Tumor Cell Intravasation Research Articles

Abstract A059: Understanding the events that promote pancreatic cancer metastasis

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy due to overwhelming metastatic burden. PDAC cells migrate and invade by two distinct epithelial-to-mesenchymal transition phenotypes: collective (partial-EMT) or single-cell (complete-EMT). The mechanism(s) by which these invasive phenotypes intravasate is unknown. The Tumor Microenvironment of Metastasis (TMEM) doorway, which is formed by a tumor cell, macrophage and an endothelial cell in direct contact, causes opening of the vasculature in which tumor cells intravasate. Intravasation via TMEM doorway is inhibited by Tie2 blockade. Whether this occurs in PDAC and whether the EMT phenotype affects intravasation is unknown. We hypothesize that PDAC cells intravasate through TMEM-dependent mechanisms regardless of EMT phenotype. Tumor samples from murine models 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 doorway opening was assessed in murine models of PDAC treated with or without Tie-2 inhibitor by analyzing TMEM-mediated extravasation of high-molecular weight dextran (155kD-TMR dextran) by Intravital Imaging, IHC and immunofluorescence (IF) of serial slides and circulating tumor cells. An intravasation trans-endothelial migration assay was also performed to assess the intravasation event by TMEM doorway in vitro. A student t-test was used for the analyses. TMEM doorways were observed in all PDAC murine models. For the first time in PDAC, focal extravasation of dextran events followed by a tumor cell intravasation was visualized by intravital imaging. PDAC tumor bearing mice treated with Tie2 inhibitor had a 2-fold decrease in the number of circulating tumor cells compared to control (958 ± 499.8 vs 534 ± 479.9 cells/mL blood, p=0.0257). The intravasation trans-endothelial migration assay showed that C-EMT cells have a higher migration rate than P-EMT cells (17.8 ± 5.53 vs 10.94 ± 3.84 migrating cells p=0.01), and this ratio increases with the presence of macrophages (23.75 ± 5 vs 15.54 ± 3.09 migrating cells p=0.005). The Tie-2 inhibition decreases the number of C-EMT and P-EMT migrating cells (7.69 ± 1.3 vs 3.67 ± 1.2 migrating cells p=0.01). Orthotopic tumors from C-EMT phenotype grow at a faster rate than the P-EMT (0.49mg ± 0.09 vs 0.12mg ± 0.05 p<0.05). Tumors from C-EMT and P-EMT cells will be analyzed for TMEM doorway score, TMEM activity and stemness signatures around TMEM doorway to determine if TMEM doorway is a niche for stemness in PDAC. TMEM doorways are present in PDAC mouse model. Inhibition of Tie2 leads to decreased TMEM doorway function and circulating tumor cells in PDAC. In addition, regarding the EMT phenotype, TMEM doorways are functional mechanisms of intravasation in vitro. This is a novel mechanism in PDAC and suggests a promising therapeutic target for decreasing intravasation and potential metastasis in PDAC. Citation Format: Erika Pereira Zambalde, Lina Ariyan, Francisco Puerta-Martinez, Yuqin Zhu, John Condeelis, Ben Stanger, John McAuliffe. Understanding the events that promote pancreatic cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A059.

A high-cholesterol diet promotes the intravasation of breast tumor cells through an LDL–LDLR axis

Most metastases in breast cancer occur via the dissemination of tumor cells through the bloodstream. How tumor cells enter the blood (intravasation) is, however, a poorly understood mechanism at the cellular and molecular levels. Particularly uncharacterized is how intravasation is affected by systemic nutrients. High levels of systemic LDL-cholesterol have been shown to contribute to breast cancer progression and metastasis in various models, but the cellular and molecular mechanisms involved are still undisclosed. Here we show that a high- cholesterol diet promotes intravasation in two mouse models of breast cancer and that this could be reverted by blocking LDL binding to LDLR in tumor cells. Moreover, we show that LDL promotes vascular invasion in vitro and the intercalation of tumor cells with endothelial cells, a phenotypic change resembling vascular mimicry (VM). At the molecular level, LDL increases the expression of SERPINE2, previously shown to be required for both VM and intravasation. Overall, our manuscript unravels novel mechanisms by which systemic hypercholesterolemia may affect the onset of metastatic breast cancer by favouring phenotypic changes in breast cancer cells and increasing intravasation.

Abstract 1507: Involvement of GABAergic signaling in enhanced tumor cell invasion in a mechanically dynamic tumor microenvironment

Abstract Metastasis is a major contributor to cancer morbidity and mortality. However, studying this complex, multi-step, multi-organ process is challenging. There is a pressing need to dissect the contributions of the tumor microenvironment (TME) on this process in a controlled and precise manner. Fortunately, the utilization of microfluidic “organs-on-chips” (OOC) in cancer research is facilitating such investigations. Moreover, by combining organoids and organs-on-chips we can enhance the progress of these studies in a patient specific manner. In this study, we have developed a cancer-on-chip model aimed at investigating early metastatic spread within the colon milieu. To achieve this, we employ colorectal cancer (CRC) cell lines as well as patient-derived CRC organoids chosen from our biorepository to encompass diverse representations across race/ethnicity, sex, and mutational profiles. The tumor cells are introduced into the upper chamber of the OOC model, while human microvascular endothelial cells (HIMECs) are introduced into the lower chamber to establish a tube-like structure resembling a blood vessel. These two chambers are separated by a porous membrane, and the chip is flanked by vacuum chambers to introduce stretch-like motions, simulating peristalsis in the gastrointestinal track. Creating a mechanically dynamic microenvironment facilitates the exploration of neurotransmitters and their impact on tumor cell behavior. Recent studies have suggested that tumor cells may exploit physiological processes, such as neurotransmitter signaling, to their benefit. By utilizing mass spectrometry-based metabolomics we detected dynamic and patient organoid-specific changes in neurotransmitter levels (i.e., serotonin, aspartate, glutamate, γ-aminobutyric acid (GABA)) in the effluent of our CRC-on-chip model. Coupled with live-cell imaging, we discovered tumor cell-derived GABA, a major inhibitory neurotransmitter, serves as an energy source for supporting tumor cell intravasation. This finding was most pronounced in KRAS-mutant tumor cells and further supported by analysis of CRC patient samples from The Cancer Genome Atlas (TCGA) database. We were able to reverse the GABA-mediated invasion effect by inhibiting 4-aminobutyrate aminotransferase (ABAT), the enzyme responsible for GABA catabolism. In summary, our cancer-on-chip model holds promise for exploring various aspects of the metastatic process and uncovering potential therapeutic targets, such as neurotransmitters. Citation Format: Carly Strelez, Rachel Perez, John S. Chlystek, Christopher Cherry, Bethany Haliday, Ah Young Yoon, Curran Shah, Ren X. Sun, Roy Lau, Aaron Schatz, Josh Neman, Heinz Josef-Lenz, Jonathan Katz, Shannon M. Mumenthaler. Involvement of GABAergic signaling in enhanced tumor cell invasion in a mechanically dynamic tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1507.

Development of a cancer metastasis-on-chip assay for high throughput drug screening.

Metastasis is the cause of most triple-negative breast cancer deaths, yet anti-metastatic therapeutics remain limited. To develop new therapeutics to prevent metastasis, pathophysiologically relevant assays that recapitulate tumor microenvironment is essential for disease modeling and drug discovery. Here, we have developed a microfluidic metastasis-on-chip assay of the early stages of cancer metastasis integrated with the triple-negative breast cancer cell line (MDA-MB-231), stromal fibroblasts and a perfused microvessel. High-content imaging with automated quantification methods was optimized to assess the tumor cell invasion and intravasation within the model. Cell invasion and intravasation were enhanced when fibroblasts co-cultured with a breast cancer cell line (MDA-MB-231). However, the non-invasive breast cancer cell line, MCF7, remained non-invasive in our model, even in the presence of fibroblasts. High-content screening of a targeted anti-cancer therapy drug library was conducted to evaluate the drug response sensitivity of the optimized model. Through this screening, we identified 30 compounds that reduced the tumor intravasation by 60% compared to controls. Multi-parametric phenotypic analysis was applied by combining the data from the metastasis-on-chip, cell proliferation and 2D cell migration screens, revealing that the drug library was clustered into eight distinct groups with similar drug responses. Notably, MEK inhibitors were enriched in cluster cell invasion and intravasation. In contrast, drugs with molecular targets: ABL, KIT, PDGF, SRC, and VEGFR were enriched in the drug clusters showing a strong effect on tumor cell intravasation with less impact on cell invasion or cell proliferation, of which, Imatinib, a multi-kinase inhibitor targeting BCR-ABL/PDGFR/KIT. Further experimental analysis showed that Imatinib enhanced endothelial barrier stability as measured by trans-endothelial electrical resistance and significantly reduced the trans-endothelial invasion activity of tumor cells. Our findings demonstrate the potential of our metastasis-on-chip assay as a powerful tool for studying cancer metastasis biology, drug discovery aims, and assessing drug responses, offering prospects for personalized anti-metastatic therapies for triple-negative breast cancer patients.

S6K1 deficiency in tumor stroma impairs lung metastasis of melanoma in mice

Accumulating data suggest that ribosomal protein S6 kinase 1 (S6K1), an effector in the mammalian target of rapamycin (mTOR) pathway, plays pleiotropic roles in tumor progression. However, to date, while the tumorigenic function of S6K1 in tumor cells has been well elucidated, its role in the tumor stroma remains poorly understood. We recently showed that S6K1 mediates vascular endothelial growth factor A (VEGF-A) production in macrophages, thereby supporting tumor angiogenesis and growth. As macrophage-derived VEGF-A is crucial for both tumor cell intravasation and extravasation across the vascular endothelium, our previous findings suggest that stromal S6K1 signaling is required for tumor metastatic spread. Therefore, we aimed to determine the impact of host S6K1 depletion on tumor metastasis using a murine model of pulmonary metastasis (S6k1−/− mice implanted with B16F10 melanoma). The ablation of S6K1 in the host microenvironment significantly reduced the metastasized B16F10 melanoma cells on the lung surface in both spontaneous and intravenous lung metastasis mouse models without affecting the incidence of metastasis to distant lymph nodes. In addition, stromal S6K1 loss decreased the number of tumor cells circulating in the peripheral blood of mice bearing B16F10 xenografts without affecting the vascular leakage induced by VEGF-A in vivo. These observations demonstrate that S6K1 signaling in host cells other than endothelial cells is required to modulate the host microenvironment to facilitate the metastatic spread of tumors via blood circulation, thus revealing its novel role in the tumor stroma during tumor progression.

Influence of Macrophages on Vascular Invasion of Inflammatory Breast Cancer Emboli Measured Using an In Vitro Microfluidic Multi-Cellular Platform.

Inflammatory breast cancer (IBC) is an aggressive disease with a poor prognosis and a lack of effective treatments. It is widely established that understanding the interactions between tumor-associated macrophages (TAMs) and the tumor microenvironment is essential for identifying distinct targeting markers that help with prognosis and subsequent development of effective treatments. In this study, we present a 3D in vitro microfluidic IBC platform consisting of THP1 M0, M1, or M2 macrophages, IBC cells, and endothelial cells. The platform comprises a collagen matrix that includes an endothelialized vessel, creating a physiologically relevant environment for cellular interactions. Through the utilization of this platform, it was discovered that the inclusion of tumor-associated macrophages (TAMs) led to an increase in the formation of new blood vessel sprouts and enhanced permeability of the endothelium, regardless of the macrophage phenotype. Interestingly, the platforms containing THP-1 M1 or M2 macrophages exhibited significantly greater porosity in the collagen extracellular matrix (ECM) compared to the platforms containing THP-1 M0 and the MDA-IBC3 cells alone. Cytokine analysis revealed that IL-8 and MMP9 showed selective increases when macrophages were cultured in the platforms. Notably, intravasation of tumor cells into the vessels was observed exclusively in the platform containing MDA-IBC3 and M0 macrophages.

On-chip modeling of tumor evolution: Advances, challenges and opportunities.

Tumor evolution is the accumulation of various tumor cell behaviors from tumorigenesis to tumor metastasis and is regulated by the tumor microenvironment (TME). However, the mechanism of solid tumor progression has not been completely elucidated, and thus, the development of tumor therapy is still limited. Recently, Tumor chips constructed by culturing tumor cells and stromal cells on microfluidic chips have demonstrated great potential in modeling solid tumors and visualizing tumor cell behaviors to exploit tumor progression. Herein, we review the methods of developing engineered solid tumors on microfluidic chips in terms of tumor types, cell resources and patterns, the extracellular matrix and the components of the TME, and summarize the recent advances of microfluidic chips in demonstrating tumor cell behaviors, including proliferation, epithelial-to-mesenchymal transition, migration, intravasation, extravasation and immune escape of tumor cells. We also outline the combination of tumor organoids and microfluidic chips to elaborate tumor organoid-on-a-chip platforms, as well as the practical limitations that must be overcome.

Selected Articles from This Issue

Highlights| May 04 2023 Selected Articles from This Issue Author & Article Information Online ISSN: 1538-8514 Print ISSN: 1535-7163 ©2023 American Association for Cancer Research2023American Association for Cancer Research Mol Cancer Ther (2023) 22 (5): 553. https://doi.org/10.1158/1535-7163.MCT-22-5-HI Related Content A commentary has been published: Discovery of a Small-Molecule Inhibitor Targeting the Androgen Receptor N-Terminal Domain for Castration-Resistant Prostate Cancer A commentary has been published: Novel Arginase Inhibitor, AZD0011, Demonstrates Immune Cell Stimulation and Antitumor Efficacy with Diverse Combination Partners A commentary has been published: Preclinical Evaluation of ON203, A Novel Bioengineered mAb Targeting Oxidized Macrophage Migration Inhibitory Factor as an Anticancer Therapeutic Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Tools Icon Tools Get Permissions Cite Icon Cite Search Site Article Versions Icon Versions Version of Record May 4 2023 Citation Selected Articles from This Issue. Mol Cancer Ther 1 May 2023; 22 (5): 553. https://doi.org/10.1158/1535-7163.MCT-22-5-HI Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest Search Advanced Search The oxidized macrophage migration inhibitory factor (oxMIF) is an emerging therapeutic target for many cancers due to oxMIF's selective occurrence in tumor lesions. Here, Rossmueller and colleagues describe ON203, a novel anti-oxMIF antibody with optimized physicochemical characteristics, in-vitro safety, and enhanced ADCC and ADCP effector functions. ON203 significantly reduced tumor growth and tumor cell proliferation as well as tumor cell intravasation and neo-angiogenesis in xenograft models. These findings highlight the potential of ON203 as a standalone immunotherapy or for enhancing efficacy in combination with angiogenesis and immune checkpoint-inhibitors in patients with solid tumors. Current mainstay treatment for prostate cancer is to suppress the androgen receptor (AR) signaling by castration and antiandrogens that target the ligand-binding domain (LBD). However, emergence of AR variants, such as AR-V7 lacking the LBD, have conferred severe drug resistance to all LBD-targeting AR inhibitors. AR-V7 has been established as a driver for the sustained AR signaling... You do not currently have access to this content.

Matrix stiffness regulates tumor cell intravasation through expression and ESRP1-mediated alternative splicing of MENA

During intravasation, cancer cells cross the endothelial barrier and enter the circulation. Extracellular matrix stiffening has been correlated with tumor metastatic potential; however, little is known about the effects of matrix stiffness on intravasation. Here, we utilize invitro systems, a mouse model, specimens from patients with breast cancer, and RNA expression profiles from The Cancer Genome Atlas Program (TCGA) to investigate the molecular mechanism by which matrix stiffening promotes tumor cell intravasation. Our data show that heightened matrix stiffness increases MENA expression, which promotes contractility and intravasation through focal adhesion kinase activity. Further, matrix stiffening decreases epithelial splicing regulatory protein 1 (ESRP1) expression, which triggers alternative splicing of MENA, decreases the expression of MENA11a, and enhances contractility and intravasation. Altogether, our data indicate that matrix stiffness regulates tumor cell intravasation through enhanced expression and ESRP1-mediated alternative splicing of MENA, providing a mechanism by which matrix stiffness regulates tumor cell intravasation.

A timeline of tumour-associated macrophage biology.

Tumour progression is modulated by the local microenvironment. This environment is populated by many immune cells, of which macrophages are among the most abundant. Clinical correlative data and a plethora of preclinical studies in mouse models of cancers have shown that tumour-associated macrophages (TAMs) play a cancer-promoting role. Within the primary tumour, TAMs promote tumour cell invasion and intravasation and tumour stem cell viability and induce angiogenesis. At the metastatic site, metastasis-associated macrophages promote extravasation, tumour cell survival and persistent growth, as well as maintain tumour cell dormancy in some contexts. In both the primary and metastatic sites, TAMs are suppressive to the activities of cytotoxic T and natural killer cells that have the potential to eradicate tumours. Such activities suggest that TAMs will be a major target for therapeutic intervention. In this Perspective article, we chronologically explore the evolution of our understanding of TAM biology put into the context of major enabling advances in macrophage biology.

Abstract IA017: The elusive ‘metastasis driver’: Is it hiding in plain sight

Abstract Recurrent single nucleotide mutations are a common feature of primary tumor evolution, where mutations in genes such as KRAS, APC, TP53, and BRAF are important drivers of malignancy. Efforts to identify comparable mutations that are exclusive to metastatic lesions - so-called 'metastasis drivers' - have been harder to find. However, the search for such metastasis-enabling genetic events has largely been limited to single nucleotide variants rather than chromosomal alterations (copy number gains and losses), which are far more common in advanced tumors. In this study, we used a stochastic mouse model of pancreatic cancer which gives rise to both highly metastatic and poorly metastatic tumors to interrogate the molecular correlates of metastatic heterogeneity. Our analysis revealed that focal amplifications of the MYC oncogene, but not other oncogenic drivers, were associated with the highly metastatic phenotype. Moreover, highly metastatic tumors exhibited elevated levels of MYC protein and greater expression of MYC target genes. This relationship between MYC copy number and metastasis was also observed in human samples, where MYC amplifications were found in 11% of metastases but fewer than 2% of primary tumors. Mechanistically, MYC enhances metastasis by recruiting pro-invasive macrophages, leading to greater vascular intravasation of tumor cells. These results nominate MYC as a bona fide metastasis driver gene and highlight the importance of considering copy number alterations in the evaluation of genetic mediators of metastasis. Citation Format: Ben Stanger. The elusive ‘metastasis driver’: Is it hiding in plain sight [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr IA017.

Challenges and Opportunities Modeling the Dynamic Tumor Matrisome.

We need novel strategies to target the complexity of cancer and, particularly, of metastatic disease. As an example of this complexity, certain tissues are particularly hospitable environments for metastases, whereas others do not contain fertile microenvironments to support cancer cell growth. Continuing evidence that the extracellular matrix (ECM) of tissues is one of a host of factors necessary to support cancer cell growth at both primary and secondary tissue sites is emerging. Research on cancer metastasis has largely been focused on the molecular adaptations of tumor cells in various cytokine and growth factor environments on 2-dimensional tissue culture polystyrene plates. Intravital imaging, conversely, has transformed our ability to watch, in real time, tumor cell invasion, intravasation, extravasation, and growth. Because the interstitial ECM that supports all cells in the tumor microenvironment changes over time scales outside the possible window of typical intravital imaging, bioengineers are continuously developing both simple and sophisticated in vitro controlled environments to study tumor (and other) cell interactions with this matrix. In this perspective, we focus on the cellular unit responsible for upholding the pathologic homeostasis of tumor-bearing organs, cancer-associated fibroblasts (CAFs), and their self-generated ECM. The latter, together with tumoral and other cell secreted factors, constitute the "tumor matrisome". We share the challenges and opportunities for modeling this dynamic CAF/ECM unit, the tools and techniques available, and how the tumor matrisome is remodeled (e.g., via ECM proteases). We posit that increasing information on tumor matrisome dynamics may lead the field to alternative strategies for personalized medicine outside genomics.

Agent-based modeling predicts RAC1 is critical for ovarian cancer metastasis.

Experimental and computational studies pinpoint rate-limiting step(s) in metastasis governed by Rac1. Using ovarian cancer cell and animal models, Rac1 expression was manipulated, and quantitative measurements of cell-cell and cell-substrate adhesion, cell invasion, mesothelial clearance, and peritoneal tumor growth discriminated the tumor behaviors most highly influenced by Rac1. The experimental data were used to parameterize an agent-based computational model simulating peritoneal niche colonization, intravasation, and hematogenous metastasis to distant organs. Increased ovarian cancer cell survival afforded by the more rapid adhesion and intravasation upon Rac1 overexpression is predicted to increase the numbers of and the rates at which tumor cells are disseminated to distant sites. Surprisingly, crowding of cancer cells along the blood vessel was found to decrease the numbers of cells reaching a distant niche irrespective of Rac1 overexpression or knockdown, suggesting that sites for tumor cell intravasation are rate limiting and become accessible if cells intravasate rapidly or are displaced due to diminished viability. Modeling predictions were confirmed through animal studies of Rac1-dependent metastasis to the lung. Collectively, the experimental and modeling approaches identify cell adhesion, rapid intravasation, and survival in the blood as parameters in the ovarian metastatic cascade that are most critically dependent on Rac1.

Fibroblast-derived CXCL12 increases vascular permeability in a 3-D microfluidic model independent of extracellular matrix contractility.

Cancer-associated fibroblasts (CAFs) play an active role in remodeling the local tumor stroma to support tumor initiation, growth, invasion, metastasis, and therapeutic resistance. The CAF-secreted chemokine, CXCL12, has been directly implicated in the tumorigenic progression of carcinomas, including breast cancer. Using a 3-D in vitro microfluidic-based microtissue model, we demonstrate that stromal CXCL12 secreted by CAFs has a potent effect on increasing the vascular permeability of local blood microvessel analogues through paracrine signaling. Moreover, genetic deletion of fibroblast-specific CXCL12 significantly reduced vessel permeability compared to CXCL12 secreting CAFs within the recapitulated tumor microenvironment (TME). We suspected that fibroblast-mediated extracellular matrix (ECM) remodeling and contraction indirectly accounted for this change in vessel permeability. To this end, we investigated the autocrine effects of CXCL12 on fibroblast contractility and determined that antagonistic blocking of CXCL12 did not have a substantial effect on ECM contraction. Our findings indicate that fibroblast-secreted CXCL12 has a significant role in promoting a leakier endothelium hospitable to angiogenesis and tumor cell intravasation; however, autocrine CXCL12 is not the primary upstream trigger of CAF contractility.

Abstract 959: Non-junctional connexin43 complexing with microtubules promotes epithelial-mesenchymal transition

Abstract Epithelial-mesenchymal transition (EMT) is a normal process of embryonic development, stem cell differentiation, and wound healing. During EMT, epithelial cells lose apical-basal polarity, remodel intercellular junctions, and acquire migratory and invasive abilities as they become mesenchymal. Pathologically, EMT has been demonstrated to contribute to intravasation of metastatic tumor cells into the vasculature. Among the activators of EMT, TGF-β has been shown to be most prevalent and is sufficient for experimental induction of EMT. Gap junctions, comprising connexin proteins, allow direct mechanical and metabolic coupling between adjacent cells, and represent a major intercellular structure altered during EMT. Connexin43 (Cx43) is a gap junction protein that has been implicated as both a tumor suppressor and enhancer depending on cancer stage. Our previous work revealed that despite losses in Cx43 gap junctions during EMT, mRNA and cytosolic levels of Cx43 protein increased. Such a shift of Cx43 to cytoplasmic pools has previously been observed in cancer progression and research suggests that non-junctional Cx43 can regulate the cytoskeleton, including microtubule stability. The C-terminus of Cx43 encompasses a tubulin binding domain which, while implicated in Cx43 trafficking, remains functionally poorly understood. We have identified a tumorigenic role for non-junctional Cx43 through its interaction with microtubules in glioma cancer stem cells. This led us to hypothesize that increased cytosolic Cx43 during EMT promotes Cx43-microtubule interaction to facilitate mobility and invasion of metastatic cancer cells. Using several independent TGF-β-induced EMT models, we observe increased complexing of Cx43 and microtubules during EMT by super-resolution microscopy. To study the role of Cx43 interaction with microtubules during EMT, we generated Cx43 harboring mutations in the tubulin binding domain which were ectopically expressed by adenoviral transduction in Cx43 knockout CRISPR/Cas9 epithelial cells. We find these Cx43 mutants fail to interact with microtubules compared to wild-type Cx43. Turning to TGF-β-induced EMT, ablation of the Cx43/microtubules interaction with these mutants is sufficient to decrease expression of mesenchymal markers and inhibit cell migration. We are currently investigating the effects of disrupting Cx43/microtubule complexing in vivo with a breast cancer mouse model to further elucidate effects on tumor progression and metastasis. Given the complex relationship between connexins and cancer progression, our findings support targeting this pathological ‘non-junctional’ Cx43 population specifically to limit metastatic disease, as Cx43 gap junction function is critical to normal tissue function and contributes to tumor suppression. Citation Format: Aryo Sorayya, Christina Wheeler, Stacie Deaver, Kenneth Young, Michael Zeitz, James Smyth, Samy Lamouille. Non-junctional connexin43 complexing with microtubules promotes epithelial-mesenchymal transition [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 959.

Phenotypic and transcriptomic profiles of tumor-conditioned macrophages in dogs, and the translational value of canine cancer immunology research.

Abstract Tumor-associated macrophages (TAMs) are heterogeneous and abundantly present in the tumor stroma. They derive from tissue-resident macrophages and recruited monocytes and play a central role in several aspects of cancer. In murine models, TAMs promote invasion, intravasation, circulatory survival and extravasation of tumor cells. TAMs also promote tumor progression and angiogenesis, remodel the tumor microenvironment, and modulate the adaptive immune system. Evidence suggests TAMs are vital for establishing the premetastatic niche. Historically, family-owned dogs have proven to be reliable cancer models. In contrast to laboratory animals, dogs develop cancer naturally and commonly, co-inhabit our environment, are genetically outbred and immunologically experienced. We hypothesized that canine tumor conditioned monocyte-derived macrophages (TCMs) share many characteristics with M2 macrophages and have a tumor-promoting phenotype as in humans. We collected blood from healthy dogs, isolated peripheral blood monocytes and differentiated them in vitro using tumor conditioned media from three canine cancer lines and M-CSF. Three TCM populations were generated and compared with M1 and M2 macrophages. We assessed the macrophages using multicolor flow cytometry and RNA-sequencing. All TCM showed an increased expression of two M2 markers (CD209a and FcɛRI), while 2/3 TCM populations had increased expression of two additional M2 markers (CD206 and CD11d). Transcriptomic analysis is ongoing. Results so far support the hypothesis that canine TCMs share many similarities with human TCMs, further warranting the use of dogs as cancer immunology models. Supported by grant from Agria Animal Insurances (N-2020-0061) and The Research fund for cancer in dogs (Oslo, Norway)

Mechanistic insights into the interplays between neutrophils and other immune cells in cancer development and progression.

Cancer is considered a major public health concern worldwide and is characterized by an uncontrolled division of abnormal cells. The human immune system recognizes cancerous cells and induces innate immunity to destroy those cells. However, sustained tumors may protect themselves by developing immune escape mechanisms through multiple soluble and cellular mediators. Neutrophils are the most plenteous leukocytes in the human blood and are crucial for immune defense in infection and inflammation. Besides, neutrophils emancipate the antimicrobial contents, secrete different cytokines or chemokines, and interact with other immune cells to combat and successfully kill cancerous cells. Conversely, many clinical and experimental studies signpost that being a polarized and heterogeneous population with plasticity, neutrophils, particularly their subpopulations, act as a modulator of cancer development by promoting tumor metastasis, angiogenesis, and immunosuppression. Studies also suggest that tumor infiltrating macrophages, neutrophils, and other innate immune cells support tumor growth and survival. Additionally, neutrophils promote tumor cell invasion, migration and intravasation, epithelial to mesenchymal transition, survival of cancer cells in the circulation, seeding, and extravasation of tumor cells, and advanced growth and development of cancer cells to form metastases. In this manuscript, we describe and review recent studies on the mechanisms for neutrophil recruitment, activation, and their interplay with different immune cells to promote their pro-tumorigenic functions. Understanding the detailed mechanisms of neutrophil-tumor cell interactions and the concomitant roles of other immune cells will substantially improve the clinical utility of neutrophils in cancer and eventually may aid in the identification of biomarkers for cancer prognosis and the development of novel therapeutic approaches for cancer treatment.

Novel antiangiogenic therapy targeting biglycan using tumor endothelial cell-specific liposomal siRNA delivery system.

Tumor blood vessels play important roles in tumor progression and metastasis. Targeting tumor endothelial cells (TECs) is one of the strategies for cancer therapy. We previously reported that biglycan, a small leucine‐rich proteoglycan, is highly expressed in TECs. TECs utilize biglycan in an autocrine manner for migration and angiogenesis. Furthermore, TEC‐derived biglycan stimulates tumor cell migration in a paracrine manner leading to tumor cell intravasation and metastasis. In this study, we explored the therapeutic effect of biglycan inhibition in the TECs of renal cell carcinoma using an in vivo siRNA delivery system known as a multifunctional envelope‐type nanodevice (MEND), which contains a unique pH‐sensitive cationic lipid. To specifically deliver MEND into TECs, we incorporated cyclo(Arg–Gly–Asp–d–Phe–Lys) (cRGD) into MEND because αVβ3 integrin, a receptor for cRGD, is selective and highly expressed in TECs. We developed RGD‐MEND‐encapsulating siRNA against biglycan. First, we confirmed that MEND was delivered into OS‐RC‐2 tumor‐derived TECs and induced in vitro RNAi‐mediated gene silencing. MEND was then injected intravenously into OS‐RC‐2 tumor‐bearing mice. Flow cytometry analysis demonstrated that MEND was specifically delivered into TECs. Quantitative RT‐PCR indicated that biglycan was knocked down by biglycan siRNA‐containing MEND. Finally, we analyzed the therapeutic effect of biglycan silencing by MEND in TECs. Tumor growth was inhibited by biglycan siRNA‐containing MEND. Tumor microenvironmental factors such as fibrosis were also normalized using biglycan inhibition in TECs. Biglycan in TECs can be a novel target for cancer treatment.

In This Issue

In This Issue

Lymphatic Endothelial Markers and Tumor Lymphangiogenesis Assessment in Human Breast Cancer

Metastasis via lymphatic vessels or blood vessels is the leading cause of death for breast cancer, and lymphangiogenesis and angiogenesis are critical prerequisites for the tumor invasion–metastasis cascade. The research progress for tumor lymphangiogenesis has tended to lag behind that for angiogenesis due to the lack of specific markers. With the discovery of lymphatic endothelial cell (LEC) markers, growing evidence demonstrates that the LEC plays an active role in lymphatic formation and remodeling, tumor cell growth, invasion and intravasation, tumor–microenvironment remodeling, and antitumor immunity. However, some studies have drawn controversial conclusions due to the variation in the LEC markers and lymphangiogenesis assessments used. In this study, we review recent findings on tumor lymphangiogenesis, the most commonly used LEC markers, and parameters for lymphangiogenesis assessments, such as the lymphatic vessel density and lymphatic vessel invasion in human breast cancer. An in-depth understanding of tumor lymphangiogenesis and LEC markers can help to illustrate the mechanisms and distinct roles of lymphangiogenesis in breast cancer progression, which will help in exploring novel potential predictive biomarkers and therapeutic targets for breast cancer.