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.

Signaling events at TMEM doorways provide potential targets for inhibiting breast cancer dissemination.

Tumor cell intravasation is essential for metastatic dissemination, but its exact mechanism is incompletely understood. We have previously shown that in breast cancer, the direct and stable association of a tumor cell expressing Mena, a Tie2hi/VEGFhi macrophage, and a vascular endothelial cell, creates an intravasation portal, called a "tumor microenvironment of metastasis" (TMEM) doorway, for tumor cell intravasation, leading to dissemination to distant sites. The density of TMEM doorways, also called TMEM doorway score, is a clinically validated prognostic marker of distant metastasis in breast cancer patients. Although we know that tumor cells utilize TMEM doorway-associated transient vascular openings to intravasate, the precise signaling mechanisms involved in TMEM doorway function are only partially understood. Using two mouse models of breast cancer and an in vitro assay of intravasation, we report that CSF-1 secreted by the TMEM doorway tumor cell stimulates local secretion of VEGF-A from the Tie2hi TMEM doorway macrophage, leading to the dissociation of endothelial junctions between TMEM doorway associated endothelial cells, supporting tumor cell intravasation. Acute blockade of CSF-1R signaling decreases macrophage VEGF-A secretion as well as TMEM doorway-associated vascular opening, tumor cell trans-endothelial migration, and dissemination. These new insights into signaling events regulating TMEM doorway function should be explored further as treatment strategies for metastatic disease.

FAK family proteins regulate in vivo breast cancer metastasis via distinct mechanisms.

Breast cancer is the most commonly diagnosed malignancy and the major leading cause of tumor-related deaths in women. It is estimated that the majority of breast tumor-related deaths are a consequence of metastasis, to which no cure exists at present. The FAK family proteins Proline-rich tyrosine kinase (PYK2) and focal adhesion kinase (FAK) are highly expressed in breast cancer, but the exact cellular and signaling mechanisms by which they regulate in vivo tumor cell invasiveness and consequent metastatic dissemination are mostly unknown. Using a PYK2 and FAK knockdown xenograft model we show here, for the first time, that ablation of either PYK2 or FAK decreases primary tumor size and significantly reduces Tumor MicroEnvironment of Metastasis (TMEM) doorway activation, leading to decreased intravasation and reduced spontaneous lung metastasis. Intravital imaging analysis further demonstrates that PYK2, but not FAK, regulates a motility phenotype switch between focal adhesion-mediated fast motility and invadopodia-dependent, ECM-degradation associated slow motility within the primary tumor. Furthermore, we validate our in vivo and intravital imaging results with integrated transcriptomic and proteomic data analysis from xenograft knockdown tumors and reveal new and distinct pathways by which these two homologous kinases regulate breast tumor cell invasiveness and consequent metastatic dissemination. Our findings identify PYK2 and FAK as novel mediators of mammary tumor progression and metastasis and as candidate therapeutic targets for breast cancer metastasis.

Tracking Tumor Cell Dissemination from Lung Metastases Using Photoconversion.

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional process wherein cells from the primary tumor disseminate and seed metastases, tumor cells in existing metastases can also redisseminate and give rise to new lesions in tertiary sites in a process known as "metastasis-from-metastases" or "metastasis-to-metastasis seeding." Metastasis-to-metastasis seeding may increase the metastatic burden and decrease the patient's quality of life and survival. Therefore, understanding the processes behind this phenomenon is crucial to refining treatment strategies for patients with metastatic cancer. Little is known about metastasis-to-metastasis seeding, due in part to logistical and technological limitations. Studies on metastasis-to-metastasis seeding rely primarily on sequencing methods, which may not be practical for researchers studying the exact timing of metastasis-to-metastasis seeding events or what promotes or prevents them. This highlights the lack of methodologies that facilitate the study of metastasis-to-metastasis seeding. To address this, we have developed - and describe herein - a murine surgical protocol for the selective photoconversion of lung metastases, allowing specific marking and fate tracking of tumor cells redisseminating from the lung to tertiary sites. To our knowledge, this is the only method for studying tumor cell redissemination and metastasis-to-metastasis seeding from the lungs that does not require genomic analysis.

Racial disparity in tumor microenvironment and distant recurrence in residual breast cancer after neoadjuvant chemotherapy.

Black, compared to white, women with residual estrogen receptor-positive (ER+) breast cancer after neoadjuvant chemotherapy (NAC) have worse distant recurrence-free survival (DRFS). Such racial disparity may be due to difference in density of portals for systemic cancer cell dissemination, called TMEM doorways, and pro-metastatic tumor microenvironment (TME). Here, we evaluate residual cancer specimens after NAC from 96 Black and 87 white women. TMEM doorways are visualized by triple immunohistochemistry, and cancer stem cells by immunofluorescence for SOX9. The correlation between TMEM doorway score and pro-metastatic TME parameters with DRFS is examined using log-rank and multivariate Cox regression. Black, compared to white, patients are more likely to develop distant recurrence (49% vs 34.5%, p = 0.07), receive mastectomy (69.8% vs 54%, p = 0.04), and have higher grade tumors (p = 0.002). Tumors from Black patients have higher TMEM doorway and macrophages density overall (p = 0.002; p = 0.002, respectively) and in the ER+/HER2- (p = 0.02; p = 0.02, respectively), but not in the triple negative disease. Furthermore, high TMEM doorway score is associated with worse DRFS. TMEM doorway score is an independent prognostic factor in the entire study population (HR, 2.02; 95%CI, 1.18-3.46; p = 0.01), with a strong trend in ER+/HER2- disease (HR, 2.38; 95%CI, 0.96-5.95; p = 0.06). SOX9 expression is not associated with racial disparity in TME or outcome. In conclusion, higher TMEM doorway density in residual breast cancer after NAC is associated with higher distant recurrence risk, and Black patients are associated with higher TMEM doorway density, suggesting that TMEM doorway density may contribute to racial disparities in breast cancer.

Macrophages Promote Tumor Cell Extravasation across an Endothelial Barrier through Thin Membranous Connections.

Macrophages are important players involved in the progression of breast cancer, including in seeding the metastatic niche. However, the mechanism by which macrophages in the lung parenchyma interact with tumor cells in the vasculature to promote tumor cell extravasation at metastatic sites is not clear. To mimic macrophage-driven tumor cell extravasation, we used an in vitro assay (eTEM) in which an endothelial monolayer and a matrigel-coated filter separated tumor cells and macrophages from each other. The presence of macrophages promoted tumor cell extravasation, while macrophage conditioned media was insufficient to stimulate tumor cell extravasation in vitro. This finding is consistent with a requirement for direct contact between macrophages and tumor cells. We observed the presence of Thin Membranous Connections (TMCs) resembling similar structures formed between macrophages and tumor cells called tunneling nanotubes, which we previously demonstrated to be important in tumor cell invasion in vitro and in vivo. To determine if TMCs are important for tumor cell extravasation, we used macrophages with reduced levels of endogenous M-Sec (TNFAIP2), which causes a defect in tunneling nanotube formation. As predicted, these macrophages showed reduced macrophage-tumor cell TMCs. In both, human and murine breast cancer cell lines, there was also a concomitant reduction in tumor cell extravasation in vitro when co-cultured with M-Sec deficient macrophages compared to control macrophages. We also detected TMCs formed between macrophages and tumor cells through the endothelial layer in the eTEM assay. Furthermore, tumor cells were more frequently found in pores under the endothelium that contain macrophage protrusions. To determine the role of macrophage-tumor cell TMCs in vivo, we generated an M-Sec deficient mouse. Using an in vivo model of experimental metastasis, we detected a significant reduction in the number of metastatic lesions in M-Sec deficient mice compared to wild type mice. There was no difference in the size of the metastases, consistent with a defect specific to tumor cell extravasation and not metastatic outgrowth. Additionally, with an examination of time-lapse intravital-imaging (IVI) data sets of breast cancer cell extravasation in the lungs, we could detect the presence of TMCs between extravascular macrophages and vascular tumor cells. Overall, our data indicate that macrophage TMCs play an important role in promoting the extravasation of circulating tumor cells in the lungs.

Cooperative NF-κB and Notch1 signaling promotes macrophage-mediated MenaINV expression in breast cancer.

Metastasis is a multistep process that leads to the formation of clinically detectable tumor foci at distant organs and frequently to patient demise. Only a subpopulation of breast cancer cells within the primary tumor can disseminate systemically and cause metastasis. To disseminate, cancer cells must express MenaINV, an isoform of the actin regulatory protein Mena, encoded by the ENAH gene, that endows tumor cells with transendothelial migration activity, allowing them to enter and exit the blood circulation. We have previously demonstrated that MenaINV mRNA and protein expression is induced in cancer cells by macrophage contact. In this study, we discovered the precise mechanism by which macrophages induce MenaINV expression in tumor cells. We examined the promoter of the human and mouse ENAH gene and discovered a conserved NF-κB transcription factor binding site. Using live imaging of an NF-κB activity reporter and staining of fixed tissues from mouse and human breast cancer, we further determined that for maximal induction of MenaINV in cancer cells, NF-κB needs to cooperate with the Notch1 signaling pathway. Mechanistically, Notch1 signaling does not directly increase MenaINV expression, but it enhances and sustains NF-κB signaling through retention of p65, an NF-κB transcription factor, in the nucleus of tumor cells, leading to increased MenaINV expression. In mice, these signals are augmented following chemotherapy treatment and abrogated upon macrophage depletion. Targeting Notch1 signaling in vivo decreased NF-κB signaling activation and MenaINV expression in the primary tumor and decreased metastasis. Altogether, these data uncover mechanistic targets for blocking MenaINV induction that should be explored clinically to decrease cancer cell dissemination and improve survival of patients with metastatic disease.

Cooperative NF-κB and Notch1 signaling promotes macrophage-mediated MenaINV expression in breast cancer.

Metastasis is a multistep process that leads to the formation of clinically detectable tumor foci at distant organs and frequently patient demise. Only a subpopulation of breast cancer cells within the primary tumor can disseminate systemically and cause metastasis. To disseminate, cancer cells must express MenaINV, an isoform of the actin-regulatory protein Mena encoded by the ENAH gene that endows tumor cells with transendothelial migration activity allowing them to enter and exit the blood circulation. We have previously demonstrated that MenaINV mRNA and protein expression is induced in cancer cells by macrophage contact. In this study, we discovered the precise mechanism by which macrophages induce MenaINV expression in tumor cells. We examined the promoter of the human and mouse ENAH gene and discovered a conserved NF-κB transcription factor binding site. Using live imaging of an NF-κB activity reporter and staining of fixed tissues from mouse and human breast cancer we further determined that for maximal induction of MenaINV in cancer cell NF-κB needs to cooperate with the Notch1 signaling pathway. Mechanistically, Notch1 signaling does not directly increase MenaINV expression, but it enhances and sustains NF-κB signaling through retention of p65, an NF-κB transcription factor, in the nucleus of tumor cells, leading to increased MenaINV expression. In mice, these signals are augmented following chemotherapy treatment and abrogated upon macrophage depletion. Targeting Notch1 signaling in vivo decreased NF-κB signaling and MenaINV expression in the primary tumor and decreased metastasis. Altogether, these data uncover mechanistic targets for blocking MenaINV induction that should be explored clinically to decrease cancer cell dissemination and improve survival of patients with metastatic disease.

Intravital imaging to study cancer progression and metastasis.

Navigation through the bulk tumour, entry into the blood vasculature, survival in the circulation, exit at distant sites and resumption of proliferation are all steps necessary for tumour cells to successfully metastasize. The ability of tumour cells to complete these steps is highly dependent on the timing and sequence of the interactions that these cells have with the tumour microenvironment (TME), including stromal cells, the extracellular matrix and soluble factors. The TME thus plays a major role in determining the overall metastatic phenotype of tumours. The complexity and cause-and-effect dynamics of the TME cannot currently be recapitulated in vitro or inferred from studies of fixed tissue, and are best studied in vivo, in real time and at single-cell resolution. Intravital imaging (IVI) offers these capabilities, and recent years have been a time of immense growth and innovation in the field. Here we review some of the recent advances in IVI of mammalian models of cancer and describe how IVI is being used to understand cancer progression and metastasis, and to develop novel treatments and therapies. We describe new techniques that allow access to a range of tissue and cancer types, novel fluorescent reporters and biosensors that allow fate mapping and the probing of functional and phenotypic states, and the clinical applications that have arisen from applying these techniques, reporters and biosensors to study cancer. We finish by presenting some of the challenges that remain in the field, how to address them and future perspectives.

Interleukin 4 Controls the Pro-Tumoral Role of Macrophages in Mammary Cancer Pulmonary Metastasis in Mice.

Metastasis is the systemic manifestation of cancer and the main cause of death from breast cancer. In mouse models of lung metastases, recruitment of classical monocytes from blood to the lung and their differentiation to metastasis-associated macrophages (MAMs) facilitate cancer cell extravasation, survival and growth. Ablation of MAMs or their monocytic progenitors inhibits metastasis. We hypothesized that factors controlling macrophage polarization modulate tumor cell extravasation in the lung. We evaluated whether signaling by Th1 or Th2 cytokines in macrophages affected transendothelial migration of tumor cells in vitro. Interferon gamma and LPS inhibited macrophage-dependent tumor cell extravasation while the Th2 cytokine interleukin-4 (IL4) enhanced this process. We demonstrated that IL4 receptor (IL4rα)-null mice developed fewer and smaller lung metastasis in E0771-LG mammary cancer models of this disease. Adoptive transfer of wild-type monocytes to IL4rα-deficient mice partially rescued this phenotype. IL4 signaling in macrophages controlled the expression of the chemokine receptor CXCR2, necessary for IL4-mediated tumor cell extravasation in vitro. Furthermore, IL4 signaling in macrophages regulated the transcript abundance of several other genes already causally associated with mammary cancer lung metastasis including Ccl2, Csf1, Ccr1, Hgf and Flt1. The central role of IL4 signaling in MAMs was confirmed by high-resolution intravital imaging of the lung in mice at the time of metastatic seeding, which showed reduced physical interaction between tumor cells and IL4rα-deficient macrophages. This interaction with wild-type MAMs enhanced tumor cell survival and seeding, which was lost in the IL4rα mice. These data indicate that IL4 signaling in monocytes and macrophages is key during seeding and growth of breast metastasis in the lung, as it regulates pro-tumoral paracrine signaling between cancer cells and macrophages.

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.

SWIP-a stabilized window for intravital imaging of the murine pancreas.

Pancreatitis and pancreatic ductal adenocarcinoma (PDAC) are grave illnesses with high levels of morbidity and mortality. Intravital imaging (IVI) is a powerful technique for visualizing physiological processes in both health and disease. However, the application of IVI to the murine pancreas presents significant challenges, as it is a deep, compliant, visceral organ that is difficult to access, easily damaged and susceptible to motion artefacts. Existing imaging windows for stabilizing the pancreas during IVI have unfortunately shown poor stability for time-lapsed imaging on the minutes to hours scale, or are unable to accommodate both the healthy and tumour-bearing pancreata. To address these issues, we developed an improved stabilized window for intravital imaging of the pancreas (SWIP), which can be applied to not only the healthy pancreas but also to solid tumours like PDAC. Here, we validate the SWIP and use it to visualize a variety of processes for the first time, including (1) single-cell dynamics within the healthy pancreas, (2) transformation from healthy pancreas to acute pancreatitis induced by cerulein, and (3) the physiology of PDAC in both autochthonous and orthotopically injected models. SWIP can not only improve the imaging stability but also expand the application of IVI in both benign and malignant pancreas diseases.

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

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.

Racial disparity in distant recurrence-free survival in patients with localized breast cancer: A pooled analysis of National Surgical Adjuvant Breast and Bowel Project trials.

BACKGROUND: Black race is associated with worse outcome in patients with breast cancer. The distant relapse-free survival (DRFS) between Black and White women with localized breast cancer who participated in National Cancer Institute-sponsored clinical trial was evaluated. METHODS: Pooled data were analyzed from 8 National Surgical Adjuvant Breast and Bowel Project (NSABP) trials including 9702 women with localized breast cancer treated with adjuvant chemotherapy (AC, n = 7485) or neoadjuvant chemotherapy (NAC, n = 2217), who self-reported as Black (n = 1070) or White (n = 8632) race. The association between race and DRFS was analyzed using log-rank tests and multivariate Cox regression. RESULTS: After adjustment for covariates including age, tumor size, nodal status, body mass index and taxane use, and treatment (AC vs NAC), Black race was associated with an inferior DRFS in estrogen receptor-positive (ER+; hazard ratio [HR], 1.24; 95% CI, 1.05-1.46; P = .01), but not in ER- disease (HR, 0.97; 95% CI, 0.83-1.14; P = .73), and significant interaction between race and ER status was observed (P = .03). There was no racial disparity in DRFS among patients with pathologic complete response (pCR) (log-rank P = .8). For patients without pCR, Black race was associated with worse DRFS in ER+ (HR, 1.67; 95% CI, 1.14-2.45; P = .01), but not in ER- disease (HR, 0.91; 95% CI, 0.65-1.28; P = .59). CONCLUSIONS: Black race was associated with significantly inferior DRFS in ER+ localized breast cancer treated with AC or NAC, but not in ER- disease. In the NAC group, racial disparity was also observed in patients with residual ER+ breast cancer at surgery, but not in those who had pCR. LAY SUMMARY: Black women with breast cancer have worse outcomes compared with White women. We investigated if this held true in the context of clinical trials that provide controlled treatment setting. Black women with cancer expressing estrogen receptors (ERs) had worse outcome than White women. If breast cancers did not express ERs, there was no racial disparity in outcome. We also observed racial disparity in women who received chemotherapy before their cancer was removed, but only if they had cancer expressing ERs and residual disease on completion of treatment. If the cancer disappeared with presurgical chemotherapy, there was no racial disparity.

Primary tumor associated macrophages activate programs of invasion and dormancy in disseminating tumor cells.

Metastases are initiated by disseminated tumor cells (DTCs) that colonize distant organs. Growing evidence suggests that the microenvironment of the primary tumor primes DTCs for dormant or proliferative fates. However, the manner in which this occurs remains poorly understood. Here, using the Window for High-Resolution Intravital Imaging of the Lung (WHRIL), we study the live lung longitudinally and follow the fate of individual DTCs that spontaneously disseminate from orthotopic breast tumors. We find that spontaneously DTCs have increased levels of retention, increased speed of extravasation, and greater survival after extravasation, compared to experimentally metastasized tumor cells. Detailed analysis reveals that a subset of macrophages within the primary tumor induces a pro-dissemination and pro-dormancy DTC phenotype. Our work provides insight into how specific primary tumor microenvironments prime a subpopulation of cells for expression of proteins associated with dissemination and dormancy.

Live tumor imaging shows macrophage induction and TMEM-mediated enrichment of cancer stem cells during metastatic dissemination.

Cancer stem cells (CSCs) play an important role during metastasis, but the dynamic behavior and induction mechanisms of CSCs are not well understood. Here, we employ high-resolution intravital microscopy using a CSC biosensor to directly observe CSCs in live mice with mammary tumors. CSCs display the slow-migratory, invadopod-rich phenotype that is the hallmark of disseminating tumor cells. CSCs are enriched near macrophages, particularly near macrophage-containing intravasation sites called Tumor Microenvironment of Metastasis (TMEM) doorways. Substantial enrichment of CSCs occurs on association with TMEM doorways, contributing to the finding that CSCs represent >60% of circulating tumor cells. Mechanistically, stemness is induced in non-stem cancer cells upon their direct contact with macrophages via Notch-Jagged signaling. In breast cancers from patients, the density of TMEM doorways correlates with the proportion of cancer cells expressing stem cell markers, indicating that in human breast cancer TMEM doorways are not only cancer cell intravasation portals but also CSC programming sites.

Targeting Tie2 in the Tumor Microenvironment: From Angiogenesis to Dissemination.

The Tie2 receptor tyrosine kinase is expressed in vascular endothelial cells, tumor-associated macrophages, and tumor cells and has been a major focus of research in therapies targeting the tumor microenvironment. The most extensively studied Tie2 ligands are Angiopoietin 1 and 2 (Ang1, Ang2). Ang1 plays a critical role in vessel maturation, endothelial cell migration, and survival. Ang2, depending on the context, may function to disrupt connections between the endothelial cells and perivascular cells, promoting vascular regression. However, in the presence of VEGF-A, Ang2 instead promotes angiogenesis. Tie2-expressing macrophages play a critical role in both tumor angiogenesis and the dissemination of tumor cells from the primary tumor to secondary sites. Therefore, Ang-Tie2 signaling functions as an angiogenic switch during tumor progression and metastasis. Here we review the recent advances and complexities of targeting Tie2 signaling in the tumor microenvironment as a possible anti-angiogenic, and anti-metastatic, therapy and describe its use in combination with chemotherapy.

SUN-MKL1 Crosstalk Regulates Nuclear Deformation and Fast Motility of Breast Carcinoma Cells in Fibrillar ECM Microenvironment.

Aligned collagen fibers provide topography for the rapid migration of single tumor cells (streaming migration) to invade the surrounding stroma, move within tumor nests towards blood vessels to intravasate and form distant metastases. Mechanisms of tumor cell motility have been studied extensively in the 2D context, but the mechanistic understanding of rapid single tumor cell motility in the in vivo context is still lacking. Here, we show that streaming tumor cells in vivo use collagen fibers with diameters below 3 µm. Employing 1D migration assays with matching in vivo fiber dimensions, we found a dependence of tumor cell motility on 1D substrate width, with cells moving the fastest and the most persistently on the narrowest 1D fibers (700 nm-2.5 µm). Interestingly, we also observed nuclear deformation in the absence of restricting extracellular matrix pores during high speed carcinoma cell migration in 1D, similar to the nuclear deformation observed in tumor cells in vivo. Further, we found that actomyosin machinery is aligned along the 1D axis and actomyosin contractility synchronously regulates cell motility and nuclear deformation. To further investigate the link between cell speed and nuclear deformation, we focused on the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex proteins and SRF-MKL1 signaling, key regulators of mechanotransduction, actomyosin contractility and actin-based cell motility. Analysis of The Cancer Genome Atlas dataset showed a dramatic decrease in the LINC complex proteins SUN1 and SUN2 in primary tumor compared to the normal tissue. Disruption of LINC complex by SUN1 + 2 KD led to multi-lobular elongated nuclei, increased tumor cell motility and concomitant increase in F-actin, without affecting Lamin proteins. Mechanistically, we found that MKL1, an effector of changes in cellular G-actin to F-actin ratio, is required for increased 1D motility seen in SUN1 + 2 KD cells. Thus, we demonstrate a previously unrecognized crosstalk between SUN proteins and MKL1 transcription factor in modulating nuclear shape and carcinoma cell motility in an in vivo relevant 1D microenvironment.

Hematogenous Dissemination of Breast Cancer Cells From Lymph Nodes Is Mediated by Tumor MicroEnvironment of Metastasis Doorways.

In primary breast tumors, cancer cells hematogenously disseminate through doorways in the vasculature composed of three-cell complexes (known as Tumor MicroEnvironment of Metastasis) comprising a perivascular macrophage, a tumor cell overexpressing the actin-regulatory protein Mammalian Enabled (Mena), and an endothelial cell, all in direct physical contact. It has been previously shown that once tumor cells establish lymph node metastases in patients, TMEM doorways form in the metastatic tumor cell nests. However, it has not been established if such lymph node-TMEM doorways actively transit tumor cells into the peripheral circulation and on to tertiary sites. To address this question in this short report, we used a mouse model of lymph node metastasis to demonstrate that TMEM doorways: (1) exist in tumor-positive lymph nodes of mice, (2) are restricted to the blood vascular endothelium, (3) serve as a mechanism for further dissemination to peripheral sites such as to the lungs, and (4) their activity can be abrogated by a pharmaceutical intervention. Our data suggest that cancer cell dissemination via TMEM doorways is a common mechanism of breast cancer cell dissemination to distant sites and thus the pharmacological targeting of TMEM may be necessary, even after resection of the primary tumor, to suppress cancer cell dissemination.

A small-molecule allosteric inhibitor of BAX protects against doxorubicin-induced cardiomyopathy.

Doxorubicin remains an essential component of many cancer regimens, but its use is limited by lethal cardiomyopathy, which has been difficult to target, owing to pleiotropic mechanisms leading to apoptotic and necrotic cardiac cell death. Here we show that BAX is rate-limiting in doxorubicin-induced cardiomyopathy and identify a small-molecule BAX inhibitor that blocks both apoptosis and necrosis to prevent this syndrome. By allosterically inhibiting BAX conformational activation, this compound blocks BAX translocation to mitochondria, thereby abrogating both forms of cell death. When co-administered with doxorubicin, this BAX inhibitor prevents cardiomyopathy in zebrafish and mice. Notably, cardioprotection does not compromise the efficacy of doxorubicin in reducing leukemia or breast cancer burden in vivo, primarily due to increased priming of mitochondrial death mechanisms and higher BAX levels in cancer cells. This study identifies BAX as an actionable target for doxorubicin-induced cardiomyopathy and provides a prototype small-molecule therapeutic.