Describing the Mosquito Effect: Intercellular Transfer of Cytoplasm in the Tumor Microenvironment and Its Potential Impact on T Cell Function

Immune cells communicate through various means, including soluble factors, direct cell-cell contact, and more recently recognized methods such as the exchange of cellular components. Traditional methods like in vitro systems have provided valuable insights but often fail to replicate the microenvironment and interactions occurring within living organisms. Additionally, the limited tools available for visualizing and tracking these exchanges in vivo have limited comprehensive studies on their functional consequences. To this end, new study published in Frontiers in Immunology and conducted by graduate students Kaito Hioki, Daniel Ryan, Iris Thesmar, Adam Lynch, and led by Professors Leonid Pobezinsky and Elena Pobezinskaya from the University of Massachusetts developed a novel tumor mouse model which allows for the detection and measurement of intercellular transfer of cytoplasm from tumor cells to infiltrating immune cells in vivo. The researchers generated a B16-F10 melanoma cell line expressing the ultra-bright fluorescent protein ZsGreen (ZsG) to investigate the phenomenon of intercellular transfer within the tumor microenvironment (TME). They subcutaneously injected B16ZsG cells into wild-type C57BL/6 mice, and expression of ZsG was induced using doxycycline. Tumors were harvested on day eight post-inoculation, and tumor-infiltrating lymphocytes (TILs) were isolated for analysis. Flow cytometry revealed that the majority of CD45-positive cells within the TME acquired ZsG fluorescence from the tumor cells. The researchers performed single-cell RNA sequencing (scRNA-seq) on sorted ZsG-positive and ZsG-negative cells to identify the specific immune cell types involved in intercellular transfer. They found that except for plasmacytoid dendritic cells, which remained Zs-G-negative, majority of myeloid lineage cells (monocytes, macrophages, and conventional dendritic cells (DC2 and most of DC1)) had significant ZsG fluorescence which indicated active participation in intercellular transfer. Intriguingly, the authors also revealed that a substantial number of lymphoid cells (CD4 and CD8 T cells) acquired tumor-derived ZsG fluorescence and their flow cytometry analysis confirmed these findings, showing that CD4 T cells had significantly higher ZsG signal intensity compared to CD8 T cells. The team then wanted to know the distinct subcellular localization patterns of ZsG in TILs and to this, they used ImageStream flow cytometry which showed monocytes to exhibit a punctate pattern indicative of phagocytic or endocytic granules, in contrast, T cells showed mostly homogeneous distribution which suggested that T cells might possess a unique mechanism for incorporating tumor cytoplasm into their own. From the primary T cell subsets involved in intercellular transfer, they identified Tregs and exhausted CD8 T cells (CD8-Tex) using scRNA-seq analysis.

The team investigated the correlation between T cell activation status and the degree of transfer to better understand the mechanisms of intercellular transfer using Gene ontology and Gene Set Enrichment Analysis of scRNA-seq data which showed that ZsG-positive T cells were metabolically active but exhibited suppressed immune response signatures. Moreover, they used antigen-specific in vivo model to study the role of cell-cell contact and antigen specificity in intercellular transfer and found that intercellular transfer occurs even without direct antigen specificity. Furthermore, the researchers performed a modified transwell assay to determine the contribution of extracellular vesicles to intercellular transfer by plating B16ZsG tumor cells on the basolateral side of inserts with either 0.4 μm or 12 μm pore sizes which allow direct cell-cell contact only in the larger pore size and observed a higher frequency of ZsG-positive T cells in the 12 μm pore-size inserts compared to the 0.4 μm inserts which indicated that direct cell-cell contact is the predominant mechanism of intercellular transfer.

In conclusion, Professor Elena Pobezinskaya and colleagues successfully developed a novel in vivo model using ZsGreen-expressing tumor cells that can provide a valuable research tool for tracking and quantifying the transfer of cellular components between tumor cells and infiltrating immune cells. They proposed a new conceptual framework for understanding intercellular transfer in the context of tumor-immune cell interactions, which they termed the “mosquito effect”. The discovery that T cells can integrate tumor-derived cytoplasm into their own highlights a previously unrecognized aspect of immune cell-tumor cell interactions. This knowledge enhances our understanding of how immune cells adapt and respond within the TME, which is important for developing more effective immunotherapies. Moreover, the reported intercellular transfer and its impact on T cell function suggest potential mechanisms by which tumors might evade immune surveillance and may inform the design of new therapeutic strategies to prevent or reverse the detrimental effects of intercellular transfer on T cell function. For example, targeting pathways involved in the transfer process or enhancing T cell resistance to such transfers could improve the efficacy of cancer immunotherapies. Additionally, the authors’ findings on the altered transcriptomes of T cells that acquire tumor cytoplasm offer potential biomarkers for immune modulation and discovery of genes and pathways differentially expressed in these T cells may provide new diagnostic tools to monitor immune cell status and functionality within the TME.