Elevated mPer1 gene expression in tumor stroma imaged through bioluminescence

Authors

  • Michael E. Geusz,

    Corresponding author
    1. Department of Biological Sciences, Bowling Green State University, Bowling Green, OH
    • Department of Biological Sciences, Bowling Green State University, 217 Life Sciences Bldg, Bowling Green, OH 43403-0208, USA
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    • Tel: 419-372-2433, Fax: 419-372-2024

  • Kenneth T. Blakely,

    1. Department of Biological Sciences, Bowling Green State University, Bowling Green, OH
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  • Daniel J. Hiler,

    1. Department of Biological Sciences, Bowling Green State University, Bowling Green, OH
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  • Roudabeh J. Jamasbi

    1. Department of Biological Sciences, Bowling Green State University, Bowling Green, OH
    2. Department of Public and Allied Health, Bowling Green State University, Bowling Green, OH
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Abstract

The tumor stroma has significant effects on cancer cell growth and metastasis. Interactions between cancer and stromal cells shape tumor progression through poorly understood mechanisms. One factor regulating tumor growth is the circadian timing system that generates daily physiological rhythms throughout the body. Clock genes such as mPer1 serve in molecular timing events of circadian oscillators and when mutated can disrupt circadian rhythms and accelerate tumor growth. Stimulation of mPer1 by cytokines suggests that the timing of circadian oscillators may be altered by these tumor-derived signals. To explore tumor and stromal interactions, the pattern of mPer1 expression was imaged in tumors generated through subcutaneous injection of Lewis lung carcinoma (LLC) cells. Several imaging studies have used bioluminescent cancer cell lines expressing firefly luciferase to image tumor growth in live mice. In contrast, this study used non-bioluminescent cancer cells to produce tumors within transgenic mice expressing luciferase controlled by the mPer1 gene promoter. Bioluminescence originated only in host cells and was significantly elevated throughout the tumor stroma. It was detected through the skin of live mice or by imaging the tumor directly. No effects on the circadian timing system were detected during three weeks of tumor growth according to wheel-running rhythms. Similarly, no effects on mPer1 expression outside the tumor were found. These results suggest that mPer1 activity may play a localized role in the interactions between cancer and stromal cells. The effects might be exploited clinically by targeting the circadian clock genes of stromal cells.

Increasing evidence shows that cancer cells are influenced by the circadian rhythms of the body.1–4 Disruption of circadian clock genes that serve in the circadian clock mechanism can accelerate tumor growth in mice.5 Similarly, suppression by light of nightly melatonin release from the pineal gland appears to increase the risk of non-Hodgkin lymphoma and breast cancer in humans working on nighttime schedules, possibly because of the timing irregularities that follow.6, 7 Products of clock genes also regulate the timing of mitosis in normal and transformed cells.8, 9 Cancer cells in culture or in tumors display circadian rhythms in gene expression, but the specific function of these cycles in relation to cancer is not well understood.10, 11

Circadian rhythms depend on daily transcription of clock genes, such as the mouse period genes mPer1 and mPer212 and their orthologs in other species. Circadian rhythms in the expression of these and other genes have been imaged in transgenic mice that express the firefly luciferase gene luc controlled by clock gene promoters.13 Similarly, the dynamics of tumor growth have been imaged in tissues of transgenic mice by injecting them with tumorigenic cancer cells that express luciferase.14 In these experiments, the tumor cell bioluminescence of live mice can be recorded repeatedly over several days to assay the progression of growth or metastasis, and multiple anti-tumor treatments can be tested in the same animal. Nevertheless, the tumor stroma is not luminescent and cannot be monitored directly with this approach.

Stromal cells are critical for tumor expansion as they are recruited from non-transformed tissues to become the tumor vasculature and other structures.15 In a meta-analysis of the literature, the microvessel density in lung tumors was found to be an effective prognostic indicator for patient survival.16 Vaccination against stroma-specific markers may become a powerful strategy against cancer in the near future.17 Experimental models are needed that can help in monitoring the development of tumor neovascularization and stromal components. Angiogenesis and stromal components of tumors have been examined effectively using Lewis lung carcinoma cells (LLC, also known as LL/2) injected subcutaneously (s.c.) into syngeneic C57BL/6 mice.18, 19 Inhibition of LLC tumors by angiostatin20 and growth of LLC tumors in response to endothelial cell-selective adhesion molecule21 have also been described.

Even without considering the circadian properties of tumors, the biology and physiology of the stroma is complex and involves many normal and altered cell types. For example, microvessels of tumors can arise through differentiation of cancer stem cells and these can connect functionally with the blood circulation.22 Metastasis can be facilitated by growth of microvessels or lymphatic vessels or by the proliferation of leukocytes attracted to the tumor site.16 Cancer cells can convert macrophages, neutrophils, and lymphocytes into forms that stimulate rather than suppress tumor growth.23 These tumor residents are joined by invading nerve processes that may also facilitate tumor expansion and metastasis.24 Finally, smooth muscle cells and fibroblasts comprise a substantial portion of the stroma of some tumors. In many cases, a major portion of the tumor nodule consists of stromal cells. Interactions between stromal and cancer cells have not been widely explored through current bioluminescence imaging methods despite the importance of this intercellular communication in cancer development and metastasis.

In this study, subcutaneous tumors developed by the injection of non-bioluminescent LLC cells were tested for their effects on the tissues of syngeneic bioluminescent transgenic mice that express luc controlled by the mPer1 promoter (mPer1::luc).25 The tumor stroma showed significantly elevated bioluminescence relative to the opposite thigh. Furthermore, mPER1 and mPER2 protein expression was detected in the stromal and LLC cells by immunohistochemistry. The significance of clock gene activity in the stroma in relation to the role of tumor compartments in cancer progression and metastasis needs further investigation.

Material and Methods

Animals

Transgenic mPer1::luc mice made by oocyte injection on a C57BL/6 background were provided by Dr. Hajime Tei of Mitsubishi Kagaku Institute of Life Sciences, Tokyo.25 A breeding colony was maintained in cycles of 12 h light and 12 h dark (LD 12:12) to entrain their circadian system. Both male and female two-to-eight-month-old mice were transferred from the breeding colony to cages equipped with running wheels and placed in LD 12:12 to remain entrained or in constant darkness or constant red light so that they would express their free-running locomotor rhythms. Circadian rhythms were recorded with a computer data acquisition system that counted wheel revolutions continuously within 1-min intervals. Wheels were 24 cm in diameter. Mice were removed from the LD cycle during the last 7 h of their daytime, while remaining in their cages, and placed in a darkened room during luciferin injection. The mouse tested during its night was moved, while in its cage, to the imaging room at the time of the light offset in the animal room and then left in darkness until imaging began. Mice were fed ad libitum and efforts were made to minimize any discomfort. Procedures were approved by the Bowling Green State University Institutional Animal Care and Use Committee and met National Institutes of Health guidelines.

Tumor cell line

The Lewis lung carcinoma (LLC) cell line was provided by Dr Stephen Kennel of the University of Tennessee Medical Center, Knoxville, TN. Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 incubator. During the animal's daytime, the upper rear leg was injected subcutaneously (s.c.) with 106 LLC cells in 0.1 ml phosphate-buffered saline (PBS) to produce tumors that were imaged after one or three weeks of growth.

Bioluminescence imaging

Mice were imaged under two different conditions—euthanized or anesthetized. Twenty-eight mice were anesthetized with isoflurane briefly until sedation and then injected intraperitoneally (i.p.) with 0.1 ml of 10 mM potassium luciferin (Caliper Life Sciences, Hopkinton, MA) in PBS. The mice were allowed to recover in dim light as luciferin was distributed through their circulatory system for 10 min. Eleven mice were then overdosed with 0.2 ml Nembutal (Abbott Laboratories, Abbott Park, IL) delivered i.p. and imaged immediately after they were no longer responsive. These euthanized mice were used to provide images without the spontaneous movements occasionally observed in anesthetized mice. Previously, we showed that mice imaged immediately after a Nembutal overdose produce bioluminescence signals like those of live mice.26

All whole-mouse imaging experiments used ten sequential 1-min exposures captured with a 50-mm, f/1.2 Nikkor lens (Nikon, Melville, NY) and a liquid nitrogen-cooled CCD camera containing a back-illuminated sensor (CH360, Roper Scientific, Tucson, AZ). To increase signal-to-noise ratios, the images were collected with 2 × 2 on-chip binning, resulting in 256 × 256-pixel, 16-bit images. Bioluminescence intensity was presented in analog-to-digital units (ADUs), the units of the camera's sensor. One ADU is equivalent to about two photons incident at the sensor. Bioluminescence intensity of the euthanized mice was measured using the 1-min frame that occurred 25 min after luciferin injection.

Seventeen of the 28 mice were instead imaged while anesthetized by isoflurane inhalation that was administered 8–10 min after the luciferin injection. Mice were anesthetized with 3% isoflurane in oxygen and imaged while in a Plexiglas induction chamber (E-Z anesthesia, Euthanex, Palmer, PA). They were imaged through a removable clear glass window (20 cm × 20 cm × 4 mm thick) mounted in the lid of the chamber. Body temperature was maintained with a DeltaPhase isothermal pad (Braintree Scientific, Braintree, MA). Mice were positioned on black felt adhered to a wire mesh frame that was shaped to fit the mouse body. After imaging, they were returned to their cages and circadian locomotor activity was monitored. Tumor bioluminescence intensity of these live anesthetized mice was measured using the 1-min frame that occurred approximately 15 min after luciferin injection.

After imaging, mice were killed with an overdose of Nembutal i.p. and then decapitated. Tumors were excised, measured, and prepared for histology. Some tumors of euthanized mice were imaged directly in situ after removing the skin from the lower back and upper legs. Other tumors were excised, placed in Petri dishes, and imaged separately to eliminate any scattered bioluminescence originating in neighboring tissues.

Histology

The tumors were excised from euthanized mice three weeks after LLC cell injection. Tumor size was determined from the average of the length along two major dimensions. Tumors were then prepared for histology by fixation in Histochoice (Amresco, Solon, OH) and embedding in paraffin at 57°C. Cells in 10 μm-thick sections were stained with hematoxylin and eosin or immunostained with affinity-purified rabbit polyclonal antibodies directed against mPER1 or mPER2 proteins (ab3443 from Abcam and PER21-A from Alpha Diagnostics International, San Antonio, TX). For these steps, the paraffin sections were mounted on glass slides, dewaxed, rehydrated through a series of ethanol dilutions, treated with an epitope-retrieval buffer according to the manufacturer's instructions (Bethyl Laboratories, Montgomery, TX). The sections were then immunostained with a kit that uses a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (IHC-101, Bethyl Laboratories). Sections were developed in diaminobenzidine and photographed with 400 ASA color print film or imaged with a cooled-CCD camera (Micromax Y1300, Princeton Instruments, Trenton, NJ) attached to an Axiophot microscope (Carl Zeiss Microimaging, Thornwood, NY). Cells in histological sections were examined at intervals of 4 mm or less.

To prepare brain sections for use in immunostaining as a positive control, adult mPer1::luc mice (n = 5) were anesthetized with Nembutal (0.13–0.16 ml, i.p.) and transcardially perfused with phosphate buffered saline (PBS) followed by 4% formaldehyde in PBS, pH 7.4. Fixed brains were removed, blocked, and immersed in a 4% formaldehyde solution in PBS overnight, followed by 30% sucrose in PBS overnight before being cryosectioned at 25–50 μm the following day. Tissue sections made through the hypothalamus were mounted on glass slides (Plus Gold, Fisher Scientific) and air-dried for 3 h prior to rehydration through an ethanol series and immunostaining as with paraffin sections of the tumors.

Data analysis

Bioluminescence images were analyzed with Metamorph (Molecular Devices, Downington, PA), V++ (Roper Scientific, Tucson, AZ), and ImageJ (NIH) software. Cosmic ray-type artifacts were removed by taking the minimum intensity at each pixel of two consecutive images or with a minimum filter when only a single image was available. Period estimates of running-wheel records were determined using a standard method in which lines are eye-fit to activity onsets of circadian rhythms in actograms for intervals of 10 consecutive days and by periodogram analysis using Actiview software (Mini-Mitter, Bend, OR).

Results

Bioluminescence imaging of tumors in euthanized mice

The mPer1::luc mice showed bioluminescence from elevated transgene expression in the paws, tail, snout, ears, and testes along with a signal from internal organs that is visible to a limited extent through the fur, whereas the rear thighs produced little or no signal (Fig. 1a). To test whether bioluminescence from a tumor in the upper leg would be adequately bright to detect through the fur and skin, LLC cells were injected subcutaneously into one thigh of 11 mice. The cells generated dense tumors in 10 of these mice and these were detectable by palpation about one week after cell injection (Fig. 1b and c).

Figure 1.

Bioluminescence from the mPer1::luc transgenic mice. (a) The pattern of luciferase bioluminescence from one mPer1::luc transgenic mouse prior to tumor cell injection. (b,c) Bioluminescence from the tumor site (arrow) on the left upper leg of two mice imaged three weeks after LLC cell injection (s.c.). Other bright areas are paws and tail. (d) The leg with the tumor is significantly brighter than the opposite leg. Shown is the average log intensity ± S.E. at three weeks after cell injection (n = 5). Intensity is in analog-to-digital units (ADU) of the camera. (e) Bioluminescence from the tumor site (arrow) of an anesthetized mouse. Images are shown as overlays of a pseudocolor representation of intensity on top of the reference image. Scale indicates intensity as ADU. (f) The percent change in signal in the tumor and control legs between early and late imaging sessions. Each symbol represents the same mouse.

The tumors produced bioluminescence that was visible by imaging immediately after an overdose of Nembutal administered 3 weeks after cell injection. To evaluate tumor size, tumors were excised from four mice and measured. Average tumor size was 12.6 ± 2.7 mm (±SD). Six of the 10 mice with tumors showed a significantly higher bioluminescence in the thigh with the tumor than in the opposite thigh. The signal was considered significant when maximal intensity was more than three standard deviations above the maximum intensity of the control thigh. The average of the log maximum intensity was significantly higher in the thighs with the tumor than in the opposite (control) thighs (paired t-test, p =0.044, n = 7). Tumors were then harvested for histology by dissection, fixed, and embedded in paraffin.

Imaging live, anesthetized mice

To determine whether anesthetized mice would show a significant bioluminescence in the tumor stroma like that in euthanized mice, five mPer1::luc mice were injected with LLC cells and imaged 3 weeks later (Fig. 1d). All of the mice produced a tumor and the area of the tumor was significantly brighter than the corresponding opposite leg (p = 0.0359, paired t-test).

In an attempt to improve tumor imaging, the upper rear legs of six mice were shaved and the LLC cells were injected into one leg. The mice were imaged seven days (early session) and 21–23 days (late session) after cell injection. These mice were imaged 15 min after luciferin injection while anesthetized. The majority of the shaved area continued to have little or no hair growth and was visibly distinct from areas that had never been shaved. Ten consecutive 1-min images were collected and then the mice were moved from the chamber to their cage to revive.

As with the mice euthanized before imaging, anesthetized, shaved mice showed significant bioluminescence from the tumor-bearing thigh (Fig. 1e). All of the six injected mice produced tumors. Four of these were imaged during both the early and late sessions. When the change in bioluminescence over this two-week interval was compared, the thighs with tumors showed a greater percent change in signal than their corresponding control thighs (Fig. 1f, one-tailed, paired t-test, p = 0.040, n = 4).

The mPer1 gene is expressed in a circadian rhythm in many tissues and was likely modulated in the stroma in this manner by the circadian clock. Although we imaged the mice during the light portion of the light/dark cycle, the transgene might be expressed at a much higher level during the animal's night. Many peripheral circadian oscillators, in tissues throughout the body, show peak mPer1 expression during the night. The mice were imaged during the interval between 5 hrs after light onset to the time of light offset, “dusk”. One mouse, however, was imaged during the night, 5 hrs after dusk, to test whether it would show greater bioluminescence from the stroma than mice imaged during their day. At three weeks after cell injection, this unshaved, anesthetized mouse produced a signal of 1856 ADUs in the tumor, compared with 107 ADUs in the opposite leg. Comparable unshaved, anesthetized mice produced an average tumor signal of 385.8 ADUs (±318.9 SD, n = 5) when imaged during the second half of the light portion of the animal's light cycle.

Bioluminescence imaging of exposed tumors in mice

To image the tumor stroma directly, four of the mice used for whole-animal imaging and two additional mice were used to image exposed tumors. The mice were euthanized by Nembutal injected i.p. and then decapitated. After removing the skin over both the tumor-bearing thigh and the corresponding opposite thigh, the mice were imaged with two consecutive 5-min exposures (Fig. 2a and b). The average tumor bioluminescence was significantly higher than the corresponding tissue of the opposite side (Fig. 2c, t-test, p = 0.012, p = 6).

Figure 2.

Bioluminescence from the tumor after the skin is removed. (a) Control mouse injected with saline. (b) Mouse injected with LLC cells. Arrow: tumor. (c) Intensities of the tumor and control regions of all six mice. Each symbol represents bioluminescence from the same mouse. In all cases the tumor area was brighter than the opposite side. Open symbols: measurements from anesthetized mice. Filled symbols: euthanized mice. The mean intensity and standard error are shown for the two sides. (d) Bioluminescence from an excised tumor. Note the dark central regions that are likely due to LLC cells which lack the transgene. (e) Reference image captured in red light to focus the camera. Bright areas are surface reflections. (f) Pseudocolor overlay of the bioluminescence on the reference image. A 5-min camera exposure was used to capture bioluminescence of the animal or the tumor. All intensity scales show bioluminescence according to the camera's analog-to-digital units (ADUs).

In addition, three tumors were harvested immediately after the final (late) imaging session and placed into three Petri dishes for direct imaging. Two of these tumors were imaged at higher magnification by using the 50-mm lens with two close-up lenses in combination (+10 with either +4 or +1-diopter). The aim was to determine how broadly the bioluminescence was distributed within the stroma. All three tumors showed a diffuse light in almost all areas. There were also many distinct spots showing higher signal and darker areas near the center (Fig. 2d–f).

Effects of tumors on peripheral gene expression

Bioluminescence was examined in several body areas of the euthanized mice to determine whether mPer1 expression was altered by tumor growth. Because the mPer1 gene is a component of the circadian oscillator, any effect of the tumor on the transgene might provide insight into how the circadian timing system is altered by cancer. The rear thigh opposite the tumor and the tail, paws, snout, and ears were examined in the euthanized mice. No area was significantly brighter than any of the others (ANOVA, F =1.07, p = 0.42, n = 10 mice), indicating that mPer1 gene expression on the side of the animal with the tumor was not different from the opposite side.

When the anesthetized mice were examined, certain body areas were significantly brighter than others by three weeks after cell injection. Of all 10 body regions (excluding the thigh with the tumor) the rear paws and tail were significantly brighter than the rest (ANOVA, F = 7.04, p < 0.001 by Scheffe post hoc test, (n = 6 mice). There was, however, no significant difference between the right and left rear paws suggesting that, other than in the stroma, the tumor did not have local effects on mPer1 expression in nearby tissues within the same appendage.

Effects of tumors on circadian locomotor rhythms

Circadian locomotor activity was used as an assay of the circadian timing system by monitoring voluntary wheel-running activity of mice with and without tumors (Fig. 3). Tumor growth did not produce a significant change in the period of the circadian rhythm of mPer1::luc mice (Table 1). The average periods of LLC-injected and sham-injected mice were not significantly different during either the early or late intervals, defined as the first 10 days after cell injection and the last ten days of tumor growth, respectively (t-test, p>0.05, n = 9 mice each). There was, however, a significant difference between the early and late 10-day intervals of both the LLC and the control groups (t-test, p = 0.0076 and p = 0.022, respectively). Clearly these circadian rhythms persisted without major disruption, suggesting that factors released from the tumors or neural signals arising within the tumors were of little consequence, if any, at this tumor stage.

Figure 3.

Circadian running-wheel records from mice with LLC tumors. (a) The locomotor rhythm of one control mPer1::luc mouse as shown by an actogram during 20 days in constant darkness. (b) The circadian locomotor rhythm of a second mouse in constant darkness after injection of LLC cells at day zero. Prior to day zero, both mice were in a light cycle of 12 h light and 12 h darkness. Vertical marks indicate wheel revolutions collected within one-minute intervals. The data are double plotted. (Each row displays wheel revolutions during two consecutive days.) The previous light/dark cycle is shown above along with local time. Data shown in B are from the same mouse as in Fig. 1b.

Table 1. Periods of circadian locomotor rhythms recorded in darkness
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LLC tumor histology

We characterized the composition of LLC tumors through hematoxylin and eosin staining as well as immunohistochemistry with antibodies directed against mPER1 and mPER2. The tumors showed positive immunostaining for both of the circadian clock proteins (Fig. 4a–c). This staining was found in many cell types including the nuclei of LLC cells (Fig. 4d). From staining intensity alone it was not possible to identify concentrated areas of high expression from a single cell type that was responsible for a large portion of the total bioluminescence observed in the tumors. Positive staining in neurons in the suprachiasmatic nucleus (SCN) of the hypothalamus detected the expected mPER1 protein known to be widely expressed in this circadian oscillator of the brain (Fig. 4e, positive control).

Figure 4.

Histological sections of tumors generated by LLC cells in mPer1::luc mice. (a) A tumor section immunostained for mPER1. The cancer cells are in the upper region of the image and the tumor stroma begins near the arrow. (b) Control section with primary antibody omitted. (c) Immunostaining for mPER2. Both mPer clock genes are expressed throughout the tumors in A & C. (d) The anti-mPER1 antibody is concentrated in the nuclei of LLC cells in tumor sections (at arrow, for example). (e) The suprachiasmatic nucleus (SCN) of the hypothalamus, a positive control for the anti-mPER1 antibody, also shows staining in cell nuclei. (f) H & E-stained paraffin section. Most tumor areas contained densely packed LLC cells. Box at lower left encloses typical LLC cells near the center of the tumor. Box at top right corner indicates one area of the stroma showing host cells that were observed in tumors. These cells were identified as macrophages (large arrow) and neutrophils (small arrow). Scale bars indicate 100 μm (a–c) and 50 μm (d–f).

LLC cells comprised the largest part of the central region of the tumors as identified by their characteristic size and nuclear staining. Other cells, distinguished by their strong basophilic staining, were scattered throughout the LLC cell population at densities of about 1% or less of the cells but were also found in clusters occasionally (Fig. 4f). These cells were most likely immune or inflammatory cells that had originated from the host and migrated into the tumor mass, and they clearly lacked the morphology of LLC cells (Fig. 4f). The characteristically shaped cell nuclei of neutrophils were readily indentified.

Discussion

Stromal imaging

LLC cells readily produced solid tumors when injected subcutaneously into mPer1::luc mice, as described previously.18 Over 95% of the mice had palpable tumors at the time of imaging (21 of 22 mice). Nevertheless, not all of these mice showed detectable luminescence from the area of the tumor mass. Some light was likely blocked by the dark fur, filtered by skin pigments, or scattered by the skin to an extent that limited easy detection. Even in the anesthetized mice the shaved tumor areas did not always reveal bright luminescence from the tumor stroma, suggesting that the skin is also an impediment to imaging. Although imaging conditions were not ideal, significant gene expression in the tumor stroma could be monitored reproducibly under three different conditions—euthanized, anesthetized, or shaved and anesthetized mice (Fig. 1). One advantage of stromal imaging is that it may be effective with a wide range of syngeneic cancer lines without needing to transfect the cancer cells with a fluorescent or bioluminescent reporter gene. In addition, the same host mice could be used to compare the effects of different cancer cells on the stromal response to these cells.

Without direct imaging of the tumor, it seemed possible that distension of the skin by the tumor may have made the bioluminescence of the tissues more visible than in the corresponding region of the opposite leg. To verify that the signal did indeed originate in the tumor stroma and not in the surrounding tissue, which produces a faint background signal, the tumor was exposed and imaged in euthanized mice. This procedure revealed significant bioluminescence in all tumors examined in this manner. In every case the tumor stroma was brighter than the corresponding area of the opposite leg, after removing skin from this area as well. The center of the tumor mass was often darker than the surrounding cells, because LLC cells cannot express luciferase. Considering that there was little necrosis in the tumor core, the best interpretation seems to be that the lack of signal near the center of the tumor was due to an absence of stromal cells. Finally, tumors removed from the mice showed a bioluminescence throughout much of the stroma, indicating that the light was not generated only in the outer edges of the tumor or in a few distinct structures such as blood vessels.

Source of the stromal bioluminescence

It was in some ways surprising that the exposed tumors did not show visibly luminescent blood vessels. It is possible that microvessels were present but these were below the resolution limit of the bioluminescence imaging system. Circadian rhythms in rPer1 expression have been recorded in explant cultures made from several veins and arteries along with the atrium and ventricle,27 in which case ex vivo bioluminescence recordings were made from tissues harvested from transgenic rats expressing the firefly luciferase gene regulated by the rPer1 promoter. The optimal scheduling of antiangiogenic treatments is an important factor in the control of LLC tumors in mice to avoid drug resistance.15 The specific role of core clock genes such as Per1 in the cardiovascular system is not known, and higher resolution imaging of tumors might reveal circadian rhythms in these structures.

Tumor histology was used to help characterize the distribution of mPer1::luc expression in the stroma. Cells scattered throughout the population of LLC cells likely were immune cells that had migrated into the tumor. Some neutrophils were also present near the perimeter. Macrophages have been identified in LLC tumors and these appear to alter tumor growth through neovascularization.28 Tumor-activated macrophages (TAMs) and tumor-infiltrating lymphocytes (TILs) are known to facilitate growth of other tumors as well.23 These cells may not be adequate for generating the entire stromal signal because of their scarcity in the tumors examined here (Fig. 4f). Nearly all of the cells were closely packed LLC cells, as described previously by others.19

LLC cells and a diverse set of stromal cells showed mPER1 and mPER2 immunoreactivity (Fig. 4). Therefore, the mPer1 expression assayed in tumor stroma by bioluminescence imaging represents gene activity in several different cell types. Additional imaging and cell phenotyping would be needed to determine the relative contribution of each of these host-derived components. The capsule surrounding the tumor was also positive for mPER1 and mPER2 suggesting that it is another source of bioluminescence.

Although the immunohistochemistry suggests that two of the approximately seven core clock genes29 are present in the stroma and LLC cells, molecular approaches targeting clock genes and clock-driven genes are needed. We are confident that the two commercial antibodies used in this study provided good localization of mPER proteins based on the observed mPER1-like immunostaining of neurons in the SCN of the hypothalamus, a positive control (Fig. 4),30, 31 and the many published reports using the affinity-purified anti-mPER2 antibody that was applied here.32–37

The appearance of the capsule tissue was suggestive of muscle-like cells. Many tumors contain cells with myofilaments that are evident at the ultrastructural level.38 Circadian clock genes are expressed in muscle cells,39, 40 and it appears that the abnormal muscle-like cells of the tumor capsule also express these genes. The immunolabeling results indicating clock gene expression in LLC cells and stromal cells suggest that the tumor may contain one or more circadian oscillators, although mPER1 and mPER2 expression alone is not sufficient evidence of a biological clock. Nevertheless, circadian rhythms have been described in other tumors,3 and these could be generated by clock cells within the tumor rather than driven by a circadian oscillator located elsewhere in the body. We were not able to image the mice repeatedly to identify circadian rhythms in bioluminescence in the stroma because of the limitations of our imaging methods and the rapid expansion of the tumors during the last two weeks of growth. Alternatively, methods in which bioluminescence imaging is performed continuously with freely-moving mice (without anesthesia) might identify rhythms in the stroma. Similarly, molecular approaches in which stromal tissue is harvested from many mice at time points across the circadian cycle could be effective as well.

Tumor cell communication with other body regions

An additional question addressed by this study is whether tumor growth alters mPer1::luc expression in areas well outside the tumor region such as the paws, tail, ears, and snout. All of these areas showed spontaneous mPer1::luc expression in all normal and LLC-injected mice. If we had observed induction or suppression of peripheral gene expression by the tumor, this would suggest that growth of the LLC tumor can have a systemic impact on mPer1 and therefore an influence on the circadian oscillators in peripheral tissues and, by extrapolation, the clock cells of the major circadian pacemaker in the SCN of the hypothalamus. One reasonable set of candidate mediators of this effect are the pro-inflammatory cytokines that are known to be elevated in the bloodstream as tumors grow.41 Cytokines can shift the phase of circadian rhythms,42 suggesting that they could act in signaling between clock cells within the tumor, by inducing clock genes, or they could act between the tumor and oscillators in the body.

We did not, however, find any evidence of communication between the tumor and other host cells located farther from the tumor site than the stroma. The mPer1::luc expression in the rest of the animal was not affected in a way that could be detected by bioluminescence. Similarly, the behavioral assay used here—the circadian rhythm in locomotor activity—did not show an altered free-running period. We did observe a decline in the average free-running period of both the tumor and control groups over time. A similar decrease in period during constant darkness has been described in other circadian studies using the same mouse species.43 The wheel-running rhythm primarily reflects the circadian rhythms of the SCN and is sensitive to subtle changes in the intensity of light, food availability, reproductive cycles, aging, and disruptions of metabolic state. For example, Vipr2−/− mice, which lack a key neuropeptide receptor in the SCN, show concomitant disruptions of both circadian wheel-running rhythms and circadian rhythms in SCN gene expression.44

Several studies have shown that the circadian system alters tumor growth,9, 45–51 and disrupted circadian rhythms in DNA synthesis have been described in mice carrying LLC tumors.52 The SCN interacts with other circadian oscillators of the body, thereby altering their timing, and it is conceivable that tumors might also communicate with the circadian timing system as circadian oscillators. Our results suggest, however, that any interactions between the circadian system and these tumors are mostly unidirectional if at all. Therefore, effects of the circadian system on tumor growth might be studied using the LLC tumor model without confounding effects of the tumor acting on the body's circadian clocks.

It seems most likely that the observed elevated expression of the mPer1::luc transgene is associated with the accelerated growth of the stroma relative to surrounding tissues. We observed that rapid tumor expansion occurs mostly during the last two weeks of the three weeks of tumor growth. Cell growth is associated with cell signaling pathways that can also induce mPer1 expression, e.g., ones acting through cAMP response element-binding protein (CREB).53–58 The timing ability of circadian oscillators is disrupted when Per1 expression is elevated experimentally, in which case the circadian period lengthens and the animal is less able to entrain to light cycles.59 If the elevated mPer1 expression we detected in the stroma was due to the effects of the LLC cells, then tumor growth might have disrupted the circadian clock gene functions in these cells.

Alternatively, the stromal signal may be generated in a circadian rhythm with a peak occurring during the night like many peripheral circadian oscillators (those located outside the SCN). Our preliminary experiment using a single mouse yielded a signal more than four standard deviations above the average stromal signal recorded during the day and agrees with this hypothesis. Any future stromal imaging experiments planned with these mice should take into consideration the extra requirements for imaging at night, including maintaining the mice in darkness up to the time of imaging to avoid phase shifts, and whether this effort is offset by the possibly higher signal available at this phase. What is most important for cancer studies examining the intensity of the stromal bioluminescence is to perform imaging as close to the same time of day as possible, thereby minimizing effects from circadian or daily oscillations. The phases used here for most imaging, during the second half of the daytime, may be optimal for identifying both increases and decreases in the day-to-day signal, perhaps in response to an anti-tumor treatment.

It will remain unresolved whether mPer1 expression is circadian in the stroma until a more extensive study can be performed examining mice, ideally during at least six phases of their circadian activity rhythms displayed while in constant darkness. As an alternative possibility, it should also be considered that increased mPER1 protein levels from persistent induction might disrupt the circadian oscillation. Evidence of this effect has been identified in transgenic rats.59 A key event in the circadian cycle is suppression of the period genes by their own protein products, followed by a decline in PER protein levels through degradation. If signals from the tumor act as strong inducers of mPer1, then the gene might remain active independent of circadian modulation. If mPER1 protein remains elevated in the tumor, then it should be asked whether it can serve a non-circadian role in this particular location. Interestingly, evidence indicates that mPER1 protein remains cytoplasmic in a subset of retinal cells in mice,60 and without entering the nucleus it is difficult to argue that the protein serves in the circadian timing mechanism. Although we observed nuclear mPER1-like immunostaining in the LLC cells of tumors, nuclear staining in the many stromal cells was not confirmed. Further studies are needed to determine specifically whether mPer1 plays a circadian or non-circadian role in the stroma and tumor growth.

Overall, the importance of circadian effects on cancer development or growth is not well understood. Growth of tumors from LLC cell injections in mice is inhibited when they are made to over express mPER2.4 The circadian system regulates inflammatory cytokines, such as interferon-gamma,61 and may also control natural killer cell responses.62 It is interesting to consider how the pattern of mPer1 gene expression described here could be part of an ongoing defense mechanism between the host and cancer cells, particularly if this process cycles throughout the day. Circadian clock gene expression might play an important role in these tumor-infiltrating immune cells. Additional exploration of circadian timing within the stroma is needed.

Acknowledgements

The authors thank Dr. Hajime Tei for providing mPer1::luc mice; Dr. Stephen Kennel for providing LLC cells; Denise Hook of the BGSU Animal Care Facility for help with mouse breeding; BGSU undergraduate students Nicole Dusseau, Brent Fuller, Victoria Klein, Britiany Sheard, Joseph Smith Jr., and Josh Waldman for their help.

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