In vivo assessment of vascular-targeted photodynamic therapy effects on tumor microvasculature using ultrahigh-resolution functional optical coherence tomography

1. Introduction

Angiogenesis in tumor is essential for maintaining rapid tumor cell proliferation [1]. Inhabitation of newly-formed blood vessels in tumor can prevent tumor growth [2]. Vascular-targeted photodynamic therapy (VTP) based on local destruction of vasculature is known to be an effective therapeutic modality for treating tumors [3,4]. The process involves an intravenous injection of a photosensitizer followed by light irradiation at a specified wavelength to activate photosensitizer [5,6]. The activation of the photosensitizers triggers a cascade of photodynamic reaction and generates the intravascular reactive oxygen species (ROS), damaging the blood vessels and eventually leading to tumor necrosis. Although it is well-known that the effectiveness of VTP is related to multiple factors, such as the concertation of photosensitizers and the oxygen level in the targeted vasculatures as well as the choices of light wavelength, power density and illumination duration [6–8], the exact dosages of photosensitizer and light are difficult to determine for an optimal treatment. Consequently, accurate assessment of the response of tumor vasculatures to the treatment in vivo becomes a valuable alternative [9–12].

Several noninvasive, label-free imaging techniques have been proposed for in vivo assessment of VTP induced vascular responses, including laser Doppler imaging (LDI) [13,14], laser speckle contrast imaging (LSCI) [15], photoacoustic imaging (PAI) [16,17], and optical coherence tomography (OCT) [9–12]. LDI and LSCI have been demonstrated to measure blood flow changes during VTP [13–15]. However, both techniques only offer two-dimensional information. Changes in blood oxygen saturation can be evaluated with PAI [16]. The challenge with PAI is the need for a coupling medium (such as water or ultrasound gel), making it difficult to use for in vivo monitoring during VTP. Recently OCT has been increasingly used for non-contact volumetric imaging of tumor vasculature [18]. Visible-light OCT (vis-OCT) has shown to be capable of non-contact oximetry measurement and high-resolution vasculature imaging [19,20]. This attractive, functional capability of vis-OCT is achieved at the cost of a limited penetration depth of visible light in bio-tissues. Previous studies have shown that photodynamic therapy-induced blood vessel changes in a mouse ear tumor model can be detected with an M-mode OCT operating at 1300 nm [10,11]. One challenge with 1300-nm OCT is the suboptimal resolution (∼10 µm), difficult for imaging microvasculature, particularly the newly formed ones in tumor with a diameter of around 7 µm [21,22]. In contrast, OCT operating at 800 nm affords a much higher resolution of better than 3 µm, thus potentially offering a unique capability for assessing the response of tumor vasculature to VTP.

In this study, we employed an 800-nm ultrahigh-resolution functional OCT (UHR-FOCT) to assess the microvascular alterations during VTP with Verteporfin on an A431 tumor-bearing window chamber model in mice. Microvascular metrics, such as blood vessel diameter and density, were quantified before, during, and after VTP to demonstrate the potential of UHR-FOCT for in vivo evaluation of VTP treatment of tumor in vivo and in real time.

2. Methods

2.1 800-nm UHR-FOCT system

Figure 1(a) shows the schematic of the UHR-FOCT system, the details of which have been described elsewhere [23,24]. An X-Y galvo-scanning mirrors were adopted in the sample arm to facilitate 3D imaging at an A-scan rate of 70 kHz. By using a Ti:sapphire laser with a central wavelength of ∼825 nm and a full width at half-maximum bandwidth of ∼150 nm, we were able to achieve an axial resolution of ∼2.4 µm (in air) [25,26]. A scan lens (LAC896-B, Thorlabs Inc., Newton, USA) was used to achieve a lateral resolution of ∼8.0 µm. The incident power of the imaging beam on the tissue surface was about 15 mW.

 

Fig. 1. The VTP study setup. (a) Schematic of the UHR-FOCT system, which consists of a Ti:sapphire laser and a linear-k spectrometer. A 680 nm LED was used for activating the photosensitizer to treat a mouse dorsal skinfold tumor model with VTP. M: mirror, C: multi-element achromatic collimator, FC: fiber coupler, PC: polarization controller, PP: prism pair, GSM: galvo-scanning mirrors, SL: scan lens. (b) Photograph of the platform for holding the mouse model. The mouse was placed on a temperature-controlled heating pad (37 °C). The window chamber was secured to the imaging platform plate through screws.

Download Full Size | PPT Slide | PDF

2.2 Cell culture and animal model

In this study, an A431 cell line was used for establishing the tumor model on mice dorsal. The tumor cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Grand Island, USA) supplemented with 10% (v/v) of fetal bovine serum (Sigma-Aldrich., St. Louis, USA), and kept in a humidified atmosphere at 37 °C and 5% CO₂.

Dorsal skinfold chambers (APJ Trading Co., Ventura, CA, USA) were implanted in five NCr nude mice (6-8 weeks, male, Taconic biosciences, NY, USA) by adopting the surgical procedures described by Palmer et al. [27]. In essence, the skin on one side of the window was excised, allowing subdermal microvasculature of the opposite side skin to be visualized through the window. At the center of the window chamber, 10-µl suspension of about 10⁶ A431 cells was injected in between the superficial fascia layer and the subcutaneous fat layer. During surgery, mice were anesthetized by intraperitoneal administration of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight). The animal procedures were approved by the Johns Hopkins Animal Care and Use Committee.

2.3 Study protocols

The window chamber was first imaged with UHR-FOCT right before tumor cell injection. The chamber was imaged daily after tumor cell injection to monitor the tumor growth and the angiogenesis for 6-10 days. Animals were randomly divided into 2 groups, i.e., VTP group (n = 4) and light only control group (n = 1). For the VTP group, the animals received an intravenous injection (through tail vein) of a 200 µl saline solution of Verteporfin (2 mg/ml stock in DMSO; Sigma-Aldrich., St. Louis, USA) with a dose of 2 mg (Verteporfin)/kg body weight. Following the existing protocol of Verteporfin-VTP, light irradiation was performed 15 min after the injection of photosensitizer [28,29]. For the control group, the mouse was injected with saline solution of the same amount. A high-power 680 nm LED (M680L4, Thorlabs Inc., Newton, USA) with a bandwidth of ∼ 22 nm was used to activate Verteporfin with an irradiance rate of about 100 mW/cm² and an exposure time of 20 min [Fig. 1(a)]. The LED light was collimated with a condenser lens. The whole window area was illuminated with the LED light, and the tumor area was in the middle of the beam spot during treatment. The tumor area was closely monitored with UHR-FOCT before, during, and after light irradiation. For each imaging procedure, the mouse was placed on a heating pad (37 °C) and wore a rodent mask to maintain the anesthesia with the isoflurane-oxygen mixture (1-2% at a rate of 1.0 L/min). The window chamber was stabilized secured to the imaging platform plate through screws [Fig. 1(b)].

2.4 Optical coherence tomography angiography (OCTA)

Volumetric images were collected with each 3D dataset consisting of 2048 B-scans over a field of view of 3.0 mm × 3.0 mm in X and Y directions, respectively [ Fig. 2(a)] and 1.46 µm separation between two adjacent B-scans. Each B-scan of 1024 pixels × 2048 pixels corresponded to a cross-section of 3.0 mm × 1.23 mm in X and Z directions, respectively [Fig. 2(b)]. Layer structures of dorsal skin, including fascia (F), muscle (M), adipose (A), dermis (D), and epidermis (ED), could be readily identified [30–33]. An averaging window of 4 consecutive B-scans was used to obtain an image with reduced noise [Fig. 2(c)]. The well-established phase-resolved Doppler variance (PRDV) algorithm was adopted to calculate the interframe variance [34]. The calculated interframe variance forms the OCTA image of the vasculature [Fig. 2(d)]. Overlaying OCTA on OCT structural image clearly shows that the large blood vessels mainly locate in the muscular and adipose layers [Fig. 2(e)]. Volumetric vascular image [Fig. 2(f)] can be reconstructed with OCTA images. En face vascular image [Fig. 2(g)] was constructed by projecting the mean value of each A-line in the volumetric OCTA images along Z direction. Vascular density and diameter of blood vessels were chosen as metrics. For vessel density quantification, the en face OCTA vascular images were binarized by using the well-established Phansalkar local thresholding method implemented in ImageJ (Fiji) with a default window radius of 15 pixels [35–39]. Vessel density was calculated as the ratio of the total number of white pixels (with a value of 255) (i.e., pixels of on blood vessels) to the total number of pixels in the area on the binarized image [11,12]. The diameter of blood vessels was obtained by measuring the full-width at half-maximum of the intensity profile through blood vessels in the en face image. En face image [Fig. 2(g)] clearly shows the densely distributed nearly parallel capillaries (as indicated by white arrowheads). The intensity profile was taken from of a representative capillary as indicated by the light blue line in Fig. 2(g), and the average size of three representative capillaries (as indicated by white arrowheads) was measured to be around 10 µm (i.e., the full-width at half-maximum of the intensity distribution across the capillary), which is consistent with the previous reports [40,41].

 

Fig. 2. Imaging of microvasculature in the skinfold window chamber with UHR-FOCT. (a) Volumetric image with a field of view of 3 × 3 mm² (x-y). (b) An averaging window of 4 consecutive B-scans along Y-direction for noise reduction. (c) Two adjacent averaged B-scans for calculating interframe variance. (d) Cross-sectional OCTA vascular image obtained with the PRDV algorithm. (e) OCTA overlaid on OCT structural image. (f) The reconstructed volumetric vascular image. (g) En face vascular image reconstructed by projecting the mean value along the Z direction. Insets show the enlarged view of capillaries (white arrowhead) and the diameter of a representative capillary (light blue line). F: fascia, M: muscle, A: adipose, D: dermis, ED: epidermis. The skin microanatomy was imaged from inside to the surface (epidermis) through the window. White arrowhead: capillary. Scale bars: 500 µm.

Download Full Size | PPT Slide | PDF

2.5 Data analysis

All data were processed and analyzed using Matlab (The MathWorks, Inc.) and ImageJ (Fiji). Data are presented as means with the standard deviations (SD). The paired Student’s t-test was used to compare the differences between immediately before VTP and 24 hours after VTP shown in Fig. 5. P-values of <0.05 were considered statistically significant.

 

Fig. 3. Study of angiogenesis during tumor development (from Day 0 to Day 6) in the skinfold window chamber model. Tumor cells were injected at Day 0. (a) White light images of the window chamber, with the blue rectangle indicating the area imaged with UHR-FOCT. (b) En face vascular images (corresponding to the blue rectangle area in (a)). (c) Representative cross-sectional merged OCTA/OCT images (corresponding to the location indicated by the white dashed line in (b)). (d) Normalized vascular density versus tumor development time. (e) Changes of diameters for representative veins and arteries during tumor development (as marked in (a, b)). The diameter of each selected blood vessel was measured three times within the rectangle. The vascular density and vessel diameters were normalized to the corresponding baseline values measured before tumor cells injection. Scale bars are 2 mm in (a) and 500 µm in (b) and (c). Error bars: representing the SDs. A: artery, V: vein, Green arrowhead: newly generated blood microvessel. The green dash lines indicate the tumor area.

Download Full Size | PPT Slide | PDF

3. Results

3.1 Imaging tumor angiogenesis

By directly comparing the white light images [ Fig. 3(a)] of the window chamber with the corresponding en face OCTA images [Fig. 3(b)] of the A431 cells injection area (blue rectangle), the progressive tissue vascularization could be clearly appreciated. The newly-generated blood vessels with a diameter ranging from 10 µm to 60 µm (as indicated by green arrowheads) in the tumor area (Day 4, 5 and 6) were significantly different from these in the normal skin (Day 0), where blood vessels were straight, well organized and evenly distributed. In contrast, the new blood vessels in tumor were tortuous, poorly organized, and irregular in diameter. As shown in Fig. 3(c), an obvious bulge was formed due to the development of the tumor, along with the increased vessel density. This was further verified by quantifying vascular density for the OCTA images, as shown in Fig. 3(d), which increased by almost 65% on Day 6. We also observed that the tumor growth resulted in a significantly increased diameter of the veins (as indicated by blue V’s) by about 70% and increased diameter of the arteries (as indicated by white A’s) by around 90% at Day 6 [Fig. 3(e)].

 

Fig. 4. Responses of tumor microvasculature to VTP (100 mW/cm², 20 min). (a) En face vascular (OCTA) images immediately before VTP (0 min), during VTP (10 min, 20 min), and after VTP (40 min, 60 min and 80 min). (b) White light images immediately before and 60 min after VTP, and the merged cross-sectional OCTA/OCT images corresponding to the location indicated by the white dashed line in the white light images. (c) Variation of the vascular density with time. (d) Changes of diameters for the representative veins, arteries, and tumor vessels (as marked in (a)) with time. The diameter of each selected blood vessel was measured three times within the rectangle. The vascular density and vessel diameters were normalized to the corresponding baseline values measured before VTP. Scale bars: 500 µm. Error bars: representing the SDs. A: artery, V: vein, T: tumor vessels.

Download Full Size | PPT Slide | PDF

3.2 In vivo assessment of VTP effects on tumor microvasculature

Figure 4(a) shows the changes of microvasculature by comparing the en face OCTA images immediately before, during, and after Verteporfin-mediated VTP treatment on tumor. This change can also be clearly observed from the white light images and the representative cross-sectional OCTA/OCT images acquired before and after VTP [Fig. 4(b)]. As the OCTA signal is sensitive to blood flow, the disappearance of blood vessels in the en face image and the cross-sectional image may indicate coagulation of blood or closure of blood vessels (white arrows). Vascular density and blood vessel diameter were further quantified [Figs. 4(c), 4(d)]. We found that both the vascular density within the whole field of view (FOV) and the region of interest (ROI, dashed green circle) remained almost unchanged within the first 10 min during the VTP treatment, followed by an obvious decrease after VTP [Fig. 4(c)]. This suggests a persistent VTP effect, leading to the closure of the majority of microvasculature in tumor 60 min post VTP. In addition, we found that the different types of blood vessels in the window chamber responded to VTP in a different manner for this mouse. While only a slight change was identified on arteries, VTP caused a significant decrease in diameter of veins and tumor vessels after treatment [Fig. 4(d)].

A control study was performed to further demonstrate the VTP effects on tumor microvascular. For both the VTP group (n = 4) and control group (n = 1), en face vascular (OCTA) images were acquired at different time points, i.e., before the injection of tumor cells, immediately before VTP/light irradiation and 24 hours after VTP/light irradiation [Figs. 5(a), 5(b)]. While the tumor angiogenesis was found in both groups, only the VTP group showed significant photodynamic effects, leading to a complete disappearance of blood flow within the field of view [Fig. 5(a)], suggesting the fatal damage to the blood vessels. This observation was verified by the statistically significant change (P<0.05) in vascular density for the VTP group, versus the subtle change in vascular density for the control group [Figs. 5(b), 5(d)].

 

Fig. 5. Comparison study of the VTP effects on tumor microvasculature. En face vascular (OCTA) images of baseline (i.e., before the injection of tumor cells), immediately before VTP and 24 hours after VTP for the VTP group (a) and the control group (b). The corresponding vascular densities were also calculated for the VTP group (c) and the control group (d). The vascular densities were normalized to the corresponding baseline values measured before tumor cell injection. Scale bars: 500 µm. Error bars: representing SDs. (*: P < 0.05).

Download Full Size | PPT Slide | PDF

4. Discussions and conclusion

In this study, we demonstrated in vivo imaging assessment of microvascular in tumor with an ultrahigh-resolution 800-nm OCT system. The newly formed blood vessels of about 10 µm in diameter in tumor could be readily identified. We further studied the angiogenesis of a mouse tumor model to demonstrate the capability of UHR-FOCT for providing quantitative spatial and temporal information of tumor microvasculature. By imaging and quantifying the responses of tumor microvasculature to VTP in vivo, this technique could potentially advance our understanding on VTP and help achieve an optimal tumor treatment outcome in the future.

It was noticed that different responses to VTP were observed for different types of blood vessels. In our study, VTP caused a significant decrease in diameter to veins and tumor vessels after treatment, while only a slight change was observed on arteries. This finding is consistent with the previous reports on photodynamic therapy-induced vasoconstriction in the chorioallantoic membrane (CAM) model, which demonstrated the higher vulnerability of venules than arterioles to photodynamic therapy [42]. This might be attributed to the different microstructures of blood vessels’ wall [21,43]. The artery wall is usually thicker than that of a vein of an equal size. Moreover, some of the sprouting tumor vessels have no lining of endothelial cells or fibrous tissues [21]. We might anticipate that photodynamic reaction causes less immediate damage to the thicker and more resilient vessel wall of the artery when comparing to the vein and sprouting tumor vessels of a thin wall. In addition, the differences in the blood flow (related to cooling during photodynamic therapy) and oxygen level in arteries, veins, and tumor vessels may also lead to different rates in platelet aggregation, thrombus formation and vessel occlusion, triggered by the photodynamic reaction. Furthermore, the sequence of vascular events may last for some time after the VTP treatment. More studies are needed to eventually elucidate this observation.

The current study is limited to a small number of animals. Future work should systematically study the correlation between the tumor microvascular responses and the treatment outcome of VTP. The dorsal skinfold chamber model is commonly used for the study of tumor angiogenesis and treatment responses [27]. A major advantage of this technique is its ability to track dynamic changes of the tumor microvasculature over a period of 2-3 weeks. This dorsal window chamber technique is invasive and can be replaced with a non- or less invasive model (such as the mouse ear tumor model), which also enables the study of tumor angiogenesis and its response to VTP. Moreover, motion artifacts were observed in the OCTA images. In principle, the motion artifacts would be further minimized by increasing the imaging speed.