The natural sulfoglycolipid derivative SQAP improves the therapeutic efficacy of tissue factor-targeted radioimmunotherapy in the stroma-rich pancreatic cancer model BxPC-3

Highlights • SQAP enhanced tumor uptake and the therapeutic efficacy of radiolabeled anti-tissue factor antibody 1849.• SQAP allows for a reduction of the dose of the therapeutic agent 90Y-labeled 1849 to half.• SQAP did not affect hematologic parameters, or gastrointestinal and respiratory systems in mice.• 90Y-labeled 1849 with SQAP potentially increases exposure of tumors to radiation.


Introduction
Pancreatic cancer is a highly lethal cancer with a 5-year survival rate of 10% for all stages of the disease [1]. It is projected to be the second leading cause of cancer death in the United States by 2030 [2]. The disease progresses asymptomatically in 80% of patients and is thus usually detected in an advanced stage with local invasion and/or metastasis, resulting in unresectable cancer [3]. Among the 10%-15% of patients who present with resectable disease, 80% experience a relapse [4]. Various chemotherapeutic agents are applied for locally advanced and metastatic disease, but with limited outcomes [5]. Therefore, new systemic treatment strategies are needed.
A malignant cycle of blood coagulation is postulated to generate versatile cancer stroma, leading to cancer invasion into vessels, tumor proliferation, and replacement with collagenous tissue [6]. Cancer coagulopathy is triggered by tissue factor (TF), which is a transmembrane glycoprotein (47 kDa) present on the cell surface [7]. Abnormally high TF expression is found in various tumors, including pancreatic cancer [8][9][10]. High TF expression in pancreatic cancer correlates with tumor grade, extent, metastasis, and invasion, in contrast to normal pancreas with low TF expression [7,9,11]. TF is expressed not only on the tumor cell surface but also in the tumor stroma and on tumor-associated vascular endothelial cells [8,12]. Therefore, TF is a potential target for cancer diagnostic imaging and therapy for pancreatic cancer with a rich stroma.
We developed high-affinity anti-TF antibodies and demonstrated that radiolabeled anti-TF monoclonal antibody 1849 has high potential as a noninvasive imaging probe [13]. In glioma and pancreatic cancer xenograft models, 111 In-labeled 1849 exhibits high uptake in tumors and low uptake in normal organs [14,15]. Radiolabeled 1849 in which the imaging radionuclide 111 In was replaced with a β-emitting therapeutic radionuclide, such as 90 Y and 177 Lu, is a promising radioimmunotherapy (RIT) agent for metastatic pancreatic cancer. The pancreatic cancer model BxPC-3, however, exhibits resistance to monotherapy with 90 Y-labeled antibodies [16][17][18]. RIT with 90 Y-labeled antibodies achieved complete remission in a stroma-poor model, MIA-PaCa-2, whereas the stroma-rich pancreatic cancer model BxPC-3 showed a limited efficacy of progressive disease [14,[19][20][21][22][23]. The rich stroma is involved in the resistance, which contributes to the inhibition of antibody penetration as the main barrier for RIT [23]. BxPC-3 shows the most radioresistant to RIT with 90 Y-labeled antibodies [23] and is the only clinically relevant stroma-rich pancreatic cancer model available to our knowledge. Even though the highest level of TF expression on both tumor parenchyma and stroma in BxPC-3, TF-targeted RIT with 90 Y-labeled antibodies stated only moderate effect [24]. If additional strategies to enhance the therapeutic efficacy of RIT beyond such therapeutic resistance can be established, those would serve as a superior therapeutic option even for the highest class of stroma-rich refractory pancreatic cancer. Sulfoquinovosylacylglycerol (SQAG) is a sulfoglycolipid originally isolated from natural sources, including higher plants [25,26], sea urchins [27], and marine algae [28]. SQAG has tumor-radiosensitizing properties [29,30]. A synthetic analog of SQAG, α-sulfoquinovosylacyl-1,3-propanediol (SQAP or CG-0321) ( Fig. 1) was synthesized for clinical studies [31] and acts as a radiosensitizer like SQAG [32,33]. This compound induces pharmacologic alterations of the tumor microenvironment; low-dose (2 mg/kg) intravenous administration of SQAP increases tumor perfusion and oxygenation, thereby enhancing external-beam radiotherapy by boosting the oxygen effect, which significantly delays the growth of murine SCCVII and human A549 xenografts [32]. This finding has attracted attention to SQAP as a potentially useful option for improving the therapeutic efficacy of internal radiotherapy for cancer.
The present study evaluated whether SQAP improves the therapeutic efficacy of RIT in the stroma-rich refractory pancreatic cancer model BxPC-3. An anti-TF monoclonal antibody 1849 labeled with the γ-emitter 111 In with 171 keV and 245 keV, and a half-life of 67.4 h [34], was produced. The effect of SQAP on the biodistribution of 111 In-labeled 1849 was assessed. The therapeutic efficacy of the antibody labeled with a β-emitter 90 Y (maximum energy, 2.3 MeV; half-life, 64.1 h [34]) substituted for 111 In was examined in combination use with SQAP.

Biodistribution and absorbed dose estimation
The Animal Care and Use Committee of the National Institute of Radiological Sciences approved the protocol for the animal experiments, and all animal experiments were conducted by following the Institutional Guidelines regarding Animal Care and Handling. When   were administered intravenously 20 min before injection of the radiolabeled antibody, and 1 and 2 days after the injection. At 1, 6, 24, 48, 96, and 168 h after injection of 111 In-1849, the mice were humanely killed by overexposure to isoflurane. Tumors, and tissues and organs of interest (blood, lung, liver, spleen, pancreas, intestine, kidney, muscle, and bone) were removed and weighed, and radioactivity was measured using a gamma-counter (2470 WIZARD 2 , PerkinElmer, Waltham, MA, USA). The data were expressed as the percentage of injected dose per gram of tissue (% ID/g) normalized to a mouse with a body weight of 20 g. The absorbed dose for 90 Y-labeled 1849 was estimated using the area under the curve of each organ based on the biodistribution data of 111 In-1849 and the mean energy emitted per transition of 90 Y, 1.495 × 10 − 13 Gy kg (Bq s) − 1 [36] as described previously [37].

Therapeutic experiments
When subcutaneous tumors reached a diameter of approximately 8 mm, therapeutic experiments were conducted. The mice (n = 5/dose) were injected into a tail vein with 0.925, 1.85, or 3.7 MBq of 90 Y-labeled 1849. The protein dose was adjusted to 10 µg for each preparation by adding intact antibody. Saline and SQAP (2 mg/kg body weight) were administered intravenously 20 min before injection of the radiolabeled antibody, and 1 and 2 days after the injection. Untreated mice (n = 5) were used as a control. Body weight and tumor size were measured at least 3 times a week for 8 weeks. When the tumor reached 12 mm in diameter, the mouse was humanely killed by overexposure to isoflurane. Tumor volume (mm 3 ) was calculated as (length × width 2 )/2.

Histologic analysis of tumors
As a separate experiment, tumor samples (n = 3/time-point) were extirpated at days 1, 2, and 5 after intravenous injection of intact 1849 (0 MBq) or 3.7 MBq of 90 Y-1849 with saline or SQAP (2 mg/kg body weight). Tumor sections were stained with hematoxylin and eosin (Sakura Finetek USA, Torrance, CA, USA). Apoptotic cells in tumors were stained by TUNEL staining with a DeadEnd Colorimetric TUNEL system (Promega, Madison, WI, USA). Ki-67 antigen was detected using an anti-human Ki-67 polyclonal antibody (Agilent Technologies Japan, Tokyo, Japan) as described previously [38]. CD31 antigen was detected using an anti-CD31 polyclonal antibody (Abcam, Cambridge, UK) as described previously [23]. Details are provided in the Supplementary information.

Statistical analysis
All quantitative data are expressed as the means ± standard deviation (SD). The data were analyzed with GraphPad Prism 7 software (GraphPad Software, La Jolla, CA, USA). Details are provided in the Supplementary information.

Effect of SQAP on hematologic parameters
To test the pharmacologic effects of SQAP on the blood system after a single-dose administration, hematologic tests were conducted. Compared with the saline-injected control mice, intravenous injection of 2 mg/kg of SQAP had no effect on the number of hemocytes, cell concentrations, hematocrit, or other hematologic parameters tested ( Table 1). This trend was also observed in mice injected with a higher dose (up to 32 mg/kg) of SQAP. No noticeable damage to the stomach or tail vein was observed in mice injected with less than 6 mg/kg of SQAP (data not shown). For extra-beam radiotherapy, a 2 mg/kg dose was injected intravenously 20-30 min before irradiation to evaluate effects for sensitizing radiation [32]. This dose was used in the following experiments.

Biodistribution of combined 111 In-labeled 1849 and SQAP in mice bearing BxPC-3 tumors
As the schedule shows in Fig. 2, biodistribution experiments of 111 Inlabeled 1849 were conducted in nude mice bearing BxPC-3 subcutaneous tumors at 1 to 168 h after injection of the antibody (n = 5 each timepoint). To evaluate the effect of SQAP on biodistribution, the mice were injected intravenously with saline or SQAP at 20 min before 111 In-1849 injection, and 24 and 48 h after the injection. Tumor uptake of 111 In-1849 at 1 h after injection was 3.2 ± 1.5% ID/g in the saline-injected group and 4.4 ± 1.4% ID/g in the SQAP-injected group, and uptake increased until 48 h after injection (Fig. 3). The peak values were 37.8 ± 12.5% ID/g in the saline group and 38.8 ± 12.4% ID/g in the SQAP group at 48 h after injection, and these values were almost maintained for up to 96 h after the injection and then decreased (Fig. 3). Tumor uptake differed significantly between the saline and SQAP groups at 24 h after injection (P < 0.05; Fig. 3). The time-activity curves in the blood and major organs were similar between groups with no significant difference (Fig. 3). On the basis of these results, the absorbed dose was estimated by replacing 111 In with the therapeutic radioisotope 90 Y, as shown in Table 2. The dose absorbed by tumors in the saline group injected with 0.925, 1.85, and 3.7 MBq of 90 Y-labeled 1849 was estimated to be 10.9, 21.8, and 43.7 Gy, respectively, and that in the SQAP group was 12.0, 23.9, and 47.9 Gy, respectively ( Table 2). The absorbed doses to normal organs were similar between the saline and SQAP groups ( Table 2).

Treatment with 90 Y-labeled 1849 and SQAP in mice bearing BxPC-3 tumors
The treatment effects of 90 Y-labeled 1849 with saline or SQAP were evaluated based on tumor volume and body weight in mice bearing BxPC-3 tumors (Fig. 4). Tumor growth curves are shown in Fig. 4A and representative mouse photos are shown in Fig. 4B. No statistically significant difference was observed in the 3 control groups (Fig. 4A). By contrast, treatment with 90 Y-1849 (0.925, 1.85, and 3.7 MBq) significantly suppressed tumor growth compared with no treatment, or treatment with saline or SQAP alone (P < 0.01; Fig. 4A and 4B). Treatment with 3.7 MBq 90 Y-1849 with SQAP had the greatest tumor suppression effect: BxPC-3 tumor growth was suppressed until around 28 days after treatment (Fig. 4A, 4B, and 4C), and tumor volumes gradually increased thereafter (Fig. 4A). In 2 of the 5 mice treated with 3.7 MBq 90 Y-1849 with SQAP, tumor growth suppression continued to around day 56 ( Fig. 4A and 4B). The therapeutic effect of 1.85 MBq 90 Y-1849 with SQAP on tumor growth suppression was comparable to that of 3.7 MBq 90 Y-1849 with saline (Fig. 4C). Survival was most prolonged in mice treated with 3.7 MBq of 90 Y-1849 with SQAP: 100% until day 33 and then decreasing to 40% at day 56 (end of the observation period), followed by 20% at day 56 in the groups receiving 0.925 MBq of 90 Y-1849 with SQAP, 1.85 MBq of 90 Y-1849 with saline and SQAP, and 3.7 MBq of 90 Y-1849 with saline (Supplementary Figure 1). Fig. 4D shows temporal body weight changes in mice. Compared with day 0, significant transient body weight loss was observed in the following treatments groups: SQAP alone (P < 0.05), 1.85 MBq of 90 Y-1849 with saline (P < 0.01) and SQAP (P < 0.01 and P < 0.05), and 3.7MBq of 90 Y-1849 with saline (P < 0.01 and P < 0.05) and SQAP (P < 0.01). The decreased body weight recovered within several days (Fig. 4D). No visible adverse effects, such as diarrhea and dyspnea, were observed at any dose level.

Histologic analysis of BxPC-3 tumors treated with 90 Y-labeled 1849 and SQAP
Histologic changes were evaluated in groups of mice treated with unlabeled 1849 (0 MBq) and saline or SQAP, or with 3.7 MBq of 90 Y-   labeled 1849 and saline or SQAP. Hematoxylin and eosin-stained sections of BxPC-3 tumors did not exhibit marked differences in any treatment groups compared with the untreated group as a control (Fig. 5A). As shown in Fig. 5B, TUNEL-stained sections showed few apoptotic cells in any treatment groups.
To evaluate the effect on cell proliferation, Ki-67 staining was conducted (Fig. 6A) and the positive cells were quantified (Fig. 6B).
There was no significant difference between saline and SQAP treatments in any antibody treatments and on any days ( Fig. 6A and 6B). Significant differences were observed between mice treated with 0 MBq and 3.7 MBq 90 Y-1849: 3.7 MBq with SQAP increased the number of Ki-67positive swollen cells compared with 0 MBq with SQAP at day 1 (P < 0.05) and day 2 (P < 0.01) as shown in Fig. 6B. Although there was no statistical difference, treatment using 3.7 MBq 90 Y-1849 with saline tended to increase the number of Ki-67-positive cells (Fig. 6B).
To evaluate the blood vessels in tumors after the treatments, tumors were stained with the endothelial cell marker CD31. Most vessels were observed in stromal tissues but some were found in tumor cell regions of untreated BxPC-3 tumors (Fig. 7A and 7B). Although the number of vessels was markedly increased at day 2 in the group treated using 3.7 MBq 90 Y-1849 with SQAP, the difference was not statistically significant ( Fig. 7A and 7B).

Discussion
The findings of the present study revealed that SQAP enhanced the efficacy of RIT with 90 Y-labeled 1849 against BxPC-3 tumors. This is likely because SQAP enhanced the tumor uptake of radiolabeled antibodies by increasing tumor perfusion, as observed on photoacoustic imaging and dynamic contrast-enhanced magnetic resonance imaging of tumors [32]. Radiolabeled 1849 showed high uptake in BxPC-3 by active targeting in addition to passive targeting (i.e., enhanced permeability and retention effect; EPR [39]). SQAP also increases tumor oxygenation, which can enhance the cell-killing effects of radiation [32]. These functions of SQAP would enhance the antitumor effect of 90 Y-labeled 1849, further suppressing tumor growth beyond that of 90 Y-1849 alone by increasing the absorbed dose from 43.7 to 47.9 Gy when injected with 3.7 MBq. In addition to increased perfusion, enhanced tumor oxygenation may play an important role in creating a radiosensitizing effect [32]. Noteworthy, 1.85 MBq of 90 Y-1849 with SQAP provided an antitumor effect equivalent to 3.7 MBq of 90 Y-1849 without SQAP; hence, SQAP administration can decrease the injected dose of radiotherapeutic agents to half. That means radiation doses absorbed by organs and tissues are reduced by half, suggesting that SQAP would contribute to reducing radiologic toxicity in patients. Our findings suggest that SQAP is a potentially powerful tool to enhance the efficacy of RIT with lower toxicity, and TF-targeted RIT with SQAP is a promising therapeutic option for pancreatic cancer, even those that have metastasized.
The increased perfusion and oxygenation induced by the addition of SQAP continue for only a short period [32]. Nevertheless, the enhanced antitumor effects were observed for a long period in the present study. Brief effects on perfusion were supported in the present study: the largest difference in the tumor uptake of the radiolabeled antibody was detected at 1 h (1.4-fold tumor uptake), and thereafter no significant difference was detected. Tumor growth suppression was observed for 28 days, although SQAP injection terminated on day 2. Tumor vascular formation increased in tumor cell regions treated with 90 Y-1849, especially in treatment groups also treated with SQAP. SQAP itself has no angiogenetic effects, thus the increased tumor uptake of 90 Y-1849 by SQAP enhances the absorbed radiation dose and cognate vascular formation, as is the case with neoangiogenesis induced by external-beam radiation [40]. This causes a wider intratumoral biodistribution of 90 Y-1849, enhancing tumor cell damage. Indeed, our histologic analysis revealed an increase in the number of Ki-67-positive cells, which was reported as an index after external-beam radiation [41], providing evidence for increased irradiation to BxPC-3 cells. Both increased tumor uptake by SQAP and enhanced vascular formation at early time-points could contribute to increase radiation exposure to tumors, resulting in tumor cell damage for a long period. Further investigations are necessary to better understand the mechanism of SQAP-induced radiosensitization in RIT.
SQAP enhanced the efficacy of RIT; unfortunately, the combination therapy did not lead to complete remission. Additional strategies are needed to improve the efficacy. First, SQAP dose escalation may further enhance treatment. In the present study, we used a low dose of 2 mg/kg for combination therapy, but higher doses from 4 to 18 mg/kg were tolerable in mice. Higher doses would enhance perfusion and oxygenation, perhaps conferring higher antitumor effects. Further investigation to determine the optimal dose and schedule of SQAP is required to maximize the efficacy of the combination therapy with few side effects. Second, a new SQAP derivative with long blood circulation could be an option. External-beam radiotherapy includes short-time radiation with a high dose rate, while RIT includes continuous radiation with a low dose rate. The SQAP effects do not continue for a long period [32], and SQAP was injected 3 times for a single shot of 90 Y-1849. The short-term SQAP effects are due to its short half-life in the blood (unpublished data). Extending the half-life might enhance the efficacy of RIT. Because a long half-life could increase hematologic toxicity, it is necessary to consider the balance of toxicity and benefit.
The present study has several limitations. First, although SQAP improved the efficacy of RIT, complete remission was not achieved. Further studies are needed to optimize the dose and schedule for RIT, and develop a new derivative with a long half-life suitable for RIT, as mentioned above. Second, RIT can target metastatic cancer, but the present study includes only a subcutaneous tumor model. Most pancreatic cancers are diagnosed at advanced stages with invasive and metastatic cancer cells. Our TF-targeted RIT with SQAP has the potential to treat such cancer cells. It is necessary to evaluate the efficacy in metastatic models. Such investigations will increase the potential benefit of SQAP in RIT to provide better outcomes.

Conclusion
The findings of the present study indicated that SQAP enhanced the antitumor effect of RIT as well as external-beam radiotherapy. Administration of SQAP improved the efficacy of RIT with 90 Y-labeled 1849 in the stroma-rich pancreatic cancer model BxPC-3 by increasing tumor uptake and cognate biologic actions in the early phase of the treatment. Combining 90 Y-labeled 1849 with SQAP produced a similar antitumor effect with only a half dose of 90 Y-labeled 1849 without increasing adverse effects. The use of SQAP for clinical RIT is a promising therapeutic option for malignant pancreatic cancer. Because high TF expression is observed in many types of cancer other than pancreatic cancer [9,10,42], SQAP could be applicable to enhance the therapeutic efficacy of TF-targeted RIT to different cancers. Our findings warrant further investigations for clinical application.

Declaration of competing interest
Fumio Sugawara and Kengo Sakaguchi are employees of MT3 Technologies Inc. The other authors have no financial or other competing interests to declare in relation to this study.