Biology subjects

Alon, R.

Publications and source records attributed to Alon, R..

8 recordsLinked to original sources

bioRxiv2026

Sub-endothelial platelet activation amplifies neutrophil transmigration within venular walls

Neutrophil recruitment into inflamed tissues requires coordinated transmigration across the multilayered venular wall, yet how this process is regulated beyond the endothelium remains poorly understood. Here, we identify a previously unrecognized platelet-neutrophil circuit that controls this post-endothelial phase. Intravital microscopy of inflamed cremasteric venules showed that pioneer neutrophil transmigration enabled platelet entry into the sub-endothelial compartment of venular walls. There, platelets became activated and released CXCL7 in a GPVI- and GPIb-dependent manner, generating spatially confined chemokine microdomains. These platelet-derived cues directed follower neutrophil migration and promoted their exit across the pericyte layer into the interstitial tissue. Collectively, these findings identify extraluminal platelets as spatial organizers of neutrophil trafficking and uncover a localized intramural feedback mechanism in which neutrophils amplify their own recruitment within venular walls. Graphical summary O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/739803v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1d22216org.highwire.dtl.DTLVardef@bc0279org.highwire.dtl.DTLVardef@1465d7dorg.highwire.dtl.DTLVardef@a3e755_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Inducible re-epithelialization of cancer cells increases autophagy and DNA damage: implications for breast cancer dormancy

Epithelial lineage differentiation is pivotal to mammary gland development and it can pause metastasis of breast cancer (BC) by inducing tumor dormancy. To simulate this, we expressed epithelial genes in mesenchymal BC cells. Inducible expression of the epithelial OVOL genes in metastatic BC cells suppressed proliferation and migration. We found that C1ORF116, an OVOLs target, is susceptible to genetic and epigenetic aberrations in BC. It is regulated by steroids and functions as a putative autophagy receptor that inhibits antioxidants like thioredoxin. Accordingly, boosting epithelialization lowered glutathione, elevated reactive oxygen species and increased both DNA oxidation and double strand breaks. Epithelialization also associated with redistribution of NRF2 and an altered interplay among p38, ATM, and the other kinases regulating the DNA damage response. Hence, hormonal regulation of OVOLs and chronic stress might permit epithelial differentiation and retard exit from dormancy, while altering redox homeostasis and permitting DNA damage accumulation, which may awaken dormant tumors.

cancer biology↗

bioRxiv2024

Intratumoral CXCL12 Gradients Contextualize Tumor Cell Invasion, Migration and Immune Suppression in Breast Cancer

Although the CXCL12/CXCR4 pathway has been prior investigated for its prometastatic and immuno- suppressive roles in the tumor microenvironment, evidence on the spatiotemporal regulation of these hallmarks has been lacking. Here, we demonstrate that CXCL12 forms a gradient specifically around cancer cell intravasation doorways, also known as Tumor Microenvironment of Metastasis (TMEM) doorways, thus facilitating the chemotactic translocation of prometastatic tumor cells expressing CXCR4 toward the perivascular TMEM doorways for subsequent entry into peripheral circulation. Fur- thermore, we demonstrate that the CXCL12-rich micro-environment around TMEM doorways may cre- ate immunosuppressive niches, whereby CD8+ T cells, despite being attracted to these regions, often exhibit reduced effector functions, limiting their efficacy. While the CXCL12/CXCR4 pathway can mini- mally influence the overall composition of immune cell populations, it biases the distribution of CD8+ T cells away from TMEM doorways, justifying its prior-established role as immunosuppressive factor for CD8+ T cells. Our research suggests that the complex interactions between CXCL12 and the various tumor and immune cell types contributes not only to the completion of the initial steps of the metastatic cascade, but also offers an immunological "sanctuary" to prometastatic tumor cells homed around TMEM doorways. Overall, our study enhances our current understanding on the mechanisms, via which CXCL12 orchestrates tumor cell behavior and immune dynamics, potentially guiding future thera- peutic strategies to combat breast cancer metastasis and improve anti-tumor immunity.

cancer biology↗

bioRxiv2023

Murine classical monocyte subsets display distinct functions and fates

Monocytes are short-lived myeloid immune cells that arise from adult hematopoiesis and circulate for a short time in the blood. They comprise two main subsets, in mice defined as classical Ly6Chigh and non-classical Ly6Clow monocytes (CM, NCM). Recent fate mapping and transcriptomic analyses revealed that CM themselves are heterogeneous. Here, we report surface markers that allow segregation of murine GMP- and MDP-derived CM in the BM and blood. Functional characterization, including fate definition following adoptive cell transfer, established that GMP-Mo and MDP-Mo could equal rise to homeostatic CM progeny, such as NCM in blood and gut macrophages, but differentially seeded selected other tissues. Specifically, GMP-Mo and MDP-Mo gave rise to distinct interstitial lung macrophages, thus linking CM dichotomy to previously reported pulmonary macrophage heterogeneity. Collectively, we provide comprehensive evidence for the existence of two functionally distinct CM subsets in the mouse, which differentially contribute to peripheral tissue macrophage populations in homeostasis and following challenge. Our findings are indicative of impact of monocyte ontogeny on in situ differentiation.

immunology↗

bioRxiv2021

Dendritic cell ICAM-1 strengthens immune synapses but is dispensable for effector and memory responses

Lymphocyte priming in lymph nodes (LNs) depends on the formation of functional TCR specific immune synapses (ISs) with antigen (Ag) presenting dendritic cells. The high affinity LFA-1 ligand ICAM-1 has been implicated in different ISs studied in vitro. The in vivo roles of DC ICAM-1 in Ag stimulated T cell differentiation have been unclear. In newly generated DC conditional ICAM-1 knockout mice, we report that under Th1 polarizing conditions, ICAM-1 deficient DCs could not engage in stable conjugates with newly generated CD8 blasts. Nevertheless, these DCs triggered normal lymphocyte priming, proliferation and differentiation into functional cytotoxic T cells (CTLs) and central memory lymphocytes (Tcm) in both vaccinated and virus infected skin. Single cell RNAseq analysis confirmed that Tcm were normally generated in these mice and gave rise to normal T effectors during a recall skin response. Our results suggest that although CD8 T cell blasts tightly bind DC-ICAM-1, strongly adhesive DC-T ISs are not necessary for functional TCR dependent DC mediated CD8 T cell proliferation and differentiation into productive effector and memory lymphocytes. SummarySapoznikov et al generated a new genetic murine model deficient in dendritic cell expression of the key adhesion molecule ICAM-1 and found that CD8 lymphocytes do not require strong adhesion to dendritic cells for antigen-dependent differentiation into effector and memory T cells.

immunology↗

bioRxiv2020

Reduced nuclear lamin A/C enhances cancer cell squeezing through rigid barriers, does not facilitate endothelial crossing, and impairs experimental metastasis

The mechanisms by which the nuclear lamina of tumor cells controls their migration and survival are poorly understood. Lamin A and its variant lamin C are key nuclear lamina proteins that control nucleus stiffness and chromatin conformation. Downregulation of lamin A/C levels in two metastatic lines, B16F10 melanoma and E0771 breast carcinoma, facilitated cell squeezing through rigid pores, elevated nuclear deformability and reduced heterochromatin. Unexpectedly, the transendothelial migration of both cancer cells in vitro and in vivo, through lung capillaries, was not elevated by lamin A/C knockdown. Both cancer cells with lamin A/C knockdown grew normally in primary tumors and in vitro on rigid surfaces. Strikingly, however, both lamin A/C deficient melanoma and breast cancer cells grew poorly in 3D spheroids expanded in soft agar cultures. Experimental lung metastasis of both lamin A/C knockdown cells was also markedly reduced. Taken together, our results suggest that high content of lamin A/C in multiple cancer cells promotes cancer cell survival and ability to generate lung metastasis without compromising cancer cell emigration from lung vessels.

cancer biology↗

bioRxiv2020

Microvillar cartography: a super-resolution single-molecule imaging method to map the positions of membrane proteins with respect to cellular surface topography

We introduce Microvillar Cartography (MC), a method to map proteins on cellular surfaces with respect to the membrane topography. The surfaces of many cells are not smooth, but are rather covered with various protrusions such as microvilli. These protrusions may play key roles in multiple cellular functions, due to their ability to control the distribution of specific protein assemblies on the cell surface. Thus, for example, we have shown that the T-cell receptor and several of its proximal signaling proteins reside on microvilli, while others are excluded from these projections. These results have indicated that microvilli can function as key signaling hubs for the initiation of the immune response. MC has facilitated our observations of particular surface proteins and their specialized distribution on microvillar and non-microvillar compartments. MC combines membrane topography imaging, using variable-angle total internal microscopy, with stochastic localization nanoscopy, which generates deep sub-diffraction maps of protein distribution. Since the method is based on light microscopy, it avoids some of the pitfalls inherent to electron-microscopy-based techniques, such as dehydration, carbon coating and immunogold clustering, and is amenable to future developments involving e.g. live-cell imaging. This Protocol details the procedures we developed for MC, which can be readily adopted to study a broad range of cell surface molecules and dissect their distribution within distinct surface assemblies under multiple cell activation states.

biophysics↗

bioRxiv2019

ERM-Dependent Assembly of T-Cell Receptor Signaling and Co-stimulatory Molecules on Microvilli Prior to Activation

SummaryT-cell surfaces are covered with microvilli, actin-rich and flexible protrusions. We use super-resolution microscopy to show that [≥]90% T-cell receptor (TCR) complex molecules TCR{beta} and TCR{zeta}, as well as the co-receptor CD4 and the co-stimulatory molecule CD2 reside on microvilli of human T cells. Furthermore, TCR proximal signaling molecules involved in the initial stages of the immune response, such as the protein tyrosine kinase Lck and the key adaptor molecule LAT, are also enriched on microvilli. Notably, phosphorylated proteins of the ERM (ezrin, radixin, moesin) family colocalize with these heterodimers as well as with actin filaments within the microvilli of resting T cells. This finding implies a role for one or more phosphorylated ERMs in linking the TCR complex to the actin cytoskeleton within microvilli. Indeed, expression of a dominant-negative ezrin fragment effectively redistributes TCR molecules over the whole T cell surface. Our results establish microvilli as key signaling hubs, in which the TCR complex and its proximal signaling molecules and adaptors are pre-assembled prior to activation in an ERM-dependent manner. The preformed positioning of these actin-binding TCR assemblies on individual microvilli can facilitate the local transmission of TCR signals seconds after TCR occupancy and impacts the slower subsequent events that lead to the assembly of immunological synapses.

immunology↗