Next, I would like to thank my collaborators and comrades, without whom this work would have taken much longer. Finally, I would like to thank my family and friends for all their endless support over the past five years.
Specific Aims
Therefore, an organoid-based platform was developed to enable high-throughput screening of drug response. The second part of this thesis (Chapters 5-6, Appendices 4-5) characterizes OMI in excised tissues and organoids, and validates an organoid-OMI screen for predicting drug response for breast cancer patients.
Characterize subpopulation analysis of OMI data to quantify heterogeneous cell populations. Tumor heterogeneity contributes to therapy resistance; and this aim develops
Preliminary (Appendix C) and early studies (Chapters 3-4) have demonstrated the unique sensitivity of OMI to drug-induced metabolic changes. Organoids are macrosuspensions obtained from primary tumors and contain all innate cells and the tumor microenvironment (10, 11).
Determine the sensitivity of OMI to breast cancer subtypes and therapeutic response in vitro and validate OMI measures of response in human breast cancer xenografts
Dissertation Outline
Monitoring neoadjuvant chemotherapy using multiparametric, (23)Na-sodium MR and multimodality (PET/CT/MRI) imaging in locally advanced breast cancer. Genetic, hormonal, and nutritional factors are believed to contribute to the likelihood of developing breast cancer (4).
Current Breast Cancer Treatments
A biopsy first provides the histology of the breast cancer by revealing ER status, progesterone receptor status, and HER2 status. A high concentration of HER2 receptors expressed by breast cancer cells is associated with increased tumor progression and metastasis (9).
Tumor Heterogeneity and Resistance
Many new drugs, including HER3, mTOR, and PI3K inhibitors, are being developed to overcome therapy resistance (31–39). In HER2-overexpressing cancers, possible mechanisms to overcome trastuzumab resistance include inhibition of downstream effectors of HER2 that can be activated even in the absence of HER2 signaling.
Cellular Metabolism
The optical redox ratio is used to investigate changes in cellular metabolism due to receptor expression and cellular response to targeted therapies. In addition, the OMI endpoints provide dynamic readouts of cellular metabolism and detect pre-malignant transformations in tissues (68, 69), classify subtypes of breast cancer cells (78, 79), and detect.
Prediction of clinical outcome from primary tamoxifen by expression of biological markers in breast cancer patients. A functional genetics approach identifies the PI3K pathway as a major determinant of trastuzumab resistance in breast cancer.
Abstract
Introduction
Primary tumors are heterogeneous and contain subpopulations of cells with different genetic profiles and protein expression (1, 4, 5). Subpopulations of cells with stem cell-like behavior may contribute to drug resistance and recurrent tumor growth (6).
Methods
Six representative fields of view were imaged for each dish, for a total number of ~200 cells per group. A fluorescence intensity image was generated by integrating the decay of the fluorescence lifetime over time for each pixel in the lifetime image.
Results
OMI-SPA behavior for modeling data simulated from mean and standard deviation of FAD m of SKBr3 and MDA-MB-231 cells. MDA-MB-231 cells grown independently of SKBr3 cells had a mean redox ratio of 1 and a standard deviation of 0.23.
Acknowledgements
SPA can be improved to account for different data distributions by using additional distributions that better represent the homogeneous population. The results of this experiment show that OMI-SPA can be used to identify cancer cell subpopulations based on OMI endpoints: redox ratio, NAD(P)H mean lifetime, FAD mean lifetime, and OMI index. Our previously published analyzes of cell subpopulations within tumors in vivo ( 9 ) and patient-derived tumor organoids ( 14 ) also show that OMI-SPA can accurately identify cell subpopulations with a sample of up to 300 cells.
In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia. In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia. Walsh AJ, Cook RS, Manning HC, Hicks DJ, Lafontant A, Arteaga CL, Skala MC, “Optical metabolic imaging identifies breast cancer glycolytic levels, subtypes, and early treatment response,” Cancer Research.
Abstract
Introduction
Resolution is sufficiently high to isolate single cells, enabling the identification of inflammatory infiltrates in the stroma and tumor epithelia. This single-cell-level resolution may be useful for identifying resistant subpopulations of cells that pre-exist in the tumor and are responsible for cancer relapse. This work represents a significant advance in the tools available to study cellular metabolism and tumor response to treatment in living systems.
Materials and Methods
Fluorescence lifetime images were obtained using time correlated single photon counting (TCSPC) electronics (SPC-150, Becker and Hickl). NADH and FAD fluorescence lifetime images of three different tumor locations were acquired each day. A two-component decay was used to represent the lifetimes of the free and bound configurations of NADH and FAD.
Results
The portion of free NADH (1) was reduced in the HER2+ cells compared to the non-malignant cells (Appendix A Fig. A.3c). Tumor size measurements of the HR6 xenografts showed similar growth of both control and trastuzumab-treated HR6 tumors (Fig. 4.6a). No difference was observed in the redox ratio between control and treated HR6 tumors at any time point (Fig. 4.6f, g).
Discussion
OMI is sensitive to metabolic behavior induced by ER and HER2 (Fig. 4.3), known oncogenic drivers of glycolytic metabolism in breast cancer cells. Modeling the intercellular variation of MDA-MB-361 xenograft tumors identified two subpopulations in response to trastuzumab for optical redox ratio and FAD m (Fig. 4.7), indicative of in vivo cell-to-cell. We speculate that the greater intracellular variation of both redox ratio and FAD m observed in the MDA-MB-361 tumors treated with trastuzumab (Fig. 4.7h) is due to heterogeneous responses of individual mitochondria (43).
Tumor metabolism and blood flow as assessed by positron emission tomography varies by tumor subtype in locally advanced breast cancer. Human breast cancer tumor models: molecular imaging of drug sensitivity and dosing during HER2/neu-targeted therapy. A small animal positron emission tomography study of the effect of chemotherapy and hormonal therapy on 2-deoxy-2-[F-18]fluoro-D-glucose uptake in mouse models of breast cancer.
Abstract
We hypothesized that, for a distinct period after harvest, excised tissue maintained in chilled tissue culture media would provide metabolic measurements representative of the in vivo metabolic state. Subsequently, two 6-mm biopsies of the cheek pouch were obtained immediately after the in vivo measurements of the same region imaged in vivo. A Wilcoxon rank sum test was used to test for differences between the in vivo mean values of the redox ratio, NADH and FAD1, 2, m, and 1/2 ratio and each cultured biopsy time point.
Results
The mean redox ratio of the cultured biopsy up to 4 hours after excision is within 8% of the in vivo value. However, the redox ratio of the frozen-thawed sample is 85% of the in vivo tissue measurement and this difference is significant (p < 0.01). During the first 4 h, the mean FAD m of the cultured biopsy increases slightly but remains within 8% of the in vivo FAD.
Discussion
However, no previous study has confirmed that this live tissue culture approach provides optical metabolic measurements that are consistent with in vivo values, as we have shown in the present study. NADH and FAD fluorescence intensity did not change significantly in live tissue culture over 4 hours (p > 0.05). Several significant differences, including a reduced redox ratio, increased NADH m and increased FAD m were observed in frozen-thawed samples.
Acknowledgments
To validate live tissue culture as an effective protocol for maintaining in vivo metabolic characteristics in excised tissue, the optical redox ratio, NADH fluorescence lifetime and FAD fluorescence lifetime were quantified from hamster cheek epithelia in vivo, in live cultured biopsies followed for 48 h , and in frozen-thawed samples. The live tissue culture approach resulted in no significant change in any optical endpoint relative to in vivo measures until 12 h, when a decreased NADH m was observed. These results indicate that the live tissue culture method represents the in vivo condition more accurately than the frozen-thawed procedure.
Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH. Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastases and the tumor microenvironment. Lifetime fluorescence imaging of endogenous fluorophores in histopathological sections reveals differences between normal and tumor epithelium in carcinoma in situ of the breast.
Abstract
Introduction
Macrosuspension solutions were combined with Matrigel in a ratio of 1:2 and 100 l of the solution were applied to coverslips. The OMI index is a linear combination of the norm-centered redox ratio, NADH m and FAD m with coefficients calculated for each cell. The signs of the coefficients were chosen to maximize the difference between control and drug-responsive cells.
Results
The trastuzumab-treated organoids have two populations at 72 hours, both lower than the mean OMI index of the control organoids (Fig. 1L). Subpopulation analysis revealed two subpopulations in the OMI index for all treated groups except for trastuzumab at 24 hours (Fig. 6.2K, Appendix B Fig. B.4). Subpopulation analysis revealed shifts in the mean OMI index values with these treatments within a single population of cells (Fig. 6.6B).
Discussion
The OMI index was first evaluated as a reporter of tumor response in organoids derived from BT474 (ER+/HER2+) xenografts. Significant reductions in the OMI index of HR6 organoids treated for 72 hours identified drug combinations (H+X, H+P and H+P+X) that induced a sustained reduction in tumor growth in vivo (Fig. 6.2). Organoids derived from HER2+/ER- and TNBC primary tumors have OMI responses consistent with their clinical characteristics: reduced OMI index with trastuzumab treatment and.
Acknowledgements
The results of this study support the validity of OMI for monitoring organoid response to anticancer drugs. We demonstrate the high selectivity of the OMI index to directly measure the drug response of breast cancer xenograft-derived organoids to single anticancer drugs and their combinations and validate the response measured by OMI with the gold standard of tumor growth in two xenograft models. Taken together, these results suggest that OMI of primary tumor organoids may be a powerful test for predicting the efficacy of anticancer drugs and tailoring treatment decisions accordingly.
The primary objective of this thesis is to characterize and develop optical metabolic imaging endpoints as biomarkers of drug response in breast cancer. Organoid OMI screening has the potential to guide therapy selection for other diseases and need not be limited to breast cancer. The potential impact of the live tumor (organoid) and optical metabolic imaging (OMI) system on breast cancer care.
Population density modeling of the mean OMI index per cell in control and treated organoids derived from patient sample #1 (ER+/HER2+) at 72 hours. Population density modeling of the mean OMI index per cell in control and treated organoids derived from patient sample #2 (ER+/HER2+) at 72 hours. Population density modeling of the mean OMI index per cell in control and treated organoids derived from patient sample #3 (ER+/HER2+) at 24 hours.
Abstract
The optical redox ratio (NADH fluorescence intensity divided by FAD fluorescence intensity) is a proven method for determining cellular metabolism and has been used to distinguish cancerous from noncancerous tissues in a variety of models, including oral cancer and breast cancer (21–27). For the cyanide experiment to verify the redox ratio measurement, MCF10A cells were plated at 1 x 10 5 cells per plate 48 h before imaging. Mitotic cells were first labeled with Phospho-Histone H3 (Ser10) antibody (Cell Signaling Technology, Danvers, MA).
Results
The standard error was calculated from the mean of the redox ratio across all images from each cell line.
