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The “oligometastasis” hypothesis proposes that metastases exist as a spectrum and are not always disseminated. According to this theory, a subset of patients with metastatic disease could benefit from aggressive local therapies. However, the identification of patients most likely to exhibit an oligometastatic phenotype remains challenging. Recent literature focusing on basic and translational studies has identified novel epigenetic regulators of epithelial–mesenchymal transition (EMT) and the emergence of a spectrum of metastatic behavior. Herein, we review these scientific advances and suggest that the spectrum of metastatic virulence produced by these epigenetic mechanisms broadly contributes to the emergence of clinically evident “oligometastases.” Epigenetic regulation of EMT programs can result in a spectrum of cell trajectories (e.g., quasi-mesenchymal and highly mesenchymal states) with differential propensity to develop metastases. We propose that quasi-mesenchymal cell states may be associated with a polymetastatic phenotype, whereas highly mesenchymal cell states may be associated with a more oligometastatic phenotype. The mechanisms governing epigenetic regulation of EMT and its array of intermediate states are multifaceted and may contribute to the development of the metastatic spectrum observed clinically. Within this context, translational studies that support the role of EMT and its epigenetic regulation are discussed. Continued translation of these mechanistic discoveries into novel biomarkers may help optimally select patients most likely to exhibit an oligometastatic phenotype and benefit from aggressive local therapies, such as surgery, radiotherapy, and other ablative procedures.

Over 500 clinical trials are investigating combination radiotherapy and immune checkpoint blockade (ICB) as cancer treatments; however, the majority of trials have found no positive interaction. Here we perform a comprehensive molecular analysis of a randomized phase I clinical trial of patients with non-small cell lung cancer (NSCLC) treated with concurrent or sequential ablative radiotherapy and ICB. We show that concurrent treatment is superior to sequential treatment in augmenting local and distant tumor responses and in improving overall survival in a subset of patients with immunologically cold, highly aneuploid tumors, but not in those with less aneuploid tumors. In addition, radiotherapy alone decreases intratumoral cytotoxic T cell and adaptive immune signatures, whereas radiotherapy and ICB upregulates key immune pathways. Our findings challenge the prevailing paradigm that local ablative radiotherapy beneficially stimulates the immune response. We propose the use of tumor aneuploidy as a biomarker and therapeutic target in personalizing treatment approaches for patients with NSCLC treated with radiotherapy and ICB.

The clinical utility of stimulator of interferon genes (STING) agonists has been limited due to poor tumour-targeting and unwanted toxicity following systemic delivery. Here we describe a robust tumour-targeted STING agonist, ZnCDA, formed by the encapsulation of bacterial-derived cyclic dimeric adenosine monophosphate (CDA) in nanoscale coordination polymers. Intravenously injected ZnCDA prolongs CDA circulation and efficiently targets tumours, mediating robust anti-tumour effects in a diverse set of preclinical cancer models at a single dose. Our findings reveal that ZnCDA enhances tumour accumulation by disrupting endothelial cells in the tumour vasculature. ZnCDA preferentially targets tumour-associated macrophages to modulate antigen processing and presentation and subsequent priming of an anti-tumour T-cell response. ZnCDA reinvigorates the anti-tumour activity of both radiotherapy and immune checkpoint inhibitors in immunologically 'cold' pancreatic and glioma tumour models, offering a promising combination strategy for the treatment of intractable human cancers.

Metastasis, the major cause of cancer death, represents one of the major challenges in oncology. Scientists are still trying to understand the biological basis underlying the dissemination and outgrowth of tumor cells, why these cells can remain dormant for years, how they become resistant to the immune system or cytotoxic effects of systemic therapy, and how they interact with their new microenvironment. We asked experts to discuss some of the unknowns, advances, and areas of opportunity related to cancer metastasis.

Supplementary Figure Legends

Supplementary Table 2. Gene expression patterns correlating with SAR sensitivity.

Suppl. Figure 1. A. Tumors that escape an equilibrium caused by CD8 T cells targeting tumor stroma are not Ag-loss variants. B. The Ag-specific lytic function of 2C cells in vivo has not declined in mice bearing escaping tumors. C. Equilibrium can be achieved in mice lacking perforin Suppl. Figure 2. PD-L1 but not Tim3 blocking in vitro partially reverses the ability of tumor-infiltrating CD8+ T cells to produce cytokines. Suppl. Figure 3. A second transfer of CD8+ T cells following tumor irradiation or anti-Gr1 antibody therapy cannot prevent tumor escape. Suppl. Figure 4. Cancer cells are negative for MHC-II in vitro but can up-regulate MHC-II in tumors in vivo.

Small molecules used in this study.

Adoptively transferred CD8+ T cells can stabilize the size of solid tumors over long periods of time by exclusively recognizing antigen cross-presented on tumor stroma. However, these tumors eventually escape T-cell–mediated growth control. The aim of this study was to eradicate such persistent cancers. In our model, the SIYRYYGL antigen is expressed by cancer cells that lack the MHC-I molecule Kb needed for direct presentation, but the antigen is picked up and cross-presented by tumor stroma. A single injection of antigen-specific 2C CD8+ T cells caused long-term inhibition of tumor growth, but without further intervention, tumors started to progress after approximately 3 months. Escape was associated with reduced numbers of circulating 2C cells. Tumor-infiltrating 2C cells produced significantly less TNFα and expressed more of the “exhaustion” markers PD-1 and Tim-3 than T cells from lymphoid organs. High-dose local ionizing radiation, depletion of myeloid-derived suppressor cells, infusions of additional 2C cells, and antibodies blocking PD-L1 did not prevent tumor escape. In contrast, adoptive transfer of allogeneic CD4+ T cells restored the numbers of circulating Ag-specific CD8+ T cells and their intratumoral function, resulting in tumor eradication. These CD4+ T cells had no antitumor effects in the absence of CD8+ T cells and recognized the alloantigen cross-presented on tumor stroma. CD4+ T cells might also be effective in cancer patients when PD-1/PD-L1 blockade does not rescue intratumoral CD8+ T-cell function and tumors persist. Cancer Immunol Res; 5(2); 127–36. ©2017 AACR.

Supplementary Table 1. shRNAs used

Histone demethylases mediate effects of alpha-KG.

Cytokine production by 2C CD8+ T cells from mice treated or not with allogeneic CD4+ T cells

Lung cancer is the leading cause of cancer-related deaths worldwide. Approximately 85% of all lung cancers are non–small cell histology [non–small cell lung cancer (NSCLC)]. Modern treatment strategies for NSCLC target driver oncogenes and immune checkpoints. However, less than 15% of patients survive beyond 5 years. Here, we investigated the effects of SAR302503 (SAR), a selective JAK2 inhibitor, on NSCLC cell lines and tumors. We show that SAR is cytotoxic to NSCLC cells, which exhibit resistance to genotoxic therapies, such as ionizing radiation, cisplatin, and etoposide. We demonstrate that constitutive IFN-stimulated gene expression, including an IFN-related DNA damage resistance signature, predicts for sensitivity to SAR. Importantly, tumor cell–intrinsic expression of PD-L1 is IFN-inducible and abrogated by SAR. Taken together, these findings suggest potential dual roles for JAK2 inhibitors, both as a novel monotherapy in NSCLCs resistant to genotoxic therapies, and in tandem with immune checkpoint inhibition. Mol Cancer Ther; 17(4); 732–9. ©2018 AACR.

Supplementary Methods

Despite significant advances in combinations of radiotherapy and chemotherapy, altered fractionation schedules and image-guided radiotherapy, many cancer patients fail to benefit from radiation. A prevailing hypothesis is that targeting repair of DNA double strand breaks (DSB) can enhance radiation effects in the tumor and overcome therapeutic resistance without incurring off-target toxicities. Unrepaired DSBs can block cancer cell proliferation, promote cancer cell death, and induce cellular senescence. Given the slow progress to date translating novel DSB repair inhibitors as radiosensitizers, we have explored drug repurposing, a proven route to improving speed, costs, and success rates of drug development. In a prior screen where we tracked resolution of ionizing radiation-induced foci (IRIF) as a proxy for DSB repair, we had identified pitavastatin (Livalo), an HMG-CoA reductase inhibitor commonly used for lipid lowering, as a candidate radiosensitizer. Here, we report that pitavastatin and other lipophilic statins are potent inhibitors of DSB repair in breast and melanoma models both in vitro and in vivo. When combined with ionizing radiation, pitavastatin increased persistent DSBs, induced senescence, and enhanced acute effects of radiation on radioresistant melanoma tumors. shRNA knockdown implicated HMG-CoA reductase, farnesyl diphosphate synthase, and protein farnesyl transferase in IRIF resolution, DSB repair, and senescence. These data confirm on-target activity of statins, although via inhibition of protein prenylation rather than cholesterol biosynthesis. In light of prior studies demonstrating enhanced efficacy of radiotherapy in patients taking statins, this work argues for clinical evaluation of lipophilic statins as nontoxic radiosensitizers to enhance the benefits of image-guided radiotherapy. Mol Cancer Ther; 17(2); 407–18. ©2017 AACR.See all articles in this MCT Focus section, “Developmental Therapeutics in Radiation Oncology.”

Supplementary Fig. S1. Identification of pitavastatin in IRIF persistence screen of NIH Clinical Collection in MCF7GFP-IBD and PANC02GFP-IBD cell lines; Supplementary Fig. S2. Mevalonic acid abrogates radiosensitization effects of pitavastatin in irradiated MCF7 GFP-IBD cells. Supplementary Fig. S3. Reduction in targeted protein expression by shRNA; Supplementary Fig. S4. Targeting mevalonate pathway affects protein prenylation; Supplementary Fig. S5. Farnesyl pyrophosphate abrogates radiosensitization effects of statins in irradiated MCF7 GFP-IBD cells; Supplementary Fig. S6. Silencing GGPS impacts DNA damage response; Supplementary Fig. S7. Clonogenic survival of B16.SIY cells treated with 5 μmol/L or 10 μmol/L veliparib 1 hour prior to the indicated irradiation dose; Supplementary Fig. S8. Flow cytometry co-staining assay for apoptosis and necrosis in tumor cells treated with pitavastatin and/or radiation.

Purpose: Solid tumors that have grown two weeks or longer in mice and have diameters larger than 1 cm are histologically indistinguishable from autochthonous human cancers. When experimental tumors reach this clinically relevant size, they are usually refractory to most immunotherapies but may be destroyed by adoptive T-cell transfer. However, TCR-transgenic T cells and/or tumor cells overexpressing antigens are frequently used in these experiments. Here we studied the requirements for destroying clinical size, unmanipulated 8101 tumors by adoptive cell therapy.Experimental Design: 8101 arose in an old mouse after chronic exposure to UV light. A cancer line was established, which was never serially transplanted. The immunodominant CD8+ T cell-recognized antigen of this tumor is caused by a somatic tumor–specific mutation in the RNA helicase p68. 8101 tumors were treated with spleen cells from young naive, or young and old immunized mice to ascertain the characteristics of immune cells that lead to rejection.Results: Here we show that the mutant p68 peptide has an exceptionally high affinity to the presenting MHC class I molecule Kb and that spleen cells from immunized young syngeneic mice adoptively transferred to Rag−/− or cancer-suppressed euthymic mice eradicate 8101 tumors larger than 1 cm in average diameter and established for several weeks. Spleen cells from naive young mice or from old and boosted (reimmunized) mice were ineffective.Conclusions: Relapse-free destruction of large and long-established tumors expressing a genuine very high-affinity tumor-specific antigen can be achieved by using adoptive transfer of lymphocytes from immunized young individuals. Clin Cancer Res; 18(9); 2526–33. ©2012 AACR.

Supplementary Figure S3. Pathway annotation for HNSCC super-groups. Each horizontal bar graph represents enriched pathways in different subtypes. The number at the end of bar is the total number of genes in a pathway. Percentage of genes in the dataset that overlap with the pathway are shown in grey colour of the bar. The trend line represents the -log(p-value).

Viability of HCT116-L2T cells after treatment with 5-aza-dC

Supplementary Tables S1-S4 show primers and gRNAs used in CRISPR/cas9 experiments and differential expression of 14q32 microRNAs in DKO mice and after treatment by 5-Aza-dC.

(A) Quantification of bands from Western gel shown in Main Figure 3A using Image J. The pixel units obtained for each protein band was normalized to the pixel units calculated from their respective b-Actin loading control. (B-G) Clonogenic survival of various human and mice tumor cell lines with stable shSTING knockdown in response to increasing dose of IR. (H-I) Cell viability assays of primary and immortalized MEFs isolated from WT and STING-/- mice in response to increasing IR dose. Data are representative of at least three experiments. P-values were determined using unpaired Student's t-test. Error bars are SEM. *P < 0.05, **P < 0.01, ***P < 0.005.

PDF file - 75K, ISGs associated with genotoxic stress

Alkylating chemotherapy is a central component of the management of glioblastoma (GBM). Among the factors that regulate the response to alkylation damage, NF-κB acts to both promote and block cytotoxicity. In this study, we used genome-wide expression analysis in U87 GBM to identify NF-κB–dependent factors altered in response to temozolomide and found the long noncoding RNA (lncRNA) MALAT1 as one of the most significantly upregulated. In addition, we demonstrated that MALAT1 expression was coregulated by p50 (p105) and p53 via novel κB- and p53-binding sites in the proximal MALAT1 coding region. Temozolomide treatment inhibited p50 recruitment to its cognate element as a function of Ser329 phosphorylation while concomitantly increasing p53 recruitment. Moreover, luciferase reporter studies demonstrated that both κB and p53 cis-elements were required for efficient transactivation in response to temozolomide. Depletion of MALAT1 sensitized patient-derived GBM cells to temozolomide cytotoxicity, and in vivo delivery of nanoparticle-encapsulated anti-MALAT1 siRNA increased the efficacy of temozolomide in mice bearing intracranial GBM xenografts. Despite these observations, in situ hybridization of GBM specimens and analysis of publicly available datasets revealed that MALAT1 expression within GBM tissue was not prognostic of overall survival. Together, these findings support MALAT1 as a target for chemosensitization of GBM and identify p50 and p52 as primary regulators of this ncRNA.Significance:These findings identify NF-κB and p53 as regulators of the lncRNA MALAT1 and suggest MALAT1 as a potential target for the chemosensitization of GBM.

PDF file, 33KB, Percentages and absolute numbers of regulatory CD4+ T cells (Treg) in the peripheral blood of young (5 month-old) and old (15 monthold) mice.

(A) Quantification of bands from Western gel shown in Main Figure 5D using Image J. The pixel units obtained for each protein band was normalized to the pixel units calculated from their respective b-Actin loading control. (B-E) Kinetic analysis of WT MEFs (B) as well as shScrambled D54 (C), HCT116 (D), and SCC61 (E) proliferation in vitro were measured over time in response to WEE1 inhibitor MK1775 {plus minus} IR (6 Gy) using the IncuCyte live cell imaging system. In vitro growth curve data are representative of at least three experiments, each with n = 3 per group. P-values were determined using unpaired Student's t-test. Error bars are SEM. *P < 0.05, **P < 0.01, ***P < 0.005.

PDF file, 61KB.

(A-D) Quantification of bands from Western gels shown in Main Figures 3H (A), 3I (B), 3J (C), and 3K (D) using Image J. The pixel units obtained for each protein band was normalized to the pixel units calculated from their respective b-Actin loading control. (E) Western analysis of lysates harvested from HEK293 cells that were transiently transfected with either an empty vector of recombinant STING at 48 hours following exposure to 3Gy IR show that the protein levels of p21 and cyclin D1 were restored following overexpression of STING in HEK293 cells. (F) Western analysis of lysates harvested from siCDKN1A HCT116 cells at 48 hours following exposure to increasing dose of IR demonstrate that depletion of p21 did not affect STING expression, and only affected proteins that it regulates downstream of the pathway. (G) Western analysis of lysates harvested from MEFs that were transiently transfected with siRNA targeting STING and/or p21 at 48 hours reveal that depletion of STING led to down-regulation of p21, but not vice versa. Quantified bands are shown right next to each Western blot panels. (H-J) Kinetic analysis of STING-depleted human tumor cell lines D54 (H), HCT116 (I), and SCC61 (J) that were transiently transfected with siRNA targeting p21. Proliferation of nuclei-stained cells were measured in vitro over time using the IncuCyte live cell imaging system. The number of nuclei-stained cells were calculated using the Incucyte software and data are representative of three experiments, each with n = 3 per group. P-values were determined using unpaired Student's t-test. Error bars are SEM. *P < 0.05, **P < 0.01, ***P < 0.005.

Purpose: Current classification of head and neck squamous cell carcinomas (HNSCC) based on anatomic site and stage fails to capture biologic heterogeneity or adequately inform treatment.Experimental Design: Here, we use gene expression-based consensus clustering, copy number profiling, and human papillomavirus (HPV) status on a clinically homogenous cohort of 134 locoregionally advanced HNSCCs with 44% HPV+ tumors together with additional cohorts, which in total comprise 938 tumors, to identify HNSCC subtypes and discover several subtype-specific, translationally relevant characteristics.Results: We identified five subtypes of HNSCC, including two biologically distinct HPV subtypes. One HPV+ and one HPV− subtype show a prominent immune and mesenchymal phenotype. Prominent tumor infiltration with CD8+ lymphocytes characterizes this inflamed/mesenchymal subtype, independent of HPV status. Compared with other subtypes, the two HPV subtypes show low expression and no copy number events for EGFR/HER ligands. In contrast, the basal subtype is uniquely characterized by a prominent EGFR/HER signaling phenotype, negative HPV-status, as well as strong hypoxic differentiation not seen in other subtypes.Conclusion: Our five-subtype classification provides a comprehensive overview of HPV+ as well as HPV− HNSCC biology with significant translational implications for biomarker development and personalized care for patients with HNSCC. Clin Cancer Res; 21(4); 870–81. ©2014 AACR.

Table S1 shows the included solid tumor histologies for enrolled patients. Table S2 shows at least possibly treatment-related grade 2 or greater adverse events among patients remaining on study for at least 3 months. Table S3 shows all adverse events among patients remaining on study for at least 3 months. Table S4 shows the proportion of patients receiving stereotactic body radiation therapy to all RECIST target lesions and all clinically or radiographically apparent metastases by response category.

Supplemental Figures S5-S6. Distribution of CpG sites, transcription start site and methylation-dependent CTCF binding region in DLK1-MEG9 region.

Supplemental Methods, Figures, and Tables