Small Extracellular Vesicles Promote Stiffness-mediated Metastasis.

Tissue stiffness is a critical prognostic factor in breast cancer and is associated with metastatic progression. Here we show an alternative and complementary hypothesis of tumor progression whereby physiologic matrix stiffness affects the quantity and protein cargo of small extracellular vesicles (EV) produced by cancer cells, which in turn aid cancer cell dissemination. Primary patient breast tissue released by cancer cells on matrices that model human breast tumors (25 kPa; stiff EVs) feature increased adhesion molecule presentation (ITGα2ß1, ITGα6ß4, ITGα6ß1, CD44) compared with EVs from softer normal tissue (0.5 kPa; soft EVs), which facilitates their binding to extracellular matrix proteins including collagen IV, and a 3-fold increase in homing ability to distant organs in mice. In a zebrafish xenograft model, stiff EVs aid cancer cell dissemination. Moreover, normal, resident lung fibroblasts treated with stiff and soft EVs change their gene expression profiles to adopt a cancer-associated fibroblast phenotype. These findings show that EV quantity, cargo, and function depend heavily on the mechanical properties of the extracellular microenvironment. SIGNIFICANCE: Here we show that the quantity, cargo, and function of breast cancer-derived EVs vary with mechanical properties of the extracellular microenvironment.

E-cadherin interacts with EGFR resulting in hyper-activation of ERK in multiple models of breast cancer.

The loss of intercellular adhesion molecule E-cadherin is a hallmark of the epithelial-mesenchymal transition (EMT), during which tumor cells transition into an invasive phenotype. Accordingly, E-cadherin has long been considered a tumor suppressor gene; however, E-cadherin expression is paradoxically correlated with breast cancer survival rates. Using novel multi-compartment organoids and multiple in vivo models, we show that E-cadherin promotes a hyper-proliferative phenotype in breast cancer cells via interaction with the transmembrane receptor EGFR. The E-cad and EGFR interaction results in activation of the MEK/ERK signaling pathway, leading to a significant increase in proliferation via activation of transcription factors, including c-Fos. Pharmacological inhibition of MEK activity in E-cadherin positive breast cancer significantly decreases both tumor growth and macro-metastasis in vivo. This work provides evidence for a novel role of E-cadherin in breast tumor progression and identifies a new target to treat hyper-proliferative E-cadherin-positive breast tumors, thus providing the foundation to utilize E-cadherin as a biomarker for specific therapeutic success.

Small extracellular vesicles promote stiffness-mediated metastasis.

Tissue stiffness is a critical prognostic factor in breast cancer and is associated with metastatic progression. Here we show an alternative and complementary hypothesis of tumor progression whereby physiological matrix stiffness affects the quantity and protein cargo of small EVs produced by cancer cells, which in turn drive their metastasis. Primary patient breast tissue produces significantly more EVs from stiff tumor tissue than soft tumor adjacent tissue. EVs released by cancer cells on matrices that model human breast tumors (25 kPa; stiff EVs) feature increased adhesion molecule presentation (ITGα 2 ß 1 , ITGα 6 ß 4 , ITGα 6 ß 1 , CD44) compared to EVs from softer normal tissue (0.5 kPa; soft EVs), which facilitates their binding to extracellular matrix (ECM) protein collagen IV, and a 3-fold increase in homing ability to distant organs in mice. In a zebrafish xenograft model, stiff EVs aid cancer cell dissemination through enhanced chemotaxis. Moreover, normal, resident lung fibroblasts treated with stiff and soft EVs change their gene expression profiles to adopt a cancer associated fibroblast (CAF) phenotype. These findings show that EV quantity, cargo, and function depend heavily on the mechanical properties of the extracellular microenvironment.

Serine synthesis pathway upregulated by E-cadherin is essential for the proliferation and metastasis of breast cancers.

The loss of E-cadherin (E-cad), an epithelial cell adhesion molecule, has been implicated in the epithelial-mesenchymal transition (EMT), promoting invasion and migration of cancer cells and, consequently, metastasis. However, recent studies have demonstrated that E-cad supports the survival and proliferation of metastatic cancer cells, suggesting that our understanding of E-cad in metastasis is far from comprehensive. Here, we report that E-cad upregulates the de novo serine synthesis pathway (SSP) in breast cancer cells. The SSP provides metabolic precursors for biosynthesis and resistance to oxidative stress, critically beneficial for E-cad-positive breast cancer cells to achieve faster tumor growth and more metastases. Inhibition of PHGDH, a rate-limiting enzyme in the SSP, significantly and specifically hampered the proliferation of E-cad-positive breast cancer cells and rendered them vulnerable to oxidative stress, inhibiting their metastatic potential. Our findings reveal that E-cad adhesion molecule significantly reprograms cellular metabolism, promoting tumor growth and metastasis of breast cancers.

Tumor proliferation and invasion are intrinsically coupled and unraveled through tunable spheroid and physics-based models.

Proliferation and invasion are two key drivers of tumor growth that are traditionally considered independent multicellular processes. However, these processes are intrinsically coupled through a maximum carrying capacity, i.e., the maximum spatial cell concentration supported by the tumor volume, total cell count, nutrient access, and mechanical properties of the tissue stroma. We explored this coupling of proliferation and invasion through in vitro and in silico methods where we modulated the mechanical properties of the tumor and the surrounding extracellular matrix. E-cadherin expression and stromal collagen concentration were manipulated in a tunable breast cancer spheroid to determine the overall impacts of these tumor variables on net tumor proliferation and continuum invasion. We integrated these results into a mixed-constitutive formulation to computationally delineate the influences of cellular and extracellular adhesion, stiffness, and mechanical properties of the extracellular matrix on net proliferation and continuum invasion. This framework integrates biological in vitro data into concise computational models of invasion and proliferation to provide more detailed physical insights into the coupling of these key tumor processes and tumor growth. STATEMENT OF SIGNIFICANCE: Tumor growth involves expansion into the collagen-rich stroma through intrinsic coupling of proliferation and invasion within the tumor continuum. These processes are regulated by a maximum carrying capacity that is determined by the total cell count, tumor volume, nutrient access, and mechanical properties of the surrounding stroma. The influences of biomechanical parameters (i.e., stiffness, cell elongation, net proliferation rate and cell-ECM friction) on tumor proliferation or invasion cannot be unraveled using experimental methods alone. By pairing a tunable spheroid system with computational modeling, we delineated the interdependencies of each system parameter on tumor proliferation and continuum invasion, and established a concise computational framework for studying tumor mechanobiology.

Precision-engineered biomimetics: the human fallopian tube.

The fallopian tube has an essential role in several physiological and pathological processes from pregnancy to ovarian cancer. However, there are no biologically relevant models to study its pathophysiology. The state-of-the-art organoid model has been compared to two-dimensional tissue sections and molecularly assessed providing only cursory analyses of the model's accuracy. We developed a novel multi-compartment organoid model of the human fallopian tube that was meticulously tuned to reflect the compartmentalization and heterogeneity of the tissue's composition. We validated this organoid's molecular expression patterns, cilia-driven transport function, and structural accuracy through a highly iterative platform wherein organoids are compared to a three-dimensional, single-cell resolution reference map of a healthy, transplantation-quality human fallopian tube. This organoid model was precision-engineered to match the human microanatomy. One sentence summary: Tunable organoid modeling and CODA architectural quantification in tandem help design a tissue-validated organoid model.

Engineered bispecific antibodies targeting the interleukin-6 and -8 receptors potently inhibit cancer cell migration and tumor metastasis.

Simultaneous inhibition of interleukin-6 (IL-6) and interleukin-8 (IL-8) signaling diminishes cancer cell migration, and combination therapy has recently been shown to synergistically reduce metastatic burden in a preclinical model of triple-negative breast cancer. Here, we have engineered two novel bispecific antibodies that target the IL-6 and IL-8 receptors to concurrently block the signaling activity of both ligands. We demonstrate that a first-in-class bispecific antibody design has promising therapeutic potential, with enhanced selectivity and potency compared with monoclonal antibody and small-molecule drug combinations in both cellular and animal models of metastatic triple-negative breast cancer. Mechanistic characterization revealed that our engineered bispecific antibodies have no impact on cell viability, but profoundly reduce the migratory potential of cancer cells; hence they constitute a true anti-metastatic treatment. Moreover, we demonstrate that our antibodies can be readily combined with standard-of-care anti-proliferative drugs to develop effective anti-cancer regimens. Collectively, our work establishes an innovative metastasis-focused direction for cancer drug development.

Effect of an alkyl spacer on the morphology and internalization of MUC1 aptamer-naphthalimide amphiphiles for targeting and imaging triple negative breast cancer cells.

Despite decades of research, there are few targeted treatment options available for triple negative breast cancer (TNBC), leaving chemotherapy, and radiation treatment regimes with poor response and high toxicity. Herein aptamer-amphiphiles were synthesized which selectively bind to the mucin-1 (MUC1) glycoprotein that is overexpressed in TNBC cells. These amphiphiles have a fluorescent tail (1,8-naphthalimide or 4-nitro-1,8-naphthalimide) which enables self-assembly of the amphiphiles and allows for easy visualization without the requirement for further conjugation of a fluorophore. Interestingly, the length of the alkyl spacer (C4 or C12) between the aptamer and tail was shown to influence the morphology of the self-assembled structure, and thus its ability to internalize into the TNBC cells. While both the MUC1 aptamer-C4-napthalimide spherical micelles and the MUC1 aptamer-C12-napthalimide long cylindrical micelles showed internalization into MDA-MB-468 TNBC cells but not the noncancerous MCF-10A breast cells, the cylindrical micelles showed greatly enhanced internalization into the MDA-MB-468 cells. Similar patterns of enhanced binding and internalization were observed between the MUC1 aptamer-C12-napthalimide cylindrical micelles and SUM159 and MDA-MB-231 TNBC cells. The MUC1 aptamer cylindrical micelles were not toxic to the cells, and when used to deliver doxorubicin to the TNBC cells, were shown to be as cytotoxic as free doxorubicin. Moreover, a pharmacokinetic study in mice showed a prolonged systemic circulation time of the MUC1 aptamer cylindrical micelles. There was a 4.6-fold increase in the elimination half-life of the aptamer cylindrical micelles, and their clearance decreased 10-fold compared to the MUC1 aptamer spherical micelles. Thus, the MUC1 aptamer-C12-napthalimide nanofibers represent a promising vehicle that could be used for easy visualization and targeted delivery of therapeutic loads to TNBC cells.