A Multi-modal Optical Toolkit to Characterize Spatiotemporal Metabolic Heterogeneity of Breast Cancer and its Microenvironment

Abstract

Breast cancer is a global challenge, accounting for 670,000 recorded deaths in 2022. It is the most prevalent cancer and the cancer with the highest incident rate, with 2.3 million new cases annually. Incidence of breast cancer has increased since 1990 and is projected to continue to rise through 2030. While this is true, great strides have been made in the patient outcomes. Better treatment and widespread mammography screening have contributed to a decrease in breast cancer deaths, with age adjusted mortality rate decreasing from 48 per 100,000 women in 1975 to 27 per 100,000 women in 2019. However regardless of early detection, mortality from recurrent or metastatic disease has not improved in the past 20 years, presenting an outstanding clinical challenge in breast cancer care. Over 90% of breast cancer deaths occur from recurrent disease after treatment of the primary tumor.Breast cancer is a heterogenous disease, resulting in a stratification of prognoses based on molecular subtype. Hormone receptor positive subtypes (progesterone, PR+ and estrogen, ER+) have the best prognoses and are expected to respond well to chemotherapy. Patients positive for enrichment of the human epidermal growth factor 2 (HER2+) or negative for all three receptors (triple negative breast cancer, TNBC) suffer shorter time to recurrence and have greater likelihood of developing visceral metastases than those with ER+ and PR+ disease. However, HER2+ patients have benefitted from the advent of antibody-based therapies that target HER2 on the cell surface. Conversely, as TNBC lacks receptor targets, this aggressive and highly metastatic subtype of breast cancer currently does not benefit from targeted therapies and accumulates resistance to chemotherapy. Thus far, no targeted therapies have been developed that prove efficacious against TNBC. Metabolic heterogeneity is emerging as a promising area of study that can lead to translational benefits in breast cancer survival. Dysregulated cellular energetics was recognized as a hallmark of cancer in 2011 after being an area of study for nearly 100 years. In 1923, Dr. Otto Warburg observed that cancers rely heavily on the uptake of glucose and the glycolytic pathway to support rapid proliferation. This propensity of tumors to rely on aerobic glycolysis as a primary energy source is known as the Warburg effect. The catabolism of glucose and subsequent conversion of pyruvate to lactate provides byproducts that can be used for biosynthesis, paramount to tumor biomass production. An increased production of lactate modifies the tumor microenvironment to be more acidic, advantageous to tumors. Additionally, as tumors rapidly increase in size, angiogenesis lags, creating a tortuous, incomplete, non-uniform vascular network resulting in areas of hypoxia. Hypoxia triggers transcription factor HIF1-alpha to further upregulate glycolysis. Reprogramming of glucose metabolism provides only a partial picture of the complex and dynamic metabolic landscape of tumor cellular energetics. Oxidative phosphorylation (OXPHOS) has been shown to support the energetic needs of tumors alongside aerobic glycolysis, especially in TNBC. OXPHOS has been reported by our group and others to be upregulated in tumor tissue following treatment, through regression and ultimately recurrence. Recently, metabolism of fats has emerged as a key driver of aggressive breast cancer metabolism. Situated in the adipocyte-replete breast environment, tumors uptake fatty acids from nearby lipids, even communicating with adipocytes through gap junctions to stimulate the release of free fatty acids for tumor consumption. Upregulation of the oncogene MYC in TNBC has been shown to significantly upregulate fatty acid β-oxidation (FAO), and the inhibition of FAO in preclinical models of MYC-driven TNBC has been shown to be lethal to the tumor. The metabolic pathways discussed above are intrinsically linked with the tumor microenvironment, with vasculature and tumor-associated adipocytes both playing a role. Molecular heterogeneity as well as heterogeneity in the tumor microenvironment can cause differential response to treatment spatially across a tumor. Sub-populations of cells within a tumor that survive treatment and remain as residual disease threaten to return as recurrent disease. It is likely that just as molecular subtype can provide prognostic information and guide therapeutic strategies that are likely to be effective, understanding heterogeneity in a patient’s specific cancer can predict risk of recurrence and effectiveness of different interventions. Taken together, this body of evidence points to a strong correlation between heterogeneous tumor metabolism and vasculature which drives treatment resistance and patient mortality. OXPHOS, glycolysis, and fatty acid metabolism are key metabolic phenotypes that contribute to the aggression of breast cancer across a tumor’s lifespan. These phenomena have been observed by myriad technologies such as immunohistochemistry, LC-MS and metabolomics, FDG-PET, MR(S)I, optical spectroscopy, and optical imaging. Gaps in our knowledge remain due to a dearth of technology able to longitudinally measure multiple metabolic pathways along with vasculature concurrently in a living animal while preserving spatial information. Thus, the overarching goal of this project was to develop a multi-modal optical toolkit using both fluorescence spectroscopy and microscopy that can measure OXPHOS, glucose uptake, fatty acid uptake, and vascular parameters in a single living animal. This goal was achieved through three specific aims. Aim 1 establishes a technique to measure fatty acid oxidation in vivo and characterizes fatty acid oxidation longitudinally in multiple metastatic preclinical models, and in healthy tissue controls. Previously, the fluorophore Bodipy FL C16 had been shown by our group to be a robust marker of fatty acid uptake, and the fluorophore TMRE had been used as a marker of OXPHOS. Combining a measurement of fatty acid uptake with a measurement of OXPHOS can provide information on whether the fatty acids are being oxidized via fatty acid β-oxidation. Chemical, optical, and biological crosstalk between TMRE and Bodipy FL C16 was assessed, and the two were shown to be compatible. Application of the technique developed in this aim established the utility of multiple metabolic endpoints for discrimination between healthy and tumor-bearing mammary tissue, and between TNBC models with varying metastatic potential. Aim 2 establishes a technique to measure OXPHOS and the uptake of fats and sugars that drive it concurrently in vivo using both fluorescence spectroscopy and fluorescence imaging. Fluorophores Bodipy FL C16 (uptake of fats) and 2-NBDG (uptake of sugars) had not previously been used in combination due to challenges posed by both optical and biological crosstalk. Optical crosstalk between spectrally overlapping fluorophores Bodipy FL C16 and 2-NBDG was mitigated using a spectral unmixing technique developed using spectroscopy and adapted for imaging. The success of this technique was validated for both modalities using tissue-mimicking optical phantoms. To mitigate biological crosstalk, this aim validates a concurrent injection scheme using both spectroscopy and imaging. Leveraging the unique endpoints and datasets captured with each modality, relationships between metabolic and vascular endpoints were explored and the utility of combining metabolic and vascular information exploited. Spectroscopy is a non-invasive technique that enables longitudinal study, and collection of oxygen saturation (SO2) and total hemoglobin concentration ([THB]) improves discrimination between tumor and normal tissue. The addition of spatial information preserved with imaging allows intra-tissue heterogeneity to be investigated. Aim 2 shows increased intra-tissue heterogeneity in tumor tissue compared to normal tissue. Aim 2 also demonstrates the observation of compartments within tumor tissue displaying differing metabolic phenotypes, glycolysis and FAO). The oncogene MYC is known to drive tumor aggression in part by regulating metabolism. Studies have shown that the upregulation of MYC increases both glycolysis and FAO. Inhibition of FAO in MYC-driven tumors lead to tumor regression in multiple preclinical models. Having developed and robustly validated an optical toolkit capable of imaging OXPHOS, glucose uptake, fatty acid uptake, and vasculature concurrently, in Aim 3 we sought to investigate the relationship between the oncogene MYC and metabolism in preclinical TNBC models. We showed increased fatty acid uptake in two MYC-driven models compared to normal tissue. We characterized a metabolic switch from a FAO signature in growing, primary MYC-driven tumors to oxidation of glucose in regressing tumors. We captured inter- and intra-tumoral heterogeneity across tumors, showing different metabolic signatures exist in separate regions of a tumor, contributing to heterogeneity that drives aggressive disease. Finally, we identified correlation between proximity to vasculature and metabolic phenotype across different fields of view in a single tumor. This optical toolkit has translational potential and is poised to enable wide-reaching clinical and pre-clinical studies. Patient-derived organoids (PDOs) have been shown to preserve phenotypes observed in the individual patients’ cancers they are derived from; imaging of PDOs can provide real-time data on a patient’s likelihood to respond to treatment. This is critical information to inform treatment and can be implemented in an adaptive clinical trial. Preclinically, this optical toolkit can leverage the non-invasive and longitudinal spectroscopy modality to identify temporal windows of particular interest, such as early or late regression, dormancy, residual disease, or early recurrence. Imaging can then be used at those timepoints to capture holistic information efficiently. Finally, imaging of tumor-immune cell interactions, immune cell metabolism, and cancer-associated adipocytes will enhance our understanding of the dynamic interplay of the tumor and its microenvironment. Broadly, this technology may be applied across many cancers.