Ultrasound controlled mechanophore activation in hydrogels for cancer therapy.

Mechanophores are molecular motifs that respond to mechanical perturbance with targeted chemical reactions toward desirable changes in material properties. A large variety of mechanophores have been investigated, with applications focusing on functional materials, such as strain/stress sensors, nanolithography, and self-healing polymers, among others. The responses of engineered mechanophores, such as light emittance, change in fluorescence, and generation of free radicals (FRs), have potential for bioimaging and therapy. However, the biomedical applications of mechanophores are not well explored. Herein, we report an in vitro demonstration of an FR-generating mechanophore embedded in biocompatible hydrogels for noninvasive cancer therapy. Controlled by high-intensity focused ultrasound (HIFU), a clinically proven therapeutic technique, mechanophores were activated with spatiotemporal precision to generate FRs that converted to reactive oxygen species (ROS) to effectively kill tumor cells. The mechanophore hydrogels exhibited no cytotoxicity under physiological conditions. Upon activation with HIFU sonication, the therapeutic efficacies in killing in vitro murine melanoma and breast cancer tumor cells were comparable with lethal doses of H2O2 This process demonstrated the potential for mechanophore-integrated HIFU combination as a noninvasive cancer treatment platform, named "mechanochemical dynamic therapy" (MDT). MDT has two distinct advantages over other noninvasive cancer treatments, such as photodynamic therapy (PDT) and sonodynamic therapy (SDT). 1) MDT is ultrasound based, with larger penetration depth than PDT. 2) MDT does not rely on sonosensitizers or the acoustic cavitation effect, both of which are necessary for SDT. Taking advantage of the strengths of mechanophores and HIFU, MDT can provide noninvasive treatments for diverse cancer types.

Spectral-based Quantitative Ultrasound Imaging Processing Techniques: Comparisons of RF Versus IQ Approaches.

Quantitative ultrasound (QUS) is an imaging technique which includes spectral-based parameterization. Typical spectral-based parameters include the backscatter coefficient (BSC) and attenuation coefficient slope (ACS). Traditionally, spectral-based QUS relies on the radio frequency (RF) signal to calculate the spectral-based parameters. Many clinical and research scanners only provide the in-phase and quadrature (IQ) signal. To acquire the RF data, the common approach is to convert IQ signal back into RF signal via mixing with a carrier frequency. In this study, we hypothesize that the performance, that is, accuracy and precision, of spectral-based parameters calculated directly from IQ data is as good as or better than using converted RF data. To test this hypothesis, estimation of the BSC and ACS using RF and IQ data from software, physical phantoms and in vivo rabbit data were analyzed and compared. The results indicated that there were only small differences in estimates of the BSC between when using the original RF, the IQ derived from the original RF and the RF reconverted from the IQ, that is, root mean square errors (RMSEs) were less than 0.04. Furthermore, the structural similarity index measure (SSIM) was calculated for ACS maps with a value greater than 0.96 for maps created using the original RF, IQ data and reconverted RF. On the other hand, the processing time using the IQ data compared to RF data were substantially less, that is, reduced by more than a factor of two. Therefore, this study confirms two things: (1) there is no need to convert IQ data back to RF data for conducting spectral-based QUS analysis, because the conversion from IQ back into RF data can introduce artifacts. (2) For the implementation of real-time QUS, there is an advantage to convert the original RF data into IQ data to conduct spectral-based QUS analysis because IQ data-based QUS can improve processing speed.

Noninvasive and Spatiotemporal Control of DNAzyme-Based Imaging of Metal Ions In Vivo Using High-Intensity Focused Ultrasound.

Detecting metal ions in vivo with a high spatiotemporal resolution is critical to understanding the roles of the metal ions in both healthy and disease states. Although spatiotemporal controls of metal-ion sensors using light have been demonstrated, the lack of penetration depth in tissue and in vivo has limited their application. To overcome this limitation, we herein report the use of high-intensity focused ultrasound (HIFU) to remotely deliver on-demand, spatiotemporally resolved thermal energy to activate the DNAzyme sensors at the targeted region both in vitro and in vivo. A Zn2+-selective DNAzyme probe is inactivated by a protector strand to block the formation of catalytic enzyme structure, which can then be activated by an HIFU-induced increase in the local temperature. With this design, Zn2+-specific fluorescent resonance energy transfer (FRET) imaging has been demonstrated by the new DNAzyme-HIFU probes in both HeLa cells and mice. The current method can be applied to monitor many other metal ions for in vivo imaging and medical diagnosis using metal-specific DNAzymes that have either been obtained or can be selected using in vitro selection.

High-intensity focused ultrasound-induced mechanochemical transduction in synthetic elastomers.

While study in the field of polymer mechanochemistry has yielded mechanophores that perform various chemical reactions in response to mechanical stimuli, there is not yet a triggering method compatible with biological systems. Applications such as using mechanoluminescence to generate localized photon flux in vivo for optogenetics would greatly benefit from such an approach. Here we introduce a method of triggering mechanophores by using high-intensity focused ultrasound (HIFU) as a remote energy source to drive the spatially and temporally resolved mechanical-to-chemical transduction of mechanoresponsive polymers. A HIFU setup capable of controlling the excitation pressure, spatial location, and duration of exposure is employed to activate mechanochemical reactions in a cross-linked elastomeric polymer in a noninvasive fashion. One reaction is the chromogenic isomerization of a naphthopyran mechanophore embedded in a polydimethylsiloxane (PDMS) network. Under HIFU irradiation evidence of the mechanochemical transduction is the observation of a reversible color change as expected for the isomerization. The elastomer exhibits this distinguishable color change at the focal spot, depending on ultrasonic exposure conditions. A second reaction is the demonstration that HIFU irradiation successfully triggers a luminescent dioxetane, resulting in localized generation of visible blue light at the focal spot. In contrast to conventional stimuli such as UV light, heat, and uniaxial compression/tension testing, HIFU irradiation provides spatiotemporal control of the mechanochemical activation through targeted but noninvasive ultrasonic energy deposition. Targeted, remote light generation is potentially useful in biomedical applications such as optogenetics where a light source is used to trigger a cellular response.

Ultrasonic backscatter coefficient estimation in nonlinear regime using an in situ calibration target.

Tissue characterization based on the backscatter coefficient (BSC) can be degraded by acoustic nonlinearity. Often, this degradation is due to the method used for obtaining a reference spectrum, i.e., using a planar reference in water compared to a reference phantom approach resulted in more degradation. We hypothesize that an in situ calibration approach can improve BSC estimates in the nonlinear regime compared to using the reference phantom approach. The in situ calibration target provides a reference within the medium being interrogated and, therefore, nonlinear effects would already be contained in the in situ reference signal. Simulations and experiments in phantoms and in vivo were performed. A 2 mm diameter titanium bead was embedded in the interrogated media. An L9-4/38 probe (BK Ultrasound, Peabody, MA) and an analysis bandwidth from 4.5 to 7.4 MHz were used in experiments. Radiofrequency data from the sample, bead, and reference phantoms were acquired at a quasi-linear baseline power level and at further increments of output power. Better agreement between the BSC obtained at low power compared to high power was observed for the in situ calibration compared to the reference phantom approach.

Effects of acoustic nonlinearity on communication performance in soft tissues.

Acoustic communication has been gaining traction as an alternative communication method in nontraditional media, such as underwater or through tissue. Acoustic propagation is known to be a nonlinear phenomenon; nonlinear propagation of acoustic waves in soft tissues at biomedical frequencies and intensities has been widely demonstrated. However, the effects of acoustic nonlinearity on communication performance in biological tissues have not yet been examined. In this work, nonlinear propagation of a communication signal in soft tissues is analyzed. The relationship between communication parameters (signal amplitude, bandwidth, and center frequency) and nonlinear distortion of the communication signal propagating in soft tissues with different acoustic properties is investigated. Simulated experiments revealed that, unlike linear channels, bit error rates increase as signal amplitude and bandwidth increase. Linear and decision feedback equalizers fail to address the increased error rates. When tissue properties and transmission parameters can be estimated, receivers based on maximum likelihood sequence estimation approach the performance of an ideal receiver in an ideal additive white Gaussian noise channel.

Effects of acoustic nonlinearity on pulse-echo attenuation coefficient estimation from tissue-mimicking phantoms.

The ultrasonic attenuation coefficient (ACE) can be used to classify tissue state. Pulse-echo spectral-based attenuation estimation techniques, such as the spectral-log-difference method (SLD), account for beam diffraction effects using a reference phantom having a sound speed close to the sound speed of the sample. Methods like SLD assume linear propagation of ultrasound and do not account for potential acoustic nonlinear distortion of the backscattered power spectra in both sample and reference. In this study, the ACE of a sample was computed and compared using the SLD with two independent references (high attenuating and low attenuating phantoms but with similar B/A values) and over several pressure levels. Both numerical and physical tissue-mimicking phantoms were used in the study. The results indicated that the biases in ACE increased when using a reference having low attenuation, whereas the high attenuating reference produced more consistent ACE. Furthermore, increments in ACE vs input pressure were correlated to the log-ratio of Gol'dberg numbers between the sample and reference (R2=0.979 in simulations and R2=0.734 in experiments). Therefore, the results suggest that to reduce bias in ACE using spectral-based methods, both the sound speed and the Gol'dberg number of the reference phantom should be matched to the sample.

Effects of acoustic nonlinearities on the ultrasonic backscatter coefficient estimation.

The backscatter coefficient (BSC) is a fundamental property of tissues and can be used to classify tissues. Two BSC calibration methods are the planar reflector method and the reference phantom method. In both methods, linear acoustic propagation is assumed. In this study, the calibration methods were evaluated when acoustic nonlinear distortion was present. Radio frequency data were acquired from two physical phantoms using a 5 MHz single-element transducer and low power (one excitation level) and high power (six increasing excitation levels) excitation signals. BSCs estimated from the high power settings were compared to the BSCs estimated using the low power by calculating the root mean square error (RMSE). The BSCs were parameterized by fitting the BSC curve to a power law and estimating the power law exponent and by estimating the effective scatterer diameter (ESD). When using the planar reflector method, estimates of the exponent were observed to monotonically increase in value versus increasing excitation level and the ESD decreased with increasing excitation level. The RMSE increased monotonically versus excitation level using the planar reflector method but did not increase using the reference phantom method. The results suggest that the effects of nonlinear distortion are minimized using the reference phantom method.

Effects of the container on structure function with impedance map analysis of dense scattering media.

Quantitative ultrasound (QUS) can be used to estimate acoustic properties of tissue microstructure. In one approach to QUS, the backscatter coefficient (BSC) is utilized to quantify and classify tissue state. From the BSC, parametric models can be constructed to relate the frequency-dependent BSC to geometrical properties of the underlying tissue. However, most of these parametric models are based on analytic expressions (e.g., Gaussian function) and not on actual tissue morphology. Impedance map analysis has been proposed to help identify sources of ultrasonic scattering in tissues and to develop improved models of scattering. Previously, two-dimensional impedance maps (2DZMs) were demonstrated to provide tissue models of three-dimensional (3D) structures for sparse scattering media. In the current study, 2DZMs analysis of dense scatterer media combining the structure function with impedance map analysis was studied through a series of simulations. The simulation analysis demonstrated that the correlation coefficient and power spectrum could be estimated for a dense collection of spheres using 2DZMs. The current finding implies that 2DZMs can capture information about the 3D spatial positions of scatterers in addition to information about the size and shape of the scatterers for a dense scattering media, which is expected to be encountered in many tissues.

Limitations on estimation of effective scatterer diameters.

The influence of spatial diversity in acoustic scattering properties on estimates of the effective scatterer diameter (ESD) applied to soft biological tissues is investigated. This study is based on two-dimensional simulations of scattering media, beginning with random distributions of simple disk structures where all scattering features are known exactly. It concludes with an analysis of histology maps from healthy and fatty rabbit liver. Further, the liver histology is decomposed using an orthonormal basis to separate acoustic scattering at various spatial scales and observe how it influences ESD estimates. Overall, the goal is to quantitatively interpret ESD results for diagnostic assessments despite wide variations in tissue scatterer properties.

Using two-dimensional impedance maps to study weak scattering in sparse random media.

Impedance maps (ZMs) have been proposed as a tool for modeling acoustic properties of tissue microstructure. Three-dimensional (3D) ZMs are constructed from a series of adjacent histological tissue slides that have been stained to emphasize acoustic impedance structures. The power spectrum of a 3DZM can be related to the ultrasound backscatter coefficient, which can be further reduced to a form factor. The goal of this study is to demonstrate the ability to estimate form factors using two-dimensional (2D) ZMs instead of 3DZMs, which have reduced computational and financial cost. The proposed method exploits the properties of isotropic media to estimate the correlation coefficient from slices before estimating the 3D volume power spectrum. Simulated sparse collections of objects were used to study the method by comparing the results obtained using 2DZMs to those predicted by theory. 2DZM analysis was conducted on normal rabbit liver histology and compared to 3DZM analysis of the same histology. The mean percent error between effective scatterer diameter estimates from 2DZMs and 3DZMs of rabbit liver histology was <10% when using three 2DZMs. The results suggest that 2DZMs are a feasible alternative to 3DZMs when estimating form factors for sparse collections of objects.

Quantitative Ultrasound Comparison of MAT and 4T1 Mammary Tumors in Mice and Rats Across Multiple Imaging Systems.

OBJECTIVES: Quantitative ultrasound estimates such as the frequency-dependent backscatter coefficient (BSC) have the potential to enhance noninvasive tissue characterization and to identify tumors better than traditional B-mode imaging. Thus, investigating system independence of BSC estimates from multiple imaging platforms is important for assessing their capabilities to detect tissue differences. METHODS: Mouse and rat mammary tumor models, 4T1 and MAT, respectively, were used in a comparative experiment using 3 imaging systems (Siemens, Ultrasonix, and VisualSonics) with 5 different transducers covering a range of ultrasonic frequencies. RESULTS: Functional analysis of variance of the MAT and 4T1 BSC-versus-frequency curves revealed statistically significant differences between the two tumor types. Variations also were found among results from different transducers, attributable to frequency range effects. At 3 to 8 MHz, tumor BSC functions using different systems showed no differences between tumor type, but at 10 to 20 MHz, there were differences between 4T1 and MAT tumors. Fitting an average spline model to the combined BSC estimates (3-22 MHz) demonstrated that the BSC differences between tumors increased with increasing frequency, with the greatest separation above 15 MHz. Confining the analysis to larger tumors resulted in better discrimination over a wider bandwidth. CONCLUSIONS: Confining the comparison to higher ultrasonic frequencies or larger tumor sizes allowed for separation of BSC-versus-frequency curves from 4T1 and MAT tumors. These constraints ensure that a greater fraction of the backscattered signals originated from within the tumor, thus demonstrating that statistically significant tumor differences were detected.

Enhancing cell kill in vitro from hyperthermia through pre-sensitizing with ultrasound-stimulated microbubbles.

Ultrasound-stimulated microbubbles (MBs) were demonstrated to enhance cell kill from hyperthermia. Definity MBs were injected into wells containing 4T1 cells in culture media and scanned with 1-MHz ultrasound, an exposure duration of 30 s and a negative pressure of 0.5 or 1.3 MPa. Some cell samples were placed in a water bath heated to 42 °C for 5 min. Cell death was quantified. When combining MBs, ultrasound at 1.3 MPa and hyperthermia, more than 58.8% ± 7.21% of cells were nonviable. When exposed to hyperthermia alone or exposure to MBs and ultrasound but no hyperthermia, cell death was less than 10.1% ± 6.96% and 30.1% ± 10.8%, respectively.

Scattering by single physically large and weak scatterers in the beam of a single-element transducer.

Quantitative ultrasound techniques are generally applied to characterize media whose scattering sites are considered to be small compared to a wavelength. In this study, the backscattered response of single weakly scattering spheres and cylinders with diameters comparable to the beam width of a 2.25 MHz single-element transducer were simulated and measured in the transducer focal plane to investigate the impact of physically large scatterers. The responses from large single spherical scatterers at the focus were found to closely match the plane-wave response. The responses from large cylindrical scatterers at the focus were found to differ from the plane-wave response by a factor of f(-1). Normalized spectra from simulations and measurements were in close agreement: the fall-off of the responses as a function of lateral position agreed to within 2 dB for spherical scatterers and to within 3.5 dB for cylindrical scatterers. In both measurement and simulation, single scatterer diameter estimates were biased by less than 3% for a more highly focused transducer compared to estimates for a more weakly focused transducer. The results suggest that quantitative ultrasound techniques may produce physically meaningful size estimates for media whose response is dominated by scatterers comparable in size to the transducer beam.

Backscatter coefficient estimation using tapers with gaps.

When using the backscatter coefficient (BSC) to estimate quantitative ultrasound parameters such as the effective scatterer diameter (ESD) and the effective acoustic concentration (EAC), it is necessary to assume that the interrogated medium contains diffuse scatterers. Structures that invalidate this assumption can affect the estimated BSC parameters in terms of increased bias and variance and decrease performance when classifying disease. In this work, a method was developed to mitigate the effects of echoes from structures that invalidate the assumption of diffuse scattering, while preserving as much signal as possible for obtaining diffuse scatterer property estimates. Backscattered signal sections that contained nondiffuse signals were identified and a windowing technique was used to provide BSC estimates for diffuse echoes only. Experiments from physical phantoms were used to evaluate the effectiveness of the proposed BSC estimation methods. Tradeoffs associated with effective mitigation of specular scatterers and bias and variance introduced into the estimates were quantified. Analysis of the results suggested that discrete prolate spheroidal (PR) tapers with gaps provided the best performance for minimizing BSC error. Specifically, the mean square error for BSC between measured and theoretical had an average value of approximately 1.0 and 0.2 when using a Hanning taper and PR taper respectively, with six gaps. The BSC error due to amplitude bias was smallest for PR (Nω = 1) tapers. The BSC error due to shape bias was smallest for PR (Nω = 4) tapers. These results suggest using different taper types for estimating ESD versus EAC.

Quantitative ultrasound imaging for monitoring in situ high-intensity focused ultrasound exposure.

Quantitative ultrasound (QUS) imaging is hypothesized to map temperature elevations induced in tissue with high spatial and temporal resolution. To test this hypothesis, QUS techniques were examined to monitor high-intensity focused ultrasound (HIFU) exposure of tissue. In situ experiments were conducted on mammary adenocarcinoma tumors grown in rats and lesions were formed using a HIFU system. A thermocouple was inserted into the tumor to provide estimates of temperature at one location. Backscattered time-domain waveforms from the tissue during exposure were recorded using a clinical ultrasonic imaging system. Backscatter coefficients were estimated using a reference phantom technique. Two parameters were estimated from the backscatter coefficient (effective scatterer diameter (ESD) and effective acoustic concentration (EAC). The changes in the average parameters in the regions corresponding to the HIFU focus over time were correlated to the temperature readings from the thermocouple. The changes in the EAC parameter were consistently correlated to temperature during both heating and cooling of the tumors. The changes in the ESD did not have a consistent trend with temperature. The mean ESD and EAC before exposure were 120 ± 16 µm and 32 ± 3 dB/cm3, respectively, and changed to 144 ± 9 µm and 51 ± 7 dB/cm3, respectively, just before the last HIFU pulse was delivered to the tissue. After the tissue cooled down to 37 °C, the mean ESD and EAC were 126 ± 8 µm and 35 ± 4 dB/cm3, respectively. Peak temperature in the range of 50-60 °C was recorded by a thermocouple placed just behind the tumor. These results suggest that QUS techniques have the potential to be used for non-invasive monitoring of HIFU exposure.

Machine-to-Machine Transfer Function in Deep Learning-Based Quantitative Ultrasound.

A transfer function approach was recently demonstrated to mitigate data mismatches at the acquisition level for a single ultrasound scanner in deep learning (DL)-based quantitative ultrasound (QUS). As a natural progression, we further investigate the transfer function approach and introduce a machine-to-machine (M2M) transfer function, which possesses the ability to mitigate data mismatches at a machine level. This ability opens the door to unprecedented opportunities for reducing DL model development costs, enabling the combination of data from multiple sources or scanners, or facilitating the transfer of DL models between machines. We tested the proposed method utilizing a SonixOne machine and a Verasonics machine with an L9-4 array and an L11-5 array. We conducted two types of acquisitions to obtain calibration data: stable and free-hand, using two different calibration phantoms. Without the proposed method, the mean classification accuracy when applying a model on data acquired from one system to data acquired from another system was 50%, and the mean average area under the receiver operator characteristic (ROC) curve (AUC) was 0.405. With the proposed method, mean accuracy increased to 99%, and the AUC rose to the 0.999. Additional observations include the choice of the calibration phantom led to statistically significant changes in the performance of the proposed method. Moreover, robust implementation inspired by Wiener filtering provided an effective method for transferring the domain from one machine to another machine, and it can succeed using just a single calibration view. Lastly, the proposed method proved effective when a different transducer was used in the test machine.

Grating lobe mitigation on large-pitch arrays using null subtraction imaging.

Null Subtraction Imaging (NSI) is a novel beamforming technique that can produce B-mode images resulting in high spatial resolution and low computational cost compared to other beamforming techniques. Previous work has demonstrated that in addition to a beam pattern with a narrow main lobe and low side lobes, NSI can also reduce or mitigate grating lobes, which can appear when the array pitch is larger than one half the wavelength of the transmitted pulse. These grating lobes can result in imaging artifacts that produce clutter and lower contrast. By lowering grating lobe levels, a larger pitch array could be used, which could allow arrays with a larger field of view while maintaining a standard element count. This could have important benefits for specific applications such as ultrasonic abdominal imaging. Experiments were conducted to examine the feasibility of using NSI with large pitch, wide field-of-view arrays. Grating lobe reduction was measured against array pitch, DC offset, and f-number. Experiments were conducted on wire targets and contrast targets in a phantom and results were further verified in vivo by imaging the liver of a rabbit. The results demonstrated that NSI was able to reduce grating lobe brightness by up to 45 dB compared to delay-and-sum (DAS) beamforming when using planewave transmissions, reduce the generalized contrast-to-noise ratio (gCNR) of grating lobe regions from 0.60 to 0.08, and maintain a similar speckle quality to DAS. The gCNR of anechoic regions also improves, increasing from 0.09 to 0.15 on an array with a pitch of 5 wavelengths. Due to significant grating lobe level reduction, NSI shows potential to improve image quality over DAS on a large pitch, wide field-of-view array.

In Vivo Validation of an In Situ Calibration Bead as a Reference for Backscatter Coefficient Calculation.

OBJECTIVE: The study described here was aimed at assessing the capability of quantitative ultrasound (QUS) based on the backscatter coefficient (BSC) for classifying disease states, such as breast cancer response to neoadjuvant chemotherapy and quantification of fatty liver disease. We evaluated the effectiveness of an in situ titanium (Ti) bead as a reference target in calibrating the system and mitigating attenuation and transmission loss effects on BSC estimation. METHODS: Traditional BSC estimation methods require external references for calibration, which do not account for ultrasound attenuation or transmission losses through tissues. To address this issue, we used an in situ Ti bead as a reference target, because it can be used to calibrate the system and mitigate the attenuation and transmission loss effects on estimation of the BSC. The capabilities of the in situ calibration approach were assessed by quantifying consistency of BSC estimates from rabbit mammary tumors (N = 21). Specifically, mammary tumors were grown in rabbits and when a tumor reached ≥1 cm in size, a 2 mm Ti bead was implanted in the tumor as a radiological marker and a calibration source for ultrasound. Three days later, the tumors were scanned with an L-14/5 38 array transducer connected to a SonixOne scanner with and without a slab of pork belly placed on top of the tumors. The pork belly acted as an additional source of attenuation and transmission loss. QUS parameters, specifically effective scatterer diameter (ESD) and effective acoustic concentration (EAC), were calculated using calibration spectra from both an external reference phantom and the Ti bead. RESULTS: For ESD estimation, the 95% confidence interval between measurements with and without the pork belly layer was 6.0, 27.4 using the in situ bead and 114, 135.1 with the external reference phantom. For EAC estimation, the 95% confidence intervals were -8.1, 0.5 for the bead and -41.5, -32.2 for the phantom. These results indicate that the in situ bead method has reduced bias in QUS estimates because of intervening tissue losses. CONCLUSION: The use of an in situ Ti bead as a radiological marker not only serves its traditional role but also effectively acts as a calibration target for QUS methods. This approach accounts for attenuation and transmission losses in tissue, resulting in more accurate QUS estimates and offering a promising method for enhanced disease state classification in clinical settings.

In vivo validation of an in situ calibration bead as a reference for backscatter coefficient calculation.

Objectives: The study aims to assess the capability of Quantitative Ultrasound (QUS) based on the backscatter coefficient (BSC) for classifying disease states, such as breast cancer response to neoadjuvant chemotherapy and quantifying fatty liver disease. We evaluate the effectiveness of an in situ titanium (Ti) bead as a reference target in calibrating the system and mitigating attenuation and transmission loss effects on BSC estimation. Methods: Traditional BSC estimation methods require external references for calibration, which do not account for ultrasound attenuation or transmission losses through tissues. To address this issue, we use an in situ titanium (Ti) bead as a reference target, because it can be used to calibrate the system and mitigate the attenuation and transmission loss effects on estimation of the BSC. The capabilities of the in situ calibration approach were assessed by quantifying consistency of BSC estimates from rabbit mammary tumors (N=21). Specifically, mammary tumors were grown in rabbits and when a tumor reached 1 cm or greater in size, a 2-mm Ti bead was implanted into the tumor as a radiological marker and a calibration source for ultrasound. Three days later, the tumors were scanned with a L-14/5 38 array transducer connected to a SonixOne scanner with and without a slab of pork belly placed on top of the tumors. The pork belly acted as an additional source of attenuation and transmission loss. QUS parameters, specifically effective scatterer diameter (ESD) and effective acoustic concentration (EAC), were calculated using calibration spectra from both an external reference phantom and the Ti bead. Results: For ESD estimation, the 95% confidence interval between measurements with and without the pork belly layer was (6.0,27.4) using the in situ bead and (114, 135.1) with the external reference phantom. For EAC estimation, the 95% confidence interval were (-8.1, 0.5) for the bead and (-41.5, -32.2) for the phantom. These results indicate that the in situ bead method shows reduced bias in QUS estimates due to intervening tissue losses. Conclusions: The use of an in situ Ti bead as a radiological marker not only serves its traditional role but also effectively acts as a calibration target for QUS methods. This approach accounts for attenuation and transmission losses in tissue, resulting in more accurate QUS estimates and offering a promising method for enhanced disease state classification in clinical settings.