While STING-activating agents have shown limited efficacy in early-phase clinical trials, multiple lines of evidence suggest the importance of tumor cell-intrinsic STING function in mediating ...antitumor immune responses. Although STING signaling is impaired in human melanoma, its restoration through epigenetic reprogramming can augment its antigenicity and T cell recognition. In this study, we show that reversal of methylation silencing of STING in murine melanoma cell lines using a clinically available DNA methylation inhibitor can improve agonist-induced STING activation and type-I IFN induction, which, in tumor-bearing mice, can induce tumor regression through a CD8
T cell-dependent immune response. These findings not only provide mechanistic insight into how STING signaling dysfunction in tumor cells can contribute to impaired responses to STING agonist therapy, but also suggest that pharmacological restoration of STING signaling through epigenetic reprogramming might improve the therapeutic efficacy of STING agonists.
Background
We previously described the properties of a targeted drug delivery system (DDS) in a cell-free system. Here, in this comparative cell-based study (normal and tumor cells), we provide a ...quantitative analysis of the extracellular diffusion and intracellular localization of this DDS. This DDS consists of fluorescence-labeled paclitaxel encapsulated in non-ionic surfactant vesicles/niosomes embedded in a thermo-sensitive cross-linked chitosan hydrogel with an affinity for the MUC1 mucin surface antigen overexpressed on tumor cells, and designed for a sustained and controlled, localized delivery of embedded drugs. We evaluated DDS in our novel in vitro model using MatTek’ glass-bottom culture plates and compared human cancer cell lines (OV2008 epithelial origin carcinoma and U373 glioma, both overexpressing MUC1) with human normal epithelial control cell lines (IMMC3 and IOSE-121 using differential contrast and confocal microscopy.
Results
Tumor cells incubated in the presence of chitosan alone or DDS-containing chitosan–niosome–paclitaxel–BODIPY 564/570, showed a prominent granular accumulation on their surface when compared to the normal cells. Quantitation of gray value light intensity of the extracellular region of chitosan alone treated OV2008 and IOSE-121 controls done by analysis of multiple radial line segments, 4 µm each, using ImageJ software showed 2 times higher intensity around the OV2008 than around normal IOSE-121 controls (
p
< 0.05). In the DDS-treated OV2008 cells, extracellular fluorescence intensity measured at different diffusion distances outside of the cells, in three different zones showed the difference in means of fluorescence intensity in these zones (
p
< 0.05) with the highest level of fluorescence near the cell surface indicating a concentration gradient, most likely driven by the high affinity of chitosan to the MUC1 receptor. Also, as chitosan alone accumulated two times more along the edge of tumor cells compared to normal cells, we found intracellular fluorescence intensity quantified at time intervals to be also 2 times higher in OV2008 than in normal IMCC3 cells (
p
< 0.05).
Conclusion
Based on the observation of the DDS preferentially targeting tumor cells, there is a potential implication for the localized delivery of therapeutic drug doses to solid tumors or post-surgical solid tumors cavities containing residual tumor cells.
BackgroundDespite its common epigenetic suppression in multiple cancers, STING signaling has emerged as a major pathway for augmenting tumor cell antigenicity and initiation of T cell responses.1 2 ...Another aspect of intact activation of STING signaling in tumor cells is downstream induction of T cell-homing chemokines including CXCL10 and CCL5. These chemokines are also among our earlier reported 12-chemokine (12-CK) gene expression signature (GES) predicting the presence of tumor-localized tertiary lymphoid structures (TLSs), which are increasingly shown to correlate with improved survival in certain solid tumor types.3 4 Based on these findings, we hypothesized that epigenetic silencing of STING signaling genes through promoter hypermethylation would be inversely associated with the presence of TLSs.MethodsWe assessed the correlation between the expression of STING signaling genes and the chemokines present in the 12-CK GES across melanomas and urothelial bladder carcinomas using cBioPortal datasets. To extend these studies beyond these tumor types, we performed correlative and survival analyses using the TCGA PanCancer Atlas. Additionally, we determined the correlation between the promoter methylation levels of STING signaling genes and the 12-CK GES score. We also evaluated STING expression in TLS+ and TLS- melanoma samples in situ by immunohistochemistry (IHC).ResultsWe identified a distinct correlation between STING-expressing tumors and each of the twelve chemokines among melanoma and urothelial bladder carcinoma samples. In particular, STING expression was positively correlated with secondary lymphoid organ-associated chemokines, CCL19 (p=0.0077), CCL21 (p=0.0046), and CXCL13 (p=0.0034) in urothelial bladder carcinomas. The presence of TLSs in STING-expressing melanomas was further confirmed by IHC. Using TCGA PanCancer datasets, we observed a strong correlation between the expression of cGAS (Pearson’s r=0.46) and STING (Pearson’s r=0.37) with the 12-CK GES score. In contrast, the methylation levels of cGAS and STING were inversely correlated with the 12-CK GES score (Pearson’s r=-0.37 and -0.41, respectively). Similarly, hypermethylation of STING was correlated with inferior disease-specific survival (DSS) (p<0.0001) in lung adenocarcinomas. Survival analysis on the TCGA skin cutaneous melanoma (SKCM) dataset also indicated significant DSS advantage in 12-CK GES scoreHigh cGASHighpatients (p<0.0001).ConclusionsWe provide evidence that epigenetic state of cGAS and STING cannot only shape tumor antigenicity but is also associated with the 12-CK GES and the presence of TLSs. Considering the well-established prognostic value of TLSs, these findings argue that targeting epigenetic suppression of STING signaling should be considered as a strategy to guide effective immunotherapy-based interventions.AcknowledgementsThis work was supported by the Moffitt Cancer Center Tissue Core and Analytic Microscopy Core Facilities, all comprehensive cancer center facilities designated by the National Cancer Institute (P30 CA076292). This work was funded by: NCI-NIH (1R01 CA148995, 1R01 CA184845, P30 CA076292, P50 CA168536), CJG Fund, Chris Sullivan Fund, V Foundation, Melanoma Research Foundation, and Dr. Miriam and Sheldon G. Adelson Medical Research Foundation.ReferencesFalahat R, Berglund A, Putney RM, Perez-Villarroel P, Aoyama S, Pilon- Thomas S, Barber GN, Mulé JJ. Epigenetic reprogramming of tumor cell-intrinsic STING function sculpts antigenicity and T cell recognition of melanoma. PNAS. 2021;118(15).Falahat R, Berglund A, Perez-Villarroel P, Putney RM, Hamaidi I, Kim S, Pilon-Thomas S, Barber GN, Mulé JJ. Epigenetic state determines the in vivo efficacy of STING agonist therapy. Nature Communications. 2023;14(1):1573.Messina JL, Fenstermacher DA, Eschrich S, Qu X, Berglund AE, Lloyd MC, Schell MJ, Sondak VK, Weber JS, Mulé JJ. 12-Chemokine gene signature identifies lymph node-like structures in melanoma: potential for patient selection for immunotherapy?. Scientific reports. 2012;2(1):765.Schumacher TN, Thommen DS. Tertiary lymphoid structures in cancer. Science. 2022;375(6576):eabf9419.
Tertiary lymphoid structures (TLSs) have been identified in the parenchyma and/or in the peripheral margins of human solid tumors. Uncovering the functional nature of these structures is the subject ...of much intensive investigation. Studies have shown a direct correlation of the presence of human tumor-localized TLS and better patient outcome (e.g., increase in overall survival) in certain solid tumor histologies, but not all. We had identified a tumor-derived immune gene-expression signature, encoding 12 distinct chemokines, which could reliably identify the presence of TLSs, of different degrees, in various human solid tumors. We are focused on understanding the influence of TLSs on the tumor microenvironment and leveraging this understanding to both manipulate the antitumor immune response and potentially enhance immunotherapy applications. Moreover, as not all human solid tumors show the presence of these lymphoid structures, we are embarking on bioengineering approaches to design and build "designer" TLSs to address, and potentially overcome, an unmet medical need in cancer patients whose tumors lack such lymphoid structures.
Tertiary lymphoid structures (TLSs) associate with better prognosis in certain cancer types, but their underlying formation and immunological benefit remain to be determined. We established a mouse ...model of TLSs to study their contribution to antitumor immunity. Because the stroma in lymph nodes (sLN) participates in architectural support, lymphogenesis, and lymphocyte recruitment, we hypothesized that TLSs can be created by sLN. We selected a sLN line with fibroblast morphology that expressed sLN surface markers and lymphoid chemokines. The subcutaneous injection of the sLN line successfully induced TLSs that attracted infiltration of host immune cell subsets. Injection of MC38 tumor lysate-pulsed dendritic cells activated TLS-residing lymphocytes to demonstrate specific cytotoxicity. The presence of TLSs suppressed MC38 tumor growth
by improving antitumor activity of tumor-infiltrating lymphocytes with downregulated immune checkpoint proteins (PD-1 and Tim-3). Future engineering of sLN lines may allow for further enhancements of TLS functions and immune cell compositions.
BackgroundWhile STING-activating agents have shown limited efficacy in early phase clinical trials, multiple lines of evidence suggest the importance of the so far unappreciated tumor cell-intrinsic ...STING function in antitumor immune responses. Accordingly, we have shown that although there is a widespread impairment of STING signaling among human melanomas, its restoration through epigenetic reprogramming can augment antigenicity and T cell recognition of melanoma cells.1 2 In this study, we determined if rescue of tumor cell-intrinsic STING signaling using a DNA methyltransferase inhibitor can improve the therapeutic efficacy of a STING agonist in mouse models of melanoma.MethodsWe subjected three distinct murine melanoma cell lines (B16-F10, B16-ISG and Yumm1.7) to treatment with 5-aza-2'-deoxycytidine (5AZADC) and evaluated their activation of STING following stimulation with the STING agonist ADU-S100. Using a B16-F10 subcutaneous model, we assessed the effect of 5AZADC treatment on the efficacy of intratumorally administered ADU-S100 in STINGgt/gt mice. Additionally, we performed mechanistic studies using T-cell depletion experiments as well as phenotypic and gene expression profiling.ResultsWe observed reconstitution of cGAS in all three 5AZADC-pretreated cell lines as well as up to a 46-fold increase in induction of IFN-beta (p < 0.001) and a 4.5-fold increase in MHC class I surface expression (p < 0.01) compared to untreated controls following stimulation with ADU-S100. In B16-F10 tumor-bearing mice, while treatment with a combination of 5AZADC plus ADU-S100 resulted in a marked increase in Ifnb1 transcripts within tumors (p < 0.001), it significantly delayed tumor growth compared to treatment with ADU-S100 alone (p = 0.0244 on day 22). Antibody-mediated depletion studies in mice receiving the combination therapy further indicated that this antitumor activity depends on the generation of functional tumor antigen-specific CD8+ T cells (p = 0.0111 on day 22); however, tumor growth remained unaltered by the depletion of CD4+ T cells.ConclusionsWe show that reversal of methylation silencing of cGAS in murine melanoma cell lines using a clinically available DNA methylation inhibitor can improve agonist-induced STING activation and type I IFN induction, which in tumor-bearing mice is capable of inducing tumor regression through a CD8+ T cell-dependent immune response. These findings not only provide mechanistic insight into how STING signaling dysfunction in tumor cells can contribute to impaired responses to STING agonist therapy, but also suggest, depending on tumor cell-intrinsic STING signaling status, its pharmacologic restoration should be considered for improving therapeutic efficacy of STING agonists in future clinical studies.AcknowledgementsFunding: NCI P50 CA168536, Cindy and Jon Gruden Fund, Chris Sullivan Fund, V Foundation, Dr. Miriam and Sheldon G. Adelson Medical Research Foundation.ReferencesFalahat R, Perez-Villarroel P, Mailloux AW, Zhu G, Pilon-Thomas S, Barber GN, Mulé JJ. STING signaling in melanoma cells shapes antigenicity and can promote antitumor T-cell activity. Cancer Immunol Res 2019;7(11):1837–48.Falahat R, Berglund A, Putney RM, Perez-Villarroel P, Aoyama S, Pilon-Thomas S, Barber GN, Mulé JJ. Epigenetic reprogramming of tumor cell–intrinsic STING function sculpts antigenicity and T cell recognition of melanoma. PNAS 2021;118(15).
BackgroundIt is becoming more evident that STING activity in tumor cells can have a functional role in mediating antitumor immune responses. We have recently shown that activation of STING signaling ...in human melanoma cell lines enhances their antigenicity and susceptibility to lysis by human melanoma tumor infiltrating lymphocytes (TIL) through the augmentation of MHC class I molecules.1 However, the frequent impairment of this pathway through loss of cGAS and/or STING expression in melanoma cell lines limits their antigen presentation and subsequently their sensitivity to cytotoxic T cell mediated killing. In this study, we asked if this suppression is, in part, epigenetically regulated and if it is indeed a driver of melanoma resistance to T cell-based immunotherapies.MethodsTo determine the role of DNA methylation in melanoma STING and cGAS silencing, we performed genome-wide DNA methylation profiling across a panel of 16 human melanoma cell lines. We subjected melanoma cell lines that indicated STING and/or cGAS promoter hypermethylation to treatment with 5-aza-2’-deoxycytidine (5AZADC) and evaluated their protein expression by immunoblot. We next assessed phosphorylation of IRF3 and induction of IFN-β and CXCL10 in 5AZADC-treated melanoma cells following their stimulation with dsDNA or 2’3’-cGAMP. We also co-cultured 5AZADC-pretreated melanoma cell lines with their HLA-matched human melanoma TIL in the presence or absence of dsDNA or 2’3’-cGAMP and assessed TIL production of IFN-γ.ResultsUsing whole genome methylation profiling, we identified a distinct correlation between promoter hypermethylation and loss of STING and cGAS expression in human melanoma cell lines. Reconstitution of STING and cGAS expression through DNA demethylation reinstated functional STING signaling in at least half of the examined cell lines as indicated by STING-dependent phosphorylation of IRF3, induction of CXCL10 (~300 pg/ml, P < 0.0001) and IFN-β (~900 pg/ml, P < 0.0001) and upregulation of MHC class I. We also observed up to a 8-fold increase in TIL production of IFN-γ in co-culture studies using 5AZADC-pretreated melanoma cells compared to untreated controls in the presence of dsDNA or 2’3’-cGAMP (~2,000 pg/ml, P < 0.001).ConclusionsWe provide evidence that methylation silencing of cGAS and STING is not only a notable mechanism of STING signaling dysfunction in melanoma, but also plays a role in tumor antigen presentation and recognition by TIL. Collectively, these observations argue that targeting epigenetic loss of STING signaling in melanomas should be considered as a strategy to improve the efficacy of clinical interventions using T cell-based immunotherapies.AcknowledgementsFunding: NCI P50 CA168536, Cindy and Jon Gruden Fund, Chris Sullivan Fund, V Foundation, Dr. Miriam and Sheldon G. Adelson Medical Research Foundation.ReferenceFalahat R, Perez-Villarroel P, Mailloux AW, Zhu G, Pilon-Thomas S, Barber GN, Mulé JJ. STING signaling in melanoma cells shapes antigenicity and can promote antitumor T-cell activity. Cancer Immunology Research 2019; 7(11):1837–48.
Lack or loss of tumor antigenicity represents one of the key mechanisms of immune escape and resistance to T cell-based immunotherapies. Evidence suggests that activation of stimulator of interferon ...genes (STING) signaling in tumor cells can augment their antigenicity by triggering a type I IFN-mediated sequence of autocrine and paracrine events. Although suppression of this pathway in melanoma and other tumor types has been consistently reported, the mechanistic basis remains unclear. In this study, we asked whether this suppression is, in part, epigenetically regulated and whether it is indeed a driver of melanoma resistance to T cell-based immunotherapies. Using genome-wide DNA methylation profiling, we show that promoter hypermethylation of
and
genes mediates their coordinated transcriptional silencing and contributes to the widespread impairment of the STING signaling function in clinically-relevant human melanomas and melanoma cell lines. This suppression is reversible through pharmacologic inhibition of DNA methylation, which can reinstate functional STING signaling in at least half of the examined cell lines. Using a series of T cell recognition assays with HLA-matched human melanoma tumor-infiltrating lymphocytes (TIL), we further show that demethylation-mediated restoration of STING signaling in STING-defective melanoma cell lines can improve their antigenicity through the up-regulation of MHC class I molecules and thereby enhance their recognition and killing by cytotoxic T cells. These findings not only elucidate the contribution of epigenetic processes and specifically DNA methylation in melanoma-intrinsic STING signaling impairment but also highlight their functional significance in mediating tumor-immune evasion and resistance to T cell-based immunotherapies.
STING (stimulator of IFN genes) signaling is an innate immune pathway for induction of a spontaneous antitumor T-cell response against certain immunogenic tumors. Although antigen-presenting cells ...are known to be involved in this process, insight into the participation of tumor cell-intrinsic STING signaling remains weak. In this study, we find diversity in the regulation of STING signaling across a panel of human melanoma cell lines. We show that intact activation of STING signaling in a subset of human melanoma cell lines enhances both their antigenicity and susceptibility to lysis by human melanoma tumor-infiltrating lymphocytes (TIL) through the augmentation of MHC class I expression. Conversely, defects in the STING signaling pathway protect melanoma cells from increased immune recognition by TILs and limit their sensitivity to TIL lysis. Based on these findings, we propose that defects in tumor cell-intrinsic STING signaling can mediate not only tumor immune evasion but also resistance to TIL-based immunotherapies.
Chlorotoxin (CTX) is a small peptide with unique targeting properties towards gliomas and other tumors of the neuroectodermal origin. Despite being investigated in many in vivo and in vitro targeted ...drug delivery and imaging studies, the mechanism of interaction between CTX and tumor cells has not been well understood. This study presents a new approach in monitoring the biochemical and biophysical changes in glioma cells after being exposed to CTX using Attenuated Total Reflection Fourier Transform Infra-Red (ATR-FTIR) spectroscopy. In the first part, we characterized the signature spectra of CTX and U87 cells. Next, we evaluated the differences in biochemical compositions of the spectra of the glioma cells treated with and without CTX over different incubation time periods. Our results indicated biochemical changes in U87 cells at different stages of incubation with CTX, with the most notable changes occurring after 30min of incubation. Detailed comparisons of the spectra of the U87 cells treated with and without CTX for 30min revealed distinct changes and shifts of the bands associated with proteins and lipids. In particular, a downshift of the band assigned to CH2 scissoring of lipid membranes from 1454cm−1 to 1448cm−1 followed by an appearance of a new shoulder at wavenumber of 1465cm−1 revealed conformational changes in hydrocarbon chain packing in lipid bilayers of U87 cells due to CTX. Other band shifts indicated additional changes in conformation and orientation of lipids and proteins in U87 cells exposed to CTX.
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