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Supplementary Figure S5 shows the immunohistochemical evaluation of hypoxia in NCI-H1703 subcutaneous tumor model

Dysregulation of lipid metabolism affects the behavior of cancer cells, but how this happens is not completely understood. Neutral sphingomyelinase 2 (nSMase2), encoded by SMPD3, catalyzes the breakdown of sphingomyelin to produce the anti-oncometabolite ceramide. We found that this enzyme was often downregulated in human metastatic melanoma, likely contributing to immune escape. Overexpression of nSMase2 in mouse melanoma reduced tumor growth in syngeneic wild-type but not CD8-deficient mice. In wild-type mice, nSMase2-overexpressing tumors showed accumulation of both ceramide and CD8+ tumor-infiltrating lymphocytes, and this was associated with increased level of transcripts encoding IFNγ and CXCL9. Overexpressing the catalytically inactive nSMase2 failed to alter tumor growth, indicating that the deleterious effect nSMase2 has on melanoma growth depends on its enzymatic activity. In vitro, small extracellular vesicles from melanoma cells overexpressing wild-type nSMase2 augmented the expression of IL12, CXCL9, and CCL19 by bone marrow–derived dendritic cells, suggesting that melanoma nSMase2 triggers T helper 1 (Th1) polarization in the earliest stages of the immune response. Most importantly, overexpression of wild-type nSMase2 increased anti–PD-1 efficacy in murine models of melanoma and breast cancer, and this was associated with an enhanced Th1 response. Therefore, increasing SMPD3 expression in melanoma may serve as an original therapeutic strategy to potentiate Th1 polarization and CD8+ T-cell–dependent immune responses and overcome resistance to anti–PD-1.

Although understanding of T-cell exhaustion is widely based on mouse models, its analysis in patients with cancer could provide clues indicating tumor sensitivity to immune checkpoint blockade (ICB). Data suggest a role for costimulatory pathways, particularly CD28, in exhausted T-cell responsiveness to PD-1/PD-L1 blockade. Here, we used single-cell transcriptomic, phenotypic, and functional approaches to dissect the relation between CD8+ T-cell exhaustion, CD28 costimulation, and tumor specificity in head and neck, cervical, and ovarian cancers. We found that memory tumor–specific CD8+ T cells, but not bystander cells, sequentially express immune checkpoints once they infiltrate tumors, leading, in situ, to a functionally exhausted population. Exhausted T cells were nonetheless endowed with effector and tumor residency potential but exhibited loss of the costimulatory receptor CD28 in comparison with their circulating memory counterparts. Accordingly, PD-1 inhibition improved proliferation of circulating tumor–specific CD8+ T cells and reversed functional exhaustion of specific T cells at tumor sites. In agreement with their tumor specificity, high infiltration of tumors by exhausted cells was predictive of response to therapy and survival in ICB-treated patients with head and neck cancer. Our results showed that PD-1 blockade–mediated proliferation/reinvigoration of circulating memory T cells and local reversion of exhaustion occur concurrently to control tumors.

supplementary materials & methods

TCGA dataset

Supplementary Figure S8 shows that EVT801 doesn’t modify blood pressure in rat while sorafenib increases it

Supplementary Figure S9 provides an overview of the mechanisms by which EVT801 exerts its anti-tumor effects

Supplementary Table S2 shows EVT801 metabolite potency and selectivity profile

Supplementary Figure S4 shows the evaluation of EVT801’s pharmacokinetics and pharmacodynamics

Supplementary Table S1 shows EVT801 potency and selectivity profile

Supplementary Figure S2 shows that EVT801 inhibits tubule formation

Supplementary Figure S7 shows that EVT801 decreases immunosuppressive cell infiltration in VEGFR3-positive BNL hepatoma tumors

Supplementary Figure S6 shows that EVT801 inhibits growth of VEGFR3-positive BNL hepatoma tumors, while decreasing vasculature and hypoxia

Supplementary Table S3 shows pharmacokinetic parameters of EVT801 in mice and rats

Gating Strategies

This file contains supplementary table 1 and supplementary figures 1-7.

Supplementary Figures S1-S10 and Tables S1-S3

Supplementary Figure S1 shows that EVT801 metabolite is a selective VEGFR-3 inhibitor

Supplementary Figure S3 shows the expression of VEGFR3 altogether with vascular and lymphatic markers in primary kidney tumors

Supplementary Data from Identification of a New Panel of Serum Autoantibodies Associated with the Presence of In situ Carcinoma of the Breast in Younger Women

Supplementary Figure S1 and Supplementary Table S1.

Purpose: We examined the feasibility of using a panel of autoantibodies to multiple tumor-associated proteins as a method for early detection of breast cancer and, more particularly, carcinoma in situ (CIS).Experimental Design: PPIA, PRDX2, and FKBP52 were identified as early-stage breast cancer autoantigens by proteomic approaches. The seroreactivity of a panel of antibodies consisting of these three antigens and two previously described autoantigens, HSP60 and MUC1, was tested on 235 samples (60 from primary breast cancer patients, 82 from CIS patients, and 93 from healthy controls) with the use of specific ELISAs. FKBP52, PPIA, and PRDX2 mRNA and protein expression levels were evaluated by reverse transcription-PCR and immunohistochemistry in early-stage breast tumors.Results: Three of five autoantibodies, FKBP52, PPIA, and PRDX2, showed significantly increased reactivity in primary breast cancer and CIS compared with healthy controls. When combined, the five markers significantly discriminated primary breast cancer [receiver operating characteristic area under the curve, 0.73; 95% confidence interval (95% CI), 0.60-0.79] and CIS (receiver operating characteristic area under the curve, 0.80; 95% CI, 0.71-0.85) from healthy individuals. Importantly, the receiver operating characteristic–area under the curve value of the autoantibody panel was able to distinguish CIS, including high grades, from healthy controls in women under the age of 50 years (receiver operating characteristic area under the curve, 0.85; 95% CI, 0.61-0.92). Finally, only FKBP52 mRNA and protein levels were found to be increased in CIS and primary breast cancer compared with healthy breast tissue.Conclusions: This autoantibody assay against a panel of five antigens allows for an accurate discrimination between early-stage breast cancer, especially CIS, and healthy individuals. These results could be of interest in detecting early breast cancer as an aid to mammography, especially in women under the age of 50 years with aggressive cancers.

Purpose: There is a clinical need to identify predictive markers of the responses to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI). Deoxy-2-[18F]fluoro-d-glucose positron emission tomography with computed tomography (18FDG-PET/CT) could be a tool of choice for monitoring the early effects of this class of agent on tumor activity.Experimental Design: Using models of human head and neck carcinoma (CAL33 and CAL166 cell lines), we first tested in vitro and in vivo whether the in vivo changes in 18FDG-PET/CT uptake were associated with the molecular and cellular effects of the EGFR-TKI erlotinib. Then, the pathologic and morphologic changes and the 18FDG-PET/CT uptake before and after erlotinib exposure in patients were analyzed.Results: Erlotinib strongly inhibited extracellular signal-regulated kinase-1/2 (ERK-1/2) phosphorylation both in the preclinical models and in patients. Western blotting, immunofluorescence, and immunohistochemistry showed that erlotinib did not modify Glut-1 expression at the protein level either in cell line models or in tumor tissue from mouse xenografts or in patients. Phospho-ERK-1/2 inhibition was associated with a reduction in 18FDG uptake in animal and human tumors. The biological volume was more accurate than the standardized uptake value for the evaluation of the molecular responses.Conclusion: These results show that the 18FDG-PET/CT response is a reliable surrogate marker of the effects of erlotinib in head and neck carcinoma. Clin Cancer Res; 16(17); 4434–45. ©2010 AACR.

Supplementary Figure S4. Analysis of the immune response in draining lymph nodes from WT and TNF-deficient mice.

PDF file - 140K, Vessel stabilization

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Supplementary Figure S6. Accumulation of CD8+ TIL in TNF-R1-deficient mice.

PDF file - 139, ER� is efficiently suppressed in BM and BM-derived cells using chimeric hematopoietic approach

PDF file, 47KB

Supplementary Figure S1. TNF does not modulate in vitro B16 proliferation.

Supplementary Figure S7. Effect of Etanercept injection in mice.

PDF file - 364K, B16K1, LL2 and 4T1 characterization

Supplementary information. This file contains supplementary materials and methods, and legends to supplementary figures.

PDF file - 162K, Vessel diameter quantification by granulometry

PDF file - 122K, B16K1 time- and size-dependent tumor harvesting

Estrogens directly promote the growth of breast cancers that express the estrogen receptor α (ERα). However, the contribution of stromal expression of ERα in the tumor microenvironment to the protumoral effects of estrogen has never been explored. In this study, we evaluated the molecular and cellular mechanisms by which 17β-estradiol (E2) impacts the microenvironment and modulates tumor development of ERα-negative tumors. Using different mouse models of ER-negative cancer cells grafted subcutaneously into syngeneic ovariectomized immunocompetent mice, we found that E2 potentiates tumor growth, increases intratumoral vessel density, and modifies tumor vasculature into a more regularly organized structure, thereby improving vessel stabilization to prevent tumor hypoxia and necrosis. These E2-induced effects were completely abrogated in ERα-deficient mice, showing a critical role of host ERα. Notably, E2 did not accelerate tumor growth when ERα was deficient in Tie2-positive cells, even in mice grafted with wild-type bone marrow. These results were extended by clinical evidence of ERα-positive stromal cell labeling in the microenvironment of human breast cancers. Together, our findings therefore show that E2 promotes the growth of ERα-negative cancer cells through the activation of stromal ERα (extra-hematopoietic Tie-2 positive cells), which normalizes tumor angiogenesis and allows an adaptation of blood supply to tumors, thereby preventing hypoxia and necrosis. These findings significantly deepen mechanistic insights into the impact of E2 on tumor development with potential consequences for cancer treatment. Cancer Res; 72(12); 3010–9. ©2012 AACR.

Supplementary Figure S2. Tumor growth of B16-BL6 and Yumm melanoma cell lines in TNF-deficient mice.

PDF file - 204K, B16K1 Tumor necrosis in C57BL/6 mice

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Supplementary Figure S5. Analysis of Treg and MDSC content in the tumors from WT and TNF-deficient mice.

Genomic instability (GI) influences treatment efficacy and resistance, and an accurate measure of it is lacking. Current measures of GI are based on counts of specific structural variation (SV) and mutational signatures. Here, we present a holistic approach to measuring GI based on the quantification of the steady-state equilibrium between DNA damage and repair as assessed by the residual breakpoints (BP) remaining after repair, irrespective of SV type. We use the notion of Hscore, a BP “hotspotness” magnitude scale, to measure the propensity of genomic structural or functional DNA elements to break more than expected by chance. We then derived new measures of transcription- and replication-associated GI that we call iTRAC (transcription-associated chromosomal instability index) and iRACIN (replication-associated chromosomal instability index). We show that iTRAC and iRACIN are predictive of metastatic relapse in Leiomyosarcoma (LMS) and that they may be combined to form a new classifier called MAGIC (mixed transcription- and replication-associated genomic instability classifier). MAGIC outperforms the gold standards FNCLCC and CINSARC in stratifying metastatic risk in LMS. Furthermore, iTRAC stratifies chemotherapeutic response in LMS. We finally show that this approach is applicable to other cancers.

In leiomyosarcoma (LMS), a very aggressive disease, a relatively transcriptionally uniform subgroup of well-differentiated tumors has been described and is associated with poor survival. The question raised how differentiation and tumor progression, two apparently antagonist processes, coexist and allow tumor malignancy. We first identified the most transcriptionally homogeneous LMS subgroup in three independent cohorts, which we named ‘hLMS’. The integration of multi-omics data and functional analysis suggests that hLMS originate from vascular smooth muscle cells and show that hLMS transcriptional program reflects both modulations of smooth muscle contraction activity controlled by MYOCD/SRF regulatory network and activation of the cell cycle activity controlled by E2F/RB1 pathway. We propose that the phenotypic plasticity of vascular smooth muscle cells coupled with MYOCD/SRF pathway amplification, essential for hLMS survival, concomitant with PTEN absence and RB1 alteration, could explain how hLMS balance this uncommon interplay between differentiation and aggressiveness.

As key regulators of the actin cytoskeleton, RHO GTPase expression and/or activity are deregulated in tumorigenesis and metastatic progression. Nevertheless, the vast majority of experiments supporting this conclusion was conducted on cell lines but not on human tumor samples that were mostly studied at the expression level only. Up to now, the activity of RHO proteins remains poorly investigated in human tumors. In this article, we present the development of a robust nanobody-based ELISA assay, with a high selectivity that allows an accurate quantification of RHO protein GTP-bound state in the nanomolar range (1 nM; 20 μg/L), not only in cell lines after treatment but also in tumor samples. Of note, we present here a fine analysis of RHOA-like and RAC1 active state in tumor samples with the most comprehensive study of RHOA-GTP and RHOC-GTP levels performed on human breast tumor samples. We revealed increased GTP-bound RHOA and RHOC protein activities in tumors compared to normal tissue counterparts, and demonstrated that the RHO active state and RHO expression are two independent parameters among different breast cancer subtypes. Our results further highlight the regulation of RHO protein activation in tumor samples and the relevance of directly studying RHO GTPase activities involvement in molecular pathways.

Leiomyosarcoma (LMS) is an aggressive smooth muscle cancer with few therapeutic options. LMSs show a high level of genomic instability (GI) and the mechanisms underlying their oncogenic processes are poorly understood. While the level of GI influences treatment efficacy and resistance, an accurate measure of it is lacking. Current measures of GI are based on counts of specific structural variation (SV) and mutational signatures. Here, we present a holistic approach to measuring GI based on the quantification of the steady-state equilibrium between DNA damage and repair as assessed by the residual breakpoints (BP) remaining after repair, irrespective of SV type. We use the notion of Hscore, a BP “hotspotness” magnitude scale, to measure the propensity of genomic structural or functional DNA elements to break more than expected by chance. We then derived new measures of transcription- and replication-associated GI that we call iTRAC (Transcription-Associated Chromosomal instability index (iTRAC) and iRACIN (Replication-Associated Chromosomal INstability index). We show that iTRAC and iRACIN are predictive of metastatic relapse in LMS and that they may be combined to form a new classifier called MAGIC (Mixed transcription-and replication-Associated Genomic Instability Classifier). MAGIC outperforms the gold standards FNCLCC and CINSARC in stratifying metastatic risk in LMS. Furthermore, iTRAC stratifies chemotherapeutic response in LMS. We finally show that this approach is applicable to other cancers.

Introduction: EVT801 is a highly selective small molecule inhibitor of VEGFR3 and acts by inhibiting lymphangiogenesis and tumor angiogenesis in and around the tumor. It has shown compelling activity in a wide range of cancer models and is well-tolerated in preclinical toxicology studies. A phase I study is underway and will focus primarily on understanding the safety, tolerability, and pharmacokinetics of EVT801 across a range of doses. Methods: The first stage of the study is designed to determine the maximum tolerated dose (MTD) and recommended Phase II dose (RP2D) for EVT801, with a second stage aiming to confirm RP2D in selected cancer patient populations. Clinical samples from these patients will be used to explore preliminary signals of clinical efficacy and investigate the biological activity of the drug using several biomarkers. Efficacy biomarkers will include imaging approaches (DCE-MRI and CEUS) to characterize tumor vascularization, as well as analysis of protein and mRNA quantification in on-treatment biopsies vs pre-treatment biopsies. Moreover, the relationship between activity of EVT801 and expression of key markers at protein and mRNA level will be investigated to potentially establish biomarkers for patient stratification and selection. Lastly, target engagement and pharmacodynamics effects will be investigated by immuno-monitoring as well as assessment of a defined set of proteins as markers of inflammation and angiogenesis as identified in preclinical in vivo models. We expect that these analyses will help to better understand the effects of the drug in human subjects and may also help to select the most responsive patients and provide early signs of clinical efficacy. Citation Format: Michael R. Paillasse, James Garner, Michael Fitzgerald, Lise Davenne, Pierre-Benoit Ancey, celine Poussereau-Pomie, Michael Esquerre, Gaelle Badet, Joel Tuyaret, Marie Mandron, Philippe Rochaix, Maha Ayyoub, Clara Maria Scarlata, Christine Ménétrier-Caux, Chrsitophe CAUX, Philippe Cassier, Jean-Pierre Delord, Carlos Gomez Roca, Pierre Fons. Cutting edge biomarkers strategy to provide early insights into activity of EVT-801, a novel selective VEGFR-3 inhibitor that targets tumor angiogenesis during the FIH clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3203.

Leiomyosarcoma is a very aggressive tumor with poor prognosis since half of the patients will develop metastasis. Today, neither chemotherapy nor targeted or immune therapies have demonstrated efficacy. These tumors show highly rearranged genomes and appear to form a heterogenous group. Understanding their complex oncogenesis either as a single disease or by subtypes has already been challenged but the underlying mechanisms driving leiomyosarcoma development which could lead to a patient care improvement remain to be deciphered. We identified a group of tightly clustered leiomyosarcomas due to their gene expression homogeneity, named "hLMS". We derived a transcriptional signature able to consistently stratify patients from two independent cohorts. In all cohorts, hLMS were preferentially carried by women, located in the internal trunk, highly differentiated, and similarly altered at the genomic level. Through the multi-omics integrative bioinformatic analysis, we show that hLMS originate from vascular smooth muscle cells presenting both contractile and synthetic characteristics, while the other group could derive from fibroblasts. We identified the MYOCD overexpression as a hLMS-specific driver candidate and functionally showed that the MYOCD/SRF axis is only essential for hLMS survival. Identification of hLMS could become standard clinical practice, leading to the development of specific effective treatments with MYOCD/SRF inhibitors.

Aims The diagnosis of malignant peripheral nerve sheath tumour (MPNST) may be challenging, especially in the sporadic setting. Owing to the lack of specific histological criteria, immunohistochemical and molecular diagnostic markers, several differential diagnoses must be considered, especially melanoma. Indeed, although S100 protein usually stains melanoma, other melanocytic markers are often negative, especially in spindle cell/desmoplastic types. This pattern of immunoreactivity resembles that of some nerve-derived tumours such as MPNST. Owing to their different clinical behaviours and therapeutic implications, accurate identification of these two different tumours is crucial. Methods and results S100, SOX10, KBA62, MITF, HMB45, Melan-A, tyrosinase PNL2 and BRAF-V600E immunostaining was performed in a pathologically and genetically well-characterised cohort of primary MPNST (n = 124), including 66 (53%) NF1-associated tumours. Sox10 and KBA62 expression were found, respectively, in 102 (84%) and in 101 (83%) MPNST, whereas S100 was expressed in 64 cases (52%). We observed an increased loss of S100 with increasing histological grade (P = 0.0052). We found Melan-A expression in 14% (n = 17) of all MPNST, occurring in 82% (n = 14) of cases in an NF1 context. Six per cent (n = 8) of MPNST showed tyrosinase positivity, including seven (87%) NF1-associated. MITF expression was found in 10 (8%) MPNST. None expressed PNL2, HMB45 or BRAF-V600E. Conclusion MPNST (in NF1 and a sporadic setting) can quite often be positive for Melan-A, tyrosinase and MITF. Pathologists should be cognisant of these exceptions to prevent confusion with melanoma.