Vessel Co-option Research Articles

Abstract 1927: Cancer cells induce apoptosis in hepatocytes as one of the mechanisms to displace hepatocytes in vessel co-opted colorectal cancer liver metastases

Abstract Vessel co-option in colorectal cancer liver metastases (CRCLM) has been recognized as one of the mechanistic pathways of resistance against anti-angiogenic therapy. The cancer cells are highly motile in co-opted lesions, which move toward and along the pre-existing sinusoidal vessels and hijack them to gain access to nutrient. The movement of cancer cells is accompanied by displacement of the hepatocytes. However, the molecular mechanisms underlying this displacement are unclear yet. To examine whether apoptosis involved in hepatocytes displacement by cancer cells in co-opted lesions, we performed immunohistochemical staining for pro-apoptotic markers, such as cleaved caspase-3 and cleaved PARP-1. We observed overexpression of pro-apoptotic markers in liver parenchyma of co-opted lesions compared to angiogenic lesions, specifically the hepatocytes that are in close proximity to the cancer cells. In vitro, we found that culturing hepatocytes with either colorectal cancer cells or conditioned media of co-opted CRCLM organoids induces apoptosis. Importantly, our results also suggested proprotein convertase subtilisin/kexin type 9 (PCSK-9 or PC-9) as a potential mediator of cancer cells-driven hepatocytes apoptosis. Altogether, these results confirm that cancer cells exploit apoptosis to establish vessel co-option in CRCLM. Citation Format: Miran Rada, Migmar Tsamchoe, Audrey Kapelanski-Lamoureux, Jessica Bloom, Stephanie Petrillo, Sébastien Tabariès, Diane H. Kim, Peter Younan, Alex Gregorieff, Peter Siegel, Anthoula Lazaris, Peter Metrakos. Cancer cells induce apoptosis in hepatocytes as one of the mechanisms to displace hepatocytes in vessel co-opted colorectal cancer liver metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1927.

Using Zebrafish to Elucidate Glial-Vascular Interactions During CNS Development.

An emerging area of interest in Neuroscience is the cellular relationship between glia and blood vessels, as many of the presumptive support roles of glia require an association with the vasculature. These interactions are best studied in vivo and great strides have been made using mice to longitudinally image glial-vascular interactions. However, these methods are cumbersome for developmental studies, which could benefit from a more accessible system. Zebrafish (Danio rerio) are genetically tractable vertebrates, and given their translucency, are readily amenable for daily live imaging studies. We set out to examine whether zebrafish glia have conserved traits with mammalian glia regarding their ability to interact with and maintain the developing brain vasculature. We utilized transgenic zebrafish strains in which oligodendrocyte transcription factor 2 (olig2) and glial fibrillary acidic protein (gfap) identify different glial populations in the zebrafish brain and document their corresponding relationship with brain blood vessels. Our results demonstrate that olig2+ and gfap+ zebrafish glia have distinct lineages and each interact with brain vessels as previously observed in mouse brain. Additionally, we manipulated these relationships through pharmacological and genetic approaches to distinguish the roles of these cell types during blood vessel development. olig2+ glia use blood vessels as a pathway during their migration and Wnt signaling inhibition decreases their single-cell vessel co-option. By contrast, the ablation of gfap+ glia at the beginning of CNS angiogenesis impairs vessel development through a reduction in Vascular endothelial growth factor (Vegf), supporting a role for gfap+ glia during new brain vessel formation in zebrafish. This data suggests that zebrafish glia, akin to mammalian glia, have different lineages that show diverse interactions with blood vessels, and are a suitable model for elucidating glial-vascular relationships during vertebrate brain development.

Tumor vessel co-option probed by single-cell analysis.

Tumor vessel co-option is poorly understood, yet it is a resistance mechanism against anti-angiogenic therapy (AAT). The heterogeneity of co-opted endothelial cells (ECs) and pericytes, co-opting cancer and myeloid cells in tumors growing via vessel co-option, has not been investigated at the single-cell level. Here, we use a murine AAT-resistant lung tumor model, in which VEGF-targeting induces vessel co-option for continued growth. Single-cell RNA sequencing (scRNA-seq) of 31,964 cells reveals, unexpectedly, a largely similar transcriptome of co-opted tumor ECs (TECs) and pericytes as their healthy counterparts. Notably, we identify cell types that might contribute to vessel co-option, i.e., an invasive cancer-cell subtype, possibly assisted by a matrix-remodeling macrophage population, and another M1-like macrophage subtype, possibly involved in keeping or rendering vascular cells quiescent.

Alternative Vascularization Mechanisms in Tumor Resistance to Therapy.

Simple SummaryTumors rely on blood vessels to grow and metastasize. Malignant tumors can employ different strategies to create a functional vascular network. Tumor cells can use normal processes of vessel formation but can also employ cancer-specific mechanisms, by co-opting normal vessels present in tissues or by turning themselves into vascular cells. These different types of tumor vessels have specific molecular and functional characteristics that profoundly affect tumor behavior and response to therapies, including drugs targeting the tumor vasculature (antiangiogenic therapies). In this review, we discuss how vessels formed by different mechanisms affect the intrinsic sensitivity of tumors to therapy and, on the other hand, how therapies can affect tumor vessel formation, leading to resistance to drugs, cancer recurrence, and treatment failure. Potential strategies to avoid vessel-mediated resistance to antineoplastic therapies will be discussed.Blood vessels in tumors are formed through a variety of different mechanisms, each generating vessels with peculiar structural, molecular, and functional properties. This heterogeneity has a major impact on tumor response or resistance to antineoplastic therapies and is now emerging as a promising target for strategies to prevent drug resistance and improve the distribution and efficacy of antineoplastic treatments. This review presents evidence of how different mechanisms of tumor vessel formation (vasculogenesis, glomeruloid proliferation, intussusceptive angiogenesis, vasculogenic mimicry, and vessel co-option) affect tumor responses to antiangiogenic and antineoplastic therapies, but also how therapies can promote alternative mechanisms of vessel formation, contributing to tumor recurrence, malignant progression, and acquired drug resistance. We discuss the possibility of tailoring treatment strategies to overcome vasculature-mediated drug resistance or to improve drug distribution and efficacy.

Different Forms of Tumor Vascularization and Their Clinical Implications Focusing on Vessel Co-option in Colorectal Cancer Liver Metastases.

In modern anti-cancer therapy of metastatic colorectal cancer (mCRC) the anti-angiogenic treatment targeting sprouting angiogenesis is firmly established for more than a decade. However, its clinical benefits still remain limited. As liver metastases (LM) represent the most common metastatic site of colorectal cancer and affect approximately one-quarter of the patients diagnosed with this malignancy, its treatment is an essential aspect for patients' prognosis. Especially in the perioperative setting, the application of anti-angiogenic drugs represents a therapeutic option that may be used in case of high-risk or borderline resectable colorectal cancer liver metastases (CRCLM) in order to achieve secondary resectability. Regarding CRCLM, one reason for the limitations of anti-angiogenic treatment may be represented by vessel co-option (VCO), which is an alternative mechanism of blood supply that differs fundamentally from the well-known sprouting angiogenesis and occurs in a significant fraction of CRCLM. In this scenario, tumor cells hijack pre-existing mature vessels of the host organ independently from stimulating new vessels formation. This represents an escape mechanism from common anti-angiogenic anti-cancer treatments, as they primarily target the main trigger of sprouting angiogenesis, the vascular endothelial growth factor A. Moreover, the mechanism of blood supply in CRCLM can be deduced from their phenotypic histopathological growth pattern (HGP). For that, a specific guideline has already been implemented. These HGP vary not only regarding their blood supply, but also concerning their tumor microenvironment (TME), as notable differences in immune cell infiltration and desmoplastic reaction surrounding the CRCLM can be observed. The latter actually serves as one of the central criteria for the classification of the HGP. Regarding the clinically relevant effects of the HGP, it is still a topic of research whether the VCO-subgroup of CRCLM results in an impaired treatment response to anti-angiogenic treatment when compared to an angiogenic subgroup. However, it is well-proved, that VCO in CRCLM generally relates to an inferior survival compared to the angiogenic subgroup. Altogether the different types of blood supply result in a relevant influence on the patients' prognosis. This reinforces the need of an extended understanding of the underlying mechanisms of VCO in CRCLM with the aim to generate more comprehensive approaches which can target tumor vessels alternatively or even other components of the TME. This review aims to augment the current state of knowledge on VCO in CRCLM and other tumor entities and its impact on anti-angiogenic anti-cancer therapy.

Angiogenesis as a hallmark of solid tumors - clinical perspectives.

Angiogenesis is a key and early step in tumorigenesis, and is known as a hallmark of solidtumors and a key promoter of tumor recurrence. Unlike normal tissue vessels, the architecture of the tumor vasculature is abnormal, being leaky, tortuous, fragile and blind-ended. Perivascular cells are either detached or absent, causing reduction of vascular integrity, an increase in vessel immaturity, incoherent perfusion, defective functionality and enhanced tumor dissemination and metastasis. The abnormal tumor vasculature along with the defective tumor vessel functionality finally causes bouts of hypoxia and acidity in the tumor microenvironment (TME), further reinvigorating tumor aggression. Interstitial hypertension or high interstitial fluid pressure (IFP) is an outcome of tumor hyper-permeability. High IFP can be a barrier for either effective delivery of anti-cancer drugs toward the TME or accumulation of drugs within the tumor area, thus promoting tumor resistance to therapy. Some tumors do, however, not undergo angiogenesis but instead undergo vessel co-option or vascular mimicry, thereby adding another layer of complexity to cancerdevelopment and therapy. Combination of anti-angiogenesis therapy with chemotherapy and particularly with immune checkpoint inhibitors (ICIs) is a promising strategy for a number of advanced cancers. Among the various approaches for targeting tumor angiogenesis, vascular normalization is considered as the most desired method, which allows effective penetration of chemotherapeutics into the tumor area, thus being an appropriate adjuvantto other cancer modalities.

Efficient delivery of oncolytic enterovirus by carrier cell line NK-92

Many members of the enterovirus family are considered as promising oncolytic agents; however, their systemic administration is largely inefficient due to the rapid neutralization of the virus in the circulation and the barrier functions of the endothelium. We aimed to evaluate natural killer cells as carriers for the delivery of oncolytic enteroviruses, which would combine the effects of cell immunotherapy with virotherapy. We tested four strains of nonpathogenic enteroviruses against the glioblastoma cell line panel and evaluated the produced infectious titers. Next, we explored whether these virus strains could be delivered to the tumor by natural killer cell line NK-92, which is being actively evaluated as a clinically acceptable therapeutic. Several strains of enteroviruses demonstrated oncolytic properties, but only coxsackievirus A7 (CVA7) could replicate in NK-92 cells efficiently. We compared the delivery efficiency of CVA7 in vivo, using NK-92 cells and direct intravenous administration, and found significant advantages of cell delivery even after a single injection. This suggests that the NK-92 cell line can be utilized as a vehicle for the delivery of the oncolytic strain of CVA7, which would improve the clinical potential of this viral oncolytic for the treatment of glioblastoma multiforme and other forms of cancer.

Pathogenetic Features and Current Management of Glioblastoma.

Simple SummaryGlioblastoma (GBM) is the most common form of primary malignant brain tumor with a devastatingly poor prognosis. Tumor heterogeneity (cellular, molecular and immune) is the major obstacle to current treatment failure. We revisited the recent literature to understand the heterogeneous features of GBM and their potential role in treatment resistance. This review provides a comprehensive overview covering the GBM’s pathogenetic features, currently available treatment options and the treatments currently under development in the clinic.Glioblastoma (GBM) is the most common form of primary malignant brain tumor with a devastatingly poor prognosis. The disease does not discriminate, affecting adults and children of both sexes, and has an average overall survival of 12–15 months, despite advances in diagnosis and rigorous treatment with chemotherapy, radiation therapy, and surgical resection. In addition, most survivors will eventually experience tumor recurrence that only imparts survival of a few months. GBM is highly heterogenous, invasive, vascularized, and almost always inaccessible for treatment. Based on all these outstanding obstacles, there have been tremendous efforts to develop alternative treatment options that allow for more efficient targeting of the tumor including small molecule drugs and immunotherapies. A number of other strategies in development include therapies based on nanoparticles, light, extracellular vesicles, and micro-RNA, and vessel co-option. Advances in these potential approaches shed a promising outlook on the future of GBM treatment. In this review, we briefly discuss the current understanding of adult GBM’s pathogenetic features that promote treatment resistance. We also outline novel and promising targeted agents currently under development for GBM patients during the last few years with their current clinical status.

Fishing for Contact: Modeling Perivascular Glioma Invasion in the Zebrafish Brain.

Glioblastoma multiforme (GBM) is a highly invasive, central nervous system (CNS) cancer for which there is no cure. Invading tumor cells evade treatment, limiting the efficacy of the current standard of care regimen. Understanding the underlying invasive behaviors that support tumor growth may allow for generation of novel GBM therapies. Zebrafish (Danio rerio) are attractive for genetics and live imaging and have, in recent years, emerged as a model system suitable for cancer biology research. While other groups have studied CNS tumors using zebrafish, few have concentrated on the invasive behaviors supporting the development of these diseases. Previous studies demonstrated that one of the main mechanisms of GBM invasion is perivascular invasion, i.e., single tumor cell migration along blood vessels. Here, we characterize phenotypes, methodology, and potential therapeutic avenues for utilizing zebrafish to model perivascular GBM invasion. Using patient-derived xenolines or an adherent cell line, we demonstrate tumor expansion within the zebrafish brain. Within 24-h postintracranial injection, D54-MG-tdTomato glioma cells produce fingerlike projections along the zebrafish brain vasculature. As few as 25 GBM cells were sufficient to promote single cell vessel co-option. Of note, these tumor-vessel interactions are CNS specific and do not occur on pre-existing blood vessels when injected into the animal's peripheral tissue. Tumor-vessel interactions increase over time and can be pharmacologically disrupted through inhibition of Wnt signaling. Therefore, zebrafish serve as a favorable model system to study perivascular glioma invasion, one of the deadly characteristics that make GBM so difficult to treat.

Resistance Mechanisms of Anti-angiogenic Therapy and Exosomes-Mediated Revascularization in Cancer.

Anti-angiogenic therapies (AATs) have been widely used for cancer treatment. But the beneficial effects of AATs are short, because AAT-induced tumor revascularization facilitates the tumor relapse. In this mini-review, we described different forms of tumor neovascularization and revascularization including sprouting angiogenesis, vessel co-option, intussusceptive angiogenesis, and vasculogenic mimicry, all of which are closely mediated by vascular endothelial growth factor (VEGF), angiopoietins, matrix metalloproteinases, and exosomes. We also summarized the current findings for the resistance mechanisms of AATs including enhancement in pro-angiogenic cytokines, heterogeneity in tumor-associated endothelial cells (ECs), crosstalk between tumor cells and ECs, masking of extracellular vesicles, matrix stiffness and contributions from fibroblasts, macrophages and adipocytes in the tumor microenvironment. We highlighted the revascularization following AATs, particularly the role of exosome stimulating factors such as hypoxia and miRNA, and that of exosomal cargos such as cytokines, miRNAs, lncRNAs, and circRNAs from the tumor ECs in angiogenesis and revascularization. Finally, we proposed that renormalization of tumor ECs would be a more efficient cancer therapy than the current AATs.

MicroRNAs Biogenesis, Functions and Role in Tumor Angiogenesis.

microRNAs (miRNAs) are small non-coding RNA molecules, evolutionary conserved. They target more than one mRNAs, thus influencing multiple molecular pathways, but also mRNAs may bind to a variety of miRNAs, either simultaneously or in a context-dependent manner. miRNAs biogenesis, including miRNA transcription, processing by Drosha and Dicer, transportation, RISC biding, and miRNA decay, are finely controlled in space and time.miRNAs are critical regulators in various biological processes, such as differentiation, proliferation, apoptosis, and development in both health and disease. Their dysregulation is involved in tumor initiation and progression. In tumors, they can act as onco-miRNAs or oncosuppressor-miRNA participating in distinct cellular pathways, and the same miRNA can perform both activities depending on the context.In tumor progression, the angiogenic switch is fundamental. miRNAs derived from tumor cells, endothelial cells, and cells of the surrounding microenvironment regulate tumor angiogenesis, acting as pro-angiomiR or anti-angiomiR.In this review, we described miRNA biogenesis and function, and we update the non-classical aspects of them. The most recent role in the nucleus, as transcriptional gene regulators and the different mechanisms by which they could be dysregulated, in tumor initiation and progression, are treated. In particular, we describe the role of miRNAs in sprouting angiogenesis, vessel co-option, and vasculogenic mimicry. The role of miRNAs in lymphoma angiogenesis is also discussed despite the scarcity of data.The information presented in this review reveals the need to do much more to discover the complete miRNA network regulating angiogenesis, not only using high-throughput computational analysis approaches but also morphological ones.

Vascular Heterogeneity With a Special Focus on the Hepatic Microenvironment.

Utilizing single-cell sequencing, recent studies were able to analyze at a greater resolution the heterogeneity of the vasculature and its complex composition in different tissues. Differing subpopulations have been detected, distinguishable only by their transcriptome. Designed to provide further insight into the heterogeneity of the functional vascular tissue, endothelial cells have been the main target of those studies. This review aims to present a synopsis of the variability of the different vascular beds, their endothelial variety, and the supporting cells that allow the vessels to serve their various purposes. Firstly, we are going to chart vascular tissue heterogeneity on a cellular level, describing endothelial diversity as well as stromal microenvironmental variety and interaction in a physiological setting. Secondly, we will summarize the current knowledge of pathological vessel formation in the context of cancer. Conventional anti-tumor therapeutic targets as well as anti-angiogenetic therapy is frequently limited by poor response of the tumor tissue. Reasons for moderate response and resistance to treatment can be found through different drivers of angiogenesis, different mechanisms of blood supply, but also in poorly understood tissue diversity. Based on this, we are comparing how pathologies alter the normal structure of vascular tissues highlighting the involved mechanisms. Lastly, illustrating the concept above, we will focus on the hepatic microenvironment, an organ of frequent metastatic spreading (e.g., from colorectal, breast, and lung cancers). We will address how the hepatic vasculature usually develops and subsequently we will describe how common liver metastases vary in their vasculature and the way they supply themselves (e.g., angiogenesis versus vessel co-option).

Angiogenesis and vessel co-option in a mathematical model of diffusive tumor growth: The role of chemotaxis

This work considers the propagation of a tumor from the stage of a small avascular sphere in a host tissue and the progressive onset of a tumor neovasculature stimulated by a pro-angiogenic factor secreted by hypoxic cells. The way new vessels are formed involves cell sprouting from pre-existing vessels and following a trail via a chemotactic mechanism (CM). Namely, it is first proposed a detailed general family of models of the CM, based on a statistical mechanics approach. The key hypothesis is that the CM is composed by two components: i) the well–known bias induced by the angiogenic factor gradient; ii) the presence of stochastic changes of the velocity direction, thus giving rise to a diffusive component. Then, some further assumptions and simplifications are applied in order to derive a specific model to be used in the simulations. The tumor progression is favored by its acidic aggression towards the healthy cells. The model includes the evolution of many biological and chemical species. Numerical simulations show the onset of a traveling wave eventually replacing the host tissue with a fully vascularized tumor. The results of simulations agree with experimental measures of the vasculature density in tumors, even in the case of particularly hypoxic tumors.

Tumor microenvironment conditions that favor vessel co-option in colorectal cancer liver metastases: A theoretical model.

Vessel co-option is an alternative strategy by which tumour cells vascularize and gain access to nutrients to support tumour growth, survival and metastasis. In vessel co-option, the cancer cells move towards the pre-existing vasculature and hijack them. Vessel co-option is adopted by a wide range of human tumours including colorectal cancer liver metastases (CRCLM) and is responsible for the effectiveness of treatment in CRCLM. Furthermore, vessel co-option is an intrinsic feature and an acquired mechanism of resistance to anti-angiogenic treatment. In this review, we describe the microenvironment, the molecular players, discovered thus far of co-opting CRCLM lesions and propose a theoretical model. We also highlight key unanswered questions that are critical to improving our understanding of CRCLM vessel co-option and for the development of effective approaches for the treatment of co-opting tumours.

Abstract 3935: Runt related transcription factor-1 (runx1) plays a central role in vessel co-option of colorectal cancer liver metastases

Abstract Colorectal cancer liver metastasis (CRCLM) has two major morphological growth pattern subtypes including desmoplastic (DHGP) and replacement (RHGP). The DHGP tumors depend on angiogenesis for their growth. Conversely, RHGP tumors are non-angiogenic and obtain their blood supply through vessel co-option, which the cancer cells hijacking pre-existing blood vessels of the surrounding liver tissue. Therefore, anti-angiogenic therapies have shown modest efficacy in CRCLM patients with RHGP lesions. In this study, we found overexpression of Runt Related Transcription Factor-1 (RUNX1) in the cancer cells of RHGP lesions. Upregulation of RUNX1 is positively correlated with cancer cell motility and epithelial mesenchymal transition (EMT) in vitro. Our results also showed that RUNX1 regulated by Transforming Growth Factor Beta-1 (TGFβ1) through TGFβ Receptor II (TGFβRII). Significantly, RUNX1 knockdown induced conversion RHGP lesions to DHGP in vivo. Collectively, this study suggests RUNX1 as a potential target therapy to overcome resistance to anti-angiogenic therapy in CRCLM. Citation Format: Miran Rada, Audrey Kapelanski-Lamoureux, Stephanie Petrillo, Sébastien Tabariès, Peter Siegel, Anthoula Lazaris, Peter Metrakos. Runt related transcription factor-1 (runx1) plays a central role in vessel co-option of colorectal cancer liver metastases [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3935.

Abstract 3331: Role of innate immune cells in the development of vessel co-opting CRC liver metastases

Abstract Approximatively 50 % of patients who undergo resection for primary colorectal cancer will develop liver metastases. Liver metastases present as two major histopathological growth patterns (HGP) the desmoplastic and the replacement HGP. In the desmoplastic HGP, the cancer cells are surrounded by a desmoplastic stromal ring preventing their contact with the normal liver. In the replacement HGP, the cancer cells infiltrate the normal liver and replace hepatocytes near the tumour periphery. We have found that the desmoplastic HGP tumors derive their blood supply through angiogenesis while the replacement HGP tumors are non-angiogenic and co-opt the blood vessel of the liver they invade. Furthermore, we have published that patients under chemotherapy presenting with a vessel co-opting phenotype have a worse pathological response and five-year overall survival compared to patients presenting with angiogenic liver metastases. Their prognosis is worse when treated with bevacizumab an anti-VEGF antibody used alongside chemotherapy. CRCLM are considered immunologically cold lesions, with low numbers of infiltrating T-cells, and thought to be unlikely to benefit from immunotherapies. We have recently found that neutrophils and macrophages but not lymphocytes infiltrate vessel co-opting lesions to a greater extent than angiogenic lesions. Using flow cytometry and the Nanostring GeoMx spatial protein profiling platform capable of detecting up to 96 proteins simultaneously on FFPE tissue slices, we established the immunoprofile of immune cells recruited to angiogenic or vessel co-opting CRCLM lesions in chemonaive and treated patients. Many studies have demonstrated that neutrophils and macrophages play an important role in tumor progression, modification of the ECM, angiogenesis, and immunosuppression. We hypothesize that innate immune cells can facilitate blood vessel co-option of CRCLM lesions and contribute to an immunosupressive environment favorable to tumor growth. Fully understanding the role of the innate immune system in the developmend of vessel co-opting CRCLM tumors could allow for the development of immunotherapies capable of converting vessel co-opting lesions into angiogenic lesions that are more successfully treated with chemotherapy and anti-angiogenic therapies. Citation Format: Thomas Z. Mayer, Diane H. Kim, Miran Rada, Stephanie Petrillo, Anthoula Lazaris, Peter Metrakos. Role of innate immune cells in the development of vessel co-opting CRC liver metastases [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3331.

Abstract P3-01-13: Association between the histopathological growth patterns (HGP) of liver metastases (LM) and survival after hepatic surgery in patients with oligometastatic breast cancer (BC)

Abstract Background. Liver is the 3rd most common site for metastatic disease in BC. The proportion of patients with BC LM presenting with so-called oligometastatic disease is estimated to be ~10%. Some of these patients have an indolent disease course after hepatic surgery. However, as of today, it is impossible to identify the ideal candidates for hepatic surgery based on standard clinico-pathological characteristics. Two main HGP have previously been identified in LM from colorectal cancer patients1: (i) the desmoplastic HGP with angiogenesis, inflammation and a rim of fibrous tissue that separates the cancer cells from the liver parenchyma, and (ii) the replacement HGP, with vessel co-option, no angiogenesis, and cancer cells growing into the liver parenchyma while replacing the hepatocytes, with minimal inflammation. This replacement HGP was associated with worse survival and lack of benefit from bevacizumab in patients operated for colorectal LM2. Here, we aimed at characterizing HGP of LM from BC patients who underwent hepatic surgery. Patients and methods. We considered a series of 36 consecutive patients with invasive BC from Institut Jules Bordet and Hôpital Erasme, Brussels, who underwent surgical resection of LM. Patients were operated between April 2000 and October 2017, and the median follow-up was 10.7 years. The HGPs of resected LM were scored according to international guidelines on H&E stained section by light microscopy3, by a pathologist (PV) blinded to outcome data. All sections of all LM were evaluated for each patient. Associations between HGP and clinico-pathological characteristics were assessed using the Fisher exact test, and associations with progression-free (PFS) and overall survival (OS) using Cox proportional hazard regressions considering date of hepatic surgery as the starting time. Age at hepatic surgery, ER and HER2 status of the LM, and presence of extra-hepatic metastasi(e)s were considered as adjustment variables and centre as stratification factor. Results. In 16 patients (44%) the LM exclusively presented the replacement HGP (pure-replacement group) while in the remaining 20 patients (56%) the LM were at least partly desmoplastic (any-desmoplastic group). There was no association of HGP with ER or HER2 status of primary BC or LM. LM subtypes were as follows: 2 ER-/HER2- (7%), 11 HER2+ (38%), 16 ER+/HER2- (55%)- information is currently missing for 7 patients. In 10/30 (33.3%) patients, ER-status changed in the LM compared with the primary BC (8 losses, 2 gains). Significantly more patients in the any-desmoplastic group had a primary BC without lymph node metastases (56% versus 21% in the replacement group, p=.02). Survival analyses showed that any-desmoplastic HGP was independently associated with increased PFS after liver surgery when compared with pure-replacement HGP (Hazard Ratio, HR, 0.34; 95% CI: 0.12-0.96; p=.041). All patients with pure-replacement HGP relapsed within the first two years after hepatic surgery. The other factors in the model were not significantly associated with PFS. Similar results were observed for OS (adjusted HR for HGP: 0.26; 0.07-1.01, p=.052). Conclusion. In this study, we showed evidence that the pure-replacement HGP of BC LM is related to worse outcome after hepatic surgery, corroborating our findings in patients with colorectal LM1. These findings, which are currently being validated in additional series, suggest that HGP of LM could represent a promising candidate marker of oligometastatic progression in BC. Refs: 1. doi:10.1007/s10456-019-09661-5; 2. doi:10.1038/s41571-019-0181-9; 3. doi:10.1038/bjc.2017.334. Citation Format: Peter B Vermeulen, Ali Bohlok, Sophia Leduc, François Richard, Lara Botzenhart, Michail Ignatiadis, Philippe Aftimos, Christos Sotiriou, Martine Piccart, Alain Hendlisz, Steven Van Laere, Luc Y Dirix, Jean-Christophe Noël, Valerio Lucidi, Elia Biganzoli, Denis Larsimont, Christine Desmedt, Vincent Donckier. Association between the histopathological growth patterns (HGP) of liver metastases (LM) and survival after hepatic surgery in patients with oligometastatic breast cancer (BC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-13.

Bevacizumab treatment of meningeal melanoma metastases

BackgroundMelanoma patients with metastatic growth in the meninges have poor prognosis and few treatment options. Although treatment with BRAF inhibitors or immune checkpoint inhibitors has provided promising results, most patients with advanced melanoma are resistant to these treatments and develop severe side effects. Novel treatment strategies are needed for patients with meningeal melanoma metastases, and the potential of antiangiogenic therapy was investigated in this preclinical study.MethodsTwo GFP-transfected melanoma models (A-07 and D-12) differing substantially in VEGF-A expression were included in the study, and the anti-VEGF-A antibody bevacizumab was used as therapeutic agent. Meningeal metastases were initiated in BALB/c nu/nu mice by intracranial inoculation of melanoma cells, and bevacizumab treatment was given twice a week in i.p. doses of 10 mg/kg until the mice became moribund. Therapeutic effects were evaluated by determining tumor host survival time, assessing tumor growth and angiogenic activity by quantitative analyses of histological preparations, and measuring the expression of angiogenesis-related genes by quantitative PCR.ResultsMeningeal A-07 melanomas showed higher expression of VEGF-A than meningeal D-12 melanomas, whereas the expression of ANGPT2 and IL8, two important angiogenesis drivers in melanoma, was much higher in D-12 than in A-07 tumors. Bevacizumab treatment inhibited tumor angiogenesis and prolonged host survival in mice with A-07 tumors but not in mice with D-12 tumors. Meningeal A-07 tumors in bevacizumab-treated mice compensated for the reduced VEGF-A activity by up-regulating a large number of angiogenesis-related genes, including ANGPT2 and its receptors TIE1 and TIE2. Melanoma cells migrated from meningeal tumors into the cerebrum, where they initiated metastatic growth by vessel co-option. In the A-07 model, the density of cerebral micrometastases was higher in bevacizumab-treated than in untreated mice, either because bevacizumab treatment increased mouse survival or induced increased tumor gene expression.ConclusionsThe development of antiangiogenic strategies for the treatment of meningeal melanoma metastases is a challenging task because the outcome of treatment will depend on the angiogenic signature of the tumor tissue, treatment-induced alterations of the angiogenic signature, and the treatment sensitivity of metastatic lesions in other intracranial sites.

Three-Dimensional Histologic, Immunohistochemical, and Multiplex Immunofluorescence Analyses of Dynamic Vessel Co-Option of Spread Through Air Spaces in Lung Adenocarcinoma.

Spread through air spaces (STAS) is a method of invasion in lung adenocarcinoma and is associated with tumor recurrence and poor survival. The spatial orientation of STAS cells in the lung alveolar parenchyma is not known. The aim of this study was to use high-resolution and high-quality three-dimensional (3D) reconstruction of images from immunohistochemical (IHC) and multiplex immunofluorescence (IF) experiments to understand the spatial architecture of tumor cell clusters by STAS in the lung parenchyma. Four lung adenocarcinomas, three micropapillary-predominant and one solid predominant adenocarcinoma subtypes, were investigated. A 3D reconstruction image was created from formalin-fixed, paraffin-embedded blocks. A total of 350 serial sections were obtained and subjected to hematoxylin and eosin (100 slides), IHC (200 slides), and multiplex IF staining (50 slides) with the following antibodies: cluster of differentiation 31, collagen type IV, thyroid transcription factor-1, and E-cadherin. Whole slide images were reconstructed into 3D images for evaluation. Serial 3D image analysis by hematoxylin and eosin, IHC, and IF staining revealed that the micropapillary clusters and solid nests of STAS are focally attached to the alveolar walls, away from the main tumor. Our 3D reconstructions found that STAS tumor cells can attach to the alveolar walls rather than appearing free floating, as seen on the two-dimensional sections. This suggests that the tumor cells detach from the main tumor, migrate through air spaces, and reattach to the alveolar walls through vessel co-option, allowing them to survive and grow. This may explain the higher recurrence rate and worse survival of patients with STAS-positive tumors who undergo limited resection than those who undergo lobectomy.

Vessel co-option and resistance to anti-angiogenic therapy.

Vessel co-option is a non-angiogenic mechanism of tumour vascularisation in which cancer cells utilise pre-existing blood vessels instead of inducing new blood vessel formation. Vessel co-option has been observed across a range of different tumour types, in both primary cancers and metastatic disease. Importantly, vessel co-option is now implicated as a major mechanism that mediates resistance to conventional anti-angiogenic drugs and this may help to explain the limited efficacy of this therapeutic approach in certain clinical settings. This includes the use of anti-angiogenic drugs to treat advanced-stage/metastaticdisease, treatment in the adjuvant setting and the treatment of primary disease. In this article, we review the available evidence linking vessel co-option with resistance to anti-angiogenic therapy in numerous tumour types, including breast, colorectal, lung and pancreatic cancer, glioblastoma, melanoma, hepatocellular carcinoma, and renal cell carcinoma. The finding that vessel co-option is a significant mechanism of resistance to anti-angiogenic therapy may have important implications for the future of anti-cancer therapy, including (a) predicting response to anti-angiogenic drugs, (b) the need to develop therapies that target both angiogenesis and vessel co-option in tumours, and (c) predicting the response to other therapeutic modalities, including immunotherapy.