Hyaluronan-Derived Swelling of Solid Tumors, the Contribution of Collagen and Cancer Cells, and Implications for Cancer Therapy

AbstractDespite the important role that mechanical forces play in tumor growth and therapy, the contribution of swelling to tumor mechanopathology remains unexplored. Tumors rich in hyaluronan exhibit a highly negative fixed charge density. Repulsive forces among these negative charges as well as swelling of cancer cells due to regulation of intracellular tonicity can cause tumor swelling and development of stress that might compress blood vessels, compromising tumor perfusion and drug delivery. Here, we designed an experimental strategy, using four orthotopic tumor models, to measure swelling stress and related swelling to extracellular matrix components, hyaluronan and collagen, as well as to tumor perfusion. Subsequently, interventions were performed to measure tumor swelling using matrix-modifying enzymes (hyaluronidase and collagenase) and by repurposing pirfenidone, an approved antifibrotic drug. Finally, in vitro experiments on cancer cell spheroids were performed to identify their contribution to tissue swelling. Swelling stress was measured in the range of 16 to 75 mm Hg, high enough to cause vessel collapse. Interestingly, while depletion of hyaluronan decreased swelling, collagen depletion had the opposite effect, whereas the contribution of cancer cells was negligible. Furthermore, histological analysis revealed the same linear correlation between tumor swelling and the ratio of hyaluronan to collagen content when data from all tumor models were combined. Our data further revealed an inverse relation between tumor perfusion and swelling, suggesting that reduction of swelling decompresses tumor vessels. These results provide guidelines for emerging therapeutic strategies that target the tumor microenvironment to alleviate intratumoral stresses and improve vessel functionality and drug delivery

Accumulation of mechanical forces in tumors is related to hyaluronan content and tissue stiffness

<div><p>Hyaluronan is abundant in the extracellular matrix of many desmoplastic tumors and determines in large part the tumor biochemical and mechanical microenvironment. Additionally, it has been identified as one of the major physiological barriers to the effective delivery of drugs to solid tumors and its targeting with the use of pharmaceutical agents has shown to decompress tumor blood vessels, and thus improve tumor perfusion and efficacy of cytotoxic drugs. In this study, we investigated the contribution of hyaluronan to the accumulation of mechanical forces in tumors. Using experimental data from two orthotopic breast tumor models and treating tumors with two clinically approved anti-fibrotic drugs (tranilast and pirfenidone), we found that accumulation of growth-induced, residual forces in tumors are associated with hyaluronan content. Furthermore, mechanical characterization of the tumors revealed a good correlation of the accumulated forces with the elastic modulus of the tissue. Our results provide important insights on the mechano-pathology of solid tumors and can be used for the design of therapeutic strategies that target hyaluronan.</p></div

Schematic of tumor opening experiment and calculations.

<p><b>A</b>: Typical experimental procedure showing the tumor before and after the cut has been made. The measured tumor opening appears in the figure. <b>B</b>: Representative computational results in the beginning and at the end of the simulation. In the model, the tumor consists of two domains, the tumor and a peripheral layer with thickness 5% of the tumor diameter. The simulations were used for the calculation of the growth-induced stress from the measured displacement/opening of the tumor.</p

Swelling stress agrees well with growth-induced stress.

<p>The good correlation of the two types of stress is given by the good fit along the y = x dash line. Error bars represent the range of estimated growth-induced stress.</p

Schematic of growth-induced stress and swelling stress.

<p><b>A:</b> Growth-induced stress, <b>σ</b>^g, is equal to the stress required to close the tumor after the tumor relaxes and the stress is released. <b>B:</b> Swelling stress, <b>σ</b>^<b>c</b>, is the stress required to compress tumor to initial radius from swelled tissue condition.</p

Effect of ECM composition on tumor opening.

<p>(<b>A</b>) Collagen area fraction does not correlate with opening, whereas there is a correlation of tumor opening with hyaluronan (HA) area fraction (<b>B</b>) and the ratio of HA/collagen area fraction (<b>C</b>). Five tumor specimens (n = 5) from each tumor type were used.</p

Effect of ECM composition and mechanical properties on growth-induced stress.

<p>(<b>A</b>) Collagen or (<b>B</b>) hyaluronan (HA) area fraction is not associated with growth-induced stress, whereas a relation seems to exist when (<b>C</b>) the ratio of HA/collagen area fraction is employed. (<b>D</b>) Growth-induced stress does not depend on tumor opening but there is a good correlation between growth-induced stress and tumor elastic modulus (<b>E</b>) as well as with the product of tumor opening and elastic modulus (<b>F</b>). Error bars represent the range of estimated growth-induced stress.</p

Normalizing Tumor Microenvironment with Nanomedicine and Metronomic Therapy to Improve Immunotherapy

Nanomedicine offered hope for improving the treatment of cancer but the survival benefits of the clinically approved nanomedicines are modest in many cases when compared to conventional chemotherapy. Metronomic therapy, defined as the frequent, low dose administration of chemotherapeutics – is being tested in clinical trials as an alternative to the conventional maximum tolerated dose (MTD) chemotherapy schedule. Although metronomic chemotherapy has not been clinically approved yet, it has shown better survival than MTD in many preclinical studies. When beneficial, metronomic therapy seems to be associated with normalization of the tumor microenvironment including improvements in tumor perfusion, tissue oxygenation and drug delivery as well as activation of the immune system. Recent preclinical studies suggest that nanomedicines can cause similar changes in the tumor microenvironment. Here, by employing a mathematical framework, we show that both approaches can serve as normalization strategies to enhance treatment. Furthermore, employing murine breast and fibrosarcoma tumor models as well as ultrasound shear wave elastography and contrast-enhanced ultrasound, we provide evidence that the approved nanomedicine Doxil can induce normalization in a dose-dependent manner by improving tumor perfusion as a result of tissue softening. Finally, we show that pretreatment with a normalizing dose of Doxil can improve the efficacy of immune checkpoint inhibition

Silencing of Growth Differentiation Factor-15 Promotes Breast Cancer Cell Invasion by Down-regulating Focal Adhesion Genes.

As metastasis accounts for most breast cancer (BC)-related deaths, identifying key players becomes research priority. Growth differentiation factor-15 (GDF15), a member of the transforming growth factor-β superfamily, is affected by the actin cytoskeleton and has been associated with cancer. However, its exact role in BC cell invasiveness is vague. GDF15 short-hairpin (shRNA)-mediated silencing was used to inhibit GDF15 expression in MCF-7 and MDA-MB-231 BC cells and gene expression of relevant focal adhesion (FA) genes, cell migration, invasion and tumor spheroid invasion were subsequently analyzed. GDF15 silencing promoted cell migration, cell invasion as well as tumor spheroid invasion and up-regulated urokinase plasminogen activator (uPA) and FA genes, integrin-linked kinase (ILK), LIM zinc finger domain containing 1 (LIMS1), α-parvin (PARVA), and RAS suppressor-1 (RSU1). Computational analysis of Cancer Genome Atlas BC dataset however, revealed no significant correlation between GDF15 expression and metastasis pointing towards a more complex molecular interplay between GDF15, actin cytoskeleton and FA-related genes which ultimately affects their expression pattern, in vivo. GDF15 suppresses BC cell invasion in vitro through down-regulation of FA genes but its role in BC is more complicated in vivo and warrants further investigation.The study was funded by the European Research Council, European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement no. 336839-ReEngineeringCancer

Oct4 is a gatekeeper of epithelial identity by regulating cytoskeletal organization in skin keratinocytes

Summary: Oct4 is a pioneer transcription factor regulating pluripotency. However, it is not well known whether Oct4 has an impact on epidermal cells. We generated OCT4 knockout clonal cell lines using immortalized human skin keratinocytes to identify a functional role for the protein. Here, we report that Oct4-deficient cells transitioned into a mesenchymal-like phenotype with enlarged size and shape, exhibited accelerated migratory behavior, decreased adhesion, and appeared arrested at the G2/M cell cycle checkpoint. Oct4 absence had a profound impact on cortical actin organization, with loss of microfilaments from the cell membrane, increased puncta deposition in the cytoplasm, and stress fiber formation. E-cadherin, β-catenin, and ZO1 were almost absent from cell-cell contacts, while fibronectin deposition was markedly increased in the extracellular matrix (ECM). Mapping of the transcriptional and chromatin profiles of Oct4-deficient cells revealed that Oct4 controls the levels of cytoskeletal, ECM, and differentiation-related genes, whereas epithelial identity is preserved through transcriptional and non-transcriptional mechanisms