in-vivo-imaging-to-measure-spontaneous-lung-metastasis-orthotopically

<em>In Vivo</em> Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple ...

Virginia Commonwealth University School of Medicine

Regulatory T (Treg) cells reside in lymphoid organs and barrier tissues where they control different types of inflammatory responses. Treg cells are also found in human cancers, and studies in animal models suggest that they contribute to cancer progression. However, properties of human intratumoral Treg cells and those present in corresponding normal tissue remain largely unknown. Here, we analyzed features of Treg cells in untreated human breast carcinomas, normal mammary gland, and peripheral blood. Tumor-resident Treg cells were potently suppressive and their gene-expression pattern resembled that of normal breast tissue, but not of activated peripheral blood Treg cells. Nevertheless, a number of cytokine and chemokine receptor genes, most notably CCR8, were upregulated in tumor-resident Treg cells in comparison to normal tissue-resident ones. Our studies suggest that targeting CCR8 for the depletion of tumor-resident Treg cells might represent a promising immunotherapeutic approach for the treatment of breast cancer.

Regulatory T Cells Exhibit Distinct Features in Human Breast Cancer.

Characterization of individual cell populations from the tumor microenvironment is critical to understand their functional contribution to tumor progression. Magnetic bead enrichment and fluorescence-activated cell sorting (FACS) allow for the isolation of specific cell types that can be used in downstream applications, including in vitro and in vivo functional studies and molecular profiling. In this chapter, we describe the process of isolation of tumor-associated macrophages (TAMs) from primary murine breast tumors subsequent to therapeutic or experimental intervention. Additionally, we further detail how to analyze their ability to support tumor cell growth by co-injecting isolated TAMs with tumor cells orthotopically into the mammary gland of immune-deficient hosts, and monitoring tumor progression by live imaging and caliper measurement.

Tumor-Associated Macrophage Isolation and In Vivo Analysis of Their Tumor-Promoting Activity.

Ductal carcinoma  (DCIS) is a non-obligate precursor of breast cancer, and it only progresses to invasive breast cancer in around 40% of patients. While immune infiltrates have been observed in these early cancer lesions, their potential prognostic value is still unclear. Regulatory T (Treg) cells accumulate in advanced breast cancers, and predict poor outcome. We have shown before that ablation of Treg cells in established tumors leads to significant decrease in primary and metastatic tumor burden. In this work, we sought to investigate Treg cell function in the progression from non-invasive to invasive breast cancer lesions. To this end, we used the murine mammary tumor virus polyoma middle T (MMTV-PyMT) murine model of spontaneous, stage-wise breast carcinogenesis crossed to Foxp3  knock in mice, allowing Treg cell ablation by administration of diphtheria toxin. Transient targeting of Treg cells at the  carcinoma stage resulted in a significant increase in the number of tumor-bearing mammary glands and size of growing tumors compared with control mice. Whole mammary gland mounts and histological examination confirmed larger emergent tumor area in Treg cell-ablated mice, and revealed that these tumors were characterized by a more advanced tumor staging, with presence of early invasion, increased desmoplasia and collagen deposition. Furthermore, Treg cell ablation increased the percentage of cancer stem/progenitor cells in the mammary compartment. Interestingly, Treg cell ablation resulted in increased inflammatory cytokines IL-4 and IL-5 with a concomitant reduction in classically activated tumor associated macrophages. This TH2-biased immune regulatory mammary inflammation was consistent with the enhancement in tumor promotion that we observed. Overall, our study demonstrates that Treg cells oppose breast cancer progression at early stages, raising a cautionary note regarding the consideration of immune intervention targeted at boosting immune responses for DCIS.

Regulatory T Cells Control the Switch From  to Invasive Breast Cancer.

Microglia are the resident macrophages in the central nervous system (CNS), and they constitute 15-20% of the total glial populations. They have wide developmental and protective functions during brain injury, infection and tumorigenesis. Originally thought to derive from postnatal hematopoietic progenitors, it has recently been demonstrated that microglia originate from primitive myeloid progenitor cells that arise during early development from the embryonic yolk sac. Circulating monocytes infiltrate the CNS upon inflammatory conditions, such as cancer, primarily differentiating into macrophages and dendritic cells. Both resident and recruited microglia respond to environmental cues and actively participate in pathogenic processes, albeit their transcriptomic profiles contain significant differences suggesting distinctive roles. Metastatic brain tumors are the most common intracranial neoplasm in adults, with an estimate incidence 10 times higher than all primary brain neoplasms combined, and with dismal prognosis. Microglia is a major immune population associated with brain metastatic tumors in patients. They are proposed to play multiple, and sometimes opposing roles, in tumor progression. However, our ability to evaluate individual contribution of resident and recruited populations is hindered by the fact that they express overlapping sets of surface markers. Tracking and interrogating tissue-resident vs recruited microglia in the brain tumor microenvironment becomes critical to dissect their respective roles and gain a better understanding of the mechanism governing their interaction. In this chapter, we describe the utilization of genetic reporter mice to identify recruited brain microglia, offer a comparison between the genetic method and the most widely used flow cytometric approach, and discuss potential downstream applications to interrogate BMDM function in brain metastatic disease.

Tracing bone marrow-derived microglia in brain metastatic tumors.

Spread of cancer to the brain remains an unmet clinical need in spite of the increasing number of cases among patients with lung, breast cancer, and melanoma most notably. Although research on brain metastasis was considered a minor aspect in the past due to its untreatable nature and invariable lethality, nowadays, limited but encouraging examples have questioned this statement, making it more attractive for basic and clinical researchers. Evidences of its own biological identity (i.e., specific microenvironment) and particular therapeutic requirements (i.e., presence of blood-brain barrier, blood-tumor barrier, molecular differences with the primary tumor) are thought to be critical aspects that must be functionally exploited using preclinical models. We present the coordinated effort of 19 laboratories to compile comprehensive information related to brain metastasis experimental models. Each laboratory has provided details on the cancer cell lines they have generated or characterized as being capable of forming metastatic colonies in the brain, as well as principle methodologies of brain metastasis research. The Brain Metastasis Cell Lines Panel (BrMPanel) represents the first of its class and includes information about the cell line, how tropism to the brain was established, and the behavior of each model . These and other aspects described are intended to assist investigators in choosing the most suitable cell line for research on brain metastasis. The main goal of this effort is to facilitate research on this unmet clinical need, to improve models through a collaborative environment, and to promote the exchange of information on these valuable resources.

Brain Metastasis Cell Lines Panel: A Public Resource of Organotropic Cell Lines.

Regulatory T (Treg) cell infiltration of solid tumors often correlates with poor prognosis, but their tumor-suppressive function lacks mechanistic understanding. Through a combination of transgenic mice, cell fate mapping, adoptive transfer, and co-injection strategies, we demonstrate that Treg cell ablation-dependent anti-tumor effects in murine breast cancer require intratumoral recruitment of CCR2 inflammatory monocytes, which primarily differentiate into tumor-associated macrophages (TAMs), and lead to reprogramming of their function in an IFN-γ-dependent manner. Furthermore, transcriptomic signatures from murine TAMs in Treg cell-ablated conditions correlate with increased overall survival in human breast cancer. Our studies highlight the strong myeloid dependency of breast cancer and provide the basis for the development of therapeutic strategies based on manipulation of the IFN-γ signaling pathway in monocytes.

Regulatory T Cells Support Breast Cancer Progression by Opposing IFN-γ-Dependent Functional Reprogramming of Myeloid Cells.

Epigenetic regulators can modulate the effects of cancer therapeutics. To further these observations, we discovered that the bromodomain PHD finger transcription factor subunit (BPTF) of the nucleosome remodeling factor (NURF) promotes resistance to doxorubicin, etoposide, and paclitaxel in the 4T1 breast tumor cell line. BPTF functions in promoting resistance to doxorubicin and etoposide, but not paclitaxel, and may be selective to cancer cells, as a similar effect was not observed in embryonic stem cells. Sensitization to doxorubicin and etoposide with BPTF knockdown (KD) was associated with increased DNA damage, topoisomerase II (TOP2) crosslinking and autophagy; however, there was only a modest increase in apoptosis and no increase in senescence. Sensitization to doxorubicin was confirmed  with the syngeneic 4T1 breast tumor model using both genetic and pharmacologic inhibition of BPTF. The effects of BPTF inhibition  are autophagy dependent, based on genetic autophagy inhibition. Finally, treatment of 4T1, 66cl4, 4T07, MDA-MB-231, but not ER-positive 67NR and MCF7 breast cancer cells with the selective BPTF bromodomain inhibitor, AU1, recapitulates genetic BPTF inhibition, including  sensitization to doxorubicin, increased TOP2-DNA crosslinks and DNA damage. Taken together, these studies demonstrate that BPTF provides resistance to the antitumor activity of TOP2 poisons, preventing the resolution of TOP2 crosslinking and associated autophagy. These studies suggest that BPTF can be targeted with small-molecule inhibitors to enhance the effectiveness of TOP2-targeted cancer chemotherapeutic drugs. IMPLICATIONS: These studies suggest NURF can be inhibited pharmacologically as a viable strategy to improve chemotherapy effectiveness.

Autophagy-Dependent Sensitization of Triple-Negative Breast Cancer Models to Topoisomerase II Poisons by Inhibition of the Nucleosome Remodeling Factor.

Article	Year
Regulatory T Cells Exhibit Distinct Features in Human Breast Cancer. Immunity\| PubMed ID: 27851913	2016
Tumor-Associated Macrophage Isolation and In Vivo Analysis of Their Tumor-Promoting Activity. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 30465201	2019
Regulatory T Cells Control the Switch From to Invasive Breast Cancer. Frontiers in immunology\| PubMed ID: 31555258	2019
Tracing bone marrow-derived microglia in brain metastatic tumors. Methods in enzymology\| PubMed ID: 32122556	2020
Brain Metastasis Cell Lines Panel: A Public Resource of Organotropic Cell Lines. Cancer research\| PubMed ID: 32641416	2020
Regulatory T Cells Support Breast Cancer Progression by Opposing IFN-γ-Dependent Functional Reprogramming of Myeloid Cells. Cell reports\| PubMed ID: 33296659	2020
Autophagy-Dependent Sensitization of Triple-Negative Breast Cancer Models to Topoisomerase II Poisons by Inhibition of the Nucleosome Remodeling Factor. Molecular cancer research : MCR\| PubMed ID: 33811160	2021

Paula D. Bos

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