Computational modeling of the physical features that influence breast cancer invasion into adipose tissue.

Breast cancer invasion into adipose tissue strongly influences disease progression and metastasis. The degree of cancer cell invasion into adipose tissue depends on numerous biochemical and physical properties of cancer cells, adipocytes, and other key components of adipose tissue. We model breast cancer invasion into adipose tissue as a physical process by carrying out simulations of active, cohesive spherical particles (cancer cells) invading into confluent packings of deformable polyhedra (adipocytes). We quantify the degree of invasion by calculating the interfacial area At between cancer cells and adipocytes. We determine the long-time value of At versus the activity and strength of the cohesion between cancer cells, as well as mechanical properties of the adipocytes and extracellular matrix (ECM) in which the adipocytes are embedded. We show that the degree of invasion collapses onto a master curve by plotting it versus a dimensionless energy scale Ec, which grows linearly with mean-square fluctuations and persistence time of the cancer cell velocities, is inversely proportional to the pressure of the system, and has an offset that increases with the cancer cell cohesive energy. The condition, Ec≫1, indicates that cancer cells will invade the adipose tissue, whereas for Ec≪1, the cancer cells and adipocytes remain demixed. We also show that constraints on adipocyte positions by the ECM decrease At relative to that obtained for unconstrained adipocytes. Finally, spatial heterogeneity in structural and mechanical properties of the adipocytes in the presence of ECM impedes invasion relative to adipose tissue with uniform properties.

Immunoengineering can overcome the glycocalyx armour of cancer cells.

Cancer cell glycocalyx is a major line of defence against immune surveillance. However, how specific physical properties of the glycocalyx are regulated on a molecular level, contribute to immune evasion and may be overcome through immunoengineering must be resolved. Here we report how cancer-associated mucins and their glycosylation contribute to the nanoscale material thickness of the glycocalyx and consequently modulate the functional interactions with cytotoxic immune cells. Natural-killer-cell-mediated cytotoxicity is inversely correlated with the glycocalyx thickness of the target cells. Changes in glycocalyx thickness of approximately 10 nm can alter the susceptibility to immune cell attack. Enhanced stimulation of natural killer and T cells through equipment with chimeric antigen receptors can improve the cytotoxicity against mucin-bearing target cells. Alternatively, cytotoxicity can be enhanced through engineering effector cells to display glycocalyx-editing enzymes, including mucinases and sialidases. Together, our results motivate the development of immunoengineering strategies that overcome the glycocalyx armour of cancer cells.

COLLAGEN MINERALIZATION DECREASES NK CELL-MEDIATED CYTOTOXICITY OF BREAST CANCER CELLS VIA INCREASED GLYCOCALYX THICKNESS.

Skeletal metastasis is common in patients with advanced breast cancer, and often caused by immune evasion of disseminated tumor cells (DTCs). In the skeleton, tumor cells not only disseminate to the bone marrow, but also to osteogenic niches in which they interact with newly mineralizing bone extracellular matrix (ECM). However, it remains unclear how mineralization of collagen type I, the primary component of bone ECM, regulates tumor-immune cell interactions. Here, we have utilized a combination of synthetic bone matrix models with controlled mineral content, nanoscale optical imaging, and flow cytometry to evaluate how collagen type I mineralization affects the biochemical and biophysical properties of the tumor cell glycocalyx, a dense layer of glycosylated proteins and lipids decorating their cell surface. Our results suggest that collagen mineralization upregulates mucin-type O-glycosylation and sialylation by tumor cells, which increased their glycocalyx thickness while enhancing resistance to attack by Natural Killer (NK) cells. These changes were functionally linked as treatment with a sialylation inhibitor decreased mineralization-dependent glycocalyx thickness and made tumor cells more susceptible to NK cell attack. Together, our results suggest that interference with glycocalyx sialylation may represent a therapeutic strategy to enhance cancer immunotherapies targeting bone-metastatic breast cancer.

Collagen Mineralization Decreases NK Cell-Mediated Cytotoxicity of Breast Cancer Cells via Increased Glycocalyx Thickness.

Skeletal metastasis is common in patients with advanced breast cancer and often caused by immune evasion of disseminated tumor cells (DTCs). In the skeleton, tumor cells not only disseminate to the bone marrow but also to osteogenic niches in which they interact with newly mineralizing bone extracellular matrix (ECM). However, it remains unclear how mineralization of collagen type I, the primary component of bone ECM, regulates tumor-immune cell interactions. Here, a combination of synthetic bone matrix models with controlled mineral content, nanoscale optical imaging, and flow cytometry are utilized to evaluate how collagen type I mineralization affects the biochemical and biophysical properties of the tumor cell glycocalyx, a dense layer of glycosylated proteins and lipids decorating their cell surface. These results suggest that collagen mineralization upregulates mucin-type O-glycosylation and sialylation by tumor cells, which increases their glycocalyx thickness while enhancing resistance to attack by natural killer (NK) cells. These changes are functionally linked as treatment with a sialylation inhibitor decreased mineralization-dependent glycocalyx thickness and made tumor cells more susceptible to NK cell attack. Together, these results suggest that interference with glycocalyx sialylation may represent a therapeutic strategy to enhance cancer immunotherapies targeting bone-metastatic breast cancer.

Endothelial cells metabolically regulate breast cancer invasion toward a microvessel.

Breast cancer metastasis is initiated by invasion of tumor cells into the collagen type I-rich stroma to reach adjacent blood vessels. Prior work has identified that metabolic plasticity is a key requirement of tumor cell invasion into collagen. However, it remains largely unclear how blood vessels affect this relationship. Here, we developed a microfluidic platform to analyze how tumor cells invade collagen in the presence and absence of a microvascular channel. We demonstrate that endothelial cells secrete pro-migratory factors that direct tumor cell invasion toward the microvessel. Analysis of tumor cell metabolism using metabolic imaging, metabolomics, and computational flux balance analysis revealed that these changes are accompanied by increased rates of glycolysis and oxygen consumption caused by broad alterations of glucose metabolism. Indeed, restricting glucose availability decreased endothelial cell-induced tumor cell invasion. Our results suggest that endothelial cells promote tumor invasion into the stroma due, in part, to reprogramming tumor cell metabolism.

Phosphoproteomic Changes Induced by Cell-Derived Matrix and Their Effect on Tumor Cell Migration and Cytoskeleton Remodeling.

Increased fibrotic extracellular matrix (ECM) deposition promotes tumor invasion, which is the first step of the metastatic cascade. Yet, the underlying mechanisms are poorly understood as conventional studies of tumor cell migration are often performed in 2D cultures lacking the compositional and structural complexity of native ECM. Moreover, these studies frequently focus on select candidate pathways potentially overlooking other relevant changes in cell signaling. Here, we combine a cell-derived matrix (CDM) model with phosphotyrosine phosphoproteomic analysis to investigate tumor cell migration on fibrotic ECM relative to standard tissue culture plastic (TCP). Our results suggest that tumor cells cultured on CDMs migrate faster and in a more directional manner than their counterparts on TCP. These changes in migration correlate with decreased cell spreading and increased cell elongation. While the formation of phosphorylated focal adhesion kinase (pFAK)+ adhesion complexes did not vary between TCP and CDMs, time-dependent phosphoproteomic analysis identified that the SRC family kinase LYN may be differentially regulated. Pharmacological inhibition of LYN decreased tumor cell migration and cytoskeletal rearrangement on CDMs and also on TCP, suggesting that LYN regulates tumor cell migration on CDMs in combination with other mechanisms. These data highlight how the combination of physicochemically complex in vitro systems with phosphoproteomics can help identify signaling mechanisms by which the fibrotic ECM regulates tumor cell migration.

Bone-matrix mineralization dampens integrin-mediated mechanosignalling and metastatic progression in breast cancer.

In patients with breast cancer, lower bone mineral density increases the risk of bone metastasis. Although the relationship between bone-matrix mineralization and tumour-cell phenotype in breast cancer is not well understood, mineralization-induced rigidity is thought to drive metastatic progression via increased cell-adhesion forces. Here, by using collagen-based matrices with adjustable intrafibrillar mineralization, we show that, unexpectedly, matrix mineralization dampens integrin-mediated mechanosignalling and induces a less proliferative stem-cell-like phenotype in breast cancer cells. In mice with xenografted decellularized physiological bone matrices seeded with human breast tumour cells, the presence of bone mineral reduced tumour growth and upregulated a gene-expression signature that is associated with longer metastasis-free survival in patients with breast cancer. Our findings suggest that bone-matrix changes in osteogenic niches regulate metastatic progression in breast cancer and that in vitro models of bone metastasis should integrate organic and inorganic matrix components to mimic physiological and pathologic mineralization.

Selective Accumulation of Near Infrared-Labeled Multivalent Quinidine Copolymers in Tumors Overexpressing P-Glycoprotein: Potential for Noninvasive Diagnostic Imaging.

P-glycoprotein (P-gp) is a promiscuous small molecule transporter whose overexpression in cancer is associated with multidrug resistance (MDR). In these instances, anticancer drugs can select for P-gp-overexpressing cells, leading to cancer recurrence with an MDR phenotype. To avoid selection for MDR cancers and inform individual patient treatment plans, it is critical to noninvasively identify P-gp-overexpressing tumors prior to administration of chemotherapy. We report the facile free radical copolymerization of quinidine, a competitive inhibitor of P-gp, and acrylic acid to generate multiplexed polymeric P-gp-targeted imaging agents with tunable quinidine content. Copolymer targeting was demonstrated in a nude mouse xenograft model. In xenografts overexpressing P-gp, copolymer distribution was enhanced over two-fold compared to the negative control of poly(acrylic acid) regardless of quinidine content. In contrast, accumulation of the copolymers in xenografts lacking P-gp was equivalent to poly(acrylic acid). This work forms the foundation for a unique approach toward the phenotype-specific noninvasive imaging of MDR tumors and is the first in vivo demonstration of copolymer accumulation through the active targeting of P-gp.

Biomineralogical signatures of breast microcalcifications.

Microcalcifications, primarily biogenic apatite, occur in cancerous and benign breast pathologies and are key mammographic indicators. Outside the clinic, numerous microcalcification compositional metrics (e.g., carbonate and metal content) are linked to malignancy, yet microcalcification formation is dependent on microenvironmental conditions, which are notoriously heterogeneous in breast cancer. We interrogate multiscale heterogeneity in 93 calcifications from 21 breast cancer patients using an omics-inspired approach: For each microcalcification, we define a "biomineralogical signature" combining metrics derived from Raman microscopy and energy-dispersive spectroscopy. We observe that (i) calcifications cluster into physiologically relevant groups reflecting tissue type and local malignancy; (ii) carbonate content exhibits substantial intratumor heterogeneity; (iii) trace metals including zinc, iron, and aluminum are enhanced in malignant-localized calcifications; and (iv) the lipid-to-protein ratio within calcifications is lower in patients with poor composite outcome, suggesting that there is potential clinical value in expanding research on calcification diagnostic metrics to include "mineral-entrapped" organic matrix.

Convergent Approaches to Delineate the Metabolic Regulation of Tumor Invasion by Hyaluronic Acid Biosynthesis.

Metastasis is the leading cause of breast cancer-related deaths and is often driven by invasion and cancer-stem like cells (CSCs). Both the CSC phenotype and invasion are associated with increased hyaluronic acid (HA) production. How these independent observations are connected, and which role metabolism plays in this process, remains unclear due to the lack of convergent approaches integrating engineered model systems, computational tools, and cancer biology. Using microfluidic invasion models, metabolomics, computational flux balance analysis, and bioinformatic analysis of patient data, the functional links between the stem-like, invasive, and metabolic phenotype of breast cancer cells as a function of HA biosynthesis are investigated. These results suggest that CSCs are more invasive than non-CSCs and that broad metabolic changes caused by overproduction of HA play a role in this process. Accordingly, overexpression of hyaluronic acid synthases (HAS) 2 or 3 induces a metabolic phenotype that promotes cancer cell stemness and invasion in vitro and upregulates a transcriptomic signature predictive of increased invasion and worse patient survival. This study suggests that HA overproduction leads to metabolic adaptations to satisfy the energy demands for 3D invasion of breast CSCs highlighting the importance of engineered model systems and multidisciplinary approaches in cancer research.

IGF2BP2 promotes cancer progression by degrading the RNA transcript encoding a v-ATPase subunit.

IGF2BP2 binds to a number of RNA transcripts and has been suggested to function as a tumor promoter, although little is known regarding the mechanisms that regulate its roles in RNA metabolism. Here we demonstrate that IGF2BP2 binds to the 3' untranslated region of the transcript encoding ATP6V1A, a catalytic subunit of the vacuolar ATPase (v-ATPase), and serves as a substrate for the NAD+-dependent deacetylase SIRT1, which regulates how IGF2BP2 affects the stability of the ATP6V1A transcript. When sufficient levels of SIRT1 are expressed, it catalyzes the deacetylation of IGF2BP2, which can bind to the ATP6V1A transcript but does not mediate its degradation. However, when SIRT1 expression is low, the acetylated form of IGF2BP2 accumulates, and upon binding to the ATP6V1A transcript recruits the XRN2 nuclease, which catalyzes transcript degradation. Thus, the stability of the ATP6V1A transcript is significantly compromised in breast cancer cells when SIRT1 expression is low or knocked-down. This leads to a reduction in the expression of functional v-ATPase complexes in cancer cells and to an impairment in their lysosomal activity, resulting in the production of a cellular secretome consisting of increased numbers of exosomes enriched in ubiquitinated protein cargo and soluble hydrolases, including cathepsins, that together combine to promote tumor cell survival and invasiveness. These findings describe a previously unrecognized role for IGF2BP2 in mediating the degradation of a messenger RNA transcript essential for lysosomal function and highlight how its sirtuin-regulated acetylation state can have significant biological and disease consequences.

Regulation of Tumor Invasion by the Physical Microenvironment: Lessons from Breast and Brain Cancer.

The success of anticancer therapies is often limited by heterogeneity within and between tumors. While much attention has been devoted to understanding the intrinsic molecular diversity of tumor cells, the surrounding tissue microenvironment is also highly complex and coevolves with tumor cells to drive clinical outcomes. Here, we propose that diverse types of solid tumors share common physical motifs that change in time and space, serving as universal regulators of malignancy. We use breast cancer and glioblastoma as instructive examples and highlight how invasion in both diseases is driven by the appropriation of structural guidance cues, contact-dependent heterotypic interactions with stromal cells, and elevated interstitial fluid pressure and flow. We discuss how engineering strategies show increasing value for measuring and modeling these physical propertiesfor mechanistic studies. Moreover, engineered systems offer great promise for developing and testing novel therapies that improve patient prognosis by normalizing the physical tumor microenvironment.

Tetrathiomolybdate (TM)-associated copper depletion influences collagen remodeling and immune response in the pre-metastatic niche of breast cancer.

Tetrathiomolybdate (TM) is a novel, copper-depleting compound associated with promising survival in a phase II study of patients with high-risk and triple-negative breast cancer. We sought to elucidate the mechanism of TM by exploring its effects on collagen processing and immune function in the tumor microenvironment (TME). Using an exploratory cohort, we identified markers of collagen processing (LOXL2, PRO-C3, C6M, and C1M) that differed between those with breast cancer versus controls. We measured these collagen biomarkers in TM-treated patients on the phase II study and detected evidence of decreased collagen cross-linking and increased degradation over formation in those without disease compared to those who experienced disease progression. Preclinical studies revealed decreased collagen deposition, lower levels of myeloid-derived suppressor cells, and higher CD4+ T-cell infiltration in TM-treated mice compared with controls. This study reveals novel mechanisms of TM targeting the TME and immune response with potential applications across cancer types.

Engineering strategies to capture the biological and biophysical tumor microenvironment in vitro.

Despite decades of research and advancements in diagnostic and treatment modalities, cancer remains a major global healthcare challenge. This is due in part to a lack of model systems that allow investigating the mechanisms underlying tumor development, progression, and therapy resistance under relevant conditions in vitro. Tumor cell interactions with their surroundings influence all stages of tumorigenesis and are shaped by both biological and biophysical cues including cell-cell and cell-extracellular matrix (ECM) interactions, tissue architecture and mechanics, and mass transport. Engineered tumor models provide promising platforms to elucidate the individual and combined contributions of these cues to tumor malignancy under controlled and physiologically relevant conditions. This review will summarize current knowledge of the biological and biophysical microenvironmental cues that influence tumor development and progression, present examples of in vitro model systems that are presently used to study these interactions and highlight advancements in tumor engineering approaches to further improve these technologies.

Contractility, focal adhesion orientation, and stress fiber orientation drive cancer cell polarity and migration along wavy ECM substrates.

Contact guidance is a powerful topographical cue that induces persistent directional cell migration. Healthy tissue stroma is characterized by a meshwork of wavy extracellular matrix (ECM) fiber bundles, whereas metastasis-prone stroma exhibit less wavy, more linear fibers. The latter topography correlates with poor prognosis, whereas more wavy bundles correlate with benign tumors. We designed nanotopographic ECM-coated substrates that mimic collagen fibril waveforms seen in tumors and healthy tissues to determine how these nanotopographies may regulate cancer cell polarization and migration machineries. Cell polarization and directional migration were inhibited by fibril-like wave substrates above a threshold amplitude. Although polarity signals and actin nucleation factors were required for polarization and migration on low-amplitude wave substrates, they did not localize to cell leading edges. Instead, these factors localized to wave peaks, creating multiple "cryptic leading edges" within cells. On high-amplitude wave substrates, retrograde flow from large cryptic leading edges depolarized stress fibers and focal adhesions and inhibited cell migration. On low-amplitude wave substrates, actomyosin contractility overrode the small cryptic leading edges and drove stress fiber and focal adhesion orientation along the wave axis to mediate directional migration. Cancer cells of different intrinsic contractility depolarized at different wave amplitudes, and cell polarization response to wavy substrates could be tuned by manipulating contractility. We propose that ECM fibril waveforms with sufficiently high amplitude around tumors may serve as "cell polarization barriers," decreasing directional migration of tumor cells, which could be overcome by up-regulation of tumor cell contractility.

Engineered ECM models: Opportunities to advance understanding of tumor heterogeneity.

Intratumoral heterogeneity is a negative prognostic factor for cancer and commonly attributed to microenvironment-driven genetic mutations and/or the emergence of cancer stem-like cells. How aberrant extracellular matrix (ECM) remodeling regulates the phenotypic diversity of tumor cells, however, remains poorly understood due in part to a lack of model systems that allow isolating the physicochemical heterogeneity of malignancy-associated ECM for mechanistic studies. Here, we review the compositional, microarchitectural, and mechanical hallmarks of cancer-associated ECM and highlight biomaterials and engineering approaches to recapitulate these properties for in vitro and in vivo studies. Subsequently, we describe how such engineered platforms may be explored to define the spatiotemporal dynamics through which cancer-associated ECM remodeling regulates intratumoral heterogeneity and the cancer stem-like cell phenotype. Finally, we highlight future opportunities and technological advances to further elucidate the relationship between tumor-associated ECM dynamics and intratumoral heterogeneity.

Breast cancer-secreted factors perturb murine bone growth in regions prone to metastasis.

Breast cancer frequently metastasizes to bone, causing osteolytic lesions. However, how factors secreted by primary tumors affect the bone microenvironment before the osteolytic phase of metastatic tumor growth remains unclear. Understanding these changes is critical as they may regulate metastatic dissemination and progression. To mimic premetastatic bone adaptation, immunocompromised mice were injected with MDA-MB-231-conditioned medium [tumor-conditioned media (TCM)]. Subsequently, the bones of these mice were subjected to multiscale, correlative analysis including RNA sequencing, histology, micro-computed tomography, x-ray scattering analysis, and Raman imaging. In contrast to overt metastasis causing osteolysis, TCM treatment induced new bone formation that was characterized by increased mineral apposition rate relative to control bones, altered bone quality with less matrix and more carbonate substitution, and the deposition of disoriented mineral near the growth plate. Our study suggests that breast cancer-secreted factors may promote perturbed bone growth before metastasis, which could affect initial seeding of tumor cells.

Computational 4D-OCM for label-free imaging of collective cell invasion and force-mediated deformations in collagen.

Traction force microscopy (TFM) is an important family of techniques used to measure and study the role of cellular traction forces (CTFs) associated with many biological processes. However, current standard TFM methods rely on imaging techniques that do not provide the experimental capabilities necessary to study CTFs within 3D collective and dynamic systems embedded within optically scattering media. Traction force optical coherence microscopy (TF-OCM) was developed to address these needs, but has only been demonstrated for the study of isolated cells embedded within optically clear media. Here, we present computational 4D-OCM methods that enable the study of dynamic invasion behavior of large tumor spheroids embedded in collagen. Our multi-day, time-lapse imaging data provided detailed visualizations of evolving spheroid morphology, collagen degradation, and collagen deformation, all using label-free scattering contrast. These capabilities, which provided insights into how stromal cells affect cancer progression, significantly expand access to critical data about biophysical interactions of cells with their environment, and lay the foundation for future efforts toward volumetric, time-lapse reconstructions of collective CTFs with TF-OCM.

Engineering Modular Half-Antibody Conjugated Nanoparticles for Targeting CD44v6-Expressing Cancer Cells.

Gastric cancer (GC) remains a major cause of death worldwide mainly because of the late detection in advanced stage. Recently, we proposed CD44v6 as a relevant marker for early detection of GC, opening new avenues for GC-targeted theranostics. Here, we designed a modular nanoscale system that selectively targets CD44v6-expressing GC cells by the site-oriented conjugation of a new-engineered CD44v6 half-antibody fragment to maleimide-modified polystyrene nanoparticles (PNPs) via an efficient bioorthogonal thiol-Michael addition click chemistry. PNPs with optimal particle size (200 nm) for crossing a developed biomimetic CD44v6-associated GC stromal model were further modified with a heterobifunctional maleimide crosslinker and click conjugated to the novel CD44v6 half-antibody fragment, obtained by chemical reduction of full antibody, without affecting its bioactivity. Collectively, our results confirmed the specific targeting ability of CD44v6-PNPs to CD44v6-expressing cells (1.65-fold higher than controls), highlighting the potential of CD44v6 half-antibody conjugated nanoparticles as promising and clinically relevant tools for the early diagnosis and therapy of GC. Additionally, the rational design of our nanoscale system may be explored for the development of several other nanotechnology-based disease-targeted approaches.