Biology subjects

Pally, D.

Publications and source records attributed to Pally, D..

11 recordsLinked to original sources

bioRxiv2026

SNED1 fibrillar assembly in the extracellular matrix requires fibronectin and collagen I

The extracellular matrix (ECM) is a meshwork of proteins that orchestrates a broad range of cellular phenotypes, including proliferation, adhesion, migration, and differentiation. SNED1 is a newly characterized ECM glycoprotein that promotes cell adhesion and is essential for embryonic development. Its upregulation is also associated with breast cancer metastasis and poor prognosis for breast cancer patients. We recently showed that SNED1 assembles into fibrillar structures, but the mechanisms guiding its incorporation into the ECM scaffold remain unknown. Combining biochemical assays and confocal immunofluorescence imaging, we found that SNED1 assembly in the ECM occurs early in the process of ECM building and is concomitant and overlaps with the deposition of fibronectin and collagen I, two major ECM proteins. By knocking down fibronectin or destabilizing collagen I fibers, we further demonstrate that SNED1 requires the presence of these proteins for its assembly. Last, using biolayer interferometry, we identify collagen I as the first direct binding partner of SNED1. Altogether, our results lay the foundation for future studies aimed at determining the mechanisms by which SNED1 fibers contribute to SNED1 pathophysiological functions. SUMMARY STATEMENTThe novel protein SNED1 requires the presence of fibronectin and collagen I to assemble into fibrillar structures in the extracellular matrix scaffold.

cell biology↗

bioRxiv2026

SNED1 modulates ECM architecture and cell proliferation via LDV-binding integrins

The extracellular matrix (ECM) is a complex scaffold of proteins that supports multicellular structures. Interactions between cells and the ECM via receptors, like integrins, govern cellular phenotypes (e.g., proliferation, adhesion), but also contribute to ECM assembly. Understanding how ECM-receptor interactions regulate matrix assembly is critical to uncover how alterations of the ECM cause or accompany congenital diseases, cancer, or fibrosis. SNED1 is a novel ECM protein with roles in development and metastasis. However, the mechanisms governing its assembly and signaling functions remain largely unknown. SNED1 contains two integrin-binding motifs, RGD and LDV, and we recently showed that its interaction with RGD-integrins mediates cell adhesion. Here, we investigated the role of SNED1/integrin interactions in SNED1 ECM assembly. While SNED1/integrin interactions were not necessary for its initial incorporation in the ECM, interaction with LDV-, but not RGD-, integrins, was required for ECM build-up and the patterning of SNED1 and the fibrillar proteins fibronectin and collagen I. Moreover, SNED1/LDV-integrin interaction promoted ECM alignment, cell alignment, and cell proliferation, processes essential to SNED1-driven neural crest cell migration during craniofacial development and breast cancer invasion. SUMMARY STATEMENTInteraction of SNED1 with LDV-binding integrins, but not RGD-binding integrins, mediates ECM remodeling and controls cytoskeletal rearrangement and cell proliferation.

cell biology↗

bioRxiv2025

In-depth proteomic profiling of the extracellular matrix of pancreatic ductal adenocarcinomas identifies signatures correlating with lymphocyte infiltration

The extracellular matrix (ECM) is a complex assembly of proteins surrounding cells. It is a critical component of the tumor microenvironment that plays an active role in tumor progression and modulation of tumor response to treatment. Pancreatic ductal adenocarcinoma (PDAC) is a cancer type characterized by one of the worst prognoses, as it is often diagnosed at an advanced stage. It is also characterized by a very dense ECM, which hinders efficient drug delivery. In addition, PDACs are considered "cold" tumors as they fail to elicit a strong immune response, challenging the use of immunotherapy for PDAC cancer patients. Yet, the interplay between the ECM and immune cells within the PDAC tumor microenvironment remains poorly understood. Here, we employed ECM-focused proteomics to profile the ECM compositions of PDAC mouse models characterized by different levels of CD8+ T-cell infiltration. We found that CD8lo, or "cold" tumors, and CD8hi, or "hot" tumors, exhibited different ECM compositions. Interrogation of publicly available single-cell RNA-sequencing datasets of human PDACs further revealed that the ECM proteins distinguishing hot and cold PDACs are secreted by multiple stromal cell populations, including cancer-associated fibroblasts, stellate cells, and macrophages. Last, we found that the expression of a subset of the genes encoding ECM proteins characteristic of the CD8lo phenotype correlated with CD8+ T-cell infiltration in human PDAC samples and patient survival. This study paves the way for the development of ECM-modulating interventions to enhance immune cell infiltration and responsiveness to immunotherapy. SignificanceWe report the identification of ECM protein signatures correlating with the level of CD8+ lymphocyte infiltration in murine models of pancreatic ductal adenocarcinomas and human samples. This work paves the way for the development of ECM-modulating therapeutic strategies to enhance lymphocyte infiltration and, hence, the efficacy of immunotherapies.

cancer biology↗

bioRxiv2025

Comparative proteomic analysis of the ECM composition of the human omentum and mesentery, the main sites of ovarian cancer metastasis

Due to its limited symptoms, high-grade serous ovarian cancer (HGSOC) has frequently metastasized extensively throughout the peritoneal cavity prior to its diagnosis, resulting in an overall five-year survival rate of less than 50%. The greater omentum and the small bowel mesentery are two of the most common metastatic sites in advanced HGSOC. However, the mechanisms underlying HGSOC metastatic tropism remain unknown. The extracellular matrix is a complex and dynamic meshwork of proteins that provides biochemical and mechanical signals to surrounding cells and has been shown to drive the dissemination of several cancer types to preferential distant sites. Here, using histological assessment and proteomics, we examined the composition of the extracellular matrix of paired omentum and mesentery samples from disease-free adult females. We found that the fibrillar collagen content of the mesothelial layer of the omentum was significantly higher than that of the mesentery. Using ECM-focused proteomics, we further defined the ECM composition - or matrisome - of these two tissues. We found that over 90% of the proteins detected were shared between the omentum and mesentery. Our analysis also revealed small subsets of tissue-specific ECM proteins. Future work will aim to test the possible functional contributions of these ECM proteins to HGSOC metastatic tropism. To facilitate the reuse of our dataset, we have deposited the raw mass spectrometry data and accompanying metadata files to the ProteomeXchange Consortium with the dataset identifier PXD061586.

cancer biology↗

bioRxiv2025

Stromal LOX/FAK/beta catenin pathway locks mammary fibroblasts into a tumor-promoting myCAF state

BackgroundCancer-associated fibroblasts (CAFs) sustain tumor progression, yet the soluble cues that maintain their myofibroblast (myCAF) state are poorly defined. Transforming growth factor beta (TGF-{beta}) is a canonical CAF activator. This study aims to identify TGF-{beta}-induced secreted mediators that reinforce the myCAF phenotype in breast cancer and map the downstream signaling cascade. Methods and ResultsSecretome profiling of primary patient-derived myCAFs and human mammary fibroblasts (HMF3s) engineered to over-express TGF-{beta}1 revealed 20 extracellular-matrix remodelers shared exclusively by both activated states; lysyl oxidase (LOX) was the top-ranked hit. LOX knockdown abrogated TGF-{beta}-driven -smooth-muscle actin (-SMA) induction, collagen-gel contraction and migration in HMF3s, and reduced constitutive -SMA and {beta}-catenin in myCAFs. Mechanistically, TGF-{beta} upregulated LOX, which activated focal-adhesion kinase (FAK), leading to p38 MAPK- and Akt-mediated Ser9 phosphorylation (inactivation) of GSK3{beta} and consequent {beta}-catenin stabilization. In HCC1806-luciferase orthotopic xenografts, CAFs accelerated tumor growth, whereas LOX-deficient CAFs lost this pro-tumoral effect. ConclusionLOX is a pivotal autocrine effector of TGF-{beta} that locks breast CAFs into a pro-tumoral myCAF state through a LOX/FAK/GSK3{beta}/{beta}-catenin axis. Targeting stromal LOX may disrupt CAF activation and curb breast cancer progression.

cancer biology↗

bioRxiv2024

The novel ECM protein SNED1 mediates cell adhesion via α5β1 integrin

The extracellular matrix (ECM) is a complex meshwork comprising over 100 proteins. It serves as an adhesive substrate for cells and, hence, plays critical roles in health and disease. We have recently identified a novel ECM protein, SNED1, and have found that it is required for neural crest cell migration and craniofacial morphogenesis during development and in breast cancer, where it is necessary for the metastatic dissemination of tumor cells. Interestingly, both processes involve the dynamic remodeling of cell-ECM adhesions via cell surface receptors. Sequence analysis revealed that SNED1 contains two amino acid motifs, RGD and LDV, known to bind integrins, the largest class of ECM receptors. We thus sought to investigate the role of SNED1 in cell adhesion. Here, we report that SNED1 mediates breast cancer and neural crest cell adhesion via its RGD motif. We further demonstrate that cell adhesion to SNED1 is mediated by the RGD integrins 5{beta}1 and v{beta}3. These findings are a first step toward identifying the signaling pathways activated downstream of the SNED1-integrin interactions guiding craniofacial morphogenesis and breast cancer metastasis. SUMMARY STATEMENTWe report that the novel extracellular matrix protein SNED1 promotes the adhesion of breast cancer cells and neural crest cells via interaction with 5{beta}1 and v{beta}3 integrins, the first SNED1 receptors identified to date.

cell biology↗

bioRxiv2021

Galectin-9 signaling drives breast cancer invasion through matrix

Aberration in expression and function of glycans and their binding proteins (lectins) in transformed cells constitutes one of the earliest discovered hallmarks of cancer. Galectins are a conserved family of lectins that can bind to {beta}-galactosides. Among them, the role of Galectin-9, a galectin with two carbohydrate binding domains in immune-tumor cell interactions has been well-established, although its effect on cancer cell behavior remains as yet unclear. In this study, we used a spectrum of cell lines from homeostatic breast cells to transformed non-invasive and invasive cell lines cultured in microenvironment-diverse conditions to show that Galectin-9 expression shows an elevation in association with invasiveness of breast cancer epithelia. Our observations were supported by immunohistochemical studies of breast tumors and adjacent normal-tissues from patients. Genetic perturbation of Galectin-9 as well as the pharmacological inhibition of activity using cognate inhibitors confirmed a positive correlation between Galectin-9 levels and the adhesion of the aggressive triple negative breast cancer cells MDA-MB-231 to- and their invasion through-extracellular matrices (ECM). Within a constituted organomimetic multiECM microenvironment, Galectin-9 enhanced both the solitary and the collective invasion of cancer cells. Quantitative proteomics led us to uncover the inductive role of Galectin-9 in the expression of the proinvasive protein S100A4. In addition, Galectin-9 expression correlated with FAK signaling, the inhibition of which decreased S100A4 mRNA levels. Our results provide crucial signaling insights into how the elevation in Galectin-9 expression in breast cancer cells potentiates their invasiveness through ECM during early steps of metastasis.

cancer biology↗

bioRxiv2020

ST6GAL1-mediated heterogeneity in sialic acid levels potentiates invasion of breast cancer epithelia

Heterogeneity in phenotypes of malignantly transformed cells and aberrant glycan expression on their surface are two prominent hallmarks of cancers that have hitherto not been linked to each other. In this paper, we identify heterogeneity in a specific glycan linkage: 2,6-linked sialic acids within breast cancer cells in vivo and in culture. Upon sorting out two populations with moderate and relatively higher cell surface 2,6-linked sialic acid levels from the triple negative breast cancer cell line MDA-MB-231, both populations (denoted as medium and high-2,6-Sial cells respectively) stably retained their levels in early passages. Upon continuous culturing, medium 2,6-Sial cells recapitulated the heterogeneity of the unsorted line whereas high 2,6-Sial cells showed no such tendency. Compared with the high 2,6-Sial, the medium 2,6-Sial cells showed greater adhesion to reconstituted extracellular matrices (ECM) as well as invaded faster as single cells. The level of 2,6-linked sialic acids in the two sublines was found to be consistent with the expression of a specific glycosyl transferase, ST6GAL1. Stably knocking down ST6GAL1 in the high 2,6-Sial cells, enhanced their invasiveness. When cultured together, medium 2,6-Sial cells differentially migrated to the edge of growing tumoroid-like cultures, whereas high 2,6-Sial cells formed the central bulk. Simulations in a Cellular Potts model-based computational environment that is calibrated to our experimental findings suggest that the heterogeneity of cell-ECM adhesion, likely regulated by 2,6-linked sialic acids facilitates niches of highly invasive cells to efficiently migrate centrifugally as the invasive front of a malignant tumor. Significance StatementCell-surface sugars are aberrantly expressed in cancer but their contributions to tumor heterogeneity are not known. In this study, we uncover and separate breast cancer populations with distinct 2,6-linked sialic acid levels. The moderately expressing population shows stronger adhesion to extracellular matrix than the high expressing population. It also invades faster through the matrix as single cells. Combining experiments with computational modelling, we show that the heterogeneity in matrix adhesion is vital to accentuating cell invasion. In some conditions, invasion of heterogeneous populations may compare with, or exceed that of, homogeneous moderately expressing populations. Our findings are vital to furthering our understanding of how cancers spread and potentially qualify efforts to manage the disease through glycan-editing or immunotherapeutic approaches.

cancer biology↗

bioRxiv2019

An interplay between reaction-diffusion and cell-matrix adhesion regulates multiscale invasion in early breast carcinomatosis

The progression of cancer in the breast involves multiple reciprocal interactions between malignantly transformed epithelia, surrounding untransformed but affected stromal cells, and the extracellular matrix (ECM) that is remodelled during the process. A quantitative understanding of the relative contribution of such interactions to phenotypes associated with cancer cells can be arrived at through the construction of increasingly complex experimental and computational models. Herein, we introduce a multiscale 3D organo-and patho-typic model that approximates, to an unprecedented extent, the histopathological complexity of a tumor disseminating into its surrounding stromal milieu via both bulk and solitary motility dynamics. End-point and time-lapse microscopic observations of this model allow us to study the earliest steps of cancer invasion as well as the dynamical interactions between the epithelial and stromal compartments. We then construct an agent-based Cellular Potts model that incorporates constituents of the experimental model, as well as places them in similar spatial arrangements. The computational model, which comprises adhesion between cancer cells and the matrices, cell proliferation and apoptosis, and matrix remodeling through reaction-diffusion-based morphogen dynamics, is first trained to phenocopy controls run with the experimental model, wherein one or the other matrices have been removed. The trained computational model successfully predicts phenotypes of the experimental counterparts that are subjected to pharmacological treatments (inhibition of N-linked glycosylation and matrix metalloproteinase activity) and scaffold modulation (alteration of collagen density). Our results suggest that specific permissive regimes of cell-cell and cell-matrix adhesions operating in the context of a reaction-diffusion-regulated ECM dynamics, promote multiscale invasion of breast cancer cells and determine the extent to which they migrate through their surrounding stroma.

cancer biology↗

bioRxiv2018

Mutually exclusive locales for N-linked glycans and disorder in glycoproteins

Several post-translational modifications of proteins lie within regions of disorder, stretches of amino acid residues that exhibit a dynamic tertiary structure and resist crystallization. Such localization has been proposed to expand the binding versatility of the disordered regions, and hence, the repertoire of interacting partners for the proteins. However, investigating a dataset of 500 human N-linked glycoproteins, we observed that the sites of N-linked glycosylations, or N-glycosites, lay predominantly within the regions of predicted order rather than their unstructured counterparts. This mutual exclusivity between disordered stretches and N-glycosites could not be reconciled merely through asymmetry in distribution of asparagines, serines or threonines residues, which comprise the minimum-required signature for conjugation by N-linked glycans, but rather by a contextual enrichment of these residues next to each other within the ordered portions. In fact, N-glycosite neighborhoods and disordered stretches showed distinct sets of enriched residues suggesting their individualized roles in protein phenotype. N-glycosite neighborhood residues also showed higher phylogenetic conservation than disordered stretches within amniote orthologs of glycoproteins. However, a universal search for residue-combinations that are putatively domain-constitutive ranked the disordered regions higher than the N-glycosite neighborhoods. We propose that amino acid residue-combinations bias the permissivity for N-glycoconjugation within ordered regions, so as to balance the tradeoff between the evolution of protein stability, and function, contributed by the N-linked glycans and disordered regions respectively.

bioinformatics↗

bioRxiv2018

Galectin-1 promotes the invasion of bladder cancer urothelia through their matrix milieu

The progression of carcinoma of the urinary bladder involves migration of cancer epithelia through their surrounding tissue matrix microenvironment. This was experimentally confirmed when a gender- and grade-diverse set of bladder cancer cell lines were cultured in pathomimetic three-dimensional laminin-rich environments. The high-grade cells, particularly female, formed multicellular invasive morphologies in 3D. In comparison, low- and intermediate-grade counterparts showed growth-restricted phenotypes. A proteomic approach combining mass spectrometry and bioinformatics analysis identified the estrogen-driven lactose-binding lectin Galectin-1 (GAL-1) as a putative candidate that could drive this invasion. Expression of LGALS1, the gene encoding GAL-1 showed an association with tumor grade progression in bladder cell lines. Immunohisto- and cyto-chemical experiments suggested greater extracellular levels of GAL-1 in 3D cultures of high-grade bladder cells and cancer tissues. High levels of GAL-1 associated with increased proliferation- and adhesion- of bladder cancer cells when grown on laminin-rich matrices. Pharmacological inhibition and Gal-1 knockdown in high-grade female cells decreased their adhesion to, and viability on, laminin-rich substrata. Higher GAL-1 also correlated with reduced E-cadherin and increased N-cadherin levels in consonance with a mesenchymal-like phenotype that we observed in 3D culture. The inhibition of GAL-1 reversed the stellate invasive phenotype to a more growth-restricted one in high-grade cells embedded within both basement-membrane-like and stromal collagenous matrix scaffolds. Finally, inhibition of GAL-1 specifically altered cell surface sialic acids, suggesting the mechanism by which the levels of GAL-1 may underlie the aggression and poor prognosis of invasive bladder cancer, especially in women.

cancer biology↗