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Introduction: The HER2 + tumor immune microenvironment is composed of macrophages, natural killer cells, and tumor infiltrating lymphocytes, which produce pro-inflammatory cytokines. Determining the effect of T-cells on HER2 + cancer cells during therapy could guide immunogenic therapies that trigger antibody-dependent cellular cytotoxicity. This study utilized longitudinal in vitro time-resolved microscopy to measure T-cell influence on trastuzumab in HER2 + breast cancer., Methods: Fluorescently-labeled breast cancer cells (BT474, SKBR3, MDA-MB-453, and MDA-MB-231) were co-cultured with CD4 + T-cells (Jurkat cell line) and longitudinally imaged to quantify cancer cell viability when treated with or without trastuzumab (10, 25, 50 and 100 μg/mL). The presence and timing of T-cell co-culturing was manipulated to determine immune stimulation of trastuzumab-treated HER2 + breast cancer. HER2 and TNF-α expression were evaluated with western blot and ELISA, respectively. Significance was calculated using a two-tailed parametric t-test., Results: The viability of HER2 + cancer cells significantly decreased when exposed to 25 μg/mL trastuzumab and T-cells, compared to cancer cells exposed to trastuzumab without T-cells (p = 0.01). The presence of T-cells significantly increased TNF-α expression in trastuzumab-treated cancer cells (p = 0.02). Conversely, cancer cells treated with TNF-α and trastuzumab had a similar decrease in viability as trastuzumab-treated cancer cells co-cultured with T-cells (p = 0.32)., Conclusions: The presence of T-cells significantly increases the efficacy of targeted therapies and suggests trastuzumab may trigger immune mediated cytotoxicity. Increased TNF-α receptor expression suggest cytokines may interact with trastuzumab to create a state of enhanced response to therapy in HER2 + breast cancer, which has potential to reducing tumor burden.

The use of biomarkers is integral to the routine management of cancer patients, including diagnosis of disease, clinical staging and response to therapeutic intervention. Advanced imaging metrics with computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET) are used to assess response during new drug development and in cancer research for predictive metrics of response. Key components and challenges to identifying an appropriate imaging biomarker are selection of integral vs integrated biomarkers, choosing an appropriate endpoint and modality, and standardization of the imaging biomarkers for cooperative and multicenter trials. Imaging biomarkers lean on the original proposed quantified metrics derived from imaging such as tumor size or longest dimension, with the most commonly implemented metrics in clinical trials coming from the Response Evaluation Criteria in Solid Tumors (RECIST) criteria, and then adapted versions such as immune-RECIST (iRECIST) and Positron Emission Tomography Response Criteria in Solid Tumors (PERCIST) for immunotherapy response and PET imaging, respectively. There have been many widely adopted biomarkers in clinical trials derived from MRI including metrics that describe cellularity and vascularity from diffusion-weighted (DW)-MRI apparent diffusion coefficient (ADC) and Dynamic Susceptibility Contrast (DSC) or dynamic contrast enhanced (DCE)-MRI (K trans , relative cerebral blood volume (rCBV)), respectively. Furthermore, Fluorodexoyglucose (FDG), fluorothymidine (FLT), and fluoromisonidazole (FMISO)-PET imaging, which describe molecular markers of glucose metabolism, proliferation and hypoxia have been implemented into various cancer types to assess therapeutic response to a wide variety of targeted- and chemotherapies. Recently, there have been many functional and molecular novel imaging biomarkers that are being developed that are rapidly being integrated into clinical trials (with anticipation of being implemented into clinical workflow in the future), such as artificial intelligence (AI) and machine learning computational strategies, antibody and peptide specific molecular imaging, and advanced diffusion MRI. These include prostate-specific membrane antigen (PSMA) and trastuzumab-PET, vascular tumor burden extracted from contrast-enhanced CT, diffusion kurtosis imaging, and CD8 or Granzyme B PET imaging. Further excitement surrounds theranostic procedures such as the combination of 68 Ga/ 111 In- and 177 Lu-DOTATATE to use integral biomarkers to direct care and personalize therapy. However, there are many challenges in the implementation of imaging biomarkers that remains, including understand the accuracy, repeatability and reproducibility of both acquisition and analysis of these imaging biomarkers. Despite the challenges associated with the biological and technical validation of novel imaging biomarkers, a distinct roadmap has been created that is being implemented into many clinical trials to advance the development and implementation to create specific and sensitive novel imaging biomarkers of therapeutic response to continue to transform medical oncology., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Therapy targeted to the human epidermal growth factor receptor type 2 (HER2) is used in combination with cytotoxic therapy in treatment of HER2+ breast cancer. Trastuzumab, a monoclonal antibody that targets HER2, has been shown pre-clinically to induce vascular changes that can increase delivery of chemotherapy. To quantify the role of immune modulation in treatment-induced vascular changes, this study identifies temporal changes in myeloid cell infiltration with corresponding vascular alterations in a preclinical model of HER2+ breast cancer following trastuzumab treatment., Methods: HER2+ tumor-bearing mice (N = 46) were treated with trastuzumab or saline. After extraction, half of each tumor was analyzed by immunophenotyping using flow cytometry. The other half was quantified by immunohistochemistry to characterize macrophage infiltration (F4/80), vascularity (CD31 and α-SMA), proliferation (Ki67) and cellularity (H&E). Additional mice (N = 10) were used to quantify differences in tumor cytokines between control and treated groups., Results: Immunophenotyping showed an increase in macrophage infiltration 24 h after trastuzumab treatment (P ≤ 0.05). With continued trastuzumab treatment, the M1 macrophage population increased (P = 0.02). Increases in vessel maturation index (i.e., the ratio of α-SMA to CD31) positively correlated with increases in tumor infiltrating M1 macrophages (R = 0.33, P = 0.04). Decreases in VEGF-A and increases in inflammatory cytokines (TNF-α, IL-1β, CCL21, CCL7, and CXCL10) were observed with continued trastuzumab treatment (P ≤ 0.05)., Conclusions: Preliminary results from this study in a murine model of HER2+ breast cancer show correlations between immune modulation and vascular changes, and reveals the potential for anti-HER2 therapy to reprogram immunosuppressive components of the tumor microenvironment. The quantification of immune modulation in HER2+ breast cancer, as well as the mechanistic insight of vascular alterations after anti-HER2 treatment, represent novel contributions and warrant further assessment for potential clinical translation.

The goal of this study is to experimentally and computationally investigate combination trastuzumab-paclitaxel therapies and identify potential synergistic effects due to sequencing of the therapies with in vitro imaging and mathematical modeling. Longitudinal alterations in cell confluence are reported for an in vitro model of BT474 HER2+ breast cancer cells following various dosages and timings of paclitaxel and trastuzumab combination regimens. Results of combination drug regimens are evaluated for drug interaction relationships based on order, timing, and quantity of dose of the drugs. Altering the order of treatments, with the same total therapeutic dose, provided significant changes in overall cell confluence (p < 0.001). Two mathematical models are introduced that are constrained by the in vitro data to simulate the tumor cell response to the individual therapies. A collective model merging the two individual drug response models was designed to investigate the potential mechanisms of synergy for paclitaxel-trastuzumab combinations. This collective model shows increased synergy for regimens where trastuzumab is administered prior to paclitaxel and suggests trastuzumab accelerates the cytotoxic effects of paclitaxel. The synergy derived from the model is found to be in agreement with the combination index, where both indicate a spectrum of additive and synergistic interactions between the two drugs dependent on their dose order. The combined in vitro results and development of a mathematical model of drug synergy has potential to evaluate and improve standard-of-care combination therapies in cancer.

The purpose of this study is to investigate imaging and histology-based measurements of intratumoral heterogeneity to evaluate early treatment response to targeted therapy in a murine model of HER2+ breast cancer. BT474 tumor-bearing mice (N = 30) were treated with trastuzumab or saline and imaged longitudinally with either dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) or 18 F-fluoromisonidazole (FMISO) positron emission tomography (PET). At the imaging study end point (day 4 for MRI or 7 for PET), each tumor was excised for immunohistochemistry analysis. Voxel-based histogram analysis was performed on imaging-derived parametric maps (i.e., K trans and v e from DCE-MRI, SUV from 18 F-FMISO-PET) of the tumor region of interest to measure heterogeneity. Image processing and histogram analysis of whole tumor slice immunohistochemistry data were performed to validate the in vivo imaging findings. Trastuzumab-treated tumors had increased heterogeneity in quantitative imaging measures of cellularity (v e ), with a mean Kolmogorov-Smirnov (K-S) distance of 0.32 (P = .05) between baseline and end point distributions. Trastuzumab-treated tumors had increased vascular heterogeneity (K trans ) and decreased hypoxic heterogeneity (SUV), with a mean K-S distance of 0.42 (P < .01) and 0.46 (P = .047), respectively, between baseline and study end points. These observations were validated by whole-slice immunohistochemistry analysis with mean interquartile range of CD31 distributions of 1.72 for treated and 0.95 for control groups (P = .02). Quantitative longitudinal changes in tumor cellular and vascular heterogeneity in response to therapy may provide evidence for early prediction of response and guide therapy for patients with HER2+ breast cancer., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: To discuss standard-of-care and emerging imaging techniques employed for screening and detection, diagnosis and staging, monitoring response to therapy, and guiding cancer treatments., Data Sources: Published journal articles indexed in the National Library of Medicine database and relevant websites., Conclusion: Imaging plays a fundamental role in the care of cancer patients and specifically, breast cancer patients in the neoadjuvant setting, providing an excellent opportunity for interprofessional collaboration between oncologists, researchers, radiologists, and oncology nurses. Quantitative imaging strategies to assess cellular, molecular, and vascular characteristics within the tumor is needed to better evaluate initial diagnosis and treatment response., Implications for Nursing Practice: Nurses caring for patients in all settings must continue to seek education on emerging imaging techniques. Oncology nurses provide education about the test, ensure the patient has appropriate pre-testing instructions, and manage patient expectations about timing of results availability., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Conventional methods for histopathologic tissue diagnosis are labor- and time-intensive and can delay decision-making during diagnostic and therapeutic procedures. We report the development of an automated and biocompatible handheld mass spectrometry device for rapid and nondestructive diagnosis of human cancer tissues. The device, named MasSpec Pen, enables controlled and automated delivery of a discrete water droplet to a tissue surface for efficient extraction of biomolecules. We used the MasSpec Pen for ex vivo molecular analysis of 20 human cancer thin tissue sections and 253 human patient tissue samples including normal and cancerous tissues from breast, lung, thyroid, and ovary. The mass spectra obtained presented rich molecular profiles characterized by a variety of potential cancer biomarkers identified as metabolites, lipids, and proteins. Statistical classifiers built from the histologically validated molecular database allowed cancer prediction with high sensitivity (96.4%), specificity (96.2%), and overall accuracy (96.3%), as well as prediction of benign and malignant thyroid tumors and different histologic subtypes of lung cancer. Notably, our classifier allowed accurate diagnosis of cancer in marginal tumor regions presenting mixed histologic composition. Last, we demonstrate that the MasSpec Pen is suited for in vivo cancer diagnosis during surgery performed in tumor-bearing mouse models, without causing any observable tissue harm or stress to the animal. Our results provide evidence that the MasSpec Pen could potentially be used as a clinical and intraoperative technology for ex vivo and in vivo cancer diagnosis., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Purpose: Evaluation of [ 18 F]fluoromisonidazole ([ 18 F]FMISO)-positron emission tomography (PET) imaging as a metric for evaluating early response to trastuzumab therapy with histological validation in a murine model of HER2+ breast cancer., Procedures: Mice with BT474, HER2+ tumors, were imaged with [ 18 F]FMISO-PET during trastuzumab therapy. Pimonidazole staining was used to confirm hypoxia from imaging., Results: [ 18 F]FMISO-PET indicated significant decreases in hypoxia beginning on day 3 (P < 0.01) prior to changes in tumor size. These results were confirmed with pimonidazole staining on day 7 (P < 0.01); additionally, there was a significant positive linear correlation between histology and PET imaging (r 2  = 0.85)., Conclusions: [ 18 F]FMISO-PET is a clinically relevant modality which provides the opportunity to (1) predict response to HER2+ therapy before changes in tumor size and (2) identify decreases in hypoxia which has the potential to guide subsequent therapy.

To employ in vivo imaging and histological techniques to identify and quantify vascular changes early in the course of treatment with trastuzumab in a murine model of HER2+ breast cancer. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was used to quantitatively characterize vessel perfusion/permeability (via the parameter K (trans) ) and the extravascular extracellular volume fraction (v e ) in the BT474 mouse model of HER2+ breast cancer (N = 20) at baseline, day one, and day four following trastuzumab treatment (10 mg/kg). Additional cohorts of mice were used to quantify proliferation (Ki67), microvessel density (CD31), pericyte coverage (α-SMA) by immunohistochemistry (N = 44), and to quantify human VEGF-A expression (N = 29) throughout the course of therapy. Longitudinal assessment of combination doxorubicin ± trastuzumab (N = 42) tested the hypothesis that prior treatment with trastuzumab will increase the efficacy of subsequent doxorubicin therapy. Compared to control tumors, trastuzumab-treated tumors exhibited a significant increase in K (trans) (P = 0.035) on day four, indicating increased perfusion and/or vessel permeability and a simultaneous significant increase in v e (P = 0.01), indicating increased cell death. Immunohistochemical and ELISA analyses revealed that by day four the trastuzumab-treated tumors had a significant increase in vessel maturation index (i.e., the ratio of α-SMA to CD31 staining) compared to controls (P < 0.001) and a significant decrease in VEGF-A (P = 0.03). Additionally, trastuzumab dosing prior to doxorubicin improved the overall effectiveness of the therapies (P < 0.001). This study identifies and validates improved perfusion characteristics following trastuzumab therapy, resulting in an improvement in trastuzumab-doxorubicin combination therapy in a murine model of HER2+ breast cancer. This data suggests properties of vessel maturation. In particular, the use of DCE-MRI, a clinically available imaging method, following treatment with trastuzumab may provide an opportunity to optimize the scheduling and improve delivery of subsequent cytotoxic therapy.

Microbubbles triggered with localized ultrasound (US) can improve tumor drug delivery and retention. Termed US-stimulated drug delivery, this strategy was applied to head and neck cancer (HNC) in a post-surgical tumor resection model. Luciferase-positive HNC squamous cell carcinoma (SCC) was implanted in the flanks of nude athymic mice (N = 24) that underwent various degrees of surgical tumor resection (0%, 50% or 100%). After surgery, animals received adjuvant therapy with cetuximab-IRDye alone, or cetuximab-IRDye in combination with US-stimulated drug delivery or saline injections (control) on days 4, 7 and 10. Tumor drug delivery was assessed on days 0, 4, 7, 10, 14 and 17 with an in vivo fluorescence imaging system, and tumor viability was evaluated at the same times with in vivo bioluminescence imaging. Tumor caliper measurements occurred two times per week for 24 d. Optical imaging revealed that in the 50% tumor resection group, US-stimulated drug delivery resulted in a significant increase in cetuximab delivery compared with administration of drug alone on day 10 (day of peak fluorescence) (p = 0.03). Tumor viability decreased in all groups that received cetuximab-IRDye in combination with US-stimulated drug delivery, compared with the group that received only the drug. After various degrees of surgical resection, this novel study reports positive improvements in drug uptake in the residual cancer cells when drug delivery is stimulated with US., (Copyright © 2014 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Improving the efficiency of adenovirus (Ad) delivery to target tissues has the potential to advance the translation of cancer gene therapy. Ultrasound (US)-stimulated therapy uses microbubbles (MBs) exposed to low-intensity US energy to improve localized delivery. We hypothesize that US-stimulated gene therapy can improve Ad infection in a primary prostate tumor through enhanced tumor uptake and retention of the Ad vector. In vitro studies were performed to analyze the degree of Ad infectivity after application of US-stimulated gene therapy. A luciferase-based Ad on a ubiquitous cytomegalovirus (CMV) promoter (Ad5/3-CMV-Luc) was used in an animal model of prostate cancer (bilateral tumor growth) to evaluate Ad transduction efficiency after US-stimulated therapy. Bioluminescence imaging was employed for in vivo analysis to quantify Ad infection within the tumor. In vitro studies revealed no difference in Ad transduction between groups receiving US-stimulated therapy using high, low or sham US intensity exposures at various multiplicities of infection (MOIs) (p = 0.80). In vivo results indicated that tumors receiving US-stimulated therapy after intra-tumoral injection of Ad5/3-CMV-Luc (1 × 10(6) plaque-forming units) exhibited a 95.1% enhancement in tumor delivery compared with control tumors receiving sham US (p = 0.03). US-stimulated therapy has significant potential to immediately affect Ad-based cancer gene therapy by improving virus bioavailability in target tissues., (Copyright © 2013 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)