Biology subjects

Donia, M.

Publications and source records attributed to Donia, M..

8 recordsLinked to original sources

bioRxiv2026

Acquired resistance to immune checkpoint inhibitors is associated with hypoxia and ECM remodeling in colorectal cancer

Acquired resistance to immune checkpoint inhibitors (ICIs) limits the durability of therapeutic responses across multiple cancer types, yet the underlying mechanisms remain poorly defined. Using the murine MC38 colorectal cancer model, we established an in vivo model recapitulating clinical response patterns, including complete regression, primary resistance, and acquired resistance. Tumors with acquired resistance progressed after initial benefit from combined anti-PD-1 and anti-CTLA-4 therapy and maintained resistance upon retransplantation into naive hosts, indicating a cancer cell-intrinsic driver of resistance. Whole-genome sequencing showed no mutations in antigen presentation or IFN-{gamma} signaling pathways previously described in single clinical cases of acquired resistance, and functional assays confirmed preserved antigenicity and IFN-{gamma} responsiveness. Transcriptomic and metabolic profiling of resistant cancer cells instead revealed metabolic reprogramming characterized by enhanced mitochondrial respiration and enrichment of hypoxia-related gene signatures, suggesting cancer cell-intrinsic adaptations that reshape the tumor microenvironment. Tumors with acquired resistance exhibited an increase in tumor-associated macrophages, and these macrophages displayed enriched transcriptional signatures of hypoxia, angiogenesis, and extracellular matrix (ECM) remodeling. Proteomic analysis of ECM-enriched tumor fragments showed accumulation of proteoglycans and enzymes such as lysyl oxidase, consistent with active matrix-remodeling. These changes coincided with altered T cell activation characterized by reduced cytotoxic gene expression and decreased CD44 expression on tumor-infiltrating T cells, suggesting impaired effector functions within the resistant microenvironment. This study identifies non-genetic mechanisms of acquired resistance to ICIs and highlights metabolic and ECM remodeling programs as promising therapeutic targets to prevent or reverse acquired resistance to ICIs.

cancer biology↗

bioRxiv2025

Acute IFN-γ elicits long-term proteomic IFN-γ signature in melanoma cells

Interferon-{gamma} (IFN-{gamma}) is a central mediator of antitumor immunity, and its downstream effects are widely assumed to require continuous cytokine exposure. Here, we applied state-of-the-art mass spectrometry (MS)-based proteomics to patient-derived melanoma cells exposed either continuously to IFN-{gamma} or to a 3-hour pulse followed by cytokine removal. A transient IFN-{gamma} exposure was sufficient to establish a stable proteomic state indistinguishable from continuous stimulation, demonstrating that IFN-{gamma}-induced remodeling is rapidly triggered and durably maintained independently of ligand persistence. Proximal JAK-STAT signaling was rapid, saturable at low cytokine concentrations, and fully reversible upon washout, yet downstream proteomic programs remained durably imprinted. In contrast, functional consequences such as cytostasis remained dose-dependent, revealing a disconnect between signaling activation and phenotypic sensitivity. Together, these findings challenge long-standing assumptions about IFN-{gamma} signaling, redefine the temporal requirements for cytokine-driven tumor cell programming, and provide a mechanistic framework for improving immunotherapies primarily working via IFN-{gamma}-signaling and physiologically relevant in vitro models of tumor-immune interactions.

biochemistry↗

bioRxiv2025

The proteomics and phosphoproteomics landscape of melanoma under T cell attack

Understanding how tumor cells interact with tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment (TME) is crucial for identifying targetable immune checkpoints and predictive biomarkers for immunotherapy. While transcriptional responses have been characterized, protein-level changes remain largely unexplored. Here, we used a system reproducing the interaction of TILs with cancer cells occurring in the TME, by co-culturing patient-derived cancer cells with matched autologous TILs at sub-lethal ratios. Using this system, we profiled the early response that cancer (melanoma) cells and TILs activate following autologous T cell attack. To distinguish melanoma from TIL proteomes, we applied stable isotope labeling by amino acids in cell culture (SILAC) combined with Orbitrap Astral-based data-independent acquisition (DIA) mass spectrometry, enabling cell type-specific profiling of protein and phosphorylation dynamics without FACS sorting. This approach also captured the global newly synthesized proteome of the mixed cultures. Our analyses resolved interferon-{gamma}-dependent proteome changes occurring in melanoma cells, identified the cytotoxic and regulatory T-cell molecule (CRTAM) as a selective marker of reactive cytotoxic T lymphocytes, and revealed tumor-intrinsic kinase activation signatures. Among these, multiple DNA damage response-associated kinases were activated during immune attack, suggesting potential therapeutic vulnerabilities. Overall, this framework enables proteomic dissection of tumor-immune interactions and provides a resource for guiding biomarker discovery and therapeutic strategies to improve immunotherapy outcomes.

cancer biology↗

bioRxiv2025

Checkpoint receptors in circulating γδ T cells can discern the outcome of cancer immunotherapy

Gamma delta ({gamma}{delta}) T cells are innate-like lymphocytes that in humans can be broadly classified into two main subtypes based on their unique TCR{delta} chain, V{delta}1 and V{delta}2. Both subsets have potent anti-tumor properties and the presence of V{delta}1 cells in the tumor is often associated with positive prognosis. Herein, we investigated the molecular interplay between immune checkpoint receptors (ICRs), IC blockade (ICB) therapy and {gamma}{delta} T cells. We show that ICRs display differential expression and regulation by the JAK-STAT pathway in circulating V{delta}1 and V{delta}2 cells and identify constitutive (e.g. TIGIT, PD-1) and inducible (e.g. TIM-3, LAG-3, CTLA-4) ICRs. In melanoma, V{delta}1 cells, especially in patients who did not respond to ICB or required combination therapy, expressed high levels of ICR, TOX and inhibitory killer Ig-like receptors (KIR) transcripts, reminiscent of an exhaustion transcriptional signature. At the same time all {gamma}{delta} T cells had a prominent downregulation in AP-1 transcription factors. Patient derived cells were functionally competent, although induction of LAG-3 and CTLA-4 was impaired. In the context of anti-PD-1 monotherapy, V{delta}1 cells specifically bound high levels of therapeutic antibody but only in patients who responded to treatment, revealing a potential new prognostic marker for evaluating the efficacy of ICB therapy. Finally, expression of KIR genes in V{delta}1 cells was downregulated in response to successful ICB therapy. Collectively, our data indicate an intricate relationship between ICRs, putatively also KIRs, and {gamma}{delta} T cells and reveal novel approaches by which these cells can be harnessed in order to discern or improve cancer immunotherapy.

immunology↗

bioRxiv2024

Analyzing functional heterogeneity of effector cells for enhanced adoptive cell therapy applications

Cellular effector function assays traditionally rely on bulk cell populations that mask complex heterogeneity and rare subpopulations. The Xdrop(R) droplet technology facilitates high-throughput compartmentalization of viable single cells or single-cell pairs in double-emulsion droplets, enabling the study of single cells or cell-cell interactions at an individual level. Effector cell molecule secretion and target cell killing can be evaluated independently or in combination. Compatibility with a wide range of commercial assay reagents allows for single-cell level readouts using common laboratory techniques such as flow cytometry or microscopy. Moreover, individual cells of interest can be viably isolated for further investigation or expansion. Here we demonstrate the application of the double-emulsion droplet technology with a range of cell types commonly utilized for adoptive cell therapy of cancer: peripheral blood mononuclear cells, natural killer cells, tumor-infiltrating lymphocytes, and chimeric antigen receptor T cells. Single-cell compartmentalization offers unparalleled resolution, serving as a valuable tool for advancing the development and understanding of cellular therapy products.

immunology↗

bioRxiv2018

Collagen density regulates the activity of tumor-infiltrating T cells

BackgroundTumor progression is accompanied by dramatic remodeling of the surrounding extracellular matrix leading to the formation of a tumor-specific ECM, which is often more collagen-rich and of increased stiffness. The altered ECM of the tumor supports cancer growth and metastasis, but it is unknown if this effect involves modulation of T cell activity. To investigate if a high-density tumor-specific ECM could influence the ability of T cells to kill cancer cells, we here studied how T cells respond to 3D culture in different collagen densities.\n\nMethodsT cells cultured in 3D conditions surrounded by a high or low collagen density were imaged using confocal fluorescent microscopy. The effects of the different collagen densities on T cell proliferation, survival, and differentiation were examined using flow cytometry. Cancer cell proliferation in similar 3D conditions was also measured. Triple-negative breast cancer specimens were analyzed for the number of infiltrating CD8+ T cells and for the collagen density. Whole-transcriptome analyses were applied to investigate in detail the effects of collagen density on T cells. Computational analyses were used to identify transcription factors involved in the collagen density-induced gene regulation. Observed changes were confirmed by qRT-PCR analysis.\n\nResultsT cell proliferation was significantly reduced in a high-density matrix compared to a low-density matrix and prolonged culture in a high-density matrix led to a higher ratio of CD4+ to CD8+ T cells. The proliferation of cancer cells was unaffected by the surrounding collagen-density. Consistently, we observed a reduction in the number of infiltrating CD8+ T-cells in mammary tumors with high collagen-density indicating that collagen-density has a role in regulating T cell abundance in human breast cancer.\n\nWhole-transcriptome analysis of 3D-cultured T cells revealed that a high-density matrix induces downregulation of cytotoxic activity markers and upregulation of regulatory T cell markers. These transcriptional changes were predicted to involve autocrine TGF-B signaling and they were accompanied by an impaired ability of tumor-infiltrating T cells to kill autologous cancer cells.\n\nConclusionsOur study identifies a new immune modulatory mechanism, which could be essential for suppression of T cell activity in the tumor microenvironment.

immunology↗

bioRxiv2018

Influence of mutagenic versus non-mutagenic pre-operative chemotherapy on the immune infiltration of breast cancer

BackgroundChemotherapeutic agents are often mutagenic. Induction of mutation associated neo-epitopes is one of the mechanisms by which chemotherapy is thought to increase the number of tumor-infiltrating lymphocytes, but the clinical relevance of this triggered immune response is not known.\n\nWe decided to investigate, whether treatment with various chemotherapeutic agents with significantly different mutagenic capacity induce a significantly different number of stromal tumor-infiltrating lymphocytes (StrTIL) in the clinical setting.\n\nMethods112 breast carcinoma cases treated with pre-operative chemotherapy were selected for the study. According to chemotherapy regimen 28/112 patients received platinum-based, 42/112 cyclophosphamide-based and 42/112 anthracycline-based chemotherapy. The percentage of stromal tumor-infiltrating lymphocytes (StrTIL) was evaluated on hematoxylineosin stained slides of pre-treatment core biopsy (pre-StrTIL) and post-treatment surgical tumor samples (post-StrTIL), according to the most recent recommendation of International TILs Working Group. In survival analyses, TIL changes ({Delta}StrTIL) were calculated from the difference between post-StrTIL and pre-StrTIL.\n\nResultsOf the 112 cases, 58.0% (n=65) were hormone receptor (HR) positive and 42.0% (n=47) were HR negative. There was a trend of higher post-StrTIL compared to pre-StrTIL (median 6.25% vs. 3.00%; p<0.001). When analyzing the pre-StrTIL and post-StrTIL among the three treatment groups, we experienced significant StrTIL increase independently of the treatment applied. Based on the results of survival analyses both post-StrTIL and {Delta}StrTIL was found to be independent prognostic factor in HR negative cases. Each 1% increase in post-StrTIL reduced the hazard of distant metastases development by 2.6% (hazard ratio: 0.974; CI: 0.948-1.000; p=0.05) and for each 1% {Delta}StrTIL increment, the risk of distant metastases was reduced by 4.3% (hazard ratio: 0.957; CI: 0.932-0.983; p=0.001). The prognostic role of StrTIL in HR positive cases could not be proven.\n\nConclusionsStrTIL expression might be stimulated by highly (platinum), moderately (cyclophosphamide) and marginally (taxane, anthracycline) mutagenic chemotherapeutic agents. Increase in StrTIL in residual cancer compared to pre-treatment tumor tissue is associated with improved distant metastasis-free survival in cases with HR negative breast carcinoma.

cancer biology↗

bioRxiv2018

Differential effects of corticosteroids and anti-TNF on tumor-specific immune responses - implications for the management of irAEs

Up to 60% of patients treated with cancer immunotherapy develop severe or life threatening immune-related adverse events (irAEs). Immunosuppression with high doses of corticosteroids or, in refractory cases, with tumor necrosis factor (TNF) antagonists, are the mainstay of treatment for irAEs. It is currently unknown what is the impact of corticosteroids and anti-TNF on the activity of antitumor T cells. In this study, the influences of clinically relevant doses of dexamethasone (corresponding to an oral dose of 10 to 125 mg prednisolone) and infliximab (anti-TNF) on the activation and killing ability of tumor-infiltrating lymphocytes (TILs) was tested in vitro. Overall, dexamethasone at low or intermediate/high dose impaired the activation (respectively -46% and -62%) and tumor-killing ability (respectively -48% and -53%) of tumor-specific TILs. In contrast, a standard clinical dose of infliximab only had a minor effect on T cell activation (-20%) and tumor killing (-10%). A brief resting following exposure to dexamethasone was sufficient to rescue the in vitro activity of TILs. In conclusion, clinically-relevant doses of infliximab only influenced to a lesser extent the activity of tumor-specific TILs in vitro, whereas even low doses of corticosteroids markedly impaired the antitumor activity of TILs. These data support steroid-sparing strategies and early initiation of anti-TNF for the treatment of irAEs in immuno-oncology.

cancer biology↗