Biology subjects

Lakshmanachetty, S.

Publications and source records attributed to Lakshmanachetty, S..

4 recordsLinked to original sources

bioRxiv2025

Differential Phagocytosis induces Diverse Macrophage Activation States in Malignant Gliomas

Diffuse midline glioma (DMG) and Glioblastoma are malignant brain tumors in pediatric and adult patients. The current standard-of-care treatment for DMG is radiotherapy (RT), whereas GBM treatment includes surgery, followed by RT and chemotherapy. Although RT is known to modulate immune responses in cancer and enhance the effectiveness of myeloid checkpoint blockade, the downstream macrophage responses to differential phagocytosis induction remain poorly understood. This study examined macrophage-mediated phagocytosis caused by either RT, anti-CD47 checkpoint blockade, or their combination. We found that RT increased the expression of several damage-associated molecular patterns on the surface of glioma cell lines. Furthermore, RT enhanced anti-CD47-mediated macrophage phagocytosis of glioma cell lines in vitro. Single-cell RNA-sequencing revealed the diverse transcriptional and functional signatures of human macrophage subsets that either promoted or inhibited phagocytosis of glioma cells pretreated with RT, anti-CD47 therapy, or both. Consistent with these results, the combination therapy significantly reduced tumor growth, prolonged survival in glioma-bearing mice, and induced distinct macrophage activation states in vivo compared to either treatment alone. These findings highlight the plasticity and heterogeneity of macrophage responses during phagocytosis and provide compelling evidence for combining RT with anti-CD47 therapy as a promising therapeutic strategy for glioma treatment.

cancer biology↗

bioRxiv2023

Challenges in the discovery of tumor-specific alternative splicing-derived cell-surface antigens in glioma

BackgroundDespite advancements in cancer immunotherapy, solid tumors remain formidable challenges. In glioma, profound inter-and intra-tumoral heterogeneity of antigen landscape hampers therapeutic development. Therefore, it is critical to consider alternative sources to expand the repertoire of targetable (neo-)antigens and improve therapeutic outcomes. Accumulating evidence suggests that tumor-specific alternative splicing (AS) could be an untapped reservoir of neoantigens. ResultsIn this study, we investigated tumor-specific AS events in glioma, focusing on those predicted to generate major histocompatibility complex (MHC)-presentation-independent, cell-surface neoantigens that could be targeted by antibodies and chimeric antigen receptor (CAR)-T cells. We systematically analyzed bulk RNA-sequencing datasets comparing 429 tumor samples (from The Cancer Genome Atlas [TCGA]) and 9,166 normal tissue samples (from the Genotype-Tissue Expression project [GTEx]), and identified 13 AS events in 7 genes predicted to be expressed in more than 10% of the patients, including PTPRZ1 and BCAN, which were corroborated by an external RNA-sequencing dataset. Subsequently, we validated our predictions and elucidated the complexity of the isoforms using full-length transcript amplicon sequencing on patient-derived glioblastoma cells. However, analyses of the RNA-sequencing datasets of spatially mapped and longitudinally collected clinical tumor samples unveiled remarkable spatiotemporal heterogeneity of the candidate AS events. Furthermore, proteomics analysis did not reveal any peptide spectra matching the putative neoantigens. ConclusionsOur investigation illustrated the diverse characteristics of the tumor-specific AS events and the challenges of antigen exploration due to their notable spatiotemporal heterogeneity and elusive nature at the protein levels. Redirecting future efforts toward intracellular, MHC-presented antigens could offer a more viable avenue.

cancer biology↗

bioRxiv2023

Tumor-wide RNA splicing aberrations generate immunogenic public neoantigens

T-cell-mediated immunotherapies are limited by the extent to which cancer-specific antigens are homogenously expressed throughout a tumor. We reasoned that recurrent splicing aberrations in cancer represent a potential source of tumor-wide and public neoantigens, and to test this possibility, we developed a novel pipeline for identifying neojunctions expressed uniformly within a tumor across diverse cancer types. Our analyses revealed multiple neojunctions that recur across patients and either exhibited intratumor heterogeneity or, in some cases, were tumor-wide. We identified CD8+ T-cell clones specific for neoantigens derived from tumor-wide and conserved neojunctions in GNAS and RPL22, respectively. TCR-engineered CD8+ T-cells targeting these mutations conferred neoantigen-specific tumor cell eradication. Furthermore, we revealed that cancer-specific dysregulation in splicing factor expression leads to recurrent neojunction expression. Together, these data reveal that a subset of neojunctions are both intratumorally conserved and public, providing the molecular basis for novel T-cell-based immunotherapies that address intratumoral heterogeneity.

cancer biology↗

bioRxiv2023

Glioma-neuronal circuit remodeling induces regional immunosuppression

Neuronal activity-driven mechanisms impact glioblastoma cell proliferation and invasion1-7, and glioblastoma remodels neuronal circuits8,9. Distinct intratumoral regions maintain functional connectivity via a subpopulation of malignant cells that mediate tumor-intrinsic neuronal connectivity and synaptogenesis through their transcriptional programs8. However, the effects of tumor-intrinsic neuronal activity on other cells, such as immune cells, remain unknown. Here we show that regions within glioblastomas with elevated connectivity are characterized by regional immunosuppression. This was accompanied by different cell compositions and inflammatory status of tumor-associated macrophages (TAMs) in the tumor microenvironment. In preclinical intracerebral syngeneic glioblastoma models, CRISPR/Cas9 gene knockout of Thrombospondin-1 (TSP-1/Thbs1), a synaptogenic factor critical for glioma-induced neuronal circuit remodeling, in glioblastoma cells suppressed synaptogenesis and glutamatergic neuronal hyperexcitability, while simultaneously restoring antigen-presentation and pro-inflammatory responses. Moreover, TSP-1 knockout prolonged survival of immunocompetent mice harboring intracerebral syngeneic glioblastoma, but not of immunocompromised mice, and promoted infiltrations of pro-inflammatory TAMs and CD8+ T-cells in the tumor microenvironment. Notably, pharmacological inhibition of glutamatergic excitatory signals redirected tumor-associated macrophages toward a less immunosuppressive phenotype, resulting in prolonged survival. Altogether, our results demonstrate previously unrecognized immunosuppression mechanisms resulting from glioma-neuronal circuit remodeling and suggest future strategies targeting glioma-neuron-immune crosstalk may open up new avenues for immunotherapy.

cancer biology↗