Optical imaging of the peri-tumoral inflammatory response in breast cancer
© Sista et al; licensee BioMed Central Ltd. 2009
Received: 24 June 2009
Accepted: 11 November 2009
Published: 11 November 2009
Peri-tumoral inflammation is a common tumor response that plays a central role in tumor invasion and metastasis, and inflammatory cell recruitment is essential to this process. The purpose of this study was to determine whether injected fluorescently-labeled monocytes accumulate within murine breast tumors and are visible with optical imaging.
Materials and methods
Murine monocytes were labeled with the fluorescent dye DiD and subsequently injected intravenously into 6 transgenic MMTV-PymT tumor-bearing mice and 6 FVB/n control mice without tumors. Optical imaging (OI) was performed before and after cell injection. Ratios of post-injection to pre-injection fluorescent signal intensity of the tumors (MMTV-PymT mice) and mammary tissue (FVB/n controls) were calculated and statistically compared.
MMTV-PymT breast tumors had an average post/pre signal intensity ratio of 1.8+/- 0.2 (range 1.1-2.7). Control mammary tissue had an average post/pre signal intensity ratio of 1.1 +/- 0.1 (range, 0.4 to 1.4). The p-value for the difference between the ratios was less than 0.05. Confocal fluorescence microscopy confirmed the presence of DiD-labeled cells within the breast tumors.
Murine monocytes accumulate at the site of breast cancer development in this transgenic model, providing evidence that peri-tumoral inflammatory cell recruitment can be evaluated non-invasively using optical imaging.
The intimate association between cancer and inflammation was first identified over a century ago. The role of the immune system in modulating carcinogenesis is complex; some aspects of the immune response are protective, while others are pro-tumorigenic. Several findings support the suggestion that inflammation plays a role in promoting breast cancer. From an epidemiologic perspective, immunocompromised individuals, such as organ transplant recipients, have a lower incidence of breast cancer [1, 2]. It has also been noted that as breast cancer progresses, there is a corresponding increase in the number of leukocytes, both of lymphoid and myeloid origin, surrounding the tumor .
There are several proposed mechanisms by which the immune response may promote breast cancer development. Infiltrating immune cells elaborate cytokines, chemokines, metalloserine and metallocysteine proteases, reactive oxygen species, and histamine, all of which augment tumor remodeling and angiogenesis [4–6]. Chronic B-cell activation and helper T-cell polarity towards the Th2 subtype are also thought to play roles in supporting tumorigenesis [7–10].
Tumor associated macrophages/monocytes are also thought to promote tumor development through the elaboration of tumor growth factors, proangiogenic substances, matrix degrading proteins, and DNA-disrupting reactive oxygen species [11–15]. In the mouse mammary tumor virus - polyomavirus middle T antigen (MMTV-PymT) transgenic mouse model, macrophage infiltration into premalignant breast lesions is associated with tumor progression . Moreover, limiting macrophage infiltration reduces tumor invasion and metastasis in this model . In humans, elevated levels of CSF-1 and exuberant macrophage recruitment are associated with poor prognosis [13, 15, 18].
The MMTV-PymT transgenic murine model of breast cancer is a well characterized model which recapitulates human disease, with progression from hyperplasia to invasive carcinoma and metastatic disease at ~115 days of life [3, 18]. As described above, a significant inflammatory response, populated by B and T lymphocytes, macrophages/monocytes, and mast cells, accompanies breast tumor development.
With this background, the purpose of this study was to use optical imaging to non-invasively monitor the peri-tumoral inflammatory response in the MMTV-PymT transgenic mouse by tracking monocyte recruitment. A technique based on the detection of fluorescence, optical imaging (OI) is a relatively new modality in the clinical setting. Compared with other imaging modalities, optical imaging is inexpensive, easy and fast to perform, highly sensitive, and radiation-free. In addition, breast cancer patients have been previously scanned using optical imaging; initial results indicate that this technique may supplement mammography and magnetic resonance imaging in breast cancer detection [19, 20]. Our group and others have established optical imaging-based "leukocyte scans" by labeling leukocytes with fluorochromes ex vivo, intravenously injecting them into experimental animals, and subsequently tracking the labeled cells with optical technology. These scans have been used to detect and monitor treatment of arthritis  and to track cytotoxic lymphocytes to implanted tumors .
Optically tracking monocytes to breast tumors in the MMTV-PymT model has several potential utilities. First, the temporal relationship between breast tumor development and inflammation could be better characterized, without having to sacrifice animals. Second, evaluating the extent of monocyte recruitment may have prognostic implications, as described previously. Third the effect of anti-inflammatory and chemotherapeutic regimens on peri-tumoral inflammation and monocyte recruitment could be assessed.
Materials and methods
Murine monocytes were obtained from the continuously growing leukemic cell line, 416B (Cell Culture Facility, University of California, San Francisco, ECACC equivalent 85061103) and cultured in Dulbecco's Modified Eagle Medium (DMEM) high glucose medium supplemented with 10% fetal bovine serum and 1% Penicillin/Streptomycin. 416B monocytes were grown in this medium as a non-adherent suspension culture at 37°C in a humidified 5% CO2 atmosphere.
In vitro cell labeling
Triplicate samples of 1, 2, and 4 million monocytes/mL of serum-free DMEM were incubated with a solution of the fluorochrome DiD at a ratio of 5 μL DiD/1 mL DMEM for 15 minutes at 37 degrees C. DiD (C67H103CIN2O3S,: Vybrant cell labeling solution, Invitrogen) is a non-targeted, lipophilic, carbocyanine fluorochrome with a molecular weight of 1052.08DA and excitation and emission maximum of 644 nm and 665 nm respectively. The labeled cells were washed 3 times with phosphate-buffered saline (PBS) (pH 7.4) by sedimentation (5 min, 400 rcf, 25°C). The labeled monocytes were placed in the Xenogen IVIS 50 optical imager (Xenogen Corporation, Alameda, CA) and scanned. Flow cytometry using Cytomics FC500 flow cytometer (Beckman-Coulter Inc., Fullerton, CA) was performed on labeled cells to confirm integration of DiD. Triplicate samples of 2 million cells labeled with 5 microliters of DiD were optically imaged at 24 hours to determine persistence of labeling.
2 million 416B monocytes in 2 mL DMEM were incubated for 15 minutes with 0-20 microliters of DiD, with the total volume of 20 microliters being completed with ethanol. Trypan blue testing of the labeled cells was then performed to determine viability. Additionally, 2 million 416B monocytes in 2 mL DMEM were incubated for 15 minutes with 5 microliters of DiD, and viability of cells was assessed 24 hours after labeling with trypan blue staining.
Ex vivo cell labeling
Samples of 107 monocytes were incubated for 15 minutes with 25 μl of DiD in 5 ml (Concentration: 5 microliters DiD/1 ml DMEM) of serum free DMEM and then washed 3 times with phosphate-buffered saline (PBS) (pH 7.4) by sedimentation (5 min, 400 rcf, 25°C) prior to intravenous injection.
This study was approved by the animal care and use committee at our institution. All imaging procedures as well as monocyte injections were performed under general anesthesia with 1.5-2% isoflurane in oxygen, administered via face mask. Studies were carried out in twelve mice: six MMTV-PymT trangenic mice (age range 95-115 days) and six FVB/n control mice. For cell injections, either an internal jugular or femoral vein direct cannulation was performed with a 30-guage needle. Labeled cells were suspended in a total volume of 350 microliters of PBS prior to injection. The cell-free DiD infusion was performed by injecting a solution consisting of 5 microliters of DiD and 345 microliters of PBS intravenously. Peripheral blood for flow cytometry analysis was obtained via cardiac puncture.
All optical imaging studies were performed using the IVIS 50 small animal scanner (Xenogen, Alemeda, CA) and Cy5.5 (excitation: 615-665 nm and emission: 695-770 nm passbands) filter set. For in vitro studies, cell samples were placed in a non-fluorescing container. For in vivo studies, mice were anesthetized with isofluorane and placed in the light-tight heated (37 degrees celsius) chamber. After being shaved, the animals were imaged in three positions at all time points: (1) anterior (facing the CCD camera), (2) left lateral decubitus, and (3) right lateral decubitus. Identical illumination parameters (exposure time = 2 seconds, lamp level = high, filters = Cy5.5 and Cy5.5 bkg, f/stop = 2, field of view = 12, binning = 4) were selected for each acquisition. Gray scale reference images were also obtained under low-level illumination. Optical imaging scans were obtained before and at 1, 2, 6, and 24 hours after intravenous monocyte injection. After completion of the scans, the animals were sacrificed via a combination of cardiac puncture and cervical dislocation while under anesthesia. Tissues were immediately harvested for sectioning and microscopic analysis.
OI Images were analyzed using Living Image 2.5 software (Xenogen, Alameda, Ca) integrated with Igorpro (Wavemetrics, Lake Oswego, OR, USA). Images were measured in units of average efficiency (fluorescent images are normalized by a stored reference image of the excitation light intensity and thus images are unitless) and corrected for background signal. For in vitro image analysis, regions-of-interest (ROI) were defined as the circular area of the tube. For in vivo image analysis, ROIs were placed around breast tumors (MMTV-PymT mice) and mammary tissue (FVB/n controls). The post to pre-injection fluorescence signal intensity (SI post/pre) was then calculated for each ROI.
All in vitro experiments were performed in triplicates. Data were displayed as means plus/minus the standard error of the mean (SEM). Student t-tests were used to detect significant differences between labeled and unlabeled monocytes (in vitro data) and breast tumor and control mammary tissue (in vivo data). Statistical significance was assigned for p values < 0.05.
Immune Fluorescence and Confocal Analysis
Tumors were explanted 24 hrs after monocyte injection and preserved in OCT at -80°C. 5 μm thick slides were prepared which were then processed for immunostaining. CD45 immunostaining (eBioscience, San Diego, CA) was performed to visualize murine monocytes in the tumor, while the tumor nuclei were mounted with a mounting medium containing DAPI (Vectashield Mounting medium with DAPI, Vector Laboratories, Burlingame, CA). Confocal analysis was performed using a Zeiss LSM510 confocal microscopy system equipped with krypton-argon (488, 568 and 633 nm) and ultraviolet (365 nm) lasers; images were acquired using LSM version 5. Images are magnified to 10×. The images presented are representative of four independent experiments. All images were converted to TIFF format and arranged using Adobe Photoshop CS2.
DiD labeled and unlabeled murine monocytes were resuspended in PBS/BSA and incubated for 10 min at 4°C with rat anti-mouse CD16/CD32 mAb (BD Biosciences, San Diego, CA) at a 1:100 dilution in FACS buffer to prevent nonspecific antibody binding. After incubation and washing, the cells were incubated with anti-CD45-PE (pan-leukocyte marker), anti-CD11b-PE (monocyte and macrophage marker), anti-Gr1-FITC (granulocyte marker), and anti-F4/80-FITC (macrophage marker) (eBioscience) for 20 min with 50 μl of 1:100 dilution of primary antibody followed by two washes with PBS/BSA. 7-AAD (BD Biosciences) was added (1:10) to discriminate between viable and dead cells. Data acquisition and analysis were performed on a FACSCalibur using CellQuestPro software (BD Biosciences). DiD was visualized using the FL4 channel.
In vitro optical imaging
Cell viability as a function of DiD concentration.
Amount of DiD added
Cell Viability (%)
In vivo optical imaging
The above results demonstrate that after intravenous injection of fluorochrome-labeled monocytes, there was progressive fluorescence within the breast tumors of MMTV-PymT mice, a phenomenon not seen in the mammary tissue of FVB/n control mice. Fluorescence microscopy confirmed that DiD-labeled monocytes were present within breast tumors, though the lack of uniform DiD-fluorescence distribution in the tumor specimens is likely a reflection of the heterogenous distribution of tumor-associated macrophage recruitment within the tumor microenvironment. The scattered presence of CD45 positive but DiD negative regions may either be reflective of endogenous murine monocytes recruited to the tumor simultaneously, or, alternatively, exogenous monocytes that were ineffectively labeled with DiD before intravenous injection. Nonetheless, taken together, it can be concluded that intravenously injected, fluorescently-labeled monocytes accumulate within breast tumors in this transgenic murine model of breast cancer, where they can be visualized with optical imaging technology. Flow cytometry validated the murine monocyte cell line 416B as being a legitimate and relevant cell line for this study, as these cells have expression patterns similar to monocytes isolated from the peripheral blood of control mice.
Thus far, molecular imaging techniques have focused on imaging cancer cells themselves, proteins that are overexpressed by cancer cells, angiogenic markers, or the extracellular matrix surrounding cancer [23–25]. The inflammatory component of cancer biology, on the other hand, has not been a major target of molecular imaging technologies. Inflammation has been evaluated in other contexts, such as in a mouse model of type 1 diabetes , and a rat model of arthritis . Inflammatory macrophages in atherosclerotic plaques have also been imaged with magnetic resonance using superparamagentic iron oxide particles . Genetically engineered T lymphocytes have been tracked to animal tumors using microPET technology [28, 29]. Superparamagnetic iron-oxide labeling and subsequent MR imaging of immune cells have been employed as a strategy to monitor anti-cancer cellular therapy . Monocytes have been labeled with MR contrast agents and tracked to rat gliomas . However, to our knowledge, this is the first demonstration of tracking fluorescently labeled monocytes to breast cancer using optical imaging.
The mechanism by which these cells are recruited to breast tumors in MMTV-PymT mice is multifactorial, and may be related to vascular permeability and local factors released by tumor cells, stromal cells, and inflammatory cells. Elaboration by these cells of the inflammatory chemokine CCL2 (MCP-1) is associated with both monocyte recruitment and poor prognosis [32, 33]. Jin et al. demonstrated a role for integrin alpha 4 beta 1 in the homing of monocytes to tumors. Specifically, the group noted that blocking this integrin in a mouse model of implanted lung cancer suppressed the number of macrophages within tumors and also stunted tumor growth . CSF-1 release by tumor cells is also thought to play a role [35, 36]. The relative contributions of these various factors to monocyte recruitment may potentially be further characterized using the imaging technique described here.
There are several limitations to the current study. As this was a proof of principle study, a limited number of animals was used to obtain statistical significance. A larger sample size would provide further characterization of the inflammatory response and monocyte recruitment. Second, while the pathogenesis of breast cancer seen in this animal model closely resembles that in humans, there may be significant differences between the two species. Third, while this technique has potential clinical applications, DiD has not received FDA approval. Given that other cyanine dyes have significant toxicity, further studies will be required to determine the safety of DiD. It should be noted, however, that another cyanine fluorescent dye, Indocyanine Green (ICG), has received FDA approval.
In conclusion, tracking monocytes non-invasively will lead to a better temporal and pathophysiological understanding of the in vivo inflammatory response around breast cancers. Moreover, this imaging technique could be used as a supplemental prognostic tool, given the aforementioned inverse correlation between the degree of monocyte recruitment and prognosis. In addition, the presented technique could streamline the development of novel chemotherapeutic and anti-inflammatory pharmaceuticals for breast cancer treatment . For example, following intravenous injection of fluorophore labeled leukocytes, the efficacy of such agents could be assessed by the degree of monocyte accumulation within tumors. Given the recent development of handheld OI scanners and dedicated OI breast scanners, the imaging technique described here has the potential to directly impact clinical decision making and drug development in the breast cancer arena.
Dr. Sista was supported by a T32 training grant from the National Institute of Biomedical Imaging and Bioengineering (NIBIB). Dr. Coussens was supported by grants from the National Institutes of Health (CA72006, CA94168, CA098075) and a Department of Defense Era of Hope Scholar Award (BC051640). The project described was also supported by Award Number R21CA129725 from the National Cancer Institute and a University of California San Francisco, Department of Radiology and Biomedical Imaging seed grant, #07-02.
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