The therapeutic efficacy of adoptive transfer of T cells transduced with chimeric antigen receptors (CARs) has been limited in the treatment of solid cancers, partly due to tumor antigen heterogeneity. Overcoming lack of universal tumor antigen expression would be achieved if CAR T cells could induce bystander effects. To study this process, we developed a system where CAR T cells targeting mesothelin could cure tumors containing 100% antigen-positive cells in immunocompetent mice. Using this model, we found that the CAR T cells were unable to cure tumors, even when only 10% of the tumor cells were mesothelin negative. A bystander effect was not induced by co-administration of anti-PD-1, anti-CTLA-4, or anti-TGF-β (transforming growth factor β) antibodies; agonistic CD40 antibodies; or an IDO (indoleamine 2,3-dioxygenase) inhibitor. However, pretreatment with a non-lymphodepleting dose of cyclophosphamide (CTX) prior to CAR T cells resulted in cures of tumors with up to 25% mesothelin-negative cells. The mechanism was dependent on endogenous CD8 T cells but not on basic leucine zipper transcription factor ATF-like 3 (BATF3)-dependent dendritic cells. These data suggest that CAR T cell therapy of solid tumors, in which the targeted antigen is not expressed by the vast majority of tumor cells, will not likely be successful unless combination strategies to enhance bystander effects are used.
Cooperation of cancer cells with stromal cells, such as cancer-associated fibroblasts (CAFs), has been revealed as a mechanism sustaining cancer cell survival and growth. In the current study, we focus on the metabolic interactions of MRC5 lung fibroblasts with lung cancer cells (A549 and H1299) using co-culture experiments and studying changes of the metabolic protein expression profile and of their growth and migration abilities. Using western blotting, confocal microscopy and RT-PCR, we observed that in co-cultures MRC5 respond by upregulating pyruvate dehydrogenase (PDH) and the monocarboxylate transporter MCT1. In contrast, cancer cells increase the expression of glucose transporters (GLUT1), LDH5, PDH kinase and the levels of phosphorylated/inactivated pPDH. H1299 cells growing in the same culture medium with fibroblasts exhibit a 'metastasis-like' phenomenon by forming nests within the fibroblast area. LDH5 and pPDH were drastically upregulated in these nests. The growth rate of both MRC5 and cancer cells increased in co-cultures. Suppression of LDHA or PDK1 in cancer cells abrogates the stimulatory signal from cancer cells to fibroblasts. Incubation of MRC5 fibroblasts with lactate resulted in an increase of LDHB and of PDH expression. Silencing of PDH gene in fibroblasts, or silencing of PDK1 or LDHA gene in tumor cells, impedes cancer cell's migration ability. Overall, a metabolic cooperation between lung cancer cells and fibroblasts has been confirmed in the context of direct Warburg effect, thus the fibroblasts reinforce aerobic metabolism to support the intensified anaerobic glycolytic pathways exploited by cancer cells.
Given the growing interest and promising preliminary results of immunotherapy in malignant pleural mesothelioma (MPM), it has become important to more fully understand the immune landscape in this tumor. This may be especially relevant in deciding who might benefit most from checkpoint blockade or agonist antibody therapy. Since the phenotype of tumor infiltrating lymphocytes (TILs) in MPM has not been fully described and their function has not been carefully assessed, we collected fresh tumor and blood from 22 patients undergoing surgical resection and analysed single cell suspensions by flow cytometry. The functionality of TILs was assessed by measurement of cytokine expression (IFN-γ) following overnight stimulation ex vivo. Results showed low numbers of CD8+ TILs whose function was either moderately or severely suppressed. The degree of TIL hypofunction did not correlate with the presence of co-existing macrophages or neutrophils, nor with expression of the inhibitory receptors PD-1, CD39 and CTLA-4. Hypofunction was associated with higher numbers of CD4 regulatory T cells (Tregs) and with expression of the inhibitory receptor TIGIT. On the other hand, presence of tissue-resident memory (Trm) cells and expression of TIM-3 on CD8+ cells were positively associated with cytokine production. However, Trm function was partially suppressed when the transcription factor Eomesodermin (Eomes) was coexpressed. Understanding the function of TILs in malignant mesothelioma may have clinical implications for immunotherapy, especially in choosing the best immunotherapy targets. Our data suggests that Treg cell blocking agents or TIGIT inhibitor antibodies might be especially valuable in these patients.
Background:The cellular autophagic response to radiation is complex. Various cells and tissues respond differentially to radiation, depending on both the dose of exposure and the time post irradiation. In the current study, we determined the autophagosomal and lysosomal response to radiation in lung cancer cell lines by evaluating the expression of the associated proteins, as well as the effect of relevant gene silencing in radio and chemosensitisation. Furthermore, tumour sensitisation was evaluated in in vivo autophagic gene silencing model after irradiation.Methods:A549 and H1299 cell lines were utilised as in vitro cancer models. Both cell lines were transfected with various small-interfering RNAs, silencing auto-lysosomal genes, and irradiated with 4 Gy. Cell growth response was evaluated with AlamarBlue assay. Western blot and confocal microscopy were utilised for the characterisation of the auto-lysosomal flux. Also, the H1299 cell line was stable transfected with small-hairpin RNA of the MAP1LC3A gene, and the tumour radiosensitisation in Athymic Nude-Foxn1nu was evaluated.Results:Following exposure to 4 Gy of radiation, A549 cells exhibited a significant induction of the autophagic flux, which was not supported by transcriptional activation of auto-lysosomal genes (LC3A, LC3B, p62, TFEB and LAMP2a), resulting in aggresome accumulation. Recovery of transcriptional activity and autophagy efficacy occurred 7 days post irradiation. Alternatively, H1299 cells, a relatively radio-resistant cell line, sharply responded with an early (at 2 days) transcriptional activation of auto-lysosomal genes that sustained an effective autophagosomal flux, resulting in adequate aggresome clearance. Subsequently, we tested the silencing of four genes (LC3A, LC3B, TFEB and LAMP2a), confirming a significant radiosensitisation and chemosensitisation to various chemotherapeutic agents, including cisplatin and taxanes. In mouse xenografts, exposure to radiation significantly reduced tumour growth (P<0.001), which was exacerbated among shLC3A-H1299 transfected tumours.Conclusions:The ability of lung cancer cells to survive after irradiation at 4 Gy depends on their ability to sustain a functional autophagic flux. Abrogation of such ability results in increased radiosensitivity and susceptibility to various chemotherapy agents. Selective inhibitors of cancer cell autophagic function may prove important for the eradication of lung cancer.
Fibroblast activation protein (FAP), a cell surface serine protease, is upregulated on a subset of activated fibroblasts (often distinct from α-smooth muscle actin-expressing myofibroblasts) associated with matrix remodeling, including fibroblasts in idiopathic pulmonary fibrosis (Acharya PS, Zukas A, Chandan V, Katzenstein AL, Puré E. Hum Pathol 37: 352–360, 2006.). As FAP+ fibroblasts could be pivotal in either breakdown and/or production of collagen and other matrix components, the goal of this study was to define the role of FAP+ cells in pulmonary fibrosis in two established, but different, mouse models of chronic lung fibrosis: repetitive doses of intratracheal bleomycin and a single dose of an adenoviral vector encoding constitutively active TGF-β1 (Ad-TGFβ). To determine their role in fibrotic remodeling, FAP-expressing cells were depleted by injection of T cells expressing a chimeric antigen receptor specific for murine FAP in mice with established fibrosis. The contribution of FAP to the function of FAP-expressing cells was assessed in FAP knockout mice. Using histological analyses, quantification of soluble collagen content, and flow cytometry, we found that loss of FAP+ cells exacerbated fibrosis in the bleomycin model, a phenotype largely recapitulated by the genetic deletion of FAP, indicating that FAP plays a role in this model. In contrast, depletion of FAP+ cells or genetic deletion of FAP had little effect in the Ad-TGFβ model highlighting the potential for distinct mechanisms driving fibrosis depending on the initiating insult. The role of FAP in human lung fibrosis will need to be well understood to guide the use of FAP-targeted therapeutics that are being developed.
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