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Chronic T-cell receptor (TCR) signaling in the tumor microenvironment is known to promote T-cell dysfunction. However, we reasoned that poorly immunogenic tumors may also compromise T cells by impairing their metabolism. To address this, we assessed temporal changes in T-cell metabolism, fate, and function in models of B-cell lymphoma driven by Myc, a promoter of energetics and repressor of immunogenicity. Increases in lymphoma burden most significantly impaired CD4+ T-cell function and promoted regulatory T cell (Treg) and Th1-cell differentiation. Metabolomic analyses revealed early reprogramming of CD4+ T-cell metabolism, reduced glucose uptake, and impaired mitochondrial function, which preceded changes in T-cell fate. In contrast, B-cell lymphoma metabolism remained robust during tumor progression. Finally, mitochondrial functions were impaired in CD4+ and CD8+ T cells in lymphoma-transplanted OT-II and OT-I transgenic mice, respectively. These findings support a model, whereby early, TCR-independent, metabolic interactions with developing lymphomas limits T cell–mediated immune surveillance.

Previous research suggests that inflammation triggers cancer-treatment-related symptoms (i.e., fatigue, depression, and disruptions in sleep and physical activity), but evidence is mixed. This study examined relationships between inflammatory biomarkers and symptoms in patients with gynecologic cancer compared to age-matched women with no cancer history (i.e., controls). Patients (n = 121) completed assessments before chemotherapy cycles 1, 3, and 6, and 6 and 12 months later. Controls (n = 105) completed assessments at similar timepoints. Changes in inflammation and symptomatology were evaluated using random-effects mixed models, and cross-sectional differences between patients and controls in inflammatory biomarkers and symptoms were evaluated using least squares means. Associations among inflammatory biomarkers and symptoms were evaluated using random-effects fluctuation mixed models. The results indicated that compared to controls, patients typically have higher inflammatory biomarkers (i.e., TNF-alpha, TNFR1, TNFR2, CRP, IL-1ra) and worse fatigue, depression, and sleep (ps < 0.05). Patients reported lower levels of baseline physical activity (p = 0.02) that became more similar to controls over time. Significant associations were observed between CRP, depression, and physical activity (ps < 0.05), but not between inflammation and other symptoms. The results suggest that inflammation may not play a significant role in fatigue or sleep disturbance among gynecologic cancer patients but may contribute to depression and physical inactivity.

BackgroundAlthough the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combinations are effective in advanced melanoma it remains unclear whether their mechanisms of action and resistance overlap.MethodsWe used single cell (sc) RNA-seq, flow cytometry and IHC analysis of responding SM1 and B16 melanoma flank tumors and SM1 brain metastases to explore the mechanism of action of the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combination. CD4+ and CD8+ T cell depletion and ELISPOT assays were used to demonstrate the unique role of CD4+ T cell help in the anti-tumor effects of the anti-PD-1+LAG-3 combination. Tetramer assays confirmed the loss of CD8+ tumor-reactive T cells in brain tumors resistant to the anti-PD-1+LAG-3 combination.ResultsThe anti-PD-1+CTLA-4 combination was associated with the infiltration of FOXP3+ regulatory CD4+ cells (Tregs), fewer activated CD4+ T cells and the accumulation of a subset of IFNγ secreting cytotoxic CD8+ T cells, whereas the anti-PD-1+LAG-3 combination led to the accumulation of CD4+ T helper cells that expressed CXCR4, TNFSF8, IL21R and a subset of CD8+ T cells with reduced expression of cytotoxic markers. T cell depletion studies showed a requirement for CD4+ T cells for the anti-PD-1+LAG-3 combination, but not the PD-1-CTLA-4 combination at both flank and brain tumor sites. In anti-PD-1+LAG-3 treated tumors, CD4+ T cell depletion was associated with fewer activated (CD69+) CD8+ T cells, impaired IFNγ release and increased numbers of myeloid-derived suppressor cells (MDSCs) but, conversely, increased numbers of activated CD8+ T cells and IFNγ release in anti-PD-1+CTLA-4 treated tumors. Analysis of relapsing melanoma brain metastases from anti-PD-1+LAG-3 treated mice showed an increased accumulation of MDSCs and a loss of gp100+ tumor reactive CD8+ T cells. An analysis of the inferred cell-cell interactions from the scRNA-seq data suggested the MDSCs interacted with multiple subsets of T cells in a bi-directional manner.ConclusionsTogether these studies suggest that these two clinically relevant ICI combinations have differential effects upon CD4+ T cell polarization, which in turn, impacted cytotoxic CD8+ T cell function. Further insights into the mechanisms of action/resistance of these clinically-relevant ICI combinations will allow therapy to be further personalized.

Supplementary Table from TCR-Independent Metabolic Reprogramming Precedes Lymphoma-Driven Changes in T-cell Fate

Immunotherapy (IT) and targeted therapy (TT) are both effective against melanoma, but their combination is frequently toxic. Here, we investigated whether the sequence of IT (anti–PD-1)→ TT (ceritinib–trametinib or dabrafenib–trametinib) was associated with improved antitumor responses in mouse models of BRAF- and NRAS-mutant melanoma. Mice with NRAS-mutant (SW1) or BRAF-mutant (SM1) mouse melanomas were treated with either IT, TT, or the sequence of IT→TT. Tumor volumes were measured, and samples from the NRAS-mutant melanomas were collected for immune-cell analysis, single-cell RNA sequencing (scRNA-seq), and reverse phase protein analysis (RPPA). scRNA-seq demonstrated that the IT→TT sequence modulated the immune environment, leading to increased infiltration of T cells, monocytes, dendritic cells and natural killer cells, and decreased numbers of tumor-associated macrophages, myeloid-derived suppressor cells, and regulatory T cells. Durable responses to the IT→TT sequence were dependent on T-cell activity, with depletion of CD8+, but not CD4+, T cells abrogating the therapeutic response. An analysis of transcriptional heterogeneity in the melanoma compartment showed the sequence of IT→TT enriched for a population of melanoma cells with increased expression of MHC class I and melanoma antigens. RPPA analysis demonstrated that the sustained immune response induced by IT→TT suppressed tumor-intrinsic signaling pathways required for therapeutic escape. These studies establish that upfront IT improves the responses to TT in BRAF- and NRAS-mutant melanoma models.

Cutaneous T-cell lymphoma (CTCL) is a rare cancer of skin-homing T cells. A subgroup of patients develops large cell transformation with rapid progression to an aggressive lymphoma. Here, we investigated the transformed CTCL (tCTCL) tumor ecosystem using integrative multiomics spanning whole-exome sequencing (WES), single-cell RNA sequencing, and immune profiling in a unique cohort of 56 patients. WES of 70 skin biopsies showed high tumor mutation burden, UV signatures that are prognostic for survival, exome-based driver events, and most recurrently mutated pathways in tCTCL. Single-cell profiling of 16 tCTCL skin biopsies identified a core oncogenic program with metabolic reprogramming toward oxidative phosphorylation (OXPHOS), cellular plasticity, upregulation of MYC and E2F activities, and downregulation of MHC I suggestive of immune escape. Pharmacologic perturbation using OXPHOS and MYC inhibitors demonstrated potent antitumor activities, whereas immune profiling provided in situ evidence of intercellular communications between malignant T cells expressing macrophage migration inhibitory factor and macrophages and B cells expressing CD74.Significance:Our study contributes a key resource to the community with the largest collection of tCTCL biopsies that are difficult to obtain. The multiomics data herein provide the first comprehensive compendium of genomic alterations in tCTCL and identify potential prognostic signatures and novel therapeutic targets for an incurable T-cell lymphoma.This article is highlighted in the In This Issue feature, p. 1171

S1: IT monotherapy in SW1 model S2: IT->dabrafenib-trametinib therapy in BRAF mutant melanoma. S3: IT->TT sequence in NRAS#5 model S4: Expanded heatmap of all immune cells S5: IT->TT increases accumulation of activated CD8+ T cells S6: The TT->IT sequence on T cells, Tregs and MDSCS S7: Expanded heatmap of T and NK cells S8: IT->TT increases accumulation of activated CD8+ T cells, pathway analysis. S9: Expanded heatmap of myeloid cell data S10: IT->TT sequence increases expression of MHC I mRNAs S11: TT affects melanoma antigens S12: IC50 curves showing resistance to TT S13: Model showing mechanism of IT->TT sequence ST1: tumor weights at the end of the experiment

Supplementary Data from Genomic and Single-Cell Landscape Reveals Novel Drivers and Therapeutic Vulnerabilities of Transformed Cutaneous T-cell Lymphoma

Supplementary Table from Genomic and Single-Cell Landscape Reveals Novel Drivers and Therapeutic Vulnerabilities of Transformed Cutaneous T-cell Lymphoma

Supplementary Figure from Genomic and Single-Cell Landscape Reveals Novel Drivers and Therapeutic Vulnerabilities of Transformed Cutaneous T-cell Lymphoma

Chronic T-cell receptor (TCR) signaling in the tumor microenvironment is known to promote T-cell dysfunction. However, we reasoned that poorly immunogenic tumors may also compromise T cells by impairing their metabolism. To address this, we assessed temporal changes in T-cell metabolism, fate, and function in models of B-cell lymphoma driven by Myc, a promoter of energetics and repressor of immunogenicity. Increases in lymphoma burden most significantly impaired CD4+ T-cell function and promoted regulatory T cell (Treg) and Th1-cell differentiation. Metabolomic analyses revealed early reprogramming of CD4+ T-cell metabolism, reduced glucose uptake, and impaired mitochondrial function, which preceded changes in T-cell fate. In contrast, B-cell lymphoma metabolism remained robust during tumor progression. Finally, mitochondrial functions were impaired in CD4+ and CD8+ T cells in lymphoma-transplanted OT-II and OT-I transgenic mice, respectively. These findings support a model, whereby early, TCR-independent, metabolic interactions with developing lymphomas limits T cell–mediated immune surveillance.

Supplementary Figure from TCR-Independent Metabolic Reprogramming Precedes Lymphoma-Driven Changes in T-cell Fate

Supplemental Figure 2: Etomoxir lowers CPT1 enzymatic activity but does not alter the generation of MDSC subsets, induce apoptosis, or affect proliferation in BM-MDSCs.

Supplemental Figure 3: Etomoxir decreases CPT-1 enzymatic activity in vivo, decreases Treg accumulation in spleens.

Figure S1. KSHV infection up-regulates SphK2 expression from endothelial cells. Figure S2. ABC294640 significantly increases endogenous total ceramides/dh-ceramides from TIVE-LTC but not TIVE cells. Figure S3. Chloroquine effectively blocks autophagy induced by ABC294640 in TIVE-LTC cells. Figure S4. The top 2 scored (by the number of pathways) AN networks from 'common' gene sets altered within ABC294640-treated TIVE-LTC. Figure S5. ABC294640 causes G0/G1 cell cycle arrest in TIVE-LTC. Figure S6. Directly silencing of SphK2 by RNAi increases EGR1 expression in TIVE-LTC cells. Figure S7. Successful "knock-down" LC3B and Atg5 from TIVE-LTC cells. Table S1. Primer sequences for qRT-PCR.

There are two supplementary figures. Figure S1 shows Western blot analysis of polyps from control and sildenafil treated mice, for cGMP signaling pathways (VASP, beta-catenin, AKT). Figure S2 shows RT-qPCR analysis of the mucosa and polyps from control and sildenafil treated animals for the expression of phosphodiesterases.

Supplemental Figure 1: FAO is increased in immunosuppressive T-MDSCs.

Myeloid-derived suppressor cells (MDSC) promote tumor growth by inhibiting T-cell immunity and promoting malignant cell proliferation and migration. The therapeutic potential of blocking MDSC in tumors has been limited by their heterogeneity, plasticity, and resistance to various chemotherapy agents. Recent studies have highlighted the role of energy metabolic pathways in the differentiation and function of immune cells; however, the metabolic characteristics regulating MDSC remain unclear. We aimed to determine the energy metabolic pathway(s) used by MDSC, establish its impact on their immunosuppressive function, and test whether its inhibition blocks MDSC and enhances antitumor therapies. Using several murine tumor models, we found that tumor-infiltrating MDSC (T-MDSC) increased fatty acid uptake and activated fatty acid oxidation (FAO). This was accompanied by an increased mitochondrial mass, upregulation of key FAO enzymes, and increased oxygen consumption rate. Pharmacologic inhibition of FAO blocked immune inhibitory pathways and functions in T-MDSC and decreased their production of inhibitory cytokines. FAO inhibition alone significantly delayed tumor growth in a T-cell–dependent manner and enhanced the antitumor effect of adoptive T-cell therapy. Furthermore, FAO inhibition combined with low-dose chemotherapy completely inhibited T-MDSC immunosuppressive effects and induced a significant antitumor effect. Interestingly, a similar increase in fatty acid uptake and expression of FAO-related enzymes was found in human MDSC in peripheral blood and tumors. These results support the possibility of testing FAO inhibition as a novel approach to block MDSC and enhance various cancer therapies. Cancer Immunol Res; 3(11); 1236–47. ©2015 AACR.

Supplemental Figure 5: FAO inhibitors do not affect the activation and function of T cells.

An impaired antitumor immunity is found in patients with cancer and represents a major obstacle in the successful development of different forms of immunotherapy. Signaling through Notch receptors regulates the differentiation and function of many cell types, including immune cells. However, the effect of Notch in CD8+ T-cell responses in tumors remains unclear. Thus, we aimed to determine the role of Notch signaling in CD8+ T cells in the induction of tumor-induced suppression. Our results using conditional knockout mice show that Notch-1 and Notch-2 were critical for the proliferation and IFNγ production of activated CD8+ T cells and were significantly decreased in tumor-infiltrating T cells. Conditional transgenic expression of Notch-1 intracellular domain (N1IC) in antigen-specific CD8+ T cells did not affect activation or proliferation of CD8+ T cells, but induced a central memory phenotype and increased cytotoxicity effects and granzyme B levels. Consequently, a higher antitumor response and resistance to tumor-induced tolerance were found after adoptive transfer of N1IC-transgenic CD8+ T cells into tumor-bearing mice. Additional results showed that myeloid-derived suppressor cells (MDSC) blocked the expression of Notch-1 and Notch-2 in T cells through nitric oxide–dependent mechanisms. Interestingly, N1IC overexpression rendered CD8+ T cells resistant to the tolerogenic effect induced by MDSC in vivo. Together, the results suggest the key role of Notch in the suppression of CD8+ T-cell responses in tumors and the therapeutic potential of N1IC in antigen-specific CD8+ T cells to reverse T-cell suppression and increase the efficacy of T cell–based immunotherapies in cancer. Cancer Immunol Res; 2(8); 800–11. ©2014 AACR.

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Supplemental Figure Legends

Supplemental Figure 4: FAO inhibitors do not affect the growth of tumor cells, cancer stem cells (spheres) or the expression of cancer stem-cell markers.

Supplemental Figure 6. The combined use of Etomoxir and CTX in vivo inhibits all immunosuppressive activity in T-MDSCs.

Mitochondria and endoplasmic reticulum (ER) share structural and functional networks and activate well-orchestrated signaling processes to shape cells’ fate and function. While persistent ER stress (ERS) response leads to mitochondrial collapse, moderate ERS promotes mitochondrial function. Strategies to boost antitumor T-cell function by targeting ER–mitochondria cross-talk have not yet been exploited. Here, we used carbon monoxide (CO), a short-lived gaseous molecule, to test whether engaging moderate ERS conditions can improve mitochondrial and antitumor functions in T cells. In melanoma antigen-specific T cells, CO-induced transient activation of ERS sensor protein kinase R-like endoplasmic reticulum kinase (PERK) significantly increased antitumor T-cell function. Furthermore, CO-induced PERK activation temporarily halted protein translation and induced protective autophagy, including mitophagy. The use of LC3-GFP enabled differentiation between the cells that prepare themselves to undergo active autophagy (LC3-GFPpos) and those that fail to enter the process (LC3-GFPneg). LC3-GFPpos T cells showed strong antitumor potential, whereas LC3-GFPneg cells exhibited a T regulatory–like phenotype, harbored dysfunctional mitochondria, and accumulated abnormal metabolite content. These anomalous ratios of metabolites rendered the cells with a hypermethylated state and distinct epigenetic profile, limiting their antitumor activity. Overall, this study shows that ERS-activated autophagy pathways modify the mitochondrial function and epigenetically reprogram T cells toward a superior antitumor phenotype to achieve robust tumor control. Significance: Transient activation of ER stress with carbon monoxide drives mitochondrial biogenesis and protective autophagy that elicits superior antitumor T-cell function, revealing an approach to improving adoptive cell efficacy therapy.

Recent studies suggest that B cells could play an important role in the tumor microenvironment. However, the role of humoral responses in endometrial cancer remains insufficiently investigated. Using a cohort of 107 patients with different histological subtypes of endometrial carcinoma, we evaluated the role of coordinated humoral and cellular adaptive immune responses in endometrial cancer. Concomitant accumulation of T, B, and plasma cells at tumor beds predicted better survival. However, only B-cell markers corresponded with prolonged survival specifically in high-grade endometrioid type and serous tumors. Immune protection was associated with class-switched IgA and, to a lesser extent, IgG. Expressions of polymeric immunoglobulin receptor (pIgR) by tumor cells and its occupancy by IgA were superior predictors of outcome and correlated with defects in methyl-directed DNA mismatch repair. Mechanistically, pIgR-dependent, antigen-independent IgA occupancy drove activation of inflammatory pathways associated with IFN and TNF signaling in tumor cells, along with apoptotic and endoplasmic reticulum stress pathways, while thwarting DNA repair mechanisms. Together, these findings suggest that coordinated humoral and cellular immune responses, characterized by IgA:pIgR interactions in tumor cells, determine the progression of human endometrial cancer as well as the potential for effective immunotherapies. Significance: This study provides new insights into the crucial role of humoral immunity in human endometrial cancer, providing a rationale for designing novel immunotherapies against this prevalent malignancy. See related commentary by Osorio and Zamarin, p. 766

Figure showing representative and merged results of the depletion of MDSCs and MDSC-LC using anti-Gr-1 antibody.

Supplementary Data from Single-cell Characterization of the Cellular Landscape of Acral Melanoma Identifies Novel Targets for Immunotherapy

PI3K-delta KD in murine cells and PI3K-delta inhibition in human cell

Supplemental Table 5: Cox proportional hazard model analysis identifies immune cell subpopulations associated with better survival outcomes

To modulate T-cell function for cancer therapy, one challenge is to selectively attenuate regulatory but not conventional CD4+ T-cell subsets [regulatory T cell (Treg) and conventional T cell (Tconv)]. In this study, we show how a functional dichotomy in Class IA PI3K isoforms in these two subsets of CD4+ T cells can be exploited to target Treg while leaving Tconv intact. Studies employing isoform-specific PI3K inhibitors and a PI3Kδ-deficient mouse strain revealed that PI3Kα and PI3Kβ were functionally redundant with PI3Kδ in Tconv. Conversely, PI3Kδ was functionally critical in Treg, acting there to control T-cell receptor signaling, cell proliferation, and survival. Notably, in a murine model of lung cancer, coadministration of a PI3Kδ-specific inhibitor with a tumor-specific vaccine decreased numbers of suppressive Treg and increased numbers of vaccine-induced CD8 T cells within the tumor microenvironment, eliciting potent antitumor efficacy. Overall, our results offer a mechanistic rationale to employ PI3Kδ inhibitors to selectively target Treg and improve cancer immunotherapy. Cancer Res; 77(8); 1892–904. ©2017 AACR.

Supplement Methods

Recent studies suggest that B cells could play an important role in the tumor microenvironment. However, the role of humoral responses in endometrial cancer remains insufficiently investigated. Using a cohort of 107 patients with different histological subtypes of endometrial carcinoma, we evaluated the role of coordinated humoral and cellular adaptive immune responses in endometrial cancer. Concomitant accumulation of T, B, and plasma cells at tumor beds predicted better survival. However, only B-cell markers corresponded with prolonged survival specifically in high-grade endometrioid type and serous tumors. Immune protection was associated with class-switched IgA and, to a lesser extent, IgG. Expressions of polymeric immunoglobulin receptor (pIgR) by tumor cells and its occupancy by IgA were superior predictors of outcome and correlated with defects in methyl-directed DNA mismatch repair. Mechanistically, pIgR-dependent, antigen-independent IgA occupancy drove activation of inflammatory pathways associated with IFN and TNF signaling in tumor cells, along with apoptotic and endoplasmic reticulum stress pathways, while thwarting DNA repair mechanisms. Together, these findings suggest that coordinated humoral and cellular immune responses, characterized by IgA:pIgR interactions in tumor cells, determine the progression of human endometrial cancer as well as the potential for effective immunotherapies.Significance:This study provides new insights into the crucial role of humoral immunity in human endometrial cancer, providing a rationale for designing novel immunotherapies against this prevalent malignancy.See related commentary by Osorio and Zamarin, p. 766

Response to vaccines in WT type and PI3K-delta KO mice

This file contains a figure showing the expression of CD31 in tumors from mice treated with anti-Jagged, compared to controls treated with an isotype

IC50 values for inhibition of Class IA PI3K isoform

Flow cytometry gating strategy for pAkt (S473) and pS6 (pS244) and proliferation determination.

Myeloid-derived suppressor cells (MDSC) represent a primary mechanism of immune evasion in tumors and have emerged as a major obstacle for cancer immunotherapy. The immunoinhibitory activity of MDSC is tightly regulated by the tumor microenvironment and occurs through mechanistic mediators that remain unclear. Here, we elucidated the intrinsic interaction between the expression of AMP-activated protein kinase alpha (AMPKα) and the immunoregulatory activity of MDSC in tumors. AMPKα signaling was increased in tumor-MDSC from tumor-bearing mice and patients with ovarian cancer. Transcription of the Ampkα1-coding gene, Prkaa1, in tumor-MDSC was induced by cancer cell–derived granulocyte–monocyte colony-stimulating factor (GM-CSF) and occurred in a Stat5–dependent manner. Conditional deletion of Prkaa1 in myeloid cells, or therapeutic inhibition of Ampkα in tumor-bearing mice, delayed tumor growth, inhibited the immunosuppressive potential of MDSC, triggered antitumor CD8+ T-cell immunity, and boosted the efficacy of T-cell immunotherapy. Complementarily, therapeutic stimulation of AMPKα signaling intrinsically promoted MDSC immunoregulatory activity. In addition, Prkaa1 deletion antagonized the differentiation of monocytic-MDSC (M-MDSC) to macrophages and re-routed M-MDSC, but not granulocytic-MDSC (PMN-MDSC), into cells that elicited direct antitumor cytotoxic effects through nitric oxide synthase 2-mediated actions. Thus, our results demonstrate the primary role of AMPKα1 in the immunosuppressive effects induced by tumor-MDSC and support the therapeutic use of AMPK inhibitors to overcome MDSC-induced T-cell dysfunction in cancer.Significance:AMPKα1 regulates the immunosuppressive activity and differentiation of tumor-MDSC, suggesting AMPK inhibition as a potential therapeutic strategy to restore protective myelopoiesis in cancer.

This file contains the Supplementary Figures 1-5. Supplementary Figure 1. Phospho-AMPKα1 levels increase in tumor-MDSC. Supplementary Figure 2. Tumor cell-derived GM-CSF triggers Ampkα expression in MDSC through Stat-5 activation. Supplementary Figure 3. MDSC in human myeloid progenitors treated with GM-CSF plus IL-6 for 7 days. Supplementary Figure 4. Extended survival of Prkaa1-null mice bearing ovarian tumors. Supplementary Figure 5. Effects of Prkaa1KO PMN-MDSC in tumor growth.

Supplemental Figure 1. Expression of T, NK, B and Plasma cell markers by metastatic site. Supplemental Figure 2. Expression of myeloid cell markers by metastatic site and gene expression associated with classical and alternative macrophage activation. Supplemental Figure 3. Baseline and therapy induced changes in the LMM immune landscape. Supplemental Figure 4. Baseline and therapy-mediated changes to the MBM immune landscape. Supplemental Figure 5. Expression of MHC class I and II in melanoma cells from samples with and without DC3s. Supplemental Figure 6. Cell-cell interactions between DC3s and T cells defined by SingleCellR. Supplemental Figure 7. Detection of DC3 cells in clinical samples using multiplexed IHC. Supplemental Figure 8. Validation analysis of samples from melanoma metastases in a publicly available validation dataset. Supplemental Figure 9. Expression of key markers that distinguish the major subsets of myeloid cells. Supplemental Figure 10. Correlation of DC3 gene signature and PC loading for DC3 gene signature in TCGA. Supplemental Figure 11. Overall survival for melanoma patients with LMM metastases whose CSF samples contained high DC3 cells vs. low DC3 cells. Supplemental Figure 12. Correlation of individual genes from the DC3 gene signature to overall survival and their univariate cox proportional-hazards models in TCGA dataset.