Mapping of retardance,diattenuation and polarizance vector on Poincare sphere for diagnosis and classification of cervical precancer

The mapping of diattenuation, polarizance and retardance vector (normalized Stokes vector) on Poincare sphere, evaluated from Mueller matrix of optically anisotropic stromal region of cervical tissues, is presented for cervical precancer detection and its staging. This reveals that the changes in the polarization states shown by these normalized Stokes vectors corresponds to the degradation of linearly arranged collagen fibers, breakage of the collagen cross links in the stromal region and change in the density of scattering sites when cervical cancer evolves. The distinct nature of real and imaginary parts of the refractive index for linear, linear‐45 and circular polarization from the optically anisotropic stromal region underscore the various polarization structures of the connective tissue region which get modified during the pathological changes. It has been found that versatility of these vectors for normal and precancerous cervical tissue of various grades may be utilized as a key distinction for qualitative staging of cervical precancer tissue. Quantitative classification of precancerous stages of cervical precancer has been determined with 95%–100% sensitivity and 93%–100% specificity through the evaluation of linear and circular diattenuation, linear polarizance and linear birefringence from the components of the respective vectors.     

Spatial autocorrelation analysis on two‐dimensional images of Mueller matrix for diagnosis and differentiation of cervical precancer

The spatial autocorrelation and correlation map of amplitude and phase anisotropy along with depolarization parameter from the stroma of uterine cervix utilizing their Mueller matrix (MM) images have been reported for early diagnosis of cervical cancer and differentiation of precancerous stages. The comparative results of the evaluation of the spatial autocorrelation over MM images of optically anisotropic collagen structures from normal and various grades of cervical precancer reflect significant alterations which are correlated with the pathological changes. The spatially varying polarizance from different region of anisotropic stromal region gets correlated within a given spatial lag during the precancerous changes. The diattenuation governing elements M12, M13 and M14 clearly discriminate normal and various grades of precancerous cervical tissue through their autocorrelation profile and correlation map. Evaluation of autocorrelation of spatially varying linear birefringence and linear‐45 birefringence characterized by MM elements M34 and M43 and M24 and M42 are not found to differ between the precancer grades, indicating that these changes may be arising from highly directional collagen network while the changes displayed by MM elements M23 and M32 faithfully represent that the chirality of the stromal region is compromised as the cervical cancer evolves and only one type of nature dominates.     

Prostate epithelial differentiation is dictated by its surrounding stroma

Prostate epithelial differentiation is dictated by its surrounding stroma which determines androgen induced growth responsiveness and expression of specific secretory proteins in normal prostate gland. During neoplastic progression, organ specific stroma has been shown to determine the rate of neoplastic progression from androgen-dependent to androgen-independent and metastatic states. Although growth factors and extracellular matrix are recognized as important contributors to prostate epithelial growth, hormonal responsiveness, and neoplastic progression, the exact mechanism of intercellular communication between stromal and epithelial cells remains undefined. In addition to the importance of defining the reciprocal interaction between stromal and epithelial interaction in the prostate, clonal interaction between two dissimilar prostate epithelial cells is also recognized to contribute to disease progression. In this review, we summarized recent advances made in delineating molecular mechanisms underlying stromal epithelial interaction and clonal interaction between androgen-dependent and androgen-independent prostate cancer cells in vivo and in culture. Understanding cellular interaction between prostate epithelium and its surrounding stroma could help us in developing metastatic models of prostate carcinogenesis. This concept will allow us to define epithelial-specific markers, markers induced as the result of stromal-epithelial interaction, and stroma-associated markers. These markers together will assist us in diagnosing, preventing, prognosing and treating prostate cancer more efficaciously in the future.     

From Single Cells to Tissues: Interactions between the Matrix and Human Breast Cells in Real Time

Background

Mammary gland morphogenesis involves ductal elongation, branching, and budding. All of these processes are mediated by stroma - epithelium interactions. Biomechanical factors, such as matrix stiffness, have been established as important factors in these interactions. For example, epithelial cells fail to form normal acinar structures in vitro in 3D gels that exceed the stiffness of a normal mammary gland. Additionally, heterogeneity in the spatial distribution of acini and ducts within individual collagen gels suggests that local organization of the matrix may guide morphogenesis. Here, we quantified the effects of both bulk material stiffness and local collagen fiber arrangement on epithelial morphogenesis.

Results

The formation of ducts and acini from single cells and the reorganization of the collagen fiber network were quantified using time-lapse confocal microscopy. MCF10A cells organized the surrounding collagen fibers during the first twelve hours after seeding. Collagen fiber density and alignment relative to the epithelial surface significantly increased within the first twelve hours and were a major influence in the shaping of the mammary epithelium. The addition of Matrigel to the collagen fiber network impaired cell-mediated reorganization of the matrix and increased the probability of spheroidal acini rather than branching ducts. The mechanical anisotropy created by regions of highly aligned collagen fibers facilitated elongation and branching, which was significantly correlated with fiber organization. In contrast, changes in bulk stiffness were not a strong predictor of this epithelial morphology.

Conclusions

Localized regions of collagen fiber alignment are required for ductal elongation and branching suggesting the importance of local mechanical anisotropy in mammary epithelial morphogenesis. Similar principles may govern the morphology of branching and budding in other tissues and organs.     

Preneoplastic changes in ovarian tissues

OBJECTIVE: To test whether histologically normal epithelium within ovarian inclusion cysts and stroma exhibit changes in nuclear chromatin pattern that indicate the presence of occult ovarian lesions. STUDY DESIGN: Ovaries were collected from 10 low-risk women,from 7 high-risk women and from 3 women with ovarian cancer. Histologic sections were cut at 5 microm and hematoxylin and eosin stained. High-resolution images were recorded from the epithelium lining inclusion cysts and from the underlying stroma of ovaries from these 20 subjects. A total of 2860 epithelial nuclei and 3610 stromal nuclei were recorded. Karyometric features and nuclear abnormality were computed. Discriminant analyses and unsupervised learning algorithms defined deviations from normal that were designated "above threshold" and used to compute average nuclear abnormality of a second nuclear phenotype. RESULTS: Histologically normal epithelium from inclusion cysts of ovaries harboring a malignant lesion was shown to exhibit changes in the nuclear chromatin pattern that were statistically significant using quantitative image analysis procedures. Similar changes were seen in the inclusion cyst epithelia of high-risk ovaries. A subpopulation of cells representing a new phenotype was detected in the underlying stroma of women harboring a malignant ovarian lesion and in women at high risk of ovarian cancer. CONCLUSION: The karyometric changes observed in the epithelia lining inclusion cysts and in the underlying stroma of ovaries either with ovarian cancer or at high risk of ovarian cancer suggest the presence of preneoplastic changes in histologically normal tissue.     

Alpha smooth muscle actin (α-SM actin) in normal human ovaries,in ovarian stromal hyperplasia and in ovarian neoplasms

An immunohistochemical investigation of alpha-smooth muscle actin (alpha-SM actin) using the monoclonal anti-alpha-SM-1 antibody was carried out in 15 normal ovaries, in three ovaries with stromal hyperplasia and in 27 neoplastic ovaries. In selected cases the pattern of actin isoforms was examined by means of 2 D-gel electrophoresis. In addition, the tissues were stained for vimentin and desmin. In normal ovaries alpha-SM actin was found in the inner cortex and in the theca externa. In ovarian stromal hyperplasia expression of alpha-SM actin was minimal or absent. In primary and metastatic epithelial tumors there was positive stromal staining for alpha-SM actin, especially in the vicinity of epithelial elements. This tended to be more widespread in malignant neoplasms. Thecomas did not express alpha-SM-actin and could thus be differentiated from leiomyomas which stained intensely for alpha-SM actin. Only focal stromal staining of alpha-SM actin was observed in granulosa and germ cell tumors. In all the tissues studied blood vessels were strongly positive for alpha-SM actin. Desmin, although present in the stroma of most of the specimens, was less abundant than alpha-SM actin. We concluded that alpha-SM actin is a component of the normal human ovary where it may contribute to the contractility of its stroma. Its absence in the normal outer cortex and theca interna, and in stromal hyperplasia and thecoma implies that sex hormones do not constitute a stimulus for alpha-SM actin production in the ovary. Among neoplasms it is most widely represented in the stroma of epithelial tumors in which it may reflect stromal stimulation mediated by neoplastic epithelium.     

SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland

SHARPIN is a widely expressed multifunctional protein implicated in cancer, inflammation, linear ubiquitination and integrin activity inhibition; however, its contribution to epithelial homeostasis remains poorly understood. Here, we examined the role of SHARPIN in mammary gland development, a process strongly regulated by epithelial–stromal interactions. Mice lacking SHARPIN expression in all cells (Sharpin^cpdm), and mice with a stromal (S100a4‐Cre) deletion of Sharpin, have reduced mammary ductal outgrowth during puberty. In contrast, Sharpin^cpdm mammary epithelial cells transplanted in vivo into wild‐type stroma, fully repopulate the mammary gland fat pad, undergo unperturbed ductal outgrowth and terminal differentiation. Thus, SHARPIN is required in mammary gland stroma during development. Accordingly, stroma adjacent to invading mammary ducts of Sharpin^cpdm mice displayed reduced collagen arrangement and extracellular matrix (ECM) stiffness. Moreover, Sharpin^cpdm mammary gland stromal fibroblasts demonstrated defects in collagen fibre assembly, collagen contraction and degradation in vitro. Together, these data imply that SHARPIN regulates the normal invasive mammary gland branching morphogenesis in an epithelial cell extrinsic manner by controlling the organisation of the stromal ECM.     

Differential roles for two gelatinolytic enzymes of the matrix metalloproteinase family in the remodelling cornea.

We have documented changes in collagenolytic/gelatinolytic enzymes of the matrix metalloproteinase family (MMP) in remodelling rabbit cornea. MMP-2 (65 kDa gelatinase) in the proenzyme form is synthesized by the cells of the normal corneal stroma. After keratectomy the level of MMP-2 is increased in the stroma and enzyme appears in both pro- and activated forms. In addition, corneal cells synthesize MMP-9 (92 kDa gelatinase) in the proenzyme form after keratectomy; expression occurs in both the epithelial as well as stromal corneal layers. Changes in expression of both enzymes are precisely localized to the repairing portion of cornea, but demonstrate important differences in timing that correlate with the timing of specific events of matrix remodelling. Our data suggest that each of the gelatinases plays a different role in tissue remodelling after injury. We hypothesize that MMP-2 performs a surveillance function in normal cornea, catalyzing degradation of collagen molecules that occasionally become damaged. After wounding, this enzyme appears to participate in the prolonged process of collagen remodelling in the corneal stroma that eventually results in functional regeneration of the tissue. MMP-9 expression does not correlate with stromal remodelling, but we suggest that the enzyme might play a part in controlling resynthesis of the epithelial basement membrane.     

A cell line, ROSE 199, derived from normal rat ovarian surface epithelium

A cell line, ROSE 199, derived from rat ovarian surface epithelium (ROSE) formed papillary structures which resembled, histologically, serous papillary cystadenomas of borderline malignancy seen in the human ovary. Crowded cultures produced two layers of cells separated by a thick layer of collagen fibers. Such cultures shed viable cells into the growth medium, while no cells were shed by short-term ROSE cultures. The resemblance to ovarian tumors exhibited by ROSE 199 cells in culture, reinforces the hypothesis that the common epithelial tumors of the ovary are derived from the ovarian surface epithelium. ROSE 199 cells, while retaining their epithelial morphology and ultrastructural characteristics, express stromal activity such as abundant collagen production. Perhaps this ability to express epithelial and stromal behavior is a contributing factor to the ready neoplastic transformation of the ovarian surface epithelium.     

The Role of the Microenvironment in Mammary Gland Development and Cancer

The mammary gland is composed of a diverse array of cell types that form intricate interaction networks essential for its normal development and physiologic function. Abnormalities in these interactions play an important role throughout different stages of tumorigenesis. Branching ducts and alveoli are lined by an inner layer of secretory luminal epithelial cells that produce milk during lactation and are surrounded by contractile myoepithelial cells and basement membrane. The surrounding stroma comprised of extracellular matrix and various cell types including fibroblasts, endothelial cells, and infiltrating leukocytes not only provides a scaffold for the organ, but also regulates mammary epithelial cell function via paracrine, physical, and hormonal interactions. With rare exceptions breast tumors initiate in the epithelial compartment and in their initial phases are confined to the ducts but this barrier brakes down with invasive progression because of a combination of signals emitted by tumor epithelial and various stromal cells. In this article, we overview the importance of cellular interactions and microenvironmental signals in mammary gland development and cancer.The mammary gland is composed of a combination of multiple cell types that together form complex interaction networks required for the proper development and functioning of the organ. The branching milk ducts are formed by an outer myoepithelial cell layer producing the basement membrane (BM) and an inner luminal epithelial cell layer producing milk during lactation. The ducts are surrounded by the microenvironment composed of extracellular matrix (ECM) and various stromal cell types (e.g., endothelial cells, fibroblasts, myofibroblasts, and leukocytes). Large amount of data suggest that cell-cell and cell-microenvironment interactions modify the proliferation, survival, polarity, differentiation, and invasive capacity of mammary epithelial cells. However, the molecular mechanisms underlying these effects are poorly understood. The purification and comprehensive characterization of each cell type comprising normal and neoplastic human breast tissue combined with hypothesis testing in cell culture and animal models are likely to improve our understanding of the role these cells play in the normal functioning of the mammary gland and in breast tumorigenesis. In this article, we overview cellular and microenvironmental interactions that play important roles in the normal functioning of the mammary gland and their abnormalities in breast cancer.     

Secretion of collagen types I and II by epithelial and endothelial cells in the developing chick cornea demonstrated by in situ hybridization and immunohistochemistry

Cells involved in the synthesis of collagen types I and II in the cornea of developing chick embryos have been studied by using in situ hybridization and immunohistochemistry. Corneas processed for in situ hybridization with the type I and II collagen probes demonstrated specific mRNAs in the epithelium of embryos at stage 18 with an increase at stages between 26 and 31, and then gradual decrease to the background level in the next several days. In the endothelium, a small amount of specific mRNA was recognized through these stages. In the stroma, only sections hybridized with the type I probe demonstrated mRNA in fibroblasts. Immunostaining demonstrated specific collagen types in the stroma at sites which were closely associated with cells containing specific mRNAs. Both collagens type I and II were present beneath the epithelium as narrow bands at stage 18; as the thicker primary stroma at stages 20 and 26; and as subepithelial, subendothelial and stromal staining at stage 31. Thereafter, type I collagen was increased in the stroma but it was also noted in the subepithelial and, to a lesser degree, subendothelial regions, whereas type II collagen was gradually confined to the subendothelial matrix. Electron microscopic examination of sections from 5-day-old (stage-27) embryo corneas using antibodies against the carboxyl propeptides of type I and II procollagens revealed the presence of these procollagens within the cisternae of the endoplasmic reticulum and Golgi vesicles in both epithelial and endothelial cells. In the epithelial cells both the periderm and basal cells contained these procollagens within the cytoplasmic organelles. These results indicate that not only the epithelial cells, but also the endothelial cells secrete collagen types I and II during the formation of the primary corneal stroma and for several days after invasion of fibroblasts.     

Tissue remodeling in Guinea pig lateral prostate at different ages after estradiol treatment

Estrogen seems to have an essential role in the fibromuscular growth characteristic of benign prostatic hyperplasia (BPH). This paper describes the effects of chronic estradiol treatment on Guinea pig prostatic stroma at different ages. Tissues from experimental animals were studied by histological and histochemical procedures, morphometric-stereological analysis and transmission electron microscopy (TEM). Marked fibromuscular hypertrophy was observed after estradiol treatment in animals of pre-pubertal and adult ages. Increases in the density and thickness of the collagen and elastic fibers were observed by histochemistry. TEM revealed wide distributions of collagen fibrils and large elastic fibers adjacent to the epithelial basal lamina and between the stromal cells, establishing contacts between them. These results indicate that the Guinea pig prostate simulates the stromal modifications observed in BPH in some aged animals after estrogen treatment at different ages, making it a good model for this disease.     

Identification of remodeled collagen fibers in tumor stroma by FTIR Micro-spectroscopy: A new approach to recognize the colon carcinoma

The tumor stroma plays a pivotal role in colon cancer genesis and progression. It was observed that collagen fibers in the extracellular matrix (ECM) of cancer stroma, undergo a strong remodeling. These fibrous proteins result more aligned and compact than in physiological conditions, creating a microenvironment that favors cancer development.In this work, micro-FTIR spectroscopy was applied to investigate the chemical modifications in the tumor stroma. Using Fuzzy C-means clustering, mean spectra from diseased and normal stroma were compared and collagen was found to be responsible for the main differences between them. Specifically, the modified absorptions at 1203, 1238, 1284 cm^?1 and 1338 cm^?1 wavenumbers, were related to the amide III band and CH₂ bending of side chains. These signals are sensitive to the interactions between the α-chains in the triple helices of collagen structure. This provided robust chemical evidence that in cancer ECM, collagen fibers are more parallelized, stiff and ordered than in normal tissue. Principal Component Analysis (PCA) applied to the spectra from malignant and normal stroma confirmed these findings. Using LDA (Linear Discriminant Analysis) classification, the absorptions 1203, 1238, 1284 and 1338 cm^?1 were examined as spectral biomarkers, obtaining quite promising results. The use of a PCA-LDA prediction model on samples with moderate tumor degree further showed that the stroma chemical modifications are more indicative of malignancy compared to the epithelium. These preliminary findings have shown that micro-FTIR spectroscopy, focused on collagen signals, could become a promising tool for colon cancer diagnosis.     

Bone matrix osteonectin limits prostate cancer cell growth and survival

There is considerable interest in understanding prostate cancer metastasis to bone and the interaction of these cells with the bone microenvironment. Osteonectin/SPARC/BM-40 is a collagen binding matricellular protein that is enriched in bone. Its expression is increased in prostate cancer metastases, and it stimulates the migration of prostate carcinoma cells. However, the presence of osteonectin in cancer cells and the stroma may limit prostate tumor development and progression. To determine how bone matrix osteonectin affects the behavior of prostate cancer cells, we modeled prostate cancer cell-bone interactions using the human prostate cancer cell line PC-3, and mineralized matrices synthesized by wild type and osteonectin-null osteoblasts in vitro. We developed this in vitro system because the structural complexity of collagen matrices in vivo is not mimicked by reconstituted collagen scaffolds or by more complex substrates, like basement membrane extracts. Second harmonic generation imaging demonstrated that the wild type matrices had thick collagen fibers organized into longitudinal bundles, whereas osteonectin-null matrices had thinner fibers in random networks. Importantly, a mouse model of prostate cancer metastases to bone showed a collagen fiber phenotype similar to the wild type matrix synthesized in vitro. When PC-3 cells were grown on the wild type matrices, they displayed decreased cell proliferation, increased cell spreading, and decreased resistance to radiation-induced cell death, compared to cells grown on osteonectin-null matrix. Our data support the idea that osteonectin can suppress prostate cancer pathogenesis, expanding this concept to the microenvironment of skeletal metastases.     

Characterization and co-culture of novel nontransformed cell lines derived from rat endometrial epithelium and stroma

Summary Normal and neoplastic growth of epithelial cells depends on mutual interactions between epithelial and stromal cells. As a tool for the study of the underlying molecular mechanisms, we have developed temperature-sensitive, nontransformed cell lines derived from rat uterine epithelium and stroma by transfecting primary cultures with a temperature-sensitive mutant of the SV40 large T antigen. The epithelial and stromal cell lines obtained shared relevant morphological characteristics with the primary cells from which they were derived. Immunocytochemical analysis showed that the epithelial cell lines expressed the intermediate filament cytokeratin, whereas the stromal lines expressed the intermediate filament vimentin. Alkaline phosphatase activity was present in all cell lines examined. All cell lines were anchorage dependent and did not form foci. One epithelial cell line expressed oxytocin mRNA, a gene product recently shown to be highly expressed in vivo in the uterine epithelium at term. If grown on Matrigel, this cell line formed domelike structures, a further characteristic of its differentiated phenotype. In an attempt to reconstitute an endometrium in vitro, epithelial cells were seeded on top of a layer of stromal cells. Paraffin cross sections showed that this in vitro system consisted of a bilayer structure. Four to five cuboidal epithelial cells were typically anchored atop one stromal cell, forming an endometriumlike tissue. The present in vitro system should provide a useful model for further studies on endometrial functions and epithelial/stromal cell interactions at a molecular level.     

Stroma in breast development and disease

It is increasingly apparent that normal and malignant breast tissues require complex local and systemic stromal interactions for development and progression. During development, mammary cell fate specification and differentiation require highly regulated contextual signals derived from the stroma. Likewise, during breast carcinoma development, the tissue stroma can provide tumor suppressing and tumor-promoting environments that serve to regulate neoplastic growth of the epithelium. This review focuses on the role of the stroma as a mediator of normal mammary development, as well as a critical regulator of malignant conversion and progression in breast cancer. Recognition of the important role of the stroma during the progression of breast cancers leads to the possibility of new targets for treatment of the initial breast cancer lesion as well as prevention of recurrence.     

Basement membrane collagen requirements for attachment and growth of mammary epithelium.

In vivo mammary epithelial cells rest upon a basement membrane composed in part of type IV collagen which is synthesized by these cells. In this study, basement membrane collagen is shown to be selectively recognized by normal mammary ducts and alveoli for attachment and growth when compared to the types of collagen derived from stroma (types I or III) or cartilage (type II). Cell attachment and growth on type I collagen is inhibited by the proline analogue, cis-hydroxyproline, which blocks normal collagen production. These effects of cis-hydroxyproline are not apparent when a basement membrane collagen substratum is provided. Unlike normal mammary epithelium, mammary fibroblasts show little preference for the collagen to which they will attach. A requirement of type IV collagen synthesis for normal mammary epithelial cell attachment and growth on stromal collagen in vitro may have significance in vivo where a basement membrane scaffold may be necessary for normal mammary morphogenesis and growth.     

The fiber system of the choroidocapillaris

In the central choroid of three cynomolgus monkeys (Macaca irus) and a baboon (Papio anubis) the shape of the choroidocapillary sinus is determined by a system of interstitial collagen fibers, the "fiber system of the choroidocapillaris". The inner leaflet of this system is Bruch's membrane. The outer leaflet consists of interwoven collagen bundles, covering the roof of the capillary sinus. Straight bundles of collagen fibers passing through connective tissue columns in the choroidocapillary sinus connect both leaflets. Forces created by changes in the arterial tone in the vascular stroma may be transmitted by the choroidocapillary fiber system to the elastic layer of Bruch's membrane.