Biology subjects

Battistella, A.

Publications and source records attributed to Battistella, A..

8 recordsLinked to original sources

bioRxiv2025

Adipose-mimetic granular hydrogels uncover biophysical cues driving breast cancer invasion

1.Breast cancer cells invade mammary adipose tissue during initial stages of metastasis but how the physical properties of adipose tissue regulate this process remains unclear. Here, we combined single cell mechanical characterization of primary adipocytes with microfluidic hydrogel fabrication, quantitative multiparametric imaging, Discrete Element Method (DEM) simulations, and in vivo experiments to elucidate these connections. First, we quantified the heterogeneous size and stiffness of primary adipocytes, and replicated these properties by fabricating adipocyte-sized polyacrylamide (PAAm) beads with tunable elasticity. Subsequently, we embedded these beads into type I collagen, the primary fibrillar extracellular matrix (ECM) component of breast adipose tissue, to form 3D granular hydrogels mimicking aspects of native adipose tissue architecture. Granular hydrogels embedded with beads demonstrated increased breast cancer cell invasion relative to bead-free controls, an effect that was more pronounced with soft versus stiff beads and correlated with increased collagen fiber alignment and hierarchical organization. In addition, live cell imaging and DEM simulations revealed that soft beads promoted invasion relative to stiff beads by deforming in response to confined cancer cell migration. Fiber alignment and adipocyte deformation trends were validated in vivo via intravital imaging of cancer cell migration in mammary fat pads of mice, and suggest that adipocyte mechanics regulate breast cancer invasion by coordinating both ECM architecture and cellular confinement. Ultimately, this work highlights the utility of tunable PAAm bead-collagen composites as micromechanical models to study the effect of adipose tissue structure on cancer cell invasion. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/684224v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@d3ce0org.highwire.dtl.DTLVardef@117abb1org.highwire.dtl.DTLVardef@10917faorg.highwire.dtl.DTLVardef@55f3e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO Polyacrylamide bead granular hydrogels recapitulate adipose tissue structure and alter cancer cell invasion in a stiffness-dependent manner. The invasion of primary breast cancer cells through mammary adipose is influenced by the heterogeneous mechanical properties of native stromal components, namely adipocytes and extracellular matrix (left). Customizable granular hydrogels provide insight into the physical regulation of breast cancer progression by mimicking adipose tissue extracellular matrix structure (collagen I, i.) and stromal cell mechanics (polyacrylamide bead, ii.), whereby the inclusion of soft beads and/or dilute matrix promotes cancer cell migration compared to stiffer hydrogels (right). C_FIG

bioengineering↗

bioRxiv2025

Transcriptional coregulation in cis around a contact insulation site revealed by single-molecule microscopy

Chromosome conformation in mammals is closely related to gene regulation. Within topologically associating domains, where genomic contacts are enriched, genes tend to show correlated expression across tissues and conditions, suggesting domain-wide mechanisms coregulating multiple genes, such as enhancer sharing or local histone mark spreading. At the single-cell level, where transcription occurs in sporadic bursts, transcriptional coordination has been observed between proximal genes, but how the local folding of mammalian chromosomes influences gene coregulation in cis at individual alleles remains unclear. Using single-molecule microscopy, we imaged nascent transcription from three adjacent genes located around a strong contact insulation site at the FOS locus, during the estrogen response in human breast cancer cells. To interpret this data, we developed two new analysis approaches to dissect the sources of (co)variation in gene activities: one to separate allele-extrinsic, allele-intrinsic, and gene-autonomous components; and another one to quantify the contributions of burst co-occurrence and burst size correlations. We find that transcriptional variability is largely gene-autonomous, yet correlations between genes display distinct patterns and occur almost exclusively in cis. Correlations are stronger between proximal and less insulated genes. However, unexpectedly, substantial correlations also occur across the strong insulation site and, under certain conditions, two proximal genes on the same side can exhibit uncorrelated burst occurrences. By disentangling burst co-occurrence from burst size correlations, we reveal transcriptional patterns suggesting two distinct coregulatory mechanisms influenced by local chromosome folding.

biophysics↗

bioRxiv2025

Dual inhibition of lactate transporters MCT1 and MCT4 in pancreatic neuroendocrine tumors targets metabolic heterogeneity and functional redundancy

Our current understanding of the metabolic landscape of pancreatic neuroendocrine tumors (PanNETs) is very limited. Such knowledge could lead the development of novel therapeutic strategies for subgroups of PanNET patients based on the metabolic profile of their tumor. Here, we investigated the expression of lactate transporters MCT1 and MCT4 in two independent PanNET cohorts (n=93; n=70) and analyzed their association with tumor aggressiveness and therapeutic vulnerability in cell lines, spheroids and patient-derived tumoroids of PanNET. Immunohistochemistry revealed four expressor types: MCT1/4-negative, MCT1-positive, MCT4-positive, and MCT1/4-double positive with frequent regional co-expression. Both homogenous and heterogenous expression patterns were observed, indicating metabolic heterogeneity within the latter subset of PanNETs. MCT4 expression correlated with the hypoxia marker CA9, suggesting a hypoxic and acidic tumor microenvironment. Mechanistic studies revealed that MCT1 and MCT4 operate both as lactate efflux systems in PanNET cell lines, providing functional redundancy to their glycolytic roles. Inhibition of lactate efflux in normoxia and hypoxia using the dual MCT1/4 inhibitor syrosingopine significantly impaired lactate secretion, glycolysis, and proliferation across PanNET cell lines and 3D spheroid and patient-derived tumoroid models. In contrast, selective MCT1 or MCT4 inhibitors showed limited efficacy, underscoring the therapeutic need for co-targeting MCT1 and MCT4 due to functional redundancy and heterogenous expression. This work demonstrates MCT1 and MCT4 as metabolic markers and promising therapeutic targets of a subset of PanNETs with clinical features of aggressiveness.

cancer biology↗

bioRxiv2024

The DivisionCounter, a method for counting large ranges of cell divisions in vivo, reveals cell dynamics of leukemic cell killing via CAR-T therapy

Cell division drives multicellular growth and its dysregulation can cause disease. While approximately 44 divisions are needed to produce without death all 1013 cells in the human body, current methods are limited to count 10 cell divisions in vivo across diverse mammalian cell types. Here we introduce the DivisionCounter, a method to count cell division in vivo over large division ranges ([~]70 divisions) using an easy fluorescence readout. We demonstrate that the DivisionCounter accurately measures the average cell division count of cells both in vitro and in vivo. Its use revealed that leukemia tumor division rates are independent of the organs specific microenvironment and CAR-T cell treatment, providing an estimate of tumor death rates in vivo. The DivisionCounter method holds unique potential for quantifying contributions of cell division, death, and migration to the growth of healthy and pathological mammalian tissues.

immunology↗

bioRxiv2022

Cell adhesion and spreading on fluid membranes through microtubules-dependent mechanotransduction

During cell adhesion, integrins form clusters that transmit mechanical forces to the substrate (mechanotransduction) and regulate biochemical signaling depending on substrate stiffness. Studies on mechanotransduction significantly advanced our understanding of cell adhesion and were mostly performed on rigid substrates. In contrast to rigid substrates, integrins ligands on fluid supported lipid bilayers (SLBs) are mobile and adhesive complexes cannot serve as anchoring points promoting cell spreading. Here, we demonstrate that cells spread on SLBs coated with Invasin, a high-affinity integrin ligand. We show that in contrast to SLBs functionalized with RGD peptides, integrin clusters grow in size and complexity on Invasin-SLBs to a similar extent as on glass. While actomyosin contraction dominates adhesion maturation on stiff substrates, we find that integrin mechanotransduction and cell spreading on fluid SLBs rely on dynein pulling forces along microtubules perpendicular to membranes and microtubules pushing on adhesive complexes, respectively. These forces that may also occur on non-deformable surfaces are revealed in fluid substrate set ups. Our findings, supported by a theoretical model, demonstrate a new mechanical role for microtubules in integrin clustering.

cell biology↗

bioRxiv2022

Arp2/3 inhibition switches Eps8's network associations to favour longer actin filament formation necessary for tunneling nanotubes

Tunneling nanotubes (TNTs) connect distant cells and mediate cargo transfer for intercellular communication in physiological and pathological contexts. How cells generate these actin-mediated protrusions to span lengths beyond those attainable by canonical filopodia remains unknown. Through a combination of micropatterning, microscopy and optical tweezer-based approaches, we demonstrate that TNTs forming through the outward extension of actin (not through cellular dislodgement) achieve distances greater than the mean length of filopodia, and that branched Arp2/3-dependent pathways attenuate the extent to which actin polymerizes in nanotubes, limiting TNT occurrence. Proteomic analysis using Epidermal growth factor receptor kinase substrate 8 (Eps8) as a positive effector of TNTs showed that upon Arp2/3 inhibition, proteins enhancing filament turnover and depolymerization were reduced and Eps8 instead exhibited heightened interactions with the inverted Bin/Amphiphysin/Rvs (I-BAR) domain protein IRSp53 that provides a direct connection with linear actin polymerases. Our data reveals how common protrusion players (Eps8 and IRSp53) form TNTs, and that when competing pathways overutilizing such proteins and monomeric actin in Arp2/3 networks are inhibited, processes promoting linear actin growth dominate to favour TNT formation. Thus, this work reinforces a general principle for actin network control for cellular protrusions where simple shifts in the balance between processes that inhibit actin growth versus those that promote growth dictate protrusion formation and the ultimate length scales protrusions achieve.

cell biology↗

bioRxiv2021

Coupling between protrusion dynamics and polarized trafficking steers persistent cell migration

Migrating cells present a variety of paths, from random to highly directional ones. While random movement can be explained by basal intrinsic activity, persistent movement requires stable polarization. Here, we quantitatively address emergence of persistent migration in RPE1 cells over long timescales. By live-cell imaging and dynamic micropatterning, we demonstrate that the Nucleus-Golgi axis aligns with direction of migration leading to efficient cell movement. We show that polarized trafficking is directed towards protrusions with a 20 min delay, and that migration becomes random after disrupting internal cell organization. Eventually, we prove that localized optogenetic Cdc42 activation orients the Nucleus-Golgi axis. Our work suggests that polarized trafficking stabilizes the protrusive activity of the cell, while protrusive activity orients this polarity axis, leading to persistent cell migration. Using a minimal physical model, we show that this feedback is sufficient to recapitulate the quantitative properties of cell migration in the timescale of hours.

biophysics↗

bioRxiv2019

Actin accumulates nesprin-2 at the front of the nucleus during confined cell migration

The mechanisms by which cells exert forces on their nuclei to migrate through openings smaller than the nuclear diameter remain unclear. In microfluidic devices, the hourglass shape of the nucleus and its strain patterns as it translocates through narrow constrictions suggest pulling forces. We use CRISPR/Cas9 to fluorescently label nesprin-2 giant, a protein that links the cytoskeleton to the interior of the nucleus. We demonstrate that nesprin-2 giant accumulates at the front of the nucleus during nuclear deformation through narrow constrictions, independently of the nuclear lamina. We find that nesprins are more mobile than lamin A/C, at time scales similar to that of the accumulation. Using artificial constructs, we show that the actin-binding domain of nesprin-2 is necessary and sufficient to generate this accumulation, and that microtubules are not necessary. Actin filaments are organized in a barrel structure around the moving nucleus in the direction of movement, suggesting that this structure is responsible for redistribution of nesprins towards the front of the nucleus. Two-photon ablation and the use of drugs inhibiting the cytoskeleton demonstrate a pulling force on the nucleus from the front of the cell that is dependent on formin and actomyosin contractility. This elastic recoil is significantly reduced when nesprins are reduced at the nuclear envelope. We thus show that actin redistributes nesprin-2 giant towards the front of the nucleus and contributes to pulling the nucleus through narrow constrictions, in concert with myosin.

cell biology↗