The association between the tumor immune microenvironments and clinical outcome in low-grade, early-stage endometrial cancer patients.

Endometrial tumors show substantial heterogeneity in their immune microenvironment. This heterogeneity could be used to improve the accuracy of current outcome prediction tools. We assessed the immune microenvironment of 235 patients diagnosed with low-grade, early-stage endometrial cancer. Multiplex quantitative immunofluorescence was carried out to measure CD8, CD68, FOXP3, PD-1, and PD-L1 markers, as well as cytokeratin (CK), on tissue microarrays. Clustering results revealed five robust immune response patterns, each associated with specific immune populations, cell phenotypes, and cell spatial clustering. Most samples (69%) belonged to the immune-desert subtype, characterized by low immune cell densities. Tumor-infiltrating lymphocyte (TIL)-rich samples (4%) displayed high CD8+ T-cell infiltration, as well as a high percentage of CD8/PD-1+ cells. Immune-exclusion samples (19%) displayed the lowest CD8+ infiltration combined with high PD-L1 expression levels in CK+ tumor cells. In addition, they demonstrated high tumor cell spatial clustering as well as increased spatial proximity of CD8+ /PD-1+ and CK/PD-L1+ cells. FOXP3 and macrophage-rich phenotypes (3% and 4% of total samples) displayed relatively high levels of FOXP3+ regulatory T-cells and CD68+ macrophages, respectively. These phenotypes correlated with clinical outcomes, with immune-exclusion tumors showing an association with tumor relapse. When compared with prediction models built using routine pathological variables, models optimized with immune variables showed increased outcome prediction capacity (AUC = 0.89 versus 0.78) and stratification potential. The improved prediction capacity was independent of mismatch repair protein status and adjuvant radiotherapy treatment. Further, immunofluorescence results could be partially recapitulated using single-marker immunohistochemistry (IHC) performed on whole tissue sections. TIL-rich tumors demonstrated increased CD8+ T-cells by IHC, while immune-exclusion tumors displayed a lack of CD8+ T-cells and frequent expression of PD-L1 in tumor cells. Our results demonstrate the capability of the immune microenvironment to improve standard prediction tools in low-grade, early-stage endometrial carcinomas. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Weakly supervised deep learning to predict recurrence in low-grade endometrial cancer from multiplexed immunofluorescence images.

Predicting recurrence in low-grade, early-stage endometrial cancer (EC) is both challenging and clinically relevant. We present a weakly-supervised deep learning framework, NaroNet, that can learn, without manual expert annotation, the complex tumor-immune interrelations at three levels: local phenotypes, cellular neighborhoods, and tissue areas. It uses multiplexed immunofluorescence for the simultaneous visualization and quantification of CD68 + macrophages, CD8 + T cells, FOXP3 + regulatory T cells, PD-L1/PD-1 protein expression, and tumor cells. We used 489 tumor cores from 250 patients to train a multilevel deep-learning model to predict tumor recurrence. Using a tenfold cross-validation strategy, our model achieved an area under the curve of 0.90 with a 95% confidence interval of 0.83-0.95. Our model predictions resulted in concordance for 96,8% of cases (κ = 0.88). This method could accurately assess the risk of recurrence in EC, outperforming current prognostic factors, including molecular subtyping.

Overcoming microenvironmental resistance to PD-1 blockade in genetically engineered lung cancer models.

Immune checkpoint blockade (ICB) with PD-1 or PD-L1 antibodies has been approved for the treatment of non-small cell lung cancer (NSCLC). However, only a minority of patients respond, and sustained remissions are rare. Both chemotherapy and antiangiogenic drugs may improve the efficacy of ICB in mouse tumor models and patients with cancer. Here, we used genetically engineered mouse models of Kras G12D/+;p53 -/- NSCLC, including a mismatch repair-deficient variant (Kras G12D/+;p53 -/-;Msh2 -/-) with higher mutational burden, and longitudinal imaging to study tumor response and resistance to combinations of ICB, antiangiogenic therapy, and chemotherapy. Antiangiogenic blockade of vascular endothelial growth factor A and angiopoietin-2 markedly slowed progression of autochthonous lung tumors, but contrary to findings in other cancer types, addition of a PD-1 or PD-L1 antibody was not beneficial and even accelerated progression of a fraction of the tumors. We found that antiangiogenic treatment facilitated tumor infiltration by PD-1+ regulatory T cells (Tregs), which were more efficiently targeted by the PD-1 antibody than CD8+ T cells. Both tumor-associated macrophages (TAMs) of monocyte origin, which are colony-stimulating factor 1 receptor (CSF1R) dependent, and TAMs of alveolar origin, which are sensitive to cisplatin, contributed to establish a transforming growth factor-ß-rich tumor microenvironment that supported PD-1+ Tregs Dual TAM targeting with a combination of a CSF1R inhibitor and cisplatin abated Tregs, redirected the PD-1 antibody to CD8+ T cells, and improved the efficacy of antiangiogenic immunotherapy, achieving regression of most tumors.

Proteomic Analysis of Low-Grade, Early-Stage Endometrial Carcinoma Reveals New Dysregulated Pathways Associated with Cell Death and Cell Signaling.

Low-grade, early-stage endometrial carcinoma (EC) is the most frequent malignant tumor of the uterine corpus. However, the molecular alterations that underlie these tumors are far from being fully understood. The purpose of this study is to describe dysregulated molecular pathways from EC patients. Sixteen samples of tumor tissue and paired healthy controls were collected and both were subjected to mass spectrometry (MS)/MS proteomic analysis. Gene ontology and pathway analysis was performed to discover dysregulated pathways and/or proteins using different databases and bioinformatic tools. Dysregulated pathways were cross-validated in an independent external cohort. Cell signaling, immune response, and cell death-associated pathways were robustly identified. The SLIT/ROBO signaling pathway demonstrated dysregulation at the proteomic and transcriptomic level. Necroptosis and ferroptosis were cell death-associated processes aberrantly regulated, in addition to apoptosis. Immune response-associated pathways showed a dominance of innate immune responses. Tumor immune infiltrates measured by immunofluorescence demonstrated diverse lymphoid and myeloid populations. Our results suggest a role of SLIT/ROBO, necroptosis, and ferroptosis, as well as a prominent role of innate immune response in low-grade, early-stage EC. These results could guide future research in this group of tumors.

Periostin Limits Tumor Response to VEGFA Inhibition.

Resistance to antiangiogenic drugs limits their applicability in cancer therapy. Here, we show that revascularization and progression of pancreatic neuroendocrine tumors (PNETs) under extended vascular-endothelial growth factor A (VEGFA) blockade are dependent on periostin (POSTN), a matricellular protein expressed by stromal cells. Genetic deletion of Postn in RIP1-Tag2 mice blunted tumor rebounds of M2-like macrophages and αSMA+ stromal cells in response to prolonged VEGFA inhibition and suppressed PNET revascularization and progression on therapy. POSTN deficiency also impeded the upregulation of basic fibroblast growth factor (FGF2), an adaptive mechanism previously implicated in PNET evasion from antiangiogenic therapy. Higher POSTN expression correlated with markers of M2-like macrophages in human PNETs, and depleting macrophages with a colony-stimulating factor 1 receptor (CSF1R) antibody inhibited PNET revascularization and progression under VEGFA blockade despite continued POSTN production. These findings suggest a role for POSTN in orchestrating resistance to anti-VEGFA therapy in PNETs.

Dual angiopoietin-2 and VEGFA inhibition elicits antitumor immunity that is enhanced by PD-1 checkpoint blockade.

Pathological angiogenesis is a hallmark of cancer and a therapeutic target. Vascular endothelial growth factor A (VEGFA) and angiopoietin-2 (ANGPT2; also known as ANG2) are proangiogenic cytokines that sustain tumor angiogenesis and limit antitumor immunity. We show that combined ANGPT2 and VEGFA blockade by a bispecific antibody (A2V) provided superior therapeutic benefits, as compared to the single agents, in both genetically engineered and transplant tumor models, including metastatic breast cancer (MMTV-PyMT), pancreatic neuroendocrine tumor (RIP1-Tag2), and melanoma. Mechanistically, A2V promoted vascular regression, tumor necrosis, and antigen presentation by intratumoral phagocytes. A2V also normalized the remaining blood vessels and facilitated the extravasation and perivascular accumulation of activated, interferon-γ (IFNγ)-expressing CD8+ cytotoxic T lymphocytes (CTLs). Whereas the antitumoral activity of A2V was, at least partly, CTL-dependent, perivascular T cells concurrently up-regulated the expression of the immune checkpoint ligand programmed cell death ligand 1 (PD-L1) in tumor endothelial cells. IFNγ neutralization blunted this adaptive response, and PD-1 blockade improved tumor control by A2V in different cancer models. These findings position immune cells as key effectors of antiangiogenic therapy and support the rationale for cotargeting angiogenesis and immune checkpoints in cancer therapy.

The Selective Tie2 Inhibitor Rebastinib Blocks Recruitment and Function of Tie2Hi Macrophages in Breast Cancer and Pancreatic Neuroendocrine Tumors.

Tumor-infiltrating myeloid cells promote tumor progression by mediating angiogenesis, tumor cell intravasation, and metastasis, which can offset the effects of chemotherapy, radiation, and antiangiogenic therapy. Here, we show that the kinase switch control inhibitor rebastinib inhibits Tie2, a tyrosine kinase receptor expressed on endothelial cells and protumoral Tie2-expressing macrophages in mouse models of metastatic cancer. Rebastinib reduces tumor growth and metastasis in an orthotopic mouse model of metastatic mammary carcinoma through reduction of Tie2+ myeloid cell infiltration, antiangiogenic effects, and blockade of tumor cell intravasation mediated by perivascular Tie2Hi/Vegf-AHi macrophages in the tumor microenvironment of metastasis (TMEM). The antitumor effects of rebastinib enhance the efficacy of microtubule inhibiting chemotherapeutic agents, either eribulin or paclitaxel, by reducing tumor volume, metastasis, and improving overall survival. Rebastinib inhibition of angiopoietin/Tie2 signaling impairs multiple pathways in tumor progression mediated by protumoral Tie2+ macrophages, including TMEM-dependent dissemination and angiopoietin/Tie2-dependent angiogenesis. Rebastinib is a promising therapy for achieving Tie2 inhibition in cancer patients. Mol Cancer Ther; 16(11); 2486-501. ©2017 AACR.

Cancer Metastasis: Perivascular Macrophages Under Watch.

TIE2-expressing macrophages cluster around blood vessels and sustain tumor angiogenesis. Harney and colleagues now use live imaging of mouse mammary tumors to show that these perivascular macrophages also promote the transient opening of tumor blood vessels to facilitate hematogenous cancer cell dissemination and metastasis.

Role of angiopoietin-2 in adaptive tumor resistance to VEGF signaling blockade.

Angiopoietin-2 (ANG2/ANGPT2) is a context-dependent TIE2 receptor agonist/antagonist and proangiogenic factor. Although ANG2 neutralization improves tumor angiogenesis and growth inhibition by vascular endothelial growth factor (VEGF)-A signaling blockade, the mechanistic underpinnings of such therapeutic benefits remain poorly explored. We employed late-stage RIP1-Tag2 pancreatic neuroendocrine tumors (PNETs) and MMTV-PyMT mammary adenocarcinomas, which develop resistance to VEGF receptor 2 (VEGFR2) blockade. We found that VEGFR2 inhibition upregulated ANG2 and vascular TIE2 and enhanced infiltration by TIE2-expressing macrophages in the PNETs. Dual ANG2/VEGFR2 blockade suppressed revascularization and progression in most of the PNETs, whereas it had only minor additive effects in the mammary tumors, which did not upregulate ANG2 upon VEGFR2 inhibition. ANG2/VEGFR2 blockade did not elicit increased PNET invasion and metastasis, although it exacerbated tumor hypoxia and hematopoietic cell infiltration. These findings suggest that evasive tumor resistance to anti-VEGFA therapy may involve the adaptive enforcement of ANG2-TIE2 signaling, which can be reversed by ANG2 neutralization.