Difference between revisions of "Team:NJU-China/test"

Latest revision as of 03:48, 20 October 2016

Background
- Project Design
Modeling
- Results
  - Parts
  - Validations
  - Safety
  - Conclusions
Human Practices
Team
Attributions
Collaborations
- Notebook
  - Calendar
  - Protocol

1. Overview of cancer

Undeniably, no matter what we pursue, it is the health and wellness that people care about most. Everyone goes for a healthier and stronger body, but we are all clear about that there are numerous diseases around us, and it's still clueless for us to completely resist and cure a large quantity of them. People usually feel uncomfortable and scared by intractable and severe diseases. And cancer must be one of the nightmares.

Cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells. Once the spread, also known as metastasis, isn’t well controlled, it can result in death.^[1] As one of the most frightening death threat, cancer can be aggressive and malignant. The harmful effects of cancer on individual, family and society are enormous and appalling.

Figure 1. Estimated number of new cancer cases by area.

According to WHO and the latest global cancer statistics (2015), there were 14.1 million new cancer cases in 2012 worldwide and the corresponding estimates for total cancer deaths were 8.2 million (Fig. 1). Literally, about 22,000 cancer deaths happened a day. Besides, 1 in 7 deaths was related to cancer, and cancer caused more deaths than AIDS, tuberculosis and malaria combined. By 2030, the global burden is expected to grow to 21.7 million new cancer cases and 13 million cancer deaths on account of rapid growth and aging of population.^[1] Due to the adoption of lifestyles that are known to increase cancer risk, such as smoking, poor diet, physical inactivity and reproductive changes (including lower parity and later age at first birth) in developing country, it’s reasonable to estimate that the actual figures will be considerably larger.^[2]

Therefore, cancer is becoming one of the leading causes of death and the major public health problem around the world (Fig. 2).

Figure 2. Cancer incidence and mortality by region.^{[3] [4]}

2. Global and Chinese cancer statistics

Cancer statistics describe what happens in large groups of people and provide a picture of the burden of cancer on society. Although statistical trends may not be directly applicable to individual patients, they provide researchers, health professionals, and policy makers with essential information, helping them understand the impact of cancer on population and thus develop appropriate strategies to address the challenges that cancer poses to the society at large. Also, statistical trends are important for measuring the success of efforts in controlling and managing cancer.

2.1 Global cancer statistics

According to the latest Worldwide Cancer Statistics from Cancer Research UK, the top 5 most common cancer sites globally are lung, breast, colorectum, prostate and stomach, with more than 6.9 million new cases reported in 2012^[3](Fig. 3a). Correspondingly, the top 5 most common cancer deaths are lung, liver, stomach, colorectum and breast, accounting for more than half of total cancer death.^[4](Fig. 3b) Over the past 40 years, the most common cancer sites have changed little. Lung, liver, stomach and bowel cancers have been the four most common causes of cancer death since 1975.^[5]

Figure 3. Top 10 most common cancers (a) and causes of cancer death (b)^{[3] [4]}

More specifically, as noted in Global Cancer Statistics (2015) and Global Cancer Facts & Figures (3rd edition, 2015), worldwide, the top 5 most common cancer in male are lung, prostate, colon & rectum, stomach and liver cancer, while in female, they are breast, colon & rectum, lung, cervix uteri, stomach. And the five most common cancer sites are also the five leading causes of cancer death (Fig. 4).^{[1] [2]}

Figure 4. Top 5 most common cancers and most common causes of cancer death in male/female (2012)

2.2 Chinese cancer statistics

Cancer incidence and mortality have maintained vigorous growth in China, making cancer the leading cause of death since 2010, corresponding to global trend. Because of China's massive population and difficulties in data collection, previous national estimates were limited and inaccurate. With high-quality data from an additional number of population-based registries now being available through the National Central Center Registry of China (NCCR), it's possible for us to have a general understanding of cancer incidence and mortality in China.

According to Cancer Statistics in China (2015), there were about 4,292,000 newly diagnosed invasive cancer cases in 2015, meaning that almost 12,000 new cancer diagnoses averagely per day. It is estimated that approximately 2,814,000 Chinese died from cancer in 2015, corresponding to more than 7,500 cancer deaths on average per day (Fig. 5)

Figure 5. Top 5 most common cancers and most common causes of cancer death in China (2015)^[6]

Two line charts below showed trends of cancer incidence and death rates from year 2000 to 2011, as well as trends of new cancer cases and deaths in China (Fig. 6a, 6b). For all cancers combined, the age-standardized incidence rates were stable from 2000 to 2011 for males, while significant upward trends were observed among females. On the contrary, the age-standardized mortality rates decreased considerably for both sexes. However, it is still worth noting that despite this favorable trend, the number of cancer deaths substantially increased (73.8% increase) during the corresponding period because of the aging and growth of the population.

Figure 6. a) Trends in Number of New cases and Deaths for All Cancer Combined by Sex: China, 2000 to 2011.^[6]

Figure 6. b) Trends in Cancer Incidence and Death Rates (Age-standardized to the Segi Standard Population) for All Cancer Combined by Sex: China, 2000 to 2011.^[6]

These striking figures deserve our attention and contemplation, driving us to dig out deeper understanding of cancer, develop appropriate policies for cancer control and design effective treatments for various cancer types.

3. Lung cancer

3.1 Lung cancer statistics

Lung cancer is one of the most frequently diagnosed cancer types and the leading cause of cancer-related death around the world.^[7] Globally, an estimated 1.8 million new cases occurred in 2012, accounting for approximate 13% of total cancer diagnoses.^[2] An estimated 1.6 million deaths (1.1 million in men and 491,200 deaths in women) were reported in 2012.^[1] In some countries, for women, it is now the leading cause of cancer death surpassing breast cancer. The figure below reveals international variation in lung cancer incidence published in Global Cancer Facts & Figures (3rd edition, 2015) (Fig. 7).

Figure 7. International Variation in Lung Cancer Incidence Rates, 2012.^[1]

Take the United States as an example. According to National Cancer Institute, 224,390 new cases of lung and bronchus cancer are estimated in 2016, accounting for 13.3% of all new cancer cases, in line with the global trends.^[8] Despite improvements in surgical techniques and combined therapies over the past several decades, lung cancer is still one of the most lethal cancers. Five-year net survival rate is generally similar worldwide, ranging from 10% to 20%.^[1] According to data from SEER 18 2006-2012, present 5-year survival rate of lung cancer in the US is merely 17.7% (Fig. 8).^[8] The table below shows 5-year relative surviving of lung cancer from year 1975 to 2008 in the US (Table. 1).

Figure 8. Present Five-Year Survival Rate of Lung Cancer in the US^[8]

Table 1. Five-Year Survival Rates of Lung Cancer in the US from 1975 to 2008^[8]

Cancer stage at diagnosis determines treatment options and is strongly related to the length of survival. In general, five-year survival situation will be more optimistic when patients being diagnosed lung and bronchus cancer at an earlier stage. For lung cancer, 15.7% are diagnosed at the local stage, the 5-year survival for which is 55.2%^[8] (Fig. 9).

Figure 9. Percent of Cases and Five-Year Survival Rates by Stage at Diagnosis^[8]

3.2 Basic information of lung cancer

3.2.1 Symptoms, risk factors and classification

Symptoms of lung cancer do not usually occur until the cancer is advanced, and may include persistent cough, sputum streaked with blood, chest pain, voice change, worsening shortness of breath, and recurrent pneumonia or bronchitis. By far, smoking is widely recognized as the primary contributor to lung cancer. And for nonsmokers, passive smoking can be a silent killer. Studies from the US, Europe and the UK have consistently shown significantly increased risk among those exposed to secondhand smoke.^[7] Besides, lung cancer is also attributable to increasing age, radon gas, asbestos, air pollution, as well as inherited factors.^[1]

Two main categories of lung cancer are classified according to histological types: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). The most common types of NSCLC are squamous cell carcinoma, large cell carcinoma and adenocarcinoma, and all the types can occur in unusual histologic variants. Although NSCLC is associated with smoking, adenocarcinomas may be found in non-smokers. As a class, NSCLC is relatively insensitive to chemotherapy and radiation therapy compared with SCLC.^[9]

3.2.2 Treatments of lung cancer

Treatments of lung cancer include surgery, radiation therapy, chemotherapy and targeted therapies, based on types and stages. For early-stage NSCLC, surgery is usually the ideal choice, while chemotherapy, sometimes in combination with radiation therapy, may be given as well. Advanced-stage NSCLC patients are usually treated with chemotherapy, targeted drugs, or some combination of both. Chemotherapy alone or combined with radiation is the usual treatment for SCLC.^[10]

Discovery of driver oncogenes has revolutionized the field of lung cancer therapeutics. The identification of distinctive mutations in lung cancer has led to the booming development of molecular targeted therapy. In particular, subsets of adenocarcinoma now can be defined by specific mutations in genes encoding components of the epidermal growth factor receptor (EGFR) and downstream mitogen-activated protein kinases (MAPK) and phosphatidylinositol 3-kinases (PI3K) signaling pathways. These mutations may define mechanisms of drug sensitivity and primary or acquired resistance to kinase inhibitors.^[10] KRAS-mutant lung cancer accounts for approximately 25% of NSCLC, representing an enormous burden of cancer worldwide. KRAS mutations are frequently observed in adenocarcinoma. Since KRAS mutation in lung cancer was first reported in mid-1980s and remained the most commonly mutated oncogenes in unselected patients, they have risen to fame as elusive targets in a rapidly-evolving field of molecular therapeutics. In the ever-changing world of targeted therapy for molecular drivers of lung cancer growth, KRAS mutations continue to represent a significant challenge for drug development.^[11]

4. Traditional cancer therapies and their defects

Cancer destroy both patients' body and willpower. Traditional therapies for cancers vary from surgery, radiotherapy to chemotherapy.

Surgery is the primary method of treatment for most isolated, solid cancer and may play a role in palliation of pain and prolongation of survival.^[12] Namely, feasibility and efficacy of surgery is based on the type and stage of cancer, and for certain types, surgery is far from sufficient to eliminate the cancer.

Radiation therapy is a therapy using ionizing radiation, generally as part of cancer treatment to control or kill malignant cells. It may be curative in a number of types of cancer if they are localized to one area of the body. And it may also be used as part of adjuvant therapy, to prevent tumor recurrence after the surgery to remove a primary malignant tumor. Radiation therapy is synergistic with chemotherapy as well, and has been used before, during, and after chemotherapy in susceptible cancer cases.^[13]

Chemotherapy is another conventional cancer treatment that uses one or more anti-cancer drugs, called chemotherapeutic agents, as part of a standardized chemotherapy regimen. It is always given with a curative intent and aims to prolong life or to reduce uncomfortable symptoms.^[14]

While radiotherapy and chemotherapy are effective to some extent, the adverse effects remain inevitable, the main kinds of which are fatigue and skin irritation. Acute side effects such as nausea, vomiting, appetite loss, swelling, intestinal discomfort and late side effects such as fibrosis, epilation, dryness and sleeping problem are very common. Immunosuppression, myelosuppression, typhlitis, gastrointestinal discomfort, anemia can also occur after administration of chemotherapy.^[15] ^[16] Since these therapies will inevitably and indiscriminately damage the normal cells, patients always show fragile health condition after treatment, leading to low prognosis.

5. Leading edge of cancer treatment

Nowadays, clinical trials have broadened their ways to immunotherapy, hormone therapy, etc. Take immunotherapy for example: it is one kind of treatment that could induce, enhance, or suppress immune response. Active immunotherapy directs the immune system to attack tumor cells by targeting tumor-associated antigens. Passive immunotherapies enhance existing anti-tumor responses and include the use of monoclonal antibodies, lymphocytes and cytokines.^[17]Despite much exiting progress, the therapeutic effect isn’t always satisfactory. Due to the heterogeneity and complexity, tumor cells are good liars playing trick on immune system. Till now, relapse and drug resistance are unable to be avoided effectively and immunotherapy remains controversial and immature. Moreover, it can be very expensive in some cases.

Since cancer is an extremely heterogeneous disease with variants having different genetic and biological properties, drugs targeting a specific molecule that interferes with cancer growth or metastasis are much helpful and effective to ensure that patients will receive optimum management and can be an ideal choice for further exploration. The identification of distinctive mutations in various cancer has led to the booming development of molecular targeted therapy. Small molecule targeted drugs like Gefitinib (Iressa) and Erlotinib (Tarceva) have already been launched into market.^[11] However, these drugs have drawbacks as high cost of development and inefficiency due to relatively high off-target rate. Furthermore, their depending on the same chemical entity leads to the difficulty of transplanting to another mutation or cancer case. For instance, Gefitinib, as an EGFR inhibitor, is unable to deal with KRAS mutation in NSCLCs.

6. Ideal drug system

In view of serious situation of cancer incidence and mortality, and drawbacks of current treatments, it’s worth developing a much more effective drug system and this is what our project focus on.

Our ideal drug system will contain some basic features as follows:

First, it is designed to be targeting and systemic. Namely, our drug aims at tumor cells and once metastasis appears, it can be a treatment for the whole body as well. In our project, we chose KRAS as our target (插入介绍KRAS的link), and exosome as our delivery agent (插入介绍exosome的link).

Second, precision is one of our goals. To be exact, the targeting peptides on the surface of our delivery agent guarantee the accurate arrival in tumor cells by ligand-receptor interaction. Thus, tumor cell's escape and normal cell's damage will be avoided as much as possible, giving rise to efficiency and minimizing side effects.

In addition, we aim to construct a drug system of high transplantability. There are different types of cancer cases and each case has its distinctive mutations in specific oncogenes. And our system consists of delivery agent (exosome), drug (RNAi) (插入介绍RNAi的link) and targeting tool (iRGD-lamp 2b fusion protein). Hence, it is much easier for us to design a derived drug based on the same silencing and delivery system. By replacing appropriate siRNA and corresponding targeting peptides, it can specifically down-regulate different disease-related genes and thus treat various diseases pertinently. To wit, our drug owns general applicability, leading to remarkable reduce in cost of drug development.

Now you can click three links below to get more detailed information in our drug system.

Target: KRAS

KRAS: A Significant Oncogene

Cancer is caused by alterations in oncogenes, tumor-suppressor genes, and microRNA genes. Oncogenes encode proteins that control cell proliferation, apoptosis, or both. They can be activated by structural alterations resulting from mutation or gene fusion [18].

The two most commonly mutated oncogenes in lung cancer are the epidermal growth factor receptor (EGFR) and KRAS. In terms of EGFR, EGFR mutants were only recently identified, but they have already been established in the clinic as valid predictors of increased sensitivity to EGFR kinase inhibitors (gefitinib and erlotinib). By contrast, even though KRAS mutations were identified in NSCLC tumors more than 20 years ago, the clinic importance of KRAS mutant in cancer therapy just began to be paid more attention. Recent studies show that lung cancer patients with KRAS mutant do not respond to either EGFR inhibitor or adjuvant chemotherapy. Therefore, KRAS-targeting therapeutics are sorely needed [19].

K-ras protein is a small GTPase (~21 kDa) encoded by KRAS gene [20]. K-ras possesses an essential role in normal tissue signaling. It can be activated by signals from cell surface receptors, subsequently switches on other proteins and ultimately turns on genes, turning on genes involved in cell growth, differentiation, and survival. K-ras uses a bound guanine nucleotide (GDP/GTP) to toggle between its “on” and “off” states. It binds GDP in its neutral state. When a signal arrives, GTP will take place of GDP with the help of guanine nucleotide exchange factors (GEFs), causing a rearrangement of the protein, thus, switching itself “on” to deliver the signal to the final destination [21]. Normally, K-ras has an intrinsic ability to hydrolyze GTP transiently and turn itself “off”. Mutations of KRAS oncogene lead to a strongly reduced GTPase activity of K-ras protein, making KRAS stay at a constitutively activated “on” state. Thereby, the signal is passed continuously, allowing the cell to proliferate without control [22].

Figure10. K-ras is part of the MAP kinase signaling cascade (RAS/RAF/MEK/ERK) that relays chemical signals from outside the cell to the cell's nucleus and is primarily involved in controlling cell division.

KRAS in Lung Cancer

The two main types of lung cancer are non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) [23]. NSCLC accounts for about 85% of lung cancers. As a class, NSCLCs are relatively insensitive to chemotherapy, compared to small cell carcinoma. Lung adenocarcinoma is the most common subtype of NSCLC, and it accounts for approximately 40% of lung cancers [24].

Figure 11. Subtype and mutation frequency of NSCLC [25]

It has been found that the activation of the KRAS oncogene is specifically associated with the histological features of adenocarcinoma and does not or rarely occur in other types of NSCLC [26]. Approximately 15–25% of the patients with lung adenocarcinoma have tumor-associated KRAS mutations. In the majority of cases, these mutations are missense mutations which introduce a single amino acid substitution at position 12, 13, or 61. The result of these mutations is constitutive activation of KRAS signaling pathways [27]. In the vast majority of cases, KRAS mutations are found in tumors with wild type for EGFR or ALK; in other words, they are non-overlapping with other oncogenic mutations found in NSCLC. Therefore, KRAS mutation defines a distinct molecular subset of the disease.

Figure 12. Frequency of major driver mutations in signaling molecules in lung adenocarcinomas [28].

Clinical Importance

far the most important single prognostic factor. But the role of KRAS as either a prognostic or predictive factor in NSCLC still remains mostly unclear at the moment [29].

The KRAS point mutation may not only be valuable as a prognostic marker. Valiant attempts have been made to develop cancer therapeutics targeting mutant KRAS signaling. However, to date, there are no direct anti-KRAS therapies available.

The therapeutic potential of systemically delivered RNAi had been demonstrated as a novel treatment for cancer. Thanks to many prominent advantages like nontoxicity and targeting, KRAS siRNA delivery is a promising choice for further drug development.

Reference

[1] Global Cancer Facts & Figures (3rd edition), 2015. American Cancer Society.

[2] Lindsey A. Torre, Freddie Bray, Rebecca L. Siegel, Jacques Ferlay, Joannie Lortet-Tieulent, Ahmedin Jemal. Global Cancer Statistics, 2012. CA CANCER J CLIN. 2015; 00:

[3] Worldwide Cancer Incidence. International Agency for Research on Cancer of WHO and Cancer Research UK.

[4] Worldwide Cancer Mortality. International Agency for Research on Cancer of WHO and Cancer Research UK.

[5] Worldwide cancer mortality statistics. Cancer Research UK. http://www.cancerresearchuk.org/health-professional/cancer-statistics/worldwide-cancer/mortality

[6] Wanqing Chen, Rongshou Zheng, Peter D. Baade, Siwei Zhang, Hongmei Zeng, Freddie Bray, Ahmedin Jemal, Xue Qin Yu, Jie He. Cancer Statistics in China, 2015. CA CANCER J CLIN. 2016; 66:115–132.

[7] Lung cancer. Wikipedia. https://en.wikipedia.org/wiki/Lung_cancer

[8] Cancer Statistics. National Cancer Institute. https://www.cancer.gov/about-cancer/understanding/statistics

[9] Fact Sheets About Lung and Bronchus Cancer. National Cancer Institute. http://seer.cancer.gov/statfacts/html/lungb.html

[10] Non-Small Cell Lung Cancer Treatment (PDQ®)–Health Professional Version. National Cancer Institute. https://www.cancer.gov/types/lung/hp/non-small-cell-lung-treatment-pdq

[11] Admir Ahmad, Shirish Gadgeel. Lung Cancer and Personalized Medicine. Springer, 2016; 127, 155-156, 172-173.

[12] Cancer. Wikipedia. https://en.wikipedia.org/wiki/Cancer

[13] Radiation therapy. Wikipedia. https://en.wikipedia.org/wiki/Radiation_therapy

[14] Chemotherapy. Wikipedia. https://en.wikipedia.org/wiki/Chemotherapy

[15] Cancer Treatment. National Cancer Institute. https://www.cancer.gov/about-cancer/treatment

[16] Side Effects. National Cancer Institute. https://www.cancer.gov/about-cancer/treatment/side-effects

[17] Cancer immunotherapy. Wikipedia. https://en.wikipedia.org/wiki/Cancer_immunotherapy

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-                 <div class="nav-wrapper"><a class="page-title">Results</a></div>
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+                         <span class="card-title">1. Overview of cancer</span>
-                            <li class="tab col s4"><a class="active" href="#Parts">Parts</a></li>
+                        <p>Undeniably, no matter what we pursue, it is the health and wellness that people care about most. Everyone goes for a healthier and stronger body, but we are all clear about that there are numerous diseases around us, and it's still clueless for us to completely resist and cure a large quantity of them. People usually feel uncomfortable and scared by intractable and severe diseases. And cancer must be one of the nightmares.</p>
-                            <li class="tab col s4"><a href="#Validations">Validations</a></li>
+                        <p>Cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells. Once the spread, also known as metastasis, isn’t well controlled, it can result in death.<sup>[1] </sup> As one of the most frightening death threat, cancer can be aggressive and malignant. The harmful effects of cancer on individual, family and society are enormous and appalling.</p>
-                            <li class="tab col s4"><a href="#Safety">Safety</a></li>
+                        <img data-caption="Figure 1. Estimated number of new cancer cases by area." src="https://static.igem.org/mediawiki/2016/0/06/NJU_China_2016_iGEM_Background_Background_Figure_1.jpg" class="responsive-img">
-                            <li class="tab col s4"><a href="#Conclusions">Conclusions</a></li>
+                        <div align="center">Figure 1. Estimated number of new cancer cases by area.</div>
-                         </ul>
+                        <p>According to WHO and the latest global cancer statistics (2015), there were 14.1 million new cancer cases in 2012 worldwide and the corresponding estimates for total cancer deaths were 8.2 million (Fig. 1). Literally, about 22,000 cancer deaths happened a day. Besides, 1 in 7 deaths was related to cancer, and cancer caused more deaths than AIDS, tuberculosis and malaria combined. By 2030, the global burden is expected to grow to 21.7 million new cancer cases and 13 million cancer deaths on account of rapid growth and aging of population.<sup>[1] </sup> Due to the adoption of lifestyles that are known to increase cancer risk, such as smoking, poor diet, physical inactivity and reproductive changes (including lower parity and later age at first birth) in developing country, it’s reasonable to estimate that the actual figures will be considerably larger.<sup>[2] </sup></p>
+                        <p>Therefore, cancer is becoming one of the leading causes of death and the major public health problem around the world (Fig. 2).</p>
+                        <img data-caption="Figure 2. Cancer incidence and mortality by region." src="https://static.igem.org/mediawiki/2016/8/81/NJU_China_2016_iGEM_Background_Background_Figure_2.jpg" class="responsive-img">
+                        <div align="center">Figure 2. Cancer incidence and mortality by region.<sup>[3] [4] </sup></div>
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-                                    <p>Anti-KRAS siRNA (siRNA for KRAS gene silencing)</p>
+                        <span class="card-title">2. Global and Chinese cancer statistics</span>
-                                    <h4>Introduction</h4>
+                        <p>Cancer statistics describe what happens in large groups of people and provide a picture of the burden of cancer on society. Although statistical trends may not be directly applicable to individual patients, they provide researchers, health professionals, and policy makers with essential information, helping them understand the impact of cancer on population and thus develop appropriate strategies to address the challenges that cancer poses to the society at large. Also, statistical trends are important for measuring the success of efforts in controlling and managing cancer.</p>
-                                    <p style="padding-top: 0;">This part is an artificially designed RNA strand. It serves as an element of the Team NJU-CHINA RNAi module which can be used for down-regulation of KRAS expression in lung adenocarcinoma cells. We designed specific KRAS siRNA with algorithm based on a software developed by SYSU-Software team. This tool can find the best siRNA sequence on target gene KRAS to ascertain the maximum gene-specificity and silencing efficacy and also designs the pair of oligonucleotides needed to generate short hairpin RNAs (shRNAs) in the plasmid. Then we synthesize the shRNA sequence from a DNA synthesis company (Genscript).</p>
+                        <h5>2.1 Global cancer statistics</h5>
-                                    <div align="middle">
+                        <p>According to the latest Worldwide Cancer Statistics from Cancer Research UK, the top 5 most common cancer sites globally are lung, breast, colorectum, prostate and stomach, with more than 6.9 million new cases reported in 2012<sup>[3] </sup>(Fig. 3a). Correspondingly, the top 5 most common cancer deaths are lung, liver, stomach, colorectum and breast, accounting for more than half of total cancer death.<sup>[4] </sup>(Fig. 3b) Over the past 40 years, the most common cancer sites have changed little. Lung, liver, stomach and bowel cancers have been the four most common causes of cancer death since 1975.<sup>[5] </sup></p>
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+                        <img data-caption="Figure 3. Top 10 most common cancers (a) and causes of cancer death (b)<sup>[3] [4]</sup>" src="https://static.igem.org/mediawiki/2016/7/7f/NJU_China_2016_iGEM_Background_Background_Figure_3.jpg" class="responsive-img">
-                                    </div>
+                        <div align="center">Figure 3. Top 10 most common cancers (a) and causes of cancer death (b)<sup>[3] [4] </sup></div>
-                                    <div align="middle">Figure 1. The sequence of KRAS shRNA</div>
+                        <br>
-                                    <h4>Usage and Biology</h4>
+                        <p>More specifically, as noted in Global Cancer Statistics (2015) and Global Cancer Facts & Figures (3rd edition, 2015), worldwide, the top 5 most common cancer in male are lung, prostate, colon & rectum, stomach and liver cancer, while in female, they are breast, colon & rectum, lung, cervix uteri, stomach. And the five most common cancer sites are also the five leading causes of cancer death (Fig. 4).<sup>[1]  [2] </sup></p>
-                                    <p style="padding-top: 0;">We packaged KRAS siRNA into exosomes by transfecting HEK293 cells with a plasmid expressing KRAS siRNA and then collected siRNA-encapsulated exosomes. When modified exosomes being intravenously injected, they will specifically recognize integrin receptors and fuse with lung adenocarcinoma cells under the direction of the iRGD peptide. Once inside cells, KRAS siRNA will bind to KRAS mRNA through base-pairing and digest the mRNA, resulting in sharp decrease of K-ras in lung cancer cells. As a consequence, K-ras protein’s reduction and disturbed function will both result in the inhabitation of the proliferation of cancer cells, which ultimately have some therapeutic effects on lung cancer (non-small cell lung cancer in this case).</p>
+                        <img data-caption="Figure 4. Top 5 most common cancers and most common causes of cancer death in male/female (2012)" src="https://static.igem.org/mediawiki/2016/5/56/NJU_China_2016_iGEM_Background_Background_Figure_4.jpg" class="responsive-img">
-                                    <h4>Characterization</h4>
+                        <div align="center">Figure 4. Top 5 most common cancers and most common causes of cancer death in male/female (2012)</div>
-                                    <h5>Interference efficiency of anti-KRAS siRNA plasmid</h5>
+                        <br>
-                                    <p style="padding-top: 0;">To ensure the interference efficiency of anti-KRAS siRNA plasmid, we transfected it into human lung adenocarcinoma cell line A549 and then extracted protein from these cells to perform western blot. Significant down-regulation of K-ras can be observed in A549 cells treated with anti-KRAS siRNA, demonstrating that anti-KRAS siRNA has the gene silencing effect on lung cancer cells.</p>
+                        <h5>2.2 Chinese cancer statistics</h5>
-                                    <div align="middle">
+                        <p>Cancer incidence and mortality have maintained vigorous growth in China, making cancer the leading cause of death since 2010, corresponding to global trend. Because of China's massive population and difficulties in data collection, previous national estimates were limited and inaccurate. With high-quality data from an additional number of population-based registries now being available through the National Central Center Registry of China (NCCR), it's possible for us to have a general understanding of cancer incidence and mortality in China.</p>
-                                        <img src="https://static.igem.org/mediawiki/2016/0/05/NJU_China_2016_iGEM_Result-2.jpg" class="responsive-img">
+                        <p>According to Cancer Statistics in China (2015), there were about 4,292,000 newly diagnosed invasive cancer cases in 2015, meaning that almost 12,000 new cancer diagnoses averagely per day. It is estimated that approximately 2,814,000 Chinese died from cancer in 2015, corresponding to more than 7,500 cancer deaths on average per day (Fig. 5)</p>
-                                    </div>
+                        <img src="https://static.igem.org/mediawiki/2016/e/ea/NJU_China_2016_iGEM_Background_Background_Figure_5.jpg" class="responsive-img">
-                                    <div align="middle" style="padding-bottom: 30px;">Figure 2. Protein quantitatively analysis of K-ras extracted from cells without any treatment (Nude) and cells transfected with control siRNA (NC, siRNA targeting a random sequence) or anti-KRAS siRNA, which was made for intuitively support that anti-KRAS siRNA can suppress K-ras expression.</div>
+                        <div data-caption="Figure 5. Top 5 most common cancers and most common causes of cancer death in China (2015)<sup>[6]</sup>" align="center">Figure 5. Top 5 most common cancers and most common causes of cancer death in China (2015)<sup>[6]</sup></div>
-                                </div>
+                        <br>
-                            </li>
+                        <p>Two line charts below showed trends of cancer incidence and death rates from year 2000 to 2011, as well as trends of new cancer cases and deaths in China (Fig. 6a, 6b). For all cancers combined, the age-standardized incidence rates were stable from 2000 to 2011 for males, while significant upward trends were observed among females. On the contrary, the age-standardized mortality rates decreased considerably for both sexes. However, it is still worth noting that despite this favorable trend, the number of cancer deaths substantially increased (73.8% increase) during the corresponding period because of the aging and growth of the population.</p>
-                            <li>
+                        <img src="https://static.igem.org/mediawiki/2016/a/a9/NJU_China_2016_iGEM_Background_Background_Figure_6.jpg" class="responsive-img">
-                                <div class="collapsible-header active"><i class="material-icons">settings</i>BBa_K1942002</div>
+                        <br>
-                                <div class="collapsible-body">
+                        <div align="center">Figure 6. a) Trends in Number of New cases and Deaths for All Cancer Combined by Sex: China, 2000 to 2011.<sup>[6]</sup></div>
-                                    <p>A coding sequence of iRGD peptide and position it outside the membrane.</p>
+                        <div align="center">Figure 6. b) Trends in Cancer Incidence and Death Rates (Age-standardized to the Segi Standard Population) for All Cancer Combined by Sex: China, 2000 to 2011.<sup>[6]</sup></div>
-                                    <h4>Usage and Biology</h4>
+                        <br>
-                                    <p style="padding-top: 0;">iRGD is a tumor-penetrating peptide that can increase vascular and tissue permeability. Importantly, this effect did not require the drugs to be chemically conjugated to the peptide. To enhance the accuracy of drug delivery system and improve targeting index of drugs, iRGD peptide was displayed on the surface of the exosome containing our previously designed siRNA, allowing us to target recipient cells in vivo efficiently. Lamp-2b is a protein found specifically abundant in exosomal membranes. So we connect iRGD with Lamp2b by a glycine-linker, and promote the expression using cmv promoter. We engineered our chassis, human embryonic kidney 293 (HEK293) cells, to express iRGD-Lamp2b fusion protein. Therefore, the iRGD exosomes (iRGD-Exos) are endowed with site-specific recognition ability and were purified from cell culture supernatants and loaded with Dox by electroporation.</p>
+                         <p>These striking figures deserve our attention and contemplation, driving us to dig out deeper understanding of cancer, develop appropriate policies for cancer control and design effective treatments for various cancer types.</p>
-                                    <h4>Characterization</h4>
-                                    <p style="padding-top: 0;">The iRGD-Lamp2b expressing vector was thoroughly described in Tian’s article (Yanhua Tian, et al. Biomaterials, 2013). He showed that exosomes, endogenous nano-sized membrane vesicles secreted by most cell types, could deliver chemotherapeutics such as doxorubicin (Dox) to tumor tissue in BALB/c nude mice. To reduce immunogenicity and toxicity, mouse immature dendritic cells (imDCs) were used for exosome production. Tumor targeting was facilitated by engineering the imDCs to express a well-characterized exosomal membrane protein (Lamp2b) fused to αν integrin-specific iRGD peptide (CRGDKGPDC). Purified exosomes from imDCs were loaded with Dox via electroporation, with an encapsulation efficiency of up to 20%. iRGD exosomes showed highly efficient targeting and Dox delivery to αν integrin-positive breast cancer cells in vitro as demonstrated by confocal imaging and flow cytometry. Intravenously injected targeted exosomes delivered Dox specifically to tumor tissues, leading to inhibition of tumor growth without overt toxicity. The results suggested that exosomes modified by targeting ligands could be used therapeutically for the delivery of Dox to tumors, thus having great potential value for clinical applications in our project.</p>
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                      </div>
-                    <div id="Validations" class="col s12">
+                </div>
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+            </div>
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+            <div class="section">
-                                <div class="collapsible-header active"><i class="material-icons">filter_drama</i>Silencing capability validation in vitro</div>
+                <div class="card z-depth-3">
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+                    <div class="card-content">
-                                    <h5>1.1 KRAS siRNA interference efficiency verification in vitro</h5>
+                        <span class="card-title">3. Lung cancer</span>
-                                    <p style="padding-top: 0;">To ensure the interference efficiency of anti-KRAS siRNA, we transfected the plasmid loaded with this siRNA into human lung adenocarcinoma cell line A549 and extracted protein to perform western blot. Significant down-regulation of K-ras can be observed in A549 cells treating with anti-KRAS siRNA plasmid, compared with the control group, demonstrating that anti-KRAS siRNA has a gene silencing effect on lung cancer cells.</p>
+                        <h5>3.1 Lung cancer statistics</h5>
-                                    <div align="middle">
+                        <p>Lung cancer is one of the most frequently diagnosed cancer types and the leading cause of cancer-related death around the world.<sup>[7] </sup> Globally, an estimated 1.8 million new cases occurred in 2012, accounting for approximate 13% of total cancer diagnoses.<sup>[2] </sup> An estimated 1.6 million deaths (1.1 million in men and 491,200 deaths in women) were reported in 2012.<sup>[1] </sup> In some countries, for women, it is now the leading cause of cancer death surpassing breast cancer. The figure below reveals international variation in lung cancer incidence published in Global Cancer Facts & Figures (3rd edition, 2015) (Fig. 7).</p>
-                                        <img src="https://static.igem.org/mediawiki/2016/d/d1/NJU_China_2016_iGEM_Result-3.jpg" class="responsive-img">
+                        <img data-caption="Figure 7. International Variation in Lung Cancer Incidence Rates, 2012.<sup>[1]</sup>" src="https://static.igem.org/mediawiki/2016/e/ea/NJU_China_2016_iGEM_Background_Background_Figure_7.jpg" class="responsive-img">
-                                    </div>
+                        <div align="center">Figure 7. International Variation in Lung Cancer Incidence Rates, 2012.<sup>[1]</sup></div>
-                                    <div align="middle" style="padding-left: 30px; padding-right: 30px;">
+                        <br>
-                                        Figure 3. Anti-KRAS siRNA transfected into A549 cells successfully reduced KRAS expression. Left panel: western blot analysis of KRAS protein levels in cells without any treatment (Nude) or treated with negative control siRNA (NC, siRNA targeting at a random sequence) and transfected with anti-KRAS siRNA using Lipo2000. Right panel: protein quantitative analysis made for intuitive support that anti-KRAS siRNA can suppress KRAS expression.
+                        <p>Take the United States as an example. According to National Cancer Institute, 224,390 new cases of lung and bronchus cancer are estimated in 2016, accounting for 13.3% of all new cancer cases, in line with the global trends.<sup>[8] </sup> Despite improvements in surgical techniques and combined therapies over the past several decades, lung cancer is still one of the most lethal cancers. Five-year net survival rate is generally similar worldwide, ranging from 10% to 20%.<sup>[1] </sup> According to data from SEER 18 2006-2012, present 5-year survival rate of lung cancer in the US is merely 17.7% (Fig. 8).<sup>[8] </sup> The table below shows 5-year relative surviving of lung cancer from year 1975 to 2008 in the US (Table. 1). </p>
-                                    </div>
+                        <img data-caption="Figure 8. Present Five-Year Survival Rate of Lung Cancer in the US" src="https://static.igem.org/mediawiki/2016/6/60/NJU_China_2016_iGEM_Background_Background_Figure_8.jpg" class="responsive-img">
-                                    <h5 style="padding-top: 30px;">1.2 TEM imaging of exosomes carrying KRAS siRNA and expressing iRGD peptide on their membrane</h5>
+                        <div align="center">Figure 8. Present Five-Year Survival Rate of Lung Cancer in the US<sup>[8]</sup></div>
-                                    <p style="padding-top: 0;">After co-transfection of the two plasmids mentioned above, we performed a transmission electron microscopy (TEM) to characterize the iRGD-exosomal KRAS siRNA. The TEM image showed that the exosomes presented normal morphological characteristics after outside modification and siRNA loading, with a diameter of approximately 200 nanometer and a double-layer membrane. </p>
+                        <br>
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+                        <!-- <img src="" class="responsive-img"> -->
-                                        <img src="https://static.igem.org/mediawiki/2016/0/0c/NJU_China_2016_iGEM_Result-4.png" class="responsive-img">
+                        <img src="https://static.igem.org/mediawiki/2016/1/1a/NJU_China_2016_iGEM_Background_Background_Table_1.jpg" class="responsive-img">
-                                    </div>
+                        <br>
-                                    <div align="middle">Figure 4. TEM image of iRGD-modified exosomes packaging KRAS siRNA</div>
+                        <div align="center">Table 1. Five-Year Survival Rates of Lung Cancer in the US from 1975 to 2008<sup>[8]</sup></div>
-                                    <h5 style="padding-top: 30px;">1.3 Nanoparticle tracking analysis to ascertain the relationship between protein concentration and exosomes quantity </h5>
+                        <br>
-                                    <p style="padding-top: 0;">We then asked NUDT_CHINA to help us perform nanoparticle tracking analysis (NTA) for a further evaluation of the quantity and size of secreted exosomes. The use of Nanosight enabled quantification and size determination of the extracellular vesicles, as nanoparticles can be automatically tracked and sized based on Brownian motion and the diffusion coefficient. The size of exosomes attained ranged around 270nm. Basing the particle size and relative intensity, we also created a 3D plot for a visual explanation. Under measurement condition listed, the exosomes secreted by HEK293 cells were assayed for 2.95 E8 particles each milliliter. Then the relationship between particle number and protein was determined that exosomes in 1 ng protein were equivalent to 6277.95 particles, according to the dilution multiple (24) and protein concentration (1127.756 ng/ul) we have tested. All the data collected helped us decide the transfection dosage of siRNA and dosing of treatment prepared for animal experiment.</p>
+                        <p>Cancer stage at diagnosis determines treatment options and is strongly related to the length of survival. In general, five-year survival situation will be more optimistic when patients being diagnosed lung and bronchus cancer at an earlier stage. For lung cancer, 15.7% are diagnosed at the local stage, the 5-year survival for which is 55.2%<sup>[8] </sup> (Fig. 9). </p>
-                                    <div align="middle">
+                        <img data-caption="Figure 9. Percent of Cases and Five-Year Survival Rates by Stage at Diagnosis<sup>[8]</sup>" src="https://static.igem.org/mediawiki/2016/b/b6/NJU_China_2016_iGEM_Background_Background_Figure_9.jpg" class="responsive-img">
-                                        <img src="https://static.igem.org/mediawiki/2016/b/bb/NJU_China_2016_iGEM_Result-5.png" class="responsive-img">
+                        <div align="center">Figure 9. Percent of Cases and Five-Year Survival Rates by Stage at Diagnosis<sup>[8]</sup></div>
-                                    </div>
+                        <br>
-                                    <div align="middle">Figure 5. Nanoparticle tracking analysis (NTA) for Characterization of secreted exosomes. (a) Concentration of different particle sizes of exosomes. (b) 3D plot of particle size and relative intensity. (c) Experiment condition for our measurement. (d) Results attained after measurement of exosomes.</div>
+                        <h5>3.2 Basic information of lung cancer</h5>
-                                    <h5 style="padding-top: 30px;">1.4 The iRGD-exosomal KRAS siRNA suppressed KRAS expression in A549 cells in vitro</h5>
+                        <h6>3.2.1 Symptoms, risk factors and classification</h6>
-                                    <p style="padding-top: 0;">We next evaluate the effect of iRGD-exosomal KRAS siRNA on KRAS expression in vitro. The KRAS expression level was assayed in A549 cells after co-cultured with exosomal KRAS siRNA. Non-loaded iRGD-exosomes were used as control to ascertain that any RNAi response observed did not derive from the exosomes per se. The western electrophoresis and knockdown data obtained from qPCR analysis of KRAS gene expression indicated that KRAS protein and mRNA levels both dramatically decreased in the cells incubating with iRGD-exosomal KRAS siRNA compared with cells treating with nude exosomes or without any treatment. This result suggests that iRGD-exosomal KRAS siRNA can deliver siRNA into target cells and finally reduce the KRAS expression.</p>
+                        <p>Symptoms of lung cancer do not usually occur until the cancer is advanced, and may include persistent cough, sputum streaked with blood, chest pain, voice change, worsening shortness of breath, and recurrent pneumonia or bronchitis. By far, smoking is widely recognized as the primary contributor to lung cancer. And for nonsmokers, passive smoking can be a silent killer. Studies from the US, Europe and the UK have consistently shown significantly increased risk among those exposed to secondhand smoke.<sup>[7] </sup> Besides, lung cancer is also attributable to increasing age, radon gas, asbestos, air pollution, as well as inherited factors.<sup>[1] </sup></p>
-                                    <div align="middle">
+                        <p>Two main categories of lung cancer are classified according to histological types: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). The most common types of NSCLC are squamous cell carcinoma, large cell carcinoma and adenocarcinoma, and all the types can occur in unusual histologic variants. Although NSCLC is associated with smoking, adenocarcinomas may be found in non-smokers. As a class, NSCLC is relatively insensitive to chemotherapy and radiation therapy compared with SCLC.<sup>[9] </sup></p>
-                                        <img src="https://static.igem.org/mediawiki/2016/8/89/NJU_China_2016_iGEM_Result-6.jpg" class="responsive-img">
+                        <h6>3.2.2 Treatments of lung cancer</h6>
-                                    </div>
+                        <p>Treatments of lung cancer include surgery, radiation therapy, chemotherapy and targeted therapies, based on types and stages. For early-stage NSCLC, surgery is usually the ideal choice, while chemotherapy, sometimes in combination with radiation therapy, may be given as well. Advanced-stage NSCLC patients are usually treated with chemotherapy, targeted drugs, or some combination of both. Chemotherapy alone or combined with radiation is the usual treatment for SCLC.<sup>[10] </sup></p>
-                                    <div align="middle">Figure 6. Quantitative RT-PCR analysis of KRAS mRNA levels in A549 cells without any treatment (NC), transfected with non-loaded exosomes (exosome) and transfected with iRGD-exosomal KRAS siRNA (siRNA-exosome) shows that exosomal KRAS siRNA can down-regulate KRAS expression in transcription level.</div>
+                        <p>Discovery of driver oncogenes has revolutionized the field of lung cancer therapeutics. The identification of distinctive mutations in lung cancer has led to the booming development of molecular targeted therapy. In particular, subsets of adenocarcinoma now can be defined by specific mutations in genes encoding components of the epidermal growth factor receptor (EGFR) and downstream mitogen-activated protein kinases (MAPK) and phosphatidylinositol 3-kinases (PI3K) signaling pathways. These mutations may define mechanisms of drug sensitivity and primary or acquired resistance to kinase inhibitors.<sup>[10] </sup> KRAS-mutant lung cancer accounts for approximately 25% of NSCLC, representing an enormous burden of cancer worldwide. KRAS mutations are frequently observed in adenocarcinoma. Since KRAS mutation in lung cancer was first reported in mid-1980s and remained the most commonly mutated oncogenes in unselected patients, they have risen to fame as elusive targets in a rapidly-evolving field of molecular therapeutics. In the ever-changing world of targeted therapy for molecular drivers of lung cancer growth, KRAS mutations continue to represent a significant challenge for drug development.<sup>[11] </sup></p>
-                                    <h5 style="padding-top: 30px;">1.5 The iRGD-exosomal KRAS siRNA efficiently arrests cancer cell proliferation in vitro</h5>
-                                    <p style="padding-top: 0;">KRAS over-expression has been demonstrated to promote the growth of lung adenocarcinoma cells and be involved in migration and invasion of lung cancer. For further verification, we examined the role of iRGD-exosomal KRAS siRNA in cell proliferation. An EDU assay using Cell-Light™ EdUTP Apollo®567 TUNEL Cell Detection Kit, was carried out after siRNA-iRGD-exosome incubation and as a control, nude exosomes were also treated to A549 cells. The result indicated that KRAS knockdown had an anti-proliferation effect on lung tumor cells while nude exosomes were not capable of inhibiting the growth of tumor cells. </p>
-                                    <div align="middle">
-                                        <img src="https://static.igem.org/mediawiki/2016/c/cf/NJU_China_2016_iGEM_Result-7.jpg" class="responsive-img">
-                                    </div>
-                                    <div align="middle" style="padding-bottom: 30px;">Figure 7. Cell proliferation assay for A549 cells treated with PBS or transfected with iRGD-exosomal KRAS siRNA (exosome). Left panel: Fluorescence microscope photos of A549 cells. The red points represent divided cells for cell proliferation rate calculation. Right panel: Quantitative analysis of cell proliferation rate, indicating that iRGD-exosomal KRAS siRNA can effectively suppress cell proliferation. (**: p < 0.01)</div>
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-                            <li>
-                                <div class="collapsible-header active"><i class="material-icons">filter_drama</i>Silencing capability validation in vivo</div>
-                                <div class="collapsible-body">
-                                    <h5>2.1 Establishment of none-small cell lung cancer mouse model with 40 mice to perform validation experiment in vivo</h5>
-                                    <p style="padding-top: 0;">The iRGD-exosomal KRAS siRNA can be released into A549 cells and suppress the expression of KRAS in vitro. To examine the consequence of KRAS knockdown by anti-KRAS siRNA in vivo, we built a non-small cell lung cancer mouse model for in vivo experiment. Forty mice were subcutaneously injected A549-LUC cells to realize tumor implantation. Then tumor volume were measured through bioluminescent imaging several times after injection. The areas that emit fluorescence in the mice bodies represent labeled tumors (tumors developed from the implanted A549-LUC cells), which helps us to monitor the tumor growth, location and metastasis.</p>
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-                                        <img src="https://static.igem.org/mediawiki/2016/1/1f/NJU_China_2016_iGEM_Result-8.jpg" class="responsive-img">
-                                    </div>
-                                    <div align="middle">
-                                        Figure 8. In vivo imaging of tumor-bearing mice. The parts in the mice body representing the tumors xenografted with A549-Luc indicates that the tumors are relatively uniform in size. First panel: the imaging of tumor-bearing mice injected with PBS. Second panel: the imaging of tumor-bearing mice treated with iRGD-exosomal KRAS siRNA.
-                                    </div>
-                                    <div align="middle">
-                                        <img src="https://static.igem.org/mediawiki/2016/9/93/NJU_China_2016_iGEM_Result-9.png" class="responsive-img">
-                                    </div>
-                                    <div align="middle">
-                                        Figure 9. Quantitative analysis of fluorescence intensity of tumors in mice after treatment of PBS or KRAS-siRNA-iRGD-exosomes (exosome). The intensity of tumor fluorescence in mice treated with KRAS-siRNA-iRGD-exosomes showed dramatically decrease compared with the group treated with PBS. (****: p < 0.0001)
-                                    </div>
-                                    <h5 style="padding-top: 30px">2.2 Mice were divided to receive different treatment, testing the function of iRGD-exosomal KRAS siRNA</h5>
-                                    <p style="padding-top: 0;">Mice were randomly assigned to 2 groups (n=20 per group) and treated differently. One group received PBS injections, the other group is treated with iRGD-exosomal KRAS siRNA via tail-vein injections. The administrations were given five times for 2 successive weeks since contamination of HEK293 cells resulted in exosomes shortage and the treatment of mice were delayed.</p>
-                                    <h5 style="padding-top: 30px;">2.3 The measurement of tissues harvested indicates the function of KRAS siRNA on the tumor treatment</h5>
-                                    <p style="padding-top: 0;">Subsequently, mice were sacrificed for tumor harvest after in vivo imaging. All the tumors were measured for length, volumes and weight. Small pieces from every tumor was cut independently and fixed by paraformaldehyde to prepare for histopathological examination. </p>
-                                    <div align="middle">
-                                        <img src="https://static.igem.org/mediawiki/2016/9/96/NJU_China_2016_iGEM_Result-10.png" class="responsive-img">
-                                    </div>
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-                                        Figure 10. Quantitative analysis of tumor weight measurement for mice treated with PBS or KRAS-siRNA-iRGD-exosomes (exosome).
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-                                    <h5 style="padding-top: 30px;">2.4 Pathological section to observe tumor cells</h5>
-                                    <p style="padding-top: 0;">After measurement of tumors, HE staining was conducted to verify the function of iRGD-exosomal siRNA. HE staining enabled better visualization of tissue structure and cell morphology, which can be used for morphological observation of normal and diseased tissue. The detection result showed that tumor cell necrosis rate in experimental group was much higher than control group. In other words, iRGD exosomes KRAS siRNA can effectively induce tumor cell necrosis and suppress cell proliferation</p>
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-                                    <p>Figure 11. Pathological section of tumor tissues from model mice treated with PBS or iRGD-exosomal KRAS siRNA (exosome).</p>
-                                    <h5 style="padding-top: 30px">2.5 iRGD-exosomal KRAS siRNA reduce the KRAS expression in tumor cells in vivo</h5>
-                                    <p style="padding-top: 0;">Then, total protein and RNA were extracted from the rest tumor tissues to evaluate the expression level of KRAS in vivo. Results showed both KRAS protein and mRNA level were reduced in tumor cells of mice injected with KRAS-siRNA-iRGD-exosomes compared with mice treated with PBS. Though the down-regulation is not that evident, it still demonstrated that iRGD-exosomal KRAS siRNA can efficiently be delivered into tumor cells and regulate target gene expression, taking the short time of treatment into account.</p>
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-                                    <p>Figure 12. Quantitative RT-PCR analysis of KRAS mRNA level in tumor cells after treatment of PBS and KRAS-siRNA-iRGD-exosomes (exosome). (***: p < 0.001)</p>
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-                                <div class="collapsible-header active"><i class="material-icons">youtube_searched_for</i>Endotoxin detecting of anti-KRAS siRNA-loaded exosomes</div>
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-                                    <p>Endotoxin is a type of natural pyrogen that was found in outer cell membrane of Gram-negative bacteria and can make impact on over 30 biological activities. To ensure the safety of our drug system, avoid toxicities, thus prove that our achievement is of great value for clinical application, a detecting assay was carried out using an endotoxin test kit. The result was negative, demonstrating that our drug system satisfies the safety requirement.</p>
+                        <span class="card-title">4. Traditional cancer therapies and their defects</span>
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+                        <p>Cancer destroy both patients' body and willpower. Traditional therapies for cancers vary from surgery, radiotherapy to chemotherapy.</p>
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+                        <p>Surgery is the primary method of treatment for most isolated, solid cancer and may play a role in palliation of pain and prolongation of survival.<sup>[12] </sup> Namely, feasibility and efficacy of surgery is based on the type and stage of cancer, and for certain types, surgery is far from sufficient to eliminate the cancer.</p>
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+                        <p>Radiation therapy is a therapy using ionizing radiation, generally as part of cancer treatment to control or kill malignant cells. It may be curative in a number of types of cancer if they are localized to one area of the body. And it may also be used as part of adjuvant therapy, to prevent tumor recurrence after the surgery to remove a primary malignant tumor. Radiation therapy is synergistic with chemotherapy as well, and has been used before, during, and after chemotherapy in susceptible cancer cases.<sup>[13] </sup></p>
-                                    <div align="middle" style="padding-bottom: 30px;">Figure 13. Endotoxin detecting of anti-KRAS siRNA-loaded exosomes</div>
+                        <p>Chemotherapy is another conventional cancer treatment that uses one or more anti-cancer drugs, called chemotherapeutic agents, as part of a standardized chemotherapy regimen. It is always given with a curative intent and aims to prolong life or to reduce uncomfortable symptoms.<sup>[14] </sup></p>
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+                        <p>While radiotherapy and chemotherapy are effective to some extent, the adverse effects remain inevitable, the main kinds of which are fatigue and skin irritation. Acute side effects such as nausea, vomiting, appetite loss, swelling, intestinal discomfort and late side effects such as fibrosis, epilation, dryness and sleeping problem are very common. Immunosuppression, myelosuppression, typhlitis, gastrointestinal discomfort, anemia can also occur after administration of chemotherapy.<sup>[15] </sup> <sup>[16] </sup> Since these therapies will inevitably and indiscriminately damage the normal cells, patients always show fragile health condition after treatment, leading to low prognosis. </p>
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-                                <div class="collapsible-header active"><i class="material-icons">check</i>An efficient drug delivery system</div>
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+                    <div class="card-content">
-                                    <p>KRAS mutation was identified in NSCLC more than 20 years ago, but its clinical importance in cancer therapy just began to be appreciated. Our project aimed to develop a drug system that employed modified exosomes with iRGD peptide on its surface to deliver KRAS-siRNA into lung cancer cells specifically, thus target KRAS gene and down-regulate K-ras protein expression to treat lung cancer cases. Our results had demonstrated that iRGD-exosomal KRAS siRNA can be delivered into tumor cells and efficiently down-regulate KRAS expression both in vitro and in vivo. In silencing validation, we transfected KRAS siRNA into A549 cells using Lipo 2000 and performed western blot to test the function of our siRNA. Subsequently, we collected iRGD-modified exosomes loaded with KRAS siRNA, evaluating its effect on lung cancer cells (A549) by examining KRAS expression after transfection. The result confirmed that iRGD-exosomal KRAS siRNA could effectively down-regulate KRAS transcription level and reduce its protein expression. Later, the EDU assay further ascertained the biological role of KRAS siRNA in cell proliferation suppression in vitro.</p>
+                         <span class="card-title">5. Leading edge of cancer treatment</span>
-                                    <p style="padding-bottom: 30px;">To perform in vivo validation, none-small cell cancer mouse model was established after implanting tumor in 40 mice by subcutaneous injection with A549-LUC cells. After a short-time treatment, tumors harvested from killed mice were measured, then protein and RNA extracted from these tissues were examined, results supporting that KRAS siRNA can efficiently suppress KRAS expression, inhibit cell proliferation and thus have its potential to be taken as a cancer treatment. Besides, endotoxin detecting validated that our drug system satisfies the safety requirement and won’t impact on biological activities. To be more meaningful, we purified the batch of iRGD-exosomal KRAS siRNA for liquid fill, completing a full process including experiment design, compounds synthesis, effect test and drug production.</p>
+                        <p>Nowadays, clinical trials have broadened their ways to immunotherapy, hormone therapy, etc. Take immunotherapy for example: it is one kind of treatment that could induce, enhance, or suppress immune response. Active immunotherapy directs the immune system to attack tumor cells by targeting tumor-associated antigens. Passive immunotherapies enhance existing anti-tumor responses and include the use of monoclonal antibodies, lymphocytes and cytokines.<sup>[17] </sup>Despite much exiting progress, the therapeutic effect isn’t always satisfactory. Due to the heterogeneity and complexity, tumor cells are good liars playing trick on immune system. Till now, relapse and drug resistance are unable to be avoided effectively and immunotherapy remains controversial and immature. Moreover, it can be very expensive in some cases.</p>
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+                        <p>Since cancer is an extremely heterogeneous disease with variants having different genetic and biological properties, drugs targeting a specific molecule that interferes with cancer growth or metastasis are much helpful and effective to ensure that patients will receive optimum management and can be an ideal choice for further exploration. The identification of distinctive mutations in various cancer has led to the booming development of molecular targeted therapy. Small molecule targeted drugs like Gefitinib (Iressa) and Erlotinib (Tarceva) have already been launched into market.<sup>[11] </sup> However, these drugs have drawbacks as high cost of development and inefficiency due to relatively high off-target rate. Furthermore, their depending on the same chemical entity leads to the difficulty of transplanting to another mutation or cancer case. For instance, Gefitinib, as an EGFR inhibitor, is unable to deal with KRAS mutation in NSCLCs.</p>
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-                                    <div align="middle" style="padding-bottom: 30px;">Figure 14. Exosome final products</div>
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+                        <span class="card-title">6. Ideal drug system</span>
+                        <p>In view of serious situation of cancer incidence and mortality, and drawbacks of current treatments, it’s worth developing a much more effective drug system and this is what our project focus on.</p>
+                        <p>Our ideal drug system will contain some basic features as follows:</p>
+                        <p>First, it is designed to be targeting and systemic. Namely, our drug aims at tumor cells and once metastasis appears, it can be a treatment for the whole body as well. In our project, we chose KRAS as our target (插入介绍KRAS的link), and exosome as our delivery agent (插入介绍exosome的link). </p>
+                        <p>Second, precision is one of our goals. To be exact, the targeting peptides on the surface of our delivery agent guarantee the accurate arrival in tumor cells by ligand-receptor interaction. Thus, tumor cell's escape and normal cell's damage will be avoided as much as possible, giving rise to efficiency and minimizing side effects.</p>
+                        <p>In addition, we aim to construct a drug system of high transplantability. There are different types of cancer cases and each case has its distinctive mutations in specific oncogenes. And our system consists of delivery agent (exosome), drug (RNAi) (插入介绍RNAi的link) and targeting tool (iRGD-lamp 2b fusion protein). Hence, it is much easier for us to design a derived drug based on the same silencing and delivery system. By replacing appropriate siRNA and corresponding targeting peptides, it can specifically down-regulate different disease-related genes and thus treat various diseases pertinently. To wit, our drug owns general applicability, leading to remarkable reduce in cost of drug development. </p>
+                        <p>Now you can click three links below to get more detailed information in our drug system.</p>
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+                <h4>Target: KRAS</h4>
+                <h5>KRAS: A Significant Oncogene</h5>
+                <p>Cancer is caused by alterations in oncogenes, tumor-suppressor genes, and microRNA genes. Oncogenes encode proteins that control cell proliferation, apoptosis, or both. They can be activated by structural alterations resulting from mutation or gene fusion [18].</p>
+                <p>The two most commonly mutated oncogenes in lung cancer are the epidermal growth factor receptor (EGFR) and KRAS. In terms of EGFR, EGFR mutants were only recently identified, but they have already been established in the clinic as valid predictors of increased sensitivity to EGFR kinase inhibitors (gefitinib and erlotinib). By contrast, even though KRAS mutations were identified in NSCLC tumors more than 20 years ago, the clinic importance of KRAS mutant in cancer therapy just began to be paid more attention. Recent studies show that lung cancer patients with KRAS mutant do not respond to either EGFR inhibitor or adjuvant chemotherapy. Therefore, KRAS-targeting therapeutics are sorely needed [19]. </p>
+                <p>K-ras protein is a small GTPase (~21 kDa) encoded by KRAS gene [20]. K-ras possesses an essential role in normal tissue signaling. It can be activated by signals from cell surface receptors, subsequently switches on other proteins and ultimately turns on genes, turning on genes involved in cell growth, differentiation, and survival. K-ras uses a bound guanine nucleotide (GDP/GTP) to toggle between its “on” and “off” states. It binds GDP in its neutral state. When a signal arrives, GTP will take place of GDP with the help of guanine nucleotide exchange factors (GEFs), causing a rearrangement of the protein, thus, switching itself “on” to deliver the signal to the final destination [21]. Normally, K-ras has an intrinsic ability to hydrolyze GTP transiently and turn itself “off”. Mutations of KRAS oncogene lead to a strongly reduced GTPase activity of K-ras protein, making KRAS stay at a constitutively activated “on” state. Thereby, the signal is passed continuously, allowing the cell to proliferate without control [22].</p>
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+                    Figure10. K-ras is part of the MAP kinase signaling cascade (RAS/RAF/MEK/ERK) that relays chemical signals from outside the cell to the cell's nucleus and is primarily involved in controlling cell division.
+                </div>
+                <h5>KRAS in Lung Cancer</h5>
+                <p>The two main types of lung cancer are non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) [23]. NSCLC accounts for about 85% of lung cancers. As a class, NSCLCs are relatively insensitive to chemotherapy, compared to small cell carcinoma. Lung adenocarcinoma is the most common subtype of NSCLC, and it accounts for approximately 40% of lung cancers [24]. </p>
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+                    Figure 11. Subtype and mutation frequency of NSCLC [25]
+                </div>
+                <p>It has been found that the activation of the KRAS oncogene is specifically associated with the histological features of adenocarcinoma and does not or rarely occur in other types of NSCLC [26]. Approximately 15–25% of the patients with lung adenocarcinoma have tumor-associated KRAS mutations. In the majority of cases, these mutations are missense mutations which introduce a single amino acid substitution at position 12, 13, or 61. The result of these mutations is constitutive activation of KRAS signaling pathways [27]. In the vast majority of cases, KRAS mutations are found in tumors with wild type for EGFR or ALK; in other words, they are non-overlapping with other oncogenic mutations found in NSCLC. Therefore, KRAS mutation defines a distinct molecular subset of the disease. </p>
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+                    Figure 12. Frequency of major driver mutations in signaling molecules in lung adenocarcinomas [28].
+                </div>
+                <h5>Clinical Importance</h5>
+                <p>far the most important single prognostic factor. But the role of KRAS as either a prognostic or predictive factor in NSCLC still remains mostly unclear at the moment [29]. </p>
+                <p>The KRAS point mutation may not only be valuable as a prognostic marker. Valiant attempts have been made to develop cancer therapeutics targeting mutant KRAS signaling. However, to date, there are no direct anti-KRAS therapies available. </p>
+                <p>The therapeutic potential of systemically delivered RNAi had been demonstrated as a novel treatment for cancer. Thanks to many prominent advantages like nontoxicity and targeting, KRAS siRNA delivery is a promising choice for further drug development. </p>
+            </div>
+            <div class="divider"></div>
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+                    <div class="card-content white-text">
+                        <span class="card-title">Reference</span>
+                        <p class="reference">[1] Global Cancer Facts & Figures (3rd edition), 2015. American Cancer Society.</p>
+                        <p class="reference">[2] Lindsey A. Torre, Freddie Bray, Rebecca L. Siegel, Jacques Ferlay, Joannie Lortet-Tieulent, Ahmedin Jemal. Global Cancer Statistics, 2012. CA CANCER J CLIN. 2015; 00: </p>
+                        <p class="reference">[3] Worldwide Cancer Incidence. International Agency for Research on Cancer of WHO and Cancer Research UK.</p>
+                        <p class="reference">[4] Worldwide Cancer Mortality. International Agency for Research on Cancer of WHO and Cancer Research UK.</p>
+                        <p class="reference">[5] Worldwide cancer mortality statistics. Cancer Research UK. <a href="http://www.cancerresearchuk.org/health-professional/cancer-statistics/worldwide-cancer/mortality">http://www.cancerresearchuk.org/health-professional/cancer-statistics/worldwide-cancer/mortality</a></p>
+                        <p class="reference">[6] Wanqing Chen, Rongshou Zheng, Peter D. Baade, Siwei Zhang, Hongmei Zeng, Freddie Bray, Ahmedin Jemal, Xue Qin Yu, Jie He. Cancer Statistics in China, 2015. CA CANCER J CLIN. 2016; 66:115–132.</p>
+                        <p class="reference">[7] Lung cancer. Wikipedia. <a href="https://en.wikipedia.org/wiki/Lung_cancer">https://en.wikipedia.org/wiki/Lung_cancer</a></p>
+                        <p class="reference">[8] Cancer Statistics. National Cancer Institute. <a href="https://www.cancer.gov/about-cancer/understanding/statistics">https://www.cancer.gov/about-cancer/understanding/statistics</a></p>
+                        <p class="reference">[9] Fact Sheets About Lung and Bronchus Cancer. National Cancer Institute. <a href="http://seer.cancer.gov/statfacts/html/lungb.html">http://seer.cancer.gov/statfacts/html/lungb.html</a></p>
+                        <p class="reference">[10] Non-Small Cell Lung Cancer Treatment (PDQ®)–Health Professional Version. National Cancer Institute. <a href="https://www.cancer.gov/types/lung/hp/non-small-cell-lung-treatment-pdq">https://www.cancer.gov/types/lung/hp/non-small-cell-lung-treatment-pdq</a></p>
+                        <p class="reference">[11] Admir Ahmad, Shirish Gadgeel. Lung Cancer and Personalized Medicine. Springer, 2016; 127, 155-156, 172-173.</p>
+                        <p class="reference">[12] Cancer. Wikipedia. <a href="https://en.wikipedia.org/wiki/Cancer">https://en.wikipedia.org/wiki/Cancer</a></p>
+                        <p class="reference">[13] Radiation therapy. Wikipedia. <a href="https://en.wikipedia.org/wiki/Radiation_therapy">https://en.wikipedia.org/wiki/Radiation_therapy</a></p>
+                        <p class="reference">[14] Chemotherapy. Wikipedia. <a href="https://en.wikipedia.org/wiki/Chemotherapy">https://en.wikipedia.org/wiki/Chemotherapy</a></p>
+                        <p class="reference">[15] Cancer Treatment. National Cancer Institute. <a href="https://www.cancer.gov/about-cancer/treatment">https://www.cancer.gov/about-cancer/treatment</a></p>
+                        <p class="reference">[16] Side Effects. National Cancer Institute. <a href="https://www.cancer.gov/about-cancer/treatment/side-effects">https://www.cancer.gov/about-cancer/treatment/side-effects</a></p>
+                         <p class="reference">[17] Cancer immunotherapy. Wikipedia. <a href="https://en.wikipedia.org/wiki/Cancer_immunotherapy">https://en.wikipedia.org/wiki/Cancer_immunotherapy</a></p>
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