Recent advances in clinical image guidance have enabled techniques like SBRT to deliver large doses of ionizing radiation to pancreatic tumors, while minimizing damage to healthy tissues. Despite its clear benefits, the ultimate success of SBRT in clinical trials for pancreatic cancer has been suboptimal38. While several studies have shown that SBRT improves overall survival for pancreatic cancer patients4–6, treatment failure remains a significant issue17. One potential explanation for treatment failure following SBRT is the presence of hypoxia within the pancreatic TME39. Tumor hypoxia is known to contribute to treatment resistance to both chemo- and radiotherapy40,41 and pancreatic tumors have been shown to exhibit significant levels of inherent hypoxia42. In addition, hypoxia has been shown to predict more aggressive growth and spontaneous metastasis formation in pancreatic tumor models43. Thus, understanding the radiobiological response of pancreatic tumors to SBRT, including its effects on tumor hypoxia within the TME, can help optimize SBRT and improve overall treatment response. In this study, we combined an orthotopic mouse model of pancreatic cancer with an IVFM platform to directly monitor the effects of 5×8Gy SBRT on the pancreatic tumor and TME in vivo. Using this technique, we were able to simultaneously image DsRed fluorescent human pancreatic cancer cells, tumor cell hypoxia (using a 5xHRE/GFP reporter), and tumor microvasculature before and (1, 4, 7, and 14 days) after SBRT. Ex vivo IHC staining was performed on excised tumors 1 and 14 days after SBRT to validate in vivo findings.
It is well known that DNA damage is the main mechanism by which radiotherapy kills tumor cells44. By performing IHC staining on tumor tissue sections, we were able to quantify significant DNA damage in treated tumors 1 day post-SBRT (Fig. 4b,c), confirming the accurate delivery and treatment effect of ionizing radiation in our orthotopic tumor model. Then, using our mouse intravital imaging platform, we were able to directly monitor the effects of SBRT in orthotopically grown BxPC3-DsRed tumor cells in vivo before and up to two weeks after SBRT. Indeed, other studies have shown that constitutively expressed fluorescent proteins are suitable for monitoring total gene expression in cells45 as well as overall cell viability46. In our experiment, we found that the DsRed MFI persistently decreased for up to two weeks post-SBRT, compared to non-irradiated tumors (Fig. 4a), suggesting, unsurprisingly, that SBRT had a significant impact on tumor cell viability. This was further evidenced by a decrease in tumor cell proliferation (Ki67 expression) post-SBRT (Fig. 4d,e). These results are consistent with clinical studies showing decreased Ki67 expression in pancreatic tumors following SBRT47, which is associated with progression-free survival48. Overall, our experiment demonstrated a significant treatment response in orthotopic BxPC3 tumors following SBRT, resulting in both DNA damage and decreased cellular activity (i.e., viability and proliferation), compared with non-irradiated tumors.
Hypoxia is also an important component of the TME49. It is a major contributor to treatment resistance in pancreatic tumors41 and also promotes a more invasive and malignant phenotype50. In this study, we demonstrate that 5×8Gy SBRT significantly decreased tumor hypoxia in our orthotopic pancreatic tumor model within the first 2 weeks after treatment completion. Using IVFM to monitor HRE-driven GFP fluorescence (a biomarker of tumor hypoxia in BxPC3 tumors35) we found that the GFP MFI decreased as early as 1 day after SBRT, compared to non-irradiated tumors (Fig. 5a). To corroborate these observations, IHC staining of BxPC3 tumors showed a significant decrease in CA9 expression at both 1 and 14 days after SBRT, compared to non-irradiated tumors (Fig. 5b,c). CA9 is another biomarker of hypoxia and has previously been used to assess hypoxic fraction in tumors51,52. These findings demonstrate that 5×8Gy SBRT produced a reoxygenation effect as early as 1 day post-SBRT in our pancreatic tumor model, and persisted for at least 14 days.
Reoxygenation is one of the ‘four Rs’ of conventional radiotherapy30. As ionizing radiation kills tumor cells, it allows more oxygen to reach previously hypoxic cells, reoxygenating the tumor for additional fractions of radiotherapy53. However, studies using higher doses of radiotherapy (e.g., SBRT) have sometimes produced the opposite effect, causing acute radiation-induced vascular damage and increased tumor hypoxia54–57. Other preclinical studies of SBRT have produced similar results to our own, showing decreased tumor hypoxia, either by conventional reoxygenation of the tumor or by promoting improved blood vessel perfusion39,58–60. The varying effects of SBRT on tumor oxygenation are likely dependent on the total radiation dose and fractionation schedule as well as tumor model, type, site, and stage61. Nevertheless, this inconsistency in the literature requires a better understanding of the radiobiological mechanisms impacting tumor (re)oxygenation following SBRT. Optimizing SBRT to reoxygenate the pancreatic TME may help prevent occurrences of spontaneous metastasis and relapse following SBRT, improving treatment outcomes.
Tumor oxygenation is controlled by two main factors: the demand for oxygen from proliferating tumor cells and the supply of oxygen from (tumor) blood vessels62. Decreased oxygen consumption within a tumor, either from decreased tumor cell density or decreased cell metabolic activity, can improve tumor oxygenation63. Conversely, improving the functionality of tumor microvasculature can increase blood perfusion and oxygen delivery to tumor tissue64. To better understand the underlying cause of reoxygenation in our pancreatic tumor model following SBRT, we examined whether changes in oxygenation were caused by either a decrease in oxygen demand or an increase in oxygen supply (or both).
In general, tissue oxygen concentrations are inversely proportional to cell density65. Once cells lose their contact inhibition (a key step in malignant transformation) and experience uncontrolled proliferation, the physical constraints of the surrounding tissue can lead to overcrowding of cells66. This increasing density of tumor cells has been shown to limit the penetration of anticancer drugs67 and increase local O2 consumption leading to localized regions of hypoxia68 and upregulation of HIF activity as well as other hypoxia response pathways. In our experiment, we used hematoxylin and eosin (H&E) staining to show that tumor cell density decreased significantly by an average of 22% in tumors 1-day post-SBRT, compared to non-irradiated tumors (Fig. 6). This dramatic decrease in cell density may have reduced O2 demand within the tumors, explaining the corresponding decrease in tumor hypoxia 1 day post-SBRT. However, given that the difference in cell density was not statistically significant 14 days after SBRT, despite a persistent decrease in CA9 expression (Fig. 5b), it is likely that other factors contributed to the sustained reoxygenation of the tumor at later time points. For example, cellular proliferation (Ki67 expression) and viability (DsRed fluorescence) were both significantly decreased at 7 and 14 days post-SBRT, respectively. Cellular proliferation and metabolic activity have previously been shown to have a direct impact on oxygen consumption rates in tumor cells69. Thus, our results suggest that the observed reoxygenation of tumors following SBRT in our study was likely driven by a decrease in oxygen demand within the tumor tissue.
A functional vascular network is essential to supply oxygen to the pancreatic TME70. Pancreatic tumors with a high microvascular density (MVD) tend to be less hypoxic than those with low MVD71. Indeed, we have demonstrated this relationship using our intravital animal model in a previous study35. Thus, to understand how pancreatic tumor microvasculature responded to SBRT (and potentially influenced tumor hypoxia), we analyzed the blood vessel density of tumors using both in vivo IVFM (Fig. 7a) and ex vivo IHC staining of CD31-positive vascular endothelial cells (Fig. 7b,c). From both data, no significant differences in blood vessel density were observed between SBRT-treated and non-irradiated tumors at any of our imaging timepoints. Interestingly, our group72 and others54,73 have previously shown that high doses of radiotherapy—typically greater than 10 Gy per fraction61—can cause severe vascular damage in tumors. Thus, it is likely that the dosing schedule in our experiment (5 daily fractions of 8 Gy) was below the threshold to inflict significant vascular damage. Conversely, other studies have shown that using similar doses of radiotherapy can potentially improve tumor vasculature function through multiple mechanisms. For example, Sonveaux et al.74 found that irradiating hepatocarcinoma xenografts with 1×6Gy increases tumor blood flow and oxygenation through a vascular nitric oxide (NO)-dependent pathway. Tong et al.59 also found that irradiating non-small cell lung cancer xenografts with 1×12Gy resulted in a brief window of ‘vascular normalization’ and subsequent reoxygenation 7- and 14-days post-radiotherapy. This effect coincided with a downregulation of the p-STAT3/HIF-1α pathway (as well as its downstream angiogenic factors: VEGFA and CXCL12), resulting in decreased tumor blood vessel diameter and tortuosity, and increased pericyte coverage. In our experiment, we were able to observe a similar phenomenon in CD31-stained tumor tissue sections with a significant decrease in total vascular area 1 day post-SBRT (Fig. 7d). Given that tumor microvasculature is often pathologically dilated and irregular in size75,76, a transient decrease in tumor vascular area following 5×8Gy SBRT may suggest that there was a brief window of vascular normalization. However, future studies are needed to validate whether this phenomenon occurred and if it played a significant role in the reoxygenation of our pancreatic tumor model after SBRT.
In summary, using both in vivo IVFM and ex vivo IHC staining, this study demonstrated that 5×8Gy SBRT significantly decreased tumor cell viability, proliferation, density, and hypoxia in our pancreatic tumor model. Hypoxia is a major contributor to treatment resistance in pancreatic tumors41 and promotes a more invasive and malignant phenotype50. Thus, understanding the extent (and time course) of tumor reoxygenation following SBRT can help optimize radiation dose and fractionation schemes in the clinical setting and improve treatment response. While our intravital imaging platform allowed us to monitor the short-term effects (days to weeks) of SBRT on the pancreatic TME, follow-up studies are needed to appreciate the long-term impact of these changes on tumor control, metastasis, and overall survival. As previously mentioned, the effects of SBRT on solid tumors are likely dependent on several factors, including treatment regimen, tumor type, site, and stage61. Future work can continue to explore how SBRT-induced reoxygenation effect occurs in a dose-dependent manner, and if this effect is generalizable to other pancreatic tumor models, in order to better predict treatment response.