IMD-0354 Targets Breast Cancer Stem Cells: A Novel Approach for an Adjuvant to Chemotherapy to Prevent Multidrug Resistance in a Murine Model

Although early detection of breast cancer improved in recent years, prognosis of patients with late stage breast cancer remains poor, mostly due to development of multidrug resistance (MDR) followed by tumor recurrence. Cancer stem cells (CSCs), with higher drug efflux capability and other stem cell-like properties, are concentrated in a side population (SP) of cells, which were proposed to be responsible for MDR and tumor repopulation that cause patients to succumb to breast cancer. Therefore, targeting of CSCs as an adjuvant to chemotherapy should be able to provide a more effective treatment of this disease. Here, we used IMD-0354, an inhibitor of NF-κB, identified for targeting CSCs, in a combination therapy with doxorubicin encapsulated in targeted nanoparticles. IMD-0354 did target CSCs, evidenced by a decrease in the SP, demonstrated by the inhibition of the following: dye/drug efflux, reduction in ABC transporters as well as in colony formation in soft agar and low attachment plates. Decrease of stem-like gene expression of Oct4, Nanog and Sox2, and apoptosis resistance related to the Survivin gene also was observed after treatment with this compound. In addition, IMD-0354 targeted non-CSCs as indicated by reducing viability and increasing apoptosis. Targeted drug delivery, achieved with a legumain inhibitor, proved to enhance drug delivery under hypoxia, a hallmark of the tumor microenvironment, but not under normoxia. Together, this allowed a safe, non-toxic delivery of both anticancer agents to the tumor microenvironment of mice bearing syngeneic metastatic breast cancer. Targeting both bulk tumor cells with a chemotherapeutic agent and CSCs with IMD-0354 should be able to reduce MDR. This could eventually result in decreasing tumor recurrences and/or improve the outcome of metastatic disease.     

Cancer stem cells: Road to therapeutic resistance and strategies to overcome resistance

Unlike other normal cells, a subpopulation of cells often termed as “stem cells” are long-lived and generate cellular progeny throughout life. Cancer stem cells (CSCs) are rare immortal cells within a tumor that can both self-renew by dividing and giving rise to many cell types that constitute the tumor. CSCs also have been shown to be involved in fundamental processes of cell proliferation and metastatic dissemination. CSCs are generally resistant to chemotherapy and radiotherapy, a subset of remaining CSCs after therapy can survive and promote cancer relapse and resistance to therapies. Understanding the biological characteristics of CSCs, the pathways leading to their sustainability and proliferation, and the CSCs role in drug resistance is crucial for establishing novel tumor diagnostic and therapeutic strategies. In this review, we address the pathways that regulate CSCs, the role of CSCs in the resistance to therapy, and strategies to overcome therapeutic resistance.     

Drug-resistant cancer cell-derived exosomal EphA2 promotes breast cancer metastasis via the EphA2-Ephrin A1 reverse signaling

Tumor metastasis induced by drug resistance is a major challenge in successful cancer treatment. Nevertheless, the mechanisms underlying the pro-invasive and metastatic ability of drug resistance remain elusive. Exosome-mediated intercellular communications between cancer cells and stromal cells in tumor microenvironment are required for cancer initiation and progression. Recent reports have shown that communications between cancer cells also promote tumor aggression. However, little attention has been regarded on this aspect. Herein, we demonstrated that drug-resistant cell-derived exosomes promoted the invasion of sensitive breast cancer cells. Quantitative proteomic analysis showed that EphA2 was rich in exosomes from drug-resistant cells. Exosomal EphA2 conferred the invasive/metastatic phenotype transfer from drug-resistant cells to sensitive cells. Moreover, exosomal EphA2 activated ERK1/2 signaling through the ligand Ephrin A1-dependent reverse pathway rather than the forward pathway, thereby promoting breast cancer progression. Our findings indicate the key functional role of exosomal EphA2 in the transmission of aggressive phenotype between cancer cells that do not rely on direct cell–cell contact. Our study also suggests that the increase of EphA2 in drug-resistant cell-derived exosomes may be an important mechanism of chemotherapy/drug resistance-induced breast cancer progression.Subject terms: Breast cancer, Metastasis     

BMP4 enhances anoikis resistance and chemoresistance of breast cancer cells through canonical BMP signaling

Bone morphogenetic proteins (BMPs) regulate cell fate during development and mediate cancer progression. In this study, we investigated the role of BMP4 in proliferation, anoikis resistance, metastatic migration, and drug resistance of breast cancer cells. We utilized breast cancer cell lines and clinical samples representing different subtypes to understand the functional effect of BMP4 on breast cancer. The BMP pathway was inhibited with the small molecule inhibitor LDN193189 hydrochloride (LDN). BMP4 signaling enhanced the expression of stem cell genes CD44, ALDH1A3, anti-apoptotic gene BCL2 and promoted anoikis resistance in MDA-MB-231 breast cancer cells. BMP4 enhanced self-renewal and chemoresistance in MDA-MB-231 by upregulating Notch signaling while LDN treatment abrogated anoikis resistance and proliferation of anoikis resistant breast cancer cells in the osteogenic microenvironment. Conversely, BMP4 downregulated proliferation, colony-forming ability, and suppressed anoikis resistance in MCF7 and SkBR3 cells, while LDN treatment promoted tumor spheroid formation and growth. These findings indicate that BMP4 has a context-dependent role in breast cancer. Further, our data with MDA-MB-231 cells representing triple-negative breast cancer suggest that BMP inhibition might impair its metastatic spread and colonization.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12079-021-00649-9.     

抗血管生成治疗联合免疫检查点抑制剂 在NSCLC中的研究进展

血管生成是非小细胞肺癌(NSCLC)生长、复发和转移的关键环节。抗血管生成治疗可以通过使肿瘤血管及微环境正常化,改善肿瘤血供和含氧量,增强放、化疗效果。也可以抑制肿瘤内毛细血管生长,使肿瘤细胞进入休眠状态,并诱导其凋亡。因此,靶向抗血管生成已成为NSCLC治疗研究的主要方向。贝伐珠单抗和雷莫芦单抗已被批准用于联合一线标准化疗治疗局部晚期或转移性NSCLC。然而,在这一治疗过程中,肿瘤会逐渐对抗血管生成药物产生耐药,这可能与肿瘤微环境(tumor microenvironment,TME)的改变有关。最近,免疫检查点抑制剂(immune checkpoint inhibitors,ICI)已经取得了相当大的成功,但是反应率仍然被认为不是最佳的。因此,为了提高疗效,各种组合疗法正在测试中。临床前数据表明促血管生成因子具有免疫抑制作用,为ICI和抗血管生成药物联合使用提供了合理的解释。并且有研究认为,抗血管生成治疗与肿瘤免疫治疗相联合可能是一种相互增益的治疗策略。     

Murine mammary tumor cells with a claudin-low genotype

In adults, bone is the preferential target site for metastases from primary cancers of prostate, breast, lungs and thyroid. The tendency of these cancers to metastasize to bone is determined by the anatomical distribution of the blood vessels, by the genetic profile of the cancer cells and by the biological characteristics of the bone microenvironment that favour the growth of metastatic cells of certain cancers. Metastases to bone may have either an osteolytic or an ostoblastic phenotype. The interaction in the bone microenvironment between biological factors secreted by metastatic cells, and by osteoblasts and osteoclasts, and the osteolytic and osteoblastic factors released from the organic matrix mediate a vicious cycle characterized by metastatic growth and by ongoing progressive bone destruction. This interaction determines the phenotype of the metastatic bone disease.     

Breast cancer stem cells

Breast cancer stem cells (BCSCs) constitute a subpopulation of tumor cells that express stem cell-associated markers and have a high capacity for tumor generation in vivo. Identification of BCSCs from tumor samples or breast cancer cell lines has been based mainly on CD44(+)/CD24(-/low) or ALDH(+) phenotypes. BCSCs isolation has allowed the analysis of the molecular mechanisms involved in their origin, self-renewal, differentiation into tumor cells, resistance to radiation therapy and chemotherapy, and invasiveness and metastatic ability. Molecular genetic analysis using knockout animals and inducible transgenics has identified NF-κB, c-Jun, p21(CIP1), and Forkhead-like-protein Dach1 involvement in BCSC expansion and fate. Clinical analyses of BCSCs in breast tumors have found a correlation between the proportion of BCSCs and poor prognosis. Therefore, new therapies that specifically target BCSCs are an urgent need. We summarize recent evidence that partially explain the biological characteristics of BCSCs.     

Glioblastoma: Therapeutic challenges, what lies ahead

Glioblastoma (GBM) is one of the most aggressive human cancers. Despite current advances in multimodality therapies, such as surgery, radiotherapy and chemotherapy, the outcome for patients with high grade glioma remains fatal. The knowledge of how glioma cells develop and depend on the tumor environment might open opportunities for new therapies. There is now a growing awareness that the main limitations in understanding and successfully treating GBM might be bypassed by the identification of a distinct cell type that has defining properties of somatic stem cells, as well as cancer-initiating capacity - brain tumor stem cells, which could represent a therapeutic target. In addition, experimental studies have demonstrated that the combination of antiangiogenic therapy, based on the disruption of tumor blood vessels, with conventional chemotherapy generates encouraging results. Emerging reports have also shown that microglial cells can be used as therapeutic vectors to transport genes and/or substances to the tumor site, which opens up new perspectives for the development of GBM therapies targeting microglial cells. Finally, recent studies have shown that natural toxins can be conjugated to drugs that bind to overexpressed receptors in cancer cells, generating targeted-toxins to selectively kill cancer cells. These targeted-toxins are highly effective against radiation- and chemotherapy-resistant cancer cells, making them good candidates for clinical trials in GBM patients. In this review, we discuss recent studies that reveal new possibilities of GBM treatment taking into account cancer stem cells, angiogenesis, microglial cells and drug delivery in the development of new targeted-therapies.     

Epithelial plasticity, cancer stem cells, and the tumor-supportive stroma in bladder carcinoma

High recurrence rates and poor survival rates of metastatic bladder cancer emphasize the need for a drug that can prevent and/or treat bladder cancer progression and metastasis formation. Accumulating evidence suggests that cancer stem/progenitor cells are involved in tumor relapse and therapy resistance in urothelial carcinoma. These cells seem less affected by the antiproliferative therapies, as they are largely quiescent, have an increased DNA damage response, reside in difficult-to-reach, protective cancer stem cell niches and express ABC transporters that can efflux drugs from the cells. Recent studies have shown that epithelial-to-mesenchymal transition (EMT), a process in which sessile, epithelial cells switch to a motile, mesenchymal phenotype may render cancer cells with cancer stem cells properties and/or stimulate the expansion of this malignant cellular subpopulation. As cancer cells undergo EMT, invasiveness, drug resistance, angiogenesis, and metastatic ability seem to increase in parallel, thus giving rise to a more aggressive tumor type. Furthermore, the tumor microenvironment (tumor-associated stromal cells, extracellular matrix) plays a key role in tumorigenesis, tumor progression, and metastasis formation. Taken together, the secret for more effective cancer therapies might lie in developing and combining therapeutic strategies that also target cancer stem/progenitor cells and create an inhospitable microenvironment for highly malignant bladder cancer cells. This review will focus on the current concepts about the role of cancer stem cells, epithelial plasticity, and the supportive stroma in bladder carcinoma. The potential implications for the development of novel bladder cancer therapy will be discussed. Mol Cancer Res; 10(8); 995-1009. ?2012 AACR.     

Doxorubicin in Combination with a Small TGFβ Inhibitor: A Potential Novel Therapy for Metastatic Breast Cancer in Mouse Models

Background

Recent studies suggested that induction of epithelial-mesenchymal transition (EMT) might confer both metastatic and self-renewal properties to breast tumor cells resulting in drug resistance and tumor recurrence. TGFβ is a potent inducer of EMT and has been shown to promote tumor progression in various breast cancer cell and animal models.

Principal Findings

We report that chemotherapeutic drug doxorubicin activates TGFβ signaling in human and murine breast cancer cells. Doxorubicin induced EMT, promoted invasion and enhanced generation of cells with stem cell phenotype in murine 4T1 breast cancer cells in vitro, which were significantly inhibited by a TGFβ type I receptor kinase inhibitor (TβRI-KI). We investigated the potential synergistic anti-tumor activity of TβR1-KI in combination with doxorubicin in animal models of metastatic breast cancer. Combination of Doxorubicin and TβRI-KI enhanced the efficacy of doxorubicin in reducing tumor growth and lung metastasis in the 4T1 orthotopic xenograft model in comparison to single treatments. Doxorubicin treatment alone enhanced metastasis to lung in the human breast cancer MDA-MB-231 orthotopic xenograft model and metastasis to bone in the 4T1 orthotopic xenograft model, which was significantly blocked when TβR1-KI was administered in combination with doxorubicin.

Conclusions

These observations suggest that the adverse activation of TGFβ pathway by chemotherapeutics in the cancer cells together with elevated TGFβ levels in tumor microenvironment may lead to EMT and generation of cancer stem cells resulting in the resistance to the chemotherapy. Our results indicate that the combination treatment of doxorubicin with a TGFβ inhibitor has the potential to reduce the dose and consequently the toxic side-effects of doxorubicin, and improve its efficacy in the inhibition of breast cancer growth and metastasis.     

血小板在肿瘤转移中的作用

血小板是巨核细胞产生的无核细胞碎片,其主要功能是参与凝血和止血。近年来,越来越多的研究和临床证据表明,血小板还参与并促进了肿瘤转移。当肿瘤细胞从原位肿瘤组织脱落进入血管后,血小板是其第一个接触到的宿主细胞。作为肿瘤转移微环境中的重要成员,血小板与肿瘤细胞之间相互作用和相互影响。一方面,肿瘤细胞能通过诱导血小板活化和聚集来调节血小板功能;另一方面,血小板能够通过直接接触和释放生物活性介质的方式,促进肿瘤转移。大量的研究结果表明,血小板主要通过以下几条途径促进肿瘤转移:1)降低血液流体剪切力对肿瘤细胞造成的机械损伤;2)帮助肿瘤细胞逃避免疫监视;3)促进肿瘤细胞在血管内的迁移和停滞;4)促进肿瘤细胞上皮间质转化;5)促进肿瘤细胞外渗;6)构建适合肿瘤细胞生存的转移生态位。因此,靶向血小板与肿瘤细胞相互作用成为潜在的肿瘤治疗策略。本文以国内外最新研究进展为基础,综述血小板在肿瘤转移不同阶段发挥的作用,以及抗血小板药物在肿瘤治疗中的应用。     

Influence of adjuvant radiotherapy on circulating epithelial tumor cells and circulating cancer stem cells in primary non-metastatic breast cancer

Background: There is an unmet need to identify biomarkers that directly reflect response to adjuvant radiotherapy (RT). Circulating epithelial tumor cells (CETCs) represent the liquid component of solid tumors and are responsible for metastatic relapse. CETC subsets with cancer stem cell characteristics, circulating cancer stem cells (cCSCs), play a pivotal role in the metastatic cascade. Monitoring the most aggressive subpopulation of CETCs could reflect the aggressiveness of the remaining tumor burden. There is limited data on the detection and monitoring changes in CETC and cCSC numbers during RT in early breast cancer.Methods: CETC numbers were analyzed prior to, at midterm and at the end of RT in 52 primary non-metastatic breast cancer patients. Hormone receptor status was determined in CETCs prior to and at the end of RT. For the identification of cCSCs cell suspensions from the peripheral blood of patients were cultured in vitro under conditions favoring growth of tumorspheres.Results: Hormone receptor status in CETCs before RT was comparable to that in primary tumor tissue. Prior to RT numbers of CETCs correlated with aggressiveness of primary tumors. cCSCs could be successfully identified and monitored during RT. Prior to RT patients treated with neoadjuvant chemotherapy had significantly higher numbers of CETCs and tumorspheres compared to patients after adjuvant chemotherapy. During RT, the number of CETCs decreased continuously in patients after neoadjuvant chemotherapy but not after adjuvant chemotherapy.Conclusion: Monitoring the number of CETCs and the CETC subset with cancer stem cell properties during RT may provide additional clinically useful prognostic information.     

Human colonic fibroblasts regulate stemness and chemotherapy resistance of colon cancer stem cells

There is increasing evidence that cancers are heterogeneous and contain a hierarchical organization consisting of cancer stem cells and their differentiated cell progeny. These cancer stem cells are at the core of the tumor as they represent the clonogenic cells within a tumor. Moreover, these cells are considered to contain selective therapy resistance, which suggests a pivotal role in therapy resistance and tumor relapse. Here we show that differentiated cells can re-acquire stemness through factors secreted from fibroblasts. This induced CSC state also coincides with re-acquisition of resistance to chemotherapy. Resistance induced in newly formed CSCs is mediated by the anti-apoptotic molecule BCLXL and inhibition of BCLXL with the BH3 mimetic ABT-737 sensitizes these cancer cells toward chemotherapy. These data point to an important interplay between tumor cells and their microenvironment in the regulation of stemness and therapy resistance.     

Extracellular vesicle-mediated transport: Reprogramming a tumor microenvironment conducive with breast cancer progression and metastasis

Breast cancer metastatic progression to critical secondary sites is the second leading cause of cancer-related mortality in women. While existing therapies are highly effective in combating primary tumors, metastatic disease is generally deemed incurable with a median survival of only 2, 3 years. Extensive efforts have focused on identifying metastatic contributory targets for therapeutic antagonism and prevention to improve patient survivability. Excessive breast cancer release of extracellular vesicles (EVs), whose contents stimulate a metastatic phenotype, represents a promising target. Complex breast cancer intercellular communication networks are based on EV transport and transference of molecular information is in bulk resulting in complete reprogramming events within recipient cells. Other breast cancer cells can acquire aggressive phenotypes, endothelial cells can be induced to undergo tubule formation, and immune cells can be neutralized. Recent advancements continue to implicate the critical role EVs play in cultivating a tumor microenvironment tailored to cancer proliferation, metastasis, immune evasion, and conference of drug resistance. This literature review serves to frame the role of EV transport in breast cancer progression and metastasis. The following five sections will be addressed: (1) Intercellular communication in developing a tumor microenvironment & pre-metastatic niche. (2) Induction of the epithelial-to-mesenchymal transition (EMT). (3). Immune suppression & evasion. (4) Transmission of drug resistance mechanisms. (5) Precision medicine: clinical applications of EVs.     

Cancer Associated Fibroblasts Promote Tumor Growth and Metastasis by Modulating the Tumor Immune Microenvironment in a 4T1 Murine Breast Cancer Model

Background

Local inflammation associated with solid tumors commonly results from factors released by tumor cells and the tumor stroma, and promotes tumor progression. Cancer associated fibroblasts comprise a majority of the cells found in tumor stroma and are appealing targets for cancer therapy. Here, our aim was to determine the efficacy of targeting cancer associated fibroblasts for the treatment of metastatic breast cancer.

Methodology/Principal Findings

We demonstrate that cancer associated fibroblasts are key modulators of immune polarization in the tumor microenvironment of a 4T1 murine model of metastatic breast cancer. Elimination of cancer associated fibroblasts in vivo by a DNA vaccine targeted to fibroblast activation protein results in a shift of the immune microenvironment from a Th2 to Th1 polarization. This shift is characterized by increased protein expression of IL-2 and IL-7, suppressed recruitment of tumor-associated macrophages, myeloid derived suppressor cells, T regulatory cells, and decreased tumor angiogenesis and lymphangiogenesis. Additionally, the vaccine improved anti-metastatic effects of doxorubicin chemotherapy and enhanced suppression of IL-6 and IL-4 protein expression while increasing recruitment of dendritic cells and CD8⁺ T cells. Treatment with the combination therapy also reduced tumor-associated Vegf, Pdgfc, and GM-CSF mRNA and protein expression.

Conclusions/Significance

Our findings demonstrate that cancer associated fibroblasts promote tumor growth and metastasis through their role as key modulators of immune polarization in the tumor microenvironment and are valid targets for therapy of metastatic breast cancer.     

Basement Membrane-Rich Organoids with Functional Human Blood Vessels Are Permissive Niches for Human Breast Cancer Metastasis

Metastasic breast cancer is the leading cause of death by malignancy in women worldwide. Tumor metastasis is a multistep process encompassing local invasion of cancer cells at primary tumor site, intravasation into the blood vessel, survival in systemic circulation, and extravasation across the endothelium to metastasize at a secondary site. However, only a small percentage of circulating cancer cells initiate metastatic colonies. This fact, together with the inaccessibility and structural complexity of target tissues has hampered the study of the later steps in cancer metastasis. In addition, most data are derived from in vivo models where critical steps such as intravasation/extravasation of human cancer cells are mediated by murine endothelial cells. Here, we developed a new mouse model to study the molecular and cellular mechanisms underlying late steps of the metastatic cascade. We have shown that a network of functional human blood vessels can be formed by co-implantation of human endothelial cells and mesenchymal cells, embedded within a reconstituted basement membrane-like matrix and inoculated subcutaneously into immunodeficient mice. The ability of circulating cancer cells to colonize these human vascularized organoids was next assessed in an orthotopic model of human breast cancer by bioluminescent imaging, molecular techniques and immunohistological analysis. We demonstrate that disseminated human breast cancer cells efficiently colonize organoids containing a functional microvessel network composed of human endothelial cells, connected to the mouse circulatory system. Human breast cancer cells could be clearly detected at different stages of the metastatic process: initial arrest in the human microvasculature, extravasation, and growth into avascular micrometastases. This new mouse model may help us to map the extravasation process with unprecedented detail, opening the way for the identification of relevant targets for therapeutic intervention.     

减毒单核细胞增生李斯特氏菌作为疫苗载体在肿瘤治疗中的应用

肿瘤免疫疗法已成为继手术、化疗和放疗之后的第四种肿瘤治疗方法,是当今肿瘤治疗的新希望。将细菌疫苗应用于肿瘤治疗的研究已经历了多方面的探索。单核细胞增生李斯特氏菌因其能够趋向肿瘤病灶,在肿瘤微环境保护下激起肿瘤浸润细胞的免疫反应,削弱免疫抑制作用而受到研究者的广泛关注。并且,其在乳腺癌、肝癌、黑素瘤、胰腺癌等癌症中都已具有较为广泛的研究。本文就单核细胞增生李斯特氏菌的免疫应答方式、作为肿瘤载体的优势、减毒策略以及在多种肿瘤免疫治疗中的应用进行综述。     

Pancreatic cancer stem cells and relevance to cancer treatments

Pancreatic cancer continues to be a malignancy with few therapeutic options. The majority of patients that present for an evaluation have locally advanced or metastatic disease that is incurable by surgical approaches. Chemotherapy and radiotherapy resistance of pancreatic adenocarcinomas limits the efficacy of these therapeutic approaches. Recent evidence supports the existence of human pancreatic cancer stem cells, which appear to drive tumor initiation and progression and are particularly resistant to cell death induced by radiation or chemotherapy. Understanding the mechanisms of pancreatic cancer stem cell self‐renewal and resistance to standard therapies may lead to new, more effective therapies to treat this dismal disease. J. Cell. Biochem. 107: 40–45, 2009. © 2009 Wiley‐Liss, Inc.     

Periostin Contributes to the Acquisition of Multipotent Stem Cell-Like Properties in Human Mammary Epithelial Cells and Breast Cancer Cells

Periostin (POSTN), a recently characterised matricellular protein, is frequently dysregulated in various malignant cancers and promotes tumor metastatic growth. POSTN plays a critical role in the crosstalk between murine breast cancer stem cells (CSCs) and their niche to permit metastatic colonization. However, whether pro-metastatic capability of POSTN is associated with multipotent potentials of mesenchymal stem cells (MSCs) has not been documented. Here we demonstrate that POSTN promotes a stem cell-like trait and a mesenchymal phenotype in human mammary epithelial cells and breast cancer cells. Interestingly, ectopic overexpression of POSTN or recombinant POSTN treatment can induce human mammary epithelial cells and breast cancer cells differentiation into multiple cell lineages that recapitulate part of the multilineage differentiation potentials of MSCs. Moreover, POSTN is highly expressed in bone marrow-derived MSCs and their derived adipocytes, chondrocytes, and osteoblasts in vitro. Furthermore, POSTN promotes the growth of xenograft tumors in vivo. POSTN-overexpressing human mammary epithelial cells enhance breast tumor growth and metastasis. These data thus provide evidence of a new role for POSTN in mammary epithelial neoplasia and metastasis, suggesting that epithelial cancer cells might acquire CSC-like traits and a mesenchymal phenotype, as well as the multipotent potentials of MSCs to promote tumorigenesis and metastasis. Therefore, targeting POSTN and other extracellular matrix components of tumor microenvironment may help to develop new therapeutical strategies to inhibit tumor metastasis.