With the widespread adoption of mammograms for cancer detection, modern research has principally pivoted towards a focus on how to predict recurrence and ultimately to prevent it. Approximately 30% of breast cancer patients lacking any clinical or pathological signs of metastasis have disseminated tumor cells present at the time of their diagnosis. Thus, residual disease at the primary tumor site after systemic therapy could be viewed as a surrogate for micro-metastasis elsewhere in the body that may have evaded the drug and could potentially return as recurrent disease. Given that residual disease is an important determinant of clinical outcome, it is important to predict the potential for and mitigate distant recurrence. Through the emergence of complex and robust animal models, like genetically engineered mouse models, we can study common oncogenes and key features of dormancy, like regression to a non-proliferative state following therapy and ultimately recurrence, similar to human cancers to better inform future therapy options for these resilient diseases. We are creating a combination of metabolic and vascular imaging strategies and algorithms to provide an unprecedented window into metabolic reprogramming of residual disease and recurrence and facilitate understanding of how these tumors evade therapeutic stress. This can inform the development and application of new therapies to mitigate tumor recurrence or eliminate residual disease altogether.

Tumor metabolism reprograms during the different stages of a tumor's life cycle. Mammospheres stained for either 2-NBDG (glucose uptake) or TMRE (mitochondrial activity) were imaged while overexpressing the oncogene HER2 (primary), following HER2 downregulation for 2 days prior to imaging (early regression), for 4 days prior to imaging (late regression), for 14 days prior to imaging (early residual disease), and for 28 days prior to imaging (late residual disease). Also, following 21 days of HER2 downregulation, a culture of cells was re-conditioned to overexpress HER2 for 7 days (recurrence). Representative mean projection images of 2-NBDG and TMRE intensities.

While the focus in high resource settings is recurrence from breast cancer treatment, the challenge in low resource settings is early and effective diagnosis of breast cancer. With the advent of portable ultrasound systems it is now possible to perform imaging at the point of care for breast cancer. However, access to pathology is the bottleneck. In high- resource settings, pathology is readily accessible for breast cancer diagnosis, albeit with a delay between diagnosis and treatment because of the labor-intensive nature of pathology. In low-resource settings, this is simply not an option due to the dearth of pathologists in fragile health care systems. We have ushered in an era of molecular diagnostics given the plethora of molecular targeting agents that have been developed for cancer treatment. We have harnessed this capability to make molecular diagnostics the potential new face of pathology, using a contrast agent, fluorescein, tethered to a small molecule inhibitor to heat shock protein 90 (HSP90), which is overexpressed in the different subtypes of breast cancer. We have designed a strategy to use ultrasound imaging to perform imaging, needle-based tissue excision and molecular diagnostics as a new strategy for breast cancer diagnostics at the point of care.

A rapid assay for Hsp90 imaging in clinical breast specimens. ER + /PR + (A) an ultrasound-guided core-needle biopsy of tumor (B) and non-tumor (C) biopsy. Biopsies are treated ex vivo with 100 µM HS-27 for one minute. Tissue types were determined by a trained pathologist using the histology images shown to the left of the fluorescence images. The top right corner of each tumor image shows the percentage of total tumor involvement in that region along with the percent cellularity



Madonna MC, Fox DB, Crouch BT, Lee J, Zhu C, Martinez AF, Alvarez JV, Ramanujam N. Optical imaging of glucose uptake and mitochondrial membrane potential to characterize Her2 breast tumor metabolic phenotypes. Molecular Cancer Research. 2019 molcanres-0618.


Zhu C, Li M, Vincent T, Martin HL, Crouch BT, Martinez AF, Madonna MC, Palmer GM, Dewhirst MW, Ramanujam N. Simultaneous in vivo optical quantification of key metabolic and vascular endpoints reveals tumor metabolic diversity in murine breast tumor models. Journal of biophotonics. 2019 Apr;12(4):e201800372.


Crouch BT, Gallagher J, Wang R, Duer J, Hall A, Soo MS, Hughes P, Haystead T, Ramanujam N. Exploiting heat shock protein expression to develop a non-invasive diagnostic tool for breast cancer. Scientific reports. 2019 Mar 5;9(1):3461.


Zhu C, Martin HL, Crouch BT, Martinez AF, Li M, Palmer GM, Dewhirst MW, Ramanujam N. Near-simultaneous quantification of glucose uptake, mitochondrial membrane potential, and vascular parameters in murine flank tumors using quantitative diffuse reflectance and fluorescence spectroscopy. Biomedical optics express. 2018 Jul 1;9(7):3399-412.


Martinez AF, McCachren SS, Lee M, Murphy HA, Zhu C, Crouch BT, Martin HL, Erkanli A, Rajaram N, Ashcraft KA, Fontanella AN. Metaboloptics: Visualization of the tumor functional landscape via metabolic and vascular imaging. Scientific reports. 2018 Mar 8;8(1):4171.


Hu F, Martin H, Martinez A, Everitt J, Erkanli A, Lee WT, Dewhirst M, Ramanujam N. Distinct Angiogenic Changes during Carcinogenesis Defined by Novel Label-Free Dark-Field Imaging in a Hamster Cheek Pouch Model. Cancer research. 2017 Dec 15;77(24):7109-19.


Crouch B, Murphy H, Belonwu S, Martinez A, Gallagher J, Hall A, Soo MS, Lee M, Hughes P, Haystead T, Ramanujam N. Leveraging ectopic Hsp90 expression to assay the presence of tumor cells and aggressive tumor phenotypes in breast specimens. Scientific reports. 2017 Dec 13;7(1):17487.


Zhu C, Martinez AF, Martin HL, Li M, Crouch BT, Carlson DA, Haystead TA, Ramanujam N. Near-simultaneous intravital microscopy of glucose uptake and mitochondrial membrane potential, key endpoints that reflect major metabolic axes in cancer. Scientific reports. 2017 Oct 23;7(1):13772.


Hu F, Vishwanath K, Salama JK, Erkanli A, Peterson B, Oleson JR, Lee WT, Brizel DM, Ramanujam N, Dewhirst MW. Oxygen and perfusion kinetics in response to fractionated radiation therapy in FaDu head and neck cancer xenografts are related to treatment outcome. International Journal of Radiation Oncology* Biology* Physics. 2016 Oct 1;96(2):462-9.


Hu F, Morhard R, Murphy HA, Zhu C, Ramanujam N. Dark field optical imaging reveals vascular changes in an inducible hamster cheek pouch model during carcinogenesis. Biomedical optics express. 2016 Sep 1;7(9):3247-61.


Rajaram N, Reesor AF, Mulvey CS, Frees AE, Ramanujam N. Non-invasive, simultaneous quantification of vascular oxygenation and glucose uptake in tissue. PloS one. 2015 Jan 30;10(1):e0117132.


Frees AE, Rajaram N, McCachren III SS, Fontanella AN, Dewhirst MW, Ramanujam N. Delivery-corrected imaging of fluorescently-labeled glucose reveals distinct metabolic phenotypes in murine breast cancer. PloS one. 2014 Dec 19;9(12):e115529.


Rajaram N, Frees AE, Fontanella AN, Zhong J, Hansen K, Dewhirst MW, Ramanujam N. Delivery rate affects uptake of a fluorescent glucose analog in murine metastatic breast cancer. PloS one. 2013 Oct 18;8(10):e76524.

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