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Short-term anti-vascular endothelial growth factor treatment elicits vasculogenic mimicry formation of tumors to accelerate metastasis
Yuan Xu1,
Qin Li1,
Xiao-Yu Li2,
Qiu-Ya Yang1,
Wei-Wei Xu2 &
Gao-Lin Liu1
Antiangiogenic therapy is one of the most significant advances in anticancer treatment. The benefits of antiangiogenic therapies of late-stage cancers have been investigated but are still too limited.
We used an ovarian cancer model to test the effect of short-term bevacizumab treatment on metastasis as measured by bioluminescence. Western blotting and CD34-PAS dual staining were performed to assess hypoxia-inducible transcription factor-1α (HIF-1α) expression and vasculogenic mimicry(VM) formation. Cell viability was examined by a CCK8 assay.
Bevacizumab demonstrated antitumor effects in models of ovarian cancer, but also accelerated metastasis together, with marked hypoxia and VM formation in mice receiving short-term therapy. Bevacizumab treatment did not affect SKOV3 cell viability and the amount of VM in three-dimensional culture.
These results suggest that antiangiogenic therapy may potentially influence the progression of metastatic disease, which has been linked to the hypoxic response and VM formation.
Tumors can grow to a maximum diameter of between 1 and 2 mm before their metabolic demands are restricted due to the diffusion limit of oxygen and lack of essential nutrients. To exceed this size or spread to other organs, tumors require an independent blood supply. In the 1970s, Folkman et al was the first to propose the concept of antiangiogenesis as a therapeutic approach to treat solid tumors [1]. Targeting the blood supply by inhibiting the formation of blood vessel will lead to tumor growth arrest. Numerous angiogenesis inhibitors have been therapeutically used in both preclinical and clinical settings [2]. Vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitors and a VEGF-neutralizing antibody have been clinically validated to target VEGF or its receptors as an anticancer treatment. However, a number of limitations are observed in current antiangiogenic therapies. Many clinical benefits are short-lived, and enduring clinical responses are rare. While numerous trials have shown an increase in survival after patients are treated with antiangiogenic therapy, the increase for many was only a matter of months [3]. Moreover, single-agent use of antiangiogenesis appears to be insufficient to improve patient survival [4]. While any improvement in overall survival for patients should be regarded as advancement, it is importart to understand why such clinical improvements are sometimes transitory so that new therapies result in more enduring benefits.
One explanation for these limitations is a potential link between antiangiogenic therapy and increased metastasis [5]. In RIP-Tag2 mice treated with the VEGF receptor 2-inhibitor DC101, although tumors were smaller, they showed significantly more invasive and malignant phenotypes, with most showing wide fronts of invasion into urrounding acinar tissues [6]. Rodents treated with an anti-VEGF antibody showing a striking increase in the number and total area of small satellite tumors compared with those that had not received antiangiogenic therapy, and tumor cells often had migrated over long distances [7, 8]. Together, these results suggest that antiangiogenic therapy may influence the progression of metastatic disease. To understand the reasons for these observations and to enable enduring benefits of antiangiogenic therapies, we examined the effect of a VEGF-neutralizing antibody on metastasis in mice after short-term administration. Furthermore, the hypoxic response and vasculogenic mimicry (VM) formation were assessed in this study.
For western blotting and histopathological analyses, a mouse anti-HIF-1α monoclonal antibody was purchased from Novus Biologicals (Littleton, CO, USA), CD34 monoclonal antibody from Abgent (San Diego, CA, USA).
The human ovarian cancer cell line SKOV3 was purchased from the ATCC and transfected with a luciferase-expressing lentivirus containing an independent open-reading frame of GFP. After 72 hours, cells were examined by fluorescence microscopy to confirm infection. Luciferase expression was determined using luciferin and an in vivo imaging system (Xenogen). Cells were maintained in RPMI-1640 medium supplemented with 10% heatinactivated fetal bovine serum (Gibco Invitrogen Corp), and incubated at 37°C in a humidified atmosphere containing 5% CO2.
Three-dimensional(3D) cultures
Matrigel (BD Biosciences) was placed dropwise onto glass coverslips in 12-well culture plates and allowed to polymerize for 30 min at 37°C. SKOV3 cells were then seeded onto the 3D matrix in complete medium.
SKOV3LUC+ cells (1.2 × 106 cells) were directly injected into the tail vein of 6- 8-week-old female nude mice. Forty mice were assigned into four groups(A, B, C and D). Group A was treated with phosphate-buffered saline (PBS) bi-weekly for 3 weeks. Group B was treated with 40 mg/kg bevacizumab bi-weekly for 3 weeks. Group C was treated with 3 mg/kg cisplatin weekly for 3 weeks. Group D was treated with both bevacizumab bi-weekly and cisplatin weekly for 3 weeks. Bevacizumab and cisplatin were administered intraperitoneally. Body weight was measured and recorded weekly. Metastatic disease progression in SKOV3LUC+ tumor-bearing mice was monitored. Before mice were anesthetized with Forane, an aqueous solution of luciferin (150 mg/kg) was intraperitoneally injected at 10 min prior to imaging. Mice were placed into the light-tight chamber of a CCD camera system (Xenogen), and photons emitted from luciferase-expressing cells within mice were quantified for 1 min, using the software program living. Four weeks after initial treatment, all mice were sacrificed to assess the effects of drug treatments. All procedures involving mice complied with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health).
The tissues were homogenized in 0.5 ml Hepes (50 mM, pH 7.5) containing 100 mM NaCl, 1 mM CaCl2, 1 mM dithiothreitol, 1% ethylene glycol-bis(aminoethyl ether)-tetraacetic acid 1% Triton X- 100 and proteinase inhibitors. Protein extracts were kept in ice for 30 min and then centrifuged at 14,000 g at 4°C for 30 min. Protein concentrations were determined using a bicinchoninic acid protein assay reagent kit. Protein samples (20 mg) were mixed with equal volumes of loading buffer (20% glycerol, 4% sodium dodecyl sulfate, and 100 mM Tris-HCl, pH 6.8) and then boiled for 5 min in the presence of β-mercaptoethanol. Proteins were separated in 8% sodium dodecyl sulfate-polyacrylamide gels at 100 V for 2 h and then electrotransferred to nitrocellulose membranes at 270 mA for 2 h. Membranes were blocked with 5% non-fat dry milk in PBS with 0.1% Tween 20 for 1 h at room temperature. Then, membranes were incubated with anti- HIF-1α (1:500) overnight at 4°C and finally with a horseradish peroxidase-conjugated anti-mouse IgG for 1 h at room temperature after washing with TBS containing 0.1% Tween 20. Proteins were visualized by enhanced chemiluminescence reagents after washing. Protein expression was semi-quantified using an image analysis system.
CD34-PAS dual staining
Four micrometer paraffin sections were routinely deparaffinized and dehydrated. First, CD34 immunohistochemical staining was applied to the sections. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in 50% methanol for 10 min at room temperature. Sections were rehydrated and washed with PBS and then pretreated with citrate buffer (0.01 M citric acid, pH 6.0) for 20 min at 100°C in a microwave oven. Non-specific binding sites were blocked with 2% normal goat serum in PBS for 20 min at 37°C. Sections were then incubated overnight at 4°C with anti-CD34 at a 1:200 dilution. Then, sections were rinsed with PBS and incubated with biotinylated goat anti-mouse IgG for 20 min at 37°C, followed by incubation with 3,3'-diaminobenzidine(DAB) chromogen for 10 min at room temperature. Sections were then rinsed with water for 1 min to stop the DAB-staining reaction. Formalin and melanin granules were then removed using the methods mentioned above. Finally, sections were treated with 0.5% periodic acid solution for 10 min and rinsed with distilled water for 2-3 min. In a dark chamber, sections were treated with Schiff solution for 15-30 min. After rinsing with distilled water, sections were counterstained with hematoxylin [9].
Cell counting kit 8 (CCK8) assay
Cell viability was measured by conversion of Dojindo's highly water-soluble tetrazolium salt WST-8 to a yellow-colored watersoluble formazan (CCK8 assay). The amount of formazan dye generated by the activity of mitochondrial dehydrogenases in cells is directly proportional to the number of living cells. CCK8 is more sensitive than the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay [10]. SKOV3 cells were trypsinized and seeded at 5 × 103 cells/well in 96 well plates in 3D cultures. After 24 h, various concentrations of bevacizumab were added, followed by incubation for another 48 h. Then, 10 μL CCK8 (Sigma, USA) solution in PBS was added to each well. Plates were incubated for an additional 2 h. The optical density of each well was measured using a microculture plate reader at a 490 nm wavelength.
All results were evaluated using the SPSS 13.0 statistical software package. Data were analyzed using one-way ANOVA. Results were expressed as the mean ± standard deviation, and P < 0.05 was considered statistically significant.
Increased metastasis after short-term treatment with the angiogenesis Inhibitor bevacizumab
In our study, a model of metastasis was used to test the effect of short-term bevacizumab treatment. SKOV3LUC+ cells expressing luciferase were directly injected into the tail vein of female nude mice and then received bevacizumab and/or cisplatin treatment for 3 weeks. Forty mice were equally divided into four groups at random (PBS, bevacizumab, cisplatin and bevacizumab + cisplatin groups). Tumor growth and metastasis were monitored by bioluminescence at 1 and 4 weeks after treatment. Mean photon counts of each group were quantified, and the ratio of metastasis was measured. The pulmonary metastasis rate was 100%. Tumor growth delay was observed at 1 week after bevacizumab and/or cisplatin treatments, without extrapulmonary metastasis. Short-term bevacizumab treatment resulted in accelerated extrapulmonary metastasis at 4 weeks after treatment. Extrapulmonary metastases were found in livers and legs. Cisplatin and bevacizumab + cisplatin treatment inhibited tumor growth, compared with that of PBS treatment.. While no significant difference in tumor growth was observed between bevacizumab and control groups (Figure 1).
Increased metastasis after short-term treatment with bevacizumab. Forty mice were assigned into four groups (PBS, bevacizumab, cisplatin and bevacizumab + cisplatin). Mean photon counts of each group were quantified. (A) Tumor growth and metastasis were monitored by bioluminescence at 1 and 4 weeks after treatment. A representative experiment is shown. (B) Short-term bevacizumab treatment resulted in accelerated extrapulmonary metastasis at 4 weeks after treatment. Extrapulmonary metastases were found in the livers and legs. The ratio of metastasis of each group was measured. (C) Quantification of bioluminescence showed inhibited tumor growth in bevacizumab and/or cisplatin treatment groups at 1 week after treatment, compared with that of the PBS group. Bevacizumab + cisplatin treatment inhibited tumor growth, compared with that of cisplatin at 1 week after treatment. (D) Quantification of bioluminescence showed no significant difference in tumor growth between bevacizumab and PBS groups 4 weeks after treatment. Bevacizumab + cisplatin treatment inhibited tumor growth compared with that of cisplatin at 4 weeks after treatment. *P < 0.05, **P < 0.01.
Hypoxia is implicated in the adaptive response
To gain an insight into possible molecular mechanisms of the increased metastasis, we determined whether hypoxia development was concomitant with metastasis. Mice were assigned into four groups (PBS, bevacizumab, cisplatin and bevacizumab + cisplatin) and received bevacizumab and/or cisplatin treatments for 3 weeks. Four weeks after initial treatment, five mice from each group were sacrificed for examination. Expression of HIF-1α in pulmonary tumor nodules was analyzed by western blotting. In PBS and cisplatin groups, most tumors showed little hypoxia. In contrast, mice that received bevacizumab and bevacizumab + cisplatin therapy showed a markedly increased level of HIF-1α expression (Figure 2). Differences in HIF-1α protein levels in each group were considered statistically significant.
Hypoxia is implicated in the adaptive response after short-term bevacizumab treatment. Expression of HIF-1α in pulmonary tumor nodules of the four groups. (A) A representative western blot is shown. β-actin was used as a loading control. (B) While most tumors showed little expression of HIF-1α protein in PBS and cisplatin groups, mice that received bevacizumab and bevacizumab + cisplatin therapy showed a markedly increased level of HIF-1α expression.. *P < 0.05, **P < 0.01.
Anti-VEGF treatment also induces increased VM
The definition of VM is that tumor cells mimic endothelial cells and form vasculogenic networks. CD34-PAS double staining was used to distinguish VM and endothelial-dependent vessels. CD34 is a marker of endothelial cells, and the basement membrane is positive for PAS. Therefore, we counted PAS-positive and CD34-negative vessels for indicate. Mice were assigned into four groups (PBS, bevacizumab, cisplatin and bevacizumab + cisplatin) that received bevacizumab and/or cisplatin treatments for 3 weeks. Four weeks after initial treatment, five mice from each group were sacrificed for examination. Tumors in the bevacizumab group formed more VM channels than those of PBS and cisplatin, and bevacizumab + cisplatin groups (Figure 3).
Anti-VEGF treatment induces increased VM. Comparison of VM channels in mice with various treatments. VM channels were positive for PAS staining and negative for CD34 staining in sections (arrow, ×400). (A) PBS (B) bevacizumab (C) cisplatinp and(D) bevacizumab + cisplatin groups. (E) Comparison of VM channels in A, B, C and D. Tumors in the bevacizumab group formed more VM channels than that of PBS and cisplatin, and bevacizumab + cisplatin groups. **P < 0.01.
In vitro experiment demonstrating the effect of bevacizumab on VM
SKOV3 cells were cultured in 3D culture, which formed VM channels. Then, we compared the cell viability and the ability to form VM in 3D culture after treatment with bevacizumab (0, 1, 10, 100 and 1000 μg/ml)for up to 48 h. Cell viability was examined by a CCK8 assay. Bevacizumab treatment did not affect SKOV3 cell viability and the number of tubules (Figures 4 and 5).
Bevacizumab treatment did not affect SKOV3 cell viability. Bevacizumab treatment (0, 1, 10, 100 and 1000 μg/ml) does not affect SKOV3 cell viability in 3D culture. There were no statistically significant difference (P > 0.05).
Bevacizumab treatment did not affect the number of tubules. The effect of bevacizumab (0, 10 and 1000 μg/ml) on the formation of VM channels (× 100). (A) Bevacizumab at 0/(B) 10/(C) 1000 μg/ml. (D) Bevacizumab treatment did not affect the number of tubules (P > 0.05).
Antiangiogenic therapy is one of the most significant advances in cancer treatment. Its clinical value has been investigated, but is still too limited. A number of recent clinical and preclinical observations have been reported. In a neoadjuvant phase II trial of advanced epithelial ovarian cancer patients treated with the combinational therapy of carboplatin/paclitaxel with the angiogenesis inhibitor sorafenib, Pölcher M et al. reported that progressive disease was diagnosed in two patients out of four, and surgical exploration showed an increased number of peritoneal tumor implants [11]. Furthermore, after short-term treatment, varous forms of antiangiogenic therapy can lead to increased metastasis in mouse models of multiple tumor types [12, 13]. Thus, there is a strong need to improve treatment strategies and to better understand the mechanisms of failure that hinder targeted antiangiogenic therapies. Here, we address the effect of short-term bevacizumab treatment using ovarian cancer xenografts. The data show that short-term bevacizumab treatment induces a reduction in tumor growth and an increase in distant tumor metastasis as measured by bioluminescence. Importantly, similar results were obtained when nu/nu mice were treated with bevacizumab + cisplatin and cisplatin alone. It should be noted that in mouse models of ovarian cancer, antiangiogenic therapy can elicit an adaptive response involving increased dissemination and the emergence of distant metastasis.
To investigate this metastatic "conditioning" effect, a better understanding of the biological effects of anti-VEGF treatment is required. Antiangiogenic therapy inhibits the development of new blood vessels, i.e. angiogenesis, resulting in reduced perfusion and an increase in intratumoral hypoxia, which may indirectly affect the tumor cells by inducing a more invasive phenotype in response to hypoxia, leading to increased invasion and metastatic dissemination [14, 15]. HIF-1α is a main regulator of the transcriptional response of cancer cells to hypoxia. By analyzing HIF-1α expression using western blotting we showed that treatment with bevacizumab increases intratumoral hypoxia in metastasis models of ovarian cancer. While most tumors showed little or no expression of HIF-1α protein in groups without bevacizumab treatment, HIF-1α expression markedly increased both in bevacizumab and bevacizumab + cisplatin groups. In summary, short-term bevacizumab treatment results in increased of HIF-1α expression. Interestingly, HIF-1α regulates genes that are involved in angiogenesis, cell survival, invasion and metastasis [16]. Therefore, downstream pathways of HIF-1α gene may contribute to metastatic phenotypes.
Current antiangiogenic strategies are mainly directed against tumor endothelial cells. However, tumours do not only rely on host blood vessels for nourishment, they can also form their own vasculature. The term "VM" has been used to describe the manner in which tumor cells mimic endothelial cells to form vasculogenic networks. VM has been described in ovarian cancer and some other highly aggressive tumors such as melanoma, prostatic carcinoma, breast cancer, soft tissue sarcomas and lung cancer [17–22]. The presence of VM correlates to an increased risk of metastasis and poor clinical outcome [23–26]. Several key molecules, including VE-cadherin, matrix metalloproteinases, laminin-5 γ2 chain and EphA2, have been implicated in VM. Moreover, the tumor microenvironment, including hypoxia, ischemia and acidosis, plays a major role in trans-endothelial differentiation of aggressive tumor cells [27–30]. In the hypoxic microenvironment, melanoma cells increase HIF-1α expression and induce the formation of VM channels to acquire an adequate blood supply [31]. In 3D culture, bevacizumab treatment for up to 48 h did not affect SKOV3 cell viability and the ability to form VM. Moreover, our data showed more VM channels in short-term bevacizumab treatment groups than those in control groups. This feature suggests that VM channels, which cannot be inhibited by bevacizumab, may satisfy the vascular requirements of ovarian cancer growth, invasion and metastasis during hypoxia. Thus, the increased of VM formation as a result of bevacizumab-induced hypoxia may increase dissemination and the emergence of distant metastasis. These findings offer a possible explanation for why antiangiogenesis only shows transitory clinical benefits.
VEGF inhibition causes hypoxia, induces HIF-1α expression and the formation of VM, which may be associated with tumor invasion and metastasis. Antiangiogenesis inhibits endothelium-dependent vessels, and then causes hypoxia in tumors. To compensate for tumor hypoxia, VM may increase to maintain the tumor blood supply and provide a convenient route for tumor metastasis. Another question raised by our results is whether other mechanisms linked to the hypoxic response could contribute to the metastatic phenotype, such as angiogenesis, growth factor signaling and apoptosis. The relationship between antiangiogenic therapy and metastasis remains to be determined and is an important topic for future research. Further study may provide additional drug targets, resulting in adjuvant therapies that can enhance the clinical benefits of antiangiogenic treatment.
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We thank Jing Zhou for technical assistance.
Department of Pharmacy, Shanghai First People's Hospital, School of medicine, Shanghai Jiao Tong University, No.100 Haining Road, Shanghai, 200080, China
Yuan Xu
, Qin Li
, Qiu-Ya Yang
& Gao-Lin Liu
Department of Orthopaedics, Yangzhou First People's Hospital, Jiangsu, China
Xiao-Yu Li
& Wei-Wei Xu
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Correspondence to Gao-Lin Liu.
XY carried out the design of the experiments, performed most of experiments and drafted the manuscript. LQ participated in the design of the experiments, western blot and cell culture. LXY participated in statistical analysis and interpretation. YQY participated in animal experiments. XWW participated in the statistical analysis and helped drafting the manuscript. LGL participated in the design of the experiments and helped drafting the manuscript. All authors read and approved the final manuscript.
Yuan Xu, Qin Li contributed equally to this work.
Xu, Y., Li, Q., Li, X. et al. Short-term anti-vascular endothelial growth factor treatment elicits vasculogenic mimicry formation of tumors to accelerate metastasis. J Exp Clin Cancer Res 31, 16 (2012). https://doi.org/10.1186/1756-9966-31-16
Accepted: 23 February 2012
Antiangiogenic therapies
Vasculogenic mimicry


WOMEN'S FICTION: Willow Armstrong is the CEO of a weight loss company Pound Busters and is caught eating food not in the diet plan. Everything has gone wrong in Willow's life. Her husband cheating on her has caused their divorce, and Willow possibly losing her beloved company has left Willow's life in shambles. When Willow finds a will in her deceased mother's things, it sparks her curiosity. Apparently, Willow had grandparents and is now the owner of their house in England. Willow goes to England thinking she can sell the house and make some money. When she gets to England, she discovers the house also has a cottage where the caretaker, Owen Hughes, lives in with his daughter. As Willow gets closer to Owen and his daughter, will she be able to sell the house along with the cottage without repercussions?
This story is amazingly true to life, filled with funny and poignant moments. The ups and downs of losing and gaining weight, plus the horrible things that happen in life, will definitely strike a chord with readers. The buildup of the heroine's relationships is slow, though, and the family drama does need some clarification. Readers will find Willow's daily struggles with her weight and her deep dark family secrets easily relatable. Owen is the nice perfect English gentleman, brimming with warmth and charm, making it hard not to love him, even with his dogged attempts to go on tour. England is described vividly, as a magical yet heart-wrenching place as the story that unfolds there.


text:
PARALYMPIC RESULTS
Greg Polychronidis targets first world title
Greek boccia player ranked No. 1 ahead of the 2018 World Champs 17 Jun 2018
Greg Polychronidis will aim for his first individual world title at Liverpool 2018
ⒸGetty Images
By BISFed 2018 World Boccia Championships LOC
"Personally, I feel great competing in the UK! The greatest title of my career, the Paralympic gold medal, was won at the London 2012 Paralympic Games. I have also won three more medals in the UK, so I am hoping I can secure another great achievement."
Greece's Greg Polychronidis moved into the world No. 1 spot and is among the ones to watch in the BC3 class at the BISFed 2018 World Boccia Championships.
Get to know the four-time Paralympic medallist, who will go for his first individual gold medal at the Worlds, set for 12-18 August in Liverpool, Great Britain.
How is your season going so far?
GP: The season is going great. I just got ranked world No. 1 in my category and I won gold in the Madrid Regional Open (in March), as well as two gold medals at the Greek National Championships. I am now the Greek champion for 17 consecutive years.
How tough do you expect the competition to be in Liverpool?
The World Championships in Liverpool will be as tough as the Paralympic Games. The greatest athletes from all over the world will give their best performance and fight for the World Champion title. I expect very difficult matches, so I train hard in order to deliver my best performance.
How important are the Worlds in the calendar?
It is the second major event after the Paralympics. Only the best athletes are competing in the Worlds. Also, it's where we can collect the most qualification points for the Tokyo 2020 Paralympic Games so it's a really important tournament.
Apart from yourself, who do you think are the ones to watch?
First of all, thank you for the honour of including me in the ones to watch list! There are so many elite athletes in each category, that it is almost impossible and unfair to choose just a few names.
Are you excited about returning to the United Kingdom?
Boccia UK in collaboration with the organisers are always creating great events. Taking in mind the London 2012 Paralympics as well as the National Paralympic Days that I have competed at, the experience was amazing. I am sure [Great Britain] will raise the bar even more in Liverpool.
Personally, I feel great competing in the UK! The greatest title of my career, the Paralympic gold medal, was won at the London 2012 Paralympic Games. I have also won three more medals in the UK, so I am hoping I can secure another great achievement.
How did you get started in the sport?
The Athens 2004 Paralympics were my inspiration. I had a dream to participate in these Games in my home city. I started playing boccia in 2001 and made my dream come true.
Do you have any routines or superstitions before you compete?
I always pray. I mostly listen to classical music, in order to calm myself and have a clear mind before entering the court.
You married your assistant Katerina last August. How crucial is the relationship between player and assistant? And how special is it to share these moments with your wife?
My relationship with Katerina is one of my strongest advantages. We have a great, effective and very fast communication since we are together 24/7. Also, because we are husband and wife we don't face any training schedule limits and it is important in order to train as much as needed in such a competitive sport as boccia.
Regarding the moments that we share together, I feel the luckiest man to have the opportunity to experience the greatest achievements of my life with the person that I love most and is my wife.
How do you feel about the growth of boccia?
I started competing internationally in 2003 and it has been wonderful to witness the sport grow so much. The number of countries developing boccia, and the number of athletes has been greatly increased. The standard of competition has also grown. The foundation of the Boccia International Sport Federation (BISFed) has totally changed the competition system which has upgraded boccia's status amongst the other Paralympic sports.
Also, the creation of different committees by BISFed for the development of the sport provides efficient results. I am honoured to be a member of the Competition and Rules Committee. My role is to share my sport experience as well as to gather feedback from other athletes, in collaboration with my colleagues, in order for our committee to solve technical problems and create fair rules.
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redpajama_set_name: RedPajamaCommonCrawl


Haven uses the regular features of a standard Android smartphone to detect changes in the environment around the device. The app, which adapts the camera for use as a motion tracker, can also pair with other devices to record sounds or changes in light before sending an alert to the owner – an ideal feature for those looking to ward off unwelcome snoopers.
In a video posted online, Snowden appealed for funds to further develop the open-source project while hailing it as a means for activists to protect their data without fear of it being hacked or stolen. "We designed Haven as a tool for investigative journalists, human rights defenders and people at risk," the whistleblower said.
None of the data collected by Haven is sent to outside servers, meaning people remain in full control of their data. It was developed in conjunction with the Guardian Project, a global collective of software developers and activists who create open-source mobile security technology.
Micah Lee, a journalist who helped develop the app along with Snowden, said it could be used to prevent so-called "evil maid" attacks, in which an intruder attempts to physically tamper with a machine.
Writing for The Intercept, Lee said: "Here's how Haven might work. You lock your laptop in a hotel safe — not a secure move on its own — and place your Haven phone on top of it. If someone opens the safe while you're away, the phone's light meter might detect a change in lighting, its microphone might hear the safe open, its accelerometer might detect motion if the attacker moves the laptop, and its camera might capture a snapshot of the attacker's face."
A user can also choose to have alerts sent via Signal, a Tor onion service website, or even a simple SMS text message. It is not without its flaws, however. Critics believe that if an attacker were to block WiFi, SMS or mobile data, they could prevent an alert being logged. For Snowden, however, the device he calls a "safe room that fits in your pocket" is a major step forward for privacy.
Android phone users have been up in arms in recent times following revelations that Google has been collecting their location data even while they have that feature turned off. Last month, Quartz discovered that Android devices have been sending "Cell ID codes" to the search engine giant via cellular towers across the US.


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You are here: Home / TV / Watching Sports and Special Events / How to Watch LSU vs. Georgia in the SEC Championship
How to Watch LSU vs. Georgia in the SEC Championship
Published 2022-12-01 By Dennis Restauro
The 2022 College Football SEC Championship is between LSU and Georgia this Saturday, December 3rd, 2022 at 4:00 pm EST on CBS. Read on to learn how to watch the game for free!
Watch LSU vs. Georgia Free: You can Watch The SEC College Football Championship live on CBS with a Free Trial to Paramount+.
Watch LSU vs. Georgia Live Without Cable
Watch The SEC Football Championship on Paramount+
Watch The SEC College Football Championship Live on Hulu
Watch The SEC College Football Championship on YouTube TV
Watch The SEC College Football Championship on fuboTV
Watch The SEC College Football Championship on DIRECTV STREAM
The SEC Football Championship Game will be decided this Saturday on CBS when LSU faces off against Georgia. The following summarizes your streaming options as seen in our guide to watching CBS without cable and will let you watch The SEC College Football Championship as it airs live on CBS. You can also watch for free on CBS using a TV antenna.
Paramount+ (formerly CBS All Access) streams your local CBS station so you can watch the SEC Championship. They offer a free trial so that you can watch the game for free! After the trial, the service costs $4.99 per month with ads and $9.99 per month to watch without ads. However, if you cancel during the trial period you will not be charged. Paramount+ is available on Roku Players, Apple TV, Xbox, Chromecast, Android TV, Amazon Fire TV, and Fire TV Stick. To Watch UECL matches, simply sign-up for this seven-day free trial to create a username and password, then download the Paramount+ app to a supported device. Then log in with the account you just created. Then use the search menu to search for "SEC Championship" and the game will be available for you to watch live and on-demand.
See our review of Paramount+ for more information on this streaming service.
Hulu offers a live TV service that carries CBS in nearly all US TV markets along with cable TV and broadcast networks. Therefore, you can watch The SEC College Football Championship live as it airs on CBS. To see if live CBS is available in your area, you can use this tool: Hulu Live TV Channels by Zip code.
Hulu + Live TV is supported on Roku, Amazon Fire TV, iOS, Android, and more. You can read more about Hulu in our review of Hulu. There isn't a month-to-month contract, so you can sign up to test the service without fear of a long-term commitment. You can sign up for Hulu Live TV using this link: Sign up for Hulu + Live TV.
How Much: $69.99 per month.
YouTube TV is another great option to watch The SEC College Football Championship live. YouTube TV offers live CBS with its streaming service in nearly ALL markets in the US. The only market where YouTube TV doesn't offer CBS is in the Rochester-Mason City-Austin market, which covers parts of Minnesota and Iowa. (YouTube TV does offer CBS in Rochester, NY, and Austin, TX.)
YouTube TV supports Android, iOS, Apple TV, Fire TV, Chromecast, and more. The YouTube TV app has also returned to Roku devices after settling a dispute between the two companies. Be sure to check out our review of YouTube TV for more information. You can sign up for their free trial.
FuboTV recently added CBS in the following major markets:
Fresno-Visalia, CA
Raleigh-Durham, NC (Fayetteville)
San Francisco-Oakland-San Jose, CA
You can check out a free trial to FuboTV here. Fubo TV supports the following streaming devices:
Apple TV (4th Generation only)
Amazon Fire TV and Fire TV Stick
For more information on FuboTV features, please see our Fubo TV review.
How Much: FuboTV costs $74.99.
DIRECTV STREAM offers live CBS streaming in many markets. CBS is available through its "Entertainment" package for $69.99 a month, along with a ton of cable TV and other broadcast networks. DIRECTV STREAM offers CBS in over half the markets in the US. They offer a free trial to allow you to make sure CBS is offered in your area. For more information, check out our review of DIRECTV STREAM.
How Much: $69.99 per month through the "Entertainment" package.