Targeting Tumor Acidosis and Regulatory T Cells Unmasks Anti-Metastatic Potential of Local Tumor Ablation in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an immunologically heterogenous disease that lacks clinically actionable targets and is more likely to progress to metastatic disease than other types of breast cancer. Tumor ablation has been used to increase response rates to checkpoint inhibitors, which remain low for TNBC patients. We hypothesized that tumor ablation could produce an anti-tumor response without using checkpoint inhibitors if immunosuppression (i.e., Tregs, tumor acidosis) was subdued. Tumors were primed with sodium bicarbonate (200 mM p.o.) to reduce tumor acidosis and low-dose cyclophosphamide (100-200 mg/kg i.p.) to deplete regulatory T cells, as has been shown independently in previous studies. A novel injectable ablative was then used to necrose the tumor, release tumor antigens, and initiate an immune event that could create an abscopal effect. This combination of bicarbonate, cyclophosphamide, and ablation, called "BiCyclA", was tested in three syngeneic models of TNBC: E0771 (C57BL/6), 67NR (BALB/c), and 4T1-Luc (BALB/c). In E0771 and 67NR, BiCyclA therapy significantly reduced tumor growth and cured 5/7 and 6/10 mice 50 days after treatment respectively. In the metastatic 4T1-Luc tumors, for which surgery and checkpoint inhibitors fail, BiCyclA cured 5/10 mice of primary tumors and lung metastases. Notably, CD4+ and CD8+ T cells were found to be crucial for the anti-metastatic response, and cured mice were able to resist tumor rechallenge, suggesting production of immune memory. Reduction of tumor acidity and regulatory T cells with ablation is a simple yet effective therapy for local and systemic tumor control with broad applicability as it is not limited by expensive supplies.

Optical imaging reveals chemotherapy-induced metabolic reprogramming of residual disease and recurrence.

Fewer than 20% of triple-negative breast cancer patients experience long-term responses to mainstay chemotherapy. Resistant tumor subpopulations use alternative metabolic pathways to escape therapy, survive, and eventually recur. Here, we show in vivo, longitudinal metabolic reprogramming in residual disease and recurrence of triple-negative breast cancer xenografts with varying sensitivities to the chemotherapeutic drug paclitaxel. Optical imaging coupled with metabolomics reported an increase in non-glucose-driven mitochondrial metabolism and an increase in intratumoral metabolic heterogeneity during regression and residual disease in resistant MDA-MB-231 tumors. Conversely, sensitive HCC-1806 tumors were primarily reliant on glucose uptake and minimal changes in metabolism or heterogeneity were observed over the tumors' therapeutic life cycles. Further, day-matched resistant HCC-1806 tumors revealed a higher reliance on mitochondrial metabolism and elevated metabolic heterogeneity compared to sensitive HCC-1806 tumors. Together, metabolic flexibility, increased reliance on mitochondrial metabolism, and increased metabolic heterogeneity are defining characteristics of persistent residual disease, features that will inform the appropriate type and timing of therapies.

Adaptive Design of Fluorescence Imaging Systems for Custom Resolution, Fields of View, and Geometries.

Objective and Impact Statement: We developed a generalized computational approach to design uniform, high-intensity excitation light for low-cost, quantitative fluorescence imaging of in vitro, ex vivo, and in vivo samples with a single device. Introduction: Fluorescence imaging is a ubiquitous tool for biomedical applications. Researchers extensively modify existing systems for tissue imaging, increasing the time and effort needed for translational research and thick tissue imaging. These modifications are application-specific, requiring new designs to scale across sample types. Methods: We implemented a computational model to simulate light propagation from multiple sources. Using a global optimization algorithm and a custom cost function, we determined the spatial positioning of optical fibers to generate 2 illumination profiles. These results were implemented to image core needle biopsies, preclinical mammary tumors, or tumor-derived organoids. Samples were stained with molecular probes and imaged with uniform and nonuniform illumination. Results: Simulation results were faithfully translated to benchtop systems. We demonstrated that uniform illumination increased the reliability of intraimage analysis compared to nonuniform illumination and was concordant with traditional histological findings. The computational approach was used to optimize the illumination geometry for the purposes of imaging 3 different fluorophores through a mammary window chamber model. Illumination specifically designed for intravital tumor imaging generated higher image contrast compared to the case in which illumination originally optimized for biopsy images was used. Conclusion: We demonstrate the significance of using a computationally designed illumination for in vitro, ex vivo, and in vivo fluorescence imaging. Application-specific illumination increased the reliability of intraimage analysis and enhanced the local contrast of biological features. This approach is generalizable across light sources, biological applications, and detectors.

A Spectroscopic Technique to Simultaneously Characterize Fatty Acid Uptake, Mitochondrial Activity, Vascularity, and Oxygen Saturation for Longitudinal Studies In Vivo.

Aggressive breast cancer has been shown to shift its metabolism towards increased lipid catabolism as the primary carbon source for oxidative phosphorylation. In this study, we present a technique to longitudinally monitor lipid metabolism and oxidative phosphorylation in pre-clinical tumor models to investigate the metabolic changes with mammary tissue development and characterize metabolic differences between primary murine breast cancer and normal mammary tissue. We used optical spectroscopy to measure the signal of two simultaneously injected exogenous fluorescent metabolic reporters: TMRE (oxidative phosphorylation surrogate) and Bodipy FL C16 (lipid catabolism surrogate). We leverage an inverse Monte Carlo algorithm to correct for aberrations resulting from tissue optical properties and to extract vascular endpoints relevant to oxidative metabolism, specifically oxygen saturation (SO2) and hemoglobin concentration ([Hb]). We extensively validated our optical method to demonstrate that our two fluorescent metabolic endpoints can be measured without chemical or optical crosstalk and that dual measurements of both fluorophores in vivo faithfully recapitulate the measurements of each fluorophore independently. We then applied our method to track the metabolism of growing 4T1 and 67NR breast tumors and aging mammary tissue, all highly metabolic tissue types. Our results show the changes in metabolism as a function of mammary age and tumor growth, and these changes can be best distinguished through the combination of endpoints measured with our system. Clustering analysis incorporating both Bodipy FL C16 and TMRE endpoints combined with either SO2 or [Hb] proved to be the most effective in minimizing intra-group variance and maximizing inter-group differences. Our platform can be extended to applications in which long-term metabolic flexibility is important to study, for example in tumor regression, recurrence following dormancy, and responses to cancer treatment.

Ethanol Ablation Therapy Drives Immune-Mediated Antitumor Effects in Murine Breast Cancer Models.

Ethanol ablation is a minimally invasive, cost-effective method of destroying tumor tissue through an intratumoral injection of high concentrations of cytotoxic alcohol. Ethyl-cellulose ethanol (ECE) ablation, a modified version of ethanol ablation, contains the phase-changing polysaccharide ethyl-cellulose to reduce ethanol leakage away from the tumor. Ablation produces tissue necrosis and initiates a wound healing process; however, the characteristic of the immunologic events after ECE ablation of tumors has yet to be explored. Models of triple-negative breast cancer (TNBC), which are classically immunosuppressive and difficult to treat clinically, were used to characterize the immunophenotypic changes after ECE ablation. In poorly invasive TNBC rodent models, the injury to the tumor induced by ECE increased tumor infiltrating lymphocytes (TILs) and reduced tumor growth. In a metastatic TNBC model (4T1), TILs did not increase after ECE ablation, though lung metastases were reduced. 4T1 tumors secrete high levels of granulocytic colony stimulating factor (G-CSF), which induces a suppressive milieu of granulocytic myeloid-derived suppressor cells (gMDSCs) aiding in the formation of metastases and suppression of antitumor immunity. We found that a single intratumoral injection of ECE normalized tumor-induced myeloid changes: reducing serum G-CSF and gMDSC populations. ECE also dampened the suppressive strength of gMDSC on CD4 and CD8 cell proliferation, which are crucial for anti-tumor immunity. To demonstrate the utility of these findings, ECE ablation was administered before checkpoint inhibitor (CPI) therapy in the 4T1 model and was found to significantly increase survival compared to a control of saline and CPI. Sixty days after tumor implant no primary tumors or metastatic lung lesions were found in 6/10 mice treated with CPI plus ECE, compared to 1/10 with ECE alone and 0/10 with CPI and saline.

In vivo metabolic imaging identifies lipid vulnerability in a preclinical model of Her2+/Neu breast cancer residual disease and recurrence.

Recurrent cancer cells that evade therapy is a leading cause of death in breast cancer patients. This risk is high for women showing an overexpression of human epidermal growth factor receptor 2 (Her2). Cells that persist can rely on different substrates for energy production relative to their primary tumor counterpart. Here, we characterize metabolic reprogramming related to tumor dormancy and recurrence in a doxycycline-induced Her2+/Neu model of breast cancer with varying times to recurrence using longitudinal fluorescence microscopy. Glucose uptake (2-NBDG) and mitochondrial membrane potential (TMRE) imaging metabolically phenotype mammary tumors as they transition to regression, dormancy, and recurrence. "Fast-recurrence" tumors (time to recurrence ~55 days), transition from glycolysis to mitochondrial metabolism during regression and this persists upon recurrence. "Slow-recurrence" tumors (time to recurrence ~100 days) rely on both glycolysis and mitochondrial metabolism during recurrence. The increase in mitochondrial activity in fast-recurrence tumors is attributed to a switch from glucose to fatty acids as the primary energy source for mitochondrial metabolism. Consequently, when fast-recurrence tumors receive treatment with a fatty acid inhibitor, Etomoxir, tumors report an increase in glucose uptake and lipid synthesis during regression. Treatment with Etomoxir ultimately prolongs survival. We show that metabolic reprogramming reports on tumor recurrence characteristics, particularly at time points that are essential for actionable targets. The temporal characteristics of metabolic reprogramming will be critical in determining the use of an appropriate timing for potential therapies; namely, the notion that metabolic-targeted inhibition during regression reports long-term therapeutic benefit.

Understanding the sources of errors in ex vivo Hsp90 molecular imaging for rapid-on-site breast cancer diagnosis.

Overexpression of heat shock protein 90 (Hsp90) on the surface of breast cancer cells makes it an attractive molecular biomarker for breast cancer diagnosis. Before a ubiquitous diagnostic method can be established, an understanding of the systematic errors in Hsp90-based imaging is essential. In this study, we investigated three factors that may influence the sensitivity of ex vivo Hsp90 molecular imaging: time-dependent tissue viability, nonspecific diffusion of an Hsp90 specific probe (HS-27), and contact-based imaging. These three factors will be important considerations when designing any diagnostic imaging strategy based on fluorescence imaging of a molecular target on tissue samples.

Polymer-assisted intratumoral delivery of ethanol: Preclinical investigation of safety and efficacy in a murine breast cancer model.

Focal tumor ablation with ethanol could provide benefits in low-resource settings because of its low overall cost, minimal imaging technology requirements, and acceptable clinical outcomes. Unfortunately, ethanol ablation is not commonly utilized because of a lack of predictability of the ablation zone, caused by inefficient retention of ethanol at the injection site. To create a predictable zone of ablation, we have developed a polymer-assisted ablation method using ethyl cellulose (EC) mixed with ethanol. EC is ethanol-soluble and water-insoluble, allowing for EC-ethanol to be injected as a liquid and precipitate into a solid, occluding the leakage of ethanol upon contact with tissue. The aims of this study were to compare the 1) safety, 2) release kinetics, 3) spatial distribution, 4) necrotic volume, and 5) overall survival of EC-ethanol to conventional ethanol ablation in a murine breast tumor model. Non-target tissue damage was monitored through localized adverse events recording, ethanol release kinetics with Raman spectroscopy, injectate distribution with in vivo imaging, target-tissue necrosis with NADH-diaphorase staining, and overall survival by proxy of tumor growth. EC-ethanol exhibited decreased localized adverse events, a slowing of the release rate of ethanol, more compact injection zones, 5-fold increase in target-tissue necrosis, and longer overall survival rates compared to the same volume of pure ethanol. A single 150 µL dose of 6% EC-ethanol achieved a similar survival probability rates to six daily 50 µL doses of pure ethanol used to simulate a slow-release of ethanol over 6 days. Taken together, these results demonstrate that EC-ethanol is safer and more effective than ethanol alone for ablating tumors.

In Vivo Optical Metabolic Imaging of Long-Chain Fatty Acid Uptake in Orthotopic Models of Triple-Negative Breast Cancer.

Targeting a tumor's metabolic dependencies is a clinically actionable therapeutic approach; however, identifying subtypes of tumors likely to respond remains difficult. The use of lipids as a nutrient source is of particular importance, especially in breast cancer. Imaging techniques offer the opportunity to quantify nutrient use in preclinical tumor models to guide development of new drugs that restrict uptake or utilization of these nutrients. We describe a fast and dynamic approach to image fatty acid uptake in vivo and demonstrate its relevance to study both tumor metabolic reprogramming directly, as well as the effectiveness of drugs targeting lipid metabolism. Specifically, we developed a quantitative optical approach to spatially and longitudinally map the kinetics of long-chain fatty acid uptake in in vivo murine models of breast cancer using a fluorescently labeled palmitate molecule, Bodipy FL c16. We chose intra-vital microscopy of mammary tumor windows to validate our approach in two orthotopic breast cancer models: a MYC-overexpressing, transgenic, triple-negative breast cancer (TNBC) model and a murine model of the 4T1 family. Following injection, Bodipy FL c16 fluorescence increased and reached its maximum after approximately 30 min, with the signal remaining stable during the 30-80 min post-injection period. We used the fluorescence at 60 min (Bodipy60), the mid-point in the plateau region, as a summary parameter to quantify Bodipy FL c16 fluorescence in subsequent experiments. Using our imaging platform, we observed a two- to four-fold decrease in fatty acid uptake in response to the downregulation of the MYC oncogene, consistent with findings from in vitro metabolic assays. In contrast, our imaging studies report an increase in fatty acid uptake with tumor aggressiveness (6NR, 4T07, and 4T1), and uptake was significantly decreased after treatment with a fatty acid transport inhibitor, perphenazine, in both normal mammary pads and in the most aggressive 4T1 tumor model. Our approach fills an important gap between in vitro assays providing rich metabolic information at static time points and imaging approaches visualizing metabolism in whole organs at a reduced resolution.

Exploiting heat shock protein expression to develop a non-invasive diagnostic tool for breast cancer.

Leveraging the unique surface expression of heat shock protein 90 (Hsp90) in breast cancer provides an exciting opportunity to develop rapid diagnostic tests at the point-of-care setting. Hsp90 has previously been shown to have elevated expression levels across all breast cancer receptor subtypes. We have developed a non-destructive strategy using HS-27, a fluorescently-tethered Hsp90 inhibitor, to assay surface Hsp90 expression on intact tissue specimens and validated our approach in clinical samples from breast cancer patients across estrogen receptor positive, Her2-overexpressing, and triple negative receptor subtypes. Utilizing a pre-clinical biopsy model, we optimized three imaging parameters that may affect the specificity of HS-27 based diagnostics - time between tissue excision and staining, agent incubation time, and agent dose, and translated our strategy to clinical breast cancer samples. Findings indicated that HS-27 florescence was highest in tumor tissue, followed by benign tissue, and finally followed by mammoplasty negative control samples. Interestingly, fluorescence in tumor samples was highest in Her2+ and triple negative subtypes, and inversely correlated with the presence of tumor infiltrating lymphocytes indicating that HS-27 fluorescence increases in aggressive breast cancer phenotypes. Development of a Gaussian support vector machine classifier based on HS-27 fluorescence features resulted in a sensitivity and specificity of 82% and 100% respectively when classifying tumor and benign conditions, setting the stage for rapid and automated tissue diagnosis at the point-of-care.

Simultaneous in vivo optical quantification of key metabolic and vascular endpoints reveals tumor metabolic diversity in murine breast tumor models.

Therapeutically exploiting vascular and metabolic endpoints becomes critical to translational cancer studies because altered vascularity and deregulated metabolism are two important cancer hallmarks. The metabolic and vascular phenotypes of three sibling breast tumor lines with different metastatic potential are investigated in vivo with a newly developed quantitative spectroscopy system. All tumor lines have different metabolic and vascular characteristics compared to normal tissues, and there are strong positive correlations between metabolic (glucose uptake and mitochondrial membrane potential) and vascular (oxygen saturations and hemoglobin concentrations) parameters for metastatic (4T1) tumors but not for micrometastatic (4T07) and nonmetastatic (67NR) tumors. A longitudinal study shows that both vascular and metabolic endpoints of 4T1 tumors increased up to a specific tumor size threshold beyond which these parameters decreased. The synchronous changes between metabolic and vascular parameters, along with the strong positive correlations between these endpoints suggest that 4T1 tumors rely on strong oxidative phosphorylation in addition to glycolysis. This study illustrates the great potential of our optical technique to provide valuable dynamic information about the interplay between the metabolic and vascular status of tumors, with important implications for translational cancer investigations.

Optical Imaging of Glucose Uptake and Mitochondrial Membrane Potential to Characterize Her2 Breast Tumor Metabolic Phenotypes.

With the large number of women diagnosed and treated for breast cancer each year, the importance of studying recurrence has become evident due to most deaths from breast cancer resulting from tumor recurrence following therapy. To mitigate this, cellular and molecular pathways used by residual disease prior to recurrence must be studied. An altered metabolism has long been considered a hallmark of cancer, and several recent studies have gone further to report metabolic dysfunction and alterations as key to understanding the underlying behavior of dormant and recurrent cancer cells. Our group has used two probes, 2-[N-(7-nitrobenz-2-oxa-1, 3-diaxol-4-yl) amino]-2-deoxyglucose (2-NBDG) and tetramethyl rhodamine ethyl ester (TMRE), to image glucose uptake and mitochondrial membrane potential, respectively, to report changes in metabolism between primary tumors, regression, residual disease, and after regrowth in genetically engineered mouse (GEM)-derived mammospheres. Imaging revealed unique metabolic phenotypes across the stages of tumor development. Although primary mammospheres overexpressing Her2 maintained increased glucose uptake ("Warburg effect"), after Her2 downregulation, during regression and residual disease, mammospheres appeared to switch to oxidative phosphorylation. Interestingly, in mammospheres where Her2 overexpression was turned back on to model recurrence, glucose uptake was lowest, indicating a potential change in substrate preference following the reactivation of Her2, reeliciting growth. Our findings highlight the importance of imaging metabolic adaptions to gain insight into the fundamental behaviors of residual and recurrent disease. IMPLICATIONS: This study demonstrates these functional fluorescent probes' ability to report metabolic adaptations during primary tumor growth, regression, residual disease, and regrowth in Her2 breast tumors.

Near-simultaneous quantification of glucose uptake, mitochondrial membrane potential, and vascular parameters in murine flank tumors using quantitative diffuse reflectance and fluorescence spectroscopy.

The shifting metabolic landscape of aggressive tumors, with fluctuating oxygenation conditions and temporal changes in glycolysis and mitochondrial metabolism, is a critical phenomenon to study in order to understand negative treatment outcomes. Recently, we have demonstrated near-simultaneous optical imaging of mitochondrial membrane potential (MMP) and glucose uptake in non-tumor window chambers, using the fluorescent probes tetramethylrhodamine ethyl ester (TMRE) and 2-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG). Here, we demonstrate a complementary technique to perform near-simultaneous in vivo optical spectroscopy of tissue vascular parameters, glucose uptake, and MMP in a solid tumor model that is most often used for therapeutic studies. Our study demonstrates the potential of optical spectroscopy as an effective tool to quantify the vascular and metabolic characteristics of a tumor, which is an important step towards understanding the mechanisms underlying cancer progression, metastasis, and resistance to therapies.

Metaboloptics: Visualization of the tumor functional landscape via metabolic and vascular imaging.

Many cancers adeptly modulate metabolism to thrive in fluctuating oxygen conditions; however, current tools fail to image metabolic and vascular endpoints at spatial resolutions needed to visualize these adaptations in vivo. We demonstrate a high-resolution intravital microscopy technique to quantify glucose uptake, mitochondrial membrane potential (MMP), and SO2 to characterize the in vivo phentoypes of three distinct murine breast cancer lines. Tetramethyl rhodamine, ethyl ester (TMRE) was thoroughly validated to report on MMP in normal and tumor-bearing mice. Imaging MMP or glucose uptake together with vascular endpoints revealed that metastatic 4T1 tumors maintained increased glucose uptake across all SO2 ("Warburg effect"), and also showed increased MMP relative to normal tissue. Non-metastatic 67NR and 4T07 tumor lines both displayed increased MMP, but comparable glucose uptake, relative to normal tissue. The 4T1 peritumoral areas also showed a significant glycolytic shift relative to the tumor regions. During a hypoxic stress test, 4T1 tumors showed significant increases in MMP with corresponding significant drops in SO2, indicative of intensified mitochondrial metabolism. Conversely, 4T07 and 67NR tumors shifted toward glycolysis during hypoxia. Our findings underscore the importance of imaging metabolic endpoints within the context of a living microenvironment to gain insight into a tumor's adaptive behavior.

Near-simultaneous intravital microscopy of glucose uptake and mitochondrial membrane potential, key endpoints that reflect major metabolic axes in cancer.

While the demand for metabolic imaging has increased in recent years, simultaneous in vivo measurement of multiple metabolic endpoints remains challenging. Here we report on a novel technique that provides in vivo high-resolution simultaneous imaging of glucose uptake and mitochondrial metabolism within a dynamic tissue microenvironment. Two indicators were leveraged; 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) reports on glucose uptake and Tetramethylrhodamine ethyl ester (TMRE) reports on mitochondrial membrane potential. Although we demonstrated that there was neither optical nor chemical crosstalk between 2-NBDG and TMRE, TMRE uptake was significantly inhibited by simultaneous injection with 2-NBDG in vivo. A staggered delivery scheme of the two agents (TMRE injection was followed by 2-NBDG injection after a 10-minute delay) permitted near-simultaneous in vivo microscopy of 2-NBDG and TMRE at the same tissue site by mitigating the interference of 2-NBDG with normal glucose usage. The staggered delivery strategy was evaluated under both normoxic and hypoxic conditions in normal tissues as well as in a murine breast cancer model. The results were consistent with those expected for independent imaging of 2-NBDG and TMRE. This optical imaging technique allows for monitoring of key metabolic endpoints with the unique benefit of repeated, non-destructive imaging within an intact microenvironment.