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SF1: Tumor growth assays for lung and HN tumors. SF2: The complete set of MCR-derived pure component spectra and tables listing their peak assignments. SF3: Representative MCR score maps for lung and HN tumor datasets. SF4: Histologic images for HN tumor dataset. TS1-4: Results of binary leave-one-mouse-out SVM analyses.

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.

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.

Radiation resistance can halt a well-curated treatment plan in its tracks, but it may take weeks to realize this lack of efficacy resulting in lost time and unnecessary side effects. In this study, we sought to elucidate novel biomarkers of radiation resistance to predict treatment response in head and neck squamous cell carcinoma (HNSCC) at much earlier timepoints. We grew tumor xenografts from radiation-resistant UM-SCC-47 (n=25) and -sensitive UM-SCC-22B (n=37) cells in nude athymic mice. Each group was randomly divided into treatment and control groups, which were further subdivided into four timepoints for tumor excision: pre-treatment baseline, 1-, 24-, and 48-hours post-treatment. Prior to excision, we injected pimonidazole to quantify tumor hypoxia. We determined the two-photon excited fluorescence intensity and lifetime of NADH and FAD and quantified the optical redox ratio, ratio of free to bound NADH, and mean NADH lifetime of frozen sections of each tumor. We observed spatial and temporal heterogeneity in these high-resolution optical maps that indicated different metabolic programs in radiation-resistant and -sensitive tumors. A histogram-based heterogeneity analysis revealed time dependent shifts toward glycolysis in the radiation-resistant UM-SCC-47 treatment group, while the sensitive UM-SCC-22B treatment group showed the opposite trend. Proteomics performed on both cell lines identified multiple dysregulated proteins that are often implicated in HNSCC and associated with metabolic reprogramming — specifically, EGFR and SMAD4. We also found elevated levels of hypoxia and HIF-1 in the UM-SCC-47 tumors compared with the UM-SCC-22B tumors prior to and post-radiation. Using spatially co-registered optical imaging and immunohistochemical maps of the same field of view, we will present correlations between our optical markers and EGFR, SMAD4, and HIF-1. These studies will provide evidence of how protein dysregulation causes changes to optical metabolic imaging markers. Citation Format: Jesse D. Ivers, Sina Dadgar, Narasimhan Rajaram. Multimodal metabolic imaging and proteomics of radiation resistance in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3703.

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.

We used diffuse reflectance spectroscopy to assess the effects of different radiation therapy doses on tumor microenvironment. We injected 4T1 cells into the flanks of 25 mice to develop tumor xenografts, and randomly distributed the animals into control and radiation groups, which received a dose of either 1, 2, or 4 Gy. Treatment started when tumors reached a volume of 200 mm3. DRS measurements were obtained prior to and 1 hour after radiation on the five days of treatment, and once a day thereafter. Our data demonstrates that DRS is sensitive to tumor microenvironmental changes after low doses of radiation.

We present functional and biomolecular changes in head and neck tumor xenografts when exposed to radiation therapy and discuss how these changes can be used to monitor tumor response to therapy.

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.

Global rise in incidence of woody breast (WB) syndrome imposes a significant economic burden on the poultry industry. The increase in WB is due to the large increase in the weight of chickens these days within a very short period. An early determination of WB can significantly reduce losses to the poultry industry. Diffuse reflectance spectroscopy provides a noninvasive and rapid method to interrogate tissue function. The sensitivity of DRS to the distinct absorption spectra of oxygenated and deoxygenated hemoglobin allows accurate quantification of average hemoglobin concentration and vascular oxygenation within the sampled tissue. In this study, we used diffuse reflectance spectroscopy to monitor breast hemoglobin concentration (THb) and vascular oxygen saturation (sO2) of 16 chickens that were exposed to heat stress (HS). HS is an important cause of WB myopathy in chickens. Animals were exposed to heat-stress (HS) and optical data were acquired at three time points: at baseline prior to heat stress, 2 days, and 21 days after initiation of HS. Our results show that animals from control and HS groups had a steady decay in optically derived breast hemoglobin concentration consistent with independent i-STAT measurements made on blood sampled from the femoral artery and could provide a noninvasive technology for monitoring tissue function in the poultry industry.

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.

Currently, anatomical assessment of tumor volume performed several weeks after completion of treatment is the clinical standard to determine whether a cancer patient has responded to a treatment. However, functional changes within the tumor could potentially provide information regarding treatment resistance or response much earlier than anatomical changes. We have used diffuse reflectance spectroscopy to assess the short and long-term re-oxygenation kinetics of a human head and neck squamous cell carcinoma xenografts in response to radiation therapy. First, we injected UM-SCC-22B cell line into the flank of 50 mice to grow xenografts. Once the tumor volume reached 200 mm 3 (designated as Day 1), the mice were distributed into radiation and control groups. Members of radiation group underwent a clinical dose of radiation of 2 Gy/day on Days 1, 4, 7, and 10 for a cumulative dose of 8 Gy. DRS spectra of these tumors were collected for 14 days during and after therapy, and the collected spectra of each tumor were converted to its optical properties using a lookup table-base inverse model. We found statistically significant differences in tumor growth rate between two groups which is in indication of the sensitivity of this cell line to radiation. We further acquired significantly different contents of hemoglobin and scattering magnitude and size in two groups. The scattering has previously been associated with necrosis. We furthermore found significantly different time-dependent changes in vascular oxygenation and tumor hemoglobin concentration in post-radiation days.