Extracellular Matrix Stiffness Research Articles

Abstract B041: Investigating the impact of extracellular matrix stiffness on macrophage function and organoid growth in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of only 11%. Current treatments mostly target the cancer cells themselves and have not been able to significantly improve survival rates, meaning there is an urgent need for novel therapies. PDAC is unique among solid tumors because it exhibits extensive desmoplasia that is characterized by an abundance of extracellular matrix (ECM) components. In recent years, an increasing body of work has shown that cancer tissue is stiffer than normal tissue by a factor of 3 or more. This increase in ECM stiffness has been shown to play an important role in affecting cell-to-cell communication as well as cancer progression and chemoresistance . However, how ECM stiffness affects tumor-associated macrophages, an important component of the pancreatic tumor microenvironment (TME), is still understudied. Previous studies have demonstrated that macrophages are highly responsive to mechanical cues, with the mechanical environment influencing macrophage polarization and function in a context- and disease-dependent manner. Elucidating these potential changes in PDAC can provide us with insights on how stiffness affects therapeutic outcome. Healthy pancreatic tissue typically exhibits a storage modulus of approximately 0.5-1 kPa, whereas PDAC tissue can range from 4-10 kPa. To study macrophage responses to varying stiffness, we cultured RAW264.7 mouse macrophage cell lines on 2D substrates with stiffness levels of 0.2, 2, and 8 kPa. We observed that macrophages cultured on stiffer matrices exhibited a trend toward a pro-inflammatory phenotype, with increased expression of M1 markers such as iNOS, IL-1β, and IL-6, compared to those cultured on softer matrices. However, conditioned media from macrophages cultured on both soft and stiff substrates similarly reduced the size of PDAC organoids, indicating a lack of functional difference between these groups. These findings suggest the need for more sophisticated models that better replicate the PDAC TME to fully capture the complex cell-to-cell interactions that are not evident in 2D cultures. To address this limitation, we developed a 3D tri-culture tunable biomimetic mouse model comprising PDAC organoids, host-matched cancer-associated fibroblasts (CAFs), and macrophages. This novel model allows us to modulate the stiffness of the 3D co-culture system through the addition of collagen I and CAFs. Rheometry data confirmed that the stiff tri-culture model had a significantly higher storage modulus than the soft tri-culture model. Using qPCR, we investigated the role of mechanosensitive ion channels, specifically Transient Receptor Potential Vanilloid-type 4 (TRPV4) and Piezo1, in mediating macrophage mechanosensing in response to varying levels of matrix stiffness. This study highlights the importance of 3D models that can recapitulate biophysical cues of cancer and will allow us to better understand how macrophages respond to stiffness, which may give rise to new therapeutic targets and development of new treatments in the future. Citation Format: Bayan Mahgoub, Weikun Xiao, Eileen Fung, Shannon Mumenthaler, Reginald Hill. Investigating the impact of extracellular matrix stiffness on macrophage function and organoid growth in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B041.

Abstract A006: Investigating the effects of fibroblasts derived from the primary versus the metastatic organ on 3D biomimetic models of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer death in the United States with a 5-year survival rate of only 13%. Early diagnosis is very difficult and thus 53% of patients are diagnosed after metastasis has already occurred, with the liver being the most frequently affected site. Both primary tumors and metastases have dense desmoplastic stroma due to a high percentage of fibroblasts within the tumor microenvironment (TME). Previously, we developed a tunable 3D biomimetic model of the liver metastatic niche (LMN) to investigate how local fibroblasts affect tumor growth and chemoresistance in PDAC liver metastasis. This study aims to investigate the contributions of fibroblasts at the primary versus metastatic sites. We used matched primary PDAC and liver metastatic (LM) organoids derived from the Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre (“KPC”) genetically engineered mouse model. This allows us to compare the effects of primary PDAC cancer associated fibroblasts (CAFs) and liver metastasis associated fibroblasts (MAFs) on tumor cell growth and chemoresistance. Rheometry analyses were conducted to study the impact of local fibroblasts on the extracellular matrix (ECM) stiffness. Our results showed that both storage and loss moduli were enhanced only when PDAC CAFs were present in the co-cultured gels compared to gels with MAFs. Additionally, the 3D biomimetic model showed fibroblasts increased tumor growth and chemoresistance to gemcitabine, only when they were derived from the same site as the tumor cells. This highlighted the importance of studying the organ-specific microenvironment to develop better treatment options for patients with metastatic PDAC. To delve deeper into the molecular basis of these observations, we conducted RNA-sequencing to identify and compare the gene expression profiles in primary and metastatic tumor organoids exposed to conditioned media from CAFs and MAFs using a transwell membrane co-culture model. Our results showed significant differences in gene expression based on the type of fibroblasts co-cultured with the organoids. Moreover, we investigated the role of MAF-derived exosomes on LM growth and chemoresistance using exosome depletion and direct exosome addition to LM organoids. Lastly, we performed exosomes size and distribution characterizations using dynamic light scattering (DLS) analysis. Taken together, these findings highlight the critical role of the organ-specific microenvironment in tumor progression and the necessity of targeted therapies to improve patient outcomes in metastatic PDAC. Our results underscore the importance of developing a tunable 3D biomimetic model of the LMN, as it enables the testing of novel therapeutic strategies designed to disrupt interactions between MAFs and tumor cells, and potentially advancing treatments for metastatic PDAC. Citation Format: Mahsa Pahlavanneshan, Weikun Xiao, Eileen Fung, Vaidhyanathan Mahaganapathy, Chae Young Eun, Chang-Il Hwang, Shannon Mumenthaler, Reginald Hill. Investigating the effects of fibroblasts derived from the primary versus the metastatic organ on 3D biomimetic models of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A006.

Abstract A041: Deciphering the crosstalk between cancer cells and stroma that drives EMT- coupled fibrosis

Abstract In the tumor microenvironment (TME) cancer cells engage dynamically with the stromal component in promoting tumor progression and metastasis formation that are accompanied by intratumor fibrosis. Extensive extracellular matrix (ECM) remodeling and stiffness boost fibrosis which co-occurs with poor immune infiltration. Very little is known about the cross-interactions between tumor cells and stroma. A better understanding of this dynamic relationship may advance the development of new therapies. Transforming growth factor (TGF)-β signaling in both cancer epithelial cells and stroma is crucial for the establishment of intratumor fibrosis that enhances tumor progression. We previously discovered that in cancer cells, TGF-β signaling synergizes with RAS signal to regulate classical EMT transcription factors (Snail, Zeb1) and fibrogenic factors (Il11, Pdgf, Ctgfb, Wisp and others). This synergistic program is required for metastasis formation in pancreatic and lung adenocarcinoma (LUAD) models. However, how this dual program is affecting the stromal population in vivo remains unknown. Additionally, the role of each TGF-β dependent fibrogenic factor in regulating the stroma is still unclear. Using metastasis in vivo assays, we tested the metastatic ability of LUAD cells wild-type in comparison to Knock-Out cells targeting each one of the TGF-β dependent fibrogenic factors. We found that these factors are drivers of metastasis. More interestingly, these fibrogenic factors have a preferential effect depending on the metastatic site, suggesting that survival of the cancer cells in a certain tissue is dependent on the stromal niche within the site, which will act in favor or not of their growth. We also deeply characterize the stroma of micro-metastatic lesions by using high-throughput multiplex immunofluorescence to analyze the whole tissue TME at single cell resolution and found that lack of any of these fibrogenic factors compromise the establishment of a robust ECM at the very beginning of the metastatic colonization, to favor immune infiltration and consequently lead to cancer cell clearance. Together, these findings shed light on the potential role of these specific fibrogenic factors in metastasis, how their absence impacts the TME and how targeting these factors could be beneficial in metastatic disease. Citation Format: Elena Spina, Liangji Li, Junho Lee, Joan Massaguè. Deciphering the crosstalk between cancer cells and stroma that drives EMT- coupled fibrosis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A041.

Modulating Fibrotic Mechanical Microenvironment for Idiopathic Pulmonary Fibrosis Therapy.

Idiopathic pulmonary fibrosis (IPF) is exacerbated by injurious mechanical forces that destabilize the pulmonary mechanical microenvironment homeostasis, leading to alveolar dysfunction and exacerbating disease severity. However, given the inherent mechanosensitivity of fibrotic lungs, where type II alveolar epithelial cells (AEC IIs) are subjected to persistent stretching and overactivated myofibroblasts experience malignant interactions during mechanotransduction, it becomes imperative to develop effective strategies to modulate the pulmonary mechanical microenvironment. Herein, cyclo (RGDfC) peptide-decorated zeolitic imidazolate framework-8 nanoparticles (named ZDFPR NPs) are constructed to target and repair the aberrant mechanical force levels in pathological lungs. Specifically, reduces mechanical tension in AEC IIs by pH-responsive ZDFPR NPs that release zinc ions and 7, 8-dihydroxyflavone to promote alveolar repair and differentiation. Meanwhile, malignant interactions between myofibroblast contractility and extracellular matrix stiffness during mechanotransduction are disrupted by the fasudil inhibition ROCK signaling pathway. The results show that ZDFPR NPs successfully restored pulmonary mechanical homeostasis and terminated the fibrosis process in bleomycin-induced fibrotic mice. This study not only presents a promising strategy for modulating pulmonary mechanical microenvironment but also pioneers a novel avenue for IPF treatment.

TMIC-67. EMERGING ROLE OF EXTRACELLULAR MATRIX STIFFNESS AS A HALLMARK OF GLIOBLASTOMA: INSIGHT TO MECHANICAL MEMORY FORMATION AND PROLONGED INVASION IN THE TUMOR PERIPHERY

Abstract The extracellular matrix (ECM) plays a critical role in cancer progression, with its biophysical properties influencing cancer behavior. ECM stiffness can induce long-lasting adaptations in cancer cells, promoting sustained invasion through a process known as “mechanical memory” (MM). Glioblastoma (GBM) exhibits spatial heterogeneity in stiffness, with stiffer ECM found at the tumor periphery where invasive cells reside and promote recurrence. We hypothesize that increased stiffness at the tumor periphery primes GBM cells for sustained invasion through MM. METHODS Cell lines were established from image-guided human GBM samples from tumor margin and core (n=10 patients, 20 cell lines). Cells from random locations were conditioned in physiologically relevant extracellular matrices for two weeks. Human organotypic assay (n=3), Migration Assays, Atomic Force Microscopy (AFM), Immunocytochemistry, Western Blot, and RNA-sequencing (n=6) were performed. RESULTS GBM cells at higher stiff surfaces displayed increased migration (p<0.01). Magnetic Resonance Elastography (n=9) confirmed GBM heterogeneity, with softer cores (0.2-0.5kPa) transitioning to stiffer margins (>2kPa). Organotypic scaffolds mimicking the tumor margin promoted enhanced migration of GFP-labeled GBM cells. Margin cells and cells conditioned on stiffer matrices demonstrated higher migratory potential compared to their core and soft-conditioned counterparts (p<0.05). These trends persist over time and across multiple passages. ECM stiffness exposure causes nanomechanical, cytoskeletal, and transcriptional adaptations. AFM shows that cells from the margin (n=6) are softer (p<0.01) and more deformable (p<0.01). RNAseq identified upregulation of MM-specific genes in GBM margin cells. The mechanosensitive transcription factor YAP1 emerged as a key driver of MM. Notably, YAP1 is targetable by the FDA-approved drug Verteporfin. YAP1 expression was elevated in margin and stiff-conditioned cells (p<0.05). Stiff-conditioned cells and margin cells displayed increased sensitivity to Verteporfin treatment (p<0.05), with treatment significantly reducing margin migration compared to the core (p<0.001). CONCLUSION Stiffer GBM margins promote the formation of aggressive phenotypes through MM. Targeting YAP1 with Verteporfin, a readily available drug, presents a promising strategy to halt GBM invasion and improve patient outcomes.

Sex Differences in Response to Diet Enriched with Glutathione Precursors in the Aging Heart.

Common features of the aging heart are dysregulated metabolism, inflammation, and fibrosis. Elevated oxidative stress is another hallmark of cardiac aging that can exacerbate each of these conditions. We hypothesize that by increasing natural antioxidant levels (glutathione), we will improve cardiac function. Twenty-one-month-old mice were fed Glycine and N-Acetyl Cysteine (GlyNAC) (glutathione precursors)-supplemented or control diets for 12 weeks. Heart function was monitored longitudinally, and the exercise performance was determined at the end of the study. We found that the GlyNAC diet was beneficial for old male but not old female mice, leading to an increase of Ndufb8 expression (a subunit of the mitochondrial respiratory chain complex), and higher enzymatic activity for CPT1b and CrAT, two carnitine acyltransferases that are critical to cardiomyocyte metabolism. Although no quantifiable change of collagen turnover was detected, hearts from GlyNAC-fed old males exhibited a slight but significant enrichment in Fmod, a protein that can inhibit collagen fibril formation, possibly reducing extracellular matrix (ECM) stiffness and thus improving diastolic function. Cardiac diastolic function was modestly improved in males but not females, and surprisingly GlyNAC-fed female mice showed a decline in exercise performance. In summary, our work supports the concept that aged male and female hearts are phenotypically different. These basic differences may affect the response to pharmacological and diet interventions, including antioxidants.

Mitochondrial mechanotransduction through MIEF1 coordinates the nuclear response to forces.

Tissue-scale architecture and mechanical properties instruct cell behaviour under physiological and diseased conditions, but our understanding of the underlying mechanisms remains fragmentary. Here we show that extracellular matrix stiffness, spatial confinements and applied forces, including stretching of mouse skin, regulate mitochondrial dynamics. Actomyosin tension promotes the phosphorylation of mitochondrial elongation factor 1 (MIEF1), limiting the recruitment of dynamin-related protein 1 (DRP1) at mitochondria, as well as peri-mitochondrial F-actin formation and mitochondrial fission. Strikingly, mitochondrial fission is also a general mechanotransduction mechanism. Indeed, we found that DRP1- and MIEF1/2-dependent fission is required and sufficient to regulate three transcription factors of broad relevance-YAP/TAZ, SREBP1/2 and NRF2-to control cell proliferation, lipogenesis, antioxidant metabolism, chemotherapy resistance and adipocyte differentiation in response to mechanical cues. This extends to the mouse liver, where DRP1 regulates hepatocyte proliferation and identity-hallmark YAP-dependent phenotypes. We propose that mitochondria fulfil a unifying signalling function by which the mechanical tissue microenvironment coordinates complementary cell functions.

Substrate stiffness-dependent activation of Hippo pathway in cancer associated fibroblasts

The tumor microenvironment (TME) comprises a heterogenous cell population within a complex three-dimensional (3D) extracellular matrix (ECM). Stromal cells within this TME are altered by signaling cues from cancer cells to support uncontrolled tumor growth and invasion events. Moreover, the ECM also plays a fundamental role in tumor development through pathological remodeling, stiffening and interaction with TME cells. In healthy tissues, Hippo signaling pathway actively contributes to tissue growth, cell proliferation and apoptosis. However, in cancer, the Hippo signaling pathway is highly dysregulated, leading to nuclear translocation of the YAP/TAZ complex, which directly contributes to uncontrolled cell proliferation and tissue growth, and ECM remodeling and stiffening processes. Here, we compare the effect of increasing cell culture substrate stiffness, derived from tumor progression, upon the dysregulation of the Hippo signaling pathway in colorectal cancer-associated fibroblasts (CAFs) and normal colorectal fibroblasts (NFs). We correlate the dysregulation of Hippo pathway with the magnitude of the traction forces exerted by healthy and malignant stromal cells. We found that ECM stiffening is crucial in Hippo pathway dysregulation in CAFs, but not in normal fibroblasts.

YAP/TAZ drives Notch and angiogenesis mechanoregulation in silico

Endothelial cells are key players in the cardiovascular system. Among other things, they are responsible for sprouting angiogenesis, the process of new blood vessel formation essential for both health and disease. Endothelial cells are strongly regulated by the juxtacrine signaling pathway Notch. Recent studies have shown that both Notch and angiogenesis are influenced by extracellular matrix stiffness; however, the underlying mechanisms are poorly understood. Here, we addressed this challenge by combining computational models of Notch signaling and YAP/TAZ, stiffness- and cytoskeleton-regulated mechanotransducers whose activity inhibits both Dll4 (Notch ligand) and LFng (Notch-Dll4 binding modulator). Our simulations successfully mimicked previous experiments, indicating that this YAP/TAZ-Notch crosstalk elucidates the Notch and angiogenesis mechanoresponse to stiffness. Additional simulations also identified possible strategies to control Notch activity and sprouting angiogenesis via cytoskeletal manipulations or spatial patterns of alternating stiffnesses. Our study thus inspires new experimental avenues and provides a promising modeling framework for further investigations into the role of Notch, YAP/TAZ, and mechanics in determining endothelial cell behavior during angiogenesis and similar processes.

Calciprotein particles induce arterial stiffening ex vivo and impair vascular cell function

Calciprotein particles (CPPs) are an endogenous buffering system, clearing excessive amounts of Ca2+ and PO43- from the circulation and thereby preventing ectopic mineralization. CPPs circulate as primary CPPs (CPP1), which are small spherical colloidal particles, and can aggregate to form large, crystalline, secondary CPPs (CPP2). Even though it has been reported that CPPs are toxic to vascular smooth muscle cells (VSMC) in vitro, their effect(s) on the vasculature remain unclear. Here we have shown that CPP1, but not CPP2, increased arterial stiffness ex vivo. Interestingly, the effects were more pronounced in the abdominal infrarenal aorta compared to the thoracic descending aorta. Further, we demonstrated that CPP1 affected both endothelial and VSMC function, impairing vasorelaxation and contraction respectively. Concomitantly, arterial glycosaminoglycan accumulation was observed as well, which is indicative of an increased extracellular matrix stiffness. However, these effects were not observed in vivo. Hence, we concluded that CPP1 can induce vascular dysfunction.

The biomechanics of turgor pressure

If you ever forget to water your houseplant, you may find its leaves getting soft and droopy - if you water it again in time, the leaves may stiffen, spring back up, and resist gravity. During this recovery, plant cells absorb water and build up an intracellular pressure, called turgor pressure, similar to inflating a balloon. Turgor pressure is an intrinsic component of plant physiology, and its biomechanical role as the 'hydroskeleton' is generally appreciated either statically in structural stability, like leaves resisting gravity, or dynamically in rapid motions, like Venus flytrap snapping, Mimosa closing, or stomatal opening. Slow but non-static processes like plant cell expansion also rely on turgor pressure, and it has been increasingly realized that turgor pressure and water fluxes in plant tissues play active roles in plant growth and morphogenesis. But where does turgor pressure come from, and how does it interact with other biomechanical properties of a plant cell? This primer aims to answer these questions by taking a brief tour from osmosis, to pressure vessel theory, then plant cell rheology. Although this primer is centered around intracellular pressure in plant cells, the biomechanical concepts are generally applicable to other organisms like bacteria and fungi, and even animal cells, which do not have cell walls, but are either embedded in stiff extracellular matrix (ECM) or are contracting (see the following section). To explain some of these concepts, we included a few equations, most of which are not hard to derive at all. We encourage readers to check the included examples, try for themselves, and look up derivations in suggested further reading materials.

Geometric constraint of mechanosensing by modification of hydrogel thickness prevents stiffness-induced differentiation in bone marrow stromal cells.

Extracellular matrix (ECM) stiffness is fundamental in cell division, movement and differentiation. The stiffness that cells sense is determined not only by the elastic modulus of the ECM material but also by ECM geometry and cell density. We hypothesized that these factors would influence cell traction-induced matrix deformations and cellular differentiation in bone marrow stromal cells (BMSCs). To achieve this, we cultivated BMSCs on polyacrylamide hydrogels that varied in elastic modulus and geometry and measured cell spreading, cell-imparted matrix deformations and differentiation. At low cell density BMSCs spread to a greater extent on stiff compared with soft hydrogels, or on thin compared with thick hydrogels. Cell-imparted matrix deformations were greater on soft compared with stiff hydrogels or thick compared with thin hydrogels. There were no significant differences in osteogenic differentiation relative to hydrogel elastic modulus and thickness. However, increased cell density and/or prolonged culture significantly reduced matrix deformations on soft hydrogels to levels similar to those on stiff substrates. This suggests that at high cell densities cell traction-induced matrix displacements are reduced by both neighbouring cells and the constraint imposed by an underlying stiff support. This may explain observations of the lack of difference in osteogenic differentiation as a function of stiffness.

Matrix stiffening from collagen fibril density and alignment modulates YAP-mediated T-cell immune suppression

T-cells are essential components of the immune system, adapting their behavior in response to the mechanical environments they encounter within the body. In pathological conditions like cancer, the extracellular matrix (ECM) often becomes stiffer due to increased density and alignment of collagen fibrils, which can have a significant impact on T-cell function. In this study, we explored how these ECM properties—density and fibrillar alignment—affect T-cell behavior using three-dimensional (3D) collagen matrices that mimic these conditions. Our results show that increased matrix stiffness, whether due to higher density or alignment, significantly suppresses T-cell activation, reduces cytokine production, and limits proliferation, largely through enhanced YAP signaling. Individually, matrix alignment appears to lower actin levels in activated T-cells and changes migration behavior in both resting and activated T-cells, an effect not observed in matrices with randomly oriented fibrils. Notably, inhibiting YAP signaling was able to restore T-cell activation and improve immune responses, suggesting a potential strategy to boost the effectiveness of immunotherapy in stiff ECM environments. Overall, this study provides new insights into how ECM characteristics influence T-cell function, offering potential avenues for overcoming ECM-induced immunosuppression in diseases such as cancer.

Substrate stiffness modulates extracellular vesicles’ release in a triple-negative breast cancer model

Aim: The microenvironment effect on the tumoral-derived Extracellular Vesicle release, which is of significant interest for biomedical applications, still represents a rather unexplored field. The aim of the present work is to investigate the interrelation between extracellular matrix (ECM) stiffness and the release of small EVs from cancer cells. Here, we focus on the interrelation between the ECM and small extracellular vesicles (sEVs), specifically investigating the unexplored aspect of the influence of ECM stiffness on the release of sEVs. Methods: We used a well-studied metastatic Triple-Negative Breast Cancer (TNBC) cell line, MDA-MB-231, as a model to study the release of sEVs by cells cultured on substrates of different stiffness. We have grown MDA-MB-231 cells on two collagen-coated polydimethylsiloxane (PDMS) substrates at different stiffness (0.2 and 3.6 MPa), comparing them with a hard glass substrate as control, and then we isolated the respective sEVs by differential ultracentrifugation. After checking the cell growth conditions [vitality, morphology by immunofluorescence microscopy, stiffness by atomic force microscopy (AFM)], we took advantage of a multi-parametric approach based on complementary techniques (AFM, Nanoparticle Tracking Analysis, and asymmetric flow field flow fractionation with a multi-angle light scattering detector) to characterize the TNBC-derived sEV obtained in the different substrate conditions. Results: We observe that soft substrates induce TNBC cell softening and rounding. This effect promotes the release of a high number of larger sEVs. Conclusion: Here, we show the role of ECM physical properties in the regulation of sEV release in a TNBC model. While the molecular mechanisms regulating this effect need further investigation, our report represents a step toward an improved understanding of ECM-cell-sEVs crosstalk.

Evaluating ECM stiffness and liver cancer radiation response via shear-wave elasticity in 3D culture models

BackgroundThe stiffness of the tumor microenvironment (TME) directly influences cellular behaviors. Radiotherapy (RT) is a common treatment for solid tumors, but the TME can impact its efficacy. In the case of liver cancer, clinical observations have shown that tumors within a cirrhotic, stiffer background respond less to RT, suggesting that the extracellular matrix (ECM) stiffness plays a critical role in the development of radioresistance.MethodsThis study explored the effects of ECM stiffness and the inhibition of lysyl oxidase (LOX) isoenzymes on the radiation response of liver cancer in a millimeter-sized three-dimensional (3D) culture. We constructed a cube-shaped ECM-based millimeter-sized hydrogel containing Huh7 human liver cancer cells. By modulating the collagen concentration, we produced two groups of samples with different ECM stiffnesses to mimic the clinical scenarios of normal and cirrhotic livers. We used a single-transducer system for shear-wave-based elasticity measurement, to derive Young’s modulus of the 3D cell culture to investigate how the ECM stiffness affects radiosensitivity. This is the first demonstration of a workflow for assessing radiation-induced response in a millimeter-sized 3D culture.ResultsIncreased ECM stiffness was associated with a decreased radiation response. Moreover, sonoporation-assisted LOX inhibition with BAPN (β-aminopropionitrile monofumarate) significantly decreased the initial ECM stiffness and increased RT-induced cell death. Inhibition of LOX was particularly effective in reducing ECM stiffness in stiffer matrices. Combining LOX inhibition with RT markedly increased radiation-induced DNA damage in cirrhotic liver cancer cells, enhancing their response to radiation. Furthermore, LOX inhibition can be combined with sonoporation to overcome stiffness-related radioresistance, potentially leading to better treatment outcomes for patients with liver cancer.ConclusionsThe findings underscore the significant influence of ECM stiffness on liver cancer’s response to radiation. Sonoporation-aided LOX inhibition emerges as a promising strategy to mitigate stiffness-related resistance, offering potential improvements in liver cancer treatment outcomes.

Abstract C025: Enhanced efficacy of gemcitabine analogs and AZD 1775 combination therapy in patient-derived organoids

Abstract Pancreatic cancer (PCa), specifically Pancreatic Adenocarcinoma (PDAC), is projected to become the second leading cause of cancer related death by 2040. Currently, the 5-year survival rate for pancreatic cancer is between 7% - 12%. The standard treatment options for PDAC are chemotherapy and surgical resection. However, pancreatic cancer presents multiple challenges to successful treatment. First, PDAC is usually diagnosed in the advanced stages of the disease, even if diagnosed early only a small fraction of patients are eligible for surgical resection, about 13% - 15%. Second, PDAC is characterized by a stiff extracellular matrix. This stiffness can lead to poor vascularization, diminished immune responses and cell migration/metastasis. Finally, PDAC is notorious for being extremely resistant to chemotherapy regiments. In addition to these challenges, health disparities add to the dismal prognosis of pancreatic cancer. Examples of these disparities include unequal access to healthcare facilities and screenings, awareness of disease risk factors and symptoms and socioeconomic factors. Differences in genetics can influence the various outcomes seen in PDAC in terms of health disparities. Certain genetic mutations, some more prevalent in certain ethnic groups, can increase the risk for PDAC. Variation in tumor heterogeneity and genetic factors impacting drug metabolism can influence patient outcomes. To overcome some of these challenges, we have developed and tested novel gemcitabine analogs, conjugated with various fatty acid chains in collagen-based 3D gel, Patient Derived Organoid (PDO) models. The collagen-based gel allows tuning of the physical stiffness by varying the gelatin percentage, creating a more realistic tumor microenvironment with the intratumoral pressure can be upwards of 10 kPa. We have tested the gemcitabine analogs on patient derived cells, G43 and G46, from black and white patients respectively, in both soft and stiff matrices. Our results indicated that medium to long fatty acid chains (8, 10 and 18 carbon chains) have increased effectiveness, especially in softer matrices. Comparatively, G46 cells exhibited higher drug resistance across all environments than G43 cells. Recent studies have shown that combining Gemcitabine with AZD 1775, a WEE1 inhibitor may have synergistic effects. We tested the combinations of Gemcitabine analogs and AZD-1775 on both G43 and G46 patient derived cells in 2D and 3D environments. Our data suggests that short to medium length fatty acid chains (2-8 carbons) provide optimal combinational effectiveness. The combined treatment also demonstrated promising results in increasing treatment effectiveness in G46, which are typically chemoresistant. Further studies will examine the influence that tumor microenvironment may have on PDAC response to the combination therapy, as well as intrinsic difference between black patient-derived cells and white patient-derived cells and their sensitivity or resistance to treatment. Citation Format: Jonathan Barajas, Edward Agyare, Xueyou Zhu, Sherise Rogers, Ba Xuan Hoang, Bo Han. Enhanced efficacy of gemcitabine analogs and AZD 1775 combination therapy in patient-derived organoids [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C025.

Genetic variation drives cancer cell adaptation to ECM stiffness

The progression of many solid tumors is accompanied by temporal and spatial changes in the stiffness of the extracellular matrix (ECM). Cancer cells adapt to soft and stiff ECM through mechanisms that are not fully understood. It is well known that there is significant genetic heterogeneity from cell to cell in tumors, but how ECM stiffness as a parameter might interact with that genetic variation is not known. Here, we employed experimental evolution to study the response of genetically variable and clonal populations of tumor cells to variable ECM stiffness. Proliferation rates of genetically variable populations cultured on soft ECM increased over a period of several weeks, whereas clonal populations did not evolve. Tracking of DNA barcoded cell lineages revealed that soft ECM consistently selected for the same few variants. These data provide evidence that ECM stiffness exerts natural selection on genetically variable tumor populations. Soft-selected cells were highly migratory, with enriched oncogenic signatures and unusual behaviors such as spreading and traction force generation on ECMs with stiffness as low as 1 kPa. Rho-regulated cell spreading was found to be the directly selected trait, with yes-associated protein 1 translocation to the nucleus mediating fitness on soft ECM. Overall, these data show that genetic variation can drive cancer cell adaptation to ECM stiffness.

High Mechanical Conditioning by Tumor Extracellular Matrix Stiffness Is a Predictive Biomarker for Antifibrotic Therapy in HER2-Negative Breast Cancer.

Tumor progression has been linked to stiffening of the extracellular matrix caused by fibrosis. Cancer cells can be mechanically conditioned by stiff extracellular matrix, exhibiting a 1,004-gene signature [mechanical conditioning (MeCo) score]. Nintedanib has demonstrated antifibrotic activity in idiopathic pulmonary fibrosis. This study explores nintedanib's antifibrotic effect on breast cancer outcomes. We present long-term follow-up and analysis of a neoadjuvant randomized phase II trial in early HER2-negative breast cancer. Patients (N = 130) underwent a baseline biopsy and received 12 paclitaxel courses alone (control arm) or in combination with nintedanib (experimental arm). The tumor MeCo score was determined by RNA sequencing. The primary aim was to assess nintedanib's impact on event-free survival based on MeCo scores. Follow-up data were retrieved from 111 patients; 75 baseline and 24 post-run-in phase samples were sequenced. After median follow-up of 9.67 years, median event-free survival was not statistically different between arms (P = 0.37). However, in the control arm, high- versus low-MeCo patients had a statistically higher relapse risk: HR = 0.21; P = 0.0075. This risk was corrected by nintedanib in the experimental arm: HR = 0.37; P = 0.16. Nintedanib demonstrated pharmacodynamic engagement, reducing the MeCo score by 25% during the run-in phase (P < 0.01). Patients with low MeCo after run-in had the best long-term prognosis (HR = 0.087; P = 0.03). High MeCo is predictive of poor outcomes in HER2-negative early breast cancer, although this risk can be mitigated by nintedanib, which is able to specifically reduce MeCo.

The modulation of lung cancer cell motility by Calponin 3 is achieved through its ability to sense and respond to changes in substrate stiffness.

The influence of extracellular matrix (ECM) stiffness on cell behavior is a well-established phenomenon. Tumor development is associated with the stiffening of the ECM. However, the understanding of the role of biomechanical behavior and mechanotransduction pathways in the oncogenesis of tumor cells remains limited. In this study, we constructed in vitro models using Polydimethylsiloxane substrates to create soft and stiff substrates. We then evaluated the migration of lung cancer cells A549 using video-microscopy and transwell assays. The mechanical properties were assessed through the utilization of atomic force microscopy, Optical Magnetic Twisting Cytometry, and traction force analysis. Additionally, the expression of Calponin 3 (CNN3) was evaluated using reverse transcription‑quantitative PCR and immunofluorescence techniques. Our observations indicate that the presence of a stiff substrate enhances A549 motility, as evidenced by increased stiffness and traction force in A549 cells on the stiff substrate. Furthermore, we observed a decrease in CNN3 expression in A549 cells on the stiff substrate. Notably, when CNN3 was overexpressed, it effectively inhibited the migration and invasion of A549 cells on the stiff substrate. The results of our study provide novel perspectives on the mechanisms underlying cancer cell migration in response to substrate mechanical properties.

Melatonin mitigates matrix stiffness-induced intervertebral disk degeneration by inhibiting reactive oxygen species and melatonin receptors mediated PI3K/AKT/NF-κB pathway.

Intervertebral disk degeneration (IVDD) may lead to an increase in extracellular matrix (ECM) stiffness, potentially contributing to the progression of the disease. Melatonin reportedly mitigates IVDD; however, its potential to attenuate elevated matrix stiffness-induced IVDD remains unexplored. Therefore, we aimed to investigate whether melatonin can alleviate the progression of IVDD triggered by increased matrix stiffness and elucidate its underlying mechanisms. Nucleus pulposus (NP) tissues were collected from patients, and ECM stiffness, reactive oxygen species (ROS) levels, apoptosis rates, and P65 expression in these tissues with varying Pfirrmann scores were determined. In vitro experiments were conducted to investigate the effects of melatonin on various pathophysiological mechanisms within the NP cells cultured on soft substrates with differing stiffness levels. Our findings revealed a positive correlation between ECM stiffness in human NP tissue and degree of IVDD. In addition, phosphorylation of P65 exhibited a strong association with matrix stiffness. Enhanced levels of ROS and cellular apoptosis were observed within degenerated intervertebral disks. In vitro experiments demonstrated that melatonin significantly inhibited catabolism and apoptosis induced by stiff matrices, along with elevated ROS levels. Furthermore, we observed that melatonin inhibited NP cell catabolism and apoptosis by reducing the melatonin receptors mediated activation of the PI3K/AKT and nuclear factor-kappa B (NF-κB) pathways. Also, we found that the reduction of ROS by melatonin can assist in inhibiting the activation of the NF-κB pathway. The outcomes of the in vivo experiments corroborated the results of the in vitro experiments, illustrating that melatonin treatment could alleviate the compression-induced upregulation of matrix stiffness in NP and IVDD. Collectively, melatonin can potentially alleviate high matrix stiffness-induced IVDD by reducing intracellular ROS levels and inhibiting the PI3K/AKT/NF-κB pathway.NEW & NOTEWORTHY Melatonin mitigates intervertebral disk degeneration (IVDD) induced by matrix stiffness through reactive oxygen species (ROS) reduction. Matrix stiffness is related to increased nucleus pulposus cell ROS, apoptosis, and degeneration. Melatonin inhibits PI3K/AKT/NF-κB pathways via melatonin receptors in a stiff matrix environment. In vivo, melatonin restores disk height and alleviates IVDD progression.