Nanomedicine for Cancer Immunotherapy: Tracking Cancer-Specific T‑Cells <i>in Vivo</i> with Gold Nanoparticles and CT Imaging

Application of immune cell-based therapy in routine clinical practice is challenging due to the poorly understood mechanisms underlying success or failure of treatment. Development of accurate and quantitative imaging techniques for noninvasive cell tracking can provide essential knowledge for elucidating these mechanisms. We designed a novel method for longitudinal and quantitative <i>in vivo</i> cell tracking, based on the superior visualization abilities of classical X-ray computed tomography (CT), combined with state-of-the-art nanotechnology. Herein, T-cells were transduced to express a melanoma-specific T-cell receptor and then labeled with gold nanoparticles (GNPs) as a CT contrast agent. The GNP-labeled T-cells were injected intravenously to mice bearing human melanoma xenografts, and whole-body CT imaging allowed examination of the distribution, migration, and kinetics of T-cells. Using CT, we found that transduced T-cells accumulated at the tumor site, as opposed to nontransduced cells. Labeling with gold nanoparticles did not affect T-cell function, as demonstrated both <i>in vitro</i>, by cytokine release and proliferation assays, and <i>in vivo</i>, as tumor regression was observed. Moreover, to validate the accuracy and reliability of the proposed cell tracking technique, T-cells were labeled both with green fluorescent protein for fluorescence imaging, and with GNPs for CT imaging. A remarkable correlation in signal intensity at the tumor site was observed between the two imaging modalities, at all time points examined, providing evidence for the accuracy of our CT cell tracking abilities. This new method for cell tracking with CT offers a valuable tool for research, and more importantly for clinical applications, to study the fate of immune cells in cancer immunotherapy

Isolation of neoantigen-specific T cells from tumor and peripheral lymphocytes

Adoptively transferred tumor-infiltrating T lymphocytes (TILs) that mediate complete regression of metastatic melanoma have been shown to recognize mutated epitopes expressed by autologous tumors. Here, in an attempt to develop a strategy for facilitating the isolation, expansion, and study of mutated antigen-specific T cells, we performed whole-exome sequencing on matched tumor and normal DNA isolated from 8 patients with metastatic melanoma. Candidate mutated epitopes were identified using a peptide-MHC-binding algorithm, and these epitopes were synthesized and used to generate panels of MHC tetramers that were evaluated for binding to tumor digests and cultured TILs used for the treatment of patients. This strategy resulted in the identification of 9 mutated epitopes from 5 of the 8 patients tested. Cells reactive with 8 of the 9 epitopes could be isolated from autologous peripheral blood, where they were detected at frequencies that were estimated to range between 0.4% and 0.002%. To the best of our knowledge, this represents the first demonstration of the successful isolation of mutation-reactive T cells from patients' peripheral blood prior to immune therapy, potentially providing the basis for designing personalized immunotherapies to treat patients with advanced cancer

Loss of Protein Kinase C δ Gene Expression in Human Squamous Cell Carcinomas: A Laser Capture Microdissection Study

Protein kinase C delta (PKC-δ) protein levels are frequently low in chemically and UV-induced mouse skin tumors as well as in human cutaneous squamous cell carcinomas (SCCs). Furthermore, overexpression of PKC-δ in human SCC lines and mouse epidermis is sufficient to induce apoptosis and suppress tumorigenicity, making PKC-δ a potential tumor suppressor gene for SCCs. Here we report that PKC-δ is lost in human SCCs at the transcriptional level. We used laser capture microdissection to isolate cells from three normal human epidermis and 14 human SCCs with low PKC-δ protein. Analysis by quantitative reverse transcription-PCR revealed that PKC-δ RNA was reduced an average of 90% in the SCCs tested, consistent with PKC-δ down-regulation at the protein level. Analysis of DNA from nine of the same tumors revealed that PKC-δ gene was deleted in only one tumor. In addition, Ras-transformed human keratinocytes, which have selective down-regulation of PKC-δ at both protein and mRNA levels, had significantly repressed human PKC-δ promoter activity. Together, these results indicate that PKC-δ gene expression is suppressed in human SCCs, probably via transcription repression. Our results have implications for the development of topical therapeutic strategies to trigger the re-expression of pro-apoptotic PKC-δ to induce apoptosis in SCCs

Modulating serine palmitoyl transferase (SPT) expression and activity unveils a crucial role in lipid-induced insulin resistance in rat skeletal muscle cells

International audienceSaturated fatty acids, such as palmitate, promote accumulation of ceramide which impairs activation and signaling of protein kinase B (PKB/Akt) to important end-points such as glucose transport. Serine Palmitoyl Transferase (SPT) is a key enzyme regulating ceramide synthesis from palmitate and represents a potential molecular target in curbing lipid-induced insulin resistance. Here we explore effects of palmitate upon insulin action in L6 muscle cells in which SPT expression/activity have been reduced by shRNA or sustained incubation with myriocin, a SPT inhibitor. Incubation of L6 myotubes with palmitate (16h) increases intramyocellular ceramide and reduces insulin-stimulated PKB activation and glucose uptake. PKB inhibition was not associated with impaired IRS signaling and was ameliorated by short-term treatment with myriocin. Silencing SPT expression (~90%) by shRNA or chronic cell incubation with myriocin (7 days) markedly suppressed SPT activity and palmitate-driven ceramide synthesis. However, challenging these muscle cells with palmitate still inhibited the hormonal activation of PKB. This inhibition was associated with reduced IRS1/p85-PI3-kinase coupling that arises from diverting palmitate towards greater DAG synthesis, which elevates IRS1 serine phosphorylation via activation of DAG-sensitive PKCs. Treatment of SPT-shRNA cells or those treated chronically with myriocin with PKC inhibitors antagonised palmitate-induced loss in insulin signaling. The findings indicate that SPT plays a crucial role in desensitizing muscle cells to insulin in response to incubation with palmitate. Whilst short-term inhibition of SPT ameliorates palmitate/ceramide-induced insulin resistance, sustained loss/reduction in SPT expression/activity promotes greater partitioning of palmitate towards DAG synthesis, which impacts negatively upon IRS1-directed insulin signaling