Triple-negative breast cancer (TNBC) is one of the most lethal subtypes of breast cancer that has limited treatment options. Its high rates of recurrence and metastasis have been associated, in part, ...with a subpopulation of breast cancer stem-like cells that are resistant to conventional therapies. A compendium of markers such as CD44(high)/CD24(low), and increased expression of the ABCG2 transporter and increased aldehyde dehydrogenase (ALDH1), have been associated with these cells. We developed a CD44-targeted monoclonal antibody photosensitizer conjugate for combined fluorescent detection and photoimmunotherapy (PIT) of CD44 expressing cells in TNBC. The CD44-targeted conjugate demonstrated acute cell killing of breast cancer cells with high CD44 expression. This cell death process was dependent upon CD44-specific cell membrane binding combined with near-infrared irradiation. The conjugate selectively accumulated in CD44-positive tumors and caused dramatic tumor shrinkage and efficient elimination of CD44-positive cell populations following irradiation. This novel phototheranostic strategy provides a promising opportunity for the destruction of CD44-positive populations that include cancer stem-like cells, in locally advanced primary and metastatic TNBC.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The poor prognosis of metastatic prostate cancer continues to present a major challenge in prostate cancer treatment. The tumor extracellular matrix (ECM) plays an important role in facilitating ...metastasis. Here, we investigated the structure and function of an ECM that facilitates prostate cancer metastasis by comparing orthotopic tumors that frequently metastasize to poorly metastatic subcutaneous tumors.
Both tumors were derived from a human prostate cancer PC3 cell line engineered to fluoresce under hypoxia. Second harmonic generation (SHG) microscopy was used to characterize collagen 1 (Col1) fiber patterns in the xenografts as well as in human samples. MRI was used to determine albumin-Gd-diethylenetriaminepenta-acetate (alb-GdDTPA) transport through the ECM using a saturation recovery MR method combined with fast T
SNAPSHOT-FLASH imaging. Cancer-associated fibroblasts (CAF) were also quantified in these tumors.
Significant structural and functional differences were identified in the prometastatic orthotopic tumor ECM compared to the less metastatic subcutaneous tumor ECM. The significantly higher number of CAFs in orthotopic tumors may explain the higher Col1 fiber volumes in these tumors.
, alb-GdDTPA pooling was significantly elevated in metastatic orthotopic tumors, consistent with the increased Col1 fibers.
Developing noninvasive MRI indices of macromolecular transport, together with characterization of Col1 fiber patterns and CAFs can assist in stratifying prostate cancers for aggressive treatments or active surveillance. These results highlight the role of CAFs in supporting or creating aggressive cancers, and the importance of depleting CAFs to prevent metastatic dissemination in prostate cancer.
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Cancer stem cells (CSCs) have the ability to self-renew and induce drug resistance and recurrence in colorectal cancer (CRC). As current chemotherapy doesn't eliminate CSCs completely, there is a ...need to identify novel agents to target them. We investigated the effects of cucurbitacin B (C-B) or I (C-I), a natural compound that exists in edible plants (bitter melons, cucumbers, pumpkins and zucchini), against CRC. C-B or C-I inhibited proliferation, clonogenicity, induced G
/M cell-cycle arrest and caspase-mediated-apoptosis of CRC cells. C-B or C-I suppressed colonosphere formation and inhibited expression of CD44, DCLK1 and LGR5. These compounds inhibited notch signaling by reducing the expression of Notch 1-4 receptors, their ligands (Jagged 1-2, DLL1,3,4), γ-secretase complex proteins (Presenilin 1, Nicastrin), and downstream target Hes-1. Molecular docking showed that C-B or C-I binds to the ankyrin domain of Notch receptor, which was confirmed using the cellular thermal shift assay. Finally, C-B or C-I inhibited tumor xenograft growth in nude mice and decreased the expression of CSC-markers and notch signaling proteins in tumor tissues. Together, our study suggests that C-B and C-I inhibit colon cancer growth by inhibiting Notch signaling pathway.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Many cancer cells are characterized by increased glycolysis and decreased respiration, even under aerobic conditions. The molecular mechanisms underlying this metabolic reprogramming are unclear. ...Here we show that hypoxia-inducible factor 1 (HIF-1) negatively regulates mitochondrial biogenesis and O
2 consumption in renal carcinoma cells lacking the von Hippel-Lindau tumor suppressor (VHL). HIF-1 mediates these effects by inhibiting C-MYC activity via two mechanisms. First, HIF-1 binds to and activates transcription of the
MXI1 gene, which encodes a repressor of C-MYC transcriptional activity. Second, HIF-1 promotes MXI-1-independent, proteasome-dependent degradation of C-MYC. We demonstrate that transcription of the gene encoding the coactivator PGC-1β is C-MYC dependent and that loss of PGC-1β expression is a major factor contributing to reduced respiration in VHL-deficient renal carcinoma cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Coronavirus disease‐2019 (COVID‐19), caused by the novel severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2), has lead to a global pandemic with a rising toll in infections and deaths. ...Better understanding of its pathogenesis will greatly improve the outcomes and treatment of affected patients. Here we compared the inflammatory and cardiovascular disease‐related protein cargo of circulating large and small extracellular vesicles (EVs) from 84 hospitalized patients infected with SARS‐CoV‐2 with different stages of disease severity. Our findings reveal significant enrichment of proinflammatory, procoagulation, immunoregulatory and tissue‐remodelling protein signatures in EVs, which remarkably distinguished symptomatic COVID‐19 patients from uninfected controls with matched comorbidities and delineated those with moderate disease from those who were critically ill. Specifically, EN‐RAGE, followed by TF and IL‐18R1, showed the strongest correlation with disease severity and length of hospitalization. Importantly, EVs from COVID‐19 patients induced apoptosis of pulmonary microvascular endothelial cells in the order of disease severity. In conclusion, our findings support a role for EVs in the pathogenesis of COVID‐19 disease and underpin the development of EV‐based approaches to predicting disease severity, determining need for patient hospitalization and identifying new therapeutic targets.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Vascular endothelial growth factor (VEGF) plays key roles in angiogenesis, vasculogenesis, and wound healing. In cancers, including triple negative breast cancer (TNBC), VEGF has been associated with ...increased invasion and metastasis, processes that require cancer cells to traverse through the extracellular matrix (ECM) and establish angiogenesis at distant sites. To further understand the role of VEGF in modifying the ECM, we characterized VEGF-mediated changes in the ECM of tumors derived from TNBC MDA-MB-231 cells engineered to overexpress VEGF. We established that increased VEGF expression by these cells resulted in tumors with reduced collagen 1 (Col1) fibers, fibronectin, and hyaluronan. Molecular characterization of tumors identified an increase of MMP1, uPAR, and LOX, and a decrease of MMP2, and ADAMTS1. α-SMA, a marker of cancer associated fibroblasts (CAFs), increased, and FAP-α, a marker of a subset of CAFs associated with immune suppression, decreased with VEGF overexpression. Analysis of human data from The Cancer Genome Atlas Program confirmed mRNA differences for several molecules when comparing TNBC with high and low VEGF expression. We additionally characterized enzymatic changes induced by VEGF overexpression in three different cancer cell lines that clearly identified autocrine-mediated changes, specifically uPAR, in these enzymes. Unlike the increase of Col1 fibers and fibronectin mediated by VEGF during wound healing, in the TNBC model, VEGF significantly reduced key protein components of the ECM. These results further expand our understanding of the role of VEGF in cancer progression and identify potential ECM-related targets to disrupt this progression.
A critical event in the pathogenesis of invasive and metastatic cancer is E-cadherin loss of function. Renal clear cell carcinoma (RCC) is characterized by loss of function of the von Hippel-Lindau ...tumor suppressor (VHL), which negatively regulates hypoxia-inducible factor-1 (HIF-1). Loss of E-cadherin expression and decreased cell-cell adhesion in VHL-null RCC4 cells were corrected by enforced expression of VHL, a dominant-negative HIF-1alpha mutant, or a short hairpin RNA directed against HIF-1alpha. In human RCC biopsies, expression of E-cadherin and HIF-1alpha was mutually exclusive. The expression of mRNAs encoding TCF3, ZFHX1A, and ZFHX1B, which repress E-cadherin gene transcription, was increased in VHL-null RCC4 cells in a HIF-1-dependent manner. Thus, HIF-1 contributes to the epithelial-mesenchymal transition in VHL-null RCC by indirect repression of E-cadherin.
Photoimmunotherapy (PIT), carried out using an Ab conjugated to the near infrared dye IRDye700DX, is achieving significant success in target‐specific elimination of cells. Fibroblast activation ...protein alpha (FAP‐α) is an important target in cancer because of its expression by cancer‐associated fibroblasts (CAFs) as well as by some cancer cells. Cancer‐associated fibroblasts that express FAP‐α have protumorigenic and immune suppressive functions. Using immunohistochemistry of human breast cancer tissue microarrays, we identified an increase of FAP‐α+ CAFs in invasive breast cancer tissue compared to adjacent normal tissue. We found FAP‐α expression increased in fibroblasts cocultured with cancer cells. In proof‐of‐principle studies, we engineered human FAP‐α overexpressing MDA‐MB‐231 and HT‐1080 cancer cells and murine FAP‐α overexpressing NIH‐3T3 fibroblasts to evaluate several anti‐FAP‐α Abs and selected AF3715 based on its high binding affinity with both human and mouse FAP‐α. After conjugation of AF3715 with the phthalocyanine dye IR700, the resultant Ab conjugate, FAP‐α‐IR700, was evaluated in cells and tumors for its specificity and effectiveness in eliminating FAP‐α expressing cell populations with PIT. Fibroblast activation protein‐α‐IR700‐PIT resulted in effective FAP‐α‐specific cell killing in the engineered cancer cells and in two patient‐derived CAFs in a dose‐dependent manner. Following an intravenous injection, FAP‐α‐IR700 retention was three‐fold higher than IgG‐IR700 in FAP‐α overexpressing tumors, and two‐fold higher compared to WT tumors. Fibroblast activation protein‐α‐IR700‐PIT resulted in significant growth inhibition of tumors derived from FAP‐α overexpressing human cancer cells. A reduction of endogenous FAP‐α+ murine CAFs was identified at 7 days after FAP‐α‐IR700‐PIT. Fibroblast activation protein‐α‐targeted near infrared PIT presents a promising strategy to eliminate FAP‐α+ CAFs.
In our studies we first demonstrated increased expression of FAP‐α CAFs in human breast cancer tissue microarrays and in human mammary fibroblasts (HMFs) co‐cultured with human breast cancer cells. Next, in proof‐of‐principle studies, we engineered FAP‐α overexpressing human breast cancer MDA‐MB‐231 and human fibrosarcoma HT‐1080 cells to demonstrate successful elimination of FAP‐α expressing cell populations in tumors in vivo with NIR‐PIT. NIR‐PIT resulted in significant and selective growth inhibition of FAP‐α overexpressing tumors, and effective depletion of endogenous FAP‐α murine CAFs. The combination of target‐specific antibody binding with localized NIR exposure of the tumor avoided the off‐target toxicity associated with systemic FAP‐α‐targeted therapies.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The CD44 transmembrane glycoproteins play multifaceted roles in tumor progression and metastasis. CD44 expression has also been associated with stem-like breast cancer cells. Hypoxia commonly occurs ...in tumors and is a major cause of radiation and chemo-resistance. Hypoxia is known to inhibit differentiation and facilitates invasion and metastasis. Here we have investigated the effect of hypoxia on CD44 and two of its isoforms in MDA-MB-231 and SUM-149 triple negative human breast cancer cells and MDA-MB-231 tumors using imaging and molecular characterization.
The roles of hypoxia and hypoxia inducible factor (HIF) in regulating the expression of CD44 and its variant isoforms (CD44v6, CD44v7/8) were investigated in human breast cancer cells, by quantitative real-time polymerase chain reaction (qRT-PCR) to determine mRNA levels, and fluorescence associated cell sorting (FACS) to determine cell surface expression of CD44, under normoxic and hypoxic conditions. In vivo imaging studies with tumor xenografts derived from MDA-MD-231 cells engineered to express tdTomato red fluorescence protein under regulation of hypoxia response elements identified co-localization between hypoxic fluorescent regions and increased concentration of (125)I-radiolabeled CD44 antibody.
Our data identified HIF-1α as a regulator of CD44 that increased the number of CD44 molecules and the percentage of CD44 positive cells expressing variant exons v6 and v7/8 in breast cancer cells under hypoxic conditions. Data from these cell studies were further supported by in vivo observations that hypoxic tumor regions contained cells with a higher concentration of CD44 expression.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Metastatic dissemination continues to be a major cause of prostate cancer (PCa) mortality, creating a compelling need to understand factors that play a role in the metastatic cascade. Since hypoxia ...plays an important role in PCa aggressiveness, we characterized patterns of hypoxia in the primary tumor and metastatic environments of a human PCa xenograft. We previously developed and characterized an imaging strategy based on the hypoxia response element (HRE)–driven expression of long-lived enhanced green fluorescent protein (EGFP) and short-lived luciferase (luc) fused to the oxygen-dependent degradation domain in human PCa PC-3 cells. Both reporter proteins were placed under the transcriptional control of a five–tandem repeat HRE sequence. PC-3 cells also constitutively expressed the tdTomato red fluorescent protein, allowing cancer cell detection in vivo. This “timer” strategy can provide information on the temporal evolution of HIF activity and hypoxia in tumors. Here, for the first time, we performed in vivo and ex vivo imaging of this dual HIF reporter system in PC-3 metastatic tumors implanted orthotopically in the prostate and PC-3 nonmetastatic tumors implanted subcutaneously. We observed distinct patterns of EGFP and luc expression in subcutaneous and orthotopic tumors, and in metastatic nodules, that provide new insights into the presence of hypoxia at primary and metastatic tumor sites, and of the role of hypoxia in metastasis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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