Biology subjects

Rafat, M.

Publications and source records attributed to Rafat, M..

15 recordsLinked to original sources

bioRxiv2026

Radiotherapy-induced vascular dysfunction alters neutrophil phenotype to promote tumor cell colonization of mammary adipose tissue

Recurrence remains a significant clinical challenge for triple-negative breast cancer patients and radiotherapy (RT) is a key therapeutic tool that significantly reduces the risk of relapse. Despite this, patients with chronic lymphopenia and a high neutrophil:lymphocyte ratio (NLR) experience recurrence at significantly higher rates than those whose hematological biomarkers return to baseline post-RT, and there are currently no treatments to prevent this relapse. Here, we utilized in vivo mouse models of lymphocyte-deficiency to identify the mechanism behind systemic immune status and recurrence. Using a model of RT-induced circulating tumor cell (CTC) colonization, we found that neutrophils are sustained in the irradiated, lymphocyte-deficient mammary tissue up to 10 days post-RT. Persistent neutrophil accumulation was accompanied by excessive vascular remodeling and tumor cell colonization of the mammary tissue, both of which were abated upon systemic neutrophil depletion. Neutrophils in the colonization-permissive microenvironment exhibited a state of pathological activation consistent with a polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) phenotype that was not seen in systemic compartments, highlighting a microenvironment-induced phenotype shift. This phenotype was recapitulated in vitro when naive neutrophils were exposed to the secretome of RT-induced senescent endothelial cells (ECs). We found that these activated neutrophils then regulated the dysfunction of irradiated ECs in a canonical NF-kB-dependent manner, increasing their barrier permeability and activating downstream inflammatory signals. Our findings not only establish neutrophils as a critical component of tumor cell colonization in lymphocyte-deficient subjects but also uncover a positive feedback loop between neutrophils and irradiated vasculature that leads to aberrant tissue remodeling and ultimately tumor cell colonization. This work deepens our understanding of how systemic factors influence treatment-damaged microenvironments, which could ultimately lead to identification of molecular targets for prevention of RT failure.

cancer biology↗

bioRxiv2026

A Latent Inflammatory Tissue-State Variable Mechanistically Links Radiotherapy-Induced Immune Remodeling to Recurrent Tumor Permissiveness

Triple-negative breast cancer recurrence following radiotherapy is associated with a microenvironment characterized by immune dysfunction and persistent inflammation. We developed an experimentally constrained agent-based model to investigate how transient immune remodeling becomes a persistent recurrence-permissive tissue state. The model reproduced experimentally observed macrophage recruitment and phenotype dynamics but demonstrated that recurrent recruitment, impaired inflammatory resolution, and adaptive immune bias were insufficient to reproduce the recurrent macrophage ecology. We therefore introduced recurrence-associated microenvironmental inflammation (RAMI), a latent tissue-state variable representing accumulated unresolved inflammatory remodeling. Coupling RAMI to the emergence of experimentally constrained interleukin-6 signaling generated tissue-to-cell feedback that reinforced recurrence-associated macrophage phenotypes and increased tumor establishment, supporting inflammatory tissue memory as a mechanistic intermediary between transient immune perturbation and persistent recurrent tumor permissiveness.

systems biology↗

bioRxiv2026

A mammary-specific microfluidic device for studying post-radiotherapy vascular-immune cell interactions

Radiotherapy (RT) significantly improves outcomes and reduces the risk of recurrence in breast cancer. However, in patients that experience recurrence despite treatment, the irradiated breast tissue may create a pre-metastatic niche that promotes tumor cell infiltration. Because current models of metastasis lack key physiological and microenvironmental cues, we developed a mammary vasculature-on-chip (MVoC) to model how the vasculature responds to RT and impacts immune and tumor cell behavior in mammary tissue. Murine and human MVoCs incorporated endothelial cells (ECs) cultured under physiologic shear stress and mammary-specific fibroblasts to mimic the vascular-fibrous stroma interface. MVoCs exhibited a transient, acute response to RT that led to a persistent phenotypic shift. Neutrophils, which play key roles in pre-metastatic niche formation, adhered more to irradiated ECs and induced persistent damage to the endothelial barrier, preventing recovery. Neutrophils also significantly increased tumor cell adhesion to the endothelium in MVoCs. These findings demonstrate that MVoCs can be used to study RT-induced pre-metastatic niche formation and suggest a positive feedback loop between damaged ECs and immune cell activation that may facilitate tumor cell colonization. MVoCs provide a biologically relevant approach to probe molecular and cellular interactions, improving translation toward developing novel therapies that improve patient outcomes.

cancer biology↗

bioRxiv2026

CD4+ T-Cells Drive Triple Negative Breast Cancer Recurrence via Non-Canonical TGFβ Signaling

Radiation therapy is a cornerstone of breast cancer treatment and reduces recurrence overall. However, patients with triple negative breast cancer (TNBC) continue to experience recurrence at higher rates than patients with other subtypes, especially when immunocompromised. While CD8 T-cells are known to mitigate recurrence, the role of CD4+ T-cell subsets in shaping the irradiated microenvironment remains unclear. We show that irradiated mammary tissue from mice accumulates CD4+ T-cells and exhibits a TGF{beta}-enriched cytokine milieu coincident with macrophage infiltration. We demonstrate that Th2-polarized CD4+ T-cells promote invasion of TNBC cells and macrophages through secretion of TGF{beta}. Neutralization of TGF{beta} significantly reduces this invasive phenotype. Mechanistically, Th2-conditioned media induces Tgfb1 expression in both TNBC cells and macrophages, establishing a TGF{beta}-dependent feed-forward amplification loop. In TNBC cells, Th2-derived TGF{beta} activates non-canonical signaling characterized by increased p38 MAPK and NF-{kappa}B phosphorylation, linking cytokine exposure to pro-invasive behavior. Together, these findings identify Th2-derived TGF{beta} as a driver of pro-invasive tumor reprogramming and suggest that interruption of Th2-TGF{beta} signaling may prevent recurrence following therapy.

cancer biology↗

bioRxiv2026

Development of Shelf-Stable Reagents and Assay Kits for Bioluminescence Applications using the Capillary-Assisted Vitrification Platform Stabilization Technology

Luminescence is a powerful method for detecting trace analytes and monitoring biological processes. However, most bioluminescence reagents, including luciferase and its substrates, are sensitive to temperature, limiting their useable shelf lives, and resulting in inconsistent performance. Enhancing the stability of these reagents could improve data quality, simplify workflows, and address cold chain storage issues. In this study, we demonstrate the application of the platform stabilization technology, capillary-assisted vitrification (CAV), as a tool to stabilize different luciferases and their substrates, and the application of the stabilized reagents in both in vitro and in vivo bioluminescent assays. We demonstrate that CAV-stabilized reagents can be stored and shipped ambiently, maintain consistent performance over time, and are suitable for use in cell viability quantification, tumor monitoring, in vivo imaging, microbial detection, and immunoassays. Additionally, different reagents can be co-formulated to make ready-to-use assay kits that can also be shipped and stored ambiently. Our results demonstrate that CAV stabilization is a viable alternative to traditional storage methods, with broad potential to improve bioluminescence workflows.

biochemistry↗

bioRxiv2025

Mammary Fibroblasts Secrete Damage Associated Molecular Patterns through Extracellular Vesicles in Response to Ionizing Radiation

Ionizing radiation (IR) is an integral component of cancer therapy. Cellular exposure to IR typically leads to major biological consequences including cell death and senescence. Furthermore, tissue injury in known to involve the release of damage-associated molecular patterns (DAMPs) into the extracellular space, which trigger inflammation and wound healing. However, DAMP release in the context of radiation injury remains to be fully characterized. Evidence suggests that extracellular vesicle (EV) secretion and associated cargo components are part of the cellular response to IR, but the mechanisms integrating cellular damage and EV secretion post-IR are largely unexplored. In this study, we show that acute IR-induced damage in mammary fibroblasts results in a senescence-like phenotype and substantially increased EV secretion. Quantitative proteomic analysis revealed that IR-induced EVs are enriched with extracellular and intracellular DAMPs, along with other pro-inflammatory mediators. We show that knockdown of the GTPase Rab27a abrogates IR-induced EV secretion and inhibits the enrichment of key DAMPs in EVs. By examining the integration of cellular damage and senescence with the release of inflammatory signals, this study elucidates a potentially critical role for EV-associated proteins in the radiation response.

cancer biology↗

bioRxiv2025

Encoded cell-material interactions to reroute cytokine signaling for regenerative medicine

Regenerative engineering harnesses materials science and stem cell biology to develop strategies to repair damaged and diseased tissue. Despite advances in designer materials, few techniques effectively provide auto-regulated feedback mechanisms that govern how cells sense and respond to discrete microenvironmental changes. Here, we demonstrate that the artificial, juxtacrine-like receptor synthetic Notch (synNotch) can be activated by endogenous multimeric cytokines in solution, without immobilizing materials, revealing a previously unreported activation modality and yielding up to 24-fold dynamic range. To broaden synNotch sensing to monomeric cytokines, we developed nMATRIX, a co-engineered material-cell platform that detects endogenous, soluble ligands and routes them to programmed gene circuits with spatially confined effects. nMATRIX can be tuned to recognize the interleukins IL-1{beta} and IL-6 using synNotch receptors plus cognate biomaterials, yielding up to 68-fold dynamic range and converting these inflammatory inputs into orthogonal outputs that reprogram nearby cell phenotypes. nMATRIX functions across multiple cell types and can incorporate the synNotch-related SNIPR synthetic receptor platform. nMATRIX repurposed inflammatory signals and converted them into anti-inflammatory cues to polarize macrophages (increased CD163, CD206; decreased CD86). Thus, nMATRIX couples native soluble cues to customized cellular responses with tunable sensitivity, offering a flexible materials-based approach for self-regulating regenerative therapies.

bioengineering↗

bioRxiv2024

miR-100 and miR-125b Contribute to Enhanced Growth and Invasiveness

Extracellular communication via the transfer of vesicles and nanoparticles is now recognized to play an important role in tumor microenvironment interactions. Cancer cells upregulate and secrete abundant levels of miR-100 and miR-125b that can alter gene expression by both cell- and non-cell-autonomous mechanisms. We previously showed that these miRNAs activate Wnt signaling in colorectal cancer (CRC) through noncanonical pairing with 5 negative regulators of Wnt signaling. To identify additional targets of miR-100 and miR-125b, we used bioinformatic approaches comparing multiple CRC cell lines, including knockout lines lacking one or both of these miRNAs. From an initial list of 96 potential mRNA targets, we tested 15 targets with 8 showing significant downregulation in the presence of miR-100 and miR-125b. Among these, Cingulin (CGN) and Protein tyrosine phosphatase receptor type-R (PTPRR) are downregulated in multiple cancers, consistent with regulation by increased levels of miR-100 and miR-125b. We also show that increased cellular levels of miR-100 and miR-125b enhance 3D growth and invasiveness in CRC and glioblastoma cell lines. Lastly, we demonstrate that extracellular transfer of miR-100 and miR-125b can silence both reporter and endogenous mRNA targets in recipient cells and also increase the invasiveness of recipient spheroid colonies when grown under 3D conditions in type I collagen.

cancer biology↗

bioRxiv2023

Ultrasound Imaging Enables Longitudinal Tracking of Vascular Changes that Correlate with Immune Cell Infiltration After Radiotherapy

BackgroundWhile immunotherapy shows great promise in patients with triple negative breast cancer, many will not respond to treatment. Radiotherapy has the potential to prime the tumor-immune microenvironment for immunotherapy. However, predicting response is difficult due to tumor heterogeneity across patients, which necessitates personalized medicine strategies that incorporate tumor tracking into the therapeutic approach. Here, we investigated the use of ultrasound (US) imaging of the tumor vasculature to monitor the tumor response to treatment. MethodsWe utilized ultrafast power doppler US to track the vascular response to radiotherapy over time. We used 4T1 (metastatic) and 67NR (non-metastatic) breast cancer models to determine if US measurements corroborate conventional immunostaining analysis of the tumor vasculature. To evaluate the effects of radiation, tumor volume and vascular index were calculated using US, and the correlation between vascular changes and immune cell infiltration was determined. ResultsUS tumor measurements and the quantified vascular response to radiation were confirmed with caliper measurements and immunostaining, respectively, demonstrating a proof-of-principle method for non-invasive vascular monitoring. Additionally, we found significant infiltration of CD8+ T cells into irradiated tumors 10 days after radiation, which followed a sustained decline in vascular index and an increase in splenic CD8+ T cells that was first observed 1 day post-radiation. ConclusionsOur findings reveal that ultrafast power doppler US can evaluate changes in tumor vasculature that are indicative of shifts in the tumor-immune microenvironment. This work may lead to improved patient outcomes through observing and predicting response to therapy.

cancer biology↗

bioRxiv2022

Irradiated Mammary Spheroids Elucidate Mechanisms of Macrophage-Mediated Breast Cancer Recurrence

While most patients with triple negative breast cancer receive radiotherapy to improve outcomes, a significant subset of patients continue to experience recurrence. Macrophage infiltration into radiation-damaged sites has been shown to promote breast cancer recurrence in pre-clinical models. However, the mechanisms that drive recurrence are unknown. Here, we developed a novel spheroid model to evaluate macrophage-mediated tumor cell recruitment. We first characterized infiltrating macrophage phenotypes into irradiated mammary tissue to inform our model. We then established spheroids consisting of fibroblasts isolated from mouse mammary glands. We observed that tumor cell motility toward irradiated spheroids was enhanced in the presence of a 2:1 ratio of pro-healing:pro-inflammatory macrophages. We also measured a significant increase in interleukin 6 (IL-6) secretion after irradiation both in vivo and in our model. This secretion increased tumor cell invasiveness, and invasion was mitigated by neutralizing IL-6. Taken together, our work suggests that interactions between infiltrating macrophages and damaged stromal cells facilitates breast cancer recurrence through IL-6 signaling.

cancer biology↗

bioRxiv2022

Radiation-Induced Metabolic Reprogramming of Fibroblasts Regulates the Breast Cancer Microenvironment

Patients with triple-negative breast cancer (TNBC) experience high recurrence rates despite current interventions, which includes radiation therapy (RT). Tumor cells thought to be involved in recurrence survive in part due to their interactions with irradiated fibroblasts following treatment. How fibroblasts metabolically respond to RT and influence the behavior of TNBC cells is poorly understood. In this study, we demonstrate that irradiated fibroblasts undergo a mitochondrial stress response that is regulated by autophagy, resulting in a metabolic profile characterized by high levels of mitochondrial respiration and fatty acid oxidation. This stress response in fibroblasts induces an aggressive phenotype in TNBC cells that is mitigated when fibroblast autophagy is blocked. Our work reveals how a metabolic stress response in irradiated fibroblasts and crosstalk with TNBC cells leads to a microenvironment conducive to recurrence. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/492249v3_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@181680aorg.highwire.dtl.DTLVardef@d58a1forg.highwire.dtl.DTLVardef@15f04eforg.highwire.dtl.DTLVardef@13cf5f9_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2022

Mammary Tissue-Derived Extracellular Matrix Hydrogels Reveal the Role of the Irradiated Microenvironment in Breast Cancer Recurrence

Radiation therapy (RT) is essential for triple negative breast cancer (TNBC) treatment. However, patients with TNBC continue to experience recurrence after RT. The role of the extracellular matrix (ECM) of irradiated breast tissue in tumor recurrence is still unknown. In this study, we evaluated the structure, molecular composition, and mechanical properties of irradiated murine mammary fat pads (MFPs) and developed ECM hydrogels from decellularized tissues (dECM) to assess the effects of RT-induced ECM changes on breast cancer cell behavior. Irradiated MFPs were characterized by increased ECM deposition and fiber density compared to unirradiated controls, which may provide a platform for cell invasion and proliferation. ECM component changes in collagens I, IV, and VI, and fibronectin were observed following irradiation in both MFPs and dECM hydrogels. Encapsulated TNBC cell proliferation and invasive capacity was enhanced in irradiated dECM hydrogels. In addition, TNBC cells co-cultured with macrophages in irradiated dECM hydrogels induced M2 macrophage polarization and exhibited further increases in proliferation. Our study establishes that the ECM in radiation-damaged sites promotes TNBC invasion and proliferation as well as an immunosuppressive microenvironment. This work represents an important step toward elucidating how changes in the ECM after RT contribute to breast cancer recurrence.

cancer biology↗

bioRxiv2022

Non-Invasive monitoring of normal tissue radiation damage using quantitative ultrasound spectroscopy

BackgroundWhile radiation therapy (RT) is an critical component of breast cancer therapy and is known to decrease overall local recurrence rates, recent studies have shown that normal tissue radiation damage may increase recurrence risk. Fibrosis is a well-known consequence of RT, but the specific sequence of molecular and mechanical changes induced by RT remains poorly understood. PurposeThere is currently a need to understand the contribution of the irradiated tissue microenvironment to recurrence to improve cancer therapy outcomes. This study seeks to evaluate the use of quantitative ultrasound spectroscopy (QUS) for real time determination of the normal tissue characteristic radiation response and to correlate these results to molecular features in irradiated tissues. MethodsMurine mammary fat pads (MFPs) were irradiated to 20 Gy, and QUS was used to analyze tissue physical properties pre-irradiation as well as at 1, 5, and 10 days post-irradiation. Tissues were processed for scanning electron microscopy imaging as well as histological and immunohistochemical staining to evaluate morphology and structure. ResultsTissue morphological and structural changes were observed non-invasively following radiation using mid-band fit (MBF), spectral slope (SS), and spectral intercept (SI) measurements obtained from QUS. Statistically significant shifts in MBF and SI indicate structural tissue changes in real time, which matched histological observations. Radiation damage was indicated by increased adipose tissue density and extracellular matrix (ECM) deposition. ConclusionsOur findings demonstrate the potential of using QUS to non-invasively evaluate normal tissue changes resulting from radiation damage. This supports further pre-clinical studies to determine how the tissue microenvironment and physical properties change in response to therapy, which may be important for improving treatment strategies.

cancer biology↗

bioRxiv2019

FLASH irradiation enhances the therapeutic index of abdominal radiotherapy in mice

Peritoneal metastases are the leading cause of morbidity and mortality in ovarian cancer. Despite current surgery, chemotherapy and targeted therapies, the majority of patients diagnosed with advanced epithelial ovarian cancer develop recurrent disease and overall survival rates remain poor. It is known that ovarian cancer is a radiosensitive tumor. Historically, total abdominal irradiation (TAI) was used as an effective postsurgical adjuvant therapy in the management of chemotherapy sensitive and resistant ovarian cancer. However, TAI fell out of favor due to high toxicity, particularly of the gastrointestinal tract. We have developed a preclinical irradiation platform that allows for total abdominal ultrahigh dose rate FLASH irradiation. We demonstrate that TAI-FLASH reduces radiation-induced intestinal injury in both healthy and tumor-bearing mice compared to conventional dose rate (CONV) irradiation. Single high dose TAI-FLASH reduced mortality from gastrointestinal syndrome, spared gut function and epithelial integrity, and decreased cell death in crypt base columnar cells. Importantly, FLASH and CONV irradiation had similar efficacy in the reduction of ovarian cancer peritoneal metastases. These findings suggest that FLASH irradiation may be an effective strategy to enhance the therapeutic index of radiotherapy for the treatment of metastatic ovarian cancer in women.

cancer biology↗

bioRxiv2019

2'3'-cGAMP is an immunotransmitter produced by cancer cells and regulated by ENPP1

23-cyclic GMP-AMP (cGAMP) is characterized as an intracellular second messenger that is synthesized in response to cytosolic dsDNA and activates the innate immune STING pathway. Our previous discovery of its extracellular hydrolase ENPP1 hinted at the existence of extracellular cGAMP. Here, using mass spectrometry, we detected that cGAMP is continuously exported as a soluble factor by an engineered cell line but then efficiently cleared by ENPP1, explaining why it has escaped detection until now. By developing a potent, specific, and cell impermeable ENPP1 inhibitor, we detected cGAMP export in cancer cell lines commonly used for mouse tumor models. In tumors, depletion of extracellular cGAMP using neutralizing proteins decreased tumor-associated dendritic cells. Boosting extracellular cGAMP by genetic knockout and pharmacological inhibition of ENPP1 increased tumor-associated dendritic cells, shrunk tumors, and synergized with ionizing radiation and anti-CTLA-4 to cure tumors. In conclusion, cGAMP is an anti-cancer immunotransmitter released by tumors and detected by host innate immunity.

biochemistry↗