pelvic malignant tumors for clinical treatment reference through data collection of tumor volume and weight change.

In this example 2, the drugs to be screened include: FOLFIRI regimen (calcium folinate, irinotecan and fluorouracil, see Group02 in fig. 6), FOLFOX regimen (calcium folinate, oxaliplatin and fluorouracil, see Group03 in fig. 6), voritinib (savolitinib, see Group04 in fig. 6), FOLFOX regimen in combination with bevacizumab (see Group05 in fig. 6), Group01 in fig. 6, 0.5% MC as control.

The screening results are shown in fig. 6, and the FOLFIRI scheme, the FOLFOX scheme and bevacizumab have the best inhibition effect on the abdominal and pelvic malignant tumors in combination among the screened medicaments.

The method for constructing the tumor model of the abdominal cavity flushing fluid for screening the medicines for the abdominal cavity and pelvic cavity malignant tumors, disclosed by the embodiment of the application, can be used for testing various medicines and combined medicines and screening the medicines for the abdominal cavity and pelvic cavity malignant tumors to obtain the human source allograft animal model with the characteristics of the abdominal cavity and pelvic cavity malignant tumors.

The technical solutions and the technical details disclosed in the embodiments of the present application are only examples to illustrate the inventive concept of the present application, and do not constitute a limitation on the technical solutions of the present application, and all the conventional changes, substitutions, combinations, and the like made to the technical details disclosed in the present application have the same inventive concept as the present application and are within the protection scope of the claims of the present application.

Claims

1. A method for constructing a tumor model of an abdominal cavity flushing fluid for screening medicines of an abdominal cavity malignant tumor is characterized by comprising the following steps of:

purifying the peritoneal washing solution by a magnetic bead method to obtain non-immune cells;

inoculating the obtained non-immune cells into a high immunodeficiency animal;

2. The method for constructing a tumor model of an abdominal cavity washing liquid for screening drugs with characteristics of pelvic malignant tumors according to claim 1, wherein the non-immune cells are human CD45 cells obtained by magnetic bead negative screening.

3. The method for constructing an intraperitoneal washing liquid tumor model for screening an intraperitoneal malignant tumor medicament as claimed in claim 1, wherein the animal is an NPI mouse.

4. The method for constructing an abdominal cavity washing liquid tumor model for screening of drugs for pelvic cavity malignant tumors according to claim 1, wherein the volume of the tumor body is controlled to be 600-800 mm³In the meantime.

5. The method for constructing a tumor model of an abdominal cavity washing fluid for screening drugs for pelvic and abdominal malignancies according to claim 1, wherein animals with tumor bodies are screened by two-color flow analysis.

6. The method of claim 5, wherein the screening of the animal further comprises STR detection.

7. The method for constructing an abdominal cavity washing liquid tumor model for screening the medicines for the pelvic cavity malignant tumor according to claim 1, wherein the feeding period of the animals is controlled to be 60-90 days.

8. The method for constructing a tumor model of an abdominal cavity washing liquid for screening an abdominal cavity malignant tumor medicament according to claim 1, wherein the obtained non-immune cells are inoculated to the subcutaneous part of the high immunodeficiency animal.

9. The peritoneal irrigation solution tumor model for screening the peritoneal and pelvic cavity malignant tumor medicaments is an animal model with the characteristics of the peritoneal and pelvic cavity malignant tumor, which is obtained by the construction method of the peritoneal irrigation solution tumor model for screening the peritoneal and pelvic cavity malignant tumor medicaments as claimed in any one of claims 1 to 8.

10. The use of the tumor model of peritoneal irrigation solution for screening of drugs for pelvic and abdominal malignancies of claim 9 in screening of drugs for pelvic and abdominal malignancies.