Diffuse Reflectance Spectroscopy of Changes in Tumor Microenvironment in Response to Different Doses of Radiation.

Radiation therapy plays an important role in cancer treatment, as it is an established method used as part of the treatment plan for the majority of cancer patients. Real-time monitoring of the effects of radiation on the tumor microenvironment can contribute to the development of better treatment plans. In this study, we use diffuse reflectance spectroscopy, a non-invasive optical fiber-based technique, to determine the effects of different doses of radiation on the tumor microenvironment, as well as to determine the sensitivity of diffuse reflectance spectroscopy to low doses of radiation that are used in the treatment of certain cancers. We injected 4T1 cells into 50 Balb/c mice to generate tumor xenografts. When the tumors grew to 200 mm3, we distributed the mice into a control group or one of three radiation groups: 1, 2, or 4 Gy/fraction, and they underwent treatment for five consecutive days. We measured the tumor volume and collected diffuse reflectance spectra every day, with optical measurements being acquired both before and one h postirradiation on the five days of treatment. Based on the diffusely reflected light, we quantified vascular oxygenation, total hemoglobin content, and tissue scattering within these tumors. There was a significant increase in tumor vascular oxygenation, which was primarily due to an increase in oxygenated hemoglobin, in response to a 1 Gy/fraction of radiation, while there was a decrease in tissue scattering in response to all doses of radiation. Immunohistochemical analysis revealed that tumor cell proliferation and apoptosis were higher in irradiated groups compared to the control group. Our findings show that diffuse reflectance spectroscopy is sensitive to microenvironmental changes in tumors treated with doses of radiation as low as 1 Gy/fraction.

Diffuse reflectance spectroscopy reveals heat stress-induced changes in hemoglobin concentration in chicken breast.

Heat stress (HS) is devastating to the poultry industry due to its adverse effects on animal well-being and performance. The effects of heat stress are typically measured using a portable i-STAT blood analyzer that quantifies circulatory hemoglobin concentration and other blood chemistry parameters. Here, we used diffuse reflectance spectroscopy (DRS) as a novel non-invasive method to directly determine changes in hematological parameters in the breast tissues of live heat-stressed broilers. Three-week-old male broilers were randomly subjected to two environmental conditions (thermoneutral, TN, 24 °C vs. cyclic heat stress, HS, 35 °C, 12 h/day). Optical spectra were acquired using DRS to monitor breast hemoglobin (Hb) concentration and vascular oxygen saturation (sO2) at three time points: at baseline prior to heat stress, 2 days, and 21 days after initiation of HS. While i-STAT did not demonstrate a discernible change due to HS in circulatory hemoglobin, DRS found a significant decrease in breast Hb and sO2 after exposure to chronic HS. The decrease in sO2 was found to be due to a decrease in oxygenated hemoglobin concentration, indicating a large increase in oxygen consumption in heat-stressed broilers. Our results demonstrate that DRS could potentially be used to study the effects of HS directly in specific organs of interest, such as the breast and thigh, to improve meat quality.

Spectroscopic investigation of radiation-induced reoxygenation in radiation-resistant tumors.

Fractionated radiation therapy is believed to reoxygenate and subsequently radiosensitize surviving hypoxic cancer cells. Measuring tumor reoxygenation between radiation fractions could conceivably provide an early biomarker of treatment response. However, the relationship between tumor reoxygenation and local control is not well understood. We used noninvasive optical fiber-based diffuse reflectance spectroscopy to monitor radiation-induced changes in hemoglobin oxygen saturation (sO2) in tumor xenografts grown from two head and neck squamous cell carcinoma cell lines - UM-SCC-22B and UM-SCC-47. Tumors were treated with 4 doses of 2 Gy over 2 consecutive weeks and diffuse reflectance spectra were acquired every day during the 2-week period. There was a statistically significant increase in sO2 in the treatment-responsive UM-SCC-22B tumors immediately following radiation. This reoxygenation trend was due to an increase in oxygenated hemoglobin (HbO2) and disappeared over the next 48 h as sO2 returned to preradiation baseline values. Conversely, sO2 in the relatively radiation-resistant UM-SCC-47 tumors increased after every dose of radiation and was driven by a significant decrease in deoxygenated hemoglobin (dHb). Immunohistochemical analysis revealed significantly elevated expression of hypoxia-inducible factor (HIF-1) in the UM-SCC-47 tumors prior to radiation and up to 48 h postradiation compared with the UM-SCC-22B tumors. Our observation of a decrease in dHb, a corresponding increase in sO2, as well as greater HIF-1α expression only in UM-SCC-47 tumors strongly suggests that the reoxygenation within these tumors is due to a decrease in oxygen consumption in the cancer cells, which could potentially play a role in promoting radiation resistance.

Optical Imaging Approaches to Investigating Radiation Resistance.

Radiation therapy is frequently the first line of treatment for over 50% of cancer patients. While great advances have been made in improving treatment response rates and reducing damage to normal tissue, radiation resistance remains a persistent clinical problem. While hypoxia or a lack of tumor oxygenation has long been considered a key factor in causing treatment failure, recent evidence points to metabolic reprogramming under well-oxygenated conditions as a potential route to promoting radiation resistance. In this review, we present recent studies from our lab and others that use high-resolution optical imaging as well as clinical translational optical spectroscopy to shine light on the biological basis of radiation resistance. Two-photon microscopy of endogenous cellular metabolism has identified key changes in both mitochondrial structure and function that are specific to radiation-resistant cells and help promote cell survival in response to radiation. Optical spectroscopic approaches, such as diffuse reflectance and Raman spectroscopy have demonstrated functional and molecular differences between radiation-resistant and sensitive tumors in response to radiation. These studies have uncovered key changes in metabolic pathways and present a viable route to clinical translation of optical technologies to determine radiation resistance at a very early stage in the clinic.

Quantum Blue Reduces the Severity of Woody Breast Myopathy via Modulation of Oxygen Homeostasis-Related Genes in Broiler Chickens.

The incidence of woody breast (WB) is increasing on a global scale representing a significant welfare problem and economic burden to the poultry industry and for which there is no effective treatment due to its unknown etiology. In this study, using diffuse reflectance spectroscopy (DRS) coupled with iSTAT portable clinical analyzer, we provide evidence that the circulatory- and breast muscle-oxygen homeostasis is dysregulated [low oxygen and hemoglobin (HB) levels] in chickens with WB myopathy compared to healthy counterparts. Molecular analysis showed that blood HB subunit Mu (HBM), Zeta (HBZ), and hephaestin (HEPH) expression were significantly down regulated; however, the expression of the subunit rho of HB beta (HBBR) was upregulated in chicken with WB compared to healthy counterparts. The breast muscle HBBR, HBE, HBZ, and hypoxia-inducible factor prolyl hydroxylase 2 (PHD2) mRNA abundances were significantly down regulated in WB-affected compared to normal birds. The expression of HIF-1α at mRNA and protein levels was significantly induced in breasts of WB-affected compared to unaffected birds confirming a local hypoxic status. The phosphorylated levels of the upstream mediators AKT at Ser473 site, mTOR at Ser2481 site, and PI3K P85 at Tyr458 site, as well as their mRNA levels were significantly increased in breasts of WB-affected birds. In attempt to identify a nutritional strategy to reduce WB incidence, male broiler chicks (Cobb 500, n = 576) were randomly distributed into 48 floor pens and subjected to six treatments (12 birds/pen; 8 pens/treatment): a nutrient adequate control group (PC), the PC supplemented with 0.3% myo-inositol (PC + MI), a negative control (NC) deficient in available P and Ca by 0.15 and 0.16%, respectively, the NC fed with quantum blue (QB) at 500 (NC + 500 FTU), 1,000 (NC + 1,000 FTU), or 2,000 FTU/kg of feed (NC + 2,000 FTU). Although QB-enriched diets did not affect growth performances (FCR and FE), it did reduce the severity of WB by 5% compared to the PC diet. This effect is mediated by reversing the expression profile of oxygen homeostasis-related genes; i.e., significant down regulation of HBBR and upregulation of HBM, HBZ, and HEPH in blood, as well as a significant upregulation of HBA1, HBBR, HBE, HBZ, and PHD2 in breast muscle compared to the positive control.

Label-Free Raman Spectroscopy Reveals Signatures of Radiation Resistance in the Tumor Microenvironment.

Delay in the assessment of tumor response to radiotherapy continues to pose a major challenge to quality of life for patients with nonresponsive tumors. Here, we exploited label-free Raman spectroscopic mapping to elucidate radiation-induced biomolecular changes in tumors and uncovered latent microenvironmental differences between treatment-resistant and -sensitive tumors. We used isogenic radiation-resistant and -sensitive A549 human lung cancer cells and human head and neck squamous cell carcinoma (HNSCC) cell lines (UM-SCC-47 and UM-SCC-22B, respectively) to grow tumor xenografts in athymic nude mice and demonstrated the molecular specificity and quantitative nature of Raman spectroscopic tissue assessments. Raman spectra obtained from untreated and treated tumors were subjected to chemometric analysis using multivariate curve resolution-alternating least squares (MCR-ALS) and support vector machine (SVM) to quantify biomolecular differences in the tumor microenvironment. The Raman measurements revealed significant and reliable differences in lipid and collagen content postradiation in the tumor microenvironment, with consistently greater changes observed in the radiation-sensitive tumors. In addition to accurately evaluating tumor response to therapy, the combination of Raman spectral markers potentially offers a route to predicting response in untreated tumors prior to commencing treatment. Combined with its noninvasive nature, our findings provide a rationale for in vivo studies using Raman spectroscopy, with the ultimate goal of clinical translation for patient stratification and guiding adaptation of radiotherapy during the course of treatment. SIGNIFICANCE: These findings highlight the sensitivity of label-free Raman spectroscopy to changes induced by radiotherapy and indicate the potential to predict radiation resistance prior to commencing therapy.

Optical spectroscopic sensing of tumor hypoxia.

Tumor hypoxia is a critical indicator of poor clinical outcome in patients with cancers of the breast, cervix, and oral cavity. The ability to noninvasively and reliably monitor tumor oxygenation both prior to and during therapy can aid in identifying poor treatment response earlier than is currently possible and lead to effective changes in treatment regimen. Diffuse reflectance spectroscopy (DRS) has been used in several studies to measure tissue scattering, total hemoglobin content (THb), and vascular oxygenation (sO2) in tissue. In this study, we validate in vivo DRS-based measurements of vascular oxygenation using immunohistochemical staining of tumor hypoxia using pimonidazole, an established hypoxia marker. Using tumor xenografts grown from two different head and neck cell lines-UM-SCC-22B and UM-SCC-47-we demonstrate statistically significant negative correlations between tumor hypoxic fraction (HF) and THb (r = - 0.45; p = 0.04) and sO2 (r = - 0.50; p = 0.02). In addition, we also found a statistically significant positive correlation between HF and mean reduced scattering coefficient (r = 0.60; p = 0.005). Our results demonstrate that DRS-based measures of sO2 can provide reliable indirect measurements of tumor hypoxia that can be of significant utility in preclinical and clinical studies.