Cell-derived nanoplatforms for cancer therapy

BackgroundTumor is a new organism formed by abnormal hyperplasia of local tissue cells under the action of various tumorigenic factors. Inflammation plays a decisive role in inducing tumorigenesis, promoting tumor development, invasion and migration. More and more evidence indicate that exosomes are involved in regulating the formation of tumor microenvironment in the process of proinflammatory carcinogenesis, leading to the stimulation of anti-tumor immune response or systemic immunosuppression, and exosomes play a crucial role in the development of tumor.MethodsThe articles on tumor-derived exosomes and inflammatory responses from January 2005 to January 2024 were collected through Web of Science (WOS), and the inclusion criteria were “Article”, “Review Article” and “Early Access”. Articles obtained after excluding “Book Chapters”, “Editorial Material”, “Proceeding Paper”, “Meeting ” and “Retracted Publication”. Bibliometrics and visualization analysis were carried out on the obtained articles using CiteSpace6.2.R6 and VOSviewer1.6.20.ResultsTotal of 703 articles were included. The number of published documents showed a fluctuating growth trend year by year. A total of 61 countries have participated in the research on the effects of exosomes and inflammatory responses on tumors, among which China and the United States have the largest influence in this field. The obtained articles have been published in 60 journals around the world, among which PLOS ONE and NAT REV IMMUNOL are the journals with the most published articles and the highest co-citations respectively. The article from French author THERY C was cited the most (202 times). As a major researcher on the basic function of exosomes, THERY C established the gold standard for extraction, separation and identification of exosomes, and found that exosomes promote tumor metastasis through direct regulation of miRNA. Her research has had a huge impact on the field. Keyword co-occurrence analysis indicate that extracellular vesicles, inflammation, cancer, miRNAs, mesenchymal stem cells, drug delivery, gastric cancer and circulating endothelial microparticles are the research hotspot at present stage. The main keywords of the cluster analysis show that extracellular vesicles, human papilloma virus, myeloid cells, tumor macro-environment are the current research hotspots and frontier. The research hotspots have developed over time from the time chart of keywords and clustering, especially after 2016, exosomes have established extensive links with drug delivery, cancer treatment, inflammatory response and other fields. Tumor-derived exosomes stimulate receptor cells to secrete pro-inflammatory cytokines and growth factors, enabling immune and inflammatory cells to perceive the intracellular environment of cancer cells even when cancer cells do not express any tumor-specific antigens. For example, in anoxic environment, cancer cells can secrete exosomes containing pro-inflammatory factors to promote the invasion and metastasis of cancer cells. In the complex tumor microenvironment, both tumor cells and various stromal cells will secrete specific exosomes, and promote the development of tumors through various ways, so that tumor cells have drug resistance, and bring adverse effects on the clinical treatment of tumor patients. MicroRNAs and long noncoding RNA as hot keywords play important roles in regulating and mediating tumor development, and their specificity makes them important biomarkers for cancer prediction and diagnosis. Highlighting word analysis shows that microRNAs secreted by leukemia patients can effectively promote the proliferation of malignant cells and the development of cardiovascular diseases. At the same time, exosomes can induce the secretion of some microRNAs in patients, leading to cardiac repair and regeneration. Therefore, the detection and screening of microRNAs plays a crucial role in predicting the incidence of cardiovascular diseases in patients.ConclusionExosomes have attracted increasing attention due to their significant heterogeneity and ability to regulate the tumor immune microenvironment. However, tumor cell-derived exosomes accelerate tumor progression by enhancing immunosuppression and inflammation, increasing oxidative stress, and promoting angiogenesis, which may lead to poor prognosis.Graphical abstract

Chronic cancer-associated inflammation and immunosuppression are common features in most patients with solid malignancies. The causes of this chronic inflammatory-immunosuppressive state are still largely undefined. We hypothesized that selective RNAs can be secreted by cancer cells in extracellular vesicles (EVs) and may trigger proinflammatory responses in target cells, leading to chronic inflammation linked to immune cell dysfunction and immunosuppression. We found that tumor cell lines from pancreatic ductal adenocarcinoma (PDAC), prostate cancer (PCa) and from pediatric cancer such as Ewing sarcoma (EwS) continuously secrete large numbers of small (40-200 nm) EVs. In contrast to non-transformed fibroblasts, cancer cell-derived EVs are enriched with large subsets of retroelement and pericentromeric transcripts, including LINE, SINE and HERV retroelements, and human satellite 2 and 3 (HSAT2,3) RNAs. These virus-like RNAs were highly elevated in plasma EVs from EwS patients but not in healthy donors. Some of them, including HERV-K and HSAT2, were detected in peripheral blood myeloid cells with CD33+HLA-DR− immunosuppressive phenotypes, and these cell populations were expanded in EwS patients compared to healthy donors. Using mouse xenografts and in vitro models, we also found that at least some of these RNAs, such as HSAT2, are transmitted to stromal fibroblasts and immune cells in the tumor microenvironment. They also accumulated in fibroblasts after treatment with EwS EVs, coincident with the induction of proinflammatory and DNA damage responses. Prolonged exposure of fibroblasts to EwS EVs also led to mitotic defects and senescence. Expression and dissemination of these highly immunogenic virus-like RNAs in EVs may thus be a common feature of multiple human malignancies, potentially affecting host cells in the local and systemic tumor environment. This, in turn, may induce chronic inflammation contributing to an overall immunosuppressed state in patients. Citation Format: Valentina Evdokimova, Peter Ruzanov, Hendrik Gassmann, Lincoln D. Stein, Poul H. Sorensen, Stefan Burdach, Laszlo Radvanyi. Tumor-derived extracellular vesicles transmit retroelement and pericentromeric RNAs to drive proinflammatory and DNA damage responses in stromal fibroblasts and immune cells. [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 4468.

Tumor-secreted extracellular vesicles (EVs) play an important role as mediators of intercellular communication. Hypoxia is a common feature of solid tumors frequently associated with an aggressive clinical behavior. This study aimed to gain a deeper understanding into the functions of EVs in intercellular communication between primary and metastatic cancer cells under hypoxic conditions. EVs were isolated from two isogenic colorectal cancer (CRC) cell lines SW480 and SW620 cultured under normoxic and hypoxic conditions. Their uptake and effects in SW480 and SW620 cells were studied using EV uptake, proliferation, spheroid-formation, wound healing and invasion assays. Our data showed that hypoxia enhanced the release of EVs from CRC cells in a Hypoxia Induced Factor (HIF)-1-dependent manner. Hypoxic EVs were taken up by CRC cells more efficiently than normoxic EVs. Hypoxic EVs stimulated motility, invasiveness and stemness of primary tumour-derived SW480 cells, whereas they had a little effect on metastasis-derived SW620 cells. Hypoxic colorectal cancer-derived EVs confer aggressiveness and invasiveness to hypoxia-naïve cancer cells.

Metastatic breast cancer still lacks effective treatment, and remains the primary cause of mortality. Therefore, new effective strategies to inhibit breast cancer metastasis are needed. The Rho family GTPase Rac is an ideal target for anti-metastatic cancer therapy, because Rac is a key molecular switch that can promote cancer cell migration/invasion and survival. Previously, we reported the design and development of EHop-016, a small molecule compound, which inhibits Rac activation by the oncogene Vav in metastatic cancer cells with an IC50 of 1 microM. Consequently, EHop-016 inhibits the activity of the Rac downstream effector p21-activated kinase (PAK) and cell migration in metastatic breast cancer cells; and significantly reduces tumor growth, metastasis, and angiogenesis in a mouse model of metastatic breast cancer. The reduced tumor angiogenesis in response to EHop-016 treatment may be attributed to the inhibition of the Vav/Rac activity of endothelial cells, as shown by the ability of EHop-016 to inhibit the Rac activation and capillary tube formation of endothelial cells, in vitro. Recent studies have also shown a pivotal role for tumor associated stromal cells in signaling to the cancer cells to leave the primary tumor to initiate metastasis. Because Ehop-016 acts as a specific inhibitor of the Vav/Rac interaction, we hypothesize that EHop-016 inhibits the Vav/Rac signaling in not only cancer cells, but also in fibroblasts and leukocytes. To determine whether EHop-016 treatment affects the tumor microenvironment (TME), athymic nude mice bearing mammary fatpad tumors were treated with vehicle or 30 mg/kg BW EHop-016 by IP, 3X a week, for 52 days. Immunofluorescence microscopy of mammary tumor tissue sections demonstrated a statistically significant 40% decrease in Ki-67 expression, a 60% decrease in capillary tubes as quantified by CD-31 staining, and a 33% decrease in F4/80 positive macrophages in the tumors from mice following EHop-016 treatment, compared to those treated with vehicle. To investigate the effect of EHop-016 on the cytokine milieu, plasma recovered from the mice in this study were subjected to a cytokine array for common inflammatory mediators. EHop-016 treatment demonstrated a statistically significant decrease in mouse plasma levels of the major inflammatory mediator interleukin-6 (IL-6) that has been implicated with cancer progression in the TME, as well as a trend in reduced monocyte chemoattractant protein and tumor necrosis factor alpha. A PCR array analysis of the mammary tumors from this study also demonstrated a decrease in IL-6 levels in tumor tissue. Reduced macrophages and IL-6 signaling in the TME, in response to EHop-016 treatment, is expected to decrease the cytokine-mediated malignant signaling cross talk between cancer cells and stromal cells. Therefore, inhibition of Vav/Rac signaling in the TME may additionally enhance the anti-metastatic effects of EHop-016. This study was supported by National Institute on Minority Health and Health Disparities of the National Institutes of Health (NIMHHD/NIH) U54MD008149 and the Puerto Rico Science and Technology Trust to SD; Title V PPOHA P031M10505 and Title V Cooperative P031S130068 from U.S. Department of Education, and RCMI 8G12MD007583 to UCC; and UPR RCM NIH/NIMHHD grants 5U54CA096297 andR25GM061838 to THB. Citation Format: Linette Castillo-Pichardo, Cristina Del Valle, Jessica Morales, Valance Washington, Krizia Rohena-Rivera, Magaly Martinez, Luis Cubano, Suranganie Dharmawardhane. Anti-breast cancer effects of the Vav/Rac inhibitor Ehop-016 in the tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B29.

Metastasis is associated with poor prognosis in breast cancer patients. Not all cancer cells within a tumor are capable of metastasizing. The microRNA-200 (miR-200) family, which regulates the mesenchymal-to-epithelial transition, is enriched in the serum of patients with metastatic cancers. Ectopic expression of miR-200 can confer metastatic ability to poorly metastatic tumor cells in some settings. Here, we investigated whether metastatic capability could be transferred between metastatic and nonmetastatic cancer cells via extracellular vesicles. miR-200 was secreted in extracellular vesicles from metastatic murine and human breast cancer cell lines, and miR-200 levels were increased in sera of mice bearing metastatic tumors. In culture, murine and human metastatic breast cancer cell extracellular vesicles transferred miR-200 microRNAs to nonmetastatic cells, altering gene expression and promoting mesenchymal-to-epithelial transition. In murine cancer and human xenograft models, miR-200-expressing tumors and extracellular vesicles from these tumors promoted metastasis of otherwise weakly metastatic cells either nearby or at distant sites and conferred to these cells the ability to colonize distant tissues in a miR-200-dependent manner. Together, our results demonstrate that metastatic capability can be transferred by the uptake of extracellular vesicles.

Simple SummaryThe tumor microenvironment (TME) is composed of various cell types and extracellular components and plays an important role in cancer development and progression, as well as in therapeutic resistance and metastasis. Cancer-associated fibroblasts (CAFs) are a key cell type in the TME and interact with cancer cells as well as with other TME cells, such as immune cells. Immunocompetent mouse cancer models that recapitulate the interactions of cancer cells with the TME have provided a better understanding of the TME network and support the development of new therapeutic strategies. In this review, we focus on cancer cell–TME interactions in heterogeneous tumor tissue, and we provide an overview of therapeutic strategies that target the TME.The tumor microenvironment (TME) plays a key role in cancer development and progression, as well as contributes to the therapeutic resistance and metastasis of cancer cells. The TME is heterogeneous and consists of multiple cell types, including cancer-associated fibroblasts (CAFs), endothelial cells, and immune cells, as well as various extracellular components. Recent studies have revealed cross talk between cancer cells and CAFs as well as between CAFs and other TME cells, including immune cells. Signaling by transforming growth factor-β, derived from CAFs, has recently been shown to induce remodeling of tumor tissue, including the promotion of angiogenesis and immune cell recruitment. Immunocompetent mouse cancer models that recapitulate interactions of cancer cells with the TME have provided insight into the TME network and support the development of new anticancer therapeutic strategies. Recent studies based on such models have revealed that the antitumor action of molecularly targeted agents is mediated in part by effects on the tumor immune environment. In this review, we focus on cancer cell–TME interactions in heterogeneous tumor tissue, and we provide an overview of the basis for anticancer therapeutic strategies that target the TME, including immunotherapy.

Ovarian cancer has high mortality among all gynecologic malignancies due to the lack of clear symptoms at early stage and the metastasis of cancer cells when diagnosed with advanced stages. The disseminated ovarian cancer cells have to overcome the immune surveillance in abdominal environment, which is rich in many types of immune cells, to survive and proliferate. However, the key genes and mechanisms preventing the metastasis of ovarian cancer cells into abdominal sites remain largely unexplored. Here, through an in vivo, genome-scale CRISPR/Cas9 knockout screening, we identified IL-20/IL24-IL20RA as a key checkpoint preventing the abdominal metastasis of ovarian cancer cells. In ovarian cancer patients, the level of IL20RA is dramatically decreased in metastatic cancer cells when compared with that in primary sites. Overexpression of IL20RA in highly metastatic ovarian cancer cells greatly suppressed the abdominal metastasis of ovarian cancer and the formation of ascites. In contrast, silencing IL20RA could increase the metastasis of ovarian cancer cells. We further examined IL02RA-mediated crosstalk between the disseminated cancer cells and the immuno-microenvironment in mouse in vivo ovarian cancer models and in vitro coculture experiments. Silencing IL20RA in ovarian cancer cells resulted in a dramatic decrease of M1 subtype of macrophages and increase of the M2 subtype of macrophages without affecting the population of T and B lymphocytes in ascites. Further mechanistic studies revealed that the ovarian cancer cells, when invaded into the abdominal sites, greatly induced the mesothelial cells in peritoneum to produce IL20RA ligands, i.e., IL-20 and IL-24, which in turn activated the IL20RA downstream signaling in ovarian cancer cells to trigger the production and maturation of IL-18. IL-18, when secreted into the microenvironment, was able to promote the polarization of macrophages into M1-subtype to clear the cancer cells. Thus, we identified a novel immune checkpoint IL20/IL24-IL20RA that was always downregulated in metastatic ovarian cancer cells to prevent the formation of an antitumor immune microenvironment, which could be utilized to develop new strategies for the immunotherapy of ovarian cancers. Citation Format: Jia Li, Xuan Qin, Mengyao Xu, Jie Yan, Longlong Wang, Yi Shi, Rong Xiang. The systematic identification of novel immune checkpoint averting the metastasis of ovarian cancer: The role of IL-20/IL24-IL20RA axis [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B52.

Modulating the activation of hepatic stellate cells: a cunning way for metastatic cells to create a permissive soil for seeding in the liver?

Extracellular vesicles (EVs) mediate intercellular signaling and communication, allowing the intercellular exchange of proteins, lipids, and genetic material. Their recognized role in the maintenance of the physiological balance and homeostasis seems to be severely disturbed throughout the carcinogenesis process. Indeed, the modus operandi of cancer implies the highjack of the EV signaling network to support tumor progression in many (if not all) human tumor malignancies. We have reviewed the current evidence for the role of EVs in affecting cancer hallmark traits by: (i) promoting cell proliferation and escape from apoptosis, (ii) sustaining angiogenesis, (iii) contributing to cancer cell invasion and metastasis, (iv) reprogramming energy metabolism, (v) transferring mutations, and (vi) modulating the tumor microenvironment (TME) by evading immune response and promoting inflammation. Special emphasis was given to the role of EVs in the transfer of drug resistant traits and to the EV cargo responsible for this transfer, both between cancer cells or between the microenvironment and tumor cells. Finally, we reviewed evidence for the increased release of EVs by drug resistant cells. A timely and comprehensive understanding of how tumor EVs facilitate tumor initiation, progression, metastasis and drug resistance is instrumental for the development of innovative EV-based therapeutic approaches for cancer.

The mortality rate due to metastasis in cancer is as high as 90%. The underlying mechanisms have been studied, however, the process of metastasis is complicated, current therapies still cannot inhibit metastasis efficiently. In recent years, studies have mentioned that extracellular vesicles play an important role in cancer cell metastasis and tumor microenvironment. Extracellular vesicles can be divided into microvesicles, apoptotic bodies and exosomes, which can carry different substances into the tumor microenvironment. Especially exosomes, whose diameter is around 30-160nm, can carry DNA, RNA, miRNA, membrane proteins, lipids and other intracellular molecules into the cell through endocytosis for regulation. The process of metastasis and the drug resistance were also been demonstrated can be regulated by exosomes. At present, the physiological functions of exosomes are not clear. HT29 colon cancer cells previously discovered in our laboratory can have different responses to dasatinib treatment, and cells can be isolated to dasatinib selected cells(DSTS) and resistant cells(DSTR). In this study, the role of exosome upon dasatinib treatment in HT29 cell is investigated. In the experiment, exosomes from HT29, DSTS, and DSTR were isolated by ExoQuick-TC. Transmission electron microscope was used to observe the difference in exosome size. Exosomes from different cells were treated in HT29 cells, and differences at the protein and mRNA levels were observed using western blot and reverse transcription-polymerase chain reaction(RT-PCR). In my results, the diameter of exosomes in DSTS cells was smaller than that in DSTR and HT29 cells. The protein expression levels of Cavoelin-1 and Clathrin in HT29 were also increased in the treatment of exosome that from three types of cells. In addition, the DSTR derived-exosomes can induce the expression of pFAK and Clathrin mRNA in HT29 cells. The preliminary results suggest that DSTR derived-exosomes may affect cell migration. This study provides the clues that through understanding exosomes that secreted from different cells, cancer diagnosis and treatment can be conducted according to their characteristics, so as to reduce the possibility of cell metastasis precisely. Citation Format: Hui Min Koo, Yi-Wen Lu, Xiang-Ling Hou, Wei-Ting Chao. Investigates the role of exosomes in dasatinib treated HT29 colon cancer cells [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 6973.

Background Although breast cancer liver metastases (BCLM) are common events, with 40-50% of patients with metastatic breast cancer developing liver metastases, their development indicates particularly poor prognosis. Targeted treatments for liver metastases are virtually non-existent, partly because the key events of early liver metastasis formation remain unknown. The liver is a dense organ, and metastatic breast cancer cells (MCC) must first clear space among hepatocytes, the primary cell population in the liver, to form. Cell competition is a process that explains how organs or primary tumors form from two populations of competing cells with varied fitness levels. The expansion of one population of cells creates space through the induction of apoptosis in the other. Objectives In this study, we tested the hypothesis that MCC use cell competition with hepatocytes to create space and expand within the liver, resulting in hepatocyte apoptosis. Methods We develop new in-vitro and mouse lineage-traced models to assay cell competition between MCC and hepatocytes. These models directly capture MCC and hepatocyte interactions and we can assess modes of cell competition, including apoptosis, through immunostaining. Because prior literature suggests that the developing mammary gland informs both cell competition and metastasis, we performed a bioinformatics screen to identify potential signals that contribute to cell competition between MCC and hepatocytes. We generate a new single-cell RNA-seq dataset that incorporates information from the developing mammary gland and breast cancer cells and identify genes enriched in both. Next, we validated genes by testing for their high expression in MCC when compared to hepatocytes. Then, we determined the impact of target genes on cell competition in our models. Results/Discussion Our models demonstrate that MCC use cell competition to create space in the liver by inducing apoptosis in neighboring hepatocytes. MCC induce apoptosis in neighboring hepatocytes through cell competition in in-vitro co-cultures by 11.7-fold compared to MCC apoptosis. Hydrodynamic tail-vein engraftment in NOD-scid-gamma mice with MCC also resulted in increased neighboring hepatocyte apoptosis. Using our bioinformatics screen, we identified SerpinE2, a protease inhibitor, as a target that could drive breast cancer metastasis and cell competition. Knock-out of SerpinE2 in MCC did not affect their viability or growth but resulted in MCC apoptosis adjacent to hepatocyte neighbors by 16.9-fold when compared to wild-type (WT) conditions. Further, co-culture of MCC that lacked SerpinE2 with hepatocytes resulted in decreased hepatocyte apoptosis by 2.3-fold compared to hepatocytes co-cultured with WT MCC. These findings suggest that perturbation of SerpinE2 successfully modulates competition between MCC and hepatocytes: Hepatocytes can selectively outcompete and kill MCC that lack SerpinE2. Future work focused on the perturbation of SerpinE2 may lead to novel therapies for patients at risk for or with BCLM. Citation Format: Katherine E Lake, Sakshi Mohta, Clayton A Smith, Venkata Repaka, Lily Xu, Kaitlyn Saunders, Vrushali Pandit, Emily Goff, Jacob Pena, Luise Chinea, Luisa Froessl, Sangeetha M Reddy, Heather L McArthur, Christine Hodges, Julia Maues, Elizabeth Chen, Isaac S Chan. Treating liver metastases by reversing cell competition between metastatic cancer cells and hepatocytes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A041.

Background: Although breast cancer liver metastases (BCLM) are common events, with 40-50% of patients with metastatic breast cancer developing liver metastases, their development indicates poor prognosis. Patients with BCLM have a 5-year survival rate of 8%. Specific treatments for liver metastases are virtually non-existent, partly because the key events of early liver metastasis formation remain unknown. The liver is a dense organ, and metastatic breast cancer cells (MCC) must clear space among hepatocytes, the primary cell population in the liver, during metastatic formation. Cell competition is a developmentally conserved process that explains how organs or primary tumors form when cells within those tissues have varying fitness and compete with one another. This competition can result in the survival and expansion of one cell population, and the induction of cell death through apoptosis in the other. We hypothesized that MCC use cell competition with hepatocytes to create space and expand within the liver. Objectives: In this study, we tested the hypothesis that MCC use cell competition with hepatocytes to create space and expand within the liver, resulting in hepatocyte apoptosis. Methods: We develop new in-vitro and mouse lineage-traced models to assay cell competition between MCC and hepatocytes, initially using balb/c derived breast cancer (4T1) and hepatocyte (H2.35) cells and syngeneic mouse models of liver metastasis. These models directly capture MCC and hepatocyte interactions, and we can assess modes of cell competition, including apoptosis, through immunostaining. Because prior literature suggests that the developing mammary gland informs both cell competition and metastasis, we next performed a bioinformatics screen to identify potential signals that contribute to cell competition between MCC and hepatocytes. We generated a new single-cell RNA-seq dataset that incorporates information from the developing mammary gland and breast cancer cells. This dataset includes 3264 primary breast cancer cells across 3 models of aggressive breast cancers, encompassing HER2+ and TNBC subtypes, as well as 526 embryonic mammary gland cells. Using this platform, we identify genes enriched in both breast cancer and the developing mammary gland. Next, we validate genes by testing for their high expression in MCC when compared to hepatocytes. Then, we determine the impact of target genes on cell competition in our models. Results/Discussion: Our models demonstrate that MCC use cell competition to create space in the liver by inducing apoptosis in neighboring hepatocytes. MCC induce apoptosis in neighboring hepatocytes through cell competition in in-vitro co-cultures by 11.7-fold compared to MCC apoptosis. Hydrodynamic tail-vein engraftment in NOD-scid-gamma mice livers with MCC also resulted in increased neighboring hepatocyte apoptosis. Using our bioinformatics screen, we identified 5 potential targets that could drive breast cancer metastasis and cell competition. Using our competition models, perturbation of SerpinE2, a protease inhibitor which was one of the top gene targets, successfully modulated competition between MCC and hepatocytes. Knock-out of SerpinE2 in MCC did not affect their viability or growth but resulted in MCC apoptosis adjacent to hepatocyte neighbors by 16.9-fold when compared to wild type conditions. Further, co-culture of MCC that lacked SerpinE2 with hepatocytes resulted in decreased hepatocyte apoptosis by 2.3-fold compared to hepatocytes co-cultured with wild-type (WT) MCC. These findings suggest that hepatocytes cannot outcompete or kill WT MCC, but they can outcompete and kill MCC that lack SerpinE2. Future work focused on the perturbation of SerpinE2 may lead to novel therapies for patients at risk for or with BCLM. Our findings suggest that restoring the competitiveness of the liver parenchyma could be a new treatment modality for treating liver metastases. Citation Format: Katherine Lake, Sakshi Mohta, Clayton Smith, Venkata Repaka, Lily Xu, Kaitlyn Saunders, Vrushali Pandit, Emily Goff, Christine Zhang, Jacob Pena, Christine Hodgdon, Julia Maues, Elizabeth Chen, Isaac Chan. Treating liver metastases by reversing cell competition between metastatic cancer cells and hepatocytes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-25-01.

The metastasis of breast cancer mainly occurs through the axillary lymph node and blood circulation systems. It is extremely difficult to know when the cancer cells start to metastasize; however, early detection of breast cancer metastasis is crucial and challenging to enable surgical removal of the primary tumor and perform a systematic lymphadenectomy to eliminate invasion of the tumor. Herein, we report real-time tracking of the metastasis of orthotopic breast cancer with background-free near-infrared long-persistent luminescence (NIR-PL) imaging, and its guidance for the surgical removal of lymph nodes. The NIR-PL imaging is based on Cr3+/Nd3+ codoped ZnGa2O4 (A-ZGCN) nanoparticles with a superlong afterglow time of more than 15 days. We show that the detection sensitivity of metastasis of cancer cells with the NIR-PL imaging is higher than the classic bioluminescence imaging. We find that the metastasis of breast cancer cells to lymph nodes occurred as early as on the third day after orthotopic inoculation of breast cancer cells and followed an order of the proper axillary lymph node (PALN, day 3) > accessory axillary lymph node (AALN, day 6) > accessory mandibular lymph node (AMLN, day 9) > mandibular lymph node (MLN, day 25). In addition, we show that the NIR-PL nanoprobes (i.e., A-ZGCN NPs) displayed 17% radioenhancement, which was used for radiotherapy of orthotopic breast cancer to further prevent and reduce its metastasis to other organs. The radiotherapy treatment is superior to surgery for removal of the tumor accompanied by a NIR-PL imaging-guided lymphadenectomy. Our work demonstrates the great potential of NIR-PL imaging and the corresponding nanoprobes for tracking metastasis of cancer cells and for radiotherapy.

Tumor cells secrete elevated amounts of extracellular vesicles (EVs) as a mean of communication between them and other cells in the tumor microenvironment. Here, we established a series of breast cancer cell lines releasing fluorescent EVs to identify the common cell types that receive tumor EVs within the tumor microenvironment. Among the different cell types, cancer-associated fibroblasts consistently take up the most EVs from cancer cells. Using next generation sequencing, we find that miR-125b is among the top enriched miRNAs in EVs released by mouse isogenic triple-negative breast cancer 4T1 and 4TO7 cell lines. Co-injection of 4TO7 cells with 4T1 EVs results in an upregulation of fibroblast activation in vivo while blockage of miR-125b in 4T1 EVs rescues the effect indicating that EV-delivery of miR-125b is crucial for the fibroblast activation in mouse breast cancer models. Moreover, EVs from human breast cancer cells also contain abundant miR-125b and fibroblasts within the xenografted tumor that take up these EVs gain higher levels of miR-125b and upregulation of cancer-associated fibroblast markers. Both mouse and human fibroblasts that are transfected with miR-125b mimics show an activated phenotype similar to the knockdown of established miR-125b targets. In summary, normal fibroblasts within the tumor microenvironment actively take up the miR-125b-containing EVs from breast cancer cells and develop into cancer-associated fibroblasts. Citation Format: Luyen Tien Vu, Boya Peng, Daniel Xin Zhang, Victor Ma, Andrew Grimson, William C. Cho, Judy Lieberman, Minh TN Le. Activation of cancer-associated fibroblasts via the transfer of miR-125b containing extracellular vesicles from tumor 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 2034.

Irradiation of Glioblastoma Cells Increases CD147 Levels in their Extracellular Vesicles: Contribution to Increased MMP9 Activity in the Tumor Microenvironment