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Background: Black women with residual estrogen receptor (ER)-positive breast cancer after neoadjuvant chemotherapy (NAC) have worse distant recurrence free survival (DRFS) compared to White women. Distant metastases result from motile breast cancer stem cells (BCSCs) disseminating through intravasation portals on blood vessels called tumor microenvironment of metastasis (TMEM) doorways. Each TMEM doorway is composed of an actin-regulatory protein (Mena) expressing cancer cell, a macrophage expressing high levels of receptor tyrosine kinase TIE2, and an angiopoietin-expressing endothelial cell. High TMEM doorway density predicts metastatic potential in patients with ER+/HER2- breast cancer. The function of TMEM doorways can be pharmacologically blocked using TIE2 inhibitors. We hypothesized that racial disparity in DRFS in patients with residual ER-positive disease post-NAC is due to difference in density of TMEM doorways, BCSCs, and motile cells in the residual breast tumor microenvironment (TME). Methods: We performed a retrospective, multi-institutional study of the TME in the residual disease post-NAC from 200 patients. Triple immunohistochemistry of formalin-fixed paraffin-embedded (FFPE) tissue sections was used to visualize TMEM doorways. Immunofluorescent staining of FFPE tissue sections for SOX9 and MenaINV was used to visualize BCSCs and motile cells, respectively. The density of TMEM doorways, BCSC and motile cells was determined by automated image analysis. The relationship between the density of TMEM doorways, stem and motile cells, and DRFS was examined using Kaplan Meier log-rank test. Comparisons were made for the entire cohort, and separately for the two most prevalent molecular subtypes: ER+/HER2- and triple negative (TN). The analysis included 200 samples of residual breast cancer post-NAC: 100 from Black and 100 from White women. Results: Black compared to White women were more likely to develop distant recurrence (49% vs 34%, p=0.04), to receive mastectomy (70% vs 51%, p=0.009), and to have TN subtype (35% vs 21%, p Citation Format: Gina Kim, Saeed Asiry, Isabelle Oktay, Yu Lin, Xianjun Ye, Esther Cheng, Nurfiza Ladak, John Condeelis, Esther Adler, Paula Ginter, Timothy D'Alfonso, David Entenberg, Maja Oktay. Racial disparity in post-neoadjuvant chemotherapy residual breast tumor microenvironment [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-36.

Supplementary Movie 2 from Direct Visualization of Macrophage-Assisted Tumor Cell Intravasation in Mammary Tumors

Legends to Supplementary Figures & Movies from Direct Visualization of Macrophage-Assisted Tumor Cell Intravasation in Mammary Tumors

Supplementary Movie from Epidermal Growth Factor Receptor Overexpression Results in Increased Tumor Cell Motility In vivo Coordinately with Enhanced Intravasation and Metastasis

Supplementary Figure 1 from An EGFR–Src–Arg–Cortactin Pathway Mediates Functional Maturation of Invadopodia and Breast Cancer Cell Invasion

Supplementary Movie 4 from Direct Visualization of Macrophage-Assisted Tumor Cell Intravasation in Mammary Tumors

Supplementary Figure 3 from Direct Visualization of Macrophage-Assisted Tumor Cell Intravasation in Mammary Tumors

Supplementary Figures Legends 1-6 from An EGFR–Src–Arg–Cortactin Pathway Mediates Functional Maturation of Invadopodia and Breast Cancer Cell Invasion

Supplementary Movie 1 from Direct Visualization of Macrophage-Assisted Tumor Cell Intravasation in Mammary Tumors

Although overexpression of the epidermal growth factor receptor (EGFR; ErbB1) has been correlated with poor prognosis in breast and other cancers, clinical trials of ErbB1 inhibitors have shown limited efficacy in inhibiting tumor proliferation. To evaluate other possible roles of ErbB1 in tumor malignancy besides proliferation, we have developed a series of tools for analysis of intravasation. Overexpression of ErbB1 in MTLn3 mammary adenocarcinoma cells results in increased intravasation and lung metastasis from tumors formed by injection of cells in the mammary fat pad. However, increased ErbB1 expression has no effect on primary tumor growth and lung seeding efficiency of cells injected i.v. Chemotactic responses to low concentrations of EGF in vitro and cell motility in vivo in the primary tumor measured using intravital imaging are significantly increased by ErbB1 overexpression. The increased cell motility is restricted to ErbB1-overexpressing cells in tumors containing mixtures of cells expressing different ErbB1 levels, arguing for a cell-autonomous effect of increased ErbB1 expression rather than alteration of the tumor microenvironment. In summary, we propose that ErbB1 overexpression makes more significant contributions to intravasation than growth in some tumors and present a novel model for studying ErbB1 contributions to tumor metastasis via chemotaxis and intravasation. (Cancer Res 2006; 66(1): 192-7)

Supplementary Figure 5 from An EGFR–Src–Arg–Cortactin Pathway Mediates Functional Maturation of Invadopodia and Breast Cancer Cell Invasion

Supplementary Figure 3 from An EGFR–Src–Arg–Cortactin Pathway Mediates Functional Maturation of Invadopodia and Breast Cancer Cell Invasion

Invasion of tumor cells into the surrounding connective tissue and blood vessels is a key step in the metastatic spread of breast tumors. Although the presence of macrophages in primary tumors is associated with increased metastatic potential, the mechanistic basis for this observation is unknown. Using a chemotaxis-based in vivo invasion assay and multiphoton-based intravital imaging, we show that the interaction between macrophages and tumor cells facilitates the migration of carcinoma cells in the primary tumor. Gradients of either epidermal growth factor (EGF) or colony-stimulating factor 1 (CSF-1) stimulate collection into microneedles of tumor cells and macrophages even though tumor cells express only EGF receptor and macrophages express only CSF-1 receptor. Intravital imaging shows that macrophages and tumor cells migrate toward microneedles containing either EGF or CSF-1. Inhibition of either CSF-1– or EGF-stimulated signaling reduces the migration of both cell types. This work provides the first direct evidence for a synergistic interaction between macrophages and tumor cells during cell migration in vivo and indicates a mechanism for how macrophages may contribute to metastasis.

Supplementary Figure 4 from An EGFR–Src–Arg–Cortactin Pathway Mediates Functional Maturation of Invadopodia and Breast Cancer Cell Invasion

Supplementary Figure 2 from Direct Visualization of Macrophage-Assisted Tumor Cell Intravasation in Mammary Tumors

Supplementary Movie 3 from Direct Visualization of Macrophage-Assisted Tumor Cell Intravasation in Mammary Tumors

Although the presence of macrophages in tumors has been correlated with poor prognosis, until now there was no direct observation of how macrophages are involved in hematogenous metastasis. In this study, we use multiphoton microscopy to show, for the first time, that tumor cell intravasation occurs in association with perivascular macrophages in mammary tumors. Furthermore, we show that perivascular macrophages of the mammary tumor are associated with tumor cell intravasation in the absence of local angiogenesis. These results show that the interaction between macrophages and tumor cells lying in close proximity defines a microenvironment that is directly involved in the intravasation of cancer cells in mammary tumors. [Cancer Res 2007;67(6):2649–56]

Supplementary Figure 1 from Direct Visualization of Macrophage-Assisted Tumor Cell Intravasation in Mammary Tumors

Background: Black, compared to White women with localized breast cancer have higher mortality and worse distant recurrence free survival (DRFS). This has been attributed to social determinants of health and higher prevalence of triple negative breast cancer (TNBC) in Black compared to White women. Recent studies indicate that racial disparity in outcome is present in patients with estrogen receptor-positive (ER+), but not ER- disease, in particular in patients with residual disease after neoadjuvant chemotherapy (NAC). It has been shown that in some patients NAC may induce pro-metastatic changes in tumor microenvironment, such as increased density of tumor associated macrophages and portals for cancer cell dissemination to distant sites called TMEM doorways (TMEM score). TMEM score correlates with metastasis in patients with ER+/HER2- breast cancer. We hypothesized that racial disparity in DRFS in patients with residual ER+/HER2- disease is due to enhanced pro-metastatic components (macrophage and TMEM doorway density) in the tumor microenvironment post-chemotherapy in Black compared to White women. Methods: We performed a retrospective, multi-institutional study of TMEM score and macrophage density in the residual disease after NAC from 196 patients diagnosed with unilateral invasive ductal cancer of breast between 2004 and 2014. 99 patients self-identified as Black and 97 as White. TMEM doorways were visualized by triple immunohistochemistry for macrophages (CD68), tumor cells (panMena), and endothelial cells (CD31). The evaluation of TMEM score and macrophage density was done using automated image analysis. Tumor characteristics and patient survival were compared between Black and White patients. The relationship between TMEM score, macrophage density and DRFS was examined by log-rank test and multivariate Cox regression model. The covariates in Cox model included TMEM score, age (continuous), race (Black vs White), surgery type (mastectomy vs lumpectomy), tumor stage (T3 vs T1; T2 vs T1), lymph node status (positive vs negative), and tumor subtype (triple negative [TN] vs ER+/HER2-; other vs ER+/HER2-). Results: Black compared to White women were more likely to develop distant recurrence (49.5% vs 34%, p=0.04), receive mastectomy (69.7% vs 51.5%, p=0.014), and have higher grade (p=0.001). Tumors from Black patients had more macrophages and a higher TMEM score in the entire cohort (p=0.004; p=0.001 respectively) and in the ER+/HER2- subset (p=0.008; p=0.008 respectively), but not in the TNBC subset. High TMEM score was associated with worse DRFS in all patients (p=0.004) and in the ER+/HER2- (p=0.03), but not in TNBC. In multivariate Cox model, TMEM score was an independent prognostic factor in the entire cohort (HR, 1.92; 95%CI, 1.15-3.22; p=0.01) and trended towards significance in ER+/HER2- disease (HR, 2.13; 95%CI, 0.96-4.71; p=0.06). TN, compared to ER+/HER2- cancers had higher TMEM score (p=0.01), and macrophage density (p=0.001). Conclusion: Racial disparity in outcome in patients with localized breast cancer may be due to a more pronounced pro-metastatic response to chemotherapy in Black, compared to White patients with ER+/HER2- disease. Thus, higher prevalence of TNBC in Black patients may not be the controlling factor in racial disparity. Citation Format: Burcu Karadal, Gina Kim, Ved Sharma, Jessica Pastoriza, Isabelle Oktay, Yu Lin, Xianjun Ye, Jiyue Qin, Esther Cheng, Nurfiza Ladak, John Condeelis, Esther Adler, Paula Ginter, Timothy D’Alfonso, Xiaonan Xue, David Enterberg, Joseph Sparano, Maja Oktay. Racial disparity in tumor microenvironment and outcomes in residual breast cancer treated with neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr GS1-02.

Cell invasion into the surrounding extracellular matrix or across tissue boundaries and endothelial barriers occurs in both physiological and pathological scenarios such as immune surveillance or cancer metastasis. Podosomes and invadopodia, collectively called 'invadosomes', are actin-based structures that drive the proteolytic invasion of cells, by forming highly regulated platforms for the localized release of lytic enzymes that degrade the matrix. Recent advances in high-resolution microscopy techniques, in vivo imaging and high-throughput analyses have led to considerable progress in understanding mechanisms of invadosomes, revealing the intricate inner architecture of these structures, as well as their growing repertoire of functions that extends well beyond matrix degradation. In this Review, we discuss the known functions, architecture and regulatory mechanisms of podosomes and invadopodia. In particular, we describe the molecular mechanisms of localized actin turnover and microtubule-based cargo delivery, with a special focus on matrix-lytic enzymes that enable proteolytic invasion. Finally, we point out topics that should become important in the invadosome field in the future.

Introduction: Pancreatic adenocarcinoma (PDAC) is highly lethal due to overwhelming metastatic burden [1]. The mechanisms underpinning metastasis in PDAC have not been identified. The TMEM doorway is the sole portal of intravasation in breast cancer [2, 3]. Importantly, intravasation and dissemination via TMEM doorways is inhibited by Tie2 blockade in breast cancer [4]. We evaluated whether TMEM Doorways are a common targetable mechanism of dissemination in PDAC. Methods: Tumor samples from treatment-naïve patients who underwent curative-intent resection of PDAC were stained for TMEM doorways using a triple immunohistochemistry (IHC) stain (tumor cells expressing the actin regulatory protein Mena, macrophages expressing CD68, and endothelial cells expressing CD31). TMEM doorways were manually counted in the highest TMEM doorway-bearing 400x field (hotspot) identified at 100x scanning magnification for each case. TMEM doorway function was assessed in a murine model of PDAC treated with or without rebastinib (an investigational oral Tie2 inhibitor [4]) by analyzing; (1) TMEM doorway mediated opening using high-molecular weight dextran (155kD-TMR dextran); (2) circulating tumor cells and (3) disseminated tumor cells in murine livers. The Wilcoxon Test was used for the human TMEM doorway scoring comparisons. A student t-test was used for the murine analyses. Results: TMEM doorways were observed in all PDAC tumors from 50 unique treatment-naïve patients. The TMEM doorway score was ≈6x higher than observed in published breast cancer patient samples. Mean TMEM doorway score in “hotspots” from resected human PDAC was 19 and 34 in low- and high-grade tumors, respectively (p-value < 0.01). TMEM doorway mediated opening was decreased by rebastinib treatment (159.8 µm2 ± sd26.5 vs 22.6 µm2 ± sd3.5, p Conclusions: TMEM doorways appear to be a common mechanism of dissemination in PDAC and breast cancer. TMEM doorway concentration is significantly associated with PDAC tumor grade. Inhibition of Tie2 with the selective inhibitor, rebastinib, preclinically leads to decreased TMEM doorway function and dissemination in PDAC. Tie2 is a promising therapeutic target for decreasing dissemination and potential metastasis in PDAC that warrants further clinical development in the PDAC space. 1.Siegel, R.L., et al. CA: A Cancer Journal for Clinicians, 2022. 72(1): p. 7-33.2.Harney, A.S., et al. Cancer Discovery, 2015. 5(9): p. 932-943.3.Karagiannis, G.S., et al., Science Translational Medicine, 2017. 9(397): p. eaan0026.4.Harney, A.S., et al. Molecular Cancer Therapeutics, 2017. 16(11): p. 2486-2501. Citation Format: Jakeb Petersen, Robert Eddy, Xianjun Ye, Christian Adkisson, David Entenberg, Maja Oktay, John Condeelis, Nicole Panarelli, John Christopher McAuliffe. TMEM doorways are an actionable target in pancreatic adenocarcinoma [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 2512.

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease. Tumors are poorly immunogenic and immunosuppressive, preventing T cell activation in the tumor microenvironment. Here, we present a microbial-based immunotherapeutic treatment for selective delivery of an immunogenic tetanus toxoid protein (TT856-1313) into PDAC tumor cells by attenuatedListeria monocytogenes. This treatment reactivated preexisting TT-specific memory T cells to kill infected tumor cells in mice. Treatment of KrasG12D,p53R172H, Pdx1-Cre (KPC) mice withListeria-TT resulted in TT accumulation inside tumor cells, attraction of TT-specific memory CD4 T cells to the tumor microenvironment, and production of perforin and granzyme B in tumors. Low doses of gemcitabine (GEM) increased immune effects ofListeria-TT, turning immunologically cold into hot tumors in mice. In vivo depletion of T cells fromListeria-TT + GEM–treated mice demonstrated a CD4 T cell–mediated reduction in tumor burden. CD4 T cells from TT-vaccinated mice were able to kill TT-expressing Panc-02 tumor cells in vitro. In addition, peritumoral lymph node–like structures were observed in close contact with pancreatic tumors in KPC mice treated withListeria-TT orListeria-TT + GEM. These structures displayed CD4 and CD8 T cells producing perforin and granzyme B. Whereas CD4 T cells efficiently infiltrated the KPC tumors, CD8 T cells did not.Listeria-TT + GEM treatment of KPC mice with advanced PDAC reduced tumor burden by 80% and metastases by 87% after treatment and increased survival by 40% compared to nontreated mice. These results suggest thatListeria-delivered recall antigens could be an alternative to neoantigen-mediated cancer immunotherapy.

Palpation, as already mentioned in the ancient Egyptian medical text Ebers Papyrus, utilizes that solid tumors are stiffer than the surrounding tissue. However, cancer cell lines tend to soften, which may intuitively foster invasion by enhancing the ability of cancer cells to squeeze through dense tissue. This paradox raises questions besides the oxymoron itself: Does softness emerge from adaptation to the external microenvironment? Or are soft cells already present inside a rigid primary tumor mass to support cancer cell unjamming? We investigate primary tumor explants from patients with breast and cervix carcinomas on multiple length scales from the tissue level down to single cells. We find that primary tumors are highly heterogeneous in their mechanical properties. From the tissue level this heterogeneity persists down to the scale of individual cells in cancer cell clusters, resulting in a broad distribution of cell rigidities with a higher fraction of softer, more squeezable cells. Plus, squeezed cell shapes correlate with cancer cell motility. Mechanical modelling based on patient data reveals that a tumor mass as a whole is able to maintain a rigid, solid behavior even when it contains a significant fraction of very soft cells. Cell softening induced cancer cell unjamming generates heterogeneous cancer cell clusters with a solid backbone of rigid cells surrounded by soft motile cells.

Metastatic dissemination is the major cause of cancer mortality and is responsible for over 1/2 million deaths each year in the U.S. alone. Over the past decades, metastasis has been speculated to be an inefficient process, as the majority of disseminated tumor cells (DTCs) are not believed to complete all the steps of the metastatic cascade. This conclusion has been reached by studies in which TCs are intravenously injected in mice - a process called experimental metastasis (EM) - and then attempt to quantify the number of disseminated cells in the lung over time using low resolution and indirect methods. To date, very little is known about the efficiency of each step in metastatic cascade due to the inability to track metastatic cancer cells in an intact organ over time. Using a new cutting-edge technology, the Window for High Resolution Imaging of the Lung1, we are able for the first time to (1) track in real-time the fate of each DTC and (2) quantitatively assess the efficiency of each step of the metastatic cascade in the lung, from DTC arrival and retention in the lung vasculature, extravasation, interaction with the host microenvironment, survival, dormancy, to growth into micro-metastases. All steps are studied at single-cell resolution, longitudinally, in the same animal, and using the EM model as well as a more clinically relevant model called Spontaneous Metastasis (SM) in which tumor cells spontaneously disseminate from an orthotopic primary tumor. A comparative analysis of the two models (EM vs. SM) demonstrated that DTCs in SM have a drastically increased metastatic efficiency compared to DTCs in EM. In particular, we found that DTCs are retained in the lung 10-times more efficiently compared to EM (62% vs. 6%). We further observe that in SM, DTCs extravasate in the lung very rapidly compared to EM (8 hrs vs. 24 hrs) and that the vast majority of DTCs after extravasation died in EM compared to SM where DTCs are dormant and express a stem-like phenotype. These data indicate that dissemination is indeed a very efficient process, but that growth at the secondary site is a rate-limiting step in the SM model. In conclusion, the ability to observe both spontaneous and experimental metastasis, with single cell resolution, and longitudinally, has provided new insight into the efficiency of each step of the metastatic cascade in the lung. This approach additionally gives the ability to investigate the molecular mechanisms underlying seeding and dormancy of metastatic tumor cells, in the presence of the full tumor microenvironment, as well as to directly evaluate the response of disseminated tumor cells to therapeutic treatment in real time in a live mouse. (1). Entenberg D, et al., (2017). A permanent window for the murine lung enables high-resolution imaging of cancer metastasis. Nat Methods. 15(1):73-80. Citation Format: Lucia Borriello, Anouchka Coste, Yarong Wang, Maja Oktay, David Entenberg, John Condeelis. Intravital imaging at single-cell resolution reveals, for the first time, the mechanism of cancer cell dissemination and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4514.

Tumor cell intravasation is an essential step in the metastatic cascade, but its exact mechanism is not completely understood. We have previously shown that the direct physical association of a tumor cell over-expressing Mena, a perivascular Tie2hi/Vegfhi macrophage and an endothelial cell, forming a cell triad termed “tumor microenvironment of metastasis” (TMEM), increases vascular permeability, facilitating intravasation of tumor cells. It is only at the TMEM site that intravasation occurs leading to breast tumor metastasis. TMEM density is a clinically validated prognostic marker of distant metastasis in breast cancer patients. The precise molecular mechanisms relating TMEM function had not been elucidated. Here we describe the molecular mechanism. We show here that TMEM function involves the three cells in TMEM: firstly endothelial cell-secreted Ang2 stimulates VEGF build up in the TMEM macrophage, secondly a tumor cell secretes CSF1 which, third, stimulates the TMEM macrophage VEGF secretion, leading to vascular opening and metastasis. In addition, we show that acute blockage of CSF1R and Tie2-Ang2 signaling by inhibitors and blocking antibodies both in vitro and in mammary tumors leads to decreased macrophage VEGF production and secretion, decreased trans-endothelial migration of tumor cells, and decreased TMEM-dependent vascular permeability, circulating tumor cells and lung metastases. We conclude that dynamic interaction between the cells associated with TMEM leads to Ang2 and CSF1-mediated stimulation of macrophage VEGF expression and secretion leading to vascular opening, resulting in tumor cell intravasation. This is the first description of the molecular mechanism behind the predictive power of the clinically used prognostic marker TMEM and represents a major step in defining new biomarkers and targets for the treatment of metastatic tumors. Citation Format: Chinmay R. Surve, Allison Harney, Yarong Wang, Xiaoming Chen, Ved Sharma, Richard Stanley, Maja Oktay, John Condeelis. Regulation of breast tumor metastasis by the dynamic interaction between the TMEM macrophage, tumor, and endothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 976.

Neoadjuvant chemotherapy (NAC) is used for treatment of localized breast cancer to decrease tumor size and improve surgical outcome. However, Karagiannis et al. (2017) has uncovered a previously unrecognized side-effect, which involves the induction of pro-metastatic changes in the primary breast cancer microenvironment, in response to NAC treatment. In particular, NAC promotes the assembly of structures that serve as doorways for intravasation of tumor cells called tumor microenvironment of metastasis (TMEM), and increases the proportion of the highly invasive and migratory MenaINV-hi/Mena11alo (MenaCalc+) tumor cells, which utilize the TMEM sites for hematogenous dissemination (Karagiannis et al., 2017). MenaINV expression in particular is associated with decreased sensitivity of cancer cells to receptor tyrosine-kinase (RTK) and tyrosine-kinase (TK) inhibitors and with dramatically increased TMEM-dependent intravasation. The mechanism behind these pro-metastatic changes was not known until recently. Here, we report that NAC induces an influx of macrophages in the tumors, resulting in increased contact between macrophages and tumor cells leading to MenaINV expression in the cancer cells, and MenaINV- and TMEM-dependent tumor cell intravasation. Multichannel IF imaging and distance analysis algorithms demonstrate that MENAINV-hi cells have a higher probability of being in direct cell contact with infiltrating macrophages in chemotherapy-treated tumors. Specific depletion of the macrophage lineage in transgenic MMTV-PyMT mice developing spontaneous breast carcinoma and patient-derived xenografts results in a significant suppression of the prometastatic MenaINV-hi cancer cell subpopulation. Moreover, chemotherapy-induced MenaINV expression in tumor cells is not seen in such macrophage-depleted mice, indicating that macrophage influx is both necessary and sufficient for the generation of the de novo prometastatic tumor cell subpopulation. These data provide an explanation as to why mice and patients with breast cancer do not always respond with a decrease in circulating tumor cells (CTCs), or even respond with a slight increase, following cytotoxic NAC treatment. Our work has uncovered a previously unrecognized mechanism behind pro-metastatic changes in response to cytotoxic chemotherapy and the markers that predict these changes (TMEM, MenaCalc and MenaINV). Reference: Karagiannis GS, et al. Neoadjuvant chemotherapy induces breast cancer metastasis through aTMEM-mediated mechanism. Sci Transl Med, 2017; 9 aeen0026. Citation Format: George S. Karagiannis, Luis Rivera Sanchez, Yarong Wang, Ved P. Sharma, Joseph Burt, Sara Brizio, David Entenberg, Maja H. Oktay, John S. Condeelis. MENAINV expression identifies a de novo chemotherapy-induced prometastatic subpopulation in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 913.

As molecular labels for cells and tissues, fluorescent probes have shaped our understanding of biological structures and processes. However, their capacity for quantitative analysis is limited because photon emission rates from multicolour fluorophores are dissimilar, unstable and often unpredictable, which obscures correlations between measured fluorescence and molecular concentration. Here we introduce a new class of light-emitting quantum dots with tunable and equalized fluorescence brightness across a broad range of colours. The key feature is independent tunability of emission wavelength, extinction coefficient and quantum yield through distinct structural domains in the nanocrystal. Precise tuning eliminates a 100-fold red-to-green brightness mismatch of size-tuned quantum dots at the ensemble and single-particle levels, which substantially improves quantitative imaging accuracy in biological tissue. We anticipate that these materials engineering principles will vastly expand the optical engineering landscape of fluorescent probes, facilitate quantitative multicolour imaging in living tissue and improve colour tuning in light-emitting devices., Quantum dots with different size emit light at different wavelengths but also different brightness, which complicates analysis of fluorescence images. Here, the authors synthesize multicolour brightness-equalized quantum dots by controlling the composition and structure of core-shell HgCdSeS-CdZnS nanocrystals.

Heterogeneity within and between primary tumors (PTs) is reflected by genetic and epigenetic diversity and varying PT microenvironments. Further, whether PT microenvironments might influence the fate of disseminating tumor cells (DTC) has never been explored. We found that breast tumors enriched for a specific dormancy signature (DS+) displayed longer metastasis-free periods than those poor (DS-) for the signature. Key genes in the DS induce quiescence and are also regulated by hypoxia. Clinical evidence links hypoxic tumors to increased therapy resistance and a worse outcome. However, a main response of tumor cells to hypoxia is growth arrest, but how this response is linked to the clinical outcome is unknown. We hypothesized that hypoxic PT microenvironments may spawn a subpopulation of DTCs that, by virtue of becoming dormant, might escape therapies and eventually fuel incurable metastasis. We used H2B-GFP inducible HEp3 HNSCC and photo-switchable (green-to-red fluorescence) H2B-Dendra2 expressing MDA-MB-231 and ZR-75-1 human breast cancer cell lines to identify cells from hypoxic microenvironments. To initiate spatially defined hypoxic microenvironments in primary tumors we implanted induction NANo IntraVItal Devices (iNANIVIDs) carrying a hypoxia-mimetic agent (desferrioxamine - DFOM) in T-HEp3 tumors in vivo or exposed cultured MDA-MB-231 or ZR-75-1 cells in vitro to either 21% or 1% O2. The regions influenced by the DFOM-iNANIVID displayed significant upregulation of p27, NR2F1 and DEC2 (dormancy genes), as well as induction of hypoxia markers (GLUT1, HIF1α). Human HNSCC PT samples showed the same link between spontaneous hypoxic regions and up regulation of dormancy markers. We further found a significant increase in quiescent lung DTCs of hypoxia induced H2B-GFP T-HEp3 or H2B-Dendra2 MDA-MB-231 cells, traceable > 2 weeks after extravasation using H2B-GFP and H2B-Dendra2-RED label retention. Using human Vimentin to screen for HEp3 tumor cells in lungs, we found that single, unproliferating DTCs originating from the iNANIVID induced hypoxic regions showed a dormant profile (upregulation of p27, NR2F1, DEC2 and TGFβ2) compared to DTCs originating from a normoxic milieu. Simultaneously, only the hypoxic pre-treated group was able to form micro-metastasis at 10 days after injection, suggesting the presence of a more aggressive sub clone in this group. Further, analysis in 3D culture models revealed that ER+/DS+ breast cancer cells (ZR-75-1) are more prone to enter a prolonged quiescent state after a brief exposure to hypoxia (1% O2) in an NR2F1-dependent manner. This response is not observed in triple negative/DS- breast cancer cells. Lastly, using a spontaneously metastatic PyMT driven Dendra2-tagged breast cancer model, we found that ~75% of dormant DTCs up regulate the dormancy marker NR2F1 at or soon after reaching the lung, suggesting a rapid induction of dormancy upon reaching target organs. We propose that hypoxic primary tumor stress microenvironments increase phenotypic heterogeneity of DTCs and leads to the expression of the DS. Upon spreading, these DTCs may be more prone to enter dormancy, evade anti-proliferative therapies and eventually fuel metastasis. Citation Format: Georg Fluegen, Alvaro Avivar-Valderas, Yarong Wang, Michael R. Padgen, James K. Williams, Vladislav Verkhusha, Julie F. Cheung, David Entenberg, James Castracane, Patricia J. Keely, John Condeelis, Julio A. Aguirre-Ghiso. Phenotypic heterogeneity of disseminated tumor cells is predetermined by primary tumor hypoxic microenvironments. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A43.

Analysis of the individual steps in metastasis is crucial if insights at the molecular level are to be linked to the cell biology of cancer. A technical hurdle to achieving the analysis of the individual steps of metastasis is the fact that, at the gross level, tumors are heterogeneous in both animal models and patients. Human primary tumors show extensive variation in all properties ranging from growth and morphology of the tumor through tumor-cell density in the blood and formation and growth of metastases. Methods capable of the direct visualization and analysis of tumor-cell behavior at single-cell resolution in vivo have become crucial in advancing the understanding of mechanisms of metastasis, the definition of microenvironment, and the markers related to both. High-resolution multiphoton imaging of tumors in vivo is a valuable tool in this regard. Because tumor cells have been found to be attracted to blood vessels, the in vivo invasion assay was developed to analyze which factors may stimulate invasion of these cells into the vessels. This protocol describes the preparation and handling of collection needles for the assay. A set of 33-gauge needles is used to create artificial or surrogate blood vessels that are injected into tumors, using a special holding device attached to a micromanipulator to stabilize the needle positions during and after insertion into the anesthetized animal. The needles are filled with Matrigel and various growth factors to determine which of these factors may influence the invading tumor cells.

This is a correction of an earlier published article.

Herein we analyze how the aging process affects the structure and functionality of the lymphatic collectors (LCs) with reference to their ability to maintain pathogen clearance. Ultrastructural, biochemical and proteomic analysis indicated a loss of extracellular matrix proteins, an increase in protein oxidative modifications as well as activation of nuclear factor-κB signaling as sign of “low grade” inflammation in aged LCs. This resulted in a decrease in contractile and pumping activity of LCs, as measured in vivo. Functionally, this impairment also translated into a reduced ability for in vivo bacterial transport as determined by time-lapse microscopy. Ultrastructural and proteomic analysis also indicated a decrease in the thickness of the endothelial cell glycocalyx and loss of gap-junction proteins in aged LCs. Redox proteomic analysis mapped an aging-related increase in the glycation and carboxylation of endothelial cell glycocalyx structural proteins. Functionally, these modifications translated into higher ability of the pathogen to escape from aged LCs into the surrounding tissue. Altogether, our analysis mapped the complexity of the aging-related anatomical, biochemical and functional changes in LCs. The decreased ability to transport bacteria to the draining nodes, associated with increased bacterial escape in the surrounding tissue can contribute to the decreased ability of the immune system to clear pathogens in the elderly, as observed in immunosenescence.

During metastasis from a primary tumor, cell motility is believed to be important for dissemination of tumor cells from the primary. An understanding of the mechanisms of cell motility in metastatic tumor cells in vivo, in particular, would be important for a more rational system of diagnosis and treatment of metastatic cancers. We have developed a new method for imaging tumor cells in live animals and have identified subpopulations of moving cells within primary breast tumors. Previous to this approved application, we described only a metastatic cell-line (MTLn3) using this technique. Since then, we have described a non-metastatic cell line (MTC) and reported the observable differences between the two cell lines. We proposed to use this novel imaging method to collect the motile subpopulation of tumor cells in live primary tumors which can then be used for further analysis to define how these cells differ from their non-motile neighbors in the same tumor and to define how cell motility contributes to metastasis. We have been able to differentially collect the motile sub-population of metastatic cells and shown that due to the differing chemotactic response to EGF, we were able to selectively collected the metastatic cell

Metastasis is the leading cause of the death in cancer patients. Cell motility is believed to be a necessary step in the metastatic process. Currently, most methods available to study the behavior of metastatic tumor cells are indirect, e.g., cell motility is examined in vitro and the results are correlated with metastatic capability. We have developed a model that directly examines the motility of metastatic primary tumor cells in situ. A metastatic rat breast cancer cell line was established that constitutively expresses the green fluorescent protein (GFP). Upon subcutaneous injection of these cells into the mammary fat pad of female Fischer 344 rats, primary tumors and subsequent metastases form that fluoresce when excited with light passed through an FITC filter set. Animations of metastatic tumor cells moving in live rats were generated by intravital imaging of the primary tumor, in situ, on a laser scanning confocal microscope. With this model, the behavioral phenotype of tumor cells can be described and the effects of genetic manipulations or therapeutic treatments on this phenotype can be determined. This is the first report of visualization of cells in a primary tumor in live animals in a clinically relevant model.

A new actin binding protein has been purified to homogeneity from amoebae of Dictyostelium discoideum. This protein is a single polypeptide with a molecular weight of 120,000 upon sodium dodecyl sulfate gel electrophoresis. It is soluble and trypsin-sensitive, contains no carbohydrate, increases the viscosity and sedimentation rate of F actin, and inhibits the actin-stimulated Mg ATPase of rabbit muscle heavy meromyosin. The interaction of 120,000-dalton protein with F actin is not inhibited by millimolar ATP, pyrophosphate, or micromolar calcium. The 120,000-dalton actin binding protein increases the initial rate of actin polymerization and decreases the critical concentration of actin at steady state. These properties demonstrate that 120,000-dalton protein from Dictyostelium discoidum is not a myosinlike protein. Rather, this protein is probably involved in regulating the assembly of the actin cytoskeletion.

Myosin has been isolated from the large carnivorous amoebae Chaos carolinensis and Amoeba proteus to 90% purity with the aid of microtechniques. These techniques for electrophoresis, dialysis, colorimetric protein, and ATPase assays and centrifugation permitted the isolation, purification, and characterization of microgram quantities of myosin from less than 1 ml of packed cells. The purification utilized sucrose extraction of the actomyosin, low ionic strength precipitation, and final separation of the actin and myosin by gel filtration in the presence of KI. The properties of carnivorous amoeba myosin are similar to those of several other cytoplasmic myosins. Its heavy chain molecular weight is greater than that of the heavy chain of rabbit skeletal muscle myosin. The ATPase activities of myosin from C. carolinensis and A. proteus were almost identical and demonstrated inhibition by EDTA and Mg++ and activation by Ca++ in the presence of 0.6 m KCl and the absence of actin. Actin activated the Mg++ ATPase activity by an average factor of 6. No cofactor protein was required for this activity. These properties are similar to those of myosin isolated from Starfish eggs, Physarum, and Dictyostelium. Carnivorous amoeba myosin bound rabbit muscle F actin in the absence but not in the presence of Mg++ and ATP.