Cancer Cell Intravasation Research Articles

Intratumoral CXCL12 Gradients Contextualize Tumor Cell Invasion, Migration and Immune Suppression in Breast Cancer.

Although the CXCL12/CXCR4 pathway has been prior investigated for its prometastatic and immuno- suppressive roles in the tumor microenvironment, evidence on the spatiotemporal regulation of these hallmarks has been lacking. Here, we demonstrate that CXCL12 forms a gradient specifically around cancer cell intravasation doorways, also known as Tumor Microenvironment of Metastasis (TMEM) doorways, thus facilitating the chemotactic translocation of prometastatic tumor cells expressing CXCR4 toward the perivascular TMEM doorways for subsequent entry into peripheral circulation. Fur- thermore, we demonstrate that the CXCL12-rich micro-environment around TMEM doorways may cre- ate immunosuppressive niches, whereby CD8 + T cells, despite being attracted to these regions, often exhibit reduced effector functions, limiting their efficacy. While the CXCL12/CXCR4 pathway can mini- mally influence the overall composition of immune cell populations, it biases the distribution of CD8 + T cells away from TMEM doorways, justifying its prior-established role as immunosuppressive factor for CD8 + T cells. Our research suggests that the complex interactions between CXCL12 and the various tumor and immune cell types contributes not only to the completion of the initial steps of the metastatic cascade, but also offers an immunological "sanctuary" to prometastatic tumor cells homed around TMEM doorways. Overall, our study enhances our current understanding on the mechanisms, via which CXCL12 orchestrates tumor cell behavior and immune dynamics, potentially guiding future thera- peutic strategies to combat breast cancer metastasis and improve anti-tumor immunity.

PDGFRα+ITGA11+ fibroblasts foster early-stage cancer lymphovascular invasion and lymphatic metastasis via ITGA11-SELE interplay

Cancer-associated fibroblasts (CAFs) exhibit considerable heterogeneity in advanced cancers; however, the functional annotation and mechanism of CAFs in early-stage cancers remain elusive. Utilizing single-cell RNA sequencing and spatial transcriptomic, we identify a previously unknown PDGFRα+ITGA11+ CAF subset in early-stage bladder cancer (BCa). Multicenter clinical analysis of a 910-case cohort confirms that PDGFRα+ITGA11+ CAFs are associated with lymphovascular invasion (LVI) and poor prognosis in early-stage BCa. These CAFs facilitate LVI and lymph node (LN) metastasis in early-stage BCa, as evidenced in a PDGFRα+ITGA11+ CAFs-specific deficient mouse model. Mechanistically, PDGFRα+ITGA11+ CAFs promote lymphangiogenesis via recognizing ITGA11 surface receptor SELE on lymphatic endothelial cells to activate SRC-p-VEGFR3-MAPK pathway. Further, CHI3L1 from PDGFRα+ITGA11+ CAFs aligns the surrounding matrix to assist cancer cell intravasation, fostering early-stage BCa LVI and LN metastasis. Collectively, our study reveals the crucial role of PDGFRα+ITGA11+ CAFs in shaping metastatic landscape, informing the treatment of early-stage BCa LVI.

Unveiling breast cancer metastasis through an advanced X-ray imaging approach

Breast cancer is a significant global health burden, causing a substantial number of deaths. Systemic metastatic tumour cell dissemination is a major cause of poor outcomes. Understanding the mechanisms underlying metastasis is crucial for effective interventions. Changes in the extracellular matrix play a pivotal role in breast cancer metastasis. In this work, we present an advanced multimodal X-ray computed tomography, by combining Small-angle X-ray Scattering Tensor Tomography (SAXS-TT) and X-ray Fluorescence Computed Tomography (XRF-CT). This approach likely brings out valuable information about the breast cancer metastasis cascade. Initial results from its application on a breast cancer specimen reveal the collective influence of key molecules in the metastatic mechanism, identifying a strong correlation between zinc accumulation (associated with matrix metalloproteinases MMPs) and highly oriented collagen. MMPs trigger collagen alignment, facilitating breast cancer cell intravasation, while iron accumulation, linked to angiogenesis and vascular endothelial growth factor VEGF, supports cell proliferation and metastasis. Therefore, these findings highlight the potential of the advanced multimodal X-ray computed tomography approach and pave the way for in-depth investigation of breast cancer metastasis, which may guide the development of novel therapeutic approaches and enable personalised treatment strategies, ultimately improving patient outcomes in breast cancer management.

VEGF-C-expressing TAMs rewire the metastatic fate of breast cancer cells

The expression of pro-lymphangiogenic VEGF-C in primary tumors is associated with sentinel lymph node metastasis in most solid cancer types. However, the impact of VEGF-C on distant organ metastasis remains unclear. Perivascular tumor-associated macrophages (TAMs) play a crucial role in guiding hematogenous spread of cancer cells by establishing metastatic pathways within the tumor microenvironment. This process supports breast cancer cell intravasation and metastatic dissemination. We show here that VEGF-C-expressing TAMs reduce the dissemination of mammary cancer cells to the lungs while concurrently increasing lymph node metastasis. These TAMs express podoplanin and interact with normalized tumor blood vessels expressing VEGFR3. Moreover, clinical data suggest inverse association between VEGF-C-expressing TAMs and breast cancer malignancy. Thus, our study elucidates the paradoxical role of VEGF-C-expressing TAMs in redirecting cancer cells to preferentially disseminate to lymph nodes rather than to lungs, partially achieved by normalizing tumor blood vessels and promoting lymphangiogenesis.

Exact and solitary wave structure of the tumor cell proliferation with LQ model of three dimensional PDE by newly extended direct algebraic method

An essential stage in the spread of cancer is the entry of malignant cells into the bloodstream. The fundamental mechanism of cancer cell intravasation is still completely unclear, despite substantial advancements in observing tumor cell mobility in vivo. By creating therapeutic methods in conjunction with control engineering or by using the models for simulations and treatment process evaluation, tumor growth models have established themselves as a crucial instrument for producing an engineering backdrop for cancer therapy. Because tumor growth is a highly complex process, mathematical modeling has been essential for describing it because a carefully crafted tumor growth model constantly describes the measurements and the physiological processes of the tumors. This article discusses the exact and solitary wave behavior of a tumor cell with a three-dimensional linear-quadratic model. Exact solutions have been discussed in detail using the newly extended direct algebraic method, which presents a variety of answers to this issue based on the conditions applied. This article also illustrates its graphical behavior with surface and contour plots of several solitons.

Gut microbiota: key facilitator in metastasis of colorectal cancer.

Colorectal cancer (CRC) ranks third in terms of incidence among all kinds of cancer. The main cause of death is metastasis. Recent studies have shown that the gut microbiota could facilitate cancer metastasis by promoting cancer cells proliferation, invasion, dissemination, and survival. Multiple mechanisms have been implicated, such as RNA-mediated targeting effects, activation of tumor signaling cascades, secretion of microbiota-derived functional substances, regulation of mRNA methylation, facilitated immune evasion, increased intravasation of cancer cells, and remodeling of tumor microenvironment (TME). The understanding of CRC metastasis was further deepened by the mechanisms mentioned above. In this review, the mechanisms by which the gut microbiota participates in the process of CRC metastasis were reviewed as followed based on recent studies.

MUC13 drives cancer aggressiveness and metastasis through the YAP1-dependent pathway.

Anchorage-independent survival after intravasation of cancer cells from the primary tumor site represents a critical step in metastasis. Here, we reveal new insights into how MUC13-mediated anoikis resistance, coupled with survival of colorectal tumor cells, leads to distant metastasis. We found that MUC13 targets a potent transcriptional coactivator, YAP1, and drives its nuclear translocation via forming a novel survival complex, which in turn augments the levels of pro-survival and metastasis-associated genes. High expression of MUC13 is correlated well with extensive macrometastasis of colon cancer cells with elevated nuclear YAP1 in physiologically relevant whole animal model systems. Interestingly, a positive correlation of MUC13 and YAP1 expression was observed in human colorectal cancer tissues. In brief, the results presented here broaden the significance of MCU13 in cancer metastasis via targeting YAP1 for the first time and provide new avenues for developing novel strategies for targeting cancer metastasis.

Travelling under pressure - hypoxia and shear stress in the metastatic journey.

Cancer cell invasion, intravasation and survival in the bloodstream are early steps of the metastatic process, pivotal to enabling the spread of cancer to distant tissues. Circulating tumor cells (CTCs) represent a highly selected subpopulation of cancer cells that tamed these critical steps, and a better understanding of their biology and driving molecular principles may facilitate the development of novel tools to prevent metastasis. Here, we describe key research advances in this field, aiming at describing early metastasis-related processes such as collective invasion, shedding, and survival of CTCs in the bloodstream, paying particular attention to microenvironmental factors like hypoxia and mechanical stress, considered as important influencers of the metastatic journey.

Mechanobiology and survival strategies of circulating tumor cells: a process towards the invasive and metastatic phenotype.

Metastatic progression is the deadliest feature of cancer. Cancer cell growth, invasion, intravasation, circulation, arrest/adhesion and extravasation require specific mechanical properties to allow cell survival and the completion of the metastatic cascade. Circulating tumor cells (CTCs) come into contact with the capillary bed during extravasation/intravasation at the beginning of the metastatic cascade. However, CTC mechanobiology and survival strategies in the bloodstream, and specifically in the microcirculation, are not well known. A fraction of CTCs can extravasate and colonize distant areas despite the biomechanical constriction forces that are exerted by the microcirculation and that strongly decrease tumor cell survival. Furthermore, accumulating evidence shows that several CTC adaptations, via molecular factors and interactions with blood components (e.g., immune cells and platelets inside capillaries), may promote metastasis formation. To better understand CTC journey in the microcirculation as part of the metastatic cascade, we reviewed how CTC mechanobiology and interaction with other cell types in the bloodstream help them to survive the harsh conditions in the circulatory system and to metastasize in distant organs.

Cover Image

IUBMB LifeVolume 75, Issue 6 COVER IMAGEFree Access Cover Image First published: 02 May 2023 https://doi.org/10.1002/iub.2731AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Graphical Abstract On the Cover: Examples of the roles of proteases in prostate cancer development and progression. A simplified scheme of the roles of the major proteases discussed in this review is illustrated. The schematic representation of tumor growth and early steps of metastatic dissemination, shown in the center panel, include: 1) Proliferative and EMT signaling (interaction of cancer and stromal cells and inflammatory responses); 2) Cancer cell migration and invasion, involving ECM remodeling and signaling; 3) Stimulation of (lymph)angiogenesis and intravasation of invasive cancer cells, involving ECM remodeling, degradation of anti-angiogenic factors, production of angiogenic peptides and growth-factor activation; 4) Stromal cells, including cancer-associated fibroblasts (CAFs), are also involved in tumorigenic processes; 5) A small fraction of circulating tumor cells (CTCs) survive and eventually form distant metastases. These processes often involve autocrine-paracrine loops in the prostate cancer microenvironment, which due to simplicity have been omitted here by Koistinen et al.; Article first published: 04 Jan 2023; https://doi.org/10.1002/iub.2700 Volume75, Issue6June 2023 RelatedInformation

Abstract 60: An emerging paradigm of Cxcl12 & Cxcr4 involvement in breast cancer metastasis

Abstract The Cxcl12/Cxcr4 signaling axis has been shown to promote metastasis in multiple mouse models of breast carcinoma, and has been linked to breast cancer cell seeding, homing, survival, and proliferation at future metastatic sites. The precise mechanism by which Cxcr4+ breast cancer cells escape the primary tumors (which also highly express Cxcl12), remains poorly understood. By using a novel multichannel immunofluorescence (mIF) based methodology for quantifying chemotactic gradients in fixed tissue, we here demonstrate that Cxcl12 gradients in mouse primary breast tumors are concentrically expressed around sites of cancer cell intravasation known as Tumor Microenvironment of Metastasis (TMEM) doorways. Via distance analysis algorithms, we additionally demonstrate that TMEM doorway-mediated Cxcl12 gradients associate with Cxcr4+ breast cancer cells migrating towards the underlying TMEM doorways. Consistent with this observation, pharmacological inhibition of the Cxcl12/Cxcr4 pathway significantly abrogates the translocation of Cxcr4+ cancer cells to TMEM doorways, suppressing TMEM doorway-mediated metastatic dissemination. However, targeted elimination of the Cxcr4 gene specifically from breast cancer cells, paradoxically results in a suboptimal response, thus suggesting the existence of a bypass or compensatory mechanism. Previously, it was shown that Cxcr4+ tumor-associated macrophages (TAMs) support the invasive and migratory properties of tumor cells that utilize TMEM doorways. We thus hypothesized that, absent Cxcr4 expression in tumor cells, accompanying Cxcr4+ TAMs may still “read” TMEM-generated Cxcl12 chemotactic gradients. Indeed, clodronate-mediated TAM depletion results in the significant suppression of Cxcr4+ cancer cell translocation to TMEM doorways and their subsequent dissemination to the peripheral circulation and future metastatic sites. Finally, we used a variety of stromal and immune cell lineage markers to identify the precise source of TMEM doorway-generated Cxcl12 gradients in mouse primary breast cancers. Despite the fact that blood vessels (irrespective of presence of TMEM doorways) were primarily lined by Pdgfrb+ stromal cells with basal Cxcl12 expression, TMEM-generated Cxcl12 gradients were specifically linked to a subset of Cxcl12+Iba1+ perivascular TAMs. Pharmacological inhibition of Pdgfrb depletes Pdgfrb+Cxcl12+ stromal cells, but does not significantly affect Cxcl12/Cxcr4- mediated translocation of Cxcr4+ tumor cells to TMEM doorways. Overall, our data support a new paradigm implicating the Cxcl12/Cxcr4 axis during the early stages of the metastatic cascade, and point to a new avenue for rationalized antimetastatic treatments for breast cancer. Citation Format: Dimitra P. Anastasiadou, Luis R. Sanchez, Camille L. Duran, Joseph Burt, Xiaoming Chen, Yu Lin, Robert Eddy, Allison S. Harney, David Entenberg, John S. Condeelis, Maja H. Oktay, George S. Karagiannis. An emerging paradigm of Cxcl12 & Cxcr4 involvement in breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 60.

Intravital imaging of Wnt/β-catenin and ATF2-dependent signalling pathways during tumour cell invasion and metastasis.

Wnt signalling has been implicated as a driver of tumour cell metastasis, but less is known about which branches of Wnt signalling are involved and when they act in the metastatic cascade. Here, using a unique intravital imaging platform and fluorescent reporters, we visualised β-catenin/TCF-dependent and ATF2-dependent signalling activities during human cancer cell invasion, intravasation and metastatic lesion formation in the chick embryo host. We found that cancer cells readily shifted between states of low and high canonical Wnt activity. Cancer cells that displayed low Wnt canonical activity showed higher invasion and intravasation potential in primary tumours and in metastatic lesions. In contrast, cancer cells showing low ATF2-dependent activity were significantly less invasive both at the front of primary tumours and in metastatic lesions. Simultaneous visualisation of both these reporters using a double-reporter cell line confirmed their complementary activities in primary tumours and metastatic lesions. These findings might inform the development of therapies that target different branches of Wnt signalling at specific stages of metastasis.

The tumour microenvironment modulates cancer cell intravasation

Development of three dimensional (3D) in vitro models to realistically recapitulate tumor microenvironment has the potential to improve translatability of anti-cancer drugs at the preclinical stage. To capture the in vivo complexity, these in vitro models should minimally incorporate the 3D interactions between multiple cell types, cellular structures such as vasculature and extracellular matrices. Here, we utilized microfluidic platforms to study the effect of various natural hydrogels (fibrin, collagen, Matrigel) and presence of tumor spheroids on the 3D vascularisation morphology. Various ECM compositions impacted the vessel morphology while near the tumor spheroids the vessel diameter was considerably smaller for all different ECM compositions. Strikingly, cancer cells could enter the microvessel lumens (intravasation) only when the ECM was comprised of all the three types of hydrogels which increased the physical contact between the microvessels and the tumour spheroids. Our findings highlight the role of ECM composition in modulating the intravasation capacity of tumors. • A human in vitro microfluidics model for visualising cancer cell intravasation was developed. • Composite hydrogel matrices emulate in vivo like complexities of the acellular tumour microenvironment. • Three-dimensional multicellular culture system recreates the complex tumour-stromal-vascular interactions. • Combination of collagen, Matrigel and fibrin hydrogels increased physical cellular interactions and enhanced cellular cross-talk.

Assessment of MRI to estimate metastatic dissemination risk and prometastatic effects of chemotherapy

Metastatic dissemination in breast cancer is regulated by specialized intravasation sites called “tumor microenvironment of metastasis” (TMEM) doorways, composed of a tumor cell expressing the actin-regulatory protein Mena, a perivascular macrophage, and an endothelial cell, all in stable physical contact. High TMEM doorway number is associated with an increased risk of distant metastasis in human breast cancer and mouse models of breast carcinoma. Here, we developed a novel magnetic resonance imaging (MRI) methodology, called TMEM Activity-MRI, to detect TMEM-associated vascular openings that serve as the portal of entry for cancer cell intravasation and metastatic dissemination. We demonstrate that TMEM Activity-MRI correlates with primary tumor TMEM doorway counts in both breast cancer patients and mouse models, including MMTV-PyMT and patient-derived xenograft models. In addition, TMEM Activity-MRI is reduced in mouse models upon treatment with rebastinib, a specific and potent TMEM doorway inhibitor. TMEM Activity-MRI is an assay that specifically measures TMEM-associated vascular opening (TAVO) events in the tumor microenvironment, and as such, can be utilized in mechanistic studies investigating molecular pathways of cancer cell dissemination and metastasis. Finally, we demonstrate that TMEM Activity-MRI increases upon treatment with paclitaxel in mouse models, consistent with prior observations that chemotherapy enhances TMEM doorway assembly and activity in human breast cancer. Our findings suggest that TMEM Activity-MRI is a promising precision medicine tool for localized breast cancer that could be used as a non-invasive test to determine metastatic risk and serve as an intermediate pharmacodynamic biomarker to monitor therapeutic response to agents that block TMEM doorway-mediated dissemination.

Abstract 3124: An emerging paradigm of Cxcl12/Cxcr4 involvement in breast cancer metastasis

Abstract The Cxcl12/Cxcr4 signaling axis has been shown to promote metastasis in multiple mouse models of breast carcinoma and to be associated with increased metastatic risk in humans. Indeed, prior studies have specifically linked Cxcl12/Cxcr4 to breast cancer cell seeding, homing, survival and proliferation at future metastatic sites, due to the aberrant Cxcl12 expression in these sites (e.g. lung, liver and bone marrow). Interestingly however, the precise mechanism via which Cxcr4+ breast cancer cells escape the primary tumors in the first place (which also highly express Cxcl12), remains poorly understood. By using a novel methodology for quantifying chemotactic gradients using fixed tissue multichannel immunofluorescence (mIF), here, we demonstrate in mouse primary breast tumors that Cxcl12 gradients are concentrically expressed around cancer cell intravasation sites, known as Tumor Microenvironment of Metastasis (TMEM) doorways. Via distance analysis algorithms using mIF, we also demonstrate that TMEM-mediated Cxcl12 gradients contextually associate with Cxcr4+ breast cancer cells migrating towards the underlying TMEM doorways. As such, pharmacological inhibition of the Cxcl12/Cxcr4 pathway significantly abrogates the translocation of Cxcr4+ cancer cells to TMEM doorways, suppressing TMEM-mediated metastatic dissemination. However, targeted elimination of the Cxcr4+ gene specifically from breast cancer cells, paradoxically results in a suboptimal response, thus suggesting the existence of a bypass or compensatory mechanism. Previously, it was shown that Cxcr4+ tumor-associated macrophages (TAMs) support the invasive and migratory properties of tumor cells utilizing TMEM doorways. We thus theorized that, in the absence of Cxcr4 expression in tumor cells, the accompanying Cxcr4+ TAMs may still “read” TMEM-generated Cxcl12 chemotactic gradients. Indeed, clodronate-mediated TAM depletion results in the significant suppression of Cxcr4+ cancer cell translocation to TMEM doorways and their subsequent dissemination to the peripheral circulation and future metastatic sites. Finally, we used a variety of stromal and immune cell lineage markers to identify the precise source of TMEM-generated Cxcl12 gradients in mouse primary breast cancers. Despite that blood vessels (irrespective of presence of TMEM doorways) were primarily lined by Pdgfrb+ stromal cells with basal Cxcl12 expression, TMEM-generated Cxcl12 gradients were specifically linked to a subset of Cxcl12+Iba1+ perivascular TAMs. Pharmacological inhibition of Pdgfrb depletes Pdgfrb+Cxcl12+ stromal cells, but does not significantly affect Cxcl12/Cxcr4- mediated translocation of Cxcr4+ tumor cells to TMEM doorways. Overall, our data support a new paradigm for the implication of the Cxcl12/Cxcr4 axis during the early stages of the metastatic cascade, and propose a new avenue for rationalized antimetastatic treatments for breast cancer. Citation Format: Maria K. Lagou, Luis G. Rivera, Camille E. Duran, Joseph Burt, Xiaoming Chen, Yu Lin, Robert Eddy, Allison S. Harney, David Entenberg, John S. Condeelis, Maja H. Oktay, George S. Karagiannis. An emerging paradigm of Cxcl12/Cxcr4 involvement in breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3124.

An Emerging and Previously Unrecognized Paradigm of Cxcl12/Cxcr4 Pathway Implication in Breast Cancer Metastasis

Abstract The Cxcl12/Cxcr4 signaling axis promotes metastasis in mouse models of breast carcinoma and is linked with increased metastatic risk in humans. Prior studies have linked Cxcl12/Cxcr4 to breast cancer cell homing and survival at metastatic sites. However, the precise mechanism via which Cxcr4+ breast cancer cells escape primary tumors, which also express Cxcl12, is poorly understood. By using a novel imaging method for visualizing chemokine gradients, we show that Cxcl12 is concentrically expressed around cancer cell intravasation doorways, known as Tumor Microenvironment of Metastasis (TMEM). Using distance analysis algorithms, we also show that TMEM-mediated Cxcl12 gradients contextually associate with Cxcr4+ breast cancer cells migrating towards TMEM doorways. As such, pharmacological inhibition of the Cxcl12/Cxcr4 axis significantly abrogates translocation of Cxcr4+ cancer cells to TMEM doorways, thus suppressing metastatic dissemination. However, the targeted elimination of Cxcr4 from breast cancer cells paradoxically results in a suboptimal response compared to pharmacologic inhibition, implying the existence of a compensatory mechanism. Using clodronate-mediated macrophage depletion, we demonstrate that, in the absence of Cxcr4 expression in tumor cells, accompanying Cxcr4+ macrophages may still “read” TMEM-generated Cxcl12 chemotactic gradients. Indeed, Pdgfrb+ stromal cells may express low levels of Cxcl12, but TMEM-generated Cxcl12 gradients are primarily linked to a subset of Iba1+ perivascular tumor-associated macrophages. Overall, our data reveal a new paradigm for the implication of Cxcl12/Cxcr4 in early stages of metastasis and propose an avenue for rationalized antimetastatic treatments. Supported by Recruitment Funds RECR 3A3218

Combining TMEM Doorway Score and MenaCalc Score Improves the Prediction of Distant Recurrence Risk in HR+/HER2- Breast Cancer Patients.

Simple Summary90% of breast cancer mortality is caused by distant metastasis, a process that involves both dissemination of cancer cells to distant sites as well as their proliferation after arrival. However, prognostic assays currently used in the clinic are based on proliferation and do not measure tumor cell dissemination potential. We previously reported that the density of Tumor Microenvironment of Metastasis (TMEM) doorways (portals for cancer cell intravasation and dissemination) is a prognostic biomarker for the development of distant metastasis in hormone receptor positive/human epidermal growth factor receptor 2 negative (HR+/HER2−) patients. We have shown further that MenaCalc, a mechanistically linked (but independent) biomarker for distant metastasis, is prognostic in some cohorts of triple-negative patients. Here, we develop and compare several digital pathology-based machine vision algorithms to investigate if a combined TMEM-MenaCalc biomarker could provide improved prognostic information over and above that of either biomarker alone.Purpose: to develop several digital pathology-based machine vision algorithms for combining TMEM and MenaCalc scores and determine if a combination of these biomarkers improves the ability to predict development of distant metastasis over and above that of either biomarker alone. Methods: This retrospective study included a subset of 130 patients (65 patients with no recurrence and 65 patients with a recurrence at 5 years) from the Calgary Tamoxifen cohort of breast cancer patients. Patients had confirmed invasive breast cancer and received adjuvant tamoxifen therapy. Of the 130 patients, 86 cases were suitable for analysis in this study. Sequential sections of formalin-fixed paraffin-embedded patient samples were stained for TMEM doorways (immunohistochemistry triple staining) and MenaCalc (immunofluorescence staining). Stained sections were imaged, aligned, and then scored for TMEM doorways and MenaCalc. Different ways of combining TMEM doorway and MenaCalc scores were evaluated and compared to identify the best performing combined marker by using the restricted mean survival time (RMST) difference method. Results: the best performing combined marker gave an RMST difference of 5.27 years (95% CI: 1.71–8.37), compared to 3.56 years (95% CI: 0.95–6.1) for the associated standalone TMEM doorway analysis and 2.94 years (95% CI: 0.25–5.87) for the associated standalone MenaCalc analysis. Conclusions: combining TMEM doorway and MenaCalc scores as a new biomarker improves prognostication over that observed with TMEM doorway or MenaCalc Score alone in this cohort of 86 patients.

Further Evidence that Gradients of Extracellular pH Direct Migration of MDA-MB-231 Cells In Vitro.

We hypothesised that concentration gradients of O2/H+ within tissue guide migration of primary cancer cells toward intra-tumour microvessels, thus promoting intravasation and eventual haematogenous metastasis of cancer cells. Previously, we demonstrated in vitro that MDA-MB-231 cells under pH and O2 gradients (0.2-0.3units/mm and ~6%/mm, respectively) migrate toward higher pH/O2 regions. The present study was designed to address questions yet unanswered in the previous one, i.e., (1) whether extracellular O2 gradients could be a cue for directional cell migration in physiologically relevant O2 environments, and (2) whether average pH level in the bulk extracellular medium affects directional cell migration. In the absence of pH gradients, directional cell migration was not demonstrated at a physiological O2 level (<5%). We demonstrated that both the migration velocity and directionality are significantly affected by the average extracellular pH level. This result is consistent with our model for directional cell migration that does not necessitate sensing of pH gradient at a single cell level. Thus, in this study, we demonstrated further evidence that gradients of extracellular pH direct migration of MDA-MB-231 cells in vitro.

Establishment of Embryonic Zebrafish Xenograft Assays to Investigate TGF-β Family Signaling in Human Breast Cancer Progression.

Transforming growth factor-β (TGF-β) family members have pivotal functions in controlling breast cancer progression, acting not only on cancer cells but also on other cells within the tumor microenvironment. Here we describe embryonic zebrafish xenograft assays to investigate how TGF-β family signaling controls breast cancer cell intravasation, extravasation and regulates tumor angiogenesis. Fluorescently mCherry-labeled breast cancer cells are injected in the perivitelline space or Duct of Cuvier of Tg (fli:EGFP) transgenic Casper zebrafish embryos, in which the zebrafish express enhanced green fluorescent protein in the entire vasculature. The dynamic responses of migratory and invasive human cancer cells, and the induction of new blood vessel formation by the cancer cells in zebrafish host, are visualized using a fluorescent microscope. These assays provide efficient, reliable, low-cost models to investigate the effect of (epi)genetic modulators and pharmacological compounds that perturb the activity of TGF-β family signaling components on breast cancer cell metastasis and angiogenesis.

EMT-cancer cells-derived exosomal miR-27b-3p promotes circulating tumour cells-mediated metastasis by modulating vascular permeability in colorectal cancer.

BackgroundMetastasis is the main cause of death in colorectal cancer (CRC). Circulating tumour cells (CTCs) are regarded as the precursor cells of metastasis. The CTCs, which underwent epithelial‐mesenchymal transition (EMT), are associated with metastasis and responsible for poor prognosis. EMT cancer cells modulate endothelial permeability in the invasive front and facilitate cancer cell intravasation, resulting in CTCs‐mediated distant metastasis. Exosomes derived from cancer cells are key mediators of cancer‐host intercommunication. However, the mechanism by which EMT‐tumour cells‐derived exosomes modulate vascular permeability and promote CTCs generation has remained unclear.MethodsExosomes isolation and purification were conducted by ultra‐centrifugation. Exosomal miRNA was identified by sequencing followed by quantitative PCR. In vitro co‐culture assay experiments were conducted to evaluate the effect of exosomal miR‐27b‐3p on the permeability of blood vessel endothelium. Dual‐luciferase reporter assay, chromatin immunoprecipitation (ChIP) and RNA immunoprecipitation (RIP) were performed to investigate the underlying mechanism by which miR‐27b‐3p is packaged into exosomes. A mouse model was established to determine the role of exosomal miR‐27b‐3p in blood vessel permeability modulation in vivo.ResultsWe found that EMT‐CRC cells attenuate the blood vessel barrier by transferring miR‐27b‐3p to human umbilical vein endothelial cells (HUVECs) in exosomes. Mechanically, miR‐27b‐3p atteuated the expression of vascular endothelial cadherin (VE‐Cad) and p120 at the post‐transcriptional level by binding to 3′‐untranslated region of VE‐Cad and p120 directly. The packaging of miR‐27b‐3p into exosomes was induced by heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), which activated by STAT3. Clinically, miR‐27b‐3p up‐regulated in CRC tissues. Plasma exosomal miR‐27b‐3p was positively correlated with malignant progression and CTC count in CRC patients. Our study reveals a novel mechanism by which EMT‐CRC cells promote metastasis, increasing blood vessel permeability and facilitating the generation of CTCs.ConclusionExosomal miR‐27b‐3p secreted by EMT‐CRC cells increases blood vessel permeability and facilitates the generation of CTCs. Exosomal miR‐27b‐3p may become a promising biomarker for CRC metastasis.