Targeting Tumor Acidosis and Regulatory T Cells Unmasks Anti-Metastatic Potential of Local Tumor Ablation in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is an immunologically heterogenous disease that lacks clinically actionable targets and is more likely to progress to metastatic disease than other types of breast cancer. Tumor ablation has been used to increase response rates to checkpoint inhibitors, which remain low for TNBC patients. We hypothesized that tumor ablation could produce an anti-tumor response without using checkpoint inhibitors if immunosuppression (i.e., Tregs, tumor acidosis) was subdued. Tumors were primed with sodium bicarbonate (200 mM p.o.) to reduce tumor acidosis and low-dose cyclophosphamide (100–200 mg/kg i.p.) to deplete regulatory T cells, as has been shown independently in previous studies. A novel injectable ablative was then used to necrose the tumor, release tumor antigens, and initiate an immune event that could create an abscopal effect. This combination of bicarbonate, cyclophosphamide, and ablation, called “BiCyclA”, was tested in three syngeneic models of TNBC: E0771 (C57BL/6), 67NR (BALB/c), and 4T1-Luc (BALB/c). In E0771 and 67NR, BiCyclA therapy significantly reduced tumor growth and cured 5/7 and 6/10 mice 50 days after treatment respectively. In the metastatic 4T1-Luc tumors, for which surgery and checkpoint inhibitors fail, BiCyclA cured 5/10 mice of primary tumors and lung metastases. Notably, CD4+ and CD8+ T cells were found to be crucial for the anti-metastatic response, and cured mice were able to resist tumor rechallenge, suggesting production of immune memory. Reduction of tumor acidity and regulatory T cells with ablation is a simple yet effective therapy for local and systemic tumor control with broad applicability as it is not limited by expensive supplies.     

Tumor-Derived Small Extracellular Vesicles Involved in Breast Cancer Progression and Drug Resistance

Breast cancer is one of the most serious and terrifying threats to the health of women. Recent studies have demonstrated that interaction among cancer cells themselves and those with other cells, including immune cells, in a tumor microenvironment potentially and intrinsically regulate and determine cancer progression and metastasis. Small extracellular vesicles (sEVs), a type of lipid-bilayer particles derived from cells, with a size of less than 200 nm, are recognized as one form of important mediators in cell-to-cell communication. sEVs can transport a variety of bioactive substances, including proteins, RNAs, and lipids. Accumulating evidence has revealed that sEVs play a crucial role in cancer development and progression, with a significant impact on proliferation, invasion, and metastasis. In addition, sEVs systematically coordinate physiological and pathological processes, such as coagulation, vascular leakage, and stromal cell reprogramming, to bring about premetastatic niche formation and to determine metastatic organ tropism. There are a variety of oncogenic factors in tumor-derived sEVs that mediate cellular communication between local stromal cells and distal microenvironment, both of which are important in cancer progression and metastasis. Tumor-derived sEVs contain substances that are similar to parental tumor cells, and as such, sEVs could be biomarkers in cancer progression and potential therapeutic targets, particularly for predicting and preventing future metastatic development. Here, we review the mechanisms underlying the regulation by tumor-derived sEVs on cancer development and progression, including proliferation, metastasis, drug resistance, and immunosuppression, which coordinately shape the pro-metastatic microenvironment. In addition, we describe the application of sEVs to the development of cancer biomarkers and potential therapeutic modalities and discuss how they can be engineered and translated into clinical practice.     

The Complex Biology of the Obesity-Induced,Metastasis-Promoting Tumor Microenvironment in Breast Cancer

Breast cancer is one of the most prevalent cancers in women contributing to cancer-related death in the advanced world. Apart from the menopausal status, the trigger for developing breast cancer may vary widely from race to lifestyle factors. Epidemiological studies refer to obesity-associated metabolic changes as a critical risk factor behind the progression of breast cancer. The plethora of signals arising due to obesity-induced changes in adipocytes present in breast tumor microenvironment, significantly affect the behavior of adjacent breast cells. Adipocytes from white adipose tissue are currently recognized as an active endocrine organ secreting different bioactive compounds. However, due to excess energy intake and increased fat accumulation, there are morphological followed by secretory changes in adipocytes, which make the breast microenvironment proinflammatory. This proinflammatory milieu not only increases the risk of breast cancer development through hormone conversion, but it also plays a role in breast cancer progression through the activation of effector proteins responsible for the biological phenomenon of metastasis. The aim of this review is to present a comprehensive picture of the complex biology of obesity-induced changes in white adipocytes and demonstrate the relationship between obesity and breast cancer progression to metastasis.     

ANXA1 Contained in EVs Regulates Macrophage Polarization in Tumor Microenvironment and Promotes Pancreatic Cancer Progression and Metastasis

The tumor microenvironment (TME) is a dynamic system where nontumor and cancer cells intercommunicate through soluble factors and extracellular vesicles (EVs). The TME in pancreatic cancer (PC) is critical for its aggressiveness and the annexin A1 (ANXA1) has been identified as one of the oncogenic elements. Previously, we demonstrated that the autocrine/paracrine activities of extracellular ANXA1 depend on its presence in EVs. Here, we show that the complex ANXA1/EVs modulates the macrophage polarization further contributing to cancer progression. The EVs isolated from wild type (WT) and ANXA1 knock-out MIA PaCa-2 cells have been administrated to THP-1 macrophages finding that ANXA1 is crucial for the acquisition of a protumor M2 phenotype. The M2 macrophages activate endothelial cells and fibroblasts to induce angiogenesis and matrix degradation, respectively. We have also found a significantly increased presence of M2 macrophage in mice tumor and liver metastasis sections previously obtained by orthotopic xenografts with WT cells. Taken together, our data interestingly suggest the relevance of ANXA1 as potential diagnostic/prognostic and/or therapeutic PC marker.     

The Novel Association of Early Apoptotic Circulating Tumor Cells with Treatment Outcomes in Breast Cancer Patients

Stemness and epithelial–mesenchymal plasticity are widely studied in the circulating tumor cells of breast cancer patients because the roles of both processes in tumor progression are well established. An important property that should be taken into account is the ability of CTCs to disseminate, particularly the viability and apoptotic states of circulating tumor cells (CTCs). Recent data demonstrate that apoptosis reversal promotes the formation of stem-like tumor cells with pronounced potential for dissemination. Our study focused on the association between different apoptotic states of CTCs with short- and long-term treatment outcomes. We evaluated the association of viable CTCs, CTCs with early features of apoptosis, and end-stage apoptosis/necrosis CTCs with clinicopathological parameters of breast cancer patients. We found that the proportion of circulating tumor cells with features of early apoptosis is a perspective prognosticator of metastasis-free survival, which also correlates with the neoadjuvant chemotherapy response in breast cancer patients. Moreover, we establish that apoptotic CTCs are associated with the poor response to neoadjuvant chemotherapy, and metastasis-free survival expressed at least two stemness markers, CD44 and CD133.     

Roles of Lysyl Oxidase Family Members in the Tumor Microenvironment and Progression of Liver Cancer

The lysyl oxidase (LOX) family members are secreted copper-dependent amine oxidases, comprised of five paralogues: LOX and LOX-like l-4 (LOXL1-4), which are characterized by catalytic activity contributing to the remodeling of the cross-linking of the structural extracellular matrix (ECM). ECM remodeling plays a key role in the angiogenesis surrounding tumors, whereby a corrupt tumor microenvironment (TME) takes shape. Primary liver cancer includes hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), ranked as the seventh most common cancer globally, with limited therapeutic options for advanced stages. In recent years, a growing body of evidence has revealed the key roles of LOX family members in the pathogenesis of liver cancer and the shaping of TME, indicating their notable potential as therapeutic targets. We herein review the clinical value and novel biological roles of LOX family members in tumor progression and the TME of liver cancers. In addition, we highlight recent insights into their mechanisms and their potential involvement in the development of target therapy for liver cancer.     

CXC Chemokine Signaling in Progression of Epithelial Ovarian Cancer: Theranostic Perspectives

Patients with epithelial ovarian cancer (EOC) are often diagnosed at an advanced stage due to nonspecific symptoms and ineffective screening approaches. Although chemotherapy has been available and widely used for the treatment of advanced EOC, the overall prognosis remains dismal. As part of the intrinsic defense mechanisms against cancer development and progression, immune cells are recruited into the tumor microenvironment (TME), and this process is directed by the interactions between different chemokines and their receptors. In this review, the functional significance of CXC chemokine ligands/chemokine receptors (CXCL/CXCR) and their roles in modulating EOC progression are summarized. The status and prospects of CXCR/CXCL-based theranostic strategies in EOC management are also discussed.     

Breast Cancer Metastasis: Mechanisms and Therapeutic Implications

Breast cancer is the most common malignancy in women worldwide. Metastasis is the leading cause of high mortality in most cancers. Although predicting the early stage of breast cancer before metastasis can increase the survival rate, breast cancer is often discovered or diagnosed after metastasis has occurred. In general, breast cancer has a poor prognosis because it starts as a local disease and can spread to lymph nodes or distant organs, contributing to a significant impediment in breast cancer treatment. Metastatic breast cancer cells acquire aggressive characteristics from the tumor microenvironment (TME) through several mechanisms including epithelial–mesenchymal transition (EMT) and epigenetic regulation. Therefore, understanding the nature and mechanism of breast cancer metastasis can facilitate the development of targeted therapeutics focused on metastasis. This review discusses the mechanisms leading to metastasis and the current therapies to improve the early diagnosis and prognosis in patients with metastatic breast cancer.     

Current Advancements of Plant-Derived Agents for Triple-Negative Breast Cancer Therapy through Deregulating Cancer Cell Functions and Reprogramming Tumor Microenvironment

Triple-negative breast cancer (TNBC) is defined based on the absence of estrogen, progesterone, and human epidermal growth factor receptor 2 receptors. Currently, chemotherapy is the major therapeutic approach for TNBC patients; however, poor prognosis after a standard chemotherapy regimen is still commonplace due to drug resistance. Abnormal tumor metabolism and infiltrated immune or stromal cells in the tumor microenvironment (TME) may orchestrate mammary tumor growth and metastasis or give rise to new subsets of cancer cells resistant to drug treatment. The immunosuppressive mechanisms established in the TME make cancer cell clones invulnerable to immune recognition and killing, and turn immune cells into tumor-supporting cells, hence allowing cancer growth and dissemination. Phytochemicals with the potential to change the tumor metabolism or reprogram the TME may provide opportunities to suppress cancer metastasis and/or overcome chemoresistance. Furthermore, phytochemical intervention that reprograms the TME away from favoring immunoevasion and instead towards immunosurveillance may prevent TNBC metastasis and help improve the efficacy of combination therapies as phyto-adjuvants to combat drug-resistant TNBC. In this review, we summarize current findings on selected bioactive plant-derived natural products in preclinical mouse models and/or clinical trials with focus on their immunomodulatory mechanisms in the TME and their roles in regulating tumor metabolism for TNBC prevention or therapy.     

A Single Variant in Pri-miRNA-155 Associated with Susceptibility to Hereditary Breast Cancer Promotes Aggressiveness in Breast Cancer Cells

Variants in genes encoding for microRNAs have been associated with their deregulation in breast cancer (BC). Sequencing of microRNAs deregulated in BC was performed using DNA from Chilean patients with a strong family history and negative for mutations in BRCA1/BRCA2. Seventeen variants were identified, three of which were selected for a case-control association study: rs376491654 (miR-335), rs755634302 (miR-497), and rs190708267 (miR-155). For rs190708267 C>T, the heterozygous T allele was detected in four BC cases and absent in controls, while homozygous TT cases were not detected. Variants were modelled in silico, cloned in a plasmid, expressed in BC cell lines, and functional in vitro assays were performed. Overexpression of the miR-155-T allele increased mature miR-155-5p levels in both BC cell lines, suggesting that its presence alters pre-miR-155 processing. Moreover, BC cells overexpressing the miR-155-T allele showed increased proliferation, migration, and resistance to cisplatin-induced death compared to miR-155-C overexpressing cells. Of note, the 3′UTR of APC, GSK3β, and PPP1CA genes, all into the canonical Wnt signaling pathway, were identified as direct targets. APC and GSK3β mRNA levels decreased while PP1 levels increased. These results suggest a pathogenic role of the variant rs190708267 (miR-155) in BRCA 1/2 negative BC, conferring susceptibility and promoting traits of aggressiveness.     

Lymphatics in Tumor Progression and Immunomodulation

The lymphatic system consists of a unidirectional hierarchy of vessels responsible for fluid homeostasis, lipid absorption, and the transport of immune cells and antigens to secondary lymphoid organs. In cancer, lymphatics play complex and heterogenous roles that can promote or inhibit tumor growth. While lymphatic proliferation and remodeling promote tumor dissemination, functional lymphatics are necessary for generating an effective immune response. Recent reports have noted lymphatic-dependent effects on the efficacy of immunotherapy. These findings suggest that the impact of lymphatic vessels on tumor progression is organ- and context-specific and that a greater understanding of the interaction of tumor cells, lymphatics, and the tumor microenvironment can unveil novel therapies.     

Generation and Characterization of a Tumor Stromal Microenvironment and Analysis of Its Interplay with Breast Cancer Cells: An In Vitro Model to Study Breast Cancer-Associated Fibroblast Inactivation

Breast cancer-associated fibroblasts (BCAFs), the most abundant non-cancer stromal cells of the breast tumor microenvironment (TME), dramatically sustain breast cancer (BC) progression by interacting with BC cells. BCAFs, as well as myofibroblasts, display an up regulation of activation and inflammation markers represented by α-smooth muscle actin (α-SMA) and cyclooxygenase 2 (COX-2). BCAF aggregates have been identified in the peripheral blood of metastatic BC patients. We generated an in vitro stromal model consisting of human primary BCAFs grown as monolayers or 3D cell aggregates, namely spheroids and reverted BCAFs, obtained from BCAF spheroids reverted to 2D cell adhesion growth after 216 h of 3D culture. We firstly evaluated the state of activation and inflammation and the mesenchymal status of the BCAF monolayers, BCAF spheroids and reverted BCAFs. Then, we analyzed the MCF-7 cell viability and migration following treatment with conditioned media from the different BCAF cultures. After 216 h of 3D culture, the BCAFs acquired an inactivated phenotype, associated with a significant reduction in α-SMA and COX-2 protein expression. The deactivation of the BCAF spheroids at 216 h was further confirmed by the cytostatic effect exerted by their conditioned medium on MCF-7 cells. Interestingly, the reverted BCAFs also retained a less activated phenotype as indicated by α-SMA protein expression reduction. Furthermore, the reverted BCAFs exhibited a reduced pro-tumor phenotype as indicated by the anti-migratory effect exerted by their conditioned medium on MCF-7 cells. The deactivation of BCAFs without drug treatment is possible and leads to a reduced capability of BCAFs to sustain BC progression in vitro. Consequently, this study could be a starting point to develop new therapeutic strategies targeting BCAFs and their interactions with cancer cells.     

Heterogeneous Manifestations of Epithelial–Mesenchymal Plasticity of Circulating Tumor Cells in Breast Cancer Patients

To date, there is indisputable evidence of significant CTC heterogeneity in carcinomas, in particular breast cancer. The heterogeneity of CTCs is manifested in the key characteristics of tumor cells related to metastatic progression – stemness and epithelial–mesenchymal (EMT) plasticity. It is still not clear what markers can characterize the phenomenon of EMT plasticity in the range from epithelial to mesenchymal phenotypes. In this article we examine the manifestations of EMT plasticity in the CTCs in breast cancer. The prospective study included 39 patients with invasive carcinoma of no special type. CTC phenotypes were determined by flow cytometry before any type of treatment. EMT features of CTC were assessed using antibodies against CD45, CD326 (EpCam), CD325 (N-cadherin), CK7, Snail, and Vimentin. Circulating tumor cells in breast cancer are characterized by pronounced heterogeneity of EMT manifestations. The results of the study indicate that the majority of heterogeneous CTC phenotypes (22 out of 24 detectable) exhibit epithelial–mesenchymal plasticity. The variability of EMT manifestations does not prevent intravasation. Co-expression of EpCAM and CK7, regardless of the variant of co-expression of Snail, N-cadherin, and Vimentin, are associated with a low number of CTCs. Intrapersonal heterogeneity is manifested by the detection of several CTC phenotypes in each patient. Interpersonal heterogeneity is manifested by various combinations of CTC phenotypes in patients (from 1 to 17 phenotypes).     

The Role of MicroRNAs in Breast Cancer Stem Cells

The concept of the existence of a subset of cancer cells with stem cell-like properties, which are thought to play a significant role in tumor formation, metastasis, resistance to anticancer therapies and cancer recurrence, has gained tremendous attraction within the last decade. These cancer stem cells (CSCs) are relatively rare and have been described by different molecular markers and cellular features in different types of cancers. Ten years ago, a novel class of molecules, small non-protein-coding RNAs, was found to be involved in carcinogenesis. These small RNAs, which are called microRNAs (miRNAs), act as endogenous suppressors of gene expression that exert their effect by binding to the 3′-untranslated region (UTR) of large target messenger RNAs (mRNAs). MicroRNAs trigger either translational repression or mRNA cleavage of target mRNAs. Some studies have shown that putative breast cancer stem cells (BCSCs) exhibit a distinct miRNA expression profile compared to non-tumorigenic breast cancer cells. The deregulated miRNAs may contribute to carcinogenesis and self-renewal of BCSCs via several different pathways and can act either as oncomirs or as tumor suppressive miRNAs. It has also been demonstrated that certain miRNAs play an essential role in regulating the stem cell-like phenotype of BCSCs. Some miRNAs control clonal expansion or maintain the self-renewal and anti-apoptotic features of BCSCs. Others are targeting the specific mRNA of their target genes and thereby contribute to the formation and self-renewal process of BCSCs. Several miRNAs are involved in epithelial to mesenchymal transition, which is often implicated in the process of formation of CSCs. Other miRNAs were shown to be involved in the increased chemotherapeutic resistance of BCSCs. This review highlights the recent findings and crucial role of miRNAs in the maintenance, growth and behavior of BCSCs, thus indicating the potential for novel diagnostic, prognostic and therapeutic miRNA-based strategies.     

Crosstalk between Pancreatic Cancer Cells and Cancer-Associated Fibroblasts in the Tumor Microenvironment Mediated by Exosomal MicroRNAs

Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant digestive tumors, characterized by a low rate of early diagnosis, strong invasiveness, and early metastasis. The abundant stromal cells, dense extracellular matrix, and lack of blood supply in PDAC limit the penetration of chemotherapeutic drugs, resulting in poor efficacy of the current treatment regimens. Cancer-associated fibroblasts (CAFs) are the major stromal cells in the tumor microenvironment. Tumor cells can secrete exosomes to promote the generation of activated CAFs, meanwhile exosomes secreted by CAFs help promote tumor progression. The aberrant expression of miRNAs in exosomes is involved in the interaction between tumor cells and CAFs, which provides the possibility for the application of exosomal miRNAs in the diagnosis and treatment of PDAC. The current article reviews the mechanism of exosomal miRNAs in the crosstalk between PDAC cells and CAFs in the tumor microenvironment, in order to improve the understanding of TME regulation and provide evidence for designing diagnostic and therapeutic targets against exosome miRNA in human PDAC.