Quantitative diffuse reflectance spectroscopy of short-term changes in tumor oxygenation after radiation in a matched model of radiation resistance

: There is a critical need to identify patients with radiation-resistant tumors early after treatment commencement. In this study, we use diffuse reflectance spectroscopy (DRS) to investigate changes in vascular oxygenation and total hemoglobin concentration in A549 radiation-sensitive and resistant tumors treated with a clinically relevant dose fraction of 2 Gy. DRS spectra were acquired before, immediately after, 24, and 48 hours after radiation. Our data reveals a significantly higher reoxygenation (sO 2 ) in the radiation-resistant tumors 24 and 48h after treatment, and provides promising evidence that DRS can discern between the reoxygenation trends of radiation-sensitive and resistant tumors.


Introduction
Radiation therapy is typically delivered in several fractions to treat cancers because dose fractionation is believed to reoxygenate and hence radiosensitize the tumor. The central hypothesis of this theory is that the death of oxygenated cells in response to a radiation dose fraction improves the oxygenation of previously hypoxic cells, thus radiosensitizing these cells to subsequent radiation fractions [1]. The oxygen-sensing pO 2 microelectrode has established a wealth of knowledge related to tumor oxygenation before and during radiation therapy, and its association with treatment outcome [2][3][4][5][6][7]. Gatenby et al. assessed pretreatment oxygen tension in advanced head and neck tumors in the clinical setting and observed that complete responders were characterized by a significantly higher oxygen content compared to those in the non-responder group. Their findings strongly suggested that hypoxia within metastatic lesions was linked to a poor prognosis after radiotherapy [5]. Similar findings were later reported by Brizel et al. [2], who showed that hypoxia adversely affected the short term clinical response to radiation, disease-free survival and overall survival in patients with head and neck squamous cell carcinoma (HNSCC). Ressel et al. monitored changes in pO 2 in HNSCC patients, and found that tumors with the best long-term outcome displayed a significant increase in the median pO 2 during therapy [8]. In paclitaxeltreated MCA-4 murine mammary carcinoma tumors, Milas et al. observed that reoxygenation was the dominant mechanism by which paclitaxel greatly enhanced tumor radioresponse [9]. Although these studies and others [10,11] have offered compelling evidence that tumor reoxygenation before and between radiation fractions is critically associated with treatment response, others have shown that reoxygenation after radiation is also associated with tumor recurrence [12,13]. In a clinical study, Dietz et al. measured pO 2 in cervical lymph node metastases of 37 patients with advanced HNSCC before and one week after chemoradiation. Their findings revealed that a higher degree of reoxygenation was correlated with a poor Vol. 9, No. 8 | 1 Aug 2018 | BIOMEDICAL OPTICS EXPRESS 3794 treatment outcome, and also suggested that pretreatment hypoxia held a poor prognostic significance for advanced HNSCC [12].
Given the invasive nature of the oxygen-sensing electrode and its inability to perform frequent longitudinal measurements of the same tissue, we sought to determine if a noninvasive approach involving diffuse reflectance spectroscopy (DRS) was sensitive to radiation-induced changes in tumor oxygenation, and whether such changes were different in radiation-sensitive and resistant tumor xenografts. DRS is an optical fiber-based technique that uses visible or near-infrared light to interrogate tissue; the sampling depth within tissue is dependent on the separation of source and detector optical fibers, and the reflected light is used to quantify the underlying scatterers and absorbers, a combination of which is used for recognition of tissue pathology. The vascular oxygenation (sO 2 ) in tumors can be quantified by determining the individual contributions of major absorbers, such as oxygenated (HbO 2 ) and deoxygenated (dHb) hemoglobin within the blood vessels [14][15][16][17]. We recently demonstrated that vascular oxygenation was inversely correlated with immunohistochemical quantification of tumor hypoxic fraction [18]. In addition, such measurements have been shown to be concordant with microelectrode-based determinations of tumor pO 2 [19]. DRS has previously been shown to be sensitive to radiation dose-dependent changes in oxygenation during therapy [13]. Furthermore, such optical measurements of oxygenation were shown to be predictive of local control and treatment failure after radiation in a human head and neck tumor xenograft model [13,20]. However, studies examining the sensitivity of optical spectroscopy to changes in oxygenation after clinically relevant radiation dose fractions have been lacking. The goal of this study was to determine the sensitivity of DRS to changes in tumor oxygenation in response to dose fractions that were closer to the clinical paradigm. To perform these investigations, we used a matched model of radiation resistance to generate an isogenic radiation-resistant cell line from human A549 lung cancer cells. Whereas most investigations studying the physiologic and molecular changes associated with radiotherapy establish responder and non-responder groups following a high radiation dose, this study was conducted with tumor xenografts generated from a pair of isogenic radiationresistant and sensitive cancer cell lines, where the radioresistance had already been established. The tumor xenografts developed from these cell lines in athymic mice were radiated with 4 doses of 2 Gy X-ray radiation therapy. DRS spectra were acquired every day during the course of treatment, allowing the evaluation of short-term changes in tumor oxygenation, and the cumulative effects of radiation following each dose. Our results point to radiation-induced changes in oxygenation in both sensitive and resistant tumors.

Cell culture and development of tumor xenografts
A549 lung cancer cells were obtained from American Type Culture Collection (ATCC; CCL185). A matched radiation-resistant clone (rA549) was developed from the A549 cells by repeated exposure to radiation (25 fractions of 2 Gy each every 3 days) as described previously [21]. Swiss athymic (nu/nu) mice that were 8-10 weeks old and weighing between 20 and 25 g were injected on the left or right flank with a subcutaneous bolus (10 million cells suspended in serum-and media-free saline) of A549 or rA549 cells to grow tumor xenografts. Once tumor volumes reached 200 mm 3 , mice were assigned to 1 of 4 groups depending on whether they received radiation therapy (A549-XT and rA549XT) or sham radiation (A549-NT and rA549-NT). A total of 30 mice and 32 tumors were used across all four groups. The number of mice allocated to each group can be seen in Table 1. Tumor volumes were monitored using Vernier calipers calculated according to the equation V = (π/6) x (length) x (width) x (height).

Radi
Radi Rad Rad Mice in the X two weeks [ Radiotherapy Branford, CT collimated X-Mice were an into the radia shielded using 90 post-radiat

Diffuse r
The DRS set Florida), a fib probe (dia. = separation of and five perip 80% reflectan lamp through wavelength ra 100, 150, and on days 1, 4, also acquired performed to radiation thera   Based on the LUT model fits to the DRS spectra, Figs. 3(c) and 3(d) present the corresponding wavelength-dependent absorption coefficients. In both radiation-sensitive (A549) and resistant (rA549) tumors, the flatter HbO 2 absorption bands before radiation (0h) indicate a lower oxygenation when compared to the 24h and 48h time points. Post-radiation, the absorption peaks of oxygenated hemoglobin appear more prominent, indicating an increase in sO 2 in both tumors. We also observed a higher magnitude in the absorption coefficient 24h after radiation in the radiation-sensitive tumors, when compared to the preradiation and 48h time points. Based on these apparent differences, we proceeded to quantify vascular oxygenation and THb in both the radiation-sensitive and resistant groups.  Figure 4 illus four groups. F (0h), 1 hour a data revealed For the radia increase in sO observed that at the pre-rad [ Fig. 4  Given the absence of any significant short-term changes in THb, we investigated the changes in oxygenated (HbO 2 ) and deoxygenated hemoglobin (dHb) at the same time points (before, 1h after, 24h, and 48h) to identify the source responsible for driving the changes in sO 2 . Figure 6 presents mean values of HbO 2 and dHb between the treated and untreated groups for both radiation-sensitive and resistant tumors. We found that radiation caused a significant change in in HbO 2 and dHb in the radiation-resistant tumors. Specifically we noted that HbO 2 was significantly greater 48h post-radiation compared with the pre-radiation measurement (p = 0.04) [ Fig. 6(b)]. We also observed that dHb was significantly lower 48h post-radiation when compared with the pre-radiation measurement. (p = 0.047) [ Fig. 6(d)]. No significant changes in either HbO 2 or dHb were observed in the untreated and treated radiation-sensitive tumors [Figs. 6(a) and 6(c)].

Discussion
Although previous studies have investigated tumor reoxygenation following radiation in preclinical animal models, the data available covers tumor oxygenation changes following either significantly high radiation doses on consecutive days, or a single high dose [13,20]. Our findings demonstrate that diffuse optical spectroscopy is sensitive to radiation-induced changes in vascular oxygenation when therapeutically relevant doses are administered. Previous studies have shown that reoxygenation depends on both tumor model and radiation dose [23,24]. For the first time, we report changes in reoxygenation kinetics measured in tumors established from a matched model of radiation resistance following a 2Gy radiation dose. By generating a radiation-resistant cancer cell line through the repeated exposure of a parental line to radiation, we avoid the underlying genetic differences present between two different tumor models, and further reduce the contributions that intrinsic cellular properties of different cell lines could be making to the changes we observed.
The mechanism of reoxygenation due to fractionated radiotherapy has been attributed to various processes over the years. Kallman et al. in 1972 proposed four mechanisms by which reoxygenation occurs: reduced O 2 metabolism, improved circulation, shrinkage, and migration of surviving cells [25]. Subsequent studies have further investigated the factors responsible for reoxygenation and found that a decrease in oxygen consumption by the tumor cells, and/or an increase in oxygen delivery play a role in reoxygenation [26]. We did not observe significant difference in tumor growth between the radiation-sensitive and radiationresistant tumors (data not shown). This was most likely due to the lower radiation dose used and the frequency of administration (every 72 hours). However, even when significant changes in tumor size were not present, we were still able to detect functional changes within the tumor with DRS, following such a low radiation dose.
We observed significant differences in oxygenation for the radiation-resistant tumors at time points that we did not observe in the radiation-sensitive tumors. Specifically, we noted a significant increase in oxygenation in the resistant tumors 24 hours after radiation (Fig. 5). An increase in reoxygenation was also observed in the radiation-sensitive tumors; however, this increase was not significant. These findings are in agreement with the studies showing that reoxygenation after radiation can also be a characteristic of radiation-resistant tumors [12,13,20]. In a clinical study with advanced HNSCC patients, Dietz et al. [12] investigated the link between reoxygenation during therapy and the clinical outcome, and observed that the lower the degree of reoxygenation, the better the initial response and clinical outcome; i.e. a higher degree of reoxygenation was more likely to lead to a poor outcome. This is similar to the reoxygenation trend we observed, where the radiation-resistant tumors would be analogous to the poor clinical outcome observed in their study. Similarities in reoxygenation trends between the radiation-resistant and sensitive tumors were also observed. Forty-eight hours after radiation, both radiation-sensitive and resistant tumors display a significant increase in reoxygenation, although the radiation-sensitive tumors present a rather marginal increase in reoxygenation at this time point compared to the resistant tumors [ Fig. 5].
Although we see changes in oxygenation, most pointedly in the radiation-resistant tumors, we do not see changes in THb for either radiation-resistant or sensitive tumors. This is particularly noteworthy because THb has been used as a marker for perfusion [27], and changes in perfusion following radiation therapy were observed by several investigators [26,28]. Previous studies have investigated changes in tumor perfusion and oxygenation following radiation therapy and found that an increase in tumor perfusion takes place 24 and 48h after irradiation [28][29][30]. Crokart et al. characterized changes in the microenvironment of FSaII tumors after a 2Gy radiation dose and observed an increase in tumor perfusion as early as 4h following irradiation [26]. This is very different from what we observed in our study, given that we failed to see any changes in perfusion (THb). Goda et al. investigated changes in oxygen tension and blood perfusion in MTG-B and RIF-1 tumors before and after a single 20Gy radiation dose, and observed a significant decrease in both oxygen tension and tumor blood perfusion 24h after radiation. This observation led them to conclude that changes in perfusion after radiation are one of the main causes of reoxygenation within the tumor [28]. Considering we administered a 2Gy dose, this difference in dosage could explain why we do not observe the same changes in tumor perfusion that they did 24h post-radiation.
Given that our data did not reveal any changes in THb, we considered possible changes in oxygen consumption. From one of our previous studies investigating the early metabolic changes in the A549 and rA549 cell lines in culture, we observed that the radiation-resistant cells displayed decreased levels of oxygen consumption compared to the radiation-sensitive cells both at baseline and 24h after radiation (2Gy) [21]. Following a 2Gy dose, the radiationresistant cells appear to decrease their oxygen consumption even more and resort to increased glycolysis to counter-act the ROS-induced toxicity [31]. Furthermore, we found that hypoxia-inducible factor (HIF-1α) protein content was significantly greater in the radiation-resistant cells after radiation compared to the radiation-sensitive cells. Coupling this data from the cells with our in vivo studies, we believe that the reoxygenation observed in the radiation-resistant tumors could be mediated by a decrease in oxygen consumption, which leads to the higher oxygen saturation we were able to detect with DRS. Hu et al. observed very similar results in the inconsistent relationship between THb and changes in sO 2 . They suggested that the improvement in oxygenation they observed after radiation might be due to the decreased oxygen consumption in the tumor due to tumor cell death. They observed these effects before they noted any anatomical changes in the tumor burden. Their results, just as ours, suggest that changes in perfusion are not directly responsible for changes observed in reoxygenation [13]. The significant changes in HbO 2 and dHb in the radiation-resistant tumors [ Fig. 6] lead us to believe that the primary drivers of changes in sO 2 are local changes in cellular oxygen consumption due to either cell death or as a method to avoid ROS-induced toxicity. Immunohistochemical (IHC) assessments of perfusion, cell death, and DNA damage are necessary to further tease apart these two phenomena, which are part of our ongoing work.
In conclusion, we report the short term reoxygenation trends in a matched model of radiation resistance immediately after, 24h, and 48h after radiation. We demonstrate that DRS was sensitive to changes in oxygenation following a clinically relevant dose of radiation. Although we see a clear distinction in reoxygenation at the 48h post radiation mark between the radiation-resistant and radiation-sensitive tumors, both tumors display a reoxygenation trend following radiation. This suggests that although DRS is sensitive to radiation-induced changes in oxygenation between resistant and sensitive tumors, monitoring of reoxygenation alone may not provide a holistic understanding of the underlying functional changes taking place during radiation therapy to predict therapy response. Other molecular and metabolic markers could shed more light on the pathways involved in the development and maintenance of radiation resistance.