System and method for tumor treatment

Abstract

An apparatus used for increasing the level of CD4.sup.+T cells in a mammal. The mammal has a cancerous tissue. The apparatus comprises a cold treatment unit and a heat treatment unit. The cold treatment unit is used for cooling the cancerous tissue. The heat treatment unit is used for heating the cancerous tissue. A method for increasing the level of CD4.sup.+ T cells in the body of a cancer patient, comprising steps for cold treatment of the cancerous tissue and for heat treatment of the cancerous tissue.

Claims

1. A method for increasing a level of CD4+ T cells in peripheral blood in a mammal, wherein the mammal has a cancerous tissue, and the method comprises the following steps: (1) cold treatment: treating at least one or more cancerous tissues of the mammal with a cold treatment to obtain a cold-treated cancerous tissue, wherein the cold treatment comprises cooling the cancerous tissue to a temperature of −10° C. or below and maintaining the temperature for 2-20 minutes; (2) rewarming treatment: naturally warming the cold-treated cancerous tissue to a temperature of 5-25° C. to obtain a rewarmed cancerous tissue; (3) heat treatment: treating the rewarmed cancerous tissue with heat treatment via irradiation of radio frequency or microwave, wherein the heat treatment comprises heating the rewarmed cancerous tissue to a temperature of 45-60° C. and maintaining the temperature for 2-20 minutes; and (4) measuring a level of the CD4+T cells in spleen and the peripheral blood on day 0, day 7, day 14, day 21, and day 28 after step (3) via a flow cytometry wherein a fluorescent antibody is used to label cell surface molecules to detect the specific CD4+T cell population; wherein the CD4+T cells secrete a cytotoxic cytokine IFN-γ and have a strong cytotoxic effect; wherein the cancerous tissue is a superficial cancerous tissue; the method further comprises monitoring temperature of the cancerous tissue in steps (1) to (3), wherein the monitoring temperature of the cancerous tissue includes non-invasive temperature monitoring; wherein the cold treatment and the heat treatment are operated with a non-invasive method; wherein in step (1) of cold treatment, the cold treatment is carried out by contacting the skin with a transfer device; wherein in step (3) of heat treatment, the heat treatment is carried out by contacting the skin with the transfer device; wherein the transfer device includes a contact head which comprises a contact surface for closely contacting a surface of the cancerous tissue, wherein a cold source cools the cancerous tissue with the contact head, and a heat source heats the cancerous tissue with the contact head; wherein the contact surface is a circular plane or a spherical surface; and the contact surface of the contact head has an area of 0.25 cm.sup.2 to 10 cm.sup.2.

2. The method according to claim 1, wherein the cancerous tissue comprises a tumor, a tissue invaded by cancer cells, and/or a tumor metastasis focus.

3. The method according to claim 1, wherein the non-invasive temperature monitoring is temperature detection via infrared image analysis, nuclear magnetic resonance temperature detection, or ultrasonic temperature detection.

4. The method according to claim 1, wherein the method further comprises a step of: repeating steps (1) to (3) one or more times.

5. A method for releasing immunogen of a cancerous tissue of a mammal comprising: (1) cold treatment: treating the cancerous tissues to obtain a cold-treated cancerous tissue, wherein the treating comprises cooling the cancerous tissue to a temperature of −10° C. or below and maintaining the temperature for 2-20 minutes; (2) rewarming treatment: naturally warming the cold-treated cancerous tissue to a temperature of 5-25° C. to obtain a rewarmed cancerous tissue; (3) heat treatment: treating the rewarmed cancerous tissue via irradiation of radio frequency or microwave, wherein the heat treatment comprises heating the rewarmed cancerous tissue to a temperature of 45-60° C. and maintaining the temperature for 2-20 minutes, and (4) measuring a level of CD4+T cells in spleen and peripheral blood on day 0, day 7, day 14, day 21, and day 28 after step (3) via flow cytometry wherein a fluorescent antibody is used to label cell surface molecules to detect the specific CD4+T cell population; the method further comprises monitoring temperature of the cancerous tissue in steps (1) to (3), wherein the monitoring temperature of the cancerous tissue includes non-invasive temperature monitoring: wherein the cold treatment and the heat treatment are operated with a non-invasive method; wherein the CD4+T cells secrete cytotoxic cytokine IFN-γ and have a strong cytotoxic effect; wherein the cold treatment in step (1) and the heat treatment in step (3) are conducted with a system comprising a treatment device; wherein the treatment device comprises a cold treatment unit, a heat treatment unit, and a contact head; wherein the cold treatment unit comprises a cold source; wherein the heat treatment unit comprises a heat source; wherein the contact head comprises a contact surface for closely contacting a surface of the cancerous tissue; wherein the cold source cools the cancerous tissue by the contact head, and the heat source heats the cancerous tissue by the contact head; wherein the contact surface is a circular plane or a spherical surface; and wherein the contact surface of the contact head has an area of 0.25 to 10 cm.sup.2.

6. The method according to claim 5, wherein the cancerous tissue has a volume of 0.1 cm.sup.3 to 100 cm.sup.3.

7. The method according to claim 5, wherein in step (1), the cancerous tissue is cooled to a temperature of −18° C. to −20° C.

8. The method according to claim 5, wherein in step (1), the temperature is maintained for a period of 15 to 20 minutes.

9. The method according to claim 5, wherein in step (3), the rewarmed cancerous tissue is heated to a temperature of 45° C. to 55° C. during the heat treatment.

10. The method according to claim 5, wherein in step (3), the rewarmed cancerous tissue is heated to a temperature of 50° C. to 55° C., and the temperature is maintained for 5 to 15 minutes during the heat treatment.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows changes of immune cells in spleen induced in different treatment groups, wherein Figure A shows changes of level of CD8.sup.+ T cells in spleen; and Figure B shows changes of level of CD4.sup.+ T cells in spleen.

(2) FIG. 2 shows the overall survival percentage and survival time in different groups of mice (6 mice in each group).

(3) FIG. 3 shows tumor metastasis on surface of mice three weeks after treatment, wherein Figure A shows the treatment group of surgical resection, Figure B shows treatment group only with cold treatment, and Figure C shows the treatment group with only heat treatment.

(4) FIG. 4 shows the status of the mice 6 weeks after cold and heat treatment. Figure A shows the mice before the treatment; Figure B shows the mice after the treatment; Figure C shows the mice 1 week after treatment; Figure D shows the mice 3 weeks after treatment; and Figure E shows the mice 6 weeks after treatment.

(5) FIG. 5 shows lung metastases detected via H&E staining 28 days after treatment. Figure A shows microscopic observation via H&E staining; and Figure B shows lung metastases in each treatment group.

(6) FIG. 6 illustrates a treatment device for increasing a level of CD4.sup.+T cells in a mammal having a tumor, in accordance with an embodiment.

DETAILED DESCRIPTION OF THE INVENTION

(7) Through extensive and intensive studies, the inventors of the present invention have obtained a method for improving level of CD4.sup.+ T cells in tumor patients. The experimental results have shown that the treatment of cancerous tissue by alternate cold and heat can significantly improve level of CD4.sup.+ T cells in tumor patients. The present invention also provides a system useful in said method.

(8) Treatment System or Device:

(9) In a preferred embodiment, the device of the present invention for improving level of CD4.sup.+ T cells of mammals comprises: a cold treatment unit and a heat treatment unit, wherein the cold treatment unit is used to cool the cancerous tissue; and the heat treatment unit is used to heat the cancerous tissue. The cold treatment unit comprises a cold source which can be liquid nitrogen, liquid oxygen or liquid nitrous oxide. The cold treatment unit cools the cancerous tissue with liquid nitrogen, liquid oxygen or liquid nitrous oxide. The heat treatment unit heats the cancerous tissue by using irradiation of radio frequency, microwave, infrared radiation or laser radiation, electric heating or the like.

(10) In another preferred embodiment, the device further includes a temperature monitoring unit for monitoring temperature of the cancerous tissue.

(11) In another preferred embodiment, the device further comprises a CD4.sup.+ T cell level monitoring unit for detecting level of CD4.sup.+ T cells in mammals.

(12) In another preferred embodiment, the device further comprises a contact head which comprises a contact surface for closely fitting the cancerous tissue, and the cold source cools the cancerous tissue with the contact head, and/or the heat source heats the cancerous tissue with the contact head.

(13) In another preferred embodiment, the contact surface is a circular plane or the contact surface is a curved surface of a spherical or nearly spherical surface.

(14) In another preferred embodiment, the means for conduct the cold treatment (low temperature treatment) of cancerous tissue in the present invention includes:

(15) (1) using the transportation and accurate control of flow of liquid nitrogen, liquid oxygen, or liquid nitrous oxide so as to obtain the desired temperature of the invention;

(16) (2) using throttle nozzle design and throttle effect of argon so as to obtain the desired temperature of the invention.

(17) In preferred embodiments, the means for conducting heat treatment (heating) of cancerous tissue in the present invention includes: using irradiation of radio frequency, microwave, infrared radiation or laser radiation, or direct electric heating so as to obtain the desired temperature of the invention.

(18) In a preferred embodiment of the present invention, the device used as the cold and heat alternating treatment system is described in Chinese Patent application of CN2005100343.8 and CN200410017864.0.

(19) The main advantages of the present invention includes:

(20) (1) In the present invention, a method for effectively improving level of CD4.sup.+ T cells in tumor patients is disclosed for the first time. The experimental results have shown that treating tumor-bearing mice by the method of the invention can significantly improve level of CD4.sup.+ T cells in mice;

(21) (2) Treating the tumor-bearing mice by the method of the invention can significantly improve the survival percentage.

(22) (3) Treating the tumor-bearing mice by the method of the invention can significantly reduce metastasis of tumor cells in tumor-bearing mice.

(23) The invention is further illustrated by the following examples. It should be appreciated that these examples are only intended to illustrate the invention, but not to limit the scope of the invention. Unless indicated otherwise, all percentage and parts are calculated by weight.

EXAMPLES

(24) 1. Materials and Methods

(25) 1.1 Animals, Cell Lines and Main Reagents

(26) SPF Balb/c female mice aged 6-8 weeks (Shanghai Slac Animal Center) are kept in a separate ventilation box cage. An artificially control light of 12 hrs day light and 1.2 hrs night light is used. The mice can freely take feed sterilized with .sup.60Co radiation and water sterilized with high temperature. Mouse 4T1 breast cancer cells (obtained from Shanghai First People's Hospital. In other embodiments, other breast cancer cells such as T47D cell line can be used.) are cultured in RPMI 1640 medium (Hyclone Co., USA) supplemented with 10% newborn fetal bovine serum (HangZhou Sijiging Co., Ltd.) and double antibiotics (100 U/mL penicillin and 100 g/mL streptomycin) (Shanghai Biotechnology Engineering Co., Ltd.). Ultra-fine precipitation of barium sulfate particles is purchased from Shanghai Zewen Trade Co., Ltd. FITC-labeled CD4 and PE-labeled CD8 for flow cytometry and immunofluorescence are purchased from Biolegend. Hematoxylin and eosin solution are purchased from Shanghai Hongqiao Le Xiang Medical Reagent Co., Ltd.

(27) 1.2 Establishment of 4T1 Breast Cancer Model and Determination of Tumor Size

(28) The 4T 1 mouse breast cancer cell used in this study is capable of metastasis to lungs, liver, bone marrow and brain via hematogenous pathways, and is a model of highly metastatic breast cancer. 1×10.sup.6 U/0.1 mL of 4T1 cells suspension was prepared and placed on ice. Animals were anesthetized by intraperitoneal injection of 0.016 g/mL pentobarbital sodium in a dose of 0.5 mL/100 g mice, and 0.1 mL cell suspension was subcutaneously injected into the back of mice. 21 days after tumor inoculation, the tumor volume was measured with a vernier caliper, and calculated according to the following formula: V (cm.sup.3)=p×long axis of tumor (cm)×short axis of tumor (cm)×tumor height (cm)/6. The tumor-bearing mice were randomly divided into control group, surgical resection treatment group, heat treatment group, and heat and cold combination treatment group. In 4T1 mouse breast cancer animal model established by the inventors, the mice were found to have micro-metastasis in the liver or the like 21 days after inoculation of tumor cells by using PET/CT scans and F18-labeling in vivo. Therefore, the present inventors selected 21 days after tumor inoculation as a time window for treatment.

(29) 1.3 Alternate Heat and Cold Treatment

(30) 1.3.1 Experimental Scheme

(31) The control group (the tumor-bearing mice, Control, referred as C), the surgical resection treatment group (Resection treatment, referred as R), the heat treatment group (Heat treatment, referred as H) and heat and cold combination treatment group (Alternate cooling and heating treatment, referred to as A). The research was as follows: (1) 21 days after inoculation, the mice were randomly divided into four groups and treated respectively: control group (C), surgical resection treatment group (R), heat treatment group (H) and heat and cold combination treatment group (A). After treatment, six mice from each group were selected to observe long-term therapeutic effect. In addition, three mice from each group were selected, and the lungs of the mice were taken 28 days after the treatment to detect lung metastasis of mice after treatment. (2) At 0 day before the treatment (that is 21 days after inoculation) and 7 days, 14 days, 21 days and 28 days after treatment, the mice were sacrificed, and the spleen and peripheral blood of mice in each group were taken to analyze the immunized cells. In addition, in order to further observe changes in immune environment of the metastatic lungs, 14 days after treatment, additional 3-5 mice from each group were taken to conduct lung analysis.

(32) 1.3.2 Alternate Heat and Cold Treatment System

(33) A device for improving level of CD4.sup.+ T cells in mammals in the present embodiment comprises: a cold treatment unit and a heat treatment unit; the cold treatment unit is used to cool the cancerous tissue; and the heat treatment unit is used to heat the cancerous tissue. The cold treatment unit comprises a cold source liquid nitrogen, and the liquid nitrogen is used to cool the cancerous tissue. The heat treatment unit heats the cancerous tissue by irradiation of radio frequency. The device includes a contact head which comprises a contact surface for closely fitting the cancerous tissue. The cold source cools the cancerous tissue with the contact head, and the heat source heats the cancerous tissue with the contact head. The contact surface is a circular plane which is particularly suitable for model of superficial tumor used in this experiment.

(34) 1.3.3 Experimental Process

(35) The mice were treated 21 days after tumor inoculation. The tumor volume before treatment was measured, and the mice with average tumor volume of less than 0.2 cm.sup.3 were selected as treatment subject. Before treatment, the mice were randomly divided into four groups and treated respectively: control group, surgical resection treatment group, heat treatment group, and heat and cold combination treatment group. First, mice to be treated were anesthetized, and sterilized with alcohol and iodine tincture at the tumor site.

(36) During treatment, the probe was placed on surface of tumor and a thermocouple for temperature measurement was inserted into base of tumor. During heating treatment, radiofrequency was used to heat the tumor so that the tumor was heated to a temperature of 50° C. (the temperature which was measured with the thermocouple) and it was maintained for 15 minutes. Alternate heat and cold treatment was divided into three processes: (1) cold treatment wherein tumor was lowered to a temperature of −20° C. by means of liquid nitrogen cooling and maintained at such temperature for 5 minutes; (2) rewarming wherein the tumor after cold treatment was naturally rewarmed to about 10° C.; (3) heat treatment wherein at the end of rewarming process, the temperature of was raised to 50° C. by the radiofrequency heat and such temperature was maintained for 10 mins

(37) 1.3.4 Evaluation on Efficacy of Cold and Heat Treatment

(38) After treatment, the survival status of mice was observed comprehensively. This part of study mainly includes: observation of in situ tumor growth in control group, in situ tumor ablation and recurrence in treatment group and metastasis of mice in each group (daily), records of change of body weight of mice before and after treatment (twice a week), statistics of long-term survival percentage and survival time of mice and so on. These indicators could effectively reflect survival of mice, wherein statistics of survival percentage and survival time are the most important indicators for evaluation of treatment

(39) 1.3.5 Lung Analysis Based on H&E Staining

(40) At 28 days after treatment, the mice were sacrificed. After perfusion of 4% formaldehyde into lungs though the weasand, the lungs were removed, and placed in formalin for 48 hours for fixation. After the lungs were embedded with paraffin, the specimen was cut into 8 microns of paraffin slices by using a paraffin slicing machine and the slices were stored at room temperature. The slices were dewaxed with xylene and a gradient from alcohol (100% −90% −80% −70%) to water was used. The paraffin slices was stained with hematoxylin-eosin (H&E), incubated in hematoxylin solution for about 10 minutes, washed with tap water for about 20-30 mins. A gradient of alcohol (70% −80% −90% −100%) was used for dehydration. The slices was fixed with xylene for two hours, sealed with neutral gum and air dried. After completion of staining, the slide was placed under a microscope to observe lung metastasis. The regions of interest were observed, photographed and recorded.

(41) 1.3.6 Flow Cytometry Analysis

(42) At 0 day before the treatment (i.e., 21 days after inoculation) and 7 days, 14 days, 21 days and 28 days after treatment, mice were sacrificed, and spleen and peripheral blood of mice in each group were taken to analyze immunized cells. The spleen of mice was taken and placed on a 70 micrometer nylon cell filter, milled with a 5 ml syringe and rinsed with DMEM into a 50 ml centrifuge tube. After centrifuging at 2000 rpm for 10 minutes, the supernatant was discarded, and 2 ml erythrocyte lysate was added and blown evenly, at room temperature for 5 minutes. Add PBS and after dilution as much as possible abandon the supernatant, then get the spleen white blood cells. After resuscitation, add 1 μl corresponding fluorescent antibody (label the cell surface molecules to detect the specific cell populations, specific labeling in table 7) and incubate for 30 minutes at 4° C. After washed by PBS and then resuspended, carry into the flow tube on the machine to detect. In addition, the function of T cells was further identified in this experiment, and labeled CD4.sup.+ T cells and CD8.sup.+ T cells were sorted by flow cytometry.

(43) 1.3.7 Data Statistics

(44) The statistics and analysis of all experimental data in experiments were performed by using Graph pad Prism software, and Student's t test was used to analyze the difference between groups. The results were shown as mean±standard deviation. Image Pro Plus software was used to analyze area data of lungs metastasis. The FlowJo software was used to process image of flow cytometry.

(45) 2 Results

(46) 2.1 Change of Level of CD4.sup.+ T Cells in Spleen

(47) It was found in the study that the anti-tumor immune cells (CD4.sup.+/CD8.sup.+ T cells) in spleen in only-heat-treatment group and resection group were similar to those in alternate cold and heat treatment group within 14 days after treatment. All showed a tendency of increase. 14 days after treatment, CD4.sup.+/CD8.sup.+ T cells began to decrease gradually in spleen in only-heat-treatment group and resuscitation group, and at day 28, CD4.sup.+/CD8+ T cells in only-heat-treatment group and surgery resection group decreased almost to the same level as that in control group. The levels of CD4.sup.+/CD8.sup.+ T cells were significantly increased in alternate heat and heat treatment group within 21 days, and the level of CD8.sup.+ T cells began to decrease after 21 days, but the level of CD4.sup.+ T cells was still significantly increased. The results of experiment are shown in FIG. 1. FIG. 1 shows change of immune cells in spleen induced in different treatment groups.

(48) 2.2 Cold and Heat Treatment Improves Survival Percentage of Mice

(49) The mice were observed for 3 months after treatment, and there were 6 mice in each group. After alternate cold and heat treatment, 4 mice of them had a good living condition, and almost all of mice died in control group, only-heat-treatment group and surgery resection group. Therefore, alternate cold and heat treatment of tumor could greatly improve survival percentage of mice, and achieve a good therapeutic effect. FIG. 6 shows the overall survival percentage and survival time of mice in different groups (6 mice in each group).

(50) 2.3 Cold and Heat Treatment Inhibits Tumor Metastasis

(51) The mice in surgery resection group, only-cold-treatment group and only-heat-treatment group began to appear metastasis in their body surface three weeks after treatment (see FIG. 3). After alternate cold and heat treatment, tumor began to subside, to scab, and even to ablation (as shown in FIG. 4). Especially after 6 weeks of alternate cold and heat treatment, the in situ tumors in tumor-bearing mice were completely ablated and there was no metastasis tumor on their body surface, and the mice lived well. FIG. 3 shows tumor metastasis of mice three weeks after treatment wherein (A) is the surgical resection treatment group, (B) is the only-cold-treatment group, and (C) is the only-heat-treatment group. FIG. 4 shows the mice within 6 weeks after alternate cold and heat treatment wherein (A) is before treatment; (B) is after treatment; (C) is 1 week after treatment; (D) is 3 weeks after treatment; and (E) is 6 weeks after treatment.

(52) Then the paraffin sections of lung of mice in different treatment groups were stained with hematoxylin and observed. The lung metastasis was compared between the control group and different treatment groups (see FIG. 5). It was found in the study that in the surgical resection group, there was abnormal lung structure and significant tumor metastasis, and there was no significant difference of tumor metastasis area when compared with the control group. There were some small metastatic nodules in the only-heat-treatment group and lung tissue structure was relatively normal, and the area of lung tumor metastasis was significantly decreased. In the alternate cold and heat treatment group, some small metastatic nodules were observed, the lung tissue structure was relatively normal, and the area of tumor metastasis was decreased most significantly. FIG. 5 shows lung metastases of tumor 28 days after treatment, and it can be seen that the metastatic area in the alternate cold and heat treatment group is significantly lower than those in other groups.

(53) Referring to FIG. 6, FIG. 6 illustrates a treatment device 1 for increasing a level of CD4.sup.+T cells in a mammal having a tumor in accordance with an embodiment. The treatment device 1 can include a CD4.sup.+T cell level monitor unit 5 for detecting the level of CD4.sup.+T cells in peripheral blood 30, a cold treatment unit 10, a heat treatment unit 15, a contact head 25, and a temperature monitoring unit for monitoring the temperature of the cancerous tissue 35.

3. CONCLUSION AND DISCUSSION

(54) In this study, 4T1 breast cancer was used as a model to study efficacy of alternate cold and heat treatment of local tumor on CD4.sup.+ and CD8.sup.+ T cells in peripheral blood and spleen. The results surprisingly showed that after alternate cold and heat treatment of tumor tissue, CD4.sup.+ and CD8.sup.+ T cells in mice were significantly increased within 21 days, but after 21 days, the level of CD8.sup.+ T cells in mice began to decrease, while the level of CD4.sup.+ T cells began to increase significantly with the decrease of CD8.sup.+ T cells.

(55) Studies have shown that CD4.sup.+ T cells also have cytotoxic effects.sup.[1]. In specific cases, such as when lymphopenia cells decrease, CD4.sup.+ T cells can obtain an ability to directly kill cells.sup.[2, 3]. In CD4.sup.+ T cells, there are mainly Fas pathway and another INF-related apoptosis-inducing ligand pathway to mediate their cytotoxic effects.sup.[1]. In this study, it has been found that, compared with CD8.sup.+ T cells, CD4.sup.+ T cells in spleen and blood are more significantly increased after alternate cold and heat treatment, and also can secrete cytotoxic cytokines such as IFN-γ, so they have strong cytotoxic effects. Further, in the peripheral blood, 14 days after treatment, when compared with other groups in which CD4.sup.+ T cells decreased significantly, the same cells in alternate cold and heat group maintained a high level for a long period. These experimental data suggest that CD4.sup.+ T cells play a more important role. The present invention can induce CD4.sup.+ T cells by cold and heat treatment, enhance anti-tumor immunity in the body, significantly reduce mortality of tumor-bearing mice, and extend survival time of mice in the experiment.

(56) All the documents cited herein are incorporated into the invention as reference, as if each of them is individually incorporated. Further, it would be appreciated that, in light of the above described teaching of the invention, the skilled in the art could make various changes or modifications to the invention, and these equivalents would still be in the scope of the invention defined by the appended claims of the application.

REFERENCES

(57) [1]. Martorelli, D., Muraro, E., Merlo, A., et al., Role of CD4.sup.+ cytotoxic T lymphocytes in the control of viral diseases and cancer [J]. International reviews of immunology, 2010, 29 (4), pp 371-402. [2]. Hirschhorn-Cymerman, D., Budhu, S., Kitano, S., et al., Induction of tumoricidal function in CD4.sup.+ T cells is associated with concomitant memory and terminally differentiated phenotype [J]. The Journal of experimental medicine, 2012, 209 (11), pp 2113-2126. [3]. Quezada, S. A., Simpson, T. R., Peggs, K. S., et al., Tumor-reactive T cells develop cytotoxic activity and eradicate large established melanoma after transfer into lymphopenic hosts [J]. The Journal of experimental medicine, 2010, 207 (3), pp 637-650.