Alex J. Walsh

Quantitative Optical Imaging of Primary Tumor Organoid Metabolism Predicts Drug Response in Breast Cancer

There is a need for technologies to predict the efficacy of cancer treatment in individual patients. Here, we show that optical metabolic imaging of organoids derived from primary tumors can predict the therapeutic response of xenografts and measure antitumor drug responses in human tumor-derived organoids. Optical metabolic imaging quantifies the fluorescence intensity and lifetime of NADH and FAD, coenzymes of metabolism. As early as 24 hours after treatment with clinically relevant anticancer drugs, the optical metabolic imaging index of responsive organoids decreased (P < 0.001) and was further reduced when effective therapies were combined (P < 5 × 10(-6)), with no change in drug-resistant organoids. Drug response in xenograft-derived organoids was validated with tumor growth measurements in vivo and staining for proliferation and apoptosis. Heterogeneous cellular responses to drug treatment were also resolved in organoids. Optical metabolic imaging shows potential as a high-throughput screen to test the efficacy of a panel of drugs to select optimal drug combinations. Cancer Res; 74(18); 5184-94. ©2014 AACR.

Optical imaging of metabolism in HER2 overexpressing breast cancer cells

The optical redox ratio (fluorescence intensity of NADH divided by that of FAD), was acquired for a panel of breast cancer cell lines to investigate how overexpression of human epidermal growth factor receptor 2 (HER2) affects tumor cell metabolism, and how tumor metabolism may be altered in response to clinically used HER2-targeted therapies. Confocal fluorescence microscopy was used to acquire NADH and FAD auto-fluorescent images. The optical redox ratio was highest in cells overexpressing HER2 and lowest in triple negative breast cancer (TNBC) cells, which lack HER2, progesterone receptor, and estrogen receptor (ER). The redox ratio in ER-positive/HER2-negative cells was higher than what was seen in TNBC cells, but lower than that in HER2 overexpressing cells. Importantly, inhibition of HER2 using trastuzumab significantly reduced the redox ratio in HER2 overexpressing cells. Furthermore, the combinatorial inhibition of HER2 and ER decreased the redox ratio in ER+/HER2+ breast cancer cells to a greater extent than inhibition of either receptor alone. Interestingly, trastuzumab had little impact upon the redox ratio in a cell line selected for acquired resistance to trastuzumab. Taken together, these data indicate that the optical redox ratio measures changes in tumor metabolism that reflect the oncogenic effects of HER2 activity within the cell, as well as the response of the cell to therapeutic inhibition of HER2. Therefore, optical redox imaging holds the promise of measuring response and resistance to receptor-targeted breast cancer therapies in real time, which could potentially impact clinical decisions and improve patient outcome.

Optical Metabolic Imaging of Treatment Response in Human Head and Neck Squamous Cell Carcinoma

Optical metabolic imaging measures fluorescence intensity and lifetimes from metabolic cofactors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). These molecular level measurements provide unique biomarkers for early cellular responses to cancer treatments. Head and neck squamous cell carcinoma (HNSCC) is an attractive target for optical imaging because of easy access to the site using fiber optic probes. Two HNSCC cell lines, SCC25 and SCC61, were treated with Cetuximab (anti-EGFR antibody), BGT226 (PI3K/mTOR inhibitor), or cisplatin (chemotherapy) for 24 hours. Results show increased redox ratio, NADH α1 (contribution from free NADH), and FAD α1 (contribution from protein-bound FAD) for malignant cells compared with the nonmalignant cell line OKF6 (p<0.05). In SCC25 and SCC61 cells, the redox ratio is unaffected by cetuximab treatment and decreases with BGT226 and cisplatin treatment (p<0.05), and these results agree with standard measurements of proliferation rates after treatment. For SCC25, NADH α1 is reduced with BGT226 and cisplatin treatment. For SCC61, NADH α1 is reduced with cetuximab, BGT226, and cisplatin treatment. Trends in NADH α1 are statistically similar to changes in standard measurements of glycolytic rates after treatment. FAD α1 is reduced with cisplatin treatment (p<0.05). These shifts in optical endpoints reflect early metabolic changes induced by drug treatment. Overall, these results indicate that optical metabolic imaging has potential to detect early response to cancer treatment in HNSCC, enabling optimal treatment regimens and improved patient outcomes.

Development of spectral markers for the discrimination of ulcerative colitis and Crohn's disease using Raman spectroscopy.

BACKGROUND: Ulcerative colitis and Crohns disease are 2 distinct forms of IBD that can overlap radiologically, endoscopically, and pathologically. This difficulty complicates surgical options. The development of new technologies providing accurate diagnosis of IBD is needed. Raman spectroscopy is a noninvasive method that uses the intrinsic properties of tissue and that tissues vibrational energy in reaction to light. PURPOSE: We hypothesize that Raman spectroscopy can detect the structural and compositional changes that occur in the tissue during the development of inflammatory bowel disease, and thus may offer increased diagnostic certainty in the differentiation between Crohns disease and ulcerative colitis. METHODS: Fresh frozen colon tissue biopsies from patients with ulcerative colitis (n = 12) and with Crohns disease (n = 9) were measured in vitro using a custom-designed Raman fiber-optic probe. For spectra collection, the probe was placed in gentle contact with the mucosa surface for 3 seconds, with excitation power at 150 mW. Five spectra were acquired from each biopsy to increase the signal-to-noise ratio and to ensure repeatability of data collection. Mean spectra were analyzed for peak difference and molecular origin. RESULTS: Significant difference was observed between the spectra from each disease in the spectral regions assigned to nucleic acid, phenylalanine, and lipids. Tissue samples from patients with ulcerative colitis demonstrated higher content of lipid and lower amount of phenylalanine and nucleic acid. These characteristic Raman features could serve as spectral markers that can potentially be applied to distinguish ulcerative colitis and Crohns disease. CONCLUSIONS: This study presents the only application of Raman spectroscopy in the diagnosis of inflammatory bowel disease. The feasibility of this technique in differentially detecting molecular alterations in ulcerative colitis and Crohns disease has been demonstrated, indicating the potential to improve diagnostic accuracy of inflammatory bowel disease.

In Vivo Autofluorescence Imaging of Tumor Heterogeneity in Response to Treatment

Subpopulations of cells that escape anti-cancer treatment can cause relapse in cancer patients. Therefore, measurements of cellular-level tumor heterogeneity could enable improved anti-cancer treatment regimens. Cancer exhibits altered cellular metabolism, which affects the autofluorescence of metabolic cofactors NAD(P)H and FAD. The optical redox ratio (fluorescence intensity of NAD(P)H divided by FAD) reflects global cellular metabolism. The fluorescence lifetime (amount of time a fluorophore is in the excited state) is sensitive to microenvironment, particularly protein-binding. High-resolution imaging of the optical redox ratio and fluorescence lifetimes of NAD(P)H and FAD (optical metabolic imaging) enables single-cell analyses. In this study, mice with FaDu tumors were treated with the antibody therapy cetuximab or the chemotherapy cisplatin and imaged in vivo two days after treatment. Results indicate that fluorescence lifetimes of NAD(P)H and FAD are sensitive to early response (two days post-treatment, P < .05), compared with decreases in tumor size (nine days post-treatment, P < .05). Frequency histogram analysis of individual optical metabolic imaging parameters identifies subpopulations of cells, and a new heterogeneity index enables quantitative comparisons of cellular heterogeneity across treatment groups for individual variables. Additionally, a dimensionality reduction technique (viSNE) enables holistic visualization of multivariate optical measures of cellular heterogeneity. These analyses indicate increased heterogeneity in the cetuximab and cisplatin treatment groups compared with the control group. Overall, the combination of optical metabolic imaging and cellular-level analyses provide novel, quantitative insights into tumor heterogeneity.

Ex vivo optical metabolic measurements from cultured tissue reflect in vivo tissue status

Abstract. Optical measurements of metabolism are ideally acquired in vivo; however, intravital measurements are often impractical. Accurate ex vivo assessments would greatly broaden the applicability of optical measurements of metabolism. We investigate the use of live tissue culture experiments to serve as a surrogate for in vivo metabolic measurements. To validate this approach, NADH and FAD fluorescence intensity and lifetime images were acquired with a two-photon microscope from hamster cheek pouch epithelia in vivo, from biopsies maintained in live tissue culture up to 48 h, and from flash-frozen and thawed biopsies. We found that the optical redox ratio (fluorescence intensity of NADH/FAD) of the cultured biopsy was statistically identical to the in vivo measurement until 24 h, while the redox ratio of the frozen-thawed samples decreased by 15% (p<0.01). The NADH mean fluorescence lifetime (τm) remained unchanged (p>0.05) during the first 8 h of tissue culture, while the NADH τm of frozen-thawed samples increased by 13% (p<0.001). Cellular morphology did not significantly change between in vivo, cultured, and frozen-thawed tissues (p>0.05). All results were consistent across multiple depth layers in this stratified squamous epithelial tissue. Histological markers for proliferation and apoptosis also confirm the viability of tissues maintained in culture. This study suggests that short-term ex vivo tissue culture may be more appropriate than frozen-thawed tissue for optical metabolic and morphologic measurements that approximate in vivo status.

Optical metabolic imaging quantifies heterogeneous cell populations

The genetic and phenotypic heterogeneity of cancers can contribute to tumor aggressiveness, invasion, and resistance to therapy. Fluorescence imaging occupies a unique niche to investigate tumor heterogeneity due to its high resolution and molecular specificity. Here, heterogeneous populations are identified and quantified by combined optical metabolic imaging and subpopulation analysis (OMI-SPA). OMI probes the fluorescence intensities and lifetimes of metabolic enzymes in cells to provide images of cellular metabolism, and SPA models cell populations as mixed Gaussian distributions to identify cell subpopulations. In this study, OMI-SPA is characterized by simulation experiments and validated with cell experiments. To generate heterogeneous populations, two breast cancer cell lines, SKBr3 and MDA-MB-231, were co-cultured at varying proportions. OMI-SPA correctly identifies two populations with minimal mean and proportion error using the optical redox ratio (fluorescence intensity of NAD(P)H divided by the intensity of FAD), mean NAD(P)H fluorescence lifetime, and OMI index. Simulation experiments characterized the relationships between sample size, data standard deviation, and subpopulation mean separation distance required for OMI-SPA to identify subpopulations.

Drug response in organoids generated from frozen primary tumor tissues.

Primary tumor organoids grown in three-dimensional culture provide an excellent platform for studying tumor progression, invasion, and drug response. However, organoid generation protocols require fresh tumor tissue, which limits organoid research and clinical use. This study investigates cellular morphology, viability, and drug response of organoids derived from frozen tissues. The results demonstrate that viable organoids can be grown from flash-frozen and thawed tissue and from bulk tissues slowly frozen in DMSO supplemented media. While the freezing process affects the basal metabolic rate of the cells, the optical metabolic imaging index correlates between organoids derived from fresh and frozen tissue and can be used to detect drug response of organoids grown from frozen tissues. The slow, DMSO frozen tissue yielded organoids with more accurate drug response than the flash frozen tissues, and thus bulk tissue should be preserved for subsequent organoid generation by slow freezing in DMSO supplemented media.

In vivo hyperspectral imaging of microvessel response to trastuzumab treatment in breast cancer xenografts.

HER2-amplified (HER2 + ) breast cancers are treated with the anti-HER2 monoclonal antibody trastuzumab. Although trastuzumab reduces production of the angiogenic factor VEGF in HER2 + tumors, the acute and sustained effects of trastuzumab on the tumor vasculature are not understood fully, particularly in trastuzumab-resistant tumors. We used mouse models of trastuzumab sensitive and trastuzumab-resistant HER2 + breast cancers to measure dynamic changes in tumor microvessel density and hemoglobin oxygenation (sO2) in vivo using quantitative hyperspectral imaging at 2, 5, 9, and 14 days after antibody treatment. Further analysis quantified the distribution of microvessels into low and high oxygenation groups, and monitored changes in these distributions with trastuzumab treatment. Gold standard immunohistochemistry was performed to validate complementary markers of tumor cell and vascular response to treatment. Trastuzumab treatment in both responsive and resistant tumors resulted in decreased sO2 5 days after initial treatment when compared to IgG-treated controls (p<0.05). Importantly, responsive tumors showed significantly higher vessel density and significantly lower sO2 than all other groups at 5 days post-treatment (p<0.05). Distribution analysis of vessel sO2 showed a significant (p<0.05) shift of highly oxygenated vessels towards lower oxygenation over the time-course in both trastuzumab-treated responsive and resistant tumors. This study suggests that longitudinal hyperspectral imaging of microvessel sO2 and density could distinguish trastuzumab-responsive from trastuzumab-resistant tumors, a finding that could be exploited in the post-neoadjuvant setting to guide post-surgical treatment decisions.

Optical Imaging of Drug-Induced Metabolism Changes in Murine and Human Pancreatic Cancer Organoids Reveals Heterogeneous Drug Response.

Objectives Three-dimensional organoids derived from primary pancreatic ductal adenocarcinomas are an attractive platform for testing potential anticancer drugs on patient-specific tissue. Optical metabolic imaging (OMI) is a novel tool used to assess drug-induced changes in cellular metabolism, and its quantitative end point, the OMI index, is evaluated as a biomarker of drug response in pancreatic cancer organoids. Methods Optical metabolic imaging is used to assess both malignant cell and fibroblast drug response within primary murine and human pancreatic cancer organoids. Results Anticancer drugs induce significant reductions in the OMI index of murine and human pancreatic cancer organoids. Subpopulation analysis of OMI data revealed heterogeneous drug response and elucidated responding and nonresponding cell populations for a 7-day time course. Optical metabolic imaging index significantly correlates with immunofluorescence detection of cell proliferation and cell death. Conclusions Optical metabolic imaging of primary pancreatic ductal adenocarcinoma organoids is highly sensitive to drug-induced metabolic changes, provides a nondestructive method for monitoring dynamic drug response, and presents a novel platform for patient-specific drug testing and drug development.