Physicochemical Properties and Route of Systemic Delivery Control the In Vivo Dynamics and Breakdown of Radiolabeled Gold Nanostars.

The in vivo dynamics of nanoparticles requires a mechanistic understanding of multiple factors. Here, for the first time, the surprising breakdown of functionalized gold nanostars (F-AuNSs) conjugated with antibodies and 64 Cu radiolabels in vivo and in artificial lysosomal fluid ex vivo, is shown. The short-term biodistribution of F-AuNSs is driven by the route of systemic delivery (intravenous vs intraperitoneal) and long-term fate is controlled by the tissue type in vivo. In vitro studies including endocytosis pathways, intracellular trafficking, and opsonization, are combined with in vivo studies integrating a milieu of spectroscopy and microcopy techniques that show F-AuNSs dynamics is driven by their physicochemical properties and route of delivery. F-AuNSs break down into sub-20 nm broken nanoparticles as early as 7 days postinjection. Martini coarse-grained simulations are performed to support the in vivo findings. Simulations suggest that shape, size, and charge of the broken nanoparticles, and composition of the lipid membrane depicting various tissues govern the interaction of the nanoparticles with the membrane, and the rate of translocation across the membrane to ultimately enable tissue clearance. The fundamental study addresses critical gaps in the knowledge regarding the fate of nanoparticles in vivo that remain a bottleneck in their clinical translation.

Extracellular vesicles: mediators of intercellular communication in tissue injury and disease.

Intercellular communication is a critical process that ensures cooperation between distinct cell types and maintains homeostasis. EVs, which were initially described as cellular debris and devoid of biological function, are now recognized as key components in cell-cell communication. EVs are known to carry multiple factors derived from their cell of origin, including cytokines and chemokines, active enzymes, metabolites, nucleic acids, and surface molecules, that can alter the behavior of recipient cells. Since the cargo of EVs reflects their parental cells, EVs from damaged and dysfunctional tissue environments offer an abundance of information toward elucidating the molecular mechanisms of various diseases and pathological conditions. In this review, we discuss the most recent findings regarding the role of EVs in the progression of cancer, metabolic disorders, and inflammatory lung diseases given the high prevalence of these conditions worldwide and the important role that intercellular communication between immune, parenchymal, and stromal cells plays in the development of these pathological states. We also consider the clinical applications of EVs, including the possibilities for their use as novel therapeutics. While intercellular communication through extracellular vesicles (EVs) is key for physiological processes and tissue homeostasis, injury and stress result in altered communication patterns in the tissue microenvironment. When left unchecked, EV-mediated interactions between stromal, immune, and parenchymal cells lead to the development of disease states Video Abstract.

Effects of radiation on metastasis and tumor cell migration.

It is well known that tumor cells migrate from the primary lesion to distant sites to form metastases and that these lesions limit patient outcome in a majority of cases. However, the extent to which radiation influences this process and to which migration in turn alters radiation response remains controversial. There are preclinical and clinical reports showing that focal radiotherapy can both increase the development of distant metastasis, as well as that it can induce the regression of established metastases through the abscopal effect. More recently, preclinical studies have suggested that radiation can attract migrating tumor cells and may, thereby, facilitate tumor recurrence. In this review, we summarize these phenomena and their potential mechanisms of action, and evaluate their significance for modern radiation therapy strategies.

Mammary tissue-derived extracellular matrix hydrogels reveal the role of irradiation in driving a pro-tumor and immunosuppressive microenvironment.

Radiation therapy (RT) is essential for triple negative breast cancer (TNBC) treatment. However, patients with TNBC continue to experience recurrence after RT. The role of the extracellular matrix (ECM) of irradiated breast tissue in tumor recurrence is still unknown. In this study, we evaluated the structure, molecular composition, and mechanical properties of irradiated murine mammary fat pads (MFPs) and developed ECM hydrogels from decellularized tissues (dECM) to assess the effects of RT-induced ECM changes on breast cancer cell behavior. Irradiated MFPs were characterized by increased ECM deposition and fiber density compared to unirradiated controls, which may provide a platform for cell invasion and proliferation. ECM component changes in collagens I, IV, and VI, and fibronectin were observed following irradiation in both MFPs and dECM hydrogels. Encapsulated TNBC cell proliferation and invasive capacity was enhanced in irradiated dECM hydrogels. In addition, TNBC cells co-cultured with macrophages in irradiated dECM hydrogels induced M2 macrophage polarization and exhibited further increases in proliferation. Our study establishes that the ECM in radiation-damaged sites promotes TNBC invasion and proliferation as well as an immunosuppressive microenvironment. This work represents an important step toward elucidating how changes in the ECM after RT contribute to breast cancer recurrence.

miR-100 and miR-125b Contribute to Enhanced 3D Growth and Invasiveness and can be Functionally Transferred to Silence Target Genes in Recipient Cells.

Extracellular communication via the transfer of vesicles and nanoparticles is now recognized to play an important role in tumor microenvironment interactions. Cancer cells upregulate and secrete abundant levels of miR-100 and miR-125b that can alter gene expression by both cell- and non-cell-autonomous mechanisms. We previously showed that these miRNAs activate Wnt signaling in colorectal cancer (CRC) through noncanonical pairing with 5 negative regulators of Wnt signaling. To identify additional targets of miR-100 and miR-125b , we used bioinformatic approaches comparing multiple CRC cell lines, including knockout lines lacking one or both of these miRNAs. From an initial list of 96 potential mRNA targets, we tested 15 targets with 8 showing significant downregulation in the presence of miR-100 and miR-125b . Among these, Cingulin (CGN) and Protein tyrosine phosphatase receptor type-R (PTPRR) are downregulated in multiple cancers, consistent with regulation by increased levels of miR-100 and miR-125b. We also show that increased cellular levels of miR-100 and miR-125b enhance 3D growth and invasiveness in CRC and glioblastoma cell lines. Lastly, we demonstrate that extracellular transfer of miR-100 and miR-125b can silence both reporter and endogenous mRNA targets in recipient cells and also increase the invasiveness of recipient spheroid colonies when grown under 3D conditions in type I collagen.

Technical note: Noninvasive monitoring of normal tissue radiation damage using spectral quantitative ultrasound spectroscopy.

BACKGROUND: While radiation therapy (RT) is a critical component of breast cancer therapy and is known to decrease overall local recurrence rates, recent studies have shown that normal tissue radiation damage may increase recurrence risk. Fibrosis is a well-known consequence of RT, but the specific sequence of molecular and mechanical changes induced by RT remains poorly understood. PURPOSE: To improve cancer therapy outcomes, there is a need to understand the role of the irradiated tissue microenvironment in tumor recurrence. This study seeks to evaluate the use of spectral quantitative ultrasound (spectral QUS) for real time determination of the normal tissue characteristic radiation response and to correlate these results to molecular features in irradiated tissues. METHODS: Murine mammary fat pads (MFPs) were irradiated to 20 Gy, and spectral QUS was used to analyze tissue physical properties pre-irradiation as well as at 1, 5, and 10 days post-irradiation. Tissues were processed for scanning electron microscopy imaging as well as histological and immunohistochemical staining to evaluate morphology and structure. RESULTS: Tissue morphological and structural changes were observed non-invasively following radiation using mid-band fit (MBF), spectral slope (SS), and spectral intercept (SI) measurements obtained from spectral QUS. Statistically significant shifts in MBF and SI indicate structural tissue changes in real time, which matched histological observations. Radiation damage was indicated by increased adipose tissue density and extracellular matrix (ECM) deposition. CONCLUSIONS: Our findings demonstrate the potential of using spectral QUS to noninvasively evaluate normal tissue changes resulting from radiation damage. This supports further pre-clinical studies to determine how the tissue microenvironment and physical properties change in response to therapy, which may be important for improving treatment strategies.

Development of an alginate-Matrigel hydrogel system to evaluate cancer cell behavior in the stiffness range of the bone marrow.

Bone metastasis is highly prevalent in breast cancer patients with metastatic disease. These metastatic cells may eventually form osteolytic lesions and affect the integrity of the bone, causing pathological fractures and impairing patient quality of life. Although some mechanisms have been determined in the metastatic cascade to the bone, little is known about how the mechanical cues of the bone marrow microenvironment influence tumor cell growth and invasion once they have homed to the secondary site. The mechanical properties within the bone marrow range from 0.5 kPa in the sinusoidal region to 40 kPa in the endosteal region. Here, we report an alginate-Matrigel hydrogel that can be modulated to the stiffness range of the bone marrow and used to evaluate tumor cell behavior. We fabricated alginate-Matrigel hydrogels with varying calcium sulfate (CaSO4) concentrations to tune stiffness, and we demonstrated that these hydrogels recapitulated the mechanical properties observed in the bone marrow microenvironment (0.7-16 kPa). We encapsulated multiple breast cancer cell lines into these hydrogels to assess growth and invasion. Tumor cells in stiffer hydrogels exhibited increased proliferation and enhanced elongation compared to lower stiffness hydrogels, which suggests that stiffer environments in the bone marrow promote cellular invasive capacity. This work establishes a system that replicates bone marrow mechanical properties to elucidate the physical factors that contribute to metastatic growth.

Irradiated Mammary Spheroids Elucidate Mechanisms of Macrophage-Mediated Breast Cancer Recurrence.

Introduction: While most patients with triple negative breast cancer receive radiation therapy to improve outcomes, a significant subset of patients continue to experience recurrence. Macrophage infiltration into radiation-damaged sites has been shown to promote breast cancer recurrence in pre-clinical models. However, the mechanisms that drive recurrence are unknown. Here, we developed a novel spheroid model to evaluate macrophage-mediated tumor cell recruitment. Methods: We characterized infiltrating macrophage phenotypes into irradiated mouse mammary tissue via flow cytometry. We then engineered a spheroid model of radiation damage with primary fibroblasts, macrophages, and 4T1 mouse mammary carcinoma cells using in vivo macrophage infiltration results to inform our model. We analyzed 4T1 infiltration into spheroids when co-cultured with biologically relevant ratios of pro-healing M2:pro-inflammatory M1 macrophages. Finally, we quantified interleukin 6 (IL-6) secretion associated with conditions favorable to tumor cell infiltration, and we directly evaluated the impact of IL-6 on tumor cell invasiveness in vitro and in vivo. Results: In our in vivo model, we observed a significant increase in M2 macrophages in mouse mammary glands 10 days post-irradiation. We determined that tumor cell motility toward irradiated spheroids was enhanced in the presence of a 2:1 ratio of M2:M1 macrophages. We also measured a significant increase in IL-6 secretion after irradiation both in vivo and in our model. This secretion increased tumor cell invasiveness, and tumor cell invasion and recruitment were mitigated by neutralizing IL-6. Conclusions: Our work suggests that interactions between infiltrating macrophages and damaged stromal cells facilitate breast cancer recurrence through IL-6 signaling. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00775-x.

Ultrasound Imaging Enables Longitudinal Tracking of Vascular Changes that Correlate with Immune Cell Infiltration After Radiotherapy.

While immunotherapy shows great promise in patients with triple negative breast cancer, many will not respond to treatment, and predicting response is made difficult by significant tumor heterogeneity. Non-invasive imaging of the tumor vasculature enables the monitoring of treatment and has potential to aid in predicting therapeutic response. Here, we use ultrafast power doppler ultrasound (US) to track longitudinal changes in the vascular response to radiotherapy in two breast cancer models to correlate vascular and immune changes in the tumor microenvironment. Tumor volume and vascular index were calculated to evaluate the effects of radiation using US imaging. US tumor measurements and the quantified vascular response to radiation were confirmed with caliper measurements and immunohistochemistry observations, respectively, demonstrating a proof-of-principle method for non-invasive vascular monitoring. Additionally, we found significant infiltration of CD8+ T cells into irradiated tumors 10 days after radiation, which followed a sustained decline in vascular index that was first observed 1 day post-radiation. Taken together, our findings reveal the potential for ultrafast power doppler US to evaluate changes in tumor vasculature that may be indicative of the tumor-immune microenvironment and ultimately improve patient outcomes by predicting response to immunotherapy.

Multifunctional magnetoliposomes as drug delivery vehicles for the potential treatment of Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease. Therefore, development of novel technologies and strategies to treat PD is a global health priority. Current treatments include administration of Levodopa, monoamine oxidase inhibitors, catechol-O-methyltransferase inhibitors, and anticholinergic drugs. However, the effective release of these molecules, due to the limited bioavailability, is a major challenge for the treatment of PD. As a strategy to solve this challenge, in this study we developed a novel multifunctional magnetic and redox-stimuli responsive drug delivery system, based on the magnetite nanoparticles functionalized with the high-performance translocating protein OmpA and encapsulated into soy lecithin liposomes. The obtained multifunctional magnetoliposomes (MLPs) were tested in neuroblastoma, glioblastoma, primary human and rat astrocytes, blood brain barrier rat endothelial cells, primary mouse microvascular endothelial cells, and in a PD-induced cellular model. MLPs demonstrated excellent performance in biocompatibility assays, including hemocompatibility (hemolysis percentages below 1%), platelet aggregation, cytocompatibility (cell viability above 80% in all tested cell lines), mitochondrial membrane potential (non-observed alterations) and intracellular ROS production (negligible impact compared to controls). Additionally, the nanovehicles showed acceptable cell internalization (covered area close to 100% at 30 min and 4 h) and endosomal escape abilities (significant decrease in lysosomal colocalization after 4 h of exposure). Moreover, molecular dynamics simulations were employed to better understand the underlying translocating mechanism of the OmpA protein, showing key findings regarding specific interactions with phospholipids. Overall, the versatility and the notable in vitro performance of this novel nanovehicle make it a suitable and promising drug delivery technology for the potential treatment of PD.

Nanoengineering the heart: conductive scaffolds enhance connexin 43 expression.

Scaffolds that couple electrical and elastic properties may be valuable for cardiac cell function. However, existing conductive materials do not mimic physiological properties. We prepared and characterized a tunable, hybrid hydrogel scaffold based on Au nanoparticles homogeneously synthesized throughout a polymer templated gel. Conductive gels had Young's moduli more similar to myocardium relative to polyaniline and polypyrrole, by 1-4 orders of magnitude. Neonatal rat cardiomyocytes exhibited increased expression of connexin 43 on hybrid scaffolds relative to HEMA with or without electrical stimulation.

Cross-linked, heterogeneous colloidosomes exhibit pH-induced morphogenesis.

Inspired by morphogenesis in biology, we present a strategy for developing functional 3D materials with the capacity to morph based on environmental cues. We utilized local mechanical stresses to cause global shape changes in colloidosomes. Colloidosomes were assembled from pH-sensitive calcium alginate particles (CAPs) with high and low swelling ratios. Colloidosomes were subsequently cross-linked via diamine compounds with varying carbon chain lengths. New colloidosome isoforms were generated from heterogeneous mixtures of CAPs, which resulted in nonuniform stresses. Our study demonstrated that coordinated networks of heterogeneous subunits may be used to design programmable materials.

Hypoxia inducible factor signaling in breast tumors controls spontaneous tumor dissemination in a site-specific manner.

Hypoxia is a common feature in tumors and induces signaling that promotes tumor cell survival, invasion, and metastasis, but the impact of hypoxia inducible factor (HIF) signaling in the primary tumor on dissemination to bone in particular remains unclear. To better understand the contributions of hypoxia inducible factor 1 alpha (HIF1α), HIF2α, and general HIF pathway activation in metastasis, we employ a PyMT-driven spontaneous murine mammary carcinoma model with mammary specific deletion of Hif1α, Hif2α, or von Hippel-Lindau factor (Vhl) using the Cre-lox system. Here we show that Hif1α or Hif2α deletion in the primary tumor decreases metastatic tumor burden in the bone marrow, while Vhl deletion increases bone tumor burden, as hypothesized. Unexpectedly, Hif1α deletion increases metastatic tumor burden in the lung, while deletion of Hif2α or Vhl does not affect pulmonary metastasis. Mice with Hif1α deleted tumors also exhibit reduced bone volume as measured by micro computed tomography, suggesting that disruption of the osteogenic niche may be involved in the preference for lung dissemination observed in this group. Thus, we reveal that HIF signaling in breast tumors controls tumor dissemination in a site-specific manner.

Y box binding protein 1 inhibition as a targeted therapy for ovarian cancer.

Y box binding protein 1 (YB-1) is a multifunctional protein associated with tumor progression and the emergence of treatment resistance (TR). Here, we report an azopodophyllotoxin small molecule, SU056, that potently inhibits tumor growth and progression via YB-1 inhibition. This YB-1 inhibitor inhibits cell proliferation, resistance to apoptosis in ovarian cancer (OC) cells, and arrests in the G1 phase. Inhibitor treatment leads to enrichment of proteins associated with apoptosis and RNA degradation pathways while downregulating spliceosome pathway. In vivo, SU056 independently restrains OC progression and exerts a synergistic effect with paclitaxel to further reduce disease progression with no observable liver toxicity. Moreover, in vitro mechanistic studies showed delayed disease progression via inhibition of drug efflux and multidrug resistance 1, and significantly lower neurotoxicity as compared with etoposide. These data suggest that YB-1 inhibition may be an effective strategy to reduce OC progression, antagonize TR, and decrease patient mortality.

The HIF target MAFF promotes tumor invasion and metastasis through IL11 and STAT3 signaling.

Hypoxia plays a critical role in tumor progression including invasion and metastasis. To determine critical genes regulated by hypoxia that promote invasion and metastasis, we screen fifty hypoxia inducible genes for their effects on invasion. In this study, we identify v-maf musculoaponeurotic fibrosarcoma oncogene homolog F (MAFF) as a potent regulator of tumor invasion without affecting cell viability. MAFF expression is elevated in metastatic breast cancer patients and is specifically correlated with hypoxic tumors. Combined ChIP- and RNA-sequencing identifies IL11 as a direct transcriptional target of the heterodimer between MAFF and BACH1, which leads to activation of STAT3 signaling. Inhibition of IL11 results in similar levels of metastatic suppression as inhibition of MAFF. This study demonstrates the oncogenic role of MAFF as an activator of the IL11/STAT3 pathways in breast cancer.

Effects of Ultra-high doserate FLASH Irradiation on the Tumor Microenvironment in Lewis Lung Carcinoma: Role of Myosin Light Chain.

PURPOSE: To investigate whether the vascular collapse in tumors by conventional dose rate (CONV) irradiation (IR) would also occur by the ultra-high dose rate FLASH IR. METHODS AND MATERIALS: Lewis lung carcinoma (LLC) cells were subcutaneously implanted in mice. This was followed by CONV or FLASH IR at 15 Gy. Tumors were harvested at 6 or 48 hours after IR and stained for CD31, phosphorylated myosin light chain (p-MLC), γH2AX (a surrogate marker for DNA double strand break), intracellular reactive oxygen species (ROS), or immune cells such as myeloid and CD8α T cells. Cell lines were irradiated with CONV IR for Western blot analyses. ML-7 was intraperitoneally administered daily to LLC-bearing mice for 7 days before 15 Gy CONV IR. Tumors were similarly harvested and analyzed. RESULTS: By immunostaining, we observed that CONV IR at 6 hours resulted in constricted vessel morphology, increased expression of p-MLC, and much higher numbers of γH2AX-positive cells in tumors, which were not observed with FLASH IR. Mechanistically, MLC activation by ROS is unlikely, because FLASH IR produced significantly more ROS than CONV IR in tumors. In vitro studies demonstrated that ML-7, an inhibitor of MLC kinase, abrogated IR-induced γH2AX formation and disappearance kinetics. Lastly, we observed that CONV IR when combined with ML-7 produced some effects similar to FLASH IR, including reduction in the vasculature collapse, fewer γH2AX-positive cells, and increased immune cell influx to the tumors. CONCLUSIONS: FLASH IR produced novel changes in the tumor microenvironment that were not observed with CONV IR. We believe that MLC activation in tumors may be responsible for some of the microenvironmental changes differentially regulated between CONV and FLASH IR.

Organoids as Complex In Vitro Models for Studying Radiation-Induced Cell Recruitment.

Patients with triple negative breast cancer (TNBC) typically receive chemotherapy, surgery, and radiation therapy. Although this treatment improves prognosis for most patients, some patients continue to experience recurrence within 5 years. Preclinical studies have shown that immune cell infiltration at the irradiated site may play a significant role in tumor cell recruitment; however, little is known about the mechanisms that govern this process. This lack of knowledge highlights the need to evaluate radiation-induced cell infiltration with models that have controllable variables and maintain biological integrity. Mammary organoids are multicellular three-dimensional (3D) in vitro models, and they have been used to examine many aspects of mammary development and tumorigenesis. Organoids are also emerging as a powerful tool to investigate normal tissue radiation damage. In this review, we evaluate recent advances in mammary organoid technology, consider the advantages of using organoids to study radiation response, and discuss future directions for the applications of this technique.

Lipids in the tumor microenvironment: From cancer progression to treatment.

Over the past decade, the study of metabolic abnormalities in cancer cells has risen dramatically. Cancer cells can thrive in challenging environments, be it the hypoxic and nutrient-deplete tumor microenvironment or a distant tissue following metastasis. The ways in which cancer cells utilize lipids are often influenced by the complex interactions within the tumor microenvironment and adjacent stroma. Adipocytes can be activated by cancer cells to lipolyze their triglyceride stores, delivering secreted fatty acids to cancer cells for uptake through numerous fatty acid transporters. Cancer-associated fibroblasts are also implicated in lipid secretion for cancer cell catabolism and lipid signaling leading to activation of mitogenic and migratory pathways. As these cancer-stromal interactions are exacerbated during tumor progression, fatty acids secreted into the microenvironment can impact infiltrating immune cell function and phenotype. Lipid metabolic abnormalities such as increased fatty acid oxidation and de novo lipid synthesis can provide survival advantages for the tumor to resist chemotherapeutic and radiation treatments and alleviate cellular stresses involved in the metastatic cascade. In this review, we highlight recent literature that demonstrates how lipids can shape each part of the cancer lifecycle and show that there is significant potential for therapeutic intervention surrounding lipid metabolic and signaling pathways.

Emerging Biomimetic Materials for Studying Tumor and Immune Cell Behavior.

Cancer is one of the leading causes of death both in the United States and worldwide. The dynamic microenvironment in which tumors grow consists of fibroblasts, immune cells, extracellular matrix (ECM), and cytokines that enable progression and metastasis. Novel biomaterials that mimic these complex surroundings give insight into the biological, chemical, and physical environment that cause cancer cells to metastasize and invade into other tissues. Two-dimensional (2D) cultures are useful for gaining limited information about cancer cell behavior; however, they do not accurately represent the environments that cells experience in vivo. Recent advances in the design and tunability of diverse three-dimensional (3D) biomaterials complement biological knowledge and allow for improved recapitulation of in vivo conditions. Understanding cell-ECM and cell-cell interactions that facilitate tumor survival will accelerate the design of more effective therapies. This review discusses innovative materials currently being used to study tumor and immune cell behavior and interactions, including materials that mimic the ECM composition, mechanical stiffness, and integrin binding sites of the tumor microenvironment.

Systemic Inflammation After Radiation Predicts Locoregional Recurrence, Progression, and Mortality in Stage II-III Triple-Negative Breast Cancer.

PURPOSE: Patients with triple-negative breast cancer experience high rates of recurrence after radiation, which may be facilitated by the recruitment of circulating tumor cells to proinflammatory microenvironments in the absence of lymphocytes. We hypothesized that patients with lymphopenia and elevated inflammatory hematologic markers after radiation therapy would have an increased risk of locoregional failure. METHODS AND MATERIALS: With approval, we retrospectively studied a cohort of women treated with adjuvant radiation therapy for stage II-III triple-negative breast cancer. We analyzed the relationship between post-radiation therapy neutrophil:lymphocyte ratio (NLR) and locoregional recurrence by using Cox regression. RESULTS: One-hundred thirty patients met inclusion criteria, and median follow-up time was 7.6 years. Patients with an NLR ≥3 had a higher rate of locoregional failure (P = .04) and lower overall survival (P = .04). After adjusting for stage (hazard ratio [HR], 5.5; P < .0001) and neoadjuvant chemotherapy (HR, 2.5; P = .0162), NLR was highly predictive of locoregional failure (HR, 1.4; P = .0009). NLR was also highly predictive of overall survival (HR, 1.3; P = .0007) after adjustment for stage and neoadjuvant chemotherapy. CONCLUSIONS: Innate peripheral inflammation after radiation therapy for triple-negative breast cancer in an immunocompromised setting may be a novel prognostic biomarker for locoregional recurrence, progression, and survival. This finding supports preclinical studies of post-radiation therapy inflammation-mediated tumor progression. Further studies are needed to confirm this finding and develop treatment strategies.