Mild Hyperthermia Enhanced Liposomal Doxorubicin Delivery and CD8+ T cell Infiltration in Triple Negative Breast Cancer.

Mild hyperthermia (MHTh) is often used in combination with chemotherapy and radiotherapy for cancer treatment. In the current study, the effect of MHTh on the enhanced uptake of the FDA-approved chemotherapy drug, liposomal doxorubicin (dox) in syngeneic 4T1 tumors was investigated. Doxorubicin has inherent fluorescence properties having an emission signal at 590 nm upon excitation with a 480 nm laser. A group of mice administered with doxorubicin (dox) were exposed to MHTh (42 °C) for 30 minutes whereas control group given dox did not receive MHTh. Ex vivo optical imaging of harvested tumors confirmed higher uptake of dox in treated versus the control untreated tumors. Confocal microscopy of tumor sections indicates higher fluorescent intensity due to increased accumulation of dox in MHTh-treated compared to untreated tumors. We examined the effect of MHTh to enhance CD8 tumor infiltration, production of interferon-γ (IFN-γ) and expression of programmed death ligand-1 (PD-L1). mRNA in situ hybridization was performed to test for transcripts of CD8, IFN-γ and PD-L1. Results showed that higher expression of CD8 mRNA was observed in MHTh-administered tumors versus untreated cohorts. The signal for IFN-γ and PD-L1 in both groups were not significantly different. Taken together, our findings imply that MHTh can improve tumor uptake of dox. Importantly, our data suggests that MHTh can boost CD8+ T cell infiltration.

Wavelength-dependent error minimization for quantitative spectroscopic photoacoustic tomography with a ring-array system.

PURPOSE: Photoacoustic tomography (PAT) is a non-invasive and high-resolution imaging technique that can provide functional and molecular information from the optical properties of pathological tissues, such as cancer. Spectroscopic PAT (sPAT) is capable of supplying information such as oxygen saturation (sO2), which is an important biological indicator for diseases such as cancer. However, the wavelength dependent nature of sPAT makes it challenging to provide accurate quantitative measurements of tissue oxygenation beyond shallow depths. We have previously reported the utility of combined ultrasound tomography and PAT to achieve optical and acoustic compensated PAT images at a single wavelength and for enhanced PAT images at larger depths. In this work we further explore the utility of the optical and acoustic compensation PAT algorithm to minimize the wavelength dependency in sPAT by showcasing improvements in spectral unmixing. MATERIALS AND METHODS: Two optically and acoustically characterized heterogenous phantoms were manufactured to test the ability of the system and developed algorithm to minimize the wavelength-dependence driven error in sPAT spectral unmixing. The PA inclusions within each phantom were composed of a mixture of two sulfate dyes, copper sulfate (CuSO4) and nickel sulfate (NiSO4), with known optical spectra. Improvements between uncompensated and optically and acoustically compensated PAT (OAcPAT) were quantified as the relative percent error between the measured results and the ground truth. RESULTS: The results of our phantom studies demonstrate that OAcPAT can significantly improve the accuracy of sPAT measurements in a heterogenous medium and especially at larger inclusions depths which can reach to up to 12% improvement in measurement errors. This significant improvement can play a vital role in reliability of future in-vivo biomarker quantifications. CONCLUSIONS: Utilizing UST for model-based optical and acoustic compensation of PAT images was proposed by our group previously. In this work, we further demonstrated the efficacy of the developed algorithm in sPAT by minimizing the error caused by the tissue's optical heterogeneity on improving spectral unmixing, which is a major limiting factor in reliability of sPAT measurements. Such synergistic combination of UST and PAT provides a window of opportunity to achieve bias-free quantitative sPAT measurements, which plays an important role in future pre-clinical and clinical utility of PAT.

Feasibility of ultrasound tomography-guided localized mild hyperthermia using a ring transducer: Ex vivo and in silico studies.

BACKGROUND: As of 2022, breast cancer continues to be the most diagnosed cancer worldwide. This problem persists within the United States as well, as the American Cancer Society has reported that ∼12.5% of women will be diagnosed with invasive breast cancer over the course of their lifetime. Therefore, a clinical need continues to exist to address this disease from a treatment and therapeutic perspective. Current treatments for breast cancer and cancers more broadly include surgery, radiation, and chemotherapy. Adjuncts to these methods have been developed to improve the clinical outcomes for patients. One such adjunctive treatment is mild hyperthermia therapy (MHTh), which has been shown to be successful in the treatment of cancers by increasing effectiveness and reduced dosage requirements for radiation and chemotherapies. MHTh-assisted treatments can be performed with invasive thermal devices, noninvasive microwave induction, heating and recirculation of extracted patient blood, or whole-body hyperthermia with hot blankets. PURPOSE: One common method for inducing MHTh is by using microwave for heat induction and magnetic resonance imaging for temperature monitoring. However, this leads to a complex, expensive, and inaccessible therapy platform. Therefore, in this work we aim to show the feasibility of a novel all-acoustic MHTh system that uses focused ultrasound (US) to induce heating while also using US tomography (UST) to provide temperature estimates. Changes in sound speed (SS) have been shown to be strongly correlated with temperature changes and can therefore be used to indirectly monitor heating throughout the therapy. Additionally, these SS estimates allow for heterogeneous SS-corrected phase delays when heating complex and heterogeneous tissue structures. METHODS: Feasibility to induce localized heat in tissue was investigated in silico with a simulated breast model, including an embedded tumor using continuous wave US. Here, both heterogenous acoustic and thermal properties were modeled in addition to blood perfusion. We further demonstrate, with ex vivo tissue phantoms, the feasibility of using ring-based UST to monitor temperature by tracking changes in SS. Two phantoms (lamb tissue and human abdominal fat) with latex tubes containing varied temperature flowing water were imaged. The measured SS of the water at each temperature were compared against values that are reported in literature. RESULTS: Results from ex vivo tissue studies indicate successful tracking of temperature under various phantom configurations and ranges of water temperature. The results of in silico studies show that the proposed system can heat an acoustically and thermally heterogenous breast model to the clinically relevant temperature of 42°C while accounting for a reasonable time needed to image the current cross section (200 ms). Further, we have performed an initial in silico study demonstrating the feasibility of adjusting the transmit waveform frequency to modify the effective heating height at the focused region. Lastly, we have shown in a simpler 2D breast model that MHTh level temperatures can be maintained by adjusting the transmit pressure intensity of the US ring. CONCLUSIONS: This work has demonstrated the feasibility of using a 256-element ring array transducer for temperature monitoring; however, future work will investigate minimizing the difference between measured SS and the values shown in literature. A hypothesis attributes this bias to potential volumetric average artifacts from the ray-based SS inversion algorithm that was used, and that moving to a waveform-based SS inversion algorithm will greatly improve the SS estimates. Additionally, we have shown that an all-acoustic MHTh system is feasible via in silico studies. These studies have indicated that the proposed system can heat a tumor within a heterogenous breast model to 42°C within a narrow time frame. This holds great promise for increasing the accessibility and reducing the complexity of a future all-acoustic MHTh system.

Ultrasound and Photoacoustic Imaging of Breast Cancer: Clinical Systems, Challenges, and Future Outlook.

Presently, breast cancer diagnostic methods are dominated by mammography. Although drawbacks of mammography are present including ionizing radiation and patient discomfort, not many alternatives are available. Ultrasound (US) is another method used in the diagnosis of breast cancer, commonly performed on women with dense breasts or in differentiating cysts from solid tumors. Handheld ultrasound (HHUS) and automated breast ultrasound (ABUS) are presently used to generate reflection images which do not contain quantitative information about the tissue. This limitation leads to a subjective interpretation from the sonographer. To rectify the subjective nature of ultrasound, ultrasound tomography (UST) systems have been developed to acquire both reflection and transmission UST (TUST) images. This allows for quantitative assessment of tissue sound speed (SS) and acoustic attenuation which can be used to evaluate the stiffness of the lesions. Another imaging modality being used to detect breast cancer is photoacoustic tomography (PAT). Utilizing much of the same hardware as ultrasound tomography, PAT receives acoustic waves generated from tissue chromophores that are optically excited by a high energy pulsed laser. This allows the user to ideally produce chromophore concentration maps or extract other tissue parameters through spectroscopic PAT. Here, several systems in the area of TUST and PAT are discussed along with their advantages and disadvantages in breast cancer diagnosis. This overview of available systems can provide a landscape of possible intersections and future refinements in cancer diagnosis.

Model-based optical and acoustical compensation for photoacoustic tomography of heterogeneous mediums.

Photoacoustic tomography (PAT) is a non-invasive, high-resolution imaging modality, capable of providing functional and molecular information of various pathologies, such as cancer. One limitation of PAT is the depth and wavelength dependent optical fluence, which results in reduced PA signal amplitude from deeper tissue regions. These factors can therefore introduce errors into quantitative measurements such as oxygen saturation (sO2) or the localization and concentration of various chromophores. The variation in the speed-of-sound between different tissues can also lead to distortions in object location and shape. Compensating for these effects allows PAT to be used more quantitatively. We have developed a proof-of-concept algorithm capable of compensating for the heterogeneity in speed-of-sound and depth dependent optical fluence. Speed-of-sound correction was done by using a straight ray-based algorithm for calculating the family of iso-time-of-flight contours between the transducers and every pixel in the imaging grid, while fluence compensation was done by utilizing the graphics processing unit (GPU) accelerated software MCXCL for Monte Carlo modeling of optical fluence variation. This algorithm was tested on a polyvinyl chloride plastisol (PVCP) phantom, which contained cyst mimics and blood inclusions to test the algorithm under relatively heterogeneous conditions. Our results indicate that our PAT algorithm can compensate for the speed-of-sound variation and depth dependent fluence effects within a heterogeneous phantom. The results of this study will pave the way for further development and evaluation of the proposed method in more complex in-vitro and ex-vivo phantoms, as well as compensating for the wavelength-dependent optical fluence in spectroscopic PAT.

All-reflective ring illumination system for photoacoustic tomography.

Given that breast cancer is the second leading cause of cancer-related deaths among women in the United States, it is necessary to continue improving the sensitivity and specificity of breast imaging systems that diagnose breast lesions. Photoacoustic (PA) imaging can provide functional information during in vivo studies and can augment the structural information provided by ultrasound (US) imaging. A full-ring, all-reflective, illumination system for photoacoustic tomography (PAT) coupled to a full-ring US receiver is developed and tested. The US/PA tomography system utilizes a cone mirror and conical reflectors to optimize light delivery for PAT imaging and has the potential to image objects that are placed within the ring US transducer. The conical reflector used in this system distributes the laser energy over a circular cross-sectional area, thereby reducing the overall fluence. This, in turn, allows the operator to increase the laser energy achieving better cross-sectional penetration depth. A proof-of-concept design utilizing a single cone mirror and a parabolic reflector is used for imaging cylindrical phantoms with light-absorbing objects. For the given phantoms, it has been shown that there was no restriction in imaging a given targeted cross-sectional area irrespective of vertical depth, demonstrating the potential of mirror-based, ring-illuminated PAT system. In addition, the all-reflective ring illumination method shows a uniform PA signal across the scanned cross-sectional area.

Photoacoustic Tomography with a Ring Ultrasound Transducer: A Comparison of Different Illumination Strategies.

Photoacoustic (PA) imaging is a methodology that uses the absorption of short laser pulses by endogenous or exogenous chromophores within human tissue, and the subsequent generation of acoustic waves acquired by an ultrasound (US) transducer, to form an image that can provide functional and molecular information. Amongst the various types of PA imaging, PA tomography (PAT) has been proposed for imaging pathologies such as breast cancer. However, the main challenge for PAT imaging is the deliverance of sufficient light energy horizontally through an imaging cross-section as well as vertically. In this study, three different illumination methods are compared for a full-ring ultrasound (US) PAT system. The three distinct illumination setups are full-ring, diffused-beam, and point source illumination. The full-ring system utilizes a cone mirror and parabolic reflector to create the ringed-shaped beam for PAT, while the diffuse scheme uses a light diffuser to expand the beam, which illuminates tissue-mimicking phantoms. The results indicate that the full-ring illumination is capable of providing a more uniform fluence irrespective of the vertical depth of the imaged cross-section, while the point source and diffused illumination methods provide a higher fluence at regions closer to the point of entry, which diminishes with depth. In addition, a set of experiments was conducted to determine the optimum position of ring-illumination with respect to the position of the acoustic detectors to achieve the highest signal-to-noise ratio.