Stromal Organization Research Articles

Abstract P6-04-11: Improved quantitative fibrosis indices reveal diverse survivals of triple negative breast cancer patients

Abstract Background: The ability of cancer cells to metastasise influences the mortality rate of patients with cancer. Extracellular matrix (ECM) from stromal organization is associated with tumorigenesis and metastasis in breast cancer. It is proposed that morphological features of collagen, based on second harmonic generation (SHG) microscopy, within the tumour environment can be a quantitative image-based biomarker for to predict the survival rate of triple-negative breast cancer (TNBC) patients. To this, we have developed quantitative fibrosis indices by combining multiple collagen features in breast tumour to reveal the diverse survivals of TNBC patients. Method: The patients (n = 216) in this study were diagnosed with TNBC in Singapore from 2003 to 2015. Disease-free survival (DFS) and overall survival (OS) were defined as time from the point of diagnosis to recurrence or to death/the date of the last follow-up, respectively. The constructed tissue microarrays (TMA) of breast tumours were scanned by the SHG microscope (Genesis, HistoIndex Pte. Ltd., Singapore). 33 collagen parameters were quantified from each sample. These collagen parameters were used to build disease free survival (DFS) index, overall survival (OS) index for prediction of early recurrence (DFS< 1 year) and early death (OS< 4 years), respectively. Kaplan-Meier survival analysis was further performed to assess long-term survival of TNBC patients with high and low risk as stratified by the indices, tumour grade and tumour size. The indices were validated using leave-one-out method. Results: Both DFS-index and OS-index were created using 10 collagen parameters chosen by sequential selection methods. Due to insufficient follow-up time, 12 patients and 81 patients were excluded from the early recurrence analysis and early death analysis, respectively. The DFS-index could differentiate low-risk patients with DFS≥1 year (n=179) and high-risk patients with DFS< 1 year(n=25) (training p< 0.001; validation p=0.157) with a cut-off value DFS-index=0.880. The OS-index could differentiate the low-risk patients with OS≥4 years (n=101) and high-risk patients with OS< 4 years (n=34) (training p< 0.001; validation p=0.011) with a cut-off value OS-index=0.703. The log-rank test showed DFS-index (training p = 0.001; validation p=0.025) and OS-index (training p < 0.001; validation p=0.011) could be used for the prediction of disease-free survival and overall survival. Kaplan-Meier survival analysis revealed tumour size>20mm (DFS, p=0.605; OS, p=0.136) and tumour grade=3 (DFS, p=0.328; OS, p=0.768) had poor predictive value in this study. Conclusion: Quantitative assessment of fibrosis in breast cancer correlates with long-term survival of TNBC patients. This study used DFS-index and OS-index combined complex morphological collagen features and obtained better prediction results than tumour size and tumour grade. Table. The difference between low and high risk groups differentiated by developed indices, tumour size and tumour grade. Low-risk group has a longer DFS and OS months based on DFS-index and OS-index. Citation Format: Yayun Ren, Dean Tai, Ying Zhao, Puay Hoon Tan. Improved quantitative fibrosis indices reveal diverse survivals of triple negative breast cancer patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-04-11.

Abstract 1563: Spatial transcriptomics reveals heterogeneity and pathway dependencies of cancer associated fibroblasts in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDA) is a deadly disease characterized by an immunosuppressive microenvironment and a dense stroma that encapsulates the tumor, making therapeutic targeting particularly challenging. One of the main cellular components of PDA tumor microenvironment (TME) is cancer associated fibroblasts (CAFs), whose function is to sustain tumor growth and provide structural support to the TME. Previously, we established the existence of three transcriptional subtypes of CAFs, myCAF, iCAF and apCAF, each with distinct functions and location in respect to cancer cells. Two of these subtypes, myCAF and iCAF, display a highly plastic potential in vitro and can transdifferentiate into one another upon activation of specific signaling pathways. The plasticity observed supports the existence of transitional CAF sub-states that have previously eluded observation due to technical limitations to their isolation. Through a combination of spatial transcriptomics and fluorescent in situ hybridization (FISH) on tumor samples obtained from KPC (KrasLSL-G12D; p53LSL-R172H; PDX-CRE) and FPC (KrasFrt-LSL-G12V-Frt; p53LSL-R172H; PDX-CRE; Rosa26FlpOERT2) mice, two PDA mouse models displaying a Kras G12D and G12V mutation respectively, we were able to confirm the presence of the previously identified CAF subtypes and two additional CAF sub-states. These sub-states are associated with the expression of specific markers and display different pathway enrichment. Notably, Kras G12D and G12V mutations generate similar organization of the stroma and heterogeneity of the CAF population. Subsequently, to understand the contribution of activating Kras mutation to these CAF phenotypes, we utilized the unique feature of the FPC mouse model which allows the excision of Kras G12V once the tumor has fully developed. Through FISH analysis, we observed deep reorganization of the stroma and a shift in marker expressions within the CAF populations upon Kras excision. Overall, the data here presented offers insight into the diversity of PDA CAFs and the role of mutated Kras in regulating CAF subtypes and stromal organization. Citation Format: Giuseppina Caligiuri, Jennifer Thalappillil, Juliene Hinds, Elise T. Courtois, William F. Flynn, Paul Robson, Alexander Dobin, Youngkyu Park, David A. Tuveson. Spatial transcriptomics reveals heterogeneity and pathway dependencies of cancer associated fibroblasts in pancreatic ductal adenocarcinoma [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 1563.

The complex role of telocytes in female prostate tumorigenesis in a rodent model.

The prostate is not an organ exclusive to the male. It is also found in females of several species, including humans, in which part of the Skene gland is homologous to the male prostate. Evidence is accumulating that changes in the stroma are central to tumorigenesis. Equally, telocytes, a recently discovered type of interstitial cell, are essential for the maintenance of stromal organization. However, it is still uncertain whether there are telocytes in the female prostate and if they play a role in tumorigenesis. The present study used ultrastructural and immunofluorescence techniques to investigate the presence of telocytes in the prostate of Mongolian gerbil females, a rodent model that often has a functional prostate in females, as well as to assess the impact of a combination of N-ethyl-N-nitrosourea, testosterone, and estradiol on telocytes. The results point to the presence of telocytes in the female prostate in the perialveolar and interalveolar regions, and reveal that these cells are absent in regions of benign and premalignant lesions in the gland, in which the perialveolar smooth muscle is altered. Additionally, telocytes are also closely associated with infiltrated immune cells in the stroma. Our data suggest that telocytes are important for both the maintenance of smooth muscle and prostatic epithelium integrity, which indicates a protective role against the advancement of tumorigenesis. But telocytes are also associated with immune cells and a proinflammatory/proangiogenic role for these cells cannot be ruled out, implying that telocytes have a complex role in prostatic tumorigenesis in females.

Collagen XIV Is an Intrinsic Regulator of Corneal Stromal Structure and Function

Collagen XIV is poorly characterized in the body, and the current knowledge of its function in the cornea is limited. The aim of the current study was to elucidate the role(s) of collagen XIV in regulating corneal stromal structure and function. Analysis of collagen XIV expression, temporal and spatial, was performed at different postnatal days (Ps) in wild-type C57BL/6 mouse corneal stromas and after injury. Conventional collagen XIV null mice were used to inquire the roles that collagen XIV plays in fibrillogenesis, fibril packing, and tissue mechanics. Fibril assembly and packing as well as stromal organization were evaluated using transmission electron microscopy and second harmonic generation microscopy. Atomic force microscopy was used to assess stromal stiffness. Col14a1 mRNA expression was present at P4 to P10 and decreased at P30. No immunoreactivity was noted at P150. Abnormal collagen fibril assembly with a shift toward larger-diameter fibrils and increased interfibrillar spacing in the absence of collagen XIV was found. Second harmonic generation microscopy showed impaired fibrillogenesis in the collagen XIV null stroma. Mechanical testing suggested that collagen XIV confers stiffness to stromal tissue. Expression of collagen XIV is up-regulated following injury. This study indicates that collagen XIV plays a regulatory role in corneal development and in the function of the adult cornea. The expression of collagen XIV is recapitulated during wound healing.

Defining the Tumor Microenvironment by Integration of Immunohistochemistry and Extracellular Matrix Targeted Imaging Mass Spectrometry.

Simple SummaryExtracellular matrix within the tumor microenvironment influences signaling, controls molecular diffusion of nutrients and growth factors, alters immunogenicity, and contributes to disease progression and therapeutic response. ECM is secreted by multiple cell types, including tumor cells, fibroblasts, and immune cells, yet there are limited approaches that link the cell type to the ECM proteins within the heterogeneous tumor microenvironment. Here, we show that integrating immunohistochemistry (IHC) with extracellular matrix (ECM) imaging mass spectrometry allows ECM proteomic profiling based on patterns of diverse cell types and proteins in tissue. The developed approach is demonstrated using phosphatase and tensin homolog (PTEN) staining and ECM imaging/proteomics on the same tissue sections in normal breast and in a tissue microarray of breast tumor and normal adjacent tissue. The data suggests that PTEN expression in tumor and in normal adjacent tissue may be associated with different collagen types and regulation by post-translational sites of modification.Breast stroma plays a significant role in breast cancer risk and progression yet remains poorly understood. In breast stroma, collagen is the most abundantly expressed protein and its increased deposition and alignment contributes to progression and poor prognosis. Collagen post-translation modifications such as hydroxylated-proline (HYP) control deposition and stromal organization. The clinical relevance of collagen HYP site modifications in cancer processes remains undefined due to technical issues accessing collagen from formalin-fixed, paraffin-embedded (FFPE) tissues. We previously developed a targeted approach for investigating collagen and other extracellular matrix proteins from FFPE tissue. Here, we hypothesized that immunohistochemistry staining for fibroblastic markers would not interfere with targeted detection of collagen stroma peptides and could reveal peptide regulation influenced by specific cell types. Our initial work demonstrated that stromal peptide peak intensities when using MALD-IMS following IHC staining (αSMA, FAP, P4HA3 and PTEN) were comparable to serial sections of nonstained tissue. Analysis of histology-directed IMS using PTEN on breast tissues and TMAs revealed heterogeneous PTEN staining patterns and suggestive roles in stromal protein regulation. This study sets the foundation for investigations of target cell types and their unique contribution to collagen regulation within extracellular matrix niches.

Detailed Morphological Analysis of Cryoinjury in Human Ovarian Tissue Following Vitrification or Slow Freezing.

Cryopreservation of human ovarian tissue represents a key procedure for fertility preservation. The two most widely used cryopreservation methods for human ovarian cortex samples are slow freezing\thawing (SF\T) and vitrification\warming (V\W). The aim of the present study was to analyze the effects of SF\T and V\W using a metal chamber, on specific follicle and oocyte structures and on the stromal organization post-cryopreservation. We did histology analysis of SF\T and V\W ovarian fragments from nine healthy subjects. Overall results showed that cryopreserved tissues presented significant rates of damage in primordial and primary follicles. Altered nuclear structure of primordial follicles and cell detachment from primordial and primary follicles were the main injuries observed after V/W and SF/T. The stromal components were similarly well preserved after cryopreservation. We conclude that both cryopreservation methods may be used for fertility preservation purposes with similar outcomes in terms of follicular and stromal integrity. Detachment of follicle cells from basal membrane represents an important cryoinjury that deserves further investigation.

Stromal cell interplay in prostate development, physiology, and pathological conditions.

Advances in prostatic stroma studies over the past few decades have demonstrated that the stroma not only supports and nourishes the gland's secretory epithelium but also participates in key aspects of morphogenesis, in the prostate's hormonal metabolism, and in the functionality of the secretory epithelium. Furthermore, the stroma is implicated in the onset and progression of prostate cancer through the formation of the so-called reactive stroma, which corresponds to a tumorigenesis-permissive microenvironment. Prostatic stromal cells are interconnected and exchange paracrine signals among themselves in a gland that is highly sensitive to endocrine hormones. There is a growing body of evidence that telocytes, recently detected interstitial cells that are also present in the prostate, are involved in stromal organization, so that their processes form a network of interconnections with both the epithelium and the other stromal cells. The present review provides an update on the different types of prostate stromal cells, their interrelationships and implications for prostate development, physiology and pathological conditions.

Stromal Organization as a Predictive Biomarker of Response to Neoadjuvant Therapy in Locally Advanced Rectal Cancer

Stromal Organization as a Predictive Biomarker of Response to Neoadjuvant Therapy in Locally Advanced Rectal Cancer

A Novel Therapeutic Approach to Corneal Alkaline Burn Model by Targeting Fidgetin-Like 2, a Microtubule Regulator.

PurposeThe purpose of this study was to determine the efficacy of nanoparticle-encapsulated Fidgetin-like 2 (FL2) siRNA (FL2-NPsi), a novel therapeutic agent targeting the FL2 gene, for the treatment of corneal alkaline chemical injury.MethodsEighty 12-week-old, male Sprague-Dawley rats were divided evenly into 8 treatment groups: prednisolone, empty nanoparticles, control-NPsi (1 µM, 10 µM, and 20 µM) and FL2-NPsi (1 µM, 10 µM, and 20 µM). An alkaline burn was induced onto the cornea of each rat, which was then treated for 14 days according to group assignment. Clinical, histopathologic, and immunohistochemical analyses were conducted to assess for wound healing. FL2-NPsi-mediated knockdown of FL2 was confirmed by in vitro quantitative polymerase chain reaction (qPCR). Toxicity assays were performed to assess for apoptosis (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling [TUNEL] assay) and nerve damage (whole mount immunochemical staining). Statistical analyses were performed using Student's t-test and ANOVA.ResultsCompared with controls, FL2-NPsi-treated groups demonstrated enhanced corneal wound healing, with the 10 and 20 µM FL2-NPsi-treated groups demonstrating maximum rates of corneal re-epithelialization as assessed by ImageJ software, enhanced corneal transparency, and improved stromal organization on histology. Immunohistochemical analysis of vascular endothelial cells, macrophages, and neutrophils did not show significant differences between treatment groups. FL2-NPsi was not found to be toxic to nerves or induce apoptosis (p = 0.917).ConclusionsDose-response studies found both 10 and 20 µM FL2-NPsi to be efficacious in this rat model. FL2-NPsi may offer a novel treatment for corneal alkaline chemical injuries.Translational RelevanceBasic cell biology findings about the microtubule cytoskeleton were used to design a therapeutic to enhance corneal cell migration, highlighting the promise of targeting microtubules to regulate corneal wound healing.

Molecular description of ANGPT2 associated colorectal carcinoma

Angiopoietin-2 (ANGPT2) is a prognostic factor in metastatic colorectal cancer (CRC). Nevertheless, it remains to be elucidated which molecular characteristics make up the ANGPT2-related poor-prognosis CRC subset. Public transcriptomic datasets were collected from Gene Expression Omnibus GEO and with the TCGAbiolinks R-package for the TCGA. After appropriate normalization, differential expression analysis was performed using Benjamini and Hochberg method for false discovery rate. Plasma from two prospective clinical trials were used to investigate the clinical impact of ANGPT2-related biomarkers. In the 935 samples included in four annotated platforms (GPL) and derived from localized CRC, ANGPT2hi expression conferred a worst overall survival (HR = 1.20; p = 0.02). CRC stage, ANGPT2hi expression but not Consortium Molecular Subtype (CMS) predict overall survival in multivariate analysis. ANGPT2 expression was not correlated with a specific CMS nor to RAS, RAF, MSI, p53, CIN, CIMP genomic alterations. Gene expression analysis revealed that ANGPT2hi CRC subset is characterized by angiogenesis-related gene expression, presence of myeloid cells, stromal organization and resistance to chemotherapy. A prognostic model was proposed using seric levels of ANGPT2, STC1 and CD138 in 97 mCRC patients. Our results provide evidence that ANGPT2 is a prognostic factor in localized CRC and defined a specific CRC subset with potential clinical implementation.

Integrated-omics of MRI-visible and -invisible prostate cancer identifies molecular correlations with clinical outcome.

e17506 Background: The inclusion of multiparametric MRI (mpMRI) to prostate cancer (PCa) diagnostics has increased the detection rate and has improved the prediction rate of clinically significant PCa. Yet, mpMRI has a false negative rate of 12.6%, missing 6-13% of clinically significant PCa. The mechanisms underlying MRI visibility are poorly understood; therefore, to probe the molecular and cellular underpinnings of PCa MRI visibility, we profiled tissue from Gleason score and clinically matched patients with MRI-visible and MRI-invisible PCa who underwent radical prostatectomy. Methods: MRI-invisible (n = 7) and MRI-visible (n = 8) PCa tumors were evaluated with multiplex immunofluorescence (MxIF; 14 markers) and were subjected to gene expression profiling using the HTG EdgeSeq Oncology Biomarker Panel (2,549 genes). Gene expression analysis was also performed using The Cancer Genome Atlas (TCGA), including normal prostate (n = 52) and PCa (n = 387) tissue. Analyses were performed using the BostonGene automated pipeline. Results: MpMRI-visible PCa tumors (62.5%) displayed compact epithelial tumor architecture compared with mpMRI-invisible PCa tumors (28.5%). mpMRI-visible PCa had higher malignant cell density (p = 0.04) and increased neighboring malignant cells (p = 0.07), correlating with MRI-visible PCa complex tumor architecture (r = 0.49 for neighboring malignant cells vs tumor cell density). Tumor stromal organization differences were determined by measuring Wasserstein distances between distributions, and mpMRI-invisible PCa stroma appeared more similar to normal tissue. The visible group exhibited lower expression of stroma-enriched genes such as filamin C (FLNC) (FDR < 0.1) and cellular adhesion-related genes (FDR < 0.4), with gene expression signatures markedly different compared to normal prostate tissue. Higher malignant cell density, neighboring malignant cells, and Wasserstein distances, and low FLNC expression – all mpMRI visibility characteristics – were associated with patient relapse (p = 0.02). Low stroma signature expression in the TCGA cohort correlated with inferior PCa PFS (p = 0.005). Conclusions: This is the first integrated multi-omics analysis of clinically matched mpMRI-visible and -invisible PCa. mpMRI-invisible tumors exhibited molecular, cellular, and structural characteristics more akin to normal prostate tissue, which may render them undetectable by imaging. A stroma-associated gene signature, a mpMRI-invisible tumor feature, correlated with better PCa clinical outcomes.

Stromal Collagen Arrangement Correlates with Stiffness of the Canine Cornea.

The cornea is the most external layer of the eye and serves two important roles in (1) the refraction of light and (2) protection from the outside environment, both of which are highly dependent on the collagen assembly of the corneal stroma. This study sought to determine the collagen fiber arrangement of the canine corneal stroma and correlate the stromal organization with tissue stiffness in the anterior and posterior cornea. Collagen organization of the canine cornea was visualized through second-harmonic generation (SHG) imaging, and tissue stiffness of the anterior and posterior corneal stroma was determined by atomic force microscopy. Analysis of the canine anterior corneal stroma using SHG imaging documented intertwining of the collagen fibers with a high degree of fiber branching, with a more lamellar and non-branching posterior stroma. The anterior stroma had significantly higher tissue stiffness in both dogs and humans, when compared with the posterior corneal stroma (canine median: 1.3 kPa vs. 0.3 kPa; human median: 14.6 kPa vs. 2.1 kPa, respectively). There was a direct correlation between corneal collagen stromal organization and tissue stiffness in the dog, which was consistent with other mammalian species previously examined and likely reflects the need for maintenance of rigidity and corneal curvature.

Abstract SY35-02: Bone marrow remodeling in response to distant tumor starts early in transformation

Abstract Bone marrow (BM) stromal cell are sensors of immunological stress signals from peripheral tissue. We showed that chronicity of such signals induces persistent changes in the stromal architecture providing favorable conditions for the establishment and progression of a myeloproliferative spur (Blood 2012, 120: 3541). A key regulator of bone marrow stromal changes is the secreted protein acidic and rich in cysteine (SPARC) a matricellular protein with both structural and regulatory functions on extracellular matrix (ECM) organization. Up- and downregulation of SPARC has been indeed associated with different hematologic neoplasms including myeloid and lymphoid malignancies (Cancer Res. 2017, 77: 3685; Cancer Discovery 2014, 4: 110). We identified SPARC as a common stromal trait of the BM osteoblastic niche and SLO germinal center (GC) microenvironments, where it regulates physiological B lymphopoiesis and B cell development, as well as B cell lymphomagenesis (Oncoimmunology 2014). Like infections and autoimmunity, also the presence of solid tumors involves the establishment of a chronic state of inflammation that can act locally but also be sensed systemically, particularly by the BM. The last responds with an expansion of accessory cells further fostering tumor growth and dissemination.Others and we have indeed demonstrated in tumor mouse models the expansion in the BM of the myeloid compartment that parallels tumor progression. In this context we contributed to identify some of the key molecules involved in the BM-tumor cross talk, such as VEGF, MMP-9 (Cancer Res. 2007, 67:11438). Tumor-expanded myeloid cells are generally endowed with immune suppressive features, which actively contribute to cancer-associated systemic immune suppression.We have recently described two additional mechanisms that contribute to the shaping of the myeloid cell phenotype and suppressive activity in mammary tumors and involve SPARC (Cell Report 2016, 17: 233) and another matricellular protein, i.e. osteopontin (Cancer Res. 2014, 74: 4706). New data indicate that tumor-induced myeloid cell expansion in the BM is associated with profound changes not only in the hematopoietic compartment, but also in the stromal niche organization. In a spontaneous model of mammary carcinogenesis we show significant alterations in the density and localization of Nestin+ mesenchymal stromal elements and in the expression of the prototypical stroma-derived chemotactic factor CXCL12, which were both increased in the hematopoietic interstitium of mammary tumor bearing mice. How early during transformation these hematopoietic and stromal changes are detectable, and the identification of soluble factors involved in these BM modification are the focus of our present research and will be presented in this symposium.Understanding the molecular determinants of cancer-driven hematopoietic adaptation would offer a new prospect on cancer early detection and prognostication, eventually prompting novel strategies to interfere with such detrimental changes besides the specific targeting of functional hematopoietic subsets. Citation Format: Mario P. Colombo. Bone marrow remodeling in response to distant tumor starts early in transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr SY35-02.

A Novel Tissue-Engineered Corneal Stromal Equivalent Based on Amniotic Membrane and Keratocytes.

To investigate a novel strategy in constructing tissue-engineered corneal stromal equivalent based on amniotic membrane and keratocytes. The ultrathin amniotic membrane (UAM) was laminated, with corneal stromal cells (CSCs) distributed between the space of the layered UAMs. Calcein AM staining was used to evaluate cellular viability, morphology, and arrangement. Immunostaining, qRT-PCR, and Western blot were performed to detect gene and protein expression in keratocytes. Optical coherence tomography visualized the cross sections and thickness of the UAM construction. The microstructure of the CSC-secreted extracellular matrix (ECM) was investigated by scanning electron microscopy and transmission electron microscopy (TEM). To evaluate the feasibility of the multilayer UAM-CSC lamination for surgery, the corneal substitute was used to perform lamellar keratoplasty. Slit lamp microscopy and corneal fluorescein staining were performed in postsurgery observation. The CSCs maintained their keratocyte phenotype and secreted well-organized ECM on the aligned UAM surface. The four-layer UAM-CSC lamination attained half thickness of the human cornea (250 ± 18 μm) after 8 weeks' culture, which also showed promising optimal transparency. In TEM images, the CSC-generated ECM displayed stratified, multilayered lamellae with orthogonal fibril arrangement, which was similar to the human cornea microstructure. Furthermore, the stromal equivalent was successfully preformed in lamellar keratoplasty. Four weeks post surgery, the substitute was well integrated into the recipient cornea and completely epithelialized without myofibroblast differentiation. Our study established a novel 3D biomimetic corneal model to replicate the corneal stromal organization with multilayer UAM, which was capable of promoting the development of corneal stroma-like tissues in vitro, establishing a new avenue for basic research and therapeutic potential.

Cell regulation of collagen fibril macrostructure during corneal morphogenesis

While tissue form and function is highly dependent upon tissue-specific collagen composition and organization, little is known of the mechanisms controlling the bundling of collagen fibrils into fibers and larger structural designs that lead to the formation of bones, tendons and other tissues. Using the cornea as a model system, our previous 3 dimensional mapping of collagen fiber organization has demonstrated that macrostructural organization of collagen fibers involving interweaving, branching and anastomosing plays a critical role in controlling mechanical stiffness, corneal shape and refractive power. In this work, the cellular and mechanical mechanisms regulating critical events in the assembly of collagen macrostructure are analysed in the developing chicken cornea. We elucidated the temporal events leading to adult corneal structure and determined the effects of intraocular pressure (IOP) on the organization of the collagen macrostructure. Our findings indicate that the complex adult collagen organization begins to appear on embryonic day 10 (E10) after deposition of the primary stroma and full invasion of keratocytes. Importantly, organizational changes in keratocytes appearing at E9 preceded and predicted later changes in collagen organization. Corneal collagen organization remained unaffected when the development of IOP was blocked at E4. These findings support a primary role for keratocytes in controlling stromal organization, mechanical stiffness and corneal shape that are not regulated by the IOP. Our findings also suggest that the avian cornea represents an excellent experimental model for elucidating key regulatory steps and mechanisms controlling the collagen fiber organization that is critical to determining tissue form and function. Statement of significanceThis work by using an ex ovo model system, begins to investigate the potential mechanisms controlling collagen fibril macrostructure. In particular, this work highlights a convergent role for the corneal keratocytes in organizing the complex collagen macrostructure, necessary to support high visual acuity. Our data supports that the intraocular pressure does not influence collagen fibril macrostructure and suggest that the avian cornea represents an excellent experimental model for elucidating key regulatory steps and mechanisms controlling the collagen fiber organization that is critical to determining tissue form and function. Clearly understanding the cellular and molecular mechanisms that underlie collagen fibril macrostructure will be highly beneficial for future tissue engineering and regenerative medicine applications.

Degradation of silk films in multipocket corneal stromal rabbit models.

The need for human cornea tissues continues to grow as an alternative option to donor tissues. Silk protein has been successfully used as a substrate to engineer corneal epithelium and stroma in vitro. Herein, we investigated the in vivo response and the effect of silk crystalline structure (beta sheet) on degradation rate of silk films in rabbit multipocket corneal models. Three different surgical techniques (peripheral-median P-M, central-superficial C-S, central-deep C-D) were used to assess the in vivo response as well as the degradation profile of silk films with low, medium and high beta sheet (crystalline) content at 2 and 3 months after surgery. Approach C-D showed signs of sample degradation without inflammation, with one single incision and a pocket created by flushing air two thirds deep in the corneal stroma. In comparison, approaches P-M and C-S with multiple incisions presented manually dissected surgical pockets resulted in inflammation and possible extrusion of the samples, respectively. Low beta sheet samples lost structural integrity at 2 months after surgery C-D, while medium and high beta sheet content films showed initial evidence of degradation. The in vivo response to the silk films was dependent on the location of the implant and pocket depth. Crystallinity content in silk films played a significant role in the timing of material degradation, without signs of inflammation and vascularization or changes in stromal organization.

Identifying the Spatial Relationships of Thymic Stromal and Thymocyte Subsets by Immunofluorescence Analysis.

Immunofluorescence analysis of thymic tissue sections is an indispensable technique for visualizing spatial relationships among thymocyte and stromal cell subsets. The thymus is organized into distinct microenvironmental zones in which particular thymic epithelial cell (TEC) subsets support specific stages of thymocyte maturation. Conversely, thymocytes and lymphoid tissue inducer cells support functional maturation of TECs. The composition and organization of TECs change during ontogeny to generate a maximally functional organ in the young adult. Deterioration of thymic architecture and stromal organization occurs with age as the thymus undergoes involution. Such changes can be monitored by immunofluorescent staining of thymic sections obtained at different ages throughout the life-span. Here we describe methods to generate frozen or paraffin-embedded thymic tissue sections for multicolor immunofluorescence staining using antibodies to surface and/or cytoplasmic antigens.

Type III Collagen Directs Stromal Organization and Limits Metastasis in a Murine Model of Breast Cancer

Breast cancer metastasis is the leading cause of cancer-related deaths in women worldwide. Collagen in the tumor microenvironment plays a crucial role in regulating tumor progression. We have shown that type III collagen (Col3), a component of tumor stroma, regulates myofibroblast differentiation and scar formation after cutaneous injury. During the course of these wound-healing studies, we noted that tumors developed at a higher frequency in Col3(+/-) mice compared to wild-type littermate controls. We, therefore, examined the effect of Col3 deficiency on tumor behavior, using the murine mammary carcinoma cell line 4T1. Notably, tumor volume and pulmonary metastatic burden after orthotopic injection of 4T1 cells were increased in Col3(+/-) mice compared to Col3(+/+) littermates. By using murine (4T1) and human (MDA-MB-231) breast cancer cells grown in Col3-poor and Col3-enriched microenvironments in vitro, we found that several major events of the metastatic process were suppressed by Col3, including adhesion, invasion, and migration. In addition, Col3 deficiency increased proliferation and decreased apoptosis of 4T1 cells both in vitro and in primary tumors in vivo. Mechanistically, Col3 suppresses the procarcinogenic microenvironment by regulating stromal organization, including density and alignment of fibrillar collagen and myofibroblasts. We propose that Col3 plays an important role in the tumor microenvironment by suppressing metastasis-promoting characteristics of the tumor-associated stroma.

Disorganised stroma determined on pre-treatment breast cancer biopsies is associated with poor response to neoadjuvant chemotherapy: Results from the NEOZOTAC trial

IntroductionThe tumor-associated stroma is of importance for tumor progression and is generally accepted to have a significant influence on patient prognosis. However, little is known regarding specific features of tumor-associated stromal tissues and response to (neoadjuvant) chemotherapy. This study investigated the predictive value of extracellular matrix organization on response to chemotherapy in patients treated in the NEOZOTAC trial. MethodsStromal organisation was analyzed via a simple method using image analysis software on hematoxylin and eosin (H&E)-stained slides from primary tumor biopsies collected as part of the NEOZOTAC trial. Heidenhain's AZAN trichrome-stained slides were also analyzed for comparison of collagen evaluation. Sections were stained for phospho-Smad2 (pS2) in order to determine the relationship of TGF-β signaling with stromal organization. ResultsA statistically significant relationship was observed between stroma consisting of organised collagen and pathological response to neoadjuvant chemotherapy (Odds Ratio 0.276, 95%CI 0.124–0.614, P = 0.002). This parameter was also related to ER-status (P = 0.003), clinical tumor -status (P = 0.041), nodal status (P = 0.029) and pS2 status (P = 0.025). Correlation between stromal organisation determined on H&E-stained and AZAN-stained tissue sections was high (Pearson's correlation coefficient = 0.806). ConclusionIntratumoral stromal organisation determined using pre-treatment breast cancer biopsies was related to pathological response to chemotherapy. This parameter might play a role in the management of breast cancer for identifying those patients that are likely to benefit from neoadjuvant chemotherapy.

Interclass small leucine-rich repeat proteoglycan interactions regulate collagen fibrillogenesis and corneal stromal assembly

The corneal stroma is enriched in small leucine-rich proteoglycans (SLRPs), including both class I (decorin and biglycan) and class II (lumican, keratocan and fibromodulin). Transparency is dependent on the assembly and maintenance of a hierarchical stromal organization and SLRPs are critical regulatory molecules. We hypothesize that cooperative interclass SLRP interactions are involved in the regulation of stromal matrix assembly. We test this hypothesis using a compound Bgn−/0/Lum−/− mouse model and single Lum−/− or Bgn−/0 mouse models and wild type controls. SLRP expression was investigated using immuno-localization and immuno-blots. Structural relationships were defined using ultrastructural and morphometric approaches while transparency was analyzed using in vivo confocal microscopy. The compound Bgn−/0/Lum−/− corneas demonstrated gross opacity that was not seen in the Bgn−/0 or wild type corneas and greater than that in the Lum−/− mice. The Bgn−/0/Lum−/− corneas exhibited significantly increased opacity throughout the stroma compared to posterior opacity in the Lum−/− and no opacity in Bgn−/0 or wild type corneas. In the Bgn−/0/Lum−/− corneas there were abnormal lamellar and fibril structures consistent with the functional deficit in transparency. Lamellar structure was disrupted across the stroma with disorganized fibrils, and altered fibril packing. In addition, fibrils had larger and more heterogeneous diameters with an abnormal structure consistent with abnormal fibril growth. This was not observed in the Bgn−/0 or wild type corneas and was restricted to the posterior stroma in Lum−/− mice. The data demonstrate synergistic interclass regulatory interactions between lumican and biglycan. These interactions are involved in regulating both lamellar structure as well as collagen fibrillogenesis and therefore, corneal transparency.