Fibrillar Collagen Research Articles

In vivo multiphoton multiparametric 3D quantification of human skin aging on forearm and face

Quantifying skin aging changes and characterizing its 3D structure and function in a non-invasive way is still a challenging area of research, constantly evolving with the development of imaging methods and image analysis tools. In vivo multiphoton imaging offers means to assess skin constituents in 3D, however prior skin aging studies mostly focused on 2D analyses of dermal fibers through their signals’ intensities or densities. In this work, we designed and implemented multiphoton multiparametric 3D quantification tools for in vivo human skin pigmentation and aging characterization. We first demonstrated that despite the limited field of view of the technic, investigation of 2 regions of interest (ROIs) per zone per volunteer is a good compromise in assessing 3D skin constituents in both epidermis and superficial dermis. We then characterized skin aging on different UV exposed areas—ventral and dorsal forearms, face. The three major facts of aging that are epidermal atrophy, the dermal–epidermal junction (DEJ) flattening and dermal elastosis can be non-invasively quantified and compared. Epidermal morphological changes occur late and were only objectified between extreme age groups. Melanin accumulation in suprabasal layers with age and chronic exposure on ventral and dorsal forearms is less known and appears earlier. Superficial dermal aging changes are mainly elastin density increase, with no obvious change in collagen density, reflected by SHGto2PEF ratio and SAAID index decrease and ImbrN index increase on all skin areas. Analysis of the z-dermal distribution of these parameters highlighted the 2nd 20 µm thickness normalized dermal sub-layer, that follows the DEJ shape, as exhibiting the highest aging differences. Moreover, the 3D ImbrN index allows refining the share of photoaging in global aging on face and the 3D SAAID index on forearm, which elastin or fibrillar collagens densities alone do not allow. Photoaging of the temple area evolves as a function of chronic exposure with a more pronounced increase in elastin density, also structurally modified from thin and straight elastic fibers in young volunteers to dense and compact pattern in older ones. More generally, multiphoton multiparametric 3D skin quantification offers rich spatial information of interest in assessing normal human skin condition and its pathological, external environment or product induced changes.

The complex genetic basis of fibromuscular dysplasia, a systemic arteriopathy associated with multiple forms of cardiovascular disease.

Artery stenosis is a common cause of hypertension and stroke and can be due to atherosclerosis accumulation in the majority of cases and in a small fraction of patients to arterial fibromuscular dysplasia (FMD). Artery stenosis due to atherosclerosis is widely studied with known risk factors (e.g. increasing age, male gender, and dyslipidemia) to influence its etiology, including genetic factors. However, the causes of noninflammatory and nonatherosclerotic stenosis in FMD are less understood. FMD occurs predominantly in early middle-age women, a fraction of the population where cardiovascular risk is different and understudied. FMD arteriopathies are often diagnosed in the context of hypertension and stroke and co-occur mainly with spontaneous coronary artery dissection, an atypical cause of acute myocardial infarction. In this review, we provide a comprehensive overview of the recent advances in the understanding of molecular origins of FMD. Data were obtained from genetic studies using complementary methodological approaches applied to familial, syndromic, and sporadic forms of this intriguing arteriopathy. Rare variation analyses point toward mechanisms related to impaired prostacyclin signaling and defaults in fibrillar collagens. The study of common variation, mainly through a recent genome-wide association study, describes a shared genetic link with blood pressure, in addition to point at potential risk genes involved in actin cytoskeleton and intracellular calcium homeostasis supporting impaired vascular contraction as a key mechanism. We conclude this review with future strategies and approaches needed to fully understand the genetic and molecular mechanisms related to FMD.

Three-dimensional fine structure of fibroblasts and other mesodermally derived tissues in the dermis of adults of the Bahamas lancelet (Chordata, Cephalohordata), as seen by serial block-face scanning electron microscopy.

Tissues of adult cephalochordates include sparsely distributed fibroblasts. Previous work on these cells has left unsettled such questions as their developmental origin, range of functions, and even their overall shape. Here, we describe fibroblasts of a cephalochordate, the Bahamas lancelet, Asymmetron lucayanum, by serial block-face scanning electron microscopy to demonstrate their three-dimensional (3D) distribution and fine structure in a 0.56-mm length of the tail. The technique reveals in detail their position, abundance, and morphology. In the region studied, we found only 20 fibroblasts, well separated from one another. Each was strikingly stellate with long cytoplasmic processes rather similar to those of a vertebrate telocyte, a possibly fortuitous resemblance that is considered in the discussion section. In the cephalochordate dermis, the fibroblasts were never linked with one another, although they occasionally formed close associations of unknown significance with other cell types. The fibroblasts, in spite of their name, showed no signs of directly synthesizing fibrillar collagen. Instead, they appeared to be involved in the production of nonfibrous components of the extracellular matrix-both by the release of coarsely granular dense material and by secretion of more finely granular material by the local breakdown of their cytoplasmic processes. For context, the 3D structures of two other mesoderm-derived tissues (the midline mesoderm and the posteriormost somite) are also described for the region studied.

Mechanical Confinement and DDR1 Signaling Synergize to Regulate Collagen-Induced Apoptosis in Rhabdomyosarcoma Cells.

Fibrillar collagens promote cell proliferation, migration, and survival in various epithelial cancers and are generally associated with tumor aggressiveness. However, the impact of fibrillar collagens on soft tissue sarcoma behavior remains poorly understood. Unexpectedly, this study finds that fibrillar collagen‐related gene expression is associated with favorable patient prognosis in rhabdomyosarcoma. By developing and using collagen matrices with distinct stiffness and in vivo‐like microarchitectures, this study uncovers that the activation of DDR1 has pro‐apoptotic and of integrin β1 pro‐survival function, specifically in 3D rhabdomyosarcoma cell cultures. It demonstrates that rhabdomyosarcoma cell‐intrinsic or extrinsic matrix remodeling promotes cell survival. Mechanistically, the 3D‐specific collagen‐induced apoptosis results from a dual DDR1‐independent and a synergistic DDR1‐dependent TRPV4‐mediated response to mechanical confinement. Altogether, these results indicate that dense microfibrillar collagen‐rich microenvironments are detrimental to rhabdomyosarcoma cells through an apoptotic response orchestrated by the induction of DDR1 signaling and mechanical confinement. This mechanism helps to explain the preference of rhabdomyosarcoma cells to grow in and metastasize to low fibrillar collagen microenvironments such as the lung.

MT1-MMP and ADAM10/17 exhibit a remarkable overlap of shedding properties.

Membrane-type-I matrix metalloproteinase (MT1-MMP) is one of six human membrane-bound MMPs and is responsible for extracellular matrix remodelling by degrading several substrates like fibrillar collagens, including types I-III, or fibronectin. Moreover, MT1-MMP was described as a key player in cancer progression and it is involved in various inflammatory processes, as well as in the pathogenesis of Alzheimer's disease (AD). The membrane-tethered metalloprotease meprin β as well as a disintegrin and metalloproteinase 10 (ADAM10) and ADAM17 are also associated with these diseases. Interestingly, meprin β, ADAM10/17 and MT1-MMP also have a shared substrate pool including the interleukin-6 receptor and the amyloid precursor protein. We investigated the interaction of these proteases, focusing on a possible connection between MT1-MMP and meprin β, to elucidate the potential mutual regulations of both enzymes. Herein, we show that besides ADAM10/17, MT1-MMP is also able to shed meprin β from the plasma membrane, leading to the release of soluble meprin β. Mass spectrometry-based cleavage site analysis revealed that the cleavage of meprin β by all three proteases occurs between Pro602 and Ser603 , N-terminal of the EGF-like domain. Furthermore, only inactive human pro-meprin β is shed by MT1-MMP, which is again in accordance with the shedding capability observed for ADAM10/17. Vice versa, meprin β also appears to shed MT1-MMP, indicating a complex regulatory network. Further studies will elucidate this well-orchestrated proteolytic web under distinct conditions in health and disease and will possibly show whether the loss of one of the above-mentioned sheddases can be compensated by the other enzymes.

Electroacupuncture remodels the extracellular matrix and promotes synaptic plasticity in a mouse model of depression

Depression is a chronic and recurrent disease without satisfactory treatment strategies. The therapeutic effect of acupuncture is well known in Traditional Chinese medicine (TCM) due to its unique non-pharmacological nature. Electroacupuncture (EA) for antidepressant therapy has been widely recognized and used in clinic. In this study, the lipopolysaccharide (LPS)-induced depression mice model was used to evaluate the anti-depressant effects of EA treatment. Open Field Test (OFT), Force Swimming Test (FST), and Sucrose Preference Test (SPT) were utilized to detect the ethological alterations in mice. The transcriptology technique was used to evaluate the changes in the hippocampal transcriptome in different groups. We measured protein levels using Western blot and immunohistochemistry. Our data showed that LPS induced ethological alterations in mice and enhanced the gene expression related to gene ontology such as the banded collagen fibril, fibrillar collagen trimer, and collagen fibril organization, pathways such as collagen chain trimerization, collagen biosynthesis and modifying enzymes, and collagen formation. EA could reverse the ECM deposition by inhibiting collagen type Ⅳ trimer, extracellular matrix organization, and collagen formation. EA could enhance the MMP1 and MMP9 expression and promote synaptic plasticity. These data indicated that EA possesses an antidepressant effect, this may achieve by increasing MMPs expression and then remodeling the ECM surrounding the neurons, ultimately repairing neural circuits and promoting synaptic plasticity.

Suppression of resistance to aminolevulinic acid-based photodynamic therapy in esophageal cell lines by administration of iron chelators in collagen type I matrices

Purpose Photodynamic therapy (PDT) utilizes visible light to activate the cytotoxic effects of photosensitizing drugs. PDT protocols require optimization to overcome treatment resistance and induce a beneficial anti-tumor immune response. The aim of this study was to examine the possibility to suppress the resistance of esophageal cell lines to aminolevulinic acid (ALA)-PDT by administration of iron chelators to induce sufficient cell cytotoxicity under pathophysiologically relevant conditions, mimicking the advanced stages of cancer. Materials and methods Effects of ALA-PDT in combination with iron chelators were compared in three esophageal cell lines in conventional monolayers and in 3 D cultures based on collagen type I. Modified colony assay and fluorescence-based live cell imaging, respectively were applied. The latter was used also to test the capability of pre-polarized macrophages to interact with cancer cells subjected to ALA-PDT with or without iron chelators. Results Iron chelators were effective in the enhancement of ALA-PDT in all cell lines under both culture conditions. Fluorescence evaluation of cell viability in 3 D cultures indicated the contribution of apoptotic cell death after ALA-PDT, both with and without iron chelators. Engulfment of remnants of dead cancer cells by macrophages in 2 D cultures was indicated, however, the interaction between macrophages and cancer cells in 3 D cultures subjected to ALA-PDT with or without iron chelators was not present. Conclusions The potential of iron chelators to enhance ALA-PDT was maintained in 3 D collagen matrices. Although PDT dose (ALA concentration, light exposure time) required modification in a cell line-dependent manner to achieve a comparable effect of PDT alone in conventional monolayers and in collagen matrices, the potential of iron chelators to suppress the resistance of esophageal cells to ALA-PDT was not influenced by a fibrillar collagen matrix.

A Biomimetic High Throughput Model of Cancer Cell Spheroid Dissemination onto Aligned Fibrillar Collagen.

Cell dissemination during tumor development is a characteristic of cancer metastasis. Dissemination from three-dimensional spheroid models on extracellular matrices designed to mimic tissue-specific physiological microenvironments may allow us to better elucidate the mechanism behind cancer metastasis and the response to therapeutic agents. The orientation of fibrillar collagen plays a key role in cellular processes and mediates metastasis through contact-guidance. Understanding how cells migrate on aligned collagen fibrils requires in vitro assays with reproducible and standardized orientation of collagen fibrils on the macro-to-nanoscale. Herein, we implement a spheroid-based migration assay, integrated with a fibrillar type I collagen matrix, in a manner compatible with high throughput image acquisition and quantitative analysis. The migration of highly proliferating U2OS osteosarcoma cell spheroids onto an aligned fibrillar type I collagen matrix was quantified. Cell dissemination from the spheroid was polarized with increased invasion in the direction of fibril alignment. The resulting area of cell dissemination had an aspect ratio of 1.2 ± 0.1 and an angle of maximum invasion distance of 5° ± 44° relative to the direction of collagen fibril alignment. The assay described here can be applied to a fully automated imaging and analysis pipeline for the assessment of tumor cell migration with high throughput screening.

Pharmacologic inhibition of discoidin domain Receptor 2 (DDR2) sensitizes homologous recombination proficient ovarian cancer models to treatment with olaparib (245)

Pharmacologic inhibition of discoidin domain Receptor 2 (DDR2) sensitizes homologous recombination proficient ovarian cancer models to treatment with olaparib (245)

Low Methylation of Matrix Metalloproteinase 1 (MMP1) is Associated with Preterm Labour in Malaysian Mothers

Preterm births comprise 10.6% of livebirths worldwide and account for 35% of deaths among newborn babies. Understanding DNA methylation may offer basic knowledge in the understanding of pathogenesis of preterm labour. The study was undertaken to determine DNA methylation of matrix metalloproteinase 1 (MMP1) promoter in term and preterm labour using methylation-specific polymerase chain reaction (MSP). Thirty maternal venous blood samples (n=15 each) of term and preterm labour was subjected to bisulfite treatment prior to MSP. This result was then validated using DNA sequencing. Evaluation of the sequencing results by CpG islands analysis was performed using the ClustalW and SPSS software. Primers for MMP1 were located between -1226 and -1378 upstream from the transcription start site (TSS) that consisted five CpG islands. Preterm labour group had significantly lower methylated CpG islands with 39 out of total 75 (52%) compared to the term labour that has 49 out of 75 methylated CpG islands (65.33%) (t=0.694, p<0.05). Methylation occurred in 4 out of 5 methylated CpG islands in the MMP1 promoter where it only involved 2 preterm samples (13.33%) and 7 term samples (46.47%). This data suggested there were significant lower percentage of methylated MMP1 in preterm labour. Higher percentage of methylated MMP1 as observed in the term labour, will probably reduce the expression of MMP1, thus maintaining fibrillar collagen strength on the amniotic membrane and subsequently maintain the pregnancy till term. In conclusion, preterm labour has higher percentage of methylated CpG compared with term labour in MMP1 gene.

Using sequence data to predict the self-assembly of supramolecular collagen structures

Collagen fibrils are the major constituents of the extracellular matrix, which provides structural support to vertebrate connective tissues. It is widely assumed that the superstructure of collagen fibrils is encoded in the primary sequences of the molecular building blocks. However, the interplay between large-scale architecture and small-scale molecular interactions makes the ab initio prediction of collagen structure challenging. Here, we propose a model that allows us to predict the periodic structure of collagen fibers and the axial offset between the molecules, purely on the basis of simple predictive rules for the interaction between amino acid residues. With our model, we identify the sequence-dependent collagen fiber geometries with the lowest free energy and validate the predicted geometries against the available experimental data. We propose a procedure for searching for optimal staggering distances. Finally, we build a classification algorithm and use it to scan 11 data sets of vertebrate fibrillar collagens, and predict the periodicity of the resulting assemblies. We analyzed the experimentally observed variance of the optimal stagger distances across species, and find that these distances, and the resulting fibrillar phenotypes, are evolutionary well preserved. Moreover, we observed that the energy minimum at the optimal stagger distance is broad in all cases, suggesting a further evolutionary adaptation designed to improve the assembly kinetics. Our periodicity predictions are not only in good agreement with the experimental data on collagen molecular staggering for all collagen types analyzed, but also for synthetic peptides. We argue that, with our model, it becomes possible to design tailor-made, periodic collagen structures, thereby enabling the design of novel biomimetic materials based on collagen-mimetic trimers.

Stress-controlled shear flow alignment of collagen type I hydrogel systems

Disease research and drug screening platforms require in vitro model systems with cellular cues resembling those of natural tissues. Fibrillar alignment, occurring naturally in extracellular matrices, is one of the crucial attributes in tissue development. Obtaining fiber alignment in 3D, in vitro remains an important challenge due to non-linear material characteristics. Here, we report a cell-compatible, shear stress-based method allowing to obtain 3D homogeneously aligned fibrillar collagen hydrogels. Controlling the shear-stress during gelation results in low strain rates, with negligible effects on the viability of embedded SH-SY5Y cells. Our approach offers reproducibility and tunability through a paradigm shift: The shear-stress initiation moment, being the critical optimization parameter in the process, is related to the modulus of the developing gel, whereas state of the art methods often rely on a predefined time to initiate the alignment procedure. After curing, the induced 3D alignment is maintained after the release of stress, with a linear relation between the total acquired strain and the fiber alignment. This method is generally applicable to 3D fibrillar materials and stress/pressure-controlled setups, making it a valuable addition to the fast-growing field of tissue engineering. Statement of significanceControlling fiber alignment in vitro 3D hydrogels is crucial for developing physiologically relevant model systems. However, it remains challenging due to the non-linear material characteristics of fibrillar hydrogels, limiting the scalability and repeatability.Our approach tackles these challenges by utilizing a stress-controlled rheometer allowing us to monitor structural changes in situ and determine the optimal moment for applying a shear-stress inducing alignment. By careful parameter control, we infer the relationship between time, induced strain, alignment and biocompatibility.This tunable and reproducible method is both scalable and generally applicable to any fibrillar hydrogel, therefore, we believe it is useful for research investigating the link between matrix anisotropy and cell behavior in 3D systems, organ-on-chip technologies and drug research.

Molecular Basis of Pathogenic Variants in the Fibrillar Collagens.

The fibrillar collagen family is comprised of the quantitatively major types I, II and III collagens and the quantitatively minor types V and XI. These form heterotypic collagen fibrils (composed of more than a single collagen type) where the minor collagens have a regulatory role in controlling fibril formation and diameter. The structural pre-requisites for normal collagen biosynthesis and fibrillogenesis result in many places where this process can be disrupted, and consequently a wide variety of phenotypes result when pathogenic changes occur in these fibrillar collagen genes. Another contributing factor is alternative splicing, both naturally occurring and as the result of pathogenic DNA alterations. This article will discuss how these factors should be taken into account when assessing DNA sequencing results from a patient.

Collagen XI Alpha 1 Chain, a Novel Therapeutic Target for Cancer Treatment.

The extracellular matrix (ECM) plays an important role in the progression of cancer. Collagen is the most abundant component in ECM, and is involved in the biological formation of cancer. Although type XI collagen is a minor fibrillar collagen, collagen XI alpha 1 chain (COL11A1) expression has been found to be upregulated in a variety of human cancers including colorectal, esophagus, glioma, gastric, head and neck, lung, ovarian, pancreatic, salivary gland, and renal cancers. High levels of COL11A1 usually predict poor prognosis, owing to its association with angiogenesis, invasion, and drug resistance in cancer. However, little is known about the specific mechanism through which COL11A1 regulates tumor progression. Here, we have organized and summarized recent developments regarding the interactions between COL11A1 and intracellular signaling pathways and selected therapeutic agents targeting COL11A1, as these indicate its potential as a target for treatment of cancers, especially epithelial ovarian cancer.

Assessment of Optimal Conditions for Marine Invertebrate Cell-Mediated Mineralization of Organic Matrices.

Cellular strategies and regulation of their crystallization mechanisms are essential to the formation of biominerals, and harnessing these strategies will be important for the future creation of novel non-native biominerals that recapitulate the impressive properties biominerals possess. Harnessing these biosynthetic strategies requires an understanding of the interplay between insoluble organic matrices, mineral precursors, and soluble organic and inorganic additives. Our long-range goal is to use a sea anemone model system (Nematostella vectensis) to examine the role of intrinsically disordered proteins (IDPs) found in native biomineral systems. Here, we study how ambient temperatures (25–37 °C) and seawater solution compositions (varying NaCl and Mg ratios) will affect the infiltration of organic matrices with calcium carbonate mineral precursors generated through a polymer-induced liquid-precursor (PILP) process. Fibrillar collagen matrices were used to assess whether solution conditions were suitable for intrafibrillar mineralization, and SEM with EDS was used to analyze mineral infiltration. Conditions of temperatures 30 °C and above and with low Mg:Ca ratios were determined to be suitable conditions for calcium carbonate infiltration. The information obtained from these observations may be useful for the manipulation and study of cellular secreted IDPs in our quest to create novel biosynthetic materials.

Tissue mechanics coevolves with fibrillar matrisomes in healthy and fibrotic tissues

Fibrillar proteins are principal components of extracellular matrix (ECM) that confer mechanical properties to tissues. Fibrosis can result from wound repair in nearly every tissue in adults, and it associates with increased ECM density and crosslinking as well as increased tissue stiffness. Such fibrotic tissues are a major biomedical challenge, and an emerging view posits that the altered mechanical environment supports both synthetic and contractile myofibroblasts in a state of persistent activation. Here, we review the matrisome in several fibrotic diseases, as well as normal tissues, with a focus on physicochemical properties. Stiffness generally increases with the abundance of fibrillar collagens, the major constituent of ECM, with similar mathematical trends for fibrosis as well as adult tissues from soft brain to stiff bone and heart development. Changes in expression of other core matrisome and matrisome-associated proteins or proteoglycans contribute to tissue stiffening in fibrosis by organizing collagen, crosslinking ECM, and facilitating adhesion of myofibroblasts. Understanding how ECM composition and mechanics coevolve during fibrosis can lead to better models and help with antifibrotic therapies.

Dietary Moringa oleifera leaf powder improves jejunal permeability and digestive function by modulating the microbiota composition and mucosal immunity in heat stressed rabbits.

Heat stress (HS) has detrimental effects on intestinal health by altering digestive and immune responses in animals. Dietary Moringa oleifera leaf powder (MOLP) has been implicated in ameliorating the impact of HS, but its effects in terms of intestinal function improvement under HS remain poorly characterized. Therefore, the current study investigated the impact of HS and MOLP supplementation on tight junction barriers, intestinal microbiota (jejunal digesta), and differentially expressed genes (DEGs) in jejunal mucosa of heat-stressed rabbits by using the next-generation sequencing techniques. A total of 21 male New Zealand White rabbits (32weeks old mean body weight of 3318 ± 171g) were divided into three groups (n = 7/group) as control (CON, 25°C), heat stress (HS, 35°C for 7h daily), and HS with MOLP supplementation (HSM, 35°C for 7h daily) gavage at 200mg/kg body weight per day for 4weeks. The results indicated that MOLP supplementation increased mRNA expression of tight junction proteins and glutathione transferase activity, while the malonaldehyde concentration was decreased in the jejunal mucosa compared to HS group (P < 0.05). Furthermore, MOLP decreased the concentrations of lipopolysaccharide, pro-inflammatory cytokines, and myeloperoxidase compared with HS group (P < 0.05). Intestinal microbiota analysis revealed that at phyla level, the relative abundance of Bacteroidetes was higher in HSM group compared to CON and HS groups. MOLP supplementation also resulted in higher abundance of putatively health-associated genera such as Christensenellaceae R-7 gut group, Ruminococcaceae NK4A214 group, Ruminococcus 2, Lachnospiraceae NK4A136 group, and Lachnospiraceae unclassified along with higher butyrate levels in HSM group as compared to HS group. The analysis of DEGs revealed that MOLP reversed inflammatory response by downregulation of genes, such as TNFRSF13C, LBP, and COX2 in enriched KEGG pathway of NF-kβ pathway. MOLP supplementation also significantly upregulated the expression of genes in protein digestion and absorption pathway, including PRSS2, LOC100349163, CPA1, CPB1, SLC9A3, SLC1A1, and SLC7A9 in HSM group. Three genes of fibrillar collagens, i.e., COL3A1, COL5A3, and COL12A1 in protein digestion were also down-regulated in HSM group. In conclusion, MOLP supplementation could improve jejunal permeability and digestive function, positively modulate microbiota composition and mucosal immunity in heat-stressed rabbits.

Abstract 6096: The microarchitecture and fibrillar collagen expression of leiomyosarcoma are associated with malignancy

Abstract The expression, abundance, and microarchitecture of fibrillar collagens are associated with tumor development and aggressiveness in various carcinomas. However, the impact of fibrillar collagens on mesenchymal tumors is less understood. While uterine leiomyomas, also known as fibroids, are characterized by high fibrillar collagen deposition and depend on ECM signaling for cell proliferation, the impact of fibrillar collagens on malignant uterine leiomyosarcomas has not been explored. Thus, identifying malignancy and aggressiveness-associated features of the fibrillar collagen-leiomyosarcoma crosstalk may provide novel biomarkers and therapeutic targets for these aggressive tumors. We used publicly available RNAseq data and performed RNAseq and picrosirius red analysis of fiber microarchitecture in a cohort of normal myometrium (MM; n =68 ), leiomyoma (LM; n = 66), and leiomyosarcoma (LMS; n = 67) tissues. Furthermore, we cultured patient-derived primary cells (4 MM, 3 LM, and 4 LMS) on collagen I-functionalized polyacrylamide gels at stiffness ranging from 0.5 to 115 kPa, covering the physiological and pathological stiffness, to investigate distinct behaviors between cell types, including proliferation, migration, and activity of the ECM stiffness molecular rheostat YAP/TAZ. At the protein level, analysis of fibrillar collagen microarchitecture revealed that LMS tumors present reduced fibrillar collagen density and hyphal growth units and enhanced fiber endpoints compared to both MM and LM. At the gene expression level, however, LMS tumors did not show reduced fibrillar collagen expression, instead they exhibited enhanced matrix metalloproteinase expression, particularly of MMP14. Furthermore, COL11A1 was specifically upregulated in LMS tumors and its expression was associated with poor prognosis. Finally, in vitro response of MM, LM, and LMS cells to collagen I at defined stiffness showed that LMS cell migration, proliferation, and subcellular localization of YAP/TAZ are less sensitive to substrate stiffness than in MM and LM cells, although the response varied between distinct donors. In conclusion, we show that LMS tumors typically present low fibrillar collagen protein expression likely due to enhanced degradation. In addition, collagen I adhesion and stiffness have a lower impact on malignant LMS cells than on MM and LM, which may explain their ability to grow in low-collagen microenvironments. Furthermore, this study shows that COL11A1 is a potential biomarker with prognostic value in leiomyosarcoma. Citation Format: Jordi Gonzalez-Molina, Paula Hahn, Raul Maia Falcão, Georgia Kokaraki, Jorge Estefano de Souza, Tirzah Braz Petta Lajus, Kaisa Lehti, Joseph W. Carlson. The microarchitecture and fibrillar collagen expression of leiomyosarcoma are associated with malignancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6096.

Abstract 3840: Novel aspects of tumor fibrosis: Non-invasively assessed type XXII collagen, and not type I collagen, depicts highy elevated levels in patients with various solid tumor types

Abstract Background: The major constituents of the tumor microenvironment (TME), are besides the tumor cells themselves, cancer associated fibroblasts (CAFs), stromal cells and the extracellular matrix (ECM). CAFs synthesize excess amounts of collagens leading to a fibrotic TME with poor response to anti-cancer treatment. Traditionally, tumor fibrosis is defined by quantification of fibrillar collagens such as type I collagen (COL1). So-called Fibril-Associated Collagen with Interrupted Triple helices (FACITs) are less studied in this context. Type XXII collagen (COL22) is a minor FACIT located at the basement membrane. COL22 may therefore provide a different value compared to standard COL1 assessment traditionally associated with tumor fibrosis. Methods: Levels of COL1 (PRO-C1) and COL22 (PRO-C22) was measured non-invasively using competitive enzyme-linked immunosorbent assays in serum from patients diagnosed with bladder-, breast-, colorectal-, head and neck-, kidney-, lung-, melanoma-, pancreatic-, prostate-, and stomach cancer (n=220) and compared to healthy controls (n=33) (Cohort 1). Based on results from Cohort 1, PRO-C22 was evaluated in an independent cohort (Cohort 2) comprising patients with pancreatic ductal adenocarcinoma (PDAC) (n=39) and healthy controls (n=20). Receiver operating characteristic (ROC) curve analysis was performed to assess the diagnostic performance of PRO-C22. Kaplan-Meier curves and Cox proportional hazard models were used to assess the association between PRO-C22 and overall survival (OS) after dichotomizing PRO-C22 at the median cutpoint. Results: In Cohort 1, PRO-C22 was significantly higher in patients with all individual cancer types compared to healthy controls (p&amp;lt;0.01 to p&amp;lt;0.0001). In support, the area under the ROC (AUROC) for separation of patients with cancer from healthy controls using PRO-C22 ranged from 0.89 to 0.98 (p&amp;lt;0.0001). As comparison PRO-C1 assesment showed no significant difference. PRO-C22 was also significantly higher in patients with PDAC (Cohort 2) when compared to healthy (p&amp;lt;0.0001) with an AUROC of 0.87 (p&amp;lt;0.0001). Furthermore, high PRO-C22 levels were associated with shorter OS (median OS days 162 and 1363 for ‘high’ and ‘low’ PRO-C22 subgroups, respectively, p=0.0011). Likewise, high PRO-C22 levels at baseline predicted increased risk of mortality (HR=3.53, 95% CI 1.59-7.86, p=0.0020). Conclusion: These proof-of-concept data indicate that PRO-C22 may exhibit potential as a novel diagnostic and prognostic tumor fibrosis biomarker in cancer. PRO-C22 may be superior to type PRO-C1. Larger comprehensive clinical cohorts are needed to fully understand the biology associated with COL22 in cancer as well as the biomarker potential of PRO-C22 in various cancer types. Citation Format: Emilie Albrecht Madsen, Jeppe Thorlacius-Ussing, Christina Jensen, Neel I. Nissen, Astrid Z. Johansen, Inna M. Chen, Julia S. Johansen, Tina Manon-Jensen, Hadi M. Diab, Lars N. Jørgensen, Morten Karsdal, Nicholas Willumsen. Novel aspects of tumor fibrosis: Non-invasively assessed type XXII collagen, and not type I collagen, depicts highy elevated levels in patients with various solid tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3840.

Abstract 2475: Visualization of cell to cell and cell to matrix interactions in the disruption of tumor-promoting desmoplasia by adoptive transfer of fibroblast activation protein targeted chimeric antigen receptor (CAR) T cells

Abstract The efficacy of chimeric antigen receptor (CAR) T-cell therapy in solid tumors is limited by inefficient T-cell infiltration, localization and hypofunctionality within the tumor microenvironment. Prior studies demonstrated that stromal cell targeted CAR T cells, in particular CAR T cells that target fibroblast activation protein (FAP), can effectively infiltrate, and inhibit tumor growth in mouse models of pancreatic ductal adenocarcinomas (PDAC) due to their capacity to deplete stromal cells and extracellular matrix (ECM) that otherwise present a barrier to adoptive cell therapies. However, the kinetics of tumor infiltration by FAP-CAR T cells, and the cell-cell and cell-matrix interactions that contribute to their anti-tumor activity are poorly understood. A better understanding FAP-CAR T-cell kinetics and intratumoral interactions should provide insights into the requirements for successful adoptive cell therapies in solid tumors. By combining immunostaining and real-time two-photon microscopy in tumor fragments of PDAC, we found that stromal cell targeted FAP-CAR T cells can efficiently traffic into tumors. Initially (days 1-3) FAP-CAR T cells were enriched in the stroma surrounding tumor nests but excluded from the tumor nests per se. While associated with the stroma rich region, the CAR T cells appeared activated and T cell motility was observed in loose collagen regions, but T cells migrated poorly in dense matrix areas. Aligned collagen fibers in the tumor boarder dictated the migratory trajectory of T cells and restricted them from entering tumor nests. By day 5 we observed depletion of immunosuppressive stromal cells, dissolution of the fibrillar collagen network surrounding tumor nests and infiltration of FAP-CAR T cells into tumor nests. Treatment with hyaluronidase increased the ability of T cells to infiltrate the tumor nest. Thus, FAP-CAR T cell-mediated ablation of immunosuppressive FAP+ stromal cells and disruption of the desmoplastic stroma they generate, can enhance accumulation and functionality of CAR-T cell therapy in the context of highly desmoplastic solid tumors. Citation Format: Zebin Xiao, Leslie A. Hopper, Meghan C. Kopp, Emily McMillan, Yue Li, Richard L. Barrett, Ellen Puré. Visualization of cell to cell and cell to matrix interactions in the disruption of tumor-promoting desmoplasia by adoptive transfer of fibroblast activation protein targeted chimeric antigen receptor (CAR) T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2475.