Alterations in the tumor microenvironment not only modulate cancer cell progression and invasiveness but also affect the viscoelastic properties of immune cells therein.

It is well established that reciprocal communication between cancer cells and other cells in the tumor microenvironment plays a crucial role in cancer progression and therapy response. There are multiple ways by which cells communicate, including direct cell-cell contact and the secretion of soluble mediators. The secretome of cancer cells contains valuable information to disentangle the complex conversation that is happening between cancer cells and neighboring or distant cells such as immune cells, fibroblasts and endothelial cells. Here, we provide a workflow of mapping the cancer cell secretome in an unbiased way using amino acid-analog labeling in combination with mass spectrometry. The generation of single cells from fresh tumors, isolation of primary cancer cells from a complex multi-cellular pool, and the detection of newly synthesized proteins that are secreted into the medium is described in detailed protocols. Using this experimental pipeline the secretome of cancer cells across different tumors can be determined, paving the way to unravel cell-cell communication networks in the tumor microenvironment, which may uncover novel therapeutic targets.

Abstract Immunotherapy has been far less effective in breast cancer compared to other cancer types, with only a small fraction of patients having clinical benefit. Historically, invasive lobular carcinomas (ILC), the second most common histological subtype of breast cancer, have been categorized as immunological cold tumors, but recent studies suggest that ILCs may recruit a myeloid-rich immunosuppressive immune landscape. Cancer cell intrinsic features such as the genetic makeup of tumor cells can dictate the spatial distribution, composition and activation state of immune cells, which can exert both anti-tumor and pro-tumor activities and affect disease progression and immunotherapy response. Genetic aberration in key genes of the phosphoinositide-3 kinase (PI3K) signaling pathway are observed in >50% of primary ILCs. The overall aim of this project is to determine whether genetic aberrations in key genes of the PI3K pathway regulate the composition and functional state of the immune landscape, and whether this influences the clinical benefit of immunomodulatory strategies in ILC patients. To dissect the immune regulatory mechanisms of distinct PI3K alterations we have used transgenic ILC mouse models, in which immune profiling studies by flow cytometry and immunohistochemistry revealed that distinct PI3K alterations in cancer cells differentially affect the tumor immune landscape. With some mutations driving a myeloid-rich tumor microenvironment, whereas the immune landscape of tumors harboring other PI3K mutations is dominated by lymphoid cells. To understand the observed cancer genotype-immunophenotypes mechanistically, we have performed single-cell RNA sequencing and secretome analysis to assess cellular communication between the cancer cells and the immune system. We identified key differences in ligand-receptor interactions and secreted signaling that can explain the influx of different immunosuppressive immune cell types in the tumor microenvironment of murine ILCs. We are currently investigating whether therapeutic targeting of immunosuppression is a feasible strategy for PI3K-altered ILC tumors with a tumor-promoting immune landscape. Shedding light on the mechanisms that govern the diversity and function of immune cells in the tumor microenvironment will provide a greater understanding of interpatient heterogeneity, and will pave the way for the development of novel and more tailored immunomodulatory strategies for ILC patients. Citation Format: Antoinette van Weverwijk, Michael Schubert, Joseph Siefert, Martine van Miltenburg, Kim Vrijland, Julia Houthuijzen, Anne Stunnenberg, Cheei-Sing Hau, Lodewyk Wessels, Jos Jonkers, Karin E de Visser. Dissecting the immune regulatory mechanisms of distinct PI3K alterations in invasive lobular carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A052.

PDF file 325K, Additional validation of ST6GalNAc2 as a metastasis suppressor including in a spontaneous metastasis assay using the parental 4T1 cells

PDF file 299K, Additional characterization of human breast cancer cell lines; immunofluorescence analysis and lung retention assays

Tumours display an astonishing variation in the spatial distribution, composition and activation state of immune cells, which impacts their progression and response to immunotherapy. Shedding light on the mechanisms that govern the diversity and function of immune cells in the tumour microenvironment will pave the way for the development of more tailored immunomodulatory strategies for the benefit of patients with cancer. Cancer cells, by virtue of their paracrine and juxtacrine communication mechanisms, are key contributors to intertumour heterogeneity in immune contextures. In this Review, we discuss how cancer cell-intrinsic features, including (epi)genetic aberrations, signalling pathway deregulation and altered metabolism, play a key role in orchestrating the composition and functional state of the immune landscape, and influence the therapeutic benefit of immunomodulatory strategies. Moreover, we highlight how targeting cancer cell-intrinsic parameters or their downstream immunoregulatory pathways is a viable strategy to manipulate the tumour immune milieu in favour of antitumour immunity.

Supplementary Tables (Tables S1 - S6) and Supplementary Figures (Figures S1 - S7)

PDF file 149K, In vivo validation of 3 hits identified in the screen (Mre11a, Fen1, Wwc1)

PDF file 174K, Additional characterization of the 4T1-Luc cells in lung retention assays

PDF file 107K, Inventory of supplementary material. Supplementary Table S1 listing primers and siRNAs. Legends for Supplementary Fig. S1 - S7 Legends for Supplementary Movies S1 and S2 Supplementary Figures S1 - S6 legends Supplementary Movies S1 and S2 legends

MOV file 2886K, Video showing dynamic flow adhesion assays of ZR75.1 shNTC and shST6 cells on a HUVEC monolayer in the presence of the galectin-3 inhibitor GCS-100

PDF file 60K, Characterization of the MDA-MB-231 cells stably expressing vector or mouse ST6GalNAc2

PDF file 95K, Data demonstrating that siRNA mediated downregulation of ST6GalNAc2 expression in human and mouse cell lines promotes tumor cell aggregation in vivo

Breast cancer is accompanied by systemic immunosuppression, which facilitates metastasis formation, but how this shapes organotropism of metastasis is poorly understood. Here, we investigate the impact of mammary tumorigenesis on regulatory T cells (Tregs) in distant organs and how this affects multi-organ metastatic disease. Using a preclinical mouse mammary tumor model that recapitulates human metastatic breast cancer, we observe systemic accumulation of activated, highly immunosuppressive Tregs during primary tumor growth. Tumor-educated Tregs show tissue-specific transcriptional rewiring in response to mammary tumorigenesis. This has functional consequences for organotropism of metastasis, as Treg depletion reduces metastasis to tumor-draining lymph nodes, but not to lungs. Mechanistically, we find that Tregs control natural killer (NK) cell activation in lymph nodes, thereby facilitating lymph node metastasis. In line, an increased Treg/NK cell ratio is observed in sentinel lymph nodes of breast cancer patients compared with healthy controls. This study highlights that immune regulation of metastatic disease is highly organ dependent.

Profiling studies have revealed considerable phenotypic heterogeneity in cancer-associated fibroblasts (CAFs) present within the tumour microenvironment, however, functional characterisation of different CAF subsets is hampered by the lack of specific markers defining these populations. Here we show that genetic deletion of the Endo180 ( MRC2 ) receptor, predominantly expressed by a population of matrix-remodelling CAFs, profoundly limits tumour growth and metastasis; effects that can be recapitulated in 3D co-culture assays. This impairment results from a CAF-intrinsic contractility defect and reduced CAF viability which, coupled with the lack of phenotype in the normal mouse, demonstrates that upregulated Endo180 expression by a specific, potentially targetable CAF subset is required to generate a supportive tumour microenvironment. Further, characterisation of a tumour subline selected via serial in vivo passage for its ability to overcome these stromal defects provides important insight into how tumour cells adapt to a non-activated stroma in the early stages of metastatic colonisation.

Oncogenic transformation is associated with profound changes in cellular metabolism, but whether tracking these can improve disease stratification or influence therapy decision-making is largely unknown. Using the iKnife to sample the aerosol of cauterized specimens, we demonstrate a new mode of real-time diagnosis, coupling metabolic phenotype to mutant PIK3CA genotype. Oncogenic PIK3CA results in an increase in arachidonic acid and a concomitant overproduction of eicosanoids, acting to promote cell proliferation beyond a cell-autonomous manner. Mechanistically, mutant PIK3CA drives a multimodal signaling network involving mTORC2-PKCζ-mediated activation of the calcium-dependent phospholipase A2 (cPLA2). Notably, inhibiting cPLA2 synergizes with fatty acid-free diet to restore immunogenicity and selectively reduce mutant PIK3CA-induced tumorigenicity. Besides highlighting the potential for metabolic phenotyping in stratified medicine, this study reveals an important role for activated PI3K signaling in regulating arachidonic acid metabolism, uncovering a targetable metabolic vulnerability that largely depends on dietary fat restriction. VIDEO ABSTRACT.

BackgroundDissemination of breast cancers to the brain is associated with poor patient outcome and limited therapeutic options. In this study we sought to identify novel regulators of brain metastasis by profiling mouse mammary carcinoma cells spontaneously metastasising from the primary tumour in an immunocompetent syngeneic host.Methods4T1 mouse mammary carcinoma sublines derived from primary tumours and spontaneous brain and lung metastases in BALB/c mice were subject to genome-wide expression profiling. Two differentially expressed genes, Id2 and Aldh3a1, were validated in in-vivo models using mouse and human cancer cell lines. Clinical relevance was investigated in datasets of breast cancer patients with regards to distant metastasis-free survival and brain metastasis relapse-free survival. The role of bone morphogenetic protein (BMP)7 in regulating Id2 expression and promoting cell survival was investigated in two-dimensional and three-dimensional in-vitro assays.ResultsIn the spontaneous metastasis model, expression of Id2 and Aldh3a1 was significantly higher in 4T1 brain-derived sublines compared with sublines from lung metastases or primary tumour. Downregulation of expression impairs the ability of cells to colonise the brain parenchyma whereas ectopic expression in 4T1 and human MDA-MB-231 cells promotes dissemination to the brain following intracardiac inoculation but has no impact on the efficiency of lung colonisation. Both genes are highly expressed in oestrogen receptor (ER)-negative breast cancers and, within this poor prognosis sub-group, increased expression correlates with reduced distant metastasis-free survival. ID2 expression also associates with reduced brain metastasis relapse-free survival. Mechanistically, BMP7, which is present at significantly higher levels in brain tissue compared with the lungs, upregulates ID2 expression and, after BMP7 withdrawal, this elevated expression is retained. Finally, we demonstrate that either ectopic expression of ID2 or BMP7-induced ID2 expression protects tumour cells from anoikis.ConclusionsThis study identifies ID2 as a key regulator of breast cancer metastasis to the brain. Our data support a model in which breast cancer cells that have disseminated to the brain upregulate ID2 expression in response to astrocyte-secreted BMP7 and this serves to support metastatic expansion. Moreover, elevated ID2 expression identifies breast cancer patients at increased risk of developing metastatic relapse in the brain.

The different stages of the metastatic cascade present distinct metabolic challenges to tumour cells and an altered tumour metabolism associated with successful metastatic colonisation provides a therapeutic vulnerability in disseminated disease. We identify the aldo-keto reductase AKR1B10 as a metastasis enhancer that has little impact on primary tumour growth or dissemination but promotes effective tumour growth in secondary sites and, in human disease, is associated with an increased risk of distant metastatic relapse. AKR1B10 High tumour cells have reduced glycolytic capacity and dependency on glucose as fuel source but increased utilisation of fatty acid oxidation. Conversely, in both 3D tumour spheroid assays and in vivo metastasis assays, inhibition of fatty acid oxidation blocks AKR1B10 High -enhanced metastatic colonisation with no impact on AKR1B10 Low cells. Finally, mechanistic analysis supports a model in which AKR1B10 serves to limit the toxic side effects of oxidative stress thereby sustaining fatty acid oxidation in metabolically challenging metastatic environments.

Recent studies have revealed a role for macrophages and neutrophils in limiting chemotherapy efficacy; however, the mechanisms underlying the therapeutic benefit of myeloid-targeting agents in combination with chemotherapy are incompletely understood. Here, we show that targeting tumour-associated macrophages by colony-stimulating factor-1 receptor (CSF-1R) blockade in the K14cre;Cdh1F/F;Trp53F/F transgenic mouse model for breast cancer stimulates intratumoural type I interferon (IFN) signalling, which enhances the anticancer efficacy of platinum-based chemotherapeutics. Notably, anti-CSF-1R treatment also increased intratumoural expression of type I IFN-stimulated genes in patients with cancer, confirming that CSF-1R blockade is a powerful strategy to trigger an intratumoural type I IFN response. By inducing an inflamed, type I IFN-enriched tumour microenvironment and by further targeting immunosuppressive neutrophils during cisplatin therapy, antitumour immunity was activated in this poorly immunogenic breast cancer mouse model. These data illustrate the importance of breaching multiple layers of immunosuppression during cytotoxic therapy to successfully engage antitumour immunity in breast cancer.