Animal surface temperature profile captured using infrared camera is helpful for the assessment of physiological responses associated with the regulation of body temperature. Diagnosing breast cancer in early stage itself has a greater effect on the prognosis. In this work, asymmetrical temperature distribution analysis of chemical carcinogen 7,12-dimethyl benz(a)anthracene-induced in the lower right flank region of Wistar rats (
Breast cancer is an uncontrolled growth of breast cells originating from breast tissue, most commonly from the inner lining of milk ducts or the lobules as a result of mutations in the genes responsible for regulating the growth of cells and keeping them healthy. Worldwide 23% (1.38 million) of the total new cancer cases and 14% (458,400) of the total cancer deaths occurred in 2008.The estimated number of new breast cancer cases has been raised from about 641,000 cases in 1980 to 1.6 million cases in 2010 and 625,000 deaths in 2010 [
Thermographic imaging could detect temperature changes as small as 0.1°C decrease on the skin surface at an early stage of tumor and it was proven in xenografts (MDAMB-231, MCF7) induced nude mice model [
Female Wister rats (
20 mg of 7,12-dimethylbenz(a)anthracene (DMBA) purchased from Sigma Chemicals was mixed with 0.5 ml sunflower oil and 0.5 ml saline to induce breast cancer in each rat. A single dose injection of DMBA was given subcutaneously into the right flanks only in test group I. 0.5 ml of sunflower oil and 0.5 ml of saline alone injected in the left flanks control side in all six rats and group II animals (control) were injected with 0.5 ml of sunflower oil and 0.5 ml of saline in both right and left flank region.
Infrared camera (FLIR T400, FLIR Systems, Boston, MA, USA) with wide temperature range of −4 to 2192°F (−20 to 1700°C), FOV 25° × 19°, and thermal sensitivity of 0.1°C was used in this study. As a baseline anterior to posterior view of whole body, thermal images of all the rats were taken under standard conditions at constant distance of (12 cm). Then immediately after chemical carcinogen injection starting from the first day thermal images was taken every day over a period of nine weeks.
When the images are relatively symmetrical, small asymmetries may indicate suspicious region. Based on this physiology, tumor region can be identified by the analysis of asymmetrical temperature distribution. For asymmetrical temperature distribution analysis, the whole animal thermal image was divided into six region of interests (ROI), with size approximately 2 cm
2 ml of blood was collected from each rat and serum was separated. Carcinoembryonic antigen levels (ngm/ml) in the serum of experimental animals were estimated by the ELISA kit after the MC has developed to a palpable size in the ninth week.
Tumors were removed from sacrificed rats and immediately fixed in 10% formalin fixative for 24 h. The tissues were then dehydrated in ascending series of alcohol, kept in 1 : 1 mixture of absolute alcohol and benzene, and then in benzene for 1 h each. Finally, tissue pieces were embedded in paraffin wax and 7 micron thick sections were cut and spread on glass slides, stained with hematoxylin and eosin, slides mounted in DPX, and viewed under light microscope and photographed.
Mean and standard deviation of skin temperature of ROIs for all the rats in group I were calculated for every week. Student’s
Mean and standard deviation of temperatures for different regions were calculated every week and tabulated. Table
Comparison of mean skin temperature region-wise (flank region, abdomen region, and shoulder region).
Week | Tumor induced site | Nontumor site | Nontumor site | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Lower right flank region temperature (°C)—mean ± SD | Lower left flank region temperature (°C)—mean ± SD | Difference in temperature between right flank and left flank region (°C) | Percentage of asymmetrical temperature difference | Right abdomen region temperature (°C) —mean ± SD | Left abdomen region temperature (°C) —mean ± SD | Difference in temperature between right abdomen and left abdomen region (°C) | Percentage of asymmetrical temperature difference | Right shoulder region temperature (°C)—mean ± SD | Left shoulder region temperature (°C)—mean ± SD | Difference in temperature between right shoulder and left shoulder region (°C) | Percentage of asymmetrical temperature difference | |
Base line (Before induction) | 38.98 ± 0.28 | 39 ± 0.13 | 0.02 | 0.05 | 38.62 ± 0.19 | 38.72 ± 0.07 | 0.1 | 0.26 | 38.5 ± 0.46 | 38.64 ± 0.37 | 0.14 | 0.36 |
Immediately after induction | 36.62 ± 0.2 | 36.8 ± 0.24 | 0.18 | 0.49 | 36.52 ± 0.24 | 36.58 ± 0.39 | 0.06 | 0.16 | 36.46 ± 0.34 | 36.62 ± 0.29 | 0.16 | 0.44 |
Week 1 | 36.32 ± 0.6 | 36.53 ± 0.55** | 0.21 | 0.58 | 36.19 ± 0.5 | 36.38 ± 0.66 | 0.18 | 0.51 | 36.86 ± 0.67 | 36.88 ± 0.6 | 0.01 | 0.04 |
Week 2 | 35.61 ± 0.08 | 35.84 ± 0.11* | 0.24 | 0.65 | 35.74 ± 0.55 | 35.74 ± 0.64 | −0.00 | −0.009 | 36.33 ± 0.4 | 36.26 ± 0.58 | −0.07 | −0.18 |
Week 3 | 35.18 ± 0.59 | 35.52 ± 0.66** | 0.34 | 0.95 | 35.44 ± 0.74 | 35.53 ± 0.62 | 0.09 | 0.25 | 35.93 ± 0.60 | 35.86 ± 0.50 | −0.07 | −0.19 |
Week 4 | 35.54 ± 0.74 | 36.1 ± 0.74* | 0.56 | 1.56 | 35.94 ± 0.64 | 36.12 ± 0.60 | 0.19 | 0.52 | 36.14 ± 0.86 | 36.28 ± 0.69 | 0.14 | 0.38 |
Week 5 | 36.41 ± 0.65 | 36.82 ± 0.57** | 0.42 | 1.13 | 36.89 ± 0.58 | 36.88 ± 0.54 | −0.02 | −0.04 | 37.14 ± 0.60 | 37.14 ± 0.59 | 0 | 0 |
Week 6 | 37.09 ± 0.64 | 37.33 ± 0.56* | 0.24 | 0.63 | 37.48 ± 0.57 | 37.38 ± 0.51 | −0.1 | −0.27 | 37.61 ± 0.53 | 37.46 ± 0.53 | −0.15 | −0.40 |
Week 7 | 36.71 ± 0.32 | 37.05 ± 0.35** | 0.34 | 0.92 | 37.14 ± 0.38 | 37.19 ± 0.38 | 0.05 | 0.12 | 37.51 ± 0.35 | 37.51 ± 0.38 | 0 | 0 |
Week 8 | 36.55 ± 1.06 | 36.81 ± 1.07* | 0.26 | 0.71 | 36.77 ± 0.38 | 36.81 ± 0.37 | 0.04 | 0.11 | 37.15 ± 0.35 | 37.14 ± 0.34 | −0.01 | −0.027 |
Temperature distribution was symmetrical (difference in temperature between right and left side was not significant) in the base line image before induction and also immediately after induction on zeroth day in all the regions and the value of asymmetrical temperature difference percentage was <0.5%.
Similarly temperature distributions in the right and left abdomen region were also compared. From Table
Temperature distributions in the right and left shoulder region were also compared and tabulated in Table
Average skin temperature of all the tumor induced groups calculated weekly was plotted separately in all the three regions (flank, abdomen, and shoulder region) to visualize right and left side temperature variations. From Figures
Average skin temperature in different regions (a) in the tumor induced lower flank region, (b) in the abdomen region, and (c) in the shoulder region. Mean ± SD temperature values (week wise) for 6 tumor induced rats from the specific ROIs of right and left side for all regions (lower flank region, abdomen region, and shoulder region) were plotted. There was difference in temperature observed between right and left flank regions. Tumor induced right side (lower right flank side) temperature was less in all the weeks. There was no difference in temperature observed between right and left side of abdomen and shoulder regions.
Percentage of asymmetrical temperatures distribution over the period of study still ninth week of the entire region were plotted. From Figure
Percentage asymmetrical difference in skin temperature comparison between lower flank (tumor induced region), abdomen, and shoulder regions. The value of asymmetrical difference in skin temperature between left and right side ranges from 0.5 to 2.0% in the tumor induced flank region, whereas in the nontumor site (abdomen and shoulder regions) it was less than 0.5%.
To see the progress of tumor development, the thermogram ion images were converted into medical images which assign different colors to different span of temperatures using FLIR software. Figure
Whole body digital infrared images of a single rat taken on different weeks ((a) week 1, (b), week 2, (c) week 3, and (d) week 4) after tumor cancer induction by DMBA which show the tumor growth by color change from thick orange (a), then light orange (b), then red color(c), and then to red color with pink center in the fourth week indicating a reduction in skin temperature progressively.
The color image was separated into different components as red, green, and blue with different bins and the respective histogram was plotted using MATLAB program for all the six ROIs and compared symmetrically. Asymmetrical green component, that is, the green pixel distribution was between 100 and 150 (Figure
RGB histogram of single rat thermal image in different regions (a) lower right (tumor induced) flank region, (b) lower left flank region, (c) right abdomen region, (d) left abdomen region, (e) right shoulder region, and (f) left shoulder region. Red and blue pixel distributions were symmetrical between right and left sides in tumor induced flank region except green pixel distribution (asymmetrical). All red, green, and blue pixel distributions were symmetrical between corresponding right and left sides in nontumor sites (abdomen and shoulder regions).
RGB histogram of single rat (control group II) thermal image in different regions (a) lower right flank region, (b) lower left flank region, (c) right abdomen region, (d) left abdomen region, (e) right shoulder region, and (f) left shoulder region. Red, blue, and green pixel distributions were symmetrical between right and left sides in all the regions (flank, abdomen, and shoulder).
In the RGB histogram analysis, asymmetry was observed only in the green components, whereas in red and blue components there was no predictable change.
Serum CEA level measured using ELISA kit also showed significant difference between mammary cancers induced group I and control group II. The mean tumor size was about 1.35 cm in group I and the value of CEA level was 0.426 ± 0.05 (ngm/ml). In control group II CEA level was 0.186 ± 0.01 (ngm/ml).
Histopathological results showed ductal carcinoma (grade III) in (
(a) Single rat with ductal carcinoma slides at different magnification. Arrow indicates ductal cells arranged in tubular pattern and tumor with 50% ductal differentiation, (b) Single rat with metastasis stage carcinoma slides at different magnifications. Arrow indicates lymph node with metastasis deposits.
In this study, whole-body thermal images of the rats with induced tumor were obtained dynamically without anesthesia in order to rule out heat loss associated with anesthetic drugs. D. Colman et al. reported a significant amount of heat loss in an anesthetized Wister rat. The decrease in skin temperature, compared to the base line measurement after 30 minutes of induction, were found to be 1.75°C, 2.17°C, and 1.9°C for the anesthetic drugs halothane, isofluorane, and sevoloflurane, respectively, [
In a xenograft induced breast tumor nude mice model using MDA-MB-231 and MCF7, the induced breast tumor had a lesser skin temperature on 6th day of tumor induction, when comparing to nontumor parts of the mice. It was reported that the measured skin temperatures at the tumor site were lesser by 1.5°C and 3°C in MDA-MB-231 and MCF7 induced tumor mice models, respectively, [
In a study of breast tumor in women using thermography, it was reported that the measured skin temperature at the tumor site was higher than the non-tumor part of the breast [
In the present study, the baseline measurements (before tumor induction) showed that the mean and SD value of skin temperature of whole-body rat was 38.98 ± 0.28. After tumor induction by chemical carcinogen, the measured mean and SD skin temperatures of the whole-body rat was found to be decreased progressively and it was 35.18 ± 0.59 during third week after tumor induction. The reason for reduction in mean whole-body skin temperature of the tumor bearing rat comparing to the baseline measurements may be due to the following: (i) poor functioning of newly developed blood vessels, and (ii) decreased blood flow at the tumor site by an autonomic nervous system [
Increase in temperature was observed from fourth week. This may be due to metastasis condition, that is, because of the spreading of cancer to other regions. This increase was because of angiogenesis and higher metabolic rate of well-developed cancerous cells.
From the first week to fourth week of the study, asymmetrical variation of skin temperature was visually observed (by noting changes in colors of the thermogram) in all the rats (Figures
thermal texture mapping (TTM) was found to be superior method for detection of malignant among the benign diseases of breast than mammography and ultrasound images [
The non-tumor sites of abdomen and shoulder region of the rat showed symmetrical red, green, and blue pixel distributions between right and left sides of the rats. The measured red, green, and blue pixel distributions at abdomen region of the rat were found to be 240 to 255, 175 to 220, and 0 to 10, respectively, and these values were the same in both right and left sides. Similarly, in the shoulder region of the rat, the measured red, green, and blue pixel distributions were found to be 240 to 255, 200 to 240, and 0 to 50, respectively, which were the same in both right and left sides.
This study had few limitations. In this study, the number of rats used in this model was limited. Additional clinical and animal studies are required to prove whether thermography is useful in predicting the breast tumor well before. The finding of this study should be taken with the caution that it was related with small animal breast tumor model.
The findings of the study indicate that asymmetrical analysis of thermal images might have considerable potential in monitoring progress of tumor and also diagnosis of cancer in early stage itself. Significant (<0.01) difference in asymmetrical skin temperature distribution was observed in tumor induced lower region, whereas in other regions temperature distribution was symmetrical. This was proven by asymmetrical pixel distribution in RGB histogram. And also green component of the infrared image plays an important role in the asymmetrical histogram analysis for diagnosing tumor in animal model with decrease in skin temperature. Further this study can be extended for studying the response to anticancer drugs.